Human Reproduction vol.14 no.2 pp.465–469, 1999 Effect of oxygen concentration on human in-vitro fertilization and embryo culture* John C.M.Dumoulin1,3, Chantal J.J.Meijers1, Marijke Bras1, Edith Coonen1, Joep P.M.Geraedts2 and Johannes L.H.Evers1 1Department of Obstetrics & Gynaecology and 2Department of Molecular Cell Biology & Genetics, Academic Hospital, University of Maastricht, Maastricht, The Netherlands 3To In this prospective randomized study on 1380 consecutive in-vitro fertilization (IVF) treatments, the results were compared of culture of human oocytes and embryos for the first 2 or 3 days of development in microdroplets of medium under oil using a gas phase containing either atmospheric (~20%) or reduced (5%) O2 concentrations. No significant differences were found between the two groups cultured under either 5% or 20% O2 in rates of fertilization (60 versus 61%, respectively), embryo development at day 2 or 3, pregnancy (26.6 versus 25.4%, respectively), and implantation (13.4 versus 14.0%, respectively). Culture of surplus embryos under 5% O2 resulted in a significantly higher mean incidence of blastocyst formation per cycle as compared to the 20% O2 group (25.8 K 2.0 versus 20.4 K 1.9, respectively). The mean number of cells of embryos classified as blastocysts by microscopic observation of a blastocoel was significantly higher in the 5% O2 group as compared to the 20% O2 group, both in blastocysts fixed on day 5 (39.8 K 1.7 versus 31.9 K 1.9, respectively), as well as those fixed on day 6 (45.6 K 2.6 versus 33.7 K 3.4, respectively). This difference was due to the fact that significantly more blastocysts of the 20% O2 group had an abnormal low cell number of < 25 as compared to the 5% O2 group, both in blastocysts fixed on day 5 (39 versus 22%, respectively), as well as those fixed on day 6 (43 versus 22%, respectively). To conclude, although culture under 5% O2 leads to slightly improved preimplantation embryonic viability, this effect is either too marginal to result in higher pregnancy rates, or low O2 concentrations exert an effect during the later stages of preimplantation development only. Key words: embryo culture/oxygen/preimplantation development *Data presented in part at the 14th Annual Meeting of the European Society for Human Reproduction and Embryology (ESHRE), Göteborg, Sweden, June 21–24, 1998. © European Society of Human Reproduction and Embryology Mammalian embryos can be cultured successfully using a gas phase containing either atmospheric (~20%) or reduced (5%) O2 concentrations. For human in-vitro fertilization (IVF) treatments, both O2 concentrations are widely used and similar success rates have been reported. In a previous study, we evaluated the effect of both of these O2 concentrations on IVF and embryo culture in the human and found no beneficial effect of culturing under 5% O2 as compared to culturing under atmospheric O2 concentrations (Dumoulin et al., 1995). It has been demonstrated, however, that embryos from several species (mouse, sheep, goat, cattle) show improved development when cultured under reduced O2 tension (Bavister, 1995). The use of small, static droplets of medium covered with an oil overlay has been speculated to result in marginally hypoxic culture conditions at lower O2 concentrations (ByattSmith et al., 1991). Furthermore, it has been shown in several animal studies that different culture systems may require different O2 concentrations for optimal results (Fukui et al., 1991; Voelkel and Hu, 1992; Ali et al., 1993). Therefore, the results from our previous study, in which human embryos were cultured in 1 ml of medium without an oil overlay, may not be applicable to a culture system of droplets of medium under oil. In the present study, we compared the results of human IVF and embryo culture under both of these gas phases, using 20 µl droplets under oil. Materials and methods Patients 1 During a period of 32 years, 1380 consecutive IVF and intracytoplasmic sperm injection (ICSI) treatment cycles were included in this study. Oocytes and embryos were alternately allocated per set of two treatment cycles to fertilization and culture either under ambient 1 (~20%) or reduced (5%) O2. During the last 22 years of the present study, a second study was performed, in which oocytes and embryos were alternately allocated to the use of either of two culture media: either IVF-50™ or human tubal fluid (HTF). By this allocation procedure, a random distribution of treatments over the different culture techniques was ensured and the two studies were independent. The results of this second study will be published separately. ICSI treatment was performed in cases of male subfertility (621 cycles), and when no fertilization had occurred in two previous IVF cycles (93 cycles). Male subfertility was defined as a progressive motile sperm concentration of ,33106/ml in combination with ,5% morphologically normal spermatozoa, evaluated using strict criteria (Enginsu et al., 1992). For all other indications (666 cycles), a conventional in-vitro insemination procedure with 50 000 motile spermatozoa/ml was used. The stimulation protocol used has been described previously (Land 465 Downloaded from http://humrep.oxfordjournals.org/ by guest on March 14, 2016 whom correspondence should be addressed at: IVF Laboratory, Department of Obstetrics and Gynaecology, Academic Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands Introduction J.C.M.Dumoulin et al. et al., 1996). In summary, the gonadotrophin-releasing hormone (GnRH) agonist nafarelin (Synarel; Searle BV, Maarssen, The Netherlands) was used in combination with human menopausal gonadotrophin (HMG, Pergonal; Serono, Amsterdam, The Netherlands; or Humegon, Organon, Oss, The Netherlands) to stimulate multiple follicular development. Follicle growth was monitored by ultrasound and 5000 IU of human chorionic gonadotrophin (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 34–35 h after HCG administration. Insemination or ICSI was performed ~5 h after oocyte retrieval. Culture of human surplus embryos If cryopreservation was deemed unfeasible, surplus embryos were used in one of the studies running in our centre. These studies have been approved by the local Ethics Committee. To avoid selection bias, surplus embryos of all treatment cycles during prearranged periods were used in only one study. In the present study, surplus embryos were left in their original culture medium for another 2 or 3 days. Developmental stages were recorded at each day of in-vitro development. On the morning of day 5 after ovum retrieval, surplus embryos that cavitated to form blastocyst-like structures (defined as 466 Gas phase 5% O2 20% O2 Number of cycles Number of patients Age (years)a 690 400 33.0 6 0.1 690 402 33.0 6 0.1 Indicationb Tubal infertility Idiopathic infertility Male subfertility Other 101 89 193 17 84 96 197 25 First attemptsc 352 (51) Duration of infertilitya (25) (22) (48) (4) 5.4 6 0.1 (21) (24) (49) (6) 338 (49) 5.5 6 0.1 Type of infertilityb Primary Secondary Standard IVFc ICSI 272 128 324 366 (68) (32) (47) (53) 285 (71) 117 (29) 342 (50) 348 (50) Culture mediumc IVF-50™ HTF 262 (38) 428 (62) 260 (38) 430 (62) Day of embryo transferd Day 2 Day 3 519 (80) 130 (20) 513 (81) 121 (19) aMean 6 SEM with regard to the female partner at the time of ovum retrieval. bValues in parentheses are percentages of total number of patients. cValues in parentheses are percentages of total number of treatment cycles. dValues in parentheses are percentages of total number of embryo transfers. a rim of cells surrounding a large cavity of extracellular fluid accumulated within the embryo) were fixed and stained with 49,6diamidino-2-phenylindole (DAPI) as described earlier (Coonen et al., 1994), provided that the patients had given consent. The number of nuclei stained with DAPI was taken as the number of cells of the embryo. All other embryos, including those that had only just started to form a small blastocoelic cavity, were cultured for another day and were subsequently fixed on day 6 when they had developed to the full blastocyst stage. Analysis of results Data were analysed by χ2-test or unpaired Student’s t-test, where appropriate. Results The patient’s characteristics and other factors are summarized in Table I. No significant differences for any of the parameters studied were found between the two groups. In Tables II and III, the results in terms of fertilization, embryo development, implantation rates and pregnancy rates of the culture under either 5% or 20% O2 are summarized. None of the investigated parameters was significantly different between the two groups. In 501 of the 1380 cycles, at least one surplus embryo was cultured to day 5 or 6. In the other 889 cycles, either no fertilization took place, available embryos were all transferred, surplus embryos were cryopreserved, or they were used in another study. In Table IV, development of surplus embryos to the blastocyst stage is summarized. The mean incidence of blastocyst formation per cycle in which surplus embryos were Downloaded from http://humrep.oxfordjournals.org/ by guest on March 14, 2016 Culture procedures Oocytes and embryos were cultured in 50 and 20 µl droplets, respectively, under mineral oil (Sigma, cat. no. M-8410). HTF medium was ‘in-house’ prepared according to Quinn et al. (1985), and supplemented with 8% (v/v) of a pasteurized human plasma protein solution (PPS) obtained from the Central Laboratory of the Blood Transfusion Service (Amsterdam, The Netherlands) as described by Huisman et al. (1992). IVF-50™ is a ready-to-use commercially available medium (Scandinavian IVF Science AB, Göteborg, Sweden). The incubator used was a Napco double-chamber 7300 model with separate gas controlling systems for both chambers (Boom BV, Meppel, The Netherlands). In one chamber, an atmosphere of 5% CO2 in air (~20% O2) was used, while in the other chamber a gas mixture of 5% CO2, 5% O2 and 90% N2 was used. The CO2 and O2 concentrations of both incubator chambers were regularly checked using a Servomex-570A Oxygen Analyzer (Servomex, Zoetermeer, The Netherlands) and a Normocap 200 CO2-Analyzer (Datex Medical Electronics, Hoevelaken, The Netherlands), and, if necessary, the incubator chambers were recalibrated. After incubation for 18–20 h, 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. At the second and third day after oocyte recovery, the developmental stage and morphological aspect of all embryos were assessed under an inverted microscope at 3200 magnification according to the criteria of Bolton et al. (1989). For each embryo, an embryo score was calculated by multiplying the morphological grade by the number of blastomeres (Steer et al., 1992). For each treatment cycle, the score of all embryos was averaged to obtain a mean embryo score (MES) (Steer et al., 1992). Embryo transfer was routinely performed on day 2 after ovum retrieval, or, in a minority of the cases, on day 3 for reasons of convenience to avoid transfers on Sundays. If available, two or three embryos, depending on the developmental stage and morphological appearance of the embryos, as well as on the age of the patient, were transferred. After transfer, any supernumerary embryos were cultured until the third day after ovum retrieval. Cryopreservation of supernumerary embryos was performed on the morning of the third day after insemination if one or more embryos had reached the 8-cell stage, and if they were of good morphological quality (grades 3 and 4; Bolton et al., 1989). Table I. Characteristics of the two study groups cultured under either 5% or 20% O2 Effect of oxygen on human IVF results Table II. Summary of results of the culture under either 5% or 20% O2: fertilization and embryo development at day 2 and day 3 of in-vitro development Table IV. Development to the blastocyst stage of surplus embryos cultured under either 5% or 20% O2 Gas phase Gas phase 5% O2 20% O2a Cycles No. of oocytes per cycleb Fertilization rate per cycleb,c 690 10.0 6 0.2 0.60 6 0.01 690 9.6 6 0.2 0.61 6 0.01 Embryonic development at day 2 Cleavage to ù4-cell stageb,d Morphologically normal embryosb,e Embryo score per cycle (MES)b 49 6 2 56 6 1 8.3 6 0.2 45 6 2 56 6 2 8.1 6 0.1 Embryonic development at day 3f Cleavage to ù6-cell stageb,g Morphologically normal embryosb,h Embryo score per cycle (MES)b 42 6 3 52 6 3 15.4 6 0.6 36 6 3 56 6 4 14.7 6 0.6 aNo Table III. Summary of results of the culture under either 5% or 20% O2: pregnancy and implantation rates Gas phase Cycles Embryo transfers Pregnancies (%)b,c Ongoing (ù12 weeks) pregnancies (%)b,d Multiple pregnancies (%)d,e Mean no. of embryos transferred per cycle (6 SEM) Total no. of transferred embryos Implantation sites (%)d,f Viable fetuses (ù12 weeks) (%)d,f 5% O2 690 649 (94.1) 184 (26.7) 155 (22.5) 41 (26.5) 2.62 6 0.04 1762 236 (13.4) 196 (11.1) 20% O2a 690 634 (91.9) 175 (25.4) 147 (21.3) 50 (34.0) 2.55 6 0.04 1757 246 (14.0) 204 (11.6) aNo significant differences were noted between the two groups. bValues in parentheses are percentages of total number of treatment cycles. cPositive urinary pregnancy test (sensitivity 50 IU/l HCG) at 16–18 days after ovum retrieval. dAs determined by ultrasound at 5 and ù12 weeks after ovum retrieval, respectively. eValues in parentheses are percentages of total number of ongoing pregnancies. fValues in parentheses are percentages of total number of embryos replaced. cultured was significantly higher in the group cultured under 5% O2. Of the embryos that developed a blastocoel, 236 of 310 (76%) embryos cultured under 5% O2, and 157 of 231 (68%) embryos cultured under 20% O2 were fixed. Reasons for not fixing were either that no consent was obtained from the patients, or embryos that were classified as early blastocysts on day 5 and subsequently cultured for another day, were found to be degenerated on day 6. The mean number of cells per blastocyst (as determined by the observation of a clear blastocoel under the microscope) was significantly higher in Fixation of blastocysts on day 5 Blastocysts fixed Fixation successfulc Cells per blastocystd Blastocysts consisting of at least 25 cellse Cells per confirmed blastocystf Fixation of blastocysts on day 6 Blastocysts fixed Fixation successfulc Cells per embryod Blastocysts consisting of at least 25 cellse Cells per confirmed blastocystf 247 1034 310 (30.0) 20% O2 254 1006 231 (23.0)h 25.8 6 2.0 20.4 6 1.9i 112 103 (92) 39.8 6 1.7 80 (78) 103 89 (86) 31.9 6 1.9j 54 (61)g 46.2 6 1.6 42.2 6 2.1 124 117 (94) 45.6 6 2.6 91 (78) 54 44 (81) 33.7 6 3.4j 25 (57)g 54.9. 6 2.6 49.1 6 4.6 aValues in parentheses are percentages of total number of surplus embryos cultured. bExpressed as mean percentage 6 SEM of number of embryos developing to the blastocyst stage on day 5 or 6/total number of surplus embryos cultured per cycle. cValues in parentheses are percentages of total number of blastocysts fixed. dExpressed as mean number 6 SEM of cells per successfully fixed embryo. eValues in parentheses are percentages of successfully fixed blastocysts. fOnly blastocysts with a minimum of 25 cells are taken into account. g,h,i,jSignificant difference when compared with culture under 5% O : χ2-test 2 (gP , 0.05; hP , 0.01), or unpaired Student’s t-test (iP , 0.05; jP , 0.001). the group cultured under 5% O2, both in blastocysts fixed on day 5, as well as those fixed on day 6. This difference was for the largest part due to the fact that significantly more blastocysts of the 20% O2 group consisted of ,25 cells. Discussion This prospective randomized study on 1380 consecutive IVF treatments shows that culture of human oocytes and embryos for the first 2 or 3 days of development under a gas phase of either 5% CO2/95% air (20% O2) or 5% CO2/90% N2/5% O2 does not result in significant differences between the two groups, neither in pregnancy rates, nor in implantation rates. Rates of fertilization and cleavage of the embryos also did not differ significantly between the two groups. These results are comparable to those reported in an earlier study in which oocytes and embryos were cultured in 1 ml medium instead of in microdroplets under an oil overlay (Dumoulin et al., 1995). It has been speculated that the use of small medium droplets under an oil phase could result in O2 levels in the medium close to detrimentally low levels, especially when large numbers of embryos or other cells (e.g. cumulus cells) are cultured together (Baltz and Biggers, 1991; Byatt-Smith et al., 1991). From these studies employing mathematical models it was calculated that human embryos possibly could become marginally hypoxic at lower O2 concentrations (Byatt-Smith et al., 1991). In our 467 Downloaded from http://humrep.oxfordjournals.org/ by guest on March 14, 2016 significant differences were noted between the two groups. 6 SEM. oocytes/total number inseminated oocytes per cycle. dPercentage of embryos developed to the 4-cell stage or more at 39–42 h after insemination or injection per cycle. ePercentage of embryos of good morphological quality (grades 3 and 4; Bolton et al., 1989) at 39–42 h after insemination or injection per cycle. fOnly cycles of which the embryo transfer was performed on day 3 (i.e. all embryos were cultured until day 3) were taken into account. gPercentage of embryos developed to the 6-cell stage or more at 63–66 h after insemination or injection per cycle. hPercentage of embryos of good morphological quality (grades 3 and 4; Bolton et al., 1989) at 63–66 h after insemination or injection per cycle. bResults are expressed as means cPercentage of number fertilized Cycles with at least one surplus embryo cultured Total no. of surplus embryos cultured Total no. of blastocysts observed on day 5 or 6a Incidence of blastocyst formation per cycleb 5% O2 J.C.M.Dumoulin et al. 468 O2 concentrations exert an effect during the later stages of preimplantation development only. To conclude, in a culture system of microdroplets of medium under oil, no beneficial effect on fertilization, embryonic development and pregnancy rates of culturing human oocytes and embryos for 2 or 3 days under 5% O2 as compared to atmospheric O2 concentrations was found. Only when embryos were cultured in vitro during the total preimplantation period, was a slight but significant improvement of development to the blastocyst stage found when 5% O2 was used. Acknowledgements 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-Jansen, MT; Gerard A.J.Dunselman, MD; Germaine Kengen, RN; Renate Kuijper, RN; Jacques Maas, MD; Nienke Muntjewerff, MD; Jolande A.Land, MD; Anja Winkens-Tersteeg, RN; Lucie C.P.van Wissen, MT. References Ali, J., Whitten, W.K. and Shelton, J.N. (1993) Effect of culture systems on mouse early embryo development. Hum. Reprod., 8, 1110–1114. Baltz, J.M. and Biggers, J.D. (1991) Oxygen transport to embryos in microdrop cultures. Mol. Reprod. Dev., 28, 351–355. Batt, P.A., Gardner, D.K. and Cameron, A.W.N. (1991) Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro. Reprod. Fertil. Dev., 3, 601–607. Bavister, B.D. (1995) Culture of preimplantation embryos: facts and artifacts. Hum. Reprod. Update, 1, 91–148. Berthelot, F. and Terqui, M. (1996) Effects of oxygen, CO2/pH and medium on the in vitro development of individually cultured porcine one- and twocell embryos. Reprod. Nutr. Dev., 36, 241–251. Betterbed, B. and Wright, R.W. (1985) Development of one-cell ovine embryos in two culture media under two gas atmospheres. Theriogenology, 23, 547–553. Bolton, V.N., Hawes, S.M., Taylor, C.T. and Parsons, J.H. (1989) 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 Fertil. Embryo Transfer, 6, 30–35. Byatt-Smith, J.G., Leese, H.J. and Gosden, R.G. (1991) An investigation by mathematical modelling of whether mouse and human preimplantation embryos in static culture can satisfy their demands for oxygen by diffusion. Hum. Reprod., 6, 52–57. Coonen, E., Dumoulin, J.C.M., Ramaekers, F.C.S. et al. (1994) Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in-situ hybridization. Hum. Reprod., 9, 533–537. Dumoulin, J.C.M., Vanvuchelen, R.C.M., Land, J.A. et al. (1995) Effect of oxygen concentration on in vitro fertilization and embryo culture in the human and the mouse. Fertil. Steril., 63, 115–119. Enginsu, M.E., Pieters, M.H.E.C., Dumoulin, J.C.M. et al. (1992) Male factor as determinant of in-vitro fertilization outcome. Hum. Reprod., 7, 1136–40. Fischer, B. and Bavister, B.D. (1993) Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J. Reprod. Fertil., 99, 673–679. Fukui, Y., McGowan, L.T., James, R.W. et al. (1991) Factors affecting the invitro development to blastocysts of bovine oocytes matured and fertilized in vitro. J. Reprod. Fertil., 92, 125–131. Gardner, D.K. and Lane, M. (1996) Alleviation of the ‘2-cell block’ and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum. Reprod., 11, 2703–2712. Gardner, D.K., Vella, P., Lane, M. et al. (1998) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil. Steril., 69, 84–88. Goto, Y., Noda, Y., Mori, T. and Nakano, M. (1993) Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radic. Biol. Med., 15, 69–75. Downloaded from http://humrep.oxfordjournals.org/ by guest on March 14, 2016 study, however, no evidence was found of any adverse effect when embryos were cultured under oil at 5% O2. In the present study we show that culture under 5% O2 results in significantly more human surplus embryos reaching the blastocyst stage, as well as a higher proportion of blastocysts consisting of a normal number of cells. In a study from Hardy et al. (1989) it was shown that the minimum number of cells in normally fertilized human blastocysts on day 5 of development was ~25 cells. The mean number of cells per blastocyst in their study was 58 on day 5, which is somewhat higher than in the present study (42–46 cells, Table IV). A possible reason for this discrepancy is the fact that all surplus embryos used in our study were of poor morphological quality. Another reason could be the fact that the culture media used in the present study are not as suitable as the recently developed sequential media to support the development of viable blastocysts in vitro (Gardner et al., 1998). The use of a low O2 concentration has been shown to be beneficial for embryo development in vitro to the blastocyst stage in various animal species, e.g. mouse (Quinn and Harlow, 1978; Pabon et al., 1989; Umaoka et al., 1992; Gardner and Lane, 1996); hamster (McKiernan and Bavister, 1990); rabbit (Li and Foote, 1993); rat (Kishi et al., 1991); pig (Berthelot and Terqui, 1996); sheep (Thompson et al., 1990); cow (Fukui et al., 1991; Liu and Foote, 1995), and goat (Batt et al., 1991). Also in the human, indirect evidence exists that a low O2 tension of 5% appears to enhance the blastulation rate of surplus embryos (Noda et al., 1994). However, other studies have failed to demonstrate the beneficial effect of low O2 on the development to the blastocyst stage in the mouse (NasrEsfahani et al., 1992; Ali et al., 1993), cat (Johnston et al., 1991), and sheep (Betterbed and Wright, 1985). Also it appears that different culture systems may require different O2 concentrations for optimal results. When cow or sheep embryos are co-cultured with other cells, 20% O2 is found to give better results as compared to 5% O2 in several studies (Fukui et al., 1991; Voelkel and Hu, 1992; Watson et al., 1994). Several other lines of evidence would suggest that low O2 concentrations are more suitable culture conditions for preimplantation embryos. Embryos develop in vivo under low O2 levels, as in the oviduct and uterus of various mammalian species O2 concentrations have been reported to be ~11–60 mmHg, which corresponds to ~1.5–9% O2 (Fischer and Bavister, 1993). Furthermore, a reduced O2 concentration of 5% has been shown to result in a slightly decreased formation of reactive oxygen species in mouse embryos as compared to 20% O2 (Goto et al., 1993). Reactive oxygen species have been implicated in the retardation of early embryo development in vitro (Johnson and Nasr-Esfahani, 1994; Tarı́n, 1996). The majority of studies thus indicate that culture under low O2 is beneficial for complete preimplantation embryonic development in vitro. However, little is known about the effect of a lower O2 concentration during the first 2 or 3 days of invitro development on postimplantation embryonic development and pregnancy rates. 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(1992) Effect of gas atmosphere on the development of one-cell bovine embryos in two culture systems. Theriogenology, 37, 1117–1131. Watson, A.J., Watson, P.H., Warnes, D. et al. (1994) Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere. Biol. Reprod., 50, 715–724. Received on June 26, 1998; accepted on October 15, 1998 469