Evaluation of a long-acting recombinant bovine FSH for multiple ovulation and embryo transfer in dromedary camels.

The present study was conducted to test a new super-agonist recombinant bovine FSH (rbFSH) to induce superovulation (SOV) in dromedary camels. In experiment I, a single IM injection of 40, 60, 80, 100, or 120 µg rbFSH was administered (4 donors per group) to determine the effective dose resulting in acceptable multiple ovulation and embryo yield. Administration of 40 µg was ineffective, while 100 and 120 µg were associated with increased numbers of developed follicles, corpora lutea, and recovered embryos compared to administration of 60 and 80 µg. In experiment II, donors were divided into treatment groups to compare rbFSH with two conventional protocols for SOV. Donors received a single dose of 2000 IU eCG in combination with 400 mg porcine follicle-stimulating hormone (pFSH; Folltropin-V®; Group 1, n = 29) or 500 µg of pFSH with 100 µg of pLH (Stimufol®; Group 2, n = 16). Group 3 (n = 19) received a single dose of 100 µg rbFSH. No difference was found in the size and number of follicles per donor. Response time, ovulation rate, and the number of corpora lutea and recovered embryos per donor were similar in all groups. The number of medium-sized and transparent embryos decreased while the number of small-sized and semi-transparent embryos increased in Group 3 (rbFSH) compared to the other two groups. The pregnancy rate of the recipients at 10 days post-ET, at two months of gestation, and the rate of early pregnancy loss (EPL) did not differ among the groups. In conclusion, a single IM administration of 100 µg rbFSH induces a successful superovulation in dromedary camels and has the advantage of reducing stress associated with multiple FSH administration of the conventional protocols.

Long-term effect of chemotherapy after ovarian decortication on the ovarian function in women surviving cancer.

PURPOSE: Ovarian decortication may affect ovarian function. We investigated the status of ovarian reserve after ovarian decortication plus chemotherapy at a stage of presumed stabilized recovery in women surviving cancer. METHODS: We searched our database for cancer survivors subjected to ovarian decortication and chemotherapy at least 3 years previously. Ovarian function was explored for levels of anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), and estradiol (E2), and menstrual pattern. RESULTS: Forty women (mean age 29.6 (SD, 6.1) years) were assessed at a mean of 4.7 (1.5) years after surgery. The predecortication levels of AMH and FSH changed at post-treatment from 2.2 (1.4) to 0.5 (1.3) ng/mL for AMH (p < 0.001) and from 4.7 (2.1) to 16.7 (21. 6) IU/L for FSH (p < 0.001). Amenorrhea consistent with primary ovarian insufficiency (POI) was diagnosed in 11 women, and normal ovarian reserve (AMH ≥ 1.0 ng/mL) was found in 4 of the 21 women who recovered regular cycles. Logistic regression confirmed AMH as an independent predictor of diminished ovarian reserve (OR = 0.24, 95% CI: 0.04-0.63, p = 0.025) and POI (OR = 0.11, 95% CI: 0.01-0.52, p = 0.027), and age was predictive of POI (OR = 1.36, 95% CI: 1.08-1.96, p = 0.035) and of irregular menstrual cycle (OR = 1.20, 95% CI: 1.03-1.46, p = 0.034). CONCLUSION: Ovarian decortication plus chemotherapy had a deleterious effect when assessed at a stage of stabilized ovarian recovery, but whether ovarian decortication had a specific impact cannot be revealed from our data.

Oral follicle-stimulating hormone receptor agonist affects granulosa cells differently than recombinant human FSH.

OBJECTIVE: To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing in vitro fertilization. DESIGN: Basic science research with a preclinical allosteric FSHR agonist. SETTING: University hospital. PATIENT(S): Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS. INTERVENTION(S): Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. MAIN OUTCOME MEASURE(S): Estradiol (E2) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells. RESULT(S): TOP5300 consistently stimulated E2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS. CONCLUSION(S): TOP5300 appears to stimulate E2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E2 production.

Effects of follicle-stimulating hormone, insulin-like growth factor 1, fibroblast growth factor 2, and fibroblast growth factor 9 on sirtuins expression and histone deacetylase activity in bovine granulosa cells.

Granulosa cells (GC) are critical regulators of fertility. During the process of ovarian folliculogenesis, these cells undergo profound changes while producing steroid hormones that are important to control follicular growth, oocyte maturation, and ovulation. Sirtuins are enzymes that regulate several biological processes and have been associated with control of GC function. However, how sirtuins are regulated in GC during ovarian folliculogenesis remains to be unveiled. The present study was designed to investigate effects of hormones that control GC proliferation, differentiation, and steroidogenesis on expression of the seven members of the mammalian sirtuins family (SIRT1-7) and on histone deacetylase activity of nuclear sirtuins (SIRT1, 6, and 7) in GC. Bovine granulosa cells were isolated from small antral follicles (1-5 mm) and were treated with or without follicle-stimulating hormone (FSH), insulin-like growth factor 1 (IGF-1), and fibroblast growth factors 2 (FGF2) and 9 (FGF9). Following treatments, cell proliferation was determined via a cell analyzer, estradiol synthesis and histone deacetylase activity were determined via ELISA, and sirtuins mRNA expression was determined via qPCR. Treatments with FSH and IGF-1 stimulated cell proliferation while addition of FGF2 or FGF9 suppressed estradiol production stimulated by FSH plus IGF-1. In terms of treatments that regulated sirtuins expression in GC, fibroblast growth factors were the most impactful: FGF2 alone increased SIRT1 mRNA expression in comparison to several treatments and increased mRNA abundance of SIRT2 and SIRT7 when added to the combination of FSH and IGF-1; the addition of FGF9 to the combination of FSH and IGF-1 increased mRNA expression of SIRT2, SIRT3, SIRT4, SIRT6, and SIRT7 and increased mRNA expression of SIRT5 in comparison to the negative control group that received no treatment. Also, FGF2 alone increased histone deacetylase activity of sirtuins in comparison to all treatments that contained FSH and/or IGF-1. Furthermore, several correlations were observed between treatments and sirtuins expression and activity, between estradiol or GC numbers and sirtuins expression, and between expression of sirtuins. As FGF2 and FGF9 are considered anti-differentiation factors of GC that stimulate GC proliferation while suppressing estradiol production in combination with FSH and IGF-1, data of this study suggest that sirtuins are associated with control of differentiation of bovine GC.

Follicle-Stimulating Hormone Biological Products: Does Potency Predict Clinical Efficacy?

Follicle-stimulating hormone (FSH), together with luteinizing hormone (LH) and human chorionic gonadotropin (hCG), plays a fundamental role in human reproduction. The discovery of FSH and other gonadotropins was a defining moment in our understanding of reproduction and led to the development of many treatments for infertility. In this regard, exogenous FSH has been used to treat infertility in women for decades. Today, several recombinant and highly purified urinary forms of FSH are used in medically assisted reproduction (MAR). However, differences in the macro- and micro-heterogeneity of FSH result in a variety of FSH glycoforms, with glycoform composition determining the bioactivity (or potency), pharmacokinetic/pharmacodynamic (PK/PD) profiles, and clinical efficacy of the different forms of FSH. This review illustrates how the structural heterogeneity of FSH glycoforms affects the biological activity of human FSH products, and why potency does not predict effects in humans in terms of PK, PD, and clinical response.

Effects of Follicle-Stimulating Hormone on Human Sperm Motility In Vitro.

To evaluate whether the follicle-stimulating hormone (FSH) receptor (FSHR) is expressed in human spermatozoa and the effects of FSH incubation on sperm function. Twenty-four Caucasian men were recruited. Thirteen patients had asthenozoospermia, and the remaining 11 had normal sperm parameters (controls). After confirming FSHR expression, spermatozoa from patients and controls were incubated with increasing concentrations of human purified FSH (hpFSH) to reassess FSHR expression and localization and to evaluate progressive and total sperm motility, the mitochondrial membrane potential, and protein kinase B (AKT) 473 and 308 phosphorylation. FSHR is expressed in the post-acrosomal segment, neck, midpiece, and tail of human spermatozoa. Its localization does not differ between patients and controls. Incubation with hpFSH at a concentration of 30 mIU/mL appeared to increase FSHR expression mainly in patients. Incubation of human spermatozoa with hpFSH overall resulted in an overall deterioration of both progressive and total motility in patients and controls and worse mitochondrial function only in controls. Finally, incubation with FSH increased AKT473/tubulin phosphorylation to a greater extent than AKT308. FSHR is expressed in the post-acrosomal region, neck, midpiece, and tail of human spermatozoa. Contrary to a previous study, we report a negative effect of FSH on sperm motility and mitochondrial function. FSH also activates the AKT473 signaling pathway.

Role of aquaporin 3 in reproductive performance of dairy goats after repeated estrus synchronization stimulation.

This study explored the specific molecular mechanisms through which repeated estrus synchronization (ES) treatments reduce the reproductive performance of dairy goats. Ninety-six goats (n = 24/group) were randomly assigned to two groups receiving ES treatments thrice every fortnight (3-equine chorionic gonadotropin [eCG] and 3-follicle stimulating hormone [FSH] groups) and two groups receiving one ES treatment (1-eCG and 1-FSH groups). ES treatments of 1- and 3-eCG goats were performed via the intravaginal insertion of a controlled internal drug release (CIDR) device containing 300 mg progesterone (P4), followed by 300 IU eCG injections 48 h before CIDR withdrawal. The 1- and 3-FSH goats received CIDR for 10 days, followed by 50 IU FSH and 100 µg PGF2α within 12 h of CIDR withdrawal. Ovaries of three goats in estrus from both groups were harvested for analysis. Subsequently, all the goats in estrus were artificially inseminated twice. Consequently, 3-eCG and 3-FSH goats showed a considerably reduced estrus rate and litter size than 1-eCG and 1-FSH goats. AQP3 mRNA and protein expression were significantly higher in the 3-eCG and 3-FSH groups than in the 1-eCG and 1-FSH groups. AQP3 overexpression led to cell apoptosis and decreased steroid hormone secretion ability of ovarian granulosa cells. Moreover, it resulted in a decrease in maturation and cleavage rates after parthenogenetic activation and in vitro fertilization, respectively. AQP3 gene was involved in reducing the reproductive performance of repeated ES-treated dairy goats. These findings provide a theoretical foundation for the effective use of reproductive hormones in breeding techniques for livestock.

AKAP13 Enhances CREB1 Activation by FSH in Granulosa Cells.

Granulosa cells (GCs) must respond appropriately to follicle-stimulating hormone (FSH) for proper follicle maturation. FSH activates protein kinase A (PKA) leading to phosphorylation of the cyclic AMP response element binding protein-1 (CREB1). We identified a unique A-kinase anchoring protein (AKAP13) containing a Rho guanine nucleotide exchange factor (RhoGEF) region that was induced in GCs during folliculogenesis. AKAPs are known to coordinate signaling cascades, and we sought to evaluate the role of AKAP13 in GCs in response to FSH. Aromatase reporter activity was increased in COV434 human GCs overexpressing AKAP13. Addition of FSH, or the PKA activator forskolin, significantly enhanced this activity by 1.5- to 2.5-fold, respectively (p < 0.001). Treatment with the PKA inhibitor H89 significantly reduced AKAP13-dependent activation of an aromatase reporter (p = 0.0067). AKAP13 physically interacted with CREB1 in co-immunoprecipitation experiments and increased the phosphorylation of CREB1. CREB1 phosphorylation increased after FSH treatment in a time-specific manner, and this effect was reduced by siRNA directed against AKAP13 (p = 0.05). CREB1 activation increased by 18.5-fold with co-expression of AKAP13 in the presence of FSH (p < 0.001). Aromatase reporter activity was reduced by inhibitors of the RhoGEF region, C3 transferase and A13, and greatly enhanced by the RhoGEF activator, A02. In primary murine and COV43 GCs, siRNA knockdown of Akap13/AKAP13 decreased aromatase and luteinizing hormone receptor transcripts in cells treated with FSH, compared with controls. Collectively, these findings suggest that AKAP13 may function as a scaffolding protein in FSH signal transduction via an interaction with CREB, resulting in phosphorylation of CREB.

Progestin primed ovarian stimulation using corifollitropin alfa in PCOS women effectively prevents LH surge and reduces injection burden compared to GnRH antagonist protocol.

Utilizing corifollitropin alfa in GnRH antagonist (GnRHant) protocol in conjunction with GnRH agonist trigger/freeze-all strategy (corifollitropin alfa/GnRHant protocol) was reported to have satisfactory outcomes in women with polycystic ovary syndrome (PCOS). Although lessening in gonadotropin injections, GnRHant were still needed. In addition to using corifollitropin alfa, GnRHant was replaced with an oral progestin as in progestin primed ovarian stimulation (PPOS) to further reduce the injection burden in this study. We try to investigate whether this regimen (corifollitropin alfa/PPOS protocol) could effectively reduce GnRHant injections and prevent premature LH surge in PCOS patients undergoing IVF/ICSI cycles. This is a retrospective cohort study recruiting 333 women with PCOS, with body weight between 50 and 70 kg, undergoing first IVF/ICSI cycle between August 2015 and July 2018. We used corifollitropin alfa/GnRHant protocol prior to Jan 2017 (n = 160), then changed to corifollitropin alfa/PPOS protocol (n = 173). All patients received corifollitropin alfa 100 µg on menstruation day 2/3 (S1). Additional rFSH was administered daily from S8. In corifollitropin alfa/GnRHant group, cetrorelix 0.25 mg/day was administered from S5 till the trigger day. In corifollitropin alfa/PPOS group, dydrogesterone 20 mg/day was given from S1 till the trigger day. GnRH agonist was used to trigger maturation of oocyte. All good quality day 5/6 embryos were frozen, and frozen-thawed embryo transfer (FET) was performed on subsequent cycle. A comparison of clinical outcomes was made between the two protocols. The primary endpoint was the incidence of premature LH surge and none of the patients occurred. Dydrogesterone successfully replace GnRHant to block LH surge while an average of 6.8 days of GnRHant injections were needed in the corifollitropin alfa/GnRHant group. No patients suffered from ovarian hyperstimulation syndrome (OHSS). The other clinical outcomes including additional duration/dose of daily gonadotropin administration, number of oocytes retrieved, and fertilization rate were similar between the two groups. The implantation rate, clinical pregnancy rate, and live birth rate in the first FET cycle were also similar between the two groups. In women with PCOS undergoing IVF/ICSI treatment, corifollitropin alfa/PPOS protocol could minimize the injections burden with comparable outcomes to corifollitropin alfa/GnRHant protocol.

The Effect of Luteinising Hormone Suppression in In Vitro Fertilisation Antagonist Cycles.

Use of GnRH antagonists in IVF stimulation protocols shortens controlled ovarian hyperstimulation (COH) and reduces the risk of ovarian hyperstimulation syndrome (OHSS). However, profound reduction in LH levels has been associated with use of GnRH antagonists. This study aims to determine if LH suppression during GnRH antagonist cycles results in poorer IVF outcomes. This was a prospective pilot longitudinal study where serum LH levels were measured on day 2/3 of the menstrual cycle before COH, 1/2 days following institution of GnRH antagonist and at the day of ovulation trigger. A threshold of LH <0.5 IU/L was used to define profound LH suppression. Data on IVF outcomes was collected. Logistic regression analysis was used to investigate risk factors associated with LH suppression following GnRH antagonist IVF treatment. Ninety-one eligible women were recruited. Women underwent a standard antagonist cycle with Puregon 200u and Ganirelix. No participant had LH <0.5 IU/L prior to GnRH antagonist treatment, and 27 participants (29.7%) had significant LH suppression at either time point. Predictors of profound LH suppression following GnRH antagonist treatment identified (P < 0.20) were age (OR = 0.80, P = 0.013), no previous ovulation induction (OR = 0.26, P = 0.033) and previous GnRH antagonist IVF cycle (OR = 4.32, P = 0.125). Numbers of oocytes, embryos and ongoing pregnancy rates at 12 weeks gestation in patients with and without LH suppression did not differ significantly. We found associations between clinical characteristics and risk of profound LH suppression in women undergoing GnRH antagonist IVF cycles, but no significant differences in IVF and pregnancy outcomes between women with and without significant LH suppression.

The Impact of Controlled Ovarian Stimulation Hormones on the Metabolic State and Endocannabinoid System of Human Cumulus Cells.

Different Follicle Stimulating Hormone (FSH) formulation and Luteinizing Hormone (LH) are used in Assisted Reproductive Technology (ART) to induce follicles development and oocytes maturation, but it is still under debate which protocol is to be preferred. In the present study, the different effects on cumulus cells (CCs) of three controlled ovarian stimulation (COS) protocols, based on urinary FSH, recombinant FSH, or human Menopausal Gonadotropin (hMG) administration, were assessed. CCs were obtained from 42 normal-responders women undergoing COS, randomly divided into three groups according to the used gonadotropin formulation. Differences were found in the expression of genes belonging to the endocannabinoid system (the receptors CNR1, CNR2 and TRPV1, and the enzymes involved in the metabolisms of anandamide, NAPE-PLD and FAAH, and 2-acylglycerol, DAGL and MAGL); consistently, changes in lipid (PPARα, and FASN) and carbohydrate (GLUT1 and GLUT9) metabolisms, in CCs' macromolecules composition (highlighted by Fourier Transform Infrared Microspectroscopy, FTIRM), and in the number of retrieved oocytes were found. For the first time, statistically significant evidence on the differences related to each COS protocol on the endocannabinoid system, metabolism and macromolecular composition of CCs was found, representing a proof of concept to be further confirmed in a larger cohort of patients.

Bioactivity of recombinant hFSH glycosylation variants in primary cultures of porcine granulosa cells.

Previous studies have reported hypo-glycosylated FSH and fully-glycosylated FSH to be naturally occurring in humans, and these glycoforms exist in changing ratios over a woman's lifespan. The precise cellular and molecular effects of recombinant human FSH (hFSH) glycoforms, FSH21 and FSH24, have not been documented in primary granulosa cells. Herein, biological responses to FSH21 and FSH24 were compared in primary porcine granulosa cells. Hypo-glycosylated hFSH21 was significantly more effective than fully-glycosylated hFSH24 at stimulating cAMP accumulation and protein kinase A (PKA) activity, leading to the higher phosphorylation of CREB and ß-Catenin. Compared to fully-glycosylated hFSH24, hypo-glycosylated hFSH21 also induced greater levels of transcripts for HSD3B, STAR and INHA, and higher progesterone production. Our results demonstrate that hypo-glycosylated hFSH21 exerts more robust activation of intracellular signals associated with steroidogenesis than fully-glycosylated hFSH24 in primary porcine granulosa cells, and furthers our understanding of the differing bioactivities of FSH glycoforms in the ovary.

Development of a robust reporter gene-based assay for the bioactivity determination of recombinant human follicle stimulating hormone (rhFSH) pharmaceutical products.

FSH plays a key role in the function of the reproductive system of human beings and is widely used both diagnostically and therapeutically in reproductive medicine. With the growing incidence of infertility, the demand for FSH pharmaceutical products is increasing. For this reason, the quality control process for FSH products is becoming more stringent. An accurate determination of bioactivity is crucial for the safety and efficacy of recombinant human follicle stimulating hormone (rhFSH). Up to now, in-vivo bioassay based on FSH-induced increases in rat ovarian weight has been the only method widely accepted by different pharmacopoeias. However this method has such drawbacks as the complex procedures, long assay period and high variability. Here, we established a reporter gene assay (RGA) based on the CHO-K1-FSHR-CRE-Luc cell line that stably expresses human follicle stimulating hormone receptor (hFSHR), as well as a luciferase reporter under the control of cyclic adenosine monophosphate (cAMP) response elements (CRES). Our study showed that our new assay not only has good dose-dependent responsiveness to rhFSH, but it also performs excellently in terms of specificity, precision, linearity, and simplicity compared with in-vivo rat bioassays. These results implied that this robust reporter gene assay may be a viable supplement to the animal in-vivo bioassay and may be employed in potency determination of rhFSH pharmaceutical products.

A pilot study comparing corifollitropin alfa associated with hp-HMG versus high dose rFSH antagonist protocols for ovarian stimulation in poor responders.

Does corifollitropin alfa associated with hp-HMG protocol from the beginning of ovarian stimulation perform better than high dose rFSH alone for ovarian stimulation with GnRH antagonist in poor responders? This retrospective, monocentric, case-control pilot study was conducted in 65 poor responders (Bologna criteria) undergoing 2 consecutive IVF cycles. All patients underwent a first ovarian stimulation cycle with high dose rFSH (≥300 IU/day) alone in antagonist protocol, unfortunately leading to poor ovarian response and no pregnancy. The following cycle was performed with 150 µg of corifollitropin alfa associated with daily injections of hp-HMG from the beginning of the cycle. The primary outcome was the number of mature oocytes retrieved. The secondary outcomes were ovarian stimulation cancellation and embryo transfer rate per initiated cycle. The number of mature oocytes was not significantly different between the 2 groups. However, cycle cancellation rate was significantly lower and the proportion of cycles with embryo transfer was significantly higher with corifollitropin + hp-HMG protocol, leading to an encouraging clinical pregnancy rate of 24.1% per oocyte retrieval. This pilot study based on corifollitropin alfa associated with hp-HMG from the onset of stimulation appears to be promising for ovarian stimulation in poor responders.

Quercetin protects human granulosa cells against oxidative stress via thioredoxin system.

Granulosa Cells (GCs) are sensitive to excessive production of reactive oxygen species (ROS). Quercetin (QUR) is a free radical scavenger which can alleviate oxidative stress through nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/antioxidant response element (ARE) pathway and thioredoxin (Trx) system. We aimed to explore the probable protective role of QUR on cultured human GCs treated with hydrogen peroxide (H2O2) as an inducer of oxidative stress. MTT assay was applied for evaluating the cell cytotoxicity of QUR and H2O2. The rate of apoptotic cells and intracellular ROS generation were determined by Annexin V-FITC/PI staining and 2'-7'-dichlorodihydrofluorescein diacetate fluorescent probes (DCFH-DA), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blot analysis were used to evaluate the gene and protein expression of Nrf2 and kelch-like ech-associated protein 1 (Keap1)1. The Nrf2 and Trx activities were measured by Enzyme-linked Immunosorbent Assay (ELISA). The results indicated that QUR pretreatment can decrease ROS production and apoptosis induced by H2O2. In addition, QUR increased Nrf2 gene and protein expression, as well as its nuclear translocation. Moreover, in QUR-treated group, a lower level of Keap1 protein was observed, which was not reported as significant. The results also indicated a significant correlation between the expression of Nrf2 and Keap1 in QUR-treated group. Further, QUR protected GCs from oxidative stress by increasing Trx gene expression and activity. This study suggests that QUR as a supplementary factor may protect GCs from oxidative stress in diseases related to this condition.

A single injection of corifollitropin alfa supplemented with human chorionic gonadotropin increases follicular recruitment and transferable embryos in the rabbit.

Superovulation protocols are designed to achieve maximum embryo yields. Nevertheless, ovarian response control and the quality of obtained embryos are still a challenge. On the other hand, to save the superovulated embryos until their subsequent use, it is usual to cryopreserve them, so it is also crucial to assess their cryotolerance. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (FSH-CTP) alone or supplemented with human chorionic gonadotropin (hCG) and to determine the impact of this stimulation on in vitro and in vivo development of fresh or devitrified embryos. Our outcomes showed that ovulation rate and recovered embryos were significantly increased when hCG was used. In vitro development of fresh and devitrified embryos and survival at birth were not significantly affected by superstimulation treatment. Results of this study suggest that a single injection of long-acting FSH-CTP supplemented with hCG can be effectively used in rabbits to elicit an increase in ovulation rate and number of recovered embryos. Furthermore, we demonstrated that hCG supplementation had no negative effects in embryo cryosurvival and development, showing similar survival rate at birth than FSH-CTP alone group.

In vitro culture of isolated preantral and antral follicles of goats using human recombinant FSH: Concentration-dependent and stage-specific effect.

The present study aimed to investigate a concentration-response curve of human recombinant FSH (hrFSH) for in vitro culture of isolated preantral and early antral follicles of goats. Isolated follicles were cultured for 18 days using the following treatments: basic culture medium (control); or control medium supplemented with 10, 50, and 100 mIU/mL of hrFSH. At the end of the culture, cumulus-oocyte complexes were recovered and subjected to in vitro maturation. The following endpoints were evaluated: follicle morphology, growth rate and antrum formation, oocyte viability and meiotic stage, and estradiol production, as well as relative expression of FSH receptor (FSHR), and steroidogenic enzyme (3ß-HSD, CYP17, and CYP19A1) genes. In antral follicles, the FSH addition at 50 mIU/mL increased follicular diameter and growth rate, percentage of fully developed oocytes, and oocyte diameter (P < 0.05), and tended to increase the percentage of MII oocytes when compared to the control (P = 0.07). With preantral follicles, FSH addition at 100 mIU/mL increased relative abundance of mRNA for CYP19A1 when compared to the control (P < 0.05). At the same FSH concentrations of 100 and 50 mIU/mL, there was a greater relatively abundance of mRNA for 3ß-HSD and CYP17 in preantral than in antral follicles (P < 0.05). For preantral and antral follicle comparisons when the same treatments were imposed, there were greater concentrations of estradiol for antral follicles (P < 0.05). In conclusion, hrFSH enhanced in a concentration-dependent manner the in vitro development of caprine antral follicles; however, there was no positive effect in the culture of preantral follicles.

Sequential clomiphene/corifollitrophin alpha as a technique for mild controlled ovarian hyperstimulation in IVF: a proof of concept study.

PURPOSE: Mild controlled ovarian hyperstimulation (COH), combined with oocyte retrieval (OR) under local anaesthesia (LA), may provide low-impact IVF. Since a single injection of corifollitrophin alfa (CFA) provides 7 days of COH, we hypothesised that clomiphene-citrate (CC) followed by CFA may provide adequate COH response from one single FSH injection. Therefore, the aim was to assess IVF outcomes after a novel clomiphene citrate/CFA (CC/CFA) protocol, compared to women undergoing standard rFSH COH protocols (good prognosis comparative cohort:GPCC) in a 1:2 matched design. MATERIALS AND METHODS: In this pilot study of 25 patients (ANZCTR id:ACTRN12612000740897, MINIVA:Minimal_Stimulation_in_IVF), we examined the effectiveness of oral clomiphene (100 mg-days 2-6) followed by CFA in a GnRH antagonist protocol producing a single injection COH stimulation regime. All OR were conducted under LA pre-ovarian block. Cycle outcomes were compared to a matched good prognosis comparative cohort (GPCC) undergoing standard rFSH COH. RESULTS: Mild stimulation was achieved with less oocytes being collected compared to the GPCC (6.4 ± 0.7 vs. 10.7 ± 0.9, p < 0.001), resulting in a reduced number of good quality embryos available for transfer/cryopreservation (3.7 ± 0.6 vs. 5.7 ± 0.5, p = 0.01). While embryo quality was similar between the two groups, endometrial thickness was significantly lower in the group receiving CC/CFA. Pregnancy rates were significantly lower in the CC/CFA cohort compared to GPCC (31.8 vs. 57.1%, p = 0.04) and 44% of CC/CFA participants required supplemental rFSH in order to achieve the hCG trigger criteria. CONCLUSION: Sequential clomiphene CFA protocol does not appear to be an optimal regime for low impact IVF treatment as it does not provide adequate COH from a single CFA injection and results in lower fresh embryo transfer pregnancy rates and fewer embryos for cryopreservation.

Cryosurvival of rabbit embryos obtained after superovulation with corifollitropin alfa with or without LH.

The efficiency of an embryo bank depends on provision of optimal conditions for recovery, cryopreservation and transfer to a breed or strain. In this sense, increasing the number of embryos available using superovulation should improve the cryobank efficiency. However, vagueness of response to conventional protocols to control or increase ovarian response and the quality of oocytes and embryos and their cryotolerance remain a challenge. The aim of our study was to evaluate the effect of corifollitropin alpha (CTP) and a recombinant human FSH (rhFSH), alone or supplemented with rhLH, on embryo cryosurvival by in vitro development and OCT4 and NANOG mRNA abundance at blastocyst stage and offspring rate. In vitro development of vitrified embryos was not significantly affected by superstimulation with or without rhLH supplementation, resulting in similar development rates to those of the control groups (fresh and vitrified embryos from non-superstimulated donor does). Blastocysts developed from vitrified embryos showed higher levels of OCT4 transcript abundance than fresh control, while NANOG transcript abundance was only higher in the blastocysts developed from vitrified embryos after superstimulation treatment in comparison with control groups. The implantation and offspring rates at birth were negatively affected by supplementation with rhLH. Both rhFSH or CTP vitrified embryo groups showed an implantation rate similar to those of the control groups, but an offspring rate lower than control. In conclusion, embryos produced using corifollitropin alpha did not compromise the cryosurvival of vitrified embryos in the rabbit. In addition, this study points out the negative effect of rhLH supplementation in terms of offspring rate on embryo vitrification.

Role of FSH glycan structure in the regulation of Sertoli cell inhibin production.

Variations in follicle-stimulating hormone (FSH) carbohydrate composition and structure are associated with important structural and functional changes in Sertoli cells (SCs) during sexual maturation. The aim of the present study was to investigate the impact of FSH oligosaccharide structure and its interaction with gonadal factors on the regulation of monomeric and dimeric inhibin production at different maturation stages of the SC. Recombinant human FSH (rhFSH) glycosylation variants were isolated according to their sialylation degree (AC and BA) and complexity of oligosaccharides (CO and HY). Native rhFSH stimulated inhibin α-subunit (Pro-αC) but did not show any effect on inhibin B (INHB) production in immature SCs isolated from 8-day-old rats. Activin A stimulated INHB and had a synergistic effect on FSH to stimulate Pro-αC. The less acidic/sialylated rhFSH charge analogues, BA, were the only charge analogue mix that stimulated INHB as well as the most potent stimulus for Pro-αC production. Native rhFSH stimulated both Pro-αC and INHB in SCs at a more advanced maturation stage, isolated from 20-day-old rats. In these cells, all rhFSH glycosylation variants increased INHB and Pro-αC production, even in the presence of growth factors. The BA preparation exerted a more marked stimulatory effect on INHB and Pro-αC than the AC. Glycoforms bearing high mannose and hybrid-type oligosaccharides, HY, stimulated INHB and Pro-αC more effectively than those bearing complex oligosaccharides, CO, even in the presence of gonadal growth factors. These findings demonstrate the modulatory effect of FSH oligosaccharide structure on the regulation of inhibin production in the male gonad.