WO2016036697A1 - Methods of detecting embryo mosaicism - Google Patents

Abstract

Non-invasive methods, compositions and kits for determining the relative risk that one or more embryos is mosaic are provided. These methods, compositions and kits find use in identifying embryos in vitro that are most useful in treating infertility in humans.

Description

METHODS OF DETECTING EMBRYO MOSAICISM REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 62/046,828 filed on September 5, 2014, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to the field of biological and clinical testing, and particularly the imaging and evaluation of zygotes/embryos from both humans and animals.

BACKGROUND OF THE INVENTION

[0003] Infertility is a common health problem that affects 10-15% of couples of reproductive-age. In the United States alone in the year 2006, approximately 140,000 cycles of in vitro fertilization (IVF) were performed (cdc.gov/art). This resulted in the culture of more than a million embryos annually with variable, and often ill-defined, potential for implantation and development to term. The live birth rate, per cycle, following IVF was just 29%, while on average 30% of live births resulted in multiple gestations (cdc.gov/art).

Multiple gestations have well-documented adverse outcomes for both the mother and fetuses, such as miscarriage, pre-term birth, and low birth rate. Potential causes for failure of IVF are diverse; however, since the introduction of IVF in 1978, one of the major challenges has been to identify the embryos that are most suitable for transfer and most likely to result in term pregnancy.

[0004] The understanding in the art of basic embryo development is limited as studies on human embryo biology remain challenging and often exempt from research funding.

Consequently, most of the current knowledge of embryo development derives from studies of model organisms. Embryos from different species go through similar developmental stages, however, the timing varies by species. These differences and many others make it inappropriate to directly extrapolate from one species to another. (Taft, R.E. (2008)

Theriogenology 69(1):10-16). The general pathways of human development, as well as the fundamental underlying molecular determinants, are unique to human embryo development. For example, in mice, embryonic transcription is activated approximately 12 hours post- fertilization, concurrent with the first cleavage division, whereas in humans embryonic gene activation (EGA) occurs on day 3, around the 8-cell stage (Bell, C. E., et al. (2008) Mol. Hum. Reprod. 14:691-701; Braude, P., et al. (1988) Nature 332:459-461; Hamatani, T. et al. (2004) Proc. Natl. Acad. Sci. 101:10326-10331; Dobson, T. et al. (2004) Human Molecular Genetics 13(14):1461–1470). In addition, the genes that are modulated in early human development are unique (Dobson, T. et al. (2004) Human Molecular Genetics 13(14):1461– 1470). Moreover, in other species such as the mouse, more than 85% of embryos cultured in vitro reach the blastocyst stage, one of the first major landmarks in mammalian development, whereas cultured human embryos have an average blastocyst formation rate of approximately 30-50%, with a high incidence of mosaicism and aberrant phenotypes, such as fragmentation and developmental arrest (Rienzi, L. et al. (2005) Reprod. Biomed. Online 10:669-681; Alikani, M., et al. (2005) Mol. Hum. Reprod. 11:335-344; Keltz, M. D., et al. (2006) Fertil. Steril. 86:321-324; French, D. B., et al. (2009) Fertil. Steril.). In spite of such differences, the majority of studies of preimplantation embryo development derive from model organisms and are difficult to relate to human embryo development (Zernicka-Goetz, M. (2002) Development 129:815-829; Wang, Q., et al. (2004) Dev Cell. 6:133-144; Bell, C. E., et al. (2008) Mol. Hum. Reprod. 14:691-701; Zernicka-Goetz, M. (2006) Curr. Opin. Genet. Dev. 16:406-412; Mtango, N. R., et al. (2008) Int. Rev. Cell. Mol. Biol. 268:223-290).

[0005] Traditionally in IVF clinics, human embryo viability has been assessed by simple morphologic observations such as the presence of uniformly-sized, mononucleate blastomeres and the degree of cellular fragmentation (Rijinders PM, Jansen CAM. (1998) Hum Reprod 13:2869-73; Milki AA, et al. (2002) Fertil Steril 77:1191-5). More recently, additional methods such as extended culture of embryos (to the blastocyst stage at day 5) and analysis of chromosomal status via preimplantation genetic diagnosis (PGD) have also been used to assess embryo quality (Milki A, et al. (2000) Fertil Steril 73:126-9; Fragouli E, (2009) Fertil Steril Jun 21; El-Toukhy T, et al. (2009) Hum Reprod 6:20; Vanneste E, et al. (2009) Nat Med 15:577-83). However, potential risks of these methods also exist in that they prolong the culture period and disrupt embryo integrity (Manipalviratn S, et al. (2009) Fertil Steril 91:305-15; Mastenbroek S, et al. (2007) N Engl J Med. 357:9-17). Additionally, PGD analyzes chromosomal content of a single blastomere and therefore is not able to detect mosaicism.

[0006] Single-embryo transfer (SET) is currently the preferred practice in in vitro fertilization (IVF) treatment in order to reduce the risk for adverse outcomes associated with multiple gestation pregnancy. For SET, embryologists need a reliable embryo selection method that allows for consistent identification of embryos with the highest developmental potential. Recently, time-lapse analysis of embryo development kinetics has been shown to provide valuable information to improve embryo selection and subsequent pregnancy outcomes.

[0007] The use of time lapse imaging through day 3 of development provides insight into the probability a cleavage stage embryo will blastulate. This raises the question of whether these parameters and/or other features extracted through time-lapse imaging during this time or other stages of development can be used in a calculation to determine whether or not an embryo is at high risk for mosaicism. Mosaicism is a particularly problematic abnormality as it may result in misdiagnoses from pre-implantation genetic screening (PGS) and is a potential reason why some embryos designated as euploid fail to implant. Parameters identifying embryos at increased or decreased risk for mosaicism would, therefore, be useful during embryo selection. [0008] Notwithstanding the recent developments in time lapse imaging that allow clinicians to select embryos with greater developmental potential based on timing parameters of the first few cell cycles, current embryo selection relies primarily on morphological evaluations which are very subjective and offer limited predictive value of embryo viability. Failure to correctly identify the most viable embryos can lead to unsuccessful IVF treatment or multiple gestation pregnancy. Time-lapse imaging technology allows real time non-invasive embryo monitoring and provides additional insight into human embryo developmental biology. This technology has allowed for the non-invasive identification and measurement of new timing parameters that identify embryos at increased or decreased risk for mosaicism as described herein.

SUMMARY OF THE INVENTION

[0009] The invention provides for the non-invasive quantitative assessment of human embryo morphology using imaging features to measure parameters to determine the relative risk that an embryo is mosaic or non-mosaic. In particular, methods for measuring time from insemination to first cytokinesis and the interval between the five cell stage and cavitation are provided. These methods are useful in methods of treating infertility in humans and other animals. [0010] One aspect of the invention provides non-invasive methods for detecting mosaicism in an in vitro fertilized human embryo by in vivo culturing one or more human embryos under conditions sufficient for embryo development and time lapse imaging the one or more human embryos for a time period from at least insemination to at least the first cytokinesis and measuring the time period of at least one timing parameter. In one embodiment, the time period from insemination to the onset of the first cytokinesis (P0) is measured. In another embodiment, the time period from the 5 cell stage to the onset of cavitation (Pcav₅) is measured. In still another embodiment both P0 and Pcav₅ are measured. In one embodiment, the method provides for identification of human embryos with an increased risk of mosaicism when P0 is more than about 24 hours and/or Pcav₅ is more than about 76 hours. In one embodiment the measuring of P0 and/or Pcav₅ is automated. In another embodiment, the identification of human embryos with an increased risk of mosaicism is automated. In still another embodiment both the measurement and identification are automated. In another embodiment, the non-invasive time lapse imaging method is used in conjunction with PGS to identify mosaicism in one or more human embryos. In one embodiment, the non-invasive time-lapse imaging method is used for PGS patients to select embryos for biopsy and genetic analysis. In still another embodiment, the non-invasive time-lapse imaging method is used in conjunction with PGS to identify euploid embryos with lowest risk of mosaicism for transfer.

[0011] Another aspect of the invention provides non-invasive methods for deselecting one or more human embryos that is at increased risk for mosaicism by in vivo culturing one or more human embryos under conditions sufficient for embryo development and time lapse imaging the one or more human embryos for a time period from at least insemination to at least the first cytokinesis and measuring the time period of at least one timing parameter. In one embodiment, the time period from insemination to the onset of the first cytokinesis (P0) is measured. In another embodiment, the time period from the 5 cell stage to the onset of cavitation (Pcav₅) is measured. In still another embodiment both P0 and Pcav₅ are measured. In one embodiment, an embryo at increased risk for mosaicism is deselected when P0 is more than about 24 hours and/or Pcav₅ is more than about 76 hours. In one embodiment the measuring of P0 and/or Pcav₅ is automated. In another embodiment, the deselection of human embryos with an increased risk of mosaicism is automated. In still another embodiment both the measurement and deselection are automated. In another embodiment, the non-invasive time lapse imaging method is used in conjunction with PGS to deselect human embryos with an increased risk of mosaicism.

[0012] Another aspect of the invention provides a non-invasive method for selecting one or more human embryos at low risk for mosaicism by in vivo culturing one or more human embryos under conditions sufficient for embryo development and time lapse imaging the one or more human embryos for a time period from at least insemination to at least the first cytokinesis and measuring the time period of at least one timing parameter. In one embodiment, the time period from insemination to the onset of the first cytokinesis (P0) is measured. In another embodiment, the time period from the 5 cell stage to the onset of cavitation (Pcav₅) is measured. In still another embodiment both P0 and Pcav₅ are measured. In one embodiment, an embryo at low risk for mosaicism is selected when P0 is less than about 23 hours and/or Pcav₅ is less than about 75hours. In one embodiment the measuring of P0 and/or Pcav₅ is automated. In another embodiment, the selection of human embryos with a decreased risk of mosaicism is automated. In still another embodiment both the measurement and selection are automated. In another embodiment, the non-invasive time lapse imaging method is used in conjunction with PGS to select one or more human embryos with a decreased risk of mosaicism.

[0013] In another aspect, the invention provides for a non-invasive method of determining the relative risk for mosaicism in a human embryo by in vivo culturing one or more human embryos under conditions sufficient for embryo development and time lapse imaging the one or more human embryos for a time period from at least insemination to at least the first cytokinesis and measuring the time period of at least one timing parameter. In one embodiment, the time period from insemination to the onset of the first cytokinesis (P0) is measured. In another embodiment, the time period from the 5 cell stage to the onset of cavitation (Pcav₅) is measured. In still another embodiment both P0 and Pcav₅ are measured. In one embodiment the relative risk for mosaicism is decreased in embryos with a shorter P0 and Pcav₅. In another embodiment, the relative risk for mosaicism is increased in embryos with a longer P0 and Pcav₅. In one embodiment the measuring of P0 and/or Pcav₅ is automated. In another embodiment the determination of relative risk of mosaicism is automated. In still another embodiment both the measurement and determination of relative risk are automated. In another embodiment, the non-invasive time lapse imaging method is used in conjunction with PGS to determine the relative risk of mosaicism in one or more human embryos.

[0014] In one embodiment, the one or more human embryos are produced by fertilization of oocytes in vitro. In one embodiment, the oocytes are matured in in vitro. In still another embodiment oocytes matured in vitro are supplemented with growth factors. In one embodiment, the one or more human embryos are not frozen prior to culturing. In still another embodiment, the one or more human embryos are frozen prior to culturing. [0015] In one embodiment the time lapse imaging acquires images that are digitally stored. In one embodiment, the time lapse imaging employs darkfield illumination. In another embodiment, the time lapse imaging employs brightfield illumination. In still another embodiment, the time lapse imaging employs a combination of darkfield and brightfield illumination.

[0016] In one embodiment, the one or more human embryos is placed in a culture disk prior to culturing under conditions suitable for embryo development. In a further embodiment the culture disk comprises a plurality of microwells. In still a further embodiment, the one or more human embryos is placed within a microwell prior to culturing under conditions sufficient for embryo development.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Before the present methods and compositions are described, it is to be understood that this invention is not limited to any particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0018] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0020] It must be noted that as used herein and in the appended claims, the singular forms “a”,“an”, and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of such cells and reference to“the peptide” includes reference to one or more peptides and equivalents thereof, e.g.

polypeptides, known to those skilled in the art, and so forth.

[0021] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0022] Methods are provided herein for quantitatively assessing the relative risk of a human embryo to exhibit mosaicism using time lapse imaging. The disclosed methods provide an objective, standardized measurement guideline for assessing the relative risk of mosaicism in human embryos.

[0023] The term“AUC” or“area under curve” when used herein with respect to prediction and/or evaluation methods is used to refer to the performance of the prediction method (i.e. the probability that the predication method will rank a randomly chosen euploid and/or non- mosaic embryo or euploid and/or non-mosaic blastocyst higher than a randomly chosen aneuploid and/or mosaic embryo or aneuploid and/or mosaic blastocyst). AUC is calculated from the receiver operating characteristic (ROC) and is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one.

[0024] The term odds ratio (OR) when used herein with respect to prediction and/or evaluation methods is used to refer to a measure of the effectiveness of the prediction method. OR = (TP/FN)/(FP/TN); where TP, FN, FP and TN are the number of true positives, false negatives, false positives and true negatives respectively.

[0025] The term“embryo” is used herein to refer both to the zygote that is formed when two haploid gametic cells, e.g. an unfertilized secondary oocyte and a sperm cell, unite to form a diploid totipotent cell, e.g. a fertilized ovum, and to the embryo that results from the immediately subsequent cell divisions, i.e. embryonic cleavage, up through the morula, i.e. 16-cell stage and the blastocyst stage (with differentiated trophectoderm and inner cell mass).

[0026] The term“blastocyst” is used herein to describe all embryos that reach cavitation (i.e., the formation of cavities).

[0027] The terms“born live” or“live birth” are used herein to include but are not limited to healthy and/or chromosomally normal (normal number of chromosomes, normal chromosome structure, normal chromosome orientation, etc.) births.

[0028] The term“arrested” is used herein to refer to any embryo that does not meet the definition of blastocyst. [0029] The term“oocyte” is used herein to refer to an unfertilized female germ cell, or gamete. Oocytes of the subject application may be primary oocytes, in which case they are positioned to go through or are going through meiosis I, or secondary oocytes, in which case they are positioned to go through or are going through meiosis II. [0030] By“meiosis” it is meant the cell cycle events that result in the production of gametes. In the first meiotic cell cycle, or meiosis I, a cell’s chromosomes are duplicated and partitioned into two daughter cells. These daughter cells then divide in a second meiotic cell cycle, or meiosis II, that is not accompanied by DNA synthesis, resulting in gametes with a haploid number of chromosomes. [0031] By a“mitotic cell cycle”, it is meant the events in a cell that result in the duplication of a cell’s chromosomes and the division of those chromosomes and a cell’s cytoplasmic matter into two daughter cells. The mitotic cell cycle is divided into two phases: interphase and mitosis. In interphase, the cell grows and replicates its DNA. In mitosis, the cell initiates and completes cell division, first partitioning its nuclear material, and then dividing its cytoplasmic material and its partitioned nuclear material (cytokinesis) into two separate cells. [0032] By a“first mitotic cell cycle” or“cell cycle 1” it is meant the time interval from fertilization/insemination to the completion of the first cytokinesis event or first mitosis, i.e. the division of the fertilized oocyte into two daughter cells. In instances in which oocytes are fertilized in vitro, the time interval between the injection of human chorionic gonadotropin (HCG) (usually administered prior to oocyte retrieval) to the completion of the first cytokinesis event may be used as a surrogate time interval.

[0033] “P0” or“P0 duration” is used herein to refer to the time interval from insemination to the onset of first cytokinesis (i.e. the appearance of the first cleavage furrow).

[0034] P1” or“P1 duration” is used herein to refer to the time interval between the appearance of the first cleavage furrow to completion of the 1^st cell division or first cytokinesis event.

[0035] “1^st cytokinesis phenotype” or“P1 phenotype” is used herein to refer to the cellular, biochemical and/or morphological characteristics of an embryo prior to completing P1 (i.e. the cellular, physical, biochemical and/or morphological characteristics of an embryo prior to completing the 1^st cell division or first cytokinesis event).

[0036] “Abnormal P1 phenotype” or“A1^cyt” is used herein to refer to uncharacteristic cellular, biochemical and/or morphological events of an embryo prior to completing P1 (i.e. prior to completing the 1^st cell division or first cytokinesis event) when compared to a reference or control embryo having a high likelihood of reaching blastocyst, becoming a good quality blastocyst and/or implanting into the uterus.“Abnormal P1 phenotype” or“A1^cyt” as used herein includes oolemma ruffling, membrane ruffling, and/or formation of one or more pseudo cleavage furrows before the initiation and/or completion of P1 (the time interval between the appearance of the first cleavage furrow to completion of the 1^st cell division or first cytokinesis event).

[0037] By a“second mitotic cell cycle” or“cell cycle 2” or“P2” it is meant the second cell cycle event observed in an embryo, the time interval between the production of daughter cells from a fertilized oocyte by mitosis and the production of a first set of granddaughter cells from one of those daughter cells (the“leading daughter cell”, or daughter cell A) by mitosis. P2 also encompasses the duration of time that the embryo is a 2 cell embryo, that is, the duration of the 2 cell stage. Cell cycle 2 may be measured using several morphological events including the end of cytokinesis 1 and the beginning of cytokinesis 2, or the end of cytokinesis 1 and the end of cytokinesis 2 or the beginning of cytokinesis 1 and the beginning of cytokinesis 2 or the beginning of cytokines 1 and the end of cytokinesis 2 or the end of mitosis 1 and the beginning of mitosis 2 or the end of mitosis 1 and the end of mitosis 2 or the beginning of mitosis 1 and the beginning of mitosis 1 or the beginning of mitosis 1 and the end of mitosis 2. Upon completion of cell cycle 2, the embryo consists of 3 cells. In other words, cell cycle 2 can be visually identified as the time between the embryo containing 2- cells and the embryo containing 3-cells.

[0038] By a“third mitotic cell cycle” or“cell cycle 3” or“P3” it is meant the third cell cycle event observed in an embryo, typically the time interval from the production of a first set of granddaughter cells from the first daughter cell and the production of a second set of granddaughter cells from the second daughter cell (the“lagging daughter cell” or daughter cell B) by mitosis. Cell cycle 3 may be measured using several morphological events including the end of cytokinesis 2 and the beginning of cytokinesis 3, or the end of cytokinesis 2 and the end of cytokinesis 3 or the beginning of cytokinesis 2 and the beginning of cytokinesis 3 or the beginning of cytokinesis 2 and the end of cytokinesis 3 or the end of mitosis 3 and the beginning of mitosis3 or the end of mitosis 2 and the end of mitosis3 or the beginning of mitosis 2 and the beginning of mitosis 3 or the beginning of mitosis 2 and the end of mitosis 3. In other words, cell cycle 3 can be visually identified as the time between the embryo containing 3-cells and the embryo containing 4-cells.

[0039] By a“fourth mitotic cell cycle” or“cell cycle 4” or“P4” it is meant the fourth cell cycle event observed in an embryo, typically the time interval from the production of a second set of granddaughter cells from the second daughter cell and the production of a first set of great-granddaughter cells from one of the granddaughter cells by mitosis. Cell cycle 4 may be measured using several morphological events including the end of cytokinesis 3 and the beginning of cytokinesis 4, or the end of cytokinesis 3 and the end of cytokinesis 4 or the beginning of cytokinesis 3 and the beginning of cytokinesis 4 or the beginning of cytokinesis 3 and the end of cytokinesis 4 or the end of mitosis 3 and the beginning of mitosis 4 or the end of mitosis 3 and the end of mitosis 4 or the beginning of mitosis 3 and the beginning of mitosis 4 or the beginning of mitosis 3 and the end of mitosis 4. In other words, cell cycle 4 can be visually identified as the time between the embryo containing 4-cells and the embryo containing 5-cells. [0040] By“first cleavage event” or“first cleavage”, it is meant the first division, i.e. the division of the oocyte into two daughter cells, i.e. cell cycle 1. Upon completion of the first cleavage event, the embryo consists of 2 cells.

[0041] By“second cleavage event” or“second cleavage”, it is meant the second set of divisions, i.e. the division of leading daughter cell into two granddaughter cells and the division of the lagging daughter cell into two granddaughter cells. In other words, the second cleavage event consists of both cell cycle 2 and cell cycle 3. Upon completion of second cleavage, the embryo consists of 4 cells.

[0042] By“third cleavage event”, it is meant the third set of divisions, i.e. the divisions of all of the granddaughter cells. Upon completion of the third cleavage event, the embryo typically consists of 8 cells.

[0043] By“cytokinesis” or“cell division” it is meant that phase of mitosis in which a cell undergoes cell division. In other words, it is the stage of mitosis in which a cell’s partitioned nuclear material and its cytoplasmic material are divided to produce two daughter cells. The period of cytokinesis is identifiable as the period, or window, of time between when a constriction of the cell membrane (a“cleavage furrow”) is first observed and the resolution of that constriction event, i.e. the generation of two daughter cells. The initiation of the cleavage furrow may be visually identified as the point in which the curvature of the cell membrane changes from convex (rounded outward) to concave (curved inward with a dent or indentation). This is illustrated for example in Fig.4 of US Patent No. 7,963,906 top panel by white arrows pointing at 2 cleavage furrows. The onset of cell elongation may also be used to mark the onset of cytokinesis, in which case the period of cytokinesis is defined as the period of time between the onset of cell elongation and the resolution of the cell division.

[0044] By“first cytokinesis” or“cytokinesis 1” it is meant the first cell division event after fertilization/insemination, i.e. the division of a fertilized oocyte to produce two daughter cells. First cytokinesis usually occurs about one day after fertilization/insemination.

[0045] By“second cytokinesis” or“cytokinesis 2”, it is meant the second cell division event observed in an embryo, i.e. the division of a daughter cell of the fertilized oocyte (the “leading daughter cell”, or daughter A) into a first set of two granddaughters. [0046] By“third cytokinesis” or“cytokinesis 3”, it is meant the third cell division event observed in an embryo, i.e. the division of the other daughter of the fertilized oocyte (the “lagging daughter cell”, or daughter B) into a second set of two granddaughters.

[0047] The term“fiduciary marker” or“fiducial marker,” is an object used in the field of view of an imaging system which appears in the image produced, for use as a point of reference or a measure. It may be either something placed into or on the imaging subject, or a mark or set of marks in the reticle of an optical instrument.

[0048] The term“micro-well” refers to a container that is sized on a cellular scale, preferably to provide for accommodating eukaryotic cells or a single oocyte or embryo. [0049] The term“selecting” or“selection” refers to any method known in the art for moving one or more embryos, blastocysts or other cell or cells as described herein from one location to another location. This can include but is not limited to moving one or more embryos, blastocysts or other cell or cells within a well, dish or other compartment or device so as to separate the selected one or more embryos, blastocysts or other cell or cells of the invention from the non- or deselected one or more embryos of the invention (such as for example moving from one area of a well, dish, compartment or device to another area of a well, dish, compartment or device). This can also include moving one or more embryos, blastocysts or other cell or cells from one well, dish, compartment or device to another well, dish, compartment or device. Any means known in the art for separating or distinguishing the selected one or more embryos, blastocysts or other cell or cells from the non- or deselected one or more embryos, blastocysts or other cell or cells can be employed with the methods of the present invention. In one embodiment, selected embryos are selected for transfer to a recipient for gestation. In another embodiment, selected embryos are selected for freezing for potential future implantation. In another embodiment, embryos are selected for continued culture. In another embodiment, embryos are selected for further evaluation by other methods such as preimplantation genetic testing, genomics, proteonomics, and/or secretomics.

[0050] The term“deselected” or“deselection” as used herein refers to embryos with poor developmental potential which are not chosen for implantation or are chosen for non- implantation. In some embodiments, deselected embryos are not transferred or implanted into the uterus. For example, an embryo at high risk for aneuploidy and/or mosaicism is deselected. [0051] After fertilization both gametes contribute one set of chromosomes (haploid content), each contained in a structured referred to herein as a“pronucleus” (“PN”) After normal fertilization, each embryo shows two pronuclei (PNs), one representing the paternal genetic material and one representing the maternal genetic material.“Syngamy” as used herein refers to the breakdown of the pronuclei (PNs) when the two sets of chromosomes unite, occurring within a couple hours before the first cytokinesis.

[0052] The term“compaction” refers to the joining of the outer cells of the embryo by intercellular connections, such as tight junctions, gap junctions and desmosomes, beginning at about the 8 cell stage. Compaction results in a compact sphere of tightly bound cells of the morula stage. During compaction, the boundaries of individual cells become less distinguishable.

[0053] The term“cavitation” refers to the process that begins when the outer layer of cells of the morula begin secreting fluid that creates a cavity, thus forming a blastocyst comprising an outer trophectoderm and an inner cell mass.

[0054] The term“blastocyst expansion” or“expansion” refers to the process wherein the volume, or size, of the blastocyst increases. In contrast, the term“blastocyst collapse” or “collapse” refers to a decrease in the volume or the size of a blastocyst. A blastocyst may repeat expansion and collapse one or more times.

[0055] The term“euploid” is used herein to refer to a cell that contains an integral multiple of the haploid, or monoploid, number. For example, a human autosomal cell having 46 chromosomes is euploid, and a human gamete having 23 chromosomes is euploid. By “euploid embryo” it is meant that the cells of the embryo are euploid.

[0056] The term“aneuploid” is used herein to refer to a cell that contains an abnormal number of chromosomes. For example, a cell having an additional chromosome, or a part of a chromosome, and a cell missing a chromosome, or a part of a chromosome, are both aneuploid. By“aneuploid embryo” it is meant that one or more cells of an embryo are aneuploid. By“aneuploid chromosome” is meant a chromosome that has more or less than two copies. For example, chromosome 21 is aneuploid in embryos with trisomy 21.

Aneuploid embryos are not chromosomally normal and have low developmental potential. [0057] By the term“monosomy” is meant an aneuploidy where a specific chromosome is present in only one copy. By“trisomy” is meant an aneuploidy where a specific chromosome is present in three copies.

[0058] The term“complex aneuploidy” is used herein to refer to aneuploidies in which 2 or more, for example 3 or more, or 4 or more or 5 or more or more than 6, chromosomes are affected. A complex aneuploidy can be a mixture of monosomies and trisomy. For example, a complex aneuploidy may comprise both trisomy 21 and monosomy 17, or any other combination of monosomies or trisomies.

[0059] The term“non-viable aneuploidy” is used herein to refer to aneuploidies which, when present, do not result in a viable embryo. Examples of“non-viable aneuploidies” include, without limitation, trisomy 2 and/or monosomy 1.

[0060] The term“disease causing aneuploidy” is meant those aneuploidies which may, but not necessarily, give rise to viable embryos, but for which a disease is associated. Examples of disease causing aneuploidies include, without limitation, trisomy 21 (Down syndrome), Trisomy 18 (Edward’s syndrome), Trisomy 13 (Patau syndrome), Trisomy 16, Trisomy 22, and sex chromosome aneuploidies including, but not limited to monosomy X or 45X (Turner syndrome) and XXY or 47XXY (Klinefelter syndrome).

[0061] The term“mosaicism” or“mosaic” is used herein to refer to an individual organism that has two or more chromosomally distinct cell lineages. Examples of mosaicism include, without limitation, trisomies that only occur in a selection of cells (e.g., 46/47 XY/XXY Klinefelter syndrome mosaic). By“mosaic embryo” or“embryo exhibiting mosaicism” it is meant that one or more cells of an embryo have a different chromosomal composition compared to another cell in the embryo. By“non-mosaic embryo” it is meant that all the cells of an embryo have the same chromosomal composition.

[0062] By“high likelihood of mosaicism” or“increased likelihood of mosaicism” or “increased risk of mosaicism” or“relative increased risk of mosaicism” or“increased relative risk of mosaicism” it is meant that the likelihood or relative risk that an embryo is mosaic is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90% or more. By“low likelihood of mosaicism” or“decreased likelihood of mosaicism” or “decreased risk of mosaicism” or“relative decreased risk of mosaicism” or“decreased relative risk of mosaicism” it is meant that the likelihood or relative risk that an embryo is mosaic is less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or lower.

[0063] The focus of prior patents and applications including US Patent No.: 7,963,906; 8,323,177; 8,337,387 and PCT Appl. No. WO 2012/163363 each center primarily around selection criteria for human embryos in in vitro fertilization. While these patents/applications each discuss determining whether embryos have good or poor developmental potential (i.e. are likely or not to develop as desired), the timing parameters described therein are typically used in the clinic in large part to select embryos with good developmental potential. In contrast, the methods of the current invention center around two timing parameters, P0 and the novel time parameter Pcav₅ (i.e. interval from the 5 cell stage to onset of cavitation), that may be used to deselect human embryos and deprioritize their use for transfer in in vitro fertilization treatment. Alternatively, these two timing parameters may be used select human embryos for transfer into the uterus. These parameters may be used alone or in combination with the selection parameters described in US Patent No.: 7,963,906; 8,323,177; 8,337,387 and PCT Appl. No. WO 2012/163363. For example, once an embryo is determined to have good developmental potential by the methods of US Patent No.: 7,963,906; 8,323,177;

8,337,387 and PCT Appl. No. WO 2012/163363, that embryo may be further analyzed for one or both of the P0 and Pcav₅ parameters described herein to further increase the sensitivity and specificity of the claimed methods. Similarly, the parameters Pcav₅ and P0 can be used in conjunction with PGS to better assess the relative risk that an embryo will exhibit mosaicism. [0064] The deselection criteria of the current invention include: prolonged duration from insemination to first cytokinesis and/or prolonged duration from the 5 cell stage to cavitation as determined according to the disclosed methods. These P0 and Pcav₅ parameters may be used alone or in combination with PGS and/or with each other or other parameters including the previously described criteria parameters including, but not limited to those described in Table 1.

Table 1: List of Parameters

[0065] Mosaicism is a biological phenomenon that can impact embryo selection and subsequent pregnancy outcomes in multiple ways. First, mosaicism may affect the developmental potential of the embryo. Second, mosaicism affects the diagnostic accuracy of pre-implantation genetic screening (PGS), which is used to diagnose embryo chromosomal health and select a chromosomally normal embryo for transfer. There is currently no non- invasive way to assess risk of mosaicism. Current methods to assess mosaicism require multiple biopsies or destruction of the embryo in a research setting. As a result, mosaicism is not assessed in regular IVF clinical practice. Mosaicism is a particularly problematic abnormality as it may result in misdiagnoses from PGS and is a potential reason why some embryos designated as euploid fail to deliver. In contrast, the methods of the current invention provide a more quantitative and non-invasive measurement of embryo

developmental kinetics to identify embryos with at increased risk of being mosaic.

[0066] The methods of the current invention also provide for novel selection or deselection parameters for human embryos that can be measured by time lapse microscopy.

[0067] In methods of the invention, one or more embryos is assessed for its relative risk of being non-mosaic, and/or reach the blastocyst stage and/or become a good quality blastocyst and/or implant into the uterus and/or be born live by measuring one or more timing parameters, including P0 and/or Pcav₅ (time to cavitation), and employing these

measurements to determine the relative risk that the embryo(s) will be non-mosaic and/or reach the blastocyst stage and/or implant into the uterus. Such parameters have been described, for example, in US Patent Nos. 7,963,906; 8,323,177, and 8,337,387 and PCT Appl. No.: WO 2012/163363, the disclosure of each of which is incorporated herein by reference in their entirety. The information thus derived may be used to guide clinical decisions, e.g. whether or not to transfer an in vitro fertilized embryo, whether or not to transplant a cultured cell or cells, whether or not to freeze an embryo for later implantation, whether or not to continue to culture the embryo, or whether or not to evaluate the embryo by other methods such as preimplantation genetic testing, genomics, proteonomics, and/or secretomics.

[0068] Examples of embryos that may be assessed by the methods of the invention include 1-cell embryos (also referred to as zygotes), 2-cell embryos, 3-cell embryos, 4-cell embryos, 5-cell embryos, 6-cell embryos, 8-cell embryos, etc. typically up to and including 16-cell embryos, morulas, and blastocysts, any of which may be derived by any convenient manner, e.g. from an oocyte that has matured in vivo or from an oocyte that has matured in vitro.

[0069] Embryos may be derived from any organism, e.g. any mammalian species, e.g. human, primate, equine, bovine, porcine, canine, feline, etc. Preferably, they are derived from a human. They may be previously frozen, e.g. embryos cryopreserved at the 1-cell stage and then thawed. Alternatively, they may be freshly prepared, e.g., embryos that are freshly prepared (not frozen prior to culturing) from oocytes by in vitro fertilization techniques (fresh or previously frozen oocytes); oocytes that are freshly harvested and/or freshly matured through in vitro maturation techniques (including, e.g., oocytes that are harvested from in vitro ovarian tissue). They may be cultured under any convenient conditions (including different types of culture media) known in the art to promote survival, growth, and/or development of the sample to be assessed, e.g. for embryos, under conditions such as those used in the art of in vitro fertilization; see, e.g., US Patent No. 6,610,543, US Patent No. 6,130,086, US Patent No. 5,837,543, the disclosures of which are incorporated herein by reference; for oocytes, under conditions such as those used in the art to promote oocyte maturation; see, e.g., US Patent No. 5,882,928 and US Patent No. 6,281,013, the disclosures of which are incorporated herein by reference; for stem cells under conditions such as those used in the art to promote maintenance, differentiation, and proliferation, see, e.g. US Patent No. 6,777,233, US Patent No. 7,037,892, US Patent No. 7,029,913, US Patent No. 5,843,780, and US Patent No. 6,200,806, US Application No. 2009/0047263; US Application No.

2009/0068742, the disclosures of which are incorporated herein by reference. Often, the embryos are cultured in a commercially available medium such as KnockOut DMEM, DMEM-F12, or Iscoves Modified Dulbecco’s Medium that has been supplemented with serum or serum substitute, amino acids, growth factors and hormones tailored to the needs of the particular embryo being assessed.

[0070] In some embodiments, the embryos are assessed by measuring timing parameters by time-lapse imaging. The embryos may be cultured in standard culture dishes. Alternatively, the embryos may be cultured in custom culture dishes, e.g. custom culture dishes with optical quality micro-wells as described herein. In such custom culture dishes, each micro-well holds a single fertilized egg or embryo, and the bottom surface of each micro-well has an optical quality finish such that the entire group of embryos within a single dish can be imaged simultaneously by a single miniature microscope with sufficient resolution to follow the cell mitosis processes. The entire group of micro-wells shares the same media drop in the culture dish, and can also include an outer wall positioned around the micro-wells for stabilizing the media drop, as well as fiducial markers placed near the micro-wells. The media drops can have different volumes. The hydrophobicity of the surface can be adjusted with plasma etching or another treatment to prevent bubbles from forming in the micro-wells when filled with media. Regardless of whether a standard culture dish or a custom culture dish is utilized, during culture, one or more developing embryos may be cultured in the same culture medium, e.g. between 1 and 30 embryos may be cultured per dish.

[0071] Images are acquired over time, and are then analyzed to arrive at measurements of the one or more timing parameters. Time-lapse imaging may be performed with any computer-controlled microscope that is equipped for digital image storage and analysis, for example, inverted microscopes equipped with heated stages and incubation chambers, or custom built miniature microscope arrays that fit inside a conventional incubator. The array of miniature microscopes enables the concurrent culture of multiple dishes of samples in the same incubator, and is scalable to accommodate multiple channels with no limitations on the minimum time interval between successive image capture. Using multiple microscopes eliminates the need to move the sample, which improves the system accuracy and overall system reliability. The individual microscopes in the incubator can be partially or fully isolated, providing each culture dish with its own controlled environment. This allows dishes to be transferred to and from the imaging stations without disturbing the environment of the other samples.

[0072] The imaging system for time-lapse imaging may employ brightfield illumination, darkfield illumination, phase contrast, Hoffman modulation contrast, differential interference contrast, polarized light, fluorescence, single or multiplane or combinations thereof. In some embodiments, darkfield illumination may be used to provide enhanced image contrast for subsequent feature extraction and image analysis. In addition, red or near-infrared light sources may be used to reduce phototoxicity and improve the contrast ratio between cell membranes and the inner portion of the cells. [0073] Images that are acquired may be stored either on a continuous basis, as in live video, or on an intermittent basis, as in time lapse photography, where a subject is repeatedly imaged in a still picture. Images may also be acquired in response to a detected event or a scheduled event in order to, for example, obtain a more granular image based features from increased or decreased sampling of an image sequence. Preferably, the time interval between images should be between 1 to 30 minutes, or between 1 to 20 minutes or between 1 to 15 minutes, or between 1 to 10 minutes or between 1 to 5 minutes in order to capture significant morphological events as described below. In an alternative embodiment, the time interval between images could be varied depending on the amount of cell activity. For example, during active periods images could be taken as often as every few seconds or every minute, while during inactive periods images could be taken every 10 or 15 minutes or longer. Real- time image analysis on the captured images could be used to detect when and how to vary the time intervals. In our methods, the total amount of light received by the samples is estimated to be equivalent to approximately 52 seconds of continuous low-level light exposure for 5- days of imaging. The light intensity for a time-lapse imaging system is significantly lower than the light intensity typically used on an assisted reproduction microscope due to the low- power of the LEDs (for example, using a 1W LED compared to a typical 100W Halogen bulb) and high sensitivity of the camera sensor. Thus, the total amount of light energy received by an embryo using the time-lapse imaging system is comparable to or less than the amount of energy received during routine handling at an IVF clinic. In addition, exposure time can be significantly shortened to reduce the total amount of light exposure to the embryo. For 2-days of imaging, with images captured every 5 minutes at 0.5 seconds of light exposure per image, the total amount of low-level light exposure is less than 21 seconds.

[0074] Following image acquisition, the embryos are localized and analyzed for different cellular parameters or image based parameters, for example, zygote size, blastomere size, thickness of the zona pellucida, smoothness or ruffling of the plasma membrane, smoothness or ruffling of the oolemma, formation of one or more pseudo cleavage furrows, degree of fragmentation, symmetry of daughter cells resulting from a cell division, time intervals between the first few mitoses, duration of cytokinesis, timing and quality of syngamy, area of outer boundary segmentation, boundary segment distribution at the center of the embryo, changes in standard deviation of segment distribution at the center of the embryo, embryo shape, and texture at the edge or center of the embryo. Image analysis methods that may be used to analyze cellular and image based parameters include, for example, shape based methods (e.g., thresholding, blob extraction, template matching and Hough transforms (lines, ellipses, arbitrary shape, etc.)), low level methods (e.g., detecting edges, texture, ridges, corners, blobs; local image feature detectors such as scale-invariant feature transform (SIFT) and speeded up robust features (SURF), local binary patterns (LBP), SIFT-like GLOH features, PCA-SIFT, and SIFT-Rank detector), and curvature methods (e.g., edge direction, changing intensity, and correlation). Analysis can involve measuring features, e.g., texture, for the entire embryo, or at specific regions of the embryo such as the embryo edge and/or at the embryo center. These cellular or image based parameters may be used in conjunction with classifiers, cluster methods and like to produce the intended prediction of clinical variables. Methods for image-based classification and automated cell tracking are described in co-pending application numbers 14/194,386 and 14/194,391, both of which are incorporated by reference in their entireties.

[0075] Cellular parameters that may be measured by time-lapse imaging are usually morphological events. For example, in assessing embryos, time-lapse imaging may be used to visualize syngamy. Additionally, time-lapse imaging may be used to measure the duration of time between insemination and/or syngamy and the onset or resolution of first cytokinesis. Additionally, time-lapse imaging may be used to measure the duration of time between the 5 cell stage and the onset of cavitation. Similarly, time lapse imaging may be used to measure the time between insemination and/or syngamy and the onset or resolution of the second cytokinesis or the third cytokinesis or the fourth cytokinesis. Time lapse imaging may also be used to determine any combination of the time between the onset or resolution of the first cytokinesis and the onset or resolution of the second cytokinesis, or third cytokinesis or fourth cytokinesis. Time lapse imaging can also be used to determine any combination of the time between the onset or resolution of the second cytokinesis and the onset or resolution of the third cytokinesis or fourth cytokinesis. Time lapse imaging may also be used to determine any combination of the time between the onset or resolution of the third cytokinesis and the onset or resolution of the fourth cytokinesis.

[0076] While it is often considered advantageous that the embryo be transferred to the uterus early in development to reduce embryo loss due to disadvantages of culture conditions relative to the in vitro environment, and to reduce potential adverse outcomes associated with epigenetic errors that may occur during culturing, (Katari et al. (2009) Hum Mol Genet. 18(20):3769-78; Sepúlveda et al. (2009) Fertil Steril. 91(5):1765-70) cavitation generally occurs at about day 5. Therefore, embryos assessed by the current methods are transferred at day 5 or later.

[0077] Parameters can be measured manually, or they may be measured automatically, e.g. by image analysis software. When image analysis software is employed, image analysis algorithms may be used that employ a probabilistic model estimation technique. The probabilistic model estimation technique may be based on a sequential Monte Carlo method, e.g. generating distributions of hypothesized embryo models, simulating images based on a simple optical model, and comparing these simulations to the observed image data. When such probabilistic model estimations are employed, cells may be modeled as any appropriate shape, e.g. as collections of ellipses in 2D space, collections of ellipsoids in 3D space, and the like. To deal with occlusions and depth ambiguities, the method can enforce geometrical constraints that correspond to expected physical behavior. To improve robustness, images can be captured at one or more focal planes. A more detailed description of such a technique is provided in US Patent No. 7,963,906. Alternatively or in addition, the image analysis algorithm may leverage observable cell features such as boundary segments in a conditional random field (CRF) model over which multi-pass data driven approximate inference may be performed. A more detailed description of such a technique is provided in Moussavi, et al. (2014)“A Unified Graphical Models Framework for Automated Human Embryo Tracking in Time Lapse Microscopy,” International Symposium on Biomedical Imaging, In Press.

Methods other than probabilistic models may also be employed that do not track cells individually but extract features directly from the images and use those features for classification or analysis. For example, image-based cell classification and/or outcome-based classification applied to series of images may be employed. The classification may be based on features including handcrafted and/or machine learned features. A more detailed description of such a classification approach is provided in Automated embryo stage classification in time-lapse microscopy video of early human embryo development, Yu Wang, Farshid Moussavi, Peter Lorenzen, International Conference on Medical Image

Computing and Computer Assisted Intervention (MICCAI), vol. 8150 of Lecture Notes in Computer Science, pages 460-467, Springer, 2013. Classification-based approaches may also be used in conjunction with tracking-based approaches, as described in Moussavi, et al. (2014)“A Unified Graphical Models Framework for Automated Human Embryo Tracking in Time Lapse Microscopy,” International Symposium on Biomedical Imaging, In Press. [0078] In some embodiments, the timing parameter measurement is used directly to determine the relative risk that an embryo will be mosaic, and/or the likelihood that an embryo will reach the blastocyst stage or will become a good quality embryo. In some embodiments, the timing parameter measurement is used directly to determine the relative risk that an embryo will be mosaic and/or the likelihood that an embryo will successfully implant into the uterus and/or will be born live. In other words, the absolute value of the measurement itself is sufficient to determine the relative risk that an embryo will be mosaic and/or the likelihood that an embryo will reach the blastocyst stage and/or implant into the uterus and/or be born live. Examples of this in embodiments using time-lapse imaging to measure timing parameters include, without limitation, the following, which in combination are indicative of the relative risk that an embryo will be mosaic and/or the likelihood that an embryo will successfully implant into the uterus and/or be born live: (a) a duration of P0 of about 0 to about 23 hours wherein a shorter duration of P0 is indicative of a decreased risk for mosaicism; and (b) a time interval from the 5 cell stage to the onset of cavitation of about 0 to about 75 hours wherein a shorter duration between the 5 cell stage and the onset of cavitation is indicative of a decreased risk for mosaicism. In some embodiments, determining the relative risk that an embryo will be non-mosaic and/or the likelihood that an embryo will timing parameters, including but not limited to: a duration of cytokinesis 1 that is about 0 to about 33 hours, time from fertilization/insemination to the 5 cell stage that is about 47 hours to about 57 hours, and a duration of Psyn that is more than about 1 hour.

[0079] Examples of direct measurements, any of which alone or in combination are indicative of an increased risk that an embryo will be mosaic and/or a higher likelihood that an embryo will not successfully implant into the uterus and/or will not be born live, include, without limitation: (a) a duration of P0 that is more than about 24 hours, wherein a longer duration of P0 is indicative of an increased risk for mosaicism and (b) a time interval from the 5 cell stage to the onset of cavitation that lasts more than about 76 hours, wherein a longer interval from the 5 cell stage to the onset of cavitation is indicative of an increased risk for mosaicism. In some embodiments, determining the risk that an embryo will be mosaic and/or will not successfully implant into the uterus and/or will not be born live can include additionally measuring timing parameters, including, but not limited to: a duration of cytokinesis 1 that is more than about 33 minutes, a time interval between

fertilization/insemination and the 5 cell stage that is less than about 47 hours or more than about 57 hours, or a duration of Psyn that is less than about 1 hour. [0080] For example, embryos that have a P0 that is longer than about 24 hours are at increased risk of being mosaic. Similarly, embryos that have a time interval between the 5 cell stage and the onset of cavitation that is longer than about 76 hours are at an increased risk of being mosaic. Conversely, embryos that have a P0 that is less than about 23 hours and/or a time interval between the 5 cell stage and onset of cavitation that is less than about 75 hours are have a decreased risk of mosaicism and are more likely to be non-mosaic.

[0081] In some embodiments, the timing parameter measurement is employed by comparing it to a respective timing parameter measurement from a reference, or control, embryo, and using the result of this comparison to provide a determination of the relative risk of an embryo being aneuploid or mosaic and/or the likelihood that the embryo will reach or not reach the blastocyst stage, and/or become a good quality blastocyst and/or implant into the uterus and/or be born live. The terms“reference” and“control” as used herein mean a standardized embryo or cell to be used to interpret the timing parameter measurements of a given embryo and assign a determination of the relative risk of the embryo to be aneuploid and/or mosaic, and/or the likelihood the embryo will reach the blastocyst stage, and/or become a good quality blastocyst and/or implant into the uterus and/or be born live. The reference or control may be an embryo that is known to have a desired phenotype, e.g., euploid and/or non-mosaic, likely to reach the blastocyst stage, and/or become a good quality blastocyst and/or implant into the uterus and/or be born live, and therefore may be a positive reference or control embryo. Alternatively, the reference/control embryo may be an embryo known to not have the desired phenotype, e.g., an aneuploid and/or a mosaic embryo, and therefore be a negative reference/control embryo.

[0082] In certain embodiments, timing parameters are first employed to determine whether an embryo will be euploid and/or likely to reach the blastocyst stage, and/or become a good quality blastocyst and/or implant into a uterus and/or be born live. In such embodiments, embryos that fall within one or more of the above referenced timing parameter time frames is selected to have good developmental potential and/or be euploid. These embryos are then analyzed to determine their relative risk of mosaicism by measuring P0 and Pcav₅. Embryos previously selected to have good developmental potential are deselected when they are determined to be at increased risk for mosaicism by having a prolonged P0 and/or duration of the time interval between the 5 cell stage and the onset of cavitation, thereby selecting for implantation or freezing for potential future implantation, only those embryos that fall within the selection criteria and outside the deselection criteria. [0083] In certain embodiments embryos are analyzed to determine their relative risk of mosaicism by measuring P0 and Pcav₅. Embryos determined to be at high risk for mosaicism (e.g. those embryos with a P0 of greater than about 24 hours and/or a Pcav₅ of greater than about 76 hours) are deselected for further PGS. This narrowing of the candidate pool for biopsy and PGS analysis has the added benefit of not only selecting the most viable embryos for implantation but also reducing the cost of further genetic screening.

[0084] In certain embodiments, the obtained timing parameter measurement(s) is compared to a comparable timing parameter measurement(s) from a single reference/control embryo to obtain information regarding the phenotype of the embryo/cell being assayed. In yet other embodiments, the obtained timing parameter measurement(s) is compared to the comparable timing parameter measurement(s) from two or more different reference/control embryos to obtain more in depth information regarding the phenotype of the assayed embryo/cell. For example, the obtained timing parameter measurements from the embryo(s) being assessed may be compared to both a positive and negative embryo to obtain confirmed information regarding whether the embryo/cell has the phenotype of interest. In another embodiment, the timing parameter is used in conjunction with PGS to confirm the presence of mosaicism. For example, an embryo determined to be at high risk for mosaicism by the non-invasive methods of the current invention (i.e. those embryos having a P0 of greater than about 24 hours and/or a Pcav₅ of greater than about 76 hours) are deselected for PGS screening. Alternatively, the non-invasive method of the current invention may be employed with embryos determined by PGS to be euploid to select embryos with the lowest risk of mosaicism (embryos with a P0 of less than about 23 hours and/or a Pcav₅ of less than about 75 hours and a determination of euploiody by PGS).

[0085] As discussed above, one or more parameters may be measured and employed to determine the relative risk of being aneuploid and/or mosaic, and/or the likelihood of reaching the blastocyst stage and/or becoming a good quality blastocyst and/or implant into the uterus and/or be born live for an embryo. In some embodiments, a measurement of two parameters may be sufficient to arrive at a determination of the relative risk of being aneuploid and/or mosaic, and/or the likelihood of reaching the blastocyst stage and/or becoming a good quality blastocyst and/or implant into the uterus and/or be born live. In some embodiments, it may be desirable to employ measurements of more than two parameters, for example, 3 parameters or 4 or more parameters. In some embodiments, it may be desirable to measure one or more parameters for selecting an embryo with good developmental potential and one or more parameters for deselecting embryos with poor developmental potential. In certain embodiments, 1 selection parameter and 1 deselection parameter is measured. In another embodiment, 1 selection parameter and 2 deselection parameters are measured. In another embodiment, 1 selection parameter and 3 deselection parameters are measured. In another embodiment, 2 selection parameters and 1 deselection parameter are measured. In another embodiment, 3 selection parameter and 1 deselection parameter are measured. In another embodiment, more than 3 selection parameters and 1 deselection parameter are measured. In another embodiment, 2 selection parameters and 2 deselection parameters are measured. In another embodiment, 2 selection parameters and 3 deselection parameters are measured. In another embodiment, 3 selection parameters and 2 deselection parameters are measured. In another embodiment, more than 3 selection parameters and 2 deselection parameters are measured. In another embodiment, more than 3 selection parameters and 3 deselection parameters are measured. In another embodiment timing parameters are used in conjunction with non-timing parameters and/or PGS to determine the relative risk of mosaicism in a human embryo.

[0086] In certain embodiments, assaying for multiple parameters may be desirable as assaying for multiple parameters may provide for greater sensitivity and specificity. By sensitivity it is meant the proportion of actual positives which are correctly identified as being such. This may be depicted mathematically as:

[0087] Thus, in a method in which“positives” are the embryos that have good

developmental potential, i.e. that will become a good quality blastocyst and/or implant into the uterus and/or euploid and/or non-mosaic, and“negatives” are the embryos that have poor developmental potential, i.e. that will not develop into good quality blastocysts and/or implant into the uterus and/or will be euploid and/or mosaic, a sensitivity of 100% means that the test recognizes all embryos that will become good quality blastocysts or implant in to the uterus or be chromosomally normal as such. In some embodiments, the sensitivity of the assay may be about 70%, 80%, 90%, 95%, 98% or more, e.g. 100%. By specificity it is meant the proportion of“negatives” which are correctly identified as such. As discussed above, the term“specificity” when used herein with respect to prediction and/or evaluation methods is used to refer to the ability to predict or evaluate an embryo for determining the likelihood that the embryo will not become a good quality blastocyst or implant into the uterus or will be euploid and/or mosaic by assessing, determining, identifying or selecting embryos that are not likely to become a good quality blastocyst and/or implant into the uterus and/or will be euploid and/or mosaic. This may be depicted mathematically as:

[0088] Thus, in a method in which positives are the embryos that are likely to become good quality blastocysts and/or implant into the uterus and/or be euploid and/or non-mosaic, and negatives are the embryos that are likely not to develop into good quality blastocysts and/or implant into the uterus and/or will be euploid and/or mosaic, a specificity of 100% means that the test recognizes all embryos that will not develop into good quality blastocysts and/or implant into the uterus and/or will be euploid and/or mosaic. In some embodiments, the specificity can be a“high specificity.” In addition, the specified mean values and/or cut-off points may be modified depending upon the data set used to calculate these values as well as the specific application. [0089] As discussed above, methods of the invention may be used to assess embryos or cells to determine the relative risk of the embryos to be aneuploid and/or mosaic, and/or the likelihood of the embryos to reach the blastocyst stage, and/or develop into good quality blastocysts and/or implant into the uterus and or be born live. This determination of the relative risk of the embryos to be aneuploid and/or mosaic, and/or the likelihood of the embryos to reach the blastocyst stage, and/or develop into good quality blastocysts and/or implant into the uterus and or be born live may be used to guide clinical decisions and/or actions. For example, in order to increase pregnancy rates, clinicians often transfer multiple embryos into patients, potentially resulting in multiple pregnancies that pose health risks to both the mother and fetuses. Using results obtained from the methods of the invention, the relative risk of the embryos being aneuploid and /or mosaic and/or the likelihood of reaching the blastocyst stage, and/or developing into good quality blastocysts and/or implanting into the uterus and/or be born live can be determined for embryos being transferred. As the embryos that are at decreased risk of being aneuploid and/or mosaic, and more likely to reach the blastocyst stage, and/or develop into good quality blastocysts and/or implant into the uterus are more likely to develop into fetuses, the determination of the risk of the embryo to be aneuploid and/or mosaic, and/or the likelihood of the embryos to reach the blastocyst stage, and/or develop into good quality blastocysts and/or implant into the uterus prior to transplantation allows the practitioner to decide how many embryos to transfer so as to maximize the chance of success of a full term pregnancy while minimizing risk.

[0090] Assessments made by following methods of the invention may also find use in ranking embryos in a group of embryos for the relative risk that the embryos will be aneuploid and/or mosaic and/or the likelihood that the embryo will reach the blastocyst stage as well as for the quality of the blastocyst that will be achieved (e.g., in some embodiments this would include the likelihood of implanting into the uterus and/or being chromosomally normal). For example, in some instances, multiple embryos may be determined to be at low risk for mosaicism and have a high likelihood of to reach the blastocyst stage and/or implant into the uterus. In this case, embryos selected with the highest developmental potential (likelihood to reach blastocyst and/or implant into the uterus) by, for example, the measurement of P2 and/or P3 are then analyzed for the relative risk of mosaicism. In the event that multiple embryos display a P0 of less than 23 hours and a Pcav₅ of less than 75 hours, embryos are selected to have the lowest risk of mosaicism with a shorter P0 and/or Pcav₅. For example, if one embryo displays a P0 of 22 hours and another displays a P0 if 19 hours, the embryo with the P0 of 19 hours is determined to be at lower risk than the embryo with a P0 of 22 hours even though both embryos are within the“low risk” window of 0-23 hours. Similarly, and embryo with a Pcav₅ of 70 hours is selected to be at lower risk for mosaicism than an embryo with a Pcav₅ of 73 hours. [0091] In this way, a practitioner assessing, for example, multiple zygotes/embryos, can choose only the best quality embryos, i.e. those with the best likelihood of implanting into the uterus and/or being euploid and/or non-mosaic to transfer so as to maximize the chance of success of a full term pregnancy while minimizing risk.

[0092] Also provided are reagents, devices and kits thereof for practicing one or more of the above-described methods. The subject reagents, devices and kits thereof may vary greatly. Reagents and devices of interest include those mentioned above with respect to the methods of measuring any of the aforementioned parameters, where such reagents may include culture plates, culture media, microscopes, imaging software, imaging analysis software, nucleic acid primers, arrays of nucleic acid probes, antibodies, signal producing system reagents, etc., depending on the particular measuring protocol to be performed. [0093] In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits. [0094] Some of the methods described above require the ability to observe embryo development via time-lapse imaging. This can be achieved using a system comprised of a miniature, multi-channel microscope array that can fit inside a standard incubator. This allows multiple samples to be imaged quickly and simultaneously without having to physically move the dishes. One illustrative prototype, shown in Fig. 22 of US Patent No. 7,963,906, consists of a 3-channel microscope array with darkfield illumination, although other types of illumination could be used. By“three channel,” it is meant that there are three independent microscopes imaging three distinct culture dishes simultaneously. A stepper motor is used to adjust the focal position for focusing or acquiring 3D image stacks. White- light LEDs are used for illumination, although we have observed that for human embryos, using red or near-infrared (IR) LEDs can improve the contrast ratio between cell membranes and the inner portions of the cells. This improved contrast ratio can help with both manual and automated image analysis. In addition, moving to the infrared region can reduce phototoxicity to the samples. Images are captured by low-cost, high-resolution webcams, but other types of cameras may be used.

[0095] As shown in Fig. 22 of US Patent No. 7,963,906, each microscope of the prototype system described above is used to image a culture dish which may contain anywhere from 1- 30 embryos. The microscope collects light from a white light LED connected to a heat sink to help dissipate any heat generated by the LED, which is very small for brief exposure times. The light passes through a conventional dark field patch for stopping direct light, through a condenser lens and onto a specimen labeled“petri dish,” which is a culture dish holding the embryos being cultured and studied. The culture dish may have wells that help maintain the order of the embryos and keep them from moving while the dish is being carried to and from the incubator. The wells can be spaced close enough together so that embryos can share the same media drop. The scattered light is then passed through a microscope objective, then through an achromat doublet, and onto a CMOS sensor. The CMOS sensor acts as a digital camera and is connected to a computer for image analysis and tracking as described above.

[0096] This design is easily scalable to provide significantly more channels and different illumination techniques, and can be modified to accommodate fluidic devices for feeding the samples. In addition, the design can be integrated with a feedback control system, where culture conditions such as temperature, CO2 (to control pH), and media are optimized in real- time based on feedback and from the imaging data. This system was used to acquire time- lapse videos of human embryo development, which has utility in determining embryo viability for in vitro fertilization (IVF) procedures. Other applications include stem cell therapy, drug screening, and tissue engineering. [0097] In one embodiment of the device, illumination is provided by a Luxeon white light- emitting diode (LED) mounted on an aluminum heat sink and powered by a BuckPuck current regulated driver. Light from the LED is passed through a collimating lens. The collimated light then passes through a custom laser-machined patch stop, as shown in Fig. 22 of US Patent No. 7,963,906, and focused into a hollow cone of light using an aspheric condenser lens. Light that is directly transmitted through the sample is rejected by the objective, while light that is scattered by the sample is collected. In one embodiment, Olympus objectives with 20X magnification are used, although smaller magnifications can be used to increase the field-of-view, or larger magnifications can be used to increase resolution. The collected light is then passed through an achromat doublet lens (i.e. tube lens) to reduce the effects of chromatic and spherical aberration. Alternatively, the collected light from the imaging objective can be passed through another objective, pointed in the opposing direction, that acts as a replacement to the tube lens. In one configuration, the imaging objective can be a 10X objective, while the tube-lens objective can be a 4X objective. The resulting image is captured by a CMOS sensor with 2 megapixel resolution (1600 x 1200 pixels). Different types of sensors and resolutions can also be used.

[0098] For example, Fig. 23A of US Patent No. 7,963,906 shows a schematic of the multi- channel microscope array having 3 identical microscopes. All optical components are mounted in lens tubes. In operation of the array system, Petri dishes are loaded on acrylic platforms that are mounted on manual 2-axis tilt stages, which allow adjustment of the image plane relative to the optical axis. These stages are fixed to the base of the microscope and do not move after the initial alignment. The illumination modules, consisting of the LEDs, collimator lenses, patch stops, and condenser lenses, are mounted on manual xyz stages for positioning and focusing the illumination light. The imaging modules, consisting of the objectives, achromat lenses, and CMOS sensors, are also mounted on manual xyz stages for positioning the field-of-view and focusing the objectives. All 3 of the imaging modules are attached to linear slides and supported by a single lever arm, which is actuated using a stepper motor. This allows for computer-controlled focusing and automatic capture of image- stacks. Other methods of automatic focusing as well as actuation can be used. [0099] The microscope array was placed inside a standard incubator, as shown in, for example, Fig. 23B of US Patent No. 7,963,906. The CMOS image sensors are connected via USB connection to a single hub located inside the incubator, which is routed to an external PC along with other communication and power lines. All electrical cables exit the incubator through the center of a rubber stopper sealed with silicone glue.

[0100] The above described microscope array, or one similar, can be used to record time- lapse images of early human embryo development and documented growth from zygote through blastocyst stages. In some embodiments, images can be captured every 5 minutes with roughly 1 second of low-light exposure per image. The total amount of light received by the samples can be equivalent to 52 seconds of continuous exposure, similar to the total level experienced in an IVF clinic during handling. The 1 second duration of light exposure per image can in some embodiments be reduced. Prior to working with the human embryos, extensive control experiments were performed with mouse pre-implantation embryos to ensure that both the blastocyst formation rate and gene expression patterns were not affected by the imaging process.

[0001] Individual embryos can be followed over time, even though their positions in the photographic field shifted as the embryos underwent a media change, in some cases the media was changed at day 3. The use of sequential media may be needed to meet the stage- specific requirements of the developing embryos. During media change, the embryos were removed from the imaging station for a few minutes and transferred to new petri dishes. The issue of tracking embryo identity can be mitigated by using wells to help arrange the embryos in a particular order.

[0101] When transferring the petri dishes between different stations, the embryos can sometimes move around, thereby making it difficult to keep track of embryo identity. This poses a challenge when time-lapse imaging is performed on one station, and the embryos are subsequently moved to a second station for embryo selection and transfer. One method is to culture embryos in individual petri dishes. However, this requires each embryo to have its own media drop. In a typical IVF procedure, it is usually desirable to culture all of a patient’s embryos on the same petri dish and in the same media drop. To address this problem, we have designed a custom petri dish with micro-wells. This keeps the embryos from moving around and maintains their arrangement on the petri dish when transferred to and from the incubator or imaging stations. In addition, the wells are small enough and spaced closely together such that they can share the same media drop and all be viewed simultaneously by the same microscope. The bottom surface of each micro-well has an optical quality finish. For example, Fig. 27A in US Patent No. 7,963,906 shows a drawing with dimensions for one exemplary embodiment. In this version, there are 25 micro-wells spaced closely together within a 1.7 x 1.7 mm field-of-view. Fig. 27B of US Patent No. 7,963,906 shows a 3D-view of the micro-wells, which are recessed approximately 100 microns into the dish surface. The petri dish may have 1 to 25 or more micro-wells. For example, in one embodiment, a petri dish with 12 wells is utilized. Fiducial markers, including letters, numbers, and other markings, are included on the dish to help with identification. All references cited herein are incorporated by reference in their entireties. EXAMPLES

[0102] The following examples are put forth so as to provide those of ordinary skill in the art with a disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. EXAMPLE 1

PROLONGED TIME TO FIRST CYTOKINESIS AND THE INTERVAL BETWEEN THE FIVE CELL STAGE AND EARLY CAVITATION ARE ASSOCIATED WITH EMBRYONIC MOSAICISM [0103] Embryos that tested aneuploid and had undergone time-lapse imaging from the 2PN to the blastocyst stage were studied. The embryos were biopsied 4 times, placed into individual PCR tubes, blinded as to their origin, and submitted for NextGen based comprehensive chromosomal screening (CCS). If any of the biopsies yielded different CCS results, the embryo was deemed mosaic. If all biopsies agreed, the embryo was deemed non- mosaic. Traditional time lapse parameters shown to predict blastulation and an additional parameter, time from 5-cell stage to the first sign of cavitation, were compared between mosaic and non-mosaic embryos. [0104] Twelve of the 53 (23%) embryos were mosaic. As shown in Table 2, mosaic embryos took longer to undergo first cytokinesis (P0) (p< 0.02). Other time-lapse parameters evaluated through the cleavage stage of development were equivalent. Interestingly, the time to early cavitation from the 5-cell stage was longer in mosaics (P<0.03). Table 2: Time-Lapse Parameters in Mosaic and Non-Mosaic Embryos

[0105] These data demonstrate that two time-lapse parameters related to first cytokinesis and time to first cavitation are associated with embryonic mosaicism. The former may reflect abnormalities in formation of the first mitotic spindle. The latter may indicate that cellular processes leading to mosaicism also result in delayed rates of development after genomic activation. With further investigation, such parameters may help avoid the transfer of mosaic embryos.

Claims

Claims 1. A non-invasive method of detecting mosaicism in an in vitro fertilized human embryo comprising:

(a) in vitro culturing one or more human embryos under conditions sufficient for embryo development;

(b) time lapse imaging said one or more embryos for a time period from at least insemination to at least the first cytokinesis;

(c) measuring the time period of at least one timing parameter comprising:

(i) a time period from insemination to the onset of first cytokinesis (P0); and

(ii) a time period from the 5-cell stage to the onset of cavitation (Pcav₅); and/or

(d) identifying an embryo with an increased risk of mosaicism when:

(i) the time period from insemination to the onset of first cytokinesis is more than about 24 hours; and/or

(ii) the time period from the 5-cell stage to the onset of cavitation is more than about 76 hours.

2. The non-invasive method of claim 1 wherein said one or more embryos are produced by fertilization of oocytes in vitro.

3. The non-invasive method of claim 2 wherein said oocytes are matured in vitro.

4. The non-invasive method of claim 3 wherein said oocytes matured in vitro are supplemented with growth factors.

5. The non-invasive method of claim 1 wherein said one or more embryos have not been frozen prior to culturing.

6. The non-invasive method of claim 1 wherein said one or more embryos have been frozen prior to culturing.

7. The non-invasive method of claim 1 wherein said measuring and/or identifying is automated.

8. The non-invasive method of claim 1 wherein said imaging acquires images that are digitally stored.

9. The non-invasive method of claim 1 wherein said imaging employs darkfield illumination, brightfield illumination or a combination of darkfield and brightfield illumination.

10. The non-invasive method of claim 1 wherein said one or more human embryos are placed in a culture dish prior to culturing under conditions sufficient for embryo development.

11. The non-invasive method of claim 10 wherein said culture dish comprises a plurality of microwells.

12. The non-invasive method of claim 11 wherein one or more human embryos is placed within a microwell prior to culturing under conditions sufficient for embryo development.

13. The non-invasive method of claim 1 wherein the determining is carried out at an imaging station.

14. A non-invasive method for deselecting one or more human embryos that is at increased risk of mosaicism comprising:

(a) in vitro culturing one or more human embryos under conditions sufficient for embryo development;

(b) time lapse imaging said one or more embryos for a time period from at least insemination to at least the first cytokinesis;

(c) measuring the time period of at least one timing parameter comprising:

(i) a time period from insemination to the onset of first cytokinesis (P0); and

(ii) a time period from the 5-cell stage to the onset of cavitation (Pcav₅); and

(d) deselecting an embryo with an increased risk mosaicism when said at least one parameter for said embryo falls within:

(i) the time period from insemination to the onset of first cytokinesis is more than about 24 hours; or Ĩii) the time period from the 5-cell stage to the onset of cavitation is more than about 76 hours,

thereby deselecting an embryo at increased risk of mosaicism.

15. The non-invasive method of claim 14 wherein said one or more embryos are produced by fertilization of oocytes in vitro.

16. The non-invasive method of claim 15 wherein said oocytes are matured in vitro.

17. The non-invasive method of claim 16 wherein said oocytes matured in vitro are supplemented with growth factors.

18. The non-invasive method of claim 14 wherein said one or more embryos have not been frozen prior to culturing.

19. The non-invasive method of claim 14 wherein said one or more embryos have been frozen prior to culturing.

20. The non-invasive method of claim 14 wherein said measuring is automated.

21. The non-invasive method of claim 14 wherein said deselecting an embryo likely to exhibit mosaicism is automated.

22. The non-invasive method of claim 14 wherein said imaging acquires images that are digitally stored.

23. The non-invasive method of claim 14 wherein said imaging employs darkfield illumination, brightfield illumination or a combination of darkfield and brightfield illumination.

24. The non-invasive method of claim 14 wherein said one or more human embryos are placed in a culture dish prior to culturing under conditions sufficient for embryo development.

25. The non-invasive method of claim 24 wherein said culture dish comprises a plurality of microwells.

26. The non-invasive method of claim 25 wherein one or more human embryos is placed within a microwell prior to culturing under conditions sufficient for embryo development.

27. The non-invasive method of claim 14 wherein the determining is carried out at an imaging station.

28. A non-invasive method for selecting one or more human embryos that is at low risk for mosaicism comprising:

(a) in vitro culturing one or more human embryos under conditions sufficient for embryo development;

(b) time lapse imaging said one or more embryos for a time period from at least insemination to at least the first cytokinesis;

(c) determining the time period of at least one timing parameter comprising:

(i) a time period from insemination to the onset of first cytokinesis (P0); and

(ii) a time period from the 5-cell stage to the onset of cavitation (Pcav₅); and

(d) selecting an embryo at low risk for mosaicism when:

(i) the time period from insemination o the onset of first cytokinesis is about 0 to about 23 hours; or

(ii) the time period from the 5-cell stage to the onset of cavitation is about 0 to about 75 hours,

thereby selecting an embryo with a low risk for mosaicism.

29. The non-invasive method of claim 28 wherein said one or more embryos are produced by fertilization of oocytes in vitro.

30. The non-invasive method of claim 29 wherein said oocytes are matured in vitro.

31. The non-invasive method of claim 30 wherein said oocytes matured in vitro are supplemented with growth factors.

32. The non-invasive method of claim 28 wherein said one or more embryos have not been frozen prior to culturing.

33. The non-invasive method of claim 28 wherein said one or more embryos have been frozen prior to culturing.

34. The non-invasive method of claim 28 wherein said measuring is automated.

35. The non-invasive method of claim 28 wherein said selecting an embryo at low risk for mosaicism is automated.

36. The non-invasive method of claim 28 wherein said imaging acquires images that are digitally stored.

37. The non-invasive method of claim 28 wherein said imaging employs darkfield illumination, brightfield illumination or a combination of darkfield and brightfield illumination.

38. The non-invasive method of claim 28 wherein said one or more human embryos are placed in a culture dish prior to culturing under conditions sufficient for embryo development.

39. The non-invasive method of claim 38 wherein said culture dish comprises a plurality of microwells.

40. The non-invasive method of claim 39 wherein one or more human embryos is placed within a microwell prior to culturing under conditions sufficient for embryo development.

41. The non-invasive method of claim 28 wherein the determining is carried out at an imaging station.

42. The method according to any one of the preceding claims wherein the non-invasive time lapse imaging of human embryos is used in conjunction with PGS to determine the relative risk of mosaicism in one or more human embryos.

43. A non-invasive method for determining the relative risk for mosaicism in a human embryo comprising:

(a) in vitro culturing one or more human embryos under conditions sufficient for embryo development; (b) time lapse imaging said one or more human embryos for a time period from at least insemination to at least the first cytokinesis;

(c) measuring the time period of at least one timing parameter comprising:

(i) a time period from insemination to the onset of the first cytokinesis (P0); and

(ii) a time period from the 5-cell stage to the onset of cavitation (Pcav₅); and

(d) determining the relative risk for mosaicism wherein a shorter P0 and/or a shorter Pcav₅ are indicative of a lower risk for mosaicism.