Effect of oxygen concentration on in vitro fertilization and embryo culture in the human and the mouse

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1 FERTILITY AND STERILITY Copyright c 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Effect of oxygen concentration on in vitro fertilization and embryo culture in the human and the mouse John C. M. Dumoulin, M.Sc.*t Rosie C. M. Vanvuchelen, M.T.* Jolande A. Land, M.D.* Math H. E. C. Pieters, M.D.* Joep P.M. Geraedts, Ph.D.:j: Johannes L. H. Evers, M.D.* Academisch Ziekenhuis Maastricht, University of Limburg, Maastricht, The Netherlands Objective: To compare the effect of culturing oocytes, zygotes and embryos under low (5%) versus ambient (20%) oxygen conditions on human IVF results and on mouse blastocyst formation. Design: A prospective, randomized study of 257 consecutive IVF treatment cycles in 186 couples undergoing oocyte retrieval for various reasons of infertility. Gametes and resulting embryos after IVF were alternately allocated to fertilization and culture either under a gas phase of 5% C0 2/90% N 2 /5% 0 2, or 5% C0 2/95% air (20% 0 2). Oocytes and embryos from randomly bred and hybrid mouse strains were randomly allocated to culture under either of the two gas phases. Setting: A university hospital-based IVF-ET program. Main Outcome Measure: In the human, rates of fertilization, embryonic development at the time of embryo replacement (42 to 46 hours after insemination), pregnancy, and implantation were compared. In the mouse, the rates of blastocyst formation were compared. Results: Clinical pregnancies occurred in 24.2% versus 19.4% of retrievals when culture took place under low oxygen versus ambient oxygen conditions. Fertilization, embryonic development, pregnancy, and implantation rates did not differ significantly between the groups. Slightly higher blastocyst rates occurred when mouse embryos from hybrid strains were cultured under low oxygen compared with culture under ambient oxygen conditions, whereas no such difference in blastocyst rates was found in randomly bred mouse embryos. Conclusions: This study failed to demonstrate any improvement in human IVF results associated with the use of a gas mixture of 5% C0 2/90% N 2/5% 0 2 during the first two days of development compared with the use of 5% C0 2 in air. Fertil Steril 1995;63:115-9 Key Words: Preimplantation development, embryo culture, oxygen Received February 2, 1994; revised and accepted July 29, *Department of Obstetrics and Gynecology. t Reprint requests: John C.M. Dumoulin, M.Sc., IVF Laboratory, Department of Obstetrics and Gynaecology, Academisch Ziekenhuis Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands (FAX: ). :j: Department of Molecular Cell Biology and Genetics. Vol. 63, No.1, January 1995 Nonhuman mammalian embryos can be successfully cultured using a gas phase of either 5% C0 2 / 95% air (20% 0 2 ) or 5% C0 2 /90% N 2 /5% 0 2 Also for human IVF treatments, both oxygen concentrations are widely used (1), and no apparent differences in success rate are noted. However, embryos from several animal species show improved development when cultured under reduced oxygen tension. Concerning human IVF, data in the literature regarding the effect of the oxygen concentration are fragmentary and limited. In this prospective randomized study we compared the results of human IVF when using either of the two gas phases. Rates of fertilization, embryonic development at the time of embryo replacement, pregnancy, and implantation were compared. Although several studies show that mouse embryonic development benefits from the use of low oxygen concentrations (2, 3), other studies could not confirm this beneficial effect (4, 5). To have a comparison group for our human experiments regarding the effect of oxygen on embryonic development, Dumoulin et al. Effect of oxygen on human IVF results 115

2 we studied the blastocyst formation in the mouse when culturing under a low oxygen concentration of 5% compared with culturing under the atmospheric concentration of 20%, using either zygotes from a hybrid strain or from a randomly bred strain. MATERIALS AND METHODS Human IVF Procedures Oocytes and embryos from 257 consecutive IVF treatment cycles in 186 couples attending our IVF clinic were alternately allocated to fertilization and culture either under ambient (20%) or reduced (5%) 0 2 Indications for IVF treatment were tubal factor in 137, unexplained infertility in 96, male subfertility in 10, and other indications in 14. Male subfertility was defined as having a progressive motile sperm density of <3 X 10 6 /ml in combination with <5% morphologically normal spermatozoa, evaluated using strict criteria (6). The stimulation protocols used have been described previously (6). In summary, the GnRHagonist buserelin acetate (Suprefact; Hoechst B.V., Amsterdam, The Netherlands) was used in combination with hmg (Pergonal; Serono, Geneva, Switzerland, or Humegon; Organon, Oss, The Netherlands) to stimulate multiple follicular development. Follicle growth was monitored by ultrasound and 10,000 U of hcg (Pregnyl; Organon) was given as soon as the dominant follicle was judged to be mature (> 18 mm) to induce final follicular and oocyte maturation. Ultrasound-guided oocyte retrieval was performed 34 to 35 hours after hcg administration. Five hours after ovum pick-up, the oocytes were inseminated using a suspension of highly motile spermatozoa prepared by a swim-up technique as described by Enginsu et al. (6). Oocytes and embryos were cultured in Multidish 4-well dishes (Nunc, Life Technology BV, Breda, The Netherlands) containing in each well 1 ml human tubal fluid (HTF) culture medium (7) supplemented with 8% of a human serum protein solution (Central Laboratory of the Blood Transfusion Service, Amsterdam, The Netherlands) as described by Huisman et al. (8). The incubator used was a Napco double-chamber 7300 model with separate gas controlling systems for either chamber (Wilten W oltil, De Meern, The Netherlands). In one chamber, an atmosphere of 5% C0 2 in air was used, while in the other chamber a gas mixture of 5% C0 2, 5% 0 2, and 90% N 2 was used. The C0 2 and 0 2 concentrations of both incubator chambers were regularly checked using a Servomex-570A Oxygen Analyzer (Servomex, Zoetermeer, The Netherlands) and a Normocap 200 C0 2 - Analyzer (Datex Medical Electronics, Hoevelaken, The Netherlands), and, if necessary, the incubator chambers were recalibrated. After incubation for 18 to 20 hours, the oocytes were checked for the presence of pronuclei as proof of fertilization, washed once, and after transfer to fresh medium, cultured for another day. Before embryo replacement at 42 to 46 hours after insemination, the developmental stage and the morphological aspect were assessed according to the criteria of Bolton et al. (9). Up to three embryos, or, in exceptional cases, four embryos, with the best morphological appearance were transferred to the uterus of the patient. After embryo transfer, supernumerary good quality embryos were frozen. Mouse IVF Procedures Two experiments were performed using either (C57B1 X DBA)F1 hybrid or randomly bred Swiss mice. The IVF and embryo culture methods used were as described previously (10). Briefly, oocytes were obtained after superovulating female mice with 10 U pregnant mare serum gonadotropin and 10 U hcg. In both experiments, oocytes from each female were inseminated with 2 X 10 6 sperm per ml from a Swiss male and incubated under an oxygen concentration of 5%. HTF medium was used in all experiments. For IVF, the medium contained 30 mg bovine serum albumin (BSA)/mL; 5 mg BSA/mL was used for culturing of the embryos. Five hours after insemination, the oocytes were washed once and distributed over two treatment groups for further culture to the blastocyst stage. One group was cultured under ambient (20%) 0 2, whereas for the other group a reduced (5%) 0 2 concentration was used. Oocytes and embryos were cultured per female separately and not pooled. Twenty hours after insemination, two-cell embryos were counted and at 120 hours after insemination, blastocysts were counted. Statistics In the human, pregnancy and implantation rates were compared using the x 2 test. The 95% confidence intervals (Cis) and the power of these determinations were calculated. Statistical significance was accepted if differences had 80% power at the 5% level of significance. Data on mouse embryonic 116 Dumoulin et al. Effect of oxygen on human IVF results Fertility and Sterility

3 development were analyzed by paired Student's t test. RESULTS Effect of the Oxygen Concentration on Human IVF and Embryonic Development Insemination and embryo culture of 128 ovum pick-ups was performed using a gasphase of 5% 0 2, 5% C0 2, 90% N 2, whereas for the other 129 ovum pick-ups 5% C0 2 in air (20% 0 2 ) was used. None of the parameters investigated, e.g., the fertilization rate (Table 1), the embryonic development until the time of embryo replacement (Table 1), the pregnancy rate, nor the implantation rate (Table 2), were significantly different between the two groups cultured in a gasphase of either 5% 0 2 or 20% 0 2 The 95% confidence Cis of the pregnancy rates were 17.1 to 32.6 in the 5% 0 2 group and 13.0 to 27.3 in the 20% 0 2 group. Effect of the Oxygen Concentration on Mouse Embryonic Development In the first experiment, 560 oocytes were obtained from 14 hybrid females, whereas in the second experiment, 584 oocytes were obtained from 15 randomly bred females (mean number of oocytes (± SD) per female 40.0 ± 8.6 in the first experiment and 38.9 ± 6.4 in the second experiment). In both experiments, oocytes were divided at 5 hours after Table 1 Fertilization and Development of Human Oocytes Cultured Under Different Gas Mixtures No. of ovum pick-ups No. of oocytes inseminated Fertilization at 18 to 20 hours after insemination Fertilization rate No. of zygotes showing 2PNt No. of zygotes with >2PNt Embryonic stages at the time of transfer ( 42 to 46 hours after insemination):j: No. of zygotes not cleaved No. of embryos completely fragmented No. of 2-cell embryos No. of 3-cell embryos No. of 4-cell embryos No. of 5- to 8-cell embryos Gas phase 5% o. 20% o , ± ,009 (67.8) 70 (4.7) 40 (4.0) 20 (2.0) 287 (28.4) 113 (11.2) 418 (41.4) 131 (13.0) 129 1, ± (70.1) 73 (5.3) 42 (4.4) 27 (2.8) 231 (24.1) 117 (12.2) 382 (39.8) 160 (16.7) *Values are fertilization per oocyte per ovum pick-up (means± SD). t Values in parentheses are percents of zygotes relative to the number of oocytes inseminated. :j: Values in parentheses are percents of embryos relative to the number of zygotes showing 2PN at 18 to 20 hours after insemination. Table 2 Comparison of Results of IVF-ET Treatments Cultured Under Different Gas Mixtures No. of ovum pick-ups (OPU) No. of embryo transfers No. ofpregnanciest Pregnancy rate per OPU (%) No. of ongoing pregnancies (> 12 weeks) Ongoing pregnancy rate per OPU (%) No. of embryos replaced No. of implantation sites:j: Implantation rate per embryo replaced(%) No. of fetuses with heart activity:j: Rate of viable fetuses per embryo replaced (%) 5% o Gas phase 20% o * 25* * * * 7.3 *Not significant when compared with 5% 0 2 t Positive test on urinary hcg (sensitivity 50 IU/L) at 16 to 18 days after ovum pick-up. :j: Confirmed by ultrasound, 5 weeks after OPU. insemination in two groups and subsequently cultured under either 5% 0 2, or 20% 0 2 The mean fertilization rate(± SD) per female was 0.70 ± 0.13 in the first experiment and 0.85 ± 0.09 in the second experiment. The results with respect to embryonic development are summarized in Table 3. As can be seen from the data, we found a significantly higher incidence of blastocyst formation when in vitrofertilized zygotes from hybrid females were cultured under 5% 0 2, compared with those cultured under 20% 0 2, whereas this difference in the randomly bred strain did not reach significance. DISCUSSION This prospective randomized study on 257 consecutive IVF treatments shows that culture of human oocytes and embryos for the first two days of development under a gas phase of either 5% C0 2/ 95% air (20% 0 2) or 5% C0 2/90% N 2/5% 0 2 does Table 3 Mouse Embryonic Development Using Different Gas Mixtures Gase phase No. of blastocysts/ no. of 2-cell embryos* Hybrid (C57Bl x DBA)Fl mouse strain 171/190 (0.90 ± 0.03)t 140/198 (0.70 ± 0.03) Randomly bred Swiss mouse strain 42/248 (0.18 ± 0.05) 20/250 (0.08 ± 0.02) * Mean incidence of blastocyst formation (±SE) per two-cell embryos per female in parentheses. t Significantly different from 20% 0 2 group (paired Student's t-test: P < 0.01). Dumoulin et al. Effect of oxygen on human IVF results 117

4 not result in significant differences in pregnancy rates between the two groups. If the observed difference of 4.8% in pregnancy rate between the 5% 0 2 and 20% 0 2 group would have persisted, more than 1,162 cycles in each group would be required to show a significant difference between the pregnancy rates with 80% power. Rates of fertilization and cleavage and developmental potential of the embryos after replacement also did not differ significantly between the two groups. These results are comparable with those reported earlier (Dorfmann AD, Bender S, Robinson P, Fugger E, Bustillo M, Schulman JD, abstract), which showed no differences in fertilization rates and cleavage rates when 406 oocytes from 70 patients were cultured either under 5% 0 2 or under 20% 0 2 Also Feichtinger et al. (11) found comparable fertilization rates in a small series of human oocytes fertilized under either of the oxygen concentrations. Many animal studies show the superiority of the use of reduced oxygen tension compared with ambient tension. Embryos from the rabbit (12, 13), goat (14), sheep and cattle (15), all show improved development when cultured under reduced oxygen tension. Also in the human, an improved blastulation rate was found when human surplus embryos were cultured under 5% 0 2 compared with embryos cultured under 20% 0 2 (Noda Y, Narimoto K, Umaoka Y, Takakura K, Kanzaki H, Taii S, et al., abstract). However, in the cat, comparable percentages of in vitro-fertilized zygotes developed to the blastocyst stage in 5% and in 20% 0 2 (16). In the mouse, significantly higher blastocyst rates were found in some studies when 5% oxygen was used compared with 20% (2, 3), whereas in other studies no such clear positive effect of 5% 0 2 was found (4, 5). In our experiments, only embryos from hybrid females benefitted from the culture under low oxygen. A reduced oxygen tension of 8% 0 2, which was shown to be optimal for rabbit fertilization in vitro (12), corresponds to the low oxygen tension in the rabbit oviduct, which was shown to be approximately 60 mm Hg, remaining unchanged at the time of ovulation and in the first 3 days after ovulation (17). Also, in the rat uterus, the oxygen tension was shown to vary between 38 and 42 mm Hg (5.2% to 5.9% 0 2 ) during the first 4 days of pregnancy (18). Interestingly, the oviduct of the Rhesus monkey becomes relatively well oxygenated during the period when fertilization and preimplantation embryo development occur, as the oxygen tension was found to rise from very low levels ( <10 mm Hg) to high levels of 80 mm Hg (11.2% 0 2 ) at the time of ovulation to approximately 5 to 7 days thereafter (19). It has been suggested that the detrimental effect of the high atmospheric oxygen tension of 150 mm Hg (20%) on the development of embryos is due to an excess production of toxic reactive oxygen species (e.g., hydrogen peroxyde, superoxide anions, and hydroxy radicals) (3). In the presence of traces of transitional metals such as iron, H 2 0 2, and auperoxide can be converted into hydroxy radicals, which are even more toxic (20). These highly reactive oxygen species have been shown to cause severe cellular damage through membrane lipid peroxidation, enzyme inactivation, and DNA damage in many cell types (21). They also have been implicated in loss of motility and function of spermatozoa (22, 23). Furthermore, it has been hypothesized that the in vitro two-cell block in mice is due to the damage caused by oxygen radicals (3). This hypothesis is in agreement with the findings that supplementing the culture medium with compounds that scavenge the free oxygen radicals, like superoxide dismutase (3), or iron chelators that prevent the formation of hydroxy radicals, like ethylenediaminetetraacetic acid (EDTA) or transferrin (20), results in higher percentages of mouse embryos reaching the blastocyst stage. Also the beneficial effect of taurine on mouse embryonic development, as was described recently by our group (10), could be hypothesized to be the result of the scavenging of oxygen radicals by taurine. This scavenging effect of taurine was demonstrated by Alvarez and Storey (22). Although it is clear that reactive oxygen species lead to impaired embryonic development in the mouse, it is less clear whether a gasphase of 20% 0 2 indeed is a major contributor to a higher concentration of these reactive oxygen species in the microenvironment of the embryo. It has been demonstrated that mouse embryos show a marked rise in H production from the mid-two-cell stage until the early four-cell stage, when cultured in vitro from the one-cell stage onwards, regardless of whether they were cultured under 20% or 5% oxygen (4). Thus, it could be argued that culture under 20% oxygen has only a minor effect on the reactive oxygen concentration, whereas the embryo itself is the major source. To conclude, there seems to be no advantage in the use of either 5% 0 2 or 20% 0 2 in the gasphase for the fertilization and embryo culture in human 118 Dumoulin et al. Effect of oxygen on human IVF results Fertility and Sterility

5 IVF. However, in view of the data provided in the literature regarding the toxic effects of high oxygen levels to cells and embryos in culture with respect to membrane and DNA damage, it seems prudent to protect human embryos from oxidative stress when they are cultured in vitro. Acknowledgments. The authors thank the following members of the IVF-team, Academic Hospital Maastricht, Maastricht, The Netherlands, for their clinical and technical assistance: J. Marij Bergers-Janssen, M.T.; Marijke Bras, M.T.; Gerard A.J. Dunselman, M.D.; M. Engin Enginsu, M.D.; Paul J.Q. Vander Linden, M.D.; Anja Tersteeg, R.N. REFERENCES 1. Braude PR. Fertilization of human oocytes and culture of human preimplantation embryos in vitro. In: Monk M, editor. Mammalian development, a practical approach. Oxford: IRL Press, 1987: Pabon JE Jr, Findley WE, Gibbons WE. The toxic effect of short exposures to the atmospheric oxygen concentration on early mouse embryonic development. Fertil Steril 1989;51: Umaoka Y, Noda Y, Narimoto K, Mori T. Effects of oxygen toxicity on early development of mouse embryos. Mol Reprod Dev 1992;31: Nasr-Esfahani M, Winston NJ, Johnson MH. Effects of glucose, ethylenediaminetetraacetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryo in vitro. J Reprod Fert 1992;96: Ali J, Whitten WK, Shelton JN. Effect of culture systems on mouse early embryo development. Hum Reprod 1993;8: Enginsu ME, Pieters MHEC, Dumoulin JCM, Evers JLH, Geraedts JPM. Male factor as determinant of in-vitro fertilization outcome. Hum Reprod 1992;7: Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44: Huisman GJ, Lo-A-Njoe NM, Alberda ATh, Leerentveld RA, Verhoeff A, Zeilmaker GH. Comparison of results obtained with human serum and a protein solution as a supplement for in vitro fertilization culture medium. Fertil Steril 1992;58: Bolton VN, Hawes SM, Taylor CT, Parsons JH. Development of spare human preimplantation embryos in vitro: an analysis of the correlations among gross morphology, cleavage rates, and development to the blastocyst. J In Vitro Fert Embryo Transfer 1989;6: Dumoulin JCM, Evers JLH, Bras M, Pieters MHEC, Geraedts JPM. Positive effect of taurine on preimplantation development of mouse embryos in vitro. J Reprod Fertil 1992;94: Feichtinger W, Kemeter P, Szalay S. The Vienna program of in vitro fertilization and embryo-transfer-a successful clinical treatment. Eur J Obstet Gynecol Reprod Bioi 1983;15: Brackett BG, Williams WL. Fertilization of rabbit ova in a defined medium. Fertil Steri11968;19: Li J, Foote RH. Culture of rabbit zygotes into blastocysts in protein-free medium with one to twenty per cent oxygen. J Reprod Fertil1993;98: Batt PA, Gardner DK, Cameron AWN. Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro. Reprod Fertil Dev 1991;3: Thompson TGE, Simpson AC, Pugh PA, Donelly PE, Tervit HR. Effect of oxygen concentration on in vitro development of preimplantation sheep and cattle embryos. J Reprod Fertil 1990;89: Johnston LA, Donoghue AM, O'Brien SJO, Wildt DE. Influence of temperature and gas atmosphere on in-vitro fertilization and embryo development in domestic cats. J Reprod Fertil 1991;92: Mastroianni L, Jones R. Oxygen tension within the rabbit fallopian tube. J Reprod Fertil 1965;9: Yochim JM, Mitchell JA. Intrauterine oxygen tension in the rat during progestation: its possible relation to carbohydrate metabolism and the regulation of nidation. Endocrinology 1968;83: Maas DHA, Storey BT, Mastroianni L Jr. Oxygen tension in the oviduct of the Rhesus monkey (Macaca mulatta). Fertil Steril1976;27: Nasr-Esfahani M, Johnson MH, Aitken RJ. The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryos in vitro. Hum Reprod 1990;5: Sohal RS, Allen RG. Oxidative stress as a causal factor in differentiation and aging: a unifying hypothesis. Exp Gerontal 1990;25: Alvarez JG, Storey BT. Taurine, hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against loss of motility. Bioi Reprod 1983;29: Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Bioi Reprod 1989;40: Dumoulin et al. Effect of oxygen on human IVF results 119