Human Epididymis-specific Protein Research Articles

Prognostic Role of Human Epididymis Protein4 (HE4) in Endometrial Lesions: Study in a Tertiary Care Centre.

Endometrial cancer accounts for 20% of malignant tumours in the female reproductive system. A novel biological marker human epididymis protein4 (HE4) represents an important alternative indicator which may benefit patient mortality. To correlate the immunohistochemical expression of HE4 in different non-neoplastic and neoplastic endometrial lesions and with the WHO grade of the tumours. Our study was a cross-sectional, observational study done in a tertiary care hospital from December 2019 to June 2021 on the hysterectomy sample of 50 patients with a clinical history of abnormal uterine bleeding and pelvic pain. The study showed strong positivity of HE4 in cases of endometrial carcinoma, weak positivity in cases of atypical endometrial hyperplasia, and negativity in cases of endometrial hyperplasia without atypia group. WHO grade 3(50%) and grade 2 (29%) endometrioid adenocarcinoma NOS in our study showed strong positivity for HE4 which was statistically significant (P value = 0.001). In recent studies using overexpression of HE4-related genes, the malignant biological behaviour such as cell adhesion, invasion, and proliferation was enhanced. It was seen in our study that strong positivity of HE4 was seen in all endometrial carcinoma groups and with higher WHO grade. So, HE4 may become a potential therapeutic target for advanced-stage endometrial carcinoma which requires further research. Thus, human epididymis-specific protein 4 (HE4) has been shown to be a promising marker for the detection of endometrial carcinoma patients who could be benefitted from targeted therapy.

Two-phase dual-signal-readout immunosensing platform based on multifunctional carbon nano-onions for ovarian cancer biomarker detection.

In order to detect early tumor markers and gain valuable time for treatment, there is an urgent need to develop a fast, cheap, and ultrasensitive multi-reading sensing platform. Herein, a solid/liquid two-phase dual-output biosensor was explored based on a sensitized sonochemiluminescence (SCL) strategy and a multifunctional carbon nano-onion (CNO) probe. It is clear that ultrasonic radiation caused the formation of hydroxyl radicals (˙OH), triggering the SCL signal of the emitter lucigenin (Luc2+). Meanwhile, titanium carbide nanodots and ethanol were used to enhance the SCL signal, and an astonishingly linear enhancement of the SCL intensity was produced with increasing ethanol concentration. More importantly, the CNOs, with their excellent photothermal properties and adsorption capacity, can output both the temperature signal and an enhanced SCL strength from the solid-liquid phase. Through inter-calibration of the signals from the two-phases, this biosensor shows excellent analytical performance for the detection of the ovarian cancer biomarker, human epididymis-specific protein 4, from 10-5 to 10 ng mL-1 with a low detection limit of 3.3 fg mL-1. This work not only provides a novel two-phase signal-output mode that broadens the scope of multiperformance joint applications of CNOs, but also enriches the quantitative detection of point-of-care testing.

Biomarkers in Ovarian Pathology: From Screening to Diagnosis. Review of the Literature.

Background: Ovarian cancer has a low incidence, but high mortality due to a habitual diagnosis in advanced cancer stages. Currently, used biomarkers have good sensitivity, but low specificity. Aim: To determine the usefulness of the biomarkers and algorithms used up to now in the screening, diagnosis, response to treatments and identification of recurrence in patients with ovarian masses. Methodology: Systematic search of publications in English in the Medline-PubMed database with the terms: “biomarkers”, “tumour”, “tumour biomarkers”, “marker”, “tumour marker”, “ovarian cancer”, “ovarian”, “Neoplasms”, “cancer”, CA-125 Antigen; Human Epididymis-specific Protein E4; Risk of Malignancy Index (RMI); Risk of Ovarian Malignancy Algorithm (ROMA); Ovarian Neoplasms. Original articles, clinical trials, reviews, systematic reviews and meta-analyses, published between January 2000 and November 2020, were selected to determine the usefulness (among others) of CA 125 and HE4 antigen in ovarian cancer. Results: Finally, 39 transcendental publications were selected to write this article to determine the usefulness of tumour markers and algorithms in ovarian cancer. Conclusions: The usefulness of the tumour markers antigen CA125 and antigen HE4 individually or as a basis for decision-making algorithms has low specificity; however, there is little evidence that confirms their usefulness as markers in ovarian cancer screening.

Substrate-Induced Growth of Micro/Nanostructured Zn(OH)F Arrays for Highly Sensitive Microfluidic Fluorescence Assays.

To date, ZnO array-based microfluidic fluorescence assays have been widely investigated and have exhibited excellent performance in the detection of cancer biomarkers. However, the requirements of highly sensitive detection necessitate further improvement of current Zn-based fluorescence detection devices. Here, a rhombus-like Zn(OH)F array-based microfluidic fluorescence detection device is proposed. Construction of Zn(OH)F arrays on the inner wall of a microchannel is carried out via a microfluidic chemical method. A substrate-induced growth strategy for Zn(OH)F arrays is proposed, and various micro/nanostructured Zn(OH)F arrays are successfully obtained. Zn(OH)F nanorod arrays with a high aspect ratio can be constructed on the columnar ZnO nanorod arrays, and the results indicate that the fluorescence enhancement factor (EF) of the Zn(OH)F arrays toward Cy3 is approximately 4-fold that of the ZnO nanorod arrays, which can be attributed to the higher excitation light absorption and evanescent electric field. In human epididymis-specific protein 4 (HE4) detection, the limit of detection (LOD) reaches 9.3 fM, and the dynamic linear range is 10 fM to 100 pM. It has been demonstrated that Zn(OH)F nanorod array-based microfluidic devices are excellent fluorescence assay platforms that also provide a new design and construction strategy for fluorescence enhancement substrates for the detection of biomarkers.

Design and application of proximity hybridization-based multiple stimuli-responsive immunosensing platform for ovarian cancer biomarker detection

The development of convenient and sensitive multi-readout immunoassay is crucial but highly challenged for meeting the demand of exactness and diversity in early clinical diagnosis. Herein, a split-type multiple stimuli-responsive biosensor was outlined combined with the outstanding superiority of luminol probe-based electrochemiluminescence (ECL) strategy, mimicking enzyme-mediated colorimetric system and portable photothermal effect-induced temperature sensing. Especially, versatile MoS2 nanosheets (MoS2 NSs) with distinguished property not only acted as dual-promoter to improve the cathodic ECL of luminol because of its good electrocatalytic activity for dissolved O2 and favorable photothermal effect for elevating electrode temperature, but also used as nanozyme to regulate subsequent split-type visual colorimetric sensing due to its peroxidase-like activity for the generation of oxidized 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) in ABTS-H2O2 colorimetric system. More importantly, the green oxidized ABTS (ABTS•+) also exhibited strong near-infrared (NIR) laser-triggered photothermal performance, which can be innovatively employed as sensitive photothermal agent for converting biological signals into temperature under the irradiation of NIR laser, accomplishing more simpler temperature quantitative detection by a portable thermometer. Furthermore, on account of the affinity discrepancy of MoS2 NSs to single-stranded and double-stranded nucleic acids, a label-free proximity hybridization-based multifunctional assay platform was proposed for target detection with human epididymis-specific protein 4 (HE4) as model protein, demonstrating good analytical performances. Significantly, this innovative work not only enriches the foundational study of multi-model biosensing based on the unitary material but also provides an unambiguous guideline for exploring more accurate and simpler point-of-care diagnosis.

Ultrasound Features Improve Diagnostic Performance of Ovarian Cancer Predictors in Distinguishing Benign and Malignant Ovarian Tumors.

To determine whether ultrasound features can improve the diagnostic performance of tumor markers in distinguishing ovarian tumors, we enrolled 719 patients diagnosed as having ovarian tumors at Nanfang Hospital from September 2014 to November 2016. Age, menopausal status, histopathology, the International Federation of Gynecology and Obstetrics (FIGO) stages, tumor biomarker levels, and detailed ultrasound reports of patients were collected. The area under the curve (AUC), sensitivity, and specificity of the bellow-mentioned predictors were analyzed using the receiver operating characteristic curve. Of the 719 patients, 531 had benign lesions, 119 had epithelial ovarian cancers (EOC), 44 had borderline ovarian tumors (BOT), and 25 had non-EOC. AUCs and the sensitivity of cancer antigen 125 (CA125), human epididymis-specific protein 4 (HE4), Risk of Ovarian Malignancy Algorithm (ROMA), Risk of Malignancy Index (RMI1), HE4 model, and Rajavithi-Ovarian Cancer Predictive Score (R-OPS) in the overall population were 0.792, 0.854, 0.856, 0.872, 0.893, 0.852, and 70.2%, 56.9%, 69.1%, 60.6%, 77.1%, 71.3%, respectively. For distinguishing EOC from benign tumors, the AUCs and sensitivity of the above mentioned predictors were 0.888, 0.946, 0.947, 0.949, 0.967, 0.966, and 84.0%, 79.8%, 87.4%, 84.9%, 90.8%, 89.1%, respectively. Their specificity in predicting benign diseases was 72.9%, 94.4%, 87.6%, 95.9%, 86.3%, 90.8%, respectively. Therefore, we consider biomarkers in combination with ultrasound features may improve the diagnostic performance in distinguishing malignant from benign ovarian tumors.

HE4 is a novel tissue marker for therapy response and progestin resistance in medium- and low-risk endometrial hyperplasia.

The aim of the present study was to investigate whether changes in the tissue expression of human epididymis-specific protein 4 (HE4) could predict therapy resistance and relapse after progestin hormone therapy for medium- and low-risk endometrial hyperplasia. Endometrial biopsies were obtained from women participating in a multicentre RCT performed according to the CONSORT guidelines; the women were randomly assigned to either LNG-IUS; 10 mg of oral medroxyprogesterone acetate (MPA) administered for 10 days per cycle; or 10 mg of oral MPA administered daily for 6 months. Of the 153 women who completed therapy, 141 had adequate material for immunohistochemistry in pre- and post-treatment biopsies. An antibody to HE4 (clone 12A2 monoclonal IgG1 antibody, Fujirebio Diagnostics, Inc.) was used for the immunohistochemical staining of the pre- and post-treatment biopsies from each participant. The expression of HE4 staining was evaluated by the histological score (H-score) using light microscopy. Changes in the expression of HE4 (H-score) during therapy were related to the therapy group (P 0.05). Changes in the intracellular bodies were shown to predict both the therapy response (P=0.038) and relapse (P=0.014). Changes in the expression of HE4 during progestin therapy regimens can predict therapy response or indicate progestin resistance for medium- and low-risk endometrial hyperplasia.

HE4 is a novel tissue marker for therapy response and progestin resistance in medium- and low-risk endometrial hyperplasia

Background:The aim of the present study was to investigate whether changes in the tissue expression of human epididymis-specific protein 4 (HE4) could predict therapy resistance and relapse after progestin hormone therapy for medium- and low-risk endometrial hyperplasia.Methods:Endometrial biopsies were obtained from women participating in a multicentre RCT performed according to the CONSORT guidelines; the women were randomly assigned to either LNG-IUS; 10 mg of oral medroxyprogesterone acetate (MPA) administered for 10 days per cycle; or 10 mg of oral MPA administered daily for 6 months. Of the 153 women who completed therapy, 141 had adequate material for immunohistochemistry in pre- and post-treatment biopsies. An antibody to HE4 (clone 12A2 monoclonal IgG1 antibody, Fujirebio Diagnostics, Inc.) was used for the immunohistochemical staining of the pre- and post-treatment biopsies from each participant. The expression of HE4 staining was evaluated by the histological score (H-score) using light microscopy.Results:Changes in the expression of HE4 (H-score) during therapy were related to the therapy group (P<0.001) and therapy response (P<0.001) of the individuals but could not predict relapse (P>0.05). Changes in the intracellular bodies were shown to predict both the therapy response (P=0.038) and relapse (P=0.014).Conclusions:Changes in the expression of HE4 during progestin therapy regimens can predict therapy response or indicate progestin resistance for medium- and low-risk endometrial hyperplasia.

Electrochemical sandwich immunoassay for human epididymis-specific protein 4 using a screen-printed electrode modified with graphene sheets and gold nanoparticles, and applying a modular magnetic detector device produced by 3D laser sintering

The authors describe an electrochemical sandwich immunoassay for the human epididymis-specific protein 4 (HE4). A commercially available electromagnetic detector device and a screen-printed electrode (SPE) modified with graphene sheets and gold nanoparticles were used to fabricate the detector. First, a nanocomposite suspension consisting of graphene sheets and gold nanoparticles was deposited onto the SPE. This results in an enlarged electrochemically active area and improves electron transfer. Next, biotinylated monoclonal antibody against HE4 (anti-HE4) was bound to streptavidin-modified magnetic beads via biotin-avidin binding. Nonspecific binding sites were blocked with bovine serum albumin. Stepwise changes in the microscopic features of the surfaces and electrochemical properties upon the formation of each layer were studied by scanning electron microscopy, transmission electron microscopy and cyclic voltammetry. After magnetic separation and washing, the biotinylated anti-HE4 beads were deposited on the SPE, which then was inserted into the electromagnetic modular detector. Following the immunoreaction with HE4, a sandwich-type immunoassay was performed on the SPE using horseradish peroxidase (HRP)-labeled HE4 as a tracer label. Electrochemical detection was carried out after addition of H2O2 as a substrate for HRP. Under optimal conditions, differential pulse voltammetry can detect HE4 in the 0 to 400 pM concentration range with a detection limit of 0.5 pM (at an S/N ratio of 3). The system was applied to the determination of HE4 with good accuracy and selectivity.

Ultrasensitive electrochemical immunosensor for HE4 based on rolling circle amplification

A novel electrochemical immunoassay system for the detection of human epididymis-specific protein 4 (HE4) was developed. A chitosan–titanium carbide (TiC) nanocomposition film was first electrodeposited onto a tin-doped indium oxide (ITO) electrode at a constant potential. Gold (Au) nanoparticles were then electrodeposited on the surface of the chitosan–TiC film by cyclic voltammetry (CV). The capture antibody (anti-HE4) was adsorbed onto the Au and TiC nanoparticles. After a specific sandwich immunoreaction among the capture antibody, HE4, and biotinylated secondary antibody, biotinylated primer DNA was immobilized on the secondary antibody by biotin–streptavidin system. Appropriate amounts of circular template DNA and biotinylated primer DNA were used for rolling circle amplification (RCA) under optimal conditions. The RCA products provided a large number of sites to link DNA detection probes. Doxorubicin hydrochloride intercalated the CG–GC steps between the RCA products and the DNA detection probes, which was monitored by differential pulse voltammetry (DPV) based on the current signal of doxorubicin hydrochloride. With the above-mentioned amplification factors, the current responded to HE4 linearly in the concentration range of 3–300pM under optimal detection conditions, with a detection limit of 0.06pM. Stepwise changes in the microscopic features of the surfaces and electrochemical properties upon the formation of each layer were confirmed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and electrochemical impedance spectroscopy (EIS). This system was successfully employed for the detection of HE4 with good accuracy and renewable ability.

Diagnostic value of HE4 for ovarian cancer: a meta-analysis

Ovarian cancer (OC) is the second most common gynecological cancer and the first cause of death from gynecological malignancy in Western Europe and the USA. While human epididymis-specific protein 4 (HE4) has been reported as a predictive diagnostic index, it has not been widely accepted because of inconsistent conclusions. The aim of this study was to evaluate the diagnostic value of HE4 systematically for ovarian cancer. All relevant original studies about HE4 in the diagnosis of ovarian cancer published from January 1974 to May 2011 were retrieved. By measuring methodological qualities, 12 papers were selected for this study, while 531 articles were searched. The overall diagnostic sensitivity, specificity, positive likelihood ratio (LR+) and negative likelihood ratio (LR-), and area under the receiver operating characteristic curve (AUC-ROC) were used to evaluate the diagnostic value of HE4 for ovarian cancer using the Meta-DiSc statistical software. There were 2607 subjects included in this meta-analysis. The sensitivity, specificity, LR+ and LR- (95% confidence interval) of HE4 was 0.800 (0.770-0.827), 0.916 (0.902-0.929), 10.271 (6.982-15.109) and 0.228 (0.181-0.287), respectively. The area under the summary receiver operating characteristic (sROC) curve of HE4 was 0.946. The index of Q* was 0.885. HE4 was found to be better than CA125 as an auxiliary indicator for the diagnosis of ovarian cancer in terms of better sensitivity, specificity, LR+ and LR-.

Ovarian tumor marker HE4 is differently expressed during the phases of the menstrual cycle in healthy young women

The objective of the present study was to investigate in healthy young women the fluctuations in serum concentration of human epididymal secretory protein human epididymis-specific protein 4 (HE4) and CA125 during the phases of the menstrual cycle and the correlation between HE4 and CA125 values and age. Forty women with regular menstrual cycles were included in the study. Pelvic and transvaginal ultrasound were performed in order to exclude ovarian pathologies. Blood samples were collected at follicular (FP), ovulatory (OP), and luteal (LP) phases of the hormonal cycle. The values of HE4 (expressed as picomoles per liter) observed were (mean ± SEM) 39.1 ± 1.1 (FP), 45.3 ± 1.19 (OP), and 42.0 ± 1.3 (LP). The difference between FP and OP was statistically significant (p = 0.0002). By contrast, serum CA125 levels (expressed as units per milliliter) were 14.35 ± 0.66 (FP), 13.15 ± 0.54 (OP), and 13.70 ± 0.54 (LP), respectively. The levels of HE4 observed in serum samples of women below 35 years were 37.5 ± 1.28 in the FP, 46.6 ± 1.4 in the OP, and 42.8 ± 1.49 in the LP. In this group, a statistically significant difference was observed in the FP compared with the OP (p < 0.0001), whereas no statistically significant difference was observed during the different hormonal phases in the group of women over 35. In conclusion, the correct interpretation of laboratory data is essential to define a threshold of normality, and for what concerns HE4 levels, the menstrual cycle phase-dependent variability appears indicated in the interpretation of the results.

HE6, a two‐subunit heptahelical receptor associated with apical membranes of efferent and epididymal duct epithelia

Human Epididymis-specific protein 6 [HE6 (GPR64)] is a highly conserved, tissue-specific seven-transmembrane receptor of the human epididymis. The rodent counterparts were cloned and 5'-inverse PCR employed to confirm that the cDNA sequences were full length. Downstream from the highly conserved signal peptide-coding sequence, the 5'-regions contained at least six mini-exons of less than 50 nucleotides. Multiple splice variants involving these mini-exons were cloned in the human, the majority of which was also found in rodents. Northern blot analysis showed that the tissue distribution of the mRNA was very similar in human and rodents. The human HE6 gene was assigned to the X chromosome in a region, which is syntenic to the mouse. The HE6 sequence predicted a two-subunit receptor of the LNB-TM7 subfamily. A membrane preparation and protein solubilization method was adopted to identify the endogenous epididymal proteins. Two sets of peptides were chosen for antibody production, assuming that protein scission had occurred within the conserved GPS-motif. Western blot analysis revealed abundant two-subunit proteins in human and rodents, comprising an approximately 180 kDa hydrophilic ectosubunit and a <40 kDa hydrophobic endosubunit. Deglycosylation experiments showed that the large ectosubunits were highly glycosylated, the carbohydrate side chains dramatically increasing the apparent molecular mass. Immunohistochemical studies revealed that both subunits were associated with apical membranes of efferent ductule and proximal epididymal duct epithelia.

Epididymal G protein-coupled receptor (GPCR): two hats and a two-piece suit tailored at the GPS motif.

G protein-coupled receptors (GPCRs) are involved in cell recognition and signaling and their function has been experimentally determined by ligand activation and site-directed mutagenesis. Structurally, GPCRs consist of an extracellular N-terminus and an intracellular C-terminus separated by seven helical transmembrane domains (TM7). The extracellular region is highly glycosylated. The intracellular region binds to G proteins. An epididymal GPCR, designated HE6 (for human epididymis-specific protein 6), is present in the stereocilia projecting from the apical domain of principal cells into the epididymal lumen. In conceptual terms, HE6 wears two hats: an unusually long extracellular region characteristic of cell adhesion proteins, and an intracellular region with binding affinity to G protein. The binding partner to the long extracellular region has not been identified. HE6 has another remarkable feature comparable to the GPCR calcium-independent receptor of alpha-latrotoxin, designated CIRL. Both HE6 and CIRL are endogenously cleaved into two pieces at the GPCR proteolytic site (GPS) located adjacent to TM1, the first of the seven transmembrane helices. One fragment of the heterodimer wears the cell adhesion hat; the other retains the typical characteristics of GPCRs. This proteolytic processing may be regarded as a mechanism of molecular compartmentalization of cell adhesion and G protein activation functions. The latter may engage a beta-arrestin-driven endocytic trafficking mechanism independent from the adhesive properties of the mucin extracellular domain. It is also conceivable that events taking place in the epididymal lumen can be surveyed by the long adhesive rod and subsequently coupled inside principal cells to a signaling cascade.

A Major Human Epididymis-Specific cDNA Encodes a Protein with Sequence Homology to Extracellular Proteinase Inhibitors1

The amino acid sequence of a major human epididymis-specific protein was deduced from the nucleotide sequence of its cloned cDNA. The encoded product showed characteristics of a secretory protein, with a signal peptide followed by a small (approximately 10-kDa), acidic (pI 4.3), and cysteine-rich polypeptide. The positions of half-cysteines suggested that it was a two-domain member of the family of 'four-disulfide core' proteins to which a number of proteinase inhibitors belong. Southern blot analyses of human genomic DNA showed that the transcripts originated from a single copy gene. Northern blot and in situ transcript hybridization specifically localized the HE4 (human epididymis gene product) mRNA to the epithelial cells of the epididymal duct, predominantly within the distal sections. A possible function in sperm maturation as indicated by amino acid similarities to extracellular proteinase inhibitors of genital tract mucous secretions is discussed in the context of its tissue-specific transcription.