Distinct Post-Insemination Dynamics Between Fresh and Frozen-Derived Gametes Using Testicular Sperm

The use of testicular sperm extraction (TESE) has revolutionized treatment for men with non-obstructive azoospermia (NOA). However, whether cryopreserved testicular sperm perform equivalently to fresh TESE in terms of fertilization, embryo development, and chromosomal normality remains uncertain. This retrospective analysis included 72 ICSI cycles from 30 couples, each contributing to at least one cycle with fresh and one with frozen-thawed TESE sperm, enabling the first intra-patient comparison of post-insemination and morphokinetic dynamics between the two sperm types under controlled male and female conditions.

A total of 980 metaphase-II oocytes were inseminated using either fresh or frozen-thawed TESE sperm, with embryos cultured in time-lapse incubators and analyzed for preimplantation genetic testing for aneuploidy (PGT-A) by next-generation sequencing (NGS). Fertilization and blastulation rates per inseminated oocyte were significantly lower when frozen sperm were used, though euploidy rates per biopsied blastocyst were comparable between groups.

Time-lapse morphokinetic analyses revealed distinct post-fertilization dynamics. Embryos from frozen-TESE sperm exhibited a prolonged pronuclear (2PN) phase, with a mean delay of 2.1 hours (P = 0.008), suggesting potential nuclear repair mechanisms or cryo-induced DNA damage responses. Similar prolongation was observed in embryos from vitrified oocytes. Beyond this early lag, embryos fertilized with frozen testicular sperm progressed to the 6-cell stage more quickly but required slightly more time to reach full blastocyst expansion than embryos generated with fresh sperm.

These findings emphasize that synchronizing fresh-TESE with oocyte retrieval, when clinically feasible, optimizes fertilization outcomes without compromising euploidy. Cryopreserved TESE remains a viable option but may introduce subtle developmental delays that warrant further study. The observed 2PN lag could represent a novel morphokinetic biomarker reflecting gamete integrity after freezing.

Overall, this intra-patient controlled study provides critical insights into how cryopreservation of testicular sperm affects early embryonic events, guiding clinical decision-making for TESE timing and cryostorage strategies in severe male infertility.

Written by: Daniela Nogueira, MD, IVF Department, ART Fertility Clinics, Abu Dhabi, UAE; INOVIE Fertilité, Toulouse, France

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