Follicular Fluid Progesterone Research Articles

Expression of interleukin-1β and its receptor in human granulosa cells and their association with steroidogenesis

This study aimed to investigate whether interleukin 1β (IL-1β) and soluble IL-1 receptor 2 (sIL-1R2) are expressed in human granulosa cells (GCs) and relate to ovarian steroidogenesis. Ninety-six women undergoing in vitro fertilization (IVF) were recruited. RT-PCR and immunocytochemistry were used to detect mRNAs and proteins of IL-1β and IL-1R2, respectively. The steroidogenesis of primary cultured GCs was evaluated following treatment with either IL-1β alone or IL-1β and FSH in combination. There were positive correlations between serum IL-1β and serum progesterone (r = 0.220, p = 0.032) and follicular fluid (FF) estradiol (r = 0.242, p = 0.018). Additionally, serum and FF sIL-1R2 were negatively and positively correlated with FF estradiol (r = −0.376, p = 0.005) and FF progesterone (r = 0.434, p = 0.001), respectively. The mRNA and protein expression of IL-1β and IL-1R2 became evident in GCs. IL-1β alone significantly increased estradiol secretion from GCs, but in the presence of FSH, it could notably promote progesterone secretion in addition to estradiol. In conclusion, IL-1β and sIL-1R2 are expressed in human GCs and substantially contribute to ovarian steroidogenesis, suggesting that the IL-1β system may be a potential target for optimizing ovarian hyperstimulation and steroidogenesis in IVF cycles.

O-233 Follicular fluid extracellular vesicles as a potential tool for human spermatozoa selection in vitro

Abstract Study question What characteristics and features of follicular fluid (FF) extracellular vesicles (EVs) of women of different ages can affect the functional characteristics of spermatozoa? Summary answer The size, lipid composition, and progesterone levels of the FF EVs change with a woman's age and affect the fertilizing ability of spermatozoa. What is known already Aging influences on molecular composition of the FF, reducing fertilization processes occurring in the fallopian tubes. FF is an important factor in activating spermatozoa during oocyte fertilization, enhancing the process of sperm hyperactivation and capacitation. FF progesterone is one of the main chemoattractants in the female genital tract, attracting sperm to the oocyte and starting the fertilization processes. Our previous studies have shown a significant effect of FF EVs of young women compared to advanced maternal age women (AMA) on the hyperactivation and motility of spermatozoa. The binding of FF EVs to the acrosoma of the sperm also was detected. Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 10*6/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Next steps: immunofluorescent staining, test for acrosomal reaction of spermatozoa after incubation, NTA analysis, the lipid profile and progesterone measuring of FF EVs of women of different ages. Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 21) aged 28-36. FF EVs were obtained from young (n = 14) aged 22-30 and AMA patients (n = 14) aged 42-47. The methods used in this work also include immunofluorescence microscopy, nanoparticle tracking analysis, flow cytometry, liquid chromatography–mass spectrometry, statistical data analysis. Lipids were extracted from EVs using a modified Folch extraction protocol. Main results and the role of chance Progesterone levels were significantly higher in FF EVs samples from young women compared to FF EVs samples from AMA women and controls (3951000 a.u., 598500 a.u., 6701 a.u.). This may explain the increase in sperm hyperactivation and motility after incubation with FF EVs. The difference in size (144.78 nm ± 9.92 VS 127.03 nm ± 17.17) and lipid composition of young women's FF EVs may explain the more significant functional and therapeutic effect on spermatozoa compared to AMA women FF EVs. A total of 389 vesicle lipids were identified. The level of cholesterol esters, cholesterol, lysophosphatidylcholines, phosphatidylcholines was 1,5 times higher in FF EVs of young women. The level of di- and triglycerides, ceramides, oxidized forms of phosphatidylcholines was higher in FF EVs of AMA women. The level of spontaneous acrosomal reaction was almost 3 times lower in sperm samples after incubation with FF EVs compared to the control (11,21% VS 32,88%). This supports the hypothesis that FF EVs may restrain premature acrosomal reaction in the fallopian tubes. Age-related changes are reflected in reproductive important biological processes, which leads to a change in the composition and functions of FF and affect the functional role of FF EVs. Limitations, reasons for caution Small amount of data. To be completely confident in the importance of the participation of FF EVs in the activation of spermatozoa, it is necessary to perform proteomic analyses of FF EVs to discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics. Wider implications of the findings Further studies may help not only to complement the fundamental understanding of the processes of fertilization and the impact on spermatozoa morphofunctional characteristics, but also to develop a tool for improving ART outcomes in couples with male infertility. The solutions could be the use of FF EVs from young donors. Trial registration number not applicable

Follicular fluid and plasma concentrations of progesterone following hCG administration before ultrasound-guided ablation of dominant follicles ≥25mm in mares

Ultrasound-guided transvaginal follicle ablation of dominant follicles ≥25mm during the follicular phase results in luteinization and an increase in circulating concentrations of progesterone (Ignácio et al., Theriogenology. 2021; 160:128-133). Although LH receptors are present in early antral follicles of 5mm diameter (Goudet et al., Biol Reprod. 1999; 60:1120-7) and increase after deviation (Bastos et al., Theriogenology. 2014; 82:1131-6), the ablation-induced luteinization rate may be less for smaller-sized dominant follicles <30mm and progesterone may not achieve concentrations >2ng/mL in a timely manner (Mozzaquatro et al., Reprod Domest Anim. 2012; 47:288-92). The objective of the present study was to determine if hCG administered 24h before ablation of dominant follicles <30 or ≥30mm increases the luteinization rate and results in progesterone concentrations >2ng/mL to support pregnancy. Cycling mares were randomly assigned to four ablation groups: 1) ≥25 (ablation of follicles 25-29mm, n=9), 2) hCG+≥25 (2500 IU hCG, i.v. 24h before ablation of follicles 25-29mm, n=11), 3) ≥30 (ablation of follicles ≥30mm, n=13), and 4) hCG+≥30mm (2500 IU hCG, i.v. 24h before ablation of follicles ≥30mm, n=10). Subsequent to PGF2α – induced luteolysis in all mares and administration of hCG to respective groups, D0 marked the day whenthe dominant follicle reached 25-29mm or ≥30mm and follicular fluid was collected. Blood samples were collected daily until D15, fresh embryos were transferred when plasma plasma progesterone (PP4) was >2ng/mL on D7. Follicular fluid progesterone (FFP4) and PP4 were measured by a commercial RIA kit. Wilcoxon test was used to evaluate FFP4. Repeated measures models and Tuckey test were used to evaluate PP4. Regardless of follicle size on D0, mean FFP4 but not PP4 was higher (P<0.05) in mares that received hCG (115.0ng/mL) vs those that did not (53.0ng/mL). Based on PP4 >2ng/mL, luteinization rate was 43 vs 75% in the ≥25 vs hCG+≥25 group (P<0.05) and 77 vs 67% in the ≥30 vs hCG+≥30 group (P>0.05). From D0, daily PP4 gradually increased and on D7 mean concentrations did not differ among the ≥25, hCG+≥25, ≥30, and hCG+≥30 groups, respectively. Regardless of follicle size, embryo transfer resulted in pregnancy rates of 43% (3/7) for the follicle ablation alone and 43% (6/14) for the hCG + ablation groups. In conclusion, administration of hCG before ablation of follicles 25-29mm but not ≥30mm enhanced the luteinization rate with PP4 >2ng/mL at D7 among groups. This was sufficient to support pregnancy after embryo transfer.

Follicular fluid progesterone downregulated HPGD and COX2 in granulosa cells via suppressing NF-ĸB in endometriosis†.

Increasing evidences showed that ovulatory dysfunction, possibly caused by luteinized unruptured follicular follicle syndrome (LUFS), is one of the reasons for endometriosis-related infertility. The present study was conducted to explore the potential effect of elevated progesterone in follicular fluid (FF) on ovulation in endometriosis. A prospective study including 50 ovarian endometriosis patients and 50 control patients with matched pairs design was conducted with alterations in FF and peritoneal fluid (PF) components identified by metabolomics analyses and differentially expressed genes in granulosa cells (GCs) identified by transcriptome analysis. Patients with endometriosis exhibited a significantly higher progesterone level in serum, FF, and PF. Granulosa cells from endometriosis patients revealed decreased expression of HPGD, COX-2, and suppressed NF-ĸB signaling. Similarly, progesterone treatment in vitro downregulated HPGD and COX2 expression and suppressed NF-ĸB signaling in granulosa tumor-like cell line KGN (Bena Culture Collection, China) and primarily cultured GCs, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIĸBα/IĸBα ratio, and nucleus translocation of p65. On the contrary, TNF-α treatment increased expression of IL1R1, IRAK3, pIĸBα, p65, and HPGD in GCs. One potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation. In conclusion, we found that intrafollicular progesterone might downregulate HPGD and COX-2 in GCs via suppressing the NF-ĸB signaling pathway, shedding light on the mechanism underlying the endometriosis-related ovulatory dysfunction.

Lnc2300 is a cis-acting long noncoding RNA of CYP11A1 in ovarian granulosa cells.

The high level of progesterone and 17β-estradiol ratio (P4/E2) in follicular fluid has been considered as a biomarker of follicular atresia. CYP11A1, the crucial gene encoding the rate-limiting enzyme for steroid hormone synthesis, has been reported differently expressed in the ovary during follicular atresia. However, the regulation mechanism of CYP11A1 expression during follicular atresia still remains unclear. Here, we have demonstrated that lnc2300, a novel pig ovary-specific highly expressed cis-acting long noncoding RNA (lncRNA) transcribed from chromosome 7, has the ability to induce the expression of CYP11A1 and inhibit the apoptosis of porcine granulosa cells (GCs). Mechanistically, lnc2300, mainly located in the cytoplasm of porcine GCs, sponges and suppresses the expression of miR-365-3p through acting as a competing endogenous RNA (ceRNA), which further relieves the inhibitory effects of miR-365-3p on the expression of CYP11A1. Besides, CYP11A1 is validated as a direct functional target of miR-365-3p in porcine GCs. Functionally, lnc2300 is an antiapoptotic lncRNA that reduces porcine GC apoptosis by inhibiting the proapoptotic function of miR-365-3p. In summary, our findings reveal a cis-acting regulation mechanism of CYP11A1 through lncRNA, and define a novel signaling pathway, lnc2300/miR-365-3p/CYP11A1 axis, which is involved in the regulation of GC apoptosis and follicular atresia.

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography–high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Progesterone, Myo-Inositol, Dopamine and Prolactin Present in Follicular Fluid Have Differential Effects on Sperm Motility Subpopulations.

Considering the challenges surrounding causative factors in male infertility, rather than relying on standard semen analysis, the assessment of sperm subpopulations and functional characteristics essential for fertilization is paramount. Furthermore, the diagnostic value of sperm interactions with biological components in the female reproductive tract may improve our understanding of subfertility and provide applications in assisted reproductive techniques. We investigated the response of two sperm motility subpopulations (mimicking the functionality of potentially fertile and sub-fertile semen samples) to biological substances present in the female reproductive tract. Donor semen was separated via double density gradient centrifugation, isolated into high (HM) and low motile (LM) sperm subpopulations and incubated in human tubal fluid (HTF), capacitating HTF, HD-C medium, progesterone, myo-inositol, dopamine and prolactin. Treated subpopulations were evaluated for vitality, motility percentages and kinematic parameters, hyperactivation, positive reactive oxygen species (ROS), intact mitochondrial membrane potential (MMP) and acrosome reaction (AR). While all media had a significantly positive effect on the LM subpopulation, dopamine appeared to significantly improve both subpopulations’ functional characteristics. HD-C, progesterone and myo-inositol resulted in increased motility, kinematic and hyperactivation parameters, whereas prolactin and myo-inositol improved the LM subpopulations’ MMP intactness and reduced ROS. Furthermore, progesterone, myo-inositol and dopamine improved the HM subpopulations’ motility parameters and AR. Our results suggest that treatment of sub-fertile semen samples with biological substances present in follicular fluid might assist the development of new strategies for IVF treatment.

P–012 A new sperm preparation technique for ICSI

Abstract Study question Is there any difference in this technique(swim-in) according to clinical results in ICSI cycles? Summary answer Fertilization rates are significantly better with this new technique(swim-in). There is no difference in pregnancy and ongoing pregnancy rates between swim-in and DGS techniques. What is known already Sperm separation is crucial in assisted reproductive technologies, based on different principles like migration, filtration, or density gradient centrifugation techniques. However, there are some studies that centrifugation steps and using gradient solutions may increase sperm DNA fragmentation. On the other hand, multiple contacts to different plastic surfaces such as pipettes, plastic tubes, etc., and also pipetting the ejaculate in sperm preparation may have detrimental effects. The ideal sperm separation technique should be non-invasive, not time-consuming, easy, and cost-effective, not cause sperm damage or non-physiological alterations of the separated sperm cells, eliminate dead spermatozoa and other cells, including leukocytes. Study design, size, duration This is a prospective randomized study between 01/02/2019...01/01/2021. Sperms were prepared either with this new technique that we call “swim-in”(n = 359) or with density gradient centrifugation(n = 404) before microinjection (ICSI). Fertilization rates, clinical pregnancy rates, and on-going pregnancy rates were compared between groups. Sperm motility is less than%20 patients are not included in the study. t-test and Chi-square test used. p &amp;lt; 0.05 significant. Participants/materials, setting, methods For this new centrifugation free technique(swim-in), a Braun injector, 21G green needle, and sperm washing medium are used. First, we pulled a 0.5 ml sperm wash medium in a Braun injector. After sperm liquefaction, we put Braun injector with the needle in the sperm cap perpendicularly and waited 30 minutes at 37-degree Celcius. Sperms swam through the needle to the injector used for ICSI. For DGC sperms were prepared according to the 2015 WHO manual. Main results and the role of chance: We try to make a centrifugation-free, gentle, cost-effective, and easy sperm preparation technique and called it a swim-in technique. Groups were comparable according to mean age, collected oocyte numbers, and oocyte maturation ratios. Fertilization rates are%73,2(1786/2438) vs%66,9(2167/3237) respectively for swim-in and DGC groups. Clinical pregnancy rates are%45,1 vs%42,3 respectively for swim-in and DGC groups. On-going pregnancy rates are%41,5 vs%39,6 for swim-in and DGC respectively. There is a significant difference between the two techniques according to fertilization rates but no difference according to clinical pregnancy and ongoing pregnancy rates. Limitations, reasons for caution Sperms can move by their own motility from ejaculate to the sperm washing medium for that reason with severe sperm problem patients are not suitable for this technique. Wider implications of the findings: This technique is centrifugation free so there are no detrimental g-forces. The needle used may mimic the physiological environment like Uterine Tube. Alternative sperm washing mediums like follicular fluid or chemical attractor progesterone rich medium can be used.DNA fragmentation rates and live birth rates should be established for future aspect. Trial registration number Not applicable

Effects of lipopolysaccharide on beta-catenin, aromatase, and estrogen production in bovine granulosa cells in vivo and in vitro

Diseases resulting from Gram-negative bacterial infection can induce an immune response by releasing a lipopolysaccharide (LPS) endotoxin that may lead to impaired fertility in cows. To evaluate the effects of LPS on follicular dynamics in a subacute inflammatory disease state, 14 Angus heifers (BW = 413 kg±14) were blocked by weight and assigned to vehicle (n = 7) or LPS treated (n = 7) groups. Heifers received subcutaneous injections of saline (CON) or 2.0 μg/kg LPS on d 2, 5, and 8 of a select synch plus controlled internal drug release device (CIDR) follicular wave synchronization protocol. Fifty hours following CIDR withdrawal, ovaries were harvested, and follicular fluid was collected for hormone and LPS analysis. Daily blood samples were collected from d 0 to d 7. Beginning on d 8 blood samples were collected at 0, 16, 24, 32, 40, and 50 h following LPS challenge. Rectal temperatures were recorded prior to treatment and at regular intervals after each LPS challenge. Heifers treated with LPS exhibited mild (+0.5 °C) hyperthermia (P < 0.05) at 3, 4, and 8 h after the initial LPS challenge (d 2) when compared to vehicle-treated controls. Follicular fluid concentrations of estradiol (E2) increased (P = 0.04) in LPS-treated heifers compared to controls (1,595 ng/mL and 808 ng/mL±240, respectively), while follicular fluid progesterone (P4) concentrations did not differ (P = 0.27) between treatment groups. Additionally, LPS concentrations tended to be increased (P = 0.59) in dominant follicles of LPS-treated heifers, but no difference was detected (P = 0.81) in small developing follicles. To further delineate the impact of LPS on ovarian signaling pathways, a granulosa cell line (KGN) was incubated in the presence or absence of LPS (10 μg/mL) for 48 h. Cells were then collected for gene expression and protein analysis. Cells in both treatment groups expressed toll-like receptor 4, myeloid differentiation factor-2 receptor, and CD-14 complex genes required for LPS signaling. Cells treated with LPS exhibited decreased mRNA expression of aromatase (P = 0.03) and beta-catenin (P = 0.02). However, no change (P > 0.10) was detected in abundance of total beta-catenin protein or beta-catenin phosphorylated isoforms at serine 552 or 675. Based on results from this in vivo experiment, these investigators concluded that low doses of LPS can alter E2 concentrations and this effect may be modulated in part through beta-catenin regulation of aromatase transcription.

Steroid Hormones of Follicular Fluid and the Outcome of in Vitro Fertilization

Abstract One of the succes factors of biomedically assisted fertilization is the regular maturation of one or more oocytes. The quality of the oocytes is significantly influenced by the environment in which it is located, the so-called „microenvironment” that includes cumulus cells, follicular fluid in which hormones and growth factors involved in its growth and development are secreted. The main aim was to examine whether the concentration of steroid hormones in the follicular fluid affects the rate of fertilization and the outcome of the in vitro fertilization process itself. The study included 31 patients who were included in vitro fertilization procedure at the Department for Biomedically Assisted Fertilization, Clinic for Gynecology and Obstetrics, Clinical Center Kragujevac. We used follicular fluid as biological material for analysis. Examination of the obtained follicular fluid and collection of oocytes under a stereomicroscope was done in the embryological laboratory at the Department. Biochemical parameters of follicular fluid were analyzed in the Department for Laboratory Diagnostics, Clinical Center Kragujevac. In vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) methods were used as the fertilization method. The criteria of the Istanbul Consensus of Clinical Embryologists were used as a reference framework for embryo quality assessment. Pregnancy was confirmed by a positive serum level of the hormone β-hCG 14 days after embryo transfer. A software package ЅРЅЅ 20 was used for statistical data processing. The results of the analysis of follicular fluid samples show that there was no statistically significant difference in the concentration of estradiol, progesterone and testosterone in follicular fluid in relation to fertilization rate and the outcome of in vitro fertilization. Based on our results, it can be concluded that the concentration of steroid hormones did not affect fertilization rate and the outcome of in vitro fertilization.

Follicular fluid progesterone in thyroid autoantibodies positive women and assisted reproductive technology outcome

Follicular fluid progesterone in thyroid autoantibodies positive women and assisted reproductive technology outcome

Altrenogest affects the development and endocrine milieu of ovarian follicles in prepubertal and mature gilts†.

Altrenogest with gonadotropins is commonly used to synchronize the estrous cycle, but it can also lead to follicular cyst formation, especially in prepubertal gilts. Here, we aimed to investigate how maturity and altrenogest treatment affect the development, endocrine milieu, and molecular control of ovarian follicles. Crossbred prepubertal and mature gilts were challenged or not (control) with altrenogest, and ovaries were collected in the morning on the first day of behavioral estrus. In prepubertal gilts, altrenogest decreased the percentage of primordial and atretic small follicles, but increased large antral follicles when compared with controls. In mature gilts, altrenogest reduced the percentage of primary follicles and elevated the total number of antral follicles. Maturity affected the estradiol level in the follicular fluid of preovulatory follicles, luteinizing hormone (LH)-stimulated cyclic adenosine monophosphate (cAMP) generation, and LH receptor messenger RNA (mRNA) expression in granulosa. Moreover, cytochrome P45017A1 (CYP17A1) mRNA levels in the theca layer were affected and correlated with follicular androstendione and estradiol concentration. Altrenogest negatively affected follicular fluid progesterone concentration and decreased levels of prostaglandin (PG) E2 in prepubertal gilts and PGF2alpha metabolite in mature gilts. LH-stimulated cAMP release in granulosa cells of mature gilts as well as human chorionic gonadotropin- and forskolin-induced cAMP were also affected. In addition, altrenogest downregulated CYP17A1 mRNA in the prepubertal theca layer and PGF2alpha synthase expression in the granulosa and theca layer of mature gilts. To the best of our knowledge, this is the first study to report multiple effects of maturity and altrenogest on the endocrine milieu and molecular regulations governing ovarian follicle development in gilts.

Effects of follicular ablation and induced luteolysis on LH and follicular fluid factors during the periovulatory period in mares

Haemorrhagic anovulatory follicles (HAFs) are the most common pathological anovulatory condition in the mare. To enhance understanding of the physiopathology of HAFs, the aim of the present study was to determine the effects of an induced-follicular wave on LH concentrations and follicular fluid factors relevant to the ovulatory process. Mares were allocated to treatment or control groups (n = 7/group) in a crossed over design during 14 oestrous cycles with a period of one cycle occurring when there were no treatments between the times when treatments were administered. In the treatment group, all antral follicles ≥8 mm were ablated on Day 10 after ovulation followed by administration of a luteolytic dose of PGF2α. All mares of both groups were treated with 1500 IU of hCG when a follicle ≥32 mm was detected (Hour 0), and follicular fluid was aspirated 35 h later. Blood samples were collected every 48 h from Day 10 until Hour 0 from all mares. Follicular fluid was assayed for PGE2, estradiol and progesterone. Plasma was assayed for LH concentrations. A follicular wave followed follicle ablation in the treated mares. Concentrations of LH were greater (P = 0.05) in mares ot the treatment compared with control group. Concentrations of PGE2, estradiol and progesterone in follicular fluid did not differ between groups (P > 0.05). Treatment resulted in an earlier increase in circulating LH, however, there was no effect on concentrations of intra-follicular PGE2, estradiol or progesterone in hCG-stimulated preovulatory follicles.

Follicular fluid progesterone concentration is associated with fertilization outcome after IVF: a systematic review and meta-analysis.

Follicular fluid is a key biochemical environment for oocyte development. The potential effect of follicular progesterone level on successful fertilization is a subject of debate, and so the aim of this study was to provide a summary of the currently available evidence on the association between follicular fluid progesterone level and fertilization outcome. To do so, a systematic review and a meta-analysis were performed, with the literature searches being conducted in three databases (PubMed, Embase and the Cochrane Library) to identify all relevant studies published up to 19 August 2017. Data were available from 13 studies (four intracytoplasmic sperm injection [ICSI] and nine conventional IVF) and 1009 individually aspirated follicular fluid samples were included in the analysis. The progesterone levels in follicular fluid were significantly higher in normal fertilization than in failed fertilization, both in conventional IVF (33% difference, P<0.001) and ICSI (34% difference, P=0.004). Although these data show that fertilized oocytes are derived from follicles with higher levels of progesterone, the results must be interpreted with caution, because of various progesterone measurement methods and different treatment protocols and it is too early to state that follicular fluid progesterone level could be considered as a marker for oocyte quality.

High copper levels in follicular fluid affect follicle development in polycystic ovary syndrome patients: Population-based and in vitro studies

Although the adverse effects of copper overexposure on the liver, kidney, spleen and intestinal organs are well known, information about the impact of copper toxicity on human reproduction is limited. A total of 348 infertile patients were enrolled in our present study, including 89 with polycystic ovary syndrome (PCOS), 145 with fallopian tube obstruction and 114 controls. The follicular fluid concentrations of 22 trace elements were measured by inductively coupled plasma mass spectrometry (ICP-MS). Principal component analysis was used to identify trace element profile alterations in different groups. The mRNA levels of steroidogenesis-related genes were measured by real-time PCR. Our results showed that the trace element profile in follicular fluid was obviously altered in PCOS patients. Copper concentrations were significantly (p < .05) higher in the PCOS group than in the other two groups. Increased copper levels in follicular fluid were associated with a higher number of retrievable oocytes in the PCOS group (B = 1.785, p = .001) but a lower rate of high-quality embryos (B = −6.360, p = .050). Moreover, follicular fluid copper levels were positively correlated with follicular fluid progesterone levels (r = 0.275, p = .010) and testosterone levels (r = 0.250, p = .022). Cultured human granulosa cells overexposed to copper showed significantly (p < .05) increased estradiol secretion and decreased testosterone levels. Real-time quantitative PCR revealed a significant (p < .05) increase in CYP19A1 and HSD3b mRNA expression. Our results indicate that increased copper levels in follicular fluid could affect follicle development in PCOS patients, and the mechanism may be related to copper-induced abnormalities in steroidogenesis.

Biochemical constituents and steroid hormones in follicular fluid from antral follicles of cross-bred Malabari goats of Kerala

ABSTRACT A study was conducted in cross-bred (CB) Malabari goats of Southern India, Kerala to assess the pattern of change in biochemical constituents of follicular fluid during final stages of follicle development. One hundred and twenty goat ovaries were collected from local slaughter houses. After classifying the antral follicles based on size i.e. small (1–3 mm) and large (3–6 mm), the follicular fluid was aspirated and analysed to estimate the concentration of various biochemical constituents including total protein, albumin, globulin, glucose, triglycerides, cholesterol, high-density lipoprotein (HDL) and steroid hormones. The results showed that the concentration of glucose in follicular fluid was significantly higher (p < 0.05) in small follicles compared the large ones. However, the level of HDL and oestrogen was significantly higher (p < 0.05) in large follicles. Follicular fluid protein, albumin, globulin, triglycerides, cholesterol, and progesterone did not differ significantly between the small and large follicles. The results of the present study suggest that the biochemical constituents in the antral follicular fluid vary with the follicular size and the data would contribute to better understanding of the follicular fluid metabolite dynamics of goat ovarian follicles which imparts oocyte competence for fertilization during later developmental stages.

Intrafollicular level of steroid hormones and the expression of androgen receptor in the equine ovary at puberty

Steroidogenic activity in the equine ovary from birth to puberty has been poorly investigated. This study aimed to examine the capability of the ovarian follicles of prepubertal and pubertal fillies to produce steroid hormones and to evaluate the expression and cellular localization of androgen receptor (AR) in their ovaries. The ovaries of 6–18 month-old fillies were divided into two groups: prepubertal (PrP) – without preovulatory follicle (pF) and corpus luteum (CL), and ovulating/postpubertal (Ov/pB) – with pF and/or CL in at least one of the gonads. Adult mares (Me) were used as a control. The concentration of progesterone (P4), testosterone (T) and estradiol (E2) in follicular fluid (FF) was measured by radioimmunoassay. AR distribution was assessed by immunohistochemistry, while AR protein expression was examined by Western blot analysis. In the female groups, E2 concentration in FF of small follicles (<10 mm) was low and increased with the diameter of the follicle reaching the greatest value in pF (Ov/pB and Me group). In follicles (11–30 mm) of PrP fillies, the concentration of E2 was similar to that from Ov/pB fillies, but less than half (P < 0.05) than in Me follicles. In FF from all classes of follicles of Ov/pB fillies, the concentration of all steroids was similar to that in Me. AR immunolocalization, predominantly nuclear, was observed in all types of follicular cells (granulosa and theca cells) as well as in stroma and luteal cells. The pattern of staining was dependent on the follicle size and the group of females. In smaller antral follicles and in pF, the nuclear AR staining in granulosa cells was stronger than that found in follicles of 21–25 mm. In theca interna cells of pF, both nuclear and faint cytoplasmic reactions were seen. In luteal cells, AR labeling was noted in the nuclei and the cytoplasm: the strongest one in the early CL and almost negative in the late CL. AR protein expression in filly and mare ovarian tissues was confirmed by Western blot analysis and detected as a single band at approximately 110 kDa. In summary, the ovaries of fillies aged at least 6 months are capable of active steroidogenesis. ARs are present either in the cell nuclei or cytoplasm of all compartments of the equine ovary. AR expression in follicular and stroma cells may indicate the sensitivity of the filly ovarian tissue to androgens, the impact of androgens on folliculogenesis and the development of the equine ovary via a receptor-mediated pathway.

Impaired insulin signaling pathways affect ovarian steroidogenesis in cows with COD

Cystic ovarian disease (COD) represents an important cause of infertility in dairy cattle and is associated with multiple physiological disorders. Steroidogenesis, which is necessary to ensure normal ovarian functions, involves multiple enzymatic pathways coordinated by insulin and other proteins. We have previously shown that cows with COD have an altered insulin response. Therefore, in the present study, we evaluated further alterations in intermediates downstream of the PI3K pathway and pathways mediated by ERK as critical signals for the expression of steroidogenic enzymes in the ovaries of control cows and cows with spontaneous COD. To this end, we evaluated the gene and protein expression of pan-AKT, mTOR, ERK1/2, and steroidogenic enzymes by real-time PCR and immunohistochemistry. Steroid hormone concentrations were assessed at systemic and intrafollicular level. Results showed altered expression of intermediate molecules of the insulin signaling pathway, whose action might modify the synthetic pathway of steroidogenic hormones. Similarly, the expression of steroidogenic enzymes and the concentration of progesterone in serum and follicular fluid were altered. These alterations support the hypothesis that systemic factors contribute to the development and/or maintenance of COD, and that metabolic hormones within follicles such as insulin exert determinant effects on ovarian functionality in cows with COD.

Differential insulin and steroidogenic signaling in insulin resistant and non-insulin resistant human luteinized granulosa cells—A study in PCOS patients

Insulin resistance (IR) is one of the significant aberrations in polycystic ovarian syndrome (PCOS), however is only observed in 70%–80% of obese PCOS and 20%–25% of lean PCOS. Hyperinsulinemia accompanies PCOS-IR along with hyperandrogenemia against normal insulin and androgen levels in PCOS-non insulin resistance (NIR). This could possibly be due to defects in the downstream signaling pathways. The study thus aims to unravel insulin and steroidogenic signaling pathways in luteinized granulosa cells isolated from PCOS-IR and NIR vs matched controls. Luteinized granulosa cells from 30 controls and 39 PCOS were classified for IR based on a novel method of down regulation of protein expression of insulin receptor-β (INSR- β) as shown in our previous paper. We evaluated expression of molecules involved in insulin, steroidogenic signaling and lipid metabolism in luteinized granulosa cells followed by analysis of estradiol, progesterone and testosterone in follicular fluid. Protein expression of INSR- β, pIRS (ser 307), PI(3)K, PKC-ζ, pAkt, ERK1/2, pP38MAPK and gene expression of IGF showed differential expression in the two groups. Increased protein expression of PPAR-γ was accompanied by up regulation in SREBP1c, FAS, CPT-1 and ACC-1 genes in PCOS-IR group. Expression of StAR, CYP19A1, 17 β- HSD and 3 β- HSD demonstrated significant decrease along with increase in CYP11A1, FSH-R and LH-R in both the groups. Follicular fluid testosterone increased and progesterone decreased in PCOS-IR group. This study shows how candidate molecules that were differentially expressed, aid in designing targeted therapy against the two phenotypes of PCOS.

Complex CatSper-dependent and independent [Ca2+]i signalling in human spermatozoa induced by follicular fluid.

STUDY QUESTIONDoes progesterone in human follicular fluid (hFF) activate CatSper and do other components of hFF modulate this effect and/or contribute separately to hFF-induced Ca2+ signaling?SUMMARY ANSWERhFF potently stimulates CatSper and increases [Ca2+]i, primarily due to high concentrations of progesterone, however, other components of hFF also contribute to [Ca2+]i signaling, including modulation of CatSper channel activity and inhibition of [Ca2+]i oscillations.WHAT IS KNOWN ALREADYCatSper, the principal Ca2+ channel in spermatozoa, is progesterone-sensitive and essential for fertility. Both hFF and progesterone, which is present in hFF, influence sperm function and increase their [Ca2+]i.STUDY DESIGN, SIZE, DURATIONThis basic medical research study used semen samples from >40 donors and hFF from >50 patients who were undergoing surgical oocyte retrieval for IVF/ICSI.PARTICIPANTS/MATERIALS, SETTING, METHODSSemen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service REC1. Activities of CatSper and KSper were assessed by patch clamp electrophysiology. Sperm [Ca2+]i responses were examined in sperm populations and single cells. Computer-assisted sperm analysis (CASA) parameters and penetration into viscous media were used to assess functional effects.MAIN RESULTS AND THE ROLE OF CHANCEhFF and progesterone significantly potentiated CatSper currents. Under quasi-physiological conditions, hFF (up to 50%) failed to alter membrane K+ conductance or current reversal potential. hFF and progesterone (at an equivalent concentration) stimulated similar biphasic [Ca2+]i signals both in sperm populations and single cells. At a high hFF concentration (10%), the sustained (plateau) component of the [Ca2+]i signal was consistently greater than that induced by progesterone alone. In single cell recordings, 1% hFF-induced [Ca2+]i oscillations similarly to progesterone but with 10% hFF generation of [Ca2+]i oscillations was suppressed. After treatment to ‘strip’ lipid-derived mediators, hFF failed to significantly stimulate CatSper currents but induced small [Ca2+]i responses that were greater than those induced by the equivalent concentration of progesterone after stripping. Similar [Ca2+]i responses were observed when sperm pretreated with 3 μM progesterone (to desensitize progesterone responses) were stimulated with hFF or stripped hFF. hFF stimulated viscous media penetration and was more effective than the equivalent does of progesterone.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis was an in vitro study. Caution must be taken when extrapolating these results in vivo.WIDER IMPLICATIONS OF THE FINDINGSThis study directly demonstrates that hFF activates CatSper and establishes that the biologically important effects of hFF reflect, at least in part, action on this channel, primarily via progesterone. However, these experiments also demonstrate that other components of hFF both contribute to the [Ca2+]i signal and modulate the activation of CatSper. Simple in vitro experiments performed out of the context of the complex in vivo environment need to be interpreted with caution.STUDY FUNDING/COMPETING INTEREST(S)Funding was provided by MRC (MR/K013343/1, MR/012492/1) (S.G.B., S.J.P., C.L.R.B.) and University of Abertay (sabbatical for S.G.B.). Additional funding was provided by TENOVUS SCOTLAND (S.M.D.S.), Chief Scientist Office/NHS Research Scotland (S.M.D.S). C.L.R.B. is EIC of MHR and Chair of the WHO ESG on Diagnosis of Male infertility. The remaining authors have no conlicts of interest.