[Guidlines for the good laboratory practice in assisted reproduction - clean environment]. / Smernice ke správné laboratorní praxi pri asistované reprodukci - cistota prostredí

The guidlines are the recommendation for good laboratory practice in embryological laboratories. In this first part the requirements of the clean environment from the point of view of the oocytes, sperm, and embryos protection against infection and from point of view of the embryological laboratory staff health protection were described.

[Activities and responsibilities of workers in embryologic and andrologic laboratories in assisted reproduction centers]. / Cinnosti a odpovednosti pracovníku embryologické a andrologické laboratore v centrech asistované reprodukce.

This paper presents the current status and rules for the laboratory staff activities and their competences in the centers of assisted reproduction. The rules were processed by the members of the Association of Reproductive Embryology (ARE) committee under the current legislation. Committee members of the Czech Sterility and Assisted Reproduction Society and Czech Gynecology and Obstetric Society approved these rules as obligatory for assisted reproduction centres in Czech Republic.

[Theoretic and practical content of the clinical embryology]. / Teoretická a praktická nápln oboru klinická embryologie.

Clinical embryology is in whole Europe respected health care specialization. The specialists in this field are certified by the European Society of Human Reproduction and Embryology (ESHRE). In the Czech Republic the specialization has been formally established by the Government Order No 31/ 2010 after 30 years unformal existence. Concurrently with the wide development of new techniques in assisted reproduction, there is necessary to define the content of clinical embryology as a health care, scientific and educational discipline. The definition authorized by the Committee of Association of Reproductive Embryology (ARE) is given in this article..

[Prerequisites for preimplantation genetic diagnosis (PGD in carriers of mutations responsible for hereditary cancers]. / Predpoklady pro preimplantacní genetickou diagnostiku (PGD) u nosicu mutací v nádorových predispozicních genech.

BACKGROUNDS: Carriers of hereditary mutations in cancer susceptibility genes represent a limited but high-risk population characterized by a high probability of cancer development, frequently with its manifestation in early age and with a 50% chance of pathogenic allele inheritance by offspring. In case of monogenic disorders, preimplantation genetic diagnosis (PGD) could be used for characterization of the DNA region affected by pathogenic mutation in the early stages of an embryo created by in vitro fertilization (IVF). Therefore, the transfer of unaffected embryos could be performed based on the results of PGD genotyping, enabling the development of offspring not carrying the pathogenic alteration. AIM: Here we present the consensus of the collaborative group of the Society for Medical Genetics, the Czech Society for Oncology and other professionals for use of PGD in the Czech Republic for carriers of mutations in cancer susceptibility genes. We address the conditions, prerequisites, and limits of practical application of this method. We also point out specific issues of ovarian hyperstimulation in carriers of mutations in BRCA1, BRCA2, and p53, anticipating the increased risk of hormonally dependent breast and ovarian cancers development. CONCLUSIONS: We assume that a narrow but non-negligible subgroup of cancer susceptibility gene mutation carriers may benefit from PGD.They are mainly individuals deciding to undergo IVF and PGD recruited from mutation carriers with extreme concerns about transmitting the mutation to their children. The PGD in these individuals should be managed by a closely cooperating multidisciplinary team of professionals responsible for indication of PGD, giving complete information regarding the IVF and PGD procedures including their limits, evaluating individual risks and performing instrumental and laboratory procedures with respect to up-to-date good laboratory and clinical practice.

[How can the haematopoietic stem cells from the umbilical cord blood be de-differentiated in vitro? Our first results using the co-cultivation systems]. / Jak dediferencovat hematopoetické kmenové bunky z umbilikální krve in vitro? Nase první výsledky s pouzitím kokultivacních systému.

OBJECTIVE: Aim of this study was to de-differentiate the haematopoietic stem cells (HSCs) that originated from the umbilical cord blood. One of the ways to do it is to use a co-cultivation system. DESIGN: Prospective experimental study. SETTING: Laboratory study - Institute of reproductive medicine and endocrinology, Pilsen. METHODS: HSCs were co-cultivated with mouse embryonic stem cells (mESC) with and without feeder cells. After co-cultivation HSCs were analyzed using flow-cytometry for presence of haematopoietic markers (CD34, CD45, CD133) and using immunohistochemistry for presence of embryonic stem cell markers (SSEA-4, Tra-1-60, Tra-1-81). RESULTS: No de-differentiation was detectable in any our experiment, only the intensity of the HSC cell markers decreased. CONCLUSION: We suppose that there were two major reasons for the experiment failure: there was no direct cell to cell contact and there was a mixture of cell types that originated from two different species. To reach our goal of in vitro de-differentiation we will need to change our strategy towards a pure human culture system without any animal additives and with cell to cell contact.

[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.

[Medical genetics in reproductive medicine]. / Vyuzití lékarské genetiky v reprodukcní medicíne.

Reproductive genetics (RG) is another new field of medical genetics, integrated with reproductive medicine, assisted reproduction and developmental genetic. RG is closely linked to the perioconceptional prevention, perinatology, ultrasound and biochemical screening in the end of the first and beginning of the second trimesters. RG is based on the system of specialized genetic counseling, clinical cytogenetics, molecular cytogenetics and molecular genetics to provide prefertilization, preimplantation and classical prenatal diagnosis in the Ist to IIIrd trimesters. Thus, RG is part of the fetal medicine and therapy. The six years experience with RG is summarized. A system of the specialized health care, organized, if possible in one integrated center of RG and reproductive medicine (RM) is presented. Reproductive medicine provides all necessary clinical gynecological and andrological surveillance, with assisted reproduction and further obstetrical ultrasound examinations, including nuchal translucency measurements and 2D, 3D ultrasound, echocardiography examinations, if indicated, as well as the invasive method of prenatal diagnosis and perinatology care. Specialized genetic counseling and cytogenetic analysis, if indicated, should be offered to all partners with reproductive disorders as well as to oocyte donors. Chromosome anomalies are disclosed in 6% of men with abnormal sperm analysis as well as in women with severe reproductive disorders. In males with severe oligo, azoospermia, the sperm aneuploidy analysis by molecular cytogenetic methods is recommended. Advised is also the molecular genetic detection of Y chromosome microdeletions, which is detected in 9% of our azoospermic men with deletions in AZFb region. CFTR gene mutations and intron 8 and 10 polymorphism examination is provided not only in men with obstructive azoospermia (CBAVD), but also if severe oligospermy with less than 1 x 10(6) sperm/ml is detected. Molecular genetic analysis of thrombophilic mutations of factor II., V. (Leiden) and MTHFR gene in unexplained recurrent abortions and in cases with unsuccessful IVF is part of the diagnostic strategy. The population frequencies of carriers of mutations of factor II. (2.3%), factor V.-Leiden (5.7%) and MTHFR gene (38%) were determined. The laser biopsy of the first polar body and of blastomeres was introduced for FISH analysis of chromosome aneuploidies. Quantitative fluorescent PCR (QFPCR) detection is used for testing of the most frequent delta F508 CFTR gene mutation and the most frequent aneuploidies of chromosome 13, 18, 21, X and Y. QFPCR was successfully tested for male fetal sex examination from partially purified fetal cells in the maternal blood. The first trimester ultrasound and biochemical screening is recommended to all successful pregnancies after different IVF methods. If borderline levels of first trimester biochemical screening of PAPP-A protein and beta hCG are detected without pathological ultrasound findings, classical triple test of biochemical screening in 16th week of gestation is recommended. If pathological results of ultrasound and biochemical screening are disclosed, invasive prenatal genetic diagnosis is indicated as well as in pregnancies after ICSL, if there is not any obstetrical contraindication.

Activation of pig oocytes using calcium ionophore: effect of the protein kinase inhibitor 6-dimethyl aminopurine.

The aim of our study was to investigate the parthenogenetic activation of in vitro matured pig oocytes after their combined treatment with calcium ionophore A 23187 and the inhibitor of protein kinases, 6-dimethylaminopurine (6-DMAP) and to study the further embryonic development of oocytes activated using this treatment. The oocytes were exposed to ionophore (10, 25 or 50 microM) for 0.5, 1, 3, 5 or 7 min and then cultured with 6-DMAP (0 or 2 microM) The highest activation rate (up to 88% of the activated eggs reached the pronuclear stage) was observed after combined treatment of the oocytes with 50 microM ionophore and 6-DMAP. The highest rate of embryonic development was observed after treatment with 25 microM ionophore without 6-DMAP, when up to 51% of the eggs developed beyond two-cell stage, 2% of the eggs developed up to the stage of morula and up to 3% of the eggs reached the stage of blastocyst. When 50 microM ionophore was used, the embryonic development of the activated eggs was arrested before the morula and blastocyst stage. After treatment of the activated eggs with 6-DMAP, we did not observe any development beyond the stage of 16 blastomeres. We can conclude that combined treatment with calcium ionophore A 23187 and 6-DMAP increases the activation rate in pig oocytes matured in vitro, but this combined treatment exerts a detrimental effect on further embryonic development of the activated eggs.

[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.

Multi-fetal pregnancy reduction does not influence perinatal results in twin pregnancies.

The objective of this study was to compare perinatal results in multifetal pregnancies where the reduction to twins was performed with non-reduced twin pregnancies. Perinatal results in 99 sets of twins after transabdominal multifetal pregnancy reduction of triple and higher-order multiple pregnancies performed in a single center were compared with a control group consisting of 151 twin pregnancies conceived in the same time period after infertility treatment, which were not a result of reduction. The main outcome measures were length of pregnancies, weight of the newborns, percentage of miscarriages and the mode of the delivery were analyzed. No significant difference could be found at a 5% level of significance regarding the average duration of pregnancy or average weight of the twins. 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 a 5% level of statistical significance, no significant difference in probability of perinatal death of the fetus or delivery of a stillborn fetus was found. The percentage of cesarean sections did not differ significantly in both groups. The analysis of both groups demonstrated that reduction of multifetal pregnancies to twins may not influence perinatal results in comparison to twin pregnancies where reduction was not performed.

[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.

Activation of pig oocytes using calcium ionophore: effect of protein synthesis inhibitor cycloheximide.

In vitro matured pig oocytes were activated using a combined treatment of calcium ionophore A 23187 with cycloheximide. The oocytes were exposed to ionophore (10, 25 or 50 microM) for 0.5, 1, 3, 5 or 7 min and then cultured with cycloheximide (0 or 10 microg/ml) for 6 h. Cycloheximide treatment significantly increased the activation rate of oocytes and the percentage of oocytes that were able to develop after activation. The highest activation rate was observed after treatment with 50 microM ionophore. The highest percentage of developing eggs was observed after combined treatment of ionophore (25 microM) with cycloheximide. The percentage of oocytes developing up to the morula and blastocyst stage was not significantly increased after cycloheximide treatment.

The influence of different types of antibodies on in vitro fertilization results.

PROBLEM: The influence of anti-sperm (ASA), anti-phospholipid (APA), and antizonal (AZA) antibodies on in vitro fertilization (IVF) results and the need for intracytoplasmic sperm injection (ICSI) were assessed. METHOD OF STUDY: Forty-four couples with infertility of immunologic origin were investigated. ASA in serum and ovulatory mucus were studied by a tray agglutination test (TAT) and indirect mixed anti-globulin reaction test (MAR) test, AZA were studied by passive hemagglutination and commercial enzyme-linked immunosorbent assay (ELISA; BioGen, Germany), and APA were tested by ELISAs in immunoglobulin isotypes IgG and IgM. RESULTS: Because of failed or very low fertilization after standard IVF in the previous cycle, ICSI had to be used in five out of 15 cases with ASA (33.3%), in 16 out of 18 couples with AZA (89.4%), and in only one case if APA were present (9%). Clinical pregnancy rate was 60% in cases with ASA, 38.5% with AZA, and 27.3% per embryo transfer (ET) if APA were detected. CONCLUSIONS: Immunologic infertility can be treated by IVF with very good results. The most important group are women with AZA, in whom IVF ICSI without any delay is recommended.

[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.

[Effect of oocyte transportation (IVF transport) on effectiveness of the IVF and ET program]. / Sledování vlivu transportu oocytu (transportní IVF) na efektivitu programu IVF + ET.

The programme of in vitro fertilization helps at present an ever increasing number of married couples to resolve successfully the problem of sterility. It is however a method pretentions technically, as regards time, material and in particular funds. This applies above all to embryological laboratories. The transport system of the IVF programme makes possible care of patients indicated for sterility treatment by the IVF method in departments without their own embryological laboratory and thus it is possible to include more patients in the programme and save money. In the author's department the first two stages of the IVF programme are implemented, i.e. stimulation of the ovaries and aspiration of follicular fluid. Then this material is taken in a transport box of the K-system Co., Denmark, type G-81E to a centre with full accreditation where the subsequent stages of the IVF programme take place (selection of the oocyte, fertilization, cultivation of embryos and embryo transfer). The transport system has operated now for three years (1995-1997) and at present the authors evaluate the system. The fertility rate and pregnancy rate in individual years was compared and the following conclusions were reached: The fertility rate is significantly (at the 1% level) lower in the transport centre in 1995 and 1996 (47%, 54%) as compared with the other centre the laboratory of which is used by the authors (57%, 61%). In 1997 the difference is no longer significant (63% x 64%). The difference in the pregnancy rate in centres for aspiration and embryo transfer in both centres is insignificant in the years of investigation. It may thus be stated that the oocyte transport does not have a negative influence on the effectiveness of the IVF + ET programme (same pregnancy rate) and thus makes it possible to include a greater and ever increasing number of patients in the IVF programme by making use of reserve capacities of embryological laboratories and thus reduce the total costs of this programme.