BackgroundAlthough 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.Aims, Methods & FindingsTo 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.ConclusionIn conclusion, our results support the recent findings of association of LOY in blood cells with carcinogenesis in males.

Seminal plasma is a potential source of biomarkers for many disorders of the male reproductive system including male infertility. The identification and characterisation of differentially expressed proteins in seminal plasma of man with normal and impaired spermatogenesis can help in the elucidation of the molecular basis of male infertility. We compared the protein expression profiles of seminal plasma from four different groups of men as follows: normozoospermic, asthenozoospermic, oligozoospermic and azoospermic groups, using two-dimensional differential gel electrophoresis (2-D DIGE). We found eight proteins with statistically significant increased expression in azoospermia compared with at least one of the other studied groups. The differentially expressed spots were fibronectin, prostatic acid phosphatase (PAP), proteasome subunit alpha type-3, beta-2-microglobulin, galectin-3-binding protein, prolactin-inducible protein and cytosolic nonspecific dipeptidase. Notably, PAP was increased in patients with azoospermia compared with that of all other groups. We have observed no statistically significant differences in protein expression between three of the groups: normozoospermic, oligozoospermic and asthenozoospermic. We suggest that the identified panel of proteins in our study especially PAP have a strong potential to be used as azoospermia markers. However, further investigations will be necessary to validate these markers in samples of larger and independent patient cohorts and to clarify their role in the pathogenesis of male infertility.

Infertility is a major health problem today, affecting about 15% of couples trying to conceive a child. Impaired fertility of the male factor is causative in 20% of infertile couples and contributory in up to another 30%-40%. Based on association studies, an increasing number of gene polymorphisms have been proposed to modulate the efficiency of spermatogenesis. Here, we have investigated the possible association of 9 single-nucleotide polymorphisms (SNP) in 8 different genes-FASLG, JMJDIA, LOC203413, TEX15, BRDT, OR2W3, INSR, and TAS2R38-with male infertility. We analyzed a total of 136 men with idiopathic infertility (60 azoospermic and 76 oligozoospermic) and 161 fertile controls. Our study group included individuals of different ethnic origin: 93 of the infertile men were Macedonians, 32 were Albanians, and 11 were of other origin. The control group was composed of 125 Macedonian and 36 Albanian men. The methodology included multiplex polymerase chain reaction/SNaPshot analyses, followed by capillary electrophoresis on an ABI3130 Genetic Analyzer. Of the 9 SNPs evaluated, 3 are significantly associated (P , .05) with male infertility: SNPs rs5911500 in LOC203413, rs3088232 in BRDT, and rs11204546 in OR2W3. SNP rs5911500 showed the strongest association with infertility among Albanians (P 5 .0001), whereas rs3088232 was most significantly associated with azoospermia among Macedonians (P 5 .0082). Moreover, the frequency of cooccurrence of LOC203413 minor T allele with either homozygosity or heterozygosity for the BRDT minor G allele was significantly higher among both azoospermic (6 of

Spermatogenesis is a complex process that involves thousands of genes whose expression during different stages is strictly regulated. Small non-coding microRNAs play an important role in the posttranscriptional regulation of mRNA processing during spermatogenesis. Using Agilent SurePrint v16 microRNA 8 × 60 K microarray kit, we investigated the microRNA expression profiles of 24 formalin-fixed paraffin-embedded testicular biopsies from patients with hypospermatogenesis (n = 10), hypospermatogenesis and azoospermia factor c region on the Y chromosome (AZFc) deletion (n = 3), Sertoli cell-only syndrome (n = 3) and maturation arrest (n = 2), in comparison with subjects with normal spermatogenesis (n = 6). After adjusting for multiple testing, six deregulated miRNAs were detected in the patients with AZFc deletion, 30 in maturation arrest group, 52 in Sertoli cell-only syndrome group of patients, and none in the group of patients with hypospermatogenesis. Some of the deregulated microRNAs were shared between groups, resulting in 58 unique differentially expressed microRNAs. The expression of five microRNAs (hsa-miR-34b, hsa-miR-449b, hsa-miR-517c, hsa-miR-181c, and hsa-miR-605) was validated by qRT-PCR in a total of 74 samples. Using mRNA expression profiles of subjects with matching histopathological patterns of impaired spermatogenesis from publically available Gene Expression Omnibus data sets, we have performed integrated mRNA-microRNA regulatory network analysis. Pathway analysis revealed significantly enriched set of genes for tumor necrosis factor-related apoptosis-inducing ligand signaling pathway, previously shown to be involved in regulation of apoptosis in normal functioning testis. Our results should be considered as preliminary as we have analyzed only a small number of patients in each studied group. Further studies with larger number of patients with impaired spermatogenesis as well as more targeted approaches with parallel microRNA and mRNA expression profiling in isolated subpopulations of somatic or germ cells from different stages of spermatogenesis are needed to clarify the role of the microRNAs in the process of spermatogenesis.

The most common genetic causes of spermatogenic failure are sex chromosomal abnormalities (most frequently Klinefelter's syndrome) and deletions of the azoospermia factor (AZF) regions (AZFa, AZFb, and AZFc) of the Y chromosome. Several studies have proposed that partial AZFc deletions/duplications may be a risk factor for spermatogenic impairment. We describe a multiplex quantitative fluorescent-polymerase chain reaction (QF-PCR) method that allows simultaneous detection of these genetic causes and risk factors of male infertility. The 11-plex QF-PCR permitted the amplification of the amelogenin gene, four polymorphic X-specific short tandem repeat (STR) markers (XHPRT, DXS6803, DXS981, and exon 1 of the androgen receptor gene), nonpolymorphic Y-specific marker (SRY gene), polymorphic Y-specific STR marker (DYS448), and coamplification of DAZ/DAZL, MYPT2Y/MYPT2, and two CDY2/CDY1 fragments that allow for determination of the DAZ, MYPT2Y, and CDY gene copy number. A total of 357 DNA samples from infertile/subfertile men (n = 205) and fertile controls (n = 152) was studied. We detected 14 infertile males with sex chromosome aneuploidy (10 with Klinefelter's syndrome, 2 XX, and 2 XYY males). All previously detected AZF deletions, that is, AZFc (n8), AZFb (n1), AZFb + c (n1), gr/gr (n11), gr/gr with b2/b4 duplication (n3), and b2/b3 (n5), gave a specific pattern with the 11-plex QF-PCR. In addition, 32 DNA samples showed a pattern consistent with presence of gr/gr or b2/b4 and 4 with b2/b3 duplication. We conclude that multiplex QF-PCR is a rapid, simple, reliable, and inexpensive method that can be used as a first-step genetic analysis in infertile/subfertile patients.

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Balkan endemic nephropathy (BEN) is a disease that affects people that live in the alluvial plains along the tributaries of the Danube River in the Balkan region. BEN is a chronic tubulointerstitial disease with a slow progression to terminal renal failure and has strong association with upper tract urothelial carcinoma (UTUC). There are several hypotheses about the etiology of BEN, but only the toxic effect of aristolochic acid has been confirmed as a risk factor in the occurrence of the disease. Aberrantly expressed miRNAs have been shown to be associated with many types of cancers. A number of studies have investigated the expression of microRNAs in urothelial carcinoma, mainly on urothelial bladder cancer, and only a few have included patients with UTUC. Here we present the first study of microRNA profiling in UTUC tissues from patients with BEN (BEN-UTUC) and patients with UTUC from nonendemic Balkan regions (non-BEN-UTUC) in comparison to normal kidney tissues. We found 10 miRNAs that were differentially expressed in patients with BEN-UTUC and 15 miRNAs in patients with non-BEN-UTUC. miRNA signature determined in BEN-UTUC patients differs from the non-BEN-UTUC patients; only miR-205-5p was mutual in both groups.

Globozoospermia is a rare but severe teratozoospermia, characterized by ejaculates consisting completely of round-headed spermatozoa that lack an acrosome or, in partial globozoospermia, containing a variable proportion (20.0–90.0%) of acrosomeless spermatozoa. Men that are affected with total globozoospermia are infertile, and even the application of intracytoplasmic sperm injection (ICSI) has met with disappointingly low success rates. In humans, several case reports of globozoospermia have demonstrated that two or more siblings were affected in each family, which suggested a genetic component to this disease. Currently, three genes are known to be associated with total globozoospermia in humans, SPATA16 , PICK1 and DPY19L2 genes. Mutations in SPATA16 and PICK1 are rare causes of globozoospermia, found in only one patient each. Several studies have suggested that DPY19L2 mutations are the major cause of globozoospermia in patients from different ethnic origins and different geographic regions. The most common DPY19L2 mutation is the 200 kb deletion arising from a nonallelic homologous recombination (NAHR) between the flanking low copy repeats (LCRs). Here we describe the presence of a homozygous deletion of the DPY19L2 gene in two infertile Macedonian patients with 100.0% round headed spermatozoa, thus suggesting that this deletion represents a major cause of globozoospermia among Macedonian men.