反复种植失败患者阴道菌群特征研究

doi:10.12280/gjszjk.20230430

摘要/Abstract

摘要：

目的：探讨反复种植失败（repeated implantation failure，RIF）患者的阴道菌群特征。方法：纳入49例接受冻融胚胎移植（frozen-thawed embryo transfer，FET）患者。其中观察组患者（n=29）有RIF病史且本次FET周期妊娠失败；对照组患者（n=20）首次行FET且成功妊娠。2组均采取人工周期准备内膜、移植第5天优质囊胚。于FET日收集患者阴道分泌物，通过16S rRNA测序对2组阴道菌群多样性、群落组成以及差异菌属进行分析。用受试者工作特征（receiver operating characteristic，ROC）曲线评估阴道乳杆菌属丰度诊断RIF的效能。结果：α多样性分析显示，观察组阴道菌群多样性（Shannon指数：P=0.049 8）和丰富度（Chao指数：P=0.047 1）均显著高于对照组。β多样性分析显示，2组阴道菌群结构差异有统计学意义（P=0.016）。在属水平上，2组阴道的优势菌均为乳杆菌属，与对照组相比，观察组乳杆菌属丰度显著降低，而不动杆菌属和假单胞菌属丰度显著增加（LDA>3，P<0.05）。阴道乳杆菌属丰度诊断RIF的最佳截断值为70%，曲线下面积（area under curve，AUC）为0.77。结论：RIF患者阴道菌群显示差异，以乳杆菌属丰度降低为主要特征。阴道乳杆菌属低丰度可能与RIF相关。

关键词: 反复种植失败, 阴道, 阴道菌群, 16S rRNA测序, 高通量测序

Abstract:

Objective: To explore the characteristics of vaginal microbiota in patients with repeated implantation failure (RIF). Methods: A total of 49 patients undergoing the frozen-thawed embryo transfer (FET) cycles were recruited. In the observation group, patients (n=29) with a history of RIF have failed pregnancy during the FET cycles. In the control group, patients (n=20) underwent the FET for the first time and achieved clinical pregnancy. In both groups, artificial hormone cycles were performed, and the top-quality blastocysts cultured on Day 5 were tranferred. Vaginal samples were collected on the FET day. Microbial diversity, microbial community composition and differential bacterial genera between the two groups were analyzed by 16S rRNA sequencing. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of the abundance of Lactobacillus for RIF. Results: The α diversity analysis showed that the microbial diversity (Shannon: P=0.049 8) and richness (Chao: P=0.047 1) of the observation group were significantly higher than those of the control group. The β diversity analysis showed that there was significant difference in the vaginal microbial community composition between the two groups (P=0.016). At the genus level, Lactobacillus was dominant in both groups. The abundance of Lactobacillus of the observation group was significantly lower than that of the control group, while the abundances of Acinetobacter and Pseudomonas of the observation group were significantly higher than those of the control group (LDA>3, P<0.05). The optimal cutoff value for diagnosing RIF with the abundance of Lactobacillus was 70%, and the area under curve (AUC) was 0.77. Conclusions: Vaginal microbiota was showed differences in RIF patients, including the decreased abundances of Lactobacillus. The low abundance of vaginal Lactobacillus may be related with the pathogenesis of RIF.

Key words: Repeated implantation failure, Vagina, Vaginal flora, 16S rRNA sequence, High throughput sequencing

张荣雪, 王苗苗, 贾媛媛, 薛惠英. 反复种植失败患者阴道菌群特征研究[J]. 国际生殖健康/计划生育杂志, 2024, 43(2): 89-94.

ZHANG Rong-xue, WANG Miao-miao, JIA Yuan-yuan, XUE Hui-ying. Study on the Vaginal Microbiota in Patients with Repeated Implantation Failure[J]. Journal of International Reproductive Health/Family Planning, 2024, 43(2): 89-94.

图/表 6

表1 2组一般资料比较

组别	n	年龄（岁）	BMI （kg/m²）	不孕年限（年）	不孕类型		内膜准备方案		移植胚胎数（枚）	移植日内膜厚度（mm）
组别	n	年龄（岁）	BMI （kg/m²）	不孕年限（年）	原发性	继发性	HRT	促排卵方案	移植胚胎数（枚）	移植日内膜厚度（mm）
观察组	29	31.24±4.00	24.43±4.32	2.48±1.33	18（62.1）	11（37.9）	22（75.9）	7（24.1）	1.48±0.50	9.77±1.95
对照组	20	31.55±2.80	23.51±3.24	2.80±1.58	14（70.0）	6（30.0）	14（70.0）	6（30.0）	1.15±0.36	9.27±1.52
t或χ²		0.29	0.79	0.76	0.33		0.12		2.51	0.95
P		0.77	0.43	0.45	0.57		0.73		0.02	0.35

图1 测序数据分析注：A OTU数目；B 稀释曲线。

图2 阴道菌群多样性分析注：A 2组Shannon和Chao指数比较；B 2组阴道菌群PCoA图。

图3 2组阴道菌群丰度分布堆叠图注：A 科水平； B 属水平。仅展示在2组样本中丰度>1%的菌科/属。

图4 2组菌群LEfSe差异性分析（LDA>3）注：红色表示在对照组富集，绿色表示在观察组富集；f 科（Family），g 属（Genus）；norank提示菌科/属缺乏分类学信息。

图5 乳杆菌属丰度预测RIF的ROC曲线注：黑色圆点表示最佳截断值。

参考文献 17

[1]	Integrative HMP(iHMP) Research Network Consortium. The Integrative Human Microbiome Project[J]. Nature, 2019, 569(7758):641-648. doi: 10.1038/s41586-019-1238-8.
[2]	Gao XS, Laven J, Louwers Y, et al. Microbiome as a predictor of implantation[J]. Curr Opin Obstet Gynecol, 2022, 34(3):122-132. doi: 10.1097/GCO.0000000000000782.
[3]	Koedooder R, Singer M, Schoenmakers S, et al. The vaginal microbiome as a predictor for outcome of in vitro fertilization with or without intracytoplasmic sperm injection: a prospective study[J]. Hum Reprod, 2019, 34(6):1042-1054. doi: 10.1093/humrep/dez065.
[4]	中国医师协会生殖医学专业委员会, 中国女医师协会生殖医学专业委员会. 反复种植失败临床诊治中国专家共识[J]. 中华医学杂志, 2023, 103(2):89-100. doi: 10.3760/cma.j.cn112137-20221105-02317.
[5]	Gardner DK, Surrey E, Minjarez D, et al. Single blastocyst transfer: a prospective randomized trial[J]. Fertil Steril, 2004, 81(3):551-555. doi: 10.1016/j.fertnstert.2003.07.023. pmid: 15037401
[6]	Liptáková A, Čurová K, Záhumensky J, et al. Microbiota of female genital tract - functional overview of microbial flora from vagina to uterine tubes and placenta[J]. Physiol Res, 2022, 71(Suppl 1):S21-S33. doi: 10.33549/physiolres.934960.
[7]	Sirota I, Zarek SM, Segars JH. Potential influence of the microbiome on infertility and assisted reproductive technology[J]. Semin Reprod Med, 2014, 32(1):35-42. doi: 10.1055/s-0033-1361821. pmid: 24390919
[8]	Punzón-Jiménez P, Labarta E. The impact of the female genital tract microbiome in women health and reproduction: a review[J]. J Assist Reprod Genet, 2021, 38(10):2519-2541. doi: 10.1007/s10815-021-02247-5.
[9]	Haahr T, Jensen JS, Thomsen L, et al. Abnormal vaginal microbiota may be associated with poor reproductive outcomes: a prospective study in IVF patients[J]. Hum Reprod, 2016, 31(4):795-803. doi: 10.1093/humrep/dew026.
[10]	Hyman RW, Herndon CN, Jiang H, et al. The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization-embryo transfer[J]. J Assist Reprod Genet, 2012, 29(2):105-115. doi: 10.1007/s10815-011-9694-6.
[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]	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.
[13]	Ichiyama T, Kuroda K, Nagai Y, et al. Analysis of vaginal and endometrial microbiota communities in infertile women with a history of repeated implantation failure[J]. Reprod Med Biol, 2021, 20(3):334-344. doi: 10.1002/rmb2.12389. pmid: 34262402
[14]	Fu M, Zhang X, Liang Y, et al. Alterations in Vaginal Microbiota and Associated Metabolome in Women with Recurrent Implantation Failure[J]. mBio, 2020, 11(3):e03242-19. doi: 10.1128/mBio.03242-19.
[15]	郑晶晶, 宋静慧, 托娅, 等. 蒙古族细菌性阴道病病人阴道乳杆菌及SIgA含量的相关性研究[J]. 内蒙古医科大学学报, 2016, 38(4):293-297. doi: 10.16343/j.cnki.issn.2095-512x.2016.04.004.
[16]	陈仝, 杨晓葵. 反复种植失败的体外受精-胚胎移植免疫学因素研究进展[J]. 中国计划生育和妇产科, 2020, 12(1):16-19. doi: 10.3969/j.issn.1674-4020.2020.01.04.
[17]	Al-Nasiry S, Ambrosino E, Schlaepfer M, et al. The Interplay Between Reproductive Tract Microbiota and Immunological System in Human Reproduction[J]. Front Immunol, 2020,11:378. doi: 10.3389/fimmu.2020.00378.