Materials
Ovestinon® 0.1% (Estriol) (hereinafter referred to as estriol cream) is a common and commercially available pharmaceutical product for treating genital tract atrophy. It contains 15 mg of estriol per 15 g of ointment. The vaginal flora in vaginal colony transplantation was derived from control C57 mice. Control and colony transplantation mice were randomly matched and fixed for transplantation.
Establishment of menopausal model
Female SPF-grade C57BL/6J mice (eight-week-old) were purchased from Hangzhou Ziyuan Laboratory Animal Science and Technology Co. Ltd. and housed in the Animal Experiment Center of Anhui Medical University. Mice were housed under specific pathogen-free (SPF) conditions and maintained under standard laboratory conditions with 12h:12h light-dark cycles, and fed regular chow. Food and water were freely available. After one week of acclimatization, 30 mice were randomly divided into five groups as follows: (a) control group (CON group, n=6); (b) bilateral ovariectomy with estriol-treated group (OVX+EST group, n=6); (c) bilateral ovariectomy with VMT group (OVX+VMT group, n=6); (d) bilateral ovariectomy with normal saline lavage group (OVX+NST group, n=6); and (e) bilateral ovariectomy group (OVX group, n=6). Except for the control group, all groups underwent bilateral ovariectomy to simulate patients undergoing oophorectomy. After injecting sodium pentobarbital to anesthetize the mice, they were placed in the supine position. A 2 cm incision was made in the midline of the abdomen to explore the ovaries and surrounding fat pads. The ovaries were ligated, while in the control group, only bilateral fat pads were removed without ligating the ovaries. Finally, the muscles and skin were sutured. The study's overall design and protocol were approved by the Laboratory Animal Ethics Committee of Anhui Medical University (LLSC20241413). The animal experiments conducted in this study were performed in accordance with the ARRIVE guidelines, and all methods were performed in accordance with the relevant guidelines and regulations.
Animal treatment
Four weeks after removing bilateral ovaries, the vaginas of the four groups were closed. For each group, the following treatment was administered:
(a) Only as the normal microbiota donor in OVX+VMT group;
(b) We vaginally administered estriol cream 0.33 mg (20 g mouse body weight/60 kg human body weight) according to human dosage recommendations;
(c) 100 μl of fresh flora lavage solution was given;
(d) 100 μl of normal saline was given;
(e) No treatment was administered;
The treatments described above were administered once daily for eight weeks.
Sample collection
Collection of vaginal lavage solution: To collect vaginal lavage solution, first we ensured that the researchers are wearing protective gear. With one hand, they gently held the mouse and used a sterile cotton swab to stimulate the outlet of the reproductive tract. We encouraged the mouse to urinate, and after urination, we used the cotton swab to absorb the urine. Next, we disinfected the mouse's vulva with iodophor, avoiding its contact with the vagina. A 0.5 ml disposable sterile dropper was used to suck up 200 μl of sterile saline, which was then inserted into the mouse's vagina for about 5 mm. The saline was blown repeatedly for 50 times to ensure adequate washing of the vagina. An equal amount of the saline lavage was then injected into the freezing tube and stored [26].
Mice were injected intraperitoneally with sodium pentobarbital (30-90 mg/kg) and blood was collected from the heart for plasma collection. After obtaining vaginal tissue, mice were sacrificed by cervical dislocation and observed for loss of respiration and heartbeat to confirm death. The weight and length of the isolated vagina were measured using an electronic balance and ruler. The upper third of the vagina was fixed in formalin, dehydrated, and paraffin-embedded for histological analysis.
Histological and immunohistochemical analysis
The vaginal wall was treated with formalin, dehydrated, and embedded. A cross-section of the specimen (5 μm) was cut and HE staining was conducted. Using Image J software, the thickness of the vaginal mucosa was measured in the vaginal cross-section. Immunohistochemical techniques were utilized to measure the expression of ER and proliferating cell nuclear antigen (PCNA) in the vaginal epithelium and endometrium of mice. The mean value of ER-positive and PCNA-positive vaginal mucosal cells and the total number of epithelial cells in each field were calculated.
The concentration of estrogen and inflammatory factors in serum and vaginal lavage fluid were measured by ELISA
ELISA kits (Elabscience, Wuhan, China) were used to measure the levels of estriol and estradiol in mouse serum and the levels of inflammatory factors IL-1β and TNF-α in mouse serum and vaginal lavage fluid. Each sample was diluted five-fold and the instructions of the product manual were followed for measurement. We added the diluted samples to wells. Thereafter, an appropriate amount of enzyme labeling reagent was added. The mixture was then incubated for a specified duration. Next, the well plate was washed and the substrate solution was added, followed by another incubation period. Finally, the termination solution was added to complete the process. The absorbance was measured at 450 nm using an enzyme-labeling apparatus. The concentration of each sample was calculated based on the standard curve provided by the kit.
Analysis of 16S RNA in vaginal lavage flui
The OMEGA Soil DNA Kit (D5625-01) (Omega BioTek, Norcross, GA, USA) was used to extract genomic DNA from samples and assess the purity and concentration of DNA. According to the selection of sequencing region, the selected V3-V4 variable region was amplified by PCR using specific primers with Barcode and high-fidelity DNA polymerase. PCR products were detected by 2% agarose gel electrophoresis, and the target fragments were cut and recovered by Quant-iT PicoGreen dsDNA Assay Kit. Based on the quantitative results of electrophoresis, the PCR amplification recovered products were detected and quantified using a microplate reader (BioTek, FLx800) fluorescence quantitative system. The corresponding proportions were mixed according to the sequencing requirements of each sample. The library was constructed using the TruSeq Nano DNA LT Library Prep Kit from Illumina. The constructed library was inspected by Agilent Bioanalyzer 2100 and Promega QuantiFluor. After qualifying the library, it was sequenced.
Data analysis: Raw sequencing data were obtained in FASTQ format. Paired-end reads were then preprocessed using Cutadapt software to detect and cut off the adapter. After trimming, paired-end reads were filtered for low-quality sequences, denoised, merged, and detected. The chimera reads were cut off using DADA2 with the default parameters of QIIME2. The software provided the representative reads and the ASV abundance table.
Annotation: The representative reads of each ASV were selected using the QIIME 2 package. All representative reads were annotated and blasted against Silva database Version 138 (16s/18s rDNA) using classify-sklearn with the default parameters.
Diversity analysis: We calculated the α and β diversity indices using Qiime2 software. α diversity is used to analyze the diversity of microbial communities in each sample. The richness and diversity of microbial communities in the sample can be reflected through the diversity analysis of each sample (α diversity). Diversity analysis includes the use of species accumulation box chart, species diversity curve, and a series of statistical analysis indexes to measure the differences in species richness and diversity of microbial communities in each sample. β diversity is a comparative analysis of microbial community composition in different samples.
Difference analysis: LEfSe (Linear discriminant analysis effect size, linear discriminant analysis, and influence factor) can be used to identify species characteristics that can best explain the differences between groups in two or more groups of samples, and determine the influence degree of these characteristics on the differences between groups. Using the inter-group difference test method, species differences between two groups/multiple groups of samples were measured, and the information of species with significant differences between the two groups was obtained.
The STAMP difference analysis was used to compare the species abundance between two groups (Wilcox test) or multiple groups (Kruskal Wallis test). Species with significant differences were obtained.
Functional analysis: PICRUSt2 (Phylogenetic Investigation of Communities by Recommendation of Unobserved States) is a software that predicts functional abundance only based on the sequence of the marker gene (16S/18S). It provides the gene function spectrum of their common ancestors based on the tree of OTU from the Greengene database and the gene information from OTU. It provides the gene function spectrum of other undetected species in the Greengenes database, builds the gene function prediction spectrum of the whole pedigree of archaea and bacteria domain, and finally "maps" the composition of the flora obtained by sequencing into the database to predict the metabolic function of flora.
High-resolution off-target metabolomics of the vaginal microbiota
LC-MS/MS analysis
The analysis was performed using a UHPLC (1290 Infinity LC, Agilent Technologies) coupled to a quadrupole time-of-flight (AB Sciex TripleTOF 6600, Shanghai Applied Protein Technology Co., Ltd.).
For HILIC separation, samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH Amide 1.7 µm column (waters, Ireland). In both ESI-positive and ESI-negative modes, the mobile phase contained A=25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B=acetonitrile. The gradient was 95% B for 0.5 min and linearly reduced to 65% in 6.5 min. Then, the gradient was reduced to 40% in 1 min and kept for 1 min. Then, the gradient increased to 95% in 0.1 min, with a 3 min re-equilibration period.
The ESI source conditions were as follows: Ion Source Gas1 (Gas1) as 60, Ion Source Gas2 (Gas2) as 60, curtain gas (CUR) as 30, source temperature: 600℃, IonSpray Volt-age Floating (ISVF): ± 5500 V. In MS-only acquisition, the instrument was set to ac-quire over the m/z range 60-1000 Da, and the accumulation time for TOF MS scan was set at 0.20 s/spectra. In auto MS/MS acquisition, the instrument was set to acquire over the m/z range 25-1000 Da, and the accumulation time for product ion scan was set at 0.05 s/spectra. The product ion scan was acquired using information-dependent acquisition (IDA) with high sensitivity mode. The parameters were as follows: the collision energy (CE) was fixed at 35 V with ± 15 eV; declustering potential (DP) was 60 V (+) and −60 V (−); isotopes within 4 Da were excluded, and 10 candidate ions were used to monitor per cycle.
The analysis was performed using a UHPLC (Vanquish UHPLC, Thermo) coupled to an Orbitrap Exploris™ 480 (Shanghai Applied Protein Technology Co., Ltd.).
For HILIC separation, samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH Amide 1.7 µm column (Waters, Ireland). In both ESI-positive and ESI-negative modes, the mobile phase contained A=25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B= acetonitrile. The gradient was 95% B for 0.5 min and was linearly reduced to 65% in 6.5 min. Then, the gradient was reduced to 40% in 1 min and kept for 1 min. Thereafter, it increased to 95% in 0.1 min and was kept at 95% for 2.9 min.
The ESI source conditions were set as follows: Ion Source Gas1 (Gas1) as 50, Ion Source Gas2 (Gas2) as 2, source temperature: 350℃, IonSpray Voltage Floating (ISVF): +3500 V/-2800V. In MS-only acquisition, the instrument was set to acquire over the m/z range 70-1200 Da, the resolution was set at 60000, and the accumulation time was set at 100 ms. In auto MS/MS acquisition, the instrument was set to acquire over the m/z range 70-1200 Da, the resolution was set at 60000, and the accumulation time was set at 100 ms, excluding time within 4 s.
Raw MS data were converted to MzXML files using ProteoWizard MSConvert before importing them into freely available XCMS software. The following parameters were used for peak picking: centWave m/z = 10 ppm, peak width = c (10, 60), and prefilter = c (10, 100). For peak grouping, bw = 5, mzwid = 0.025, and minfrac = 0.5 were used. CAMERA (Collection of Algorithms of MEtabolite pRofile Annotation) was used for annotation of isotopes and adducts. In the extracted ion features, only variables with more than 50% of the nonzero measurement values in at least one group were kept. The compounds of metabolites were identified by comparing the accuracy m/z value (<10 ppm), and MS/MS spectra with an in-house database were established with available authentic standards.
After sum-normalization, the processed data were analyzed using R package (ropls). Multivariate data analysis, including Pareto-scaled principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA), were conducted. The 7-fold cross-validation and response permutation test were used to evaluate the robustness of the model. The variable importance in the projection (VIP) value of each variable in the OPLS-DA model was calculated to indicate its contribution to the classification. Student’s t-test was applied to determine the significance of differences between two groups of independent samples. VIP > 1 and p value < 0.05 were used to screen significantly changed metabolites. Pearson’s correlation analysis was performed to measure the correlation between two variables.