Evidence of reduced single-stranded testicular sperm DNA from obstructive azoospermic men after 3 days of in-vitro culture

Abstract

The aim of the present study was to verify whether culturing testicular tissue, to obtain a higher percentage of motile spermatozoa and a better post-thaw recovery rate, affected the ratio between single/double-stranded sperm DNA and, consequently, DNA sensitivity to damage. Testicular biopsy samples from men with obstructive and secretory azoospermia, candidates for assisted reproductive treatment, were cultured for 72 h. The percentage of motile spermatozoa and the single/double stranded DNA ratio were assessed on the day of retrieval (day 0) and again on day 3. The single/double stranded DNA ratio was measured by the acridine orange (AO) staining method. Spermatozoa were classified as green (double-stranded chromatin) or red fluorescing (single-stranded chromatin). In obstructive azoospermia, median motility was 22% (range 10-44%) on day 0 and 50% (range 38-63%) on day 3 (P < 0.01). The median percentage of red stained spermatozoa was 53.5% (range 0.1-88%) on day 0 and 20% (range 2.7-99.9%) on day 3 (P < 0.05). No changes were observed in secretory azoospermia. The culture procedure from obstructive azoospermia not only increased the post-thaw recovery rate, as previously observed, but also reduced the portion of spermatozoa containing single-stranded DNA, thereby increasing the availability of double-stranded DNA spermatozoa for ICSI use.

Comparison of reproductive outcome in oligozoospermic men with high sperm DNA fragmentation undergoing intracytoplasmic sperm injection with ejaculated and testicular sperm

“…Additionally, the SCD test is more sensitive than TUNEL for the evaluation of SDF (4), while flow-cytometry techniques and the Comet assay are not fully operative in tissues because spermatozoa are mixed with somatic cells (18). Hence, protocol modifications will be required to first remove nonsperm cells, which may also eliminate spermatozoa, thus biasing the correct estimate of the true nature of SDF (34).…”

Development of a new and efficient laboratory method for processing testicular sperm.

“…Recent studies suggest that it is beneficial to incubate testicular sperm for 48-72 h before ICSI (2,18,26). In vitro incubation was found to increase the percentage of morphologically normal and motile sperm (2,18) and sperm with double-stranded DNA (26).…”

“…In vitro incubation was found to increase the percentage of morphologically normal and motile sperm (2,18) and sperm with double-stranded DNA (26). However, implementing a 2-3 day culture period prior to ICSI is logistically difficult.…”

Current Assessment of Sperm DNA Integrity

“…Association with single-stranded DNA will cause the dye to bind externally and fluoresce red (Fig. 1b) [Emiliani et al 2001;Evenson and Wixon 2006].…”