The two-shot protocol. A randomized controlled trial of a novel two-injection ovarian stimulation strategy.

STUDY QUESTION: Is the number of MII (metaphase II) oocytes comparable in the two-shot protocol versus the conventional corifollitropin alfa (CFA) followed by follitropin-beta protocol? SUMMARY ANSWER: Although the mean number of MII oocytes did not differ significantly between the two-shot protocol and the control group, non-inferiority was not statistically met. WHAT IS KNOWN ALREADY: Controlled ovarian stimulation (COS) protocols are often associated with significant side effects, largely due to the burden of multiple daily injections. Efforts to develop more patient-friendly protocols include the shift from GnRH-agonist to antagonist protocols and the introduction of progestin protocol for pituitary suppression (PPS). While such patient-friendly approaches benefit infertile patients, they are even more appealing for women undergoing COS for elective fertility preservation. The current study aims to compare a novel ovarian stimulation protocol involving two sequential CFA injections (two-shot protocol) with a conventional approach using CFA and follitropin-beta in elective fertility preservation. STUDY DESIGN, SIZE, DURATION: Phase III, randomized, controlled, single-center, non-inferiority clinical trial conducted at a Fertility Unit within a University-Affiliated Hospital between December 2023 and July 2025. A total of 194 women were enrolled with a 1:1 allocation. Eligible participants were ≤40, with antral follicular count <20, and anti-Mullerian hormone (AMH) ≤ 3 ng/ml. Participants were randomized to receive either two sequential shots of CFA (150 mcg on Day 1 + 100 mcg on Day 5) or CFA (150 mcg) on Day 1, followed by recombinant FSH (200 IU/day) from Day 8. PARTICIPANTS/MATERIALS, SETTING, METHODS: In both groups, the LH surge was suppressed using a PPS protocol with daily oral desogestrel 75 mcg from the beginning of stimulation until the trigger day. Final oocyte maturation was triggered with GnRH-agonist (triptorelin 0.2 mg). The primary outcome was to compare the two ovarian stimulation protocols in terms of the number of MII oocytes. Comparisons will be made using the estimated mean difference (MD) and corresponding 95% CI. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 194 patients were assessed for eligibility and randomized into two groups: 97 patients were assigned to the two-shot protocol (Group A), while 97 patients followed the conventional protocol involving CFA followed by rFSH (Group B). No statistically significant differences were found in the mean number of oocytes nor MII oocytes between the two-shot protocol and the control group, respectively, 8.09 ± 5.87 vs 9.11 ± 6.86; MD -1.02 [95% CI: -2.83; 0.79] and 6.14 ± 4.59 vs 7.19 ± 5.67, MD -1.04 [95% CI: -2.27; 0.18]. However, because the lower bound of the CI for the primary endpoint (-2.27) exceeded the pre-specified non-inferiority margin of -1.5 MII oocytes, non-inferiority of the two-shot protocol could not be demonstrated. Duration of stimulation (10.27 ± 1.69 vs 10.19 ± 1.47 days; MD 0.09 [95% CI -0.39; 0.56]) and number of follicles ≥14 mm on trigger day (10.67 ± 5.36 vs 11.14 ± 5.46; MD -0.47 [95% CI -2.11; 1.17]) were comparable between groups. The hormonal assessment performed during the stimulation revealed no clinically meaningful differences, except for the FSH concentrations, which were markedly higher in Group A on Day 8 (53.43 ± 13.28 vs 24.60 ± 8.33 IU/l; MD 28.82 [95% CI: 25.39; 32.25]) and remained higher on the day of ovulation trigger (31.28 ± 9.50 vs 24.46 ± 5.01 IU/l; MD 6.82 [95% CI: 4.32; 9.32]). LIMITATIONS, REASONS FOR CAUTION: The current study focused only in elective fertility preservation women, preventing conclusions on embryological differences between the protocols. Furthermore, since the sample size was powered to detect differences in the number of MII oocytes, findings for secondary outcomes, such as the hormonal profile, should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: The two-shot protocol yielded a comparable number of MII oocytes to conventional stimulation; however, the pre-specified non-inferiority margin was not met, as the CI could not exclude a true reduction exceeding 1.5 MII oocytes. The point estimate of approximately one fewer MII oocyte is unlikely to be clinically meaningful, but larger studies are required before the protocol can be formally established as non-inferior. FUNDING: The study was supported by Fundación Santiago Dexeus Font and an unrestricted grant by Organon. DISCLOSURES: N.P.P. has received research grant funding paid to the institution and personal honoraria for lectures from Organon. V.D. has received payment or honoraria for lectures from Organon. N.S.-G., M.D., S.G.-M., and I.R. have not conflict of interest to declare. TRIAL REGISTRATION NUMBER: NCT06134479. TRIAL REGISTRATION DATE: 18 November 2023. DATE OF FIRST PATIENT’S ENROLMENT: 18 December 2023.