Age and menopausal status are important factors influencing the serum human epididymis secretory protein 4 level: a prospective cross-sectional study in healthy Chinese people / 中华医学杂志(英文版)

BACKGROUND@#Human epididymis secretory protein 4 (HE4) is a new ovarian cancer biomarker. The factors influencing HE4 levels are not clear, and the reference data in China are limited. Here, we aim to evaluate the effects of menopause and age on HE4 levels and to provide a possible reference value for HE4 in healthy Chinese people.@*METHODS@#A total of 2493 healthy females aged 40 years or older were recruited from March 2013 to March 2017 with the cooperation of four medical institutions across Beijing, China. The serum levels of HE4 and cancer antigen 125 (CA125) were measured by enzyme-linked immunosorbent assay. The Wilcoxon rank-sum test of variance and a stratified analysis were used to analyze the relationships among age, menopausal status, and levels of HE4 or CA125. Confidence intervals (5%-95%) were determined for reference ranges in different populations.@*RESULTS@#There was a statistically significant difference in median HE4 levels between the post-menopausal (n = 2168) and pre-menopausal groups (n = 325) (36.46 vs. 24.04 pmol/L, Z = -14.41, P < 0.001). HE4 increased significantly with age in the post-menopausal groups (H = 408.18, P < 0.001) but not in the pre-menopausal subjects (Z = -0.43, P = 0.67). The upper 95th percentile of HE4 levels were 44.63 pmol/L for pre-menopausal women, 78.17 pmol/L for post-menopausal women, and 73.3 pmol/L for all women. In the post-menopausal population, the HE4 reference ranges were 13.15 to 47.31, 14.31 to 58.04, 17.06 to 73.51, 24.50 to 115.25, and 35.71 to 212.37 pmol/L for different age groups from forty divided by decade. The CA125 level was affected mainly by menopausal status and not age.@*CONCLUSIONS@#Menopausal status and age were both important factors influencing the level of HE4, and age affected HE4 levels mainly in post-menopausal women. The HE4 level was higher in the post-menopausal population than in the pre-menopausal population and increased with age.

Current biomarkers for the detection of endometriosis / 中华医学杂志(英文版)

A clinically reliable non-invasive test for endometriosis is expected to reduce the diagnostic delay. Although varieties of biomarkers have been investigated for decades, and cancer antigen-125, cancer antigen-199, interleukin-6, and urocortin were the most studied ones among hundreds of biomarkers, no clinically reliable biomarkers have been confirmed so far. Some emerging technologies including "omics" technologies, molecular imaging techniques, and microRNAs are promising in solving these challenges, but their utility to detect endometriosis has yet to be verified. New combinations of researched indicators or other non-invasive methods and further exploration of the emerging technologies may be new targets and future research hotspots for non-invasive diagnosis of endometriosis. In conclusion, researches of biomarkers for the detection of endometriosis are still ongoing and may benefit from novel molecular biology, bioinformatics methods and a combination of more diverse monitoring methods. Though it will be a daunting task, the identification of a specific set of diagnostic biomarkers will undoubtedly improve the status of endometriosis.

Effects of Previous Laparoscopic Surgical Diagnosis of Endometriosis on Pregnancy Outcomes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The association between the previous history of endometriosis and obstetric outcomes is still ambiguous. This study aimed to evaluate the effects of previous history of operatively diagnosed endometriosis on pregnancy outcomes.</p><p><b>METHODS</b>A total of 98 primiparous women who had been diagnosed with endometriosis by previous laparoscopic surgery were included in this retrospective cohort study. Pregnancy outcomes were compared between these women (study group) who had a live birth and 300 women without endometriosis (control group) who had a live birth. In the study group, the pregnancy outcomes of 74 women who conceived naturally (no assisted reproductive technology [ART] subgroup) were simultaneously compared with 24 women who conceived by ART (ART subgroup).</p><p><b>RESULTS</b>Miscarriage was observed in 23 of 98 women with endometriosis (23.5%). There were 75 women who had a live birth after laparoscopic diagnosis of endometriosis in the study group eventually. On multivariate analysis, the postpartum hemorrhage rate increased significantly in the study group when compared with the control group (adjusted odds ratio: 2.265, 95% confidence interval: 1.062, 4.872; P = 0.034). There was an upward tendency of developing other pregnancy-related complications, such as preterm birth, placental abruption, placenta previa, cesarean section, fetal distress/anemia, and others in the study group than in the control group. However, the differences showed no statistical significance. Within the study group, the occurrence rate of postpartum hemorrhage and preterm birth was both higher in the ART subgroup than in the no ART subgroup. The differences both had statistical significance (44.4% vs. 17.5%, P = 0.024 and 27.8% vs. 1.8%, P = 0.010, respectively). At the same time, median (interquartile range) for gestational age at delivery in the ART subgroup was significantly shorter than that in the no ART subgroup (38 weeks [36-39 weeks] vs. 39 weeks [38-40 weeks]; P = 0.005).</p><p><b>CONCLUSIONS</b>Endometriosis may affect obstetric outcomes. Women with endometriosis have a higher risk of postpartum hemorrhage. Women with endometriosis who conceived by ART may have a higher risk of postpartum hemorrhage and preterm birth than those conceived naturally.</p>

Evaluation of Circulating Endometrial Cells as a Biomarker for Endometriosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Circulating endometrial cells (CECs) have been reported to be present in the peripheral blood of women with endometriosis (EM), providing clear and specific evidence of the presence of ectopic lesions. In this study, we established a method with a high detection rate of CECs, assessed the diagnostic value of CECs for EM and compared with serum CA125, and proposed a hypothesis for the pathogenesis of EM from the new perspective of CECs.</p><p><b>METHODS</b>The participants were enrolled prospectively from October 2015 to July 2016. The peripheral blood samples were collected from 59 participants, and the blood cells were isolated for immunofluorescence staining via microfluidic chips. The cells that were positive for vimentin/cytokeratin and estrogen/progesterone receptor and negative for CD45 were identified as CECs. The serum CA125 level was tested with electrochemiluminescence immunoassay.</p><p><b>RESULTS</b>The detection rate of CECs reached 89.5% (17/19) in the EM group, which was significantly higher than that of the control group (15.0% [6/40], P < 0.001) and was independent of menstrual cycle phases. Furthermore, a positive CEC assay detected 4/5 cases of Stage I-II EM. In contrast, a positive CA125 test had limited value in detecting EM (13/19, 68.4%) and detected only one case of Stage I-II EM.</p><p><b>CONCLUSION</b>CECs are promising biomarkers for EM with great potential for a noninvasive diagnostic assay.</p>

Aberrant Alternative Polyadenylation is Responsible for Survivin Up-regulation in Ovarian Cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Survivin is an oncoprotein silenced in normal mature tissues but reactivated in serous ovarian cancer (SOC). Although transcriptional activation is assumed for its overexpression, the long 3'-untranslated region (3'-UTR) in survivin gene, which contains many alternate polyadenylation (APA) sites, implies a propensity for posttranscriptional control and therefore was the aim of our study.</p><p><b>METHODS</b>The abundance of the coding region, the proximal and the distal region of survivin mRNA 3'-UTR, was evaluated by real-time polymerase chain reaction (PCR) in SOC samples, cell lines, and normal fallopian tube (NFT) tissues. The APA sites were confirmed by rapid amplification of cDNA 3' ends and DNA sequencing. Real-time PCR were used to screen survivin-targeting microRNAs (miRNAs) that were inversely correlated with survivin. The expression of an inversely correlated miRNA was restored by pre-miRNA transfection or induction with a genotoxic agent to test its inhibitory effect on survivin overexpression.</p><p><b>RESULTS</b>Varying degrees of APA were observed in SOC by comparing the abundance of the proximal and the distal region of survivin 3'-UTR, and changes of 3'-UTR correlated significantly with survivin expression (r = 0.708, P< 0.01). The main APA sites are proved at 1197 and 1673 of survivin 3'-UTR by DNA sequencing. Higher level of 3'-UTR proximal region than coding region was observed in NFT, as well as in SOC and cell lines. Among the survivin-targeting miRNAs, only a few highly expressed miRNAs were inversely correlated with survivin levels, and they mainly targeted the distal part of the 3'-UTR. However, in ovarian cancer cells, restoration of an inversely correlated miRNA (miR-34c) showed little effect on survivin expression.</p><p><b>CONCLUSIONS</b>In NFT tissues, survivin is not transcriptionally silenced but regulate posttranscriptionally. In SOC, aberrant APA leads to the shortening of survivin 3'-UTR which enables it to escape the negative regulation of miRNAs and is responsible for survivin up-regulation.</p>

Identification of biomarkers for endometriosis using clinical proteomics / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We investigated possible biomarkers for endometriosis (EM) using the ClinProt technique and proteomics methods.</p><p><b>METHODS</b>We enrolled 50 patients with EM, 34 with benign ovarian neoplasms and 40 healthy volunteers in this study. Serum proteomic spectra were generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) combined with weak cationic exchange (WCX) magnetic beads. Possible biomarkers were analyzed by a random and repeat pattern model-validation method that we designed, and ClinProtools software, results were refined using online liquid chromatography-tandem MS.</p><p><b>RESULTS</b>We found a cluster of 5 peptides (4210, 5264, 2660, 5635, and 5904 Da), using 3 peptides (4210, 5904, 2660 Da) to discriminate EM patients from healthy volunteers, with 96.67% sensitivity and 100% specificity. We selected 4210 and 5904 m/z, which differed most between patients with EM and controls, and identified them as fragments of ATP1B4, and the fibrinogen alpha (FGA) isoform 1/2 of the FGA chain precursor, respectively.</p><p><b>CONCLUSIONS</b>ClinProt can identify EM biomarkers, which - most notably - distinguish even early-stage or minimal disease. We found 5 stable peaks at 4210, 5264, 2660, 5635, and 5904 Da as potential EM biomarkers, the strongest of which were associated with ATP1B4 (4210 Da) and FGA (5904 Da); this indicates that ATP1B4 and FGA are associated with EM pathogenesis.</p>

Low programmed cell death 5 expression is a prognostic factor in ovarian cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Ovarian cancer is a leading gynecological malignancy. We investigated the prognostic value of programmed cell death 5 (PDCD5) in patients with ovarian cancer.</p><p><b>METHODS</b>Expression levels of PDCD5 mRNA and protein were examined in six ovarian cancer cell lines (SKOV3, CAOV3, ES2, OV1, 3AO, and HOC1A) and one normal ovarian epithelial cell line (T29) using reverse transcription polymerase chain reaction, Western blotting, and flow cytometry. After inducing PDCD5 induction in SKOV3 cells or treating this cell line with taxol or doxorubicin (either alone or combined), apoptosis was measured by Annexin V-FITC/propidium iodide staining. Correlations between PDCD5 protein expression and pathological features, histological grade, FIGO stage, effective cytoreductive surgery, and serum cancer antigen-125 values were evaluated in patients with ovarian cancer.</p><p><b>RESULTS</b>PDCD5 mRNA and protein expression were downregulated in ovarian cancer cells. Recombinant human PDCD5 increased doxorubicin-induced apoptosis in SKOV3 cells (15.96 ± 2.07%, vs. 3.17 ± 1.45% in controls). In patients with ovarian cancer, PDCD5 expression was inversely correlated with FIGO stage, pathological grade, and patient survival (P < 0.05, R = 0.7139 for survival).</p><p><b>CONCLUSIONS</b>PDCD5 expression is negatively correlated with disease progression and stage in ovarian cancer. Therefore, measuring PDCD5 expression may be a good method of determining the prognosis of ovarian cancer patients.</p>

Effect of human epididymis protein 4 gene silencing on the malignant phenotype in ovarian cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human epididymis secretory protein 4 (HE4) has been proved to be a promising novel biomarker for the detection of epithelial ovarian carcinomas. Compared with CA125, HE4 assay demonstrated an improved ability to discriminate between pelvic mass with malignant and benign disease. Though it is well known that HE4 is overexpressed in ovarian cancer, however, the role of HE4 in the carcinogenesis and progression of ovarian cancer remains unkown.</p><p><b>METHODS</b>In this study, we explored the role of HE4 in the carcinogenesis and progression of ovarian cancer. We screened nine ovarian cancer cell lines for HE4 expression, and using RNA interference (RNAi), we silenced HE4 gene expression in CaoV3 and SKOV3.ip1 ovarian cancer cell lines. We assessed the effect of HE4 gene silencing on the transformed phenotype by examining the cell cycle, apoptosis, proliferation and transwell migration/invasion in vitro.</p><p><b>RESULTS</b>HE4 gene silencing induces G0/G1 arrest and blocks the progression from the G1 to S phase in CaoV3 and SKOV3.ip1 cells. HE4 knockdown also inhibited cell proliferation, migration and invasion in SKOV3.ip1 cells in vitro.</p><p><b>CONCLUSION</b>HE4 may be involved in the regulation of the cell cycle and promote ovarian cancer migration and invasion.</p>

Predictive validity of BODE index for anxious and depressive symptoms in patients with chronic obstructive pulmonary disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Anxiety and depression are two of the commonest and most modifiable comorbidities of chronic obstructive pulmonary disease (COPD) and have an independent effect on health and prognosis. FEV1% has been shown to be a poor predictor of anxiety and depression. The body mass index, degree of airflow obstruction, dyspnea, and exercise capacity (BODE) index is a multidimensional assessment system which may predict health outcome in COPD patients. The purpose of this study was to investigate the predictive validity of the BODE index for anxious and depressive symptoms in COPD patients.</p><p><b>METHODS</b>This was a multicenter prospective cross-sectional study in 256 patients with stable COPD. Anxious and depressive symptoms were assessed using the Hospital Anxiety and Depression Scale (HADS). The relationships between anxiety, depression and potential predictors (including the BODE index) were analyzed by a binary Logistic regression model.</p><p><b>RESULTS</b>Subjects who were anxious and depressive walked a shorter six-minute walking distance (6MWD), had more dyspnea, a higher BODE index, and lower health-related quality of life (P < 0.01). Anxiety and depression score was significantly correlated with BODE index, respectively (r = 0.335, P < 0.001; r = 0.306, P < 0.001). The prevalence of anxiety and depression increased with BODE stage increasing (P < 0.05). On the basis of binary Logistic regression, the BODE index was a good and independent predictor of anxiety and depression because it comprised dyspnea and 6MWD, which were shown to be the main determinants.</p><p><b>CONCLUSIONS</b>The predictive validity of the BODE index for anxiety and depression was demonstrated. We propose that the BODE index should be included in assessment of COPD severity.</p>

Screening for genes associated with ovarian cancer prognosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human epithelial ovarian cancer cell line SKOV3.ip1 is more invasive and metastatic compared with its parental line SKOV3. A total of 17 000 human genome complementary DNA microarrays were used to compare the gene expression patterns of the two cell lines. Based on this, the gene expression profiles of 22 patients with ovarian cancer were analyzed by cDNA microarray, and screened the 2-fold differentially expressed genes compared with the normal ones. We screened genes relevant to clinical prognosis of serous ovarian cancer by determining the expression profiles of ovarian cancer genes to investigate cell receptor and immunity-associated genes, and as groundwork, identify ovarian cancer-associated antigens at the gene level.</p><p><b>METHODS</b>Total RNA was extracted from 22 patients with ovarian cancer and DNA microarrays were prepared. After scanning, hybridization signals were collected and the genes that were differentially expressed twice as compared with the normal ones were screened.</p><p><b>RESULTS</b>We screened 236 genes relevant to the prognosis of ovarian cancer from the 17 000 human genome cDNA microarrays. According to gene classification, 48 of the 236 genes were cell receptor or immunity-associated genes, including 2 genes related to the International Federation of Gynecology and Obstetrics (FIGO) stage, 4 genes to histological grade, 18 genes to lymph node metastasis, 11 genes to residual disease, and 13 genes to the reactivity to chemotherapy. Several functionally important genes including fibronectin 1, pericentriolar material 1, beta-2-microglobulin, PPAR binding protein were identified through review of the literature.</p><p><b>CONCLUSIONS</b>The cDNA microarray of ovarian cancer genes developed in this study was effective and high throughput in screening the ovarian cancer-associated genes differentially expressed. Through the studies of the cell receptor and immunity-associated genes we expect to identify the molecular biology index of ovarian cancer-associated antigens.</p>

Effect of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry on identifing biomarkers of endometriosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endometriosis is a common gynecological disease. This study aimed to screen proteins that were expressed differently in patients with endometriosis versus normal controls using proteomic techniques, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS).</p><p><b>METHODS</b>Protein chip SELDI-TOF-MS combines the advantages of microarray and mass spectrometry, and can screen latent markers in sera of patients with endometriosis. Serum samples from patients and normal volunteers were analyzed by SELDI-TOF-MS.</p><p><b>RESULTS</b>After comparing the serum protein spectra of 36 patients with 24 normal controls, 24 differently expressed potential biomarkers (P < 0.01) were identified. Using Biomarker Pattern software, we established a tree model of the 60 serum protein spectra. When using the three biomarkers to classify the samples, the sensitivity for diagnosing endometriosis was 91.7%, specificity was 95.8%, and coincidence rate was 93.3%. Then we used serum samples from 12 patients and 8 normal controls to validate the tree model and report the sensitivity for diagnosing endometriosis was 91.7%, specificity was 75%, and coincidence rate was 85%.</p><p><b>CONCLUSIONS</b>SELDI-TOF-MS may be a useful tool in high-risk population screening for endometriosis. The identification and application of the biomarkers need to further study.</p>

Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166 - 9 and whose corresponding monoclonal antibody is COC166 - 9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice.</p><p><b>METHODS</b>The fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively.</p><p><b>RESULTS</b>The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F (ab) 2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week.</p><p><b>CONCLUSION</b>The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.</p>

Immuno-therapeutic study of anti-idiotype minibody (single chain Fv-CH3) on ovarian carcinoma bearing mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>An anti-idiotypic minibody with optimal antigenicity which mimicking ovarian cancer antigen was used for therapeutic research in mice model bearing ovarian cancer.</p><p><b>METHODS</b>Using gene engineering technique, prokaryotic expression vector was constructed by genetic fusion of 6B11scFv to human IgG1 hinge and CH3 region. The fusion protein named minibody was induced with IPTG in E. coli and analyzed with Western blot and inhibition ELISA tests respectively. Twenty human-PBL-SCID mice bearing i.p. Skov3.ip1 cells were divided into two groups (10 per-group), 10 mice were immunized repeatedly by minibody every two weeks for three times. Indirect ELISA test was employed for analyzing the characterization of anti-anti-idiotypic scFv (Ab3). The latent period of ascites growth and the mean survival time were observed respectively. CD4+ and CD8+ T cells from the spleen of immunized mice were assayed by flow cytometry.</p><p><b>RESULTS</b>SDS-PAGE gel electrophoresis showed that a protein band with molecular weight of 50,000 appeared as the expected size after transformation and induction the host bacteria BL21 (DE3). The expressed minibody could be reacted with COC166-9 (Ab1 of 6B11) and binding goat anti-human IgG1 antibody in Western blot. Inhibition ELISA showed minibody had the capacity of binding ovarian cancer monoclonal antibody COC166-9 instead of primal antigen. Ab3 could be detected in the sera of immunized mice with minibody by ELISA test. Ab3 reached the highest at the 14th day after last vaccination and lasted for 6 weeks. The ratio of CD4+/CD8+ was the highest at the 13th day after last vaccination. The latent period of ascites growth were (37.7 +/- 5.5) days and (48.6 +/- 14.3) days (P = 0.04) respectively; while the mean survival time were (42.5 +/- 1.8) days and (59.4 +/- 16.8) days (P = 0.011) in the control and minibody group respectively.</p><p><b>CONCLUSIONS</b>These results demonstrate the successful construction and expression minibody with good immune activities of 6B11scFv and human IgG1 molecules function. Antigenicity is increased without adjuvants and partial humanization is realized. Minibody can induce humoral anti-idiotypic immunity responses against ovarian carcinoma in vivo. When ascites formation was delayed or prevented and the survival was prolonged in minibody group. We expect that minibody may be used as tumor vaccine to ovarian carcinoma in the future clinical trails.</p>