Ovarian Follicular Fluid Research Articles

Lipopolysaccharide alters CEBPβ signaling and reduces estradiol production in bovine granulosa cells

BackgroundBacterial infection of the uterus in postpartum dairy cows limits ovarian follicle growth, reduces blood estradiol concentrations, and leads to accumulation of bacterial lipopolysaccharide (LPS) in ovarian follicular fluid. Although treating granulosa cells with LPS in vitro decreases the expression of the estradiol synthesis enzyme CYP19A1 and reduces estradiol secretion, the molecular mechanisms are unclear. The transcription factor CCAAT enhancer binding protein beta (CEBPβ) not only facilitates the transcription of LPS regulated cytokines, but also binds to the promoter region of CYP19A1 in humans, mice, and buffalo. We hypothesized that LPS alters CEBPβ signaling to reduce CYP19A1 expression, resulting in decreased estradiol secretion.MethodsBovine granulosa cells were isolated from small/medium or large follicles and treated with LPS in the presence of FSH and androstenedione for up to 24 h.ResultsTreatment with LPS increased CXCL8 and IL6 gene expression and reduced estradiol secretion in granulosa cells from both small/medium and large follicles. However, LPS only reduced CYP19A1 expression in granulosa cells from large follicles. Treatment with LPS increased CEBPB expression and reduced CEBPβ nuclear localization in granulosa cells from small/medium follicles, but not granulosa cells from large follicles.ConclusionsAlthough LPS reduces estradiol synthesis in bovine granulosa cells, the effects of LPS on CYP19A1 and CEBPβ are dependent on follicle size.

Distinct proteomic profile of ovarian follicular fluid in ewes from small versus large developing follicles

This study aimed to describe the proteins of the ovarian follicular fluid from ewes. The follicular fluid was taken from 294 ovaries collected from slaughterhouses and split into two groups based on follicle size: Group 1 (< 4 mm); and Group 2 (≥ 4 to 6 mm). The samples for ESI-Q-TOF MS/MS mass spectrometry were prepared through Tryptic in-gel digestion. For comparisons between groups, the data were analyzed using uni and multivariate statistical analysis, represented by principal component analysis (PCA), the measure of a variable's importance (VIP score) in the partial least squares-discriminant analysis (PLS-DA), the fold change, and t-test, using the exponentially modified protein abundance index (emPAI). Moreover, gene ontology was performed to identify molecular function, biological process, and cellular component. Fifty-five major proteins were identified in the 2 groups. The separation of the formed clusters was clearly observed in the PCA. Twelve proteins had a VIP score with α ≥ 1.5. Only 2 proteins were found in higher abundance in Group 1, the serum amyloid A-4 protein isoform X1 and trinucleotide repeat-containing gene 6c protein isoform X2. However, in Group 2, 10 proteins were in higher abundance: complement component C6, serotransferrin isoform X2, fibrinopeptide A, tetranectin, inter-alpha-trypsin inhibitor heavy chain H4 isoform X1, serpin B9 isoform X2, alpha-enolase isoform X6, serum paraoxonase/arylesterase 1, complement factor B, and complement C3 isoform X2, partial. We concluded, therefore, that a distinct profile of protein abundance is observed in small vs large growing follicles, including proteins especially related to ovulation, follicular metabolism, and oocyte development and quality.

Follicular fluid lipidomic profiling reveals potential biomarkers of polycystic ovary syndrome: A pilot study.

BackgroundPolycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder associated with multiple metabolic conditions including obesity, insulin resistance, and dyslipidemia. PCOS is the most common cause of anovulatory infertility; however, the molecular diversity of the ovarian follicle microenvironment is not fully understood. This study aimed to investigate the follicular fluid (FF) lipidomic profiles in different phenotypes of PCOS and to explore novel lipid biomarkers.MethodsA total of 25 women with PCOS and 12 women without PCOS who underwent in vitro fertilization and embryo transfer were recruited, and their FF samples were collected for the lipidomic study. Liquid chromatography-tandem mass spectrometry was used to compare the differential abundance of FF lipids between patients with different PCOS phenotypes and controls. Subsequently, correlations between specific lipid concentrations in FF and high-quality embryo rate (HQER) were analyzed to further evaluate the potential interferences of lipid levels with oocyte quality in PCOS. Candidate biomarkers were then compared via receiver operating characteristic (ROC) curve analysis.ResultsIn total, 19 lipids were identified in ovarian FF. Of these, the concentrations of ceramide (Cer) and free fatty acids (FFA) in FF were significantly increased, whereas those of lysophosphatidylglycerol (LPG) were reduced in women with PCOS compared to controls, especially in obese and insulin-resistant groups. In addition, six subclasses of ceramide, FFA, and LPG were correlated with oocyte quality. Twenty-three lipid subclasses were identified as potential biomarkers of PCOS, and ROC analysis indicated the prognostic value of Cer,36:1;2, FFA C14:1, and LPG,18:0 on HQER in patients with PCOS.ConclusionsOur study showed the unique lipidomic profiles in FF from women with PCOS. Moreover, it provided metabolic signatures as well as candidate biomarkers that help to better understand the pathogenesis of PCOS.

Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity

Endometriosis (EMs) occurs in approximately 50% of women with infertility. The main causes of EMs-related infertility are follicle dysplasia and reduced oocyte quality. Iron overload occurs in ovarian follicular fluid (FF) of patients with EMs, and this condition is associated with oocyte maturation disorder. However, the underlying molecular mechanism remains largely unknown. In the present study, we identified the mechanism underlying ferroptosis in ovarian granulosa cells and oocyte maturation failure in EMs based on a retrospective review of in vitro fertilization/intracytoplasmic sperm injection-frozen embryo transfer outcomes in infertile patients with EMs. Mouse granulosa cells were treated with EMs-related infertile patients' follicular fluid (EMFF) in vitro. Western blot analysis, quantitative polymerase chain reaction, fluorescence staining, and transmission electron microscopy were used to assess granulosa cells ferroptosis. The effects of exosomes were examined by nanoparticle tracking analysis, RNA-seq, and Western blot analysis. Finally, the therapeutic values of vitamin E and iron chelator (deferoxamine mesylate) in vivo were evaluated in an EMs-related infertility model. Patients with ovarian EMs experienced poorer oocyte fertility than patients with non-ovarian EMs. We observed that EMFF with iron overload-induced granulosa cell ferroptosis in vitro and in vivo. Mechanically, nuclear receptor coactivator four-dependent ferritinophagy was involved in this process. Notably, granulosa cells undergoing ferroptosis further suppressed oocyte maturation by releasing exosomes from granulosa cells. In therapeutic studies, vitamin E and iron chelators effectively alleviated EMs-related infertility models. Our study indicates a novel mechanism through which EMFF with iron overload induces ferroptosis of granulosa cells and oocyte dysmaturity in EMs-related infertility, providing a potential therapeutic strategy for EMs-related infertility.

Arsenic exposure diminishes ovarian follicular reserve and induces abnormal steroidogenesis by DNA methylation

Arsenic contamination is a worldwide public health problem, and the effect of arsenic on male reproduction has been extensively studied; however, data on the biotoxicity of arsenic in terms of female reproduction are more scarce. In this study, a human-cell-animal translational strategy was applied to explore the effect of arsenic exposure on ovarian steroidogenesis and its potential mechanism. We conducted a 1:1 propensity score matched case–control study involving 127 diminished ovarian reserve (DOR) cases and 127 healthy controls. The ovarian follicular fluid levels of 21 metal elements, including arsenic, were measured. The results showed that there were significant differences in follicular fluid metal profiles between DOR patients and controls and that arsenic, molybdenum, and strontium played important roles in DOR progression [OR (95 % CI): 2.203 (1.385, 3.503), 2.308 (1.490, 3.575) and 2.922 (1.864, 4.580), respectively]. In the primary ovarian granulosa cell culture model, we found that treatment with 8 μM arsenic for 24 and 48 h induced a decrease in human granulosa cell viability. The estradiol (E2) level was significantly decreased after arsenic exposure (P < 0.05), which was dependent on significant alterations (P < 0.05) in key enzymes in steroidogenesis. In addition, a model for sodium arsenite exposure through water in rats from weaning to sexual maturity was established. We evaluated ovarian development by monitoring the estrous cycle, observing ovarian pathology, and calculating the follicular proportion. RT–qPCR, Western blotting, and bisulfite-sequencing PCR were used to investigate the effect of arsenic exposure on ovarian steroidogenesis and its possible mechanism. The results indicated that steroidogenic factor-1 (SF-1) was an important target of the steroidogenesis disorder induced by arsenic exposure. Arsenic significantly increased the DNA methylation level (P < 0.05) in the promoter region of SF-1 to reduce its expression, subsequently decreasing the levels of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (CYP11A1), and aromatase (CYP19A1) (P < 0.05), leading to premature depletion of ovarian follicles.

P-655 Insulin like growth factor-1 levels in follicular fluids of patients with poor ovarian response undergoing in vitro fertilization

Abstract Study question Does follicular fluid insulin like growth factor level (IGF-1) in poor responders (PR) differ from normoresponders? Is there a correlation between IGF-1 and IVF outcomes? Summary answer Follicular IGF-1 levels are decreased in poor-responders A positive correlation exists between IGF-1 and number of metaphase II (MII) oocytes, fertilized oocytes and transferred embryos What is known already IGFs are mainly synthesized in liver with growth hormone stimulation. IGF-1 component in follicular fluid has been reported to play a crucial role in follicular and embryonic development. In addition to previously reported positive correlation between follicular IGF-I levels and number of high quality embryos, a significant decrease in IGF-1 levels with PR and advanced maternal age has also been reported. GH supplementation in poor responders undergoing IVF has been suggested to improve IVF outcomes. A recent Cochrane analysis reported that GH slightly increases the number of oocytes retrieved and there is an uncertain effect on live birth rates (LBR). Study design, size, duration Study was designed prospectively. A power analysis, of 91% determined a need for 186 patients. Therefore 186 women undergoing IVF between October 2019 and December 2020 were included. 72 (%38,7) patients were assigned to PRs group according to Bologna criteria (BC) and 114 patients (%61.3) were recruited as controls. Antagonist protocol was used for controlled ovarian hyperstimulation. hCG was administered for ovulation trigger. Participants/materials, setting, methods Following the ovarian hyperstimulation follicular fluid samples were collected during oocyte retrieval. When the total desired number of patients was reached, frozen and preserved follicle fluids were thawed at room temperature. IGF-1 levels in samples were measured using hIGF-1 ELISA kit (BT®, China). Follicular IGF-1 levels in PRs were compared to normoresponders and correlation between IGF-1 levels and AMH levels, number of retrieved oocytes, MII and fertilized oocytes, transferred embriyos and LBR were evaluated. Main results and the role of chance Mean IGF-1 levels of normoresponders and Bologna subgroups (B1, B2, B3, B4) were 13.79±14.50 ng/ml vs 5.75±1.86, 5.47±2.83, 5.96±2.16 and 5.03±1.85 ng/ml respectively (p &amp;lt; 0.001). There was no statistically significant difference in clinical pregnancy (CP) per transfer and LBR per transfer between normoresponders and PRs (CPR/ET: 55.1% vs 43.7%, p:0.375; 26.5% vs 12.5% , p: 0.257). In the ROC analysis for the prediction of collection of fewer than 4 oocytes at retrieval (according to BC definition of PR), statistically determined IGF-1 value was 7.02 with a sensitivity of 83% and a specificity of 38.6%. Patients with an IGF-1 value below 7.02 had a higher risk of having an oocyte number less than 4According to the results of Spearman's correlation analysis, IGF-1 levels were negatively correlated with age and number of previous failed cycles (r:-0.250, p:0,001; r:-0.153, p:0,039 respectively). IGF-1 levels were positively correlated with AMH levels, oocyte, MII oocyte, fertilized oocyte numbers and number of transferred embriyos (r: 0.221, p: 0.007; r: 0.283, p &amp;lt; 0.001; r: 0.293, p &amp;lt; 0.001; r: 0.282, p &amp;lt; 0.001; r: 0.194, p: 0.008 respectively). There was a positive correlation between IGF-1 levels and cycle cancellation rates (r: 0.637, p &amp;lt; 0,001). There was no correlation between IGF-1 levels and LBR (p:0.118). Limitations, reasons for caution Small number of patients was the main limitation of study.Results were not interpreted as LBR in all cases, as 21% (n:24) of the cases had still ongoing pregnancies Wider implications of the findings Detecting lower follicular levels of IGF-1 which plays a crucial role in GH activity may highligt the effect of GH supplementation in poor responders undergoing IVF in terms of improved outcomes. However, there is need for more solid data to employ GH in IVF treatment modalities of poor-responders. Trial registration number not applicable

Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24–38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART.

Proteomic Analysis of the Alterations in Follicular Fluid Proteins During Oocyte Maturation in Humans.

Many components in ovarian follicles (follicular fluid, cumulus cells, granular cells, etc.) dynamically change during folliculogenesis and play a positive or negative role in oocyte maturation. Infertile women who underwent intracytoplasmic sperm injection (ICSI) treatment in the reproductive medicine centre of Hangzhou Women’s Hospital between October 2018 and October 2021 were included. The ovarian follicular fluid and cumulus cells of diminished ovarian response (DOR) patients and control subjects with medical records of clinical data were collected. In total, 31 differentially expressed proteins, including 10 upregulated proteins (>1.50-fold, P<0.05) and 21 downregulated proteins (<0.67-fold, P<0.05), were identified in mature vs. immature oocytes by iTRAQ labelling coupled with 2D LC-MS/MS. GO analysis revealed that ‘cell population proliferation’ was the most diverse enrichment trend between up/downregulated proteins, while phagosome process and the PI3K-Akt signaling pathway were the two most significant pathways revealed by KEGG enrichment classification. Human prostatic acid phosphatase (PAP, ACPP) and CD5 antigen-like (CD5L) were two proteins verified by ELISA to be differentially expressed between MII and Gv oocytes (P<0.0001 and P<0.0001, respectively). Further measurement found significantly lower level of ACPP in follicular fluids and cumulus cells of DOR patients (P=0.028 and P=0.004, respectively), as an indicator of oocyte quality. Otherwise, CD5L level is upregulated in follicular fluid of DOR patients (P<0.0001). Our study provided experimental data to establish the objective indicator of oocyte maturation in the microenvironment of ovarian follicles, and also provided new insight into the measurement of oocyte quality.

Persistent organic pollutants affect steroidogenic and apoptotic activities in granulosa cells and reactive oxygen species concentrations in oocytes in the mouse.

The destruction of granulosa cells (GCs), the main functional cell type in the ovary, prevents steroid hormone production, which in turn may damage oocytes, resulting in ovarian failure. The accumulation of a number of persistent organic pollutants (POPs) in the ovarian follicular fluid (FF) has been documented, which raises serious questions regarding their impact on female fertility. We aimed to determine whether a mixture of POPs reflecting the profile found in FF influences mouse GCs or oocyte function and viability. A mixture of POPs, comprising perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, polychlorinated biphenyl 153, and hexachlorobenzene, was used. In addition to using the exact concentration of POPs previously measured in human FF, we tested two other mixtures, one with10-fold lower and another with 10-fold higher concentrations of each POP. Steroidogenesis was disrupted in GCs by the POP mixture, as demonstrated by lower oestradiol and progesterone secretion and greater lipid droplet accumulation. Furthermore, the POP mixture reduced GC viability and increased apoptosis, assessed using caspase-3 activity. The POP mixture significantly increased the number of oocytes that successfully progressed to the second meiotic metaphase and the oocyte reactive oxygen species (ROS) concentration. Thus, a mixture of POPs that are typically present in human FF has detrimental effects on ovarian function: it reduces the viability of GCs, and increases the oocyte concentrations of ROS. These results indicate that chronic exposure to POPs adversely affects female reproductive health.

Sperm-Guiding Unconventional Prostaglandins in C. elegans: Synthesis and Signaling.

Prostaglandins comprise a family of lipid signaling molecules derived from polyunsaturated fatty acids and are involved in a wide array of biological processes, including fertilization. Prostaglandin-endoperoxide synthase (a.k.a. cyclooxygenase or Cox) initiates prostaglandin synthesis from 20-carbon polyunsaturated fatty acids, such as arachidonic acid. Oocytes of Caenorhabditis elegans (C. elegans) have been shown to secrete sperm-guidance cues prostaglandins, independent of Cox enzymes. Both prostaglandin synthesis and signal transduction in C. elegans are environmentally modulated pathways that regulate sperm guidance to the fertilization site. Environmental factors such as food triggers insulin and TGF-β secretion and their levels regulate tissue-specific prostaglandin synthesis in C. elegans. This novel PG pathway is abundant in mouse and human ovarian follicular fluid, where their functions, mechanism of synthesis and pathways remain to be established. Given the importance of prostaglandins in reproductive processes, a better understanding of how diets and other environmental factors influence their synthesis and function may lead to new strategies towards improving fertility in mammals.

Perfluorooctane sulfonate (PFOS) exposure of bovine oocytes affects early embryonic development at human-relevant levels in an in vitro model

Perfluorooctane sulfonate (PFOS) has been added to Stockholm Convention for global phase out, but will continue to contribute to the chemical burden in humans for a long time to come due to extreme persistence in the environment. In the body, PFOS is transferred into to the ovarian follicular fluid that surrounds the maturing oocyte. In the present study, bovine cumulus oocyte complexes were exposed to PFOS during 22 h in vitro maturation. Concentrations of 2 ng g-1 (PFOS-02) representing average human exposure and 53 ng g-1 (PFOS-53) relevant to highly exposed groups were used. After exposure, developmental competence was recorded until day 8 after fertilisation. Blastocysts were fixed and either stained to evaluate blastomere number and lipid distribution using confocal microscopy or frozen and pooled for microarray-based gene expression and DNA methylation analyses.PFOS-53 delayed the first cleavage to two-cell stage and beyond at 44 h after fertilisation (p < .01). No reduction of proportion blastocysts were seen at day 8 in either of the groups, but PFOS-53 exposure resulted in delayed development into more advanced stages of blastocysts seen as both reduced developmental stage (p = .001) and reduced number of blastomeres (p = .04). Blastocysts showed an altered lipid distribution that was more pronounced after exposure to PFOS-53 (increased total lipid volume, p=.0003, lipid volume/cell p < .0001) than PFOS-02, where only decreased average lipid droplet size (p=.02) was observed. Gene expression analyses revealed pathways differently regulated in the PFOS-treated groups compared to the controls, which were related to cell death and survival through e.g., P38 mitogen-activated protein kinases and signal transducer and activator of transcription 3, which in turn activates tumour protein 53 (TP53). Transcriptomic changes were also associated with metabolic stress response, differentiation and proliferation, which could help to explain the phenotypic changes seen in the blastocysts. The gene expression changes were more pronounced after exposure to PFOS-53 compared to PFOS-02. DNA-methylation changes were associated with similar biological functions as the transcriptomic data, with the most significantly associated pathway being TP53. Collectively, these results reveal that brief PFOS exposure during oocyte maturation alters the early embryo development at concentrations relevant to humans. This study adds to the evidence that PFOS has the potential to affect female fertility.

Effects of Bushen-Tiaojing-Fang on the pregnancy outcomes of infertile patients with repeated controlled ovarian stimulation

Bushen-Tiaojing-Fang (BSTJF) is commonly used to treat infertility. This study investigated the effects of BSTJF on the pregnancy outcomes of patients with repeated controlled ovarian stimulation (COS), on mitochondrial function, and on oxidative stress in ovarian granulosa cells (GCs) and follicular fluid (FF). The samples and clinical data of 97 patients, including 35 in the control group, 29 in the placebo group and 33 in the BSTJF group, were collected for this study. The mitochondrial ultrastructure, ATP content, mitochondrial DNA (mtDNA) number, 8-hydroxy-2-deoxyguanosine (8-OHdG), Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GSH-Px) activity levels, and mRNA expression levels of Mn-SOD, GSH-Px, and nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) were analyzed. The high-grade embryo (P < 0.001), implantation (P = 0.033), and clinical pregnancy (P = 0.031) rates, as well as the ATP content (P = 0.014), mtDNA number (P = 0.035), GSH-Px activity (P = 0.004 in GCs and P = 0.008 in FF) and mRNA expression levels (P = 0.019), were significantly lower in the placebo group than in the control group, whereas the 8-OHdG content was significantly (P = 0.006 in FF) higher in the placebo group than in the control group. Compared with those in the placebo group, the high-grade embryo rate (P = 0.007), antioxidant enzyme activity (P = 0.037 and 0.036 in Mn-SOD; P = 0.047 and 0.030 in GSH-Px) and mRNA level (P < 0.001 in Nrf2, P = 0.039 in Mn-SOD and P = 0.002 in GSH-Px) were significantly higher in the BSTJF group, as were changes in mitochondrial ultrastructure, ATP (P = 0.040) and mtDNA number (P = 0.013). In conclusion, BSTJF can improve oxidative stress in patients with repeated COS and pregnancy outcomes.

Complex combined steroid mix of the female tract modulates human sperm

Human spermatozoa interact with a complex biochemical environment in the female reproductive tract en route to the site of fertilisation. Ovarian follicular fluid contributes to this complex milieu and is known to contain steroids such as progesterone, whose effects on sperm physiology have been widely characterised. We have previously reported that progesterone stimulates intracellular calcium concentration ([Ca2+]i) signalling and acrosome reaction in human spermatozoa. To characterise the effects of the unified complete follicular fluid steroid hormone complement on human spermatozoa, a comprehensive, data-based, ‘physiological standard’ steroid hormone balance of follicular fluid (shFF) was created from individual constituents. shFF induced a rapid biphasic [Ca2+]i elevation in human spermatozoa. Using population fluorimetry, we compared [Ca2+]i signal amplitude in cells exposed to serial applications of shFF (6 steps from 10-5X up to 1X shFF) with responses to the equivalent progesterone component alone (6 steps from 135 pM - 13.5μM). Threshold for the response to shFF was right-shifted (≈10-fold) compared to progesterone alone, but the maximum response to shFF was greatly enhanced. An acrosome reaction assay was used to assess functional effects of shFF-induced sperm calcium signalling. shFF as well as progesterone-treated spermatozoa showed a significant increase in % acrosome reaction (P < 0.01). All of this evidence suggests the modulation of progesterone-mediated responses by other follicular fluid steroids.

Hyaluronan and Collagen Are Prominent Extracellular Matrix Components in Bovine and Porcine Ovaries.

The extracellular matrix (ECM) is a major component of the ovarian stroma. Collagen and hyaluronan (HA) are critical ovarian stromal ECM molecules that undergo age-dependent changes in the mouse and human. How these matrix components are regulated and organized in other mammalian species with reproductive characteristics similar to women such as cows and pigs, has not been systematically investigated. Therefore, we performed histological, molecular, and biochemical analyses to characterize collagen and HA in these animals. Bovine ovaries had more collagen than porcine ovaries when assessed biochemically, and this was associated with species-specific differences in collagen gene transcripts: Col3a1 was predominant in cow ovaries while Col1a1 was predominant in pig ovaries. We also observed more HA in the porcine vs. bovine ovary. HA was distributed across three molecular weight ranges (<100 kDa, 100–300 kDa, and >300 kDa) in ovarian tissue and follicular fluid, with tissue having more >300 kDa HA than the other two ranges. Transcripts for HA synthesis and degradation enzymes, Has3 and Hyal2, respectively, were predominant in cow ovaries, whereas Has2, Kiaa1199, and Tmem2 tended to be predominant in pig ovaries. Together, our findings have implications for the composition, organization, and regulation of the ovarian ECM in large mammalian species, including humans.

FOLLICULAR FLUID OF POLYCYSTIC OVARIAN SYNDROME SUBJECTS SHOW SPECIFIC MIRNA PROFILES WHEN COMPARED TO CONTROLS WITH THE SAME STAGE OF OOCYTE MATURATION

Altered expression of microRNAs in ovarian follicular fluid (FF) was proposed to be one of the factors affecting folliculogenesis and steroidogenesis. Extracellular vesicles (EV), carriers of miRNAs, are protected from degradation compared to free-circulating miRNAs. As a result, EV miRNAs emerge as promising biomarker candidates for various diagnostic or response to treatment paradigms.

Assisted Reproductive Technology in Neotropical Deer: A Model Approach to Preserving Genetic Diversity.

Simple SummaryDeer species in the Neotropical region have undergone a decline of their populations. Although conservation of their natural habitat is considered the best way to assist the species, the speed of occupation of these areas and the anthropic actions are so fast that the efforts are, at times, insufficient. As free-living populations decrease, there is a descent in the genetic diversity and an increase in crossbreeding between related individuals (inbreeding). Genetic diversity is essential for survival, since it enables natural selection to occur, providing adaptation and maintenance of the species. To protect the genetic diversity, it is possible to use reproductive techniques and conserve different types of cells, which can be used in the future to reestablish any alleles that have been lost by the populations.One of the most significant challenges in deer is the ability to maintain genetic diversity, avoiding inbreeding and sustaining population health and reproduction. Although our general knowledge of reproductive physiology is improving, it appears that the application of assisted reproductive technology (ART) will more efficiently advance wildlife conservation efforts and preserve genetic diversity. The purpose of this review is to present the most important results obtained with the use of ART in Neotropical deer. Thus, the state-of-the-art for estrus synchronization, semen technology, artificial insemination, and in vivo embryo production will be presented. In vitro embryo production (IVP) is also a biotechnology that is taking initial steps in deer. In this aspect, the approach with the proteomics of ovarian follicular fluid is being used as a tool for a better understanding of oocyte maturation. Finally, cell banks and the use of interspecific somatic cell nuclear transfer (iSCNT) as well as the use of stem cells for gametes differentiation are promising techniques.

Toxic elements in follicular fluid adversely influence the likelihood of pregnancy and live birth in women undergoing IVF.

STUDY QUESTIONAre follicular fluid (FF), arsenic (As), mercury (Hg), cadmium (Cd) and lead (Pb) concentrations associated with IVF outcomes among women undergoing IVF?SUMMARY ANSWERThere was a non-linear association between higher FF Hg concentration and a lower likelihood of biochemical pregnancy and live birth. Higher FF Pb concentration was also associated with a lower probability of live birth.WHAT IS KNOWN ALREADYPrevious research suggests that toxic elements may affect fertility among couples conceiving with and without assistance. However, the results have been inconsistent, possibly related in part to exposure misclassification. Very few studies have used ovarian FF to measure toxic elements, as it requires an invasive collection procedure, yet it may offer a more accurate estimate of a biologically effective dose than blood or urine.STUDY DESIGN, SIZE, DURATIONThis is a prospective study of 56 women undergoing IVF, from October 2015 to June 2017. FF was collected for analysis on the day of oocyte retrieval.PARTICIPANTS/MATERIALS, SETTING, METHODSAs, Cd, Hg and Pb were determined in 197 FF specimens, using inductively coupled plasma tandem mass spectrometry. FF glutathione peroxidase, glutathione reductase, total glutathione-S-transferase, superoxide dismutase, arylesterase and paraoxonase (PON1p) activities were measured using kinetic enzyme assays.MAIN RESULTS AND THE ROLE OF CHANCENon-linear associations were detected, in which the probabilities of biochemical pregnancy (P = 0.05) and live birth (P = 0.05) were lower in association with FF Hg greater than ∼0.51 µg/l Hg, adjusted for age, race, cigarette smoking and recent seafood consumption. Higher FF Pb was also associated with a lower likelihood of live birth (relative risk (RR) = 0.68, 95% CI: 0.46, 1.00; P = 0.05). We also found a suggestive, although imprecise, antagonizing mediating effect of PON1p activity on the association between FF Pb and live birth (−28.3%; 95% CI: −358%, 270%).LIMITATIONS, REASONS FOR CAUTIONThe results should be interpreted judiciously given the limited sample size and difficulty accounting for correlated data in generalized additive models and mediation analyses. Additionally, women undergoing IVF are highly selected with respect to age and socioeconomic status, and so the generalizability of the results may be limited.WIDER IMPLICATIONS OF THE FINDINGSOverall, the results suggest that FF Hg was associated with a lower likelihood of biochemical pregnancy and live birth, with a potential threshold effect, and that higher FF Pb was associated with a lower probability of live birth. These results may help to guide clinical recommendations for limiting the exposure of patients to Hg and Pb and ultimately improve IVF success rates.STUDY FUNDING/COMPETING INTEREST(S)This work was funded in part by the National Institute of Environmental Health Sciences (NIEHS), grant number 1R56ES023886-01, to the University at Albany (M.S.B.), and in part by the National Institute of Environmental Health Sciences (NIEHS), grant number 1U2CES026542-01, to the Wadsworth Center (P.J.P.). The authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A

Protegrin-1 Regulates Porcine Granulosa Cell Proliferation via the EGFR-ERK1/2/p38 Signaling Pathway in vitro.

Antimicrobial peptides (AMPs) are traditionally known to be essential components in host defense via their broad activities against bacteria, fungi, viruses, and protozoa. Their immunomodulatory properties have also recently received considerable attention in mammalian somatic tissues of various species. However, little is known regarding the role of AMPs in the development and maturation of ovarian follicles. Protegrin-1 (PG-1) is an antimicrobial peptide which is known to have potent antimicrobial activity against both gram positive and negative bacteria. Here we report that the PG-1 is present in the porcine ovarian follicular fluid. Treatment of granulosa cell with PG-1 enhanced granulosa cell proliferation in a dose-dependent manner. This is accompanied by increased expression of cell-cycle progression-related genes such as cyclin D1(CCND1), cyclin D2 (CCND2), and cyclin B1(CCNB1). Additionally, Western blot analysis showed that PG-1 increased phosphorylated epidermal growth factor receptor (EGFR), and the phosphorylated-/total extracellular signal-regulated kinase (ERK)1/2 ratio. Pretreatment with either U0126, a specific ERK1/2 phosphorylation inhibitor, or EGFR kinase inhibitor, AG1478, blocked the PG-1 induced proliferation. Moreover, luciferase reporter assay revealed that ETS domain-containing protein-1 (Elk1) C/EBP homologous protein (CHOP), and the transcription activators downstream of the MAPK pathway, were activated by PG-1. These data collectively suggest that PG-1 may regulate pig granulosa cell proliferation via EGFR-MAPK pathway., Hence, our finding offers insights into the role of antimicrobial peptides on follicular development regulation.

The Release of Peripheral Immune Inflammatory Cytokines Promote an Inflammatory Cascade in PCOS Patients via Altering the Follicular Microenvironment.

BackgroundHormones and immune imbalance are critical factors in polycystic ovary syndrome (PCOS). The alternation of immune microenvironment of oocytes may play a significant role in infertility of PCOS patients.ObjectiveThis study explores the role of follicular fluid microenvironment change in inflammatory pathways activation of granulosa cells (GCs) in PCOS women infertility.MethodsWe enrolled 27 PCOS patients and 30 controls aged 22 to 38 years who underwent IVF and collected their luteinized granulosa cells (LGCs). Meanwhile, a granulosa-like tumor cell line (KGN) as a cell-model assisted this study. Key inflammatory markers in human ovarian GCs and follicular fluid were detected by RT-qPCR, Western blotting, or ELISA. The KGN cells were treated with follicle supernatant mixed with normal medium to simulate the microenvironment of GCs in PCOS patients, and the inflammation indicators were observed. The assembly of NLRP3 inflammasomes was detected by immunofluorescence techniques. Dihydroethidium assay and EdU proliferation assay were used to detect ROS and cell proliferation by flow cytometry.ResultsCompared with normal controls (n = 19), IL-1β (P = 0.0005) and IL-18 (P = 0.021) in the follicular fluid of PCOS patients (n = 20) were significantly increased. The NF-κB pathway was activated, and NLRP3 inflammasome was formatted in ovarian GCs of PCOS patients. We also found that inflammation of KGN cells was activated with LPS irritation or stimulated by follicular fluid from PCOS patients. Finally, we found that intracellular inflammation process damaged mitochondrial structure and function, which induced oxidative stress, affected cellular metabolism, and impaired cell proliferation.ConclusionInflammatory microenvironment alteration in the follicular fluid of PCOS patients leads to activated inflammatory pathway in GCs, serving as a crucial factor that causes adverse symptoms in patients. This study provides a novel mechanism in the inflammatory process of PCOS.

The Effects of Leptin and Irisin on Steroidogenic Enzyme Gene Expression in Human Ovarian Granulosa Cells - Initial Studies

Fertility and energy metabolism are closely associated, and the cytokines produced by the adipose and muscle tissue play a role in this association. Leptin, predominantly produced by the white adipose tissue, and irisin, produced by the brown adipose and skeletal muscle tissues, are cytokines that are important in balancing energy metabolism. This study aimed to investigate the effects of leptin and irisin on steroidogenic enzyme gene expression in human ovarian granulosa cells in vitro. Granulosa cells were retrieved and isolated from ovarian follicular fluid during in vitro fertilization (IVF) procedures. Cells were placed in primary in vitro cultures and treated with increasing concentrations of leptin (25, 50, 100, 200, and 400 ng/ml) or irisin (125, 250, 500, 1,000, and 2,000 ng/ml) for 24, 48, and 72 hours. mRNA expression levels of CYP11A1, CYP19A1, CYP21A2, HSD3B1, and HSD17B3 were measured by qRT-PCR analysis. Leptin treatment of granulosa cells resulted in significant upregulation of CYP21A2 mRNA levels, while irisin significantly downregulated mRNA levels of CYP11A1, CYP19A1, and HSD3B1. Taken together, these early experiments demonstrate that leptin and irisin may affect steroid hormone production in the ovary by targeting the gene expression of key steroidogenic enzymes. Additional experiments are in progress.