NLRP1 restricts porcine deltacoronavirus infection via IL-11 inhibiting the phosphorylation of the ERK signaling pathway.

Continuously emerging highly pathogenic coronaviruses remain a major threat to human and animal health. Porcine deltacoronavirus (PDCoV) is a newly emerging enterotropic swine coronavirus that causes large-scale outbreaks of severe diarrhea disease in piglets. Unlike other porcine coronaviruses, PDCoV has a wide range of species tissue tropism, including primary human cells, which poses a significant risk of cross-species transmission. Nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 1 (NLRP1) has a key role in linking host innate immunity to microbes and the regulation of inflammatory pathways. We now report a role for NLRP1 in the control of PDCoV infection. Overexpression of NLRP1 remarkably suppressed PDCoV infection, whereas knockout of NLRP1 led to a significant increase in PDCoV replication. A mechanistic study revealed that NLRP1 suppressed PDCoV replication in cells by upregulating IL-11 expression, which in turn inhibited the phosphorylation of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor U0126 effectively hindered PDCoV replication in pigs. Together, our results demonstrated that NLRP1 exerted an anti-PDCoV effect by IL-11-mediated inhibition of the phosphorylation of the ERK signaling pathway, providing a novel antiviral signal axis of NLRP1-IL-11-ERK. This study expands our understanding of the regulatory network of NLRP1 in the host defense against virus infection and provides a new insight into the treatment of coronaviruses and the development of corresponding drugs.IMPORTANCECoronavirus, which mainly infects gastrointestinal and respiratory epithelial cells in vivo, poses a huge threat to both humans and animals. Although porcine deltacoronavirus (PDCoV) is known to primarily cause fatal diarrhea in piglets, reports detected in plasma samples from Haitian children emphasize the potential risk of animal-to-human spillover. Finding effective therapeutics against coronaviruses is crucial for controlling viral infection. Nucleotide-binding oligomerization-like receptor (NLR) family pyrin domain-containing 1 (NLRP1), a key regulatory factor in the innate immune system, is highly expressed in epithelial cells and associated with the pathogenesis of viruses. We demonstrate here that NLRP1 inhibits the infection of the intestinal coronavirus PDCoV through IL-11-mediated phosphorylation inhibition of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor can control the infection of PDCoV in pigs. Our study emphasizes the importance of NLRP1 as an immune regulatory factor and may open up new avenues for the treatment of coronavirus infection.

Activation of the NLRP1 Inflammasome and Its Role in Transmissible Gastroenteritis Coronavirus Infection.

The inflammasome pathway is a critical early response mechanism of the host that detects pathogens, initiates the production of inflammatory cytokines, and recruits effector cells to the infection site. Nonetheless, the mechanism of inflammasome activation in coronavirus infection and its biological functions in host defense remain unclear. Transmissible gastroenteritis virus (TGEV), a member of the genus Alphacoronavirus, is a significant pathogen that mainly infects piglets and causes intestinal inflammation and inflammatory cell infiltration. Here, we investigated the mechanism of inflammasome activation in intestinal epithelial cells (IECs) infected with TGEV. We observed a substantial increase in interleukin 1ß (IL-1ß) and IL-18 levels in both IECs and TGEV-infected porcine intestinal tissues. Furthermore, TGEV infection resulted in increased activation of caspase-1 and the NLRP1 (NOD-like receptor [NLR]-containing pyrin domain [PYD]) inflammasome. Our findings revealed that TGEV infection impeded the interaction between porcine NLRP1 (pNLRP1) and porcine dipeptidyl peptidases 9 (pDPP9), yet it did not reduce the expression of pDPP9. Importantly, the ZU5 domain, not the function-to-find domain (FIIND) reported in human NLRP1, was identified as the minimal domain of pNLRP1 for pDPP9 binding. In addition, the robust type I IFN expression induced by TGEV infection also upregulated pNLRP1 expression and pNLRP1 itself acts as an interferon-stimulated gene to counteract TGEV infection. Our data demonstrate that pNLRP1 has antiviral capabilities against coronavirus infection, which highlights its potential as a novel therapeutic target for coronavirus antiviral therapy. IMPORTANCE Coronavirus primarily targets the epithelial cells of the respiratory and gastrointestinal tracts, leading to damage in both humans and animals. NLRP1 is a direct sensor for RNA virus infection which is highly expressed in epithelial barrier tissues. However, until recently, the precise molecular mechanisms underlying its activation in coronavirus infection and subsequent downstream events remained unclear. In this study, we demonstrate that the alphacoronavirus TGEV induces the production of IL-1ß and IL-18 and upregulates the expression of pNLRP1. Furthermore, we found that pNLRP1 can serve as an interferon-stimulated gene (ISG) to inhibit the infection of enterovirus TGEV. Our research highlights the crucial role of NLRP1 as a regulator of innate immunity in TGEV infection and shows that it may serve as a potential therapeutic target for the treatment of coronavirus infection.

Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

Different Mechanisms Are Utilized by Coronavirus Transmissible Gastroenteritis Virus To Regulate Interferon Lambda 1 and Interferon Lambda 3 Production.

Type III interferons (IFN-λ) are shown to be preferentially produced by epithelial cells, which provide front-line protection at barrier surfaces. Transmissible gastroenteritis virus (TGEV), belonging to the genus Alphacoronavirus of the family Coronaviridae, can cause severe intestinal injuries in porcine, resulting in enormous economic losses for the swine industry, worldwide. Here, we demonstrated that although IFN-λ1 had a higher basal expression, TGEV infection induced more intense IFN-λ3 production in vitro and in vivo than did IFN-λ1. We explored the underlying mechanism of IFN-λ induction by TGEV and found a distinct regulation mechanism of IFN-λ1 and IFN-λ3. The classical RIG-I-like receptor (RLR) pathway is involved in IFN-λ3 but not IFN-λ1 production. Except for the signaling pathways mediated by RIG-I and MDA5, TGEV nsp1 induces IFN-λ1 and IFN-λ3 by activating NF-κB via the unfolded protein responses (UPR) PERK-eIF2α pathway. Furthermore, functional domain analysis indicated that the induction of IFN-λ by the TGEV nsp1 protein was located at amino acids 85 to 102 and was dependent on the phosphorylation of eIF2α and the nuclear translocation of NF-κB. Moreover, the recombinant TGEV with the altered amino acid motif of nsp1 85-102 was constructed, and the nsp1 (85-102sg) mutant virus significantly reduced the production of IFN-λ, compared with the wild strain. Compared to the antiviral activities of IFN-λ1, the administration of IFN-λ3 showed greater antiviral activity against TGEV infections in IPEC-J2 cells. In summary, our data point to the significant role of IFN-λ in the host innate antiviral responses to coronavirus infections within mucosal organs and in the distinct mechanisms of IFN-λ1 and IFN-λ3 regulation. IMPORTANCE Coronaviruses cause infectious diseases in various mammals and birds and exhibit an epithelial cell tropism in enteric and respiratory tracts. It is critical to explore how coronavirus infections modulate IFN-λ, a key innate cytokine against mucosal viral infection. Our results uncovered the different processes of IFN-λ1 and IFN-λ3 production that are involved in the classical RLR pathway and determined that TGEV nsp1 induces IFN-λ1 and IFN-λ3 production by activating NF-κB via the PERK-eIF2α pathway in UPR. These studies highlight the unique regulation of antiviral defense in the intestine during TGEV infection. We also demonstrated that IFN-λ3 induced greater antiviral activity against TGEV replication than did IFN-λ1 in IPEC-J2 cells, which is helpful in finding a novel strategy for the treatment of coronavirus infections.

Development and validation of a novel survival model for head and neck squamous cell carcinoma based on autophagy-related genes.

BACKGROUND: In view of the critical role of autophagy-related genes (ARGs) in the pathogenesis of various diseases including cancer, this study aims to identify and evaluate the potential value of ARGs in head and neck squamous cell carcinoma (HNSCC). METHODS: RNA sequencing and clinical data in The Cancer Genome Atlas (TCGA) were analyzed by univariate Cox regression analysis and Lasso Cox regression analysis model established a novel 13- autophagy related prognostic genes, which were used to build a prognostic risk model. A multivariate Cox proportional regression model and the survival analysis were used to evaluate the prognostic risk model. Moreover, the efficiency of prognostic risk model was tested by receiver operating characteristic (ROC) curve analysis based on data from TCGA database and Gene Expression Omnibus (GEO). Besides, the other independent datasets from Human Protein Atlas dataset (HPA) also applied. RESULTS: 13 ARGs (GABARAPL1, ITGA3, USP10, ST13, MAPK9, PRKN, FADD, IKBKB, ITPR1, TP73, MAP2K7, CDKN2A, and EEF2K) with prognostic value were identified in HNSCC patients. Subsequently, a prognostic risk model was established based on 13 ARGs, and significantly stratified HNSCC patients into high- and low-risk groups in terms of overall survival (OS) (HR = 0.379，95% CI: 0.289-0.495, p < 0.0001). The multivariate Cox analysis revealed that this model was an independent prognostic factor (HR = 1.506, 95% CI = 1.330-1.706, P < 0.001). The areas under the ROC curves (AUC) were significant for both the TCGA and GEO, with AUC of 0.685 and 0.928 respectively. Functional annotation revealed that model significantly enriched in many critical pathways correlated with tumorigenesis, including the p53 pathway, IL2 STAT5 signaling, TGF beta signaling, PI3K Ak mTOR signaling by gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA). In addition, we developed a nomogram shown some clinical net could be used as a reference for clinical decision-making. CONCLUSIONS: Collectively, we developed and validated a novel robust 13-gene signatures for HNSCC prognosis prediction. The 13 ARGs could serve as an independent and reliable prognostic biomarkers and therapeutic targets for the HNSCC patients.

Amide-sialoside protein conjugates as neomucin bioshields prevent influenza virus infection.

We report the preparation of multivalent amide-sialoside-decorated human serum albumin (HSA) and bovine serum albumin (BSA) as mimics of natural mucin and bioshields against influenza virus infection. Free sialic acid with an amine on C-2 was covalently attached to the protein scaffolds using di-(N-succinimidyl) adipate. Dynamic light scattering (DLS) showed that the synthetic neomucins were able to act as bioshields and aggregate the influenza virion particles. The dissociation constants (KD) of the interactions between the prepared glycoconjugates and three different viral strains were measured by isothermal titration calorimetry (ITC) indicating the multivalent presentation of sialyl ligands on the HSA and BSA backbones can dramatically enhance the adsorbent capability compared to the corresponding monomeric sialoside. Hemagglutinin inhibition (HAI) and neuraminidase inhibition (NAI) assays showed that the glycoconjugates acted as moderate HA and NA inhibitors, thus impeding viral infection. Moreover, the different binding affinities of the glycoproteins to HA and NA proteins from different influenza viruses demonstrated the importance of HA/NA balance in viral replication and evolution. These findings provide a foundation for the development of antiviral drugs and viral adsorbent materials based on mimicking the structure of mucin.

Transcriptome Landscape of Human Folliculogenesis Reveals Oocyte and Granulosa Cell Interactions.

The dynamic transcriptional regulation and interactions of human germlines and surrounding somatic cells during folliculogenesis remain unknown. Using RNA sequencing (RNA-seq) analysis of human oocytes and corresponding granulosa cells (GCs) spanning five follicular stages, we revealed unique features in transcriptional machinery, transcription factor networks, and reciprocal interactions in human oocytes and GCs that displayed developmental-stage-specific expression patterns. Notably, we identified specific gene signatures of two cell types in particular developmental stage that may reflect developmental competency and ovarian reserve. Additionally, we uncovered key pathways that may concert germline-somatic interactions and drive the transition of primordial-to-primary follicle, which represents follicle activation. Thus, our work provides key insights into the crucial features of the transcriptional regulation in the stepwise folliculogenesis and offers important clues for improving follicle recruitment in vivo and restoring fully competent oocytes in vitro.

Multivalent oleanolic acid human serum albumin conjugate as nonglycosylated neomucin for influenza virus capture and entry inhibition.

We report the synthesis of multivalent oleanolic acid (OA) protein conjugates as nonglycosylated neomucin mimic for the capture and entry inhibition of influenza viruses. Oleanolic acid derivatives bearing an amine-terminated linker were synthesized by esterification of carboxylic acid and further grafted onto the human serum albumin (HSA) via diethyl squarate method. The binding of hemagglutinin (HA) on the virion surface to the synthetic neomucin was evaluated by hemagglutination inhibition assay. The influenza virus capture ability of the PEGylated OA-HSA conjugate was further investigated by Dynamic Light Scattering (DLS), virus capture assay and Isothermal Titration Calorimeter (ITC) techniques. The pronounced agglutination of viral particles, the high capture efficiency and affinity constant indicate that this neoprotein is comparable to natural glycosylated mucin, suggesting that this material could potentially be used as anti-infective barriers to prevent virus from invading host cells. The study also rationalizes the feasibility of antiviral drug development based on OA or other antiviral small molecules conjugated protein strategies.

Multivalent S-sialoside protein conjugates block influenza hemagglutinin and neuraminidase.

A new class of S-sialoside Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) conjugates were prepared to enhance the binding affinity to hemagglutinin (HA) and neuraminidase (NA). The valency of glycoconjugates was controlled by the reaction ratio of the S-sialoside monomer and protein. Hemagglutination inhibition assay showed that these synthetic glycoproteins have higher affinity to HA than the small clusters of sialosides with lower valency, due to multivalent effect and optimized three dimensional presentation of sialosides on the protein platform. The results of fluorescent NA inhibition assay showed that some of the conjugates have moderate NA inhibitory activity, in comparison to the monomer and low valent conjugates with weak or none inhibitory activity. These synthetic sialylated proteins were not cytotoxic with concentrations up to 100 µM, since the sialylation did not change the secondary structure of protein. This new kind of conjugates can be used as lead compounds for antiviral drug design and the construction of pseudo sialoside-protein conjugates library to investigate the carbohydrate-HA/NA recognition process and a platform for the influenza virus capturing.

Quantitative proteomic analysis of mitochondria from human ovarian cancer cells and their paclitaxel-resistant sublines.

Paclitaxel resistance is a major obstacle for the treatment of ovarian cancer. The chemoresistance mechanisms are partly related to the mitochondria. Identification of the relevant proteins in mitochondria will help in clarifying the possible mechanisms and in selecting effective chemotherapy for patients with paclitaxel resistance. In the present study, mitochondria from two paclitaxel-sensitive human ovarian cancer cell lines (SKOV3 and A2780) and their corresponding resistant cell lines (SKOV3-TR and A2780-TR) were isolated. Guanidine-modified acetyl-stable isotope labeling and liquid chromatography-hybrid linear ion trap Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) were performed to find the expressed differential proteins. Comparative proteomic analysis revealed eight differentially expressed proteins in the ovarian cancer cells and their paclitaxel-resistant sublines. Among them, mimitin and 14-3-3 ζ/δ were selected for further research. The effects of mimitin and 14-3-3 ζ/δ were explored using specific siRNA interference in ovarian cancer cell lines and immunohistochemistry in human tissue specimens. The downregulation of mimitin and 14-3-3 ζ/δ using specific siRNA in paclitaxel-resistant ovarian cancer cells led to an increase in the resistance index to paclitaxel. Multivariate analyses demonstrated that lower expression levels of the mimitin and 14-3-3 ζ/δ proteins were positively associated with shorter progression-free survival (PFS) and overall survival (OS) in patients with primary ovarian cancer (mimitin: PFS: P = 0.041, OS: P = 0.003; 14-3-3 ζ/δ: PFS: P = 0.031, OS: P = 0.011). Mimitin and 14-3-3 protein ζ/δ are potential markers of paclitaxel resistance and prognostic factors in ovarian cancer.

[Relationship between endometriosis stage, characteristics of enodmetriotic lesions and severity of dysmenorrhoea].

OBJECTIVE: To explore the association between endometriosis stage, characteristics of endometriotic lesions and severity of dysmenorrhoea. METHODS: The clinical data were collected from 140 patients with laparoscopically diagnosed endometriosis between May 2013 and December 2013. They were scored by visual analogue scale (VAS) according to their preoperative dysmenorrhoea. Endometriotic lesions were recorded by their anatomical distributions. And endometriosis was staged and scored according to the score of Revised American Fertility Society (r-AFS). The relationship between dysmenorrhoea and endometriosis stage as well as endometriotic foci was analyzed. Chi-square test and Logistic regression were used for statistical analyses. RESULTS: Among them, there were 95 (67.86%) patients with dysmenorrhoea and 45(32.14%) without dysmenorrhoea. No significant inter-group difference existed in age (P > 0.05). The interval from menarche to the onset of dysmenorrhoea was (8 ± 9) years and duration of dysmenorrhoea (2.3 ± 1.5) days each month. A correlation existed between endometriosis stage and severity of dysmenorrhoea (χ² = 20.677, P < 0.05). A strong association was found between posterior cul-de-sac obliteration and severity of dysmenorrhoea (χ² = 8.471, P < 0.05). No significant difference was found for ovarian endometriomas, ovarian adhesion, superficial peritoneal lesions and deep infiltrating endometriosis in non- and minimal dysmenorrhoea groups with moderate and severe dysmenorrhoea (P > 0.05). Posterior cul-de-sac obliteration was an independent influencing factor for dysmenorrhoea. The odds ratio (OR) was 3.291 and 95% confidence interval (CI) 1.453-7.454. However, no relevance existed between ovarian endometriomas and dysmenorrhoea by Logistic analysis. CONCLUSION: The severity of dysmenorrhoea has close correlation with posterior cul-de-sac obliteration. However, there is a weak relevance with ovarian endometreaiomas.

An atresia cervix removal, lower uterine segment substitute for cervix and uterovaginal anastomosis: a case report and literature review.

PURPOSE: The management of cervical dysgenesis is still a big challenge, mostly due to the complexity of the malformation and the difficulty to conserve the patient's fertility during the surgery. The objective of this article is to report a new successful approach for treating cervical dysgenesis as well as conserving patients' fertility. MATERIALS AND METHODS: We presented a case of a 22-year-old girl with vaginal agenesis and cervical dysgenesis, who after an initial failure at reconstructive surgery was successfully treated by removing a cord-like cervix and a central muscular cylinder of her lower uterine segment and lining the channel with peritoneum as the reconstructed cervix. RESULTS: The patient has had normal menstruation without dysmenorrhea for one and a half years since the surgery. CONCLUSIONS: Several steps should be followed in managing patients with congenital cervical atresia: (1) a sufficient imaging evaluation before operation is necessary; (2) a falling-off-proof cervical catheter such as a mushroom catheter (also referred to as pezzers self-retaining catheter), and a proper vaginoplasty are key to a successful cervical canalization; (3) smooth grafted tissues such as peritoneum can be used to line the cervical canal to avoid adhesions.

[Left ovarian vein thrombosis after cesarean section: a case report].

To investigate the clinical presentation, diagnosis and therapy of the postpartum ovarian vein thrombosis. Retrospective analysis was made of one case in our hospital of postpartum ovarian vein thrombosis. Literature was reviewed to investigate the clinical presentation,diagnosis and therapy of postpartum ovarian vein thrombosis. The patient presented with fever, abdominal pain, lower back pain, and ultrasound showed pyelectasis. Her blood and urine bacterial culture was negative, and the antibiotic treatment had no significant effect, which was diagnosed by CT finally. The patient's blood routine returned to normal 3 days after anti-inflammatory and anticoagulant therapy, and thrombosis was significantly reduced. She was followed-up and her condition was stable. Postpartum ovarian vein thrombosis patients often present with high temperature with unknown causes and one side abdominal pain, and CT diagnosis is needed. Timely and effective anti-inflammatory and anticoagulant therapy can significantly improve the prognosis.

Mitochondrial comparative proteomics of human ovarian cancer cells and their platinum-resistant sublines.

Resistance to platinum-based chemotherapy is the major obstacle to successful treatment of ovarian cancer. It is evident that mitochondrial defects and the dysfunctions of oxidative phosphorylation and energy production in ovarian cancer cells were directly related to their resistance to platinum drugs. Using 2-D DIGE, we compared mitochondrial proteins from two platinum-sensitive human ovarian cancer cell lines (SKOV3 and A2780) with that of four platinum-resistant sublines (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP). Among the 236 differentially expressed spots, five mitochondrial proteins (ATP-α, PRDX3, PHB, ETF, and ALDH) that participate in the electron transport respiratory chain were identified through mass spectrometry. All of them are downregulated in one or two of the platinum-resistant cell lines. Three proteins (ATP-α, PRDX3, and PHB) were validated by using western blot and immunohistochemistry. There is a significant decrease of PHB in tumor tissues from ovarian cancer patients who were resistant to platinum-based chemotherapies. This is the first direct mitochondrial proteomic comparison between platinum-sensitive and resistant ovarian cancer cells. These studies demonstrated that 2-D DIGE-based proteomic analysis could be a powerful tool to investigate limited mitochondrial proteins, and the association of PHB expression with platinum resistance indicates that mitochondria defects may contribute to platinum resistance in ovarian cancer cells.

[Clinical analysis of the effect of cervical conization on fertility and pregnancy outcome].

OBJECTIVE: To evaluate the effect of conization for cervical intraepithelial neoplasia on the outcome of subsequent fertility and pregnancy. METHODS: A retrospective study was performed of patients who underwent conization from 1999 to 2005 in Peking Union Medical College Hospital. The nullipara patients below 40-years old without primary or secondary infertility were followed up. RESULTS: Thirty-five patients aged from 23 to 40 years, who had pregnancy desire, were included in the study. The mean age of patients was 31 years. Twenty-five women with 26 pregnancies were identified among the 35 patients, and the pregnancy rate was 74% (26/35), which was not significantly different from that of the same patients before conization (69%, P > 0.05). Among the 26 pregnancies, there occurred one ectopic pregnancy and four cases of spontaneous abortion. In the 13 delivery cases, there were one premature delivery, two cases of premature rupture of the membrane, and ten cases of cesarean section. The sample height of the cone was less than 2.0 cm in the nine delivery cases, and the mean width of the cone was over 2.5 cm. CONCLUSION: No evidence of secondary infertility caused by cervical conization was found. There was also no significant increase in the number of either premature delivery cases or low birth weight infants. The sample height of the cone might play a more important role in the pregnancy outcome than the width, which still needs to be further verified by larger studies.

[Value of hysteroscopy and laparoscopy in differential diagnosis of gestational trophoblastic neoplasia].

OBJECTIVE: To determine the efficacy of hysteroscopy and laparoscopy in differential diagnosis of pregnancy-related diseases, including gestational trophoblastic neoplasia (GTN), incomplete abortion and ectopic pregnancy. METHODS: Twenty-seven patients with a suspected diagnosis of GTN were transferred to Peking Union Medical College Hospital from September 2003 to March 2006, and underwent hysteroscopy and laparoscopy. Clinical data of patients were reviewed retrospectively. Most patients had abnormal vaginal bleeding and persistently elevated plasma beta human chorionic gonadotropin (beta-hCG) level for a median (53 +/- 37) days (range, 15 - 125 days) after evacuation. Ultrasound revealed a lesion with affluent blood flow in intrauterine, unilateral horn of uterus, or myometrium. No positive findings were revealed by computerized tomography or X-ray of the chest in all patients. Eleven patients underwent evacuation under hysteroscope, 10 patients were diagnosed and treated by laparoscopy, and 6 by hysteroscopy and laparoscopy. RESULTS: Choriocarcinoma was diagnosed in 4 patients, who achieved complete remission by chemotherapy later. The diagnosis of GTN was ruled out in the other 23 patients, including cornual pregnancy in 12, pregnancy in rudimentary horn in 1, and incomplete abortion in 10, who were cured by hysteroscopic and laparoscopic surgery and postoperative adjuvant single dose methotrexate. CONCLUSIONS: The major causes of pregnancy-related abnormal bleeding include incomplete abortion, ectopic pregnancy, and GTN. Hysteroscopy and laparoscopy are effective alternative of diagnosis for differentiation of GTN from non-GTN and can also offer therapeutic treatment.