FSH Stimulation Research Articles

VCAM-1 might be a potential angiogenic biomarker for Metal-on-Metal prosthetic device and FSH-induced inflammation

Abstract Follicle stimulating hormone (FSH) rises in peri-menopausal women and remains elevated during menopause. Bone loss, previously attributed to estrogen, may be due to direct effect of FSH. On this background of decorticated bone, hips are replaced due to fractures or osteoarthritis. Metal-on-metal (MoM) hips replacements were developed as they were thought to be longer lasting and robust implants. However, in women, they are failing at a significantly higher rate when compared to men. Failure modes include pain, device loosening due to osteolysis and exuberant blood vessel formation resulting in pseudotumor formation. Recent work from our lab has demonstrated the molecular interaction of the wear particles from MoM with immune cells and the release of inflammatory and pro-angiographic factors such as IL-1b, TNF-a and IL-8. Therefore we hypothesize that binding of monocytes to the endothelium induced via FSH and MoM wear particles should stimulate angiogenesis that would increase detectable inflammatory biomarkers. Vascular inflammatory markers were investigated with FSH and MoM particle stimulation of endothelial cells and bound monocytes. From these data, soluble VCAM-1 protein levels increased during monocyte-endothelial co-culture induced by FSH and MoM wear particle co-stimulation. FSH stimulation was also associated with increased monocyte binding and endothelial tube thickness. These data indicate that FSH promotes vascular endothelial cell cohesion and monocyte recruitment, which may lead to increased VCAM-1 expression towards combined MoM wear particle and FSH stimulation. Our findings suggest that sVCAM-1 may be a biomarker of pseudotumor formation elicited by MoM wear particles in women with elevated FSH.

Testosterone and FSH modulate Sertoli cell extracellular secretion: Proteomic analysis

Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and by juxtacrine testicular cross-talk. Sertoli cells (SC) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors, but many of the molecular pathways of SC action remain controversial and unclear. Over the last two decades, research has focused on extracellular vesicles as an important mechanism of intercellular communication. The aim of this study was to investigate the presence of extracellular vesicles (EVs) in SC and the modulation of their content in SC after FSH and testosterone stimulation. Highly purified porcine pre-pubertal Sertoli cells were isolated according to previously established methods. After 48h of culture with FSH or FSH+testosterone stimulation, we identified sertolian EVs containing specific mRNAs. Proteomic analysis of EVs content identified 29 proteins under non-stimulatory conditions, most of which were related to receptor binding activity. FSH stimulation induced increases in inhibin-alpha, inhibin-beta, plakoglobin, haptoglobin, D-3-phosphoglycerate dehydrogenase and sodium/potassium-transporting ATPase in sertolian EVs. Testosterone stimulation enhanced the abundance of inhibin-alpha, inhibin-beta, tissue-type plasminogen activator, epidermal growth factor-like protein 8, elongating factor 1-gamma and D-3-phosphoglycerate dehydrogenase. These results are likely to help determine the unknown molecular secretion of Sertoli cells.

ASAS-SSR Triennial Reproduction Symposium: Looking Back and Moving Forward-How Reproductive Physiology has Evolved: WNTs role in bovine folliculogenesis and estrogen production.

Appreciation of mechanisms that affect steroidogenesis is critical to identifying compromising signals that may decrease reproductive efficiency. Follicle maturation and steroidogenesis requires coordinated actions from the pituitary gonadotropins and local ovarian signaling molecules. β-Catenin (CTNNB1), the lynchpin molecule of canonical wingless-type mouse mammary tumor virus integration site (WNT) signaling, is required for maximal gonadotropin stimulation of steroid production from granulosa (GC) and luteal cells. WNTs are locally secreted glycoproteins involved in ovarian development and folliculogenesis. In cultured bovine GC, WNT2 and AKT mRNAs and CTNNB1 protein increase after FSH stimulation. Likewise, CTNNB1 protein is greater in large antral follicles with high intrafollicular estradiol concentrations, suggesting the hormonal milieu responsible for increased estradiol content modulates CTNNB1 accumulation. In addition, concurrent treatment of FSH and WNT3A in GC results in reduced steroidogenic enzymes and ovarian differentiation factors. It is likely that FSH regulation of WNT signaling establishes a negative feedback loop to ensure CTNNB1 remains controlled. To explore the mechanism resulting in this inhibitory effect, AKT pathway modulators were utilized and unveiled a requirement for AKT activity in FSH-mediated CTNNB1 accumulation. Cells treated with FSH, IGF-1, and IGF-1 + FSH had increased CTNNB1 protein accumulation compared with controls. Similarly, estradiol medium concentrations increased in treated cells compared with non-treated controls, while co-treatment of FSH and IGF-1 with the AKT inhibitor LY294002 reduced CTNNB1 and estradiol production. Subsequent studies evaluated whether FSH regulation of CTNNB1 occurs through a specific phosphorylation event. In bovine GC, phosphorylation of CTNNB1 at Ser-552 was demonstrated in FSH-treated cells, whereas IGF-1 treatment did not phosphorylate CTNNB1 Ser-552. Data indicate that in cattle phosphorylation on CTNNB1 Ser-552 is a protein kinase A (PKA) dependent, protein kinase B (AKT) independent event. Data suggest that CTNNB1 regulated by AKT is a fundamental component of FSH-induced estrogen production. However, AKT's role in estradiol synthesis does not appear to be through phosphorylation of CTNNB1 Ser-552. The complex interplay between FSH and ovarian WNT/CTNNB1 signaling is key to regulation of follicle maturation and steroidogenesis.

Luteinizing Hormone Facilitates Antral Follicular Maturation and Survival via Thecal Paracrine Signaling in Cattle

LH supplementation in assisted reproductive technology cycles improves the ongoing pregnancy rate in women with poor ovarian response (POR). However, our knowledge of the precise role of LH during the follicular phase of the menstrual cycle is incomplete. To explore the role of LH in the maturation of small antral follicles, we used an in vitro two-cell system that involved coculturing bovine granulosa cells (GCs) and theca cells (TCs) on a collagen membrane. Treatment of TCs with LH stimulated androgen production in TCs by inducing the expression of androgenic factors, subsequently increasing estrogen biosynthesis in GCs by providing androgen substrates, and inducing aromatase expression. LH stimulation of TCs induced functional LH receptor expression in GCs, a response modulated by the synthesis and action of estrogen. In the presence of TCs, LH stimulation of TCs and FSH stimulation of GCs increased the expression of IGF-1, IGF-2, and IGF-1 receptor in GCs. LH-induced expression of thecal IGF-1 protected GCs from apoptosis and promoted GC survival. Furthermore, LH stimulation of TCs increased FSH sensitivity in GCs. Thus, the LH-TC axis may be involved in the acquisition of LH dependence and the survival of small antral follicles by upregulating androgen/estrogen biosynthesis and activating the IGF system. The use of LH supplementation in ovarian stimulation may increase gonadotropin sensitivity in small antral follicles and promote follicular growth and survival by suppressing GC apoptosis and follicular atresia, resulting in multiple follicular development, even in patients with POR.

Reproductive and metabolic determinants of granulosa cell dysfunction in normal-weight women with polycystic ovary syndrome

To determine the degree to which E2 hyperresponsiveness to FSH and antimüllerian hormone (AMH) overproduction in normal-weight women with polycystic ovary syndrome (PCOS) correlate with increased antral follicle number (AFN), hyperandrogenism, and/or metabolic dysfunction. Prospective cohort study. Academic medical center. Seven normal-weight women with PCOS (1990 National Institutes of Health criteria) ages 20-34years and 13 age- and body mass index- (BMI-; 18.5-25kg/m2) matched normoandrogenic ovulatory women were studied. All women underwent basal serum hormone and metabolic measurements, FSH stimulation testing with transvaginal ovarian sonography, frequently sampled IV glucose tolerance testing, and whole-body dual-energy x-ray absorptiometry. Serum hormone/metabolite levels, 24-hour serum E2 response to 150 IU recombinant human (rh) FSH infusion, AFN, insulin sensitivity, and body mass measurements. Serum E2 responsiveness to rhFSH and AMH levels were greater in women with PCOS than in BMI- and age-matched control women, as were serum androgen levels, AFN, and abdominal fat mass. In all women combined, serum E2 responsiveness to rhFSH was associated with AFN. Serum AMH levels, however, positively correlated with AFN but remained positively correlated with serum LH and free T levels and negatively correlated with total body fat and percent body fat, adjusting for AFN. In normal-weight women with PCOS, serum E2 hyperresponsiveness to rhFSH represents increased AFN, while elevated serum AMH levels reflect opposing effects of stimulatory reproductive (hyperandrogenism and increased AFN) versus inhibitory metabolic (body fat) factors. Given the small number of subjects reported, additional follow-up studies are required to confirm these data.

Acute effects of lead on porcine neonatal Sertoli cells in vitro

Environmental pollution is one of the main factors responsible for reducing fertility in males. Lead is one of the major heavy metal contaminants that impairs several organs; it preferentially accumulates in male reproductive organs and alters sperm quality both in vivo and in vitro. However, the underlying mechanisms remain unclear. Sertoli cells (SCs) provide structural and physiological support to spermatogenic cells within seminiferous tubules. Therefore, changes in SCs affect the developing germ cells and alter spermatogenesis. This study aimed to assess whether exposure to subtoxic doses of adversely affects SC functioning in higher mammals. Purified and functional porcine neonatal SCs were exposed to lead acetate at three different concentrations. Lead exposure decreased the mRNA expression and protein levels of inhibin B and anti-Mullerian hormone (AMH) compared to control, indicating loss of FSH-r integrity in terms of 17-β-oestradiol production under FSH stimulation. In addition, we observed an increase in the mRNA levels of Akt and mTOR, and the phosphorylation of p38 and Akt in SCs exposed to lead at all concentrations compared to unexposed control SCs. In conclusion, lead is toxic to SCs, even at low concentrations, and is expected to alter spermatogenesis.

4 Relationship Between Ovarian Vascularity, Cumulus–Oocyte Morphology and Luteal Development in Four-Month-Old Calves After FSH Stimulation

The objectives of this study were to determine the effect of LH on the blood flow to the ovaries of 4-month-old calves after 2 FSH stimulation protocols, and to examine the relationship between ovarian vascularity after superstimulation to the morphology of the cumulus–oocyte complexes (COC) and luteal function. We hypothesise that ovarian vascularity (detected by 3-dimensional (3D) analysis of Doppler ultrasound cineloops) will increase in response to LH, and the magnitude of change in vascularity would be predictive of (1) a greater proportion of expanded COC, (2) greater development of luteal tissue volume and vascularity at 3 and 7 days after follicle aspiration, and (3) higher levels of plasma progesterone. Ovarian superstimulation was initiated at the beginning of an induced follicular wave in 4-month-old beef calves (n = 16), and beef cattle >16 months of age (control group, adults; n = 8) using either a traditional 4-day or an extended 7-day FSH protocol (n = 8 calves and n = 4 controls per group). Power Doppler ultrasound cineloops were recorded immediately before (i.e. 12 h after the last FSH treatment) and 24 h after LH treatment (before ultrasound-guided follicular aspiration for oocyte collection) to assess ovarian vascularity, and 3 and 7 days after follicular aspiration to assess luteal tissue volume and vascularity. Video segments were analysed in Fiji and Imaris software to obtain the 3D ovarian vascularity index (ratio of blood flow volume to tissue volume). The ovarian vascularity index tended to increase >1.7-fold in response to exogenous LH in both prepubertal calves (pre-LH 1.5 ± 0.4% v. post-LH 2.6 ± 0.7%; P = 0.08) and adult cattle (pre-LH 2.2 ± 0.6% v. post-LH 4.7 ± 0.9%; P = 0.07). Calves with a recovery of >75% of expanded COC had a higher ovarian vascularity index (10.7 ± 2.6% v. 4.8 ± 1.6%; P = 0.06) and luteal vascularity index (15.7 ± 4.5% v. 5.7 ± 2.1%; P < 0.05) 7 days after aspiration than those with <75% expanded COC. Calves in the 7-day FSH protocol had >10-fold higher concentration of plasma progesterone on Day 3 (12.7 ± 7.3 ng mL−1 v. 1.2 ± 0.4 ng mL−1; P < 0.05) and Day 5 (50.6 ± 28.0 ng mL−1 v. 4.5 ± 1.0 ng mL−1; P < 0.05), and ~2-fold higher luteal vascularity index at 7 days after follicle aspiration (13.7 ± 4.6% v. 7.7 ± 2.8%; P < 0.05) than calves in the 4-day FSH protocol, whereas no difference (P > 0.05) was found in control (adult) animals. In conclusion, there was an increase in ovarian vascularity resulting from LH treatment in prepubertal calves and adult cattle. A greater proportion of expansion of COC at 24 h after LH treatment (an indicator of follicular maturation) was related to higher ovarian and luteal vascularity on Day 7 after collection in prepubertal calves, but not in adults. Luteal vascularity on Day 3 was reflective of plasma progesterone concentration, and prepubertal calves in the 7-day FSH protocol had greater plasma progesterone than calves in the 4-day FSH protocol. The use of FSH in calves allows a greater number of follicles for oocyte collection as it does in adult cattle. Research was supported by an NSERC grant.

A novel homozygous mutation in the FSHR gene is causative for primary ovarian insufficiency

To identify the potential FSHR mutation in a Chinese woman with primary ovarian insufficiency (POI). Genetic and functional studies. University-based reproductive medicine center. A POI patient, her family members, and another 192 control women with regular menstruation. Ovarian biopsy was performed in the patient. Sanger sequencing was carried out for the patient, her sister, and parents. The novel variant identified was further confirmed with the use of control subjects. Sanger sequencing and genotype analysis to identify the potential variant of the FSHR gene; hematoxylin and eosin staining of the ovarian section to observe the follicular development; Western blotting and immunofluorescence to detect FSH receptor (FSHR) expression; and cyclic adenosine monophosphate (cAMP) assay to monitor FSH-induced signaling. Histologic examination of the ovaries in the patient revealed follicular development up to the early antral stage. Mutational screening and genotype analysis of the FSHR gene identified a novel homozygous mutation c.175C>T (p.R59X) in exon 2, which was inherited in the autosomal recessive mode from her heterozygous parents but was absent in her sister and the 192 control women. Functional studies demonstrated that invitro the nonsense mutation caused the loss of full-length FSHR expression and that p.R59X mutant showed no response to FSH stimulation in the cAMP level. The mutation p.R59X in FSHR is causative for POI by means of arresting folliculogenesis.

Does ovarian reserve affect outcomes in single ideal blastocyst transfers in women less than 40years of age?

There is much debate whether diminished ovarian reserve is purely a quantitative issue, or if quality as determined by pregnancy potential of the ensuing oocytes is also affected. The purpose of this study was to determine whether diminished ovarian reserve, as established by one of three ways described below, affects pregnancy outcomes of women under 40years old undergoing a single ideal blastocyst transfer. This was a retrospective cohort study, including 507 women undergoing an ideal quality single embryo transfer between August 2010 and March 2014. Logistic regression was used to control for age, duration of infertility, parity, body mass index, and smoking status. For analysis, women were stratified for: antral follicle counts (≤5 vs.>5), basal serum FSH levels (<13 vs.≥13IU/L), and quartile of total FSH dose required for stimulation. In stratifying women by antral follicle count (AFC)≤5 vs.>5, the pregnancy rate (40 vs. 53%, p=0.04), clinical pregnancy rate (29 vs. 46%, p=0.02), and live birth rate (13 vs. 43%, p=0.001) were superior with AFC>5. Using FSH levels (<13 vs.≥13IU/L), the pregnancy rate (50 vs. 31%, p=0.27), clinical pregnancy rate (40 vs. 13%, p=0.45), and live birth rate (38 vs. 13%, p=0.48) were similar. Examining quartiles of FSH stimulation, the pregnancy rates were similar (from lowest to highest: 45, 52, 54, 41%, p=0.13); however, clinical pregnancy rate (36, 43, 47, 25%, p=0.003) and live birth rate (32, 38, 44, 20%, p=0.005) were superior in lower quartiles vs. the highest quartile. Ovarian reserve may affect embryo pregnancy potential and outcomes when measured by AFC and exogenous stimulation but not by basal FSH levels.

Association between FSH dose and AMH level on oocyte quantity and quality in GnRH antagonist cycles

Accumulating data suggest that high FSH doses during IVF may impair follicular number and function, as well as oocyte and embryo quality. It is unknown whether this detrimental relationship holds true for all patients regardless of AMH level. The objective of this study was to test the hypothesis that high FSH dosing has a detrimental effect on oocyte quantity and quality in patients with low and normal AMH, but may be necessary for patients with a high AMH to overcome the inhibitory effect of AMH on gonadotropin receptor expression and action in granulosa cells. Retrospective cohort study All first GnRH antagonist IVF/ICSI cycles between 1/2013 and 5/2016 (n=994) in patients with a documented AMH at a single academic institution were included. The cohort was stratified by AMH (ng/mL) into tertiles (<1.8, 1.8-4.2, >4.2). The primary exposure was daily FSH dose on cycle days 2-7: <225IU, 225-449IU, and >450IU. Primary outcomes were total and mature (MII) oocyte yield. Secondary outcomes were 2PN and usable (transferred plus frozen) embryo yield, as well as % cycles with at least 1 degenerating oocyte or embryo, and/or arrested embryo. Poisson regression adjusted a priori for age and AMH was used to model the association between FSH dosing and stimulation outcomes across AMH tertiles. Other covariates, including diagnosis, donor status, BMI, day 3 FSH, antral follicle count, use of hMG, trigger type, and length of OCP lead-in were tested for confounding. High FSH dosing was associated with a significantly lower yield of MII and total oocytes for both low and medium AMH tertiles, but the opposite was observed for the high AMH tertile (Table). In contrast to the lack of association between high FSH dose and usable embryos in the low AMH tertile, a significant association was observed in the high AMH tertile. There was no significant association between FSH dose and 2PN yield, or % cycles with oocyte/embryo degeneration or arrested embryos. These findings support the hypothesis that early high FSH doses in GnRH antagonist cycles have a negative effect on total and mature oocyte yield in patients with an expected low or normal response. However, in patients with a high AMH (>4.2 ng/mL), the effect is opposite, suggesting that high gonadotropin dosing may be necessary to overcome the inhibitory effect of AMH on early folliculogenesis.Tabled 1Ovarian stimulation outcomes as a function of day 2-7 FSH dosing (daily IU) and AMH (ng/mL) tertilesAMH<1.8AMH 1.8-4.2AMH>4.2N=323N=337N=334Mean no. MII oocytesFSH dose <225 IU9.2 (6.2)12.2 (6.1)14.2 (8.1)225-449 IU7.5 (4.1)11.5 (6.5)15.9 (9.5)>450 IU5.3 (3.0)7.2 (4.0)16.7 (10.2)aRR (95% CI)1.00 (Ref)1.00 (Ref)1.00 (Ref)aRR (95% CI)0.95 (0.81-1.12)1.11 (1.04-1.20)1.17 (1.07-1.27)aRR (95% CI)0.80 (0.67-0.96)0.73 (0.60-0.89)1.21 (0.99-1.48)P-value<0.0001<0.00010.01Mean no. oocytes retrievedFSH dose <225 IU11.6 (6.8)15.1 (7.2)18.8 (10.2)225-449 IU9.6 (5.0)14.8 (7.6)20.8 (10.8)>450 IU7.2 (3.8)11.4 (4.7)26.7 (16.5)aRR (95% CI)1.00 (Ref)1.00 (Ref)1.00 (Ref)aRR (95% CI)0.96 (0.83-1.11)1.15 (1.08-1.23)1.18 (1.09-1.27)aRR (95% CI)0.86 (0.73-1.01)0.91 (0.78-1.07)1.49 (1.27-1.75)P-value0.02<0.0001<0.0001Mean no. usable embryosFSH dose <225 IU4.1 (2.7)5.0 (3.1)5.8 (4.0)225-449 IU3.2 (2.0)4.9 (3.9)6.3 (4.5)>450 IU2.9 (1.9)4.5 (2.8)7.0 (3.4)aRR (95% CI)1.00 (Ref)1.00 (Ref)1.00 (Ref)aRR (95% CI)0.88 (0.68-1.14)1.17 (1.04-1.31)1.25 (1.09-1.43)aRR (95% CI)0.91 (0.69-1.21)1.10 (0.83-1.47)1.43 (1.01-2.02)P-value0.600.030.001Values represent mean +/- SD Open table in a new tab

The testicular transcriptome associated with spermatogonia differentiation initiated by gonadotrophin stimulation in the juvenile rhesus monkey (Macaca mulatta).

What is the genetic landscape within the testis of the juvenile rhesus monkey (Macaca mulatta) that underlies the decision of undifferentiated spermatogonia to commit to a pathway of differentiation when puberty is induced prematurely by exogenous LH and FSH stimulation? Forty-eight hours of gonadotrophin stimulation of the juvenile monkey testis resulted in the appearance of differentiating B spermatogonia and the emergence of 1362 up-regulated and 225 down-regulated testicular mRNAs encoding a complex network of proteins ranging from enzymes regulating Leydig cell steroidogenesis to membrane receptors, and from juxtacrine and paracrine factors to transcriptional factors governing spermatogonial stem cell fate. Our understanding of the cell and molecular biology underlying the fate of undifferentiated spermatogonia is based largely on studies of rodents, particularly of mice, but in the case of primates very little is known. The present study represents the first attempt to comprehensively address this question in a highly evolved primate. Global gene expression in the testis from juvenile rhesus monkeys that had been stimulated with recombinant monkey LH and FSH for 48 h (N = 3) or 96 h (N = 4) was compared to that from vehicle treated animals (N = 3). Testicular cell types and testosterone secretion were also monitored. Precocious testicular puberty was initiated in juvenile rhesus monkeys, 14-24 months of age, using a physiologic mode of intermittent stimulation with i.v. recombinant monkey LH and FSH that within 48 h produced 'adult' levels of circulating LH, FSH and testosterone. Mitotic activity was monitored by immunohistochemical assays of 5-bromo-2'-deoxyuridine and 5-ethynyl-2'-deoxyuridine incorporation. Animals were bilaterally castrated and RNA was extracted from the right testis. Global gene expression was determined using RNA-Seq. Differentially expressed genes (DEGs) were identified and evaluated by pathway analysis. mRNAs of particular interest were also quantitated using quantitative RT-PCR. Fractions of the left testis were used for histochemistry or immunoflouresence. Differentiating type B spematogonia were observed after both 48 and 96 h of gonadotrophin stimulation. Pathway analysis identified five super categories of over-represented DEGs. Repression of GFRA1 (glial cell line-derived neurotrophic factor family receptor alpha 1) and NANOS2 (nanos C2HC-type zinc finger 2) that favor spermatogonial stem cell renewal was noted after 48 and 96 h of LH and FSH stimulation. Additionally, changes in expression of numerous genes involved in regulating the Notch pathway, cell adhesion, structural plasticity and modulating the immune system were observed. Induction of genes associated with the differentiation of spermatogonia stem cells (SOHLH1(spermatogenesis- and oogenesis-specific basic helix-loop-helix 1), SOHLH2 and KIT (V-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog)) was not observed. Expression of the gene encoding STRA8 (stimulated by retinoic acid 8), a protein generally considered to mark activation of retinoic acid signaling, was below our limit of detection. The entire mRNA data set for vehicle and gonadotrophin treated animals (N = 10) has been deposited in the GEO-NCBI repository (GSE97786). The limited number of monkeys per group and the dilution of low abundance germ cell transcripts by mRNAs contributed from somatic cells likely resulted in an underestimation of the number of differentially expressed germ cell genes. The findings that expression of GDNF (a major promoter of spermatogonial stem cell renewal) was not detected in the control juvenile testes, expression of SOHLH1, SOHLH2 and KIT, promoters of spermatogonial differentiation in mice, were not up-regulated in association with the gonadotrophin-induced generation of differentiating spermatogonia, and that robust activation of the retinoic acid signaling pathway was not observed, could not have been predicted. These unexpected results underline the importance of non-human primate models in translating data derived from animal research to the human situation. The work described was funded by NIH grant R01 HD072189 to T.M.P. P.A. was supported by an Endocrine Society Summer Research Fellowship Award and CONICET (Argentine Research Council), S.N. by a grant from Vali-e-Asr Reproductive Health Research Center of Tehran University of Medical Sciences (grant #24335-39-92) to Dr Batool Hosseini Rashidi, and M.P.H. by grants from the National Health and Medical Research Council of Australia, and the Victorian State Government's Operational Infrastructure Support Program. The authors have nothing to disclose.

Deregulation of WNT2/FZD3/β-catenin pathway compromises the estrogen synthesis in cumulus cells from patients with polycystic ovary syndrome

Mechanistic insight into estrogen deficiency by polycystic ovary syndrome (PCOS) remains a longstanding challenge in reproductive medicine. Recent advance suggest that Wingless-type MMTV integration site family members (WNTs), in concert with its Frizzled (FZD) receptors, regulate normal folliculogenesis, luteogenesis and ovarian steroidogenesis. However, no studies have so far investigated any causality between WNT-FZDs interactions and disrupted estrogen synthesis under certain pathological conditions. Here, we show that (i) FZD3 expression was significantly up-regulated in the cumulus cells (CCs) from PCOS patients. This up-regulation, along with the activation of WNT2/β-Catenin pathway, was tightly associated with insulin resistance and estrogen deficiency, two hallmarks of PCOS. (ii) Overexpression of exogenous FZD3 in human granulosa cell COV434 impaired long-term FSH incubation-induced CYP19A1 transactivation and the recruitment of β-Catenin onto CYP19A1 promoter, and subsequently compromised FSH-stimulated estrogen production. (iii) Conversely, inhibition of FZD3 expression exhibited a therapeutic effect on estrogen synthesis in PCOS CCs. Thus, excessive FZD3 expression in CCs may act as a brake on steroidogenic activation that is normally overcome by FSH stimulation. Future endeavor in this field should help to elucidate the complicated crosstalk between energy metabolism and endocrine cells through WNT/FZD signaling molecules.

Bio-equivalent doses of recombinant HCG and recombinant LH during ovarian stimulation result in similar oestradiol output: a randomized controlled study

In nature, HCG is secreted by the implanting embryo from peri-implantation and onwards. In contrast, LH is mandatory for steroidogenesis and follicular development during the follicular phase, working in synergy with FSH. Moreover, LH is mandatory for the function of the corpus luteum. Although LH and HCG bind to the same receptor, significant molecular, structural and functional differences exist, inducing differences in bioactivity. This randomized controlled study compared the effect of recombinant FSH stimulation combined with daily either micro-dose recombinant HCG or recombinant LH supplementation in a 1:1 bioactivity ratio from day 1 of stimulation in a long gonadotrophin releasing hormone agonist down regulation protocol. A total of 100 patients from a public clinic completed the study. The primary end-point was the oestradiol level on the day of ovulation trigger and the median oestradiol level in the HCG supplemented group was 8662 pmol/l versus 9203 pmol/l in the recombinant LH supplemented group; therefore, no significant difference was found. Moreover, no differences were observed in the number of oocytes retrieved or in the live birth rate. We conclude that recombinant HCG and recombinant LH are equally effective in boosting oestradiol synthesis during ovarian stimulation when used in a 1:1 bioactivity ratio.

The effect of estradiol on granulosa cell responses to FSH in women with polycystic ovary syndrome

BackgroundThe influence of estradiol (E2) on granulosa cell (GC) function has not been tested clinically in women with polycystic ovary syndrome (PCOS). The objective of this study is to determine if E2 influences GC responses to FSH in women with PCOS.MethodsThis is a two phase, single cohort study conducted over a 2-year period at a single academic center. Nine women with PCOS according to NIH criteria. In Phase 1, FSH stimulation of GC responses as measured by E2 and Inhibin B (Inh B) were assessed before and at 5 and 6 weeks after GnRH agonist administration. In Phase 2, the same protocol was employed with the addition of an aromatase inhibitor (letrozole, LET) administered daily beginning at week 4 for 2 weeks.ResultsIn Phase 1, recovery of FSH, E2 and Inh B from ovarian suppression occurred at 5 and 6 weeks after GnRH agonist injection and preceded resumption of LH and androgen secretion. In Phase 2, hormone recovery after GnRH agonist was characterized by elevated FSH and suppressed E2 levels whereas recovery of LH and androgen levels were unchanged. In Phase 1, FSH stimulated E2 and Inh B responses were unaltered during recovery from ovarian suppression. In Phase 2, E2 and Inh B fold changes after FSH were significantly reduced at weeks 5 (p < 0.04) and 6 (p < 0.01), respectively.ConclusionIn anovulatory women with PCOS, chronic, unopposed E2 secretion may contribute, at least in part, to enhanced ovarian responsiveness to FSH.Trial registrationNCT02389088

FSH Stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization.

Can granulosa cells produce progesterone (P) in response to FSH stimulation? FSH actively promotes P synthesis and output from granulosa cells without luteinization by up-regulating the expression and increasing enzymatic activity of 3β-hydroxysteriod dehydrogenoase (3β-HSD), which converts pregnenolone to P. Serum P level may rise prematurely prior to ovulation trigger in stimulated IVF cycles and adversely affect implantation and clinical pregnancy rates by impairing endometrial receptivity. A translational research study. Human ovarian cortical samples (n=15) and non-luteinizing FSH-responsive human mitotic granulosa cell line (HGrC1) were stimulated with rec-FSH at 12.5, 25 and 50 mIU/ml concentrations for 24 and 48 h. FSH receptor expression was knocked-down and up-regulated in the granulosa cells using short hairpin RNA (shRNA) technology and activin-A administration, respectively. The expressions of the steroidogenic enzymes were analyzed at mRNA level by real-time quantitative RT-PCR, and protein level by western blot and immunoprecipitation assay. The enzymatic activity of 3β-HSD was measured using a spectrophotometric method. In vitro estradiol (E2) and P productions of the cells before and after FSH stimulation were measured by electro-chemiluminescence immunoassay method. Stimulation of the HGrC1 cells with FSH resulted in a dose-dependent increase in the mRNA and protein level of 3β-HSD. Overall, when all time points and FSH doses were analyzed collectively, FSH significantly up-regulated the mRNA expression of its own receptor (3.73±0.06-fold, P<0.001), steroidogenic acute regulatory protein (stAR, 1.7±0.03-fold, P<0.01), side-chain cleavage enzyme (SCC, 1.75±0.03-fold, P<0.01), aromatase (4.49±0.08-fold, P<0.001), 3β-HSD (1.68±0.02-fold, P<0.01) and 17β-hydroxy steroid dehydrogenase (17β-HSD, 2.16±0.02-fold, P<0.01) in the granulosa cells. Expression of 17α-hydroxylase (17α-OH, 1.03±0.01-fold P>0.05) did not significantly change. Similar changes were observed in the protein expression analysis of these enzymes on western blotting after FSH stimulation. FSH significantly increased 3β-HSD, 17β-HSD and aromatase in a dose-dependent manner but did not affect 17α-OH. Protein expression of P was increased along with 3β-HSD after FSH stimulation, which was further evidenced by immunoprecipitation assay. Enzymatic activity of 3β-HSD was significantly enhanced by FSH administration in the HGrC1 cells in a dose-dependent manner. In line with these findings P output (1.05±0.3 vs. 0.2±0.1 ng/ml, respectively, P<0.001) from the samples stimulated with FSH were significantly increased along with E2 (1918±203 vs. 932±102 pg/ml, respectively, P<0.001) compared to unstimulated controls. FSH-induced increase in 3β-HSD expression was amplified and reversed in the HGrC1 cells when FSH receptor expression was up-regulated by activin-A and down-regulated with shRNA, respectively. As only the effect of FSH was studied we cannot extrapolate our findings to the potential effects of HMG and recombinant LH. This data provides a molecular explanation for the largely unexplained phenomenon of P rise during the follicular phase of gonadotropin stimulated IVF cycles. Our findings may progress the research to uncover potential mechanisms for preventing premature P rise that appears to be associated with inferior outcomes in women undergoing IVF. Funded by the School of Medicine and the Graduate School of Health Sciences of Koc University. All authors declare no conflict of interest. None.

Recombinant LH supplementation in patients with a relative reduction in LH levels during IVF/ICSI cycles: A prospective randomized controlled trial

PurposeThe aim of this study was to assess the use of recombinant luteinizing hormone (rLH) supplementation in patients who experience a reduction in LH concentration during controlled ovarian hyperstimulation (COH) for IVF/ICSI. MethodsA multi-center prospective randomized controlled trial (RCT) was performed over three years. Two hundred and forty patients aged between 24 and 42 years undergoing IVF/ICSI treatment with a long down regulation (LDR) protocol were recruited. LH was measured on the day FSH was started and again 6days later. 100 patients had a 50% or greater reduction in LH levels and these were randomized to receive either recombinant LH (rLH) supplementation (group 1, n=43) or no additional rLH supplementation (group 2, n=57). Group 1 received rLH 75IU daily from day 7 of FSH stimulation to the day of HCG trigger. ResultsThere were no differences in either live birth or clinical pregnancy rates per embryo transfer between the two groups (27.8% vs. 37.0%, p=0.39, RR=0.75, 95%CI 0.39–1.44 and 36.1% vs. 43.5% p=0.51, RR=0.84, 95%CI 0.5–1.48, respectively). ConclusionIn conclusion the addition of rLH in patients with a relative reduction in serum LH concentration during COH for IVF/ICSI did not improve live birth or clinical pregnancy rates. However the results were not conclusive and further large well-designed RCTs are required to confirm these findings.

Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density

BackgroundIn previous studies it has been shown that bovine granulosa cells (GC) cultured at a high plating density dramatically change their physiological and molecular characteristics, thus resembling an early stage of luteinization. During the present study, these specific effects on the GC transcriptome were comprehensively analysed to clarify the underlying mechanisms.MethodsGC were cultured in serum free medium with FSH and IGF-1 stimulation at different initial plating density. The estradiol and progesterone production was determined by radioimmunoassays and the gene expression profiles were analysed by mRNA microarray analysis after 9 days. The data were statistically analysed and the abundance of selected, differentially expressed transcripts was re-evaluated by qPCR. Bioinformatic pathway analysis of density affected transcripts was done using Ingenuity Pathway Analysis.ResultsThe data showed that at high plating density the expression of 1510 annotated genes, represented by 1575 transcript clusters, showed highly altered expression levels. Nearly two-thirds were up- and one third down-regulated. Within the top up-regulated genes VNN2, RGS2 and PTX3 could be identified, as well as HBA or LOXL2. Down-regulated genes included important key genes of folliculogenesis like CYP19A1 and FSHR. Ingenuity pathway analysis identified “AMPK signaling” as well as “cAMP-mediated signaling” as major pathways affected by the alteration of the expression profile. Main putative upstream regulators were TGFB1 and VEGF, thus indicating a connection with cell differentiation and angiogenesis. A detailed cluster analysis revealed one single cluster that was highly associated with the upstream regulator beta-estradiol. Within this cluster key genes of steroid biosynthesis were not included, but instead, other genes importantly involved in follicular development, like OXT and VEGFA as well as the three most down-regulated genes TXNIP, PAG11 and ARRDC4 were identified.ConclusionsFrom these data we hypothesize that high density conditions induce a stage of differentiation in cultured GC that is similar to early post-LH conditions in vivo. Furthermore we hypothesize that specific cell-cell-interactions led to this differentiation including transformations necessary to promote angiogenesis.

Pb effects on an experimental model of porcine prepubertal Sertoli cells.

The environmental pollution is one of the main factors implicated in the world’s fertility decline. Lead (Pb) is one of the major heavy metal contaminants that impairs several organs but preferentially accumulates in male reproductive organs and alters in vivo and in vitro sperm quality [1]. Nowadays, the underlying mechanisms remain unclear. Sertoli cells (SC) provides structural and metabolic support to the spermatogenic cells within the seminiferous tubules, therefore, metabolic and structural changes in SC affect the developing germ cells and consequently alter spermatogenesis. This study aimed to assess whether exposure to subtoxic doses of Pb would adversely affect superior mammalian SC function. Highly purified and functional porcine pre-pubertal SC were isolated [2] and treated with three different Pb acetate concentrations. Parameters of SC functionality, such as inhibin B and anti-Mullerian hormone (AMH) mRNAs and proteins were decreased by Pb exposure respect to the control, such as the FSH-r integrity in terms of 17-β-estradiol production, under FSH stimulation. In addition, we observed an increase of AKT and mTOR mRNAs, p38 phosphorylation ratio and Akt phosphorylation ratio in all experimental conditions, respect to the control. In conclusion, the Pb-related toxicity on SC, even at low concentrations, is expected to alter spermatogenesis.

The impact of ovarian stimulation protocol on oocyte quality, subsequent in vitro embryo development, and pregnancy after transfer to recipients in Eld's deer (Rucervus eldii thamin)

Propagating genetically valuable individuals through oocyte collection, in vitro fertilization (IVF) and embryo transfer is critical to maintain sustainable populations of the endangered Eld's deer. The objectives of this study were to assess the impact of exogenous FSH injections on (1) the number and in vitro competence of oocytes collected and (2) the developmental potential of resulting IVF embryos after transfer into recipients during the breeding season (February–April). In a pilot experiment, estrus synchronization was conducted in three surplus females (using intravaginal progesterone-releasing devices, CIDRG for 14 days and injections of buserelin (a GnRH agonist). Five days after CIDR removal, ovaries were excised, minced and a total of 133 oocytes were recovered. Following in vitro maturation (IVM) and IVF, 63% of the oocytes formed embryos but only 5% reached the blastocyst stage. In a subsequent study, follicle numbers and diameters were compared between synchronized does stimulated with 0 or 80 mg FSH (-FSH and +FSH; n = 8 does in each group) and oocytes collected either by laparoscopic ovum pick-up or ovariectomy. FSH stimulation increased the main follicular diameter from 2–3 mm to 4–5 mm (P < 0.05) but not the oocyte number (∼20/donor) or the percentage of good quality oocytes (57%) regardless of the treatment. FSH stimulation did not either affect the percentage of cleaved embryos after IVF (25–35%; P > 0.05). Lastly, embryos at the 2-to 8-cell stage (from either + FSH or –FSH groups) were transferred into the oviducts of 11 synchronized recipients. With the +FSH embryos, three pregnancies failed between 90 and 120 days of gestation and two fawns that were born preterm (Days 215 and 224 of gestation) died at birth. In the -FSH group one healthy female fawn was born on Day 234 of gestation. This is the first report of a successful in vitro embryo production and subsequent birth of a live Eld's deer fawn. Further investigations are required to improve IVM/IVF success and the developmental potential of the embryos.

Molecular regulation of LHCGR expression by miR-122 during follicle growth in the rat ovary

We have previously reported that LHCGR expression in the ovary is regulated through a post-transcriptional mechanism involving an mRNA binding protein designated as LRBP, which is regulated, at least in part, by a non-coding RNA, miR-122. Our present study examined the regulatory role of miR-122 in FSH-induced LHCGR expression during follicle development. Treatment of rat granulosa cells concurrently with FSH and 17β estradiol showed, as expected, a time-dependent increase in LHCGR mRNA levels as well as hCG-induced progesterone production. However, miR-122 expression was decreased during the early time periods, which preceded the increased expression of LHCGR mRNA. The role of miR-122 in FSH-induced LHCGR mRNA expression was then examined by overexpressing miR-122 prior to FSH stimulation by infecting granulosa cells with an adenoviral vector containing a miR-122 insert (AdmiR-122). Pretreatment with AdmiR-122 resulted in complete abrogation of FSH- mediated upregulation of LHCGR. AdmiR-122 also blocked FSH-induced decrease in LRBP expression and increased the binding of LHCGR mRNA to LRBP. Based on these results, we conclude that miR-122 plays a regulatory role in LHCGR expression by modulating LRBP levels during FSH-induced follicle growth.