In vitro culture under physiologic oxygen concentration improves blastocyst yield and quality: a prospective randomized survey on sibling oocytes
2008; Elsevier BV; Volume: 91; Issue: 4 Linguagem: Inglês
10.1016/j.fertnstert.2008.07.1707
ISSN1556-5653
AutoresH. Nadir Çıray, T. Aksoy, Kursat Yaramanci, Ilyas Karayaka, Mustafa Bahçeci,
Tópico(s)Ovarian function and disorders
ResumoA prospective randomized study was carried on sibling oocytes in which human gametes and embryos were cultured at physiologic (6% CO2 + 5% O2 + 89% N2) condition from insemination to day 6 or at atmospheric (6% CO2 in air) condition until day 3 and at physiologic environment afterward. The results showed that physiologic oxygen concentration throughout culture period improved total blastocyst yield and day5 embryo quality. A prospective randomized study was carried on sibling oocytes in which human gametes and embryos were cultured at physiologic (6% CO2 + 5% O2 + 89% N2) condition from insemination to day 6 or at atmospheric (6% CO2 in air) condition until day 3 and at physiologic environment afterward. The results showed that physiologic oxygen concentration throughout culture period improved total blastocyst yield and day5 embryo quality. Embryos are exposed to an oxygen concentration of 2% to 8% under in vivo conditions (1Mastroianni Jr., L. Jones R. 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Physiologic (5%) oxygen concentration resulted in a higher blastocyst yield with a higher mean number of cells in one study (20Dumoulin J.C.M. Meijers C.J.J. Bras M. Coonen E. Geraedts J.P.M. Evers J.L.H. Effect of oxygen concentration on human in vitro fertilization and embryo culture.Hum Reprod. 1999; 14: 465-469Crossref PubMed Scopus (137) Google Scholar) and similar blastocyst quality in another (21Kea B. Gebhardt J. Watt J. Westphal L.M. Milki A.A. Behr B. Effect of reduced oxygen concentrations on the outcome of in vitro fertilization.Fertil Steril. 2007; 87: 213-216Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The latter study has shown a trend toward improved pregnancy rate when embryos that have been cultured under physiologic environment were transferred (21Kea B. Gebhardt J. Watt J. Westphal L.M. Milki A.A. Behr B. Effect of reduced oxygen concentrations on the outcome of in vitro fertilization.Fertil Steril. 2007; 87: 213-216Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In cleavage stage transfers, whereas some found significant improvement in the pregnancy and implantation rates in favor of physiologic oxygen concentration (22Catt J.W. Henman M. Toxic effects of oxygen on human embryo development.Hum Reprod. 2000; 15: 199-206Crossref PubMed Scopus (142) Google Scholar), others reported similar outcome (20Dumoulin J.C.M. Meijers C.J.J. Bras M. Coonen E. Geraedts J.P.M. Evers J.L.H. Effect of oxygen concentration on human in vitro fertilization and embryo culture.Hum Reprod. 1999; 14: 465-469Crossref PubMed Scopus (137) Google Scholar, 21Kea B. Gebhardt J. Watt J. Westphal L.M. Milki A.A. Behr B. Effect of reduced oxygen concentrations on the outcome of in vitro fertilization.Fertil Steril. 2007; 87: 213-216Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Dumoulin J.C.M. Vanvuchelen R.C.M. Land J.A. Pieters M.H. Geraedts J.P. Evers J.L. Effect of oxygen concentration on in vitro fertilization and embryo culture in the human and the mouse.Fertil Steril. 1995; 63: 115-119Abstract Full Text PDF PubMed Scopus (59) Google Scholar, 24Ulug U. Jozwiak E.A. Mesut A. Berksoy M.M. Bahceci M. Survival rates during the first trimester of multiple gestations achieved by ICSI: a report of 1448 consecutive multiples.Hum Reprod. 2004; 19: 360-364Crossref PubMed Scopus (21) Google Scholar). The present study tested the hypothesis that physiologic oxygen concentration improved blastocyst yield and quality in human embryo culture. A sibling oocyte model was employed to minimize potential variations between female (and male) gametes obtained from different patients that may obscure the impact of culture conditions on embryo development. A prospectively randomized study was performed for a period of 6 months (June to December 2007) in the IVF unit of the German Hospital in Istanbul. Approval of the ethics committee of the hospital was obtained. All cycles in which at least 16 oocytes retrieved were included in the survey regardless of sperm characteristics and the number of previous trials. Patients were hyperstimulated according to the ovulation induction protocol that has been described elsewhere (25Bahceci M. Ciray H.N. Karagenc L. Ulug U. Bener F. Effect of oxygen concentration during the incubation of embryos of women undergoing ICSI and embryo transfer: a prospective randomized study.Reprod BioMed Online. 2005; 4: 438-443Abstract Full Text PDF Scopus (33) Google Scholar). All metaphase II oocytes were subjected to ICSI. After microinjection, oocytes from each patient's cycle were randomly divided into incubators (ThermoForma, Model 3141; Forma Scientific Inc., Marietta, OH), which were gassed with 6% CO2 in air (control group) and 6% CO2 + 5% O2 + 89% N2 (study group) according to the protocol obtained from the internet (www.randomization.com). The CO2 and O2 measurements (and calibrations, if necessary) were checked daily before incubators' doors were opened using an IR sensor (Labotect InControl 1050, Göttingen, Germany). The embryology procedures of the present study have been described elsewhere (26Ciray H.N. Karagenc L. Ulug U. Bener F. Bahceci M. Use of both early cleavage and day 2 moonucleation to predict embryos with high implantation potential in intracytoplasmic sperm injection cycles.Fertil Steril. 2005; 84: 1411-1416Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Briefly, embryos were cultured individually in 20-μL drops of Quinn's Advantage Plus Cleavage Medium (until day 3) and Quinn's Advantage Plus Blastocyst Medium (from day 3 to day 6) under sterile oil (Sage IVF, Trumbull, CT). Fertilization was assessed at 18 to 20 hours after insemination. Zygotes displaying two pronuclei were subsequently inspected at 24 to 27 hours and scored as early cleaved (two cells), syngamy (absence of pronuclei without cleavage) or two pronuclei. Between 44 to 46 and 66 to 68 hours, embryos were scored according to the following parameters; number of blastomeres, nuclear morphology, degree of fragmentation and blastomere symmetry. At day 3, embryos in the control group were transferred into incubators with physiologic oxygen concentration. Embryos were scored at day 5 and 6 according to Gardner et al. (27Gardner D.K. Lane J.D. Stevens J. Schlenker T. Schoolcraft W.B. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.Fertil Steril. 2000; 73: 1155-1158Abstract Full Text Full Text PDF PubMed Scopus (1088) Google Scholar). One or two blastocysts that were at the most advanced stage of development and displayed the highest quality were chosen for transfer. All transfers were performed at day 5 using a Wallace catheter under ultrasound guidance. Surplus blastocysts that were of good quality (≥4 BB) were frozen at day 5 or 6. The luteal phase was supported by daily administration of progesterone vaginal gels (Crinone, Serono, Switzerland). The outcome parameter of the study was the blastocyst yield after day 6. To obtain a 5% increase in the blastocyst yield from 40%, it was calculated that with 5% type 1 and 20% type 2 errors, a total of 1,227 oocytes were needed. The blastocyst quality was also assessed in the groups. The values were compared using a paired t test or Wilcoxon matched pairs test according to the normality of distribution. A probability of p<.05 was considered as statistically significant. As the pregnancy and implantation data were not the focus of this particular study, these data were not analyzed. A total of 75 oocyte retrieval cycles were studied, of which 74 resulted in embryo transfer. In one cycle, all embryos were arrested at an earlier stage of development, resulting in cancellation of transfer. The age of women was 29.6 ± 4.4 years (mean ± standard deviation). Total number of retrieved oocytes was 2,061, of which 1,737 were at metaphase II (0.85 ± 0.11). There were 869 and 868 oocytes that were subjected to intracytoplasmic sperm injection (ICSI) in the control and study groups, respectively (Table 1). The results showed a significant improvement in day 3 and day 5 embryo qualities and blastocyst yield in the study group (P<.05).Table 1The outcome parameters of prolonged embryo culture under atmospheric (6% CO2 in air) and physiological (6% CO2+5% O2) oxygen concentrations of sibling oocytes.% (N)6% CO2 in air6% CO2+5% O2P valueTwo pronuclei1Per Metaphase II oocytes.71.2 ± 15.6 (622)71.8 ± 16.7 (620)0.933Wilcoxon matched pairs test.Early cleavage2Per two pronuclei oocytes.39.6 ± 32.9 (242)43.1 ± 32.5 (251)0.133Wilcoxon matched pairs test.Syngamy2Per two pronuclei oocytes.29.8 ± 21.2 (186)25.9 ± 20.9 (172)0.113Wilcoxon matched pairs test.Top quality day 2 embryo2Per two pronuclei oocytes.AEmbryo displaying 4 mononucleated blastomeres and less than 20% fragmentation.50.7 ± 28.4 (305)46.3 ± 24.6 (288)0.154Paired t-test.Top quality day 3 embryo2Per two pronuclei oocytes.BEmbryo displaying 8 mononucleated blastomeres and less than 20% fragmentation or compaction.32.9 ± 21.4 (204)39.6 ± 25.1 (247)0.014Paired t-test.Day 5 blastocyst yield2Per two pronuclei oocytes.C≥2 according to Gardner's score.33.2 ± 23.7 (206)36.6 ± 23.3 (226)0.234Paired t-test.Day 6 blastocyst yield2Per two pronuclei oocytes.C≥2 according to Gardner's score.42.4 ± 26.0 (263)47.7 ± 23.9 (291)0.044Paired t-test.Good quality day 5 blastocyst2Per two pronuclei oocytes.D≥4BB according to Gardner's score.11.4 ± 15.2 (75)16.5 ± 16.6 (100)0.0063Wilcoxon matched pairs test.1 Per Metaphase II oocytes.2 Per two pronuclei oocytes.3 Wilcoxon matched pairs test.4 Paired t-test.A Embryo displaying 4 mononucleated blastomeres and less than 20% fragmentation.B Embryo displaying 8 mononucleated blastomeres and less than 20% fragmentation or compaction.C ≥2 according to Gardner's score.D ≥4BB according to Gardner's score. Open table in a new tab The findings of the present study showed that use of physiologic oxygen concentration during the entire prolonged culture period yield improvement in embryo quality after day 3. Therefore, it is in accordance to the results of the study by Dumoulin et al. (20Dumoulin J.C.M. Meijers C.J.J. Bras M. Coonen E. Geraedts J.P.M. Evers J.L.H. Effect of oxygen concentration on human in vitro fertilization and embryo culture.Hum Reprod. 1999; 14: 465-469Crossref PubMed Scopus (137) Google Scholar) and contradicts that of Kea et al. (21Kea B. Gebhardt J. Watt J. Westphal L.M. Milki A.A. Behr B. Effect of reduced oxygen concentrations on the outcome of in vitro fertilization.Fertil Steril. 2007; 87: 213-216Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Dumoulin et al. (20Dumoulin J.C.M. Meijers C.J.J. Bras M. Coonen E. Geraedts J.P.M. Evers J.L.H. Effect of oxygen concentration on human in vitro fertilization and embryo culture.Hum Reprod. 1999; 14: 465-469Crossref PubMed Scopus (137) Google Scholar) suggested that the beneficial effect of physiologic oxygen concentration could have been observed in extended embryo culture, although data have been obtained from limited number of surplus embryos after day 3 transfers. In that study a higher blastocyst yield with more cells have been reported at physiologic oxygen concentration, but day 3 embryo qualities did not differ between groups that contradicted to the findings of Kea et al. (21Kea B. Gebhardt J. Watt J. Westphal L.M. Milki A.A. Behr B. Effect of reduced oxygen concentrations on the outcome of in vitro fertilization.Fertil Steril. 2007; 87: 213-216Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) as well as our earlier (24Ulug U. Jozwiak E.A. Mesut A. Berksoy M.M. Bahceci M. Survival rates during the first trimester of multiple gestations achieved by ICSI: a report of 1448 consecutive multiples.Hum Reprod. 2004; 19: 360-364Crossref PubMed Scopus (21) Google Scholar) and present studies. It should be considered that culture media and embryo scoring parameters have been advanced since then, which may explain the difference between results. In contrast to day 3, day 5 embryo quality remained similar in the study by Kea et al. (21Kea B. Gebhardt J. Watt J. Westphal L.M. Milki A.A. Behr B. Effect of reduced oxygen concentrations on the outcome of in vitro fertilization.Fertil Steril. 2007; 87: 213-216Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). However, as the investigators reported, patient demographics varied between groups. In the present study, using a sibling oocyte model, it has been found that the impact of physiologic oxygen concentration became evident at day 3 and afterward. Increased blastocyst yield with better quality may cause improved pregnancy, implantation, and delivery rates in a physiologic oxygen environment; however, data with homogeneous transfers are needed. In conclusion, the results of the present study favor culture of embryos in 5% O2, in which mammalian physiologic conditions of the reproductive tract are mimicked.
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