Follicular metabolic alterations are associated with obesity in mares and can be mitigated by dietary supplementation.

Obesity is a growing concern in human and equine populations, predisposing to metabolic pathologies and reproductive disturbances. Cellular lipid accumulation and mitochondrial dysfunction play an important role in the pathologic consequences of obesity, which may be mitigated by dietary interventions targeting these processes. We hypothesized that obesity in the mare promotes follicular lipid accumulation and altered mitochondrial function of oocytes and granulosa cells, potentially contributing to impaired fertility in this population. We also predicted that these effects could be mitigated by dietary supplementation with a combination of targeted nutrients to improve follicular cell metabolism. Twenty mares were grouped as: Normal Weight [NW, n = 6, body condition score (BCS) 5.7 ± 0.3], Obese (OB, n = 7, BCS 7.7 ± 0.2), and Obese Diet Supplemented (OBD, n = 7, BCS 7.7 ± 0.2), and fed specific feed regimens for ≥ 6 weeks before sampling. Granulosa cells, follicular fluid, and cumulus-oocyte complexes were collected from follicles ≥ 35 mm during estrus and after induction of maturation. Obesity promoted several mitochondrial metabolic disturbances in granulosa cells, reduced L-carnitine availability in the follicle, promoted lipid accumulation in cumulus cells and oocytes, and increased basal oocyte metabolism. Diet supplementation of a complex nutrient mixture mitigated most of the metabolic changes in the follicles of obese mares, resulting in parameters similar to NW mares. In conclusion, obesity disturbs the equine ovarian follicle by promoting lipid accumulation and altering mitochondrial function. These effects may be partially mitigated with targeted nutritional intervention, thereby potentially improving fertility outcomes in the obese female.

Enhanced branched-chain amino acid metabolism improves age-related reproduction in C. elegans.

Reproductive ageing is one of the earliest human ageing phenotypes, and mitochondrial dysfunction has been linked to oocyte quality decline; however, it is not known which mitochondrial metabolic processes are critical for oocyte quality maintenance with age. To understand how mitochondrial processes contribute to Caenorhabditis elegans oocyte quality, we characterized the mitochondrial proteomes of young and aged wild-type and long-reproductive daf-2 mutants. Here we show that the mitochondrial proteomic profiles of young wild-type and daf-2 worms are similar and share upregulation of branched-chain amino acid (BCAA) metabolism pathway enzymes. Reduction of the BCAA catabolism enzyme BCAT-1 shortens reproduction, elevates mitochondrial reactive oxygen species levels, and shifts mitochondrial localization. Moreover, bcat-1 knockdown decreases oocyte quality in daf-2 worms and reduces reproductive capability, indicating the role of this pathway in the maintenance of oocyte quality with age. Notably, oocyte quality deterioration can be delayed, and reproduction can be extended in wild-type animals both by bcat-1 overexpression and by supplementing with vitamin B1, a cofactor needed for BCAA metabolism.

FGF2, LIF, and IGF-1 supplementation improves mouse oocyte in vitro maturation via increased glucose metabolism†.

Chemically defined oocyte maturation media supplemented with FGF2, LIF, and IGF-1 (FLI medium) enabled significantly improved oocyte quality in multiple farm animals, yet the molecular mechanisms behind such benefits were poorly defined. Here, we first demonstrated that FLI medium enhanced mouse oocyte quality assessed by blastocyst formation after in vitro fertilization and implantation and fetal development after embryo transfer. We then analyzed the glucose concentrations in the spent media; reactive oxygen species concentrations; mitochondrial membrane potential; spindle morphology in oocytes; and the abundance of transcripts of endothelial growth factor-like factors, cumulus expansion factors, and glucose metabolism-related genes in cumulus cells. We found that FLI medium enabled increased glucose metabolism through glycolysis, pentose phosphate pathway, and hexosamine biosynthetic pathway, as well as more active endothelial growth factor-like factor expressions in cumulus cells, resulting in improved cumulus cell expansion, decreased spindle abnormality, and overall improvement in oocyte quality. In addition, the activities of MAPK1/3, PI3K/AKT, JAK/STAT3, and mTOR signaling pathways in cumulus cells were assessed by the phosphorylation of MAPK1/3, AKT, STAT3, and mTOR downstream target RPS6KB1. We demonstrated that FLI medium promoted activations of all these signaling pathways at multiple different time points during in vitro maturation.

Jiawei Buzhong Yiqi decoction ameliorates polycystic ovary syndrome via oocyte-granulosa cell communication.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiawei Buzhong Yiqi Decoction (JWBZYQ), from records of FuqingzhuNvke, is a classical formula for treating obese women related infertility. JWBZYQ has been shown to be effective in treating polycystic ovary syndrome (PCOS) in both clinical studies and practical practice, with the pharmacological mechanism remaining unknown. AIM OF THE STUDY: To explore the potential therapeutic effects and mechanistic insights of JWBZYQ in PCOS. MATERIALS AND METHODS: An overweight PCOS rat model was established via testosterone propionate (TP) injection and 45% high-fat diet (HFD). Then they were categorized into five distinct groups: Control group, Model group, low-dose of JWBZYQ (JWBZYQ1) group, high-dose of JWBZYQ (JWBZYQ2) group, and metformin (Met) group. Body weight, estrous cycle, and sex hormone levels were observed. Hematoxylin-Eosin staining was employed to investigate the histological characteristics of the ovaries. To identify the pathways that changed significantly, transcriptome analysis was performed. The protein and mRNA levels of key molecules in ovarian zona pellucida (ZP) organization, transzonal projections (TZPs) assembly, steroid hormone receptors, and steroidogenesis were assessed using phalloidin staining, immunohistochemistry, Western blot, and polymerase chain reaction. RESULTS: RNA-seq analysis demonstrated that regulation of hormone secretion, cilium assembly, cell projection assembly, and ZP production may all have crucial impact on the etiology of PCOS and therapeutic effect of JWBZYQ. In particular, PCOS rats exhibited elevated expressions of ZP1-3, which can be reversed by JWBZYQ2 particularly. Simultaneously, TZPs assembly was totally disrupted in PCOS rats, evidenced by the phalloidin staining, upregulated calcium-/calmodulin-dependent protein kinase II beta (CaMKIIß), and deficient p-CaMKIIß, myosin X (MYO10), proline-rich tyrosine kinase 2 (PTK2), and Fascin. Nonetheless, JWBZYQ or metformin treatment revived the disturbance, repairing the oocyte-granulosa cell communication, regulating steroidogenesis in PCOS rats. In this way, JWBZYQ and metformin exerted remarkable effects in alleviating altered ovarian morphology and function in PCOS rats, with JWBZYQ2 revealing the best effect. CONCLUSIONS: JWBZYQ restored the altered ovarian morphology and function by regulating the oocyte-granulosa cell communication, which was related with ZP organization and TZPs assembly in the ovary.

In vitro maturation in synthetic oviductal fluid increases gene expression associated with quality and lipid metabolism in bovine oocytes.

Traditionally, in vitro oocyte and embryo culture progresses through a series of varying culture medium. To investigate simplifying the in vitro production of bovine cumulus-oocyte complexes (COCs), this study used synthetic oviductal fluid (SOF) supplemented with conjugated linoleic acid (CLA). Special interest was placed on gene expression linked to lipid metabolism and oocyte maturation. COCs were matured in different media: Medium 199 (M199 group), M199 with 100 µM CLA (M199 + CLA group), SOF (SOF group), and SOF with 100 µM CLA (SOF + CLA group). COCs matured with SOF showed a higher relative abundance of mRNA of quality indicators gremlin 1 (GREM1) and prostaglandin-endoperoxide synthase 2 (PTGS2) in oocytes, and GREM1 in cumulus cells compared with in the M199 group. SOF medium COCs had a higher relative abundance of fatty acid desaturase 2 (FADS2) compared with the M199 group, which is essential for lipid metabolism in oocytes. Furthermore, the abundance of stearoyl-coenzyme A desaturase 1 (SCD1) in oocytes matured with SOF was not influenced by the addition of CLA, whereas the relative abundance of SCD1 was reduced in M199 medium with CLA. We concluded that maturation in SOF medium results in a greater abundance of genes linked to quality and lipidic metabolism in oocytes, regardless of the addition of CLA.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

STUDY QUESTION: Can in vitro maturation (IVM) and developmental competence of human oocytes be improved by co-culture with ovarian support cells (OSCs) derived from human-induced pluripotent stem cells (hiPSCs)? SUMMARY ANSWER: OSC-IVM significantly improves the rates of metaphase II (MII) formation and euploid Day 5 or 6 blastocyst formation, when compared to a commercially available IVM system. WHAT IS KNOWN ALREADY: IVM has historically shown highly variable performance in maturing oocytes and generating oocytes with strong developmental capacity, while limited studies have shown a positive benefit of primary granulosa cell co-culture for IVM. We recently reported the development of OSCs generated from hiPSCs that recapitulate dynamic ovarian function in vitro. STUDY DESIGN, SIZE, DURATION: The study was designed as a basic science study, using randomized sibling oocyte specimen allocation. Using pilot study data, a prospective sample size of 20 donors or at least 65 oocytes per condition were used for subsequent experiments. A total of 67 oocyte donors were recruited to undergo abbreviated gonadotropin stimulation with or without hCG triggers and retrieved cumulus-oocyte complexes (COCs) were allocated between the OSC-IVM or control conditions (fetal-like OSC (FOSC)-IVM or media-only IVM) in three independent experimental design formats. The total study duration was 1 April 2022 to 1 July 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte donors between the ages of 19 and 37 years were recruited for retrieval after informed consent, with assessment of anti-Mullerian hormone, antral follicle count, age, BMI and ovarian pathology used for inclusion and exclusion criteria. In experiment 1, 27 oocyte donors were recruited, in experiment 2, 23 oocyte donors were recruited, and in experiment 3, 17 oocyte donors and 3 sperm donors were recruited. The OSC-IVM culture condition was composed of 100 000 OSCs in suspension culture with hCG, recombinant FSH, androstenedione, and doxycycline supplementation. IVM controls lacked OSCs and contained either the same supplementation, FSH and hCG only (a commercial IVM control), or FOSCs with the same supplementation (Media control). Experiment 1 compared OSC-IVM, FOSC-IVM, and a Media control, while experiments 2 and 3 compared OSC-IVM and a commercial IVM control. Primary endpoints in the first two experiments were the MII formation (i.e. maturation) rate and morphological quality assessment. In the third experiment, the fertilization and embryo formation rates were assessed with genetic testing for aneuploidy and epigenetic quality in blastocysts. MAIN RESULTS AND THE ROLE OF CHANCE: We observed a statistically significant improvement (∼1.5×) in maturation outcomes for oocytes that underwent IVM with OSCs compared to control Media-IVM and FOSC-IVM in experiment 1. More specifically, the OSC-IVM group yielded a MII formation rate of 68% ± 6.83% SEM versus 46% ± 8.51% SEM in the Media control (P = 0.02592, unpaired t-test). FOSC-IVM yielded a 51% ± 9.23% SEM MII formation rate which did not significantly differ from the media control (P = 0.77 unpaired t-test). Additionally, OSC-IVM yielded a statistically significant ∼1.6× higher average MII formation rate at 68% ± 6.74% when compared to 43% ± 7.90% in the commercially available IVM control condition (P = 0.0349, paired t-test) in experiment 2. Oocyte morphological quality between OSC-IVM and the controls did not significantly differ. In experiment 3, OSC-IVM oocytes demonstrated a statistically significant improvement in Day 5 or 6 euploid blastocyst formation per COC compared to the commercial IVM control (25% ± 7.47% vs 11% ± 3.82%, P = 0.0349 logistic regression). Also in experiment 3, the OSC-treated oocytes generated blastocysts with similar global and germline differentially methylated region epigenetic profiles compared commercial IVM controls or blastocysts after either conventional ovarian stimulation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: While the findings of this study are compelling, the cohort size remains limited and was powered on preliminary pilot studies, and the basic research nature of the study limits generalizability compared to randomized control trials. Additionally, use of hCG-triggered cycles results in a heterogenous oocyte cohort, and potential differences in the underlying maturation state of oocytes pre-IVM may limit or bias findings. Further research is needed to clarify and characterize the precise mechanism of action of the OSC-IVM system. Further research is also needed to establish whether these embryos are capable of implantation and further development, a key indication of their clinical utility. WIDER IMPLICATIONS OF THE FINDINGS: Together, these findings demonstrate a novel approach to IVM with broad applicability to modern ART practice. The controls used in this study are in line with and have produced similar to findings to those in the literature, and the outcome of this study supports findings from previous co-culture studies that found benefits of primary granulosa cells on IVM outcomes. The OSC-IVM system shows promise as a highly flexible IVM approach that can complement a broad range of stimulation styles and patient populations. Particularly for patients who cannot or prefer not to undergo conventional gonadotropin stimulation, OSC-IVM may present a viable path for obtaining developmentally competent, mature oocytes. STUDY FUNDING/COMPETING INTEREST(S): A.D.N., A.B.F., A.G., B.P., C.A., C.C.K., F.B., G.R., K.S.P., K.W., M.M., P.C., S.P., and M.-J.F.-G. are shareholders in the for-profit biotechnology company Gameto Inc. P.R.J.F. declares paid consultancy for Gameto Inc. P.C. also declares paid consultancy for the Scientific Advisory Board for Gameto Inc. D.H.M. has received consulting services from Granata Bio, Sanford Fertility and Reproductive Medicine, Gameto, and Buffalo IVF, and travel support from the Upper Egypt Assisted Reproduction Society. C.C.K., S.P., M.M., A.G., B.P., K.S.P., G.R., and A.D.N. are listed on a patent covering the use of OSCs for IVM: U.S. Provisional Patent Application No. 63/492,210. Additionally, C.C.K. and K.W. are listed on three patents covering the use of OSCs for IVM: U.S. Patent Application No. 17/846,725, U.S Patent Application No. 17/846,845, and International Patent Application No.: PCT/US2023/026012. C.C.K., M.P.S., and P.C. additionally are listed on three patents for the transcription factor-directed production of granulosa-like cells from stem cells: International Patent Application No.: PCT/US2023/065140, U.S. Provisional Application No. 63/326,640, and U.S. Provisional Application No. 63/444,108. The remaining authors have no conflicts of interest to declare.

FGF2, LIF, and IGF1 (FLI) supplementation during human in vitro maturation enhances markers of gamete competence.

STUDY QUESTION: Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. WHAT IS KNOWN ALREADY: During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. STUDY DESIGN, SIZE, DURATION: This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. WIDER IMPLICATIONS OF THE FINDINGS: FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Melatonin ameliorates histone modification disorders in mammalian aged oocytes by neutralizing the alkylation of HDAC1.

Aging-associated histone modification changes in oocytes have been sporadically reported, but the underlying mechanisms remain elusive. Here, we systematically characterize multiple histone modifications in oocytes during aging. We find that maternal and postovulatory aging markedly alter the status of histone modifications, specifically H4K12ac and H3K4me3, in both mouse and porcine oocytes. Meanwhile, we identify a substantial reduction in HDAC1 (histone deacetylase 1) protein in aged oocytes, which contributes to the changes in H4K12ac and H3K4me3. Moreover, by employing methylglyoxal (MG) and site-directed mutagenesis, we demonstrate that the elevated reactive carbonyl species (RCS) level induces HDAC1 degradation, likely through attacking the cysteine residues, thereby influences histone modification state. Importantly, supplementation of melatonin not only prevents the loss of HDAC1 protein, but also partially corrects the H4K12ac and H3K4me3 status in aged oocytes. To sum up, this study established the link between redox disequilibrium and histone modification alterations during mammalian oocyte aging.

Inhibition of checkpoint kinase prevents human oocyte apoptosis induced by chemotherapy and allows enhanced tumour chemotherapeutic efficacy.

STUDY QUESTION: Could inhibition of the checkpoint kinase (CHEK) pathway protect human oocytes and even enhance the anti-tumour effects, during chemotherapy? SUMMARY ANSWER: CHEK inhibitors prevented apoptosis of human oocytes induced by chemotherapy and even enhanced the anti-tumour effects. WHAT IS KNOWN ALREADY: CHEK inhibitors showed ovarian protective effects in mice during chemotherapy, while their role in human oocytes is unclear. STUDY DESIGN, SIZE, DURATION: This experimental study evaluated the ovarian reserve of young patients (120 patients) with cancer, exposed or not exposed to taxane and platinum (TP)-combined chemotherapy. Single RNA-sequencing analysis of human primordial oocytes from 10 patients was performed to explore the mechanism of oocyte apoptosis induced by TP chemotherapy. The damaging effects of paclitaxel (PTX) and cisplatin on human oocytes were also evaluated by culturing human ovaries in vitro. A new mouse model that combines human ovarian xenotransplantation and patient-derived tumour xenografts was developed to explore adjuvant therapies for ovarian protection. The mice were randomly allocated to four groups (10 mice for each group): control, cisplatin, cisplatin + CK1 (CHEK1 inhibitor, SCH 900776), and cisplatin + CK2 (CHEK2 inhibitor, BML277). PARTICIPANTS/MATERIALS, SETTING, METHODS: In the prospective cohort study, human ovarian follicles were counted and serum AMH levels were evaluated. RNA-sequencing analysis was conducted, and staining for follicular damage (phosphorylated H2AX histone; γH2AX), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) assays and assessments of apoptotic biomarkers (western blot and immunofluorescence) were conducted in human ovaries. After the treatments, histological analysis was performed on human ovarian samples to investigate follicular populations, and oocyte damage was measured by γH2AX staining, BAX staining, and TUNEL assays. At the same time, the tumours were evaluated for volume, weight, and apoptosis levels. MAIN RESULTS AND THE ROLE OF CHANCE: Patients who received TP chemotherapy showed decreased ovarian reserves. Single RNA-sequencing analysis of human primordial oocytes indicated that TP chemotherapy induced apoptosis of human primordial oocytes by causing CHEK-mediated TAp63α phosphorylation. In vitro culture of human ovaries showed greater damaging effects on oocytes after cisplatin treatment compared with that after PTX treatment. Using the new animal model, CHEK1/2 inhibitors prevented the apoptosis of human oocytes induced by cisplatin and even enhanced its anti-tumour effects. This protective effect appeared to be mediated by inhibiting DNA damage via the CHEK-TAp63α pathway and by generation of anti-apoptotic signals in the oocytes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was a preclinical study performed with human ovarian samples, and clinical research is required for validation. WIDER IMPLICATIONS OF THE FINDINGS: These findings highlight the therapeutic potential of CHEK1/2 inhibitors as a complementary strategy for preserving fertility in female cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This work was financially supported by the National Natural Science Foundation of China (nos. 82001514 and 81902669) and the Fundamental Research Funds for the Central Universities (2021yjsCXCY087). The authors declare no conflict of interest.

C-X-C motif chemokine ligand 12 improves the developmental potential of bovine oocytes by activating SH2 domain-containing tyrosine phosphatase 2 during maturation†.

In vitro maturation of mammalian oocytes is an important means in assisted reproductive technology. Most bovine immature oocytes complete nuclear maturation, but less than half develop to the blastocyst stage after fertilization. Thus, inefficient in vitro production is mainly caused by a suboptimal in vitro culture process, in which oocyte quality appears to be the limiting factor. In our study, a potential maternal regulator, C-X-C motif chemokine ligand 12, was identified by analyzing transcriptome data. C-X-C motif chemokine ligand 12 supplementation promoted the developmental potential of oocytes by improving protein synthesis and reorganizing cortical granules and mitochondria during in vitro maturation, which eventually increased blastocyst formation efficiency and cell number after parthenogenesis, fertilization, and cloning. All these promoting effects by C-X-C motif chemokine ligand 12 were achieved by activating SH2 domain-containing tyrosine phosphatase 2, thereby promoting the mitogen-activated protein kinase signaling pathway. These findings provide an in vitro maturation system that closely resembles the maternal environment to provide high-quality oocytes for in vitro production.

Investigating the Role of Ferroptosis-Related Genes in Ovarian Aging and the Potential for Nutritional Intervention.

With advancing age, women experience irreversible deterioration in the quality of their oocytes, resulting in reduced fertility. To gain a deeper understanding of the influence of ferroptosis-related genes on ovarian aging, we employed a comprehensive approach encompassing spatial transcriptomics, single-cell RNA sequencing, human ovarian pathology, and clinical biopsy. This investigation revealed the intricate interactions between ferroptosis and cellular energy metabolism in aging germ cells, shedding light on the underlying mechanisms. Our study involved 75 patients with ovarian senescence insufficiency, and we utilized multi-histological predictions of ferroptosis-related genes. Following a two-month supplementation period with DHEA, Ubiquinol CoQ10, and Cleo-20 T3, we examined the changes in hub genes. Our results showed that TFRC, NCOA4, and SLC3A2 were significantly reduced and GPX4 was increased in the supplement group, confirming our prediction based on multi-omic analysis. Our hypothesis is that supplementation would enhance the mitochondrial tricarboxylic acid cycle (TCA) or electron transport chain (ETC), resulting in increased levels of the antioxidant enzyme GPX4, reduced lipid peroxide accumulation, and reduced ferroptosis. Overall, our results suggest that supplementation interventions have a notable positive impact on in vitro fertilization (IVF) outcomes in aging cells by improving metal ion and energy metabolism, thereby enhancing oocyte quality in older women.

Cytokine supplemented maturation medium improved development to term following somatic cell nuclear transfer (SCNT) in cattle.

CONTEXT: In vitro maturation is an important process in the production of embryos. It has been shown that three cytokines, fibroblast growth factor 2, leukemia inhibitory factor and insulin-like growth factor 1 (FLI), increased efficiency of in vitro maturation, somatic cell nuclear transfer (SCNT) blastocyst production, and in vivo development of genetically engineered piglets. AIMS: Assess effects of FLI on oocyte maturation, quality of oocytes, and embryo development in bovine in vitro fertilisation (IVF) and SCNT. KEY RESULTS: Cytokine supplementation resulted in significant increases in maturation rates and decreased levels of reactive oxygen species. Oocytes matured in FLI had increased blastocyst rates when used in IVF (35.6%vs 27.3%, P <0.05) and SCNT (40.6%vs 25.7%, P <0.05). SCNT blastocysts contained significantly more inner cell mass and trophectodermal cells when compared to the control group. Importantly, SCNT embryos derived from oocytes matured in FLI medium resulted in a four-fold increase in full-term development compared to control medium (23.3%vs 5.3%, P <0.05). Relative mRNA expression analysis of 37 genes associated with embryonic and fetal development revealed one gene had differential transcript abundance in metaphase II oocytes, nine genes at the 8-cell stage, 10 genes at the blastocyst stage in IVF embryos and four genes at the blastocyst stage in SCNT embryos. CONCLUSIONS: Cytokine supplementation increased efficiency of in vitro production of IVF and SCNT embryos and in vivo development of SCNT embryos to term. IMPLICATIONS: Cytokine supplementation is beneficial to embryo culture systems, which may shed light on requirements of early embryo development.

Boosting mitochondrial function and metabolism in aging female germ cells with dual ROCK/ROS inhibition.

The decline in oocyte quality with age is an irreversible process that results in low fertility. Reproductive aging causes an increase in oocyte aneuploidy leading to a decrease in embryo quality and an increase in the incidence of miscarriage and congenital defects. Here, we show that the dysfunction associated with aging is not limited to the oocyte, as oocyte granulosa cells also show a range of defects related to mitochondrial activity. The addition of Y-27632 and Vitamin C combination drugs to aging germ cells was effective in enhancing the quality of aging cells. We observed that supplement treatment significantly decreased the production of reactive oxygen species (ROS) and restored the balance of mitochondrial membrane potential. Supplementation treatment reduces excessive mitochondrial fragmentation in aging cells by upregulating mitochondrial fusion. Moreover, it regulated the energy metabolism within cells, favoring oxygen respiration and reducing anaerobic respiration, thereby increasing cellular ATP production. In an experiment with aged mice, supplement treatment improved the maturation of oocytes in vitro and prevented the buildup of ROS in aging oocytes in culture. Additionally, this treatment resulted in an increased concentration of anti-mullerian hormone (AMH) in the culture medium. By improving mitochondrial metabolism in aging females, supplement treatment has the potential to increase quality of oocytes during in vitro fertilization.

Attenuation of Oxidative Stress and Regulation of AKT Signaling by Vanillic Acid during Bovine Pre-Implantation Embryo Development.

Vanillic acid (VA) has shown antioxidant and anti-inflammatory activities in different cell types, but its biological effects in the context of early embryo development have not yet been clarified. In the current study, the impact of VA supplementation during in vitro maturation (IVM) and/or post-fertilization (in vitro culture; IVC) on redox homeostasis, mitochondrial function, AKT signaling, developmental competence, and the quality of bovine pre-implantation embryos was investigated. The results showed that dual exposure to VA during IVM and late embryo culture (IVC3) significantly improved the blastocyst development rate, reduced oxidative stress, and promoted fatty acid oxidation as well as mitochondrial activity. Additionally, the total numbers of cells and trophectoderm cells per blastocyst were higher in the VA-treated group compared to control (p < 0.05). The RT-qPCR results showed down-regulation of the mRNA of the apoptosis-specific markers and up-regulation of AKT2 and the redox homeostasis-related gene TXN in the treated group. Additionally, the immunofluorescence analysis showed high levels of pAKT-Ser473 and the fatty acid metabolism marker CPT1A in embryos developed following VA treatment. In conclusion, the study reports, for the first time, the embryotrophic effects of VA, and the potential linkage to AKT signaling pathway that could be used as an efficacious protocol in assisted reproductive technologies (ART) to improve human fertility.

Establishment and optimization of an in vitro guinea pig oocyte maturation system.

Guinea pigs are a valuable animal model for studying various diseases, including reproductive diseases. However, techniques for generating embryos via embryo engineering in guinea pigs are limited; for instance, in vitro maturation (IVM) technique is preliminary for guinea pig oocytes. In this study, we aimed to establish and optimize an IVM method for guinea pig oocytes by investigating various factors, such as superovulation induced by different hormones, culture supplementation (e.g., amino acids, hormone, and inhibitors), culture conditions (e.g., oocyte type, culture medium type, and treatment time), and in vivo hCG stimulation. We found that oocytes collected from guinea pigs with superovulation induced by hMG have a higher IVM rate compared to those collected from natural cycling individuals. Moreover, we found that addition of L-cysteine, cystine, and ROS in the culture medium can increase the IVM rate. In addition, we demonstrated that in vivo stimulation with hCG for 3-8 h can further increase the IVM rate. As a result, the overall IVM rate of guinea pig oocytes under our optimized conditions can reach ~69%, and the mature oocytes have high GSH levels and normal morphology. In summary, we established an effective IVM method for guinea pig oocytes by optimizing various factors and conditions, which provides a basis for embryo engineering using guinea pigs as a model.

Alpha-ketoglutarate supplementation ameliorates ovarian reserve and oocyte quality decline with aging in mice.

Assisted reproductive technology is widely accepted as an effective treatment to improve female fertility, but the decline of aging oocyte quality remains an important factor in the decrease of female fecundity. However, the effective strategies for improving oocyte aging are still not well understood. In the study, we demonstrated that ROS content and abnormal spindle proportion were increased and mitochondrial membrane potential was decreased in aging oocytes. However, supplementation of α-ketoglutarate (α-KG), an immediate metabolite in the tricarboxylic acid cycle (TCA), for 4 months to aging mice, significantly increased the ovarian reserve showed by more follicle numbers observed. In addition, the oocyte quality was significantly improved, as demonstrated by reduced fragmentation rate and decreased reactive oxygen species (ROS) levels, in addition to a lower rate of abnormal spindle assembly, thereby improving the mitochondrial membrane potential. Consistent with the in vivo data, α-KG administration also improved the post-ovulated aging oocyte quality and early embryonic development by improving mitochondrial functions and reducing ROS accumulation and abnormal spindle assembly. Our data revealed that α-KG supplementation might be an effective strategy to improve the quality of aging oocytes in vivo or in vitro.

Nonylphenol Exposure-Induced Oocyte Quality Deterioration Could be Reversed by Boric Acid Supplementation in Rats.

In this study, we reported boric acid's protective effects on the quality of nonylphenol (NP)-exposed oocytes. Female rats were classified into 4 groups: control, boric acid, NP, and NP+boric acid. Histopathological studies and immunohistochemical analysis of anti-müllerian hormone (AMH), mechanistic target of rapamycin (mTOR), Sirtuin1 (SIRT1), stem cell factor (SCF) studies were done. The comet assay technique was utilized for DNA damage. The ELISA method was used to determine the concentrations of oxidative stress indicators (SOD, CAT, and MDA), ovarian hormone (INH-B), and inflammation indicators (IL-6 and TNF-α). Boric acid significantly reduced the histopathological alterations and nearly preserved the ovarian reserve. With the restoration of AMH and SCF, boric acid significantly improved the ovarian injury. It downregulated SIRT1 and upregulated the mTOR signaling pathway. It provided DNA damage protection. Ovarian SOD, CAT levels were decreased by boric acid. Boric acid co-administration significantly reduced NP's MDA, IL-6, and TNF-activities. This results imply that boric acid has a protective role in ovarian tissue against NP-mediated infertility.

Effects of n-3 PUFA supplementation on oocyte in vitro maturation in mice with polycystic ovary syndrome.

n-3 PUFAs are classic antioxidant that can be used to treat follicular dysplasia and hyperinsulinemia caused by excessive oxidative stress in PCOS women. To investigate the effect of n-3 PUFA supplementation on the oocyte quality of polycystic ovary syndrome (PCOS) mice during in vitro maturation, a PCOS mouse model was established by dehydroepiandrosterone (DHEA). The GV oocytes of the control and PCOS groups were collected and cultured in vitro with or without n-3 PUFAs. After 14 h, the oocytes were collected. Our data demonstrated that the oocyte maturation rate of PCOS mice significantly increased after the addition of 50 µM n-3 PUFAs. The results of immunofluorescence showed that the abnormal rates of spindles and chromosomes in the PCOS + n-3 PUFA group were lower than those in the PCOS group. The mRNA expression of an antioxidant-related gene (Sirt1) and DNA damage repair genes (Brca1/Msh2) was found to be significantly rescued after n-3 treatment. Additionally, the results of living cell staining showed that the addition of n-3 PUFAs could reduce the levels of reactive oxygen species and mitochondrial superoxide in PCOS oocytes. In conclusion, the addition of 50 µM n-3 PUFAs during the in vitro maturation of PCOS mouse oocytes can improve the maturation rate by reducing the level of oxidative stress and the rate of spindle/chromosome abnormalities, providing valuable support during the IVM process.

Tumor-Derived Oxidative Stress Triggers Ovarian Follicle Loss in Breast Cancer.

Breast cancer is a common indication for ovarian cryopreservation. However, whether the grafting ovarian tissue meets functional requirements, as well as the need for additional interventions, remains unclear. The current study demonstrates abnormal serum hormones in breast cancer in humans and breast cancer cell line-derived tumor-bearing mice, and for the first time shows tumor-induced loss of primordial and growing follicles, and the number of follicles being lost to either growth or atresia. A gene signature of tumor-bearing mice demonstrates the disturbed regulatory network of steroidogenesis, which links to mitochondria dysfunction in oocytes and granulosa cells via the phosphatidylinositol 3-kinase signaling pathway. Notably, increased reactive oxygen species were identified in serum and ovarian tissues in tumor-bearing mice. Furthermore, supplementation with vitamin C promoted follicular quiescence, repairing tumor-induced follicle loss via inactivation of the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway, indicating the potential of antioxidants as a fertility therapy to achieve higher numbers of healthy follicles ready for ovarian cryopreservation.

Mitochondrial DNA Deficiency and Supplementation in Sus scrofa Oocytes Influence Transcriptome Profiles in Oocytes and Blastocysts.

Mitochondrial DNA (mtDNA) deficiency correlates with poor oocyte quality and fertilisation failure. However, the supplementation of mtDNA deficient oocytes with extra copies of mtDNA improves fertilisation rates and embryo development. The molecular mechanisms associated with oocyte developmental incompetence, and the effects of mtDNA supplementation on embryo development are largely unknown. We investigated the association between the developmental competence of Sus scrofa oocytes, assessed with Brilliant Cresyl Blue, and transcriptome profiles. We also analysed the effects of mtDNA supplementation on the developmental transition from the oocyte to the blastocyst by longitudinal transcriptome analysis. mtDNA deficient oocytes revealed downregulation of genes associated with RNA metabolism and oxidative phosphorylation, including 56 small nucleolar RNA genes and 13 mtDNA protein coding genes. We also identified the downregulation of a large subset of genes for meiotic and mitotic cell cycle process, suggesting that developmental competence affects the completion of meiosis II and first embryonic cell division. The supplementation of oocytes with mtDNA in combination with fertilisation improves the maintenance of the expression of several key developmental genes and the patterns of parental allele-specific imprinting gene expression in blastocysts. These results suggest associations between mtDNA deficiency and meiotic cell cycle and the developmental effects of mtDNA supplementation on Sus scrofa blastocysts.