- 學位論文
藉由植入前胚胎基因篩檢及胚胎縮時攝影監控來提升試管嬰兒之臨床結果
Optimizing IVF outcomes by applying preimplantation genetic screening and time-lapse monitoring technologies
摘要
為使試管嬰兒療程成功率更為良善，近年來多種侵入式或非侵入式之胚胎篩選技術蓬勃發展，冀望以更先進之技術於移植前鑑別個別胚胎的懷孕能力。而最常見之試管嬰兒療程失敗因素多與胚胎非整倍體有關，特別是針對重複著床失敗或不明原因重複流產之不孕症患者，為鑑定胚胎之非整倍體細胞於胚胎中之比例，多種著床前基因篩檢平台因應而生，其中以全面性染色體篩選相關平台最具優勢，用以判定全染色體之異常狀態。另一方面，基於縮時攝影培養系統之發展與成熟，非侵入式之胚胎篩選技術已從傳統之靜態形態學評估衍伸至最新穎之形態動力學分析，而發育時間參數與臨床結果間之相關性研究陸續開發多種形態動力學胚胎篩選模型，用以預測胚胎之懷孕潛能。本論文之目標乃探討利用全面性染色體篩選與縮時攝影胚胎觀察等相關技術進一步改善試管嬰兒療程成功率之可能性。據此，本論文以茂盛醫院之資料回溯性探討兩個研究主題，其一為探討全面性染色體篩選之微陣列晶片比較性基因體雜合分析是否可改善好發非整倍體胚胎患者之試管嬰兒療程活產率；其二為探討是否與單胚胎移植之臨床結果相關。 本論文研究一結果顯示，以微陣列晶片比較性基因體雜合分析進行著床前基因篩檢後，相較於未處理組 (32.8%) 可顯著改善高齡婦女不孕症療程之活產率(54.1%) (P = 0.018)，且經篩檢之高齡 (54.1%)、重複著床失敗 (51.6%) 與重複性流產(55.9%) 患者之活產率均相近於接受卵子捐贈之患者(57.1%)。研究二結果顯示，低度鑲嵌體胚胎之形態學或形態動力學特徵無異於整倍體胚胎，然而高度鑲嵌體胚胎則具明顯延遲之t5 (median 51.9 hpi, P = 0.034) 與t8 (median 58.6 hpi, P = 0.032) 並伴隨較長之CC3 (median 14.7 h, P = 0.012)，在形態學上，高度鑲嵌體胚胎具顯著較高之多核發生率 (P = 0.011)。非整倍體胚胎則是具延遲之tB (median 106.0 hpi, P = 0.039)，形態分析發現其優等囊胚形成比率顯著低於整倍體胚胎者 (42.6% versus 65.7%, P < 0.001)。再者，利用形態動力學分析模組KIDScore D5將整倍體胚胎依據所預測之著床能力分成三個等級，其中C等級胚胎相較於A等級 (76.2%, 79.4%, 68.3%) 與B等級 (62.5%, 66.7%, 62.5%) 胚胎具顯著較低之臨床懷孕率、著床率與持續懷孕率 (25%, 25%, 10%)(P = 0.0171 to < 0.0001)。 本論文證明利用全面性染色體篩選技術可顯著改善好發非整倍體胚胎患者之試管嬰兒療程活產率，特別是針對高齡之不孕症患者。隨著篩選技術之進步，高解析度次世代定序技術可更準確地分離低度鑲嵌體與整倍體胚胎。基於此篩選平台，整併縮時攝影培養系統與形態動力學篩選模型後，可知不同染色體狀態之胚胎雖具相異的形態動力學特徵，仍無法確切地預測染色體狀態。再者，形態動力學模型可進一步篩選具高懷孕潛能之整倍體胚胎。
並列摘要
In order to optimize success rates in in-vitro fertilization (IVF) cycles, advanced identification of the embryos with a higher potential to establish pregnancy has been attempted over years by using invasive or non-invasive techniques. The most common cause of human IVF failures, especially for the patients with repeated implantation failures (RIF) or recurrent miscarriages (RM), has been known to associate with embryonic aneuploidy. Several preimplantation genetic test for aneuploidy (PGT-A) platforms have been developed for detection of diploid-aneuploid levels in the embryos, and comprehensive chromosome screening (CCS) was the most powerful method to assess the entire chromosome complement. On the other hand, non-invasive techniques for embryo selection range from traditional static morphological evaluation to dynamic morphokinetic assessment. The development of time-lapse system provides not only a non-disturbance culture environment but also the practical possibility to monitor the embryo through the full course of in vitro development. Based on the recorded morphokinetic parameters, various selection models were then established for prediction of pregnancy outcomes. The overall aim of this thesis was to investigate the possibility of further improvement of IVF outcomes by applying and combining these novel technologies. Two independent studies were conducted using retrospective analysis of clinic data (1) to investigate whether preimplantation genetic testing for aneuploidy (PGT-A) of blastocysts through array comparative genomic hybridization (aCGH) improves live birth rates (LBR) in IVF cycles for patients with high prevalence of aneuploidy; (2) to test the hypothesis that the morphokinetics of euploid blastocysts evaluated by a generally applicable algorithm is associated with the clinical outcomes of single-embryo transfer (SET) with a hrNGS-identified euploid blastocyst. The results of each aim were as follows: (1) for the patient with advanced maternal age (AMA), a significant increase in LBRs was found in the PGT-A group compared with the non-PGT-A group (54.1% vs. 32.8%, P = 0.018). Consistent LBRs (54.1%, 51.6%, 55.9%, and 57.1%, in the group of AMA, repeated implantation failure, recurrent miscarriage, and oocyte donation, respectively) were obtained for all the indications; (2) compared with euploid blastocysts, low-level mosaic blastocysts presented comparable morphokinetic and morphological features. However, high-level mosaic blastocysts exhibited significant delays in t5 (median 51.9 h post insemination (hpi), P = 0.034) and t8 (median 58.6 hpi, P = 0.032) accompanied by a prolonged time period for the third cell cycle (median 14.7 h, P = 0.012). A significantly higher incidence (P = 0.011) of multinucleation indicated a susceptibility of high-level mosaic blastocysts to mitotic errors. Only a delay in the time for the embryo to reach the full blastocyst stage (median 106.0 hpi, P = 0.039) was revealed in aneuploid blastocysts, reflecting the reduced formation of good-quality blastocysts (42.6% versus 65.7%, P < 0.001). Euploid blastocysts with specific morphokinetic characteristics were graded using the KIDScore D5 algorithm. Grade C embryos achieved significantly lower rates of clinical pregnancy, implantation and ongoing pregnancy (25%, 25% and 10%, respectively) compared with the grade A (76.2%, 79.4% and 68.3%, respectively) or grade B (62.5%, 66.7% and 62.5%, respectively) embryos (P = 0.0171 to < 0.0001). In conclusion, the current thesis demonstrates that LBRs can be improved using aCGH-based CCS in IVF cycles for patients with a high rate of aneuploidy, especially for patients with AMA. The hr-NGS-based CCS was then applied for further distinguishing embryos with euploidy or mosaicism. When culturing the embryos in a time-lapse incubator, hr-NGS results reveals dissimilar morphokinetic features in embryos with different diploid–aneuploid mosaic levels. However, predicting chromosomal abnormalities using morphokinetics alone is still insufficient. Importantly, the use of the generally applicable KIDScore D5 algorithm has the potential to discriminate euploid blastocysts with different developmental competence.