Does LH supplementation in poor responders affect granulosa cells apoptosis rate in ART? A prospective randomised controlled trial.

The aim was to compare granulosa cell's (GCs) apoptosis rate with (group A) or without (group B) luteinising hormone (LH) supplementation in poor ovarian responders (PORs) during controlled ovarian stimulation (COS). After oocyte retrieval, the follicular fluid was analysed by cytoflowmetry. Primary outcomes were GCs apoptosis rate in terms of viability, early apoptosis, late apoptosis and necrosis. Secondary outcome was clinical pregnancy rate. The viability was 96.7{IQR: 8} and 83.5{IQR: 20} for groups A and B, respectively (p < .001). Late apoptosis rates were significantly lower in group A (median 1.5, {IQR: 3.1}) than group B (median 9.5, {IQR: 20.6}) (p < .001). Median early apoptosis rates were 1.4 {IQR: 2.9} and 5.2 {IQR: 6.5} for group A and B respectively (p = .04). No significant difference was observed in the clinical pregnancy rate. Although LH seems necessary in PORs to decrease late granulosa apoptosis rates, this does not improve clinical pregnancy rates.IMPACT STATEMENTWhat is already known on this subject? LH supplementation during COS has long been an issue in PORs to overcome the rFSH responsiveness due to the LH polymorphism. LH receptors have also been on GCs and their expression increases in preovulatory follicles. GCs apoptosis rates may show the oocyte quality and reproductive potential of oocyte retrieved and the requirement for LH supplementation.What do the results of this study add? The present study shows that LH supplementation during COS for PORs promotes the GC viability and reduces early/late apoptosis rates. Similarly, the number of MII oocytes was significantly higher in the LH regimen group. However, there was no significant difference in terms of clinical pregnancy rates.What are the implications of these findings for clinical practice and/or further research? The oocyte quality parameters such as higher GC viability and lower GC early/late apoptosis rates verify the LH supplementation in PORs during COS. However, the limited size of this study requires further multi-centre research in a larger cohort of patients. Results obtained with a sensitive and validated method will help clinicians to make better decisions in patient care.

TMT-based proteomic and bioinformatic analyses of human granulosa cells from obese and normal-weight female subjects.

BACKGROUND: Increasing evidence supports a relationship between obesity and either infertility or subfertility in women. Most previous omics studies were focused on determining if the serum and follicular fluid expression profiles of subjects afflicted with both obesity-related infertility and polycystic ovary syndrome (PCOS) are different than those in normal healthy controls. As granulosa cells (GCs) are essential for oocyte development and fertility, we determined here if the protein expression profiles in the GCs from obese subjects are different than those in their normal-weight counterpart. METHODS: GC samples were collected from obese female subjects (n = 14) and normal-weight female subjects (n = 12) who were infertile and underwent in vitro fertilization (IVF) treatment due to tubal pathology. A quantitative approach including tandem mass tag labeling and liquid chromatography tandem mass spectrometry (TMT) was employed to identify differentially expressed proteins. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were then conducted to interrogate the functions and pathways of identified proteins. Clinical, hormonal, and biochemical parameters were also analyzed in both groups. RESULTS: A total of 228 differentially expressed proteins were noted, including 138 that were upregulated whereas 90 others were downregulated. Significant pathways and GO terms associated with protein expression changes were also identified, especially within the mitochondrial electron transport chain. The levels of free fatty acids in both the serum and follicular fluid of obese subjects were significantly higher than those in matched normal-weight subjects. CONCLUSIONS: In GCs obtained from obese subjects, their mitochondria were damaged and the endoplasmic reticulum stress response was accompanied by dysregulated hormonal synthesis whereas none of these changes occurred in normal-weight subjects. These alterations may be related to the high FFA and TG levels detected in human follicular fluid.

Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cells derived from human follicular fluid to oocyte-like cell.

Follicular fluid (FF) is essential for developing ovarian follicles. Besides the oocytes, FF has abundant undifferentiated somatic cells containing stem cell properties, which are discarded in daily medical procedures. Earlier studies have shown that FF cells could differentiate into primordial germ cells via forming embryoid bodies, which produced oocyte-like cells (OLC). This study aimed at isolating mesenchymal stem cells (MSC) from FF and evaluating the impacts of bone morphogenetic protein 15 (BMP15) on the differentiation of these cells into OLCs. Human FF-derived cells were collected from 78 women in the assisted fertilization program and cultured in human recombinant BMP15 medium for 21 days. Real-time polymerase chain reaction and immunocytochemistry staining characterized MSCs and OLCs. MSCs expressed germline stem cell (GSC) markers, such as OCT4 and Nanog. In the control group, after 15 days, OLCs were formed and expressed zona pellucida markers (ZP2 and ZP3), and reached 20-30 µm in diameter. Ten days after induction with BMP15, round cells developed, and the size of OLCs reached 115 µm. A decrease ranged from 0.04 to 4.5 in the expression of pluripotency and oocyte-specific markers observed in the cells cultured in a BMP15-supplemented medium. FF-derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in promoting the differentiation of these cells, which may give an in vitro model to examine germ cell development.

Compatibility of Distinct Label-Free Proteomic Workflows in Absolute Quantification of Proteins Linked to the Oocyte Quality in Human Follicular Fluid.

We present two separate label-free quantitative workflows based on different high-resolution mass spectrometers and LC setups, which are termed after the utilized instrument: Quad-Orbitrap (nano-LC) and Triple Quad-TOF (micro-LC) and their directed adaptation toward the analysis of human follicular fluid proteome. We identified about 1000 proteins in each distinct workflow using various sample preparation methods. With assistance of the Total Protein Approach, we were able to obtain absolute protein concentrations for each workflow. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 455 and 215 proteins were quantified by the Quad-Orbitrap and Triple Quad-TOF workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 20 and 22 proteins linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis without the need of extensive fractionation, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples after the initial effort of building a comprehensive spectral library.

Influence of Extracellular Vesicles of the Follicular Fluid on Morphofunctional Characteristics of Human Sperm.

We studied the effect of extracellular vesicles of the follicular fluid on morphofunctional characteristics of human spermatozoa using CASA (computer-assisted sperm analysis) analytical system. The vesicles were obtained by sequential centrifugation at different rotational speeds and frozen at -80°C in the Sydney IVF Gamete Buffer medium. The sperm fraction was isolated from the seminal fluid of 21 patients aged 27-36 years by differential centrifugation in a density gradient. The precipitate was suspended in Sydney IVF Gamete Buffer to a concentration of 106/ml and incubated with vesicles (1:2) at 37°C in a CO2 incubator for 30 min and 1 h. Sperm fraction incubated without vesicles served as the control. After incubation, some sperm samples were centrifuged at 700g for 5 min and fixed in 2.5% glutaraldehyde in 0.1 M buffer for transmission electron microscopy. After 30-min and 1-h incubation, the progressive and total sperm motility improved, the curvilinear and linear velocity of spermatozoa did not change significantly. Incubation with vesicles significantly changed the trajectory of sperm movement, which can attest to an increase in their hyperactivation and, probably, fertilizing capacity. Analysis of the effect of extracellular vesicles of follicular fluid on sperm motility will help to improve the effectiveness of assisted reproductive technology programs with male infertility factor by improving sperm characteristics in patients with asthenozoospermia and increasing the fertilizing ability of the sperm.

Extracellular vesicles as a potential diagnostic tool in assisted reproduction.

PURPOSE OF REVIEW: Extracellular vesicles have emerged as a promising field of research for their potential to serve as biomarkers. In the pathophysiology of reproduction, they have attracted significant attention because of their diverse roles in gametogenesis and embryo-endometrial cross-talk. Advances in extracellular vesicle translational potential are herein reviewed with a particular focus in oocyte competence, semen quality diagnostics, embryo selection and detection of endometrial receptivity. RECENT FINDINGS: Specific miRNAs present in follicular fluid-derived extracellular vesicles have been associated with follicle development and oocyte maturation. Some proteins known to regulate sperm function and capacitation such as glycodelin, and CRISP1 have been found as overrepresented in semen exosomes isolated from severe asthenozoospermic compared to normozoospermic men. In vitro developed human embryos can secrete extracellular vesicles whose propitiousness for preimplantation genetic testing is being increasingly investigated. Endometrial cell-derived extracellular vesicles recovered from uterine flushings might represent a reservoir of molecular markers potentially exploited for monitoring the endometrial status. SUMMARY: Accumulated knowledge on extracellular vesicles deriving from endometrium, follicular fluid, embryos or male reproductive system may be translated to clinical practice to inform diagnostics in assisted reproduction technology (ART). Validation studies and technology developments are required to implement the profiling of extracellular vesicles as diagnostic tests in ART.

NMR metabolic profiling of follicular fluid for investigating the different causes of female infertility: a pilot study.

INTRODUCTION: Several metabolomics studies have correlated follicular fluid (FF) metabolite composition with oocyte competence to fertilization, embryo development and pregnancy but there is a scarcity of research examining the metabolic effects of various gynaecological diseases. OBJECTIVES: In this study we aimed to analyze and correlate the metabolic profile of FF from women who were following in vitro fertilization (IVF) treatments with their different infertility pathologies. METHODS: We selected 53 women undergoing IVF who were affected by: tubal diseases, unexplained infertility, endometriosis, polycystic ovary syndrome (PCOS). FF of the study participants was collected at the time of oocytes retrieval. Metabolomic analysis of FF was performed by nuclear magnetic resonance (NMR) spectroscopy. RESULTS: FF presents some significant differences in various infertility pathologies. Although it was not possible to discriminate between FF of control participants and women with tubal diseases and unexplained infertility, comparison of FF metabolic profile from control women with patients with endometriosis and PCOS revealed significant differences in some metabolites that can be correlated to the causes of infertility. CONCLUSION: NMR-based metabolic profiling may be successfully applied to find diagnostic biomarkers for PCOS and endometriosis and it might be also used to predict oocyte developmental potential and subsequent outcome.

Mitochondrial cell-free DNA secreted from porcine granulosa cells.

Several studies have proposed that cell-free DNA (cfDNA) is a potential biomarker present in follicular fluid (FF) for oocyte quality. Recently we reported that mitochondria-derived cfDNA (mt-cfDNA) closely reflects the amount of cfDNA in FFs. The present study investigated the mechanism regulating mt-cfDNA secretion from porcine granulosa cells. Oocytes and cumulus cell complexes or granulosa cells (GCs) were cultured in maturation medium for 24 or 48 h respectively. Then, nuclear-derived cell-free DNA (n-cfDNA) or mt-cfDNA contents in the spent medium were examined using real-time polymerase chain reaction. When 10 µM of MG132, a proteasome inhibitor, was added to the culture medium, cellular viability of both COCs and GCs decreased and n-cfDNA significantly increased in the culture medium, whereas mt-cfDNA significantly decreased. Supplementation of the culture medium with GW4869, an inhibitor of intracellular vesicle formation, significantly decreased the mt-cfDNA, whereas no effect was observed on n-cfDNA in the medium of both COCs and GCs. Furthermore, the addition of bafilomycin, an inhibitor of autophagy to the culture medium significantly increased mt-cfDNA in the culture medium. After filtration (0.22 µm) and centrifugation (23,000 g), the mt-cfDNA content of the medium decreased significantly. In conclusion, the proteasomal mitochondrial quality control system is upstream of mt-cfDNA secretion and autophagy plays a role in cellular digestion of mitochondrial DNA in the cytoplasm. It is further suggested that dsDNA is enclosed in certain vesicles or associated with small molecules and secreted into the medium.

Fertility preservation in women with malignancies: the accuracy of antral follicle count collected randomly during the menstrual cycle in predicting the number of oocytes retrieved.

PURPOSE: To evaluate the capacity of random antral follicle count (AFC), i.e., AFC recorded at any time during the menstrual cycle, to predict the number of retrieved mature oocytes in women with malignancies undergoing random start ovarian hyperstimulation METHODS: A consecutive series of 72 women with malignancies who underwent ovarian hyperstimulation aimed at egg freezing between July 2014 and December 2016 was retrospectively reviewed. A standardized random start protocol was used for all women. AFC and serum AMH were systematically assessed prior to initiating ovarian hyperstimulation. The main outcome was the retrieval of ≥ 10 mature oocytes. The accuracy of random AFC was tested with the c-statistics (area under the ROC curve). RESULTS: For the whole cohort, the c-statistics for the prediction of ≥ 10 mature oocytes using AFC and serum AMH were similar. Specifically, the areas under the curve were 0.76 (95%CI 0.66-0.87) and 0.82 (95%CI 0.72-0.92), respectively (p = ns). Moreover, when considering the subgroup of women recruited after day 5 of the cycle (proper random start, n = 52), the areas under the curve did not also differ. Specifically, they resulted in 0.77 (95%CI 0.64-0.89) and 0.83 (95%CI 0.72-0.95), respectively (p = ns). CONCLUSIONS: AFC collected at any time during the menstrual cycle can provide valuable information for the counseling of women with malignancies scheduled for oocyte cryopreservation. Its reliability appears to be non-inferior to that of serum AMH.

Advanced glycation end products alter steroidogenic gene expression by granulosa cells: an effect partially reversible by vitamin D.

STUDY QUESTION: Does vitamin D attenuate the adverse effects of advanced glycation end products (AGEs) on steroidogenesis by human granulosa cells (GCs)? SUMMARY ANSWER: AGEs alter the expression of genes important in steroidogenesis while 1,25-dihydroxyvitamin D3 (vit D3) in vitro attenuates some of the actions of AGEs on steroidogenic gene expression, possibly by downregulating the expression of the pro-inflammatory cell membrane receptor for AGEs (RAGE). WHAT IS KNOWN ALREADY: Vitamin D attenuates the pro-inflammatory effects of AGEs in non-ovarian tissues. STUDY DESIGN, SIZE, DURATION: Women who were undergoing IVF were enrolled. Follicular fluid samples (n = 71) were collected and cumulus GCs (n = 12) were treated in culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicular fluid levels of the anti-inflammatory soluble RAGE (sRAGE), AGEs and 25-hydroxyvitamin D (25-OHD) were quantified for possible correlations. GCs of each participant were split equally and treated with either media alone (control) or with human glycated albumin (HGA as a precursor for AGEs) with or without vit D3 after which RT-PCR and immunofluorescence were performed and cell culture media estradiol (E2) levels were compared. MAIN RESULTS AND THE ROLE OF CHANCE: In follicular fluid, sRAGE levels were positively correlated with 25-OHD levels. HGA treatment (i) increased CYP11A1 (by 48%), 3ß-HSD (by 38%), StAR (by 42%), CYP17A1 (by 30%) and LHR (by 37%) mRNA expression levels (P < 0.05 for all) but did not alter CYP19A1 or FSHR mRNA expression levels; and (ii) increased E2 release in cell culture media (P = 0.02). Vit D3 treatment (i) downregulated RAGE mRNA expression by 33% and RAGE protein levels by 44% (P < 0.05); (ii) inhibited the HGA-induced increase in CYP11A1, StAR, CYP17A1 and LHR mRNA levels, but not the increase in 3ß-HSD mRNA levels; and (iii) did not inhibit the HGA-induced E2 release in cell culture media. LIMITATIONS REASONS FOR CAUTION: This study used luteinized GCs that were collected from women who received gonadotropins thus the results obtained may not fully extrapolate to non-luteinized GCs in vivo. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that there is a relationship between AGEs and their receptors (RAGE and sRAGE) with vitamin D. Understanding the interaction between AGEs and vitamin D in ovarian physiology could lead to a more targeted therapy for the treatment of ovarian dysfunction. STUDY FUNDING/COMPETING INTEREST(S): Funding was received from NIH (R01 NS045940), American Society for Reproductive Medicine, Ferring Pharmaceuticals Inc., and University of Vermont College of Medicine Bridge Funds. All authors have nothing to disclose.

Influence of follicular fluid and cumulus cells on oocyte quality: clinical implications.

An equilibrium needs to be established by the cellular and acellular components of the ovarian follicle if developmental competence is to be acquired by the oocyte. Both cumulus cells (CCs) and follicular fluid (FF) are critical determinants for oocyte quality. Understanding how CCs and FF influence oocyte quality in the presence of deleterious systemic or pelvic conditions may impact clinical decisions in the course of managing infertility. Given that the functional integrities of FF and CCs are susceptible to concurrent pathological conditions, it is important to understand how pathophysiological factors influence natural fertility and the outcomes of pregnancy arising from the use of assisted reproduction technologies (ARTs). Accordingly, this review discusses the roles of CCs and FF in ensuring oocyte competence and present new insights on pathological conditions that may interfere with oocyte quality by altering the intrafollicular environment.

Beta endorphin in serum and follicular fluid of PCOS- and non-PCOS women.

PURPOSE: To compare the concentrations of beta endorphin in serum and follicular fluid (FF) of PCOS- and non-PCOS women. Secondarily, to investigate associations between beta endorphin and other parameters. METHODS: Fifty-nine women undergoing in vitro fertilization (IVF) were included in the study. Sixteen were stratified to the PCOS group using the Rotterdam criteria. The remaining 43 women served as controls. Follicular fluid was collected during oocyte retrieval and peripheral blood sampling was performed on the same day. Beta endorphin concentrations in serum and follicular fluid, serum levels of insulin, glucose, LH, estradiol and progesterone were measured. Additionally, testosterone was measured before starting the stimulation protocol. RESULTS: There was no difference in beta endorphin levels between PCOS- and non-PCOS women. The concentration of the peptide was higher in serum than in FF, likely due to collection of FF after ovulation induction and corresponding to the early luteal phase. We found a significant correlation between the number of mature Metaphase II (MII) oocytes retrieved and beta endorphin concentration in FF. In women with biochemical hyperandrogenemia, beta endorphin levels in FF correlated with testosterone levels. CONCLUSION: Beta Endorphin concentrations in serum and FF do not differ between PCOS- and non PCOS-women undergoing IVF. However, together with sex hormones, beta endorphin might play a key role in oocyte maturation.

Expression of mRNA and protein of IL-18 and its receptor in human follicular granulosa cells.

PURPOSE: There is no information available about the IL-18 receptor in ovarian follicles, so the present study attempts to demonstrate the expression of IL-18 and its receptor in human granulosa cells (GCs). METHODS: To evaluate the concentration of IL-18 in serum and follicular fluid (FF), we collected serum and FF from 102 women undergoing oocyte retrieval. Also, to detect expression of IL-18 and its receptor by luteinized GCs, these cells were pooled six times from a total of twenty individual patients with 5-16 follicles each. The IL-18 concentration was determined by ELISA and the expression of IL-18 and its receptor by immunocytochemistry and reverse transcription polymerase chain reaction. RESULTS: Our results showed that the median IL-18 concentration in serum, 159.27 pg/ml (IQR 121.41-210.1), was significantly higher than in FF, 142.1 pg/ml (IQR 95.7-176.5), p < 0.001. Moreover, we found that IL-18 and its receptor are expressed by GCs. CONCLUSION: The presence of IL-18 in FF and the expression of IL-18 and its receptor by GCs suggest an important role for this cytokine in ovarian function.

Biobanking of different body fluids within the frame of IVF-a standard operating procedure to improve reproductive biology research.

PURPOSE: The aim of the present study was to develop a standard operating procedure (SOP) for the collection, transport, and storage of human cumulus cells, follicular fluid, blood serum, seminal plasma, embryo culture supernatant, and embryo culture supernatant control obtained within the IVF process under approved protocols and written informed consent from participating patients. The SOP was developed at the Kinderwunsch Institut Schenk, Dobl, Austria, together with Biobank Graz of the Medical University of Graz, Austria. METHODS: The SOP provides comprehensive details of laboratory procedures and sampling of the different fluids within the IVF process. Furthermore, information on sample coding, references of involved laboratory techniques (e.g., oocyte retrieval with a Steiner-TAN needle), ethical approvals, and biobanking procedures are presented. RESULTS: The result of the present study is a standard operating procedure. CONCLUSIONS: The SOP ensures a professional way for collection and scientific use of IVF samples by the Kinderwunsch Institut Schenk, Dobl, Austria, and Biobank Graz of the Medical University of Graz, Austria. It can be used as a template for other institutions to unify specimen collection procedures in the field of reproductive health research.

Cellular and extracellular vesicular origins of miRNAs within the bovine ovarian follicle.

The ovarian follicle components must provide an ideal environment to ensure the success of reproductive processes, and communication between follicular cells is crucial to support proper oocyte growth. Recently, it has been demonstrated that the presence of extracellular vesicles (EVs) carrying microRNAs (miRNAs) in follicular fluid represents an important autocrine and paracrine communication mechanism inside the ovarian follicle. In this study, we tested the hypothesis that the miRNA content of EVs isolated from ovarian follicular (granulosa and cumulus-oocyte complexes) cell-conditioned culture media is dependent upon cell type. We initially screened bovine granulosa cells (GCs) and cumulus-oocyte complexes (COCs), as well as their derived EVs for 348 miRNAs using real-time PCR, and detected 326 miRNAs in GCs and COCs cells and 62 miRNAs in GCs and COCs EVs. A bioinformatics analysis of the identified cell-specific and differentially expressed miRNAs predicted that they likely modulate important cellular processes, including signalling pathways such as the PI3K-Akt, MAPK and Wnt pathways. By investigating the origins of miRNAs within the follicular fluid, the results of this study provide novel insights into follicular miRNA content and intercellular communication that may be of invaluable use in the context of reproductive technologies, diagnostic of ovarian-related diseases and/or the identification of biomarkers for oocyte and embryo quality.

Protein pathways working in human follicular fluid: the future for tailored IVF?

The human follicular fluid (HFF) contains molecules and proteins that may affect follicle growth, oocyte maturation and competence acquiring. Despite the numerous studies, an integrated broad overview on biomolecular and patho/physiological processes that are proved or supposed to take place in HFF during folliculogenesis and oocyte development is still missing. In this review we report, for the first time, all the proteins unambiguously detected in HFF and, applying DAVID (Database for Annotation, Visualization and Integrated Discovery) and MetaCore bioinformatic resources, we shed new lights on their functional correlation, delineating protein patterns and pathways with reasonable potentialities for oocyte quality estimation in in vitro fertilisation (IVF) programs. Performing a rigorous PubMed search, we redacted a list of 617 unique proteins unambiguously-annotated as HFF components. Their functional processing suggested the occurrence in HFF of a tight and highly dynamic functional-network, which is balanced by specific effectors, primarily involved in extracellular matrix degradation and remodelling, inflammation and coagulation. Metalloproteinases, thrombin and vitamin-D-receptor/retinoid-X-receptor-alpha resulted as the main key factors in the nets and their differential activity may be indicative of ovarian health and oocyte quality. Despite future accurate clinical investigations are absolutely needed, the present analysis may provide a starting point for more accurate oocyte quality estimation and for defining personalised therapies in reproductive medicine.

Transcriptional characteristics of different sized follicles in relation to embryo transferability: potential role of hepatocyte growth factor signalling.

STUDY HYPOTHESIS: We hypothesized that a better discrimination between follicles containing oocytes with high developmental competence and those containing oocytes with low competence, based on a combination of a follicle's size and transcriptomic signature, will provide a reliable method to predict embryonic outcome of IVF. STUDY FINDING: This study provides new insights on the impact of follicular size on oocyte quality as measured by embryonic development and demonstrates that medium follicles yield a better percentage of transferable embryos. WHAT IS KNOWN ALREADY: Although it is generally accepted that large ovarian follicles contain better eggs, other studies report that a better follicular size subdivision and a better characterization are needed. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Individual follicles (n = 136), from a total of 33 women undergoing IVF, were aspirated and categorized on the basis of their follicular liquid volume (small, medium or large) and the embryonic outcome of the enclosed oocyte: poor or good development. Comprehensive gene expression analysis between cells from the different sized follicles was performed using microarrays and quantitative RT-PCR to find molecular markers associated with follicular maturity and oocyte developmental competence. MAIN RESULTS AND THE ROLE OF CHANCE: The analysis of embryonic outcome in relation to follicular size indicates that the medium-sized follicles category yielded more transferable embryos (35%) compared with the largest follicles (30%) (NS). Gene expression analysis revealed expression markers with significant (P < 0.05) discrimination between the poor development groups for all three follicle sizes, and good development medium-size follicles, including up-regulation of thrombomodulin, transforming growth factor, beta receptor II and chondrolecti, and those associated with hyaluronan synthesis, coagulation and hepatocyte growth factor signalling. LIMITATIONS, REASONS FOR CAUTION: These analyses were performed in a single cohort of patients coming from a single clinic and the biomarkers generated will require validation in different geographical and biological contexts to ensure their global applicability. WIDER IMPLICATIONS OF THE FINDINGS: Medium-size follicles seem to be the optimal size for a positive embryonic outcome and are associated with competence markers that may help in understanding the ideal differentiation status during late folliculogenesis. LARGE SCALE DATA: The data discussed in this publication have been deposited in The National Center for Biotechnology Information Gene Expression Omnibus database and are accessible through GEO Series accession number GSE52851. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada (NSERC) to M.A.S. There are no competing interests to declare.

Extracellular vesicles in human follicular fluid do not promote coagulation.

Body fluids contain extracellular vesicles expressing tissue factor on their surface and serve as an additional trigger for coagulation. During the menstrual cycle ovarian tissue restoration is mandatory and it is unknown whether follicular fluid might provide procoagulant substances. Within an observational study, follicular fluid from women undergoing IVF/intracytoplasmic sperm injection (ICSI) was analysed by fluorescence-activated cell sorting (FACS), electron microscopy, resistive pulse sensing (RPS), nanoparticle-tracking analysis (NTA) and fibrin generation tests (FGT). The presence of extracellular vesicles, especially CD9-positive extracellular vesicles in follicular fluid, was proven. However, clotting tests revealed no procoagulant properties of the detected extracellular vesicles.

Inhibition of angiopoietin-1 (ANGPT1) affects vascular integrity in ovarian hyperstimulation syndrome (OHSS).

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotrophins following human chorionic gonadotrophin (hCG) administration. The relationship between hCG and OHSS is partly mediated via the production of angiogenic factors, such as vascular endothelial growth factor A (VEGFA) and angiopoietins (ANGPTs). Here, we investigated the effect of ANGPT1 inhibition on ovarian angiogenesis in follicular fluid (FF) from women at risk of OHSS, using the chorioallantoic membrane (CAM) of quail embryos as an experimental model. We also analysed cytoskeletal changes and endothelial junction protein expression induced by this FF in the presence or absence of an ANGPT1-neutralising antibody in endothelial cell cultures. The presence of this antibody restored the number of vascular branch points and integrin αvß3 levels in the CAMs to control values. ANGPT1 inhibition in FF from OHSS patients also restored the levels of claudin-5, vascular endothelial cadherin and phosphorylated ß-catenin and partially reversed actin redistribution in endothelial cells. Our findings suggest that ANGPT1 increases pathophysiological angiogenesis in patients at risk of OHSS by acting on tight and adherens junction proteins. Elucidating the mechanisms by which ANGPT1 regulates vascular development and cell-cell junctions in OHSS will contribute to identifying new therapeutic targets for the treatment of human diseases with aberrant vascular leakage.

Exosome-mediated communication in the ovarian follicle.

Cells are able to produce and release different types of vesicles, such as microvesicles and exosomes, in the extracellular microenvironment. According to the scientific community, both microvesicles and exosomes are able to take on and transfer different macromolecules from and to other cells, and in this way, they can influence the recipient cell function. Among the different macromolecule cargos, the most studied are microRNAs. MicroRNAs are a large family of non-coding RNAs involved in the regulation of gene expression. They control every cellular process and their altered regulation is involved in human diseases. Their presence in mammalian follicular fluid has been recently demonstrated, and here, they are enclosed within microvesicles and exosomes or they can also be associated to protein complexes. The presence of microvesicles and exosomes carrying microRNAs in follicular fluid could represent an alternative mechanism of autocrine and paracrine communication inside the ovarian follicle. The outcomes from these studies could be important in basic reproductive research but could also be useful for clinical application. In fact, the characterization of extracellular vesicles in follicular fluid could improve reproductive disease diagnosis and provide biomarkers of oocyte quality in ART (Assisted Reproductive Treatment).