A study of the effect of an extremely low oxygen concentration on the development of human embryos in assisted reproductive technology

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1 Reprod Med Biol (2010) 9: DOI /s ORIGINAL ARTICLE A study of the effect of an extremely low oxygen concentration on the development of human embryos in assisted reproductive technology Koji Nakagawa Asako Shirai Yayoi Nishi Rie Sugiyama Yasushi Kuribayashi Rikikazu Sugiyama Masato Inoue Received: 9 January 2010 / Accepted: 17 March 2010 / Published online: 17 April 2010 Ó Japan Society for Reproductive Medicine 2010 Abstract Purpose To determine whether embryos cultured with a low oxygen level (2%) brought about beneficial effects on the outcome of ART. Methods This is a sequential case control embryo-culture study. Embryos were cultured either with a gas mixture containing 2% O 2, 5% CO 2, and 93% N 2 (low-oxygen group) or 5% O 2,5%CO 2, and 90% N 2 (conventional group). From January 2008 to September 2008, 873 fertilized oocytes were obtained from 250 patients in the lowoxygen group and from October 2008 to March 2009, 730 fertilized oocytes were obtained from 213 patients in the conventional group. The outcomes of ART were compared between two groups. Results The cleavage rate in the low-oxygen group (94.4%) was similar to that (94.7%) in the conventional group. The mean number of blastomeres on Day 3 in the low-oxygen group (mean ± SE) was 6.5 ± 1.9, and this was significantly lower than in the conventional group (6.8 ± 1.9, p \ 0.05). Moreover, the low-oxygen group produced worse quality embryos, on the basis of the significantly higher embryo grade 2.1 ± 0.6 versus 1.9 ± 0.6, p \ 0.001, in 5% oxygen. The pregnancy and miscarriage rates in the low-oxygen group were 22.3 and 20.8%, respectively, which were statistically similar to the outcomes in the conventional group. Conclusions Overall, culture of embryos at the low oxygen level did not significantly improve ART results compared with embryos grown in 5% oxygen. The study K. Nakagawa (&) A. Shirai Y. Nishi R. Sugiyama Y. Kuribayashi R. Sugiyama M. Inoue Division of Reproductive Medicine, Sugiyama Clinic, Ohara, Setagaya, Tokyo , Japan nakagawa-jiko@spice.ocn.ne.jp suggests that a low oxygen level worsens embryo morphology but does not impair embryo viability. Keywords ART Embryo development Embryo quality High oxygen Low oxygen Introduction In assisted reproductive technology, a gas phase containing either atmospheric oxygen (*20%) or a reduced (5%) oxygen level is widely used for the culture of both gametes and embryos. Similar success has been reported for both oxygen levels. For example, a previous report showed that in the human no beneficial effect was found when embryos were cultured in 5% O 2 as compared with an atmospheric O 2 concentration [1]. Moreover, a prospective randomized study on 1,380 consecutive IVF treatments showed that culture of human oocytes and embryos for 2 or 3 days using a gas phase containing either 5% CO 2 /95% air (20% O 2 )or5%co 2 /90% N 2 /5% O 2 did not result in significant differences between the two groups [2]. However, it has been demonstrated that development of embryos from several mammalian species, for example mouse, sheep, goat, and cattle, is improved by culture in 5% oxygen [3]. Compared with in-vivo genital tract conditions, one of the important differences during in-vitro culture is the oxygen concentration in which oocytes are fertilized and embryos develop. The most commonly used system for cell culture contains 5% CO 2 in air. This system approaches 20% O 2 in the gaseous phase, which is much higher than the oxygen concentration reported in the mammalian oviduct. Fischer and Bavister [4] demonstrated that intraluminal oxygen tension in the oviducts and uteri of rhesus monkeys, hamsters, and rabbits was less than half that of

2 164 Reprod Med Biol (2010) 9: atmospheric concentration, ranging from *1.5 to 9%. Furthermore, an O 2 concentration of 5% has been shown to reduce the formation of reactive oxygen species in mouse embryos compared with cultures in 20% O 2 [5]. On the basis of these reports, it is possible that even 5% O 2 may be too high for embryo culture, and it may be postulated that even lower O 2 levels may be more suitable for in-vitro development. In this study, we prepared a gas mixture which contained 2% O 2,5%CO 2, and 93% N 2 and compared fertilization and embryo development, and ART outcomes, with results obtained using a conventional gas mixture containing 5% O 2,5%CO 2, and 90% N 2. Materials and methods Patients and ovarian stimulation For this study, we enrolled 463 patients who had 463 treatment cycles during ART treatment at the Division of Reproductive Medicine at the Sugiyama Clinic between January 2008 and March The number of previous treatment cycles was not used as a criterion for participant selection. Approval of the institutional review board and informed consent from all patients undergoing ART treatment were obtained. All patients were treated with either a mild stimulation protocol or a gonadotropin-releasing hormone agonist (GnRH-a) long protocol. The mild stimulation protocol [6] and GnRH-a long protocol with recombinant-fsh [7] have been fully described elsewhere, and a brief description follows. The mild stimulation protocol consisted of 50 mg clomiphene citrate (Serophen, Merck-Serono, Tokyo, Japan) each day for 5 days from the 3rd to the 7th day of the patient s menstrual cycle; 150 international units (IU) of recombinant (rec) follicle-stimulating hormone (rec- FSH; Follistim, Schering-Plough, Tokyo, Japan) per day were administered to the patients on the 3rd, 5th, and 7th day of their menstrual cycles; and additional rec-fsh (150 IU/day) was administered on the basis of follicular growth assessed by ultrasonography. For ovarian hyperstimulation using the GnRH-a long protocol, 600 lg/day buserelin acetate (Buserecur, Fuji Pharma, Tokyo, Japan) was administered intranasally starting in the midluteal phase of the pretreatment cycle and this was continued until the day of human chorionic gonadotropin (hcg) injection; on days 3 and 4 of the menstrual cycle, 225 IU of rec-fsh were administered; and IU rec-fsh were administered on subsequent days until a dominant follicle reached 16 mm in diameter. Regardless of the ovarian stimulation protocol, when the dominant follicles reached C17 mm in diameter, 10,000 IU human chorionic gonadotropin (hcg; Gonadotropin, Mochida, Tokyo, Japan) was administrated, and oocyte retrieval was performed 35 h later. IVF/ICSI procedure and embryo culture The IVF procedures used in this study have been described elsewhere [8]. Oocytes were retrieved transvaginally using a needle-guide technique, controlled by ultrasonography. All follicles with a mean diameter [15 mm were aspirated individually, using a 17-gauge needle connected to a tube and a machine for suction (Flush pump/oocyte Incubator; Airey, Fukushima, Japan). The needle was removed after aspiration of each follicle. The aspiration was interrupted and a new syringe was used if blood appeared in the tube connected to the syringe, thus avoiding contamination by blood. All follicles were irrigated with culture medium. Semen was produced by masturbation and, after washing with culture medium, motile sperm were separated using a min swim-up period. In vitro insemination was performed by incubation of each oocyte with motile sperm within 5 6 h after oocyte collection. When there was evidence of male factor infertility, intra-cytoplasmic sperm injection (ICSI) was performed instead of in-vitro insemination [9]. Depending on the study period, oocytes retrieved from their follicles were incubated either under a low oxygen gas mixture that contained 2% O 2,5%CO 2, and 93% N 2 or a conventional (control) gas mixture of 5% O 2,5%CO 2, and 90% N 2. These gas mixtures were prepared commercially (Saisan, Saitama, Japan). Between January 2008 and September 2008, all oocytes were incubated under low O 2 and between October 2008 and March 2009, all oocytes were incubated under the conventional 5% O 2. Oocytes and embryos were cultured without a covering of mineral oil in standard culture dishes (BD Falcon , BD Bioscience, NC, USA) using an Airtech incubator (CGI K, Tokyo, Japan). In each group oocytes were incubated for several hours before insemination or ICSI was performed. Oocytes were examined using a dissecting microscope h after insemination or ICSI to identify fertilization by the presence of two pronuclei and extrusion of the second polar body. For both fertilization and embryo culture, the commercially available medium known as HUCUM, (Nipro, Osaka, Japan) was used. All embryos were cultured for 72 h after insemination or ICSI until either embryo transfer or cryopreservation was performed. Embryo assessment and ART results Embryos were assessed 72 h after insemination or ICSI and one or two embryos were transferred into the uterus of each patient. Embryos containing seven or eight mononucleated

3 Reprod Med Biol (2010) 9: blastomeres that were regular, approximately equal in size, with no fragmentation, were classified as grade 1. Those with similar mononucleated blastomeres and less than 10% fragmentation were evaluated as grade 2. Embryos containing fewer than seven or unequal blastomeres, with no fragmentation, were grade 3. Those with unequal blastomeres and more than 15% fragmentation were assigned to grade 4. This system of embryo assessment was modified from the classification described by Veeck [10]. Residual embryos, of sufficient quality, were cryopreserved using the vitrification method. On the day of embryo transfer, and 3 and 7 days after embryo transfer, each patient received an injection of 100 mg progesterone for luteal support. In addition, a combination of estrogen and progesterone was administered orally for 12 days after embryo transfer. A pregnancy was recognized when the development of a gestational sac was detected by transvaginal ultrasound imaging on the 21st day after oocyte retrieval. In this study a miscarriage was defined as a pregnancy loss before 22 weeks of gestation. Statistical analysis was performed using an unpaired t test and a chi-squared test, and statistical significance was set at P \ Results In the low-oxygen concentration gas phase group 250 cycles yielded 873 fertilized oocytes whereas in the conventional oxygen concentration group 213 cycles produced 730 fertilized oocytes. The clinical features of the patients in each group are summarized in Table 1. The average age, the indications for ART, the type of ovarian stimulation, and fertilization methods were similar in both groups of patients (Table 1). The mean number of retrieved oocytes per patient, and the mean number of fertilized oocytes per patient were also similar in both groups. The fertilization and cleavage rates in the low-oxygen group were 73.2 and 94.4%, respectively and these results were not significantly different (80.2 and 94.7%) from those in the conventional group (Table 2). Although the fertilization rates of ICSI in the low-oxygen and conventional groups were similar, this rate of conventional insemination in the low-oxygen groups was significantly lower than that in the conventional group (Table 2). The mean number of blastomeres per embryo on Day 3 in the low-oxygen group was 6.5 ± 1.9 (mean ± SE), and this was significantly lower than that in the group cultured in 5% O 2 (6.8 ± 1.9, p \ 0.05). Moreover, the mean embryo grade on day 3 in the low-oxygen group (2.1 ± 0.6) was significantly higher than that in the 5% O 2 group (1.9 ± 0.6, p \ 0.001) (Table 2). The percentage of morphologically good quality embryos on day 3 in the lowoxygen group was 60.4%, and this was similar to that in the higher O 2 group (59.6%). However, the mean number of blastomeres and the embryo grade of transferred embryos on day 3 in the low-oxygen group (7.1 ± 0.1 and 2.0 ± 0.03, respectively) were equal to those in the 5% O 2 group (7.1 ± 0.1 and 1.8 ± 0.03, respectively), and the pregnancy rate and miscarriage rate in the low-oxygen group was 22.3 and 20.8%, respectively, and these outcomes were not significantly different from those in the conventional group (20.0 and 10.0%, respectively) at the current stage of the study. Discussion More than 30 years have elapsed since the first success of human in vitro fertilization and embryo-transfer treatment [11], and in that period the oocytes and embryos of several mammalian species have been cultured using a gas phase Table 1 A summary of clinical data for the 2% O 2 and the 5% O 2 study groups OPU follicular oocyte pick up, CC clomiphen citrate, recfsh recombinant follicle-stimulating hormone, GnRH gonadotropinreleasing hormone a Mean age and range 2% Oxygen group 5% Oxygen group Patient number OPU cycles Mean age (years) a 37.9 (28 46) 37.9 (29 45) n.s. Indications for ART Tubal factor n.s. Male factor n.s. Endometriosis n.s. Unexplained infertility n.s. IVF/ICSI 112/138 93/120 n.s. Ovarian stimulation (cycles) CC with recfsh n.s. RecFSH with GnRH-antagonist n.s. Long protocol/short protocol 28/19 19/15 n.s.

4 166 Reprod Med Biol (2010) 9: Table 2 A summary of the results obtained in the 2% O 2 and 5% O 2 study groups a Morphologically good quality embryos at the 7-cell stage or more and grade 1 or 2 of Veeck s classification b To compare the pregnancy and miscarriage rates between the 2% O 2 and the 5% O 2 groups under the same conditions, only the cycles with embryo transfer on Day 3 were used for evaluation of both groups 2% Oxygen group 5% Oxygen group Total number of oocytes collected 1,257 1,177 Mean number of retrieved oocytes n.s. Total number of oocyte fertilized Mean number of fertilized oocytes n.s. Fertilization rate (%) n.s. Fertilization rates in conventional insemination (%) p \ Fertilization rates in ICSI (%) n.s. Mean number of cleaved oocytes n.s. Cleavage rate (%) n.s. Mean number of morphologically good quality n.s. embryos on day 3 a The percentage of good quality embryos on day % 59.6% n.s. Mean number of blastomeres per embryo on Day 3 ±SE 6.5 ± ± 1.9 p \ 0.05 Mean grade of embryo on Day 3 ±SE 2.1 ± ± 0.6 p \ Mean number of transferred embryos n.s. Mean number of blastomeres per embryo transferred 7.1 ± ± 0.1 n.s. on Day 3 ±SE a Mean grade of transferred embryo on Day 3 ±SE 2.0 ± ± 0.03 n.s. Number of pregnancies/number of embryo transfers b 23/103 20/100 Pregnancy rate (%) n.s. Number of miscarriage/number of pregnancies b 5/23 2/20 Miscarriage rate (%) n.s. containing atmospheric O 2 (approximately 20%). Advances in oocyte and embryo culture have suggested that a lower oxygen concentration may improve embryo development. To provide evidence for this proposal, studies comparing atmospheric with reduced (5 7% O 2 ) have been reported for the culture of mouse, bovine, goat and human embryos [2, 12, 13]. Reducing the oxygen concentration from 20 to 7% significantly increased the number of blastomeres and enabled most goat embryos to develop into expanded and hatched blastocysts in vitro [14]. In an earlier study, Quinn and Harlow [15] found that reduced oxygen levels increased blastocyst formation in the mouse, and that concentrations of 2.5 5% were optimum. Moreover, a recent report indicated that a reduced oxygen concentration between 1 and 5% enhanced blastocyst development in bovine and pig embryos [16, 17]. In this study, the fertilization rates of ICSI in the lowoxygen and conventional groups were similar, but the rate of conventional insemination in the low-oxygen groups was significantly lower than that in the conventional group. We speculated that this difference occurred because both cumulus cells and sperm consume O 2 during conventional insemination, especially under extremely low O 2 condition. The mean number of blastomeres per embryo was lower, and the mean embryo grade on Day 3, was significantly higher in the low-oxygen group. These results indicate that human embryos cultured in the presence of 2% oxygen developed at a slower rate and were of worse quality, as judged by their morphology, than embryos cultured in the presence of 5% oxygen. However, if only grades 1 and 2 were considered, then there was no significant difference in the percentage of morphologically good quality embryos in the 2 and 5% oxygen groups. Perhaps the better quality embryos survive and progress similarly to day 3 of development at low and conventional levels of oxygen. Indeed, this study has shown that embryos cultured at a low oxygen tension (2% O 2 ) did not result in reduced pregnancy rates when compared with embryos cultured at the conventional 5% O 2 level. In contrast, embryos developing in low oxygen seemed to miscarry at twice the rate although the markedly different proportions were not statistically significant at the present stage. However, if this trend continued with higher numbers of pregnancies, the results would indicate that the low oxygen level impaired implantation survival. It is widely believed that the best ART outcomes are obtained with human oocytes and embryos when they are cultured under conditions that mimic the environment in the fallopian tube. The paper by Fischer and Bavister in 1993 provided basic information on the optimum culture of gametes and zygotes in vitro. Their studies showed that embryos developed in vivo at low oxygen levels, because they reported that in the oviduct and uterus of various

5 Reprod Med Biol (2010) 9: mammalian species O 2 tensions ranged from 11 to 60 mmhg, which corresponds to 1.5 9% O 2 [4]. It should be noted that in the experiments mentioned above, O 2 levels were measured in rhesus monkeys, hamsters, and rabbits, but not in humans. Thus, the oxygen concentration in the human fallopian tube, where fertilization and early embryo development occurs, has not yet been determined. This new knowledge, based on animal studies, was applied in ART clinics although it is possible that the real oxygen concentration in the human fallopian tube may be lower than 5%. Towards the end of the 1990s, many groups working in ART clinics, reduced the oxygen concentration for embryo culture from atmospheric to the 5% O 2 level based on the animal studies. However, IVF results derived from the reduced (5%) O 2 concentration were similar to those obtained with atmospheric O 2 concentrations [1, 2]. But recently, it was established that low-oxygen culture resulted in an improved blastocyst formation rate compared with cultures under atmospheric O 2 concentration [18 20]. On the basis of these results, many groups began to use a gas phase containing 5% CO 2,5%O 2 and 90% N 2, and consider this to be optimum for culture of human embryos. From the findings in this study, showing the onset of embryo deterioration at the 2% oxygen level, it may be postulated that optimum oxygen levels are in the vicinity of 2 5%, however, further study is necessary in order to determine the optimum oxygen level for embryo culture. At the lower levels, reduced oxidative stress may be counterbalanced by the onset of an anoxic stress. It would also worth determining whether better quality embryos are being selected by their ability to retain viability under the lower oxygen environment. However, a number of questions still remain. Is this conventional gas phase optimum for culture of the human gamete and zygote? Is the real oxygen tension in the human fallopian tube and uterine cavity actually 5%? Or is the real oxygen tension in the human fallopian tube and uterine cavity less than 5%? A theoretical basis for this could be that there is a need to maintain low levels of free-radical generation, to minimize intracellular damage in vivo. This proposal is supported by reports that the inclusion in media of free radical scavengers, for example hypotaurine and penicillamine, increased the number of blastocysts formed from cultured mouse zygotes [21]. It was this rationale that encouraged us to evaluate a very low oxygen level for embryo culture in our laboratory. A reduction of the oxygen concentration has been shown to slightly reduce the formation of reactive oxygen species in mouse embryo cultures compared with the use of atmospheric gas conditions [5]. In this study, however, the actual concentration of the reactive oxygen species was not measured, but it can be postulated that their formation decreased at low oxygen levels. The reasons why embryo quality was poor when cultured under extremely low O 2 concentration were unclear, but we plan to measure the real concentration of reactive oxygen species at various oxygen levels. It is also important to measure mitochondrial activity in embryos cultured under extremely low O 2 conditions. In our clinic, embryo transfer was routinely performed on day 3, and residual embryos were cryopreserved on the same day. Thus, embryos were not routinely cultured to the blastocyst stage, so the number of embryos cultured for extended periods was small in each group. But even in the small sample size, blastocyst formation rates were similar in both the 2 and 5% O 2 groups (data not shown). In conclusion, the culture of human embryos at a low oxygen tension (2%) did not have a beneficial effect on our IVF results compared with cultures at the conventional 5% oxygen level. Moreover, it is possible that the culture of embryos at lower oxygen levels may be useful for selecting viable embryos. We are planning studies to evaluate this idea in choosing embryos for day 3 single-embryo transfers. Acknowledgments We wish to thank Dr Alex Lopata for his helpful advice and for editing the paper. References 1. Dumoulin JC, Vanvuchelen RC, Land JA, Pieters MH, Geraedts JP, Evers JL. Effect of oxygen concentration on in vitro fertilization and embryo culture in the human and the mouse. Fertil Steril. 1995;63: Dumoulin JC, Meijers CJ, Bras M, Coonen E, Geraedts J, Evers JL. Effect of oxygen concentration on human in vitro fertilization and embryo transfer. Hum Reprod. 1999;14: Bavister BD. Culture of preimplantation embryos: facts and artifacts. 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J Assist Reprod Genet. 2006;23: Nakagawa K, Yamano S, Moride N, Yamashita M, Yoshizawa M, Aono T. Effect of activation with Ca ionophore A23187 and puromycin on the development of human oocytes that failed to

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