Currently, a number of studies in the world have shownthat embryo culture in IVF in low oxygen levels 5% increases therate of embryos on the third day to the blastocyst stage and increas
Trang 1NGUYEN THI MINH
STUDY ON THE EFFECTIVENESS OF BLASTOCYST CULTURE METHOD AT LOW OXYGEN CONCENTRATION OF 5%
Trang 21 Assoc Prof Quan Hoang Lam, MD, PhD
2 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 MedicalAcademy at …… …… …… 2019
The thesis can be found at following library:
- National library
- Vietnam Military Medical Academy library
Trang 3INTRODUCTION OF THE THESIS
1 Background
The introduction of in vitro fertilization has brought happinessand hope to be a father and mother to infertile couples around theworld However, in the early years of the birth from the 1980s to themid-1990s, the success rate of this method was only about 20% Inrecent years, thanks to the development of ovarian stimulationmethods, embryo transfer techniques and especially blastocystculture techniques, the annual worldwide pregnancy rate hasimproved Blastocyst transfer not only increases the pregnancy ratebut also reduces the rate of multiple pregnancies However,blastocyst culture still exists the risk of no embryo transfer forpatients It has been found that oocytes and embryos exist, develop inoviduct and uterine environment at 2-8% oxygen While, in theculture system normally 20% oxygen concentration like in the air islikely to be toxic to the embryos Reducing the concentration ofoxygen provided to the embryo culture system, facilitating embryodevelopment Currently, a number of studies in the world have shownthat embryo culture in IVF in low oxygen levels (5%) increases therate of embryos on the third day to the blastocyst stage and increasesimplantation 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 2
Trang 42 New contributions of the thesis
There is no domestic studies in the aspect of embryo culture inlow oxygen concentration
This is a dedicated study, in the long term from ovariancontrolled stimulation to live birth rate with fresh embryo transferand frozen embryo transfer as well
The results of this thesis again confirm the value of lowoxygen concentration embryo culture; help other ART centersorienting culture strategy It is recommended to transfer blastocyststhat were cultured in low oxygen in order to improve pregnancyoutcomes and reduce multiple pregnancies
3 Structure of the thesis
The thesis consists of 122 pages, including: Introduction (2pages); Chapter 1 - Backgraound (33 pages); Chapter 2: Materialsand Methodology (22 pages); Chapter 3: Results (23 pages); Chapter4: Discussion (39 pages); Conclusion (2 pages); Recommendation: 1page; Contributions and limitations of the topic (2 pages); The thesishas 46 tables of data, 10 images of embryos; 129 References (4Vietnamese documents, 125 English documents), appendices ofresearch form and patient list
Trang 5Chapter 1 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 gettingclose to human natural physiology to improve pregnancy rate, livebirths rate and limiting the number of transfer embryos to reduce therate of multiple pregnancies
The blastocyst culture is also significant in the technique of implantation diagnosis and screening Recently, top quality blastocystsplay an important role in the culture of human stem cells throughtrophectoderm biopsy for the treatment of some medical diseases
pre-1.3.2 Limitations of blastocyst culture
Beside the above benefits, prolonged blastocyst culture is havingrisk that there will be no embryo to the blastocyst stage even theembryo is degenerated, leading to no embryo for transfer
1.4 Effect of high oxygen concentration on embryo development
Oxidative phosphorylation reactions consume oxygen to producethe energy supplied to the cell, while also secreting free radicals(generated from the release of high energy electrons when breakingdown the electronic transport chain) These free radicals are extremelyreactive 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 1 - 4% of oxygen molecules turninto free radicals, which destroy cellular macromolecules such asDNA, protein and lipids The accumulation of damagedmacromolecules leads to cell aging
All changes at the molecular level can contribute to theexpression of oxygen toxicity at the cell level
Trang 61.4.2 Solutions for reducing the toxicity of oxygen in the embryo culture system
(1) Reducing the oxygen during embryo culture and embryodevelopment: use tri-gas embryo culture incubators or use an embryoculture incubators with premixed low oxygen
(2) Reduce the culturing time to reduce the negative effects ofoxygen produced by sperm metabolism
(3) Change the enviroment culture fomula including ingredientsthat 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 2011compared 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 studieswas not different
+ The implantation rate of embryo transfer on day 2, day 3 andblastocyst in other studies had no statistically significance (4 studies).+ The implantation rate of blastocyst transfer on day 2, day 3 betweenthe 2 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 3 andblastocyst in the 5% oxygen concentration group tended to be higherthan that of the 20% oxygen concentration group However, thedifference is not statistically significant
Trang 7+ The overall of ongoing pregnancy of embryos transfer on day
2, day 3 between both groups had no statistically different
+ The overall of ongoing pregnancy of embryos transferbetween both groups had no statistically different
The author argued that more RCT studies are needed to comparethe embryo culture at low oxygen concentrations with culturedembryos of oxygen concentration like in the air to confirm the effect
of embryo culture on low oxygen
Chapter 2 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 normaluterus without polyps, having at least 3 good embryos on the third day,easy transferation, without blood on catheter, agreed to participate in ourstudy The patients were divided to 2 randomly groups:
- Group I: Patients having embryos that were cultured in anincubators with oxygen concentration like in the air (20% O2, 6% CO2)
- Group II: Patients having embryos that were cultured inincubators with 3 tri-gas and low oxygen concentration (5% O2, 6%CO2, 89 % N2)
Trang 82.2.2 Sampling
2
2 2 2 1 1 2
/ 2 (1 ) (1 ) (1 )(
∆
−+
−+
N: Number of patients in each group
p1 = 70% pregnancy rate of patients who had embryo culture at5% oxygen concentration (Kea B et al (2007))
p2 = 33% pregnancy rate of patients who had embryo culture at20% 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 = 1,645After calculatation, the final sample size is 61,7 The minimumnumber of patients for each study group is 62 In this study, wecollected n = 86 patient samples for each group, ensuring a minimumsample size
2.2.3 Study protocol
Figure 2.1 describes the study protocol for 172 patients in 2 seperatedgroups
Trang 9Diagram 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 150litters, 2-gas for 20% oxygen concentration and Heracell embryoculture cabinet 150 litters, tri-gas for 5% oxygen concentration
3.4 Evaluate embryo morphology
Morphology assessment and embryo classification at day 2, day
3 and day 5 which have been using in our Assisted ReproductiveTechnology Center followed Landuyt L V (2010) criteria
Trang 103.4.1 Evaluating the quality of cleavage stage:
Evaluation of embryonic morphology in the early division stagebased on the following criteria: speed of cleavage, size of
blastomeres, fragments, granularity, syncronization of blastomere
divisions, multi-nucleation, vacuoles Embryos on day 2, day 3 are
divided into 4 grade:
+ Grade 4 top embryo+ Grade 3 good embryo + Grade 2 moderate embryo + Grade 1 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 4 categories as follows:
Table of blastocysts classification
Top (Grade 4) • Embryo expansion grade ≥ 3
• ICM & TE Grade: AA; ABGood (Grade 3) • Embryo expansion grade ≥ 3
• ICM & TE Grade: BA; BB;
Moderate (Grade 2)
• Embryo expansion grade <3
• Or Embryo expansion grade ≥ 3, with ICM & TE Grade: AC; BC; CB; CC;
Trang 11Chapter 3: RESULTS
3.1 Characteristics of research subjects
3.1.1 Characteristics of infertile patients in two groups
Causes of infertility of patients
Chart 3.1 Causes of infertility of patients in two groups
The cause of infertility due blockage of Fallopian tubes is the maincause The causes of infertility in the two groups are similar with p > 0.05
Type of infertility of patients in two groups
Chart 3.2 Type of infertility of patients in two groups
The rate of primary infertility and secondary infertility in bothgroups was not statistically significant with p> 0.05
Infertility period of patients in two groups
Average age, infertility time of patients in two groups werecomparable The 5% oxygen concentration group had an average age
of 30.16 ± 3.60, and the average infertility time was 4.15 ± 2.81 years.The oxygen concentration group of 20% has an average age of 30.85 ±3.41 years; the average infertility period was 4.12 ± 3.01 years
Table 3.2 Infertility time of patients in two groups
Oxygen 5%
(n=86)
Oxygen 20%
30,85 ± 3,41 (23 – 38) 0,2
Trang 123.1.2 Characteristics of ovarian reserve in two groups
Table 3.3 Indicators assessing ovarian reserve of patients in two groups
0,3
LH day 3 (mIU/mL) 4,53 ± 3,03
(1,3 – 15,9)
5,12 ± 3,13(1 – 15,6)
0,1E2 day 3 (ng/mL) 44,34 ± 40,8
(10 – 379)
37,13 ± 14,5(2,5 – 83)
0,3Secondary follicle 11,40 ± 5,21
(4 – 32)
10,70 ± 4,51(2 – 20)
0,8Ovarian reserve of both groups was based on indicators of averageFSH (day 3 of the menstrual cycle), LH (day 3 of the menstrualcycle), E2 (day 3 of the menstrual cycle), AFC (day 3 of themenstrual cycle) is evaluated normally These indicators in the twogroups are not statistically significant
3.1.3 Characteristics of ovarian stimulation of patients in the two groups
Characteristics of ovarian stimulation of patients in the two groups
The total dose of FSH (IU), total days of FSH, E2 level on the day
of hCG injection (pg / ml), average number of oocytes, fertilizationrate and average number of embryos between the two groups werecomparable, the difference was no statistical significance with p> 0.05
Table 3.4 Characteristics of ovarian stimulation of patients in the
two groups Characteristics
2166,6 ± 584,3(1000 – 3850) 0,9
Trang 13Total days of using
FSH
9,70 ± 0,68(8 – 11)
9,8 ± 0,90(8 – 12) 0,4E2 level on the day of
hCG injection (ng/mL)
6020,1 ± 3185,1(1447 – 19171)
6705,7 ± 7538,7(1132 – 6705,65) 0,9
The protocol of ovarian stimulation in the two groups
Chart 3.3 The protocol of ovarian stimulation in the two groups
The regimen of ovarian stimulation in two main groups is mostlylong-term regimen, the rate of using regimens in the two groups issimilar (with p> 0.05)
3.1.4 The total number of oocytes retrieved and the fertilization rate in two groups
The average number of oocytes retrieved and the fertilization rate
in the two groups were same Likely, the average number of the
embryos day 2 in the two groups is similar (P > 0,05)
Table 3.6 Classification of oocytes retrieved and the fertilization
rate in two groups Characteristics
(4 – 21)
10,8 ± 3,9(4 – 20)The fertilization rate (%) 89,3 ± 14,4 91,1 ± 11,9 0,5
0,2 The mean of embryos obtained
(day 2)
8,7 ± 3,5(3 – 19)
9,4 ± 3,5(3 – 18)
3.2 Comparing the quality of embryos between the two study groups
3.2.1 The quality of embryos day 2 in the two study groups
Table 3.7 The quality of embryos day 2 in the two study groups
Trang 14The quality of embryos
The results showed that the Grade 1 of ICSI group wassignificantly higher with p <0.05 The quality of embryos from levelGrade 2 to Grade 4 has no difference between the two groups
3.2.3 The number of patients who had cryopreserved embryos day
2 and the mean of cryopreserved embryos day 2
Table 3.9 The number of patients had cryopreserved embryos day 2
and the mean of cryopreserved embryos day 2
(n=86)
Oxy 20%
Trang 15The number of patients 72 (83,7%) 77 (89,5%) 0,2The mean of
cryopreserved embryos
4,9 ± 3,5(0 – 16)
5,1 ± 3,5(0 – 14) 0,7The number of patients who had cryopreserved embryos day 2and the mean of cryopreserved embryos day 2 between 2 groups had
no significant differences (P > 0,05)
3.2.4 The mean of day-3 embryos to day-5 blastocysts, rate of blastulation
Trang 16Table 3.10 The mean of day-3 embryos to day-5 blastocysts, rate of
day-5 blastocysts
3,7 ± 0,8(3 – 6)
4,0 ± 1,1( 3 – 8)Blastulation rate (%) 90,04 ±
17,86
74,44 ±26,63
0,01The rate of top and good quality
blastocysts (%)
79,90 ±26,04
73,61 ±35,30
0,02The blastulation rate and the number of top and good qualityblastocysts (Grade 3 – 4) in the group of 5% oxygen concentration issignificantly higher with p < 0,05
3.2.5 The quality of embryos on day 5 between the two groups Table 3.11 The quality of embryos on day 5 in the two groups