Elevated plasma levels of lysophosphatidic acid and aberrant expression of lysophosphatidic acid receptors in adenomyosis

This study was approved by the Clinical Research Ethics Committee of the Jiangxi Provincial Maternal and Child Health Hospital (Registration number: JXFBEC005–2014, registered on 20th of july 2014). All subjects were given consent forms and the study protocols, and they provided written, informed consent. Plasma samples during the proliferative phase were obtained from thirty patients (age range, 30–42 years) who had been diagnosed with adenomyosis by laparoscopy and confirmed by pathological examination. Another thirty age matched fertile women (proliferative phase) who were selected from the health examination center were regarded as the control group. The sampling conditions between adenomyosis patients and healthy controls are identical. These control women were healthy (with neither clinical symptoms of adenomyosis nor any abnormality detected during the clinical examination) according to physical examination, transvaginal ultrasound, and biochemical blood tests. Women with irregular cycling, amenorrheic postmenopausal women and those who had received steroid hormone therapy in the last six months were excluded from the study.

After informed consent was obtained, eutopic endometrial tissue and ectopic endometrial tissue (Endometrial samples were placed in 4% paraformaldehyde before paraffin embedding. Paraffin blocks were sectioned, representative slides stained with hematoxy and eosin. Ectopic endometrial tissue, with subsequent histological verification of endometriotic lesions in myometrium.) samples were obtained during the proliferative phase of the menstrual cycle from ten patients (age range, 30–42 years) with both laparoscopically and histologically diagnosed adenomyosis. Healthy endometrial tissues were collected during the proliferative phase of the menstrual cycle from ten women (age range, 30–42 years) who underwent surgery for benign indications other than adenomyosis or endometriosis. Both these ten control patients and adenomyosis subjects exhibited normal hormonal profiles.

None of the patients had received steroid hormone therapy within the past six months, and none exhibited any obvious internal medicine or surgical comorbidities. Women with irregular cycling, endometrial abnormalities, fibroids, ovarian cysts, or lesions were excluded. The fertility status of the patients used in this study was unknown.

After informed consent was obtained, endometrial tissues were collected during hysterectomy from five women (proliferative phase, age range, 30–42 years) who had been diagnosed with intramural leiomyoma, with no evidence of endometrial abnormalities, adenomyosis, or endometriosis, and who had not taken any hormonal medication in the past six months. The endometrial tissues were placed in Hank’s balanced salt solution (HBSS) and transported to the laboratory for endometrial stromal cell (ESC) isolation and culture.

Blood samples were collected in EDTA-containing tubes and centrifuged at 1750×g for 15 min at room temperature. Plasma samples were frozen and stored at −80 °C until used. LPA were extracted as described previously [10]. HPLC-ESI-MS/MSanalysis of LPA species were performed using a hybrid triple quadrupole/linear ion trap mass spectrometer (AB SCIEX Qtrap, Framingham, MA, USA) and interfaced with a Shimadzu HPLC system (Shimadzu, Tokyo, Japan). LPA species were separated with a reversed phase C18 column (20 mm × 2.1 mm, 5 μm, Shiseido, Japan). 300 μM ammonium phosphate buffer, pH 5.4 solution was used for mobile phase A, while 9:1 (V/V) methanol-acetonitrile was used as mobile phase B. The gradient was 70% B (30% A) at 0.2 mL/min, and 8 min for each sample. A [¹³C₁₆] labeled 16:0 LPA is used as the internal standard. Mass spectrometric analyses were performed online using electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. Monitoring ions were m/z 425 → 153 for [¹³C₁₆] 16:0 LPA, m/z 481 → 153 for 22:6 LPA, m/z 457 → 153 for 22:4 LPA, m/z 437 → 153 for 18:0 LPA, m/z 435 → 153 for 18:1 LPA, m/z 433 → 153 for 18:2LPA, m/z 409 → 153 for 16:0 LPA. All standard LPA forms and heavy isotope-labeled [¹³C₁₆] 16:0 LPA were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Quantitative analysis was performed as described previously. We established standard curves for each LPA form by mixing different concentrations of a particular form of LPA with the same concentration of internal standard, and then performing ESI-MS analyses. Data processing was highly automated by using the MS software.

Representative paraffin-embedded soft tissues were cut into 4-μm-thick sections. Tissue sections were deparaffinized in xylene solutions at room temperature, hydrated in serial diluted alcohols. Antigen retrieval was then performed in citrate buffer (pH 6.0, 15 min), and endogenous peroxidase activity was eliminated by incubation in 3% hydrogen peroxide. Slides were then incubated with an anti-LPA1 antibody (LS-B1498/63066; Lifespan Bioscience Inc., Seattle, WA, 1:500 dilution, rabbit), an anti-LPA2 antibody (LS-B512/10150; Lifespan Bioscience Inc., Seattle, WA, 1:300 dilution, rabbit), an anti-LPA3 antibody (LS-A1014/40949; Lifespan Bioscience Inc., Seattle, WA, 1:50 dilution, rabbit), an anti-LPA4 antibody (LS-A872/36703; Lifespan Bioscience Inc., Seattle, WA, 1:100 dilution, rabbit), an anti-LPA5 antibody (LS-A428/42622; Lifespan Bioscience Inc., Seattle, WA, 1:50 dilution, rabbit), an anti-LPA6 antibody (LS-A852/52093; Lifespan Bioscience Inc., Seattle, WA, 1:50 dilution, rabbit) overnight at 4 °C. The primary antibody was replaced with phosphate-buffered saline (PBS) for negative control slides. On the following day, the slides were incubated with a biotin-labeled goat anti-rabbit secondary antibody (Zhongshanjinqiao Biotec, Beijing, China). To visualized protein expression, a chromogenic reaction was performed using a 3,3′-diaminobenzidine color reagent kit (ZSGB-BIO, Beijing, China) according to the manufacturer’s instructions. Image-Pro plus software was used to quantitative analyze the results as described previously [11]. With reference to Weidner’s counting method, any endothelial cell or endothelial cell cluster that was clearly separate from an adjacent cluster was considered to be a single, countable microvessel. The microvessel density (MVD) values are expressed as the largest number of microvessels.

Endometrial tissue was minced with a sterile surgical blade and digested in HBSS (Sigma-Aldrich, St. Louis, USA) containing collagenase B (1 mg/mL, 15 IU/mg), deoxyribonuclease I (0.1 mg/mL, 1500 IU/mg), penicillin (200 U/mL), and streptomycin (200 mg/mL) for 1 h at 37 °C with gentle shaking. The dispersed endometrial cells were separated by filtration through a 75-μm wire sieve, and the resultant supernatant was centrifuged at 1000 rmp for 5 min, the pellets (ESCs) was resuspended and cultured in a solution of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 (1:1 v/v; Invitrogen Corp. Shanghai, China), containing fetal bovine serum (10% v/v; ExCell Biology Inc. Shanghai, China). The cultures were maintained under a standard 95% atmosphere, in a 5% CO₂ incubator at 37 °C, and allowed to replicate to confluence. ESCs were cultured in DMEM supplemented with 10% fetal bovine serum in a humidified atmosphere of 95% air-5% CO₂ at 37 °C. After 1 passage, ESCs were assayed immunocytochemically, using vimentin and cytokeratin as specific cell-surface markers, and confirmed to have a purity of >95%.

Cell proliferation was determined by using a cell count-8 kit (Dojindo Lab. Tokyo, Japan) based on the protocol described previously [12]. ESCs were seeded at 1× 10⁴ per well in 200 μL medium. Cells were exposed to 0.1, 1, 10 μM LPA for 24 h or 48 h. WST-8 reagent solution (10 μL) was added to each well, and then the mixture was incubated for 2 h at 37 °C under an atmosphere of 5% CO₂. Absorbance of formazan in cell suspension was measured at 450 nm using a microplate spectrophotometer (BioRad, CA, USA).

ESCs were grown in 6-well plates (1× 10⁶ per well) in 2 ml of medium. Cells were exposed to 0.1, 1, 10 μM LPA for 24 h or 48 h. After treatment, the cell suspensions were centrifuged, and the supernatant was collected and stored at −80 °C until analysis. IL-8 and VEGF levels in cell-free supernatants were measured using commercially available ELISA kit (R&DSystems, Minneapolis, MN, USA) according to manufacturer’s instructions.

Using HPLC-ESI-MS/MS method, we quantify the levels of six LPA species (16:0, 18:2, 18:1, 18:0, 20:4 and 22:6 LPA) in plasma samples collected from adenomyosis patients and healthy controls. As shown in Table. 1, the adenomyosis patients group showed significantly higher plasma levels of total LPA and LPA species 16:0-, 18:2-, 18:1-, 20:4-, 22:6-LPA, compared with controls. 18:2 LPAwas the predominant form in all plasma samples. We also find that the levels of LPA species in human plasma generally follow the order: 18:2 LPA > 16:0 LPA, 20:4 LPA >18:1 LPA, 22:6 LPA >18:0 LPA.

To investigate the potential role of LPA receptors in adenomyosis, we used IHC to examine the expression of the LPA receptors LPA1–6 in the ectopic endometrium and paired eutopic endometrium in patients with adenomyosis and in the endometrial of healthy controls. Herein, we show endometrial express LPA receptors LPA1–5, while LPA6 was undetectable in the endometria of both patients with adenomyosis and healthy controls (data of LPA6 not shown).As shown in Fig. 1, In endometrium in healthy control group, LPA1, LPA4 and LPA5 staining were little in both stroma and epithelial cells, LPA2 and LPA3 staining were localized to the nuclei of both stromal and glandular cells. In eutopic endometrium in patients with adenomyosis group, LPA1 staining was localized to the cytoplasm, membrances of the epithelial cells of the endometrial glands, LPA2, LPA3 and LPA5 were localized to the nuclei of both stromal and glandular cells, LPA4 was little in both stroma and epithelial cells. In ectopic endometrium in patients with adenomyosis group, LPA1 staining was localized to the cytoplasm, membrances of the epithelial cells of the endometrial glands, LPA2, LPA4 and LPA5 staining were localized to the nuclei of both stromal and glandular cells, LPA3 was little in both stroma and epithelial cells. As shown in Fig. 1 and Fig. 2, LPA1, LPA4 and LPA5 immunoreactivity were significantly higher in the adenomyosis group than in the control group, while LPA2 and LPA3 immunoreactivity were significantly lower in the adenomyosis group than in the control group. In addition, the levels of LPA1, LPA4 and LPA5 in the ectopic endometrium were much higher than in the eutopic endometrium in the adenomyosis group, while the levels of LPA2 and LPA3 in the ectopic endometrium were much lower than in the eutopic endometrium in the adenomyosis group.

As shown in Fig. 3, LPA increase the release of IL-8 from ESCs, while no differences in the release of VEGF from ESCs were detected in LPA treatment group compared with control. LPA at all concentrations did not affect cell proliferation after 24 h or 48 h stimulation.