Follicular Fluid Concentrations Research Articles

Induction of Collagen I by CXCL10 in Ovarian Theca-Stroma Cells via the JNK Pathway.

Premature ovarian insufficiency (POI) poses a great threat to reproductive-age women. Ovarian fibrogenesis is a basic histologic feature of POI. Ovarian theca–stroma cells are responsible for ovarian fibrosis, but few studies have focused on the ovarian microenvironment. The role and mechanism of chemokines in the development of POI remain unclear. Here, we evaluated C-X-C motif chemokine ligand 10 (CXCL10) in biochemical POI patients, POI patients, and a POI mouse model. CXCL10 levels in serum and follicular fluid were higher in both bPOI and POI patients than in controls. An increased level of CXCL10 was also observed in a POI mouse model. CXCL10 concentrations in serum and follicular fluid were positively associated with follicle-stimulating hormone and negatively associated with antral follicle count. Our study for the first time found that CXCL10 induced COL1A1 and COL1A2 production, two subunits of collagen I in mouse theca–stroma cells by activating the JNK/c-Jun pathway. Inhibition of JNK and c-Jun attenuated the increases of COL1A1 and COL1A2 caused by CXCL10. Moreover, CXCL10 had no effects on hormone synthesis, proliferation, and apoptosis in human luteinized granulosa (hGL) cells. Our findings revealed a potential diagnostic value of CXCL10 in the early stage of POI and shed new insights into the biological function of CXCL10 in ovarian fibrosis.

Enrichment of boron element in follicular fluid and its potential effect on the immune function

The blood–follicle barrier (BFB) between the blood and follicular fluid (FF) can maintain the microenvironment balance of oocyte. Boron, an exogenous environmental trace element, has been found to possibly play an important role in oocyte maturation. This study aimed to examine the distribution characteristics of boron across the BFB and find the potential effect of boron on FF microenvironment. We analyzed the concentration of boron in paired FF and serum collected from 168 women undergoing in vitro fertilization and embryo transfer in Beijing City and Shandong Province, China. To explore the potential health impact of boron enrichment in oocyte maturation, a global proteomics analysis was conducted to tentatively correlate the protein levels with the boron enrichment. Interestingly, the results showed that the concentration of boron in FF (34.5 ng/mL) was significantly higher than that in serum (22.0 ng/mL), with a median concentration ratio of 1.52. Likewise, the concentrations of boron in FF and serum were positively correlated (r = 0.446), suggesting that boron concentration in serum can represent its concentration in follicular fluid to a large extent.. This is the first time to observe the enrichment of boron in the FF to our knowledge. It is interesting to observe a total of 13 proteins, which mainly belong to immunoglobulin class, were positively correlated with boron concentration in FF. We concluded that boron, as one environmental trace element, was enriched in FF from blood validated by two area in north china, which may be involved in an increased level of immune processes of immunoglobulins.

Women with polycystic ovary syndrome (PCOS) have reduced melatonin concentrations in their follicles and have mild sleep disturbances

BackgroundPolycystic ovary syndrome (PCOS) is a common gynecologic disorder related to abnormal circadian rhythm. Therefore, we aimed to find whether the level of melatonin, a rhythm regulating hormone changed in the ovarian microenvironment in this disease.MethodsThe melatonin concentrations in follicular fluid (FF) were measured in 35 PCOS and 36 non-PCOS women undergoing in vitro fertilization (IVF) treatment.ResultsThe FF melatonin concentration was significantly lower in PCOS women than non-PCOS women (p = 0.045) and it was found positively correlated with serum basal FSH level (r = 0.308, p = 0.013). In IVF procedures, there was no significant difference in the fertilization rate of oocytes between the two groups, but the high-quality embryogenesis rate on the third day of the PCOS group was significantly lower than that of the control group (p = 0.042), which showed a weak positive correlation with the FF melatonin concentration (rs = 0.240, p = 0.044). Furthermore, there was no significant difference in overall pregnancy outcome. The PSQI questionnaire showed that sleep disorders were more likely to exist in the PCOS group, though there was no significant difference.ConclusionThe obtained results suggested PCOS women had lower melatonin concentrations in the ovarian microenvironment.

A Simple and Feasible Quantification of Metabolites in the Human Follicular Fluid Using 1H HR-MAS NMR Spectroscopy

This study presents a reliable method for the quantification of metabolite concentrations in follicular fluid with the high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and the ERETIC2 (Electronic REference To access In vivo Concentrations) based on PULCON (pulse length based concentration determination) principle. The positive effect of the HR-MAS probe technology on spectral quality and its ability to perform analyses with very low sample amounts were the most important factors of proposing this method. In evaluating the performance of the proposed method, standard creatine solutions in different concentrations containing DSS (2,2-dimethyl-2-silapentane-5-sulfonate sodium salt) as an internal reference standard were analyzed using different pulse programs (cpmgpr1d and zg30). The results obtained with the ERETIC2 were compared with the classical internal standard NMR quantification method (DSS method). The relative standard deviation (RSD) values for ERETIC2 were in the range of 0.3% - 5.7% and recovery values were calculated as minimum 90.3%, while RSD values for DSS method were in the range of 0.1% - 3.1% and recovery values were minimum 97.0%. Besides, it was observed that the metabolite concentration values calculated using the ERETIC2 procedure of follicular fluid samples obtained from the women with endometriosis and healthy controls were compatible with the values those obtained using different methodologies. The obtained results showed that the proposed quantification method based on the HR-MAS spectroscopy can easily be used in biological fluids and therefore it can be utilized as a good alternative to the internal standard method considering its accuracy and precision.

Bone morphogenetic protein 15 intrafollicular injection inhibits ovulation in cattle

Oocyte-derived bone morphogenetic protein 15 (BMP15) is one of the main local regulators of ovarian physiology, but its role in the regulation of preovulatory follicles and ovulation is not well established. Therefore, this study was conceived to determine the effect of intrafollicular injection (IFI) of BMP15 on final follicular growth, ovulation and luteinization in cattle. Initially, it was observed that relative mRNA abundance of the BMP15 receptor BMPR1B in granulosa cells was regulated by GnRH treatment, and it was negatively correlated (R2 = 0.5; P < 0.001) to progesterone concentration in follicular fluid (FF) from preovulatory follicles. The IFI of recombinant human BMP15 tended to inhibit the growth of dominant follicles, as evidenced by an average increase of only 7.7% in the follicular diameter (from 8.8 mm to 9.1 mm) at 36 h post injection compared to 36.4% increase (from 8.9 mm to 14 mm) in the control group. Injection of BMP15 into preovulatory follicles (12–14 mm), simultaneously to im GnRH treatment, inhibited ovulation compared to control group, but did not prevent luteinization and progesterone production. Most of preovulatory follicles injected with BMP15 became luteinized cysts. Collectively, these findings indicate a suppressive role of BMP15 on later follicular development and ovulation in cattle, but not on luteogenesis and progesterone secretion.

Excessive follicle-stimulating hormone during ovarian stimulation of cattle may induce premature luteinization of most ovulatory-size follicles†.

High follicle-stimulating hormone (FSH) doses during ovarian stimulation are detrimental to ovulatory follicle function and decrease live birth rate in cattle and women. However, the mechanism whereby excessive FSH causes ovarian dysfunction is unknown. This study tested the hypothesis that excessive FSH during ovarian stimulation induces premature luteinization of ovulatory-size follicles. Small ovarian reserve heifers were injected twice daily for 4 days with 70IU (N = 7 heifers) or 210IU (N = 6 heifers) Folltropin-V [commercial FSH-enriched preparation of porcine pituitary glands with minor (<1%) luteinizing hormone (LH) contamination, cpFSH]. Ovulatory-size (≥10mm) follicles were excised from ovaries after the last cpFSH injection and hormone concentrations in follicular fluid (FF) were determined using ELISA. Luteinization was monitored by assessing cumulus cell-oocyte complex (COC) morphology and measuring concentrations of estradiol (E), progesterone (P), and oxytocin (O) in FF. COCs were classified as having compact (cCOC) or expanded (eCOC) cumulus cell layers, and as estrogen-active (E:P in FF ≥1), estrogen-inactive (EI, E:P in FF ≤1 > 0.1), or extreme-estrogen-inactive (EEI, E:P in FF ≤0.1). A high proportion (72%) of ovulatory-size follicles in 210IU, but not 70IU, dose heifers displayed eCOCs. The high doses also produced higher proportions of EI or EEI follicles which had lower E:P ratio and/or E but higher P and/or O concentrations compared with the 70IU dose heifers. In conclusion, excessive cpFSH doses during ovarian stimulation may induce premature luteinization of most ovulatory-size follicles in heifers with small ovarian reserves.

Assessment of RANTES, MIP4A, MMP7, MMP9, MMP14, TIMP 1, TIMP 2 and TIMP 3 concentration in the follicular fluid of patients undergoing in vitro fertilization/embryo transfer procedure.

Matrix metalloproteinases (MMP) degrade extracellular matrix. Some studies show that MMP9 concentration in follicular fluid (FF) may play a role in oocyte maturation and in vitro fertilization (IVF) success. The immunology of follicular fluid is still not fully understood. Assessment of RANTES, MIP4A, MMP7, MMP9, MMP14, TIMP 1, TIMP 2 and TIMP 3 concentration in the follicular fluid of patients undergoing in vitro fertilization/embryo transfer procedure. This case-control study included 20 randomly selected patients with a positive pregnancy (PPG) test and 20 patients with a negative pregnancy (NPG) test after IVF/ET. In FF obtained during oocyte retrieval, the concentrations of MIP4A, MMP7, MMP14, TIMP 1, TIMP 2, TIMP 3, RANTES, IL-12p40, and IL-17A were measured. Their effect on the characteristics of follicles, embryos, and the efficiency of IVF and ET were analysed. There was no statistically significant relationship between a positive pregnancy test and the results of the immunoassay performed. The number of COC-1 correlates significantly and positively with RANTES (r = 0.34; p = 0.038) and IP-10 (r = 0.329; p = 0.038). MII correlates significantly and positively with RANTES (r = 0.341, p = 0.031). The number of top-quality embryos correlates significantly and positively with IL-17A (r = 0.451, p = 0.004) and TIMP 1 (r = 0.44, p = 0.005). The concentration of IL-17A and TIMP 1 may predict IVF/ET success. Further studies are required on the influence of the follicular fluid immunological environment on oocyte maturation and quality and, subsequently, embryo development.

Progesterone-induced blocking factor 1 and cytokine profile of follicular fluid of infertile women qualified to in vitro fertilization: The influence on fetus development and pregnancy outcome.

Introduction: Progesterone is essential for both the initiation and the maintenance of pregnancy. The immunological effects of progesterone are mediated by the progesterone-induced blocking factor (PIBF), which is an immunomodulatory factor with anti-abortive properties. The aim of the research was to establish the cytokine profile and PIBF1 concentration in follicular fluid (FF) of patients undergoing in vitro fertilization (IVF).Methods: Seventy-eight patients who qualified for IVF underwent a detailed medical interview, including the course of fertility treatment and physical, gynecological, and cytological examinations. The concentration of PIBF1, IL-18, IL-2, IL-4, IL-6, IL-10, interferon-γ (IFN-γ), IL-1α, IL-1β, IL-5, IL-8, and IL-15 in FF during ovarian puncture was measured using commercially available ELISA kits.Results: IL-1 beta concentration was lower in the FF of patients with successful IVF. IL-8 concentration in FF correlated with the number of cumulus-oocyte complexes (COC-1), metaphase II (MII), and top-quality embryos. PIBF1 concentration had a positive correlation with the number of MII and top-quality embryos. IL-2 and IL-6 concentrations were positively correlated with the number of COC-1 and MII. An important parameter in assessing the chances of successful IVF is the number of top-quality embryos achieved.Conclusion: Higher PIBF1 concentration in FF may indicate a greater possibility of successful IVF due to the higher number of top-quality embryos. IL-1 beta concentration was found to be lower in the FF of patients with successful IVF. Therefore, PIBF1 and IL-1 beta in FF could be candidates for a marker of successful IVF.

The Influence of the Corpus Luteum Location on Hormonal and Vitamin C Composition of Follicular Fluid and Serum in Dromedary Camels (Camelus dromedarius)

The aim of this study was to compare hormonal composition of the predominant antral follicle coexisting with or without a corpus luteum (CL) in dromedary camels. Forty-seven genitalia and blood samples were collected from clinically healthy adult (7–12 years of age) non-pregnant female camels during the breeding season at a local abattoir. Follicles (0.7–1.8 cm in diameter) did not coexist with a CL were classified as follicles F1 and their blood serum labeled F1S (n=12), follicles coexist with CL on the same ovary were labeled F2 and their blood serum labeled as F2S (n=9), and follicles with contralateral CL on the other ovary labeled F3 and their blood serum named F3S (n=11). Follicular fluid (FF) and sera were subjected to biochemical and hormonal analysis. Results revealed greater (P<0.05) concentrations of progesterone in the FF of F1 follicles and F1S serum than that found in F3 follicles and F3S serum. The concentrations of cortisone and T3 were higher (P<0.05) in FF of F1 and F3, and serum of F1S and F3S in comparison to FF of follicles type F2 and serum of F2S. The thyroxine and vitamin C concentrations in FF from F1 and F2 follicles, and serum of F1S and F2S were higher (P<0.01) in comparison with the FF from F3 and serum of F3S. The presence of CL might exert a local effect on hormonal composition of FF and could indirectly influence follicular development and oocyte quality. It is better to use oocytes originated from ovaries without a CL in in-vitro fertilisation (IVF) in dromedary camels.

Diet crude protein reduction on follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows.

This study evaluated the effect of crude protein (CP) reduction in four diets (156, 139, 132, and 127 g Kg-1 DM) maintaining constant metabolizable protein (188 g/day) on the follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows. Twenty-two Girolando cows with average of 21.55 ±3.19 L daily milk yield, 105.30 ±22.62 days in lactation and 3.22 ±0.03 body condition score were selected. To reduce CP in diets and maintain constant metabolizable protein, urea and soybean meal were gradually replaced by lignosulfonate-treated soybean meal (SoyPass®, Cargill), resulting in an increase in rumen-undegradable protein and a reduction in rumen degradable protein. A linear and quadratic reduction was observed in the plasma and follicular fluid urea nitrogen concentration following CP reduction, with the most intense reduction occurring in the 127 g Kg-1 DM group (p<0.001). As CP reduced, there was a tendency for a linear increase in the follicular growth rate (P=0.0696), on the number and proportion of viable oocytes (P<0.09), and also a linear increase for the number (P=0.0397) and proportion (P<0.09) of grade I viable oocytes. Plus, there was a linear effect for the number of cumulus oophorus cells. Cows fed with the lowest amount of CP had cumulus-oocyte complexes with higher numbers of cumulus oophorus cells (P=0.0238). Also, the reduction of diet crude protein was followed by a decrease in the probability of oocytes’ DNA degradation. In conclusion, the reduction of CP in the diet of mid-lactating Girolando cows, reduces urea nitrogen concentration in both blood plasma and follicular fluid, and, as a consequence, increases the viability of oocytes and the number of cumulus oophorus cells while reducing oocytes’ DNA degradation of follicular included cumulus-oocyte complex. The reduction on dietary CP may improve in vivo oocytes’ embryo development impacting fertility of lactating dairy cows.

Quantitative analyses of insulin-like peptide 3 and sex steroid hormones in dominant follicles and corpora lutea during the estrus cycle and in follicular cysts in beef heifers.

Insulin-like peptide 3 (INSL3) and sex steroids were measured in bovine dominant follicles and corpora lutea during the estrus cycle and in follicular cysts. Paired ovaries from beef heifers (n = 47) were classified, by their morphological features, either into four stages of the estrus cycle (Day 1 = day of ovulation, Day 20 = day of estrus) as Stage I (Days 1-4; n = 8), Stage II (Days 5-10; n = 10), Stage III (Days 11-17; n = 8), and Stage IV (Days 18-20; n = 11) or follicular cystic (n = 10). Cysts (n = 15) were subdivided into estrogen-active (n = 7) and estrogen-inactive (n = 8) cysts. INSL3, testosterone, and estradiol-17β concentrations in the dominant follicular fluid of Stage IV were higher than those in Stages II and III (P < 0.05). INSL3 concentrations in the cystic fluid were similar to those in dominant follicles at Stage IV, whereas testosterone and estradiol-17β concentrations were lower in cysts (P < 0.05). INSL3 content per estrogen-inactive cyst was higher than that of Stage IV (P < 0.05). INSL3 and progesterone concentrations in luteal tissue and contents per corpus luteum were higher in Stages II and III (P < 0.05). In conclusion, INSL3 secretion in bovine dominant follicles increased with maturation. Follicular cysts may retain the production of INSL3 during their formation but tend to lose the capacity for testosterone secretion. Estrogen-inactive cysts subjected to advanced atresia may accumulate more INSL3. INSL3 production in bovine corpora lutea is enhanced during maturation.

Follicle-stimulating hormone regulates glycolysis of water buffalo follicular granulosa cells through AMPK/SIRT1 signalling pathway.

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<.05). Furthermore, we found that expression trend of AMPK/Sirtuin1 (SIRT1) pathway-related genes in GCs was consistent with the expression trend of glycolysis-related genes and was positively correlated with FSH concentrations in vivo or cultured in vitro. Activation of SIRT1 increased the expression level of glycolytic key proteins and lactic acid production in GCs, while inhibition of SIRT1 showed the opposite effect. In general, glycolysis in water buffalo GCs in vivo or cultured in vitro was positively correlated with FSH concentration. AMPK/SIRT1 pathway plays an important role in the regulation of FSH on glycolysis in GCs. Our findings will enrich the understanding of FSH regulating the development of water buffalo follicles.

Positive effects of amphiregulin on human oocyte maturation and its molecular drivers in patients with polycystic ovary syndrome.

Does use of medium containing amphiregulin improve meiotic maturation efficiency in oocytes of women with polycystic ovary syndrome (PCOS) undergoing in vitro maturation (IVM) preceded by a capacitation culture step capacitation IVM (CAPA-IVM)? Use of medium containing amphiregulin significantly increased the maturation rate from oocytes retrieved from follicles with diameters <6 or ≥6 mm pre-cultured in capacitation medium. Amphiregulin concentration in follicular fluid is correlated with human oocyte developmental competence. Amphiregulin added to the meiotic trigger has been shown to improve outcomes of IVM in a range of mammalian species. This prospective, randomized cohort study included 30 patients and was conducted at an academic infertility centre in Vietnam from April to December 2019. Patients with PCOS were included. In the first stage, sibling oocytes from each patient (671 in total) were allocated in equal numbers to maturation in medium with (CAPA-AREG) or without (CAPA-Control) amphiregulin 100 ng/ml. After a maturation check and fertilization using intracytoplasmic sperm injection (ICSI), all good quality Day 3 embryos were vitrified. Cumulus cells (CCs) from both groups were collected at the moment of ICSI denudation and underwent a molecular analysis to quantify key transcripts of oocyte maturation and to relate these to early embryo development. On return for frozen embryo transfer (second stage), patients were randomized to have either CAPA-AREG or CAPA-Control embryo(s) implanted. Where no embryo(s) from the randomized group were available, embryo(s) from the other group were transferred. The primary endpoint of the study was meiotic maturation efficiency (proportion of metaphase II [MII] oocytes; maturation rate). In the per-patient analysis, the number of MII oocytes was significantly higher in the CAPA-AREG group versus the CAPA-Control group (median [interquartile range] 7.0 [5.3, 8.0] versus 6.0 [4.0, 7.0]; P = 0.01). When each oocyte was evaluated, the maturation rate was also significantly higher in the CAPA-AREG group versus the CAPA-Control group (67.6% versus 55.2%; relative risk [RR] 1.22 [95% confidence interval (CI) 1.08-1.38]; P = 0.001). No other IVM or embryology outcomes differed significantly between the two groups. Rates of clinical pregnancy (66.7% versus 42.9%; RR 1.56 [95% CI 0.77-3.14]), ongoing pregnancy (53.3% versus 28.6%; RR 1.87 [95% CI 0.72-4.85]) and live birth (46.7% versus 28.6%; RR 1.63 [95% CI 0.61-4.39]) were numerically higher in the patients who had CAPA-AREG versus CAPA-Control embryos implanted, but each fertility and obstetric outcome did not differ significantly between the groups. In the CAPA-AREG group, there were significant shifts in CC expression of genes involved in steroidogenesis (STAR, 3BHSD), the ovulatory cascade (DUSP16, EGFR, HAS2, PTGR2, PTGS2, RPS6KA2), redox and glucose metabolism (CAT, GPX1, SOD2, SLC2A1, LDHA) and transcription (NRF2). The expression of three genes (TRPM7, VCAN and JUN) in CCs showed a significant correlation with embryo quality. This study included only Vietnamese women with PCOS, limiting the generalizability. Although 100 ng/ml amphiregulin addition to the maturation culture step significantly improved the MII rate, the sample size in this study was small, meaning that these findings should be considered as exploratory. Therefore, a larger patient cohort is needed to confirm whether the positive effects of amphiregulin translate into improved fertility outcomes in patients undergoing IVM. Data from this study confirm the beneficial effects of amphiregulin during IVM with respect to the trigger of oocyte maturation. The gene expression findings in cumulus indicate that multiple pathways might contribute to these beneficial effects and confirm the key role of the epidermal growth factor system in the stepwise acquisition of human oocyte competence. This work was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED; grant number FWO.106-YS.2017.02) and by the Fund for Research Flanders (FWO; grant number G.OD97.18N). L.N.V. has received speaker and conference fees from Merck, grants, speaker and conference fees from Merck Sharpe and Dohme, and speaker, conference and scientific board fees from Ferring. T.M.H. has received speaker fees from Merck, Merck Sharp and Dohme and Ferring. J.S. reports speaker fees from Ferring Pharmaceuticals and Biomérieux Diagnostics and grants from FWO Flanders, is co-inventor on granted patents on CAPA-IVM methodologies in USA (US10392601B2), Europe (EP3234112B1) and Japan (JP 6806683 registered 08-12-2020) and is a co-shareholder of Lavima Fertility Inc., a spin-off company of the Vrije Universiteit Brussel (VUB, Brussels, Belgium). NA, TDP, AHL, MNHN, SR, FS, EA and UDTH report no financial relationships with any organizations that might have an interest in the submitted work in the previous three years, and no other relationships or activities that could appear to have influenced the submitted work. NCT03915054.

Ovulation sources coagulation protease cascade and hepatocyte growth factor to support physiological growth and malignant transformation

• Ovulatory follicular fluid exerts a long-lasting transformation activity covering throughout the ovulation cycle. • The ovulation injury-coagulation proteases-hepatocyte growth factor (HGF) cascade is responsible for the sustained activity. • Ovulation sources HGF into the peritoneal cavity, then into the blood circulation. • This coagulation-HGF cascade promotes the transformation of fallopian tube epithelial cells and ovarian cancer cells. • Physiologically, it promotes the growth of the corpus luteum and injured epithelium after ovulation. The fallopian tube fimbrial epithelium, which is exposed to the follicular fluid (FF) contents of ovulation, is regarded as the main origin of ovarian high-grade serous carcinoma. Previously, we found that growth factors in FF, such as IGF2, are responsible for the malignant transformation of fallopian tube epithelium. However, ovulation is a monthly transient event, whereas carcinogenesis requires continuous, long-term exposure. Here, we found the transformation activity of FF sustained for more than 30 days after drainage into the peritoneal fluid (PF). Hepatocyte growth factor (HGF), activated through the ovulation injury-tissue factor–thrombin–HGF activator (HGFA)–HGF cleavage cascade confers a sustained transformation activity to fallopian tube epithelium, high-grade serous carcinoma. Physiologically, the high reserve of the coagulation-HGF cascade sources a sustained level of HGF in PF, then to the blood circulation. This HGF axis promotes the growth of the corpus luteum and repair of tissue injury after ovulation.

243 Relationship Among Granulosa Cell Gnrh-i, -II, and Receptor Mrna Abundance and Follicular Fluid Steroid Hormone Concentrations of Bovine Antral Follicles at Specific Stages of Follicular Development

Abstract Transcript abundance of two forms of GnRH and its receptor have been characterized in bovine ovaries. The objective was to investigate the relationship of GnRH-1, GnRH-2, GnRH-R, intrafollicular estradiol (E2) and progesterone (P4) during follicular development. Ovaries were collected from beef cows at specific stages of follicular development [pre-selection (PRE, n = 9), post-selection (POST, n = 9), and post-selection 24h after luteal regression (PG, n = 9)]. The largest follicle per stage was aspirated to obtain granulosa cells (GC) and follicular fluid (FF). Total cellular RNA was extracted from GC and RT-PCR was performed for GnRH-1, GnRH-2, GnRH-R and GAPDH. Radioimmunoassays were performed to determine FF concentrations of E2 and P4. Data were analyzed using the MIXED and REG procedures in SAS. There was no difference (P ≥ 0.23) in mRNA abundance of GnRH-2, GnRH-R or FF concentrations of P4 across follicular stages. There was an effect of stage on FF E2 (PRE: 17,925±20,273, POST: 28,458±21,503, and PG: 252,616±21,503 pg/mL; P &amp;lt; 0.01). Stage affected GnRH-1 mRNA abundance (PRE: 2.28±0.55, POST: 0.92±0.55, and PG: 0.11±0.55; P = 0.03). There was no relationship of GnRH-1 and GnRH-2 mRNA abundance or effect of FF P4 on GnRH-R mRNA abundance (P ≥ 0.23). There was no effect of FF P4 on GnRH-1 mRNA abundance (P = 0.68). There was no effect of FF E2 on GnRH-2 mRNA abundance (P = 0.66). As FF E2 increased GnRH-R mRNA abundance tended to increase (P = 0.10;r2=0.13). As FF P4 concentrations increased GnRH-2 mRNA abundance tended to decrease (P = 0.09;r2=0.12). As FF E2 increased GnRH-1 mRNA abundance decreased (P = 0.02;r2=0.20). In conclusion, there were differences in peptide and receptor mRNA abundance of GnRH in relation to FF E2 and P4 at specific stages of follicular development. This is supportive of a regulatory relationship between ovarian GnRH and steroidogenesis. This work is supported by AFRI Grant No.2018-67016-27578 from USDA. USDA is an equal opportunity provider and employer.

Monocyte chemotactic protein-1 concentrations and expression of women with endometriosis undergoing IVF cycles

To research the local action of endometriosis on follicular inflammatory microenvironment of patients undergoing in vitro fertilization (IVF) cycles. A prospective case control study was organized at Kamal AL-Samarai IVF center (Baghdad/Iraq), January 2021 - May 2021. The study included (80) infertile women undergoing IVF cycles were split to two groups: Patients with endometriosis group (n = 40) and Patients without endometriosis group (n = 40). Follicular fluid (FF) and blood samples were gathered at the period of oocyte pick-up. Levels of MCP-1 mRNA were investigated with the reverse transcription polymerase chain reaction (RT-PCR) technique. Concentrations of cytokines (monocyte chemo attractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10)) were investigated in FF by sandwich enzyme-linked immunosorbent assay (ELISA). The mRNA expression of MCP-1 was significantly (P = 0.00) higher in patients with endometriosis group (1.95 ± 0.69) compared with patients without endometriosis group (0.40 ± 0.15). The studied cytokines concentrations in FF of women with endometriosis group were significantly (P = 0.00) higher than those without endometriosis group: MCP-1 (548.07 ± 199.43 pg/ml vs. 45.98 ± 18.72 pg/ml), TNF-α (912.15 ± 316.12 pg/ml vs. 261.89 ± 90.69 pg/ml), IL-10 (258.94 ± 68.82 pg/ml vs. 52.12 ± 14.46 pg/ml). The immune milieu of FF of patients with endometriosis is modified, which could explain the effect of endometriosis on infertility. Furthermore, elevated MCP-1 expression in endometriosis patients suggesting systemic inflammatory changes.

The effect of follicular fluid selenium concentration on oocyte maturation in women with polycystic ovary syndrome undergoing in vitro fertilization/Intracytoplasmic sperm injection: A cross-sectional study.

Background A high level of free radicals and oxidative substances in women with polycystic ovary syndrome (PCOS) can affect the ovaries through oxidative stress. Antioxidants such as selenium, a vital trace element in human health, can improve the prognosis of PCOS by reducing oxidative stress.ObjectiveThis study was performed due to the lack of comprehensive information about selenium concentration in follicular fluid and its effect on the oocyte count and quality in infertile women with PCOS.Materials and MethodsIn this cross-sectional study, 78 women with PCOS referred to Umm-al-Banin Infertility Clinic Center, Ganjavian Hospital, Dezful, Iran for in-vitro fertilization from March to November 2019 were enrolled. After ovarian stimulation with the antagonist protocol, the oocytes were retrieved under transvaginal ultrasound in in-vitro fertilization/intracytoplasmic sperm injection cycles, and selenium concentrations were measured in the follicular fluid using an atomic absorption method by spectrophotometer device. Oocyte count and morphology were evaluated using inverted optical microscopy.ResultsThere were no significant differences between follicular fluid selenium concentrations in terms of the total number of oocytes and immature oocytes in the metaphase I and germinal vesicle stages. However, a significantly reduced number of metaphase II oocytes was observed at selenium levels 40 μg/dL (p = 0.001). ConclusionBased on our results, low levels of follicular selenium concentration in infertile women with PCOS can reduce the quality and potency of oocyte maturation.

Impact of porcine follicular fluid during folliculogenesis as a supplement on the primary cell culture of oviductal epithelial cells

The impact of varying concentrations of porcine follicular fluid (pFF) derived from three ovarian follicle types and produced during folliculogenesis on porcine oviductal epithelial cell (pOEC) culture was studied for 24 h using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cells treated with pFF at a protein concentration of 500 µg/mL (from small- and large-sized ovarian follicles) and 600 µg/mL (from medium-sized ovarian follicles) showed the highest viability, which was significantly different from that observed in the control group (p&lt;0.05) and not significantly higher than that observed in the positive control group.This study demonstrated that the impact of pFF on pOECs can be used as a model for biotechnological studies. Further, the study showed that instead of costly fetal calf serum, pFF produced during folliculogenesis can be used as a supplement in culture media to promote porcine oviductal epithelium cell growth and development.

SERUM 24,25-DIHYDROXYVITAMIN D3 IS AN ACCURATE INDICATOR OF FOLLICULAR VITAMIN D STATUS AND OOCYTE AGING IN IVF PATIENTS

There is increasing evidence supporting the role of vitamin D in telomere biology and thus the potential need for normal vitamin D status to develop a competent oocyte. The unreliable results obtained with immunoassay-based methods have supported the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and other vitamin D metabolites measurement. We previously reported that 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), the main catabolite of 25-hydroxyvitamin D3 (25OHD3), is a more accurate and stable indicator of systemic and ovarian vitamin D status in young egg donors. Consequently, we aimed to explore if 24,25(OH)2D3 is a suitable indicator of systemic and follicular vitamin D status in a population of women undergoing IVF PGT-A cycles. Then, we aimed to evaluate the correlation between telomere length in cumulus cells (CCTL) and vitamin D metabolites concentrations in serum (S) and follicular fluid (FF) in this IVF patient population. 27 women between 37 and 40-years old undergoing IVF PGT-A cycle were included in this prospective proof of concept study. On the pick-up day, S and pooled FF from mature follicles were evaluated for 24,25(OH)2D3 and 25OHD3 levels via LC-MS/MS. Cumulus cells (CC) were obtained after oocyte stripping for CCTL measurement. Genomic DNA from CC was isolated and average delta cycle threshold (ΔCt) was determined using a SYBR green quantitative real-time PCR protocol with Taqman probes for relative telomere length (TL) normalized by multicopy Alu. Difference in means between S and FF vitamin D metabolites and correlation between 24,25(OH)2D3 and ΔCtCC, were evaluated by t-test and Pearson correlation test. 25OHD3 and 24,25(OH)2D3 mean concentrations were, respectively, 43.15 ± 10.28 ng/ml and 14.30 ± 4.95 in S, and 33.67 ± 6.87 ng/ml and 11.67 ± 3.53 ng/ml in FF. Both metabolites highly correlated between them in S (r=0.78, p=1.5e-06) and in FF (r=0.73, p=1.8e-05). When analyzing each metabolite separately, 24,25(OH)2D3 showed the strongest association between S and FF levels (r=0.96, p=1.9e-15) followed by 25OHD3 (r=0.92, p=2.2e-11). ΔCtCC mean value was 7.54 ± 1.04, and we found a significant negative correlation with 24,25(OH)2D3 levels in S (r=-0.51, p=0.0068) and FF (r=-0.42, p=0.0307). Since ΔCt values are inversely proportional to the amount of nucleic acids amplified, a higher ΔCt value indicates a shorter telomere. Our results show serum 24,25(OH)2D3 is a better indicator of ovarian vitamin D status than serum 25OHD3. Further, the observed significant inverse correlation between this metabolite and CCTL, indicates that, the higher the vitamin D concentrations in S and FF, the longer the telomeres in the cumulus cells in this homogenous population of women undergoing IVF PGT-A cycle.

Effect of a dietary and exercise intervention in women with overweight and obesity undergoing fertility treatments: protocol for a randomized controlled trial

BackgroundDistinct molecular, inflammatory, and metabolic signatures are present in oocytes and follicular fluid derived from women with obesity when compared to those derived from normal weight women, which suggest existing signals that may program future offspring for metabolic diseases. This study aims to assess the feasibility and efficacy of a peri-conception nutrition and exercise intervention on mitigating obesity-associated changes in oocyte gene expression profiles and follicular fluid metabolites.MethodsThis single blinded randomized control trial will include 120 women with a BMI of 25–45 kg/m2, ≥21 years of age, and undergoing in vitro fertilization (IVF) treatments. Participants will be randomized to standard of care (N = 60) or an intervention group (N = 60) in a block design by polycystic ovary syndrome status. The intervention will combine a dietary component (Mediterranean meal plan) with exercise prescription following the Physical Activity Guidelines for Americans. Participants will be assessed pre- and post-intervention. The standard of care group will be offered to join the intervention group if the IVF treatments are unsuccessful as a cross over design. Recruitment is anticipated to start in July of 2021. Primary outcomes will include single oocyte gene expression profiles and follicular fluid metabolites. Mann-Whitney U nonparametric tests will be used to assess potential differences for each stratum. Follicular fluid and serum metabolites will be analyzed using a one-factor Analysis of Covariance (ANCOVA) at four levels, pair-wise comparisons using Tukey-Kramer post-hoc tests will be used to identify groups whose means differ significantly while retaining the family-wise error rate at 5%. When the design is balanced, two-way Analysis of Variance (ANOVA), or non-parametric Friedman test will be used in data analysis. Additionally, general linear models and ANCOVA may be used to control for covariates. Significance will be set at p < 0.05. Findings will be disseminated via peer-reviewed manuscripts and presentations at scientific conferences.DiscussionThis study will provide novel data and key information on the impact of a dietary and exercise intervention on oocyte gene expression and follicular fluid content. Results will demonstrate the potential of such intervention in mitigating obesity-induced changes in oocyte gene expression and follicular fluid metabolites.Trial registrationClinicalTrials.gov (NCT04273048): submitted November 13, 2019; posted February 17, 2020.