Assessment of Gene Expression on the Gap Junction Connexin of Cumulus Cells on Infertile Women With Polycystic Ovary Syndrome and Poor Ovarian Response: The Novel Role of Propranolol

The in vitro maturation (IVM) of human oocytes for in vitro fertilization (IVF): is it time yet to switch to IVM-IVF?

Is poor ovarian response associated with a change in predicted age based on a DNA methylation-derived age prediction model (the Horvath algorithm) in white blood cells (WBCs) or cumulus cells (CCs)? In young women, poor ovarian response is associated with epigenetic age acceleration within WBC samples but is not associated with age-related changes in CC. The majority of human tissues follow predictable patterns of methylation which can be assessed throughout a person's lifetime. DNA methylation patterns may serve as informative biomarkers of aging within various tissues. Horvath's 'epigenetic clock', which is a DNA methylation-derived age prediction model, accurately predicts a subject's true chronologic age when applied to WBC but not to CC. A prospective cohort study was carried out involving 175 women undergoing ovarian stimulation between February 2017 and December 2018. Women were grouped according to a poor (≤5 oocytes retrieved) or good (>5 oocytes) response to ovarian stimulation. Those with polycystic ovary syndrome (PCOS) (n = 35) were placed in the good responder group. DNA methylation patterns from WBC and CC were assessed for infertile patients undergoing ovarian stimulation at a university-affiliated private practice. DNA was isolated from peripheral blood samples and CC. Bisulfite conversion was then performed and a DNA methylation array was utilized to measure DNA methylation levels throughout the genome. Likelihood ratio tests were utilized to assess the relationship between predicted age, chronologic age and ovarian response. The Horvath-predicted age for WBC samples was consistent with patients' chronologic age. However, predicted age from analysis of CC was younger than chronologic age. In subgroup analysis of women less than 38 years of age, poor ovarian response was associated with an accelerated predicted age in WBC (P = 0.017). Poor ovarian response did not affect the Horvath-predicted age based on CC samples (P = 0.502). No alternative methylation-based calculation was identified to be predictive of age for CC. To date, analyses of CC have failed to identify epigenetic changes that are predictive of the aging process within the ovary. Despite the poor predictive nature of both the Horvath model and the novel methylation-based age prediction model described here, it is possible that our efforts failed to identify appropriate sites which would result in a successful age-prediction model derived from the CC epigenome. Additionally, lower DNA input for CC samples compared to WBC samples was a methodological limitation. We acknowledge that a universally accepted definition of poor ovarian response is lacking. Furthermore, women with PCOS were included and therefore the group of good responders in the current study may not represent a population with entirely normal methylation profiles. The process of ovarian and CC aging continues to be poorly understood. Women who demonstrate poor ovarian response to stimulation represent a common clinical challenge, so clarifying the exact biological changes that occur within the ovary over time is a worthwhile endeavor. The data from CC support a view that hormonally responsive tissues may possess distinct epigenetic aging patterns when compared with other tissue types. Future studies may be able to determine whether alternative DNA methylation sites can accurately predict chronologic age or ovarian response to stimulation from CC samples. Going forward, associations between epigenetic age acceleration and reproductive and general health consequences must also be clearly defined. No external funding was obtained for the study and there are no conflicts of interest. N/A.

Gene expression of cumulus cells in women with poor ovarian response after dehydroepiandrosterone supplementation

Study question To investigate if the anti-ovarian antibody (AOA) is associated with poor ovarian response (POR) and pro-inflammatory immune responses in women undergoing assisted reproductive technology (ART) cycles. Summary answer The POR patients have a higher prevalence of AOAs. Women with autoimmune POR (POR(+)/AOA(+)) have dysregulated pro-inflammatory immune responses and metabolic factors. What is known already It has been proved that AOAs play important role in diseases that related to human reproduction such as premature ovarian failure (POF) which also termed as premature ovarian insufficiency (POI), infertility, polycystic ovary syndrome (PCOS), in vitro fertilization (IVF) implantation failure, and in poor ovarian response in IVF stimulation. The POR women had elevated inflammatory immune responses: increased NK cell count and cytotoxicity, B cell counts, Th1/Th2 ratio and elevated metabolic factors such as higher homocysteine and plasminogen activator inhibitor–1 (PAI–1) level. Study design, size, duration This study is a retrospective cohort study between December 2015 and February 2019. 248 women who underwent ART cycles were included. Study patients were divided into four groups based on AOA test and POR diagnose defined by the European Society of Human Reproduction and Embryology consensus: POR(-)/AOA (-) group (N = 148), POR(+)/AOA(-) group (N = 34), POR (-)/AOA (+) group (N = 44), POR(+)/AOA(+) group (N = 22). Peripheral blood was collected during the early follicular phase when they enter the program. Participants/materials, setting, methods The natural killer (NK) cell levels and cytotoxicity, T helper (Th) 1/Th2 cell ratios were measured by flowcytometry. Anti-phospholipid Antibodies (APA) was tested by enzyme linked immunosorbent assay (ELISA). AOA, 25 (OH) vitamin D level, homocysteine, PAI–1 level was tested by Immunofluorescence Assay.One way ANOVA was applied to compare the continuous variables among study groups. Chi-squared analysis or Fisher’s exact test were performed to compare the categorical variables. Main results and the role of chance The POR patients have a significantly higher prevalence of AOA than non-POR patients (39.3% vs. 22.9%, P = 0.017, OR 2.176 95% CI 1.156–4.099). Peripheral blood CD56+ NK cell level (%), NK cytotoxicity, CD19+CD5+ B–1 cell level (%) and IFN-g/IL–10 producing Th1/Th2 cell ratios were significantly higher in POR(+)/AOA(+) group than those of other groups (P < 0.05, P < 0.05, P < 0.05, P < 0.05, respectively). TNF-a/IL–10 producing Th1/Th2 cell ratio of POR(+)/AOA (+) group was significantly higher than those of POR(+)/AOA(-) and POR(-)/AOA(-) groups (P < 0.05, respectively). Peripheral blood homocysteine and vitamin D levels of the POR(+)/AOA (+) group were significantly lower than those of other groups (P < 0.005, respectively). Peripheral blood PAI–1 level of POR(+)/AOA(+) group was significantly higher than that of POR(-)/AOA(-) group (P < 0.05). In POR(+)/AOA(+) group, the prevalence of antiphospholipid antibody was significantly higher than that of POR(+)/AOA(-) group (54.5% vs 20.5%, P = 0.005, OR 4.667, 95% CI 1.532–14.216). Limitations, reasons for caution This was a single center study, results need to be validated across different center and study population. Wider implications of the findings: The diagnostic and therapeutic approaches for AOA (+) autoimmune POR patients should be differentiated from those for non-autoimmune POR. Trial registration number Not applicable

Relationship between embryo development and apoptotic gene expression of cumulus cells in poor responders and polycystic ovary syndrome

Study question Is telomerase machinery altered in cumulus and granulosa cells of patients with Polycystic ovary syndrome (PCOS) undergoing assisted reproductive treatment (ART)? Summary answer Patients with PCOS presented an altered expression of telomerase inhibitor factors TRF1 and TRF2 and shorter telomeres in cumulus cells (CCs) and granulosa cells (GCs) What is known already PCOS affects 6%-20% of fertile women; one of the most common features is represented by abnormal folliculogenesis, showing a higher number of follicles at all developmental stages. In PCOS, increased levels of reactive oxygen species (ROS) have been related to abnormal follicular development due to oxidative stress-induced DNA damage. Telomeres, present at the ends of the chromosomes, are essential for maintaining their stability during somatic cell proliferation. When guanine-rich telomeric repeats are exposed to oxidative stress including ROS, they undergo oxidative DNA damage that shortens telomeres in non-dividing cells like follicular cells, thereby potentially altering folliculogenesis. Study design, size, duration A prospective experimental study was conducted at IVIRMA Rome and University Unicamillus from September 2022 to January 2024. CCs and GCs were collected from the follicular fluid of patients undergoing in vitro fertilization (IVF). Twenty-eight patients with PCOS and. Thirty-one normal responders were included in the pilot study. Participants/materials, setting, methods Women with PCOS (n = 28), diagnosed according to the Rotterdam criteria, were included in the study. The healthy control group (CONT) included normal responders (n = 31) undergoing IVF. CCs and GCs were collected on the day of oocyte retrieval and subsequently used for immunofluorescence spot counting of TRF1 and TRF2. In addition, telomere length was measured using a specific kit. qRT-PCRs for TRF1, TRF2 and apoptotic marker BAX genes were performed and expressed as 2^-ΔCT values. Main results and the role of chance Immunofluorescence for TRF1 in the PCOS group presented a higher spot number in CCs compared to the CONT (9.90±0.23 vs 7.96±0.33, p = 0.0009) while TRF2 spots significantly decreased (8.85 ±0.24 vs 11.23±0.28; p = 0.0007). An increase of TRF2 gene expression was observed in CCs of the PCOS group when compared to CONT (0.0052±0.0006 vs 0.0030±0.0003; p = 0.062) no differences were found in the expression of TRF1 (0.0343±0.006 vs 0.026±0.047p=0.68; Bax was significantly increased (0.0139±0.001 vs 0.0102±0,0007 p = 0.007) and telomeres have a shortening trend (0.36±0.36 vs 1±0 p = 0.74). In GC a lower number of TRF1 spots in PCOS vs CONT was observed while TRF2 had just an increasing trend (11.16± 0.586 vs 11.70 ± 1.99, p = 0.61; 16.79 ± 3,85 vs 11.34 ± 0.588; p = 0.68). In GCs of PCOS group vs CONT there was an increase in TRF2 gene expression (0,007 ± 0,001 vs 0.003 ± 0.0004; p = 0.03) and no differences were found in TRF1 (0.032± 0.003 vs 0.003± 0.012 p = 0.25). Ultimately Bax was increased in GCs of PCOS samples vs CONT (0.018 ±0.002 vs 0.011 ±0.0005 p = 0.0006) and the telomeres were significantly shorter (0.0246± 0.0349 vs 1± 0 p < 0.0001). Limitations, reasons for caution A limited number of samples were analyzed in the study and different PCOS sub-phenotypes were not considered. The analysis of CCs could be uninformative due to inter-individual variability in the PCOS sub-phenotypes within the same study group. Wider implications of the findings These findings demonstrate that PCOS patients present a higher expression of telomerase-inhibiting factors TRF1 and TRF2, undergo apoptosis and have significantly shorter telomeres supporting the theory that abnormal folliculogenesis in PCOS affects telomere length, therefore shedding new light on this medical condition and possibly explaining its clinical and biochemical features. Trial registration number not applicable

Background and Objectives: Polycystic ovary syndrome (PCOS) is a major cause of anovulatory infertility, and ovulation induction is the first-line treatment. If this fails, laparoscopic ovarian drilling (LOD) is used to induce mono-ovulations. There have been implications, that LOD can cause destruction of ovarian tissue and therefore premature ovarian failure. Furthermore, unexpected poor ovarian response (POR) to gonadotrophins can occur in PCOS women after LOD. There have been reports about FSH receptor polymorphisms found in women with PCOS that are related to higher serum FSH levels and POR to gonadotrophins. Materials and Methods: In the present study, we retrospectively analyzed data of 144 infertile PCOS women that had LOD performed before IVF. Results: Thirty of included patients (20.8%) had POR (≤3 oocytes) to ovarian stimulation with gonadotrophins. Women with POR had significantly higher median levels of basal serum FSH (7.2 (interquartile range (IQR), 6.0–9.2) compared to women with normal ovarian response (6.0 (IQR, 5.0–7.4); p = 0.006). Furthermore, women with POR used a significantly higher median cumulative dose of gonadotrophins (1875 IU (IQR, 1312.5–2400) for ovarian stimulation compared to women with normal ovarian response (1600 IU (IQR, 1200–1800); p = 0.018). Conclusion: Infertile PCOS women who experience POR after LOD have significantly higher serum FSH levels compared to women with normal ovarian response after LOD. As these levels are still within the normal range, we speculate that LOD is not the cause of POR. We presume that women with PCOS and POR after LOD could have FSH-R genotypes associated with POR and higher serum FSH levels.

Clinical-pregnancy outcome after vitrification of blastocysts produced from in vitro maturation cycles

Background and Aims: Controlled Ovarian Stimulation (COS) is a crucial first step in the In Vitro Fertilization (IVF) or Intra-Cytoplasmic Sperm Injection (ICSI) process. Progestin-primed ovarian stimulation (PPOS) has been recently introduced as an alternative to traditional COS regimens. However, the efficacy of PPOS regimen among infertile women with poor ovarian response (POR) remains unclear. This study aims to evaluate the effect of PPOS regimen on the chance of success throughout the IVF process in Vietnamese infertile women with POR. Method: This randomized controlled trial included 120 infertile women with POR (according to POSEIDON criteria) who received IVF at Tam Anh Fertility Center (Hanoi, Vietnam). PPOS and GnRH_ant regimens were randomly applied to the participants (n=60 for each group). A chain of 4 successive Binomial regression models has been applied to estimate the chance of success for 4 steps: oocyte maturation, fertilization and achieving good quality day-3 and day-5 embryos. The effect of PPOS in comparison with GnRH_ant protocol was evaluated by marginal risk-ratio (RR) from each model. Results: Chances of successfully achieving the MII oocytes, fertilization, high-quality D3 and D5 embryos were all moderately higher when using the PPOS regimen, compared with those of GnRH_ant protocol (95% confidence intervals of RR were respectively 0.88–1.07, 0.89–1.14, 0.96–1.14 and 0.74–1.73). Conclusion: Although not yet reached a statistical significance, those results indicated that using PPOS protocol may slightly improve the chance of success in 4 first critical steps of the IVF process for infertile women with POR.

Polycystic ovary syndrome (PCOS) is a metabolic and endocrine disorder which affects women of reproductive age with prevalence of 8–18%. The oocyte within the follicle is surrounded by cumulus cells (CCs), which connect with mural granulosa cells (MGCs) that are responsible for secreting steroid hormones. The main aim of this study is comparing gene expression profiles of MGCs and CCs in PCOS and control samples to identify PCOS-specific differentially expressed genes (DEGs). In this study, two microarray databases were searched for mRNA expression microarray studies performed with CCs and MGCs obtained from PCOS patients and control samples. Three independent studies were selected to be integrated with naive meta-analysis since raw meta-data from these studies were found to be highly correlated. DEGs in these somatic cells were identified for PCOS and control groups. This study enabled us to reveal dysregulation in MAPK (mitogen activated protein kinase), insulin and Wnt signaling pathways between CCs and MGCs in PCOS. The meta-analysis results together with qRT-PCR validations provide evidence that molecular signaling is dysregulated through MGCs and CCs in PCOS, which is important for follicle and oocyte maturation and may contribute to the pathogenesis of the syndrome.

Background Polycystic ovarian syndrome (PCOS) is a leading cause of infertility and metabolic dysfunction in women, characterized by hyperandrogenism, anovulation, and insulin resistance. Cumulus cells play a crucial role in folliculogenesis and oocyte maturation, necessitating a deeper understanding of their molecular alterations impact in PCOS. Method This study investigates transcriptomic differences in cumulus cells between PCOS and non-PCOS women using high-throughput RNA sequencing data obtained from the NCBI Gene Expression Omnibus (GEO) database (accession number: GSE277906). The RNA sequencing data from 23 PCOS and 17 non-PCOS women were analyzed to identify differentially expressed genes (DEGs) using R-based computational pipelines. Results Differential gene expression analysis identified 3245 significantly dysregulated genes, comprising 1723 upregulated and 1522 downregulated genes in PCOS samples. Functional enrichment analysis revealed that key DEGs (CDH5, CLEC4D, and GNAT1) were associated with follicular development, insulin signaling, and immune response. Gene Set Enrichment Analysis (GSEA) further identified dysregulation in metabolic and reproductive pathways, including ribonucleoprotein complex biogenesis and vascular endothelial growth factor (VEGF) signaling. Conclusion This study highlights that altered gene expression in cumulus cells may impair oocyte competence, potentially influencing fertility outcomes in PCOS patients. GNAT1, previously linked to diabetes, emerged as a novel gene potentially involved in PCOS pathophysiology. However, these findings are derived from a single-center dataset which requires experimental validation. Future studies should incorporate qRT-PCR validation and functional assays in larger and ethically diverse cohorts as means for development of targeted therapeutic interventions to mitigate the reproductive consequences of PCOS.

Oocyte maturation and competence to development depends on its close relationship with cumulus cells (CCs). However, the maturation conditions of human cumulus-oocyte complexes (COCs) might affect gene expression in both oocyte and CCs. We thus compared the transcriptome profiles of CCs isolated from in vivo and in vitro matured COCs at different nuclear maturation stages. Three groups of CCs from patients who underwent ICSI were included: CCs of patients with polycystic ovary syndrome (PCOS) referred for in vitro maturation (IVM), CCs from patients with PCOS for in vivo maturation (used as controls) and CCs from normal responders referred for in vivo maturation. CCs were isolated from COCs at the germinal vesicle, metaphase I and metaphase II stages. Microarray technology was used to analyse the global gene expression and significance analysis of microarray to compare the expression profiles of CCs from COCs at different nuclear maturation stages following IVM or in vivo maturation. Selected genes were validated by RT-qPCR. In CCs isolated after IVM, genes related to cumulus expansion and oocyte maturation, such as EREG, AREG and PTX3, were down-regulated, while cell cycle-related genes were up-regulated in comparison with CCs from in vivo matured COCs from PCOS and normal responder patients. Moreover, irrespective of the stage of oocyte maturation, genes involved in DNA replication, recombination and repair were up-regulated in CCs after IVM. The CC transcriptomic signature varies according to both the oocyte maturation stage and the maturation conditions. Our findings suggest a delay in the acquisition of the mature CC phenotype following IVM, opening a new perspective for the improvement in IVM conditions.

To detect expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) in oocytes, and their receptor type 2 receptor for BMPs (BMPR2) in cumulus cells in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF), and determine if BMPR2, BMP15, and GDF9 expression correlate with hyperandrogenism in FF of PCOS patients. Prospective case-control study. Eighteen MII-oocytes and their respective cumulus cells were obtained from 18 patients with PCOS, and 48 MII-oocytes and cumulus cells (CCs) from 35 controls, both subjected to controlled ovarian hyperstimulation (COH), and follicular fluid (FF) was collected from small (10-14 mm) and large (>18 mm) follicles. RNeasy Micro Kit (Qiagen) was used for RNA extraction and gene expression was quantified in each oocyte individually and in microdissected cumulus cells from cumulus-oocyte complexes retrieved from preovulatory follicles using qRT-PCR. Chemiluminescence and RIA assays were used for hormone assays. BMP15 and GDF9 expression per oocyte was higher among women with PCOS than the control group. A positive correlation was found between BMPR2 transcripts and hyperandrogenism in FF of PCOS patients. Progesterone values in FF were lower in the PCOS group. We inferred that BMP15 and GDF9 transcript levels increase in mature PCOS oocytes after COH, and might inhibit the progesterone secretion by follicular cells in PCOS follicles, preventing premature luteinization in cumulus cells. BMPR2 expression in PCOS cumulus cells might be regulated by androgens.

Background: Growing studies have demonstrated that dehydroepiandrosterone (DHEA) may improve fertility outcomes in poor ovarian responders (PORs). The aim of this study was to compare clinical outcomes and cumulus cell (CC) expression before and after DHEA treatment in PORs undergoing in vitro fertilization (IVF) cycles.Methods: Six patients with poor ovarian response were enrolled in the study according to Bologna criteria. DHEA was supplied at least 2 months before patients entered into the next IVF cycle. Expression of apoptosis-related genes in CCs was determined by quantitative real-time PCR. Mitochondrial dehydrogenase activity of CCs was assessed by cell counting kit-8 assay.Results: Metaphase II oocytes, maturation rate, embryos at Day 3, and fertilization rate significantly increased following DHEA treatment. Expression of cytochrome c, caspase 9, and caspase 3 genes in CCs were significantly reduced after DHEA therapy. Additionally, increased mitochondrial activity of CCs was observed following DHEA supplementation.Conclusions: DHEA supplementation may protect CCs via improved mitochondrial activity and decreased apoptosis, leading to better clinical outcomes in PORs.

This study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.