Kidney-tonifying and Blood-activating Prescription Regulates Wnt/β-catenin Signaling Pathway to Improve Uterine Spiral Artery Remodeling in Mouse Model of Recurrent Miscarriage / 中国实验方剂学杂志

ObjectiveTo observe the effects of the kidney-tonifying and blood-activating prescription on the Wnt/β-catenin signaling pathway and uterine spiral artery remodeling in a mouse model of recurrent miscarriage and to explore its underlying mechanism. MethodA mouse model of normal pregnancy was established by mating CBA/J mice with BALB/c mice. A mouse model of recurrent miscarriage was established by mating CBA/J mice with DBA/2 mice. The modeled mice of recurrent miscarriage were randomized into model， dydrogesterone， and low- and high-dose Chinese medicine groups. The mice in normal pregnancy were used as the control group. Each group consisted of 10 mice， and the drug administration lasted for 14 days. After the treatment， the embryo absorption rate of each group was recorded. Hematoxylin-eosin （HE） staining was employed to observe the pathological morphology of the uterine decidua， and the physiological transformation rate of spiral arteries （SPA） was evaluated. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to determine the mRNA and protein levels， respectively， of matrix metalloproteinases （MMP）-2， MMP-9， vascular endothelial growth factor （VEGF）， and Wnt/β-catenin signaling pathway. ResultCompared with the control group， the model group presented increased embryo absorption rate （P<0.05）， decreased physiological transformation rate of uterine SPA （P<0.05）， cellular swelling， degeneration， and disordered arrangement in the uterine decidua tissue， and down-regulated mRNA and protein levels of key factors involved in SPA remodeling （MMP-2， MMP-9， VEGF） and the Wnt/β-catenin signaling pathway （Wnt2， β-catenin， Cyclin D1， c-Myc） （P<0.05）. Compared with the model group， both the low- and high-dose Chinese medicine reduced embryo absorption rate （P<0.05）， increased SPA physiological transformation rate （P<0.05）， improved uterine decidua tissue morphology， and increased decidua vessel count. Furthermore， they up-regulated the mRNA and protein levels of MMP-2， MMP-9， VEGF， and proteins in the Wnt/β-catenin signaling pathway （P<0.05）. ConclusionRecurrent miscarriage is associated with impaired uterine spiral artery remodeling. The kidney-tonifying and blood-activating prescription can promote uterine spiral artery remodeling by activating the Wnt/β-catenin signaling pathway and promoting the expression of VEGF， MMP-2， and MMP-9， thus treating recurrent miscarriage.

Risk Factors Related to Gestational Trophoblastic Disease

Background: Gestational trophoblastic diseases (GTD) consist of a group of neoplastic disorders arising from placental trophoblastic tissue after normal or abnormal fertilization. The WHO classification of GTD includes hydatidiform mole, invasive mole, choriocarcinoma, placental site trophoblastic tumor, and miscellaneous and unclassified trophoblastic lesions. This study aimed to analyze the risk factors related to the gestational trophoblastic disease. Material & Methods: This prospective study was conducted at the Department of Obstetrics & Gynecology in Uttara Adhunik Medical College & Hospital, Dhaka, Bangladesh for 1 year; from April 2020 to March 2021. A total of 100 subjects were included in this study. Informed written consent was taken from the study subjects. Data was collected using a pre-formed data sheet. Data processing and analysis were done by using SPSS version 17. The test statistics used to analyze the data were descriptive statistics, the McNemar Chi-square test, and Repeated Measure ANOVA statistics. All patients underwent necessary investigations. All information was kept confidential and used only for this study purpose. The ethical Clearance Certificate was obtained from Bangladesh Medical College. Results: The majority of the patients were more than of 38 years age (53, 53.0%). Out of these patients, 50 (50.0%) were para one, while 40 (40.0%) were para more than four, most of the patients (63, 63.9%) were illiterate and 5 (5.0%) were graduates, most of the subjects (73, 73.0%) belonged to the low socioeconomic group. The most common presenting symptom was bleeding per vagina (35, 35.0%) followed by pain in the lower abdomen (24, 24.0%), the passage of moles (16, 16.0%), hyperemesis gravidarum (14, 14.0%) and dyspnea in 11 (11.0%) subjects. Conclusion: The disease was common in extremes of ages, low para, and grand multiparous women. The hydatidiform mole was the commonest type of trophoblastic disease in these patients. The most common presenting complaint was bleeding per vagina followed by pain in the lower abdomen. The hydatidiform mole was diagnosed in 65 (65.0%) patients, the invasive mole in 28 subjects (28.0%), and choriocarcinoma in 7 (7.0%) patients. No patient had a placental site trophoblastic tumor.

Role of ROS/TXNIP/NLRP3 signaling pathway in high glucose-induced pyroptosis of human embryonic trophoblast cells / 中华内分泌外科杂志

Objective:To investigate the changes of ROS/TXNIP/NLRP3 signaling pathway in pyroptosis of human embryonic trophoblast cells induced by high glucose.Methods:Human embryonic trophoblast cells were cultured in vitro to establish high glucose injury model, and they were randomly divided into control group, high glucose (HG) group and HG + ROS inhibitor N-acetyl-L-cysteine (HG + NAC) group. MTT assay was used to detect the cell survival rate. The level of ROS in each group was detected by dihydroethidine ROS fluorescence probe. Expression of TXNIP and NLRP3 mRNA was detected by real-time quantitative PCR (RT-qPCR). Western blot analysis was used to detect the expression levels of TXNIP, NLRP3, Caspase-1, interleukin (IL) -1β, tumor necrosis factor-α (TNF-α) and GSDMD proteins. In addition, pyroptosis was detected by flow cytometry.Results:The optimal glucose concentration for high glucose-induced injury of human embryonic trophoblast cells was 30 mmol/L. Compared with the control group (96.27±3.10) %, the survival rate of human embryonic trophoblast cells in HG group (55.44±2.15) % was significantly lower ( P<0.05), while the fluorescence intensity (ROS level) of 7 'dichlorofluorescein (DCF), the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly higher ( P<0.05) ; Compared with HG group, the survival rate of human embryonic trophoblast cells in HG+NAC group (84.75±2.33) % was significantly higher ( P<0.05), the fluorescence intensity (ROS level) of DCF, the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, and expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly lower ( P<0.05) . Conclusion:Inhibition of ROS level in human embryonic trophoblast cells induced by high glucose may promote cell proliferation and reduce the occurrence of pyroptosis by inhibiting TXNIP/NLRP3 signaling pathway.

Exaggerated placental site gestational trophoblastic disease: A case report / Philippine Journal of Obstetrics and Gynecology

@#Gestational trophoblastic diseases (GTDs) represent a unique group of lesions with an abnormal proliferation of trophoblasts. GTD can be divided into molar lesions and nonmolar lesions. Partial and complete hydatidiform moles and invasive moles are under molar lesions, whereas non‑molar lesions include choriocarcinomas and lesions that are derived from intermediate trophoblasts (ITs). These IT can be from the implantation site (exaggerated placental site [EPS] and placental site trophoblastic tumor) or from the chorionic type (placental site nodule and epithelioid trophoblastic tumor). EPS is a relatively uncommon form of GTD. It is a challenging condition for clinicians to diagnose because of the limited number of reported cases. From 1990 to April 2022, there were only 25 case reports published internationally, and this is the first local case report. Implantation site ITs (ISITs) are difficult to distinguish histologically. Immunohistochemical staining such as Ki‑67 can improve diagnostic accuracy by differentiating ISIT. Ki 67 will show staining of <1% in EPS. This is the case of a 25‑year‑old patient, G6P5 (5005), who experienced vaginal bleeding associated with pelvic and hypogastric pain after 13 weeks of missed menses. She was diagnosed with a molar pregnancy and underwent an emergency total abdominal hysterectomy with bilateral salpingectomy due to severe uterine bleeding. Histopathologic studies in this case showed diffuse and infiltrative growth of atypical monomorphic ITs arranged in sheets and cords, infiltrating and separating myometrial fibers. The uterine blood vessel wall was replaced with fibrinoid deposition, with areas of hemorrhages and necrosis. There were also chorionic villi. The histopathological findings revealed GTD arising from ITs, specifically EPS. This article describes the clinical presentation, diagnostic procedure, and management, together with histopathological observations and a review of related literature, of this rare GTD.

Inhibition of LAMP3 mediates the protective effect of vitamin D against hypoxia/reoxygenation in trophoblast cells

Inadequate invasion and excessive apoptosis of trophoblast cells are associated with the development of preeclampsia. Vitamin D deficiency in pregnant women may lead to an increased risk of preeclampsia. However, the underlying mechanisms by which vitamin D is effective in preventing preeclampsia are not fully understood. The objectives of this study were to investigate the role of lysosome-associated membrane glycoprotein 3 (LAMP3) in the pathogenesis of preeclampsia and to evaluate whether vitamin D supplementation would protect against the development of preeclampsia by regulating LAMP3 expression. Firstly, the mRNA and protein levels of LAMP3 were significantly upregulated in the placentas of preeclampsia patients compared to normal placentas, especially in trophoblast cells (a key component of the human placenta). In the hypoxia/reoxygenation (H/R)-exposed HTR-8/Svneo trophoblast cells, LAMP3 expression was also upregulated. H/R exposure repressed cell viability and invasion and increased apoptosis of trophoblast cells. siRNA-mediated knockdown of LAMP3 increased cell viability and invasion and suppressed apoptosis of H/R-exposed trophoblast cells. We further found that 1,25(OH)2D3 (the hormonally active form of vitamin D) treatment reduced LAMP3 expression in H/R exposed trophoblast cells. In addition, 1,25(OH)2D3 treatment promoted cell viability and invasion and inhibited apoptosis of H/R-exposed trophoblast cells. Notably, overexpression of LAMP3 abrogated the protective effect of 1,25(OH)2D3 on H/R-exposed trophoblast cells. Collectively, we demonstrated trophoblast cytoprotection by vitamin D, a process mediated via LAMP3.

Diagnóstico y tratamiento quirúrgico de una paciente con una neoplasia trofoblástica gestacional / Diagnosis and surgical treatment of a patient with gestational trophoblastic neoplasia

Resumen ANTECEDENTE: La neoplasia trofoblástica gestacional forma parte del grupo de afecciones derivadas de la proliferación anómala del trofoblasto con capacidad para invasión y metástasis. CASO CLÍNICO: Paciente de 42 años, asintomática, con sospecha ecográfica de mola hidatiforme. El legrado uterino y el estudio anatomopatológico confirmaron el diagnóstico de mola hidatiforme completa. Con la cuantificación consecutiva de tres elevaciones de la β-HCG se diagnosticó: neoplasia trofoblástica gestacional. Se estadificó en estadio I, bajo riesgo y ante el deseo genésico satisfecho la paciente aceptó la histerectomía más salpingectomía bilateral. En el seguimiento posterior la paciente se encontró asintomática, con determinaciones seriadas de b-HCG negativa y ecografías vaginales sin hallazgos. CONCLUSIÓN: La histerectomía con salpingectomía bilateral puede ser el tratamiento definitivo en casos seleccionados de neoplasia trofoblástica. La evidencia disponible es escasa, por lo que es necesario seguir investigando en este campo.

Abstract BACKGROUND: Gestational trophoblastic neoplasia is one of a group of conditions resulting from abnormal trophoblast proliferation with capacity for invasion and metastasis. CLINICAL CASE: 42-year-old asymptomatic patient with ultrasound suspicion of hydatidiform mole. Uterine curettage and anatomopathological study confirmed the diagnosis of complete hydatidiform mole. With the consecutive quantification of three elevations of β-HCG a diagnosis of gestational trophoblastic neoplasia was made. It was staged as stage I, low-risk, and the patient agreed to hysterectomy plus bilateral salpingectomy. At subsequent follow-up the patient was found to be asymptomatic, with negative serial determinations of β-HCG and vaginal ultrasound scans without findings. CONCLUSION: Hysterectomy with bilateral salpingectomy may be the definitive treatment in selected cases of trophoblastic neoplasia. The available evidence is scarce and further research is needed in this field.

Enzyme-linked immunosorbent assays for quantification of MMMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera / 药物分析学报

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosor-bent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were suc-cessfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation be-tween serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias％ranged from-12.2％to-5.2％,precision ranged from-12.4％to-1.4％,and the relative standard deviation(RSD)was less than 6.6％and 8.7％,respectively.The total error was less than 20.4％.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

Role of miR-145 and PTEN/AKT/mTOR pathway in rat abortion and damage of trophoblast cells induced by arsenic exposure / 环境与职业医学

Background Exposure to arsenic can damage trophoblast cells and thus induce abortion, but the mechanism is not known. Objective To investigate the role of miR-145 and PTEN/AKT/mTOR pathway in arsenic-induced abortion and trophoblast cell damage in rats. Methods In the animal experiment, twenty SD pregnant rats were randomly divided into a normal control group (saline gavage) and an arsenic-induced abortion group (10.65 mg·kg−1 sodium arsenite solution was administered by gavage, and the gavage volume was 10 mL·kg−1), with 10 rats in each group. After the miscarriage occurred in the arsenic-induced abortion group (5-6 d after exposure), placental tissues were collected from the two groups. The mRNA expression levels of microRNA-145 (miR-145), phosphatase and tensin homologue (PTEN), kinase B (AKT), mammalian target of rapamycin (mTOR) were detected by real-time quantitative PCR (RT-PCR), and the protein expression levels of PTEN, AKT, mTOR, p-AKT, and p-mTOR were detected by Western blotting. For the in vitro study with immortalized human trophoblast cell line (HTR-8/SVneo cells), a control group, an arsenic exposure group, an miR-145 overexpression group, and an arsenic exposure+miR-145 overexpression group were prepared and cultured for 72 h with 37 °C and 5% CO2, at cell density of 5×105 cells per well, and the arsenic exposure concentration was 20 μmol·L−1. The MTT method was applied to detect cell viability, crystal violet staining to detect the number of monoclonal formation, flow cytometry to detect the level of apoptosis, Image J Angiogenesis Analyzer 1.8.0 plug-in to evaluate total blood vessel length and total blood vessel number; the detection indexes and methods of genes and proteins were the same as "animal experiment". Results (1) In the animal experiment, compared with the normal control group, the expression level of miR-145 mRNA in the placenta tissues of the arsenic-induced abortion group was increased (P<0.05), and the expression levels of PTEN, AKT, mTOR mRNA and proteins, and p-AKT and p-mTOR proteins were decreased (P<0.05). (2) For the in vitro study, compared with the control group, the cell viability rate, number of monoclonal formation, total vessel length, and total vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the arsenic exposure group and the miR-145 overexpression group, the cell viability rate, number of monoclonal formation, total vessel length, and vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the control group, the levels of miR-145 mRNA in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group increased (P<0.05), the expression levels of PTEN, AKT, mTOR mRNA and protein and the expression levels of p-AKT and p-mTOR protein were decreased (P<0.05); compared with the arsenic exposure group and the miR-145 overexpression group, the level of miR-145 mRNA in the arsenic exposure+miR-145 overexpression group was increased (P<0.05), and the levels of PTEN, AKT, mTOR mRNA and protein as well as p-AKT and p-mTOR protein were decreased (P<0.05). Conclusion miR-145 might be related to abortion due to arsenic exposure. miR-145 could inhibit the proliferation and angiogenesis of trophoblast HTR-8/SVNEO cells, and promotes their apoptosis; the mechanism may be related to the inhibition of PTEN/AKT/mTOR pathway.

Comparative analysis of trophoblasts and angiogenesis in human placental compartments / Análisis comparativo de trofoblastos y angiogénesis en compartimentos placentarios humanos

SUMMARY: Trophoblasts perform different functions depending on their location. This study aimed to obtain structural clues about the functions of villous and extravillous trophoblasts by using light and electron microscopy. Term placenta samples were obtained from 10 healthy pregnant women following cesarean sections. Frozen sections were stained with hematoxylin-eosin, semi- thin sections were stained with toluidine blue and examined with a light microscope, while thin sections were contrasted using uranyl acetate-lead citrate and evaluated under an electron microscope. Fine structural features of villous trophoblasts overlapped some villous stromal cells. In addition to the usual appearance of mature capillaries in villous stroma, we demonstrated and reported maturational stages of angiogenetic sprouts in term placenta. Extravillous trophoblasts were classified according to their location: fibrinoid, chorion, trophoblastic, column, maternal vascular endothelium, or decidua. All of these trophoblasts shared some ultrastructural features but also were distinct from each other. In decidua, it was noted that the endothelial lining of some vessels was invaded by a few endovascular trophoblasts with irregular microvilli. These cells shared some ultrastructural properties with both villous trophoblasts and stromal cells. Examination showed that angiogenesis was still present in term placentas and that trophoblasts, endothelial and stromal cells have very similar properties ultrastructurally, suggesting they represent transformational forms.

RESUMEN: Los trofoblastos dependiendo de su ubicación realizan diferentes funciones. Este estudio tuvo como objetivo obtener pistas estructurales sobre las funciones de los trofoblastos vellosos y extravellosos mediante el uso de microscopía óptica y electrónica. Se obtuvieron muestras de placenta a término de 10 mujeres embarazadas sanas después de cesáreas. Las secciones congeladas se tiñeron con hematoxilina-eosina, las secciones semidelgadas se tiñeron con azul de toluidina y se examinaron con un microscopio óptico, mientras que las secciones delgadas se contrastaron con acetato de uranilo-citrato de plomo y se evaluaron con un microscopio electrónico. Las finas características estructurales de los trofoblastos vellosos se superponen a algunas células estromales vellosas. Además de la apariencia habitual de capilares maduros en el estroma velloso, demostramos e informamos etapas de maduración de brotes angiogenéticos en la placenta a término. Los trofoblastos extravellosos se clasificaron según su localización: fibrinoide, corion, trofoblástico, columna, endotelio vascular materno o decidua. Todos estos trofoblastos compartían algunas características ultraestructurales, pero también eran distintos entre sí. En decidua se observó que el revestimiento endotelial de algunos vasos estaba invadido por unos pocos trofoblastos endovasculares con microvellosidades irregulares. Estas células compartían algunas propiedades ultraestructurales tanto con los trofoblastos vellosos como con las células del estroma. El examen mostró que la angiogénesis todavía estaba presente en las placentas a término y que los trofoblastos, las células endoteliales y estromales tienen propiedades ultraestructurales muy similares, lo que sugiere que representan formas de transformación.

SNHG22 promotes migration and invasion of trophoblasts via miR-128-3p/ PCDH11X axis and activates PI3K/Akt signaling pathway

Abstract Objectives Long non-coding RNAs (LncRNAs) act as an indispensable role in the Preeclampsia (PE)-related trophoblast function, while its relationship with Small Nucleolar RNA Host Gene 22 (SNHG22) remains unknown. Hence, this study aimed to investigate the roles of lncRNA SNHG22 in the Preeclampsia (PE)-related trophoblasts function and the underlying mechanism. Methods Normal placentas and placentas from PE patients were collected to detect the expression of lncRNA SNHG22. Then, trophoblasts HTR-8/Svneo and JEG-3 were purchased, cultured, and treated to investigate the roles of lncRNA SNHG22 on cell migration and invasion as well as its underlying regulatory mechanism. Results The SNHG22 was downregulated in PE patients, and it was found that SNHG22 overexpression could drive migration and invasion of trophoblasts, while SNHG22 depletion exerted a suppressive effect. Mechanistically, SNHG22 was validated to regulate microRNA-128-3p (miR-128-3p), and Protocadherin 11 X-Linked (PCDH11X) was identified as the target gene of miR-128-3p. Furthermore, it was found that SNHG22 acted as a promoter in the migration and invasion of trophoblast cells in a miR-128-3p/PCDH11X dependent manner, and SNHG22 silencing weakened the activation of PCDH11X-mediated PI3K/Akt signaling pathways through inhibiting miR-128-3p, thereby preventing migration and invasion of trophoblasts. Conclusion SNHG22 acted as a driver in the migration and invasion of trophoblasts and may be considered a candidate for the amelioration of PE.

Chagas disease affects the human placental barrier's turnover dynamics during pregnancy

BACKGROUND Trypanosoma cruzi crosses the placental barrier and produces the congenital transmission of Chagas disease (CD). Structural alterations of the chorionic villi by this parasite have been described in vitro, but little is known about trophoblast turnover in placentas from women with CD. OBJECTIVE To analyze the proliferation and fusion processes in placentas from women with CD. METHODS Archived human term placenta paraffin-embedded blocks were used, from women with CD (CDP), and no pathology (NP). Immunohistochemistry tests were performed for Ki67 to calculate the proliferation index (PI) of cytotrophoblast (CTB) and Syncytin-1, a fusion marker of syncytiotrophoblast (STB). Hematoxylin/Eosin stained sections were employed to analyze STB percentages, STB detachment areas and syncytial knots quantity. Non parametric Student's t-tests were performed (p < 0.05). RESULTS Syncytial knots and STB detachment significantly increased in placental villi from the CDP group. STB percentage was significantly lower in the CDP group as well as the PI and Syncytin-1 expression significantly decreased in these placentas, compared with control (NP). CONCLUSION Dynamic of trophoblast turnover is altered in placentas from women with CD. These changes may lead into a gap in the placental barrier possibly allowing the parasite entry into the chorionic villi.

Participação dos neutrófilos durante a implantação embrionária no desenvolvimento da placenta em camundongos / Participation of neutrophils during embryonic implantation in the placental development in mice

A implantação do embrião na parede uterina é um processo complexo que consiste na interação do blastocisto com as células epiteliais do útero, e depende de diferentes tipos celulares do microambiente uterino. Embora a literatura mostre a participação de neutrófilos neste processo, os dados ainda são incipientes para proposição da função exata destas células nos períodos iniciais da gestação. Dados do nosso grupo de pesquisa mostraram que neutrófilos pró-angiogênicos induzem a tolerância gestacional, e que a depleção de neutrófilos durante as fases iniciais da gestação prejudica a implantação do blastocisto e a progressão da gestação. Com base nestes resultados, o presente estudo visou investigar se a depleção de neutrófilos na fase pré-receptiva da janela de implantação do blastocisto altera a morfologia placentária. Para tanto, foi utilizado o modelo de gestação alogênica, onde camundongos fêmeas C57BL/6, após cruzamento com machos Balb/C foram tratadas com anticorpo anti-Ly6G ou isotipo no dia 1,5 da gestação (24 horas após a detecção do plug vaginal) em dose suficiente para manter a depleção de neutrófilos circulantes por 48 horas (200µg/ 500µL; i.p). No final da gestação (dia 18,5), o sangue periférico foi coletado e, em seguida, os animais foram submetidos a laparotomia para retirada da placenta, a qual foi submetida à análise histológica. As análises dos leucócitos circulantes evidenciaram a efetividade do tratamento para depleção de neutrófilos periféricos. A análise histológica mostrou alterações significativas na morfologia da placenta nos animais tratados com anti-Ly6G. Foram detectadas a redução da zona juncional, de células trofoblásticas e de fatores angiogênicos, como fator de crescimento do endotélio vascular (VEGF), e das moléculas de adesão intracelular-1 (ICAM-1) e de plaqueta e endotélio (PECAM-1). Esses dados evidenciam a importância dos neutrófilos nos primeiros dias de gestação para o desenvolvimento da placenta

Blastocyst implantation is a complex process, consisting of the interaction between blastocyst and uterine epithelial cells. Also, it is well known that the implantation site resembles an inflammatory response, with a profusion of recruited immune cells into the endometrial stroma and lumen from the blood. The role of macrophages, natural killers, and dendritic cells have been extensively studied, however, the participation of neutrophils in this process remains unclear. Data from our research group showed that pro-angiogenic neutrophils induced gestation tolerance, also peripheral neutrophils depletion at the time of active placental development led to smaller embryo sizes and abnormal placentation in mice. In this context, the present study aimed to investigate whether pharmacological depletion of neutrophils in mice in the blastocyst implantation phase alters placental morphology. Therefore, C7/BL/6 female mice, after mating with Balb/C males, were treated with an anti-Ly6G antibody or isotype on day 1 of gestation (after detection of the vaginal plug) at a dose sufficient to maintain the depletion of circulating neutrophils for 48 hours (200 µg/500µL; i.p). At the end of the gestational day (day 18), peripheral blood was collected, and then the animals were submitted to laparotomy for the placenta removal and subsequent histological analysis. The analysis of circulating leukocytes from neutrophils depleted mice showed a reduction of peripheral neutrophils up to 48 hours after antibody injection. The histological analysis showed significant alterations in the placenta morphology of the animals treated with anti-Ly6G. The morphometric analyses showed a reduction in the size of neutrophils depleted placenta due to diminished junctional zone and reduction of trophoblast cells. Also, it was observed a reduction of vascular endothelial growth factors (VEGF), reduction of adhesion molecules intracell-1 (ICAM-1), and platelets and endothelium (PECAM-1) positive cells in the junctional zone. In conclusion, these data show the importance of neutrophils on the first days of pregnancy for the development of the placenta

miR-146a-5p-mediated suppression on trophoblast cell progression and epithelial-mesenchymal transition in preeclampsia

OBJECTIVE: This study aims to identify the effect of miR-146a-5p on trophoblast cell invasion as well as the mechanism in preeclampsia (PE). METHODS: Expression levels of miR-146a-5p and Wnt2 in preeclamptic and normal placentae were quantified. Trophoblast cells (HTR-8) were separately transfected with miR-146a-5p mimic, miR-146a-5p inhibitor, pcDNA3.1-Wnt2 or sh-Wnt2, and then the expression levels of miR-146a-5p, Wnt2, and epithelial-mesenchymal transition (EMT)-related proteins (Vimentin, N-cadherin and E-cadherin) were measured. Moreover, the proliferative, migratory and invasive capacities of trophoblast cells were detected, respectively. Dual luciferase reporter assay determined the binding of miR-146a-5p and Wnt2. RESULTS: Compared with normal placental tissues, the placentae from PE patients showed higher miR-146a-5p expression and lower Wnt2 expression. Transfection of miR-146a-5p inhibitor or pcDNA3.1-Wnt2 exerted pro-migratory and pro-invasive effects on HTR-8 cells and encouraged EMT in HTR-8 cells; transfection with miR-146a-5p mimic or sh-Wnt2 weakened the proliferative, migratory and invasive capacities as well as reduced EMT process of HTR-8 cells. Moreover, Wnt2 overexpression could partially counteract the suppressive effects of miR-146a-5p overexpression on the progression and EMT of HTR-8 cells. CONCLUSION: miR-146a-5p mediates trophoblast cell proliferation and invasion through regulating Wnt2 expression.

Does Enoxaparin treatment have any effects on the placenta in women with unexplained histories of habitual abortion? A case control study

ABSTRACT BACKGROUND: It is very common to offer low molecular weight heparin (LMWH) medications to women with unexplained habitual abortion, to increase the livebirth rate. Although no benefit from LMWH has been clearly demonstrated, examination of the effects of enoxaparin on placental structure is lacking. OBJECTIVE: To assess placental structural changes in pregnancies treated with enoxaparin, compared with controls. DESIGN AND SETTING: Case-control study in an obstetrics and gynecology unit of a tertiary-level university hospital in Turkey. METHODS: Forty patients who had had term pregnancies and live births but also histories of habitual abortion were recruited for this study. Placentas were sampled using a systematic random sampling method. Tissue samples were obtained, embedded and sectioned for routine histological analyses. Hematoxylin and eosin staining was used. Surface area and length estimates from placental components were evaluated by using Image J. Cell proliferation and apoptosis were also assessed via immunohistochemistry. RESULTS: There were no significant differences between the groups regarding maternal age, abortion rate, birth weight or gestational age. Comparison of the enoxaparin and control groups showed that there were no significant differences in terms of surface area and ratios of placental components. We found that Bcl-2 was generally expressed at high levels in the enoxaparin group, while there was no difference in terms of Ki-67 between the groups. CONCLUSIONS: This study demonstrates that enoxaparin did not show any significant effect on the placental structure of cases that had histories of habitual abortion.

Expresión de calreticulina en placenta canina / Calreticulin expression in canine placenta

La placenta es un anexo embrionario de los mamíferos que tiene por función principal el intercambio de nutrientes y gases y proteger al concepto de un potencial daño inmune provocado por diferencias alogénicas en los Complejos Principales de Histocompatibilidad paternos. Se han descrito diversas proteínas asociadas a su función, siendo Calreticulina una de ellas. Si bien existen estudios de la presencia de Calreticulina en placenta humana, no existen reportes de esta proteína en la placenta canina. Se obtuvieron muestras de placenta canina de las que se extrajo el contenido proteico total y se determinó la presencia de Calreticulina por western blot e inmunohistoquímica. Los resultados mostraron presencia de Calreticulina en placenta canina con un peso molecular aparente de 60 kDa, concordante con lo descrito para la molécula por otros autores. El análisis inmunohistoquímico mostró que Calreticulina canina está presente principalmente en el trofoblasto de las vellosidades, no existiendo diferencias en cuanto a su localización al compararla con placenta humana, pese a sus diferencias morfológicas e histológicas. Esta información permitirá establecer un protocolo estandarizado de extracción de Calreticulina desde placenta, así como orientar acerca de los posibles roles de esta molécula en la placenta.

The placenta is an embryonic organ present in mammals, whose main functions are the exchange of nutrients and gases and to protect the fetus from potential immune damage mediated by paternal and maternal allogeneic differences in the Major Histocompatibility Complex. Several proteins associated with its function have been described, being Calreticulin one of them. Although there are studies on the presence of Calreticulin in human placenta, there are no reports of this protein in canine placenta. Samples from canine placenta were obtained, proteins extracted and Calreticulin was subsequently detected by western blot and immunohistochemistry. The results showed the presence of Calreticulin in canine placenta with an apparent molecular weight of 60 kDa, in agreement with the results from other authors. The immunohistochemical analysis showed that canine Calreticulin is present mainly in the trophoblast of the villi, and there is no difference in its localization when compared with a blood-filled placenta such as human one, despite its morphological and histological differences. We also propose a standardized protocol for the extraction of Calreticulin from placenta, given its abundant expression in this organ. Future studies are aimed at elucidating possible roles of this protein in placenta.

Doença trofoblástica gestacional: correlação ßhCG sérico quantitativo, ultrassonografia e anatomopatologia em serviço de obstetrícia do Ceará / Gestational trophoblastic disease: ßhCG quantitative serum correlation, ultrasonography and pathology in obstetrics service in Ceará

Doença trofoblástica gestacional (DTG) é uma anomalia que engloba formas clínicas benignas (mola hidatiforme completa e parcial) e malignas (mola invasora, coriocarcinoma, tumor trofoblástico do sítio placentário e tumor trofoblástico epitelioide). O objetivo deste estudo é realizar levantamento epidemiológico retrospectivo de prontuários de 40 pacientes internadas entre abril de 2014 e fevereiro de 2016 com hipótese diagnóstica de DTG atendidas no Hospital Regional Norte/ Centro de Apoio à Saúde Reprodutiva da Mulher em Sobral, no Ceará, traçando o perfil de cada paciente (idade, paridade), além de fazer correlação dos parâmetros clínicos, laboratoriais e anatomopatológico. Entre as pacientes que obtiveram o diagnóstico de DTG, observou-se que em torno de 93,33% possuíam exame ultrassonográfico evidenciando possível mola hidatiforme; o anatomopatológico confirmou doença trofoblástica em aproximadamente 52,5% da população estudada. Este estudo é inédito, por ser o primeiro a realizar um levantamento de dados em pacientes com DTG na cidade de Sobral.(AU)

Gestational trophoblastic disease (GTD) is an anomaly that encompasses benign clinical forms (complete and partial hydatidiform mole) and malignant (invasive mole, choriocarcinoma, placental site trophoblastic tumor and epithelioid trophoblastic tumor). The objective of this study was to carry out a retrospective epidemiological survey of medical records of 40 hospitalized patients between April 2014 and February 2016 with diagnostic hypothesis of GTD attended at the Regional Hospital Norte/Center for Support to Women's Reproductive Health in Sobral, Ceará, drawing the profile of each patient (age, parity), in addition to correlating the clinical, laboratory and anatomopathological parameters. Among the patients who had the diagnosis of GTD, it was observed that about 93.33% had ultrasonographic examination evidencing a possible hydatidiform mole; the anatomopathological confirmed trophoblastic disease in about 52.5% of the study population. This study is unprecedented because it is the first to perform a data collection in patients with GTD in the city of Sobral.(AU)

Interacción endometrio trofoblasto, en la implantación humana: revisión de la literatura / Endometrium-trophoblast interaction in human implantation: review of literature

Un embarazo exitoso requiere de una serie de interacciones mediadas por factores hormonales, moleculares y fenómenos de inmunomodulación. Una de estas interacciones es la que ocurre entre el endometrio y el blastocito, previo y durante el proceso de implantación. El objetivo de esta revisión bibliográfica es complementar lo descrito en la literatura clásica de embriología humana sobre interacción de endometrio-blastocito. La búsqueda bibliográfica se realizó en la base de datos MEDLINE usando los términos en inglés "implantation", "endometrium" y "embryo"; además se realizó una búsqueda manual, que incluyó artículos de revistas no indexadas, libros de texto y atlas. Se consideraron criterios de inclusión y exclusión para la selección de los artículos y otros recursos bibliográficos. Entre los criterios de inclusión se consideraron estudios realizados en humanos, artículos de revisión y experimentación, publicados en los últimos 5 años. Como criterios de exclusión se consideraron artículos que utilizaran animales, estudios sobre fertilidad in vitro, patologías asociadas y artículos no relacionados al tema. Una vez completada la selección, se examinaron los textos completos, en los cuales se aplicaron nuevamente los criterios de exclusión. La búsqueda arrojó un total de 560 artículos, cuyo análisis de los títulos y resúmenes resultó en 475 trabajos excluidos, a partir de los diferentes criterios de exclusión antes descritos. Por lo tanto, se obtuvieron 85 artículos, en los cuales se realizó el análisis del texto completo. De estos artículos, se obtuvieron un total de 34 estudios y los contenidos seleccionados en esta revisión fueron: Endometrio, Interacción endometrio trofoblasto, Aposición, Adhesión y Migración-Invasión. Durante la implantación se genera una interacción entre el endometrio y el trofoblasto, con la participación de moléculas reguladoras de proliferación y diferenciación, como factores hormonales, moleculares y de expresión génica. Sin embargo, los mecanismos específicos de acción e interacción deben continuar siendo investigados, para responder interrogantes en el ámbito del crecimiento y desarrollo humano.

A successful pregnancy requires a series of interactions, mediated by hormonal, molecular and immunomodulation phenomena. One of these interactions is between the endometrium and the blastocyst, before and during the implantation process. The objective of this literature review is to complement what is described in the classic human embryology literature on endometrial-blastocyst interaction. The bibliographic search was carried out in the MEDLINE database using the terms "implantation", "endometrium" and "embryo", and a manual search was carried out, which included articles from non-indexed journals, textbooks and atlases. Inclusion and exclusion criteria were considered for the selection of articles and other bibliographic resources, including human studies, review and experimentation articles, published in the last 5 years. Articles with animals as experimental subjects, in vitro fertility studies, associated pathologies and articles not related to the subject were excluded. When the selection was completed, the complete texts were examined, in which the exclusion criteria were applied again The search yielded a total of 560 articles, whose analysis of titles and abstracts resulted in 475 excluded works, in relation to different exclusion criteria described above. Therefore, 85 articles were obtained, in which the complete text analysis was performed. From these articles, a total of 34 studies were obtained and the contents selected in this review were: Endometrium, Endometrium trophoblast, Aposition, Adhesion and Migration-Invasion. During the implantation, aninteraction between the endometrium and the trophoblast is generated, with the participation of regulatory molecules of proliferation and differentiation, such as hormonal, molecular and gene expression factors. However, the specific mechanisms of action and interaction must continue to be investigated, to answer questions in the field of human growth and development.

Clinical significance of expression of MnSOD/SIRT3 in pernicious placenta previa combined with placenta implantation / 医学研究生学报

Objective Dangerous placenta previa(PPP) combined with placenta implantation seriously threatens maternal life safety. This paper aim to explore the changes of MnSOD and SIRT3,the expression of SIRT3 in maternal placenta PPP combined with placenta implantation, and the relationship between trophoblast invasion and placental implantation. Methods 90 cases with placenta implantation of pernicious placenta previa were collected from January 2014 to June 2018 in Anhui Maternal and Child Health Hospital. According to the depth of placental villus invading uterine myometrium, 30 cases of placenta adhesion, 30 cases of placental implantation, and 30 cases of placenta penetration, 30 cases of normal control group．Immunohistochemical SP and Western blot were used to detect the expression of MnSOD and SIRT3 in placental tissues of the study group and the control group, then compared and analyzed. Results Compared with the control group, the expression of MnSOD and SIRT3 in the placental implantation group were increased. With the increasing of placental implantation degree, the level of MnSOD and SIRT3 decreased significantly (P<0.05). Western blot showed that , the relative protein expressions of MnSOD/β-actin and SIRT3/β-actin in the control group were (0.39±0.05) and (0.41±0.08), which were higher than those in the adhesion group[(0.35±0.04), (0.32±0.02)], the implantion group[(0.28±0.02), (0.20±0.03)], and the penetration group[(0.23±0.01), (0.17±0.02)]. The difference was statistically significant(P<0.05). Conclusion The expressions of MnSOD and SIRT3 incytoplasm or nucleus of invasive trophoblasts and placental tissues of pregnant women with placental implantation is significantly decreased, both of which are involved in the occurrence and development of placental implantation, but the specific pathogenesis still needs to be further explored.

Vitamin D down-regulates microRNA-21 expression to promote human placental trophoblast cell migration and invasion / 南方医科大学学报

OBJECTIVE@#To investigate the effect of vitamin D on microRNA-21(miR-21) expression and migration and invasion of human placental trophoblast cells.@*METHODS@#The changes in the expression of miR-21 were detected using RT-qPCR in HTR-8/SVneo cells following stimulation by vitamin D at different doses for 24, 48 and 72 h.HTR-8/SVneo cells transfected with miR-21 mimic or inhibitor with or without vitamin D treatment were examined for changes in cell migration and invasion abilities using Transwell assay, and Western blotting was used to detect protein expressions of E-cadherin, fibronectin, and MMP9.@*RESULTS@#Vitamin D obviously inhibited the expression of micoRNA-21 in HTR-8/SVneo cells in a concentration-and time-dependent manner.Transfection with the miR-21 mimic significantly inhibited the migration and invasion of HTR-8/SVneo cells, and this inhibitory effect was abolished by treatment with vitamin D; transfection with miR-21 inhibitor obviously promoted the migration and invasion of HTR-8/SVneo cells, and these effects were not significantly affected by vitamin D treatment.@*CONCLUSIONS@#Vitamin D may promote trophoblast cell migration and invasion to accelerate the development of preeclampsia by down-regulating the expression of miR-21.

Effect of ASPP2 on regulating GRP78 in apoptosis of placental trophoblast cells induced by L-NAME / 中国药理学通报

Aim: To explore the role of apoptosis stimulating protein 2 of p53 (ASPP2) on L-NAME induced apoptosis of placental trophoblast cells by regulating glucose-regulated protein78(GRP78), and provide a theoretical basis for the study of clinical pregnancy-induced hypertension. Methods: The HTR-8/SVneo human placental trophoblast cells were cultured in vitro, and in the absence (control group) or presence of 100 μmol · L-1 L-NAME (L-NAME group) for 48 h. The effects of L-NAME on placental trophoblast cell apoptosis were tested using flow cytometry and AO/EB assay. The expressions of caspase-12, GRP78 and ASPP2 were detected by Western blot. The ASPP2 interference with adenovirus was used to transfect the cells, and the mRNA expression level of ASPP2 and the protein expression level of GRP78 were detected by qRT-PCR and Western blot, respectively. After treated with 100 (xrnol · L-1 L-NAME for 48 h, the protein expression of caspase-12 and GRP78 was detected by Western blot and immunofluorescence. Results: Compared with control group, the placental trophoblast cell apoptosis significantly increased in L-NAME group (P < 0. 05). AO/EB staining showed that compared with control group, the majority of cells in L-NAME group showed bright orange and the number of late apoptotic cells increased significantly. At the same time, caspase-12, GRP78 and ASPP2 protein expression increased (P < 0. 05, P < 0. 01). After interfering with ASPP2, caspase-12 and GRP78 protein expressions decreased (P < 0. 05). Conclusions: Down-regulation of ASPP2 could decrease GRP78 expression and inhibit L-NAME-induced apoptosis in placental trophoblast cells.