Article Clinical consequences of microdeletions of the Y chromosome: the extended Münster experience

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1 RBMOnline - Vol 16 No Reproductive BioMedicine Online; on web 12 December 2007 Article Clinical consequences of microdeletions of the Y chromosome: the extended Münster experience Manuela Simoni obtained her MD in 1982, followed by a specialisation in Endocrinology and Metabolism in 1985, from the University of Modena, Italy. In 1991 she completed a PhD in Endocrinology and Metabolism. Since 1990 she has been working at the Institute of Reproductive Medicine of the University of Münster, Germany, where she holds a Professorship for Endocrinology and Molecular Biology of Reproduction. Her research interests are gonadotropin and androgen action, testicular function, genetics of male infertility, endocrinology and pathophysiology of reproduction. She is a member of several international societies and serves on the editorial board of several journals in the fields of endocrinology and reproduction. Dr Manuela Simoni Manuela Simoni 1, Frank Tüttelmann, Jörg Gromoll, Eberhard Nieschlag Institute of Reproductive Medicine, University Hospital, Domagkstr. 11, D Münster, Germany 1 Correspondence: Tel: ; Fax: ; manuela.simoni@ukmuenster.de Abstract A total of 3179 patients were screened for Y-chromosome microdeletions and 821 patients for partial AZFc deletions. Thirtynine Y-chromosomal microdeletions were found (2.4% of men with < /ml spermatozoa): two AZFa, two AZFb, one AZFbc, one partial AZFb, one partial AZFb+c and 32 AZFc (b2/b4). Partial AZFc deletions were found in 45 patients (5.5%), mostly gr/gr deletions (n = 28). In patients with AZFc deletion, azoospermia was found in 53.1% and sperm concentrations of mostly < /ml were found in 46.9%. Semen analyses and FSH measurements showed no trend over time. Elongated spermatids were seen in 6/15 AZFc patients and bilateral Sertoli cell-only was found in 4/15. Testicular sperm extraction (TESE) was attempted in 10 patients and spermatozoa were found in six. Compared with infertile men matched by sperm concentration, no differences in hormonal and seminal parameters could be found in patients with AZFc or gr/gr deletions. It is concluded that: (i) frequency of AZF deletions in Germany is much lower than in other countries; (ii) AZFc deletions are associated with severe disturbances of spermatogenesis and TESE is not possible in half of these patients; (iii) AZFc and gr/ gr deletions are not associated with any clinical diagnostic parameter; (iv) and no trend is apparent over time. Keywords: AZF, deletion, gr/gr, infertility, Y chromosome Introduction Microdeletions of the Y chromosome represent one of the few, well-recognized genetic causes of spermatogenetic failure resulting in male infertility (Ferlin et al., 2007). After the original description of the deleted in azoospermia (DAZ) gene (Reijo et al., 1995) and the characterization of discrete deletion patterns in infertile men (Vogt et al., 1996), molecular diagnosis of microdeletions became available to most andrological centres. In the following decade, the occurrence of microdeletions of the Y chromosome in infertile but not in control men was reported from various countries, although with different frequency, possibly depending on the selection criteria of the patients and on the ethnic background (Krausz and Degl Innocenti, 2006). The molecular diagnosis of Y-chromosomal microdeletions is relatively easy and cheap, justifying its popularity, which now makes it one of the most frequently performed diagnostic tests in molecular genetics. Laboratory guidelines have been issued (Simoni et al., 1999, 2004) and an external quality assessment scheme is currently offered (European Molecular Genetics Quality Network, 2007). Knowledge of the complete sequence of the Y chromosome has allowed the molecular mechanism of microdeletions to be identified as homologous recombination between identical sequences in palindromes (Kuroda-Kawaguchi et al., 2001; Repping et al., 2002; Skaletsky et al., 2003). The breakpoints of deletions are well characterized and five main microdeletion patterns have been identified, named AZFa, P5-proximal P1 (AZFb), P5-distal P1 (AZFbc), P4-distal P1 (AZFbc) and b2/b4 (AZFc) (Repping et al., 2002). Even with this detailed knowledge, it has been Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 290 estimated that the current basic protocol for routine microdeletion screening is sufficient to detect over 95% of clinically relevant deletions (Simoni et al., 2004), although rare exceptions of partial deletions within the above-mentioned regions might occur and warrant further ad hoc characterization (Krausz et al., 2006). In addition, smaller, partial deletions and rearrangements within the b2/b4 (AZFc) region have been identified (Repping et al. 2003), although the pathogenetic meaning of such deletions (gr/gr and similar) is still debated (Noordam and Repping, 2006). A recent survey of 10 years of experience in northern Italy showed a prevalence of 8.3% for microdeletions in non-obstructive azoospermia and 5.5% in severe oligozoospermia, confirming the significant diagnostic and prognostic value of microdeletion analysis in the work-up of male infertility. In addition, some interesting features of men with deletions have been presented, such as the frequent occurrence of partial deletion within the AZFa region and significantly lower serum FSH concentrations in men with microdeletions compared with infertile men without microdeletions (Ferlin et al., 2007). Molecular diagnosis of microdeletions of the Y chromosome have been performed in the authors institute since 1995, and analysis of partial deletions of the AZFc regions since Over 3000 patients have been analysed for microdeletions and over 800 patients for partial deletions. This report presents the experience, based on 39 patients with AZF deletions and 33 patients with partial deletions of the AZFc region (gr/gr and similar). The experience extends over a decade, allowing some important clinical conclusions, and highlights some differences from findings in other countries. In particular, the clinical data of patients with AZFc deletions or with gr/gr deletions are compared with those of carefully selected patients without deletions and matched by semen characteristics in order to define whether any clinical parameter routinely assessed in the work-up of male infertility may help to identify those patients in whom this genetic testing is indicated. Materials and methods Patients In a prospective study design from November 1995 to May 2007, 3179 patients were screened for microdeletions of the Y chromosome. Among them, 1609 men had a sperm concentration of < /ml, and 2115 had a sperm concentration of < / ml. Part of the data has been presented previously (Simoni et al., 1997; Kamischke et al., 1999; Maurer and Simoni, 2000; Maurer et al. 2001; Luetjens et al., 2002; Rolf et al., 2002). Selection criteria for AZF screening changed over time, as described in detail in a previous report (Maurer and Simoni, 2000). Briefly, from November 1995 to December 1996, only patients with non-obstructive azoospermia or severe oligozoospermia were analysed (n = 228). From January 1997 to May 1997, all patients consulting the infertility clinic were tested, irrespective of their semen analysis (n = 378). After May 1997, only patients with severe oligozoospermia or azoospermia and intracytoplasmic sperm injection (ICSI) candidates were screened (n = 2573). In addition, after the publication of a report of high incidence of AZF deletions in Klinefelter syndrome (Mitra et al., 2006), an additional cohort of 208 patients with Klinefelter syndrome (karyotype 47,XXY) was retrospectively analysed. Screening of partial deletions of the AZFc region began in 2004 in patients with moderate and severe oligozoospermia. From January 2004 until May 2007, 821 patients were analysed. The results concerning the first group of patients (n = 348) have been reported previously (Hucklenbroich et al., 2005). All subjects gave informed consent for genetic analysis of their donated DNA samples according to a protocol approved by the Ethics Committee of the Medical Faculty and State Medical Board. Procedures All patients underwent a complete physical examination including ultrasonographic analysis of the scrotal content. Sonographic (ultrasound scanner type 2002 ADI; BK Medical, Gentofte, Denmark) measurements of the testes were performed by applying a high-frequency 7.5 MHz convex scanner. Volume was calculated using the ellipsoid method (Behre et al., 2000). Bilateral testicular biopsies were performed after scrotal incision and preparation of the tunica albuginea of the testis. Each biopsy sample (about 5 mm in length and 2 mm in diameter) was immediately fixed in Bouin s solution for h and then washed in 70% alcohol. Sections of 4 5 µm were prepared and stained with periodic acid Schiff, and histological analysis was performed. The number of elongated spermatids in 100 or more individual tubuli was counted in each section from each side. A 5 ml volume of EDTA blood was collected from each study subject and genomic DNA was extracted by using the Wizard DNA extraction kit (Promega, Madison, WI, USA) according to the manufacturer s protocol. Microdeletion analysis of the Yq regions was performed according the European Academy of Andrology (EAA)/European Molecular Genetics Quality Network (EMQN) guidelines (Simoni et al., 2004) using the recommended first choice primers and the Qiagen Multiplex PCR Kit (Qiagen, Hilden, Germany), as described previously (Fernando et al., 2006). Analysis of partial deletions within the AZFc region was carried out using two multiplex systems, one including the sequence tagged site primers sy1291, sy1191 and sy1161, and the second including sy1201 and sy1206, as described previously (Hucklenbroich et al., 2005). Deletions were confirmed by repeating the single primer (simplex) reactions. In a subset of 231 patients with negative AZF screening using the EAA/EMQN kit, the two AZFa genes DBY and USP9Y were additionally amplified using the primers DBY forward: 5 -TATTGGCAATCGTGAAA GAC-3, DBY reverse: 5 -TGCCGGTTGCCTCTACTGGT-3, USP9Y forward: 5 -GAGCCCATCTTTGTCAGTTTAC-3, USP9Y reverse: 5 -CTGCCAATTTTCCACATCAACC-3 (annealing temperature 57 C). Conventional karyotype analyses were performed in selected patients at the Institute of Human Genetics of the University of Münster, after Giemsa banding, i.e. staining on GTP-banded metaphase peripheral blood lymphocytes according to standard methods as reported elsewhere (Therman et al., 1980) Analysis of sperm concentration was performed with Neubauer improved chambers and motility was assessed on a heated (37 C) microscope stage. Slides for morphology assessment were stained via the modified Papanicolaou method and examined with phasecontrast optics. All values were determined in accordance with the current World Health Organization criteria (1999). Progressive

3 motility comprises World Health Organization categories a and b. Total sperm count was calculated by multiplying semen volume by sperm concentration. In the authors laboratory, semen analysis is under constant internal and external quality control (Cooper et al., 2002). Serum concentrations of LH, FSH, sex-hormone-binding globulin (SHBG), prolactin, oestradiol and prostatic-specific antigen were determined by immunofluorometric assays (Autodelfia; Perkin Elmer, Freiburg, Germany) and serum testosterone by a commercial direct solid-phase enzyme immunoassay (DRG Aurica Elisa Testosterone kit; DRG Instruments, Marburg, Germany). Free testosterone was calculated from total testosterone and SHBG concentrations (Vermeulen et al., 1999). External quality control for hormones followed the Deutsche Gesellschaft für Klinische Chemie e.v. guidelines (renamed Deutsche Vereinte Gesellschaft für Klinische Chemie und Laboratoriumsmedizin e.v. in 2004). All hormone assays were continuously under internal quality control. Control groups Control groups were set up by retrospectively selecting patients without deletions of the Y chromosome using the Institute s database (Androbase ; Tüttelmann et al., 2006). It is well documented that patients with microdeletions of the Y chromosome can have accompanying infertility-related symptoms such as maldescended testes, varicocoele, etc., and that such symptoms can affect per se the endocrine and clinical parameters. In order to investigate whether Y-chromosomal deletions have independent effects on endocrine and clinical parameters, control groups were established based on patients without microdeletions matched by semen parameters with (control group 1) or without (control group 2) accompanying symptoms. A script was programmed as extension to Androbase thatsystematically searched for matching patients using the following criteria: presence of an accompanying symptom (e.g. maldescensus, varicocoele, infection, etc.) and allowed a range of sperm concentration in each sperm concentration category (set at ±100% for sperm concentrations between / ml and /ml; ±50% for concentrations between / ml and /ml; ±10% for concentrations between / ml and /ml; ±15% for concentrations between /ml and /ml; ±20% for concentrations over /ml). These ranges roughly correspond to the coefficient of variation of sperm concentration in each category in the entire patient cohort archived in Androbase (over 18,000 patients) from 1976 to No range of sperm concentration was applied for azoospermic patients and patients with a concentration below /ml. From all suitable patients with complete clinical information extracted from the database (n = 4347), up to 10 control subjects per study patient were selected using random numbers and formed the control groups. Statistics Data were statistically analysed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA). Frequency distributions were compared by chi-squared tests. Hormone values and semen parameters were compared by nonparametric Kruskal Wallis tests. Results Type and incidence of deletions Using the EAA/EMQN kit, microdeletions of the Y chromosome were found in a total of 54 patients (Table 1). This group included 14 men with AZFabc deletions. Eleven of these were SRY positive men with karyotype 46,XX, while one man had a deletion of the entire long arm of the Y chromosome. The karyotype abnormality was already known in nine of the XX males, in which the AZF diagnosis was performed retrospectively to complete the data set for a separate study (Vorona et al., 2007). However, in two patients it was the AZF diagnosis that permitted the discovery of the chromosomal abnormality. Two additional patients with complete AZFabc deletion and karyotype 46,XX were, in fact, recipients of bone marrow transplantation (von Eckardstein et al., 1999). A partial deletion involving only sy134 was detected in one patient with Klinefelter syndrome (see below). In one patient with a chromosomal translocation [46, X (Y) (q11)(48)/45, X (2)] the AZFbc regions were missing. Among the infertile patients without known karyotype abnormalities, 39 genuine Y-chromosomal microdeletions were identified (Table 2): two AZFa, two AZFb, one AZFbc, one with AZFc deletion partially extending into the AZFb region (sy134, sy254 and sy255 missing), and 32 AZFc (b2/b4) deletions. In addition, one partial AZFb deletion involving only sy130 and sy143 was identified in one oligozoospermic man who spontaneously fathered a son. This case has been reported in detail previously (Rolf et al., 2002). The overall frequency of microdeletions among men without known karyotype abnormalities is 1.2% of all patients screened, corresponding to 1.8% of men with sperm concentration < /ml, and 2.4% of patients with sperm concentration < /ml. The ethnic background of the patients with microdeletions was relatively heterogeneous: 30 patients (77% of all patients with microdeletions) were German, four (10%) were Turkish, two were from the Russian/Slavic area (5%) and one each (2.5%) were Italian, Hispanic and Polish. This distribution corresponds roughly to the ethnic composition of the patients attending the Institute as registered over the last 2.5 years, consisting of about 83% Germans, 5% Turks, 4.4% Russians and Slavs, 1.4% southern Europeans and 6.2% from other areas. It is of note that the two patients with AZFa deletion were not of German origin. Partial deletions within the AZFc region were found in 45 patients, as reported in Table 1. This corresponds to a frequency of 5.5% of all patients screened. Fourteen of these cases have been reported previously (Hucklenbroich et al., 2005). Most partial deletions (62%) were gr/gr deletions (n = 28). In turn, the majority of gr/ gr deletions were found in patients with sperm concentrations of between /ml and /ml (n = 19, 68%); five (18%) were in patients with severe oligozoospermia (< /ml) and 4 (14%) were in patients with azoospermia. Semen analysis, testis histology and assisted reproduction techniques results Table 2 reports the andrological data of the 39 patients with genuine Y-chromosomal microdeletions. Some of these cases 291

4 Table 1. Summary of microdeletions of the Y chromosome found from Deletion n Comment AZFa 2 Partial AZFb 2 One patient with Klinefelter syndrome AZFb 2 AZFc 32 AZFbc 2 One patient with translocation AZFabc 14 One patient with entire long arm of Y chromosome deleted, two recipients of bone marrow transplantation, 11 men with karyotype 46,XX Total AZF 54 sy1191, sy1161, sy sy1291, sy sy1291, sy1201, sy1206, sy b1/b3 3 b2/b3 10 gr/gr 28 Total partial AZFc deletions have been reported previously, as indicated in the table (Simoni et al., 1997; Kamischke et al., 1999; Maurer et al., 2001; Luetjens et al., 2002; Rolf et al., 2002). With the exception of the partial AZFb deletion (ID 11326; Rolf et al., 2002) all patients had azoospermia or severe oligozoospermia, with no case exceeding a sperm concentration of /ml. In all cases with complete AZFa or complete AZFb deletion, azoospermia was consistently present. A few spermatozoa were occasionally present in the ejaculate of the patient with complete AZFbc deletion. Very low sperm concentrations, mostly < / ml, were found in 15/32 (46.9%) patients with AZFc deletion. Repeated semen analysis was performed in 21 patients with AZFc deletion, showing only minor oscillations of sperm concentrations without any particular trend over time. In seven patients at least four semen samples or more (up to 20) were obtained over 2 5 years, again showing no trend. In four of such patients, azoospermia alternated with the occasional finding of a few spermatozoa in the ejaculate. In four patients, serum FSH concentrations were measured repeatedly over up to 5 years and no significant increase over time was observed (data not shown). Similarly, no significant difference was found in the age of the patients at first presentation between men with consistent azoospermia (n = 18, 32 ± 5.2 years) and men with spermatozoa present in the ejaculate (n = 15, 36 ± 7.3 years). Open bilateral testicular biopsy was performed in 22 cases. Testicular sperm extraction (TESE) was performed in 16 patients. In both patients with AZFa deletion, the biopsy sample showed a complete bilateral Sertoli cell-only (SCO) picture and no spermatozoa could be recovered upon TESE. The two patients with AZFb deletion showed spermatogenetic arrest at the level of spermatocytes and round spermatids. Interestingly, 2% of the tubuli showed elongated spermatids in one patient (ID 14163) but no spermatozoa were recovered upon TESE. Biopsy data were available from 15 patients with AZFc deletion and one with partial AZFb+AZFc. Elongated spermatids were the most advanced germ cells found in 6/15 (40%) AZFc patients, in two patients few spermatozoa were present in the ejaculate. Spermatozoa were recovered upon TESE in three patients (ID 11675, and 15796), and ICSI was tried twice for one man (ID 16675) without success. Complete bilateral SCO was found in 4/15 (27%) AZFc patients. One of these was orchiectomized because of a Leydig cell tumour in the left testis. Two patients (ID 9528 and 11911) had complete SCO on one side and spermatocytes or spermatogonia as the most advanced germ cell type in only 5 and 3% of the tubuli in the contralateral biopsy, respectively. Adding these two cases to those of complete bilateral SCO makes up 40% (6/15) of patients with no or only very few immature germ cells in the testis. The remaining three patients had a bilateral arrest at the level of spermatocytes (two cases with a few spermatozoa in the ejaculate) or round spermatids (one case with azoospermia). TESE was attempted in these three patients but spermatozoa were found only in the two with spermatocytic arrest and spermatozoa present in the ejaculate (ID and 13238). In one patient (ID 11227) ICSI was performed three times with ejaculated spermatozoa and once with TESE spermatozoa but no pregnancy was achieved. Taken together, the biopsy and TESE data show that in patients with azoospermia and AZFc deletion the histological picture is variable, but the spermatogenetic impairment is very severe in the majority of the cases. The probability of finding spermatozoa upon TESE is 60% (6/10). In three of the six TESE-positive cases ICSI was attempted without success. The only case in which ICSI was successfully performed concerned a patient with AZFc deletion and severe oligozoospermia (ID 9954). A pregnancy was obtained by ICSI with ejaculated spermatozoa and resulted in the birth of a healthy boy who was carrier of the same mutation; this has been reported previously (Kamischke et al., 1999). Clinical and hormonal parameters in patients with microdeletions of the Y chromosome In order to answer the question whether any clinical parameter could help to identify patients with AZFc (n = 33, including

5 Table 2. Sperm concentration, testis histology and assisted reproduction treatment results in 39 patients with AZF deletions. Pregnancy/ART (ejaculate/tese) Testis histology (% of tubuli) Sperm found upon TESE (right/left) Right Left Sperm concentration ( 10 6 /ml) (range or maximum) Deletion No. of semen analyses Patient ID Age (years) 9980 a,b 30 AZFa SCO (complete SCO) 100 SCO (complete SCO) 0/0 ND/ND AZFa SCO (complete SCO) 95 SCO, 5 TS (complete SCO) 0/0 ND/ND a,b,c 30 Partial AZFb Natural pregnancy (sy130, sy143) 0/0 ND/ND 9816 a,b 33 AZFb SG, 99 SCO (arrest: SG) 55 SC, 23 SG, 22 SCO (arrest: SC) 5 RS, 92 SC, 0 SG, 3 SCO (arrest: 0/0 ND/ND RS) 100 SC, 0 SG, 0 SCO (arrest: SC) 0/0 ND/ND AZFb ES, 1 RS, 2 SC, 1 SG, 8 SCO, 86 TS (residual spermatogenesis) a,b 31 AZFbc 5 0 < SC, 0 SG, 0 SCO, 22 TS (arrest: SC) b 34 Partial AZFb SC, 0 SG, 96 SCO, 3 TS (arrest: 87 SCO, 13 TS (complete SCO) 0/2 ND/ND AZFc SC) 6892 b,d 39 AZFc SCO (complete SCO) 100 SCO (complete SCO) ND 8091 b,d 30 AZFc 1 0 ND 9484 b,d 28 AZFc ES, 1 RS, 6 SC, 0 SG, 90 SCO 100 SCO (complete SCO) ND (residual spermatogenesis) 9528 b,d 25 AZFc SC, 0 SG, 95 SCO (arrest: SC) 100 SCO (complete SCO) ND 9781 b,d 35 AZFc % SCO (complete SCO) 100% SCO (complete SCO) ND 9954 a,b,e 28 AZFc ICSI, one son a,b 46 AZFc 2 0 ND a,b 23 AZFc SC, 3 SG, 87 SCO (arrest: SC) 100 SCO (complete SCO) 0/0 ND/ND 0/3 ND/2 ICSI, no pregnancy 5 SC, 8 SG, 68 SCO, 19 TS (arrest: SC) a,b 32 AZFc ES, 7 RS, 24 SC, 9 SG, 40 SCO (reduced spermatogenesis) a,b 31 AZFc Anamnestic SCO (complete SCO) 100 SCO (complete SCO) a,b 35 AZFc SC, 2 SG, 96 SCO (arrest: SC) 5 RS, 62 SC, 10 SG, 24 SCO 0/1 3 ICSI/1 ICSI, no (arrest: RS) pregnancy a,b 32 AZFc ND a,b 31 AZFc 2 <0.1 ND a,b 33 AZFc SCO, 13 TS (complete SCO) 3 SG, 79 SCO, 18 TS (arrest: SG) 0/0 ND/ND Continued on page

6 294 Table 2 continued from page 293 Pregnancy/ART (ejaculate/tese) Testis histology (% of tubuli) Sperm found upon TESE (right/left) Sperm concentration ( 10 6 /ml) (range or maximum) Deletion No. of semen analyses Patient ID Age (years) Right Left b 26 AZFc SC, 3 SG, 92 SCO (arrest: SC) 10 SC, 10 SG, 79 SCO (arrest: SC) 0/0 ND/ND b 37 AZFc 2 0 ND b 35 AZFc 3 0 ND b 35 AZFc ES, 29 RS, 11 SC, 2 SG, 2 SCO 1 RS, 49 SC, 20 SG, 27 SCO, 2 TS 1 ICSI, no pregnancy (reduced spermatogenesis) (residual spermatogenesis) b 30 AZFc 9 0 <0.1 5 RS, 82 SC, 3 SG, 8 SCO, 3 TS 2 RS, 88 SC, 0 SG, 4 SCO, 7 TS 4/2 1 ICSI/ND, no pregnancy (arrest: RS) (arrest: RS) b 29 AZFc 2 <0.1 ND 2/0 ND/ND AZFc SCO, 3 TS (complete SCO) 100 SCO (Leydig cell tumour + complete SCO) AZFc 1 <0.1 ND AZFc 2 < ES, 7 RS, 56 SC, 10 SG, 96 SCO, 4 TS (complete SCO) 4/3 ND/ND 13 SCO, 3 TS (reduced spermatogenesis) AZFc 1 0 ND AZFc ICSI, no pregnancy 4/8 ND/ND 2 ES, 1 RS, 21 SC, 1 SG, 75 SCO (residual spermatogenesis) AZFc ES, 6 RS, 17 SC, 6 SG, 56 SCO (reduced spermatogenesis) AZFc 1 0 ND 0/0 ND/ND 2 ES, 0 RS, 67 SC, 2 SG, 26 SCO, 3 TS (residual spermatogenesis) AZFc SC, 3 SG, 51 SCO, 3 TS(arrest: SC) AZFc ND AZFc ND AZFc 2 <0.1 ND a Maurer et al., 2001; b Luetjens et al., 2002; c Rolf et al., 2002; d Simoni et al., 1997; e Kamischke et al., ART = assisted reproduction treatment; ES = elongated spermatids; ICSI = intracytoplasmic sperm injection; RS = round spermatids; SC = spermatocytes; SG = spermatogonia; SCO = Sertoli cell only; TESE = testicular sperm extraction; TS = tubule shadows; ND = not done.

7 the one patient with partial AZFb+c) and gr/gr microdeletions (n = 28), their clinical and hormonal data were compared with those of two groups of patients without microdeletions matched by sperm concentration. The two control groups consisted of patients with infertility and associated disorders such as varicocoele, maldescended testes, infections, etc. (control group 1) and patients with complete idiopathic infertility (control group 2). As shown in Tables 3 and 4, differences could be found neither in the incidence of associated symptoms nor in the hormonal and seminological parameters in patients with AZFc and gr/gr deletions. Therefore, these data obtained from the comparison to patients displaying exactly the same seminological phenotype, exclude that either low (Foresta et al., 2001; Ferlin et al., 2007) or elevated (Wang et al., 2005) FSH concentrations might be characteristic of patients with deletions. Klinefelter syndrome Only one partial deletion involving sy134 in the AZFb region was found in one out of 208 patients with Klinefelter syndrome. This marker was confirmed to be absent in repeated amplification attempts at lower annealing temperature. These data exclude that patients with Klinefelter syndrome have a high incidence of microdeletions of the Y chromosome, as suggested by Mitra et al. (2006). Further screening of the AZFa region in negative patients using the EAA/EMQN kit Since partial deletions within the AZFa region not detected by Table 3. Clinical and hormonal parameters in patients with AZFc deletions compared with patients matched by semen parameters with (control group 1) or without (control group 2) other concomitant symptoms. Diagnosis AZFc Control group 1 Control group 2 (n = 33) (n = 330) (n = 66) Testicular tumour (%) a 6.1 (2) 10.0 (33) 0.0 (0) Malignant disease (e.g. lymphoma, leukaemia) (%) a 3.0 (1) 5.2 (17) 0.0 (0) Maldescensus (current or former) (%) a 12.1 (4) 22.4 (74) 0.0 (0) Varicocele (current or former) (%) a 27.3 (9) 20.0 (66) 0.0 (0) Infections (current or former) (%) a 21.2 (7) 16.4 (54) 0.0 (0) Endocrine disorders (e.g. thyroid, adrenal) (%) a 3.0 (1) 7.0 (23) 0.0 (0) Metabolic disorders (e.g. diabetes) (%) a 12.1 (4) 9.7 (32) 0.0 (0) Chronic disease (e.g. asthma, hypertension) (%) a 9.1 (3) 9.1 (30) 0.0 (0) Age (years) b 33 (30 36) 32 (28 35) 32 (27,75 37) Height (cm) b 180 ( ) 182 ( ) 182 ( ) Weight (kg) b 78 (70 89) 83 (75 93) 84 (75 95) Body mass index (kg/m 2 ) b 24.7 ( ) 25.2 ( ) 25.5 ( ) Combined testicular volume (ml) b 29 (21 38) 27 (19 38) 28 (20 39) LH (IU/l) b 4.4 ( ) 5.1 ( ) 4.5 ( ) FSH (IU/l) b 12.8 ( ) 13.2 ( ) 11.1 ( ) Testosterone (nmol/l) b 15.1 ( ) 14.8 ( ) 14.4 ( ) SHBG (nmol/l) b 35 (22 44) 32 (24 42) 31 (21 42) Free testosterone (pmol/l) b 331 ( ) 303 ( ) 298 ( ) Prolactin (miu/l) b 224 ( ) 196 ( ) 187 ( ) Oestradiol (pmol/l) b 63 (54 92) 70 (55 85) 66 (55 82) PSA (µg/l) b 0.6 ( ) 0.6 ( ) 0.5 ( ) No. with azoospermia c 18 (56) 185 (56) 37 (56) No. with sperm concentration < /ml c 9 (28) 90 (27) 18 (27) No. with sperm concentration /ml c 5 (16) 55 (17) 11 (17) Abstinence (days) b 3.5 (3 5) 4 (3 5) 4 (2 5) Sperm concentration ( 10 6 /ml) b 0.2 ( ) 0.6 ( ) 0.7 ( ) Semen volume (ml) b 4.2 ( ) 3.5 ( ) 3.6 ( ) Total sperm count ( 10 6 ) b 0.8 ( ) 1.6 ( ) 1.9 ( ) a+b Motility (%) b 18 (11 27) 30 (18 41) 36 (27 49) Normal morphology (%) b 4 (1 7) 6 (2 11) 5 (1 13) Results are given as frequency (%) and number of observations (n) or median and range. a Values in parentheses are numbers of patients; b values in parentheses are ranges; c values in parentheses are percentages. There were no statistically significant differences between the patients with deletions and the controls. PSA = prostatic-specific antigen; SHBG = sex hormone-binding globulin. 295

8 Table 4. Hormonal parameters in patients with gr/gr deletions compared with patients matched by semen parameters with (control group 1, matched controls) or without (control group 2, idiopathic controls) other concomitant symptoms. Diagnosis AZF gr/gr Matched controls Idiopathic controls (n = 28) (n = 280) (n = 112) Testicular tumour (%) a 0.0 (0) 0.0 (0) 0.0 (0) Malignant disease (e.g. lymphoma, leukaemia) (%) a 0.0 (0) 0.0 (0) 0.0 (0) Maldescensus (current or former) (%) a 25.0(7) 36.4 (102) 0.0 (0) Varicocele (current or former) (%) a 25.0 (7) 35.0 (98) 0.0 (0) Infections (current or former) (%) a 21.4 (6) 27.1 (76) 0.0 (0) Endocrine disorders (e.g. thyroid, adrenal) (%) a 3.6 (1) 11.1 (31) 0.0 (0) Metabolic disorders (e.g. diabetes) (%) a 21.4 (6) 14.6 (41) 0.0 (0) Chronic disease (e.g. asthma, hypertension) (%) a 10.7 (3) 6.4 (18) 0.0 (0) Age (years) b 34 (30 37) 32 (29 37) 32 (29 36) Height (cm) b 181 ( ) 182 ( ) 183 ( ) Weight (kg) b 86 (76 94) 85 (76 96) 88 (78 96) Body mass index (kg/m 2 ) b 25.3 ( ) 25.6 ( ) 26.5 ( ) Combined testicular volume (ml) b 40 (27 45) 34 (25 42) 36 (26 46) LH (IU/l) b 4.5 ( ) 4.1 ( ) 3.5 ( ) FSH (IU/l) b 8.4 ( ) 7.0 ( ) 5.7 ( ) Testosterone (nmol/l) b 16.1 ( ) 15.5 ( ) 15.5 ( ) SHBG (nmol/l) b 32 (25 40) 32 (23 41) 29 (21 43) Free testosterone (pmol/l) b 310 ( ) 312 ( ) 308 ( ) Prolactin (miu/l) b 212 ( ) 179 ( ) 192 ( ) Oestradiol (pmol/l) b 66 (54 73) 67 (55 82) 69 (54 82) PSA (µg/l) b 0.7 ( ) 0.6 ( ) 0.6 ( ) No. with azoospermia c 4 (14) 40 (14) 16 (14) No. with sperm concentration < /ml c 5 (18) 50 (18) 20 (18) No. with sperm concentration /ml c 19 (68) 190 (68) 76 (68) Abstinence (days) c 3 (3 4) 4 (3 5) 4 (3 5) Sperm concentration ( 10 6 /ml) b 3.6 ( ) 2.6 ( ) 2.8 ( ) Semen volume (ml) b 3.6 ( ) 3.6 ( ) 3.5 ( ) Total sperm count ( 10 6 ) b 8.2 ( ) 8.2 ( ) 10.1 ( ) a+b Motility (%) b 39 (24 49) 38 (26 49) 36 (26 50) Normal morphology (%) b 8 (3 12) 8 (5 16) 8 (4 16) Results are given as frequency (%) and number of observations (n) or median and range. a Values in parentheses are numbers of patients; b values in parentheses are ranges; c values in parentheses are percentages. There were no statistically significant differences between the patients with deletions and the controls. PSA = prostatic-specific antigen; SHBG = sex hormone-binding globulin. 296 the EAA/EMQN kit are found in some populations even in patients with oligozoospermia (Ferlin et al., 2007; Krausz et al., 2006), two gene-specific markers (DBY, USP9Y) were analysed in a subgroup of patients with negative AZFa screening. This group consisted of 46 men with azoospermia, 51 men with sperm concentration < /ml and 134 men with a sperm concentration between /ml and /ml. No additional deletion was found. Discussion Microdeletions of the Y chromosome are one of the bestestablished genetic causes of male infertility and their molecular diagnosis is one of the most popular tests in molecular genetics and in the work-up of the infertile male (Vogt, 2005; Jequier, 2006). In the last decade several groups have reported large collections of infertile patients with microdeletions from all over the world, and valuable review articles have been published analysing important aspects such as the frequency of microdeletions, the genotype/phenotype correlations, the indication for diagnosis and the prognostic value of various clinical parameters (Maurer and Simoni, 2000; Foresta et al., 2001; Krausz and Degl Innocenti, 2006). From the data in the literature the overall incidence of microdeletions was originally estimated to be around 8% of men with nonobstructive azoospermia, and between 3 and 5% in severely oligozoospermic men (Simoni et al., 1998; Foresta et al., 2001). It was also established that microdeletions removing the AZFa or the AZFb region are invariably associated with azoospermia with virtually no possibility of recovering spermatozoa upon

9 TESE (Oates et al. 2002; Simoni et al., 2004). Other aspects, such as the possibility that sperm parameters deteriorate over time in men with AZFc deletions (Simoni et al., 1997; Ferlin et al., 2007), require more experience and longer observation. Recently a very comprehensive overview of a large collection of Italian patients reported valuable clinical data of patients with Y-chromosomal microdeletions that should help clinicians to define the diagnostic and prognostic value of this investigation (Ferlin et al., 2007). The independent extensive experience of over one decade in Germany provided by the present study highlights the heterogeneity of clinical aspects of Y-chromosomal microdeletions in different populations. In the experience of the authors, microdeletions of the Y chromosome are rare and the vast majority (over 80%) are deletions involving the AZFc region. With an overall frequency of only 2.4% of patients with sperm concentration < /ml, the incidence is much lower than that of Klinefelter syndrome. Of the 8800 patients who visited the authors institute from 1996, the time when the molecular screening of microdeletions began, only 39 patients with microdeletions were observed compared with 225 patients with Klinefelter syndrome. This might in part reflect the particular character of the institute, which is a tertiary referral centre for male infertility and hypogonadism, while other centres might have a less-selected clientele. However, microdeletion frequency seems to be lower in the German and Scandinavian areas compared with other countries of the world (Figure 1 and Appendix). It is unclear whether the Y-chromosomal background, e.g. the haplogroup, plays any role in the propensity of the Y chromosome to undergo deletions, and the literature is not unanimous on this subject (Carvalho et al., 2003; Arredi et al., 2007). In this study, haplogroup analysis was not performed. Moreover, the ethnic composition of the patients with microdeletions did not provide enough statistical power to clarify this point. In addition, other genetic/epigenetic factors might also be involved. Finally, a more detailed analysis of the AZFa region in over 200 patients confirmed that the EAA/ EMQN kit is appropriate for routine analysis in the authors region and that an underestimation of clinically relevant deletions is improbable. It is concluded that the prevalence of AZF microdeletions in the authors centre and in Germany is much lower than that reported in most papers published from other regions (see Appendix). Similarly, a high incidence of complete SCO (40%) was observed in men with AZFc, with a very poor prognosis for TESE/ICSI (see below). These findings suggest that geographical differences in the incidence of deletions and in the severity of the phenotype exist (Hopps et al., 2003). The data confirm that complete deletions of AZFa or AZFb are associated with azoospermia. Complete, bilateral SCO is the histological feature of AZFa deletions and of 50% of AZFb deletions. Patients with AZFc deletions have an only slightly milder phenotype and it is shown here that SCO is also present in the majority of these patients. This is reflected by the semen parameters, which show azoospermia in about half the patients, and only a few spermatozoa in the ejaculate occasionally present in the other half. Repeated semen analysis might be useful in such patients, since spermatozoa may occasionally appear in the ejaculate, but in our experience the prognosis for ICSI is unfavourable. When the hormonal data of the patients with AZFc deletion were compared with those of patients matched by semen characteristics, no differences could be found. Special attention was paid to selecting the control groups so that they consisted of patients with exactly the same clinical characteristics, except for the microdeletion. In this way it was possible to exclude that the microdeletions per se would affect spermatogenesis and hormonal parameters differently than other known andrological conditions such as maldescended testes, varicocoele, etc. Moreover, the analysis shows that the concomitance of deletion and other andrological problems does not seem to worsen seminal and hormonal parameters. Indeed serum FSH concentrations were not lower in patients with AZFc deletions compared with properly matched infertile men without deletion. Therefore, it was not possible to identify any clinical parameter that would help to identify patients with microdeletions. Considering that microdeletions are quite rare and the phenotype was severe in our German cohort of patients, molecular diagnosis of Y chromosome is currently being restricted to men with azoospermia or sperm concentration < /ml, irrespective of the presence of other symptoms and serum FSH concentrations. It had been suggested that semen parameters might worsen with time in men with AZFc deletions. In the authors first study there was a report of two patients who were azoospermic when attending the institute but had had spermatozoa in the ejaculate assessed earlier in other institutions (Simoni et al., 1997). From this observation it was speculated that sperm parameters could change with time. Some indirect observations such as the difference in age between microdeleted patients with azoospermia and oligozoospermia or the increase of FSH concentrations over time in some subjects further substantiated the experience of Ferlin et al. (2007). In the present study it was not possible to document any of these changes and neither any decrease in sperm concentration nor increases in FSH concentrations over time was observed. However, it cannot be excluded that spermatogenesis was already so damaged at the time of consultation in the institute that it could not worsen any further. If a trend towards deterioration of spermatogenesis over time exists, this cannot be demonstrated in the patients who already had extremely low sperm count at the time of first presentation in the department. A previous report suggested a high incidence of Y-chromosomal microdeletions in a small group of 14 Indian patients with Klinefelter syndrome (7/14, 50%) (Mitra et al., 2006). These deletions were rather unconventional, involving only isolated markers of the AZFa and/or the AZFb region. Since these deletions were not confirmed with an independent method such as Southern blotting, they should probably be regarded as methodological artefacts. Indeed, based on much larger cohorts of patients, it is confirmed here that deletions of Y chromosome do not occur in patients with Klinefelter syndrome. While AZF deletions play a causal role in spermatogenetic failure, the impact of gr/gr deletions on spermatogenesis is still debated. gr/gr deletions have been identified as a polymorphism significantly associated with infertility, especially oligozoospermia (Repping et al., 2003). They are found in about 3 5% of infertile men, but also in controls. In the authors experience gr/gr deletions are not significantly more frequent in infertile compared with control subjects (Hucklenbroich et al., 2005). However, a meta-analysis of the literature demonstrates a significant association with infertility 297

10 Figure 1. Worldwide frequencies of AZF deletions among patients with non-obstructive azoospermia (< /ml) studied so far. The data utilised can be found in the Appendix. Percentages are coded in colours according to the legend. Note: Sweden, Germany and Austria show the lowest incidence. (Tüttelmann et al., 2007). The present study investigated whether any clinical parameter would characterize patients with gr/gr deletions. No significant difference was found in seminal and hormonal parameters compared with patients matched by sperm concentration, independently of whether other symptoms were present or not. Therefore, it is concluded that the phenotype associated with gr/gr deletions in infertile men does not differ from that of infertility without gr/gr deletions. Genes belonging to five families are removed by gr/gr deletions and it is possible that gene dosage influences spermatogenesis quantitatively more than qualitatively, but no correlation between sperm concentration/number and number or type of deleted copies has been observed so far. Even if gr/gr deletions are a risk factor for spermatogenetic failure in certain individuals, at present there seems to be no advantage gained from this type of diagnosis in male infertility work-up. Acknowledgements We thank the physicians of the Institute of Reproductive Medicine who were involved in patient care and the many technical assistants who performed semen analysis over the years of the study. S Borchert and N Terwort performed the molecular screening. We are grateful to Professor S Kliesch and Dr CM Luetjens for taking and analysing testicular biopsies, respectively. We thank S Nieschlag, MA, for language editing of the manuscript. The authors report no financial or commercial conflicts of interest. 298 In conclusion, the results demonstrate that in north-west Germany the frequency of AZF deletions is much lower than in other countries, that AZFc deletions are associated with very severe testicular phenotypes and that TESE is not possible in almost half of these patients. Neither AZFc nor gr/gr deletions are associated with any clinical or diagnostic parameter, including FSH concentration. Finally, no trend in sperm concentration and FSH concentration over time is apparent.

11 Appendix Frequency of Y chromosomal microdeletions in patients (n = 16,316) with non-obstructive azoospermia and severe oligozoospermia (sperm concentration < /ml) and normal karyotype in different countries. Country Frequency No. of Reference Comment (%) a patients Australia Lynch et al., 2005 Austria Gruber et al., 2003 No deletion using 18 STS (Promega) Belgium Van Landuyt et al., 2000 Brazil (São Paulo) Pina-Neto et al., 2006 Brazil (São Paulo) SaoPedro et al., 2003 Brazil (São Paulo) Pieri Pde et al., 2002 Croatia Medica et al., 2005 Croatia 4.5? Sertic et al., 2001 Cyprus Ioulianos et al., 2002 Czech Republic Machatková et al., 2003 Czech Republic Kolárová et al., 2001 Denmark Cruger et al., 2003 Denmark Bor et al., 2002 Denmark Krausz et al., 2001 Egypt El Awady et al., 2004 Single STS deletions found Finland Aho et al., 2001 France (Paris) Le Bourhis et al., 2000 France (Paris) Krausz et al., 1999b France (Clermont-Ferrand) Seifer et al., 1999 Number of patients with sperm concentration > unclear France (Kremlin Bicetre) Selva et al., 1997 Germany (Bonn) van der Ven et al., 1997 One (out of two) partial deletion with moderate oligozoospermia Germany (Leipzig) Tzschach et al., 2001 Germany (multicentre) Vogt et al., 1996 Germany (Westfalia) This report Greece Giannouli et al., 2004 India (Varanasi) Ambasudhan et al., 2003 Partial deletions in some cases India (Hyderabad) Thangaraj et al., 2003 India (Andhra Pradesh) Swarna et al., 2004 India (New Dehli) Dada et al., 2006 India (New Delhi) Dada et al., 2004 India (New Delhi) Dada et al., 2003 At least one STS in some cases India (New Delhi) Dada et al., 2002 India (North) Mittal et al., 2004 India (South) Rao et al., 2004 India (South) Babu et al., 2002 Only one marker/region screened Iran Omrani et al., 2006 Several partial deletions Ireland Friel et al., 2001 Israel Madgar et al., 2002 Israel Kleiman et al., 1999 Number of patients with sperm concentration > unclear (but <15) Italy (Florence) Krausz et al., 1999a Italy (Padua) Ferlin et al., 2007 Italy (Rome) Grimaldi et al., 1998 Japan (Kanazawa) Fukushima et al., 2006 Non-contiguous deletions found Japan (Oita) Kihaile et al., 2004 Japan (Ishikawa) Nakashima et al., 2002 Five patients with single deletions using 58 STS Japan (Chiba) Shimizu et al., 2002 Only azoospermic men screened Japan (Nishinomiya) Sawai et al., 2002 Several inconsistent deletion patters Japan (Kobe) Fujisawa et al., 2001 Only azoospermic patients studied Japan (Tokyo) Nakahori et al., 1996 Some inconsistent deletions Korea Lee et al., 2003 One or more deleted STS of 13 loci Continued on page

12 Appendix continued from page 299 Country Frequency No. of Reference Comment (%) a patients Mexico Calleja Macías et al., 2003 New Zealand Kerr et al., 2000 PR China (Nanjing) Song et al., 2006 PR China (Chengdu) Zhou-Cun et al., 2006 PR China (Nanjing) Song et al., 2005 PR China (Shaanxi) Cui et al., 2005 PR China (Guandong) Cai et al., 2005 PR China (Changsha) Fu et al., 2002 PR China (Beijing) Yao et al., 2001 Several inconsistent deletion patterns PR China (Nanjing) Wu et al., 2007 PR China (Hong Kong) Tse et al., 2002 PR China (Shangai) PR China (Hong Kong) Tse et al., 2000 Poland Sobczynska-Tomaszewska et al., 2006 R. Macedonia Plaseski et al., 2006 Romania Raicu et al., 2003 Russia Chernykh et al., 2006 Saudi Arabia Hellani et al., 2006 Singapore Liow et al., 1998 Slovenia Peterlin et al., 2002 Spain (Valencia) Martinez et al., 2000 Spain (Barcelona) Oliva et al., 1998 Sri Lanka Fernando et al., 2006 Sweden Osterlund et al., 2000 Taiwan Chen et al., 2003 Taiwan Lin et al., 2002 Taiwan Lin et al., 2001 Only two partial AZFa found Taiwan Lin et al., 2000 Some non-contiguous deletions Thailand Vutyavanich et al., 2007 The Netherlands Dohle et al., 2002 The Netherlands Kremer et al., 1997 Mainly oligozoospermic men studied Tunisia (Sfax) Abdelmoula et al., 2006 Same city as Hadj-Kacem et al., 2006 Tunisia (Sfax) Hadj-Kacem et al., 2006 Same city as Abdelmoula et al., 2006 Turkey (Ankara) Vicdan et al., 2004 Turkey (Izmir) Okutman-Emonts et al., 2004 Turkey (Antalya) Sargin et al., 2004 USA (St Louis, MO) Silber et al., 1998 USA (New York) Rucker et al., 1998 Five deletions with chromosomal abnormalities USA (TX) Vereb et al., 1997 Deletions only in azoospermic men. Number of patients with sperm concentration > unclear USA (CA) Najmabadi et al., 1996 Venezuela Fernández-Salgado et al., 2006 a Mean frequency = 7.84; STS = sequence tagged site. 300

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