Csilla Krausz

EAA/EMQN best practice guidelines for molecular diagnosis of Y‐chromosomal microdeletions: state‐of‐the‐art 2013

The molecular diagnosis of Y‐chromosomal microdeletions is a common routine genetic test which is part of the diagnostic workup of azoospermic and severe oligozoospermic men. Since 1999, the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) have been actively involved in supporting the improvement of the quality of the diagnostic assays by publication of the laboratory guidelines for molecular diagnosis of Y‐chromosomal microdeletions and by offering external quality assessment trials. The present revision of the 2004 laboratory guidelines summarizes all the clinical novelties related to the Y chromosome (classic, partial and gene‐specific deletions, genotype–phenotype correlations, methodological issues) and provides an update on the results of the quality control programme. These aspects also reflect the consensus of a large group of specialists present at a round table session during the recent Florence‐Utah‐Symposium on ‘Genetics of male infertility’ (Florence, 19–21 September, 2013). During the last 10 years the gr/gr deletion has been demonstrated as a significant risk factor for impaired sperm production. However, the screening for this deletion type in the routine diagnostic setting is still a debated issue among experts. The original basic protocol based on two multiplex polymerase chain reactions remains fully valid and appropriate for accurate diagnosis of complete AZF deletions and it requires only a minor modification in populations with a specific Y chromosome background. However, in light of novel data on genotype–phenotype correlations, the extension analysis for the AZFa and AZFb deletions is now routinely recommended. Novel methods and kits with excessively high number of markers do not improve the sensitivity of the test, may even complicate the interpretation of the results and are not recommended. Annual participation in an external quality control programme is strongly encouraged. The 12‐year experience with the EMQN/EAA scheme has shown a steep decline in diagnostic (genotyping) error rate and a simultaneous improvement on reporting practice.

European Association of Urology Guidelines on Male Infertility: The 2012 Update

CONTEXT New data regarding the diagnosis and treatment of male infertility have emerged and led to an update of the European Association of Urology (EAU) guidelines for Male Infertility. OBJECTIVE To review the new EAU guidelines for Male Infertility. EVIDENCE ACQUISITION A comprehensive work-up of the literature obtained from Medline, the Cochrane Central Register of Systematic Reviews, and reference lists in publications and review articles was developed and screened by a group of urologists and andrologists appointed by the EAU Guidelines Committee. Previous recommendations based on the older literature on this subject were taken into account. Levels of evidence and grade of guideline recommendations were added, modified from the Oxford Centre for Evidence-based Medicine Levels of Evidence. EVIDENCE SUMMARY These EAU guidelines are a short comprehensive overview of the updated guidelines of male infertility as recently published by the EAU (http://www.uroweb.org/guidelines/online-guidelines/), and they are also available in the National Guideline Clearinghouse (http://www.guideline.gov/).

Y-chromosome lineages trace diffusion of people and languages in southwestern Asia

The origins and dispersal of farming and pastoral nomadism in southwestern Asia are complex, and there is controversy about whether they were associated with cultural transmission or demic diffusion. In addition, the spread of these technological innovations has been associated with the dispersal of Dravidian and Indo-Iranian languages in southwestern Asia. Here we present genetic evidence for the occurrence of two major population movements, supporting a model of demic diffusion of early farmers from southwestern Iran-and of pastoral nomads from western and central Asia-into India, associated with Dravidian and Indo-European-language dispersals, respectively.

Y CHROMOSOME AND MALE INFERTILITY: UPDATE, 2006

Male factor infertility accounts for about half the cases of couple infertility and in around 50% of cases its etiology remains unknown. Molecular genetic techniques have unveiled a number of etiopathogenetic factors, including microdeletions of the Yq. Y chromosome microdeletions removing the AZoospermia Factor (AZF) regions are the most frequent molecular genetic causes of oligo/azoospermia. The intense effort of many laboratories contributed to a better understanding of the clinical significance of this genetic anomaly and to the identification of fertility candidate genes in the AZF regions. Important progress has been made on the structure of the Y chromosome and the mechanism of deletion. Studies aimed to define a predisposing genetic background for Yq deletions were not successful, perhaps due to the low number of patients analyzed so far. The screening for Yq deletions became a routine diagnostic test that provides an etiology for spermatogenic disturbances, and assess in the prognosis for testicular sperm retrieval according to the type of deletion. Assisted reproductive techniques represent an efficient symptomatic therapy for men bearing Y microdeletions, however, this genetic defect is transmitted to the male offsprings, affecting their fertility. Future studies should focus on understanding the biological function of AZF genes which is an essential step for the development of more appropriate and knowledge-based therapies.

Gene polymorphisms/mutations relevant to abnormal spermatogenesis

Despite the identification of an increasing number of candidate genes involved in spermatogenesis, the armamentarium of diagnostic genetic tests in male infertility remains extremely limited. A number of new causative mutations have been reported for hypogonadotrophic hypogonadism but still the genetic diagnosis in this pathological condition is made only in about 20% of cases. The sole molecular genetic test that is routinely proposed in severe spermatogenic disturbances is screening for Yq microdeletion. The search for causative mutations in the Y chromosome, and in autosomal and X-linked genes, has mostly been unsuccessful. The paucity of gene mutations raises questions about the appropriateness of the currently used screening approaches. Among the proposed genetic risk factors, gr/gr deletion of the Y chromosome seems to be the most promising polymorphism. Other polymorphisms are awaiting further confirmation, whereas for some (POLG, DAZL, USP26, FSHR) a lack of association with abnormal spermatogenesis has now been ascertained. It is likely that some polymorphisms lead to testicular dysfunction only when in association with a specific genetic background or with environmental factors. Future large-scale studies with stringent study design may provide a more efficient way to identify clinically relevant genetic factors of male infertility.

Stimulation of oxidant generation by human sperm suspensions using phorbol esters and formyl peptides: relationships with motility and fertilization in vitro.

OBJECTIVES To investigate the influence of reactive oxygen species generated by human spermatozoa and contaminating leukocytes on sperm movement and fertilization in vitro. DESIGN A chemiluminescence technique, using luminol and peroxidase, was used to monitor the generation of reactive oxygen species by human sperm suspensions and the results were correlated with sperm movement and the fertilization of human ova in vitro. SETTING Diagnostic Andrology Laboratory and IVF Clinic. PATIENTS Infertile couples undergoing IVF therapy. RESULTS An N-formyl-methionyl-leucyl-phenylalanine (FMLP) provocation test was used to demonstrate that the presence of leukocytes in 28.5% of the sperm preparations was associated with elevated levels of spontaneous reactive oxygen species production, impaired movement, and a reduced capacity for fertilization in vitro. In the absence of leukocytes, exposure to phorbol ester stimulated a burst of reactive oxygen species generation by human spermatozoa, the magnitude of which was correlated highly with a loss of sperm motility but not with fertilization rates observed in the concurrent IVF cycle. CONCLUSION Leukocyte contamination of human sperm preparations can be detected readily by FMLP-induced, luminol-dependent chemiluminescence and the results have an important bearing on the fertilizing capacity of the spermatozoa in vitro.

The gr/gr deletion(s): a new genetic test in male infertility?

Y chromosome microdeletions are the most frequent genetic cause of severe oligozoospermia ( 99.9%) nucleotide identity, organised in massive palindromes. These repeated sequences may undergo genetic exchange through gene conversion—that is, non-reciprocal transfer of sequence information occurring between duplicated sequences within the chromosome, a process that could account for the >99.9% nucleotide identity between the arms of a palindrome. Although this mechanism may serve to preserve Y chromosome genes from the gradual accumulation of deleterious mutations and thus prolong their genetic fitness,6 this peculiar organisation also provides the structural basis for deletions and rearrangements. The classical AZFc deletion, which removes 3.5 Mb between the b2/b4 amplicons, is the most frequent type of deletion. Taking into consideration the Y chromosome structure and the suggested deletion mechanism, a number of other possible partial deletions have been proposed in both the AZFb and AZFc regions.7,8 The frequency and the pathological significance of these partial deletions is not yet clear, although recently a partial deletion termed gr/gr has been described specifically in infertile men with varying degrees of spermatogenic failure.9,10 This deletion removes half the AZFc gene content, including two copies of the major AZFc …

Effects of transmission of Y chromosome AZFc deletions

Deletions of specific regions on the Y chromosome cause male infertility. Recent advances in infertility treatment allow Y chromosome deletions to be transmitted to male offspring with the assumption that there will be no clinical consequences other than infertility in adult life. We screened 12 patients, who had a 45X/46XY karyotype and presented with Turner stigmata or sexual ambiguities, or both, for Y chromosome microdeletions with PCR. A third of these patients had Y chromosome microdeletions of distal Yq, the most common microdeletion seen in infertile men with azoospermia or severe oligozoospermia. Transmission of Y chromosome microdeletions could potentially have severe clinical consequences other than male infertility, such as the development of sexual ambiguities and Turner stigmata.

Evaluation of 172 candidate polymorphisms for association with oligozoospermia or azoospermia in a large cohort of men of European descent

BACKGROUND In spite of tremendous efforts by a number of groups, the search for single nucleotide polymorphisms (SNPs) strongly associated with male factor infertility by means of gene re-sequencing studies has yielded few likely candidates. A recent pilot, genome-wide SNP association study (GWAS) identified a list of SNPs associated with oligozoospermia and azoospermia. This is an expanded follow-up study of the SNPs identified by the GWAS as well as other SNPs from previously published gene re-sequencing studies. METHODS On the basis of the pilot GWAS and SNPs with published associations with male infertility, 172 SNPs were genotyped in men with idiopathic azoospermia or oligozoospermia using the Illumina BeadXpress platform. RESULTS Several SNPs were identified or confirmed to be significantly associated with oligozoospermia and/or azoospermia. More importantly, this follow-up study indicates that, at least in Caucasian men, no single common SNP accounts for a significant proportion of spermatogenic failure cases. CONCLUSIONS The associations reported in this study are promising, but much larger genome-wide studies will be necessary to confidently validate these SNPs and identify novel SNPs associated with male infertility.

Genetic Risk Factors in Male Infertility

The etiopathogenesis of testicular failure remains unknown in about half of the cases and is referred to as “idiopathic infertility”. “Idiopathic” testicular failure is of probable genetic origin since the number of genes involved in human spermatogenesis is likely thousands and only a small proportion of them have been identified and screened in infertile men. In parallel with studies aimed to identify mutations with a clear cause-effect relationship in spermatogenesis candidate genes, there is an increasing interest towards genetic susceptibility factors to male infertility. Despite many efforts, only a few clinically relevant polymorphisms have been identified. This is mainly related to the multifactorial nature of male infertility and to the inappropriate study design of the majority of the studies. The most promising polymorphisms are in genes involved in the endocrine regulation of spermatogenesis and on the Y chromosome, the “gr/gr” deletions. Polymorphisms are generally considered as co-factors. Their final effect on testis function and fertility is probably modulated by the genetic background of each individual and/or by the presence of certain environmental factors. In this review, recent findings concerning some of the most widely studied polymorphisms and male infertility will be discussed.