Screening for Y-chromosome microdeletions in infertile Indian males: Utility of simplified multiplex PCR

Background & objectives: Analysis of the microdeletions in the azoospermia factor (AZF) region of Y chromosome by PCR is an important screening tool in the work-up of infertile males opting for assisted reproductive techniques. In the present study, the Y chromosome microdeletions were analyzed by PCR using primers corresponding to 16 sequence tagged sites (STS) and three genes of the AZF region in infertile Indian men. Feasibility of developing a simplified multiplex PCR for screening of the Y chromosome microdeletions has been explored.

Methods: A total of 271 male subjects were analyzed, of which, 170 were infertile patients (51 oligospermic and 119 azoospermic) and 101 were fertile controls. Subjects showing normal karyotype only were included in the study. The semen analysis was done and plasma follicle stimulating hormone (FSH) concentrations were determined by radioimmunoassay. Testicular histopathology was analyzed by fine needle aspiration cytology (FNAC).

Results: Y chromosome microdeletions were observed in nine out of 170 (5.29%) infertile males all of whom were azoospermic. Of the nine subjects, two had deletions in AZFa, one in AZFb, three in AZFc and three in AZFb+c regions. No deletions were observed in the infertile severe oligospermic men (

Interpretation & conclusions: The multiplex PCRs described in the present study may be a suitable, cost-effective and less time consuming method for screening the Y chromosome deletions in infertile males in routine clinical diagnosis and counselling prior to assisted reproduction.

Key words AZF - FNAC - FSH - multiplex polymerase chain reaction - testicular phenotype - Y chromosome microdeletions

Infertility is a major health problem affecting 10-15 per cent of married couples and a male factor can be identified in half of these cases'. Spermatogenesis may be affected by systemic disease, cryptorchidsm, endocrinological disorders, obstruction in seminal pathways, and infection or immunological factors. However, the cause of male infertility is unknown in up to 50 per cent of cases1. In these cases, genetic aetiology may be associated with abnormal spermatogenesis. A genetic factor located at Yq11 has been established to be important for male germ cell development and the gene cluster is referred to as azoospermia factor (AZF)2. In the AZF region, three loci termed as AZFa, AZFb and AZFc have been identified. In the Y chromosome, 300 sequence tagged sites (STS) have been generated and mapped for the above three AZF regions1. STS are known sequences of genomic DNA that can be amplified by PCR. The role of Y chromosome microdeletions in male infertility has been well established3-6. The frequency of reported Y chromosome microdeletions varies from 1 per cent in a cohort of men undergoing intracytoplasmic sperm injection (ICSI) for varying reasons3 to 55 per cent in men with Sertoli cell only (SCO) syndrome4. The variations among populations, the method of designing a particular study and the validation of real deletions may explain this wide range of variability. The recommendations of European Academy of Andrology (EAA) guidelines7, suggest that over 90 per cent microdeletions can be detected by the use of 2 STS markers for each AZF loci. A study of Y chromosome deletions in 340 azoospermic Indian men employing 30 STS markers revealed an overall deletion of 8.5 per cent6. Importantly, this study failed to find any deletion in the sY86 and sY84 STSs as recommended by EAA7 for analysis. On the other hand, the deleted STSs were sY746, sY741, sY742, sY615 and the gene DFFRY; which are inter-spread between or around sY86 and sY84. The authors thus suggested that the chances of frequenting a deletion are more, if more sets of primers are used. These findings raise questions about the validity of the primer sets recommended in the EAA guidelines7 in all racial populations.

The phenotypes associated with deletions in the different AZF regions are variable8,9. Complete deletions of AZFb and AZFb+c are characterized by a histological picture of Sertoli cell only (SCO) or spermatogenic arrest resulting in azoospermia9 and deletions in AZFa region have been associated with SCO I (complete absence of germ cells in the seminiferous tubules and presence of Sertoli cells only) syndrome10,11. However, deletion of only AZFc region is found to be associated with a wide range of phenotypes ranging from hypospermatogenesis to SCO II (isolated foci of spermatogenesis along with Sertoli cells) syndrome9. A study by Machev et al12 represents the evidence that even partial AZFc deletion or gene conversion may frequently be the basis for spermatogenic failure. A plausible explanation for the variable phenotype is progressive regression of germinal epithelium over time which has been reported in patients with AZFc deletions13. Among the three AZF regions, deletion of AZFc is more frequent, followed by AZFb and AZFa14. This lack of accurate association of genotype with testicular histology has resulted in the absence of any clear clinical parameters that can be used to predict the presence of microdeletions in a given infertile male15.