A systematic investigation into the cervical bacterial communities in pregnant women with premature cervical dilation using massively parallel genomic sequencing

• Other Authors: Cheung, Chee Yin (author.)
• Format: Others
• Language: English; Chinese
• Published: 2014
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292741

Normally, the cervix remains long and closed, until the third trimester. However, in certain pregnancies, the cervix shortens and dilates prematurely during or before the second trimester, without pain and regular uterine contractions. Pregnant women with this condition, often known as premature/advanced cervical dilation (ACD), which is a severe form of cervical insufficiency (CI), are at increased risk of intra-amniotic infection, preterm prelabor rupture of membrane, preterm labor and preterm birth. Preterm birth (birth before 37 gestational weeks)is one of the leading causes of neonatal mortality or morbidity. Annually, among the 15 million preterm births worldwide, 1 million die because of this complication. === Cervical cerclage is an intervention to prevent preterm birth for women with ACD, but is inappropriate for cases with infection. Thus, a sensitive detection of microorganisms may result in more appropriate intervention. However, the detection methods for microorganisms using culture, microscopy or species-specific polymerase chain reaction (PCR) are only moderately sensitive or target only a few species. A comprehensive view of the microorganisms in women with ACD is lacking. === This study has embarked on a systematic investigation into the bacterial communities in theACD cervix, using massively parallel genomic sequencing (MPGS) of the 16S ribosomal RNA (rRNA) gene. Each cervical swab sample was subjected to DNA extraction and PCR amplification using universal primers targeting evolutionary-conserved regions of the 16S rRNAgenes possessed by all bacteria. These primers could amplify 99.8% of 9,244 species of sequenced, bacteria. The amplified hypervariable regions of 16S rRNA was sequenced by MPGS at an average of 28,800 raw sequencing reads per sample, which should be enough to capture majority of bacterial species within a sample. Flowgram-denoised, high-quality and non-chimeric reads were taxonomically classified by matching against the most updated 16S rRNA sequence databases of sequenced bacteria, including non-culturable species. The relative abundance of each identified bacterium was calculated in samples from women with ACD (n=19), andcompared with that from women without ACD (n=13). === In this project, the first comprehensive view of the bacterial communities in the cervix of pregnant women with ACD was presented. I have identified 9 and 7 bacteria that were significantly over-represented and under-represented, respectively, in the dilated cervix (ACD) group, compared withthe closed cervix (no ACD) group. The presence of any of the 9 over-represented bacteria was significantly associated with ACD, spontaneous preterm birth <28 weeks,preterm birth <28 weeks and intraventricular hemorrhage. === Among the 32 pregnancies analyzed, 13 second-trimester cervices had been colonized by one or more of these over-represented 9 bacteria.Intriguingly, all of them resulted in preterm birth <34 weeks.Of these, 7 cases resulted in spontaneous preterm birth <34weeks. Notably, using ≥1 of the 9 over-represented bacteria as a detection threshold, all these 7 spontaneous preterm births could be detected. === The data presented in this thesis form the groundwork for developing species-specific PCR assays to detect ACD-associated bacteria. The relationship between these ACD-associated bacteria and certain adverse pregnancy outcomes warrants further investigationin prospective population-based studies. === 在一個正常的妊娠，宮頸保持封閉狀態且维持一定長度，直到孕晚期。然而，在某些異常妊娠裡，宮頸會過早地在孕中期縮短和擴張，子宮無疼痛和規律收縮，這被稱為宮頸機能不全（CI）。孕婦有這種情況，會大大增加從陰道進入子宮腔而受的感染，早產和胎膜早破的風險。早產，即在孕37週前分娩，是新生兒死亡或發病的主要原因之一。每年，全球約有1500萬早產嬰兒（約佔全部活產嬰兒的10％），其中約100萬嬰幼兒死於早產並發症。 === 宮頸環扎術，是一種有效防止因宮頸機能不全導致早產的治療手段。但宮頸環扎術並不適合用於宮內感染的病例。因此，一個有敏感且全面的檢測微生物方法可以幫助臨床醫生正確治療宮頸機能不全的孕婦。常規用以檢測微生物的方法主要包括細菌培養，顯微鏡學或針對個別細菌種類的聚合酶鏈反應(PCR)。然而，這些方法敏感度低或只能測度有限數量的幾種微生物。到目前為止，仍沒有一個全面篩查導致不成熟宮頸擴張的有關微生物的研究。 === 在我的哲學碩士學習中，我採用16S核糖體核糖核酸(16S rRNA) 的基因組序列的高通量二代測序，系統地調查與孕婦過早宮頸擴張有關的微生物群落。針對所有的細菌具有的16S rRNA基因的進化保守區通用PCR引物，以各宮頸拭子提取的DNA為模板擴增相應產物。這些通用引物可以擴增多達九千種已經明確基因組序列的細菌。二代測序針對每個樣品擴增的16S rRNA高度變異區產生平均二萬八千八百測序片段。這個測序數量足夠捕捉樣本中大多數細菌類群。高質量，修剪和非嵌合的測序片段根據最新的16S rRNA基因序列數據庫匹配已知的細菌。細菌種類的相對豐度計算根據有過早子宮頸擴張的孕婦（N=19）與無該病例現象的孕婦（N=13）進行比較。 === 我的論文率先展示有關導致過早宮頸擴張的孕婦的細菌群落的全面圖譜。並在這些有過早宫颈擴張的孕婦宫頸拭子中鑒定出九種細菌相對豐度顯著增加，七種相對豐度顯著减少（p<0.05）。其中，這九種相對豐度顯著增加的細菌與自然早產（早於28孕週），早產（早於28孕週）和早產兒腦室內出血等臨床表現有顯著關係。另外，在三十二個病例中，有十三個早產病例〈早於34週〉對那九種細菌相對豐度顯著增加的細菌類群中至少一種呈陽性。而其中，有七個病例屬於自然早產〈早於34週〉。值得注意的是，這七個早產病例都對那九種細菌相對豐度顯著增加的細菌類群中至少一種呈陽性。 === 這篇論文中的相關數據為開發有關特異性實時定量聚合酶鏈反應實驗來檢測與過早宮頸擴張相關的細菌種類提供了堅實基礎。與過早宮頸擴張相關的細菌種類和如何針對不同細菌導致的過早宮頸擴張的治療和臨床療效值得進一步深入研究。 === Cheung, Chee Yin. === Thesis M.Phil. Chinese University of Hong Kong 2014. === Includes bibliographical references (leaves 100-106). === Abstracts also in Chinese. === Title from PDF title page (viewed on 13 March, 2018). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.