male infertility is generally characterized by dysfunctions in processes of adhesion , penetration , and fusion of sperm with oocyte . during spermatogenesis , the haploid round spermatids undergo an elongation phase , cytoplasmic droplet rejection , and eventually differentiate into mature spermatozoa . the formation of mature spermatozoa is a unique process involving a series of meiotic and mitotic changes in the cytoplasmic architecture , replacement of somatic cell - like histones with transition proteins , and the addition of protamines leading to a highly packaged chromatin . the sperm chromatin is normally a highly organized , compact structure consisting of dna and heterogeneous nucleoproteins . this condensed and insoluble nature protects the genetic integrity and facilitates the transport of the paternal genome through female reproductive tract . several studies have demonstrated that failure of fertilization can be attributed to the defects which lie in chromatin decondensation in infertile cases.[57 ] during spermiogenesis , the structure of chromatin is permanently modified . the first step of this process takes place in haploid round spermatids and includes replacement of somatic histones by transition proteins 1 and 2 ( tnp1 and tnp2 ) . in the phase of elongated spermatids , tnp1 and tnp2 are replaced by protamines . after binding of protamines to nuclear sperm chromatin , the process of gene transcription is completely inactivated . , an attempt was made to compare the in vitro sperm nuclear chromatin decondensation between fertile and infertile males . a total of 65 infertile males who referred to the mediwave ivf and fertility hospital ( mysore , india ) for further evaluation and treatment were recruited as subjects for this study . on the other hand , institutional ethical clearance was taken from university ethical clearance committee and written informed consent letters were obtained from all participants before entering the study . the semen samples were collected from the infertile subjects as well as the control group through masturbation after 3 - 5 days of ejaculatory abstinence . the samples were collected in a sterile plastic container in a room especially provided for this purpose by following the world health organization ( who ) guidelines . the collected semen samples were allowed to liquefy at 37c for 30 minutes and were analyzed within 1 hour after collection . physical examination such as liquefaction time , color , odor , and ph basic microscopic examination was carried out to record the count , vitality , density , morphology , and motility of the sperm according to the who guidelines . semen samples were diluted with ethylenediaminetetraacetic acid ( edta ) and sodium dodecyl sulfate ( sds ) mixture and incubated with equal volume of glutaraldehyde / borate buffer at 37c for 60 minutes . then , 10 microliters of the incubated mixture were placed on a clean glass slide and 200 spermatozoa were examined under a microscope using a 40x objective . the obtained values were recorded and subjected to statistical analyses using the statistical software called prism3.0 . the mean values of the groups were analyzed by analysis of variance ( anova ) . the semen samples were collected from the infertile subjects as well as the control group through masturbation after 3 - 5 days of ejaculatory abstinence . the samples were collected in a sterile plastic container in a room especially provided for this purpose by following the world health organization ( who ) guidelines . the collected semen samples were allowed to liquefy at 37c for 30 minutes and were analyzed within 1 hour after collection . physical examination such as liquefaction time , color , odor , and ph basic microscopic examination was carried out to record the count , vitality , density , morphology , and motility of the sperm according to the who guidelines . semen samples were diluted with ethylenediaminetetraacetic acid ( edta ) and sodium dodecyl sulfate ( sds ) mixture and incubated with equal volume of glutaraldehyde / borate buffer at 37c for 60 minutes . then , 10 microliters of the incubated mixture were placed on a clean glass slide and 200 spermatozoa were examined under a microscope using a 40x objective . the obtained values were recorded and subjected to statistical analyses using the statistical software called prism3.0 . the mean values of the groups were analyzed by analysis of variance ( anova ) . table 1 shows the spermiogram which includes sperm count , viability , motility , and semen fluid volume . the asthenospermic condition revealed decreased semen volume ( 1.2  0.4 ) compared to the controls . in fact , oat and oligospermic conditions were accompanied with less sperm viability which was followed by asthenospermia and teratozoospermia . decreased motility was recorded in asthenospermic ( 25.4  7 ) and oligoasthenoteratozoospermic ( 17  3 ) conditions . figure 1 depicts the response of sperms to ncd test in different infertile conditions . decreased level of ncd was observed in oat condition , oligospermia , and idiopathic conditions whereas asthenospermic cases presented nearer values to the control groups . statistical analysis ( anova ) recorded evident significant variation ( p < 0.05 ) between different infertility conditions and the control group for ncd test . the response of spermatozoa for ncd test in different infertile conditions and the control group in figure 2 . spermiogram details of different infertile conditions response of spermatozoa in different infertile conditions for nuclear chromatin decondensation test ( one - way analysis of variance : p < 0.05 ; f = 10.42 ; r2 = 0.38 ) o : oligosepermia , t : teratozoospermia , a : asthenospermia , i : idiopathic , oat : oligoasthenoteratozoospermia , c : controle sperm nuclear chromatin decondensation ( ncd ) response in different infertile subgroups ( white arrows indicate negative response and black arrows indicate positive response . ) plate a : ncd response in teratozoospermic cases plate b : ncd response in oligospermic cases plate c : ncd response in asthenospermic cases plate d : ncd response in oligoasthenoteratozoospermic cases plate e : ncd response in the control group successful fertilization and formation of pronucleus depends on the sperm ncd ability in the oocyte . the failure of sperm decondensation in the oocyte due to sperm abnormalities is unrecognizable by conventional semen analysis . this study demonstrated reduced sperm chromatin decondensation in different infertile subgroups compared to the control group . cho et al . established that a subset of infertile men ( 5 - 15% ) possessed complete protamine deficiency . by genetic analysis , they confirmed that it was due to a mutation of protamine gene cluster . environmental stress , gene mutations , and chromosomal abnormalities can disturb the highly refined biochemical events that occur during spermatogenesis , and this can ultimately lead to an abnormal chromatin structure . in our study , oligospermia and oat condition accompanied with very less chromatin decondensation which indicates abnormal protamine package and susceptibility to dna damage . since such protamines are small , i.e. only half of the size of the core histones which are extremely basic , around 55 - 70% of the amino acids among them are arginine . furthermore , the sperm protamines also contain numerous cysteine residues , which are used to generate disulfide cross - links between adjacent protamine molecules during chromatin condensation . the 4 levels of organization for packaging in the spermatozoa are ( i ) chromosomal anchoring , which refers to the attachment of the dna to the nuclear annulus ; ( ii ) formation of dna loop domains as the dna attaches to the newly added nuclear matrix ; ( iii ) replacement of histones by protamines , which condenses the dna into compact ; and ( iv ) chromosomal positioning . dna stability of sperm also depends on the amount and ratio of disulfide bonds ( s - s ) , non - covalent bonds between zinc and sulfhydryl groups ( sh = zn = sh ) , and free thiol group which reflects the epididymal maturation and more or less normal function of male genital glands . moreover , the ability of ncd depends on the sh = zn = sh bonds . in this study , although we expected negative response of teratozoospermic condition to ncd due to abnormal shaped sperm heads , our results were positive which indicates that dna damages were not evident in the teratozoospermic subjects assessed in this study . other infertile conditions in our study subjects showed poor or no response in ncd test which could be due to low levels of protamines . low levels of protamine ( pn1 , pn2 ) may lead to accumulation of dna damages in sperm , morphological abnormalities , initiation of apoptotic pathway , inactivation of mitochondria , and decreased sperm motility . several studies have suggested that poor chromatin packaging and/or damaged dna may contribute to failure of sperm decondensation after intracytoplasmic sperm injection ( icsi ) and thus failure of fertilization . hence , ncd of spermatozoa and subsequent male pronucleus formation are essential for fertilization and normal embryonic development . the knowledge of sperm ncd is important in the field of assisted reproductive technique ( art ) . this study helped in examining the levels of fertilization capacity and nuclear stability of the spermatozoa in different subgroups of infertile population