Mostafa K. El Awady¹, Sohair F. El Shater¹, Ehab Ragaa², Khaled Atef¹, Ibrahim M. Shaheen³, Nagwa A. Megiud²

Keywords: AZF; Y microdeletions; multiplex polymerase chain reaction; male infertility; azoospermia

Abstract

Aim: To determine the frequency of genetic deletions within the azoospermia factors in Egyptian infertile males. Methods: The Yq microdeletions in 33 infertile males with undetectable chromosomal anomalies were examined by mutiplex polymerase chain reaction (PCR). Deletions were confirmed using single PCR amplifications. Results: Four out of the total 33 (12 %) men had Yq11 microdeletions, thus supporting the average reported figures in other populations. Three of those 4 cases had single short tandem sequence deletions with discrete histological findings of their testes. Single sY272 deletion within AZFc was associated with Sertoli cell only syndrome, whereas a patient with isolated sY84 deletion within AZFa had immature testicular structure. The remaining case had a large deletion in AZFa-c and short stature. Conclusion: The present study supports the hypothesis that the Yq11 encompasses genetic determinants of stature besides genes controlling spermatogenesis.

1 Introduction

Among couples with infertility problems, male factor is the etiology in almost 50 % of cases [1] and nearly 20 % of all infertile men have idiopathic oligozoospermia [2]. Recent studies reported that DNA microdeletions on the long arm of the Y chromosome may be responsible for some cases of idiopathic male infertility [3-4] and the most important regions are within intervals 5 and 6, which are designated azoospermic factor (AZF) regions. The hypothesis that the existence of a gene or a group of genes on Y chromosome is involved in the control of human spermatogenesis was first reported by Tiepolo & Zuffardi [5]. To date, 3 non-overlapping subregions, AZFa, AZFb and AZFc, have been described. These regions are located in the proximal, central and distal segments of Yq11, respectively. Vogt et al. [6] hypothesized a correlation between specific spermatogenetic defects and a well defined Yq11 microdeletion pattern, suggesting that deletion occurring in AZFa or AZFb could predispose to Sertoli cell-only syndrome (SCOS) with subsequent spermatogenic arrest.

The purpose of this study was to determine the frequency of Y chromosome microdeletions using multiplex polymerase chain reaction (PCR) analysis in a group of well-characterized Egyptian men with idiopathic and non-obstructive oligozoo-/azoospermia.

2 Materials and methods

A total of 180 adult males (28 years - 40 years) were referred to the Human Genetics Clinic from different IVF Centers and Andrology Clinics of Egypt. For each patient two complete semen analysis were performed based on the World Health Organization guidelines for semen analysis [7]. In all the patients, GTG-banded Karyotype was performed, testicular size evaluated with ultrasonography and serum follicle-stimulating hormone (FSH), leutenizing hormone (LH) and testosterone levels were determined. Testicular biopsy was performed with conventional window technique under patient's consent. Those with defective spermatogenesis secondary to obstruction of seminal tract, injury, mumps or other infections or having chromosomal anomaly were excluded. Thirty-three azoospermic and severe oligozoospermic (sperm count less than 510⁶ mL) infertile males fulfilled the inclusion criteria and were included in the study.

Genomic DNA was extracted from the peripheral blood after digestion with proteinase K and subsequent salting out of cellular proteins using sodium chloride followed by ethanol precipitation. Alternatively, the genomic DNA extraction kit (QIA amp, cat. # 51104, QIAGEN GmbH, Germany) was also used. Both methods proved reproducible giving rise to high quality genomic DNA for PCR reactions. Eleven STS on Yq11 were utilized to identify submicroscopic deletions. In addition a single STS located within the SRY gene was used as an internal control testing for the presence of Y chromosome. Four multiplex PCR reactions were used as follows: multiplex (1) contained primers for (sY272, sY109, sY143), multiplex (2) for (sY 255(DAZ), sY158, sY84), multiplex (3) for (sY130, sY127, sY134) and multiplex (4) for (sY 146, sY14 (SRY), sY155). Whenever failure of amplification occurred, three additional separate PCR reactions were performed using the unmixed pair of primers to confirm deleted STS. The STS primer sequences were previously published [8-9].

3 Results

Using our in-house developed protocol for Y-chromosome microdeletion screening (Figure 1) only 4 men had Yq microdeletions (Table 2) at sub-regions AZFa (patient B), AZFc (patients C and D) and almost the entire AZF region (patient A). The latter patient had an apparent shortening of the long arm of Y-chromosome as revealed by the G-banding method. This finding confirmed the failure of PCR amplification at all the 11 Yq11-specific loci tested. Succesful amplification of SRY gene confirms, besides AZF deletion, the histological findings of intact interstitial tissue and total absence of germ cells within the tubules. These data altogether agree with the elevated plasma levels of gonadotrophins and normal plasma T levels in this patient. The presence of gene deletion at sY272 within AZFc sub-region in 2 out of 4 patients (C and D) suggests that this locus is a hotspot for Y-chromosome microdeletions in Egyptian population. Another interesting finding (Table 2) is the detection of the rare microdeletion within AZFa sub-region (sY84). This patient seems to have a unique etiology of male infertility with premature testicular structure and reduced levels of LH, FSH and T.

Numerous studies detected Yq11-chromosome microdeletions at various prevalence (3 % - 30 %) among men with idiopathic azoospermia or severe oligozoospermia [10-14]. This wide variability most likely arises from differences in the inclusion criteria of patients in different studies. Oliva et al [14] reviewed the published series of azoospermia and demonstrated that the worldwide frequency of microdeletions was 13 %. This is in accordance with the results of the present study as in this series Yq11 microdeletions were detected in approximately 12 % of idiopathic infertile 46, XY men. The reason why we did not detect Yq11 microdeletions at higher prevalence is partially due to our laboratory efforts to repeatedly confirm the presence of microdeletions by single pair primers in 3 separate PCR reactions so that we could avoid technical failure of amplification of Yq11 loci during multiplex reactions. Yq11 microdeletions were detected in 3 azoospermic and only one oligozoospermic men. Our data did not correspond with other studies which reported Yq11 microdeletions at low prevalence in severe oligozoospermic men [14-16]. This is explained on the bases of either different inclusion criteria and/or the relatively smaller population sample in the present study.

It is now becoming evident that genes localized on the long arm of Y chromosome are closely linked to spermatogenesis in human [3, 16]. Recent studies suggest that various phases of spermatogenesis are controlled by specific loci in Yq11 [2, 6], since disruption of spermatogenesis at specific stages correlated with Yq11 microde-letions within the same sub-region. The mechanisms of these microdeletions are still debatable. Earlier studies assume that recombination events between homologous sequence repeats (e.g. Alu repeats) on the X and Y chromosomes may produce Y-microdeletions [17]. Alternatively unbalanced sister chromatid exchange may give rise to Y-microdeletions. Both assumptions are related to the high frequency of repetitive sequences and instability of the Y chromosome [18]. Since sequence instability is closely associated with spontaneous suspected de novo genetic abnormality, both mechanisms of Y-microdele-tions are supported by data from the present study. However, PCR analysis for their father(s) is highly recommended. In at least 2 patients (A and D) de novo Yq11 microdeletions were detected, since male infertility has never been documented in their families.

In conclusion the present study contributes to the current understanding of the complicated function of Yq11 genes in spermatogenesis. The detection of only isolated STS deletions in patients B, C and D allowed us to correlate structure with function at levels as minor as a single AZF locus.

References

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Correspondence to: Mostafa K. El Awady, Ph.D., Department of Biomedical Technology, National Research Center, Dokki, Cairo 12622, Egypt.
Tel: +20-2-313 2640, Fax: +20-2-337 0931
Email: melawady@hotmail.com
Received 2003-03-10 Accepted 2004-01-24