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標題: | 胚胎細胞中粒線體DNA含量與胚胎發育能力之關係 The investigation of mitochondrial DNA copy number as a biomarker of embryo developmental competency |
作者: | Shao-Ying Wu 吳劭穎 |
指導教授: | 李妮鍾(Ni-Chun Lee) |
關鍵字: | 粒線體,試管嬰兒,胚胎品質,次世代定序,染色體, mitochondria,in-vitro fertilization,embryo viability,next-generation sequencing,chromosomes, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 研究背景: 全世界首例試管嬰兒問世至今已超過 40 年。雖然目前人工生殖的技術已十分發達,傳統試管嬰兒的成功率仍僅有三成左右。造成胚胎植入後著床失敗的主因為胚胎染色體套數異常。植入前胚胎染色體套數檢查 (Preimplantation Genetic Screening; PGS) 是一種在植入前對胚胎進行切片和染色體數目分析的技術,目的為挑選出染色體數目正常的胚胎來進行植入。即使如此,植入經由 PGS 篩選過的的胚胎仍有近三成左右仍無法順利著床。由此可見除了染色體的數目是否正確之外,應還有其他可用於輔助判斷胚胎著床能力的因素存在。粒線體是一種存在於人體大部分細胞內的胞器。它是細胞內能量分子「三磷酸腺苷」(Adenosine Triphosphate; ATP)合成製造的主要場所,所以粒腺體可被視為「細胞能量中心」。近年來已陸續有研究發現胚胎細胞內的粒線體 DNA 含量等對胚胎品質是有影響的,不過亦有其他研究結果抱持的相反結果。所以胚胎內的粒腺體 DNA 含量是否可用於預測胚胎品質、著床率、懷孕率等目前仍具爭議性。
研究目的: 探討胚胎滋養層細胞內的粒線體 DNA 含量與 (1)胚胎染色體套數 [euploid vs. aneuploid]、(2)胚胎發育速度 [Day 5囊胚vs.Day 6囊胚] 及 (3)母體年齡之間的關係。另外亦有針對染色體套數正常的但是具有不同發育速度的胚胎之間的懷孕率及粒線體含量進行分析。希望此研究結果能讓粒線體DNA含量在臨床上成為篩選胚胎著床能力的另一重要指標。 研究方法: 我們進行回溯性病例研究,收集自2016 年11月至2018年04月在愛群婦產科診所進行人工生殖 (試管嬰兒與植入前胚胎染色體數目篩檢) 的胚胎及母親年齡之資訊。我們從NGS數據中分析滋養層細胞內的粒腺體 DNA (mitochondrial DNA copy number; mtDNA) 與細胞核 DNA (nuclear DNA copy number; nDNA) 的相對含量 (mtDNA score)。同時也探討胚胎染色體套數、胚胎發育速度、胚胎植入後結果以及母親年齡等資料與mtDNA score之相關性。 結果: 總共有來自460 名女性不孕症患者中共 1301 顆有接受次世代定序 PGS 的胚胎被分析。染色體套數異常的胚胎和染色體套數正常的胚胎相比在滋養層細胞中含有量較多的 mtDNA,且此差異在統計學上具有顯著性。在第五天發育為成熟囊胚的胚胎 [Day 5 囊胚] 和於第六天才發育為成熟囊胚的胚胎 [Day 6 囊胚] 相比在滋養層細胞中含有量較多的 mtDNA,且此差異在統計學上具有顯著性。Day 5 囊胚不論是在胚胎染色體正常比例,臨床懷孕率都顯著優於 Day 6 囊胚。母體年齡對於胚胎滋養層細胞中的 mtDNA 含量無直接影響。根據本研究結果所界定用於預測胚胎著床力的相對 mtDNA含量為 0.002,即所有 mtDNA 高於此數值的胚胎在植入後均未著床成功。 結論: 雖然在染色體套數異常的胚胎中 mtDNA 的量會顯著上升,不過這並非代表在染色體套數正常的胚胎中 mtDNA含量越高胚胎品質是越差的。更大型且採用雙盲隨機分配的相關研究仍須持續進行來使我們能更了解粒線體量的多寡在胚胎中的意義以及臨床運用於挑選植入成功率的最高的胚胎之可行性。 Introduction: The primary goals of modern-day assisted reproductive technology (ART) is to increase pregnancy and live birth rates, while at the other hand decrease complications related to preterm births from multiple pregnancies. Scientists have tested many novel technologies that could reliably assess embryo viability and quality, which ultimately would enable transferring only a single competent embryo that is suffice in creating a healthy live birth. Recently, mitochondria DNA quantity within the embryonic trophectoderm cells have been suggested to be a predictor of embryo viability in several studies, while others have found no such correlation. Objective(s): To investigate the relationship between the quantity of embryonic trophectoderm cells’ mitochondria DNA and (1) embryo ploidy (euploid vs. aneuploid) 、 (2) embryo developmental rate (Day 5 vs. Day 6 blastocyst formation) and (3) maternal age. Secondly, to explore the difference in mtDNA quantity and transfer outcomes between euploid embryos of different development rates. cells have implications in respect to the reproductive potential of the embryos. Lastly, to assess the accuracy of mtDNA quantity in predicting implantation potential of euploid embryos. Study Design: This is an observational retrospective analysis consisted of medical records review of infertile patients that underwent in-vitro fertilization and preimplantation genetic screening at IHMED Fertility Center from 2016/11 to 2018/04. Primary intervention was the calculation of “mtDNA score”, which was the relative quantification of embryo mitochondria DNA copy number with regard to nuclear DNA copy number extracted from the NGS data for PGS. The relationship between mtDNA score to embryo ploidy, embryo developmental rate, maternal age, and embryo transfer outcomes were also analyzed. Result(s): A total of 1,301 embryos from 460 infertile women undergoing in-vitro fertilization (IVF) with intracyoplasmic sperm injection (ICSI) and next generation sequence (NGS) based pre-implantation genetic screening (PGS) were included for the analysis. Aneuploid embryos had significantly greater quantity of mtDNA compared to eupoid embryos. When considering the embryo development rate, day 5 blastocysts had a significantly higher mtDNA quantity than day 6 blastocysts, independent of embryo ploidy. Thirdly, when factoring the effects of maternal age, no differences in the mtDNA quantity was noted when analyzing blastocysts pooled from subjects aged < 40 and > 40 years-old, irrespective of embryo ploidy and development rate. The comparison of embryo euploidy rates, clinical pregnancy rates, and mtDNA score of euploid embryos that resulted in clinical pregnancy between day 5 blastocysts and day 6 blastocysts also showed significant difference favoring day 5 blastocysts. The optimal cut-off values of mtDNA score that predicts the viability of euploid blastocysts was 0.002. Conclusion(s): mtDNA quantity is a useful indicator that accurately predicts the implantation potential of euploid blastocysts. Increased mtDNA content was frequently observed in aneuploid blastocysts or euploid blastocysts of reduced implantation potential, indicating possible embryonic stress or intrinsic mitochondrial dysfunction. Nevertheless, a minimal mtDNA quantity must also be maintained in order for proper embryonic development and establishment of viable pregnancy. The delicate balance between mtDNA quantity and embryo viability warrants further large scale, randomized controlled studies. |
URI: | http://tdr.lib.ntu.edu.tw/handle/123456789/646 |
DOI: | 10.6342/NTU201903046 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 分子醫學研究所 |
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