A prospective, randomized study comparing day 2 and day 3 embryo transfer in human IVF.

It is believed that delayed transfer of embryos after IVF allows for a better selection of good quality embryos. Hence, the number of embryos and all other prognostic factors being equal, transfer of day 3 embryos should be associated with higher implantation and pregnancy rates than transfer of day 2 embryos. To investigate this hypothesis, a prospective randomized study was carried out to compare implantation and pregnancy rates between day 2 and day 3 transfers. The relationship between the embryo quality score of day 2 and day 3 embryos and their respective implantation rates was also analysed. In a 2 year period all patients undergoing infertility treatment and in whom at least seven normally fertilized oocytes were obtained were included in the study. A minimization procedure was performed taking into account the patient's age and the method of fertilization (IVF or intracytoplasmic sperm injection). By using a uniform policy of embryo transfer, the number of embryos transferred was similar in both groups. The outcome parameters were embryo quality, implantation and pregnancy rates. No difference was observed in implantation and pregnancy rates between transfers on day 2 versus day 3 (23.8 versus 23.8% and 47.9 versus 46.8% respectively). The incidence of embryos of moderate to poor quality was higher in embryos cultured for 3 days compared with those cultured for 2 days. It is concluded that the outcomes of embryo transfer in terms of implantation and pregnancy rates are comparable for day 2 and day 3 embryos, although the overall embryo quality score decreases when embryos are kept in culture till day 3.

Effect of post-ovulatory age and calcium in the injection medium on the male pronucleus formation and metaphase entry following injection of human spermatozoa into golden hamster oocytes.

The occurrence of parthenogenetic activation is a major hurdle in obtaining sperm chromosome metaphases after heterospecific intracytoplasmic sperm injection (ICSI) of golden hamster oocytes with human spermatozoa. We addressed two potential contributors to parthenogenetic activation namely, post-ovulatory age of the oocyte and Ca2+ content of the injection medium. In serial experiments, hamster oocytes were retrieved at 11.5, 13, 16 and 21 h after the ovulatory dose of human chorionic gonadotrophin (HCG) and microinjected with human spermatozoa suspended alternately in a regular (1.9 mM Ca2+) or a Ca2+-free medium. A progressive decrease in the rates of male pronucleus (MPN) formation and metaphase entry and increase in the rates of parthenogenetic activation without male pronucleus occurred with increasing post-ovulatory age. The favourable influence of Ca2+-free injection medium on the mean rates of MPN and metaphase entry was restricted to the relatively older oocytes (MPN 16 h: 49.5 versus 32.3%, P< 0.008; 21 h: 22.2 versus 11.1%, P< 0.001; metaphase entry 16 h: 36.8 versus 25.1%, P< 0.02; 21 h: 13.3 versus 5.2%, P< 0.01 in the Ca2+-free and regular groups respectively). Our data confirm the increased activation sensitivity with post-ovulatory ageing and its adverse influence on the MPN formation and metaphase entry after heterospecific ICSI of hamster oocytes.

In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium.

In-vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotrophin stimulation for in-vitro fertilization (IVF). The pregnancy rates from oocytes matured in vitro are much lower than those of in-vivo stimulation cycles indicating that optimization of IVM remains a challenge. Therefore, we investigated the effect of supplementation of the medium with gonadotrophins, oestradiol and epidermal growth factor (EGF) and the effect of retaining or removing the cumulus cells on nuclear and cytoplasmic maturation of immature oocytes. Human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation for intracytoplasmic sperm injection (ICSI) were cultured in a complex defined medium either supplemented with gonadotrophins, oestradiol and physiological concentrations of EGF (2 ng/ml) or gonadotrophins and oestradiol alone. The cumulus cells were either removed or kept intact. In GV stage oocytes cultured without cumulus (group I) significantly more oocytes reached the metaphase II (MII) stage at 30 h in media supplemented with EGF (64.3 versus 33.9%, P < 0.003). For oocytes cultured with intact cumulus (group II), more oocytes reached MII at 30 h than in group I, but there was no difference in medium with or without EGF supplementation (81.8 and 79.8% respectively). Cytoplasmic maturation of MII oocytes was judged from their capability to activate and fertilize after ICSI. In group I, the rates of activation and normal fertilization were similar. However, in group II, significantly more oocytes underwent normal fertilization in the EGF-supplemented than the unsupplemented group (71.7 versus 45.6%, P < 0.05). The cleavage rates of the fertilized oocytes were similar in the sibling oocyte subgroups cultured with or without EGF supplementation, but the overall cleavage rates were higher in cumulus-intact compared to cumulus-denuded oocytes (88.9 versus 47.8%, P < 0.001). Thus, supplementation of the maturation medium with EGF and maintenance of the cumulus during culture improve the nuclear and cytoplasmic maturation of human oocytes in vitro.

Fluorescent in-situ hybridization on human embryos showing cleavage arrest after freezing and thawing.

Our current freezing-thawing policy is to transfer only embryos that cleave further in the 24 h following thawing. The purpose of our study was to investigate the incidence of numerical abnormalities for chromosomes X, Y and 1 in blastomeres of human preimplantation embryos that survived cryopreservation but did not cleave further after thawing. A total of 63 embryos surviving a freezing-thawing cycle but not cleaving further within 24 h after thawing were screened. Of the 63 screened embryos that showed cleavage arrest 24 h after thawing, 56 embryos (88.9%) remained arrested 48 h after thawing and slightly more than half of these (29/56; 51.8%) showed further deterioration in morphological quality. Seven embryos (11.1%) showed signs of further cleavage; five embryos showed additional cleavage of one blastomere and two developed a blastocoelic cavity. Fluorescent in-situ hybridization (FISH) with three specific probes for simultaneous detection of chromosomes X, Y and 1 was performed and was successful in 60 out of 63 embryos. Of these successfully labelled embryos, 26 (43.3%) were in the diploid range: 12 (20%) were uniformly diploid for the chromosomes X, Y and 1; three embryos showed aneuploidy in all their blastomeres (two were XXY-karyotype and one was monosomy 1) and in 11 embryos nondisjunction was detected. Thirteen embryos were categorized as being either haploid, triploid, tetraploid or hexaploid. Nine embryos were classified as mosaic and 12 as being highly abnormal or chaotic. These preliminary results suggest that a large proportion of embryos that do not cleave further after freezing and thawing carry chromosomal aberrations. This finding supports our policy of not transferring cryopreserved embryos which do not cleave further 24 h following thawing.

Prospective randomized study comparing human serum albumin with fetal cord serum as protein supplement in culture medium for in-vitro fertilization.

The use of human serum albumin (HSA) instead of fetal cord serum (FCoS) as protein supplement highly simplifies the preparation of culture medium for human in-vitro fertilization (IVF) but whether they are equivalent in sustaining embryo development is still controversial. We performed a prospective randomized study of patients undergoing IVF or intracytoplasmic sperm injection (ICSI) where embryos were cultured in Earle's balanced salt solution containing either 8% (v/v) FCoS or 0.4% (w/v) HSA as protein source. Fertilization rates, morphological embryonic quality and pregnancy rates were compared. A total of 2189 oocytes from 210 cycles were cultured in medium supplemented with HSA in patient group 1 and 2109 oocytes from 203 cycles in medium supplemented with FCoS in patient group 2. The fertilization rate, defined as the presence of two nuclei, for microinjected oocytes was similar in both patient groups (77.4 and 76.7%, respectively). The fertilization rate for inseminated oocyte-cumulus complexes was significantly higher in the HSA group than in the FCoS group (62.9 versus 53.8%, P < 0.025). The embryonic quality was significantly better after culture in medium supplemented with HSA than with FCoS (13.7 versus 9.9% morphologically excellent embryos, P < 0.001). Implantation rates per transferred embryo were not significantly different (22.5 versus 18.2%), but there was a significantly higher pregnancy rate per embryo transfer in the HSA group (45.7 versus 35.9%, P < 0.05, respectively). Non-evolutive pregnancy rates were significantly different (27.4 and 16.7%). Our data demonstrate that the use of human serum albumin as a protein supplement for culture medium in human IVF programmes is associated with improved embryonic quality and significantly higher pregnancy rates. For this reason as well as the additional benefits of being virus-free and being purified, HSA is preferable to FCoS for the preparation of culture media in human IVF.

Triple colour fluorescent in-situ hybridization for chromosomes X,Y and 1 on spare human embryos.

The potential for implantation of human embryos obtained by in-vitro fertilization is presumably determined to a large extent by their chromosomal constitution but cytogenetic analysis of preimplantation embryos has been hampered by a number of practical and technical problems. With the advent of fluorescent in-situ hybridization (FISH) a practical method for numerical chromosomal analysis has become available. A limited amount of data has been obtained with FISH on human embryos using probes binding to chromosomes X, Y, 16, 18 and 13/21 combined or for chromosomes X and Y or 1 and 17. It was our purpose to extend these data by the combined analysis of chromosomes X, Y and 1 in spare human embryos. A short fluorescent in-situ hybridization procedure involving the simultaneous use of three deoxyribonucleic acid probes detected with red, green, and a mixture of red and green was used to determine chromosomal abnormalities in 116 spare embryos with a poor morphological score and/or displaying one or more multinucleated blastomeres. The majority of the embryos was obtained by intracytoplasmic sperm injection. Less than half of the embryos (n = 54) were diploid and only 39 of them were uniformly XY11 or XX11; two embryos showed a non-disjunction and 13 embryos were aneuploid. Of the remainder, 22 were mosaic, nine were either haploid, triploid or tetraploid and 12 embryos were classified as chaotic. The latter pattern was particularly frequent in multinucleated blastomeres. Our data are comparable with those obtained with FISH using other chromosomal probes and confirm that the majority of preimplantation embryos carry a numerical chromosomal defect. Aneuploidy for chromosome 1 does not appear to be more common in preimplantation embryos than is reported for other chromosomes. Although the high incidence of chromosomal anomalies is presumably biased by the fact that only embryos with a poor morphological score were analysed, it nevertheless indicates that natural selection is the foremost reason for the low implantation rates of human preimplantation embryos in in-vitro fertilization (IVF) programmes.