Sperm vacuolization and mitochondrial activity in raw versus prepared semen: effect on embryological outcomes

Can specific conventional and advanced sperm characteristics more accurately predict fertilization and blastulation rates after sperm preparation compared with raw semen?

Prospective study of 90 infertile couples undergoing intracytoplasmic sperm injection (ICSI) with fresh autologous gametes. Semen parameters were obtained from the fresh ejaculate collected on the day of oocyte retrieval and used for ICSI. Sperm concentration and motility were assessed according to WHO 2021 guidelines; morphology was evaluated using Kruger classification with Spermoscan staining. Parameters were reassessed in processed samples following sperm preparation techniques. Acrosomal status, mitochondrial activity and DNA fragmentation were evaluated in raw and prepared semen. Fertilization and blastulation rates were subsequently calculated. Women aged under 36 years, with a BMI over 30 kg/m² and at least three oocytes retrieved, were included; cycles with testicular or embryo biopsies were excluded. Statistical methods included Spearman and Pearson correlations, linear regression, bootstrap analyses and FDR adjustment.

Among analysed parameters, sperm vacuolization in prepared semen and mitochondrial activity in raw semen emerged as significant predictors of fertilization rate and blastulation rate, respectively. A higher percentage of vacuolated spermatozoa after preparation was associated with lower fertilization rate (R = -0.28, P = 0.008). Linear regression confirmed this association, described by the equation y = 79.78 - 1.39x, with an R² of 0.115 (P = 0.001). In raw semen, the percentage of spermatozoa with active mitochondria (SAM-RS%) was positively correlated with blastulation rate (R = 0.24, P = 0.035). Linear regression showed that higher SAM-RS% predicted increased blastulation rate, as described by the equation y = 21.14 + 0.37x, with an R² of 0.059 (P = 0.03). These associations remained statistically significant after bias-corrected bootstrap analyses and FDR adjustment.

In prepared semen, vacuolated sperm percentage may predict fertilization rate, whereas the percentage of spermatozoa with active mitochondria in raw semen may predict blastulation rate.