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Study question Influence of sperm selector separation of sperms on their translocation load, segregation pattern, motility and occurrence of interchromosomal effects Summary answer Sperm selector separation led to reduction of the translocation load, shift in segregation pattern and lower rates of interchromosomal effects within sperm samples What is known already Balanced translocations in men are known to be one of the main causes of reproductive failure. The segregation pattern in sperms is determined by the distribution of the chromosomes during meiosis. Interchromosomal effects can also influence the distribution of chromosomes that are not involved in the translocation. The sperm selector used consists of two concentric chambers, which are overlaid by a U-ring and a cover glass. Motile sperms migrate from the native ejaculate in the medium filled inner chamber by using a capillary bridge created by the U-ring. This avoids potential harmful centrifugation and allows accumulation of motile sperms. Study design, size, duration Twenty-one carriers of balanced translocations participated in the study. In addition, 15 patients were involved as control. All participants signed an informed consent (F–8–15). Samples of three patients did not meet the internal quality criteria and had to be excluded from analysis. The study started in 2015 and is still ongoing. Participants/materials, setting, methods Liquefied native ejaculate was processed with a sperm selector. Native ejaculate, non-migrated sperms from the outer chamber and migrated sperms from the inner chamber were transferred onto glass slides, fixed and underwent a decondensation treatment. For segregation analysis FISH translocation specific FISH probe mixes were used and tested on patient’s blood. Interchromosomal effects were analysed with FISH probes for the chromosomes X, Y, 18 and 13, 21. Evaluation was done manually using fluorescence microscopy. Main results and the role of chance Segregation analysis was done for more than 25,000 sperms from men carrying a balanced translocation (18 patients with reciprocal and 3 patients with Robertsonian translocation). Separation via sperm selector led to a reduction in translocation load (native to separated approach 49,1±11,5% to 34,8±9,4% (P = Limitations, reasons for caution The number of accumulated strandbreak-free sperms depended on the motility and sperm count of the native ejaculate. Examinations are not reproducible, as each sample delivery is influenced by external circumstances Wider implications of the findings: Sperm selector separation can be used before ART to reduce the translocation load and rate of maldistribution in sperms from carriers of balanced translocations. This could have a considerable impact on PGT results after trophectoderm biops. Trial registration number Not applicable

Mehr als in anderen Fachgebieten gehoren angeborene Erkrankungen zum Alltag des Facharztes fur Kinder- und Jugendmedizin. Die zunehmende Verfugbarkeit der Next Generation Sequencing-Methoden in den letzten 5 Jahren zeigt, dass die meisten dieser Erkrankungen genetischen bzw. monogenen Ursprungs sind, und eroffnet ungeahnte Moglichkeiten, aber auch Herausforderungen in Diagnostik und Therapie. Dieser Beitrag bietet einen Uberblick uber die Bedeutung genetischer Erkrankungen in der Padiatrie, erlautert die Moglichkeiten, aber auch Limitationen moderner genetischer Diagnostik wie der Exomsequenzierung und ihre Auswirkungen auf die tagliche Patientenversorgung.

Introduction Sexually transmitted infections (STIs) continue to be a significant public health problem with a high burden in women of reproductive age. Rates of Chlamydia trachomatis , Neisseria gonorrhoeae and Trichomonas vaginalis are frequently tested for and rates of infection are generally high in African settings, but the prevalence of other genital STIs is largely unknown. The aim of this study was to determine the prevalence of genital mycoplasmas ( Mycoplasma genitalium , M. hominis , Ureaplasma parvum and U. urealyticum ) in women visiting the infertility clinic of a tertiary academic hospital in South Africa. Methods In this pilot evaluation self-collected vaginal swabs were obtained from 51 women visiting the infertility clinic. The genomic DNA was extracted from the swabs using the ZR Fungal/Bacterial DNA Miniprep (Thermo Scientific, USA) and analysed using the Anyplex II STI-7 (Seegene, Korea) real-time PCR assay for the simultaneous detection and identification of seven STIs including the four mycoplasma species. Results The real-time PCR assay detected the following genital mycoplasmas and co-infections in the 51 women: U. parvum [55% (28/51)], M. hominis [20% (10/51)] and U. urealyticum [16% (8/51)]; none of the specimens tested positive for M. genitalium . Among the nine patients where mixed infections were observed, M. hominis and Ureaplasma spp. were frequently detected together [67% (6/9)]. In addition to the mycoplasmas, one woman tested positive for C. trachomatis; N. gonorrhoeae and T. vaginalis were not detected. Conclusion This pilot study demonstrated an unexpectedly high rate of genital mycoplasma infections among women visiting an infertility clinic. The burden of genital mycoplasma infection is largely unknown and warrants further investigation, in particular with regards to the prevalence and clinical significance in different population groups. Support: Anyplex II STI-7 kits provided by Seegene, Korea

A case of prenatal diagnosis of de novo mosaic deletion of the long arm of chromosome 13 (del(13)(q13.3)) is presented. Routine scanning in a 27-year-old primigravida at 25 weeks' gestation showed fetal bilateral hydronephrosis. Detailed anomaly scanning in our tertiary referral center further demonstrated posterior meningoencephalocele, sloping forehead, microcephaly, syndactyly and hypoplastic thumbs. Both genetic amniocentesis and cordocentesis revealed a mosaic karyotype, 46,XY/46,XY,del(13)(q13.3). Sonographic findings were confirmed by postmortem autopsy and additional abnormalities such as agenesis of corpus callosum, hypoplastic cerebellum and macroglossia were diagnosed. Detailed sonography in this case thus revealed multiple malformations that prompted fetal karyotyping at 25 weeks' gestation.

In this explorative study it should be evaluated how the introduction of non invasive first trimester diagnosis (nuchal translucency measurement, Combined Test, first trimester ultrasound screening) has influenced the indications and cytogenetic results of chorion villi samplings.Between 1989 and 2008 3337 pregnancies with CVS between 11 and 14 weeks of gestation were examined retrospectively. They were divided in two groups: CVS 1989 - 2001 before introduction of non invasive first trimester diagnosis (n = 1698) and CVS 2002 - 2008 after introducing non invasive testing at the end of 2001 (n = 1639). In both groups the indications for CVS (maternal age, sonographic findings, past history, maternal anxiety, and abnormal results of the Combined Test only in the second group) and the cytogenetic results were evaluated.In the first group (1989 - 2001, n = 1698) 85,6% (n = 1454) of all CVS were performed because of maternal age and only 3% (n = 51) due to sonographic findings. In the second group (2002 - 2008, n = 1639) there was a distinct increase of sonographic findings leading to CVS (33,9%, n = 555) with a clear decrease of maternal age to 37,9% (n = 621). Abnormal cytogenetic results were found in 10,5% (n = 172) in the second group, in the first group only in 4,5% (n = 76), respectively. The parameter with the highest rate of chromosomal disorders was fetal hydrops (66,1%), follwed by hygroma colli (48,2%), malformations (12,9%) and increased nuchal translucency (11,2%). Regarding maternal age alone the rate of abnormal chromosomes was 3,1%.It could be shown that non invasive first trimester diagnosis has lead to a more specific indication for invasive fetal testing (sonographic findings 33,9 vs. 3%, maternal age 37,9 vs. 85,6%) with a higher rate of chromosomal disorder in this group (10,5 vs. 4,5%).

Background and aims Germline mutations in the E-cadherin (CDH1) gene have been found in families with hereditary diffuse gastric cancer (HDGC). These families are characterized by a highly penetrant susceptibility to diffuse gastric cancer with an autosomal dominant pattern of inheritance. We describe the clinical presentation of three sibling cases with advanced gastric cancer, the way of confirming the suspicion of underlying HDGC and the clinical management of the other healthy family members. Methods Screening for CDH1 germline mutation was carried out by denaturing high-performance liquid chromatography and automated DNA sequencing. The clinical suspicion of HDGC has been confirmed by identifying a frameshift mutation in exon 9 (1302_1303insA, 1306_1307delTT) of the E-cadherin gene. Results Eight of nine tested family members were positive for the CDH1 germline mutation. Prophylactic laparoscopic gastrectomies were performed in five mutation carriers. After pathological examination, we could identify intramucosal malignant signet-ring cell carcinoma in all resected stomachs. Conclusion This report underlines that prophylactic gastrectomy remains the only option to eliminate the high risk for gastric cancer in CDH1 mutation carriers.

In 1988, a 27-year-old male patient received an allogeneic BMT for leukemic relapse 8 months after ABMT for AML (M2) in first complete remission. Because of chronic GVHD of the liver CsA was administered until 1994. Nine months after discontinuation of CsA, locally advanced gastric granulocytic sarcoma (GS) was diagnosed without evidence of systemic relapse. The patient was treated with two courses of polychemotherapy (ICE, NOVIA). Granulocyte colony-stimulating factor (G-CSF)-mobilized donor buffy coat cells were reinfused after each chemotherapy cycle in an attempt to accelerate hematopoietic regeneration and to induce a graft-versus-leukemia (GVL) effect. Local irradiation and surgical resection of residual leukemic cells resulted in complete remission. Seventeen months from diagnosis of GS the patient relapsed again with multiple lesions and died of generalized bleeding during aplasia after a third course of polychemotherapy (ICE). In our patient donor peripheral blood stem cell support did not accelerate hematopoietic regeneration (time to neutrophil recovery > 0.5 x 10(9) g/l from the start of chemotherapy was 27 days after ICE and 36 days after NOVIA) and did not result in long-term disease-free survival.

Die Haufigkeit einer Krebserkrankung mit unbekanntem Primartumor betragt bis zu 15% aller Malignome und ist abhangig davon, wie intensiv die Diagnostik betrieben wird (Abbruzzese u. Raber 1995; Altman u. Cadman 1986). Das mediane Alter der Patienten liegt bei etwa 60 Jahren, wobei sich eine leichte Pradominanz des mannlichen Geschlechts findet. Die Entdeckung des Primartumors gelingt bei 8–31% der Patienten im weiteren Krankheitsverlauf. Bei 60–80 % wird erst bei der Autopsie eine definitive Diagnose gestellt, oft findet sich hierbei eine andere Lokalisation des Primartumors, als nach den Krebsstatistiken zu erwarten ware: Die Inzidenz an Lungenkarzinomen ist doppelt so hoch, Kolonkarzinome sind ungewohnlich selten und primare Lebertumoren finden sich zehnfach haufiger als in den Krebsstatistiken angegeben. Daruber hinaus zeigt sich beim CUP-Syndrom oft ein ungewohnliches Metastasierungsmuster: Lebermetatasen stammen selten von Kolonkarzinomen, Knochenmetastasen durch Primartumoren in der Mamma oder der Lunge sind relativ ungewohnlich. Als Grund fur das Nichtauffinden des Primartumors sind die spontane Regression, die Zerstorung durch immunologische oder vaskulare Mechanismen oder eine sehr geringe Grose des Tumors denkbar. Eine Auswertung der Obduktionsbefunde von 436 Patienten mit CUP-Syndrom ist in Tabelle 80.1 dargestellt. Die diagnostische Sicherheit der postmortalen Untersuchung ist abhangig vom Differenzierungsgrad des Tumors und ist am hochsten bei gut differenzierten Neoplasien.

A case of prenatal diagnosis of de novo mosaic deletion of the long arm of chromosome 13 (del(13)(q13.3)) is presented. Routine scanning in a 27-year-old primigravida at 25 weeks' gestation showed fetal bilateral hydronephrosis. Detailed anomaly scanning in our tertiary referral center further demonstrated posterior meningoencephalocele, sloping forehead, microcephaly, syndactyly and hypoplastic thumbs. Both genetic amniocentesis and cordocentesis revealed a mosaic karyotype, 46,XY/46,XY,del(13)(q13.3). Sonographic findings were confirmed by postmortem autopsy and additional abnormalities such as agenesis of corpus callosum, hypoplastic cerebellum and macroglossia were diagnosed. Detailed sonography in this case thus revealed multiple malformations that prompted fetal karyotyping at 25 weeks' gestation.

Counting the repetitive kringle-IV repeats in the gene encoding human apolipoprotein(a) by fibre-FISH

Dendritic cells (DC) are professional antigen-presenting cells specialized in the initiation of primary immune responses. We were interested to know whether mature DC can be grown in vitro from peripheral blood mononuclear cells (PBMC) of patients with chronic myelogenous leukemia (CML), and whether they carry the Philadelphia (Ph) translocation. Using a method recently described, DC were generated from PBMC precursors of 12 patients with CML using GM-CSF, IL-4, and monocyte-conditioned medium. DC exhibited the typical morphology with thin cytoplasmatic processes and expressed high levels of MHC class II, CD86, and CD83 typical for mature DC. After sorting with the monoclonal antibody CD83, a cell population of more than 95% CD83 positive cells was obtained. The presence of the Ph translocation was analyzed in these cells, in PBMC, lymphoblastoid cell lines (LCL), and in phytohemagglutinin (PHA)-induced T blasts from the same patients by fluorescence in situ hybridization (FISH). In contrast to all other cells analyzed, the vast majority of DC (95.9 +/- 0.7%) displayed the Ph translocation, irrespective of disease stage or therapy. PBMC were predominantly positive for the Ph chromosome (67.6 +/- 7.3%), whereas only 11.4 +/- 1% of the B cells and 4.4 +/- 1.1% of the PHA blasts carried the Ph translocation. Using such leukemic DC as antigen-presenting cells, a primary CML-directed cytotoxic immune response in vitro was obtained, as shown by the specific recognition of Ph chromosome positive cells. We conclude that DC can be generated from blood progenitors of CML patients in vitro and exhibit, to a large extent, the Ph translocation. Such DC, which in a preliminary experiment have been able to induce a primary CML-directed cytotoxic immune response in vitro, might be ideal candidates for adoptive immunotherapy either by direct transfer of DC for in vivo generation of a T-cell response or by in vitro generation of CML-specific cytotoxic autologous or HLA-matched normal T-cell clones for use in vivo.

In a prospective multicenter phase-II trial 80 patients with Philadelphia (Ph)-positive chronic myelogenous leukemia (CML) were treated with recombinant interferon (IFN) alpha-2c, administered subcutaneously at an absolute dose of 3.5 megaunits (MU)/day. Complete hematological remission was achieved in 29 (39%) and partial hematological remission in 26 (35%) of the 74 patients evaluable for response. Major cytogenetic responses were observed in ten (13%) and minor cytogenetic responses in 11 patients (15%). Median duration of cytogenetic response was 33 months (range, 2-90); relapses were seen in all of the 11 patients with minor and in three of the ten patients with major cytogenetic responses. Median survival estimates for pretreated (n = 19) and untreated (n = 58) patients were 51 months (95% confidence interval [CI], 30-72) and 77 months (95% CI, 43-111), and the survival probabilities at 5 years were 45% and 54% for the two groups, respectively. Hematological response after 3 months of treatment demonstrated a clear-cut discriminative capacity with 5-year survival probabilities of 100%, 67% and 24% for patients achieving CHR (n = 6), PHR (n = 34), and less than PHR (n = 35), respectively. Landmark analysis at 12, 18, and 24 months after start of IFN therapy and an analysis treating time to cytogenetic response as a time-dependent covariate showed that cytogenetic response was associated with longer survival. The impact of a low-dose IFN regimen on survival in CML patients is unclear and requires further clarification by randomized clinical trials. Early hematological and cytogenetic response to IFN-alpha treatment identifies patients with a favorable long-term prognosis.

At the ob/gy department of the Innsbruck University routine, genetic amniocentesis has been offered from the 13/14 week of gestation since 1991. In 1991 44.5% of all amniocenteses were performed as early amniocentesis, it rose in 1992 to 61.5%, the rest being performed in the 16th week of gestation. In a total of 346 genetic amniocenteses, 8 pathological karyotypes were obtained (six trisomies, one 47 XXY, one marker chromosome), six from samples obtained at early amniocentesis and two obtained at the 16th week. Three weeks following the procedure, a spontaneous abortion rate of 0.87% occurred. Cultivation took 1-2 days longer with samples from early amniocentesis than with samples collected at the 16th week. In two cases the cultures failed and a repeat procedure had to be performed at the 16th week. With early amniocentesis performed at week 13/14 results are known of gestation. The long period of anxiety, which many women see as a serious disadvantage of amniocentesis is thus significantly reduced.

We present two patients, a 12 year old Turkish and a 7 year old Italian girl, with severe mental retardation, multiple congenital malformations and a constitutional interstitial deletion of the short arm of chromosome 17, del(17) (11.2). The main clinical features of this syndrome which is also referred to as the Smith-Magenis syndrome consist of a broad flat midface with brachycephaly, broad nasal bridge, brachydactyly, speech delay, hoarse deep voice and peripheral neuropathy. Behavioural abnormalities include hypermotility, self mutilation and sleep disturbances. The remarkable consistency of the main clinical features of the 59 patients documented so far permits the clinical diagnosis of this syndrome; a fact which is of importance, since many of the reported patients--including ours--were considered normal on earlier cytogenetic studies.

In a Phase II study, 82 patients with Philadelphia chromosome(Ph)-positive chronic myelogenous leukaemia were treated with 3.5 million I.U./d of recombinant interferon alpha-2c (rIFN). 73 patients have so far been evaluated (42 male, 31 female, mean age 50 [12-87] years). At the start of therapy, 10 were in the accelerated phase (group 1) and 63 in the chronic phase, of whom 19 had received previous treatment with cytotoxics (group 2), while the remainder (group 3, n = 44) had received primary treatment with rIFN. There was short-term stabilization in 7 of the 10 group 1 patients, but none had complete haematological or cytogenetic remission. In contrast, the remission rate (complete or partial haematological remission) was 63% in the previously treated (group 2) and 66% in the previously untreated chronic phase patients (group 3). There was a reduction in the proportion of Ph-positive metaphases in 7 group 2 patients (11%) and in 10 group 3 patients (23%). Complete cytogenetic remission has so far been seen in 2 patients. Cytogenetic improvement occurred after 3 months at the earliest, and in some patients only after 12 to 19 months treatment. Differences in response to treatment were related to stage (prognostic staging system of Kantarjian et al.) in group 3 patients: complete or partial haematological remission was seen in 22 out of 25 patients with stage 1 disease, in 4 out of 7 in stage 2, and in only 3 out of 12 in stages 3 and 4.

Four male volunteers inhaled for 4 h 70 and 140 ppm trichloroethylene (TRI) at rest and also at rest combined with exercise. To estimate the amount retained in the body (dose), minute volume and concentration in exhaled air were determined. Concentrations of TRI, trichloroethanol (TCE) and trichloroacetic acid (TCA) were determined in blood. Exhaled air was analysed for TRI and TCE; urine for TCE and TCA.

In 1963 Berg described lipoprotein (a) as a genetic variant of the low-density lipoprotein (LDL) and supposed it to be transmitted as an autosomal dominant trait [2]. Later investigators have shown that lipoprotein (a) represents a quantitative rather than a qualitative genetic marker, and that it is under polygenic control probably with a major gene effect for high concentrations [9, 10, 15]. As early as 1967 Renninger et al. [13] recognized a positive association with myocardial infarction. A series of subsequent investigations have confirmed and extended this observation [3, 11, 14]. Therefore it has been postulated that high concentrations of lipoprotein (a) in plasma represents an independent risk factor for the development of coronary heart disease.

The Lp(a) lipoprotein represents a quantitative genetic trait. It contains two different polypeptide chains, the Lp(a) glycoprotein and apo B-100. We have demonstrated the Lp(a) glycoprotein directly in human sera by sodium dodecyl sulfate-gel electrophoresis under reducing conditions after immunoblotting using anti-Lp(a) serum and have observed inter- and intraindividual size heterogeneity of the glycoprotein with apparent molecular weights ranging from approximately 400,000-700,000 D. According to their relative mobilities compared with apo B-100 Lp(a) patterns were categorized into phenotypes F (faster than apo B-100), B (similar to apo B-100), S1, S2, S3, and S4 (all slower than apo B-100), and into the respective double-band phenotypes. Results from neuraminidase treatment of isolated Lp(a) glycoprotein indicate that the phenotypic differences do not reside in the sialic acid moiety of the glycoprotein. Family studies are compatible with the concept that Lp(a) glycoprotein phenotypes are controlled by a series of autosomal alleles (Lp[a]F, Lp[a]B, Lp[a]S1, Lp[a]S2, Lp[a]S3, Lp[a]S4, and Lp[a]0) at a single locus. Comparison of Lp(a) plasma concentrations in different phenotypes revealed a highly significant association of phenotype with concentration. Phenotypes B, S1, and S2 are associated with high and phenotypes S3 and S4 with low Lp(a) concentrations. This suggests that the same gene locus is involved in determining Lp(a) glycoprotein phenotypes and Lp(a) lipoprotein concentrations in plasma and is the first indication for structural differences underlying the quantitative genetic Lp(a)-trait.