br Fig Immunohistochemical IHC analysis of hCG
Fig. 2. Immunohistochemical (IHC) analysis of hCG and LHCGR expression in tissue microarrays (TMA) collected from patients with epithelial ovarian cancer (EOC). (A–C) IHC staining for hCG expression produced (A) no, (B) weak, and (C) strong staining in normal, benign, and malignant ovarian tissues, respectively. (D–F) IHC staining for LHCGR expression produced (D) strong, (E) weak, and (F) no staining in normal, benign, and cancerous ovarian tissues, respectively. Original magnification, ×200. Scale bar, 100 μm.
Fig. 3. hCG and LHCGR expression in diﬀerent epithelial ovarian cancer (EOC) samples of varying histological classifications. (A–C) Positive im-munohistochemical (IHC) staining of hCG in (A) serous, (B) endometrioid, and (C) clear-cell ovarian cancer. (D–F) Negative IHC staining of LHCGR in (D) serous, (E) endometrioid, and (F) clear-cell ovarian cancer. Original magnification ×200. Scale bar, 100 μm).
[12,13]. Similarly, Noci et al.  previously showed that LHCGR ex-pression correlates with human primary endometrial cancer invasive-ness in vitro, and LHCGR protein is produced by both normal and neoplastic endometrium. Moreover, the presence of LHCGR mRNA and protein has also been documented in breast cancer cell lines and en-dometrioid, adrenal, and prostate tumors [8,15].
Nevertheless, neither LHCGR expression in EOC, nor its correlation with patient clinical parameters, has yet been directly evaluated; thus, it is not clear whether hCG and/or LHCGR expression directly modulate tumor growth and/or disease progression in EOC [16–18]. Our research group previously showed that treating OVCAR-3 QC6352 with diﬀerent hCG concentrations in vitro does not significantly aﬀect the expression of the hCG receptor . In the present study, we performed quanti-tative polymerase chain reaction (qPCR) and western blot assays to
analyze the mRNA and protein expression levels of hCG and LHCGR, respectively, in fresh ovarian epithelial cancer tissues, and in tissue arrays collected from patients with EOC. We furthermore evaluated whether changes of hCG and LHCGR expression patterns correlate with the clinical/pathological features (FIGO, grade, histological classifica-tion, ascites cell, serum CA125(U/ml) and metastasis), and/or the prognosis of patients with EOC. The Federation of Gynecology and Obstetrics (FIGO) stage system is widely used for grading endometrioid carcinomas which is based on architecture. Tumours with ≤50% solid glandular component are grade low, and tumours with > 50% of solid glandular component are classified as grade high. The FIGO, grade, histological classification and metastasis are associated the che-motherapy and therapy method, serum CA-125 levels may be a specific biomarker for EOC .
Fig. 4. Correlation between hCG and LHCGR expression in ovarian cancer.
There was an obvious negative correlation between hCG and LHCGR expression levels in the analyzed ovarian cancer tissues (R = −0.249, P = 0.003).
2. Materials and methods
2.1. Clinical data and tissue samples
The present study enrolled 242 patients who were admitted to the Aﬃliated Hospital of Nantong University, China, between January 2005–December 2012. The median age of the patients was 55.05 years (range 24–80 years). Samples collected from the patients included 24 normal ovarian and 24 normal fallopian tube (control) samples, as well as 12 benign, 43 borderline, and 139 EOC ovarian tumor samples. The inclusion criteria were as follows: all patients provided complete clin-ical information to the study, were diagnosed by the pathologist, and had undergone no preoperative chemotherapy, radiotherapy, or im-munotherapy. Tumor histological grades and clinical stages were de-termined pathologically after surgery. All fresh tissues samples (in-cluding 18 ovarian cancers and 5 noncancerous ovarian tissues: ovarian cyst and serous cystadenoma) for real-time (RT)-PCR and western blot assays were collected from the patient cohort at the Aﬃliated Hospital of Nantong University between March 2017–February 2018. The study
protocol was approved by the Ethics Committee of the Aﬃliated Hospital of Nantong University, and all experiments were performed in line with approved guidelines of the Aﬃliated Hospital of Nantong University. All study patients provided written informed consent for their participation in the study.
2.2. Immunohistochemical (IHC) staining and evaluation
All IHC analyses were performed using the tissue microarray system (Quick-Ray, UT06, UNITMA, Korea) utilized by the Department of Clinical Pathology, Nantong University Hospital, Jiangsu, China. Tissue MicroArray (TMA) specimens were cut into 4-μm sections and placed on super frost-charged glass microscope slides. These were then twice incubated in a xylene liquid tank for 15 min, before being heated in a constant temperature oven at 60 °C for 6–8 h. The sections were then passed through an ethanol series comprising 100% (4 min), 95% ethanol (4 min), 80% (2 min), and 70% (2 min) ethanol (diluted in ddH2O, which is double distilled H2O), before being rinsed in tap (2 min) and ddH2O (2 min). They were then immersed in citrate buﬀer, and pressure-cooked (3 min) to facilitate antigen retrieval. After cooling to room temperature, the sections were blocked (20 min) with 3% H2O2, washed three times (5 min) in PBS, spin-dried, and incubated with primary hCG (ab187285, 1:100 dilution) and LHCGR (ab204950, 1:100 dilution) antibodies (4 °C, overnight). After a further three (5 min) PBS washes, the sections were incubated (30 min) with sec-ondary antibody reagents. The three-step immunohistochemistry kit was bought by Beijing Zhongshan Jinqiao Company. Color was devel-oped using a coloring solution, DAB (containg concentrate and di-luents), concentrate: 1:20 dilution before the sections were counter-stained with hematoxylin (10–20 s) and dehydrated in an ethanol series comprising 70% (3 min), 80% (3 min), and 95% ethanol (5 min), and anhydrous ethanol (5 min). Finally, the sections were made transparent with xylene, and sealed using neutral resin. All scores were assigned by two independent pathologists, without knowledge of patient clinical or pathological factors, using a Leica fluorescence microscope (Wetzlar, Germany). If the judgment results of the two pathologists were dif-ferent, a third person evaluated again. hCG and LHCGR expression was visualized via immunohistochemical staining (SP method), the intensity of which was scored as either 0 (negative), 1 (weakly positive), 2 (moderately positive), or 3 (strongly positive). The percentage of