Y-chromosome haplogroup architecture confers susceptibility to azoospermia factor c microrearrangements: a retrospective study

Aim To assess the association between azoospermia factor c microrearrangements and semen quality, and between Y-chromosome background with distinct azoospermia factor c microrearrangements and semen quality impairment. Methods This retrospective study, carried out in the Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov,” involved 486 men from different ethnic backgrounds referred for couple infertility from 2002- 2017: 338 were azoospermic/oligozoospermic and 148 were normozoospermic. The azoospermia factor c microrearrangements were analyzed with sequence tagged site and sequence family variant markers, quantitative fluorescent polymerase chain reaction, and multiplex ligation probe amplification analysis. The Y-haplogroups of all participants were determined with direct single nucleotide polymorphism typing and indirect prediction with short tandem repeat markers.Results Our participants had two types of microdeletions: gr/gr and b2/b3; three microduplications: b2/b4, gr/gr, and b2/b3; and one complex rearrangement gr/gr deletion + b2/b4 duplication. Impaired semen quality was not associated with microrearrangements, but b2/b4 and gr/ gr duplications were significantly associated with haplogroup R1a (P < 0.001 and P = 0.003, respectively) and b2/b3 deletions with haplogroup E (P = 0.005). There were significantly more b2/b4 duplication carriers in Albanians than in Macedonians with haplogroup R1a (P = 0.031). Conclusion Even though azoospermia factor c partial deletions/duplications and Y-haplogroups were not associated with impaired semen quality, specific deletions/ duplications were significantly associated with distinct haplogroups, implying that the Y chromosome background may confer susceptibility to azoospermia factor c microrearrangements

Comparative proteomics analysis of human FFPE testicular tissues reveals new candidate biomarkers for distinction among azoospermia types and subtypes.

Understanding molecular mechanisms that underpin azoospermia and discovery of biomarkers that could enable reliable, non-invasive diagnosis are highly needed. Using label-free data-independent LC-MS/MS acquisition coupled with ion mobility, we compared the FFPE testicular proteome of patients with obstructive (OA) and non-obstructive azoospermia (NOA) subtypes hypospermatogenesis (Hyp) and Sertoli cell-only syndrome (SCO). Out of 2044 proteins identified based on ≥2 peptides, 61 proteins had the power to quantitatively discriminate OA from NOA and 30 to quantitatively discriminate SCO from Hyp and OA. Among these, H1-6, RANBP1 and TKTL2 showed superior potential for quantitative discrimination among OA, Hyp and SCO. Integrin signaling pathway, adherens junction, planar cell polarity/convergent extension pathway and Dectin-1 mediated noncanonical NF-kB signaling were significantly associated with the proteins that could discriminate OA from NOA. Comparison with 2 transcriptome datasets revealed 278 and 55 co-differentially expressed proteins/genes with statistically significant positive correlation. Gene expression analysis by qPCR of 6 genes (H1-6, RANBP1, TKTL2, TKTL1, H2BC1, and ACTL7B) with the highest discriminatory power on protein level and the same regulation trend with transcriptomic datasets, confirmed the proteomics results. In summary, our results suggest some underlying pathways in azoospermia and broaden the range of potential novel candidates for diagnosis. SIGNIFICANCE: Using a comparative proteomics approach on testicular tissue we have identified several pathways associated with azoospermia and a number of testis-specific and germ cell-specific proteins that have the potential to pinpoint the type of spermatogenesis failure. Furthermore, comparison with transcriptomics datasets based on genome-wide gene expression analyses of human testis specimens from azoospermia patients identified proteins that could discriminate between obstructive and non-obstructive azoospermia subtypes on both protein and mRNA levels. Up to our knowledge, this is the first integrated comparative analysis of proteomics and transcriptomics data from testicular tissues. We believe that the data from our study contributes significantly to increase the knowledge of molecular mechanisms of azoospermia and pave the way for new investigations in regards to non-invasive diagnosis

Association of IL-10 (rs1800872) and IL-4R (rs1805010) polymorphisms with cervical intraepithelial lesions and cervical carcinomas

Summary Purpose: Genetic characteristic of cytokines may influence cervical intraepithelial neoplasia (CIN) and cervical cancer (CCa) susceptibility. We analysed an association of IL-10- 592 A/C, IL-4R I75VA/G polymorphisms with susceptibility to human papillomavirus (HPV) positive CIN and CCa. Methods: Using multiplex PCR- SNaPShot analysis, 134 cases (HPV positive CINs and CCa) and 113 controls (HPV negative NILM) were genotyped for these two cytokine variants. Results: Data analyzed using odds ratio (OR) and chisquare (x2 ) test showed that the frequency of CC of IL-10-592 genotype was significantly higher in cases (67.2%) than in controls (49.6%) [CC vs CA+AA; p=0.005, OR=2.08 (95%CI: 1.24-3.49)] as well as the allelic frequency of major C allele (82.1%) in cases than in controls (72.6%) [p=0.01, OR=1.73 (95%CI: 1.13-2.66)]. Furthermore, AA genotype of IL-4RI75V had significantly lower frequency in CIN1 (25.0%) compared with CIN2+ group (30.8%) (p=0.03, OR=0.39, 95%CI: 0.14- 1.11) after the stratifications of the cases in low grade and high grade with CCa as separate groups. Conclusion: We concluded that IL-10-592 A/AA variant indicates a protective role in cervical cancer development and the GG genotype of IL-4RI75V conferred protection against progression of CIN1 to CIN2+ or CCa among women from Republic of North Macedonia. Key words: cervical ca

SNaPshot assay for the detection of the most common CFTR mutations in infertile men.

Congenital bilateral absence of vas deferens (CBAVD) is the most common CFTR-related disorder (CFTR-RD) that explains about 1-2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G->A and IVS8polyT variants. The assay was validated on 50 previously genotyped samples and was used to screen a total of 369 infertile men with different impairment of spermatogenesis and 136 fertile controls. Our results show that double heterozygosity of cystic fibrosis (CF) and CFTR-related disorder (CFTR-RD) mutations are found in a high percentage (22.7%) of infertile men with obstructive azoospermia, but not in other studied groups of infertile men. The SNaPshot assay described here is an inexpensive, fast and robust method for primary screening of the most common CFTR mutations both in patients with classical CF and CFTR-RD. It can contribute to better understanding of the role of CFTR mutations in impaired spermatogenesis, ultimately leading to improved management of infertile men

Multilevel regression modeling for aneuploidy classification and physical separation of maternal cell contamination facilitates the QF-PCR based analysis of common fetal aneuploidies.

BackgroundThe quantitative fluorescent polymerase chain reaction (QF-PCR) has proven to be a reliable method for detection of common fetal chromosomal aneuploidies. However, there are some technical shortcomings, such as uncertainty of aneuploidy determination when the short tandem repeats (STR) height ratio is unusual due to a large size difference between alleles or failure due to the presence of maternal cell contamination (MCC). The aim of our study is to facilitate the implementation of the QF-PCR as a rapid diagnostic test for common fetal aneuploidies.MethodsHere, we describe an in-house one-tube multiplex QF-PCR method including 20 PCR markers (15 STR markers and 5 fixed size) for rapid prenatal diagnosis of chromosome 13, 18, 21, X and Y aneuploidies. In order to improve the aneuploidy classification of a given diallelic STR marker, we have employed a multilevel logistic regression analysis using "height-ratio" and "allele-size-difference" as fixed effects and "marker" as a random effect. We employed two regression models, one for the 2:1 height ratio (n = 48 genotypes) and another for the 1:2 height ratio (n = 41 genotypes) of the trisomic diallelic markers while using the same 9015 genotypes with normal 1:1 height ratio in both models. Furthermore, we have described a simple procedure for the treatment of the MCC, prior DNA isolation and QF-PCR analysis.ResultsFor both models, we have achieved 100% specificity for the marker aneuploidy classification as compared to 98.60% (2:1 ratio) and 98.04% (1:2 ratio) specificity when using only the height ratio for classification. Treatment of the MCC enables a successful diagnosis rate of 76% among truly contaminated amniotic fluids.ConclusionsAdjustment for the allele size difference and marker type improves the STR aneuploidy classification, which, complemented with appropriate treatment of contaminated amniotic fluids, eliminates sample re-testing and reinforces the robustness of the QF-PCR method for prenatal testing

Loss of Y Chromosome in Peripheral Blood of Colorectal and Prostate Cancer Patients.

Although age-related loss of chromosome Y (LOY) in normal hematopoietic cells is a well-known phenomenon, the phenotypic consequences of LOY have been elusive. However, LOY has been found in association with smoking, shorter survival and higher risk of cancer. It was suggested that LOY in blood cells could become a predictive biomarker of male carcinogenesis.To investigate the association of LOY in blood cells with the risk for development of colorectal (CC) and prostate cancers (PC), we have analyzed DNA samples from peripheral blood of 101 CC male patients (mean age 60.5±11.9 yrs), 70 PC patients (mean age 68.8±8.0 yrs) and 93 healthy control males (mean age 65.8±16.6 yrs). The methodology included co-amplification of homologous sequences on chromosome Y and other chromosomes using multiplex quantitative fluorescent (QF) PCR followed by automatic detection and analysis on ABI 3500 Genetic Analyzer. The mean Y/X ratio was significantly lower in the whole group of cancer patients (0.907±0.12; p = 1.17x10-9) in comparison to the controls (1.015±0.15), as well as in CC (0.884±0.15; p = 3.76x10-9) and PC patients (0.941±0.06; p = 0.00012), when analyzed separately. Multivariate logistic regression analysis adjusting for LOY and age showed that LOY is a more significant predictor of cancer presence than age, and that age probably does not contribute to the increased number of subjects with detectable LOY in cancer patients cohort.In conclusion, our results support the recent findings of association of LOY in blood cells with carcinogenesis in males

Strategy for detection of IVS8polyT alleles with single base extension aproach.

<p>a) ‘5T/7T/9T extension primer’ in the presence of 5T allele is extended with dideoxyadenine while in the presence of 7T/9T alleles is extended with dideoxythymidine; b) ‘7T/9T extension primer’ in the presence of 7T allele is extended with dideoxyadenine while in the presence of 9T allele is extended with dideoxythymidine. Results of the two primers extension reactions give the final genotype. Extension primer sequences are given in 5′->3′ orientation, while the alleles represent the complementary (minus) strand.</p

Primers used for PCR amplification of seven <i>CFTR</i> exons and intron 8 fragment.

a<p>Legacy name.</p>b<p>The exon/intron numbering is based on legacy exon intron nomenclature (<a href="http://www.genet.sickkids.on.ca/" target="_blank">http://www.genet.sickkids.on.ca/</a>).</p><p>Primers used for PCR amplification of seven <i>CFTR</i> exons and intron 8 fragment.</p

<i>CFTR</i> multiplex SNaPshot primer extension mix with primer orientation, size and concentrations.

a<p>Data generated on ABI PRISM 3130 Genetic Analyzer with POP-4 polymer, 36-cm capillary array and sized against GeneScan-120 LIZ size standard.</p>b<p>Concentration in the SNaPshot extension primer mix adjusted to produce relatively equal peak heights.</p><p><i>CFTR</i> multiplex SNaPshot primer extension mix with primer orientation, size and concentrations.</p