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Introduction: Treatment with antibodies directed against programed-cell death ligand 1 (PD-L1) is a novel therapy for patients with gestational trophoblastic disease. Assessment of PD-L1 expression in tumor tissue is commonly used to identify patients who might benefit from anti-PD-L1 treatment. Multiple antibodies are available to detect PD-L1-expressing cells, and percentages of PD-L1-expressing cells in samples of patients with gestational trophoblastic disease indicated by these antibodies differ substantially. This raises the question which PD-L1 antibody best reflects PD-L1 expression to select patients for treatment., Material and Methods: Seven commercially available antibodies for PD-L1 staining (E1L3N, 73-10, 22C3, CAL10, SP142, 28-8, SP263) were validated on Chinese hamster ovarian (CHO) cells transfected with PD-L1, PD-L2, wildtype CHO cells and tonsil tissue. Next, four complete hydatidiform moles and four choriocarcinomas were stained. Samples were independently assessed by two pathologists., Results: All seven antibodies showed membranous staining in the PD-L1-transfected CHO cells. E1L3N and 22C3 scored the highest percentages of PD-L1-positive cells (70%-90% and 60%-70%, respectively). E1L3N stained the cytoplasm of non-transfected CHO cells and was excluded from analysis. The remaining six antibodies predominantly stained syncytiotrophoblast cells of both complete hydatidiform moles and choriocarcinomas. The percentage of PD-L1-stained trophoblast cells and staining intensity varied substantially per used PD-L1 antibody and between complete hydatidiform moles and choriocarcinomas. Agreement between pathologists was best with 22C3 (intraclass correlation coefficient 0.94-0.96)., Conclusions: Based on staining results of the CHO cells, gestational trophoblastic disease samples and intraclass correlation coefficient, 22C3 seems the most suitable for adequate detection of PD-L1-expressing trophoblast cells. All antibodies detected PD-L1-expressing cells in the gestational trophoblastic disease samples, though with great variability, hampering comparison of results between studies in this rare disease and emphasizing the need for uniformity in detecting PD-L1-expressing cells., (© 2022 The Authors. Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).)

Objectives: The primary aim of the study was to identify miRNAs that were differentially expressed between complete hydatidiform moles (CHMs) that turned out to be gestational trophoblastic neoplasia (GTN) [GTN moles] and CHMs that regressed spontaneously after evacuation [remission moles]. The secondary aim was to study the profiles of miRNA expressions in CHMs. Methods: A case-control study was conducted on GTN moles and remission moles. We quantitatively assessed the expression of 800 human miRNAs from molar tissues using Nanostring nCounter. Results: From a pilot study, 21 miRNAs were significantly downregulated in GTN moles compared to the remission moles. Five of them (miR-566, miR-608, miR-1226-3p, miR-548ar-3p and miR-514a-3p) were downregulated for >4 folds. MiR-608 was selected as a candidate for further analysis on 18 CHMs (9 remission moles and 9 GTN moles) due to its striking association with malignant formation. MiR-608 expression was slightly lower in GTN moles compared to the remission moles, that is, 2.22 folds change [p = 0.063]. Conclusion: We identified 21 miRNAs that were differentially expressed between GTN moles and remission moles suggesting that miRNA profiles can distinguish between the two groups. Although not reaching statistically significant, miR-608 expression was slightly lower in GTN moles compared to remission moles.

Gestational trophoblastic diseases are a group of pregnancy-related disorders, originated from trophoblast cells. They include benign and aggressive tumors, such as the invasive mole, the choriocarcinoma, the placental site trophoblastic tumor (PSTT), and the epithelioid trophoblastic tumor (ETT). These malignancies are characterized as gestational trophoblastic neoplasm (GTN), rarer, although more dangerous. The diagnosis of GTN is made in most cases by monitoring serum chorionic gonadotropin (hCG) with histological confirmation. The use of specific tissue biomarkers has been increasingly employed as a differential diagnosis, leading to more accurate results and different therapy protocols and prognosis for each GTN. The treatment is based on the International Federation of Gynecology and Obstetrics anatomical staging system and the World Health Organization prognostic score system. If an accurate diagnosis is made and the guidelines followed, the cure for choriocarcinoma and invasive mole cases can reach 98%, whereas PSTT and ETT still present mild success rates. The improved knowledge about GTN and its peculiarities allows physicians to efficiently achieve the differential diagnosis and choose the best available therapy protocol, thus increasing the overall survival of affected women. Nevertheless, obtaining epidemiological data and improving knowledge through basic and translational research are essential to answer open questions on GTN physiopathology, their causes, and cellular behavior., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Gestational trophoblastic disease (GTD) is a group of interrelated but distinct diseases and has a serious impact on the reproductive health of women. To analyze the expression of Nanog in GTD and to evaluate its potential to predict the development of gestational trophoblastic neoplasia (GTN)., Methods: The study included 41 normal first-trimester placentas matched by gestational age to 53 regressed-hydatidiform-moles (rHMs), 56 malignant-HMs (mHMs) and 17 choriocarcinomas (CCAs) and evaluated the Nanog expression by immunohistochemistry. The chi-square test, ANOVA, Fisher's exact test and logistic regression were performed to assess the Nanog expression and clinical prognostic factors in GTD., Results: Compared to normal placenta levels, the Nanog expression was increased in GTD samples ( p  < .05). In HMs, Nanog expression was positively correlated with serum β-hCG levels,uterine size and theca-lutein cysts ( p  < .05). Compared with the low-risk metastatic group (Federation of Gynecology and Obstetrics (FIGO) score ≤ 6), the high-risk metastatic group (FIGO score >7) had higher Nanog expression ( p  = .030). Moreover, logistic regression analysis showed that the positive expression of Nanog had the highest risk of developing into GTN (OR = 4.764, p  < .001)., Conclusions: Nanog is an independent predictor of clinical outcomes. It can also be a reliable predictor for GTN development from GTD.

A healthy pregnancy requires the development of maternal-fetal immune tolerance against the semi-allogeneic fetus. The interactions between the trophoblastic cells and the maternal immune cells (p.e., natural killer cells, T cells, macrophages, dendritic cells and B-cells) are important for the development of the maternal-fetal immune tolerance and the placental growth and function. These interactions are mediated by cell to cell contact and secreted molecules such as cytokines, chemokines, angiogenic factors and growth factors. The maternal immune cells are present in normal non-pregnant and pregnant endometrium and there are several lines of evidence based on immunohistochemical and RNA sequencing data that the decidual immune cells and immune-related pathways display alterations in GTD, which may have pathogenetic and clinical significance. The present review focuses on the usefulness of the immunohistochemical analysis which provides multiparametric in situ information regarding the numbers, the immunophenotypes and the immunotopographical distributions of the decidual immune cells in tissue sections from normal pregnancy and GTD. We also discuss the significance of the immunohistochemical information in order to gain insight in the putative mechanisms explaining the alterations of the decidual immune cells in GTD and the potential implications of these alterations in the pathogenesis and the clinical behavior of GTD., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Low-risk gestational trophoblastic neoplasia (GTN) is generally treated with single agent chemotherapy and methotrexate (MTX) as a first-line therapy. Vitamin A helps to increase trophoblast cell regression, as well as to decrease β-hCG levels. Vitamin A also increases the effectiveness of MTX by inducing more malignant cell death than MTX alone. Therefore, the aim of the current study was to analyze the changes in β-hCG levels in low-risk GTN patients following vitamin A administration., Methods: This study was a randomized clinical trial, which examined initial serum vitamin A and β-hCG levels in GTN patients before and after three cycles of MTX therapy. Patients were given vitamin A supplementation of 6,000 IU (1.8 mg RAEs) per day, and the changes in serum β-hCG were observed after three cycles. Patients were grouped by β-hCG levels (decreased or stagnant)., Results: A total of 32 low-risks GTN patients were divided into the intervention group (16 patients who received vitamin A supplementation) and the control group (16 patients who did not receive vitamin A supplementation). In the intervention group, the average initial β-hCG level was 170,949.3 ± 354,452.1 mIU/mL, and the average β-hCG post-cycle level was 1,611.9 ± 3,652.5 mIU/mL. In the control group, the average initial β-hCG level was 178,834.1 ± 2913844.6 mIU/mL, and the average β-hCG post-cycle level was 25,388.5 ± 58,437.7 mIU/mL., Conclusion: In patients with low-risk GTN who underwent MTX chemotherapy, the levels of β-hCG and the incidence of chemo resistance in the intervention group were lower than those in the control group. Older age may also influence the incidence of chemo resistance in GTN patients. Oral administration of 6,000 IU vitamin A could help to reduce β-hCG levels in low-risk GTN patients who receive MTX chemotherapy., .

Gestational trophoblastic diseases (GTD) are group of pregnancy-related tumors characterized by abnormal levels of 'β-hCG' with higher incidence in South-East Asia, especially India. Our laboratory has reported that wild-type BRCA1 transcriptionally regulates β-hCG in triple negative breast cancers (TNBCs). These factors culminated into analysis of BRCA1 status in GTD, which would emanate into elucidation of BRCA1- β-hCG relationship and unraveling etio-pathology of GTD. BRCA1 level in GTD is down-regulated due to the over-expression of DNMT3b and subsequent promoter hypermethylation, when compared to the normal placentae accompanied with its shift in localization. There is an inverse correlation of serum β-hCG levels with BRCA1 mRNA expression. The effects of methotrexate (MTX), which is the first-line chemotherapeutic used for GTD treatment, when analyzed in comparison with plumbagin (PB) revealed that PB alone is efficient than MTX alone or MTX-PB in combination, in showing selective cytotoxicity against GTD. Interestingly, PB increases BRCA1 levels post-treatment, altering DNMT3b levels and resultant BRCA1 promoter methylation. Also, cohort study analyzed the incidence of GTD at Sree Avittom Thirunal (SAT) Hospital, Thiruvananthapuram, which points out that 11.5% of gestational trophoblastic neoplasia (GTN) cases were referred to Regional Cancer Centre, Thiruvananthapuram, for examination of breast lumps. This has lend clues to supervene the risk of GTD patients towards BRCA1-associated diseases and unveil novel therapeutic for GTD, a plant-derived naphthoquinone, PB, already reported as selectively cytotoxic against BRCA1 defective tumors., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Problem: To investigate EMT phenotype and SALL4 expression of circulating tumour cells (CTCs) in patients with gestational trophoblastic neoplasm (GTN)., Method of Study: CanPatrol CTC detection system in combination with SALL4 RNA in situ hybridization was used to investigate the profile of CTCs in different types of gestational trophoblastic disease (GTD). Circulating CTCs were phenotyped and annotated with SALL4 expression in 41 GTD patients, including 12 HM and 29 GTN, as well as 22 pregnant volunteers., Results: A positive correlation between the number of CTC and serum β-hCG concentration was found among the GTN patients. The number of E/M-CTC was positively correlated with serum β-hCG, while M-CTC was positively correlated with prognostic score. Comparison among malignant GTD, benign GTD and healthy pregnant women revealed a significant difference in the number of total CTC, E/M-CTC, and M-CTC but not in E-CTC. ROC analysis was conducted to evaluate the performance of CTC phenotypes in distinguishing GTD patients from healthy pregnant women yielding an AUC as 0.826. Youden's index was maximal at the cutoff value of 8.5/4 mL with sensitivity and specificity at 53.66% and 100%, respectively. SALL4 expression was evaluated in GTD patients with CTC count greater than cutoff value. SALL4 high expressing CTCs (>2 signal dots) were detected in 66.67% (10/15) of malignant GTD patients but not in benign patients (0/5)., Conclusion: Differential expression of SALL4 was seen in CTCs derived from hydatidiform moles and GTN. CTC profiling may be developed as an adjunct marker to diagnose GTN., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different proportions. The N-glycan repertoire is more diverse than previously reported. Bisected and LewisX structures are potential targets for diagnostics. hCG isolated from pregnancy urine inhibits NK cell cytotoxicity in vitro at nanomolar levels and bisected type glycans have previously been implicated in the suppression of NK cell cytotoxicity, suggesting that hCG-related bisected type N-glycans may directly suppress NK cell cytotoxicity., Competing Interests: Affiliation with Mologic LTD UK I can confirm that this commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in guide for authors http://journals.plos.org/plosone/s/competing-interests).

Increasing evidence indicates the pivotal role of long noncoding RNAs in a variety of cancers, but there is limited focus on the link between long noncoding RNAs and gestational choriocarcinoma. This study aimed to examine the role of long noncoding RNA OGFRP1 in JEG-3 and JAR cells. Small interfering RNA was used to downregulate long noncoding RNA OGFRP1 level. Cell proliferation was measured by cell counting kit-8 and clone formation assays. Cell cycle and apoptosis were analyzed by flow cytometry. Cell invasion was examined by transwell assay. Protein expression was determined by Western blot. A double-effect inhibitor (BEZ235) that inhibits AKT and mTOR phosphorylation was used as a positive control. Knockdown of long noncoding RNA OGFRP1 significantly inhibited the proliferation of JEG-3 and JAR cells. Knockdown of long noncoding RNA OGFRP1 induced cell cycle arrest in G1 phase and apoptosis. On the other hand, knockdown of long noncoding RNA OGFRP1 inhibited the invasion of JEG-3 and JAR cells. Finally, knockdown of long noncoding RNA OGFRP1 resulted in the inactivation of AKT/mTOR signaling pathway. In addition, knockdown of long noncoding RNA OGFRP1 caused changes in the expression of intracellular cell cycle-related proteins and apoptosis-related proteins, including downregulation of CDK4, CDK6, Cyclin D1, Nusap1, and Bcl2 protein expression and upregulation of Bax protein expression. In conclusion, we found that downregulation of long noncoding RNA OGFRP1 inhibited cell proliferation, cell cycle progression, and invasion of JEG-3 and JAR cells and induced apoptosis through AKT/mTOR pathway. This study extends the understanding of the function of long noncoding RNA OGFRP1 in tumorigenesis, and these findings may be important for developing a potential therapeutic target for gestational choriocarcinoma therapy.

Background : A clinical risk score has been introduced into the management of persistent trophoblastic disease to allow individualized therapy. However, this risk scoring system lacks histopathologic predictors. The hypothesis is that there are prognostic histological markers that might contribute to the detection of those cases that will have persistent trophoblastic disease. Methods : Trophoblastic proliferation and apoptosis were investigated via immunohistochemical expression of Ki67 and caspase in 24 complete moles. These were divided into two groups; group A represented cases with persistent trophoblastic disease and group B represented cases with no persistent trophoblastic disease. Sections were immunostained with a monoclonal antibody for both caspase and Ki67. Results : No statistically significant difference was found between either group regarding the expression of Ki67 or caspase. Conclusion : Neither proliferation or apoptosis are reliable markers for progression of molar pregnancy.

Objective: To determine the optimal duration of human chorionic gonadotrophin (hCG) surveillance following treatment for low and high risk gestational trophoblastic neoplasia (GTN) and establish whether the current surveillance protocol that recommends life-long hCG monitoring requires revision., Methods: A population-based cohort study was undertaken using a national registry, comprising patients from both tertiary trophoblastic disease treatment units in the UK (London and Sheffield). All patients who received chemotherapy for low or high risk GTN in the UK between 1958 and 2014 in London and 1973 and 2015 in Sheffield (n = 4201) were included in the study. Patients with placental site trophoblastic tumours and epithelioid trophoblastic tumours were excluded due to their distinct clinical behavior, treatment and follow-up requirements. The risk of recurrence with time following completion of chemotherapy for low or high risk GTN was measured., Results: The overall risk of relapse in this national cohort of 4201 patients was 4.7% (198/4201) with a median time to recurrence of 117.5 days (range 9 days to 6.54 years). The greatest risk of recurrence occurred in the first year after completing treatment for either low or high risk GTN measuring 72.7% (n = 112) or 86.4% (n = 38), respectively. The subsequent recurrence risk reduced over time with none observed beyond 7 years., Conclusions: The absence of any recurrences beyond seven years following completion of chemotherapy for GTN indicates that the UK policy of life-long hCG surveillance is unnecessary. Our revised conservative protocol recommends stopping after 10 years., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: The B7 family check-point molecules are potential therapeutic targets in cancer immunotherapy. However, their expression status in human gestational trophoblastic neoplasia (GTN) remains unknown. We investigated the expression profiles of the B7 family check-point proteins PD-L1, PD-L2, B7-H3, B7-H4, VISTA and B7-H6 in GTN and their clinical significance., Methods and Results: We identified 112 patients with GTN, including 68 with choriocarcinoma, 33 with placental-site trophoblastic tumour (PSTT) and 11 with epithelioid trophoblastic tumour (ETT). Immunohistochemical staining was performed on whole-tissue GTN sections using anti-B7 family antibodies. VISTA expression was immunohistochemically analysed using microarrays of normal human tissues and of 20 common cancers. PD-L1 and B7-H3 were highly expressed in all GTN tumours, while PD-L2 was expressed in 87.5% of the samples. B7-H4 and B7-H6 were negative in 100% and 98.2% of the samples, respectively. PD-L1, B7-H3 and VISTA levels were significantly higher in choriocarcinomas and PSTTs than in ETTs. There was no association between B7 family check-point expression in tumour cells and disease stage, prognostic score or patient outcomes (complete remission versus death). VISTA protein was widely overexpressed in 98.2% of all the GTN, but its expression varied in other cancer types and was negative in normal adult and fetal tissues except placental trophoblasts and splenic lymphocytes., Conclusions: The GTN trophoblast cells show high expression of PD-L1, B7-H3 and VISTA in a manner that is independent of clinical outcomes. These proteins may be potential immunotherapeutic targets when treating GTN., (© 2019 John Wiley & Sons Ltd.)

Versican is a proteoglycan known to interact with cells to influence their ability to proliferate, differentiate, migrate, invade and assemble extracellular matrix, with all of these cell functions present during placentation. In the placenta, cytotrophoblast cells have the ability to differentiate into the syncytiotrophoblast, a mechanism that is greatly increased in gestational trophoblastic diseases (GTD). Nevertheless, the molecular signaling underlying the increased syncytiotrophoblast differentiation are still being unveiled and may result in novel therapeutic targets for GTD. Versican expression was investigated to establish its differential expression among GTD (partial moles, complete moles, invasive moles and choriocarcinoma) and the possible functional outcomes from versican gene silencing. Tissue samples had their versican expression evaluated using immunohistochemistry and RT-PCR. BeWo cells were employed for versican silencing with siRNA and the efficiency was confirmed by RT-PCR, immunofluorescence and flow cytometry. Cell death and forskolin-induced syncytialization were analyzed by a morphological analysis and human chorionic gonadotropin (hCG) production using immunofluorescence. Versican V0 and V1 isoforms were mainly expressed in the syncytiotrophoblast and they were the most expressed in benign rather than in malignant tumors. BeWo cells also expressed V0 and V1 isoforms, but only in cells undergoing syncytial fusion. After versican silencing, cell death was greatly increased, whereas spontaneous and forskolin-induced syncytialization decreased as well as hCG production. Versican is differentially expressed in GTD and is important for hydatidiform moles pathophysiology, protecting trophoblast cells from death and playing a role in their differentiation and functionality.

The immune checkpoint proteins, programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1), are crucial for maintaining fetomaternal immune tolerance and immune escape in cancers. In this study, we performed a comprehensive immunohistochemical study of PD-L1 expression in a large cohort of trophoblastic tissues and tumors. We found that normal villi and hydatidiform moles showed a heterogeneous PD-L1 staining among trophoblast (strong in syncytiotrophoblast, moderate in intermediate trophoblast, and weak/negative in cytotrophoblast). Eleven exaggerated placental sites (100%) showed variable PD-L1 staining, whereas 7 (36.8%) of 19 placental site nodules/plaques were weakly positive for PD-L1 (P < .001). All gestational choriocarcinomas (CCs; n = 63), epithelioid trophoblastic tumors (n = 12), and placental site trophoblastic tumors (n = 41) were PD-L1 positive, with most showing strong staining. However, PD-L1 expression was lower in epithelioid trophoblastic tumors compared with placental site trophoblastic tumors and CCs (P = .004). Three presumably germ cell-derived pure CCs, the CC elements in 13 mixed germ cell tumors, and 4 gastric/rectal CCs were also positive for PD-L1, with widespread staining. The background nontrophoblastic tissues, such as endometrial glands, squamous cells, and adenocarcinomas, were PD-L1 negative. Western blot analysis showed that PD-L1 was expressed in all 3 trophoblastic cell lines. We conclude that PD-L1 is a sensitive but nonspecific marker for trophoblast and related tumors. The frequent strong PD-L1 expression suggests that immune checkpoint blockade could be a promising approach in treating trophoblastic tumors that merits further investigation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Protein expression in cells are associated with oncogenesis. This study aims to explore proteomic profiles and discover potential biomarkers that can predict malignant transformation of hydatidiform mole., Methods: Retrospective analysis was done in 14 cases of remission hydatidiform mole and 14 cases of hydatidiform mole who later developed malignancy (GTN group). Molar tissues were retrieved from -70 °C frozen tissue. Subsequently, a large-scale proteomic analysis was performed to identify proteins and compare their abundance levels in the preserved molar tissues from these two groups using a dimethyl-labeling technique coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: A total of 2,153 proteins were identified from all samples. 22 and 10 proteins were significantly up-regulated and down-regulated, respectively, in the GTN group compared with the mole group. These altered proteins were found in several biological groups such as cell-cell adhesion, secreted proteins, and ribonucleoproteins. Several hormone-related proteins were among the most up-regulated proteins in the GTN group including choriogonadotropin subunit beta (β-hCG) and alpha (α-hCG), growth/differentiation factor 15, as well as both pregnancy-specific beta-1-glycoproteins 2 and 3. In contrast, protein S100-A11 and l-lactate dehydrogenase A chain, were down-regulated in molar tissue from most patients in the GTN group., Discussion: This study identified a set of differentially expressed proteins in molar tissues that could potentially be further examined as predictive biomarkers for the malignant transformation of CHMs. A molar proteome database was constructed and can be accessible online at http://sysbio.chula.ac.th/Database/GTD&#95;DB/Supplementary&#95;Data.xlsx., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Growth hormone (GH), an endocrine hormone, primarily secreted from the anterior pituitary, stimulates growth, cell reproduction, and regeneration and is a major regulator of postnatal growth. Humans have two GH genes that encode two versions of GH proteins: a pituitary version (GH-N/GH1) and a placental GH-variant (GH-V/GH2), which are expressed in the syncytiotrophoblast and extravillous trophoblast cells of the placenta. During pregnancy, GH-V replaces GH-N in the maternal circulation at mid-late gestation as the major circulating form of GH. This remarkable change in spatial and temporal GH secretion patterns is proposed to play a role in mediating maternal adaptations to pregnancy. GH-V is associated with fetal growth, and its circulating concentrations have been investigated across a range of pregnancy complications. However, progress in this area has been hindered by a lack of readily accessible and reliable assays for measurement of GH-V. This review will discuss the potential roles of GH-V in normal and pathological pregnancies and will touch on the assays used to quantify this hormone.

Objective: To define the factors associated with methotrexate (MTX) resistance in patients with low-risk gestational trophoblastic neoplasia (GTN)., Methods: A total of 63 patients with low-risk GTN according to International Federation of Gynecology and Obstetrics (FIGO) criteria were included. A total of 37 (58.7%) patients were treated with successive doses of 1 mg/kg intramuscular (IM) MTX on days 1, 3, 5, and 7, and 0.1 mg/kg IM folinic acid (FA) on days 2, 4, 6, and 8, until β-human chorionic gonadotropin (hCG) levels were normalized. After the β-hCG value dropped to the normal level, an additional cycle of MTX/FA was administered. This protocol is defined as the standard protocol. In a watchful waiting protocol, the same 8-day IM MTX/FA regimen was given only once (n = 8) or twice (n = 18) to 26 (41.3%) patients and patients in whom β-hCG values declined were subjected to follow-up and no additional cycles were administered as long as there was a decrease in β-hCG value. Clinical response and factors affecting therapeutic outcomes were analyzed retrospectively., Results: Of 63 patients, 47 (74.3%) were cured with primary MTX/FA treatment irrespective of any protocol. Of the 16 patients who were not able to be treated with primary MTX/FA, 3 were treated with single-agent actinomycin-D and 11 were treated with multi-agent chemotherapy. Univariate analysis showed that a pretreatment β-hCG level of ≥5000 IU/L was related to reduced therapeutic response (p = 0.001). The FIGO score, antecedent gestational pathology, and treatment with standard or watchful waiting protocol were not related to treatment response., Conclusions: The level of β-hCG prior to therapy is an important factor for predicting therapeutic outcomes. It should be noted that the success of the therapy decreases notably in case of high β-hCG level.

One of the major immune checkpoints responsible for immune evasion in cancer cells is the interaction between programmed cell death-1 (PD-1) and its ligand (PD-L1). As human trophoblastic cells display many of the features of malignant cells such as the ability to invade normal tissue including blood vessels and are apparently not eradicated by the host immune system, we undertook the present study to determine whether PD-L1 was upregulated in different types of trophoblastic cells during normal pregnancy and in gestational trophoblastic diseases. Immunohistochemistry using an anti-PD-L1-specific antibody demonstrated that in early and term normal placentas, PD-L1 was highly expressed in syncytiotrophoblast and to a much lower extent in intermediate trophoblastic cells located in the chorion laeve and implantation site. PD-L1 immunoreactivity was undetectable in cytotrophoblastic cells. This staining pattern in normal placenta was recapitulated in various types of gestational trophoblastic disease. PD-L1 was highly expressed by syncytiotrophoblast in complete moles and choriocarcinomas. The intermediate trophoblastic neoplasms, placental site trophoblastic tumors, and epithelioid trophoblastic tumors showed variable PD-L1 immunoreactivity but at a lower intensity than in the neoplastic syncytiotrophoblast in choriocarcinoma. In addition, we observed PD-1-positive lymphocytes located within the implantation site and in trophoblastic tumors. In summary, this study describes a novel mechanism for trophoblastic cells to create a tolerogenic feto-maternal interface by upregulating PD-L1 in syncytiotrophoblast and in intermediate trophoblast. Trophoblastic tumors may also use PD-L1 expression to evade the host immune response thereby promoting their survival.

The invasion cascade exhibited by placental trophoblasts and cancerous cells bears many similarities, and it is attributed to extracellular matrix degradation mediated by matrix metalloproteinases (MMPs). Although proper and controlled invasion by trophoblasts into the maternal uterus is an essential requirement for maintenance of normal pregnancy, any abnormality in this phenomenon results in the development of invasion-related disorders such as gestational trophoblastic diseases (GTDs) and preeclampsia. We studied the epigenetic basis of differential expression of two placental MMPs (MMP2 and MMP9) and tissue inhibitors of metalloproteinases (TIMP2 and TIMP1) during normal gestation and invasion-related disorders, i.e., preeclampsia and GTDs. Our study suggests the association of H3K9/27me3 with differential expression of these MMPs and their inhibitors, which regulate the placental invasion during normal pregnancy, whereas no role of CpG methylation was observed in the differential expression of MMPs/TIMPs. Further, development of GTDs was associated with abnormally higher expression of these MMPs and lower levels of their inhibitors, whereas the reverse trends were observed for MMPs and their TIMPs in case of preeclampsia, in association with abnormal changes in H3K9/27me3. These results suggest the involvement of higher levels of MMPs in an aggressive invasive behavior depicted by GTDs, whereas lower levels of these MMPs in shallow and poor invasive phenotype associated with preeclampsia. Thus, our study shows the significance of a proper balance regulated by histone trimethylation between differential expression of MMPs and their TIMPs for maintaining normal pregnancy and its deregulation as a contributing factor for pathogenesis of invasive disorders during pregnancy., (© 2016 Society for Reproduction and Fertility.)

Aims: GATA3 is a zinc-finger transcription factor that is important for trophoblast differentiation. GATA3 is sensitive for urothelial and breast carcinomas, but the specificity is low. The aim of this study was to investigate the expression of GATA3 in trophoblast-related tissues and neoplasia., Methods and Results: GATA3 immunohistochemistry was performed on 33 placentas, one atypical placental site nodule, 25 hydatidiform moles (HMs), and 13 gestational trophoblastic tumours (GTTs). One hundred and sixty endometrial adenocarcinomas were also stained. Western blotting was performed on trophoblastic cell lines and compared to other cancer cell lines. Immature placentas were characterized by strong, diffuse nuclear GATA3 staining. Mature placentas showed less expression with scattered positive cells in the villous cytotrophoblast. HMs showed diffuse expression in cytotrophoblast and implantation site trophoblast, and heterogeneous expression in extravillous trophoblast. All GTTs were positive for GATA3. All endometrial adenocarcinomas were GATA3-negative. Western blotting demonstrated GATA3 in choriocarcinoma, whereas the placenta, and cervical and endometrial cancer cell lines, were negative., Conclusions: All trophoblast lineages were positive for GATA3. The extent of GATA3 expression varied between immature and mature placentas, suggesting a role in trophoblast maturation. GATA3 does not distinguish normal placenta, HMs, or GTTs. Nevertheless, GATA3 may help in distinguishing trophoblastic tumors from Mullerian epithelial malignancies and a subset of tumours of unknown origin., (© 2015 John Wiley & Sons Ltd.)

Objective: To evaluate the potential effects of race on clinical characteristics, extent of disease, and response to chemotherapy in women with postmolar low-risk gestational trophoblastic neoplasia (GTN)., Methods: This non-concurrent cohort study was undertaken including patients with FIGO-defined postmolar low-risk GTN treated with comparable doses and schedules of chemotherapy at the New England Trophoblastic Disease Center (NETDC) between 1973 and 2012. Racial groups investigated included whites, African American and Asians. Information on patient characteristics and response to chemotherapy (need for second line chemotherapy, reason for changing to an alternative chemotherapy, number of cycles/regimens, need for combination chemotherapy, and time to hCG remission) was obtained., Results: Of 316 women, 274 (86.7%) were white, 19 (6%) African American, and 23 (7.3%) Asian. African Americans were significantly younger than white and Asian women (p=0.008). Disease presentation, and extent of disease, including antecedent molar histology, median time to persistence, median hCG level at persistence, rate of D&C at persistence, presence of metastatic disease, and FIGO stage and risk score were similar among races. Need for second line chemotherapy (p=0.023), and median number of regimens (p=0.035) were greater in Asian women than in other races., Conclusions: Low-risk GTN was more aggressive in Asian women, who were significantly more likely to need second line chemotherapy and a higher number of chemotherapy regimens to achieve complete remission than women of African American and Asian descent. Further studies involving racial differences related to clinical, biological and environmental characteristics are needed., (Copyright © 2015. Published by Elsevier Inc.)

Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objectives: Gestational trophoblastic disease (GTD) involves a spectrum of abnormal proliferations arising from the placental villous trophoblast. Although the incidence is low, a biomarker with short serum half-life would be a major clinical advance to monitor surgical and medical treatment reducing the socioeconomic burden of multiple control visits as well as patient's anxiety. Placental growth hormone (hGH-V) plays an important role in the regulation of normal placental growth and has shown angiogenic effects. We aimed to determine by immunohistochemistry (IHC) whether hGH-V is expressed in GTD and whether it can be detected in the patient's blood for potential monitoring of surgical or medical treatment procedures., Methods: Tissue and sera were collected from women undergoing treatment for GTD in a tertiary care university hospital. We evaluated partial and complete hydatidiform moles, invasive moles and choriocarcinoma, n=16. Trophoblast specimens were examined by a newly developed IHC set-up for hGH-V in addition to gross morphologic and histopathological examination. Serum samples were analyzed by a highly sensitive hGH-V specific immunoassay., Results: hGH-V was localized in all entities of GTD to the syncytiotrophoblast by immunohistochemistry. Serum hGH-V was detected for the first time in GTD and was present in a high percentage of all analyzed entities., Conclusions: hGH-V can be detected in all entities of GTD by IHC as well as by serum analysis and may therefore serve as a novel biomarker for the disease. Its clinical utility in diagnosis of GTD and monitoring surgical or medical treatment needs to be determined in further studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Hyperglycosylated human chorionic gonadotropin (hCG) is a variant of hCG with large oligosaccharide side chains. Although hCG is produced by syncytiotrophoblast cells, hyperglycosylated hCG marks cytotrophoblast cell. Hyperglycosylated hCG signals placental implantation., Methods: Total hCG in serum and urine is measured by the Siemens Immulite hCG pregnancy test; the result is in milli-international unit per milliliter. Hyperglycosylated hCG is determined by the B152 microtiter plate assay; the result is in nanogram per milliliter. Hyperglycosylated hCG results can be converted to milli-international unit per milliliter equivalents by multiplying by 11. The test measures proportion hyperglycosylated hCG, hyperglycosylated hCG / total hCG., Results: Proportion hyperglycosylated hCG marks cases intent on developing persistent hydatidiform mole (68% detection at 17% false detection). Proportion hyperglycosylated hCG also marks persistent hydatidiform mole (100% detection at 5.1% false detection). Proportion hyperglycosylated hCG distinguishes choriocarcinoma and gestational trophoblastic neoplasm cases, absolutely discriminating aggressive cases and minimally aggressive cases. Proportion hyperglycosylated hCG identifies quiescent gestational trophoblastic disease cases. It recognizes quiescent cases that become persistent disease (100% detection at 0% false positive)., Discussion: Proportion hyperglycosylated hCG is an invaluable test for discriminating gestational trophoblastic diseases.

Objective: To analyze the expression and clinicopathologic features of epithelial to mesenchymal transition (EMT) associated protein of cytokeratin-18 (CK-18) and vimentin in gestational trophoblastic disease (GTD)., Methods: Immunohistochemistry was used to determine theexpression of CK-18 and vimentin in normal pregnancy villis (39 cases), hydatidiform mole (HM) (33 cases) and gestational trophoblastic neoplasia (GTN) (invasive mole 27 cases and choriocarcinoma 5 cases)., Results: The GTN group had significantly lower levels of CK-18 compared with the normal pregnancy villis group and the HM group (P<0. 01). Vimentin was weakly positive in all kinds of trophocyte. There was no significance among normal pregnancy villis, HM and GTN groups (P> 0. 05). In HM, expression level of CK18 was significantly lower with the increase of the pathological malignancy (P<0. 01). And in GTN, the expression of CK-18 was significantly lower with the increase of the tumor anatomy stage (P<0. 001)., Conclusion: The expression of CK-18 decreases with the increase of tropohblast malignancy, which indicates that CK-18 may be involved in malignant transformation of trophocytes and EMT may play an important role in the malignant transformation of GTN.

Introduction: Gestational trophoblastic disease (GTD) is a heterogeneous group of pregnancy-related disorders. Hydatidiform mole (HM) is the most common type of GTD, whereas gestational choriocarcinoma is the most aggressive. Non-receptor tyrosine kinase c-Src contributes to the transformation to a malignant phenotype in various cancers. However, the role of c-Src in the pathogenesis of GTD remains largely unknown., Methods: The expression level of phosphorylated c-Src was determined by immunohistochemistry and Western blotting assay. JAR and JEG-3 cells were treated with hCG, specific c-Src inhibitor saracatinib and PP2, and PKA specific inhibitor, PKI. Cell growth rate and cell migration/invasion ability was determined by cell proliferation and transwell assays respectively., Results: c-Src was highly activated in HM tissues and choriocarcinoma cells (JAR and JEG-3). c-Src was activated by hCG in a time and concentration-dependent manner, which was abrogated by specific c-Src and PKA inhibitors. Inhibition of c-Src activity in JAR and JEG-3 cells by saracatinib leaded to a decrease in the rate of cell growth and cell migration/invasion ability. Furthermore, inhibition of c-Src phosphorylation induced cell cycle arrest and reduced expressions of cyclin A2, cyclin B1, cyclin E1, FOXD3 and NANOG. Moreover, inhibition of c-Src activity resulted in decreased p-FAK(Tyr397) phosphorylation., Discussion and Conclusion: Our findings indicate an important role of c-Src in the pathogenesis of GTD, and we propose that c-Src inhibitors are potential adjuvant chemotherapeutic drugs for the treatment of GTD., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Context: The ability of intermediate trophoblasts to invade maternal tissue during placentation depends on how well they can degrade the extracellular matrix. Invasion into the extracellular matrix requires many complex proteases. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is a novel family of secreted metalloproteinases. The ADAMTS-1, -4, -5, and -14 subtypes are known to be expressed in human placenta, but little is understood about their expression patterns., Objective: To examine the expression patterns of ADAMTS-1, -4, -5, and -14 in specific human placenta cell types during gestation and in gestational trophoblastic diseases., Design: Placental tissues were obtained from 25 pregnant women and 21 cases of gestational trophoblastic diseases (10 early complete moles, 3 placental site trophoblastic tumors, 4 invasive moles, and 4 choriocarcinomas). The expression of the 4 ADAMTS was analyzed by immunohistochemistry., Results: ADAMTS-1, -4, -5, and -14 were differentially expressed by the human placenta throughout gestation in a time-specific and cell type-specific manner, as well as in gestational trophoblastic diseases. ADAMTS-1 showed gradually strong staining intensity in gestational trophoblastic diseases according to the invasive potential but showed consistent strong intensity throughout normal placenta. ADAMTS-4 and ADAMTS-5 exhibited higher and restricted expression in first-trimester intermediate trophoblasts. They also exhibited comparably strong expression in gestational trophoblastic diseases. However, ADAMTS-14 expression remained unchanged throughout gestation., Conclusions: The restricted expression pattern of ADAMTS-4 and ADAMTS-5 and their increased expression in gestational trophoblastic diseases suggest that these 2 ADAMTS subtypes are associated with a biological phenotype of trophoblasts involved in human placentation and the development of gestational trophoblastic diseases.

Objectives: To delineate the potential role of p21-activated kinases (PAKs) in the pathogenesis of gestational trophoblastic diseases (GTD) by defining the expression pattern of PAK-1, -4 and -6 and their potential implication in estrogen receptor (ER) regulation of normal placental tissue and GTD., Methods: We evaluated immunohistochemically 10 normal first-trimester placentas (NP), 10 partial moles (PM), 15 complete moles (CM) and 3 choriocarcinomas (CCA) for PAK-1, PAK-4, PAK-6 and ER expression intensity and localization. Staining outcomes were assessed utilizing non-parametric Kruskal-Wallis one-way analysis of variance test followed by pairwise Wilcoxon Rank Sum tests. Statistical significance was determined by two-sided p-value of <0.05., Results: In NP, PAK-6 immunoreactivity was predominantly cytoplasmic. Compared to NP, PM and CM demonstrated significant increase of cytoplasmic PAK-6 in cytotrophoblast (p=0.012, p=0.033 respectively), accompanied by significantly increased nuclear immunoreactivity in cytotrophoblast (p=0.008, p=0.045 respectively) and intermediate trophoblast (p=0.003, p=0.015 respectively). PAK-4 was found significantly upregulated in both cytoplasmic and nuclear compartments of cytotrophoblast and syncytiotrophoblast in PM (p=0.004 and p=0.002 for cytotrophoblast; p=0.018 and p=0.002 for syncytiotrophoblast, respectively) and CM (p=0.001 and p=0.001 for cytotrophoblast; p=0.002 and p=0.001 for syncytiotrophoblast, respectively) when compared to NP, whereas PAK-1 expression was significantly reduced in the syncytiotrophoblast of PM (p=0.025 for cytoplasm and p=0.008 for nucleus). Nuclear expression of ER was undetectable in all stained samples., Conclusion: Our results reveal PAK-6 upregulation in GTD compared to NP. The absence of nuclear expression of ER might stem in part from the repressive effect of PAK-6 in trophoblastic tissue., (© 2013.)

The histologic features that permit the identification of complete mole (CM) and partial mole (PM) as well as hydropic abortion (HA) may be overlapping. The cyclin-dependent kinase inhibitor p57Kip2 protein (p57) has been included among the paternally imprinted genes in humans that commonly used for diagnosis of CM. P27 is one of the cell-cycle controlling molecules that may be involved in the proliferation, differentiation and oncogenesis of trophoblastic cells. The current study tried to test the diagnostic validity of several histopathological parameters together with p57 and p27 immunostaining in differentiation between different gestational trophoblastic diseases. The current study was carried out on 13 cases product of conception, 13 cases PM, 25 cases CM and 8 cases choriocarcinoma. Maximal villous diameter at 1.5 mm cut-off point was found to be the most reliable factor in discrimination between PM and product of conception followed by presence of villous cistern, p57 expression by extravillous cytotrophoblasts at 1.5% cut-off point and p27 expression by villous cytotrophoblasts at 25% cut-off point. P27 expression by stromal cells and total trophoblastic population at 7.5% cut-off point could discriminate between PM and CM. P57 and p27 are co-parallely expressed in non-molar as well as partial molar gestations, but they did not show this coordination in choriocarcinoma. This study demonstrated diagnostic values for several cut-off points for p57 and p27 in discrimination between different categories of gestational trophoblastic diseases. The co-expression of p27 and p57 by extravillous cytotrophoblasts and their positive correlation in non-molar gestations may indicate its suppressive role on the proliferation of these cells to provide them the capacity for differentiation and invasion.

A total of 297 samples of hydropic villi were classified according to DNA polymorphisms as androgenetic moles, dispermic triploids, or biparental diploids. A subset of 267 appropriate samples was included in the study. Most of the macroscopically diagnosed complete mole cases were genetically androgenetic in origin. The partial mole cases consisted of 30 androgenetic moles and 12 dispermic triploids. For the 59 cases macroscopically categorized as hydropic abortion, the genetic analysis revealed 38 androgenetic moles, seven dispermic triploids and 14 biparental diploids. These results showed that a new diagnostic method was required for the management of patients with hydropic villi. We identified the TSSC imprint gene of which expression was shown in normal and partial mole villi but was silenced in complete mole villi. Immunohistochemistry using the TSSC3 antibody demonstrated its efficacy as the differential diagnostic method. TSSC3 play an important role in the differentiation from trophoblast stem cells to progenitors and/or labyrinth trophoblast through the TSSC3/PI3K/Akt/Mash2 signaling pathway., (© 2013 The Authors. Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology.)

Abnormal trophoblast differentiation is the main cause of gestational trophoblast diseases in the case of hydatidiform moles and choriocarcinomas. Here we investigated the expression patterns of two gene products, p16 and Bcl-2, implicated in cell cycle regulation and apoptosis, respectively, using immunohistochemistry during normal placenta differentiation, hydatidiform moles (partial, complete and invasive) and post-molar choriocarcinomas. The p16 protein shows a gradual expression in cytotrophoblast of normal villous, from a p16 weak proliferative phenotype to a p16 strong invasive phenotype reaching a maximum around 17 weeks of gestation. The expression pattern in cytotrophoblast was similar in moles in contrast to the villous mesenchyme of invasive moles where p16 was strongly expressed. Bcl-2 expression was syncytiotrophoblast specific in normal placenta and moles and increased gradually during normal differentiation. The results explain the persistence of normal and molar villous fragments during their development and their dramatic invasion in the uterine arteries in case of invasive moles. In choriocarcinomas the weak Bcl-2 expression is associated with weak p16 expression indicating a great apoptotic and proliferative potentials. The results suggest that strong p16 expression in the villous mesenchyme may be responsible in part of the morbidity of the moles, and the key of cancer progression in the choriocarcinomas would be a fast cell-cycle turnover.

Aim: Considering that Ki-67 is a proliferative marker in molar pregnancies and the possible progression of these kinds of pregnancies to gestational trophoblastic neoplasia (GTN), we decided to evaluate the rate of expression of this marker in patients with uneventful hydatidiform moles and GTN. Moreover, we determined the predictive value of this factor for the progression of molar pregnancies to GTN., Methods: In two groups of patients, including 30 patients with uneventful molar pregnancies and 30 patients with GTN, an immunohistochemical staining technique using the Envision method was performed. To evaluate nuclear immunoreactivity of trophoblastic cells for Ki67 on paraffin sections obtained from molar pregnancy products, the percentage of the stained cells was used. Semi-quantitative evaluation was also performed. Data were analyzed using Mann-Whitney tests and receiver operating characteristic curve analysis., Results: The expression of Ki67 in cytotrophoblastic and syncytiotrophoblastic cells of patients with GTN was significantly more than for patients with an uneventful molar pregnancy (P<0.05). We considered a 12.5% cut-off value for Ki67 in cytotrophoblastic cells and a sensitivity of 90%, specificity 93%, positive predictive value of 93.1% and negative predictive value of 90.3% were obtained. Similarly, considering a cut-off value of 6% for Ki67 in syncytiotrophoblastic cells, results of 90% were obtained for all diagnostic indices., Conclusion: Our findings suggest that expression of the Ki67 oncogene in trophoblastic cells in patients with GTN are found far more frequently than in patients with an uneventful molar pregnancy, and demonstrate a high predictive value of progression to GTN., (© 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.)

Sialic acid immunoglobulin-like lectin (Siglec)-6 is a transmembrane receptor that binds leptin. Leptin is an obesity-associated peptide hormone overexpressed in gestational trophoblastic disease (GTD). GTD encompasses several placental abnormalities that range from benign to malignant. Among GTD, molar placentas are characterized by excess proliferation, whereas gestational trophoblastic neoplasias (GTN) have characteristically aggressive invasion. We hypothesized that in GTD, Siglec-6 expression would increase with disease severity and that Siglec-6 and leptin would promote proliferation, inhibit apoptosis and/or promote invasion. Siglec-6 expression patterns were evaluated with particular attention to the diagnostic utility of Siglec-6 in GTD (controls: normal placentas (n=32), hydropic abortus placentas (n=7), non-GTD reproductive tract cancers (n=2); GTD: partial moles (PM; n=11), complete moles (n=24), GTN (n=6)). In normal placentas, Siglec-6 expression dramatically decreased after 8 weeks gestation. Complete molar placentas had significantly higher Siglec-6 expression than controls, but expression was not significantly different from PM. In GTN, Siglec-6 expression was low. These data suggest that Siglec-6 may have diagnostic utility for distinguishing complete moles from normal and hydropic abortus placentas. Functional studies in choriocarcinoma-derived BeWO cells demonstrated a complex interplay between Siglec-6 expression and leptin exposure. In cells lacking Siglec-6, leptin treatment promoted invasion, likely through interaction with LepR leptin receptor, without affecting proliferation or apoptosis. Siglec-6 expression promoted proliferation in a leptin-dependent manner, but protected cells from apoptosis and promoted invasion in a leptin-independent manner. We propose that Siglec-6 and leptin play a role in the aberrant properties characteristic of GTD, namely excess proliferation and invasion.

Aim: To investigate the roles of nuclear factor κB p65 (NF-κBp65) and inhibitor of nuclear factor κB α (IκBα) in the development and metastasis of gestational trophoblastic neoplasia (GTN) by analyzing the expressions of NF-κBp65 and IκBα in normal early pregnancy villi and gestational trophoblastic diseases, and to reveal the relationship of NF-κBp65 and IκBα with age and clinical stage of GTN patients., Methods: The expressions of NF-κBp65 and IκBα were detected by immunohistochemistry in normal pregnancy villi (20 cases), hydatidiform moles (HM, 30 cases), invasive hydatidiform moles (IHM, 13 cases) and chorionic carcinomas (CCA, 15 cases)., Results: NF-κBp65 expression was statistically different (P<0.05) between normal pregnancy villi and IHM (P=0.013), normal pregnancy villi and CCA(P=0.018), HM and IHM(P=0.026), HM and CCA (P=0.035). Differences in IκBα expression were statistically significant between normal pregnancy villi and IHM, normal pregnancy villi and CCA, HM and IHM, HM and CCA (P<0.01). The expressions of NF-κBp65 and IκBα were correlated to clinical stage (P=0.043, 0.042, P<0.05), but not to patients' ages. Spearman correlation analysis revealed that there was a negative association between the protein expressions of NF-κBp65 and IκBα in GTN (r=-0.403, P=0.034, P<0.05)., Conclusion: Up-regulated expression of NF-κBp65 and down-regulated expression of IκBα may be related to the development, invasion and metastasis of GTN. The expressions of NF-κBp65 and IκBα are negatively correlated in gestational trophoblastic tumor tissues.

Objective: To investigate the role of PTEN and MDM2 expression in hydatidiform mole in the prediction of postmolar gestational trophoblastic neoplasia (GTN)., Study Design: A total of 145 cases of hydatidiform mole were diagnosed at the King Chulalongkorn Memorial Hospital from 1999 to 2010. Patients were classified into 2 groups: spontaneous remission and patients with postmolar GTN. Clinicopathologic features and immunohistochemical staining by PTEN and MDM2 were reviewed. The results were analyzed in correlation to the clinical characteristics and outcomes., Results: Of 145 recruited cases, 128 (88.3%) cases were complete hydatidiform mole (CHM) and 17 (11.7%) cases were partial hydatidiform mole (PHM). Postmolar GTN was clinically diagnosed in 42 cases (28.9%). The incidences of postmolar GTN following CHM and PHM were 29.7% and 23.5%, respectively. Mean age of the postmolar GTN group was significantly older than that of the remission group (31.2 vs. 27.8, p = 0.04). There was no significant difference of pathologic features between cases with remission and postmolar GTN. Immunoreactivity of PTEN and MDM2 were similarly present in trophoblastic cells of both CHM and PHM groups without significantly different patterns., Conclusion: Immunohistochemical expression of PTEN and MDM2 in hydatidiform mole cannot predict molar malignant transformation.

Introduction: We have previously defined a new syndrome "Minimally-aggressive gestational trophoblastic neoplasms" in which choriocarcinoma or persistent hydatidiform mole has a minimal growth rate and becomes chemorefractory. Previously we described a new treatment protocol, waiting for hCG rise to >3000 mIU/ml and disease becomes more advanced, then using combination chemotherapy. Initially we found this treatment successful in 8 of 8 cases, here we find this protocol appropriate in a further 16 cases. Initially we used hyperglycosylated hCG, a limited availability test, to identify this syndrome. Here we propose also using hCG doubling rate to detect this syndrome., Methods: Minimally aggressive gestational trophoblastic disease can be detected by chemotherapy resistance or low hyperglycosylated hCG, <40% of total hCG. It can also be identified by hCG doubling rate, with doubling time greater than 2 weeks., Results: Nineteen new cases were identified as having minimally aggressive gestational trophoblastic disease by hyperglycosylated hCG and by hCG doubling test. All were recommended to hold off further chemotherapy until hCG >3000mIU/ml. One case died prior to the start of the study, one case withdrew because of a lung nodule and one withdrew refusing the suggested combination chemotherapy. The remaining 16 women were all successfully treated., Discussion: A total of 8 plus 16 or 24 of 24 women were successfully treated using the proposed protocol, holding back on chemotherapy until hCG >3000mIU/ml., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objectives: This study aimed to investigate the expression of recently identified matrix metalloproteinases (MMPs), their inhibitors (TIMPs), and inducer (CD147) in a wide range of gestational trophoblastic diseases (GTD) thereby expanding our understanding of the potential role of MMPs in GTD., Methods: Paraffin sections of 10 normal first-trimester placentas (NP), 10 partial moles (PM), 10 complete moles (CM), 5 choriocarcinomas (CCA) and 5 placental site trophoblastic tumors (PSTT) were studied immunohistochemically for expression of MMP-7, MMP-14, MMP-21, MMP-28, TIMP-3, TIMP-4 and CD147. Immunolocalization of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 and TIMP-1 was performed on 5 CCA and 5 PSTTs., Results: CCA showed stronger intensity for MMP-14 and MMP-28 than PSTT (p<0.05, p<0.05). CCA and PSTT had stronger expression of MMP-21 than NP, PM and CM (p<0.05, p<0.05, p<0.01). PSTT (p<0.05, p<0.05), NP (p<0.01, p<0.01) and CM (p<0.01, p<0.05) showed stronger staining for TIMP-3 and TIMP-4 than CCA., Conclusion: Choriocarcinoma's high expression of MMPs and low expression of MMP inhibitors may contribute to its invasiveness and metastatic potential. Similarly, PSTT's lower expression of MMPs and high expression of MMP inhibitors may partly explain its lower invasiveness. Agents that inhibit MMP may prove useful in treating GTD., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To investigate the expression of low molecular mass polypeptide-2 (LMP2) and protein phosphatase 1A (PPM1A) in gestational trophoblastic disease and elucidate their predictive value in malignant transformation of hydatidiform mole., Methods: The expressions of LMP2 and PPM1A protein in 196 complete hydatidiform moles (in which 28 cases with malignant transformation), 7 invasive moles, 5 choriocarcinomas and 20 normal chorionic villus were detected with the method of EnVision immunohistochemistry. Their clinicopathologic data were retrospectively analyzed., Results: LMP2 and PPM1A protein expressed in cytotrophocytes, syncytiotrophoblast and extravillous trophoblast. The level of LMP2 expression in deteriorative hydatidiform mole was significantly higher than that in non-deteriorative hydatidiform mole or normal chorionic villus (6.79±2.38, 5.26±2.63 and 3.10±1.65, all P<0.01), while there were no difference compared with gestational trophoblastic neoplasms (6.42±2.68, P=0.113). The level of PPM1A expression was highest in normal chorionic villus, and decreased gradually in hydatidiform mole (non-deteriorative and deteriorative) and gestational trophoblastic neoplasms (6.30±2.98, 4.93±2.50, 4.43±2.04 and 3.33±2.06, all P<0.01); the level of PPM1A expression in deteriorative hydatidiform mole was significantly lower than that in non-deteriorative hydatidiform mole (P=0.001). The expression of LMP2 protein was correlated to theca lutein ovarian cyst, the expression of PPM1A protein was related with uterine size (P<0.05). While, there was no correlation between the expressions of the two proteins (P>0.05)., Conclusions: High expression of LMP2 and low expression of PPM1A might play an important role in the motility and invasiveness of trophoblast cells and malignant transformation of hydatidiform mole. Testing the expression of LMP2 and PPM1A in hydatidiform mole tissues of initial uterine evacuation might be have some reference significance in judging outcomes of hydatidiform mole.

DJ-1 is found to be important in human neurodegenerative diseases and cancers by regulating oxidative damage and apoptosis. DJ-1 is also a regulator of PTEN, a frequently mutated tumor suppressor gene in cancers. In this study, we investigated the expression of DJ-1 and PTEN in normal placentas and gestational trophoblastic disease in relation to apoptotic indices and p53 status. A total of 95 trophoblastic samples were retrieved for immunohistochemical study whereas 79 trophoblastic samples, 3 normal trophoblastic and 2 choriocarcinoma cell lines were collected for quantitative real time reverse transcription polymerase chain reaction detection. There was a significant correlation between DJ-1 and PTEN immunostaining indices in the trophoblastic samples (P=0.013). Significantly higher DJ-1 and PTEN immunoreactivity indices were found in the complete mole (P<0.01) and choricarcinoma (P<0.01) compared with the first trimester placenta. Quantitative real time reverse transcription polymerase chain reaction also detected significantly higher messenger ribonucleic acid expressions of DJ-1 and PTEN in hydatidiform moles (P<0.05) and choriocarcinomas (P<0.05) compared with the first trimester placentas. A significant negative correlation was found between DJ-1 and the apoptosis resistant gene Bcl-2 (P=0.031), whereas a positive correlation was shown between PTEN and wild-type p53 (P=0.019). Significant correlations between PTEN and embryonic stem cell transcription factors, Stat3 and Nanog, were also displayed (P=0.001, 0.015). Our findings showed, for the first time, overexpression of DJ-1 at both transcriptional and protein levels in gestational trophoblastic disease. Overexpressed DJ-1 may play a role in regulating apoptotic activities of trophoblasts in relation to PTEN and p53.

Gestational trophoblastic disease (GTD) includes hydatidiform mole (HM), which can develop persistent gestational trophoblastic neoplasia requiring chemotherapy; choriocarcinoma, which is a frankly malignant tumor; placental site trophoblastic tumor; and epithelioid trophoblastic tumor. p21-Activated kinases (PAKs) promote malignant tumor progression. Therefore, this study investigated PAK1, PAK2, and p-PAK2 Ser(20) in the pathogenesis of GTD. By real-time PCR, PAK1 mRNA was significantly higher in HMs, particularly metastatic HMs (P = 0.046) and HMs that developed persistent disease (P = 0.011), when compared with normal placentas. By immunohistochemistry, significantly increased cytoplasmic PAK1 immunoreactivity in cytotrophoblasts was also detected in HMs (P = 0.042) and choriocarcinomas (P = 0.003). In addition, HMs that developed persistent disease displayed higher PAK1 immunoreactivity than those that regressed (P = 0.016), and elevated PAK1 immunoreactivity was observed in placental site trophoblastic tumors. Indeed, there was significant positive correlation between PAK1 expression and the proliferative indices Ki-67 (P = 0.016) and MCM7 (P = 0.026). Moreover, higher PAK1 mRNA and protein expression was confirmed in the choriocarcinoma cell-lines JEG-3 and JAR; however, PAK2 mRNA and p-PAK2 immunoreactivity showed a similar expression pattern in normal first trimester placentas and GTD. Knockdown of PAK1 in JEG-3 and JAR reduced cell proliferation, migration, and invasion ability, up-regulated p16, and down-regulated vascular endothelial growth factor and MT1-MMP expression. This is the first report revealing the involvement of PAK1 in the pathogenesis and clinical progress of GTD.

Introduction: The literature shows that hyperglycosylated hCG is the invasion stimulus in malignant gestational trophoblastic diseases. The USA hCG Reference Service has characterized 2 gestational trophoblastic disease conditions marked by low proportion of hyperglycosylated hCG. These are quiescent gestational trophoblastic disease, defined as inactive or benign invasive disease, and minimally invasive gestational trophoblastic disease, defined as slow growing or chemorefractory disease with hCG increasing very slowly (doubling rate 2-6 weeks). Here we examine the USA hCG Reference Service experience with both diseases., Methods: Patient were referred to the USA hCG Reference Service, 133 cases shown to have quiescent gestational trophoblastic disease, 35 cases with aggressive and 30 with minimally invasive gestational trophoblastic disease., Results: Quiescent or inactive gestational trophoblastic disease was demonstrated in 133 women. In 127 of these cases, no hyperglycosylated hCG was detected, and in 6 cases 4-27% hyperglycosylated hCG was detected. This is quiescent or inactive disease. Only 1 of 35 cases with aggressive gestational trophoblastic disease (>40% hyperglycosylated hCG) was chemorefractory. In contrast, 30 of 30 minimally invasive cases (<40% hyperglycosylated hCG) were chemorefractory. In chemorefractory cases hyperglycosylated hCG ranged from <1% to 39% of total hCG. The USA hCG Reference Service showed that proportions hyperglycosylated hCG significantly increases as total hCG rises. They recommended in minimally invasive cases to wait to hCG was >3000 IU/L before commencing chemotherapy. This was successful in 10 of 10 minimally invasive cases., Discussion: Measurement of hyperglycosylated hCG or invasiveness is a critical step in management of invasive gestational trophoblastic disease. Quiescent of inactive gestational trophoblastic disease requires no therapy. Minimally invasive disease in chemorefractory. The USA hCG Reference Service experience suggests waiting until hCG exceeds 3000 IU/L before commencing any chemotherapy.

HtrA1 is a secreted protein which behaves as a molecular chaperone at low temperatures and as a serine protease at high temperatures. When the placenta escapes the normal growth control mechanisms, which are present during normal pregnancy, it may develop trophoblastic diseases, such as hydatidiform mole and choriocarcinoma. The aim of the study is to investigate the expression of HtrA1 in these gestational trophoblastic diseases and evaluate whether different HtrA1 expression might be associated with increasingly severe forms of disease. We used immunohistochemistry to assess the expression of HtrA1 in normal human placenta, hydatidiform mole (partial and complete) and choriocarcinoma. In addition to that we used the western blotting technique to quantify HtrA1 immunoreaction in normal human placentas. The most striking finding of our investigation is the decrease in immunostaining of this protease with increasing severity of gestational trophoblastic disease. For instance, in partial and complete moles HtrA1 is weakly expressed in the trophoblast. Moreover, absence of immunoreaction for HtrA1 is observable in the choriocarcinoma cells. In conclusion, we suggest that HtrA1 may play an important role in the pathogenesis and progression of hydatidiform moles and choriocarcinomas, and that HtrA1 may play an important role during the normal development of the placenta, as well as in trophoblastic diseases.

Nonthrombotic pulmonary embolism (NTPE) is defined as embolisation to the pulmonary circulation of different cell types (adipocytes, haematopoietic, amniotic, trophoblastic or tumour), bacteria, fungi, foreign material or gas. The purpose of this article is to describe the clinical signs, pathogenesis, diagnosis and treatment of the different NTPE subtypes. The complex and diverse pathogenesis of different subtypes of emboli is subject to continuing speculation and is certainly far more complex than "simple" mechanical obstruction after embolisation of vascular thrombi. Nonthrombotic emboli may also lead to a severe inflammatory reaction both in the systemic and pulmonary circulation, as well as in the lung. NTPE presents a formidable diagnostic challenge, as the condition often presents with very unusual and peculiar clinical signs that are frequently overlooked. They range from very dramatic acute presentations such as acute respiratory distress syndrome to signs observed late in the disease course. Pathological observations play a key role in the exact diagnosis, and sometimes carefully aspirated blood from the pulmonary artery or specific staining of cells recovered from bronchoalveolar lavage fluid may be helpful. Frequently, lung biopsies revealing severe granulomatous reaction or unfortunate post-mortem pathological investigations of pulmonary tissue are necessary to confirm the diagnosis. Here, we also aim to familiarise the reader with the atypical radiological features of NTPE. Thin-section computed tomography of the lungs showing peculiar radiographic findings, such as a feeding vessel, the so-called tree-in-bud pattern or the appearance of micronodules distributed at the termination of bronchovascular bundles, may be observed in certain forms of NTPE. Increased awareness of NTPE as an underestimated cause of acute and chronic embolism, which may result in acute and chronic pulmonary hypertension, is needed. Despite the fact that detailed descriptions of several forms of NTPE have existed for nearly 100 years, well-designed trials have never been performed to evaluate therapy in the different subsets of these patients.

Objectives: To investigate the expression of laminin receptor 1 (LR1), a non-integrin-type laminin receptor, in gestational trophoblastic diseases and normal first-trimester placenta, since it may play a role in controlling trophoblast invasion in normal and molar pregnancies., Methods: Paraffin sections from 24 gestational age controlled normal first-trimester placentas, 47 partial moles, 56 complete moles, 3 invasive moles, 4 gestational choriocarcinomas, and 1 placental-site trophoblastic tumor were studied immunohistochemically for expression of LR1., Results: In complete and partial moles, decidual cells showed significantly stronger LR1 protein staining compared to the normal placenta (p<0.01). When compared to the partial moles, weak staining in less than 33% of decidual cells was also more prominent in the normal placenta (p<0.05). Complete and partial moles, invasive moles, choriocarcinomas, and placental-site tumors did not differ from each other with respect to staining intensity. Strong immunostaining for LR1 in decidual cells, cytotrophoblasts, syncytiotrophoblasts, and extracellular matrix cells of partial and complete moles was not significantly correlated with the development of persistent postmolar gestational trophoblastic tumors., Conclusions: LR1 may be important in the pathogenesis of gestational trophoblastic diseases. The increased expression of LR1 in decidual cells of partial and complete moles may not influence the development of persistent gestational trophoblastic tumor. Since they are seen rarely, multicentric studies should be planned to study LR1 expression in invasive moles and gestational trophoblastic neoplasms.

Oct4 is a transcription factor that plays a crucial role in maintaining pluripotency of embryonic stem cells. Down-regulation of Oct4 is associated with the differentiation of trophectoderm cell lineage, from which the normal placenta derives. We investigated the methylation and expression status of Oct4 in normal placenta and gestational trophoblastic disease (GTD) as attempts to investigate the role of Oct4 in the pathogenesis of GTD. By methylation-specific PCR, we observed both methylated and unmethylated Oct4 alleles in all 25 first trimester and 10 term placentas while 33% (18/54) of hydatidiform moles, and two choriocarcinoma cell line (JEG3 and JAR), only displayed methylated Oct4 allele. By quantitative TaqMan real-time PCR, Oct4 mRNA was significantly reduced in hydatidiform moles (P=0.04), JEG3 and JAR (P=0.024) when compared with normal placentas. Oct4 methylation was significantly correlated with Oct4 mRNA expression in placenta and GTD (P=0.012). Hypermethylation in minimal promoter and exon 1 region of Oct4 were confirmed in JEG3 and JAR by bisulfite genomic sequencing. The Oct4 mRNA expression in JEG3 and JAR increased after treatment with 5-aza-2'-deoxycytidine and/or trichostatin A. Our findings suggest that Oct4 is down-regulated by hypermethylation in normal placenta and GTD and such process is important in pathogenesis of GTD.