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

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

New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

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

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

Safety of early cumulus cell removal combined with early rescue ICSI in the prevention of fertilization failure.

RESEARCH QUESTION: What are the clinical outcomes and safety implications of early cumulus cell removal after short-term insemination combined with early rescue intracytoplasmic sperm injection (ICSI) in preventing fertilization failure? DESIGN: In this retrospective study, a total of 14,360 cycles were divided into four groups based on insemination method and fertilization ability: conventional IVF group (n = 5519); early cumulus cell removal group (n = 4107); conventional ICSI group (n = 4215); and early rescue ICSI group (where failed or low fertilization was predicted, n = 519). Fertilization outcomes, pregnancy outcomes, neonatal outcomes and birth defects were analysed by comparing the early cumulus cell removal group with the conventional IVF group, and the early rescue ICSI group with the conventional ICSI group. RESULTS: There were no significant differences in the outcomes of fertilization, pregnancy, neonates or birth defects between the conventional IVF group and the early cumulus cell removal group (P > 0.05). When compared with the conventional ICSI group, the early rescue ICSI group had similar rates of two pronuclei (2PN) at fertilization, clinical pregnancy, miscarriage, ectopic pregnancy, live birth, sex, mean gestational age, very low birthweight, macrosomia and birth defects (P > 0.05) but a higher polyploidy rate, lower high-quality embryo rate (both P < 0.001), lower twin pregnancy rate (P < 0.01), lower rate of low birthweight, and a higher rate of normal birthweight (both P = 0.024). CONCLUSIONS: Early cumulus cell removal combined with early rescue ICSI led to good pregnancy and neonatal outcomes without an increase in birth defects. This approach could therefore be an effective and safe method for patients with fertilization failure in conventional IVF.

RFRP-3 Influences Apoptosis and Steroidogenesis of Yak Cumulus Cells and Compromises Oocyte Meiotic Maturation and Subsequent Developmental Competence.

RF amide-related peptide 3 (RFRP-3), a mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), is identified to be a novel inhibitory endogenous neurohormonal peptide that regulates mammalian reproduction by binding with specific G protein-coupled receptors (GPRs) in various species. Herein, our objectives were to explore the biological functions of exogenous RFRP-3 on the apoptosis and steroidogenesis of yak cumulus cells (CCs) and the developmental potential of yak oocytes. The spatiotemporal expression pattern and localization of GnIH/RFRP-3 and its receptor GPR147 were determined in follicles and CCs. The effects of RFRP-3 on the proliferation and apoptosis of yak CCs were initially estimated by EdU assay and TUNEL staining. We confirmed that high-dose (10-6 mol/L) RFRP-3 suppressed viability and increased the apoptotic rates, implying that RFRP-3 could repress proliferation and induce apoptosis. Subsequently, the concentrations of E2 and P4 were significantly lower with 10-6 mol/L RFRP-3 treatment than that of the control counterparts, which indicated that the steroidogenesis of CCs was impaired after RFRP-3 treatment. Compared with the control group, 10-6 mol/L RFRP-3 treatment decreased the maturation of yak oocytes efficiently and subsequent developmental potential. We sought to explore the potential mechanism of RFRP-3-induced apoptosis and steroidogenesis, so we observed the levels of apoptotic regulatory factors and hormone synthesis-related factors in yak CCs after RFRP-3 treatment. Our results indicated that RFRP-3 dose-dependently elevated the expression of apoptosis markers (Caspase and Bax), whereas the expression levels of steroidogenesis-related factors (LHR, StAR, 3ß-HSD) were downregulated in a dose-dependent manner. However, all these effects were moderated by cotreatment with inhibitory RF9 of GPR147. These results demonstrated that RFRP-3 adjusted the expression of apoptotic and steroidogenic regulatory factors to induce apoptosis of CCs, probably through binding with its receptor GPR147, as well as compromised oocyte maturation and developmental potential. This research revealed the expression profiles of GnIH/RFRP-3 and GPR147 in yak CCs and supported a conserved inhibitory action on oocyte developmental competence.

Transcriptome Signature of Immature and In Vitro-Matured Equine Cumulus-Oocytes Complex.

Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes. Thus, the aim of this study was to elucidate the transcriptome dynamics of oocyte maturation by comparing transcriptomic changes during in vitro maturation in both oocytes and their surrounding cumulus cells. Cumulus-oocyte complexes were obtained from antral follicles and divided into two groups: immature and in vitro-matured (MII). RNA was extracted separately from oocytes (OC) and cumulus cells (CC), followed by library preparation and RNA sequencing. A total of 13,918 gene transcripts were identified in OC, with 538 differentially expressed genes (DEG) between immature OC and in vitro-matured OC. In CC, 13,104 genes were expressed with 871 DEG. Gene ontology (GO) analysis showed an association between the DEGs and pathways relating to nuclear maturation in OC and GTPase activity, extracellular matrix organization, and collagen trimers in CC. Additionally, the follicle-stimulating hormone receptor gene (FSHR) and luteinizing hormone/choriogonadotropin receptor gene (LHCGR) showed differential expressions between CC-MII and immature CC samples. Overall, these results serve as a foundation to further investigate the biological pathways relevant to oocyte maturation in horses and pave the road to improve the IVP outcomes and the overall clinical management of equine assisted reproductive technologies (ART).

Peroxiredoxin 4, a new favorable regulator, can protect oocytes against oxidative stress damage during in vitro maturation.

BACKGROUND: Finding an effective regulator to avoid harmful effects caused by excessive reactive oxygen species (ROS) is a bottleneck during oocyte in vitro maturation (IVM). Previously, we found that peroxiredoxin 4 (Prdx4) expression is significantly higher in mature cumulus cell-oocyte complexes (COCs) than in immature COCs. Prdx4 belongs to the antioxidant enzyme family and can catalyze the reduction of H2O2. RESULTS: In this study, we established an oxidative stress model with mouse COCs cultured in vitro. Treatment with H2O2 decreased cumulus expansion indexes and oocyte maturation in a concentration-dependent manner, indicating follicular development dysplasia. Infection with a Prdx4-overexpressing adenovirus significantly attenuated H2O2-induced changes, exhibiting effects similar to those of the intracellular ROS scavenger tiron (the positive control). Furthermore, the results confirmed that the protective effect of Prdx4 on oocyte maturation may be due to reductions in ROS levels and apoptosis. However, when the gap junctions between cumulus cells (CCs) and oocytes were destroyed, Prdx4 overexpression did not exert antiapoptotic effects. The expression levels of the gap junction marker protein CX43 were significantly recovered in the Prdx4-overexpressing group. CONCLUSIONS: These results demonstrate that Prdx4 in CCs may be a new favorable regulator that improves in vitro-matured oocyte quality and enhances oocyte developmental competence by preventing CC apoptosis caused by oxidative stress through gap junctions. The findings expand the body of knowledge regarding follicle development, and the identification of Prdx4 as a new favorable regulator will aid in immature oocyte IVM.

MTOR-mediated interaction between the oocyte and granulosa cells regulates the development and function of both compartments in mice†.

Coordinated development of the germline and the somatic compartments within a follicle is an essential prerequisite for creating a functionally normal oocyte. Bi-directional communication between the oocyte and the granulosa cells enables the frequent interchange of metabolites and signals that support the development and functions of both compartments. Mechanistic target of rapamycin (MTOR), a conserved serine/threonine kinase and a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation, is emerging as a major player that regulates many facets of oocyte and follicle development. Here, we summarized our recent observations on the role of oocyte- and granulosa cell-expressed MTOR in the control of the oocyte's and granulosa cell's own development, as well as the development of one another, and provided new data that further strengthen the role of cumulus cell-expressed MTOR in synchronizing oocyte and follicle development. Inhibition of MTOR induced oocyte meiotic resumption in cultured large antral follicles, as well as cumulus expansion and the expression of cumulus expansion-related transcripts in cumulus-oocyte complexes in vitro. In vivo, the activity of MTOR in cumulus cells was diminished remarkably by 4 h after hCG administration. These results thus suggest that activation of MTOR in cumulus cells contributes to the maintenance of oocyte meiotic arrest before the LH surge. Based on the observations made by us here and previously, we propose that MTOR is an essential mediator of the bi-directional communication between the oocyte and granulosa cells that regulates the development and function of both compartments.

Excessive follicle-stimulating hormone during ovarian stimulation of cattle may induce premature luteinization of most ovulatory-size follicles†.

High follicle-stimulating hormone (FSH) doses during ovarian stimulation are detrimental to ovulatory follicle function and decrease live birth rate in cattle and women. However, the mechanism whereby excessive FSH causes ovarian dysfunction is unknown. This study tested the hypothesis that excessive FSH during ovarian stimulation induces premature luteinization of ovulatory-size follicles. Small ovarian reserve heifers were injected twice daily for 4 days with 70 IU (N = 7 heifers) or 210 IU (N = 6 heifers) Folltropin-V [commercial FSH-enriched preparation of porcine pituitary glands with minor (<1%) luteinizing hormone (LH) contamination, cpFSH]. Ovulatory-size (≥10 mm) follicles were excised from ovaries after the last cpFSH injection and hormone concentrations in follicular fluid (FF) were determined using ELISA. Luteinization was monitored by assessing cumulus cell-oocyte complex (COC) morphology and measuring concentrations of estradiol (E), progesterone (P), and oxytocin (O) in FF. COCs were classified as having compact (cCOC) or expanded (eCOC) cumulus cell layers, and as estrogen-active (E:P in FF ≥1), estrogen-inactive (EI, E:P in FF ≤1 > 0.1), or extreme-estrogen-inactive (EEI, E:P in FF ≤0.1). A high proportion (72%) of ovulatory-size follicles in 210 IU, but not 70 IU, dose heifers displayed eCOCs. The high doses also produced higher proportions of EI or EEI follicles which had lower E:P ratio and/or E but higher P and/or O concentrations compared with the 70 IU dose heifers. In conclusion, excessive cpFSH doses during ovarian stimulation may induce premature luteinization of most ovulatory-size follicles in heifers with small ovarian reserves.

Oocyte-derived growth factors promote development of antrum-like structures by porcine cumulus granulosa cells in vitro.

Oocytes communicate with the surrounding somatic cells during follicular development. We examined the effects of two oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the development of porcine oocyte-cumulus cell complexes (OCCs) in vitro. We collected OCCs from early antral follicles (1.2-1.5 mm) and prepared oocytectomized cumulus cell complexes (OXCs), which were then cultured in a growth medium supplemented with 0-100 ng/ml GDF9 and/or BMP15 for 7 days. In the medium without GDF9 or BMP15, OCCs developed during culture, and approximately 30% of them formed antrum-like structures. GDF9 promoted OCC development and structure formation in a dose-dependent manner. However, OXCs did not form antrum-like structures without growth factors. GDF9 promoted the development of OXCs, and 50 and 100 ng/ml GDF9 promoted the formation of the structures by 8% and 26%, respectively; however, BMP15 did not promote the formation of these structures. OXCs were then cultured with 100 ng/ml GDF9 and various concentrations of BMP15 to investigate their cooperative effects on the formation of antrum-like structures. BMP15 promoted the formation of antrum-like structures in a dose-dependent manner. In conclusion, GDF9 derived from oocytes is probably important for the formation of antrum-like structures in porcine OXCs, and BMP15 cooperates with GDF9 to form these structures.

High expression of CFTR in cumulus cells from mature oocytes is associated with high-quality of oocyte and subsequent embryonic development.

OBJECTIVE: The purpose of this study was to explore the association of expression of cystic fibrosis transmembrane conductance regulator (CFTR) in cumulus cells (CCs) from mature oocytes with oocyte quality and embryonic development. METHODS: A total of 338 infertile women who underwent ovarian stimulation cycle of oocyte retrieval in Zhejiang University School of Medicine were retrospectively enrolled in this study. The relative mRNA expression levels of CFTR, bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) in CCs were detected by qPCR technology. ROC curve was applied for the diagnosis of oocyte maturation. The serum levels of anti-Müllerian hormone (AMH), E2, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and androstenedione were measured. Oocyte maturation rate, fertilization rate, cleavage rate, high-quality embryo formation rate, and implantation rate after embryo transfer were also determined. RESULTS: The mRNA expression levels of CFTR in CCs were significantly increased in metaphase II (MII) oocytes compared to that in metaphase I (MI) or germinal vesicle (GV) oocytes. The ROC curve analysis illustrated that CFTR mRNA expression could efficiently discriminate MII oocytes from MI or GV oocytes (AUC = 0.954), and revealed that 0.695 RQU is the optimal cut-off value for diagnosis. So the cut-off value of 2-ΔΔCT = 0.70 was used to divide the patients into two groups: low- (n = 114) and high-CFTR group (n = 224). The mRNA expression of CFTR in CCs was positively correlated with the antral follicular count (AFC), number of oocytes retrieved, number of MII oocytes, serum E2 level on hCG day, and BMP15 and GDF9 expression in CCs. Under continuous stimulation with the same dose of recombinant follicle-stimulating hormone (rFSH), the number of follicles, average recovered oocytes, recovered oocytes, MII oocytes, as well as the oocyte recovery rate, fertilization rate, oocyte cleavage rate, high-quality embryo formation rate, and implantation rate were decreased in patients with lower CFTR. CONCLUSIONS: This study suggests that CFTR expression in CCs is associated with the developmental potential of human oocytes.

Knockdown of CLAUDIN-6 Inhibited Apoptosis and Induced Proliferation of Bovine Cumulus Cells.

This study aims to investigate the effects of CLAUDIN-6 (CLDN6) on cell apoptosis and proliferation of bovine cumulus cells (CCs). Immunofluorescence staining was used to localize CLDN6 protein in CCs. Three pairs of siRNA targeting CLDN6 and one pair of siRNA universal negative sequence as control were transfected into bovine CCs. Then, the effective siRNA was screened by real-time quantitative PCR (RT-qPCR) and Western blotting. The mRNA expression levels of apoptosis related genes (CASPASE-3, BAX and BCL-2) and proliferation related genes (PCNA, CDC42 and CCND2) were evaluated by RT-qPCR in CCs with CLDN6 knockdown. Cell proliferation, apoptosis and cell cycle were detected by flow cytometry with CCK-8 staining, Annexin V-FITC staining and propidium iodide staining, respectively. Results showed that the CLDN6 gene was expressed in bovine CCs and the protein was localized in cell membranes and cytoplasms. After CLDN6 was knocked down in CCs, the cell apoptosis rate significantly decreased and the pro-apoptotic genes BAX and CASPASE-3 were down-regulated significantly, whereas the anti-apoptotic gene BCL-2 was markedly up-regulated (p < 0.05). Additionally, CLDN6 knockdown significantly enhanced cell proliferation of CCs at 72 h after siRNA transfection. The mRNA levels of proliferation-related genes PCNA, CCND2 and CDC42 increased obviously in CCs with CLDN6 knockdown (p < 0.05). After CLDN6 was down-regulated, the percentage of CCs at S phase was significantly increased (p < 0.05). However, there was no remarkable difference in the percentages of cells at the G0/G1 phase and G2/M phase between CCs with or without CLDN6 knockdown (p > 0.05). Therefore, the expression of CLDN6 and its effects on cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. CLDN6 low expression inhibited cell apoptosis, induced cell proliferation and cell cycle arrest of bovine CCs.

The first report of clonidine in vivo/in vitro effects on infertile women with polycystic ovary syndrome (in vivo/in vitro study).

The sympathetic nervous system (SNS) is hyperactive in women with polycystic ovary syndrome (PCOS). This study was designed in two sections: in vivo/in vitro with clonidine as the alpha-2 adrenoceptor (ADR-α2) agonist for modulating this hyperactivity. Eighty women with PCO participated in this randomised clinical trial (in vivo). A clonidine (0.1 mg) tablet was given twice a day for two months. Polycystic ovary morphology (PCOM) and pregnancy rate were the main outcome measurements. In the candidates for in vitro fertilisation (IVF), clonidine was added to the culture medium during IVF for two study groups (PCO-clonidine/PCO-without) and two control groups (egg donors-clonidine/egg donors-without). Our results showed that the pregnancy rate significantly was higher in the study group (p = .002). The mRNA expression of ADR-α1 and ADR-ß2 in PCO was higher than control group (p value <.001). But ADR-α1 expression in PCO-clonidine group decreased (p value = .042), the same as ADR-α2 expression. The intensity of this effect showed a pattern for ADR-α1

Expression of ADR-α1, 2 and ADR-ß2 in cumulus cell culture of infertile women with polycystic ovary syndrome and poor responder who are a candidate for IVF: the novel strategic role of clonidine in this expression.

OBJECTIVE: Alpha and beta-adrenoceptors (ADR-α1, 2, and ß2) play a regulatory role in the folliculogenesis and steroidogenesis in the ovarian follicles. This study aimed to measure these adrenoceptors mRNA and its protein levels in cumulus cells (CCs) culture of poor ovarian reserve (POR) and polycystic ovarian syndrome (PCOS) infertile women (IVF candidate) and the effect of clonidine treatment at CCs culture. METHODS: This case/control study was conducted in 2017 includes a control (donation oocytes) and two studies (PCO and POR) groups. The ovulation induction drugs were prescribed in all groups. After the oocyte puncture, the follicular fluid was collected and CCs were isolated were cultured. RNA was extracted and cDNA was synthesized and designed the primer for the ADR-α1, 2 and ADR-ß2 gene expression. The protein levels were investigated by Western Blot. RESULTS: The results showed a high level of three adrenergic expressions in PCO women compared to the control group (p-value <.001), which can be reduced by clonidine. POR group showed a significant decrease in the gene expression of ADR-α1 (p-value = .004) and ADR-α2 (p-value = .003) compared to the control group and clonidine treatment had no effect. CONCLUSION: The significant increase of three adrenoceptors gene expression and protein levels in CCs culture indicate to the hyperactivity of the ovarian sympathetic nervous system at the receptor levels in women with PCOS, and clonidine confirmed it by reducing this expression. In POR women, the reduction of ADR-α1, 2 expressions maybe lead to the aging process in the ovary.

Mitochondrial DNA copy number of cumulus cells is not linked to embryo implantation in good prognosis IVF patients.

RESEARCH QUESTION: Could the mitochondrial DNA (mtDNA) copy number of cumulus cells be used as a biomarker of the potential of embryo implantation in good prognosis IVF patients? DESIGN: A prospective cohort study on good prognosis IVF patients from a large reproductive medicine centre. A total of 392 embryos from 61 cycles (including 31 implanted and 30 non-implanted cycles) were enrolled in the study. The corresponding cumulus cell mtDNA copy number of embryos was tested by real-time quantitative polymerase chain reaction. The corresponding cumulus cell mtDNA copy numbers were compared between implanted and non-implanted embryos and also compared between high quality and poor quality embryos. Then, a mitochondrial function assay including mitochondrial membrane potentials, concentration of reactive oxygen species (ROS) and ATP content of the corresponding cumulus cells were compared between high quality and poor quality embryos to verify the above experimental findings. RESULTS: For the same population, the mean cumulus cell mtDNA copy numbers for implanted and non-implanted embryos were 255.61 ± 81.02 and 254.50 ± 73.29 (P = 0.47), and those for high quality and poor quality embryos were 266.02 ± 98.56 and 295.71 ± 70.64 (P = 0.99), respectively. There was no significant difference in cumulus cell mtDNA copy number between implanted and non-implanted embryos or between high quality and poor quality embryos. The mitochondrial membrane potential, ROS levels and ATP content of the corresponding cumulus cells did not differ significantly between high quality and poor quality groups. CONCLUSIONS: Measurement of cumulus cell mtDNA copy number might not provide any advantage to embryo prioritization in good prognosis IVF patients. Any suggested link between cumulus cell mtDNA copy number and embryo implantation requires further validation.

Altered transcriptome in cumulus cells of infertile women with advanced endometriosis with and without endometrioma.

RESEARCH QUESTION: Is the transcriptome of cumulus cells of infertile women with advanced endometriosis (EIII/IV), with and without endometrioma, altered? DESIGN: In this prospective case-control study, next-generation RNA sequencing was used to compare the transcript profile of cumulus cells among infertile patients undergoing ovarian stimulation for intracytoplasmic sperm injection with EIII/IV, with (n = 9) and without endometrioma (n = 9), and controls (n = 9). An in-silico enrichment analysis was conducted to establish the possibly altered pathways in cumulus cells of patients with endometriosis. RESULTS: Most of the differentially expressed genes (DEG) were found when cumulus cells from women with EIII/IV with endometrioma were compared with controls (DEG, n = 461). In women with EIII/IV without endometrioma, only 66 DEG were verified compared with controls. The enrichment analysis showed that some DEG in cumulus cells of endometriosis are involved in important pathways for the oocyte competence acquisition, such as oxidative phosphorylation, metabolism, mitochondrial function, acetylation and steroid biosynthesis. No DEG were found when cumulus cells from women with EIII/IV with and without endometrioma were compared. CONCLUSION: RNA sequencing results suggest that cumulus cells of infertile women with EIII/IV have an altered transcriptome, regardless of endometrioma. The present findings offer a better understanding of the genes and molecular mechanisms that may be involved in endometriosis-related infertility, mostly in the oocyte competence acquisition process.

CFP1-dependent histone H3K4 trimethylation in murine oocytes facilitates ovarian follicle recruitment and ovulation in a cell-nonautonomous manner.

CxxC-finger protein 1 (CFP1)-mediated trimethylated histone H3 at lysine-4 (H3K4me3) during oocyte development enables the oocyte genome to establish the competence to generate a new organism. Nevertheless, it remains unclear to which extent this epigenetic modification forms an instructive component of ovarian follicle development. We investigated the ovarian functions using an oocyte-specific Cxxc1 knockout mouse model, in which the H3K4me3 accumulation is downregulated in oocytes of developing follicles. CFP1-dependent H3K4 trimethylation in oocytes was necessary to maintain the expression of key paracrine factors and to facilitate the communication between an oocyte and the surrounding granulosa cells. The distinct gene expression patterns in cumulus cells within preovulatory follicles were disrupted by the Cxxc1 deletion in oocytes. Both follicle growth and ovulation were compromised after CFP1 deletion, because Cxxc1 deletion in oocytes indirectly impaired essential signaling pathways in granulosa cells that mediate the functions of follicle-stimulating hormone and luteinizing hormone. Therefore, CFP1-regulated epigenetic modification of the oocyte genome influences the responses of ovarian follicles to gonadotropin in a cell-nonautonomous manner.

In vitro growth of bovine oocytes in oocyte-cumulus cell complexes and the effect of follicle stimulating hormone on the growth of oocytes.

Several successful in vitro culture experiments have used oocyte-cumulus cell-mural granulosa cell complexes (OCGCs) from early antral follicles (0.5-0.7 mm) for the growth of bovine oocytes. However, in studies related to in vitro oocyte maturation and in vitro embryo production, oocyte-cumulus cell complexes (OCCs) that have no mural granulosa cells have been widely used instead of OCGCs. The purpose of this study was to determine whether cumulus cells alone support oocyte growth. First, OCCs and OCGCs were cultured in vitro for 14 days to compare the integrity of the complexes as well as antrum formation. After 14 days, the diameter and meiotic competence of oocytes in OCCs and OCGCs were examined. Oocytes in OCCs grew fully and acquired meiotic competence similar to OCGCs, whereas antrum formation occurred later in OCCs as compared to OCGCs. Subsequently, the effects of follicle stimulating hormone (FSH) on in vitro growth of OCCs were examined for 14 days. When FSH was added to the culture medium, OCCs formed antrum-like structures one day earlier than those cultured without FSH. Oocytes cultured with 1 mIU/ml FSH grew fully and acquired meiotic competence. In contrast, when oocytes were cultured in media containing high concentrations of FSH, some of the OCCs collapsed and the number of degenerated oocytes increased. In conclusion, bovine oocytes in OCCs grow and acquire meiotic competence similar to OCGCs and, 1 mIU/ml FSH supports the development of OCCs and oocyte growth as observed in our culture system.

Ghrelin antagonist D-Lys3-GHRP-6 counteract ghrelin effects in bovine cumulus-oocytes complexes matured in vitro.

Ghrelin is a gut hormone related to energy balance and reproductive functions. The aim of this study was to evaluate the effect of ghrelin antagonist D-Lys3-GHRP-6 (GA) as a potential agent that prevents ghrelin effects during bovine oocyte maturation on progesterone production, cumulus cell (CC) viability, CC DNA damage and embryo development and hatching rates. Ghrelin's potential to induce oxidative stress in cumulus-oocyte complexes (COC) was also evaluated. COCs were cultured for 24 hr in medium without supplementation (C) or supplemented with 60 pM ghrelin (Ghrelin60), Ghrelin60 + 20 pM GA (GA20), Ghrelin60 + 60 pM GA (GA60) or Ghrelin60 + 100 pM GA (GA100) for experiment I. For experiment II, C and Ghrelin60 treatments were used. Differences between C and Ghrelin60 and the linear or quadratic association between GAs on Ghrelin60 were evaluated. Results demonstrated that Ghrelin60 increased progesterone concentration, reduced CC viability, induced CC DNA damage and decreased blastocyst and hatching rate compared with C (p < .05). GA20, GA60 and GA100 had a linear effect on CC genetic damage index (p ≤ .05) and a quadratic effect on CC viability (p < .01). GA20 counteracted the low hatching rate produced by Ghrelin60. However, GAs did not counteract progesterone concentration and blastocyst rate (p ≥ .21). GRH60 did not differ from C in the oxidative status (p ≥ .19). Our study highlights that GA could prevent the negative effects of ghrelin during bovine IVM.

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.