Downregulation of decidual SKP2 is associated with human recurrent miscarriage.

BACKGROUND: Recurrent miscarriage (RM) is a very frustrating problem for both couples and clinicians. To date, the etiology of RM remains poorly understood. Decidualization plays a critical role in implantation and the maintenance of pregnancy, and its deficiency is closely correlated with RM. The F-box protein S-phase kinase associated protein 2 (SKP2) is a key component of the SCF-type E3 ubiquitin ligase complex, which is critically involved in ErbB family-induced Akt ubiquitination, aerobic glycolysis and tumorigenesis. SKP2 is pivotal for reproduction, and SKP2-deficient mice show impaired ovarian development and reduced fertility. METHODS: Here, we investigated the expression and function of SKP2 in human decidualization and its relation with RM. A total of 40 decidual samples were collected. Quantitative PCR analysis, western blot analysis and immunohistochemistry analysis were performed to analyze the differential expression of SKP2 between RM and control cells. For in vitro induction of decidualization, both HESCs (human endometrial stromal cells) cell line and primary ESCs (endometrial stromal cells) were used to analyze the effects of SKP2 on decidualization via siRNA transfection. RESULTS: Compared to normal pregnant women, the expression of SKP2 was reduced in the decidual tissues from individuals with RM. After in vitro induction of decidualization, knockdown of SKP2 apparently attenuated the decidualization of HESCs and resulted in the downregulation of HOXA10 and FOXM1, which are essential for normal human decidualization. Moreover, our experiments demonstrated that SKP2 silencing reduced the expression of its downstream target GLUT1. CONCLUSIONS: Our study indicates a functional role of SKP2 in RM: downregulation of SKP2 in RM leads to impaired decidualization and downregulation of GLUT1 and consequently predisposes individuals to RM.

Dysfunction of B-cell lymphoma 2/adenovirus E1B 19KD interacting protein 3 in decidua is involved in the pathogenesis of preeclampsia.

OBJECTIVES: Abnormal decidualization is a contributing factor for the development of preeclampsia. BCL-2/adenovirus E1B 19KD interacting protein 3 (BNIP3) has been identified as an apoptosis regulator in many tumors. Furthermore, our previous studies showed that both BNIP3 and cleaved-caspase 3 were significantly decreased in the decidual tissue of preeclampsia. Therefore, we hypothesized that BNIP3 might affect the development of preeclampsia by regulating both decidualization and apoptosis of decidual cells. METHOD: BNIP3 expression in human decidua and its function during decidualization were investigated using in-vitro cultured human endometrial stromal cells (hESCs) and primary hESCs using real-time PCR, western blotting, immunohistochemistry, siRNA techniques, and flow cytometry. RESULTS: The levels of BNIP3 mRNA and protein in the decidua of female preeclampsia patients were lower than those of women with normal pregnancy. The expression of BNIP3 was upregulated after in-vitro decidualization and knock down of BNIP3 with small interfering RNA (siRNA) significantly reduced the transcription of decidualization markers. In addition, BNIP3 knockdown upregulated p-mTOR and p-p70s6k as well as decreased apoptosis, whereas rapamycin (which is an inhibitor of mTOR) reversed apoptosis. CONCLUSION: This study indicates that BNIP3 is particularly important for decidualization and may contribute to both the occurrence and development of preeclampsia via mTOR/p70s6k/BCL-2 signaling pathways.

Impaired decidualization caused by downregulation of circadian clock gene BMAL1 contributes to human recurrent miscarriage†.

Recurrent miscarriage (RM) is characterized by two or more consecutive losses of a clinically established intrauterine pregnancy at early gestation. To date, the etiology of RM remains poorly understood. Impaired decidualization is thought to predispose women to subsequent pregnancy failure. The transcriptional factor brain and muscle aryl hydrocarbon receptor nuclear translocator-like (BMAL1) controls circadian rhythms and regulates a very large diversity of physiological processes. BMAL1 is essential for fertility. Here, we investigated the expression and function of BMAL1 in human decidualization and its relation with RM. A total of 39 decidua samples were collected. We also examined human endometrial stromal cells (HESCs) and primary endometrial stromal cells (ESCs), and primary decidual stromal cells (DSCs) isolated from decidua of first-trimester pregnancies. Compared to normal pregnant women, the expression of BMAL1 was reduced in the decidual tissues from individuals with RM. After in vitro induction of decidualization, the transcription of BMAL1 in both HESCs and primary ESCs was increased. This is in line with the relatively higher expression of BMAL1 in DSCs than in ESCs. Silencing of BMAL1 resulted in impaired decidualization. Moreover, levels of tissue inhibitors of metalloproteinases (TIMPs) increased significantly upon decidualization. Further experiments demonstrated that BMAL1 silencing curtails the ability of DSCs to restrict excessive trophoblast invasion via downregulation of TIMP3. Our study demonstrates a functional role for BMAL1 during decidualization: the downregulation of BMAL1 in RM leads to impaired decidualization and aberrant trophoblast invasion by regulating TIMP3 and consequently predisposing individuals for RM.

The Attenuation of Trophoblast Invasion Caused by the Downregulation of EZH2 Is Involved in the Pathogenesis of Human Recurrent Miscarriage.

Recurrent miscarriage (RM) is currently defined as two or more losses of a clinically established intrauterine pregnancy. Despite years of research, RM continues to be a clinically frustrating challenge for patients and physicians, and its etiology remains poorly understood. Accumulating evidence has suggested that epigenetic modifications are involved in early embryogenesis, and defects in epigenetic patterning contribute to the development of RM. Here, we studied the role of enhancer of zeste homolog 2 (EZH2) in the pathogenesis of RM and found that the EZH2 expression was significantly decreased in the villi from women with RM compared with that in control villi. EZH2 promoted the invasion of trophoblast cells. Moreover, EZH2 could promote epithelial-mesenchymal transition by epigenetically silencing CDX1. Both chromatin immunoprecipitation (ChIP)-PCR and dual-luciferase report assays demonstrated that EZH2 repressed CDX1 transcription via direct binding to its promoter region and then trimethylating Histone3-Lysine27. Furthermore, we discovered that progesterone, which is used extensively in the treatment of miscarriage and RM, increased the expression of EZH2 via the extracellular signaling-regulated kinase (ERK1/2) pathway. These findings revealed that EZH2 may regulate trophoblast invasion as an epigenetic factor, suggesting that EZH2 might be a potential therapeutic target for RM.

Dysfunction of pseudogene PGK1P2 is involved in preeclampsia by acting as a competing endogenous RNA of PGK1.

OBJECTIVES: Normal decidualization is essential for normal pregnancy and abnormal decidualization is thought to cause preeclampsia (PE). Phosphoglycerate kinase 1 (PGK1) is an enzyme involved in the glycolytic pathway which is the main metabolism process decidual cells exhibit. Phosphoglycerate kinase 1, pseudogene 2 (PGK1P2), which is also a long non-coding RNA (lncRNA), has a high sequence similarity to PGK1 and therefore acquires an ability for sequence-specific regulation. METHODOLOGY: The expression of PGK1 and PGK1P2 in human decidua, as well as their relationship and functions during decidualization was investigated using in vitro cultured human endometrial stromal cell lines (hESCs) and primary ESCs by real-time PCR, immunohistochemistry, western blotting, siRNA techniques and miRNA inhibitor or mimic transfection. RESULTS: The levels of PGK1 and PGK1P2 mRNA and PGK1 protein in severe preeclamptic decidua were lower than those in normal pregnant controls. PGK1 and PGK1P2 mRNAs were both induced after in vitro decidualization and their deficiency caused impaired decidualization in turn. We also found PGK1P2 acted as a competing endogenous RNA (ceRNA) to regulate PGK1 expression through miR-330-5p. CONCLUSIONS: We proved that PGK1 and PGK1P2 are a pair of ceRNAs against miR-330-5p and they play a vital role in human decidualization by regulating angiogenesis and glycolysis metabolism. The deficiency of PGK1 and PGK1P2 in the decidua jeopardizes the decidualization process and subsequently might lead to the occurrence of PE. These findings may help in promoting novel predictive, diagnostic and prognostic biomarkers of PE in future.

Dysregulated Pseudogene HK2P1 May Contribute to Preeclampsia as a Competing Endogenous RNA for Hexokinase 2 by Impairing Decidualization.

Preeclampsia is a pregnancy-specific hypertensive disorder, which seriously undermines the health of maternity and fetus. However, its cause and pathogenesis remain elusive. Flawed decidualization is considered to be related to preeclampsia. Increasing evidence indicates that long noncoding RNAs are correlated with a variety of diseases, including preeclampsia. In this study, we verified the expression of long noncoding RNA HK2P1 (hexokinase 2 pseudogene 1) and its cognate gene HK2 (hexokinase 2), which were found by our previous RNA-sequencing analysis in the decidua of severe preeclampsia patients and matched control subjects. Besides that, we also investigated the function and the mechanism of HK2P1 and HK2 during decidualization. HK2 is the crucial enzyme involved in glycolysis. The HK2P1 and HK2 genes are homologous to each other. The results demonstrated that HK2P1, like HK2, stimulated the glucose uptake and lactate production of human endometrial stromal cells. In addition, HK2P1 and HK2 are indispensable for endometrial decidualization. Downregulated HK2P1 or HK2 inhibited human endometrial stromal cells proliferation and differentiation. Furthermore, there was a significant positive correlation between the expression of HK2P1 and HK2, and HK2P1 regulated the HK2 expression via competition for the shared miR-6887-3p. Taken together, our results indicated that the reduced expression of HK2P1 and HK2 may have contributed to the occurrence and development of preeclampsia by suppressing glycolysis and impairing decidualization. Our study would be helpful to understand the pathogenesis and the regulatory network of preeclampsia.

Downregulation of decidual SP1 and P300 is associated with severe preeclampsia.

Preeclampsia (PE) is a pregnancy-induced disorder characterized by hypertension and proteinuria after 20 weeks of gestation, affecting 5-7% of pregnancies worldwide. So far, the etiology of PE remains poorly understood. Abnormal decidualization is thought to contribute to the development of PE. SP1 belongs to the Sp/KLF superfamily and can recruit P300 to regulate the transcription of several genes. SP1 is also very important for decidualization as it enhances the expression of tissue factor. In this study, we investigated the expression of SP1 and P300 in deciduae and their relationship with PE. A total of 42 decidua samples were collected, of which 21 were from normal pregnant (NP) and 21 from severe PE. SP1 and P300 expression in deciduae and the levels of SP1 and P300 in cultured human endometrial stromal cells (hESCs) and primary hESCs during decidualization were determined. To further investigate the role of SP1 and P300 in human decidualization, RNA interference was used to silence SP1 and P300 in hESCs and primary hESCs. The following results were obtained. We found that the expressions of SP1 and P300 were reduced in decidual tissues with PE compared to those from NP. In the in vitro model of induction of decidualization, we found an increase in both SP1 and P300 levels. Silencing of SP1 and P300 resulted in abnormal decidualization and a significant reduction of decidualization markers such as insulin-like growth factor-binding protein1 and prolactin. Furthermore, the expression of vascular endothelial growth factor was also decreased upon SP1 and P300 silencing. Similar results were observed in primary hESCs. Our results suggest that SP1 and P300 play an important role during decidualization. Dysfunction of SP1 and P300 leads to impaired decidualization and might contribute to PE.

Diagnosis and treatment of retroperitoneal ectopic pregnancy: review of the literature.

Retroperitoneal ectopic pregnancies (REP) are extremely rare, and early diagnosis and treatment is very difficult. We completed an English literature search in MEDLINE through PubMed for articles on REP. We identified 14 articles (all case reports) but selected only 12 because of unavailable data in the other 2 articles. We also report the case of an REP which was misdiagnosed as ectopic choriocarcinoma. The 33-year-old woman was admitted via the outpatient department with a history of 54 days of amenorrhea and persistent elevated serum ß-human chorionic gonadotropin (hCG) levels. The presumed diagnosis ectopic choriocarcinoma was made based on imaging findings. Single-drug chemotherapy with methotrexate (MTX; 20-mg intramuscular injection daily for 5 consecutive days) was administered. An upper abdominal mass was noticed by the patient and laparotomy was performed. A retroperitoneal pregnant lesion was found and removed successfully. The diagnosis, treatment and mechanisms of REP are discussed. We believe REP should be considered in patients with elevated serum ß-hCG levels when the uterus and adnexa appear to be normal. Systemic administration of MTX in nonruptured REP before operation may prove to be helpful.

CD82 gene suppression in endometrial stromal cells leads to increase of the cell invasiveness in the endometriotic milieu.

Tetraspanin CD82 is a wide-spectrum tumor metastasis suppressor that inhibits motility and invasiveness of cancer cells. Endometriosis is a benign gynecological disorder, but appears malignant behaviors including invasion, ectopic implantation and recurrence. This study is to elucidate the role of CD82 expression regulation in the pathogenesis of endometriosis. The short interfering RNA silence was established to analyze the roles of CD82, chemokine CCL2, and its receptor CCR2 in the invasiveness of endometrial stromal cells (ESCs). We have found that the mRNA and protein levels of CD82 in the primary normal ESCs from endometrium without endometriosis are significantly higher than that of the primary ESCs from eutopic endometrium and ectopic tissue. CD82 inhibits the invasiveness of ESCs by downregulating CCL2 secretion and CCR2 expression via mitogen-activated protein kinase (MAPK) and integrinß1 signal pathway, and in turn upregulating the expression of TIMP1 and TIMP2 in an autocrine manner. The combination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with 17ß-estradiol can promote the invasion of ESCs via suppressing CD82 expression and stimulating CCL2 secretion and CCR2 expression, and the enhanced interaction of CCL2-CCR2 recruits more macrophages into the ectopic milieu in a paracrine manner, which further downregulates CD82 expression in the ectopic ESCs. Our study has demonstrated for the first time that the abnormal lower CD82 expression in ESCs induced by TCDD and estrogen may be an important molecular basis of endometriosis pathogenesis through enhancing the CCL2 secretion and CCR2 expression and the invasion of ESCs via MAPK and integrinß1 signal pathway.