Compositions comprising histone acetyltransferase inhibitors or reverse transcriptase inhibitors and uses thereof

Abstract

A cell culture medium comprising luteinizing hormone, follicle stimulating hormone and at least one histone acetyltransferase inhibitor or reverse transcriptase inhibitor is provided. Also provided are methods of maturing human oocytes in vitro, determining in vitro maturing suitability, and kits comprising a culture medium, luteinizing hormone, follicle stimulating hormone, and at least one histone acetyltransferase inhibitor or reverse transcriptase inhibitor.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 317,975 filed on March 9, 2022 and U.S. Provisional Patent Application No. 63 / 436,912 filed on January 4, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The invention belongs to the field of in vitro oocyte maturation and fertilization. Background Art

[0004] Reproductive aging is characterized by an age-related loss of fertility due to increasing damage to the reproductive and other systems. Oocytes accumulate damage in an age-related manner and deteriorate to the point where they are no longer able to fertilize, mature, or generate a suitable environment for early embryogenesis. In female humans, this occurs at a relatively early age, before other organs and tissues begin to age. Since the 1970s, the average age of childbearing in the United States (and other countries) has risen from 21 to over 28 years, with more women having children in their 40s (CDC / NCHS data). This worldwide increase, which occurred in a very short period of time, has resulted in an overall decline in female fertility (3.5 children per woman in the United States in 1960, compared to 1.8 today), and an increase in the incidence of aneuploidy-related syndromes, infertility, and infertility-related syndromes (such as primary ovarian insufficiency, POI). For example, between 2003 and 2012, the miscarriage rate recorded in Sweden increased from 40 to 73 per 100,000, and the number of IVF cases recorded in the United States almost tripled between 1997 and 2009. Therefore, it is crucial to understand the mechanisms underlying reproductive aging in order to improve the survival of aged oocytes and bring hope of parenthood to millions of patients worldwide.

[0005] Oocyte aging is a multifactorial phenomenon. Some of the factors found to be associated with oocyte aging are reactive oxygen species (ROS)-related damage, accumulated DNA damage, age-related changes in the hormonal and regulatory environment of the oocyte, and loss of cohesion with age leading to oocyte aneuploidy.

[0006] Changes in epigenetic regulation of gene expression and altered chromosome dynamics have been implicated as causes of aging, and epigenetic changes during aging have been listed among the “hallmarks of aging.” Loss of heterochromatin histone marks has been implicated in the aging process in many systems and tissues. The consequences of heterochromatin dysregulation in aging are associated with derepression of many genes, but also with activated transcription of transposable elements (TEs), which constitute approximately 40% of the genome. Elevated expression of TEs leads to significant DNA damage, which has a negative impact on genome stability and cellular integrity. This has been shown to occur in several aging organisms and systems. However, little is known about the epigenetic changes that oocytes undergo with age. Therefore, methods to improve oocyte maturation and in vitro conception are urgently needed. Summary of the invention

[0007] The present invention provides a cell culture medium comprising luteinizing hormone, follicle stimulating hormone and at least one histone acetyltransferase (HAT) inhibitor or reverse transcriptase (RT) inhibitor. Also provided are methods for in vitro mature human oocytes and determining the suitability of in vitro maturation (IVM). Also provided are kits comprising culture medium, luteinizing hormone, follicle stimulating hormone and at least one histone acetyltransferase inhibitor or reverse transcriptase inhibitor.

[0008] According to a first aspect, a cell culture medium is provided, comprising luteinizing hormone (LH), follicle stimulating hormone (FSH) and at least one histone acetyltransferase (HAT) inhibitor or reverse transcriptase (RT) inhibitor.

[0009] According to some embodiments, the culture medium is a human oocyte in vitro maturation (IVM) medium comprising at least a HAT or RT inhibitor.

[0010] According to some embodiments, the HAT is selected from E1A-associated protein p300 (p300), cyclic AMP response element binding protein binding protein (CREBBP / CBP), histone acetyltransferase KAT5 (KAT5 / Tip60), KAT6A and N-acetyltransferase 10 (NAT10).

[0011] According to some embodiments, the HAT inhibitor is not a P300 / CBP-associated factor (PCAF / KAT2B) inhibitor.

[0012] According to some embodiments, the HAT inhibitor is selected from the group consisting of curcumin, garcinol, spermidine-CoA, A, Spd-CoA), Lys-CoA, histone H4 lysine 16 (H4K16)-coenzyme A (CoA), MG149, EML264, LTK-14, cyanidin-B3, epicatechin gallate (EGCG), anacardic acid, gambogic acid, delphinidin, remodelin, butyrolactone 3 (MB-3), NK13650A, taxodione (NP-15), Δ12-prostaglandin J2 (PGJ2), plumbaginone (RTK1), NU9056, DC-M01-7, CCT077791, NSC694614, PU141, C646, TH1834, L002, WM-8014, DC-G16-11 and their derivatives.

[0013] According to some embodiments, the HAT inhibitor is a naturally occurring molecule.

[0014] According to some embodiments, the HAT inhibitor is selected from curcumin, plumbagin and mangosteen.

[0015] According to some embodiments, the RT inhibitor is selected from zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, truvada, azvudine, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, doravirine, islatravir and derivatives thereof.

[0016] According to some embodiments, the RT inhibitor is selected from zidovudine (azidothymidine, AZT) and lamivudine (3TC).

[0017] According to some embodiments, the culture medium includes both a HAT inhibitor and a RT inhibitor.

[0018] According to some embodiments, the culture medium comprises Curcumin and AZT.

[0019] According to some embodiments, Curcumin is present at a concentration of about 5-20 μM.

[0020] According to some embodiments, Curcumin is present at a concentration of about 10 μM.

[0021] According to some embodiments, AZT is present at a concentration of about 0.5-5 μM.

[0022] According to some embodiments, AZT is present at a concentration of about 1 μM.

[0023] According to some embodiments, the cell culture medium comprises a pH value between 6.8 and 7.8.

[0024] According to some embodiments, the culture medium is for IVM of human oocytes.

[0025] According to some embodiments, the human oocytes are obtained from a female aged 35 years or older.

[0026] According to another aspect, a method for maturing human oocytes in vitro is provided, the method comprising receiving an immature oocyte from a human subject, and culturing the received immature oocyte in a culture medium comprising at least one HAT inhibitor or RT inhibitor for a period of time sufficient to achieve extrusion of a first polar body from the oocyte, thereby maturing the human oocyte in vitro.

[0027] According to some embodiments, the human subject is at least 35 years old or older.

[0028] According to some embodiments, the human subject is at least 38 years old or older.

[0029] According to some embodiments, the human subject does not receive hormone injections prior to harvesting the immature oocytes.

[0030] According to some embodiments, the hormone injection comprises injecting at least one of FSH, LH, human menopausal gonadotropin (HMG), and human chorionic gonadotropin (HCG).

[0031] According to some embodiments, the culture medium is IVM medium supplemented with at least one gonadotropin.

[0032] According to some embodiments, the IVM medium is supplemented with LH and FSH.

[0033] According to some embodiments, the HAT is selected from E1A-associated protein p300 (p300), cyclic AMP response element binding protein binding protein (CREBBP / CBP), histone acetyltransferase KAT5 (KAT5 / Tip60), KAT6A and N-acetyltransferase 10 (NAT10).

[0034] According to some embodiments, the HAT inhibitor is not a P300 / CBP-associated factor (PCAF / KAT2B) inhibitor.

[0035] According to some embodiments, the HAT inhibitor is selected from the group consisting of curcumin, garcinol, spermidine-coenzyme A (SpD-CoA), Lys-CoA, histone H4 lysine 16 (H4K16)-coenzyme A, MG149, EML264, LTK-14, cyanidin-B3, epicatechin gallate (EGCG), anacardic acid, gambogic acid, delphinidin, Remodelin, butyrolactone 3 (MB-3), NK13650A, balsam pear dione (NP-15), Δ12-prostaglandin J2 (PGJ2), plumbaginone (RTK1), NU9056, DC-M01-7, CCT077791, NSC694614, PU141, C646, TH1834, L002, WM-8014, DC-G16-11 and derivatives thereof.

[0036] According to some embodiments, the HAT inhibitor is a naturally occurring molecule.

[0037] According to some embodiments, the HAT inhibitor is selected from the group consisting of curcumin, plumbagin and mangosteen. According to some embodiments, the HAT inhibitor is selected from the group consisting of curcumin and plumbagin.

[0038] According to some embodiments, the RT inhibitor is selected from zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, Truvada, azithromycin, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, doravirine and islavirine.

[0039] According to some embodiments, the RT inhibitor is selected from zidovudine (azidothymidine, AZT) and lamivudine (3TC).

[0040] According to some embodiments, the culture medium includes both a HAT inhibitor and a RT inhibitor.

[0041] According to some embodiments, the culture medium comprises Curcumin and AZT.

[0042] According to some embodiments, the culture medium comprises a pH between 6.8 and 7.8.

[0043] According to some embodiments, the culture medium is the culture medium of the present invention.

[0044] According to some embodiments, the time is about 18-72 hours.

[0045] According to some embodiments, the HAT inhibitor is present at a concentration sufficient to increase the level of constitutive heterochromatin in the oocyte.

[0046] According to some embodiments, the level of constitutive heterochromatin is increased by at least a predetermined threshold.

[0047] According to some embodiments, the predetermined threshold is an increase of 25%.

[0048] According to some embodiments, the HAT inhibitor or RT inhibitor is present at a concentration sufficient to reduce the amount of retrotransposon expression in the oocyte.

[0049] According to some embodiments, the amount of retrotransposon expression is reduced by at least a predetermined threshold.

[0050] According to some embodiments, the predetermined threshold is a 25% reduction.

[0051] According to some embodiments, the culturing further comprises adding a meiotic entry inhibitor.

[0052] According to some embodiments, the method improves the maturation rate compared to IVM performed in the absence of a HAT inhibitor or a RT inhibitor.

[0053] According to some embodiments, the HAT inhibitor or RT inhibitor is present at a concentration sufficient to increase first polar body extrusion compared to culturing in a medium without the HAT inhibitor or RT inhibitor.

[0054] According to another aspect, there is provided a method of determining the suitability of a subject in need of IVM to be treated by the method of the present invention, the method comprising:

[0055] a. receiving at least one germinal vesicle (GV) arrested oocyte from a human female subject; and

[0056] b. measuring heterochromatin in GV-arrested oocytes, wherein heterochromatin levels below a predetermined threshold indicate that the subject is suitable for IVM by the method of the present invention;

[0057] The suitability of the subject to be treated by the methods of the invention is thereby determined.

[0058] According to some embodiments, a GV-arrested oocyte is harvested for in vitro fertilization (IVF) but is found to be insufficiently mature.

[0059] According to some embodiments, measuring heterochromatin comprises measuring histone 3 lysine 9 (H3K9) trimethylation, H4K20 trimethylation, heterochromatin protein 1 (HP1) levels, 5-methylcytosine levels, or a combination thereof.

[0060] According to some embodiments, the subject has undergone at least one unsuccessful round of IVF, and the level of heterochromatin above a predetermined threshold indicates that the subject is suitable for another round of IVF.

[0061] According to another aspect, there is provided a kit comprising:

[0062] a. Cell culture medium;

[0063] b. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH); and

[0064] c. at least one histone acetyltransferase (HAT) inhibitor or reverse transcriptase (RT) inhibitor.

[0065] According to some embodiments, the culture medium is a human oocyte in vitro maturation (IVM) medium.

[0066] According to some embodiments, the HAT is selected from E1A-associated protein p300 (p300), cyclic AMP response element binding protein binding protein (CREBBP / CBP), histone acetyltransferase KAT5 (KAT5 / Tip60), KAT6A and N-acetyltransferase (NAT10).

[0067] According to some embodiments, the HAT inhibitor is not a P300 / CBP-associated factor (PCAF / KAT2B) inhibitor.

[0068] According to some embodiments, the HAT inhibitor is selected from the group consisting of curcumin, garcinol, spermidine-coenzyme A (SpD-CoA), Lys-CoA, histone H4 lysine 16 (H4K16)-coenzyme A, MG149, EML264, LTK-14, cyanidin-B3, epicatechin gallate (EGCG), anacardic acid, gambogic acid, delphinidin, Remodelin, butyrolactone 3 (MB-3), NK13650A, balsam pear dione (NP-15), Δ12-prostaglandin J2 (PGJ2), plumbaginone (RTK1), NU9056, DC-M01-7, CCT077791, NSC694614, PU141, C646, TH1834, L002, WM-8014, DC-G16-11 and derivatives thereof.

[0069] According to some embodiments, the HAT inhibitor is a naturally occurring molecule.

[0070] According to some embodiments, the HAT inhibitor is selected from curcumin, plumbagin and mangosteen.

[0071] According to some embodiments, the RT inhibitor is selected from zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, Truvada, azithromycin, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, doravirine, islavirine and derivatives thereof.

[0072] According to some embodiments, the RT inhibitor is selected from zidovudine (azidothymidine, AZT) and lamivudine (3TC).

[0073] According to some embodiments, the kit includes both a HAT inhibitor and a RT inhibitor.

[0074] According to some embodiments, the kit comprises Curcumin and AZT.

[0075] According to some embodiments, the cell culture medium comprises a pH between 6.8 and 7.8 and is sufficiently buffered such that the addition of a HAT inhibitor or a RT inhibitor does not drop the pH below 6.8.

[0076] According to some embodiments, the kit is for IVM of human oocytes.

[0077] According to some embodiments, the human oocytes are obtained from a female aged 35 years or older.

[0078] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figures 1A-1C : (1A) 9-month-old oocytes show maturation defects but not aneuploidy: In vitro maturation results of naive young (2-month-old) and old (9-month-old) oocytes collected at prophase I arrest and released simultaneously into meiosis. Oocytes were fixed and DNA stained to assess entry into MII (Z-test for 2 proportions p=0.00135, N of animals used for this experiment 年轻 =3, N 年老 =3), see Methods. (1B) Chromosomes of young (2 months old) and old (9 months old) oocytes at MII prophase 00. Sister chromatid pairs were counted for each cell to assess ploidy (Z test p = 0.554, N animals used for the experiment 年轻 =3, N 年老=4). (1C) Representative images of metaphase II chromosome spreads with or without CREST staining.

[0080] Figures 2A-2D : 9-month-old oocytes lose heterochromatin marks: (2A-2D) In ​​situ staining images and staining intensity analysis of chromatin marks and proteins in oocytes from young (2-month-old) and old (9-month-old) females. Oocytes were stained and imaged during prophase I arrest by in situ immunofluorescence (see Methods). Staining intensity was quantified for each image and values ​​were statistically analyzed (see Methods). (2A) H3K9me2 (t-test p<0.0001, N animals used for experiments) was detected by staining. 年轻 =4,N 年老 =4) and (2B) HP1γ (t test p = 0.0146, animals used in the experiment N 年轻 =3,N 年老 =3) to measure constitutive heterochromatin marks and proteins. (2C) To examine facultative heterochromatin, oocytes were stained and imaged for H3K27me3 (t-test p=0.0005, N animals used for experiments 年轻 =3,N 年老 =5). (2D) Euchromatin staining by staining for the marker H3K4me3 (t-test p=0.657, N animals used for the experiment 年轻 =3,N 年老 =6).

[0081] Figures 3A-3D : Heterochromatin loss is uniformly present in different oocyte populations: IHF staining of ovarian cryosections from young (2 months old) and old (9 months old) female mice. (3A) H3K9me2 (t test < 0.0001, N animals used for experiments) 年轻 =3,N 年老 =3) and (3B) H3K27me3 staining, the loss of heterochromatin in intact oocytes was detected. (MW = 0.0008, animal N used for the experiment 年轻 =3,N 年老 =3). MI metaphase chromosomes from young (2 months old) and old (9 months old) females were stained for the following markers and analyzed for staining intensity (see Methods): (3C) H3K9me2 (t-test p=0.008, N animals used for the experiment 年轻 =3,N 年老 =5) and (3D) H3K27me3 (MW p<0.0001, animals used for experiments N 年轻 =3,N 年老 =4).

[0082] Figures 4A-4H Aged oocytes show elevated activity of L1 and IAP retrotransposons and increased DNA damage: (4A) Expression of L1 retrotransposon in young (2-month-old) and aged (9-month-old) prophase I arrested mouse oocytes (MW p=0.01, animals used for experiments N 年轻 =10, N 年老 =10) and IAP element mRNA (MW p = 0.03, animals used for the experiment N 年轻 =10, N 年老 =10) Transcriptional retrotransposon activity was measured using qRT-PCR (see Methods). (4B) Upregulation of L1 retrotransposon protein was measured by Western blotting of L1-ORF1p in young (2 months old) and old (9 months old) prophase I arrested mouse oocytes. (N 年轻 =6, N 年老 =8). (4C) L1 retrotransposon activity was measured by in situ staining of L1-ORF1p in oocytes of young (2 months old) and old (9 months old) prophase I arrested mice (Shapiro Wilk p<0.05, t-test p<0.0001, N 年轻 =3,N 年老 =3). (4D) The accumulation of DNA damage with age was assessed by in situ staining of Rad51 in young (2 months old) and old (9 months old) prophase I arrested mouse oocytes and quantification of its nuclear localization (see Methods). 年轻 =3,N 年老 =5). (4E) In situ staining of γH2AX quantified by the number of nuclear foci in oocytes of old and young prophase I arrested mice (MW p=0.0085, N 年轻 =3,N 年老 =5), another measure of DNA damage was performed (see Methods). (4F) To investigate the phenotype of aged oocytes after inhibition of retroviral activity, oocytes were treated with AZT and then stained with γH2A, and the number of nuclear foci in AZT-treated and untreated control aged prophase I arrested oocytes was quantified (MW p<0.0274, N=3 animals for experiment). (4G) The maturation efficiency of aged oocytes treated with two different doses of AZT and matured in vitro was measured by the proportion of oocytes that successfully completed MII after treatment (Z test p=0.0102) (see Methods). Since the use of drug incubation in IBMX requires longer incubation than normal, extended incubation without AZT was also added as a control. (4H) Quantification of H3K9me2 in situ staining intensity in control aged oocytes and aged oocytes treated with two different doses of AZT (see Methods).

[0083] Figures 5A-5E : Aged oocytes show elevated retrotransposon RNA processing activity: To quantify the differential presence of small RNAs in young (2 months old) and old (9 months old) oocytes, small RNA-seq studies were performed (see Methods). (5A) Identification of the 10 repeat types that were most up- or down-regulated in aged oocytes. Note that the major repeat type overexpressed is retrotransposon RNA molecules, while the list of under-expressed repeat types also includes other types of repeats. (5B) Family-based smRNA analysis. The ratio of sequencing reads between old and young in the transcripts of each repeat family is plotted. tRNA and rRNA were used as controls. (5C-5D) To quantify global RNA processing activity in aged oocytes, we performed (5C) dsRNA (MW p<0.0001, N 年轻 =3,N 年老 =5) and (5D)Dicer (MW p<0.0001, N 年轻 =3,N 年老 =5) were used for staining. (5E) Western blotting confirmed the increase of Dicer (N 年轻 =6, N 年老 =8).

[0084] Figures 6A-6H: Human oocytes show a decrease in the H3K9me2 heterochromatin mark and the REC8 meiotic cohesion mark with age: To investigate changes in heterochromatin in human oocytes with age, human GV-arrested oocytes were stained by immunofluorescence for H3K9me2 (see text and methods). Oocytes were obtained from 33 women, with an average of 2.2 oocytes per woman. Oocytes stained during prophase I arrest or arrested in anaphase of meiosis were analyzed separately. (6A) Examples of prophase I arrested oocytes stained (H3K9me2) from patients under 35 years old and over 35 years old. (6B) Oocytes arrested during meiosis also show a decrease in signal intensity with age, which is linearly correlated, (N=28, F test p=0.0008). The color of the oocytes of each patient is different (for a given age, several patients with more than one oocyte can be represented). (6C) Examples of prophase I oocytes after meiotic resumption from patients under 35 years of age and over 35 years of age stained for (H3K9me2). (6D) Prophase I oocytes show a linear decrease in signal intensity with age. (N=21, F test p=0.03). (6E) Examples of in situ staining of prophase I arrested human oocytes (N=21) for the Rec8 meiotic cohesion subunit. (6F) These oocytes show a linear decrease in signal intensity with age (F test p=0.02). (6G) Examples of in situ staining of Rec8 in human prophase I oocytes after meiotic resumption (N=47). (6H) These oocytes show a linear decrease in signal intensity with age (F test p=0.01).

[0085] Figures 7A-7C : Treatment of oocytes with chaetocin mimics natural aging: Young prophase I arrested mouse oocytes (nine animals were used in this experiment) were released to resume meiosis in medium supplemented with chaetocin, which inhibits heterochromatin formation (see methods). (7A) The resulting phenotype was measured by assessing the maturation efficiency of treated oocytes compared to untreated controls (Z test p = 0.008). For biosafety reasons, incubation with chaetocin requires sealing of the plates. Therefore, additional controls with sealed plates but without drug were added (see methods). (7B) Activation of the L1 retrotransposon after chaetocin treatment was measured by immunofluorescence of L1-ORF1p in young MII mouse oocytes with or without chaetocin (MW p = 0.0007). (7C) To measure RNA processing activity in chaetocin-treated oocytes, in situ staining of dsRNA was performed in young MII mouse oocytes with or without chaetocin (MW p = 0.02).

[0086] Figures 8A-8H: Recovery of aged oocytes occurs when heterochromatin is enhanced: Overexpression of chromatin modifying enzymes was performed in aged (9-month-old) prophase I arrested oocytes. (8A) Recovery of heterochromatin in EZH2 electroporated oocytes was measured by H3K27me3 staining (MW p=0.03, 4 animals were used for the experiment). (8B) Reduction of retrotransposon activity in treated oocytes was observed by L1-ORF1 staining (MW p=0.03, 3 animals were used for the experiment). (8C) Cell functionality was measured by successful MII completion in EZH2 electroporated oocytes compared to empty electroporation controls. (Z test p=0.019, 7 animals were used for the experiment). (8D) Recovery of heterochromatin in SIRT1 electroporated oocytes was measured by H3K9me2 staining (MW p=0.0026, 3 animals were used for the experiment). (8E) Reduction in retrotransposon activity in treated oocytes was observed by L1-ORF1 staining (MW p=0.0013, 5 animals were used for the experiment). (8F) Cell functionality was measured by successful MII completion in oocytes electroporated with SIRT1 compared to empty electroporation controls. (Z test p=0.0086, 7 animals were used for the experiment). (8G) Quantification of H3K9me2 staining intensity in control aged oocytes and aged oocytes treated with SRT-1720 (MW p=0.0079). (8H) The maturation efficiency of aged oocytes treated with SRT-1720 and matured in vitro was measured by the proportion of oocytes that successfully completed MI after treatment. (Z test p=0.0078, N=6 animals used for the experiment). As mentioned above, since incubation with the drug requires longer IVM incubation than normal, extended incubation without SRT-1720 was also added as a control.

[0087] Figures 9A-9D : Bar graph of maturation rates in IVM cultures of aged mouse oocytes supplemented with (9A) curcumin, (9B) mangosteen, (9C) various HAT inhibitors and a RT inhibitor and (9D) a combination of HAT inhibitors or curcumin and AZT.

[0088] Figures 10A-10B : (10A-10B) In situ staining of H3K9me2 in old and young (10A) GV and (10B) GVBD human oocytes (left). Line graphs comparing biological age and H3K9me2 staining intensity are provided, as well as a regression line showing an inverse correlation (GV pval = 0.03, GBVD pval = 0.0008) (right).

[0089] Fig.11 : Bar graph of oocyte maturation of oocytes from a 20 year old fertility preservation patient in the IVM protocol with AZT supplementation. DETAILED DESCRIPTION

[0090] In some embodiments, the present invention provides a cell culture medium comprising luteinizing hormone, follicle stimulating hormone and at least one histone acetyltransferase inhibitor or reverse transcriptase inhibitor. Also provided are methods for maturing human oocytes in vitro and determining suitability for in vitro maturation.

[0091] In vitro maturation method

[0092] In a first aspect, a method for maturing an oocyte is provided, the method comprising receiving an immature oocyte and culturing the received immature oocyte in a culture medium comprising at least one histone acetyltransferase (HAT) inhibitor, a reverse transcriptase (RT) inhibitor, or both, thereby maturing the oocyte.

[0093] In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is implemented in culture. In some embodiments, the method is a method of in vitro maturation (IVM). In some embodiments, the method is a part of in vitro fertilization (IVF). In some embodiments, the method further produces a mature oocyte. In some embodiments, the mature oocyte is suitable for fertilization. In some embodiments, the mature oocyte is suitable for IVF. In some embodiments, the method further includes fertilizing the mature oocyte. In some embodiments, the method further includes transferring the fertilized mature oocyte to a woman. In some embodiments, the method further includes IVF. In some embodiments, the mature oocyte is used for IVF. In some embodiments, the fertilized mature oocyte is used for transplantation to a woman.

[0094] The term oocyte refers to a female gamete prior to fertilization. In some embodiments, the oocyte is a mammalian oocyte. In some embodiments, the mammal is a human. In some embodiments, the subject is an older subject. In some embodiments, the subject is a reproductively older subject. In some embodiments, the human subject is at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 years old. Each possibility represents a separate embodiment of the present invention. In some embodiments, the human subject is at least 35 years old. In some embodiments, the human subject is at least 38 years old. In some embodiments, the human subject is at least 40 years old.

[0095] In some embodiments, oocyte is at least one oocyte. In some embodiments, oocyte is a plurality of oocytes. In some embodiments, oocyte is a cumulus oocyte complex. In some embodiments, oocyte is at least 1,2,3,4,5,6,7,8,9 or 10 oocytes. Every possibility represents an independent embodiment of the present invention. In some embodiments, oocyte is 1-40,5-40,10-40,1-30,5-30,5-10,10-40 or 10-30 oocyte. Every possibility represents an independent embodiment of the present invention. In some embodiments, oocyte is 1-40 oocyte.

[0096] In some embodiments, the method includes extracting immature oocytes from a subject. In some embodiments, extraction is harvesting. In some embodiments, the subject receives hormones before extraction. In some embodiments, the subject does not receive hormone injections before harvesting. In some embodiments, the immature oocytes are from subjects who have received hormone injections. In some embodiments, the immature oocytes are from subjects who have not received hormone injections. In some embodiments, the hormone is a hormone that induces maturation. In some embodiments, the hormone is a gonadotropin. In some embodiments, the hormone is selected from follicle stimulating hormone (FSH), luteinizing hormone (LH), human menopausal gonadotropin (HMG), human chorionic gonadotropin (HCG) and combinations thereof. In some embodiments, the hormone includes or is FSH. In some embodiments, the hormone includes or is LH. In some embodiments, the hormone includes or is HMG. In some embodiments, the hormone includes or is HCG.

[0097] In some embodiments, the immature oocyte is an oocyte that has not reached meiotic competence. In some embodiments, the immature oocyte is an oocyte that has not reached the first stage of meiosis. In some embodiments, the immature oocyte is an oocyte that has not yet split into MI. In some embodiments, the immature oocyte is an oocyte without a polar body. In some embodiments, the immature oocyte is an MI oocyte. In some embodiments, the immature oocyte is a germinal vesicle oocyte. In some embodiments, the immature oocyte is an oocyte enclosed by a cumulus ovum. In some embodiments, the immature oocyte is a cumulus ovum-oocyte complex. In some embodiments, the immature oocyte is an oocyte that is not suitable for fertilization.

[0098] In some embodiments, the culture is in a culture medium. In some embodiments, the culture medium is a tissue culture medium. In some embodiments, the culture is a tissue culture. In some embodiments, the culture is an in vitro culture. In some embodiments, the culture medium is an IVM culture medium. In some embodiments, the culture medium is an IVF culture medium. IVM culture medium and IVF culture medium are well known in the art and are commercially available. Any such culture medium may be used as part of the present invention. Examples of IVM culture medium include, but are not limited to, SAGE in vitro maturation (IVM) culture medium, MediCult, human fallopian tube fluid, and blastocyst culture medium. In some embodiments, the culture medium is a SAGE culture medium. In some embodiments, the SAGE culture medium is a SAGE IVM culture medium.

[0099] In some embodiments, the culture medium is supplemented with at least one hormone. In some embodiments, the hormone is a gonadotropin. In some embodiments, the gonadotropin is LH. In some embodiments, the gonadotropin is FSH. In some embodiments, the culture medium is supplemented with LH and FSH. In some embodiments, the LH concentration supplemented is 0.075 IU / mL. In some embodiments, the FSH concentration supplemented is 0.075 IU / mL.

[0100] Histone acetyltransferases are well known in the art, and a list of known or inferred HATs can be found, for example, in Sterner and Berger, "Acetylation of Histones and Transcription-Related Factors", Microbiol Mol Biol Rev. 2000 Jun; 64 (2): 435-459, the entire contents of which are incorporated herein by reference. There are two main HAT families: the Gcn5-related N-acetyltransferase (GNAT) family and the MYST HAT family. However, some HATs do not belong to either of these two families, such as p300, CBP, nuclear receptor coactivators (e.g., ACTR / SRC-1), TAFII250, TFIIIC, Rtt109, and CLOCK. In some embodiments, the HAT is E1A-related protein p300 (p300). In some embodiments, the HAT is cyclic adenosine monophosphate response element binding protein binding protein (CREBBP / CBP). In some embodiments, the HAT is histone acetyltransferase KAT5 (KAT5 / Tip60). In some embodiments, the HAT is KAT6A. In some embodiments, the HAT is N-acetyltransferase 10 (NAT10). In some embodiments, the HAT is a steroid receptor coactivator (SRC-1). In some embodiments, the HAT is ACTR. In some embodiments, the HAT is TAFII250. In some embodiments, the HAT is TFIIIC. In some embodiments, the HAT is Rtt109. In some embodiments, the HAT is CLOCK. In some embodiments, the HAT is not a P300 / CBP-associated factor (PCAF / KAT2B).

[0101] As used herein, the term "HAT inhibitor" refers to a molecule that at least partially blocks, inhibits, reduces or in any way disturbs the deposition of acetyl groups onto lysine residues in the histone tail. HAT inhibitors do not need to completely eliminate HAT activity, but only need to inhibit or reduce its activity. In some embodiments, HAT inhibitors are direct inhibitors. As used herein, "direct HAT inhibitors" are molecules that directly interact with HAT and inhibit its acetyltransferase function. In some embodiments, the acetyltransferase function is a lysine acetyltransferase function. In some embodiments, the HAT inhibitor is a lysine acetyltransferase inhibitor. In some embodiments, the histone is H2A. In some embodiments, the histone is H2B. In some embodiments, the histone is H3. In some embodiments, the histone is H4. In some embodiments, the HAT inhibitor is not a P300 / CBP-related factor (PCAF / KAT2B) inhibitor. In some embodiments, the HAT inhibitor does not directly inhibit the P300 / CBP-related factor (PCAF / KAT2B). In some embodiments, HAT suppresses the increase including heterochromatin. In some embodiments, HAT suppresses the reduction including euchromatin. In some embodiments, HAT suppresses the suppression including RT. It will be appreciated by the skilled artisan that histone acetylation and euchromatin formation are the upstream of repetitive elements and retrotransposon reverse transcription. Therefore, HAT suppresses the generation of heterochromatin, which will suppress the RT of transposable elements, but RT suppresses the absence of influence on chromatin state. HAT inhibitors are well known in the art, and any inhibitor can be used as a part of the present invention. In addition, the analysis of assessing HAT activity is well known, and the skilled artisan can easily determine whether the molecule is a HAT inhibitor by performing HAT analysis in the presence of molecules. In some embodiments, the method includes determining whether the compound is a HAT inhibitor. In some embodiments, the method includes determining whether the compound is a HAT inhibitor. In some embodiments, the method includes selecting the compound as a HAT inhibitor.

[0102] In some embodiments, the HAT inhibitor is a small molecule inhibitor. In some embodiments, the HAT inhibitor is an inhibitory nucleic acid molecule. In some embodiments, the nucleic acid molecule is RNA. In some embodiments, the nucleic acid molecule is an antisense oligonucleotide. In some embodiments, the HAT inhibitor is selected from the group consisting of curcumin, garcinol, spermidine-CoA (Spd-CoA), Lys-CoA, histone H4 lysine 16 (H4K16)-CoA, MG149, EML264, LTK-14, cyanidin-B3, epicatechin gallate (EGCG), anacardic acid, gambogic acid, delphinidin, Remodelin, butyrolactone 3 (MB-3), NK13650A, balsam pear dione (NP-15), Δ12-prostaglandin J2 (PGJ2), plumbaginone (RTK1), NU9056, DC-M01-7, CCT077791, NSC694614, PU141, C646, TH1834, L002, WM-8014, DC-G16-11 and derivatives thereof. In some embodiments, the HAT inhibitor is selected from the group consisting of curcumin, garcinol, spermidine-coenzyme A (SpD-CoA), Lys-CoA, histone H4 lysine 16 (H4K16)-CoA, MG149, EML264, LTK-14, cyanidin-B3, epicatechin gallate (EGCG), delphinidin, Remodelin, butyrolactone 3 (MB-3), NK13650A, bald cyperus dione (NP-15), Δ12-prostaglandin J2 (PGJ2), plumbagin (RTK1), NU9056, DC-M01-7, CCT077791, NSC694614, PU141, C646, TH1834, L002, WM-8014, DC-G16-11 and derivatives thereof. In some embodiments, the HAT inhibitor is selected from: curcumin, garcinol, spermidine coenzyme A (SpD-CoA), Lys-CoA, histone H4 lysine 16 (H4K16)-coenzyme A, MG149, EML264, LTK-14, cyanidin-B3, delphinidin, Remodelin, butyrolactone 3 (MB-3), NK13650A, bald cyperus dione (NP-15), Δ12-prostaglandin J2 (PGJ2), plumbagin (RTK1), NU9056, DC-M01-7, CCT077791, NSC694614, PU141, C646, TH1834, L002, WM-8014, DC-G16-11 and derivatives thereof. In some embodiments, the HAT inhibitor is a naturally occurring molecule. In some embodiments, the HAT inhibitor is an artificial molecule. In some embodiments, the HAT inhibitor is selected from curcumin and mangosteen.In some embodiments, the HAT inhibitor is curcumin. In some embodiments, the HAT inhibitor is garcinone. In some embodiments, the HAT inhibitor is plumbagin. In some embodiments, the HAT inhibitor is selected from the group consisting of curcumin, garcinone, and plumbagin. In some embodiments, the HAT inhibitor is selected from the group consisting of curcumin and garcinone. In some embodiments, the HAT inhibitor is selected from the group consisting of curcumin and plumbagin.

[0103] In some embodiments, the HAT inhibitor is not an acid. In some embodiments, the HAT inhibitor is not epi-epicatechin gallate (EGCG), anacardic acid or gambogic acid. EGCG is also acidic, and it, together with anacardic acid and gambogic acid, will reduce the pH of the culture medium to which it is added. In some embodiments, the pH of the culture medium is neutral. In some embodiments, before the addition of the HAT inhibitor or RT inhibitor, the pH of the culture medium is neutral. In some embodiments, after the addition of the HAT inhibitor, the pH of the culture medium is neutral. In some embodiments, neutral pH is pH from about 6.8 to 7.8. In some embodiments, neutral pH is pH from about 6.9 to 7.6. In some embodiments, neutral pH is pH about 7. In some embodiments, neutral pH is pH about 7.3. In some embodiments, neutral pH is pH about 7.4. In some embodiments, neutral pH is physiological pH.

[0104] In some embodiments, the culture medium after adding the HAT inhibitor or RT inhibitor does not include an acidic pH. In some embodiments, the acidic pH is a pH lower than 7, 6.9, 6.8, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 or 5. Each possibility represents a separate embodiment of the present invention. In some embodiments, the acidic pH value is a pH lower than 6.9. In some embodiments, the acidic pH value is a pH lower than 6.8. In some embodiments, the acidic pH is a pH lower than 6. In some embodiments, the culture medium after adding a HAT inhibitor or RT inhibitor comprises a pH higher than 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7. Each possibility represents a separate embodiment of the present invention. In some embodiments, the culture medium after adding a HAT inhibitor or RT inhibitor comprises a pH higher than 6. In some embodiments, the culture medium after adding a HAT inhibitor or RT inhibitor comprises a pH higher than 6.8. In some embodiments, the culture medium after adding a HAT inhibitor or RT inhibitor comprises a pH higher than 6.9.

[0105] In some embodiments, the culture medium is pH buffered. In some embodiments, the culture medium before adding the HAT inhibitor or RT inhibitor is sufficiently buffered so that the addition of the HAT inhibitor or RT inhibitor does not make the culture medium acidic. In some embodiments, the culture medium before adding the HAT inhibitor or RT inhibitor is sufficiently buffered so that the addition of the HAT inhibitor or RT inhibitor does not reduce the pH value to less than 6.8. In some embodiments, the culture medium before adding the HAT inhibitor or RT inhibitor is sufficiently buffered so that the addition of the HAT inhibitor or RT inhibitor does not reduce the pH value to less than 6.9. In some embodiments, the culture medium before adding the HAT inhibitor or RT inhibitor is sufficiently buffered so that the addition of the HAT inhibitor or RT inhibitor does not reduce the pH to less than 6.

[0106] In some embodiments, the HAT inhibitor is present in a concentration sufficient to increase the level of constitutive heterochromatin in the oocyte. In some embodiments, the increase is a significant increase. In some embodiments, significant is statistically significant. In some embodiments, the increase is at least an increase by a predetermined threshold. In some embodiments, the threshold is an increase of 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%. Each possibility represents a separate embodiment of the present invention. In some embodiments, the threshold is a 25% increase. In some embodiments, the threshold is a 50% increase.

[0107] In some embodiments, HAT inhibitors are present in a concentration sufficient to induce maturation. In some embodiments, inducing maturation is to increase maturation. In some embodiments, increasing is compared with cultivating in a culture medium without HAT inhibitors. In some embodiments, increasing is at least 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% increase. Each possibility represents a separate embodiment of the present invention. In some embodiments, increasing is at least 25% increase. In some embodiments, increasing is at least 50% increase. In some embodiments, increasing is at least twice.

[0108] In some embodiments, maturation includes first polar body extrusion. In some embodiments, maturation includes chromosome splitting. In some embodiments, chromosome splitting is chromosome separation. In some embodiments, chromosome splitting is appropriate or functional chromosome splitting. In some embodiments, functional splitting refers to the splitting of fertilizable oocytes. In some embodiments, induced maturation is induced maturation in oocytes exposed to HAT inhibitors at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%. Each possibility represents a separate embodiment of the present invention. In some embodiments, induced maturation is to induce at least 30% oocyte maturation. In some embodiments, induced maturation is to induce at least 40% oocyte maturation.

[0109] In some embodiments, Curcumin is present in the culture or culture medium at a concentration of about 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 2-50, 2-40, 2-30, 2-20, 2-25, 2-10, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 7-50, 7-40, 7-30, 7-20, 7-15, 7-10, 10-50, 10-40, 10-30, 10-20 or 10-15 μM. Each possibility represents a separate embodiment of the present invention. In some embodiments, Curcumin is present in the culture or culture medium at a concentration of about 5-20 μM. In some embodiments, Curcumin is present in the culture or culture medium at a concentration of about 10 μM.

[0110] In some embodiments, plumbagin is present in an amount of about 0.1-50, 0.1-40, 0.1-30, 0.1-20, 0.1-15, 0.1-10, 0.1-5, 0.1-2.5, 0.5-50, 0.5-40, 0.5-30, 0.5-20, 0.5-15, 0.5-10, 0.5-5, 0.5-2.5, 1-50, 1-4 In some embodiments, plumbagin is present in a culture or culture medium at a concentration of about 0.5-5 μM. In some embodiments, plumbagin is present in a culture or culture medium at a concentration of about 1-2.5 μM.

[0111] The term "RT inhibitor" as used herein refers to a molecule that at least partially blocks, inhibits, reduces or in any way disrupts the reverse transcription or action of a reverse transcriptase. RT inhibitors do not need to completely eliminate RT activity, but only need to inhibit or reduce its activity. In some embodiments, the RT inhibitor is a direct inhibitor. As used herein, a "direct RT inhibitor" is a molecule that directly interacts with RT and inhibits its reverse transcriptase function. In some embodiments, RT inhibition includes reducing the transcription of a retrotransposon. In some embodiments, RT inhibition includes reducing the expression of a retrotransposon. In some embodiments, RT inhibition includes reducing retrotransposon hope. In some embodiments, RT inhibition includes reducing retrotransposon silencing. RT inhibitors are well known in the art, and any inhibitor can be used as part of the present invention. In addition, assays for evaluating RT activity are also well known, and a skilled artisan can easily determine whether the molecule is an RT inhibitor by performing an RT assay in the presence of a molecule. In some embodiments, the method includes determining whether a compound is an RT inhibitor. In some embodiments, the method includes determining whether a compound is an RT inhibitor. In some embodiments, the method comprises selecting a compound that is an inhibitor of RT.

[0112] In some embodiments, the RT inhibitor is present in a concentration sufficient to induce maturation. In some embodiments, inducing maturation is to increase maturation. In some embodiments, the increase is compared to cultivating in a culture medium without the RT inhibitor. In some embodiments, the increase is at least 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% increase. Each possibility represents a separate embodiment of the present invention. In some embodiments, the increase is at least 25% increase. In some embodiments, the increase is at least 50% increase. In some embodiments, the increase is at least twice.

[0113] In some embodiments, maturation includes first polar body extrusion. In some embodiments, maturation includes chromosome splitting. In some embodiments, chromosome splitting is chromosome separation. In some embodiments, chromosome splitting is proper or functional chromosome splitting. In some embodiments, functional splitting refers to the splitting of oocytes that can be fertilized. In some embodiments, induced maturation is induced maturation in oocytes exposed to RT inhibitors at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%. Each possibility represents a separate embodiment of the present invention. In some embodiments, induced maturation is to induce at least 30% oocyte maturation. In some embodiments, induced maturation is to induce at least 40% oocyte maturation.

[0114] In some embodiments, the RT inhibitor is a small molecule inhibitor. In some embodiments, the RT inhibitor is an inhibitory nucleic acid molecule. In some embodiments, the nucleic acid molecule is RNA. In some embodiments, the nucleic acid molecule is an antisense oligonucleotide. In some embodiments, the RT inhibitor is selected from: zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, Truvada, azithromycin, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, doravirine, islavirine and its derivatives. In some embodiments, the RT inhibitor is a naturally occurring molecule. In some embodiments, the RT inhibitor is an artificial molecule. In some embodiments, the RT inhibitor is an antiviral agent. In some embodiments, the RT inhibitor is zidovudine (azidothymidine, AZT). In some embodiments, the RT inhibitor is AZT. In some embodiments, the RT inhibitor is lamivudine. In some embodiments, lamivudine is 3TC lamivudine. In some embodiments, the RT inhibitor is selected from AZT and lamivudine.

[0115] In some embodiments, the HAT inhibitor is present in a concentration sufficient to reduce the expression of the retrotransposon in the oocyte. In some embodiments, the RT inhibitor is present in a concentration sufficient to reduce the expression of the retrotransposon in the oocyte. In some embodiments, the reduction is a significant reduction. In some embodiments, significant is statistically significant. In some embodiments, the reduction is at least a predetermined threshold reduction. In some embodiments, the threshold is a reduction of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97 or 100%. Each possibility represents a separate embodiment of the present invention. In some embodiments, the threshold is a 25% reduction. In some embodiments, the threshold is a 50% reduction.

[0116] In some embodiments, AZT is present in the culture or culture medium at a concentration of about 0.1-10, 0.1-7, 0.1-5, 0.1-3, 0.1-2, 0.1-1, 0.2-10, 0.2-7, 0.2-5, 0.2-3, 0.2-25, 0.2-1, 0.5-10, 0.5-7, 0.5-5, 0.5-3, 0.5-2, 0.5-1, 0.7-10, 0.7-7, 0.7-5, 0.7-3, 0.7-2, 0.7-1, 1-10, 1-7, 1-5, 1-3, or 1-2 μM. Each possibility represents a separate embodiment of the invention. In some embodiments, AZT is present in the culture or culture medium at a concentration of about 0.5-5 μM. In some embodiments, AZT is present in the culture or culture medium at a concentration of about 1 μM.

[0117] In some embodiments, lamivudine is present in the culture or medium at a concentration of about 0.1-10, 0.1-7, 0.1-5, 0.1-3, 0.1-2, 0.1-1, 0.2-10, 0.2-7, 0.2-5, 0.2-3, 0.2-2, 0.2-1, 0.5-10, 0.5-7, 0.5-5, 0.5-3, 0.5-2, 0.5-1, 0.7-10, 0.7-7, 0.7-5, 0.7-3, 0.7-2, 0.7-1, 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, or 5-7 μM. Each possibility represents a separate embodiment of the invention. In some embodiments, lamivudine is present in the culture or culture medium at a concentration of about 1-10 μM. In some embodiments, lamivudine is present in the culture or culture medium at a concentration of about 5 μM.

[0118] As used herein, the term "derivative" refers to any compound based on a known inhibitor and retaining an inhibitory function. Assays for testing HAT inhibition and RT inhibition are well known in the art, so it is easy to test whether a derivative retains this function. In some embodiments, the method includes testing whether a molecule has a HAT or RT inhibitory function.

[0119] In some embodiments, the culture medium includes both a HAT inhibitor and a RT inhibitor. In some embodiments, the culture medium contains multiple HAT inhibitors. In some embodiments, the culture medium includes multiple RT inhibitors. In some embodiments, the culture medium includes curcumin and AZT. In some embodiments, the culture medium includes garcinone and AZT. In some embodiments, the culture medium includes plumbagin and AZT. In some embodiments, the culture medium includes curcumin and lamivudine. In some embodiments, the culture medium includes garcinone and lamivudine. In some embodiments, the culture medium includes plumbagin and lamivudine. In some embodiments, the culture medium is a culture medium of the present invention.

[0120] In some embodiments, the culture is sufficient for the time that the oocyte matures. In some embodiments, the time that is sufficient for maturation is the time that is sufficient for the oocyte to no longer be immature. In some embodiments, the culture is sufficient for the time that the first polar body extrude. In some embodiments, the culture is until the first polar body extrude. In some embodiments, the time is at least 12, 15, 16, 18, 20, 22 or 25 hours. Each possibility represents a separate embodiment of the present invention. In some embodiments, the time is at least 18 hours. In some embodiments, the time is at most 60, 65, 70, 72, 74, 75 or 80 hours. Each possibility represents a separate embodiment of the present invention. In some embodiments, the time is at most 72 hours. In some embodiments, the time is about 18-48, 18-54, 18-60, 18-66, 18-72, 24-48, 24-54, 24-60, 24-66, 24-72, 36-48, 36-54, 36-60, 36-66, 36-72, 48-54, 48-60, 48-66 or 48-72 hours. Each possibility represents a separate embodiment of the present invention. In some embodiments, the time is about 18-72 hours. In some embodiments, the time is about 48 hours.

[0121] In some embodiments, the culture further comprises adding a meiotic inhibitor. In some embodiments, the meiotic inhibitor is a meiotic entry inhibitor. In some embodiments, the method comprises a pre-culture before the culture, wherein the pre-culture comprises a meiotic inhibitor. In some embodiments, the culture is a biphasic culture, which comprises a first culture comprising a meiotic inhibitor and a second culture comprising a HAT inhibitor or a RT inhibitor. In some embodiments, the culture medium is replaced between the pre-culture and the culture. In some embodiments, the culture medium is replaced between the first culture and the second culture. In some embodiments, the oocyte is rinsed between two culture stages or between the pre-culture and the culture.

[0122] Meiotic inhibitors are well known in the art, and any such inhibitor can be used for pre-culture / first culture. These inhibitors include various CDK inhibitors, oocyte maturation inhibitors (OMI), WEE2 inhibitors, phosphodiesterase inhibitors, adenosine receptor antagonists and many other inhibitors. In some embodiments, meiotic inhibitors are C-type natriuretic peptides (CNP). In some embodiments, meiotic inhibitors are isobutyl methyl xanthine, 1-methyl-3-isobutyl xanthine (IBMX). In some embodiments, meiotic inhibitors exist with a concentration sufficient to suppress entry into meiosis.

[0123] In some embodiments, the method is increased compared to the IVM implemented in the absence of the HAT inhibitor. In some embodiments, the method is increased compared to the IVM implemented in the absence of the RT inhibitor. In some embodiments, the method is increased compared to the IVM implemented in the absence of the HAT inhibitor and the RT inhibitor. In some embodiments, the increase is an increase. In some embodiments, the method further comprises selecting a mature oocyte. In some embodiments, the method further comprises selecting an oocyte with increased heterochromatin. In some embodiments, the method further comprises selecting an oocyte with reduced retrotransposon expression.

[0124] Culture medium

[0125] In another aspect, a culture medium is provided that includes at least one gonadotropin and at least one HAT inhibitor or RT inhibitor.

[0126] In another aspect, a kit is provided, comprising:

[0127] a. Culture medium;

[0128] b. at least one gonadotropin; and

[0129] c. at least one HAT inhibitor or RT inhibitor.

[0130] In some embodiments, the culture medium is a culture medium. In some embodiments, the culture medium is a cell culture medium. In some embodiments, the culture medium is a tissue culture medium. In some embodiments, the culture medium is an IVM culture medium. In some embodiments, the culture medium is sterile. In some embodiments, the culture medium is chemically defined. In some embodiments, the culture medium does not contain animal products. In some embodiments, the animal is non-human.

[0131] In some embodiments, the gonadotropin is a mammalian gonadotropin. In some embodiments, the mammal is a human. In some embodiments, the mammal is a veterinary animal. In some embodiments, the mammal is a domesticated animal. In some embodiments, the mammal is a livestock. In some embodiments, the veterinary animal is selected from cats, dogs, horses, cows, pigs, sheep and goats. In some embodiments, the gonadotropin is human gonadotropin. In some embodiments, the gonadotropin is LH. In some embodiments, the gonadotropin is FSH. In some embodiments, the gonadotropin is LH and FSH.

[0132] In some embodiments, the culture medium is for IVM. In some embodiments, the kit is for IVM. In some embodiments, the IVM is IVM of an oocyte. In some embodiments, the culture medium is for IVF. In some embodiments, the kit is for IVF. In some embodiments, the IVF is IVF of an oocyte. In some embodiments, the oocyte is a mammalian oocyte. In some embodiments, the oocyte is a human oocyte.

[0133] Patient selection

[0134] In another aspect, a method of determining the suitability of a subject to be treated by the method of the present invention is provided, the method comprising:

[0135] a. receiving at least one oocyte from a female subject; and

[0136] b. measuring heterochromatin in the oocyte, wherein the heterochromatin level is below a predetermined threshold value indicating that the subject is suitable for IVM by the method of the present invention;

[0137] The suitability of a subject to be treated by the methods of the invention is thereby determined.

[0138] In some embodiments, the oocyte is an immature oocyte. In some embodiments, the oocyte is a germinal vesicle (GV) oocyte. In some embodiments, the oocyte is a GV-arrested oocyte. In some embodiments, the oocyte is harvested for IVF. In some embodiments, the method further comprises harvesting oocytes from females. In some embodiments, the subject is human. In some embodiments, it is found that the oocyte received is immature. In some embodiments, the oocyte received is immature. In some embodiments, immature refers to insufficient maturity. In some embodiments, the oocyte received is not suitable for IVF. In some embodiments, the subject has undergone at least one round of unsuccessful IVF. In some embodiments, unsuccessful refers to a round of IVF that does not produce implantation. In some embodiments, implantation is embryo implantation. In some embodiments, unsuccessful refers to a round of IVF that does not produce pregnancy. In some embodiments, unsuccessful refers to a round of IVF that does not produce a fetus. In some embodiments, unsuccessful refers to a round of IVF that does not produce birth. In some embodiments, unsuccessful refers to a round of IVF that does not produce a baby. A skilled technician in the field of IVF can determine whether an oocyte is suitable for IVF. Unused oocytes are suitable for diagnostic methods. In some embodiments, a heterochromatin level above a predetermined threshold indicates that the subject is suitable for another round of IVF. In some embodiments, a heterochromatin level below a predetermined threshold indicates that the subject is not suitable for another round of IVF. In some embodiments, a heterochromatin level below a predetermined threshold indicates that the subject is suitable for the method of the present invention. In some embodiments, a heterochromatin level below a predetermined threshold indicates that the subject is suitable for the method of the present invention, and then IVF is performed. In some embodiments, the method further includes performing the IVM method of the present invention on a suitable subject. In some embodiments, the method further includes performing another round of IVF on a subject whose chromatin level is above a predetermined threshold.

[0139] Assays for measuring heterochromatin are well known in the art, and any such assays may be used. Methods include those disclosed below: Western blot, immunostaining, chromatin immunoprecipitation, sequencing, methyl sequencing, bisulfite sequencing, and the like. In some embodiments, measuring heterochromatin comprises measuring histone 3 lysine 9 (H3K9) methylation. In some embodiments, methylation comprises monomethylation. In some embodiments, methylation comprises dimethylation. In some embodiments, methylation comprises trimethylation. In some embodiments, measuring heterochromatin comprises measuring H3K9me3. In some embodiments, measuring heterochromatin comprises measuring H4K20 methylation. In some embodiments, measuring heterochromatin comprises measuring H4K20me3. In some embodiments, measuring heterochromatin comprises measuring heterochromatin protein 1 (HP1). In some embodiments, measuring HP1 comprises measuring HP1 levels. In some embodiments, the levels are mRNA levels. In some embodiments, the levels are protein levels. In some embodiments, measuring heterochromatin comprises measuring methylcytosine. In some embodiments, methylcytosine is 5-methylcytosine. In some embodiments, the methylcytosine is the level of methylcytosine.

[0140] As used herein, the term "about" when combined with a numerical value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm±100 nm.

[0141] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "a polynucleotide" includes a plurality of such polynucleotides, "the polypeptide" includes one or more polypeptides and equivalents thereof known to those of ordinary skill in the art, and so forth. It is further noted that claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as antecedent authority for use of exclusive terminology such as "solely," "only," and the like in connection with reciting claim elements, or for use of a "negative" limitation

[0142] Where a convention similar to “at least one of A, B, and C, etc.” is used, generally speaking, such interpretation is intended to be in the sense that a person of ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). A person of ordinary skill in the art would further understand that almost any non-connective word and / or phrase that presents two or more optional terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one term, either term, or both terms. For example, the phrase “A or B” would be understood to include the possibility of “A” or “B” or “A and B”.

[0143] It will be appreciated that certain features of the invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for the sake of brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments related to the present invention are specifically encompassed by the present invention and are disclosed herein, as if each combination were individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed by the present invention and are disclosed herein, as if each such sub-combination were individually and explicitly disclosed.

[0144] Additional objects, advantages and novel features of the present invention will become apparent to those skilled in the art after studying the following examples, but these examples are not intended to be limiting. In addition, each of the various embodiments and aspects of the present invention as described above and claimed in the claims section finds experimental support in the following examples.

[0145] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0146] Example

[0147] In general, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and DNA recombinant techniques. These techniques are explained in detail in the literature. For example, see "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methods such as those described in U.S. Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated herein by reference. Other general references are provided in this document. .

[0148] Materials and methods

[0149] animal :RCC-C57BL / 6JHsd female mice were used for the experiments. For the young group, mice aged 7-11 weeks were used, and for the old group, mice aged 8-10 months were used. Mice were used after an acclimatization period of at least 3 days after transportation. The experiments were approved by the institutional ethics committee.

[0150] Mouse oocyte in vitro maturation : After euthanasia, ovaries were collected and dissected in L-15 medium (011151A) and supplemented with 200um IBMX (I7018) to arrest meiotic progression. Prophase I arrested oocytes were identified by observation of typical germinal vesicles and collected under binocular using a stripper (MD-MXL3-STR-CGR). After collection, oocytes were transferred to α-MEM medium (22561021) supplemented with IBMX, covered with mineral oil (M8410-1l) to prevent evaporation, recovered for 25min in an incubator at 37°C with 5% CO2, and then rinsed with α-MEM medium without IBMX to initiate meiosis. Oocytes were incubated in α-MEM under oil in the incubator for 6 hours (for prophase I) or 19 hours (for metaphase II). To assess maturation rate, oocytes were incubated for 19 hours after release and then fixed and stained by Hoechst to examine the entry into the second meiotic division. See below for modifications to drug treatment.

[0151] Collection of mouse oocytes for in situ immunofluorescence: After euthanasia, ovaries were collected and dissected in M2 culture medium (M7167). Oocytes were collected from the pool of at least 3 animals (the quantity of each experiment is indicated in the legend). Strippers were used to collect the prophase I arrested oocytes under binoculars, and rinsed with hyaluronidase (H4272-30MG) to remove follicle cells, and rinsed with acidic Tyrode's solution (T1788) to remove zona pellucida. PFA 4% (15710) was used to fix oocytes for 20 min, then quenched in the PBS supplemented with 10 mM glycine and 1% BSA.

[0152] Chromosome spreading and immunofluorescence As described above, prophase I arrested oocytes were collected from ovaries and incubated for 6 hours to reach the first prophase, or 17 hours to reach the second meiotic prophase. Mature oocytes were rinsed with M2 medium and then rinsed with acidic Tyrode's solution to remove the zona pellucida. 15 min before the desired time for chromosome expansion, oocytes were stored in a hypotonic solution consisting of FBS (F7524) diluted in DDW at a ratio of 1:1. Oocytes were then expanded in expansion solution (1% PFA buffered to pH 9.2, supplemented with 0.15% Triton X-100 and 0.03% DDT) on Superfrost plus slides (32090003). To assess ploidy, prophase II was extended with Hoechst staining to visualize chromatin, and CREST staining was used to visualize centromeres, and imaged chromosomes were manually counted.

[0153] In situ immunofluorescence : Permeabilization was performed using 0.1% Triton X-100. Cells were cultured in PBS containing 5% BSA for blocking, and primary and secondary antibodies were diluted in 0.1Tween 20 (P9416) / PBS containing 5% BSA. Immunostained cells were mounted in Vectashield (H-1000) mounting medium containing 80nM Hoechst (33342) and sealed on a slide with an imaging gasket (GBL654002).

[0154] Mouse ovarian IHF: Ovaries were collected, rinsed with PBS, and fixed in 4% PFA at 4° with shaking for 24 hours. After fixation, the ovaries were placed in 30% sucrose and shaken at 4° overnight. Tissues were stored at -80° in Tissue-Tek OCT compound (4583). Cryosectioning was performed on a Leica CM 1950 cryostat, and the sections were applied to superfrost plus slides. In order to capture most of the oocyte chromatin spanning approximately 30um in the Z plane of each cell, each section was 20um thick. In order to avoid having the same oocyte (50-70um) in two different sections on the same slide, each slide contained sections at least 80μm apart. Before staining, the slides were dried overnight at room temperature and rinsed extensively with PBS to remove OCT. Sections were incubated in PBS containing 1% FBS and 0.1% Triton X-100 for blocking. All antibodies were diluted in blocking solution at a concentration of 1:100. Slides were mounted in Vectashield mounting medium containing 80 nM Hoechst and sealed with nail polish.

[0155] Antibody : For heterochromatin staining, H3K9me2 (Abcam, ab1220, working dilution 1:400), H3K27me3 (Abcam, ab205728, 1:100), HP1γ (Abcam, ab227478, 1:50) antibodies were used. For euchromatin marks, H3K27ac (Cell signaling, 8173s, 1:100) and H3K4me3 (Cell signaling, 11960s, 1:50) antibodies were used. In order to track retroviral activity and loss of transcriptional regulation, L1-ORF1p (Abcam, ab216324, 1:100), dsRNA (SCICONS, 10020200, 1:500) and Dicer (Abcam, ab167444, 1:200) antibodies were used. Meiotic cohesion protein was stained with REC8 antibody (Abcam, ab192241, 1:200), and DNA damage was assessed using RAD51 (Sigma, PC130, 1:200) and γH2Ax (Sigma, 0563625UG, 1:500) antibodies. GFP expression was measured using anti-GFP antibody (Rockland 600-101-215, 1:250). Centromere visualization was achieved using CREST antibody (INC 15-235-0001, 1:100).

[0156] For western blot, primary antibodies included: GAPDH (Abcam, ab8245 1:3000), REC8 (Abcam, ab192241, 1:4000), L1-ORF1p (Abcam, ab216324, 1:1000), Dicer (Abcam, ab167444, 1:1000) and Tubulin (T5168 1:4000). Anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG H&L (HRP) ab6721 (1:10,000) and rabbit anti-mouse IgG H&L (HRP) ab6728 (1:10,000) or goat anti-mouse IgG H&L (HRP) ab97040 (1:10,000) were used.

[0157] Imaging and quantification : Oocytes were imaged using a Ti-Eclipse Nikon system with an Andor Zyla nsc05537 camera. When light imaging could not obtain clear images, confocal imaging was performed using a Yokogawa W1 spinning disk on a motorized fluorescence microscope Ti2E with a 2sCMOS ZYLA camera or a Zeiss LSM710. Each cell was imaged in multiple planes. For quantitative analysis of images, maximum intensity projections were created. To measure staining intensity, each evaluation area was normalized to an equal-sized area of ​​the background (outside the target area). An intensity score was generated by dividing the intensity of the target area by the intensity of the background.

[0158] Imaging and quantification : For quantitative analysis of the images, maximum intensity projections were created. To measure staining intensity, each evaluation area was normalized to an equal-sized area of ​​the background (outside the target area). The intensity of the target area was divided by the intensity of the background to generate an intensity score.

[0159] Small RNA Sequencing About 50 oocytes from young and old females (triplicates per group) were collected from ovaries as above (numbers matched between each experimental group) and dissected in M2 medium, and after removal of follicular cells and zona pellucida (as described above), oocytes were inserted into 1 ml TRIzol (15596026). RNA extraction was performed according to the TRIzol reagent user guide in 10 μl of nuclease-free water. -12Rxns Amplification was performed using the smRNA-Seq kit (TAKARA 635029). Using Agencourt AMPure XP beads, 50ul of sample was added to 100ul of bead solution, and DNA bound to the beads was eluted (selecting transcripts below 200bp) and size selected. Sequencing was performed on a NextSeq machine at the Interdepartmental Equipment Park on the Ein Kerem campus.

[0160] Trimming and filtering raw reads NextSeq base call files were converted to fastq files using the bcl2fastq program with default parameters (no trimming or filtering was applied at this stage). Raw reads (fastq files) were checked for quality issues using FastQC. Reads were then quality trimmed at both ends, poly-G sequences (no signal for NextSeq), adapter sequences, and poly-A sequences were removed from the 3' end according to the SMARTer smRNA-Seq library construction protocol, and finally low-quality reads were filtered out. Cutadapt was used to trim sequences at the ends of reads, with parameters including using a minimum overlap of 1, allowing read wildcards, and filtering out reads shorter than 15 nt. Final filtering by quality was performed using the fastq_quality_filter program in the FASTX software package, with a quality threshold of 20 at 90% or more of the read positions.

[0161] Alignment and counting : Processed reads were aligned to the mouse transcriptome and genome using TopHat. The genome version was GRCm38, and annotations were from Ensembl release 99. Quantification was done using htseq-count.

[0162] Differential expression :DESeq2 software package was used for normalization and differential expression analysis. The results were corrected for batch effects and further normalized to control gene expression (gene Rian (Hatada et al., 2001, PMID: 11481034) between samples. The original data were submitted to GEO, accession number GSE159789. The sequencing results are shown in Wasserzug-Pash et al., "Loss of heterochromatin and retrotransposon silencing as determinants inoocyte aging", Aging Cell, 2022Mar; 21(3): e13568, which is incorporated herein by reference in its entirety.

[0163] qRT-PCR: As above, after euthanasia, mouse ovaries were collected and dissected in M2 medium (M7167) containing 200um IBMX (I7018) to arrest meiotic progression. 40 to 60 (numbers matched between groups in each experiment) prophase I arrested oocytes were collected and rinsed with hyaluronidase (H4272-30MG) to remove follicle cells, followed by extensive washing with M2 until all follicle cells were removed from the oocytes. Oocytes were transferred to TRIzol (15596026) and chloroform solution for RNA precipitation, treated with RNase-free DNase (E0013-1D3) to remove DNA, and then RNA was purified by using Ampure beads (A63881). Using iScript TM Reverse transcription was performed using Reverse transcription Supermix for RT-qPCR (1708891) according to the manufacturer's recommendations. Amplification reactions were performed using iTaq TM Universal Green Supermix (1725124). Quantitative PCR measurements were performed using a CFX96 C1000 BioRad machine.

[0164] Mouse oocyte western blot : After euthanasia, ovaries were collected and dissected in L-15 medium (011151A) supplemented with 200 μM IBMX (I7018) to arrest meiotic progression. 101 prophase I arrested oocytes were collected from each ovary, rinsed with hyaluronidase (H4272-30MG) to remove follicle cells, and then rinsed extensively with L-15 until all follicle cells were removed from the oocytes. The clean oocytes were then rinsed with PBS until all the medium was washed off and transferred to RIPA buffer (1% NP-40, 0.1% SDS, 50mM Tris, 150mM NaCl, 0.5% sodium deoxycholate, 1mM EDTA, 1X complete TMProtease Inhibitor Cocktail 11836145001) and stored on ice. Protein extraction was performed by boiling the lysate in Laemmli buffer (1610747) 335mM 2-mercaptoethanol. Under reducing conditions, the lysate was fractionated by 10% acrylamide SDS gel and transferred to a nitrocellulose membrane (Millipore) using a transfer device according to the manufacturer's (Bio-Rad) protocol. The blot was developed using an ECL system according to the manufacturer's (Bio-Rad) protocol. Results were collected using Bio-Rad's ChemiDoc XRS+ system. After euthanasia, ovaries were collected and dissected in L-15 medium (011151A) supplemented with 200um IBMX (I7018) to prevent meiotic progression. 101 prophase I arrested oocytes were collected from each ovary, washed with hyaluronidase (H4272-30MG) to remove follicle cells, and then washed extensively with L15 until all follicle cells were removed from the oocytes. The clean oocytes were then rinsed with PBS until all the culture medium was washed away and transferred to RIPA buffer (1% NP-40, 0.1% SDS, 50mM Tris, 150mMNaCl, 0.5% sodium deoxycholate, 1mM EDTA, 1X cOmplete TM Protease Inhibitor Cocktail 11836145001) and placed on ice. Protein extraction was performed by boiling the lysate in Laemmli buffer (1610747) 335mM 2-mercaptoethanol. Under reducing conditions, the lysate was fractionated by 10% acrylamide SDS gel and transferred to a nitrocellulose membrane (Millipore) using a transfer device according to the manufacturer's (Bio-Rad) protocol. The blot was developed using an ECL system according to the manufacturer's (Bio-Rad) protocol. Results were collected using the ChemiDocXRS+ system from Bio-Rad.

[0165] Drug treatmentOvaries were collected and dissected in L-15 medium supplemented with 200 um IBMX to arrest meiotic progression as described above. Prophase I arrested oocytes were collected using a stripper as described above. After collection, oocytes were transferred to α-MEM medium supplemented with IBMX and the desired drugs, covered with mineral oil to prevent evaporation, and incubated at 37° in a 5% CO2 incubator for 4 hours (no pause was performed in the chaetocin experiment because chaetocin and IBMX are cross-reactive with each other). After the pause, oocytes were rinsed with α-MEM without IBMX containing drugs to initiate meiosis. 19 hours after the oocytes were released from IBMX, the oocytes were fixed with PFA, permeabilized, and sealed as described above. Chaetocin is a hazardous substance. For biosafety reasons, the chaetocin-supplemented plates and matched controls were placed in a sealed chamber during the incubation time. The drugs used were Trichostatin A (TSA) T1952, Chaetocin (sc-200893), SRT1720 (biovision 2772) and Zidovudine (AZT) (PHR1292-1G).

[0166] Oocyte electroporation As mentioned above, ovaries were collected and dissected in L-15 culture medium supplemented with 200um IBMX to stop meiotic progression. Prophase I oocytes were rinsed with acidic Tyrode's solution and transferred to EC-002 electroporation cuvettes, which contained 100ul clean L-15 culture medium for the control group and 50-200ng / ullonza pmaxGFP (DMC00054) or gene expression plasmid for the experimental group. Electroporation was performed in Nepagene NEPA21 electroporators. Electroporated oocytes were simply rinsed and then cultivated in the α-MEM supplemented with IBMX. After electroporation for 24 hours, oocytes were fixed and stained with reporter antibodies.

[0167] Human In Vitro Fertilization (IVF) Program : Patients were treated with one of two ovarian stimulation regimens determined by their physicians. The long-acting agonist regimen began in the luteal phase of the menstrual cycle, or the antagonist regimen began on the second day of the menstrual cycle. For controlled ovarian hyperstimulation in both regimens, recombinant FSH preparations, recombinant FSH and LH, or HMG (human menopausal gonadotropin) were used. The dose was determined individually for each patient based on BMI and ovarian reserve parameters. The ovarian response was monitored by serial ultrasound examinations and serum E2 level assessments, and gonadotropin dose adjustments were then made as needed. Human chorionic gonadotropin (HCG) was injected when at least 3 leading follicles were 17-18 mm in diameter. Oocyte retrieval was performed 36 hours after HCG administration using a transvaginal ultrasound-guided approach.

[0168] Human oocyte Human prophase I arrested oocytes retrieved during IVF treatment were incubated for 24 hours and then determined to remain in this state without maturation into fully grown MII oocytes. After written informed consent (following IRB approval 0020-16-SZMC), embryologists processed oocytes by puncturing the zona pellucida to increase permeability. The day after retrieval, oocytes were fixed in 4% PFA for 20 min at room temperature and then quenched in PBS supplemented with 10 mM glycine and 1% BSA. Immunofluorescence was performed as described above. Prophase I arrested oocytes, identified by typical germinal vesicles and dispersed chromatin configuration, and oocytes arrested after meiotic resumption, identified by lack of germinal vesicles and chromatin condensation, were analyzed separately. Oocytes that entered the second meiotic division, identified by chromatin fragmentation and polar body extrusion, were excluded from the data.

[0169] Specificity of REC8 antibody :HCT116 human colon cancer cell line cells were maintained in Dulbecco's Modified Eagle's medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS), 1% PenStrep (100U / mL penicillin and 100μg / mL streptomycin) in a 37°C incubator (5% CO2). The cell line was identified at the Biomedical Core Facility of the Technion, Haifa, Israel. REC8 and EGFP alone (vector pEGFP-N1, plasmid number 170528HM8137-6, from BD Biosciences) labeled with enhanced green fluorescent protein (EGFP) were used. The expression vectors were obtained from Qiagen. Spin miniprep kit (cat-27104, Germany) was used to extract plasmids. All cells were transfected 24 hours after initial plating. Transfection was performed using PolyJet TM In vitro DNA transfection reagent (cat-SL100688, USA) was used at a 3:1 (transfection reagent: DNA) ratio. 48 hours after transfection, adherent cells were lysed using hot sample buffer (10% glycerol, 50mmol / L Tris-HCl pH 6.8, 20% SDS and 5% 2-mercaptoethanol) and subjected to Western blot analysis. The proteins were analyzed by SDS PAGE (Criterion TM TGX(Tris-Glycine eXtended)Stain-Free TMLysate was fractionated using precast gels (Bio-Rad) and transferred to nitrocellulose membranes (Millipore) using a transfer device according to the manufacturer's (Bio-Rad) protocol. The blots were developed using an ECL system according to the manufacturer's (Bio-Rad) protocol. Results were collected using the ChemiDoc XRS+ system from Bio-Rad.

[0170] Statistical analysis : Statistical analysis was performed using Excel, R, and GraphPad prism. For comparison of means, parametric tests (t-test for single comparisons and one-way Anova for group comparisons) were used if N>30 or if the Shapiro Wilk test returned no significance. Otherwise, nonparametric tests were used (Mann-Whitney (MW) for single comparisons and Kruskal Wallis (KW) for group comparisons). To compare differences in the presence of transcripts between groups, a ratio paired T-test was used. For comparison of proportions, a Z-test of two proportions was used - when normality of the parameters was confirmed by N>30 or N*P>5 for each comparison group. Correlations were calculated using the Person correlation coefficient, and the significance of the calculated R was determined using the F-test.

[0171] Human IVM Protocol :GV oocytes are subjected to IVM overnight in the Sage culture medium (Al-rad Medical) supplemented with 0.075IU / mL luteinizing hormone (LH) and 0.075IU / mL follicle stimulating hormone (FSH). In some experiments, HAT and / or RT inhibitors are added to overnight cultures. All naked oocytes found are transferred and cultured 48 hours in the oocyte maturation culture medium (Sage In-Vitro Fertilization) supplemented with 0.075IU / mL human menopausal gonadotropin (hMG) (Menopur, Ferring Pharmaceuticals). In some experiments, HAT and / or RT inhibitors are added to 48 hours cultures. After incubation for 24 hours, mature oocytes are searched for in culture medium and frozen immediately. Then culture medium is incubated for another 24 hours, other mature oocytes are searched again.

[0172] Improved human IVF protocol:GV oocytes are incubated in oocyte maturation medium (Sage In-Vitro Fertilization) supplemented with 0.075IU / mL hMG (Menopur, Ferring Pharmaceuticals) and HAT and / or RT inhibitors. All MII oocytes (retrieved or obtained by IVM incubation) are stored in oocyte preservation medium supplemented with HAT and / or RT inhibitors and ICSI is performed. Fertilized dipronuclear zygotes are cultured separately for 2 or 3 days in 20mL cleavage stage medium (Sage), and blastocyst culture is also carried out using commercially available culture medium (Quinn's Advantage; Sage). All embryos are cultured with full humidity in a water-jacketed small multi-gas incubator (Astec) at 37°C under the gas phase conditions of 5% O2, 5% CO2 and 90% N2. Blastocyst culture, selective vitrification and subsequent freezing-thawing ET are all routinely performed. Embryos showing better quality are transferred at the cleavage stage. The other embryos were cultured to the blastocyst stage.

[0173] Embodiment 1:

[0174] To investigate the status of heterochromatin histone marks in oocytes at ages prior to the onset of aneuploidy, we compared the levels of specific epigenetic marks in prophase I arrested oocytes from unstimulated (i.e., not superovulated) 2-month-old (young) and 9-month-old (old) female mice by immunofluorescence (IF). In accordance with previous studies, oocytes from 9-month-old mice had lower aneuploidy and were not significantly different from those of young mice. However, other traits related to fertility, such as the maturation level of 9-month-old oocytes by IVM (in vitro maturation), the number of oocytes found in the oviduct after superovulation, and fecundity, had clearly decreased. The mouse strain we used in this study (RCC-C57BL / 6JHsd, see Methods) showed findings consistent with this decrease, namely that the maturation rate of old mice was 27% lower than that of young mice, while the aneuploidy rate was the same between the two age groups ( Figures 1A-1C We then immunostained oocytes for the constitutive heterochromatin markers H3K9me2 and HP1γ. When assessed by in situ immunofluorescence staining, the levels of both markers decreased significantly with age ( Figure 2A-2B , see Methods for details). In addition, levels of the facultative heterochromatin mark H3K27me3 also decrease with age when assessed by in situ immunofluorescence staining ( Figure 2C ).

[0175] There are two main chromatin configuration forms in mouse and human prophase I oocytes, which characterize two developmental stages. The first chromatin configuration in mature oocytes is called non-surrounded nucleolus (NSN), which is characterized by a diffuse chromatin configuration. As oocytes grow, chromatin gradually organizes in a ring structure around the nucleolus, and this configuration is called surrounded nucleolus (SN). The transition from NSN to SN is accompanied by a variety of cellular changes, including comprehensive histone modification changes and transcriptional silencing, which lasts throughout the entire oocyte maturation and early embryogenesis process. Interestingly, with age, defects in heterochromatin modifications are much more obvious when oocytes enter the SN stage-when oocyte transcription is turned off. However, facultative heterochromatin reduction has consistently occurred in oocytes (both NSN and SN) at all prophase I arrest stages, showing that some age-related epigenetic defects can be seen in the early stages of oocyte maturation. Importantly, transcription remains active in prophase I arrested oocytes, as shown by BrU incorporation into RNA, and complete transcriptional shutdown occurs only later during oocyte maturation. This was confirmed by plasmid electroporation experiments showing eGFP expression in electroporated prophase I arrested oocytes.

[0176] To investigate whether the reduction in repressive marks occurs due to loss of nucleosomes or histones, and to control for differences in oocyte staining capacity between young and aged oocytes, we stained oocytes for the active chromatin marks H3K27Ac and H3K4me3 by in situ immunofluorescence. Importantly, no significant differences were detected between young and aged oocytes for these two chromatin marks ( Figure 2D ). These results show that the loss of histone marks is specific to heterochromatin modifications and that no detectable histone loss occurs in aged oocytes.

[0177] To achieve information about a wider range of oocyte populations and to avoid possible confounding effects of the immunofluorescence staining procedure, we performed IHF on mouse ovarian cryosections. Using this approach, we measured heterochromatin levels in a wider range of oocyte stages and sizes than could be achieved with manual collection of oocytes. Age-dependent changes in heterochromatin loss in ovarian cryosections were also evident, as evidenced by lower levels of H3K9me2 and H3K27me3 in aged ovarian oocytes compared to young groups ( Figure 3A-3B). This trend was observed in all oocytes of all sizes. Furthermore, having characterized the epigenetic changes in aged immature oocytes, we also explored whether epigenetic defects persist into later stages of oocyte maturation. To this end, we investigated heterochromatin marks in mid-MI oocytes by performing chromosome spreads followed by immunostaining. The spreads were stained for H3K9me2 and H3K27me3 ( Figure 3C-3D ). In these experiments, a significant decrease in the signal was observed in older oocytes. This result shows that the epigenetic defects that occur in older oocytes persist into meiosis. Since the extended experiments enable observation of entire chromosomes, we also asked whether the reduction in repressive chromatin marks occurs at specific loci in the genome or is uniformly reduced along the entire chromosome. In our analysis, no significant specific preferential patterns were found. Despite the limited ability to discern specific regions of the genome by microscopy, this result suggests that the reduction in heterochromatin is widespread and includes many regions of the genome.

[0178] Histone post-translational modifications are key components of transcriptional regulation, particularly in the silencing of transposable elements (TEs), which comprise approximately 50% of the mammalian genome. Activation of TEs can pose a significant threat to the cellular genome, and regulation and silencing of TE activity are increasingly being recognized as hallmarks of cellular integrity in health and disease. To investigate the consequences of heterochromatin loss on transcription of TEs in aged oocytes, we examined RNA expression of two families of retrotransposons: long interspersed nuclear elements-1 (L1) and intracisternal A particles (IAPs), which have been shown to be expressed in the germline. As assessed by quantitative RT-PCR, expression of L1 and IAP transcripts was increased approximately 2-fold in oocytes from older females compared with young oocytes ( Figure 4A By Western blotting for the L1 retrotransposon protein, L1-encoded ORF1p, elevated synthesis of retrotransposon-derived proteins in aged oocytes was detected ( Figure 4B We confirmed these findings by immunostaining oocytes. Low levels of L1-ORF1p were detectable in young oocytes, whereas the intensity increased in older oocytes ( Figure 4C ). With the increase in activity of these TEs, older oocytes show increased recruitment of the DNA repair machinery indicative of DNA damage. This is indicated by elevated nuclear localization of Rad51 and more presence of γH2Ax foci in older oocytes ( Figure 4D-4E γH2Ax foci are considered a true marker of DNA damage and repair. Elevated nuclear localization of Rad51 is also a marker of DNA damage and has been observed in HeLa and HCT116 cells after treatment with ionizing radiation.

[0179] We wondered whether inhibiting the reverse transcription activity of retrotransposons, and therefore retrotransposition in older oocytes, would improve their cellular integrity and maturation capacity. To this end, we matured older oocytes in vitro with or without the addition of azidothymidine (AZT), a reverse transcriptase inhibitor that has been shown to inhibit retrotransposon activation in fetal oocytes. As a thymidine kinase 1 inhibitor, AZT also exhibits toxic effects on cell growth in some cell lines. However, previous studies in oocytes have not reported toxic effects in this system. Nevertheless, we used lower concentrations than those reported to be toxic (see Methods). Our results ( Figure 4G ) showed that the relatively low maturation level of older oocytes in vitro could be partially rescued and showed that the maturation of mature oocytes was increased up to 28.6% after incubation in 4 μM AZT. We also showed that DNA damage was also reduced by AZT treatment, as manifested by a reduction in the γH2Ax locus in these oocytes ( Figure 4F Despite these effects, AZT treatment did not alter heterochromatin levels in older oocytes ( Figure 4H ), suggesting that heterochromatin loss may be upstream of retrotransposon elevation.

[0180] In addition to retrotransposon expression in older oocytes, we investigated whether we also saw evidence for increased retrotransposon RNA processing. Increased retrotransposon expression can lead to increased retrotransposon mRNA expression and its translation into protein, but can also lead to increased processing. RNA processing systems, such as RNAi, regulate gene expression by post-transcriptionally targeting mRNAs from regulatory regions. This processing system inhibits unregulated transcriptional activity through Dicer-mediated miniaturization of transcripts into small RNAs and activation of various downstream targeting and silencing complexes such as Argonaute proteins. Increased activity of the RNA processing machinery suggests that a loss of transcriptional regulation has occurred, and by analyzing small RNA repertoires it may be possible to identify specific loci where this loss has occurred. We therefore performed small RNA sequencing in oocytes from young and old females. We sequenced (in duplicate) RNA molecules ranging from 17 to 167 bp (median 20–80 percentiles, see Methods), with a median molecule size of 18 bp in young oocytes and 106 bp in older oocytes. Measurement of the size of small RNA fragments was done computationally (see Methods). A clear peak at 155 bp for ribosomal RNA was observed in both samples. The difference in small RNA size between young and old oocytes shows an increase in the presence of RNA fragments of this size, which are not typical Dicer products and may originate from spurious transcription due to the loss of genomic repression by heterochromatin. Small RNAs can originate from different regions of the genome. However, RNA expression differences between young and old oocytes were significant, especially in the group of small RNAs from genomic repetitive sequences (usually marked by heterochromatin). Most repeat types remained unchanged between young and older oocytes (83%). In older oocytes, 4.6% of the repeat types were overexpressed, and 12.1% were more than 2-fold underexpressed. However, when looking at which repeat types changed, the list of overexpressed repeat types in older oocytes ( Figure 5A ) showed that the majority of RNA molecules were derived from retrotransposons, while within the low-expressing repeats, there were additional repeat types. When we compared the fold changes in expression between the different families of RNA molecules in the data, we found that while in tRNA and rRNA, there was no increase in older compared to younger oocytes, but in genomic repeats in general, and in retrotransposons in particular, transcripts were significantly increased in older compared to younger oocytes ( Figure 5B Consistent with the greater abundance of retrotransposon mRNAs and retrotransposon-derived small RNAs in some repetitive sequences in older oocytes, we also found that the signal of dsRNA molecules was significantly increased in older oocytes ( Figure 5CIn addition, Dicer protein (an enzyme involved in retrotransposon processing) also showed an increase by both immunostaining and Western blot analysis ( Figure 5D-5E ). We therefore conclude that loss of epigenetic silencing in aged oocytes is accompanied by transcriptional activity and processing of retrotransposons in the genome.

[0181] Since there are some significant differences in epigenetic regulation between mice and humans, we wanted to investigate whether heterochromatin loss with maternal age also occurs in human oocytes. To this end, we investigated heterochromatin in oocytes from IVF treatments. We retrieved oocytes from 33 patients, with an average of 2.2 oocytes per patient. Generally, oocytes arrested in prophase I are not used for fertilization in the clinic, but to ensure that the oocytes we used for our studies could not mature in vitro, we kept the oocytes in culture for an additional 24 h, and then we fixed and stained the immature oocytes. Therefore, these oocytes most likely represent a subset of human oocytes that are meiotically incompetent. Indeed, fixed oocytes stained for DNA visualization were arrested at several developmental stages (Fig. 6). In addition, we stained the oocytes by in situ immunofluorescence staining for H3K9me2. Strict QC criteria were applied to the staining results in order to exclude oocytes with fragmented or damaged genomes (see Methods). We found that the H3K9me2 signal decreased in an age-dependent manner in oocytes arrested during prophase I and after release from meiotic arrest, which was fitted with a linear decline curve ( Figures 6A-6D As a control, we also stained human oocytes for the Rec8 meiotic cohesion protein. This staining also showed ( Figure 6E-6H ) an age-dependent downward trend, as shown previously for mouse and human cohesin.

[0182] To show a causal link between heterochromatin degradation and maturation defects in aged oocytes, we treated young mouse oocytes with chemicals known to affect epigenetic regulation. The Suvar39h1 / 2 enzymes are essential for heterochromatin formation, and chaetocin has been characterized as a specific and potent inhibitor of Suvar39h1 / 2. Therefore, we treated 2-month-old oocytes with 0.5 μM chaetocin in vitro for 18 h. After treatment, staining for H3K9me2 was reduced by staining for chromosome spreads at metaphase MI. However, in situ staining for H3K4me3 in MI-treated oocytes did not show significant changes, indicating that heterochromatin manipulation with chaetocin does not interfere with other aspects of chromatin regulation. Assessment of the efficiency of oocyte maturation after treatment with chaetocin showed an 18% decrease in the number of normally matured oocytes after treatment ( Fig. 7A). After treatment, treated oocytes were stained by in situ immunofluorescence. L1-encoded ORF1p showed an increase in staining intensity ( Figure 7B Consistently, dsRNA staining was significantly increased in treated oocytes compared to untreated control oocytes ( Figure 7C ).

[0183] Since heterochromatin characterized by H3K9me2 and binding to HP1γ is also associated with histone deacetylation at H3K27, we sought to investigate whether inhibition of histone deacetylation in oocytes would result in similar effects. Previous reports have also shown that treatment with histone deacetylase (HDAC) inhibitors affects oocyte maturation in vitro. Therefore, we treated young oocytes with the HDAC inhibitor trichostatin A (TSA, 100 nM, 4 h arrest, then 18 h until MII). In vitro treated oocytes showed an increase in H3K27Ac by in situ staining, as well as a decrease in H3K9me2, linking different heterochromatin pathways in oocytes, such as H3K27 deacetylation and H3K9 methylation. TSA-treated young oocytes showed similar maturation defects as chaetocin-treated young oocytes, and H3K4me3 staining was unchanged. Interestingly, TSA-treated oocytes did not show an increase in the overall signal level of L1-encoded ORF1p. In contrast, L1-ORF1p accumulated in the nuclei of treated oocytes, which may indicate that deacetylation activity is central to a specific stage in the maturation of retrotransposons, leading to enhanced nuclear recruitment of L1 proteins when deacetylation does not occur. However, in TSA-treated oocytes, the DNA damage response was elevated, as shown by increased nuclear localization of Rad51 in treated oocytes. Together, these results show that epigenetic manipulation in the H3K9 methylation or H3K27 acetylation pathways results in a failure to mature young oocytes and mimics natural aging.

[0184] To further strengthen the causal link between heterochromatin loss, retroviral activity, and oocyte maturation defects in older oocytes, we tested whether increasing heterochromatin by genetic overexpression could reverse the oocyte aging phenotype. To this end, we used plasmid electroporation as above to overexpress EZH2, an enzyme subunit of the PRC2 complex that catalyzes H3K27 methylation, and SIRT1, an NAD + EZH2-electroporated oocytes showed elevated H3K27 methylation staining compared to empty electroporated controls ( Fig. 8A ), oocytes electroporated with SIRT1 plasmid showed increased H3K9 methylation ( Fig.8DIn both groups, the increase in heterochromatin resulted in a significant decrease in L1-ORF1 staining ( Figure 8B , 8E ), showing that when heterochromatin was restored in these oocytes, they regained the ability to silence retroviral activity. Finally, treated oocytes exhibited an increased maturation rate compared to empty electroporated controls ( Figure 8C , 8F ). To confirm these results with another manipulation method targeting sirtuins, we cultured aged oocytes with the SIRT1 activating molecule SRT-1720. The treated oocytes had a phenotype similar to the overexpression experiments described above. SRT-1720 treatment triggered an increase in heterochromatin, which led to the restoration of maturation competence of the treated cells ( Figure 8G-8H ). Notably, previous studies have shown that supplementation of SIRT1-associated metabolic cofactor nicotinamide adenine dinucleotide (NAD + ) also showed similar results, and this cofactor rejuvenated oocyte quality in aged animals. Taken together, these results show that heterochromatin loss occurs upstream of retrotransposon activity and that heterochromatin loss may be reversible in older oocytes.

[0185] Example 2: IVM with HATI and RTI

[0186] We next tested whether similar results could be achieved without genetic manipulation. Aged oocytes were treated with two different naturally occurring histone acetyltransferase (HAT) inhibitors: curcumin and mangosteen. As a negative control, oocytes were given vehicle alone. Curcumin produced a dose-dependent increase in maturation rate, such that at the highest dose (10 μM), maturation rates of over 80% ( Fig. 9A ). Mangosteen also increases the rate of maturity, but to a lesser extent ( Fig. 9B Next, a broader panel of natural HAT inhibitors was tested along with another reverse transcriptase (RT) inhibitor ( Fig. 9C ). As before, aged oocytes were incubated in IVM medium (α-MEM) supplemented with inhibitors for 24 h, and then maturity was measured. Oocytes with both first polar body extrusion and normal chromosome division (as assessed by Hoechst staining) were considered mature. Curcumin was used as a positive control. It was found that the new RT inhibitor lamivudine (3TC) and one of the HAT inhibitors plumbagin ( Fig. 9C ) is comparable to curcumin in its ability to induce maturation. The other three HAT inhibitors tested—garcinic acid, anacardic acid ( Fig. 9C ) and EGCG( Fig.9D )—neither produced increased maturity.

[0187] Garcinia and anacardic acid are highly acidic, and EGCG is more weakly acidic. There is evidence in the literature that lower pH levels are detrimental to oocyte maturation. The pH of the medium used was approximately 7.3, with a buffering capacity between 6.9 and 7.6. The medium does not have a particularly strong buffering capacity, so the addition of these three molecules makes it acidic (more so for gambogic and anacardic acids). This acidity interferes with maturation and, in fact, inhibits maturation in the case of the two strongly acidic molecules. Culture media with greater buffering capacity or different concentrations of these acidic molecules would allow HAT inhibition-induced maturation improvements. It was further noted that gambogic and anacardic acid strongly inhibit PCAF, while curcumin and plumbagin do not, which may lead to differences in maturation. Taken together, these results show that heterochromatin loss occurs upstream of retrotransposon activity and that heterochromatin loss may be reversible in older oocytes, including by HAT inhibitors.

[0188] Finally, the combination of a HAT inhibitor and a RT inhibitor was tested. Aged oocytes were cultured as before but in the presence of both curcumin and AZT, and maturity was measured 24 hours later. Interestingly, the effect of this combination was superior to that of the HAT inhibitor alone ( Fig.9D This was somewhat unexpected, as heterochromatin loss is upstream of retrotransposon activity, so one might have assumed that once curcumin was added, AZT would have no further effect, yet a significant increase was observed with this combination.

[0189] Example 3: Human oocyte IVM with HATI and RTI

[0190] We wanted to investigate whether heterochromatin loss with maternal age is constant in human oocytes. To this end, we investigated epigenetic marks in prematurely arrested oocytes from IVF treatment. Patients were admitted to an IVF clinic and stimulated according to a protocol determined by the physician (see Methods). The antagonist protocol started on the second day of the menstrual cycle. The specific dose was determined individually for each patient, based on BMI and ovarian reserve parameters. When at least 3 leading follicles reached 17–18 mm in diameter, a gonadotropin-releasing hormone (GnRH) antagonist or human chorionic gonadotropin (hCG) or a combination of both was administered for final oocyte maturation. 36 h after final maturation, oocyte retrieval was performed using a transvaginal ultrasound-guided approach. These oocytes (>150 μm) represent a subset of human oocytes that are indeed arrested at several developmental stages ( Figures 10A-10B ).

[0191] In addition, we performed H3K9me2 staining of oocytes by in situ immunofluorescence. After analyzing the ratio between DNA and chromatin staining and using the shape of the genomic material, oocytes with fragmented or damaged genomes were excluded. Our preliminary results showed an age-dependent decrease in H3K9me2 signals in oocytes arrested at the GV stage (GV clearly present) or after GV breakdown (GVBD), which was fitted with a linear decline curve (GV R2 = 0.21, GBVD R2 = 0.35, Figures 10A-10B ). As a positive control, we also stained human oocytes for the Rec8 meiotic cohesion protein. This staining also showed an age-dependent downward trend, as previously shown for mouse and human cohesion proteins. Therefore, epigenetic degradation and loss of heterochromatin also occur in human oocytes. In addition, it should be noted that oocytes from some patients showed significantly less heterochromatin than expected for their age (points in the lower left corner below the trend line). Oocytes from these women are epigenetically older than their physiological age and can particularly benefit from the maturation methods provided herein.

[0192] To also observe non-arrested human oocytes, we studied the behavior of another batch of human oocytes extracted from patients who had undergone gonadotoxic treatments and therefore fertility preservation. In these cases, surgical retrieval of the ovaries was performed when hormonal induction and oocyte retrieval were not possible due to the young age of the patients or social reasons. After retrieval, the ovaries were dissected in culture medium in the IVF laboratory and the ovarian tissue was cryopreserved. After remission, the ovarian tissue could be transplanted and IVF performed. During ovarian dissection, immature oocytes were released into the culture medium. These are usually discarded. After obtaining ethical committee approval and informed consent from the patients, we retrieved oocytes of 150 μm and above from the culture medium and matured them in vitro overnight using Sage medium supplemented with 0.075 IU / mL luteinizing hormone (LH) and 0.075 IU / mL follicle-stimulating hormone (FSH). Oocyte denudation was performed 24 h later. Thereafter, polar body extrusion, chromatin status, and immunostaining of epigenetic markers were evaluated. Incubation of young oocytes with AZT, mangosteen or curcumin did not impair their maturation capacity and even enhanced it ( Fig.11 ). Therefore, by supplementing with HAT inhibitors, RT inhibitors or a combination thereof, immature oocytes can be matured in a superior manner.

[0193] Although the present invention has been described in conjunction with its specific embodiments, many alternatives, modifications and variations are apparent to those skilled in the art. Therefore, the present invention is intended to encompass all such alternatives, modifications and variations within the spirit and broad scope of the appended claims.

Claims

1. A cell culture medium comprising luteinizing hormone (LH), follicle stimulating hormone (FSH) and at least one histone acetyltransferase (HAT) inhibitor or reverse transcriptase (RT) inhibitor.

2. The culture medium according to claim 1, wherein the culture medium is a human oocyte in vitro maturation (IVM) medium comprising at least a HAT or RT inhibitor.

3. The culture medium according to claim 1 or 2, wherein the HAT is selected from E1A-associated protein p300 (p300), cyclic AMP response element binding protein binding protein (CREBBP / CBP), histone acetyltransferase KAT5 (KAT5 / Tip60), KAT6A and N-acetyltransferase 10 (NAT10).

4. The culture medium according to any one of claims 1 to 3, wherein the HAT inhibitor is not a P300 / CBP-associated factor (PCAF / KAT2B) inhibitor.

5. The culture medium according to any one of claims 1 to 3, wherein the HAT inhibitor is selected from the group consisting of curcumin, garcinol, spermidine-coenzyme A (SpD-CoA), Lys-CoA, histone H4 lysine 16 (H4K16)-coenzyme A, MG149, EML264, LTK-14, cyanidin-B3, epicatechin gallate (EGCG), anacardic acid, gambogic acid, delphinidin, Remodelin, butyrolactone 3 (MB-3), NK13650A, balsam pear dione (NP-15), Δ12-prostaglandin J2 (PGJ2), plumbagin (RTK1), NU9056, DC-M01-7, CCT077791, NSC694614, PU141, C646, TH1834, L002, WM-8014, DC-G16-11 and derivatives thereof.

6. The culture medium according to any one of claims 1 to 4, wherein the HAT inhibitor is a naturally occurring molecule.

7. The culture medium according to claim 5, wherein the HAT inhibitor is selected from the group consisting of curcumin, plumbagin and mangosteen.

8. The culture medium according to any one of claims 1 to 7, wherein the RT inhibitor is selected from the group consisting of zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, Truvada, azithromycin, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, doravirine, islavirine and derivatives thereof.

9. The culture medium according to claim 8, wherein the RT inhibitor is selected from zidovudine (azidothymidine, AZT) and lamivudine (3TC).

10. The culture medium according to any one of claims 1 to 9, comprising both a HAT inhibitor and a RT inhibitor.

11. The culture medium according to claim 10, comprising curcumin and AZT.

12. The culture medium according to any one of claims 5 to 11, wherein the curcumin is present at a concentration of about 5-20 μM.

13. The culture medium of claim 12, wherein the curcumin is present at a concentration of about 10 μM.

14. The culture medium according to any one of claims 8 to 13, wherein the AZT is present at a concentration of about 0.5-5 μM.

15. The culture medium of claim 14, wherein the AZT is present at a concentration of about 1 μM.

16. The culture medium according to any one of claims 1 to 15, wherein the cell culture medium comprises a pH value between 6.8 and 7.

8.

17. The culture medium according to any one of claims 1 to 16, for use in IVM of human oocytes.

18. The culture medium of claim 17, wherein the human oocytes are obtained from a female aged 35 years or older.

19. A method for maturing human oocytes in vitro, the method comprising receiving an immature oocyte from a human subject, and culturing the received immature oocyte in a culture medium comprising at least one HAT inhibitor or RT inhibitor for a period of time sufficient to achieve extrusion of a first polar body from the oocyte, thereby maturing the human oocyte in vitro.

20. The method of claim 19, wherein the human subject is at least 35 years of age or older.

21. The method of claim 20, wherein the human subject is at least 38 years old or older.

22. The method of any one of claims 19 to 21, wherein the human subject does not receive hormone injections prior to harvesting the immature oocytes.

23. The method of claim 22, wherein the hormone injection comprises injecting at least one of FSH, LH, human menopausal gonadotropin (HMG), and human chorionic gonadotropin (HCG).

24. The method according to any one of claims 19 to 23, wherein the culture medium is IVM medium supplemented with at least one gonadotropin.

25. The method of claim 24, wherein the IVM medium is supplemented with LH and FSH.

26. The method according to any one of claims 19 to 25, wherein the HAT is selected from E1A-associated protein p300 (p300), cyclic AMP response element binding protein binding protein (CREBBP / CBP), histone acetyltransferase KAT5 (KAT5 / Tip60), KAT6A and N-acetyltransferase 10 (NAT10).

27. The method of any one of claims 19 to 26, wherein the HAT inhibitor is not a P300 / CBP-associated factor (PCAF / KAT2B) inhibitor.

28. according to the method described in any one of claims 19 to 26, wherein the HAT inhibitor is selected from: curcumin, garcinol, spermidine coenzyme A (SpD-CoA), Lys-CoA, histone H4 lysine 16 (H4K16)-coenzyme A, MG149, EML264, LTK-14, cyanidin-B3, epicatechin gallate (EGCG), anacardic acid, gambogic acid, delphinidin, Remodelin, MB-3, NK13650A, NP-15, Δ12-prostaglandin J2 (PGJ2), RTK1, NU9056, DC-M01-7, CCT077791, NSC694614, PU141, C646, TH1834, L002, WM-8014, DC-G16-11 and their derivatives.

29. The method of any one of claims 19 to 28, wherein the HAT inhibitor is a naturally occurring molecule.

30. The method of claim 29, wherein the HAT inhibitor is selected from the group consisting of curcumin, plumbagin and mangosteen.

31. The method of any one of claims 19 to 30, wherein the RT inhibitor is selected from zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, Truvada, azithromycin, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, doravirine and islavirine.

32. The method of claim 31, wherein the RT inhibitor is selected from zidovudine (azidothymidine, AZT) and lamivudine.

33. The method of any one of claims 19 to 32, wherein the culture medium comprises both a HAT inhibitor and a RT inhibitor.

34. The method of claim 33, wherein the culture medium comprises curcumin and AZT.

35. The method according to any one of claims 19 to 34, wherein the culture medium comprises a pH between 6.8 and 7.

8.

36. The method according to any one of claims 19 to 35, wherein the culture medium is the culture medium of any one of claims 1 to 18.

37. The method of any one of claims 19 to 36, wherein the time is about 18-72 hours.

38. The method of any one of claims 19 to 37, wherein the HAT inhibitor is present at a concentration sufficient to increase the level of constitutive heterochromatin in the oocyte.

39. The method of claim 38, wherein the level of constitutive heterochromatin is increased by at least a predetermined threshold.

40. The method of claim 39, wherein the predetermined threshold is an increase of 25%.

41. The method of any one of claims 19 to 40, wherein the HAT inhibitor or RT inhibitor is present at a concentration sufficient to reduce the amount of retrotransposon expression in the oocyte.

42. The method of claim 41, wherein the amount of retrotransposon expression is reduced by at least a predetermined threshold.

43. The method of claim 42, wherein the predetermined threshold is a 25% reduction.

44. The method of any one of claims 19 to 43, wherein the culturing further comprises adding a meiotic entry inhibitor.

45. The method of any one of claims 19 to 44, wherein the method increases maturation rate compared to IVM performed in the absence of the HAT inhibitor or the RT inhibitor.

46. ​​The method of any one of claims 19 to 45, wherein the HAT inhibitor or the RT inhibitor is present at a concentration sufficient to increase first polar body extrusion compared to culturing in a medium without the HAT inhibitor or the RT inhibitor.

47. A method of determining the suitability of a subject in need of IVM to be treated by the method of any one of claims 19 to 46, the method comprising: a. receiving at least one germinal vesicle (GV) arrested oocyte from a human female subject; and b. measuring heterochromatin in the GV-arrested oocyte, wherein a level of heterochromatin below a predetermined threshold indicates that the subject is suitable for IVM by the method of any one of claims 19 to 46; The suitability of the subject to be treated by the method of any one of claims 19 to 46 is thereby determined.

48. The method of claim 47, wherein the GV-arrested oocyte is harvested for in vitro fertilization (IVF) but is found to be insufficiently mature.

49. The method of claim 47 or 48, wherein the measuring heterochromatin comprises measuring histone 3 lysine 9 (H3K9) trimethylation, H4K20 trimethylation, heterochromatin protein 1 (HP1) levels, 5-methylcytosine levels, or a combination thereof.

50. The method of claim 47 or 48, wherein the subject has undergone at least one unsuccessful round of IVF, and a heterochromatin level above the predetermined threshold indicates that the subject is suitable for another round of IVF.

51. A kit comprising: a. Cell culture medium; b. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH); and c. at least one histone acetyltransferase (HAT) inhibitor or reverse transcriptase (RT) inhibitor.

52. The kit of claim 51, wherein the culture medium is human oocyte in vitro maturation (IVM) medium.

53. The kit according to claim 51 or 52, wherein the HAT is selected from E1A-associated protein p300 (p300), cyclic AMP response element binding protein binding protein (CREBBP / CBP), histone acetyltransferase KAT5 (KAT5 / Tip60), KAT6A and N-acetyltransferase (NAT10).

54. A kit according to any one of claims 51 to 53, wherein the HAT inhibitor is not a P300 / CBP-associated factor (PCAF / KAT2B) inhibitor.

55. The kit according to any one of claims 51 to 54, wherein the HAT inhibitor is selected from the group consisting of curcumin, garcinol, spermidine-CoA (SpD-CoA), Lys-CoA, histone H4 lysine 16 (H4K16)-CoA, MG149, EML264, LTK-14, cyanidin-B3, epicatechin gallate (EGCG), anacardic acid, gambogic acid, delphinidin , Remodelin, MB-3, NK13650A, NP-15, Δ12-prostaglandin J2 (PGJ2), RTK1, NU9056, DC-M01-7, CCT077791, NSC694614, PU141, C646, TH1834, L002, WM-8014, DC-G16-11 and their derivatives.

56. A kit according to any one of claims 51 to 55, wherein the HAT inhibitor is a naturally occurring molecule.

57. The kit of claim 56, wherein the HAT inhibitor is selected from the group consisting of curcumin, plumbagin and mangosteen.

58. The kit of any one of claims 51 to 57, wherein the RT inhibitor is selected from the group consisting of zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, Truvada, azithromycin, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, doravirine, islavirine, and derivatives thereof.

59. The kit of claim 58, wherein the RT inhibitor is selected from zidovudine (azidothymidine, AZT) and lamivudine.

60. A kit according to any one of claims 51 to 59 comprising both a HAT inhibitor and a RT inhibitor.

61. The kit of claim 60 comprising Curcumin and AZT.

62. The kit of any one of claims 51 to 61, wherein the cell culture medium comprises a pH between 6.8 and 7.8 and is sufficiently buffered such that addition of the HAT inhibitor or the RT inhibitor does not lower the pH below 6.

8.

63. A kit according to any one of claims 51 to 62 for use in IVM of human oocytes.

64. The kit of claim 63, wherein the human oocytes are obtained from a female 35 years of age or older.

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