The Assessment of Cryopreservation on the Quality of Endangered Oravka Rooster Spermatozoa Using Casa and Cytometry.

BACKGROUND: Preservation of genetic resources in gene bank is necessary for conservation of endangered poultry species. OBJECTIVE: This study is to characterize Oravka rooster semen quality. MATERIALS AND METHODS: Heterospermic pool was diluted (1:1 by volume) in a freeze medium composed of a commercial diluent (Kobidil+, K), 8% dimethyl sulphoxide (DMSO) or 8% ethylene glycol (EG) or 8% glycerol (GL), and then frozen in liquid nitrogen vapour. RESULTS: Spermatozoa in the GL/K+ had significantly higher number of motile and progressively moving spermatozoa (p < 0.05) than in DMSO/K+ and EG/K+ groups. The percentage of apoptotic and necrotic spermatozoa were significantly higher in the DMSO/K+ and EG/K+ groups compared with the GL/K+ group. Based on the total motility, progressive movement parameters and viability, our study showed that 8% GL diluted in Kobidil+ provided the highest cryoprotective effect on the Oravka rooster spermatozoa.

Incidental tubal endometrioid adenocarcinoma - case report.

The fallopian tubes represent a highly important structure for ovarian carcinogenesis. They provide the passage of eutopic endometrium from the uterus, as well as metastatic cells from the ovaries. A direct source of tumor cells for ovarian cancer was also recently confirmed in the fallopian tubes. The authors present a case report of an incidental tubal endometrioid carcinoma with the coexistence of adenomyosis.

Changes in urine autofluorescence in ovarian cancer patients.

Ovarian cancer is the type of cancer with the highest mortality rate among gynaecologic malignancies. Due to lack of screening tools, this disease is mainly diagnosed at a progressed stage, when it is too late to adequate therapy. Despite many attempts, enough sensitive and specific biomarker was not still uncovered. Fluorescence spectroscopy has proven to be a useful diagnostic tool with high efficiency. Fluorescence detection has three major advantages over other light-based investigation methods: high sensitivity, high speed, and reliability. Biological materials consist of a number of intrinsic fluorescent compounds -autofluorophores, which are associated with cardinal metabolic pathways. It is well known, that cancerous tissue metabolism is altered compared to healthy one, what influence also intrinsic fluorophores composition of bodily fluids. Urine is one of the biological fluids that could be obtained most easily and displays a blue - green fluorescence that can change in case of pathological process. Analysis of urine autofluorescence is non invasive and simple technique. Using fluorescent spectroscopy, ovarian cancer patients and healthy control group were discerned with high significance, so we predict that fluorescence analysis of urine could be a potential means of ovarian cancer screening.

Ultrastructure of vitrified rabbit transgenic embryos.

The aim of the study was to determine the viability of rabbit transgenic (enhanced green fluorescent protein (EGFP)-positive) embryos cultured in vitro and compare with gene-microinjected (Mi) non-transgenic (EGFP-negative) embryos following vitrification. Non-microinjected and non-vitrified embryos were used as the control. Morphological signs of injury to embryo organelles were determined at the ultrastructural level using transmission electron microscopy (TEM). Morphometric evaluation was performed on cellular organelles using microphotographs obtained by TEM. Intact and Mi embryos recovered from in vivo fertilized eggs at 19-20 hours post coitum (hpc) were cultured for up to 72 hpc (morula stage), evaluated for the EGFP gene integration and then vitrified in 0.25 ml insemination straws in modified EFS (40% ethylene glycol + 18% Ficoll 70 + 0.3 M sucrose) vitrification solution. After 1-3 days the embryos were devitrified, a representative selection of embryos was analyzed by TEM and the remaining embryos were subjected to additional in vitro culture. Observations by TEM showed that the vitrified/warmed EGFP-positive and EGFP-negative embryos had a slight accumulation of cellular debris and lipid droplets compared with the control intact embryos. More severe changes were detected in the membrane structures of the treated embryos, mostly in the cytoplasmic envelope, trophoblastic microvilli, junctional contacts and mitochondria. We suggest that the higher proportion of deteriorated cell structures and organelles in the treated embryos may be due to the vitrification process rather than to mechanical violation (the gene-microinjection procedure), as a detailed inspection of ultrastructure revealed that most damage occurred in the cell membrane structures.

Borderline ovarian tumor - a case report with genetic testing.

Despite an accurate classification, the borderline category of ovarian tumors is one of the most controversial topics in gynecologic oncology and is confusing to both clinicians and patients. The treatment is often confronted with the necessity of a fertility-sparing approach, although under-treatment increases the risk of disease recurrence. The clear definition of a group of patients with low or high risk of relapse requiring more or less extensive treatment is lacking. Currently, the main criteria affecting the treatment extent include histopathological features, particularly the presence of microinvasion, and invasive implants. Expansion of knowledge about genetic nature of the tumor characteristics may more closely specify the scope of therapeutic approach for individual patients. The authors report a case report of serous borderline ovarian tumor patient with tumor cell dispersion into the gastrointestinal tract. The genes of tumor vascular markers GPM6B and DR6 were also studied and compared to a group of healthy patients.

Molecular diagnosis of Toxoplasma gondii in pregnant women.

BACKGROUND: Novel approaches in the diagnostics of T. gondii have enabled a detection of the parasite in the amniotic fluid or blood of pregnant women. OBJECTIVE: The high titres of IgM and IgG antibodies against T. gondii are not always indicative of the presence of this parasite in pregnant women, therefore the molecular assays can be used to diagnose and genetically characterise T. gondii in amniotic fluids and blood samples. METHODS: The study analysed 15 samples of amniotic fluid and 1 sample of the blood from pregnant women suspected for toxoplasmosis. The serological ELISA test was used for the immunological study and molecular analyses, PCR at SAG2 locus followed by RFLP analysis were used for Toxoplasma gondii genotyping. CONCLUSION: Using PCR assay with TGR1E gene we have confirmed the presence of T. gondii in the blood of a pregnant woman. The parasite was typed as genotype I, belonging to virulent strains (Tab. 1, Fig. 2, Ref. 30).

[New and clinically used oncomarkers of bladder cancer]. / Nové a klinicky vyuzívané onkomarkery karcinómu mocového mechúra.

Bladder cancer is the fourth most common cancer in men and the eighth most common cancer in women. Oncomarkers play a crucial role in early detection of bladder cancer, as well as in treatment response monitoring and prognosis. Search for a new marker by molecular analysis is in progress because any diagnostic sensitivity and specificity enhancement is a great benefit for clinical practice.

[Risk of the prolapse "de novo" in primary unaffected compartment by using the syntethic mesh in the surgery treatment of the pelvic organe prolapse]. / Riziko vzniku prolapsu "de novo" v primárne nepostihnutom kompartmente pri pouzití syntetického implantátu v rámci operacnej liecby prolapsu panvových orgánov.

OBJECTIVE: The aim of this retrospective trial was to evaluate the risk of the prolapse "de novo" in primary unaffected compartment by using the syntethic mesh. DESIGN: Rerospective multicentric trial. SETTING: 2nd department of Gynaecology and Obstetrics, Medical Faculty of Safarik's University and L. Pasteur Hospital, Kosice and Gynaecology department, Gynpor, s.r.o., Sliac, Slovakia. POPULATION AND METHODS: One hundred and eleven women with stage > or = 2 prolapse according to the Pelvic Organe Prolapse - Quantification (POP-Q) system was enrolled into the study. In group MESH A (anterior compartment) was 86 (17x Prolift Anterior; 69x Prolift Anterior like MESH and group MESH P (posterior compartment) was 25 (9x Prolift Posterior; 16x Prolift posterior like MESH) of enrolled women. The primary outcome was the risk of POP > or = 2 "de novo" prolapse in primary unaffected compartment at 12 months follow-up. The second was the efficacy of the treatment by using synhetic mesh. Third was: Did uterus sparing operation increase the risk of reccurance of the prolapse. Complications was also reported. RESULTS: For subject attendigs the 12-month follow-up, the risk of "de novo" prolapse in MESH A group was 20.9% (17/86) compared with only 16% (4/25) in the MESH P group and was not significantly different (p<0.13). The efficacy was 91.6% in the MESH A group and 84% in the MESH P group and was not significantly different. Uterus sparing operation did not increase the risk the prolapse,,de novo" in primary unaffected compartment. Vaginal mesh exposure occured in 14.4%. CONCLUSION: Vaginal repair by using MESH is effective in the treatment of the pelvic organe prolapse, but is posible to provoke the development of prolapse in primary unaffected compartment.

[The impact of exogenous luteinizing hormone on IVF/ICSI cycles parameters]. / Vplyv exogénneho luteinizacného hormónu na parametre IVF/ICSI cyklov.

OBJECTIVE: To evaluate the effect of exogenous luteinizing hormone on IVF/ICSI success. DESIGN: Retrospective clinical study. SETTING: 2nd Department of Gynaecology and Obstetrics, Medical Faculty and L. Pasteur University Hospital Kosice, Slovak Republic. METHODS: Individual parameters and clinical pregnancy rate of 232 IVF/ICSI cycles were compared in dependence on stimulation protocol (pure rFSH vs. rFSH and 75 IU rLH from S7). RESULTS: There was no statistically significant difference in IVF/ICSI success between individual groups and subgroups, but we demonstrated the increase in clinical pregnancy rate per ET in agonist cycles group with exogenous LH activity totally above 11% (27.6% vs. 38.6%), in patients under 35 years above 12% (30.4% vs. 42.6%) and in patients over 35 years above 14% (16.7% vs. 30.8%). In antagonist cycles group there was demonstrated the increase in clinical pregnancy rate only in women over 35 years - above 14% (13.3% vs. 30.8%). CONCLUSIONS: Despite of negative statistical analyses, from the clinical point of view we can strongly recommend the LH addition during COH, especially in agonist cycles and in older women.

[Overview of potential oncomarkers for detection of early stages of ovarian cancer]. / Prehl'ad potenciálnych onkomarkerov detekcie skorých fáz rakoviny vajecníkov.

The causes of ovarian cancer have not been fully elucidated yet but genetic predisposition is found in approximately 10% of patients. When the disease is detected at an early stage, up to 90% of patients have a hope of recovery. However, no preventive measures or precise screening tests to detect early stages of this disease are known yet. Standard tumor markers (CA125) are usually investigated in women with an increased risk. Nevertheless, due to low sensitivity and specificity during the first stage of the cancer, CA125 determination showed a very low efficacy (less than 26%). There has been a considerable progress over the recent years in understanding the molecular mechanisms leading to tumor formation and metastasis. Gradually, 46 genes were identified, initially named tumor endothelial markers (TEM), the expression of which is increased in tumors compared to normal endothelial cells. Death receptor 6 (DR6) and glycoprotein M6B (GPM6B), both detectable from patients serum, are among the most promising candidates for a marker of an early stage of ovarian cancer. This review aims to clearly describe potential as well as clinically used tumor markers useful in an early detection of ovarian cancer. Search for new markers, characterized by increased expression in patients'blood is a highly topical issue.

In vitro antiproliferative and antiangiogenic effects of Flavin7.

Flavin7 (F7) is a nutritional supplement often taken by cancer patients in Central Europe during chemo- and radiation therapy. In this study, investigation of the antiproliferative and antiangiogenic activities of this supplement were performed. Flavin7 showed antiproliferative activity in Jurkat as well as in HeLa cells. It significantly reduced the growth of both cancer cell lines at the doses of 200 microg/ml to 20 microg/ml (p<0.001 and p<0.01, respectively). In F7-treated Jurkat cells we found a significant increase in the fraction of cells with sub-G(0)/G(1) DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V staining and DNA fragmentation. Furthermore, F7 at the doses of 100 microg/ml to 4 microg/ml inhibited endothelial cell migration and capillary tube formation what indicates its potential antiangiogenic properties. Flavin7 also inhibited the activity of matrix metalloproteinases (MMPs), preferentially MMP-9, at the doses of 100 microg/ml to 4 microg/ml. Our data suggest that F7 possesses marked antiproliferative and antiangiogenic properties in vitro. Further research is needed to elucidate also its in vivo activities.

Post-thaw survival, cell death and actin cytoskeleton in gene-microinjected rabbit embryos after vitrification.

The aim of this study was to compare two vitrification procedures (VPs), using either ethylene glycol (EG) in combination with dimethylsulfoxide (DMSO, vitrification protocol (VPI)) or Ficoll 70 (vitrification protocol II (VPII)), for rabbit embryo cryopreservation based on their post-thaw survival, cell death and actin cytoskeleton. The pronuclear stage eggs were flushed from the oviducts of the slaughtered New Zealand White rabbit does 19-20h post coitum (hpc) and randomly divided into two groups: intact (control) and microinjected (Mi). Mi embryos or intact embryos were cultured for up to 72hpc (morula stage), and then vitrified using either VPI (VPI+Mi, VPI) or VPII (VPII+Mi, VPII). After 2-3 days at -196 degrees C, the embryos were thawed and cultured until 96-100hpc to assess their development to blastocyst, apoptotic rate (TUNEL assay) and state of actin cytoskeleton (phalloidine-TRITC). Mi procedure reduced blastocyst yield, but it was higher than in either vitrified (VPI) or Mi vitrified (VPI+Mi) embryos. VPI compromised, whereas VPII did not significantly affect blastocyst development compared to intact embryos. Mi and VP both affected the embryo quality increasing TUNEL-index and decreasing the ratio of embryos with high quality actin cytoskeleton compared to control. A higher apoptotic index was recorded in VPI group. A combination of Mi and VP induced an increase in apoptotic rate (10.35% and 7.54% for VPI+Mi and VPII+Mi, respectively) as compared to Mi alone (5.7%). Ratio of embryos belonging to best actin quality (grade I) was different among groups and most of the embryos with grade I actin were in intact (84%), Mi (71%) or VPII (70%) groups. A significantly lower number of embryos with grade I actin quality was observed in VPI (58%), VPI+Mi (54%) or VPII+Mi (66%). These observations indicate that of the vitrification schemes tested, the VPII using EG and ficoll 70 as cryoprotectants, was less harmful than VPI (EG combined with DMSO in vitrification medium).

[Prediction of intrauterine growth retardation using the integrated test markers]. / Predikcia intrauterinnej rastovej retardácie plodu pomocou markerov integrovaného testu.

OBJECTIVE: Author's objective was to ascertain the option for prediction of intrauterine growth retardation using the integrated test markers on the population of pregnant women in Kosice city. DESIGN: Retrospective, longitudinal, multicenter study. SETTING: 2nd Department of Obstetrics and Gynecology University of P. J. Safárik, Kosice, Slovak Republic. METHODS: The study group comprised 578 pregnant patients of 2nd Department of Obstetrics and Gynecology, in the period from 01.11.2001 until 31.01.2005 for integrated test consisting of (examination of the gestation age using CRL, concentration of PAPP-A, NT, AFP, uE3, hCG). Information on birth weight, gender, gestation age at the time of delivery, occurrence of preeclampsia and other pregnancy complications were found in 578 patients retrospectivelly. We have designed two outcome criteria: 1. intrauterine growth retardation <5th fetal weight percentile - IUGR 5 - and intrauterine growth retardation <10th percentile - IUGR 10. In order to make use of the summary information provided by the markers, we have used the method of logistic regression to identify statistically significant predictors of individual binary outcomes (IUGR 5 and IUGR 10). First we developed an equation based on all available predictors. Statistically insignificant predictors were gradually eliminated from the model (Stepwise regression) until the final model remained, for which a ROC curve was developed and values of sensitivity, specificity, positive and negative predictive values at 5% FP (false positivity) were calculated. RESULTS: The final model of IUGR 5, containing only statistically significant markers of integrated test achieved - at 5% FP level and 95% specificity level - a 34.8% sensitivity, 24.1% positive and 97.0% negative predictive value - in prediction of birth weight under 5th percentile with respect to gestation age and gender. The final model of IUGR 10, containing only statistically significant markers achieved - at 5% FP level and 95% specificity level - a 36.4% sensitivity, 32.1% positive and 95.8% negative predictive value - in prediction of birth weight under 10th percentile with respect to gestation age and gender. CONCLUSION: Our results, as well as the results of other studies, lead to the conclusion, that in spite of the multitude of biochemical markers and various options for their combination with ultrasound markers, so far it has been impossible to find a combination which would meet the following criteria: 1. high sensitivity and specificity at low false positive values and high positive and negative predictive value, 2. application as a screening method for prediction of intrauterine growth retardation and preeclampsia in unselected population of pregnant women.

[Prediction of preeclampsia using the integrated test markers]. / Predikcia preeklampsie pomocou markerov integrovaného testu.

OBJECTIVE: Author's objective was to ascertain the option for prediction of preeclampsia using the integrated test markers on the population of pregnant women in Kosice city. DESIGN: Retrospective, longitudinal, multicenter study. SETTING: 2nd Department of Obstetrics and Gynecology University of P. J. Safárik, Kosice, Slovak Republic. METHODS: The study group comprised 578 pregnant patients of 2nd Department of Obstetrics and Gynecology, the period from 01.11.2001 until 31.01.2005 for integrated test consisting of (CRL, NT, concentration of PAPP-A, uE3, hCG, AFP). Primary outcome was defined as incidence of preeclampsia during pregnancy. Data on incidence of preeclampsia was collected from birth journals and records in 578 patients retrospectivelly. In order to make use of the summary information provided by the markers we have used the method of logistic regression to identify statistically significant predictors of the binary outcome measure of preeclampsia. First we developed an equation based on all available predictors. Statistically insignificant predictors were gradually eliminated from the model (Stepwise regression) until the final model remained, for which a ROC curve was developed and values of sensitivity, specificity, positive and negative predictive values at 5% FP (false positivity) were calculated. RESULTS: The final model of preeclampsia, which is based only on statistically significant markers of integrated test achieved--at 5% FP and 95% specificity--36.4% sensitivity and 22.7% positive and 97,4 negative predictive value for prediction of preeclampsia in pregnant women. CONCLUSION: Our results, as well as the results of other studies, lead to the conclusion, that in spite of the multitude of biochemical markers and various options for their combination with ultrasound markers for prediction of preeclampsia, so far it has been impossible to find a combination which would meet the following criteria: (1) high sensitivity and specificity at low false positive values and high positive and negative predictive value, (2) application as a screening method for prediction of preeclampsia in unselected population of pregnant women.

Ultrastructure of Cell Organelles in Pre-implantation Embryos from Cows with Different Body Condition Score.

Morphology of important cell organelles (mitochondria, lipid droplets, vacuoles, inclusion bodies and apoptotic bodies) in embryos derived from cows with different body condition score (BCS) was analysed by transmission electron microscopy (TEM). Embryos were recovered on 7th day after the insemination by a standard non-surgical flushing of the uterine horns from superovulated Holstein Friesian cows with BCS 2, 3, 4 and 5. Thereafter, the good quality blastocysts were processed for TEM. The electronograms were evaluated by stereological analysis. The relative volume of lipid droplets in BCS4 and BCS5 embryos increased significantly (18.53 and 22.40%) when compared to BCS3 embryos (5.46%). In the embryos from the BCS4 or BCS5 cows, we observed different morphological patterns of mitochondria, as well as the mitochondria containing vacuoles. BCS4 and BCS5 embryo cell nuclei showed the structure typical for low transcription activity (none or very few reticular nucleoli); also dilated inter-cellular spaces were often observed in these embryos. In conclusion, differences in the ultrastructural morphology of embryos from over-conditioned cows (BCS4 and BCS5), particularly the higher lipid content in the cytoplasm, can be a marker of their low quality, and this fact can be a contributing factor to subfertility in over-conditioned cows.

Survival of mouse embryos after vitrification depending on the cooling rate of the cryoprotectant solution.

The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196 degrees C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitro culturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitro without freezing in a culturing medium in an environment consisting of 95% air and 5% CO2. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitro culturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival.

[Basal concentrations of FSH (follicle stimulating hormone) as a predictor of succes of IVF-ICSI cycle]. / Bazálne koncentrácie FSH (folikulostimulacného hormónu) ako prediktor úspesnosti IVF-ICSI cyklu.

OBJECTIVE: To evaluate the predictive value of basal follicle-stimulating hormone (FSH) levels in relation to the ovarian reserve and to establish their effect on IVF success. DESIGN: Retrospective clinical study. SETTING: 2nd Department of Gynaecology and Obstetrics, Medical Faculty and L. Pasteur University Hospital Kosice. METHODS: 155 IVF cycles were evaluated in relation to the basal FSH levels. Several parameters were regarded--duration of stimulation, gonadotrophins dosage, number of oocytes, oocyte quality, fertilization ratio, number of embryos, pregnancy rate and miscarriage rate. RESULTS: Statistically significant correlation in relation to basal FSH levels was established in case of number of oocytes and number of embryos (p < 0.05). Other parameters were not correlated to basal FSH levels. There was not established lowerpregnancy rate in patients with elevated basal FSH levels. CONCLUSIONS: On basis of this study results we can observe that basal FSH concentrations give us some information about ovarian reserve, but they neither inform about oocyte quality nor predict IVF success.

[Influence of exogenous supplementation with luteinizing hormone during controlled ovarian hyperstimulation on the results of IVF cycle]. / Vplyv exogénnej suplementácie luteinizacného hormónu pocas kontrolovanej ovariálnej hyperstimulácie na výsledky cyklu IVF.

OBJECTIVE: To evaluate the effect of exogenous luteinizing hormone in in vitro fertilization cycles with depot GnRH agonist and low residual LH levels. DESIGN: Prospective randomized clinical trial. SETTING: 2nd Department of Gynecology and Obstetrics, Medical Faculty and L. Pasteur University Hospital Kosice METHODS: In the prospective randomized clinical trial the effect of exogenous LH on outcomes of assisted reproduction was evaluated. The total number of 68 in vitro fertilization cycles in normogonadotrophic women undergoing assisted reproduction with GnRH agonist down-regulation and recombinant FSH controlled ovarian stimulation were included. In all cycles the low residual LH level in the middle of stimulation was detected. The cycles were randomized into three groups. The first group was stimulated with pure recombinant FSH. In the second group the exogenous LH activity in the form of human menotrophin was added. And in the third group the human recombinant LH was added. RESULTS: Better outcomes of assisted reproduction were detected in both groups with exogenous LH activity. But these results, except the dosage of FSH, were not statistically significant. The increase of pregnancy rate by more than one fifth in these both groups can be considered as clinically relevant. CONCLUSIONS: It can not be positively proved at the base of our results that exogenous LH activity in cycles with low residual LH level can improve outcomes of assisted reproduction. It could be appropriate in the future to select women that can profit from exogenous LH activity according to other parameters not only to the LH serum level.

[Clinical experience with a modified method of human embryo vitrification]. / Klinické skúisenosti s modifikovanou metódou vitrifikáicie l'udských embryí.

OBJECTIVE: To obtain clinical experience with a modified method of vitrification used to freeze supernumerary human embryos following in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). SETTING: Centre of Assisted Reproduction, 2nd gynaecological-obstetrical clinic of Faculty hospital of L. Pasteur and Medical Faculty of the University of P. J. Safáirik, Kosice. METHODS: A modified method of vitrification was used to freeze 215 human embryos after IVF-ICSI, obtained in 42 cycles. The embryos were frozen at 48 h after in vitro culturing. The modified method of vitrification consisted in the use of a series of solutions with increasing cryoprotectant concentrations (ethylene glycol and sucrose) up to the final concentration of 40% ethylene glycol and 1 M sucrose. A 100 microl pipetting "tip" was used as a freezing container. The embroys were thawed at room temperature and the cryoprotectant was washed out three consecutive steps with sucrose solutions of decreasing concentrations (1 M, 0.5 M, and the pure medium). The morphology of embryos was evaluated before and after thawing. After thawing, the embryos were cultured in vitro for 24 hours and only the embryos with at least one cleaved blastomere were used for transfer. RESULTS: The percentage of embryos which survived thawing and were capable of further development was 69.8% and 48%, respectively. The mean number of transferred and developing embryos was 2.9 +/- 1.4. The percentage of clinical pregnancies per thawing cycle and per transfer was 19.0% and 27.6%, respectively. Seven patients delivered 8 children. CONCLUSION: The results showed that our modification of vitrification procedure of human embryos is reliable and represents a simpler, shorter and cheaper alternative to slow-rate freezing.

[Influence of serum levels of luteinizing hormone during controlled ovarian hyperstimulation on the results of IVF cycle]. / Vplyv sérových hladín luteinizacného hormónu pocas kontrolovanej ovariálnej hyperstimulácie na výsledky cyklu IVF.

OBJECTIVE: To evaluate the effect of endogenous luteinizing hormone level in the middle of in vitro fertilization cycle with depot GnRH agonist on the outcomes of assisted reproduction. DESIGN: Prospective clinical trial. SETTING: 2nd Department of Gynecology and Obstetrics of Medical Faculty and L. Pasteur University Hospital Kosice METHODS: In the prospective clinical trial the effect of endogenous LH level during ovarian hyperstimulation with recombinant follicle stimulating hormone on outcomes of IVF was evaluated. The total number of 364 in vitro fertilization cycles in normogonadotrophic women undergoing assisted reproduction with GnRH agonist down-regulation and recombinant FSH controlled ovarian stimulation were included. A part of cycles with low residual LH levels were not included into statistical analysis, because of stimulation protocol adaptation. 315 cycles were taken into final statistical analysis. These cycles were divided into three groups according to middle stimulation LH level: under 0.5 IU/L, 0.5-2.0 IU/L and over 2.0 IU/L. RESULTS: The best results were achieved in the middle group. The differences in most of parameters were statistically significant: FSH dosage, days of stimulation, number of oocytes, estradiol level, number of embryos and fertilization rate. Pregnancy rate and early pregnancy loss differences were not significant. CONCLUSIONS: It was statistically evaluated that the best outcomes of assisted reproduction were in the group with LH level 0.5-2.0 IU/L. Correlation lines proved that the LH level 0.5 IU/L is the point under that the outcomes can worsen. It can be explained that the differences between pregnancy rates were not significant, because of low number of cycles with low residual LH levels included.