子宫内膜容受性不良不孕症女性宫腔菌群特征研究

doi:10.12280/gjfckx.20230442

摘要/Abstract

摘要：

目的：分析子宫内膜容受性不良（poor endometrial receptivity，PER）不孕症女性宫腔菌群特征，找出其生物标志物。方法：选取2021年7月—2022年5月于天津中医药大学第二附属医院（我院）妇科就诊的不孕症女性共63例。根据超声评估子宫内膜容受性的结果分为PER组（40例）和子宫内膜容受性良好的对照组（23例）。采集2组患者子宫内膜组织进行16S rRNA高通量测序，比较2组宫腔菌群特征及差异。结果：2组患者宫腔菌群α多样性比较，差异无统计学意义（均P>0.05）。PER组宫腔菌群个体间差异显著高于对照组（P<0.05）。PER组宫腔优势菌门变形菌门相对丰度高于对照组（50.52% vs. 19.35%）；2组优势菌属均为乳杆菌属，PER组宫腔优势菌属乳杆菌属相对丰度低于对照组（21.76% vs. 45.96%），罗尔斯通菌属相对丰度高于对照组（18.01% vs. 0.48%）；PER组宫腔优势菌种未分类的罗尔斯通菌相对丰度高于对照组（18.01% vs. 0.48%），卷曲乳杆菌相对丰度低于对照组（3.82% vs. 23.78%）。PER组宫腔菌群门、科、属水平的生物标志物分别为变形菌门；伯克霍尔德菌科、弧菌科等；罗尔斯通菌属、弧菌属等。结论：PER不孕症女性存在宫腔菌群失调，其中乳杆菌相对丰度的下降以及特征致病菌（伯克霍尔德菌、罗尔斯通菌等）相对丰度的增加可能与PER有关。

关键词: 不育，女（雌）性, 子宫内膜, 胚胎植入, 高通量核苷酸序列分析, 微生物群, 伯克霍尔德杆菌科, 乳杆菌属, 子宫内膜容受性, 宫腔菌群

Abstract:

Objective: To analyze the characteristics of intrauterine microbiota in infertile women with poor endometrial receptivity (PER) and identify its biomarkers. Methods: A total of 63 infertile women who visited our gynecology department from July 2021 to May 2022 were selected. According to ultrasound evaluation of endometrial receptivity, 40 cases were assigned to the PER group, while 23 cases with good endometrial receptivity were assigned to the control group. Endometrial tissue samples were collected from two groups of patients for 16S rRNA sequencing, to compare the characteristics and differences of intrauterine microbiota between the two groups. Results: There was no significant difference in the α diversity of intrauterine microbiota between the two groups (P>0.05). However, the individual difference of intrauterine microbiota in PER group was significantly higher than that in control group (P<0.05). The relative abundance of the dominant phylum Proteobacteria in the intrauterine of the PER group was significantly higher than that in the control group (50.52% vs. 19.35%). The dominant genus in both groups was Lactobacillus, but the relative abundance of Lactobacillus in the intrauterine of the PER group was significantly lower than that of the control group (21.76% vs. 45.96%), while the relative abundance of Ralstonia was significantly higher than that in the control group (18.01% vs. 0.48%). The relative abundance of Ralstonia_unclassified in the intrauterine microbiota of the PER group was significantly higher than that in the control group (18.01% vs. 0.48%), while the relative abundance of Lactobacillus_crispatus was significantly lower than that in the control group (3.82% vs. 23.78%). The Biomakers at the phylum, family, and genus levels of the intrauterine microbiota in the PER group are Proteobacteria; Burkholderiaceae, Vibrionaceae, etc; Ralstonia, Vibrio, etc. Conclusions: Infertility women with PER may have dysbiosis of the intrauterine microbiota, with a decrease in the relative abundance of Lactobacillus and an increase in the relative abundance of characteristic pathogenic bacteria such as Burkholderia and Ralstonia possibly related to PER.

Key words: Infertility, female, Endometrium, Embryo implantation, High-throughput nucleotide sequencing, Microbiota, Burkholderiaceae, Lactobacillus, Endometrial receptivity, Intrauterine microbiota

任姝晴, 宋殿荣, 张继雯, 怀其娟, 赵琳, 张崴. 子宫内膜容受性不良不孕症女性宫腔菌群特征研究[J]. 国际妇产科学杂志, 2023, 50(6): 704-710.

REN Shu-qing, SONG Dian-rong, ZHANG Ji-wen, HUAI Qi-juan, ZHAO Lin, ZHANG Wei. Study on the Characteristics of Intrauterine Microbiota in Infertile Women with Poor Endometrial Receptivity[J]. Journal of International Obstetrics and Gynecology, 2023, 50(6): 704-710.

图/表 11

表1 2组患者超声检测参数比较

组别	n	子宫内膜厚度（mm）	子宫内膜分型（A型/B型/C型）	子宫内膜容积（mL）	子宫动脉PI	子宫动脉RI	子宫动脉S/D
PER组	40	5.65±0.37	10/7/23	1.71±0.15	3.02±0.32	0.92±0.18	8.64±0.40
对照组	23	9.70±0.48	17/4/2	3.03±0.28	1.67±0.33	0.66±0.16	6.68±0.49
t或χ²		38.240	16.922	23.450	15.810	5.736	17.210
P		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

表2 2组患者一般情况比较

组别	n	年龄（岁）	月经周期（d）	BMI （kg/m²）	不孕时间（年）	不孕类型（原发性/继发性）
PER组	40	30.23±3.98	30.18±3.12	22.03±1.70	2.21±0.65	24/16
对照组	23	29.26±4.38	29.61±4.26	21.66±1.37	2.06±0.58	16/7
t或χ²		0.892	0.606	0.892	0.861	0.576
P		0.376	0.547	0.376	0.601	0.448

表3 2组患者宫腔菌群结构组成分析表

组别	有效序列（条）	Features （个）	门（种）	纲（种）	目（种）	科（种）	属（种）	种（种）
PER组	2 588 185	6 389	33	83	190	328	782	1 122
对照组	1 122 461	2 423	25	58	139	252	585	789

图1 2组患者宫腔ASV数目比较韦恩图注：韦恩图中红色区域代表PER组宫腔菌群（poor endometrial receptivity group intrauterine microbiota，PEM），绿色区域代表对照组宫腔菌群（control group intrauterine microbiota，CEM），重叠区域代表2组宫腔共有的ASV。

图2 2组患者宫腔菌群α多样性指数小提琴图注：小提琴图中黄色代表PER组宫腔菌群（poor endometrial receptivity group intrauterine microbiota，PEM），紫色区域代表对照组宫腔菌群（control group intrauterine microbiota，CEM）。基于Observed（A）、Chao 1（B）、Shannon（C）、Simpson（D）指数比较PER组与对照组宫腔菌群α多样性。

图3 2组患者宫腔菌群主坐标分析图注：β多样性分析图中蓝色代表PER组宫腔菌群（poor endometrial receptivity group intrauterine microbiota，PEM），红色代表对照组宫腔菌群（control group intrauterine microbiota，CEM）。同一组样品使用同一种颜色表示。PCoA为主坐标分析（principal Co-ordinates analysis）。

图4 2组患者门水平宫腔菌群相对丰度分布堆叠图注：图中纵坐标表示各个菌所占总体的相对丰度的百分比。

图5 2组患者属水平宫腔菌群相对丰度分布堆叠图注：图中纵坐标表示各个菌所占总体的相对丰度的百分比。

图6 2组患者种水平宫腔菌群相对丰度分布堆叠图注：图中纵坐标表示各个菌所占总体的相对丰度的百分比。

表4 2组患者宫腔门、属、种水平菌群相对丰度表达量变化程度的比较

项目	优势菌	相对丰度（PER组/对照组）	Log₂ fold change （PER组/对照组）	调节	P
门	厚壁菌门	36.77%/62.01%	-0.75	下调	0.02
	变形菌门	50.52%/19.35%	1.38	上调	0.00
属	乳杆菌属	21.76%/45.96%	-0.40	下调	0.40
	罗尔斯通菌属	18.01%/0.48%	5.23	上调	0.00
种	卷曲乳杆菌	3.82%/23.78%	-2.64	下调	0.34
	未分类的罗尔斯通菌	18.01%/0.48%	5.23	上调	0.00

图7 2组患者宫腔菌群差异分析LDA得分图（Biomarker）注：上图展示了LDA值>3.5的显著差异微生物菌群，即具有统计学差异的Biomarker；柱状图的颜色代表不同的组别，图中红色代表PER组宫腔菌群（poor endometrial receptivity group intrauterine microbiota，PEM），绿色代表对照组宫腔菌群（control group intrauterine microbiota，CEM）。柱状图长短代表LDA值，即不同组间显著差异微生物菌群的影响程度，柱状图越长，表示影响程度越高。p 门（Phylum），c 纲（Class），o 目（Order），f 科（Family），g 属（Genus），s 种（Species）。

参考文献 18

[1]	Qiao J, Wang Y, Li X, et al. A Lancet Commission on 70 years of women′s reproductive, maternal, newborn, child, and adolescent health in China[J]. Lancet, 2021, 397(10293):2497-2536. doi: 10.1016/S0140-6736(20)32708-2. pmid: 34043953
[2]	杨仁祥, 武泽. 子宫内膜容受性的研究进展[J]. 实用妇产科杂志, 2019, 35(1):19-23.
[3]	谢幸, 孔北华, 段涛. 妇产科学[M]. 9版. 北京: 人民卫生出版社, 2018.
[4]	谢宛裔, 李蓉. 辅助生殖治疗中子宫内膜容受性评估相关临床指标的研究进展[J]. 中华生殖与避孕杂志, 2021, 41(5):413-418. doi: 10.3760/cma.j.cn101441-20200226-00086.
[5]	李芷舒, 鲁海鸥, 张宇明, 等. 不孕患者移植当日子宫内膜容受性评估对临床妊娠率影响研究[J]. 中国实用妇科与产科杂志, 2020, 36(3):264-266. doi: 10.19538/j.fk2020030118.
[6]	Kitaya K, Nagai Y, Arai W, et al. Characterization of Microbiota in Endometrial Fluid and Vaginal Secretions in Infertile Women with Repeated Implantation Failure[J]. Mediators Inflamm, 2019, 2019:4893437. doi: 10.1155/2019/4893437.
[7]	Hashimoto T, Kyono K. Does dysbiotic endometrium affect blastocyst implantation in IVF patients?[J]. J Assist Reprod Genet, 2019, 36(12):2471-2479. doi: 10.1007/s10815-019-01630-7.
[8]	Benner M, Ferwerda G, Joosten I, et al. How uterine microbiota might be responsible for a receptive, fertile endometrium[J]. Hum Reprod Update, 2018, 24(4):393-415. doi: 10.1093/humupd/dmy012. pmid: 29668899
[9]	Kyono K, Hashimoto T, Kikuchi S, et al. A pilot study and case reports on endometrial microbiota and pregnancy outcome: An analysis using 16S rRNA gene sequencing among IVF patients, and trial therapeutic intervention for dysbiotic endometrium[J]. Reprod Med Biol, 2019, 18(1):72-82. doi: 10.1002/rmb2.12250. pmid: 30655724
[10]	蔺凯丽, 郭洁, 任姝晴, 等. 复发性流产患者生殖道菌群特征的初步研究[J]. 国际生殖健康/计划生育杂志, 2022, 41(3):177-183. doi: 10.12280/gjszjk.20220153.
[11]	程阳, 赵敏. 复发性流产患者阴道菌群特征[J]. 国际生殖健康/计划生育杂志, 2022, 41(3):184-188. doi: 10.12280/gjszjk.20220143.
[12]	Saito S, Nakashima A, Shima T, et al. Th1/Th2/Th17 and regulatory T-cell paradigm in pregnancy[J]. Am J Reprod Immunol, 2010, 63(6):601-610. doi: 10.1111/j.1600-0897.2010.00852.x.
[13]	陶址, 廖秦平. 妊娠期阴道微生态变化[J]. 中国实用妇科与产科杂志, 2021, 37(10):992-994. doi: 10.19538/j.fk2021100102.
[14]	Odawara K, Akino R, Sekizawa A, et al. Examination of clinical factors affecting intrauterine microbiota[J]. Reprod Fertil, 2021, 2(1):1-6. doi: 10.1530/RAF-20-0030. pmid: 35128428
[15]	林佳惠. 微生态结合宫腔镜辅助诊断慢性子宫内膜炎[D]. 福州: 福建医科大学, 2020.
[16]	Jacobson JC, Turok DK, Dermish AI, et al. Vaginal microbiome changes with levonorgestrel intrauterine system placement[J]. Contraception, 2014, 90(2):130-135. doi: 10.1016/j.contraception.2014.04.006. pmid: 24835828
[17]	Nernsai P, Sophonsritsuk A, Lertvikool S, et al. A case report of Tubo-ovarian abscess caused by Burkholderia pseudomallei[J]. BMC Infect Dis, 2018, 18(1):73. doi: 10.1186/s12879-018-2986-z. pmid: 29422033
[18]	Cui L, Wang H, Lin J, et al. Progesterone inhibits inflammatory response in E.coli- or LPS-Stimulated bovine endometrial epithelial cells by NF-κB and MAPK pathways[J]. Dev Comp Immunol, 2020, 105:103568. doi: 10.1016/j.dci.2019.103568.