Association of Two Polymorphisms in H2B.W Gene with
Azoospermia and Severe Oligozoospermia in
An Iranian Population
During spermatogenesis, the H2B family, member W (
Materials and Methods
This case control study was conducted in Royan institute during
four-year period (2010–2013). Genetic alteration of two SNPs loci, −9C>T and 368A>G, in
The frequency of allele −9T was significantly higher in CMA group than in patients with SCOS (P<0.05). The haplotype TA (corresponding to simultaneous occur- rence of −9T and 368A) compared with haplotype CA (corresponding to simultaneous occurrence of −9C and 368A) in patients suffering from CMA significantly increased, compared with patients had SCOS (P<0.05). However, statistical studies indicated that in general, the distribution frequencies of −9C>T and 368A>G had no significant difference between the infertile groups and control (P=0.859 and P=0.812, respectively).
This investigation showed that SNP −9C>T might be contribute to CMA in azoo- spermic patients and SNP 368A>G had no correlation with male infertility in Iranian population.
One of the most common causes of male infertility is impaired spermatogenesis. It is an intricate, temporal process whereby adult stem cells either self-renew or generate daughter cells that are transformed into a specialized testicular spermatozoon (1-3).
Dramatic chromatin remodeling and chromosomes rearrangement can occur during spermatogenesis. These structural alterations are involved in the normal formation of sperm pronuclei that subsequently ensure the successful fertilization.
Telomeric sequences play an important role in the reorganization and integration of sperm chromosomes (4, 5). They also conduct proper arrangement and separation of chromosomes during cell division, mitosis and meiosis (6, 7). Probably migration of telomeric chromatin to the cell membrane during spermatogenesis establishes unique architecture in the human sperm nucleus that are important in early chromatin remodeling at fertilization and early stages of fetal development (8-14).
To fulfill these roles, some features clearly distinguish telomeres of somatic cells with sperm. For example, unlike other mammals, 10-15% of the histones remain in human sperm (15-17). It is assumed that the remaining histones in human sperm tag specific genes for early expression in embryo (18); however, no evidence of nucleosomal ladder has been observed yet (19, 20).
The H2B family, member W (
According to recent studies, copy number variations
In addition, several studies indicated that genetic
polymorphisms may also increase susceptibility to
some forms of male infertility; for example, two recent
allelic association studies on −9C>T (rs7885967)
and 368A>G (rs553509) polymorphisms in
In this study, two single nucleotide polymorphisms
(SNPs), -9C> T and 368A> G of
Materials and Methods
In this case controlled study conducted in Royan institute, ninety-two infertile men, aged from 25 to 46 years, presenting azoospermia and severe oligozoospermia were enrolled. Comprehensive characterizations of all patients including at least two semen analyses, physical examination, chromosome analysis and molecular tests were performed. Azoospermic group (n=46) were divided into three subgroups according to their testicular biopsy including patients with SCOS (n=21), complete maturation arrest (CMA, n=17) and hypo spermatogenesis (n=8). Severe oligozoospermic group (n=46) were defined with sperm count less than 5 million cells/mL.
Patients with history of cystic fibrosis, trauma, malignancies, varicocele, diabetes mellitus, hypertension, and chemotherapy were not included. Patients with Klinefelter syndrome, azoospermia factor (AZF) genes micro deletions or any identifiable cause of male infertility, including congenital bilateral absence of vas deference (CBAVD), were also excluded from the study groups by review of their records. Controls included healthy, fertile men, with at least one child within 3 years by spontaneous pregnancy and no history of miscarriage. The mean age of control group was 24 to 46 years. All donors gave an informed consent form before participation. The nationality of all groups was Iranian. All samples were collected during four-year period (2010-2013). This study was approved by the Ethical Committee of Royan Reproductive and Biomedicine Research Center.
The genomic DNA was extracted from the peripheral blood samples of each patient using salting- out method, according to the protocol (33).
Choice of SNPs
Two SNPs in
Polymerase chain reaction
Amplification of a fragment containing each of these SNPs was carried out by PCR according to the protocol of Ying et al. (32). PCR amplifications were performed in a final volume of 25 μl containing about 100 ng of extracted DNA, 200 μmol/L dNTPs, 10 pmol of each primer, 2.5 μl 10X PCR buffer, 1.5 mmol/l MgCl2 and 1.5 U Taq polymerase (CinnaGen, Tehran, Iran). PCR reaction consisted of an initial denaturing step at 95˚C for 5 minutes followed by denaturation at 95˚C for 45 seconds; annealing at 54˚C (−9C>T) and 60˚C (368A>G), respectively, for 45 seconds; extension at 72˚C for 35 seconds for 30 cycles; and a final extra extension at 72˚C for 10 minutes. Specific primer pairs used in these reactions are shown in table 1,.
|- The sequence of 5′UTR and exon 1 of H2B.W gene. The position of the SNPs −9C>T and 368A>G are highlighted in green. SNP; Single nucleotide polymorphisms and UTR; Un-translated region.|
|Forward primer||Reverse primer|
UTR; Un-translated region.
Restriction enzyme treatment
For the next step, amplified fragments were digested overnight with position specific restriction enzymes. Restriction enzyme Tsp451 was used for genotyping analysis of −9C>T and Eco911 for 368A>G loci according to the manufacturer’s protocols (Fermentas, Vilnius, Lithuania).
Electrophoretic separation were done by 3% agarose
gel which indicated 212 bp band for allele T and
two bands including 182 bp and 30 bp for allele C
of −9C>T locus. Also visualization of 368A>G locus
suggested two bands (320 bp and 126 bp) for Allele
A and one band (446 bp) for allele G. The representative
results of allele analysis for 368A>G and −9C>T
Subsequently genotype alterations of some samples were confirmed performing direct DNA sequencing (Pishgam Biotech, Tehran, Iran). The reaction was carried out by Sanger method using ABI 3730xl capillary DNA sequencer (Fig.4,).
|- Restriction enzyme digestion of single nucleotide polymorphisms (SNP) −9C>T of polymerase chain reaction (PCR) product (30 bp band for −9C allele not shown in figure). The marker is a 50bp ladder.|
|- Restriction enzyme digestion of single nucleotide polymorphisms (SNP) 368A>G of PCR product. The marker is a 50bp ladder.|
All the statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, SPSS Inc., Chicago, IL, USA). In this study, the genotype frequencies of control and patient groups were compared using the chi-square test. The P value lower than 0.05 was set as statistically significant. A logistic regression analysis was performed to calculate the odds ratio (OR) and 95% confidence interval (95% CI) for measuring the relation of alleles and haplotypes of two SNPs with male infertility.
This study investigated genetic alterations of
two SNPs loci, −9C>T and 368A>G, in
In subgroups of azoospermia patients, the frequency of allele −9T in patients suffering from CMA was significantly higher compared with patients suffering from SCOS (P=0.015). The distribution frequencies of the two SNPs loci in azoospermic subgroups are listed in table 3,. Also the allele frequency distributions between azoospermic subgroups and controls are shown in table 4,.
Four kinds of haplotypes of the two SNPs (CA, TA, CG, and TG) were observed in both infertile patients and controls. Accordingly the haplotype TA compared with haplotype CA significantly increased in patients suffering from CMA, compared with men had SCOS (P=0.029). Tables 5,,-7, show the results of haplotypes observations.
|Locus||Fertile men||Infertile patients||P valuea||OR (95% CI)a|
|Allele||Total (n=60)||Total (n=92)||Azoospermia(n=46)||Severe oligo zoospermia(n=46)||1||2||3||1||2||3|
|-9C>T||C||58.30%(n=35)||59.80%(n=55)||65.2%(n=30)||54.3% (n=25)||0.859||0.471||0.682||0.942(0.486-1.824)||0.747 (0.337-1.653)||1.17(0.542-2.550)|
|G||36.70%(n=22)||34.80%(n=32)||34.7%(n=16)||34.8% (n=16)||0.921(0.468-1.815)||0.921 (0.413-2.055)||0.921(0.413-2.055)|
SNP; Single nucleotide polymorphisms, OR; Odd ratio, CI; Confidence interval, a; Controls vs. 1; Total infertile patients, 2; Azoospermia,
and 3; Severe oligozoospermia. Due to the fact that
|Locus||Azoospermia (n=46)||P valuea||OR (95% CI)a|
|Allele||Men with hypospermatogenesis(n=8)||CMA(n=17)||SCOS (n=21)||1||2||3||1||2||3|
SNP; Single nucleotide polymorphisms, CMA; Complete maturation arrest, SCOS; Sertoli cell only syndrome, OR; Odd ratio, CI; Confidence interval, a; Men with hypo spermatogenesis vs. 1; CMA, 2; SCOS and 3 SCOS vs. CMA.
|Locus||Fertile men||Azoospermia (n=46)||P valuea||OR (95% CI)a|
|Allele||Total(n=60)||Men with hypospermatogenesis(n=8)||CMA(n=17)||SCOS (n=21)||1||2||3||1||2||3|
SNP; Single nucleotide polymorphisms, CMA; Complete maturation arrest, SCOS; Sertoli cell only syndrome, OR; Odd ratio, CI; Confidence interval, a; Controls vs. 1; Men with hypo spermatogenesis, 2; CMA and 3; SCOS.
|Haplotype||Fertile men||Infertile patients||P valuea||OR (95% CI)a|
|Total (n=60)||Total (n=92)||Azoospermia(n=46)||Severe oligozoo spermia (n=46)||1||2||3||1||2||3|
SNP; Single nucleotide polymorphisms, OR; Odd ratio, CI; Confidence interval, a; Controls vs. 1; Total infertile patients, 2; Azoospermia and 3; Sever oligozoospermia.
|Haplotype||Azoospermia (n=46)||P valuea||OR (95% CI)a|
|Men with hypospermatogenesis(n=8)||CMA(n=17)||SCOS (n=21)||1||2||3||1||2||3|
SNP; Single nucleotide polymorphisms, CMA; Complete maturation arrest, SCOS; Sertoli cell only syndrome, OR; Odd ratio, CI; Confidence interval, a; Men with hypo spermatogenesis vs. 1; CMA, 2; SCOS and 3; SCOS vs. CMA.
|Haplotype||Fertile men||Azoospermia (n=46)||P valuea||OR (95% CI)a|
|Total(n=60)||Men with hypospermatogenesis(n=8)||CMA(n=17)||SCOS (n=21)||1||2||3||1||2||3|
SNP; Single nucleotide polymorphisms, CMA; Complete maturation arrest, SCOS; Sertoli cell only syndrome, OR; Odd ratio, CI; Confidence interval, a; Controls vs. 1; Men with hypo spermatogenesis, 2; CMA and 3; SCOS vs. CMA.
Study mutations in human X-linked genes with
a testis-specific pattern in view of male infertility
are considered to be remarkable. Firstly this
chromosome is enriched for genes expressed in
reproduction-related tissues and secondly it is due
to its hemizygous exposure in men (34).
In this study, the prevalence of two SNPs −9C>T
and 368A>G, in
The present study showed that the frequency of
−9T at the −9C>T locus was significantly higher
in CMA group than in patients with SCOS (Table 3,), suggesting that the mutation of allele C to T in
As shown in table 2,, in general, no significant differences are found in the frequencies of −9T allele between two groups of controls and patients, proposing that the alteration of allele C to T may be insufficient reasons for infertility in Iranian men. In contrast to previous studies, −9C>T polymorphism is associated with spermatogenic impairment in South Korean and Chinese populations (31, 32) which may be related to the following factors: environmental factors, characteristics of subjects, as well as X chromosome haplogroups in different ethnic populations.
In addition no notable association between SNP 368A>G and the risk of male infertility in Iranian population was found in this investigation. These results are similar to study on South Korean population (31).
Finally, haplotype analysis of patient and control
groups was performed in
Therefore, it may be suggested that SNPs −9C>T
and 368A>G of
To further study, it would be better to investigate
the expression level of
The present study showed no significant correlation
of SNPs −9C>T and 368A>G in
We thank all of the men who generously agreed to participate in this study. Also we are thankful to personnel of the Genetic Laboratory of Royan Reproductive Biomedicine Research Center for providing technical help, supplies and services. This study was supported by Royan Institute for Reproductive Biomedicine, ACECR. There is no conflict of interest in this study.