Follicular Fluid Concentrations Research Articles

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

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

FF-MAS prevents porcine ovarian granulosa cells from hypoxia-induced apoptosis via inhibiting STAT4 expression.

Follicular fluid meiosis-activating sterol (FF-MAS) is a small molecule compound found in follicular fluid, named for its ability to induce oocyte resumption of meiosis. Granulosa cells (GCs) within the follicle are typically located in a hypoxic environment under physiologic conditions due to limited vascular distribution. Previous research suggests that hypoxia-induced cell cycle arrest and apoptosis in GCs may be crucial triggering factors in porcine follicular atresia. However, the impact of FF-MAS on GCs within follicles has not been explored so far. In this study, we uncovered a novel role of FF-MAS in facilitating GC survival under hypoxic conditions by inhibiting STAT4 expression. We found that STAT4 expression was upregulated in porcine GCs exposed to 1% O2. Both gain and loss of function assays confirmed that STAT4 was required for cell apoptosis under hypoxia conditions, and that the GC apoptosis caused by hypoxia was markedly attenuated following FF-MAS treatment through inhibition of STAT4 expression. Correlation analysis in vivo revealed that GC apoptosis was associated with increased STAT4 expression, while the FF-MAS content in follicular fluid was negatively correlated with STAT4 mRNA levels and cell apoptosis. These findings elucidate a novel role of FF-MAS-mediated protection of GCs by inhibiting STAT4 expression under hypoxia, which might contribute to the mechanistic understanding of follicular development.

Hyperandrogenic environment regulates the function of ovarian granulosa cells by modulating macrophage polarization in PCOS.

Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder characterized by oligo-anovulation, hyperandrogenism, and polycystic ovaries, with hyperandrogenism being the most prominent feature of PCOS patients. However, whether excessive androgens also exist in the ovarian microenvironment of patients with PCOS, and their modulatory role on ovarian immune homeostasis and ovarian function, is not clear. Follicular fluid samples from patients participating in their first in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment were collected. Androgen concentration of follicular fluid was assayed by chemiluminescence, and the macrophage M1:M2 ratio was detected by flow cytometry. In an in vitro model, we examined the regulatory effects of different concentrations of androgen on macrophage differentiation and glucose metabolism levels using qRT-PCR, Simple Western and multi-factor flow cytometry assay. In a co-culture model, we assessed the effect of a hyperandrogenic environment in the presence or absence of macrophages on the function of granulosacells using qRT-PCR, Simple Western, EdU assay, cell cycle assay, and multi-factor flow cytometry assay. The results showed that a significantly higher androgen level and M1:M2 ratio in the follicular fluid of PCOS patients with hyperandrogenism. The hyperandrogenic environment promoted the expression of pro-inflammatory and glycolysis-related molecules and inhibited the expression of anti-inflammatory and oxidative phosphorylation-related molecules in macrophages. In the presence of macrophages, a hyperandrogenic environment significantly downregulated the function of granulosa cells. There is a hyperandrogenic microenvironment in the ovary of PCOS patients with hyperandrogenism. Hyperandrogenic microenvironment can promote the activation of ovarian macrophages to M1, which may be associated with the reprogramming of macrophage glucose metabolism. The increased secretion of pro-inflammatory cytokines by macrophages in the hyperandrogenic microenvironment would impair the normal function of granulosa cells and interfere with normal ovarian follicle growth and development.

Endocrine-disrupting chemical concentrations in follicular fluid and follicular reproductive hormone levels.

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

Ovarian overexpression of ASMT gene increases follicle numbers in transgenic sheep: Association with lipid metabolism

Melatonin plays an important role in mammalian reproductive activities, to further understand the effects of endogenous melatonin on functions of ovary, the transgenic sheep with overexpression of melatonin synthetic enzyme gene ASMT in ovary were generated. The results showed that total melatonin content in follicular fluid of transgenic sheep was significantly greater than that in the wild type. Accordingly, the follicle numbers of transgenic sheep were also significantly greater than those in the WT. The results of follicular fluid metabolites sequencing showed that compared with WT, the differential metabolites of the transgenic sheep were significantly enriched in several signaling pathways, the largest number of metabolites was lipid metabolism pathway and the main differential metabolites were lipids and lipoid molecules. SMART-seq2 were used to analyze the oocytes and granulosa cells of transgenic sheep and WT sheep. The main differential enrichment pathway was metabolic pathway, in which lipid metabolism genes accounted for the majority. In conclusion, this is the first report to show that ovary overexpression of ASMT increased local melatonin production and follicle numbers. These results may imply that ASMT plays an important role in follicle development and formation, and melatonin intervention may be a potential method to promote this process.

Effect of COVID-19 infection and vaccination on SARS-CoV-2 antibody titer change following ovarian stimulation: Prospective analysis of IVF outcomes.

The coronavirus disease 2019 (COVID-19) outbreak caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has affected various medical fields worldwide. However, relatively few studies have examined the impact of COVID-19 infection and vaccination on in vitro fertilization (IVF) outcomes and changes in SARS-CoV-2 antibody concentration in follicular fluid (FF). A total of 45 women were prospectively recruited and assigned to 3 groups: uninfected and non-vaccinated control group (Control group), infected group (COVID + group), and vaccinated group (Vaccination group). Serum and follicular fluid (FF) estradiol, progesterone, and SARS-CoV-2 antibody concentrations were measured. There were no statistical differences in the total number of retrieved oocytes (P = .291), mature oocytes (P = .416), and good-quality embryos (P = .694) among the 3 groups. In the vaccination group, BNT162b2 exhibited a significantly lower trigger-day serum estradiol/MII oocyte level (110.6 pg/mL) than other vaccines (289.5 pg/mL) (P = .006). No statistical differences in serum (P = .687) and FF (P = .108) SARS-CoV-2 antibody changes were noted among the 3 groups. Only FF antibody changes exhibited statistically significant differences between the BNT162b2 and other vaccine subgroups (P = .047). COVID-19 infection and vaccination do not affect IVF outcomes. However, the effect of BNT162b2 on steroidogenesis of the mature oocyte and FF SARS-CoV2 antibody titer should be further investigated.

Biochemical profiling of the follicular environment to predict oocyte competence in cattle.

To identify markers of oocyte competence, we compared the biochemical characteristics of fluid and cells from follicles containing oocytes with different capacities to form an embryo. Follicles (5-6 mm) were dissected, and follicular fluid (FF), granulosa cells (GC), cumulus cells (CC) from immature and mature cumulus-oocyte-complexes (COC) were individually collected. The oocytes were matured, fertilized, and cultured individually until day 8 (D8) of development. On D8, the samples were grouped according to embryo production into those that gave rise to blastocysts (EMB) and those that did not reach the blastocyst stage (NEMB). In CCs from immature and mature COCs and GCs, expression of CASP3, SERPINE2, VCAN, LUM, FSHR, EGFR, PGR, and GHR genes was quantified. Cell-free DNA (cfDNA), progesterone, and estradiol concentrations in the FF were determined. Data were analyzed by Mann-Whitney U test (GraphPad Prism 9). GHR was highly expressed in immature CCs from the EMB group, whereas CASP3 was highly expressed in mature CCs from the NEMB group (P<0.05). During maturation, the expression of CASP3 and GHR genes increased only in the NEMB group. ART2 cfDNA was highly detected in FF of the NEMB compared to the EMB group. Progesterone concentration was similar between the groups, whereas estradiol concentration was higher (P<0.05) in the EMB than in the NEMB group. It was concluded that a higher level of GHR transcripts in immature CCs, lower CASP3 expression in CCs from matured COCs, lower levels of ART2, and higher estradiol concentrations in FF may indicate oocytes with greater potential for development.

Morphological Characteristics of Ovarian Tissues and Follicular Fluid Metabolites of Female Lambs and Ewes in Subtropics

Background: Oocyte and follicular fluid recovery and evaluation are a crucial step in assisted reproductive technologies. The aims of the current study were to evaluate the ovarian tissues and their resulting oocytes and follicular fluid of Awassi female lambs and ewes in subtropics of Saudi Arabia. Methods: Thirty reproductive systems of female lambs in addition to forty reproductive systems of ewes of follicular and luteal phases were collected. The ovarian tissues were evaluated through ovarian follicle numbers, oocytes recovery in addition to determination of follicular fluid (FF) metabolites. The ovarian follicles were categorized into small ( less than 3 mm), medium (3-5 mm) and large follicles ( greater than 5 mm). The medium and large ovarian follicles were aspirated for FF collection and oocytes recovery. The FF concentrations of total protein (g/dl), urea (mg/dl), glucose (mg/dl), total cholesterol (mg/dl) and triglycerides (mg/dl) were determined. Result: The weight of reproductive system and ovary were increased of ewes compared to female lambs. The number of small follicles was not differed among groups whereas the numbers of medium and large follicles increase in ewes groups. The highest number of medium follicles was found in ewes of follicular phase compared to other groups. Although the percentage of oocyte recovery increased in ewes compared to lambs, the oocytes quality was not differed among groups. The total protein values were high (P less than 0.05) in FF of ewes compared to that of female lambs. On the other, the values of urea and triglycerides were low (P less than 0.05) in FF of ewes compared to FF of female lambs. Moreover, the values of total protein, urea and triglycerides were comparable in FF of ewes during follicular or luteal phase. Additionally, there is slight increase of glucose and slight decrease of total cholesterol values in FF of ewes compared to FF of female lambs. In conclusion, follicles of ovarian tissues, oocyte recovery and quality and follicular fluid biochemistry of female lambs and ewes showed differences. The recovered oocytes and FF are available for assisted reproductive techniques.

Omentin expression in the ovarian follicles of Large White and Meishan sows during the oestrous cycle and in vitro effect of gonadotropins and steroids on its level: Role of ERK1/2 and PI3K signaling pathways.

Omentin (ITLN1) is a novel adipokine mainly expressed in the white adipose tissue. It plays a crucial role in the metabolic homeostasis and insulin sensitivity. Our last study documented that ITLN1 levels in the adipose tissue and plasma are lower in fat Meishan (MS) compared to normal weight Large White (LW) pigs. The aim of this study was to investigate transcript and protein concentrations of ITLN1 as well as its immunolocalisation in the ovarian follicles and examine the molecular mechanism involved in the regulation of its expression in response to gonadotropins (FSH, LH) and steroids (P4, T, E2). Ovarian follicles were collected from LW and MS sows on days 2-3, 10-12, and 14-16 of the oestrous. We found the elevated ITLN1 expression in the ovarian follicles and the increase of concentrations in follicular fluid (FF) of LW pigs vs MS pigs; in both breeds of pigs, the levels of ITLN1 increased with the oestrous progression. We noted ITLN1 signals in oocyte, granulosa and theca cells. Gonadotropins and steroids increased ITLN1 levels in the ovarian follicle cells of LW pigs, while in MS pigs, we observed only the stimulatory effect of LH and T. Both extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol 3'-kinase (PI3K) were involved in the regulation of ITLN1. Our study demonstrated the levels and regulation of ITLN1 in the porcine ovarian follicles through ERK1/2 and PI3K signaling pathways.

Activity of enzymes in the follicular fluid and outcome of in vitro fertilization.

Defining certain parameters of the oocyte microenvironment that can be simply and quickly detected and that enable differentiation of oocytes that have better or worse quality could potentially increase the efficacy of the in vitro fertilization (IVF) method. Follicular fluid represents the microenvironment for the development of oocytes during different stages of maturation. Enzymes present in the follicular fluid may affect the quality of oocytes. There are inconsistencies in the literature concerning enzyme concentration in follicular fluid. The main aim of this study was to examine whether the concentration of enzymes aspartate aminotransferase, alanine aminotransferase (ALT), alkaline phosphatase, and lactate dehydrogenase in follicular fluid can influence the outcome of IVF. The study included 120 patients who were involved in the process of IVF. We used the follicular fluid obtained after follicular aspiration in the ovaries as biological material for the analysis. The values of enzymes LDL (method-kinetic UV test), aspartate aminotransferase (method-kinetic UV test), ALT (method-kinetic UV test) and alkaline phosphatase (method-colorimetric kinetic test) in the follicular fluid were determined on the analyzer АU 680, Becman Coulter. The criteria of the Istanbul consensus of clinical embryologists were used for the embryo assessment. The software package SPSS 20 was used for the statistical processing of data. The results of follicular fluid sample analysis showed a correlation between ALT concentration in the follicular fluid and IVF outcome. Based on our results, we can conclude that ALT concentration affects the outcome of IVF.

Association between Perfluoroalkyl Substances in Follicular Fluid and Polycystic Ovary Syndrome in Infertile Women.

In recent years, perfluoroalkyl substances (PFASs), a family of fluorinated organic com pounds, have garnered much attention due to their reproductive and developmental toxicity in humans. Polycystic ovary syndrome (PCOS) is a prevalent endocrine disease that affects women of reproductive age and is a significant contributor to female infertility. A previous study suggested that PFASs play a possible role in PCOS. We conducted a clinical study investigating the relationship between PCOS and PFAS in follicular fluid. A total of 73 infertile patients with PCOS and 218 controls were recruited from the International Peace Maternity and Child Health Hospital, affiliated with the Shanghai Jiao Tong University School of Medicine. The concentrations of 12 PFASs in follicular fluid samples and sex hormones in serum were measured. Correlation analysis and multiple linear regression revealed a positive relationship between perfluorooctanoic acid (PFOA) and testosterone (T) concentrations. The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for each PFAS were estimated using multivariable logistic regression and quantile-based g-computation (QGC). The PFOA concentrations in follicular fluid were correlated with increased odds of PCOS (second vs. first quartile: OR = 3.65, 95% CI: 1.47-9.05, p = 0.005; third vs. first quartile: OR = 2.91, 95% CI: 1.17-7.26, p = 0.022; fourth vs. first quartile: OR = 3.13, 95% CI: 1.21-8.09, p = 0.019; P for trend = 0.032). This association was confirmed with QGC. Mediation analysis suggested that the mediation effect of T in association with PFOA and PCOS was not statistically significant. Our study suggests that PFOA may be a risk factor for PCOS.

The intrafollicular concentrations of biologically active cortisol in women rise abruptly shortly before ovulation and follicular rupture.

What is the temporal activity and the concentration in follicular fluid (FF) of the anti-inflammatory steroid cortisol during the ovulatory process in humans? Intrafollicular concentrations of cortisol become massively upregulated close to ovulation concomitant with an exceptionally high biological activity securing a timely and efficient termination of inflammatory processes. Ovulation has been described as a local, controlled inflammatory process resulting in the degeneration of the follicle wall which facilitate oocyte extrusion. Ovulation also affects the glucocorticoid metabolism of granulosa cells (GCs) and although de novo synthesis of cortisol only occurs in the adrenal cortex, the mid-cycle surge has been shown to induce a change from high expression of HSD11B2, inactivating cortisol to cortisone, to high expression of HSD11B1 which reversibly catalyses cortisol production from cortisone. Furthermore, high concentrations of progesterone and 17OH-progesterone within follicles may cause dislodging of cortisol from cortisol binding protein (CBP) thereby activating the biological activity of cortisol. This prospective cohort study included 50 women undergoing fertility treatment according to a standard antagonist protocol at a university hospital-affiliated fertility clinic in Denmark. Women donated FF and GCs from one follicle for research purpose aspirated at one of four time points during the process of final maturation of follicles: T = 0 h, T = 12 h, T = 17 h, T = 32 h. A second sample was collected at oocyte pick up at T = 36 h. The concentration of cortisol and cortisone together with a range of sex steroids was measured by LC-MS/MS in FF collected at the five time points mentioned above. Whole genome microarray data, validated by q-PCR analysis, was used to evaluate gene expression of CYP11B1, CYP21A2, HSD11B1, HSD11B2, and NR3C1 in GCs at the same time points. The concentration of cortisol was significantly increased from a few nM at 0 h to around 100-140 nM (P ≤ 0.0001) at 32-36 h, whilst cortisone was almost constant from 0 to 17 h at a concentration of between 90 and 100 nM being significantly reduced to 25-40 nM (P ≤ 0.0001) at 32-36 h. This was paralleled by a 690-fold upregulation of HSD11B1 from 0 to 12 h increasing to a more than 20.000-fold change at 36 h. HSD11B2 was quickly downregulated 15- to 20-fold after ovulation induction. Concentrations of progesterone and 17OH-progesterone increased during the ovulatory process to high levels which in essence displaces cortisol from its binding protein CBP due to similar binding affinities. Furthermore, a significant decrease in 11-deoxycortisol expression was seen, but CYP11B1 expression was below detection limit in GCs. The study included women undergoing ovarian stimulation and results may differ from the natural cycle. More observations at each specific time point may have strengthened the conclusions. Furthermore, we have not been able to measure the actual active biological concentration of cortisol. For the first time, this study collectively evaluated the temporal pattern of cortisol and cortisone concentrations during human ovulation, rendering a physiological framework for understanding potential dysregulations in the inflammatory reaction of ovulation. This research was supported by the University Hospital of Copenhagen, Rigshospitalet, and Novo Nordisk Foundation grant number NNF21OC00700556. Interreg V ÔKS through ReproUnion (www.reprounion.eu); Region Zealand Research Foundation. The funders had no role in study design, collection of data, analyses, writing of the article, or the decision to submit it for publication. The authors have no conflicts of interest to declare. N/A.

Elevated levels of Zn, Cu and Co are associated with an increased risk of endometriosis: Results from a case[sbnd]control study

BackgroundEndometriosis is a common gynecological disease that affects approximately 5 %∼10 % of reproductive-aged women. Zinc (Zn), selenium (Se), copper (Cu), cobalt (Co) and molybdenum (Mo) are essential trace elements and are very important for human health. However, studies on the relationship between mixtures of essential trace elements and the risk of endometriosis are limited and inconsistent. In particular, studies confirming the association via different sample types are limited. ObjectiveThis study aimed to investigate the associations between Zn, Se, Cu, Co and Mo concentrations in blood and follicular fluid (FF) and endometriosis risk in a Chinese population. MethodsA total of 609 subjects undergoing in vitro fertilization (IVF) were recruited; 836 samples were analyzed, including 451 blood samples (234 controls and 217 cases) and 385 FF samples (203 controls and 182 cases). In addition, 227 subjects provided both blood and FF samples. Zn, Se, Cu, Co and Mo concentrations in blood and FF were quantified via inductively coupled plasma−mass spectrometry (ICP−MS). The associations between the levels of Zn, Se, Cu, Co and Mo and the risk of endometriosis were assessed using single-element models (logistic regression models), and the combined effect of the trace elements on endometriosis risk was assessed using multielement models (Bayesian kernel machine regression (BKMR) and weighted quantile sum (WQS) regression). ResultsBased on the single-element models, significant associations of Zn concentrations in blood (high-level vs. low-level group: aOR = 14.17, 95 % CI: 7.31, 27.50) and FF (first tertile vs. second tertile group: aOR = 0.34, 95 % CI: 0.16, 0.71; third tertile vs. second tertile group: aOR = 2.32, 95 % CI: 1.38, 3.91, respectively) and Co concentrations in blood (first tertile vs. second tertile group, aOR = 0.24, 95 % CI: 0.12, 0.48) and FF (third tertile vs. second tertile group: aOR = 3.87, 95 % CI: 2.19, 6.84) with endometriosis risk were found after adjustment for all confounders. In FF, Cu and Mo levels were significantly greater among the cases than among the controls, with a positive association with endometriosis risk (Cu (first tertile vs. second tertile group: aOR = 0.39, 95 % CI: 0.19, 0.81; third tertile vs. second tertile group: aOR = 2.73, 95 % CI: 1.61, 4.66, respectively) and Mo (high-level vs. low-level group: aOR = 14.93, 95 % CI: 7.16, 31.12)). However, similar associations between blood Cu and Mo levels and endometriosis risk were not found. In addition, the levels of these five essential trace element mixtures in blood and in FF were significantly and positively associated with endometriosis risk according to the BKMR analyses; the levels of Zn and Cu in blood and the levels of Mo in FF were significantly related to the risk of endometriosis, and the posterior inclusion probabilities (PIPs) were 1.00, 0.99 and 1.00 for Zn and Cu levels in blood and Mo levels in FF, respectively. Furthermore, Zn and Mo were the highest weighted elements in blood and FF, respectively, according to WQS analyses. ConclusionThe risk of endometriosis was associated with elevated levels of several essential trace elements (Zn, Cu and Co). Elevated levels of these elements may be involved in the pathomechanism of endometriosis. However, further studies with larger sample sizes will be necessary to confirm these associations.

Follicular fluid concentration of soluble Human-G Leukocytic Antigen (sHLA-G) in in vitro fertilization cycles of women with and without peritoneal endometriosis.

The objective of this research is to investigate the association between the concentrations of soluble human leukocyte G antigen (sHLA-G) in the follicular fluid (FF) in infertile patients with peritoneal endometriosis submitted to in vitro fertilization. We performed a cross-sectional study, including ninety-six women undergoing in vitro fertilization (IVF) ageing ≤ 40 years. Infertile patients were classified into two groups: with endometriosis diagnosed by laparoscopy and without endometriosis due to tubal factor. ELISA measured soluble HLA-G in the FF of a pool of punctured (more than 17mm) follicles from women with endometriosis and without endometriosis who were subjected to ovulation induction for IVF. Embryos obtained after fertilization were classified according to the graduated embryo score (GES). Groups were comparables in terms of age, the number of follicles, AMH, FSH and all included reproductive outcomes. There was no association between sHLA-G concentrations and the average score of the generated embryos (p>0.05). Measurement of sHLA-G in the follicle fluid in women with endometriosis and without endometriosis (tubal factor) showed no significant difference (p>0.05). We also compared sHLA-G per follicle and per embryo, which were not different between both groups (p>0.05). Patients with peritoneal endometriosis submitted to IVF did not demonstrate an altered sHLA-G in the follicular fluid compared to the follicular fluid sHLA-G concentration in tubal factor patients. Also, this molecule was not linked to any other reproductive outcome.

Effects of stem cell factor in follicular fluid and granulosa cells on oocyte maturity and clinical pregnancy.

Stem cell factor (SCF) is implicated in cell growth, proliferation, differentiation, migration, and apoptosis. SCF in follicular fluid (FF) and granulosa cells (GCs) plays a key role in oocyte maturation and clinical pregnancy; however, the exact mechanism is unclear. We aimed to investigate SCF potential in predicting oocyte maturity and clinical pregnancy. We collected 60 FF and 60 GCs samples from different patients with infertility. Real-time polymerase chain reaction and cellular immunofluorescence analyses were used to quantitatively and qualitatively determine SCF concentration in GCs; enzyme-linked immunosorbent assay was used to determine SCF concentration in FF. GC and FF SCF concentrations were positively correlated with metaphase (M)II oocyte proportion and clinical pregnancy (R = 0.280, 0.735 vs R = 0.257, 0.354). SCF concentrations in GCs were significantly higher in the clinical pregnancy group than in the nonclinical pregnancy group. Immunofluorescence analysis showed that SCF expression was higher in the clinical pregnancy and high-MII -oocyte proportion groups. Receiver operating characteristic curve analysis showed that combined SCF and serum anti-Müllerian hormone levels could predict oocyte maturity and clinical pregnancy better than either of these factors alone. SCF concentration in GCs and FF can serve as a predictor of oocyte maturity and clinical pregnancy.

Analysis of functional activity of granulosa cells of porcine ovarian follicles

Functioning of the granulosa cells of animal ovarian follicles (OF) determines the growth and development of the OF and the oocyte formed in it, which determines their use in modeling the composition of media for oocyte maturation in vitro in cell reproductive technologies. The purpose of the study is to determine the peculiarities of functional activity of granulosa cells in follicles of different diameters (&lt; 3, 3–5, 5–8 mm) using complex testing of granulosa cell viability parameters (apoptosis level, production of reactive oxygen species, functional activity of mitochondria and lipidome) and hormonal status of follicle fluid (estradiol, testosterone, anti-Müllerian hormone level) in order to optimize the composition of the culture media for in vitro maturation of pig oocytes. The experiments found that the proportion of apoptotic cells in the OF increased as the diameter of the OF increased (by 11% in OFs 5–8 mm in diameter, by 4% in OFs &lt; 3 mm in diameter). There was an increase in the number of high reactive oxygen species cells (by 10% in 5–8 mm diameter OF compared with &lt; 3 mm diameter OF) and high mitochondrial activity (by 12% in 5–8 mm diameter OF compared with &lt; 3 mm diameter OF). Correlations were found between the group of cells with high generation of reactive oxygen species and the level of apoptosis (0.500, p &lt; 0.05), as well as with high levels of mitochondrial activity (0.500, p &lt; 0.05). Estradiol concentration in follicular fluid increased as the OF grew (1.50 ng/ml in OF &lt; 3 mm diameter and 2.09 ng/ml in OF 3–5 mm diameter, p &lt; 0.01), being almost unchanged in OF 5–8 mm diameter (2.11 ng/ml), as was the testosterone concentration. Correlations were found between the proportion of the cells with high NileRed/LD FI (a marker of lipid droplet functional activity) and the levels of estradiol (0.998, p &lt; 0.05), testosterone (0.500, p &lt; 0.05), and anti-Müllerian hormone (–0.500, p &lt; 0.05) in the dynamics of folliculogenesis, as well as between estradiol concentration and the proportion of cells with high mitochondrial activity (0.746, p &lt; 0.05). Correlations were detected between the concentrations of estradiol and anti-Müllerian hormone, testosterone and anti-Müllerian hormone (–1.000, p &lt; 0.05), between the level of apoptotic cells and the concentration of anti-Müllerian hormone in follicular fluid (–1.000, p &lt; 0.05). The results of the monitoring of the viability and functional activity of porcine OF granulosa cells supplement the available data on the peculiarities of their functioning in the dynamics of folliculogenesis and can be used in modeling the systems of in vitro maturation of donor oocytes of Sus Scrofa Domesticus.

Severe body condition loss lowers hepatic output of IGF1 with adverse effects on the dominant follicle in dairy cows

The severe loss of body condition score (BCS) during the early lactation period has been associated with infertility in cows. However, the mechanisms are not fully understood. The aim of this study was to examine the effect of BCS loss on liver health, and ovarian functions in cows during early lactation. Retrospectively multiparous cows from two farms were categorized based on units of BCS (1–5 scale) loss as Moderate (MOD, <0.75 units; n = 11) or Severe (SEV, ≥0.75 units; n = 9) loss groups. From Weeks −3 to 7, relative to calving, MOD and SEV cows lost on average 0.4 and 1.0-unit BCS, respectively. All data except hepatic transcriptomes were analyzed with PROC MIXED procedure of SAS. The plasma concentration of non-esterified fatty acids at Week 0 and 1, ß-hydroxy butyrate at Week 1, and γ-glutamyl transferase at Weeks 1 and 7 relative to calving were higher in SEV cows. Hepatic transcriptome analysis showed that 1 186 genes were differentially expressed in SEV (n = 3) compared to MOD (n = 3) cows at Week 7 after calving. Pathway analysis revealed that significant DEGs in SEV cows enriched in lipid metabolisms including, lipid metabolic process, ether lipid metabolism, fatty acid beta-oxidation, fatty acid biosynthetic process, fatty acid metabolic process, fat digestion and absorption, linoleic acid metabolism, alpha-linolenic acid metabolism. The impaired liver function in SEV cows was associated with 1.5-fold reduction of hepatic IGF1 gene expression and lower serum IGF1 concentrations. At the ovarian level, SEV cows had lower IGF1 concentration in the follicular fluid of the dominant follicle of the synchronized follicular wave compared to that of MOD cows at 7 weeks after calving. Further, the follicular fluid concentration of estradiol-17β was lower in SEV cows along with lower transcript abundance of genes from granulosa cells associated with dominant follicle competence, including CYP19A1, NR5A2, IGF1, and LHCGR. These data show that SEV loss of BCS during early lactation leading up to the planned start of breeding is associated with liver dysfunction, including lower IGF1 secretion, and impaired function of the dominant follicle in the ovary.

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

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

Ovarian follicular fluid levels of phthalates and benzophenones in relation to fertility outcomes

BackgroundMany endocrine disrupting chemicals (EDCs), for instance phthalates and benzophenones, are associated with adverse fertility outcomes and semen quality parameters. ObjectiveTo evaluate if concentrations of selected phthalate metabolites and benzophenones measured in follicular fluid are associated with fertility outcomes (i.e., reproductive hormones, antral follicle count, detected heartbeat at gestational week 7, and live birth) and, in a supplementary study, if measured concentrations of chemicals in follicular fluid can exert biological effects on human spermatozoa. MethodsOverall, 111 couples from a fertility clinic in Denmark contributed with 155 follicular fluid samples. Concentrations of 43 metabolites from 19 phthalates and phthalate substitutes and six benzophenones were measured in follicular fluid using liquid chromatography-tandem mass spectrometry. Multiple linear and logistic regression with an applied generalized estimating equation model allowing more than one measurement per woman assessed the association between follicular EDC levels and fertility outcomes. The assessment of biological effects of individual and mixtures of EDCs on human spermatozoa was conducted through a human sperm cell based Ca2+-fluorimetric assay. ResultsBenzophenone-3 (BP-3) and seven metabolites of five phthalates were detectable in follicular fluid. Women with metabolites of dibutyl phthalate isomers in the highest tertiles had lower antral follicle count (MiBP: β = −5.35 [95 % CI: −9.06; −2.00], MnBP: β = −5.25 [95 % CI: −9.00; −2.00]) and lower odds for detecting a heartbeat at gestational week 7 (MiBP: OR = 0.35 [95 % CI: 0.14; 0.91], MnBP: OR = 0.39 [95 % CI: 0.13; 1.15]). Mixtures of the measured concentrations of BP-3 and the seven phthalate metabolites induced a small significant increase in the intracellular calcium ion concentration in human spermatozoa from healthy donors (n = 3). DiscussionPhthalate metabolites and BP-3 were detectable in follicular fluid and high concentrations of some phthalate metabolites were linked with lower chance of successful fertility treatment outcomes. Chemical mixture concentrations in follicular fluid induced a calcium response in human spermatozoa highlighting possible biological effects at physiologically relevant concentrations.

151 Comparison of prostanoid concentrations in pig plasma and follicular fluid

151 Comparison of prostanoid concentrations in pig plasma and follicular fluid