Recombinant Follicle-stimulating Hormone Research Articles

Resistant ovary syndrome: Two case reports and a literature review of effective controlled ovarian stimulation in IVF.

Resistant ovary syndrome (ROS) represents a rare reproductive endocrine disorder that is predominantly associated with infertility, characterized by heightened endogenous gonadotropin levels in the presence of a normal ovarian reserve. Patients with ROS typically exhibit a poor response to exogenous gonadotropins during controlled ovarian stimulation (COS). Due to the absence of a universally accepted effective COS protocol, this study aims to contribute to the existing body of literature by detailing 2 successful pregnancies achieved through conventional in vitro fertilization (c-IVF) in patients with ROS, and through retrospective analysis, seeks to elucidate the factors contributing to the successful ovarian stimulation in these cases, with the ultimate goal of establishing clinical guidelines for ROS management. The central challenge addressed in this study pertains to the effective induction of oocyte maturation during c-IVF COS in ROS patients. The study focuses on 2 infertile women diagnosed with ROS who sought to conceive via c-IVF. The patients were subjected to a COS protocol involving pituitary downregulation followed by ovarian stimulation using recombinant follicle-stimulating hormone (r-FSH) and human menopausal gonadotropin (HMG), preceded by 3 cycles of hormone replacement therapy (HRT) pretreatment. The proposed protocol elicited a favorable ovarian response, culminating in the retrieval of numerous mature oocytes and the development of multiple viable embryos via c-IVF, ultimately leading to successful live births post-embryo transfer. Our study suggests that the outlined COS protocol may serve as a viable treatment option for ROS patients aspiring to conceive through c-IVF, thereby potentially expanding the therapeutic repertoire for this challenging condition.

Randomized, Assessor-Blinded Trial Comparing Highly Purified Human Menotropin and Recombinant Follicle-Stimulating Hormone in High Responders Undergoing Intracytoplasmic Sperm Injection

(Abstracted from Fertil Steril 2020;114:321–330 There exists considerable variation in patient’s response to gonadotropin stimulation when undergoing assisted reproductive technologies (ARTs). Serum anti-Müllerian hormone (AMH) level has been shown to be a robust predictor of ovarian response and is used to guide gonadotropin dosing.

Outcomes of Ovulation Induction Aimed to Pregnancy in Eight Hypopituitarism Patients.

Female sex hormones work in concert. Gonadotropin-releasing hormone and ovulation-inducing agents are required in female patients with infertility owing to hormone dysregulation. Although drug-induced follicular development can be expected in patients with endogenous female hormone deficiency, data are lacking on the protocols and drugs used. We retrospectively examined the success rates of ovulation induction, assisted reproductive technology, and pregnancy outcomes in 66 cycles of eight patients with pituitary insufficiency at our hospital. Ovulation occurred in 75.4% (49/66); 82.6% (38/46) of patients <40 years and 57.9% (11/19) of patients ≥40 years of age. Five of the eight patients became pregnant, and three delivered babies. The fertilization rate was 78% with in vitro fertilization, and the recombinant follicle-stimulating hormone usage was 3,717.1 ± 1,528.9 International Unit in hypopituitarism patients. Hypopituitarism patients can achieve ovulation, pregnancy, and delivery after optimal gonadotropin administration. Further studies are needed to determine the effects of gonadotropins on other pituitary hormones, such as growth hormones.

Effects of Vaccination against Recombinant FSH or LH Receptor Subunits on Gonadal Development and Functioning Male Rats.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) play key roles in regulating testosterone secretion and spermatogenesis in male mammals, respectively, and they maintain the fertility of male animals by binding to their corresponding receptors. We designed and prepared a recombinant LH receptor (LHR) subunit vaccine and a recombinant FSH receptor (FSHR) subunit vaccine and used male Sprague Dawley (SD) rats as a model to examine their effects on testicular development, spermatogenesis, and testosterone secretion in prepubertal and pubertal mammals. Both vaccines (LHR-DTT and FSHR-DTT) significantly decreased the serum testosterone level in prepubertal rats (p < 0.05) but had no effect on the testosterone secretion in pubertal rats; both vaccines decreased the number of cell layers in the seminiferous tubules and reduced spermatogenesis in prepubertal and pubertal rats. Subunit vaccine FSHR-DTT decreased the sperm density in the epididymis in both prepubertal and pubertal rats (p < 0.01) and lowered testicular index and sperm motility in pubertal rats (p < 0.05), whereas LHR-DTT only reduced the sperm density in the epididymis in pubertal rats (p < 0.05). These results indicate that the FSHR subunit vaccine may be a promising approach for immunocastration, but it still needs improvements in effectiveness.

Expression and functional characterization of chimeric recombinant bovine follicle-stimulating hormone produced in Leishmania tarentolae.

Assisted reproductive techniques are routinely used in livestock species to increase and enhance productivity. Ovarian hyperstimulation is a process that currently relies on administering pituitary-derived follicle-stimulating hormone (FSH) or equine chorionic gonadotropin in combination with other hormones to promote the maturation of multiple follicles and thereby achieve superovulation. The use of partially purified preparations of FSH extracted from natural sources is associated with suboptimal and variable results. Recombinant FSH (rFSH) has been produced in a variety of heterologous organisms. However, attaining a bioactive rFSH of high quality and at low cost for use in livestock remains challenging. Here we report the production and characterization of a single chain bovine rFSH consisting of the β- and α-subunit fused by a polypeptide linker (scbFSH) using Leishmania tarentolae as heterologous expression system. This unicellular eukaryote is non-pathogenic to mammals, can be grown in bioreactors using simple and inexpensive semisynthetic media at 26°C and does not require CO2 or bovine serum supplementation. Stable cell lines expressing scbFSH in an inducible fashion were generated and characterized for their productivity. Different culture conditions and purification procedures were evaluated, and the recombinant product was biochemically and biologically characterized, including bioassays in an animal model. The results demonstrate that L. tarentolae is a suitable host for producing a homogeneous, glycosylated and biologically active form of scbFSH with a reasonable yield.

Experience in using biosimilars in IVF programs with donor oocytes in protocols with micronized progesterone

Relevance: Stimulation of oocyte donors' superovulation occupies a key place in the development of general assisted reproduction technology (ART). Oocyte donation programs involve young and healthy women undergoing severe ovarian stimulation protocols to obtain good-quality oocytes. Advances in recombinant DNA technology led to the development of recombinant follitropin alfa.The study aimed to evaluate the effectiveness of the biosimilar folitropin alfa in the superovulation stimulation cycles of oocyte donors in comparison with the original medication in Progestin primed ovarian stimulation (PPOS) protocols.Materials and Methods: A prospective cohort study included 25 oocyte donors who underwent superovulation induction. The first group consisted of 25 oocyte donors in whom superovulation was stimulated with a bio-analogue of follitropin alfa. The second group (control) included the same 25 oocyte donors stimulated with the original rFSH medication. The donors of the leading group were stimulated with biosimilar recombinant follicle-stimulating hormone and human menopausal gonadotropin. For the control group, a protocol was used using the original drug rFSH and CHMG in the same doses. The obtained donor oocytes were fertilized with ICSI or frozen. The program's result was determined by taking a blood test for b-hCG 14 days after embryo transfer.Results: The number of mature oocytes in the study groups did not differ significantly, amounting to 20.6 ± 1.1 in group 1 and 21.2 ± 1.3 in group 2. The average number of fertilized oocytes in group 1 was 5.6 ± 0.8, and in group 2 – 6.1 ± 1.1; thus, the fertilization rate in the two groups did not differ (67,5% in group 1 vs 79,2% in group 2, р&gt; 0,05). The blastulation rate (49.1% in group 1 and 52.2% in group 2) did not differ significantly in both the biosimilar group and the original medication. The Top Quality Blastocysts in Group 1 were 1.5 ± 0.7 (55%) vs 1.8 ± 0.5 (48%) in Group 2. At the same time, the level of NNB in both groups also had no statistically significant differences.Conclusion: Sinnal-F was not inferior to the original medication in terms of the proportion of mature oocytes in oocyte donors. The study's results confirm that Synnal-f demonstrates favorable and comparable efficacy to the original medication in oocyte maturation in IVF/ICSI cycles.

(192) Evaluating Effect of hCG and hCG+rFSH Therapy on Ejaculation Disorders in Middle-Aged Men: A Case Study

Abstract Introduction The prevalence of various ejaculation disorders affecting middle-aged and older men has gained increasing attention due to their significant impact on quality of life. One of them, Late-Onset Hypogonadism (LOH) syndrome, can result in a decline in overall well-being. Testosterone replacement therapy (TRT) is commonly used to address LOH; however, it presents challenges such as diminishing endogenous testosterone secretion and incomplete symptom relief. Objective This study aimed to investigate the effects of human chorionic gonadotropin (hCG) or hCG plus recombinant follicle-stimulating hormone (rFSH) therapy in patients experiencing ejaculation disorders characterized by low semen volume and decreased pleasure during ejaculation. Methods Four male patients, aged 55 to 59 years, complaining of decreased ejaculatory pleasure and low semen volume, were enrolled in the study. One patient received hCG+rFSH therapy, while three patients received hCG alone. The initial dose of hCG was 5,000 units administered twice a week, and if deemed insufficient, it was increased to three times a week. rFSH was administered at a dosage of 150 units three times a week. Levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and estradiol (E2) were measured monthly. Anastrozole (1 mg) was added for patients exhibiting elevated E2 levels. Results Case 1: A 58-year-old male. Prior to treatment, testosterone and E2 levels were 4.84 ng/mL and 34.2 pg/mL, respectively. hCG+rFSH therapy resulted in increased testosterone levels (10.2 ng/mL) and elevated E2 levels (109.0 pg/mL) after three months of treatment. Addition of anastrozole resulted in testosterone levels of 10.90 ng/mL and E2 levels of 57.9 pg/mL in the following month. The patient experienced improved sexual desire, increased semen volume, and hair loss as a side effect. Treatment was discontinued after six months. Case 2: A 57-year-old male with diabetes mellitus. Pre-treatment testosterone and E2 levels were 2.19 ng-mL and 21.9 pg/mL, respectively. After changing the hCG dosage to 5,000 units three times a week, testosterone levels reached 5.33 ng/mL and E2 levels increased to 72.8 pg/mL after two months. With the addition of anastrozole four months into treatment, E2 levels were 44.4 pg/mL after five months. The patient reported improved morning erections, increased libido, increased semen volume, and improved glycemic control (HbA1c decreased from 9.0 to 7.0). Case 3: A 55-year-old male. Pre-treatment testosterone and E2 levels were 4.99 ng/mL and 32.3 pg/mL, respectively. In the first month of hCG therapy, testosterone levels increased to 13.6 ng/mL, while E2 levels rose to 75.5 pg/mL. Then, despite testosterone remaining in the 9.0–10.0 ng/mL range, E2 levels increased to 100 pg/mL. With the addition of anastrozole in the fifth month, the patient reported increased semen volume thereafter. Case 4: A 59-year-old male with a history of bipolar disorder. Prior to treatment, testosterone and E2 levels were 2.45 ng/mL and 23.0 pg/mL, respectively. hCG therapy resulted in testosterone levels of 6.71 ng/ml and E2 levels of 51.0 pg/mL after two months. Conclusions This case study highlights the outcomes of hCG or hCG+rFSH therapy in middle-aged men experiencing ejaculation disorders. Disclosure No.

P-03 A report of recurring pregnancy-induced Cushing's Syndrome

Abstract Introduction The incidence of pregnancy-induced Cushing's syndrome (CS) is very low. However, the diagnosis of CS in pregnant patients is very difficult because of an overlapping signs and symptoms. Clinical Case A 20-year-old woman with polycystic ovarian syndrome (PCOS) was successfully treated with clomiphene for infertility and pregnant in September of 2016 for the first time. She experienced out of proportion abdominal obesity, excessive striosis, exacerbation of hirsutism and myasthenia during the first trimester of her pregnancy. Initial diagnosis of ovarian hyperstimulation syndrome (OHSS) was made and due to concomitant intrauterine growth retardation (IUGR), the pregnancy was electively terminated at the 8th week. All associated symptoms spontaneously regressed within 2-3 months after the abortion. The second pregnancy was induced with a pulse treatment of letrozole 5 mg, followed by 3 every other day injections of 100 units recombinant follicle-stimulating hormone. Within the first 4 weeks into the second pregnancy, the patient developed recurrence of OHSS symptoms. In addition to the prior symptoms and progressive proximal myasthenia, the patient developed hypertension, round moon-like face, back hump, and suprasternal fat pad consistent with the typical features of CS. In week 14 of pregnancy, the patient complained from acute-onset bilateral blindness and hypertension (blood pressure of 180/120 mmHg), which soon progressed into loss of consciousness and grand-mal seizure. She was admitted to the intensive care unit (ICU). Admission laboratory testing included those to assess the function of the pituitary-adrenal axis (HPA) due to the presence of symptoms related to CS. Based on available clinical information a diagnosis of hemolysis, elevated liver enzymes, and a low platelet count (HELLP) syndrome was made and emergency termination of pregnancy was carried out at the gestational age of 14 weeks. The patient`s clinical signs such as hypertension and acute blindness were totally subsided soon after the therapeutic abortion. Diagnosis of ACTH independent CS was made based on elevated 8 a.m. serum cortisol level unresponsive to low-dose dexamethasone (45-41 µg/ dL). Conclusion Diagnosis of CS in pregnancy is challenging as the physiological changes during pregnancy may mimic those of CS. Long-term treatment of gestational CS has not been as successful. However, in the case of adrenal hyperplasia, bilateral adrenalectomy offers a certain cure. Our patient had a therapeutic abortion due to severe maternal complications and development of CS during the early gestational age. The patient was reeducated on the subject of contraception and the possibility of adrenalectomy for future pregnancies. Due to the temporal relation between the occurrence of CS after each pregnancy the diagnosis of gestational hypercortisolism was made, and for further pregnancies bilateral adrenalectomy was recommended.

Management of Normogonadotropic males with idiopathic infertility with bilateral varicocelectomy with and without intramuscular recombinant FSH and HCG course(s): A twenty-year- experience in a prospective, controlled, and randomized clinical study

This study presents a clinical prospective double-blinded study that contains three groups of infertile males with normogonadotropic oligoasthenospermia. All groups A, B, and C were operated on for their bilateral varicoceles. The first included 152 patients who were only operated for bilateral varicocele (nominated group A). The second group of 237 patients constituted those who were operated on, with fertile female partners, and whose preoperative spermogram showed a count lower than 15 million/ml and a 3-hour-motility lower than 20%. This group constituted two categories based on their treatment. Therefore, groups B and C received postoperative courses of intramuscular injections of recombinant follicle-stimulating hormone (rFSH) and human chorionic gonadotropin (HCG), i.e., group B received one course (3 months), and group C received two courses (6 months). The main objective is to determine the improvement in the postoperative seminal parameters in the different groups. The sample constituted 391 infertile males who were recruited conveniently based on their willingness to share their progress information and were also informed that they could stop their participation freely with no questions asked. In our treated patients, in both groups B and C, who received 1 or 2 courses of rFSH and HCG, spontaneous pregnancy occurred in 69 patients and 52 patients, respectively. Likewise, assisted pregnancy occurred in 28 and 19 patients, respectively. So treated males of groups B and C resulted in 91/140 (65%) and 61/96 (63.54%) live offspring, respectively.

#21 : Letrozole Can Be a Good Adjuvant for In Vitro Fertilization (IVF) Procedure in Patient with Ovarian Endometriosis

Background and Aims: To evaluation the pregnancy rate in IVF procedure with letrozole in patient with ovarian endometrioma. Method: Prospective study performed by single physician at primary fertility center. From August 2016 to November 2019, 109 cycles of IVF were performed from couples who were diagnosed as endometrioma. 40 women were diagnosed during ovarian cystectomy, 69 were by transvaginal ultrasonography. In 64 cycles, gonadotrophic releasing hormone (GnRH) antagonist protocol was used, and in another 45 cycles, gonadotrophin releasing hormone agonist (GnRHa) protocol were applied. In every cycle, 10 mg of letrozole was added from the start of gonadotrophin till the end of ovarian stimulation. Results: Women’s age was 36.10±3.67, and the number of their previous IVF cycle was 2.66±1.63. AMH was 2.30±2.31, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) on the third day of menstruation were 9.40±5.26, 7.81±4.60 and 54.1±44.2. Start dosages and total dosages of recombinant FSH were 314.32±74.99 and 2541.37±992.77, and serum E2 and progesterone on trigger day were 1220.06±1072.49 and 0.92±0.79. The endometrial thickness was 9.20±1.46. The number of obtained oocytes was 9.05±5.94 and metaphase II(M2) oocytes were 6.71±4.12. 1.91±0.42 embryos were transferred to the uterine cavity and 2.68±2.58 surplus embryos were cryopreserved. Pregnancy rate was diagnosed if serum beta human chorionic gonadotrophin (hCG) level was above 5 ng/ml on 14th day after oocyte retrieval, and the result was 44.95% (49/109). Clinical pregnancy rate which was defined if gestational sac was visualized by transvaginal ultrasonography after at least 21st days after oocyte retrieval, and the result was 24.77% (27/109). Conclusion: In IVF procedure for the patients with ovarian endometrioma, just adding letrozole to conventional controlled ovarian hyperstimulation protocol can reduce serum peak E2 level without make harmful effect on uterine endometrium, and result in comparable pregnancy rate.

Duphaston as a suitable medication for women taking intracytoplasmic sperm injection to treat infertility

Objective: Duphaston is used as an alternative to gonadotropin-releasing hormone (GnRH) antagonists for suppressing premature luteinizing hormone (LH) surges. This study aimed to compare the efficacy of Duphaston protocols with that of Cetrotide in suppressing the LH surge. Methods: In this retrospective cross-sectional study, 669 infertile women (aged 25–40 years) were administered recombinant follicle-stimulating (rFSH) hormone from the third day of their menstrual cycle. After the follicular diameter reached–13-14 mm, Cetrotide was prescribed orally to the control subjects, also case subjects took Duphaston orally. The retrieved oocytes were fertilized by an intracytoplasmic sperm injection (ICSI). Results: The levels of FSH, LH, and estradiol did not differ significantly between the groups (P &gt;0.05). However, the findings indicated that Duphaston led to greater oocyte retrieval than Cetrotide (P=0.006). However, the number of mature oocytes and viable embryos in the case subjects did not reach a significant difference compared to that in the control subjects (P &gt;0.05). The number of good-quality embryos (stage1) was significantly higher in the case subjects (P =0.04). In addition, there is no significant difference in the chemical, clinical pregnancy, implantation rate, and miscarriage rate between both groups (P &gt;0.05) . Conclusion: Duphaston could be utilized as a suitable medication rather than a GnRH antagonist in women subjected to controlled ovarian hyperstimulation.

The effect of letrozole as an adjunct in GnRH-antagonist protocol on IVF/ICSI outcome in women with endometriosis: a randomized clinical trial

BackgroundTo evaluate the effect of adding letrozole to the antagonist ovarian stimulation protocol (COS) on in-vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcome in endometriosis patients.MethodsThis randomized clinical trial was carried out in the department of infertility treatment at Arash Women’s Hospital from May 2019 to May 2021. The eligible women with normal ovarian reserve tests who had endometriosis diagnosis and underwent IVF/ICSI cycles were evaluated. A flexible regimen of GnRH-antagonist protocol was used for COS. In the experimental (n = 34), the patients received 5 mg letrozole daily for the first 5 days in combination with 150 IU of recombinant follicle-stimulating hormone (rFSH). In the control group (n = 30), the patients received only the same dose of rFSH. The treatment cycle was compared between groups.ResultsAnalysis of demographic characteristics, severity of endometriosis, and baseline hormonal tests of patients showed that the two groups were similar and comparable. The means of total used gonadotropins ampoules and serum E2 level on oocyte trigger day in the letrozole group were significantly lower than those of in the control group (P = 0.03 and P = 0.004, respectively). No statistically significant difference in terms of the total number of retrieved and MII oocytes as well as the total numbers of obtained and top-quality embryos, and cryopreserved embryos was found.ConclusionThe co-treatment of letrozole with gonadotropins during the antagonist protocol was associated with a reduction in the total dose of gonadotropins, although it had no effect on the oocyte or embryo yield in patients with endometriosis.Trial registrationThe study was registered in the Iranian Registry of Clinical Trials on 2018 -07-13 (IRCT20150310021420N4 at www.irct.ir, registered while recruiting).

Maternal MicroRNA Profile Changes When LH Is Added to the Ovarian Stimulation Protocol: A Pilot Study.

While the use of follicle-stimulating hormone (FSH) in ovarian stimulation for in vitro fertilization (IVF) is an established practice, the use of luteinizing hormone (LH) remains debatable. MicroRNAs (miRNAs) are short, endogenous, non-coding transcripts that control a variety of cellular functions, such as gonadotrophin production and follicular development. The goal of this pilot study was to investigate whether the employment of recombinant LH (rLH) in ovarian stimulation protocols results in changes in the miRNA profiles in human oocytes. Patients were divided into two groups: seven received recombinant FSH (rFSH, 225 IU), and six received rFSH (150 IU) plus rLH (75 IU). MiRNA predesigned panels and real-time PCR technology were used to analyze the oocytes retrieved from the follicular ovarian retrieval. Among the miRNAs evaluated, a series of them evidenced upregulation or downregulation in their expression in the FSH plus LH group compared to the FSH group. Considering the results obtained from the functional and network analysis, the different maternal miRNA profiles in the two groups revealed a differential modulation of pathways involved in numerous biological functions. Overall, based on the pathways associated with most of these maternal miRNAs, the presence of LH may result in a different modulation of pathways regulating survival under the control of a Tp53-related mechanism. Interestingly, among the miRNAs differentially expressed in oocytes of the two groups, we have found miRNAs already investigated at ovarian, follicular, oocyte, and embryonic levels: hsa-miR-484, hsa-miR-222, hsa-miR-520d-5p, hsa-miRNA-17, hsa-miR-548, and hsa-miR-140. Thus, investigation into the role of these miRNAs in oocyte molecular pathways may help determine how LH affects oocyte competence and eventually leads to the clinical improvement of IVF.

SAT336 A Successful Ovulation Induction And Embryo Generation In A Woman With P450-oxidoreductase Deficiency From Mutation G539R, Which Preferentially Impairs 17,20-lyase Activity

Abstract Disclosure: S. Charoensri: None. M. Thakur: None. R.J. Auchus: None. Introduction: Cytochrome P450 oxidoreductase (POR) deficiency is an autosomal recessive disorder causing decreased activity of several P450 enzymes, including CYP21A2, CYP17A1, and CYP19A1 that are essential for steroidogenesis in the adrenals and gonads. Female POR deficiency patients are typically manifested with genital ambiguity, abnormal menstruation, and infertility. Very few pregnancies have been documented from these patients. Case presentation: During an evaluation for infertility, a 35-year-old woman with late menarche, irregular cycles, and unruptured ovarian follicles was diagnosed with POR deficiency due to compound heterozygous pathogenic variant c.1615G&amp;gt;C (G539R) and a large deletion c.1669+5_1669+32del in the POR gene. Because mutation G539R preferentially impairs the 17,20-lyase activity of CYP17A1, she underwent in-vitro fertilization (IVF) with the protocol that has been used successfully to achieve pregnancy in incomplete 17-hydroxylase deficiency. Oral dexamethasone of 0.5 mg/day was given to suppress the adrenal-derived progesterone. After one menstrual cycle induced with estradiol and progesterone priming, leuprolide acetate was used to down-regulate endogenous gonadotropin secretion, then the oocyte maturation was induced with recombinant follicle-stimulating hormone (rFSH) and highly purified menotrophin. In the first cycle, 6 eggs were retrieved, 2 that were mature and fertilized with intracytoplasmic sperm injection (ICSI); however, none advanced to the blastocyst stage. She underwent the same protocol in a second IVF cycle with higher rFSH dosing. Despite increasing dexamethasone to 1.5 mg/day, progesterone rose to 4.9 ng/mL, while estradiol peaked at 120 pg/mL, consistent with progesterone accumulation in the developing ovarian follicles due to POR deficiency. From 6 eggs retrieved, 5 eggs were mature and all fertilized, and 3 developed past day 3. On day 6, one embryo (grading 5BB) was biopsied for preimplantation genetic testing and frozen. The second phase of the treatment plan is to lower adrenal-derived progesterone with a combination of hydrocortisone and prednisolone, prepare the endometrium with estradiol followed by progesterone, and perform embryo transfer. Conclusions: Very few examples of pregnancy in women with POR deficiency have been reported, and we describe the first example of successful ovulation induction and embryo generation in a woman with POR deficiency caused by the G539R mutation, which preferentially impairs 17,20-lyase activity. The complex array of steroid excess and deficiencies, from both adrenal and ovarian origin, must be systematically managed during assisted reproduction treatment for these women. Presentation: Saturday, June 17, 2023

OR03-01 Functional Rescue Of Trafficking-Defective Mutant Follicle-Stimulating Hormone Receptors (FSHR) Leading To Premature Ovarian Failure With The Pharmacoperone CAN1405

Abstract Disclosure: A. Ulloa-Aguirre: None. T. Zariñán: None. R. Gutiérrez-Sagal: None. S. Nataraja: None. H.N. Yu: None. E. Jardón-Valadez: None. Inactivating mutations in the follicle-stimulating hormone (FSH) receptor (FSHR) cause premature ovarian failure. Approximately 30% of mutant FSHRs (mutFSHR) are misfolded proteins unable to route to the plasma membrane (PM). We herein tested the effect of a small molecule FSHR ago-allosteric modulator (CAN1405) on the functional rescue of several inactivating mutFSHR. Wild-type (WT) and mutFSHRs A189V, A191I and D224V (at the ectodomain), and four FSHRs with mutations in the serpentine region (SR) of the receptor (D408Y, A419I, I423T, and P519T) were expressed in HEK-293 cells and stimulated with recombinant FSH in the presence of vehicle or CAN1405. Changes in plasma membrane expression and function provoked by CAN1405 exposure were analyzed by immunoblotting and by activation of the cAMP-sensitive pSOMLuc reporter plasmid and ERK1/2 phosphoryation, respectively. Molecular interactions between FSHR and CAN1405, were predicted in silico by computational docking. Exposure of WT and mutFSHR-expressing cells to CAN1405 increased the ratio of mature (PM-expressed) to immature (intracellular) FSHR forms (m/i ratio) of all SR mutant FSHRs (from 0.5 to 3.0) as disclosed by immunoblotting. Similar results were observed when activation of the cAMP-sensitive pSOMLuc reporter and ERK1/2 phosphorylation were examined in FSH-stimulated cells. PM expression of FSHR WT and the activation-defective mutFSHR I423T, but not the FSHRs with mutations in the ectodomain, were also increased by CAN1405. Interesting, in the presence of CAN1405, signaling of the mutFSHR I423T was marginal in terms of activation of the pSOMLuc reporter but not of pERK1/2, suggesting that binding of CAN1405 to the mutFSHR changed the conformation of the receptor allowing functional selectivity on FSH-stimulated signaling. CAN1405 was located in a binding pocket at interhelical upper half of the FSHR. Side chains of residues Y530, A450, T449 S453, L415, H615, M585, V612, I588, and K608 were detected in close interaction with the CAN1045 and the FSHR-CAN1405 complex was likely stabilized by hydrophobic interactions. It is concluded that the small molecule CAN1405 with ago-allosteric properties is an effective pharmacological chaperone that corrects trafficking of SR misfolded mutFSHRs and also increases WT FSHR expression and function. These data demonstrate that allosteric modulators of the FSHR may serve as an effective therapeutic approach for the treatment of infertility in women bearing the WT FSHR or in heterozygous carriers of mutations at the transmembrane domain of this receptor. Presentation: Thursday, June 15, 2023

Development of an enzyme-linked immunosorbent assay for determining FSH plasma concentrations in green sea turtle (Chelonia mydas), using recombinant gonadotropins

Follicle-stimulating hormone (FSH) is involved in the regulation of essential reproductive processes such as gametogenesis and follicular growth. There are presently no immunoassays for measuring FSH in turtles. Recently we produced green sea turtle (Chelonia mydas) recombinant (r) FSH as a single-chain polypeptide using the methylotrophic yeast Pichia pastoris expression system, and polyclonal antibodies for the recombinant FSH. In this work we developed a competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of FSH concentrations in plasma samples from green sea turtles. We used the rFSHβα for standard, rFSHβ for coating and a cmFSHβ antibody. The sensitivity of the assay was 0.13 ng/ml and the intra-assay and inter-assay coefficients of variation were 5.54% and 13.52% respectively. Parallelism was observed between the linearized FSH standard curves and the corresponding serial dilutions of green sea turtle plasma samples. We also observed parallelism between the linearized standard and serial dilutions of plasma samples from the loggerhead sea turtle (Caretta caretta), hawksbill sea turtle (Eretmochelys imbricate), and African softshell turtle (Trionyx triunguis). The ELISA was used to study the FSH plasma concentrations during the reproductive cycles and was compared to hormonal steroid concentrations (Testosterone, Estradiol and Progesterone). This revealed a positive correlation between FSH and estradiol concentrations in females; estradiol concentrations were increased immediately after FSH elevation. In addition, nested females presented an increase in FSH concentrations prior to progesterone elevation in January to April, slightly before egg laying. This ELISA will increase our understanding of gonadotropin functions, and their effects on reproduction in the green sea turtle.

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

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

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 invitro fertilization. Basic science research with a preclinical allosteric FSHR agonist. University hospital. 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. Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. 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. 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. 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.

Conventional follicular-phase ovarian stimulation vs. luteal-phase stimulation in suboptimal responders: a randomized controlled trial

To compare the oocyte yield between follicular-phase stimulation (FPS) and luteal-phase stimulation (LPS) in suboptimal responders. Prospective, randomized, crossover clinical trial. Forty-one patients with infertility according to the POSEIDON (Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number) criteria (1b/2b). Crossover study on 2 assigned ovarian stimulations that started randomly in the follicular or luteal phase. The invitro fertilization cycles were not consecutive but separated in time (45 days to 6 months). The random crossover design ensured that all subjects received the first treatment by chance. The primary objective was the number of cumulus-oocyte complexes retrieved in each cycle. Secondary objectives were number of metaphase II and fertilized oocytes, additional doses of recombinant follicle-stimulating hormone, and the duration of ovarian stimulation (days). The mean number of cumulus-oocyte complexes retrieved was similar between the FPS and LPS groups (7.5 ± 4.6 vs. 7.0 ± 4.1; 95% confidence interval [CI] for the mean, 5.8-8.7 vs. 5.6-8.3, respectively; the difference between means, -0.5; 95% CI, -1.8 to +1.5). Similarly, the mean number of metaphase II oocytes retrieved was not different between the FPS and LPS groups (5.4 ± 3.6 vs. 5.2 ± 2.8; 95% CI for the mean, 4.2-6.5 vs. 4.3-6.1, respectively; the difference between means, -0.2; 95% CI, -1.2 to +1.1). Moreover, the secondary objectives were similar between FPS and LPS groups. In this study, the oocyte yield in LPS did not increase in suboptimal responders compared with that in FPS when the onset of LPS was separated in time from FPS. NCT039393990 https://beta.clinicaltrials.gov/study/NCT03939390.

Highly Purified Human Menopausal Gonadotropin (HP-hMG) Versus Recombinant Follicle-Stimulating Hormone (rFSH) for Controlled Ovarian Stimulation in USPredicted High-Responder Patients: A Cost-Comparison Analysis.

Infertility imposes considerable clinical and economic burden, and the high costs of fertility care are a major barrier to payers. This study assessed the cost differences of highly purified human menopausal gonadotropin (HP-hMG) versus recombinant follicle stimulating hormone (rFSH) for controlled ovarian stimulation (COS) protocols in predicted high-responders from the US payer perspective. A discrete event simulation model was built to perform a cost-comparison analysis of HP-hMG versus rFSH in a cohort of predicted high-responders undergoing up to three embryo transfer cycles, informed by efficacy data from the MEGASET-HR trial. The model considered an event-based treatment pathway and transition probabilities were derived from MEGASET-HR. A variable time horizon was employed, and deterministic and probabilistic sensitivity analyses conducted. Subjects undergoing COS with HP-hMG and rFSH demonstrated comparable live birth rates following three in vitro fertilization (IVF) cycles, with 161 live births with HP-hMG and 152 live births with rFSH, per 310 high-responders. The total cost saving per live birth in subjects receiving HP-hMG versus rFSH was US$3024. These cost savings were largely driven by the need for fewer embryo transfers to achieve similar efficacy outcomes and a reduced rate of ovarian hyperstimulation syndrome. Following deterministic sensitivity analysis, HP-hMG remained cost saving in all baseline parameter variations. No parameters led to rFSH providing cost savings when compared with HP-hMG. Comparable clinical outcomes can be achieved at a lower cost when using HP-hMG versus rFSH based COS protocols in a cohort of predicted high-responders. Such cost savings may reduce the economic burden infertility currently presents to US healthcare providers and those seeking fertility care.