育龄期女性宫颈菌群及其与鳞状上皮内瘤变的相关性

Characteristics of cervical microbiota and its relationship with squamous intraepithelial lesion among reproductive women

目的研究育龄期妇女从HPV感染到宫颈鳞状上皮内瘤变(squamous intraepithelial lesion,SIL)过程中的宫颈菌群组成和变化。方法对40名参与者(年龄2149岁)的宫颈分泌物菌群16S rDNA进行了特征分析,其中包括高级别鳞状上皮内瘤变组(HSIL)、低级别鳞状上皮内瘤变组(LSIL)、单纯HPV感染组[Ctrl HPV(+)]和健康对照组[Ctrl HPV(-)],每组各10例。样本内的α多样性采用Species和Shannon指数,样本间的β多样性采用非度量多维尺度分析(NMDS)。利用线性判别分析效应大小(LEfSe)比较菌群的相对丰度。通过共现分析确定标记属之间的相关性,并通过PICRUSt探索宫颈菌群的功能特征和途径。结果宫颈分泌物pH值随疾病进展显著上升(P<0.05),且HPV感染状态及亚型在4组间显著不同(P<0.05)。16S rDNA分析宫颈分泌物菌群结果表明,与Ctrl HPV(-)和Ctrl HPV(+)组相比,LSIL和HSIL组ASVs减少,4组间仅有少量共有ASVs(n=107)。菌群多样性分析显示,各组α多样性差异无统计学意义(P>0.05),但β多样性差异有统计学意义(P<0.05),且LSIL和HSIL组菌群结构明显偏离Ctrl HPV(-)和Ctrl HPV组。LEfSe分析获得11个标记属,包括HSIL的Bifidobacterium、Megasphaera、Gemella和Mucispirillum,以及LSIL的Klebsiella、Capnocytophaga和NB1-j。共现性网络分析显示,Helicobacter、Clade_Ⅲ和Aureimarina与其他标记属共现相关性最强,且绝大多数呈现正相关。相关性分析显示,上述11个差异属与差异KEGG途径相关,而其他差异属无相关结果;与Klebsiella相关的差异功能途径,大多数与Mucispirillum的关联性相反。结论育龄期女性在SIL发生的不同阶段,宫颈菌群结构存在差异。标记属可能参与病变进展,其或将有助于SIL的诊断、预防和治疗。

Objective To characterize cervical microbial feature during the progression from chronic cervicitis to squamous intraepithelial lesion(SIL)in women of childbearing age.Methods We characterized the 16S rDNA of cervical secretion from 40 participants aged 21-49 years.Participants were divided into HSIL group(high-grade SIL),LSIL group(low-grade SIL),Ctrl HPV(+)group(histopathologically normal while HPV-positive)and Ctrl HPV(-)group(healthy control)with 10 cases in each group.Species and Shannon indices were used forαdiversity within samples,and non-metric multidimensional scaling(NMDS)was used forβdiversity among samples.Linear discriminant analysis effect size(LEfSe)was used to compare the relative abundance of the microbiota.Correlation between marker genera was determined by co-occurrence analysis,and functional characteristics and pathways of cervical microbiota were explored by PICRUSt.Results The pH of cervical secretions increased significantly with disease progression(P<0.05),and HPV infection status and subtypes were significantly different among the 4 groups(P<0.05).16S rDNA analysis of cervical secretions showed that ASVs were reduced in the LSIL and HSIL groups compared with the Ctrl HPV(-)and Ctrl HPV(+)groups,and only a small number of ASVs(n=107)were shared among the four groups.Bacterial diversity analysis showed that there was no significant difference inαdiversity among all groups(P>0.05),whereas the difference ofβdiversity was statistically significant(P<0.05),and the microbial structure of LSIL and HSIL groups was significantly different from that of Ctrl HPV(-)and Ctrl HPV groups.Eleven marker genera were identified by LEfSe analysis,including Bifidobacterium,Megasphaera,Gemella and Mucispirillum from HSIL,and Klebsiella,Capnocytophaga and NG1-J from LSIL.Co-occurrence network analysis showed that Helicobacter,Clade_Ⅲand Aureimarina had the strongest co-occurrence correlation with other marker genera,and most of them showed positive correlation.Further correlation analysis showed that the above 11 difference genera were correlated with differential KEGG pathway,while the other difference genera had no correlation results.Klebsiella and Mucispirillum were inversely associated with most KEGG pathways.Conclusion In women of childbearing age,the structure of cervical microbiota is different at different stages of SIL.Marker genera may be involved in the progression of lesions,which might be used for diagnosis,prevention and treatment of SIL.