Rational approach to genetic testing of cystic fibrosis (CF) in infertile men.

Male infertility as a result of isolated congenital bilateral absence of the vas deferens (CBAVD) is one primary genital form of cystic fibrosis (CF) and occurs in 1-2% of infertile men. Assisted fertilization in patients with CBAVD increases the risk of transmitting mutations in the CF gene. We developed a rational approach to genetic CF testing in infertile men. A total of 282 infertile male patients were screened for the most common CF mutations (DeltaF508, R117H, IVS8-5T). Clinical data including medical history, examination, semen analysis, sweat tests, karyotypes and hormonal values were analysed. We identified 23 patients carrying mutations in the CF gene (DeltaF508: 10 patients; R117H: six patients; IVS8-5T: 11 patients). Two patients were compound heterozygote for DeltaF508/R117H, two others for DeltaF508/IVS8-5T. Correlating these molecular analyses with the clinical data pertaining to serum follicle-stimulating hormone concentration, semen pH, sperm count and total testicular volume, we were able to develop a score with a high specificity (98.4) for the presence of a cystic fibrosis transmembrane conductance regulator (CFTR) mutation, but only with a low sensitivity (positive post-test likelihood: 62.5%; negative post-test likelihood: 6.3%). With regard to the low sensitivity and the high number of CFTR mutations found in this heterogeneous group of infertile men, we still recommend genetic CF testing before assisted fertilization.

Increased frequency of X-bearing sperm in males from an infertility clinic: analysis by two-color fluorescence in situ hybridization.

Semen samples from 34 men visiting the Lübeck infertility clinic were investigated using a two-color FISH method to determine the ratio of X- and Y-bearing sperm. The overall ratio was significantly shifted to a preponderance of X-containing sperm. A statistical comparison with seven reports from the literature which included 53 normal probands demonstrated in our patients a significant tendency of a preponderance of X-bearing sperm and significantly less Y-bearing sperm. Furthermore, the Lübeck sperm samples are remarkably more heterogeneous in respect to their variability of X- and Y-bearing spermatozoa than in the other mentioned studies with normal probands. These phenomena have to be evaluated in further studies on groups of infertile males showing similar infertility histories.

A new technique for cyclic in situ amplification and a case report about amplification of a single copy gene sequence in human metaphase chromosomes through PCR-PRINS.

Since the introduction of PRimed IN Situ labeling (PRINS) as a rapid and extremely sensitive alternative method to conventional fluorescence in situ hybridization (FISH), its application in clinical cytogenetics has been limited to the detection of highly repeated sequences, such as centromeric and telomeric regions. In the original PRINS method, unlabeled oligonucleotide probes are annealed to their repeated complementary target sequences in fixed human metaphase chromosomes on a slide. The probes serve as primers for subsequent in situ chain elongation with Taq DNA polymerase and labeled nucleotides. In contrast to conventional PCR, cyclic in situ amplification of the chromosomal target DNA with paired primers remained both difficult and strictly limited to highly repeated sequences, since the maintenance of constant reaction conditions on the slide during temperature and pressure shifts presents a major problem. We developed a new system for in situ PCR that allows the amplification of target sequences analogous to PCR in the test tube. We applied this method successfully for the detection of highly repeated sequences, for the detection of low copy repeats, and in one case, for the detection of a single-copy DNA sequence. The significance of this development for further in situ PCR applications will be discussed.

Primed in situ labeling: sensitivity and specificity for detection of alpha-satellite DNA in the centromere regions of chromosomes 13 and 21.

The centromeric alpha-satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence. So far it has proven difficult to discriminate between sequence variations in the chromosome 13 and 21 alpha-satellite regions using in situ techniques. To analyze whether the method of modified single-color and double-color PRINS could be used to detect single nucleotide polymorphisms within this region, we used previously published primers D13Z and D21Z that differ in the terminal 3'-nucleotide and an additionally constructed primer "D13/21-test" lacking the final nucleotide at the 3' end. The results show that a one-base pair mismatch at the 3' end is sufficient to be detected by PRINS. Surprisingly, only about 35% of our samples exhibited the expected combination of two chromosomes 13 specifically labeled with only primer D13Z and two chromosomes 21 specifically labeled with only primer D21Z. The rest of the samples showed a polymorphic distribution of the target sequence for the primers, therefore these primers are not suited for routine detection of chromosomes 13 and 21 during interphase. Our data indicate that an interchromosomal exchange of alpha-satellite DNA takes place between chromosomes 13 and 21, possibly due to a concerted evolution process.

Comparative study of disomy and diploidy rates in spermatozoa of fertile and infertile men: a donor-adapted protocol for multi-colour fluorescence in situ hybridization (FISH).

Spermatozoa from seven healthy donors (two of whom had already fathered children) and five infertile patients taking part in the local programme of intracytoplasmic sperm-injection (ICSI) were investigated for the disomy rates of chromosomes 13/21, 18, X and Y as well as for the diploidy rates. Two- and three-colour fluorescence in situ hybridization (FISH) was applied after a donor-adapted decondensation pre-treatment: in a preliminary decondensation series the optimum fluorescence signals were individually determined by variation of the concentration of the decondensation reagents and the duration of incubation with these reagents. Strict scoring criteria were applied. The average disomy rates ranged from 0.10% (chromosomes 13/21) to 0.44% (disomy XY) in the infertile donors and from 0.07% (disomy XX) to 0.36% (disomy XY) in the controls. The average diploidy rates were 0.22% and 0.20% for the infertile donors and the controls respectively. There was no statistically significant difference between the two groups with respect to the disomy and diploidy rates. Within the two groups there were inter-individual differences which were partly statistically significant, indicating considerable inter-donor variation of the aneuploidy rates.

Ketotifen and cardiovascular effects of xamoterol following single and chronic dosing in healthy volunteers.

AIMS: To study whether desensitization occurs after long-term administration of the 1-adrenoceptor partial agonist xamoterol and, if so, whether this can be influenced by ketotifen. METHODS: In a double-blind, randomized design 10 young, healthy males received ketotifen (2 x 1 mg day(-1) p.o.) or placebo for 3 weeks with xamoterol (2 x 200 mg day(-1) p.o.) administered concomitantly during the last 2 weeks. 'l1-adrenoceptor mediated responses were assessed as exercise-induced tachycardia and isoprenaline-induced shortening of heart rate corrected electromechanical systole (QS2c); isoprenaline-induced tachycardia was measured as a mixed beta1-/beta2-adrenoceptor-mediated effect. RESULTS: The first dose of xamoterol significantly increased resting heart rate and systolic blood pressure and significantly shortened QS2c. The last dose of xamoterol after 2 weeks of treatment still produced the same responses. Ketotifen did not influence these effects of xamoterol on resting haemodynamics. The first dose of xamoterol caused a rightward shift of the exercise- and isoprenaline-induced tachycardia (mean dose ratios+/-s.e.mean: 1.20+/-0.05 and 2.46+/-0.23) and the isoprenaline-evoked shortening of QS2c (dose ratio 3.59+/-0.68). This rightward shift was even more pronounced after 2 weeks xamoterol treatment. This additional rightward shift after 2 weeks of xamoterol was not affected by ketotifen (mean difference (95% CI) of log transformed dose ratios between placebo and ketotifen: exercise tachycardia 0.001 (-0.03; 0.04); isoprenaline tachycardia 0.03 (-0.15; 0.21); isoprenaline induced shortening of QS2c 0.13 (-0.22; 0.48)). CONCLUSIONS: In humans xamoterol is a partial beta1-adrenoceptor agonist with positive chrono- and inotropic effects at rest and antagonistic properties under conditions of beta-adrenoceptor stimulation. These effects were well maintained after chronic dosing with no signs of beta1-adrenoceptor desensitization. Ketotifen does not change the beta-adrenoceptor mediated responses of xamoterol after chronic dosing.

Molecular cytogenetic diagnostics in sperm.

Among other factors contributing to male infertility, chromosomal anomalies are a frequent finding. Investigating 440 males with reduced sperm counts (< 20 x 10(6)/mL) we found 19 males with chromosome abnormalities (aneuploidies and translocations). This frequency of chromosomal aberrations (4.3%) is much higher than in the normal population. We were able to investigate sperm nuclei from seven out of these 19 men with molecular cytogenetic methods. There were two carriers of Robertsonian translocations, two had a constitutional reciprocal translocation and three were XYY males. In addition, one other man with a long history of infertility and a marker chromosome 15 was included. Using fluorescence in-situ hybridization, it was possible to investigate a great number of sperm nuclei in each case. Each translocation case showed a specific mode of chromosomal segregation; confirming the dependence on chromosomes involved and the individual segregation pattern in each patient. The well known elimination of the supernumerary Y-chromosome in XYY males during meiosis was confirmed in our study. Molecular cytogenetic investigations in sperm provide a more reliable risk estimate with respect to the possible injection of chromosomally unbalanced sperm during intracytoplasmatic sperm injection and can be valuable in genetic counselling.