Revealing the Disturbed Vaginal Micobiota Caused by Cervical Cancer Using High-Throughput Sequencing Technology

Abstract

Cervical cancer is the fourth most prevalent cancer type among all malignancies, so it is of great significance to find its actual pathogenesis mechanisms. In the present study, 90 women were enrolled, and high-throughput sequencing technology was firstly used to analyze the vaginal microbiota of healthy women (C group), cervical intraepithelial neoplasia patients (CIN group) and cervical cancer patients (CER group). Our results indicates that compared with C group, a higher HPV infection rate as well as increased Neutrophil ratio and tumor marker squamous cell carcinoma antigen (SCCA) were obtained, and a decrease in Lymphocyte ratio and Hemoglobin were also present. In addition, the cervical cancer showed a strong association with reduced probiotics Lactobacillus, increased pathogens Prevotella spp., Sneathia spp. and Pseudomonas spp. These results prove that the immunological changes generated by the cervical cancer and the vaginal microbiota can interact with each other. However, further study investigating the key bacteria for cervical cancer is still needed, which can be a clue for the diagnosis or treatment of cervical cancer.

Keywords: Lactobacillus; cervical cancer; cervical squamous intraepithelial neoplasia; vaginal microbial disorders; vaginal microbiota.

Figure 1

This study initially included 90 volunteers, of which 18 were excluded due to lack of clinical test data (3 in group C, 8 in CIN group, and 7 in CER group). Thereafter, the vaginal posterior fornix secretions of these 72 volunteers were collected for bacterial and microbial DNA extraction. During this process, 10 volunteers failed to extract DNA and were excluded (2 in group C, 3 in CIN group and 5 in the CER group).

Figure 2

The Pap smear of the cervical cell from five different health status people. The normal one (A), low squamous intraepithelial lesion (B), high squamous intraepithelial lesion (C), atypical squamous cell (D) and squamous cell carcinoma (E) are from the participants.

Figure 3

Differences in clinical blood test indicators. In the comparison of neutrophil ratio (C) (P = 0.0249) and squamous cell carcinoma antigen (SCCA) (D) (P = 0.0001), CER group is higher than CIN group, and CIN group is much higher than C group. To the contrary, the hemoglobin (A) (P = 0.0024) and lymphocyte ratio (B) (P < 0.0001) in C group is much higher than CIN group and CER group. P value less than 0.05 is statistically significant. * means p < 0.05; * means P < 0.01; ** means P < 0.001; *** means P < 0.001; p < 0.05 indicates significant difference. C, Control group, a group of women who have no problem in gynecology; CER, Cervical cancer group, a group of women who were diagnosed a cervical cancer by pathological biopsy; CIN, Cervical intraepithelial neoplasis group, a group of women who were diagnosed by pathological biopsy or cytological test; ns, not significant.

Figure 4

Differences in the alpha diversity and beta diversity of the vaginal flora among these three groups. The Shannon index (A) (P = 0.2609), Simpson index (B) (P = 0.2245), and Scalar-Venn (D) reveal the alpha diversity of the vaginal flora. The difference in alpha diversity has no statistical significance. The Venn result shows that there are 6573 OTUs in the C group, 6516 OUTs in the CIN group and 6517 OUTs in CER group. The PCoA analysis (C) reveals the PCoA beta diversity index. C: Control group, a group of women who have no problem in gynecology. CER: Cervical cancer group, a group of women who were diagnosed a cervical cancer by pathological biopsy. CIN: Cervical intraepithelial neoplasis group, a group of women were diagnosed a by pathological biopsy or cytological test.

Figure 5

The difference in the construction of the vaginal flora at the level of phylum. The most abundant 20 phyla in each group (A), the differences among the main member (B), and the most common pathogens in the vaginal flora (C–E). C: Control group, a group of women who have no problem in gynecology. CER: Cervical cancer group, a group of women who were diagnosed a cervical cancer by pathological biopsy. CIN: Cervical intraepithelial neoplasis group, a group of women were diagnosed a by pathological biopsy or cytological test. * means p <0.05; ** means P <0.01; *** means P <0.001; p < 0.05 indicates significant difference. ns, no significance.

Figure 6

Comparison of the influence on the host cell metabolism and DNA status. Differences in cell growth and death (A) (P = 0.0075), and cell motility (B) (P = 0.1437), are shown above. As the same, differences in the DNA folding, sorting and degradation (C) (P = 0.0184), and DNA replication and repair (D) (P = 0.5705). P value less than 0.05 is statistically significant.

Figure 7

Correlation analysis among Lactobacillus, Pseudomonas, Prevotella and SCCA and Neutrophil ratio using stratified analysis. (A, F, K) for the overall level, (B, G, L) for the CINII group, (C, H, M) for the CINIII group, (D, I, N) for the cervical cancer staged earlier than Ib1 and (E, J, O) for the cervical cancer staged later or equal to Ib1. C: Control group, a group of women who have no problem in gynecology. CER: Cervical cancer group, a group of women who were diagnosed a cervical cancer by pathological biopsy. CIN: Cervical intraepithelial neoplasis group, a group of women were diagnosed a by pathological biopsy or cytological test. * means p <0.05; *** means P <0.001; p <0.05 indicates significant difference. ns, no significance.