Preimplantation genetic testing: method validation and outcome analysis

Ph.D.Chromosomal aneuploidy has been reported in 40−50% blastocysts. Preimplantation genetic testing for aneuploidy (PGT-A) has been successfully applied to improve IVF pregnancy outcomes by selecting euploid embryos for transfer. One of the challenges in PGT-A is chromosomal mosaicism, which manifest marginal ploidy status and unknown pregnancy outcome. In addition, embryo biopsy is an invasive but inevitable procedure in PGT-A. Non-invasive PGT-A (niPGT-A) using genome sequencing and metabolic footprint analysis in embryonic spent culture medium (SCM) has been proposed as an alternative approach. The objectives of this project were to improve the clinical value of PGT by studying the pregnancy outcomes of mosaic embryo transfers (MET) and establishing novel PGT approaches, including niPGT-A and concurrent PGT.In the first study, we compared the pregnancy outcomes of 137 mosaic and 476 euploid embryos. MET group showed significantly worse pregnancy outcomes. Subsequent meta-analysis demonstrated consistency among current and previous studies. We further conducted a longitudinal follow-up study on one MET case by investigating multiple placenta biopsies after full term delivery, which provided additional evidence of aneuploidy depletion in humans. This part of my thesis thus highlighted MET as an alternative option to euploid embryo transfer when pregnancy outcome can be explained by genetic counselling thoroughly and prenatal genetic diagnosis can be monitored for the ploidy status of baby intensively.To explore novel methodologies for non-invasive PGT-A (niPGT-A), we first studied 168 SCMs from PGT-A embryos by using next-generation sequencing-based niPGT-A. Of the 116 SCMs with sequencing results of sufficient quality, the sensitivity reached 91.0% by excluding trophectoderm-mosaic cases. To further evaluate the potential of SCM for niPGT-A, we established Raman spectroscopy combining machine learning and conducted a clinical applicability study on different SCMs. A total of 319 SCMs from 120 PGT patients were analysed in this Raman-based niPGT-A. Phase I was a reproducibility study using 99 SCMs, with relatively low sensitivity (67.9%). Therefore, we refined the Phase I algorithms, resulted in improving sensitivity to 80.4% and specificity to 100%. In phase II, Raman-based niPGT-A robustness was tested by analysing 165 G-2 Plus and 55 G-TL media, both demonstrated over 90% sensitivity and specificity. In addition, Raman is a novel niPGT-A assay with the merits of low cost, highly sensitive and fast turnaround time. This part of my thesis demonstrated the broad clinical feasibility of two novel niPGT-A methods.With the promising PGT-A findings, we aimed to further enhance the clinical utility of PGT by developing a novel concurrent PGT method (OneNGS) that can facilitate PGT for monogenic disease (PGT-M) and PGT for structural rearrangement (PGT-SR) and PGT-A simultaneously using genome-wide haplotyping. The method was established by retrospectively testing re-biopsied embryos with diverse genetic diseases and comparing to conventional PGT results. With 100% concordance rate for PGT-M and 80% for PGT-A/-SR, further validation was prospectively performed on 59 embryos, with 100% concordance. Our novel genome-wide haplotyping approach enabled concurrent PGT with promising robustness, surpassing conventional PGT by the shortened turnaround time and minimized diagnostic failure.In conclusion, my thesis demonstrated MET can increase the chance of acquiring transferable embryo per cycle in IVF patients. We have also established, refined, and validated innovative non-invasive PGT-A and concurrent PGT methods with promising clinical application potential.染色體非整倍體囊胚占總數的40% - 50%。胚胎植入前遗传学筛查（PGTA）作為非整倍體篩查手段通過選擇整倍體胚胎移植，已成功應用於提高IVF周期的妊娠結局。因此，是改善妊娠結局的關鍵。PGT-A 的挑戰之一是胚胎染色體嵌合現象，表現為臨界倍性及未知的妊娠結局。此外，胚胎活檢具有侵入性。因此，在胚胎培養液（SCM）中使用基因組測序和代謝分析的非侵入性PGT-A（niPGT-A）被提出作為一種替代方法。本課題的目的是研究嵌合胚胎移植（MET）的妊娠結局，並且建立新的PGT 方法，其中包括niPGT-A 和合併PGT，來優化PGT 的臨床應用。在第一項研究中，我們比較了137 個嵌合體和476 個整倍體胚胎進行胚胎移植的妊娠結局。MET組妊娠結局明顯比整倍體移植組較差。隨後，薈萃分析證實了當前研究與以往研究的一致性。我進一步對一個MET 病例進行了縱向隨訪研究，對足月分娩後的胎盤進行了多點活檢，這為非整倍體缺失提供了更加詳盡的證據。本前瞻性研究證明MET 可作為生殖遺傳諮詢過程中整倍體胚胎移植的替代選擇。妊娠結局需對病人進行詳盡的解釋，並且以產前遺傳學檢查密切監測胎兒的染色體整倍性。為了建立一種無創的PGT-A 方法，我們首先使用基於下一代測序技術的niPGT-A 對168 個胚胎培養液進行研究。在測序質量足够的116 個SCMs 中，除滋養外胚層嵌合病例外，niPGT-A 的敏感性達到了91.0%。為了進一步評估SCM用於niPGT-A的潜力，我們建立了機器學習結合拉曼光譜的創新方法，對不同類型的胚胎培養液進行了臨床適用性研究。對120 例PGT 患者的319 個SCMs 進行分析，以建立基於Raman 的niPGT-A。第一階段是在99 個SCMs 中觀察到的低靈敏度（67.9%）的重複性研究。因此，我們開發了新的算法並對Phase I 算法進行了優化，提高了靈敏度至80.4%，特异性至100%。在第二階段，為了測試基於Raman 的niPGT-A 穩定性，對165 個G-2 Plus 和55 個G-TL培養液進行了分析，均顯示了超過90%的敏感性和特异性。此外，拉曼是一種新型的niPGT-A 檢測方法，具有成本低、靈敏度高、檢測週期短等優點。我們的研究證明了兩種新型niPGT-A 方法廣泛的臨床可行性。隨著PGT-A 的發現， 我們希望通過開發一種新的合併PGT 方法（OneNGS）來進一步提高PGT 的臨床應用，該方法可以利用全基因組單倍體分型的方法，通過單一檢測完成PGT- M /PGT- SR 和PGT-A。我通過回顧性驗證，將攜帶不同遺傳疾病的胚胎與常規PGT 的結果進行比較。PGT-M 的一致率為100%，PGT-A/-SR 的一致率為80%，我們對59 個胚胎進行了進一步的前瞻性驗證，結果一致率為100%。我們的新方法驗證了合併PGT 的可行性和穩定性。其通過縮短診斷週期、降低診斷失敗率，從而使其臨床價值超過傳統的PGT。綜上所述，MET 可以提高IVF 患者每週期獲得可轉移胚胎的機會。我們建立、完善並驗證了無創及合併PGT，並且證明了其具有良好臨床應用潜力。Zhang, Yingxin.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 154-173).Abstracts also in Chinese.Title from PDF title page (viewed on 20, September 2021)