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Background: Polycystic ovary syndrome (PCOS) is a disease associated with inflammation and the follicular fluid of this patient contains proinflammatory cytokines. Abdominal obesity (AO) is also linked to increased levels of proinflammatory cytokines. This study investigated the induction of inflammation and decidualization of in vitro cultured endometrial stromal cells (ESCs) obtained from women with a normal uterus using the follicular fluid of PCOS and non-PCOS patients with or without abdominal obesity. Methods and results: Forty patients under 35 years old, referred to the Royan Institute, were divided into four groups: PCOS with AO, PCOS without AO, non-PCOS with AO, and non-PCOS without AO. Follicular fluid samples were added to the culture medium of ESCs for each group. The rate of decidualization was measured by examining decidual markers. The study also investigated morphological changes in uterine endometrial cells, cell migration rates, and gene expression across all groups. We found that the non-PCOS group without AO had the highest decidualization potential and the highest expression of decidualization markers (P ≤ 0.05). Groups with an inflammatory phenotype of PCOS or abdominal obesity showed the highest expression of decidual pathway markers. The expression levels of inflammatory and proliferative markers in the PCOS group with AO were significantly higher than in the other groups (P ≤ 0.05). Conclusions: The inflammatory profile present in the follicular fluid may trigger the decidualization process. Consequently, in the future, follicular fluid could be utilized as a natural supplement with human cells to promote decidualization and enhance endometrial receptivity in assisted reproductive technology. [ABSTRACT FROM AUTHOR]

This study explores the proteomic composition of follicular fluid (FF) from women undergoing oocyte retrieval for in vitro fertilisation (IVF), with a focus on the effects of polycystic ovary syndrome (PCOS). FF samples were collected from 74 patients, including 34 with PCOS and 40 oocyte donors. Proteomic profiling using machine learning identified significant differences in protein abundance between the PCOS and control groups. Of the 484 quantified proteins, 20 showed significantly altered levels in the PCOS group. Functional annotation and pathway enrichment analysis pointed to the involvement of protease inhibitors and immune-related proteins in the pathophysiology of PCOS, suggesting that inflammation and immune dysregulation may play a key role. Additionally, HDL assembly was identified as a significant pathway, with apolipoprotein-AI (APOA1) and alpha-2-macroglobulin (A2M) as the major proteins involved. Notably, myosin light polypeptide 6 was the most downregulated protein, showing the highest absolute fold change, and may serve as a novel independent biomarker for PCOS. [ABSTRACT FROM AUTHOR]

The development of the ovarian follicle requires the presence of several factors that come from the blood and follicular cells. Among these factors, extracellular vesicles (EVs) represent an original communication pathway inside the ovarian follicle. Recently, EVs have been shown to play potential roles in follicular development and reproduction‐related disorders, including the polycystic ovary syndrome (PCOS). The proteomic analysis of sEVs isolated from FF in comparison to sEVs purified from plasma has shown a specific pattern of proteins secreted by ovarian steroidogenic cells such as granulosa cells. Thus, a human granulosa cell line exposed to sEVs from FF of normal patients increased their progesterone, estradiol, and testosterone secretion. However, if the sEVs were derived from FF of PCOS patients, the activity of stimulating progesterone production was lost. Stimulation of steroidogenesis by sEVs was associated with an increase in the expression of the StAR gene. In addition, sEVs from FF increased cell proliferation and migration of granulosa cells, and this phenomenon was amplified if sEVs were derived from PCOS patients. Interestingly, STAT3 is a protein overexpressed in sEVs from PCOS patients interacting with most of the cluster of proteins involved in the phenotype observed (cell proliferation, migration, and steroid production) in granulosa cells. In conclusion, this study has demonstrated that sEVs derived from FF could regulate directly the granulosa cell activity. The protein content in sEVs from FF is different in the case of PCOS syndrome and could perturb the granulosa cell functions, including inflammation, steroidogenesis, and cytoskeleton architecture. [ABSTRACT FROM AUTHOR]

The increase in female reproductive disorders, such as polycystic ovary syndrome, endometriosis, and diminished ovarian reserve that lead to subfertility and infertility, has encouraged researchers to search and discover their underlying causes and risk factors. One of the crucial factors that may influence the increasing number of reproductive issues is environmental pollution, particularly exposure to man-made endocrine-disrupting chemicals (EDCs). EDCs can interfere with the ovarian microenvironment, impacting not only granulosa cell function but also other surrounding ovarian cells and follicular fluid (FF), which all play essential roles for oocyte development, maturation, and overall reproductive function. FF surrounds developing oocytes within an ovarian follicle and represents a dynamic milieu. EDCs are usually found in biological fluids, and FF is therefore of interest in this respect. This narrative review examines the current knowledge on specific classes of EDCs, including industrial chemicals, pesticides, and plasticizers, and their known effects on hormonal signaling pathways, gene expression, mitochondrial function, oxidative stress induction, and inflammation in FF. We describe the impact of EDCs on the development of reproductive disorders, oocyte quality, menstrual cycle regulation, and their effect on assisted reproductive technique outcomes. The potential transgenerational effects of EDCs on offspring through animal and first-human studies has been considered also. While significant progress has been made, the current understanding of EDCs' effects on ovarian function, particularly in humans, remains limited, underscoring the need for further research to clarify actions and effects of EDCs in the ovary. [ABSTRACT FROM AUTHOR]

Introduction: Bisphenol A (BPA) is a common contaminant widely used in many industrial sectors. Because of its wide use and dispersion, it can be accumulated in living human bodies through both oral assumption and nondietary routes. BPA exhibits hormone-like properties, falling under the class of endocrine disruptors; therefore, it can alter relevant physiological functions. In particular, in women, it can affect folliculogenesis and therefore reproduction, contributing not only to infertility, but also to endometriosis and premature puberty. Methods: We conducted a multicenter study on 91 women undergoing a first in vitro fertilization (IVF) treatment in the Campania region (Southern Italy). We investigated the presence and concentration of BPA in serum and follicular fluids to assess the effects of airborne BPA contamination. The analysis was conducted on 32 women living in a low environmental impact (LEI) area, from the Sele Valley River and Cilento region, and 59 women living in a high environmental impact (HEI) area, the so-called "Land of Fires", a highly contaminated territory widely exposed to illegal waste practices. Results: A higher average BPA content in both blood serum and follicular fluid was revealed in the HEI group when compared with the LEI group. In addition, we revealed higher average BPA content in blood serum than in folliclular fluid in the HEI area, with opposite average content in the two fluids in the LEI zone. In addition, our results also showed a lack of correlation between BPA content in follicular and serum fluids both in the overall population and in the HEI and LEI groups, with peculiar trends in different subsets of women. Conclusion: From our results, we revealed a heterogeneity in the distribution of BPA content between serum and follicular fluid. Further studies are needed to unravel the bioaccumulation mechanisms of BPA in highly polluted and nonpolluted areas. [ABSTRACT FROM AUTHOR]

Background: Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. Results: Ultrasonographic monitoring and follicular fluid aspiration of preovulatory follicles in horses during the anovulatory (spring: non-breeding) and ovulatory (spring, summer, and fall: breeding) seasons and subsequent EV isolation and miRNA profiling identified significant variation in EV-miRNA cargo content. We identified 97 miRNAs with differential expression among the groups and specific clusters of miRNAs involved in the spring transition (miR-149, -200b, -206, -221, -328, and -615) and peak breeding period (including miR-143, -192, -451, -302b, -100, and let-7c). Bioinformatic analyses showed enrichments in various biological functions, e.g., transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Cluster analyses revealed distinct sets of significantly up- and down-regulated miRNAs associated with spring anovulatory (Cluster 1) and summer ovulation–the peak breeding season (Clusters 4 and 6). Conclusions: The findings from the current study shed light on the dynamics of FF-EV-coupled miRNAs in relation to equine ovulatory and anovulatory seasons, and their roles in understanding the mechanisms involved in seasonal shifts and ovulation during the breeding season warrant further investigation. [ABSTRACT FROM AUTHOR]

Background and Aim: In females of various species and experimental animals, iron (Fe) status in follicular fluid (FF) is associated with local physiological reproductive events related to follicle development, steroidogenesis, and oocyte maturation. However, these mechanisms remain unknown. This study aimed to determine and compare the intrafollicular and plasma concentrations of Fe, ferritin (Ferr), and transferrin (TRF) in cycling mares. Materials and Methods: Sixty ovaries were collected during the breeding season from 30 clinically normal mares raised for slaughterhouse meat production. Blood samples were collected before slaughter. Follicles were classified into three categories according to size: Small (20-30 mm; n = 20), medium (=31-40 mm; n = 20), and large (=41 mm; n = 20). The FF samples, after collection, were immediately taken to the laboratory for processing and were centrifuged, and the Fe and Ferr concentrations in the supernatant and plasma were determined by spectrophotometry. Results: Although intrafollicular Fe and Ferr were similar to plasma, TRF was significantly higher in FF than in systemic circulation (p < 0.05). Follicular development does not modify the status of Fe in the mare. Conclusions: Based on this evidence, it is possible that the acquisition of this molecule possibly originated from a local de novo source, whereas their diffusion through ultrafiltration does not play a relevant role. These results provide new scientific insights into the status of follicle Fe, suggesting its involvement in normal ovarian functions in mares. [ABSTRACT FROM AUTHOR]

To locate and fertilize the egg, sperm probe the varying microenvironment prevailing at different stages during their journey across the female genital tract. To this end, they are equipped with a unique repertoire of mostly sperm-specific proteins. In particular, the flagellar Ca2+ channel CatSper has come into focus as a polymodal sensor used by human sperm to register ligands released into the female genital tract. Here, we provide the first comprehensive study on the pharmacology of the sperm-specific human Slo3 channel, shedding light on its modulation by reproductive fluids and their constituents. Weshow that seminal fluid and contained prostaglandins and Zn2+ do not affect the channel, whereas human Slo3 is inhibited in a non-genomic fashion by diverse steroids as well as by albumin, which are released into the oviduct along with the egg. This indicates that not only CatSper but also Slo3 harbours promiscuous ligandbinding sites that can accommodate structurally diverse molecules, suggesting that Slo3 is involved in chemosensory signalling in human sperm. [ABSTRACT FROM AUTHOR]

Objective: To assess the associations between phthalates (PAEs) exposure and the levels of inflammatory cytokines in the ovarian microenvironment. Methods: 64 females who were undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) from April to December in 2020 were included in this cross-sectional study. The levels of 10 kinds of phthalate metabolites (mPAEs) and 2 kinds factors in follicular fluid were measured. Multivariable linear regression model and further stratified analysis by age were conducted to analyze the associations between mPAEs and inflammatory factors. Results: There were significant positive associations between mono-butyl phthalate (MBP) and tumor necrosis factor-α (β=0.446, P=0.003) and interleukin-6 (β=0.425, P=0.003) in the follicular fluid. Similarly, there was a significant positive relationship between mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) and tumor necrosis factor-α（β=0.411, P=0.019). In the stratified analysis, MBP still showed significantly the positive association with tumor necrosis factor-α (β=0.667, P= 0.000) and interleukin-6 (β=0.407, P=0.028) in the group aged≥30 years. In contrast, there were no correlations between mPAEs and inflammatory factors within the group aged ＜30 years. Conclusions: Our results suggest that there is strongly association between PAEs exposure and the altered levels of inflammatory factor, and that PAEs exposure may bring changes of inflammatory burden in the ovarian microenvironment. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Are markers of epigenetic age acceleration in follicular fluid associated with outcomes of ovarian stimulation? SUMMARY ANSWER Increased epigenetic age acceleration of follicular fluid using the Horvath clock, but not other epigenetic clocks (GrimAge and Granulosa Cell), was associated with lower peak estradiol levels and decreased number of total and mature oocytes. WHAT IS KNOWN ALREADY In granulosa cells, there are inconsistent findings between epigenetic age acceleration and ovarian response outcomes. STUDY DESIGN, SIZE, DURATION Our study included 61 women undergoing IVF at an academic fertility clinic in the New England area who were part of the Environment and Reproductive Health Study (2006–2016). PARTICIPANTS/MATERIALS, SETTING, METHODS Participants provided a follicular fluid sample during oocyte retrieval. DNA methylation of follicular fluid was assessed using a genome-wide methylation screening tool. Three established epigenetic clocks (Horvath, GrimAge, and Granulosa Cell) were used to predict DNA-methylation-based epigenetic age. To calculate the age acceleration, we regressed epigenetic age on chronological age and extracted the residuals. The association between epigenetic age acceleration and ovarian response outcomes (peak estradiol levels, follicle stimulation hormone, number of total, and mature oocytes) was assessed using linear and Poisson regression adjusted for chronological age, three surrogate variables (to account for cellular heterogeneity), race, smoking status, initial infertility diagnosis, and stimulation protocol. MAIN RESULTS AND ROLE OF CHANCE Compared to the median chronological age of our participants (34 years), the Horvath clock predicted, on an average, a younger epigenetic age (median: 24.2 years) while the GrimAge (median: 38.6 years) and Granulosa Cell (median: 39.0 years) clocks predicted, on an average, an older epigenetic age. Age acceleration based on the Horvath clock was associated with lower peak estradiol levels (−819.4 unit decrease in peak estradiol levels per standard deviation increase; 95% CI: −1265.7, −373.1) and fewer total (% change in total oocytes retrieved per standard deviation increase: −21.8%; 95% CI: −37.1%, −2.8%) and mature oocytes retrieved (% change in mature oocytes retrieved per standard deviation increase: −23.8%; 95% CI: −39.9%, −3.4%). The age acceleration based on the two other epigenetic clocks was not associated with markers of ovarian response. LIMITATIONS, REASONS FOR CAUTION Our sample size was small and we did not specifically isolate granulosa cells from follicular fluid samples so our samples could have included mixed cell types. WIDER IMPLICATIONS OF THE FINDINGS Our results highlight that certain epigenetic clocks may be predictive of ovarian stimulation outcomes when applied to follicular fluid; however, the inconsistent findings for specific clocks across studies indicate a need for further research to better understand the clinical utility of epigenetic clocks to improve IVF treatment. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by grants ES009718, ES022955, ES000002, and ES026648 from the National Institute of Environmental Health Sciences (NIEHS) and a pilot grant from the NIEHS-funded HERCULES Center at Emory University (P30 ES019776). RBH was supported by the Emory University NIH Training Grant (T32-ES012870). TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Background and Aim: In females of various species and experimental animals, iron (Fe) status in follicular fluid (FF) is associated with local physiological reproductive events related to follicle development, steroidogenesis, and oocyte maturation. However, these mechanisms remain unknown. This study aimed to determine and compare the intrafollicular and plasma concentrations of Fe, ferritin (Ferr), and transferrin (TRF) in cycling mares. Materials and Methods: Sixty ovaries were collected during the breeding season from 30 clinically normal mares raised for slaughterhouse meat production. Blood samples were collected before slaughter. Follicles were classified into three categories according to size: Small (20–30 mm; n = 20), medium (≥31–40 mm; n = 20), and large (≥41 mm; n = 20). The FF samples, after collection, were immediately taken to the laboratory for processing and were centrifuged, and the Fe and Ferr concentrations in the supernatant and plasma were determined by spectrophotometry. Results: Although intrafollicular Fe and Ferr were similar to plasma, TRF was significantly higher in FF than in systemic circulation (p < 0.05). Follicular development does not modify the status of Fe in the mare. Conclusion: Based on this evidence, it is possible that the acquisition of this molecule possibly originated from a local de novo source, whereas their diffusion through ultrafiltration does not play a relevant role. These results provide new scientific insights into the status of follicle Fe, suggesting its involvement in normal ovarian functions in mares.

Abstract Background Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. Results Ultrasonographic monitoring and follicular fluid aspiration of preovulatory follicles in horses during the anovulatory (spring: non-breeding) and ovulatory (spring, summer, and fall: breeding) seasons and subsequent EV isolation and miRNA profiling identified significant variation in EV-miRNA cargo content. We identified 97 miRNAs with differential expression among the groups and specific clusters of miRNAs involved in the spring transition (miR-149, -200b, -206, -221, -328, and -615) and peak breeding period (including miR-143, -192, -451, -302b, -100, and let-7c). Bioinformatic analyses showed enrichments in various biological functions, e.g., transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Cluster analyses revealed distinct sets of significantly up- and down-regulated miRNAs associated with spring anovulatory (Cluster 1) and summer ovulation–the peak breeding season (Clusters 4 and 6). Conclusions The findings from the current study shed light on the dynamics of FF-EV-coupled miRNAs in relation to equine ovulatory and anovulatory seasons, and their roles in understanding the mechanisms involved in seasonal shifts and ovulation during the breeding season warrant further investigation.

The purpose of our study was to determine the influence of lead and cadmium in concentrations commonly found in the environment on the redox system of the follicular fluid (FF) and on the results of assisted reproduction. A prospective study of 113 patients with unexplained infertility who qualified for intracytoplasmic sperm injection (ICSI). Patients with moderate or severe endometriosis or poor ovarian reserve were excluded from the study. Biochemical analyses and heavy metal assays of follicular fluid and serum (blood) were followed by statistical analyses of dependencies between lead and cadmium and the components of redox system and results of assisted reproduction. A highly significant linear correlation of lead (Pb) and cadmium (Cd) concentrations in serum and in FF was stated. The number of retrieved oocytes and MII (metaphase II stage) oocytes depended on the malondialdehyde (MDA), catalase (CAT), catalase/g of protein (CAT/g of protein), and glutathione reductase (GR) concentrations. Among biochemical factors, MDA was the only factor that correlated negatively with cadmium concentration in serum and FF and simultaneously influenced the number of retrieved oocytes and MII oocytes. The fertilization rate of MII oocytes was influenced by thiol groups—SH, SH/g of protein, CAT, CAT/g of protein, and glutathione peroxidase/g of protein (GPx/g of protein). The Pb and Cd concentrations in FF did not significantly influence the fertilization rates. Lead as well as cadmium at concentrations commonly found in women of reproductive age despite some adaptive changes in the redox system in follicular fluid do not cause large changes in the ovarian follicular environment as a whole and do not significantly worsen the final results of assisted reproduction. [ABSTRACT FROM AUTHOR]

Background: This research investigates the metabolic profiles of follicular fluid (FF) samples from patients with polycystic ovary syndrome (PCOS) undergoing in vitro fertilisation and aims to identify diagnostic and therapeutic biomarkers for PCOS through lipidomic analysis. Methods: We performed non-targeted lipid analysis of FF samples from women with PCOS (n = 6) and normal controls (n = 6) using ultra-high-performance liquid chromatography-tandem mass spectrometry. Differential lipids between the two groups were screened using multidimensional statistical analysis, followed by fold change analysis and t-tests to identify potential PCOS biomarkers. Results: Multivariate statistical analysis revealed significant differences in FF lipid levels between the PCOS and control groups. Five different lipids were selected as standards, with p < .05. Phosphatidylcholine (PC), the main differentially expressed lipid, was significantly increased in the FF of the POCS group and was closely related to other lipids. Conclusions: Using ultra-high-performance liquid chromatography-tandem mass spectrometry, we investigated lipid biomarkers based on FF lipidomics to provide useful information for the discovery of diagnostic markers for PCOS. Our study identified five distinct lipids as potential markers of PCOS, with PC being the primary aberrant lipid found in the FF of patients with PCOS. [ABSTRACT FROM AUTHOR]

Abstract Secretory cells in the fallopian tube fimbria epithelium (FTE) are regarded as the main cells of origin of ovarian high-grade serous carcinoma (HGSC). Ovulation is the main cause of FTE oncogenesis, which proceeds through a sequence of TP53 mutations, chromosomal instability due to Rb/cyclin E aberration, in situ carcinoma (STIC), and metastasis to the ovary and peritoneum (metastatic HGSC). Previously, we have identified multiple oncogenic activities of the ovulatory follicular fluid (FF), which exerts the full spectrum of transforming activity on FTE cells at different stages of transformation. After ovulation, the FF is transfused into the peritoneal fluid (PF), in which the FTE constantly bathes. We wondered whether PF exerts the same spectrum of oncogenic activities as done by FF and whether these activities are derived from FF. By using a panel of FTE cell lines with p53 mutation (FT282-V), p53/CCNE1 aberrations (FT282-CCNE1), and p53/Rb aberrations plus spontaneous transformation, and peritoneal metastasis (FEXT2), we analyzed the changes of different transformation phenotypes after treating with FF and PF collected before or after ovulation. Similar to effects exhibited by FF, we found that, to a lesser extent, PF promoted anchorage-independent growth (AIG), migration, anoikis resistance, and peritoneal attachment in transforming FTE cells. The more transformed cells were typically more affected. Among the transforming activities exhibited by PF treatment, AIG, Matrigel invasion, and peritoneal attachment growth were higher with luteal-phase PF treatment than with the proliferative-phase PF treatment, suggesting an ovulation source. In contrast, changes in anoikis resistance and migration activities were similar in response to treatment with PF collected before and after ovulation, suggesting an ovulation-independent source. The overall transforming activity of luteal-phase PF was verified in an i.p. co-injection xenograft mouse model. Co-injection of Luc-FEXT2 cells with either FF or luteal-phase PF supported early peritoneal implantation, whereas co-injection with follicular-phase PF did not. This study, for the first time, demonstrates that PF from ovulating women can promote different oncogenic phenotypes in FTE cells at different stages of malignant transformation. Most of these activities, other than anoikis resistance and cell migration, are sourced from ovulation.

Background: The follicular fluid (FF) of mature oocytes contains a high concentration of growth factors and cytokinesthat have the potential to influence implantation in either a paracrine or autocrine manner. During the physiologicalprocesses of ovulation, FF enters the fallopian tubes in conjunction with the oocyte. The purpose of this studyis to evaluate implantation and clinical pregnancy rates following uterine flushing with FF and granulosa cells ininfertile women with moderate male factor infertility after ovum retrieval for intracytoplasmic sperm injection (ICSI).Materials and Methods: This phase III randomised clinical trial enrolled 140 women with moderate male factorinfertility who intended to undergo ICSI at Royan Infertility Clinic (Tehran, Iran). A computer-generated program andopaque sealed envelopes were used to randomly allocate patients to either an intervention group (n=70) or a controlgroup (n=70). Participants in the intervention group received 2 ml of clear FF (without blood contamination) from 2to 3 dominant follicles after oocyte retrieval. The control group only underwent uterine cavity catheterisation.Results: The intervention group had a clinical pregnancy rate of 38.5% (25/65) compared to the control group [42.9%(27/63); P=0.719] and an implantation rate of 24.1% compared to the control group (27%; P=0.408). These rates did notdiffer between the groups. There were no statistically significant differences between the intervention and control groupsin terms of pregnancy-related complications-ectopic pregnancy, blighted ovum or anembryonic pregnancy, and abortion.Conclusion: Uterine cavity flushing with FF from mature follicles following oocyte retrieval had no effect, eitherpositively or negatively, on clinical pregnancy or implantation rates in women with moderate male factor infertility(registration number: NCT04077970).

Ageing populations, mass "baby-free" policies and children born to mothers at the age at which they are biologically expected to become grandmothers are growing problems in most developed societies. Therefore, any opportunity to improve the quality of infertility treatments seems important for the survival of societies. The possibility of indirectly studying the quality of developing oocytes by examining their follicular fluids (hFFs) offers new opportunities for progress in our understanding the processes of final oocyte maturation and, consequently, for predicting the quality of the resulting embryos and personalising their culture. Using mass spectrometry, we studied follicular fluids collected individually during in vitro fertilisation and compared their composition with the quality of the resulting embryos. We analysed 110 follicular fluids from 50 oocyte donors, from which we obtained 44 high-quality, 39 medium-quality, and 27 low-quality embryos. We identified 2182 proteins by Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) using a TripleTOF 5600+ hybrid mass spectrometer, of which 484 were suitable for quantification. We were able to identify several proteins whose concentrations varied between the follicular fluids of different oocytes from the same patient and between patients. Among them, the most important appear to be immunoglobulin heavy constant alpha 1 (IgA1hc) and dickkopf-related protein 3. The first one is found at higher concentrations in hFFs from which oocytes develop into poor-quality embryos, the other one exhibits the opposite pattern. None of these have, so far, had any specific links to fertility disorders. In light of these findings, these proteins should be considered a primary target for research aimed at developing a diagnostic tool for oocyte quality control and pre-fertilisation screening. This is particularly important in cases where the fertilisation of each egg is not an option for ethical or other reasons, or in countries where it is prohibited by law. [ABSTRACT FROM AUTHOR]

To date, not many studies have presented evidence of SARS-CoV-2 infecting the female reproductive system. Furthermore, so far, no effect of the administration of anti-COVID 19 vaccines has been reported to affect the quality of oocytes retrieved from women who resorted to assisted reproduction technology (ART). The FF metabolic profiles of women who had been infected by SARS-CoV-2 before IVF treatments or after COVID-19 vaccination were examined by 1H NMR. Immunochemical characterization of proteins and cytokines involved in the redox and inflammatory pathways was performed. The increased expression of SOD2 and NQO1, the lack of alteration of IL-6 and CXCL10 levels, as well as the increased expression of CD39, suggested that, both sharing similar molecular mechanisms or proceeding along different routes, the redox balance is controlled in the FF of both vaccinated and recovered women compared to controls. The lower amount of metabolites known to have proinflammatory activity, i.e., TMAO and lipids, further supported the biochemical results, suggesting that the FF microenvironment is controlled so as to guarantee oocyte quality and does not compromise the outcome of ART. In terms of the number of blastocysts obtained after ICSI and the pregnancy rate, the results are also comforting. [ABSTRACT FROM AUTHOR]

Background: This study aimed to examine the differential variations in the metabolic composition of follicular fluid (FF) among normal-weight patients with polycystic ovary syndrome (PCOS) and controls and to identify potential biomarkers that may offer insights into the early identification and management of these patients. Methods: We collected FF samples from 45 normal-weight women with PCOS and 36 normal-weight controls without PCOS who were undergoing in vitro fertilization–embryo transfer. An untargeted metabolomic study of collected FF from infertile women was performed using high-performance liquid chromatography–tandem spectrometry (LC-MS). The tendency of the two groups to separate was demonstrated through multivariate analysis. Univariate analysis and variable importance in projection were used to screen out differential metabolites. Metabolic pathway analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG), and a diagnostic model was established using the random forest algorithm. Results: The metabolomics analysis revealed an increase in the expression of 23 metabolites and a decrease in that of 10 metabolites in the FF of normal-weight women with PCOS. According to the KEGG pathway analysis, these differential metabolites primarily participated in the metabolism of glycerophospholipids and the biosynthesis of steroid hormones. Based on the biomarker combination of the top 10 metabolites, the area under the curve value was 0.805. The concentrations of prostaglandin E2 in the FF of individuals with PCOS exhibited an inverse association with the proportion of high-quality embryos (p < 0.05). Conclusions: Our research identified a distinct metabolic profile of the FF from normal-weight women with PCOS. The results offer a broader comprehension of the pathogenesis and advancement of PCOS, and the detected differential metabolites could be potential biomarkers and targets for the treatment of PCOS. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women at childbearing age. Anti-Müllerian hormone (AMH) is a widely accepted sensitive marker of ovarian reserve, which has been suggested that could also act as biomarker of ovarian morphology for PCOS diagnosis. Oxidative stress (OS) is known to be associated and have a negative impact factor in several reproductive conditions, including PCOS. However, the relationship between circulating AMH and OS within the follicular fluid (FF), and its potential impact on in vitro fertilization (IVF) outcomes of women with PCOS, remains largely unexplored. A total of 84 women, with PCOS (n = 30) or ovulatory controls (n = 54), were enrolled in this study. Women underwent individualized controlled ovarian stimulation for oocyte retrieval. Blood and FF obtained from mature follicles were collected at the time of oocyte retrieval, for measuring total testosterone, Î"4-androstenedione, progesterone, sex hormone binding globulin (SHBG) and AMH. OS in the FF was assessed by measuring total antioxidant capacity (TAC) through the ferric reducing antioxidant power (FRAP) and lipid peroxidation (LPO) by quantification of malondialdehyde (MDA) levels. Our results demonstrated that women with PCOS had significantly higher plasma levels of AMH, Î"4-androstenedione, total testosterone and a free androgen index (FAI) than observed in non-PCOS controls. In women with PCOS, total testosterone and AMH levels in the FF were also higher, while TAC was lower compared to non-PCOS. Furthermore, circulating AMH levels were positively correlated with Î"4-androstenedione, albeit negatively correlated with TAC. In this study we demonstrated that the susceptibility to OS, as assessed by the total antioxidant capacity in the FF, is higher in women with PCOS and inversely related to AMH levels. This study results lead us to forge the reasonable hypothesis that the greater susceptibility to OS within the follicle microenvironment is potentially at the end of a roadway that starts with elevated Î"4-androstenedione and AMH within the FF, which in turn are mirrored by circulating AMH and androgen levels. Thus, suggesting that circulating AMH levels could act as a surrogate biomarker of follicular fluid oxidative stress in women with PCOS. [ABSTRACT FROM AUTHOR]

Secretory cells in the fallopian tube fimbria epithelium (FTE) are regarded as the main cells of origin of ovarian high-grade serous carcinoma (HGSC). Ovulation is the main cause of FTE oncogenesis, which proceeds through a sequence of TP53 mutations, chromosomal instability due to Rb/cyclin E aberration, in situ carcinoma (STIC), and metastasis to the ovary and peritoneum (metastatic HGSC). Previously, we have identified multiple oncogenic activities of the ovulatory follicular fluid (FF), which exerts the full spectrum of transforming activity on FTE cells at different stages of transformation. After ovulation, the FF is transfused into the peritoneal fluid (PF), in which the FTE constantly bathes. We wondered whether PF exerts the same spectrum of oncogenic activities as done by FF and whether these activities are derived from FF. By using a panel of FTE cell lines with p53 mutation (FT282-V), p53/CCNE1 aberrations (FT282-CCNE1), and p53/Rb aberrations plus spontaneous transformation, and peritoneal metastasis (FEXT2), we analyzed the changes of different transformation phenotypes after treating with FF and PF collected before or after ovulation. Similar to effects exhibited by FF, we found that, to a lesser extent, PF promoted anchorage-independent growth (AIG), migration, anoikis resistance, and peritoneal attachment in transforming FTE cells. The more transformed cells were typically more affected. Among the transforming activities exhibited by PF treatment, AIG, Matrigel invasion, and peritoneal attachment growth were higher with luteal-phase PF treatment than with the proliferative-phase PF treatment, suggesting an ovulation source. In contrast, changes in anoikis resistance and migration activities were similar in response to treatment with PF collected before and after ovulation, suggesting an ovulation-independent source. The overall transforming activity of luteal-phase PF was verified in an i.p. co-injection xenograft mouse model. Co-injection of Luc-FEXT2 cells with either FF or luteal-phase PF supported early peritoneal implantation, whereas co-injection with follicular-phase PF did not. This study, for the first time, demonstrates that PF from ovulating women can promote different oncogenic phenotypes in FTE cells at different stages of malignant transformation. Most of these activities, other than anoikis resistance and cell migration, are sourced from ovulation. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Do the molecular signatures of cumulus cells (CCs) and follicular fluid (FF) of adolescents undergoing fertility preservation differ from that of oocyte donors? SUMMARY ANSWER The microenvironment immediately surrounding the oocyte, including the CCs and FF, is altered in adolescents undergoing fertility preservation compared to oocyte donors. WHAT IS KNOWN ALREADY Adolescents experience a period of subfecundity following menarche. Recent evidence suggests that this may be at least partially due to increased oocyte aneuploidy. Reproductive juvenescence in mammals is associated with suboptimal oocyte quality. STUDY DESIGN, SIZE, DURATION This was a prospective cohort study. Adolescents (10–19 years old, n = 23) and oocyte donors (22–30 years old, n = 31) undergoing ovarian stimulation and oocyte retrieval at a single center between 1 November 2020 and 1 May 2023 were enrolled in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS Patient demographics, ovarian stimulation, and oocyte retrieval outcomes were collected for all participants. The transcriptome of CCs associated with mature oocytes was compared between adolescents (10–19 years old, n = 19) and oocyte donors (22–30 years old, n = 19) using bulk RNA-sequencing. FF cytokine profiles (10–19 years old, n = 18 vs 25–30 years old, n = 16) were compared using cytokine arrays. MAIN RESULTS AND THE ROLE OF CHANCE RNA-seq analysis revealed 581 differentially expressed genes in CCs of adolescents relative to oocyte donors, with 361 genes downregulated and 220 upregulated. Genes enriched in pathways involved in cell cycle and cell division (e.g. GO: 1903047, P  = 3.5 × 10−43; GO: 0051983, P  = 4.1 × 10−30; GO: 0000281, P  = 7.7 × 10−15; GO: 0044839, P  = 5.3 × 10−13) were significantly downregulated, while genes enriched in several pathways involved in cellular and vesicle organization (e.g. GO: 0010256, P  = 1.2 × 10−8; GO: 0051129, P  = 6.8 × 10−7; GO: 0016050, P  = 7.4 × 10−7; GO: 0051640, P  = 8.1 × 10−7) were upregulated in CCs of adolescents compared to oocyte donors. The levels of nine cytokines were significantly increased in FF of adolescents compared to oocyte donors: IL-1 alpha (2-fold), IL-1 beta (1.7-fold), I-309 (2-fold), IL-15 (1.6-fold), TARC (1.9-fold), TPO (2.1-fold), IGFBP-4 (2-fold), IL-12-p40 (1.7-fold), and ENA-78 (1.4-fold). Interestingly, seven of these cytokines have known pro-inflammatory roles. Importantly, neither the CC transcriptomes nor FF cytokine profiles were different in adolescents with or without cancer. LARGE SCALE DATA Original high-throughput sequencing data have been deposited in Gene Expression Omnibus (GEO) database with the accession number GSE265995. LIMITATIONS, REASONS FOR CAUTION This study aims to gain insights into the associated gamete quality by studying the immediate oocyte microenvironment. The direct study of oocytes is more challenging due to sample scarcity, as they are cryopreserved for future use, but would provide a more accurate assessment of oocyte reproductive potential. WIDER IMPLICATIONS OF THE FINDINGS Our findings have implications for the adolescent fertility preservation cycles. Understanding the expected quality of cryopreserved eggs in this age group will lead to better counseling of these patients about their reproductive potential and may help to determine the number of eggs that is recommended to be banked to achieve a reasonable chance of future live birth(s). STUDY FUNDING/COMPETING INTEREST(S) This project was supported by Friends of Prentice organization SP0061324 (M.M.L. and E.B.), Gesualdo Family Foundation (Research Scholar: M.M.L.), and NIH/NICHD K12 HD050121 (E.B.). The authors have declared that no conflict of interest exists. [ABSTRACT FROM AUTHOR]

Objectives: ABO blood type was hypothesised to be related to a number of infertility processes. There is still an open debate on ABO blood group's incompatibility and infertility. It was associated with ovarian reserve in women with subfertility. There is still not enough information on the influence of blood type and the immunology of follicular fluid (FF). Material and methods: 78 patients were selected, who underwent in vitro fertilization (IVF) between April 2021 and January 2022. FF samples from each individual patient were taken on the day of ovarian puncture and stored at -80°C until immunological assessment. Concentration of chosen interleukins - IL-1a, IL-2, IL-4, IL-5, IL-6, IL-8 IL-10, IL-15, IL-1ß, IL-18, IFN, LIF, TNFa, GCSF and PIBF-1 were measured using commercially available ELISA kits. Results: All assessed cytokines were present in the FF of exanimated patients. The concentration was compared to the blood type ABO of all women undergoing in vitro fertilization. No statistical relevance was found between blood type ABO and the concentration of GCSF, PIBF1, LIF, IL-15, IL-5, IL-8, IL-1 alfa, IL-1 beta, INF gamma, IL-2HS, IL-4HS, IL-6HS, IL-10HS in the FF obtained during ovarian puncture (p > 0,05). There was no statistically significant correlation between blood type ABO and the quality of embryo, and the positive pregnancy test in patients undergoing IVF/ET. Conclusions: The blood type ABO does not influence the wide cytokine profile of FF obtained during ovarian puncture in women with infertility of different origin, as well as embryo quality and pregnancy rate. [ABSTRACT FROM AUTHOR]

Background: Polycystic ovary syndrome with insulin resistance (PCOS-IR) is the most common endocrine and metabolic disease in women of reproductive age, and low fertility in PCOS patients may be associated with oocyte quality; however, the molecular mechanism through which PCOS-IR affects oocyte quality remains unknown. Methods: A total of 22 women with PCOS-IR and 23 women without polycystic ovary syndrome (control) who underwent in vitro fertilization and embryo transfer were recruited, and clinical information pertaining to oocyte quality was analyzed. Lipid components of follicular fluid (FF) were detected using highcoverage targeted lipidomics, which identified 344 lipid species belonging to 19 lipid classes. The exact lipid species associated with oocyte quality were identified. Results: The number (rate) of two pronuclear (2PN) zygotes, the number (rate) of 2PN cleaved embryos, and the number of high-quality embryos were significantly lower in the PCOS-IR group. A total of 19 individual lipid classes and 344 lipid species were identified and quantified. The concentrations of the 19 lipid species in the normal follicular fluid (control) ranged between 10-3 mol/L and 10-9 mol/L. In addition, 39 lipid species were significantly reduced in the PCOS-IR group, among which plasmalogens were positively correlated with oocyte quality. Conclusions: This study measured the levels of various lipids in follicular fluid, identified a significantly altered lipid profile in the FF of PCOS-IR patients, and established a correlation between poor oocyte quality and plasmalogens in PCOS-IR patients. These findings have contributed to the development of plasmalogen replacement therapy to enhance oocyte quality and have improved culture medium formulations for oocyte in vitro maturation (IVM). [ABSTRACT FROM AUTHOR]

One of the succes factors of biomedically assisted fertilization is the regular maturation of one or more oocytes. The quality of the oocytes is significantly influenced by the environment in which it is located, the so-called „microenvironment" that includes cumulus cells, follicular fluid in which hormones and growth factors involved in its growth and development are secreted. The main aim was to examine whether the concentration of steroid hormones in the follicular fluid affects the rate of fertilization and the outcome of the in vitro fertilization process itself. The study included 31 patients who were included in vitro fertilization procedure at the Department for Biomedically Assisted Fertilization, Clinic for Gynecology and Obstetrics, Clinical Center Kragujevac. We used follicular fluid as biological material for analysis. Examination of the obtained follicular fluid and collection of oocytes under a stereomicroscope was done in the embryological laboratory at the Department. Biochemical parameters of follicular fluid were analyzed in the Department for Laboratory Diagnostics, Clinical Center Kragujevac. In vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) methods were used as the fertilization method. The criteria of the Istanbul Consensus of Clinical Embryologists were used as a reference framework for embryo quality assessment. Pregnancy was confirmed by a positive serum level of the hormone β-hCG 14 days after embryo transfer. A software package SPSS 20 was used for statistical data processing. The results of the analysis of follicular fluid samples show that there was no statistically significant difference in the concentration of estradiol, progesterone and testosterone in follicular fluid in relation to fertilization rate and the outcome of in vitro fertilization. Based on our results, it can be concluded that the concentration of steroid hormones did not affect fertilization rate and the outcome of in vitro fertilization. [ABSTRACT FROM AUTHOR]

Purpose: To determine correlations between chemicals in follicular fluid (FF) and follicular reproductive hormone levels. Methods: The analysis was part of a larger cohort study to determine associations between exposure to EDCs and in vitro fertilization (IVF) outcomes. FF was aspirated from a single leading follicle per participant. Demographics and data on exposure to EDCs were self-reported by the participants using a questionnaire. The concentrations of estradiol (E2), progesterone (PG), anti-Mullerian hormone (AMH), and inhibin B, as well as that of 12 phthalate metabolites and 12 phenolic chemicals were measured in each FF sample. Multivariate linear regression model was used to identify the drivers of hormone levels based on participant's age, BMI, smoking status, and chemical exposure for the monitored chemicals detected in more than 50% of the samples. Benjamini–Hochberg false discovery rate (FDR) correction was applied on the resulting p values (q value). Results: FF samples were obtained from 72 women (mean age 30.9 years). Most of the phthalates and phenolic substances monitored (21/24, 88%) were identified in FF. Ten compounds (7 phthalate metabolites, 3 phenols) were found in more than 50% of samples. In addition, there were positive associations between E2 levels and mono-n-butyl phthalate (MnBP) (beta = 0.01) and mono-isobutyl phthalate (MiBP) (beta = 0.03) levels (q value < 0.05). Conclusion: Higher concentrations of several phthalate metabolites, present among others in personal care products, were associated with increased E2 levels in FF. The results emphasize the need to further investigate the mechanisms of action of such EDCs on hormonal cyclicity and fertility in women. [ABSTRACT FROM AUTHOR]

Global methylation levels differ in in vitro- and in vivo-developed embryos. Follicular fluid (FF) contains extracellular vesicles (EVs) containing miRNAs that affect embryonic development. Here, we examined our hypothesis that components in FF affect global DNA methylation and embryonic development. Oocytes and FF were collected from bovine ovaries. Treatment of zygotes with a low concentration of FF induced global DNA demethylation, improved embryonic development, and reduced DNMT1/3A levels. We show that embryos take up EVs containing labeled miRNA secreted from granulosa cells and the treatment of zygotes with EVs derived from FF reduces global DNA methylation in embryos. Furthermore, the methylation levels of in vitro-developed blastocysts were higher than those of in their vivo counterparts. Based on small RNA-sequencing and in silico analysis, we predicted miR-29b, -199a-3p, and -148a to target DNMTs and to induce DNA demethylation, thereby improving embryonic development. Moreover, among FF from 30 cows, FF with a high content of these miRNAs demethylated more DNA in the embryos than FF with a lower miRNA content. Thus, miRNAs in FF play a role in early embryonic development. [ABSTRACT FROM AUTHOR]

Background: Cortisol, is a glucocorticoid hormone produced by the adrenal glands, performs many vital functions in the body, including the regulation of energy, metabolism, and immunity. The body also releases cortisol to activate our "flightor-fight" responses to counteract acutely stressful situations. If cortisol levels stay elevated for extended periods, however, they can lead to chronic stress. This long-term activation of the stress-response system can interfere with the normal functioning of other body systems, including reproductive health. Several studies demonstrate a relationship between stress, cortisol and infertility – higher levels of stress and cortisol result in a decreased fertility rate [1]. Aim of the study: to detect the follicular fluid and blood concentration of the cortisol hormone on subfertility in women undergoing Intracytoplasmic Sperm Injection (ICSI) for fertility treatment. Method: This case control study carried out on eighty women, from these, forty female were subfertile patient defined as the cases, other fourty female as control included fertile female came to fertility center due to male facter of subfertility. A detailed medical history, physical examination was recorded. At the same day of ovum pickup blood and follicular fluid were collected, for hormonal analysis of cortisol. Results: Show significantly higher levels of serum and follicular cortisol in subfertile women (cases) than control group ( p < 0.05), and significant positive correlations between serum and follicular fluid. Conclusion: Cortisol hormone level in blood and follicular fluid were significantly higher in cases group than control and there is a positive correlations between serum and follicular fluid of cortisol. [ABSTRACT FROM AUTHOR]

IntroductionBisphenol A (BPA) is a common contaminant widely used in many industrial sectors. Because of its wide use and dispersion, it can be accumulated in living human bodies through both oral assumption and nondietary routes. BPA exhibits hormone-like properties, falling under the class of endocrine disruptors; therefore, it can alter relevant physiological functions. In particular, in women, it can affect folliculogenesis and therefore reproduction, contributing not only to infertility, but also to endometriosis and premature puberty.MethodsWe conducted a multicenter study on 91 women undergoing a first in vitro fertilization (IVF) treatment in the Campania region (Southern Italy). We investigated the presence and concentration of BPA in serum and follicular fluids to assess the effects of airborne BPA contamination. The analysis was conducted on 32 women living in a low environmental impact (LEI) area, from the Sele Valley River and Cilento region, and 59 women living in a high environmental impact (HEI) area, the so-called “Land of Fires”, a highly contaminated territory widely exposed to illegal waste practices.ResultsA higher average BPA content in both blood serum and follicular fluid was revealed in the HEI group when compared with the LEI group. In addition, we revealed higher average BPA content in blood serum than in folliclular fluid in the HEI area, with opposite average content in the two fluids in the LEI zone. In addition, our results also showed a lack of correlation between BPA content in follicular and serum fluids both in the overall population and in the HEI and LEI groups, with peculiar trends in different subsets of women.ConclusionFrom our results, we revealed a heterogeneity in the distribution of BPA content between serum and follicular fluid. Further studies are needed to unravel the bioaccumulation mechanisms of BPA in highly polluted and nonpolluted areas.

The increase in female reproductive disorders, such as polycystic ovary syndrome, endometriosis, and diminished ovarian reserve that lead to subfertility and infertility, has encouraged researchers to search and discover their underlying causes and risk factors. One of the crucial factors that may influence the increasing number of reproductive issues is environmental pollution, particularly exposure to man-made endocrine-disrupting chemicals (EDCs). EDCs can interfere with the ovarian microenvironment, impacting not only granulosa cell function but also other surrounding ovarian cells and follicular fluid (FF), which all play essential roles for oocyte development, maturation, and overall reproductive function. FF surrounds developing oocytes within an ovarian follicle and represents a dynamic milieu. EDCs are usually found in biological fluids, and FF is therefore of interest in this respect. This narrative review examines the current knowledge on specific classes of EDCs, including industrial chemicals, pesticides, and plasticizers, and their known effects on hormonal signaling pathways, gene expression, mitochondrial function, oxidative stress induction, and inflammation in FF. We describe the impact of EDCs on the development of reproductive disorders, oocyte quality, menstrual cycle regulation, and their effect on assisted reproductive technique outcomes. The potential transgenerational effects of EDCs on offspring through animal and first-human studies has been considered also. While significant progress has been made, the current understanding of EDCs’ effects on ovarian function, particularly in humans, remains limited, underscoring the need for further research to clarify actions and effects of EDCs in the ovary.

The plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to have endocrine-disrupting properties mediated by its many metabolites that form upon exposure in biological systems. In a previous study, we reported an inverse association between DEHP metabolites in the human ovarian follicular fluid (FF) and the responsiveness of the follicles to controlled ovarian stimulation during in vitro fertilization (IVF) treatments. Here, we explored this association further through molecular analysis of the ovarian FF samples.Ninety-six IVF patients from Swedish (N = 48) and Estonian (N = 48) infertility clinics were selected from the previous cohort (N = 333) based on the molar sum of DEHP metabolites in their FF samples to arrive at “high” (mean 7.7 ± SD 2.3 nM, N = 48) and “low” (0.8 ± 0.4 nM, N = 48) exposure groups. Extracellular miRNA levels and concentrations of 15 steroid hormones were measured across FF samples. In addition, FF somatic cells, available for the Estonian patients, were used for RNA sequencing.Differential expression (DE) and interactions between miRNA and mRNA networks revealed that the expression levels of genes in the cholesterol biosynthesis and steroidogenesis pathways were significantly decreased in the high compared to the low DEHP group. In addition, the DE miRNAs were predicted to target key enzymes within these pathways (FDR

Endocrine-disrupting chemicals (EDCs) exhibited the detriment in female reproductive health. Our objective was to investigate the individual and mixture effects of EDCs present in follicular fluid, the environment in which oocytes grow and develop, on early reproductive outcomes. We recruited 188 women seeking reproduction examination from the Study of Exposure and Reproductive Health (SEARCH) cohort between December 2020 and November 2021. We assessed the concentrations of 7 categories of 64 EDCs in follicular fluid, and measured early reproductive outcomes, including retrieved oocytes, mature oocytes, normal fertilized oocytes, and high-quality embryos. In this study Monomethyl phthalate (MMP) (2.17 ng/ml) were the compounds found in the highest median concentrations in follicular fluid. After adjusting for multiple testing, multivariate regression showed that multiple EDCs were significantly negatively associated with early assisted reproduction outcomes. For example, MMP showed a significant negative correlation with the number of high quality embryos (β: −0.1, 95 % CI: −0.15, −0.04). Specifically, eight types of EDCs were significantly negatively associated with four early assisted reproductive outcomes (β range: −0.2 ∼ -0.03). In the mixed exposure model, we found that mixtures of EDC were significantly negatively correlated with all four outcomes. In the quantile g-computation (QGCOMP) model, for each interquartile range increase in the concentration of EDC mixtures, the number of oocytes retrieved, mature oocytes, normally fertilized oocytes, and high-quality embryos decreased by 0.46, 0.52, 0.77, and 1.2, respectively. Moreover, we identified that phthalates (PAEs) predominantly contributed to the negative effects. Future research should validate our findings.

Abstract Background Imbalances in alkali elements (AEs) and alkaline earth elements (AEEs) cause reproductive disorders. However, it remains unclear whether AEs/AEEs in follicular fluid have a relationship with the serious reproductive disorder known as diminished ovarian reserve (DOR). Methods A nested case‒control study was carried out in China. Follicular fluid samples from 154 DOR patients and 154 controls were collected and assessed for nine AEs/AEE levels. Both the mixed and single effects of the elements on DOR were estimated with a Bayesian kernel machine (BKMR) and logistic regressions. Results The DOR group had higher median concentrations of Li, Na, and K in follicular fluid (all P values

Abstract Background Polycystic ovary syndrome (PCOS) is an endocrinological and metabolic disorder that can lead to female infertility. Lipid metabolomics and proteomics are the new disciplines in systems biology aimed to discover metabolic pathway changes in diseases and diagnosis of biomarkers. This study aims to reveal the features of PCOS to explore its pathogenesis at the protein and metabolic level. Methods We collected follicular fluid samples and granulosa cells of women with PCOS and normal women who underwent in vitro fertilization(IVF) and embryo transfer were recruited. The samples were for the lipidomic study and the proteomic study based on the latest metabolomics and proteomics research platform. Results Lipid metabolomic analysis revealed abnormal metabolism of glycerides, glycerophospholipids, and sphingomyelin in the FF of PCOS. Differential lipids were strongly linked with the rate of high-quality embryos. In total, 144 differentially expressed proteins were screened in ovarian granulosa cells in women with PCOS compared to controls. Go functional enrichment analysis showed that differential proteins were associated with blood coagulation and lead to follicular development disorders. Conclusion The results showed that the differential lipid metabolites and proteins in PCOS were closely related to follicle quality,which can be potential biomarkers for oocyte maturation and ART outcomes.

Background: Oxidative/nitrosative stress in the oocyte microenvironment could have an impact on intracytoplasmicsperm injection (ICSI) outcomes. The presence of reactive oxygen species (ROS) and reactive nitrogen species (RNS) canstimulate the secretion of pro-inflammatory cytokines, leading to chronic inflammation and potentially affecting embryo aswell as oocyte quality. This study aimed to examine the relationship of lipid peroxidation [measured by the malondialdehyde(MDA) assay] with protein carbonyl [measured by the 2,4 dinitrophenylhydrazine (DNPH) assay] levels in cumulus cells(CCs), as well as nitric oxide (NO), peroxynitrite (ONOO-), and C-reactive protein (CRP) levels in follicular fluid (FF).The potential relationship of these levels with ICSI outcomes was also evaluated.Materials and Methods: In this prospective study, 63 FF samples and their corresponding CCs were collected forICSI procedures. Spectrophotometry was used to assess levels of DNPH, MDA, NO, and ONOO-. CRP levels wereevaluated using an immunoturbidimetric assay.Results: The patients under 37 years with normal ovarian reserve had significantly lower levels of MDA, DNPH,NO, ONOO-, and CRP compared to those over 37 years. Additionally, we observed higher levels of MDA, DNPH,NO, ONOO-, and CRP in the group with an oocyte maturity rate of less than 60%. No significant difference was observedbetween the DNPH levels and factors such as infertility duration, embryo quality, pregnancy, or the number ofretrieved oocytes. A higher level of MDA, NO, ONOO-, and CRP was found to be significantly related to the lowernumber of retrieved oocytes, longer periods of infertility, poor embryo quality, and negative pregnancy outcomes.Conclusion: Oxidative/nitrosative stress, linking to inflammation in the oocyte microenvironment, can be consideredas a potentially useful biomarker for assessing the development and competence of oocytes and embryos and predictingICSI outcomes.

Introduction: Polycystic ovary syndrome (PCOS) is a common multifactorial and polygenic disorder of the endocrine system, affecting up to 20% of women in reproductive age with a still unknown etiology. Follicular fluid (FF) represents an environment for the normal development of follicles rich in metabolites, hormones and neurotransmitters, but in some instances of PCOS the composition can be different. Vasoactive intestinal peptide (VIP) is an endogenous autonomic neuropeptide involved in follicular atresia, granulosa cell physiology and steroidogenesis. Methods: ELISA assays were performed to measure VIP and estradiol levels in human follicular fluids, while AMH, FSH, LH, estradiol and progesterone in the plasma were quantified by chemiluminescence. UHPLC/QTOF was used to perform the untargeted metabolomic analysis. Results: Our ELISA and metabolomic results show: i) an increased concentration of VIP in follicular fluid of PCOS patients (n=9) of about 30% with respect to control group (n=10) (132 ± 28 pg/ml versus 103 ± 26 pg/ml, p=0,03) in women undergoing in vitro fertilization (IVF), ii) a linear positive correlation (p=0.05, r=0.45) between VIP concentration and serum Anti-Müllerian Hormone (AMH) concentration and iii) a linear negative correlation between VIP and noradrenaline metabolism. No correlation between VIP and estradiol (E2) concentration in follicular fluid was found. A negative correlation was found between VIP and noradrenaline metabolite 3,4-dihydroxyphenylglycolaldehyde (DOPGAL) in follicular fluids. Conclusion: VIP concentration in follicular fluids was increased in PCOS patients and a correlation was found with noradrenaline metabolism indicating a possible dysregulation of the sympathetic reflex in the ovarian follicles. The functional role of VIP as noradrenergic modulator in ovarian physiology and PCOS pathophysiology was discussed. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the molecular composition of follicular fluid (FF) extracellular vesicles (EVs) in women of different reproductive ages and its possible relationship to sperm fertilizing ability. FF EVs were obtained by differential centrifugation. The concentration and size distribution of FF EVs were analyzed by nanoparticle tracking analysis. The lipidome and proteome were analyzed by liquid chromatography–mass spectrometry. The isolated FF EVs had a variety of shapes and sizes; their concentration and size distribution did not differ significantly between the age groups. In women younger than 35 years, the concentration of vesicular progesterone was 6.6 times higher than in women older than 35 years, and the total levels of the main lipid classes were increased in younger women. A proteomic analysis revealed that not only FF EV-specific proteins, but also proteins involved in sperm activation were present. New data were obtained on the composition of FF EVs, confirming their importance as molecular indicators of age-related changes in the female reproductive system. In addition, these results shed light on the possible interaction between the FF EVs of women in different age groups and male germ cells. Therefore, studying the transcriptomic and metabolomic profile of FF EVs may be a crucial approach to evaluate the efficacy of ART. [ABSTRACT FROM AUTHOR]

Follicular fluid is the internal environment where follicular cells grow and proliferate, and the changes of its components are closely related to the functional status of follicular cells. In recent years, it has been found that exosome, a double⁃layer membrane vesicle with a diameter of about 30-150 nm, exists in many biological fluids, and its mediating role in many physiological and pathological processes has been revealed. The researchers also identified exosomes in follicular fluid and found that they are closely related to the growth and proliferation of follicular cells, indirectly influencing oocyte status, which is important for assessing oocyte quality. [ABSTRACT FROM AUTHOR]

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Female subfertility has been a growing concern for reproductive health. Assisted reproductive technologies make pregnancy possible, but the outcome rate is still suboptimal. Zinc is an essential factor for fertility and development. Zinc levels in follicular fluids were measured by electrochemical method, and we found that zinc in the follicular fluids was related to high-quality embryo rate (R = 0.39, p = 0.01). Basal estradiol levels and estradiol levels on the day of HCG injection were negatively correlated with zinc concentrations in the follicular fluid (R = − 0.53, p < 0.001; R = − 0.32, p < 0.05), and estradiol promoted ZnT 9 protein expression in cumulus granulosa cells in vitro and in vivo. When the zinc level was at 3.63–3.85 μg/mL, follicular fluid samples had the highest SOD activity. Therefore, zinc played an important role in improving oocyte development by increasing antioxidant capacity. Our results suggested that estradiol affected zinc homeostasis in follicles by controlling the expression of ZnT 9, which in turn influenced the potential of oocytes to develop into good-quality embryos. This study to provide tangible improvements to patient outcomes will make it a focus of both scientific and translational efforts in the future. [ABSTRACT FROM AUTHOR]

Background: Polycystic ovary syndrome (PCOS) is an endocrinological and metabolic disorder that can lead to female infertility. Lipid metabolomics and proteomics are the new disciplines in systems biology aimed to discover metabolic pathway changes in diseases and diagnosis of biomarkers. This study aims to reveal the features of PCOS to explore its pathogenesis at the protein and metabolic level. Methods: We collected follicular fluid samples and granulosa cells of women with PCOS and normal women who underwent in vitro fertilization(IVF) and embryo transfer were recruited. The samples were for the lipidomic study and the proteomic study based on the latest metabolomics and proteomics research platform. Results: Lipid metabolomic analysis revealed abnormal metabolism of glycerides, glycerophospholipids, and sphingomyelin in the FF of PCOS. Differential lipids were strongly linked with the rate of high-quality embryos. In total, 144 differentially expressed proteins were screened in ovarian granulosa cells in women with PCOS compared to controls. Go functional enrichment analysis showed that differential proteins were associated with blood coagulation and lead to follicular development disorders. Conclusion: The results showed that the differential lipid metabolites and proteins in PCOS were closely related to follicle quality,which can be potential biomarkers for oocyte maturation and ART outcomes. [ABSTRACT FROM AUTHOR]

Purpose: Decreased ovarian reserve function is mainly characterized by female endocrine disorders and fertility decline. Follicular fluid (FF) exosomal microRNAs (miRNAs) have been shown to regulate the function of granulosa cells (GCs). The present study explored differentially expressed miRNAs (DEmiRNAs) in patients with diminished ovarian reserve (DOR). Methods: FF was collected from 12 DOR patients and 12 healthy controls. DEmiRNAs between the two groups were identified and analyzed using high-throughput sequencing technology and validated by real-time quantitative PCR (RT-qPCR). Results: A total of 592 DEmiRNAs were identified using high-throughput miRNA sequencing, of which 213 were significantly upregulated and 379 were significantly downregulated. The sequencing results were further validated by RT-qPCR. These DEmiRNA target genes were mainly involved in the cancer pathway, phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, regulation of actin cytoskeleton signaling pathway, and biological processes related to protein binding, nucleoplasm, cytoplasm, and cell membrane. Conclusion: FF exosomal miRNAs are significantly differentially expressed in DOR patients versus non-DOR patients, underscoring their crucial role in regulating the pathogenesis of DOR. [ABSTRACT FROM AUTHOR]

Background: Oxidative/nitrosative stress in the oocyte microenvironment could have an impact on intracytoplasmic sperm injection (ICSI) outcomes. The presence of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can stimulate the secretion of pro-inflammatory cytokines, leading to chronic inflammation and potentially affecting embryo as well as oocyte quality. This study aimed to examine the relationship of lipid peroxidation [measured by the malondialdehyde (MDA) assay] with protein carbonyl [measured by the 2,4 dinitrophenylhydrazine (DNPH) assay] levels in cumulus cells (CCs), as well as nitric oxide (NO), peroxynitrite (ONOO-), and C-reactive protein (CRP) levels in follicular fluid (FF). The potential relationship of these levels with ICSI outcome was also evaluated. Materials and Methods: In this prospective study, 63 FF samples and their corresponding CCs were collected for ICSI procedures. Spectrophotometry was used to assess levels of DNPH, MDA, NO, and ONOO-. CRP levels were evaluated using an immunoturbidimetric assay. Results: The patients under 37 years with normal ovarian reserve had significantly lower levels of MDA, DNPH, NO, ONOO-, and CRP compared to those over 37 years. Additionally, we observed higher levels of MDA, DNPH, NO, ONOO-, and CRP in the group with an oocyte maturity rate of less than 60%. No significant difference was observed between the DNPH levels and factors such as infertility duration, embryo quality, pregnancy, or the number of retrieved oocytes. A higher level of MDA, NO, ONOO-, and CRP was found to be significantly related to the lower number of retrieved oocytes, longer periods of infertility, poor embryo quality, and negative pregnancy outcomes. Conclusion: Oxidative/nitrosative stress, linking to inflammation in the oocyte microenvironment, can be considered as a potentially useful biomarker for assessing the development and competence of oocytes and embryos and predicting ICSI outcomes. [ABSTRACT FROM AUTHOR]

Aim/Background: Throughout oogenesis and folliculogenesis, the follicular fluid's composition alters physiologically to fit the needs of particular microenvironmental demands. This study's main goal was to compare the effect of follicular fluid collected from endometriotic and non-endometriotic patients on systemic body functioning of female mice. Materials and Methods: Both healthy and endometriotic participants' follicular fluid was collected and pooled separately. Female Swiss albino mice were injected with 1 and 2 mL/kg/day endometrial fluid in the intraperitoneal region and monitored for 21 days. Change in body weight, hormonal profile, glucose profile and hematological profile was monitored and recorded on regular basis. Results: On day 21, the blood glucose level increased from 100.2±0.96 mg/dL to 138.4±3.32 mg/dL. Endometriotic follicular fluid had a dose-dependently decreased serum estradiol from 28.80±0.37 to 27.00±1.0 ng/mL and progesterone form 24.17±0.7 to 1.72±0.21 pg/mL and a rise in testosterone levels from normal 3.95±0.81 nmol/mL to 9.4±0.92. It has elevated serum LH levels to approximately three times normal levels. In contrast, the serum FSH level decreased from 19.40±0.74 mIU/mL to 2.5±0.22. As a result, the LH to FSH ratio increased from 0.18±0.01 to 3.9±0.19. There was a dose-dependent rise in serum insulin level significantly (p<0.001) from normal 0.74±0.02 IU/mL to 1.63±0.05 and 2.09±0.1 respectively. In a similar manner, HOMA-IR also showed increase in insulin resistance from normal 0.17 to 0.57±0.02 and 0.71±0.04. HOMA-Beta normal level was 8.17±0.3 increased to 10.05±0.4 beta cell dysfunction. Nevertheless, QUICKI were both dosages dependently decreased insulin sensitivity from 1.31±0.07 dose dependently to 0.9±0.01and 0.83±0.02. Conclusion: Female mice treated with endometriotic follicular fluid of endometriosis patients displayed pancreatic abnormality. It has been concluded that endometriotic patients' follicular fluid not only has a localized effect but also contains elements that enter the systemic circulation, have negative effects, and may be connected to significant clinical symptoms of endometriotic condition. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with infertility and poor reproductive outcomes. The follicular fluid (FF) microenvironment plays a crucial role in oocyte development. This review summarizes evidence elucidating the alterations in FF composition in PCOS. Various studies demonstrated a pronounced proinflammatory milieu in PCOS FF, characterized by increased levels of cytokines, including but not limited to interleukin-6 (IL-6), tumor necrosis factor α, C-reactive protein, and IL-1β, concomitant with a reduction in anti-inflammatory IL-10. T lymphocytes and antigen-presenting cells are dysregulated in PCOS FF. PCOS FF exhibit heightened reactive oxygen species production and the accumulation of lipid peroxidation byproducts, and impaired antioxidant defenses. Multiple microRNAs are dysregulated in PCOS FF, disrupting signaling critical to granulosa cell function. Proteomic analysis reveals changes in pathways related to immune responses, metabolic perturbations, angiogenesis, and hormone regulation. Metabolomics identify disturbances in glucose metabolism, amino acids, lipid profiles, and steroid levels with PCOS FF. Collectively, these pathological alterations may adversely affect oocyte quality, embryo development, and fertility outcomes. Further research on larger cohorts is needed to validate these findings and to forge the development of prognostic biomarkers of oocyte developmental competence within FF. Characterizing the follicular environment in PCOS is key to elucidating the mechanisms underlying subfertility in this challenging disorder. [ABSTRACT FROM AUTHOR]

To examine the effects of clomiphene citrate (CC) on follicular fluid metabolites and related metabolic pathways in rats with polycystic ovary syndrome (PCOS) using non-targeted metabolomics and determine how CC treats ovulation disorder in PCOS. The Sprague Dawley rats were randomly divided into control, model, and CC groups. A PCOS model was established with letrozole. Body weight, ovarian weight, estrus cycles, serum hormone levels, and ovary histopathology of the rats were collected for further evaluation. Moreover, through ultra-performance liquid chromatography-mass spectrometry, the study of follicular fluid metabolites revealed the mechanism of action of CC. CC reduced ovarian weight and regulated estrous cycles and serum hormone levels in PCOS rats but did not affect their body weight. Moreover, the metabolomic results showed that CC adjusted 153 metabolites, among which 16 cross metabolites like testosterone, androstenedione, 17α-hydroxyprogesterone, and cholic acid were considered as potential biomarkers for CC to improve ovulation disorders in PCOS rats. Kyoto Encyclopedia of Genes and Genomes pathway enrichment also showed that the CC group mainly engaged in tryptophan metabolism and steroid hormone biosynthesis. CC can improve ovulation disorders in rats, and its mechanism is related to the regulation of the secretion of serum hormone and follicular fluid metabolites and the amelioration of multi-metabolic pathways. [ABSTRACT FROM AUTHOR]