High-throughput sequencing study on the abundance of placenta and fetal membrane flora in pregnant women with premature rupture of membranes

doi:10.3877/cma.j.issn.1673-5250.2024.05.011

Objective

To investigate the results of high-throughput sequencing of placenta and membrane flora abundance in premature rupture of membranes （PROM）and normal pregnant women，and to analyze the differences.

Methods

Fifteen pregnant women who gave birth in the Department of Obstetrics，the First Affiliated Hospital of Kunming Medical University from January 2019 to February 2020 were selected as study subjects.According to whether PROM occurred or not and their gestational age at the time of PROM occurrence，they were included into preterm PROM（PPROM）group（n=5，PPROM pregnant women），term PROM （TPROM）group（n=6，TPROM pregnant women）and control group（n=4，pregnant women without PROM）.Immediately after placenta delivery，one sample of tissue from fetal face and maternal face placenta and fetal membrane was collected from pregnant women in three groups.The flora DNA in placenta and fetal membrane tissues was extracted，and the high variable region V3-V4 of 16S ribosomal RNA（r RNA）gene was sequenced with high throughput，and the flora structure of phylum and genus in the placenta and fetal membrane tissue samples of pregnant women in three groups was analyzed.Metastats software was used to compare the relative abundance of bacteria in placenta and fetal membranes tissues in PPROM group and control group，TPROM group and control group，PPROM group and TPROM group by parameter T test.There were no significant differences in the general clinical data such as age and weight of pregnant women during delivery among three groups （P ＞0.05）.The procedure followed in this study met the requirements of the WorldMedicalAssociationDeclaration ofHelsinki revised in 2013.

Results

①The difference in gestational age of pregnant women among three groups was statistically significant（F=18.65，P＜0.001）.②At the phylum level，there were 7 phyla with relative abundance＞0.1%in the placental and fetal membrane tissue samples of pregnant women in three groups：Firmicutes，Bacteroidetes，Spirochaetes，Proteobacteria，Actinobacteria，Cyanobacteria，and Fusobacteria.③At the genus level，there were 6 species of bacteria with relative abundance＞0.1%in the placental and fetal membrane tissue samples of pregnant women in three groups：uncultured bacterium，Lactobacillus，Clostridium，Streptococcus，Catenibacterium，Treponema.④The difference analysis of genus level bacterial flora in placenta and fetal membrane tissue samples of pregnant women in three groups showed that the relative abundance of Clostridium，Streptococcus，unclassified bacteria and Treponema in placenta and fetal membrane tissue samples of pregnant women in PPROM and TPROM groups were significantly higher than those in control group，while the relative abundance of Streptobacillus in PPROM and TPROM groups were significantly lower than that in control group，and all the differences were statistically significant （P＜0.05）.

Conclusions

The placenta and fetal membranes of normal pregnant women and PPROM and TPROM pregnant women are not sterile，and the distribution of placenta and fetal membrane flora among them is diverse.Compared with normal pregnant women，the diversity of placenta and fetal membrane flora of PROM pregnant women is consistent，but the abundance of some flora is obviously different.

Key words: Fetal membranes，premature rupture, High-throughput nucleotide sequencing, RNA，ribosomal，16S, Placenta, Bacteria, Biodiversity, Flora abundance, Pregnant women

表1 3组孕妇一般临床资料比较（¯x±s）

组别	例数	分娩时年龄（岁）	分娩时体重（kg）	分娩孕龄（周）
PPROM组	5	33.2±4.4	71.6±4.5	33.6±1.5
TPROM组	6	29.3±5.8	72.0±9.7	38.5±1.8
对照组	4	32.8±3.9	75.2±5.5	39.2±1.0
F值		1.01	0.33	18.65
P值		0.393	0.723	＜0.001

表1 3组孕妇一般临床资料比较（¯x±s）

组别	例数	分娩时年龄（岁）	分娩时体重（kg）	分娩孕龄（周）
PPROM组	5	33.2±4.4	71.6±4.5	33.6±1.5
TPROM组	6	29.3±5.8	72.0±9.7	38.5±1.8
对照组	4	32.8±3.9	75.2±5.5	39.2±1.0
F值		1.01	0.33	18.65
P值		0.393	0.723	＜0.001

图1 3组孕妇胎膜、胎儿面与母体面胎盘组织样本菌群OTU 数量柱状图注：横坐标中，PP表示PPROM 组孕妇，TP表示TPROM 组孕妇，N 表示对照组孕妇；1～6表示每组患者编号，A～C分别表示胎膜、胎儿面胎盘、母体面胎盘组织样本；Total表示3组孕妇胎膜与胎儿面及母体面胎盘组织样本。OTU 数量是根据菌群高通量测序DNA 序列相似度＞97%定义为1个OTU 进行计算的。OTU 为操作分类单元，PPROM 为未足月胎膜早破，TPROM 为足月胎膜早破

表2 3组孕妇胎膜、胎儿面与母体面胎盘组织样本中菌群OTU 数量比较（个，¯x±s）

组别	例数	胎膜	胎儿面胎盘	母体面胎盘
PPROM组	5	408.6±42.0	392.6±37.6	395.6±38.5
TPROM组	6	390.8±27.8	386.2±25.2	389.8±31.1
对照组	4	416.0±37.8	422.8±38.5	430.5±31.9
F值		0.69	1.56	1.88
P值		0.521	0.250	0.194

表2 3组孕妇胎膜、胎儿面与母体面胎盘组织样本中菌群OTU 数量比较（个，¯x±s）

组别	例数	胎膜	胎儿面胎盘	母体面胎盘
PPROM组	5	408.6±42.0	392.6±37.6	395.6±38.5
TPROM组	6	390.8±27.8	386.2±25.2	389.8±31.1
对照组	4	416.0±37.8	422.8±38.5	430.5±31.9
F值		0.69	1.56	1.88
P值		0.521	0.250	0.194

图2 3组孕妇胎儿面与母体面胎盘及胎膜组织样本中各类菌门相对丰度柱状图注：横坐标中，PP表示PPROM 组孕妇，TP表示TPROM 组孕妇，N 表示对照组孕期未发生PROM 孕妇；1～6表示每组患者编号，A～C分别表示胎膜、胎儿面胎盘、母体面胎盘组织样本。Firmicutes 为厚壁菌门，Bacteroidetes 为拟杆菌门，Spirochaetes 为螺旋菌门，Proteobacteria 为变形菌门，Actinobacteria 为放线菌门，Cyanobacteria 为蓝藻菌门，Fusobacteria 为梭杆菌门，Tenericutes 为软壁菌门，Verrucomicrobia 为疣微菌门，others指其他菌门。PPROM 为未足月胎膜早破，TPROM 为足月胎膜早破，PROM 为胎膜早破

表3 3组孕妇胎儿面与母体面胎盘及胎膜组织样本中各类菌门平均相对丰度比较（%）

组别	例数	厚壁菌门	拟杆菌门	变形菌门	放线菌门	蓝藻菌门	梭杆菌门	合计
PPROM组	5	69.7	24.1	2.2	0.9	0.5	0.1	97.5
TPROM组	6	66.0	27.9	1.4	0.8	0.4	1.2	97.7
对照组	4	79.9	13.0	1.7	0.2	1.3	0.1	96.2

表3 3组孕妇胎儿面与母体面胎盘及胎膜组织样本中各类菌门平均相对丰度比较（%）

组别	例数	厚壁菌门	拟杆菌门	变形菌门	放线菌门	蓝藻菌门	梭杆菌门	合计
PPROM组	5	69.7	24.1	2.2	0.9	0.5	0.1	97.5
TPROM组	6	66.0	27.9	1.4	0.8	0.4	1.2	97.7
对照组	4	79.9	13.0	1.7	0.2	1.3	0.1	96.2

图3 3组孕妇胎儿面与母体面胎盘及胎膜组织样本中菌属相对丰度的柱状图注：横坐标中，PP表示PPROM 组孕妇，TP表示TPROM 组孕妇，N 表示对照组孕期未发生PROM 孕妇；1～6表示每组患者编号，A～C分别表示胎膜、胎儿面胎盘、母体面胎盘组织样本。uncultured_bacterium_f_Muribaculaceae 为其他未分类菌属，Lactobacillus 为乳酸菌属，Clostridium_sensu_stricto_1 为梭菌属，Streptococcus 为链球菌属，Catenibacterium 为链杆菌属，Terrisporobacter 为土孢杆菌属，Lachnospiraceae_XPB1014_group 为毛足灰蝶属，Ruminococcaceae_UCG-002 为疣微菌UCG-002，Ruminococcaceae_UCG-014 为疣微菌UCG-014，Treponema_2 为密螺旋体属，others为其他菌属。PPROM 为未足月胎膜早破，TPROM 为足月胎膜早破，PROM 为胎膜早破

表4 3组孕妇胎儿面与母体面胎盘及胎膜组织样本中细菌菌属平均相对丰度比较（%）

组别	例数	未分类菌属	乳酸杆菌属	链杆菌属	梭菌属	链球菌属	密螺旋体属	合计
PPROM组	5	19.8	12.8	6.3	11.5	10.8	7.6	68.8
TPROM组	6	22.8	12.6	3.0	12.0	11.5	8.2	70.1
对照组	4	9.1	17.2	21.1	5.3	5.3	3.6	61.6

表4 3组孕妇胎儿面与母体面胎盘及胎膜组织样本中细菌菌属平均相对丰度比较（%）

组别	例数	未分类菌属	乳酸杆菌属	链杆菌属	梭菌属	链球菌属	密螺旋体属	合计
PPROM组	5	19.8	12.8	6.3	11.5	10.8	7.6	68.8
TPROM组	6	22.8	12.6	3.0	12.0	11.5	8.2	70.1
对照组	4	9.1	17.2	21.1	5.3	5.3	3.6	61.6

表5 3组孕妇胎儿面与母体面胎盘及胎膜组织样本中菌属差异性比较（%，¯x±s）

组别	例数	乳酸菌属	梭菌属	链球菌属	未分类菌属	链杆菌属	密螺旋体属
PPROM组	5	10.3±2.1	12.4±0.3	11.0±0.2	21.0±0.9	5.1±1.0	1.9±0
TPROM组	6	9.2±2.9	12.9±1.0	12.3±0.9	24.6±0.7	2.2±0.4	2.0±0
对照组	4	15.8±2.1	6.4±0.4	6.2±1.5	10.7±0.9	19.2±2.1	1.8±0
PPROM组 vs 对照组（q值/P值）		0.50/0.390	0.04/0.017	0.04/0.008	0.04/0.006	0.04/0.008	0.04/0.013
TPROM组 vs 对照组（q值/P值）		0.46/0.383	0.02/0.007	0.01/0.005	0.01/0.003	0.01/0.002	0.03/0.017
PPROM组 vs TPROM（q值/P值）		0.98/0.780	0.98/0.759	0.98/0.716	0.93/0.339	0.93/0.366	0.98/0.852

表5 3组孕妇胎儿面与母体面胎盘及胎膜组织样本中菌属差异性比较（%，¯x±s）

组别	例数	乳酸菌属	梭菌属	链球菌属	未分类菌属	链杆菌属	密螺旋体属
PPROM组	5	10.3±2.1	12.4±0.3	11.0±0.2	21.0±0.9	5.1±1.0	1.9±0
TPROM组	6	9.2±2.9	12.9±1.0	12.3±0.9	24.6±0.7	2.2±0.4	2.0±0
对照组	4	15.8±2.1	6.4±0.4	6.2±1.5	10.7±0.9	19.2±2.1	1.8±0
PPROM组 vs 对照组（q值/P值）		0.50/0.390	0.04/0.017	0.04/0.008	0.04/0.006	0.04/0.008	0.04/0.013
TPROM组 vs 对照组（q值/P值）		0.46/0.383	0.02/0.007	0.01/0.005	0.01/0.003	0.01/0.002	0.03/0.017
PPROM组 vs TPROM（q值/P值）		0.98/0.780	0.98/0.759	0.98/0.716	0.93/0.339	0.93/0.366	0.98/0.852

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