The alteration and potential relationship of vaginal microbiota and chemokines for unexplained recurrent spontaneous abortion

Abstract

The diagnosis and treatment of unexplained recurrent spontaneous abortion (URSA) is an important and hot topic in the field of obstetrics and gynecology. During our clinical investigation (observation), we have found that URSA patients usually experience recurrent vaginitis or vaginal dysbacteriosis during periods of non-pregnancy, pregnancy, and post-abortion. However, there is no research on vaginal dysbacteriosis's influence on URSA. Using women with normal induced abortion as a control group, and using 16S rRNA sequencing, which helps to screen differentially expressed flora, this study discusses the relevance between differential bacteria at the genus level and the incidence of URSA. Another aim of this study is to determine whether certain pathogenic genera can cause an imbalance in immune tolerance of the maternal and fetal interface through regulatory chemokines, which leads to recurrent spontaneous abortion. This article has explored URSA pathogenesis from the perspective of differentially expressed vaginal flora, which has great theoretical significance for the early diagnosis and treatment of URSA.

Figure 1

RSA patients from Guangzhou Women and Children's Medical Center in recent 5 years.

Figure 2

Comparison of ACE diversity index of vaginal flora through Alpha diversity analysis. (A) Regarding the comparison on the ACE abundance estimation index of vaginal flora between the natural pregnancy induced abortion (NPSA) and recurrent spontaneous abortion (URSA), the vaginal flora diversity of recurrent spontaneous abortion is higher than that of natural pregnancy abortion, ^∗P < .05; (B) Regarding the comparison on the ACE abundance estimation index of vaginal flora among natural pregnancy induced abortion (NPSA), recurrent spontaneous abortion (URSA) and patients who have successful fetus protection, the vaginal flora diversity of recurrent spontaneous abortion patients who have successful fetus protection has decreased, ^∗P < .05.

Figure 3

The relative flora abundance of NPSA and URSA groups on phylum level. A. Actinobacteria of NPSA group has higher expressive abundance compared with URSA group. B. Verrucomicrobia of NPSA group has higher expressive abundance compared with URSA group. Proteobacteria of URSA group has higher expressive abundance compared with NPSA group.

Figure 4

The relative flora abundance of NPSA and URSA groups on genus level. A, B, C, D. Expressive abundance of Pseudomonas, Roseburia, Collinsella aerofaciens, and Arthrobacter is higher in URSA group than NPSA group.

Figure 5

LEfSe analysis of vaginal flora differences in NPSA and URSA groups. A. logarithmic scores of LDA difference analysis are used to describe the expressive abundance of flora with significant differences between groups. High expressive flora in NPSA group include: Actinomycetes, Bifidobacteria, Gardnerella, Veillonococcus, Megacoccus, Peptostreptococcaceae, Comamonadaceae, Anaerolineae, Rhodospirillaceae, Verrucomicrobiaceae, LDA score>2; High expressive flora in URSA group include: Gammaproteobacteria, Proteobacteria , Pseudomonas, Moraxella, Rumenococcus, Collinsella aerofaciens, Alteromonadaceae, Cellvibrio, Arthrobacter, Roseburia, Micrococcaceae, LDA score>2. B. The classification level tree displayed by cladogram describes the hierarchical relationship of all the flora from phylum to genus (from the inner circle to the outer circle) in the sample community, and the average relative abundance of the corresponding flora. On phylum, the differential flora mainly includes Actinobacteria, Proteobacteria, and Verrucomicrobia. On genus, the differential flora mainly includes Pseudomonas, Gardnerella, Bifidobacterium, Megacoccus, Akkermansia, Roseburia, Collinsella aerofaciens, Arthrobacter, Ruminococcus, and Cellvibrio, among which the Pseudomonas, Roseburia, Collinsella aerofaciens, Arthrobacter, Ruminococcus, and Cellvibrio have high expressive abundance in URSA group and the Gardnerella, Bifidobacterium, Megacoccus, and Akkermansia have high expressive abundance in NPSA group.

Figure 6

(A) By using Unweighted UniFrac PCoA analysis, each dot represents a sample with the red ones representing NPSA patients and blue ones representing URSA patients; most NPSA samples can be separated from URSA samples, indicating a certain difference of sample composition of the 2 groups. (B) Box plot of Unweighted UniFrac distance of multiple groups. (A) stands for NPSA group and (B) for URSA group. The difference in (A) vs (B) is significantly higher than the difference within (B) group, indicating the statistical difference between NPSA and URSA samples.

Figure 7

The scanning result of chemokine chip. (A) The chemiluminescence detection results of chemokine in NPSA group; (B) The chemiluminescence detection results of chemokine in URSA group. The size and brightness of the dot reflected the expression level of chemokine. The larger and the brighter of the dot, the higher the expression of chemokine, and the lower on contrast. From left to right, the red circle marks the expression quantity of CCL8/CCL5/CCL3/CCL4/CCL2 and the expression quantity of chemokine in URSA group in higher than NPSA. (C) The comparison of the chemokine expression quantity between NPSA group and URSA group. The expression quantity of CCL2/CCL8/CCL3/CCL4/CCL5 in URSA is significant higher than that of NPSA (∗P < .05).