Study On Y Chromosome Microdeletions In Patients With Severe Oligozoospermia And Azoospermia

Background:Idiopathic male infertility is a kind of disease that is involved in complicated genetic factors. The cause of these patients,in approximately half ,is defective spermatogenesis.In recent years,there have been intensive studies on the genetic causes underlying male infertility and several genetic etiology were found,such as numeral and structural abnormality of chromosome,microdeletion of azoospermia factor(AZF) on the Y chromosome and mutations of some known genes.After other obvious urological reasons and klinefelter syndrome have been ruled out,particular attention had been given to the Yq11 region.1976, Tiepolo and zuffardi first reported the presense of azoospermia factors(AZF) in the human Y chromosome.Through the use of molecular markers that have been mapped at density on the Ychromosome,numerous deletion-mapping studies have defined at least three distinct non-overlapping intervals each associated with variable degrees of spermatogenic impairment.Three regions located on the long arm of the Y chromosome were named azoospermia factor(AZF)-a,AZFb,AZFc . Kent-First have recently proposed the existence of a fourth region,AZFd,in closely proximity to AZFc.Several candidate genes responsible for spermatogenesis have been identified in these regions and some of them are thought to be functional in human spermatogenesis.With the advances in assisted 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. This possibility should be conveyed to infertile couples with an explanation of the analysis of microdeletion of the Y chromosome.Object:This study aimed at assessing the frequency of Y chromosome microdeletions and the frequency of the sites deletions in Chinese men with idiopathic and nonidiopathic infertility with varicocele and cryptorchidism and discussing the clinical significance of the AZF region.Material and Method:135 azoospermia and severe oligozoospermia patients were included in this study.We studied 60 age-matched healthy fertile men.Of the 135 subjects,one had a history of unilateral cryptorchidism orchidopexied, 37 underwent varicocelectomy because of varicocele,and 87 were classified as idiopathic patients.All patients above are have karyotype 46,XY.Complete semen analyses were performed according to WHO guidelines.Plasma concentrations of hormone were determined by radio immunoassay(RIA).All selected patients underwented 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.Genomic DNA was prepared from peripheral blood samles 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 -20℃.11 STSs were analysed in multiplex PCR: sY84 and sY86 for AZFa,sY127/sY134 and sY143 for AZFb,sY157/sY254 and sY255 for AZFc,sY152 for AZFd.As internal controls,SRY and ZFX/Y were included in PCR multiplexes.Several external controls are used for each PCR reaction:blank;female DNA;and fertile male DNA. The experiments were done in triplicate.A STS was condidered as absent only after 3 amplification failures in the presence of successful amplification of internal control.Result:1. Y-Chromosome microdeletions analysis with the normal karyotype:(1) Successful amplification of SRY and ZFX/Y gene in patients confirmed good quality of DNA used in PCR reactions.Among normal karyotype infertile men,we found 15 microdeletions (15/125,12.00%). PCR analysis in 60 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 87 idiopathic patients (12/87,13.79%),And in 3 out of the 38 nonidiopathic patients (3/38,8.11%).Among the idiopathic patients,one patient had microdeletions in the AZFb+c+d region (1/15,6.67%); 4 patients presented with deletionsAZFc+d region; 7 patients had deletion in the AZFc region involving the DAZ gene (7/15,46.67%). Three deletions were in nonidiopathic patients with varicocele.one had microdeletions in the AZFb+c region (1/15);Two patients had deletions in the AZFc+d region (2/15).(2) Compmarison of the frequency of Y chromosome microdoeletions: Incidence of Y chromosome microdeletions involved in idiopathic, nonidiopathic and control group was 13.79%(12/87), 10.42%(5/48) and 0.00%(0/60),respectively.There was no significant difference of microdeletion incidences between idiopathic and nonidiopathic groups,but there was significant difference between idiopathic or nonidiopathic with control groups. (3)The deletions of STS in the 15 patients with Y chromosome microdeletions: The sY84 and sY86 of AZFa region,sY134 of AZFb region were not found deletions in the patients;The deletions of sY254, sY255 account the mostly proportion of all patients. 2. microdeletions analysis with the abnormal karyotype: Among 10 patients with abnormal karyotype,we found 2 patients had Y chromosome microdeletions (2/10,20.00%).Conclusions:1. Y chromosome microdeletion is one of the major causes of dyszooospermia. To screen for Y chromosome deletions to patients with idiopathic severe oligozoospermia or nonobstructive azoospermia is important.2. DAZ gene are one of the important candidates for azoospermia factor (AZF).Microdeletion on DAZ is a major cause of azoospermia and severe oligozoospermia leading to male infertility.3. On the basis of our data,we recommend a more extended screening program to idiopathic and nonidiopathic, especially seeking ICSI treatment.4. Chromosome karyotype analysis is essential to patients with bnormal fertile;To find the exactly causes ,it is important to screen for Y chromosome deletions to patients with sex chromosomal abnormality.