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The complex composition of the follicular fluid (FF), the intimate proximity to the oocyte, and the continual changes in their composition have a major effect on folliculogenesis and oogenesis. To date, the profiling of FF proteomes during follicle selection, development, and ovulation has not been comprehensively investigated. Therefore, a shotgun proteomics approach and bioinformatics analyses were used to profile the proteomes of equine FF harvested in vivo from follicles at the following development stages: predeviation (18-20 mm), deviation (22-25 mm), postdeviation (26-29 mm), preovulatory (30-35 mm), and impending ovulation. A total of 294 proteins were detected in FF (FDR 1%), corresponding to 65 common proteins and 124, 142, 167, 132, and 142 proteins in the predeviation, deviation, postdeviation, preovulatory, and impending ovulation groups, respectively. The higher expression of properdin and several other proteins belonging to the complement system during the deviation time and ovulation suggested their contribution in the selection of the future dominant follicle and ovulation. Apolipoprotein A-1 and antithrombin-III appeared to be important throughout folliculogenesis. The "complement and coagulation cascades" was the major KEGG pathway across all stages of follicle development. The significant expression of several proteins belonging to the serine-type endopeptidase indicated their likely contribution to follicle and oocyte development. Our data provide an extensive description and functional analyses of the equine FF proteome during follicle selection, development, and ovulation. This information will help improve understanding of the ovarian function and ovulatory dysfunctions and might serve as a reference for future biomarker discovery for oocyte quality assessment.

Glycolysis in follicular granulosa cells (GCs) is the primary source of energy metabolism substrate of oocytes and is closely related to follicular development in mammals. Many physiological functions of GCs are regulated by follicle-stimulating hormone (FSH). In contrast, whether FSH regulates the glycolysis of GCs and its mechanism remains unclear. This study explored the correlation between FSH concentration and glycolysis level of GCs from different diameters of water buffalo follicles, and further explored the mechanism of FSH regulation in glycolysis in vitro cultured GCs. Results showed the variation trend of lactic acid concentration in follicular fluid and the expression level of glycolysis-related genes in GCs were consistent with the variation trend of FSH concentration in follicular fluid from follicles with different diameters. When GCs were treated with FSH in vitro, the expression level of glycolysis-related genes, lactate production and glucose uptake increased correspondingly (p

We aimed to investigate the value of follicular fluid fetuins-A and -B to predict successful IVF and pregnancy outcomes in infertile women with poor, normal, and high ovarian reserve.The follicular fluid of 96 infertile women who underwent intra-cytoplasmic sperm injection (ICSI) procedure was analyzed. Fetuins-A and -B levels were examined and compared in those who could achieve pregnancy and those who could not. Receiver operating characteristic curve analyzes were used to determine cut-off and statistically significant associations for fetuins-A and -B.Follicular fluid fetuin-A levels were higher in cases with weak ovarian reserve (OR) (p 0.05) and higher in patients who did not achieve clinical pregnancy (p 0.05). Conversely, the follicular fluid fetuin-B levels were lower in cases with poor OR (p 0.05) and were lower in patients who did not achieve a clinical pregnancy (p 0.05). A follicular fluid fetuin-A concentration ≤ 19.12 ng/mL had a sensitivity and specificity of 94.74% and 93.1%, respectively, at predicting clinical pregnancy. While the follicular fluid fetuin-B concentration24.7 ng/mL had sensitivity and specificity of 71.1% and 51.7%, respectively, for clinical pregnancy prediction.Overall, high levels of follicular fluid fetuin-A may be independently associated with unsuccessful IVF irrespective of OR grouping. A low level of follicular fetuin-B was also associated with failed IVF. The sensitivity and specificity were found to be higher for fetuin-A in predicting clinical pregnancy. Therefore, the follicular fluid fetuin-A may be more predictive for successful IVF and clinical pregnancy outcomes than follicular fluid fetuin-B.

The aim of this study was to investigate the effects of extracellular vesicles (EVs) on the proliferation and steroid hormone synthesis of bovine ovarian cortical stromal cells in vitro. The release and uptake of EVs are the new mechanisms of cell-to-cell communication. Using reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, TUNEL and other experiments, we found that EVs in bovine follicular fluid can promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. Moreover, 100 μg/ml EVs caused the most significant effect. We conclude that EVs at 100 μg/ml can significantly promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. This research is of great significance for further elucidating the regulatory role of follicular fluid EVs in follicular development and atresia and for research on the interaction of ovarian stromal cells, granulosa cells and oocytes.

Objective In this study, we investigated the effect of thyroid-stimulating hormone (TSH) level on the outcomes of in vitro fertilization (IVF) in patients with polycystic ovary syndrome (PCOS). Methods Data pertaining to 60 patients who underwent IVF between May 2017 and May 2018 were included in the study. Thirty-two patients were diagnosed as PCOS (PCOS group) and 28 patients had tubal infertility (control group). Serum and follicular fluid TSH levels and follicular cyclic AMP (cAMP) level were detected by ELISA. TSH receptor (TSHR) expression level in granulosa cells was quantified by RT-PCR and Western blot. Results In the PCOS group, oocyte maturation rate and fertilization rate were significantly lower than in the control group. Serum and follicular fluid TSH levels and ovarian cAMP level were higher in the PCOS group with an upregulation of ovarian TSHR. On multivariate linear regression analysis, fertilization rate showed a negative correlation with TSH levels in serum (B = -0.106, P = 0.005) and follicular fluid (B = -0.107, P = 0.001). Conclusion In PCOS patients, TSH levels, both in serum and follicular fluid, were negatively correlated with IVF oocyte maturation rate and fertilization rate. The effect of TSH on controlled ovarian hyperstimulated oocyte growth was likely mediated by the TSHR/cAMP signaling pathway.

Ovarian hyperstimulation syndrome (OHSS) is one of the most dangerous iatrogenic complications in controlled ovarian hyperstimulation (COH). The exact molecular mechanism that induces OHSS remains unclear. In recent years, accumulating evidence found that exosomal miRNAs participate in many diseases of reproductive system. However, the specific role of miRNAs, particularly the follicular fluid‐derived exosomal miRNAs in OHSS remains controversial. To identify differentially expressed follicular fluid exosomal miRNAs from OHSS and non‐OHSS patients, the analysis based on miRNA‐sequence was conducted. The levels of 291 miRNAs were significantly differed in exosomes from OHSS patients compared with normal control, and exosomal miR‐27 was one of the most significantly down‐regulated miRNAs in the OHSS group. By using MiR‐27 mimic, we found it could increase ROS stress and apoptosis by down‐regulating the expression of p‐ERK/Nrf2 pathway by negatively regulating SPRY2. These data demonstrate that exosomal miRNAs are differentially expressed in follicular fluid between patients with and without OHSS, and follicular fluid exosomal miR‐27 may involve in the pathological process of OHSS development.

Comparative proteomic profiling of human follicular fluid (HFF) from polycystic ovary syndrome (PCOS) and non-PCOS patients who displayed low levels of fertility was carried out via two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry. HFF, an important reproductive fluid, was used for the proteomic analysis of PCOS patients to determine the effect of PCOS on folliculogenesis. HFF was obtained from 10 women (average age, 35 years) undergoing in vitro fertilization at two hospitals. The proteins of HFF were separated using 2-DE analysis and validated by Western blot assay. Approximately 250 protein spots were separated on the 2-DE gel. Among them, the expression levels of seven proteins were found to change at least 1.5-fold in the PCOS patient group. Three protein spots, albumin, uncharacterized protein 1, and uncharacterized protein 2, were downregulated in PCOS patients. However, four protein spots, gelsolin, vitamin D binding protein, serum albumin, and complement factor B, were upregulated in PCOS patient group. These proteins may serve as a panel of potential pathological biomarkers during fertilization and oocyte development.

Research has been focused on determining the follicular microenviroment produced by the theca and granulosa cells since the molecular characterisation of this body fluid could lead to the understanding of several fertility problems. Oxidative stress may be one of the factors involved in female infertility since it plays a key role in the modulation of oocyte maturation and finally pregnancy. An increase in oxidative stress is correlated with inflammation and intense research was developed to understand the interaction between inflammation and adiponectin, based on the fact that many adipokines are inflammation related proteins linked to reactive oxygen species production. The aim of this study is to investigate the correlation between total adiponectin levels and oxidative stress amount in the serum and follicular fluid (FF) of women who undergone in vitro fertilization. Moreover we verified the expression of adiponectin in granulosa and cumulus cells. To clarify the predictive value of steroid hormones in human assisted reproduction, twelve steroid hormones in FF and serum, were quantified in a single run liquid chromatography/mass spectrometry, by using a multiple reaction monitoring mode and we related the serum and follicular fluids adiponectin levels with the concentration of the investigated steroid hormones.

To explore the effect of anti-nuclear antibodies (ANAs) on the outcome of in vitro fertilization-embryo transplantation (IVF-ET) and to study the effect of ANAs in follicular fluid (FF) on embryonic development.The expression of ANAs in FF of patients treated with IVF-ET and healthy group. The patients were divided into ANAs-positive group and ANA-negative group. The age, duration of infertility, body mass index (BMI), basic follicle stimulating hormone (bFSH), anti-Mullerian hormone (AMH), number of retrieved oocytes, portion of metaphase II oocytes (MII), number of embryos in the cleavage stage, bipronuclear (2PN), number of embryos, number of high-quality embryos and the outcome of IVF-ET were compared between the two groups. In vitro, HTR8/SVneo trophoblast cells were cultivated and divided into ANAs-negative group (control group) and ANAs-positive group (ANAs-positive FF was added to cytotrophoblasts). The ANAs titer in the serum and FF of patients who treated with IVF-ET was detected using ELSIA method. CCK-8 assay and flow cytometry (at 24 h and at 48 h) were used to detect the cell proliferation and apoptosis frequency of the two groups, respectively.Among those who underwent IVF-ET treatment, the number of retrieved oocytes, the number of fertilization and the portion of MII oocytes in the FF-positive group were significantly lower than those in the FF-negative group. Furthermore, the implantation rate and the clinical pregnancy rate were decreased, and early miscarriage rate was increased in the FF-positive patients than those in the FF-negative patients. In vitro, the cytotrophoblasts proliferation activity in the ANAs group was significantly lower than that in the control group. Moreover, the cytotrophoblasts apoptosis rate in the ANAs group was significantly higher than that in the control group.Our data suggested that ANAs in FF might become an obstacle to embryonic development through promoting trophoblast apoptosis and inhibiting trophoblast proliferation. ANAs in FF might be an unfavorable factor for the outcome of those who undergo IVF-ET treatment.

The COVID-19 pandemic has many clinical manifestations. Rapid vaccine development raised concerns and speculations about future fertility outcomes and vaccine safety. We evaluated the effect of Pfizer-BioNTech mRNA SARS-CoV-2 vaccine on IVF treatment, oocyte and embryo quality, and pregnancy outcomes.This prospective, observational cohort study was conducted in a referral IVF Unit, 3/2021-5/2021. We aimed to recruit all women undergoing IVF/ICSI cycles from 3/1-4/30/2021, 2-8 weeks after the second vaccination, and to analyze 50-60 samples in the 2-month period. Patients were categorized according to serum antibody levels: positive for spike (S), positive for nucleotide (N), or negative for both. On the day of ovum pick-up, follicular fluid and blood samples were analyzed for anti-nucleotide (anti-N) antibodies, and anti-spike (anti-S) antibodies, hormonal profile, C-reactive protein (CRP) and other metabolic parameters.Of 59 women enrolled, 37 reported being vaccinated and 22 were not. We found 97% correlation between anti-S and anti-N in the blood and the follicular fluid. Follicular fluid was analyzed based on antibody categorization. All IVF treatment parameters in the follicular fluids and serum were comparable, except CRP was significantly elevated among patients with anti-N antibodies (2.29 [1.42-6.08] vs. 4.11 [1.62-5.75] vs. 1.44 [.36-8.33]; p .001). Pregnancy outcomes were comparable (44% vs. 33% vs. 50%; p = .97).mRNA SARS-CoV-2 vaccine did not appear to affect treatment outcomes or ovarian reserves in the subsequent IVF cycle.

Sperm velocity is a key trait that predicts the outcome of sperm competition. By promoting or impeding sperm velocity, females can control fertilization via postcopulatory cryptic female choice. In Chinook salmon, ovarian fluid (OF), which surrounds the ova, mediates sperm velocity according to male and female identity, biasing the outcome of sperm competition towards males with faster sperm. Past investigations have revealed proteome variation in OF, but the specific components of OF that differentially mediate sperm velocity have yet to be characterized. Here we use quantitative proteomics to investigate whether OF protein composition explains variation in sperm velocity and fertilization success. We found that OF proteomes from six females robustly clustered into two groups and that these groups are distinguished by the abundance of a restricted set of proteins significantly associated with sperm velocity. Exposure of sperm to OF from females in group I had faster sperm compared to sperm exposed to the OF of group II females. Overall, OF proteins that distinguished between these groups were enriched for vitellogenin and calcium ion interactions. Our findings suggest that these proteins may form the functional basis for cryptic female choice via the biochemical and physiological mediation of sperm velocity.

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

Introduction Human ovulation is a biologically complex process that involves several biochemical factors, promoting follicular rupture and release of a fertilizable oocyte. Proteins which are present in follicular fluid at high concentrations during ovulation are likely to be active participants in the biochemical pathways of ovulation. The aim of the study was to identify, by use of a modern proteomic technique, proteins of human follicular fluid which are differentially regulated during ovulation of the natural menstrual cycle. Material and methods This prospective experimental study over 3 years included women planned for laparoscopic sterilization. During surgery, retrieval of the dominant follicle was performed either at the preovulatory stage or during ovulation. Four women of preovulatory phase and four women of ovulatory phase met the predetermined criteria of hormone levels for respective phases, and samples of these were finally included out of the 15 women operated. Follicular fluid was aspirated from the excised follicle and subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ) technology for isobaric tagging of peptides. This enables simultaneous identification and quantification of proteins. The protein profiles of the follicular fluid of the preovulatory phase and the ovulatory phase were analyzed, and proteins that were present were identified. Results A total of 502 proteins were identified, several of which previously have not been identified in human follicular fluid. Of the 115 proteins that were found in all samples, 20 proteins were at higher levels during ovulation. These were inflammatory-related proteins, coagulation factors, proteins in lipid metabolism, complement factors and antioxidants. Five proteins were present in lower levels during ovulation, with three being enzymes and the other two proteins of lipid metabolism and iron transport. Conclusions Twenty-five follicular fluid proteins, with differential regulation during ovulation, were identified in human follicular fluid of the natural menstrual cycle. These proteins may have essential roles in the ovulatory cascade.

Introduction Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders among women of reproductive age. Chemerin, a novel adipokine, is involved in inflammation, energy metabolism, adipogenesis, angiogenesis and insulin secretion in the adipose cells and ovary. This systematic review with meta‐analysis aimed to compare serum and follicular fluid (FF) chemerin and ovarian chemerin mRNA expression among women with PCOS and non‐PCOS. Methods Electronic databases including Web of Science, PubMed, Google Scholar, Scopus, Cochrane and CINAHL were used for a comprehensive search through April 2021. Of the 174 articles initially identified, 22 studies met the eligibility criteria. A random‐effects model with a weighted mean difference (WMD) and 95% confidence interval (CI) was performed to compare the outcomes between groups. Subgroup and sensitivity analyses were performed to detect the sources of heterogeneity. Results Women with PCOS compared to without PCOS showed significantly higher serum chemerin [WMD: 12.02 pg/ml (95% CI: [10.92, 13.13]), p, This systematic review with meta‐analysis was shown serum, and follicular fluid chemerin levels and chemerin mRNA expression were higher in the PCOS group compared to the controls. Further, serum chemerin was higher in women with PCOS and a more BMI. The present findings illustrated that chemerin may be associated with both PCOS status and BMI, independently.

Hepatitis B virus (HBV) can be found in ovarian tissues. This study compared HBV DNA levels in follicular fluid collected during oocyte retrieval with paired serum samples in HBV carriers after ovarian stimulation during IVF treatment for infertility. Sixty-four HBV carrier women referred to the Assisted Reproductive Units of two Hong Kong hospitals were recruited. At oocyte retrieval, the follicular fluid aspirated from the first follicle was collected for study. In 22 women, the first follicular fluid sample from both ovaries was similarly collected and studied. These women were also tested for liver function test and HBeAg. In 28 (43.8%) women, HBV DNA was detected in follicular fluid and the level correlated with serum levels (Spearman's correlation P .001). There was concordant detection of HBV DNA in both ovaries, and the levels were significantly correlated (Spearman's correlation P = .029). In 40% of women with FF HBV DNA, the follicular fluid:serum ratio was1.0, suggesting stimulation of HBV replication. These women also had significantly different liver function test results. Increased HBV replication exists in 40% of women with HBV DNA detected in follicular undergoing ovarian stimulation during IVF treatment.

Cows experiencing high levels of inflammation and specific metabolic conditions tend to have slower follicular growth and lower serum and follicular concentrations of oestradiol (E2). Paraoxonase1 (PON1) activity decreases during inflammatory processes. Therefore, the aim of this study was to evaluate the association between serum and intrafollicular (FF) PON1 activity and the serum and intrafollicular levels of E2 and progesterone (P4), as well as the mRNA expression of genes related to steroidogenesis, metabolism and inflammation in the first post-partum dominant follicle of Holstein cows. No correlation was found between PON1 activity, the expression of the analysed genes and levels of follicular E2 and P4, except for a negative correlation between serum E2 and follicular PO1 activity. Also, no correlation was found between serum and follicular PON1 during the first post-partum follicular wave.

Purpose This study aimed to examine the relationship between granulosa cells (GCs), number of follicles, and the ability of follicular fluid to support in vitro growth of oocytes. Methods The culture medium was supplemented with follicular fluid (FF) collected from GC‐rich ovaries and GC‐poor ovaries, and its effect on in vitro growth and quality of oocytes derived from early antral follicles (EAFs) was assessed. Results GC‐rich FF treatment enhanced oocyte growth and augmented changes in the chromatin configuration and lipid content of oocytes when compared to oocytes treated with GC‐poor FF. Moreover, oocytes treated with GC‐rich FF had a higher ability to progress to the blastocyst stage, than oocytes derived from large antral follicles (3‐5 mm in diameter). In addition, supplementation of the culture medium with either GC‐rich or GC‐poor FF enhanced histone acetylation in oocytes grown in vitro. Conclusion GC‐rich FF contains key factors that support in vitro oocyte growth; hence, oocytes grown in GC‐rich FF medium had high developmental competence, which was comparative to the oocytes grown in vivo.

Purpose To study the association between stromal cell‐derived factor‐1 (SDF‐1/CXCL12) and vascular endothelial growth factor (VEGF) concentrations in individual human ovarian follicles and IVF outcomes. Methods Concentrations of SDF‐1 and VEGF in 261 follicular fluid samples were measured with enzyme‐linked immunosorbent assay. IVF outcome parameters were included in fertilization rate, cleavage rate, embryo morphology on day 3, and blastocyst morphology on day 5. Results The follicular concentration of SDF‐1 and VEGF was not significantly associated with fertilization and cleavage outcome, and embryo morphology. The rates of full blastocysts and good‐quality blastocysts were significantly higher in follicles with an SDF‐1 concentration of 275‐350 pg/mL than in the follicles with SDF‐1 concentrations of

Objective To investigate the role of the transforming growth factor-β1 (TGF-β1)/Smad3 signaling pathway in development of ovarian follicle by promoting apoptosis of granulosa cells in polycystic ovary syndrome (PCOS). Methods A total of 54 female rats were obtained and randomly assigned into the PCOS (n = 27) and control groups ( n = 27). PCOS rat models were constructed using the dehydroepi-androsterone method to observe ovarian morphology and ultrastructure. Chemiluminescence immunoassay was performed to detect the levels of luteinizing hormone, follicle stimulating hormone, estradiol, progesterone, and testosterone. The release of TGF-β1 in follicular fluid and serum was tested by the enzyme-linked immunosorbent assay. Results Expression of p-Smad3 significantly increased in granulosa cells of PCOS group. In terms of the Smad2 protein, there was no significant difference between the PCOS and control group. In comparison to the control group, the number of normal secondary follicles and normal antral follicles were significantly decreased, whereas the number of hypogenetic secondary follicles undergoing atresia and antral follicles were significantly elevated in the PCOS group. Furthermore, the thickness of granulosa cells decreased and the apoptosis of granulosa cells increased in the PCOS group compared with the control one. Conclusion These results indicate that p-Smad3 may have a close relationship with apoptosis of granulosa cells, the mechanism is that the TGF-β1/Smad3 signaling pathway may inhibit development of ovarian follicle of PCOS by regulating the apoptosis of granulosa cells.

Background Bovine leptospirosis is mainly characterized by reproductive disorders. Contamination of the oocyte was previously demonstrated in vitro, resulting in some apparent damage. However, it is not clear whether it occurs under natural conditions. The present study aimed to characterize the presence of pathogenic Leptospira DNA in the ovarian follicles of non-pregnant cows. Methods Follicular fluid samples were collected from 65 animals and subjected to lipL32 PCR and secY sequencing. Results In total, seven of 65 (10.8%) were positive, indicating a possible early infection of the oocyte. Moreover, secY sequencing identified L. interrogans and L. santarosai, both very closely related to bovine strains from the Sejroe serogroup (Hardjoprjitno and Guaricura). We demonstrated that ovarian follicles can also be infected. Conclusions It was hypothesised that ovarian infection can contribute to embryonic death, causing reproductive failure and estrus repetition. In the present study, we show that the organism identified in the follicle is closely related to one that is known to be associated with reproductive disorders.

Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder in women of reproductive age. Some studies have investigated metabolic alterations in plasma and follicular fluid from PCOS patients, but they did not control for obesity or insulin resistance (IR); additionally, correlation analysis of metabolites is sparse. Accordingly, in this study, we aimed to examine metabolic differences owing to the pathogenesis of PCOS, identify the hub metabolites and investigate its associations with androgens. We applied GC-MS platform coupled with a correlation network approach to analyze follicular fluid samples from 32 PCOS patients without obesity and IR and 31 healthy women. Thirty significantly altered metabolites in PCOS patients were enriched in amino acid metabolism. l-Phenylalanine, l-tryptophan, pyroglutamic acid, l-tyrosine, l-leucine and l-valine were screened as hub metabolites in metabolic correlation network. Among them, increased l-tryptophan and l-tyrosine were altered hub metabolites, and they had a more significant impact on the metabolic change of PCOS. In addition, l-tryptophan and l-tyrosine were significantly positively associated with serum androgens levels in the PCOS. Our results suggest that disorders of amino acid metabolism, especially tryptophan and tyrosine metabolism, might play an important role in the development of PCOS in predisposed women without obesity and IR.

Purpose To investigate impact of the one‐carbon metabolism (OCM) on oocyte maturity and embryo development. Methods This prospective study analyzed 18 women who agreed to participate. We measured the OCM biomarkers’ concentrations including Vitamin B12 (VB12), folic acid (FA), and homocysteine (Hcy) in serum and follicular fluid (FF), and assessed their correlation. We also evaluated the influence of such OCM biomarker concentrations in mono‐FF on oocyte maturation, fertilization, embryo quality, and consequent pregnancy after embryo transfers. Results All biomarkers showed a high concentration variability in different follicles of each woman, but their mean levels correlated with the serum levels. Among the 106 collected oocytes, 92 were mature, 59 were fertilized, and 16 yielded good‐quality embryos. We performed 26 single embryo transfers, and 7 patients achieved clinical pregnancies. VB12 concentration (FF) was significantly lower in fertilized than unfertilized oocytes by univariate analysis. In multivariate logistic analysis, a significant correlation was found between FA concentration (FF), Imbalances in one‐carbon metabolism (OCM), a metabolic pathway composed of folic acid and methionine metabolism, cause hyperhomocysteinemia. OCM in follicular fluid may affect fertilization, embryo quality, and clinical pregnancy. In addition, folic acid and homocysteine at certain levels were found to be potential indicators of good‐quality embryos and clinical pregnancy, respectively

Problem The mechanism(s) by which antinuclear antibodies (ANA) induce implantation failure are not clear, and little information regarding the function of autoantibodies in reproductive tissues is available. Methods of study A total of 380 patients who underwent in vitro fertilization and embryo transfer (IVF-ET) were divided into control, serum positive, and follicular fluid (FF) positive groups based on the results of indirect immunofluorescence assay for ANA in the serum and FF. Immunofluorescence assay was performed to evaluate the existence of ANA in granular cells and endometrial tissues. Presence in FF of soluble apoptotic markers, including Bcl-2, Caspase-3, cleaved PARP, Cytochrome C, GAPDH, and p53, was assessed using magnetic bead based assays. Results The patients in the FF positive group had the lowest numbers of retrieved oocytes, fertilizations, and high-quality embryos. The fertilization rate, and the proportion of two pronuclear (2PN) embryos in patients in the FF positive group were significantly lower than those in the other two groups. The FF positive group also had the lowest clinical pregnancy rate, and the highest early miscarriage rate. Granulosa cells and endometrial tissues in patients in the FF positive group were ANA positive. High levels of BCL-2, Caspase-3, Cytochrome C, GAPDH, and p53 were found in the FF of patients in the FF positive group. Conclusions Antinuclear antibodies in FF and endometrial tissues may cause imbalanced apoptosis, resulting in poor IVF-ET treatment outcomes. Local autoimmunity and cell apoptosis in reproductive tissues could be considered new therapeutic targets for improving IVF-ET treatment efficacy.

The nutritional alteration of amino acids (AAs) profile in physiological fluid was poorly characterized in livestock. After oestrus synchronization, 24 ewes were randomly assigned to two groups based on the nutrient requirement recommended for maintenance (M): the feed-supplemented group (S, 1.5 × M, N = 12) and feed-restricted group (R, 0.5 × M, N = 12) on days 6-12 of their oestrous cycle, which occurred shortly before ovulation. The concentration of 30 AAs in peripheral blood (PB) and follicular fluid (FF) was quantified to calculate the PB-to-FF concentration gap for each AA and determine its correlation with metabolites and hormones in PB and FF. Results showed that the feed restriction enlarged the oestrous cycle length, decreased the number of follicles 2.5-3.5 mm, increased the number of follicles >3.5 mm and augmented the volume of follicles >2.5 mm. Nineteen AAs from PB were significantly different between the groups. The phosphoethanolamine (PEtN) and ration of essential AAs to nonessential AAs (EAA/NEAA) in FF significantly (p < 0.05) increased and decreased in the R group, respectively. Most AAs, except aspartate (Asp) and carnosine (Car) in the R group and alanine (aAla) in both groups, were significantly lower within FF than those within PB. The correlation of AAs with FSH and progesterone (P4 ) was more significant than that of AAs with other endocrine milieu characteristics. In conclusion, our results revealed that the influence of short-term nutritional manipulation during luteal phase on folliculogenesis might not be due to the variation of intrafollicular AAs profile but rather attribute to the peripheral blood AAs profile alteration.

Purpose Antioxidant status and oxidative stress markers in human follicular fluid (FF) surrounding oocytes may be related to the outcomes of in vitro fertilization and embryo transfer (IVF‐ET). Therefore, we herein examined the relationship between antioxidant status and oxidative stress markers in FF and the outcomes of IVF‐ET. Methods One hundred and seventeen infertile women were included in this study. FF was obtained from mature follicles at the time of oocyte retrieval. The total antioxidant capacity (TAC) and total glutathione (GSH), vitamin C, and 8‐ hydroxy‐2′‐deoxyguanosine (8‐OHdG) concentrations were measured. Results Total GSH levels were lower in patients who had a low fertilization rate after intracytoplasmic sperm injection (ICSI). In addition, 8‐OHdG levels were higher in patients who had a low fertilization rate after ICSI and low rate of good quality blastocysts. Total GSH activity was lower in patients with endometriosis. No significant differences were noted in pregnancy outcomes. Conclusions Total GSH and 8‐OHdG in human FF may be potential markers for fertilization in ART. Also, our findings may suggest that oxidative stress in women with infertility is associated with endometriosis.

The quality of oocytes depends on interactions with surrounding granulosa cells. Granulosa cells are essential in normal follicular maturation process since they produce steroidal hormones and growth factors, and they play a crucial role in follicular atresia. The success in reproductive biology and medicine depends on reliable assessment of oocyte and embryo viability which presently mainly bases on oocyte and embryo morphology. Recent investigations have tried to establish an evaluation system for oocyte quality and to predict outcome of in vitro fertilization (IVF) based on the incidence of granulosa cells and cumulus cells apoptosis. Apoptosis of granulosa cells seems to have a negative effect on conception and pregnancy rates in IFV programs. Thus, in this review we present a brief outline of clinical correlation of apoptosis in human granulosa cells and cumulus cells, and its influence upon oocyte quality and IFV outcome. Taken together, understanding the influence of granulosa cell apoptosis on oocyte quality and maturity as well as on embryo health may ultimately allow scientists and clinicians to harness better protocols of ovarian stimulation for infertility treatments.

The follicular fluid exerts an effect on the sperm capacitation of several species; however, these effects vary according to species, both in the sperm motility and in the subsequent acrosome reaction. In this study, the effect of alpaca follicular fluid (aFF) on the motility and acrosome reaction of alpaca spermatozoa was observed, using follicular fluid of three follicle sizes: small (3 mm), medium (3-6 mm) and large (6 mm), in a concentration of 30%. Sperm motility at the first hour of incubation with aFF of small follicles was 48.0%, with aFF of medium follicles it was 43.33% and with aFF of large follicles, it was 34.53%, while control averaged 26.00%. At the second hour, control achieved an average of 28.13%, treatment with aFF from small follicles showed an average of 46.53%, with aFF from medium follicles it was 40.00% and with aFF from large follicles it was 35.60%. The acrosome reaction after 4 hours of incubation was 30.06% for control, whereas for aFF of small follicles it was 66.3%, with aFF of medium follicles it was 58.86% and for aFF of large follicles, it was 67.63%. In the case of sperm motility, a significant difference is demonstrated for all treatments in relation to the control at the first hour, whereas only the treatments with aFF of small and medium follicles show a significant difference with respect to the control at the second hour. In the case of the acrosome reaction, all treatments with follicular fluid show a significant difference with respect to the control. It was concluded that alpaca follicular fluid favours sperm capacitation and the acrosome reaction in alpaca spermatozoa.

Early endometriosis is associated with infertility, and oxidative stress may play a role in the pathogenesis of disease-related infertility. This prospective case-control study aimed to compare the presence of oxidative stress markers in the follicular microenvironment and systemic circulation of infertile women with minimal/mild endometriosis (EI/II) versus individuals undergoing controlled ovarian stimulation for intracytoplasmic sperm injection (ICSI). Seventy-one blood samples (27 from infertile women with EI/II and 44 controls with tubal and/or male infertility factor) and 51 follicular fluid samples (19 EI/II and 32 controls) were obtained on the day of oocyte retrieval. Total hydroperoxides (FOX1 ), reduced glutathione, vitamin E, Superoxide dismutase, total antioxidant capacity, malondialdehyde, advanced oxidation protein products, and 8-hydroxy-2'-deoxyguanosine (8OHdG) concentrations were measured in both fluids. Women with EI/II showed higher FOX1 (8.48 ± 1.72 vs. 7.69 ± 1.71 μmol/g protein) and lower total antioxidant capacity (0.38 ± 0.18 vs. 0.46 ± 0.15 mEq Trolox/L) concentrations in serum, and higher 8OHdG concentrations (24.21 ± 8.56 vs. 17.22 ± 5.6 ng/ml) in follicular fluid compared with controls. These data implicate both systemic and follicular oxidative stress may in infertile women with EI/II undergoing controlled ovarian stimulation for ICSI. Furthermore, the elevated 8OHdG concentrations in follicular fluid of women with EI/II may be related to compromised oocyte quality.

Follicular fluid (FF) accumulates in the antrum of the ovarian follicle. In addition, FF provides the microenvironment for oocyte development, oocyte maturation and competence, which are acquired during follicular development. Superstimulatory treatment of 1-month-old lambs can achieve synchronous development of numerous growing follicles. However, these growing follicles are unable to completely mature and ovulate. Furthermore, the oocytes exhibit lower competence compared with those of ewes. In this study, we utilized an isobaric tag for relative and absolute quantification (iTRAQ)-based proteomics analysis and compare protein composition between pre-pubertal and adult superstimulated follicle FF in sheep. In total, 243 differentially expressed proteins were identified, including 155 downregulated and 88 upregulated between lamb and ewe. Gene ontology (GO) and KEGG pathway analysis indicated that the differentially expressed proteins are involved in signal transduction, anatomical structure development, stress response, metabolic pathways, and the complement and coagulation cascades. Many of the proteins known to affect follicle development were observed in lower abundance in FF of lamb (e.g. ADAMTS9, CD14, CTNNB1, FST, GCLC, HSPG2, IGFBP2, IGFBP6, INHBA, PRL, PAPPA, POSTN, PRDX1, SERPINA1, SOD3, STC1, VEGFC, etc.). However, a higher abundance was observed for proteasome proteins. Inadequate amounts of these proteins in FF may be lead to the unique characteristics of follicular development in lamb. These differentially expressed proteins illuminate the age-dependent changes in protein expression in the follicle microenvironment.

Purpose To assess the kisspeptin concentrations in follicular fluid and their relationship with clinical outcomes during assisted reproductive technology. Methods Thirty‐nine patients who were aged 24‐40 years and underwent oocyte retrieval for in vitro fertilization/intracytoplasmic sperm injection participated in this study. In 65 follicular fluid samples that had been obtained from 30 patients and their blood samples, the kisspeptin levels were measured in order to investigate the correlations with their gonadal hormone levels. Venous blood samples were collected from 14 patients to investigate their plasma kisspeptin levels across different phases of assisted reproductive technology. Results The follicular fluid kisspeptin level was significantly higher than that of the plasma level and was positively associated with the follicular fluid estradiol concentration and with the serum estradiol and number of mature oocytes. In the plasma, the maximum concentration of kisspeptin was observed on the day of ovum pick‐up and on the day of embryo transfer during ovarian stimulation for assisted reproductive technology. Conclusion Kisspeptin was present in the follicular fluid and the plasma kisspeptin concentration was affected by ovarian stimulation. Kisspeptin appears to affect oocyte maturation and ovulation.

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa. Fil: Breininger, Elizabeth. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina Fil: Dubois, D.. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina Fil: Pereyra, Vanina Eliana. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina Fil: Rodriguez, P. C.. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina Fil: Satorre, María Mercedes. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina Fil: Cetica, Pablo Daniel. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina

AimTo examine the potential of bovine follicular fluid (BFF) to attract bull spermatozoa. MethodsThe ability of the BFF to attract bull sperm was evaluated by observing changes in sperm migration after being placed in a cross-column chamber. The movement parameters of the heads and flagella of the sperm that were attracted to the BFF were analyzed by using the Computer Assisted Sperm Analysis system. ResultsIt was observed that 61.6% of the bull sperm migrated toward the BFF when the BFF was used at a concentration of 0.1%, but 67.2% of the sperm did not migrate toward the BFF at a concentration of 10%. Relatively larger numbers of both precapacitated and postcapacitated bull sperm migrated toward the BFF (0.1%). The ability of the 0.1% BFF to attract sperm probably affected both the normal artificial insemination (AI) fertility sperm and the poor AI fertility spermatozoa. The flagellar curvilinear ratio of the sperm winding to the 0.1% BFF was significantly higher than that of the prewinding sperm. ConclusionThese results could suggest that BFF potentially attracts bull sperm at a certain concentration, irrespective of the capacitation status of the sperm. Although the mechanism by which this attraction occurs remains unclear, these data imply that it could be related to BFF-dependent changes in the sperm flagellar curvilinear ratio., Article, REPRODUCTIVE MEDICINE AND BIOLOGY.16(2):133-138(2017)

In vitro maturation (IVM) in serum causes hampered expansion of porcine cumulus-oocyte complexes (COCs) due to excessive alpha2 -macroglobulin (A2M). This study investigated two hypotheses that could explain the effect of A2M: (i) binding of epidermal growth factor (EGF) to A2M, followed by its decreased availability; and (ii) inhibition of zinc-dependent metalloproteases. Cumulus expansion was evaluated based on the diameter of the COCs, the proportion of COCs participating in a floating cloud and the proportion of COCs with loss of cumulus cells. The first hypothesis of decreased EGF availability was tested by increasing the EGF concentration (20 and 50 ng/mL vs. 10 ng/mL), but was not confirmed because cumulus expansion did not improve. To verify the second hypothesis of inhibited zinc-dependent metalloproteases, the effect of tissue inhibitor of metalloproteases-3 (TIMP-3) on cumulus expansion during IVM with and without A2M was investigated. To immuno-neutralize A2M, serum was pre-incubated with A2M antibodies. Impaired cumulus expansion because of TIMP-3 could only be observed during IVM in 10% of serum with A2M antibodies. No effect of TIMP-3 was observed in medium without A2M antibodies. These results indicate that A2M and TIMP-3 share a common target, a zinc-dependent metalloprotease. Future research is directed toward the identification of the protease involved.

Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured in α-MEM+ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte-like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.

Polycystic ovary syndrome (PCOS) is closely related with the onset and development of metabolic abnormalities. However, the correlation between PCOS and kidney injury has not been clarified, and the underlying mechanism remains unknown. Herein, we performed a prospective survey in 55 PCOS and 69 healthy participants. Furthermore, the correlation analyses between serum testosterone and renal functional manifestations of patients and healthy subjects, including urinary albumin to creatinine ratio (UACR), urinary κ‐light chains (KapU), urinary λ‐light chains (LamU), urinary α1‐microglobulin (α1‐MU), and urinary β2‐microglobulin (β2‐MU), were analyzed. Compared with that in normal subjects, the levels of serum testosterone and UACR were significantly higher in PCOS patients. Serum testosterone is significantly correlated with the disease severity of PCOS. Although urinary excretions of KapU, LamU, α1‐MU, and β2‐MU did not increase in PCOS patients, they had a significantly positive correlation with the extent of serum testosterone in PCOS patients. IN vitro, primary cultured human ovary granulosa cells (GCs) were isolated from the follicular fluid (FF) extracting from PCOS patients and controls. FF, especially which extracted from PCOS patients with a high expression of serum testosterone, significantly induced cell apoptosis and inflammation in human GCs. To examine the communication between PCOS and kidney injury, a human proximal tubular epithelial cell line (HKC‐8) was cultured and administered FF. Interestingly, FF from PCOS patients with a higher level of serum testosterone induced fibrotic lesions in HKC‐8 cells. These data suggest serum testosterone plays a critical role in PCOS and PCOS‐associated kidney injury. Serum testosterone may serve as a promising indicator for kidney fibrotic injury outcomes in PCOS patients.

Communication between the oocyte and cumulus facilitates oocyte growth, cell cycle regulation, and metabolism. This communication is mediated by direct contact between oocytes and cumulus cells, and soluble secreted molecules. Secreted molecules involved in this process are known inflammatory mediators. Lipopolysaccharide (LPS) is detected in follicular fluid and is associated with reduced fertility, whereas accumulation of inflammatory mediators in follicular fluid, including tumor necrosis factor-α (TNF-α), is associated with female infertility. Maturation of oocytes in the presence of LPS or TNF-α reduces meiotic maturation and the capacity to develop to the blastocyst. Here we evaluated the abundance of 92 candidate genes involved immune function, epigenetic modifications, embryo development, oocyte secreted factors, apoptosis, cell cycle, and cell signaling in bovine cumulus cells or zona-free oocytes after exposure to LPS or TNF-α during in vitro maturation. We hypothesize that LPS or TNF-α will alter the abundance of transcripts in oocytes and cumulus cell in a cell type dependent manner. Exposure to LPS altered abundance of 31 transcripts in oocytes (including ACVR1V, BMP15, DNMT3A) and 12 transcripts in cumulus cells (including AREG, FGF4, PIK3IP1). Exposure to TNF-α altered 1 transcript in oocytes (IGF2) and 4 transcripts in cumulus cells (GJA1, PLD2, PTGER4, STAT1). Cumulus expansion was reduced after exposure to LPS or TNF-α. Exposing COCs to LPS had a marked effect on expression of targeted transcripts in oocytes. We propose that altered oocyte transcript abundance is associated with reduced meiotic maturation and embryo development observed in oocytes cultured in LPS or TNF-α.

The growth of ovarian follicles is accompanied by fluid-filled antrum formation. Water movement within the follicular wall is predominantly transcellular via membranous water channels named aquaporins (AQPs). Androgens are important regulators of mammalian folliculogenesis, and their prenatal and/or neonatal deficiency affects female fertility in adulthood. Therefore, this study was performed to determine whether gestational or neonatal exposure to the anti-androgen flutamide influences androgen-dependent AQP5 expression in pre-antral and large antral follicles of adult pigs. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 post-natally. The ovaries were collected from flutamide-treated and non-treated (control) sexually mature pigs. In pre-antral follicles, AQP5 mRNA and protein levels were both downregulated following maternal (p < 0.01 and p < 0.01, respectively) and neonatal (p < 0.01 and p < 0.01, respectively) flutamide exposure. Likewise, the expression of mRNA (p < 0.01 and p < 0.001, respectively) and protein (p < 0.05 and p < 0.01, respectively) for AQP5 were diminished in large antral follicles in both groups. Immunohistochemistry showed decreased intensity of AQP5 immunoreaction in pre-antral (p < 0.01) and large antral (p < 0.001) follicles following flutamide treatment. Moreover, radioimmunological analysis revealed that changes observed in AQP5 expression corresponded with diminished follicular androgens production after both maternal (p < 0.05 and p < 0.05, respectively) and neonatal (p < 0.05 and p < 0.01, respectively) flutamide administration. Therefore, AQP5 appears to be a potential regulator of follicular fluid accumulation, under androgen control, and may be a key factor in antral follicle growth.

Oocytes play critical roles in regulating the expression of transcripts encoding the glycolytic enzymes phosphofructokinase, platelet (PFKP) and lactate dehydrogenase A (LDHA) in granulosa cells in mice, but whether this is the case in pigs or other mammals has not been adequately investigated. Therefore, the aim of this study was to determine whether porcine oocytes regulate the expression levels of these transcripts in granulosa cells in vitro. Porcine cumulus cells expressed higher levels of PFKP and LDHA transcripts than mural granulosa cells (MGCs). However, co-culturing with oocytes had no significant effect on the isolated cumulus cells. While murine oocytes promoted the expression of both Pfkp and Ldha transcripts by murine MGCs, porcine oocytes promoted the expression of only Pfkp, but not Ldha transcripts by murine MGCs. Neither murine nor porcine oocytes affected PFKP and LDHA expression by porcine MGCs. Moreover, in the presence of porcine follicular fluid, porcine oocytes maintained the expression of PFKP, but not LDHA by porcine cumulus cells. Therefore, porcine oocytes are capable of regulating the expression of PFKP but not LDHA in granulosa cells in coordination with unknown factor(s) present in the follicular fluid.

SummaryReasons for performing study The wide variation in circulating anti-Mullerian hormone (AMH) concentrations between mares is attributed to differences in antral follicle count (AFC) which may reflect follicular function. There are few data regarding variations in AFC and associated regulatory factors for AMH in the equine follicle during follicular development. Objectives To examine molecular and hormonal differences in the equine follicle in relation to variations in AFC and circulating AMH concentrations during follicular development and to identify genes co-expressed with AMH in the equine follicle. Study design Observational study. Methods Plasma AMH concentrations and AFC were determined in 30 cyclic mares. Granulosa cells, theca cells and follicular fluid were recovered from growing (n = 17) or dominant follicles (n = 13). The expression of several genes, known to be involved in folliculogenesis and steroidogenesis, was examined using real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Intrafollicular oestradiol and AMH concentrations were determined by immunoassay. Results Within growing follicles, the expression of AMH, AMHR2, ESR2 and INHA in granulosa cells was positively correlated with AFC and plasma AMH concentrations. In addition, the expression of ESR1 and FSHR was positively associated with plasma AMH concentrations. No significant associations were detected in dominant follicles. Furthermore, there was no association between AMH or oestradiol concentrations in follicular fluid and variations in AFC. Finally, the expression of AMH and genes co-expressed with AMH (AMHR2, ESR2 and FSHR) in granulosa cells as well as intrafollicular AMH concentrations decreased during follicular development while intrafollicular oestradiol concentrations increased and were inversely related to intrafollicular AMH concentrations. Conclusions This study indicates that variations in AFC and circulating AMH concentrations are associated with molecular changes in the growing equine follicle.

Summary This study was undertaken to elucidate the effect of ammonia-generating diet on serum and follicular fluid ammonia and urea levels, serum oestrogen and progesterone concentrations and granulosa cell growth and secretion parameters in ewes (Ovis aries). Ewes were fed with 14% CP diet (control) or ammonia-generating diet or ammonia-generating diet plus soluble sugar. The serum and follicular fluid ammonia and urea level, serum oestrogen and progesterone levels and granulosa cell (obtained from ovaries of slaughtered ewes) growth parameters and secretory activities were estimated. Ammonia-generating diet (high-protein diet) increased the serum ammonia and urea concentration. Supplementation of soluble sugar significantly reduced the ammonia concentration in serum with comparable levels as in control group; however, the urea level in the same group was higher than that observed in control group. Supplementation of soluble sugar significantly reduced the follicular fluid ammonia concentration; however, the level was significantly higher compared to control group. Supplementation of soluble sugar brought down the follicular fluid urea level comparable to that observed in control group. Oestrogen and progesterone levels remained unchanged in ewes fed with different types of diet. Oestrogen and progesterone secretion were significantly lowered from granulosa cells recovered from ewes fed with high ammonia-generating diet. Low metabolic activity and high incidence of apoptosis were observed in granulosa cells obtained from ovaries of ewes fed with ammonia-generating diet.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors, such as members of the insulin-like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy-associated plasma protein A (PAPP-A). The aim of this study was to determine IGFR1 and PAPP-A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP-A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP-A present in the follicular fluid from these follicles showed no differences. Although no PAPP-A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle. Fil: Rodríguez, Fernanda Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Colombero, Fernanda de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Amweg, Ayelen Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Huber, Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Gareis, Natalia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Salvetti, Natalia Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Rey, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina