Follitropin Research Articles

Patients with low prognosis in ART: a Delphi consensus to identify potential clinical implications and measure the impact of POSEIDON criteria

BackgroundCurrently, there is no consensus on the optimal management of women with low prognosis in ART. In this Delphi consensus, a panel of international experts provided real-world clinical perspectives on a series of literature-supported consensus statements regarding the overall relevance of the POSEIDON criteria for women with low prognosis in ART.MethodsUsing a Delphi-consensus framework, twelve experts plus two Scientific Coordinators discussed and amended statements and supporting references proposed by the Scientific Coordinators (Round 1). Statements were distributed via an online survey to an extended panel of 53 experts, of whom 36 who voted anonymously on their level of agreement or disagreement with each statement using a six-point Likert-type scale (1 = Absolutely agree; 2 = More than agree; 3 = Agree; 4 = Disagree; 5 = More than disagree; 6 = Absolutely disagree) (Round 2). Consensus was reached if > 66% of participants agreed or disagreed.ResultsThe extended panel voted on seventeen statements and subcategorized them according to relevance. All but one statement reached consensus during the first round; the remaining statement reached consensus after rewording. Statements were categorized according to impact, low-prognosis validation, outcomes and patient management. The POSEIDON criteria are timely and clinically sound. The preferred success measure is cumulative live birth and key management strategies include the use of recombinant FSH preparations, supplementation with r-hLH, dose increases and oocyte/embryo accumulation through vitrification. Tools such as the ART Calculator and Follicle-to-Oocyte Index may be considered. Validation data from large, prospective studies in each POSEIDON group are now needed to corroborate existing retrospective data.ConclusionsThis Delphi consensus provides an overview of expert opinion on the clinical implications of the POSEIDON criteria for women with low prognosis to ovarian stimulation.

In-vitro fertilization experience with follitropin-delta in poor responders identified by POSEIDON Classification.

Controlled ovarian stimulation during in-vitro fertilization (IVF) is personalized based on anticipated hyper, normal, poor response. With respect to poor responders, who are often treated using higher gonadotropin dosing and combination of urinary and recombinant gonadotropins (rFSH) with marginal benefit, we report our experience with a newer, more potent rFSH (Follitropin-δ) undergoing IVF. Retrospective analysis of all IVF cycles in which follitropin-δ was used alone or combined with urinary gonadotropins over a 3-year period. Patients were grouped according to the POSEIDON Classification as expected low responders (POSEIDON 3-4; AMH<1.2; N.=45), unexpected low responders (POSEIDON 1-2; retrieval of ≤9 oocytes; N.=67) and those with a normal response (N.=93). Demographic, stimulation (including target number of retrieved oocytes [8 to14]), embryology and clinical outcome parameters (clinical pregnancy rate [CPR], live birth rate [LBR], cumulative live birth rate [cLBR]) were compared. Those categorized as POSEIDON patients were older, had lower ovarian reserve, were more likely to use a mixed protocol, less likely to reach the target oocytes retrieved (35.7% vs. 51.6%, P<0.001), and had a lower cLBR per patient (29.5% vs. 38.7%, P=0.006) when compared to non-POSEIDON patients. Expected low responders (POSEIDON 3-4) were older and had lower AMH when compared to unexpected low responders (POSEIDON 1-2), but no differences in the target of oocytes retrieved (33.3% vs. 37.3%, P=0.66) and cLBR (28.9% vs. 37.3%, P=0.06) were noted. In expected low responders, follitropin-δ can be used to optimize oocyte collection and clinical outcome though one may need to deviate from the algorithm-suggested dose. Future studies should explore stimulation modifications in unexpected low responders.

Effects of Switching FSH Preparations on Sperm Parameters and Pregnancy: A Prospective Controlled Study

Objective: To study the effect of switching to a follicle-stimulating hormone (FSH) preparation other than that to which infertile male patients have not had an effective response. Patients and methods: Seventy-four normogonadotropinemic, non-obstructive, oligozoospermic patients who were poor responders to the administration of highly purified FSH (hpFSH) (Group 1 (n = 22) and Group 3 (n = 15)) or to recombinant human FSH (rhFSH) (Group 2 (n = 22) and Group 4 (n = 15)) were selected for this prospective study. After 3 months of washout from treatment with the first FSH preparation of choice, rhFSH was administered to patients in Groups 1 and 4 and hpFSH to those in Groups 2 and 3. Serum luteinizing hormone, FSH, total testosterone levels, conventional sperm parameters, testicular volume, and the number of pregnancies were evaluated at study entry and after the first and second treatment cycles. Results: Comparing treatment groups, the greatest improvement in sperm parameters was recorded in the groups of patients prescribed the switch in FSH preparation. Group 1 had the greatest benefit from therapy, with the highest pregnancy rate after the second treatment cycle. Indeed, eight couples achieved pregnancy (36.4%), compared to Groups 2 (n = 4; 18.2%), 3 (n = 1; 6.7%), and 4 (n = 2; 13.3%) (p = 0.04). Conclusions: The results of this study suggest that a therapeutic scheme involving the “switching” of the FSH preparation yields better results than a protocol using the same FSH preparation for six months. These findings, if confirmed by further studies, will help us better design a treatment strategy with FSH for infertile patients with oligozoospermia.

Analytical Investigation of the Profile of Human Chorionic Gonadotropin in Highly Purified Human Menopausal Gonadotrophin Preparations.

Highly purified human menopausal gonadotropin (HP-hMG [Menopur®, Ferring Pharmaceuticals, Saint-Prex, Switzerland]) contains a 1:1 ratio of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This analysis aimed to assess gonadotropin (FSH, LH and hCG) abundance in HP-hMG and clarify the source of hCG by assessing the presence of sulfated glycans, which are diagnostic for pituitary hCG forms due to their distinct glycosylation patterns. Additionally, the purity of each sample, their specific components, and their oxidation levels were assessed. HP-hMG samples (three of Menopur® and two of Menogon® Ferring Pharmaceuticals, Saint-Prex, Switzerland) were included in the current analyses. Brevactid® (urinary hCG; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and Ovidrel® (recombinant hCG; Merck KGaA, Darmstadt, Germany) were used as control samples. Glycopeptide mapping and analysis of impurities were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oxidation was assessed through reducing peptide mapping using LC-MS/MS. The FSH and LH in the HP-hMG samples showed sulfated glycans, while no signals of sulfated glycopeptides were detected on any site of the beta subunit of hCG. HP-hMG test samples presented the same hCG glycan distribution as the control sample (placental hCG, Brevactid®) extracted from the urine of pregnant women, suggesting a non-pituitary source of hCG. Protein impurities were estimated to constitute approximately 20-30% of the entire HP-hMG protein content in the test samples. More than 200 non-gonadotropin proteins were identified in the HP-hMG test samples, of which several were involved in embryonic development or pregnancy. The alpha subunit of the tested samples was strongly oxidized, with a relative abundance of 20% of the total gonadotropin content. Without taking into account all the protein impurities, the beta subunit of LH was detected only in traces (0.9-1.2%) in all tested HP-HMG samples, confirming the data obtained by intact molecule analysis, while high levels of beta hCG (18-47%) were observed. Advanced molecular analysis of HP-hMG indicates a primarily placental origin of hCG, as evidenced by the absence of hCG sulfated glycans and the predominance of placental non-sulfated hCG in LH activity. The analysis revealed 20-30% of protein impurities and a significant presence of oxidized forms in the HP-hMG samples. These findings are critical for understanding the quality, safety, and clinical profile of HP-hMG.

P-602 Comparative analysis of recombinant human FSH and a biosimilar FSH preparation in controlled ovarian stimulation: a retrospective study of 12,952 oocyte donation and autologous cycles

Abstract Study question Does the type of FSH preparation used for ovarian stimulation, recombinant human FSH (r-hFSH) or biosimilar, influence reproductive success in oocyte donation and autologous cycles? Summary answer Ovarian response and reproductive outcomes, including live birth rates, are comparable in oocyte donation and autologous cycle, irrespective of the type of r-hFSH preparation used. What is known already Exogenous gonadotrophins, particularly FSH, have long been used in assisted reproduction, evolving from urinary to recombinant forms. The first generation of r-hFSH, including Puregon® and Gonal-f®, became available in the 1990s. However, interest in biosimilar FSH products has grown globally following the expiry of original patents. Approved by the European Medicines Agency, two r-hFSH biosimilars, Ovaleap® and Bemfola®, offer cost-effective alternatives for infertility treatments. Regulatory guidelines emphasize non-inferiority to the originator product, with clinical trials showing comparable outcomes between biosimilar and reference FSH preparations. However, debates persist regarding the efficacy and safety of r-hFSH biosimilars, particularly in achieving live births. Study design, size, duration In this retrospective cohort study, we compared reproductive outcomes following 8,429 controlled ovarian stimulations (COS) using r-hFSH Gonal-f® (n = 5,059) or the biosimilar FSH Bemfola® (n = 3,370), performed between January 2015 and December 2022 at a single fertility center. Overall, 242,978 mature (MII) oocytes were retrieved, utilized in 12,952 ICSI cycles (n = 11,421 heterologous and n = 1,531 autologous). We primarily evaluated ovarian response, measured by oocyte retrieval and maturation rates. Secondary outcomes included ongoing pregnancy and live birth rates. Participants/materials, setting, methods Participants included both oocyte donors (6,895 COS, 18-35 years) and patients using autologous oocytes (1,534 COS, 20-46 years). Univariate and multivariate analyses were performed separately across oocyte donor and patient groups. Outcomes in autologous cycles were further stratified by age. Regressions were adjusted for r-hFSH dose, antral follicle count (AFC), days of stimulation, age, BMI, use of menotropins, fertilization rates and number of embryos transferred, as appropriate. All p-values &amp;lt;0.05 were considered statistically significant. Main results and the role of chance Oocyte donors exhibited similar demographics (age, BMI, days of stimulation), however AFC was significantly higher in the biosimilar group compared to donors stimulated with Gonal-f® (11.31 ± 9.72 vs 7.63 ± 7.5). In patients, we observed no difference in demographic variables between study groups. A mean of 20.5 cumulus-oocyte complexes (COCs) and 15.7 MII oocytes were obtained across all COS. In oocyte donation cycles, the number of COCs retrieved was comparable between groups (22.6, Gonal-f®, 22.3, Bemfola®), however stimulation with Gonal-f® led to a slightly higher number of MII oocytes (17.6 vs 16.9, t-test p &amp;lt; 0.0001). Nevertheless, adjusted analyses revealed improved oocyte retrieval rates with Bemfola® (coeff 0.12, 95% CI: (0.05-0.18), p = 0.0006), while oocyte maturity rates were equivalent amongst the two groups (80% Gonal-f®, 77%, Bemfola®). Comparable results were observed in autologous cycles in terms of COCs (13.7, Gonal-f®, 12.7, Bemfola®) and number of MII oocytes (9.6, Gonal-f®, 9.0, Bemfola®). In autologous cycles, r-hFSH choice did not affect oocyte retrieval rates (coeff -3.3x10-04, 95% CI (-0,09-0,09), p &amp;gt; 0.05) nor oocyte maturation rates (coeff -2.7x10-04, 95% CI (-0,02-0,02), p &amp;gt; 0.05). Our adjusted analysis revealed comparable ongoing pregnancy and live birth rates between the study groups across all 12,952 ICSI cycles (p &amp;gt; 0.05 in all cases). Limitations, reasons for caution The main limitation of this study is its non-interventional retrospective cohort design, impacting generalizability to broader populations and diverse clinical settings. Despite its exploratory nature, the study’s scale, encompassing over 8,000 COS and close to 13,000 ICSI cycles, offers valuable insights into routine clinical care. Wider implications of the findings Comparable ovarian response, pregnancy and live birth rates following COS with Gonal-f® and Bemfola®, in both heterologous and autologous cycles, underscore the utility of biosimilars in ART. Our findings support the broader implications of EU’s policy on biosimilar interchangeability, ultimately enhancing patient access to a wider range of treatment options. Trial registration number not applicable

P-621 Rekovelle® vs Gonal-F®: in vitro characterization of intracellular signals induced by the two FSH preparations

Abstract Study question Do Rekovelle® and Gonal-F® modulate preparation-specific signalling? Summary answer Gonal-F® and Rekovelle® induce preparation-specific pattern of intracellular signalling. What is known already Rekovelle® and Gonal-F® are two commercial preparations of recombinant FSH used in assisted reproduction. These preparations differ in originator cells (PER.C6® cell line of human fetal retinal origin for Rekovlle®; modified Chinese Hamster Ovary cell line for Gonal-F®), likely reflecting glycosylation profiles impacting their mode of action. Study design, size, duration 3-year in vitro functional comparison of Rekovelle® and Gonal-F®, using the transfected HEK293 cell line expressing FSH receptor (FSHR). Participants/materials, setting, methods FSHR-expressing HEK293 cells were treated with increasing concentrations of GonalF® and Rekovelle® (pM-µM) to evaluate receptor-receptor interaction and trafficking, intracellular cyclic adenosine monophosphate (cAMP) and Ca2+ increase by bioimaging techniques, as well as phospho-protein by Western blotting, and genomic effects by CRE-luciferase reporter gene activity. Main results and the role of chance Both the preparations induced similar, dose-dependent increase of receptor trafficking through intracellular organelles. These data were obtained by evaluation of the interaction between FSHR and endosomal markers RAB-GTPases (Rab) 5 and 7. Rekovelle® induced more pronounced steroifogenic signals than Gonal-F®, detected as FSHR-FSHR interactions at the cell membrane, cAMP accumulation, cAMP-responsive elements binding protein (CREB) phosphorylation and CRE promoter activity. Interestingly, while cell treatment by Gonal-F® mediated complete dose-response of cAMP, consisting in the continuous increase of the second messenger together with the increase of FSH concentration, Rekovelle® had biphasic action where cAMP increased from 0 to 100 nM although decreased at 1 µM Rekovelle® dose. Moreover, Rekovelle® was 10-times more potent than Gonal-F® in activating intracellular Ca2+ activation. The two preparations induced similar pattern of extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation. In conclusion, Rekovelle® has higher potency than Gonal-F® in activating cAMP- and Ca2+-dependent intracellular events, potentially resulting in preparation-specific steroidogenic pattern. Limitations, reasons for caution Experiments were performed in the transfected HEK293 cell line in vitro and should be confirmed by clinical observations. Wider implications of the findings We described different intracellular events mediated by Gonal-F® and Rekovelle® in vitro, likely originated by different glycan profiles and possibly impacting the ovarian response during assisted reproduction. Trial registration number Not Applicable

P-671 Modelling human follicle growth and development during ovarian stimulation: a physiological computational approach

Abstract Study question Can key elements of ovarian physiology be accurately modelled, thereby providing a tool to generate novel insights and guide development of new therapeutics? Summary answer A physiological computational model was developed to describe follicle growth and follicle size distributions observed in agonist and antagonist ovarian stimulation (OS) protocols. What is known already The outcome of OS remains unpredictable despite identification of several prognostic factors. The average first cycle pregnancy rate is 30-40% across fertility centres, highlighting the need for improved stimulation protocols. This is an active area of clinical research; however, clinical trials are costly, time-consuming, and frequently have outcomes that are unexpected or difficult to interpret. We integrated available physiological knowledge of human follicle growth and development and clinical trial data into a computational model to enable in silico simulations with the aim to optimise clinical trial design. Study design, size, duration Computational model building was performed using a mechanistic approach aimed at capturing well-understood physiology that is relevant in OS protocols. Model inputs included patient characteristics (such as anti-Müllerian hormone (AMH) level), OS regimen and pituitary downregulation with gonadotrophin releasing hormone (GnRH) agonist or antagonist. Key outputs of the model included follicle number and mean size, duration of stimulation and serum hormone concentrations (oestradiol, testosterone, inhibin B and androstenedione). Participants/materials, setting, methods Published physiological data on regulation of follicle development and hormone production informed model design. Following implementation of the model in the Python platform, data from two OS clinical trials were used to calibrate (partially censored) and test the model. The two trials provided data on the effects of the recombinant follicle stimulating hormone preparation follitropin delta over a range of fixed doses and compared the effects of follitropin delta in agonist and antagonist OS protocols. Main results and the role of chance The computational model describes the interplay between pituitary gonadotrophins ovarian steroid hormones and follicle growth and development. Processes spanning the cellular level (such as steroidogenesis and receptor dynamics in theca and granulosa cells), the ovaries (follicle number and size) and the organism (pharmacokinetics and hypothalamic-pituitary-ovarian axis) are included as a system of coupled differential equations. Approximately 70 compartments, 500+ parameters and 1700+ reactions constitute the model framework. Follicles are recruited in the model at variable times, starting from 7 days prior to the start of stimulation. Follicles are initialised with individual sensitivity to gonadotrophins and growth rates. The fate of each follicle (dominance or atresia) is governed by these properties, which determine whether the balance shifts towards proliferation or atresia under the influence of gonadotrophins. Model-simulated numbers of follicles, their average size and mean serum hormone concentrations match closely with observed values in the two clinical trials. For each of these variables, their dependence on time, the dose of follitropin delta, AMH level and downregulation protocol were described with good precision. The inter-patient variability in the simulated number of follicles and in serum hormone levels matched closely with the observed variability based on comparison of the interquartile range. Limitations, reasons for caution The computational model currently describes follicle development during treatment with follitropin delta. We envision extending the model to encompass other treatment outcomes and additional preparations of exogenous gonadotrophins. While the model incorporates our current understanding of mechanisms important for follicle development, ongoing research in the field will likely drive improvements. Wider implications of the findings The computational model is hypothesis-generating and can improve the design of future clinical trials through in silico trial simulation. For instance, the effects of changing subject inclusion/exclusion criteria or of novel dosing regimens can be investigated. Trial registration number NCT01426386, NCT03809429

P-611 Quantity or quality? Comparative study on the effectiveness of recombinant, purified and mixed gonadotropins on the outcome of ICSI treatments from an embryologist perspective

Abstract Study question Are oocyte retrieval and embryo development affected by the origin of the gonadotropins used for controlled ovarian stimulation (COS), when controlling fertilization and culture conditions? Summary answer Patients stimulated with recombinant gonadotropins obtained similar number of oocytes than those using purified hormones, but more blastocysts and better embryo morphology, needing lower doses. What is known already Many authors have questioned the safety of purified urinary gonadotropins used for COS prior to an IVF treatment, and have demonstrated the presence of impurities and a high heterogeneity between batches. Another strategy involves using recombinant follicle-stimulating hormone with/without luteinizing hormone (rFSH+/-rLH) synthetized in vitro. Many studies have compared the cost-effectiveness of these drugs; however, a conclusion has not been reached. These studies have varying inclusion criteria and focus on final endpoints such as pregnancy and live birth, sometimes lacking control over the many variables introduced during the fertilization and culture procedures, which affect embryo development and potential. Study design, size, duration Preliminary retrospective observational study performed over 453 patients subjected to COS prior to autologous ICSI treatments in a private clinic over 3 years, using Time-lapse incubator. The total dose employed, the number and maturity rate of the oocytes retrieved of patients subjected to stimulation with recombinant (RH; n = 46) or mixed hormones (MH; n = 332) were compared with those using purified hormones (PH; n = 75). After ICSI, fertilization and embryo development were also compared between the three groups. Participants/materials, setting, methods The stimulation protocol was adjusted for each patient following the standard protocols of the clinic. Fertilization was performed by ICSI and embryo culture was carried out in Geri time-lapse incubators using Geri Medium (Genea Biomedx). When available (1,097 embryos), Eeva-Xtend Test was applied. Statistical analysis was performed by generalized estimating equations (significance: P≤0.05). Considered confounders include age, body mass index, days of stimulation, down-regulation protocol, and other variables relevant to embryo development. Main results and the role of chance Proper follicle stimulation was achieved with lower doses of RH (1,606.26±1,110.52 IU) than PH (2,941.73±1,011.14 IU) and MH (3,629.68±1,080.79 IU). No association was found between the use of different gonadotropins and the number of oocytes collected (Odds ratio (OR, RH vs PH)=2.51, P=0.411; OR (MH vs PH)=1.26, P=0.754) or their maturity rate (OR (RH vs PH)=2.38, P=0.328; OR (MH vs PH)=1.01, P=0.988). No significant association was found between the odds of a correct fertilization and the type of hormone used. However, the odds of reaching blastocyst stage were statistically higher for embryos from patients treated with RH: OR (RH vs PH)=1.72, P=0.006; OR (MH vs PH)=1.45, P=0.005; blastocyst rate=68.7% RH, 61.9% MH and 54.6% PH. The mean morphological classification by ASEBIR criteria of the embryos was significantly better in the RH group than in the PH group (OR = 1.55, P=0.032), but no association was found in the MH-PH comparison (OR = 1.26, P=0.092). No association was found between the type of hormone and the distribution of the Eeva-Xtend scores. Significantly lower values of the morphokinetic parameter s2 (synchrony between the second and third cell divisions) were also associated with the use of RH (OR (RH vs PH)=0.311, P=0.004). Limitations, reasons for caution The reduced sample size and the heterogeneous number of patients in the three groups limit the statistical power of the comparisons. Different hormones and brands were included in each group. Wider implications of the findings Although not altering the number of oocytes retrieved, the use of RH may improve the chances of success of ICSI cycles by increasing the number of available blastocysts. The positive outcomes paired with the lower dose needed, could make the use of recombinant hormones a cost-effective strategy. Trial registration number Not applicable

Follicle-Stimulating Hormone Receptor (FSHR) Ser680Asn Genotype Does Not Affect the Follicular Fluid Hormonal Profile in Stimulated Cycles Using Different Gonadotropin Preparations for Ovulation Induction: A Pilot Study.

The existing literature lacks consensus on the effectiveness of utilizing polymorphisms to enhance outcomes in in vitro fertilization (IVF), particularly regarding ovulation induction protocols, oocyte and embryo quality, and pregnancy rates. Therefore, the present pilot study aims to assess whether the composition of different gonadotropin preparations affects the ovarian stimulation protocol concerning follicle-stimulating hormone receptor (FSHR)Ser680Asn genotypes (Ser/Ser, Ser/Asn, and Asn/Asn), in terms of ovulation induction parameters, including oocyte maturation rate, embryo quality, and pregnancy rate. A total of 94 IVF patients underwent treatment using a GnRH antagonist protocol with four distinct gonadotropin preparations: HMG, HMG/hCG, rFSH, and rFSH/hCG. Follicular fluid (FF) samples were pooled for each patient for analysis. No statistical differences in the FFhormonal profile (progesterone, testosterone, androstenedione, estradiol, FSH, hCG) among the FSHR genotypes were reported either separately for each protocol or in combination for the four different preparations of gonadotropins. The maturation rate of MII oocytes and embryo quality did not differ among women carrying either Ser/Ser, Ser/Asn, or Asn/Asn genotype (p-value=0.475, and p-value=1.000, respectively). Moreover, no statistically significant correlation was revealed among Ser/Ser, Ser/Asn, and Asn/Asn carriers and pregnancy rate (p = 0.588). FF hormonal analysis of women undergoing IVF using different ovulation induction protocols and carrying either Ser/Ser, Ser/Asn, or Asn/Asn genotype revealed no significant correlations, in terms of maturation rate of MII oocytes, embryo quality, and pregnancy rate, indicating that the FSHR Ser680Asn genotype does not constitute a biomarker for a positive pregnancy outcome. Therefore, the existence of a different mechanism for the expression of FSHRSer680Asn genotypes in the FF hormonal profile related to stimulated cycles is implied.

Quality evaluation of highly purified human menopausal gonadotropin preparations by means of gel electrophoresis and mass spectrometry.

Human gonadotropins are glycoprotein hormones with a highly complex structure, which demands the application of sophisticated analytical methodologies to assess their quality. The principal objective of this study was a comparative evaluation of gel electrophoretic techniques and mass spectrometry-based methods for the quality study of the two urinary-derived, highly purified, human menopausal gonadotropin preparations, Menopur 75/75 I. U. and Meriofert 75 I. U. Molecular mass (Mr), isoelectric point (pI), and isoform pattern of studied compounds were estimated via SDS-PAGE and 2D gel electrophoresis, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the downstream characterization of peptides obtained after in-gel tryptic digestion of selected protein spots. Additionally, for the estimation of the glycosylation pattern of these biologics, the enzymatic release of oligosaccharides was performed, and the isoform pattern was studied. Gel electrophoresis showed a typical electrophoretic behaviour for protein biotherapeutics medicines consisting of extremely complex spot patterns migrating at different masses and pIs. MS analysis proved to be a powerful tool for the identification and detailed characterization of the gonadotropins and the relevant peptides were identified with high sequence coverages. The results of this study are not only useful for the quality assessment of this class of complex biopharmaceuticals but may also serve as a supporting platform for further development of biopharmaceuticals based on modulation of the glycosylation pattern to enhance efficacy or reduce side effects.

In vitro analysis of FSHR internalization and signalling mediated by two different FSH preparations

<i>In vitro</i> analysis of FSHR internalization and signalling mediated by two different FSH preparations

Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation

BackgroundElevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates.MethodsThe senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation.ResultsThe high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG.ConclusionsFSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis (“FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients.Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

Dynamics of gonadotropin and thienopyrimidine derivative TP03 effects on ovulation and ovarian steroidogenesis in Follimag-stimulated immature female rats

BACKGROUND: Gonadotropin preparations, particularly human chorionic gonadotropin (hCG), are commonly used to induce ovulation and treat reproductive disorders in women, albeit with associated side effects. Low-molecular-weight allosteric agonists of the luteinizing hormone receptor (LHR), such as thieno[2,3-d]pyrimidine derivatives, offer a potential alternative. AIM: This study aims to compare the effects of thieno[2,3-d]pyrimidine TP03 and hCG on ovarian weight, corpus luteum formation, and plasma levels of estradiol, progesterone, and luteinizing hormone in immature female rats pre-treated with Follimag®. It also examines their impact on ovarian gene expression related to LHR and steroidogenesis. MATERIALS AND METHODS: TP03 and hCG were administered 48 h after the Follimag® injection at a dose of 20 mg/kg (i.p.) and 15 IU/rat (s.c.), respectively. Parameters were assessed at 1, 2, 4, 8, 16, and 24 h after TP03 and hCG administration. Plasma hormone levels were measured via ELISA, and ovarian gene expression was analyzed using real-time PCR. RESULTS: TP03 increased ovarian weight, progesterone levels in the blood, and expression of steroidogenic genes encoding the cholesterol-transporting protein StAR and the cytochromes CYP11A1 and CYP17A1. TP03 also stimulated corpus luteum formation (16–24 h after treatment). The temporal dynamics of its stimulating effects were similar to those of hCG, although their magnitude was slightly inferior to those of gonadotropin. TP03-induced decrease in blood estradiol levels and aromatase gene expression in the ovaries was also more moderate. Unlike hCG, which suppressed LHR gene expression 8 h after treatment, TP03 maintained a high LHR gene expression, preserving ovarian sensitivity to endogenous luteinizing hormone. CONCLUSIONS: TP03 exhibits potential as an ovulation inducer with milder stimulating effects on ovarian steroidogenesis than hCG, which reduces the risks of developing ovarian hyperstimulation syndrome and resistance to gonadotropins.

Quantification of urinary gonadotropins by specific assays may improve the evaluation of sex-specific hormonal changes in early infancy.

The aim of this study was to investigate the feasibility of different gonadotropin assays for determining total and intact luteinizing hormone(LH), and follicle-stimulating hormone (FSH) immunoreactivity in urine (U-LH-ir and U-FSH-ir, respectively) during early infancy. Morning urine samples were obtained from 31 infants, aged between 0 and 6 months, to study the age-related course of urinary gonadotropins. Additionally, we investigated bi-hourly urine samples of a 5-day-old male neonate for 24 h to observe the course of urinary gonadotropins during a daily cycle. We employed different immunofluorometric assays for measuring total and intact U-LH-ir, and U-FSH-ir. In neonates up to 21 days of age, the U-LH-ir levels measured by the regular LH assay (also detectinghCG) were significantly higher than those determined by the total (specific) LH assays (p = .004). U-FSH-ir was higher in girls than boys during both the first and the next 5 months (p = .02 and p < .001, respectively), whereas total U-LH-ir was higher in boys until 6 months of age (p < .001). Total U-LH-ir/U-FSH-ir ratio was significantly higher in boys than girls across the first half-year (p < .001). The assessment of total U-LH-ir and U-FSH-ir, and their respective ratio constitutes a noninvasive, practical and scalable tool to investigate sex-specific changes during early infancy, with the ratio being significantly higher in boys than girls. Only highly specific LH assays detecting beta-subunit and its core fragment in addition to intact LH should be used for determining U-LH-ir in the neonatal period to avoid potential cross-reactivity with hCG of placental origin.

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.

Urinary gonadotropin assay on 24-h collections as a tool to detect early central puberty onset in girls: determination of predictive thresholds.

Is the 24-h urinary gonadotropin assay an effective diagnostic tool in central precocious puberty (CPP) in girls? This study is the first to provide 24-h urinary gonadotropin assay data, using an electrochemiluminescent immunoassay (CMIA), and to report its usefulness as a tool for the diagnosis of CPP. Data about the GnRH test in the diagnosis of CPP are variable and there is no consensus regarding its interpretation. The measurement of FSH and LH in urines was previously reported to be an alternative biological tool. This is a retrospective two-cohort study, involving a setting and a validation cohort. A total of 516 girls, included between October 2012 and July 2015, and 632 urinary collections were analyzed in the setting cohort. In the validation cohort, 39 girls were included between January 2021 and May 2023, and 49 urinary collections were analyzed. This study included girls who consulted for an investigation of disturbed growth rate or a clinical suspicion of puberty onset in different medical centres across France (setting cohort). Girls with a suspicion of precocious puberty onset were addressed at the expert centre of paediatric endocrinology of the Groupement Hospitalier Lyon Est (validation cohort). Pelvic ultrasonography was performed and enabled their classification according to clinical and morphologic changes criteria (prepubertal or pubertal groups). The parents collected 24-h urine samples (u24) according to standardized instructions. FSH and LH (urinary or plasmatic) were measured using a current and automated CMIA. The area under the ROC curves for CPP prediction was 0.709 for u24FSH (P < 0.001), 0.767 for u24LH (P < 0.001), and 0.753 for the u24LH/u24FSH ratio (P < 0.001). We retained all possible combinations of the four thresholds in the validation cohort (u24FSH = 1.1 or 2.0 IU/24h; u24LH = 0.035 or 0.08 IU/24h). The combination of u24FSH > 1.1 IU/24h and u24LH > 0.08 IU/24h had a positive PV of 85.7% and a negative PV of 94.3%, a sensitivity of 85.7% and a specificity of 94.3%, for classifying prepubertal and pubertal girls in this cohort. This is a retrospective study, in which a margin of error remains due to the inherent uncertainty regarding the clinical assessment of pubertal onset. It must be considered that the thresholds can only apply to the used reagents; measurements without extractions using other reagents are likely to show important heterogeneity. The assay performed herein is a simple, non-invasive, and analytically robust technique meeting the criteria for an alternative to the GnRH test which could be used to supplement its lack of sensitivity. No specific funding was used. All authors declared no conflict of interest. In-house #23-5214 registered study.

Effects of different gonadotropin preparations in GnRH antagonist protocol for patients with polycystic ovary syndrome during IVF/ICSI: a retrospective cohort study.

To compare the effects of recombinant FSH alfa (rFSH-alfa), rFSH-beta, highly purified human menopausal gonadotropin (HP-hMG) and urinary FSH (uFSH) in women with polycystic ovarian syndrome who have undertaken the GnRH antagonist protocol during IVF/ICSI treatment. A single-center retrospective cohort study including women with PCOS who received the GnRH antagonist protocol from January 2019 to July 2022 was conducted. Patients were divided into rFSH-alfa group, HP-hMG group, uFSH group, and rFSH-beta group, and the number of oocytes retrieved, clinical pregnancy rate of the fresh cycle (primary outcomes), embryo quality, and severe OHSS rate (secondary outcomes) were compared. No statistical differences were found among the four groups in fresh cycle clinical pregnancy rate (p=0.426), nor in the subgroup analyses. The HP-hMG group had a smaller number of oocytes retrieved and a higher high-quality D3 embryo rate than the three FSH groups (p<0.05). No statistical differences were found among the four groups in the severe OHSS rate (p=0.083). For women with PCOS undergoing the GnRH antagonist protocol, the clinical pregnancy rates of fresh IVF/ICSI-ET cycle are similar for all four types of Gn. With a lower risk of OHSS and a similar number of high-quality and available embryos, HP-hMG may have an advantage in the PCOS population.

Follitropin Alpha versus Follitropin Beta in IVF/ICSI Cycle: A Retrospective Cohort Study.

The purpose of this study was to compare the efficacy of Follitropin alpha (Gonal-F) and Follitropin beta (Puregon) on cumulative live birth rate (CLBR), defined as the percentage of the number of patients who delivered for the first time in a single ovarian stimulation cycle and the number of patients in all oocyte retrieval cycles. A retrospective cohort study including 2864 infertile patients who underwent ovarian stimulation with Puregon (group A, n=1313) and Gonal-F (group B, n=1551) was conducted between July 2015 and June 2021 at a university-affiliated reproductive medicine center. Reduce potential confounding factors between groups, propensity scores and multivariable logistic regression analyses were estimated to obtain unbiased estimates of outcomes. The primary outcome was the difference in CLBR between the two groups. Each group identified 1160 individuals after propensity score matching (PSM). Baseline characteristics were similar between groups after PSM. The total gonadotrophin (Gn) dose (2400 vs 2325), p=0.038) and cost of Gn usage (5327.9¥ vs 7547.2¥, p<0.001) between the Puregon and Gonal-F groups were statistically significant. Nevertheless, the pregnancy outcomes between the two groups were comparable after fresh embryo transfer and subsequent frozen-thawed embryo transfer. Additionally, there was also no difference observed in the primary outcome of CLBR (52.8% vs 55.7%, p=0.169). Multivariable regression analysis revealed that the type of Gn was not associated with CLBR (p = 0.912). Gonal-F may be a reasonable option for infertile patients who are hesitant to receive more Gn dosage injections. Furthermore, Puregon can eliminate unneeded anxiety and expenses while also administering more flexibility. Taken together, these findings could well be utilized in everyday clinical practice to better inform patients when deciding on an ovarian stimulation strategy.

The negative impact of levothyroxine treatment on urinary luteinizing hormone measurements in pediatric patients with thyroid disease.

Previous studies suggest urinary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) measurements by immunofluorometric assays (IFMA) as noninvasive alternatives to serum assays for puberty assessment. However, these studies excluded patients with other endocrine disorders and those taking medications. Besides, the recent discontinuation of IFMA manufacturing is a concern. We explored the utility of luminometric assays (LIA) for urinary gonadotropins and thyroid-stimulating hormone (TSH) determinations in euthyroid patients with thyroid pathologies. We used LIA and IFMA assays to measure serum and first-morning-voided (FMV) urine LH, FSH, and TSH concentrations in euthyroid patients with various thyroid disorders. Of the 47 euthyroid patients with normal serum TSH (S-TSH) levels, 14 were receiving levothyroxine therapy. FMV total urinary LH (U-LH) concentrations correlated significantly with those measured in serum using either LIA (r=0.67, P<.001) or IFMA (r=0.83, P=.003) in patients not receiving levothyroxine treatment; however, no significant correlation could be detected in patients receiving levothyroxine regardless of the assay method (for LIA: r=0.50, P=.08 and IFMA r=0.44, P=.15). Urinary TSH (U-TSH) concentrations correlated poorly with those in serum in both the untreated and the treated groups (r=-0.13, P=.49, and r=-0.45, P=.11, respectively). FMV total U-LH determinations by LIA can be used to assess pubertal development in patients with thyroid pathology, provided the euthyroid patient is not on levothyroxine treatment. U-TSH measurements by LIA cannot replace invasive S-TSH measurements at least in patients with normal S-TSH levels. Further research may reveal the utility of U-TSH determinations in patients with elevated S-TSH levels.

#243 : Comparison of IVF Outcome Following Follitropin Alfa and Follitropin Delta Utilization at Melati Clinic, Harapan Kita Women and Children Hospital

Background and Aims: Follitropin delta is the third recombinant FSH (rFSH) preparation developed with an individualized dosing regimen based on patient’s body weight and AMH level. According to the existing report, a daily dose of follitropin delta provides an equivalent ovarian response to that of follitropin alfa. However, the effect of follitropin delta on the resulting embryo and biochemical pregnancy remains to be elucidated. Hence, the present study aims to evaluate the effect of follitropin delta on embryo quality and biochemical pregnancy rates compared with that of follitropin alfa in an in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle. Methods: A retrospective cohort study was conducted between January and December 2022 at Melati Clinic, Harapan Kita Women and Children Hospital. A total of 59 IVF/ICSI cycles stimulated with follitropin alfa and 26 IVF/ICSI cycles treated with follitropin delta were subsequently analyzed. Results: After analysis, no significant difference was observed regarding the embryo quality of both treatments (P&gt;0.05). The median of the good, moderate, and poor embryos in the follitropin alfa and follitropin delta group was 2 vs. 3, 1 vs. 2, and 2 vs. 2, respectively. Similarly, the biochemical pregnancy rate between the follitropin alfa and follitropin delta group was comparable (25% vs. 15%, P&gt;0.05). Conclusions: In conclusion, our study suggests that stimulation with follitropin delta in IVF/ICSI cycles yields a similar embryo quality and biochemical pregnancy rates to those of follitropin alfa.