Relationship between reactive oxygen species production in human semen and sperm DNA damage assessed by Sperm Chromatin Structure Assay.

AIM: The aim of this prospective study was to find possible relationship between ROS production measured by chemiluminescence and flow cytometry in human semen and sperm DNA damage estimated by Sperm Chromatin Structure Assay. METHODS: Study included 39 men from infertile couples and 23 fertile volunteers who served as a control group. Aliquot of neat semen was used for ROS detection by chemiluminescence. Aliquot of sperm suspension in phosphate buffered saline was used for the detection of ROS by flow cytometry. Another aliquot of sperm suspension was used for SCSA to measure DNA fragmentation index and High DNA stainability. RESULTS: DNA fragmentation index correlated negatively with sperm morphology and motility. High DNA stainability correlated positively with ROS production and negatively with sperm morphology and concentration. Although there were similar trends of rising DNA fragmentation index and ROS production among the three groups of men, the relationship did not reach statistical significance. CONCLUSIONS: Higher values of DNA fragmentation index and high DNA stainability may also reflect developmental and/or environmental problems and not only oxidative stress.

Balanced chromosomal translocations in men: relationships among semen parameters, chromatin integrity, sperm meiotic segregation and aneuploidy.

PURPOSE: To analyse relationships between semen parameters, sperm chromatin integrity and frequencies of chromosomally unbalanced, disomic and diploid sperm in 13 Robertsonian and 37 reciprocal translocation carriers and to compare the results with data from 10 control donors. METHODS: Conventional semen analysis, Sperm Chromatin Structure Assay and FISH with probes for chromosomes involved in the individual translocations and for chromosomes X, Y, 7, 8, 13, 18 and 21. RESULTS: Normal semen parameters were found in 30.8 % of Robertsonian and 59.5 % of reciprocal translocation carriers. The rates of unbalanced sperm were 12.0 % in Robertsonian and 55.1 % in reciprocal translocation carriers with no difference between normospermic patients and those showing altered semen parameters. Significantly increased frequencies of spermatozoa showing defects in chromatin integrity and condensation, aneuploidy for chromosomes not involved in a translocation and diploidy were detected in translocation carriers with abnormal semen parameters. Normospermic reciprocal translocation carriers showed an increase in chromosome 13 disomy compared to the control group. There was no relationship between gametic and somatic aneuploidy in 12 translocation carriers studied by FISH on sperm and lymphocytes. The frequency of motile sperm was negatively correlated with the frequency of sperm showing disomy, diploidy and defective chromatin condensation. CONCLUSIONS: Abnormal semen parameters can serve as indicators of an additional risk of forming spermatozoa with defective chromatin and aneuploidy in translocation carriers.

der(4)t(Y;4): Three-generation transmission and sperm meiotic segregation analysis.

We present a family where five members (three males and two females) are carriers of der(4)t(Y;4)(q11.23;p16.3). The adult carriers are phenotypicaly normal and fertile; the boy shows macrocephaly, psychomotor retardation, and atypical autism. The FISH on cultured lymphocytes confirmed that the redundant Yq heterochromatin was attached to the 4p-subtelomeric region maintained on the der(4). Sperm FISH analysis performed in a normospermic der(4) carrier showed a significant distortion of the expected 1:1 ratio of the X- and Y-bearing spermatozoa in favor of the X chromosome and significant lack of Y,der(4)spermatozoa. The overall lack of Y spermatozoa was not balanced even by a relative excess of Y,4 sperm. The analysis of X, Y, 7, 8, 18, and 21 sperm disomy and diploidy did not indicate any interchromosomal effect. The chromosome 4 disomy was significantly increased but still very low to be of considerable reproductive significance. The neurodevelomental phenotype of the boy was probably caused by a gene mutation. The coincidental occurrence of such chromosomal aberration and boy's phenotype might lead to misinterpretation of the causal relationship between these findings. It is necessary to consider the results of chromosomal analysis and clinical records of relatives for provide genetic counseling in such families.

Sperm and embryo analysis in a carrier of supernumerary inv dup(15) marker chromosome.

We identified a small, paternally inherited, supernumerary marker chromosome, inv dup(15), in a phenotypically normal and normozoospermic male from a couple with reproductive problems. Sperm analysis by fluorescence in situ hybridization (FISH) showed that the marker was present in 26% of sperm nuclei. The disomy 15 was 10 times higher than in normal control donors. FISH analysis for aneuploidies of the other chromosomes showed an increase in nondisjunction of chromosome 21. We also examined 24 embryos by preimplantation genetic diagnosis, and 10 embryos (41.7%) contained the marker. This report provides information about inheritance of inv dup(15) from a male carrier.

Hybridization of the 18 alpha-satellite probe to chromosome 1 revealed in PGD.

Although the chromosome 18 alpha-satellite probe is considered to have a very low polymorphism rate, the routine use of this probe in prenatal diagnosis revealed rare variants in size and copy number of these sequences. A polymorphic signal was detected in preimplantation genetic diagnosis (PGD) for aneuploidy, in a patient with repeated early miscarriages. A third small signal of chromosome 18 alpha-satellite probe was observed in two of four evaluated embryos. Hybridization to the woman's metaphasic lymphocytes revealed that the small signal was localized in the pericentromeric region of chromosome 1. Reanalysis of blastomeres with telomeric probes for chromosome 18q confirmed the presence of only two copies of chromosome 18. Options for verifying PGD analysis results, to prevent misdiagnosis in cases of suspected polymorphism, are discussed. Although some authors speculate about a possible role of heterochromatin polymorphism in infertility, this rare polymorphism of 18 alpha-satellite sequences is in itself probably a normal variant. This is the third report of a cross-hybridization of the chromosome 18 alpha-satellite probe and the first report of the localization of the polymorphic 18 alpha-satellite signal to chromosome 1.

Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: detection of translocations in embryos by fluorescence in situ hybridization.

Robertsonian translocation rob(16;20) in the heterozygous state was discovered in a subfertile bull of the Czech Siemmental breed. A chromosomal analysis of its family has shown that this dicentric fusion is formed de novo. The present experiments were designed to detect rob(16;20) and determine its incidence for in vitro produced embryos, using fluorescence in situ hybridization (FISH) and rob(1;29) as a detection control. To characterize semen of both bulls with the rob translocations, their sperm was examined for DNA integrity by the sperm chromatin structure assay (SCSA). For in vitro fertilization of oocytes, spermatozoa from a rob(16;20) bull carrier (Czech Siemmental breed) and those from a rob(1;29) bull carrier (Charolais breed) were used. Embryos at the 6- to 8-cell stage were cultured in a vinblastine-supplemented medium for 17 h, and embryos at the blastocyst stage were cultured in a colcemide-supplemented medium for 4 h. The embryos were fixed in methanol and acetic acid with Tween-20. Painting probes for chromosomes 16 (Spectrum Green) and 20 (Spectrum Orange) and chromosomes 1 (Spectrum Orange) and 29 (Spectrum Green) were simultaneously hybridized. In the embryos derived from the rob(16;20) bull, the presence of this translocation was not detected. On the other hand, 52.5% of the embryos derived from the rob(1;29) bull were translocation carriers. There was no significant difference in the frequency of this translocation between early and advanced embryos.