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 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.

A modified method of intracytoplasmic sperm injection without the use of polyvinylpyrrolidone.

In a controlled study we compared the outcome of intracytoplasmic sperm injection (ICSI) performed by two different methods. The oocytes from 20 patients were equally divided into two groups and injected either by conventional ICSI using polyvinylpyrrolidone (PVP) or by a modified PVP-free ICSI procedure. While in the conventional ICSI method the spermatozoon is aspirated into the injection pipette, in the modified ICSI procedure the spermatozoon is attached to the end of the narrow micropipette by aspirating its tail. The sperm head is never drawn into the pipette. Accordingly, even a fast-moving spermatozoon can be 'caught' easily. As a result of such an aspiration the spermatozoon loses its motility. Therefore, PVP is required neither to slow down the movement of the spermatozoon nor to facilitate the movement of the solution in the injection pipette. A total of 230 mature oocytes were injected by both methods and the results were analysed. No differences were observed in survival rate between the two ICSI procedures (89% and 91%, respectively). However, the proportion of normally fertilized oocytes was significantly higher after microfertilization by modified ICSI (74%) when compared with the outcome of the conventional ICSI method (62%). The frequency of abnormal fertilization was not influenced by the method of ICSI used. The cleavage rate and quality of resulting embryos were also comparable. In conclusion, we have demonstrated a modified ICSI method which does not require the use of PVP. When compared with the conventional ICSI procedure, even better fertilization rates can be achieved. The proposed ICSI modification may provide an alternative procedure for elimination of the potentially harmful effects which may be associated with conventional ICSI.