Borut Kovačič

Developmental capacity of different morphological types of day 5 human morulae and blastocysts.

After prolonged culture of human embryos, the expanded blastocysts with oval inner cell mass (ICM) and cohesive trophectoderm (TE) are preferably selected for transfer. In cycles with poor embryonic development, the selection has to be done from among suboptimal blastocysts for which no grading system exists. In this study, 1396 transferred blastocysts and morulae were classified into eight morphologic categories. The B1 category constituted the optimal blastocysts. The other categories were characterized by different deviations from optimal blastocysts: cytoplasmic fragments and necrosis in TE (B2), unexpanded blastocoele (B3), non-compact or small ICM (B4), fragments in TE and ICM (B5), up to 20% excluded blastomeres (B6), necrotic TE and ICM (B7), and more than 20% excluded cells from blastocysts (B8). The live birth rate was calculated from blastocysts with known outcome after transfer (88.9% transferred blastocysts). The birth rate declined from B1 to B8 by the same order and was: 45.2, 32.8, 26.9, 23, 17.7, 16.7, 7.7 and 1.2% respectively. Normal ICM was recognized as the most important parameter for implantation. There was a strong relation between such ordered morphology categories and implantation capacity (P < 0.0001). Such a grading system is helpful in selecting the best of all available day-5 embryos for transfer.

A prospective, randomized trial on the effect of atmospheric versus reduced oxygen concentration on the outcome of intracytoplasmic sperm injection cycles.

OBJECTIVE To assess whether the embryo cultivation at different oxygen tensions had any effect on intracytoplasmic sperm injection (ICSI) outcome. DESIGN Prospective randomized trial. SETTING University clinical center. PATIENT(S) Women undergoing ICSI (n = 647). INTERVENTION(S) Culturing of embryos, either at 6% CO(2), 5% O(2), 89% N(2), or at 6% CO(2) in air. MAIN OUTCOME MEASURE(S) The primary outcome was ongoing pregnancy rates (PR). SECONDARY OUTCOMES cumulative PRs, implantation, and embryo quality, for both treatment groups and clinical outcomes for subgroups (optimal cycles, poor responders, older women). RESULT(S) Although low oxygen resulted in a higher proportion of good day 2 embryos (0.547 +/- 0.021 vs. 0.39 +/- 0.019) and optimal blastocysts (0.162 +/- 0.199 vs. 0.083 +/- 0.128), the ongoing PRs (31.6% vs. 27.1%) and implantation rates (28.8% vs. 25.2%) were similar in both oxygen groups. Low oxygen caused a higher cumulative PR (38% vs. 28.3%) in the main group and a higher PR in the poor responder subgroup (23% vs. 9.8%) with embryo transfers performed mostly on day 3. CONCLUSION(S) The use of reduced oxygen in IVF is reasonable, irrespective of the duration of embryo culture. It improves embryo development and cumulative PR and is also recommended in poor responding cycles.

Configuration of maternal and paternal chromatin and pertaining microtubules in human oocytes failing to fertilize after intracytoplasmic sperm injection.

The microtubules and chromosomes of 180 human oocytes failing to fertilize after intracytoplasmic sperm injection were observed in order to establish how sperm chromatin and sperm astral microtubule configuration is related to the phases of oocyte cell cycle, and to find the defects in those structures causing fertilization arrest. As many as 125 (69%) oocytes were arrested at metaphase II. In one‐fourth of them, damages of the second meiotic spindle were noted. In their cytoplasm intact sperm were found in 38 (30%) cases, a swollen sperm head in 36 (29%) and prematurely condensed sperm chromosomes (G1‐PCC)—a result of active mitosis promoting factor (MPF)—in 51 (41%) cases. G1‐PCC were mostly (73%) surrounded by the bipolar paternal spindle instead of astral microtubules. A male pronucleus was never presented in metaphase II oocytes. In 19 (11%) oocytes, arrested at anaphase II, no intact sperm were found. As many as 9 (47%) oocytes contained sperm in G1‐PCC form, which proves that anaphase II oocytes mostly retain active MPF, despite oocyte activation. As many as 78% of 36 monopronucleate oocytes contained sperm, with delay in the process of sperm nucleus decondensation. Sperm in G1‐PCC form and a bipolar paternal spindle were never found in monopronucleate oocytes. From this we conclude that sperm that does not activate the oocyte may continue decondensing the chromatin, but the oocyte prevents male pronucleus formation before the female one, mostly by causing PCC in the sperm and by duplicating the sperm centrosome. Mol. Reprod. Dev. 55:197–204, 2000.

Clinical outcome of day 2 versus day 5 transfer in cycles with one or two developed embryos.

OBJECTIVE To determine whether extended culture of embryos to blastocysts has any benefit in cycles with only one or two created embryos. DESIGN Retrospective analysis of cycles comparing outcomes of day 2 and day 5 transfers. Our day 2 group was from the year 1999 and our day 5 group, from the year 2000. SETTING Assisted reproductive technology program of a teaching hospital. PATIENT(S) All patients, irrespective of age, who had developed one or two embryos. INTERVENTION(S) Stimulated IVF, intracytoplasmic sperm injection, or testicular sperm extraction and intracytoplasmic sperm injection cycles with 2-day culture in universal IVF medium (n = 133) or 5-day culture in BlastAssist media (MediCult, Jyllinge, Denmark; n = 132). MAIN OUTCOME MEASURE(S) Pregnancy, implantation, and take-home baby rates. RESULT(S) In the groups of 2-day and 5-day culture, embryo transfer was performed in 98% and in 57% of cycles, respectively. However, the total implantation rate per created embryo (18% vs. 18%), the pregnancy rate per cycle (23% vs. 21%), and the take-home baby rate (69.4% vs. 71.4%) did not differ between the day 2 and day 5 groups. CONCLUSION(S) Extended culture of embryos does not improve or decrease their capacity for implantation but only allows for better selection and is therefore not necessary in cycles with fewer than three embryos.

Comparable effectiveness using flexible single-dose GnRH antagonist (cetrorelix) and single-dose long GnRH agonist (goserelin) protocol for IVF cycles--a prospective, randomized study.

This prospective randomized study compared the effectiveness of a flexible single-dose gonadotrophin-releasing hormone (GnRH) antagonist (cetrorelix) and a single-dose long GnRH agonist (goserelin) protocol for ovarian stimulation in IVF/intracytoplasmic sperm injection (ICSI) cycles. All patients from the waiting list were successively included in the study, pre-programmed with an oral contraceptive, and randomized into goserelin and cetrorelix groups. Depending on the date on which their menstrual period started, patients took oral contraceptives for one or two cycles. Ultimately, 236 patients in the first group received a single dose of depot preparation of goserelin and 224 patients received a single 3 mg dose of cetrorelix in the late follicular phase, when the mean follicle diameter exceeded 12 mm. The mean number of ampoules of FSH and the duration of stimulation was statistically significantly lower in the cetrorelix group than in the goserelin group (25.9 versus 34.5, and 9.6 versus 12.2 days, P < 0.01). The mean number of oocytes retrieved was similar (6.7 +/- 4.5 versus 7.2 +/- 4.6, NS). Similar results were observed in fertilization rates, blastulation rates and blastocyst transfer rates in both groups. Clinical pregnancy and delivery rates per cycle were higher in the goserelin group (34.3 and 30.1%) than in the cetrorelix group (31.9 and 28.3%), but the differences were not statistically significant. The flexible single-dose GnRH antagonist protocol is an advantageous alternative to the long GnRH agonist protocol, with similar efficacy, shorter duration, a significant reduction in the number of FSH ampoules used and without the menopause-like effects of the GnRH antagonist.

Prediction of human blastocyst development from morulas with delayed and/or incomplete compaction

OBJECTIVE To determine the influence of delayed compaction and fragmentation on the developmental capacity of morulas. DESIGN Prospective study. SETTING University IVF center. PATIENT(S) Intracytoplasmic sperm injection (ICSI) cycles with compact embryos on day 4 or day 5. INTERVENTION(S) The embryos were divided into day 4 (n = 329) and day 5 (n = 256) morulas and graded I, II, or III, according to the percentage of fragmentation (<5%, 5%-20%, or >20%). The embryos were measured using Cronus3 software. MAIN OUTCOME MEASUREMENT(S) Blastocyst development rate, blastocoel expansion rate, and optimal blastocyst rate. In an optimal blastocyst: surface area, trophectoderm cell number, inner cell mass (ICM) surface area, ICM volume and ICM shape. RESULT(S) Day 4 morulas in classes I-III developed into optimal blastocysts in 57.4%, 50%, and 35.6% of the total, respectively, and day 5 morulas in classes I-III in 43.3%, 29.1%, and 13.6% of the total, respectively. A negative association was identified between the amount of morula fragmentation, the blastocyst ICM size, and the number of trophectoderm cells. A delay of 1 day in compaction was associated with a reduced ICM volume. CONCLUSION(S) The measurement of compaction timing and cytoplasmic loss in morulas assists in predicting their ability to develop into optimal blastocysts.

Importance of Blastocyst Morphology in Selection for Transfer

Prolonged cultivation of embryos to the blastocyst stage has become a routine practice in the human in vitro fertilization program (IVF) since 1999, when the first commercial sequential media were developed. The culture systems have been improved many times and today most of the blastocyst culture media enable the embryos to reach the blastocyst stage in more than 50% of cases (Gardner et al., 1998; Kovacic et al., 2004). The advantages of blastocyst culture are in the possibilities for selection of embryos that have an activated genome (Braude et al., 1988), higher predictive values for implantation on the basis of their morphological appearance as compared with earlier embryos (Gardner and Schoolcraft, 1999; Kovacic et al., 2004) and in a reduction in the number of transferred embryos without compromising pregnancy rate (Gardner et al., 2000a). Blastocyst is also a stage that is better synchronized with endometrial receptivity for its implantation (Croxatto et al., 1978; Gardner et al., 2000c). By replacement of embryos in the blastocyst stage, their exposure to hyperstimulated milieu and consequent endometrial contractions, which could be fatal for them, is significantly shortened (Lesny et al., 1998). Blastocysts also contain a larger number of cells than early stage embryos and should therefore have a better possibility of survival of cryopreservation (Veeck, 2003).

Value of the serum estradiol level on the day of human chorionic gonadotropin injection and on the day after in predicting the outcome in natural in vitro fertilization/intracytoplasmic sperm injection cycles

OBJECTIVE To predict the risk of premature ovulation and possibility of pregnancy in natural IVF/ICSI cycles on the basis of the estradiol (E2) level on the day of hCG injection and on the day after. DESIGN A prospective study. SETTING Hospital research program. PATIENT(S) One hundred sixty-four women undergoing 305 IVF/ICSI natural cycles. INTERVENTION(S) Serum E2 levels were measured approximately 12 h before (day 0) and 12-17 h after hCG administration (day 1). MAIN OUTCOME MEASURE(S) E2 levels on day 0 and day 1, the ratio of the day 1 to day 0 levels, and cycle outcome. RESULT(S) In cycles with premature ovulation and in conception cycles, the average E2 level on day 0 was statistically significantly higher than in other cycles, whereas the E2 ratio was statistically significantly lower. Multiple logistic regression was used to determine the connection of the E2 level on day 0 (B0 = -0.742, B = 2.147, P =.01) and the E2 ratio (B0 = -0.742, B = -3.135, P<.001) with premature ovulation. Only the E2 ratio (B0 = 0.659, B = -2.209, P =.0068) was significantly connected with conception. CONCLUSION(S) In predicting the outcome of natural IVF/ICSI cycles, the importance lies not in the E2 level on the day of hCG administration or on the day after, but rather in the E2 ratio.

Artificial blastocoel collapse of human blastocysts before vitrification and its effect on re-expansion after warming – a prospective observational study using time-lapse microscopy

Vitrified human blastocysts show varied re-expansion capacity after warming. This prospective observational study compared behaviour of artificially collapsed blastocysts (study group patients, n = 69) to that of blastocysts that were vitrified without artificial collapse (control group patients, n = 72). Warmed blastocysts were monitored by time-lapse microscopy and blastocoel re-expansion speed and growth patterns compared between study and control groups. These parameters were also retrospectively compared between blastocysts that resulted in live birth and those that failed. Artificially collapsed blastocysts re-expanded on average 15.01 µm2/min faster than control blastocysts (P = 0.0013). Warmed blastocysts expressed four different patterns of blastocoel growth. The pattern showing contractions at the end of culture was observed to have a lower prevalence in control blastocysts, which coincided with the lower incidence of hatching in this group. Re-expansion speed and prevalence of growth patterns were comparable between blastocysts that did and did not result in a live birth. This was seen in the study and control groups. Despite faster re-expansion and different growth patterns of artificially collapsed blastocysts, live birth rate did not differ between groups. However, this result should be interpreted with caution due to the small sample size and high risk of bias.

Comparison of embryo transfer strategies and assisted reproduction outcome in Slovenian and cross-border patients

A reduction in the number of embryos transferred is the most important step in decreasing multiple gestation rates after medically assisted reproduction. Slovenia has implemented insurance company regulations that regulate single-embryo transfer in selected good-prognosis couples. The aim of the present study was to evaluate its effects on the Slovenian population compared with cross-border patients, who are not affected by the insurance company policy. Ultimately, 2403 couples undergoing IVF or intracytoplasmic sperm injection were included in the retrospective analysis. Patients were classified according to their origin. The decision about the number of embryos transferred and the treatment success were evaluated. The implementation of the policy favouring single-embryo transfer resulted in a significant decrease in the twin birth rate in Slovenian patients (24.4% before policy versus 6.7% after policy implementation, P<0.001). Although in cross-border patients twin birth rates have declined through the study period, they remained significantly higher compared with Slovenian patients (23.1% versus 6.7%, P<0.001). The data demonstrate that insurance company policies favouring single-embryo transfer are an effective tool in decreasing multiple gestation rates. Similar mechanisms should be implemented in the cross-border patient population.