Microdeletions in the Y chromosome of patients with idiopathic azoospermia

Akiyuki Shimizu¹, Tomohiko Ichikawa¹, Noriyuki Suzuki¹, Takako Yamazaki¹, Takashi Imamoto¹, Satoko Kojima¹, Yukio Naya¹, Akira Komiya¹, Hiroyoshi Suzuki¹, Koichi Nagao², Kazukiyo Miura², Haruo Ito¹

Asian J Androl  2002 Jun; 4:  111-115             

Keywords: male infertility; idiopathic azoospermia; azoospermia factor; deleted in azoospermia; intracytoplasmic sperm injection

Abstract

Aim: To evaluate the occurrence and prevalence of microdeletions in the Y chromosome of patients with azoospermia. Methods: DNA from 29 men with idiopathic azoospermia was screened by polymerase chain reaction (PCR) analysis with a set of Y chromosome specific sequence-tagged sites (STSs) to determine microdeletions in the Y chromosome. Results: Deletions in the DAZ (deleted in azoospermia) loci sY254 and sY255 were found in three patients with idiopathic azoospermia, resulting in an estimated frequency of deletions of 10.7% in idiopathic azoospermia men. Conclusion : We conclude that PCR analysis is useful for the diagnosis of microdeletions in the Y chromosome, which is important when deciding the suitability of a patient for assisted reproductive technology such as testicular sperm extracion-intracytoplasmic sperm injection (TESE-ICSI).

Over 50 % of all infertile men have idiopathic azoospermia or oligozoospermia. Studies on infertile men have demonstrated the existence of a gene, or gene complex, associated with spermatogenesis in the Y chromosome, known as the azoospermia factor (AZF) [1-5]. Microdeletions in this region are thought responsible for normal spermatogenesis. Recently, the development of assisted reproductive technologies (ART), such as intracytoplasmic sperm injection (ICSI) substantially improved the outlook for patients with severe male fertility problems. However, this implies that genetic defects associated with male infertility may be transmitted to offspring and result in a genetic disease. Mulhall et al.[6] reported that mature spermatozoa were found in ~50 % of azoospermic patients with AZFc deletions. Therefore, in the present study, we investigated the microdeletions in the Y chromosome using polymerase chain reaction (PCR) analysis in a group of men with idiopathic and nonobstructive azoospermia.

From April 1993 to September 1999, 59 patients with azoospermia, visiting our departments and related hospitals, were enrolled in this study. Informed consent was obtained from all participants. Azoospermia was confirmed by two separate semen analyses 3 weeks apart, each following a 3-day sexual abstinence, according to the World Health Organization criteria [7]. A complete medical history was obtained and a close physical examination was performed in each case. Patients who had obstruction in the seminal tract, hypogonadotropic hypogonadism, varicocele, chromosomal abnormality, and/or other causes of possible testicular damage, such as chemotherapy, were excluded. When a varicocele was suspected, scrotal ultrasonography, Doppler ultrasonography and thermography were performed as reported by Takahara et al [8]. Serum LH, FSH, prolactin, testo-sterone, and estradiol levels were measured by radio-immunoassay. Chromosomal analysis was performed on cultured lymphocytes using the G-banding technique. Testicular biopsies were performed in 16 patients after obtaining written informed consent. Sertoli-cell-only syndrome was diagnosed only if all seminiferous tubules contained only the Sertoli cells. The diagnosis of maturation arrest was made when tubules contained no spermatozoa but showed spermatogonia, spermatocytes, and/or spermatids. When tubules showed mosaicism of tubules with or without sperm, the diagnosis was hypospermato-genesis.

A set of 16 previously described Y-specific STSs was tested in each patient. The order of the STS primers tested is shown in Table 1 [5].

Table 1. Order of the STS primers.

2.3 Deletion analysis by polymerease chain react

Genomic DNA was extracted from peripheral blood granulocytes using the QIAamp blood maxi kit (QIAGEN, California, USA). PCR analysis was performed with 50 ng of genomic DNA in a final volume of 50 mL, comprising of the reaction buffer (0.5 % dimethylsulfoxide), 0.2 mmol/L diethylnitrophenyl thiophosphate mix, 10 pmol of each primer, and Taq DNA polymerase. Thermocy-cling consisted of an initial denaturation of 5 minutes at 94, followed by 35 cycles of 30 seconds at 94 (melting), 30 seconds at 55 (annealing), and 1 minute at 72 (extension), and finally 5 minutes at 72. PCR products were separated by 1.5 % agarose gel electro-phoresis, stained with ethidium bromide, and visualized using ultraviolet transillumination.

Of the 58 consecutive azoospermic patients, 29 showed idiopathic azoospermia and were enrolled in this study (Table 2). These 29 patients demonstrated a karyotype of 46,XY without any apparent cytogenetic abnormalities by conventional G-banding analysis. Clinical findings for these patients are summarized in Table 3.

Table 2. Characteristics of 58 patients with azoospermia

3.2 Immunohistochemical staining and TGF-b1 index

Testicular biopsy was performed in 16 of the 29 azoospermic patients. Histologic diagnosis identified 3 cases of Sertoli-cell-only syndrome, 4 maturation arrest, 8 hypospermatogenesis and 1 normal spermatogenesis.

Screening for microdeletions in the Y chromosome was performed in the 29 patients. Interstitial deletions of Yq were found in 3 (10.3 %, Figures 1 and 2). All deletions overlapped with STSs ( sY254, sY255, sY283) for the DAZ gene (Figure 1). There were no deletions in the AZFb subregion (sY142, sY143). Table 4 summarizes the hormonal and histologic characteristics of the 3 patients with idiopathic azoospermia who had Yq micro-deletions.

Figure 1. An example of PCR analysis for sY255 in patients with azoospermia. Lane a, #3 patient indicated in Table 4; Lanes b and c, patients with idiopathic azoospermia without deletion. bp = base pairs.
Figure 2. Summary of STSs and PCR data of the three patients with Yq microdeletions. Deletion intervals and Y-chromosome STSs used are listed above. The solid black boxes indicate presence of the STSs. Blank spaces indicate absence of the STS.

Table 4. Phenotypic features of azoospermic men with Yq microdeletions. aSCO, Sertoli cell only. bN.D., not determined.

About a quarter of a century ago, Tiepolo and Zuffardi [1] reported six azoospermic men with gross deletion of the Y chromosome and postulated the existence of a factor, located on the Y chromosome, that is necessary for normal spermatogenesis. Later, this factor was named azoospermia factor (AZF). More recently, small interstitial deletions (microdeletions) have been detected using molecular techniques. Two candidate genes for AZF have been suggested: RBM (RNA binding motif) [2] and DAZ [3]. Both genes are expressed in the testis and encode a presumed RNA-binding protein, the precise function of which remains to be determined. RBM seems to be a gene family and multiple copies are distributed along the Y chromosome. The DAZ gene is equivalent to AZFc, as indicated by Vogt et al[4], who suggested the presence of three spermatogenesis loci in Yq11 and designated them AZFa (proximal), AZFb (central) and AZFc (distal); They hypothesized a correlation between specific spermatogenic defects and a well-defined Yq11 microdeletion pattern, suggesting that deletions occurring in AZFa result in Sertoli-cell-only syndrome and deletions in AZFb result in spermatogenic arrest. However, in other similar studies these associations were not evident. Initially, DAZ was thought to be a single copy, however, it is now known as the DAZ gene family on Yq11 and has an autosomal copy on the short arm of chromosome 3 ( DAZL or DAZH) [9].

In the literature, the occurrence of microdeletions varies between 1 % and 55 % [10-14]. Most microdele-tions have been found in patients with idiopathic azoospermia or severe oligozoospermia. Among our cases, mi-crodeletions were detected in 3 of 29 patients with idiopathic azoospermia (10.3 %). This frequency is within the range of previous studies.

Apart from sporadic cases of natural transmission of similar or identical Y chromosome microdeletions [15], the majority of deletions are believed to be de novo [16,17], i.e. somatic events arising randomly in some germ cells of fertile men who thereby produce an infertile son with a Y chromosomal defect in his genome. Until recently, such an individual would normally be infertile and further transmission of the genetic defect would not occur. However, ICSI now offers an effective therapeutic option for these men, and Yq microdeletions can be transmitted to male offspring [16-19]. Mulhall et al.[6] demonstrated that men with nonobstructive azoospermia with only AZFc deletions may harbor spermatozoa, allowing effective fertilization, normal embryo development and term pregnancy via TESE and ICSI. Kent-First et al. [20] analyzed the AZF genes in DNA from the blood of 32 infertile men and their sons conceived through ICSI. They found microdeletions in one infertile father and three ICSI infants. The infertile father and his son had identical microdeletions in the AZF genes. Therefore, DNA screening for the AZF genes as well as conventional cytogenetic studies in infertile males have been suggested to be necessary before ICSI [21-24]. Although the pre-sent three cases with Yq microdeletions have not undergone TESE and ICSI, these couples should receive careful genetic counseling to allow them to make a well-informed choice about reproduction and family planning.

References

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Correspondence to: Tomohiko Ichikawa, M.D., Ph.D., Department of Urology (E5), Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba 260-8670, Japan.
Tel: +81-43-226-2134, Fax: +81-43-226-2136
E-mail: ichikawa@urology1.m.chiba-u.ac.jp
Received 2002-04-23      Accepted 2002-05-08