Human oocyte development was evaluated after a reduced time exposure to spermatozoa in vitro. A total of 119 patients were assigned to two study groups in a randomized prospective study in which each patient's oocytes were exposed to spermatozoa for either 1 h (group 1 - 58 patients) or the standard 16 h incubation period (group 2 - 61 patients). The fertilization rate obtained in group 1 was higher than in group 2 (285/393, 73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyte interaction occurs within 1 h. This was confirmed in a study in vitro using fluorescently labelled spermatozoa and normal oocyte-cumulus complexes. Spermatozoa enter the cumulus complex within 15 min, traverse the cumulus layer within 3 h, and first appear in the oocyte cortex at 4 h post-insemination. The incidence of polyspermy was higher in oocytes exposed to spermatozoa for 16 h (3%) than for 1 h (1%). There was no difference in the cleavage rate or morphological characteristics of embryos from both study groups. However, when evaluating the timing of embryo development, group 1 generated a significantly higher percentage of four to five cell embryos when compared to group 2 (55 versus 39%; P < 0.001), documented at 40 h post-insemination. The implantation and pregnancy rates for group 1 were 11 and 28%, while the corresponding rates for group 2 were 8 and 15%. This suggests that a reduced exposure of oocyte to spermatozoa favours embryo viability, possibly due to a decrease in potential damage from sperm metabolic waste products.
A reduced time interval of oocyte exposure to spermatozoa was investigated to assess whether it could enhance oocyte development and improve embryo viability, especially in cases of male factor infertility. A total of 167 patients were included in a prospective randomized study. They were randomly allocated to two major study groups, A (n = 85) and B (control group; n = 82). The oocytes from group A patients were exposed to spermatozoa for only 1 h; those from group B were exposed for 16 h. The two study groups were then subdivided according to semen quality for further analysis of the results. Significantly higher percentages were obtained in group A than in group B in terms of the fertilization rate (74 versus 68%, P < 0.025), cleavage rate (53 versus 41%, P < 0.005), pregnancy rate (27 versus 12%, P < 0.05) and implantation rate (11 versus 6%, P < 0.05). In addition, an increased fertilization rate was achieved in oocytes exposed to male factor spermatozoa for only 1 h compared with the conventional incubation period (78 versus 65%, P < 0.01). Advanced cellular stages (55 versus 41%, P < 0.02) and higher implantation rates (13 versus 4%, P < 0.05) were attained in the subgroup whose oocytes were exposed to normal spermatozoa for 1 h compared with the male factor spermatozoa with the standard culture interval. The higher fertilization rates, enhanced embryo development and viability achieved in group A indicate that prolonged exposure of oocytes to high concentrations of spermatozoa is detrimental, decreasing sperm-oocyte interaction and subsequent embryo implantation, particularly in male factor patients.
PGD (preimplantation genetic diagnosis) of aneuploidy for chromosomes X, Y, 13, 18 and 21 was carried out on 196 embryos from 36 infertile patients classified with a poor prognosis due to (i) maternal age, (ii) repeated in-vitro fertilization (IVF) failures and (iii) mosaic karyotype. The percentage of abnormal embryos was comparable in the three groups of patients: maternal age 63%, repeated IVF failure 57%, and mosaic karyotype 62%. The analysis of the overall data revealed an increased incidence of abnormal embryos in the older age categories (predominantly due to aneuploidy), even in embryos at the 7- to 8-cell stage. In addition, the percentage of chromosomally abnormal embryos was directly proportional to the number of IVF failures, where the increase in chromosomal abnormalities was not correlated to aneuploidy but to other aberrations such as mosaicism and polyploidy. Following PGD, 28 patients had at least one embryo transferred that appeared normal by fluorescent in-situ hybridization (FISH). Four clinical pregnancies resulted, with an implantation rate of 10% per normal embryo. In conclusion, the high rate of chromosomally abnormal embryos in poor prognosis patients may have been the cause of implantation failure in their previous IVF cycles. Therefore, the possibility of transferring embryos with a normal FISH complement could improve the chance of pregnancy in this category of patients.
Retarded development and blastomere fragmentation of human preimplantation embryos represent a common phenomenon in in-vitro culture systems. Even though media composition is generally formulated to meet embryo nutritional requirements, the influence of antibiotic supplementation has not been investigated thoroughly. The present study was performed to evaluate the effects of antibiotics on embryo morphology and growth in modified culture media. A total of 196 zygotes from 18 couples was cultured in three different media: (i) conventional medium (n = 99, control group); (ii) medium modified with half the standard antibiotic concentration (n = 54; and (iii) antibiotic-free medium (n = 43); 49 embryos from the control group were selected at the zygote stage and transferred to the patients on day 2. The remaining 147 zygotes were cultured to the blastocyst stage for cryopreservation; their morphology and cell number were assessed daily at 40, 64, 88 and 112 h post-insemination. Overall cleavage rate was 95% and embryo scoring revealed 91% grade 1 embryos throughout the culture period in the three media. Significantly higher cleavage rates were obtained in the antibiotic-free medium at each observation, including the blastocyst stage, when compared to the other two groups. In addition, no notable improvement was observed in the embryos cultured in a reduced concentration of antibiotics. In conclusion, antibiotic supplementation of media has an adverse effect on the growth rate of preimplantation embryos, even in reduced concentrations, suggesting that antimicrobial drugs may interfere with the timing of cleavage events either by delaying or blocking embryo development.
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