Pseudoautosomal abnormalities in terminal AZFb+c deletions are associated with isochromosomes Yp and may lead to abnormal growth and neuropsychiatric function.

STUDY QUESTION: Are copy number variations (CNVs) in the pseudoautosomal regions (PARs) frequent in subjects with Y-chromosome microdeletions and can they lead to abnormal stature and/or neuropsychiatric disorders? SUMMARY ANSWER: Only subjects diagnosed with azoospermia factor (AZF)b+c deletions spanning to the end of the Y chromosome (i.e. terminal deletions) harbor Y isochromosomes and/or cells 45,X that lead to pseudoautosomal gene CNVs, which were associated with abnormal stature and/or neuropsychiatric disorders. WHAT IS KNOWN ALREADY: The microdeletions in the long arm of the Y chromosome (Yq) that include the loss of one to three AZF regions, referred to as Yq microdeletions, constitute the most important known etiological factor for primary spermatogenic failure. Recently, controversy has arisen about whether Yq microdeletions are associated with gain or loss of PAR genes, which are implicated in skeletal development and neuropsychiatric function. STUDY DESIGN, SIZE, DURATION: We studied a cohort of 42 Chilean patients with complete AZF deletions (4 AZFa, 4 AZFb, 23 AZFc, 11 AZFb+c) from a university medical center, diagnosed over a period of 15 years. The subjects underwent complete medical examinations with special attention to their stature and neuropsychiatric function. PARTICIPANTS/MATERIALS, SETTING, METHODS: All subjects were characterized for Yq breakpoints by PCR, and for CNVs in PARs by multiplex ligation-dependent probe amplification (MLPA), followed by qPCR analysis for genes in PAR1 (SHOX and ZBED1), PAR2 (IL9R) and two single copy genes (SRY and DDX3Y, respectively located in Yp11.3 and AZFa). In addition, karyotypes revision and fluorescence in situ hybridization (FISH) for SRY and centromeric probes for X (DXZ1) and Y (DYZ3) chromosomes were performed in males affected with CNVs. MAIN RESULTS AND THE ROLE OF CHANCE: We did not detect CNVs in any of the 35 AZF-deleted men with interstitial deletions (AZFa, AZFb, AZFc or AZFb+c). However, six of the seven patients with terminal AZFb+c deletions showed CNVs: two patients showed a loss and four patients showed a gain of PAR1 genes, with the expected loss of VAMP-7 in PAR2. In these patients, the Yq breakpoints localized to the palindromes P8, P5 or P4. In the four cases with gain of PAR1, qPCR analysis showed duplicated signals for SRY and DDX3Y and one copy of IL9R, indicating isodicentric Yp chromosomes [idic(Y)] with breakpoint in Yq11.22. The two patients who had loss of PAR1, as shown by MLPA, had an additional reduction for SRY and DDX3Y, as shown by qPCR, associated with a high proportion of 45,X cells, as determined by FISH and karyotype. In agreement with the karyotype analysis, we detected DYZ3++ and DYZ3+ cells by FISH in the six patients, confirming idic(Y) and revealing additional monocentric Y chromosome [i(Y)]. Five patients had a history of major depressive disorders or bipolar disorder, and three had language impairment, whereas two patients showed severe short stature (Z score: -2.75 and -2.62), while a man with bipolar disorder was very tall (Z score: +2.56). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The number of males studied with Y-chromosome microdeletions and normozoospermic controls with normal karyotypes may not be enough to rule out an association between AZF deletions and PAR abnormalities. The prevalence of Y isochromosomes and/or 45,X cells detected in peripheral blood does not necessarily reflect the variations of PAR genes in target tissues. WIDER IMPLICATIONS OF THE FINDINGS: This study shows that CNVs in PARs were present exclusively in patients with terminal AZFb+c deletions associated with the presence of Y isochromosomes and 45,X cells, and may lead to neuropsychiatric and growth disorders. In contrast, we show that men with interstitial Yq microdeletions with normal karyotypes do not have an increased risk of PAR abnormalities and of phenotypical consequences. Moreover, our results highlight the importance of performing molecular studies, which are not considered in the usual screening for patients with Yq microdeletions. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Fund for Scientific and Technological Development of Chile (FONDECYT), grant no. 1120176 (A.C.). The authors declare that no conflicting interests exist.

Methylation changes in mature sperm deoxyribonucleic acid from oligozoospermic men: assessment of genetic variants and assisted reproductive technology outcome.

OBJECTIVE: To characterize a potential genetic cause for methylation errors described in oligozoospermia. DESIGN: Analysis of PEG1/MEST-DMR and H19-DMR methylation level in sperm, in parallel with the study of several genes on the Y chromosome, DNMT3A, and DNMT3L. Clinical outcome was also looked at regarding PEG1/MEST-DMR and H19-DMR methylation level in sperm. SETTING: Research and diagnostic laboratories. PATIENT(S): One hundred nineteen normospermic and 175 oligozoospermic men consulting for couple infertility. INTERVENTION(S): We studied PEG1/MEST-DMR and H19-DMR methylation profiles in 294 men. We searched for Y chromosome gene aberrations and for mutations in both DNMT3A and DNMT3L genes in men showing epimutations. Assisted reproductive technology (ART) outcomes were also investigated. MAIN OUTCOME MEASURE(S): Sperm samples were collected from 294 volunteers for genomic DNA isolation that was used to study methylation profiles in imprinted loci and Y chromosome SMCY, DNMT3A, and DNMT3L genes. Pregnancy rate was also studied after ART treatment using sperm showing epimutations. RESULT(S): Epimutations in H19-DMR and PEG1/MEST-DMR were found in 20% and 3% of oligozoospermic men, respectively. We identified an amino acid change in DNMT3A in one case and in DNMT3L in eight men with altered methylation profiles. No mutations were detected in SMCY or in selected Y chromsome genes. No correlation between ART outcome and epimutations was found. CONCLUSION(S): We observed epimethylations in spermatozoa of oligozoospermic individuals, but no association was found with genetic variants or in the ART outcome.

Cytogenetics and fluorescence in situ hybridization assessment of sex-chromosome mosaicism in Klinefelter's syndrome.

A retrospective study was carried out in 152 infertile men to determine the prevalence of sex chromosome abnormalities among non-obstructive azoospermic and severe oligospermic men (n = 51) and to evaluate the feasibility of fluorescence in situ hybridization (FISH) techniques to assess mosaicism in Klinefelter's patients in comparison with conventional cytogenetics. Cytogenetic analysis were performed for 51 infertile men and among 14 chromosomal abnormalities found, nine were compatible with Klinefelter's syndrome. FISH staining with a CEP X/CEP Y probes were performed for Klinefelter's patients and for five of them; testes were biopsied for histopathologic examination. Six Klinefelter's patients showed a non-mosaic 47,XXY and three showed a 47,XXY/46,XY mosaic by G or R banding analysis of 20 cells with a ratio of 17%, 20% and 33%, respectively. FISH analysis confirmed mosaicism in only one patient (the first) in whom a third cells population was found. There was no relationship between the ratios of mosaicism by banding and FISH analysis. Conventional histopathologic findings in five non-mosaic Klinefelter's patients confirm the diagnosis of Sertoli Only Cells syndrome. FISH is recommended in Klinefelter's syndrome to define exactly the cytogenetic statute as mosaic or non-mosaic and then discussing prognosis and decision regarding fertility counseling.

Importance of mitochondrial and nuclear sperm DNA in sperm quality assessment and assisted reproduction outcome.

Intracytoplasmic sperm injection (ICSI) has made irrelevant the conventional criteria of concentration, motility and morphology for assessment of sperm quality and so we urgently need new assays by which to gauge sperm 'health'. ICSI may be facilitating the transfer of genetic disorders to future generations by bypassing all the natural hurdles for sperm selection without imposing more pertinent criteria of selection. Sperm DNA quality is vital to the future offspring irrespective of whether the child is conceived naturally, by in vitro fertilization (IVF) or by ICSI. The DNA integrity of sperm can be determined quickly and accurately using a range of techniques that also have strong prognostic power in predicting successful IVF and ICSI outcomes with ejaculated sperm. Moreover, there is a close correlation between testicular nuclear DNA integrity and pregnancy rates in ICSI. Mitochondrial DNA can be measured using long PCR in ejaculated and testicular sperm and is also useful for predicting success in assisted conception. This review discusses how the integrity of both nuclear and mitochondrial affect the choice of sperm for assisted conception.

Assessment of DNA fragmentation of spermatozoa that were surgically retrieved from men with obstructive azoospermia.

OBJECTIVE: To determine the degree of DNA fragmentation in spermatozoa of men with obstructive azoospermia or anejaculation compared with that of ejaculated spermatozoa from fertile donors. DESIGN: Observational study. SETTING; University Medical Center St. Radboud, Nijmegen. The Netherlands. PATIENT(S): Forty-one patients with obstructive azoospermia or anejaculation and 10 fertile donors. MAIN OUTCOME MEASURE(S): Sperm samples were obtained surgically from the epididymis or testis of men with azoospermia or anejeculation and by ejaculation in fertile patients. DNA fragmentation was analyzed in the total sample and in a motile fraction that was isolated as in routine ICSI procedures. DNA breaks were measured by using the TdT-mediated dUTP nick-end labeling assay. RESULT(S): A higher percentage of cells with DNA breaks was found in men with obstructive azoospermia or anejaculation compared with donors (mean, 18.9% vs. 6.2%). A significant lower degree of DNA fragmentation was observed in the motile fraction from patients compared with donors (0.4% vs. 0.6%). CONCLUSION(S): High percentages of cells with DNA damage were found in sperm samples from men with obstructive azoospermia or anejaculation, but a very low frequency of damage to the DNA was observed in the motile fraction. In an ICSI setting, the use of motile sperm retrieved from epididymis or testis of men with obstructive azoospermia does not seem to pose a higher genetic risk to the progeny than does use of motile ejaculated sperm.

Substantial prevalence of microdeletions of the Y-chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence-tagged site-based mapping strategy.

Genes on the long arm of Y (Yq), particularly within interval 6, are believed to play a critical role in human spermatogenesis. Cytogenetically detectable deletions of this region are associated with azoospermia in men, but are relatively uncommon. It has been hypothesized that microdeletions of Yq may account for a significant proportion of men with infertility. The objective of this study was to validate a sequence-tagged site (STS)-mapping strategy for the detection of Yq microdeletions and to use this method to determine the proportion of men with idiopathic azoospermia or severe oligozoospermia who carry microdeletions in Yq. STS mapping of a sufficiently large sample of infertile men should also help further localize the putative gene(s) involved in the pathogenesis of male infertility. Genomic DNA was extracted from peripheral leukocytes of 16 normal fertile men, 7 normal fertile women, 60 infertile men (50 of whom had azoospermia and 10 of whom had severe oligozoospermia with no other recognizable cause of infertility), and 15 patients with the X-linked disorder, ichthyosis. PCR primers were synthesized for 26 STSs that span Yq interval 6. None of the 16 normal men of known fertility had microdeletions. Seven normal fertile women failed to amplify any of the 26 STSs, providing evidence of their Y specificity. No microdeletions were detected in any of the 15 patients with ichthyosis. Of the 60 infertile men typed with 26 STSs, 11 (18%; 10 azoospermic and 1 oligozoospermic) failed to amplify 1 or more STS. Interestingly, 4 of the 11 patients had microdeletions in a region that is outside the Yq region from which the DAZ (deleted in azoospermia gene region) gene was cloned. In an additional 3 patients, microdeletions were present both inside and outside the DAZ region. In 3 subjects, the microdeletions were verified by Southern analysis using labeled PCR products corresponding to the deleted STSs as probes. These data suggest a high prevalence (18%) of Yq microdeletions in men with idiopathic azoospermia/severe oligospermia. The physical locations of these microdeletions provide further support for the concept that a gene(s) on Yq deletion interval 6 plays an important role in spermatogenesis. The presence of deletions that do not overlap with the DAZ region suggests that genes other than the DAZ gene may also be implicated in the pathogenesis of some subsets of male infertility.