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Background: Multiple pregnancies are undesired consequence of infertility treatment by in vitro fertilization (IVF). In 2008 the Health Insurance Institute of Slovenia strove for lowering the number of multiple pregnancies. It widened the rights of patients by reimbursement of two additional IVF cycles, having totally six cycles reimbursed. But in women younger than 36, only one top-quality embryo in the first two IVF cycles has to be transferred. The aim of the study was to assess, how the new approach, including also the transfers of frozen-thawed embryos, influenced the IVF outcomes. Methods: In year 2008, the transfer of one optimal embryo had to be done in 47.4 % (287/650) of IVF cycles. The criteria for optimality of early cleavage stage embryos were: at least 6 blastomeres and less than 20 % of fragmentation; and for blastocysts: expanded blastocoel and oval inner-cell-mass. Embryos were cultured to blastocysts in 79.4 % (228/287) of cycles. The surplus blastocysts were vitrified in 172 cycles. The transfer of devitrified blastocysts was performed in 82 patients, which did not conceive in fresh cycle. The pregnancy rate and twins rate after fresh transfers and after transfers of devitrified embryos were analyzed. Results: Single embryo or blastocyst transfer was done in 58.9 % of women. In the remaining 41.1 % of cycles the embryos were morphologically not optimal and we decided for double embryo or blastocyst transfer. In the group in which a single and mostly the only embryo was trans- ferred, only 17.2 % of women became pregnant. After the transfer of elective single blastocyst, 51.4 % of women conceived. After double blastocyst transfer, 40.9 % of patients conceived and 50 % of them had twins. The replacement of devitrified blastocysts was successful in 23.2 %. The cumulative pregnancy rate in group of cycles with single fresh blastocyst transfer, followed by transfers of devitrified blastocysts, was 62.9 %. The cumulative pregnancy and multiple pregnancy rate in the whole group was 50.9 % and 14.4 %, respectively. Conclusions: By the policy of reduction the number of transferred embryos into the uterus in IVF pro- gramme, the multiple pregnancy rate decreased from previously 40 % down to 14.4 %.

Background: We have been sending Slovenian data on biomedically assisted procreation (BMAP) to European IVF Monitoring (EIM) registry since 2001. In order to see our place in Europe, we compare our data for 2005 to the last available EIM – 2005 report data. Methods: The summaries of all three Slovenian centres sent to EIM 2005 report : 3292 BMAP cycles (730 IVF cycles, 1495 ICSI cycles, 584 FER cycles, 470 IUI cycles) in a population of 2.003 358 inhabitants were compared to the results in 418 111 BMAP cycles reported from differ- ent European countries. Results: The calculated cumulative live birth rate per fresh IVF /ICSI cycle in Slovenia was 27.7 % . The availability in Slovenia corresponded to 1.643 assisted reproduction cycles and 1402 IVF, ICSI or FER cycles per million inhabitants .The percentage of infants born after BMAP was 3.9 % . In 2005 there were 0.4 % births of triplets and 18.9 % births of twins. Conclusions: The results in 2005 reflect our law which permits the fertilisation of all oocytes and the cryopreservation of vital remaining embryos The calculated cumulative live birth rate per fresh IVF /ICSI cycle was comparable to the best rated European countries. Considering the availability of BMAP per million inhabitants, Slovenia was on the 7th place in Europe. The percentage of infants born after BMAP was the highest in Europe . Slovenia was between 12 European countries with a successful PGD programme to prevent severe hereditary diseases.

Background. Spontaneous miscarriage isn’t an unexpected complication either in early spontaneous or in ART pregnancy. Previous studies showed that incidence of SM was slightly increased in ART pregnancies in comparison with spontaneous ones after adjusting for maternal age and previous SM. Our objective was to examine the relationship between SM and embryo quality after transfer of one or two blastocysts. Materials and methods. The total of 1433 stimulated IVF and ICSI cycles achieved in our center in the period from 2001 to 2002 after SBT or DBT were retrospectively analyzed. Of these, in the final analysis we included only cases with positive βhCG for which complete data on pregnancy outcome were available – 418 cycles in total. Results. The mean age of patients was 32.2 ± 4.5 years. IVF was performed in 133 cases and ICSI was performed in 285 cases, SBT in 69 and DBT in 349. After SBT, SM rate was 11.6 % and after DBT it was 12.0 %. In SBT group we didn’t find significant relationship between embryo quality and SM (logistic regression: c2 = 0.88; p > 0.05). In this groups, using standard statistical tests, we also couldn’t find significant difference in SM rate between subgroups where optimal or suboptimal quality blastocyst was transferred (Yates corrected c2 test: p > 0.05, Student’s t-test: p > 0.05). In DBT group, we found a strong relationship between embryo quality and SM (logistic regression: c2 = 10.12; p < 0.01). After standard analysis, we confirmed significant difference between subgroups with different combinations of blastocyst quality: after transfer of both optimal blastocysts SM rate was 8.5 %, after transfer of optimal and suboptimal blastocyst SM rate was 10.1 % and after transfer of both suboptimal blastocyst SM rate was 25.4 % (Kruskal-Wallis test: p < 0.001; one-way ANOVA: p < 0.001). We confirmed strong relationship between age of the patients and SM (logistic regression: c2 = 14.57; p < 0.0001). Conclusions. In our study SM rate was 11.9 % which was lower than in previous reports; it was even at the lower limit of expected SM rate in general population. This discrepancy was probably the consequence of longer selection period of blastocysts in in vitro conditions. We didn’t find a significant relationship between SM and blastocyst quality after SBT, which was probably due to the small sample size. A strong relationship between SM and blastocyst quality after DBT was proved. We also confirmed a strong relationship between age of the patients and SM.

Background. Vitrification is a new, simpler and more rational way of cryopreservation of redundant embryos in in vitro fertilisation. The purpose of this study was to introduce vitrification of blastocysts in clinical practice and compared two commercial cryoprotectants. Methods. We started vitrifying blastocysts in 2008. Two commercial vitrification protocols were used, Vitrification Cooling/Warming (MediCult, Denmark) – (MC), and Vitrification Freeze/Thaw (IrvineScientific, ZDA) – (IS). IS medium includes two cryoprotectants: dimethylsulfoxide (DMSO) and ethylene glycol, meanwhile MC medium contains only ethylene glycol. Among 1030 in vitro fertilizated cycles, we preserved redundant blastocysts in 404 (39.2 %) cycles in liquid nitrogen. At the begining MC vitrification protocol was used in 253 cycles and later IS protocol in 151 cycles. 127 transfers (248 devitrified blastocysts) were performed using MC protocol and 71 transfers (124 blastocysts) using IS protocol. We compared the survival rate of blastocysts, rate of clinical and ongoing pregnancies and miscarriages. Cycles with at least one optimal blastocyst were evaluated separately. Results. Survival rate of vitrified blastocysts by IS protocol was statistically significantly higher than survival rate by MC protocol (88.7 % or 77 %; P = 0.0103). Implantation rate of devitrified blastocysts by IS and MC protocol are comparable (22.7 % or 17.8 %). The rate of miscarriages does not statistically differ between the protocols (18.2 % or 16.7 %). There are no statistically significant differences between IS and MC protocols in clinical (31.8 % or 24 %) and ongoing pregnancies (26.1 % or 20 %). Pregnancy rate in cycles with at least one optimal devitrified blastocyst is 45.7 % by IS protocol and 36.4 % by MC protocol. In the same cycles, the rate of miscarriages is 5.7 % by IS and 4.5 % by MC protocol. Conclusions. Higher values in all parameters were by IS media, even though the differences were not statistically significant because of low number of cycles. The only statistically significant difference between protocols was in survival rate in favor of IS protocol. The study continues.

Background: Occurrence of early miscarriage in pregnancies accomplished by in vitro fertilisation procedures was studied, almost exclusively, in women in whom cleavage stage embryos were transferred. The primary objective of our retrospective study was to identify the factors predicting early miscarriage following the transfer of one or two blastocysts. Materials and Methods: Using the multivariate logistic regression model, we analyzed the parameters of 1020 sequential IVF/ICSI cycles with the transfer of one or two blastocysts, in which positive levels of βhCG were observed. Age of the patient, fertilization method, number of retrieved oocytes, number of developed, frozen and transferred blastocysts and their quality were analyzed as possible predictors for early spontaneous miscarriage. Results: The overall frequency of biochemical pregnancies in the observed group of patients was 6.6 % (67/1020). Biochemical pregnancies occurred somewhat more frequently following the transfer of one, rather than two blastocysts (9.05 % (23/254) vs. 5.8 % (44/763)). The rate of early clinical miscarriage was 13.3 % (136/1020), with the rate of miscarriage higher following the transfer of one as opposed to two blastocysts (15.3 % (39/254) vs. 12.7(97/763)). If biochemical pregnancies and early clinical miscarriages were observed together, the rate of early pregnancy losses in the overall sample studied was 19.9 % (203/1020), following the transfer of one blastocyst 24.4 % (62/254) and following the transfer of two 18.4 % (141/763). Using the multivariate logistic regression, we demonstrated that statistically significant predictors for early spontaneous miscarriages were the patient’s age (OR = 1.098; 95 % CI 1.057–1.140, P < 0.001), the number of blastocysts transferred (OR = 0.592; 95 % CI 0.412–0.851, P = 0.005), the quality of blastocysts transferred (OR=0.666; 95 % CI 0.468–0.949; P = 0.024), as well as the number of blastocysts frozen (OR = 0.912; 95 % CI 0.832–0.999; P = 0.048). Conclusion: The transfer of optimal quality blastocyst(s) and transfer of two blastocysts were associated with a reduced rate of spontaneous miscarriages in pregnancies achieved by ART.

Background: Pregnancy rate after the transfer of frozen / thawed human blastocysts has been lower in comparison with the pregnancy rate in in vitro fertilization (IVF) cycles with fresh embryos. There is a need for the optimization of freezing protocols. Conclusions: All freezing protocols using a simple freezing medium were more effective than protocol with a complex medium. Their application in IVF patients with frozen surplus blastocysts (these patients delivered in fresh cycles in 51.4 %) increases the cumulative delivery rate by an additional 15.2 % with every thawing cycle. The morphology of blastocysts and cycle preparation for transfer had the strongest influence on the success of freezing programme.

Background: The aim of our study was to ascertain if the acrosome functionality differs between various morphological types of spermatozoa in human. Methods: Fourteen ejaculates were resuspended in the culture medium with the aim to wash the spermatozoa from the seminal plasma and to induce sperm capacitation. Only motile spermatozoa were isolated from the washed sperm. Each sample was divided into two aliquots. The Ca2+ ionophore was added to the first aliquot to induce sperm acrosome reaction. The second aliquot served as control for evaluating the spontaneous activation of the acrosome. After the treatment, the spermatozoa were fixed in ethanol and their acrosomes were stained with FITC fluorochrome. The samples were observed under an immunofluorescence microscope. The proportion of spermatozoa with functional acrosome (ARIC – acrosome reaction to ionophore challenge) was calculated for each morphological type as a difference between the proportions of spermatozoa with induced and spontaneous acrosome reaction. Results: The mean proportion of spontaneous AR in the analysed sperm samples was 1.6 ± 1.8 %, induced AR 51.8 ± 18.3 % and ARIC 50.1 ± 19.1 %, respectively. Various morphological types of sperm differed significantly from each other by the proportion of successfully induced acrosome reactions (χ2 = 87.62, p < 0.001) and also by the proportion of functional acrosomes (ARIC; χ2 = 101.45, p0.001). All types of morphologically nonoptimal spermatozoa (except the types with round heads and nuclear abnormalities) had a significantly lower percentage of functional acrosomes (p < 0.05) in comparison with morphologically optimal type. Conclusions: The acrosome functionality is related to morphological characteristics of spermatozoa. Optimal morphology types have the highest, while elongated and amorphous types of spermatozoa have the lowest proportion of normal acrosome.

Objective: In many stimulated infertility treatment cycles some oocytes are collected immature and can be matured in vitro (IVM). However, their safe clinical use is questionable because of their apparently low morphologic and genetic quality. We investigated the influence of the IVM duration on the type and frequency of chromosome abnormalities in germinal vesicle stage (GV) oocytes from stimulated cycles. Design: In-vitro maturation of GV oocytes (from stimulated cycles) for 24 (Group-A) or 36 (Group-B) hours and subsequent fluorescent in situ hybridization analysis (FISH) of chromosomes 13, 16, 18, 21 and 22. Results: After maturation, chromosomes were undoubtedly analyzable in 102 oocyte-first polar body pairs. Aneuploidy rates in both groups did not differ statistically. However, within Group-B a significantly higher rate of hyperhaploidy over hypohaploidy was observed. Also, there was a significantly higher frequency of disomy than nullisomy in both groups, and the aneuploidy rate of chromosomes 18 and 22 was significantly increased in Group-B. Conclusion: The observed preferential excess over the loss of genetic material and the increase in chromosome non-disjunction in the oocytes from Group-B, indicate a negative influence of prolonged IVM on chromosome segregation or conversely, suggests that GV oocytes which attain maturity later, possess more intrinsic abnormalities that result in aneuploidy.Design: In-vitro maturation of GV oocytes (from stimulated cycles) for 24 (Group-A) or 36 (Group-B) hours and subsequent fluorescent in situ hybridization analysis (FISH) of chromosomes 13, 16, 18, 21 and 22. Results: After maturation, chromosomes were undoubtedly analyzable in 102 oocyte-first polar body pairs. Aneuploidy rates in both groups did not differ statistically. However, within Group-B a significantly higher rate of hyperhaploidy over hypohaploidy was observed. Also, there was a significantly higher frequency of disomy than nullisomy in both groups, and the aneuploidy rate of chromosomes 18 and 22 was significantly increased in Group-B. Conclusion: The observed preferential excess over the loss of genetic material and the increase in chromosome non-disjunction in the oocytes from Group-B, indicate a negative influence of prolonged IVM on chromosome segregation or conversely, suggests that GV oocytes which attain maturity later, possess more intrinsic abnormalities that result in aneuploidy.

Background: There is a lack of clinical trials on the success of embryo culture in different commercial culture systems in the programme of in vitro fertilization (IVF). In our prospective study, embryo development was compared in two sequential media. Methods: The oocytes from each of 62 patients, treated by IVF/ICSI, were distributed into two groups for 5-day culture in the BlastAssist® System (Medicult, Denmark) (Group 1, n = 421) and in the GIII Series™ (Vitrolife, Sweden) (Group 2, n = 415) media. The dynamics of embryo development, implantation and pregnancy rates were compared between groups. Results: The differences between Groups 1 and 2 were first noted on day 3 when GIII yielded more fast-cleaving (> 6 cells) nonfragmented embryos (31.3 % vs. 44.4 %, P < 0.01). On day 4 however, a larger number of compact embryos was found in Group 1 (42.2 % vs. 28.7 %, P < 0.005). The percentage of blastocysts or compact morulae on day 5 was the same in both groups (59.6 % vs. 55.6 %). But expanded blastocysts of optimal quality with oval inner-cell-mass developed more frequently in Group 1 (29.2 % vs. 17.7 %, P < 0.05), and therefore more transfers were performed exclusively with Group 1 embryos (28 vs. 17). Pregnancy and implantation rates were the same in both groups (53.6 % vs. 52.9 % and 34.7 % vs. 34.2 %, respectively). Conclusions: Although the GIII Series™ yields more fast-cleaving embryos on day 3, embryo compaction (morula) and cavitation (blastocoel) start earlier in the BlastAssist® System media. BlastAssist® System therefore allows easier selection of embryos for transfer on day 5.

Background. During the period between 1998 and 2002 Department for reproductive medicine, Maribor Teaching Hospital performed 31.1% of all cycles of assisted reproduction (ART) in Slovenia.Methods. Three different methods of ART (in vitro fertilization – IVF, intracytoplasmic sperm injection with ejaculated sperm – ICSI and testicular sperm TESA/TESE) were compared. Comparison of clinical results was also made between the period when up to 4 embrios were transferred and the period when up to 2 embrios were transferred.Results. During the five year period, pregnancy rate per started cycle was between 29.6% in 1998 and 36.4% in 2002. During the whole period, delivery rate per started cycle was higher than the mean average reported for European centers for the year 1999. The observed values were higher with the tendeny to slightly increase from 27.4% in 1998 to 30.8% in 2001.Conclusions. Clinical results of ART programme at Maribor Clinic for reproductive medicine in the observed five year period over the period 1998–2002 were superior to the last results published by the European register of ART.

Background. Slovene data on iatrogenic multiple pregnancy after IVF/ICSI procedures are presented. The influence of elective transfer of two high-quality embryos on the incidence of iatrogenic multiple pregnancy were evaluated.Methods. Retrospective analysis of the Slovene IVF/ICSI data in the period 1983–2000.Results. In 17436 IVF/ICSI cycles 3068 pregnancies were achieved. Of the 2308 deliveries 369 (16%) were twins and 49 (2%) triplets. Of the 2827 newborns 738 (26%) were twins and 148 (5%) triplets. With elective transfer of two high quality embryos in 1999 and 2000 in two of the three Slovene centres for assisted reproduction, the pregnancy rate per transfer increased (32%), triplet deliveries decreased to 2/492 deliveries (0.4%) (p < 0.000), and twin deliveries remained unchanged 111/492 deliveries (23%).Conclusions. After elective transfer of two high-quality embryos and cryopreservation of surplus high-quality embryos in two of the three Slovene centres in the years 1999 and 2000, the problem of iatrogenic triple and higher rate multiple pregnancies has been successfully resolved, but the twin delivery rate still remains too high. Only by elective transfer of a single high-quality embryo the high multiple pregnancy rate after IVF/ICSI procedures could be avoided.

Background. The blastocysts, obtained from in vitro fertilization, have different morphologic characteristics. The aim of the study is to investigate the effect of morphology of transferred blastocysts on pregnancy and multiple pregnancy rate.Methods. The quality of blastocysts was evaluated by four parameters: amount of embryo mass transformed into the blastocyst, blastocoel expansion, shape of inner cell mass and proliferation of trophectoderm cells. In the first group the blastocysts with four optimal parameters were arranged. The second group was formed from blastocysts with one nonoptimal parameter and the third group from blastocysts with more nonoptimal parameters. The results of 175 transfers of two blastocysts from first group, 65 transfers of two blastocysts from second and 20 transfers of two blastocysts from third group were analysed retrospectively.Results. Implantation rate was the highest in the first group of transfers and differed significantly in comparison to second and third group (54.7% vs. 31.5% vs. 7.5%; p < 0.05). Most pregnancies were achieved after the transfer of two optimal blastocysts in comparison to other groups of transfers (62.3% vs. 40% vs. 15%; p < 0.05). Multiple pregnancy rate was 56.9% in the first group, 23% in the second group, while in the third group of transfers there was no multiple pregnancy.Conclusions. By selecting the blastocysts for transfer into the uterus it is advantageous that blastocsyt morphology characteristics are taken into the consideration. Using our blastocyst scoring system it is possible to select out the high quality blastocysts. The probability of implantation by them is more than 50%, for that reason only one such blastocyst could be transferred without any effect on pregnancy rate.

Background. Among oocytes obtained during aspiration of preovulatory ovarian follicles in hormonally stimulated cycles, we ascertained the percentage of immature oocytes with the nucleus in the metaphase (M I oocytes) or even in the prophase (GV oocytes) of the first meiotic division and their capacity to mature in vitro in a simple medium without hormonal supplements.Methods. In 818 women, stimulated by gonadotropin releasing hormone agonist (GnRHa) and gonadotropins, aspiration of preovulatory size follicles yielded 4972 oocytes. From these we denuded cells of cumulus oophorus and corona, meiotic maturity was evaluated under a microscope. Cells in the metaphase of the second meiotic division (M II oocytes) and those maturing after 5 hours were used clinically in the intracytoplasmic sperm injection (ICSI) procedure. Immature cells were left in the simple medium. The degree of their nuclear maturity was evaluated after one and after two days of culture. In vitro maturation was clinically used also in 14 cycles with no mature oocytes.Results. Among 4731 oocytes with denuded corona and cumulus, 4199 (88.8%) were mature M II oocytes, 295 (6.2%) immature M I oocytes and 237 (5%) immature GV oocytes. Under in vitro conditions, 68.7% (90/131) GV oocytes attained maturity. Among M I oocytes, 63.6% (136/214) cells matured already after 5 hours and 26.6% (57/214) until the next day. In all 14 women with only immature oocytes, the embryos for embryotransfer were obtained after in vitro maturation and ICSI procedure. The result was four pregnancies and two deliveries.Conclusions. Immature oocytes, obtained in hormonally stimulated cycles, may become clinically applicable if left to mature in vitro in a simple medium without supplementation of growth factors and hormones.

Background. The aim of our study was to asses the success of the method of intrauterine insemination (IUI) and ovulation stimulation with clomiphene citrat compared to other techniques of assisted reproductive technology (AR).Methods. In retrograde, 533 cycles of IUI were analysed in 204 infertile couples. The success of IUI was compared according to the most common indications, the patients age and the successive cycle of IUI. The different values of the variables were compared using Pearson’s Chi-square test.Results. The overall pregnancy rate was 6.7%. The success was 2.35% in couples with a tubal cause of infertility (with one side tubal pathology), 12.12% in couples with male infertility only and 7.33% in couples with idiopathic infertility. The differences in the results of IUI between the age groups of female patients were not statistically significant.Conclusions. IUI is painless, noninvasive and less expensive method of AR and has therefore an important role for the treatment of infertility. IUI is suitable mainly for idiopathic infertility, some forms of male infertility and oligo – anovulations and if there are no contraindications we recommend it as treatment before IVF program.

Background. The aim of this study was to analyse the results of natural IVF/ICSI cycles and to find out which factors have an impact on the success of the cycle.Methods. 340 natural IVF/ICSI cycles in 162 infertile couples were analysed within a retrospective study. Cycles were divided in groups according to the cause of infertility and women’s age and pregnancy rates were compared. A t-test was used to obtain the different values of the variables between the cycles with pregnancies and cycles without pregnancies.Results. A premature ovulation occurred in 32 cases (9.41%), an oocyte was obtained in 244 cycles (79.2%), fertilization occurred in 175 cases (71.72%) and pregnancy in 41 cycles (the pregnancy rate per puncture = 13.31, per transfer = 25.31). The highest pregnancy rate per puncture was achieved in patients with idiopathic infertility (17.91) followed by male infertility with ICSI procedure (12.67) and female infertility (11.40%). Pregnancy rate per cycle was 13.42 in women below the age of 38 years, 8.11 in women between 38 and 40 years, and no pregnancy were achieved in women 40 and over years of age. There are statistically significant higher values of estradiol measured on the day of the HCG injection and later application of HCG in cycles that resulted in pregnancy than those which did not result in a pregnancy. There were no differences in the duration of infertility, the patients’ age, the average and maximum diameter of the follicle and the thickness of the endometrium on the day of the HCG injection as well as between the basal values of FSH and LH between the two groups.Conclusions. Satisfactory results are obtained with the natural IVF/ICSI cycles independent from the cause of infertility. A higher pregnancy rate per puncture is expected with higher levels of estradiol, a later application of HCG and in women below the age of 38 years.

Background. The aim of our study was to analyse the results of ICSI procedure with respect to the number of embryos transferred 3 or 5 days after fertilization.Methods. The outcome of 1498 day 3 embryo transfers and 364 day 5 embryo (blastocyst) transfers was analysed. The percentage of implanted embryos was compared with regard to the number and quality of embryos transferred. Differences in pregnancy rate between the groups were established with the chi-square test.Results. Implantation occurred in 21.3% (320/1498) of embryos transferred to the uterus on day 3 post fertilization. In the transfer of one, two, three or four embryos, the pregnancy rate increased from 20.0% to 22.1%, 54.5% and 59.0% respectively. In the group with one or two embryos for transfer, the pregnancy rate did not change statistically significantly in correlation with embryo quality. The twin pregnancy rate after the transfer of two good quality embryos was 66.6% (8/12). The presence of at least one good quality embryo among three or four embryos transferred had a statistically significant effect on the singleton pregnancy rate (37.8% and 41.3%). The rate of multiple pregnancies remained low (18.1% and 16.6%). Increasing the percentage of good quality embryos for transfer to three or four had a greater effect on the percentage of multiple implantations (43.1% and 54.5%) than on the pregnancy rate (55.5% and 75.8%). After the transfer of 5-day-old embryos (blastocysts), implantation occurred in 37.3% (136/364) of embryos. After the transfer of one blastocyst, the pregnancy rate was 36.2% (21/58), after the transfer of two blastocysts it was 55.7% (98/176). The twin pregnancy rate after the transfer of two blastocysts amounted to 38.7%.Conclusions. The transfer of blastocyst stage embryos results in a statistically significant increase in the success of the ICSI procedure. In the transfer of two blastocysts, the rate of twin pregnancies remains equally high as in the transfer of three good quality embryos on the third post fertilization day.