The presence of living endometrial cells in ovarian endometriotic cyst fluid may contribute to the recurrence of endometriosis after surgical excision of endometriomas

Sample collection of endometriotic cyst fluid, drainage fluid and endometrial tissue

A total of 39 ovarian endometriosis patients and 14 non-endometriosis controls who underwent laparoscopy between September 2020 and January 2021 were enrolled in this study after informed consent was obtained. Among them, 39 patients had ovarian endometrioma with or without endometriotic lesions in other places of the pelvis. The other 14 patients without endometriosis underwent surgical procedures for myoma, ovarian teratoma and hydrosalpinx.

Thirty-nine endometriotic cyst fluid and 14 postoperative drainage fluid samples were collected from the endometriosis patients. Fourteen postoperative drainage fluid samples were collected from non-endometriosis control patients. Ectopic tissues from the cyst wall were collected from 11 endometriosis patients and eutopic endometrium was collected from 3 endometriosis patients.

For endometriotic cyst fluid collection, the cyst fluid from 39 patients with endometriosis was randomly collected during surgery through incision aspiration or puncture aspiration (22 vs. 17). For incision aspiration, a small breach of the cyst wall was made with scissors, and the cyst fluid was collected with an aspirator connected to a 50 mL sterile syringe. Puncture aspiration was performed using a Transfix needle connected to a 50 mL sterile syringe. Approximately 5-100 mL of the endometriotic cyst fluid was aspirated into the syringe. Cyst fluid was obtained from both sides of the cysts in the patients with bilateral ovarian endometriomas. The cyst fluid was transported to the laboratory within 30 minutes on ice.

The drainage fluid was obtained from the pelvic drainage bag. Twenty-four hours after surgery, the drainage fluid (5-20 mL) was collected using a 50 mL sterile syringe from the bag and immediately transported to the laboratory in a cold chain.

The endometriotic cyst wall and eutopic endometrium were collected during the surgery and immediately transported to the laboratory in a cold chain. After being washed with cold sterile saline three times, the ectopic endometrial tissues from the cyst wall and the eutopic endometrium from the corresponding patients were immersed in liquid nitrogen for RNA extraction. The other ectopic tissues on the cyst wall were washed and immersed in 4% neutral buffered formalin for the histological experiment.

All the endometriomas were removed during the surgery. In addition, none of the patients had received sex-hormone therapy within the 6 months before surgery. Detailed information on the patients enrolled in the study and the samples used in each experiment is shown in Additional file 1.

Classification of cyst fluid viscosity and determination of the apparent diffusion coefficient in endometriomas

The endometriotic cyst fluid was filtered through a 100 μm filter membrane, and the viscosity of the cyst fluid was classified into three types according to the difficulty of the cyst fluid passing through the filter membrane: type 1, the cyst fluid was not viscous and could directly pass through the 100 μm filter membrane, marked as “-1”; type 2, the cyst fluid had some viscosity and thereby needed to be repeatedly blown through the pipette to pass through the filter membrane, marked as “0”; type 3, the cyst fluid was very viscous and could not pass through the filter membrane even after repeated blowing, marked as “1”. It was necessary to dilute the type 3 cyst fluid more than twice until it could pass through the sieve.

Thirty-two endometriosis patients underwent MRI examination before surgery. Apparent diffusion coefficient (ADC) values were obtained from 18 patients. Fourteen patients underwent MRI in the outpatient department, and the MRI image was unavailable for analysing when they underwent the surgery. MRI scans were performed on a 1.5 Tesla MR system (Signa HDxt; GE Healthcare, Milwaukee, WI) with a phased-array abdominal coil. The patients were in a supine position and breathed freely during acquisition. The scanning range was from the inferior pubic symphysis to the renal hilum and extended beyond the dome of the cyst in patients with large masses. Diffusion-weighted imaging (DWI) was performed in axial planes at b values of 0, 800 s/mm2 (TR/TE 4600 ms/72 ms). The scanning parameters were as follows: 5 mm slice thickness, 1.2 mm gap, 256× 256 matrix, 296 mm field of view and four excitations.

ADC values were measured on ADC maps. A circular region of interest (ROI) of at least 1 cm2 was placed at targeted areas with the possibly lowest ADC values in the cyst components of ovarian endometrial cysts, by referring to conventional MR imaging. At least three measurements were obtained and averaged.

Acquisition of endometrial fragments in endometriotic cyst fluid and drainage fluid

All cyst fluid or drainage fluid samples were filtered with 100 μm apertures sieves under sterile circumstances. To separate the debris and the endometrial fragments, the remnants in the 100 μm aperture sieves were washed with 1x phosphate-buffered saline (PBS) several times. Fresh, endometrial fragments were collected and washed with 1x PBS. Three endometrial fragments from cyst fluid were immersed in liquid nitrogen for RNA extraction. Ten endometrial fragments from the cyst fluid were immersed in 4% neutral buffered formalin for the histological experiment. Fifteen endometrial fragments from the cyst fluid were used for in vitro cell culture. All the endometrial fragments acquired from drainage fluid were immersed in 4% neutral buffered formalin for the histological experiment.

In vitro culture of endometrial cells

Fragments were collected under sterile conditions and digested with type I collagenase (Biofroxx, Germany) in a shaker at 37 °C. After 45 minutes, digestion was stopped with Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) (1:1, Gibco, US) containing 12% foetal bovine serum. A 100 μm nylon cell strainer was used to remove the debris. Stromal cells and epithelial cells were not separated. The filtered liquid was centrifuged and resuspended in DMEM/F12 containing 12% foetal bovine serum. The cells were cultured in a humidified incubator at 37 °C with 5% CO2.

Transcriptome sequencing and bioinformatic analysis

Total RNA of the endometrial fragments in the cyst fluid (Cyst), ectopic tissues on the cyst wall (Ec) and eutopic endometrium (Eu) was extracted using TRIzol reagent (Invitrogen). Cleaved RNA fragments were reverse-transcribed to create a cDNA library by the protocol for the mRNA Seq sample preparation kit (Illumina), with a resulting average insert size for paired-end libraries of 200 bp (± 50 bp). Samples were subjected to paired-end sequencing on an Illumina HiSeq 4000 (LC Sciences) by Lc-Bio Technologies (Hangzhou, China) Co., Ltd.

Mapped reads for each sample were assembled using String Tie. All transcriptomes were merged to reconstruct a comprehensive transcriptome using Perl scripts. Following generation of the final transcriptome, String Tie and edge R were used to estimate the expression levels of all transcripts. String Tie was used to determine the expression level of mRNAs by calculating the fragments per kilobase per million (FPKM). Differentially expressed mRNAs and genes were selected as log2 (fold change) > 2 or log2 (fold change) > − 2 and with statistical significance (P < 0.05). The SNP sites in coding regions were analysed based on the transcriptome level. According to the Hisat2 alignment results of each sample and the reference genome, mpileup processing was performed with SAMtools software, and the possible SNP and INDEL information of each sample was then annotated with ANOVA. Bioinformatic analysis was performed using OmicStudio tools at https://www.omicstudio.cn/tool/. We used the gene differential gene expression in the cyst, ectopic and eutopic groups as the input, and the volcano plot, heatmap, GSEA, KEGG pathway enrichment, and Venn diagram were exported from the websites. Circos graphs of INDELs were generated by Lc-Bio Technologies (Hangzhou, China) Co., Ltd.

Immunohistochemistry staining

The endometrial fragments from cyst fluid and drainage fluid were embedded in paraffin and cut into 4 μm sections. Haematoxylin and eosin (H&E) staining was utilized to confirm the endometrial structures in the fragments. To determine the epithelial and interstitial structure, the slides were incubated with anti-cytokeratin 19 (10712-1, Proteintech, China) and anti-CD10 (18008-1, Proteintech, China) overnight. For the slides of negative control, the primary antibody was replaced by PBS. The corresponding secondary antibodies were incubated for 60 min. Slides were visualized with DAB (3,3′-diaminobenzidine), counterstained with haematoxylin and observed under a microscope.

For immunofluorescence, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 10 min. Unspecific binding was blocked by using 8% bovine albumin in PBS at room temperature for 1 hour. Cells were incubated overnight at 4 °C with anti-vimentin (60330-1, 1:100, Proteintech, China) and anti-cytokeratin 19 (10712-1, 1:100, Proteintech, China) antibodies. The next day, the slides were washed 3 times in PBS and incubated with goat anti-mouse antibody (Alexa Fluor® 488, ab150117, 1:500, Abcam, USA) and donkey anti-rabbit antibody (Alexa Fluor® 647, ab150075, 1:500, Abcam, USA) at room temperature for 1 hour. Nuclear DNA was labelled in blue with DAPI (ab104139, Abcam, USA). The cells were visualized under a confocal microscope (Olympus, Japan).

For the histochemical assay, paraffin-embedded slides of endometrial fragments from cyst fluid and ectopic endometrial tissue from the cyst wall were incubated with anti-ICAM2 (10121-2, 1:100, Proteintech, China) and anti-Claudin5 (GB11290, 1:100, Servicebio, China) antibodies. A semiquantitative evaluation of the immunostaining intensity was carried out using ImageJ, and the mean optical density (MOD) was used to represent the levels of protein expression. The average MOD of five different fields of a slide was regarded as the expression of the molecule.

Animal experiment

This study was carried out in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Six-week-old female BALB/C nude mice were purchased from Shanghai Animal Center, Chinese Academy of Science. The mice were housed in an environment at 21 °C ± 0.5 °C under a 12 h light/dark cycle and with free access to food and water. The endometriotic cyst fluid used in this part was collected from 12 patients (The information of patients enrolled is shown in Additional file 1, sheet2). The endometriotic cyst fluid was filtered through a 100 μm filter membrane, and fresh, endometrial fragments were collected. The fragments from each patient were cut into smaller pieces and suspended in 500 μL filtered cyst fluid. The mixture from each patient was injected into the pelvic cavity of 4 mice. The mice were sacrificed after two (n = 16) or three (n = 27) weeks. The abdominal of the mouse was carefully probed and lesions were collected for histological examination.

Statistics

Statistical analysis was performed by using GraphPad Prism 6.0 (GraphPad Software, USA). All the results are expressed as the mean ± standard error of the mean (SEM). The normality of the data was tested with the Shapiro-Wilk test. An unpaired Student’s t-test (two-tailed) was used to identify statistically significant differences between the two groups. Pearson correlation was used to analyse the correlation between the viscosity of the cyst fluid and the ADC mean value. A P value of < 0.05 was considered statistically significant.

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