The trial, described in a study protocol published in Frontiers in Urology and registered at ClinicalTrials.gov under identifier NCT07626359, will be conducted at nine tertiary assisted reproductive technology centers in India. It pits two same-day sperm preparation strategies against each other in couples undergoing ICSI where the male partner shows elevated sperm DNA fragmentation, defined as a DNA fragmentation index above 20 percent on a sperm chromatin dispersion assay performed within 90 days before enrollment. The female partner must be between 21 and 35 years old and expected to yield at least four mature metaphase II oocytes, the only oocytes capable of being fertilized by a single injected sperm.

Why does sperm DNA fragmentation matter so much? Mature spermatozoa are remarkably vulnerable. They carry tightly packed DNA but possess almost no capacity to repair it, so damage accumulated during sperm production, epididymal transit, and storage persists until fertilization. Oxidative stress, driven by reactive oxygen species, is considered the central mechanism behind this damage. Varicocele, genital tract infections, aging, heat stress, smoking, obesity, and unhealthy lifestyles can all fuel it, as can defective chromatin remodeling during spermatogenesis and abortive apoptosis, the programmed cell death process that normally culls defective sperm. Pathological sperm DNA fragmentation has been associated with impaired fertilization, compromised embryo development, recurrent pregnancy loss, and lower pregnancy rates in assisted reproduction, even when conventional semen parameters look acceptable on a standard analysis.

Laboratories have long fought back with density gradient centrifugation, or DGC, a workhorse technique in which liquefied semen is layered over a column of 40 and 80 percent silane-coated silica gradients and spun at 400g for 15 minutes. The centrifuge pulls debris, leukocytes, and poorly motile sperm down while enriching the pellet with vigorous, morphologically better sperm, which are then washed and resuspended for injection. But centrifugation itself has a paradoxical downside: the mechanical stress can generate reactive oxygen species and potentially inflict additional DNA damage on already susceptible samples. That vulnerability has inspired a new generation of centrifugation-free sperm separation systems that mimic natural selection barriers instead of brute-force spinning.

The device at the heart of the trial is the LensHooke CA0, a microfluidic sperm separation platform. One milliliter of liquefied semen is loaded into its lower chamber, and 0.9 milliliters of pre-equilibrated flushing medium is added to the upper chamber, separated by a 10-micrometer polycarbonate membrane. After 20 minutes of incubation at 37 degrees Celsius, only the fittest motile sperm have migrated through the membrane, and half a milliliter of this enriched suspension is aspirated for ICSI. The principle is elegantly physiological: sperm must literally swim their way to selection, leaving behind the weak, the damaged, and the dead without ever touching a centrifuge. Recent systematic reviews and meta-analyses suggest such microfluidic techniques can significantly lower sperm DNA fragmentation and may improve embryological outcomes, though the evidence base remains heterogeneous, with many studies hampered by small samples, non-randomized designs, and patient populations not specifically enriched for pathological fragmentation.

The trial’s second arm takes a completely different tack: rather than new technology, it exploits a biological trick. The male partner provides a first ejaculate after two to three days of abstinence, which goes to the CA0 device. Then, one to three hours later, he provides a second ejaculate, which is processed by conventional density gradient centrifugation. The logic rests on accumulating evidence that very short abstinence intervals can reduce sperm DNA fragmentation, presumably because sperm lingering too long in the epididymis and storage ducts accumulate oxidative damage over time. A fresher, second sample may carry a younger, less damaged sperm population, and DGC can then polish it further. Both interventions are accessible, can be performed on the day of oocyte retrieval, and avoid invasive sperm retrieval procedures, making the head-to-head comparison clinically meaningful.

What makes this trial scientifically powerful is its within-patient, sibling-oocyte design. On the day of oocyte retrieval, the mature metaphase II oocytes from each woman are randomly allocated one-to-one between the two sperm sources. Because sibling oocytes from the same woman, produced in the same stimulation cycle and handled in the same laboratory, are fertilized with sperm from each arm, the design strips out confounding from female factors, ovarian response variability, and inter-cycle laboratory differences. Injected oocytes are cultured to the blastocyst stage and graded by standardized criteria. If both arms produce at least one transferable blastocyst of grade 3BB or better, the embryo chosen for the first transfer is itself randomized by arm of origin. If only one arm succeeds, a predefined rescue strategy allows transfer of the available embryo so that no patient is denied treatment because of study allocation, with the outcome attributed to the arm that produced the transferred embryo.

The primary endpoint is the ongoing pregnancy rate at 12 weeks of gestation, confirmed by fetal cardiac activity, following transfer of an embryo originating from a randomized arm. A battery of secondary outcomes will track the journey from sperm to baby: post-processing DNA fragmentation and sperm parameters, normal fertilization defined by two pronuclei and two polar bodies at 16 to 18 hours after injection, cleavage and blastocyst formation rates, usable and good-quality blastocyst yields, implantation, biochemical and clinical pregnancy, pregnancy loss, and live birth. Paired pre- and post-processing measurements of each sample will also reveal how much each strategy actually improves sperm quality before injection, offering mechanistic insight alongside clinical results.

The trial is framed as a non-inferiority study, meaning the cheaper option wins if it proves merely comparable, not superior. Density gradient processing of the second ejaculate will be declared non-inferior if its ongoing pregnancy rate is no more than 10 percentage points lower than that achieved with centrifugation-free selection. Assuming an ongoing pregnancy rate of 40 percent per transfer, 80 percent statistical power, and a one-sided alpha of 0.025, the calculations call for roughly 377 embryo transfers per arm, inflated to about 415 per arm, or 830 total, to buffer against attrition. Analyses will follow a modified intention-to-treat principle with supportive per-protocol analyses, using logistic regression and mixed-effects models that account for within-patient clustering when multiple oocytes or embryos from the same couple contribute data.

The stakes extend well beyond the laboratory. If the second-ejaculate DGC strategy proves non-inferior, clinics worldwide, especially in resource-limited settings where microfluidic devices are costly or unavailable, could adopt a low-cost, easily implementable approach to managing elevated sperm DNA fragmentation using equipment they already own. If the CA0 device instead shows superior outcomes, the results would strengthen the case for investing in centrifugation-free sperm selection for this patient group. The investigators acknowledge limitations, including restricted generalizability to older women, poor responders, donor-oocyte cycles, and severe male-factor cases, unavoidable inter-laboratory variability in fragmentation testing, and the fact that the two arms differ in both preparation method and ejaculate source. Live birth, the ultimate measure of success, is not the powered endpoint. Still, with results expected to be published regardless of direction, the trial promises to deliver exactly what the fragmented field of sperm selection has lacked: rigorous, directly comparative evidence for one of fertility medicine’s most consequential daily decisions.

Subject of Research: A randomized non-inferiority trial comparing sperm preparation strategies for ICSI in men with elevated sperm DNA fragmentation

Article Title: 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

Article References: Chandra, V., Sanagoudar, S., Shinde, J., Shah, N., Suwalka, I., & Esteves, S. C. (2026). 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. Frontiers in Urology, 6, Article 1913330. https://doi.org/10.3389/fruro.2026.1913330

Image Credits: AI Generated

DOI: 10.3389/fruro.2026.1913330

Keywords: sperm DNA fragmentation, ICSI, density gradient centrifugation, microfluidic sperm selection, male infertility, assisted reproductive technology, non-inferiority trial, sibling-oocyte design, LensHooke CA0, sequential ejaculation, ongoing pregnancy rate, study protocol