PCR analysis of Yq microdeletions in infertile males, a study from South India

Institute of Genetics & Hospital for Genetic Diseases, Scientist, Osmania University, Begumpet, Hyderabad-500 016, India

Asian J Androl 2002 Dec; 4: 265-268            

Keywords: male infertility; Y-chromosome; AZF region; RBM; DAZ; oligozoospermia; azoospermia

Abstract

Aim: To estimate the frequency of microdeletions in the long arm of Y-chromosome of 20 infertile males from South India. Methods: Polymerase chain reaction (PCR) amplification using Y-specific STS of azoospermia factor (AZF) regions i.e., SY 84 for AZFa, SY 127 for AZFb and SY 254 for AZFc. Results: Of the 20 infertile subjects 3 (15 %), one azoospermic and two oligozoospermic, showed microdeletions in the AZF region of Y-chromosome. Conclusion: The frequency of deletions involving AZF region of the Y-chromosome is 15 % in azoospermic and severely oligozoospermic infertile men. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.

1 Introduction

About 15 %~20 % of couples are infertile and 40 % of these cases, male factor is identified as the main cause of infertility [1]. Tiepolo and Zuffardi [2] have postulated the existence of a spermatogenesis locus located at Yq11. Molecular analysis of infertile men with severe oligozoospermia or azoospermia has identified three non-overlapping regions of the Y-chromosome and these regions have been termed as azoospermia factor AZFa, AZFb and AZFc. In each region candidate genes have been proposed i.e., DFFRY in AZFa region, RBM (RNA binding motif) in AZFb region and DAZ (deleted in azoospermia) in AZFc region [3]. Recent genetic studies have identified nearly 15 novel genes or gene families in the human Y-chromosome, some of which are located within AZF intervals [4]. Microdeletions in one or more of these genes could be associated with spermatogenic failure and infertility [5].

Extensive studies have been carried on Y microdeletions in different racial groups from all over the globe and the results showed vide variation (1 % to 55 %). However, no studied on the microdeletions in the Y-chromosome were reported from India. Hence, an attempt has been made to understand the role of Y-chromosome microdeletions in infertile males.

2 Materials and methods

2.1 Subjects

Couples with infertility problem referred to the Institute of Genetics and Hospital for Genetic Diseases, Hyderabad by various fertility centers and hospitals throughout Andhra Pradesh, India were thoroughly studied. Twenty males with normal female partners, as ascertained by physical, hormonal and reproductive system examinations, were selected for the study.

The subjects were examined by a clinical geneticist of the Institute of Genetics and information on their age, occupation, family pedigree, habits, illness, occupational exposure to physical and chemical agents and duration of infertility was recorded in a standard proforma. Physical examinations, including assessment of the secondary sexual characteristics, inspection of the penis and determination of the testicular size by orchidometry were carried out. Ultrasonography scanning of the scrotum and testicular biopsy were also carried out to help determining the cause of infertility.

2.2 Chromosomal analysis

Chromosomal analysis was carried out on peripheral blood lymphocytes and the males with abnormal karyotypes were excluded from the study. Every man provided a semen sample after sexual abstinence for three days. These samples were analyzed on the basis of the criteria of the World Health Organization [6] and based on the sperm count they were categorized as azoospermia and oligozoospermia. Ten proven fertile males were selected for the control data and 5 women was used as the negative controls.

2.3 PCR amplification

PCR amplifications were performed in DNA samples from 20 infertile subjects and 10 proven fertile controls according to the modified method of Simoni et al. [7]. Each reaction was performed with 200 ng of genomic DNA, 10 mmol/L dNTPS, 50 pmol/L of each primer, 5 mL buffer and 1U of Taq DNA polymerase in a reaction volume of 50 mL. The primers SY 84, SY 127 and SY 254 were amplified individually by 35 cycles at 94 for 50 seconds, 58 for 55 seconds and 72 for 1 minute. The programmes were preceded by a 5 minutes denaturing step at 94 oC and followed by a final extension step at 72 for 5 minutes.

2.4 Primers used for PCR amplification

2.4.1 SY 84 for amplification of AZFa region

Forward: 5'-AGA AGG GTC TGA AAG CAG GT-3'
Reverse: 5'-GCC TAC CTG GAG GAG GCT TC-3'
Product size: 326 bp.

2.4.2 SY 127 for amplification of AZFb region

Forward: 5'-GGC TCA CAA ACG AAA AGA AA-3'
Reverse: 5'-CTG CAG GCA GTA ATA AGG GA-3'
Product size: 274 bp.

2.4.3 SY 254 for amplification of AZFc region

Forward: 5'-GGG TGT TAC CAG AAG GCA AA-3'
Reverse: 5'-GAC CGT ATC TAC CAA AGC TGC-3'
Product size: 380 bp.

2.5 Electrophoresis

Electrophoresis of PCR amplified DNA products was carried out on 1.2 % agarose gel. The presence of deletion results in the absence of synthesis of the desired product, therefore, absence of band corresponding to any of the studied region is indicative of the presence of deletion of the particular region. Similarly the presence of band corresponding to the amplified region is indicative of absence of deletion. A positive control (fertile male DNA) and a negative control (female DNA) were also included with each set of reaction to ensure that all the primers are active and that no contamination has occurred.

3 Results

The results on sperm concentration and clinical finding are presented in Table 1. In 10 (50 %) out of 20 infertile males, no spermatozoa were observed. In the remaining 10 cases, the sperm count was between 5-20 million/mL. Out of the 10 azoospermic males, 1 had testicular atrophy, 1 varicocele, 1 congenital bilateral absence of vas deferens (CBAVD). Out of the 10 oligozoospermic infertile male subjects, 1 had testicular atrophy, 2 varicocele, 1 cryptoorchidism, 1 epididymal cyst.

Table 1. Sperm concentration and clinical findings of infertile males.

Table 2 shows that out of the 20 infertile subjects, 3 (15 %) showed microdeletions in the AZF regions of Y-chromosome. Out of the 10 azoospermic males, 1 had deletion in the AZFc region. Out of the 10 oligozoospermic males, 2 had deletions: one in the AZFb region and the other in the AZFc region (Figure 1, 2).

In the present study, 8 of the 20 infertile males showed clinical abnormality, such as testicular atrophy, varicocele, epididymal cyst, etc. and in these cases no microdeletions were detected. Three (15 %) males showed Y micro-deletions, which is in close agreement with the data (16 %) reported by Yao et al [8]. Some studies [9,10] reported 13 % of infertile males with microdeletions in the Y-chromosome, while others [7, 11, 12] less than 5%. Girardi et al [13], Mulhall et al [14] and Aho et al[15] showed an incidence of microdeletions between 5.1 % and 9.6 % in the infertile males. However, Foresta et al [16] reported 55.5 % of infertile males with Y microdeletions from Italy.

In different studies the frequency of the infertile males with microdeletions varied extensively in both the azoospermic and oligozoospermic men. In the azoosper-mic men, we indicated that 10 % (1/10) had microdeletions in the long arm of the Y-chromosome, while Mulhall et al [14] showed a value of 9.6 % and others [9, 15, 17] between 13 % and 23 %. However, Foresta et al [16] reported 55.5 % of azoospermic males with microdeletions in the Y-chromosome.

In the oligozoospermic infertile males, we indicated that 20 % (2/10) showed microdeletions in the long arm of Y-chromosome. Foresta et al [18] reported a value of 22 % and Pryor et al [17] 9.7 %. However, some studies [5, 7, 11, 13] showed that less than 6 % of these males had microdeletions.

Three genes from interval 6 (Yq11.23) of AZF, RBM (RNA binding motif), DAZ (Deleted in Azoosperma) and SPGY are thought to be closely associated with male infertility. These genes are expressed specifically in the testis and encode presumed RNA-binding proteins [19]. The RBM genes are multi-copy genes and their location on the Y-chromosome is still not clearly known. Recent studies have suggested that microdeletions of the Y-chromosome outside interval 6 may also play an important role in the regulation of spermatogenesis [20].

Vogt et al [20] proposed a close relationship between the size of deletion, their localization with respect to AZF loci and the severity of spermatogenesis impairment. Deletion of AZFa is related to the absence of germ cells in seminiferous tubules while deletion of AZFb locus is associated with maturation arrest during meiosis. Deletion of AZFc may cause variable phenotypes ranging from Sertoli cell only syndrome type II to hypospermato-genesis. In the present study, no clinical abnormality was observed in the three males who showed microdeletions.

The majority of Y microdeletions occur as denovo events. The origin of Y microdeletions is not clearly understood. Microdeletions may arise in the testis, in fertilized eggs or embryos; microdeletions prevent the formation of spermatogonia in the fetus resulting in impaired spermatogenesis in the adult [21]. The high frequency of Y microdeletions suggests that the Y chromosome is susceptible to spontaneous loss of genetic material. Aberrant recombination events occur between areas of homologous or similar sequence repeats between X and Y chromosome or within Y chromosome itself by unbalanced sister chromatid exchanges [22]. The instability of the Y chromosome may be related to a high frequency of repetitive elements clustered along the length of the chromosome [23].

Although it is very clear that microdeletions in the AZF region are responsible for spermatogenic failure, further studies are worthwhile to delineate the exact function of the genes present in AZF region and their role in spermatogenesis and fertility.

We thank, Drs. V. Dashavantha Reddy and K. Venkateswara Rao of CPMB, Osmania University, Hyderabad, A.P., India for providing lab facilities to carry out the PCR work. S. Ramesh Babu thanks the University Grants Commission, New Delhi for providing the financial support to carry out this work.

References

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Correspondence to: Prof. P.P. Reddy, Director, Institute of Genetics & Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad 500 016, India.

Fax: +91-40-709 8003

E-mail: reddypp@rediffmail.com

Received 2002-09-03 Accepted 2002-11-22