Sperm selection during ICSI treatments reduces single- but not double-strand DNA break values compared to the semen sample

Abstract

Purpose: To detect a possible bias in sperm DNA fragmentation (SDF) testing when performed on semen samples or on those few spermatozoa selected for Intracytoplasmic Sperm Injection (ICSI) treatments.

Methods: A multimethodological analysis of Single- and Double-Strand DNA Breaks (SSB and DSB, respectively) was performed through the Neutral Comet, the Alkaline Comet, the Sperm Chromatin Dispersion (SCD) and the Terminal deoxynucleotidyl transferase dUTP Nick End Labelling (TUNEL) assays. SDF was evaluated in (i) semen samples from 23 infertile patients (not achieving pregnancy or suffering recurrent miscarriage); (ii) samples after a Swim-up and (iii) spermatozoa microselected for ICSI (ICSI-S).

Results: The analysis of 3217 ICSI-S revealed a significant reduction of SSB values compared to the Ejaculate and the Swim-up samples. On the contrary, DSB values were not reduced after any sperm selection method. The No-pregnancy group presented poorer semen parameters and higher SSB values. The Recurrent miscarriage group presented better semen parameters but also higher DSB values.

Conclusion: The analysis of SDF on semen samples may not be fully representative of those few spermatozoa selected for ICSI. Since oxidative stress impairs sperm motility and causes SSB, selecting a motile sperm may intrinsically imply choosing a sperm not affected by this damage. DSB have an enzymatic origin which does not affect motility, making it difficult to select a sperm without this damage. Therefore, ICSI treatments could be effective in patients presenting high SSB values. Patients presenting high DSB values should expect bad ICSI results if this damage is not reduced through other specific methods.

Keywords: Comet assay; Double-strand breaks; ICSI; Sperm DNA fragmentation; Sperm selection.

Fig. 1

Microscopic images of ejaculated and ICSI-selected spermatozoa analysed by Neutral Comet, Alkaline Comet, SCD (a); and TUNEL assays (b)

Fig. 2

Multimethodological analysis of sperm DNA fragmentation on (a) Ejaculated, Swim-up-selected and ICSI-selected spermatozoa (ICSI-S) and (b) Ejaculate samples according to patients’ fertility background (No-pregnancy and Recurrent miscarriage). Outlier values 3 and 1.5 times out of the Q3 interquartile range are indicated with asterisks and circles in the boxplot, respectively. Horizontal bars with an asterisk symbol indicate significant differences (p value < 0.05)