Chinese Medical Journal 2005;118(17):1462-1467
Screening for Y chromosome microdeletions in idiopathic and nonidiopathic infertile men with varicocele and cryptorchidism

SONG Ning-hong,  WU Hong-fei,  ZHANG Wei,  ZHUO Zuo-min,  QIAN Li-xing,  HUA Li-xing,  GUO Lin,  FENG Ning-han

SONG Ning-hong (Department of Urology, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

WU Hong-fei (Department of Urology, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

ZHANG Wei (Department of Urology, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

ZHUO Zuo-min (Key Laboratory of Reproductive Medicine of Jiangsu Province, Nanjing Medical University, Nanjing 210029, China)

QIAN Li-xing (Department of Urology, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

HUA Li-xing (Department of Urology, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

GUO Lin (Model Animal Research Center, Nanjing University, Nanjing 210032, China)

FENG Ning-han (Department of Urology, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China)

Correspondence to:WU Hong-fei,Department of Urology, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China (Tel: 86-25-83718836-6603. Fax:.
Y chromosome;microdeletions;multiplex polymerase chain reaction;male infertility
Background Cytogenetic and molecular studies of azoospermic and oligozoospermic males have suggested the presence of azoospermia factors (AZF) in the Y chromosome. Deletion in AZF regions has been reported to disrupt spermatogenesis and cause infertility. Several candidate genes responsible for spermatogenesis have been identified in this region and some of them are thought to be functional in human spermatogenesis. And we reported clinical and molecular studies of Y chromosome microdeletions in Chinese. This study aimed at assessing the frequency of microdeletions in Chinese men with idiopathic and nonidiopathic infertility problems and dicussing the clinical significance of the AZF region.
Methods In this study, we screened 143 infertile men (62 with idiopathic infertilitas and 81 with nonidiopathic infertilitas), in whom karyotype, sperm count, hormonal parameters and fine needle aspiration cytology were evaluated. Genomic DNA was extracted from the peripheral leukocytes. Molecular analysis was performed by two multiplex polymerase chain reactions (PCR) using a set of a sequence tagged sites (STS) from 3 different regions of the Y chromosome: AZFa (sY84, sY86), AZFb (sY127, sY134), AZFc (sY254, sY255).
Results Nineteen point four percent of idiopathic males (12/62, 19.4%) had microdeletions of either the AZFa, AZFb, AZFc or AZFb+c region. Significantly, a high frequency of microdeletions (9/81, 11.1%) was found in nonidiopathic patients with varicocele and cryptorchidism. No deletions were found in healthy fertile men. There were no significant differences in the localization and extent of deletions between idiopathic and nonidiopathic patients.
Conclusions The knowledge of the presence of these deletions in idiopathic and nonidiopathic cases is important to understand the prognosis, better management and counsel these patients accordingly. Furthermore, a more extended screening for Y chromosome microdeletions in idiopathic and nonidiopathic men, particularly candidates for intracytoplasmic sperm injection, is recommended.
It has been estimated that over 10% of couples worldwide suffer from infertility and in approximately half of these cases are due to defective spermatogenesis. Spermatogenesis occurs by successive mitotic, meiotic and postmeiotic processes. Genes that are expressed during spermatogenesis encode proteins necessary for specific stages as well as for maintaining the general housekeeping functions of the cells involved. The Y chromosome comprises 2% of the genome and consists of a short (Yp) and long arm (Yq). Since 1970 it has been established that Human Y chromosome deletions are possibly related to male infertility. Chromosome banding indicated that these deletions were located in Yq11. This region was defined as the azoospermic factor (AZF). The sequence tagged sites (STS)/multiplex polymerase chain reaction (PCR) mapping approach facilitated detailed studies spanning the Yq11 region and the screening of genomic DNA samples from a wide range of infertile men. Analysis of these microdeletions resulted in the identification of three loci in Yq11 involved in the control of spermatogenesis, corresponding to three nonoverlapping regions: AZFa, AZFb, AZFc. Some authors[1] have recently proposed the existence of a fourth region, AZFd, in close proximity to AZFc. Y chromosome microdeletions are now recognized as the most important genetic etiology of idiopathic infertility.

This study aimed at assessing the frequency of microdeletions in Chinese men with idiopathic and nonidiopathic infertility with varicocele and cryptorchidism and discussing the clinical significance of the AZF region.


One hundred and forty-three azoospermia and severe oligozoospermia [<5×10(6) sperm/ml] were included in this study. We studied 40 age-matched healthy fertile men. Of the 143 subjects, 36 had a history of unilateral cryptorchidism orchidopexied in childhood, 45 underwent varicocelectomy because of varicocele, and 62 were classified as idiopathic patients. Semen samples were obtained on three different occasions, separated by a 3-week interval, following a 3-day period of sexual abstinence, and complete semen analyses were performed according to WHO guidelines. Plasma concentrations of follicle-stimulating hormone (FSH) and testosterone were determined by radio immunoassay (RIA). All selected patients underwent ultrasound scanning of the testes to evaluate testicular size. In all selected patients, idiopathic patients were studied with a comprehensive history and general investigation to exclude other possible cause of testicular damage, and varicocele or cryptorchidism was not associated with other phenotypic anomalies. The testicular structure of infertile men was analyzed by means of bilateral fine needle aspiration cytology (FNAC). The cellular material was examined under a light microscope and at least 200 spermatogenic cells were counted per smear. Spermatogenic cells were expressed as relative percentages, while the interposed Sertoli cells were expressed as the Sertoli index (SEI, the number of Sertoli cells/100 spermatogenic cells), which was found to be a reliable index of the tubular germ potential. ① Sertoli cell-only syndrome (SCOS): only Sertoli cells, but no germ cells are visible in all seminiferous tubules of testis tissue sections. ② severe hypospermatogenesis (SH): hyposper-matogenesis is characterized by a reduction in the absolute number of germ cells with respect to Sertoli cells, and it is distinguished as severe hypospermatogenesis on the basis of the Sertoli index (SEI>300, with normal values<50). ③ Maturation arrest (MA): arrest at different stage of spermatogenesis (Spermatogonia, primary sper-matocytes, secondary spermatocytes, early and late spermatids) is observed in the testis tissue of patients.

Genomic DNA was prepared from peripheral blood samples of the infertile and fertile Chinese men, after informed consent had been obtained, in order to screen for Y chromosome deletions by PCR. DNAs were stored at 4℃. The French society for human genetics (SFGH) edited practical recommendations for Y-chromosome microdeletion detection. The analysis of these recommendations was sufficient (>90%) for the deletions. The test should be accurate, reliable, easy to perform, reproducible, fast and inexpensive. Our own technique from SFGH recommendations:[2] for a first screening, eight STS were analysed in two multiplex PCR: sY84 and sY86 for AZFa (Shanghai Sangon Biological Engineering Technology Company, China), sY127 and sY134 for AZFb(Shanghai Sangon Biological Engineering Technology Company, China), sY254 and sY255 for AZFc (DaLian TaKaRa Biotechnology Company, China). The two multiplex PCR consisted of: Mix1: SRY (sY14)-ZFY-sY84-sY134-sY255 and Mix2: SRY (sY14)-ZFY-sY86 -sY127-sY254. As internal controls, SRY and ZFY were included in both PCR multiplexes. Several external controls are used for each PCR reaction: blank; female DNA; and fertile male DNA. Primers for PCR amplification are made according to the sequence reported by Sargin CF et al[3] and conditions of the PCR reaction are displayed in Table 1. PCR was performed in a 50 μl reaction volume containing 1.5 mmol/L MgCl2, 10 mmol/L Tris-HCl pH 8.0, 50 mmol/L KCl, 200 μmol/L each deoxyribonucleoside triphosphate, 1 U Taq DNA polymerase (Promega Company, USA), and 20 μmol/L of each primer, sterile distilled water 50 μl. After a preheating step at 95℃ for 5 minutes, the PCR was performed with denaturing at 95℃ for 1 minute, annealing at 56℃ for 30 seconds, and primer extension at 72℃ for 30 seconds, in 35 cycles, with final incubation at 72℃ for 5 minutes. Amplified products were separated on 1.8% agarose gels(Sino-America Biotechnology Company, China). The experiments were done in triplicate. A STS was considered as absent only after 3 amplification failures in the presence of successful amplification of internal control. To decrease the risk of contamination, a female technician performed all experiments.

Statistical analysis
Fisher’s exact text was used to test the prevalence of Y chromosome microdeletions in three groups (idiopathic infertility, nonidiopathic infertility and control group). Computations were performed with the STATA statistical package version 7.0. P values less than 0.05 were considered statistically significant.


Y-Chromosome microdeletions analysis
Successful amplification of SRY and ZFY gene in patients confirmed good quality of DNA used in PCR reactions. Among normal karyotype infertile men, we found 21 submicroscopic deletions (21/143, 14.7%). PCR analysis in 40 normal fertile men did not detect any abnormalities, while no amplification was observed in women and blank. PCR analysis with this set of Y-DNA markers showed deletions of portions of Yq in 12 of the 62 idiopathic patients (12/62, 19.4%), and in 9 out of the 81 nonidiopathic patients (9/81, 11.1%). The results of the screening are shown in Table 2 and Fig. A and B . One patient (subject 5) had a microdeletions in the AZFa region, with sY84, sY86 (1/21, 4.8%); 2 patients (subjects 16 and 63) presented with a large deletion involving sY127, sY143 from AZFb and sY254, sY255 from AZFc (2/21, 9.5%); 2 deletions (subjects 80 and 82) were located in a AZFb region (2/21, 9.5%); 16 patients (subjects 9, 11, 23, 27, 43, 49, 51, 57, 69, 75, 77, 84, 93, 96, 116 and 123) had a deletion in the AZFc region involving the DAZ(deleted in azoospermia)gene (16/21, 76.2%).

Y-Chromosome polymorphisms and de novo deletions
None of the patients mentioned familial infertility. In the majority of cases in this study, male relatives of the AZF-deleted individuals were unavailable for study, and hence, we did not know whether the deletions were inherited. However, all deletions were large in both idiopathic and nonidiopathic patients and hence were unlikely to represent polymorphisms. Fortunately, four male relatives were available for study, and no Yq deletions were found in the father or brothers of 4 patients (11, 49, 57 and 82); hence, Yq deletions were de novo.

Hormonal data of patients with microdeletions
Testosterone levels were within the normal range [(7.4-52.4) IU/L] in the 21 patients with deletions, the mean being (33.3±1.5) IU/L. Of 21 patients, 12 had high FSH plasma concentrations [>12.0 IU/L, normal range (0.7-11.1) IU/L]; however, the mean FSH level among patients with deletions [(12.3±4.3) IU/L] did not differ significantly from the mean FSH level in patients without deletion [(12.1±9.1) IU/L].

Testicular histology of patients with micro-deletions
Our results of testis biopsy samples from men with AZF deletions pointed to wide histological variations: severe hypospermatogenesis, complete testicular maturation arrest, and sertoli cell-only syndrome have been found. Among 21 infertile men, four showed a testicular cytologic picture of MA; six patients had SH; and 11 cases presented SCOS.

Comparison of the frequency of Y chromosome microdeletions
Incidence of Y chromosome microdeletions involved in idiopathic, nonidiopathic and control group was 19.35% (12/62), 11.11% (9/81) and 0.00% (0/40), respectively. There was no significant difference of microdeletion incidences between idiopathic and nonidiopathic groups (P=0.233), but there was significant difference between idiopathic or nonidiopathic with control groups (P=0.003 and P=0.029, respectively).


The Y chromosome microdeletions are the most common genetic causes of male infertility due to spermatogenesis failure and have been reported in 1%-55% of infertile men.[4] In general, about 15% azoospermic and 5%-15% oligozoospermics men show Y chromosome microdeletions. Semen analysis, clinical findings or cytogenetic analysis alone can not help in the prediction of Y chromosome deletions. Thus, PCR based on Y chromosome screening is becoming necessary both for providing accurate diagnosis as well as for proper management of the patient clinically and for counseling. In this study, we performed a multiplex PCR diagnostic analysis for AZF deletions.

Idiopathic infertility is the most common cause of male infertility. In this study, we analyzed genomic DNA from 62 patients with idiopathic infertility.Yq11 microdeletions were found in 19.4% (12 of 62) of these patients. The testicular histology of the patients varied from SCOS to maturation arrest. In these patients, isolated microdeletion of the AZFa region is relatively rare, whereas deletion of the AZFc region occurs more commonly, either in isolation or in conjunction with deletion of the AZFb. The prevalence of Yq11 microdeletions in our patients was within the range of 3%-37.5%, as reported in previous studies.[5] This confirms that Yq abnormalities are frequently associated with infertility in men diagnosed as having idiopathic infertility.

The main objective of this study was to ascertain whether there was an association between the occurrence of Y chromosome microdeletions and nonidiopathic infertility. Among the 81 nonidiopathic cases of infertility, 9 (11%) microdeletions were detected, one in AZFa (subject 5), one in AZFb+c (subject 16), and seven in AZFc (subjects 23, 49, 51, 57, 84, 93 and 123). There was a significant difference between nonidiopathic group and the control, thus suggesting that such associations exist. Varicocele is a dilatation of the panpiniform plexus that occurs at puberty. It is frequently associated with azoospermia and severe oligozoospermia. In our experiment, deletions of Yq was observed in four patients (subjects 5, 16, 23 and 57) affected by left varicocele and azoospermia by bilateral SCOS/MA. Similarly to our study, a research[6] proposed that microdeletions in the Y chromosome may be responsible for the bilateral severe testicular damage observed in these cases and that varicocele may worsen the sperm production or represent an associated finding without pathogenic role or even a phenotypic effect of the deletion itself. It was recently reported that men with microdeletions and varicoceles did not respond to varicocelectomy.[7] The finding of a genetic aetiology in infertile men with varicocele suggests that in such patients a Yq microdeletion screening should be performed for a proper diagnosis and avoiding unnecessary treatments (varicocelectomy) that will not probably improve the sperm count. Cyptorchidism represents one of the most frequent congenital anomalies. Cyptorchidism means “hidden testis” and is used to describe an undescended testis. The incidence of cryptorchidism in full-term infants is approximately 3%, whereas it may be as high as 33% in males born prematurely. Documented consequences of cryptorchidism included increasing risk for development of testicular cancer and male infertility.[8] However, it is still disputable whether the genetic factor that controls testicular descent, also directs spermatogenesis. Recently, a controlled clinical study[9] found that no causal relation exists between Y chromosome microdeletions and cryptorchidism. On the contrary, a prospective study[10] reported a similar frequency of AZF deletions both in idiopathic (13.3%) and cryptorchid men (11.6%). Subjects 49, 51, 84, 93 and 123 in our study supported a role for genetic causes. One cryptorchid patient (subject 49) showed de novo AZFc microdeletions. It was possible that the fathers were mosaic for these Yq deletions and harboured the Yq deletion only in the germ cell line whereas the peripheral blood had normal Y chromosome and thus the deletions were transmitted to the sons but were not picked up by PCR microdeletion analysis. In addition, our results showed that AZF deletions in patients with unilateral cryptorchidism were associated with bilateral testicular damage. Some authors[11-13] concluded that Yq microdeletions might lead to oligozoospermia/azoospermia, and might also lead to cryptorchidism. This may be due to the altered testicular response to mechanism regulating testicular descent. Knowledge of these deletions is necessary to determine the prognosis in these cases after orchidopexy.

With the development of artificial reproductive technology, microdeletions of the Y chromosome will be transmitted to male offspring, and these sons will be at risk of having the same infertility problem. Actually, some studies[14,15] demonstrated that microdeletions were indeed transmitted to male offspring via intracytoplasmic sperm injection (ICSI). So, detection of Yq microdeletions may play an important role in the study of male infertility. ① The study of Yq microdeletions will be helpful in the development of better diagnostic methods and the expansion of the current knowledge of spermatogenesis. There may be other genetic factors or unknown spermatogenic gene defects causing spermatogenesis impairment, such as the DAZLA gene.[16] ② A molecular analysis for AZF DNA deletions in the Y chromosome of azoospermic men is an attractive prognostic tool for finding mature sperms in the patient’s testis tissue without the need of testicular gene expression analyses. Since it has been known that patients with a complete AZFa or AZFb deletion usually suffer from a complete absence of germ cells (AZFa) or a complete absence of postmeiotic germ cells (AZFb), clinical testicular sperm extractions (TESE) for ICSI treatment are not recommended for these patient groups, because these are not successful.③ An inexpensive Y microdeletion screening test would be helpful in the management of varicocele, especially in those cases without testicular failure at the time of study. In cases of subclinical or grade one varicocele in young men, there is not always a clear indication for varicocelectomy.[17] ④ Our findings that Yq deletions can occur in 19.4% of males diagnosed as having idiopathic infertility and, significantly, in 11.1% of nonidiopathic infertile males raise important questions concerning the screening of Y deletions and genetic counseling. Current protocols[18] recommend that men with idiopathic severe oligozoospermia or nonobstructive azoospermia be screened for Y chromosome deletions. On the basis of our data, we recommend a more extended screening program to include all men, idiopathic and nonidiopathic, seeking ICSI treatment.


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  1. “135” Foundation of Jiangsu Province,No. 0151;