Basic situation of research subjects
A total of 75 patients were enrolled in the study, and the detailed information was shown in Table 1. Generally, there are 35 patients in the experimental group, with an average age of 52.7 ± 8.9, including 27 serous cell carcinoma (SC), two mucinous carcinoma (MC), three clear cell carcinoma (CCC), and three endometrioid carcinoma(EC) patients. In this group, cotton swabs samples from four parts of the same patient, including cervical, uterine cavity, fallopian tube, and ovarian, were harvested in 24 patients. For 9 cases, the fallopian tube samples were unavailable due to tumor invasion. For 1 case, the cervix was not taken. And for the other one case, the cervical uterine cavity result was unavailable. There were thirty noncancerous cases (benign cases) including cases of hysterectomy and adnexectomy due to inflammation or benign lesions, with an average age of 55.1 ± 11.2 in the control group, twenty of which did not undergo ovariectomy. Among the malignant cases, eleven patients were not ligated, and thirteen were ligated. Platinum resistant was defined as relapse 6 months after 1st line platinum-based chemotherapy[19]. Eight cases were platinum-resistant, and nineteen cases were platinum-sensitive.
General microbial distribution in the paired samples of both benign and malignant patients
After the sample data has undergone strict quality control and de-host analysis, Kraken2 + PlusPF is used for species annotation. Then the seqkit stat is used to perform a basic analysis of the quality-controlled sample data. The average read sequence length of samples from patients in each state is in the range of 61bp to 147.3 bp, and the median is in the range of 103bp to 144bp. The base quality of the read sequence of each state sample is acceptable, and the median Q30 ratio is above 96% (SuppFig1 A). The reads of samples of different parts can be effectively annotated (SuppFig1 B and C).
Further analysis of the specific composition of microorganisms, bacteria accounted for the highest proportion in each sample, with a median of 99%. The ratio of fungi, viruses, eukaryotes, and archaea was relatively small, less than 1% in most samples. At the phylum level, the top 5 relative abundance are Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Tenericutes (SuppFig1 E). The Proteobacteria phylum has the highest abundance in all samples. The proportion of Firmicutes in cervical samples is higher than that of the other three parts. The average abundance of Actinobacteria bacteria from all malignant patients is higher than that in benign patients, especially in the ovarian. At the species level, the OUTs that appear in at least 10% of the samples are conserved. The top 5 are Pseudomonas tolaasii, Klebsiella pneumoniae, Salmonella sp, Acinetobacter johnsonii, and Escherichia coli (Fig. 1A). On a species level, species-level alpha-diversity suggests no difference in microbial diversity in the cervix, uterine cavity, and fallopian tube except for ovarian tissue (Fig. 1A, SuppFig 1 D).
To analyze the migration of microorganisms from the lower reproductive tract to the upper reproductive tract, we conducted a similarity analysis of the adjacent positions of samples from the four parts. At the phylum level, the relative abundance of Firmicutes bacteria in both the benign and malignant patients generally decreased from CCT to EMT (SuppFig2 A and B). The relative abundance of Ascomycota bacteria in malignant patients from EMT to FTT was both increased, and the difference was statistically significant (p < 0.01) (SuppFig2 C and D). The relative abundance of Uroviricota gate in benign patients from FTT to OCT generally raised, but the difference was not statistically significant. (SuppFig2 E and F) At the level of CCT-EMT, there are differences in the similarity between the two groups, and the differences are apparent (Fig. 1B). There was no significant difference in the parallel of other parts between the two groups, the relative abundance of Brevundimonas sp. DS20 bacteria in group B patients generally decreased from CCT to EMT. The relative abundance of Brevundimonas mediterranea, Brevundimonas sp. scallop, Brevundimonas sp. Bb-A, Brevundimonas sp. DS20, Brevundimonas sp. GW460-12-10-14-LB2, Brevundimonas sp. SGAir0440 and Cutibacterium_acness is reduced from CCT to EMT in the malignant group (SuppFig3 A and B). Cutibacterium acnes and Komagataella phaffii generally increased in relative abundance from EMT to FTT in patients in malignant group (SuppFig3 C and D). The relative abundance of Cutibacterium acnes in benign patients from FTT to OCT has an upward trend (p > 0.05). The relative abundance of Brucella intermedia in the experiment group from FTT to OCT decreased, which has a statistical difference.
For the sample diversity, the beta diversity analysis indicated that there are significant differences in the microbial composition of patients in different groups (P < = 0.001) (Fig. 1C). To better show the species distribution of different groups in other parts, we performed a Venn diagram analysis. The result suggested that the out shared by four parts accounted for the majority, indicating that the lower reproductive tract is the primary source of microorganisms for the upper reproductive tract.
The microbial differences among the four parts of a same patient
To exclude the effects of different microbial populations between subjects, we further analyzed of the types of microorganisms in four parts of a single patient. Briefly, we analyzed the relative abundance of microbes at the species level of four parts in 10 patients of benign subjects and 24 patients of malignant subjects. The samples of these 34 cases were divided into two clusters according to the salmonella sp. abundance (> 1%), depending on the environmental control and sample classification. We then analyzed the distribution of Salmonella sp. and Pseudomonas Tolaasii in benign and malignant patients with the relative abundance of salmonella sp. more than 1% at any part (CCT, EMT, FTT, or OCT). In the benign group, the relative abundance of Pseudomonas tolaasii gradually decreased as migrated upward from the lower reproductive tract to the ovarian tissue. There was a similar trend in the malignant samples. Meanwhile, Salmonella sp. gradually enriched among the 4 sites within the malignant group. The relative abundance in the uterine cavity was significantly higher than that of the lower reproductive tract (cervix) (Fig. 2B). Interestingly, while no difference of Pseudomonas tolaasii abundance was detected in all samples of benign and malignant cases, Salmonella sp enriched in both benign and malignant uterine cavities. Moreover, in malignant patients, Salmonella sp. is further increased in the ovary, while in benign cases, no such enrichment in ovary samples (Fig. 2C). The phenomenon also supported that the microorganisms in ovarian tissue originate from the reproductive tract. These findings illustrate that the migration of the upper reproductive tract microorganisms of the malignant patients was different from that of benign controls with noticeable enriched microorganisms.
Malignant ovarian tissue had distinct microbial signatures
To clarify the distribution of microorganisms on the malignant ovarian tissues, the ovarian samples of 34 experimental subjects were compared with the samples of 10 cases of benign ovarian tissues. The two groups were different in the distribution of β-diversity (P = 0.001, and P = 0.005) at the genus level and the species level. There are 146 genus-level OTUs in all the ovarian samples. The differential bacteria enriched on the surface of malignant ovarian tissue are Salmonella, Asticcacaulis, Arthrobacter, Lactobacillus, Pseudarthrobacter, and Pseudarthrobacter. The top 5 different bacteria enriched on the surface of benign ovarian tissue are Brevundimonas, Ralstonia, Pandoraea, Streptococcus, and Corynebacterium. Total of 329 OTUs at the species level. Salmonella sp., Asticcacaulis excentricus, Acinetobacter sp. NEB 394, Acinetobacter lwoffii, and Arthrobacter sp. FB24 was enriched in malignant tissues. While Brevundimonas sp. Bb-A, Brevundimonas sp. DS20, Ralstonia pickettii, Pandoraea pnomenusa, Staphylococcus hominis, Komagataella phaffii, Finegoldia magna, Cutibacterium acnes, Prevotella intermedia, and Agrobacterium tumefaciens were enriched in benign ovarian tissues. Notably, Salmonella sp. was the most enriched species in malignant tissues, and its relative abundance was also the highest in the bacteria.
The influence of tubal ligation on the distribution of microbiota on the surface of ovarian cancer tissue
It has been proved that tubal ligation and hysterectomy lower the risk of ovarian cancer. However, the influence of the ligation on the microorganism distribution on the ovary remains unclear. In the current study, a total of 312 OTUs were found between 13 fallopian tube ligated patients and 11 non-ligation patients. Though there was no statistical difference (p = 0.146), the β-diversity showed that the two groups share unidentical compositions of microorganisms (Fig. 4A). This may be due to the insufficient sample numbers. Among the different species, the top 5 most enriched bacteria were: Arthrobacter sp. J3.40, Arthrobacter sp. UKPF54-2, Arthrobacter sp. KBS0702, Arthrobacter sp. FB24, and Acinetobacter sp. NEB 394 on the surface of the ovarian tissue in the ligated subjects. As for the ovarian tissue of the unligated subjects, the most enriched species were Brevundimonas sp. DS20, Ralstonia mannitolilytica, Brevundimonas mediterranea, Gardnerella vaginalis, Achromobacter xylosoxidans(Fig. 4B and C).
Platinum resistance and the microbiota distribution of the ovarian cancer tissues
The cancer is recognized as platinum-resistant if the cisplatin and carboplatin act no function half a year after surgery. To determine the potential relationship between postoperative platinum treatment resistance and ovarian cancer surface microbial population diversification, we compared the ovarian tissue surface microbial species between 19 postoperative platinum-sensitive and eight platinum-resistant patients. On the species level, the two groups shared 226 species (Fig. 5A). The β-diversity analysis indicated no difference in species between the two groups (p = 0.44). The top 5 enrichment in the platinum-resistant group is Pseudomonas_aeruginosa, Ralstonia mannitolilytica, Achromobacter xylosoxidans, Brevundimonas sp. DS20, and Brevundimonas sp. Bb-A (Fig. 5B and C).