The first multiple center prospective study of rhFSH CTP in patients undergoing assisted reproductive technology in China

Abstract

We assessed the safety and efficacy of rhFSH-CTP, a novel long-acting FSH agent, in controlled ovarian hyperstimulation for patients undergoing ART. A multi-center, open-label, randomized, positive-control, non-inferiority clinical trial was conducted. The study consisted of a phase III randomized design, with a 1:1 ratio favoring the rhFSH-CTP group over the control group. Eligible patients in the rhFSH-CTP group received a single dose of rhFSH-CTP at 100–150 µg for the first 7d of stimulation following a gonadotropin-releasing hormone antagonist protocol. In total, 142 and 141 patients received rhFSH-CTP and rhFSH, respectively. At a confidence interval of 95%, the difference in the number of oocytes (1.13–4.22, 2.67) suggested that rhFSH-CTP was not inferior to rhFSH. Additionally, the top-quality embryos, implantation rates, and pregnancy outcomes were similar between the two groups (P > 0.05). In the rhFSH-CTP group, no cases of severe OHSS were observed, which was a significant improvement compared to the 1.4% incidence in the rhFSH group. With regard to another safety endpoint, no patients tested positive for adenosine deaminase (ADA) in the rhFSH-CTP group. The results demonstrated that the product had a comparable safety profile to pregnancy outcomes and newborn information in the control group, indicating its suitability for use.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-86962-4.

Introduction

Infertility is an increasingly prevalent and severe reproductive health issue affecting approximately 15% of couples globally, and is defined clinically as a lack of conception after 12 months of unprotected intercourse¹. In China, 25% of couples actively attempting to conceive are affected by infertility, with incidence rates increasing among younger ages, as reported by the Chinese Women and Children Development Center and the China Population Association^2,3. Assisted reproductive technologies including in vitro fertilization and embryo transfer and intracytoplasmic sperm injection (ICSI) have emerged as effective treatments for infertility.

Advancements in ARTs have replaced urinary follicle-stimulating hormone (uFSH) with recombinant human follicle-stimulating hormone (rhFSH), now a standard in vitro fertilization treatment. RhFSH offers several advantages over uFSH, including higher bioactivity⁴, widespread availability of raw materials, absence of luteinizing hormone (LH), and lower variability between batches⁵. However, the short half-life of the rhFSH poses a considerable challenge for its clinical application, as patients require daily or twice-daily subcutaneous or intramuscular injections lasting up to 8–12 d per assisted reproductive cycle. Such frequent injections can result in decreased patient compliance and local discomfort even among highly motivated individuals. Therefore, there is an urgent need for novel long-acting follicle-stimulating hormone (FSH) agents to improve infertility treatment. FSH-C-terminal peptide (CTP), marketed as Elonva^®, represents the first corifollitropin alfa approved by the European Commission that meets this need⁶. A 28-amino acid carboxyl-terminal peptide sequence was fused to the FSH beta subunit using site-specific mutagenesis and recombination techniques. Four O-linked glycosylation sites were also identified. This strategic modification considerably prolonged the half-life of FSH and enhanced its bioactivity in physiological environments. Consequently, the single administration of FSH-CTP during the controlled ovarian stimulation treatment cycle maintains therapeutic levels throughout one reproductive cycle. This effectively eliminates the need for daily FSH injections during the initial week^7,8. Implementing FSH-CTP simplifies the care process at medical institutions, and reduces the number of required injections and patient pain. Despite its clinical benefits, FSH-CTP is not yet available in China and is currently only approved for clinical studies by the Chinese Food and Drug Administration. Therefore, Chinese scholars actively pursue the development of long-acting FSH analogs, specifically rhFSH-C-terminal peptide (rhFSH-CTP) fusion protein. This innovative approach involves linkage of a 28-amino acid human chorionic gonadotropin (HCG) CTP sequence to the C-terminus of the human FSH β subunit. This construct is subsequently co-transfected with the FSH α subunit gene into Chinese hamster ovary cells. Through extensive screening involving cell line identification and library construction, a high-purity rhFSH-CTP fusion protein, representing a novel glycoprotein-like gonadotropin, is obtained. The rhFSH-CTP functions by modifying the structure of FSH. Specifically, this alteration leads to an extension of its half-life. By maintaining a prolonged half-life, the FSH-CTP is able to keep the level of FSH above the threshold required for follicle recruitment over an extended period. This continuous presence of FSH above the threshold is crucial as it allows for a more sustained and effective stimulation of the ovarian follicles, which in turn influences follicular development. Preclinical animal studies have confirmed the safety and efficacy of rhFSH-CTP and phase I clinical trials have been successfully completed. This innovative therapeutic agent is currently advancing through the second stage of clinical testing. A recent phase I clinical trial further supports the promising outlook of this new approach, revealing favorable biosafety and tolerability outcomes, along with preliminary evidence of its clinical activity⁹.

The introduction of a single rhFSH-CTP injection to replace the initial seven daily FSH injections aims to alleviate the injection burden for patients undergoing ovarian stimulation. This study was conducted to ascertain the effectiveness and safety of this novel long-acting FSH agent for controlled ovarian hyperstimulation (COH) in individuals undergoing ART. Phase-II clinical trials have established a foundation for defining clinical endpoints and determining appropriate dosing regimens. The primary aim of this multicenter, randomized, positive-control phase III clinical trial was to comprehensively assess the safety and efficacy of rhFSH-CTP in patients with infertility. We also aimed to enhance our understanding of the potential benefits and optimal utilization of rhFSH-CTP as a therapeutic intervention for the management of infertility.

Materials and methods

Study design

The present investigation encompassed a meticulously designed, multi-center, open-label, randomized, positive-controlled clinical trial that spanned 13 Reproductive Medical Centers situated in 12 provinces throughout China (The First Affiliated Hospital of Anhui Medical University; The First Hospital of Peking University; Beijing Obstetrics and Gynecology Hospital, Capital Medical University; Tianjin Central Obstetrics and Gynecology Hospital; The Sixth Affiliated Hospital of Sun Yat-sen University; The First Affiliated Hospital of Hainan Medical University; Wuhan Tongji Reproductive Medicine Hospital; The Third Affiliated Hospital of Guangzhou Medical University; The Affiliated Hospital of Inner Mongolia Medical University; Nanchang Reproductive Hospital; Shengjing Hospital of China Medical University; Women^’s Hospital School of Medicine Zhejiang University and Suzhou municipal Hospital). The trial was conducted from October 2021 to September 2023 with the aim of evaluating the efficacy and safety of the two treatment groups under scrutiny. To ensure equitable distribution, randomization was employed in a 1:1 ratio at each center, with stratification based on participants’ body weight (≤ 60 kg or > 60 kg) using randomly permutated blocks.

Clinical trial

This intervention study has been registered in the Chinese Clinical Trial Registry (ChiCTR), reference number ChiCTR2000038030 on 08/09/2020. This study was approved by the Research Ethics Board of the First Affiliated Hospital of Anhui Medical University (PJ2019-17-06(6)) and the Ethics Committee of each participating hospital. All participants provided written informed consent prior to enrolment. This clinical trial was conducted in accordance with the guidelines of the Declaration of Helsinki and Good Clinical Practice.

Study population and eligibility criteria

Study inclusion criteria

Eligible participants had to satisfy the following predetermined inclusion criteria: females aged 20–35 undergoing IVF, ICSI, or a combination,, with serum levels of FSH, estradiol, progesterone, and LH within normal ranges. Furthermore, regular menstrual cycles lasting 25–34 d and a body mass index (BMI) of 18.5–28.0 kg/m² were required.

Study exclusion criteria

Exclusion criteria for the study included patients with a prior history of recurrent miscarriage (i.e., three or more consecutive miscarriages), OHSS, or active pelvic inflammatory disease, potentially affecting embryo implantation and pregnancy outcomes. Individuals who exhibited a low or no ovarian response or those who had undergone more than three unsuccessful cycles of ovarian stimulation since the establishment of the last sustained pregnancy were also excluded.

Randomisation and masking

The study utilized a central randomization system to assign participants in a 1:1 ratio to either the experimental group or the control group. The randomization method used was “stratified block randomization,” which accounted for two factors: participant weight(≤ 60 kg and > 60 kg) and study center. This system assigned random numbers to allocate eligible participants to the respective treatment groups. Researchers or authorized personnel entered participant information into the system; If eligibility criteria were met, they accessed the Interactive Web Response System (IWRS) using their credentials to retrieve the participant’s random number and drug code. Researchers involved in oocyte retrieval and evaluation remained blinded throughout the trial until the database was locked.

Trial procedures

The present study aimed to compare the efficacy of COH for ART using recombinant FSH with rFSH-CTP in a gonadotropin-releasing hormone antagonist setting. Following extensive screening to confirm participant eligibility, individuals were randomly allocated to two groups in a 1:1 ratio (rhFSH group: rhFSH-CTP group), as depicted in Fig. 1. All enrolled participants initiated ovarian stimulation on either day 2 or 3 of their menstrual cycle (COH day 1) with injections administered by investigators. rhFSH-CTP (Suzhou Shengji Pharmaceutical Co., Ltd. Suzhou, China) treatment was administered via a single subcutaneous injection, with a dose of 100 µg prescribed for patients weighing ≤ 60 kg and 150 µg for those weighing > 60 kg. Starting on COH D8, patients received a daily subcutaneous dose of 150 IU/d (weight ≤ 60 kg) or 225 IU/d (weight > 60 kg) of rhFSH (Gonal-F, Merck Serono S.p.A, Geneva, Switzerland) until the day of HCG. In the positive control group, participants received daily subcutaneous injections of 150 IU/d (body weight ≤ 60 kg) or 225 IU/d (body weight > 60 kg) of rhFSH until the day 7 of COH, after which the rhFSH dosage was adjusted as appropriate until the day of HCG administration. To prevent premature LH surge, a fixed dose of 0.25 mg/d of gonadotrophin-releasing hormone (GnRH) antagonist (Cetrotide, Pierre Fabre Medicament Production, Aquitaine Pharm International, Geneva, Switzerland) was administered subcutaneously from COH days 5 or 6 until the day of HCG administration. Follicular growth and development were monitored using transvaginal ultrasonography. Once any of the three dominant follicles reached a 17 mm diameter or two reached 18 mm, 250 µg of recombinant human chorionic gonadotropin (Ovidrel, Merck Serono S.p.A., Germany) or 0.2 mg of triptorelin (Decapeptyl, Ferring GmbH, Switzerland) was administered. Oocyte retrieval was performed approximately 30–36 h later, followed by standard IVF or ICSI procedures. Each patient would transfer a maximum of two embryos. Luteal support was provided using progesterone vaginal sustained-release gel (Fleet Laboratories Limited, HK) in combination with drospirenone tablets (Abbott Healthcare Products B.V., Holland). This combined approach ensured adequate support during the luteal phase of the menstrual cycle, optimizing the chances of successful implantation and ongoing pregnancy.

Fig. 1.

Graphical illustration of the treatment regimens applied in this study.

HCG, human chorionic gonadotrophin; OR, Oocyte Retrieval; ET, embryo transfer;

Assessments/Outcomes

The primary outcome measure for this study was the number retrieved oocytes, obtained via transvaginal ultrasound-guided aspiration. Secondary outcome measures included the number of retrieved metaphase II (MII) oocytes, fertilization rate, good-quality embryo rate, embryo implantation rate, and endocrine levels (E2 and FSH). Additionally, biochemical pregnancy, clinical pregnancy, ongoing pregnancy, and live birth rates were evaluated. Follicular development was monitored in real time using transvaginal ultrasound during pharmacological stimulation. Safety was assessed based on adverse events (AE), OHSS, serious adverse events, and vital signs. To assess the immunogenicity of rhFSH-CTP, blood samples were collected from patients in the trial group at baseline (COH D1) and approximately 2 weeks after ET. These samples were subsequently tested for adenosine deaminase levels. Participants who maintained an ongoing pregnancy were required to provide data on pregnancy outcomes and neonatal characteristics.

Statistical analyses

Three distinct datasets were identified for analysis in this study: the full analysis set (FAS), per-protocol set (PPS), and safety set (SS). The FAS included patients who received at least one dose of the study drug and had baseline demographic and clinical information, alongside at least one efficacy evaluation following treatment, adhering to intention-to-treat principles. The PPS included patients from the FAS who adhered to the study protocol without deviation. The SS included patients who received at least one dose of the study drug and underwent at least one safety evaluation. Descriptive statistics were used to assess all collected data, including demographic characteristics, safety measures, and efficacy outcomes. Statistical analyses were performed using SAS software v9.4. The primary efficacy endpoint analysis involved comparing the total number of retrieved oocytes between groups using analysis of covariance (ANCOVA), with group assignment, center location, and participant weight included as fixed effects and age as a covariate. Least squares mean differences and their respective two-sided 95% confidence intervals were computed between the groups. Evaluation included assessing whether the lower bound of the two-sided 95% confidence interval exceeded a predefined non-inferiority margin of -3, thereby determining if the experimental group demonstrated non-inferiority to the control group. Before performing the analysis of covariance (ANCOVA), we conducted normality tests and tests for homogeneity of variance, and also assessed the linearity assumptions. Within-group comparisons were conducted using either the t-test or Wilcoxon rank-sum test depending on data distribution (normal or non-normal, respectively). All statistical tests were two-sided, and P ≤ 0.05 was considered statistically significant. Continuous parameters were analyzed using Student’s t-test for normally distributed data, presented as the mean ± standard deviation (SD). For non-normally distributed data, the Mann–Whitney U test was applied, and results were reported as the median (minimum–maximum). Count data are expressed as rates (%) and group comparisons were assessed using χ2 tests (including CMH-χ2 tests) or Fisher’s exact test.

Results

Baseline characteristics

In total, 283 women were randomly assigned in a 1:1 ratio to either the rhFSH-CTP or rhFSH groups, with 142 and 141 participants, respectively (Fig. 2). Tubal factors were the primary cause of infertility in the study population. Baseline demographic and clinical data are presented in Table 1. The two groups exhibited comparable demographic characteristics, menstrual patterns and duration, ultrasonographic findings, and hormone levels. In the rhFSH-CTP group, the mean age of patients was 30.1 ± 2.83 years, the average length of menstrual cycles was 29.64 ± 3.005 d, and the mean BMI was 22.34 ± 2.446 kg/m². For the rhFSH group, the mean age was 30.7 ± 2.49 years, the average length of menstrual cycles was 29.38 ± 2.162 d, and the mean BMI was 22.29 ± 2.285 kg/m². During clinical data analysis, may influence outcomes, leading to the division of patients into two subgroups: ≤60 kg and > 60 kg^10,11. Additionally, no significant differences in endometrial thickness and antral follicle number were observed between the two groups (P > 0.05). Furthermore, serum levels of estradiol (31.7 ± 11.70 pg/mL vs. 31.2 ± 12.86 pg/mL, P = 0.644) and FSH (5.988 ± 1.547 IU/L vs. 5.820 ± 1.308 IU/L, P = 0.519) were equivalent and within the normal range in both groups.

Fig. 2.

Flow of Participants in the study.

Table 1.

Baseline characteristics of study patients.

	RhFSH-CTP	Control	p value
No. of cases, na	142	141	–
≤ 60 kg, n(%)	105 (73.94)	106 (75.17)	–
> 60 kg, n(%)	37 (26.06)	35 (24.82)	–
BMI, Mean ± SD, kg/m2	22.34 ± 2.446	22.29 ± 2.285	0.845
Age, Mean ± SD, y	30.1 ± 2.83	30.7 ± 2.49	0.059
Days of menstrual cycle, Mean ± SD, d	29.64 ± 3.005	29.38 ± 2.162	0.410
Endometrial thickness, Mean ± SD, mm	5.59 ± 1.822	5.96 ± 2.023	0.153
Number of left antral follicles (< 11 mm), Mean ± SD	6.7 ± 2.44	7.1 ± 2.30	0.104
Number of right antral follicles (< 11 mm), Mean ± SD	6.9 ± 2.28	7.5 ± 2.54	0.052
Basal serum E2, Mean ± SD, pg/mL	31.7 ± 11.70	31.2 ± 12.86	0.644
Basal serum FSH, Mean ± SD, IU/L	5.988 ± 1.547	5.820 ± 1.308	0.519

AMH antimullerian hormone, E2 Estradiol, FSH follicle-stimulating hormone.

^a FAS.

Efficacy

Primary outcome

Patients treated with rhFSH-CTP yielded a mean 14.6 ± 7.71 oocytes, whereas those treated with rhFSH yielded a mean of 11.7 ± 5.65 oocytes, demonstrating a difference of approximately 2.67 oocytes(95% CI:1.13–4.22). This suggested that rhFSH-CTP was not inferior to rhFSH in ovarian stimulation. Subgroup analyses were conducted according to patient weight. Moreover, the number of oocytes retrieved was 13.8 ± 7.22 and 11.6 ± 5.67 in the rhFSH-CTP and rhFSH groups, respectively, for patients weighing no more than 60 kg. The median number of oocytes retrieved was 15.0 and 12.0 in the rhFSH-CTP-treated and rhFSH groups, respectively, in patients weighing > 60 kg (Due to the non-normal distribution of the number of eggs obtained in the group weighing > 60 kg, the median value is reported). The lower limits of the 95% CI for treatment differences were >-3 in all subgroups. Overall, the stimulatory effect of rhFSH-CTP on the ovaries was considered equivalent to that of rhFSH. The detailed information is listed in Table 2.

Table 2.

Number of oocyte retrieval stratified by weight.

	RhFSH-CTP	Control	d value (95% CI)
No. of cases, na	142	141	–
Total duration of stimulation, Mean ± SD, d	8.6 ± 1.17	8.3 ± 1.51	–
Number of oocytes obtained, Mean ± SD	14.6 ± 7.71	11.7 ± 5.65	2.67 (1.13 to 4.22)
≤ 60 kg
No. of cases, n	105	106	–
Total duration of stimulation, Mean ± SD, d	8.6 ± 1.16	8.4 ± 1.64	–
Number of oocytes obtained, Mean ± SD	13.8 ± 7.22	11.6 ± 5.76	–
Min–Max	2–41	0–26	–
> 60 kg
No. of cases, n	37	35	–
Total duration of stimulation, Mean ± SD, d	8.8 ± 1.18	8.1 ± 0.97	–
Number of oocytes obtained, Median	15.0	12.0	–
Min–Max	5–38	0–25	–

^a FAS.

Secondary outcome

No significant difference was observed in baseline E2 levels between the rhFSH-CTP and control groups (P = 0.644). Following administration, both groups exhibited a gradual increase in E2 levels, peaking at 2256.2 ± 1128.95 pg/mL and 2077.8 ± 1158.10 pg/mL on the trigger day for the rhFSH-CTP and control groups, respectively. However, this difference was not statistically significant (P = 0.070). E2 fluctuation patterns were similar between the two groups (Fig. 3A). Similarly, no significant difference was observed in baseline FSH levels between the rhFSH-CTP and control groups (P = 0.519). Following the intervention, overall FSH levels increased. Specifically, in the rhFSH-CTP group, FSH levels were 25.518 ± 5.050mIU/mL on COH D6, 15.803 ± 3.573 mIU/mL on COH D8, and 15.359 ± 3.590 mIU/mL on the trigger day. In the control group, FSH levels were recorded as 11.285 ± 2.410 mIU/mL on COH D6, 10.873 ± 2.229 mIU/mL on COHD8, and 11.094 ± 2.235 mIU/mL on the trigger day. Compared to the control group, the rhFSH-CTP group showed a larger degree of change in FSH levels. This is related to the longer elimination half-life of rhFSH-CTP, which is able to maintain FSH levels during follicular development(Fig. 3B). Additionally, subgroup analyses based on weight categories showed consistent patterns of E2 (Fig. S1A and B) and FSH levels (Fig. S2A and B) during COH, aligning with the overall patterns between the two groups.

Fig. 3.

Serum concentrations of (A) oestradiol and (B) FSH for rhFSH-CTP regimen and the daily rFSH regimen. All-subjects-treated population.

In addition, we analyzed follicular development between the rhFSH-CTP and rhFSH groups. At baseline, the follicle sizes of participants in both the rhFSH-CTP and control groups were primarily in the range of 2–11 mm in diameter. Following administration of rFSH-CTP and Gonal-F, a significant increase in follicular development was observed. Overall, no statistically significant difference was observed in the follicular development status between the rhFSH-CTP and control groups during COH. Analyses of the subgroups categorized by body weight, including those weighing ≤ 60 kg and those weighing > 60 kg, were consistent with those of the overall population. From the start of COH on day 1 to trigger day, both groups experienced a gradual decline in the number of follicles with diameters between 2 and 11 mm. On trigger day, the average number of follicles within this size range was 4.1 ± 4.76 and 4.3 ± 4.77 mm in the rhFSH-CTP and control groups, respectively. Similarly, both groups initially observed an increase followed by a decrease in the number of follicles with a diameter between 11 mm and < 15 mm, peaking at 8.0 ± 4.10 and 6.0 ± 3.36 on COH day 8. On the trigger day, 6.4 ± 4.03 and 4.9 ± 3.07 follicles were observed within this size range in the rhFSH-CTP and control groups, respectively. Furthermore, both groups exhibited a consistent upward trend in the number of follicles with diameters between 15 mm and < 17 mm, and those with a diameters ≥ 17 mm, from COH day 1 to the trigger day. Specifically, on trigger day, the treatment group had 3.8 ± 2.71 follicles with diameters between 15 mm and < 17 mm, whereas the control group had 2.9 ± 1.89 follicles in the same range. Regarding follicles with a diameter ≥ 17 mm, the rhFSH-CTP group had 4.7 ± 1.96 and the control group had 4.3 ± 1.83 on trigger day. Lastly, on trigger day, the rhFSH-CTP group had 13.9 ± 5.58 follicles with a diameter ≥ 12 mm, whereas the control group had 11.2 ± 4.22 follicles in the same size range.

Moreover, the percentage of top-quality embryos was compared between the rhFSH-CTP and rhFSH groups, with values of 67.425 ± 29.533 and 73.785 ± 28.488, respectively (P = 0.052). Studies using rhFSH-CTP for follicular development demonstrated a comparable number of MII oocytes retrieved compared to rhFSH treatment (11.8 ± 6.87 vs. 9.3 ± 4.71, P = 0.260), with an overall fertilization rate reaching 61.07 ± 23.206% in the rhFSH-CTP group and 61.59 ± 21.313% in the rhFSH group (P = 0.995). Cancellation rates for ET were based on factors such as absence of viable embryos, high progesterone levels at the end of ovarian stimulation, OHSS, or retrieval of more than 25 oocytes. The cancellation rates were 26.8% (38/142) and 21.3% (30/141) in the rhFSH-CTP and rhFSH groups, respectively. Of the total patients, 104 out of 142 (73.2%) in the rhFSH-CTP group and 111 out of 141 (78.7%) in the rhFSH group underwent ET. After this phase, clinical pregnancy was observed in a total of 40 patients in the rhFSH-CTP group and 48 in the rhFSH group. A comparison of the two groups did not reveal significant differences in biochemical pregnancies (45.6% vs. 53.2%; P = 0.271), clinical pregnancies (38.5% vs. 43.2%; P = 0.476), or ongoing pregnancies (31.7% vs. 36.9%; P = 0.422). The results are summarized in Table 3.

Table 3.

Comparison of clinical parameters and outcomes of oocytes/embryos.

I	RhFSH-CTP	Control	p value
Metaphase II oocytes (in ICSI), Mean ± SD	11.8 ± 6.87	9.3 ± 4.71	0.260
Fertilization rate, Mean ± SD, %	61.07 ± 23.206	61.59 ± 21.313	0.995
Number of top-quality embryos, Mean ± SD	5.7 ± 4.78	5.6 ± 4.16	0.836
Top-quality embryos rate, Mean ± SD, %	67.425 ± 29.533	73.785 ± 28.488	0.052
Embryo implantation rate, Mean ± SD, %	36.5 ± 48.89	38.7 ± 46.56	0.613
Biochemical pregnancy, Mean, %	45.6%	53.2%	0.271
Clinical pregnancy, Mean, %	38.5%	43.2%	0.476
Survival pregnancy rate, Mean, %	35.6%	40.5%	0.454
Ongoing pregnancy, Mean, %	31.7%	36.9%	0.422

ICSI Intracytoplasmic Sperm Injection, IVF In vitro fertilization.

Primary immunogenicity analysis

In the rhFSH-CTP group, none of the participants demonstrated post administration positivity for antibodies, with an individual positivity rate of 0.0%. This findingindicated that the use of FSH-CTP carries a minimal risk of inducing the production of anti-FSH antibodies.

Safety

In this study, 283 participants were enrolled, 142 of whom were assigned to the rhFSH-CTP group and 141 to the control group. Of the participants in the rhFSH-CTP group, 85(59.9%) experienced treatment-emergent adverse events (TEAEs), 32 (22.5%) experienced adverse drug reactions (ADRs), and seven (4.9%) experienced SAE. In the control group, 79 individuals (56.0%) experienced TEAEs, 26 (18.4%) experienced ADR, and eight (5.7%) experienced SAE. Statistical analyses revealed no significant differences between the two groups (P > 0.05). The rate of AE was comparable between the two groups, as shown in Table 4. In subgroup analysis based on body weight, among those weighing ≤ 60 kg, 61 cases (58.1%) in the rhFSH-CTP group and 56 cases (52.8%) in the control group experienced TEAEs. Furthermore, 24 (22.9%) and 18 (17.0%) patients included in the rhFSH-CTP and control groups, respectively, experienced ADRs. Among individuals with a body weight > 60 kg, 24 (64.9%) in the rhFSH-CTP group and 23 (65.7%) in the control group experienced TEAEs, whereas eight (21.6%) in the rhFSH-CTP group and eight (22.9%) in the control group experienced ADRs.

Table 4.

Summary of patients with TEAE (safety population).

	RhFSH-CTP	Control	p value
Patients with TEAE, total (%)	85 (59.9%)	79 (56.0%)	0.514
TEAE (treated) n (%)	37 (26.1%)	36 (25.5%)	0.920
TEAE (special attention) n (%)	24 (16.9%)	23 (16.3%)	0.894
Patients with ADR, total (%)	32 (22.5%)	26 (18.4%)	0.393
Serious ADR n (%)	1 (0.7)	2 (1.4)	0.622
ADR (treated) n (%)	8 (5.6%)	7 (5.0%)	0.802
Patients with SAEs, total (%)	7 (4.9%)	8 (5.7%)	0.780
Severe OHSS n (%)	0 (0%)	2 (1.4%)	–

TEAE treatment-emergent adverse event, ADR adverse drug reaction, OHSS ovarian hyperstimulation syndrom, SAE serious adverse events.

In the rhFSH-CTP group, TEAEs with an incidence rate ≥ 5%, included nausea (15 cases, 10.6%), abdominal distension (15 cases, 10.6%), increased progesterone levels (12 cases, 8.5%), abdominal pain (10 cases, 7.0%), OHSS (10 cases, 7.0%), threatened abortion (10 cases, 7.0%), and vomiting (9 cases, 6.3%). The control group exhibited TEAEs with an incidence rate ≥ 5%, including nausea (15 cases, 10.6%), abdominal pain (13 cases, 9.2%), abdominal distension (12 cases, 8.5%), vomiting (eight cases, 5.7%), vaginal bleeding (eight cases, 5.7%), and biochemical pregnancy (eight cases, 5.7%). The rhFSH-CTP group had 10 (7.0%) cases of threatened abortion, which occurred in 14 cases. Among them, seven maintained their pregnancies from weeks 10 to 12 after ET. Similarly, the control group encountered six cases (4.3%) of threatened abortion, occurring in six instances. Among them, four participants maintained their pregnancies from weeks 10 to 12 after ET. ADRs with an incidence rate ≥ 3% occurred in the rhFSH-CTP group, including elevated progesterone levels (10 cases, 7.0%), OHSS (10 cases, 7.0%), and abdominal distension (nine cases, 6.3%). The control group exhibited ADRs with an incidence rate ≥ 3%, including abdominal distension (nine cases, 6.4%) and OHSS (five cases, 3.5%).

Pregnancy outcomes and neonatal characteristics

Based on the comprehensive analysis dataset, a follow-up study was conducted with 33 participants in the rhFSH-CTP group and 41 participants in the control group who maintained their pregnancies. This study evaluated various parameters such as the success rate of live births, successful deliveries post fresh ETs, live birth rates post fresh ETs, pregnancy termination rates, delivery circumstances, and incidence of multiple pregnancies. Additionally, information regarding newborns, including length, weight, head circumference, Apgar scores, and occurrence of birth defects, was analyzed. The results demonstrated excellent safety of the product regarding both pregnancy outcomes and newborn information compared to the control group (Table S1). No significant difference was observed in the delivery rate (31.7% vs. 36%) or live birth rate (31.7% vs. 36%) post fresh ET between the rhFSH-CTP and control groups (P = 0.5053). In the rhFSH-CTP group, all 33 participants (23.2%) who sustained pregnancy delivered without pregnancy termination. Among them, 29 (87.9%) had full-term deliveries, whereas four (12.1%) were premature. Of these, 13 (39.4%) had vaginal deliveries and 20 (60.6%) had cesarean sections. Among the deliveries, 29 (87.9%) resulted in single births, whereas four (12.1%) resulted in twins. In the control group, 40 participants(28.4%) who sustained pregnancy delivered, with one participant experiencing pregnancy termination due to missed abortion. Among the participants who gave birth, 32 (80.0%) had full-term deliveries, whereas eight (20.0%) were premature. Of these, 13 (32.5%) had vaginal deliveries and 27 (67.5%) had cesarean sections. Among the deliveries, 34 (85.0%) resulted in single births, whereas six (15.0%) resulted in twins. The newborn information of both the rhFSH-CTP and control groups was followed up and evaluated. Data pertaining to normal birth, neonatal length, neonatal weight, neonatal head circumference, and neonatal Apgar scores showed no significant intergroup differences. In the rhFSH-CTP group, newborns had no birth defects, with mean neonatal length, weight, head circumference, and Apgar score of 49.6 ± 1.65 cm, 2.9805 ± 0.5936 kg, 33.22 ± 1.277 cm, and 9.5 ± 0.87 (with a median score of 10), respectively. In the control group, one newborn had a birth defect (bilateral talipes equinovarus), with mean neonatal length, weight, head circumference, and Apgar score of 48.5 ± 3.44 cm, 3.0466 ± 0.6608 kg, 33.14 ± 1.049 cm, and 9.6 ± 0.58 (with a median score of 10), respectively.

Discussion

Individuals undergoing ART treatment are exposed to various external factors, such as hormones and reproductive cell manipulation, raising concerns about the health of mothers and their ART-conceived children owing to inherited reproductive issues. Ensuring the well-being of children born through assisted reproduction, especially with new drugs or technologies, is crucial. The first systematic study on a novel recombinant hormone, corifollitropin alfa, designed as a sustained follicle stimulant, was reported in 2008 ¹². Administration of a single dose during the early follicular phase of the menstrual cycle resulted in the initiation and maintenance of multiple follicular developments for 7 d. The unique half-life and changes in blood drug concentration of FSH-CTP protein make it particularly suitable for ovulation induction in assisted reproductive technology. When combined with the currently mainstream antagonist protocol, it can simplify the assisted reproductive treatment process and offer greater convenience to patients. Currently, there are only a few long-acting ovulation-stimulating drugs for ovarian stimulation. Aside from Elonva^®, most are still being developed, and their long-term safety and effectiveness are not yet confirmed. Yin-Li Zhang and his team have developed KN015, a long-acting recombinant human FSH¹³. KN015 is an FSH-Fc/Fc heterodimer protein made using recombinant DNA technology in CHO cells. Its main benefit is its long half-life, which allows it to last throughout the entire ovulation stimulation cycle. However, all studies so far have been conducted in animals, so clinical trials are needed to confirm if the results apply to humans. This study supports existing literature suggesting that rhFSH-CTP provides a simplified and potentially advantageous treatment approach for individuals undergoing controlled ovarian stimulation prior to IVF or ICSI.

In phase II, the rhFSH-CTP group had twice the number of participants compared to the control group receiving rFSH. rFSH, a reference medication commercially available for years, has established safety and efficacy. A 2:1 randomization aimed at collect more comprehensive information on the investigational drug. Preliminary results from phase II indicated that rhFSH-CTP fusion protein, with GnRH antagonists, successfully stimulated follicular development in patients undergoing ART while maintaining safety (the article has not been published). A larger sample size was used in the phase III experiment, and a 1:1 randomization ratio was implemented between the rhFSH-CTP and control groups, providing additional evidence of the safety and efficacy of this drug. The impact of age on the ovarian functional reserve among individuals undergoing ART was noteworthy. Medical literature defines advanced maternal age as ≥ 35 years, associated with higher risks of diminished ovarian reserve, compromised oocyte quality, and an increased chances of miscarriage, stillbirth, and chromosomal abnormalities in offspring^14,15. This elevated risk is primarily due to a higher probabilities of meiotic errors during oogenesis, resulting in the production of karyotypically abnormal oocytes in both 46XX women and translocation carriers¹⁶. The use of rhFSH-CTP aims to mitigate these concerns and reduce complications such as OHSS, conventionally associated with younger age¹⁷. Consequently, a cutoff age of 35 years separated the younger and older age groups in this study. In addition, the relationship among antral follicle count, sex hormone levels, and ovarian response to controlled ovarian stimulation for ovulation is well-established^17,18. In the study, no significant difference was observed in antral follicle count and hormone levels between the rhFSH-CTP and control groups, and all values were within normal ranges. Therefore, evaluating the efficacy of rhFSH-CTP fusion protein injections in patients undergoing ART based on the number of retrieved oocytes is essential for reliable outcomes. In evaluating rhFSH-CTP efficacy in patients undergoing ART, in relation to the primary endpoint, rhFSH-CTP treatment resulted in a significantly higher number of oocytes compared to the reference group (2.67 oocytes, 95% confidence interval 1.13–4.22). However, the estimated difference was within the predetermined equivalence margin. The unique half-life and mechanism of action of long-acting FSH suggest that FSH-CTP treatment may lead to an increase in the number of retrieved oocytes. However, with the antagonist protocol being the mainstream ovulation induction regimen and the implementation of the whole embryo freezing strategy, the incidence of severe OHSS in FSH-CTP treatment is controllable. Although the rhFSH-CTP group had 2.67 more oocytes retrieved (95% CI: 1.13–4.22), it is important to consider this in the context of the overall treatment approach. The increase in oocyte number may potentially improve the chances of obtaining viable embryos for transfer or freezing. At the same time, the use of the antagonist protocol and embryo freezing helps to mitigate the risks associated with OHSS that could potentially be exacerbated by a higher oocyte yield. This balance between maximizing the benefits of increased oocyte retrieval and minimizing the risks of complications is a crucial aspect of the clinical significance of our findings. The unique half-life and mechanism of action of rhFSH-CTP indicates that patients of advanced age and with low ovarian response might potentially benefit more from FSH-CTP treatment. RhFSH-CTP results in a higher number of oocytes, potentially increasing chances of viable embryos, and improving the higher live birth rates¹⁹. Additionally, it does not increase the risk of severe OHSS and could be beneficial for high-responder patients. However, further research is needed to assess its impact on cumulative live birth rates and long-term neonatal safety. Prior dose-finding trials conducted on corifollitropin alfa indicated that body weight plays a crucial role in determining rhFSH-CTP exposure and treatment outcomes^7,12,20,21. Therefore, for subgroup analysis, a body weight of 60 kg was selected as the threshold to differentiate between the administration of rhFSH-CTP and rFSH. RhFSH-CTP may have different results depending on factors like age, BMI, and ethnicity. In the future, we plan to do more research with a wider variety of patient groups. In this clinical trial, the mean stimulation duration in the rhFSH-CTP group was 8.6 d, equivalent to that of the reference group under the same GnRH antagonist protocol. After a single rhFSH-CTP injection, patients required an additional 1.6 d of rFSH stimulation to meet the criteria for inducing final oocyte maturation. These findings confirm that a single dose of either 100 µg (for individuals with a body weight ≤ 60 kg) or 150 µg (for individuals with a body weight > 60 kg) of rhFSH-CTP adequately extends the FSH threshold window for a week, facilitating the growth of multiple follicles. Notably, despite distinct pharmacokinetic differences between rhFSH-CTP and daily rFSH, their induced pharmacodynamic effects were similar. Additionally, the experimental and positive control drugs in the trial varied in formulation, specification, and administration, rendering a double-blind design impractical. Therefore, an open-label design was implemented. To minimize or eliminate bias, participants in both treatment groups underwent the same study procedures and assessments throughout the treatment period.

In immunogenicity research, literature suggests a low prevalence of antibody-positive participants in ART clinical trials treated with Elonva^®. This study found no instances of participants testing positive for antibodies post-administration, aligning with existing literature on Elonva^®22. The human immune system may recognize biologic therapies as foreign substances, and various factors can influence the immune response to these products. Immune reactions to certain biologic products can lead to serious clinical consequences. However, there is currently limited information on the immune response to therapeutic gonadotropins in women undergoing fertility treatment. To date, two reviews have reported a lack of antibody response to exogenous urine-derived or recombinant FSH^23,24. A recent Phase IV multicenter study on the immunogenicity of rhFSH found that four patients tested positive for ADA at different time points after receiving rhFSH treatment; however, all subsequent tests were negative²⁵. The overall efficacy and safety of the drug were consistent with existing literature, and no specific clinical impact related to immunogenicity was observed. OHSS is a notable complication associated with controlled ovarian hyperstimulation. During the phase III trials of Ensure and Engage, OHSS incidence rates in the Elonva^® group were 6.7% and 7.0%, respectively^7,20,22. The 10 cases of OHSS in the rhFSH-CTP group, leading to a 7.0% incidence rate, align with overseas OHSS data for Elonva. In this study, the rhFSH-CTP group had a higher incidence of mild to moderate OHSS than the control group, possibly due to the slightly higher number of oocytes retrieved in the rhFSH-CTP group. Moderate to severe OHSS can lead to risks such as acute renal failure, acute respiratory distress syndrome, and venous thromboembolism, while also imposing significant economic and psychological burdens on patients²⁶. Young age, low body weight, and polycystic ovary syndrome (PCOS) are all considered high-risk factors for OHSS. For these populations, the use of rhFSH-CTP may offer potential benefits²⁷. Future research will be conducted to validate this hypothesis. The incidence of elevated progesterone was slightly higher in the experimental group (10 cases, 7.0%) compared to the control group (2 cases, 1.4%). However, the mechanism behind the increase in progesterone remains unclear. A previous review summarized that, in women with normal ovarian response undergoing controlled ovarian hyperstimulation, the causes of elevated progesterone may include multiple follicular developments, excessive gonadotropins, and early luteinization²⁸. Recent post hoc analyses of the ENGAGE and Pursuit trials indicated that the incidence of elevated progesterone was significantly lower in the Elonva^® group compared to rFSH. This is thought to be due to Elonva^®’s pharmacokinetics, which show an initial increase in FSH activity for the first two days, followed by a decrease. This pharmacokinetic profile resembles a downregulation scheme, leading to a significantly lower progesterone rise (5.4% vs. 18.3%)²⁹. In this study, rhFSH-CTP exhibited a similar pharmacokinetic profile to Elonva^®, but the mechanism behind elevated progesterone remains unclear and requires further investigation.

Phase III study results demonstrate pregnancy outcomes and newborn data in both the experimental and control groups, consistent with previous research on a comparable products such as Elonva^®. Additionally, the rhFSH-CTP group had fewer preterm births than the control group, indicating improved safety for pregnant individuals. This lower rate of preterm birth rate may be linked to an increased number of ETs and a higher incidence of multiple births associated with Elonva^®. The risks associated with ART were evaluated by comparing the rates of chromosomal abnormalities between IVF/ICSI-conceived pregnancies and naturally conceived pregnancies. Foreign studies have suggested a higher risk of birth defects in IVF/ICSI babies than in the general population, possibly owing to factors related to parental infertility³⁰. In a pregnancy follow-up trial of rhFSH-CTP, the experimental group had no cases of birth defects, whereas the control group had a birth defect rate of 2.2%. Elonva^® showed an overall congenital malformation incidence of 14.5% in live births, with marginally elevated rates in multiple births³¹. Compared to the control drug and Elonva^®, this product exhibited no AEs on neonatal development. In summary, data from the Phase III trial suggest that this product achieves pregnancy outcomes and newborn data comparable to both the control group and Elonva^®, indicating its safety.

FSH-CTP offers several significant benefits. Firstly, it reduces the number of injections required, which is a major advantage in terms of patient comfort and convenience. This reduction in injection frequency also simplifies the overall treatment process, making it less burdensome for patients and healthcare providers alike. Secondly, it has the potential to lower the overall cost of treatment. With fewer injections and a more streamlined protocol, associated costs such as those related to injection supplies and healthcare visits can be minimized. Finally, the simplified treatment regimen leads to improved patient compliance. Notably, the results presented here are based on a limited sample size derived from a phase III study within a multi-center prospective study conducted in China. Furthermore, the pharmacokinetic profile of rhFSH-CTP under various conditions, including age, race, ovarian response, and BMI, remains unclear.

Conclusions

In summary, this study demonstrates that rhFSH-CTP and Gonal-f^® exhibited similar clinical effectiveness and safety characteristics for the first time in a multi-center setting in China. The efficacy and safety profiles of rhFSH-CTP are consistent with the information provided in the product label of Elonva^®, a similar overseas product. Consequently, rhFSH-CTP can complement GnRH antagonists during COH in ART for stimulating the development of multiple follicles. Replacing daily short-acting FSH injections with rhFSH-CTP simplifies treatment, reduces the psychological burden of frequent injections, and improves patient adherence. Further investigations, including the assessment of gametes and embryo epigenetics, multi-generational offspring development, and phenotypes, is necessary to establish the safety and efficacy of rhFSH-CTP in patients undergoing assisted reproductive technology.

Electronic supplementary material

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Abbreviations

rhFSH-CTP

Recombinant human follicle-stimulating hormone-CTP

FSH

Follicle-stimulating hormone

COH

Controlled ovarian hyperstimulation

ART

Assisted reproductive technology

E2

Estradiol

ICSI

Intracytoplasmic sperm injection

rhFSH

Recombinant human follicle-stimulating hormone

uFSH

Urinary follicle-stimulating hormone

LH

Luteinizing hormone

BMI

Body mass index

HCG

Human chorionic gonadotropin

ET

Embryo transfer

AE

Adverse event

OHSS

Ovarian hyperstimulation syndrome

FAS

Full analysis set

PPS

Per protocol set

SS

Safety set

SD

Standard deviation

CI

Confidence interval

TEAE

Treatment-emergent adverse events

ADR

Adverse drug reaction

Author contributions

L W, H Y and L G , data analysis and writing–original draft; G L, J Z and Q S, data collection; X N, C W and T W, interpretation of data and describing some of the clinical ART procedures; C X and H G, acquisition of clinical ART data; XJ H and BS contributed to review and editing the manuscript. Y C contributed to study design. All authors read and approved the final manuscript.

Funding

Financial support for this trial was provided by Suzhou Centergene Pharmaceuticals Co., Ltd.This work was also funded by The Clinical Medical Research Transformation Project of Anhui Province (202204295107020009), Foundation of Education Department of Anhui Province for Outstanding Young Teachers (gxgwfx2022009), Anhui Provincial Health and Wellness Research Project(AHWJ2023BAc20006)and Project of Anhui Institute of Translational Medicine (2021zhyx-C36).

Data availability

The original contributions presented in the study are included in the article material, further inquiries can be directed to the corresponding authors.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

The studies were approved by the Ethics Committee of all the 13 Hospital in China, including The First Affiliated Hospital of Anhui Medical University (PJ2019-17-07(6)), The First Hospital of Peking University (SZSJ-2019-001), Beijing Obstetrics and Gynecology Hospital, Capital Medical University (2020-YW-013-03), Tianjin Central Obstetrics and Gynecology Hospital (2020YS007-01), The Sixth Affiliated Hospital of Sun Yat-sen University (2021ZSLYEC-427), The First Affiliated Hospital of Hainan Medical University (2021-YW-123),Wuhan Tongji Reproductive Medicine Hospital (2020(65)-3), The Third Affiliated Hospital of Guangzhou Medical University(2021 − 157), The Affiliated Hospital of Inner Mongolia Medical University (NO.SY(2022017)), Nanchang Reproductive Hospital (2021-003), Shengjing Hospital of China Medical University (2021PS117), Women’s Hospital School of Medicine Zhejiang University (IRB-20220001-D) and Suzhou municipal Hospital (Y-2021-038-H01). The patients/participants provided their written informed consent to participate in this study.