Our study included total 160 of central Indian origin idiopathic non-obstructive infertile men and 50 normal healthy fertile controls were included in this study. All cases visited our hospital out-patient-department and referred to reproductive biology unit for semen analysis. Their seminal examination report showed azoospermia (n=160). The age of azoospermic males ranging were 25 to 45 years. Infertile men were subjected to wide-ranging questionnaires from related to their medical, surgical, sexual and family history, lifestyle habits. Clinical exclusion criteria were strictly followed for selection of cases. Patients having chromosomal abnormalities, cystic fibrosis, testicularpathy, obstructive azoospermia, hypogonadism, hormonal disbalanced, chronic diseases were excluded from this study. Two seminal reports were considered to determine their infertility status, after 3-4 days of sexual abstinence. Seminal analysis was assessed on the basis of the guidelines of World Health Organization [10]. In case of azoospermic men scrotal sonography reports were obtained when required. Informed written consent form was obtained from each infertile male. Institutional ethical clearance committee had approved the present study.

Phytohemagglutinin (PHA) stimulated peripheral lymphocyte cultures were set up for 72 hours in RPMI 1640 medium supplemented with 10% fetal bovine serum. Chromosomal slide were prepared using standard cytogenetic technique. Ten well spread G banded metaphase were observed per individual and karyotype were prepared by microphotography [11].

In this study, 148 infertile azoospermic men were subjected for genomic DNA extraction by standard methods. Simplex polymerase chain reaction was done for detection of microdeletions on the AZF loci. Twelve sets of Y specific sequence tagged sites (STSs) primer were subjected to examine three AZF locus that mapped to interval 5 and 6 of the Y chromosome. The following STS primers were used: AZFa – sY746, sY84, sY86; AZFb – sY118, sY127, sY134, RBMIY; and AZFc – sY153, sY148, sY254, sY255, sY158;. Internal control was used as the SRY-sex-determining region on the short arm of the Y chromosome (sY14). All the primer sets were used from our previous studies [9]. This STS primer is recommended by the European Academy of Andrology. Over 90% of deletion in the AZF locus is making possible to detect by above primer sets and it allow for less standardization effort. A positive control of normal fertile male DNA and two negative control: (i) normal female DNA; (ii) composition of reaction mixtures except DNA were added in every PCR assay. The PCR amplification comprised a total volume of 50μL, which contained 100 to 200 ng of human genomic DNA as template, 2.5mM dNTP’s, 10 pmol/μl each forward and reverse primers (IDT, USA),10X Taq DNA polymerase assay buffer, 25mM MgCl₂ and 5U Taq DNA polymerase (Merck Biosciences, India). The conditions for thermo-cycling were standardized for the each STS primer, utilizing a Veriti (Applied Biosystem, USA) thermo-cycler. Samples were subjected to polymerase chain reaction amplification using 30 cycles at 94^oC for 30 sec for denaturation, 55-60^oC for 45 sec for annealing and 72^oC for 60 sec for aplification. Initial denaturation was done at 94^oC for 5 min and final extension at 72^oC for 7 min. The PCR amplified products were subjected to electrophoresis on 1% agarose gels contained 0.5μg/mL ethidium bromide and product was separated on the basis of the size. In case of detecting deletions with STS primer the PCR assay was repeated thrice for conformation.

The cytogenetic abnormalities containing numerical and structural sex chromosomal aberration were observed in 12 (7.5%) individuals of 160 infertile males. However, 9 individual was 47, XXY karyotype, 1 was 46, XYq- and 2 men were 46, XY 22q+. Following the cytogenetic result, these 12 individuals were excluded from molecular studies. Polymerase chain reaction microdeletions analysis was done in 148 infertile males and 50 fertile controls. The type of cytogenetic abnormalities and AZF microdeletions are presented in [Table/Fig-1,2]. Out of 148 azoospermic men, 19 (12.8%) had microdeletions in AZFa, AZFb and AZFc regions, Three azoospermic males had microdeletions in AZFa (2.02%), here interstitial deletion was detected in sY746, sY86 STS markers in AZFa. Five azoospermic males had deletions in AZFb region (3.37%) with STS marker sY118, sY127, sY134. Nine cases had deletions in AZFc region (6.08%) with STS markers sY153, sY148, sY254, sY255 [Table/Fig.-3,4]. Deletions in both AZFb and AZFc were detected continuous in two (1.35%) azoospermic cases. No deletions were observed in the control fertile males. 12.8% (19/148) males with idiopathic non-obstructive azoospermic infertility history showed Y chromosome microdeletions with different sequence-tag-sites primers. Regionwise frequencies AZF deletions in azoospermic males were shown in Ven-diagram [Table/Fig-5]. The most frequently microdeletions (6.08%) were found within AZFc region with STS primers sY148, sY255 and sY254.

[Table/Fig-3]:

Agarose gel electrophoresis analysis shows the microdeletions in AZFc region of STS marker sY254 (389 bp) of Y chromosome in Azoospermia cases. M.W.: 100 bp of DNA ladder; Lane no 1: Internal control SRY; Lane no. 2, 4 & 11: Azoospermia with sY254 deletions; Lane 13 & 14 negative control female DNA & water sample; Lane no 15: positive control fertile male

[Table/Fig-4]:

Agarose gel electrophoresis analysis shows the microdeletions in AZFc region of STS marker sY255 (123 bp) of Y chromosome in Azoospermia cases. M.W.: 100 bp of DNA ladder; Lane no 1: Internal control SRY; Lane no. 3, 4, 6, 8 & 11: Azoospermia with sY255 deletions; Lane 13 & 14 negative control female DNA & water sample; Lane no 15: positive control fertile male

[Table/Fig-5]:

Vendiagram shows percentage of azoospermic cases having microdeletions in AZFa region (2.02%), AZFb (3.37%), AZFc (6.08%) and AZFb+c (1.35%). And also the frequency distributions of microdeletions in AZFa, AZFb and AZFc regions are 15.79%, 31.58% and 52.63% respectively

Spermatogenesis is regulated through number of genes on Y chromosome and autosomes. Microdeletions on AZF region of Y chromosome is established cause of human male infertility [12]. Spermatogenesis impairment has depended on the increasing frequency on microdeletions of Y chromosome. In male infertility, 15-30% cases involving complex genetic abnormalities can lead to different degree for spermatogenic failure [13]. By clinical findings, on the basis of semen analysis or by karyotyping Y chromosome microdeletions cannot be predicted [14]. In general, patient’s age and duration of infertility are considered important factors in making decisions regarding how to investigate, treat, and establish a reproductive prognosis [15]. Hence, in idiopathic non-obstructive azoospermic infertile male Y-chromosome microdeletions analysis has become a standard molecular test with STS markers. The prevalence of Y-chromosome microdeletions were reported in different studies, range between 10-30% of males with non-obstructive azoospermia [2,3]. Our study has evaluated the incidence of Y-chromosome microdeletions in azoospermic central Indian infertile male patients.

In present study, cytogenetic abnormalities were reported on 7.5% of azoospermia men. Nine patients were Klinefelter’s syndrome 47, XXY sex chromosomal abnormalities and in one azoospermic men had karyotype 46, XYq- which is consistent with earlier studies performed in other populations [16]. Autosomal abnormalities 46, XY (22+), found in two azoospermic patient, was already reported in azoospermic [17]. The assumption for this occurrence is that chromosomal abnormalities may interfere with normal chromosome pairing and segregation in meiosis I, leading to the potential formation of unbalanced gametes [18]. Another possibility in translocation of autosome is involved in male gametogenesis may not be regulated by breakpoint in genes [17]. The azoospermic patient with karyotype 46, XY, delY (q del) seen in the present study had lost the whole q arm with spermatogenesis involving genes. It provides the evidence for importants of Yq region for spermatogenesis and male fertility [4]. However, the relationship between chromosomal abnormalities and male infertility has been observed with a non-random distribution.

We found 19 (19/148) patients carrying microdeletions corresponding to a frequency of 12.8%. Some studies reported high frequencies of microdeletions up to more than 30% of infertile male, while others reported less than 4% in comparison to the statistical values obtained from all surveys. Another study showed an incidence of microdeletion between 5.1% and 9.6% in the infertile males. Our results are supported with these results between 3-18% [19]. In worldwide data illustrated that high frequencies of Y microdeletions are present in cases of non-obstructive azoospermia. The frequency of deletion of the AZFc region was found to be higher as compared to AZFa and AZF b region in our study, is a conformity finding with earlier studies [20]. Some earlier studies showed that deletion in the AZFc region was equal when compared with that in the AZFa and AZFb region [19]. In our present study, 3/19 (15.79%) deleted infertile males showed deletions in AZFa region, 6/19 (31.58%) deleted infertile males showed deletions in AZFb region, 10/19 (52.63%) deleted infertile males showed deletions in AZFc region and two infertile males showed deletions in both AZFb+c region. The prevalence of AZF deletion in this study population was 12.8% which is supported to previous studies of Indian population [14,21]. In another study, the prevalence of Y-chromosome microdeletions in AZF sub-regions was reported as 13.3% (12/90). A meta-analysis study has mentioned that the prevalence of Y-chromosome microdeletions was between 0.7-34.5% with an average of 8.2% [22]. However, some microdeletions are more predominant in certain populations, one has to use a more number of STS markers from each AZF region to identify Y chromosome deletions. Duplication or reduction in the copy number of DAZ genes (AZFc) was reported to have increased risk in subfertility and infertility [23].

However, aetiologies of a large number of azoospermic men are still unknown because of male infertility is caused by various genetic and non-genetic factors. New autosomal, X chromosomal or more Y chromosomal STS markers would help in identifying more genetic aetiologies for the idiopathic azoospermic individuals. There are variations in frequencies in different reports and these may be due to various diagnostic protocols, wrong diagnosis, selection criteria of the study population or heterogeneity in STS markers selection by various investigators. In light of the above, we also believe that the aetiology of male infertility may differ between ethnic populations. Therefore, researchers need to keep this in mind and define the strategies for analysing infertile samples. Our data will be useful for infertility clinics for genetic counselling of oligozoospermic men by advising them to choose a female child in case of Y chromosome microdeletions and to adopt appropriate methods for assisted reproduction.