女性生殖道菌群与细胞因子紊乱对反复着床失败的影响

Impact of female reproductive tract microbiota and cytokine disorders on recurrent implantation failure

目的探索反复着床失败(recurrent implantation failure,RIF)患者生殖道菌群的功能组成变化及其与免疫调控相关的细胞因子的关系。方法采用病例对照研究,纳入了北京大学第三医院妇产科生殖医学中心2020年8月至2021年1月期间因不孕而就诊的患者,入组患者分为对照组(单纯男性因素不孕,n=20)和RIF组(n=15)。研究通过16S rRNA测序和MSD电化学发光检测入组患者阴道和宫腔灌洗液的菌群和细胞因子表达情况。使用Spearman相关分析观察菌群和差异细胞因子之间的相关性。结果两组患者的年龄、体质量指数、月经周期或基础性激素水平差异均没有统计学意义(均P>0.05)。与对照组相比,RIF组阴道菌群和宫腔灌洗液菌群的Shannon指数差异均有统计学意义(P=0.032,P=0.015),宫腔灌洗液菌群的β多样性差异有统计学意义(P=0.020)。RIF组单核细胞趋化蛋白(monocyte chemotactic protein,MCP)-4的表达水平[(3.39±1.23)ng/L]明显高于对照组[(1.07±0.31)ng/L,P=0.044],而白细胞介素(interleukin,IL)-17A的表达水平[(1.12±0.29)ng/L]明显低于对照组[(2.70±0.52)ng/L,P=0.040]。宫腔灌洗液中RIF组的干扰素(interferon,IFN)-γ[(4.70±2.10)ng/L]、IL-10[(0.20±0.10)ng/L]、肿瘤坏死因子-α[(0.84±0.13)ng/L]以及IL-5[(0.35±0.05)ng/L]的表达水平明显高于对照组[(0.97±0.30)ng/L,P=0.049;(0.05±0.01)ng/L,P=0.009;(0.56±0.07)ng/L,P=0.045;(0.23±0.03)ng/L,P=0.029]。相关性分析显示,阴道菌群厌氧杆菌属(Anoxybacillus)与MCP-4的表达呈显著负相关(r=-0.42,P=0.025);宫腔灌洗液菌群中Muribaculaceae菌属与IL-5呈显著正相关(r=0.51,P=0.017),而Vulcaniibacterium菌属、贪铜菌属(Cupriavidus)以及厌氧杆菌属(Anoxybacillus)与IL-5呈显著负相关(r=-0.55,P=0.010;r=-0.62,P=0.003;r=-0.45,P=0.041);厌氧杆菌属(Anoxybacillus)、土芽孢杆菌属(Geobacillus)、嗜热菌属(Thermus)以及亚栖热菌属(Meiothermus)还与IFN-γ呈显著负相关(r=-0.43,P=0.015;r=-0.38,P=0.035;r=-0.39,P=0.029;r=-0.38,P=0.035)。结论RIF患者生殖道菌群和相应细胞因子发生紊乱,菌群与细胞因子之间存在相关性。

Objective To explore the changes in the functional composition of genital tract microbiota in patients with recurrent implantation failure(RIF)and its relationship with cytokines related to immune regulation.Methods This was a case-control study.The study included patients from the Reproductive Medicine Center of Peking University Third Hospital,enrolled from August 2020 to January 2021.The enrolled patients were divided into control group(n=20)and the RIF group(n=15).The study examined the expression of flora and cytokines in vaginal and uterine lavage fluid by 16S rRNA sequencing and MSD electrochemiluminescence.Spearman analysis was used to observe the correlation between flora and differently expressional cytokines.Results Compared with control group,there were statistically significant differences in the Shannon index of vaginal microbiota and uterine lavage fluid microbiota in the RIF group(P=0.032,P=0.015).There was a statistically significant difference inβdiversity of the bacterial community in uterine lavage fluid(P=0.020).The expression level of monocyte chemotactic protein-4(MCP-4)in the RIF group[(3.39±1.23)ng/L]was significantly higher than that in control group[(1.07±0.31)ng/L,P=0.044],while the expression level of interleukin(IL)-17A[(1.12±0.29)ng/L]was significantly lower than that in control group[(2.70±0.52)ng/L,P=0.040].The expression levels of interferon(IFN)-γ[(4.70±2.10)ng/L],IL-10[(0.20±0.10)ng/L],tumor necrosis factor-α[(0.84±0.13)ng/L],and IL-5[(0.35±0.05)ng/L]in the RIF group in the uterine lavage were significantly higher than those in control group[(0.97±0.30)ng/L,P=0.049;(0.05±0.01)ng/L,P=0.009;(0.56±0.07)ng/L,P=0.045;(0.23±0.03)ng/L,P=0.029].Correlation analysis revealed significant correlations between vaginal flora and MCP-4(r=-0.42,P=0.025),and in the microbial community of uterine cavity lavage fluid,there was a significant positive correlation between the Muribaculaceae genus and IL-5(r=0.51,P=0.017).Conversely,the Vulcaniibacterium genus,Cupriavidus genus,and Anoxybacillus genus exhibited a significantly negative correlation with IL-5(r=-0.55,P=0.010;r=-0.62,P=0.003;r=-0.45,P=0.041).Additionally,the genera Anoxybacillus,Geobacillus,Thermus,and Meiothermus showed a significantly negative correlation with IFN-γ(r=-0.43,P=0.015;r=-0.38,P=0.035;r=-0.39,P=0.029;r=-0.38,P=0.035).Conclusion The reproductive tract microbiota and corresponding cytokines in RIF patients are disrupted,and there is a correlation between the microbiota and cytokines.