tóm tắt luận án tiến sĩ bằng tiếng anh nghiên cứu biến đổi hình thái cấu trúc phôi người ngày 3, ngày 5 trước đông lạnh và sau rã đông bằng kỹ thuật thủy tinh hóa

Freezing human embryos for later transfer is currently anindispensible technique in every assisted reproduction center.There have been a number of researches in attempt to improve thesur

Freezing human embryos for later transfer is currently anindispensible technique in every assisted reproduction center.There have been a number of researches in attempt to improve thesurvival rate of embryos after thawing and the pregnancy rate ofthese embryos after transferring Freezing techniques have alsobeen advanced and divided into two major groups: slow-freezingtechnique and vitrification technique

Embryo can be frozen at every stage of maturity Freezingembryos at pre-nucleus stage (day 1) will make it difficult to selectembryos to freeze and identify the number of embryos to thaw.Meanwhile, it is common to freeze embryos at splitting stage (day

2 or 3) because it facilitates the selection of embryos beforefreezing and after thawing based on morphological standards Asembryos are all independent, the degenerated embryos cannotinfluence healthy ones At compacting stage (day 4), embryos arevery vulnerable It is very difficult to assess their morphology andthus, embryos cannot be frozen at this stage Freezing embryos atblastocyst stage (day 5,6,7) is more favorable than at splitting stagebecause there are more cells and the degeneration of some cellscannot influence the embryos Several researches proved that thesurvival rate and pregnancy rate of embryos frozen at day 5 arehigher than that of embryos frozen at day 6 and day 7 Embryofreezing at blastocyst stage can also reduce the rate of multiplepregnancies, yet the probability of embryos at blastocyst stage isonly 50-60% It is still a controversy over the time and technique

of freezing embryos The basis for identifying the influences offreezing procedures and freezing time on the effectiveness offreezing technique is the transformation of morphological structure

of embryos before freezing and after thawing Therefore, thisresearch is conducted with the topic: “Transformation ofmorphological structure of day-three and day-five human embryosbefore freezing and after thawing by vitrification technique.”

The research aims to:

1 Describe the transformation of morphological structure ofday-three and day-five human embryos before freezing andafter thawing by vitrification technique

2 Compare the survival rate, implantation rate, and pregnancyrate of day-three and day-five embryos before freezing andafter thawing by vitrification technique

Summary of new scientific contributions of thesis:

The research is conducted on 444 day-three and day-five humanembryos before freezing and after thawing by vitrificationtechnique, of 157 patients having in vitro fertilization from 2009 to

2012 The results show that:

- Under optical microscope, no morphological transformation

is detected between embryos before freezing and afterthawing

- Under electron microscope, it is observed that the zonapellucida of embryos is transformed and loses its porosity;glycoprotein fibers are not clearly visible Embryos thatsurvived after thawing show little damage; there are someparticle degeneration and vacuoles appearance at somepoints in the cytoplasmic The degenerative post-thawingembryos show deformed cell membrane and mitochondria,expanded endoplasmic reticulum, and shrunk nuclearmembrane

- Survival rate and the rate of intact embryos after thawing inday-three embryos are higher than those in day-fiveembryos, with statistical significance No statisticallysignificant difference is detected in between frozen day-threeembryos and frozen day-five embryos in terms of pregnancyrate and implantation rate.

Structure of the thesis

The thesis consists of 133 pages, of which there are two pages ofIntroduction, 33 pages of Literature Review, 20 pages of ResearchSubjects and Methodology, 45 pages of Research Findings, 30pages of Discussion, 2 pages of Conclusion and 1 page ofRecommendation There is also a page list of publications and 112references (16 Vietnamese and 96 English references)

CHAPTER 1: LITERATURE REVIEW 1.1 Principles and techniques of embryo freezing

1.1.1 Principles of embryo freezing

The principle of freezing is to reduce the temperature of the culturecontaining the cell or embryo to -196oC Almost all biologicalactivities inside the cell, including bio-chemical reactions andmetabolic activity, stop at this temperature The cell, thus, exists inlatent form (no growth) and can be preserved for a period of time.The materials inside the cell exist in a combined form (crystal andglass forms) and thus, there is no internal nor external effect caninfluence the cell

While embryos are frozen and thawed, certain changes in theculture can affect the structure, function, integrity, and survivalability of embryos after thawing

Like other cells, embryos can be affected by three types of damageoccurring at different ranges of temperature during freezing andthawing procedures At 15oC to -5oC, coldness can damage the cell

by destroying lipid drops within cytoplasmic and microtubulestructures From -5oC to -80oC, the intracellular and extracellularcrystallization is the main cause of cell damage, which isconsidered the most dangerous risk for frozen cells in general andfrozen embryos in particular From -50oC to -150oC, the zonapellucida or plasma membrane may be broken During the thawingprocess, the cells may suffer the same types of damage but in areverse order The cell can be recrystallized, leading to the return

of intracellular crystals when the temperature increases to a levelhigher than -120oC Therefore, it is crucial to thaw the cells quicklythrough this period and limit the possible damage to the cells

1.1.2 Techniques of embryo freezing

Techniques of embryo freezing are classified into two major typesbased on the concentration of preservatives and the speed offreezing: (1) slow freezing and (2) vitrification

1.1.2.1 Slow freezing

Slow freezing technique is also called speed-controlled freezingmethod Cells will be frozen at a slow speed (1-3oC/min), fromtheir physiological temperature to a very low temperature (-80oC),before being preserved in liquid nitrogen Besides, the thawingprocess is also conducted slowly with a number of small steps toeliminate freezing preservatives

1.1.2.2 Vitrification

This technique is based on the phenomenon that water freezeswhen the temperature reduces rapidly One major factor thataffects the survival and growth ability of frozen embryos after

thawing is the formation of ice crystals The freezing speed invitrification reaches 23000oC/min; water, hence, cannot becomeice through crystallization but transform into glass In the form ofglass, water molecules and other solutes maintain their positionstill The advantage of this technique is that it keeps the frozencells from possible damage due to crystallization The thawingprocess is also conducted very fast to limit transformation fromglass to ice.

1.2 Trend of research on embryo freezing in the world and in Vietnam

- Most researches comparing the effectiveness of freezingtechniques in the past prove that vitrification technique issuperior to slow freezing technique

- Researches investigate the effectiveness of freezing atdifferent stage of embryos’ maturity, yet it is stillcontroversial which stage is the best for freezing

- There are researches examining the transformation ofmorphological structure and ultrastructure of embryos beforefreezing and after thawing; yet the number of researchesinvestigating the morphological ultrastructure of embryos isstill limited while most of them are conducted on animals’embryos and human oocytes There has been no study onembryos’ morphological ultrastructure in Vietnam

- Many researches take into account the safety of vitrificationtechnique Most of these researches indicate all the kids bornfrom frozen embryos are able to grow as normally as thoseborn from fresh embryos

CHAPTER 2: RESEARCH DATA AND METHODOLOGY

2.1 Research Data

2.1.1 Subjects of the research

Subjects of the research consist of 444 embryos of 157 patientswhose embryos are frozen and thawed at day-three and day-five byvitrification technique at the centre for Human Reproduction andEmbryology, Vietnam Military Medical University from 2009 to

2012 The embryos are divided into two groups:

- Group I: 250 frozen day-three embryos (79 patients) whichare thawed

- Group II: 194 frozen day-five embryos (78 patients) whichare thawed

Criteria to select embryos to freeze:

- Day-three embryos: level III, IV

- Day-five embryos: A, B

Criteria to exclude:

- Day-three embryos: level I, II

- Day-five embryos: C

2.1.2 Place of the research

- The centre for Human Reproduction and Embryology,Vietnam Military Medical University

- Scanning electron microscope lab, Morphology Department,Laboratory 69, Government Command Defending Ho ChiMinh Mausoleum

2.1.3 Time of the research

From October 2009 to June 2012

2.2 Research Methodology

2.2.1 Research design

The research applies analytical descriptive study

2.2.2 Sample size and sample selection

Sample size is calculated by using the sample size formula for an

analytical descriptive study with two rates:

n=[z 1−α /2+z 1−β]

2

(p1q1 +p2q2 ) [p1– p2]2

in which: n is the least sample size, z 1−α/ 2 is the confidencecoefficient (z 1−α/ 2 = 1.96 at 5% level of significance, z 1−α/ 2 = 1.28 at10% level of significance), p1 and p2 are survival rate of day-threeand day-five after thawing, respectively

From literature review, in this research, p1 and p1is chosen as 95%(0.95) and 85% (0.85), respectively Substitute the numbers intothe formula, we have n = 184 Therefore, both the two samples ofthe research (2 groups) satisfy the least sample size requirement During the treatment time, there are patients who have no appeal topreserve excess embryos, and patients who do not need to usefrozen embryos and decide to abolish these embryos because theyhave enough children from transferring fresh embryos Theseexcess embryos are used as templates for electron microscope, inparticular:

- For transmission electron microscope: Day-three embryos (5fresh embryos, 5 post-thawing embryos), and day-fiveembryos (5 fresh embryos, 5 post-thawing embryos)

- For scanning electron microscope: Day-three embryos (5fresh embryos, 5 post-thawing embryos), and day-fiveembryos (5 fresh embryos, 5 post-thawing embryos)

2.2.3 Methods and Techniques

- In vitro fertilization

- Embryo freezing method: Applying the Kitazato freezingculture and fast freezing procedures by Kuwayama M.(Japan, 2005).

- Assisted hatching technique using Tyrode acid

- Technique to create template for electron microscopes, byNguyen Kim Giao (2004)

2.2.4 Morphological structure assessment methods

 Effectiveness of embryo preservation: The effectiveness ofembryo preservation is measured by:

- Survival rate of embryos after thawing: Embryos aremorphologically examine 1 hour after thawing Survivalindex is calculated as the ratio between survived cells andthe total number of cells An embryo is considered survived

if more than 50% of its cells survive after the embryo isthawed

- The ability of embryos to grow in culture medium: Embryosare assessed after 24 hours, based on survival rate (the ratiobetween the number of survived embryos and the totalnumber of thawed embryos).

- Implantation rate

 Pre-transferring morphological assessment:

- Day-three embryos after thawing are assessed based on thepre-freezing embryo level, degeneration level, and thecontinued split of embryos after being overnight in theculture medium

- Day-five embryos after thawing are assessed based on thenumber of embryos with intact inner cells mass andtrophoblastic cell layer, and the number of embryos that re-expanded after thawing

 Criteria for describing morphological structure of embryosunder scanning electron microscope and transmissionelectron microscope:

- Describe the changes in the zona pellucida of pre-freezingand post-thawing embryos (under scanning electronmicroscope)

- Describe the changes in the zona pellucida, cell membrane,cytoplasmic organelles, and cell nucleus of embryos beforefreezing and after thawing

2.2.5 Identification of pregnancy rate and implantation

rate in day-three and day-five frozen embryos after transfer

Fourteen days after embryo transfer, patients are appointed to theCenter to test the concentration of ß-hCG in the blood:

- Biochemical pregnancy: ß-hCG level exceeds 25mIU/ml by

14 days after transfer

- Clinical pregnancy: amniotic sac is detected in the uterus byultrasound 4-5 weeks after transfer

The implantation rate of embryos is measured by the ratio betweenthe number of implanted embryos and the total number oftransferred embryos

2.2.6 Examination of factors influencing the pregnancy

rate of frozen embryos, by Menezo, 2001

2.2.7 Data processing

Data are processed by SPSS 18.0 Software, version for Windows

2.2.8 Ethical issues in scientific research

- All research procedures are conformed to the law ofVietnam on reproduction assistance

- All patients, who are the subjects of the research, haveagreed and voluntarily participated in the research Allpersonal information is confidential

- Measurements are conducted only on photos; the shootingtime is no longer than 2 minutes to ensure that the embryoquality is not affected

- Embryos used as ultrastructure template are excess embryosthat patients voluntarily give up

CHAPTER 3: RESULTS AND FINDINGS 3.1 Characteristics of research subjects

Table 3.1: Average age and duration of infertility of patients

Group I

duration of

infertility(X´ ± SD)

According to the Table 3.1, there is no statistically significantdifference between the two groups in terms of average age andduration of infertility, with p = 0.084 and 0.362, respectively

Table 3.2: The number of periods and embryos in two research

groups

Quantity ofthawedembryos

Percentage(%) Quantity ofthawing

cycles

Percentage(%)

The number of thawing cycles in group I (250 day-three embryos)

is 86 cycles, and in group II (194 day-five embryos) is 81 cycles

3.2 Morphological structure of day-three human embryos before freezing and after thawing

3.2.1 Changes in the thickness of zona pellucida and the

diameter of embryos before freezing and after thawing

Every embryo is measured before freezing and after thawing interms of the zona pellucida thickness and the embryo diameter.The results are presented in Table 3.8 and Table 3.9

Table 3.8: The thickness of zona pellucida in day-three embryos before freezing, after thawing, and 1 day after

After thawing

After embryoculture (3) (10,50 – 20,30)14,90 ± 1,08

Table 3.8 reveals that there is no statistically significant difference

in the thickness of zona pellucida in day-three embryos at differentphases (before freezing, after thawing, and after embryo culture),with p > 0.05

Table 3.9: Diameter of day-three embryos before freezing,

after thawing, and after embryo culture

Time Average embryodiameter (µm)

(min-max)

p

Beforefreezing(1)

147,20 ± 7,82 (132,40 –

165, 30)

p1-2 =0,246

p2-3 =0,001

Afterthawing(2)

147,50 ± 8,14 (135,50 –

158,70)

Afterembryoculture (3)

149,50 ± 6,30 (135,40 –

167,90)

There is no statistically significant difference in average diameter

of day-three embryos before freezing and after thawing

Nevertheless, the embryo diameter after embryo culture for 1 day

is larger than that after thawing, with statistical significance (p =0.0001)

3.2.2 Survival index and survival rate of day-three

embryos after thawing

The viability of embryos after thawing is measured by survivalindex, which is equal to the ratio between the number of survivedblastomeres and the total number of blastomeres (1624/1711 =94.92%) The survival rate of embryos is equal to the ratio betweenthe number of survived embryos and the total number of thawedembryos (238/250 = 95.2%)

3.2.3 Morphology of day-three embryos before freezing,

after thawing and after 24 hours in culture medium, observed under stereoscopic microscope

Table 3.11: Transformation of morphology of day-three embryos before freezing and 1 hour after thawing

Quantity

Percentage(%)

Quantity

Percentage(%)

Table 3.13: The rate of embryo that continues spitting after

thawing (level 4 embryos) 83 (83/99)83,8

Continue splitting after

thawing (level 3 embryos) 70 (70/122)57,4

The number of embryo that continues splitting is 171 embryos(68.4%), of which there are 83 level 4 embryos (83.8%) and 70level 3 embryos (57.4%) continue splitting after thawing

3.2.4 Morphological ultrastructure of day-three embryos

before freezing and after thawing

3.2.4.1 Day-three embryos under scanning electron

microscope

At 5000 times magnification, alternating glycoprotein fibers areobserved on the surface of day-three embryos before freezing Thesurface of day-three embryos survived after thawing is as plump as

that of pre-freezing embryos However, at 5000 timesmagnification, the post-thawing embryos show smoother surfacethan the pre-freezing ones and it is difficult to detect theglycoprotein fibers The surface of degenerated embryos isunevenly rough because the blastomeres are cringed

3.2.4.2 Day-three embryos under transmission electron

microscope

At 800 times magnification, the zona pellucida, blastomeres, andthe distance between the zona pellucida and blastomeres can beobserved on day-three embryos At 5000 times magnification, thezona pellucida’s structure can be revealed more clearly andseparated to the blastomeres Glycoprotein fibers can also bedetected

Survived embryos after thawing: observing the zona pellucida at

5000 times magnification, we can see that the concentration ofelectron is smoother and that glycoprotein fibers are not as visible

as in fresh embryos Blastomeres’ structure: there is no significantchange in the cell membrane, nucleus and nuclear Nevertheless,vacuoles are detected and the nuclear is degenerated at some points

in cytoplasmic

Degenerated embryos after thawing: the nucleus membrane of cellsare cringed and broken at some points There are many folds, andmitochondria become a substance with light color Crestedmitochondria cannot be observed The endoplasmic reticulum hasparticles surrounding the mitochondria

3.3 Morphological structure of day-five embryos before freezing, after thawing and after embryo culture

3.3.1 Changes in the thickness of zona pellucida, embryo

diameter and the number of trophoblastic cells before freezing and after thawing

Table 3.14: The average thickness of zona pellucida of day-five embryos before freezing, after thawing and after embryo

p2-3 =0,001

Table 3.15: Average diameter of day-five embryos before freezing, after thawing, and after embryo culture

p2-3 =0,001

After thawing (2) 152,70 ± 9,80 (137,50

– 187,50)

After embryo

culture (3) 161,70 ± 19,50 (138,40 –198,20)