Multiple morphological abnormalities of the sperm flagella (MMAF) compromise intracytoplasmic sperm injection (ICSI) outcomes. Testicular sperm aspiration (TESA) may be beneficial, but its impact on early embryonic development and pregnancy in MMAF remains uncertain. This study evaluated the effect of TESA on fertilization, cleavage, on-time Day-3 embryo development, and clinical pregnancy and explored modification by female age and MMAF genotype. In this retrospective cohort, 196 MMAF patients undergoing ICSI (January 2019–December 2024) without female-factor infertility were analyzed: TESA group (n = 65) versus non-TESA group (n = 131). Only fresh embryo transfer cycles were included. Outcomes were fertilization, cleavage, on-time Day-3 embryo development, and pregnancy. Multivariable regression was complemented by inverse probability of treatment weighting, propensity score matching, and stratified analyses by female age and genotype. Robustness was examined using bootstrap resampling and outlier-sensitivity analyses. Notably, TESA was associated with higher fertilization rates (adjusted coefficient: +5.63%, P = 0.022; inverse probability of treatment weighting average treatment effect [IPTW-ATE]: +5.9%, P = 0.022; Cohen’s d: 0.371). Cleavage gains were smaller (IPTW-ATE: +3.8%, P = 0.013; Cohen’s d: 0.246) and method-sensitive. No significant differences were found for on-time Day-3 development or pregnancy; power was limited for these endpoints. TESA mostly improved fertilization in women aged 30–35 years (IPTW-ATE: +9.5%, P = 0.017) and cleavage in genotype-negative patients (IPTW-ATE: +10.1%, P = 0.012). Propensity models showed good balance (area under the receiver operating characteristic curve: 0.638, and standardized differences <0.1); power was adequate for fertilization (0.825). Overall, TESA is associated with higher fertilization rates in MMAF-ICSI, with modest and method-sensitive evidence for cleavage, supporting selective use. Prospective, multi-center validation is needed. Limited downstream effects suggest a role primarily at early stages.

Keywords: cleavage rate; effect modification; fertilization rate; ICSI; MMAF; propensity score analysis; testicular sperm aspiration