Distinct post-insemination dynamics between fresh and frozen-derived gametes using testicular sperm

To evaluate differences in post-insemination events between fresh and frozen-derived gametes when testicular sperm extraction (TESE) is used. A retrospective cohort study including 980 oocytes retrieved across 72 cycles from 30 couples, between January 2017 and September 2023. Gamete sources were fresh (fresh-TESE) or frozen-thawed-TESE (frozen-TESE) from the same patients with non-obstructive azoospermia (NOA), and fresh or vitrified (frozen) oocytes from the same female partner. Following insemination by TESE-sperm, embryos were cultured in a time-lapse (TL) incubator. Resulting blastocysts underwent trophectoderm (TE) biopsy for preimplantation genetic testing for aneuploidy (PGT-A) by next-generation sequencing (NGS). Fertilization, usable blastocysts, and euploidy rates were recorded, and morphokinetic key parameters were annotated. Oocyte/sperm dyads using frozen-TESE showed an association with lower fertilization (OR 0.64, 95% CI 0.45-0.89, P = 0.008) and lower blastulation/MII (OR 0.53, 95% CI 0.37-0.77, P = 0.001), but similar blastulation/2PN (OR 0.69, 95% CI 0.45-1.07, P = 0.09). After accounting for female age, euploidy per tested blastocyst was similar whether fresh or frozen-TESE was used, but frozen oocyte use showed no association with these outcomes. The use of frozen dyads was associated with longer duration of 2PN after tPNa to tPNf: in frozen-TESE, 2.10 h longer (95% CI 0.55-3.64, P = 0.008) and in vitrified oocytes, 2.34 h longer (95% CI 0.31-4.36, P = 0.024). Slight morphokinetic differences between fresh and frozen-TESE were observed during subsequent developmental stages. Synchronizing fresh-TESE with oocyte retrieval, when possible, appears safer to maximize fertilization rates. Regardless of the gamete type, frozen-sourced sperm or oocyte impact 2PN-lag time, possibly indicating nuclear repair mechanisms post-freezing, requiring further investigation.