Conceived and designed the experiments: FT MS BD PW. Performed the experiments: FT SL AR. Analyzed the data: FT AR. Contributed reagents/materials/analysis tools: FT SK PW AR. Wrote the paper: FT AR. Characterised and selected the subjects: FT MS SK. Approved the manuscript: FT MS SK SL BD PW AR.
The authors have declared that no competing interests exist.
A genetic origin is estimated in 30% of infertile men with the common phenotypes of oligo- or azoospermia, but the pathogenesis of spermatogenic failure remains frequently obscure. To determine the involvement of Copy Number Variants (CNVs) in the origin of male infertility, patients with idiopathic severe oligozoospermia (N = 89), Sertoli-cell-only syndrome (SCOS, N = 37)) and controls with normozoospermia (N = 100) were analysed by array-CGH using the 244A/400K array sets (Agilent Technologies). The mean number of CNVs and the amount of DNA gain/loss were comparable between all groups. Ten recurring CNVs were only found in patients with severe oligozoospermia, three only in SCOS and one CNV in both groups with spermatogenic failure but not in normozoospermic men. Sex-chromosomal, mostly private CNVs were significantly overrepresented in patients with SCOS. CNVs found several times in all groups were analysed in a case-control design and four additional candidate genes and two regions without known genes were associated with SCOS (P<1×10−3). In conclusion, by applying array-CGH to study male infertility for the first time, we provide a number of candidate genes possibly causing or being risk factors for the men's spermatogenic failure. The recurring, patient-specific and private, sex-chromosomal CNVs as well as those associated with SCOS are candidates for further, larger case-control and re-sequencing studies.
Infertility, which affects 10–15% of all couples, is attributed to a male (co-)factor in around 50%. Male infertility is mostly caused by spermatogenic failure, clinically noted as oligo- or azoospermia. However, the reasons for the decreased sperm production remain largely unclear: After a full clinical workup around 30% of cases are considered ‘idiopathic infertile’ and an additional 40% have not sufficient/uncertain causes (e.g. varicocele, infections)
The currently established genetic causes of male infertility comprise abnormalities on all genomic levels from chromosomal abnormalities, in particular Klinefelter syndrome, to Y-chromosomal (azoospermia factor, AZF) deletions, to mutations of the cystic fibrosis transmembrane conductance regulator (
A multitude of up to 1,500 genes are thought to be involved in spermatogenesis of which 300–600 are specifically expressed in the male germline
Copy Number Variants (CNVs) have been shown to be an important source of genetic diversity with remarkable differences between individuals and to play a role in complex diseases such as mental retardation, schizophrenia and cancer
Caucasian patients of German origin with idiopathic infertility were selected from the clientele of the Department of Clinical Andrology of the Centre of Reproductive Medicine and Andrology, Münster, a tertiary-referral centre, using the Androbase© database
All participants gave written informed consent for evaluation of their clinical data and genetic analysis of their donated DNA samples according to a protocol approved by the Ethics Committee of the Medical Faculty in Münster and State Medical Board.
Genomic DNA was extracted from peripheral blood by standard methods and analysed at first by the commercially available Human Genome CGH Microarray Kits 244A (Agilent Technologies, Santa Clara, California, USA). This array comprises 236,381 60-mer oligonucleotide probes with a median probe spacing of 8.9 Kb. During the course of the study, the higher-resolution 400K microarray with 411,056 oligonucleotides and a median probe spacing of 5.3 Kb became available. We switched to using this array to be able to compare the impact of smaller CNVs detected with the higher-resolution on spermatogenic failure. Finally, 78 control men and 42 with severe oligozoospermia were analysed with the 244A arrays and 22 and 47 with the 400K arrays. All azoospermic patients with SCOS were analysed with the 400K arrays. Each patient's DNA was compared to 10 pooled DNAs (Promega Human Genomic DNA: Male, Cat.-Nr. G1471). Labelling and hybridisation were performed according to the manufacturer's protocol. In brief, 1 µg of patients' DNA and the pooled control DNA were double-digested with
Comparisons between patients and controls were carried out using the two-sample t-tests if data were normally distributed (e.g. number of CNVs) and otherwise non-parametric Mann-Whitney test. Frequencies were compared by Fisher's exact test. In principle,
The clinical parameters of the normozoospermic controls and the two study groups with severe oligozoospermia and azoospermia caused by SCOS are presented in
Normozoospermic controls (N = 100) | Severe oligozoospermia (N = 89) | Sertoli-cell-only syndrome (N = 37) | |
|
40.1±4.4 (40, 30–59) | 38.7±5.8 (39, 23–58) | 40.3±5.4 (40, 29–50) |
|
58±18 (59, 22–111) | 41±13 (39, 12–89) |
29±11 (30, 10–56) |
|
3.1±1.2 (3.0, 0.7–7.1) | 4.5±2.3 |
7.8±3.2 |
|
3.7±2.0 (3.1, 1.3–9.4) | 7.7±5.4 |
15.3±4.9 |
|
16.4±5.1 (15.9, 6.9–39.0) | 15.3±4.9 (15.0, 4.6–31.3) | 14.1±5.8 (12.4, 7.6–34.4) |
|
3.9±1.6 (4, 2–7) | 3.8±1.3 (4, 2–7) | 4.2±1.2 (4, 2–10) |
|
77.0±47.0 (62.0, 20.0–231.0) | 1.4±1.2 |
0 |
|
3.9±1.5 (3.5, 2.0–9.0) | 3.6±1.4 (3.4, 1.5–9.2) | 4.1±1.8 (4.0, 1.0–10.0) |
|
287.3±182.1 (269.5, 40.0–1083.5) | 4.5±3.3 |
0 |
|
53.7±5.9 (54, 36–75) | 32.4±13.0 |
- |
|
16.7±5.2 (16, 10–34) | 6.0±5.6 |
- |
**significantly different compared to controls (
In total, 1304 CNVs were detected in 100 controls, 1297 CNVs in oligozoospermic men (N = 89) and 728 CNVs in patients with SCOS (N = 37); the numbers and categorisations of CNVs are depicted in
Around twice as many CNVs (independent of type and fitting the roughly two-fold increased resolution) were detected with the 440K array and in general more duplications than deletions. No significant differences between patient groups were found.
Normozoospermic controls | Severe oligozoospermia | Sertoli-cell-only syndrome | |||
Array | 244A | 400K | 244A | 400K | 400K |
|
10.9±3.0 | 20.6±5.7 | 10.2±2.8 | 18.5±6.3 | 19.7±5.9 |
|
5.0±2.5 | 11.3±5.0 | 4.9±2.4 | 8.8±3.8 | 9.2±4.1 |
|
5.9±2.3 | 9.4±3.0 | 5.4±2.1 | 9.6±3.8 | 10.5±3.4 |
|
2732±1335 | 3076±842 | 3015±1383 | 3313±2257 | 3087±1292 |
|
1510±1234 | 1649±1144 | 1842±1414 | 1761±1219 | 1464±850 |
|
1223±745 | 1427±730 | 1173±932 | 1551±1841 | 1624±950 |
No significant differences were found between the study groups.
To analyse whether the chromosomal distribution of CNVs was different between the groups, the number of CNVs, and duplications/deletions separately, was calculated per chromosome and normalised to 100 men (
Significantly different frequencies between the groups are marked with an asterisk.
In many cases, several differently sized CNVs spanned a common region but probably have different breakpoints. If either the gene content was identical or the CNVs spanned regions with the breakpoints distance ±10 Kb (roughly corresponding to one oligonucleotide and thereby the minimum array resolution), these CNVs were aggregated for statistical analyses. Duplications and deletions were considered as different variants because of their supposedly diverse impact. Of the 3329 CNVs, a majority of 2711 (81%) could thereby be summarised as 310 recurring variants (159 deletions and 151 duplications), of which the smallest common region is then reported. Some of these were only found in normozoospermic controls (N = 21), at most once per group (N = 58) or not in controls (N = 14, recurring, patient-specific CNVs).
The frequencies of the remaining 217 variants were compared between the study groups (
Region | Start | End | Size [Kb] | Gene symbol(s) | Type | Normozoosp. | Sev. oligozoosp. |
|
Sertoli-cell-only syndrome |
|
|
|
4q13.2 | 69069451 | 69166014 | 96.6 |
|
dup | 10/100 (10%) | 12/89 (13.5%) | 0.50132 | 18/37 (48.6%) |
|
|
yes |
4q13.2 | 69069451 | 69166014 | 96.6 |
|
del | 28/100 (28%) | 21/89 (23.6%) | 0.51072 | 1/37 (2.7%) |
|
|
yes |
7q34 | 141413152 | 141438704 | 25.6 |
|
dup | 1/22 (4.5%) | 0/47 (0%) | 0.31884 | 10/37 (27%) | 0.04059 |
|
yes |
7q34 | 141413152 | 141438704 | 25.6 |
|
del | 3/22 (13.6%) | 8/47 (17%) | 1.00000 | 4/37 (10.8%) | 1.00000 | 0.56819 | yes |
12p13.31 | 9528390 | 9610254 | 81.9 | dup | 1/100 (1%) | 9/89 (10.1%) |
|
0/37 (0%) | 1.00000 | 0.37395 | yes | |
12p13.31 | 9528390 | 9610254 | 81.9 | del | 4/100 (4%) | 11/89 (12.4%) | 0.05618 | 13/37 (35.1%) |
|
|
yes | |
14q11.2 | 19268576 | 19490830 | 222.3 | dup | 10/100 (10%) | 13/89 (14.6%) | 0.37765 | 8/37 (21.6%) | 0.08990 | 0.18733 | yes | |
14q11.2 | 19268576 | 19490830 | 222.3 | del | 36/100 (36%) | 24/89 (27%) | 0.21172 | 2/37 (5.4%) |
|
|
yes | |
14q32.33 | 105602402 | 105630289 | 27.9 | dup | 8/100 (8%) | 5/89 (5.6%) | 0.57633 | 11/37 (29.7%) |
|
|
yes | |
14q32.33 | 105602402 | 105630289 | 27.9 | del | 14/100 (14%) | 12/89 (13.5%) | 1.00000 | 7/37 (18.9%) | 0.59345 | 0.44578 | yes | |
17q21.31 | 41521344 | 41566740 | 45.4 |
|
dup | 10/100 (10%) | 11/89 (12.4%) | 0.64844 | 1/37 (2.7%) | 0.28787 | 0.13883 | yes |
17q21.31 | 41521344 | 41566740 | 45.4 |
|
del | 6/100 (6%) | 9/89 (10.1%) | 0.41980 | 16/37 (43.2%) |
|
|
yes |
For comparison, the corresponding duplication/deletion (if present in any group) is included independent of
If CNVs were only found by higher-resolution 400K-array, the number of all cases is reduced to 22 for normozoospermic controls and to 47 for patients with severe oligozoospermia (see
SCOS = Sertoli-cell-only syndrome.
Group | Region | Start | End | Size (Kb) | Number, type | Gene symbol(s) | DGV |
|
2p11.2 | 89635198 | 89902565 | 267.0 | 2xdel | - | yes |
3p11.1 | 89476719 | 89499633 | 22.0 | 4xdel |
|
yes | |
4p16.1 | 8235974 | 8261720 | 25.7 | 2xdup |
|
yes | |
6p21.31 | 35143115 | 35184210 | 41.1 | 2xdup |
|
no | |
10q23.1 | 84138134 | 84171245 | 33.1 | 2xdel |
|
no | |
10q23.33 | 96497202 | 96536412 | 39.2 | 2xdel |
|
no | |
12q13.3 | 55866674 | 55896055 | 29.4 | 2xdup | no | ||
16q22.1 | 66942648 | 66967713 | 25.1 | 2xdup | no | ||
17q12 | 30624580 | 30787596 | 163.0 | 2xdel | no | ||
18q23 | 75746093 | 75779459 | 33.0 | 1xdup, 2xdel | no | ||
Xq26.3 | 134120502 | 134157976 | 37.5 | 2xdup |
|
yes | |
|
3p11.1 | 89476719 | 89499633 | 22.9 | 3xdel |
|
yes |
8q24.3 | 145061948 | 145093349 | 31.4 | 1xdup, 1xdel | yes | ||
12p11.21 | 31132516 | 31223665 | 91.1 | 2xdel | yes | ||
12q23.1 | 98491661 | 98519308 | 27.6 | 2xdel |
|
no |
SCOS = Sertoli-cell-only syndrome. Gene information - name, location, IDs - available as
Group | Region | Start | End | Size (Kb) | Type | Gene symbol(s) | DGV |
|
Xp21.3 | 28162190 | 28214748 | 52.0 | dup | - | yes |
Xp11.4 | 38376283 | 38513841 | 137.6 | dup |
|
no | |
Xp11.22 | 52657689 | 52978139 | 320.5 | dup | yes | ||
Xp11.22 | 52842080 | 52909890 | 67.8 | dup | no | ||
Xq22.1 | 102134796 | 102496321 | 361.5 | dup | no | ||
Xq22.2 | 103066101 | 103190187 | 124.0 | dup | yes | ||
Xq22.3 | 105010614 | 105561054 | 550.4 | dup | yes | ||
Xq22.3 | 110238448 | 110260226 | 21.0 | dup |
|
no | |
Xq23 | 111598447 | 111621531 | 23.0 | del |
|
no | |
Xq25 | 123911267 | 124039708 | 128.4 | del |
|
no | |
Xq27.1 | 139706586 | 139904507 | 197.9 | dup |
|
no | |
Xq28 | 154044877 | 154079019 | 34.0 | del |
|
no | |
|
Xp22.33 | 2711073 | 2814530 | 103.5 | del | no | |
Xp22.2 | 16688233 | 16707403 | 19.2 | dup |
|
no | |
Xp21.3 | 25568263 | 25583583 | 15.3 | del |
|
no | |
Xp11.3 | 44067590 | 44084085 | 16.5 | dup |
|
no | |
Xq11.1 | 64806000 | 64854709 | 48.7 | dup |
|
no | |
Xq12 | 65385501 | 65413711 | 28.2 | dup |
|
no | |
Xq22.3–q23 | 110226892 | 110965127 | 738.2 | dup | no | ||
Xq24 | 118780844 | 118798128 | 17.3 | dup |
|
no | |
Xq25 | 122920543 | 123009115 | 88.6 | dup |
|
no | |
Xq26.2 | 131413847 | 131439663 | 25.8 | del |
|
no | |
Xq26.3 | 134600709 | 134628136 | 27.4 | dup |
|
yes | |
Yp11.2 | 7348864 | 7491480 | 142.6 | dup |
|
no | |
Y11.223 | 21964794 | 22058959 | 94.2 | dup |
|
yes | |
Y11.23 | 26870161 | 27073218 | 203.1 | dup | - | yes |
SCOS = Sertoli-cell-only syndrome. Gene information - name, location, IDs - available as
Group | Phenotype | Region | Gene symbol | OMIM | Function |
|
Normo, Oligoz., SCOS | 4q13.2 |
|
601903 | glucuronidase essential for urinary testosterone excretion |
|
4xOligoz./3xSCOS | 3p11.1 |
|
179611 | Anks proteins involved in modulating degradation of EphA receptors |
2xOligoz. | 6p21.31 |
|
608994 | ||
2xSCOS | 12q23.1 |
|
607815 | ||
2xSCOS | 8q24.3 |
|
601282 | Plectin in Sertoli cells concentrated at intercellular junctions and nuclear surface |
|
2xOligoz. | 16q22.1 |
|
610087 | protein methyltransferase, cooperates with the testis-specific factor CTCFL |
|
|
1xOligoz. | Xp11.4 |
|
300096 | interaction with SPAG11B (sperm associated antigen) isoform D, associated with spermatozoa |
1xOligoz. | Xp11.22 |
|
300542 | cancer-testis antigen, expressed in normal testis tissue |
|
1xOligoz. | Xp11.22 |
|
300668 | cancer-testis antigen, expressed in post-meiotic spermatids |
|
1xOligoz. | Xq22.1 |
|
300690 | in mice: expression in pachytene spermatocytes and spermatids |
|
1xOligoz. | Xq22.1 |
|
300316 | belongs to a family of nuclear RNA export factors (NXF), Nxf2 plays a role in spermatogenesis (meiosis and maintenance) |
|
1xOligoz. | Xq22.2 |
|
300507 | testis-specific histone, SNP in 5′ untranslated region associated with oligozoospermia |
|
1xOligoz./1xSCOS | Xq22.3 |
|
300142 | one isoform specifically expressed in testis |
|
1xSCOS | Xq23 |
|
300334 | interacts and co-localises with Enkurin in sperm |
OMIM = Online Mendelian Inheritance in Man (
Under the hypothesis that accumulating CNVs might lead to reduced sperm output, the number of total CNVs, duplications and deletions as well as amount of DNA change was correlated with sperm concentration and count. Because the number of deletions varies strongly with the array used, these were analysed separately. In the largest group, 78 normozoospermic men analysed by 244A arrays, a significant negative association (r = −0.27,
For the first time, we analysed 89 strictly selected patients with severe oligozoospermia, 37 with azoospermia due to SCOS and 100 with normal spermatogenesis as controls by array-CGH. Although interpretation of the results would have been more straightforward if only one type of microarray had been used and while knowing that amount of DNA available as well as funding would not permit us to repeat the analyses on the first set of samples, we favoured switching to the higher resolution array to detect smaller CNVs in almost half of our subjects, which in the eand appeared to be beneficial. As result, we report several genes and genomic regions on autosomes and more pronouncedly on the sex-chromosomes that might either be risk factors (also found in controls) or causative by themselves (not found in controls) for spermatogenic failure.
We hypothesised that an increased number or specific distribution of CNVs could result in defective recombination, meiotic and thereby spermatogenic failure. Structural chromosomal aberrations are found more frequently in men with oligo- and azoospermia with an emphasis on autosomal translocations in the former (3–4% compared to 0.5–1.5% in controls) and sex-chromosomal aneuploidy in the latter (13–16% compared to 0.5–1%)
In principle, CNVs may result in altered gene transcription/protein function through different mechanisms: they might encompass dosage-sensitive genes, a deletion may demask a recessive mutation on the homologous chromosome, genes overlapped by structural variation may be disrupted directly or a CNV can exert position effects
Genes may be prioritised according to a known function or indirectly through their expression in the testis. Therefore, the 4 autosomal and 8 X-chromosomal genes presented in
In two recent studies, our group analysed patients with POF and XY gonadal dysgenesis by array-CGH
With sperm counts ranging from zero to hundreds of millions per ejaculate, sperm output may be viewed as a quantitative trait and male infertility is sometimes postulated as a polygenic disease
While on the one hand the selection of our control group from the patient clientele avoids population stratification, on the other hand the conclusions drawn primarily remain limited to the phenotype of spermatogenic failure and cannot be readily extended to fertility. For this purpose, a group of proven fertile men (fathers) would be needed as additional controls. However, at least one-fifth of our normozoospermic controls had fathered a child before then presenting with either secondary infertility or infertility in a new relationship. Contrariwise, the usually utilised Database of Genomic Variants (DGV) of ‘healthy’ controls is not amenable to be used with respect to the phenotype of spermatogenic impairment, as the fertility status (let alone spermatogenesis) is unknown. Thus, as a larger group of proven fertile men was neither available to us nor has - to our knowledge - been analysed anywhere else yet, our control group may well be used to study spermatogenesis. The CNV data of the 100 controls is provided as supplement as well as accessible through dbVar and will be valuable for other studies on genetics of male infertility.
In contrast to many candidate-gene approaches, only one recently published study analysed 80 men with normozoospermia, 52 with OAT, and 40 with non-obstructive azoospermia (histology not mentioned) using whole genome SNP arrays
In conclusion, by the first CNV study in male infertility, we provide evidence that CNVs contribute to the complex origin of male infertility and present a number of candidate genes possibly causing or being risk factors for spermatogenic failure.
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The authors thank the patients and physicians who took care of them at the Centre of Reproductive Medicine and Prof. Jörg Gromoll for general support. The technical assistance of Mandy Hoffmann, Katja Hagen, Nilusha Sivapalan and Gerrit Randau is gratefully acknowledged. We thank Prof. Sören W. Perrey, University of Applied Sciences, Gelsenkirchen, for the development of a tool for automated DGV queries.