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

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

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

Distribution of <i>CFTR</i> and IVS8polyT genotypes in the two groups of patients divided according to the histopathological results: obstructive azoospermia and nonobstructive azoospermia/oligozoospermia.

<p>[−] means no CF mutation detected on single chromosome.</p><p>Distribution of <i>CFTR</i> and IVS8polyT genotypes in the two groups of patients divided according to the histopathological results: obstructive azoospermia and nonobstructive azoospermia/oligozoospermia.</p

<i>CFTR</i> genotypes in 50 DNA samples used for validation of the SNaPshot method.

<p>Note: [−] means no CF mutation detected on single chromosome.</p><p><i>CFTR</i> genotypes in 50 DNA samples used for validation of the SNaPshot method.</p

Representative electrophoreograms of <i>CFTR</i> SNaPshot multiplex assay.

<p>a) Electrophoreogram of <i>CFTR</i> SNaPshot multiplex assay for detection of 12 common <i>CFTR</i> gene mutations showing a compound heterozygote for F508del/IVS8-5T. The fluorescence intensity is represented on the Y axis of the electrophoreogram and the fragments’ size on the X axis. N = normal allele (wild type) M = mutant allele; b-d) Electrophoreograms of samples with different IVS8polyT genotypes: 5T/7T (b), 5T/9T (c) and 7T/9T (d).</p

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

Distribution of <i>CFTR</i> and IVS8polyT genotypes in infertile men with different sperm counts and fertile controls.

<p>Note: [−] means no CF mutation detected on single chromosome.</p><p>Distribution of <i>CFTR</i> and IVS8polyT genotypes in infertile men with different sperm counts and fertile controls.</p