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Background: In mammals, female fertility is influenced by the result of follicular development (ovulation or atresia). Follicular atresia is a complex physiological process that results in the degeneration of oocytes from the ovary. However, the molecular mechanisms of oocyte degeneration and key protein markers of follicular atresia remain unclear. In this study, we investigated the complex transcriptional regulatory mechanisms and protein profiles in oocytes and follicular fluid in atretic follicle stages using single-cell RNA sequencing and tandem mass tag proteomics. Results: First, through paired analysis of different follicle development stages, we identified 175 atresia-specific genes and eight candidate oocyte-secreted factors, including PKG1, YTHDF2, and MYC. Meanwhile, we also characterized unique features of the oocyte transcriptional landscape in the atretic follicle stage that displayed cell death-related transcriptional changes and mechanisms, such as autophagy (TBK1 and IRS4), necroptosis (PKR), and apoptosis (MARCKS). Moreover, we identified atresia-specific genes, namely FTH1, TF, and ACSL4, which may participate in regulation of oocyte ferroptosis in atretic follicles through a series of mechanisms including ferritinophagy, ferritin transport, and lipid metabolism. Additionally, we uncovered 333 differentially expressed proteins that may coordinate follicular atresia and revealed key pathways, such as negative regulation of angiogenesis, metabolic pathways, and transcription and mRNA splicing, that lead to oocyte degeneration. Finally, by combining transcriptome and proteomics analyses, we identified two oocyte-secreted biomarkers, PGK1 and ANGPT2, that may be associated with follicular atresia. Conclusions: In conclusion, our work offers a thorough characterization of oocyte transcription mechanism and follicular fluid protein changes in ovine atretic follicles, which offers a crucial reference for analyzing the mechanism of follicular atresia and establishing an oocyte quality assessment system in sheep. Highlights: 1. Distinct transcription regulation and protein profiles exist in healthy and atretic follicles. 2. Ferroptosis-induced oocyte degeneration occurs in atretic follicles. 3. Identification of oocyte-secreted biomarkers is associated with follicular atresia. [ABSTRACT FROM AUTHOR]

Background: To a large extent, the ovarian reserve determines a woman's reproductive potential. The etiological and pathological mechanisms of diminished ovarian reserve (DOR) remain unclear, and no reliable treatment is currently available for DOR. Adipokines and cytokines in follicular fluid (FF) play pivotal roles in follicular development and maturation. The concentrations of adipokines and cytokines in FF from individual follicles of women with DOR undergoing in vitro fertilization (IVF) were studied. In particular, we investigated the associations between the levels of adipokines and cytokines in individual FFs from women with different ovarian reserves and between the follicular levels of adipokines and cytokines and IVF outcomes in individual follicles. Methods: A total of 115 women who underwent IVF were recruited. Patients diagnosed with DOR, defined as a basal antral follicle count < 5 or an anti-Mullerian hormone concentration < 1.1 ng/mL, were assigned to the DOR group, while patients with a normal ovarian reserve (NOR) were assigned to the NOR group. FF was sampled from the first follicle with a diameter of approximately 18–20 mm from each patient, and the IVF outcome of the oocyte from the corresponding follicle was tracked. The levels of 5 adipokines (including visfatin-1, monocyte chemoattractant protein-1 [MCP-1], resistin, leptin, and chemerin) and 3 cytokines (including interleukin [IL]-6, IL-12p70, and tumor necrosis factor [TNF]-α) in FF were determined by Luminex technology. Results: The follicular levels of TNF-α, IL-6, visafatin, MCP-1, IL-12, and chemerin were significantly lower in women with NOR than in those with DOR. The follicular level of IL-6 was negatively correlated with the quality of embryos according to the binary logistic regression analysis, while the follicular levels of adipokines and other cytokines did not correlate with IVF outcomes regardless of the woman's ovarian reserve. Conclusions: Our study demonstrated that the levels of adipokines and cytokines in individual follicles in women with DOR were different from those in women with NOR, indicating that increased intrafollicular inflammation might be related to DOR. Moreover, a high follicular level of IL-6 might negatively impact embryo quality. [ABSTRACT FROM AUTHOR]

Introduction: It is well acknowledged that lipids assume a critical role in oocyte maturation and early embryonic metabolism, this study aimed to evaluate the relationship between the lipid composition of plasma and follicular fluid (FF), and the consequences of embryonic development. This study compared the lipidomic profiles of paired plasma and FF samples obtained from sixty-five Chinese women who underwent assisted reproductive technology (ART) treatments. Methods: Non-targeted lipidomics analysis. Result: Results not only indicated similarities in lipid composition between these biofluids, but also revealed a number of unique differences. The biomatrix distinction was found to be primarily driven by lipids belonging to the lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), ether PE, and triglyceride (TG) classes. In addition, specific species from these subclasses were discovered to be correlated with embryo development outcomes during ART. Notably, the composition of the fatty acyl chains appeared to play a crucial role in these associations. Furthermore, thirteen plasma lipid variables were identified, represented by Phosphatidylcholine 18:014:0 and PE P-18:020:1, which correlated with successful blastocyst formation (BF). Discussion: The present study demonstrated that FF has a distinctive lipid composition, setting it apart from plasma; and the association observed with embryonic development underscored an important role of lipid composition in the healthy development of oocytes. [ABSTRACT FROM AUTHOR]

Background: To investigate the impact of Melatonin on follicular oxidative stress and assisted reproductive technology (ART) outcomes in women with diminished ovarian reserve (DOR). Method: We put 68 women with DOR who were going through ART into a randomized controlled trial. Starting on the fifth day of their menstrual cycle, we gave them either 3 mg of Melatonin or a placebo every day before stimulating their ovaries. We obtained follicular fluid during oocyte retrieval, assessed it for oxidative stress indicators, and documented ART outcomes. Results: Melatonin administration markedly enhanced the quantity of oocytes retrieved, fertilization rates, and embryo quality. In addition, Melatonin changed markers of oxidative stress, specifically the levels of reduced glutathione (rGSH) and total antioxidant capacity (TAC). The Melatonin group exhibited significantly elevated biochemical pregnancy rates. Conclusion: Melatonin may improve the quality of oocytes and help with reproductive technology in women with low ovarian reserves, possibly by lowering oxidative stress in the follicles. [ABSTRACT FROM AUTHOR]

The objective of this study was to measure the different redox biomarker levels within the follicular fluid (FF) and evaluate correlations with embryo quality using the one follicle–one oocyte/embryo approach. The prospective study included 54 women (average age 34.6 ± 3.0 years). Out of the 235 mature metaphase II cells that underwent intracytoplasmic sperm injection, fertilization was achieved in 177 cells, producing 92 Grade I embryos, 26 Grade II embryos, 39 Grade III embryos, and 20 Grade IV embryos. The activities of antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and glutathione transferase were significantly higher in the group consisting of lower-quality (Grades II–IV) embryos in comparison with top-quality (Grade I) embryos (p = 0.011; p = 0.021; p = 0.008, respectively). The concentration of oxidative stress markers, malondialdehyde, 8-hydroxy-2′-deoxyguanosine, and thiol groups was significantly increased in the group with lower-quality embryos (Grades II–IV) compared to top-quality embryos (0.027; 0.018; 0.021, respectively). Furthermore, a significant positive correlation between each oxidative marker and the activities of antioxidant enzymes was observed (p < 0.001). According to our findings, the best embryos and, consequently, better in vitro fertilization outcomes are linked to low levels of oxidative stress and low antioxidant enzyme activity. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What molecular mechanisms underlie the decline in ovarian reserve as the number and quality of oocytes decrease in patients with ovarian endometriomas (OEM)? SUMMARY ANSWER Elevated expression of the let-7 micro(mi)RNAs in the follicular microenvironment of OEM-affected ovaries targets the expression of type 1 insulin-like growth factor receptor (IGF1R) in granulosa cell (GC) and disrupts their proliferation, steroid hormone secretion levels, adenosine triphosphate (ATP) energy metabolism, and reactive oxygen species (ROS) oxidative stress levels. WHAT IS KNOWN ALREADY Patients with OEM exhibit diminished ovarian reserve, characterized by reduced oocyte quantity and quality. Fibrotic changes in the ovarian tissue surrounding the OEM create a disruptive microenvironment for follicular growth and development. STUDY DESIGN, SIZE, DURATION This is a cross-sectional study aimed to elucidate the molecular mechanisms underlying the impact of OEM on follicular development. Initially, miRNA expression profiles in follicular fluid (FF) samples were sequenced from patients with infertility related to OEM (N   =   3) and male factor (MF) infertility (N   =   3), with the latter serving as the control group. Differentially expressed miRNAs were validated in additional samples from each group (N   =   55 in OEM group and N   =   45 in MF group) to confirm candidate miRNAs. The study also investigated indicators associated with GCs dysfunction in vitro on rat GCs. Subsequently, rat models of OEM were established through endometrial allogeneic transplantation, and fertility experiments were conducted to assess the let-7/ IGF1R axis response to OEM in vivo. Patient samples were collected between May 2018 and April 2019, and the mechanistic study was conducted over the subsequent three years. PARTICIPANTS/MATERIALS, SETTING, METHODS FF and GC samples were obtained from infertile patients undergoing IVF treatment for OEM and MF related infertility. miRNA expression profiles in FF samples were analyzed using second-generation high-throughput sequencing technology, and candidate miRNAs were validated through quantitative PCR (qPCR). In the in vitro experiments conducted with rat GCs, cell proliferation was assessed using the CCK-8 assay, while steroid hormone concentrations were measured using chemiluminescence. ATP content was determined with an ATP assay kit, and levels of ROS were quantified using flow cytometry. A dual luciferase reporter gene assay was employed to identify the target gene of let-7 based on the construction of a IGF1R reporter gene plasmid using 293T cells. Western blotting was utilized to evaluate the expression of IGF1R in GCs, as well as its downstream proteins, and changes in signaling pathways following let-7 agomir/antagomir transfection and/or Igf1r silencing. In the in vivo OEM rat models, alterations in ovarian structure and cyst morphology were observed using hematoxylin and eosin staining. The expressions of let-7 and Igf1r in GCs were evaluated through qPCR, while variations in IGF1R expression were investigated with immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE The cohort of patients with ovarian OEM in this study exhibited significantly decreased antral follicle counts, oocyte retrieval numbers, and normal fertilization rates compared to the control group with MF. The expression of the let-7 miRNA family was markedly upregulated in the FF and GCs of OEM patients. Transfection of rat GCs with let-7 agonists diminished the functions of GCs, including disrupted cell proliferation, mitochondrial oxidative phosphorylation, and steroid hormone secretion, while transfection of rat GCs with let-7 antagonists caused the opposite effects. Luciferase reporter gene experiments confirmed that let-7 complementarily bound to the 3′-untranslated regions of IGF1R. Stimulation of let-7 expression in rat GCs led to a significant decrease in IGF1R expression, while inhibition of let-7 increased IGF1R expression. The expression of IGF1R in the GCs of OEM patients was also significantly reduced compared to MF patients. Silencing of Igf1r led to the dysfunction of GCs, similar to the effects of let-7 agonization, as demonstrated by the downregulation of key proteins involved in cell proliferation (CCND2 and CCND3) and oestradiol synthesis, as well as an increase in progesterone synthesis (StAR), while implicating the PI3K-Akt and MAPK signaling pathways. The antagonistic effect of let-7 on GCs was ineffective when Igf1r was silenced. Conversely, the agonistic effect of let-7 on GCs could be reversed by stimulation with the IGF1R ligand IGF-1. These findings suggested that let-7 regulated the proliferation, differentiation, and ATP synthesis of GCs through targeting IGF1R. The OEM rat model demonstrated alterations in ovarian morphology and structure, along with reduced fertility. Let-7 expression was significantly upregulated in GCs of OEM rats compared to normal rats, while Igf1r and IGF1R expression in pre-ovulatory follicular GCs were notably downregulated, supporting the notion that elevated let-7 expression in the follicular microenvironment of OEM inhibited IGF1R, leading to abnormal GC function and impacting fertility at the molecular level. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION The synthesis and secretion mechanisms of steroid hormones are intricate and complex. Some enzymes that regulate oestrogen synthesis also play a role in progesterone synthesis. Moreover, certain receptors can respond to multiple hormone signals. Therefore, in this study, the expression patterns of key enzymes such as CYP17A, CYP11A1, HSD3B2, StAR, and receptors including AR, LHCGR, FSHR, ESR2, might be influenced by various factors and might not demonstrate complete consistency. WIDER IMPLICATIONS OF THE FINDINGS Future research will concentrate on investigating the potential impact of ovarian stromal cells on the external microenvironment of follicle growth. Additionally, screening for small molecule drugs that target let-7 and IGF1R actions can be conducted to intervene and modify the ovarian microenvironment, ultimately enhancing ovarian function. STUDY FUNDING/COMPETING INTEREST(S) This study received funding from the National Natural Science Foundation of China (grant number 82301851 to L.B.S. grant numbers U23A20403 and U20A20349 to S.Y.Z. and grant number 82371637 to Y.D.D.) and the Natural Science Foundation of Zhejiang Province (grant LTGY23H040010 to F.Z.). The authors have no conflicts of interest to declare. [ABSTRACT FROM AUTHOR]

ABSTRACT: Objectives: The optimization of polyethylene glycol (PEG)-based extracellular vesicles (EVs) extraction from human follicular fluid (FF) and serum was investigated, and their functional analysis was confirmed. The PEG-based EV results were compared to the ExoQuick (ExoQ)-based EV. Materials and Methods: FF-EVs and serum-EVs were extracted by using different concentrations of PEG (8000). Nanoparticle tracking analysis was used to count the particles, and electron microscopy of EVs was performed for visualization. Exosomes were confirmed by the western blot analysis with exosome-specific markers. RNA and microRNA were extracted from exosomes and quantitative polymerase chain reaction analysis was performed. Fallopian tube epithelial (FTE) cells were used for the EV uptake experiment and an anchorage-independent growth test to confirm that extracted EVs harbor transformation activity. Results: The PEG 8% enriched method produced the highest yield and the lowest carry-over protein. Salt containing PEG 8% produced a higher yield than nonsalted PEG 8%. Overnight enrichment increased four times and 18 times for PEG 8% and ExoQ-based EV extraction from FF. For serum EV, the same overnight enrichment moderately increased yield for both PEG 8% and ExoQ methods. Less carry-over protein resulted in more EV-promoted transformation activity. Conclusion: This study overcomes the time-consuming, expensive, laborious, and complicated machine-dependent EV extraction methods. The study highlights that longer incubation time is needed for EV extraction from FF. PEG 8000-based EV extraction provided a higher yield and less carry-over protein than ExoQ-based EV extraction. [ABSTRACT FROM AUTHOR]

(1) Background: The RoXstaTM system has been developed as a rapid, effective means of profiling different types of antioxidant activity. The purpose of this study was to examine its performance utilizing a diverse array of biological fluids including semen, blood plasma, serum, urine, saliva, follicular fluid and plant extracts. (2) Methods: The RoXstaTM system was used to assess the ability of different fluids to suppress free radical formation as well as scavenge a variety of toxic oxygen metabolites including free radicals and both hydrogen and organic peroxides. (3) Results: Human semen was shown to have significantly (p < 0.001) more peroxide scavenging power than any other fluid tested (10–14 mM vitamin C equivalent compared with 1–2 mM for blood serum or plasma), while urine was particularly effective in scavenging free radicals and preventing free radical formation (p < 0.001). The powerful antioxidant properties of human semen were shown to reside within the seminal plasma (SP) fraction, rather than the spermatozoa, and to be resistant to snap freezing in liquid nitrogen. Moreover, comparative studies demonstrated that human SP exhibited significantly (p < 0.001) higher levels of antioxidant potential than any other species examined (stallion, bull, dog) and that this intense activity reflected the relative vulnerability of human spermatozoa to peroxide attack. (4) Conclusions: The RoXstaTM system provides valuable information on the antioxidant profile of complex biological fluids, supporting its diagnostic role in conditions associated with oxidative stress. Based on the results secured in this study, human semen is identified as a particularly rich source of antioxidants capable of scavenging both hydrogen and organic peroxides, in keeping with the high susceptibility of human spermatozoa to peroxide-mediated damage. [ABSTRACT FROM AUTHOR]

Endometriosis is a chronic inflammatory condition characterized by the presence of endometrium‐like tissue outside the uterus, primarily affecting pelvic organs and tissues. In this study, we explored platelet activation in endometriosis. We utilized the STRING database to analyze the functional interactions among proteins previously identified in small extracellular vesicles (EVs) isolated from the peritoneal fluid of endometriosis patients and controls. The bioinformatic analysis indicated enriched signaling pathways related to platelet activation, hemostasis, and neutrophil degranulation. Double immunohistochemistry analysis for CD61 and MPO revealed a significant presence of neutrophils and platelets in close contact infiltrating endometriotic lesions, suggesting potential cell–cell interactions. Subsequently, we isolated small EVs from the peritoneal fluid of women diagnosed with endometriosis and from women without endometriosis who underwent surgery for non‐inflammatory benign diseases. We performed single‐particle phenotyping analysis based on platelet biomarkers GPIIb/IIIa and PF4 using nanoflow cytometry, as well as single‐particle morphological and nanomechanical characterization through atomic force microscopy. The study demonstrated that patients with endometriosis had a notably higher proportion of particles testing positive for platelet biomarkers compared to the total number of EVs. This finding implies a potential role for platelets in the pathogenesis of endometriosis. Further research is necessary to delve into the mechanisms underlying this phenomenon and its implications for disease progression. [ABSTRACT FROM AUTHOR]

AbstractPolycystic ovary syndrome (PCOS) is a complex multifactorial endocrinopathy affecting reproductive aged women globally, whose presentation is strongly influenced by genetic makeup, ethnic, and geographic diversity leaving these affected women substantially predisposed to reproductive and metabolic perturbations. Sophisticated techniques spanning genomics, proteomics, epigenomics, and transcriptomics have been harnessed to comprehensively understand the enigmatic pathophysiology of PCOS, however, conclusive markers for PCOS are still lacking today. Metabolomics represents a paradigm shift in biotechnological advances enabling the simultaneous identification and quantification of metabolites and the use of this approach has added yet another dimension to help unravel the strong metabolic component of PCOS. Reports dissecting the metabolic signature of PCOS have revealed disparate levels of metabolites such as pyruvate, lactate, triglycerides, free fatty acids, carnitines, branched chain and essential amino acids, and steroid intermediates in major biological compartments. These metabolites have been shown to be altered in women with PCOS overall, after phenotypic subgrouping, in animal models of PCOS, and also following therapeutic intervention. This review seeks to supplement previous reviews by highlighting the aforementioned aspects and to provide easy, coherent and elementary access to significant findings and emerging trends. This will in turn help to delineate the metabolic plot in women with PCOS in various biological compartments including plasma, urine, follicular microenvironment, and gut. This may pave the way to design additional studies on the quest of unraveling the etiology of PCOS and delving into novel biomarkers for its diagnosis, prognosis and management. [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. PLAIN LANGUAGE SUMMARY: Follicular fluid (FF) is a complex microenvironment involved in oocyte growth, follicular maturation and germ cell–somatic cell communication. All metabolites during oocyte growth are collected from the FF. This study used lipidomic analysis to identify differences in FF lipids between normal women and those diagnosed with polycystic ovary syndrome (PCOS). The pathogenesis of PCOS is associated with abnormal metabolism of glyceroglycolipids and sphingomyelin. Here, we found that phosphatidylcholine is the main abnormal lipid in FF in patients with PCOS. Our study informs the future research into the development of diagnostic markers for PCOS to be used in clinical practice. [ABSTRACT FROM AUTHOR]

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]

Mono-butyl phthalate (MBP), the metabolite of dibutyl phthalate (DBP), is the most abundant phthalate metabolite found in Chinese women. Extracellular vesicles (EVs) are nanoscale lipid bilayer particles produced by extensive kinds of cells, serving a key role in intercellular communication. Extracellular vesicle miRNAs (EV-miRNAs) in follicular fluid (FF) have been evidenced to be associated with female reproductive health. The objective of this study was to investigate the associations of EV-miRNAs expressed profile with DBP exposure in FF of female participants and expose its potential mechanism in impaired oocyte development. Based on participants' FF MBP concentrations and fertilization status, we compared the miRNA expression between the FF-EVs of group A (high DBP exposure and impaired fertilization) and group B (low DBP exposure and normal fertilization). Compared with group B, miR-1246, miR-3679-5p, miR-423-5p, miR-5585-3p, miR-116-5p, miR-172-5p were upregulated, while miR-34b-3p was downregulated in group A. Target genes of the differently expressed miRNAs were predicted, and the functional analysis was performed. Furthermore, we exposed human ovarian granulosa tumor cell line (KGN) to MBP (4ug/L) to isolate the EVs from the culture medium and validated the expression levels of different miRNAs. We found that MBP exposure was significantly associated with increased levels of miR-116-5p (P = 0.01). In addition, we demonstrated that the most different miRNA, miR-116-5p regulated oocyte fertilization by inhibiting FOXO3a. Our findings suggested that EV-miRNAs in the FF might mediate MBP toxicity in oocytes. [ABSTRACT FROM AUTHOR]

The astringent selection criteria for milk-oriented traits in dairy cattle have rendered these animals prone to various metabolic disorders. Postpartum lactational peak and reduced feed intake lead to negative energy balance in cattle. As a compensatory mechanism, cattle start mobilizing fat reserves to meet the energy demand for vital body functions. Consequently, diminished glucose concentrations and elevated ketone body levels lead to poor ovarian function. The impaired follicular development and subpar oocyte quality diminish the conception rates, which poses significant economic repercussions. Follicular fluid is integral to the processes of follicular growth and oocyte development. Hence, the present study was performed to identify potential alterations in metabolites in the follicular fluid under in vitro culture conditions mimicking negative energy balance. Our results revealed nine distinct metabolites exhibiting differential expression in follicular fluid under negative energy balance. The differentially expressed metabolites were predominantly associated with pathways related to amino acid metabolism, lipid metabolism, signal transduction mechanisms, and membrane transport, alongside other biological processes. The identified signature metabolites may be further validated to determine oocyte fitness subjected to in vitro fertilization or embryo production from slaughterhouse source ovaries. [ABSTRACT FROM AUTHOR]

This study aims to investigate alterations in immune cell counts within preovulatory follicles of patients with poor ovarian response (POR) during assisted reproductive technology (ART), classified according to the POSEIDON criteria. Methods: This single-centre cross-sectional study included 543 women undergoing IVF/ICSI treatment, selected based on specific inclusion and exclusion criteria: 292 with normal ovarian response and 251 with poor response. Follicular fluid (FF) was collected on the day of oocyte retrieval and analysed by flow cytometry to determine the proportions of macrophages (Ms), M1 and M2 Ms, T cells (CD4 and CD8 T cells), dendritic cells (DCs), including type 1 conventional dendritic cells (cDC1) and type 2 conventional dendritic cells (cDC2), and neutrophils. Multivariable logistic regression assessed the relationship between immune cell counts and POR, Pearson correlation determined associations with the number of retrieved oocytes, and receiver operating characteristic (ROC) curves evaluated the predictive power of immune cell counts for POR. Results: Immune cells accounted for 52.57% (± 23.90%) of the total cell population in the follicular microenvironment, which was approximately equal to that of granulosa cells, with Ms being the most abundant, followed sequentially by T cells, DCs, and neutrophils. In patients with POR, overall Ms infiltration in the follicular microenvironment decreased, whereas M1 and M2 polarization increased. T cell infiltration increased, with a decrease in the CD4/CD8 ratio. Both cDC1 and cDC2 were significantly elevated. Moreover, multivariable logistic regression revealed that the total macrophage count, CD4 T cell count, and cDC2 count were independent predictors of POR. Notably, cDC2 showed the largest area under the ROC curve, suggesting its strong potential as a biomarker for predicting POR. Conclusion: The proportion of immune cells in preovulatory follicles were significantly altered in patients with POR. These findings suggest that immune cell dynamics in the follicular microenvironment may play a crucial role in determining ovarian response and prognosis, indicating that targeted immunomodulatory strategies could be considered in future therapeutic approaches. [ABSTRACT FROM AUTHOR]

Objectives: The combination of growth hormone (GH) with gonadotropin was a prevalent method to improve clinical reproduction in adjuvant for assisted reproduction treatment (ART). However, the contradictory results from previous studies failed to confirm the benefits. The present study is focused on the mechanism analysis of GH-IGF1-gonadal axis in ART and the changes of IGF1 in follicular fluid among different types of patients. Material and methods: We recruited 136 patients and divided them into eight groups according to their ages and ovarian reserves. The baseline characteristics of the study population were summarized. The therapeutic outcomes in the study population were observed. In the meantime, concentrations of IGF1 in follicular fluids from different types of patients who underwent GH strategy were measured by Western blot. The functional mechanism of GH-IGF1-gonadal axis in ART was also analyzed. Results: We analyzed the baseline characteristics of the study population, the therapeutic outcome of GH-IGF-1-gonadal axis, as well as the relative protein level of IGF1 and IGFBP1 in follicular fluid from different groups. The chemical pregnancy rate was significantly increased in different degrees for groups with GH co-treatment compared to groups without GH co-treatment. The IGF1 in follicular fluid of patients under 35 years' old showed an upward trend compared with groups of poor, normal and high ovarian reserves. After GH induction, IGF1 in follicular fluid was significantly increased in patients over 35 years old. Conclusions: The study suggested that the application of GH might be beneficial to the pregnancy outcome in patients. GH application in patients older than 35 years might have a beneficial effect on pregnancy outcome via promoting the expression of IGF1. Our study indicates a different mechanism from GH application among younger and older patient in ART and provides a new clue for individual clinical treatment in infernity patients. [ABSTRACT FROM AUTHOR]

Objectives: The optimization of polyethylene glycol (PEG)-based extracellular vesicles (EVs) extraction from human follicular fluid (FF) and serum was investigated, and their functional analysis was confirmed. The PEG-based EV results were compared to the ExoQuick (ExoQ)-based EV. Materials and Methods: FF-EVs and serum-EVs were extracted by using different concentrations of PEG (8000). Nanoparticle tracking analysis was used to count the particles, and electron microscopy of EVs was performed for visualization. Exosomes were confirmed by the western blot analysis with exosome-specific markers. RNA and microRNA were extracted from exosomes and quantitative polymerase chain reaction analysis was performed. Fallopian tube epithelial (FTE) cells were used for the EV uptake experiment and an anchorage-independent growth test to confirm that extracted EVs harbor transformation activity. Results: The PEG 8% enriched method produced the highest yield and the lowest carry-over protein. Salt containing PEG 8% produced a higher yield than nonsalted PEG 8%. Overnight enrichment increased four times and 18 times for PEG 8% and ExoQ-based EV extraction from FF. For serum EV, the same overnight enrichment moderately increased yield for both PEG 8% and ExoQ methods. Less carry-over protein resulted in more EV-promoted transformation activity. Conclusion: This study overcomes the time-consuming, expensive, laborious, and complicated machine-dependent EV extraction methods. The study highlights that longer incubation time is needed for EV extraction from FF. PEG 8000-based EV extraction provided a higher yield and less carry-over protein than ExoQ-based EV extraction.

Abstract Follicular fluid (FF) plays a vital role in the bidirectional communication between oocytes and granulosa cells (GCs), regulating and promoting oocyte growth and development. This fluid constitutes a complex microenvironment, rich in various molecules including hormones, growth factors, cytokines, lipids, proteins, and extracellular vesicles. Understanding the composition and metabolic profile of follicular fluid is important for investigating ovarian pathologies such as polycystic ovary syndrome (PCOS) and endometriosis. Additionally, analyzing follicular fluid can offer valuable insights into oocyte quality, aiding in optimal oocyte selection for in vitro fertilization (IVF). This review provides an overview of follicular fluid composition, classification of its components and discusses the influential components of oocyte development. It also highlights the role of follicular fluid in the pathogenesis and diagnosis of ovarian diseases, along with potential follicular fluid biomarkers for assessing oocyte quality. By understanding the intricate relationship between follicular fluid and oocyte development, we can advance fertility research and improve clinical outcomes for infertility patients.

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.

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.

Follicular fluid (FF) plays a vital role in the bidirectional communication between oocytes and granulosa cells (GCs), regulating and promoting oocyte growth and development. This fluid constitutes a complex microenvironment, rich in various molecules including hormones, growth factors, cytokines, lipids, proteins, and extracellular vesicles. Understanding the composition and metabolic profile of follicular fluid is important for investigating ovarian pathologies such as polycystic ovary syndrome (PCOS) and endometriosis. Additionally, analyzing follicular fluid can offer valuable insights into oocyte quality, aiding in optimal oocyte selection for in vitro fertilization (IVF). This review provides an overview of follicular fluid composition, classification of its components and discusses the influential components of oocyte development. It also highlights the role of follicular fluid in the pathogenesis and diagnosis of ovarian diseases, along with potential follicular fluid biomarkers for assessing oocyte quality. By understanding the intricate relationship between follicular fluid and oocyte development, we can advance fertility research and improve clinical outcomes for infertility patients. [ABSTRACT FROM AUTHOR]

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: In vitro maturation (IVM) is a promising technique in assisted reproductive technologies, offering benefits such as reducing the risk of ovarian hyperstimulation syndrome. Objective: This study aimed to evaluate the effects of timed follicular fluid meiosis-activating sterol (FF-MAS) supplementation on the IVM of germinal vesicle oocytes using a dynamic microfluidic system. Materials and Methods: In this lab trial study, 266 germinal vesicle oocytes were collected from the Infertility Center of Fatemieh hospital, Hamedan, Iran between June 2023 and January 2024. The oocytes were allocated into 3 groups for dynamic microfluidic culture. Each group received culture medium at a flow rate of 0.36 µL/min for 24 hr through inlet A and FF-MAS supplementation through inlet B for 1, 2, and 6 hr. The study evaluated maturation and fertilization rates, embryo development, and mitochondrial status, which was assessed using the JC-1 mitochondrial membrane potential assay. Results: Maturation rates were significantly higher in the medium-term FF-MAS exposure (MTG) and long-term FF-MAS exposure groups compared to the short-term FF-MAS group (STG) (p < 0.05). Fertilization rates were also higher in the MTG and long-term FF-MAS group compared to the STG (p <0.05). Embryo formation rates and the proportion of good-quality embryos were higher in the MTG compared to the STG (100% vs. 75%; p = 0.03) and (83.3% vs. 33.3%; p = 0.01), respectively. Mitochondrial peripheral distribution was significantly higher in the MTG than in the STG (p = 0.04). Conclusion: Optimizing FF-MAS exposure duration enhances IVM efficiency, offering a promising strategy to increase oocyte utilization in in vitro fertilization programs. [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]

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]

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]

BackgroundMany components in follicular fluid (FF), such as peptide hormones, cytokines, and steroids, undergo dynamic changes during folliculogenesis and have important roles in follicular development. Because systemic inflammation has also been found to contribute to diminished ovarian reserve (DOR) in previous studies, do certain serum/FF inflammatory biomarkers affect both follicular development and ovarian function?MethodsSerum samples from the menstruation phase (n=26), serum samples from the ovulation phase (n=26), FF samples of mature oocytes (n=26), and FF samples of immature oocytes (n=10) were collected. Olink proteomic proximity extension assay (PEA) technology was used to compare the differentially expressed proteins (DEPs), and patients were divided into two subgroups—the normal ovarian reserve (NOR) group and the DOR group—for further bioinformatics analysis and verification by enzyme-linked immunosorbent assay (ELISA).ResultsIn total, 16 DEPs were detected between the mature group and the immature group (FF), and 11 DEPs were detected between the ovulation group and the menstruation group (serum). Further subdivision of the ovarian reserve subgroups revealed 22 DEPs in FF and 3 DEPs in serum. Among all four comparisons, only the expression of oncostatin M (OSM) significantly differed. The OSM signaling pathway, the IL-10 anti-inflammatory signaling pathway, and the PI3K−Akt signaling pathway are three notable pathways involved in affecting ovarian reserve capacity according to bioinformatics analysis. In addition, the concentration of estradiol on the hCG day was slightly but positively correlated with OSM (r=0.457, P=0.029). A significantly greater level of OSM (5.41 ± 2.65 vs. 3.94 ± 1.23 pg/mL, P=0.007) was detected in the serum of NOR patients via ELISA verification, and the sensitivity and specificity of ovarian reserve division were 50.00% and 83.33%, respectively.ConclusionThis study proposed that immunological changes assessed by PEA technology affect ovarian function in humans and that OSM may serve as a potential inflammatory biomarker for ovarian function in serum, thus revealing alterations in FF.

ABSTRACT Follicular fluid is a haven that provides a microenvironment for the development and maturation of oocytes. It is a medium that provides a means of communication within the follicular cells. The role of follicular fluid in the development and maturation of follicles and oocytes involves the actions of certain genes. Here, we examined the impact of CFF on the gene expression of camel oocytes during (IVM). Hypothetically, IVM medium was enhanced with 2.5% CFF, and we analyzed the expression of several genes including Bax, Bcl-2, P53, Acta2, TGFβ, Cx43, and Tagln. Consequently, the follicular fluid from camels had a substantial effect on the mRNA transcript level of the BCL2 gene, which is related to apoptosis. Acta2 and Tagln genes that relate to cytoskeletal protein “actin” in transforming growth factor beta (TGFβ) pathway were also modulated. However, the follicular fluid increased the expression of TGFβ in camel oocytes. This study showcases the beneficial effects of CFF on camel oocyte Gene Expression and provides a platform for studying the many mechanisms involved in oocyte meiosis, particularly those associated with the TGFβ pathway. We recommend using higher dosage and measuring the expression levels of certain genes involved in programmed cell death and inflammation.

Introduction: Epidemiological studies have implicated ovulation as a risk factor for ovarian high-grade serous carcinoma (HGSC) at the initiation stage. Precancerous lesions of HGSC commonly exhibit TP53 mutations attributed to DNA deamination and are frequently localized in the fallopian tube epithelium (FTE), a site regularly exposed to ovulatory follicular fluid (FF). This study aimed to assess the mutagenic potential of FF and investigate the expression levels and functional role of activation-induced cytidine deaminase (AID) following ovulation, along with the resulting TP53 DNA deamination. Methods: The mutagenic activity of FF toward premalignant and malignant FTE cells was determined using the hypoxanthine phosphoribosyl transferase (HPRT) mutation assay with or without AID knockdown. The sequential activation of AID, including expressional induction, nuclear localization, DNA binding, and deamination, was determined. AID inducers in FF were identified, and the times of action and signaling pathways were determined. Results: FF induced AID activation and de novo FTE cell mutagenesis in two waves of activity in accordance with post-ovulation FF exposure. The ERK-mediated early activity started at 2 min and peaked at 45 min, and the NF-κB-mediated late activity started at 6 h and peaked at 8.5 h after exposure. ROS, TNF-α, and estradiol, which are abundant in FF, all induced the two activities, while all activities were abolished by antioxidant cotreatment. AID physically bound to and biochemically deaminated the TP53 gene, regardless of known mutational hotspots. It did not act on other prevalent tumor-suppressor genes of HGSC. Conclusion: This study revealed the ROS-dependent AID-mediated mutagenic activity of the ovulatory FF. The results filled up the missing link between ovulation and the initial TP53 mutation and invited a strategy of antioxidation in prevention of HGSC.

Background: Beta-oxidation of fatty acids takes place in the mitochondria to produce energy. This process is linked to the formation of free radicals. Previous researches propose that some fatty acids may be related to mitochondrial dysfunction, as they induce oxidative stress. Objectives: To examine the correlation between follicular fluid fatty acids and relative cell-free mitochondrial DNA in the follicular fluid in women experiencing intra-cytoplasmic sperm injection (ICSI). Methods: Fifty women subjected to ICSI participated in this cross-sectional research. Follicular fluid samples were obtained during oocyte pick-up. The samples were assessed for fatty acids, utilizing gas chromatography, and for relatively cell-free mitochondrial DNA, real-time polymerase chain reaction (PCR) was used. Results: There was a strong significant positive correlation between follicular fluid margaric acid and follicular fluid relative to cell-free mitochondrial DNA, as the correlation coefficient was 0.869, and the P value was 0.025. In addition, a strong significant inverse correlation was noticed, in women with diminished ovarian reserve, between follicular fluid oleic acid and relative cell-free mitochondrial DNA in the follicular fluid, as indicated by a correlation coefficient = - 0.9 and a P value = 0.037. Conclusion: Margaric acid correlated positively with the relative cell-free mitochondrial DNA, which might reflect mitochondrial dysfunction, due to aggravation of oxidative stress. Whereas, oleic acid, in women with diminished ovarian reserve, correlated negatively with relative cell-free mitochondrial DNA. However, more studies are required in this area of research.

IntroductionIt is well acknowledged that lipids assume a critical role in oocyte maturation and early embryonic metabolism, this study aimed to evaluate the relationship between the lipid composition of plasma and follicular fluid (FF), and the consequences of embryonic development. This study compared the lipidomic profiles of paired plasma and FF samples obtained from sixty-five Chinese women who underwent assisted reproductive technology (ART) treatments.MethodsNon-targeted lipidomics analysis.ResultResults not only indicated similarities in lipid composition between these biofluids, but also revealed a number of unique differences. The biomatrix distinction was found to be primarily driven by lipids belonging to the lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), ether PE, and triglyceride (TG) classes. In addition, specific species from these subclasses were discovered to be correlated with embryo development outcomes during ART. Notably, the composition of the fatty acyl chains appeared to play a crucial role in these associations. Furthermore, thirteen plasma lipid variables were identified, represented by Phosphatidylcholine 18:014:0 and PE P-18:020:1, which correlated with successful blastocyst formation (BF).DiscussionThe present study demonstrated that FF has a distinctive lipid composition, setting it apart from plasma; and the association observed with embryonic development underscored an important role of lipid composition in the healthy development of oocytes.

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]