Cumulus Cell Gene Expression Research Articles

P-567 Recombinant hCG (choriogonadotropin beta, CG beta) exerts a positive dose-dependent effect on human follicular steroidogenesis during ovarian stimulation using a constant rFSH administration

Abstract Study question How does increasing doses of CG beta augment follicular steroidogenesis and is it associated with gene expression in cumulus cells? Summary answer With the exception of progesterone, CG beta exerts dose-dependent effects on intrafollicular steroid hormone concentrations and CYP19a1 expression during constant rFSH administration. What is known already CG beta (FE 999302) is a newly developed rhCG, produced by a human cell line (PER.C6®). This variant contains the same amino acid sequence as the endogenous hCG but presents with a different glycosylation profile when compared to urinary hCG or rhCG derived from a CHO cell line. CG beta has also been shown to have a longer half-life and higher relative potency. It is well known that hCG like LH creates an intracellular response through the LHR expressed on theca cells and mature granulosa cells and hereby augment the production of androgens, progesterone, and oestrogen. Study design, size, duration This study is part of a randomised, double-blind, placebo-controlled trial to investigate the efficacy and safety of CG beta as an add-on treatment to rFSH in women undergoing ovarian stimulation during a long GnRH agonist protocol. The primary endpoint of this study was intrafollicular steroid levels in relation to CG beta administration. Secondary outcomes were gene expression of LHR, FSHR, CYP19a1, and androgen receptor (AR) and single nucleotide polymorphisms (SNPs) in LHR(312) and FSHR(307). Participants/materials, setting, methods 619 women with AMH levels 5-35 pmol/L were randomized to receive either placebo or 1, 2, 4, 8, or 12 µg CG beta from day one of stimulation combined with a constant individualized dose of rFSH. Follicular fluid (FF) (n = 558), granulosa (n = 498) and cumulus cells (n = 368) were collected at oocyte retrieval. Steroid FF hormones were measured using ELISAs, gene expression was analysed in cumulus cells by qRT-PCR, and SNP analysis was performed in granulosa cells. Main results and the role of chance This study found that CG beta has a pronounced positive dose-dependent effect on intrafollicular concentrations of 17-OH-progesterone, androstenedione, testosterone, and oestradiol during constant rFSH administration. As compared to the placebo group without CG beta administration the concentrations of steroids were significantly dose-dependently increased (p-value<0.0001) reaching up to 10 times higher values in the highest dose group without a clear plateauing. However, for progesterone, there was no statistically significant difference between the CG beta dose groups and placebo. The gene expression level of CYP19a1 in the cumulus cells collected at oocyte retrieval increased significantly (p-value=0.0325) for the highest dose of CG beta. However, the gene expression level of FSHR, LHR, and AR in cumulus cells were not affected by CG beta administration, as no dose-response trend with increasing dose of CG beta was observed. Additionally, this study revealed that there was no overall clear difference in the number of oocytes retrieved between the different CG beta dose groups based on either the FSHR(307) or LHR(312) genotypes. Limitations, reasons for caution The gene expression was assessed in cumulus cells, but it would also have been interesting to evaluate the mural granulosa cells. Analysis of the gene expression of 3βHSD and other important steroidogenic enzymes in the mural granulosa cells could reveal further explanation to the effect of CG beta administration. Wider implications of the findings To our knowledge, this is the first study to show a clear dose-dependent effect of rhCG on human steroidogenesis during constant rFSH administration in preovulatory follicles. This reflects the importance of the combined effect of FSH and hCG/LH on granulosa cell activity, follicle health, and potentially oocyte quality. Trial registration number 000289

P-192 ATP5G3 and VEGFC expression in cumulus cells are non-invasive markers of fertilization rate

Abstract Study question Could expression of aging- and hypoxia-related genes in cumulus cells be used as non-invasive markers of fertilization rate in ART? Summary answer The expression levels of specific aging- (ATP5G3) and hypoxia-related genes (VEGFC) in human cumulus cells correlate with oocyte ability to be fertilized. What is known already Throughout a woman reproductive life, cumulus cells (CC) protect the dormant oocyte from damage, act as sensors of the follicular microenvironment and act as gatekeeper for the oocyte developmental potential. One such mechanism relays on the hypoxia-dependent response, which controls the cell metabolic and oxidative state. With age, hypoxia tolerance decreases in the whole organism, including the ovary. Recently, alterations in CC response to the follicular microenvironment have been described in advanced maternal age (AMA) women. We investigate whether expression levels of hypoxia-responsive genes in CC could be used as non-invasive marker of fertilization rate in human oocytes. Study design, size, duration Prospective study of 94 women (41 oocyte donors and 53 patients) recruited from 2018 to 2021. The overall age was 32±8.0 (range 18-46); 25±4.3 (range 18-34) for oocyte donors and 38±4.6 (range 26-46) for patients. The mean antral follicular count (AFC) was 18±10.9 (range 1-48) and the mean MII rate at ovum pick-up (OPU) was 75±17.8% (range 20-100%). Participants/materials, setting, methods Pooled CC were collected from each woman after OPU; total RNA was extracted, reverse transcribed to cDNA using random hexamers and expression of aging- (LYZ, FGF2, ATP5G3, AMH, ANXA5) and hypoxia-related genes (HIF-1A, NOS2, NOS3, HMOX1, TXNIP, CLU, FABP3, TGFBR3, VEGFC) was detected by qPCR and normalized against TBP. Expression levels were plotted against woman age, AFC, MII rate at OPU (MR), and fertilization rates (FR) and non-parametric Spearman’s correlation was applied. Main results and the role of chance Overall, MII and FR rates did not decrease with increasing woman’s age (rs= -0.2, p = 0.06 and rs= -0.1, p = 0.39, respectively). When analyzing expression levels of aging markers (LYZ, FGF2, ATP5G3, AMH, ANXA5), none of them correlate with age, AFC, or MR (rs<│0.1│, p > 0.05) in CCs. Moreover, although abundant, the expression of HIF1A, the main hypoxia-related gene, was not correlated with age, AFC, MR or FR. However, the expression of several genes regulated by HIF1A did show correlation with age, MR or FR. CLU (a secreted chaperone involved in cell death), TXNIP (a tumor suppressor gene involved in redox regulation) and NOS2 (an inducible and calcium-independent isoform of nitric oxide synthase) correlated positively with age (rs=0.25, p = 0.014; rs=0.31, p = 0.0027; and rs=0.24, p = 0.03, respectively). We further analyzed CC gene expression against FR and we found that ATP5G3 and VEGFC levels decreased with increasing FR, independently of age (rs=-0.26, p = 0.044 and rs=-0.31, p = 0.014; respectively). Limitations, reasons for caution The analysis of pooled CC allows for cohort interpretation of results, and they cannot be extrapolated at this time to single oocyte’s reproductive outcome prediction. Wider implications of the findings We found that the fertilization potential of a cohort of oocytes is related to the ability of CC to respond to oxidative stress and hypoxia, pointing at ATP5G3 and VEGFC expression as markers for fertilization success. Further research is needed to assess their association with embryo quality and pregnancy outcomes. Trial registration number NA

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

Research questionIs there a relationship between embryo quality, pregnancy rates and apoptotic gene expression in cumulus cells of oocytes collected from patients with poor ovarian response and polycystic ovary syndrome? DesignFifty infertile couples who underwent assisted reproductive technology treatment were included in the study (Approval date 4 February 2020, number 03). The patients were divided into four group: control (n = 9; 90 oocytes), unexplained infertility (n = 8; 86 oocytes), polycystic ovary syndrome (PCOS) (n = 6; 137 oocytes) and poor ovarian response (POR) (n = 27; 124 oocytes). Cumulus cells were isolated individually from 437 oocytes obtained. Intracytoplasmic sperm injection was undertaken on 365 mature oocytes. The embryos were monitored. Caspase-3, Bax and Bcl-2 gene expressions of the cumulus cells were measured by real-time polymerase chain reaction. ResultsA significant and negative correlation was found between Bax and Bcl-2 expressions of the cumulus cells of poor-quality embryos. The increase in Caspase-3 gene expression in the POR group statistically decreases the pregnancy rates. Fertilization and good-quality embryo development of 365 oocytes whose cumulus cells were examined, however, were not associated with apoptotic gene expression. The Bax/Bcl-2 ratio was found to be significantly lower in cumulus cells of mature oocytes. ConclusionsOur results demonstrated no significant associations between fertilization, quality embryo development and apoptotic gene expression. Bax expression and the Bax/Bcl-2 ratio are high in immature oocyte cumulus cells has shown us that the apoptotic process may begin when the cumulus–oocyte connection exists.

Effects of EGF and melatonin on gene expression of cumulus cells and further in vitro embryo development in bovines.

Despite previous research demonstrating the benefits of including growth factors and antioxidants to maturation medium to support embryo production, to date the effect of epidermal growth factor (EGF) and melatonin (Mel) on oocyte competency has not been studied. This study supplemented in vitro maturation (IVM) medium with EGF (10 ng/ml) and Mel (50 ng/ml) alone, or in combination, and evaluated cumulus cell (CC) gene expression and the development and quality of parthenogenetic blastocysts. No differences in CC gene expression levels indicative of developmental potential were found among the treatment groups. Antioxidant gene CuZnSOD was significantly (P < 0.05) decreased in CCs from the Mel group. Moreover, blastocyst rates on day 7 were significantly increased in EGF or Mel (P < 0.05), but not EGF+Mel. Significant decrease (P < 0.05) in GPX1, CuZnSOD, SLC2A1 and HSPA1A (P = 0.07) mRNA levels was observed in blastocysts from the Mel group. OCT4 gene expression was significantly increased (P < 0.05) in EGF+Mel and confirmed using immunofluorescence. Our results indicate that, despite the lack of changes of competence-related genes in CCs, IVM medium supplemented with Mel improved the culture environment sufficiently, resulting in improved blastocysts. Moreover, EGF and Mel combined during maturation increased OCT4 gene and protein expression in blastocysts, indicating its potential for stem cells.

PCOS and Role of Cumulus Gene Expression in Assessing Oocytes Quality.

The global infertility rate has been declining from year to year. PCOS is one of the treatable accountable causes contributing to anovulatory infertility. Nevertheless, the success rate of treatments and live-birth outcomes especially involving assisted reproductive techniques is still not very promising. There is a reduction in the development potential of oocytes and high-quality embryos in PCOS patients compared to non-PCOS patients. A critical step in IVF treatment is the assessment of oocyte and embryo competence before embryo transfer. Oocytes in metaphase II are very fragile. Repeated morphological assessment on these oocytes may directly impair the quality and affect the whole process. Identification of potential biomarkers especially in the cumulus cells oocytes complex will help to predict the outcome and may create space for improvement. This review has explored gene expression in cumulus cells with regards to oocytes quality in both normal and PCOS women. The gene expression was classified according to their physiological function such as the contribution on cumulus expansion, cumulus cells apoptosis, and glucose metabolism. Collectively, the review suggested that positive expression of HAS2, PTX3, GREM1, and VCAN may correlate with good quality oocytes and can be used as an indicator among PCOS women.

Gene expression of equine cumulus cells during in vitro and in vivo oocyte maturation

In vivo matured oocytes collected from dominant, pre-ovulatory follicles have superior developmental competence when compared to their in vitro matured counterparts, indicating that in vitro maturation (IVM) methods are sub-optimal. Cumulus cells (CC) have a critical role during oocyte maturation; thus, their gene expression holds key information regarding cellular processes associated with oocyte development. Understanding these biological events occurring during in vivo maturation of equine oocytes has the potential to improve IVM conditions. The aim of this study was to compare gene expression profiles of CC obtained from in vitro matured oocytes (IVM-CC) and in vivo matured oocytes (IVV-CC) by identifying differentially expressed genes (DEG) and significant biological pathways related to maturation. For the IVM-CC group, cumulus-oocyte complexes (COCs; n =12) were collected and matured in vitro as previously described (Meyers et al. Reprod Fertil Dev. 2019; 12:1874-1884). COCs for the IVV-CC group (n = 12) were collected after gonadotropin induction of ovulation as previously described (Foss et al. EVJ. 2013; 45:39-43). Extracted RNA from CC was sequenced, generating an average of 24 million reads/sample. The raw reads were trimmed (Trimmgalore) and mapped (STAR) to the current equine reference genome. Mapped reads were quantified (Featurecount) and compared between IVM-CC and IVV-CC groups,using the DESeq2 package in R software. A total of 14,479 genes were expressed in the cumulus cells. Principal component analysis showed a uniform pattern of gene expression among the IVV-CC, in contrast to a more scattered pattern in IVM-CC samples. We found 6,677 DEGs with 4,490 downregulated and 2,187 upregulated genes in the IVM-CC group. The enrichment pathway analysis demonstrated that downregulated genes were involved in transmembrane signaling receptors and chemical stimulus detection, while upregulated genes were associated with chromosome segregation, DNA repair, sister chromatid segregation, DNA replication, mitotic cell cycle process, and nuclear division. We found abnormal expression of genes involved in cumulus expansion and gonadotropin receptors in the IVM-CC group. Our data highlights that IVM culture conditions seem to be a distant replica of the in vivo conditions, as demonstrated by the divergent gene expression of the cumulus cells. The difference observed in gene expression and associated pathways could explain the variability in developmental competence between the in vivo and in vitro matured gametes. Lastly, this data serves as a starting point to determine modifications of current in vitro maturation culture protocols necessary to mimic in vivo conditions. This study was funded by the Theriogenology Foundation and Center for Equine Health at UC Davis.

Leptin improves in-vitro maturation of goat oocytes through MAPK and JAK2/STAT3 pathways and affects gene expression of cumulus cells

We investigated whether the recombinant leptin (1, 10, 100 ng/mL) influences the meiotic maturation of goat oocytes, whether the MAPK and JAK2/STAT3 pathways mediate the effects of leptin during in-vitro maturation, and whether leptin differently affects the abundance of mRNAs relevant to leptin signal transduction and apoptosis in oocytes and cumulus cells. The addition of leptin to the maturation medium positively affected the number of oocytes that completed nuclear maturation. Nuclear oocyte maturation stimulated by leptin was significantly impaired when we added the specific inhibitors of MAPK (U0126) and JAK2/STAT3 (AG490) to the maturation medium. The addition of leptin (10 ng/mL) during maturation did not affect the expression of AMPKα1, PPARα, Caspase 3, and BCL2 genes in oocytes or cumulus cells. The PPARγ and BAX mRNA abundances were significantly reduced in cumulus cells in the leptin group compared to the control group. Our results demonstrate that supplementation of the in-vitro maturation medium with leptin significantly improves nuclear maturation and reveal the important role of the MAPK and JAK2/STAT3 signaling pathways in establishing the leptin-mediated nuclear maturation of goat oocytes. Moreover, leptin treatment affects PPARγ and BAX gene expression in cumulus cells.

Protein source in maturation media affects gene expression in cumulus cells and embryo development in cattle

We aimed to evaluate if protein source (PS) alterations during IVM affect embryo sex/development and gene expression profile in cumulus cells (CCs). Bovine oocytes were matured and cultured in the presence of FBS or BSA. Then, the PS effect during IVM on gene expression (GPC4, VCAN, GHR, PTGS2, and ALCAM) was determined. CC biopsy was removed before and after IVM treatments. After fertilization and cultured, CCs were grouped according to their fate into CCs from immature COCs, CCs from COCs that did or did not result in embryos (according to PS). Results showed that when the culture was performed in FBS presence, blastocyst rate was higher (p < 0.05) than BSA. However, when embryos were cultured with BSA, no effect (p > 0.05) of PS during IVM was observed. PS used during IVM did not affect embryos sex (p > 0.05) but changed VCAN, GHR, PTGS2, and ALCAM genes expression. No differences (p > 0.05) were observed between immature and mature CCs groups in gene expression, regardless of their fate. Only the GHR gene was related to embryo production but just with FBS on IVM. In conclusion, PS can affect embryo development when using the serum on IVM and IVC, influences CCs gene expression, and has to be considered when studying oocyte quality markers.

Accompaniment of Time-Lapse Parameters and Cumulus Cell RNA-Sequencing in Embryo Evaluation.

The aim of this study was to investigate the use of time-lapse morphokinetic parameters and cumulus cells transcriptomic profile to achieve a more accurate and non-invasive method in embryo evaluation. Two hundred embryos from 20 couples were evaluated based on morphokinetic characteristics using time-lapse. Embryos were divided into the high-quality, moderate-quality, and bad-quality groups. Non-fertilized oocytes were considered as the fourth group. T5 (time to five cells), S2 (time from three to four cells), and CC2 (time from two to three cells) were recorded. Also, the cumulus cells of the respective oocytes were divided into high-quality, moderate-quality, bad-quality, and non-fertilized groups based on the grading of the embryos. Then their transcriptomic profiles were analyzed by RNA-sequencing. Finally, the correlation between differentially expressed genes and embryo time-lapse parameters was investigated. T5 was the only timing that showed a statistically significant difference between high-quality group and other groups. RNA-sequencing results showed that 37 genes were downregulated and 106 genes were upregulated in moderate, bad-quality, and non-fertilized groups compared to high-quality group (q value < 0.05). These genes were involved in the main biological processes such as cell cycle, DNA repair, cell signaling and communication, transcription, and cell metabolism. Embryos graded in different groups showed different transcriptomic profiles in the related cumulus cells. Therefore, it seems that embryo selection using the combination of cytokinetics and cumulus cells gene expression can improve the accuracy of the embryo selection and pregnancy rate in ART clinics.

Oocyte Morphometric Assessment and Gene Expression Profiling of Oocytes and Cumulus Cells as Biomarkers of Oocyte Competence in Sheep.

Simple SummaryIn vitro production (IVP) of embryos is an essential element of many reproductive biotechnologies. Although some remarkable achievements have been obtained for ovine IVP, the overall efficiency is still low. A limiting factor of the technique is the great variability of the oocyte quality used in IVP. The current selection criterion of oocytes is based on a morphological factor, which is insufficient to determine oocytes’ competence. Therefore, the identification of biomarkers for the selection of oocytes of high developmental competence is a major research goal. The purpose of this study was to evaluate the morphometric parameters of oocytes and the relative gene expression of oocytes and cumulus cells (CCs) as biomarkers of oocyte quality after individually culturing them in order to increase the final efficiency of the IVP technique in sheep. We observed that higher blastocyst rates were obtained from oocytes classified as intermediate and large according to total diameter, oocyte diameter, and zona pellucida thickness. Moreover, the expression of genes related to oocyte quality was higher in oocytes classified as large and in CCs from oocytes classified as large and thus able to reach the blastocyst stage. Oocyte morphometric assessments and gene expression in CCs may be used as biomarkers of oocyte quality.Oocyte quality is crucial for subsequent embryo development and so it is a major challenge in assisted reproductive technologies. The aim of the present work was to evaluate the morphometric parameters of oocytes (experiment 1) and the relative gene expression of oocytes and cumulus cells (CCs) (experiment 2) as biomarkers of oocyte quality after individually culturing them (one oocyte or embryo/drop). In experiment 1, individually matured oocytes were measured and classified into small, intermediate, and large oocytes after a cluster analysis, based on total diameter (with zona pellucida, ZP), oocyte diameter (without ZP), and ZP thickness. These oocytes were individually fertilized in vitro and cultured. The embryo development was evaluated up to the blastocyst stage. According to the total diameter, oocyte diameter, and ZP thickness, the blastocyst rate decreased in the small oocytes group (3.1 ± 3.1, 14.1 ± 9.4, and 26.7 ± 3.9, respectively) compared to the intermediate (29.4 ± 5.2, 30.5 ± 10.1, and 28.6 ± 9.6, respectively) and large oocytes groups (54.2 ± 13.5, 44.4 ± 3.9, and 67.6 ± 12.4, respectively). In addition, the probability of reaching the blastocyst stage was positively related to the total diameter (p < 0.001), oocyte diameter (p < 0.05), and ZP thickness (p < 0.001). Furthermore, the relative gene expression of BAX, BCL2, GDF9, and GJA1 was lower in oocytes classified as large. In experiment 2, the mRNA transcript relative abundance pattern of genes in CCs was evaluated according to oocyte total diameter and developmental stage reached. CCs from oocytes classified as large and oocytes capable of developing to the blastocyst stage had a lower relative expression of BAX, STAR, and PTGS2, while a higher expression of HAS2 and SDC2 transcript was observed for those oocytes. In conclusion, oocyte morphometric parameters and gene expression analysis in oocytes and CCs provide methods for the identification of the most competent oocytes for assisted reproductive technologies in sheep.

Cumulus cells of euploid versus whole chromosome 21 aneuploid embryos reveal differentially expressed genes

Research questionCan cumulus cells be used as a non-invasive target for the study of determinants of preimplantation embryo quality? DesignCumulus cells were collected from monosomy 21, trisomy 21 and euploid embryos and subjected to RNA sequencing analysis and real-time polymerase chain reaction assays. The differential gene expression was analysed for different comparisons. ResultsA total of 3122 genes in monosomy 21 cumulus cells and 19 genes in trisomy 21 cumulus cells were differentially expressed compared with euploid cumulus cells. Thirteen of these genes were differentially expressed in both monosomy and trisomy 21, compared with euploid, including disheveled segment polarity protein 2 (DVL2), cellular communication network factor 1 (CCN1/CYR61) and serum response factor (SRF), which have been previously implicated in embryo developmental competence. In addition, ingenuity pathway analysis revealed cell–cell contact function to be affected in both monosomy and trisomy 21 cumulus cells. ConclusionsThese findings support the use of cumulus cell gene expression analysis for the development of biomarkers evaluating oocyte quality for patients undergoing fertility preservation of oocytes.

Stratifying Cumulus Cell Samples Based on Molecular Profiling to Help Resolve Biomarker Discrepancies and to Predict Oocyte Developmental Competence.

To increase the efficiency of assisted reproductive techniques (ART), molecular studies have been performed to identify the best predictive biomarkers for selecting the most suitable germ cells for fertilization and the best embryo for intra-uterine transfer. However, across different studies, no universal markers have been found. In this study, we addressed this issue by generating gene expression and CpG methylation profiles of outer cumulus cells obtained during intra-cytoplasmic sperm injection (ICSI). We also studied the association of the generated genomic data with the clinical parameters (spindle presence, zona pellucida birefringence, pronuclear pattern, estrogen level, endometrium size and lead follicle size) and the pregnancy result. Our data highlighted the presence of several parameters that affect analysis, such as inter-individual differences, inter-treatment differences, and, above all, specific treatment protocol differences. When comparing the pregnancy outcome following the long protocol (GnRH agonist) of ovarian stimulation, we identified the single gene markers (NME6 and ASAP1, FDR < 5%) which were also correlated with endometrium size, upstream regulators (e.g., EIF2AK3, FSH, ATF4, MKNK1, and TP53) and several bio-functions related to cell death (apoptosis) and cellular growth and proliferation. In conclusion, our study highlighted the need to stratify samples that are very heterogeneous and to use pathway analysis as a more reliable and universal method for identifying markers that can predict oocyte development potential.

Expression of the apoptosis regulatory gene family in the long-term in vitro cultured human cumulus cells

Abstract Human cumulus cells (CCs) play a key role in the regulation of ovarian follicle maturation and oocyte fertilization. They influence the oocyte development by transferring the various molecules via the specific gap junction proteins, also known as the connexins, which provide a direct transmembrane connection between the oocyte and CCs. The human CCs were obtained in the patients diagnosed with infertility, who underwent the procedure of the controlled ovarian stimulation, and the following in vitro fertilization to elucidate the possible involvement of the CCs in the regulation of the fertilization and oocyte aging. Collected samples were long-term cultured and harvested after 7, 15, and 30 days of cultivation. Afterward, we assessed the relative expression of the following apoptosis regulatory genes - BAX, CASP9, and TP53 - using the RT-qPCR method. We noted a decrease in the expression of all above-mentioned genes in the samples harvested after 15 and 30 days, in reference to 7 days in vitro cultured CCs. In summary, our results provide precious insight into the dynamics of changes and confirm the continuous expression of the proapoptotic genes – BAX, CASP9, and TP53 in the long-term cultured CCs. Running title: Apoptotic gene expression in the human cumulus cells

Insufficient Cumulus Expansion and Poor Oocyte Retrieval in Endometriosis-Related Infertile Women.

Endometriosis (EMs) is a common cause for female infertility, leading to the need for in vitro fertilization (IVF). In clinics, we found the operative oocyte retrieval to be more or less difficult in women with EMs. We hypothesized that EMs may be involved in the insufficient cumulus expansion that partially explained the lower oocyte retrieval in EMs-related infertile women undergoing assisted reproductive technology (ART). To explore whether the insufficient cumulus expansion exists in EMs-related infertile women and whether there is a possible relationship between the insufficient cumulus expansion and the clinical phenomenon of difficulty in oocyte retrieval. Those infertile women undergoing IVF recorded in our database between January 2013 and October 2017 were included. The expression levels of cumulus expansion-related genes (HAS2/PTGS2/PTX3/TNFAIP6) in the cumulus cells (CCs) from 19 infertile women with EMs and 24 controls were analyzed by real-time PCR. After that, 635 women with EMs-associated infertility (the EMs group) and 4634 women with male factor-associated infertility (the control group) were included in the retrospective analysis. The clinical outcomes were compared between the two groups. The relative mRNA levels of cumulus expansion-related genes were significantly decreased in the CCs from those infertile women with EMs when compared to the control group (all p < 0.05), especially the expression of PTGS2. The mean oocyte retrieval rates (proportion of obtained oocytes in punctured follicles) were (76.33 ± 2.58)% and (71.80 ± 0.58)% (p < 0.01). The mean numbers of flushing times per follicle were 1.11 ± 0.65 and 3.86 ± 1.53 (p < 0.001). The lower expression of cumulus expansion-related genes in CCs suggests the insufficient cumulus expansion in EMs-related infertile women, which partially explains a possible mechanism related to poor oocyte retrieval.

Clinical and Molecular Effects of GnRH Agonist and Antagonist on The Cumulus Cells in The In Vitro Fertilization Cycle.

Background:Gonadotropin-releasing hormone (GnRH) analogues have been extensively utilized in the ovarian stimulation cycle for suppression of endogenous rapid enhancement of luteinizing hormone (LH surge). Exclusive properties and functional mechanisms of GnRH analogues in in vitro fertilization (IVF) cycles are clearly described. This study was performed to evaluate clinical and molecular impacts of the GnRH agonist and antagonist protocols in IVF cycles. For This purpose, gene expression of cumulus cells (CCs) as well as clinical and embryological parameters were evaluated and compared between two groups (GnRH agonistand antagonist) during the IVF cycle. Materials and Methods:Twenty-one infertile individuals were enrolled in this study. Subjects were selected from two groups of GnRH agonist(n=10) treated patients and GnRH antagonist (n=11) treated individuals. The defined clinical embryological parameters were compared between the two groups. Expression of BAX, BCL-2, SURVIVIN, ALCAM, and VCAN genes were assessed in the CCs of the participants using the real-time polymerase chain reaction (PCR) technique.Results:The mean number of cumulus oocyte complex (COC), percentage of metaphase II (MII) oocytes, grade A embryo and clinical parameters did not show noticeable differences between the two groups. BAX gene expression in the CCs of the group treated with GnRH agonist was remarkably higher than those received GnRH antagonist treatment (P<0.001). The mRNA expression of BCL-2 and ALCM genes were considerably greater in the CCs of patients who underwent antagonist protocol in comparison to the group that received agonist protocol (P<0.001).Conclusion:Despite no considerable difference in the oocyte quality, embryo development, and clinical outcomes between the group treated with GnRH agonist and the one treated with antagonist protocol, the GnRH antagonist protocol was slightly more favorable. However, further clinical studies using molecular assessments are required to elucidate this controversial subject.

Molecular and cellular effects of insulin-like growth factor-1 and LongR3-IGF-1 on in vitro maturation of bovine oocytes: comparative study

Addition effects of insulin-like growth factor-1 (IGF-1) and its synthetic analogue insulin-like growth factor-1 recombinant-3 (LongR3-IGF-1) after in vitro maturation (IVM) of cattle cumulus-oocyte complexes (COCs) were compared and evaluated on meiotic progression, apoptosis and profile genes of oocyte competence (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGBFP4 and IGFBP5), and their respective cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). The 739 COCs (n = 10 pools) of bovine ovaries were collected, selected and matured with IGF-1 (100 ng/mL), LongR3-IGF-1 (100 ng/mL), and in two control groups with 0.1% polyvinyl alcohol (PVA) or 10% fetal bovine serum (FBS), for 22–24 h. The statistical analysis was performed by a linear mixed effects model, ANOVA and Tukey tests. There was no statistical difference between experimental groups taken into account the meiotic progression and apoptosis (P > 0.05). Nevertheless, there were statistical differences (P ≤ 0.05) among FBS, IGF-1 and LongR3-IGF-1 groups for IGFBP4 gene expression, and among PVA, IGF-1 and LongR3-IGF-1 for COX2 gene expression in cumulus cells. Moreover, statistical difference was found for BCL2 gene expression between IGF-1, FBS and PVA groups and for IGFBP4 gene expression between LongR3-IGF-1, PVA and FBS in oocytes. There was no statistical difference between experimental groups for other genes evaluated. These results showed a good performance of IVM of bovine oocytes in the presence of LongR3-IGF-1 and the possibility of replacement of IGF-1 and FBS.

OOCYTE SECRETED FACTORS REGULATE FSHR AND AMH mRNA LEVELS IN CUMULUS CELLS

Considering that (1) FSH activity decreases cumulus-oocyte communication via retraction of transzonal processes (TZP), (2) AMH inhibits FSH signalling by decreasing FSHR expression and adenylyl cyclase activation, and (3) the oocyte is an active player in the cumulus-oocyte complex (COC) regulating cumulus cell metabolism and gene expression via oocyte-secreted factors (OSF), we tested the hypothesis that the oocyte decreases mRNA abundance of FSHR while increasing that of AMH in cumulus cells through OSF, as part of an effort to preserve its communication with cumulus cells thus safeguarding its developmental competence. The relative mRNA abundance of FSHR and AMH in cumulus cell was compared among 3 treatment-groups: (1) Intact COC subjected to in vitro maturation (IVM); (2) oocytectomized COC (OOX) subjected to IVM and (3) OOX subjected to IVM with the addition of denuded oocytes (OOX+DO; 1 DO/μL). Four culture replicates including all three treatment-groups were performed. COC were aspirated from 3-8mm follicles of bovine ovaries. Oocytectomy was achieved by aspiration of the ooplasm with a micromanipulator while preserving the structure of the COC. COC and OOX underwent IVM in pools of 20 for 22 hours. IVM was performed in 100 μL of TCM199 with Earl’s salts supplemented with 1μg/mL FSH, 0.4% BSA, 22 μg/mL sodium pyruvate, 75 μg/mL of amikacin at 38,5°C and 5.5% CO2 in humid atmosphere. Abundance of mRNA encoding FSHR and AMH in cumulus cells was assessed by real-time RT-PCR using Power SybrGreen (LifeTech®) and RPL15 (ribosomal protein 15) as the reference gene. Effects of treatments were tested by ANOVA and groups were compared with the Tukey test. Statistically significant differences were those with p<0.05, while p values >0.05 and <0.1 were considered as tendency of difference. Oocyctectomy decreased AMH mRNA levels (p=0.005) while tended to increase FSHR mRNA abundance (p=0.077) in cumulus cells. The addition of denuded oocytes (DO) reversed the effects of oocytectomy on both genes. Relative AMH mRNA levels (AMH/RPL15 mRNA abundance, mean ± EPM) were 0.875 ± 0.1, 0.3675 ± 0.02 and 0.7825 ± 0.1 for COC, OOX and OOX+DO, respectively. For FSHR, the respective values (FSHR/RPL15 mRNA abundance) were 1.495 ± 0.54, 2.575 ± 0.52 and 0.875 ± 0.29. Oocyte secreted factors seem to reduce FSHR expression while enhancing AMH expression in cumulus cells. We speculate that this is part of an effort of the oocyte to restrain FSH activity in the COC in order to preserve its communication with cumulus cells thus favouring developmental competence.

Decreased gremlin 1 expression in women with body mass index ≥35 kg/m2 is mediated by interleukin 10 and interleukin 1β in the follicular fluid

To determine how body mass index (BMI) affects the follicular fluid cytokine milieu and investigate how this inflammatory environment impacts cumulus signaling. Experimental study. Tertiary hospital based research laboratory. Women with normal (18.5 to 24.9 kg/m2) and obese (35 to 42 kg/m2) BMI undergoing controlled ovarian stimulation for intracytoplasmic sperm injection (ICSI). Cumulus cell treatment with obese follicular fluid, interleukin (IL) 10, and IL-1β. Follicular fluid cytokine concentrations between normal and obese women were compared using multiplex bead assay. Differential cumulus cell gene expression of GREM1, HAS2, PTGS2, and VCAN were measured using quantitative reverse polymerase chain reaction (RT-qPCR) while protein levels were determined by flow cytometry and confocal microscopy. Compared to women with normal BMI, women with BMI ≥35 kg/m2 undergoing ICSI had higher follicular concentrations of IL-10 (9.46 pg/mL [0.59-19.16] vs 53.39 pg/mL [14.97-236.37], p=0.004) and IL-1β (1.92 pg/mL [1.92-5.18] vs 5.18 pg/mL [1.92-16.33], p=0.017), as well as decreased relative cumulus cell expression of GREM1 (1.01 [0.66-1.40] vs 0.51 [0.38-0.74], p=0.03), a surrogate marker of positive ICSI outcomes. Furthermore, elevated IL-10 and IL-1β appear to be responsible for decreasing GREM1 expression in women with BMI ≥35 kg/m2. Our findings suggest that follicular inflammation associated with obesity impacts cumulus cell signaling. At a molecular level, derangements to the immune system resulting in decreased GREM1 expression may be a partial explanation for the suboptimal ICSI outcomes observed with obesity.

Morphokinetic analysis of cleavage stage embryos and assessment of specific gene expression in cumulus cells independently predict human embryo development to expanded blastocyst: a preliminary study.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

Cumulus cell gene expression in luteal-phase-derived oocytes after double stimulation in one menstrual cycle

Research questionDoes double stimulation (DuoStim) affect cumulus cell gene expression in luteal-phase-derived oocytes? DesignThis prospective observational study included 39 patients with reduced ovarian reserve. Fifteen patients (group 1) underwent IVF with a gonadotrophin-releasing hormone antagonist in the follicular phase and 24 patients (group 2) underwent DuoStim. A total of 149 cumulus cell samples were divided into two groups according to the phase of the cycle: group 1 included 55 follicular-phase-derived oocytes and group 2 included 94 luteal-phase-derived oocytes. The expression levels of the following genes were assessed using quantitative polymerase chain reaction: HAS2, VCAN, ALCAM, PTGS2, GREM1, ITPKA, TRPM7, SDC4, CALM2, SPSB2, TP53I3, PGR and PFKP. ResultsThe expression of 10 out of 13 genes in cumulus cells was similar between DuoStim luteal-phase-derived oocytes and follicular-phase-derived oocytes. A significant increase in the mRNA levels of VCAN (15.542 ± 6.8 versus 20.353 ± 10.58; P = 0.001), SDC4 (1.016 ± 0.65 versus 1.318 ± 0.97; P = 0.013), and TP53I3 (0.185 ± 0.09 versus 0.270 ± 0.11; P = 1.19E-05) was observed in group 2. The number of oocytes collected (5.57 ± 2.3 versus 5.7 ± 2.7; P > 0.05) and the number of blastocysts were comparable between the groups (2.1 ± 2.1 versus 2.7 ± 2.2; P > 0.05). ConclusionsThe DuoStim approach leads to changes in the follicular environment. It affects the expression levels of VCAN, SDC4, and TP53I3 in the cumulus cells of luteal-phase-derived oocytes. These results, however, did not correlate with oocyte maturation, embryo quality and pregnancy rate.