Effect of erythrocyte-sperm separation medium on nuclear, acrosomal, and membrane maturity parameters in human sperm

Abstract

Purpose: The purpose of this study is to investigate whether erythrocyte-sperm separation medium (ESSM) has effects on human sperm motility, morphology, viability, membrane maturity, acrosome integrity, and nuclear attributes before and after cryopreservation.

Methods: Semen samples from normozoospermic (n = 36) and oligozoospermic (n = 9) patients were analyzed. Samples from the same patient were divided into three aliquots: group 1 and group 2 were resuspended in sperm washing media and ESSM, respectively. Group 3 was resuspended in ESSM with blood sample to mimic the extensive number of erythrocytes in the testicular sperm extraction (TESE) material. All groups were evaluated for sperm concentration, motility, Kruger/Tygerberg strict morphology, viability by eosin-nigrosin staining, membrane maturity by hyaluronic acid-binding assay (HBA), acrosomal integrity by Pisum sativum lectin staining, chromatin maturity by aniline blue staining, and DNA integrity by TUNEL assay before and after cryopreservation.

Results: No significant difference was determined between ESSM-treated and ESSM-untreated sperm samples for the sperm parameters tested (p > 0.05). After cryopreservation, total sperm motility and viability decreased regardless of ESSM used. The percentages of sperm with Tygerberg normal morphology, intact acrosome, and HA-bound sperm were found to be lower in oligozoospermic samples before cryopreservation in each group. However, no statistically significant differences were found between oligozoospermic and normozoospermic samples when all groups were compared. Thus, ESSM treatment did not cause a significant change on sperm motility, normal morphology, viability, HA-binding capacity, chromatin maturity, and DNA fragmentation.

Conclusion: ESSM can enhance the efficiency of sperm retrieval protocol and can also decrease the time required to collect spermatozoa while not affecting sperm morphogenetic properties.

Keywords: Cryopreservation; Erythrocyte-sperm separation; Human sperm maturity; Sperm parameters; TESE.

Fig. 1

Experimental design of the study. HBA hyaluronic acid-binding assay, SW sperm washing medium

Figure 2

a Morphologically normal sperm in ESSM-treated and ESSM-untreated sperm before and after cryopreservation. No significant difference was observed between three groups before (p = 0.96 for overall, p = 0.92 for oligozoospermic, and p = 0.69 for normozoospermic samples) and after cryopreservation (p = 0.49 for overall, p = 0.60 for both oligozoospermic and normozoospermic samples). b The percentage of alive (unstained) sperm after eosin-nigrosin staining refers to sperm viability in ESSM-treated and ESSM-untreated sperm before and after cryopreservation. No significant difference was observed between three groups before and after cryopreservation. p values for before and after cryopreservation were p = 0.65 and p = 0.05 (oligozoospermic samples), p = 0.89 and p = 0.98 (normozoospermic samples), respectively. c The percentage of HA-bound sperm showing the membrane maturity of ESSM-treated and ESSM-untreated sperm before and after cryopreservation. No significant difference was observed between three groups before and after cryopreservation. p values for before and after cryopreservation were p = 0.77 and p = 0.30 (oligozoospermic sampels), p = 0.52 and p = 0.74 (normozoospermic samples), respectively. d The percentage of the integrity of acrosome reaction. No significant difference was observed between 3 groups in overall, oligozoospermic and normozoospermic before (p = 0.56, p = 0.90, and p = 0.55, respectively) and after cryopreservation (p = 0.89, p = 0.33, and p = 0.92, respectively)

Figure 3

a–b Evaluation of sperm chromatin maturity in ESSM-treated and ESSM-untreated sperm before and after cryopreservation. a Mature sperm were stained with aniline blue very lightly (light), whereas immature sperm (intermediate) and severely immature sperm (dark) were stained notably due to incomplete histone-protamine transition. b The percentage of immature sperm in ESSM-treated and ESSM-untreated sperm before (p = 0.66, p = 0.96, and p = 0.71 for overall, oligozoospermic, and normozoospermic samples, respectively) and after cryopreservation (p = 0.78, p = 0.79, and p = 0.88 for overall, oligozoospermic, and normozoospermic samples, respectively). No significant difference was observed between three groups before and after cryopreservation. c DNA integrity was assessed by TUNEL assay and the percentage of apoptotic cells in ESSM-treated and ESSM-untreated sperm before and after cryopreservation. No significant difference was observed between 3 groups before (p = 0.65 for overall, p = 0.80 for oligozoospermic, and p = 0.58 for normozoospermic samples) and after cryopreservation (p = 0.93 for overall, p = 0.45 for oligozoospermic, and p = 0.94 for normozoospermic samples)