[Implementation of arrays in first trimester prenatal diagnosis]. / Implementace array vysetrení do prenatální diagnostiky v I. trimestru.

OBJECTIVE: Array technology in chorionic villus sampling (CVS) - analysis of clinical benefit and a proposal of a more effective 1st trimester genetic testing policy. DESIGN: Retrospective study. SETTING: Gennet, Center of Medical Genetics and Reproductive Medicine, Prague. MATERIAL AND METHODS: Total of 913 CVS were performed at Gennet between 2010-2014. All 913 samples were tested by QF-PCR rapid test for aneuploidy of chromosomes 13, 18, 21, X and Y and karyotyping following standard long term culture. Microarray analysis (Illumina HumanCytoSNP12 v2.1) was performed on 179 samples with normal result from both - QF-PCR and karyotyping. RESULTS: At 229 samples the common chromosomal aneuploidy was detected using rapid QF-PCR (25% from 911 successful rapid tests). Conventional karyotyping revealed 239 unbalanced chromosome aberrations (27% from 897 successful cultivations). 227/239 (95%) positive karyotypes confirmed QF-PCR finding of common aneuploidies. 10 unbalanced chromosome aberrations were not covered by rapid QF-PCR test. Microarray analysis of samples with normal result from both- QF-PCR and karyotyping- revealed 13 clinically relevant chromosome aberrations (7.5%). CONCLUSION: New policy for chorionic villi testing at Gennet was established. Based on evaluation of the results of karyotyping, array and QF-PCR and analysis of published data we decided to replace karyotyping by microarray analysis in all cases of foetuses with normal results from QF-PCR. More effective detection of pathological and clinically relevant chromosome aberrations in examined foetuses is expected.

[Application of SNP array method in prenatal diagnosis]. / Aplikace metody SNP array v prenatální diagnostice.

OBJECTIVES: SNP array (array method using Single Nucleotide Polymorphisms) enables to detect cytogenetically undetectable submicroscopic alterations (microdeletions, microduplications), which could be also causative for ultrasonographic anomalies of fetus. This article describes the principle, advantages, disadvantages and application possibilities of the SNP array method in prenatal diagnosis. The ten month experience with SNP array use in prenatal diagnosis is presented. DESIGN: Prospective study. SETTINGS: Gennet, Prague. MATERIAL AND METHODS: During the period from April 2010 to January 2011 we performed 110 SNP array analyses of fetal DNA: 14 chorionic villi samples (CVS), 88 amniotic fluid samples (AMC), 1 cord blood sample and 7 miscarriage samples. Laboratory tests were carried out on DNA from both cultured and uncultured fetal cells. Examinations were performed in fetuses with sonographic abnormal findings having normal karyotype. In addition 14 fetal cytogenetic abnormalities were solved. SNP array analysis was performed using Illumina InfiniumHD HumanCytoSNP-12 chip. All data were analysed by Illumina KaryoStudio and GenomeStudio software. RESULTS: SNP array analysis was performed in 108 fetuses (only 2 examination failures, 1.8%). In total, we detected CNV (copy number variation) in 29 samples (29/108 = 27%). 15% (16/108) of fetuses with abnormal ultrasound findings were found to carry clinically relevant CNV. Probably benign CNVs were found in 8 samples (8/108 = 7%) and in additional 5 CNVs parental samples have not been analysed yet. Excluding karyotypically abnormal cases clinically relevant CNVs were found in 10% of fetuses (9/94). In all cases with de novo chromosomal aberration the clinical relevancy was clarified (imbalances in 50%). CONCLUSION: Our data suggest that SNP array analysis is a relevant and useful technique in prenatal diagnosis.

[The first pregnancy and birth after cryopreservation of oocytes in the Czech Republic--importance of the method for reproductive medicine]. / První tehotenství a porod po kryokonzervaci oocytu v CR--význam metodiky pro reprodukcní medicínu.

OBJECTIVE: The purpose of this work was to develop methods for successful cryopreservation of human oocytes. DESIGN: Case-controlled study and case report. SETTING: Sanatorium Pronatal, Prague. METHODS: Propanediol (PrOH)-sucrose was used as cryoprotectant medium for cumulus-free oocytes and stepwise dilution of cryoprotectant post-thaw. RESULTS: The method was used in three patients (38 denuded oocytes) and yielded excellent survival and fertilization rates (89.5% and 73.5% respectively). In all patients embryo-transfer was possible, one patient got pregnant and delivered a healthy baby. CONCLUSION: Our data show that cryopreservation may ensure that the integrity of the human oocyte is adequate for normal fertilization and embryo development.

[Effect of salpingectomy on the results of IVF in women with tubal sterility--prospective study]. / Vliv salpingektomie na výsledky IVF u zen s tubární sterilitou--prospektivní studie.

OBJECTIVE: To compare the IVF results after salpingectomy of hydrosalpinges visible on ultrasound with IVF results in women with tubal infertility but without hydrosalpinx. DESIGN: Prospective study. SETTING: Sanatorium Pronatal, Na Dlouhé mezi 4/12, 147 00, Praha 4. METHODS: 101 women with tubal factor infertility were evaluated. In a prospective study we compared the results of first IVF cycle after salpingectomy of hydrosalpinges visible on ultrasound performed in 51 women (study group) with IVF results of 50 women with tubal infertility (confirmed by HSG and laparoscopy) but without hydrosalpinges and without salpingectomy. RESULTS: The maternal age in both groups (32.4 +/- 3.9 in salpingectomy group and 33.0 +/- 4.1 in control group), maximum estradiol levels (1392.6 +/- 675.8 pg/ml in salpingectomy group vers. 1624.7 +/- 909.7 pg/ml in control group), number of oocytes (11.3 +/- 5.8 after salpingectomy vers. 11.0 +/- 6.1 in controls), number of embryos generated (7.1 +/- 4.6 vers. 7.9 +/- 4.7) and number of embryos transferred were not statistically different. In their first IVF cycle after salpingectomy has been performed 30 women out of 51 became clinically pregnant (58.8%), while in control group a clinical pregnancy could be verified in 16 women (32.0%). Implantation rate in women after salpingectomy was 28.2% vers. 12.3% in control group. Both implantation rate and pregnancy rate were statistically different at 5% level of significance. CONCLUSION: After salpingectomy of hydrosalpinges visible on ultrasound the implantation rate and clinical pregnancy rate are significantly better when compared to IVF patients with tubal infertility without the presence of hydrosalpinges and without salpingectomy. Salpingectomy should be offered to all patients with hydrosalpinges visible on ultrasound. Moreover, this radical approach should be considered even in women with highly damaged tubes but without the presence of hydrosalpinges.

[Does reduction in multifetal pregnancy increase the risk of poor outcome in twin pregnancy?]. / Zhorsuje redukce vícecetné gravidity perinatální výsledky u dvoucetných gravidit?

OBJECTIVE: To compare perinatal results for multi-foetal pregnancies where the reduction was performed with pregnancies where the reduction was not performed. SETTING: Sanatorium Pronatal, Na Dlouhé mezi 4/12, 147 00, Praha 4--Hodkovicky. METHODS: We have analyzed results of pregnancies, after delivery, for women with twin pregnancy which originated in our sanatorium, as a result of treatment with assisted reproduction methods, in the period of time from January 1st 1996 to December 31st 1998. In the group being monitored there were 122 twin pregnancies originated as a result of reduction of triple and more-foetal pregnancies. We evaluated the percentage of miscarriages, length of pregnancies, weight of the newborns and the manner of termination of the delivery. These results were compared with our control group consisting of 180 cases of twin pregnancies which were not a result of reduction. RESULTS: Analysis was performed for those mothers only where complete data were available. At a 5% level of statistical significance, it was not proved that both groups differed in average term of pregnancy or average weight of the twins. Average age of the mothers differed at 5% level of significance (average age values were 30.16 for the group with reduction and 31.73 for the group without reduction). Fisher test on 5% significance level did not ascertain any significant difference in the probability of miscarriage between the group with reduction (5.26%) and the group without reduction (12.84%). At 5% level of statistical significance, no significant difference in probability of perinatal death of the foetus or delivery of a stillborn foetus was ascertained. However, it is necessary to point out a low frequency of these phenomena in our group. The percentage of cesarean sections did not differ significantly in both groups (86.24% in the group monitored vs 87.24% in the group of twins without reduction). CONCLUSION: The analysis of both groups proves that reduction of multi-foetal pregnancies does not worsen perinatal results in comparison to pregnancies where reduction was not performed.

[Monochorionic twins after treatment of sterility using assisted reproduction methods]. / Monochoriální dvojcata po lécbe sterility metodami asistované reprodukce.

OBJECTIVE: The goal of this study has been to determine frequency of occurrence of monochorial twins within the study group consisting of 521 pregnancies conceived through the In Vitro Fertilization (IVF) programme. DESIGN: An observational study. SETTING: Sanatorium Pronatal, Na Dlouhé mezi 4/12, 147 00 Praha 4-Hodkovicky. METHODS: A condition to be included in the study group was that there was a gestation sac detected by ultrasound. The first ultrasound examination was performed transvaginally and was done between the fifth and the sixth week of each pregnancy. A multiple pregnancy has been classified as monochorial in case when gestation sac contains two yolk sacs and two fetuses. RESULTS: Within the whole study group there were 13 monochorial twins which presents 2.5% of all pregnancies. The incidence of monochorionicity among all multiple pregnancies is 6.4%, in IVF cycles without micromanipulation techniques the occurrence of monochorial twins has been 0.9%, following cryoembryotransfer 3.9%, with micromanipulation (ICSI, AH) it reaches 5.7% of all pregnancies. CONCLUSION: Monochorial twins were detected seven time more frequent in comparison within occurrence in ordinary population. The occurrence rises in connection with the number of fetuses in uterus. In comparison with simple IVF cycles we have proved higher occurrence of monochorionicity in connection with micromanipulation techniques and cryoembryotransfer.

[Hormone replacement for pregnancy in functionally agonadal women after transfer of thawed embryos in the in vitro fertilization program--problems of placental incompetence]. / Hormonální substituce tehotenství funkcne agonadálních zen po prenosu rozmrazených embryí v programu IVF--problematika placentární kompetence.

Experience with hormonal substitution of sterile women treated within the IVF programme by means of donated oocytes changed completely views on endocrinological prerequisites of successful implantations and the further development of early pregnancy. While the effect of oestrogens and progesterone on endometrial proliferation and receptivity has been elaborated in great detail in the literature, data on the substitution of early pregnancy and the onset of placental competence are much scarcer. 1. The placenta is during the 15th week of pregnancy already fully competent and hormonal substitution can be probably terminated already sooner. 2. From the shape of the curves (as compared with steroid levels in spontaneous pregnancies) the authors conclude that the used doses (in particular of progesterone were higher than physiological. 3. Pregnancy of women with primary or secondary ovarian failure is an ideal in vivo model for investigation of the receptivity of the endometrium, the problem of implantation and function of the foetoplacental unit. Available data are not uniform and therefore in this very interesting field further investigations are needed.

[Chromosomal aberrations in prenatal diagnosis--results of karyotyping 2,000 samples of amniotic fluid]. / Chromosomální aberace v prenatální diagnostice--výsledky karyotypizace témer 2,000 odberu plodové vody.

The frequencies of different types of chromosomal abnormalities are shown in a study performed during 1989-1996. 32 pathological karyotypes were found, i.e. 1.6%. The ratio of numerical/structural abnormalities is 1.67. The number of de novo or unexpected structural abnormalities is 67% from all structural ones in this study. This strongly supports the need of complete cytogenetic analysis.

Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode.

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the gram-positive methylotroph Brevibacterium methylicum by electrotransformation.

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10(5) transformants/micrograms DNA) was achieved.