Nghiên cứu hiệu quả của phương pháp nuôi cấy phôi nang ở nồng độ oxy thấp 5 trong thụ tinh ống nghiệm tt tiếng anh

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:
1. 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 Medical
Academy at …… …… …… 2019

The thesis can be found at following library:
- National library
- Vietnam Military Medical Academy library


3
INTRODUCTION OF THE THESIS
1. 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 2
purposes following:
1. To compare the quality of embryos between culture at a low
5% oxygen concentration and 20% air oxygen concentration.
2. To evaluate pregnancy outcome of transfer of blastocysts at
low 5% oxygen concentration and blastocysts at 20% oxygen
concentration.


4
2. 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.
3. Structure of the thesis
The thesis consists of 122 pages, including: Introduction (2
pages); Chapter 1 - Backgraound (33 pages); Chapter 2: Materials
and Methodology (22 pages); Chapter 3: Results (23 pages); Chapter
4: Discussion (39 pages); Conclusion (2 pages); Recommendation: 1
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.


5
Chapter 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 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 1 - 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.


6
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 3 and
blastocyst in other studies had no statistically significance (4 studies).
+ The implantation rate of blastocyst transfer on day 2, day 3 between
the 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 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.


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 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 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 normal
uterus without polyps, having at least 3 good embryos on the third day,
easy transferation, without blood on catheter, agreed to participate in our
study. The patients were divided to 2 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 3 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.


8
2.2.2. Sampling

n=

P

( zα / 2 2 P (1 − P ) + z β P1 (1 − P1 ) + P2 (1 − P2 ) ) 2
∆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 2 seperated
groups


9

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
3 and day 5 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 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
Classification
Top (Grade 4)

Description

•
•

Good (Grade 3)

Moderate (Grade 2)

•
•

Embryo expansion grade ≥ 3
ICM & TE Grade: AA; AB

•
•

Embryo expansion grade ≥ 3
ICM & TE Grade: BA; BB;
Embryo expansion grade <3
Or Embryo expansion grade ≥ 3, with ICM & TE Grade: AC;
BC; CB; CC;

Bad (Grade 1)

•

ICM grade D


11
Chapter 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 main
cause. 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 both
groups was not statistically significant with p> 0.05.
 Infertility period of patients in two groups
Average age, infertility time of patients in two groups were
comparable. 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
Oxygen 5%
20%
p-values
(n=86)
(n=86)

Infertility time
4,15 ± 2,81
4,12 ± 3,01
0,8
X
(1 – 12)
(1 – 15)
± SD)
Mean age
30,16 ± 3,60
30,85 ± 3,41
(21 – 37)
(23 – 38)
0,2
X
(
± SD)


12
3.1.2 Characteristics of ovarian reserve in two groups
Table 3.3. Indicators assessing ovarian reserve of patients in two groups
Oxygen 5%
Oxygen 20%
Characteristics
p
X
X
(
± SD)

(
± SD)
FSH day 3 (mIU/mL)
5,65 ± 1,87
5,95 ± 1,80
0,3
(2,2 – 9,8)
(2,2 – 13,5)
LH day 3 (mIU/mL)
4,53 ± 3,03
5,12 ± 3,13
0,1
(1,3 – 15,9)
(1 – 15,6)
E2 day 3 (ng/mL)
44,34 ± 40,8
37,13 ± 14,5
0,3
(10 – 379)
(2,5 – 83)
Secondary follicle
11,40 ± 5,21
10,70 ± 4,51
0,8
(4 – 32)
(2 – 20)
Ovarian reserve of both groups was based on indicators of average
FSH (day 3 of the menstrual cycle), LH (day 3 of the menstrual
cycle), E2 (day 3 of the menstrual cycle), AFC (day 3 of the
menstrual cycle) is evaluated normally. These indicators in the two

groups 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, fertilization
rate and average number of embryos between the two groups were
comparable, the difference was no statistical significance with p> 0.05.
Table 3.4. Characteristics of ovarian stimulation of patients in the
two groups
Oxy 5%
Oxy 20%
Characteristics
p
X
X
(
± SD)
(
± SD)
Total doses of FSH
2176,2 ± 504,9
2166,6 ± 584,3
0,9
(UI)
(1250 – 3600)
(1000 – 3850)


13
Total days of using

FSH
E2 level on the day of
hCG injection (ng/mL)

9,70 ± 0,68
(8 – 11)
6020,1 ± 3185,1
(1447 – 19171)

9,8 ± 0,90
(8 – 12)
6705,7 ± 7538,7
(1132 – 6705,65)

0,4
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 mostly
long-term regimen, the rate of using regimens in the two groups is
similar (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
Oxy 5%

Oxy 20%
Characteristics
p
X
X
± SD)
(
± SD)
The total number of oocytes retrieved
884
919
10,5 ± 3,9
10,8 ± 3,9
0,6
The mean of oocytes retrieved
(4 – 21)
(4 – 20)
The fertilization rate (%)
89,3 ± 14,4 91,1 ± 11,9 0,5
The number of embryos obtained
769
810
The mean of embryos obtained
8,7 ± 3,5
9,4 ± 3,5
0,2
(day 2)
(3 – 19)
(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


14
The quality of embryos
day 2

Oxy 5%
(

X

Oxy 20%
p

X

± SD)
(
± SD)
5,4 ± 2,3
6,1 ± 2,5
Grade 4
0,1
(0 – 14)
(3 – 15)
2,5 ± 1,6
2,5 ± 1,7
Grade 3

0,9
(0 – 7)
(0 – 8)
2,9 ± 2,4
2,3 ± 1,3
Grade 2
0,5
(0 – 10)
(0 – 6)
1,5 ± 0,9
3 ± 2,6
Grade 1
0,1
(0 – 4)
(0 – 8)
The quality of embryos day 2 in the two study groups were not
statistically significant (P> 0.05).
3.2.2 The relationship between fertilization techniques and embryo
quality of day 2 of the 5% Oxygen group

Table 3.8. Fertilization techniques and embryo quality of day 2 of the 5%
Oxygen group

IVF
ICSI
p
Grade 4
5,9 ± 2,2
5,0 ± 2,4
> 0,05

Grade 3
1,4 ± 1,7
1,6 ± 1,8
> 0,05
Grade 2
1,6 ± 2,3
1,5 ± 2,3
> 0,05
Grade 1
0,0 ± 0,2
0,4 ± 0,8
< 0,05
The results showed that the Grade 1 of ICSI group was
significantly higher with p <0.05. The quality of embryos from level
Grade 2 to Grade 4 has no difference between the two groups.
embryo quality of day 2

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
Day 2

Oxy 5%
(n=86)

Oxy 20%
(n=86)

p

15
The number of patients
72 (83,7%)
77 (89,5%)
0,2
The mean of
4,9 ± 3,5
5,1 ± 3,5
0,7
cryopreserved embryos
(0 – 16)
(0 – 14)
The number of patients who had cryopreserved embryos day 2
and 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


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)

( 3 – 8)

Blastulation rate (%)

90,04 ±

74,44 ±

0,0

17,86

26,63

1

79,90 ±

73,61 ±

0,0

26,04

35,30

2

blastocysts
The mean of day-3 embryos to

The rate of top and good quality
blastocysts (%)

0,0
2

The blastulation rate and the number of top and good quality
blastocysts (Grade 3 – 4) in the group of 5% oxygen concentration is
significantly 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
Oxy 5%
Oxy 20%
Grade
p
X
X
(
± SD)
(
± SD)
Grade 4
1,7 ± 0,9
1,6 ± 0,9
0,7
Grade 3
1,8 ± 0,8
1,8 ± 0,8
0,9
Grade 2
1,2 ± 0,6
1,5 ± 0,7
0,1
Grade 1
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 3 – 4
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
6
1 child
24
58,5
16
55,2
2 children
16
36,6
13
44,8 0,4
3 children
1
4,9
0
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)
%
1 male
14
16,3
9
10,5
0,3
1 female
10
11,6
7
8,1
0,4
1 male – 1 female
9
10,5
4
4,7
0,1
2 males
6
7,0

3
3,5
0,3
2 females
1
1,2
6
7,0
0,6
3 females
1
1,2
0
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

2

X

(
± SD)
3091±361,1
(1600 3700)
2429,4±459,
3
(1400–3100)
1000±412,3
(800-2700)

Oxy 20%
n
(
29
13

0

X

p
± SD)

3040±642,3
(1200–3800)

0,
6

2230,8±534,
5
(1700–3200)

0,
3


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 2 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
1
0,9
3
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 5
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 5
41
47,7
29
33,7
0,06
st
nd
Live births
1 , 2 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.

21
The rate of live births in the 5% oxygen concentration group and
the 20% oxygen concentration group was not statistically significant
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 4 embryos in day 2 is the highest one,
opposite to the mean of Grade 1 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.


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4.2.3. The relationship between fertilization techniques and embryo
quality of day 2 of the 5% Oxygen group
In our study, comparing the relationship between the fertilization
technique and the quality embryos of day 2 of the 5% Oxygen group
showed that the number of bad embryos (Grade 1) on day 2 in
patients was made by ICSI technique was significantly higher than
the rate of bad embryos (grade 1) on day 2 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 3.10). The results of our study were
similar to the results of research by Sobrinho D. et al. (2011), the rate
of blastocyst formation in the embryo culture group with 5% oxygen
concentration was significantly higher than that of embryo culture at
20% oxygen concentration.
4.2.5. The quality of blastocysts of the two groups
The ratio of top and good quality blastocysts (%) in the 5%

oxygen group (79.90 ± 26.04) significantly higher than the 20%
oxygen group (73.61 ± 35.30) (with p <0.05).
4.2.6. The mean of embryos transfer and the endometrial thickness
between two groups
The endometrial thickness between two groups is similar and in
the range of implantation (11,4 ± 2,1 và 10,9 ± 1).
4.3 Comparing the pregnancy outcomes in patients with fresh
blastocysts transfer in two groups


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4.3.1. The pregnancy outcomes in patients with fresh blastocysts
transfer in two groups
* The rate of biochemical pregnancy in blastocysts transfer
The biochemical pregnancy rate of the 5% oxygen group was higher
than that with the 20% oxygen group (60.5% compared to 46.5%),
however the difference was not statistically significant with p = 0.054.
* The rate of clinical pregnancy in blastocyst transfer
The rate of clinical pregnancy in the 5% oxygen group was
significantly higher than the 20% oxygen group (57.0% compared to
40.7%; p = 0.03). Our results were similar to results in a research by
Kovaci et al 2010 and Meintjes M. & et al 2009.
4.3.2. Implantation rate of blastocyst transfer
In our research, the 5% oxygen group transfered 289 embryos
included 72 implanted blastocysts with amniotic sac (24.9%), the 5%
oxygen group transfered 279 embryos included 58 implanted
blastocysts with amniotic sac (20.7%). So the implantation rate in the
5% oxygen concentration group is higher. However, the difference was
not statistically significant with p > 0,05. Our results were similar to
results in a research by Kovaci et al 2010 and Garcia J& cs 2010.

4.3.3. The live birth rate of embryo transfer
The live birth rate in the group had low oxygen concentration
(47.7%) was higher than the group with high oxygen concentration
(33.7%). However, the difference was not statistically significant
(with p = 0.06) (table 3.21). Our study results has the similar with the
study’s results by Marius Meintjes et al. (2009).
4.3.4. The number and weight of live births between 2 groups of blastocyst
transfer
In our study, the average weight of babies from group 1 was higher
than the average weight of babies from group 2, however the
difference was not 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


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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:
1. 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)


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+ 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.
2. 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%).