Hum. Reprod. Advance Access published online on April 25, 2008
Human Reproduction, doi:10.1093/humrep/den124
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Sons conceived by assisted reproduction techniques inherit deletions in the azoospermia factor (AZF) region of the Y chromosome and the DAZ gene copy number
1 University Department of Growth and Reproduction, Rigshospitalet, Section GR-5064, Blegdamsvej 9, DK-2100 Copenhagen, Denmark 2 Human Developmental Genetics, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris, France 3 The Fertility Clinic, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
4 Correspondence address. Tel: +45-3545-5017/8145; Fax: +45-3545-6054; E-mail: erm{at}rh.regionh.dk
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Abstract |
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BACKGROUND: Deletions in the azoospermia factor (AZF) region of the Y chromosome are frequent in infertile men. The clinical consequences and the mode of inheritance of these deletions are not yet clear.
METHODS: Y chromosome deletion mapping and quantitative PCR analysis of the DAZ-gene copy number, supplemented with haplogroup typing in deleted patients, were performed, in combination with clinical assessments in 264 fathers and their sons conceived by assisted reproduction techniques (ART), and in 168 fertile men with normal sperm concentration.
RESULTS: In the ART fathers group, a complete AZFc deletion was detected in 0.4% (1/264). AZFc rearrangements/polymorphisms were found in 6.8% (18/264; 95% CI: 4.4–10.5), which was significantly more frequent (P = 0.021) than in the controls (3/168; 1.8%, 95% CI: 0.6–5.1). All deletions were transmitted to the sons, without any clinical symptoms in early childhood. In the fathers, there was no significant correlation between the DAZ copy number and the severity of spermatogenic failure.
CONCLUSIONS: AZFc rearrangements/polymorphisms are transmitted to sons and may represent a risk factor for decreased testis function and male subfertility, which needs confirmation in further studies in larger cohorts. However, deletions of two DAZ gene copies are compatible with normal spermatogenesis and fertility.
Key words: gr–gr deletion/b2–b3 deletion/AZFc/Y chromosome/ART
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Introduction |
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The vast majority of cases of severely impaired spermatogenesis in infertile men used to be categorized as idiopathic, until the progress in cytogenetics and molecular biology led to identification of deletions on the long arm of the Y chromosome in a sizeable proportion of such patients (Tiepolo and Zuffardi, 1976
; Vogt et al., 1992; Reijo et al., 1995; Krausz, 2005). These deletions helped to delineate the azoospermia factor (AZF) region on the long arm of the Y chromosome (Yq11) where genes involved in germ cell differentiation and spermatogenesis are clustered (Vogt et al., 1992). More recently, the sequencing of this region and of the entire Y chromosome has revealed an unusual structure with repeats of variable length, known as amplicons, some of them palindromic, which apparently facilitate the occurrence of deletions in this region (called the male-specific region of the Y chromosome, MSY) (Kuroda-Kawaguchi et al., 2001). In addition to the large amplicons, such as b2/b4, smaller palindromes within the AZFc region have been identified, and their rearrangements leading to partial AZFc deletions, i.e. gr/gr, g1/g3, b2/b3 or—using another nomenclature—DAZ1/DAZ2, u3+DAZ3/DAZ4, were detected in patients with fertility problems (Fernandes et al., 2002; Ferras et al., 2004; Repping et al., 2003, 2004a,b).
The clinical consequences are most severe in patients with AZFa and -b deletions who are azoospermic, whereas patients with complete AZFc (b2/b4) deletions may present with a range of phenotypes from azoospermia to severe oligozoospermia (Krausz, 2005). The consequences of partial AZFc deletions are less clear, and still debated. Some of these partial deletions tend to occur in association with certain Y chromosome lineages, thus further complicating the studies of their pathogenic role in male infertility (for review see Vogt, 2005; McElreavey et al., 2006). Most of the published studies seem to agree that the presence of a gr/gr deletion is a risk factor for male infertility (Repping et al., 2003, 2004b; de Llanos et al., 2005; Giachini et al., 2005; Lynch et al., 2005; Vogt, 2005; Tüttelmann et al., 2007), while according to most studies, except one (Wu et al., 2007), the presence of a b2/b3 deletion does not affect testicular function and may be a polymorphism (Fernandes et al., 2004; Machev et al., 2004; Ferlin et al., 2005; Giachini et al., 2005; Hucklenbroich et al., 2005). The spermatogenic impairment observed in many patients with partial deletions in the AZFc region is mainly attributed to the loss of copies of the multicopy DAZ gene but may also be linked to a loss of other multicopy genes, such as BPY2 or CDY1 (Kuroda-Kawaguchi et al., 2001; Machev et al., 2004; Giachini et al., 2005; Lin et al., 2005).
With the development of intracytoplasmic sperm injection (ICSI), an infertile man with just a few spermatozoa can become a biological father, thus circumventing the natural selection against genetic disorders of spermatogenesis. An AZF deletion, a possible risk factor for spermatogenic failure, may thus be transmitted to the son (Cram et al., 2000; Oates et al., 2002; Silber and Repping, 2002; Lynch et al., 2005). The existing data on the prevalence of father to son transmissions are scarce and it is unknown whether or not they affect the early development of the reproductive system in the sons. In addition, some studies raised concern that ICSI children may have a greater frequency of de novo chromosomal aberrations, including Y chromosome deletions (Kent-First et al., 1996; Bonduelle et al., 2002; Georgiou et al., 2006), and that some of the inherited deletions may progressively expand (Komori et al., 2002). One study even reported that the ICSI procedure may lead to de novo microdeletions of the Y chromosome, but the reported ‘deletions’ were not compatible with the existing knowledge and the poor quality of the study rendered it inconclusive (Lee et al., 2006). Therefore, we investigated a cohort of male partners from infertile couples, who were treated either with ICSI or in vitro fertilization (IVF), and their children. We report here the molecular analysis of the Y chromosome for AZF deletions, supplemented by a quantitative analysis of the copy number of the multicopy DAZ gene, and the haplogroup analysis in the majority of individuals with deletions. We correlated the results of the genetic analysis with clinical parameters of the testicular function in both fathers and sons.
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Study subjects
The patients and controls were all from the Copenhagen area and all were recruited at the same hospital (Rigshospitalet). The patients signed an informed consent, whereas the control data were coded and anonymized. The study was approved by the local Ethics Committee and the Danish Data Protection Agency, and was performed according to the Helsinki declaration II.
The patients were recruited consecutively among infertile couples referred for assisted reproduction technique (ART) treatment. All patients were Danish Caucasians, nearly all of them ethnic Danes, with just a few fathers with foreign surnames (Turkish or British). For the adult control group, we used clinical data and DNA samples from age-matched proven fertile men, who became fathers after natural conception. All controls were Danish and born in Denmark. These men previously participated in a study of reproductive health, and underwent a full andrological examination (Jorgensen et al., 2001). For the control group, in this study, we selected 168 men with sperm concentration >20 million/ml, which is a normal value according to WHO (1992); the range was 20.0–301.9 million/ml.
A total of 265 men who fathered a child by ART in the period 1998–2005 were included in the study. Among 178 couples treated by ICSI, 141 (79.2%) had male factor infertility, 4 (2.2%) female factor infertility, 22 (12.4%) combination of infertility factors, 11 (6.2%) no cleavage or unexplained infertility. Among 87 couples treated by IVF, 13 (14.9%) had male factor infertility, 49 (56.3%) female factor infertility, 6 (6.9%) combination of infertility factors and 19 (21.8%) no cleavage or unexplained infertility. The self-reported frequency of the history of cryptorchidism in the group of all ICSI fathers was 8.1%. Nearly all patients with previous problems with testis descent were in the subgroup with male factor infertility (20/180, 11.1%, the information was unavailable from two men).
All men, except one, had a blood sample taken for DNA extraction and most of them provided a semen sample. The semen analysis revealed sperm concentrations ranging from 0.1 to 287.4 million/ml (median 15.8 million/ml). One hundred and forty-five (55%) men (132 ICSI fathers, 13 IVF fathers) underwent additional clinical investigations in our clinic for evaluation of their reproductive function, including andrological physical examination, further semen analysis and a comprehensive reproductive hormone profile in serum. In 41 (28%) of these men, a testicular biopsy was taken as a diagnostic procedure.
Couples treated by ICSI had 107 sons (23% were twins) and 102 daughters (22% twins); couples treated by IVF had 62 sons (19% twins) and 40 daughters (22% twins). None of the children included in this study were conceived with donor semen. All children conceived by ART underwent a general physical examination, as described previously (Barnes et al., 2004; Bonduelle et al., 2005; Ponjaert-Kristoffersen et al., 2005; Mau Kai et al., 2006, 2007; Wennerholm et al., 2006), including the assessment of testes size by scrotal ultrasound and serum hormone profile (from an antecubital vein). At the same time, a blood sample was taken for DNA extraction. Blood samples could not be obtained from all infants due to unsuccessful procedure or parental objection and for ethical reasons, only one attempt of venipuncture was made in each child. For this study, we had blood samples available from 113 out of 169 boys (67%).
The control group for the ART children originated from a contemporary prospective birth cohort study at the same hospital investigating genital development in naturally conceived children, as described previously (Boisen et al., 2004, 2005; Boas et al., 2006; Main et al., 2006) and a Danish cohort within an international multicentre cohort study (Barnes et al., 2004; Bonduelle et al., 2005; Mau Kai et al., 2006, 2007; Ponjaert-Kristoffersen et al., 2005; Wennerholm et al., 2006) including a total of 1787 children (1028 boys). DNA was not available in any of the naturally conceived control children. Comparisons were made within the same gender and age group (reproductive hormones were only compared in 2.5–4.5 months old children since the pituitary-gonadal axis is transiently active at this age).
Molecular analysis of the Y chromosome
DNA purification
Genomic DNA was extracted from leukocytes in whole blood samples using a manual kit (Roche, Basel, Switzerland) or Quick Gene 810 automatic nucleic acid isolation system (Fujifilm, Tokyo, Japan).
AZF deletion analysis
The mapping of deletions was based on our previously reported methodology (Frydelund-Larsen et al., 2002, 2003) and the recent guidelines of the European Molecular Quality Network, EMQN, to which our laboratory belongs (Simoni et al., 2004). In order to test for partial AZFc deletions, we added additional markers (sequence tagged sites, STS), sY1191, sY1197 and sY1291, using previously described primer sequences (Repping et al., 2003). The analyses were performed by polymerase chain reaction (PCR) amplification of STS markers and gene specific sequences, spanning the three AZF loci (a, b and c) on the q arm of the Y chromosome and control fragments from the SRY locus on Yp and ZFY/X loci on Yp/Xp. Four multiplex PCR reactions with a total of 14 primer pairs were designed as follows: multiplex A: sY114, sY1197, sY85, DBY; multiplex B: sY1291, sY1191, sY134, eIF-1AY; multiplex C: ZFY/X, sY254 (DAZ), sY152; and multiplex D: SRY, sY158, BPY2. In brief, a gr/gr deletion was detected if only sY1291 was absent, whereas the absence of sY1191 alone was indicative of a b2/b3 deletion (Repping et al., 2004a; Lin et al., 2006). For each analysis, a genomic DNA sample from a normal fertile male was used as a positive control; a female genomic DNA sample (positive only for ZFX/Y) and H2O were used as negative controls. Amplification conditions consisted of an initial denaturation step (96°C) of 5 min, followed by 25 cycles of 30 s denaturation (94°C), 30 s annealing (55°C), 4 min elongation (65°C), ended by a final extension step of 5 min and subsequent cooling to 4°C. The products of all four PCR reactions were resolved separately on agarose gels in comparison to size markers (Fig. 1). The presence of deletions was confirmed by repeating the analysis in all cases.
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Analysis of the DAZ gene dosage
To establish the copy number of the DAZ gene, a quantitative real-time PCR (QPCR) was performed using a method similar to the one previously described (Machev et al., 2004
), but which was not validated by FISH or other established method. The general QPCR protocol was established and validated for other genes, as described in detail elsewhere (Ottesen et al., 2007). For the DAZ gene dosage analysis, a 126 base-pair DAZ fragment was amplified using a set of primers for sY255, the primer sequences as published in EMQN guidelines (Simoni et al., 2004). A fragment of an autosomal biallelic gene, GAPDH, was used as an internal quantification standard. Reaction tubes contained 11.25 ng genomic DNA, the concentration being measured before each experiment using a NanoDrop ND-1000 spectrophotometer (Saveen Werner AB, Malmö, Sweden), 15 µl Brilliant SYBR Green QPCR Master Mix (Stratagene, Cedar Creek, TX, USA), 7.0 µl sY255 (DAZ) primer-mixture (final concentration: forward 300 nM, reverse 300 nM) or GAPDH (final concentration: forward 50 nM, reverse 50 nM) and topped with H2O to a total volume of 30 µl. Genomic DNA and primer dilutions were done manually, but the mixtures were distributed on PCR plates by a robotic pipetting system (Theonyx Liquid Performer, Mechatronic Systems, Ebersberg, Germany). All reactions were done in triplicate. In addition to a normal male control, also a female control (46,XX) without any DAZ gene was included in each QPCR experiment. For a blank control, DNA was substituted with H2O. QPCR was performed on the Mx3000P platform from Stratagene, with the following amplification conditions: 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 30 s/56°C for 1 min/72°C for 30 s and 1 cycle at 95°C for 1 min/56°C for 30 s/95°C for 30 s. Quantification was done using the Mx3000P-software by comparing the data from analysis of DAZ with data from analysis of the GAPDH gene of the same patient, and subsequently calibrating this ratio to the ratio of a normal male reference DNA. A normal male sample with a ratio repeatedly at 1.0 was subsequently used as the calibrator. The mean ratio 1.01 ± 0.14 (±SD) was established using 20 normospermic males without any deletion in the AZF analysis, and who had very similar ratios to each other, thus were assumed to contain the normal number of four DAZ copies. Each control sample was analysed in at least two separate experiments, and the reference range for the ratio consistent with four DAZ copies was set at 0.73–1.29 (mean ± 2SD). Subsequently, the assay was tested using DNA samples from two normal women and seven infertile patients with a complete deletion of AZFc region, for which the ratio of amplification was repeatedly 0. Further validation was performed by measuring the mean ratios for, in total, 44 patients with either gr/gr or b2/b3 deletion in the STS analysis, who repeatedly had the ratios close to 0.5, consistent with a deletion of two DAZ copies. The mean ratio for two DAZ copies was established as 0.55 ± 0.07 (mean ± SD) and the range 0.41–0.69 (mean ± 2SD).
The Y chromosome haplogroup analysis
When a partial AZFc deletion was identified, the Y chromosome haplogroup was defined using the binary markers TAT, M9, 92R7, SRY1532, YAP, LLY22 g, P2, SRY4064, M174, M172 and M81 (Y Chromosome Consortium, 2002). The marker typing was carried out as described in Rosser et al. (2000). The deep-rooting markers SRY1532, M9, 92R7, YAP and M124 were typed in all samples and, in some cases, the remaining markers were typed hierarchically; e.g. M174 and sY81 only on YAP+ chromosomes, LLY22g and TAT only on those chromosomes M9 derived and 92R7 ancestral.
Semen analysis and testicular biopsies
Fertile control men
The men were asked to abstain from ejaculation for at least 48 h, but were not given any upper limit. The analysis of semen samples was performed according to WHO guidelines (WHO, 1992) with small modifications, as previously described in detail (Jorgensen et al., 2001).
Men from infertile couples treated with ART
For this study, data on semen parameters were collected from medical records. In most cases, the semen analysis was performed before preparation on the day of fertilization. If more than one semen sample was available, the medians of sperm concentration and semen volume were used. In 10 fathers, the spermatozoa used for ICSI were retrieved from the testicle or the epididymis (five testicular sperm extractions, three testicular sperm aspirations and two microsurgical epididymal sperm aspirations). Nine fathers (seven ICSI and two IVF) had antisperm antibodies.
Testicular biopsies were performed only in selected men (38 candidates for ICSI and 3 candidates for IVF). Twenty-five men had a bilateral testicular biopsy taken for the investigation of residual spermatogenesis, two due to previous cryptorchidism, one man had an atrophic testis. The clinical examination raised a tumour suspicion in five men and eight men had abnormal ultrasound examination, which raised suspicion of carcinoma in situ. In none of the 38 patients who underwent a biopsy, a testicular malignancy was found.
Hormone analyses
Analyses of reproductive hormones were performed in our certified laboratory using validated assays. Non-fasting peripheral venous blood samples were taken from the study subjects between 9.00 a.m. and 6.15 p.m. Samples were separated by centrifugation and serum was stored at –20°C until analysis. The following hormones were measured in serum: testosterone by a radioimmunoassay (RIA, Diagnostic Products Corp., Los Angeles, CA, USA), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and sex hormone-binding globulin (SHBG) by Delfia (Wallac Inc. Turku, Finland), inhibin B by a double antibody enzyme-immunometric assay using monoclonal antibody raised against the inhibin βB subunit in combination with a labelled antibody against the 
-subunit, and estradiol by RIA (Pantex Corp.; Immunodiagnostic Systems Limited, Santa Monica, CA, USA). The detection limits, inter- and intra-assay values were as described in a previous study of the same cohorts (Mau Kai et al., 2007).
Anthropometric measurements
Fathers’ height and weight were self-reported. Birth data and obstetric history from all pregnancies were obtained from medical records. Anthropometric measurements in children, including body length in infancy, standing height at 5 years of age, body weight and penile length were done as previously reported (Mau Kai et al., 2006, 2007). Until the age of 18 months and in adult men testicular size was measured by ultrasound (Aloka SSD-500; Aloka, Tokyo, Japan) with a linear 7.5 MHz transducer (8 mm footprint, model UST-5521U-7,5) to measure length (mm) and width (mm) from which the volume was calculated. At 5 years of age, a standard orchidometer was used.
Statistical analysis
Differences in the prevalence of gr/gr and b2/b3 deletions between groups were tested using two-sided Fisher’s exact test and confidence intervals (CI) (95%) for proportions were calculated. Probability (P) values of <0.05 were considered statistically significant.
For statistical analyses of hormone profiles, results under the detection limit were assigned the detection limit. Descriptive statistics are given as median hormone values and reference ranges (2.5th and 97.5th percentiles). The statistical analyses were carried out using SPSS version 14 (SPSS, INC., Chicago, IL, USA) and Confidence Interval Analysis version 2.0, BMJ Books, London, UK.
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Analysis of the Y chromosome genotype
No deletions in AZFa or AZFb regions were found. On the basis of the analysis of STS markers alone, we initially found 19 men with presumed AZFc deletions or rearrangements in the entire cohort of 264 ART fathers. All 19 men with rearrangements were ethnic Danes. Only one father had a complete deletion of the AZFc region (b2/b4), which constituted 0.4% of the study group (1/264). The absence of sY1291 or sY1191 suggesting a partial AZFc deletion or rearrangement was detected in 6.8% (18/264) of ART fathers, which was significantly more frequent than in the control group (3/168 = 1.8%, P = 0.021). The vast majority of patients with presumed partial AZFc deletions were in the group of male factor infertility (N = 180), with the absence of sY1191 (suggestive of b2/b3 rearrangement) (4.4%, 8/180) more frequent (P = 0.04) compared with 0.6% (1/168) in the fertile controls. These frequencies are given only to allow comparison with previous studies conducted without the gene dosage analysis.
To establish whether the absence of these single STS markers was indeed associated with a deletion, we next investigated by QPCR how many copies of the DAZ gene were present in these individuals. Genomic DNA with non-degraded large fragments suitable for QPCR analysis was available from 15/18 of ART fathers, 10/12 of sons and 1/3 of fertile controls with the absence of STS markers indicative of AZFc deletions. The numbers of DAZ copies for all these subjects are listed in Tables I and II, and a representative QPCR analysis result is shown in Fig. 2. The patient with a complete AZFc deletion had no amplification of the sY255 (mean ratio 0.00, which was equal to undetectable values in two control 46, XX females), thus confirming that he had all four DAZ copies deleted. In the majority of the ART patients (15/18) with either a gr/gr or b2/b3 deletion, the mean ratio was, as expected, close to 0.5 of the normal value (0.53 ± 0.08), consistent with the presence of two copies of DAZ. In seven subjects with the absence of sY1291 or sY1191 (including three ART fathers and three sons, and one control fertile subject), the QPCR analysis showed ratios close to 1.0 (0.90 ± 0.08), suggesting that they had four copies of DAZ. In addition, one ART father with a b2/b3 deletion had in two separate measurements a mean ratio of 1.5 ± 0.09, which suggested that he might have six DAZ copies. After analysis of the DAZ copy number, the adjusted differences in the frequency of partial deletions between ICSI fathers and controls were no longer significant.
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To investigate whether the AZFc rearrangements were linked to specific variants of the Y chromosome, haplogroups (hgr) were defined using informative binary markers, where sufficient DNA was available. The results are included in Tables I and II. The patient with a complete AZFc deletion (b2/b4) belonged to hgr P(xR1a1). Gr/gr deletions were observed on hg I and P(xR1a1). Among the 10 individuals with b2/b3 deletions whose Y chromosome haplogroups could be inferred from either father's or sons' DNA, six (60%) belonged to hgr N3. The others belonged to hgr R1a1, I and P(xR1a1).
Among the 19 ART-treated men with an AZFc deletion or rearrangement/polymorphism, 17 fathered a child by ICSI (male factor infertility) and 2 by IVF (female factor infertility) (Table I). All fathers transmitted the deletion to their sons and all these boys had the same number of DAZ copies as their fathers (the transmission could not be confirmed in one child with a b2/b3 deletion and the loss of two DAZ copies, because of the lack of father’s consent for a blood sample) (Tables I and II). Among the fathers with a gr/gr deletion and two DAZ copies, two had male offspring of which one was conceived by IVF and two had daughters conceived by ICSI. Seven fathers with a b2/b3 deletion and two DAZ copies had in total five sons (one set of male twins conceived by IVF) and three daughters.
Clinical phenotype of patients and their sons with AZFc deletions
Fathers
In the father with a complete AZFc deletion, the clinical picture was consistent with severe testicular failure, which was reflected in his reproductive hormones measured in the serum (FSH 17 IU/l, undetectable inhibin B, LH 5.1 IU/l, testosterone 11.3 nmol/l). Bilateral testicular biopsy showed predominantly Sertoli-cell-only pattern, with only 5% of tubules containing late spermatids in one of the testes. His semen quality was very poor (sperm concentration <0.1 mill/ml). After the ICSI procedure, he became the biological father of a healthy girl (46, XX).
The clinical characteristics and reproductive hormones of the individuals with partial AZFc deletions/rearrangements in the ART group are shown in Table I. All patients were normally virilized and had normal heights and weights. The karyotypes of the fathers were available in 72% (13/18) and were normal (46, XY). Among four men with a gr/gr deletion and two DAZ copies present, two patients had a history of cryptorchidism. Three patients with a gr/gr deletion had very poor semen quality, only one of them without any discernible reason, but one man with haplogroup P(xR1a1) had normal sperm quality (was in the ART group due to a female factor). Among seven fathers with a b2/b3 deletion and two DAZ copies, three (43%) had a history of cryptorchidism, one had unilateral hernia, one was refertilized after vasectomy, one had sperm antibodies and only one (or possibly two) had poor semen quality without any known aetiological reason. Finally, one patient with a rearrangement of the AZFc region involving sY1191 locus and an apparent duplication resulting in six DAZ gene copies had idiopathic infertility and moderate oligozoospermia (Table I). Reproductive parameters of the control subjects with lack of sY1191 or sY1291 were normal.
Sons
All sons with a partial AZFc deletion had a normal karyotype (46, XY). There were no significant differences in the anthropometrical measurements between sons with an inherited deletion compared with sons without a deletion or compared with the control group of naturally conceived boys. The boys with a partial AZFc deletion had penile length and testicular size within the normal range for their age (Table II). One boy who inherited a gr/gr deletion (with two DAZ copies) had unilateral ascending testis and was operated at the age of 3 years. His father also had a history of cryptorchidism.
Seven ART boys and 10 naturally conceived boys had serum testosterone values below the detection limit. The sons (N = 5), who had inherited a b2/b3 deletion (all had two DAZ copies except for one boy where DNA was insufficient), had a significantly lower serum testosterone (median 1.4 nmol/l, range 0.8–3.1 nmol/l) than the naturally conceived control boys (N = 614) (3.3 nmol/l, 0.5–7.6 nmol/l), P = 0.002), but the difference compared with ART sons without a deletion (N = 93) was not significant (2.6 nmol/l, 0.2–6.7 nmol/l, P = 0.132). Including only singletons in the analysis did not change the results. Serum inhibin B was likewise only marginally lower in singleton 2.5–4.5-month-old ART boys with a b2/b3 deletion (N = 4) 297 pg/ml (98–349 pg/ml) compared with ART sons at the same age without a deletion (N = 69) (379 pg/ml, 101–711 pg/ml, Table 2). Reproductive hormone profiles in boys with a gr/gr deletion did not differ from controls.
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In this study, we have carried out a detailed analysis of clinical consequences of Y chromosome (AZF) deletions in infertile men and their children conceived by ART. All sons born after ART in our series have inherited their fathers’ deletions and DAZ-gene copy numbers, and we had not detected any deletion occurring de novo in the boys conceived by ART from whom we had a blood sample (67% of the cohort), but we cannot completely exclude the possibility of such an event in the remaining 53 boys. This is in line with earlier findings of father–son transmission where most studies, except one (Kent-First et al., 1996
), found no de novo Y chromosome deletions (Cram et al., 2000; Oates et al., 2002; Buch et al., 2004; Lynch et al., 2005). Case reports have linked microdeletions of the Y chromosome to sex chromosome mosaicism (45, X/46, XY) (Siffroi et al., 2000; Papadimas et al., 2001). In our study, all 13 karyotyped fathers with an AZFc deletion or polymorphism had a normal karyotype (46, XY), and reassuringly, we found a normal male karyotype in all sons who inherited a microdeletion.
We analysed in great detail the reproductive function of both fathers and sons with partial AZFc deletions. Reproductive hormone profiles in these fathers were similar to the values in oligozoospermic fathers without deletions, and both groups had significantly lower values compared with fertile men. Recently, we showed that boys conceived by ICSI because of male factor infertility had significantly lower serum testosterone at 3 months of age compared with naturally conceived boys (Mau Kai et al., 2007). In this study, we found a tendency (but not a significant difference) to lower serum testosterone and inhibin B levels in ART infants with a b2/b3 deletion compared with infants without a deletion. These observations are potentially interesting, but may be chance findings due to the very small number of children with partial AZFc deletions and must be confirmed in a larger cohort. In addition, a lower average serum testosterone in the whole group of ICSI boys (Mau Kai et al., 2007), including those without AZFc deletions, suggests that other susceptibility genes are likely to be involved. Alternatively, the boys may have been exposed to environmental factors that impair androgen regulation, perhaps in analogy to their fathers.
Apart from this observation, we have not identified any specific reproductive impairment in the boys who inherited partial AZFc deletions, except for one boy with a gr/gr deletion who had an unilateral ascending testis and whose father had a history of cryptorchidism. We found a history of cryptorchidism in 45% (5/11) of ART fathers with partial AZFc deletion and the loss of two DAZ copies, which is most likely a chance finding. We can conclude that there is no causal link between cryptorchidism and partial AZFc deletions, otherwise all boys who had inherited these deletions, would have also inherited problems with testicular descent from their fathers. This is consistent with a recently published study which investigated partial AZFc deletions in Italian infertile men with a history of cryptorchidism (Giachini et al., 2007). That study found partial AZFc deletions (only gr/gr deletions) in the cryptorchid group, but with a frequency similar to that in infertile men with normal testis descent, and concluded that gr/gr is not associated with cryptorchidism. It is important to stress that infertility and cryptorchidism are complex multifactorial disorders, and dissecting the relative importance of genetic versus environmental factors is difficult. The prevalence of undescended testis in Denmark is very high (Boisen et al., 2004), and the same trend is seen for testicular cancer and other manifestations of the testis dysgenesis syndrome (TDS), which is assumed to be primarily caused by environmental/lifestyle factors (Skakkebaek et al., 2001).
Not surprisingly, among ART-treated fathers we found none with large AZFa or b deletions, and only one man with a complete AZFc deletion, whose clinical picture was consistent with severe testicular failure, as previously described in other patients with this aberration (Krausz et al., 2001; Frydelund-Larsen et al., 2002; Luetjens et al., 2002). In the vast majority of patients with a deletion, the alteration was a discrete rearrangement in the AZFc region. Spermatogenic impairment in men with a partial AZFc deletion has been attributed to the loss of two copies of the DAZ-gene or other genes, i.e. CDY 1a, CDY 1b (Ferlin et al., 2005; Giachini et al., 2005; Lin et al., 2005). A recent study from the Florence group proposed that a subtype of gr/gr deletion, characterized by the loss of DAZ1/DAZ2 and CDY1a might be a more severe variant (Giachini et al., 2007). In addition, a deletion of the sequences located in between the protein-coding genes, which may contain other important non-protein regulatory factors such as miRNAs may also contribute to the clinical picture. Our DAZ gene dosage analysis revealed that in a few ART patients and in one naturally fertile control with presumed partial AZFc deletions, four DAZ copies were retained and in one case, even six copies appeared to be present. This suggests that these men had other simultaneous rearrangements, most likely duplications, but an alternative explanation could simply be a polymorphism in the sequence recognized by the two STS makers, as has been described previously in some Y haplogroups (Machev et al., 2004; Vogt, 2005). As expected, most of b2/b3 deletions in our series were linked to hgr N3, but were also observed on three other independent Y lineages, confirming that not all b2/b3 deletions are the same and are often combined with inversions and duplications. Haplogroup N3 is common in Finland, where the prevalence of TDS is much lower than in Denmark (Skakkebaek et al., 2001; Boisen et al., 2004; McElreavey et al., 2006). Although our findings in this study may be counterintuitive to the epidemiological distribution of TDS, it is conceivable that some variants of the Y chromosome that are neutral in one region may render susceptibility to an environmental factor in another region or population, and may constitute a confounding a risk factor for male reproductive problems (McElreavey et al., 2006). This hypothesis is at this point a sheer speculation and clearly more studies are needed, especially in countries with a high proportion of rare haplogroups.
In analogy with a previous study (Machev et al., 2004), we did not find an correlation between the DAZ copy number and spermatogenic failure, however, men with apparent AZFc rearrangements involving polymorphisms of loci recognized by STS markers, especially those suggestive of b2/b3 deletion, tended to cluster among infertile patients, consistent with a possible involvement of other genes in the AZFc region. This observation needs further studies, as we could not examine the gene dosage in the entire cohort. A recent study suggested that the b2/b3 deletion may be a genetic risk factor for male infertility in the Chinese population (Wu et al., 2007), although effects of population stratification cannot be excluded as contributing to this effect. Our study may also be biased in a similar manner, since hgr analysis was not performed for the entire study population and we detected a relatively small number of subjects with partial deletions.
In conclusion, in our study of ART-treated men and their children we found no significant correlation between the DAZ copy number and testis function, both in adulthood and early infancy, confirming that a deletion of two DAZ copies is compatible with normal testis development and spermatogenesis. However, our study suggests that structural rearrangements or polymorphisms in the AZFc region, including seemingly neutral deletions or duplications, which occur independently on various Y chromosome lineages, may represent a confounding risk factor for male infertility in certain populations. This remains a hypothesis but warrants further studies. In concert with previous studies, all ICSI fathers transmitted their Y chromosome deletion to their sons. The increasing use of ICSI probably will result in an increasing population prevalence of these and other genetic aberrations. This is of concern since ICSI may contribute to decreasing fertility rates in societies where this procedure is available.
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This work was supported by grants from The University of Copenhagen, The Danish Medical Research Council, The European Commission, the Svend Andersen Foundation and the Novo Nordisk Foundation.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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We are grateful to all the families, especially the children, who participated in this study. We also acknowledge the contribution of all doctors from The Nordic Cryptorchidism Study Group, who examined many of the children (M. Chellakooty, I.M. Schmidt, K. Boisen, I.N. Damgaard, A-M. Suomi and M. Boas), the skilled help of Nurse H. Kelkeland and of the laboratory technicians, especially N. Nguyen (Paris).
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Submitted on April 22, 2007; resubmitted on February 24, 2008; accepted on March 21, 2008.
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