Volume 13, Issue 8 (9-2015)                   IJRM 2015, 13(8): 495-502 | Back to browse issues page

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Ghasemzadeh J, Talebi A R, Khalili M A, Fesahat F, Halvaei I, Nabi A et al . Sperm parameters, protamine deficiency, and apoptosis in total globozoospermia. IJRM. 2015; 13 (8) :495-502
URL: http://journals.ssu.ac.ir/ijrmnew/article-1-671-en.html
1- International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
2- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran , prof_talebi@ssu.ac.ir
3- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
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Introduction
 
Globozoospermia is a severe form of teratozoospermia with very low incidence (< 0.1%) in infertile patients. Globozoospermia is classified as total owing to the absence of any sperm with acrosome (type 1) and partial in that the ratio of round-headed spermatozoa is less than 100% (i.e., 20-90%) and acrosomal vesicles are present in the rest of the spermatozoa (type 2) (1). The pathogenesis of globozoospermia syndrome is not well known. The most important morphological characteristics of these spermatozoa are including round headed, acrosomeless, round nucleus, no post-acrosomal sheath and coiled tail. Despite the persistence of other normal sperm parameters such as count, density and motility, globozoospermia is considered as one of the important etiologies of male infertility with very low ART success rates (2-4). It is shown that globozoospermia as well as other abnormal morphologic forms of human spermatozoa is associated with DNA strand breaks and abnormal chromatin structures. Some recent studies have reported high amounts of disrupted replacement of nuclear histones by sperm specific protamines (5), increased P1/P2 ratio, DNA strand breaks and other sperm chromatin abnormalities (6-8) in sperm cells of globozoospermic patients. Although, some studies have shown no significant increase in DNA denaturation or sperm DNA fragmentation in globozoospermic cases compared fertile men (6-7, 9), Sutovsky et al in 2001 with the introduction of SUTI (sperm-ubiquitin tag immunoassay) technique showed that globozoospermic are highly ubiquitinated that indicated their damaged DNA (10). In fact, these results suggest that abnormal globozoospermic spermatozoa can be carrier of abnormal chromatin as a possible source of DNA fragmentation.
Among the different hypothesis for the origin of DNA fragmentation of sperm cell, abortive apoptosis is considered as one of the main mechanisms that could explain these results. According to this hypothesis sperm with abnormal chromatin and apoptotic markers are presented in the ejaculate due to inefficient sperm removing by Sertoli cells (11, 12). Also, the chromatin with abnormal compaction can be more sensitive to external stresses such as reactive oxygen species that induce DNA fragmentation (13). The round-headed spermatozoa may be containing an abnormal chromatin with DNA strand breaks. It can cause the early pregnancies loss or increase the risk of birth malformations, cancer, and genetic disorders in newborns (14). Therefore, the evaluation of chromatin structure status and sperm DNA in globozoospermic patients should be considered as one of the basic laboratory assessments. As it is mentioned above, there are few studies indicating the changes of sperm chromatin structure in these patients, but, there are controversies in the results (6, 9-10, 15). The main reason for different reports may be the low frequency of globozoospermia among from infertile couples (15). In fact, most studies are presented as case reports using low number of semen samples to evaluate the semen parameters and sperm chromatin structure (2, 6, 9, 15). Therefore, the aim of this study was to compare the semen parameters, protamine deficiency, and apoptosis in ejaculated spermatozoa between total globozoospermic and normozoospermic men.
 
Materials and methods
 
Patient’s selection
In this case-control study, 36 semen samples of fertile and infertile men referred to andrology laboratory of Yazd Research and Clinical Center for Infertility from June 2010 to November 2014 were evaluated in two groups. The case group consisted of 15 infertile men with total globozoospermia (with more than %90 round headed spermatozoa) and the control group consisted of 21 fertile men with normal semen parameters and whose partners had successful pregnancies within the last one year and their samples were taken before vasectomy. A complete evaluation in globozoospermic patients including physical examinations, smoking, and cytogenetic, immunological and reproductive hormonal assays were done. Men with heavy smoking, drug and alcohol abuse, varicocele and age ≥45 years which may impact the sperm DNA integrity were excluded. This study was approved by institutional review board of Yazd Research and Clinical Center for Infertility and informs consent forms signed by all participants.
 
Sperm collection and analysis
Semen samples were collected by masturbation after 2-5 days of sexual abstinence. Each semen sample was allowed to undergo liquefaction, and then was evaluated for sperm motility, concentration, and morphology according to World Health Organization (WHO, 2010) criteria (16). Briefly, sperm motility including progressive motility (PR), non-progressive motility (NP), and immotile (IM) were assessed manually by phase-contrast microscopy (Zeiss, Axiostar plus, Germany) at X400 magnification. Papanicolaou staining was applied to evaluate morphological abnormalities and at least 200 sperm cells were examined per slide. Sperm count was assessed by Makler counting chamber (Sefi Medical Co., Haifa, Israel) (17). All analyses were done twice by one experienced laboratory technician blinded to the study.
 
Sperm DNA integrity tests
For the evaluation of sperm chromatin structure, two tests were used including; TUNEL assay for sperm apoptosis detection and chromomycin A3 (CMA3) for sperm protamine deficiency.
 
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay
The percentage of apoptotic spermatozoa in each sample was determined by TUNEL assay by In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) (18). In this method, the smeared samples were fixed with 4% paraformaldehyde in PBS (Phosphate buffer saline) for 15 minutes at room temperature. After washing with PBS, the samples were incubated with 0.3% H2O2 in methanol for 1 hour to quench endogenous peroxidase activity. The cell permeability was done with 0.1% Trit