Wstudy on some factors affecting embryo morphology after biopsy for genetic diagnosis before embryo transfer

Nội dung text: Wstudy on some factors affecting embryo morphology after biopsy for genetic diagnosis before embryo transfer

1. 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 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. 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 3 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 3 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 role in the field of assisted reproduction and Infertility is a problem that is growing development is increasingly widespread in interest in the world in general and Vietnam the world. However, the success rate in in particular, according to the World Health assisted reproduction only reached 30 - 40%, Organization, the rate of infertility accounts in this area, there are many issues that need to for 10% of couples. In Vietnam, by Nguyen be further studied. Currently, most of them are Viet Tien et al (2013) [3], the rate of infertility choosing embryos based on morphological in the community accounts for 7.5%. Today, criteria of the embryo. However, the morphologica there are many treatment methods for infertility lassessment does not fully reflect the true patients, in vitro fertilization plays an important quality of the embryos, * Vietnam Military Medical University Address correspondence to Nguyen Dinh Tao: Vietnam Military Medical University E.mail: ndtao@yahoo.com 50
2. Journal of military pharmaco-medicine 7-2013 if only based on morphological parameters, * Selection criteria for embryo biopsy: the results of in vitro fertilization treatment is the embryos were cultured until day 3 of IVF limited. To improve the quality of treatment culture medium. The uniform cell embryos, as well as ensuring that a generation of the percentage of cytoplasmic fragments healthy physical, spiritual insight, as well as (fragment) of less than 20% (grade III embryos, screening genetic diseases, genetic diagnosis grade IV). prior to embryo transfer (Preimplantation * Exclusion criteria: the early embryo Genetic Diagnosis - PGD) is one of the critical grade I, grade II: uneven cells, cytoplasmic requirements, urgent and practical. Principles fragments ratio of 20% or more. of PGD technique based on the performance 2. Methods. of in vitro fertilization (IVF) to create embryos, * Evaluation process of fertilization and embryo biopsy and then analyzed by embryo culture: chromosome or DNA FISH technique, CGH or PCR [1]. Approximately 16 - 18 hours after the injection of sperm into the egg, the egg is Embryo biopsy technique is invasive assessed whether or not fertilization. If techniques, many operations require meticulous fertilized embryos form will appear 2PN and and accurate. So in the process of implementation 2 polary bodies. Then the embryos were may affect the quality of the embryos. In scored at each assessment time 40 hours, parallel with the work completed embryo biopsy 68 hours and 112 hours after fertilization procedure, we evaluated embryo morphology, and embryo morphology evaluation after and survey a number of factors can affect the biopsy 24 hours, 48 hours. The number of development of embryo after embryo biopsy. blastomere and form, number fragments On that basis, we conducted a study to assess thickness of ZP, and diameter embryos embryo morphology and some related factors were collected to assess embryo quality [5]. can affect the quality of the embryos after * The criteria of evaluation of embryo biopsy. quality: Assessing the quality of day 3 embryos SUBJECTS AND METHODS cultured according to T. Ebner., (2003), 1. Subjects. and is being applied in our Center. - Including 102 surplus embryos of in vitro According to this way of evaluating the quality embryos on day 3 of culture is fertilization. based on three main criteria: the number The study subjects were divided into 3 of blastomere in an embryo, the embryo groups: evenly between embryos and the ratio (%) - Group I: 33 embryos of patients with between cytoplasmic fragments and the age ≤ 30. volume of the embryo [7]. - Group II: 39 embryos of patients with - Number of embryos (denoted by a with age > 30 years old. number). - Group II: 30 embryos of patients with - Compare uniformity between blastomere age > 35 years. (denoted by a capital letter): 51
3. Journal of military pharmaco-medicine 7-2013 + A: The even blastomere. * The number of infertile patients of the + B: The uneven blastomere. study group: - Percentage of cytoplasmic fragments Of the 102 patients, we divided into 3 (denoted by a number, is the ratio of groups, group I consists of 33 patients under cytoplasmic fragments compared to the the age of 30, accounted for 32.35%, group II volume of the embryo): included 39 patients aged 30 - 35 accounted for 38.24% and group III included 30 patients 1: There is no cytoplasmic fragments. aged over 35 accounted for 29.41%, with the 2: Percentage of cytoplasmic fragments number of targets to ensure research. < 20%. * Age and infertility duration of the study 3: Percentage of cytoplasmic fragments group patients: from 20% to 50%. The average age of all study patients 4: Percentage of cytoplasmic fragments was 29.3 ± 3.6 (20 to 42 years old), the > 50%. average age of patients in group I was 21.5 ± - Based on the quality of embryos 3.2 (lowest age is 20, the highest age is 29). according to 4 degrees is applied to Patients in group II with an average age of evaluate the quality of embryos at Center in 32.5 ± 2.2 (lowest age is 30, oldest is 35). Academy of Military Medical Author Andres Patients in group III with an average age of Salumets, (2001) [10]. 38.4 ± 3.0 (minimum age is 36, highest is 42). - Grade IV: The embryonic blastomeres Average duration of infertility patients in of even, and no or very little (negligible) group I was 3.3 ± 2.3 years, the shortest cytoplasmic fragments. period of 1 year and a maximum of 5 years. The patients in group II with an average - Grade III: The embryonic blastomeres duration of infertility was 4.1 ± 2.6 years, the of uneven and/or debris cytoplasmic ratio shortest time is 1 year and the longest is < 20% of the embryo volume. 7 years. The patients in group III with an - Grade II: The embryo fragments average duration of infertility was 4.8 ± 3.4 cytoplasmic ratio from 20% to < 50% of the in the shortest time is 1 year and the longest embryo volume. is 12 years. Over time we see figures of the - Grade I: The embryo fragments cytoplasmic infertile group I was the shortest duration ratio ≥ 50% of the embryo volume. and infertility group III is the longest. 2. The morphological changes of embryos RESULTS after biopsy. 1. Some characteristics of the study * Embryo morphology at 3 days before subjects. biopsy: Research carried out on 102 patients, Before biopsies, we measured indicators each 1 embryo patient is put into research as a basis for comparison and evaluation of subjects, the subjects were divided into 3 morphological changes of the embryo before groups as follows: and after biopsy. The evaluation criteria were: 52
4. Journal of military pharmaco-medicine 7-2013 number of blastomeres, cytoplasmic fragments whole group was 83 embryos in total of ratio, ZP thickness and diameter of embryo. 102 embryos, achieved 81.37 and 84.84%, Average number of embryos of group I, II 82.05%, 76.66% respectively. The difference and III, respectively, 7.8 ± 1.21, 7.9 ± 3.13, between group I and group II are not 8.2 ± 2.25; cytoplasmic cytoplasmic ratio statistically significant with p > 0.05, but the median respectively was 6.21 ± 1.23, 5.63 ± difference between groups I compared to III 1.31, 6.82 ± 1.27. Pellucida thickness was and II compared with III statistically significant measured by cross-sectional profiles at random with p < 0,05. two locations of membrane, the zona pellucida 3. Some factors affecting the viability thickness average of 3 groups I, II and III, of the embryo and blastocyst forming. respectively, 15.70 ± 3.14, 16.20 ± 2.22, 16.60 ± 2.42. Average diameter of embryo * The patient's age affects ability to survive of 3 groups is 150.20 ± 7.85, 150.20 ± 7.85, and forming blastocyst: 149.50 ± 8.15, respectively. Cytoplasmic Of the 102 patients, we divided into 3 fragment ratio was as high as 15% in most groups, group I consists of 33 patients distributed group of over 35 age and at least under the age of 30 accounted for 32.35%, in the age group under 30. The thickness of group II included 39 patients aged 30 - 35 ZP is the largest of the group III: 24.4 micron, accounted for 38.24% and group III included 7.2 micron thinnest group II. However, the 30 patients aged over 35 accounted for difference in the number of embryos, the 29.41%. After 24 hour biopsy, embryo rate of cytoplasmic fragments, ZP thickness, survival rate continues to grow each group I, diameter 3 embryos between 3 groups are II and III was 93.93%, 92.31%, 86.66%, not statistically significant. and by day 5 blastocyst formation rate is After that embryo biopsy continues to be 84.84%, 82.05%, 76.66%. The difference cultured in Cook Incubator, after that we between group I and group II are not measured indicator such as: diameter, statistically significant with p > 0.05, but the ZP thickness, and the rate of blastocyst difference between groups I and III, and II formation 24 hours, 36 hours and 48 hours, compared to III is statistically significant with respectively in each age group to assess p < 0.05. the development of embryos after biopsy. * The infertility duration affects ability to * Embryo survival rate continues to grow survive and blastocyst forming: of the 102 and the rate of blastocyst formation after embryos studied, 58 patients with 1 - 5 biopsy: years of infertility, 29 infertile patients from After a 24 hour biopsy, survival rate of 5 - 10 years, and 15 patients above 10 years embryos, further development of the 3 of infertility. The embryo survival rate groups is 93 embryos in total of 102 continues to develop is 91.13, 93.10, 86.66 and embryos, achieved 91.17% and the group I, blastocyst rate is 84.48, 82.76, 66.66, II and III was 93.93%; 92,31%, 86.66%, and respectively. The difference between group by day 5 blastocyst formation rate of the I and group II are not statistically significant 53
5. Journal of military pharmaco-medicine 7-2013 with p > 0.05, but the difference between DISCUSSION groups I and II compared to III is statistically 1. Morphology before and after embryo significant with p < 0.05. biopsy. * The patient's FSH on 2 cycles affect Embryo biopsy at day 3: In the study, the ability to survive and create lastocyst: before and after embryo biopsy were divided Of the 102 embryos studied, 23 patients into 4 levels based on the morphological with hormone FSH day 2 of the menstrual structure of the evaluation criteria of Salumets cycle < 5 IU/l, 72 patients FSH from 5-10 A (2001) [10]. Although with all the comments, IU/l, and 7 patients with FSH over 10 IU/l. but measurable number of embryonic cells The embryo survival rate continues to and the structure of living cells, especially develop is 91.30, 91.67, 85.71 and blastocyst those observed cytoplasmic fragments, we forming rate 84.53, 83.33, 71.42, respectively. can evaluate the embryos before and after The difference between group I and group II biopsy. So far, embryo quality assessment is are not statistically significant with p > 0.05, mainly based on embryo morphology including but the difference between groups I, II cell number, the ratio of cytoplasmic fragments compared to III is statistically significant with and uniformity between blastomeres. Numerous p < 0.05. studies worldwide have shown that good * Protocol of using hormone affect embryo quality embryos, the embryo is uneven, with viability and forming blastocyst: of the 102 little cytoplasm fragments was little or no embryos studied 53 patients used shorter morphological changes after biopsy, the regimens, 21 patients used long protocol embryos have cytoplasm fragments much and 28 patients used antagonist protocol. higher and decreases the survival rate after The embryo survival rate continues to develop biopsy [8]. The research results showed general is 92.45, 95.23, 85.71 and blastocyst that the embryo grade 4 after biopsy was rate is 84.90; 80.95; 75.00 respectively. 100% intact, level 3 after biopsy was only The difference between group I and group II 72.2% intact. This result is consistent with are not statistically significant with p > 0.05, Herhberger PE et al research (2012), the rate but the difference between groups I, II of embryo development does not increase compared to III is statistically significant with when the rate of cytoplasmic fragments over p < 0. 05. 20% [8]. * The method of fertilization (IVF or ICSI) 2. Some relevant factors affecting the affects the viability of the embryo and ability to survival and create blastocyst. blastocyst creation: The authors suggest that the higher Of the 102 embryos studied, 26 patients maternal age is, the lower success rate of applied IVF, 76 patients ICSI. The embryo IVF is, although on embryos of grade III and survival rate continues to develop is 88.46, IV, but studies on the ability to continue 92.11 and blastocyst rate is 80.76, 81.57 developing embryo, the formed blastocyst respectively. The difference between the after biopsy they found differences between groups is not statistically significant. groups was still statistically significant. 54
6. Journal of military pharmaco-medicine 7-2013 For the duration of infertility, embryonic into oocytes because these patients often development continues or not is largely carry chromosomal disorders such as: low, dependent on the quality of the early embryo. weak and deformed sperm. But when the Morphology of embryos after biopsy does embryo has reached, the standard of III not depend on the origin of embryos from and IV does not affect embryo morphology how many year infertility mothers but depends during development after biopsy [6]. on the quality of the embryos before biopsy, however in this study the infertility duration CONCLUSION also interfere with the ability to survival and create blastocyst. This study is consistent By studying 102 biopsied embryos for PGD, with studies by Munne. S [9]. FSH basically we withdraw the following conclusions: did not affect form embryos after biopsy, - Laser biopsy does not alter embryo the results are consistent with studies by morphology, survival and forming blastocyst. Munne and et al. They compared embryo morphology and FSH concentrations from - After a 24-hour biopsy, survival rate of which to assess the relationship between embryos, further development of the 3 groups FSH levels and with basic morphological are 93 embryos in total of 102 embryos, changes during embryonic development after reached 91.17%, and by day 5 blastocyst biopsy. But for women over the age of f ormation s are 83 embryos, reached 81.37%. 35 with high FSH levels, although embryo - Cytoplasm fragment ratio, ZP thickness, morphology was less dependent on FSH embryo diameter between groups did not concentrations, increased concentrations of change significantly. FSH associated with reduced ovarian reserve * Several factors affect the survival and capacity and consistent with low rate of the forming blastocyst after biopsy: pregnancy in this patients group [2]. Results of dosing regimens showed that after embryo - Maternal age, duration of infertility, biopsy and further development, generate FSH levels higher decreased embryo survival blastocyst are not dependent on which is rate. derived from patients taking the whatever - Drug regimen does not influence much regimen, however, for patients with higher age, to embryo survival rate. prolonged duration of infertility also affects - The method of IVF or ICSI does not the ability to create blastocyst, consistent affect the survival rate and continues to form with the study results by Munne et al [9]. blastocyst after biopsy. Walker. M. C, Murphy KE, Pan S (2004) compared methods of in vitro fertilized (IVF) REFERENCES with methods of cytoplasmic sperm injection into oocytes (ICSI). The results showed that 1. Trương Đình Kiệt, Hồ Mạnh Tường. Thiết lập quy trình kỹ thuật trong chẩn đoán di truyền morphology of two embryos in two groups are tiền làm tổ (PGD) các phôi thụ tinh trong ống the same. However, the rate of chromosomal nghiệm. Báo cáo đề tài cấp thành phố 4-2011. abnormalities was significantly higher in the patient group using cytoplasmic sperm injection 55
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