INTRODUCTION Freezing human embryos for later transfer is currently an indispensible technique in every assisted reproduction center. There have been a number of researches in attempt to improve the survival rate of embryos after thawing and the pregnancy rate of these embryos after transferring. Freezing techniques have also been advanced and divided into two major groups: slow-freezing technique and vitrification technique. Embryo can be frozen at every stage of maturity. Freezing embryos at pre-nucleus stage (day 1) will make it difficult to select embryos 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 before freezing and after thawing based on morphological standards. As embryos are all independent, the degenerated embryos cannot influence healthy ones. At compacting stage (day 4), embryos are very vulnerable. It is very difficult to assess their morphology and thus, embryos cannot be frozen at this stage. Freezing embryos at blastocyst stage (day 5,6,7) is more favorable than at splitting stage because there are more cells and the degeneration of some cells cannot influence the embryos. Several researches proved that the survival rate and pregnancy rate of embryos frozen at day 5 are higher than that of embryos frozen at day 6 and day 7. Embryo freezing at blastocyst stage can also reduce the rate of multiple pregnancies, yet the probability of embryos at blastocyst stage is only 50-60%. It is still a controversy over the time and technique of freezing embryos. The basis for identifying the influences of freezing procedures and freezing time on the effectiveness of freezing technique is the transformation of morphological structure 1 1 of embryos before freezing and after thawing. Therefore, this research is conducted with the topic: “Transformation of morphological structure of day-three and day-five human embryos before freezing and after thawing by vitrification technique.” The research aims to: 1. Describe the transformation of morphological structure of day-three and day-five human embryos before freezing and after thawing by vitrification technique. 2. Compare the survival rate, implantation rate, and pregnancy rate of day-three and day-five embryos before freezing and after thawing by vitrification technique. Summary of new scientific contributions of thesis: The research is conducted on 444 day-three and day-five human embryos before freezing and after thawing by vitrification technique, 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 after thawing. - Under electron microscope, it is observed that the zona pellucida of embryos is transformed and loses its porosity; glycoprotein fibers are not clearly visible. Embryos that survived after thawing show little damage; there are some particle degeneration and vacuoles appearance at some points in the cytoplasmic. The degenerative post-thawing embryos show deformed cell membrane and mitochondria, expanded endoplasmic reticulum, and shrunk nuclear membrane. 2 2 - Survival rate and the rate of intact embryos after thawing in day-three embryos are higher than those in day-five embryos, with statistical significance. No statistically significant difference is detected in between frozen day-three embryos and frozen day-five embryos in terms of pregnancy rate and implantation rate. Structure of the thesis The thesis consists of 133 pages, of which there are two pages of Introduction, 33 pages of Literature Review, 20 pages of Research Subjects and Methodology, 45 pages of Research Findings, 30 pages of Discussion, 2 pages of Conclusion and 1 page of Recommendation. There is also a page list of publications and 112 references (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 culture containing the cell or embryo to -196 o C. Almost all biological activities inside the cell, including bio-chemical reactions and metabolic activity, stop at this temperature. The cell, thus, exists in latent form (no growth) and can be preserved for a period of time. The materials inside the cell exist in a combined form (crystal and glass forms) and thus, there is no internal nor external effect can influence the cell. While embryos are frozen and thawed, certain changes in the culture can affect the structure, function, integrity, and survival ability of embryos after thawing. 3 3 Like other cells, embryos can be affected by three types of damage occurring at different ranges of temperature during freezing and thawing procedures. At 15 o C to -5 o C, coldness can damage the cell by destroying lipid drops within cytoplasmic and microtubule structures. From -5 o C to -80 o C, the intracellular and extracellular crystallization is the main cause of cell damage, which is considered the most dangerous risk for frozen cells in general and frozen embryos in particular. From -50 o C to -150 o C, the zona pellucida or plasma membrane may be broken. During the thawing process, the cells may suffer the same types of damage but in a reverse order. The cell can be recrystallized, leading to the return of intracellular crystals when the temperature increases to a level higher than -120 o C. Therefore, it is crucial to thaw the cells quickly through 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 types based on the concentration of preservatives and the speed of freezing: (1) slow freezing and (2) vitrification. 1.1.2.1.Slow freezing Slow freezing technique is also called speed-controlled freezing method. Cells will be frozen at a slow speed (1-3 o C/min), from their physiological temperature to a very low temperature (-80 o C), before being preserved in liquid nitrogen. Besides, the thawing process is also conducted slowly with a number of small steps to eliminate freezing preservatives. 1.1.2.2.Vitrification This technique is based on the phenomenon that water freezes when the temperature reduces rapidly. One major factor that affects the survival and growth ability of frozen embryos after 4 4 thawing is the formation of ice crystals. The freezing speed in vitrification reaches 23000 o C/min; water, hence, cannot become ice through crystallization but transform into glass. In the form of glass, water molecules and other solutes maintain their position still. The advantage of this technique is that it keeps the frozen cells from possible damage due to crystallization. The thawing process is also conducted very fast to limit transformation from glass to ice. 1.2. Trend of research on embryo freezing in the world and in Vietnam - Most researches comparing the effectiveness of freezing techniques in the past prove that vitrification technique is superior to slow freezing technique. - Researches investigate the effectiveness of freezing at different stage of embryos’ maturity, yet it is still controversial which stage is the best for freezing. - There are researches examining the transformation of morphological structure and ultrastructure of embryos before freezing and after thawing; yet the number of researches investigating the morphological ultrastructure of embryos is still limited while most of them are conducted on animals’ embryos and human oocytes. There has been no study on embryos’ morphological ultrastructure in Vietnam. - Many researches take into account the safety of vitrification technique. Most of these researches indicate all the kids born from frozen embryos are able to grow as normally as those born from fresh embryos. CHAPTER 2: RESEARCH DATA AND METHODOLOGY 2.1. Research Data 5 5 2.1.1. Subjects of the research Subjects of the research consist of 444 embryos of 157 patients whose embryos are frozen and thawed at day-three and day-five by vitrification technique at the centre for Human Reproduction and Embryology, Vietnam Military Medical University from 2009 to 2012. The embryos are divided into two groups: - Group I: 250 frozen day-three embryos (79 patients) which are thawed - Group II: 194 frozen day-five embryos (78 patients) which are 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 Chi Minh 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 6 6 Sample size is calculated by using the sample size formula for an analytical descriptive study with two rates: in which: n is the least sample size, is the confidence coefficient ( = 1.96 at 5% level of significance, = 1.28 at 10% level of significance), and are survival rate of day-three and day-five after thawing, respectively. From literature review, in this research, and is chosen as 95% (0.95) and 85% (0.85), respectively. Substitute the numbers into the formula, we have n = 184. Therefore, both the two samples of the research (2 groups) satisfy the least sample size requirement. During the treatment time, there are patients who have no appeal to preserve excess embryos, and patients who do not need to use frozen embryos and decide to abolish these embryos because they have enough children from transferring fresh embryos. These excess embryos are used as templates for electron microscope, in particular: - For transmission electron microscope: Day-three embryos (5 fresh embryos, 5 post-thawing embryos), and day-five embryos (5 fresh embryos, 5 post-thawing embryos) - For scanning electron microscope: Day-three embryos (5 fresh embryos, 5 post-thawing embryos), and day-five embryos (5 fresh embryos, 5 post-thawing embryos) 2.2.3. Methods and Techniques - In vitro fertilization - Embryo freezing method: Applying the Kitazato freezing culture and fast freezing procedures by Kuwayama M. (Japan, 2005). - Assisted hatching technique using Tyrode acid. 7 7 - Technique to create template for electron microscopes, by Nguyen Kim Giao (2004). 2.2.4. Morphological structure assessment methods • Pre-freezing embryos: - Day-three embryos: assessed by using method of Andres Salumets (2001). - Day-five embryos: assessed by using method of David K. Gardner (2001). • Post-thawing embryos: - Intact post-thawing embryos are classified in two periods as above. Degenerated embryos are assessed based on the level of degeneration, according to the morphological criteria developed by Brian Dale and Kay Elder (1997). - All post-thawing embryos are subjected to measurement of zona pellucida’s thickness and embryos’ size by Axio 4.8 software (Carl Zeiss). Results of measurement for post- thawing embryos are compared with those for pre-freezing embryos. • Effectiveness of embryo preservation: The effectiveness of embryo preservation is measured by: - Survival rate of embryos after thawing: Embryos are morphologically examine 1 hour after thawing. Survival index is calculated as the ratio between survived cells and the total number of cells. An embryo is considered survived if more than 50% of its cells survive after the embryo is thawed. - The ability of embryos to grow in culture medium: Embryos are assessed after 24 hours, based on survival rate (the ratio between the number of survived embryos and the total number of thawed embryos). - Implantation rate. 8 8 • Pre-transferring morphological assessment: - Day-three embryos after thawing are assessed based on the pre-freezing embryo level, degeneration level, and the continued split of embryos after being overnight in the culture medium. - Day-five embryos after thawing are assessed based on the number of embryos with intact inner cells mass and trophoblastic cell layer, and the number of embryos that re- expanded after thawing • Criteria for describing morphological structure of embryos under scanning electron microscope and transmission electron microscope: - Describe the changes in the zona pellucida of pre-freezing and post-thawing embryos (under scanning electron microscope). - Describe the changes in the zona pellucida, cell membrane, cytoplasmic organelles, and cell nucleus of embryos before freezing 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 the Center 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 by ultrasound 4-5 weeks after transfer The implantation rate of embryos is measured by the ratio between the number of implanted embryos and the total number of transferred embryos. 9 9 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 of Vietnam on reproduction assistance. - All patients, who are the subjects of the research, have agreed and voluntarily participated in the research. All personal information is confidential. - Measurements are conducted only on photos; the shooting time is no longer than 2 minutes to ensure that the embryo quality is not affected. - Embryos used as ultrastructure template are excess embryos that 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 (min – max) Group II (min – max) p Average age ( ± SD) 31,5 ± 3,4 (24 - 40) 32,0 ± 3,6 (25 - 40) 0,08 4 Average duration of infertility ( ± SD) 6,4 ± 3,6 (1 - 19) 6,7 ± 4,3 (1 – 21) 0,36 2 10 10 [...]... 17 17 Embryo maturity AA 1 AB BA BB AA 2 AB BA BB AA 3 AB BA BB Total Before freezing Quantit y 1 1 27 52 10 16 33 30 8 16 194 Survived after thawing Percentag Quantit Percentage e (%) y (%) 0 ,5 1 0,6 0 ,5 1 0,6 13,9 27 15, 6 26,8 50 28,9 5, 2 10 5, 8 8,2 11 6,4 17,1 33 19,0 15, 5 24 13,8 4,1 6 3 ,5 8,2 10 5, 8 100 173 100 After thawing, all 1AA, 2AA, 3AA embryos survived while there are two 2AB and two 3BA... embryos before freezing and after thawing: Day 3: 14,70 ± 1,13 µm and 14,80 ± 1,20 µm (p > 0, 05) ; Day 5: 13,6 0 ± 1 ,50 µm and 12,40 ± 1,30 µm (p > 0, 05) The diameter of embryos before freezing and after thawing: Day 3: 147,2 ± 7,82 µm and 147 ,5 ± 8,14 µm (p > 0, 05) ; Day 5: 1 53 ,5 ± 9,40 µm and 152 ,7 ± 9,8 µm (p > 0, 05) At ultramicroscopic level, it is observed under electron microscope that in both day-three... that in post-thawing embryos, with p = 0.001 Table 3. 15: Average diameter of day-five embryos before freezing, after thawing, and after embryo culture Average embryo p Time diameter (µm) (min-max) Before freezing 1 53 ,5 0 ± 9,40 (1 33,3 – p1-2 = (1) 1 85, 00) 0 ,53 7 p2-3 = After thawing (2) 152 ,70 ± 9,80 (137 ,50 0,001 – 187 ,50 ) After embryo 161,70 ± 19 ,50 (138,40 culture (3) –198,20) The average diameter... 70,6 Implantation rate (%) 14,7 16,6 Transfer cycle rate (%) Pregnancy rate(%) 20 10,06 ± 1,41 (6 – 14) 100 95, 1 34,9 32 ,5 20 2,41 (1,10 – 5, 36) p= 0,016 3,1 7 (1,88 – 5, 36) p= 0,001 0,86 (0,48 – 1 ,54 ) p = 0,6 p = 0,11 1,11 (0 ,55 – 2, 25) p = 0,74 Accumulated pregnancy rate (%) 34,2 32,4 1,08 (0 ,52 – 2,24) p = 0,82 From the above table, it can be seen that there is no statistically significant difference... embryos, corresponding to 177 frozen embryos, of which there are 55 embryos at early stage, 41 embryos at expanding stage, and 71 embryos at fully expanded stage The survival rate of early stage embryos, expanding embryos, and fully expanded embryos is 54 .5% , 58 .5% and 20.3%, respectively The corresponding pregnancy rate is 22.7%, 23 .5% , and 4 .5% , respectively The author shows that the survival rate and... Table 3.2: The number of periods and embryos in two research groups Quantity of Percentage Quantity of Percentage thawed (%) thawing (%) embryos cycles I 250 56 ,3 86 51 ,5 II 194 43,7 81 48 ,5 Total 444 100 167 100 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... before freezing, after thawing, and after embryo culture Average embryo p Time diameter (µm) (min-max) Before 147,20 ± 7,82 (132,40 – p1-2 = freezing 1 65, 30) 0,246 (1) After 147 ,50 ± 8,14 (1 35, 50 – p2-3 = 0,001 thawing 158 ,70) (2) After 149 ,50 ± 6,30 (1 35, 40 – embryo 167,90) culture (3) There is no statistically significant difference in average diameter of day-three embryos before freezing and after... vitrification technique Criteria DayDayOR three five ( 95% CI) 19 19 Average number of transferred embryos Average thickness of uterine lining (mm) Average preserving duration (months) Survival rate (%) embryos embryo s 2,70 ± 2,17 ± 1,10 1,02 (1 – 5) (1 – 4) ; p p= 0,001 10,13 ± 1,90 (7 – 14) p = 0, 95 7,64 ± 5, 67 (0,87 – 30 ,5) 95, 2 8,33 ± 6,17 (2,0 33,9 ) 89,2 p= 0,068 Intact embryo rate (%) 88,4 70,6 Implantation... Percentage y (%) Thawed 250 Survived 238 95, 2 (238/ 250 ) Continue splitting 171 68,4 (171/ 250 ) Continue splitting after 83 83,8 thawing (level 4 (83/99) embryos) Continue splitting after 70 57 ,4 thawing (level 3 (70/122) embryos) The number of embryo that continues splitting is 171 embryos (68.4%), of which there are 83 level 4 embryos (83.8%) and 70 level 3 embryos (57 .4%) continue splitting after thawing... day-five embryos before freezing, after thawing and after embryo culture Average thickness p Time of ZP membrane (µm) (min - max ) 15 15 Before freezing 13,6 0 ± 1 ,50 (10,20 (1) – 16, 70) After thawing (2) 12,40 ± 1,30 (9,7 – 17) After embryo 10,90 ± 1, 75 (8,30 – culture (3) 15, 70) p1-2 = 0,107 p2-3 = 0,001 The average thickness of zona pellucida of day-five embryos before freezing and after thawing are . 0 ,5 1 0,6 AB 1 0 ,5 1 0,6 BA - - - - BB - - - - 2 AA 27 13,9 27 15, 6 AB 52 26,8 50 28,9 BA 10 5, 2 10 5, 8 BB 16 8,2 11 6,4 3 AA 33 17,1 33 19,0 AB 30 15, 5 24 13,8 BA 8 4,1 6 3 ,5 BB 16 8,2 10 5, 8 Total. (µm) (min-max) p Before freezing (1) 147,20 ± 7,82 (132,40 – 1 65, 30) p 1-2 = 0,246 p 2-3 = 0,001 After thawing (2) 147 ,50 ± 8,14 (1 35, 50 – 158 ,70) After embryo culture (3) 149 ,50 ± 6,30 (1 35, 40 – 167,90) There is no statistically. of thawed embryos Percentage (%) Quantity of thawing cycles Percentage (%) I 250 56 ,3 86 51 ,5 II 194 43,7 81 48 ,5 Total 444 100 167 100 The number of thawing cycles in group I ( 250 day-three embryos) is 86 cycles, and in group