LAUSR

Is there a correlation between the area of double-stranded DNA fragmentation, known to decrease embryo competence, and the presence of human sperm head vacuoles? There is no or negligible correlation between the area of double-stranded DNA fragmentation and the presence of human sperm head vacuoles. Double-stranded DNA breaks (DSB) in sperm are linked to delayed embryo development and lower implantation rates, while single-stranded breaks have less impact. Some studies suggest a correlation between sperm head vacuoles and DNA fragmentation, but these don't distinguish between single and double-stranded breaks. Recent research reveals that sperm head vacuoles are actually nuclear envelope invaginations, termed 'nuclear invaginations,' which may not directly relate to DNA integrity. Currently, there’s no direct evidence linking these invaginations to DSB specifically, highlighting the need for further research to understand their combined impact on embryo development and fertility outcomes. This prospective observational and cellular study was conducted from March 2024 to August 2024. A total of 1,157 sperm were evaluated from 82 patients (mean age 38.79 ± 5.79, aged 26 to 50) and 19 volunteers (mean age 30.50 ± 6.34, aged 20 to 36). The study was approved by the ethics committee of Juntendo University Faculty of Medicine (E23-0207). Informed written consent was obtained from all participants. Sperm were collected from semen samples obtained from infertility patients and healthy volunteers. After standard centrifugation to isolate sperm, individual sperm were fixed on slides and observed under high-magnification microscopy (×60 oil immersion) to detect head vacuoles. DSBs were visualized by γH2AX immunofluorescence staining. Areas of γH2AX signal foci and whole sperm heads were measured for each sperm. Correlation analyses were performed using Pearson’s method. Among 1,157 sperm, sperm head vacuoles were found in 689 sperm (59.6%) and γH2AX fluorescence signal was detected in 596 sperm (51.5%). The area of γH2AX fluorescence signal(DSB area) was 0.66 ± 0.04 μm2, whereas the area of the whole sperm head was 11.73 ± 0.08 μm2 . The DSB area ratio, calculated by dividing the DSB area by the whole sperm head area in individual sperm, was 0.059 ± 0.003 (5.9 ± 0.3%). The point-biserial correlation coefficient for DSB area ratio and vacuole presence (coded as 0 for absence, 1 for presence) was 0.065 (p = 0.027), indicating no or negligible correlation between the two. The fluorescence intensity cutoff was determined visually by three embryologists, potentially introducing measurement errors due to observer variability. The limited sample size may affect generalizability. Larger confirmatory studies are warranted to address these limitations. Selecting vacuole-free sperm for ICSI doesn't eliminate the risk of using sperm with significant double-stranded DNA breaks. While morphological assessment aids selection, it can't fully exclude DNA damage. Developing a non-invasive, real-time method to evaluate double-stranded breaks during ICSI remains a crucial research goal. No