Article A genetic survey of 1935 Turkish men with severe male factor infertility

Transcription

1 RBMOnline - Vol 18 No Reproductive BioMedicine Online; on web 25 February 2009 Article A genetic survey of 1935 Turkish men with severe male factor infertility Dr Yakup Kumtepe graduated from the Faculty of Medicine, Atatürk University, Erzurum, Turkey in Upon completing his residency at the Department of Obstetrics & Gynaecology, Atatürk University in 1998, he worked in the IVF and Reproductive Endocrinology Unit, Sevgi Hospital, Ankara-Turkey between 1998 and He went on sabbatical for 1 year to the Mount Sinai School of Medicine, New York, USA and worked on minimal invasive surgery. He works as academic staff, clinician, lecturer and researcher. His research focuses on infertility, reproductive endocrinology and laparoscopic pelvic surgery. Dr Yakup Kumtepe Yakup Kumtepe 1,4, Cagrı Beyazyurek 2, Cigdem Cinar 2, Isa Ozbey 3, Semih Ozkan 2, Kadir Cetinkaya 1, Güvenc Karlikaya 2, Hale Karagozoglu 2, Semra Kahraman 2 1 Department of Obstetrics and Gynaecology, Faculty of Medicine, Atatürk University, 25240, Erzurum; 2 Istanbul Memorial Hospital, ART, Reproductive Endocrinology and Genetics Unit, Piyalepasa Blv, 80270, Okmeydani, Istanbul; 3 Department of Urology, Faculty of Medicine, Atatürk University, 25240, Erzurum, Turkey 4 Correspondence: ykumtepe@hotmail.com Abstract Male factor infertility is the sole reason in approximately 25% of couples who suffer from infertility. Genetic factors such as numerical and structural chromosomal abnormalities and microdeletions of the Y chromosome might be the cause of poor semen parameters. The results of karyotype analyses and Y-chromosome microdeletions of 1935 patients with severe male factor infertility, which is the largest series from Turkey, were assessed retrospectively. The frequency of cytogenetic abnormalities among 1214 patients with non-obstructive azoospermia (NOA) and 721 patients with severe oligoasthenoteratozoospermia (OAT) were and 5.83% respectively. The overall incidence of Y-chromosome microdeletion was 7.70%. The incidence of Y chromosome microdeletion in patients with NOA and OAT was 9.51 and 1.86% respectively. The abnormality rate increased with the severity of infertility. Some patients (n = 22) were carriers of both chromosomal abnormalities and Y-chromosome microdeletions. Results suggest the need for genetic screening and proper genetic counselling before initiation of assisted reproduction treatment. Keywords: assisted reproduction techniques, karyotype analysis, male factor infertility, Y chromosome microdeletions Introduction Infertility affects approximately 15% of couples of reproductive age (Iammarrone et al., 2003) and 20 25% of reproductive inability can be due to male factor alone [World Health Organization (WHO), 1997]. It is a dilemma that even the most comprehensive work-up covering physical examinations, serological and hormonal tests, detailed semen analysis and imaging techniques may fail to detect the aetiology of reproductive disorders (Crosignani et al., 1993). A number of genetic and non-genetic conditions have been associated with male infertility; these include numerical and structural chromosomal abnormalities, Y chromosome microdeletions, mutations in cystic fibrosis transmembrane conductance regulator (CFTR) gene, congenital bilateral absence of the vas deferens (CBAVD), gonadotrophin deficiency, varicocoele, testicular tumours, drugs, sperm autoimmunity, smoking, and exposure to toxins (Crosignani et al., 1993; WHO, 1997). Male infertility appears to have a familial occurrence, especially among brothers and maternal uncles (Van Golde et al., 2004). It is estimated that genetic factors are involved in approximately 60% of male factor infertility cases (Lilford et al., 1994). Cytogenetic abnormalities such as numerical and structural chromosomal abnormalities (Chandley, 1998) and microdeletions of the Y chromosome (Lilford et al., 1994; Reijo et al., 1995; Vogt et al., 1996) are known to be amongst the genetic causes of low sperm concentrations. Structural abnormalities such as reciprocal and Robertsonian translocations can be inherited through generations by vertical transmission (Kobayashi et al., 1994; Vogt et al., 1996; Chang et al., 1999). In the general male population the prevalence of chromosomal abnormalities ranges between 0.7 and 1.0% (Lange and Engel, Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 1991), whereas it is approximately 10.6% among azoospermic and oligozoopermic men (Dohle et al., 2002). The frequency of karyotypic abnormalities increases with the severity of the semen parameters; the incidences among mild (oligospermic) and severe (azoospermic) groups reported as 4.6 and 13.7% respectively (Van Assche et al., 1996). The entire length of the Y chromosome has been subdivided into seven deletion intervals (Figure 1). Each of these intervals is further subdivided into subintervals a, b and c. Some of the genes that are critical for spermatogenesis are located on the long arm of the Y chromosome in deletion interval 6 band and deletion interval 5. More than 14 protein encoding Y genes in azoospermia factor (AZF) regions affect spermatogenesis and fertility, for instance, the USP9Y gene in AZFa, the RNA binding motif (RBM) gene in the AZFb region, and the deleted in azoospermia (DAZ) gene in the AZFc region (Kobayashi et al., 1994; Vogt, 1995; Vogt et al., 1996). Deletions removing the entire AZFa or AZFb regions are associated with Sertoli cell only syndrome (SCOS) and spermatogenic arrest respectively (Krausz et al., 2000; Kamp et al., 2001). AZFc deletions are, however, associated with normozoospermia, oligozoospermia, severe oligozoospermia and azoospermia (Oliva et al., 1998). The deletion types of AZF a, b and c loci are the potential prognostic factors in patients recommended to undergo testicular surgery such as testicular sperm extraction (TESE) and microdissection TESE procedures (micro-tese). In more than half of men with AZFc deletions, mature spermatozoa can be obtained in the ejaculate or in the testis via surgery, whereas in patients with complete deletions in AZFa or AZFb regions, it is nearly impossible to obtain mature spermatozoa (Hopps et al., 2003). So far, several retrospective studies have tried to establish the frequency of cytogenetic abnormalities and Y chromosome microdeletions in the Turkish population (Okutman-Emonts et al., 2004; Sargin et al., 2004; Vicdan et al., 2004). However, sample sizes in these studies were inadequate to make inferences. The purpose of this study was to evaluate the genetic risks in a large number of infertile Turkish men who were referred for assisted reproduction treatment. Materials and methods Population Cytogenetic and molecular screening test results of 1935 infertile men with oligoasthenoteratozoospermia (OAT) or non-obstructive azoospermia (NOA) who had been referred to Istanbul Memorial Hospital In-Vitro Fertilization (IVF) and Reproductive Genetics Centre between July 2000 and September 2007 were analysed retrospectively. Sperm samples were harvested 2 6 days after sexual abstinence and were subjected to evaluation for total count, percent motility and forward progression (WHO, 1999). Sperm morphology was evaluated according to strict criteria as outlined by Kruger et al. (1988) following Spermac staining. Blood samples were taken for both cytogenetic analysis (n = 1935) and Y-chromosome microdeletion test (n = 1364). This study protocol was reviewed and approved by the Internal Review Board of Istanbul Memorial Hospital and written informed consent was obtained from each participant. Cytogenetic analysis Because of absence of spermatoza and low sperm concentration (< /ml) in a considerable numbers of patients, NOA and OAT were evaluated in 1214 and 721 patients respectively. Peripheral blood samples were collected from both partners and kept in lithium heparin tubes for karyotyping. Blood samples 466 Figure 1. Schematic representation of the Y chromosome showing seven intervals and pseudoautosomal region (PAR) 1 and 2. Tiepolo and Zuffardi (1976) revealed the existence of deletions in the long arm of the Y chromosome and considered these regions an essential genetic factor for spermatogenesis. Later, this factor was named as azoospermia factor (AZF) (Ma et al., 1992; Vogt et al., 1992; Reijo et al., 1996). Interstitial microdeletions occur in three discrete regions on Yq: AZFa (proximal), AZFb (central) and AZFc (distal) (Vogt et al., 1996). The distal part of AZF is deleted in azoospermia (DAZ) (Reijo et al., 1995).

3 were put into Roswell Park Memorial Institute (RPMI) 1640 medium (Biological Industries, Ltd, Israel) and added with HEPES containing fetal bovine serum albumin, l-glutamine, penicillin streptomycin and phytohemagglutinin. Forty-eight hours after incubation at 37 C, 150 µl of thymidine was added. During 17 h, culture was washed with RPMI medium without phytohaemagglutinin and left at 37 C for further incubation. Three hours later, 100 µl colcemide (10 µg/ml) was added for 50 min incubation before harvest. Following hypotonic treatment, cells were fixed by fresh cold fixative (methanol:glacial acetic acid, vol:vol, 3:1). Giemsa trypsin Giemsa (GTG) banding was performed using trypsin (0.025% trypsin EDTA) and Giemsa s solution (5%) (Merck and Co. Inc., Germany). A total of 20 metaphase plaques were analysed for each subject. The metaphase count was increased to 100 and fluorescent insitu hybridization (FISH) was applied in cases of mosaicism. Numerical and structural karyotype analyses were performed at 550 band levels using Ikaros software (MetaSystems Inc., Germany). FISH analysis FISH analysis was conducted on metaphase plaques obtained from patients with structural abnormalities or numerical mosaicisms. Centromeric (CEP), locus specific (LSI), whole chromosome painting (WCP), and telomeric (Tel) probes were used according to general FISH protocols. Probes for regions specific for sex chromosomes such as sex determining region Y (LSI SRY, Yp11.3), CEP X (DXZ1 alpha satellite) and WCP Y (Vysis Inc., Germany) were generally used to confirm the location of the SRY region. Co-denaturation of probe and DNA was performed at 72 C and hybridization was performed at 37 C in a hybridization chamber (Hybrite; Vysis Inc., Germany) for at least 6 h. Slides were washed in 0.4% saline sodium citrate for 2 min at 71 C. Following application of DAPI (4,6-diamidino- 2-phenylindole II; Vysis) counterstain, images were analysed and captured in green, red, gold, aqua and DAPI fluorescence filters with an Olympus BX50 fluorescence microscope using commercial software (Isis; Metasystems Inc., Germany). Molecular analysis Out of 1935 cases that had been screened for cytogenetic abnormalities, 1364 were also analysed for Y chromosome microdeletion. The majority of patients who screened for microdeletions were azoospermic (n = 1041) and the rest had severe OAT (n = 323). Genomic DNA was obtained from peripheral blood leukocytes and analysed using an isolation kit (QIAmp DNA mini kit; Qiagen) according to the manufacturer s protocol. Microdeletions of the Y chromosome were screened using multiplex polymerase chain reaction (PCR). Promega kit containing 18 loci (from 2000 to 2003) and a homemade kit containing primers specific for 25 loci (thereafter) were employed. The following loci were screened: in the SRY region SY14; in the AZFa region SY81, SY82, SY83, SY84, SY86, SY87, SY88, SY182; in the AZFb region SY121, SY124, SY127, SY128, SY130, SY133, SY134, SY135; in the AZFc region SY143, SY145, SY152, SY157, SY158, SY254, SY255; and in the heterochromatic region SY160. Deletions in the fourth region (between AZFb and AZFc), which used to be termed AZFd in the previous kit, were distributed into AZFb and AZFc regions as now adopted. For each sample, four different multiplex fluorescent PCR mixes were prepared, which contained primer, PCR mix with buffer, dntp and MgCl 2, Taq polymerase, 30 ng DNA sample, and H 2 O with a final volume of 25 µl. The PCR program was set at 95 C for 10 min, followed by 35 cycles at 94 C for 60 s, 60 C for 60 s, and 72 C for 60 s, which was followed by final extension at 65 C for 45 min. In each reaction, one sample of fertile male genomic DNA as positive, one sample of female DNA and one sample of distilled water without DNA were used as controls for each set of primers. After amplification, PCR products were subjected to capillary electrophoresis on genetic analyser (ABI Prism 3100; Applied Biosystems, USA) and the outputs were analysed with sequence analysis software (GeneScan, Applied Biosystems). Statistical analysis Data were subjected to the proc freq procedure of Statistical Analysis System (SAS, 1999). Differences in group frequencies were attained using the chi-squared test and significance was declared at P < Results Table 1 presents detailed peripheral karyotype analysis with respect to semen parameters. The overall incidence of cytogenetic abnormalities was 12.45%. The frequencies of abnormalities increased with the severity of semen parameters, being 5.83 and 16.40% in OAT and NOA groups respectively. Klinefelter s syndrome (47,XXY) was the most frequent abnormality (n = 138, 7.13%), especially in the NOA group (n = 133, 10.95%). Both reciprocal and Robertsonian translocations together (n = 25, 3.47%) were the most frequent abnormality in the OAT group. The incidence of 46,XX males (n = 10), was nearly 1% among NOA patients (Figure 2). Six out of 10 males with this abnormality were also screened for Y-microdeletions; four patients were positive and two were negative for the SRY region. Further analysis using FISH technique revealed that the SRY region was translocated to one of the X chromosomes in all SRY+ cases (Figure 3). Another abnormality evaluated in this group was a 45,X0 male (Figure 4), where the SRY region was associated with autosomal chromosome 2: 45,X,tas(Y;2) (p11.3;qter). So far as is known, this case is the first 45,X0 male with SRY associated with the telomeric region of chromosome 2 (Figures 5 and 6). The overall incidence of Y chromosome microdeletion was 7.70%. It was significantly different between OAT (1.86%) and NOA (9.51%) groups (P < ; Table 2). The most frequent region with deletions was the AZFc region. A minority of patients (n = 22) had both karyotypic abnormalities and Y chromosome microdeletions (Table 3). One case of Klinefelter s syndrome (47,XXY) was associated with partial deletions in the AZFa, AZFb and AZFc regions of the Y chromosome. Six 46,XX males and one 45,X male had no Y chromosome. Eight patients had mosaic forms of sex chromosomal abnormalities accompanied mostly by AZFb + c deletions. 467

4 Table 1. Distribution of normal and abnormal karyotypes in the infertile men studied. Karyotype Category of male infertility Total (n = 1935) NOA (n = 1214) OAT (n = 721) Normal (46,XY) 973 (80.15) 645 (89.46) 1618 (83.62) Normal variable features/polymorphisms 42 (3.46) 34 (4.72) 76 (3.93) Abnormal 199 (16.40) 42 (5.83) 241 (12.45) Klinefelter syndrome (47,XXY) 133 (10.96) 5 (0.69) 138 (7.13) Mosaic Klinefelter 10 (0.82) 6 (0.83) 16 (0.83) Other sex chromosome mosaicisms 13 (1.07) 2 (0.28) 15 (0.78) 45,X0 (n = 1) and 46,XX (n = 10) 11 (0.91) 0 (0.00) 11 (0.57) Other X chromosome abnormalities 10 (0.82) 1 (0.14) 11 (0.57) Reciprocal translocation 12 (0.99) 13 (1.80) 25 (1.29) Robertsonian translocation 1 (0.08) 12 (1.66) 13 (0.67) Inversions 4 (0.33) 1 (0.14) 5 (0.26) Markers 1 (0.08) 1 (0.14) 2 (0.10) Other abnormalities 4 (0.33) 1 (0.14) 5 (0.26) Values are number (%). NOA = non-obstructive azoospermia; OAT = oligoasthenoteratozoospermia. Figure 2. Karyotype analysis results of a 46,XX, SRY positive male. 468

5 Figure 3. Fluorescence in-situ hybridization technique enabled identification and localization of the SRY region on one of the X chromosomes. CEP X is in spectrum green, whereas LSI SRY is in spectrum red. Figure 4. Karyotyping revealed that in addition to the absence of the Y chromosome, only one X chromosome was present in all of the metaphase plaques analysed; 45, X0. Figure 5. Fluorescence in-situ hybridization (FISH) analysis results of 45,X,tas(Y;2)(p11.3;qter). FISH study revealed that the SRY (spectrum red) region was not translocated on the X chromosome (CEP X, spectrum green) but was on an autosomal chromosome which was presumed to be chromosome 2. Figure 6. Further fluorescence in-situ hybridization study using probes specific to centromeric (CEP 2, spectrum gold) and telomeric loci (Tel 2q, spectrum gold) of chromosome 2 revealed that the SRY region was associated with the telomeric region of chromosome 2, which was still present in its location together with SRY (evident from WCP probe, spectrum green). The WCP probe binds both SRY region and a homologous region on the X chromosome. 469

6 Table 2. Y chromosome microdeletion in severe male factor infertility. Y chromosome Category of male infertility Total (n = 1364) NOA (n = 1041) OAT (n = 323) Normal 942 (90.50) 317(98.14) 1259 (92.30) Deleted 99 (9.51) 6 (1.86) 105 (7.70) AZFa AZFb AZFc AZFbc AZFabc Values are number (%) or number. NOA = non-obstructive azoospermia; OAT = oligoasthenoteratozoospermia. Table 3. Patients having both karyotype abnormalities and Y-microdeletions (n = 22). Karyotype abnormality n Y-microdeletion n 47,XXY 1 Partial AZFa, AZFb and AZFc 1 46,XX male SRY+ 4 Y complete 7 46,XX male SRY 2 45,X male SRY+ 1 46,X,del(Y)(q11.2) 3 del complete b, c 2 del complete b, del partial c 1 idic Y(p) 3 del complete b, c, sy del Y complete 1 del partial a, del complete b, c 1 mos 46,XY(92%)/45,X(8%) 1 del complete b, c 1 mos 46,XY(78%)/45,X(22%) 1 del complete b, c 1 mos 46,XY(57%)/45,X(43%) 1 del complete b, c 1 mos 46,XY(80%)/45,X(18%)/47,XYY(2%) 1 del complete b, c, sy mos 45,X(50%)/46,Xi(Y)(p11.1)(50%) 1 del Y complete, SRY+ 1 mos 46,X,idic(Yp)(72%)/45,X(24%) 1 del complete b, c 1 mos 45,X(52%)/46,X,idicY(p)(48%) 1 del complete b, c, sy mos 46,XidicYp(70%)/45,X(30%) 1 del complete b, c, sy mos = mosaic; del = deletion. 470 Discussion The chromosomal abnormality rate in this study (12.4%), was similar to the study of Nakamura and colleagues (2001) in Japan (12.6%), but lower than reported in the study of Kleiman and colleagues (1999) in Israel (16.6%) and higher than reported in the studies of Yoshida and colleagues (1997) in Japan (6.2%) and Quilter and colleagues (2003) in the UK (9.7%). Among all cytogenetic abnormalities, Klinefelter s syndrome was the most frequent abnormality detected, which is in agreement with other studies conducted on infertile men (Nakamura et al., 2001; Elghezal et al., 2006). The second most frequent abnormality was related to chromosomal rearrangements (2.2%) such as reciprocal and Robertsonian translocations and inversions. The high rate of translocations in this population could be associated with the high proportion of patients with male infertility as well as repeated implantation failures or recurrent pregnancy losses. Patients were referred to the clinic, which is a reference clinic as well as a genetic diagnostic centre in Turkey performing more than 500 cycles per year for preimplantation genetic diagnosis (PGD), mostly for aneuploidy screening and translocations. Chromosomal rearrangements can affect fertility due to impaired gametogenesis and/or production of chromosomally unbalanced gametes. In the present study, the translocation rate was lower in the NOA group than the OAT group (1.1 versus 3.5%). Van Assche and colleagues (1996) also reported a lower incidence of translocations in patients with NOA than in those with OAT (1.06 versus 3.46%). These results show that this type of chromosomal rearrangement may not always cause spermatogenic arrest. For translocation carrier couples, poor prognosis is related to a reduced chance of producing normal or balanced gametes. Attempts have been made to reveal any possible correlation between chromosomal abnormalities in gametes and embryos. Using FISH on sperm cells, it is possible to determine the rate of chromosomally unbalanced sperm cells in the ejaculate. The rate of unbalanced spermatozoa and gametes in ejaculates has been reported as 72.1 (Estop et al., 1995) and up to 25.0% (Escudero et al., 2000) respectively. The PGD technique can be offered to couples who are translocation carriers. By analysing one cell on day 3 of embryonic development, it is possible to detect the balanced or normal embryos for transfer. With experienced staff, PGD decreases

7 Table 4. Y chromosome microdeletion rate determined in idiopathic azoospermic and severe oligoazoospermic patients from different countries. Country n Deletion Reference (%) Asia China Fu et al., 2002 China Song et al., 2005 Taiwan Lin et al., 2000 Taiwan Lin et al., 2002 Japan Kobayashi et al., 1994 India Dada et al., 2003 India Athalye et al., 2004 Oceania New Zealand Kerr et al., 2000 Europe France Seifer et al., 1999 Spain Oliva et al., 1998 Slovenia Peterlin et al., 2002 Finland Aho et al., 2001 N. America USA Girardi et al., 1997 USA Najmabadi et al., 1996 Middle East Israel Nakamura et al., 2001 Turkey Vicdan et al., 2004 Turkey Okutman-Emonts et al., 2004 Turkey Sargin et al., 2004 Total Turkey Present study abortion rates and increases pregnancy and implantation rates among couples with chromosomal rearrangements (Otani et al., 2006; Beyazyurek et al., 2007). Heterochromatin polymorphisms were observed in 3.9% of all men screened for karyotype abnormalities (Table 1). The question arises as to whether these polymorphisms are less innocent than might be expected. In support of this, the incidence of heterochromatin polymorphism is higher in infertile than in fertile men (Yakin et al., 2005). A wellknown example of this type of variation is a regular pericentric inversion on chromosome 9; inv(9)(p12;q13). Although this variation is not considered to have an effect on fertility, there have been some studies demonstrating its association with an infertility factor (Sasagawa et al., 1998; Tomaru et al., 1999; Davalos et al., 2000), the mechanism has not been identified. In addition to inv(9), heterochromatic region polymorphisms on chromosomes 1,9,16 and Y are also associated with impaired spermatogenesis (Nazarenko and Sukhanova, 1991; Bobrow, 1995). The impairment of adjacency between X and Y chromosomes during meiotic divisions has been proposed to have an effect in spermatogenic arrest in the case of microdeletions (Yogev et al., 2004). Significant impairment in X/Y alignment was observed in patients with microdeletions in AZF b + c regions compared with patients with isolated AZFc deletions, suggesting that either the deletion or the absence of the genes involved were probably responsible for meiosis impairment (Yogev et al., 2004). The deletion rate is highly variable in the literature (Table 4). In the present study, DNA analysis of AZFa, AZFb and AZFc regions showed that 7.70% of infertile patients had Y-microdeletion; with a higher incidence in the NOA group than the OAT group (9.51 versus 1.86%; Table 2). Out of 105 patients with microdeletions, 97 patients (92.38%) were carrying partial or complete deletions in AZFc region, as reported elsewhere (Schlegel, 2002; Simoni et al., 2008). Y chromosome microdeletion rates may vary from 0 to 25.90% (Simoni et al., 2008) and the rate in this study (7.70%) is at the mid-point of other reports worldwide (Girardi et al., 1997; Simoni et al., 1998; Athalye et al., 2004). This rate is higher than two previous reports (Okutman-Emonts et al., 2004; Sargin et al., 2004) and lower than one previous report (Vicdan et al., 2004) conducted in Turkey (Table 4). The variability is probably related to differences in clinical selection criteria, molecular strategies, and the number of loci used for screening as well as sample size. The deletion types of AZF a, b and c loci on Yq11 are the potential prognostic factors in patients planned to undergo TESE/micro-TESE procedures. For men carrying complete deletions in AZFa, AZFb regions, it is virtually impossible to obtain mature spermatozoa (Simoni et al., 2004). Similar to the present results, in 56% of men with deletions in the AZFc region, 471

8 472 mature spermatozoa could be retrieved via testicular biopsy (Hopps et al., 2003). Therefore, Y chromosome microdeletion screening provides clinicians with the information needed to avoid unnecessary surgical procedures and alter treatment choices. Micro-TESE was performed on 30 patients with microdeletions after informing them about the rates of sperm retrieval. Two patients with complete AZFb + c and one patient with partial AZFa + b + c deletions chose to undergo TESE. No spermatozoa were obtained in two patients with partial AZFa, two patients with complete AZFb + c and one patient with partial AZFa + b + c deletions. In the remaining 25 patients with AZFc deletions, mature spermatozoa were obtained in 14 patients (56%). Eight patients had already become fathers of eight children (five girls and three boys) via intracytoplasmic sperm injection (ICSI). Boys were not monitored for AZFc deletions, but the parents were informed about the likelihood of occurrence of the same problem in their children. Out of 30, 15 patients had microdeletions involving exactly the same regions (SY152, SY157, SY158, SY254, SY255), mature spermatozoa were, however, found in six patients and no spermatozoa were obtained in nine patients. It was noteworthy that eight cases carried sex chromosomal mosaicisms including 45,X0 cell lines and Y chromosome microdeletions (Table 3). An association between Y-chromosome microdeletions and 45,X0/46,XY chromosomal mosaicism has been proposed before (Siffroi et al., 2000; Alvarez-Nava et al., 2008). Moreover, Alvarez-Nava and colleagues (2008) found a higher incidence of Y-chromosome microdeletions on gonadal DNA than on peripheral blood lymphocyte DNA in 11 patients with 45,X0/46,XY gonadal dysgenesis, suggesting that microdeletions in the Y chromosome might be associated with Y chromosomal instability leading to mitotic loss of the Y chromosome (Siffroi et al., 2000; Alvarez-Nava et al., 2008). The PGD technique can also be considered as a treatment option for cases with both a high rate of sex chromosomal mosaicisms and Y chromosome microdeletions because there is a risk of transmitting this unstable Y chromosome that may lead to gonadal dysgenesis in the offspring. Polymorphisms (Ferlin et al., 2007) and expression profiles of several candidate genes (Kleiman et al., 2007) in testicular tissue of azoospermic patients are associated with impaired spermatogenesis. Although Y chromosome microdeletions have direct effects on spermatogenesis, microdeletions of Yq involving the DAZ gene are associated with variable phenotypic expressions that can include evidently normal fertility (Chang et al., 1999) and thereby permitting vertical transmission. In the present study, despite having deletions in the same loci in AZFc region, spermatozoa were retrieved from six patients, whereas no spermatozoa could be obtained from nine patients after micro-tese procedures. The presence of polymorphisms or mutations in other genes related to spermatogenesis may contribute to variable phenotypic expression accompanied by AZFc deletions. There is growing evidence that draws attention to the paternal contribution to embryonic development, implantation and abortion. Morphological abnormalities of head and tail as well as poor motility are associated with increased chromosomal abnormalities in spermatozoa (Calogero et al., 2001; Kahraman et al., 2004, 2006; Tempest et al., 2004) and embryos (Kahraman et al., 2004, 2006). Furthermore, FISH studies reveal that aneuploidy rate is high in sperm samples obtained from TESE and its incidence increases with the severity of infertility (Gianaroli et al., 2005b). There is a significant proportional increase in gonosomal aneuploidy in embryos derived from testicular sperm, despite no overall increase in chromosomal aneuploidy (Gianaroli et al., 2005a). Men with high levels of DNA fragmentation are also at greater risk for low blastocyst rates and failure to initiate an ongoing pregnancy (Virro et al., 2004) and have significantly higher IVF abortion rates (Lin et al., 2007). Therefore, the fragmentation and aneuploidy tests provide invaluable prognostic information to physicians counselling couples before IVF and/or ICSI cycles. In conclusion, among Turkish infertile men with idiopathic male factor infertility, the frequency of cytogenetic abnormality and Y chromosome microdeletion was and 7.70% respectively, suggesting the need for genetic screening and counselling in these particular cases. To give the appropriate treatment to couples requiring ICSI, peripheral blood karyotyping and Y chromosome microdeletion analysis should be considered before initiating IVF cycles. References Aho M, Harkonen K, Suikkari AM et al Y-chromosomal microdeletions among infertile Finnish men. Acta Obstetrica et Gynecologica Scandinavica 80, Alvarez-Nava F, Puerta H, Soto M et al High incidence of Y-chromosome microdeletions in gonadal tissues from patients with 45,X/46,XY gonadal dysgenesis. Fertility and Sterility 89, Athalye AS, Madon PF, Naik NJ et al A study of Y chromosome microdeletions in infertile Indian males. International Journal of Human Genetics 4, Beyazyurek C, Saglam Y, Sertyel S et al Factors Affecting Outcome of Preimplantation Genetic Diagnosis Cycles for Translocation Cases. In: Proceedings of the Preimplantation Genetic Diagnosis International Society (PGDIS) Conference, June 13 16, Melbourne. Bobrow M 1995 Heterochromatic chromosome variation and reproductive failure. Experimental and Clinical Immunogenetics 2, Calogero AE, De Palma A, Grazioso C et al Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters. Human Reproduction 16, Chandley A 1998 Chromosome anomalies and Y chromosome microdeletions as causal factors in male infertility. Human Reproduction 13, Chang PL, Sauer MV, Brown S 1999 Y chromosome microdeletion in a father and his four infertile sons. Human Reproduction 14, Crosignani PG, Collins J, Cooke ID et al Recommendations of the ESHRE workshop on unexplained infertility. Human Reproduction 8, Dada R, Gupta NP, Kucheria K 2003 Molecular screening for Yq microdeletion in men with idiopathic oligozoospermia and azoospermia. Journal of Bioscience 28, Davalos IP, Rivas F, Ramos AL et al Inv (9) (p24q13) in three sterile brothers. Annales de Genetique 43, Dohle GR, Halley DJJ, Hemel JOV et al Genetic risk factors in infertile men with severe oligozoospermia and azoospermia. Human Reproduction 8, Elghezal H, Hidar S, Braham R et al Chromosome abnormalities in one thousand infertile males with nonobstructive sperm disorders. Fertility and Sterility 86, Escudero T, Lee M, Carrel D et al Analysis of chromosome

9 abnormalities in sperm and embryos from two 45,XY,t(13;14) (q10;q10) carriers. Prenatal Diagnosis 20, Estop AM, Van Kirk V, Cieply K 1995 Segregation analysis of four translocations, t(2;18), t(3;15), t(5;7), and t(10;12), by sperm chromosome studies and a review of the literature. Cytogenetics and Cell Genetics 70, Ferlin A, Raicu F, Gatta V et al Male infertility: role of genetic background. Reproductive BioMedicine Online 14, Fu J, Li L, Lu G 2002 Relationship between microdeletion on Y chromosome and patients with idiopathic azoospermia and severe oligozoospermia in the Chinese. Chinese Medical Journal 115, Gianaroli L, Magli MC, Ferraretti AP 2005a Sperm and blastomere aneuploidy detection in reproductive genetics and medicine. Journal of Histochemistry and Cytochemistry 53, Gianaroli L, Magli MC, Cavallini G et al. 2005b Frequency of aneuploidy in sperm from patients with extremely severe male factor infertility. Human Reproduction 20, Girardi SK, Mielnik A, Schlegel PN 1997 Submicroscopic deletions in the Y chromosome of infertile men. Human Reproduction 12, Hopps CV, Mielnik A, Goldstein M et al Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions. Human Reproduction 18, Iammarrone E, Balet R, Lower AM et al Male infertility. Best Practice and Research Clinical Obstetrics and Gynaecology 17, Kahraman S, Findikli N, Biricik A et al Preliminary FISH studies on spermatozoa and embryos in patients with variable degrees of teratozoospermia and a history of poor prognosis. Reproductive BioMedicine Online 12, Kahraman S, Sertyel S, Findikli N et al Effect of PGD on implantation and ongoing pregnancy rates in cases with predominantly macrocephalic spermatozoa. Reproductive BioMedicine Online 9, Kamp C, Huellen K, Fernandes S et al High deletion frequency of the complete AZFa sequence in men with Sertoli-cell-only syndrome. Molecular Human Reproduction 7, Kerr NJ, Zhang J, Sin FY et al Frequency of microdeletions in the azoospermia factor region of the Y-chromosome of New Zealand men. New Zealand Medical Journal 10, Kleiman SE, Yogev L, Hauser R et al Expression profile of AZF genes in testicular biopsies of azoospermic men. Human Reproduction 22, Kleiman SE, Yogev L, Gamzu R et al Genetic evaluation of infertile men. Human Reproduction 14, Kobayashi K, Mizuno K, Hida A et al PCR analysis of the Y chromosome long arm in azoospermic patients: evidence for a second locus required for spermatogenesis. Human Molecular Genetics 3, Krausz C, Quintana-Murci L, McElreavey K 2000 Prognostic value of Y deletion analysis: what is the clinical prognostic value of Y chromosome microdeletion analysis? Human Reproduction 15, Kruger TF, Acosta AA, Simmons KF et al Predictive value of abnormal sperm morphology in in vitro fertilization. Fertility and Sterility 49, Lange R, Engel W 1991 Chromosomal aberrations as a cause of male subfertility. Journal of Fertility and Reproduction 9, Lilford R, Jones AM, Bishop DT et al Case control study of whether subfertility in men is familial. British Medical Journal 309, Lin MH, Kuo-Kuang LR, Li SH et al Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates. Fertility and Sterility 90, Lin YM, Lin YH, Teng YN et al Gene-based screening for Y chromosome deletions in Taiwanese men presenting with spermatogenic failure. Fertility and Sterility 77, Lin YM, Chen CW, Sun HS et al Y-chromosome microdeletion and its effect on reproductive decisions in Taiwanese patients presenting with nonobstructive azoospermia. Urology 56, Ma K, Sharkey A, Kirsch S et al Towards the molecular localisation of the AZF locus: mapping of micro deletions in azoospermic men within 14 subintervals of interval 6 of the human Y chromosome. Human Molecular Genetics 1, Najmabadi H, Huang V, Yen P et al Substantial prevalence of microdeletions of the Y chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence-tagged site-based mapping strategy. Journal of Clinical Endocrinology and Metabolism 81, Nakamura Y, Kitamura M, Nishimura K et al Chromosomal variants among 1790 infertile men. International Journal of Urology 8, Nazarenko SA, Sukhanova NN 1991 C-polymorphism of chromosomes in men with azoospermia. Tsitologiia Genetika 25, Okutman-Emonts O, Pehlivan S, Tavmergen E et al Screening of Y chromosome microdeletion which contains AZF regions in 71 Turkish azoospermic men. Genetic Counseling 15, Oliva R, Margarit E, Ballesca JL et al F. Prevalence of Y chromosome microdeletions in oligospermic and azoospermic candidates for intracytoplasmic sperm injection. Fertility and Sterility 70, Otani T, Roche M, Mizuike M et al Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies. Reproductive BioMedicine Online 13, Peterlin B, Kunej T, Sinkovec J et al Screening for Y chromosome microdeletions in 226 Slovenian subfertile men. Human Reproduction 17, Quilter CR, Svennevik EC, Serhal P et al Cytogenetic and Y chromosome microdeletion screening of a random group of infertile males. Fertility and Sterility 80, Reijo R, Alagappan RK, Patrizio P, Page DC 1996 Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet 347, Reijo R, Lee TY, Salo P et al Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nature Genetics 10, Sargin CF, Berker-Karaüzüm S, Manguoglu E et al AZF microdeletions on the Y chromosome of infertile men from Turkey. Annales de Genetique 47, SAS 1999 User s Guide, Statistics, Version 8. Statistical Analysis System. SAS Inst., Inc., Cary, NC. Sasagawa I, Ishigooka M, Kubota Y et al Pericentric inversion of chromosome 9 in infertile men. International Urology and Nephrology 30, Schlegel PN 2002 The Y chromosome. Reproductive BioMedicine Online 5, Seifer I, Amat S, Delgado-Viscogliosi P et al Screening for microdeletions on the long arm of chromosome Y in 53 infertile men. International Journal of Andrology 22, Siffroi JP, Le Bourhis C, Krausz C et al Sex chromosome mosaicism in males carrying Y chromosome long arm deletions. Human Reproduction 15, Simoni M, Tüttelmann F, Gromoll J, Nieschlag E 2008 Clinical consequences of microdeletions of the Y chromosome: the extended Münster experience. Reproductive BioMedicine Online 16, Simoni M, Bakker E, Krausz C 2004 EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State of the art International Journal of Andrology 27, Simoni M, Kamischke A, Nieschlag E 1998 Current status of the molecular diagnosis of Y chromosomal microdeletions in the work-up of male infertility. Initiative for international quality control. Human Reproduction 13, Song NH, Wu HF, Zhang W et al Screening for Y chromosome 473

10 microdeletions in idiopathic and nonidiopathic infertile men with varicocele and cryptorchidism. Chinese Medical Journal 118, Tempest HG, Homa ST, Dalakiouridou M et al The association between male infertility and sperm disomy: evidence for variation in disomy levels among individuals and a correlation between particular semen parameters and disomy of specific chromosome pairs. Reproductive Biology and Endocrinology 2, Tiepolo L, Zuffardi O 1976 Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Human Genetics 34, Tomaru M, Sasagawa I, Ishigooka M et al Pericentric inversion of chromosome 9 associated with Sertoli-cell-only tubule. Urology International 52, Van Assche E, Bonduelle M, Tournaye H et al Cytogenetics of infertile men. Human Reproduction 11, Van Golde RJ, Van der Avoort IA, Tuerlings JH et al Phenotypic characteristics of male subfertility and its familial occurrence. Journal of Andrology 25, Vicdan A, Vicdan K, Günalp S et al Genetic aspects of human male infertility: the frequency of chromosomal abnormalities and Y chromosome microdeletions in severe male factor infertility. European Journal of Obstetrics and Gynecology and Reproductive Biology 117, Virro MR, Larson-Cook KL, Evenson DP 2004 Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertility and Sterility 81, Vogt PH 2005 AZF deletions and Y chromosomal haplo groups: history and update based on sequence. Human Reproduction 11, Vogt PH, Edelmann A, Kirsch S et al Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Human Molecular Genetics 5, Vogt P, Chandley AC, Hargreave TB et al Microdeletions in interval 6 of the Y chromosome of males with idiopathic sterility point to disruption of AZF, a human spermatogenesis gene. Human Genetics 89, WHO 1999 World Health Organization Laboratory Manual for the Examination of Human Semen and Sperm Cervical Mucus Interaction. Cambridge University Press, UK. WHO 1997 Towards more objectivity in diagnosis and management of male fertility. International Journal of Andrology 7, Yakin K, Balaban B, Urman B 2005 Is there a possible correlation between chromosomal variants and spermatogenesis? International Journal of Urology 12, Yogev L, Segal S, Zeharia E et al Sex chromosome alignment at meiosis of azoospermic men with azoospermia factor microdeletion. Journal of Andrology 25, Yoshida A, Miura K, Shirai M 1997 Cytogenetic survey of 1007 infertile males. Urology International 58, The results of this study were presented in part at a meeting organized by Jordanian Society of Obstetrics and Gynaecology and the Royal College of Obstetricians and Gynaecologists in Declaration: The authors report no financial or commercial conflicts of interest. Received 21 May 2008; refereed 22 July 2008; accepted 20 November