Commentary: FSH receptor N680S genotype-guided gonadotropin choice increases cumulative pregnancy and live birth rates after in vitro fertilization

Introduction. This is a comment on the findings described by Hjelmér et al. 2025 (1) and their proposal of using urinary FSH (uFSH) preparations to improve assisted reproduction techniques (ART) outcome in women homozygous for the FSH receptor (FSHR) '680Ser' polymorphic variant (rs6166). We aimed to appraise methodological flaws and interpretative issues of the Hjelmér et al.'s study. Relevant concerns arose from interpretation of results, and their use to support the conclusion that uFSH should be recommended to treat genotyped FSHR 680Ser carriers.Inaccurate terminology leads to misinterpretation of results. The commercial preparation Menopur (Ferring Pharmaceuticals SA, Saint Prex, Switzerland), improperly defined by authors as a uFSH, is a mixture of FSH and LH obtained from menopausal women, with addition of hCG (2), and the drug should be indicated as 'menopausal gonadotropins' (hMG). UFSH drugs contain exclusively FSH molecules purified from urine, and this is not the case of Menopur (3). Although it could seem just a semantic issue, the inaccurate terminology may have led to the misinterpretation that FSH of urinary origin elicits a response in vivo which could be, at least in part, attributable to LH and hCG instead. This might be the origin of result misinterpretations discussed below.In vitro data do not support authors' conclusion. In their study, Hjelmér and colleagues performed in vitro experiments that should support their clinical observations. They measured cyclic adenosine monophosphate (cAMP) production in the transfected COS-1 cell line treated with rFSH and hMG drugs. However, those cells display FSHR expression only, preventing the action of LH/hCG molecules exerted through their receptor (LHCGR). In summary, it is not a model representative of the clinical ovarian stimulation, where granulosa cells have both FSHR and LHCGR and theca cells express LHCGR (4). LH/hCG are contained in Menopur whereas they are absent in the recombinant FSH (rFSH) used as a reference drug (Gonal-F, Merck KGaA, Darmstadt, Germany) (2). FSH and hCG efficacy varies considerably, depending whether LHCGR is co-expressed together with FSHR or not (see Table II of the paper (2)). However, the in vitro system proposed by Hjelmér et al. does not allow detecting the contribution of LH/hCG to cAMP activation due to lack of LHCGR expression.Once established that cell response shown in the Figure 2 (1) is mediated by FSH molecules only, it is not surprising that cAMP levels are different between hMG vs rFSH group in the FSHR-expressing COS-1 cells. These considerations pose concerns about the real FSH concentration administered to the cell line in vitro, and how these doses were established. In fact, authors used the international Units (IU), instead of molarity, introducing a bias: IU is not a measure of the amount of hormone since it indicates an activity in vivo, in rodents (5), whereas molarity suits better in vitro experiments because it indicates the number of molecules. There is no direct proportion between Menopur and Gonal-F IU, since they are determined as activity of different molecules (Table I, (2)). Using IU in in vitro experiments, authors used different concentrations of FSH, which is clearly overdosed in the group treated with Menopur. In addition, there is no information about the volume of stimulation, and it is not possible to determine the concentration of hormone used, while the quantity (up to 90 IU) seems to be huge for in vitro experiments.In vitro data have also methodological and statistical issues, since t-test is improperly used to analyze multiple groups. Authors have three groups (treatments: 'uFSH' vs 'rFSH' vs 'no FSH') with two variables (i.e. FSH dose: 0, 1, 10, 90 IU). A two-way ANOVA or a non-parametric equivalent, e.g. Kruskal Wallis test, must be used instead of t-test, which works on twogroup comparisons and may lead to 'type 1 error' when used for multiple testing (6). Finally, the number of independent replicates and buffer volume used to treat cells are not indicated, and no data from 'FSHR 680Asn' and 'FSHR 680Asn + 680Ser' COS-1 cells are shown.In summary, there are not enough data allowing in vitro experiments to be repeated and correctly interpreted. Therefore, these results do not support any of the authors' conclusions.Major flaws of clinical study. The main problem is the lack of clinical study registration number, which prevents evaluating if the experimental design has been modified a posteriori and biased by data selection, eventually performed after that analyses did not reveal any difference at first round. This observation is particularly relevant considering the retrospective nature of the study (1).Other key information is missing. For instance, drug used to treat non-genotyped women is not indicated, preventing it to be a control group. Results may be biased by gonadotropin dosing, which is higher in the hMG group (see Table 1 in the paper (1)). Details on how adjustments on BMI, age and fertilization method were performed. Other relevant data impacting prognosis of ART patients are missing, such as antral the follicular count (AFC) (7). In summary, the study design is not thought to prove the authors' hypothesis.There is no clear evidence demonstrating that uFSH elicits different responses than rFSH, weakening the rationale of the study by Hjelmér et al. Commercial FSH peparations are mixtures of different glycoforms of the same molecule, but they hardly trigger clear preparation-specific responses, due to lack of substantial diversity in the overall composition and half-life between these mixtures (3,8,9). There are many clinical studies in favor of or against the superiority of rFSH vs uFSH, and vice-versa (10)(11)(12)(13)(14)(15). However, they never achieved converging evidence for the superiority of one drug in improving clinical outcomes (14). Instead, previous findings consistently support that differences between rFSH vs hMG are due to LH/hCG (16), since they modulate the response to FSH through LHCGR activation (17), regardless of FSHR genotype. The concept was demonstrated in a large dataset in vitro (18), where human primary granulosa lutein cells were treated with rFSH, in the presence and in the absence of recombinant LH (Luveris, Merck KGaA). LH improved the cell response regardless FSHR genotype, demonstrating LH/hCG addition may have indication in poor/subresponder patients, rather than in a sub-group of FSHR 680Ser carriers ( (18), see Supplementary Tables SIII andSIV). Rigorous meta-analysis (19) and registry-based study (20) consolidated this view, revealing that LHCGR ligands may be used to personalize FSH treatments, when the ovarian response to FSH needs to be optimized.In conclusion, the study proposed by Hjelmér et al. fails to provide indications for the use of hMG, and even more uFSH, according to FSHR polymorphism. Improper study design, lack of key information, missing support by in vitro experiments and data misinterpretations undermine the authors' conclusions. Converging data suggest that LH/hCG addition to FSH may benefit ART patients with poor/sub-response to FSH, regardless FSHR genotype.