A common haplotype of protamine 1 and 2 genes is associated with higher sperm counts.

Sperm chromatin compaction in the sperm head is achieved when histones are replaced by protamines during spermatogenesis. Haploinsufficiency of the protamine 1 (PRM1) or PRM2 gene causes infertility in mice. However, the published data remain inconclusive about a role of PRM1/2 variants in male infertility and their association with semen parameters. By full sequence analysis, we assessed the frequency of sequence variations in PRM1 and PRM2 in three groups of Caucasian patients with idiopathic teratozoospermia and normal (n = 88) or reduced sperm concentration (n = 83) and in men with a high percentage of normal sperm morphology and normal concentrations (n = 77). Two rare (c.54G>A and c.102G>T) and one common SNP (c.230A>C) were identified in PRM1. In PRM2, some rare heterozygous mutations and the two common intronic SNPs 298G>C and 373C>A were detected. None of the PRM1/2 variants was associated with teratozoospermia or individually with other semen parameters. However, significant linkage disequilibrium was detected between the common SNPs of PRM1 and PRM2 which formed haplotypes. Analysis of the pooled group (n = 248) revealed that homozygous carriers of the common haplotype ACC had a twofold higher sperm concentration and count than men lacking this haplotype, with sperm counts of heterozygotes for ACC being midway between the homozygotes. This markedly decreased sperm output might either be caused by spermatozoa lacking the ACC haplotype not being viable, or subject to negative selection. In addition, a significant deviation from Hardy-Weinberg-Equilibrium of these SNPs might indicate natural selection in favour of the ACC allele which leads to higher sperm output and therefore better fertility. In conclusion, for the first time we describe an association of a common haplotype formed by PRM1 and PRM2 with sperm output in a large group of men.

Evaluation of high-resolution melting (HRM) for mutation scanning of selected exons of the CFTR gene.

Hereby we present evaluation of high-resolution melting for mutation scanning applied to the cystic fibrosis transmembrane conductance regulator gene. High resolution melting was used for mutation scanning of selected samples derived from cystic fibrosis patients with a known cystic fibrosis transmembrane conductance regulator genotype. We tested 19 different disease-causing cystic fibrosis transmembrane conductance regulator mutant genotypes located within six exons of the cystic fibrosis transmembrane conductance regulator gene (4, 7, 10, 11, 14b and 22). Normalized melting curves of tested samples were compared to sequenced-verified wildtype samples. Determined mutations are as follows: p.F508del, p.I507del, p.G551D, p.R347P, c.1717- 1G>A, c.621+1G>T, p.Y122X, p.I336K, p.R553X, c.2789+5G>A, c.574delA, c.1811+1G>C, p.L1335F, p.L1335P, p.L1324P and p.M470V and represent minimally 76.5 % of all cystic fibrosis alleles detected in the Czech cystic fibrosis population. All analysed samples with mutant genotypes were unambiguously distinguished from wild-type samples. High-resolution melting analysis enabled reliable detection of all single-nucleotide polymorphism classes and 1- or 3- base pair deletions. We examined the specificity, sensitivity and precision of this methodology. High-resolution melting analysis is an economical, sensitive and specific close-tube method and has a high utility for the detection of unknown mutations in cystic fibrosis DNA diagnostics.

The effect of the ultrashort beta-blocker esmolol on cardiac function recovery: an experimental study.

OBJECTIVE: This is an experimental work designed to determine, using the isolated perfused rat heart, the effect of the ultra-short acting beta-blocker esmolol on cardiac arrest and cardiac function recovery following esmolol withdrawal. METHODS: Changes in heart rate, coronary flow, diastolic pressure and the rate pressure product were evaluated on the isolated heart (Langendorff model). Esmolol concentrations of 125, 250, and 500 mg/l were tested. In another experiment using esmolol concentration of 250 mg/l, cardiac function recovery was assessed after 20- and 45-min arrest. RESULTS: While concentrations of 250 and 500 mg/l are necessary to produce cardiac arrest, the concentration of 500 mg/l does not result in full cardiac function recovery following esmolol withdrawal. After the highest concentration of esmolol, coronary flow, heart rate and the rate-pressure product recovered to about 80, 70 and 60% of the initial control values, respectively. When comparing 20- and 45-min arrests we found cardiac function normalization occurs later after 45-min arrest. CONCLUSION: The induction of cardiac arrest by esmolol is optimal at a concentration of 250 mg/l. A concentration of 125 mg/l does not result in cardiac arrest and produces bradycardia only, a concentration of 500 mg/l may be dangerous on account of persisting undesirable effects on the rat heart.