WO2025231097A1

WO2025231097A1 - Use of arachidonic acid for promoting endometrial epithelium regeneration and stem cell plasticity

Info

Publication number: WO2025231097A1
Application number: PCT/US2025/027029
Authority: WO
Inventors: Semir BEYAZ; Onur ESKIOCAK; Vyom SHAH
Original assignee: Cold Spring Harbor Laboratory
Current assignee: Cold Spring Harbor Laboratory
Priority date: 2024-05-02
Filing date: 2025-04-30
Publication date: 2025-11-06
Anticipated expiration: 2026-11-02

Abstract

Disclosed herein are methods and compositions for promoting fertility and/or endometrial receptivity in a subject, by providing arachidonic acid triglyceride (AA-TG) to the subject. In some embodiments, compositions are provided to the subject before or during a course of fertility treatment or process of natural conception.

Description

USE OF ARACHIDONIC ACID FOR PROMOTING ENDOMETRIAL EPITHELIUM REGENERATION AND STEM CELL PLASTICITY

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/641,642, filed May 2, 2024, the entire contents of which are incorporated herein by reference.

BACKGROUND

The endometrium is the inner lining of the uterus with remarkable regenerative capabilities, given its monthly reformation of the endometrial lining during the menstrual cycle¹. This process is managed by endometrial epithelial stem cells (ESCs), which operate under the coordination of hormonal cues and growth factors²'⁴. Malfunctions in the activities of these ESCs or their surrounding environment are linked to diseases such as infertility and endometrial cancer^5,6. Unfortunately, these disorders are not only common, but also come with a significant economic impact, with billions of dollars spent annually on diagnosis and treatment .

SUMMARY

The present disclosure exemplifies how augmented endometrial stem cell (ESC) generation or sternness-enhancing effects of oral administration or consumption of arachidonic acid triglyceride (AA-TG), described herein, provides a basis for methods and compositions for promoting fertility and/or endometrial receptivity in a subject. Disclosed herein is, among other things, the effect of fatty acids, such as administration on AA-TGs on endometrial stem cell regeneration. In some embodiments, a fatty acid (FA) screen using endometrial organoids revealed a subset of omega-6 fatty acids converging on arachidonic acid (AA) with stem cell-enhancing effect. Using tools, such as, bulk and single-cell RNA sequencing analysis, it was found that dietary AA (e.g., AA-TG) evokes stem cell plasticity and regeneration-associated gene expression programs. Thus, AA supplementation, in the form of oral administration of arachidonic acid triglyceride (AA-TG) and/or dietary elevation of AA in a sample in the subject, such as through consumption of an AA-TG-rich diet or other oral intake, augments ESC generation.

Without wishing to be bound by theory, AA (e.g., AA-TG) begets Prostaglandin E2 (PGE2) and activates the prostaglandin E receptor 4 (Ptger4) - cyclic adenosine monophosphate (cAMP) - protein kinase A (PKA) signaling axis to promote stem cell plasticity; downstream of PKA, AA-induced sternness is mediated by the transcription factor cAMP responsive element binding protein (CREB) 1; and, dietary AA (e.g., AA-TG) boosts stem cell activity in human patient-derived organoids through the Ptger4 - CREB1 axis. As disclosed herein, elevation of sternness by AA - Ptger4 - CREB1 axis correlated with endometrial receptivity and fertility. It was demonstrated that dietary AA (e.g., AA-TG) is a conserved promoter of endometrial stem cell activity and highlights the potential of dietary AA (e.g., AA-TG) as a regenerative therapeutic for augmenting endometrial receptivity and fertility.

In some embodiments, the present disclosure provides fatty acids (FAs) (e.g., dietary FAs) to a subject in need thereof to promote fertility in a subject. In some embodiments, administration of a FA, such as arachidonic acid (AA) to a subject before the subject starts a fertility treatment, after the subject starts a fertility treatment or during a fertility treatment promotes fertility in the subject. In some embodiments, providing AA promotes fertility in the subject. In some embodiments, AA is in triglyceride form (AA-TG).

In some embodiments, methods of promoting fertility in a subject are disclosed.

In some embodiments, the method comprises administering orally to a subject in need thereof at least about 2 g of arachidonic acid triglyceride (AA-TG) per day (2 g/d) for a sufficient time to promote fertility in the subject in need thereof.

In some embodiments, the sufficient time is at least about 7 days; and (a) administration starts no earlier than 28 days before the subject in need thereof starts a fertility treatment; (b) administration starts no later than 28 days after the subject starts a fertility treatment; or (c) administration starts at any time during a fertility treatment.

In some embodiments, the sufficient time is at least about 7 days; and (a) administration starts no earlier than 28 days before the subject in need thereof starts a process of natural conception; (b) administration starts no later than 28 days after the subject starts a process of natural conception; or (c) administration starts at any time during a process of natural conception.

In some embodiments, the fertility treatment is an assisted reproductive technique.

In some embodiments, the assisted reproductive technique is in vitro fertilization (IVF), gamete intrafallopian transfer (GIFT), pronuclear stage tubal transfer (PROST), tubal embryo transfer (TET), or zygote intrafallopian transfer (ZIFT).

In some embodiments, the fertility treatment is intrauterine insemination, ovulation stimulation, or intrauterine insemination and ovulation stimulation. In some embodiments, the fertility treatment or the process of natural conception lasts for at least about 3 months.

In some embodiments, the fertility treatment or the process of natural conception lasts for at least about 6 months.

In some embodiments, the fertility treatment or the process of natural conception lasts for at least about 12 months.

In some embodiments, the fertility treatment or the process of natural conception lasts from about 3 months to about 12 months.

In some embodiments, the method prevents, reduces, or reverses the incidence of infertility or incidence of miscarriage in the subject in need thereof.

In some embodiments, the sufficient time is at least about 14 days.

In some embodiments, the sufficient time is at least about 21 days.

In some embodiments, the sufficient time is at least about 28 days.

In some embodiments, at least about 3 g of AA-TG/day (3 g/d) is administered to the subject.

In some embodiments, at least about 20 g of AA-TG/day (20 g/d) is administered to the subject.

In some embodiments, at least about 30 g of AA-TG/day (30 g/d) is administered to the subject.

In some embodiments, at least about 60 g of AA-TG/day (60 g/d) is administered to the subject.

In some embodiments, at least about 90 g of AA-TG/day (90 g/d) is administered to the subject.

In some embodiments, at least about 100 g of AA-TG/day (100 g/d) is administered to the subject.

In some embodiments, from about 2 g of AA-TG/day (2 g/d) to about 100 g of AA- TG/day (100 g/d) is administered to the subject.

In some embodiments, the AA-TG is in a composition.

In some embodiments, the composition comprises at least about 2% AA-TG by weight.

In some embodiments, the composition comprises between about 20% AA-TG and about 50% AA-TG by weight.

In some embodiments, the composition comprises about 40% AA-TG by weight. In some embodiments, the composition comprises no more than 5% arachidonic acid (AA) ester by weight.

In some embodiments, the composition is an oil.

In some embodiments, the oil is extracted from a fungus.

In some embodiments, the fungus is Mortierella alpina.

In some embodiments, the composition is a liquid or a powder.

In some embodiments, the composition is in a food, in a capsule or in a pill.

In some embodiments, the AA-TG increases an endometrial tissue AA level in the subject that produces a beneficial effect.

In some embodiments, administration of AA-TG increases a plasma AA level or increases an endometrial tissue AA level in the subject in need thereof by at least 2-fold relative to a reference.

In some embodiments, the reference is a plasma AA level or endometrial tissue AA level in the subject in need thereof before administration of AA-TG, or the reference is a predetermined plasma AA level or a pre-determined endometrial tissue AA level.

In some embodiments, the subject in need thereof is a mammal.

In some embodiments, the subject in need thereof is a human.

In some embodiments, the subject in need thereof is livestock.

In some embodiments, the subject in need thereof is a mouse.

In some embodiments, methods of promoting endometrial tissue regeneration in a subject are disclosed.

In some embodiments, the method comprises administering orally to a subject in need thereof at least about 2 g of arachidonic acid triglyceride (AA-TG) per day (2 g/d) for a sufficient time to promote endometrial tissue regeneration in the subject in need thereof.

In some embodiments, the fertility treatment is an assisted reproductive technique. In some embodiments, the assisted reproductive technique is in vitro fertilization (IVF), gamete intrafallopian transfer (GIFT), pronuclear stage tubal transfer (PROST), tubal embryo transfer (TET), or zygote intrafallopian transfer (ZIFT).

In some embodiments, the fertility treatment is intrauterine insemination, ovulation stimulation, or intrauterine insemination and ovulation stimulation.

In some embodiments, the fertility treatment or the process of natural conception lasts for at least about 3 months.

In some embodiments, the sufficient time is at least about 14 days.

In some embodiments, the sufficient time is at least about 21 days.

In some embodiments, the sufficient time is at least about 28 days.

In some embodiments, the AA-TG is in a composition. In some embodiments, the composition comprises at least about 2% AA-TG by weight.

In some embodiments, the composition comprises about 40% AA-TG by weight.

In some embodiments, the composition comprises no more than 5% arachidonic acid (AA) ester by weight.

In some embodiments, the composition is an oil.

In some embodiments, the oil is extracted from a fungus.

In some embodiments, the fungus is Mortierella alpina.

In some embodiments, the composition is a liquid or a powder.

In some embodiments, the composition is in a food, in a capsule or in a pill.

In some embodiments, the subject in need thereof is a mammal.

In some embodiments, the subject in need thereof is a human.

In some embodiments, the subject in need thereof is livestock.

In some embodiments, the subject in need thereof is a mouse.

In some embodiments, methods of increasing an arachidonic acid (AA) level in a subject indicative of an endometrial tissue AA level that promotes fertility are disclosed.

In some embodiments, the method comprises: (a) measuring an AA level in a sample from a subject in need thereof and determining if the AA level is below a pre-determined AA level sufficient to promote fertility; and (b) if the AA level is below the pre-determined AA level, administering to the subject in need thereof in (a) at least about 2 g of AA-TG per day (2 g/d) for a sufficient time to increase the AA level to or above the pre-determined AA level.

In some embodiments, the method further comprises: (c) measuring the AA level resulting from administering AA-TG in (b) and determining the AA level; and (d) if the AA level in (b) is not at or above the pre-determined AA level, further administering to the subject in need thereof a sufficient amount of AA-TG per day to result in an endometrial tissue AA level at or above the pre-determined AA level.

In some embodiments, the method further comprises repeating (c)-(d) to produce in the subject in need thereof an endometrial tissue AA level at or above the pre-determined AA level.

In some embodiments, the sample is plasma.

In some embodiments, the sample is endometrial tissue.

In some embodiments, kits for use in promoting fertility in a subject are disclosed.

In some embodiments, the kit comprises: (a) one or more supplement units sufficient to provide to a subject in need thereof at least about 2 g of arachidonic acid triglyceride (AA- TG) per day (2 g/d) for at least 7 days to promote fertility in the subject in need thereof; and (b) instructions for preparation and consumption of the one or more supplement units.

In some embodiments, the one or more supplement units each comprise 500 mg of AA-TG, 1 g of AA-TG, 2 g of AA-TG, or 4 g of AA-TG.

In some embodiments, the number of supplement units to administer to a subject in need thereof is determined in consultation with a healthcare provider.

In some embodiments, the one or more supplement units are in the form of a liquid or a powder.

In some embodiments, the one or more supplement units are in the form of pills or capsules.

In some embodiments, the one or more supplement units are in one or more containers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1H show that fatty acid screens on human and mouse endometrial organoids revealed that omega-6 polyunsaturated fatty acids bolster sternness. FIG. 1A is a schematic describing endometrial organoid culture and fatty acid screen. FIG. IB shows mean organoid area ± s.e.m (pm²x 10³) in response to 20 diverse fatty acids (n = 50). FIG. 1C shows quantification of AA-treated mouse organoids (n = 128). FIG. ID shows quantification and representative images of secondary mouse organoid culture (n = 24). FIG. IE shows the proportion of nutrients in isocaloric (3.8 kcal/g) control diet (control) and AA- rich diet (ARD). Carb, indicates Carbohydrate and Prot. indicates Protein. FIG. IF shows metabolomics analysis by liquid chromatography coupled to mass spectrometry (LC-MS) depicting AA abundance in control or ARD-fed mice (n = 6). FIG. IE shows organoid formation rate from tissue derived from ARD-treated mice (n = 20). FIG. 1H shows representative images depicting enhanced organoid area in-response to ARD diet in mice. P- values are from two-tailed unpaired Student’s t-test (FIGs. 1B-1C and FIG. IF).

FIGs. 2A-2I demonstrate that AA induced a stem cell reprogramming gene expression signature in mouse and human organoids. FIG. 2A shows a heatmap depicting differentially expressed genes (Log2FC > 1 and Padj < 0.05) implicated in sternness from bulk RNA sequencing of AA and control treated mice organoids. FIG. 2B shows annotated Uniform Manifold Approximation and Projection (UMAP) of single cell RNA sequencing (scRNA-seq) of 6110 cells from AA treated and WT mice organoids. FIG. 2C shows a stacked bar plot depicting the fraction of stem cell subtypes in control and AA-treated organoids in scRNA data. FIG. 2D shows a UMAP displaying the relative difference in cellular density of single cell cells between AA and control groups. FIG. 2E shows a UMAP displaying pseudotime trajectories. Arrows denote predicted trajectories within cell clusters (n = 9353 cells). Color scale represents pseudotime. FIG. 2F shows density plots displaying density differences between AA and control along pseudotime in all cells (n = 6110 cells, Fisher test). FIGs. 2G-2I show split-violin plots depicting single-cell Ctnnbl (FIG. 2G), Krtl9 (FIG. 2H), and Ascl2 (FIG. 21) expression across clusters in control and AA treated organoids (n = 6110 cells, Wilcoxon Rank-Sum test). P-values were calculated using DESeq2 in R (FIG. 2A); Fischer test (FIG. 2F); Wilcoxon rank sum test (FIGs. 2G-2I).

FIGs. 3A-3K demonstrate that prostaglandin E2 (PGE2) recapitulated the sternnessenhancing effects of AA. FIG. 3A shows metabolomics LCMS analysis depicting abundances of AA metabolites in control or AA treated mouse organoids (n = 8). FIG. 3B shows a flow plot depicting results of metabolic interactome modeling in stem cell subtypes. Color scale indicates Log(p value), link width indicates communication score, and node size indicates number of connections. FIGs. 3C-3D show organoid area quantification (FIG. 3C, n = 50) and representative images (FIG. 3D) in response to AA metabolite or vehicle treatment. FIG. 3E shows a correlation plot depicting concordance between AA and PGE2 induced changes in gene expression in scRNA data (R² = 0.81, P < .001). FIG. 3F shows a UMAP displaying the relative difference in cellular density of single cell cells between PGE2 and control groups. FIG. 3G shows a stacked bar plot depicting the fraction of stem cell subtypes in control and PGE2-treated organoids in scRNA data. FIG. 3H shows density plots displaying density differences between PGE2 and control along pseudotime in all cells (n = 5157 cells, Fisher test). FIG. 31 shows density UMAP plots displaying Log2 fold change of PGE2 versus Control for B catenin targets. FIGs. 3J-3K show violin plots depicting single-cell Krtl9 (FIG. 3J) and Ascl2 (FIG. 3K) expression across clusters in control, AA-treated, and PGE2-treated organoids (n= 9353 cells, Wilcoxon Rank-Sum test). P-values were calculated using MEBOcost (FIG. 3B); Wilcoxon rank-sum test (FIG. 3C, FIG. 3J, and FIG. 3K); Pearson test (FIG. 3E); Fischer test (FIG. 3H).

FIGs. 4A-4L demonstrate that the Ptger4 - cAMP - PKA signaling axis regulated AA-induced sternness. FIG. 4A show split- violin plots depicting single-cell Ptger4 expression across clusters in control and AA treated organoids (n = 6110 cells, Wilcoxon Rank-Sum test). FIGs. 4B-44C show organoid area quantification (FIG. 4B) and representative images (FIG. 4C) of Ptger4i organoids in response to vehicle, AA, and PGE2 treatment. FIG. 4D shows density UMAP plots displaying log2 fold change of AA versus Control for CREB targets. FIGs. 4E-4F show organoid area quantification (FIG. 4E) and representative images (FIG. 4F) of H89+ organoids in response to vehicle, AA, and PGE2 treatment. FIGs. 4G-4H show organoid area quantification (FIG. 4F) and representative images (FIG. 4H) of CREBi organoids in response to vehicle, AA, and PGE2 treatment. FIG. 41 shows correlation between normalized average expression of CREB Target genes and Ctnnbl expression in single cell RNA sequencing data. Solid lines show linear regression. Color scale shows cell types as annotated in FIG. 2C. FIGs. 4J-4K show organoid area quantification (FIG. 4J) and representative images (FIG. 4K) of Pri organoids in response to vehicle, AA, and PGE2 treatment. FIG. 4L is a schematic depicting a proposed mechanism for how AA induced regeneration in the endometrial epithelium. P-values were calculated using Wilcoxon ranksum test (FIG. 4A, FIG. 4B, FIG. 4E, FIG. 4G, and FIG. 4J); Fischer test (FIG. 41).

FIGs. 5A-5I demonstrate that arachidonic acid bolstered stem cell plasticity and fertility outcomes in the human endometrial epithelium. FIG. 5A shows mean organoid area ± s.e.m (pm2 x 10³) in response to fatty acid treatment (n = 50). FIG. 5B shows representative differentially expressed genes (Log F C > 1 & P Value < .05) from AA treated versus Control bulk RNA-sequencing. FIG. 5C shows a barcode plot depicting differentially expressed genes within regeneration-induced score. NES and p-value were calculated using the Fgsea program. FIGs. 5D-5E show human organoid area quantification (FIG. 5D) and representative images (FIG. 5E) of Ptger4i treated organoids in response to vehicle, AA, and PGE2 treatment. FIG. 5F shows the correlation between normalized average expression of CREB Targets and receptive endometrium induced genes in single cell RNA sequencing data. Solid lines show linear regression. Color scale shows cell types as annotated in FIG. 2C. FIG. 5G shows a barcode plot depicting differentially expressed genes within receptive endometrium induced score. NES and p-value were calculated using the Fgsea program. FIG. 5H shows density UMAP plots displaying log2 fold change of AA versus Control for receptive endometrium induced genes. FIG. 51 shows enriched lipid metabolite subdivisions in fertile patients compared to infertile patients calculated using the mummichog algorithm. Color scale indicates p-values. P-values were calculated using Wilcoxon rank-sum test (FIG. 5A and FIG. 5D); DESeq2 (FIG. 5B); Fgsea (FIG. 5C and FIG. 5H); Fischer test (FIG. 5G).

FIG. 6 shows representative images of fatty acid treated mice organoids.

FIGs. 7A-7K. FIG. 7A shows principal component analysis from human bulk RNA sequencing. Variance from the first two principal components is projected. FIG. 7B shows GSEA from AA-treated and WT endometrial epitheliums in mice. Color scale represents p- values. FIGs. 7C-7F show UMAP plots portraying the density of Sox9 (FIG. 7C), Hmgb2 (FIG. 7D), Mki67 (FIG. 7E), and Anxa3 (FIG. 7F) expression (n = 9353 cells). Color scale represents density of expression. FIG. 7G is a dot plot showing expression and percentage of cells expressing marker genes across cell types in scRNA data. Color scale shows average expression and dot size depicts percentage of cells expressing the gene. FIG. 7H is a paired bar plot showing the fraction of cells in each annotated cluster for control and AA-treated groups in scRNA data. FIG. 71 shows a partition-based graph abstraction (PAGA) UMAP depicting differentiation trajectories. Gray arrows indicate trajectories exclusive to AA treatment whereas black arrows indicate trajectories present in both conditions. FIGs. 7J-7K show density UMAP plots displaying Log2 fold change of PGE2 versus control for genes implicated in Regeneration (FIG. 7 J) and Fetal spheroid functions (FIG. 7K). P-values calculated using Wilcoxon rank sum test (FIG. 7H); Fischer test (FIG. 71); (FIGs. 7J-7K).

FIGs. 8A-8H. FIG. 8A shows metabolomics LCMS analysis depicting abundances of AA metabolites in control or AA treated mouse organoids after 1 and 4 weeks (n = 4). FIG. 8B shows stacked and paired bar plots comparing concordance between the GSEA of Pge2- Ptger4 and Folate-Slcl9al signaling target genes. FIG. 8C shows gene ontology analysis conducted on targets of PGE2-Ptger4 signaling upregulated as identified by interactome modeling. FIG. 8D shows a paired bar plot showing the fraction of cells in each annotated cluster for control and PGE2-treated groups in scRNA data. FIG. 8E shows a UMAP displaying density difference of single cell cells between PGE2 and AA groups. FIG. 8F shows a line plot depicting normalized expression levels of B catenin targets in control or PGE2 treated organoids across pseudotime. FIGs. 8G-8H show violin plots depicting singlecell (FIG. 8G) Ly6a & (FIG. 8H) Wnt7a expression across clusters in control, AA-treated, and PGE2-treated organoids (n = 9353 cells). P-values from two-tailed unpaired Student’s t- test (FIGs. 8A-8D and FIG. 8K); Wilcoxon rank sum test (FIG. 8H).

FIGs. 9A-9I. FIGs. 9A-9C show violin plots depicting single-cell (FIG. 9A) Ptgerl, (FIG. 9B) Ptger2, and (FIG. 9C) Ptger3 expression across clusters in control, AA-treated, and PGE2-treated organoids (n = 9353 cells). FIGs. 9D-9E show line plot depicting normalized expression levels of CREB targets in control versus AA (FIG. 9D) or PGE2 (FIG. 9E) treated organoids across pseudotime. FIGs. 9F-9I show correlation between normalized average expression of CREB and B catenin targets (FIG. 9F), Ptger4 and Ctnnbl (FIG. 9G), CREB target genes and regeneration-induced genes (FIG. 9H), and Ptger4 and regeneration induced genes (FIG. 91) in single cell RNA sequencing data. Solid lines show linear regression. The scale shows cell types as annotated in FIG. 2C. P-values were calculated Wilcoxon-rank sum test (FIGs. 9A-9C); Fischer test (FIGs. 9G-9I and FIG. 9K); Pearson test (FIGs. 9E-9F).

FIGs. 10A-10L. FIG. 10A shows principal component analysis from human bulk RNA sequencing. Variance from the first two principal components is projected. FIG. 10B is a volcano plot showing differentially expressed genes from bulk RNA sequencing of AA and Control treated human organoids. FIG. IOC shows GSEA from AA-treated and WT endometrial epithelial organoids. The scale represents p-values. FIG. 10D shows density UMAP plots displaying Log2 fold change of AA versus control for genes implicated in representing enhanced fertility. FIGs. 10E-10I show correlation between normalized average expression of Ptger4 and fertility-induced (FIG. 10E), CREB target genes and fertility- induced (FIG. 10F), regeneration-induced and fertility-induced genes (FIG. 10G), fertility- induced and receptive endometrium induced (FIG. 10H), Ptger4 and Receptive endometrium induced (FIG. 101), regeneration induced and receptive endometrium induced (FIG. 10J), and B catenin enhanced and receptive endometrium induced in single cell RNA sequencing data (FIG. 10K). Solid lines show linear regression. The scale shows cell types as annotated in FIG. 2B. FIGs. 10J-10K show violin plots depicting single-cell scored expression of genes induced by a receptive endometrium (FIG. 10J) and enhanced fertility (FIG. 10K) expression across clusters in control, AA-treated, and PGE2-treated organoids (n = 9353 cells). FIG. 10L shows orthogonal Partial least squares-discriminant analysis of human metabolomics from uterine lining of fertile and infertile patients. Variance from the orthogonal t- scores is projected. P-values were calculated using DESeq2 (FIG. 10B); Fgsea (FIG. 10C); Pearson test (FIGs. 10E-10I); Wilcoxon-rank sum (FIGs. 10J-10K).

Unless indicated otherwise for FIGs. 1-10, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

Endometrial ailments such as endometriosis, infertility, and endometrial cancer impact 1 in 4 women in the US, accentuating the significant need to improve the understanding of endometrial biology. Endometrial stem cells regenerate the endometrium monthly, and their dysregulation is implicated in endometrial diseases. Recent studies have underscored the capacity for the metabolic reprogramming of stem cells, but little is known about the metabolic regulation of stem cell states in endometrial physiology.

Disclosed herein is the effect of fatty acids on endometrial stem cell regeneration. A fatty acid (FA) screen was performed using mouse endometrial organoids. A subset of omega-6 fatty acids converging on arachidonic acid (AA) with stem cell-enhancing effects was identified. Bulk and single-cell RNA sequencing analysis revealed that dietary AA (e.g., AA-triglyceride or AA-TG) evokes stem cell plasticity and regeneration-associated gene expression programs.

Without wishing to be bound by theory, AA begets Prostaglandin E2 (PGE2) and activates the Ptger4 - cAMP - PKA signaling axis to promote stem cell plasticity. Downstream of PKA, AA-induced sternness is mediated by the transcription factor CREB1. Finally, dietary AA boosts stem cell activity in human patient-derived organoids through the Ptger4 - CREB1 axis. Elevation of sternness by A A - Ptger4 - CREB1 axis correlated with endometrial receptivity and fertility. Dietary AA (e.g., AA-TG) is a conserved promoter of endometrial stem cell activity and, as disclosed herein, dietary AA serves a regenerative therapeutic of the endometrium, which results in augmented endometrial receptivity and fertility.

The dynamic process of endometrial tissue regeneration and repair during the menstrual cycle is orchestrated by ESCs. ESC function is mediated by numerous biological and microenvironmental factors such as hormonal changes, transcription factor signaling, and growth factors. Hormone influence can facilitate the remodeling of endometrial epithelial tissue architecture in accordance with the menstrual cycle⁸. Variable expression of transcription factors and key signaling cascades maintain stem cell identity and regulating their differentiation potential. Specifically, ESC function is dependent on Canonical Wnt/B catenin and Sox9 signaling^1,9. Altered transcription factor activity can reprogram epithelial lineages and facilitate plasticity, or the ability for cells to remain in transit between cell states^10,11. Promoting epithelial plasticity has been implicated in influencing tissue architecture through cellular processes such as trans-differentiation, dedifferentiation, cellcell fusion, and homogeneity¹². As a result, epithelial plasticity and dedifferentiation play crucial roles in endometrial regeneration and are implicated in the pathogenesis of degenerative ailments^13,14.

The role of nutrients and their metabolism in regulating stem cell function is a burgeoning area of research that has gained considerable attention in recent years. Studies have illustrated how dietary fatty acids (FAs) can modulate stem cell activity in regenerative tissues such as the intestine^15,16. There are several possible mechanisms by which FAs regulate stem cell fate. First, FAs or their metabolites bind and activate FA- sensing transcription factors (TFs) such as PPAR-c to regulate transcription directly¹⁷. FAs can also be metabolized into key ligand signaling molecules such as sphingolipids and prostaglandins that are implicated in stem cell reprogramming^18,19. FA-derived metabolites such as acetyl-CoA are also utilized for histone modifications and influence epigenetic states²⁰. Despite this growing body of evidence in other tissues, very little information exists on how FAs and their metabolism affect endometrial stem cell activity. Identifying whether specific types of FAs regulate ESC activity and defining the underlying causal mechanisms hold the promise of not only advancing our understanding of the endometrium but also of developing novel therapeutic approaches for related diseases. Through a fatty acid screen, multi-omics analysis, and an inhibition assay on murine and human organoid models, it was demonstrated that dietary FAs induce an endometrial stem cell program that can be utilized to ablate degenerative symptoms and improve fertility outcomes.

In some embodiments, the present disclosure relates to promotion of endometrial tissue regeneration in a subject by providing a subject in need thereof an amount (beneficial dose) of AA (e.g., in TG form or AA-TG) prior to starting a fertility treatment or a process of natural conception. In some embodiments, a subject in need thereof is provided an amount (beneficial dose) of AA (e.g., in TG form or AA-TG) during a fertility treatment or a process of natural conception.

In some embodiments, an amount (beneficial dose) of AA (e.g., in TG form or AA- TG) is administered after starting a fertility treatment or process of natural conception.

In some embodiments, an amount (beneficial dose) of AA (e.g., in TG form or AA- TG) is administered before starting and during a fertility treatment or before starting and during a process of natural conception.

In some embodiments, the present disclosure provides that AA (e.g., in TG form), influence sternness, for instance, in the endometrial epithelium. The findings provide basis for using AA (e.g., in TG form or AA-TG) to promote fertility or to promote endometrial tissue (e.g., epithelium) regeneration in a subject in need thereof. In some embodiments, the present disclosure relates to promoting fertility in a subject in need thereof. In some embodiments, promoting fertility refers to the treatment of infertility, favoring a process of natural conception, favoring normal reproduction, or contributing to normal reproduction in the subject in need thereof. The terms “favoring a process of natural conception,” “favoring normal reproduction” or “contributing to normal reproduction” refer to improving normal reproduction and fertility by reducing the times needed to achieve pregnancy, with “natural conception” or “normal reproduction” being understood as a state of fertility in which pregnancy is achieved under the following circumstances:

(a) in a subfertility situation: pregnancy is achieved in a period of 12 months of regular, unprotected sexual intercourse;

(b) in an infertility situation: pregnancy is achieved after a period 12 months of regular, unprotected sexual intercourse.

In some embodiments, restoring ovulation, promoting ovulation, improving oocyte and embryo quality, increasing zygote implantation on the uterine wall, regulating hypothalamic -pituitary-ovarian axis impairment, including polycystic ovarian syndrome, metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, cirrhosis, rheumatoid arthritis, celiac disease, chronic kidney failure, idiopathic causes and eating disorders, such as anorexia nervosa and bulimia, are also contemplated along with administration of AA-TG to promote fertility.

A fertility treatment, as disclosed herein, encompasses any biomedical techniques or methods facilitating or replacing at least one of the natural processes taking place during reproduction. In some embodiments, a fertility treatment is an assisted reproductive technique. In some embodiments, an assisted reproductive technique is a procedure in which either an egg, an embryo, or egg and embryo are handled, manipulated, modified or treated to help achieve a pregnancy. In some embodiments, an assisted reproductive technique involves retrieving mature eggs, fertilizing them with sperm in a laboratory, then transferring one or more embryos into a uterus. In some embodiments, an assisted reproductive technique is in vitro fertilization (IVF), gamete intrafallopian transfer (GIFT), pronuclear stage tubal transfer (PROST), tubal embryo transfer (TET), or zygote intrafallopian transfer (ZIFT). In some embodiments, an assisted reproductive technique is IVF.

In some embodiments, a fertility treatment is ovulation stimulation, intrauterine insemination (including insemination with donor’s sperm) and in vitro insemination (including insemination of a donor’s ovum or insemination with donor’s sperm). In some embodiments, ovulation stimulation (or regulation) can be achieved with a fertility drug. In some embodiments, a fertility drag works like a natural hormone — follicle-stimulating hormone (FSH) and luteinizing hormone (LH) — to trigger ovulation, to stimulate a better egg or an extra egg or eggs. Examples of fertility drugs include, but are not limited to, clomiphene citrate (by mouth), gonadotropins injected to stimulate ovary to produce multiple eggs (e.g., human menopausal gonadotropin or hMG (Menopur) and FSH (Gonal-F, Follistim AQ, Bravelie)), to mature the eggs and trigger their release at the time of ovulation (e.g., human chorionic gonadotropin (Ovidrel, Pregnyl)), Metformin (when insulin resistance is a known or suspected cause of infertility, usually in women with a diagnosis of PCOS), aromatase inhibitors (e.g., Letrozole (Femara), usually used for women younger than 39 who have PCOS), bromocriptine (e.g., Cycloset, Parlodel used when ovulation problems are caused by excess production of prolactin (hyperprolactinemia) by the pituitary gland). In some embodiments, a fertility treatment includes vitamin supplements (e.g., vitamin B, vitamin C, vitamin E and folic acid), mineral supplements (e.g., selenium, zinc or iron complexes or salts), essential fatty acids (omega-3), or extracts from plants such as chaste tree (Vitex agnus-castus), damiana, licorice, red clover flower, chasteberry, black cohosh, dong quai (Angelica sinensis), wild yam or sweet potato (Dioscorea villosa), false unicorn root, green tea, nettles (Urtica dioica), wild oats (Avena sativa), dandelion (Taraxacum officinale), etc.

In some embodiments, a fertility treatment is or comprises surgery. In some embodiments, surgery is laparoscopic or hysteroscopic surgery to correct, for instance, problems with the uterine anatomy, removing endometrial polyps and some types of fibroids that misshape the uterine cavity, or removing pelvic or uterine adhesions. In some embodiments, surgery is tubal surgery to remove adhesions, dilate a fallopian tube or create a new fallopian tubal opening. In some embodiments, tubal surgery close to the uterus can improve fertility treatment with IVF.

Therefore, a fertility treatment can be applied due to causes of infertility or reduced fertility in a female subject in need thereof. The assisted reproductive technique can also be applied in women who, despite not having any fertility problem per se, must resort to the technique for various reasons, including but not limited to, a subject in need thereof (women) without a partner who require sperm donation, couples who must resort to ovum and/or sperm donation, livestock and mice that require embryo transfer for improved productivity. In some embodiments, these fertility treatments have a success rate of less than 50% or even 40%. In some embodiments, reduced fertility or reduced reproductive efficiency relates to difficulties in embryo adhesion, embryo implantation, or in both embryo adhesion and implantation processes and administration of AA-TG (e.g., at least 2 g AA-TG per day for a sufficient time) to the subject in need thereof promotes fertility and promotes reproductive efficiency.

The term “promoting” in the phrase “promoting fertility” refers to increasing the success rate of conception and pregnancy achieved with biomedical techniques or methods facilitating or replacing at least one of the natural processes taking place during reproduction. Therefore, if the success rate in the age range of the subject in need thereof is applied was, for instance 43%, a rate from 44% and above would involve promoting fertility with the use of such technique. In some embodiments, promoting fertility is in the context of a fertility treatment, wherein AA-TG administration increases the success rate of the fertility treatment as measured by increased conception, reduced miscarriage or both increased conception and reduced miscarriage. In some embodiments, promoting fertility is in the context of a process of natural conception, wherein AA-TG administration increases the rate of conception, reduces the rate of miscarriage, or both increases the rate of conception and reduces the rate of miscarriage.

Arachidonic Acid (AA)

In some embodiments, methods of promoting fertility in a subject are disclosed. In some embodiments, the method comprises administering (e.g., orally) to a subject in need thereof AA in triglyceride form (AA-TG) for a sufficient time to promote fertility in the subject in need thereof. In some embodiments, methods of promoting endometrial tissue (e.g., epithelium) regeneration in a subject comprise administering (e.g., orally) to a subject in need thereof AA-TG (e.g., at least about 2 g of AA-TG per day) for a sufficient time to promote endometrial tissue (e.g., epithelium) regeneration in the subject in need thereof. In some embodiments, methods of increasing an AA level in a subject indicative of an endometrial tissue AA level that promotes fertility comprise (a) measuring an AA level in a sample from a subject in need thereof and determining if the AA level is below a predetermined AA level sufficient to promote fertility; and (b) if the A A level is below the predetermined AA level, administering to the subject in need thereof in (a) AA-TG (e.g., at least about 2 g of AA-TG per day) for a sufficient time to increase the AA level to or above the pre-determined AA level.

AA is a 20-carbon chain fatty acid with four methylene-interrupted cis double bonds. In some embodiments, AA is in glyceride form. In some embodiments, AA is in triglyceride (TG) form (AA triglyceride or AA-TG). In some embodiments, AA is a free fatty acid AA. In some embodiments, a free fatty acid AA is bound to a carrier protein (e.g., albumin; serum albumin). In some embodiments, AA is in phospholipid (PL) form (AA phospholipid or AA- PL). In some embodiments, an AA-PL is used in the composition, methods and kits disclosed herein. In some embodiments, AA is not associated with a TG or a PL.

AA Compositions

In some embodiments, a composition comprises AA-TG. In some embodiments, a composition is an oil. In some embodiments, the oil is extracted from an organism (e.g., plant, fungus, etc.). In some embodiments, the organism is a microorganism (See e.g., U.S. Patent No. 8,389,808, the contents of which are incorporated by reference in their entirety). In some embodiments, the microorganism belongs to the genus Mortierella, Entomophthora, Pythium, or Porphyridium. In some embodiments, the microorganism belongs to the genus Pythium. In some embodiments, the microorganism is Pythium insidiuosum. In some embodiments, the organism is a fungus. In some embodiments, the fungus belongs to the genus Mortierella. In some embodiments, the fungus is Mortierella alpina.

In some embodiments, an oil comprises about 10% or least about 10% AA-TG, about 15% or least about 15% AA-TG, about 20% or least about 20% AA-TG, about 25% or least about 25% AA-TG, about 30% or least about 30% AA-TG, about 35% or least about 35% AA-TG, about 40% or least about 40% AA-TG, about 45% or least about 45% AA-TG, about 50% or least about 50% AA-TG, about 55% or least about 55% AA-TG, about 60% or least about 60% AA-TG.

In some embodiments, an oil comprises between about 20% AA-TG and about 60% AA-TG. In some embodiments, an oil comprises between about 20% AA-TG and about 50% AA-TG. In some embodiments, an oil comprises between about 30% AA-TG and about 50% AA-TG. In some embodiments, an oil comprises at least 40% or about 40% AA-TG. In some embodiments, percent AA-TG is calculated as volume/volume percentage. In some embodiments, percent AA-TG is calculated as a weight/volume percentage. In some embodiments, percent AA-TG is calculated as weight/weight percentage.

In some embodiments, methods of promoting fertility or promoting endometrial tissue regeneration are disclosed. In some embodiments, the method comprises administering orally to a subject in need thereof a composition comprising: (a) an oil comprising arachidonic acid triglyceride (AA-TG); and (b) an oil other than the oil in (a), wherein the oil of (a) and the oil of (b) are at a ratio of about 3:4, wherein the composition is administered for at least 7 days before the subject starts a fertility treatment or a process of natural conception.

Administration

In some embodiments, AA-TG is administered to a subject in need thereof. In some embodiments, a beneficial dose of AA-TG is administered. In some embodiments, a beneficial dose is a therapeutic dose, an effective dose, or a therapeutically effective dose. In some embodiments, a beneficial dose is a clinically effective dose. In some embodiments, administration is or comprises supplementation. In some embodiments, administering is or comprises supplementing.

In some embodiments, an amount of AA-TG is administered to a subject in need thereof per day. In some embodiments, about 2 g or at least about 2 g of AA-TG/day is administered to the subject. In some embodiments, about 2.5 g or at least about 2.5 g of AA- TG/day is administered to the subject. In some embodiments, about 3 g or at least about 3 g of AA-TG/day is administered to the subject. In some embodiments, about 4 g or at least about 4 g of AA-TG/day is administered to the subject. In some embodiments, about 5 g or at least about 5 g of AA-TG/day is administered to the subject. In some embodiments, about 6 g or at least about 6 g of AA-TG/day is administered to the subject. In some embodiments, about 7 g or at least about 7 g of AA-TG/day is administered to the subject. In some embodiments, about 8 g or at least about 8 g of AA-TG/day is administered to the subject. In some embodiments, about 9 g or at least about 9 g of AA-TG/day is administered to the subject. In some embodiments, about 10 g or at least about 10 g of AA-TG/day is administered to the subject. In some embodiments, about 15 g or at least about 15 g of AA- TG/day is administered to the subject. In some embodiments, about 20 g or at least about 20 g of AA-TG/day is administered to the subject. In some embodiments, about 25 g or at least about 25 g of AA-TG/day is administered to the subject. In some embodiments, about 30 g or at least about 30 g of AA-TG/day is administered to the subject. In some embodiments, about 40 g or at least about 40 g of AA-TG/day is administered to the subject. In some embodiments, about 50 g or at least about 50 g of AA-TG/day is administered to the subject. In some embodiments, about 60 g or at least about 60 g of AA-TG/day is administered to the subject. In some embodiments, about 70 g or at least about 70 g of AA-TG/day is administered to the subject. In some embodiments, about 80 g or at least about 80 g of AA- TG/day is administered to the subject. In some embodiments, about 90 g or at least about 90 g of AA-TG/day is administered to the subject. In some embodiments, about 100 g or at least about 100 g of AA-TG/day is administered to the subject.

In some embodiments, from about 2 g to about 100 g of AA-TG/day is administered to the subject. In some embodiments, from about 2 g to about 90 g of AA-TG/day is administered to the subject. In some embodiments, from about 2 g to about 80 g of AA- TG/day is administered to the subject. In some embodiments, from about 2 g to about 70 g of AA-TG/day is administered to the subject. In some embodiments, from about 2 g to about 60 g of AA-TG/day is administered to the subject. In some embodiments, from about 2 g to about 50 g of AA-TG/day is administered to the subject. In some embodiments, from about 2 g to about 40 g of AA-TG/day is administered to the subject. In some embodiments, from about 2 g to about 30 g of AA-TG/day is administered to the subject. In some embodiments, from about 2 g to about 20 g of AA-TG/day is administered to the subject. In some embodiments, from about 2 g to about 10 g of AA-TG/day is administered to the subject.

In some embodiments, from about 5 g to about 100 g of AA-TG/day is administered to the subject. In some embodiments, from about 5 g to about 90 g of AA-TG/day is administered to the subject. In some embodiments, from about 5 g to about 80 g of AA- TG/day is administered to the subject. In some embodiments, from about 5 g to about 70 g of AA-TG/day is administered to the subject. In some embodiments, from about 5 g to about 60 g of AA-TG/day is administered to the subject. In some embodiments, from about 5 g to about 50 g of AA-TG/day is administered to the subject. In some embodiments, from about 5 g to about 40 g of AA-TG/day is administered to the subject. In some embodiments, from about 5 g to about 30 g of AA-TG/day is administered to the subject. In some embodiments, from about 5 g to about 20 g of AA-TG/day is administered to the subject. In some embodiments, from about 5 g to about 10 g of AA-TG/day is administered to the subject.

In some embodiments, AA-TG is administered to a subject in need thereof based on the weight of the subject. In some embodiments, about 50 mg or at least about 50 mg of AA- TG/kg of body weight, about 100 mg or at least about 100 mg of AA-TG/kg of body weight, about 150 mg or at least about 150 mg of AA-TG/kg of body weight, about 200 mg or at least about 200 mg of AA-TG/kg of body weight, about 300 mg or at least about 300 mg of AA- TG/kg of body weight, about 400 mg or at least about 400 mg of AA-TG/kg of body weight, about 500 mg or at least about 500 mg of AA-TG/kg of body weight, about 600 mg or at least about 600 mg of AA-TG/kg of body weight, about 700 mg or at least about 700 mg of AA- TG/kg of body weight, about 800 mg or at least about 800 mg of AA-TG/kg of body weight, about 900 mg or at least about 900 mg of AA-TG/kg of body weight, about 1 g or at least about 1 g of AA-TG/kg of body weight, about 1.5 g or at least about 1.5 g of AA-TG/kg of body weight, about 2 g or at least about 2 g of AA-TG/kg of body weight, or any range or combination thereof.

In some embodiments, an amount of AA-TG to be administered to a subject in need thereof accounts for one or more of age, sex, height, concomitant medication(s), and preexisting conditions in the subject. In some embodiments, a pre-existing condition is endometriosis. In some embodiments, a pre-existing condition is hyperplasia. In some embodiments, a pre-existing condition is adenomyosis. In some embodiments, a pre-existing condition is endometrial cancer.

In some embodiments, administration of AA-TG increases an AA level in the blood or a component of blood (e.g., plasma) of the subject relative to a reference. In some embodiments, an AA level is increased in the endometrial tissue (e.g., epithelium) of the subject relative to a reference. In some embodiments, an AA level is increased in the plasma and endometrial tissue (e.g., epithelium) of the subject relative to a reference.

In some embodiments, an AA level is measured in the blood or a component of blood (e.g., plasma) of the subject. In some embodiments, the AA level is measured in the endometrial tissue (e.g., epithelium) of a subject. In some embodiments, the AA level is measured in the endometrial tissue (e.g., epithelium) of a subject and in the blood or a component of blood (e.g., plasma) of the subject.

In some embodiments, administration of AA-TG to a subject in need thereof increases expression of a marker of sternness, such as increased expression of a gene associated with sternness, relative to a reference. In some embodiments, a gene associated with sternness is at least one of mesothelin (Msln), carnitine palmitoyltransferase 1A (Cptla), solute carrier family 7 member 3 (Slc7a3), and frizzled class receptor 10 (FzdlO). In some embodiments, a gene associated with sternness is S100 calcium binding protein P (SIOOP) or solute carrier family 7 member 1 (SLC7A1). In some embodiments, administration of AA-TG to a subject in need thereof increases expression of a dedifferentiation marker relative to a reference. In some embodiments, a dedifferentiation marker is at least one of Ctnnbl (encoding catenin beta 1), Krtl9 (encoding keratin 19), and Ascl2 (encoding Achaete-Scute family BHLH transcription factor 2; beta-catenin target gene). In some embodiments, administration of AA-TG to a subject in need thereof increases expression of a gene involved in stem cell plasticity. In some embodiments, a gene involved in stem cell plasticity is at least one of Cd55 (encoding CD55 molecule), Ereg (encoding epiregulin), Myof (encoding myoferlin) and Msln (encoding mesothelin). In some embodiments, administration of AA-TG to a subject in need thereof increases expression of a gene involved in regenerative repair response. In some embodiments, a gene involved in regenerative repair response is at least one of Fzd9 (encoding frizzled class receptor 9), SoxlO (encoding SRY-box transcription factor 10), and Cd36 (encoding CD36 molecule).

In some embodiments, administration of AA-TG increases expression of a gene associated with sternness, of a dedifferentiation marker, of a gene involved in stem cell plasticity, or a gene involved in a regenerative repair response about 75% or at least about 75%; about 100% or at least about 100%; about 125% or at least about 125%; about 150% or at least about 150%; about 175% or at least about 175%; about 200% or at least about 200%; about 300% or at least about 300%; about 400% or at least about 400%, about 500% or at least about 500%, about 600% or at least about 600%; about 700% or at least or about 700%, about 800% or at least about 800%, or any ranges or combinations thereof, relative to a reference.

In some embodiments, expression of a gene associated with sternness, of a dedifferentiation marker, of a gene involved in stem cell plasticity, or of a gene involved in a regenerative repair response is increased in a cell (e.g., epithelial cell, etc.) obtained from the subject. In some embodiments, expression of a gene associated with sternness is increased in a sample, such as blood, a component of blood (e.g., plasma, serum etc.) or a tissue (e.g., endometrial epithelium) obtained from the subject.

In some embodiments, administration of AA-TG to a subject in need thereof increases an AA level in the subject (e.g., a sample from the subject) relative to a reference. In some embodiments, administration of AA-TG to a subject in need thereof increases an AA level at about 2-fold or at least about 2-fold relative to a reference. In some embodiments, administration of AA-TG to a subject in need thereof increases an AA level about 2-fold or at least about 2-fold, about 3-fold or at least about 3-fold, about 4-fold or at least about 4-fold, about 5-fold or at least about 5-fold, about 6-fold or at least about 6-fold, about 7-fold or at least about 7-fold, about 8-fold or at least about 8-fold, about 9-fold or at least about 9-fold, about 10-fold or at least about 10-fold, about 11-fold or at least about 11-fold, about 12-fold or at least about 12-fold, about 13-fold or at least about 13-fold, about 14-fold or at least about 14-fold, about 15-fold or at least about 15-fold, or any ranges or combinations thereof, relative to a reference.

In some embodiments, administration of AA-TG increases an AA level in a subject in need thereof about 3-fold to about 20-fold relative to a reference. In some embodiments, administration of AA-TG increases an AA level in a subject in need thereof about 3-fold to about 15-fold relative to a reference. In some embodiments, administration of AA-TG increases an AA level in a subject in need thereof about 3-fold to about 10-fold relative to a reference. In some embodiments, administration of AA-TG increases an AA level in a subject in need thereof about 1.5-fold to about 3-fold relative to a reference.

In some embodiments, AA-TG is administered to promote fertility. In some embodiments, AA-TG is administered to promote endometrial tissue (e.g., epithelium) regeneration.

In some embodiments, AA-TG is administered for a total time of 7 days, about 7 days, or at least 7 days, 10 days, about 10 days, or at least 10 days, 2 weeks or about 2 weeks, or at least 2 weeks, 3 weeks, about 3 weeks, or at least 3 weeks, 4 weeks, about 4 weeks, or at least 4 weeks, 5 weeks, about 5 weeks, or at least 5 weeks, 6 weeks, about 6 weeks, or at least 6 weeks, 7 weeks, about 7 weeks, or at least 7 weeks, 8 weeks, about 8 weeks, or at least 8 weeks, 9 weeks, about 9 weeks, or at least 9 weeks, 10 weeks, about 10 weeks, or at least 10 weeks, 11 weeks, about 11 weeks, or at least 11 weeks, 12 weeks, about 12 weeks, or at least 12 weeks before a subject in need thereof starts a fertility treatment.

In some embodiments, AA-TG is administered for a total time of 7 days, about 7 days, or at least 7 days, 10 days, about 10 days, or at least 10 days, 2 weeks or about 2 weeks, or at least 2 weeks, 3 weeks, about 3 weeks, or at least 3 weeks, 4 weeks, about 4 weeks, or at least 4 weeks, 5 weeks, about 5 weeks, or at least 5 weeks, 6 weeks, about 6 weeks, or at least 6 weeks, 7 weeks, about 7 weeks, or at least 7 weeks, 8 weeks, about 8 weeks, or at least 8 weeks, 9 weeks, about 9 weeks, or at least 9 weeks, 10 weeks, about 10 weeks, or at least 10 weeks, 11 weeks, about 11 weeks, or at least 11 weeks, 12 weeks, about 12 weeks, or at least 12 weeks before a subject in need thereof starts a process of natural conception.

In some embodiments, AA-TG is administered for 2 weeks to 4 weeks before a subject in need thereof starts a fertility treatment. In some embodiments, AA-TG is administered for 1 week to 3 weeks, 3 weeks to 5 weeks, 4 weeks to 6 weeks, or 5 weeks to 7 weeks before a subject in need thereof starts a fertility treatment.

In some embodiments, the AA-TG is administered for 2 weeks to 4 weeks before a subject in need thereof starts a process of natural conception. In some embodiments, AA-TG is administered for 1 week to 3 weeks, 3 weeks to 5 weeks, 4 weeks to 6 weeks, or 5 weeks to 7 weeks before a subject in need thereof starts a process of natural conception.

A subject in need thereof may complete one cycle of fertility treatment or more than one cycle of fertility treatment. The number of cycles of fertility treatment may depend on the needs of the subject in need thereof. In some embodiments, a cycle takes several weeks, requires frequent blood tests, and daily hormone injections, such as in IVF.

In some embodiments, AA-TG is administered to a subject in need thereof for a sufficient time to promote fertility in the subject. In some embodiments, AA-TG is administered to a subject in need thereof for a sufficient time to promote endometrial tissue (e.g., epithelium) regeneration in the subject in need thereof.

In some embodiments, a sufficient time is at least about 3 days; and (a) administration starts before the subject starts a fertility treatment (e.g., one cycle of fertility treatment); (b) administration starts after the subject starts a fertility treatment (e.g., one cycle of fertility treatment); or (c) administration starts at any time during a fertility treatment (e.g., one cycle of fertility treatment). In some embodiments, a sufficient time is at least about 3 days; and (a) administration starts before the subject starts a process of natural conception; (b) administration starts after the subject starts a process of natural conception; or (c) administration starts at any time during a process of natural conception.

In some embodiments, a sufficient time is at least about 5 days; and (a) administration starts before the subject starts a fertility treatment or a process of natural conception; (b) administration starts after the subject starts a fertility treatment or a process of natural conception; or (c) administration starts at any time during a fertility treatment or a process of natural conception. In some embodiments, a sufficient time is at least about 7 days; and (a) administration starts before the subject starts a fertility treatment or a process of natural conception; (b) administration starts after the subject starts a fertility treatment or a process of natural conception; or (c) administration starts at any time during a fertility treatment or a process of natural conception. In some embodiments, a sufficient time is at least about 14 days; and (a) administration starts before the subject starts a fertility treatment or a process of natural conception; (b) administration starts after the subject starts a fertility treatment or a process of natural conception; or (c) administration starts at any time during a fertility treatment or a process of natural conception. In some embodiments, a sufficient time is at least about 21 days; and (a) administration starts before the subject starts a fertility treatment or a process of natural conception; (b) administration starts after the subject starts a fertility treatment or a process of natural conception; or (c) administration starts at any time during a fertility treatment or a process of natural conception. In some embodiments, a sufficient time is at least about 28 days; and (a) administration starts before the subject starts a fertility treatment or a process of natural conception; (b) administration starts after the subject starts a fertility treatment or a process of natural conception; or (c) administration starts at any time during a fertility treatment or a process of natural conception.

In some embodiments, a sufficient time is about 1 month, at least 1 month, about 2 months, at least 2 months, about 3 months, at least 3 months, about 4 months, at least 4 months, about 5 months, at least 5 months, about 6 months, at least 6 months, about 7 months, at least 7 months, about 8 months, at least 8 months, about 9 months, at least 9 months, about 10 months, at least 10 months, about 11 months, at least 11 months, about 1 year, or at least 1 year; and (a) administration starts before the subject starts a fertility treatment or a process of natural conception; (b) administration starts after the subject starts a fertility treatment or a process of natural conception; or (c) administration starts at any time during a fertility treatment or a process of natural conception.

In some embodiments, AA-TG is administered for one cycle which is or comprises administration of AA-TG for about 1 week or at least 1 week and followed by no administration of AA-TG for about 1 week or at least 1 week; for one cycle which is or comprises administration of AA-TG for about 2 weeks or at least 2 weeks and followed by no administration of AA-TG for about 2 weeks or at least 2 weeks; for one cycle which is or comprises administration of AA-TG for about 3 weeks or at least 3 weeks and followed by no administration of AA-TG for about 3 weeks or at least 3 weeks; for one cycle which is or comprises administration of AA-TG for about 4 weeks or at least 4 weeks and followed by no administration of AA-TG for about 4 weeks or at least 4 weeks. In some embodiments, AA- TG is administered for about 1 week or at least 1 week, about 2 weeks or at least about 2 weeks, about 3 weeks or at least about 3 weeks, about 4 weeks or at least 4 weeks, followed by no administration of AA-TG for about 1 week or at least 1 week, about 2 weeks or at least about 2 weeks, about 3 weeks or at least about 3 weeks, about 4 weeks or at least 4 weeks.

In some embodiments, AA-TG is administered daily to a subject in need thereof. In some embodiments, AA-TG is not administered daily to a subject in need thereof. In some embodiments, AA-TG is administered to a subject in need thereof every other day. In some embodiments, AA-TG is administered at least once per day to a subject in need thereof. In some embodiments, AA-TG is administered every other day to a subject in need thereof. In some embodiments, AA-TG is administered to a subject in need thereof two, three, or four times per day.

In some embodiments, an AA level in a sample from a subject in need thereof is below a pre-determined AA level in the absence of administration of an amount of AA-TG which increases the AA level in the subject in need thereof to or above a pre-determined AA level. In some embodiments, a pre-determined AA level is a two-fold increase in an AA level measured in a sample from the subject relative to an AA level measured in a sample from the subject before administering AA-TG. In some embodiments, a pre-determined AA level is a 3-fold, is a 4-fold, is a 5-fold, is a 6-fold, is a 7-fold, is an 8-fold, is a 9-fold or is a 10 fold, increase in an AA level measured in a sample from the subject relative to an AA level measured in a sample from the subject before administering AA-TG. In some embodiments, a pre-determined AA level is a clinically relevant AA level. In some embodiments, a pre-determined AA level is a clinically relevant plasma AA level or endometrial tissue (e.g., epithelium) AA level. In some embodiments, a pre-determined AA level is an AA level sufficient to promote fertility or endometrial tissue (e.g., epithelium) regeneration. In some embodiments, a pre-determined AA level is the lowest AA level at which a beneficial effect is observed in the subject.

In some embodiments, a pre-determined AA level is a percentage of a total fatty acid measured in a subject (e.g., as human), as shown or as disclosed in Turolo et al. (Int J Mol Sci (2021) 22:5452).

In some embodiments, a beneficial effect is increased expression of a marker of sternness, such as increased expression of a gene associated with sternness, relative to a reference. In some embodiments, a gene associated with sternness is at least one of mesothelin (Msln), carnitine palmitoyltransferase 1A (Cptla), solute carrier family 7 member 3 (Slc7a3), and frizzled class receptor 10 (FzdlO). In some embodiments, a gene associated with sternness is S100 calcium binding protein P (SIOOP) or solute carrier family 7 member 1 (SLC7A1). In some embodiments, a beneficial effect is increased expression of a dedifferentiation marker relative to a reference. In some embodiments, a dedifferentiation marker is at least one of Ctnnbl (encoding catenin beta 1), Krtl9 (encoding keratin 19), and Ascl2 (encoding Achaete-Scute family BHLH transcription factor 2; beta-catenin target gene). In some embodiments, a beneficial effect is increased expression of a gene involved in stem cell plasticity. In some embodiments, a gene involved in stem cell plasticity is at least one of Cd55 (encoding CD55 molecule), Ereg (encoding epiregulin), Myof (encoding myoferlin) and Msln (encoding mesothelin). In some embodiments, a beneficial effect is increased expression of a gene involved in regenerative repair response. In some embodiments, a gene involved in regenerative repair response is at least one of Fzd9 (encoding frizzled class receptor 9), SoxlO (encoding SRY-box transcription factor 10), and Cd36 (encoding CD36 molecule). In some embodiments, a beneficial effect is observed when expression of a gene associated with sternness, of a dedifferentiation marker, of a gene involved in stem cell plasticity, or a gene involved in a regenerative repair response is increased about 10% or at least about 10%; about 25% or at least about 25%; about 50% or at least about 50%; about 75% or at least about 75%; about 100% or at least about 100%; about 125% or at least about 125%; about 150% or at least about 150%; about 175% or at least about 175%; about 200% or at least about 200%; about 300% or at least about 300%; about 400% or at least about 400%, about 500% or at least about 500%; about 600% or at least about 600%; about 700% or at least about 700%, about 800% or at least about 800%; or any ranges or combinations thereof, relative to a reference in a subject in need thereof. In some embodiments, expression of a gene associated with sternness is increased in a cell (e.g., epithelial cell) obtained from the subject.

In some embodiments, a beneficial effect is promoting fertility in a subject in need thereof relative to a reference, as determined by a healthcare provider (e.g., a medical doctor). In some embodiments, the reference is a plasma AA level or endometrial tissue (e.g., epithelium) AA level in the subject in need thereof before administration of AA-TG, or the reference is a pre-determined plasma AA level or a pre-determined endometrial tissue (e.g., epithelium) AA level.

In some embodiments, a healthcare provider may recommend administration, consumption or supplementation of AA-TG if the subject in need thereof: has endometriosis; has hyperplasia; has adenomyosis; has endometrial cancer; is under 35 years old and has been trying to conceive for at least 1 year; is 35 years old or above 35 years old and has been trying to conceive for at least 6 months; is trying to conceive but the subject has irregular, painful or no periods; has had at least 1 miscarriage (e.g., 2 miscarriages, 3 miscarriages, or 4 more miscarriages); (or their partner) has a history of sexually transmitted infection(s); has a chronic medical condition (e.g., diabetes, genetic disorder, heart disease, hypertension, kidney disease, or thyroid condition).

In some embodiments, promoting fertility refers to increased ability of a subject in need thereof to conceive, carry a pregnancy to term, or both conceive and carry a pregnancy to term upon administration of, consumption or supplementation with AA-TG (e.g., at least about 2 g of AA-TG for a sufficient time) relative to the subject in need thereof’ s ability to conceive, carry a pregnancy to term, or both conceive and carry a pregnancy to term prior to administration of, consumption of or supplementation with AA-TG. In some embodiments, a method of promoting fertility refers to increased endometrial receptivity in a subject in need thereof upon administration of, consumption or supplementation with AA-TG (e.g., at least about 2 g of AA-TG for a sufficient time) relative to the subject in need thereof’ s endometrial receptivity prior to administration of, consumption of or supplementation with AA-TG (e.g., at least about 2 g of AA-TG for a sufficient time). In some embodiments, a method of promoting fertility refers to increased endometrial receptivity in a subject in need thereof upon administration of, consumption or supplementation with AA-TG (e.g., at least about 2 g of AA-TG for a sufficient time) relative to a reference.

Blanco-Breindel et al. (Endometrial Receptivity. [Updated 2023 Apr 14]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: ncbi.nlm.nih.gov/books/NBK587449/, the content of which is incorporated by reference in its entirety) defines endometrial receptivity as “the period of endometrial maturation during which the trophectoderm of the blastocyst can attach to the endometrial epithelial cells and subsequently invade the endometrial stroma and vasculature.” In some embodiments, endometrial receptivity is assessed by a healthcare provider (e.g., medical doctor). For instance, methods for assessing endometrial receptivity include, but are not limited to, transvaginal ultrasound imaging (TVUS), histologic evaluation by endometrial biopsy, endometrial receptivity array (ERA) and ReceptivaDx test (BCL6).

TVUS is a widely available tool that can be used to assess endometrial receptivity. TVUS can be used to measure the endometrial thickness, volume, and pattern.

ERA is a molecular diagnostic tool used to identify a receptive endometrium via a specific transcriptomic signature present in both natural and hormone replacement therapy cycles. The technology has been applied clinically to identify a patient- specific window of implantation, which is then used to guide a personalized timing of embryo transfer for patients with recurrent implantation failure. This is done by taking an endometrial biopsy at specific times during the mid-luteal phase (LH surge+7 days in natural cycles, progesterone starts +5 days in hormone replacement/“artificial” cycles. The results of the ERA are then used to guide shifts in the timing of progesterone administration before embryo transfer in a future cycle.

The ReceptivaDx test identifies endometrial receptivity defects associated with progesterone resistance. The BCL6 protein is overexpressed in women with endometriosis and BCL6 protein overexpression is associated with lower clinical pregnancy rates in women undergoing IVF. It is hypothesized that these women could be treated with GnRH agonists or surgery to improve fertility outcomes. A healthcare provider, using, for instance, any of the methods disclosed herein, can determine if a subject in need thereof has impaired endometrial receptivity and administration of AA-TG (e.g., at least about 2 g of AA-TG for a sufficient time) can be used to enhance endometrial receptivity.

In some embodiments, a beneficial effect is assessed in a sample obtained from a subject in need thereof. In some embodiments, a sample is a cell (e.g., epithelial cell, etc.), blood, a component of blood (e.g., serum, plasma) obtained from the subject in need thereof. In some embodiments, a beneficial effect is increased AA level in a subject in need thereof relative to a reference. In some embodiments, a beneficial effect is increased AA level in a subject in need thereof of about 2-fold or at least 2-fold about relative to a reference. In some embodiments, a beneficial effect is increased AA level in a subject in need thereof of at least or about 1.5-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold; at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 11-fold, at least or about 12- fold, at least or about 13-fold, at least or about 14-fold, or at least or about 15-fold, or any range or combination thereof, relative to a reference. In some embodiments, a beneficial effect is increased AA level in a subject in need thereof of about 3-fold to about 15-fold relative to a reference. In some embodiments, a beneficial effect is increased AA level in a subject in need thereof of about 3-fold to about 10-fold relative to a reference. In some embodiments, a beneficial effect is increased AA level in a subject in need thereof of about 1.5-fold to about 3-fold relative to a reference.

In some embodiments, a reference is an AA level in a subject before starting a fertility treatment (e.g., a cycle of fertility treatment) or a process of natural conception. In some embodiments, a reference is a level of a population of AA in a sample in a subject before starting a fertility treatment (e.g., a cycle of fertility treatment) or before starting a process of natural conception. In some embodiments, reference is an AA level in a subject before administration to the subject of AA-TG. In some embodiments, reference is an AA level in a subject who has not started a fertility treatment (e.g., a cycle of fertility treatment). In some embodiments, a reference is an AA level in a subject having the same condition as the subject who is to start a fertility treatment, but the subject having the same condition does not undergo a fertility treatment. In some embodiments, a condition is primary infertility (i.e., inability to conceive). In some embodiments, a condition is secondary infertility (i.e., inability to conceive after a first conception). In some embodiments, a condition is a condition of the endometrium. In some embodiments, reference is an A A level in a cell obtained from the subject before starting a fertility treatment (e.g., a cycle of fertility treatment). In some embodiments, AA-TG is administered orally to a subject in need thereof. In some embodiments, AA-TG is administered via a non-parenteral route. In some embodiments, the non-parenteral route is rectal, vaginal, sublingual, aerosolized, buccal or intranasal. In some embodiments, AA-TG is administered as a suppository. In some embodiments, AA-TG is administered intravaginally. In some embodiments, AA-TG is in a composition, wherein the composition is in the form of a liquid or a powder. In some embodiments, the composition is in the form of pills, capsules, hydro-gels, vaginal tablets, pessaries/suppositories, particulate systems, or in intra-vaginal rings. In some embodiments, AA, as a free fatty acid bound to a carrier protein (e.g., albumin; serum albumin), is administered.

Assessing the effects of administration of AA-TG can be carried out by comparing the extent of fertility, endometrial tissue (e.g., epithelium) regeneration, or both after administration of AA-TG with the extent of fertility, endometrial tissue (e.g., epithelium) regeneration, or both in the subject in need thereof prior to administration of AA-TG.

Subject

In some embodiments, a subject is a vertebrate. In some embodiments, a subject is a rodent. In some embodiments, a subject is a mouse. In some embodiments, a subject is a domestic animal (e.g., dog, cat, hamster, etc.). In some embodiments, a subject is a mammal. In some embodiments, a subject is a primate. In some embodiments, a subject is livestock (e.g., cow, bull, sheep, goat, pig, or horse). In some embodiments, a subject is a human.

In some embodiments, a subject is a subject in need thereof. In some embodiments, a subject in need thereof is a vertebrate. In some embodiments, a subject in need thereof is a rodent. In some embodiments, a subject in need thereof is a mouse. In some embodiments, a subject in need thereof is a domestic animal (e.g., dog, cat, hamster, etc.). In some embodiments, a subject in need thereof is a mammal. In some embodiments, a subject in need thereof is a primate. In some embodiments, a subject in need thereof is livestock (e.g., cow, bull, sheep, goat, pig, or horse). In some embodiments, a subject in need thereof is a human.

In some embodiments, a subject in need thereof is a subject before the subject starts a fertility treatment (e.g., a cycle of fertility treatment). In some embodiments, a subject in need thereof is a subject who has started a fertility treatment (e.g., a cycle of fertility treatment) or a process of natural conception. In some embodiments, a subject in need thereof is administered AA-TG before a fertility treatment (e.g., a cycle of fertility treatment) or a process of natural conception. In some embodiments, a subject in need thereof is administered AA-TG during a fertility treatment (e.g., a cycle of fertility treatment) or a process of natural conception. In some embodiments, a subject in need thereof is administered AA-TG before and during a fertility treatment (e.g., a cycle of fertility treatment) or a process of natural conception.

In some embodiments, a subject in need thereof is a subject who has been exposed to a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, a subject in need thereof is a subject having cancer that will be exposed to or treated with a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, a subject in need thereof is administered AA-TG before a course of chemotherapy, radiation therapy or chemotherapy and radiation therapy. In some embodiments, a subject in need thereof is administered AA-TG during a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, a subject in need thereof is administered AA-TG before and during a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

In some embodiments, a subject in need thereof is a subject who has not been exposed to a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, a subject in need thereof does not have cancer. In some embodiments, a subject in need thereof is not administered AA-TG before a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, a subject in need thereof is not administered AA-TG during a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy. In some embodiments, a subject in need thereof is not administered AA-TG before and during a course of chemotherapy, radiation therapy, or chemotherapy and radiation therapy.

In some embodiments, the method comprises increasing, in a subject, a plasma AA level to that indicative of an endometrial tissue AA level that promotes fertility.

In some embodiments, the method comprises (a) measuring an AA level in a sample from a subject in need thereof and determining if the AA level is below a pre-determined AA level to promote fertility; and (b) if the AA level is below the pre-determined AA level, administering to the subject in (a) at least about 2 g of AA-TG per day (2 g/d) for a sufficient time to increase the AA level to or above the pre-determined AA level.

In some embodiments, the method further comprises (c) measuring the AA level resulting from administering AA-TG in (b) and determining the AA level; and (d) if the AA level in (b) is not at or above the pre-determined AA level, further administering to the subject a sufficient amount of AA-TG per day to increase the AA level to or above the predetermined AA level.

In some embodiments, the method further comprises repeating (c)-(d) to produce in the subject an endometrial tissue (e.g., epithelium) AA level to or above the pre-determined A A level.

In some embodiments, measuring an AA level comprises collecting a sample from a subject in need thereof and measuring an AA level in the sample. In some embodiments, the sample is blood. In some embodiments, the sample is serum. In some embodiments, the sample is plasma. In some embodiments, the sample is or comprises tissue. In some embodiments, the tissue is endometrium or endometrial epithelium. In some embodiments, an AA in the AA level is a free AA fatty acid. In some embodiments, the AA fatty acid is associated with a carrier protein (e.g., albumin; serum albumin). In some embodiments, an AA in the AA level is an AA-PL.

In some embodiments, the AA in a sample obtained from a subject is measured by detection of AA in the sample. In some embodiments, methods for measuring AA levels in a sample obtained from a subject include, but are not limited to, mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry (LC-MS), gas chromatography, gas chromatography-mass spectrometry (GC-MS), thin-layer chromatography, size-exclusion chromatography, enzyme-linked immunosorbent assays (ELISA), nuclear magnetic resonance (NMR).

Endometrial Tissue Regeneration

In some embodiments, disclosed are methods of promoting endometrial tissue (e.g., epithelium) regeneration comprising administering to a subject in need thereof, to promote tissue regeneration in the subject, AA-TG which increases the level of AA at least 2-fold in the subject relative to a reference. In some embodiments, administration of AA-TG about 3- fold or at least about 3-fold, about 4-fold or at least about 4-fold, about 5-fold or at least about 5-fold, about 6-fold or at least about 6-fold, about 7-fold or at least about 7-fold, about 8-fold or at least about 8-fold, about 9-fold or at least about 9-fold, about 10-fold or at least about 10-fold, about 11-fold or at least about 11-fold, about 12-fold or at least about 12-fold, about 13-fold or at least about 13-fold, about 14-fold or at least about 14-fold, about 15-fold or at least about 15-fold, or any ranges or combinations thereof, relative to a reference.

In some embodiments, promoting endometrial tissue (e.g., epithelium) regeneration comprises measuring endometrial tissue (e.g., epithelium) thickness, administering AA-TG (e.g., an amount for a sufficient time), and observing increased endometrial tissue (e.g., epithelium) thickness. In some embodiments, endometrial tissue regeneration comprises at least about 200% or about 200%, at least about 100% or about 100%, at least about 95% or about 95%, at least about 90% or about 90%, at least about 80% or about 80%, at least about 70% or about 70%, at least about 60% or about 60%, at least about 50% or about 50%, at least about 40% or about 40%, or at least about 30% or about 30%, at least about 20% or about 20%, at least about 10% or about 10% increase in endometrial tissue (e.g., epithelium) thickness relative to a reference.

In some embodiments, a reference is an endometrial tissue (e.g., epithelium) in a subject who has not been administered AA-TG (e.g., at least about 2 g AA-TG per day for a sufficient time). In some embodiments, a reference is an endometrial tissue (e.g., epithelium) thickness in a subject who has not been administered AA-TG (e.g., at least about 2 g AA-TG per day for a sufficient time). In some embodiments, a reference is an endometrial tissue (e.g., epithelium) before administration of AA-TG (e.g., at least about 2 g AA-TG per day for a sufficient time). In some embodiments, a reference is dysfunctional endometrial tissue (e.g., epithelium) as determined by a healthcare provider (e.g., medical doctor). In some embodiments, the endometrial tissue (e.g., epithelium) has a thickness of or below 6 mm. For instance, a healthcare provider may determine that an endometrial tissue (e.g., epithelium) with thickness of 6 mm, 7 mm, or 8 mm, or below is associated with a negative pregnancy outcome, and administration of AA-TG (e.g., at least about 2 g AA-TG per day for a sufficient time) will increase endometrial tissue (e.g., epithelium) thickness, such as above 8 (e.g., at or above 8.5 mm), which will be associated with a positive pregnancy outcome, and improved fertility. (See e.g., Zhang et al. Medicine (Baltimore) 2018. 97(4): e9689).

In some embodiments, endometrial tissue (epithelium) regeneration comprises increased expression of a marker of sternness, such as increased expression of a gene associated with sternness, of a dedifferentiation marker, of a gene involved in stem cell plasticity, or a gene involved in a regenerative repair response, relative to a reference. In some embodiments, a gene associated with sternness is at least one of mesothelin (Msln), carnitine palmitoyltransferase 1A (Cptla), solute carrier family 7 member 3 (Slc7a3), and frizzled class receptor 10 (FzdlO). In some embodiments, a gene associated with sternness is SI 00 calcium binding protein P (SI OOP) or solute carrier family 7 member 1 (SLC7A1). In some embodiments, a dedifferentiation marker is at least one of Ctnnbl (encoding catenin beta 1), Krtl9 (encoding keratin 19), and Ascl2 (encoding Achaete-Scute family BHLH transcription factor 2; beta-catenin target gene). In some embodiments, a gene involved in stem cell plasticity is at least one of Cd55 (encoding CD55 molecule), Ereg (encoding epiregulin), My of (encoding myoferlin) and Msln (encoding mesothelin). In some embodiments, a gene involved in regenerative repair response is at least one of Fzd9 (encoding frizzled class receptor 9), SoxlO (encoding SRY-box transcription factor 10), and Cd36 (encoding CD36 molecule).

In some embodiments, expression of a gene associated with sternness, of a dedifferentiation marker, of a gene involved in stem cell plasticity, or a gene involved in a regenerative repair response is increased at least about 75% or about 75%; at least about 100% or about 100%; at least about 125% or about 125%; at least about 150% or about 150%; at least about 175% or about 175%; at least about 200% or about 200%; at least about 300% or about 300%; at least about 400% or about 400%, at least about 500% or about 500%, at least about 600% or about 600%; at least about 700% or about 700%, at least about 800% or about 800%, or any ranges or combinations thereof, relative to a reference.

In some embodiments, a reference is a level of expression of a gene associated with sternness in an endometrial tissue (e.g., epithelium) in a subject in need thereof without administration of AA-TG. In some embodiments, a reference is a level of expression of a gene associated with sternness in an endometrial tissue (e.g., epithelium) in a subject in need thereof before administration of AA-TG.

In some embodiments, the present disclosure relates to a method of promoting regeneration in an epithelial cell (e.g., isolated from the endometrium) comprising contacting the epithelial cell with AA-TG that increases the AA level at least 2-fold relative to a reference inside the epithelial cell or milieu surrounding the epithelial cell. In some embodiments, the epithelial cell is isolated from an endometrial tissue (e.g., epithelium) from a subject in need thereof. In some embodiments, the epithelial cell is or comprises a cultured epithelial cell. In some embodiments, the epithelial cell is or comprises a constituent of an organoid. In some embodiments, the epithelial cell is or comprises a human epithelial cell. In some embodiments, the epithelial cell is or comprises an animal epithelial cell. In some embodiments, the epithelial cell is or comprises a mammalian epithelial cell. In some embodiments, the epithelial cell is or comprises part of a tissue. In some embodiments, the tissue is or comprises epithelial tissue. In some embodiments, the tissue is or comprises endometrial tissue. In some embodiments, the epithelial cell is or comprises an epithelial cell of a living multicellular organism. In some embodiments, the epithelial cell is or comprises an epithelial cell obtained from a subject. In some embodiments, the AA level is at least or about 75%, at least or about 100%, at least or about 125%, at least or about 150%, at least or about 175%, at least or about 200%, at least or about 300%, at least or about 400%, at least or about 500%, at least or about 600%, at least or about 700%, at least or about 800%, or any ranges or combinations thereof, relative to a reference. In some embodiments, the AA level is measured inside the epithelial cell. In some embodiments, the AA level is assessed in the milieu surrounding the epithelial cell.

In some embodiments, the AA level is increased 1.5-fold, about 1.5-fold, or at least about 1.5-fold; 2-fold, about 2-fold or at least about 2-fold; 3-fold, about 3-fold, or at least about 3-fold; 4-fold, about 4-fold, or at least about 4-fold; 5-fold, about 5-fold, or at least about 5-fold; 6-fold, about 6-fold, or at least about 6-fold; 7-fold, about 7-fold, or at least about 7-fold; 8-fold, about 8-fold, or at least about 8-fold; 9-fold, about 9-fold, or at least about 9-fold; 10-fold, about 10-fold, or at least about 10-fold; 11 -fold, about 11 -fold, or at least about 11-fold; 12-fold, about 12-fold, or at least about 12-fold; 13-fold, about 13-fold, or at least about 13-fold; 14-fold, about 14-fold, or at least about 14-fold; 15-fold, about 15- fold, or at least about 15-fold in a cell (e.g., epithelial cell isolated from the endometrium) relative to a reference. In some embodiments, reference is the AA level in the cell (e.g., epithelial cell isolated from the endometrium) or an identical cell (e.g., epithelial cell isolated from the endometrium) before administration of an AA-TG. In some embodiments, a reference is the AA level in a control sample of cells.

Kits

In some embodiments, kits for use in preventing, reducing or reversing adverse side effects due to chemotherapy or radiation therapy in a subject are disclosed. In some embodiments, (a) one or more supplement units sufficient to provide to a subject in need thereof at least about 2 g of AA-TG per day (2 g/d) for at least 7 days; and (b) instructions for preparation and consumption of the one or more supplement units. In some embodiments, the number of supplement units to administer to a subject in need thereof is determined in consultation with a healthcare provider.

In some embodiments, the kit can include a preparation vial, a preparation diluent vial, AA-TG and additional agent(s). The diluent vial contains a diluent such as an edible composition for diluting what could be a solution or powder (such as a concentrated solution or lyophilized powder) of AA-TG. In some embodiments, the edible composition is a fruit or vegetable puree. In some embodiments the edible composition is a nutritional shake or the like. In some embodiments, the instructions comprise mixing a particular amount of the diluent with a particular amount of the concentrated solution or lyophilized powder, whereby a final formulation for dosing is prepared. In some embodiments, the instructions comprise use in a syringe or other administration device. In some embodiments, the instructions comprise treating a patient with an effective amount of AA-TG and an optional additional agent or agents. It also will be understood that the containers containing the preparations, whether the container is a bottle, a vial with a septum, an ampoule with a septum, sealed bottles of edible liquid, and the like, can contain indicia such as conventional markings which change color when the preparation has been autoclaved or otherwise sterilized.

In some embodiments, the kit is provided or sold as a bundled service with guidance, instructions, or recommendations from a healthcare provider for consuming one or more supplement units. In some embodiments, a healthcare provider or advisor is a physician, a nutritionist, a registered dietician, a physician’s assistant, a nurse practitioner, or a nurse. In some embodiments, the healthcare provider is an oncologist or a surgeon. In some embodiments, the guidance, instructions, or recommendations comprise oral communications with a healthcare provider. In some embodiments, the guidance, instructions, or recommendations comprise written directions.

In some embodiments, kits comprising one or more supplement units and one or more food compositions are disclosed herein. In some embodiments, the kit further comprises instructions for consuming the AA-TG and the one or more food compositions. In some embodiments, the supplement units of AA-TG are packaged separately from the one or more food compositions. In some embodiments, the AA-TG are pre-mixed with the one or more food compositions in the one or more supplement units.

Supplement Unit

In some embodiments, a supplement unit comprising AA-TG for administering to a subject in need thereof is disclosed. In some embodiments, a supplement unit comprises AA- TG. In some embodiments, one supplement unit is administered per day. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 supplement units are administered per day to provide a subject in need thereof at least about 2 g of AA-TG per day (2 g/d). In some embodiments, a supplement unit comprises an oil comprising AA-TG and a pharmaceutically-acceptable excipient.

In some embodiments, a composition comprising AA-TG and at least one pharmaceutically or veterinary acceptable excipient or vehicle, for promoting fertility in a subject in need thereof for promoting fertility is disclosed. The term “pharmaceutically or veterinary acceptable excipient or vehicle” refers to excipients or vehicles suitable for use thereof in pharmaceutical, veterinary or food technologies for preparing the compositions. These components, excipients or carriers must be compatible with other ingredients of the composition. It must also be suitable for use thereof in contact with the tissue or organ of human beings and animals without excessive toxicity, irritation, allergic response or other immunogenicity problems or complications at a reasonable benefit/risk ratio. They are substances lacking pharmacological activity at the concentrations present in a pharmaceutical form. The excipients or vehicles are used to provide the pharmaceutical or veterinary form characteristics which assure the stability, bioavailability, acceptability and ease of administration of one or more active ingredients. As regards the extent to which the excipients affect active ingredient release, they will be able to modify the magnitude and the time profile of the pharmacological activity of the drug product, by means of changes in its bioavailability. The excipients are also used to provide the preparation with suitable form or consistency. Examples of types of excipients: solubilizers, disintegrants or disintegrating agents, emulsifiers (emulsifying agents), dyes, flavorings, binders, antioxidants, lubricants, preservatives, thickeners, etc.

In some embodiments, a supplement unit is in the form of a liquid or a powder. In some embodiments, a supplement unit is in the form of a pill or a capsule. In some embodiments, the capsule comprises a soft gelatin or is a softgel capsule. In some embodiments, the capsule allows for modified release of AA-TG. In some embodiments, the capsule allows for timed-release of AA-TG.

In some embodiments, one or more supplement units are in one container (e.g., bottle, package, etc.) or more than one container. In some embodiments, one supplement unit is housed in a plastic pocket of a blister pack. In some embodiments, the blister pack is backed with a paperboard card. In some embodiments, a blister pack includes 10 plastic pockets each comprising one supplement unit. In some embodiments, one blister pack houses enough supplement units to provide a subject in need thereof at least 2 g of AA-TG per day (2 g/d).

In some embodiments, AA is commercially available to one of ordinary skill in the art. Non-limiting examples of include AA from Cargill (cargill.com/food- bev/na/arachidonic-acid), ARASCO™ as an oil from DSM, ARASCO® powder from DSM.

In some embodiments, one or more supplement units is consumed under medical supervision and intended for dietary management of a condition in a subject in need thereof. In some embodiments, one or more supplement units are capable of being the sole source of nourishment for a subject in need thereof. In some embodiments, one or more supplement units are intended to or supplement the general diet of a subject in need thereof.

In some embodiments, a supplement unit is in the form of a syrup, a liquid, a powder, a concentrated powder, a concentrated powder admixed with a liquid, a swallowable form, a dissolvable form, an effervescent, a granulated form, or an oral liquid solution. In some embodiments, a supplement unit is formulated in any convenient form. In some embodiments, a supplement unit is in the form of a beverage, mayonnaise, salad dressing, margarine, low fat spread, dairy product, cheese spread, processed cheese, dairy dessert, flavored milk, cream, fermented milk product, cheese, butter, condensed milk product, ice cream mix, soy product, pasteurized liquid egg, bakery product, confectionary product, confectionary bar, chocolate bar, high fat bar, liquid emulsion, spray-dried powder, freeze- dried powder, ultra-high-temperature (UHT) pudding, pasteurized pudding, gel, jelly, yogurt, or a food with a fat-based or water-containing filling; jellies, candies, including gummy candies, better known as “soft fruit candies.”

In some embodiments, a supplement unit further comprises water, sucrose, maltodextrin, milk protein concentrate, soy oil, canola oil, short chain fructooligosaccarides, soy protein isolate, com syrup, sodium caseinate, and potassium citrate.

In some embodiments, a supplement unit comprises a flavor, such as a natural flavor or an artificial flavor. In some embodiments, a flavor is apple, banana, blueberry, caramel, cherry, chocolate, cinnamon, coffee, cranberry, grape, honey, kiwi, lemon, lime, lemon-lime, mango, mint, orange, peach, pineapple, raspberry, strawberry, tangerine, vanilla, or watermelon.

In some embodiments, a supplement unit comprises additional source or sources of fat, such as an oil which is not an oil comprising a significant amount of AA-TG. In some embodiments, an oil which does not comprise a significant amount of AA-TG is an oil which does not comprise more than 5% AA-TG per total volume of oil. In some embodiments, the source or sources of fat promote energy metabolism. In some embodiments, the supplement unit comprises fat sources comprising one or more of saturated, mono-unsaturated, and polyunsaturated fatty acids in proportions seen in a healthy diet for a subject.

In some embodiments, a supplement unit comprises about 3% or at least about 3% of an oil comprising about 40% AA-TG. In some embodiments, a supplement unit comprises about 5% or at least about 5% of an oil comprising about 40% AA-TG, about 10% or at least about 10% of an oil comprising about 40% AA-TG, about 15% or at least about 15% of an oil comprising about 40% AA-TG, or about 20% or at least about 20% of an oil comprising about 40% AA-TG, or any range or combination thereof. In some embodiments percent AA oil is calculated as a weight/volume percentage. In some embodiments, percent AA oil is calculated as a weight/weight percentage. In some embodiments, percent AA oil is calculated as a volume/volume percentage.

In some embodiments, one or more supplement unit comprises about 5 g or at least about 5 g of an oil comprising about 40% AA-TG. In some embodiments, a supplement unit comprises about 10 g or at least about 10 g of an oil comprising about 40% AA-TG, about 30 g or at least about 30 g of an oil comprising about 40% AA-TG, about 40 g or at least about 40 g of an oil comprising about 40% AA-TG, about 50 g or at least about 50 g of an oil comprising about 40% AA-TG.

In some embodiments, one supplement unit comprises 50 mg of AA-TG, 100 mg of AA-TG, 200 mg of AA-TG, 300 mg of AA-TG, 400 mg of AA-TG, 500 mg of AA-TG, 1 g of AA-TG, 2 g of AA-TG, 4 g of AA-TG, 5 g of AA-TG, 10 g of AA-TG, 15 g of AA-TG, 20 g of AA-TG, or any ranges or combinations thereof. In some embodiments, one supplement unit comprises no more than 50 mg of AA-TG, 100 mg of AA-TG, 200 mg of AA-TG, 300 mg of AA-TG, 400 mg of AA-TG, 500 mg of AA-TG, 1 g of AA-TG, 2 g of AA-TG, 4 g of AA-TG, 5 g of AA-TG, 10 g of AA-TG, 15 g of AA-TG, 20 g of AA-TG. In some embodiments, one supplement unit comprises at least 50 mg of AA-TG, 100 mg of AA- TG, 200 mg of AA-TG, 300 mg of AA-TG, 400 mg of AA-TG, 500 mg of AA-TG, 1 g of AA-TG, 2 g of AA-TG, 4 g of AA-TG, 5 g of AA-TG, 10 g of AA-TG, 15 g of AA-TG, 20 g of AA-TG.

EXAMPLES

Example 1: Fatty acid screens revealed omega-6 fatty acids as promoters of endometrial stem cell activity.

Endometrial organoids are a 3D cell culture model that mimics the architecture and physiology of the endometrial epithelium thereby providing an effective system to investigate ESCs²¹'²³. To uncover how fatty acids alter stem cell fate and function in the endometrial epithelium, a live-image screening platform was utilized to observe variations in organoid morphology indicative of stem cell function (size) when treated with a diverse consortium of fatty acids. A panel of 20 FAs delineated by saturation (polyunsaturated, monounsaturated, and saturated), double bond position (omega-3, omega-6, omega-7, and omega-9), configuration (cis, trans), chain length (short, medium, long) and number of double bonds (1- 6). Since most FAs in the body are bound to serum albumin to enhance transport and solubility, poorly soluble FAs were conjugated to bovine serum albumin (BSA). The development of endometrial organoid models and fatty acid screen is summarized in (FIG. 1A).

The fatty acid screen on endometrial organoids revealed that treatment with co-6 fatty acids such as AA, dihomo-gamma-linoleic acid, and gamma-linoleic acid promoted organoid growth (FIGs. IB- ID and FIG. 6). Because omega-6 FAs that exhibited this effect give rise to AA through FA elongases (ElovlS) and desaturases (Fadsl and Fads2 AA was examined²⁴. Replating AA-treated primary organoids in secondary sub-culture experiments resulted in an increased organoid formation rate and larger size compared to vehicle-treated controls, which indicates enhanced stem cell activity¹⁵. However, it remained unclear how dietary perturbations in fatty acid intake translated with fatty acid abundance in the endometrium. To evaluate the effects of AA on stem cell function in vivo, an isocaloric (3.8 kcal/g) AA-rich diet (ARD) model (Teklad, TD 190641) was developed with a matched purified control diet (Teklad, TD97184). Oil extracted from the fungus M. alpina, which contains approximately 40% AA in the form of triglycerides, was utilized to formulate a 3% AA-rich oil and 4% soybean oil containing diet (7% total fat)²⁵. The ARD and its matched isocaloric control are composed of equal amounts of major nutrients (protein, carbohydrate, and fat) and minor nutrients (minerals and vitamins) (FIG. IE). Organoids were created by isolating and culturing endometrial epithelial crypt structures. Targeted metabolomics was then conducted to evaluate alterations in the abundance of AA. The ARD model was validated as AA levels increased in organoids derived from ARD treated mice (FIG. IF). Organoids derived from ARD treated mice also exhibited an expanded size and augmented formation rates suggesting enhanced stem cell activity and function (FIGs. 1G-1H). Overall, AA altered endometrial organoid development suggesting altered stem cell plasticity and function.

Example 2: AA promoted stem cell plasticity and reprogramming in the endometrial epithelium.

To understand the molecular underpinnings of the AA-induced morphology in endometrial organoids, bulk RNA sequencing was performed. Differential expression analysis of bulk RNA sequencing revealed that AA treatment alters the transcriptomic landscape by significantly altering the expression of 403 genes (FIG. 7A). Among the genes differentially expressed, the most upregulated genes described an enhanced hallmark stem cell response and epithelial plasticity (S100a6, Msln, FzdlO) and fatty acid metabolism signals (Cptla, Ptges) (FIG. 2A)²⁶'²⁹. Correspondingly, functional gene set enrichment analysis revealed ontologies implicated in proliferation, tissue repair and development to be enriched (FIG. 7B). Ontologies related to a humoral immune response were enhanced, suggesting AA enhances immune surveillance (FIG. 7B).

To evaluate the precise cellular states that characterize AA induced sternness in vitro, single cell RNA sequencing was conducted on AA treated organoids. 9353 cells were profiled post-filtering and were defined using cell types detailed previously^30,31. Distinct ESC-like cell states that are characterized by stem cell markers Sox9, Anxa3, and proliferation marker Ki67 (FIG. 2B and FIGs. 7C-7G) were identified³¹'³⁴. All ESC-like lineages were expanded in response to AA treatment suggesting a greater degree of stem cell plasticity (FIGs. 2C-2D and FIG. 7H). Previous studies attribute enhanced stem cell plasticity and abundance to several cell-mediated functions such as dedifferentiation^35,36. In fact, previous studies have detailed dietary factors to reprogram epithelial cells to promote plasticity in other tissues^15,37. To determine whether cell-mediated processes dictated the AA mediated expansion of stem cell subtypes in endometrial epithelium, a pseudotime trajectory and RNA velocity analysis were conducted on the single cell RNA sequencing dataset. It was found that Sox9+ ESCs differentiate into specialized cell types such as luminal and ciliated cells. Ki67+ proliferative and Anxa3+ ESC states emerge through a dedifferentiation trajectory, as indicated by the emergence of ESC lineages at later pseudotime points, which implies stem cell plasticity (FIGs. 2E-2F and FIG. 71). While stem cell plasticity has been studied in epithelial tissues like the intestine for tissue regeneration after damage, its role in endometrial regeneration remains largely unexplored^38,39. AA leads to significant upregulation putative dedifferentiation markers such as |3-catenin (Clnnbl), Krtl9 and a |3- catenin target gene Ascl2, which was recently found as a master regulator of stem cell plasticity upon injury in the intestinal epithelium through dedifferentiation of committed cells (FIGs. 2G-2I)^40,41. In differentiated lineages, AA also enhanced other genes implicated in stem cell plasticity such across model systems including a fetal-like state Cd55, Ereg, Myof, Mslr) and regenerative repair response (Fzd9, SoxlO, Cd36) highlighting AA’s role in eliciting a stem cell plasticity program resembling a regenerative response^40,42. Overall, these results demonstrated that AA promotes sternness and induces stem cell plasticity gene expression in endometrial organoids. Example 3: AA enhanced stem-cell features through metabolic production ofPGE2 AA functions in homeostatic processes through numerous mechanisms such as the manipulation of membrane fluidity, ion channels/signaling, reactive oxygen species levels, lipid sensing receptors, and the production of several bioactive lipid derivatives that are abundant in response to tissue damage⁴³. The data suggested dietary AA enhances stem cell plasticity and function in a manner that emulates regenerative responses, prompting an assessment as to whether AA derived metabolites were implicated in stem cell enhancing phenotype observed (FIGs. 1A-1H and FIGs. 2A-2I). Thus, a metabolomics assay targeting AA derivatives was conducted to determine whether metabolites contributed to ESC function. Downstream AA derivatives such as hydroxy-fatty acids (12-HETE, 5-HETE, 14, 15-EET) and prostaglandins (PGE2, PGDE2, PGG2) were more abundant in response to AA treatment in murine models (FIG. 3A and FIG. 8A). Previous studies have implicated prostaglandins in coordinating inflammation, hormonal signaling, and tissue repair responses⁴⁴. To ascertain the potential for AA metabolites like prostaglandins to drive the AA-induced phenotype, predictive metabolic signaling using the MEBOcost program was utilized to model metabolic cell-cell communication using single cell RNA-seq data. MEBOcost predicted that AA upregulated PGE2-Ptger4 and Folic acid-Slcl9Al activity in stem cell lineages (FIG. 3B)⁴⁵. Functional gene ontology analysis of PGE2-Ptger4 signaling targets as predicted by MEBOcost are implicated in enhancing stem cell function (FIG. 8B). Target genes of Slcl9al-Folic acid metabolism have concordant ontologies to that of PGE2-Ptger4 (FIG. 8C). As the role of PGE2-Ptger4 is corroborated by the targeted metabolomics assay and has been previously implicated in coordinating repair responses, PGE2 was investigated further⁴⁶. An in vitro screen of AA metabolites revealed PGE2 recapitulated the regenerative phenotype observed in murine organoids (FIGs. 3C-3D). To confirm whether PGE2 enhanced stem-cell function through the same mechanisms as AA, single cell RNA sequencing of PGE2 treated murine endometrial organoids was conducted. Comparing differential expression of AA versus control and PGE2 versus control in all cells revealed analogous differentially expressed genes that are strongly correlated (FIG. 3E). Correspondingly, PGE2 also expanded ESC-like lineages (FIGs. 3F-3G and FIG. 8D) and recapitulated the dedifferentiation phenotype as evidenced by the pseudotime analysis (FIG. 3H and FIGs. 8F and 8H). PGE2 led to the significant upregulation of canonical Wnt/p catenin signaling target genes which have been implicated in coordinating stem cell plasticity and regenerative responses in the endometrial epithelium (FIG. 31 and FIG. 8H). PGE2 robustly induced dedifferentiation signatures (Krtl9 and Ascl2) and stem cell plasticity signatures (Ly6a, and Wnt7a) to a greater degree than AA, suggesting PGE2 promotes stem cell expansion via a dedifferentiation program (FIGs. 3J-3K and FIGs. 8F-8G)^47,48. Overall, AA enhanced endometrial stem cell plasticity through its metabolite PGE2.

Example 4: Ptger4 - cAMP - PKA signaling axis regulates AA-induced sternness and fertility.

To further elucidate the mechanisms by which AA enhances sternness, PGE2-Ptger4 signaling was investigated, as indicated by the MEBOcost analysis (FIG. 3B). PGE2 binds to four G-protein coupled receptors (PTGER1-4) and activates diverse downstream pathways⁴⁹. Out of the PGE2 signal receptors, only PTGER4 was significantly upregulated by AA (FIG. 4A and FIGs. 9A-9C). PTGER4 was also upregulated in Anxa3+ stem and specialized lineages, suggesting its role in the AA-induced phenotype. To validate the necessity of PTGER4 in AA-induced stem cell plasticity, AA and PGE2 supplemented mouse endometrial organoids were treated with Ptger4 inhibitor, Ptger4i. It was found that inhibition dampened the stem cell enhancing phenotype in AA and PGE2-treated endometrial organoids (FIGs. 4B-4C). PGE2 signaling through PTGER4 engages with diverse downstream pathways such as activating phosphatidylinositol 3-kinase (PI3K), |3-arrestin, |3-catenin, extracellular signal regulated kinase (ERK), and adenylyl cyclase for increasing cAMP production and CREB activation⁵⁰. As several targets were differentially upregulated by AA, the pertinence of CREB signaling in AA-mediated plasticity was examined (FIG. 4D). CREB target gene expression was upregulated by AA at later (pseudo) time-points suggesting potential to promote a stem cell program in differentiated lineages (FIGs. 9D-9E). PTGER4-cAMP-PKA axis can activate CREB signaling.⁵¹ To determine whether cAMP-PKA signaling is implicated in AA-induced stem cell plasticity, PKA inhibitor H89+ and CREB inhibitor Crebi led to the loss of the stem cell enhancing phenotype in AA and PGE2-treated endometrial organoids suggesting AA promotes stem cell plasticity through the PGE2- PTGER4-CREB signaling axis (FIG. 4E and FIG. 4H). However, Crebi treatment still allowed PGE2 supplementation to enhance stem cell plasticity in endometrial epithelial organoids, suggesting potential for other mechanisms downstream of PGE2-PTGER4 to contribute to AA-induced stem cell plasticity. As previous studies have found PGE2- PTGER4 to engage in canonical Wnt signaling and its pivotal role in regulating endometrial stem cell function, it was investigated whether canonical Wnt may contribute to AA-induced stem cell plasticity (FIG. 4I)⁵². The necessity of canonical Wnt signaling in AA-induced stem cell plasticity was evaluated using canonical Wnt inhibitor Pri. Co-supplementation of Pri with AA or PGE2 to endometrial epithelial organoid led to the dampening of AA-induced stem cell plasticity, suggesting Canonical Wnt signaling is implicated in AA’s stem cell enhancing phenotype (FIGs. 4J-4K). Correspondingly, components of the PTGER4-CREB signaling axis were positively correlated with gene signatures implicated in epithelial regeneration and stem cell function (FIG. 9F and FIG. 91). Collectively, AA induced the PTGER4-PKA-CREB signaling axis and canonical Wnt signaling to enhance stem cell function (FIG. 4E).

Example 5: Arachidonic acid bolstered stem cell plasticity and fertility outcomes in the human endometrial epithelium

Given the known differences between mouse and human endometrial tissue, the translatability of AA’s effects on stem cell plasticity was assessed. To this end, an organoid model was developed from patient biopsies to simulate the endometrial epithelium. AA treatment not only enhanced organoid size 5-fold (FIG. 5A) but also induced the stem cell plasticity phenotype observed in mouse organoids. To validate the molecular basis underlying the AA-induced phenotype in human organoid models, bulk-RNA sequencing was conducted. Differential expression analysis revealed AA treatment altered the transcriptomic landscape upregulating 103 genes (FIGs. 10A-10B). Among the genes differentially expressed, genes implicated in stem cell function (WNT7A, WNT11), CREB signaling (FOS), and energy metabolism (CPT1A, PDK4) were observed (FIG. 5B)⁵³'⁵⁶. Correspondingly, gene set enrichment analysis confirmed that genes induced in response to AA treatment were consistent with stem cell plasticity and enhanced a regenerative program (FIG. 5C). Further tests with AA and PGE2 in human organoids showed that inhibiting PTGER4 negated the stem cell-enhancing effects, mirroring the findings in mouse models (FIGs. 5D-5E). Taken together, the data suggests that the AA-PGE2-PTGER4 signaling axis is conserved and is critical for stem cell plasticity and regeneration in both the mouse and human endometrium. This suggests that the AA-PGE2- PTGER4 signaling axis is conserved and critical for stem cell function and plasticity in both mice and humans.

Recent studies have highlighted the potential for stem cell-enhancing factors to improve fertility outcomes⁵⁷. The metabolite signaling modeling revealed AA to induce Folic acid-Slcl9al signaling, a mechanism widely implicated in female fertility outcomes (FIGs. 3A-3K)⁵⁸. Moreover, predicted targets of Folic acid-Slcl9al signaling corresponding with those of PGE2-PTGER4 signaling provided evidence for AA’s potential to enhance fertility outcomes (FIGs. 8A-8H). Thus, gene expression signatures implicated in murine and human endometrial receptivity were evaluated. These gene expression signatures were described in Koel et al. 2022 and He et al. 2019, respectively^59,60. In single cell RNA sequencing data, AA upregulated genes induced by a receptive endometrium which strongly correlated with constituents of the PTGER4-CREB axis and regeneration programs (FIGs. 5F-5G, FIG. 10E, and FIG. 10G). Gene set enrichment analysis of human bulk RNA sequencing data further validated that AA upregulates genes induced in a receptive endometrium (FIG. 5H). To further substantiate the translatability of AA’s effect on fertility outcomes, a publicly available untargeted metabolomics dataset of fertile control and infertile endometriosis patients was evaluated (n = 40)⁶¹. Fertility corresponded with distinct metabolomic profiles (FIG. 10E). Functional analysis revealed that fertile patients had a greater abundance of AA derivatives (prostaglandins, unsaturated fatty acids, and hydroxy fatty acids) supporting AA’s potential to enhance fertility outcomes (FIG. 51).

Example 6: Discussion of results

Despite the regenerative capacity of the endometrial epithelium, numerous disease states such as endometriosis, adenomyosis, and endometrial cancer induce endometrial degeneration and impact 10-15% of women of reproductive age and 35-50% of infertile women⁶²'⁶⁴. Degenerative ailments contribute to infertility by dysregulating the menstrual cycle and the development of the endometrial lining thereby impeding implantation⁶⁵. The prevalence of degenerative endometrial ailments in fertility accentuates the significant demand to develop regenerative therapeutics. Regenerative therapeutics can bolster implantation and fertility by thickening the endometrial lining⁶⁶. However, current regenerative therapeutic approaches are limited in their efficacy, safety, standardization, and ethics, urging for the development of novel approaches⁶⁷. Dietary factors have been previously implicated with reprogramming stem cells in other tissues, but it had yet to be elucidated in the context of the endometrial epithelium^15,19,68.

Here, using a fatty acid screening approach on in vitro human and mouse organoid models coupled with multi-omics approaches, causal mechanistic links between dietary AA and stem cell function in the endometrial epithelium were uncovered. Dietary AA supplementation was determined to perpetuate a regenerative program and stem cell plasticity within the endometrial epithelium via a conserved mechanism. Additionally, lipid peroxidation and release of AA at wounds regulate injury detection and tissue repair processes processes 70. Accumulating evidence shows that regeneration can be driven through epithelial cell plasticity⁷¹. As this has not yet been demonstrated in the endometrial epithelium, these data introduce a novel paradigm where differentiated cell subtypes are reprogrammed to exhibit stem- like properties. AA upregulates genes widely implicated in dedifferentiation and regeneration such as ASCL2, KRT19, and Canonical Wnt signaling in epithelial cell subtypes. Induction of a stem- like program in committed lineages alters cell determination giving rise to a distinct stem cell mimicking subtypes characterized by Anxa3. As these reverted stem cells exhibit the greatest upregulation of both a stem-like program and constituents of the PGE2-PTGER4-CREB axis, they likely play a critical role in driving AA- induced regeneration and epithelial plasticity. Previous studies have illustrated the effects of AA derived PGE2 on regeneration and repair responses. Paracrine PGE2-EP signaling has been implicated in tissue repair in numerous tissue types and the induction of stem cellenhancing mechanisms such as Hippo and canonical Wnt signaling¹⁹, but its role in endometrial signaling is not conclusive.

The organoid models indicated that AA derived PGE2 triggers the PTGER4-cAMP- PKA-CREB signaling axis to perpetuate sternness in the endometrial epithelium. cAMP- PKA-CREB signaling has been previously implicated in tissue repair and absolution of inflammatory responses⁷³. Inhibitors of cAMP phosphodiesterase enzymes that enhance cAMP signaling are tested in clinical trials for the treatment of degenerative and inflammatory conditions across diverse tissue types. For example, the phosphodiesterase inhibitor roflumilast is already approved by the FDA in the treatment of chronic obstructive pulmonary lung disease, despite its modest activity⁷⁴. However, tissue specific mechanisms of how the cAMP-PKA-CREB axis promotes regeneration has not yet been clarified. Downstream components of CREB transcriptional activation, CREB response binding element and its homologue P300, are histone acetyltransferases that interact with stem-cell enhancing transcription factors⁷⁵. Here the necessity of canonical Wnt signaling was illustrated in the maintenance of the AA-induced phenotype indicating downstream CREB may induce epithelial regeneration through beta catenin. Whether AA supplementation remodels the endometrial epigenome ought to be evaluated in future studies.

Regenerative therapeutics and stem cell therapy have been investigated for the potential to treat infertility⁷⁶. Degenerative ailments perpetuate infertility by decreasing the depth of the endometrial lining, compromising receptiveness. Regenerative therapeutics may benefit fertility outcomes by mitigating the morphologies attenuated by these ailments⁷⁶. Consistent with the data, it was observed that AA induced conserved regenerative programs in humans that correlated with greater receptivity in the endometrial epithelium. Several regeneration signatures upregulated by AA, such as Wnt7a, were also implicated in endometrial receptivity⁷⁷. Metabolic profiling using MEBOcost predicted PGE2-PTGER4 to induce a similar program to that of Folic acid, a metabolite that has been well established in promoting fertility outcomes⁷⁸. Previous studies suggest that PGE2 plays a substantial role in the ovulatory cascade and enhances luteal functions that facilitate implantation and embryo development⁷⁹. This is concordant with the finding that fertile control patients to have a greater abundance of prostaglandins compared to infertile endometriosis patients. These data emphasize the candidacy for the AA-PGE2-PTGER4-CREB axis as a regenerative therapeutic target that may enhance fertility outcomes.

Example 7: Mice, Diet and. Treatment

Mice were housed in the Cold Spring Harbor Laboratory. The following strains were obtained from the Jackson Laboratory: EP4flox (strain name: 6.129S6(D2)- Ptger4tml.lMatb/BreyJ, stock number: 028102), Lgr5-EGFP-IRES-CreERT2 (strain name: B6.129P2-Lgr5tml(cre/ERT2) Cle/J, stock number 008875). ARD studies were performed by using a diet consisting of 40% AA in the form of triglycerides to formulate a 3%AA-rich oil and 4% soybean oil containing diet (7% total fat) (Envigo) beginning at the age of 8-12 weeks and extending for 2 to 6 months (Tables 1A-1B). Control mice were age- and sex- matched and were fed with isocaloric control diet containing equal amounts of major nutrients and minor nutrients (Envigo). Alleles crossed with Lgr5-CreERT2 (to generate stem cell specific knockout, Lgr5-iKO) mice were excised by administration of tamoxifen suspended in com oil (Sigma) at a concentration of 20 mg/ml and 100 pl per 25g of body weight and administered by intraperitoneal injection every other day for 5 times.

Organoid treatments were performed with following compounds; dmPGE2 (5nM, Cayman), PGD2 (5nM, Sigma), Celexocib (l-45pM, Cayman), 8Bromo-cAMP (20pM, Tocris), Sesamin (20pM, Sigma), H89 (20pM, Tocris), Indomethacin (0-80pM, Sigma), Ptgerl Inh (50pM, R&D), Ptger2 Inh (25 M, R&D), Ptger3 Inh (50pM, R&D), Ptger4 Inh (50pM, R&D), 5-HETE (0.5pM, Cayman), 12-HETE (0.5pM Cayman), 15-HETE (0.5pM, Cayman), 8(9)-EET (0.5pM, Cayman), 11(12)-EET (0.5pM, Cayman), 14(15)-EET (0.5pM, Cayman), LTB4 (0.5pM, Cayman), TXB2 (5pM, Cayman). Wnt3a (10-100ng/ml, Peprotech), Amphiregulin (50ng/ml, R&D), Epiregulin (500ng/ml, R&D). Table 1A

Table IB Example 8: Endometrial epithelial isolation and flow cytometry

Organoid generation from endometrial epithelial tissue was performed as previously reported (Boretto et al., 2017). Briefly, the uterine horns of a mouse in the estrus phase were removed, separated from the ovaries, and stripped of any remaining fat. The uterine horns were then separated from each other before being lateralized and cut into small pieces. These tissue pieces were then incubated in PBS-/-/ EDTA (30 mM) with mild agitation for 60 minutes at 37°C. Epithelial fragments were mechanically dissociated from the tissue before filtration through a 70um mesh. After centrifugation, the resulting cells were resuspended in a mixture containing 70% Matrigel and 30% culture medium and plated as domes. Example 9: Culture media for isolated cells

Isolated uterine epithelial fragments were embedded in a mixture containing 70% Matrigel™ and 30% culture medium. This mixture was plated as domes and given 10 minutes at 37°C to polymerize. Culture medium of a sufficient volume to cover each dome was then added. The culture medium consisted of: Advanced DMEM (Gibco) supplemented with 25% WRN conditioned media from the L-WRN cell line (ATCC), HEPES 1% (Gibco), Glutamax 1% (Gibco), Penicillin/Streptomycin 1% (Homemade), Insulin-Transferrin- Selenium IX (Gibco), Nicotinamide ImM (Sigma Aldrich), EGF 50 ng/ml (PeproTech), FGF10 50ng/mL (Peprotech), Y-27632 lOuM (Tocris), A83-01 500nM (Sigma Aldrich), B27 IX (Gibco), and N2 IX (Gibco). Media refreshment occurred every 5 days, with organoids being maintained at 37 °C in a fully humidified chamber containing 5% CO2.

Organoids were quantified on days 1, 3 and 6 in culture, unless otherwise specified. In secondary experiments, individual primary organoids were mechanically dissociated for up to 15 minutes in TrypLE Express at 37°C, before resuspension in a new 70/30 Matrigel-media mixture with subsequent plating as new domes.

Example 10: BSA Conjugation

Fatty acids (FA) were reconstituted in ethanol. Then, fatty acid solutions were added to 0.01M NaOH to make a 12mM solution and stirred for 30 minutes at 70°C. Then, 10% fatty acid free BSA was added to the solution to have 3mM concentration and stirred for 1 hour at 37°C. BSA-conjugated FAs were filtered through 0.22pm and stored in glass containers at -20°C.

Example 11: Fatty Acid Screening / biotek

10,000 cells were seeded on 48 well plates and incubated for 6 hours at 37°C with 5% CO2 for recovery before proceeding to fatty acid treatment. The fatty acid screening library was composed of 20 different fatty acids, as given in Table 2. After 6 hours of incubation, media were changed to organoid media containing fatty acids at an indicated concentration in Table 2. After 24 hours of treatment, images (16 z-slices at 54.8 pm steps, fixed focal height at 1719 pm above plate carrier) were taken from each well with 6 hours of interval using Cytation7 and BioSpa platforms (Agilent BioTek, Winooski VT) at 37 °C with 5%CO2. Imaging was terminated at 120^th hour. Then, Z-projection was obtained with a focus stacking function. Digital phase contrast was applied, and images were filtered by a 100pm structuring element size. Spheroids were detected by defining low internal signal objects gated by the new metric above <= 0.95 and circularity >0.2 to create a new population. This subpopulation was normalized to the total biology area.

Table 2: Fatty acids that were used in screening in mouse and human organoids.

Example 12: Human study participants and isolation from patient biopsies

Human normal endometrial tissue samples were obtained from patients with informed consent undergoing hysterectomy or tissue biopsy procedures at Long Island Jewish Medical Center or a Northwell Health affiliated fertility clinic. Study protocols were reviewed and approved by the Northwell Health Biorepository.

Tissue biopsy samples were incubated in an RPMI solution containing Img/mL Collagenase (Sigma C9407) for 90 minutes at 37°C on a rocker. Dissociation occurred rapidly without any initial mincing of the tissue. After centrifugation and removal of the collagenase, the resulting endometrial epithelial tissue was further digested using TrypLE. Mechanical trituration via pipette followed after a 15-minute incubation period, until adequate dissociation was observed via brightfield microscopy. After centrifugation, the resulting cells were resuspended in a mixture containing 70% Matrigel and 30% culture medium and plated as domes.

Solid normal tissue samples obtained from patients undergoing hysterectomies were processed in a similar way to the procedure mentioned above. Tissue samples were minced into small fragments before being placed in an RPMI solution containing Img/mL Collagenase (Sigma C9407) for up to 2 hours at 37°C. Rough mechanical action via pipetting or shaking of the tube was performed every 15 minutes to aid in tissue dissociation. After centrifugation and removal of the collagenase, the resulting endometrial epithelial tissue was further digested using TrypLE. Mechanical trituration via pipette followed after a 15-minute incubation period, until adequate dissociation was observed via brightfield microscopy. After centrifugation, the resulting cells were resuspended in a mixture containing 70% Matrigel and 30% culture medium and plated as domes.

Example 13: Human Organoid. Passaging and Maintenance

Isolated cells were then embedded in Matrigel™ (Corning, Cat# 356231) and plated in droplets. The Matrigel™ was allowed to polymerize at 37°C for 10 minutes before the addition of culture medium to each well, enough to fully cover the domes. The culture medium consisted of Advanced DMEM (Life Technologies, Cat# 12634028) supplemented with 15% Rspol conditioned media (Homemade), IX Glutamax (Life Technologies, Cat# 35050061), lOmM HEPES (Thermo Fisher Scientific, Cat#15630080), IX B27 (Life Technologies, Cat# 12587010), IX N2 (Life Technologies, Cat# 17502048), IX Insulin- Transferrin-Selenium (Gibco, Cat# 41400045) 2mM Nicotinamide (Sigma Aldrich, Cat# N0636), 1.25mM N-acetyl cysteine (Sigma Aldrich, Cat# A9165), lOOug/mL Primocin (Invivogen, Cat# ant-pm-1), lOpM SB202190 (Sigma Aldrich, Cat# S7067), lOOng/mL Noggin (Peprotech Cat# 250-38), 2ng/mL FGF-basic (Peprotech, Cat# 100- 18B), lOng/mL FGF10 (Peprotech, Cat# 100-26), InM P-Estradiol (Sigma, Cat# E4389), lOpM Y-27632 (Tocris, Cat# 1254), 50ng/mL EGF (Peprotech, Cat # AF-100-15), and 500nM A83-01 (Sigma Aldrich, Cat # SML0788). Culture media were refreshed every 4-5 days and organoids were passaged roughly every 14 days. Organoids were harvested by removing Matrigel™ using Cell Recovery Solution (Corning, Cat# 354253). Once the Matrigel™ was dissolved, the organoids were spun at 500g for 5 minutes at 4°C and incubated in TryplE Express (ThermoFisher, Cat# 12604039) for 10-15 minutes. Pipetting followed until adequate dissociation was confirmed via brightfield microscopy. Cells were then centrifuged at 500g for 5 minutes at 4°C before seeding again in Matrigel™ as explained above. Organoids were commonly passaged in a 1:2 ratio.

Example 14: Ptger4 KO organoid line generation using lentiviral constructs

For Ptger4 KO organoids, lentiviral particles were produced in HEK293-FT cells using a Puro. Cre empty vector (Addgene plasmid # 17408 [AG 1]) and 2nd generation lentiviral system (pCMV-VSVG (Addgene plasmid #8454), psPAX2 (Addgene plasmid #12260). Virus containing supernatant was concentrated with Retro-X™ (Takarabio) before infection. After 3 days, organoids were selected with Ipg/ml Puromycin over the course of 2 passages. Then, organoid culture was performed as described above.

Example 15: Metabolomics analysis by liquid chromatography coupled to mass spectrometry (LC-MS)

Snap-frozen tissue specimens were cut and weighed into Precellys tubes prefilled with ceramic beads (Bertin Instruments). An exact volume of extraction solution (30% acetonitrile, 50% methanol and 20% water) was added to obtain 40 mg specimen per mL of extraction solution. Tissue samples were lysed using a Precellys 24 homogeniser (Bertin Instruments) and the suspension was incubated at -20°C for 60 minutes. Samples were mixed and incubated for 15 minutes at 4°C in a Thermomixer (Eppendorf, Germany), followed by centrifugation (16,000 g, 15 minutes at 4°C). The supernatant was collected and transferred into autosampler glass vials, which were stored at -80°C until further analysis.

For the analysis of polar metabolites and arachidonic acid derivatives, samples were randomized in order to avoid bias due to machine drift and processed blindly. LC-MS analysis was performed using a Vanquish Horizon UHPLC system coupled to a Q Exactive HF mass spectrometer (both Thermo Fisher Scientific). Sample extracts (5 pL) were injected onto a Sequant ZIC-pHILIC column (150 mm x 2.1 mm, 5 pm) and guard column (20 mm x 2.1 mm, 5 pm) from Merck Millipore kept at 45°C. The mobile phase was composed of 20 mM ammonium carbonate with 0.1% ammonium hydroxide in water (solvent A), and acetonitrile (solvent B). Analytes were eluted at 200 pl/minutc with the previously described gradient (Mackay et al. 2015). The mass spectrometer was operated in full MS and polarity switching mode. The acquired spectra were analyzed using XCalibur Qual Browser and XCalibur Quan Browser software (Thermo Fisher Scientific) by referencing an internal library of compounds. MetaboAnalystR (v5, (Pang et al. 2020)) was used for quality control and normalization (with options QuantileNorm, LogNorm, and MeanCenter). Differential abundance analysis was conducted using FC.Anal.unpaired.

Example 16: Gene sets

The data were analyzed in the context of previously identified gene signatures and target genes list. The gene lists are described in brief: Fetal-spheroid gene signature: 317 differentially upregulated genes in spheroids versus organoids, obtained from Table SI (Mustata et al. 2013); Regeneration-induced gene signature: 315 differentially expressed genes in upper crypt region upon DT exposure compared to resting ISCs at the bottom of the crypt from Supplementary Table 1, (Murata et al. 2020); Fertility-induced gene signature: 316 differentially expressed genes from receptive versus non-receptive mice endometriums (He et al. 2019); Receptivity-induced gene signature: 373 differentially expressed genes from the endometrial epithelium from receptive versus non-receptive patients, obtained from Table SI (Koel et al. 2022).

Example 17: Bulk RNA Sequencing

Total RNA was isolated from 3 days of vehicle and arachidonic acid-treated mouse and human endometrial epithelial organoids using Zymo RNA isolation kit according to manufacturer’s instructions. Starting from a total 250 ng RNA, rRNA depletion protocol was followed according to suggested guidelines from the manufacturer. Strand specific RNA seq libraries were prepared using NebNext Ultra II kit and sequenced on Illumina NextSeq.

Example 18: Bulk RNA sequencing data analysis

Raw outputs were trimmed with trim galore (vO.6.7) and aligned to GRCm38.p6/Gencode annotation (release M24) for mouse datasets and GRCh38.pl4/Genecode annotation for human datasets, and using STAR (v2.7.10a (Dobin et al. 2013)). Aligned counts were quantified using Salmon (vl.5.2 (Patro et al. 2017)). Stringtie was used for transcript assembly (v2.2.1 (Pertea et al. 2015)). Read and alignment quality were analyzed with rseqc (v3.0.1 (Ewels et al. 2016; Wang, Wang, and Li 2012)) and summarized with multiqc (vl.9 (Ewels et al. 2016)). Differential gene expression between control and experimental samples was assessed with DEseq2 (vl.28 (Love, Huber, and Anders 2014)), fitting a model with fixed effects for sequencing batch effect and treatment. Contrast for treatment were extracted and transcripts considered differentially expressed with an absolute fold change greater than log2(l) and adjusted p-value of less than 0.05.

Differential gene expression was assessed independently at each time point. For visualization purposes, heatmap plots were generated with ggplot2 yl.6.2, (Wickham et al. 2017)), and volcano plots with EnhancedVolcano (vl.8.0).

Example 19: Single Cell RNA sequencing

For single cell sequencing of the mouse endometrial organoids, organoids were collected using a cell recovery solution. Organoids were then disassociated with TrypLE into single cell suspension. After dissociation of the organoids, single cells were pelleted, washed, and resuspended in FACS buffer (IX PBS, 10 pM Y-27632, 1% FBS, 0.5 mM EDTA) and passed through a 100pm FlowMi cell strainer (Sigma). DAPI was used for viability assessment. DAPI-negative cells were sorted by Sony SH800S sorter and single cell droplets were immediately prepared on the 10X Chromium according to manufacturer instructions at Cold Spring Harbor Laboratory Single Cell Facility. Single cell libraries were prepared using a 10X Genomics Chromium Controller (cat #120223) and the 10X Genomics Chromium Next GEM Single Cell 3' Gene Expression kit (cat #1000268) according to the manufacturer's instructions. Cell suspensions were adjusted to target a yield of 8,000 cells per sample.

Example 20: Single-cell RNA-Seq data analysis

Single cell datasets for each experiment were assessed for data quality following the guidelines described by (Luecken and Theis 2019; Amezquita et al. 2020). Cells with more than 10% mitochondrial transcripts as well as cells that had fewer than 250 feature counts or expressed fewer than 500 genes were removed. After QC, Seurat (v4.0.3, (Butler et al. 2018)) was used for normalization, graph-based clustering and differential expression analysis. Each dataset was normalized using SCTransform and batch corrected using Harmony (Stuart et al. 2019). Cell cycle scoring and regression was conducted as described by (Stuart et al. 2019). MAGIC imputation was conducted on integrated data to impute missing values and account for technical noise (Van Dijk et al. 2018 Cell). RunPCA was implemented on the integrated datasets to identify the top 50 principle components (PCs) that were used for UMAP analysis and clustering. UMAP was calculated using the runUMAP function. Clustering was conducted by first constructing a nearest neighbor graph using the FindNeighbors function and then implementing the FindClusters function to perform clustering using the Louvain algorithm at a resolution of 1. Clusters were labeled in accordance with endometrial epithelial cellular subtype signatures identified by (Garcia- Alonso et al. 2021). Differential expression analysis was conducted between groups using the FindMarkers function with the MAST method to evaluate differences within the transcriptome (Finak et al. 2015). Wilcoxon ranksum tests to determine if gene expression was significant were conducted using the wilcox.test function in stats (v4.1.0, (R Core Team, 2021)). Metabolite interactome modeling was conducted using the MEBOcost package (Zheng et al. 2022). Functional gene sets scores were calculated by finding the average z-score per cell. Monocle3 (vO.2.3, ((Trapnell et al. 2014); (Qiu et al. 2017); (Cao et al. 2019); (Traag, Waltman, and van Eck 2019); (Levine et al. 2015))) was utilized for the trajectory analysis for both organoid and in vivo datasets.

Example 21: Metabolomics analysis by LC-MS and GC-MS to determine a predetermined AA level.

A pre-determined AA level can be measured in a sample (e.g., endometrial tissue) of a subject (e.g., a human) using LC-MS as follows. Snap-frozen tissue specimens are cut and weighed into Precellys tubes prefilled with ceramic beads (Bertin Instruments). An exact volume of extraction solution (30% acetonitrile, 50% methanol and 20% water) is added to obtain 40 mg specimen per mL of extraction solution. Tissue samples are lysed using a Precellys 24 homogeniser (Bertin Instruments) and the suspension is incubated at -20°C for 60 minutes. Samples are mixed and incubated for 15 minutes at 4°C in a Thermomixer (Eppendorf, Germany), followed by centrifugation (16,000 g, 15 minutes at 4°C). The supernatant is collected and transferred into autosampler glass vials, which are stored at - 80°C until further analysis.

For the analysis of polar metabolites and arachidonic acid derivatives, samples are randomized in order to avoid bias due to machine drift and processed blindly. LC-MS analysis is performed using a Vanquish Horizon UHPLC system coupled to a Q Exactive HF mass spectrometer (both Thermo Fisher Scientific). Sample extracts (5 pL) are injected onto a Sequant ZIC-pHILIC column (150 mm x 2.1 mm, 5 pm) and guard column (20 mm x 2.1 mm, 5 pm) from Merck Millipore kept at 45°C. The mobile phase is composed of 20 mM ammonium carbonate with 0.1% ammonium hydroxide in water (solvent A), and acetonitrile (solvent B). Analytes are eluted at 200 pl/minute with a previously described gradient (Mackay et al. 2015). The mass spectrometer is operated in full MS and polarity switching mode. The acquired spectra are analyzed using XCalibur Qual Browser and XCalibur Quan Browser software (Thermo Fisher Scientific) by referencing an internal library of compounds. MetaboAnalystR (v5, (Pang et al. 2020)) is used for quality control and normalization (with options QuantileNorm, LogNorm, and MeanCenter). Differential abundance analysis is conducted using FC.Anal.unpaired.

Furthermore, a pre-determined AA level can be measured in a sample (e.g., serum, plasma) of a subject (e.g., a human) using gas chromatography-mass spectrometry (GC-MS), as follows (See e.g., Okamura, et al. BMC Nephrology (2021) 22:68). The composition of fatty acids (FAs) in frozen serum samples is measured by GC-MS, Agilent 7890B/5977B (Agilent Technologies, Santa Clara, CA, USA). 25 pl of serum is methylated using a fatty acid (FA) methylation kit (Nacalai Tesque, Kyoto, Japan), and the final product is loaded onto a Varian capillary column (DB-FATWAX UI; Agilent Technologies). The capillary column for FA separation is CP-Sil 88 for FAME (lOOmx an inner diameter of 0.25mmx membrane thickness of 0.20 pm, Agilent Technologies). The temperature in the column is set at 100 °C for 4 min and then increased gradually by 3 °C/min to 240 °C and held there for 7 min. The samples are injected in split mode at a split ratio of 5:1. Each FA methyl ester is detected in the selected ion monitoring mode. All results are normalized to the peak height of the C17:0 internal standard (see e.g., Okamura, et al. Am J Physiol Liver Physiol (2020):ajpgi.00310.2019).

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EQUIVALENTS AND SCOPE

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context dictates otherwise.

In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

All references cited herein, including patents, published patent applications, and nonpatent publications, are incorporated by reference in their entirety.

Claims

CLAIMS What is claimed is:

1. A method of promoting fertility in a subject, comprising: administering orally to a subject in need thereof at least about 2 g of arachidonic acid triglyceride (AA-TG) per day (2 g/d) for a sufficient time to promote fertility in the subject in need thereof.

2. The method of claim 1, wherein the sufficient time is at least about 7 days; and

(a) administration starts no earlier than 28 days before the subject in need thereof starts a fertility treatment;

(b) administration starts no later than 28 days after the subject starts a fertility treatment; or

(c) administration starts at any time during a fertility treatment.

3. The method of claim 2, wherein the sufficient time is at least about 7 days; and

(a) administration starts no earlier than 28 days before the subject in need thereof starts a process of natural conception;

(b) administration starts no later than 28 days after the subject starts a process of natural conception; or

(c) administration starts at any time during a process of natural conception.

4. The method of claim 3, wherein the fertility treatment is an assisted reproductive technique.

5. The method of claim 4, wherein the assisted reproductive technique is in vitro fertilization (IVF), gamete intrafallopian transfer (GIFT), pronuclear stage tubal transfer (PROST), tubal embryo transfer (TET), or zygote intrafallopian transfer (ZIFT).

6. The method of claim 3, wherein the fertility treatment is intrauterine insemination, ovulation stimulation, or intrauterine insemination and ovulation stimulation.

7. The method of any one of claims 1-6, wherein the fertility treatment or the process of natural conception lasts for at least about 3 months.

8. The method of any one of claims 1-6, wherein the fertility treatment or the process of natural conception lasts for at least about 6 months.

9. The method of any one of claims 1-6, wherein the fertility treatment or the process of natural conception lasts for at least about 12 months.

10. The method of any one of claims 1-6, wherein the fertility treatment or the process of natural conception lasts from about 3 months to about 12 months.

11. The method of any one of claims 1-10, wherein the method prevents, reduces, or reverses the incidence of infertility or incidence of miscarriage in the subject in need thereof.

12. The method of any one of claims 1-10, wherein the sufficient time is at least about 14 days.

13. The method of any one of claims 1-10, wherein the sufficient time is at least about 21 days.

14. The method of any one of claims 1-10, wherein the sufficient time is at least about 28 days.

15. The method of any one of claims 1-14, wherein at least about 3 g of AA-TG/day (3 g/d) is administered to the subject.

16. The method of any one of claims 1-14, wherein at least about 20 g of AA-TG/day (20 g/d) is administered to the subject.

17. The method of any one of claims 1-14, wherein at least about 30 g of AA-TG/day (30 g/d) is administered to the subject.

18. The method of any one of claims 1-14, wherein at least about 60 g of AA-TG/day (60 g/d) is administered to the subject.

19. The method of any one of claims 1-14, wherein at least about 90 g of AA-TG/day (90 g/d) is administered to the subject.

20. The method of any one of claims 1-14, wherein at least about 100 g of AA-TG/day (100 g/d) is administered to the subject.

21. The method of any one of claims 1-20, wherein from about 2 g of AA-TG/day (2 g/d) to about 100 g of AA-TG/day (100 g/d) is administered to the subject.

22. The method of any one of claims 1-21, wherein the AA-TG is in a composition.

23. The method of claim 22, wherein the composition comprises at least about 2% AA- TG by weight.

24. The method of claim 22 or claim 23, wherein the composition comprises between about 20% AA-TG and about 50% AA-TG by weight.

25. The method of claim 22 or claim 23, wherein the composition comprises about 40% AA-TG by weight.

26. The method of any one of claims 22-25, wherein the composition comprises no more than 5% arachidonic acid (AA) ester by weight.

27. The method of any one of claims 22-26, wherein the composition is an oil.

28. The method of claim 27, wherein the oil is extracted from a fungus.

29. The method of claim 28, wherein the fungus is Mortierella alpina.

30. The method of any one of claims 22-29, wherein the composition is a liquid or a powder.

31. The method of any one of claims 27-30, wherein the composition is in a food, in a capsule or in a pill.

32. The method of any one of claims 1-31, wherein the AA-TG increases an endometrial tissue AA level in the subject that produces a beneficial effect.

33. The method of any one of claims 1-32, wherein administration of AA-TG increases a plasma AA level or increases an endometrial tissue AA level in the subject in need thereof by at least 2-fold relative to a reference.

34. The method of claim 33, wherein the reference is a plasma AA level or endometrial tissue AA level in the subject in need thereof before administration of AA-TG, or the reference is a pre-determined plasma AA level or a pre-determined endometrial tissue AA level.

35. The method of any one of claims 1-34, wherein the subject in need thereof is a mammal.

36. The method of any one of claims 1-34, wherein the subject in need thereof is a human.

37. The method of any one of claims 1-34, wherein the subject in need thereof is livestock.

38. The method of any one of claims 1-34, wherein the subject in need thereof is a mouse.

39. A method of promoting endometrial tissue regeneration in a subject, comprising: administering orally to a subject in need thereof at least about 2 g of arachidonic acid triglyceride (AA-TG) per day (2 g/d) for a sufficient time to promote endometrial tissue regeneration in the subject in need thereof.

40. The method of claim 39, wherein the sufficient time is at least about 7 days; and

(b) administration starts no later than 28 days after the subject starts a fertility treatment; or (c) administration starts at any time during a fertility treatment.

41. The method of claim 39, wherein the sufficient time is at least about 7 days; and

(c) administration starts at any time during a process of natural conception.

42. The method of claim 41, wherein the fertility treatment is an assisted reproductive technique.

43. The method of claim 42, wherein the assisted reproductive technique is in vitro fertilization (IVF), gamete intrafallopian transfer (GIFT), pronuclear stage tubal transfer (PROST), tubal embryo transfer (TET), or zygote intrafallopian transfer (ZIFT).

44. The method of claim 41, wherein the fertility treatment is intrauterine insemination, ovulation stimulation, or intrauterine insemination and ovulation stimulation.

45. The method of any one of claims 39-44, wherein the fertility treatment or the process of natural conception lasts for at least about 3 months.

46. The method of any one of claims 39-44, wherein the fertility treatment or the process of natural conception lasts for at least about 6 months.

47. The method of any one of claims 39-44, wherein the fertility treatment or the process of natural conception lasts for at least about 12 months.

48. The method of any one of claims 39-44, wherein the fertility treatment or the process of natural conception lasts from about 3 months to about 12 months.

49. The method of any one of claims 39-48, wherein the method prevents, reduces, or reverses the incidence of infertility or incidence of miscarriage in the subject in need thereof.

50. The method of any one of claims 39-48, wherein the sufficient time is at least about 14 days.

51. The method of any one of claims 39-48, wherein the sufficient time is at least about 21 days.

52. The method of any one of claims 39-48, wherein the sufficient time is at least about 28 days.

53. The method of any one of claims 39-52, wherein at least about 3 g of AA-TG/day (3 g/d) is administered to the subject.

54. The method of any one of claims 39-52, wherein at least about 20 g of AA-TG/day (20 g/d) is administered to the subject.

55. The method of any one of claims 39-52, wherein at least about 30 g of AA-TG/day (30 g/d) is administered to the subject.

56. The method of any one of claims 39-52, wherein at least about 60 g of AA-TG/day (60 g/d) is administered to the subject.

57. The method of any one of claims 39-52, wherein at least about 90 g of AA-TG/day (90 g/d) is administered to the subject.

58. The method of any one of claims 39-52, wherein at least about 100 g of AA-TG/day (100 g/d) is administered to the subject.

59. The method of any one of claims 39-58, wherein from about 2 g of AA-TG/day (2 g/d) to about 100 g of AA-TG/day (100 g/d) is administered to the subject.

60. The method of any one of claims 39-59, wherein the AA-TG is in a composition.

61. The method of claim 60, wherein the composition comprises at least about 2% AA- TG by weight.

62. The method of claim 60 or claim 61, wherein the composition comprises between about 20% AA-TG and about 50% AA-TG by weight.

63. The method of claim 60 or claim 61, wherein the composition comprises about 40% AA-TG by weight.

64. The method of any one of claims 60-63, wherein the composition comprises no more than 5% arachidonic acid (AA) ester by weight.

65. The method of any one of claims 60-64, wherein the composition is an oil.

66. The method of claim 65, wherein the oil is extracted from a fungus.

67. The method of claim 66, wherein the fungus is Mortierella alpina.

68. The method of any one of claims 60-67, wherein the composition is a liquid or a powder.

69. The method of any one of claims 65-68, wherein the composition is in a food, in a capsule or in a pill.

70. The method of any one of claims 39-69, wherein the AA-TG increases an endometrial tissue AA level in the subject that produces a beneficial effect.

71. The method of any one of claims 39-70, wherein administration of AA-TG increases a plasma AA level or increases an endometrial tissue AA level in the subject in need thereof by at least 2-fold relative to a reference.

72. The method of claim 71, wherein the reference is a plasma AA level or endometrial tissue AA level in the subject in need thereof before administration of AA-TG, or the reference is a pre-determined plasma AA level or a pre-determined endometrial tissue AA level.

73. The method of any one of claims 39-72, wherein the subject in need thereof is a mammal.

74. The method of any one of claims 39-72, wherein the subject in need thereof is a human.

75. The method of any one of claims 39-72, wherein the subject in need thereof is livestock.

76. The method of any one of claims 39-72, wherein the subject in need thereof is a mouse.

77. A method of increasing an arachidonic acid (AA) level in a subject indicative of an endometrial tissue AA level that promotes fertility, comprising:

(a) measuring an AA level in a sample from a subject in need thereof and determining if the AA level is below a pre-determined A A level sufficient to promote fertility; and

(b) if the AA level is below the pre-determined AA level, administering to the subject in need thereof in (a) at least about 2 g of AA-TG per day (2 g/d) for a sufficient time to increase the AA level to or above the pre-determined AA level.

78. The method of claim 77, further comprising:

(c) measuring the AA level resulting from administering AA-TG in (b) and determining the A A level; and

(d) if the AA level in (b) is not at or above the pre-determined AA level, further administering to the subject in need thereof a sufficient amount of AA-TG per day to result in an endometrial tissue AA level at or above the pre-determined AA level.

79. The method of claim 78, further comprising repeating (c)-(d) to produce in the subject in need thereof an endometrial tissue AA level at or above the pre-determined AA level.

80. The method of any one of claims 77-79, wherein the sample is plasma.

81. A kit for use in promoting fertility in a subject, comprising: (a) one or more supplement units sufficient to provide to a subject in need thereof at least about 2 g of arachidonic acid triglyceride (AA-TG) per day (2 g/d) for at least 7 days to promote fertility in the subject in need thereof; and

(b) instructions for preparation and consumption of the one or more supplement units.

82. The kit of claim 81, wherein the one or more supplement units each comprise 500 mg of AA-TG, 1 g of AA-TG, 2 g of AA-TG, or 4 g of AA-TG.

83. The kit of claim 81 or claim 82, wherein the number of supplement units to administer to a subject in need thereof is determined in consultation with a healthcare provider.

84. The kit of claim 81 or claim 82, wherein the one or more supplement units are in the form of a liquid or a powder.

85. The kit of claim 81 or claim 82, wherein the one or more supplement units are in the form of a liquid or a powder.

86. The kit of claim 81 or claim 82, wherein the one or more supplement units are in the form of pills or capsules.

87. The kit of claim 86, wherein the one or more supplement units are in one or more containers.