WO2024206245A1

WO2024206245A1 - Methods and compositions for producing ovarian support cell co-culture

Info

Publication number: WO2024206245A1
Application number: PCT/US2024/021368
Authority: WO
Inventors: Christian Kramme; Dina Radenkovic; Martin Varsavsky; Sabrina Amaral PIECHOTA; Kathryn POTTS; Bruna PAULSEN; Graham ROCKWELL; Alexander NOBLETT; Alexa GIOVANNINI; Maria Marchante CUEVAS
Original assignee: Gameto Inc
Current assignee: Gameto Inc
Priority date: 2023-03-24
Filing date: 2024-03-25
Publication date: 2024-10-03
Anticipated expiration: 2025-09-24

Abstract

Featured are methods and compositions for the in vitro maturation of oocytes. In particular, the disclosure features methods and compositions related to engineering a plurality of ovarian support cells (OSCs) for promoting the maturation of an oocyte in a medium. Such methods and compositions are particularly useful for assisted reproduction technology (ART) procedures.

Description

METHODS AND COMPOSITIONS FOR PRODUCING OVARIAN SUPPORT CELL CO-CULTURE

TECHNICAL FIELD

This disclosure relates to the fields of in vitro oocyte maturation and assisted reproductive technology (ART).

BACKGROUND

One in ten women struggle with infertility, requiring assisted reproductive technology (ART), such as in vitro fertilization (IVF). Challenges remain with maintaining oocyte health in culture, resulting in low oocyte quality and subsequently poor embryo quality. Furthermore, oocytes that are developmentally immature are traditionally discarded, constricting the available oocyte pool for IVF. In vitro maturation (IVM) holds the promise to mature oocytes in vitro after egg extraction, allowing for utilization of all retrieved eggs. Current methods for IVM are inefficient, using follicle-stimulating hormone (FSH) spike-in to the culture media, showing 5-40% maturation of immature eggs. Even worse, this method results in many unhealthy eggs, with an embryo viability rate under 17%, far lower than for standard IVF. Thus, there remains a need in the field for promoting oocyte maturation for a female subject undergoing an ART procedure.

SUMMARY OF THE INVENTION

In one aspect, the disclosure features an ex vivo composition that includes one or more ovarian support cells (OSCs) and one or more diluents or excipients. In some embodiments, the composition promotes the maturation of one or more oocytes.

In some embodiments, the one or more OSCs include one or more granulosa cells. In some embodiments, the one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. In some embodiments, the one or more OSCs express one or more genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB. In some embodiments, the one or more OSCs further express one or more genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4. In some embodiments, the one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. In some embodiments, the one or more OSCs further express genes CDH2 and/or NOTCH2. In some embodiments, the one or more OSCs do not exhibit significant expression of RARRES2.

In some embodiments, the one or more OSCs express NR2F2. In some embodiments, the one or more OSCs include ovarian stroma cells. In some embodiments, the one or more OSCs include granulosa cells and ovarian stroma cells.

In some embodiments, the one or more OSCs include more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

In some embodiments, the one or more OSCs are obtained by differentiation of a population of iPSCs. In some embodiments, the iPSCs are human iPSCs (hiPSCs). In some embodiments, the hiPSCs express or overexpress transcription factor RUNX2. In some embodiments, the hiPSCs express or overexpress transcription factor NR5A1 . In some embodiments, the hiPSCs express or overexpress transcription factor GATA4. In some embodiments, the hiPSCs express or overexpress transcription factor FOXL2.

In some embodiments, the hiPSCs express or overexpress transcription factors RUNX2 and NR5A1 . In some embodiments, the hiPSCs express or overexpress transcription factors RUNX2 and GATA4. In some embodiments, the hiPSCs express or overexpress transcription factors RUNX2 and FOXL2. In some embodiments, the hiPSCs express or overexpress transcription factors NR5A1 and GATA4. In some embodiments, the hiPSCs express or overexpress transcription factors NR5A1 and FOXL2. In some embodiments, the hiPSCs express or overexpress transcription factors GATA4 and FOXL2.

In some embodiments, the hiPSCs express or overexpress transcription factors RUNX2, NR5A1 , and GATA4. In some embodiments, the hiPSCs express or overexpress transcription factors RUNX2, GATA4, and FOXL2. In some embodiments, the hiPSCs express or overexpress transcription factors RUNX2, NR5A1 , and FOXL2. In some embodiments, the hiPSCs express or overexpress transcription factors NR5A1 , GATA4, and FOXL2. In some embodiments, the hiPSCs express or overexpress transcription factors RUNX2, NR5A1 , GATA4, and FOXL2.

In some embodiments, the transcription factor expression or overexpression is induced by way of a doxycycline-responsive transcription regulatory element, such as a doxycycline-responsive promoter or enhancer.

In some embodiments, the hiPSCs are contacted with a Wnt/p-catenin pathway activator. In some embodiments, the Wnt/p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

In some embodiments, at least one of the one or more OSCs are encapsulated. In some embodiments, the one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or any mixture thereof. In some embodiments, the one or more OSCs are encapsulated in laminin, such as laminin-521 . In some embodiments, the one or more OSCs are encapsulated in vitronectin.

In some embodiments, the one or more OSCs have reduced expression, or undetectable expression, of one or more genes associated with pluripotency relative to an unmodified iPSC. In some embodiments, the one or more genes associated with pluripotency include NANOG. In some embodiments, the one or more genes associated with pluripotency include POU5F1 .

In some embodiments, at least one of the one or more OSCs produce one or more growth factors. In some embodiments, the one or more growth factors include insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMPs), C-type natriuretic peptide (CNP), or any combination thereof. In some embodiments, at least a portion of the one or more growth factors is secreted.

In some embodiments, the one or more of the OSCs produce one or more steroids. In some embodiments, the one or more steroids include estradiol, progesterone, or a combination thereof. In some embodiments, the one or more steroids are produced in response to hormonal stimulation. In some embodiments, the hormonal stimulation comprises FSH, androstenedione treatment, or a combination thereof. In some embodiments, at least a portion of the one or more steroids is secreted. In some embodiments, the one or more OSCs are cryopreserved. In some embodiments, the composition further includes an in vitro maturation (IVM) media. In some embodiments, the IVM media includes a cell culture media. In some embodiments, the IVM media includes Medicult-IVM media. In some embodiments, the IVM media includes one or more supplements. In some embodiments, the one or more supplements include:

(i) human serum albumin (HSA), optionally at a concentration of about 5 to about 15 mg/mL, further optionally at a concentration of 10 mg/mL;

(ii) recombinant follicle stimulating hormone (rFSH), optionally at a concentration of about 70 mIU/mL to about 80 mIU/mL, further optionally at a concentration of 75 mIU/mL;

(iii) human chorionic gonadotropin (hCG), optionally at a concentration of about 95 mIU/mL to about 105 mIU/mL, further optionally at a concentration of 100 mIU/mL;

(iv) androstenedione, optionally at a concentration of about 495 ng/mL to about 505 ng/mL, further optionally at a concentration of 500 ng/mL;

(v) doxycycline, optionally at a concentration of about 0.5 pg/mL to about 1 .5 pg/mL, further optionally at a concentration of 1 pg/mL; or any combination of the one or more supplements.

In some embodiments, the one or more oocytes are retrieved from a donor subject. In some embodiments, the donor subject is from about 19 years old to about 45 years old. In some embodiments, the subject is undergoing ovarian stimulation. In some embodiments, the ovarian stimulation includes treatment with gonadotropin releasing hormone (GnRH). In some embodiments, the ovarian stimulation includes treatment with one or more GnRH analogs. In some embodiments, the one or more GnRH analog is a GnRH agonist or antagonist. In some embodiments, the ovarian stimulation includes one or more ovulatory triggers. In some embodiments, the one or more ovulatory triggers include human chorionic gonadotropin (hCG). In some embodiments, the one or more ovulatory trigger comprises a GnRH agonist, optionally wherein the GnRH agonist is leuprolide. In some embodiments, the ovarian stimulation includes FSH treatment. In some embodiments, the ovarian stimulation does not include FSH treatment. In some embodiments, the FSH treatment includes 300 international units (IU) to 700 IU of FSH. In some embodiments, the FSH treatment includes 400 IU to 600 IU of FSH. In some embodiments, the FSH treatment includes 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment includes a plurality of injections, wherein each injection includes a dose of about 100 IU to about 200 IU of the FSH. In some embodiments, the ovarian stimulation further includes clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, optionally wherein each dose is between 50 mg and150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 110-130 mg, 125-150 mg; e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg). In some embodiments, the ovarian stimulation further includes one or more hCG triggers. In some embodiments, the one or more hCG triggers includes 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.

In some embodiments, the one or more oocytes are present in cumulus oocyte complexes (COCs). In some embodiments, the one or more oocytes include one or more denuded immature oocytes. In some embodiments, all of the one or more oocytes are denuded immature oocytes. In some embodiments, the one or more oocytes are not denuded.

In some embodiments, the one or more oocytes include one or more germinal vesicle (GV)- containing oocytes. In some embodiments, the one or more of the oocytes include one or more oocytes in metaphase I (Ml). In some embodiments, the one or more of the oocytes include one or more oocytes in metaphase II (Mil). In some embodiments, at least a portion of the one or more oocytes include one or more previously vitrified oocytes. In some embodiments, at least a portion of the one or more oocytes include one or more previously cryopreserved oocytes.

In some embodiments, the one or more oocytes are co-cultured with the one or more OSCs.

In some embodiments, prior to and/or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV-stage to Mil-stage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Mil-stage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality. In some embodiments, the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.

In some embodiments, the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs. In some embodiments, the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes. In some embodiments, the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.

In some embodiments, the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours. In some embodiments, the one or more oocytes are co-cultured with the one or more OSCs for about 24 hours to about 28 hours.

In some embodiments, the one or more oocytes co-cultured with the one or more OSCs form one or more blastocytes following contact with one or more mature sperm cells.

In some embodiments, the one or more oocytes are co-cultured in direct contact with the one or more OSCs. In some embodiments, the one or more oocytes do not directly contact the OSCs. In some embodiments, the culture system is a suspension culture. In some embodiments, the culture system is an adherent culture.

In another aspect, the disclosure features a method of cultivating oocytes, wherein one or more immature oocytes are co-cultured with one or more OSCs.

In another aspect, the disclosure features a method of preparing one or more oocytes that have previously been retrieved from a human subject for use in an assisted reproduction technology (ART) procedure, the method including co-culturing the one or more oocytes with one or more OSCs.

In another aspect, the disclosure features a method of producing a mature oocyte for use in an ART procedure, the method including co-culturing one or more oocytes that have previously been retrieved from a human subject with a population of ovarian support cells that have been differentiated from one or more iPSCs.

In another aspect, the disclosure features a method of inducing oocyte maturation in vitro, the method including co-culturing one or more oocytes with a population of ovarian support cells that have been differentiated from one or more iPSCs, optionally wherein the co-culturing is conducted for a period of from about 6 hours to about 120 hours.

In another aspect, the disclosure features method of producing a mature oocyte for use in an ART procedure, the method including:

(a) differentiating one or more iPSCs to produce one or more OSCs;

(b) retrieving one or more immature oocytes from a subject; and

(c) co-culturing the one or more oocytes with the one or more OSCs, thereby producing one or more mature oocytes.

In another aspect, the disclosure features a method of promoting oocyte maturation for a subject undergoing an ART procedure and that has previously been administered one or more follicular triggering agents during a follicular triggering period, the method including:

(a) retrieving one or more immature oocytes from the subject;

(b) co-culturing the one or more oocytes with one or more OSCs that have been differentiated from iPSCs, thereby producing one or more mature oocytes; and

(c) isolating the one or more mature oocytes.

In some embodiments of any of the preceding aspects, prior to and/or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV-stage to Mil-stage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml- stage to Mil-stage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality. In some embodiments, the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.

In some embodiments, the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not include the one or more OSCs. In some embodiments, the one or more co-cultured oocytes have an improved maturation rate compared to oocytes matured in vivo. In some embodiments, the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes. In some embodiments, the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.

In some embodiments, the one or more OSCs express FOXL2, AMHR2, CD82 or any combination thereof. In some embodiments, the one or more OSCs express one or more of the genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB. In some embodiments, the one or more OSCs further express one or more of the genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4. In some embodiments, the one or more OSCs further express one or more of the genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. In some embodiments, the one or more OSCs further express genes CDH2 and/or NOTCH2, with no significant gene expression of RARRES2. In some embodiments, the one or more OSCs include granulosa cells.

In some embodiments, the one or more OSCs express NR2F2. In some embodiments, the one or more OSCs include ovarian stroma cells.

In some embodiments, the one or more OSCs include granulosa cells and ovarian stroma cells. In some embodiments, the one or more OSCs include more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

In some embodiments, the one or more OSCs are obtained by differentiation of a population of iPSCs. In some embodiments, the iPSCs are hiPSCs. In some embodiments, the hiPSCs express or overexpress transcription factor RUNX2. In some embodiments, the hiPSCs express or overexpress transcription factor NR5A1 . In some embodiments, the hiPSCs express or overexpress transcription factor GATA4. In some embodiments, the hiPSCs express or overexpress transcription factor FOXL2.

In some embodiments, the expression or overexpression of any of the foregoing transcription factors is induced by way of a doxycycline-responsive transcription regulatory element, such as a doxycycline-responsive promoter or enhancer.

In some embodiments, the one or more OSCs are encapsulated. In some embodiments, the one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly- D-Lactone, or a mixture thereof. In some embodiments, the one or more OSCs are encapsulated in laminin, optionally wherein the laminin is laminin-521. In some embodiments, the one or more OSCs are encapsulated in vitronectin.

In some embodiments, the one or more OSCs have low or undetectable expression of one or more genes associated with pluripotency relative to an iPSC. In some embodiments, the one or more genes associated with pluripotency include NANOG. In some embodiments, the one or more genes associated with pluripotency include POU5F1 .

In some embodiments, the one or more of the OSCs produce one or more growth factors. In some embodiments, the growth factors include IGF, SCF, EGF, LIF, VEGF, BMPs, CNP, or any combination thereof. In some embodiments, at least a portion of the one or more growth factors is secreted.

In some embodiments, the one or more OSCs produce one or more steroids. In some embodiments, the one or more steroids include estradiol, progesterone, or a combination thereof. In some embodiments, the one or more steroids are produced in response to hormonal stimulation of the OSCs. In some embodiments, the hormonal stimulation includes exposure to FSH, androstenedione, or a combination thereof. In some embodiments, at least a portion of the one or more steroids is secreted.

In some embodiments, the one or more OSCs are cryopreserved. In some embodiments, the composition further includes an in vitro maturation (IVM) media. In some embodiments, the IVM media includes a cell culture media. In some embodiments, the IVM media includes Medicult-IVM media. In some embodiments, the IVM media includes one or more supplements. In some embodiments, the one or more supplements includes:

In some embodiments, the one or more oocytes are retrieved from a donor subject. In some embodiments, the donor subject is from about 19 years old to about 45 years old. In some embodiments, the subject is undergoing ovarian stimulation. In some embodiments, the ovarian stimulation includes treatment with gonadotropin releasing hormone (GnRH). In some embodiments, the ovarian stimulation includes treatment with one or more GnRH analogs. In some embodiments, the one or more GnRH analog is a GnRH agonist or antagonist. In some embodiments, the ovarian stimulation includes one or more ovulatory triggers. In some embodiments, the one or more ovulatory triggers include human chorionic gonadotropin (hCG). In some embodiments, the one or more ovulatory trigger comprises a GnRH agonist, optionally wherein the GnRH agonist is leuprolide. In some embodiments, the ovarian stimulation includes FSH treatment. In some embodiments, the ovarian stimulation does not include FSH treatment. In some embodiments, the FSH treatment includes 300 international units (IU) to 700 IU of FSH. In some embodiments, the FSH treatment includes 400 IU to 600 IU of FSH. In some embodiments, the FSH treatment includes 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment includes a plurality of injections, wherein each injection includes a dose of about 100 IU to about 200 IU of the FSH. In some embodiments, the ovarian stimulation further includes clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, optionally wherein each dose is between 50 mg and 150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 110-130 mg, 125-150 mg; e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg). In some embodiments, the ovarian stimulation further includes one or more hCG triggers. In some embodiments, the one or more hCG triggers includes 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.

In some embodiments, the one or more oocytes are in cumulus oocyte complexes (COCs). In some embodiments, the one or more oocytes include one or more denuded immature oocytes. In some embodiments, all of the one or more oocytes are denuded immature oocytes. In some embodiments, the one or more oocytes are not denuded.

In some embodiments, the one or more oocytes are co-cultured in direct contact with the one or more OSCs. In some embodiments, the one or more oocytes do not directly contact the OSCs. In some embodiments, the culture system is a suspension culture. In some embodiments, the culture system is an adherent culture. In another aspect, the disclosure features a method of promoting differentiation of one or more induced pluripotent stem cells (iPSCs) to one or more ovarian support cells (OSCs), the method comprising:

(a) culturing the one or more iPSCs in vitro;

(b) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors comprising FOXL2, NR5A1 , RUNX2, GATA4, or any combination thereof, thereby producing differentiated cells;

(c) determining that the differentiated cells resulting from (b) exhibit a gene expression profile that is similar to that of one or more OSCs; and

(d) co-culturing the one or more OSCs with one or more oocytes previously retrieved from a subject, thereby maturing the one or more oocytes.

In another aspect, the disclosure features method of producing one or more ovarian support cells (OSCs) from one or more induced pluripotent stem cells (iPSCs), the method comprising:

(a) culturing the one or more iPSCs in vitro;

(d) co-culturing the identified one or more OSCs with one or more oocytes previously retrieved from a subject, thereby maturing the one or more oocytes.

In another aspect, the disclosure features a method of preparing a composition comprising one or more ovarian support cells (OSCs), the method comprising:

(a) culturing the one or more iPSCs in vitro;

In some embodiments of the preceding aspects, the iPSCs are human iPSCs (hiPSCs). In some embodiments, the iPSCs were previously cryopreserved. In some embodiments, the co-culturing is performed in in vitro maturation (IVM) media. In some embodiments, the IVM media includes a cell culture media. In some embodiments, the IVM media includes Medicult-IVM media. In some embodiments, the IVM media includes one or more supplements. In some embodiments, the one or more supplements include:

(ii) recombinant follicle stimulating hormone (rFSH), optionally at a concentration of about 70 mIU/mL to about 80 mIU/mL, further optionally at a concentration of 75 mIU/mL; (iii) human chorionic gonadotropin (hCG), optionally at a concentration of about 95 mIU/mL to about 105 mIU/mL, further optionally at a concentration of 100 mIU/mL;

In some embodiments, the induction of iPSCs to OSCs occurs for about 1 day to about 10 days, optionally wherein the induction occurs for about 5 days.

In some embodiments, the iPSCs are cultured in a media that includes a matrix. In some embodiments, the matrix includes alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly- D-Lactone, or a mixture thereof. In some embodiments, matrix includes laminin, optionally wherein the laminin is laminin-521 . In some embodiments, the matrix includes vitronectin.

In some embodiments, the iPSCs are reprogrammed using a transposase method to carry one or more inducible transcription factors. In some embodiments, the iPSCs are transformed via electroporation, liposome-mediated transformation, or viral-mediated gene transfer. In some embodiments, the expression or overexpression of the one or more transcription factors is induced in the presence of doxycycline. In some embodiments, the iPSCs are contacted with a Wnt/p-catenin pathway activator. In some embodiments, the Wnt/p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

In some embodiments, the one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. In some embodiments, the one or more OSCs express one or more of the following genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB. In some embodiments, the one or more OSCs further express one or more of the genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4. In some embodiments, the one or more OSCs further express one or more of the genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. In some embodiments, the one or more OSCs further express genes CDH2 and/or NOTCH2, optionally with no significant gene expression of RARRES2.

In some embodiments, the one or more OSCs include one or more granulosa cells.

In some embodiments, one or more OSCs express NR2F2. In some embodiments, the one or more OSCs include one or more ovarian stroma cells.

In some embodiments, the one or more OSCs produce one or more steroids. In some embodiments, the one or more steroids include estradiol, progesterone, or a combination thereof. In some embodiments, the one or more steroids are produced in the presence of one or more hormones. In some embodiments, the one or more hormones includes exposure to FSH, androstenedione, or a combination thereof. In some embodiments, at least a portion of the one or more steroids is secreted.

In some embodiments, the one or more oocytes retrieved from the subject are immature oocytes. In some embodiments, the co-culturing the one or more OSCs with one or more oocytes promotes the maturation of the one or more oocytes. In some embodiments, the method further includes harvesting the one or more oocytes for an assisted reproductive technology procedure.

In some embodiments, the subject is undergoing ovarian stimulation prior to the retrieval of one or more oocytes. In some embodiments, the ovarian stimulation includes treatment with gonadotropin releasing hormone (GnRH). In some embodiments, the ovarian stimulation includes treatment with one or more GnRH analogs. In some embodiments, the one or more GnRH analog is a GnRH agonist or antagonist. In some embodiments, the ovarian stimulation includes one or more ovulatory triggers. In some embodiments, the one or more ovulatory triggers includes hCG. In some embodiments, the one or more ovulatory trigger includes a GnRH agonist, optionally wherein the GnRH agonist is leuprolide. In some embodiments, the ovarian stimulation includes FSH treatment. In some embodiments, the ovarian stimulation does not include FSH treatment. In some embodiments, the FSH treatment includes 300 IU to 700 IU of FSH. In some embodiments, the FSH treatment includes 400 IU to 600 IU of FSH. In some embodiments, the FSH treatment includes 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment comprises a plurality of injections, wherein each injection comprises a dose of about 100 IU to about 200 IU of the FSH. In some embodiments, the ovarian stimulation further includes clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, wherein each dose is between 50 mg and 150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 110-130 mg, 125-150 mg; e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg). In some embodiments, the ovarian stimulation further includes one or more hCG triggers. In some embodiments, the one or more hCG triggers includes 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.

In some embodiments, the one or more oocytes are in cumulus oocyte complexes (COCs). In some embodiments, the one or more oocytes include one or more denuded immature oocytes. In some embodiments, all of the one or more oocytes are denuded immature oocytes. In some embodiments, the one or more oocytes are not denuded prior to or following co-culturing.

In some embodiments, the one or more oocytes include one or more germinal vesicle (GV)- containing oocytes. In some embodiments, the one or more oocytes include one or more oocytes in metaphase I (Ml). In some embodiments, the one or more oocytes include one or more oocytes in metaphase II (Mil). In some embodiments, at least a portion of the one or more oocytes include one or more previously vitrified oocytes. In some embodiments, at least a portion of the one or more oocytes includes one or more previously cryopreserved oocytes.

In some embodiments, prior to and/or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV-stage to Mil-stage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Mil-stage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality. In some embodiments, the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality. In some embodiments, the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs. In some embodiments, the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes. In some embodiments, the one or more co- cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.

In some embodiments, the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours. In some embodiments, the one or more oocytes are cultured with the one or more OSCs for about 24 hours to about 28 hours.

In some embodiments, method further includes isolating one or more Mil-stage oocytes from the co-culture including the one or more oocytes retrieved from the subject with the one or more OSCs.

In some embodiments, the one or more oocytes are cultured in direct contact with the one or more OSCs. In some embodiments, the one or more oocytes do not directly contact the one or more OSCs. In some embodiments, the co-culture is a suspension co-culture. In some embodiments, the coculture is an adherent co-culture.

In another aspect, the disclosure features a cell culture system including one or more ovarian support cells (OSCs), wherein the system promotes maturation of one or more oocytes.

In some embodiments, the one or more OSCs include one or more granulosa cells. In some embodiments, the one or more OSCs express FOXL2, AMHR2, CD82 or any combination thereof. In some embodiments, the one or more OSCs express one or more genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB. In some embodiments, the one or more OSCs further express one or more genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4. In some embodiments, the one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. In some embodiments, the one or more OSCs further express genes CDH2 and/or NOTCH2. In some embodiments, the one or more OSCs do not exhibit significant expression of RARRES2. In some embodiments, the one or more OSCs express NR2F2. In some embodiments, the one or more OSCs include ovarian stroma cells. In some embodiments, the one or more OSCs include granulosa cells and ovarian stroma cells.

In some embodiments, the transcription factor expression or overexpression is under the control of doxycycline.

In some embodiments, at least one of the one or more OSCs are encapsulated. In some embodiments, the one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or any mixture thereof. In some embodiments, the one or more OSCs are encapsulated in laminin, optionally wherein the laminin is laminin-521 . In some embodiments, the one or more OSCs are encapsulated in vitronectin.

In some embodiments, the one or more OSCs have lower expression, or undetectable expression, of one or more genes associated with pluripotency relative to an iPSC. In some embodiments, the one or more genes associated with pluripotency include NANOG. In some embodiments, the one or more genes associated with pluripotency include POU5F1 .

In some embodiments, at least some of the OSCs produce one or more growth factors. In some embodiments, the one or more growth factors include insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMPs), C-type natriuretic peptide (CNP), or any combination thereof. In some embodiments, at least a portion of the one or more growth factors is secreted.

In some embodiments, the one or more of the OSCs produce one or more steroids. In some embodiments, the one or more steroids include estradiol, progesterone, or a combination thereof. In some embodiments, the one or more steroids are produced in response to hormonal stimulation. In some embodiments, the hormonal stimulation comprises FSH, androstenedione treatment, or a combination thereof. In some embodiments, at least a portion of the one or more steroids is secreted.

In some embodiments, the one or more oocytes are retrieved from a donor subject. In some embodiments, the donor subject is from about 19 years old to about 45 years old. In some embodiments, the subject is undergoing ovarian stimulation. In some embodiments, the ovarian stimulation includes treatment with gonadotropin releasing hormone (GnRH). In some embodiments, the ovarian stimulation includes treatment with one or more GnRH analogs. In some embodiments, the one or more GnRH analog is a GnRH agonist or antagonist. In some embodiments, the ovarian stimulation includes one or more ovulatory triggers. In some embodiments, the one or more ovulatory triggers include human chorionic gonadotropin (hCG). In some embodiments, the one or more ovulatory trigger comprises a GnRH agonist, optionally wherein the GnRH agonist is leuprolide. In some embodiments, the ovarian stimulation includes FSH treatment. In some embodiments, the ovarian stimulation does not include FSH treatment. In some embodiments, the FSH treatment includes 300 international units (IU) to 700 IU of FSH. In some embodiments, the FSH treatment includes 400 IU to 600 IU of FSH. In some embodiments, the FSH treatment includes 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment includes a plurality of injections, wherein each injection includes a dose of about 100 IU to about 200 IU of the FSH. In some embodiments, the ovarian stimulation further includes clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, wherein each dose is between 50 mg and 150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 110-130 mg, 125-150 mg; e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg). In some embodiments, the ovarian stimulation further includes one or more hCG triggers. In some embodiments, the one or more hCG triggers includes 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose. In some embodiments, the one or more oocytes are in cumulus oocyte complexes (COCs). In some embodiments, the one or more oocytes include one or more denuded immature oocytes. In some embodiments, all of the one or more oocytes are denuded immature oocytes. In some embodiments, the one or more oocytes are not denuded.

In another aspect, the disclosure features a kit that includes any one of the preceding embodiments of the ex vivo composition and a package insert, wherein the package insert instructs a user of the kit to co-culture the population of ovarian support cells with one or more oocytes in accordance with any one of the preceding methods.

In another aspect, the disclosure features a kit that includes a vial that contains a population of iPSCs and a package insert, wherein the package insert instructs a user of the kit to differentiate the population of iPSCs to one or more ovarian support cells in accordance with any one of the preceding methods.

In another aspect, the disclosure features a kit that includes a vial that contains one or more OSCs and a package insert, wherein the package insert instructs a user of the kit to cultivate any one of the preceding embodiments of the cell culture system.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to illustrate embodiments of the disclosure and further an understanding of its implementations.

FIG. 1 A is a series of UMAP projections and bar graphs depicting single cell RNA-sequencing (scRNA-seq) data obtained from six independent batches of hiPSCs after five days of differentiating with three inducible transcription factors: NR5A1 , RUNX2, and GATA4. The resulting gene expression profiles of the batches were partitioned into clusters (Early GC, GC, Atresia/luteolysis, Mitochondrial gene enriched, and Ribosomal gene enriched) and subclusters (Early GC I, Early GC II, and Early GC III as well as GC I, GC II, and GC III).

FIG. 1B is a collection of gene expression enrichment data derived from the scRNA-seq data from (FIG. 1 A), shown as a bubble plot, violin plots, and UMAP projections, each of which depict the relative expression of genes in each cluster or subcluster.

FIG. 1C is a series of UMAP projections depicting gene expression enrichment data of the differentiated hiPSCs relative to gene expression data that corresponds to Primary GC, Secondary GC, Antral GC, and Pre-Ovulatory GC gene signatures.

FIG. 2 is a series of UMAP projections and bar graphs depicting scRNA-seq clustering data of differentiated hiPSCs that were cultured on a matrix of either laminin or vitronectin.

FIG. 3A is an image of a gel depicting genotype data from a PCR reaction to assess the relative expression levels of transcription factors NR5A1 , GATA4, and RUNX2 from individual clones following hiPSC reprogramming.

FIG. 3B is a series of representative micrographs showing the morphology of the indicated clones following hiPSC differentiation.

FIG. 3C is a graph showing relative expression levels of ovarian support cell biomarkers and an hiPSC biomarker expressed in each indicated clone based on a cut-off (dotted line) as measured by flow cytometry.

FIG. 3D is a bar graph depicting the levels of estradiol (E2) secreted by the selected clones in response to application of follicle stimulating hormone (+F), application of androstenedione (+A), or application of a combination of follicle stimulating hormone and androstenedione (+F+A) to the cell culture media (DK10).

FIG. 3E is a table characterizing the indicated clones based on the ratio of OSC:hiPSC, cell viability, relative expression level of indicated biomarkers, and expression level of progesterone (P4) and estradiol (E2). FIG. 4A is a series of fluorescent microscopy micrographs showing the relative expression of indicated hiPSC biomarkers and a karyotype graph to show the cellular identity of the selected clones prior to reprogramming.

FIG. 4B is a UMAP projection and a bar graph depicting scRNA-seq clustering data of two batches of independently reprogrammed hiPSC populations derived from the same clone.

FIG. 5 is an exemplary table of metabolite formulations.

FIG. 6 is an exemplary flow-chart for preparing a granulosa co-culture.

FIG. 7 is a schematic of an apparatus for aiding in human oocyte maturation in vitro.

FIG. 8A is a schematic of the experimental co-culture IVM approach. hiPSCs are differentiated using inducible transcription factor overexpression to form ovarian supporting cells (OSCs). Immature human cumulus oocyte complexes (COCs) are obtained from donors in the clinic after undergoing abbreviated gonadotropin stimulation. In the lab, embryology dishes are prepared including OSCs seeding as required, and COCs are introduced for IVM co-culture. Oocyte maturation and morphological quality are assessed after 24-28 hours IVM co-culture, and samples are banked for analysis or utilized for embryo formation.

FIG. 8B is a representative image of a co-culture setup at the time of plating containing human COCs (n=5) and 100,000 OSCs. Scale bar is 100 pm. COCs with expanded and unexpanded cumulus are seen with surrounding OSCs in suspension culture.

FIG. 9A shows the maturation rate of oocytes after 24-28 hour IVM experiments in Experiment 1 , including oocyte co-culture with OSCs, or in Media Control, n indicates the number of individual oocytes in each culture condition. Error bars indicate mean ± SEM. p-value is derived from unpaired t-test comparing OSC-IVM to Media Control condition.

FIG. 9B shows the Total Oocyte Score (TOS) generated from imaging analysis of Mil oocytes after 24-28 hour IVM experiments, n indicates the number of individual Mil oocytes analyzed. Median (dashed line) and quartiles (dotted line) are indicated. An unpaired t-test indicated no significant (p = 0.2909) difference between the means. Due to low numbers of retrieved oocytes per donor, oocytes could not be consistently split between both conditions analyzed. Groups contain oocytes from predominantly non-overlapping donor cohorts and pairwise comparisons are not utilized.

FIG. 10A shows the maturation rate of oocytes after 28-hour IVM experiments in Experiment 2, including oocyte co-culture with OSCs or in Commercially available IVM Control, n indicates the number of individual oocytes in each culture condition. Error bars indicate mean ± SEM. p-value derived from paired t-test comparing Experimental OSC-IVM to Control Condition (Commercial IVM Control).

FIG. 10B shows the Total Oocyte Score (TOS) generated from imaging analysis of MH oocytes after 28-hour IVM experiments, n indicates the number of individual MH oocytes analyzed. Median (dashed line) and quartiles (dotted line) are indicated. An unpaired t-test indicated no significant (p= 0.9420) difference between the means. COCs from each donor were randomly and equitably distributed between control and intervention to allow for pairwise statistical comparison.

FIG. 11 A shows the embryo formation outcomes after 28-hour IVM experiments in the subset of oocytes utilized for embryo formation in Experiment 2, including oocyte co-culture with OSCs or in Commercially available IVM Control. Error bars indicate mean ± SEM. Results are displayed as a percentage of total COCs treated in the group. Outcomes for fertilization, cleavage, blastocyst formation, high quality blastocyst formation and euploid blastocyst formation are assessed for both IVM conditions. FIG. 11B shows representative images of embryo formation in OSC-IVM versus Commercial IVM conditions at day 3 cleavage, as well as day 5, 6, and 7 of blastocyst formation. Embryos that were of suitable vitrification quality are labeled as “usable quality blast” and were utilized for trophectoderm biopsy.

FIG. 12A is a schematic of the experimental co-culture IVM approach. hiPSCs were differentiated using inducible transcription factor overexpression to form OSCs. Human oocytes were obtained from donors in the clinic after undergoing standard gonadotropin stimulation, and immature oocytes (GV and Ml) identified after denuding were allocated to this research study. In the embryology lab, dishes were prepared including OSCs seeding as required, and immature oocytes were introduced for IVM co-culture. Oocyte maturation and health were assessed after 24-28 hours IVM co-culture, and oocyte samples were banked for further analyses.

FIG. 12B is a representative image of co-culture setup at time of plating containing immature human oocytes (n=3) and OSCs. Scale bar is 200pm. Denuded GV oocytes are seen with surrounding OSCs in suspension culture.

FIG. 13A shows the maturation rate of oocytes after 24-28 hour IVM experiments, including oocyte co-culture with OSCs (OSC-IVM), or in Media Control (Media-IVM). n indicates the number of individual oocytes in each culture condition. Error bars indicate mean ± SEM. p-value derived from unpaired t-test comparing Experimental OSC-IVM to Control Media-IVM. Due to low numbers of retrieved oocytes per donor, each group contains oocytes from predominantly non-overlapping donor groups and pairwise comparisons are not utilized.

FIG. 13B shows Total Oocyte Scores (TOS) generated from imaging analysis of MH oocytes after 24-28 hour IVM experiments, n indicates the number of individual Mil oocytes analyzed. Median (dashed lines) and quartiles (dotted lines) are indicated. Unpaired t-test indicated no significant (ns, p=0.5725) difference between the means.

FIG. 14A shows representative images of Mil oocytes after 28-hour IVM co-culture with OSCs, stained with fluorescent alpha-tubulin dye to visualize the meiotic spindle. Blue lines transecting the middle of the PB1 and the spindle assembly from the oocyte center were used to derive the PB1 -spindle angle. PB1 -spindle angle ranges are indicated above. An example of an Mil with a missing spindle is provided from the Media-IVM condition.

FIG. 14B shows quantification of the angle between the PB1 and spindle, derived from oocyte fluorescence imaging analysis (as in FIG. 14A). n= indicates the number of individual oocytes analyzed from each condition. Number of Mil oocytes with no spindle assembly observed is also indicated below the axis labels. Median (dashed line) and quartiles (dotted line) are indicated. ANOVA statistical analysis found no significant difference (ns, p=0.1155) between the means of each condition.

FIG. 15A shows UMAP projections of oocyte transcriptomes with symbols colored by experimental batch, experimental condition (OSC-IVM, Media-IVM, IVF-MII), oocyte maturation state, and Leiden cluster. Each symbol represents one oocyte. n=81 oocytes.

FIG. 15B shows UMAP projections colored by scores for each of the gene marker sets (GV and IVF Mil).

FIG. 15C shows UMAP projection generated from the scores of cells for each of the two signature marker sets (GV vs IVF MH), colored by experimental condition, oocyte maturation state, and Leiden cluster. FIG. 15D shows quantification of oocytes in each maturation outcome (GV, Ml and MH) by experimental condition (IVM or IVF), with color distribution indicating percentage of population in each Leiden cluster. Striped bars are utilized to denote clusters with predominantly I VF-like characteristics.

FIG. 16A shows immunofluorescence images of human ovaroid (F66/N.R1 .G.F #4 granulosa-like cells + hPGCLCs) sections at days 2, 4, 14, and 32 of culture, stained for FOXL2 (granulosa), OCT4 (germ cell/pluripotent), and DAZL (mature germ cell). Scale bars are 40 pm.

FIG. 16B shows mouse ovaroid (fetal mouse ovarian somatic cells + hPGCLCs) sections stained as in FIG. 18A. Scale bars are 40 pm.

FIG. 16C shows the fraction of OCT4+ and DAZL+ cells relative to the total (DAPI+) over time in human ovaroids and mouse xenovaroids. Counts were performed at 11 time points on images from 2 replicates of human ovaroids (F66/N.R1 .G.F #4 and F66/N.R2 #1 granulosa-like cells + hPGCLCs) and 1 replicate of mouse xeno-ovaroids.

FIG. 16D shows immunofluorescence images of human ovaroid (F66/N.R2 #1 granulosa-like cells + hPGCLCs) sections at days 4 and 8 of culture, stained for SOX17 (germ cell), TFAP2C (early germ cell), and AMHR2 (granulosa). Scale bars are 40 pm.

FIG. 16E shows DAZL and OCT4 expression observed by immunofluorescence in day 16 ovaroids. Some DAZL+OCT4- cells (arrows) are visible, as well as DAZL+OCT4+ cells (arrows). Ovaroids are also beginning to form follicle-like morphology (arrows). Scale bars are 40 pm.

FIG. 17A shows day 35 human ovaroid (F66/N.R1 .G #7 + hPGCLC) sections stained for FOXL2, OCT4, and AMHR2. Scale bars are 40 pm. Follicle-like structures are marked with triangles.

FIG. 17B shows a whole-ovaroid view of follicle-like structures in human ovaroids (F66/N.R1 .G #7). Scale bars are 1 mm.

FIG. 17C shows a section of human ovaroid (F66/N.R1 .G.F #4 + hPGCLC) at day 70 of culture, stained for FOXL2, NR2F2, and AMHR2, showing multiple small follicles (triangles) consisting single layers of FOXL2+AMHR2+ cells. NR2F2+ cells are interspersed between these. Scale bars are 100 pm.

FIG. 17D shows a section of human ovaroid (F66/N.R2 #1 + hPGCLC) at day 70 of culture, stained for FOXL2, NR2F2, and AMHR2, showing an antral follicle consisting of FOXL2+AMHR2+ granulosa-like cells arranged in several layers around a central cavity. NR2F2 staining is visible outside of the follicle (marked ‘Stroma’). Scale bars are 100 pm.

FIG. 18A shows the expression (Iog2 CPM) of selected granulosa (FOXL2), stroma/theca (NR2F2), and germ cell (PRDM1 ) markers. Expression is from scRNA-seq analysis of ovaroids (F66/N.R1 .G.F #4 granulosa-like cells + hPGCLCs). Data from all samples (days 2, 4, 8, and 14) were combined for joint dimensionality reduction and clustering.

FIG. 18B shows Leiden clustering of four main clusters; the expression (Iog2 CPM) of marker genes is plotted for each cluster from the scRNA-seq analysis of ovaroids (as in FIG. 18A).

FIG. 18C shows the mapping of cells onto a human fetal ovary reference atlas (Garcia-Alonso et al., 2022) and assignment of cell types based on the scRNA-seq analysis described in (FIG. 18A).

FIG. 18D shows the proportion of somatic cell types, germ cells, DAZL+ cells, and DDX4+ cells in ovaroids from each day based on the scRNA-seq analysis described in (FIG. 18A).

FIG. 19A shows denuded oocytes from standard of care.

FIG. 19B shows COCs from minimal stimulation.

FIG. 19C shows OSC-IVM statistically significantly improves oocyte maturation rates. FIG. 20A shows morphological quality of oocytes grown in culture with OSCs-lVM.

FIG. 20B shows the angle between the PB1 and the spindle of oocytes grown in culture with OSCs-lVM.

FIG. 20C shows the high similarity of oocytes grown in culture with OSCs-lVM to in vivo Mil oocytes.

FIG. 20D shows the high similarity of oocytes grown in culture with OSCs-lVM to in vivo Mil oocytes.

FIG. 21A shows the oocyte degradation rate from a toxicity assessment of OSCs-lVM product.

FIG. 21 B shows the fertilization and blastocysts generation of OSCs-lVM product.

DEFINTIONS

Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. The use of the term "including," as well as other forms, such as "includes" and "included," is not limiting.

As used herein, the term “about” refers to a value that is within 10% (10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) above or below the value being described. For instance, the phrase “about 50 mg” refers to a value between and including 45 mg and 55 mg.

As used herein, the term “assisted reproductive technology” or “ART” refers to a fertility treatment in which one or more female gametocytes (oocytes) or gametes (ova) are manipulated ex vivo so as to promote the formation of an embryo that can, in turn, be implanted into a subject in an effort to achieve pregnancy. For example, in some embodiments, an oocyte retrieved from a subject undergoing an ART procedure may be matured in vitro using, e.g., co-culturing methodologies described herein. In some embodiments, upon the formation of a mature oocyte (ovum), the ovum may be treated with one or more sperm cells so as to promote the formation of a zygote and, ultimately, an embryo. The embryo may then be transferred to the uterus of a female subject, for instance, using the compositions and methods in the art. Exemplary ART procedures include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) techniques described herein and known in the art.

As used herein, the terms “subject” refers to an organism that receives treatment for a particular disease or condition as described herein. Examples of subjects and subjects include mammals, such as humans (e.g., a female human), receiving treatment for diseases or conditions that correspond to a reduced ovarian reserve or release of immature oocytes.

As used herein, the terms “controlled ovarian hyperstimulation” or more simply “ovarian stimulation” refers to a procedure in which ovulation is induced in a subject, such as a human subject, prior to oocyte or ovum retrieval for use in embryo formation, for instance, by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Controlled ovarian hyperstimulation procedures may involve administration of follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), and/or a gonadotropin-releasing hormone (GnRH) antagonist to the subject so as to promote follicular maturation. Controlled ovarian hyperstimulation methods are known in the art and are described herein as they pertain to methods for inducing follicular maturation and ovulation in conjunction with assisted reproductive technology.

As used herein, the term “derived from” in the context of a cell derived from a subject refers to a cell, such as a mammalian ovum, that is either isolated from the subject or obtained from expansion, division, maturation, or manipulation (e.g., ex vivo expansion, division, maturation, or manipulation) of one or more cells isolated from the subject. For instance, an ovum is “derived from” a subject or an oocyte as described herein if the ovum is directly isolated from the subject or obtained from the maturation of an oocyte isolated from the subject, such as an oocyte isolated from the subject from about 1 day to about 5 days following the subject receiving ovarian hyperstimulation procedures (e.g., an oocyte isolated from the subject from about 2 days to about 4 days following ovarian hyperstimulation procedures).

As used herein, the term “dose” refers to the quantity of a therapeutic agent, such as a follicle stimulating agent described herein, that is administered to a subject for the treatment of a disorder or condition, such as to enhance oocyte maturation and/or release and promote retrieval and ex vivo maturation of viable oocytes. A therapeutic agent as described herein may be administered in a single dose or in multiple doses. In each case, the therapeutic agent may be administered using one or more unit dosage forms of the therapeutic agent. For instance, a single dose of 100 mg of a therapeutic agent may be administered using, e.g., two 50 mg unit dosage forms of the therapeutic agent. Similarly, a single dose of 300 mg of a therapeutic agent may be administered using, e.g., six 50 mg unit dosage forms of the therapeutic agent or two 50 mg unit dosage forms of the therapeutic agent and one 200 mg unit dosage form of the therapeutic agent, among other combinations. Similarly, a single dose of 900 mg of a therapeutic agent may be administered using, e.g., six 50 mg unit dosage forms of the therapeutic agent and three 200 mg unit dosage forms of the therapeutic agent or ten 50 mg unit dosage form of the therapeutic agent and two 200 mg unit dosage forms of the therapeutic agent, among other combinations.

As used herein, the term “follicular triggering period” refers to the timepoint for administering a follicular triggering agent. The timepoint for administering a follicular triggering agent (i.e. , the follicular triggering period) to a female subject is on day 1 , day 2, or day 3 of her menstrual cycle, with preference for day 2 of her menstrual cycle. However, if the female subject is taking a hormonal contraceptive, then the timepoint for administering a follicular triggering agent is 4-6 days (e.g., 4 days, 5 days, or 6 days) after consuming the last oral contraception pill, with preference for 5 days following the dosing of her last oral contraception pill.

As used herein, the term “follicle-stimulating hormone” (FSH) refers to a biologically active heterodimeric human fertility hormone capable of inducing ovulation in a subject. FSH may be purified from post-menopausal human urine or produced as a recombinant protein product. Exemplary recombinant FSH products include follitropin alfa (GONAL-F, Merck Serono/EMD Serono) and follitropin beta (PUREGON/FOLLISTIM, MSD/Scherig-Plough).

As used herein, the term “human chorionic gonadotropin” (hCG) refers to the polypeptide hormone that interacts with the luteinizing hormone chorionic gonadotropin receptor (LHCGR) to induce follicle maturation and ovulation. hCG may be purified from the urine of pregnant women or produced as a recombinant protein product. Exemplary recombinant hCG products include choriogonadotropin alfa (OVIDREL®, Merck Serono/EMD Serono).

As used herein, the term “in vitro fertilization” (IVF) refers to a process in which an ovum, such as a human ovum, is contacted ex vivo with one or more sperm cells so as to promote fertilization of the ovum and zygote formation. The ovum can be derived from a subject, such as a human subject, undergoing various ARTs known in the art. For instance, one or more oocytes may be obtained from the subject following injection of follicular maturation stimulating agents for controlled ovarian hyperstimulation procedures, e.g., from about 1 day to about 5 days prior after injection of said agents (such as from about one day to about 4 days after injection of follicular maturation stimulating agents to the subject). The ovum may also be retrieved directly from the subject, for instance, by transvaginal ovum retrieval procedures known in the art.

As used herein, the term “intracytoplasmic sperm injection” (ICSI) refers to a process in which a sperm cell is injected directly into an ovum, such as a human ovum, so as to promote fertilization of the ovum and zygote formation. The sperm cell may be injected into the ovum, for instance, by piercing the oolemma with a microinjector so as to deliver the sperm cell directly to the cytoplasm of the ovum. ICSI procedures useful in conjunction with the compositions and methods described herein are known in the art and are described, for instance, in WO 2013/158658, WO 2008/051620, and WO 2000/009674, among others, the disclosures of which are incorporated herein by reference as they pertain to compositions and methods for performing intracytoplasmic sperm injection.

As used herein, the terms “ovum” and “oocyte” refer to a haploid female reproductive cell or gamete. In the context of assisted reproductive technology as described herein, ova may be produced ex vivo by maturation of one or more oocytes isolated from a subject undergoing ART. Ova may also be isolated directly from the subject, for example, by transvaginal ovum retrieval methods described herein or known in the art. Ovum or oocyte as used in this disclosure may refer to a plurality of oocytes. An oocyte may be in complex with surrounding cells such as a cumulus-oocyte complex (COC).

As used herein, the terms “mature ova” and “mature oocyte” refer to one or more ovum or oocyte in metaphase II (Mll)-stage of meiosis and typically has morphological or structural features consistent with metaphase II, such as a polar body and other features described herein.

As used herein, the terms “immature ovum” and “immature oocyte” refer to one or more ovum or oocyte that has not reached MH stage of meiosis. In some embodiments, an immature oocyte may be an oocyte including germinal vesicle (GV)-stage and/or metaphase I (Ml)-stage oocytes as determined by morphological features and/or other indications known in the art.

As used herein, the term “oocyte maturation” refers to the process by which an immature oocyte developmentally transitions to a mature oocyte. Oocyte maturation occurs as immature oocytes undergo cell signaling events incurred by external and internal stimuli. External stimuli may be produced by neighboring cells or supporting cells described herein. Oocyte maturation may occur prior to the release of an oocyte and retrieval from a subject. Oocyte maturation may occur in vitro as a result of culturing methods and culture compositions described herein.

As used herein, an “induced pluripotent stem cell (iPSC)” such as a human iPSC (hiPSC) refers to one or more cells that are self-renewing in an undifferentiated state and can differentiate into any one type of differentiated cell types found in an organism. iPSCs can be derived from non-embryonic sources such as a somatic cell and can proliferate without limit. iPSCs can differentiate into each one of the three embryonic germ layers (i.e. , the endoderm, mesoderm, and ectoderm) and further cell types therein, depending on the induction of transcription factors and/or use of methods of gene editing known in the art. In one example, iPSCs can differentiate into a population of ovarian support cells, as described herein. The pluripotency or “stem-ness” of an iPSC may be verified by the expression of one or more pluripotent cell-specific markers including 0CT4, SSEA3, SSEA4, TRA1 -60, TRA1 -81 , NANOG, SOX2, and/or POU5F1 , among other pluripotent cell-specific markers known in the art and described herein.

As used herein, an “ovarian support cell” (OSC) or “support cell” refers to one or more cells that promotes maturation of one or more oocytes. An OSC may be an ovarian granulosa cell (e.g., a type of granulosa cell described herein). Additionally or alternatively, an OSC may be an ovarian stroma cell (e.g., a type of stroma cell described herein). An OSC may form a cumulus-oocyte complex (COC) with an oocyte. An OSC may be generated from an exogenous source, such as from induced pluripotent stem cells (iPSCs), e.g., human induced pluripotent stem cells (hiPSCs), as described herein. An OSC may be applied to a retrieved oocyte using in vitro cell culture methods and compositions described herein. An OSC may be a mixture of two or more cell types. An OSC may be a mixture of stroma cells and granulosa cells such that the mixture is approximately a 1 :1 population of stroma cells and granulosa cells. An OSC may be a mixture of stroma cells and granulosa cells such that one cell type is in higher relative abundance compared to one or more cell types such that the mixture is approximately a 2:1 population, a 3:1 population, a 4:1 population, a 5:1 population, among other possible population distributions. An OSC may be a mixture of stroma cells and granulosa cells such that one cell type is more abundant in the mixture (e.g., 90% stroma cells and 10% granulosa cells, 80% stroma cells and 20% granulosa cells, 70% stroma cells and 30% granulosa cells, 60% stroma cells and 40% granulosa cells, 40% stroma cells and 60% granulosa cells, 30% stroma cells and 70% granulosa cells, 20% stroma cells and 80% granulosa cells, or 10% stroma cells and 90% granulosa cells, among other possible distributions). In some embodiments, an OSC may be a mixture of stroma cells and granulosa cells in combination with one or more additional cell types.

As used herein, an “ovarian stroma cell” or a “stroma cell” is a cumulus cell surrounding the oocyte to ensure healthy oocyte and subsequent embryo development. An ovarian stroma cell may form a COC with an oocyte. An ovarian stroma cell may express markers consistent with a stroma subtype such as nuclear receptor subfamily 2 group F member 2 (NR2F2), which can be detected by methods known in the art. An ovarian stroma cell may be a steroidogenic stroma cell. An ovarian stroma cell may be produced from differentiated hiPSCs as described herein.

As used herein, a “steroidogenic stroma cell” is a stroma cell that may produce one or more steroids such as estradiol, progesterone, or a combination thereof. One or more steroids may be produced in response to hormonal stimulation, such as by FSH, androstenedione, or a combination thereof. One or more steroids may be secreted.

As used herein, the terms “EGFR” and “ERBB1 ” are interchangeable terms for a gene or a biomarker expressed by a cell (e.g., an ovarian support cell; (e.g., a differentiated iPSC)). EGFR and ERBB1 are gene names of epidermal growth factor receptor (e.g., human epidermal growth factor receptor; NCBI Gene ID: 1956). Other names for the gene are known in the art and include ERRP, HER1 , mENA, PIG51 , and NISBD2.

As used herein, an “ovarian granulosa cell” or a “granulosa cell” is a cumulus cell surrounding the oocyte to ensure healthy oocyte and subsequent embryo development. An ovarian granulosa cell may form a COC with an oocyte. An ovarian granulosa cell may express markers consistent with a granulosa subtype such as FOXL2, CD82 and/or follicle-stimulating hormone receptor (FSHR), which can be detected by methods known in the art. An ovarian granulosa cell may be a steroidogenic granulosa cell. An ovarian granulosa cell may be produced from differentiated hiPSCs as described herein. As used herein, a “steroidogenic granulosa cell” is a granulosa cell that may produce one or more steroids such as estradiol, progesterone, or a combination thereof. One or more steroids may be produced in response to hormonal stimulation, such as by FSH, androstenedione, or a combination thereof. One or more steroids may be secreted.

As used herein, the term “biological sample” or “sample” may refer to a component of an in vitro cell culture system such as one or more isolated cells, a whole population of cells or a portion thereof, and/or cell culture media. Additionally, a biological sample or a sample may refer to a specimen (e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, hair, oocyte, ovum, and/or cells isolated from a subject.

As used herein, the term "express" refers to one or more of the following events: (1 ) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. Expression of a gene or a biomarker of interest in a sample (e.g., a biological sample; e.g., a biological sample that comprises one or more iPSCs, OSCs, oocytes, or a combination thereof) can manifest, for example, by detecting: the quantity or concentration of mRNA encoding a corresponding protein (as assessed, e.g., using RNA detection procedures such as quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RT-PCR), and RNA sequencing (RNA-seq) techniques, among other RNA detection methods known in the art), the quantity or concentration of a corresponding protein (as assessed, e.g., using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), immunofluorescence methods, Western blot, or mass spectrometry, among others), and/or the activity of a corresponding protein (e.g., in the case of an enzyme, as assessed using an enzymatic activity assay known in the art) in a sample. In any one of the preceding processes, the expression can increase or decrease relative to a control sample. In some embodiments, a control sample is a cell or population of cells that has not undergone one or more of the procedures that a sample of interest has undergone. Exemplary procedures include a reprogramming or differentiation method such as any one or more types of reprogramming or differentiation methods directed to an iPSC as described herein, an in vitro maturation method such as an in vitro maturation method directed to one or more oocytes as described herein, or an in vitro fertilization method such as an in vitro fertilization method described herein. In some embodiments, a control sample is a cell or a population of cells that is representative of a particular cell type, such as, an established cell line from a manufacturer or an otherwise characterized cell type (e.g., an iPSC or an OSC). In some embodiments, one or more cells are determined to express a gene or biomarker if the expression level is within an acceptable range as compared to the expression level of a control sample (e.g., within 20%, within 15%, within 10%, or within 5% of the expression level of a control sample). In some embodiments, significant expression refers to an expression level relative to a cut-off value or threshold, such as, e.g., a cut-off value or threshold for an RNA-sequencing or flow cytometry method. In some embodiments, a population of cells may be determined to express a gene or biomarker of interest if a significant portion of the population (e.g., about 50% of the population, about 60% of the population, about 70% of the population, about 80% of the population, about 90% of the population, about 95% of the population, or about 99% of the population) has significant expression of a gene or biomarker of interest by meeting or exceeding a particular cut-off or threshold.

In some instances, no significant expression of one or more target genes or biomarkers is observed. In some embodiments “no significant expression” or “no detectable expression” refers to an expression level that is below the limits of detection for a particular detection method (e.g., an RT-PCR or an ELISA) and/or an expression level that is less than about 95% (e.g., 95%, 96%, 97%, 98%, 99%, or less than 99%) relative to a suitable control, such as a particular cell type (e.g., an undifferentiated iPSC or a typical OSC such as an in vivo OSC). In some embodiments, “no significant expression” or “no detectable expression” refers to an expression level relative to a cut-off value or threshold, such as, e.g., a cut-off value or threshold for an RNA-sequencing or a flow cytometry method. No significant expression or no detectable expression may refer to the relative expression levels of a population of cells, in which a significant portion of the population (e.g., about 50% of the population, about 60% of the population, about 70% of the population, about 80% of the population, about 90% of the population, about 95% of the population, or about 99% of the population) does not have significant expression of a gene or biomarker of interest by having an expression level that is below a particular cut-off or threshold.

As used herein, the term “overexpress” refers to expression of a gene or a biomarker that is increased relative to a basal level of expression for a particular cell type (e.g., an iPSC). The expression of a gene or a biomarker may be increased by 5%, by 10%, by 15%, by 20%, by 25%, by 30%, by 35%, by 40%, by 45%, by 50%, by 55%, by 60%, by 65%, by 70%, by 75%, by 80%, by 85%, by 90%, by 95%, by 100%, or in some instances, greater than 100%, such as 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, or greater than 400%. Overexpression of a gene or biomarker may be detected or measured by any suitable methods for detecting or measuring expression of a gene or a biomarker, such as genotyping methods, mRNA detection methods, or protein detection methods, such as methods described herein and known in the art.

As used herein, the term “doxycycline-responsive transcription regulatory element” refers to a nucleotide sequence such as a promoter that initiates transcription of a gene in the presence of doxycycline (e.g., an effective amount of doxycycline).

As used herein, the terms “oral contraceptive treatment,” “oral contraception,” “contraception,” or “birth control pill” refer to a hormonal method of treatment typically used to prevent pregnancy. Oral contraceptive treatment may block the release of oocytes from the ovaries and may contain hormones including estrogen and progestin.

As used herein, the term “ovarian reserve” refers to the number of oocytes in a subject’s ovaries and the quality of said oocytes. The ovarian reserve naturally declines with age and/or medical conditions described herein. Subjects with a diminished ovarian reserve may seek IVF or other ARTs to achieve a successful pregnancy. Levels of anti-Mullerian hormone (AMH), as described herein, may be indicative of a subject’s ovarian reserve.

As used herein, the term “stimulation protocol” refers to the process of administering to the subject one or more follicular triggering agents during a follicular triggering period.

As used herein, the terms “follicular triggering agent” or “triggering agent” refer to a chemical or biological composition that stimulates release of oocytes from the ovaries during ovulation. Follicular triggering agents may include hormones such as human chorionic gonadotropin and follicle-stimulating hormone. As used herein, the term “induced pluripotent stem cells” (iPSCs) refer to artificial stem cells that derive from reprogrammed and otherwise manipulated harvested somatic cells. iPSCs may differentiate into other cell types including ovarian support cells or granulosa cells via methods known in the art and methods described herein. iPSCs may be human iPSCs (hiPSCs) or iPSCs from, e.g., other mammalian sources.

As used herein, the terms “Clomid” or “clomiphene citrate” are interchangeable terms that refer to a nonsteroidal, ovulatory stimulant that is designated chemically as 2-[p-(2-chloro-1 ,2- diphenylvinyl)phenoxy]triethylamine citrate (1 :1 ) with a molecular formula of C26H28CINO • CeHsO? and a molecular weight of 598.01 g/mol. Clomiphene citrate is a mixture of two geometric isomers in the cis (zuclomiphene) and trans (enclomiphene) forms, in which the mixture contains between 30% and 50% (e.g., about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or about 55%) of the cisisomer (zuclomiphene).

As used herein, the term “cell culture” refers to laboratory methods that enable in vitro cell proliferation and/or cultivation of prokaryotic or eukaryotic cell types.

As used herein, the term “matrix” in the context of cell culture or methods of in vitro cell cultivation refers to a coating such as a substrate or a membrane on a surface of a cell culture receptacle (e.g., a well plate or a Petri dish) that facilitates immobilization or adhesion of a cell to the surface. A suitable matrix may be a protein such as a glycoprotein, a proteoglycan, or a combination thereof. A suitable matrix may derive from plants or animal products or may be synthetic (e.g., a recombinant protein or a polymer). A suitable matrix may be an extracellular matrix protein, a particular isoform thereof, or a purified or partially purified fraction that is enriched for species based on their molecular weight. A suitable matrix may comprise a mixture of components. Exemplary matrices include alginate, laminin (e.g., laminin-111 , laminin-21 1 , laminin-121 , laminin-221 , laminin-332, laminin-31 1 , laminin-321 , laminin- 41 1 , laminin-421 , laminin-51 1 , laminin-521 , and/or laminin-213), vitronectin (e.g., type I, type II, and/or type III), fibronectin (e.g., type I, type II, and/or type III), collagen (e.g., type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XIII, and/or type XXIV), chitosan, hyaluronic acid, Poly-D-Lactone, and hydrogels. Selection of a matrix may depend on cell type or relative cellular composition (e.g., lipid content, surface protein expression; e.g., relative abundance of one or more adhesion receptors).

As used herein, the term “encapsulate” or variations thereof refer to a coating of a cell or a plurality of cells in an in vitro culture system, optionally wherein the cell or plurality of cells is delivered (e.g., implanted, dispensed, or deposited) to a cellular niche (e.g., a tissue or an organ of an organism). Encapsulating a cell or a plurality of cells may increase the efficacy of delivery. A suitable reagent for encapsulation may be a protein such as a glycoprotein, a proteoglycan, or a combination thereof. A reagent for encapsulation may derive from plants or animal products or may be synthetic (e.g., a recombinant protein or a polymer). A suitable reagent for encapsulation may be an extracellular matrix protein, a particular isoform thereof, or a purified or partially purified fraction that is enriched for species based on their molecular weight. A suitable reagent for encapsulation comprise a mixture of components. Exemplary encapsulation agents include alginate, laminin (e.g., laminin-1 1 1 , laminin-21 1 , laminin-121 , laminin-221 , laminin-332, laminin-31 1 , laminin-321 , laminin-41 1 , laminin-421 , laminin-51 1 , laminin-521 , and/or laminin-213), vitronectin (e.g., type I, type II, and/or type III), fibronectin (e.g., type I, type II, and/or type III), collagen (e.g., type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XIII, and/or type XXIV), chitosan, hyaluronic acid, Poly-D-Lactone, and hydrogels.

Selection of a reagent for encapsulation may depend on cell type or relative cellular composition (e.g., lipid content, surface protein expression; e.g., relative abundance of one or more adhesion receptors) of the cells to be encapsulated or of the cells in a niche that will receive the one or more encapsulated cells.

As used herein, the term “Wnt/p-catenin activator” refers to any agent (e.g., a nucleic acid, a protein, a lipid, a small molecule, or a combination thereof) that ultimately upregulates, stimulates, initiates, drives, or induces activation of the Wnt/p-catenin signaling pathway. A Wnt/p-catenin activator may directly contact a positive regulator of the pathway to upregulate said pathway. A Wnt/p-catenin activator may directly contact a negative regulator of the pathway to upregulate said pathway (i.e. , act as an inhibitor of an inhibitor of the Wnt/p-catenin pathway). Regulators of the Wnt/p-catenin signaling pathway are known in the art and can be found elsewhere such as, e.g., Nusse and Clevers, Cell. 169(6):985-999, 2017, hereby incorporated by reference.

In some embodiments, a Wnt/p-catenin activator is a Rho-associated protein kinase (ROCK) inhibitor, such as Y-27642 or an equivalent salt or derivative thereof, the structure of which is shown below:

In some embodiments, a Wnt/p-catenin activator is a glycogen synthase kinase-3 inhibitor, such as CHIR099021 , or an equivalent salt or derivative thereof, the structure of which is shown below:

As used herein, the term “co-culture” refers to a type of cell culture method in which more than one cell type or cell populations are cultivated with some degree of contact between them. In a typical coculture system, two or more cell types may share artificial growth medium.

As used herein, the terms “adherent co-culture systems” or “adherent cell culture” refer to a cell culture arrangement by which cells are attached to a surface for proper growth and proliferation.

As used herein, the terms “suspension co-culture systems” or “suspension cell culture” refer to a cell culture arrangement by which cells are cultivated via dispersion in a liquid medium for proper growth and proliferation. DETAILED DESCRIPTION

Described herein are compositions and methods for use in assisted reproductive technology (ART). For example, the compositions and methods described herein are directed to producing, engineering, and culturing one or more ovarian support cells (OSCs) (e.g., ovarian granulosa, ovarian stroma cells, or a combination thereof) and in vitro maturation of oocytes.

Advantageously, the compositions and methods described herein facilitate the harvest and use of previously discarded oocytes for purposes of in vitro fertilization (IVF) by performing in vitro maturation of immature oocytes via co-culture with ovarian support cells (e.g., ovarian support cells derived from reprogrammed iPSCs). The described in vitro maturation methods improve the ability to use these typically discarded immature oocytes in IVF procedures and may lead to a more cost-effective treatment strategy and reduced risk to a treated subject. For example, the methods can reduce the risk of systemic ovarian overstimulation for subjects seeking IVF procedures by requiring fewer hormone injections and/or lower doses of injected hormones than present IVF treatment options. Aspects of the present disclosure can be used to increase the overall pool of available healthy oocytes in women for use in IVF. Aspects of the present disclosure can also be used to significantly reduce hormone dosing in subjects during egg retrieval and improve oocyte quality in culture. This may greatly expand access to reproductive technology, make the duration of a single cycle significantly shorter and require fewer cycles overall to achieve pregnancy.

I. Ex vivo compositions and cell culture media

In some embodiments, the disclosure provides an engineered cell culture system. In some embodiments, the engineered cell culture system comprises a population of engineered ovarian supporting cells (OSCs). In some embodiments, the subject matter described herein relates to a method of differentiating a population of induced pluripotent stem cells (iPSCs) such as human iPSCs (hiPSCs) to a population of OSCs. In some embodiments, the subject matter described herein relates to in vitro maturation (IVM) methods. In some embodiments, the engineered cell culture system promotes the maturation of one or more oocytes. In some embodiments, the subject matter described herein relates to in vitro fertilization (IVF) methods. In some embodiments, the one or more mature oocytes are utilized in an ART or IVF method. In some embodiments, the engineered cell culture system is an engineered cell co-culture system. In some embodiments, the co-cultured cells are in a suspension culture. In some embodiments, the co-cultured cells are in an adherent culture.

A. Ovarian support cells from human induced pluripotent stem cells

Any one or more OSCs utilized in the methods described herein may be created from iPSCs using transcription factor (TF)-directed protocols. In some embodiments, the iPSCs are mammalian iPSCs. In preferred embodiments, the iPSCs are human iPSCs (hiPSCs). In some embodiments, hiPSCs may be transformed with any one or more plasmids encoding one or more transcription factors. In some embodiments, the differentiation of hiPSCs to OSCs is driven by overexpression of one or more transcription factors. In some embodiments, the one or more TFs comprise FOXL2, NR5A1 , RUNX2, GATA4, or any combination thereof. In some embodiments, undifferentiated hiPSCs are reprogrammed using a transposase method (e.g., a piggyBac transposase method) to carry specific inducible transcription factors (e.g., FOXL2, NR5A1 , RUNX2, and/or GATA4). In some embodiments, hiPSCs may be transformed via electroporation, liposome-mediated transformation, viral-mediated gene transfer, among other cell transformation methodologies known in the art. In some embodiments, gene expression of desired transcription factors may be induced in a doxycycline-dependent manner. In some embodiments, a plasmid or expression vector used for reprogramming hiPSCs may have a reporter gene such as a fluorescent protein. In some embodiments, hiPSCs may differentiate into stroma cells with induced expression of transcription factors including GATA4, FOXL2, or a combination thereof. In some embodiments, hiPSCs may differentiate into granulosa with induced expression of transcription factors including FOXL2, NR5A1 , GATA4, RUNX1 , RUNX2, or a combination thereof. In addition to a combination of one or more transcription factors of FOXL2, NR5A1 , GATA4, RUNX1 , and/or RUNX2, hiPSCs may differentiate into granulosa via expression of KLF2, TCF21 , NR2F2, or a combination thereof.

The OSCs utilized in the methods described herein may be produced using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. “CRISPR” is programmable technology that targets specific stretches of genetic code to edit DNA at precise locations. CRISPR technology may include CRISPR-CAS 9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence, allowing for the insertion of exogenous nucleic acids into a cell’s genome. For example, CRISPR-based gene editing techniques can be used to introduce into an iPSC genome, one or more genes encoding for factors that induce differentiation into OSCs (e.g., granulosa cells or stroma cells). These factors include, e.g., FOXL2, NR5A1 , GATA4, RUNX1 , and RUNX2.

Exemplary CRISPR systems include those that utilize a Cas9 enzyme. Cas9 enzymes, together with CRISPR sequences, form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. CRISPR technology may include Class 1 CRISPR systems including type I (cas3), type III (cas10), and type IV and 12 subtypes. CRISPR technology may include Class 2 CRISPR systems including type II (cas9), type V (cas12), type VI (cas13), and 9 subtypes. In some embodiments, CRISPR technology may involve CRISPR-Cas design tools which are computer software platforms and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR/Cas gene editing system. For example, CRISPR-Cas design tools may include: CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, Synthego Design Tool, and the like. CRISPR technology may also be used as a diagnostic tool. For example, CRISPR-based diagnostics may be coupled to enzymatic processes, such as SHERLOCK-based Profiling of in vitro Transcription (SPRINT). SPRINT can be used to detect a variety of substances, such as metabolites in subject samples or contaminants in environmental samples, with high throughput or with portable point-of-care devices.

In some embodiments, overexpression of the one or more TFs is driven by any suitable induction agent known in the art. In some embodiments, the induction lasts for 1 day, 2 days, 3, days, 4 days, 5 days or longer than 5 days. In some embodiments, transcription factors are constitutively expressed. In some embodiments, a plasmid or expression vector used for reprogramming hiPSCs may have a reporter gene such as a fluorescent protein. In some embodiments, the cells (e.g., hiPSCs) are treated with an agent that primes mesodermal induction. In some embodiments, during the initial induction of one or more transcription factors, cells (e.g., hiPSCs) are treated with an agent that activates Wnt/p-catenin signaling to prime cells for a mesodermal cell fate. In some embodiments, during the initial induction of one or more transcription factors, cells (e.g., hiPSCs) are treated with an agent that inhibits a serinethreonine protein kinase such as Rho-associated protein kinase (ROCK) (e.g., a small molecule ROCK inhibitor; e.g., Y-27642) or glycogen synthase kinase-3 (GSK3) (e.g., a GSK3 inhibitor; e.g., CHIR099021 ).

In some embodiments, the resulting OSCs comprise a population of cells with functional and biological similarity to human granulosa cells (cells that express FOXL2 and AMHR2, among other granulosa biomarkers known in the art and described herein). In some embodiments, the resulting OSCs comprise a population of cells with functional and biological similarity to ovarian stroma cells (cells that express NR2F2, among other stroma cell biomarkers known in the art and described herein). In some embodiments, the resulting OSCs comprise a population of cells comprising both granulosa and stroma cells. In some embodiments, a population of OSCs primarily comprises granulosa cells such that the population of OSCs comprise more than 50%, more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells. Reprogramming of hiPSCs to granulosa may be determined by genotyping methods described herein.

Differentiation of hiPSCs to OSCs (such as granulosa cells) may be determined by relative expression of biomarkers typical of a granulosa cell type including FOXL2, AMHR2, CD82, FSHR, IGFBP7, KRT19, STAR, WNT4, or a combination thereof among other granulosa cell biomarkers known in the art. hiPSCs that are differentiated to OSCs (such as granulosa cells) may be categorized into one or more clusters based on transcriptome profiling. In some embodiments, differentiation of hiPSCs to OSCs (such as granulosa cells) may be determined by relative expression of biomarkers or genes, such as genes associated with cell adherence, chemotaxis, growth factors and/or growth factor receptors, steroids and/or steroid receptors, or a combination thereof, among other genes or biomarkers associated with one or more types of OSCs. Differentiation of hiPSCs to OSCs may be determined by relative expression of biomarkers typical of an OSC (e.g., a granulosa cell), including GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and/or ITGB. In some embodiments, biomarkers typical of an OSC may further include FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and/or ERBB4. In some embodiments, biomarkers typical of an OSC may further include RARRES2, NOTCH2, NOTCH3, ID3, and/or BMPR2. In some embodiments, biomarkers typical of an OSC may further include CDH2 and/or NOTCH2, with no significant expression of RARRES2. In some embodiments, the functional screening of individual clones identifies a stable line harboring the optimal balance of expression of each transcription factor or biomarker.

In some embodiments, application of doxycycline to the cells or cell culture maintains activation of one or more TFs in the OSCs (e.g., for doxycycline-dependent induction).

Expression of one or more genes, transcription factors, and/or biomarkers may be detected or measured by methods of protein and/or mRNA expression that are routine in the art. Exemplary methods of detecting or measuring the relative expression of one or more transcription factors or biomarkers include flow cytometry, RNA-sequencing (RNA-seq) (e.g., single-cell RNA-seq), real-time reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), RT-qPCR, Northern blot analysis, mass spectrometry and proteomic modalities, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunofluorescence or immunodetection methods, among other detection methods known in the art. In some embodiments, reprogramming of hiPSCs to OSCs yield one or more OSCs that share strong gene expression similarity to in vivo granulosa cells (e.g., cells that express FOXL2 and AMHR2) In some embodiments, the OSCs share strong gene expression similarity to in vivo stroma cells (e.g., cells that express NR2F2). In some embodiments, the OSCs recapitulate folliculogenesis progression in vitro through follicle formation. In some embodiments, reprogramming of hiPSCs to one or more OSCs may be determined by production of growth factors and/or hormones that may adequately support in vitro maturation of retrieved oocyte via paracrine and juxtacrine cell signaling. In some embodiments, the OSCs produce one or more growth factors including insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMPs), C-type natriuretic peptide (CNP), or any combination thereof. In some embodiments, the one or more growth factors are secreted from the OSCs. In some embodiments, the OSCs are steroidogenic and produce hormones including estradiol and/or progesterone. In some embodiments, the OSCs are steroidogenic in the presence of an exogenously supplied reagent. In some embodiments, the OSCs such as granulosa cells produce estradiol and/or progesterone upon stimulation of androstenedione and FSH or forskolin. In some embodiments, secretion of estradiol and/or progesterone may be detected or measured by one or more protein detection methods known in the art. In some embodiments, application of doxycycline to the OSCs maintains cell identity and drives steroidogenic activity. In some embodiments, doxycycline is applied to the OSCs or OSC media upon thawing and seeding OSCs derived from reprogrammed iPSCs after cryopreservation and frozen storage (e.g., for doxycycline-dependent induction).

B. Cell culture media

OSCs derived from iPSCs (e.g., hiPSCs) or transgenic OSCs may be provided as a composition further containing a cell culture media. Said OSCs or precursors (e.g., hiPSCs prior to reprogramming) may be cultivated in a cell culture media. In some embodiments, the engineered OSCs can be added to a commercially available reproductive media (e.g., IVF, IVM, (e.g., MEDICULT IVM® media), or LAG media). In some embodiments, cell culture media comprises DMEM/F12 supplemented with Knockout Serum Replacement (KSR). In some embodiments, the cell culture media comprises L-glutamine analogs such as, e.g., GLUTAMAX™ (GIBCO™, Thermo Fisher Scientific, Waltham, MA), optionally wherein the GLUTAMAX™ has been adapted to use animal origin-free reagents. In some embodiments, an embryology lab procures a suitable IVF cell culture media. In some embodiments, the cell culture media comprises Medicult IVM media. In some embodiments, an embryology lab procures a suitable cell culture plate. In some embodiments, the cell culture plate is a GPS Universal dish. In some embodiments, an embryology lab procures an ART-grade mineral oil. In some embodiments, the co-culture is achieved by preparation of an IVM media. In some embodiments, the IVM media comprises a base medium formulation. In some embodiments, the base medium formulation comprises MEDICULT IVM® media.

In some embodiments, the hiPSCs and/or the resulting OSCs described herein are cultured on a matrix or encapsulated during induction, culturing, or co-culturing with an oocyte. In some embodiments, the hiPSCs and/or the OSCs are cultured on a matrix or encapsulated in a matrix comprising alginate. In some embodiments, the hiPSCs and/or the OSCs are cultured on a matrix or encapsulated in a matrix comprising laminin. In some embodiments, the hiPSCs and/or the OSCs are cultured on a matrix or encapsulated in a matrix comprising laminin-521 . In some embodiments, the hiPSCs and/or the OSCs are cultured on a matrix or encapsulated in a matrix comprising vitronectin. In some embodiments, the hiPSCs and/or the OSCs are cultured on a matrix or encapsulated in a matrix comprising collagen. In some embodiments, the hiPSCs and/or the OSCs are cultured on a matrix or encapsulated in a matrix comprising chitosan. In some embodiments, the hiPSCs and/or the OSCs are cultured on a matrix or encapsulated in a matrix comprising hyaluronic acid. In some embodiments, the hiPSCs and/or the OSCs are cultured on a matrix or encapsulated in a matrix comprising dextran hydrogel. In some embodiments, the hiPSCs and/or the OSCs are cultured on a matrix or encapsulated in matrix comprising a MATRIG EL® matrix.

In some embodiments, the cell culture media is supplemented. The cell culture media may be supplemented with human serum albumin (HSA) (e.g., at about 5-15 mg/mL, e.g., 10 mg/mL), follicle stimulating hormone (FSH) (e.g., at about 70-80 mIU/mL, e.g., 75 mIU/mL), human chorionic gonadotropin (hCG) (e.g., at about 95-105 mIU/mL, e.g., 100 mIU/mL), Androstenedione (e.g., at about 495-505 ng/mL, e.g., 500 ng/mL), Doxycycline (e.g., 0.5-1 .5 pg/mL, e.g., 1 pg/mL) and other compounds such as hyaluronidase and/or dPBS. In some embodiments, one or more supplemented proteins are recombinant proteins. In some embodiments, the cell culture media is supplemented with an agent that that activates Wnt/p-catenin signaling, such as, e.g., a ROCK inhibitor or a GSK3 inhibitor.

In some embodiments, the supplemented cell culture media forms an in vitro maturation (IVM) media. In some embodiments, the IVM media is utilized by placement of about 100 pL of the media into the suitable cell culture dish, with a mineral oil overlay the day before oocyte retrieval from a subject. In some embodiments, about 2 to about 4 hours prior to IVM culture, the engineered OSCs are thawed. In some embodiments, the thawed engineered OSCs are centrifuged. In some embodiments, the engineered OSCs are washed with IVM media. In some embodiments, the engineered OSCs are seeded to a cell culture droplet. In some embodiments, the engineered OSCs are seeded at a final concentration of about 1 ,000 cells per 1 pl (e.g., about 500-1 ,000 cells/pL, about 700-1 ,000 cells/pL, about 1 ,000-1 ,200 cells/pL, about 1 ,000-1 ,500 cells/pL, or about 1 ,000-2,000 cells/pL).

C. Production and storage of ovarian support cells

In some embodiments, one or more OSCs described herein may be produced in multiple batches. In some embodiments, the OSCs may be frozen and thawed prior to co-culture methods. In some embodiments, the OSCs are freshly reprogrammed from a population of iPSCs prior to an in vitro maturation method. In some embodiments, the OSCs may be seeded and equilibrated for 2-8 hours (e.g., 2-3 hours, 2-4 hours, 3-4 hours, 4-6 hours, 5-7 hours, 6-8 hours; e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours) before the addition of oocytes for in vitro maturation. In some embodiments, the OSCs may be seeded and equilibrated for about 25-90 minutes (e.g., about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, or about 90 minutes).

In some embodiments, a subject may donate hiPSCs. hiPSCs donation may follow an oocyte retrieval process as discussed herein. A subject participating in hiPSCs donation may be different, or the same, from the subject from which the oocyte was retrieved. In some embodiments, a hiPSC donor may undergo a stimulation protocol as disclosed herein. Select hiPSC clones may be expanded to generate an intermediate cell bank. Select hiPSC clones may be expanded and fully differentiated for immediate use.

In some embodiments, the hiPSCs (e.g., undifferentiated hiPSCs; e.g., intermediate cell banks) or the engineered OSCs described herein are provided in a cryovial. In some embodiments, the cryovial is composed of -125,000 cells (e.g., hiPSCs or OSCs). In some embodiments, the hiPSCs or the engineered OSCs are suspended in a cryoprotectant solution. In some embodiments, the cryoprotectant solution comprises CryoStor CS10. In some embodiments, the cryovial is a plastic vial. In some embodiments, the cryovial is an internal thread liquid nitrogen-suited plastic vial. In some embodiments, the hiPSCs or the engineered OSCs are provided in one or more aggregates. In some embodiments, the hiPSCs or the engineered OSCs are provided in one or more single cell suspensions.

In some embodiments, the hiPSCs or the engineered OSCs (e.g., OSCs produced from reprogrammed hiPSCs) are prepared and then frozen or cryopreserved for later use. Cryopreservation or freezing methods may include using a cryoprotective agent such as dimethyl sulfoxide and/or any other freezing method known in the art. In some embodiments, the hiPSCs or the engineered OSCs are stored in liquid nitrogen. In some embodiments, the hiPSCs or the engineered OSCs are stored in liquid nitrogen until use. In some embodiments, cryopreserved undifferentiated hiPSCs (e.g., intermediate cell banks) are stored in liquid nitrogen. Undifferentiated hiPSCs (e.g., intermediate cell banks) can be thawed for further differentiation steps. In some embodiments, the engineered OSCs are stored in liquid nitrogen until use for IVM methods and applications.

In some embodiments, frozen cells are submitted to different sets of batch release assays, in which cells undergo one or more tests to determine cell quality. In some embodiments, one or more aliquots of frozen cells undergo a panel of tests to determine cell count and/or viability upon thawing, to verify genetic stability, to verify genotype, to confirm sterility, or a combination thereof. One or more aliquots of undifferentiated cells (e.g., undifferentiated hiPSCs; e.g., an intermediate cell bank) may further undergo a panel of tests to measure the presence and/or relative abundance of one or more pluripotency markers (e.g., NANOG, POU5F1 , SOX2, OCT4). One or more aliquots of one or more OSC populations (e.g., final target cell banks) may undergo a panel of tests to measure the presence and/or relative abundance of a marker associated with one or more types of OSCs (e.g., a granulosa or a stroma cell) or the potency of secreted proteins or steroids (e.g., estradiol and/or progesterone production). In further embodiments, one or more aliquots of frozen cells undergo tests to determine risks of embryo toxicity (e.g., the presence of one or more chemicals or agents that may disrupt normal growth, development, or genetic differentiation of an embryo).

D. Ovarian support cells and oocytes for in vitro maturation

In some embodiments, the one or more oocytes are human oocytes. In some embodiments, the one or more oocytes are mammalian oocytes. In some embodiments, the one or more oocytes are mouse oocytes. In some embodiments, the one or more oocytes are rat oocytes. In some embodiments, the one or more oocytes are monkey oocytes. In some embodiments, the one or more oocytes are rhesus macaque oocytes. In some embodiments, the one or more oocytes is obtained from conventional stimulation.

In some embodiments, the engineered OSCs promote the maturation of one or more oocytes outside of the body (i.e. , in vitro maturation (IVM)) by forming a cumulus-oocyte-complex (COC) with one or more oocytes. In some embodiments, the one or more oocytes may be evaluated at any point during IVM based on oocyte scoring, as described in Section I l(C)(i). In some embodiments, the engineered OSCs widen access to in vitro fertilization (IVF) and oocyte freezing by offering a more cost-effective, more efficient, and less invasive methodology for subjects seeking fertility treatments or an ART.

In some embodiments, the engineered OSCs promote IVM of one or more oocytes, as describe in further detail in Section II. In some embodiments, the engineered OSCs do not have any continuing effect (e.g., biological or developmental effect) on the one or more mature oocytes following maturation. In some embodiments, the engineered OSCs are physically separated from the one or more mature oocytes after the IVM and are not present in a sample utilized in a further step of an IVF procedure (e.g., embryo formation and/or embryo implantation). In some embodiments, the one or more mature oocytes are utilized for subsequent IVF procedures including, but not limited to, embryo formation and embryo implantation. In some embodiments, the engineered OSCs are not separated from the one or more matured oocytes and have no biological or developmental impact on a developing embryo.

II. Methods of stimulating oocyte release

The methods described herein may be indicated for a subject who desires to increase the number of usable oocytes from any standard ART that utilizes controlled ovarian hyperstimulation (COH). The increased number of usable oocytes can result from co-culturing immature oocytes with one or more OSCs (e.g., one or more reprogrammed OSCs as described herein) to promote maturation of the immature oocytes that are commonly obtained in typical COH and oocyte retrieval procedures. Current standard of care is to discard retrieved immature oocytes. The OSCs described herein can promote maturation of immature oocytes, thus increasing the number of mature and usable oocytes from a population of retrieved oocytes.

Additionally, the methods described herein may be indicated for a subject seeking assisted reproductive technology procedures but may have limited access due to prohibitively high costs and/or risks associated with traditional methods of ovarian stimulation for oocyte retrieval (e.g., risks of ovarian hyperstimulation syndrome (OHSS)). Traditional methods typically require administering gonadotropins to a subject for ovarian stimulation and retrieval of mature oocytes. Delivery of administered gonadotropins is typically inefficient and requires high concentrations of gonadotropins to ensure that sufficient levels of gonadotropins are delivered to the ovarian follicle for oocyte maturation and release following systemic injection. By performing IVM methods described herein, reduced quantities of gonadotropin can be administered for oocyte retrieval, thereby providing a method that circumvents the costly and potentially dangerous side effects associated with systemic administration of high levels of gonadotropins. Immature oocytes can be retrieved, exposed to conditions that lead to optimized maturation ex vivo, and the resulting mature oocytes can be used for subsequent fertilization, embryo development, blastocyst formation, implantation, and gestation to ultimately become healthy offspring.

A. Subject selection

The methods of stimulating oocyte release described herein are directed to a subject seeking IVF treatment options. In general, a subject is a female with a low oocyte retrieval number or a subject with many immature oocytes. A subject may be between 20 and 45 years old, and a subject is typically 35 years of age or older. A subject may have a reduced ovarian reserve due to advancing age and/or a genetic or medical condition (e.g., polycystic ovarian syndrome (PCOS)) that leads to a reduced ovarian reserve. A subject may have an ovarian reserve of 20 or fewer oocytes such that a subject has 1 to 5 oocytes, 4 to 10 oocytes, 8 to 16 oocytes, or 15 to 20 oocytes, e.g., the subject has 1 oocyte, 2 oocytes, 3 oocytes, 4 oocytes, 5 oocytes, 6 oocytes, 7 oocytes, 8 oocytes, 9 oocytes, 10 oocytes, 11 oocytes, 12 oocytes, 13 oocytes, 14 oocytes, 15 oocytes, 16 oocytes, 17 oocytes, 18 oocytes, 19 oocytes, or 20 oocytes. A subject may have anti-Mullerian hormone (AMH) levels that are consistent with reduced ovarian reserve. A subject may have their AMH levels measured by a blood test and other methods known in the art. A subject may have AMH levels between 1 and 6 ng/mL (e.g., 1 -2 ng/mL, 2-4 ng/mL, or 4-6 ng/mL; e.g., 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, or 6 ng/mL). A subject may have measured estradiol levels between 20 and 50 pg/mL (e.g., 20-30 pg/mL, 25-35 pg/mL, 30-40 pg/mL, 35- 45 pg/mL, or 40-50 pg/mL; e.g., 20 pg/mL, 21 pg/mL, 22 pg/mL, 23 pg/mL, 24 pg/mL, 25 pg/mL, 30 pg/mL, 35 pg/mL, 40 pg/mL, 45 pg/mL, or 50 pg/mL).

A physician or skilled practitioner may evaluate a subject for the methods of stimulating oocyte release by taking a biological sample from the subject. A biological sample may include a laboratory specimen held by a biorepository for research. In some embodiments, a biological sample may include bodily fluids including blood, saliva, urine, semen (seminal fluid), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, optic fluid, gastric fluid, breast milk, cell culture supernatants, and the like. A biological sample may include a medical diagnosis, user input describing how a user is feeling and/or a symptomatic complaint, information collected from a wearable device pertaining to a user and the like. For example, a biological sample may include information obtained from a visit with a medical professional such as a health history. In yet another non-limiting example, a biological sample may include information such as data collected from a wearable device worn by a user and designed to collect information relating to a user’s sleep patterns, exercise patterns, and the like. In an embodiment, a biological sample collected at a particular date and/or time of a user’s menstrual cycle. For instance, and without limitation, a biological sample may be collected on the second day of a user’s menstrual cycle to evaluate one or more hormone levels. The biological sample may be utilized to determine markers of a subject’s ovarian reserve that may be measured by a subject’s AMH levels and/or other hormone levels or other indications. AMH levels of 1 ng/mL or less may be used to indicate a low ovarian reserve. A subject with a low ovarian reserve may have measured AMH levels of 1 .0 ng/mL, 0.9 ng/mL, 0.8 ng/mL, 0.7 ng/mL, 0.6 ng/mL, 0.5 ng/mL, 0.4 ng/mL, 0.3 ng/mL, 0.2 ng/mL, or 0.1 ng/mL. Other biological samples that may be utilized to determine one or more markers of a subject’s overall health include without limitation menstrual cycle progression, and/or monitor circulating hormone levels such as estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), estrone (E1 ), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels, and other hormones.

Other biological sample data taken from a subject includes at least an oocyte. As used in this disclosure, “biological sample data” is data that provides a characterization of the biological, genetic, biochemical and/or physiological properties, compositions, or activities of biological samples. In some embodiments, an oocyte may be an immature oocyte. An “immature oocyte” as used in this disclosure is a one or more immature reproductive cells originating in the ovaries. In some embodiments, an immature oocyte may be an oocyte including GV and/or Ml oocytes. In some embodiments, an immature oocyte may be a plurality of oocytes. An immature oocyte may be immature cumulus-oocyte complexes (COCs) taken from the subject. As used in this disclosure, a “cumulus-oocyte complex” is an oocyte surrounded by specialized granulosa cells. As used in this disclosure, a ’’specialized granulosa cell” is a cumulus cell surrounding the oocyte to ensure healthy oocyte and embryo development. In some embodiments, the immature oocyte may contain an oocyte wherein the specialized granulosa cell is added to mature the oocyte in a cell culture (e.g., a co-culture) and thus create a COC.

In some embodiments of the method, the biological sample may be extracted from the user through an extraction device. An “extraction device” is a device and/or tool capable of obtaining, recording and/or ascertaining a measurement associated with a sample. The extraction device may include a needle, syringe, vial, lancet, Evacuated Collection Tubes (ECT), tourniquet, vacuum extraction tube systems, any combination thereof and the like. For example, the extraction device may comprise a butterfly needle set. Data from a biological sample may include measurements, for example, of serum calcium, phosphate, electrolytes, blood urea nitrogen and creatinine, uric acid, and the like.

In an embodiment of the method, biological sample information of a subject may be obtained from an ultrasound. An “ultrasound,” as used in this disclosure, is any procedure that utilizes sound waves to generate one or more images of a user’s body. For example, an ultrasound may be utilized to obtain an image of a subject’s reproductive organs and/or tissues. In an embodiment, an ultrasound may be performed at a particular time of a subject’s menstrual cycle. For example, a subject may receive an ultrasound on day 2 of her cycle and this may be utilized to determine follicle size and/or follicle count. Selection of a stimulation protocol and/or adjustment to a stimulation protocol may be made utilizing this information. For example, a subject with an ultrasound that shows PCOS may have a dose adjustment made to one or more medications received and/or utilized during a stimulation protocol. In addition, the length of her stimulation protocol may be modified based on her PCOS diagnosis. In an embodiment, an ultrasound may be repeated one or more times throughout a subject’s stimulation protocol, and information obtained may be utilized to adjust her stimulation protocol in real time.

B. Oocyte stimulation protocols

A physician or skilled practitioner may determine the stimulation protocol of oocyte release directed to a subject using the described biological parameters. Such biological parameters include hormone levels (e.g., baseline hormone levels and/or hormone levels due to use of contraceptives), subject anatomy (e.g., follicle size, follicle count, ovarian morphology, and/or uterine morphology), among other biological parameters known to a skilled practitioner. A skilled practitioner may administer a stimulation protocol with any one or a combination of triggering agents, or compositions directed to stimulate follicular maturation and oocyte release, described herein.

Hormone levels or concentrations of other relevant compounds of the biological sample may include estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), estrone (E1 ), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels and the like. In some embodiments, the measurement of hormone levels may be based on blood analysis of the biological sample. For example, blood analysis may include plasma hormone analysis techniques. In some embodiments, measurement of hormone levels may be based on saliva hormone testing techniques. Measurement of hormone levels may be based on other forms of analysis such as hair, urine, and any other form of biological samples described throughout this disclosure. A subject may have a baseline serum level of estradiol from about 30 pg/mL to about 60 pg/mL (e.g., from about 30 pg/mL to about 45 pg/mL, from about 40 pg/mL to about 55 pg/mL, or from about 45 pg/mL to about 60 pg/mL; e.g., about 30 pg/mL, about 35 pg/mL, about 40 pg/mL, about 45 pg/mL, about 50 pg/mL, about 55 pg/mL, or about 60 pg/mL) prior to the follicular triggering period. A subject may have a baseline serum level of progesterone from about 0.5 ng/mL to about 2.5 ng/mL (e.g., from about 0.5 ng/mL to about 1 .0 ng/mL, from about 1 .0 ng/mL to about 1 .5 ng/mL, from about 1 .5 ng/mL to about 2.0 ng/mL, or from about 2.0 ng/mL to about 2.5 ng/mL; e.g., about 1 .0 ng/mL, about 1 .5 ng/mL, about 2.0 ng/mL, or about 2.5 ng/mL) prior to the follicular triggering period.

Additionally, a subject’s contraception (e.g., hormonal contraception) usage may affect assignment of a stimulation protocol. Consideration for contraception may aid in determining the follicular triggering period in the woman’s menstrual cycle. For instance, and without limitation, a subject who is not using any form of contraception may begin her stimulation protocol with recombinant follicle stimulating hormone (rFSH) between the first and third day of her menstrual cycle, with preference for the second day of her menstrual cycle. In yet another non-limiting example, a subject who is using contraception may begin her stimulation protocol with rFSH 4-6 days (e.g., 4 days, 5 days, or 6 days) after consuming her last oral contraception pill, with preference for 5 days following the dosing of her last oral contraception pill. In an embodiment, rFSH stimulation may be utilized for 2 to 3 days (e.g., 2 days or 3 days), depending on a subject’s tolerance, follicle size, and/or growth dynamics. After this 2- or 3-day window, a coasting period of 1 to 3 days (e.g., 1 day, 2 days, or 3 days) may be utilized to monitor follicle size and allow for further follicle maturation and development. A “coasting period,” as used in this disclosure, is any period of time when a medication used throughout a stimulation protocol is not administered and/or consumed. A coasting period may last for example for 1 day, 2 days, 3 days, or more if medically necessary. During a coasting period, a subject may continue to receive one or more ultrasounds to monitor her progression.

Once a follicle size has reached anywhere from between about 8-10 mm (e.g., 7.5 mm, 8 mm, 8.5 mm, 9mm, 9.5 mm, 10 mm, 10.5 mm, or more), a subject may be triggered with a dose of a triggering agent, such as human chorionic gonadotropin (hCG). A “follicle measurement” as used in this disclosure, is any measurement of an ovarian follicle. A follicle may include any sac found in an ovary that contains an unfertilized egg. A follicle measurement may be obtained using any methodology as described herein, including for example an ultrasound, a manual measurement, an automated measurement and the like. In an embodiment, a double hCG injection may be utilized, to induce follicle maturation to prepare one or more follicles for retrieval. A double hCG injection may be two or three injections of hCG. A blood test for one or more hormone levels such as E2, P4, and LH may be performed on the trigger day of the double dose of hCG injection to monitor hormone levels. After the day of the double dose of hCG, one or more hormone levels may be measured such as for example with a blood test to determine and examine levels of E2, P4, and LH.

A “triggering agent” is a chemical that triggers cell generation in the ovaries. A triggering agent (e.g., a follicular triggering agent) may include any substance including any non-prescription and/or prescription product. A triggering agent (e.g., a follicular triggering agent) may include any one or combination of the non-limiting examples such as LUPRON DEPOT® (Abbott Laboratories, North Chicago, IL), Ganirelix (Ferring Pharmaceuticals, Saint-Prex, Switzerland), Cetrotide (Merck Global, Readington Township, NJ), GONAL-F® (Merck Global), FOLLISTIM® (Merck Global), BRAVELLE® (Ferring Pharmaceuticals), CLOMID® (Patheon Pharmaceuticals Inc., Waltham, MA), Serephene (Teva, Tel Aviv-Yafo, Israel), GLUCOPHAGE® (Merck Global), FORTAMET® (Mylan, Canonsburg, PA), PREGNYL® (Schering Plough, Kenilworth, NJ), NOVAREL® (Ferring Laboratories, Parsippany, NJ), Repronex (Ferring Pharmaceuticals), FACTREL® (Zoetis Canada Inc., Kirkland, Canada), MENOPUR® (Ferring Pharmaceuticals), and other drugs that induce cell generation in ovaries that one skilled in the art would understand as applicable. A triggering agent (e.g., a follicular triggering agent) may include human serum albumin, FSH, hCG, androstenedione, and doxycycline among other triggering agents known in the art.

In one embodiment, a subject may not receive a triggering agent (e.g., a follicular triggering agent) to stimulate oocyte production. In one embodiment, a subject may receive multiple injections of a triggering agent over 1 to 4 days (e.g., 1 day, 2 days, 3 days, or 4 days) but no more than 5 days in the preferred stimulation protocol. A subject may receive multiple injections over multiple days such that a subject receives five dose injections of one or multiple triggering agents. For example, a subject may receive three days of stimulation using 300 IU to 700 IU of rFSH per injection (e.g., 300-500 IU, 400-600 IU, 500-700 IU, 300-350 IU, 350-400 IU, 400-450 IU, 450-500 IU, 500-550 IU, 550-600 IU, 600-650 IU, 650-700 IU; e.g., 300 IU, 325 IU, 350 IU, 375 IU, 400 IU, 425 IU, 450 IU, 475 IU, 500 IU, 525 IU, 550 IU, 575 IU, 600 IU, 625 IU, 650 IU, 675 IU, or 700 IU) with one or more injections per day. A subject may receive injections of hCG as a triggering agent (e.g., a follicular triggering agent) using 200-700 pg or 2,500-10,000 IU hCG (e.g., 200-500 pg, 300-600 pg, 400-700 pg, 200-300 pg, 300-400 pg, 400-500 pg, 500-600 pg, or 600-700 pg), with a preferred stimulation dose of 500 pg. A subject may receive one or more administrations (e.g., by oral administration or by injection) of clomiphene citrate in combination with other triggering agents with a dose of 50-150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 110- 130 mg, 125-150 mg; e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg) of clomiphene citrate per injection for up to 8 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days).

Prior to receiving a triggering agent, a subject’s serum may be evaluated for levels of hormones or other relevant compounds. A subject may have serum levels of estradiol from about 250 pg/mL to about 400 pg/mL (e.g., from about 250 pg/mL to about 275 pg/mL, from about 275 pg/mL to about 300 pg/mL, from about 300 pg/mL to about 325 pg/mL, from about 325 pg/mL to about 350 pg/mL, from about 350 pg/mL to about 375 pg/mL, or from about 375 pg/mL to about 400 pg/mL; e.g., about 250 pg/mL, about 260 pg/mL, about 270 pg/mL, about 280 pg/mL, about 290 pg/mL, about 300 pg/mL, about 310 pg/mL, about 320 pg/mL, about 330 pg/mL, about 340 pg/mL, about 350 pg/mL, about 360 pg/mL, about 370 pg/mL, about 380 pg/mL, about 390 pg/mL, or about 400 pg/mL) prior to receiving a triggering agent. A subject may have serum levels of progesterone from about 0.25 ng/mL to about 0.75 ng/mL (e.g., from about 0.25 ng/mL to about 0.35 ng/mL, from about 0.35 ng/mL to about 0.45 ng/mL, from about 0.45 ng/mL to about 0.55 ng/mL, from about 0.55 ng/mL to about 0.65 ng/mL, or from about 0.65 ng/mL to about 0.75 ng/mL; e.g., about 0.25 ng/mL, about 0.30 ng/mL, about 0.35 ng/mL, about 0.40 ng/mL, about 0.45 ng/mL, about 0.50 ng/mL, about 0.55 ng/mL, about 0.60 ng/mL, about 0.65 ng/mL, about 0.70 ng/mL, or about 0.75 ng/mL) prior to receiving a triggering agent. A subject may have serum levels of LH from about 1 .0 mIU/mL to about 2.5 mIU/mL (e.g., from about 1 .0 mIU/mL to about 1 .5 mIU/mL, from about 1 .5 mIU/mL to about 2.0 mIU/mL, or from about 2.0 mIU/mL to about 2.5 mIU/mL; e.g., about 1 .0 mIU/mL, about 1 .25 mIU/mL, about 1 .5 mIU/mL, about 1 .75 mIU/mL, about 2 mIU/mL, about 2.25 mIU/mL, or about 2.5 mIU/mL) prior to receiving a triggering agent. A subject may have serum levels of FSH from about 11 mIU/mL to about 14 mIU/mL (e.g., from about 11 mIU/mL to about 12 mIU/mL, from about 12 mIU/mL to about 13 mIU/mL, or from about 13 mIU/mL to about 14 mIU/mL; e.g., about 11 mIU/mL, about 12 mIU/mL, about 13 mIU/mL, or about 14 mIU/mL) prior to receiving a triggering agent.

The triggering agent (e.g., a follicular triggering agent) may be administered over a course of time to produce a follicle stimulation protocol that is a minimal stimulation protocol. The minimal stimulation protocol is configured by a skilled practitioner to trigger the release of a cell in the span of about 3 days. A “minimal stimulation protocol” is a stimulation process spanning over a shortened period of time, compared to average in vitro fertilization (IVF) stimulation protocols, to aid in inducing an ovary to produce an oocyte. Typically, the average span of time for a stimulation protocol using standard IVF is approximately 8-14 days. The minimal stimulation protocol may induce the release of a cell in a span of 8 days or less (e.g. 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day; e.g., between 1 -3 days, between 2-4 days, between 3-5 days, between 4-6 days, between 5-7 days, or between 6-8 days; e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days), which is a shorted period of time compared to the 8-14 days of standard IVF stimulation protocols. The average time for performing a minimal stimulation protocol may be 2 days. The average time for performing a minimal stimulation protocol may be 3 days. The average time for performing a minimal stimulation protocol may be 4 days. The average time for performing a minimal stimulation protocol may be 5 days. The average time for performing a minimal stimulation protocol may be 6 days. In an embodiment, the minimal stimulation protocol may not require administration of a follicular triggering agent for successful retrieval and subsequent maturation of an oocyte. In an embodiment, the minimal stimulation protocol may include selecting a first triggering agent (e.g., a follicular triggering agent) and selecting a second triggering agent (e.g., a follicular triggering agent) as a function of a follicle measurement and/or other biological sample data.

C. Oocyte retrieval

Following follicular stimulation, oocytes (or a group of cells containing an oocyte) are retrieved from the subject. An “oocyte,” as used in this disclosure, is a reproductive cell originating from an ovary. Approximately 24-48 hours (e.g., between 24-32 hours, between 32-40 hours, between 40-48 hours; e.g., about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours) after final dose of triggering agent (e.g., a follicular triggering agent) that is administered, a subject may undergo an oocyte retrieval. On the day of oocyte retrieval, a blood test for one or more hormone levels such as E2, LH, FSH and/or P4 may be performed to ensure quality metrics, hormone levels are within range, and/or that hCG dose was ingested. Hormone levels of E2 may be from about 300 pg/mL to about 450 pg/mL (e.g., from about 300 pg/mL to about 350 pg/mL, from about 350 pg/mL to about 400 pg/mL, or from about 400 pg/mL to about 450 pg/mL; e.g., about 300 pg/mL, about 325 pg/mL, about 350 pg/mL, about 375 pg/mL, about 400 pg/mL, about 425 pg/mL, or about 450 pg/mL) on the day of oocyte retrieval. Hormone levels of LH may be from about 3 mIU/mL to about 6 mIU/mL (e.g., from about 3 mIU/mL to about 4 mIU/mL, from about 4 mIU/mL to about 5 mIU/mL, or from about 5 mIU/mL to about 6 mIU/mL; e.g., about 3 mIU/mL, about 3.5 mIU/mL, about 4 mIU/mL, about 4.5 mIU/mL, about 5 mIU/mL, about 5.5 mIU/mL, or about 6 mIU/mL) on the day of oocyte retrieval. Hormone levels of FSH may be from about 6 mIU/mL to about 9 mIU/mL (e.g., from about 6 mIU/mL to about 7 mIU/mL, from about 7 mIU/mL to about 8 mIU/mL, or from about 8 mIU/mL to about 9 mIU/mL; e.g., about 6 mIU/mL, about 6.5 mIU/mL, about 7 mIU/mL, about 7.5 mIU/mL, about 8 mIU/mL, about 8.5 mIU/mL, or about 9 mIU/mL) on the day of oocyte retrieval. Hormone levels of P4 may be from about 0.5 ng/mL to about 1 .5 ng/mL (e.g., from about 0.5 ng/mL to about 1 .0 ng/mL, from about 0.75 ng/mL to about 1 .0 ng/mL, from about 1 .0 ng/mL to about 1 .5 ng/mL, or from about 1 .25 ng/mL to about 1 .5 ng/mL; e.g., about 0.5 ng/mL, about 0.75 ng/mL, about 1 .0 ng/mL, about 1 .25 ng/mL, or about 1 .5 ng/mL) on the day of oocyte retrieval.

Oocytes (or a group of cells containing an oocyte) are retrieved from the subject using methods known in the art. For example, oocytes may be retrieved via aspiration using a transvaginal ultrasound with a needle guide on the probe to suction released follicular contents. Follicular aspirates may then be examined using a dissection microscope and washed with HEPES media (G-MOPS Plus, VITROLIFE®) and filtered with a 70-micron cell strainer (FALCON®, Corning). Oocytes and/or COCs are then transferred to culture dishes and media to begin co-culturing and appropriate controls, as described herein. Other retrieval methods may include an extraction device, such as a needle, syringe, vial, lancet, Evacuated Collection Tubes (ECT), tourniquet, vacuum extraction tube systems, any combination thereof and the like. For example, the extraction device may comprise a butterfly needle set.

A retrieved oocyte may include but is not limited to an immature oocyte, a mature oocyte, a group of one or more oocytes, a group of one or more cells, such as a cumulus oocyte complex, among other examples. A “cumulus oocyte complex” (COC) as used in this disclosure, is an oocyte containing one or more surrounding cumulus cells. A COC may contain an immature oocyte. A COC may contain a mature oocyte.

An “immature oocyte” as used in this disclosure is one or more immature reproductive cells originating in the ovaries. In some embodiments, an immature oocyte may be an oocyte including but not limited to germinal vesicle stage (GV) and metaphase I stage (Ml) oocytes, as described further below. In some embodiments, an immature oocyte may be a plurality of oocytes. An immature oocyte may be immature cumulus-oocyte-complexes (COCs) taken from a subject. A “mature oocyte” as used in this disclosure, may be one or more mature oocytes in metaphase II stage (MH). Once retrieved, a COC may rest for 1 hour, 2 hours, 3 hours or more to allow for equilibration to in vitro conditions for in vitro maturation.

At the time of retrieval, any one or more of the retrieved oocytes or cells described herein may be appropriately frozen and stored using methods known in the art for future use, analysis, or experimentation. Additionally, any one or more of the retrieved oocytes or cells described herein may be used fresh (i.e., ready for immediate use such as use for in vitro maturation or any one or more analyses or experimentation described herein).

III. Method of oocyte rescue

A. Oocyte denudation

Following oocyte retrieval methods described above, one or more COCs may require oocyte denudation. As described in this disclosure, “oocyte denudation” refers to the removal of cumulus cells or other cell types from the oocyte by means of mechanical separation, chemical separation, or combinations thereof. Several methods of oocyte denudation are known in the art. In some embodiments, denudation may occur in a IVM well, by gently mechanically disassociating cells by pipetting to remove most cumulus and/or granulosa cells. If enzymatic disassociation is needed, the cells may be transferred to a separate dish for hyaluronidase treatment. COCs may be stripped with stripper tips and washed in IVM media or MOPS plus media to clean the oocyte for imaging and if needed inactivate hyaluronidase. Stripper tips may include 200 micron and/or 400 microns for fine cleaning. In some embodiments, germinal vesical (GV)-stage) and metaphase I (Ml)-stage oocytes may be formulated and utilized in cultivation following denudation of the COCs. Denuded COCs may be transferred to a separate culture dish for imaging.

B. Co-cultu ing oocytes for in vitro maturation

/. Co-culture contents and timing

In the methods described herein, an oocyte may be combined with one or more OSCs such as one or more granulosa or stroma cells. An OSC is a cumulus cell that surrounds the oocyte to ensure healthy oocyte and subsequent embryo development. In some embodiments, the granulosa and/or stroma co-culture cells derive from differentiated induced pluripotent stem cells (iPSCs) such as human iPSCs (hiPSCs) as described herein (see, Section 1(A)). As used in this disclosure, a “co-culture” is a cell cultivation set-up, in which two or more different populations of cells are grown with some degree of contact between them. In some embodiments, steroidogenic granulosa cells derived from human induced pluripotent stem cells hiPSCs, may be co-cultured with immature oocytes to form COCs, thereby reconstituting the follicular niche in vitro to promote rapid and efficient oocyte maturation in a manner that reinforces oocyte health and developmental competence. As used in this disclosure, a “steroidogenic granulosa cell” is a granulosa cell expressing high levels of steroidogenic enzymes that produce estradiol. For example, a steroidogenic granulosa cell may be a mural granulosa cell extracted from the antral follicle. Applying steroidogenic granulosa cells in the co-cultures of COCs may increase oocyte maturation in vitro after egg/oocyte retrieval, allowing for utilization of all retrieved eggs/oocyte by directly supplying nutrients, raw materials, and mechanical support to oocytes throughout gametogenesis and folliculogenesis. Steroidogenic granulosa cells may grow and perform oocyte maturation of immature oocytes in standard IVF and IVM media as described further below. This may increase the overall pool of available, healthy oocytes for use in IVF and reduce the number of ova/oocyte retrieval procedures a user is subjected to.

In some embodiments of the method, a cell culture may be formed by combining an immature oocyte with a population of engineered OSCs, which is added to mature the oocyte in the cell culture and thus create a COC after extraction of one or more oocytes following the minimal stimulation protocol. In an embodiment, one or more specialized granulosa cells and/or specialized stroma cells may be thawed during a resting period of one or more COCs. In an embodiment, anywhere from between 50,000- 150,000 specialized granulosa cells (e.g., 50,000-60,000 cells, 60,000-70,000 cells, 70,000-80,000 cells, 80,000-90,000 cells, 90,000-100,000 cells, 100,000-110,000 cells, 110,000-120,000 cells, 120,000- 130,000 cells, 130,000-140,000 cells, or 140,000-150,000 cells; e.g., 50,000 cells, 55,000 cells, 60,000 cells, 65,000 cells, 70,000 cells, 75,000 cells, 80,000 cells, 85,000 cells, 90,000 cells, 95,000 cells, 100,000 cells, 105,000 cells, 110,000 cells, 115,000 cells, 120,000 cells, 125,000 cells, 130,000 cells, 135,000 cells, 140,000 cells, 145,000 cells, or 150,000 cells) may be combined with a COC during culturing. In an embodiment, thawed specialized granulosa cells may be placed into a culture media prior to COC retrieval, including anywhere from about 24-120 hours beforehand (e.g., about 24-48 hours, about 48-72 hours, about 72-96 hours, about 96-120 hours; e.g., about 24-36 hours, about 30-40 hours, about 36-48 hours, about 48-56 hours, about 56-72 hours, about 72-84 hours, about 80-96 hours, about 90-100 hours about 96-108 hours, about 108-120 hours; e.g., about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 56 hours, about 60 hours, about 65 hours, about 72 hours, about 78 hours, about 86 hours, about 92 hours, about 96 hours, about 102 hours, about 110 hours, about 115 hours, about 120 hours). A COC may be transferred into culture media containing thawed specialized granulosa cells to form a group culture as described below in more detail. In an embodiment, a group culture may be cultured in an incubator ranging in time from anywhere between 12-48 hours (e.g., 12-16 hours, 12-20 hours, 18-24 hours, 18-36 hours, 24-36 hours, 36-48 hours; e.g., 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours). The co-culture may be conducted at a biologically suitable temperature, e.g., 37°C.

In some embodiments of the method, a retrieved oocyte, including immature cumulus-oocyte complexes, may be cultured in a group culture. A “group culture” is an extracted COC combined with one or more additional cells. An additional cell may include any cell grown together with an extracted COC. An additional cell may include a specialized stroma cell. An additional cell may include a specialized granulosa cell. In an embodiment, a group culture may be cultured and/or incubated for a particular length of time, such as from between 12-120 hours (e.g., 12-24 hours, 12-36 hours, 24-48 hours, 36-60 hours, 54-72 hours, 68-96 hours, 96-120 hours; e.g., 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours, or 120 hours). For example, group culturing may include culturing the COCs with a granulosa co-culture as described further below. In some embodiments, group culturing may include culturing a control group of COCs with no co-culture, as described further below. In some embodiments, a user may donate immature oocytes, such as GV-stage and Ml-stage oocytes that may be used in medium as part of the group culture to help grow COCs. Oocyte donation may follow an oocyte retrieval process as discussed above. A subject participating in oocyte donation may be different, or the same, from the subject related to the second biological sample containing immature COCs. In some embodiments, an oocyte donation subject may undergo a stimulation protocol as disclosed above.

In some embodiments, the maturity of the oocyte retrieved from the subject may dictate the length of time during which the oocyte is co-cultured with ovarian support cells (e.g., specialized granulosa cells and/or specialized stroma cells). For example, less mature oocytes (e.g., GV oocytes) may require longer co-culturing periods than oocytes at a more advanced stage of meiosis (e.g., Ml oocytes).

In some embodiments regarding the culture of oocytes, cell culture media may include LAG media (Medicult, COOPERSURGICAL®). For example, LAG media may be used for the incubation of oocytes and/or COCs post-retrieval from minimal stimulation protocol. For example, a modified-Medicult IVM media may be used as a baseline control during the culturing process. In some embodiments, the cell culture media may include metabolites as exemplified in FIG. 5. For example, the modified-Medicult IVM media may include human serum albumin, FSH, hCG, androstenedione, doxycycline, or any combination thereof. Media may be equilibrated for about 18 to 24 hours (e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours) pre-culture in a standard sterile 37°C incubator with 02 (e.g., having a 1 -10% 02 atmosphere, such as 4-8% 02 or 5-7% 02, e.g., 6% 02) and proper C02 levels, which are known in the art. Co-cultures and specialized granulosa cell cultures may be adherent cell cultures in cell culture dishes or flasks. Co-cultures and specialized granulosa cell cultures may be suspension cell cultures in cell culture flasks. Cell culture materials and methods include standard sterile cell culturing methods known in the art. Cell morphology and cell viability may be evaluated via one or more established methods known in the art.

In some embodiments, co-culturing is performed in accordance with the steps outlined in FIG. 6. For example, a population of ovarian support cells (e.g., ovarian granulosa cells) may be cryopreserved, thawed. In some embodiments, the ovarian support cells are centrifuged to form a cell pellet and are subsequently resuspended in media suitable for in vitro maturation. In some embodiments, the ovarian support cells are centrifuged one or more additional times and, each time, are resuspended in in vitro maturation media. The ovarian support cells may then be co-cultured with an oocyte obtained from the subject undergoing an ART procedure, thereby inducing oocyte maturation.

/'/. Granulosa cells from hiPSCs

Specialized granulosa cells utilized in the methods described herein may be created from hiPSCs using transcription factor (TF)-directed protocols described herein.

In some embodiments, a subject may donate hiPSCs. hiPSCs donation may follow an oocyte retrieval process as discussed above. A subject participating in hiPSCs donation may be different, or the same, from the subject from which the oocyte was retrieved. In some embodiments, a hiPSC donor may undergo a stimulation protocol as disclosed above.

In some embodiments, hiPSCs, granulosa cells, cumulus cells, oocytes, GV-stage oocytes, Ml- stage oocytes, Mil-stage oocytes and all other types of cells described through this disclosure may be lysed, extracted for genomic material and flash frozen for further manipulation and/or analysis (e.g., for analysis as part of an omics data collection technique described in Section I ll(C)(iii), below). For example, cells may undergo enzymatic cell lysis using enzymes such as lysozyme, lysostaphin, zymolase, cellulose, protease or glycanase, and the like. Other lysis methods may be applied such as chemical lysis, detergent lysis, alkaline lysis, mechanical lysis, thermal lysis, acoustic lysis, physical lysis, nonmechanical lysis and other lysis methods known in the art. In some embodiments, culture media may be flash frozen. Freezing methods may include using a cryoprotective agent such as dimethyl sulfoxide and/or any other freezing method known in the art.

C. Oocyte rescue

/. Oocyte scoring

At any stage of in vitro maturation or directly following in vitro maturation, an oocyte and/or granulosa cells may be appropriately frozen and stored for future analyses, experimentation, or for use in oocyte maturation. Oocytes may be scored with a scoring metric based on their morphology as determined by imagine analysis. In some embodiments, assignment of the scoring metric may include imaging the group cultures and analyzing the images of one or both of co-culture and no co-culture growth media-only control groups. In some embodiments, oocytes are scored and comparatively analyzed during any such stage of in vitro maturation. For example, group culture images may contain a pre-culture group COC image, a post-culture group COC image, and a post-culture denuded oocyte image. In some embodiments, oocytes subjected to scoring have never been frozen. In some embodiments oocytes subjected to scoring via image analysis may be thawed after storage by freezing. In some embodiments, oocytes subjected to scoring may be retrieved without in vitro maturation as described. In some embodiments, oocytes subjected to scoring may be cultured without described granulosa. In some embodiments, images may be sent to a qualified third party, such as an embryologist, developmental biologist, or other relevant skilled practitioner for scoring assignment.

In some embodiments of the methods described herein, oocytes may be assessed and subsequently classified by their maturation state according to the following criteria:

GV - presence of a germinal vesicle, typically containing a single nucleolus within the oocyte. Ml - absence of a germinal vesicle within the oocyte and absence of a polar body in the perivitelline space between the oocyte and the zona pellucida.

Mil - absence of a germinal vesicle within the oocyte and presence of a polar body in the perivitelline space between the oocyte and the zona pellucida.

In some embodiments of the method, the scoring metric may include total oocyte scoring (TOS) as a function of analyzing the imaged group cultures via relevant microscopy or imaging analysis software. Methods and approaches of TOS have been described in the art (Lazzaroni-Tealdi et al., PLoS One 10:e0143632, 2015). Oocyte scoring may include metrics such as shape, size, ooplasm characteristics, structure of the perivitelline space (PVS), zona pellucida (ZP), polar body (PB) morphology, among other possible qualifiers. Total oocyte scoring on both pre and post culture oocyte images for generation of the TOS metric may be based on a scale system of -6 to + 6.

Regarding oocyte shape, if oocyte morphology is poor (dark general oocyte coloration and/or ovoid shape), it may be assigned a value of -1 ; if it is almost normal (less dark general oocyte coloration and less ovoid shape), it may be assigned a value of 0; if it is judged to be normal, it may be assigned a value of + 1 . Regarding oocyte size: if oocyte size is defined as abnormally small or large, it may be assigned a value -1 if size is below 120 pm or greater 160 pm. If the size is almost normal, i.e., does not deviate from normal by more than 10 pm, a value of 0 may be assigned, and a value of + 1 may be assigned if oocyte size is within normal range > 130 pm and <150 pm. Regarding ooplasm characteristics, if the ooplasm is very granular and/or very vacuolated and/or demonstrates several inclusions, a value of -1 may assigned. If it is only slightly granular and/or demonstrates only few inclusions, a value of 0 may be assigned. Absence of granularity and inclusions may result in a +1 value. Regarding structure of the perivitelline space (PVS), the PVS may defined as -1 with an abnormally large PVS, an absent PVS or a very granular PVS. It may be assigned a value of 0 with a moderately enlarged PVS and/or small PVS and/ or a less granular PVS. A value of +1 may be assigned to a normal size PVS with no granules. Regarding, zona pellucida (ZP), if ZPs is very thin or thick (<10 pm or >20 pm) the oocyte may be assigned a -1 . If the ZP does not deviate from normal by more than 2 pm it may be assigned 0. A normal zona (> 12 pm and <18 pm) may be assigned a +1 . Regarding polar body (PB) morphology, PB morphology is defined as follows: Flat and/or multiple PBs or zero PBs, granular and/ or either abnormally small or large PBs is designated as -1 . PBs, judged as fair but not excellent may be designated as 0, and a designation of +1 may be given to PBs of normal size and shape. In some embodiments, Mil oocytes PB score may not be aggregated into TOS. In some embodiments of the method, the scoring metric may include performing an outcome analysis as a function of the TOS as defined and exemplified in FIG. 7. Parametric or non-parametric tests may be applied to determine the significance of findings during the analysis. Outcome analysis may be used to determine GV-stage to Mil-stage oocyte maturation rate; GV-stage to Ml-stage oocyte maturation rate; Ml-stage to Mil-stage oocyte maturation rate; Average Total Oocyte Score; Average Oocyte Shape; Average Oocyte Size; Average Ooplasm quality; Average PVS quality; Average ZP quality; Average Polar Body quality, and the like. In some embodiments these outcomes may reported as a as mean, median, and deviation.

/'/. In vitro fertilization and embryo culture

In some embodiments of the methods, any one or more ova or oocytes as described herein may be evaluated for quality or maturation state, such as by the scoring metrics described herein, to determine their readiness for use in in vitro fertilization and embryo formation.

In some embodiments of the method, the ova or oocytes may be matured via in vitro maturation and subsequently utilized for IVF and/or ART as described herein. Any one or more oocytes may be utilized for intracytoplasmic sperm injection (ICSI). Following fertilization of the ovum by contact with one or more sperm cells, the subsequently formed zygote can be matured ex vivo so as to produce an embryo, such as a morula or blastula (e.g., a mammalian blastocyst), which can then be transferred to the uterus of a subject (e.g., a subject from which the oocyte was initially harvested) for implantation into the endometrium. Embryo transfers that can be performed using the methods described herein include fresh embryo transfers, in which the ovum or oocyte used for embryo generation is retrieved from the subject and the ensuing embryo is transferred to the subject during the same menstrual cycle. The embryo can alternatively be produced and cryopreserved for long-term storage prior to transfer to the subject.

Hi. Omic data collection and analyses of oocytes, cells, and culture media

In some embodiments of the method, the scoring metric may include an Omics-based analysis. For example, frozen cell lysates and cell culture media may be analyzed for bulk RNA-sequencing, whole genome bisulfite sequencing (WGBS), mass spectrometry-based proteomics and metabolomics. Cell culture media may be utilized for metabolomics analysis to determine changes in molecular content of media following co-culture compared to pre-culture media controls. This may be utilized to profile dynamic changes in paracrine signaling between granulosa cells and oocytes. The data gathered may then be aggregated for downstream analysis for determination of changes in epigenetic state, metabolite presence, and gene expression between different co-culture conditions and controls.

In some embodiments of the method, an omics-based analysis may include, genomics, proteomics, transcriptomics, pharmacogenomics, epigenomics, microbiomics, lipidomics, glycomics, transcriptomics culturomics, and/or any other omics one skilled in the art would understand as applicable. In some embodiments, after cultivation, an oocyte that has failed to mature, showing GV or Ml characteristics, may be harvested for single cell RNA-sequencing, along with their associated granulosa cells from their culture. For this, oocytes and granulosa cells may be flash frozen and for library preparation. Of the oocytes that display Mil oocyte development, half may be harvested for single cell RNA-sequencing along with their associated granulosa cells using the above flash freeze methods described throughout this disclosure. The remaining half of Mil oocytes may be utilized for proteomic studies. The culture media for all conditions may additionally be flash frozen and utilized for metabolomics and proteomics to identify cholesterol metabolite levels and paracrine protein production. For example, frozen cell lysates and cell culture mediums may be analyzed for bulk RNA-sequencing, whole genome bisulfite sequencing (WGBS), mass spectrometry-based proteomics and metabolomics. Cell culture media may be utilized for metabolomics analysis to determine changes in molecular content of media following co-culture compared to pre-culture media controls to profile dynamic changes in paracrine signaling between granulosa cells and oocytes. As the media components are flash frozen, the sample is effectively quenched and amenable to metabolic assessment. The data gathered may then be aggregated for downstream analysis for determination of changes in epigenetic state, metabolite presence, and gene expression between different co-culture conditions and controls.

IV. Kits or articles of manufacture

The compositions or methods described herein can be provided in a kit for use in reprogramming iPSCs (e.g., hiPSCs) into a population of OSCs (e.g., granulosa cells and/or stroma cells). In some embodiments, the compositions and methods described herein can be provided in a kit for use in coculturing one or more oocytes with OSCs to produce one or more mature oocytes, optionally wherein the resulting mature oocytes are further fertilized to form an embryo in an ART or IVF procedure. In some embodiments, the kit may include a package insert that instructs a user of the kit to perform iPSC differentiation and/or in vitro maturation. In further embodiments, the kit may include a package insert that instructs a user of the kit to perform any one of the methods of ovarian stimulation and/or oocyte retrieval described herein. The kit may optionally include a syringe or device for administering the compositions of the present disclosure or for retrieving one or more oocytes. In some embodiments, the kit may include one or more additional cell media or agents used for cell culture. In some embodiments, the kit includes one or more antibodies or binding molecules to detect the expression of one or more genes or biomarkers described herein.

EXAMPLES

Example 1. A method of producing ovarian support cells by reprogramming induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are derived from adult cells that have been reprogrammed into stem cells with the potential to differentiate into any cell type in the body and, as a result, have widespread applications in biomedicine. We have applied an established combinatorial and technical platform to engineer iPSCs with inducible transcription factors that drive differentiation towards ovarian cell types. These cells, which are called ovarian support cells (OSCs), express protein markers similar to granulosa cells, the essential functional cells of the ovary, and exhibit similar transcriptomic and steroidogenic profiles. Our OSCs provide a much more physiologically relevant, ovarian-like dynamic environment for IVM than media alone, leading to improved maturation outcomes and therefore creating significant potential for our engineered cells as an ART to improve fertility treatments in the clinic. Detailed in this example is our work to develop and optimize approaches for consistent manufacturing at scale under good manufacturing practice conditions with animal origin-free materials without impacting product purity, efficacy, and safety. We provide a case study and methodology for clinical translation of both iPSCs in cell therapy and ART for fertility treatments, showing that a strategic plan for scalable and controlled manufacturing ultimately creates a much more consistent and functional product.

/. Methods for Example 1

Source material

We first procured an allogeneic hiPSC line (VCT-37-F35) from Reprocell USA (9000 Virginia Manor Rd #207, Beltsville, MD 20705) to serve as the starting material for our clinical-grade cell line. The stem-cell line, derived from human-skin fibroblasts, was generated under good manufacturing practice (GMP) conditions using a non-integrating, mRNA-based reprogramming technology with controlled conditions and GMP-compliant reagents used for the entirety of the manufacturing process. Regarding specific stages in that process, proper controls were implemented for fibroblast derivation according to established guidelines, while reprogramming and cell expansion took place under fully GMP conditions in compliance with regulatory standards and guidelines of the FDA, EMA, and PMDA. We performed a riskbased assessment for safety and suitability of the VCT-37-F35 parental cell line based on supporting documentation to evaluate compliance with regulatory requirements, finding donor eligibility compliant with 21 CFR 1271 , Subpart C and donor consent for broad use and indication. The sourced clonal hiPSC line was expanded in a Research Cell Bank (RCB) prior to our internal cell engineering. The Research Cell Bank was created by expanding the parental cell line for one passage and storing multiple samples cryopreserved to ensure sufficient material for engineering, as well as to secure a stock of the parental cell line in ideal culture conditions, either as a back-up or for future use. Reprocell, Inc., the provider of the original hiPSC line, executed in depth characterization to ensure conformance with established commercial release criteria, while we performed further testing in downstream steps of the process to confirm pluripotency, identity, genetic stability, and potency of cells.

Plasmid Manufacturing

Plasmids utilized for engineering were screened for identity, integrity, and purity verified by whole plasmid sequencing utilizing nanopore technologies and were stored at -20°C, while glycerol stocks of transformed bacteria are stored at -80°C. Plasmids encoding the transcription factors and piggyBac transpose were transfected into hiPSCs with a Lonza NUCLEOFECTOR™ device.

Cell Engineering

Engineering of hiPSCs with specific transcription factors was performed using a piggyBac transposase strategy, which enables integration of multiple copies of the transgene into the host genome. To increase efficiency of the engineering process, puromycin selection was utilized to eliminate cells without integration of the transcription factors. The engineered stem cells are exposed to the Rho- associated protein kinase inhibitor, Y-27632, and the WNT activator, the glycogen synthease kinase-3 inhibitor CHIR99021 , that together prime the cells into a mesodermal fate. Exposure to doxycycline throughout the entire process induces overexpression of the transcription factors, NR5A1 , RUNX2, and GATA4, directing differentiation of hiPSCs towards OSCs. This robust Dox-induced, TF-directed differentiation requires five days in culture. Cell screening, selection, and preliminary characterization

Following the preliminary round of testing on the pooled population of transfected hiPSCs, clones were established by limiting dilution in multiwell plate format. All wells were closely monitored daily until identification of single clones in each well. Wells with more than one clone identified were discarded. Each identified clone was further expanded and cryopreserved resulting in 43 seed clones. Each clone was initially assigned a unique code based on their plate location. The 43 seed clones were subjected to genotyping PCR to identify the presence of the three transcription factors. This initial screening resulted in identification of nine clones harboring all the transcription factors, each of which were then subjected to a more in-depth screening process, including identity, potency, and safety assays for the identification of a lead candidate cell line. To identify the lead candidate, each of the nine clones was individually differentiated and subjected to a series of assays to ensure identity (pluripotency markers, donor identity, and genotyping), conformance (cell count and viability), potency (OSC production and function), and most importantly safety (genomic integrity, vector copy number, mycoplasma, sterility, and adventitious agents) of the clones. The leading candidate clone selected to be used as the starting material for the clinical- grade cell line was named GTO-101 .

/'/. Transcription factor mediated differentiation consistently generates ovarian support cells in different stages of ovarian development and folliculogenesis

To evaluate the feasibility of utilizing a gene-modified hiPSC line, harboring three inducible transcription factors (NR5A1 , RUNX2, and GATA4), as a source to generate consistent and functional OSCs, we compared 6 independent batches of hiPSC-derived OSCs differentiated across 8 months by multiple operators following a standard operating procedure. Differentiation of OSCs mediated by overexpression of inducible transcription factors is a fast and straightforward process compared to standard protocols that rely on small molecules to recapitulate developmental trajectories. After 5 days of induction, hiPSCs multiply 3.12±1 .77 times, and acquire morphological features that resemble human granulosa cells, such as clusters of cells with spiky edges and granules observed in the cell body. Differentiated OSCs also express FOXL2 and CD82, two well characterized markers of granulosa cellfate, indicating successful differentiation into the desired cell type.

To further characterize the molecular phenotype of the differentiated OSCs, as well as better understand differences and similarities among independent batches, we performed single cell RNA- sequencing (scRNA-seq) on cryopreserved samples from six batches of differentiation (FIG. 1 A). We identified 15 initial Leiden clusters that were combined by molecular similarities, resulting in nine final clusters. Among the 15 initial Leiden clusters identified, 12 of them expressed markers demonstrated to be differentially expressed in human granulosa cells (GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, ITGB1 ) compared to other cell types in the developing ovary (Garcia-Alonso et al). These clusters were all identified as granulosa cells, and therefore assigned as the two major classes: Early GCs or GCs. The remaining three clusters were included in a third major class identified as Others, as expression of major markers was not evident in these groups of cells (FIG. 1 A).

Despite the expression of all the granulosa markers, the class assigned as Early GCs also shares transcriptional similarities to preGC-l and -lla/llb, including expression of the genes FOXO1 and CDH1 (FIG. 1 B). A subcluster of these cells, labeled as Early GC I expresses the aromatase gene, CYP19A1 , which has been described to be upregulated in preGC-ls in the ovarian medulla, as well as the gene for the chemotactic protein, RARRES2, which has been shown to reduce steroidogenesis and block oocyte meiotic progression in bovine models. The subcluster Early GC II also expresses the gene for RARRES2, similarly to the previous subcluster described, in addition to the receptor NOTCH2 (FIG. 1 B). The NOTCH signaling pathway is involved in the oocyte-GC crosstalk during folliculogenesis, and high levels of expression of NOTCH2 and NOTCH3 in cumulus cells have been positively correlated with IVF response. Finally, in the subcluster Early GC III expression of RARRES2 is no longer observed, as in the previous subcluster; while NOTCH2 expression continues to be detected in significant levels. Altogether, these patterns of expression suggest that the clusters in the Early GC class share transcriptional signature with both granulosa cells and preGC-l and -lla/llb, and differential expression of CYP19A1 , RARRES2, and NOTCH2, suggest a gradual developmental and functional progression from the subcluster Early GC I to Early GC III.

The class of GCs is marked by the expression of CDH2 in addition to all the other granulosa markers previously described, including the NOTCH2/3 receptors (FIG. 1 B). The subcluster GC I is enriched for the genes NRG1 , BMPR1 B, and genes of the ERBB family of receptors (FIG. 1 B). NRG1 has been identified to be differentially expressed in preGC- lla/l lb and was found to be expressed and secreted by granulosa cells in response to ovulatory surge. BMPR1 B, EGFR (ERBB1 ), and ERBB4 are all receptors identified in granulosa cells and have counterpart ligands expressed in oocytes (BMP6, TGFA, and NRG4, respectively). These interactions have been proposed to mediate follicular assembly. The subcluster labeled as GC II is enriched by expression of the gene ID3, which is a target of the receptor BMPR2, also expressed by these cells. Interestingly, although BMPR2 is expressed by all Early GCs and GC clusters, the CG II subcluster is the subcluster with the strongest enrichment of this target gene (FIG. 1 B), suggesting activation of the receptor BMPR2 in these cells. The last subcluster from the GC class, GC III is composed of cells expressing both CDH2 and NOTCH2, but this subcluster is not enriched for any of the other genes previously described in the subclusters for this class. In summary, we believe that the three subclusters of GCs represent ovarian support cells in slightly different cell states mediated by a distinct combination of active signaling pathways.

The last three subclusters identified (Atresia/Luteolysis; Ribosomal enriched; and Mitochondrial enriched) were incorporated into a third class labeled as Others. These clusters have overall lower expression of most markers including GJA1 and CDH2 (FIG. 1 B). Lower expression levels of GJA1 and CDH2 have been described in GCs undergoing early stages of atresia. Interestingly, cells on the Atresia/Luteolysis subcluster also express genes involved in steroidogenesis, such as CYP11 A1 , CYP19A1 , and HSD17B1 , as well as CGA, which is an estrogen receptor alpha-responsive gene in human breast cancer cells. The other two clusters are also enriched for the GCA gene, but the top expressed genes in each of the clusters are either mitochondrial genes in the Mitochondrial enriched subcluster or ribosomal genes for the Ribosomal enriched subcluster. Generally, enrichment of mitochondrial and/or ribosomal genes in scRNA-seq analysis is associated with poor quality cells, further suggesting that these clusters are composed of dying cells. It is important to highlight that it is unclear whether this observation is a consequence of the biology of ovarian support cells or whether it is an artifact of processing and handling the samples.

After identifying that the large majority of the cells in our analysis was classified as granulosa cells (Early GCs and GCs), we sought to understand whether our protocol gave rise to OSCs in different stages of folliculogenesis or whether cells were overrepresented by a specific follicular stage. For that, we leveraged as a reference a published transcriptome landscape of human folliculogenesis to generate gene signature scores that were then applied to our samples. We did not observe a clear representation of either the Primary GC or Secondary GC stages within our samples, and most of the genes associated to these signature scores were not enriched in the analyzed cells. Conversely, the signature scores for Antral GC and Pre-ovulatory GC were more clearly represented within the clusters identified in our analysis, and multiple genes driving these signatures seem to be enriched by multiple clusters (FIG. 1 C).

Following characterization of the cellular outcome resultant from our differentiation process, we investigated how reproducible and consistent independent batches of hiPSC-derived OSCs would be from each other. Overall, all batches analyzed consistently generated clusters from the 3 major classes previously described (Early GCs, GCs, and Others), five of which were very similar in terms of cluster distribution per batch. These results demonstrate a consistent methodology of producing OSCs from reprogrammed hiPSCs for clinical use for ART or fertility treatments.

Hi. Translation of the protocol towards clinical manufacturing leads to more reproducible cellular outcomes

As part of the strategy to translate the research manufacturing protocol towards clinical standards, we performed a risk assessment on our bill of materials, which led to the substitution of key components of the protocol by higher quality alternatives, including animal origin free reagents, GMP manufactured components, and cell-therapy grade raw materials. Among all the raw materials utilized during the generation of OSCs for research purposes, one of the reagents with the highest complexity was matrigel, which is derived from Engelbreth-Holm-Swarm mouse sarcoma cells, and contains multiple extracellular matrix components of tissue basement membranes. Due to the nature of this reagent, matrigel is also subjected to significant lot-to-lot variability, which can impact the overall reproducibility of the final product. The most commonly used alternative substrate for hiPSCs cultures are human recombinant laminin-521 and vitronectin. Laminin in particular is one of the main components of matrigel, while vitronectin can be found in trace amounts.

We proceeded by directly comparing differentiation induced onto either human recombinant laminin-521 or vitronectin. All the remaining raw materials were kept the same in both conditions. Initial assessment of cellular morphology during the differentiation process indicated subtle differences between both groups. Laminin-OSC generated smaller cells, which organized in compact clusters of cells. In contrast, vitronectin-OSCs presented a larger cell body and organized themselves in more sparse clusters of cells. Expression of FOXL2 and CD82 was also slightly different between these two groups.

To better understand the molecular profile of OSCs generated from each of these conditions, as well as understand how reproducible their cellular outcome is, we performed scRNA-seq in two batches of laminin-OSCs and two batches of vitronectin-OSCs and compared them against our previous dataset (FIG. 2). Laminin-OSCs are mostly distributed throughout the subclusters of the GC class: GC I, GC II, GC III, and have a molecular profile more similar to the cellular outcome observed in the GC3_GC_Batch_ll_2D batch that had uniquely stood out on the previous analysis. In contrast, vitronectin-OSCs are primarily represented by Early GC II, Early GC III, and GC III (FIG. 2). Interestingly, OSCs differentiated onto vitronectin had also a higher percentage of cells in the clusters of both ribosomal and mitochondrial gene enrichment (FIG. 2). Overall, this data indicates that differentiation performed onto laminin-521 and vitronectin, generated similar cell fates to the ones characterized in the preceding section, and therefore it is unlikely that these modifications will impact function of the cells. Interestingly, these results demonstrate that the final OSC fate can be modulated by not only the overexpression of the three transcription factors, but it can also be significantly influenced by the nature of the matrix utilized as the substrate (FIG. 2).

Despite the differences observed in cellular outcome generated in each of these two conditions, we did not observe major differences in cluster distribution among two different independent batches in the same condition (FIG. 2), suggesting that changes in the bill of materials to include higher quality reagents resulted in consistent and reproducible cellular outcomes independently of the matrix utilized. It is also important to highlight that each independent batch of differentiation was performed by a different operator, which strengthens the evidence of reproducibility. iv. Differentiation over laminin-521 leads to a scalable, pure, and functional population of ovarian support cells

After ensuring that transition to an overall higher quality bill of materials does not negatively affect reproducibility or final cellular outcome, we sought to investigate which of the two conditions (laminin-521 and vitronectin) led to better clinical outcomes and should be carried over through clinical manufacturing. As a measurement of successful clinical outcome, we considered a few parameters that would directly inform throughput, safety, and potency of each condition. For throughput, we compared the ratio of OSC:hiPSC for each batch analyzed per condition. The condition that allows for a higher yield of harvested viable cells, without changing the initial cell number or surface area for culturing the cells, would make the case for a more scalable alternative. Laminin-OSCs were harvested at 94.63±0.01 % viability and during differentiation were multiplied at a ratio of 14.83±4.48 OSC:hiPSCs. In contrast, vitronectin-OSCs were harvested at 85.25±0.1 1 % viability and were multiplied at a ratio of 5.86±1 .30 OSC:hiPSC.

Regarding safety for a product derived from hiPSC, it is imperative that we verify the presence of residual hiPSCs in the final cellular composition. Confirmation of presence of a significant number of residual hiPSCs in the final cellular composition without further steps of purification would impair the utilization of the product for assisted reproductive technologies or in vitro fertilization procedures due to a safety concern. To identify the presence of residual hiPSCs in our final differentiation, we utilize RT-qPCR to identify the presence of two key regulators of pluripotency, POU5F1 and NANOG. To determine the sensitivity of our assay, we generated a concentration curve by spiking different concentrations of hiPSCs (0%, 0.0001 %, 0.001 %, 0.01 %, 0.1 %, 1 %, 10%, 100%;) into fully differentiated OSCs. We confirmed that this assay allows for identification of up to 0.01 % of hiPSC into the final product. We then proceeded to characterize the presence of residual hiPSC on the different batches of differentiation. The resulting data demonstrated that there was no contamination of residual hiPSC in the final product. However, it is worth noting that from both batches of vitronectin-OSCs we were able to identify the presence of POU5F1 mRNA by scRNA-Seq, while no detection was observed in the laminin-OSC batches. Presence of NANOG was not identified in any conditions. Although POU5F1 is a key regulator and marker of hiPSC, in the absence of NANOG and SOX2, this gene is not able to maintain pluripotency. Moreover, there is evidence that POU5F1 can be expressed by a subset of granulosa cells in the human ovary.

We next investigated whether the final cellular composition was functional and could promote maturation of human oocytes. We co-cultured both laminin-OSCs and vitronectin-OSCs independently with immature human oocytes following a similar approach discussed herein and scored the yield of oocyte maturation in each group (see, e.g., Example 5). Co-culture with laminin-OSCs resulted in an increase of 1 .50 fold (64.91 ±6.64%) in MH formation compared to the control group, while co-culture with vitronectin- OSCs resulted in 1 .26 fold-increase (54.35±8.10%) in MH formation compared to control. Overall, although Mil formation rates were slightly higher in the laminin-OSCs group comparatively, both approaches generated functional OSCs that contributed to successfully increasing oocyte maturation rate compared to control conditions. v. Generation of a clinical-grade hiPSC line with the ability to generate functional ovarian support cells

The preceding experiments in this Example were performed with the hiPSC line, GC3, a cell line that is designated for research-use-only (RUO) and was not clinical grade. We next sought to generate a clinical-grade hiPSC line from an allogeneic female donor for commercial-grade materials. To minimize discrepancies between the results from the original RUO hiPSC line, which provided the foundation for initial preclinical studies and potency tests, and the clinical-grade hiPSC line, we applied the same manufacturing strategy as the RUO hiPSC line for the generation of the clinical-grade hiPSC line to ultimately demonstrate that the same strategy can be utilized.

The clinical-grade hiPSC line was engineered to harbor inducible versions of the three transcription factors that drive differentiation into OSCs, namely NR5A1 , RUNX2, and GATA4. Individual clones were generated by limiting dilution of the pooled engineered population and expanded into seed banks. All the clones successfully expanded were subjected to initial screening by genotyping PCR to confirm the integration of the three transcription factors (FIG. 3A). Nine seed clones harboring all the transcription factors were selected to proceed with a more in-depth screening process, which included assessing their identity, potency, and safety. For that, each clone was individually differentiated into OSCs (FIG. 3B), and multiple features were recorded for screening purposes and identification of the top lead candidates. To specifically assess clonal identity, we verified expression of the OSC markers, FOXL2 and CD82 after 5 days of differentiation and confirmed that despite the differences in the level of expression among clones, all clones were positive for both markers, suggesting overall successful generation of OSCs (FIG. 3C). Moreover, we confirmed that the level of OCT4 (hiPSC marker) in all clones was very low or null confirming efficient and pure OSC outcome.

As a functional readout of individual clones, cells were differentiated for 5 days and exposed to follicle stimulating hormone (FSH or F), Androstenedione (A4 or A) or a combination of both (F+A) hormones for 48 hours. Functional OSCs generate estradiol (E2) in response to FSH, and A4 is used as a substrate by the OSCs to complete the reaction. We observed that individual clones respond differently to treatment with FSHA + A4, being the ones that are more responsive, more likely to have a better performance while maturing immature human oocytes (FIG. 3D). Based on the attributes previously described in addition to the ratio OSC:hiPSC and viability at harvest, we identified the clones three as the top lead candidates, including clone GTO-101 (FIG. 3E). This decision was informed mostly based on levels of FOXL2 and CD82 expression, as well as clone responsiveness to FSH and A4 in regards to the estradiol production. Despite the low levels of E2 observed on clone 3-E3 (GTO-103), this sample was selected due to its high levels of identity markers (FOXL2 and CD82) for further analysis with IVM assays with immature oocytes (FIG. 3E). We observed higher rates of oocyte maturation (Mil formation) in the GTO-101 clone compared to other clones. Thus, in addition to presenting high levels of OSC markers upon differentiation, clone GTO-101 demonstrated high performance for OSC production and maturation of oocytes. vi. Generation of a clinical-grade hiPSC line with the ability to generate functional ovarian support cells

To further characterize clone GTO-101 for clinical applications, we assessed and confirmed the presence of hiPSC markers, as well as confirmed cell identity and normal karyotype (FIG. 4A). We then generated two independent batches of differentiated GTO-101 , leveraging the protocol previously identified as the most appropriate to be transitioned into clinical manufacturing. More specifically, GTO- 101 hiPSCs were differentiated with highest grade raw material onto laminin-521 coated dishes. As expected, cell morphology upon differentiation was characterized by small cells with granules in the cell body, tightly packed into clusters with spiky edges. We also confirmed that viability at harvest remained high, averaging 96.90±0.00%, and that the ratio of OSC:hiPSC was similar to the ratio achieved with the RUO hiPSC line when differentiated over laminin-521 (14.83±4.48), averaging at 11 .41 ±2.19. Identity of OSCs was confirmed by the expression of the markers, FOXL2 and CD82, and the hiPSC markers, POU5F1 and NANOG were not detected after performing the differentiation protocol, confirming cellular identity and further indicating no contamination with residual hiPSCs (FIG. 4B).

To further characterize the transcriptional signature of the differentiated OSCs, as well as assess reproducibility among independent lots, we performed scRNA-seq of two batches of differentiated GTO- 101 (FIG. 4B). Strikingly, when compared with previous samples analyzed, the two batches were nearly identical in terms of cluster distribution, and they were composed primarily of GC class clusters, particularly subclusters GC I and GC III. Interestingly, the transcriptomic profile of the OSCs derived from GTO-101 resembled the batch of the RUO hiPSC line generated initially (GC3_GC_Batch_ll_2D), as well as the two batches of laminin-OSCs, generated after the raw material optimization, both of which are described in the preceding sections. These results present a strong indication that GTO-101 -derived OSCs will result in successful functional outcome. Additionally, this observation further demonstrate reproducibility of successful hiPSC reprogramming into OSCs among independent batches of cells, independent genetic backgrounds (RUO and clinical-grade hiPSC lines), and when performed by different operators. These data collectively demonstrate promising clinical utility of these cells and this methodology of hiPSC differentiation for ART and IVF applications.

Example 2. A method of producing ovarian support cells from iPSCs

This example demonstrates how iPSC differentiation (e.g., reprogramming or engineering) to one or more types of ovarian support cells may be effectuated. It is to be understood that this example is a non-limiting embodiment of the present disclosure, intended to describe potential protocols for manufacturing OSCs from iPSC precursors. iPSCs (e.g., hiPSCs) from a previously freeze-stored stock or freshly sourced from a donor subject are cultured in vitro in a suitable culture dish that contains cell media (e.g., in vitro maturation (IVM) cell media) and a matrix such as a matrix that comprises laminin. The undifferentiated hiPSCs are reprogrammed using a transposase expression plasmid (e.g., a piggyBac transposase method) to carry specific inducible transcription factors (e.g., FOXL2, NR5A1 , RUNX2, and/or GATA4). The transposase expression plasmid was electroporated into the hiPSCs. The transcription factors were induced upon application of doxycycline to the media. Wnt/p-catenin pathway activators including a ROCK inhibitor (e.g., Y-27642) and a GSK3 inhibitor (e.g., CHIR099021 ) were also added to the media to prime the cellular environment for mesodermal cell fate.

The cells are induced for about 5 days (e.g., about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days). Over the course of the reprogramming, expression of genes or biomarkers that correspond to one or more types of OSCs are assessed. Expression levels of mRNA and protein as assessed by RT-PCR and flow cytometry confirm that a portion population of cells express FOXL2 and AMHR2, and a separate portion of the cells express NR2F2, as compared to a population of hiPSCs that were not differentiated, thereby confirming that the hiPSCs differentiated into a mixed population of granulosa and ovarian stroma cells. Additionally, the resulting OSCs produce steroids such as estradiol and/or progesterone upon stimulation with androstenedione and FSH or forskolin. Production of steroids is confirmed via ELISA in which the steroid levels secreted in the cell media are measured with antibodies that detect the one or more steroids and compared to media from a sample with undifferentiated hiPSCs as a negative control.

Further, cell identity and relative purity of the resulting OSCs is confirmed by RT-PCR, in which no significant detection of one or more markers of pluripotency (e.g., POU5F1 , NANOG, SOX2, and/or OCT4) are detected relative to expression levels detected in undifferentiated hiPSCs, a positive control. Detected expression levels of pluripotency markers that are less than about 5% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.10%) of the expression levels of hiPSCs confirms that the resulting OSC population are pure and the hiPSCs were successfully differentiated and reprogrammed into OSCs. These OSCs may be further clonally expanded and/or cryopreserved as stocks for use in an IVM method or ART application, such as any one of the methods or applications described herein.

Example 3. A method of follicle stimulation for ovarian release of oocytes and in vitro maturation of oocytes

This example demonstrates minimal follicle stimulation of a subject with a low ovarian reserve followed by oocyte harvest and in vitro maturation.

/. Follicle stimulation for ovarian release of oocytes

A 30-year old female subject receives a blood test that detects an anti-Mullerian hormone (AMH) level of less than or equal to 6 ng/mL (e.g., 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, or 6 ng/mL). Thus, she is determined to have a reduced ovarian reserve. Additional blood tests revealing that her estradiol level is between 20 and 50 pg/mL (e.g., 20-30 pg/mL, 25-35 pg/mL, 30-40 pg/mL, 35-45 pg/mL, or 40-50 pg/mL; e.g., 20 pg/mL, 21 pg/mL, 22 pg/mL, 23 pg/mL, 24 pg/mL, 25 pg/mL, 30 pg/mL, 35 pg/mL, 40 pg/mL, 45 pg/mL, or 50 pg/mL) reaffirms the determination of the reduced ovarian reserve.

The subject is administered a triggering agent (e.g., 50 mg of clomiphene citrate) to stimulate follicular maturation and oocyte release. Since the subject is taking a hormonal contraceptive, administration of the triggering agent begins on or about day 5 ±1 day (e.g., day 4, day 5, or day 6) after taking her last contraceptive and continues daily for 1 to 4 days (e.g., 1 day, 2 days, 3 days, or 4 days). The subject’s follicle size is monitored by an ultrasound until the average follicle size reaches about 8-10 mm (e.g., 7.5 mm, 8 mm, 8.5 mm, 9mm, 9.5 mm, 10 mm, 10.5 mm, or more), upon which the oocytes (or a group of cells containing an oocyte, e.g., COCs) are retrieved from the subject by an aspiration-based methodology. For example, oocyte retrieval may utilize a transvaginal ultrasound with a needle guide on the probe to suction release follicular contents. Oocyte-containing follicular contents (e.g., follicular aspirates) are after washed with HEPES media (G-MOPS Plus, VITROLIFE®), filtered with a 70-micron cell strainer (FALCON®, Corning), and examined on a dissection microscope. Oocytes (or a group of cells containing an oocyte, e.g., COCs) are transferred to culture dishes containing cell culture media (e.g., IVM, IVF, or LAG media) for about 1 to 3 hours (e.g., 1 hour, 2 hours, or 3 hours) before introducing granulosa cells for co-culture.

/'/. In vitro maturation of oocytes

If present, cultured COCs may be separated from their cumulus cells (and any other non-oocyte cells) in a process referred herein as oocyte denudation. Oocyte denudation is performed on COCs in an IVM well by mechanically disassociating cells by pipetting to remove the cumulus and/or granulosa cells. Additional oocyte denudation may be performed with an enzymatic disassociation (e.g., hyaluronidase treatment). COCs may be stripped with stripper tips and washed in IVM media or MOPS plus media to clean the oocyte for imaging and, if needed, to inactivate hyaluronidase. Stripper tips include 200 micron and/or 400 microns for fine cleaning.

Next, germinal vesical stage (GVs) and metaphase I stage (Ml) oocytes are co-cultured with about 50,000-100,000 (e.g., 50,000-60,000 cells, 60,000-70,000 cells, 70,000-80,000 cells, 80,000- 90,000 cells, or 90,000-100,000 cells; e.g., 50,000 cells, 55,000 cells, 60,000 cells, 65,000 cells, 70,000 cells, 75,000 cells, 80,000 cells, 85,000 cells, 90,000 cells, 95,000 cells, or 100,000 cells) granulosa cells (e.g., specialized granulosa cells, hiPSC-derived granulosa cells, or steroidogenic granulosa cells, as described herein). Metaphase II stage (Mil) oocytes (e.g., oocytes with a polar body in the perivitelline space) can be properly frozen for storage. Co-culturing of oocytes and granulosa cells is for about 12-120 hours (e.g., 12-24 hours, 12-36 hours, 24-48 hours, 36-60 hours, 54-72 hours, 68-96 hours, 96-120 hours; e.g., 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours,

110 hours, 112 hours, 114 hours, 116 hours, 118 hours, or 120 hours).

Following co-culture, any one or more oocytes are utilized for assisted reproduction technology (ART) procedures. For example, oocytes may be utilized for intracytoplasmic sperm injection (ICSI).

Example 4. Administration of a follicular triggering agent

This example demonstrates the administration of a triggering agent to a subject.

A 30-year old female subject receives a blood test that detects estradiol levels between 20 and 50 pg/mL (e.g., 20-30 pg/mL, 25-35 pg/mL, 30-40 pg/mL, 35-45 pg/mL, or 40-50 pg/mL; e.g., 20 pg/mL, 21 pg/mL, 22 pg/mL, 23 pg/mL, 24 pg/mL, 25 pg/mL, 30 pg/mL, 35 pg/mL, 40 pg/mL, 45 pg/mL, or 50 pg/mL). The subject is administered multiple injections of a triggering agent over 1 to 4 days (e.g., 1 day, 2 days, 3 days, or 4 days) but no more than 5 days. The subject may receive multiple injections over multiple days such that a subject receives five dose injections of one or multiple triggering agents. For example, a subject receives three days of stimulation using 300 IU to 700 IU of rFSH per injection (e.g., 300-500 IU, 400-600 IU, 500-700 IU, 300-350 IU, 350-400 IU, 400-450 IU, 450-500 IU, 500-550 IU, 550- 600 IU, 600-650 IU, 650-700 IU; e.g., 300 IU, 325 IU, 350 IU, 375 IU, 400 IU, 425 IU, 450 IU, 475 IU, 500 IU, 525 IU, 550 IU, 575 IU, 600 IU, 625 IU, 650 IU, 675 IU, or 700 IU) with one or more injections per day. In another example, the subject receives injections of hCG as a triggering agent using 200-700 pg or 2,500-10,000 IU hCG (e.g., 200-500 pg, 300-600 pg, 400-700 pg, 200-300 pg, 300-400 pg, 400-500 pg, 500-600 pg, or 600-700 pg). In yet another example, the subject receives one or more (e.g., 1 , 2, 3, 4, or 5) doses of clomiphene citrate at 50-150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 110-130 mg, 125-150 mg; e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg) per dose.

Example 5: Materials and Methods for Examples 6 through 8

We have developed human ovarian support cells (OSCs) generated from human induced pluripotent stem cells (hiPSCs) that hold the ability to recapitulate dynamic ovarian function in vitro. Here we investigate the potential of these OSCs to improve human oocyte maturation, retrieved from abbreviated gonadotropin stimulated cycles, as a co-culture system applied to IVM. We reveal that OSC- IVM significantly improves maturation rates compared to available IVM systems. Most importantly, we demonstrate OSC-assisted IVM oocytes are capable of robust euploid blastocyst formation, a key marker of their clinical utility. Together, these findings demonstrate a novel approach to IVM with broad applicability to modern IVF practice.

Specifically, to determine if in vitro maturation (IVM) of human oocytes can be improved by coculture with ovarian support cells (OSCs) derived from human induced pluripotent stem cells (hiPSCs), oocyte donors were recruited to undergo abbreviated gonadotropin stimulation with or without hCG triggers and cumulus oocyte complexes (COCs) were allocated between the OSC-IVM condition and media only IVM controls.

Oocyte donors between the ages of 19 to 37 years were recruited for donation under informed consent, with an anti-mullerian hormone (AMH) value of greater than 1 ng/mL as inclusion criteria. The OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione and doxycycline supplementation. IVM controls lacked OSCs and contained the same supplementation or only FSH and hCG.

Primary endpoints consisted of metaphase II (Mil) formation rate and morphological quality assessment. A limited cohort of oocytes were additionally utilized for fertilization and blastocyst formation with PGT-A analysis. OSC-IVM resulted in a statistically significant improvement in Mil formation rate compared to the media only control. OSC-IVM resulted in a statistically significant improvement in Mil formation rate compared to a commercially available IVM control. Oocyte morphological quality between OSC-IVM and controls did not significantly differ. OSC-IVM improved maturation, fertilization, cleavage, blastocyst formation, high quality blastocyst formation and euploid blastocyst formation compared to the commercially available IVM control.

As a conclusion, the novel OSC-IVM platform is an effective tool for maturation of human oocytes obtained from abbreviated gonadotropin stimulation cycles, yielding improved blastocyst formation. OSC- IVM shows broad utility for different stimulation regimens, including hCG triggered truncated IVF and untriggered traditional IVM cycles making it a highly useful tool for modern fertility treatment.

/. Collection of Cumulus Oocyte Complexes (COCs)

Subject ages, IRB and Informed Consent

Subjects were enrolled in the study through Ruber Clinic (Madrid, Spain), Spring Fertility Clinic (New York, USA) and Pranor Clinic (Lima, Peru) using informed consent (CNRHA 47/428973.9/22, IRB # 20225832, Western IRB, and Protocol #GC-MSP-01 respectively). Subject ages ranged between 19 and 37 years of age. Oocytes retrieved from the Ruber and Pranor clinics were utilized for maturation analysis endpoints only, while oocytes retrieved from Spring Fertility were utilized for embryo formation endpoints.

Stimulation characteristics

Twenty-five subjects received 3-4 days of stimulation using 300-600 IU of rFSH with an hCG trigger in preparation for immature oocyte aspiration for Experiment 1 , with an AMH value of >1 ng/mL (see below). Twenty-one subjects received three consecutive days of 200 IU of rFSH with an hCG trigger in preparation for immature oocyte aspiration for Experiment 2, with an AMH value of >1 .5ng/mL used as an inclusion criterion to enrich for donors yielding more oocytes (see below). Six subjects received three to five doses of clomiphene citrate (100 mg) with an additional one to two doses of 150 IU rFSH with or without an hCG trigger for Experiment 2 with the goal of subsequent embryo formation, and an AMH value of >2.0ng/mL was utilized as an inclusion criterion (see below). Gonadotropin injections were initiated on day 2 of a natural cycle or on the fifth day following cessation of oral contraceptive pills. A complete table of donor stimulation regimens for each donor in the study is provided in Table 1 below.

Table 1 : Donor stimulation regimens

/'/. Aspiration of small ovarian follicles to retrieve immature cumulus oocyte complexes Aspirations were performed 36 hours after the trigger injection (10,000 IU hCG) using a transvaginal ultrasound with a needle guide on the probe to retrieve oocytes for co-culture experiments. Aspiration was performed using ASP medium (VITROLIFE®) without follicular flushing using double lumen 19-gauge needles (double lumen needles were selected due to the additional stiffness provided by the second channel inside the needle). Vacuum pump suction (100 mm Hg) was used to harvest follicular contents through the aspiration needle and tubing into a 15 mL round bottom polystyrene centrifuge tube. For the conditions where the final outcome was embryo formation, aspirations were performed 36 hours after trigger injection (10,000 IU hCG) or 48 hours after last rFSH injection for untriggered cycles. Aspiration was performed without follicular flushing using a single lumen 19- or 20-gauge needle with a vacuum pump suction (-200 mm Hg) used to harvest follicular contents through the aspiration needle and tubing into a 15 mL round bottom polystyrene centrifuge tube. In all cases, rapid rotation of the aspiration needle around its long axis, when the follicle had collapsed, provided a curettage effect to assist the release of COCs into the aspirate fluid. Although follicles were not flushed, the aspiration needle was removed from the subject and flushed frequently throughout the oocyte retrieval procedure to limit clotting and needle blockages.

Follicular aspirates were examined in the laboratory using a dissecting microscope. Aspirates tended to include more blood than in typical IVF follicle aspirations, so were washed with HEPES media (G-MOPS Plus, VITROLIFE®) to minimize clotting. Often, the aspirate was additionally filtered using a 70-micron cell strainer (FALCON®, Corning) to improve the oocyte search process. COCs were transferred using a sterile Pasteur pipette to a dish containing LAG Medium (Medicult, COOPERSURGICAL®) until use in the IVM procedure. The number of COCs aspirated was equal to roughly 40% of the antral follicles seen in the subject’s ovaries on the start day. Hi. Preparation of Ovarian Supporting Cells (OSCs)

OSCs were created from human induced pluripotent stem cells (hiPSCs) according to transcription factor (TF)-directed protocols. The OSCs were produced in multiple batches and cryopreserved in vials of 120,000 to 150,000 live cells each and stored in liquid nitrogen in CryoStor CS10 Cell Freezing Medium (STEMCELL TECHNOLOGIES®).

Culture dishes (4+8 Dishes, BIRR) for oocyte maturation experiments were prepared with culture medium and additional constituents in 100 pL droplets under mineral oil the day before oocyte collection. The morning of oocyte collection, cryopreserved OSCs were thawed for 2-3 minutes at 37°C (in a heated bead or water bath), resuspended in OSC-IVM medium and washed twice using centrifugation and pelleting to remove residual cryoprotectant. Equilibrated OSC-IVM media was used for final resuspension. OSCs were then plated at a concentration of 100,000 OSCs per 100 pL droplet by replacing 50 pL of the droplet with 50 pL of the OSC suspension 2-4 hours before the addition of oocytes to allow for culture equilibration and media conditioning. iv. In vitro maturation

COCs were maintained in preincubation LAG Medium (Medicult, COOPERSURGICAL®) at 37°C for 2-3 hours after retrieval prior to introduction to in vitro maturation conditions. Two different sets of experimental comparisons were performed to address the following goals:

Experiment 1 (OSC activity): The purpose of this comparison was to determine whether the stimulated OSCs were the active ingredient of the co-culture system. For this purpose, medium in experimental and control conditions was prepared by following Medicult manufacturer’s recommendations, and further supplemented with androstenedione and doxycycline (both necessary for activation/stimulation of OSCs) in order to compare maturation outcomes with or without OSCs in the same medium formulation (see Table 2 below).

Experiment 2 (OSC clinical relevance): The purpose of this experiment was to compare the efficacy of the OSC-IVM system and the commercially available in vitro maturation system (Medicult IVM). For this purpose, the Control Group condition was prepared and supplemented by following Medicult manufacturer’s recommendations, while medium for OSC-IVM was prepared with all supplements (see Table 2 below).

Table 2: Cell culture media conditions

Subject description (Experiment 1 ): We collected 132 oocytes from 25 subjects (average age of 25) who underwent abbreviated gonadotropin stimulation, with 49 utilized in OSC-IVM co-culture, and 83 utilized in control culture. Co-culture in the Experimental and Control Conditions was performed in parallel when possible. COCs were distributed equitably when performed in parallel. Equitable distribution means that COCs with distinctly large cumulus masses, small cumulus masses, or expanded cumulus masses were distributed as equally as possible between the two conditions. Other than the selective distribution of the distinct COC sizes, the COCs were distributed as randomly as possible between one to two conditions. Due to the low number of oocytes retrieved per subject in this comparison, it was often not possible to distribute oocytes effectively between conditions simultaneously. COCs were subjected to these in vitro maturation conditions at 37°C for a total of 24-28 hours in a tri-gas incubator with CO2 adjusted so that the pH of the bicarbonate-buffered medium was 7.2-7.3 and with the O2 level maintained at 5%.

Subject description (Experiment 2): For the IVM outcome endpoint, 21 subjects were recruited for the comparison. We collected 143 COCs included in the comparison, allocating 70 utilized in IVM control and 73 utilized in the OSC-IVM condition. Co-culture in the Experimental and Control Conditions was performed in parallel for all subjects. COCs were distributed equitably between the two conditions, as described above. COCs were subjected to these in vitro maturation conditions at 37°C for a total of 28 hours in a tri-gas incubator with CO2 adjusted so that the pH of the bicarbonate-buffered medium was 7.2- 7.3 and with the O2 level maintained at 5%. In vitro maturation with subsequent embryo formation was performed to assess developmental competence of the oocytes treated in the OSC-co-culture system in comparison to oocytes treated with commercially available IVM medium. For embryo formation, a small cohort of oocyte donors were recruited and donor sperm was utilized for fertilization. For the embryo outcomes endpoint, six additional subjects were recruited for the comparison. We collected 46 COCs included in the comparison, allocating 21 utilized in Media-IVM control and 25 utilized in the OSC-IVM condition. Co-culture in the Experimental and Control Conditions was performed in parallel. COCs were distributed equitably between the two conditions, as described above. COCs were subjected to these in vitro maturation conditions at 37°C for a total of 28 hours in a tri-gas incubator with CO2 adjusted so that the pH of the bicarbonate-buffered medium was 7.2-7.3 and with the O2 level maintained at 5%. Embryo formation proceeded in parallel, with groups kept separate, with culture proceeding no longer than day 7 post-IVM, v. Assessment of in vitro maturation

Following the end of the 24 to 28 hour in vitro maturation period, COCs were subjected to hyaluronidase treatment to remove surrounding cumulus and corona cells. After hyaluronidase treatment, cumulus cells were banked for future analysis and oocytes were assessed for maturation state according to the following criteria:

MH - absence of a germinal vesicle within the oocyte and presence of a polar body in the perivitelline space between the oocyte and the zona pellucida. vi. Oocyte morphology scoring

Following IVM, oocytes were harvested from culture dishes and stripped of cumulus cells and OSCs, assessed for maturation assessment, then individually imaged using digital photomicrography. After imaging, oocytes were flash frozen in 0.2 mL PCR tubes prefilled with 5 pL DPBS. The images were later scored according to the Total Oocyte Score (TOS) grading system. Oocytes were scored by a single trained embryologist and given a score of -1 , 0, 1 for each of the following criteria: morphology, cytoplasmic granularity, perivitelline space (PVS), zona pellucida (ZP) size, polar body (PB) size, and oocyte diameter. Zona pellucida and oocyte diameter were measured using ECHO™ Revolve Microscope software and Imaged image analysis software (2.9.0/1 .53t). The sum of all categories was taken to give the oocyte a total quality score, ranging from -6 to +6 with higher scores indicating better morphological quality. v/7. Oocyte disposition following morphological scoring

For oocytes used only for evaluation of oocyte maturation, oocytes were snap frozen following assessment of in vitro maturation and any further morphology scoring. Snap freezing was performed by placing each oocyte in a 0.25 mL PCR tube with 5 pL DPBS. After capping the tube, it was submerged in liquid nitrogen until all bubbling ceased. Then the PCR tube was stored at -80°C for future molecular analysis.

For oocytes used to create embryos, matured oocytes were immediately utilized for intracytoplasmic sperm injection (ICSI) and subsequent embryo formation to the blastocyst stage. No oocytes from this study were utilized for transfer, implantation, or reproductive purposes. v/77. In vitro fertilization and embryo culture

A cohort of six subjects was utilized for in vitro maturation and subsequent embryo formation. The COCs from these subjects were subjected to the conditions used in Experiment 2 (treatment with OSC co-culture with all adjuvants versus commercially available IVM treatment as the control). All COCs were cultured for 28 hours then denuded and assessed for Mil formation and micrographed. Individual oocytes in each condition were injected with sperm (intracytoplasmic sperm injection (ICSI) on day 1 post- retrieval. After ICSI, the oocytes were cultured in a medium designed for embryo culture (Global Total, COOPERSURGICAL®, Bedminster, NJ) at 37°C in a tri-gas incubator with CO2 adjusted so that the pH of the bicarbonate-buffered medium was 7.2-7.3 and with the O2 level maintained at 5%. The following day they were assessed for fertilization 12 to 16 hours post-ICSI, and oocytes with one or two pronuclei were cultured until day 3. Cleaved embryos underwent laser-assisted zona perforation and were allowed to develop until the blastocyst stage. Blastocysts were scored according to the Gardner scale then underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy (PGT-A) and cryopreservation if deemed high quality, i.e. , greater than or equal to a 3CC rating.

Trophectoderm biopsies were transferred to 0.25 mL PCR tubes and sent to a reference laboratory (JUNO GENETICS®, Basking Ridge, NJ) for comprehensive chromosomal analysis using a single nucleotide polymorphism (SNP) based next generation sequencing (NGS) of all 46 chromosomes (pre-implantation genetic testing for aneuploidy, PGT-A). ix. Data analysis and statistics

Oocyte maturation outcome data was analyzed using Python statistical packages pandas (1 .5.0), scipy (1 .7.3), and statsmodels (0.13.2). Maturation percentages by donor group were analyzed using linear regression as functions of the IVM environment (OSC-IVM or Media control), t-test statistics were computed comparing cell line incubation outcomes versus media control, then used to calculate p-values. Bar graphs depict mean values for each population and error bars represent standard error of the mean (SEM).

Example 6. hiPSC-derived OSCs effectively promote human oocyte maturation following coculture system

In order to obtain immature COCs for IVM, we utilized similar protocols to previous studies for IVM, truncated IVF or hCG primed-IVM, namely 3-4 days of minimal gonadotropin stimulation and most often an hCG trigger. This abbreviated stimulation program, particularly when hCG was included, yielded a mixed cohort of oocytes that were mostly immature (GV and Ml), but expanded cumulus COCs were obtained as well, which may have contained MH oocytes. Oocyte donor demographics and treatment regimens are shown in Table 3 for each experimental group. Overall, the results demonstrate we were able to retrieve oocytes from non-polycystic ovarian syndrome (non-PCOS/PCOS) donors, albeit at a lower yield than traditional controlled ovarian hyperstimulation cycles. In Experiment 1 , oocytes from each donor were allocated to either the control IVM or OSC-IVM arm. Age, body mass index (BMI) and total COCs retrieved did not significantly differ between groups in Experiment 1 . For Experiment 2, the control and OSC-IVM arms for both endpoints contained identical donor groups as oocytes were split equally between culture conditions for each donor. Age and BMI significantly differed in Experiment 2 compared to Experiment 1 , and total COCs retrieved per donor was lower but not significantly. A schematic of the OSC-IVM condition is shown in FIG. 8A, with a representative image of the OSC co-culture seen in FIG. 8B. Table 3: Donor demographic and stimulation characteristics

We have previously demonstrated that hiPSC-derived OSCs are predominantly composed of granulosa cells and ovarian stroma cells. In response to hormonal stimulation treatment in vitro, these OSCs produce growth factors and steroids necessary for interaction with oocytes and cumulus cells. To investigate whether hiPSC-derived OSCs are functionally capable of promoting human oocyte maturation in vitro, we established a co-culture system of these cells with freshly retrieved cumulus enclosed oocytes and assessed maturation rates after 24-28 hours (see Materials and Methods, Experiment 1 ). In this comparison, due to low numbers of retrieved oocytes per donor, we were unable to consistently split oocytes between both conditions simultaneously, therefore each group contains oocytes from predominantly non-overlapping donor groups and pairwise comparisons are not utilized. Strikingly, we observed significant improvement (~1 .5x) in maturation outcomes for oocytes that undergo IVM with OSCs (FIG. 9A) compared to control. More specifically, the OSC-IVM group yielded a maturation rate of 68% ± 6.83% SEM versus 46% ± 8.51% SEM in the Media Control (FIG. 9A, p = 0.02592, unpaired t- test). The maturation rate for OSC-IVM compared to control was statistically significant. These results support functional activity of hiPSC-derived OSC in in vitro co-culture systems demonstrated by the significantly higher oocyte maturation rates.

We next examined whether hiPSC-derived OSCs would also affect the outcome of the Total Oocyte Score (TOS). Interestingly, the assessment scores (FIG. 9B) were not statistically significantly different for the two groups (unpaired t-test, p= 0.2909), indicating that the mature Mil oocytes outcome was of equivalent morphological quality between the two IVM conditions. Altogether, these data indicate that OSC co-culture improve maturation without a detrimental effect on morphological quality of human oocytes, and highlights the potential for the use of hiPSC-derived OSCs as a high performing system for cumulus enclosed oocyte IVM. Example 7. Oocyte maturation rates in OSC-IVM outperforms commercially available IVM system

To further examine the potential of using OSC-IVM as a viable system to mature human oocytes in a clinical setting, we compared our OSC co-culture system against a commercially available IVM standard. The commercially available IVM standard was utilized as described in its clinical instructions for use, with no modification (Medicult IVM). We performed a sibling oocyte study comparing the Mil formation rate and oocyte morphological quality after 28 hours of in vitro maturation in both systems (Materials and Methods, Experiment 2). Notably, OSC-IVM yielded ~1 .6x higher average Mil formation rate with 68% ± 6.74% of mature oocytes across donors compared to 43% ± 7.90% in the control condition (FIG. 10A, p= 0.0349, paired t-test). The maturation rate for OSC-IVM compared to the commercial IVM control was statistically significant. Similar to previous observations, co-culture with hiPSC-derived OSCs did not affect oocyte morphological quality between groups as measured by TOS, indicating equivalent oocyte visual morphological characteristics (FIG. 10B, p= 0.9420, unpaired t-test). These results show that OSC-IVM significantly outperformed the commercially available IVM culture medium in MH formation rate with no apparent detriment to oocyte morphological quality, pointing to a beneficial application for human IVM.

Example 8. Cumulus enclosed immature oocytes from abbreviated gonadotropin stimulation matured by OSC-IVM are developmentally competent for embryo formation

We sought to investigate the developmental competency of oocytes treated in the OSC-IVM system, by assessing euploid blastocyst formation, compared to the commercially available IVM control. Utilizing a limited cohort of six subjects who underwent abbreviated stimulation (see Materials and Methods Experiment 2, Tables 2 and 3) we investigated whether OSC-IVM treated oocytes were capable of fertilization, cleavage, and formation of euploid blastocysts. We compared these embryo outcomes to those found from oocytes treated in the commercially available IVM medium. OSC-IVM yielded ~1 .2X higher average MH formation rate with 60% ± 15.4% of mature oocytes across donors compared to 52% ± 8% in the control condition (FIG. 11 A, Table 4). Mature oocytes in both treatment groups were subjected to ICSI and fertilized oocytes were cultured until Day 7 of development. OSC-assisted Mils demonstrate a trend towards improved fertilization, cleavage, blastocyst and usable quality blastocyst formation rates as a proportion of the input COC number (52%, 52%, 40%, and 28%) compared to the commercial IVM control (38%, 38%, 24%, and 19%) (FIG. 11 A, Table 4). When examined on an incremental basis, OSC-IVM oocytes fertilize and form blastocysts at an improved rate, while cleavage of fertilized oocytes is similar to the commercial IVM control. Overall, in both conditions we find that all oocytes that fertilized subsequently cleaved. Strikingly, PGT-A results show that of the blastocysts of transferable quality generated by OSC-IVM, 100% are euploid versus 25% in the commercial IVM system. While these results are not statistically significant, likely due to the small underpowered sample size for each group, these findings demonstrate that OSC-IVM generates healthy matured oocytes with high quality developmental competency. These results additionally demonstrate OSC-IVM is capable of producing healthy, euploid embryos from abbreviated stimulation cycles at a higher rate than the commercially available IVM condition, highlighting the clinical relevance of this novel system for IVM ART practice. Table 4: OSC-IVM oocytes are developmentally competent for euploid embryo formation

Example 9. Materials and Methods for Examples 10-12

We have demonstrated that human ovarian support cells (OSCs) generated from human induced pluripotent stem cells (hiPSCs) exhibit the ability to recapitulate dynamic ovarian function in vitro. Here we investigate the utilization of these OSCs as a co-culture system to better mimic the ovarian environment in vitro and promote IVM to rescue denuded immature oocytes derived from conventional gonadotropin stimulated cycles. We find that OSC-IVM significantly improves oocyte maturation rates compared to spontaneous maturation in media matched controls. Additionally, oocytes matured in combination with OSC-IVM are transcriptionally more similar to conventional IVF metaphase II (MH) oocytes than oocytes that had spontaneously matured in media controls. Together, these findings demonstrate a novel approach to improve the outcome of matured Mil oocytes in modern IVF practice by leveraging an optimized IVM system that better mimics the ovarian environment in vitro.

Specifically, to determine if rescue in vitro maturation (IVM) of human oocytes can be improved by co-culture with ovarian support cells (OSCs) derived from human induced pluripotent stem cells (hiPSCs), fertility patients undergoing conventional ovarian stimulation donated denuded immature germinal vesicle (GV) and metaphase I (Ml) oocytes for research, which were allocated between either the control or intervention.

Oocyte donors between the ages of 25 to 45 years old donated immature oocytes under informed consent, with no additional inclusion criteria. The 24-28 hour OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione and doxycycline supplementation. The IVM control lacked OSCs and contained the same supplementation.

Primary endpoints consisted of MH formation rate and morphological quality assessment. Additionally, metaphase spindle assembly location and oocyte transcriptomic profiles were assessed compared to in vivo matured oocyte controls. OSC-IVM resulted in a statistically significant improvement in Mil formation rate compared to the Media-IVM control. Oocyte morphological quality between OSC- IVM and the Media-IVM control did not significantly differ. OSC-IVM resulted in Mil oocytes with no instance of spindle absence and no significant difference in position compared to in vivo matured Mil controls. OSC-IVM treated Mil oocytes display a transcriptomic maturity signature significantly more similar to IVF-MII controls than the Media-IVM control Mil oocytes. /. Collection of Immature Oocytes

Forty-seven oocyte donor subjects were enrolled in the study using informed consent (IRB# 20222213, Western IRB). Subject ages ranged between 25 and 45 years of age, with an average age of 35. Oocytes were retrieved at several in vitro fertilization and egg freezing clinics in New York City (IRB# 20222213, Western IRB). Fertility patients providing discarded immature oocytes had signed informed consents, provided by the clinic, permitting their use for research purposes. Patients underwent typical age-appropriate controlled ovarian hyperstimulation using gonadotropin releasing hormone (GnRH) analogs (agonist or antagonist) or injections with recombinant or highly purified urinary gonadotropins (recombinant FSH, human menopausal gonadotropins) followed by an ovulatory trigger (human Chorionic Gonadotropin (hCG) or GnRH agonist). 34-36 hours following the trigger injection(s), oocytes were retrieved from the patient under conscious sedation using standard clinical procedures.

Retrieved oocytes were exposed to hyaluronidase briefly then adherent cumulus cells were mechanically removed by repeatedly drawing up and expelling each cumulus-oocyte complex with a small-bore pipette. Denuded oocytes were assessed for maturation by observation of a polar body or a germinal vesicle. Immature oocytes (GV or Ml), which would usually be discarded, were instead allocated to our research study. All immature oocytes retrieved from the clinic each day were pooled and were placed in LAG Medium (Medicult, COOPERSURGICAL®) in a 5 mL round-bottom tube that was transferred from the clinic to our research laboratory in a 37°C transport incubator.

For some experiments, immature (GV and Ml) oocytes from similar IVF and egg freezing cycles were vitrified and stored at the clinics. Cryopreserved oocytes were transported from the clinic to our laboratory in liquid nitrogen and stored until use. Oocytes were then thawed using the standard Kitazato protocol for vitrified or slow frozen oocytes (VITROLIFE®, USA), evaluated for maturation status as GV or Ml, and used for comparisons of in vitro maturation conditions.

A limited number of MH oocytes obtained from conventional controlled ovarian hyperstimulation, which were previously banked for research purposes, were provided as controls for this study (IVF-MII). These oocytes were transferred to our laboratory from the tissue repository and thawed using either the standard Kitazato protocol for vitrified oocytes (KITAZATO™, USA) or slow freeze-thaw protocol for previously slow frozen oocytes (VITROLIFE®, USA) and utilized for live fluorescent imaging and transcriptomic analysis.

/'/. Preparation of Ovarian Supporting Cells (OSCs)

Human induced pluripotent stem cell (hiPSC) derived OSCs were created according to transcription factor (TF)-directed protocols described previously. OSCs were produced in multiple batches and cryopreserved in vials of 120,000 to 150,000 cells each and stored in the vapor phase of liquid nitrogen in CryoStor™ CS10 Cell Freezing Medium (STEMCELL TECHNOLOGIES®). Culture dishes (4+8 Dishes, BIRR) for oocyte maturation experiments were prepared with culture medium and additional constituents in 100pL droplets under mineral oil (LifeGuard, LIFEGLOBAL GROUP®) the day before oocyte collection. The morning of oocyte collection, cryopreserved OSCs were thawed for 2-3 minutes at 37°C (in a heated bead or water bath), resuspended in OSC-IVM medium and washed twice using centrifugation and pelleting to remove residual cryoprotectant. Equilibrated OSC-IVM medium was used for final resuspension. OSCs were then plated at a concentration of 100,000 OSCs per 100 pL droplet by replacing 50 pL of the droplet with 50 pL of the OSC suspension 2-4 hours before the addition of oocytes to allow for culture equilibration and culture medium conditioning (FIG. 12A). OSCs were cultured in suspension culture surrounding the denuded oocytes in the microdroplet under oil. IVM culture proceeded for 24 to 28 hours, after which oocytes were removed from culture, imaged, and harvested for molecular analysis.

Hi. In vitro Maturation

Immature oocytes were maintained in preincubation LAG Medium (Medicult, COOPERSURGICAL®) at 37°C for 2-3 hours after retrieval prior to introduction to in vitro maturation conditions (either Media-IVM or OSC-IVM).

A single experimental condition was examined:

Experiment (OSC activity): The purpose of this comparison was to determine whether the stimulated OSCs were the active ingredient or contributor to the co-culture system. For this purpose, medium in both experimental and control condition was prepared by following Medicult manufacturer’s recommendations, and further supplemented with androstenedione and doxycycline (both necessary for activation/stimulation of OSCs) in order to compare maturation outcomes with or without OSCs in the same medium formulation (see Table 5 below).

Table 5: Cell culture media conditions

Donated oocytes were retrieved from 56 patients and pooled into 29 independent cultures, totaling 141 oocytes, with 82 oocytes utilized in OSC-IVM and 59 oocytes utilized in Media-IVM. Culture in the Experimental and Control Conditions was performed in parallel when possible. Immature oocytes from each donor pool were distributed equitably between two conditions at a time, with no more than 15 oocytes per culture at a time. Specifically, immature oocytes (GV and Ml) were distributed as equally and randomly as possible between the two conditions. Due to low and highly variable numbers of available immature oocytes which were provided as discard donation, both conditions often could not be run in parallel from the same oocyte donation source often. Immature oocytes were subjected to in vitro maturation at 37°C for a total of 24-28 hours in a tri-gas incubator with CO2 adjusted so that the pH of the bicarbonate-buffered medium was 7.2-7.3 and with the O2 level maintained at 5%. iv. Assessment of in vitro maturation

At the end of the in vitro culture, oocytes were harvested from culture dishes and mechanically denuded and washed of any residual OSCs. Oocytes were then individually assessed for maturation state according to the following criteria:

Following assessment of in vitro maturation and morphology scoring, oocytes were individually imaged using digital photomicrography and if required, examined by fluorescent imaging for the second meiotic metaphase spindle. No oocytes from this study were utilized for embryo formation, transfer, implantation, or reproductive purpose. v. Oocyte morphology scoring

Oocytes harvested post-IVM were individually imaged using digital photomicrography on the ECHO™ Revolve inverted fluorescent microscope using phase contrast imaging. The images were later scored according to the Total Oocyte Score (TOS) grading system. A single trained embryologist was blinded and oocytes were given a score of -1 , 0, 1 for each of the following criteria: morphology, cytoplasmic granularity, perivitelline space (PVS), zona pellucida (ZP) size, polar body (PB) size, and oocyte diameter. Zona pellucida and oocyte diameter were measured using ECHO™ Revolve Microscope software and the image analysis software FIJI (2.9.0/1 .53t). The sum of all categories was taken to give the oocyte a total quality score, ranging from -6 to +6 with higher scores indicating better morphological quality. vi. Examination of the second meiotic metaphase spindle and its position relative to the polar body

Previously vitrified denuded immature oocytes were thawed and equitably distributed across OSC-IVM and Media-IVM conditions before being cultured for 28 hours. Additional donated MH oocytes were collected and stained to visualize the microtubules of the meiotic spindle apparatus by fluorescent microscopy as an IVF control (IVF-MII) (FIG. 14A-B). Mil oocytes were incubated in 2 pM of an alphatubulin dye (ABBERIOR™ Live AF610) for one hour in the presence of 10 pM verapamil (ABBERIOR™ Live AF610). Spindle position was then visualized using fluorescence microscopy (ECHO™ Revolve microscope, TxRED filter block EX:560/40 EM:630/75 DM:585). The angle of the first polar body and spindle apparatus in the IVM oocytes was determined (with the vertex at the center of the oocyte) using FIJI software. This measurement was also made on the cohort of IVF Mil oocytes (n=34) as a control reference population. v/7. Cryopreservation of oocytes for subsequent molecular analyses

Following the completion of morphological examination, oocytes were individually placed in 0.25 mL tubes containing 5 pL Dulbecco’s Phosphate Buffered Saline (DPBS) and snap frozen in liquid nitrogen. After the cessation of nitrogen bubble formation the tubes were stored at -80°C until subsequent molecular analysis. viii. Single oocyte transcriptomics library preparation and RNA sequencing

Libraries for RNA sequencing were generated using the NEBNEXT™ Single Cell/Low Input RNA Library Prep Kit for ILLUMINA® (NEB #E6420) in conjunction with NEBNEXT™ Multiplex Oligos for ILLUMINA® (96 Unique Dual Index Primer Pairs) (NEB #E6440S), according to the manufacturer’s instructions. Briefly, oocytes frozen in 5 pL DPBS and stored at -80°C were thawed and lysed in lysis buffer, then RNA was processed for reverse transcriptase and template switching. cDNA was PCR amplified with 12-18 cycles, then size purified with KAPA™ Pure Beads (Roche). cDNA input was normalized across samples. Following fragmentation and end prep, NEBNEXT™ Unique Dual Index Primer Pair adapters were ligated, and samples were enriched using 8 cycles of PCR. Libraries were cleaned up with KAPA™ Pure Beads, quantified using Quant-iT PicoGreen dsDNA Reagent and Kit (Invitrogen), then an equal amount of cDNA was pooled from each oocyte library. The pool was subjected to a final KAPA™ Pure bead size selection if required and quantified using Qubit dsDNA HS kit (Invitrogen). After verification of library size distribution (~325bp peak) using Bioanalyzer HS DNA kit (Agilent), the library pool was subjected to RNA sequencing analysis using the MiSeq Micro V2 (2x150bp) or MiSeq V2 (2x150bp) kit on an ILLUMINA® MiSeq according to the manufacturer’s instructions. ix. Oocyte transcriptomics data analysis

ILLUMINA® sequencing files (bcl-files) were converted into fastq read files using Illumina® bcl2fastq (v2.20) software deployed through BaseSpace using standard parameters for low input RNA- seq of individual oocytes. Low input RNA-seq data gene transcript counts were aligned to Homo sapiens GRCH38 (v 2.7.4a) genome using STAR (v 2.7.1 Oa) to generate gene count files and annotated using ENSEMBL. Gene counts were combined into sample gene matrix files (h5). Computational analysis was performed using data structures and methods from the Scanpy (v 1 .9.1 ) package as a basis. Gene transcript counts were normalized to 10,000 per sample and log (In) plus 1 transformed. Principal component analysis was performed using Scanpy package methods focusing on 30 PCA components. Integration and project (batch) correction was performed using BBKNN. Projection into two dimensions was performed using the Uniform Manifold Approximation and Projection (UMAP) method. Cluster discovery was performed with the Ledien methods with resolution 0.5.

To define the expected transcriptomic profile for normal MH oocytes we used the donated cohort of in vivo matured IVF-MII samples (n=34) as a reference point and compared this reference set to subsets of the post-IVM GV cells using differential gene expression. The top 50 differentially expressed genes were collected for each comparison using both the Wilcoxon ranked sum test and the cosine similarity-based marker gene identification (COSG) method. No other Ml or MH oocyte sets were used as reference points, as these marker genes were developed to ensure minimal bias for other Mil transcriptomic profiling. This method generated the failed-to-mature GV and IVF Mil signature marker gene expression profiles. Cells were scored for each marker gene set using Scanpy gene marker scoring methods.

To visualize our cells in signature marker space we plotted the marker scores as a two- dimensional space. We then manually divided the space into quadrants based on morphological maturation outcomes and Leiden clusters. Clusters are annotated taking into consideration their distribution in score space and presence in each quadrant correlating their IVM maturation outcome and whole transcriptomic profiles. x. Data analysis and statistics

Oocyte maturation outcome data was analyzed using Python statistical packages pandas (1 .5.0), scipy (1 .7.3), and statsmodels (0.13.2). Maturation percentages by donor group were analyzed using linear regression as functions of the IVM environment as OSC-IVM or Media-IVM. t-test statistics were computed comparing OSC-IVM versus Media-IVM, then used to calculate p-values using Welch's correction for unequal variance. One way ANOVA was utilized for comparisons of more than two groups for spindle apparatus location analysis. Chi-squared analysis was utilized for comparison of the Leiden group population make up in transcriptomic analysis for the three sample conditions. Bar graphs depict mean values for each population and error bars represent standard error of the mean (SEM).

Example 10. hiPSC-derived OSCs effectively promote human oocyte maturation following coculture system with denuded oocytes

We have demonstrated that hiPSC-derived OSCs are predominantly composed of granulosa cells and ovarian stroma cells. In response to hormonal stimulation treatment in vitro, namely FSH, these OSCs produce growth factors and steroids, and express adhesion molecules necessary for interaction with oocytes and cumulus cells. To investigate whether hiPSC-derived OSCs are functionally capable of promoting human oocyte maturation in vitro, as an approach to rescue immature denuded oocytes, we established a co-culture system of these cells with freshly retrieved denuded immature oocytes and assessed maturation rates after 24-28 hours (FIG. 12).

We first examined whether OSC-IVM affected the rate of maturation of denuded oocytes compared to oocytes kept in the Media-IVM condition containing the same culture medium and all supplements but no OSCs, with maturation rates determined per oocyte culture group for each condition. Strikingly, we observed significant improvement in maturation outcome rates (~1 .7X) for oocytes that underwent IVM with OSCs. Specifically, the OSC-IVM group yielded a maturation rate of 62% ± 5.57% SEM versus 37% ± 8.96% SEM in the Media-IVM (FIG. 13A, p= 0.0138, unpaired t-test). We additionally scored the morphological quality of MH oocytes obtained in both IVM conditions by assessing the Total Oocyte Score (TOS). We found no significant difference between the two groups (FIG. 13B, p= 0.5725, unpaired t-test), suggesting that in vitro maturation of denuded oocytes is not affecting the morphological features of Mils. Altogether, these data indicate that OSC co-culture increases the ratio of oocyte maturation, compared to spontaneous maturation observed in the control IVM media, without a detrimental effect on morphological quality of human oocytes, and highlights the potential for the use of hiPSC-derived OSCs for rescuing immature denuded oocytes from IVF procedures.

Example 11. OSC-IVM promotes high quality assembly of the second meiotic spindle apparatus in IVM oocytes

Second meiotic spindle assembly, more specifically both the presence of and the angle of the spindle relative to PB1 , has been implicated in previous studies as a key indicator of oocyte quality in relation to fertilization and developmental competence, with the presence of a spindle with a smaller angle relative to the PB1 as an indicator of improved quality. We sought to determine the relative position of the second meiotic spindle apparatus and first polar body in OSC-treated oocytes in comparison to Mil oocytes retrieved from IVF cycles (IVF-MII), as a comparative measure of oocyte quality (FIG. 14). We also included as a control the oocytes that spontaneously matured in the Media-IVM condition (FIG. 14A). We found that the spindle angle was not significantly different between conditions (MH OSC-IVM: 22° ± 5.2 SEM; Mil Media-IVM: 15° ± 5.7 SEM; IVF-MII: 41° ± 8.3 SEM; p = 0.1155; ANOVA), suggesting that in vitro maturation of denuded oocytes does not impair spindle position. Interestingly, the only condition in which we did not observe instances of spindle absence was the condition containing MH oocytes from OSC-IVM (FIG. 14B). More studies are needed to validate the relevance of this observation, but it is likely to be an indication of formation of high-quality oocytes. Altogether, these results indicate that Mil oocytes matured in vitro in combination with OSCs hold equivalent spindle angle values to Mil oocytes directly retrieved from IVF procedures, suggesting that IVM applied to rescue denuded immature oocytes is not detrimental to oocyte quality based on this parameter.

Example 12. OSC-IVM promotes maturation of MH oocytes with high transcriptomic similarity compared to in vivo matured MH oocytes

To further compare the quality and maturation of OSC-IVM oocytes relative to a cohort of IVF-MII control oocytes and the Media-IVM oocytes, we performed single oocyte transcriptomic analysis. Transcriptomic analyses provide a global view of oocyte gene expression, providing a strong representation of their cellular state, function, and general attributes. We started by combining our datasets that included: 1 ) denuded immature oocytes after 24-28 hours in co-culture with OSC (OSC- IVM), 2) denuded immature oocytes kept in the in vitro maturation media control (Media-IVM), and 3) Mil oocytes retrieved from regular IVF cycles (IVF-MII). We next generated UMAP plots and annotated individual oocytes by Condition (OSC-IVM, Media-IVM, and IVF-MII) and Maturation outcome (GV, Ml, Mil) (FIG. 15A). From this analysis, we observed that maturation state was the main driver of oocyte separation in whole transcriptomic space, suggesting that transcriptional profile is a good predictor of oocyte maturation state. Mil oocytes project predominantly into the large cluster on the upper right half of the plot (FIG. 15A Maturation). GV oocytes project predominantly into a smaller cluster on the lower left half of the plot. Hence the separation in the UMAP is a combination of the two projected dimensions. As expected, Mil oocytes retrieved from IVF (IVF-MII) show close grouping together with Mil from both the OSC-IVM, as well as Media-IVM. Similarly, GVs from OSC-IVM and Media-IVM show close distance among each other and apart from the Mil oocytes. In contrast, Ml oocytes were scattered among both groups, likely a consequence of being an intermediate maturation state and being present in very low numbers in comparison with the other two maturation states (GVs and Mils).

We next generated a reference transcriptomic signature of conventionally matured Mil oocytes to assess the quality of Mils rescued/matured in vitro. To set a standard, we used Mil oocytes retrieved from conventional ovarian hyperstimulation IVF samples (IVF-MII) to create a gene score for IVF Mil maturation signature. In parallel, we used the stalled GVs resultant from IVM conditions (OSC-IVM and Media-IVM) to generate a gene score for IVM GV failed maturation signature (FIG. 15B). These two gene signatures were utilized to capture a relative positive control of IVM, namely an I VF-like successful maturation outcome, as well as a negative control of IVM, namely oocytes that arrest as GVs. To better understand transcriptomic nuances amongst the mature Mil oocytes, we used the Leiden algorithm to further subcluster our samples into groups sharing closer transcriptomic profiles. We identified three clusters (0, 2, and 3) within the Mil oocytes population, and one cluster (1 ) comprised mostly GVs. As expected, the GV maturation signature was strongly represented in cluster 1 . Similarly, the MH maturation signature included Mils from both IVF and IVM, and it was more overrepresented in clusters 0 and 2. As such, we designate cluster 1 as representing the (GV) failed maturation transcriptomic profile, while clusters 0 and 2 represent a profile similar to the IVF MH maturation transcriptomic profile. Interestingly, cluster 3 shows lower expression for both the IVF Mil and IVM GV failed maturation signatures. This could indicate a transitional state between immature and mature development in which neither signature is highly upregulated, or could result from cell activity stasis, shutdown, or oocyte stalling.

In FIG. 15C, we assess the quality of individual oocytes relative to our IVF Mil maturation signature (y-axis), as well as the IVM GV failed maturation signature (x-axis). For visual clarity we divide our signature dimension plot into labeled quadrants which help denote the separation between classification groups. As expected, we observe that most of the oocytes morphologically classified as GVs clustered in the lower right quadrant (IV), holding a high score for GV failed maturation signature along with a low score for IVF Mil maturation signature. In contrast, individual oocytes from the IVF-MII condition clustered together (-91%) in the upper left quadrant (I), holding a high score for Mil maturation signature and a low score for GV failed maturation signature. Strikingly, OSC-IVM Mils (blue cross) were found mostly (-79%) in the upper left quadrant (I) along with the IVF-MII oocytes, suggesting a strong transcriptomic similarity between these two groups. In contrast, Mils from the Media-IVM were often (-46%) located on the lower left quadrant (III) depicting a low score for both Mil maturation signature and GV maturation signature. Interestingly, this lower left quadrant (III) comprises in its majority cells derived from cluster 3, which despite their weak Mil maturation signature, were morphologically classified as Mils. This divergence in morphological classification and transcriptomic profile suggests that these oocytes are in a low activity state, possibly as a transitional phase before maturation or a holding state. To assess for confounding variables in our transcriptomic analysis, we assessed expression of cell cycle, apoptosis and oxidative stress genes and did not detect any significant patterns, indicating that the oocytes were not biased or stressed (FIG. 14). Altogether this observation suggests that Mil oocytes derived from OSC- IVM were transcriptionally more similar to those from the IVF-MII condition compared to Media-IVM control.

Finally, to determine the ratio of Mil oocytes with a strong IVF Mil maturation signature in each experimental condition, we calculated the percentage of cells in clusters 0 and 2, identified as containing oocytes with a ‘IVF-like MU’ signature (FIG. 15D). As expected, the strong majority (91%) of Mil oocytes from the IVF-MII condition were classified within clusters 0 and 2. As a continued indication of positive maturation impact, co-culture with OSCs led to generation of 79% Mil oocytes with an ‘IVF-like MU’ profile (cluster 0 and 2). In comparison, just 56% of resultant Mil oocytes from the Media-IVM condition were found in ‘IVF-like MU’ profile clusters. This population distribution is significantly different from random (x² test, a = 0.00632). Altogether, we conclude that OSC-IVM supports formation of Mil oocytes with high transcriptomic similarity to IVF matured Mil oocytes and highlights the potential of using this novel approach to rescue denuded immature oocytes from IVF procedures. Example 13. Granulosa cells support germ cell development within ovaroids

Current methods for inducing and culturing human primordial germ cell-like cells (hPGCLCs) produce cells corresponding to immature, premigratory primordial germ cells (PGCs) that lack expression of gonadal PGC markers such as DAZL. During fetal development, PGCs mature through interactions with gonadal somatic cells, with DAZL playing a key role in downregulation of pluripotency factors and commitment to gametogenesis. This process has recently been recreated in vitro using mouse fetal ovarian somatic cells, which allowed the development of hPGCLCs to the oogonia-like stage. We hypothesized that in v/fro-derived human granulosa cells could perform a similar role, with the potential for eliminating interspecies developmental mismatches. Therefore, we combined our granulosa cells with hPGCLCs to form ovarian organoids, which we termed ovaroids.

To generate ovaroids, we aggregated these two cell types in low-binding U-bottom wells, followed by transfer to air-liquid interface Transwell culture. As a comparison, we followed a previously described protocol to isolate fetal mouse ovarian somatic cells and aggregate them with hPGCLCs. By immunofluorescence, we observed expression of the mature marker DAZL beginning in a subset of OCT4 + hPGCLCs at 4 days of co-culture with hiPSC-derived granulosa cells (FIG. 16A). In contrast, robust DAZL expression in co-culture with mouse cells was not observed until day 32 (FIG. 16B), with fainter expression visible at day 26. Similarly, in a previous study using the same hPGCLC line and anti-DAZL antibody, DAZL expression was observed only after 77 days of co-culture with mouse fetal testis somatic cells.

The fraction of DAZL+ cells reached its maximum at day 14 in human ovaroids and day 38 in mouse ovaroids (FIG. 16C). In human ovaroids, the fraction of OCT4+ cells declined after day 8. In mouse ovaroids, the fraction of OCT4+ cells also declined over time. At day 16 in human ovaroids, DAZL+OCT4- cells were also apparent (FIG. 16E) in addition to DAZL+OCT4+ cells, and past day 38 there were more DAZL+ cells than OCT4+ cells in total (FIG. 16C). The downregulation of OCT4 in DAZL+ oogonia occurs in vivo during the second trimester of human fetal ovarian development; however, we did not observe the transition of DAZL to exclusively cytoplasmic localization that was reported to take place at this stage. Expression of TFAP2C, an early-stage PGC marker, declined during ovaroid culture (FIG. 16D) and was almost entirely absent by day 8. By contrast, SOX17 expression was still visible on day 8, and OCT4 and DAZL expression continued to day 54 (FIG. 16A, C).

Although this system allowed the rapid development of hPGCLCs to the gonadal stage, the number of germ cells in both hiPSC- and mouse-derived ovaroids declined over prolonged culture (FIG. 16C), indicating that either they were dying or differentiating to other lineages. Unlike mouse-derived ovaroids, the hiPSC-derived ovaroids cultured on Transwells gradually flattened and widened, and by day 38 were largely collapsed.

Nonetheless, in these longer-term experiments, we observed the formation of empty follicle-like structures composed of cuboidal AMHR2+FOXL2+ granulosa cells (FIG. 17A), suggesting that the TFs could drive folliculogenesis even in the absence of oocytes. Follicle-like structure formation was first visible at day 16 (FIG. 16E), and at day 26 the largest of these structures had grown to 1-2 mm diameter (FIG. 17B). At day 70, ovaroids had developed follicles of a variety of sizes, mainly small single-layer follicles (FIG. 17C) but also including antral follicles (FIG. 17D). Cells outside of the follicles stained positive for NR2F2 (FIG. 17C, D), a marker of ovarian stromal and theca cells. To further examine the gene expression of hPGCLCs and somatic cells in this system, we performed scRNA-seq on dissociated ovaroids at days 2, 4, 8, and 14 of culture, and clustered cells according to gene expression. As expected, the largest cluster (cluster 0) contained cells expressing granulosa markers such as FOXL2, WNT4, and CD82 (FIG. 17A, B). Cells expressing markers of secondary/antral granulosa cells such as FSHR and CYP19A1 were also found within this cluster, although these were much less numerous. A smaller cluster (cluster 1 ) expressing the ovarian stromal marker NR2F2 was also present. NR2F2 is expressed by both stromal and theca cells, but the cells in cluster 1 did not express 17a-hydroxylase (CYP17A1 ), indicating that they could not produce androgens and were not theca cells.

We also observed a cluster of hPGCLCs expressing marker genes such as CD38, KIT, PRDM1 , TFAP2C, PRDM14, NANOG, and POU5F1 . Notably, X-chromosomal IncRNAs XIST, TSIX, and XACT were all more highly expressed (an average of —80-, -20-, and -2900- fold, respectively) in the hPGCLCs relative to other clusters (FIG. 18B), suggesting that the hPGCLCs were starting the process of X-reactivation, which in hPGCs is associated with high expression of both XIST and XACT. The X-chromosomal HPRT1 gene, known to be more highly expressed in cells with two active X chromosomes, was also ~3-fold upregulated.

We next compared our in v/fro-generated ovaroids to a reference atlas of human fetal ovarian development. We used scanpy ingest to integrate our samples into the atlas and annotate each cell with the closest cell type from the in vivo data (FIG. 18C). The ovaroids consisted mainly of granulosa, gonadal mesenchyme, and pre-granulosa lineages (FIG. 18D), with a small fraction of coelomic epithelium. The fraction of granulosa cells increased from day 2 through day 8, potentially representing a maturation of the somatic cell population. As expected, neural, immune, smooth muscle, and erythroid cells, which were present in fetal ovaries, were completely absent from our ovaroids. Epithelial, endothelial, and perivascular cells were detected, but at very low frequency (1% or less), possibly representing a low rate of off-target differentiation.

We additionally examined the overall fraction of germ cells, as well as the fraction of cells expressing the gonadal germ cell markers DAZL and DDX4, over the course of our experiment (FIG. 18D). We defined the germ cell population based on the fetal ovary atlas integration. This population increased from days 2 to 4 but declined thereafter. In comparison, the fraction of DAZL+ and DDX4+ cells also increased from days 2 to 4 but remained roughly constant from days 4 to 14 (FIG. 18D). We performed a differential gene expression analysis and gene ontology enrichment on DAZL+ cells relative to DAZL- cells. Upregulated genes (Iog2fc >2, n = 221 ) were most highly enriched for terms related to generic developmental processes but also included terms related to adhesion and migration (e.g., ‘ameboidal-type cell migration’), as well as reproductive system development. Downregulated genes (Iog2fc <-2, n = 6451 ) were strongly related to metabolic processes and mitotic cell. These data suggest that DAZL+ cells in our ovaroids are downregulating their metabolism and proliferation, in agreement with the known role of DAZL in suppressing PGC proliferation.

Example 14. Pre-clinical trials

Preclinical trials of the OSCs-IVM system were performed using cell culture media-matched controls in a sibling oocyte study for both human denuded immature oocytes retrieved after standard of care gonadotropin stimulation, and intact immature COCs retrieved after minimal gonadotropin stimulation. The control condition contained an identical media formulation as the OSCs-IVM condition, with the only difference between conditions being the presence of the OSCs in the OSC-IVM. Results show that the OSCs-IVM system statistically significantly improved oocyte maturation rate, determined by the presence of a polar body, by ~15% with denuded oocytes from standard of care (FIG. 19A) and by -17% in intact COCs from minimal stimulation (FIG. 19B). OSCs-IVM were compared to the clinically approved Medicult-IVM system, which is marketed for use with intact COCs after minimal stimulation. OSC-IVM statistically significantly improves oocyte maturation rates by -28% on average per study donor, compared to Medicult-IVM in an on-label, sibling oocyte study (FIG. 19C).

It was also determined if OSC co-culture could improve oocyte quality. While no universally accepted method exists yet to determine “oocyte quality”, studies have shown that certain morphological and molecular features can be used to infer oocyte quality, as these features are correlated with improvements in embryo formation and live birth rates in IVF. One such measure is a total oocyte score (TOS) generated from manual qualitative assessment of six morphological features of mature oocytes: oocyte size, zona size, color/shape, cytoplasmic granularity, polar body quality, and PVS quality. Another metric of quality is spindle assembly position, which has been shown as a reliable metric of oocyte quality by measuring the angle between polar body 1 (PB1 ) and the spindle apparatus, with a decrease in angle correlated with an improvement in oocyte quality. Lastly, certain genetic markers identified in transcriptomic analysis have been correlated with oocyte quality, measuring indications such as oxidative stress, embryogenesis competence, and DNA damage. All three of these metrics were employed here to determine if OSCs-IVM could improve oocyte quality relative to media matched controls. Using a limited set of denuded immature oocytes and IVF in vivo Mil controls, it was determined that the OSCs described herein trend towards improvement of oocyte quality compared to media-matched controls and show similarity with in vivo MH oocytes in terms of morphological quality (FIG. 20A). OSCs-IVM were likewise shown to on average decrease the angle between the PB1 and spindle compared to media-matched controls and IVF in vivo Mils, with no instance of spindle absence in OSCs-IVM Mils (FIG. 20B). Additionally, through differential gene expression analysis (DGEA), it was evidenced that the OSCs-IVM oocytes show high similarity to in vivo Mil oocytes, with expected expression of key embryogenesis competence genes (FIG. 20C-D).

Additionally, both human and porcine animal models were studied to determine toxicity of the OSCs co-culture. Utilizing human preclinical models, the OSCs-IVM condition was performed and assessed for oocyte outcomes considered as “degraded”, meaning the oocytes are undergoing a rapid state of apoptosis or cell death. The OSCs-IVM results in no significant enhancement in oocyte degradation rate in human oocytes compared to the Medicult-IVM media alone (FIG.21 A). We likewise assessed whether porcine oocytes matured in the presence of the OSC-IVM product were capable of forming blastocysts. As can be seen, we were able to successfully fertilize and generate blastocysts in the OSC-IVM condition for porcine oocytes (FIG. 21 B). While these porcine studies are not designed to test efficacy, they demonstrate that OSCs-IVM is not toxic to oocytes or preventative of embryo formation.

The foregoing has been a detailed description of illustrative embodiments of the invention. Various modifications and additions can be made without departing from the spirit and scope of this invention. Features of each of the various embodiments described above may be combined with features of other described embodiments as appropriate in order to provide a multiplicity of feature combinations in associated new embodiments. Furthermore, while the foregoing describes a number of separate embodiments, what has been described herein is merely illustrative of the application of the principles of the present invention. Additionally, although particular methods herein may be illustrated and/or described as being performed in a specific order, the ordering is highly variable within ordinary skill to achieve methods, systems, apparatuses, and software according to the present disclosure. Accordingly, this description is meant to be taken only by way of example, and not to otherwise limit the scope of this invention.

Exemplary embodiments have been disclosed above and illustrated in the accompanying drawings. It will be understood by those skilled in the art that various changes, omissions and additions may be made to that which is specifically disclosed herein without departing from the spirit and scope of the present invention.

SPECIFIC EMBODIMENTS

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations following, in general, the principles and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

Claims

1 . An ex vivo composition comprising one or more ovarian support cells (OSCs) and one or more diluents or excipients, optionally wherein the composition promotes maturation of one or more oocytes.

2. The composition of claim 1 , wherein the one or more OSCs comprise one or more granulosa cells.

3. The composition of claim 2, wherein the one or more OSCs express FOXL2, AMHR2, CD82 or any combination thereof.

4. The composition of any one of claims 1 -3, wherein the one or more OSCs express one or more genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB.

5. The composition of claim 3 or 4, wherein the one or more OSCs further express one or more genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4.

6. The composition of claim 3 or 4, wherein the one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2.

7. The composition of claim 3 or 4, wherein the one or more OSCs further express genes CDH2 and/or NOTCH2.

8. The composition of any one of claims 1 -7, wherein the one or more OSCs do not exhibit significant expression of RARRES2.

9. The composition of any one of claims 1 -8, wherein the one or more OSCs express NR2F2.

10. The composition of any one of claims 1 -9, wherein the one or more OSCs comprise ovarian stroma cells.

11 . The composition of any one of claims 1 -10, wherein the one or more OSCs comprise granulosa cells and ovarian stroma cells.

12. The composition of any one of claims 1 -11 , wherein the one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

13. The composition of any one of claims 1 -12, wherein the one or more OSCs are obtained by differentiation of a population of iPSCs, optionally wherein the iPSCs are hiPSCs.

14. The composition of claim 13, wherein the hiPSCs express or overexpress one or more of the following transcription factors:

(i) RUNX2;

(ii) NR5A1 ;

(iii) GATA4;

(iv) FOXL2;

(v) any combination of two of the transcription factors;

(vi) any combination of three of the transcription factors; or

(vii) a combination of all four of the transcription factors.

15. The composition of claim 13 or 14, wherein the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.

16. The composition of any one of claims 13-15, wherein the hiPSCs are contacted with a Wnt/p- catenin pathway activator.

17. The composition of claim 16, wherein the Wnt/p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

18. The composition of any one of claims 1 -17, wherein at least one of the one or more OSCs are encapsulated.

19. The composition of claim 18, wherein the one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or any mixture thereof.

20. The composition of claim 19, wherein the one or more OSCs are encapsulated in laminin, optionally wherein the laminin is laminin-521 .

21 . The system or composition of claim 19 or 20, wherein the one or more OSCs are encapsulated in vitronectin.

22. The composition of any one of claims 1 -21 , wherein the one or more OSCs have lower expression, or undetectable expression, of one or more genes associated with pluripotency relative to an iPSC.

23. The composition of claim 22, wherein the one or more genes associated with pluripotency comprise NANOG.

24. The composition of claim 23, wherein the one or more genes associated with pluripotency comprise POU5F1 .

25. The composition of any one of claims 1 -24, wherein at least some of the OSCs produce one or more growth factors.

26. The composition of claim 25, wherein the one or more growth factors comprise insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMPs), C-type natriuretic peptide (CNP), or any combination thereof.

27. The composition of claim 25 or 26, wherein at least a portion of the one or more growth factors is secreted.

28. The composition of any one of claims 1 -27, wherein the one or more OSCs produce one or more steroids.

29. The composition of claim 28, wherein the one or more steroids comprise estradiol, progesterone, or a combination thereof.

30. The composition of claim 28 or 29, wherein the one or more steroids are produced in response to hormonal stimulation.

31 . The composition of claim 30, wherein the hormonal stimulation comprises FSH, androstenedione treatment, or a combination thereof.

32. The composition of any one of claims 28-31 , wherein at least a portion of the one or more steroids is secreted.

33. The composition of any one of claims 1 -32, wherein the one or more OSCs are cryopreserved.

34. The composition of any one of claims 1 -33, further comprising an in vitro maturation (IVM) media.

35. The composition of claim 34, wherein the IVM media comprises a cell culture media.

36. The composition of claim 34 or 35, wherein the IVM media comprises Medicult-IVM media.

37. The composition of any one of claims 34-36, wherein the IVM media comprises one or more supplements.

38. The composition of claim 37, wherein the one or more supplements comprise:

39. The composition of any one of claims 1 -38, wherein the one or more oocytes are retrieved from a donor subject.

40. The composition of claim 39, wherein the donor subject is from about 19 years old to about 45 years old.

41 . The composition of claim 39, wherein the subject is undergoing ovarian stimulation.

42. The composition of claim 41 , wherein the ovarian stimulation comprises treatment with gonadotropin releasing hormone (GnRH).

43. The composition of claim 42, wherein the ovarian stimulation comprises treatment with one or more GnRH analogs.

44. The composition of claim 43, wherein the one or more GnRH analog is a GnRH agonist or antagonist.

45. The composition of any one of claims 41 -44, wherein the ovarian stimulation comprises one or more ovulatory triggers.

46. The composition of claim 45, wherein the one or more ovulatory triggers comprise human chorionic gonadotropin (hCG).

47. The composition of claim 45 or 46, wherein the one or more ovulatory trigger comprise a GnRH agonist, optionally wherein the GnRH agonist is leuprolide.

48. The composition of any one of claims 41 -47, wherein the ovarian stimulation comprises FSH treatment.

49. The composition of any one of claims 41 -47, wherein the ovarian stimulation does not comprise FSH treatment.

50. The composition of claim 48, wherein the FSH treatment comprises 300 international units (IU) to 700 IU of FSH.

51 . The composition of claim 50, wherein the FSH treatment comprises 400 IU to 600 IU of FSH.

52. The composition of any one of claims 48, 50, and 51 , wherein the FSH treatment comprises 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment comprises a plurality of injections, wherein each injection comprises a dose of about 100 IU to about 200 IU of the FSH.

53. The composition of any one of claims 41 -52, wherein the ovarian stimulation further comprises clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more injections, optionally wherein each injection comprises a dose of 50 mg to 150 mg.

54. The composition of claim 41 -53, wherein the ovarian stimulation further comprises one or more hCG triggers.

55. The composition of claim 54, wherein the one or more hCG triggers comprises 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.

56. The composition of any one of claims 1 -55, wherein the one or more oocytes are in cumulus oocyte complexes (COCs).

57. The composition of any one of claims 1 -56, wherein the one or more oocytes comprise one or more denuded immature oocytes.

58. The composition of claim 57, wherein all of the one or more oocytes are denuded immature oocytes.

59. The composition of any one of claims 1 -56, wherein the one or more oocytes are not denuded.

60. The composition of any one of claims 1 -59, wherein the one or more oocytes comprise one or more germinal vesicle (GV)-containing oocytes.

61 . The composition of any one of claims 1 -60, wherein the one or more of the oocytes comprise one or more oocytes in metaphase I (Ml).

62. The composition of any one of claims 1 -61 , wherein the one or more of the oocytes comprise one or more oocytes in metaphase II (MH).

63. The composition of any one of claims 1 -62, wherein at least a portion of the one or more oocytes comprise one or more previously vitrified oocytes.

64. The composition of any one of claims 1 -63, wherein at least a portion of the one or more oocytes comprise one or more previously cryopreserved oocytes.

65. The composition of any one of claims 1 -64, wherein the one or more oocytes are co-cultured with the one or more OSCs.

66. The composition of claim 65, wherein prior to and/or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV-stage to Mil-stage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Mil-stage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.

67. The composition of claim 66, wherein the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.

68. The composition of claim 66 or 67, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs.

69. The composition of any one of claims 66-68, wherein the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes.

70. The composition of any one of claims 65-69, wherein the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.

71 . The composition of any one of claims 65-70, wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours.

72. The composition of claim 71 , wherein the one or more oocytes are co-cultured with the one or more OSCs for about 24 hours to about 28 hours.

73. The composition claim 71 or 72, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more blastocytes following contact with one or more mature sperm cells.

74. The composition of any one of claims 65-73, wherein the one or more oocytes are co-cultured in direct contact with the one or more OSCs.

75. The composition of any one of claims 65-74, wherein the one or more oocytes do not directly contact the OSCs.

76. The composition of any one of claims 65-75, wherein the culture system is a suspension culture.

77. The composition of any one of claims 65-75, wherein the culture system is an adherent culture.

78. A method of cultivating oocytes, wherein one or more oocytes are co-cultured with one or more ovarian supporting cells (OSCs).

79. A method of preparing one or more oocytes that have previously been retrieved from a human subject for use in an assisted reproduction technology (ART) procedure, the method comprising coculturing the one or more oocytes with one or more OSCs.

80. A method of producing a mature oocyte for use in an ART procedure, the method comprising coculturing one or more oocytes that have previously been retrieved from a human subject with a population of ovarian support cells that have been differentiated from one or more iPSCs.

81 . A method of inducing oocyte maturation in vitro, the method comprising co-culturing one or more oocytes with a population of ovarian support cells that have been differentiated from one or more iPSCs, optionally wherein the co-culturing is conducted for a period of from about 6 hours to about 120 hours.

82. A method of producing a mature oocyte for use in an ART procedure, the method comprising:

(a) differentiating one or more iPSCs to produce one or more OSCs;

(b) retrieving one or more immature oocytes from a subject; and

83. A method of promoting oocyte maturation for a subject undergoing an ART procedure and that has previously been administered one or more follicular triggering agents during a follicular triggering period, the method comprising:

(a) retrieving one or more immature oocytes from the subject;

(c) isolating the one or more mature oocytes.

84. The method of any one of claims 78-83, wherein prior to and/or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV- stage to Mil-stage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Milstage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.

85. The method of claim 84, wherein the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.

86. The method of any one of claims 84 or 85, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs.

87. The method of claim 86, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes matured in vivo.

88. The method any one of claims 84-87, wherein the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes.

89. The method of any one of claims 78-87, wherein the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.

90. The method of any one of claims 78-89, wherein the one or more OSCs express FOXL2, AMHR2, CD82 or any combination thereof.

91 . The method of any one of claims 78-90, wherein the one or more OSCs express one or more of the genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB.

92. The method of claim 90 or 91 , wherein the one or more OSCs further express one or more of the genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4.

93. The method of claim 90 or 91 , wherein the one or more OSCs further express one or more of the genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2.

94. The method of claim 90 or 91 , wherein the one or more OSCs further express genes CDH2 and/or NOTCH2, with no significant gene expression of RARRES2.

95. The method of any one of claims 78-94, wherein the one of more OSCs comprise granulosa cells.

96. The method of any one of claims 78-90, wherein the one or more OSCs express NR2F2.

97. The method of any one of claims 78-90 and 96, wherein the one or more OSCs comprise ovarian stroma cells.

98. The method of any one of claims 78-97, wherein the one or more OSCs comprise granulosa cells and ovarian stroma cells.

99. The method of any one of claims 78-98, wherein the one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

100. The method of any one of claims 78-99, wherein the one or more OSCs are obtained by differentiation of a population of iPSCs, optionally wherein the population of iPSCs are hiPSCs.

101 . The method of claim 100, wherein the hiPSCs express or overexpress the following one or more transcription factors:

(i) RUNX2;

(ii) NR5A1 ;

(iii) GATA4;

(iv) FOXL2;

(v) any combination of two of the transcription factors;

(vi) any combination of three of the transcription factors; or

(vii) a combination of all four of the transcription factors.

102. The method of claim 101 , wherein the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.

103. The method of any one of claims 100-102, wherein the hiPSCs are contacted with a Wnt/p- catenin pathway activator.

104. The method claim 103, wherein the Wnt/p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

105. The method of any one of claims 78-104, wherein the one or more OSCs are encapsulated.

106. The method of claim 105, wherein the one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or a mixture thereof.

107. The method of claim 106, wherein the one or more OSCs are encapsulated in laminin, optionally wherein the laminin is laminin-521 .

108. The method of claim 106 or 107, wherein the one or more OSCs are encapsulated in vitronectin.

109. The method of any one of claims 78-108, wherein the one or more OSCs have low or undetectable expression of one or more genes associated with pluripotency relative to an iPSC.

110. The method of claim 109, wherein the one or more genes associated with pluripotency comprise NANOG.

111. The method of claim 110, wherein the one or more genes associated with pluripotency comprise POU5F1.

112. The method of any one of claims 78-111 , wherein the one or more of the OSCs produce one or more growth factors.

113. The method of claim 112, wherein the growth factors comprise IGF, SCF, EGF, LIF, VEGF, BMPs, CNP, or any combination thereof.

114. The method of claim 112 or 113, wherein at least a portion of the one or more growth factors is secreted.

115. The method of any one of claims 78-114, wherein the one or more OSCs produce one or more steroids.

116. The method of claim 115, wherein the one or more steroids comprise estradiol, progesterone, or a combination thereof.

117. The method of claim 115 or 116, wherein the one or more steroids are produced in response to hormonal stimulation of the OSCs.

118. The method of claim 117, wherein the hormonal stimulation comprises exposure to FSH, androstenedione, or a combination thereof.

119. The method of any one of claims 115-118, wherein at least a portion of the one or more steroids is secreted.

120. The method of any one of claims 78-119, wherein the one or more OSCs were previously cryopreserved.

121 . The method of any one of claims 78-120 further comprising culturing the one or more OSCs with the one of more oocytes in an in vitro maturation (IVM) media.

122. The method of claim 121 , wherein the IVM media comprises a cell culture media.

123. The method of claim 121 or 122, wherein the IVM media comprises Medicult-IVM media.

124. The method of any one of claims 121 -123, wherein the IVM media comprises one or more supplements.

125. The method of claim 124, wherein the one or more supplements comprise:

126. The method of any one of claims 78-125, wherein the one or more oocytes is retrieved from a donor subject.

127. The method of claim 126, wherein the subject is from about 19 years old to about 45 years old.

128. The method of claim 126, wherein the subject is undergoing ovarian stimulation.

129. The method of claim 128, wherein the ovarian stimulation comprises treatment with gonadotropin releasing hormone (GnRH).

130. The method of claim 129, wherein the ovarian stimulation comprises treatment with one or more GnRH analogs.

131 . The method of claim 130, wherein the GnRH analog is a GnRH agonist or antagonist.

132. The method of any one of claims 128-131 , wherein the ovarian stimulation comprises one or more ovulatory triggers.

133. The method of claim 132, wherein the one or more ovulatory triggers comprise hCG.

134. The method of claim 132 or 133, wherein the one or more ovulatory triggers comprise GnRH agonist, optionally wherein the GnRH agonist is leuprolide.

135. The method of any one of claims 128-134, wherein the ovarian stimulation comprises FSH treatment.

136. The method of claim 128-134, wherein the ovarian stimulation does not comprise FSH treatment.

137. The method of claim 135, wherein the FSH treatment comprises 300 IU to 700 IU of FSH.

138. The method of claim 137, wherein the FSH treatment comprises 400 IU to 600 IU of FSH.

139. The method of any one of claims 135, 137, and 138, wherein the FSH treatment comprises 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment comprises a plurality of injections, wherein each injection comprises a dose of about 100 IU to about 200 IU of the FSH.

140. The method of any one of claims 128-139, wherein the ovarian stimulation comprises clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, optionally wherein each dose is between 50 mg and150 mg.

141. The method of claim 128-140, wherein the ovarian stimulation comprises one or more hCG triggers.

142. The method of claim 141 , wherein the one or more hCG triggers comprises 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.

143. The method of any one of claims 78-142, wherein the one or more oocytes are in cumulus oocyte complexes (COCs).

144. The method of any one of claims 78-143, wherein the one or more oocytes comprise one or more denuded immature oocytes.

145. The method of claim 144, wherein all of the one or more oocytes are denuded immature oocytes.

146. The method of any one of claims 78-143, wherein the one or more oocytes are not denuded.

147. The method of any one of claims 78-146, wherein one or more oocytes comprise one or more germinal vesicle (GV)-containing oocytes.

148. The method of any one of claims 78-147, wherein one or more oocytes comprise one or more oocytes in metaphase I (Ml).

149. The method of any one of claims 78-148, wherein one or more oocytes comprise one or more oocytes in metaphase II (Mil).

150. The method of any one of claims 78-149, wherein one or more oocytes comprise one or more previously vitrified oocytes.

151 . The method of any one of claims 78-150, wherein one or more oocytes comprise one or more previously cryopreserved oocytes.

152. The method of any one of claims 78-151 , wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours.

153. The method of claim 152, wherein the one or more oocytes are cultured with the one or more OSCs for about 24 hours to about 28 hours.

154. The method of claim 152 or 153, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more blastocytes following contact with one or more mature sperm cells.

155. The method of any one of claims 78-154, wherein the one or more oocytes are cultured in direct contact with the one or more OSCs.

156. The method of any one of claims 78-155, wherein the one or more oocytes do not directly contact the one or more OSCs.

157. The method of any one of claims 78-156, wherein the co-culture is a suspension co-culture.

158. The method of claim 78-156, wherein the co-culture is an adherent co-culture.

159. A method of promoting differentiation of one or more induced pluripotent stem cells (iPSCs) to one or more ovarian support cells (OSCs), the method comprising:

(a) culturing the one or more iPSCs in vitro;

160. A method of producing one or more ovarian support cells (OSCs) from one or more induced pluripotent stem cells (iPSCs), the method comprising:

(a) culturing the one or more iPSCs in vitro; (b) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors comprising FOXL2, NR5A1 , RUNX2, GATA4, or any combination thereof, thereby producing differentiated cells;

161 . A method of preparing a composition comprising one or more ovarian support cells (OSCs), the method comprising:

(a) culturing the one or more iPSCs in vitro;

162. The method of any one of claims 159-161 , wherein the iPSCs are human iPSCs (hiPSCs).

163. The method of any one of claims 159-162, wherein the iPSCs were previously cryopreserved.

164. The method of any one of claims 159-163, wherein the co-culturing is performed in in vitro maturation (IVM) media.

165. The method of claim 164, wherein the IVM media comprises a cell culture media.

166. The method of claim 164 or 165, wherein the IVM media comprises Medicult-IVM media.

167. The method of any one of claims 164-166, wherein the IVM media comprises one or more supplements.

168. The method of claim 167, wherein the one or more supplements comprise:

(iv) androstenedione, optionally at a concentration of about 495 ng/mL to about 505 ng/mL, further optionally at a concentration of 500 ng/mL; (v) doxycycline, optionally at a concentration of about 0.5 pg/mL to about 1 .5 pg/mL, further optionally at a concentration of 1 pg/mL; or any combination of the one or more supplements.

169. The method of any one of claims 159-168, wherein the induction of iPSCs to OSCs occurs for about 1 day to about 10 days, optionally wherein the induction occurs for about 5 days.

170. The method of any one of claims 159-169, wherein the iPSCs are cultured in a media comprising a matrix.

171 . The method of claim 170, wherein the matrix comprises alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or a mixture thereof.

172. The method of claim 171 , wherein matrix comprises laminin, optionally wherein the laminin is laminin-521 .

173. The method of claim 171 or 172, wherein the matrix comprises vitronectin.

174. The method of any one of claims 159-173, wherein the iPSCs are reprogrammed using a transposase method to carry one or more inducible transcription factors.

175. The method of any one of claims 159-173, wherein the iPSCs are transformed via electroporation, liposome-mediated transformation, or viral-mediated gene transfer.

176. The method of any one of claims 159-175, wherein the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.

177. The method of any one of claims 159-176, wherein the iPSCs are contacted with a Wnt/p- catenin pathway activator.

178. The method claim 177, wherein the Wnt/p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

179. The method of any one of claims 159-178, wherein the one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof.

180. The method of any one of claims 159-179, wherein the one or more OSCs express one or more of the following genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB.

181 . The method of claim 179 or 180, wherein the one or more OSCs further express one or more of the genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4.

182. The method of claim 179 or 180, wherein the one or more OSCs further express one or more of the genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2.

183. The method of claim 179 or 180, wherein the one or more OSCs further express genes CDH2 and/or NOTCH2, optionally with no significant gene expression of RARRES2.

184. The method of any one of claims 159-179, wherein the one or more OSCs comprise one or more granulosa cells.

185. The method of any one of claims 159-179, wherein the one or more OSCs express NR2F2.

186. The method of any one of claims 159-179 and 185, wherein the one or more OSCs comprise one or more ovarian stroma cells.

187. The method of any one of claims 159-186, wherein the one or more OSCs comprise granulosa cells and ovarian stroma cells.

188. The method of any one of claims 159-187, wherein the one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

189. The method of any one of claims 159-188, wherein the one or more OSCs have low or undetectable expression of one or more genes associated with pluripotency relative to an iPSC.

190. The method of claim 189, wherein the one or more genes associated with pluripotency comprise NANOG.

191 . The method of claim 190, wherein the one or more genes associated with pluripotency comprise POU5F1 .

192. The method of any one of claims 159-191 , wherein one or more of the OSCs produce one or more growth factors.

193. The method of claim 192, wherein the growth factors comprise IGF, SCF, EGF, LIF, VEGF, BMPs, CNP, or any combination thereof.

194. The method of claim 192 or 193, wherein at least a portion of the one or more growth factors is secreted.

195. The method of any one of claims 159-194, wherein the one or more of the OSCs produce one or more steroids.

196. The method of claim 195, wherein the one or more steroids comprise estradiol, progesterone, or a combination thereof.

197. The method of claim 195 or 196, wherein the one or more steroids are produced in the presence of one or more hormones.

198. The method of claim 197, wherein the one or more hormones comprises FSH, androstenedione, or a combination thereof.

199. The method of any one of claims 194-198, wherein the one or more steroids are secreted.

200. The method of any one of claims 159-199, wherein the one or more oocytes retrieved from the subject are immature oocytes.

201 . The method of any one of claims 159-200, wherein the co-culturing the one or more OSCs with one or more oocytes promotes the maturation of the one or more oocytes.

202. The method of any one of claims 159-201 , wherein the method further comprises harvesting the one or more oocytes for an assisted reproductive technology procedure.

203. The method of any one of claims 159-202, wherein the subject is undergoing ovarian stimulation prior to the retrieval of one or more oocytes.

204. The method of claim 203, wherein the ovarian stimulation comprises treatment with gonadotropin releasing hormone (GnRH).

205. The method of claim 203 or 204, wherein the ovarian stimulation comprises treatment with one or more GnRH analogs.

206. The method of claim 205, wherein the one or more GnRH analog is a GnRH agonist or antagonist.

207. The method of any one of claims 203-206, wherein the ovarian stimulation comprises one or more ovulatory triggers.

208. The method of claim 207, wherein the one or more ovulatory triggers comprises hCG.

209. The method of claim 207 or 208, wherein the one or more ovulatory trigger comprises a GnRH agonist, optionally wherein the GnRH agonist is leuprolide.

210. The method of any one of claims 203-209, wherein the ovarian stimulation comprises FSH treatment.

211 . The method of any one of claims 203-209, wherein the ovarian stimulation does not comprise FSH treatment.

212. The method of claim 210, wherein the FSH treatment comprises 300 IU to 700 IU of FSH..

213. The method of claim 212, wherein the FSH treatment comprises 400 IU to 600 IU of FSH.

214. The method of any one of claims 210, 212, and 213, wherein the FSH treatment comprises 1 , 2,

3, or more injections of FSH, optionally wherein the FSH treatment comprises a plurality of injections, wherein each injection comprises a dose of about 100 IU to about 200 IU of the FSH.

215. The method of any one of claims 203-214, wherein the ovarian stimulation further comprises clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, optionally wherein each dose is between 50 mg and 150 mg.

216. The method of claim 203-215, wherein the ovarian stimulation further comprises one or more hCG triggers.

217. The composition of claim 216, wherein the one or more hCG triggers comprises 2,500 IU to

10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.

218. The method of any one of claims 159-217, wherein the one or more oocytes are in cumulus oocyte complexes (COCs).

219. The method of any one of claims 159-218, wherein the one or more oocytes comprise one or more denuded immature oocytes.

220. The method of claim 219, wherein all of the one or more oocytes are denuded immature oocytes.

221 . The method of any one of claims 159-217, wherein the one or more oocytes are not denuded prior to or following co-culturing.

222. The method of any one of claims 159-221 , wherein the one or more oocytes comprise one or more germinal vesicle (GV)-containing oocytes.

223. The method of any one of claims 159-222, wherein the one or more oocytes comprise one or more oocytes in metaphase I (Ml).

224. The method of any one of claims 159-223, wherein the one or more oocytes comprise one or more oocytes in metaphase II (MH).

225. The method of any one of claims 159-224, wherein at least a portion of the one or more oocytes comprise one or more previously vitrified oocytes.

226. The method of any one of claims 159-225, wherein at least a portion of the one or more oocytes comprises one or more previously cryopreserved oocytes.

227. The method of any one of claims 159-226, wherein prior to and/or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV-stage to Mil-stage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Mil-stage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.

228. The method of claim 227, wherein the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.

229. The method of claim 227 or 228, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs.

230. The method of any one of claims 227-229, wherein the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes.

231 . The method of any one of claims 159-230, wherein the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.

232. The method of any one of claims 159-231 , wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours.

233. The method of claim 232, wherein the one or more oocytes are cultured with the one or more OSCs for about 24 hours to about 28 hours.

234. The method of any one of claims 232 or 233, the method further comprising isolating one or more Mil-stage oocytes from the co-culture comprising the one or more oocytes retrieved from the subject with the one or more OSCs.

235. The method of any one of claims 232-234, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more blastocytes following contact with one or more mature sperm cells.

236. The method of any one of claims 159-235, wherein the one or more oocytes are cultured in direct contact with the one or more OSCs.

237. The method of claim 159-235, wherein the one or more oocytes do not directly contact the one or more OSCs.

238. The method of any one of claims 159-237, wherein the co-culture is a suspension co-culture.

239. The method of any one of claims 159-237, wherein the co-culture is an adherent co-culture.

240. A cell culture system comprising one or more ovarian support cells (OSCs), wherein the system promotes maturation of one or more oocytes.

241 . The system of claim 240, wherein the one or more OSCs comprise one or more granulosa cells.

242. The system of claim 241 , wherein the one or more OSCs express FOXL2, AMHR2, CD82 or any combination thereof.

243. The system of any one of claims 240-242, wherein the one or more OSCs express one or more genes selected from GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and ITGB.

244. The system of claim 242 or 243, wherein the one or more OSCs further express one or more genes selected from FOXO1 , CDH1 , CYP19A1 , RARRES2, NOTCH2, NRG1 , BMPR1 B, EGFR (ERBB1 ), and ERBB4.

245. The system of claim 242 or 243, wherein the one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2.

246. The system of claim 242 or 243, wherein the one or more OSCs further express genes CDH2 and/or NOTCH2.

247. The system of any one of claims 240-246, wherein the one or more OSCs do not exhibit significant expression of RARRES2.

248. The system of any one of claims 240-247, wherein the one or more OSCs express NR2F2.

249. The system of any one of claims 240-248, wherein the one or more OSCs comprise ovarian stroma cells.

250. The system of any one of claims 240-249, wherein the one or more OSCs comprise granulosa cells and ovarian stroma cells.

251 . The system of any one of claims 240-250, wherein the one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

252. The system of any one of claims 240-251 , wherein the one or more OSCs are obtained by differentiation of a population of iPSCs, optionally wherein the iPSCs are hiPSCs.

253. The system of claim 252, wherein the hiPSCs express or overexpress one or more of the following transcription factors:

(i) RUNX2;

(ii) NR5A1 ;

(iii) GATA4;

(iv) FOXL2;

(v) any combination of two of the transcription factors;

(vi) any combination of three of the transcription factors; or

(vii) a combination of all four of the transcription factors.

254. The system of claim 252 or 253, wherein the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.

255. The system of any one of claims 252-254, wherein the hiPSCs are contacted with a Wnt/p- catenin pathway activator.

256. The system of claim 255, wherein the Wnt/p-catenin pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

257. The system of any one of claims 240-256, wherein at least one of the one or more OSCs is encapsulated.

258. The system of claim 257, wherein the OSC(s) are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or any mixture thereof.

259. The system of claim 258, wherein the OSC(s) are encapsulated in laminin, optionally wherein the laminin is laminin-521 .

260. The system or system of claim 258 or 259, wherein the OSCs are encapsulated in vitronectin.

261 . The system of any one of claims 240-260, wherein the one or more OSCs have lower expression, or undetectable expression, of one or more genes associated with pluripotency relative to an iPSC.

262. The system of claim 261 , wherein the one or more genes associated with pluripotency comprises NANOG.

263. The system of claim 262, wherein the one or more genes associated with pluripotency comprises POU5F1 .

264. The system of any one of claims 240-263, wherein at least some of the OSCs produce one or more growth factors.

265. The system of claim 264, wherein the one or more growth factors comprise insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMPs), C-type natriuretic peptide (CNP), or any combination thereof.

266. The system of claim 264 or 265, wherein at least a portion of the one or more growth factors are secreted.

267. The system of any one of claims 240-266, wherein one or more of the OSCs produce one or more steroids.

268. The system of claim 267, wherein the one or more steroids comprise estradiol, progesterone, or a combination thereof.

269. The system of claim 267 or 268, wherein the one or more steroids are produced in response to hormonal stimulation.

270. The system of claim 269, wherein the hormonal stimulation comprises FSH, androstenedione treatment, or a combination thereof.

271 . The system of any one of claims 267-270, wherein at least a portion of the one or more steroids are secreted.

272. The system of any one of claims 240-271 , wherein the one or more OSCs are cryopreserved.

273. The system of any one of claims 240-272, further comprising an in vitro maturation (IVM) media.

274. The system of claim 273, wherein the IVM media comprises a cell culture media.

275. The system of claim 273 or 274, wherein the IVM media comprises Medicult-IVM media.

276. The system of any one of claims 273-275, wherein the IVM media comprises one or more supplements.

277. The system of claim 276, wherein the one or more supplements comprise:

278. The system of any one of claims 240-277, wherein the one or more oocytes are retrieved from a donor subject.

279. The system of claim 278, wherein the donor subject is from about 19 years old to about 45 years old.

280. The system of claim 278, wherein the subject is undergoing ovarian stimulation.

281 . The system of claim 280, wherein the ovarian stimulation comprises treatment with gonadotropin releasing hormone (GnRH).

282. The system of claim 281 , wherein the ovarian stimulation comprises treatment with one or more GnRH analogs.

283. The system of claim 282, wherein the one or more GnRH analog is a GnRH agonist or antagonist.

284. The system of any one of claims 280-283, wherein the ovarian stimulation comprises one or more ovulatory triggers.

285. The system of claim 284, wherein the one or more ovulatory triggers comprises hCG.

286. The system of claim 284 or 285, wherein the one or more ovulatory trigger comprises a GnRH agonist, optionally wherein the GnRH agonist is leuprolide.

287. The system of any one of claims 280-286, wherein the ovarian stimulation comprises FSH treatment.

288. The system of any one of claims 280-286, wherein the ovarian stimulation does not comprise FSH treatment.

289. The system of claim 287, wherein the FSH treatment comprises 300 international units (IU) to 700 IU of FSH.

290. The system of claim 289, wherein the FSH treatment comprises 400 IU to 600 IU of FSH.

291 . The system of any one of claims 287, 289, and 290, wherein the FSH treatment comprises 1 , 2, 3, or more injections of FSH, optionally wherein the FSH treatment comprises a plurality of injections, wherein each injection comprises a dose of about 100 IU to about 200 IU of the FSH.

292. The system of any one of claims 280-291 , wherein the ovarian stimulation further comprises clomiphene citrate administration, optionally wherein the clomiphene citrate is administered for up to 8 days as one or more doses, optionally wherein each dose is between 50 mg and 150 mg.

293. The system of claim 280-292, wherein the ovarian stimulation further comprises one or more hCG triggers.

294. The system of claim 293, wherein the one or more hCG triggers comprises 2,500 IU to 10,000 IU of hCG or about 200 pg to about 700 pg of hCG, optionally wherein the hCG is administered to the subject at a dose of about 400 pg to about 600 pg, further optionally wherein the hCG is administered to the subject at a dose of about 500 pg per dose.

295. The system of any one of claims 240-294, wherein the one or more oocytes are in cumulus oocyte complexes (COCs).

296. The system of any one of claims 240-295, wherein the one or more oocytes comprise one or more denuded immature oocytes.

297. The system of claim 296, wherein all of the one or more oocytes are denuded immature oocytes.

298. The system of any one of claims 240-295, wherein the one or more oocytes are not denuded.

299. The system of any one of claims 240-298, wherein one or more oocytes comprise one or more germinal vesicle (GV)-containing oocytes.

300. The system of any one of claims 240-299, wherein one or more of the oocytes comprise one or more oocytes in metaphase I (Ml).

301 . The system of any one of claims 240-300, wherein one or more of the oocytes comprise one or more oocytes in metaphase II (MH).

302. The system of any one of claims 240-301 , wherein at least a portion of the one or more oocytes comprise one or more previously vitrified oocytes.

303. The system of any one of claims 240-302, wherein at least a portion of the one or more oocytes comprise one or more previously cryopreserved oocytes.

304. The system of any one of claims 240-303, wherein the one or more oocytes are co-cultured with the one or more OSCs.

305. The system of claim 304, wherein prior to and/or after the co-culturing, the one or more oocytes are evaluated for a parameter selected from the group consisting of total oocyte score, GV-stage to Milstage oocyte maturation rate, GV-stage to Ml-stage oocyte maturation rate, Ml-stage to Mil-stage oocyte maturation rate, average oocyte shape, average oocyte size, average ooplasm quality, average perivitelline space (PVS) quality, average zona pellucida (ZP) quality, and average polar body quality.

306. The system of claim 305, wherein the one or more co-cultured oocytes have morphological quality substantially the same as in vivo matured oocytes, wherein the morphological quality comprises oocyte size, oocyte zona size, oocyte color, oocyte shape, oocyte cytoplasmic granularity, oocyte polar body quality, and oocyte PVS quality.

307. The system of claim 305 or 306, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not comprise the one or more OSCs.

308. The system of any one of claims 305-307, wherein the one or more co-cultured oocytes have a second meiotic metaphase spindle located substantially in the same position as in vivo matured oocytes.

309. The system of any one of claims 304-308, wherein the one or more co-cultured oocytes have a transcriptomic profile substantially the same as in vivo matured oocytes.

310. The system of any one of claims 304-309, wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours.

311 . The system of claim 310, wherein the one or more oocytes are co-cultured with the one or more OSCs for about 24 hours to about 28 hours.

312. The system claim 310 or 311 , wherein the one or more oocytes co-cultured with the one or more OSCs form one or more blastocytes following contact with one or more mature sperm cells.

313. The system of any one of claims 304-312, wherein the one or more oocytes are co-cultured in direct contact with the one or more OSCs.

314. The system of any one of claims 304-313, wherein the one or more oocytes do not directly contact the OSCs.

315. The system of any one of claims 304-314, wherein the culture system is a suspension culture.

316. The system of any one of claims 304-314, wherein the culture system is an adherent culture.

317. A kit comprising the composition of any one of claims 1 -77 and a package insert, wherein the package insert instructs a user of the kit to co-culture the population of ovarian support cells with one or more oocytes in accordance with the method of any one of claims 78-158.

318. A kit comprising a vial that contains a population of iPSCs and a package insert, wherein the package insert instructs a user of the kit to differentiate the population of iPSCs to one or more ovarian support cells in accordance with the method of any one of claims 159-239.

319. A kit comprising a vial that contains one or more OSCs and a package insert, wherein the package insert instructs a user of the kit to cultivate the cell culture system of any one of claims 240-316.