Objectives: To compare pregnancy rate, live birth rate between 2 groups 5% O2 and 20% O2. Subjects and method: This is a randomized clinical trial study among 172 patients who were divided into 2 groups: embryos cultured in 5% and 20% oxygen concentrationture in low oxygen concentration.
Journal of military pharmaco-medicine n08-2017 CLINICAL EFFECTS OF EMBRYOS CULTURE IN LOW OXYGEN CONCENTRATION Nguyen Thi Minh*; Nguyen Viet Tien*; Quan Hoang Lam** SUMMARY Objectives: To compare pregnancy rate, live birth rate between groups 5% O and 20% O2 Subjects and method: This is a randomized clinical trial study among 172 patients who were divided into groups: embryos cultured in 5% and 20% oxygen concentration Before culture, using FSH to stimulate ovaries Oocytes retrieval after hCG injection 36 hours Fertilization by conventional IVF or ICSI On day 3, - top quality embryos were selected to culture to day in both group Residual embryos were frozen by vitrification method Results: There were no differences of characteristics, of ovarian reserve, ovarian stimulation and embryos transfer in both groups Clinical pregnancy rate in 5% oxygen group was of 56.97%, significantly higher than 40,69% in 20% oxygen group Live birth rate in 5% oxygen group was (46.51%) higher than (32.55%) in 20% oxygen group but no significantly Conclusion: Clinical pregnancy rate on day 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 * Keywords: In vitro fertilization; Pregnancy rate; Live birth rate: Low oxygen concentration INTRODUCTION In the field of in vitro fertilization (IVF), techniques increasingly approach more physiological conditions in order to improve the success rate Many studies in the world state that blastocyst transfer is closely related to physiology and selects embryos of good quality andhigh viability, helping increase the rate of implantation, pregnancy, and rate of live births Moreover, many experimental studies showed that the concentration of oxygen is only about - 8% in the heart of tubal, and only < 2% in the uterus 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 [3, 9, 10] Many empirical studies showed 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 had demonstrated that embryo transfer at low oxygen levels will result in better embryos, higher implantation rate and pregnancy rate Beginning with the research on animal embryos which concluded positive results, some authors have cultured human blastocysts in low oxygen levels and the results are similar while some other studies conclude reversely [7, 10] * National Assisted Reproductive Technology Center ** Vietnam Military Medical University Corresponding author: Nguyen Thi Minh (minhn77@gmail.com) Date received: 20/08/2017 Date accepted: 30/09/2017 251 Journal of military pharmaco-medicine n08-2017 In Vietnam, most of the assisted reproduction centers are transforming embryonic day 2, day embryos cultured in 20% oxygen concentration Hence, this study is carried out with the aim: To compare the clinical pregnancy rate and live birth rate of transferred the day embryos cultured with 5% oxygen concentration and those of transferred day embryos cultured in the 20% oxygen concentration SUBJECTS AND METHODS Subjects 172 patients treated with IVF were randomly divided into groups of embryos: Embryos cultured with 5% oxygen concentration (team) and those cultured with 20% oxygen concentration (control group) Methods Randomized clinical trials - The patients received ovarian stimulation with recombinant FSH, then they were 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 - good quality embryos were selected among embryos cultured to day to continue growing to day 3, then - good quality embryos of which will be selected to continue growing until day - embryos will be transferred on day 5, the remaining good 252 day embryos (if any) are frozen, so are day embryos by vitrification method - Assessing the quality of embryos at day and transfering embryo with ultrasound guidance Determining pregnancy 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 were compared by test The difference was 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 n08-2017 RESULTS Characteristics of researching subjects Table 1: Characteristics Oxygen group 5% Oxygen group 20% p Age (years) 30.23 ± 3.9 30.44 ± 4.6 0.75 Cycle day FSH (IU/L) 5.53 ± 1.8 5.5 ± 2.0 0.92 Cycle day LH (IU/L) 5.1 ± 4.8 4.8 ± 2.9 0.67 Cycle day E2 (pg/ml) 39.74 ± 19.2 40.29 ± 17.0 0.84 Antral follicle count (AFC) 12.03 ± 4.8 11.52 ± 4.0 0.45 Ovarian reserve of the patients in the study was good, and there was no difference between the two groups of patients studied in terms of patient’s characteristics Features of ovarian stimulation Table 2: Characteristics and outcome of ovarian stimulation Characteristics Oxygen group 5% Oxygen group 20% p 2155.52 ± 470.9 2189.01 ± 595.6 0.68 6170.91 ± 3458.53 6663.42 ± 7570.35 0.58 Number of oocytes retrieved 10.28 ± 3.4 10.69 ± 3.9 0.47 Number of mature oocytes (MII) 9.84 ± 3.2 10.17 ± 3.8 0.55 92.44 ± 10.2 93.7 ± 9.1 0.40 8.94 ± 3.2 9.4 ± 3.4 0.36 Total dose of FSH E2 concentrations on hCG day Fertilized rate Number of day embryos The difference was not statistically significant between the two studying 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 Embryo transfer results Table 3: Embryo transfer and pregnancy results Characteristics Oxygen group 5% Oxygen group 20% p Number of embryos transfered 3.37 ± 0.53 3.27 ± 0.56 0.21 Endometrial thickness (mm) 11.15 ± 1/66 10.92 ± 1.98 0.37 Clinical pregnancy rate 56/97% (49/86) 40.69% (35/86) 0.047 Survival rate 46/51 % (40/86) 33.72% (29/86) 0.087 253 Journal of military pharmaco-medicine n08-2017 DISCUSSION Although currently, most of the embryos cultured Lab in assisted reproduction centers use cultured oxygen concentrations similar to the concentration 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 in vitro fertilization 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 studied patients were at the same age and infertility duration Considered ovarian reserve indicators, 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 254 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 showed the average number of embryos transferred and the endometrium thickness were not statistically significant between different groups, with p > 0.05 However, the clinical pregnancy rate of embryos cultured in low oxygen levels (5%) was 56.97%, statistically higher than that of embryos cultured at a concentration of 20% which was 40.69 %, with p < 0.05 The pooled analysis by Bontekoe S et al (2012) in studies with 2,422 patients also results 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 was not statistically significant (p = 0.061) The results of our study were similar to the results of research by Meintjes M et al (2009) [8] CONCLUSION Clinical pregnancy rate of day 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 culturedin 5% oxygen concentration tends to be higher than that of embryos cultured in 20% oxygen concentration Journal of military pharmaco-medicine n08-2017 REFERENCES Blake D, Farquahar C, Johnson N, Proctor M Cleavage stage versus blastocyst stage embryo transfer in assisted conception Published by John Wiley and Sons 2009 Bontekoe S, Mantikou E, van Wely 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 Chui-Yee Fong, Ariff Bongso, SoonChye 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 James W.C, Michael H Toxic effects of oxygen on human embryo development Human Reproduction 2000, Vol 15, Suppl 2, pp.199-206 Javier I.G, 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 John C.MD, Chantal J.JM, Marijke B Effect of oxygen concentration on human in vitro fertilization and embryo culture Human Reproduction 1999, Vol 14, No 2, pp.465-469 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 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 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 embryo transfer program Fertility and Sterility 2000, 74, Suppl, (3), p.256 255 ... that of embryos cultured in 20% oxygen concentration, with statistical significance The live birth rate of embryos culturedin 5% oxygen concentration tends to be higher than that of embryos cultured... > 0.05 However, the clinical pregnancy rate of embryos cultured in low oxygen levels (5%) was 56.97%, statistically higher than that of embryos cultured at a concentration of 20% which was 40.69... (2012) in studies with 2,422 patients also results 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