Katrien Stouffs

Genetic causes of spermatogenic failure

Approximately 10%-15% of couples are infertile, and a male factor is involved in almost half of these cases. This observation is due in part to defects in spermatogenesis, and the underlying causes, including genetic abnormalities, remain largely unknown. Until recently, the only genetic tests used in the diagnosis of male infertility were aimed at detecting the presence of microdeletions of the long arm of the Y chromosome and/or chromosomal abnormalities. Various other single-gene or polygenic defects have been proposed to be involved in male fertility. However, their causative effects often remain unproven. The recent evolution in the development of whole-genome-based techniques and the large-scale analysis of mouse models might help in this process. Through knockout mouse models, at least 388 genes have been shown to be associated with spermatogenesis in mice. However, problems often arise when translating this information from mice to humans.

Possible role of USP26 in patients with severely impaired spermatogenesis

The ubiquitin-specific protease 26 (USP26) gene is an X-linked gene specifically expressed in testis tissue. This gene is therefore a potential infertility gene. In order to analyse its possible involvement in spermatogenesis and infertility, 42 patients with Sertoli cell-only syndrome were analysed for mutations in this gene. We found four patients with exactly the same three changes of the nucleotide and therefore also amino acid sequence. These patients showed 370-371insACA, 494T>C and 1423C>T causing T123-124ins, L165S and H475Y, respectively. These changes were not found in 10 control samples. Furthermore, two polymorphisms were observed which do not alter the amino-acid sequence. A restriction analysis that can make a distinction between a T and a C, at position 494, was set up in order to examine more patient and control samples. Another 69 patients with Sertoli cell-only syndrome, 32 patients with maturation arrest and 142 control samples were analysed using this method. None of the control samples or patients with maturation arrest featured the change at position 494. However, four more patients with Sertoli cell-only syndrome were identified with the three alterations. The frequency of alterations in this group is therefore 7.2% (8/111). One of the patients had a deletion of the long arm of the Y chromosome, while another patient had a varicocoele. These results indicate that these alterations might be involved in male infertility or might increase the risk of male infertility.

What about gr/gr deletions and male infertility? Systematic review and meta-analysis

BACKGROUND The impact of gr/gr deletions on male fertility is unclear. These partial deletions of the AZFc region of the Y chromosome have been detected more frequently in infertile patients. However, few individual studies have demonstrated a statistically significant association. This study aims to quantify the strength of association between gr/gr deletions and male infertility, and to explore potential sources of heterogeneity, including ethnicity and geographical location. METHODS Medline was searched up to 31 December 2009 for full articles investigating the prevalence of gr/gr deletions in infertile and control men. A pooled odds ratio (OR) was estimated by a random-effects model. Heterogeneity was assessed by the Cochrans Q test, and quantified by I(2) statistic. RESULTS A total of 18 case-control studies, including 6388 cases and 6011 controls, met our inclusion criteria and showed that gr/gr deletions were present in 6.86% of cases and 4.69% of controls. The association between gr/gr deletions and infertility was significant (P < 0.001), with a pooled random-effects OR of 1.76 (1.21-2.66) for infertile men versus normozoospermic controls (13 studies). The test for heterogeneity among studies yielded a Q test P = 0.089 with I(2) value of 37%, indicating moderate heterogeneity. The association between gr/gr deletions and infertility was dependent on ethnicity and geographic region. CONCLUSIONS Our meta-analysis comprising >12 000 men demonstrates that gr/gr deletions occur more frequently in infertile than control men. The association between gr/gr deletions and infertility varies according to ethnicity and geographic region, with an association reaching significance among Caucasian men, in Europe and the Western Pacific region.

Male infertility and the involvement of the X chromosome

BACKGROUND Male infertility is a worldwide problem, keeping many researchers puzzled. Besides environmental factors, much attention is paid to single gene defects. In this view, the sex chromosomes are particularly interesting since men only have a single copy of these chromosomes. The involvement of the Y chromosome in male infertility is obvious since the detection of Yq microdeletions. The role of the X chromosome, however, remains less understood. METHODS Articles were obtained by searching PubMed until December 2008. A first search attempted to identify genes located on the X chromosome potentially important for spermatogenesis. A second part of the study was focused on those genes for which the role has already been studied in infertile patients. RESULTS Multiple genes located on the X chromosome are expressed in testicular tissues. The function of many genes, especially the cancer-testis genes, has not been studied so far. There were striking differences between mouse and human genes. In the second part of the study, the results of mutation analyses of seven genes (AR, SOX3, USP26, NXF2, TAF7L, FATE and AKAP4) are described. Except for AR, no infertility causing mutations have, thus far, been described. It cannot be excluded that some of the observed changes should be considered as risk factors for impaired spermatogenesis. CONCLUSIONS It can be concluded that, so far, the mutation analysis of X-linked genes in humans, presumed to be crucial for spermatogenesis or sperm quality, has been disappointing. Other approaches to learn more about male infertility are necessary.

Array comparative genomic hybridization in male infertility

BACKGROUND Male infertility caused by a maturation arrest of spermatogenesis is a condition with an abrupt stop in spermatogenesis, mostly at the level of primary spermatocytes. The etiology remains largely unknown. METHODS We focused on patients with a complete arrest at the spermatocyte level (n = 9) and used array comparative genomic hybridization to screen for deletions or duplications that might be associated with maturation arrest. Interesting copy number variations (CNVs) were further examined by using quantitative PCR. Where appropriate, the expression pattern was analyzed in multiple human tissues including the testis. RESULTS A total of 227 CNVs were detected in the patient group. After the elimination of CNVs that were also present in the control group or that were not likely to be involved in male infertility, the remaining 11 regions were investigated more in detail. We first determined the expression pattern of seven genes, for which expression had not been reported to be investigated in testicular tissue, after which one region could be eliminated. Next, all 10 promising candidate regions were analyzed by quantitative PCR in a control population. CONCLUSIONS Eight deletions/duplications were absent in our control group, and therefore might be linked with the male infertility in our patients. One of these alterations, however, has been detected in a proven fertile father group. Further research is necessary to determine the relationship between the observed genomic alterations and maturation arrest of spermatogenesis. Furthermore, several of the above genes have not been studied at the functional level and consequently, more research is required to determine their role in spermatogenesis.

Do we need to search for gr/gr deletions in infertile men in a clinical setting?

BACKGROUND Partial deletions of the AZFc region of the Y chromosome such as gr/gr deletions have been detected in infertile patients as well as in control groups. The impact of these gr/gr deletions on the etiology of male infertility remains unknown. In the present study, we investigated the presence of gr/gr deletions in Caucasian men. METHODS gr/gr deletions were analyzed by using markers sY1291, sY1191 and sY1197 and by investigating the presence of single nucleotide variants (SNV) in DAZ and CDY1 genes in patients with azoospermia (n = 44), cryptozoospermia (n = 51) or severe oligozoospermia (n = 92). Control groups consisted of men with normal spermatogenesis on testicular biopsy (n = 33), normozoospermia (n = 278) or proven fertility (n = 83). RESULTS We observed 20 gr/gr deletions, with eight in infertile patients (4.3%) and 12 in the control groups (3.0%), which was not significantly different. DAZ SNV analysis revealed eight different deletion patterns in patients and controls. CONCLUSIONS In the present study, no significant differences in the frequency of gr/gr deletions between different patient and control groups were observed. We concluded that the relationship between gr/gr deletions and male infertility remains unclear and that it is too early to systematically test for gr/gr deletions for infertile couples seeking assisted reproduction treatment.

Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?

Next-generation sequencing (NGS), an innovative sequencing technology that enables the successful analysis of numerous gene sequences in a massive parallel sequencing approach, has revolutionized the field of molecular biology. Although NGS was introduced in a rather recent past, the technology has already demonstrated its potential and effectiveness in many research projects, and is now on the verge of being introduced into the diagnostic setting of routine laboratories to delineate the molecular basis of genetic disease in undiagnosed patient samples. We tested a benchtop device on retrospective genomic DNA (gDNA) samples of controls and patients with a clinical suspicion of a mitochondrial DNA disorder. This Ion Torrent Personal Genome Machine platform is a high-throughput sequencer with a fast turnaround time and reasonable running costs. We challenged the chemistry and technology with the analysis and processing of a mutational spectrum composed of samples with single-nucleotide substitutions, indels (insertions and deletions) and large single or multiple deletions, occasionally in heteroplasmy. The output data were compared with previously obtained conventional dideoxy sequencing results and the mitochondrial revised Cambridge Reference Sequence (rCRS). We were able to identify the majority of all nucleotide alterations, but three false-negative results were also encountered in the data set. At the same time, the poor performance of the PGM instrument in regions associated with homopolymeric stretches generated many false-positive miscalls demanding additional manual curation of the data.

Biallelic Mutations in TMEM126B Cause Severe Complex I Deficiency with a Variable Clinical Phenotype

Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs∗2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.

Mutation analysis of three genes in patients with maturation arrest of spermatogenesis and couples with recurrent miscarriages

The primary aim of this study was to gain more insight into maturation arrest of spermatogenesis (MA) and its relationship with mutations in genes essential for meiosis. The study also investigated the possibility that mutations in human meiosis genes cause a milder phenotype and that, in such cases, meiosis could potentially be completed with the production of mature germ cells having an abnormal chromosomal constitution causing miscarriage. Among 40 patients with MA, five changes were observed that also predicted alterations at the amino acid level. However, since these changes were also present in men with normozoospermia in equal frequencies, it was assumed that these changes are single nucleotide polymorphisms. Among 46 patients with recurrent miscarriages, two additional changes were detected predicting an alteration at the amino acid level. One change was detected in controls. However, the second heterozygous change, detected in a conserved functional domain of the SYCP3 gene, was absent in >200 controls. These preliminary results stress the need to further investigate the relationship between abnormalities in meiosis genes and the formation of gametes with abnormal chromosomal constitution. More research is also necessary to determine the impact and frequency of such changes before implementing mutation screening in genetic counselling.

Is there a role for the nuclear export factor 2 gene in male infertility

OBJECTIVE To determine the presence of mutations in the NXF2 gene of patients with nonobstructive azoospermia. DESIGN Molecular analysis of male infertility. SETTING University genetic laboratory and reproductive clinic. PATIENT(S) Sixty-five patients with Sertoli cell-only syndrome (SCOS) and 20 control men. INTERVENTION(S) Polymerase chain reaction, sequencing analysis, RNA extraction, and reverse transcription polymerase chain reaction. MAIN OUTCOME MEASURE(S) Expression of NXF2 messenger RNA and analysis of the NXF2 gene for the presence of mutations and polymorphisms. RESULT(S) Messenger RNA derived from the NXF2 gene could be amplified from normal human testicular tissue. Sequencing analysis showed the presence of two polymorphisms in the NXF2 gene. A first alteration, c.1779C>T, was observed in one man who had complete SCOS. Although it is located near an intron-exon boundary, this change probably does not affect splicing. The second alteration, c.1857A>G, was detected in 22 patients with complete SCOS and in 13 patients with incomplete SCOS. Also, 15 of 20 men with normal spermatogenesis had this alteration. Neither of these alterations causes a change at the amino acid level. CONCLUSION(S) No mutations were detected in the NXF2 gene, from which we concluded that there is no need to screen for mutations in the NXF2 gene in a routine IVF program.