Preovulatory follicular fluid secretome added to in vitro maturation medium influences the metabolism of equine cumulus-oocyte complexes.

BACKGROUND: In vitro embryo production is a highly demanded reproductive technology in horses, which requires the recovery (in vivo or post-mortem) and in vitro maturation (IVM) of oocytes. Oocytes subjected to IVM exhibit poor developmental competence compared to their in vivo counterparts, being this related to a suboptimal composition of commercial maturation media. The objective of this work was to study the effect of different concentrations of secretome obtained from equine preovulatory follicular fluid (FF) on cumulus-oocyte complexes (COCs) during IVM. COCs retrieved in vivo by ovum pick up (OPU) or post-mortem from a slaughterhouse (SLA) were subjected to IVM in the presence or absence of secretome (Control: 0 µg/ml, S20: 20 µg/ml or S40: 40 µg/ml). After IVM, the metabolome of the medium used for oocyte maturation prior (Pre-IVM) and after IVM (Post-IVM), COCs mRNA expression, and oocyte meiotic competence were analysed. RESULTS: IVM leads to lactic acid production and an acetic acid consumption in COCs obtained from OPU and SLA. However, glucose consumption after IVM was higher in COCs from OPU when S40 was added (Control Pre-IVM vs. S40 Post-IVM: 117.24 ± 7.72 vs. 82.69 ± 4.24; Mean µM ± SEM; p < 0.05), while this was not observed in COCs from SLA. Likewise, secretome enhanced uptake of threonine (Control Pre-IVM vs. S20 Post-IVM vs. S40 Post-IVM: 4.93 ± 0.33 vs. 3.04 ± 0.25 vs. 2.84 ± 0.27; Mean µM ± SEM; p < 0.05) in COCs recovered by OPU. Regarding the relative mRNA expression of candidate genes related to metabolism, Lactate dehydrogenase A (LDHA) expression was significantly downregulated when secretome was added during IVM at 20-40 µg/ml in OPU-derived COCs (Control vs. S20 vs. S40: 1.77 ± 0.14 vs. 1 ± 0.25 vs. 1.23 ± 0.14; fold change ± SEM; p < 0.05), but not in SLA COCs. CONCLUSIONS: The addition of secretome during in vitro maturation (IVM) affects the gene expression of LDHA, glucose metabolism, and amino acid turnover in equine cumulus-oocyte complexes (COCs), with diverging outcomes observed between COCs retrieved using ovum pick up (OPU) and slaughterhouse-derived COCs (SLA).

Reduced anti-Müllerian hormone action in cumulus-oocyte complexes is beneficial for oocyte maturation without affecting oocyte competence.

Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory stage, AMH production tends to be limited to cumulus cells. Therefore, the present study investigated the role of cumulus cell-derived AMH in supporting maturation and competence of the enclosed oocyte. Cumulus-oocyte complexes (COCs) were isolated from antral follicles of rhesus macaque ovaries for in vitro maturation with or without AMH depletion. Oocyte meiotic status and embryo cleavage after in vitro fertilization were assessed. In vitro maturation with AMH depletion was also performed using COCs from antral follicles of human ovarian tissue. Oocyte maturation and morphology were evaluated. The direct AMH action on mural granulosa cells of the preovulatory follicle was further assessed using human granulosa cells cultured with or without AMH supplementation. More macaque COCs produced metaphase II oocytes with AMH depletion than those of the control culture. However, preimplantation embryonic development after in vitro fertilization was comparable between oocytes derived from COCs cultured with AMH depletion and controls. Oocytes resumed meiosis in human COCs cultured with AMH depletion and exhibited a typical spindle structure. The confluency and cell number decreased in granulosa cells cultured with AMH supplementation relative to the control culture. AMH treatment did not induce cell death in cultured human granulosa cells. Data suggest that reduced AMH action in COCs could be beneficial for oocyte maturation. Cumulus cell-derived AMH is not essential for supporting oocyte competence or mural granulosa cell viability.

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.

Single-cell RNA-seq reveals the effects of the FecB mutation on the transcriptome profile in ovine cumulus cells.

Genetic variations in the ovine ovulation rate, which are associated with the FecB mutation, provide useful models by which to explore the mechanisms regulating the development of mammalian antral follicles. In order to study the effects of the FecB mutation on cumulus cell differentiation, preovulatory follicles were aspirated and cumulus cells were isolated from three FecB genotypes (homozygous, heterozygous and wild type) of Small Tail Han (STH) sheep superstimulated with FSH. Transcriptome information from tens of thousands of cumulus cells was determined with the 10 × Genomics single-cell RNA-seq technology. Under the superovulation treatment, the observed number of preovulatory follicles in the ovaries of FecB carriers was still significantly higher than that in the wild-type (P < 0.05). The expression patterns of cumulus cells differed between FecB carriers and wild-type ewes. The screened cumulus cells could also be further divided into different cell clusters, and the differentiation states and fates of each group of cumulus cells also remained different, which supports the notion that heterogeneity in gene expression is prevalent in single cells. The oxidative phosphorylation pathway was significantly enriched in differentially expressed genes among the cell differentiation branch nodes of cumulus cells and among the differentially expressed genes of cumulus cells from the three genotypes. Combined with the important role of oxidative phosphorylation in the maturation of COCs, we suggest that the oxidative phosphorylation pathway of cumulus cells plays a crucial role in the differentiation process of cumulus cells and the mutation effect of the FecB gene.

Estrogen influences the transzonal projection assembly of cumulus-oocyte complexes through G protein-coupled estrogen receptor during goat follicle development.

Estrogen is an important hormone that plays a role in regulating follicle development and oocyte maturation. Transzonal projections (TZPs) act as communication bridges between follicle somatic cells and oocytes, and their dynamic changes are critical for oocyte development and maturation. However, the roles and mechanisms of estrogen in regulating TZPs during follicular development are not yet understood. We found that the proportion of oocytes spontaneously resuming meiosis increases as the follicle grows, which is accompanied by rising estrogen levels in follicles and decreasing TZPs in cumulus-oocyte complex. To further explore the effect of elevated estrogen levels on TZP assembly, additional estrogen was added to the culture system. The increased estrogen level significantly decreased the mRNA and protein expression levels of TZP assembly-related genes. Subsequent research revealed that TZP regulation by estrogen was mediated by the membrane receptor GPER and downstream ERK1/2 signaling pathway. In summary, our study suggests that estrogen may regulate goat oocyte meiosis arrest by decreasing TZP numbers via estrogen-mediated GPER activation during follicle development.

Metabolic imaging of human cumulus cells reveals associations with pregnancy and live birth.

STUDY QUESTION: Can fluorescence lifetime imaging microscopy (FLIM) detect associations between the metabolic state of cumulus cell (CC) samples and the clinical outcome of the corresponding embryos? SUMMARY ANSWER: FLIM can detect significant variations in the metabolism of CC associated with the corresponding embryos that resulted in a clinical pregnancy versus those that did not. WHAT IS KNOWN ALREADY: CC and oocyte metabolic cooperativity are known to be necessary for the acquisition of developmental competence. However, reliable CC biomarkers that reflect oocyte viability and embryo developmental competency have yet to be established. Quantitative measures of CC metabolism could be used to aid in the evaluation of oocyte and embryo quality in ART. STUDY DESIGN, SIZE, DURATION: A prospective observational study was carried out. In total, 223 patients undergoing IVF with either conventional insemination or ICSI at a tertiary care center from February 2018 to May 2020 were included, with no exclusion criteria applied. PARTICIPANTS/MATERIALS, SETTING, METHODS: This cohort had a mean maternal age of 36.5 ± 4.4 years and an average oocyte yield of 16.9 (range 1-50). One to four CC clusters from each patient were collected after oocyte retrieval and vitrified. CC metabolic state was assessed using FLIM to measure the autofluorescence of the molecules NAD(P)H and FAD+, which are essential for multiple metabolic pathways. CC clusters were tracked with their corresponding oocytes and associated embryos. Patient age, Day 3 and Day 5/6 embryo morphological grades, and clinical outcomes of embryos with traceable fate were recorded. Nine FLIM quantitative parameters were obtained for each CC cluster. We investigated associations between the FLIM parameters and patient maternal age, embryo morphological rank, ploidy, and clinical outcome, where false discovery rate P-values of <0.05 were considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 851 CC clusters from 851 cumulus-oocyte complexes from 223 patients were collected. Of these CC clusters, 623 were imaged using FLIM. None of the measured CC FLIM parameters were correlated with Day 3 morphological rank or ploidy of the corresponding embryos, but FAD+ FLIM parameters were significantly associated with morphological rank of blastocysts. There were significant differences for FAD+ FLIM parameters (FAD+ fraction engaged and short lifetime) from CC clusters linked with embryos resulting in a clinical pregnancy compared with those that did not, as well as for CC clusters associated with embryos that resulted in a live birth compared those that did not. LIMITATIONS, REASONS FOR CAUTION: Our data are based on a relatively low number of traceable embryos from an older patient population. Additionally, we only assessed CCs from 1 to 4 oocytes from each patient. Future work in a younger patient population with a larger number of traceable embryos, as well as measuring the metabolic state of CCs from all oocytes from each patient, would provide a better understanding of the potential utility of this technology for oocyte/embryo selection. WIDER IMPLICATIONS OF THE FINDINGS: Metabolic imaging via FLIM is able to detect CC metabolic associations with maternal age and detects variations in the metabolism of CCs associated with oocytes leading to embryos that result in a clinical pregnancy and a live birth versus those that do not. Our findings suggest that FLIM of CCs may be used as a new approach to aid in the assessment of oocyte and embryo developmental competence in clinical ART. STUDY FUNDING/COMPETING INTEREST(S): National Institutes of Health grant NIH R01HD092550-03 (to C.R., and D.J.N.). Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. and C.R. are inventors on patent US20170039415A1. TRIAL REGISTRATION NUMBER: N/A.

Extracellular vesicles secreted by cumulus cells contain microRNAs that are potential regulatory factors of mouse oocyte developmental competence.

The role of cumulus cells (CCs) in the acquisition of oocyte developmental competence is not yet fully understood. In a previous study, we matured cumulus-denuded fully-grown mouse oocytes to metaphase II (MII) on a feeder layer of CCs (FL-CCs) isolated from developmentally competent (FL-SN-CCs) or incompetent (FL-NSN-CCs) SN (surrounded nucleolus) or NSN (not surrounding nucleolus) oocytes, respectively. We observed that oocytes cultured on the former could develop into blastocysts, while those matured on the latter arrested at the 2-cell stage. To investigate the CC factors contributing to oocyte developmental competence, here we focused on the CCs' release into the medium of extracellular vesicles (EVs) and on their miRNA content. We found that, during the 15-h transition to MII, both FL-SN-CCs and FL-NSN-CCs release EVs that can be detected, by confocal microscopy, inside the zona pellucida (ZP) or the ooplasm. The majority of EVs are <200 nm in size, which is compatible with their ability to cross the ZP. Next-generation sequencing of the miRNome of FL-SN-CC versus FL-NSN-CC EVs highlighted 74 differentially expressed miRNAs, with 43 up- and 31 down-regulated. Although most of these miRNAs do not have known roles in the ovary, in silico functional analysis showed that seven of these miRNAs regulate 71 target genes with specific roles in meiosis resumption (N = 24), follicle growth (N = 23), fertilization (N = 1), and the acquisition of oocyte developmental competence (N = 23). Overall, our results indicate CC EVs as emerging candidates of the CC-to-oocyte communication axis and uncover a group of miRNAs as potential regulatory factors.

Electro-Metabolic Coupling of Cumulus-Oocyte Complex.

Oocyte-cumulus cell interaction is essential for oocyte maturation and competence. The bidirectional crosstalk network mediated by gap junctions is fundamental for the metabolic cooperation between these cells. As cumulus cells exhibit a more glycolytic phenotype, they can provide metabolic substrates that the oocyte can use to produce ATP via oxidative phosphorylation. The impairment of mitochondrial activity plays a crucial role in ovarian aging and, thus, in fertility, determining the success or failure of assisted reproductive techniques. This review aims to deepen the knowledge about the electro-metabolic coupling of the cumulus-oocyte complex and to hypothesize a putative role of potassium channel modulators in order to improve fertility, promote intracellular Ca2+ influx, and increase the mitochondrial biogenesis and resulting ATP levels in cumulus cells.

Physiological high temperatures alter the amino acid metabolism of bovine early antral follicles.

Heat stress reduces the developmental competence of bovine oocytes during the growth phase; however, the detailed mechanisms remain unclear. Amino acids play various critical roles in follicular development, including protein synthesis and as energy sources. We performed in vitro growth (IVG) culture of oocyte-cumulus-granulosa complexes (OCGCs) to assess the amino acid metabolism of small follicles at high temperatures. We isolated OCGCs from early antral follicles (0.5-1.0 mm) and subjected them to IVG culture for 12 days. OCGCs in the heat shock group were cultured under a temperature cycle of (38.5°C: 5 h, 39.5°C: 5 h, 40.5°C: 5 h, and 39.5°C: 9 h) to reproduce the body temperature of lactating cows under a hot environment. OCGCs in the control group were cultured at a constant temperature of 38.5°C for 24 h. Of the surviving OCGCs, those showing similar morphology and size between the groups were selected for amino acid analysis. We analyzed the free amino acids and their metabolites in the culture medium and calculated the depletion or appearance of molecular species. The depletion of three essential amino acids (isoleucine, leucine, and valine), two non-essential amino acids (aspartic acid and glycine), and ornithine was higher in the heat shock group (P < 0.05). Alanine depletion was lower in the heat shock group (P < 0.05). We concluded that heat exposure alters the amino acid metabolism of OCGCs isolated from early antral follicles, which might be involved with the diminished developmental potential of oocytes during summer.

Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Biochemical profiling of the follicular environment to predict oocyte competence in cattle.

To identify markers of oocyte competence, we compared the biochemical characteristics of fluid and cells from follicles containing oocytes with different capacities to form an embryo. Follicles (5-6 mm) were dissected, and follicular fluid (FF), granulosa cells (GC), cumulus cells (CC) from immature and mature cumulus-oocyte-complexes (COC) were individually collected. The oocytes were matured, fertilized, and cultured individually until day 8 (D8) of development. On D8, the samples were grouped according to embryo production into those that gave rise to blastocysts (EMB) and those that did not reach the blastocyst stage (NEMB). In CCs from immature and mature COCs and GCs, expression of CASP3, SERPINE2, VCAN, LUM, FSHR, EGFR, PGR, and GHR genes was quantified. Cell-free DNA (cfDNA), progesterone, and estradiol concentrations in the FF were determined. Data were analyzed by Mann-Whitney U test (GraphPad Prism 9). GHR was highly expressed in immature CCs from the EMB group, whereas CASP3 was highly expressed in mature CCs from the NEMB group (P<0.05). During maturation, the expression of CASP3 and GHR genes increased only in the NEMB group. ART2 cfDNA was highly detected in FF of the NEMB compared to the EMB group. Progesterone concentration was similar between the groups, whereas estradiol concentration was higher (P<0.05) in the EMB than in the NEMB group. It was concluded that a higher level of GHR transcripts in immature CCs, lower CASP3 expression in CCs from matured COCs, lower levels of ART2, and higher estradiol concentrations in FF may indicate oocytes with greater potential for development.

Characterization of metabolic patterns in porcine cumulus cells during meiotic maturation.

Metabolic coupling between oocytes and the surrounding somatic cells allows for normal two-way communication, and their interactions is necessary for generating developmentally competent eggs. However, the metabolic framework that support oocyte maturation in surrounding cumulus cells is still lacking. Herin, we established a temporal metabolome profile of porcine cumulus cells at three key stages during oocyte maturation, illustrating the picture of global metabolic network in cumulus cells. Importantly, we discovered the novel metabolic signature in cumulus cells during meiotic maturation, in specific, significant consumption of fatty acids, elevated activity of hexosamine biosynthetic pathway (HBP), and enhanced polyamine biosynthesis. Meanwhile, we observed the different utilization of tryptophan, active biosynthesis of progesterone, and progressive decrease in purine and pyrimidine metabolism as the oocytes progress through meiosis. Collectively, our metabolomic data serves an entree to elaborate on the dynamic changes in these metabolic pathways, which not only reveals the metabolic networks controlling oocyte development, but also lays a foundation for the discovery of biomarkers in the improvement in porcine oocyte culture system.

Mitochondrial Dysfunction in Advanced Maternal Aged Cumulus Cells: A Possible Link to ATP Synthase Impairment?

Age-related changes in the mitochondrial status of human cumulus cells (hCCs) impact oocyte quality; however, the relationship between hCC mitochondrial (dys)function and reproductive aging remains poorly understood. This study aimed to establish the interplay between hCC mitochondrial dysfunction and women's reproductive potential. In this investigation, 266 women were enrolled and categorized into two groups based on their age: a young group (<35 years old) and an advanced maternal age (AMA) group (≥35 years old). Comprehensive analysis of reproductive outcomes was conducted in our population. Various mitochondrial-related parameters were analyzed across distinct subsets. Specifically, mitochondrial membrane potential (∆Ψm) and mitochondrial mass were examined in 53 samples, mtDNA content in 25 samples, protein levels in 23 samples, bioenergetic profiles using an XF24 Extracellular Flux Analyzer in 6 samples, and levels of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in 39 and 43 samples, respectively. In our study, the reproductive potential of AMA women sharply decreased, as expected. Additionally, an impairment in the mitochondrial function of hCCs in older women was observed; however, no differences were found in terms of mitochondrial content. Regarding oxidative phosphorylation, metabolic profiling of hCCs from AMA women indicated a decrease in respiratory capacity, which was correlated with an age-dependent decrease in the ATP synthase (ATP5A1) protein level. However, intracellular ROS and ATP levels did not differ between groups. In conclusion, our study indicates that age-related dysfunction in hCCs is associated with impaired mitochondrial function, and, although further studies are required, ATP synthase could be relevant in this impairment.

Unravelling the role of HAS2, GREM1, and PTGS2 gene expression in cumulus cells: implications for human oocyte development competency - a systematic review and integrated bioinformatic analysis.

The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities - DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.

The possible regulatory role of miR-514 and miR-642b in cumulus cells on the oocyte maturation in patients with polycystic ovary syndrome.

Polycystic ovary syndrome is a common endocrine disorder in reproductive-age women. Accordingly, abnormal microenvironment may negatively influence oocyte developmental competence as a result of the altered expression profile of cumulus cells (CCs), mainly the key players of oocyte maturation, such as epidermal growth factor receptor (EGFR) and prostaglandin E receptor-2 (PTGER2). This study aimed to examine the expression levels of miR-514, miR-642b, and their candidate target genes (EGFR and PTGER2, respectively) in CCs of immature and mature oocytes in patients with PCOS. A total of 40 oocytes at germinal vesicle (GV) and 40 oocytes at metaphase II (MII) stages were retrieved from 30 PCOS women. Quantitative real-time PCR was performed to analyze the expression level of miR-514, miR-642b, EGFR, and PTGER2 in cumulus cells (CCS) of each oocyte. The expression level of miRNAs and their candidate target genes were compared between CCs of GV and MII oocytes. Our study suggests an inverse relationship exists between the expression levels of miR-514 and EGFR, and miR-642b and PTGER2. Furthermore, we observed that CCs of GV oocytes had higher levels of EGFR and PTGER2 mRNA and lower levels of miR-514 and miR-642b expression compared to those of MII oocytes. The present study demonstrated that miR-514 and miR-642b can regulate oocyte development by targeting EGFR and PTGER2, respectively. Therefore, examination of these miRNAs in CCs could be promising parameters to predict oocyte competence in PCOS patients.

Retinoic Acid Action in Cumulus Cells: Implications for Oocyte Development and In Vitro Fertilization.

In the field of human in vitro fertilization (IVF), selecting the best oocyte for freezing or embryo for transfer remains an important focus of clinical practice. Although several techniques are and have been used for this goal, results have generally not been favorable and/or are invasive such that damage to some embryos occurs, resulting in a reduced number of healthy births. Therefore, the search continues for non-invasive oocyte and embryo quality markers that signal the development of high-quality embryos. Multiple studies indicate the important positive effects of retinoic acid (RA) on oocyte maturation and function. We previously showed that a high follicular fluid (FF) RA concentration at the time of oocyte retrieval in IVF protocols was associated with oocytes, giving rise to the highest quality embryos, and that cumulus granulosa cells (CGCs) are the primary source of follicle RA synthesis. Data also demonstrated that connexin-43 (Cx43), the main connexin that forms gap junctions in CGCs, is regulated by RA and that RA induces a rapid increase in gap junction communication. Here, we hypothesize that CGC RA plays a causal role in oocyte competency through its action on Cx43 and, as such, may serve as a biomarker of oocyte competence. Multiple studies have demonstrated the requirement for Cx43 in CGCs for the normal progression of folliculogenesis, and that the increased expression of this connexin is linked to the improved developmental competence of the oocyte. The data have shown that RA can up-regulate gap junction intercellular communication (GJIC) in the cumulus-oocyte complex via a non-genomic mechanism that results in the dephosphorylation of Cx43 and enhanced GJIC. Recognizing the positive role played by gap junctions in CGCs in oocyte development and the regulation of Cx43 by RA, the findings have highlighted the possibility that CGC RA levels may serve as a non-invasive indicator for selecting high-quality oocytes for IVF procedures. In addition, the data suggest that the manipulation of Cx43 with retinoid compounds could provide new pharmacological approaches to improve IVF outcomes in cases of failed implantation, recurrent miscarriage, or in certain diseases that are characterized by reduced fecundity, such as endometriosis.

Transcriptomic responses of cumulus granulosa cells to SARS-CoV-2 infection during controlled ovarian stimulation.

Cumulus granulosa cells (CGCs) play a crucial role in follicular development, but so far, no research has explored the impact of SARS-CoV-2 infection on ovarian function from the perspective of CGCs. In the present study, we compared the cycle outcomes between infected and uninfected female patients undergoing controlled ovarian stimulation, performed bulk RNA-sequencing of collected CGCs, and used bioinformatic methods to explore transcriptomic changes. The results showed that women with SARS-CoV-2 infection during stimulation had significantly lower number of oocytes retrieved and follicle-oocyte index, while subsequent fertilization and embryo development were similar. CGCs were not directly infected by SARS-CoV-2, but exhibited dramatic differences in gene expression (156 up-regulated and 65 down-regulated). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses demonstrated a high enrichment in antiviral, immune and inflammatory responses with necroptosis. In addition, the pathways related to telomere organization and double strand break repair were significantly affected by infection in gene set enrichment analysis. Further weighted gene co-expression network analysis identified a key module associated with ovarian response traits, which was mainly enriched as a decrease of leukocyte chemotaxis and migration in CGCs. For the first time, our study describes how SARS-CoV-2 infection indirectly affects CGCs at the transcriptional level, which may impair oocyte-CGC crosstalk and consequently lead to poor ovarian response during fertility treatment.

Energy metabolism disorders during in vitro maturation of bovine cumulus-oocyte complexes interfere with blastocyst quality and metabolism.

Glucose and fatty acids (FA) metabolism disturbances during oocyte in vitro maturation (IVM) affect their metabolism and surrounding cumulus cells, but only inhibition of glucose metabolism decreases embryo culture efficiency. Therefore, the present experiment aimed to reveal if glucose or FA metabolism inhibition leads to the disruption of embryo developmental potential, and to characterize the metabolic landscape of embryos reaching the blastocyst stage. Inhibitors of glucose (IO + DHEA) or FA (ETOMOXIR) metabolism were applied during IVM, and the control group was matured under standard conditions. Blastocysts obtained from experimental and control groups were analyzed with regard to lipidome and metabolome (mass spectrometry), transcriptome (RNA-Seq) and fluorescence lipid droplets staining (BODIPY). We showed that inhibition of glucose and fatty acid metabolism leads to cellular stress response compromising the quality of preimplantation embryos. The inhibition of energy metabolism affects membrane fluidity as well as downregulates fatty acids biosynthesis and gene expression of trophectoderm cell line markers. Therefore, we conclude that oocyte maturation environment exerts a substantial effect on preimplantation development programming at cellular and molecular levels.

A Circular RNA Derived from the Pumilio 1 Gene Could Regulate PTEN in Human Cumulus Cells.

CircRNAs are a class of non-coding RNAs able to regulate gene expression at multiple levels. Their involvement in physiological processes, as well as their altered regulation in different human diseases, both tumoral and non-tumoral, is well documented. However, little is known about their involvement in female reproduction. This study aims to identify circRNAs potentially involved in reproductive women's health. Candidate circRNAs expressed in ovary and sponging miRNAs, already known to be expressed in the ovary, were selected by a computational approach. Using real time PCR, we verified their expression and identified circPUM1 as the most interesting candidate circRNA for further analyses. We assessed the expression of circPUM1 and its linear counterpart in all the follicle compartments and, using a computational and experimental approach, identified circPUM1 direct and indirect targets, miRNAs and mRNAs, respectively, in cumulus cells. We found that both circPUM1 and its mRNA host gene are co-expressed in all the follicle compartments and proposed circPUM1 as a potential regulator of PTEN, finding a strong positive correlation between circPUM1 and PTEN mRNA. These results suggest a possible regulation of PTEN by circPUM1 in cumulus cells and point out the important role of circRNA inside the pathways related to follicle growth and oocyte maturation.

Identification of Differentially Expressed mRNAs and miRNAs and Related Regulatory Networks in Cumulus Oophorus Complexes Associated with Fertilization.

Cumulus oophorus complexes (COCs) are the first extracellular barriers that sperm must pass through to fuse with oocytes, which have an important role in oocyte maturation and fertilization. However, little is known about the molecular mechanisms of COCs involved in fertilization. In this study, COCs were collected and then randomly divided into a test group that interacted with sperm and a control group that did not interact with sperm. Then, the total RNA was extracted; RNA transcriptome and small RNA libraries were prepared, sequenced, and analyzed. The results showed that 1283 differentially expressed genes (DEGs), including 560 upregulated and 723 downregulated genes. In addition, 57 differentially expressed miRNAs (DEMIs) with 35 upregulated and 22 downregulated were also detected. After the RNA-seq results were verified by RT-qPCR, 86 effective DEGs and 40 DEMIs were finally screened and a DEMI-DEG regulatory network was constructed. From this, the top ten hub target genes were HNF4A, SPN, WSCD1, TMEM239, SLC2A4, E2F2, SIAH3, ADORA3, PIK3R2, and GDNF, and they were all downregulated. The top ten hub DEMIs were miR-6876-5p, miR-877-3p, miR-6818-5p, miR-4690-3p, miR-6789-3p, miR-6837-5p, miR-6861-5p, miR-4421, miR-6501-5p, and miR-6875-3p, all of which were upregulated. The KEGG signaling pathway enrichment analysis showed that the effective DEGs were significantly enriched in the calcium, AMPK, and phospholipase D signaling pathways. Our study identified several DEGs and DEMIs and potential miRNA-mRNA regulatory pathways in COCs and these may contribute to fertilization. This study may provide novel insights into potential biomarkers for fertilization failure.