Genomic integrity of the Y chromosome sequence-taggedsites in infertile and Down syndrome Jordanian males

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1 ORIGINAL ARTICLE Genomic integrity of the Y chromosome sequence-taggedsites in infertile and Down syndrome Jordanian males S. R. Yasin, L. H. Tahtamouni, N. S. Najeeb, N. M. Issa, Z. A. Al Mazaydeh & A. A. Alfaouri Department of Biology and Biotechnology, Faculty of Science, The Hashemite University, Zarqa, Jordan Keywords Azoospermia factor Jordan male infertility multiplex PCR spermatogenesis Correspondence Lubna H. Tahtamouni, Department of Biology and Biotechnology, Faculty of Science, The Hashemite University, P.O. Box , Zarqa 13133, Jordan. Tel.: (5) ; Fax: (5) ; lubnatahtamuni@hu.edu.jo These authors contributed equally to the work. Accepted: June 5, 2013 doi: /and Summary The long arm of the Y chromosome contains nonoverlapping regions termed azoospermia factor (AZF) with great influence on male fertility. Microdeletions at these regions minimise the males ability to father offsprings. In this preliminary study, we attempted to screen the presence or absence of twenty Y chromosome s sequence-tagged sites (STS) associated with fertility in infertile and Down syndrome (DS) males. Genomic DNA from 35 fertile, 74 infertile and 22 karyotyped DS males was extracted and amplified in multiplex polymerase chain reaction (PCR) containing 20 primer pairs that amplify Y-specific STS that cover functional regions associated with AZF and spermatogenesisrelated genes. Our results indicated the integrity of the Y chromosome at the 20 fertility markers for both the fertile and Down syndrome males. However, the results of the infertile males showed the presence of microdeletions at these Y-specific STS. Three samples showed Y chromosome microdeletion when blood and seminal fluid genomic DNA were assayed, while two samples showed microdeletion only when seminal fluid genomic DNA was assayed. The current study demonstrated that the molecular genetic aspect of infertility should be given proper attention when dealing with infertility cases. Furthermore, our results indicate the importance of genetic counselling in managing infertility cases. Introduction Infertility affects approximately 15% of all couples seeking to have children (Shaqalaih et al., 2009). Male factor is evident in about 40 50% of clinical infertility cases worldwide (Kantartzi et al., 2007) and in 35% of infertility cases in Jordan (Tahtamouni et al., 2002). A correlation between male infertility and the long arm of the Y chromosome has long been suggested (Tiepolo & Zuffardi, 1976); it was shown that deletions in the long arm of the Y chromosome (i.e. the nonoverlapping regions which are termed Azoospermia Factor, AZFa, b, and c) are associated with spermatogenic failure (Kent- First et al., 1999; Sun et al., 2000; Foresta et al., 2001; Wu et al., 2007; Poongothai et al., 2009). Each region contains multiple genes such as the Drosophila fats facets-related Y (DFFRY), the RNA-binding motif (RBM) and the deleted in azoospermia () which play a critical role in controlling spermatogenesis (Mulhall et al., 1997; Reynolds & Cooke, 2005; Dinic et al., 2007; Mohammed et al., 2007; Viswambharan et al., 2007). In this context, the genetic aetiologies of infertility in Down syndrome (DS) individuals are still under speculations. Down syndrome or trisomy 21 is an autosomal abnormality caused by the presence of an extra copy of chromosome 21 (Gardner & Sutherlan, 1996; Bianca, 2002). Trisomy 21 is one of the most common chromosomal abnormalities, occurring in about 1 in 700 live births (Pradhan et al., 2006), and it is the leading chromosomal defect in the United States (Puumala et al., 2007). As trisomy 21 is the only autosomal trisomy with postpubertal survival (Pradhan et al., 2006), the question of their fertility (or infertility) is almost often there (van Dyke et al., 1995; Selikowitz, 1997; Bianca, 2002). Speculations about their fertility impairment include the effect of the extra copy of chromosome 21 on spermatogenesis, testicular cancer, abnormal sexual hormones and ignorance in the sexual acts (Sheridan et al., 1989; Ichiyanagi et al., 1998; Pradhan et al., 2006). However, there are reports of normal pubertal development with normal penile length, testicular volume and testosterone level in many patients. To date, there are three reports of four Blackwell Verlag GmbH

2 S. R. Yasin et al. Y chromosome and male infertility in Jordan pregnancies that were fathered by patients with DS. The foetal chromosomal complement in the four occasions was normal (Pradhan et al., 2006). Mapping partial deletions in male-specific region of the human Y chromosome associated with male infertility is accomplished with Y-specific sequence-tagged sites (STS) assay (Vollrath et al., 1992; Henegariu et al., 1994; Vogt et al., 1996; Kent-First et al., 1999; Skaletsky et al., 2003). Each Y-specific STS assay provides a straightforward means of determining the presence or absence, in a sample of human genomic DNA, of a specific point along the length of the Y chromosome. In this preliminary study, we aimed at screening the presence or absence of twenty Y chromosome s STS associated with fertility in two categories: normal infertile males and DS males. Materials and methods Study population The study included 74 idiopathic infertile men presented for intracytoplasmic sperm injection (ICSI) at two assisted reproduction units in Amman, Jordan, during the period from 2010 to The selection was based on primary infertility and on the presence of sever oligozoospermia or oligozoospermia ejaculate according to World Health Organisation (WHO) criteria (WHO, 1999). Obstructive azoospermic patients were excluded. For comparative purpose, 35 phenotypically normal fertile men who previously fathered children and have normozoospermic ejaculate were selected as the control group. The selection was matched with sex, age and nationality of the patients. All subjects were of Jordanian origin. For the Down syndrome sample, 22 karyotyped Down syndrome males were included. All donors (or the care taker) gave an informed consent for the analysis of the blood and/or semen samples, and the study was approved by the Hashemite University ethical committee. Semen analysis Semen samples from the fertile and infertile men were collected by masturbation into sterile jars after h of sexual abstinence. Semen analysis for the 109 men attending the infertility centre was performed according to the method described in the World Health Organisation laboratory manual (1999). Molecular study Genomic DNA was extracted from seminal fluid samples of fertile and infertile men (Sambrook & Russell, 2001) and whole blood of all groups using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). Extracted genomic DNA for each sample was amplified in a multiplex polymerase chain reaction (PCR) containing twenty primer pairs (Y Chromosome Deletion Detection System; Promega). The system amplifies twenty Y chromosome s STS that cover various key functional regions associated with the AZF and key spermatogenesis related genes namely, AZFa (DYS148 [SY86] and DYS273 [SY84]); AZFb (DYS212 [SY121], SMCY [SYPR3], DYS215 [SY124], DYS 218 [SY127], DYS219 [SY128], DYS221 [SY130], DYS 223 [SY133], DYS224 [SY134]); AZFb/AZFc (DYF51S1 [SY145], DYS236 [SY152]); AZFc ( [SY242], [SY208], [SY254], [SY255]); the gene ( [SY14]) of the short arm of the Y chromosome, which is an indicative of the testis-determining factor; DYS271 (SY81); KAL-Y (SY182); and genetic markers (Vollrath et al., 1992; Henegariu et al., 1994; Vogt et al., 1996). For each sample, a duplicate analysis of the genomic DNA was performed. The haploid allele-respective PCR product was analysed on a 3% agarose gel (NuSieve 3 : 1; FMC Bioproducts, Rockland, ME, USA) containing 0.5 lg ml 1 ethidium bromide. The absence of an expected PCR product was marked as a deletion in the Y chromosome of the respective allele or STS in the presence of amplification of two internal controls (SMCX and ZFX/ZFY). Results Seminal fluid analyses for the 109 men attending the infertility centre were performed. According to WHO criteria (1999), 35 men were considered fertile as they had normal semen, and the other 74 men were considered infertile based on their seminal fluid analyses (Table 1). The fertile and infertile groups did not differ in the volume and ph of the semen (P > 0.05). On the other hand, the two groups differed significantly in terms of sperm concentration, percentage of motile sperm and normal sperm morphology (P 0.05; Table 1). The mean age of fertile and infertile men was and years respectively. The cause of male infertility was diagnosed as idiopathic with duration of infertility of years. Table 1 Seminal fluid analysis. Values are expressed as means SEM Parameter Fertile n = 35 Infertile n = 74 P-value Volume (ml) >0.05 ph Concentration (10 6 ml 1 ) Progressive motile spermatozoa (%) Normal sperm forms (%) Blackwell Verlag GmbH 771

3 Y chromosome and male infertility in Jordan S. R. Yasin et al. Blood and/or seminal fluid genomic DNA samples of fertile, infertile and Down syndrome males were assayed using a multiplex PCR for the presence or absence of microdeletions in twenty Y chromosome STS associated with infertility in human males. Both the fertile and Down syndrome individuals showed no microdeletions when assayed for STS microdeletions (Tables 2 and 3). Of the 74 infertile males, five samples (6.8%) showed Y chromosome microdeletions when blood and/or seminal fluid genomic DNA were assayed. The five infertile males suffered from severe oligozoospermia or oligozoospermia, the ejaculate of four of them was asthenozoospermic (Table 4). The PCR amplification results of blood genomic DNA of three of these infertile males (samples Table 2 Schematic representation of the Y chromosome. The table below shows the results of blood genomic DNA of fertile, Down syndrome and 10 selected infertile males assayed for the presence (+) or absence ( ) of each STS AZFa AZFb AZFc STS SY14 SY 81 SY86 SY84 SY182 SY121 SYPR3 SY124 SY127 SY128 SY130 SY133 SY134 SY145 SY152 SY242 SY208 SY254 SY255 SY157 Fertile Locus DYS271 DYS148 DYS273 KAL-Y DYS212 SMCY DYS215 DYS218 DYS219 DYS221 DYS223 DYS224 DYF51S1 DYS236 DYS Infertile Patient No. Down Syn Blackwell Verlag GmbH

4 S. R. Yasin et al. Y chromosome and male infertility in Jordan Table 3 Results of seminal fluid genomic DNA of fertile and 10 selected infertile males assayed for the presence (+) or absence ( ) of each STS AZFa AZFb AZFc STS SY14 SY 81 SY86 SY84 SY182 SY121 SYPR3 SY124 SY127 SY128 SY130 SY133 SY134 SY145 SY152 SY242 SY208 SY254 SY255 SY157 Fertile Locus Infertile Patient No. DYS271 DYS148 DYS273 KAL-Y DYS212 SMCY DYS215 DYS218 DYS219 DYS221 DYS223 DYS224 DYF51S1 DYS236 DYS numbered 17, 19 and 43) showed microdeletions in five of the twenty Y chromosome STS tested. The microdeletions in this category included SY152 (DYS236), SY157 (DYS240), SY208, SY242 and SY254 at the locus (Table 2), where 62.5% of these microdeletions are placed in the AZFc and AZFb/c regions (Tables 2 and 4). Microdeletion in the SY157 STS (DYS240 locus) was common between the three infertile males who all suffered from severe oligozoospermia (Table 4). On the other hand, two additional infertile male samples (samples numbered 18 and 26) showed microdeletions when their seminal fluid genomic DNA was tested (Table 3). The five samples showed microdeletions in seven of the twenty Y chromosome STS. These included the SY81 (DYS271), SY130 (DYS221), SY152 (DYS236), SY157 (DYS240), SY208, SY242 and SY254 at the locus (Table 3). Sixty percentage of the fifteen microdeletions reported in this category are placed in the AZF region: AZFb (6.6%), AZFc (40.1%), AZFb/c (13.3%; Tables 3 and 4). Microdeletion in the SY157 STS (DYS240 locus) was common between the five infertile males (Table 4). Table 4 Comparison between the blood and seminal fluid genomic DNA-STS microdeletions Infertile patient no./ semen variable 17/ Sever oligoasthenozoospermia 18/ Oligozoospermia 19/ Sever oligo-asthenoteratozoospermia 26/ Oligoasthenozoospermia 43/ Sever oligo- asthenoteratozoospermia Blood Y chromosomal DNA No deletion (SY254) AZFc Seminal fluid Y chromosomal DNA DYS271 (SY81) (SY254) AZFc (SY254) AZFc (SY254) AZFc No deletion DYS221 (SY130) AZFb DYS236 (SY152) AZFb/c DYS236 (SY152) AZFb/c (SY208) AZFc (SY242) AZFc (SY254) AZFc DYS236 (SY152) AZFb/c (SY208) AZFc (SY242) AZFc (SY254) AZFc 2013 Blackwell Verlag GmbH 773

5 Y chromosome and male infertility in Jordan S. R. Yasin et al. A comparison between the blood and seminal fluid genomic DNA-STS microdeletions for the five samples showed that two patients (samples numbered 19 and 43) exhibited the same STS microdeletions in their blood and seminal fluid Y chromosomal DNA. Two patients (samples numbered 18 and 26) had STS microdeletions in their seminal fluid Y chromosomal DNA only, and one patient (sample numbered 17) had an extra two STS microdeletions in his seminal fluid Y chromosomal DNA (Table 4). Discussion Nongenetic causes such as hormonal abnormalities, environmental effects, varicocele, obstruction of the spermatic ducts, ejaculatory dysfunction and testicular tumours are of great importance in determining the core problem of infertility in many infertile men (Foresta et al., 2001; Hirsh, 2003; Raj et al., 2006). Molecular genetics should also be included in the procedures attempted by physicians who manage infertility cases, because molecular genetics could shed more light onto the real causes that lie behind some nonresponding infertility situations and determine the potential for passing on genetic or microdeletion defects to offspring through in vitro fertilisation (Henegariu et al., 1994; Tuerlings et al., 1998). The results of Y-specific STS assay indicated that 69 infertile patients of the 74 infertile men who were included in the current study showed the presence of all of the 20 assayed STS, possibly implicating other genetic aberrations and/or nongenetic causes for their infertility (Foresta et al., 2001; Hirsh, 2003; Raj et al., 2006). On the other hand, blood and seminal fluid samples obtained from five infertile Jordanian males showed various STS microdeletions with a frequency of 4.1% (3/74) and 6.8% (5/74) respectively (Tables 2 and 3). AZF microdeletions, which were reported to fluctuate between 1 55% worldwide (Foresta et al., 1997; van der Ven et al., 1997), showed a higher frequency (Table 4) in both blood and seminal fluid genetic DNA samples, 62.5% and 60.0% respectively. These results indicate a possible relationship between genetic microdeletions in the various infertility-associated regions of the Y chromosome and infertility in this sample of infertile Jordanian men. The presence of deletions at the Y chromosome STS which do not overlap with the region in the five infertile male seminal fluid samples (Tables 3 and 4) suggests that genes other than the gene may also be implicated in the pathogenesis of some cases of male infertility (Najmabadi et al., 1996; Martınez et al., 2000; de Carvalho & Santos, 2005). Moreover, the results shown in Table 4 indicate that blood DNA might not be representative of sperm DNA, which is of different embryonic origin (Dada et al., 2007). Sperm DNA might have a higher rate of deletions and DNA damage as a result of oxidative stress (Dada et al., 2007). Thus, it is more applicable and necessary to analyse germ cell DNA than blood DNA for Y chromosome microdeletions, because detection of these deletions has a prognostic value in predicting potential success of assisted reproduction techniques (Poongothai & Manonayaki, 2008). Finally, the results obtained for Down syndrome males enrolled in this study indicated the integrity of their Y chromosomes at the tested fertility markers, where absence of microdeletions was reported (Table 2). Thus, it is essential to counsel Down syndrome individuals about their reproductive ability because there is a 50% chance that the child would inherit the syndrome if one parent has Down syndrome. This chance is even higher where both parents have Down syndrome (Pradhan et al., 2006). 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