Granulosa Cell Samples Research Articles

Impact of Amphiregulin on Oocyte Maturation and Embryo Quality: Insights from Clinical and Molecular Perspectives

AbstractIdentifying non-invasive biomarkers which can predict the outcome of intracytoplasmic sperm injection (ICSI) is crucial, particularly in Germany where the challenges are intensified by the Embryo Protection Act. Recent research has highlighted biomarkers within the epidermal growth factor (EGF) family as central to follicular processes, although their predictive utility remains a subject of debate in the literature. Therefore, the primary objective of this study was to investigate the significance of amphiregulin concentrations in follicular fluid and gene expression in mural granulosa cells on oocyte maturation, fertilization, and embryo quality.A total of 33 women were recruited at the University Clinic of Saarland Fertility Center (Homburg, Germany). Follicular fluid aspiration consisted of single/individual aspiration of follicles, enabling a 1 : 1 correlation with retrieved oocytes. Follicular fluid and mural granulosa cell samples from 108 oocytes were analyzed. Amphiregulin levels were determined with enzyme-linked immunosorbent assay, while gene expression was analyzed with the StepOnePlus Real-Time PCR System using TaqMan Fast Advanced Master Mix assays.Results showed that amphiregulin concentrations affect oocyte maturation, fertilization, and embryo quality, while luteinizing hormone concentrations influence oocyte maturation, with significant differences identified between fertilized/unfertilized and good/poor embryo groups. Amphiregulin expression significantly impacts oocyte maturation, with downregulation observed in immature oocytes, while luteinizing hormone/chorionic gonadotropin receptor expression showed no significant differences between groups and did not influence maturation, fertilization, or embryo quality.These findings are very important for advancing infertility treatment, especially in Germany. The results for amphiregulin may provide prognostic insights which could be useful when selecting viable oocytes and embryos. This research underscores the importance of non-invasive biomarkers for optimizing ICSI outcomes and potentially enhancing the success rates of assisted reproductive technology.

Preliminary construction of non-coding RNAs and ceRNA regulatory networks mediated by exosomes in porcine follicular fluid

BackgroundFollicles are fundamental units of the ovary, regulated intricately during development. Exosomes and ovarian granulosa cells (OGCs) play pivotal roles in follicular development, yet the regulatory mechanisms governing exosomes remain elusive. ResultsHigh-throughput sequencing was employed to evaluate the complete transcript expression profiles of six samples (three porcine ovarian granulosa cells-exosome co-culture samples (GCE) and three porcine ovarian granulosa cells (POGCs) samples). Differential expression analysis revealed 924 lncRNAs, 35 circRNAs, 49 miRNAs, and 9823 mRNAs in the GCE group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated enrichment of differentially expressed transcripts in pathways related to cell proliferation and apoptosis. Furthermore, a ceRNA regulatory network comprising 43 lncRNAs, 6 circRNAs, 11 miRNAs, and 126 mRNAs was constructed based on intergene co-expression correlations. Seven miRNAs associated with cell proliferation and apoptosis regulation were identified within this network, encompassing 92 subnet pairs as candidate genes for further exploration of exosome regulatory mechanisms. Additionally, preliminary verification at the cellular level demonstrated that exosomal miR-200b enhances the viability of POGCs. ConclusionsTranscriptome analysis unveiled a pivotal candidate ceRNA network potentially implicated in exosome-mediated regulation of granulosa cell proliferation and apoptosis, thereby influencing porcine follicular development. These findings offer insights into the molecular mechanisms of follicular fluid exosome regulation, encompassing both coding and non-coding RNA perspectives.

P-762 Discovery and quantification of microplastics in human cumulus granulosa cells

Abstract Study question The impact of microplastics (MPs) on human health has received increasing attention; their accumulation in female reproductive system and possible harms have not been quantified. Summary answer This study firstly reveals the contamination of human cumulus granulosa cells and follicular fluid by MPs and the effects of these contaminants on ART outcomes. What is known already MPs are ubiquitous in soil, sea, air, fresh water, and other environments. MPs are ingested by humans through the food chain, air and direct contact, resulting in various health problems, such as endocrine disruption, immune disturbance and reproductive toxicity. MPs exposure has been found to have a great impact on females, as MPs may accumulate in the ovaries and reduce the number of follicles, ovarian size, pregnancy rate, embryo production and fertility. To date, one study has found evidence of MPs in human placentas for the first time, but MPs contamination in other human reproductive systems has not been identified. Study design, size, duration The inclusion criteria comprised female patients of infertile couples excluding male factors who visited the outpatient department of the Reproductive and Genetic Hospital of CITIC-Xiangya between January 2019 and January 2021.We randomly selected 16 women who met the criteria and assessed their CCs and follicular fluid samples for the presence of MPs. Participants/materials, setting, methods In the present study, 16 cumulus granulosa cell samples and 2 follicular fluid samples were collected, and MPs were detected via pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). Main results and the role of chance MPs were detected in both cumulus granulosa cells and follicular fluid, with an average abundance of 313.14±246.69 mg/kg in cumulus granulosa cellsand 12.88±15.54 mg/kg in follicular fluid. Polyethylene terephthalate was the predominant polymer in cumulus granulosa cells. There were no significant differences in the IVF fertilization rates, ICSI fertilization rates, IVF good-quality embryo rates, or ICSI good-quality embryo rates between the high-MP group and low-MP group. However, embryos in the high-MP group exhibited a lower normal cleavage rate than did those in the low-MP group, while the embryos in the high-MP group exhibited a greater proportion of noncleavage than did those in the low-MP group. Limitations, reasons for caution It is not clear how long these particles may persist in the human body. The method we used can detect only the concentration of MPs but not the size of MP particles. Moreover, our method cannot be used to quantify smaller-diameter nanoplastics, which are considered to pose greater risks. Wider implications of the findings This is the first study in which MPs were identified and characterized in human ovarian granulosa cells, providing reference data for evaluating their toxicity to the human female reproductive system. Trial registration number Not applicable

Investigating the Role of Inflammatory Response in Polycystic Ovary Syndrome Using Integrated RNA-Seq Analysis.

An important factor in the pathogenesis of polycystic ovary syndrome (PCOS) is chronic low-grade inflammation. However, the exact pathophysiology of PCOS is currently unknown, which makes clinical diagnosis and the development of effective treatments more difficult. We aimed to investigate the role of the inflammatory response in initiating and progressing PCOS. 13 control granulosa cell samples and 15 granulosa cell samples from patients with PCOS were obtained from the GSE102293, GSE34526, and GSE5850 datasets. The gene set variation analysis (GSVA) method was used to calculate the inflammatory response score. Subsequently, the genes associated with inflammation in the hub were identified using differential expression analysis and weighted gene co-expression network analysis (WGCNA). The findings were confirmed by analysis of independent datasets and examination of clinical samples by qRT-PCR analysis. A consensus cluster analysis was conducted to categorize the PCOS samples into subtypes related to inflammation. Functional enrichment and analysis of immune cell infiltration were conducted to explore the potential mechanisms involved. Additionally, the CMap database was utilized to predict potential drugs, and the results were confirmed through molecular docking. During the training cohort analysis, we identified five distinct genes (TGFBR2, ICAM3, WIPF1, SLC11A1, and NCF2) that could serve as potential diagnostic markers for PCOS. The expression levels of these genes were confirmed through validation in both the test set and clinical samples. In training cohort, two distinct inflammatory patterns (C1 and C2) were identified, and the C2 subtype exhibited activated immune- and inflammation-related pathways. Esmolol was shown to have potential as a drug to treat PCOS and it showed good results for molecular binding at TGFBR2, ICAM3, WIPF1, SLC11A1, and NCF2 proteins. Five diagnostic biomarkers and two inflammation-related molecular types associated with PCOS were identified, and esmolol was a potential drug for PCOS treatment. Our findings provided new diagnostic markers and potential small-molecule drugs for PCOS diagnosis and prevention.

RNA-seq and miRNA-seq profiling analyses reveal molecular mechanisms underlying the progression of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of childbearing age, mainly characterized by menstrual disorders, infertility, and other symptoms. Due to its complex and multifactorial nature, the pathogenesis of PCOS remains unclear. MicroRNAs (miRNA) play vital roles in regulating gene expression. In this study, twelve ovarian granulosa cell samples from six PCOS patients and six non-PCOS women were collected as research objects. We aimed to identify key genes and miRNAs involved in the progression of PCOS using RNA-seq and small RNA-seq techniques, and to construct the corresponding miRNA-mRNA regulatory network. A total of 1552 differentially expressed genes (DEGs) and 52 differentially expressed (DE)-miRNAs were detected. Functional enrichment analyses suggested that the occurrence and development of PCOS might be closely related to processes such as glucose metabolism, hormone synthesis and cell function. The miRNA-mRNA regulatory network indicated that hsa-miR-330-5p and hsa-miR-663b may be candidate miRNAs that affect the development of PCOS. This study provides new clues for the pathogenesis of PCOS and a theoretical basis for new therapeutic targets.

Dysregulation of ferroptosis-related genes in granulosa cells associates with impaired oocyte quality in polycystic ovary syndrome.

Poor oocyte quality remains one of the major challenges for polycystic ovary syndrome (PCOS) patients during in vitro fertilization (IVF) treatment. Granulosa cells (GCs) in PCOS display altered functions and could cause an unfavorable microenvironment for oocyte growth and maturation. Ferroptosis is a new form of programmed cell death, but its role in PCOS has been largely unclarified. Ferroptosis-related differentially expressed genes (DEGs) of GCs in women with PCOS were identified by bioinformatic analyses of GSE155489 and GSE168404 datasets. Functional enrichment analyses were conducted using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Core ferroptosis-related genes were further screened by random forest, and evaluated for diagnostic value by receiver operating characteristic curve analyses. Gene expression was validated by real-time quantitative polymerase chain reaction of collected GC samples, and analyzed for association with oocyte quality. In addition, gene regulatory network was constructed based on predicted RNA interactions and transcription factors, while potential therapeutic compounds were screened through molecular docking with crystallographic protein structures. A total of 14 ferroptosis-related DEGs were identified. These DEGs were mainly enriched in reactive oxygen species metabolic process, mitochondrial outer membrane, antioxidant activity as well as ferroptosis and adipocytokine signaling pathways. Eight core ferroptosis-related genes (ATF3, BNIP3, DDIT4, LPIN1, NOS2, NQO1, SLC2A1 and SLC2A6) were further selected in random forest model, which showed high diagnostic performance for PCOS. Seven of them were validated in GC samples, and five were found to be significantly and positively correlated with one or more oocyte quality parameters in PCOS patients, including oocyte retrieval rate, mature oocyte rate, normal fertilization rate, and good-quality embryo rate. Gene regulatory network revealed JUN and HMGA1 as two important transcription factors, while dicoumarol and flavin adenine dinucleotide were predicted as small molecules with therapeutic potential. This is the first comprehensive report to study the differential expression of ferroptosis-related genes in GCs of PCOS and their clinical relevance with oocyte quality. Our findings could provide novel insights on the potential role of GC ferroptosis in PCOS pathogenesis, diagnosis, and targeted treatment.

Effects of stem cell factor in follicular fluid and granulosa cells on oocyte maturity and clinical pregnancy.

Stem cell factor (SCF) is implicated in cell growth, proliferation, differentiation, migration, and apoptosis. SCF in follicular fluid (FF) and granulosa cells (GCs) plays a key role in oocyte maturation and clinical pregnancy; however, the exact mechanism is unclear. We aimed to investigate SCF potential in predicting oocyte maturity and clinical pregnancy. We collected 60 FF and 60 GCs samples from different patients with infertility. Real-time polymerase chain reaction and cellular immunofluorescence analyses were used to quantitatively and qualitatively determine SCF concentration in GCs; enzyme-linked immunosorbent assay was used to determine SCF concentration in FF. GC and FF SCF concentrations were positively correlated with metaphase (M)II oocyte proportion and clinical pregnancy (R = 0.280, 0.735 vs R = 0.257, 0.354). SCF concentrations in GCs were significantly higher in the clinical pregnancy group than in the nonclinical pregnancy group. Immunofluorescence analysis showed that SCF expression was higher in the clinical pregnancy and high-MII -oocyte proportion groups. Receiver operating characteristic curve analysis showed that combined SCF and serum anti-Müllerian hormone levels could predict oocyte maturity and clinical pregnancy better than either of these factors alone. SCF concentration in GCs and FF can serve as a predictor of oocyte maturity and clinical pregnancy.

Midkine characterization in human ovaries: potential new variants in follicles

To characterize the growth factor midkine (MDK) in the human ovary to determine whether MDK is produced locally within the ovary, examine whether different ovarian cell types are more likely to produce MDK, and determine whether there are any stage-specific variations during follicle growth. Previous studies have revealed that MDK potentially affects human follicle growth and oocyte maturation. Proteomic analyses in follicular fluid (FF) have identified MDK to functionally cluster together and follow a similar expression profile to that of well-known proteins involved in ovarian follicle development. Midkine has not yet been characterized in the human ovary. Descriptive study. University Hospital. The study included samples from 121 patients: 71 patients (aged 17-37 years) who underwent ovarian tissue cryopreservation provided granulosa cells (GC), cumulus cells, ovarian cortex, medulla tissue, and FF from small antral follicles (SAF); and 50 patients (aged 20-35 years) receiving invitro fertilization treatment provided FF from preovulatory follicles before and after induction of final follicle maturation. None. MDK relative gene expression was quantified using a real-time quantitative polymerase chain reaction in cumulus cells, GC, and medulla tissue. Additionally, immunostaining and western blotting assays were used to detect MDK protein in the ovarian cortex, which contains preantral follicles, SAF, and medulla tissue. Furthermore, enzyme-linked immunosorbent assay analyses were performed to measure the concentration of MDK in FF aspirated from SAF and preovulatory follicles both before and 36 hours after inducing the final maturation of follicles. Immunostaining and reverse transcription-quantitative polymerase chain reaction revealed a more prominent expression of MDK in GC compared with other ovarian cell types. Intrafollicular MDK concentration was significantly higher in SAF compared with preovulatory follicles. In addition, different molecular weight species of MDK were detected using western blotting in various ovarian sample types: GC and FF samples presented primarily one band of approximately 15 kDa and an additional band of approximately 13 kDa, although other bands with higher molecular weight (between 30 and 38 kDa) were detected in medulla tissue. This is the first time that MDK has been immunolocalized in human ovarian cells at the protein level and that potentially different MDK variants have been detected in human FF, GC, and ovarian medulla tissue. Future studies are needed to sequence and identify the different potential MDK variants found to determine their functional importance for ovary and oocyte competence.

Detection of zeb1 Gene in Granulosa Cells in Women Undergoing IVF Treatment.

ZEB1 plays a role in epithelial-to-mesenchymal transition and acts as a repressor of E-cadherin, TGF-β, and Wnt/β-catenin. Since ZEB1 protein is expressed in estrogen-responsive tissues, and expression of the gene in the normal ovary and endometrium is positively correlated with high estrogen levels, we performed a direct analysis of granulosa cell samples to determine whether there are any significant changes in zeb1 expression during folliculogenesis. ZEB1 expression levels were measured in the granulosa cells of 56 infertile women undergoing IVF treatment. RNA extraction from granulosa cells was performed along with reverse transcription quantitative polymerase chain reaction (RT-qPCR) with SYBR Green I to determine zeb1 gene expression levels. Statistical analysis was performed by using t-test, while possible correlations of the expression of ZEB1 protein with body mass index (BMI), age, number of oocytes, and oocyte maturation were investigated. Zeb1 gene expression levels correlate significantly with body mass index (BMI) and age, but not with oocyte number and oocyte maturation stage. Obese women demonstrate a higher expression level of zeb1 gene compared to normal and overweight women. Moreover, zeb1 gene is overexpressed in women aged 35-40 years old and is under-expressed in women >40 years old. ZEB1 expression should be further investigated as it may unveil new potential findings of the zeb1 gene's role in female fertility and its use as a biomarker in fertility workups.

P-651 The role of FMR1-associated miR-323a and its impact on SMAD-signaling pathway in relation to the regulation of ovarian function

Abstract Study question Is FMR1 (Fragile-X-Mental Retardation 1 Gene)-targeting miR-323a related to poor ovarian response and what impact does it have on the folliculogenesis-regulating SMAD-signaling pathway? Summary answer In poor ovarian responders, miR-323a is significantly upregulated, FMRP – downregulated. Human granulosa cells, transfected with a miR-323a mimic, show an upregulation of SMAD proteins. What is known already Folliculogenesis is a complex process, regulated, among others, by FMR1 and its protein FMRP. MicroRNAs are involved in translational repression, controlling ovarian function and follicular development. miR-323a suppresses FMR1 in tumor cells, but their relationship has not been evaluated in the human ovary. miR-323a also interacts with components of SMAD pathways, which regulate cell proliferation, differentiation and apoptosis. SMADs belong to the downstream signaling of bone morphogenetic protein-receptor-2, and our previous data shows that its expression is affected by FMRP. Thus, researching miR-323a together with its target molecules may provide more insight into the pathogenesis of poor ovarian response. Study design, size, duration For ex vivo experiments, we investigated human granulosa cell (HGC) samples of poor (POR, N = 22) and normal (NOR, N = 32) ovarian responders, with an ongoing sample collection which began in 2013. Our in vitro experiments were performed on COV434 cell line, transfected with a microRNA-323a-3p mimic. RNA extraction and RT-qPCR were performed to determine the relative expression levels of the targets, with NOR serving as the control group ex vivo and non-transfected COV434 cells in vitro. Participants/materials, setting, methods 54 patients who underwent IVF/ICSI treatments were recruited and divided into two groups based on their ovarian response according to the Bologna criteria. HGCs were collected during oocyte retrieval, and COV434 cell line was used for miRNA mimic transfection experiments. Briefly, we performed RNA extraction, cDNA synthesis and TaqMan RT-PCR, as well as protein extraction and ELISA. The collected data was analysed using the 2–ΔΔCtmethod and Student’s t-test. Main results and the role of chance We found that miR-323a was significantly upregulated in HGCs of POR (p = 0.0324). FMR1 expression in this patient group showed a slight downregulation that did not reach statistical significance (p = 0.1054). Protein expression level analysis of 22 patients from this collective (NNOR=11, NPOR=10) demonstrated a significant downregulation of FMRP in POR (p = 0.0373), suggesting its participation in the pathogenesis of poor ovarian response. Additionally, in vitro upregulation of miR-323a resulted in altered expressions of SMAD-signaling pathway components. Namely, SMAD3 expression increased by 5.7 fold, SMAD4 – 1.6 fold, SMAD5 – 2.5 fold, and SMAD8 – 4.1 fold, compared to non-transfected COV434 cells, with SMAD3 results requiring further investigation due to an unexpected 2 fold elevation of its expression in the negative control (scramble) sample. FMR1 and SMAD1 expression levels remained stable between transfected cells and control samples. These findings show that FMR1-targeting miR-323a may play a critical role in regulating the SMAD pathways, thus affecting folliculogenesis, HGC proliferation and, ultimately, ovarian reserve, which is reduced in case of poor ovarian response. Limitations, reasons for caution Larger patient sample size is needed to confirm the current results. An age-related influence cannot be ruled out because of the existing difference between the groups. More repeats of miR-323a-3p mimic transfection and miR-323a-3p inhibitor transfection should be carried out to support the in vitro findings. Wider implications of the findings For the first time, we researched the expression levels of miR-323a in HGCs. Our results suggest that miR-323a, which, like FMRP, is downregulated in POR, is involved in the FMR1/FMRP regulatory loop, and its upregulation affects the SMAD-signaling pathway, deepening our understanding of the pathogenesis of poor ovarian response. Trial registration number not applicable

Hsa-miR-320a-3p and hsa-miR-483-5p levels in human granulosa cells: promising bio-markers of live birth after IVF/ICSI

BackgroundMicroRNAs (miRNAs) are considered potential biomarkers for various diseases. This study investigated whether hsa-miR-320a-3p and hsa-miR-483-5p levels in human ovarian granulosa cells derived from follicular fluids are associated with embryo developmental competence.MethodsWe collected 195 granulosa cells samples and analyzed the treatment outcomes in patients undergoing in vitro fertilization (n = 147) or intracytoplasmic sperm injection (n = 48) cycles. The hsa-miR-320a-3p and hsa-miR-483-5p levels in granulosa cells were measured using quantitative reverse transcription-polymerase chain reaction.ResultsPatients were subdivided into four groups according to the granulosa cells hsa-miR-320a-3p and hsa-miR-483-5p levels quartiles (Q1–Q4). Embryo developmental competence was compared using the chi-square test. Patients in Q3 were less likely to achieve a normal fertilization rate for in vitro fertilization and blastocyst formation than those in Q1 as they expressed high levels of hsa-miR-320a-3p and hsa-miR-483-5p (P < 0.05). Patients in Q3 and Q4 were less likely to achieve a good-quality embryo as they expressed high levels of hsa-miR-483-5p and hsa-miR-320a-3p (P < 0.05). The hsa-miR-320a-3p and hsa-miR-483-5p levels were not associated with clinical pregnancy. However, multiple regression analysis indicated that in Q3 and Q4 intervals had experienced a decreased chance of live birth due to high expression levels of hsa-miR-320a-3p and hsa-miR-483-5p levels. The relative hsa-miR-320a-3p expression levels in granulosa cells were weakly and positively correlated with the patient age (P = 0.0033). Moreover, both the basal follicle stimulating hormone (P = 0.0003) and ovarian stimulation protocols (P = 0.006 and P = 0.004) significantly and positively affected hsa-miR-320a-3p levels. The days of stimulation was negatively correlated with the relative hsa-miR-320a-3p expression level (P = 0.047).ConclusionsThe hsa-miR-320a-3p and hsa-miR-483-5p levels in human granulosa cells negatively correlated with the good-quality embryo rate and live birth, indicating that hsa-miR-320a-3p and hsa-miR-483-5p can be used as potential negative indicators to predict good-quality embryos and live births.

Construction of a competing endogenous RNA network to identify drug targets against polycystic ovary syndrome.

Would the construction of a competing endogenous RNA (ceRNA) network help identify new drug targets for the development of potential therapies for polycystic ovary syndrome (PCOS)? Both Food and Drug Administartion (FDA)-approved and candidate drugs could be identified by combining bioinformatics approaches with clinical sample analysis based on our established ceRNA network. Thus far, no effective drugs are available for treating PCOS. ceRNAs play crucial roles in multiple diseases, and some of them are in current use as prognostic biomarkers as well as for chemo-response and drug prediction. For the bioinformatics part, five microarrays of human granulosa cells were considered eligible after applying strict screening criteria and were used to construct the ceRNA network for target identification. For population-based validation, samples from 24 women with and without PCOS were collected from January 2021 to July 2021. The public data included 27 unaffected women and 25 women with PCOS, according to the Rotterdam criteria proposed in 2003. The limma and RobustRankAggreg R packages were used to identify differentially expressed messenger RNAs and noncoding RNAs. Gene Ontology, Reactome and Kyoto Encyclopedia of Genes and Gemomes (KEGG) enrichment analyses were performed. A ceRNA network was constructed by integrating the differentially expressed genes and target genes. The population-based validation included human luteinized granulosa cell samples from 12 unaffected women and 12 women with PCOS. Quantitative real-time polymerase chain reaction was conducted to detect the levels of mRNAs and microRNAs (miRNAs). Connectivity map and computational model algorithms were implemented to predict therapeutic drugs from the ceRNA network. Additionally, we compared the predicted drugs with known clinical medications in DrugBank. A set of 10 mRNAs, 11 miRNAs and 53 long non-coding RNAs (lncRNAs) were differentially expressed. Functional enrichment analysis revealed the highest relevance to immune system-related biological processes and signalling pathways, such as cytokine secretion and leucocyte chemotaxis. A ceRNA consisting of two lncRNAs, two miRNAs and five mRNAs was constructed. Through network construction via bioinformatic analysis, we identified some already approved drugs (such as metformin) that could target some molecules in the network as potential drug candidates for PCOS. Public sequencing data were obtained from GSE34526, GSE84376, GSE102293, GSE106724 and GSE114419, which have been deposited in the Gene Expression Omnibus database. Experiments, such as immunoprecipitation, luciferase reporter assays and animal model studies, are needed to validate the potential targets in the ceRNA network before the identified drug candidates can be tested using cellular and animal model systems. Our findings provide new bioinformatic insight into the possible pathogenesis of PCOS from ceRNA network analysis, which has not been previously studied in the human reproductive field. Our study also reveals some potential drug candidates for the future development of possible therapies against PCOS. This study was supported by grants from the National Key Research and Development Program of China (2021YFC2700400) and the National Natural Science Foundation of China (82001498). The authors have no conflicts of interest to disclose.

Exploration of molecular features of PCOS with different androgen levels and immune-related prognostic biomarkers associated with implantation failure.

BackgroundPolycystic ovary syndrome (PCOS), the most common heterogeneous reproductive disease afflicting women of childbearing age, has been recognized as a chronic inflammatory disease recently. Most PCOS patients have hyperandrogenism, indicating a poor prognosis and poor pregnancy outcomes. The molecular mechanism underlying PCOS development is still unknown. In the present study, we investigated the gene expression profiling characteristics of PCOS with hyperandrogenism (HA) or without hyperandrogenism (NHA) and identified immune-related factors that correlated with embryo implantation failure.MethodsPCOS and recurrent implantation failure (RIF) microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. ClueGO software was used to perform enrichment analysis of differentially expressed genes (DEGs) in PCOS with varying androgen levels. The Weighted Co-Expression Network Analysis (WGCNA) was used to identify co-expressed modules and shared gene signatures between HA PCOS and RIF. Moreover, the upregulated DEGs of HA PCOS and RIF were intersected with shared gene signatures screening by WGCNA to excavate further key prognostic biomarkers related to implantation failure of HA PCOS. The selected biomarker was verified by qRT-PCR.ResultsA total of 271 DEGs were found in HA PCOS granulosa cell samples, and 720 DEGs were found in NHA PCOS. According to CuleGO enrichment analysis, DEGs in HA PCOS are enriched in immune activation and inflammatory response. In contrast, DEGs in NHA PCOS are enriched in mesenchymal cell development and extracellular space. Using WGCNA analysis, we discovered 26 shared gene signatures between HA PCOS and RIF, which were involved in corticosteroid metabolism, bone maturation and immune regulation. DAPK2 was furtherly screened out and verified to be closely related with the development of HA PCOS, acting as an independent predictor biomarker of the embryo implantation failure. DAPK2 expression was negatively correlated to the embryo implantation rate (r=-0.474, P=0.003). The immune infiltration results suggested that upregulated DAPK2 expression was closely related with NK cell infiltration and macrophage M2, playing an essential role in the pathogenesis of implantation failure in HA PCOS.ConclusionOur research revealed the expression profiling of PCOS with different androgen levels and identified DAPK2 as a critical prognostic biomarker for implantation failure in PCOS.

Metabolic and antioxidant status during transition is associated with changes in the granulosa cell transcriptome in the preovulatory follicle in high-producing dairy cows at the time of breeding

In this study, we hypothesized that early postpartum (pp) metabolic and oxidative stress conditions in dairy cows (particularly those with severe negative energy balance, NEB) are associated with long-term changes in granulosa cell (GC) functions in the preovulatory follicle at the time of breeding. Blood samples were collected at wk 2 and wk 8 pp from 47 healthy multiparous cows. Follicular fluid (FF) and GC were collected from the preovulatory follicle after estrous synchronization at wk 8. Several metabolic and antioxidant parameters were measured in blood and FF, and their correlations were studied. Subsequently, 27 representative GC samples were selected for RNA sequencing analysis. The GC gene expression data of LH-responsive genes and the estradiol:progesterone ratio in FF were used to identify pre- and post-LH surge cohorts. We compared the transcriptomic profile of subgroups of cows within the highest and lowest quartiles (Q4 vs. Q1) of each parameter, focusing on the pre-LH surge cohort (n = 16, at least 3 in each subgroup). Differentially expressed genes (DEG: adjusted P-value < 0.05, 5% false discovery rate) were determined using DESeq2 analysis and were functionally annotated. Blood and FF β-carotene and vitamin E concentrations at wk 2, but not at wk 8, were associated with the most pronounced transcriptomic differences in the GC, with up to 341 DEG indicative for lower catabolism, increased oxidoreductase activity and signaling cascades that are known to enhance oocyte developmental competence, increased responsiveness to LH, and a higher steroidogenic activity. In contrast, elevated blood NEFA concentrations at wk 2 (and not at wk 8) were associated with a long-term carryover effect detectable in the GC transcriptome at wk 8 (64 DEG). These genes are related to response to lipids and ketones, oxidative stress, and immune responses, which suggests persistent cellular stress and oxidative damage. This effect was more pronounced in cows with antioxidant deficiencies at wk 8 (up to 148 DEG), with more genes involved in oxidative stress-dependent responses, apoptosis, autophagy and catabolic processes, and mitochondrial damage. Interestingly, within the severe NEB cows (high blood NEFA at wk 2), blood antioxidant concentrations (high vs. low) at wk 8 were associated with up to 194 DEG involved in activation of meiosis and other signaling pathways, indicating a better oocyte supportive capacity. This suggests that the cow antioxidant profile at the time of breeding might alleviate, at least in part, the effect of NEB on GC functions. In conclusion, these results provide further evidence that the metabolic and oxidative stress in dairy cows early postpartum can have long-term effects on GC functions in preovulatory follicles at the time of breeding. The interplay between the effects of antioxidants and NEFA illustrated here might be useful to develop intervention strategies to minimize the effect of severe NEB on fertility.

High-Throughput Sequencing Profiles About lncRNAs and mRNAs of Ovarian Granulosa Cells in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, which is characterized by ovulatory dysfunction, clinical and/or biochemical androgen excess, polycystic ovaries on ultrasound and genetic heterogeneity. It was well-accepted that many lncRNAs and mRNAs were associated with PCOS, however, remain unclear. Therefore, the purpose of our study was to examine different expression profiles of lncRNAs and mRNAs in ovarian granulosa cells (GCs) in PCOS and Controls, and identify the correlation between lncRNAs, mRNAs and clinical parameters. Sixty five PCOS patients and 65 Controls were enrolled in this study and adopted standard long agonist protocols or GnRH antagonist protocols. Then 6 GCs samples in each group were subjected to high-thoughput sequencing and the remaining samples were used for the further verification by quantitative real-time PCR (qRT-PCR). Gene Oncology (GO), Kyoto Encyclopedia Genes and Genomes (KEGG) enrichment analysis were performed. We predicted the relationship between lncRNAs and mRNAs by Cytoscape software. According to the expression level of lncRNAs, mRNAs and the clinical parameters, we also explored their relationship and evaluate their predictive values for embryos quality and PCOS. We identified 1,049 differential expressed lncRNAs and 3,246 mRNAs (fold-change ≥2, p-value < 0.05). Seven lncRNAs (NONHSAT101926.2, NONHSAT136825.2, NONHSAT227177.1, NONHSAT010538.2, NONHSAT191377.1, NONHSAT230904.1, ENST00000607307) and 3 mRNAs (EREG, ENTPD6, YAP1) were validated consistent with sequence profile. Seven lncRNAs were related to hormone level and follicle counts, 3 mRNAs had connections with lipid metabolism. The area under curve (AUC) of 7 lncRNAs were valuable in distinguishing patients with PCOS from Controls. The AUC of NONHSAT230904.1 and NONHSAT227177.1 were 0.6807 and 0.6410, respectively, for distinguishing whether the rate of high-quality embryos exceeds 50%. Our study showed that the GCs lncRNAs and mRNAs were involved in the occurrence and development of PCOS, which contribute to clarify the pathogenesis mechanism of PCOS.

FEMALE GAMETE AND HUMAN EMBRYO SUSCEPTIBILITY TO SARS-COV-2

Objective: To determine the susceptibility of the oocytes, embryos, granulosa and cumulus cells to SARS-CoV-2 infection. Materials and Methods: To address this question, we retrospectively examined the gene expression profiles of SARS-CoV-2-associated receptors and proteases in human granulosa cells (GCs), cumulus cells (CCs), mature oocytes, day 3 embryos, blastocysts and trophectoderm cells obtained from our previously described Affymetrix microarray data from assisted reproduction patients. Human GCs and CCs (n=17), mature oocytes (n=6), and preimplantation embryos (n=20) were analyzed and gene expression levels of receptors and proteases closely related to SARS-CoV-2 infection was reported. For each gene, the number of samples with the probe set 'present', based on the detection call was studied. Each probe set was classified according to the signal intensity value median, as low ( 200). Results: ACE2, BSG, CTSL, CTSA were detectable at high expression level in all mature oocyte samples, while only CTSL was strongly expressed in all day 3 embryos. The most representative dual co-expression of SARS-CoV-2-associated receptor and protease (60% of samples) during the embryonic genome activation stage (EGA) was ACE2-CTSL and BSG-CTSL. In blastocysts, ACE2, BSG, CTSL, CTSA and FURIN were detectable in the entire cohort at high expression level, and the prevalence of the different dual co-expression of SARS-CoV-2-associated proteases and receptors was optimal (100% of samples). Interestingly, only CTSL was detectable in all trophectoderm samples and a prevalence of 60% was found for the BSG-CTSL co-expression. ACE2, BSG, CTSL and CTSA were present at high expression level in CCs samples. In contrast, ACE2 and BSG expression was very low while CTSL and CTSA showed a high expression level in GCs. A prevalence of 100% was reported for ACE2-CTSL, ACE2-CTSA co-expression for both cell types. In addition, BSG-CTSL and BSG-CTSA co-expression were also present in all CCs against ∼70% in GCs samples. This data suggests a potential risk of SARS-CoV-2 infection either GC or early embryo development. Conclusions: Transcriptomic analyses of SARS-CoV-2-associated receptors and proteases strongly suggest that blastocysts are most permissive to SARS-CoV-2 compared with mature oocytes and day 3 embryos. Impact Statement: Specimens from female genital tract may be considered as potential targets for SARS-CoV-2.

P–242 Gene expression profiles of SARS-CoV–2-associated receptors and proteases in human early embryonic development and follicular cells

Study questionAre the oocytes, embryos, granulosa and cumulus cells, used during ART, susceptible to the SARS-CoV–2 infection?Summary answerTranscriptomic analyses of SARS-CoV–2-associated receptors and proteases strongly suggest that blastocysts are most permissive to SARS-CoV–2 compared with mature oocytes and day 3 embryos.What is known alreadyVery few studies analyzed the gene expression profiles of SARS-CoV–2-associated receptors and proteases, mainly focusing on ACE2 and TMPRSS2 expression, resulting in partial knowledge in different specimens from female genital tract. To date, the gene expression profile of SARS-CoV–2 host entry candidates in the entire preimplantation embryos is scarcely available. Moreover, reports on oocyte and granulosa cells susceptibilityto SARS-CoV–2 are very sparse.Study design, size, durationTo address this question, we retrospectively examined the gene expression profiles of SARS-CoV–2-associated receptors and proteases in human granulosa cells (GCs), cumulus cells (CCs), mature oocytes, day 3 embryos, blastocysts and trophectoderm cells obtained from our previously described Affymetrix microarray data.Participants/materials, setting, methodsHuman GCs and CCs (n = 17), mature oocytes (n = 6), and preimplantation embryos (n = 20) were analyzed. The comparison of gene expression levels of receptors and proteases closely related to SARS-CoV–2 infection. For each gene, the number of samples with the probe set ‘present’, based on the detection call, was analyzed. Each probe set was classified according to the signal intensity value median, as low (<100), medium (100–200) or high expression level (>200).Main results and the role of chanceACE2, BSG, CTSL, CTSA were detectable at high expression level in all mature oocyte samples, while only CTSL was strongly expressed in all day 3 embryos. The most representative dual co-expression of SARS-CoV–2-associated receptor and protease (60% of samples) during the embryonic genome activation stage (EGA) was ACE2-CTSL and BSG-CTSL. In blastocysts, ACE2, BSG, CTSL, CTSA and FURIN were detectable in the entire cohort at high expression level, and the prevalence of the different dual co-expression of SARS-CoV–2-associated proteases and receptors was optimal (100% of samples). Interestingly, only CTSL was detectable in all trophectoderm samples and a prevalence of 60% was found for the BSG-CTSL co-expression. ACE2, BSG, CTSL and CTSA were present at high expression level in CCs samples. In contrast, ACE2 and BSG expression was very low while CTSL and CTSA showed a high expression level in GCs.A prevalence of 100% was reported for ACE2-CTSL, ACE2-CTSA co-expression for both cell types. In addition, BSG-CTSL and BSG-CTSA co-expression were also present in all CCs against ∼70% in GCs samples. This data suggest a potential risks of SARS-CoV–2 infection either GC or early embryo development.Limitations, reasons for cautionAnalyses of Affymetrix microarray gene expression data were performed in non-COVID–19 patients. Whether the SARS-CoV–2 infection change the gene expression profile of SARS-CoV–2-associated receptors and proteases is under investigation.Wider implications of the findings: Specimens from female genital tract may be considered as potential targets for SARS-CoV–2.Trial registration numberNot applicable

Corticosterone Injection Impairs Follicular Development, Ovulation and Steroidogenesis Capacity in Mice Ovary.

Simple SummaryResearchers have hitherto established hundreds of animal stress models. However, these models have some limitations due to the complexity in operation and large differences between individual animals. In particular, there are few stress models that are specifically applied in mammalian ovaries. In this study, using intraperitoneal injection of cortisol/corticosterone (CORT), we successfully established a stress model that acts on the ovarian function. Our data showed that CORT inhibits ovarian and follicular development and blocks ovulation. The establishment of this model might provide a living platform for studying ovarian stress in future research.The aim of this study is to establish an ovarian stress model, and to investigate the effects of stress on follicular development. Our data showed that continuous intraperitoneal injection of CORT successfully created a stressful environment in the ovary. To assess the effects of CORT on ovarian functions, 80 three-week-old ICR (Institute of Cancer Research) female mice were randomly divided into control group and treatment group. All mice were injected intraperitoneally with pregnant horse serum gonadotropin (PMSG). At the same time, the treatment group were injected with CORT (1 mg/mouse) at intervals of 8 h; while the control group was injected with same volume of methyl sulfoxide (DMSO). Blood, ovaries, or ovarian granulosa cell samples were collected at 24 h, 48 h, and 55 h after PMSG injection. The results showed that, compared with the control group, CORT-injected mice revealed a significant decrease in ovulation rates, ovarian weight, ovarian index, the number of secondary follicles and mature follicles, levels of estrogen and progesterone, and mRNA expression of steroid synthase-related genes. Collectively, our findings clearly demonstrated that CORT injection could represent an effective practice to simulate stresses that inhibit ovarian functions by reducing follicular development and ovulation.

MON-232 Differential Regulation of Genes Relevant to Reproductive Function in Human Granulosa Cells of Small Antral Follicles from Women with or without Polycystic Ovary Syndrome

Introduction: Polycystic Ovary Syndrome (PCOS) is the most common endocrinopathy in young women, affecting 5-10% of women of reproductive age. Despite its high prevalence, the underlying pathogenesis of PCOS remains complex and incompletely understood. Aberrant function of granulosa cells has been implicated in the aetiology of PCOS. Material and methods: Granulosa cells (GCs) were obtained from follicular fluid from (unstimulated) individual small antral follicles (SAF) isolated at the time of removal of ovarian tissue for fertility cryopreservation in young women. Granulosa luteal (GL) cells were collected from pooled follicular fluid collected at the time of oocyte retrieval in women undergoing IVF. RNA was extracted and RT-qPCR carried out to analyse expression of genes involved in granulosa cell function, including steroidogenesis and growth. 30 GC samples from individual small antral follicles from 15 women with regular cycles and 31 samples from 10 women with PCOS were included. Mean follicle size was similar between groups (mean 5.5±1mm). Luteinised GCs (GLCs) from 8 women with and without PCOS were also included. Results: 92% of GC samples from SAF expressed LH receptor (LHCGR) RNA. Expression of LHCGR was not significantly higher in PCOS GCs but there was a difference in distribution of results with a subset of PCOS samples (13%) with 4-6 times higher expression than in that of the controls. LHCGR expression was higher in GLCs compared to GCs (P<0.05). FSH receptor (FSHR) expression was significantly lower in PCOS GCs from SAFs (P<0.05), unlike PCOS GLCs, which expressed higher levels of FSHR (p<0.05). FSHR expression was 14 fold higher in GCs compared to GLCs (p<0.0001). There was no correlation between LHCGR, FSHR or follicle size. Androgen receptor (AR) expression was lower in PCOS GCs from SAF (p<0.01) unlike PCOS GLCs, which expressed more AR than GL controls (P<0.05). AR expression correlated with FSHR (p<0.0001) but not LHCGR. CYP11A1 was lower in both PCOS GCs and GLCs (p<0.05) and expression was 50 fold higher in GLCs than GCs (p<0.0001). Expression of 3BHSD2 was reduced in PCOS GCs (p=0.06). There were no significant differences in expression of STAR, CYP19, INHBA or INHBB between control and PCOS in either GCs or GLCs. Conclusions: Granulosa cells from small antral follicles from women with PCOS have a lower expression of FSHR, AR and CYP11A1 and a trend towards higher LHCGR compared to controls. This expression pattern is suggestive of early luteinisation of granulosa cells in unstimulated small antral follicles in PCOS.

Vibrational characterization of granulosa cells from patients affected by unilateral ovarian endometriosis: New insights from infrared and Raman microspectroscopy

Endometriosis is a chronic gynaecological disease characterised by the presence of endometrial cells in extra-uterine regions. One of the main factors impacting on the fertility of women affected by endometriosis is the poor oocyte quality. Granulosa Cells (GCs) regulate oocyte development and maintain the appropriate microenvironment for the acquisition of its competence; hence, the dysregulation of these functions in GCs can lead to severe cellular damages also in oocytes. In this study, luteinized GCs samples were separately collected from both ovaries of women affected by Unilateral Ovarian Endometriosis and analysed by infrared and Raman microspectroscopy. The spectral data were compared with those of GCs from women with diagnosis of tubal, idiopathic or male infertility (taken as control group). The coupling of these two spectroscopic techniques sheds new light on the alteration induced by this pathology on GCs metabolism and biochemical composition. In fact, the study revealed similar biochemical modifications in GCs from both ovaries of women affected by unilateral ovarian endometriosis, such as the alteration of the protein pattern, the induction of oxidative stress mechanisms, and the deregulation of lipid and carbohydrate metabolisms. These evidences suggest that unilateral endometriosis impairs the overall ovarian functions, causing alterations not only in the ovary with endometriotic lesions but also in the contralateral "healthy" one.