MINISTRY OF EDUCATION MINISTRY OF DEFENSE VIETNAM MILITARY MEDICAL ACADEMY NGUYEN THI MINH STUDY ON THE EFFECTIVENESS OF BLASTOCYST CULTURE METHOD AT LOW OXYGEN CONCENTRATION OF 5% IN IN-VITRO FERTILIZATION Specialization: Biomedical Sciences Code: 9720101 SUMMARY OF PHD THESIS IN MEDICINET HANOI - 2019 This thesis was conducted at: Vietnam Military Medical Academy Supervisors: Assoc Prof Quan Hoang Lam, MD, PhD Prof Nguyen Viet Tien, MD, PhD Peer-review 1: Prof Trinh Binh, MD, PhD Peer-review 2: Prof Cao Ngoc Thanh, MD, PhD Peer-review 3: Assoc Prof Nguyen Duy Anh, MD, PhD The thesis will be defensed at Council of Vietnam Military Medical Academy at …… …… …… 2019 The thesis can be found at following library: - National library - Vietnam Military Medical Academy library INTRODUCTION OF THE THESIS Background The introduction of in vitro fertilization has brought happiness and hope to be a father and mother to infertile couples around the world However, in the early years of the birth from the 1980s to the mid-1990s, the success rate of this method was only about 20% In recent years, thanks to the development of ovarian stimulation methods, embryo transfer techniques and especially blastocyst culture techniques, the annual worldwide pregnancy rate has improved Blastocyst transfer not only increases the pregnancy rate but also reduces the rate of multiple pregnancies However, blastocyst culture still exists the risk of no embryo transfer for patients It has been found that oocytes and embryos exist, develop in oviduct and uterine environment at 2-8% oxygen While, in the culture system normally 20% oxygen concentration like in the air is likely to be toxic to the embryos Reducing the concentration of oxygen provided to the embryo culture system, facilitating embryo development Currently, a number of studies in the world have shown that embryo culture in IVF in low oxygen levels (5%) increases the rate of embryos on the third day to the blastocyst stage and increases implantation rate and clinical pregnancy rate In Vietnam up to now, there has been no research on culturing blastocysts at low oxygen levels For that reason, “Study on the effectiveness of blastocyst culture method at low oxygen concentration of 5% in IVF” with purposes following: To compare the quality of embryos between culture at a low 5% oxygen concentration and 20% air oxygen concentration To evaluate pregnancy outcome of transfer of blastocysts at low 5% oxygen concentration and blastocysts at 20% oxygen concentration New contributions of the thesis There is no domestic studies in the aspect of embryo culture in low oxygen concentration This is a dedicated study, in the long term from ovarian controlled stimulation to live birth rate with fresh embryo transfer and frozen embryo transfer as well The results of this thesis again confirm the value of low oxygen concentration embryo culture; help other ART centers orienting culture strategy It is recommended to transfer blastocysts that were cultured in low oxygen in order to improve pregnancy outcomes and reduce multiple pregnancies Structure of the thesis The thesis consists of 122 pages, including: Introduction (2 pages); Chapter - Backgraound (33 pages); Chapter 2: Materials and Methodology (22 pages); Chapter 3: Results (23 pages); Chapter 4: Discussion (39 pages); Conclusion (2 pages); Recommendation: page; Contributions and limitations of the topic (2 pages); The thesis has 46 tables of data, 10 images of embryos; 129 References (4 Vietnamese documents, 125 English documents), appendices of research form and patient list Chapter OVERVIEW 1.3 Some benefits and limitations of blastocyst culture 1.3.1 Benefits of blastocyst culture: Culturing embryos to blastocyst stage with the aim of getting close to human natural physiology to improve pregnancy rate, live births rate and limiting the number of transfer embryos to reduce the rate of multiple pregnancies The blastocyst culture is also significant in the technique of preimplantation diagnosis and screening Recently, top quality blastocysts play an important role in the culture of human stem cells through trophectoderm biopsy for the treatment of some medical diseases 1.3.2 Limitations of blastocyst culture Beside the above benefits, prolonged blastocyst culture is having risk that there will be no embryo to the blastocyst stage even the embryo is degenerated, leading to no embryo for transfer 1.4 Effect of high oxygen concentration on embryo development Oxidative phosphorylation reactions consume oxygen to produce the energy supplied to the cell, while also secreting free radicals (generated from the release of high energy electrons when breaking down the electronic transport chain) These free radicals are extremely reactive factors and toxic to cell biochemistry, including the genome 1.4.1 Effect of free radicals generated from oxygen on embryo development In normal conditions, about - 4% of oxygen molecules turn into free radicals, which destroy cellular macromolecules such as DNA, protein and lipids The accumulation of damaged macromolecules leads to cell aging All changes at the molecular level can contribute to the expression of oxygen toxicity at the cell level 1.4.2 Solutions for reducing the toxicity of oxygen in the embryo culture system (1) Reducing the oxygen during embryo culture and embryo development: use tri-gas embryo culture incubators or use an embryo culture incubators with premixed low oxygen (2) Reduce the culturing time to reduce the negative effects of oxygen produced by sperm metabolism (3) Change the enviroment culture fomula including ingredients that resist the negative effects of oxygen 1.5 Studies around the world relate to the embryo culture at low oxygen concentration There were still controversies in low oxygen culture worldwide Recently, a meta-analysis from Sobrinho D.B.G et al in 2011 compared IVF outcomes between low oxygen (5%) and air oxygen (20%) culture The results were shown below: * Compare the implantation rate between studies + The rate of fertilization between the two groups in the studies was not different + The implantation rate of embryo transfer on day 2, day and blastocyst in other studies had no statistically significance (4 studies) + The implantation rate of blastocyst transfer on day 2, day between the study groups showed no statistically significance (4 studies) + However, the implantation rate of blastocyst transfer in the 5% oxygen group was higher, the difference was significance (2 studies) * Compare the rate of ongoing pregnancy rate + The overall rate of ongoing pregnancy on day 2, day and blastocyst in the 5% oxygen concentration group tended to be higher than that of the 20% oxygen concentration group However, the difference is not statistically significant + The overall of ongoing pregnancy of embryos transfer on day 2, day between both groups had no statistically different + The overall of ongoing pregnancy of embryos transfer between both groups had no statistically different The author argued that more RCT studies are needed to compare the embryo culture at low oxygen concentrations with cultured embryos of oxygen concentration like in the air to confirm the effect of embryo culture on low oxygen Chapter MATERIALS AND METHODOLOGY 2.1 Study subjectives and place of study 2.1.1 Study subjectives All IVF patients were at the age of under 38 years, had normal uterus without polyps, having at least good embryos on the third day, easy transferation, without blood on catheter, agreed to participate in our study The patients were divided to randomly groups: - Group I: Patients having embryos that were cultured in an incubators with oxygen concentration like in the air (20% O2, 6% CO2) - Group II: Patients having embryos that were cultured in incubators with tri-gas and low oxygen concentration (5% O2, 6% CO2, 89 % N2) 2.1.2 Study sites National Assisted Reproductive Technology Center -National Hospital of Obstetrics and Gyneacology 2.1.3 Study time: from 1/2011 to 5/2015 2.2 Methods 2.2.1 Study design This is a prospective randomized controlled study 2.2.2 Sampling n= P ( zα / 2 P (1 − P ) + z β P1 (1 − P1 ) + P2 (1 − P2 ) ) ∆2 = (P1+P2)/2; zα/2 is the z value of the normal distribution for the probability of α/2 zβ is the z value of the normal distribution for the probability of β ∆ = p1 - p2 N: Number of patients in each group p1 = 70% pregnancy rate of patients who had embryo culture at 5% oxygen concentration (Kea B et al (2007)) p2 = 33% pregnancy rate of patients who had embryo culture at 20% oxygen concentration (Kea B et al (2007)) α = 0,05 the statistical significance, the probability of making type I error with CI 99% and zα/2 = 2,58 β= 0,05 = the probability of making type II error and zβ = 1,645 After calculatation, the final sample size is 61,7 The minimum number of patients for each study group is 62 In this study, we collected n = 86 patient samples for each group, ensuring a minimum sample size 2.2.3 Study protocol Figure 2.1 describes the study protocol for 172 patients in seperated groups Diagram 2.1 research design 2.3 Environment and study equipments 2.3.1 Environment of embryo culture Using equipment of G5 SeriesTM, made by Vitrolife, Sweden 2.3.2 Embryo culture incubators In this study, we used Heracell embryo culture incubator 150 litters, 2-gas for 20% oxygen concentration and Heracell embryo culture cabinet 150 litters, tri-gas for 5% oxygen concentration 3.4 Evaluate embryo morphology Morphology assessment and embryo classification at day 2, day and day which have been using in our Assisted Reproductive Technology Center followed Landuyt L V (2010) criteria 10 3.4.1 Evaluating the quality of cleavage stage: Evaluation of embryonic morphology in the early division stage based on the following criteria: speed of cleavage, size of blastomeres, fragments, granularity, syncronization of blastomere divisions, multi-nucleation, vacuoles Embryos on day 2, day are divided into grade: + Grade top embryo + Grade good embryo + Grade moderate embryo + Grade bad embryo 3.4.2 Evaluation of embryos at blastocyst stage (day 5): The blastocyst is evaluated based on the following criteria: morphology of inner cell mass – ICM and trophectoderm – TE, embryo expansion The blastocyst is divided into categories as follows: Table of blastocysts classification Classification Top (Grade 4) Description • • Good (Grade 3) Moderate (Grade 2) • • Embryo expansion grade ≥ ICM & TE Grade: AA; AB • • Embryo expansion grade ≥ ICM & TE Grade: BA; BB; Embryo expansion grade 0.05) 3.2.2 The relationship between fertilization techniques and embryo quality of day of the 5% Oxygen group Table 3.8 Fertilization techniques and embryo quality of day of the 5% Oxygen group IVF ICSI p Grade 5,9 ± 2,2 5,0 ± 2,4 > 0,05 Grade 1,4 ± 1,7 1,6 ± 1,8 > 0,05 Grade 1,6 ± 2,3 1,5 ± 2,3 > 0,05 Grade 0,0 ± 0,2 0,4 ± 0,8 < 0,05 The results showed that the Grade of ICSI group was significantly higher with p 0,05) 3.2.4 The mean of day-3 embryos to day-5 blastocysts, rate of blastulation 16 Table 3.10 The mean of day-3 embryos to day-5 blastocysts, rate of blastulation Oxy 5% ( Total of day-3 embryos to day-5 X ± SD) Oxy 20% ( X p ± SD) 314 345 3,7 ± 0,8 4,0 ± 1,1 day-5 blastocysts (3 – 6) ( – 8) Blastulation rate (%) 90,04 ± 74,44 ± 0,0 17,86 26,63 79,90 ± 73,61 ± 0,0 26,04 35,30 blastocysts The mean of day-3 embryos to The rate of top and good quality blastocysts (%) 0,0 The blastulation rate and the number of top and good quality blastocysts (Grade – 4) in the group of 5% oxygen concentration is significantly higher with p < 0,05 3.2.5 The quality of embryos on day between the two groups Table 3.11 The quality of embryos on day in the two groups Oxy 5% Oxy 20% Grade p X X ( ± SD) ( ± SD) Grade 1,7 ± 0,9 1,6 ± 0,9 0,7 Grade 1,8 ± 0,8 1,8 ± 0,8 0,9 Grade 1,2 ± 0,6 1,5 ± 0,7 0,1 Grade 1 1,6 ± 0,6 0,1 The mean of embryos between Grades in the two groups is similar (P> 0.05) 17 3.2.6 The mean of embryos transfer and the endometrial thickness in fresh blastocyst transfering in two groups Table 3.13 The mean of embryos transfer and the endometrial thickness in fresh blastocyst transfering in two groups Oxy 5% Oxy 20% ( The mean of embryos transfer The mean of grade – embryos The endometrial thickness (mm) X ± SD) 3,3 ± 0,5 (2 – 4) 2,7 ± 0,9 (0 – 4) 11,4 ± 2,1 (8 – 17,8) ( X ± SD) 3,2 ± 0,6 (2 – 4) 2,5 ± 0,9 (0 – 4) 10,9 ± 1,8 (0 – 15) p 0,2 0,2 0,5 The number of mean embryos transfer and the endometrial thickness between two groups is similar (with p > 0,05) 3.3 Comparison of pregnancy outcomes of patients with fresh blastocyst transfer in two groups 3.3.1 Pregnancy outcomes of patients with fresh blastocyst transfer in two groups The rate of pregnant patients in the embryo culture group at 5% oxygen concentration is higher than the 20% oxygen concentration group but the difference has no statistical significant with p> 0.05 Table 3.14 Pregnancy rates of patients transferring blastocysts (patients with hCG ≥ 25 IU) Oxy 5% Oxy 20% Pregnancy rates p (n=86) (n=86) (hCG ≥ 25 IU) n % n % Not pregnant 34 39,4 46 53,5 Pregnant 52 60,5 40 46,5 0,054 Total 86 100 86 100 18 The biochemical pregnancy rate of the 5% oxygen culture group was higher than the embryo culture group at 20% oxygen concentration but not statistically significant (p>0.05) Table 3.16 Clinical pregnancy rate of patients transferring blastocysts Oxy 5% Oxy 20% Clinical p (n=86) (n=86) pregnancy n % n % Not pregnant 37 43,0 51 59,3 Pregnant 49 57,0 35 40,7 0,03 Total 86 100,0 86 100,0 The clinical pregnancy rate of the 5% oxygen culture group was significantly higher than the embryo culture group at 20% oxygen concentration (p>0.05) 3.3.2 Implantation rate of blastocyst transfer Table 3.17 Implantation rate of blastocyst transfer Implantation rate Oxy 5% Oxy 20% p 24,9% 20,7% Implantation > 0,05 (72/289) (58/279) The implantation rate of blastocyst in the 5% oxygen concentration group was higher than that in the 20% oxygen concentration, but there was no significant difference with p > 0,05 3.3.6 The proportion and the condition of live births in blastocyst transfer Table 3.21 Live birth rate of blastocyst transfer Oxy 5% Oxy 20% Survival rate p (n=86) (n=86) n % n % 0,0 The number of live births 41 47,7 29 33,7 child 24 58,5 16 55,2 children 16 36,6 13 44,8 0,4 children 4,9 0,0 The group of embryo culture with 5% oxygen had 41 patients with live births, the 20% oxygen concentration group had 29 patients The 19 survival rate in the 5% oxygen group was 20% higher than the oxygen group, but the difference was not statistically significant (with p = 0.06) Table 3.22 Number of live births of two blastocyst transfer groups Oxy 5% Oxy 20% Survival result p (n=86) (n=86) n (41) % n (29) % male 14 16,3 10,5 0,3 female 10 11,6 8,1 0,4 male – female 10,5 4,7 0,1 males 7,0 3,5 0,3 females 1,2 7,0 0,6 females 1,2 0,0 0,3 Total 59 47,7 42 33,7 0,06 The total number of live births in the embryo culture group with 5% oxygen concentration was 59 from 41 mothers and in the embryo culture group with 20% oxygen concentration was 42 from 29 mothers However, the difference is not statistically significant with p> 0.05 The gender ratio in live births of two distinct groups is not statistically significant Table 3.23 The mean weight of infants from blastocyst transfer The mean weight First child Oxy 5% n 41 17 Second child Third child X ( ± SD) 3091±361,1 (1600 3700) 2429,4±459, (1400–3100) 1000±412,3 (800-2700) Oxy 20% n ( 29 13 X p ± SD) 3040±642,3 (1200–3800) 0, 2230,8±534, (1700–3200) 0, 20 The weight of infants in the 5% oxygen concentration group is higher than the 20% oxygen concentration group, but the difference is not statistically significant with p>0.05 3.4 Pregnancy outcomes after first and second frozen embryos transfer The results showed the similar rate of clinical pregnancy and live birth between groups, p > 0.05 Table 3.29 Pregnancy outcomes after two times frozen embryo transfer Oxy 5% Oxy 20% p (n=52) (n=53) n % n % Frozen day-2 embryos transfer 51 98,1 50 94,3 0,9 Frozen day-5 embryos transfer 0,9 5,6 Clinical pregnancy 14 26,9 15 28,3 0,2 Live births 11 21,2 13 24,5 0,7 Average weight of a child 3112 ± 235,4 2800 ± 548 0,8 Average weight of twins 2033,3 ± 450,9 2123 ± 657 3.5 Cumulative pregnancy in the ovarian stimulation cycle of two groups in the study Table 3.30 The rate of cumulative results of pregnancy and live birth of the cycle of fresh and frozen embryo transfer Oxy 5% Oxy 20% p(n=86) (n=86) values n % n % ET day 49 57,0 35 40,7 0,03 Clinical 1st, 2nd FET 14 26,9 15 28,3 0,2 pregnancy Total 63 73,2 50 58,1 0,02 ET day 41 47,7 29 33,7 0,06 st nd Live births , FET 11 21,2 13 12,8 0,8 Total 52 60,4 42 48,8 0,1 The cumulative clinical pregnancy rate of 5% oxygen concentration group was significantly higher with p 0.05 CHAPTER 4: DISCUSSION 4.1 Characteristics of research subjects In our study, both group had similar about: The specified techniques, infertility characteristics, maternal age, ovarian stimulation characteristics, the distribution of these characteristics between the two groups were not statistically significant This ensures reliability when comparing the results obtained: number of oocytes, fertilization rate, number of embryos, number of cryopreserved embryos, number of embryos transferred, implantation rate, pregnancy rate, and the rate of multiple pregnancies, the rate of live birth between different groups 4.2 Discussion about the quality of embryos between two study groups 4.2.1 The quantity and quality of day-2 embryos of patients in two groups The mean number of day-2 embryos obtained in two groups was not statistically significant Our research results a embryosre similar to those of Kovacic B et al (2010) The quality of embryos in the two groups is the same, the difference in the mean number of embryos by Grades (Grade 4, Grade 3, Grade 2, Grade 1) is not statistically significant with p> 0.05 In both groups, the mean of Grade embryos in day is the highest one, opposite to the mean of Grade embryos This proves that our embryo culture method and embryo culture system reach the standard so the mean of good embryos is high, the mean of poor embryos is low 4.2.2 The number of patients who had cryopreserved day-2 embryos, the mean amount of cryopreserved day-2 embryos The number of patients who had cryopreserved day-2 embryos and the mean of cryopreserved day-2 embryos was similar in both group with p>0.05 22 4.2.3 The relationship between fertilization techniques and embryo quality of day of the 5% Oxygen group In our study, comparing the relationship between the fertilization technique and the quality embryos of day of the 5% Oxygen group showed that the number of bad embryos (Grade 1) on day in patients was made by ICSI technique was significantly higher than the rate of bad embryos (grade 1) on day in patients performed by IVF technique According to a randomized study of Sun H L 2017, statistically higher rates fertilization rate obtained in IVF cases compared to ICSI cases However, the quality of embryos between the two groups is not different 4.2.4 The rate of blastulation The rate of blastocyst formation in the embryo culture group with 5% oxygen concentration (90.04 ± 17.86) was higher than the rate of blastocyst formation in the cultured group of 20% oxygen concentration (74.44 ± 26.63), the difference was statistically significant (with p 0.05 (Table 3.23) Whether or not embryo culture at 5% oxygen level improves the weight of children born from the IVF method, other studies are 24 needed to answer this question, we have not seen any research in the world on this issue 4.4 The outcomes of first and second frozen embryo transfer After two cycles of frozen embryo transfer, the rate of clinical pregnancy between the 5% and 20% oxygen concentration groups was not statistically different The rate of live birth between two groups is similar with p> 0.05 (21.2 compared to 24.5) (table 3.29) 4.5 Cumulative clinical pregnancy in fresh and frozen embryo transfer In our study, the rate of cumulative clinical pregnancy in fresh and frozen embryo transfer stage from the 5% oxygen concentration group (73.2%) was significantly higher than the 20% oxygen concentration group (58.1%) p < 0.05 (table 3.30) The cumulative live birth rate of the embryo group with low oxygen concentration (60.4%) was higher than the 20% oxygen concentration group (48.8%) However, the difference is not statistically significant with p> 0.05 (table 3.30) CONCLUSION Study on 172 IVF patients divided randomly into two groups: 5% oxygen concentration culture group and 20% oxygen culture group, showed: Comparing the quality of embryos between the cultured group at low oxygen concentrations of 5% and the group cultured at oxygen concentrations of 20%: + The quality of day-2 embryos of the cultured group with low oxygen concentration of 5% was not significantly different from that of the cultured group at 20% oxygen concentration + The rate of blastocyst formation in the culture group with 5% oxygen concentration was statistically significantly higher than in the culture group with 20% oxygen concentration (90.04% ± 17.86 vs 74.44% ± 26.63) 25 + The quality of blastocysts for embryo culture at low oxygen concentrations of 5% is higher than that of cultured embryos at 20% oxygen concentration (Top and good quality blastocyst rates were 79.90% ± 26.04 compared with 73.61% ± 35.30) The difference is statistically significant Pregnancy outcomes, live birth rate of blastocysts transfer cultured at low 5% oxygen concentration and blastocysts in 20% oxygen concentration + The clinical pregnancy rate of blastocysts transfer at low 5% oxygen levels was statistically significantly higher than that of cultured blastocyst transfer of 20% oxygen concentration (57% vs 40.7%) Meanwhile, the rate of biochemical pregnancy in the 5% oxygen concentration culture group tended to be lower than that the 20% oxygen concentration group (3.5% versus 7.0%) + The live birth rate of embryo transfer culturing with low oxygen concentration group tended to be higher than that of 20% oxygen concentration group (44.7% versus 33.7%) however not statistically significant + The mean weight of infants in ET culturing with low oxygen concentration was not significantly different, but tended to be higher than that of the group with 20% oxygen concentration + The cumulative clinical pregnancy rate (fresh and frozen embryos transfer) of the 5% oxygen concentration group (73.2%) was significantly higher than that in the 20% oxygen concentration group (58.1%) + The cumulative rate of live births in the group with 5% oxygen concentration (60.4%) tended to be higher in the culture group with 20% oxygen concentration (48.8%) 26 27 RECOMMENDATONS Apply blastocyst culture by 5% low oxygen concentratrion More than one blastocyst transfer should not be concerned to avoid multiple pregnancies Research in bigger sample size, multi-center and in a long term to evaluate the effect of transfer of blastocysts in low oxygen concentrations Extend the study for post-thaw embryo culture and early embryo culture (from day 0) continuously at 5% low oxygen concentration LIST OF PUBLICATIONS FROM THE THESIS Nguyen Thi Minh, Nguyen Viet Tien, Quan Hoang Lam (2018), Clinical effects of embryos culture in low oxygen concentration, Journal of Military Phamarco-medicine, 43(5): 175 – 179 Nguyen Thi Minh, Quan Hoang Lam, Nguyen Viet Tien (2018) The rate of blastulation and embryo quality in low oxygen culture Journal of Military Phamarco-Medicine, 13(4): 110 – 114 ... groups Oxygen 5% Oxygen 20% Characteristics p X X ( ± SD) ( ± SD) FSH day (mIU/mL) 5, 65 ± 1,87 5, 95 ± 1,80 0,3 (2,2 – 9,8) (2,2 – 13 ,5) LH day (mIU/mL) 4 ,53 ± 3,03 5, 12 ± 3,13 0,1 (1,3 – 15, 9)... blastocysts (patients with hCG ≥ 25 IU) Oxy 5% Oxy 20% Pregnancy rates p (n=86) (n=86) (hCG ≥ 25 IU) n % n % Not pregnant 34 39,4 46 53 ,5 Pregnant 52 60 ,5 40 46 ,5 0, 054 Total 86 100 86 100 18 The... embryos day Oxy 5% ( X Oxy 20% p X ± SD) ( ± SD) 5, 4 ± 2,3 6,1 ± 2 ,5 Grade 0,1 (0 – 14) (3 – 15) 2 ,5 ± 1,6 2 ,5 ± 1,7 Grade 0,9 (0 – 7) (0 – 8) 2,9 ± 2,4 2,3 ± 1,3 Grade 0 ,5 (0 – 10) (0 – 6) 1 ,5 ± 0,9