Human Follicular Fluid Research Articles

Identification and validation of core genes associated with polycystic ovary syndrome and metabolic syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder affecting women of reproductive age, affecting reproductive health, and increasing the incidence of diabetes mellitus and hypertension. Metabolic syndrome (MetS) is the most common metabolic disorder. Although clinical studies have shown a close association between PCOS and MetS, the molecular mechanisms are unknown. In this study, datasets of PCOS and MetS were obtained from the Gene Expression Omnibus database; differential expression analysis and weighted gene coexpression network analysis (WGCNA) were performed; and gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also performed of differentially expressed genes (DEGs). The PCOS- and MetS-coexpressed DEGs were subsequently intersected with the coexpressed genes in the WGCNA module to obtain the core genes. By constructing receiver operating characteristic curves, we verified the predictive effects of the core genes. We also validated the expression of the core genes in the datasets. Finally, we verified the expression of the core genes by quantitative polymerase chain reaction in human follicular fluid granulosa cells. In addition, we used Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts to analyze the immune infiltration of immune cells in PCOS and MetS. Finally, we obtained 52 coexpressed DEGs of PCOS and MetS and 3 coexpressed genes in the WGCNA module. By taking the intersection of coexpressed DEGs and coexpressed genes of the WGCNA module, we get ELOVL fatty acid elongase 7 (ELOVL7) as the core gene. Receiver operating characteristic curve analysis showed that ELOVL7 is a reliable biological marker for PCOS and MetS. The expression level of ELOVL7 in human follicular fluid granulosa cells from PCOS patients was significantly higher than that of controls, as verified by quantitative polymerase chain reaction. This study provides the first evidence of the role of ELOVL7 in developing PCOS and MetS. This gene may serve as a potential diagnostic marker and therapeutic target for both conditions.

Benzo(a)pyrene exposure during pregnancy leads to germ cell apoptosis in male mice offspring via affecting histone modifications and oxidative stress levels

Infertility has gradually become a global health concern, and evidence suggests that exposure to environmental endocrine-disrupting chemicals (EDCs) represent one of the key causes of infertility. Benzo(a)pyrene (BaP) is a typical EDC that is widespread in the environment. Previous studies have detected BaP in human urine, semen, cervical mucus, oocytes and follicular fluid, resulting in reduced fertility and irreversible reproductive damage. However, the mechanisms underlying the effects of gestational BaP exposure on offspring fertility in male mice have not been fully explored. In this study, pregnant mice were administered BaP at doses of 0, 5, 10 and 20 mg/kg/day via gavage from Days 7.5 to 12.5 of gestation. The results revealed that BaP exposure during pregnancy disrupted the structural integrity of testicular tissue, causing a disorganized arrangement of spermatogenic cells, compromised sperm quality, elevated levels of histone modifications and increased apoptosis in the testicular tissue of F1 male mice. Furthermore, oxidative stress was also increased in the testicular tissue of F1 male mice. BaP activated the AhR/ERα signaling pathway, affected H3K4me3 expression and induced apoptosis in testicular tissue. AhR and Cyp1a1 were overexpressed, and the expression of key molecules in the antioxidant pathway, including Keap1 and Nrf2, was reduced. The combined effects of these molecules led to apoptosis in testicular tissues, damaging and compromising sperm quality. This impairment in testicular cells further contributed to compromised testicular tissues, ultimately impacting the reproductive health of F1 male mice.

Reduced ovarian cholesterol and steroid biosynthesis along with increased inflammation are associated with high DEHP metabolite levels in human ovarian follicular fluids

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 < 0.05). A decreased 17-OH-progesterone to progesterone ratio was observed in the FF of the high DEHP group (p < 0.05). Additionally, the expression levels of genes associated with inflammatory processes were elevated in the FF somatic cells, and a computational cell-type deconvolution analysis suggested an increased immune cell infiltration into the high DEHP follicles (p < 0.05).In conclusion, elevated DEHP levels in FF were associated with a significantly altered follicular milieu within human ovaries, involving a pro-inflammatory environment and reduced cholesterol metabolism, including steroid synthesis. These results contribute to our understanding of the molecular mechanisms of female reprotoxic effects of DEHP.

P-014 Extracellular vesicles (EVs) from female reproductive tracts positively affect sperm capacitation and progressive motility

Abstract Study question Do EVs from human follicular fluid (FF-EVs), cervicovaginal fluid (CVF-EVs), and endometrial stromal cells spent medium (ESCs-EVs) induce sperm capacitation and enhance sperm motility? Summary answer In humans, EVs from different female reproductive tracts actively contribute to sperm maturation by inducing capacitation and enhancing sperm progressive motility. What is known already In the human body, EVs represent crucial players in cell-to-cell communication. EVs presence has been detected also in the female and male reproductive systems, specifically in seminal, follicular, oviductal, endometrial and vaginal fluid, as well as in embryo secretions. Moreover, EVs have been shown to be of pivotal importance in the fertilization process, as they support the maturation of spermatozoa and oocytes, as well as embryos in their development and endometrial implantation. The majority of these studies, though, have been conducted in animal models, underscoring the necessity to replicate analogous investigations also in humans. Study design, size, duration Follicular fluid (FF, n = 8), cervicovaginal fluid (CVF, n = 20) and endometrial tissue (n = 8) samples were collected from women undergoing assisted reproductive technology (ART) cycles at San Raffaele Hospital Fertility Centre (Milan). CVF were collected 2 and 7 days after the LH peak (CVF LH + 2 and CVF LH + 7, respectively). ESCs-EVs were isolated from both decidualized (dESCs-EVs) and not decidualized (eESCs-EVs) endometrial cells. Semen samples (n = 15) were collected from normozoospermic men. Participants/materials, setting, methods EVs were isolated using differential centrifugations and characterized using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western blotting (WB). EVs capture by spermatozoa was assessed using flow cytometry. EVs impact on sperm progressive motility was evaluated following WHO guidelines. EVs potential to induce sperm capacitation was examined through sperm acrosome reaction (AR) induction using a Ca2+ ionophore, followed by Coomassie blue staining. All experiments were performed using EVs at 50 and 100 μg/mL. Main results and the role of chance Isolated EVs displayed comparable size, with mean diameters ranging from 147,9±11,6 nm to 168,1±6,2 nm (mean ± SEM) and average concentrations from 9,53E+10 ± 8,11E+09 to 2,70E+11 ± 3,49E+10 particles/ml (mean ± SEM). TEM analysis confirmed their round-shaped morphology and structural integrity, while WB confirmed their positivity for EVs markers (Alix, TSG101, CD63, CD9). Spermatozoa efficiency in capturing these fluorescently-labeled EVs, measured as the percentage of fluorescence-positive spermatozoa, reached up to 3,6±1,1% for FF-EVs, 12,1±3,7% for CVF-EVs and 9,4±1,9% for ESCs-EVs (mean ± SEM). Sperm cells co-incubated with any EVs group maintained unaffected vitality. Following a 4-hour incubation with EVs, a rise in the proportion of progressively motile spermatozoa was observed, with percentages of progressively motile spermatozoa ranging from a minimum of 45,4±3,8% for CVF-EVs LH + 7 to a maximum of 52,7±2,7% for dESCs-EVs (mean ± SEM). This increase resulted to be statistically significant (p &amp;lt; 0.05 and p &amp;lt; 0.01, respectively) compared to the untreated (UT) condition, which exhibited 26,2±0,7% of progressively motile sperm cells. Spermatozoa that completed the AR, indicating capacitation, after 4-hour incubation with EVs resulted to be up to 25,8±3,2% for FF-EVs, 35,0±6,6% for CVF-EVs and 26,9±3,0% for ESCs-EVs, compared to an average of 13% in the UT control. Limitations, reasons for caution This study constitutes a pilot investigation characterized by a limited sample size, necessitating larger cohorts to validate results. Wider implications of the findings Studied EVs may exert beneficial effects on capacitation and progressive motility also on asthenozoospermic (AS) men’s spermatozoa. Therefore, they could be used in ART cycles to prime spermatozoa from AS men and increase the percentage of sperm cells displaying progressive motility, potentially favouring fertilization via classical IVF instead of ICSI. Trial registration number not applicable

P-762 Discovery and quantification of microplastics in human cumulus granulosa cells

Abstract Study question The impact of microplastics (MPs) on human health has received increasing attention; their accumulation in female reproductive system and possible harms have not been quantified. Summary answer This study firstly reveals the contamination of human cumulus granulosa cells and follicular fluid by MPs and the effects of these contaminants on ART outcomes. What is known already MPs are ubiquitous in soil, sea, air, fresh water, and other environments. MPs are ingested by humans through the food chain, air and direct contact, resulting in various health problems, such as endocrine disruption, immune disturbance and reproductive toxicity. MPs exposure has been found to have a great impact on females, as MPs may accumulate in the ovaries and reduce the number of follicles, ovarian size, pregnancy rate, embryo production and fertility. To date, one study has found evidence of MPs in human placentas for the first time, but MPs contamination in other human reproductive systems has not been identified. Study design, size, duration The inclusion criteria comprised female patients of infertile couples excluding male factors who visited the outpatient department of the Reproductive and Genetic Hospital of CITIC-Xiangya between January 2019 and January 2021.We randomly selected 16 women who met the criteria and assessed their CCs and follicular fluid samples for the presence of MPs. Participants/materials, setting, methods In the present study, 16 cumulus granulosa cell samples and 2 follicular fluid samples were collected, and MPs were detected via pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). Main results and the role of chance MPs were detected in both cumulus granulosa cells and follicular fluid, with an average abundance of 313.14±246.69 mg/kg in cumulus granulosa cellsand 12.88±15.54 mg/kg in follicular fluid. Polyethylene terephthalate was the predominant polymer in cumulus granulosa cells. There were no significant differences in the IVF fertilization rates, ICSI fertilization rates, IVF good-quality embryo rates, or ICSI good-quality embryo rates between the high-MP group and low-MP group. However, embryos in the high-MP group exhibited a lower normal cleavage rate than did those in the low-MP group, while the embryos in the high-MP group exhibited a greater proportion of noncleavage than did those in the low-MP group. Limitations, reasons for caution It is not clear how long these particles may persist in the human body. The method we used can detect only the concentration of MPs but not the size of MP particles. Moreover, our method cannot be used to quantify smaller-diameter nanoplastics, which are considered to pose greater risks. Wider implications of the findings This is the first study in which MPs were identified and characterized in human ovarian granulosa cells, providing reference data for evaluating their toxicity to the human female reproductive system. Trial registration number Not applicable

O-073 CD163 in follicular fluids can be the novel marker to predict clinical outcome of oocyte

Abstract Study question Can we find novel marker in follicular fluid at the oocyte retrieval to evaluate oocyte condition besides karyotype by proteomics? Summary answer We found significant higher concentration of CD163 in the follicle fluids, from those oocytes resulted in pregnancy or live birth. What is known already Although preimplantation genetic testing for aneuploidy (PGT-A) is effective for embryo selection, only karyotype is considered in PGT-A. If we can evaluate other conditions of oocytes besides karyotype, the pregnancy rate can be improved. In addition to changes in the oocyte per se, factors affecting the quality of oocytes, such as ageing, change the microenvironment surrounding the oocyte. For example, it has been reported that the follicle itself becomes more fibrotic with age, resulting in increased oxidative stress, and that the concentration of cytokines, amino acids, fatty acids, and other substances in the follicle also changes. Study design, size, duration Proteomic analyses were performed on follicular fluid collected at the oocyte retrieval, and the proteins showed differences in relation to clinical outcome. We focused on one of those candidates, CD163, and evaluated the concentration by ELISA and checked for possible relevance to clinical outcome of the oocytes derived from each follicles. We also investigated the effect of this protein on oocyte maturation. Participants/materials, setting, methods Under the ethical review of Yokohama City University and informed consent with patients in Reproduction center of Yokohama City University Medical Center, we aspirated human follicular fluids individually at the oocyte retrieval. The oocytes were retrieved from the follicular fluid and remained fluid was cryopreserved for further analysis. We followed clinical outcome of oocyte from each follicle, compared the concentration of the protein in each follicle. Main results and the role of chance Proteomic analysis revealed CD163 in follicular fluids seemed to have relation with the clinical outcome. We collected 120 follicular fluid samples from 93 patients from 2017 to 2021, and evaluated the concentration of CD163 of them. Follicles which had contained oocytes derived are cryopreserved or fresh embryo transferred had higher than other follicles; 140.6 ± 73.1 vs 95.2 ± 30.7 ng/ml (mean ± SD, p &amp;lt; 0.01). Similarly, follicles, which had contained oocytes led to clinical pregnant and live birth after single embryo transfer, had higher concentrations of CD163; 140.5 ± 71.8 vs 101.6 ± 38.4 ng/ml (pregnant vs not pregnant, mean ± SD, p &amp;lt; 0.01), 138.7 ± 74.1 vs 109.2 ± 45.3 ng/ml (live birth vs not live birth, mean ± SD, p &amp;lt; 0.01). These results suggest that CD163 concentration in follicular fluid might be a novel marker of the clinical outcome. Limitations, reasons for caution Although we tried to investigate the relationship of CD163 and oocyte maturation, unfortunately we have not yet been able to clarify them. However, we believe that CD163 may act as an indirect marker representing other factors directly involved in oocyte maturation and development. We would like to investigate this further. Wider implications of the findings When CD163 concentration in follicular fluids is evaluated, we might be able to predict the clinical outcome of each oocyte at the day of oocyte retrieval. This may contribute to further improvement of pregnancy rate without invasion to embryo. Trial registration number the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research C (Grant Number 22K07920)

Follicular Fluid and Blood Monitorization of Infertility Biomarkers in Women with Endometriosis.

Infertility is recognized globally as a social disease and a growing medical condition, posing a significant challenge to modern reproductive health. Endometriosis, the third-most frequent gynecologic disorder, is one of the most common and intricate conditions that can lead to female infertility. Despite extensive research, the etiology, malignant transformation, and biological therapy of endometriosis remain unknown. Blood and follicular fluid are two matrices that have been carefully studied and can provide insights into women's health. These matrices are clinically significant because they contain metabolites closely associated with women's illness stage and reproductive outcomes. Nowadays, the application of metabolomic analysis in biological matrices may be able to predict the outcome of assisted reproductive technologies with greater precision. From a molecular viewpoint on reproductive health, we evaluate and compare the utilization of human follicular fluid and blood as matrices in analysis for diagnostic and assisted reproductive technology (ART) predictors of success for endometriosis patients. In the follicular fluid (FF), plasma, and serum of endometriosis-affected women, researchers identified dysregulations of oxidative stress, upregulation of several immune factors, and aberrations in energy metabolic pathways. The altered signatures negatively correlate with the overall oocyte and embryo quality and fertilization rate.

Systemic Effect of Human Follicular Fluid from Endometriotic and Healthy Subjects on Female Mice

Systemic Effect of Human Follicular Fluid from Endometriotic and Healthy Subjects on Female Mice

Increased Vasoactive Intestinal Peptide (VIP) in polycystic ovary syndrome patients undergoing IVF.

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. 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. 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. 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.

Consumption of hookahs, e-cigarettes, and classic cigarettes and the impact on medically assisted reproduction treatment

Smoking of classic cigarettes has been well-established as a health risk factor, including cardiovascular, neurological, and pulmonary diseases. Adverse effects on human reproduction have also been shown. Smokers are assumed to have a significantly lower chance of pregnancy, however, the impact of smoking on medically assisted reproduction (MAR) treatment outcomes is controversial. Moreover, smoking habits have changed during the last decades since e-cigarettes and hookahs, or water pipes, have become very popular, yet little is known regarding vaping or hookah-smoking patients undergoing MAR treatments. This prospective study aimed to examine the presence of benzo[a]pyrene, nicotine, and its main metabolite, cotinine, in human follicular fluid (FF) in non-smoking, smoking, and vaping/hookah-smoking patients and to evaluate the impact on female fertility. Human FF samples were collected from 320 women subjected to intracytoplasmic sperm injection (ICSI) cycles due to male subfertility. Gas chromatography combined with mass spectrometry was used to analyse the presence of benzo[a]pyrene, nicotine, and cotinine. A questionnaire was provided to assess patient consumption behaviour and to identify (1) non-smoking patients, (2) patients who consumed cigarettes, and (3) patients with exclusive consumption of e-cigarettes or hookahs. Data were analysed using linear and logistic regression, Fisher’s exact test, and the Mann–Whitney U Test. Nicotine was present in 22 (6.8%) and cotinine in 65 (20.3%) of the 320 samples. The nicotine and cotinine concentrations per sample ranged from 0 to 26.3 ng/ml and 0–363.0 ng/ml, respectively. Benzo[a]pyrene was not detectable in any of the samples analysed. Nicotine and cotinine were also present in the FF of patients with exclusive consumption of e-cigarettes or hookahs. The clinical pregnancy rate, fertilization and maturation rates, and number of oocytes per oocyte pick-up were not statistically significantly different between non-smoking, smoking, or vaping/hookah-smoking patients. Smoking and the accumulation of smoking toxins in the FF have no impact on the outcome of MAR treatments—neither the clinical pregnancy rate, maturation and fertilization rates, nor the number of retrieved oocytes were affected. For the first time, nicotine and cotinine were quantified in the FF of patients exclusively vaping e-cigarettes or smoking hookahs. Since vaping liquids and hookah tobaccos contain potentially harmful substances, other adverse effects cannot be excluded.Trial registration ClinicalTrials.gov Identifier: NCT03414567.

Fluorine-functionalized magnetic amino microporous organic network for enrichment of perfluoroalkyl substances

Perfluoroalkyl substances (PFAS) are persistent organic pollutants that pose significant risks to human health and the environment. Efficient and selective enrichment of these compounds was crucial for their accurate detection and quantification in complex matrices. Herein, we report a novel magnetic solid-phase extraction (MSPE) method using fluorine-functionalized magnetic amino-microporous organic network (Fe3O4@MONNH2@F7) adsorbent for the efficient enrichment of PFAS from aqueous samples. The core-shell Fe3O4@MONNH2@F7 nanosphere was synthesized, featuring magnetic Fe3O4 nanoparticles as the core and a porous amino-functionalized MONs coating as the shell, which was further modified by fluorination. The synthesized adsorbent material exhibited high specific surface area, hydrophobicity, and abundant fluorine groups, facilitating efficient and selective adsorption of PFAS via electrostatic attraction, hydrophobic-hydrophobic interactions, fluorine-fluorine interactions, π-CF interactions and hydrogen bonding. Furthermore, the MSPE method coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) allowed for the rapid, sensitive, and accurate determination of ultra-trace PFAS in real water samples, human serum, and human follicular fluid. Under optimal conditions, the established MSPE method demonstrated a linear range (2 to 2000 ng L−1), with a correlation coefficient exceeding 0.9977, low limits of detection ranging from 0.54 to 1.47 ng L−1, with a relative standard deviation (RSD) < 9.1%. Additionally, the method showed excellent performance in complex real samples (recovery ratio of 81.7 to 121.6 %). The adsorption mechanism was investigated through kinetic, isotherm, and molecular simulation studies, revealing that the introduction of fluorine groups enhanced the hydrophobic interaction and fluorine-fluorine attraction between the adsorbent and PFAS. This work provides a proof-of-concept strategy for designing adsorbent materials with high efficiency and selectivity by post-modification, which has great potential for the detection and analysis of PFAS in complex samples.

Former smoking associated with epigenetic modifications in human granulosa cells among women undergoing assisted reproduction

Smoking exposure during adulthood can disrupt oocyte development in women, contributing to infertility and possibly adverse birth outcomes. Some of these effects may be reflected in epigenome profiles in granulosa cells (GCs) in human follicular fluid. We compared the epigenetic modifications throughout the genome in GCs from women who were former (N = 15) versus never smokers (N = 44) undergoing assisted reproductive technologies (ART). This study included 59 women undergoing ART. Smoking history including time since quitting was determined by questionnaire. GCs were collected during oocyte retrieval and DNA methylation (DNAm) levels were profiled using the Infinium MethylationEPIC BeadChip. We performed an epigenome-wide association study with robust linear models, regressing DNAm level at individual loci on smoking status, adjusting for age, ovarian stimulation protocol, and three surrogate variables. We performed differentially methylated regions (DMRs) analysis and over-representation analysis of the identified CpGs and corresponding gene set. 81 CpGs were differentially methylated among former smokers compared to never smokers (FDR < 0.05). We identified 2 significant DMRs (KCNQ1 and RHBDD2). The former smoking-associated genes were enriched in oxytocin signaling, adrenergic signaling in cardiomyocytes, platelet activation, axon guidance, and chemokine signaling pathway. These epigenetic variations have been associated with inflammatory responses, reproductive outcomes, cancer development, neurodevelopmental disorder, and cardiometabolic health. Secondarily, we examined the relationships between time since quitting and DNAm at significant CpGs. We observed three CpGs in negative associations with the length of quitting smoking (p < 0.05), which were cg04254052 (KCNIP1), cg22875371 (OGDHL), and cg27289628 (LOC148145), while one in positive association, which was cg13487862 (PLXNB1). As a pilot study, we demonstrated epigenetic modifications associated with former smoking in GCs. The study is informative to potential biological pathways underlying the documented association between smoking and female infertility and biomarker discovery for smoking-associated reproductive outcomes.

Progestin and adipoQ receptor 7 (PAQR7) mediate the anti-apoptotic effect of P4 on human granulosa cells and its deficiency reduces ovarian function in female mice

PurposePAQR7 plays a key role in cell apoptosis as a progesterone membrane receptor. The physiological mechanism of PAQR7 in ovarian function and its anti-apoptotic action in mammals remain poorly understood.MethodsWe first added 0.2 µM aminoglutethimide (AG), an inhibitor of endogenous progesterone (P4) secretion, and transfected siPAQR7 co-incubated with P4 in human KGN cells to identify granulosa cell apoptosis, respectively. Additionally, we used Paqr7 knockout (PAQR7 KO) mice to assess the role of PAQR7 in the ovary.ResultsThe PAQR7 deficiency significantly increased apoptosis of KGN cells, and this significant difference disappeared following P4 supplementation. The Paqr7−/− female mice showed a prolonged estrous cycle, reduced follicular growth, increased the number of atresia follicles, and decreased the concentrations of E2 and AMH. The litters, litter sizes, and spontaneous ovulation in the Paqr7−/− mice were significantly decreased compared with the Paqr7+/+ mice. In addition, we also found low expression of PAQR7 in GCs from human follicular fluids of patients diagnosed with decreased ovarian reserve (DOR) and ovaries of mice with a DOR-like phenotype, respectively.ConclusionsThe present study has identified that PAQR7 is involved in mouse ovarian function and fertilization potential. One possible mechanism is mediating the anti-apoptotic effect of P4 on GC apoptosis via the BCL-2/BAX/CASPASE-3 signaling pathway. The mechanism underlying the effect of PAQR7 on ovarian development and aging remains to be identified.

MiR-6881-3p contributes to diminished ovarian reserve by regulating granulosa cell apoptosis by targeting SMAD4

BackgroundIn our previous investigation, we revealed a significant increase in the expression of microRNA-6881-3p (miR-6881-3p) in follicular fluid granulosa cells (GCs) from women with diminished ovarian reserve (DOR) compared to those with normal ovarian reserve (NOR). However, the role of miR-6881-3p in the development of DOR remains poorly understood.ObjectiveThis study aimed to elucidate the involvement of miR-6881-3p in the regulation of granulosa cells (GCs) function and the pathogenesis of DOR.Materials and methodsInitially, we assessed the expression levels of miR-6881-3p in GCs obtained from human follicular fluid in both NOR and DOR cases and explored the correlation between miR-6881-3p expression and clinical outcomes in assisted reproduction technology (ART). Bioinformatic predictions and dual-luciferase reporter assays were employed to identify the target gene of miR-6881-3p. Manipulation of miR-6881-3p expression was achieved through the transfection of KGN cells with miR-6881-3p mimics, inhibitor, and miRNA negative control (NC). Following transfection, we assessed granulosa cell apoptosis and cell cycle progression via flow cytometry and quantified target gene expression through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Finally, we examined the correlation between target gene expression levels in GCs from NOR and DOR patients and their association with ART outcomes.ResultsOur findings revealed elevated miR-6881-3p levels in GCs from DOR patients, which negatively correlated with ovarian reserve function and ART outcomes. We identified a direct binding interaction between miR-6881-3p and the 3’-untranslated region of the SMAD4. Transfection with miR-6881-3p mimics induced apoptosis in KGN cell. Furthermore, miR-6881-3p expression negatively correlated with both mRNA and protein levels of the SMAD4. The mRNA and protein levels of SMAD4 were notably reduced in GCs from DOR patients, and SMAD4 mRNA expression positively correlated with ART outcomes. In addition, the mRNA levels of FSHR, CYP11A1 were notably reduced after transfection with miR-6881-3p mimics in KGN cell, while LHCGR notably increased. The mRNA and protein levels of FSHR, CYP11A1 were notably reduced in GCs from DOR patients, while LHCGR notably increased.ConclusionThis study underscores the role of miR-6881-3p in directly targeting SMAD4 mRNA, subsequently diminishing granulosa cell viability and promoting apoptosis, and may affect steroid hormone regulation and gonadotropin signal reception in GCs. These findings contribute to our understanding of the pathogenesis of DOR.

Evaluation of the expression level of microRNA-21, microRNA-15a, microRNA-372 in human follicular fluid stem cells-derived oocyte-like cells (OLCs).

Today, researchers have succeeded in achieving oocyte-like cells through the in vitro differentiation of stem cells. MicroRNAs are key regulators of oocyte development. In this study we decided to evaluate the expression pattern of microRNA-21, microRNA-15a, and microRNA-372 in oocyte-like cells, to determine the maturation stage of oocyte-like cells. Human follicular fluid samples were collected and centrifuged, and their cells were divided into 3 groups; day 7 as control group, days 14 and 21. During this period, the cells were evaluated for their morphological appearance and viability by inverted microscopy. RNA isolation was performed and cDNA was reversely transcribed by specific stem-loop RT primers. Real-time RT-PCR was used to detect microRNA expression. The relative expression of microRNA-21 and microRNA-15a on day 21 was significantly down-regulated compared to the control group (day 7), but microRNA-372 did not show a significant difference. Also, on day 14 compared to the control group (day 7), microRNA-21 did not show a significant difference; but microRNA-15a and microRNA-372 were significantly down-regulated. MicroRNA-21 and microRNA-15a on day 21 compared to day 14 revealed down-regulated levels, but microRNA-372 revealed up-regulated levels. Our results showed significant decreases in the expression of microRNA-21 and microRNA-15a in oocyte-like cells, as well as in oocytes, which may lead to cytoplasmic maturation, germinal vesicle break down and the completion of meiosis І. In addition, down-regulation expression of microRNA-372 maybe a confirmation that mesenchymal stem cells have differentiated into germ cells, and these cells were differentiated into oocyte-like cells.

MiRNA profiling of granulosa cell-derived exosomes reveals their role in promoting follicle development.

To explore whether granulosa cell (GC)-derived exosomes (GC-Exos) and follicular fluid-derived exosomes (FF-Exos) have functional similarities in follicle development and to establish relevant experiments to validate whether GC-Exoscould serve as a potential substitute for follicular fluid-derived exosomes to improve folliculogenesis.GC-Exoswere characterized. MicroRNA (miRNA) profiles of exosomes from human GCs and follicular fluid were analyzed in depth. The signature was associated with folliculogenesis, such as phosphatidylinositol 3 kinases-protein kinase B signal pathway, mammalian target of rapamycin signal pathway, mitogen-activated protein kinase signal pathway, Wnt signal pathway, and cyclic adenosine monophosphate signal pathway. A total of five prominent miRNAs were found to regulate the above five signaling pathways. These miRNAs include miRNA-486-5p, miRNA-10b-5p, miRNA-100-5p, miRNA-99a-5p, and miRNA-21-5p. The exosomes from GCs and follicular fluid were investigated to explore the effect on folliculogenesis by injecting exosomes into older mice. The proportion of follicles at each stage is counted to help us understand folliculogenesis. Exosomes derived from GCs were isolated successfully. miRNA profiles demonstrated a remarkable overlap between the miRNA profiles of FF-Exos and GC-Exos. The shared miRNA signature exhibited a positive influence on follicle development and activation. Furthermore, exosomes derived from GCs and follicular fluid promoted folliculogenesis in older female mice. Exosomes derived from GCs had similar miRNA profiles and follicle-promoting functions as follicular fluid exosomes. Consequently, GC-Exos are promising for replacing FF-Exos and developing new commercial reagents to improve female fertility.

Extraction and preconcentration of parabens from the human follicular fluid through dispersive micro solid phase extraction using microporous MIL-68 (In) followed by in-situ effervescence-boosted dispersive liquid-liquid microextraction

For the first time in this study, a microextraction method was developed to perform follicular fluid safety assessment analysis. The drastic importance of follicular fluid safety on the proper nourishment and development of oocytes caused the development of the present method. Since women are regularly exposed to parabens through cosmetics, healthcare, and hygienic products, the infection of body fluids is probable in long-term exposures. Also, for the first time, MIL-68 (In) was applied in an analytical method. Moreover, a new method called in-situ effervescence-boosted dispersive liquid-liquid microextraction was adopted for the simultaneous derivatization and preconcentration of the target parabens. To perform the method, 25 mg of MIL-68 (In) was dispersed into the solution of follicular fluid by vortexing. Then, 1.0 mL of 2-propanol was used to elute the analytes from the absorbent via vortexing. The analyte-enriched organic phase was mixed with 100 µL of acetic anhydride (derivatization agent) and 27 µL 1,2-dibromoethane (extraction solvent) which was swiftly injected into a sodium carbonate solution. Following the centrifugation, the extraction solvent was sedimented at the bottom of a conical bottom glass test tube and an aliquot of it was injected into a gas chromatograph equipped with a flame ionization detector. Wide linear ranges (120–25000 µg L-1), satisfactory extraction recoveries (31–79%) and enrichment factors (31−79), and appreciable limits of detection (7–36 µg L-1) and quantification (25–120 µg L-1) were recorded. The high surface area of MIL-68 (In) (608.82 m2 g-1) and its significantly low average pore diameter (13.829 A°) provide an ideal platform for the extraction of parabens from the complex matrix of follicular fluid.

Bone morphogenetic protein-9 downregulates StAR expression by inducing snail expression via SMAD1/5/8 signaling in human granulosa-lutein cells

Ovarian steroidogenesis mediated by granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis. Bone morphogenetic protein-9 (BMP-9), also known as growth differentiation factor-2 (GDF-2), is a member of the transforming growth factor-beta (TGF-β) superfamily. BMP-9 induces epithelial-mesenchymal transition (EMT) that contributes to cancer progression. However, the function of BMP-9 in the female reproductive system remains largely unknown. It has been recently shown that BMP-9 is expressed in human follicular fluid and can downregulate StAR expression in human ovarian granulosa cells. However, the underlying molecular mechanisms warrant investigation. Our results show that treatment of primary granulosa-lutein (hGL) cells with BMP-9 downregulates StAR expression. In addition, two EMT-related transcription factors, Snail and Slug, are upregulated by the treatment of BMP-9. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we show that BMP-9 upregulates Snail and Slug expression by activating SMAD1/5/8 signaling. We also examine the effects of BMP-9 on SMAD-independent signaling pathways, including ERK1/2, p38, JNK, AKT, and CREB. However, none of them is affected by the BMP-9. Moreover, we use gain- and loss-of-function approaches to reveal that only Snail, not Slug, is required for the BMP-9-induced downregulation of StAR expression in hGL cells. This study increases the understanding of the physiology function of BMP-9 in hGL cells and provides important insights into the regulation of StAR expression.

PFOS and F–53B disrupted inner cell mass development in mouse preimplantation embryo

Perfluorooctane sulfonic acid (PFOS) is a perfluoroalkyl and polyfluoroalkyl substance (PFAS) widely used in daily life. As its toxicity was confirmed, it has been gradually substituted by F–53B (chlorinated polyfluoroalkyl sulfonates, Cl-PFESAs) in China. PFOS exposure during prenatal development may hinder the development of preimplantation embryos, as indicated by recent epidemiological research and in vivo assays. However, the embryotoxicity data for F–53B are scarce. Furthermore, knowledge about the toxicity of F–53B and PFOS exposure to internal follicular fluid concentrations on early preimplantation embryo development remains limited. In this study, internal exposure concentrations of PFOS (10 nM) and F–53B (2 nM) in human follicular fluid were chosen to study the effects of PFAS on early mouse preimplantation embryo development. We found that both PFOS and F–53B treated zygotes exhibited higher ROS activity in 8-cell embryos but not in 2-cell stage embryos. PFOS and F–53B significantly affected the proportion and aggregation of the inner cell mass (ICM) in the blastocyst, but not the total cell number. Mouse embryonic stem cells (mESCs, isolated from the ICM) and embryoid body (EB) assays were employed to assess the toxicity of PFOS and F–53B on the development and differentiation of embryonic pluripotent cells. These results suggested that mESCs exhibited more DNA damage and abnormal germ layer differentiation after brief exposure to PFOS or F–53B. Finally, RNA-sequencing revealed that PFOS and F–53B exposure affected mESCs biosynthetic processes and chromatin-nucleosome assembly. Our results indicate that F–53B has potential risks as an alternative to PFOS, which disrupts ICM development and differentiation.

Rheological characterization of human follicular fluid under shear and extensional stress conditions

The rheology of human follicular fluid has been empirically evinced to be related to the reproductive health status of individuals, which supports its use as an indicator for improving the success rates of in vitro fertilization. However, there is a dearth of studies investigating the viscoelastic properties of human follicular fluid. Moreover, a comprehensive elucidation of the rheological properties of complex fluids necessitates the assessment of data regarding both shear and extensional viscosities. Nonetheless, to the best of our knowledge, the extant literature does not include reports on the behavior of follicular fluid under extensional conditions. Consequently, this study aimed to analyze the shear and extensional viscosities of human follicular fluid. Primarily, the impact of oocytes on the rheology of follicular fluid was evaluated by measuring the shear viscosity of this fluid using a high-resolution coaxial cylinder viscometer. The shear viscosity of follicular fluid exhibited marked differences depending on the presence or absence of oocytes. Subsequently, a measurement system that enables the handling of minute quantities of body fluid was developed to determine the extensional viscosity of follicular fluid, which contains albumin. A comparison of the acquired follicular fluid data with that of the protein solution containing albumin demonstrated that the follicular fluid alone displayed extensional behavior, whereas the protein solution did not. Therefore, it can be inferred that the protein solution is not its sole determinant, as other constituents of the fluid, such as peptides and cumulus cells, may determine its rheological properties. This observation was not attained through the conventional technique consisting in shear viscosity measurements.