早产和足月分娩妇女中期妊娠羊水样品中细菌DNA谱的比较

Comparison of Bacterial DNA Profiles in Mid-Trimester Amniotic Fluid Samples From Preterm and Term Deliveries4.259

Front Microbiol . 2020 Mar 24;11:415. doi: 10.3389/fmicb.2020.00415. eCollection 2020.


Abstract

Infection and inflammation are well recognized causes of spontaneous preterm delivery (PTD) (<37 gestational weeks) and adverse infant outcomes. To date, there has been very little investigation into bacterial communities in amniotic fluid using next generation sequencing technology. In particular, it is important to characterize amniotic fluid bacterial profiles in complicated pregnancies as well as in asymptomatic women to identify predictive bacterial DNA signatures. Here, 1198 mid-trimester amniotic fluid samples from a cohort of Swedish women undergoing mid-trimester genetic amniocentesis were screened for bacterial DNA using qPCR protocols specifically designed to reduce the impacts of reagent contamination and human DNA mispriming. The majority of samples were devoid of detectable bacterial DNA; however, approximately a fifth of the cohort (19.9%) were 16S rRNA gene positive in duplicate screening. Among these, nine women had a spontaneous PTD. These nine women were matched with 18 healthy women with a delivery at term. We used PacBio SMRT technology, coupled with appropriate negative extraction and PCR controls, to sequence the full-length 16S rRNA gene in this subset of 27 women. The amniotic fluid samples contained low-abundance and low-diversity bacterial DNA profiles. Species typically associated with spontaneous PTD were absent. We were not able to identify any differences in the amniotic fluid bacterial DNA profiles of women with a subsequent spontaneous PTD compared to women who delivered at term. The findings suggest that, in a minor proportion of pregnancies, DNA from non-pathogenic bacteria may be present in the amniotic fluid far earlier than previously reported. Early detection of bacterial DNA in the amniotic fluid was, in this study, not associated with spontaneous PTD.

Keywords: 16S rRNA; amniotic fluid; bacteria; microbial invasion of amniotic cavity; preterm birth

研究背景

  自发性早产(PTD)是指妊娠28-37周之间的分娩者,已有研究表明羊水中病原菌的定植是引发感染并导致PTD的原因,但对于羊水中的细菌性质、起源、定植时期和意义尚不明确,这些细菌种群在胎儿的皮肤和肠道定植,可能对胎儿的发育成熟和免疫系统产生重要影响。

目前关于羊水中微生物的研究主要依靠细菌培养和靶向实时PCR,并且主要侧重于确定与自发性PTD病因有关的病原细菌,缺少关于妊娠中期羊水整个微生物生态学的高质量数据,本研究结合人群队列和全长16S rRNA基因测序技术,表征了在妊娠中期妇女的羊水细菌DNA谱。

图1. 队列流程图及受试人群选择

研究结果

1.   妊娠中期羊水中细菌DNA存在情况

qPCR结果表明,1198个妊娠中期羊水样本中,19.9%的样本检测到细菌DNA。有趣的是,16s rRNA检测呈阳性和阴性的人群,在统计学特征上没有出现显著差异(表1),说明细菌DNA的存在情况与自发性PTD没有明显关联,不能用于队列中的自发性PTD预测。先前研究表明Ureaplasma属物种引起的羊膜腔感染通常与自发PTD相关联,研究在进一步针对Ureaplasma parvumUreaplasma urealyticum进行靶向qPCR后发现所有样本均呈阴性,说明Ureaplasma spp.在妊娠中期的羊膜定植属于罕见事件。

表1. 16S rRNA基因阳性(n = 238)和阴性(n = 960)样品的妊娠人口统计学特征。


2.   妊娠中期羊水微生物特征
与先前预期的一致,妊娠中期的羊水细菌呈现低丰度和低多样性的特点。在种水平上,优势类群主要比对到Saccharibacteria菌门,包括Acidovorax temperans, Tepidimonas taiwanensisP. puraquae,其中P. puraquae以前在孕中期羊水中尚未被发现,但已有报道这类细菌存在于剖宫产期间收集的羊水样本中。

研究未发现通常与子宫内感染和早产有关的细菌。支持了此类物种入侵可能发生在妊娠20周后的观点,本研究中确定的物种潜在定殖发生的时间更早,并且是非致病性的。结合“无菌子宫”理论,研究认为羊水中存在的细菌特征不一定代表真正的“微生物组”,仅仅反映了细菌从母亲转移到胎儿的过程,这些细菌在子宫内短暂存在但不发生定殖,从而导致了细菌低丰度的状态。

图2. 早产妇女(n = 9)和足月分娩妇女(对照组,n = 18)在妊娠中期羊水样本中检测到的细菌类群丰度。

图3. 所有孕中期羊水样本中细菌丰度百分比


3.   足月与早产妇女妊娠中期微生物差异
研究发现自发性PTD和足月分娩妇女的羊水微生物不存在显著差异,PTD样本与对照组的α多样性(图3)未观察到差异(观察到的OTU数量p = 0.87,Chao1 p = 0.44),β多样性结果也没有差异(PERMANOVA p = 0.534),检测到的物种在PTD病人和对照样本之间没有大量变化。由于队列中自发性PTD的样本较少,并且缺乏胎盘病理学的数据,无法解释出现这一结果的原因,妊娠中后期收集的羊水样本可能会提供更多信息。


  结论

  研究结果表明,尽管大多数妊娠中期的羊水样本都是无菌的,但大约有五分之一样本中存在细菌DNA。妊娠中期羊水细菌DNA谱除具有低生物量和低多样性的特点外,还缺乏与PTD相关的物种,说明羊水中非病原性细菌存在可能远早于以往认为的时期。妊娠中期羊水样本中是否存在细菌DNA的发育和免疫学意义尚不清楚。

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