O-261 Frozen gametes differ in post-insemination dynamics to their fresh sibling counterparts in cycles using testicular sperm
Authors: Ferracuti V , Bayram A , Elkhatib I , Kalafat E , Abdala A , Melado L , Lawrenz B , Fatemi H , Nogueira D 
Published: July 2024

Abstract

Study question
Are there differences in post-insemination events between fresh and frozen-derived gametes when testicular sperm extraction (TESE) is used?

Summary answer
Frozen-TESE leads to decreased fertilization, but not blastocyst euploidy rates; 2PN-lag time is significantly longer when frozen gametes are utilized compared to their fresh counterparts

What is known already
Studies indicate that the origin of testicular sperm influences the development potential of cleavage-stage embryos into blastocysts. Recent morphokinetic analysis suggest that embryos from frozen-TESE exhibit prolonged pronuclear stages and more frequent uneven cleavage compared to those from ejaculated sperm. Notably, no comparison with fresh-TESE was performed. To date, no studies have explored analysis in embryo morphokinetic parameters comparing fresh and frozen-TESE, especially when derived from the same patient.Such comparisons could provide a more accurate understanding of potential differences. Given that TESE occasionally involves the use of vitrified oocytes, it is essential to thoroughly examine outcomes across all combinations.

Study design, size, duration
Retrospective cohort study in a tertiary referral IVF centre including 72 cycles of 30 couples between January 2017 to September 2023. Following insemination by TESE-sperm, embryos were cultured in time-lapse (TL) incubator. Blastocysts ≥BL3CC (Gardner’s) underwent trophectoderm (TE) biopsy for Preimplantation Genetic Testing for aneuploidy (PGT-A) by next generation sequency (NGS).

Participants/materials, setting, methods
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. Fertilization, usable blastocysts, and euploidy rates were recorded. Morphokinetics were annotated for time (t) of PB2 (tPB2), tPNa (PN-appearance), tPNf (PN-fading), t2-t9, CP (compaction), tM (Morula), tSB (start blastulation), tB (blastocyst) and timings between each developmental event.

Main results and the role of chance
Among included 980 oocyte/TESE dyads, 253 (25.8%) used fresh oocyte/sperm, 141 (14.4%) frozen oocyte/fresh sperm, 558 (56.9%) fresh oocyte/frozen sperm and 28 (2.9%) frozen oocyte/sperm. In oocyte/sperm dyads using fresh-TESE, normal fertilization (2PN) (52.0% vs. 44.5%) were higher compared to dyads using frozen-TESE (P &lt; 0.01). Mixed-effects logistic-regression accounting for dependency structure between oocytes/sperm dyads of the same couple, showed an association of frozen-TESE with lower fertilization (OR: 0.64, 95%CI: 0.45-0.89, P = 0.008) and lower blastulation (³BL3CC)/MII (OR: 0.53, 95%CI: 0.37-0.77, P = 0.001), but similar blastulation (³BL3CC)/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 sperm was used (OR: 0.91, 95% CI: 0.46-1.79, P = 0.788). Frozen oocyte use on the other hand was not significantly associated with these outcomes (P &gt; 0.05 for all).
Analysis of morphokinetic parameters revealed that use of frozen dyads was associated with longer duration of 2PN after tPNa to tPNf: in frozen-TESE, 2.10h longer (95%CI: 0.55-3.64, P = 0.008); and in frozen oocytes, 2.34h longer (95%CI: 0.31-4.36, P = 0.024).
Among embryos reaching blastulation, frozen-TESE embryos reached 6-cell stage earlier (tPNf to t6, 2.4h earlier, P = 0.009) but they were somewhat delayed in reaching expansion from there on (t6 to tEB, 4.4 hours later, P = 0.059).

Limitations, reasons for caution
In certain oocytes, insemination was conducted using either fresh or frozen immotile sperm that showed unresponsiveness to activation methods. However, the samples size hinders a comprehensive comparison.

Wider implications of the findings
Use of frozen-thawed TESE from NOA-patients may decrease fertilization and consequently lower blastocyst yield, but not euploidy rates. Synchronizing fresh-TESE with oocyte retrieval appears safer to maximize fertilization. Regardless of the gamete type, frozen-sourced sperm or oocyte impact 2PN-lag time, possibly indicating nuclear repair mechanisms post-freezing, requiring further investigation.

Trial registration number
2310-ABU-020-VF