Embryo morphology may change after biopsy for preimplantation genetic diagnosis

Nội dung text: Embryo morphology may change after biopsy for preimplantation genetic diagnosis

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