P-017 DNA fragmentation Index before and after density gradient centrifugation using flow cytometry, its reflection in sperm ultrastructure and its association with clinical outcome
Authors: Sioga A , Fleva A , Nikolopoulos T , Gianakou A , Mitsoli A , Bosdou J , Kolibianakis E , Grimbizis G , Chatzimeletiou A 
Published: July 2024

Abstract

Study question
Is DNA fragmentation Index (DFI) affected by Density Gradient Centrifugation (DGC), reflected in sperm ultrastructure and associated with clinical outcome?

Summary answer
DFI was not affected by DGC, nor associated with clinical outcome, but it was negatively correlated with sperm concentration/motility and positively correlated with ultrastructural anomalies.

What is known already
Oxidative stress induces DNA fragmentation during sperm transit through the male genital tract and it is suggested to be the main mechanism causing DNA breaks after ejaculation during in vitro manipulation and sperm selection for injection/insemination. High levels of sperm DNA fragmentation have been linked to lower fertilization rates, poor embryo quality and delayed cleavage. Limited studies, have investigated in detail the effects of DGC on DFI and the reflection in sperm ultrastructure, while the predictive value of DFI on clinical outcome still remains controversial.

Study design, size, duration
Semen samples were collected on the day of the female partener’s oocyte collection and analysed before and after DGC for standard characteristics by light microscopy, according to World Health Organization (WHO) criteria (n = 120), for DNA Fragmentation by Flow Cytometry and for ultrastructure by Transmission Electron Microscopy (TEM). The predictive value of DFI for clinical outcome was also assessed.

Participants/materials, setting, methods
Standard semen analysis and DGC using the Sil-Select 40%/ 80 % layers were performed at an IVF Laboratory of an academic Hospital. Sperm DFI before and after DGC was assessed by Flow Cytometry at the Department of Immunology and Histocompatibility of the same academic Hospital. Ultrastructure analysis by TEM was carried out at an academic Histology/Embryology Laboratory following fixation in 3% glutaraldehyde, 1% osmium tetroxide, washes in PBS and staining with 1% aqueous uranyl acetate.

Main results and the role of chance
DGC did not alter DFI significantly (p = 0.052), but lead to a statistically significant increase in Progressive Motility (p &lt; 0.001). DFI was higher in men with a male factor infertility compared to men without a male factor (p = 0.032) and Oligoasthenoteratospermia (OAT) exhibited the highest DFI levels (36.38% ± 5.36). DFI was negatively correlated with sperm concentration (rho = -0.361, p &lt; 0.01) and sperm motility (rho = -0.301, p &lt; 0.05). No significant association was observed between DFI and clinical outcome, but statistically significant differences were observed both in the age of men (37.54±1.33 vs 42.8±1.27 p = 0.017) and the age of women (35.77±1.25 vs 40.17±0.78 p = 0.007) in the group of patients who achieved a pregmamcy and those that did not. TEM showed that DGC did not lead to an increase in ultrastructural anomalies of spermatozoa, but DFI was positively correlated with ultrastructural anomalies. In teratozoospermic men TEM revealed head, neck and tail defects, including elongated forms, axonemal defects and premature initiation of the acrosome reaction.

Limitations, reasons for caution
The ultrastructure analysis was not possible to be performed in severely oligospermic samples.

Wider implications of the findings
This study shows that sperm ultrastructure and DFI are not adversely affected by DGC confirming the safety of the preparation method for sperm selection prior to insemination/injection. Larger-scale studies are needed, to elucidate any potential correlations between DFI and the probability of pregnancy.

Trial registration number
not applicable