AZF microdeletions associated with idiopathic and non-idiopathic cases with cryptorchidism and varicocele

Rima Dada¹, N.P. Gupta², K. Kucheria¹

¹Department of Anatomy, ²Department of Urology, All India Institute of Medical Sciences, New Delhi-110029, India

Asian J Androl 2002 Dec; 4: 259-263             

Keywords: azoospermia factor; oligozoospermia; azoospermia; infertility

Abstract

Aim: To identify submicroscopic interstitial deletions in azoospermia factor (AZF) loci in idiopathic and non-idiopathic cases of male infertility in Indians. Methods: One hundred and twenty two infertile males with oligozoospermia or azoospermia were included in this study. Semen analysis was done to determine the sperm density, i.e., normospermia (>20 million/mL), oligozoospermia (<20 million/mL) or azoospermia. They were subjected to detailed clinical examination and endocrinological and cytogenetic study. Thirty G-banded metaphases were analyzed in the 122 cases and polymerase chain reaction (PCR) microdeletion analysis was done in 70 cytogenetically normal subjects. For this genomic DNA was extracted using peripheral blood. The STS primers tested in each case were sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc). PCR amplifications found to be negative were repeated at least 3 times to confirm the deletion of a given marker. The PCR products were analyzed on a 1.8 % agarose gel. Results: Eight of the 70 cases (11.4 %) showed deletion of at least one of the STS markers. Deletions were detected in cases with known and unknown aetiology with bilateral severe testiculopathy and also in cryptorchid and varicocele subjects. Conclusion: AZF microdeletions were seen in both idiopathic and non-idiopathic cases with cryptorchidism and varicocele. The finding of a genetic aetiology in infertile men with varicocele and cryptorchidism suggests the need for molecular screening in non-idiopathic cases.

1 Introduction

Azoospermia factor (AZF) microdeletions are a prevalent cause of male factor infertility. Male Infertility is a major health problem today and 40 %~50 % men at the reproductive age have qualitative or quantitative abnormality in sperm production. In more than 60 % of cases the origin of reduced testicular function is unknown [1, 2]. Several studies have reported a marked decline in reproductive health and increase in population of subfertile males [3]. Both genetic and environmental factors are believed to be responsible for these changes.

The long arm of the Y chromosome is involved in spermatogenesis and contains genes and gene families critical for germ cell development and differentiation. Deletion of long arm of Y chromosome is associated with spermatogenic failure and leads to partial or complete spermatogenic arrest [4]. It is only in the last few years that the loci involved in the production and differentiation of sperm have been identified using molecular methods. The Y chromosome has been divided into seven deletion intervals (Figure 1). Each of these intervals is further subdivided into subintervals (A, B, C, etc.) [5]. In 1992, Vollrath and colleagues [6] constructed a 43 interval deletion map of human Y chromosome which contained an ordered array of sequence tagged sites (STS) which span the entire length of the Y chromosome. The AZF loci responsible for spermatogenesis is located on the long arm of Y chromosome in deletion intervals 5 and 6. The AZF region has 3 non-overlapping loci, AZFa, AZFb and AZFc and their deletion is associated with spermatogenic failure [7]. AZF microdeletions are associated with azoospermia, oligozoospermia and also with a varied testis histological profile ranging from Sertoli Cell Only (SCO), hypospermatogenesis to maturation arrest[7]. Deleted in azoospermia (DAZ) and RNA binding motif on Y (RBMY) are AZF genes which code for RNA binding proteins and may be involved in the regulation of gene expression. In order to gain information about AZF deletions in infertile males and its relationship with testicular phenotype, the DNA of normal controls and idiopathic and non-idiopathic infertile males with severe testiculopathy were analyzed.

Figure 1. Schematic representation of Y chromosome showing 7 deletion intervals and pseudoautosomal region PAR 1 and PAR 2.

2 Materials and methods

2.1 Patients and treatment

One hundred and twenty two infertile men and 25 age-matched fertile controls were included in this study. The diagnosis of azoospermia and oligozoospermia was made on the basis of semen analysis according to WHO guidelines [8]. Each patient was carefully examined to rule out other causes of infertility and a detailed medical, clinical, reproductive and family history was taken on a pre-designed performa. Peripheral blood cultures were set up for chromosomal analysis and 5 well spread G banded metaphases were karyotyped using the standard protocols. The blood levels of follicular stimulating hormone (FSH) and testosterone (T) were determined. Wherever possible testicular cytopathological details following testicular fine needle aspiration cytology (FNAC) were observed.

2.2 Polymerase chain reaction (PCR) Analysis

In 70 cytogenetically normal infertile men, PCR screening was done for AZF microdeletions. Peripheral blood sample was collected and DNA was isolated using standard protocols. Patients were examined for 6 AZF loci at intervals 5 and 6 of the Y chromosome. The STS primers used were: sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc). SRY (Sex determining region of the Y) -sY14 was used as the control and fertile male and female samples as positive and negative controls. This primer set used was as suggested by Simoni et al [9] and is prescribed by the European Academy of Andrology. It enables the detection of over 90 % deletions in the AZF loci and allows for minimal standardization and comparison of the data from different laboratories. Samples were subjected to PCR amplification using 35 cycles at 95 for 1 minute, 56 for 30 seconds and 72 for 30 seconds. Initial denaturation was done for 5 minutes at 95 and a final extension time of 7 minutes at 72 was given. STS was considered absent only after at least 3 amplification failures in the presence of successful amplification of the control (SRY-sY14).

The PCR products were analysed on a 1.8 % agarose gel containing ethidium bromide (0.5 mg/mL).

3 Results

Of the 122 infertile men, 106 were azoospermic and 16, oligozoospermic. Twenty-nine men had chromosomal abnormalities and were not further analyzed at the molecular level. These cases were Klinefelter Syndrome 47, XXY (n=11), mosaic Klinefelter (n=9), Klinefelter variants (n=5), 46, XX male (n=1), 46, XY(80 %)/47,XYY(20 %) (n=1), 46, XX(90 %)/46, XY(10 %) (n=1) and 46, XYq- (n=1). PCR analysis was done in 70 cytogenetically normal infertile patients and 25 fertile controls.

PCR amplification produced a band of expected size for all the 7 loci investigated in 62 patients and 25 fertile controls. Eight patients (11.4 %), 4 with idiopathic infertility and 4 non-idiopathic infertility, showed a deletion of one or more STS, namely sY84, sY86, sY127, sY134 (patients 1, 2), sY84, sY86, sY127 (patient 3), sY127, sY134 (patient 4) and sY254, sY255 (patients 5~8). All deletions were interstitial. PCR amplification performed on the fathers of patients 4 and 5 did not show microdeletions and thus they were de novo. The fathers of the other six patients with microdeletion were not available. It can be summarized that patients 1 and 2 had complete deletion of AZFa and AZFb loci, patient 3 had complete AZFa deletion and partial deletion of AZFb and patient 4 had AZFb deletion alone. In cases 5-8 there were deletions of AZFc loci. Seven of these 8 patients (patients 1~5, 7 and 8) were azoospermic and patient 6 was oligoasthenoteratozoospermic with a total sperm count of 1.5 million/mL, which declined to 0.2 million/mL after one year. Patients 1~3 with AZFa and AZFb microdeletions had SCO. Patients 6 and 8 with AZFc deletion showed hypospermatogenesis and patient 7 with AZFc deletion had maturation arrest at the secondary spermatocyte stage. Patients 4 and 5 with AZFb and AZFc deletions, respectively, were cryptorchid and thus FNAC was not possible. Patients 3 and 8 had left sided varicocele and deletions of AZFa + partial AZFb and AZFc. The mean FSH value in these 8 patients was 30.4 mIU/mL (normal 1.2 mIU/mL~5 mIU/mL). Clinical details of these eight patients with microdeletions are shown in Table 1.

Table 1. Phenotypic features in men with AZF deletion.

4 Discussion

Y chromosome deletions are emerging as the prevalent cause of male factor infertility. The frequency of Y chromosome deletion increases with the severity of spermatogenic defect [10,11]. About 15 % azoospermic and 5 %~10 % oligozoospermic men show Y chromosome deletions, which, however, cannot be predicted cytogenetically or on the basis of clinical findings or semen analysis. Thus PCR-based AZF screening for Yq microdeletions is a compulsory preliminary step in management of cases with severe testiculopthy.

In recent years several combined clinical and molecular studies have sought to define recurrently deleted region on the long arm of Y chromosome in infertile males. Efforts have also been made to determine the incidence of Yq microdeletions and to correlate the size and position of the deletion with the infertile phenotype [12]. Recent studies have shown a marked variation in the deletion frequency, which may be due to the selection of different patient groups and the use of different marker sets.

Vogt et al [7] correlated the position of the AZF deletion with the phase in which spermatogenesis was arrested. Each AZF locus acts at a different phase of spermatogenesis and deletion of each causes spermatogenic arrest at a particular stage. AZFa deletion was associated with SCO, AZFb deletion with spermatogenic arrest at the pachytene stage and AZFc deletion, with spermatogenic arrest at the spermatid stage, but it can also be associated with hypospermatogenesis or maturation arrest. Patients 1 and 2 had AZFa + AZFb deletion and patient 3 had AZFa + partial AZFb deletion and all showed SCO. Patient 5~8 had AZFc deletion; patients 6 and 8 showed hypospermatogenesis and patient 7, maturation arrest at the secondary spermatocyte stage. Thus in patients with AZFc deletion the testicular phenotype may vary [7,13,14]. Vogel and associates [14] reported that the heterogenous phenotype observed in AZF deletions may be due to the effect of environment, expression of various modifying genes and variable penetrance. Thus several factors, genetic, epigenetic and environ-mental, influence the testicular phenotype in men with AZF deletions. Patient 6 showed a decline in sperm count from 1.5 million/mL to 0.2 million/mL over a period of one year. Similar decline in semen quality has also been reported in a patient with AZFc deletion [15]. No AZF microdeletion was observed in 25 fertile controls, indicating that Yq microdeletions are only observed in men with azoospermia and oligozoospermia.

Two cryptorchid patients (4 and 5) showed AZFb and AZFc microdeletions, respectively. Cryptorchidism represents one of the commonest congenital anomalies with a prevalence of 4 %~5 % at birth but by one year the prevalence decreases to 1 %. Cryptorchidism is associated with impaired spermatogenesis [16] and increased incidence of testicular cancer [17]. In cryptorchid patients with AZF microdeletion the spermatogenic impairment may be more serious due to the additive effect of a high abdominal temperature. Elevation of temperature by 1 causes a depression of spermatogenesis by 14 % [13]. This preliminary findings show that AZF microdeletions may be responsible for severe testicular damage and may be phenotypically expressed by undescended testis and infertility [16]. Foresta et al [17] reported Yq microdeletions in the AZF region in 27.5 % of cryptorchid and 25.4 % of idiopathic infertile males. They proposed that in cryptorchid with AZF deletion, the testis failed to descend due to altered response to the mechanism regulating testicular descent. The present data show that AZFb and AZFc deletion may lead to not only spermatogenic arrest but also impaired testicular descent. In cryptorchid men without Yq microdeletion, orchidopexy at an early age results in improvement in semen quality but in cryptorchid with Yq microdeletion surgical intervention does not help [16]. AZF microdeletion may lead to partial or complete spermatogenic arrest and cryptorchidism may further worsen this condition and cause deterioration in semen quality; hence the knowledge of AZF deletions in cryptorchid cases is important to understand the prognosis [18].

Varicocele is pathological dilation of the pampiniform plexus of veins and its association with male infertility is well documented. In the present study there were two left varicocele cases and both (patients 3 and 8) were azoospermic. Patient 3 (SCO) had AZFa and partial AZFb deletion (STS deleted sY84, sY86, sY127) and patient 8 (hypospermatogenesis), AZFc deletion. To the best of our knowledge only one or two studies [19, 20] have shown the association of Yq microdeletions in men with varicocele. The prevalence of Yq microdeletion in patients with varicocele is similar to that in idiopathic severe testiculopathy [20]. Varicocele per se may not lead to spermatogenic arrest, but the latter may be present when AZF microdeletion coexists as shown by the present study. The finding of a genetic etiology in infertile men with varicocele and cryptorchidism suggests that in such patients Yq microdeletion screening should be performed both for a proper diagnosis and for avoiding unnecessary treatment that will not improve the spermatogenic status [19, 21]. The diagnosis of Yq micro-deletion in men with varicocele or cryptorchidism is also important to understand the possibility of transmitting the genetic etiology to male offspring in assisted reproduction practice [13, 22].

Patient 6 (hypospermatogenesis) with AZFc deletion showed a decline in total sperm concentration from 1.6 to 0.2 million/mL, a phenomenon previously reported by Chang et al [13]. As the testicular phenotype deteriorates with time the patient was advised to undergo sperm cryopreservation for assisted reproduction.

All the cases with AZF microdeletion had a normal karyotype thus proving the importance of PCR to analyse the infertile cases. These microdeletions may cause deregulation of gene expression by position effect [10], but it is postulated that the deletion may result in absence of genes critical for spermatogenesis. The Y chromosome has the highest spontaneous loss of genetic material in the human genome. This genetic instability is due to the presence of highly repetitive segments, the long and short interspersed repeats and also because a large portion of the Y chromosome (95 %) does not undergo recombination during meiosis. The incidence of microdeletions varies from 1 to more than 55 % in different studies. These differences may be due to the inclusion of different groups of subjects and the use of different STS primers. They may be related to genuine population variances, particular Y chromosome haplo-types, genetic background or environmental influences. In the present study only men with oligozoospermia and azoospermia were considered and the STS primers prescribed by the European Academy of Andrology were used.

AZF microdeletions were detected in 4 men with idiopathic infertility, 2 cryptorchid men and 2 varicocele men. Thus 11.4 % infertile men showed microdeletion of the AZF loci. This figure is similar to that reported in Italian, French and Danish populations, suggesting that world wide incidence of Y microdeletions is likely to be similar, if identical clinical criteria and similar marker set are used.

In conclusion, AZF microdeletion analysis in both idiopathic and non-idiopathic infertile cases with varicocele or cryptorchidism is a compulsory step to precisely define the etiology of infertility and to appropriately manage these patients. It also helps to determine the frequency and site of gene deletion and thus determine the testicular phenotype. Also the diagnosis of AZF microdeletion should be offered to the couples undergoing assisted reproduction, since it is possible to transmit the genetic anomaly to the male offspring.

Acknowledgement

The authors would like to thank Dr Ken McElreavey, Pasteur Institute, Paris, for his invaluable guidance and support throughout the present study. This work was supported by Indian Council of Medical Research (54/1/98), New Delhi.

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Correspondence to: Professor K Kucheria, Department of Anatomy and Genetics, All India Institute of Medical Sciences, New Delhi110029, India.

Tel: +91-11-659 3489,   Fax: +91-11-686 2663

Email: kkucheria@hotmail.com

Received 2002-07-05    Accepted 2002-11-18