There are many methods for treating infertility patients. IVF plays an important role in the field of assisted reproduction and their demands 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 assessments do not fully reflect the true quality of the embryos, if only based on morphological parameters, the results of in vitro fertilization treatment is limited.
Journal of military pharmaco-medicine 7-2013 wStudy on some factors affecting embryo morphology after biopsy for genetic diagnosis before embryo transfer Nguyen Ngoc Diep*; Nguyen Thanh Tung* Nguyen Dinh Tao*; Quan Hoang Lam* Summary There are many methods for treating infertility patients IVF plays an important role in the field of assisted reproduction and their demands 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 assessments 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 a study to evaluate embryo morphological changes and assess some factors affecting the ability of embryo survival and create blastocyst after biopsy 102 surplus embryos, which had biopsy on day were studied Quality was evaluated based 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 of 102 embryos, reached 91.17% and after 48 hours blastocyst formation is 83, reaching 81.37% The 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 higher maternal age, duration of infertility, FSH concentrations is, the less survival rate of embryos and forming blastocyst IVF or ICSI did not affect the survival rate of embryos, and further development of blastocyst after embryo biopsy * Key words: Embryo morphology; Genetic diagnosis; Embryo transfer INTRODUCTION 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 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 is 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 of them are choosing embryos based on morphological criteria of the embryo However, the morphologica lassessment does not fully reflect the true quality of the embryos, * Vietnam Military Medical University Address correspondence to Nguyen Dinh Tao: Vietnam Military Medical University E.mail: ndtao@yahoo.com 50 Journal of military pharmaco-medicine 7-2013 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 can affect the development of embryo after embryo biopsy On that basis, we conducted a study to assess embryo morphology and some related factors can affect the quality of the embryos after biopsy * 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 is 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 24 hours, 48 hours The number of blastomere and form, number fragments thickness of ZP, and diameter embryos were collected to assess embryo quality [5] * The criteria of evaluation of embryo quality: - Group I: 33 embryos of patients with age ≤ 30 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] - Group II: 39 embryos of patients with with age > 30 years old - Number of embryos (denoted by a number) - Group II: 30 embryos of patients with age > 35 years - Compare uniformity between blastomere (denoted by a capital letter): SUBJECTS AND METHODS Subjects - Including 102 surplus embryos of in vitro fertilization The study subjects were divided into groups: 51 Journal of military pharmaco-medicine 7-2013 + 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): 1: There is no cytoplasmic fragments 2: Percentage of cytoplasmic fragments < 20% 3: Percentage of cytoplasmic fragments from 20% to 50% * 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: - Grade I: The embryo fragments cytoplasmic ratio ≥ 50% of the embryo volume 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 (lowest age is 20, the highest age is 29) Patients in group II with an average age of 32.5 ± 2.2 (lowest age is 30, oldest is 35) Patients in group III with an average age of 38.4 ± 3.0 (minimum age is 36, highest is 42) Average duration 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 RESULTS The morphological changes of embryos after biopsy Some characteristics of the study subjects * Embryo morphology at days before biopsy: Research carried out on 102 patients, each embryo patient is put into research subjects, the subjects were divided into groups as follows: 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: 4: Percentage of cytoplasmic fragments > 50% - Based on the quality of embryos according to degrees is applied to evaluate the quality of embryos at Center in Academy of Military Medical 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 52 Journal of military pharmaco-medicine 7-2013 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; cytoplasmic cytoplasmic ratio median 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 Average diameter of embryo of groups is 150.20 ± 7.85, 150.20 ± 7.85, 149.50 ± 8.15, respectively Cytoplasmic fragment ratio was as high as 15% in most distributed group of over 35 age and at least in the age group under 30 The thickness of ZP is the largest of the group III: 24.4 micron, 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 that 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 24 hours, 36 hours and 48 hours, respectively in each age group to assess the development of embryos after biopsy * Embryo survival rate continues to grow and the rate of blastocyst formation after biopsy: After a 24 hour biopsy, survival rate of embryos, further development of the groups is 93 embryos in total of 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 of 102 embryos, achieved 81.37 and 84.84%, 82.05%, 76.66% respectively The difference between group I and group II are not statistically significant with p > 0.05, but the difference between groups I compared to III and II compared with III 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 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 difference 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 with - 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 is 84.48, 82.76, 66.66, respectively The difference between group I and group II are not statistically significant 53 Journal of military pharmaco-medicine 7-2013 with p > 0.05, but the difference between groups I and II compared to III is statistically significant with p < 0.05 * The patient's FSH on cycles affect ability to survive and create lastocyst: Of the 102 embryos studied, 23 patients with hormone FSH day of the menstrual cycle < IU/l, 72 patients FSH from 5-10 IU/l, and patients with FSH over 10 IU/l 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 difference 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 * Protocol of using hormone affect embryo viability and forming blastocyst: of the 102 embryos studied 53 patients used shorter regimens, 21 patients used long protocol and 28 patients used 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 difference 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 < 05 * The method of fertilization (IVF or ICSI) affects 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 54 DISCUSSION Morphology before and after embryo biopsy Embryo biopsy at day 3: In the study, the 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 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 much higher and decreases the survival rate after biopsy [8] The research results showed that the embryo grade after biopsy was 100% intact, level after biopsy was 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 survival and create blastocyst The authors suggest that the higher maternal age is, the lower success rate of IVF is, 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 was still statistically significant Journal of military pharmaco-medicine 7-2013 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 survival and create blastocyst This study is consistent with studies by Munne S [9] FSH basically did not affect form embryos after biopsy, the results are consistent with studies by Munne and et al They compared embryo morphology and FSH concentrations from which to assess the relationship between FSH levels and with basic morphological changes during embryonic development after biopsy But for women over the age of 35 with high FSH levels, although embryo morphology was less dependent on FSH concentrations, increased concentrations of FSH associated with reduced ovarian reserve capacity and consistent with low rate of pregnancy in this patients group [2] Results of dosing regimens showed that after embryo biopsy and further development, generate blastocyst are not dependent on which is derived from patients taking the whatever regimen, however, for patients with higher age, prolonged duration of infertility also affects the ability to create blastocyst, consistent with the study results by Munne et al [9] Walker M C, Murphy KE, Pan S (2004) compared methods of in vitro fertilized (IVF) with methods of cytoplasmic sperm injection into oocytes (ICSI) The results showed that morphology of two embryos in two groups are the same However, the rate of chromosomal abnormalities was significantly higher in the patient group using cytoplasmic sperm injection into oocytes because these patients often carry chromosomal disorders such as: low, weak and deformed sperm But when the embryo has reached, the standard of III and IV does not affect embryo morphology during development after biopsy [6] CONCLUSION By studying 102 biopsied embryos for PGD, we withdraw the following 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 of 102 embryos, reached 91.17%, and by day blastocyst formations are 83 embryos, reached 81.37% - Cytoplasm fragment ratio, ZP thickness, embryo diameter between groups did not change significantly * Several factors affect the survival and the forming blastocyst after biopsy: - Maternal age, duration of infertility, FSH levels higher decreased embryo survival rate - Drug regimen does not influence much to embryo survival rate - The method of 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 đoán di truyền tiền làm tổ (PGD) phôi thụ tinh ống nghiệm Báo cáo đề tài cấp thành phố 4-2011 55 Journal of military pharmaco-medicine 7-2013 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 56 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 Journal of military pharmaco-medicine 7-2013 57 ... significant 54 DISCUSSION Morphology before and after embryo biopsy Embryo biopsy at day 3: In the study, the before and after embryo biopsy were divided into levels based on the morphological... embryo morphology, and survey a number of factors can affect the development of embryo after embryo biopsy On that basis, we conducted a study to assess embryo morphology and some related factors. .. embryo morphology during development after biopsy [6] CONCLUSION By studying 102 biopsied embryos for PGD, we withdraw the following conclusions: - Laser biopsy does not alter embryo morphology,