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.

[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.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate.

The aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8-12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8-12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

[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 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.

[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.

[Syndrome of androgenic insensitivity]. / Syndróm androgénnej insenzitivity.

OBJECTIVE: A survey of present knowledge on the syndrome of androgenic intensitivity. TYPE OF STUDY: Survey of literature. SETTING: 2nd Department of Obstetrics and Gynaecology, P. J. Safarik University and L. Pasteur University Hospital, Kosice, Slovak Republic. CONCLUSIONS: The complete androgenic insensitivity is the third most frequent cause of primary amenorrhea. It is a hereditary disease associated with chromosome X. The occurrence of mild forms of androgenic insensitivity in the population is not known, but it probably represents large group of patients with azoospermia.