Randomized non-inferiority within-patient trial of density gradient processing of sequential ejaculates versus centrifugation-free sperm separation device for ICSI in men with elevated sperm DNA fragmentation- study protocol

Background Pathological sperm DNA fragmentation (SDF) has been associated with impaired fertilization, compromised embryo development, recurrent pregnancy loss, and lower pregnancy rates in assisted reproductive technology (ART). Two same-day strategies proposed to reduce SDF prior to in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) are: (i) processing a second ejaculate obtained after a short abstinence interval using density gradient centrifugation (DGC), and (ii) selecting sperm from the first ejaculate using a centrifugation-free sperm separation device (CFSS). Although both approaches may enrich sperm populations with lower DNA damage, direct comparative evidence regarding reproductive outcomes remains limited. Methods and analysis This prospective, multicenter, within-patient, sibling-oocyte randomized non-inferiority trial will be conducted at nine tertiary ART centers in India. Couples undergoing ICSI will be eligible if the male partner is aged 21–45 years with elevated SDF (DNA fragmentation index >20% by sperm chromatin dispersion assay within 90 days before enrollment) and the female partner is aged 21–35 years and expected to yield at least four metaphase II oocytes. On the day of oocyte retrieval, the first ejaculate collected after 2–3 days of abstinence will undergo processing using the LensHooke ® CA0 centrifugation-free sperm separation device, whereas a second ejaculate obtained 1–3 hours later will be processed using conventional DGC. Sibling metaphase II oocytes will be randomized 1:1 to ICSI using sperm from either intervention arm. Embryos will be cultured to the blastocyst stage and graded according to standardized criteria. If both arms generate at least one transferable blastocyst (≥3BB), the embryo selected for the first transfer will be randomized according to its arm of origin; otherwise, transfer will proceed using the available embryo(s) according to a predefined rescue strategy. The primary outcome is ongoing pregnancy rate at 12 weeks’ gestation following transfer of an embryo originating from the randomized study arm. Secondary outcomes include post-processing SDF and sperm parameters, normal fertilization, blastocyst formation and quality, implantation, biochemical and clinical pregnancy. Non-inferiority of DGC processing of the second ejaculate compared with centrifugation-free sperm selection will be assessed using a predefined absolute non-inferiority margin of 10 percentage points. Ethics and dissemination The study protocol has been approved by the institutional review boards of all participating centers. Written informed consent will be obtained from all participants. Study findings will be disseminated through peer-reviewed publications and scientific meetings. Clinical trial registration https://clinicaltrials.gov/ , identifier NCT07626359.