Exploring Mitochondrial Localization of SARS-CoV-2 RNA by Padlock Assay. A Pilot Study in Human Placenta

The ongoing COVID-19 pandemic dictated new priorities in biomedicine research. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is a single-stranded positive-sense RNA virus. In this pilot study, we optimized our padlock assay to visualize genomic/subgenomic regions using formalin-fixed paraffin-embedded placental samples obtained from a confirmed case of COVID-19. SARS-CoV-2 RNA was localized in trophoblastic cells. We also checked the presence of the virion by immunolocalization of its glycoprotein spike. In addition, we imaged mitochondria of placental villi keeping in mind that the mitochondrion has been suggested as a potential residence of the SARS-CoV-2 genome. Indeed, we observed a substantial overlapping of SARS-CoV-2 RNA and mitochondria in trophoblastic cells. This intriguing linkage correlated with an aberrant mitochondrial network. Overall, to our knowledge, this is the first study that provides the evidence of a co-localization of the SARS-CoV-2 genome and mitochondria in SARS-CoV-2 infected tissue. These findings also support the notion that SARS-CoV-2 infection could reprogram mitochondrial activity in highly specialized maternal/fetal interface.

A Rare Case of Epithelioid Trophoblastic Tumor Presenting as Hematoma of a Caesarean Scar in the Lower Uterine Segment

Epitheliod trophoblastic tumor (ETT) account for only 1–2% of all the cases of gestational trophoblastic neoplasia (GTN), with a reported mortality rate of 10–24%. ETT is derived from chorionic type intermediate trophoblastic cells, which appears to be the reason for the only slightly elevated βhCG levels in these patients. We present a case of a 42-year-old patient who was admitted to the clinic eight months after Caesarean delivery, for irregular vaginal bleed with normal values of beta-human chorionic gonadotropin (βhCG). A 6 × 5 cm hematoma was evacuated from the isthmic uterine segment during the operation, and the histopathological exam of the tissue surrounding the hematoma revealed ETT. There were no metastatic lesions on the thoracal, abdominal, and pelvic CT. The second ultrasonographic exam revealed tumefaction of 5 cm at the site from the previous surgical procedure. Color Doppler imaging revealed no central nor peripheral blood flow. The patient underwent a total abdominal hysterectomy with bilateral adnexectomy without adjuvant chemotherapy. This appears to be one of the shortest intervals from the anteceded gestational event until the diagnosis of this tumor, along with the absence of the significant ultrasonographic feature of the ETT-peripheral Doppler signal pattern. We underline that, even with normal values of βhCG, irregular vaginal bleeding following the antecedent gestational event should always arouse suspicion of GTN.

Methods of detection and isolation of trophoblast cells from trans-cervical specimens – a historical overview

Trophoblast cells can be detected and isolated from the cervical epithelial cells obtained via various techniques of trans-cervical samples collection such as a mucus aspiration, endocervical lavage, or standard cervical brushing in the early first trimester, even from the 5 weeks’ gestation. Isolated fetal cells can be used in the early prediction of fetal sex, prenatal diagnostics of the most common aneuploidies, and any other genetic abnormalities. Nevertheless, the collection of trophoblastic cells has limited efficacy compared to currently used methods of detection of free fetal DNA in maternal circulation or other protocols of invasive prenatal diagnostics available at later stages of pregnancy. In the past years, trans-cervical cell samples were collected mainly in women before planned pregnancy termination. The early trophoblastic cells isolation from women in ongoing pregnancies opens new perspectives for further studies focused on the elucidation of pathophysiology of numerous pregnancy-related complications.

An Overview of C19MC Cluster Subgroup 3 and Cancer

: The primate-specific microRNA gene cluster on chromosome 19 (C19MC) is composed of 56 mature microRNAs (miRNAs), which are divided into three subgroups according to the sequence similarity. This cluster is principally expressed in the placenta but not in other tissues. C19MC is involved in the regulation of proliferation, migration, and invasion of trophoblastic cells, which are important for the development of the placenta. There is a growing number of studies that have found an altered expression of some miRNAs of the C19MC cluster in cancer, suggesting that these could play an important role in the development of this disease. Therefore, in this work, we provided an overview of the C19MC cluster’s role in cancer through a systematic review of published articles. In particular, we focused on miRNAs of subgroup 3. These studies suggest that miRNAs such as miR-512-3p, miR-512-5p, miR-516a-5p, miR-516b-5p, and miR-498-5p could play a pivotal role in the development of therapies for cancer. Future studies are necessary to elucidate the molecular processes and pathways regulated by subgroup 3 miRNAs.

The Preeclamptic Environment Promotes the Activation of Transcription Factor Kappa B by P53/RSK1 Complex in a HTR8/SVneo Trophoblastic Cell Line

Preeclampsia is a pregnancy disorder associated with shallow placentation, forcing placental cells to live in hypoxic conditions. This activates the transcription factor kappa B (NFκB) in maternal and placental cells. Although the role of NFκB in preeclampsia is well documented, its mechanism of activation in trophoblastic cells has been never studied. This study investigates the mechanism of NFκB activation in a first trimester trophoblastic cell line (HTR8/SVneo) stimulated by a medium containing serum from preeclamptic (PE) or normotensive (C) women in hypoxic (2% O2) or normoxic (8% O2) conditions. The results indicate that in HTR8/SVneo cells, the most widely studied NFκB pathways, i.e., canonical, non-canonical and atypical, are downregulated in environment PE 2% O2 in comparison to C 8% O2. Therefore, other pathways may be responsible for NFκB activation. One such pathway depends on the activation of NFκB by the p53/RSK1 complex through its phosphorylation at Serine 536 (pNFκB Ser536). The data generated by our study show that inhibition of the p53/RSK1 pathway by p53-targeted siRNA results in a depletion of pNFκB Ser536 in the nucleus, but only in cells incubated with PE serum at 2% O2. Thus, the p53/RSK1 complex might play a critical role in the activation of NFκB in trophoblastic cells and preeclamptic placentas.

Functional Analysis of p21Cip1/CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

Preeclampsia (PE), a gestational hypertensive disease originating from the placenta, is characterized by an imbalance of various cellular processes. The cell cycle regulator p21Cip1/CDKN1A (p21) and its family members p27 and p57 regulate signaling pathways fundamental to placental development. The aim of the present study was to enlighten the individual roles of these cell cycle regulators in placental development and their molecular involvement in the pathogenesis of PE. The expression and localization of p21, phospho-p21 (Thr-145), p27, and p57 was immunohistochemically analyzed in placental tissues from patients with early-onset PE, early-onset PE complicated by the HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome as well as late-onset PE compared to their corresponding control tissues from well-matched women undergoing caesarean sections. The gene level was evaluated using real-time quantitative PCR. We demonstrate that the delivery mode strongly influenced placental gene expression, especially for CDKN1A (p21) and CDKN1B (p27), which were significantly upregulated in response to labor. Cell cycle regulators were highly expressed in first trimester placentas and impacted by hypoxic conditions. In support of these observations, p21 protein was abundant in trophoblast organoids and hypoxia reduced its gene expression. Microarray analysis of the trophoblastic BeWo cell line depleted of p21 revealed various interesting candidate genes and signaling pathways for the fusion process. The level of p21 was reduced in fusing cytotrophoblasts in early-onset PE placentas and depletion of p21 led to reduced expression of fusion-related genes such as syncytin-2 and human chorionic gonadotropin (β-hCG), which adversely affected the fusion capability of trophoblastic cells. These data highlight that cell cycle regulators are important for the development of the placenta. Interfering with p21 influences multiple pathways related to the pathogenesis of PE.

A rare case of placental site trophoblastic tumor

Placental site trophoblastic tumor (PSTT) is a very rare tumor of young women and unique form of gestational trophoblastic disease (GTD) representing a neoplastic transformation of intermediate trophoblastic cells that play a critical role in implantation. It accounts for 1-2% of all GTNs, incidence of it being 1 in 1,00,000 pregnancies. It displays a wide clinical spectrum from benign lesion within uterus to highly metastatic features with widespread features of metastasis and can be difficult to control even with surgery and chemotherapy. Unlike other forms of GTD, it is characterized by low β-hCG levels because it is a neoplastic proliferation of intermediate trophoblastic cells. PSTT can develop following all kind of pregnancies, whereas approximately 50% of PSTT cases occur after a normal pregnancy and other cases follow abortion, term delivery, ectopic pregnancy and molar pregnancy. Histopathologically, PSTT has proliferation of intermediate trophoblastic cells and absence of villi. It shows less invasion of vascular tissue and using immune histochemistry revealed that PSTT cells expressed HPL more than β-hCG, characterized by increased expression of the HPL in histological section as well as serum. The most common presenting symptoms of PSTT are irregular vaginal bleeding associated with uterine sub involution. Patient can also be presented with amenorrhea, nephritic syndrome, abdominal pain, galactorrhea and hemoptysis. Hence any case of unusual bleeding should be investigated with dilation and curettage and βHCG levels. Although the majority of patients of the non-metastatic PSTT are cured by hysterectomy but in metastatic cases, it requires aggressive treatment with chemotherapy and radiation. Due to its seldom occurrence and uncharacteristic clinical presentation, to reach a correct diagnosis and management is a surgeon’s nightmare and can be very challenging.

Cortisol Dose-Dependently Impairs Migration and Tube-like Formation in a Trophoblast Cell Line and Modulates Inflammatory and Angiogenic Genes

Several stimuli can change maternal hormone levels during pregnancy. These changes may affect trophoblastic cells and modulate the development of the embryo and the placental tissue itself. Changes in cortisol levels are associated with impaired trophoblast implantation and function, in addition to other pregnancy complications. This study aims to analyze the effects of low and high doses of cortisol on an extravillous trophoblast cell line, and the effects of various exposures to this hormone. SGHPL-4 cells were treated with cortisol at five doses (0–1000 nM) and two exposures (continuous: 24 h/day; and intermittent: 2 h/day). In intermittent treatment, cortisol acted mainly as an anti-inflammatory hormone, repressing gene expression of kinin B1 receptors, interleukin-6, and interleukin-1β. Continuous treatment modulated inflammatory and angiogenic pathways, significantly repressing angiogenic factors and their receptors. Cortisol affected cell migration and tube-like structures formation. In conclusion, both continuous and intermittent exposure to cortisol repressed the expression of inflammatory genes, while only continuous exposure repressed the expression of angiogenic genes, suggesting that a sustained increase in the levels of this hormone is more harmful than a high short-term increase. Cortisol also impaired tube-like structures formation, and kinin receptors may be involved in this response.

Massive Perivillous Fibrin Deposition and Chronic Histiocytic Intervillositis a Complication of SARS-CoV-2 Infection

An emerging complication of COVID-19 (SARS-CoV-2) infection is reported. A 23-year-old patient presented with high temperature and reduced fetal movements at 25 + 5/40 weeks of gestation. RT-PCR proved maternal COVID-19 infection. Ultrasound examination confirmed intrauterine death. Placenta histology showed necrosis of the villous trophoblast, associated with Chronic Histiocytic Intervillositis (CHI) and Massive Perivillous Fibrin Deposition (MPFD) with up to 90% - of the intervillous spaces being involved. Immunohistochemistry showed CD68 positive histiocytes in the intervillous spaces and the villous trophoblast was positive for the COVID-19 spike protein. RNA scope signal was indicative of the presence of the viral genome and active viral replication in the villous trophoblastic cells, respectively. MPFD is a gradually developing end-stage disease with various etiology, including autoimmune and alloimmune maternal response to antigens expressed at the feto-maternal interface and frequently accompanies chronic alloimmune villitis or histiocytic intervillositis. Covid-19 infection is associated with similar pattern of histological changes of the placenta leading to placental insufficiency and fetal death. This case report supports maternal- fetal vertical transmission of SARS-CoV-2 virus leading to placental insufficiency and fetal demise. MPFD and CHI appear to be the typical placental histology for SARS-CoV-2 virus infection associated fetal demise.

STOX2 Is the Target of Mir-30a to Increase Cell Proliferation and Metastasis in Hydatidiform Mole Through ERK, AKT and P38 Signaling Pathways

BackgroundHydatidiform mole is a disease caused by abnormal proliferation of trophoblastic cells. MiR-30a acts as a tumor suppressor gene in most tumors and participates in the development of various cancers. But its role in hydatidiform mole is not clear. MethodsRT-qPCR was used to verify the expression level of miR-190 and STOX2. Flow cytometry (FCM) assays were performed to detect cell cycle. CCK-8 assay, EDU assay and colony formation assay was used to detect proliferation ability. Transwell assay was used to test invasion ability. Dual-luciferase reporter assay and Western blotting were used to elucidate the potential mechanisms involved.ResultThe low expression of miR-30a promoted the ability of proliferation, migration and invasion in trophoblastic cells (JAR and HTR-8). The dual luciferase assay confirmed that STOX2 was a new target of miR-30a and resisted the effect of up-regulated miR-30a in trophoblastic cells. In addition, up-regulation of STOX2 by miR-30a could activate ERK, AKT and P38 signaling pathways. These results revealed that a new mechanism for ERK, AKT and P38 activation by miR-30a/STOX2, which could create excessive proliferation of trophoblast cells in the hydatidiform mole.ConclusionsIn this study, we found that miR-30a plays an important role in the development of HM. Taken together, these fndings indicate that miR-30a may effect the malignant transformation of human trophoblastic cells by downregulating STOX2, which strengthens our understanding of miR-30a in regulating BC cell transformation.