There are many methods for treating infertility patients, IVF plays an important role in the field of assisted reproduction and development are increasingly widespread in the world. However, the success rate in assisted reproduction only reached 30 - 40%. Currently, most are choosing embryos based on morphological criteria of the embryo.
JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 EMBRYO MORPHOLOGY MAY CHANGE AFTER BIOPSY FOR PREIMPLANTATION GENETIC DIAGNOSIS? Vu Van Tam* et al SUMMARY There are many methods for treating infertility patients, IVF plays an important role in the field of assisted reproduction and development are increasingly widespread in the world However, the success rate in assisted reproduction only reached 30 - 40% Currently, most are choosing embryos based on morphological criteria of the embryo However, the morphological assessment not fully reflect the true quality of the embryos, if only based on morphological parameters, the results of in vitro fertilization treatment is limited To improve the quality and refinement of some genetic diseases, preimplantation genetic diagnosis (PGD) is one of the critical requirements, urgent and practical On that basis, we conducted this study to evaluate embryo morphological changes and assess some factors affecting the ability of embryo survival and create blastocyst after biopsy Subjects and methods: 102 surplus embryos, biopsy on day Quality evaluated on three main criteria: number of blastomeres in an embryo, cytoplasm fragments and the embryo volume Results: After 24 hours after biopsy, survival rate of embryos, further development of the groups is 93 embryos in total 102 embryos, reached 91.17% and after 48 hours blastocyst formation is 83, reaching 81.37% Number of embryos that survived continued development (AA and AB) account for 65.68% of 67 embryos Cytoplasm fragment ratio, ZP thickness, embryo diameter between the groups did not change significantly Conclusion: Laser biopsy does not alter embryo morphology, viability and blastocyst forming The more higher maternal age, duration of infertility, FSH concentrations the less survival rate of embryos and less forming blastocyst IVF or ICSI did not affect the survival rate of embryos, further development of blastocyst after embryo biopsy * Key words: Preimplantation genetic diagnosis; Embryo morphology; Biopsy BACKGROUND Infertility is a problem that is growing interest in the world in general and Vietnam in particular, according to the World Health Organization, the rate of infertility accounts for about 10% of couples In Vietnam by Nguyen Viet Tien et al (2013) [3], the rate of infertility in the community accounts for 7.5% Today, there are many treatment methods for infertility patients, in vitro fertilization plays an important role in the field of assisted reproduction and development are increasingly widespread in the world However, the success rate in assisted reproduction only reached 30 40%, in this area there are many issues that need to be further studied Currently, most are choosing embryos based on morphological criteria of the embryo However, the morphological assessment * Haiphong IVF Centre Corresponding author: Vu Van Tam (drvuvantam@gmail.com) 26 JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 not fully reflect the true quality of the embryos, if only based on morphological parameters, the results of in vitro fertilization treatment is limited To improve the quality of treatment as well as ensuring that a generation of healthy physical, spiritual insight, as well as screening genetic diseases, genetic diagnosis prior to embryo transfer (Preimplantation Genetic Diagnosis/PGD) is one of the critical requirements, urgent and practical Principles of PGD technique based on the performance of in vitro fertilization (IVF) to create embryos, embryo biopsy and then analyzed by chromosome or DNA FISH technique, CGH or PCR [1] Embryo biopsy technique is invasive techniques, many operations require meticulous and accurate So in the process of implementation may affect the quality of the embryos In parallel with the work completed embryo biopsy procedure, we evaluated embryo morphology, and survey a number of factors that can affect the development of embryo after embryo biopsy On that basis, we conducted a study with project as "Study on some factors affecting embryo morphology after biopsy for genetic diagnosis before embryo transfer" to assess the changes of embryo morphology and some related factors can affect the quality of the embryos after biopsy OVERVIEW Genetic diagnosis before embryo transfer (Preimplantation Genetic Diagnosis/PGD) has been adopted in many other advanced countries in the world from the last decade Principles of PGD technique based on the performance of in vitro fertilization (IVF) to create embryos, embryo biopsy and then analyzed by chromosome or DNA FISH technique, CGH or PCR (polymerase chain reaction) General technique of PGD composed main steps: embryo biopsy and genetic diagnosis The findings of several authors [1] found no difference in success rates as well as the ability to develop mental and physiological comparison with young children after PGD and ICSI infants after birth from natural conception Embryo biopsy was carried out in three steps: preparation, opened the window on the zona pellucida and took cells There are three main methods used to open window on the zona pellucida: mechanical methods, chemical or laser Currently, using a laser to open the zona membrane is considered the simplest method, safe and easy to use The development of embryos after biopsy depends on many factors, the following are some relevant factors can affect embryo morphology after biopsy In 1995, using FISH, Munne found that the percentage of embryos with diploid and the number of fragments increases with maternal age: 16% of women aged 20 to 34, 37% of women aged 35 to 39 and the highest 53% in women over 40 years old This author in 2002 study based on cell (94 embryos) and the cells (304 embryos) found that the rate of diploid, fragments increases with maternal age, from 12.2% in the age group under 35, to 31% at the age of 40 [4] 27 JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 Studies comparing methods of in vitro fertilization (IVF) and intracytoplasmic sperm injection into oocytes (ICSI) that forms of embryos from the above methods are the same But Walker MC, Murphy KE, Pan S (2004) showed that the rate of chromosomal abnormalities significantly higher in the patient group using intracytoplasmic sperm injection, as these patients are carriers of chromosomal disorders, oligospermatozoa and OAT In this case, before making the diagnosis should be made when testing the blood and semen found that these patients tend to transmit the disease to chromosome disorder Munne et al published results comparing the relationship between embryo morphology and FSH concentrations from which to assess the association between FSH levels with changes of embryo morphology and genetic deviation during meiotic division and early embryonic development For women under 40, high FSH levels, with the rate of chromosomal disorders, embryos in degree III and IV increased significantly (p < 0.02), but not dependent on the concentration of FSH Rate of chromosomal abnormalities, embryos in degree III and IV in women under 40 have FSH levels above 10 mIU/mL is equivalent to the rate of chromosomal disorder in women over age of 40 have the same concentration of FSH Such increase chromosomal disorders, embryo morphology in women over age 40 not depend on the concentration of FSH, which increases the concentration of FSH associated with reduced ovarian reserve capacity and in accordance with rate low pregnancy in this group of patients [9] 28 SUBJECTS AND METHODS Subjects - Includes 102 surplus embryos of in vitro fertilization The study subjects were divided into groups: - Group I: 33 embryos of patients with age ≤ 30 - Group II: 39 embryos of patients with with age > 30 years old - Group II: 30 embryos of patients with age > 35 years old * Selection criteria for embryo biopsy: The embryos were cultured until day of IVF culture medium The uniform cell embryos, the percentage of cytoplasmic fragments (fragment) of less than 20% (grade III embryos, grade IV) * Exclusion criteria: The early embryo grade I, grade II: uneven cells, cytoplasmic fragments ratio of 20% or more Methods * Evaluation process of fertilization and embryo culture: Approximately 16 - 18 hours after the injection of sperm into the egg, the egg was assessed whether or not fertilization If fertilized embryos form will appear 2PN and polary bodies Then the embryos were scored at each assessment time 40 hours, 68 hours and 112 hours after fertilization and embryo morphology evaluation after biopsy 24h, 48h The number of blastomere and form, number fragments thickness of ZP, and diameter embryos were collected to assess embryo quality [5] JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 * The criteria of evaluation of embryo quality: Assessing the quality of day embryos cultured according to T Ebner (2003), and is being applied in our Center According to this way of evaluating the quality embryos on day of culture is based on three main criteria: the number of blastomere in an embryo, the embryo evenly between embryos and the ratio (%) between cytoplasmic fragments and the volume of the embryo [7]: - Number of embryos (denoted by a number) - Compare uniformity between blastomere (denoted by a capital letter): + A: The even blastomere + B: The uneven blastomere - Percentage of cytoplasmic fragments (denoted by a number, is the ratio of cytoplasmic fragments compared to the volume of the embryo) + There is no cytoplasmic fragments + Percentage of cytoplasmic fragments < 20% + Percentage of cytoplasmic fragments from 20 - 50% + Percentage of cytoplasmic fragments > 50% - Based on the quality of embryos according to degrees is applied to evaluate the quality of embryos at Vietnam Military Medical University, author Andres Salumets, 2001 [10]: + Grade IV: The embryonic blastomeres of even, and no or very little (negligible) cytoplasmic fragments + Grade III: The embryonic blastomeres of uneven and/or debris cytoplasmic ratio < 20% of the embryo volume + Grade II: The embryo fragments cytoplasmic ratio from 20% to < 50% of the embryo volume + Grade I: The embryo fragments cytoplasmic ratio ≥ 50% of the embryo volume RESULTS Some characteristics of the study subjects Research was carried out on 102 patients, each patient embryo is put into research subjects, subjects were divided into groups as follows: * The number of infertile patients of the study group: Of the 102 patients, we divided into groups: Group I consists of 33 patients under the age of 30 accounted for 32.35%, group II included 39 patients aged 30 - 35 accounted for 38.24% and group III included 30 patients aged over 35 accounted for 29.41%, with the number of targets to ensure research * Age and infertility duration of the study group patients: The average age of all study patients was 29.3 ± 3.6 (20 to 42 years old), the average age of patients in group I was 21.5 ± 3.2 (minimum age is 20, the highest age is 29) Patients in group II with an average age of 32.5 ± 2.2 (minimum age is 30, oldest is 35) Patients in group III with an average age of 38.4 ± 3.0 (minimum age is 36, oldest is 42) Average duration 29 JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 of infertility patients in group I was 3.3 ± 2.3 years, the shortest period of year and a maximum of years The patients in group II with an average duration of infertility was 4.1 ± 2.6 years, the shortest time is year and the longest is years The patients in group III with an average duration of infertility was 4.8 ± 3.4 in the shortest time is year and the longest is 12 years Over time we see figures of the infertile group I was the shortest duration and infertility group III is the longest 7.2 micron thinnest group II However, the difference in the number of embryos, the rate of cytoplasmic fragments, ZP thickness, diameter embryos between groups are not statistically significant After embryo biopsy continues to be cultured in Cook Incubator, after that we measured indicator such as: diameter, ZP thickness, and the rate of blastocyst formation respectively 24, 36h and 48h in each age group to assess the development of embryos after biopsy The morphological changes of embryos after biopsy * Embryo survival rate, continues to grow and the rate of blastocyst formation after biopsy: * Embryo morphology at days before biopsy: Before biopsies we measured indicators as a basis for comparison and evaluation of morphological changes of the embryo before and after biopsy The evaluation criteria were: number of blastomeres, cytoplasmic fragments ratio, ZP thickness and diameter of embryo Average number of embryos of group I, II and III, respectively, 7.8 ± 1.21, 7.9 ± 3.13, 8.2 ± 2.25; median cytoplasmic cytoplasmic ratio respectively was 6.21 ± 1.23, 5.63 ± 1.31, 6.82 ± 1.27 Pellucida thickness was measured by cross-sectional profiles at random two locations of membrane, the zona pellucida thickness average of groups I, II and III, respectively, 15.70 ± 3.14, 16.20 ± 2.22, 16.60 ± 2.42 Diameter of embryo of groups corresponding average is 151.50 ± 6.32 µm; 151.30 ± 4.92 µm; 152.50 ± 5.32 µm Cytoplasmic fragment ratio high as 15% in most distributed over 35 age group and at least in the age group under 30 The thickness of ZP is the largest of the group III 24.4 micron, 30 After a 24-hour biopsy survival rate of embryos, further development of the groups is 93 embryos in total 102 embryos, achieved 91.17% and the group I, II and III was 93.93%; 92%, 31%, 86.66%, and by day blastocyst formation rate of the whole group was 83 embryos in total 102 embryos, achieved 81.37% and 84.84%, 82.05%, 76.66%, respectively The differences between group I and group II are not statistically significant with p > 0.05, but the difference between group I compared to III and II compared with III group is statistically significant with p < 0.05 Some factors affecting the viability of the embryo and blastocyst forming * The patient's age affects ability to survive and forming blastocyst: Of the 102 patients, we divided into groups Group I consists of 33 patients under the age of 30 accounted for 32.35%, group II included 39 patients aged 30 - 35 accounted for 38.24% and group III included JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 30 patients aged over 35 accounted for 29.41% After 24-hour biopsy embryo survival rate, continues to grow each group I, II and III was 93.93%, 92.31%, 86.66%, and by day blastocyst formation rate is 84.84%, 82.05%, 76.66% The differences between group I and group II are not statistically significant with p > 0.05, but the difference between groups I and III, and II compared to III is statistically significant with p < 0.05 * The infertility duration affects ability to survive and blastocyst forming: Of the 102 embryos studied, 58 patients having - years of infertility, 29 infertile patients from - 10 years, and 15 patients above 10 years of infertility The embryo survival rate, continues to develop is 91.13, 93.10, 86.66 and blastocyst rate respectively is 84.48, 82.76, 66.66 The differences between group I and group II are not statistically significant with p > 0.05, but the difference between groups I and II compared to III is statistically significant with p < 0.05 * Protocol of using hormone affect embryo viability and forming blastocyst: Of the 102 embryos studied, 53 patients using shorter regimens, 21 patients using long protocol and 28 patients using antagonist protocol The embryo survival rate, continues to develop general is 92.45, 95.23, 85.71 and blastocyst rate is 84.90, 80.95, 75.00, respectively The differences between group I and group II are not statistically significant with p > 0.05, but the difference between groups I, II compared to III is statistically significant with p < 0.05 * The method of fertilization (IVF or ICSI) affect the viability of the embryo and blastocyst creation: Of the 102 embryos studied, 26 patients applied IVF, 76 patients ICSI The embryo survival rate, continues to develop is 88.46, 92.11 and blastocyst rate is 80.76, 81.57, respectively The difference between the groups is not statistically significant DISCUSSION * The patient's FSH on cycles affect ability to survive and creating blastocyst: Morphology before and after embryo biopsy Of the 102 embryos studied, 23 patients with hormone FSH day of the menstrual cycle < IU/L, 72 patients FSH from - 10 IU/L, and patients with FSH over 10 IU/L In the embryo survival rate, continues to develop is 91.30, 91.67, 85.71 and blastocyst forming rate 84.53, 83.33, 71.42, respectively The differences between group I and group II are not statistically significant with p > 0.05, but the difference between groups I, II compared to III is statistically significant with p < 0.05 Embryo biopsy at day 3: In the study, before and after embryo biopsy were divided into levels based on the morphological structure of the evaluation criteria of Salumets A (2001) [10] Although with all the comments, but measurable number of embryonic cells and the structure of living cells, especially those observed cytoplasmic fragments, we can evaluate the embryos before and after biopsy So far, embryo quality assessment is mainly based on embryo morphology including cell 31 JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 number, the ratio of cytoplasmic fragments and uniformity between blastomeres Numerous studies worldwide have shown that good quality embryos, the embryo is uneven, with little cytoplasm fragments was little or no morphological changes after biopsy, the embryos have cytoplasm fragments as much higher as decreases the survival rate after biopsy [8] The research results showed that the embryo grade after biopsy is 100% intact, level after biopsy is only 72.2% intact This result is consistent with Herhberger PE et al research (2012), the rate of embryo development does not increase when the rate of cytoplasmic fragments over 20% [8] Some relevant factors affecting the ability to survive and create blastocyst The authors suggest that the more higher maternal age is the lower success rate of IVF, although on embryos of grade III and IV but studies on the ability to continue developing embryo, the formed blastocyst after biopsy they found differences between groups is still statistically significant For the duration of infertility, embryonic development continues or not is largely dependent on the quality of the early embryo Morphology of embryos after biopsy does not depend on the origin of embryos from how many year infertility mothers but depends on the quality of the embryos before biopsy, however in this study the infertility duration also interfere with the ability to survive and create blastocyst, this study is consistent with studies of Munne S [9] FSH basically not affected to form embryos after biopsy, the 32 results are consistent with studies of Munne et al compared the relationship between embryo morphology and FSH concentrations from which to assess the relationship between FSH levels with basic morphological changes during embryonic development after biopsy But for women over the age of 35 with high FSH levels, although embryo morphology less dependent on FSH concentrations, but increased concentrations of FSH associated with reduced ovarian reserve capacity and consistent with low rate of pregnancy in this patients group [2] Results between the dosing regimens showed after embryo biopsy and further develop and generate blastocyst are not dependent on which is derived from patients taking the whatever regimen, however, for patients aged high, prolonged duration of infertility also affects the ability to create blastocyst, consistent with the results of the study of Munne et al [9] Walker MC, Murphy KE, Pan S (2004) studied comparing methods of in vitro fertilized normally (IVF) and methods cytoplasmic sperm injection into oocytes (ICSI) that morphology of two embryos in two groups are the same However, the rate of chromosomal abnormalities significantly higher in the patient group using method cytoplasmic sperm injection into oocytes, because these patients are often carry chromosomal disorders, low sperm, weak and deformed But when the embryo has reached the standard of III and IV does not affect embryo morphology during development after biopsy [6] JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 CONCLUSION By studying 102 biopsied embryos for PGD, we draw two conclusions: - Laser biopsy does not alter embryo morphology, survival and forming blastocyst + After a 24-hour biopsy survival rate of embryos, further development of the groups are 93 embryos in total 102 embryos, reached 91.17%, and by day blastocyst formation are 83 embryos, reached 81.37% + Cytoplasm fragment ratio, ZP thickness, embryo diameter between groups not change significantly - Several factors affect the survival and the forming blastocyst after biopsy + Maternal age, duration of infertility, FSH levels higher are resulting decrease of embryo survival rate + Drug regimen does not influence much embryo survival rate + The method of fertilization IVF or ICSI does not affect the survival rate and continues to form blastocyst after biopsy REFERENCES Trương Đình Kiệt, Hồ Mạnh Tường Thiết lập quy trình kỹ thuật chẩn đốn di truyền tiền làm tổ (PGD) phôi thụ tinh ống nghiệm Báo cáo đề tài cấp thành phố 4/2011 2011 Nguyễn Thị Ngọc Phượng Nội tiết sinh sản Nhà xuất Y học 2013 Nguyễn Viết Tiến Dịch tễ học vô sinh phương pháp điều trị Nhà xuất Y học 2012 Alfarawati S, Fragouli E, Colls P, Stevens J, Gutierrez C, Schoolcraft The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender Fertility and Sterility 2011, 95, pp.520-524 Alikani M, Cohen J et al Human embryo fragmentation in vitro and its implications for pregnancy and implantation Fertil Steril 1999, 71, pp.836-842 Ederisinghe WR, Murch AR, Yovich JL Cytogenetic analysis of human oocytes and embryos in an in vitro fertilization program Human Reproduction 1992, 7, pp 230-236 Ebner T, Moser M, Sommergruber M et al Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development Hum Reprod Update 2003, 9, pp.251-262 Hershberger PE, Gallo AM, Kavanaugh K The decision-making process of genetically at-risk couples considering preimplantation genetic diagnosis: Initial findings from a grounded theory study Soc Sci Med 2012, May, 74 (10), pp.1536-1543 Munne S, Chen S, Colls P, Garrisi J, Zheng X, Cekleniak N Maternal age, morphology, development and chromosome abnormalities in over 6000 cleavage-stage embryos Reproductive Biomedicine Online, 2007, 14, pp.628-634 10 Salumets A, Hydén-Granskog C, Suikkari AM, Tiitinen A The predictive value of pronuclear morphology of zygotes in the assessment of human embryo quality Hum Reprod 2001, Oct, 16 (10), pp.2177-2181 33 ... development of embryo after embryo biopsy On that basis, we conducted a study with project as "Study on some factors affecting embryo morphology after biopsy for genetic diagnosis before embryo transfer"... assess the changes of embryo morphology and some related factors can affect the quality of the embryos after biopsy OVERVIEW Genetic diagnosis before embryo transfer (Preimplantation Genetic Diagnosis/ PGD)... blastocyst formation after biopsy: * Embryo morphology at days before biopsy: Before biopsies we measured indicators as a basis for comparison and evaluation of morphological changes of the embryo before