Clinical effects of embryos culture in low oxygen concentration – Nguyen Thi Minh

Journal of military pharmaco-medicine no5-2018 175 CLINICAL EFFECTS OF EMBRYOS CULTURE IN LOW OXYGEN CONCENTRATION Nguyen Thi Minh*; Nguyen Viet Tien*; Quan Hoang Lam** SUMMARY In in-vitro-fertilization, human embryos have been cultured in incubator which are closely monitored level of components including temperature, humidity and especially level of CO2 and O2 concentration. Some centers are using 20% O2 while others apply only 5% O2 and with this low level of O2, IVF outcome is promising. However, there is no evidence about the optimized level of O2 concentration in human embryos culture. Objectives: To compare pregnancy rate, live birth rate between 2 groups is 5% O2 and 20% O2. Methods: This is a randomized clinical trial study among 172 patients who were divided into 2 groups: embryos cultured in 5% and 20% oxygen concentration. Before culture, using FSH to stimulate ovaries. Oocytes retrieval was done after hCG injection 36 hours. Fertilization was made by conventional IVF or ICSI. On day 3, 3 - 5 top quality embryos were selected to culture to day 5 in both groups. Residual embryos were frozen by vitrification method. Results: No differences in characteristics of ovarian reserve, ovarian stimulation and embryos transfer in both groups. Clinical pregnancy rate in 5% oxygen group was 57.0%, significantly higher than 40.7% in 20% oxygen group. Live birth rate in 5% oxygen group was 47.7%, higher than 33.7% in 20% oxygen group but no significantly. Conclusion: Clinical pregnancy rate on day 5 in 5% oxygen group was higher than that in 20% oxygen group. Live birth rate in 5% oxygen group was higher than in 20% oxygen group but no significantly. * Key words: Pregnancy rate; Live birth rate; Low oxygen concentration in IVF. INTRODUCTION The technique of in-vitro fertilization (IVF) has been increasingly advanced in order to improve the success rate. Many studies in the world state that blastocyst transfer is close to physiology and selected embryos with good quality and high viability will help increase the rate of implantation, pregnancy and rate of live births. Moreover, many experimental studies show that the concentration of oxygen is only about 5 - 8% in the uterus and only < 2% in the tubal. On the other hand, in the field of assisted reproduction, embryo culture with 20% oxygen concentration has been performed for about 30 years and until now many centers continue to use this culture system [Error! Reference source not found., 3, 9, 10]. Many empirical studies show that the oxygen concentration of the air can negatively affect the embryo because the oxidation or high concentrations of oxygen can create free radicals that are harmful to the embryo. Studies in animals have demonstrated that embryo transfer at low oxygen levels will result in better embryos, higher implantation rate and pregnancy rate. * ** Corresponding author: Nguyen Thi Minh (@gmail.com) Date received: 27/03/2018 Date accepted: 21/05/2018 Journal of military pharmaco-medicine no5-2018 176 Beginning with the research on animal embryos which concluded positive results, some authors have cultured human blastocysts in low oxygen levels and the results were similar while some other studies conclude reversely [7, 10]. In Vietnam, most of the assisted reproduction centers are transfering embryonic day 2, day 3 embryos cultured in 20% oxygen concentration. Hence, this study is carried out with the goal: To compare the clinical pregnancy rate and live birth rate of transferred the day-5 embryos cultured with 5% oxygen concentration and those of transferred day-5 embryos cultured in the 20% oxygen concentration. SUBJECTS AND METHODS - Studying in warranted randomized clinical trials. - Subjects: 172 patients treated with IVF were randomly assigned into 2 groups of embryos - embryos cultured with 5% oxygen concentration (team) and those cultured with 20% oxygen concentration (control group). - Methods: The patients received ovarian stimulation with recombinant FSH, then they are injected with hCG to mature ovum and oocyte are aspired 36 hours after hCG injection. Ovum aspiration is fertilized with sperm by classical methods or ICSI. The rating is conducted 16 -18 hours after fertilization. 3 - 5 good quality embryos were selected among embryos cultured to day 2 to continue growth to day 3, then 3 - 5 good quality embryos of which will be selected to continue growth until day 5. 2 - 3 embryos will be transferred on day 5, the remaining good day. 5 embryos (if any) are frozen, so are day-2 embryos by vitrification method. + Assessing the quality of embryos at day 5 and transferring embryo with ultrasound guidance: Determining pregnancy rate by hCG test 14 days after embryo transfer. All the cases with positive hCG will be tested by ultrasound on the 28th day after embryo transfer to determine the clinical pregnancy. + Determination of clinical pregnancy rate: the number of patients with a sac/the number of patients with transferred embryo. + Identify the live birth rate: the number of women who delivered live children/the number of patients with transferred embryo. * Data were collected in the sample and analyzed using SPSS 16.0. The average values are compared between the two groups by t test and the ratios are compared by 2 test. The difference is considered statistically significant when p < 0.05. - Study period: from June, 2011 to September, 2012. - Research area: Central Obstetrics Hospital. - Ethics in research: Studies over the world have concluded that embryo culture at low oxygen levels did not affect embryo. Before conducting this study, patients were given advice, agreed and signed a form of consent to participate in the study. The patient information is kept confidential, only for research. Journal of military pharmaco-medicine no5-2018 177 RESULTS AND DISCUSSION 1. Characteristics of study subjects. Table 1: Characteristics Oxygen group 5% Oxygen group 20% p Age (years) 30.16 ± 3.6 30.85 ± 3.41 0.202 c Cycle day 3 FSH (IU/L) 5.65 ± 1.87 5.95 ± 1.8 0.31 f Cycle day 3 LH (IU/L) 4.53 ± 3.03 5.13 ± 3.06 0.079 f Cycle day 3 E2 (pg/ml) 44.29 ± 40.79 37.1 ± 14.56 0.284 f Antral follicle count (AFC) 11.23 ± 5.28 10.66 ± 4.51 0.81 f Ovarian reserve of the patients in the study was good, and there were no differences between the two study groups. 2. Features of ovarian stimulation. Table 2: Characteristics and outcome of ovarian stimulation. Characteristics Oxygen group 5% Oxygen group 20% p Total dose of FSH 2176.2 ± 504.9 2166.6 ± 584.3 0.91 c E2 concentrations on hCG day (UI/l) 6020.1 ± 3185.1 6705.7 ± 7538.7 0.88 f Number of oocytes retrieved 10.58 ± 3.97 10.86 ± 3.9 0.64 c Number of mature oocytes (MII) 10.42 ± 3.97 10.78 ± 3.87 0.55 c Fertilized rate (%) 89.3 ± 14.4 91.1 ± 11.9 0.534 f Number of day 2 embryos 8.72 ± 3.49 9.4 ± 3.5 0.21 c The difference was not statistically significant between the two study groups in terms of total FSH dose used, E2 concentration, day hCG injection, the oocytes retrieved, the mature oocytes, fertilized rate and the number of embryos transfered, with p > 0.05. 3. Embryo transfer results. Table 3: Embryo transfer and pregnancy results. Characteristics Oxygen group 5% Oxygen group 20% p Number of embryos transfered 3.36 ± 0.57 3.24 ± 0.59 0.19 c Endometrial thickness (mm) 11.35 ± 2.12 10.99 ± 1.77 0,41 f Clinical pregnancy rate 49 (57.0%) 35 (40.7%) 0.033 a Survival birth rate 41 (47.7%) 29 (33.7%) 0.063 a Journal of military pharmaco-medicine no5-2018 178 DISCUSSION Currently, most of the embryos cultured lab in Assisted Reproduction Centers use cultured oxygen concentrations similar to that of oxygen in the air (~ 20%) because it does not require additional equipment. Recently, however, some authors suggest that low oxygen levels (~ 5%) is truly new and similar physiological oxygen concentrations in the uterine and tubal heart, so in such embryo culture, we can get more qualified embryos, thereby improving the outcome of IVF. It is from the experimental results, as well as from the hypothesis that some authors who have conducted studies comparing the pregnancy rate and fertilization rate of embryos in conditions of low oxygen levels concluded that at low oxygen concentration conditions, the pregnancy rate is higher than that at high oxygen levels [4, 5, 6]. The authors prove that the concentration of oxygen in the air can produce free radicals, which in turn affect the development of the embryo [4]. Because of this, it is possible to culture embryos in low oxygen levels for more positive results than in higher oxygen concentration. Our study showed that both groups of study patients were at the same age and infertility duration. Take ovarian reserve indicators into consideration, including FSH concentrations, LH levels and the number of secondary follicles were similar in both groups with p > 0.05 (table 1). Comparing ovarian stimulation results between the two groups, we found no difference in total FSH dose used, estradiol levels hCG day, the average number of oocyte aspiration as well as the average number of embryos obtained (table 2). This proves that the ovarian reserve of the patients was similar in both groups. The results in table 3 show the average number of embryos transferred and the endometrium thickness are not statistically significant between 2 different groups, with p > 0.05. However, the clinical pregnancy rate of embryos cultured in low oxygen levels (5%) is 57.0%, statistically higher than that of embryos cultured at a concentration of 20% which is 40.7 %, with p < 0.05. The pooled analysis by Bontekoe S et al (2012) in 7 studies with 2,422 patients also result in higher clinical pregnancy rate and live birth rate of embryo cultured at low oxygen levels compared to those of embryo cultured in the oxygen concentration of 20% [2]. In our study, the live birth rate of embryos cultured at low oxygen levels tend to be higher than that of embryos cultured at 20% oxygen concentration, although the difference is not statistically significant (p = 0.063). The results of our study are similar to those by Meintjes M et al (2009) [8]. CONCLUSION Clinical pregnancy rate of day 5 embryos cultured at 5% oxygen concentration is higher than that of embryos cultured in 20% oxygen concentration with statistical significance. The live birth rate of embryos cultured in 5% oxygen concentration tends to be higher than that of embryos cultured in 20% oxygen concentration. Journal of military pharmaco-medicine no5-2018 179 REFERENCES 1. Blake D., Farquahar C., Johnson N et al. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Published by John Wiley and Sons. 2009. 2. Bontekoe S., Mantikou E., Van W.M et al. Low oxygen concentrations for embryo culture in assisted reproductive technologies. Cochrane Database of Systematic Reviews. 2012, Issue 7. Art. No.: CD008950. DOI:10.1002/14651858.CD008950. 2012. 3. Chui-Yee Fong, AriffBongso, Soon-Chye Ng et al. Ongoing normal pregnancy after transfer of zona-free blastocysts: Implications for embryo transfer in the human. Human Reproduction. 1997, Vol.12 no.3, pp.557-560. 4. James WC, Michael H. Toxic effects of oxygen on human embryo development. Human Reproduction. 2000, Vol. 15, (Suppl. 2), pp.199-206. 5. Javier IG, Soledad S, Luis N. Beneficial effect of reduced oxygen concentration with transfer of blastocysts in IVF patients older than 40 years old. Health. 2010, Vol.2, No.9. 6. John CMD, Chantal JJM, Marijke B. Effect of oxygen concentration on human in- vitro fertilization and embryo culture. Human Reproduction. 1999, Vol.14 No.2, pp.465-469. 7. Kovacic B, Vlaisavljevic V. Influence of atmospheric versus reduced oxygen concentration on development of human blastocysts in vitro: a prospective study on sibling oocytes. Reproductive Biomedicine Online. 2008, 17 (2), pp.229-236. 8. Meintjes M, Samuel J.C et al. A controlled randomized trial evaluating the effect of lowered incubator oxygen tension on live births in a predominantly blastocyst transfer program. Hum Repod. 2009, 24, pp.300-307. 9. Mark P, Michael J.T. Fewer risks, new hope: The reality of blastocyst transfer. Copyright IVF. Com, Alanta, GA, USA. 2007. 10. Meintjes M, Hill K, Johnston S. The effect of lowered incubator oxygen tension on implantation, pregnancy and cryopreservation rates in a predominantly day-5 embryo transfer program. Fertility and Sterility. 2000, 74 Suppl, 1 (3), pp.256.