Cumulus Cells Research Articles

Diminished ovarian reserve causes adverse ART outcomes attributed to effects on oxygen metabolism function in cumulus cells

BackgroundDeclining oocyte quality in women with advanced age has been a major impediment to assisted reproductive treatments’ (ART) success rate. However, aging is often accompanied by a diminished ovarian reserve (DOR). Cumulus cells (CCs) are known to play an important role in the development and maturation of oocytes, and the quality of CCs actually reflects the quality of the oocyte. In this study, CCs were used to investigate the real reasons for the decline in oocyte quality in older women.MethodsNinety-nine CC samples were subdivided into 4 different groups according to the different age and ovarian reserve status. Other than clinical ART results, transcriptional expression profiles were performed in CCs to detect the differences.ResultsThe results were that DOR, no matter in young or advanced age group, was found to be significantly associated with adverse ART outcomes. Of note, there were no statistically significant changes in ART outcomes in the group at advanced age with normal ovarian reserve (NOR), compared to the young with NOR. DOR induced a series of transcriptional variations in CCs commonly enriched in oxygen metabolism.ConclusionOur results revealed that the ART outcomes in advanced patients were attributable to the DOR. The oxygen metabolic changes may interfere with CCs’ function of supporting oocytes. This study can provide guidance for ART practice that not age but ovarian reserve status is the main predictor for ART outcomes, and ovarian reserve status should be timely assessed when the clinical manifestations are still mild in elderly women.

PSIV-8 The Effects of Quercetin Supplementation During Oocyte Maturation on the in Vitro Production of Pig Embryos

Abstract Oocyte viability can be reduced during in vitro maturation due to oxidative stress and a lack of antioxidant presence in the environment. Additionally, oxidative stress is a culprit in the relatively low success rate of in vitro fertilization (IVF) in pigs. Quercetin is a natural antioxidant dietary flavonoid that has the ability to suppress free radical formation. The purpose of this study was to determine the effects of the quercetin supplementation during oocyte maturation on IVF embryonic development success rates in pigs. Oocytes were matured for 40 to 44 h supplemented with quercetin (0, 0.25, 0.5, 0.75 mM; Sigma-Aldrich Co.) then fertilized using frozen-thawed boar semen (1.0 x 105 sperm cells/mL) and co-incubated for 6-8 h followed by embryo culture for 144 h. After maturation, oocytes were evaluated for cumulus cell expansion (n = 300, r = 6) under a stereomicroscope (scale 0 to 4) then denuded to determine reactive oxygen species (ROS) production (n = 135) by measuring the fluorescent intensity from the oxidation of 2’,7’-dichlorodihydrofluorescein diacetate. After IVF, zygotes were fixed, permeabilized, and stained with bisBenzimide H 33342 trihydrochloride and evaluated for penetration, polyspermy, and pronucleus formation rates (n = 480) and embryos were evaluated for cleavage and blastocyst formation (n = 599) at 48 h and 144 h post-IVF, respectively. Cumulus cell expansion score percentages were determined and analyzed using the Chi-squared test. Fluorescent intensities of oocytes were analyzed using a GLM and compared using LSD after adjusting the control group value to 1. The IVF and embryo development data were reported as the percent observed/drop and mean percentages, and were analyzed using GLM. Means were analyzed using Tukey’s test. The average cumulus cell expansion post-maturation when no quercetin was supplemented was 2.78 ± 0.15. As quercetin supplementation increased, cumulus cell expansion decreased (1.33 ± 0.11 for 0.75 mM quercetin). Supplementation of 0.25 mM quercetin decreased (P < 0.05) ROS levels (0.52 ± 0.12) but 0.75 mM quercetin increased (P < 0.05) ROS levels (1.73 ± 0.11) compared with no quercetin. Supplementation of quercetin decreased (P < 0.05) oocyte penetration rates, increased (P < 0.05) polyspermic oocytes and pronuclear formation. Quercetin supplementation during maturation also reduced embryonic development success through decrease (P < 0.05) of cleavage and blastocyst formation rates compared with no quercetin (78.69 ± 0.02 and 31.18 ± 0.03, respectively). It appears that quercetin supplementation to the oocyte maturation media in concentrations greater than 0.25 mM are detrimental during in vitro production of pig embryos. Supplementation of 0.25 mM quercetin appears to not be different to supplementing quercetin to the maturation media except for reduction in ROS concentrations in the oocyte.

Differential Gene Expression Analysis of Human Ovarian Follicular Cumulus and Mural Granulosa Cells Under the Influence of Insulin in IVF Ovulatory Women and Polycystic Ovary Syndrome Patients Through Network Analysis

ABSTRACT Background Polycystic ovarian syndrome (PCOS) is a commonly occurring reproductive disorder among the reproductive-aged women. Its global occurrence varies based on diagnostic guidelines, ethnicities, and locations of concern. Insulin resistance (IR) is commonly observed around 65–70% of women diagnosed with PCOS, representing a prevalent association. Consequently, the study was designed with an objective of illustrating the effect of insulin on mural and cumulus granulosa cells (GCs) of PCOS patients in comparison to normal ovulating women. Methodology This study is a case–control design, wherein a total of 80 participants were recruited meeting criterion of inclusion and exclusion, divided into 8 groups with each group consisting of 10 samples. The process involves the isolation and culturing of mural granulosa cells (MGC) and cumulus granulosa cells (CGC) with and without exposure to insulin. The proteins released by untreated GCs and insulin-treated GCs were extracted, and complex protein mixtures were digested with trypsin, followed by tandem mass spectrometry analysis and data processing using bioinformatics. Results We found 595 proteins in both control and PCOS samples, of which 310 were contributed by MGCs and 285 by CGCs. The PCOS MGCs expressed 20%, both the normal MGCs and CGCs have equal representation of 16% by each, whereas the PCOS CGCs proteins contributed 15% of the total of the proteomic expression. However, the poor expression observed with the Insulin exposure, the Insulin treated PCOS CGCs contributes 13%, PCOS MGCs contributes 8%. The normal MGCs upon the Insulin treatment give 8% then and there only 4% of proteins expressed by normal CGCs after Insulin treatment. The Venn analysis widened on their precise expression topographies. The examination of strings exhibited important protein–protein interaction pathways. Conclusion This is a pioneering investigation aimed to establish the link between hyperinsulinemia in localized follicular GCs and PCOS mechanisms by comparing them to control group. The examination of various attributes, mechanisms, and traits shown by genes and proteins in individuals with PCOS compared to control populations, alongside the investigation of the dynamics of these genes and proteins following exposure to insulin, holds promise for the formulation of novel hypotheses and strategies in the identification of new biomarkers.

Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bovine oocytes.

In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation. To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM. The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass. Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts. These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal.

Oocyte-Specific Deletion of Slc6a9 Encoding the GLYT1 Glycine Transporter Eliminates Glycine Transport in Mouse Preimplantation Embryos and Their Ability to Counter Hypertonic Stress.

Early preimplantation mouse embryos are sensitive to increased osmolarity, which can block their development. To overcome this, they accumulate organic osmolytes to maintain cell volume. The main organic osmolyte used by early mouse embryos is glycine. Glycine is transported during the mature egg and 1-cell to 4-cell embryo stages by a transporter identified as GLYT1, encoded by the Slc6a9 gene. Here, we have produced an oocyte-specific knockout of Slc6a9 by crossing mice that have a segment of the gene flanked by LoxP elements with transgenic mice expressing iCre driven by the oocyte-specific Gdf9 promoter. Slc6a9 null oocytes failed to develop glycine transport activity during meiotic maturation. However, females with these oocytes were fertile. When enclosed in their cumulus-oocyte complex, Slc6a9 null oocytes could accumulate glycine via GLYT1 transport in their coupled cumulus cells, which may support female fertility in vivo. In vitro, embryos derived from Slc6a9 null oocytes displayed a clear phenotype. While glycine rescued complete preimplantation development of wild type embryos from increased osmolarity, embryos derived from null oocytes failed to develop past the 2-cell stage even with glycine. Thus, Slc6a9 is required for glycine transport and protection against increased osmolarity in mouse eggs and early embryos.

The effect of L-carnitine on oocyte mitochondrial health and biomarkers on cyclophosphamide chemotherapy drug in mice

Improving oocyte competence during chemotherapy is widely known as a contributing factor to increasing the probability of fertility. Additionally, the role of cumulus cells in oocyte quality is of utmost importance. Therefore, this study was designed to simultaneously probe into the relative gene expression of oocytes and cumulus cells as biomarkers of oocyte quality with cyclophosphamide and L-carnitine treatment. A total of 60 adult NMRI mice were divided into four groups: control, L-carnitine (LC), cyclophosphamide (CP), and cyclophosphamide+L-carnitine (CP+LC). The relative mRNA expression levels of oocyte quality genes including growth differentiation factor 9 (Gdf9), hyaluronan synthase 2 (Has2), and mitochondrial sirtuin 3 (Sirt3) in oocytes, and genes involved in bilateral communication between cumulus cells and between the oocyte and its neighboring cumulus cells including connexin 37 (Cx37) and connexin 43 (Cx43) were detected by Real-time-PCR. DCFH-DA staining analyzed the level of intracellular ROS in oocytes. Under the influence of L-carnitine, Gdf9, Has2, Cx43, and Cx37 were significantly up-regulated (p ≤ 0.05). However, cyclophosphamide considerably reduced the expression of all these genes (p ≤ 0.05). The expression of the Sirt3 gene in the CP group increased significantly compared to the other groups (p ≤ 0.05). Analysis of fluorescent images revealed that the level of intracellular ROS in the cyclophosphamide group was significantly increased compared to the other groups (p ≤ 0.05), while it plummeted in the L-carnitine group (p ≤ 0.05). L-carnitine as an antioxidant can reduce the destructive effects of cyclophosphamide and enhance bilateral communications between oocytes and cumulus cells, and it may ultimately lead to an increase in the fertility rate.

MiR-155-5p improves oocyte maturation in porcine cumulus cells through connexin 43-mediated regulation of MPF activity

In this study, we aimed to investigate the effect of the expression of miR-155-5p and its target genes on oocyte maturation. We analyzed the expression of miR-155-5p and its target genes in cumulus cells and oocytes using quantitative real-time reverse-transcription polymerase chain reaction. Using carboxyfluorescein, porcine cumulus cells were transfected with mimics and inhibitors of ssc-miR-155-5p to induce in vitro maturation, and subsequently, cumulus expansion, oocyte maturation, and cleavage rate were measured. We found that miR-155-5p expression in cumulus cells at the metaphase II stage was significantly higher than that at the germinal vesicle (GV) stage, whereas Cx43 expression was significantly lower than that at the GV stage (P < 0.05). Compared with those in the negative control group, the cumulus diffusion area of cumulus oocyte complexes; oocyte maturation rate; cleavage rate; HAS2, PTGS2, CD44, PTX3, and TNFAIP6 expression in cumulus cells; and GDF9, BMP15, CyclinB1, and CDK1 expression in oocytes were significantly increased in the miR-155-5p mimics group (P < 0.05), whereas the mRNA and protein expression of CX43 were significantly decreased (P < 0.05). Compared with that in the negative control group, the protein expression of CyclinB1 and p-CDK1 (Thr14, Tyr15) in the miR-155-5p mimics group was significantly increased (P < 0.05). These results suggest that miR-155-5p regulates maturation promoting factor activity by targeting Cx43, which improves the in vitro maturation and cleavage rate of porcine oocytes.

Cumulus expansion is impaired with advanced reproductive age due to loss of matrix integrity and reduced hyaluronan.

Reproductive aging is associated with ovulatory defects. Age-related ovarian fibrosis partially contributes to this phenotype as short-term treatment with anti-fibrotic compounds improves ovulation in reproductively old mice. However, age-dependent changes that are intrinsic to the follicle may also be relevant. In this study, we used a mouse model to demonstrate that reproductive aging is associated with impaired cumulus expansion which is accompanied by altered morphokinetic behavior of cumulus cells as assessed by time-lapse microscopy. The extracellular matrix integrity of expanded cumulus-oocyte complexes is compromised with advanced age as evidenced by increased penetration of fluorescent nanoparticles in a particle exclusion assay and larger open spaces on scanning electron microscopy. Reduced hyaluronan (HA) levels, decreased expression of genes encoding HA-associated proteins (e.g., Ptx3 and Tnfaip6), and increased expression of inflammatory genes and matrix metalloproteinases underlie this loss of matrix integrity. Importantly, HA levels are decreased with age in follicular fluid of women, indicative of conserved reproductive aging mechanisms. These findings provide novel mechanistic insights into how defects in cumulus expansion contribute to age-related infertility and may serve as a target to extend reproductive longevity.

Putrescine alleviates the oxidative damage of cumulus-oocyte complex via improving fatty acid oxidation

BackgroundFatty acid oxidation of cumulus-oocyte complex (COC) provides sufficient energy for oocyte maturation. But, the relationship between fatty acid oxidation and oxidative stress in aging follicles, as well as the effect of putrescine, is still unclear. MethodsThe porcine COCs were randomly divided into four groups and cultured in in vitro maturation (IVM) medium with or without 1 mmol/L putrescine, with 50 μmol/L hydrogen peroxide (H2O2) or with 50 μmol/L H2O2 plus 1 mmol/L putrescine. Oocyte maturation was assessed by the first polar body extrusion. The expressions of genes involved in fatty acid oxidation were detected, and the mitochondrial function was analyzed by themembrane potential. ResultsThe maturation rate of oocyte was significantly lower in the H2O2 group when compared with the control group (P＜0.001), and putrescine significantly increased this rate in the H2O2 plus putrescine group when compared with the H2O2 group (P＜0.001). The expressions of LKB1, STRAD, ACC2, AMPKα1 and AMPKα2 mRNAs in cumulus cells (CCs) were significantly downregulated by H2O2 treatment, and partly rescued by putrescine addition (P＜0.05–0.001). However, the changes of LKB1, STRAD, ACC2, AMPKα1 and AMPKα2 mRNAs in oocytes were inapparent. The mitochondrial membrane potential of CCs in the H2O2 group was significantly lower than that in the control group, while putrescine addition significantly increased the mitochondrial membrane potential (P＜0.001). ConclusionThe decrease of oocyte maturation due to oxidative stress is related with the decreased fatty acid oxidation, and putrescine may alleviate the COCs damage via improving fatty acid oxidation.

Smart-seq2 Technology Reveals a Novel Mechanism That Zearalenone Inhibits the In Vitro Maturation of Ovine Oocytes by Influencing TNFAIP6 Expression.

Zearalenone (ZEN), a non-steroidal estrogenic fungal toxin widely present in forage, food, and their ingredients, poses a serious threat to animal and human reproductive health. ZEN also threatens ovine, a major source of human food and breeding stock. However, the mechanisms underlying the impact of ZEN on the in vitro maturation (IVM) of ovine oocytes remain unclear. This study aimed to elucidate these mechanisms using the Smart-seq2 technology. A total of 146 differentially expressed genes were obtained, using Smart-seq2, from sheep oocytes cultured in vitro after ZEN treatment. ZEN treatment inhibited RUNX2 and SPP1 expression in the PI3K signaling pathway, leading to the downregulation of THBS1 and ultimately the downregulation of TNFAIP6; ZEN can also decrease TNFAIP6 by reducing PTPRC and ITGAM. Both inhibit in vitro maturation of ovine oocytes and proliferation of cumulus cells by downregulating TNFAIP6. These findings provide data and a theoretical basis for elucidating ZEN's toxicity mechanisms, screening therapeutic drugs, and reducing ZEN-related losses in the ovine industry.

The Role of Cyclin-Dependent Kinases (CDK) 4/6 in the Ovarian Tissue and the Possible Effects of Their Exogenous Inhibition.

The combination of cyclin-dependent kinase (CDK) 4/6 inhibitors with endocrine therapy is the standard treatment for patients with HR+/HER2- advanced breast cancer. Recently, this combination has also entered the early setting as an adjuvant treatment in patients with HR+/HER2- disease at a high risk of disease recurrence following (neo)adjuvant chemotherapy. Despite their current use in clinical practice, limited data on the potential gonadotoxicity of CDK4/6 inhibitors are available. Hence, fully informed treatment decision making by premenopausal patients concerned about the potential development of premature ovarian insufficiency and infertility with the proposed therapy remains difficult. The cell cycle progression of granulosa and cumulus cells is a critical process for ovarian function, especially for ensuring proper follicular growth and acquiring competence. Due to the pharmacological properties of CDK4/6 inhibitors, there could be a potentially negative impact on ovarian function and fertility in women of reproductive age. This review aims to summarize the role of the cyclin D-CDK4 and CDK6 complexes in the ovary and the potential impact of CDK4/6 inhibition on its physiological processes.

Midkine can not be accepted as a new biomarker for unexplained female infertility

Abstract Objectives This study aimed to investigate whether a growth factor and a cytokine midkine (MK) can be a new biomarker for the diagnosis and treatment of unexplained female infertility (UFI) cases. Methods Serum (S), follicle fluid (FF), and cumulus cells (CCs) of the patients aged 20–42 years, diagnosed with male factor (MF) and UFI were used. Patients underwent Intracytoplasmic Sperm Injection (ICSI). The anti-Müllerian hormone (AMH) and MK levels with other hormone levels (FSH, LH, E2, PRL, INHB, TSH), the oocyte and embryo qualities, the fertilization and pregnancy rates, and cumulus cells (Cell number and ultrastructure, apoptosis rate) were evaluated. Student-T-test was performed and p&lt;0.05 was considered statistically significant. Results The lowest numbers of CCs were found at UFI (p&lt;0.05). The lowest apoptosis rate with the highest CC viability rate was evaluated at MF (p&lt;0.05). The lowest AMH and MK levels (S, FF) were detected at UFI in comparison to MF (p&lt;0.05). MK and AMH levels of non-pregnant subjects were much lower than pregnant subjects (p&lt;0.05). In addition, these levels were lower in the subjects above 35 age (p&lt;0.05). Structural analysis of CCs showed that the number of lytic cells with cell remnants and apoptotic bodies was higher in non-pregnant subjects. It seems that MK did not show any resistance to both AMH and apoptosis. Conclusions MK can not be accepted as a new biomarker for the diagnosis and treatment monitoring of UFI cases.

Feline Wharton's jelly-derived mesenchymal stem cells as a feeder layer for oocytes maturation and embryos culture in vitro.

Due to their capacity to release growth factors and cytokines, co-culture using mesenchymal stem cells has been considered a good alternative to promoting the maturation of the oocytes and the embryo's development quality in vitro in different mammalian species. In this regard, we investigated the effect of feline Wharton's jelly MSCs as feeders layer in oocyte maturation-consequently, the development of resulting embryos in co-culture. Oocytes with dark cytoplasm and a few layers of cumulus cells were collected and subjected to in vitro maturation and embryo culture using commercial media with and without MSCs addition. The oocytes' nuclear maturation and the degree of cumulus expansion in different groups were assessed after 24 h; the development of the embryo was evaluated every 12 h until day eight. Although MSCs increased the proportion of cumulus cells oocytes exhibiting cumulus expansion, there were no significant differences in the percentage of matured oocytes (metaphase II) among the groups (p > 0.05). However, the embryo development differs significantly, with a higher cleavage, morula, and blastocyst percentage in oocytes matured with MSC co-culture conditions than in commercial media alone (p < 0.05). Also, we observed higher morula and blastocyst rates in the embryos co-cultured with MSCs during the in vitro culture (p > 0.05). Based on our results, the co-culture with MSCs during the oocyte maturation resulted in better embryo development, as well as the MSCs addition during embryo culture returned an increased number of morula and blastocysts. Further research is needed to fully understand and optimize the use of MSCs in oocyte maturation and embryo development.

FRI388 Follicle-Stimulating Hormone And Salt-inducible Kinases Crosstalk On The Regulation Of Genome-wide Gene Expression In Human Granulosa Cumulus Cells

Abstract Disclosure: E. Hayes: None. R. Filzen: None. M. Rodriguez Esquivel: None. N. Winston: None. M. Fierro: None. S. Sana: None. C.O. Stocco: Grant Recipient; Self; NIH R01HD097202. Infertility affects approximately 15% of couples, 40% are due to ovulatory defects. Successful ovulation requires the timely and tight regulation of folliculogenesis by gonadotropins, more crucially, follicle-stimulating hormone (FSH), which is needed to form preovulatory follicles. However, the mechanisms governing folliculogenesis are incompletely understood. We previously showed that salt inducible kinases (SIKs) are critical regulators of female fertility. Specifically, knockout of SIK2 enhances ovulation, whereas deletion of SIK3 results in anovulation and premature ovarian failure. At the molecular level, SIK2, but not SIK3, attenuates the expression of FSH-induced steroidogenic genes. This report aimed to outline the genome-wide effect of SIK inhibition on gene expression in the presence or absence of FSH. Here, we treated human cumulus cells with a SIK inhibitor, HG-9-91-01 (HG), FSH, or their combination. We then performed RNA sequencing and identified 3,472 differentially expressed genes (DEGs) across treatment groups. Interestingly, 498/646 (77%) of DEGs in FSH-treated cells were also identified as DEGs in HG-treated cells. Further, HG + FSH resulted in 865 DEGs not differentially regulated by FSH or HG alone. Analysis of DEGs by Gene Set Enrichment Analysis (GSEA) revealed top enriched gene ontology terms including processes related to the extracellular matrix, steroid hormone biosynthesis, PI3K-Akt signaling, and cAMP signaling. The data suggest that SIK activity profoundly affects gene expression in human granulosa cells, likely via regulation of FSH signaling and other mechanisms. These findings provide new insights and directions for investigating the mechanisms by which SIKs regulate ovarian function and fertility. Presentation: Friday, June 16, 2023

THE ROLE OF LUTEINIZING HORMONE (LH) RECEPTOR IN THE REGULATION OF OOCYTE MATURATION: A SYSTEMATIC LITERATURE REVIEW

The hormone luteinizing hormone (LH) is an important glycoprotein hormone that controls the activity of the gonadal organs, which in turn affects the physiology of the menstrual cycle. For LH to exert its effects, its specific receptor (LHR) must be expressed by cumulus cells. Objective: To determine the role of LH receptors (Luteinizing Hormone) in the regulation of human oocyte maturation processes Method:Systematic Literature Review (SLR). Data collection was conducted by documenting all articles based on predefined inclusion and exclusion criteria. A total of 10 national and international journal articles were retrieved from databases such as Google Scholar, PubMed, and Science Direct, using specific keywords related to the role of LH receptors and oocyte maturation. The literature search and study selection process followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Results:The highest expression of Luteinizing Hormone Receptor (LHR) observed in preovulatory follicles. Cumulus cell maturation, covering 100% of the oocytes, was 74%. FSHR and LHR mRNA expression levels increased with an increase in LH concentration in the in vitro maturation (IVM) medium. After 24 hours of IVM, there was a significant downregulation in the expression of LHR and FSHR.The mRNA and protein levels of FSHR and LHR in the cumulus-oocyte complex gradually decreased with increasing doses of FRBI in the IVM media compared to the control group. Majority of the cumulus cells from MII oocytes had RGS2/B2M gene expression of more than 50%. LH receptor expression in granulosa cells positively correlated with morphology, oocyte maturity, and fertility rate in the poor responder group. Granulosa cells from an agonist-triggered cycle with recombinant human chorionic gonadotropin (rhCG) for 24 hours were observed to increase viability, regulate mRNA expression of 3β-HSD, LH receptor, VEGF, and BCL-L2. More than 50% of the oocytes are matured after hCG injectionat an hCG concentration of 119.8 mIU/mL. Conclusion:LH needs receptor binding and expression for proper function, resulting in improved oocyte maturation with optimal LHR concentration which is 10 µg/mL LH. LHR expression is highest in preovulatory follicles. LHR expression increases during antral follicle growth. Supplementing media with hCG improves oocyte maturity with a 77 mIU/mL cutoff value. Supplementation with FRBI reduce LHR expression in COCs. The number of cumulus cells can also indicate the level of oocyte maturity, where cumulus cells covering 100% of the oocytes show the highest oocyte maturation.

Ferulic Acid Enhances Oocyte Maturation and the Subsequent Development of Bovine Oocytes.

Improving the quality of oocytes matured in vitro is integral to enhancing the efficacy of in vitro embryo production. Oxidative stress is one of the primary causes of quality decline in oocytes matured in vitro. In this study, ferulic acid (FA), a natural antioxidant found in plant cell walls, was investigated to evaluate its impact on bovine oocyte maturation and subsequent embryonic development. Bovine cumulus-oocyte complexes (COCs) were treated with different concentrations of FA (0, 2.5, 5, 10, 20 μM) during in vitro maturation (IVM). Compared to the control group, supplementation with 5 μM FA significantly enhanced the maturation rates of bovine oocytes and the expansion of the cumulus cells area, as well as the subsequent cleavage and blastocyst formation rates after in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Furthermore, FA supplementation was observed to effectively decrease the levels of ROS in bovine oocytes and improve their mitochondrial function. Our experiments demonstrate that FA can maintain the levels of antioxidants (GSH, SOD, CAT) in oocytes, thereby alleviating the oxidative stress induced by H2O2. RT-qPCR results revealed that, after FA treatment, the relative mRNA expression levels of genes related to oocyte maturation (GDF-9 and BMP-15), cumulus cell expansion (HAS2, PTX3, CX37, and CX43), and embryo pluripotency (OCT4, SOX2, and CDX2) were significantly increased. In conclusion, these findings demonstrate that FA supplementation during bovine oocyte IVM can enhance oocyte quality and the developmental potential of subsequent embryos.

Glucose and redox metabolism in meiotically blocked in vitro grown mouse antral follicles.

Glucose and redox metabolism characterization in mouse antral follicles with meiotically blocked oocytes, after in vitro follicle culture (IFC) from the early secondary stage. Following IFC (10 days), oocytes, corresponding cumulus (CC), and granulosa cells (GC) were collected from antral follicles: (i) on day 9-immature, germinal vesicle (GV) stage; (ii) on day 10, after hCG/EGF stimulation-mature, metaphase II (MII) stage and meiotically blocked (MB) immature GV stage. The metabolic profiles of all samples (GV, MII, and MB) were compared by measuring changes in metabolites involved in glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), and redox activity via enzymatic spectrophotometric assays in each cell type. Within MB follicles, GCs drive higher levels of glycolysis and lactic acid fermentation (LAF) while oocytes exert more PPP activity. MB-oocytes had significantly larger diameters compared to day 9 GVs. MB follicles revealed limited metabolic changes in the somatic compartment compared to their GV counterparts (before stimulation). MB-CCs showed increased aconitase and glucose-6-phosphate dehydrogenase activities with lower malate levels comparted to GV-CCs. MB and MII in vitro grown follicles displayed comparable metabolic profiles, suggesting culture induces metabolic exhaustion regardless of the maturation stage. Current results suggest that in addition to impaired nuclear maturation, metabolic disruption is present in MB follicles. MB follicles either compensate with high levels of TCA cycle and PPP activities in CCs, or are unable to drive proper levels of aerobic metabolism, which might be due to the current culture conditions.

A Redesigned Method for CNP-Synchronized In Vitro Maturation Inhibits Oxidative Stress and Apoptosis in Cumulus-Oocyte Complexes and Improves the Developmental Potential of Porcine Oocytes.

In vitro embryo production depends on high-quality oocytes. Compared with in vivo matured oocytes, in vitro oocytes undergo precocious meiotic resumption, thus compromising oocyte quality. C-type natriuretic peptide (CNP) is a follicular factor maintaining meiotic arrest. Thus, CNP-pretreatment has been widely used to improve the in vitro maturation (IVM) of oocytes in many species. However, the efficacy of this strategy has remained unsatisfactory in porcine oocytes. Here, by determining the functional concentration and dynamics of CNP in inhibiting spontaneous meiotic resumption, we improved the current IVM system of porcine oocytes. Our results indicate that although the beneficial effect of the CNP pre-IVM strategy is common among species, the detailed method may be largely divergent among them and needs to be redesigned specifically for each one. Focusing on the overlooked role of cumulus cells surrounding the oocytes, we also explore the mechanisms relevant to their beneficial effect. In addition to oocytes per se, the enhanced anti-apoptotic and anti-oxidative gene expression in cumulus cells may contribute considerably to improved oocyte quality. These findings not only emphasize the importance of screening the technical parameters of the CNP pre-IVM strategy for specific species, but also highlight the critical supporting role of cumulus cells in this promising strategy.

Early cumulus cell removal increases cumulative live birth rate while having no negative effect on the malformation rate in in vitro fertilization: a propensity score-matched cohort study.

The aim of this study was to investigate the efficacy and safety of early cumulus cell removal (ECCR) during human in vitro fertilization (IVF). A retrospective analysis was performed between January 2011 and December 2019. The study enrolled 1131 couples who underwent IVF treatment with ECCR. After propensity score matching at a 1:1 ratio, 1131 couples who underwent overnight coincubation of gametes were selected. The main outcome measure was the cumulative live birth rate. Secondary outcome measures included the cumulative pregnancy rate, polyspermy rate, available embryo rate, miscarriage rate, malformation rate, time to live birth, and oocyte-to-baby rate. There were no significant differences found between the two groups in the polyspermy rate, available embryo rate, miscarriage rate, time to live birth, oocyte-to-baby rate, and neonatal congenital anomalies rate. The results of the study showed that ECCR was associated with a significantly higher cumulative live birth rate and cumulative pregnancy rate, along with a significantly lower fertilization rate. ECCR tended to confer increased cumulative live birth rate and had no negative effect on the neonatal malformation rate.

Deciphering Cellular Heterogeneity and Communication Patterns in Porcine Antral Follicles by Single-Cell RNA Sequencing.

The antral follicle stage is a critical period in mammalian oocyte maturation, marked by complex interactions between oocyte development and neighboring granulosa cells. Understanding the heterogeneity and communication patterns of granulosa cells within antral follicles is crucial for deciphering their roles in follicle development and oocyte maturation. Here, we employed single-cell RNA-sequencing to explore the molecular and cellular characteristics of porcine antral follicles. Our analysis revealed distinct subpopulations within mural and cumulus granulosa cells, indicating diverse cellular states and functions within the follicles. Functional enrichment analysis unveiled the involvement of specific subpopulations in steroid biosynthesis, cumulus expansion, and cellular communication. Moreover, comparing mature and less mature follicles highlighted differences in cell distribution and functions, indicating developmental-specific variations. Our findings shed light on the intricate cellular heterogeneity and communication network within porcine antral follicles, providing valuable insights into the regulation of follicle development and oocyte maturation in pigs. These results hold promise for improving pig reproductive efficiency and advancing human reproductive medicine.