1 Department of Obstetrics & Gynaecology and 2 Department of Molecular Cell Biology & Genetics, Academic Hospital, University of Maastricht, Maastricht, The Netherlands
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Abstract |
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Key words: embryo culture/oxygen/preimplantation development
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Introduction |
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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 (Byatt-Smith 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.
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Materials and methods |
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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 <3x106/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 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 3435 h after HCG administration. Insemination or ICSI was performed ~5 h after oocyte retrieval.
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-50TM 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 1820 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 x200 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
).
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 a rim of cells surrounding a large cavity of extracellular fluid accumulated within the embryo) were fixed and stained with 4',6-diamidino-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 2-test or unpaired Student's t-test, where appropriate.
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Results |
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Discussion |
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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 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 (4246 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 (Nasr-Esfahani 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 ~1160 mmHg, which corresponds to ~1.59% 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
; Tarín, 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 in-vitro development on postimplantation embryonic development and pregnancy rates. The present study shows that, although culture under 5% O2 indeed leads to slightly improved preimplantation embryonic viability in the human, 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.
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.
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Acknowledgments |
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Notes |
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3 To 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
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References |
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Submitted on June 26, 1998; accepted on October 15, 1998.