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24 The change in the rate of chromosomal disorders according to age is different in the chromosomes * Evaluation of the effectiveness of NGS technique in screening 24 chromosomes before embryo transfe[.]
24 - The change in the rate of chromosomal disorders according to age is different in the chromosomes * Evaluation of the effectiveness of NGS technique in screening 24 chromosomes before embryo transfer IVF - With NGS technique applied on 288 embryos, the number of embryos with chromosomal disorder was 36.8%, the number of embryos without chromosomal disorder was 182 (63.2%) The NGS technique can screen for all 24 chromosome abnormalities - Results of evaluating the effectiveness of PGT-A-NGS for IVF showed that PGT-A-NGS can improve the rate of clinical pregnancy, pregnancy progression The clinical pregnancy rate was 43.3% in the PGT-A-NGS group and 25.0% in the non-PGT-A group INTRODUCTION RECOMMENDATIONS Because of this is a relatively new technique, during the research period, the data we have obtained is limited We hope that in the near future we can continue to study on a large sample size From there, fully identify risk factors for multiple chromosomes as well as evaluate the effectiveness of NGS technique in screening 24 chromosomes before embryo transfer for in vitro fertilization Continuing to study the relationship between embryo morphology and autosomal polyposis during embryonic development, on that basis, select embryos of good quality for embryo transfer, improving quality in treatment Infertility is a condition in which a couple, after a year of living together, have normal sexual intercourse, not use any contraception at the fertile stage in the menstrual cycle, but the wife still can not be pregnant According to statistics of the World Health Organization (WHO), the rate of infertile couples ranges from 6% to 12% This rate in Vietnam is 7.7% Among the infertility treatments, in vitro fertilization (IVF) is one of the most effective method Chromosomal disorders in the ovum or embryo are the leading cause of non-implantation, miscarriage, stillbirth and fetal malformations Along with the development of science, the advent of Preimplantation Genetic Testing for Aneuploidy (PGT-A) helps scientists to select relatively accurate embryos without structural and number disorders of chromosomes before transferring into the mother's uterus In addition, the advent of the Next-Generation Sequencing (NGS) technique has created a revolution in biotechnology, applied in both research and clinical diagnosis In Vietnam, the Next generation sequencing (NGS) technique to screen 24 chromosomes in embryos was initially performed at some Centers, but there have not been many studies to evaluate the clinical effectiveness of infertility treatment .Therefore, we conducted the study "Relationship between some risk factors and chromosomal disorders before embryo transfer", aiming at objectives: Objectives Analyze results of genetic diagnosis before embryo transfer by next generation sequencing techniques in IVF embryos Objective To evaluate the effectiveness of pre-embryo transfer genetic diagnosis for pregnancy outcome in IVF and some factors related to chromosomal disorders before embryo transfer 2 23 SCIENTIFIC AND PRACTICAL MEANINGS OF THE THESIS Among the infertility treatments, in vitro fertilization (IVF) is one of the most effective method However, despite good morphological screening of embryos prior to transfer, the clinical pregnancy rate of IVF remains low, only 30-35% Chromosomal disorders in the ovum or embryo are the leading cause of nonimplantation, miscarriage, stillbirth and fetal malformations The advent of Next-generation sequencing (NGS) with many outstanding advantages has created a revolution in biotechnology, applied in both research and clinical diagnosis In Vietnam, the NGS technique to screen 24 chromosomes in embryos was initially performed at some centers, but there have not been many studies to evaluate the clinical effectiveness of infertility treatment This study has provided scientific evidences for the application of NGS in genetic screening before embryo transfer in Vietnam, as a premise for its widespread application in assisted reproductive centers, to increase the efficiency IVF, infertility treatment in Vietnam NEW CONCLUSIONS FROM THE THESIS This study is one of the first studies on genetic screening before embryo transfer by next generation sequencing (NGS) in IVF with analysis on 603 embryos from many IVF centers in the North of Vietnam Research on some factors related to chromosomal disorders before embryo transfer showed that factors such as history of recurrent miscarriage, history of repeated IVF failures, history of consecutive miscarriages did not increase the rate of miscarriage chromosomal disorder Meanwhile, the mother's age is positively related to the rate of chromosomal disorders according to the equation: Abnormality of chromosome number = -0.7 + 0.01 x (mother's age) Evaluation of the pre-implantation genetic diagnostic effect on pregnancy outcomes in IVF found that PGT-A-NGS has the potential to improve clinical pregnancy rates and progression pregnancy rates STRUCTURE OF THE THESIS The thesis consists of 118 pages excluding appendixes, tables, charts, diagrams, figures and 129 references, in which: pages of introduction and research objectives, 39 pages of literature review, 20 pages of research methods, research results 23 pages, discussion 31 pages, conclusions pages and recommendations page miscarriage rate when performing IVF The need to select chromosomally normal embryos has led to the development of the PGTA method PGT-A NGS technique was established to meet the requirement of screening abnormal embryos both in terms of number and structure of chromosomes CONCLUSIONS From the research results, the research team has the following conclusions: Analysis of genetic diagnosis results before embryo transfer by Next-generation sequencing technique of IVF embryos - The rate of chromosomal disorders in 5-day-old embryos fertilized in vitro in the study was 42.7% An aneuploidy can occur in all chromosomes with different rates In which, the highest are chromosomes 15, 16, 21, 22 and sex chromosomes, rare in the group of chromosomes 7, 9, 11 and 19 The rate of structural abnormalities of chromosomes is not found in chromosome 12, 20 and 22 in the study samples Evaluation of the effectiveness of genetic diagnosis before embryo transfer on pregnancy outcomes in IVF and some factors related to chromosomal disorders before embryo transfer * Identifying some risk factors for chromosomal disorders in IVF embryos: - Factors such as history of IUI failure, consecutive implantation failure in IVF, miscarriage/stillbirth and type of infertility were not related to aneuploidy in the embryo - The percentage of embryos carrying chromosomal disorders and maternal age factors have a positive correlation with the degree of association at an average level (r=0.4783) Linear regression equation: Rate of abnormal number of chromosomes = -0.7 + 0.01 x (mother's age) 22 (accounting for 85.0%) While in the group that did not PGT-A, the majority of women were under 35 years old (61.7%) The mean age in both groups in our study is 31.93 ± 4.90 higher than that of Nguyen Thi Minh Khai (2017); Tran Thuy Anh et al (2017) have an average age of 33.1 ± 4.7; Nguyen Xuan Hoi et al (2010) for the average age of the wife is 30.4±3.1 The results of Table 3.9 show that the clinical pregnancy rate is 26/60 cases in the group doing PGT-A-NGS (43.3%), and 15/60 cases in the group not doing PGT-A (accounting for 25.0%) Table 3.9 also shows that the rate of advanced pregnancy was 18.3% in the group that did PGT-A-NGS, the rate of advanced pregnancy in the group that did not PGT-A was 15.08% Thus, although the study group has proven poor prognostic factors such as: high age of the wife, higher FSH concentration than the control group, low AMH concentration, low average number of follicles obtained, number of embryos The survival rate of each patient is low, the number of oocytes developing into embryos is low, the number of embryos with good morphology is low, but the clinical pregnancy rate results in the transferred embryos of the PGT-A-NGS group are still positive The effectiveness of assisted reproduction methods is highly dependent on the quality of the transferred embryos and their ability to implant Morphological screening methods not reflect the genetic nature of the embryo In fact, 42.8% of embryos with chromosomal abnormalities can still develop to the blastocyst stage Therefore, genetic screening of IVF embryos is necessary Furthermore, spontaneous abortion in humans has been found to be strongly associated with aneuploidy Since chromosomal abnormalities are the most common cause of spontaneous infertility, PGT-A is used to minimize chromosomal abnormalities in patients undergoing IVF Thanks to PGTA, it identifies diploid embryos for transfer to the uterus, thereby increasing both the implantation rate of the embryo and reducing the CHAPTER LITERATURE REVIEW 1.1 Overview of infertility 1.1.1 Infertility definition According to the World Health Organization (WHO), infertility is the state of not becoming pregnant after one year of marriage without using any method of contraception In cases where there is a relatively clear cause of infertility, the time calculation is no longer set or for couples where the wife is over 35 years old, after months of wanting to have a baby but not getting pregnant, it is also considered as Infertility needs to be examined and treated early 1.1.2 Causes of infertility Determining the exact rate of infertility is very difficult because a large percentage of couples still go to private medical facilities and healers Causes of infertility treatment can be due to a combination of causes (40%), male infertility (26-30%), ovarian dysfunction (2125%), fallopian tubes (14-20) %), other causes (cervical lesions, genital malformations ) account for 10-13%, up to 25-28% of infertility is unexplained 1.1.3 Infertility treatments Depending on the cause of infertility, there are different treatment measures: medical treatment to embryo implantation through Assisted Reproductive Technology (ART) There are treatments which specific to men or women and some that involve both couples Among them, assisted reproductive techniques are increasingly widely applied and completely solve most of the infertility causes Assisted reproduction are treatments that involve manipulation of the ovum, sperm, and embryo outside the body to help couples get pregnant - Intrauterine insemination (IUI) - In vitro fertilization (IVF) 1.2 Normal development of pre-implantation embryos in in vitro fertilization 1.2.1 Embryos in the pronucleus stage The fertilized ovum forms a zygote and develops into an embryo through several stages that begin with the pronucleus stage Male and 21 female pronucleus usually form at the same time Male pronuclei form near the site of sperm penetration while female pronuclei form at the cytoplasmic pole with the mitotic spindle 1.2.2 Embryos in the devision stage (2nd -3rd day old after IVF) Embryonic division involves a series of cytoplasmic mitotic cycles, although the size of the embryo does not change significantly The sperm's centrosome controls this first division 1.2.3 Mulberry embryo (4th day after IVF) The mulberry embryo is formed when the embryo is at the blastocyst stage and begins to solidify 1.2.4 Blastocyst (5th-6th after IVF) After the embryo solidifies, the embryo begins to grow and creates a fluid capsule inside, creating conditions for the development of blastomeres to differentiate into trophoblasts and embryonic germ 1.3 Chromosomal disorders in ovules and embryos before embryo transfer 1.3.1 Chromosomal disorders Chromosome disorder is a phenomenon in which the number of chromosomes in a cell increases or decreases by one or a few chromosomes compared to the diploid set of chromosomes Imbalance in chromosomes will lead to embryo cessation of development before implantation, miscarriage or stillbirth 1.3.2 Mosaic embryo Base on the technique of in vitro fertilization, chromosomal abnormalities of human embryos from the pre-implantation stage were detected early 1.3.3 Rate of chromosomal disorders of embryo and ovum at different stages of embryonic development - Chromosomal disorders in ovules: The rate of chromosomal disorders in oocytes is relatively high and increases in elderly women - Chromosomal disorders in pronuleus: the rate of chromosomal disorders is 70% - Chromosomal disorders in day embryos: More than 50% of embryos created in vitro at the division stage have chromosomal disorders, this rate increases to over 80% in older women multiple chromosomes, the higher the risk of damage to the embryo Therefore, these abnormalities are rare because the studied embryos are 5-day-old embryos, so only less than 6% of embryos have more than abnormality on ≥ different chromosomes 4.2 Evaluation of the effect of genetic diagnosis before embryo transfer on pregnancy outcome in IVF and some factors related to chromosomal disorders before embryo transfer * Some factors related to chromosomal disorders before embryo transfer The study involved a total of 603 embryo samples collected from 133 couples who underwent IVF and agreed to participate in the study with a diverse number of embryos, ranging from to 14 embryos Out of a total of 603 embryos, there were 578 embryos with successful genome amplification and analysis, including: 306 embryos with maternal age < 35 (32.06 ± 3.43) and 272 embryos with maternal age ≥ 35 years old (39) 63 ± 2.96) Although subjects in different age groups have a different number of embryos, based on the data of Table 3.5, it is clear that there is a relationship between the number of embryos and the mother's age and these numbers are equally relevant statistical significance (p of tests < 0.05) However, the law of this relationship is not clear because this interaction can depend on many factors Chromosomal disorders in oocytes and embryos have a relatively high rate About 25% of eggs obtained from women aged 30 to 35 years have chromosomal abnormalities (mostly aneuploidy), and the frequency of aneuploidies is often directly age dependent and can be to more than 80% in mothers over 40 years old * Evaluation of the effect of genetic diagnosis before embryo transfer on pregnancy outcomes in IVF The mean age of the group doing PGT-A was 38.03 ± 4.38; the mean age of the group that did not PGT-A was 33.72 ± 0.49 In the group doing PGT-A, the majority of women are over 35 years old 20 In this study, PGT-A-NGS analysis was used, allowing all chromosomes to be evaluated comprehensively and accurately Furthermore, the test is based on the analysis of several cells rather than just one, which theoretically reduces the risk of misclassification due to mosaicism Finally, the analysis was conducted at the 5th day embryonic stage, whereas almost all previous efforts regarding morphology and accumulation focused on pre-embryo development (calculation) to day 3) The most frequent chromosomes with aneuploidy are chromosomes 15, 16, 21, 22 and sex chromosomes with occurrence frequency ≥ 3.0% In which chromosome 22 is the chromosome with the highest rate of abnormal number of chromosomes (6.06%), followed by chromosome 16 (5.54%) The uncommon rate of aneuploidy is the group of chromosomes 7, 9, 11 and 19 with the frequency of occurrence ≤ 1.2% When evaluating the rate of quantitative abnormalities in chromosome 11 found 99.13% of cases without detecting chromosomal disorders This is also the only chromosome with an abnormal number of chromosomes less than 1% Thus, the chromosomes of day embryos are at risk of aneuploidy, this disorder can occur in chromosome, chromosomes, chromosomes or even more than chromosomes at the same time called complex aneuploidy This result is quite similar to the results in the study of Traversa et al in 2011, when assessing the degree of aneuploidy, they found that aneuploidy in one chromosome is the highest (55%) followed by aneuploidy of chromosomes (41%), aneuploidy in or more chromosomes accounts for only 7% Regarding structural chromosome disorders, of the detected structural abnormalities, 91.67% were single abnormal, that is, there was only one type of appearance or the addition or loss of only chromosome This is the type that has the least effect on embryo viability, although these abnormalities can cause disease or manifest phenotype Abnormal forms, whether large or small in size, if present on - Chromosomal disorders in blastocysts: over 40% of embryos have chromosomal disorders, this rate increases with maternal age 1.4 Risk factors for chromosomal disorders in IVF embryos Age of the mother; history of recurrent miscarriage; history of IVF and IUI failure; types of infertility and chromosomal disorders; physical and environmental factors affect chromosomal disorders; causes of infertility related to chromosomal disorders; in vitro fertilization techniques * Methods of using stimulating hormones * Factors from sperm and the method of sperm injection into the oocyte cytoplasm - Stimulating hormone and ovarian response * Patients with low responder * Patients with high ovarian response * Patients with reduced ovarian reserve 1.5 Genetic testing techniques before embryo transfer Preimplantation genetic testing (PGT) is a test to identify genetic abnormalities of embryos produced during in vitro fertilization, before being transferred to the uterus of mother Some genetic screening methods before embryo transfer: PGT-m (Preimplantation genetic testing for monogenic/single gene defects); PGT-sr (Preimplantation genetic testing for structural chromosomal rearrangements); PGT-A (Preimplantation Genetic Screening for Aneuploidy) 1.5.1 Embryo biopsy procedure for genetic screening before embryo transfer Embryo biopsies can be performed at three different stages: biopsies before and immediately after fertilization (first and second polar body biopsies), embryo biopsies at the cleavage stage (3rd day after fertilization) sperm) or biopsy at blastocyst stage (5th day after fertilization) Each stage of biopsy has its own advantages, disadvantages and indications 1.5.2 Genetic screening techniques before embryo transfer - Fluorescent in situ Hybridization- FISH 19 - KaryoLite BoBs (BACs - on - Beads) - Comparative Genomic Hybridization (CGH) - Array Comparative Genomic Hybridization-aCGH - Next Generation Sequencing (NGS) 1.6 Application of next-generation sequencing techniques in genetic screening before embryo transfer in IVF (NGS) Like the techniques above, PGT-A using NGS requires WGA with biopsied cells to generate sufficient amount of DNA for testing Many recent studies have demonstrated that next-generation sequencing is a breakthrough technology and will hold an important place in the clinical practice of in vitro fertilization The clinical results obtained in the study from the PGT-A technique using the NGS method are very encouraging CHAPTER METHODOLOGY 2.1 Location and duration of the study - Location: Tam Anh hospital, Military Institute of Clinical Embryology and Histology - Study duration: From December, 2018 to June, 2019 2.2 Study objects - Objective 1: The target population includes embryos of highrisk couples, performing embryo screening at IVF centers - Objective 2: Subjects including couples performing in vitro fertilization techniques agreed to participate in the study 2.3 Study methodology 2.3.1 Study design: Cross sectional study 2.3.2 Sample size: 2.2.2.2 Sample size for objective 1: ( ) n= Z2(1-/2) x In which: n = Cỡ mẫu tối thiểu Minimum sample size The NGS technique used in this study is one of the novel methods used to detect known chromosomal disorders from WGA (whole genome amplification) products obtained from biological 5-day-old in vitro fertilization Accuracy and efficiency of 24-chromosome screening through evaluation and testing of WGA products as well as quality control at each milestone in the process following Illumina's process (San Diego, USA) In this study, a total of 603 embryo samples studied, with 578 embryo samples (accounting for 95.9%) successfully amplifying the genome and analyzing the disorder in all 24 chromosomes using the NGS method Only 4.1% corresponding to 25 embryos failed in the whole-genome amplification step The results of gene amplification are similar to the study of Liu et al (2016) when amplifying over 221 embryo samples, there were only samples (equivalent to 1.3% of samples without amplification of the whole genome Genome amplification will not be further analyzed in the following steps The cause of unsuccessful amplification of the whole genome may be due to the embryo transport from the Assisted Reproductive Centers to the laboratory genetics causes the samples to be splashed onto the test tube cap, but centrifuging cannot obtain the study sample Another reason may be the manipulation in the genomic amplification step that causes the sample to stick to the tip of the cone to move to the outside of the tube Failed WGA process can also result from loss of embryos during embryo washing step at Assisted Reproductive Centers Out of 247 embryo samples with chromosomal disorders, 173 samples had quantitative abnormalities, 28 samples had structural abnormalities, 36 samples had chromosomal mosaicism, and samples had both structural and numerical abnormalities number of chromosomes and mosaic embryos with abnormal number of chromosomes 18 CHAPTER DISCUSSION Z(1-⍺/2) indicates reliability If the reliability of the study is 95%, corresponding to ⍺= 5%, then Z(1-⍺/2) = 1.96 d is the deviation of the study from reality (because when the study is the result of a population = n, but not the whole community, when applied to the community, there will be a certain deviation d this difference is only within the limit of 0.1% to 10%.We choose the value d = 0.055 to ensure the limit of statistical tolerance p is the ratio representing the research criterion (the rate of chromosomal disorder) determined at the research objective and related to the depth of the study – p represents the normal rate Applied to this study: Confidence = 95% corresponds ⍺ = 5%, then Z(1-⍺/2) = 1.96 p = 56% = 0.56 (proportion of blastocysts with chromosomal disorders according to the study of F E Fragouli D Wells q = 1- 0.56 = 0.44 d = 0.055 From the above formula, the minimum sample size for the study was calculated as 313 embryos In this study, actually 603 blastocysts were used to evaluate 2.2.2.1 Sample size for objective 2: The estimated sample size was calculated according to the formula: 4.1 Analysis of genetic diagnosis results before embryo transfer by next-generation sequencing in IVF embryos In IVF, finding and selecting embryos with a high chance of conception is very important Previously, embryo selection was done based on morphological observations: blastocyst size, number, size, blastocyst nucleus morphology, nucleus/cytoplasmic ratio However, blastocyst morphology had little association with IVF outcome Many blastomeres with high morphological scores failed to implant, while those with low morphological scores produced healthy babies In many cases of miscarriage, stillbirth, we have identified a chromosomal disorder in the embryo Many studies have demonstrated that there are chromosomal disorders in eggs and blastomeres Pre-embryo transfer chromosomal screening was introduced early because of its potential to increase IVF success rates Screening 24 chromosomes in embryos helps to increase the success rate of IVF and then confirmed through many studies on different samples, by different techniques such as qPCR, aSNP, aCGH Recently, NGS technique is the latest technique applied in PGT-A The potential of NGS in PGT-A was evaluated by Yin et al., by analyzing 38 blastocyst biopsies using both SNP array and NGS All 26 diploid embryos and homozygous aneuploid embryos were correctly identified by both SNP array and NGS Furthermore, NGS also detected all embryos with unbalanced chromosomal translocations, one of which was not detected by the SNP array Another disadvantage of the SNP array used in PGT-A is the inability of the algorithm to determine the number of copies when the husband and wife are related by blood This shows the advantage of NGS over SNP array In which: - Reliability (1-α) =0.95 8 17 - Strength test (1-β) = 80% - P1: The rate of progressive pregnancy of the group without genetic screening of 24 chromosomes before embryo transfer, in the study, P1 = 41.7% according to author Yang (2012) - P2: The rate of pregnancy progression of the group that was screened for 24 chromosomes before embryo transfer, in the study, P2 = 69.1% according to author Yang (2012) Substituting into the formula the minimum sample size for each group is 51 In fact, this study involved 60 couples per group 2.2.3 Study sampling 2.2.3.1 Objectives Analyze results of genetic diagnosis before embryo transfer by next generation sequencing techniques in IVF embryos Selected research subjects include embryos of couples undergoing in vitro fertilization with blastocysts generated by intracytoplasmic sperm injection (ICSI) at the Military Institute of Clinical Embryology and Histology during the period from October 2017 to August 2018 This number of embryos included 288 embryos that were screened for chromosomes at target All blastocysts were obtained from the time of the study until a sufficient number of studies are available Analysis of pre-transitional diagnostic results by next-generation sequencing in in vitro fertilization Selected research subjects include couples performing in vitro fertilization techniques that can be generated by contrast intracytoplasmic sperm injection (ICSI) at the Military Clinical Simulation Institute - Military Medicine Institute during the period from October 2017 to August 2018 This number of holes included 288 screened and sharpened in objective All measurements were taken from the time of the study to the full number of studies Table 3.7 describes the results of embryo culture in this study, showing that the number of couples who cultured more than blastocyst embryos in the group without PGT-A was 81.7%, in the PGT-A group was 20% Most of the couples in the PGT-A group obtained 3-5 embryos (61.7%) Table 3.8 Prevalence of chromosomal disorders of blastocyst embryos No Rate Indicators SD 95% CI embryo (%) Chromosomal disorder 106 36,8 0,021 0,397 – 0,478 Without chromosomal disorder 182 63,2 0,021 0,532 – 0,613 Total 288 100 With NGS technique applied on 288 embryos, the number of embryos with chromosomal disorder was 36.8%, the number of embryos without chromosomal disorder was 182 (63.2%) 3.3.4 Results of in vitro fertilization in two study groups Table 3.9 Pregnancy results of study groups Without PGT-A PGT-A group group IVF results Number Rate Number Rate (n) (%) (n) (%) Biomedical 39 65,0 36 60,0 pregnancy Clinical 26 43,3 15 25,0 pregnancy Development 11 18,3 15,0 Pregnancy Live birth 11 18,3 13,3 Miscarriage 15 26,7 10,0 Stillbirth 0 3,3 The clinical pregnancy rate in the group that did PGT-A was 43.3%, while this rate with the group that did not PGT-A was 25.0% The rate of pregnancy development in the group doing PGT-A was 18.3%, this rate in the group not doing PGT-A was 15% 16 The results in Table 3.5 show that, type I infertility has 14 embryos with chromosomal disorders and 12 normal embryos; Type II infertility has 92 chromosomal abnormalities and 166 normal embryos However, the type of infertility did not increase the risk of chromosomal disorders in embryos with RR=1.29, CI (0.95-1.76) with p>0.05 3.3 Evaluation of results of applying new generation sequencing (NGS) technique in screening 24 chromosomes before embryo transfer Table 3.6 Levels of some hormones Group with PGTGroup without A PGT-A Indicators ± SD X X ± SD FSH (UI/l) 6,72 ± 1,63 6,09 ± 1,59 Estradiol (pg/ml) 39,58 ± 4,65 38,27 ±5,06 LH (mUI/ml) 5,27 ± 3,02 6,31 ± 3,94 Prolactin (ng/ml) 15,52 ± 3,92 16,08 ± 4,35 TSH (mUI/l) 1,66 ± 0,66 1,65 ± 0,74 AMH (ng/ml) 1,80 ± 0,64 2,46 ± 0,95 2.2.3.2 Objective To evaluate the effectiveness of pre-embryo transfer genetic diagnosis for pregnancy outcome in IVF and some factors related to chromosomal disorders before embryo transfer + Group (Group the filter moved by PGT-A): 60 infertile couples satisfying the selection criteria are applied the PGT-A-NGS filter method + Group (Group is not be filter by PGT-A): 60 infertile couples met the Study criteria Proceedings were similar to the subject studies but without the inclusion of 24-chromosomal disordered migration filters 2.2.4 Study means and process of implementation In order to collect patient selection information into the research sample in accordance with the selection criteria and exclusion criteria, and at the same time collect research variables: age, menstrual characteristics, infertility status, type of infertility, duration of infertility, history of miscarriage, history of IUI failure, history of IVF implantation failure - Stage 1: Examination and treatment to stimulate ovulation - Stage 2: in vitro fertilization / Intracytoplasmic sperm injection - Stage 3: Gene sequencing - Stage 4: Transfer of embryos into the uterus of the wife after screening for chromosomal disorders 2.2.5 Research variables and indicators - Patient characteristics: age, type of infertility, obstetric history, basal FSH level, E2 level, endometrial thickness - Features of the embryo and embryo's chromosome disorders: the percentage of embryos with chromosomal disorders, the number of chromosomes disordered, the number or structure disorders of chromosomes, aneuploidy, polyploidy - Developmental characteristics of the transferred embryos of both groups: total number of embryos, biochemical pregnancy, clinical pregnancy, miscarriage, progressive pregnancy, multiple pregnancy, ectopic pregnancy The above results show that because the age of the mothers in the two groups ranges from 24 to 42 years old, the laboratory tests also fluctuated markedly The mean value of FSH in the PGT-A and nonPGT-A groups was 6.72 ± 1.63 (UI/l) and 6.09 ± 1.59 (UI/l), respectively The mean AMH test was 1.80 ± 0.64 ng/ml in the PGT-ANGS group, and 2.46 ± 0.95 ng/ml in the non-PGT-A group AMH levels greatly affect a woman's ovarian reserve It is especially reduced in the group of patients over 35 years of age Table 3.7 Results of embryo culture of research groups Group with Blastocyst Group without Total PGT-A embryos PGT-A n (%) n (%) n (%) 5 12 (20,0) 49 (81,7) 61 (50,4) Total 60 (100) 60 (100) 120 (100) 10 15 2.2.6 Xử lý số liệu - Dữ liệu giải trình tự phân tích phần mềm Miseq Reporter 2.6 BlueFuse Multi 4.0 hãng Illumina Mỹ - Số liệu khảo sát phân tích phần mềm STATA 12.0 - Số liệu nhập vào hệ thống sở liệu cách mã hóa cho bệnh nhân, phơi lưu file dạng file excel Đề tài sử dụng thuật tốn phân tích thống kê mơ tả, thống kê suy luận phân tích tương quan (các thuật tốn có ý nghĩa thống kê p < 0,05) Trong đó, phân tích thơng kê mơ tả biến định tính thực thơng qua dạng tần số tỷ lệ phần trăm, dạng độ tập trung (trung bình, trung vị) độ phân tán (biên độ, độ lệch chuẩn, phương sai) với biến định lượng Sử dụng kiểm định phù hợp với loại số liệu đặc điểm phân bố liệu - Xác định tương quan, liên quan biến định lượng qua hệ số tương quan hồi quy tuyến tính Sử dụng hệ số tương quan Pearson r để đánh giá mối tương quan biến định lượng có phân phối chuẩn - Hệ số tương quan r có giá trị (-1) → (+1), r > tương quan đồng biến, r < tương quan nghịch biến - Sequencing data were analyzed using Miseq Reporter 2.6 and BlueFuse Multi 4.0 software of Illumina USA - Survey data were analyzed using STATA 12.0 software - Data is entered into the database system by coding for each patient, each embryo and saved in the file as an excel file The topic uses algorithms on descriptive statistics, inferential statistics and correlation analysis (the algorithms have statistical significance when p < 0.05) In which, descriptive statistical analysis for qualitative variables is performed through the form of frequency and percentage, concentration (mean, median) and dispersion (amplitude, deviation) standard, variance) with quantitative variables Use the appropriate tests for each data type and the distribution characteristics of the data - Determine correlation, relationship between quantitative variables through correlation coefficient and linear regression Use Pearson's correlation coefficient r to evaluate the correlation between normally distributed quantitative variables - The correlation coefficient r has the value (-1) → (+1), r > chromosomes with the highest rate of structural abnormalities of extra chromosomes and deletions Of the two sex chromosomes, the X chromosome detected more structural abnormalities than the Y chromosome The group of chromosomes that did not detect any structural abnormalities in a total of 578 samples were chromosomes 14, 17, 18, 19, 20 , 21, 22 3.1.4 The relationship between maternal age and chromosomal disorders in IVF embryos Table 3.4 Maternal age and Chromosomal disorder Age < 35 35-39 > 40 Total No embryo n (%) n (%) n (%) n (%) 195 113 23 (46,0) 331 Diploid embryo (63,72) (50,90) (57,26) Chromosal 111 109 27 (54,00) 247 disorder embryo (36,28) (49,10) (42,73) Total 306 (100) 222 (100) 50 (100) 578 (100) X2=11,7; p=0,003 The rate of diploid embryos in the group of women under 35 years old is 63.72%, in the group 35 to 39 years old is 50.09%, in the group over 40 years old only 46%, but this difference is not statistically significant (p>0.05) The rate of embryos with chromosomal disorders in the age group under 35 was 36.28%, in the group 35-39 it was 49.1% and the group over 40 years old was 54.0% The difference was statistically significant with pQ30) sequencing data were used for subsequent analytical steps The results show that the NGS technique allows detecting quantitative abnormalities of all 24 chromosomes of human embryos with high confidence (confident > 75%) In particular, this technique can detect abnormalities related to chromosomal structure with large size (≥ 20 Mb) Out of a total of 578 samples analyzed, 331 embryo samples had no chromosomal disorders detected (accounting for 57.3%) and 247 embryo samples had chromosomal disorders detected (accounting for 42.7% of total embryos) Table 3.2 Characteristics of chromosomal disorders of 5-day-old IVF embryos No.emp Chromosomal disorders Rate ryo Numberal Chromosomal disorder index 173 70,0% Structural Chromosomal disorder index 28 11,3% Mosaic embryos 36 14,6% Disorders of both number and structure of 3,2% chromosomes Dạng thể khảm có kèm rối loạn số lượng NST 0,8% 100,0 Total 247 % Out of 247 embryo samples with chromosomal disorders, 173 samples had quantitative abnormalities, 28 samples had structural abnormalities, 36 samples had chromosomal mosaicism, and samples had both structural and numerical abnormalities number of chromosomes and mosaic embryos with abnormal number of chromosomes ... với loại số liệu đặc điểm phân bố liệu - Xác định tương quan, liên quan biến định lượng qua hệ số tương quan hồi quy tuyến tính Sử dụng hệ số tương quan Pearson r để đánh giá mối tương quan biến... STATA 12.0 - Số liệu nhập vào hệ thống sở liệu cách mã hóa cho bệnh nhân, phôi lưu file dạng file excel Đề tài sử dụng thuật toán phân tích thống kê mơ tả, thống kê suy luận phân tích tương quan. .. structure of 3,2% chromosomes Dạng thể khảm có kèm rối loạn số lượng NST 0,8% 100,0 Total 247 % Out of 247 embryo samples with chromosomal disorders, 173 samples had quantitative abnormalities, 28 samples