Screening for Y chromosome microdeletions in 226 Slovenian subfertile men

B. Peterlin1,4, T. Kunej1, J. Sinkovec2, N. Gligorievska1 and B. Zorn3

1 Division of Medical Genetics, 2 Department of Pathology and 3 Andrology Centre, Department of Obstetrics and Gynecology, University Medical Centre, 1000 Ljubljana, Slovenia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The objective of this study was to estimate the frequency of Y chromosome microdeletions in the Slovenian population of infertile men and to analyse the consequences of mutation in respect to clinical severity and prognosis. METHODS: In a controlled clinical study at the university-based medical genetics service and infertility clinic, 226 infertile men undergoing ICSI were tested. The main outcome measures included polymerase chain reaction amplification of 16 genes and gene families and 42 sequence-tagged sites in the non-recombining region of the Y chromosome, semen, testicular volume and testicular histological analysis, serum FSH concentrations, fertilization and respective pregnancy rates. RESULTS: The incidence of deletions was 4.4%: 8.6% in men with azoospermia and 1.5% in men with oligoasthenoteratozoospermia. Isolated gene deletions were not identified. No statistically significant differences in clinical outcome measures were found in patients with mutations versus patients without mutations. High fertilization (49%) and pregnancy (43%) rates with sperm of patients with Y chromosome deletions were obtained. CONCLUSIONS: Testing for gene-specific microdeletions does not contribute significantly to the sensitivity of microdeletion test. Fertilization and pregnancy rates obtained using sperm of patients with Y chromosome deletions were comparable with those achieved in conventional IVF.

Key words: azoospermia/ICSI/male infertility/oligozoospermia/Y chromosome microdeletions


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Infertility occurs in 10–15% of couples and male factor infertility represents >50% of the cases. Men with azoospermia and oligoasthenoteratozoospermia (OAT) represent ~40–50% of all infertile men (Thonneau et al., 1991Go). However, in ~30% of cases the origin of reduced male fertility is unknown.

Deletions of the long arm of the Y chromosome are frequently associated with a failure of spermatogenesis (Tiepolo and Zuffardi, 1976Go; Ma et al., 1992Go; Vogt et al., 1992Go; Reijo et al., 1995Go). Attempts to correlate a deletion pattern with the clinical picture of infertility have been only partially successful. Three regions of the Y chromosome, azoospermia factor (AZF), AZFa, AZFb and AZFc, have been defined based on the deletion analysis of 13 patients with interstitial deletions, and respective clinical phenotypes proposed (Vogt et al., 1996Go). Several genes have been identified in the AZF region (Ma et al., 1993Go; Reijo et al., 1995Go; Lahn and Page, 1997Go; Brown et al., 1998Go), however, a single case could have been causally related to a point mutation in the gene (Sun et al., 1999Go). The physiological function related to male fertility has been demonstrated in the mouse homologues of the Deleted in AZoospermia (DAZ) and RNA-binding motif (RBMY1) genes, yet their exact role in human male spermatogenesis remains to be established (Ma et al., 2000Go).

Although the causal relationship between Y chromosome deletions and reduced testicular function is still poorly understood, the screening for deletions has become widely used in the evaluation of male infertility, because it is important for elucidating the aetiology and possible prognosis of infertility. Additionally, testing provides information to the couple undergoing treatment by assisted reproduction techniques about the risk of infertility in their male offspring, since Y chromosome deletions may be iatrogenically transmitted to sons (Page et al., 1999Go) after intracytoplasmic sperm injection (ICSI).

For the clinical application of Y chromosome deletion testing, further data need to be elucidated: (i) reliable data on the frequency of deletions in the heterogeneous group of infertile men is still lacking and varies between 1 and 55% (Henegariu et al., 1994Go; Kobayashi et al., 1994Go; Reijo et al., 1995Go, 1996Go; Najmabadi et al., 1996Go; Qureshi et al., 1996Go; Stuppia et al., 1996Go; Vogt et al., 1996Go; Foresta et al., 1997Go, 1998Go; Girardi et al., 1997Go; Kremer et al., 1997Go; Mulhall et al., 1997Go; Peterlin et al., 1997Go; Pryor et al., 1997Go; Simoni et al., 1997Go; van der Ven et al., 1997Go; Vereb et al., 1997Go; Brandell et al., 1998Go; Grimaldi et al., 1998Go; Liow et al., 1998Go; Oliva et al., 1998Go; Silber et al., 1998Go; Kent-First et al., 1999Go; Kim et al., 1999Go; Kleiman et al., 1999Go; Krausz et al., 1999aGo,bGo; Seifer et al., 1999Go); (ii) information on the frequency of deletions in various subgroups of men with non-idiopathic infertility with reduced testicular function is still accumulating (Foresta et al., 1999Go; Krausz et al., 1999bGo; Moro et al., 2000Go); (iii) so far infertile patients have not been systematically tested for currently known genes on the long arm of the Y chromosome. A single study which partially addressed this question (Krausz et al., 1999aGo) tested six genes (DAZ, DBY, CDY, XKRY, eIF-1A and BPY2) in 90 subfertile patients; (iv) more data are needed for a reliable analysis of the correlation between the pattern of deletion and clinical severity and prognosis.

The aim of this study was to analyse infertile men for deletions of 16 genes and gene families (DAZ, SPGY, RBMY1, RBMY2, TTY1, TTY2, PRY, DFFRY, DBY, UTY, TB4Y, BPY1, CDY, XKRY, eIF-1AY, BPY2) and 42 sequence-tagged site (STS) markers of the Y chromosome. Deletion patterns were correlated with the clinical phenotype such as spermiological, hormonal and histological findings; additionally, a potential effect of these deletions on the fertilization and pregnancy rates was evaluated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
We analysed a group of 263 infertile patients, attending the outpatient infertility clinic of the Andrology Centre, Department of Obstetrics and Gynecology in Ljubljana. They were enrolled in the study because of poor sperm quality. Before an ICSI attempt, each man underwent an andrological examination. Special attention was paid to a history of hypogonadism, cryptorchidism, varicocele, genito-urinary tract surgery, testicular cancer and medication. The testicular volume was measured using Prader's orchidometer. Eleven patients with andrological history, three with a history of testicular carcinoma, seven with congenital bilateral absence of vas deferens (CABVD), one with obstructive azoospermia as a consequence of a genito-urinary tract infection and 26 patients with cytogenetic abnormalities were excluded from the study. Thus, the study group consisted of 226 patients, 92 men with azoospermia and 134 with OAT.

The control group consisted of 125 men with proven fertility; blood samples were analysed for the presence of microdeletions. As a negative control, the blood of 10 women was analysed.

Testing for the origin of microdeletions (inherited or occurring de novo) was not possible, because no blood of any patient's father or brother(s) was available. Informed consent was obtained from each patient. All patients were Slovene or of Slavonic origin. The study was approved by the national medical ethics' committee.

Semen analysis: spermiogram
In all 226 men, semen was assessed according to published guidelines (World Health Organization, 1999Go) in terms of the volume, sperm count, rapid progressive sperm motility and morphology by means of techniques described elsewhere (Zorn et al., 1999Go).

Hormonal evaluation
Plasma FSH was measured by microparticle enzyme immunoassay (AxSYM System; Abbott, Chicago, Illinois, USA) in 145 patients. Normal range was <8 mIU/ml.

Testicular biopsy, histological and cytological evaluation
Ninety-eight testicular biopsies were performed in 92 azoospermic men and six men with OAT. Testicular biopsy was performed under local anaesthesia. Because men were included in an ICSI programme, testicular biopsy was often of both diagnostic and therapeutic value. Following unilateral hemiscrototomy, a small testicular incision was made and at least two samples of testicular tissue were taken from each testis. The first sample was fixed in Bouin's solution, routinely embedded in paraffin and cut at a section thickness of 5 µm. The sections were stained with haematoxylin–eosin, Vierhoff van Gieson–Weigert and periodic acid–Schiff. A systematic histological evaluation was performed under light microscopy. More than 100 seminiferous tubules were scored for each patient. The results were expressed as a relative number of tubules showing Sertoli cells, spermatogonia, spermatocytes, round and elongated spermatids, and spermatozoa. All examinations were made by the same observer (J.S.). The diagnoses were as follows: normal spermatogenesis, germinal hypoplasia, early and late maturation arrest and Sertoli-cell-only syndrome (SCOS). Moreover, testicular histology was evaluated according to the Johnsen's modified classification (Holstein et al., 1994Go) from score 1 (no cell in sclerotic seminiferous tubules) to score 10 (sperm present). Cytological examination of the second sample was done on site to search for sperm that could be cryopreserved (cryo-testicular sperm extraction) and used later in programmed ICSI. If microscopical assessment showed no sperm, a testicular incision at another site was made. In some cases, repeated sampling was needed in order to observe sperm and a biopsy specimen was taken from the contralateral testis when necessary.

Cytogenetic methods
Cytogenetic analyses were performed on preparations from cultures of peripheral blood lymphocytes and assessed by R-, G- and C-banding.

To obtain prometaphase chromosomes, the cultures were treated with thymidine (6 mg/ml) after 48 h of incubation. The RHG (R-bands by heating using Giemsa), GTG (G-bands by trypsin using Giemsa) and CBG (C-bands by barium hydroxide using Giemsa) banding techniques were applied and >=40 metaphases were counted in each patient.

Molecular–genetic methods
Genomic DNA was prepared from peripheral blood samples with standard procedures. We detected Y chromosome microdeletions by a polymerase chain reaction (PCR) amplification of the Y chromosome-specific STS and genes. Sixteen PCR–multiplex and five single primer pair reactions were used for the detection of the presence or absence of 16 genes: RBMY1, RBMY2, DAZ, SPGY, TTY1, TTY2, PRY, DFFRY, DBY, UTY, TB4Y, BPY1, CDY, XKRY, eIF-1AY, BPY2 (Vogt et al., 1996Go; Lahn and Page, 1997Go; Krausz et al., 1999aGo) and 32 STS markers (sY14, sY18, sY19, sY70, sY78, sY81, sY82, sY83, sY86, sY85, sY84, sY87, sY165, sY182, sY151, sY100, sY128, sY131, sY132, sY134, sY136, sY139, sY153, sY152, sY155, sY147, sY156, sY149, sY254, sY157, sY202, sY243) (Vollrath et al., 1992Go; Reijo et al., 1995Go). In 10 patients with revealed deletions, we additionally analysed the presence or the absence of 10 STS markers (sY142, sY143, sY145, sY220, sY242, sY158, sY166, sY167, sY159, sY160) (Figure 1Go). Altogether we analysed 58 loci.



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Figure 1. Map of the Y chromosome in 10 infertile men with Y chromosome deletions. Deleted sequence-tagged site (STS) markers and genes: (–) = deleted gene or STS marker; (+) = gene or STS marker present. *Multicopy genes, with the copies also in the AZFc region.

 
The PCR was carried out in a total volume of 10 µl. The reaction mixture included 50 ng of each DNA sample, 1xPCR buffer, 1.5 mmol/l MgCl2, 200 µmol/l deoxy-nucleotidetriphosphate (NTP), 1 µmol/l of each primer pair and 0.5 U Taq DNA polymerase (Promega Corp., Madison, WI, USA and Gibco-BRL, Gaithersburg, MD, USA). The reactions were performed in a thermal cycler (MJ Research, Inc., Watertown, MA, USA). After an initial denaturation step at 94°C for 5 min, cycle parameters were: 29 cycles at 94°C for 60 s, 56–62°C for 80 s, and 72°C for 60 s. The programmes were followed by the final extension step at 72°C for 7 min. We achieved a specific amplification of sY159, with the addition of 1/10 V of dimethylsulphoxide at the annealing temperature of 61°C. Thirty-three PCR cycles were performed for the successful amplification of DAZ and RBMY2 genes. The reaction products were then analysed by electrophoresis at 76 V on 2–4% agarose gels (Sigma Chemical Co., St Louis, USA) containing ethidium bromide (0.1 mg/ml) and visualized under UV light. We confirmed the deletion of the loci if the product of the expected size was not obtained after three single primer pair PCR experiments. As a control, one sample of female, one sample of fertile male genomic DNA and one sample which contained all reaction components, but in which water instead of DNA was added, were run with each set of primers.

Fertilization and pregnancy rates
Three patients with Y chromosome deletion underwent ICSI. Ovarian stimulation was induced using a long protocol of gonadotrophin-releasing hormone agonist (buserelin acetate, Suprefact; Hoechst AG, Frankfurt am Main, Germany) and human menopausal gonadotrophin (Pergonal or Metrodin HP-75; Serono, Geneva, Switzerland). Human chorionic gonadotrophin (HCG, Primogonyl; Serono) was given (10 000 IU i.m.) when follicles reached a mean diameter of 21 mm. Oocyte retrieval was carried out 36 h after HCG injection. Oocyte handling and microinjection were performed according to a published technique (Van Steirteghem, 1993).

About 48 h after ICSI (day 2) each embryo was scored according to the number, size and shape of blastomeres. Type IV or excellent embryos were defined as embryos in which all blastomeres were of equal shape and size and anucleate fragments were present in <10% of the embryonic volume. Type III or good embryos had blastomeres with an equal shape and size with 10–30% of anucleate fragments. Type II or fair embryos and type I or bad embryos had non-equal shape and size of blastomeres with 30–50% and >50% of anucleate fragments respectively. Only types III and IV embryos were considered suitable for transfer. Embryos were loaded into 5 µl of IVF medium and into a TDT catheter (TDT set; C.C.D., Paris, France) and at most three embryos were transferred into the uterus ~48 h after microinjection (day 2). In three attempts, embryos were cultured for 5 days and were transferred at the blastocyst stage. As the luteal phase support, dydrogesterone was given per os, 300 mg/day (Dabroston; Belupa, Koprivnica, Croatia) starting on the day of embryo transfer. Clinical pregnancy was confirmed by ultrasound demonstration of cardiac activity at 6 weeks.

Statistical analysis
The clinical characteristics of infertile men are given as means ± SD, and were compared by t-test (symmetrically distributed data) and Mann–Whitney two-sample test (asymmetrically distributed data) for the significance of the difference between the means of patients with and without Y chromosome deletions. All computations were carried out with the SPSS statistical package (SPSS Inc., Illinois, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cytogenetic analysis
Cytogenetic analysis was performed in 181 patients; abnormalities were found in 33 patients. Two had a 46,X, del(Y) (q11) karyotype and represented 0.9% of the study group (n = 226). Five patients had low level mosaicism 46,XY/47,XXY (abnormal karyotype <2.5%).

Microdeletion analysis
All 226 subfertile men were analysed for the presence or absence of 16 genes and 32 STS markers. In 10 patients with detected deletions we additionally analysed the presence or absence of 10 STS markers (Figure 1Go). Both patients with cytogenetically detected deletions (9.7, 9.19) had regions AZFa, b and c missing. The patients 89.47 and 77.52 had AZFb and c deleted. Four of the 10 patients had the deletion of the AZFc region and showed the same deletion pattern. The patient 9.41 had a deletion in AZFa and the patient 77.37 had a deleted region distal to the DAZ gene (sY202, sY243). Patient 34.10, with AZFc deleted, had low level chromosomal mosaicism 46,XY/47,XXY. No deletions were found in the group of 125 fertile male controls.

We found deletions of the Y chromosome in 10 of the 226 (4.4%) patients, eight in the azoospermic group (8/92; 8.6%) and two in the OAT group with 0.1–0.3x106/ml sperm in the ejaculate (2/134; 1.5%). Considering the patients with sperm count <5x106 sperm/ml, the microdeletion frequency rate was 5.6% (10/178).

Andrological history and examination
In the study group, 38 men had a history of cryptorchidism, 27 had varicocele, four had cryptorchidism and varicocele and one had hypogonadotrophic hypogonadism. Therefore, the examination revealed 70 men with abnormal andrological findings. These abnormalities represented 31% of the study group. Among the patients with Y chromosome deletions, patient 9.26 had varicocele and the patient 9.19 had cryptorchidism and varicocele. The andrological findings in men with Y chromosome deletions are presented in Table IGo.


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Table I. Andrological findings in men with Y chromosome deletions
 
The mean left and right testicular volumes were 11.2 ± 3.9 and 10.7 ± 3.6 ml respectively in patients with Y chromosome deletions compared to 13.2 ± 5.4 and 12.8 ± 12.8 ml respectively in patients without Y chromosome deletions. In patients with deletions the values were below normal, but were not significantly different from the values found in infertile patients without deletions, irrespective of the underlying aetiology (P = 0.15 for the left and P = 0.11 for the right testicular volume).

Semen and testicular histology
In the group of 226 infertile men, 92 were azoospermic and 134 had OAT. Among OAT men, only 48 had >5x106 sperm/ml, whereas 70 had <1x106 sperm/ml (Table IIGo).


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Table II. Classification of patients according to the spermiogram and histological findings
 
On histological assessment we found no case of normal spermatogenesis. Thirty-three men were affected by germinal hypoplasia, 32 by SCOS and 20 by maturation arrest. In five cases, hypoplasia was associated with SCOS, in six cases with maturation arrest, and in one case with SCOS and maturation arrest. In one case SCOS was associated with maturation arrest. Mean Johnsen's score was 3.25 ± 2.4 in men with deletions compared with 4.02 ± 2.9 in men without deletions (Table IIGo), the difference not being statistically significant.

One patient with deletion of the AZFa region (9.41) and two patients (9.7, 9.19) with large, cytogenetically revealed deletions, who had deleted AZFa, b and c regions, had testicular histology of SCOS. In two patients who had AZFb and c deleted, testicular histology consisted of SCOS, germinal hypoplasia and maturation arrest (patient 77.52) and of SCOS and maturation arrest (patient 89.47). In patients with the same deletion pattern in AZFc region, testicular histology revealed either germinal hypoplasia and maturation arrest (patients 9.26, 34.45) or SCOS (patient 34.10) (Table IIIGo).


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Table III. Findings in 10 infertile men with Y chromosome deletions
 
Hypoplasia alone, which was present in 30% in the whole group of biopsied men, was not observed in men with deletions.

Hormones
The mean FSH concentration in patients with Y chromosome deletions (n = 10) was 16.1 ± 9.9 compared with 15.54 ± 13.66 mIU/ml in patients without Y chromosome deletions (n = 135) (P = 0.87) (Table IIIGo).

Fertilization and pregnancy rates
Cryo–TESE was successful in one patient with azoospermia and in one patient with oligozoospermia. Both patients had deletions in the AZFc region and testicular histology of maturation arrest and hypoplasia. Testicular recovery was unsuccessful in four azoospermic patients with SCOS: two had large deletions encompassing AZF a, b and c regions, one patient had an AZFa deletion and one patient an AZFc deletion.

After ICSI, the female partner of patient 34.45 conceived twice: the first pregnancy ended in an early miscarriage and the second in a delivery of a healthy girl. The female partner of the patient 9.44 became pregnant at the first ICSI attempt and delivered a healthy girl before term. In patient 9.26, two ICSI attempts were performed; in the first attempt, frozen–thawed testicular sperm were used, and in the second, fresh testicular sperm. The fertilization rate was 15% in the first and 50% in the second attempt, and the number of transferred embryos was two and three respectively (Table IVGo). The mean fertilization rate was 49%: 55% for fresh ejaculated sperm and 32% for testicular sperm, whereas the pregnancy rate was 43%. All obtained embryos were of excellent or good quality.


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Table IV. ICSI attempts in three men with AZFc deletions
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study we evaluated the importance of Y chromosome microdeletions in male infertility and the relationship between mutations and phenotypical consequences in a group of 226 Slovenian men, candidates for ICSI procedure.

The reported frequency of deletions varies from 1% (Van der Ven et al., 1997Go) to 55% (Foresta et al., 1998Go) which is largely related to different inclusion criteria. The cumulative data (Simoni et al., 1998Go) showed a 12.2% microdeletion rate in azoospermic men and 3.4% in oligozoospermic men. Those rates are comparable to those observed in our study.

Testing of 226 subfertile men for 16 Yq-chromosome genes and gene families did not reveal any isolated gene deletion. Similarly, one team (Krausz et al., 1999aGo) did not find isolated gene deletions when they analysed 90 subfertile patients with six Yq genes. Our results support the conclusion that including the 16 currently known genes/gene families in the diagnostic test does not contribute significantly to the increased sensitivity of Y chromosome microdeletion detection.

For identification of discrete regions with potential genes important for spermatogenesis, small deletions are of special interest. We identified a patient with a deletion distal to DAZ locus involving two STS markers: sY202 and sY243. This deletion could be regarded as a rare Y chromosome variant. Nevertheless, no deletions for sY202 (Reijo et al., 1995Go; Qureshi et al., 1996Go; Kent-First et al., 1996Go; Pryor et al., 1997Go; Kent-First et al., 1999Go) and for sY243 (Reijo et al., 1995Go; Stuppia et al., 1998Go; Kim et al., 1999Go) have so far been reported in men with proven fertility, our set of control men included. We therefore cannot exclude the possibility that the observed deletion might be related to infertility in our patient. The affected patient was azoospermic; unfortunately the testicular biopsy was not performed. Silber (Silber et al., 1998Go) found four patients with deletions involving the AZFc region, but extending beyond it, and none of those had any sperm in the testes or ejaculate, which led the authors to propose the possibility of additional genes, involved in spermatogenesis, present at the distal part of the Yq chromosome.

Of the 10 patients with deletions, eight patients demonstrated no aetiological cause of infertility, whereas we found a deletion in one patient with varicocele and in one patient with varicocele and cryptorchidism; both were azoospermic. We thus confirm previous reports (Pryor et al., 1997Go; Grimaldi et al., 1998Go; Foresta et al., 1999Go; Krausz et al., 1999bGo; Kent-First et al., 1999Go) which described Y chromosome deletions in males with non-idiopathic infertility. Coexistence of microdeletions with various andrological pathologies may be coincidental or may be causally related as proposed for unilateral cryptorchidism (Foresta et al., 1999Go). Regardless of the causal relationship, our results support the relevance of microdeletion screening also in candidates for ICSI with evident cause of infertility.

One of the 10 patients with a Y chromosome deletion was low level (<2.5%) 46,XY/47,XXY mosaic. The same mosaicism has been reported by Oliva (Oliva et al., 1998Go). Recently, Siffroi demonstrated that deletion of the Y chromosome long arm, especially in the AZFc region, can be associated with Y chromosomal instability leading to the formation of 45,X0 cell lines (Siffroi et al., 2000Go). Our data therefore extend the previous observation and suggest that somatic instability of both sex chromosomes may be a consequence of Y chromosome deletion. Clinical relevance of sex chromosome instability with resulting 45,X0 and 47,XXY cell lines remains to be established, nevertheless low level mosaicism for numerical sex chromosomal abnormalities has been reported as a frequent cytogenetic abnormality in couples undergoing ICSI (Meschede et al., 1998Go).

Additionally, our patient with a deletion of the AZFc region and low level 46,XY/47,XXY mosaicism demonstrated SCOS on testicular histology, suggesting a possible modulating role of 47,XXY cell line in the phenotype expression.

Several factors make a correlation between the deletion pattern and clinical consequences difficult. Testicular volume, plasma FSH concentrations and Johnsen's score were not discriminating between men with and without deletions. There is some difficulty in interpreting histological findings in infertile men. Multiple testicular biopsy samples can reveal different histological pictures in the same patient. Moreover, testicular histology may change, spermatogenesis quality usually decreasing with time.

It has been suggested that each subregion of Yq11 is associated with a distinct histopathological picture (Vogt et al., 1996Go): deletions in AZFa result in Sertoli-cell-only syndrome, deletions in AZFb result in spermatogenic arrest, usually at the spermatocyte stage, deletions in AZFc are associated with a variable histological picture with most tubules containing only Sertoli cells but some developing germ cells with a variable degree of differentiation. Other authors, however, have been unable to attribute a phenotype to a given deletion (Pryor et al., 1997Go).

Our results support the observations that large deletions and deletions in the AZFa region are associated with the SCOS phenotype, whereas the deletions in the AZFc region may result in less severely impaired spermatogenesis (Reijo et al., 1996Go; Qureshi et al. 1996Go; Girardi et al., 1997Go; Kleiman et al., 1999Go; Krausz et al., 1999bGo).

We may conclude that men with Y chromosome deletions are usually affected by severe impairments in spermatogenesis (no case of isolated germinal hypoplasia in our study) which depend on the deleted region involved.

Most men affected by Y chromosome deletions choose ICSI to overcome their incapacity to fertilize spontaneously (Nap et al., 1999Go). Sperm testicular recovery may be negative in azoospermics deleted for region AZFb (Brandell et al., 1998Go) or with larger Y deletions, including and extending beyond the AZFc region and encompassing more Y genes (Silber et al., 1998Go). In our study it was impossible to recover sperm in four patients: two had large deletions including AZFa, b and c regions, one had an AZFa deletion and one patient an AZFc deletion.

In three patients with AZFc deletion, who underwent seven ICSI attempts with ejaculated or testicular sperm, fertilization and pregnancy rates obtained were comparable with those achieved by conventional IVF in our group (Zorn et al., 2000Go). All obtained embryos were of excellent or good quality. These results do not support observations of van Golde (van Golde et al., 2001) who reported lower fertilization rate and poorer embryo quality in eight patients (19 ICSI treatments) with a microdeletion in the AZFc region. Unfortunately the authors did not provide exact data about the deletion breakpoints.

Our results confirm that sperm from oligozoospermic patients with Y chromosome deletions have good fertilizing ability during classical IVF (Rossato et al., 1998Go) or with ICSI, using either ejaculated or testicular sperm (Mulhall et al., 1997Go).

We conclude that Y chromosome deletions may be important contributors to infertility, with a frequency rate of ~5.6% in infertile patients with a sperm count of <5x106 sperm/ml.

Testing for gene-specific deletion does not contribute significantly to the sensitivity of a microdeletion test. In this study neither clinical (testicular volume) nor hormonal (plasma FSH concentration) and histological findings (Johnson's score) were able to distinguish between men with and those without Y chromosome deletions, whereas fertilization and pregnancy rates obtained using sperm of patients with Y chromosome deletions were comparable with those achieved in conventional IVF.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to Dr Irma Virant-Klun for performing ICSI procedure, to Ms Sabina Zitko and Mr Andrej Stegnar for technical assistance and to Ms Mojca Pirc for revising the English text.


    Notes
 
4 To whom correspondence should be addressed. E-mail: borut.peterlin{at}guest.arnes.si Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Brandell, R.A., Mielnik, A., Liotta, D. et al. (1998) AZFb deletions predict the absence of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test. Hum. Reprod., 13, 2812–2815.[Abstract/Free Full Text]

Brown, G.M., Furlong, R.A., Sargent, C.A. et al. (1998) Characterisation of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the Sxrb interval of the mouse Y chromosome of the Dffry gene. Hum. Mol. Genet., 7, 97–107.[Abstract/Free Full Text]

Foresta, C., Ferlin, A., Garolla, A. et al. (1997) Y-chromosome deletions in idiopathic severe testiculopathies. J. Clin. Endocrinol. Metab., 82, 1075–1080.[Abstract/Free Full Text]

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Grimaldi, P., Scarponi, C., Rossi, P. et al. (1998) Analysis of Yq microdeletions in infertile males by PCR and DNA hybridization techniques. Mol. Hum. Reprod., 4, 1116–1121.[Abstract]

Henegariu, O., Hirschmann, P. and Kilian, K. (1994) Rapid screening of the Y chromosome in idiopathic sterile men, diagnostic for deletions in AZF, a genetic Y factor expressed during spermatogenesis. Andrologia, 26, 97–106.[ISI][Medline]

Holstein, A.F., Schulze, W. and Breucker, H. (1994) Histopathology of human testicular and epididymal tissue. In Hargreave, T.B. (ed.), Male Infertility. Springer Verlag, London, pp. 105–148.

Kent-First, M.G., Kol, S. and Muallem, A. et al. (1996) The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers. Mol. Hum. Reprod., 2, 943–950.[Abstract]

Kent-First, M., Muallem, A., Shultz, J. et al. (1999) Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection. Mol. Reprod. Dev., 53, 27–41.[ISI][Medline]

Kim, S.W., Kim, K.D. and Paick, J.S. (1999) Microdeletions within the azoospermia factor subregions of the Y chromosome in patients with idiopathic azoospermia. Fertil. Steril., 72, 349–353.[ISI][Medline]

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Submitted on June 5, 2001; accepted on September 15, 2001.