{"paper_id":"9c8af409-9c2f-482c-9d54-178a11ad4b0c","body_text":"Recently, the approach to controlled ovarian stimulation (COS) has shifted from emphasizing maximum stimulation and maximum oocyte yield to prioritizing individualized stimulation and optimal yield. For hyper-responders, preventing ovarian hyperstimulation syndrome (OHSS) is critical; however, for poor responders, choosing a low stimulation protocol aligned with the predicted oocyte yield is considered beneficial. Although there is no universal dosage algorithm because of the involvement of multiple factors, the anti-Müllerian hormone (AMH) level is a key determinant of the appropriate dosage [ 1 - 4 ].\nRecombinant follicle-stimulating hormone (rFSH) has been clinically researched since 1985, and it became commercially available in 1996. rFSH has gained widespread use, primarily in Europe and the United States. In Europe, rFSH is the first-line gonadotropin preparation for ovulation induction after cessation of the production and supply of urinary-derived follicle-stimulating hormone (FSH) and human menopausal gonadotropin preparations because of concerns regarding bovine spongiform encephalopathy [ 5 ].\nCurrently, rFSH is the primary agent used for COS with assisted reproductive technology procedures. The first rFSH injection used for COS with in vitro fertilization was follitropin alfa, which is produced using Chinese hamster ovary cells [ 6 ]. The newest rFSH injection, follitropin delta (Rekovelle), which is expressed in the human fetal retinal cell line, received marketing authorization in Europe in 2016, and it was introduced in Japan in October 2021.\nRekovelle has characteristics that distinguish it from traditional rFSH preparations, including its human cell-based origin and dose determination based on AMH levels and body weight. According to meta-analyses, both follitropin alfa and follitropin delta demonstrated equivalent pregnancy and live birth rates, and their non-inferiority has been established. Additionally, the incidence of early OHSS with follitropin delta is lower than that with follitropin alfa [ 7 , 8 ].\nIn this study, we compared the outcomes of follitropin delta and follitropin alfa by performing crossover administration for the same patients. Based on these results, we evaluated the clinical applicability and unique characteristics of these rFSH preparations.\n\nStudy population\nPatients were treated between 2022 and June 2024. This investigation was conducted as a retrospective study. Even though this was a retrospective study, we considered IRB approval to be necessary and therefore submitted the protocol for review by our institutional review board. To protect personal information, all treatment outcomes were reported in a manner that does not allow for individual identification. As the study involved only past clinical data without any medical intervention, it was determined that there would be no associated risks or disadvantages to the participants. This study was approved by the Ethical Committee of Hanabusa Women’s Clinic (ethical review number 2025-4).\nEligible participants were aged 25-42 years, had a BMI of 18-30 kg/m 2 , and underwent COS within four months after the first oocyte retrieval cycle. Patients with AMH levels ≤2.03 ng/mL were divided into four groups based on the crossover use of two rFSH preparations. The minimum AMH level was set at 0.5 ng/mL or higher [ 9 ]. At our institution, we initiate controlled ovarian hyperstimulation (COH) even in patients with an AMH level around 0.5 ng/mL. Given the potential for a low oocyte yield in such cases, we presumed COH would be appropriate only when at least four oocytes were likely to be retrieved.\nThe same patients underwent two treatment cycles. This analysis was conducted on those who did not achieve pregnancy in the first cycle. Patients who received follitropin alfa during both cycles comprised the GG (follitropin alfa administered in both cycles) group (81 patients). Patients who received follitropin delta during both cycles comprised the RR (follitropin delta administered in both cycles) group (51 patients). Patients who received follitropin delta during the first cycle and follitropin alfa during the second cycle comprised the RG (follitropin delta in the first cycle followed by follitropin alfa) group (30 patients). Patients who received follitropin alfa during the first cycle and follitropin delta during the second cycle comprised the GR (follitropin alfa in the first cycle followed by follitropin delta) group (44 patients). Patients who underwent preimplantation genetic testing for aneuploidy were excluded.\nAMH measurements\nAMH levels were measured using the Elecsys AMH Plus assay (Basel, Switzerland: Roche Diagnostics GmbH) and a Cobas e411 analyzer (Basel, Switzerland: Roche Diagnostics GmbH), both of which employ the electrochemiluminescence immunoassay method.\nStimulation protocol\nAll patients underwent COS using the progestin-primed ovarian stimulation protocol. Follitropin alfa was administered at a daily dose of 225 IU, whereas follitropin delta was administered at 12 µg per day. Although follitropin alpha and follitropin delta are measured in different units, a study compared these two using the number of oocytes retrieved. In that study, which analyzed data from 1,591 cases, it was concluded that a clinical equivalent of 10 µg of follitropin delta corresponds to 150 IU of follitropin alpha. In our present analysis, we included cases in which 12 µg of follitropin delta (Rekovelle) was used, which is approximately equivalent to 180 IU of follitropin alpha [ 10 ]. However, as per clinical practice, the next standard dose above 150 IU is generally 225 IU; we followed this convention in our comparisons.\nStarting on day three of the menstrual cycle, patients received daily rFSH injections and 5 mg of medroxyprogesterone acetate (MPA). Follicular development was assessed by transvaginal ultrasound on days 8 and 10, and triggering was initiated when at least two dominant follicles ≥18 mm were observed. MPA was continued until two days before oocyte retrieval. Triggering was primarily achieved using a gonadotropin-releasing hormone antagonist flare protocol with nasal buserelin (buserelin nasal spray 0.15%, 10 mL) administered as two sprays (300 µg each) at 7:00 PM and 8:00 PM on the day of oocyte pick-up. For some patients, 5,000 IU of human chorionic gonadotropin (hCG5000; Tokyo, Japan: Fuji Pharma) or Ovitrelle Pen 250 µg (Rome, Italy: Merck Serono S.p.A.) was used instead of a gonadotropin-releasing hormone agonist. Follicular development was monitored using transvaginal ultrasound, and oocyte retrieval was performed between 8:30 AM and 9:30 AM. All embryos were scheduled to undergo cryopreservation because of the use of MPA.\nIn vitro fertilization and intracytoplasmic sperm injection procedures\nFor all groups, the duration of FSH and MPA administration, total FSH dose, and outcomes such as the numbers of retrieved oocytes, degenerated oocytes, fertilized oocytes, and blastocyst formation were analyzed. The intracytoplasmic sperm injection rate was consistently higher than the in vitro fertilization rate across all groups and cycles. Blastocyst formation was assessed on day five or six after retrieval, and blastocyst quality was graded using the Gardner criteria. Blastocysts graded as ≥3 with inner cell mass and trophectoderm scores of at least B or higher were defined as good-quality blastocysts.\nA standard deviation of 3.75 for the oocyte count variation of the GG and RR groups was observed. The same rFSH was used for both groups during the first and second trials, and the difference in the number of oocytes retrieved was analyzed. Therefore, a difference of four or more oocytes was considered statistically significant and applied to the results of the GR and RG groups.\nStatistical analysis\nThe statistical analysis was conducted using EZR software (Saitama, Japan: Jichi Medical University Saitama Medical Center). We used the Kruskal-Wallis test, a non-parametric method employed for comparing three or more independent groups. In another part of the analysis, the chi-square test was used. Statistical significance was set at p<0.05.\n\nBackground\nThe baseline characteristics of the GG, RR, RG, and GR groups are summarized in Table  1 . The mean ages of patients in the GG, RR, RG, and GR groups were 37.1, 37.9, 36.6, and 36.0 years, respectively, with no significant differences between groups. Similarly, there were no significant differences in height, weight, infertility causes, menstrual cycle characteristics, and infertility duration. The average antral follicle counts of the GG, RR, RG, and GR groups were 9.8, 10.2, 8.3, and 10.6, respectively, and their average AMH levels were 1.5, 1.4, 1.4, and 1.4 ng/mL, respectively, with no significant differences across groups. Baseline hormone levels (FSH, luteinizing hormone, and testosterone) were also comparable across these groups.\nGG: follitropin alfa administered in both cycles; RR: follitropin delta administered in both cycles; GR: follitropin alfa in the first cycle followed by follitropin delta; RG: follitropin delta in the first cycle followed by follitropin alfa; FSH: follicle-stimulating hormone; LH: luteinizing hormone\nControlled ovarian stimulation and oocyte retrieval\nThe duration of FSH administration, total FSH dose, and duration of MPA administration were similar among the GG, RR, RG, and GR groups. The number of retrieved oocytes increased during the second retrieval cycle across all groups (Table  2 ). The variation in the number of retrieved oocytes between the first and second cycles in the GR group appeared to be higher (1.7 oocytes) compared to other groups (0.8-1.2 oocytes); however, this difference was not statistically significant. The retrieval efficiency, which was calculated as retrieved oocytes/punctured follicles × 100, was not significantly different across groups. The number of degenerated oocytes was also similar across groups. No cases of OHSS or hospitalizations were reported because this study only included patients with AMH levels ≤2.03 ng/mL.\n*The result of the Kruskal-Wallis test was p=0.831.\nGG: follitropin alfa administered in both cycles; RR: follitropin delta administered in both cycles; GR: follitropin alfa in the first cycle followed by follitropin delta; RG: follitropin delta in the first cycle followed by follitropin alfa\nrFSH: recombinant follicle-stimulating hormone; MPA: medroxyprogesterone acetate; OPU: oocyte pick-up\nCulture outcomes\nWe collected data regarding MII oocytes, fertilized oocytes, cleaved embryos, blastocysts, and good-quality blastocysts, as well as the differences in the numbers of oocytes retrieved during the first and second retrieval cycles (Table  3 ). The number of retrieved oocytes, blastocyst formation rate, and good-quality blastocyst formation rate across groups were increased during the second retrieval cycle; however, no significant differences in these rates were observed across each group.\nGG: follitropin alfa administered in both cycles; RR: follitropin delta administered in both cycles; GR: follitropin alfa in the first cycle followed by follitropin delta; RG: follitropin delta in the first cycle followed by follitropin alfa\nART: assisted reproductive technology; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization\nChanges in the retrieved oocyte count\nFor the GG group, the rate of change in oocyte retrieval was 40.7%, which was the most significant increase between the first and second retrieval cycles (Table  4 ). For the other groups, the rate of change in oocyte retrieval was approximately 30%; no statistically significant differences were observed across these groups. The percentage of patients with increased oocyte numbers was similar across all groups. The decreased oocyte retrieval rate in the GR group was 4.5%, which was the lowest among all groups and significantly lower than that of the GG group (18.5%).\nA comparison of the oocyte retrieval reduction rates showed no significant difference between the GG and RR groups or between the GG and RG groups, whereas the GR group had a significantly lower reduction rate compared to the GG group (p=0.029).\nGG: follitropin alfa administered in both cycles; RR: follitropin delta administered in both cycles; GR: follitropin alfa in the first cycle followed by follitropin delta; RG: follitropin delta in the first cycle followed by follitropin alfa\n\nRecently, an increasing number of studies have been published comparing follitropin delta and follitropin alpha. In these reports, follitropin alpha is typically administered using a fixed-dose regimen, while follitropin delta is dosed based on an algorithm. The outcomes obtained in both groups have been found to be comparable. Several studies have demonstrated that the algorithm-based dosing of follitropin delta is not inferior to the individualized dosing of follitropin alpha/beta, and it also offers the advantage of objectivity [ 11 - 13 ].\nThis study aimed to examine the utility of follitropin alfa and follitropin delta in the same patients by altering the type of rFSH used for two oocyte retrieval cycles. While previous studies have evaluated stimulation protocols using different recombinant FSH preparations, few have examined their effects across consecutive cycles in the same individuals using a crossover design [ 14 ].\nrFSH is produced using Chinese hamster ovary cells, and both follitropin alfa and follitropin beta are derived from these cells. Follitropin alfa is administered in international units (150 IU or 225 IU). Follitropin delta is derived from human embryonic retinoblast cells. In contrast, follitropin delta is unique because its dosage (in μg) is determined using an algorithm based on AMH levels and body weight [ 2 ]. Currently, three rFSH preparations, follitropin alfa, follitropin beta, and follitropin delta, are available, and their comparisons have been published [ 15 ]. In Japan, studies have suggested that follitropin delta can improve pregnancy rates and reduce the risk of OHSS [ 15 ].\nIn this study, we evaluated oocyte yield variability associated with alternating rFSH preparations (follitropin alfa and follitropin delta) during different cycles in the same patients. Although the dosage units of follitropin alfa and follitropin delta differ, this study approximated equivalence by comparing 225 IU of follitropin alfa with 12 μg of follitropin delta according to the clinical standard of use. Prior reports have suggested that 1 μg of follitropin delta is approximately equivalent to 15 IU of follitropin alfa; therefore, 12 μg is approximately equivalent to 180 IU [ 10 ]. Although follitropin delta appeared to be slightly more potent, our results showed no significant increase in oocyte retrieval in the GR group compared to that in the other groups and no significant decrease in the RG group.\nIn the present study, the design aimed to closely reflect real clinical practice in line with the two rFSH injections. While the fixed-dose regimen for follitropin alpha was consistent with its routine clinical outcome, some studies have reported that follitropin delta offers greater objectivity, even though the clinical outcomes are comparable between the two [ 11 ].\nA previous study of 49 healthy women aged 21-39 years with endogenous FSH suppression reported that repeated daily administration of 13.5 μg (225 IU) of follitropin delta resulted in a 1.6-fold increase in the peak serum FSH concentration, a 0.6-fold reduction in FSH clearance, and a 2.10-fold increase in the peak estradiol concentration compared to those of follitropin alfa [ 10 ]. These differences highlight the need for dose adjustments when clinically applying follitropin delta. The dosing system of follitropin delta is weight-based, whereas that of follitropin alfa is not; therefore, the use of follitropin delta ensures more consistent outcomes.\nA dose-response phase II trial involving Japanese women aged 20-39 years compared follitropin delta doses of 6, 9, and 12 μg with a follitropin beta dose of 150 IU and revealed that the number of retrieved oocytes was similar across groups; 12 μg of follitropin delta yielded 11.6±5.6 oocytes, whereas 11 oocytes were yielded in the control group [ 16 ]. These results align with those of the present study in which AMH-limited cases were treated with higher doses of follitropin delta (12 μg) or moderately increased doses of follitropin alfa (225 IU).\nPhase II and III clinical trials demonstrated dose responsiveness with a follitropin delta dose range of 6-12 μg, thus establishing its efficacy and safety. Furthermore, its non-inferiority to follitropin alfa and follitropin beta was confirmed in terms of clinical pregnancy rates and oocyte retrieval outcomes. Additionally, during previous studies, follitropin delta was associated with a significantly lower incidence of OHSS than that associated with other rFSH preparations [ 17 , 18 ]. However, in those studies, no OHSS cases were observed, likely because of the inclusion of patients with AMH levels ≤2.03 ng/mL.\nAlthough variability in oocyte retrieval was observed with different rFSH preparations and administration sequences, no significant differences in blastocyst formation, good-quality blastocyst rates, or pregnancy rates were observed. These findings suggest that both rFSH preparations can be used interchangeably during assisted reproductive technology procedures without compromising outcomes.\nFinally, although the number of retrieved oocytes tended to increase during the second retrieval cycle across all groups, this tendency was greater in the GR group (follitropin alfa during the first cycle and follitropin delta during the second cycle). These findings suggest that using follitropin delta during the second cycle may improve patient outcomes. Further investigations are required to confirm these results.\n\nIn cases where the first in vitro fertilization (IVF) cycle does not result in pregnancy, it is important to evaluate whether the stimulation protocol should be repeated or modified in subsequent cycles. In recent years, recombinant FSH (rFSH) has been used in the majority of stimulation regimens and is considered indispensable. Therefore, we examined whether the type of rFSH used affects the number of oocytes retrieved and the subsequent culture outcomes. Our study concluded that in non-polycystic ovary syndrome (PCOS) patients with an anti-Müllerian hormone (AMH) level of ≤2.03 ng/mL - in intermediate responders - when follitropin delta (12 μg) was used in the second IVF cycle, the rate of change in the number of retrieved oocytes was significantly smaller compared to the first cycle. Follitropin delta may contribute to a more consistent oocyte yield compared to the first cycle.","source_license":"CC-BY-4.0","license_restricted":false}