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3 pages, 210 KiB

Open AccessEditorial

Signaling Pathways in Pregnancy

by Giovanni Tossetta and Daniela Marzioni

Cells 2022, 11(9), 1385; https://doi.org/10.3390/cells11091385 - 20 Apr 2022

Cited by 2 | Viewed by 1670

Abstract

We are pleased to present this Special Issue of Cells, entitled ‘Signaling Pathways in Pregnancy’ [...] Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

Research

Jump to: Editorial, Review

12 pages, 3217 KiB

Open AccessCommunication

Pravastatin Protects Cytotrophoblasts from Hyperglycemia-Induced Preeclampsia Phenotype

by Ahmed F. Pantho, Sara Mohamed, Janhavi V. Govande, Riddhi Rane, Niraj Vora, Kelsey R. Kelso, Thomas J. Kuehl, Steven R. Lindheim and Mohammad N. Uddin

Cells 2024, 13(18), 1534; https://doi.org/10.3390/cells13181534 - 13 Sep 2024

Viewed by 692

Abstract

There are no effective therapies to prevent preeclampsia (PE). Pravastatin shows promise by attenuating processes associated with PE such as decreased cytotrophoblast (CTB) migration, aberrant angiogenesis, and increased oxidative stress. This study assesses the effects of pravastatin on hyperglycemia-induced CTB dysfunction. Methods: Human [...] Read more.

There are no effective therapies to prevent preeclampsia (PE). Pravastatin shows promise by attenuating processes associated with PE such as decreased cytotrophoblast (CTB) migration, aberrant angiogenesis, and increased oxidative stress. This study assesses the effects of pravastatin on hyperglycemia-induced CTB dysfunction. Methods: Human CTB cells were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 h. Some cells were pretreated with pravastatin (1 µg/mL), while others were cotreated with pravastatin and glucose. The expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) mRNA, vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble endoglin (sEng) were measured. CTB migration was assayed using a CytoSelect migration assay kit. Statistical comparisons were performed using an analysis of variance with Duncan’s post hoc test. Results: The hyperglycemia-induced downregulation of uPA was attenuated in CTB cells pretreated with pravastatin at glucose levels > 200 mg/dL and cotreated at glucose levels > 300 mg/dL (p < 0.05). Hyperglycemia-induced decreases in VEGF and PlGF and increases in sEng and sFlt-1 were attenuated in both the pretreatment and cotreatment samples regardless of glucose dose (p < 0.05). Pravastatin attenuated hyperglycemia-induced dysfunction of CTB migration. Conclusions: Pravastatin mitigates stress signaling responses in hyperglycemic conditions, weakening processes leading to abnormal CTB migration and invasion associated with PE in pregnancy. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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14 pages, 4563 KiB

Open AccessArticle

Sudden Intrauterine Unexplained Death (SIUD) and Oxidative Stress: Placental Immunohistochemical Markers

by Angelo Montana, Letizia Alfieri, Raffaella Marino, Pantaleo Greco, Cristina Taliento, Ezio Fulcheri, Anastasio Tini, Francesca Buffelli and Margherita Neri

Cells 2024, 13(16), 1347; https://doi.org/10.3390/cells13161347 - 13 Aug 2024

Viewed by 914

Abstract

Background: Intrauterine fetal death and perinatal death represent one of the most relevant medical scientific problems since, in many cases, even after extensive investigation, the causes remain unknown. The considerable increase in medical legal litigation in the obstetrical field that has witnessed in [...] Read more.

Background: Intrauterine fetal death and perinatal death represent one of the most relevant medical scientific problems since, in many cases, even after extensive investigation, the causes remain unknown. The considerable increase in medical legal litigation in the obstetrical field that has witnessed in recent years, especially in cases of stillborn births, has simultaneously involved the figure of the forensic pathologist in scientific research aimed at clarifying the pathophysiological processes underlying stillbirth. Methods: our study aims to analyze cases of sudden intrauterine unexplained death syndrome (SIUD) to evaluate the role of oxidative stress in the complex pathogenetic process of stillbirth. In particular, the immunohistochemical expression of specific oxidative stress markers (NOX2, NT, iNOS, 8-HODG, IL-6) was evaluated in tissue samples of placentas of SIUDs belonging to the extensive case series (20 cases), collected from autopsy cases of the University of Ferrara and Politecnica delle Marche between 2017 and 2023. Results: The study demonstrated the involvement of oxidative stress in intrauterine fetal deaths in the placenta of the cases examined. In SIUD, the most expressed oxidative stress markers were NOX2 and 8-HODG. Conclusions: The study contributes to investigating the role of oxidative stress in modulating different pathways in unexplained intrauterine fetal death (SIUD) tissues. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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Figure 1
In the image, at 40× magnification, the immunohistochemical reaction against the anti-NOX2 antibody: (A) Group 1 (N = 15), the strong NOX2 diffuse immunopositivity localized in the placental tissue, central area. (B) Group 2 (N = 10), mild immunoreaction to NOX2 in the control tissue. In the graph at the bottom, the statistical representation, **** (statistically significant), p < 0.0001. Full article ">Figure 2
The results of the immunoreaction, at 40× magnification, to NT: (A) Group 1 (N = 15), overexpression of diffuse NT in the placenta of the cases, central part; (B) Group 2 (N = 10), low antibody reaction in the control tissue. In the lower part, the statistical comparison is represented graphically, **** (statistically significant): p < 0.0001. Full article ">Figure 3
The results of immunohistochemical staining, at 40× magnification, with the marker iNOS: (A), in Group 1 (N = 15), the placental tissue expresses intermediate immunoreactivity for iNOS; (B) Group 2 (N = 10), minimal immunoreactivity in the control. The graphical representation of the statistical analysis is placed at the bottom of the figure, **** (statistically significant): p < 0.0001. Full article ">Figure 4
8-HODG immunohistochemical results in figure at 40× magnification: (A) Group 1 (N = 15): a diffuse and intense positive immunoreaction localized in placental tissue, central part, of 8-HODG; (B) Group 2 (N = 10): basal reaction in the control case. The graphical representation of the statistical analysis is collocated in the lower part of the figure, **** (statistically significant): p < 0.0001. Full article ">Figure 5
IL-6 immunohistochemical results at 40× magnification: (A) Group 1 (N = 15), shows a diffuse immunohistochemical intermediate reaction in the placental tissue of a case for the IL-6 marker; (B) in the image appreciates the very moderate reaction in Group 2 (N = 10), in the control case. The lower part of the figure is a graph of statistical analysis, **** (statistically significant): p < 0.0001. Full article ">Figure 6
The graphical representation of the comparison of the expression between the various markers. Full article ">

12 pages, 1988 KiB

Open AccessArticle

Dilation of Pregnant Rat Uterine Arteries with Phenols from Extra Virgin Olive Oil Is Endothelium-Dependent and Involves Calcium and Potassium Channels

by Milena Esposito, Mariacarmela Gatto, Marilyn J. Cipolla, Ira M. Bernstein and Maurizio Mandalà

Cells 2024, 13(7), 619; https://doi.org/10.3390/cells13070619 - 2 Apr 2024

Viewed by 1365

Abstract

During pregnancy, uterine vasculature undergoes significant circumferential growth to increase uterine blood flow, vital for the growing feto-placental unit. However, this process is often compromised in conditions like maternal high blood pressure, particularly in preeclampsia (PE), leading to fetal growth impairment. Currently, there [...] Read more.

During pregnancy, uterine vasculature undergoes significant circumferential growth to increase uterine blood flow, vital for the growing feto-placental unit. However, this process is often compromised in conditions like maternal high blood pressure, particularly in preeclampsia (PE), leading to fetal growth impairment. Currently, there is no cure for PE, partly due to the adverse effects of anti-hypertensive drugs on maternal and fetal health. This study aimed to investigate the vasodilator effect of extra virgin olive oil (EVOO) phenols on the reproductive vasculature, potentially benefiting both mother and fetus. Isolated uterine arteries (UAs) from pregnant rats were tested with EVOO phenols in a pressurized myograph. To elucidate the underlying mechanisms, additional experiments were conducted with specific inhibitors: L-NAME/L-NNA (10⁻⁴ M) for nitric oxide synthases, ODQ (10⁻⁵ M) for guanylate cyclase, Verapamil (10⁻⁵ M) for the L-type calcium channel, Ryanodine (10⁻⁵ M) + 2-APB (3 × 10⁻⁵ M) for ryanodine and the inositol triphosphate receptors, respectively, and Paxilline (10⁻⁵ M) for the large-conductance calcium-activated potassium channel. The results indicated that EVOO-phenols activate Ca²⁺ signaling pathways, generating nitric oxide, inducing vasodilation via cGMP and BKCa²⁺ signals in smooth muscle cells. This study suggests the potential use of EVOO phenols to prevent utero-placental blood flow restriction, offering a promising avenue for managing PE. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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Figure 1

Figure 1
Effects of EVOO phenols and ethanol on uterine arteries. Uterine arteries, precontracted with phenylephrine, were tested with either EVOO phenols (Phenols, n = 15) or vehicle, ethanol (n = 6). Representative experiments are depicted in traces (A) and (B), respectively, while the cumulative results from multiple experiments are summarized in (C). Data are presented as mean ± SEM. Statistical analysis utilized the Area under the Curve (AUC) and was conducted using an unpaired t-test, xxx p < 0.001. Full article ">Figure 2
The influence of the endothelium on the vasodilatory effects of EVOO phenols in uterine arteries. EVOO phenols were tested on phenylephrine-precontracted uterine arteries with intact endothelium (+, n = 15) and without the endothelium (−, n = 5). The absence of the endothelium completely abolished the vasodilation effect of phenols. Data are presented as mean ± SEM, with n indicating the number of experiments. The Area under the curve (AUC) was considered for statistical analysis which was performed using unpaired t-test, xxxx p < 0.0001. Full article ">Figure 3
The involvement of nitric oxide in the vasodilatory effects of EVOO phenols on UAs. EVOO phenols were tested on phenylephrine-precontracted uterine arteries both in the absence (Control, n = 15) and in the presence of nitric oxide synthase enzyme inhibitors (L-NAME + L-NNA, n = 5). Data are presented as mean ± SEM, with n indicating the number of experiments conducted. The Area under the curve (AUC) was considered for statistical analysis which was performed using unpaired t-test, xx p < 0.01. Full article ">Figure 4
The involvement of L-type calcium channels in the vasodilatory effect of EVOO phenols on UAs. EVOO phenols were tested on phenylephrine-precontracted uterine arteries both in the absence (Control, n = 15) and in the presence of the L-type calcium channel blocker, Verapamil (n = 5). Data are presented as mean ± SEM, with n indicating the number of experiments conducted. The Area under the curve (AUC) was considered for statistical analysis which was performed using unpaired t-test, x p < 0.05. Full article ">Figure 5
The involvement of endoplasmic reticulum calcium release in the vasodilatory effects of EVOO phenols on uterine arteries. EVOO phenols were tested on phenylephrine-precontracted uterine arteries both in the absence (control, n = 15) and in the presence of specific inhibitors of ryanodine (Ryanodine, 10−5 M, n = 5) and inositol triphosphate (2-APB, 3 × 10−5 M, n = 5). The inhibition of endoplasmic reticulum calcium release abolished phenols-induced vasodilation. Data are presented as mean ± SEM, with n indicating the number of experiments conducted. The Area under the curve (AUC) was considered for statistical analysis which was performed using unpaired t-test, xxx p < 0.001. Full article ">Figure 6
The involvement of cyclic guanosine monophosphate (cGMP) in the vasodilatory effects of EVOO phenols on uterine arteries. EVOO phenols were tested on phenylephrine-precontracted uterine arteries both in the absence (Control, n = 15) and in the presence of the cGMP inhibitor (ODQ, n = 5). Data are presented as mean ± SEM, with n indicating the number of experiments conducted. The Area under the curve (AUC) was considered for statistical analysis which was performed using unpaired t-test, xxxx p < 0.0001. Full article ">Figure 7
The impact of large-conductance calcium-activated potassium channels on the vasodilatory effects of EVOO phenols in uterine arteries. EVOO phenols were tested on phenylephrine-precontracted uterine arteries both in the absence (control, n = 15) and in the presence of the specific BKca channel inhibitor, Paxilline (n = 5). Data are presented as mean ± SEM, with n indicating the number of experiments conducted. The Area under the curve (AUC) was considered for statistical analysis which was performed using unpaired t-test, xx p < 0.01. Full article ">

17 pages, 8291 KiB

Open AccessArticle

The Impact of SLC2A8 RNA Interference on Glucose Uptake and the Transcriptome of Human Trophoblast Cells

by Aleksandra Lipka, Łukasz Paukszto, Victoria C. Kennedy, Amelia R. Tanner, Marta Majewska and Russell V. Anthony

Cells 2024, 13(5), 391; https://doi.org/10.3390/cells13050391 - 24 Feb 2024

Cited by 1 | Viewed by 1512

Abstract

While glucose is the primary fuel for fetal growth, the placenta utilizes the majority of glucose taken up from the maternal circulation. Of the facilitative glucose transporters in the placenta, SLC2A8 (GLUT8) is thought to primarily function as an intracellular glucose transporter; however, [...] Read more.

While glucose is the primary fuel for fetal growth, the placenta utilizes the majority of glucose taken up from the maternal circulation. Of the facilitative glucose transporters in the placenta, SLC2A8 (GLUT8) is thought to primarily function as an intracellular glucose transporter; however, its function in trophoblast cells has not been determined. To gain insight into the function of SLC2A8 in the placenta, lentiviral-mediated RNA interference (RNAi) was performed in the human first-trimester trophoblast cell line ACH-3P. Non-targeting sequence controls (NTS RNAi; n = 4) and SLC2A8 RNAi (n = 4) infected ACH-3P cells were compared. A 79% reduction in SLC2A8 mRNA concentration was associated with an 11% reduction (p ≤ 0.05) in ACH-3P glucose uptake. NTS RNAi and SLC2A8 RNAi ACH-3P mRNA were subjected to RNAseq, identifying 1525 transcripts that were differentially expressed (|log2FC| > 1 and adjusted p-value < 0.05), with 273 transcripts derived from protein-coding genes, and the change in 10 of these mRNAs was validated by real-time qPCR. Additionally, there were 147 differentially expressed long non-coding RNAs. Functional analyses revealed differentially expressed genes involved in various metabolic pathways associated with cellular respiration, oxidative phosphorylation, and ATP synthesis. Collectively, these data indicate that SLC2A8 deficiency may impact placental uptake of glucose, but that its likely primary function in trophoblast cells is to support cellular respiration. Since the placenta oxidizes the majority of the glucose it takes up to support its own metabolic needs, impairment of SLC2A8 function could set the stage for functional placental insufficiency. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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28 pages, 4728 KiB

Open AccessArticle

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH

by Nune Darbinian, Nana Merabova, Gabriel Tatevosian, Mary Morrison, Armine Darbinyan, Huaqing Zhao, Laura Goetzl and Michael Edgar Selzer

Cells 2024, 13(1), 2; https://doi.org/10.3390/cells13010002 - 19 Dec 2023

Cited by 2 | Viewed by 2040

Abstract

Introduction: Children with fetal alcohol spectrum disorders (FASD) exhibit behavioral and affective dysregulation, including hyperactivity and depression. The mechanisms are not known, but they could conceivably be due to postnatal social or environmental factors. However, we postulate that, more likely, the affective dysregulation [...] Read more.

Introduction: Children with fetal alcohol spectrum disorders (FASD) exhibit behavioral and affective dysregulation, including hyperactivity and depression. The mechanisms are not known, but they could conceivably be due to postnatal social or environmental factors. However, we postulate that, more likely, the affective dysregulation is associated with the effects of EtOH exposure on the development of fetal serotonergic (5-HT) and/or dopaminergic (DA) pathways, i.e., pathways that in postnatal life are believed to regulate mood. Many women who use alcohol (ethanol, EtOH) during pregnancy suffer from depression and take selective serotonin reuptake inhibitors (SSRIs), which might influence these monoaminergic pathways in the fetus. Alternatively, monoaminergic pathway abnormalities might reflect a direct effect of EtOH on the fetal brain. To distinguish between these possibilities, we measured their expressions in fetal brains and in fetal brain-derived exosomes (FB-Es) isolated from the mothers’ blood. We hypothesized that maternal use of EtOH and/or SSRIs during pregnancy would be associated with impaired fetal neural development, detectable as abnormal levels of monoaminergic and apoptotic biomarkers in FB-Es. Methods: Fetal brain tissues and maternal blood were collected at 9–23 weeks of pregnancy. EtOH groups were compared with unexposed controls matched for gestational age (GA). The expression of 84 genes associated with the DA and 5-HT pathways was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on microarrays. FB-Es also were assayed for serotonin transporter protein (SERT) and brain-derived neurotrophic factor (BDNF) by enzyme-linked immunosorbent assay (ELISA). Results: Six EtOH-exposed human fetal brain samples were compared to SSRI- or polydrug-exposed samples and to unexposed controls. EtOH exposure was associated with significant upregulation of DA receptor D3 and 5-HT receptor HTR2C, while HTR3A was downregulated. Monoamine oxidase A (MAOA), MAOB, the serine/threonine kinase AKT3, and caspase-3 were upregulated, while mitogen-activated protein kinase 1 (MAPK1) and AKT2 were downregulated. ETOH was associated with significant upregulation of the DA transporter gene, while SERT was downregulated. There were significant correlations between EtOH exposure and (a) caspase-3 activation, (b) reduced SERT protein levels, and (c) reduced BDNF levels. SSRI exposure independently increased caspase-3 activity and downregulated SERT and BDNF. Early exposure to EtOH and SSRI together was associated synergistically with a significant upregulation of caspase-3 and a significant downregulation of SERT and BDNF. Reduced SERT and BDNF levels were strongly correlated with a reduction in eye diameter, a somatic manifestation of FASD. Conclusions: Maternal use of EtOH and SSRI during pregnancy each was associated with changes in fetal brain monoamine pathways, consistent with potential mechanisms for the affective dysregulation associated with FASD. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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20 pages, 6510 KiB

Open AccessArticle

Cellular Functions of High-Temperature Requirement Factor A4 in Placenta

by Chang-Zhu Pei, Bum-Chae Choi, Jun-Hyeok Park, Hyo Young Park, Jinyoung Paek, Kyung-Ju Lee, Bo-Seong Yun, Young Ju Kim and Kwang-Hyun Baek

Cells 2023, 12(11), 1459; https://doi.org/10.3390/cells12111459 - 24 May 2023

Cited by 3 | Viewed by 1954

Abstract

The expression of High-temperature requirement factor A4 (HtrA4) mRNA is significantly lower in the chorionic villi of patients with recurrent pregnancy loss (RPL) than in the control group. We conducted an investigation into the cellular functions of HtrA4 using the CRISPR/Cas9 system and [...] Read more.

The expression of High-temperature requirement factor A4 (HtrA4) mRNA is significantly lower in the chorionic villi of patients with recurrent pregnancy loss (RPL) than in the control group. We conducted an investigation into the cellular functions of HtrA4 using the CRISPR/Cas9 system and shRNA-HtrA4 to create knockout BeWo cells and HtrA4 knockdown JEG3 cells. Our results indicated that the knockout BeWo cells exhibited reduced capacity for invasion and fusion, but increased levels of proliferation and migration, with a significantly shortened cell cycle compared to wild-type cells. Wild-type BeWo cells highly expressed cell invasion- and fusion-related factors, while knockout BeWo cells highly expressed migration-, proliferation-, and cell cycle-related factors. The shRNA-HtrA4 JEG3 cells showed a decreased capacity for invasion, but an increased capacity for migration, accompanied by a decrease in the expression of cell invasion-related factors and an increase in migration-related factors. Moreover, our ELISA results revealed that the serum HtrA4 level was lower in patients with RPL than in the controls. These findings suggest that HtrA4 depletion may be associated with placental dysfunction. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Generation of HtrA4 knockout in BeWo cells. (A) BeWo WT cells were transfected with HtrA4-sgRNA-pX458 plasmid through electroporation. After 36 h, the transfection efficiency was determined using fluorescent microscopy. (B) HtrA4 T7E1 primers were utilized to amplify PCR products containing the sgRNA target in both BeWo WT and BeWo KO cells. (C) The T7E1 assay was performed on the PCR products to detect the cleavage of the DNA. (D) Direct sequencing of PCR products was performed on both alleles 1 and 2. The sgRNA target site is underlined in purple, PAM sequences are underlined in green, deletion sequences are underlined in red, co-present sequences near 5′ primer are framed in pink, and those near 3′ primer are framed in blue. This fragment contains all the components required for gRNA expression. RT-PCR and western blot analysis, respectively, were performed to measure HtrA4 mRNA (E) and HtrA4 protein (F) in BeWo WT and BeWo KO cells. Each experiment was performed in triplicate. Full article ">Figure 1 Cont.
Generation of HtrA4 knockout in BeWo cells. (A) BeWo WT cells were transfected with HtrA4-sgRNA-pX458 plasmid through electroporation. After 36 h, the transfection efficiency was determined using fluorescent microscopy. (B) HtrA4 T7E1 primers were utilized to amplify PCR products containing the sgRNA target in both BeWo WT and BeWo KO cells. (C) The T7E1 assay was performed on the PCR products to detect the cleavage of the DNA. (D) Direct sequencing of PCR products was performed on both alleles 1 and 2. The sgRNA target site is underlined in purple, PAM sequences are underlined in green, deletion sequences are underlined in red, co-present sequences near 5′ primer are framed in pink, and those near 3′ primer are framed in blue. This fragment contains all the components required for gRNA expression. RT-PCR and western blot analysis, respectively, were performed to measure HtrA4 mRNA (E) and HtrA4 protein (F) in BeWo WT and BeWo KO cells. Each experiment was performed in triplicate. Full article ">Figure 2
Effect of HtrA4 on cell fusion. (A) BeWo WT, BeWo KO, and BeWo KO-HtrA4 rescue cells were subjected to two conditions: they were treated with 50 μM of forskolin or equal amounts of DMSO to induce cell fusion. After 48 h, an immunocytochemistry analysis was performed to examine the expression of E-cadherin and β-hCG. nuclei were stained blue with DAPI, while E-cadherin and β-hCG were labelled with red and green, respectively. (B) Quantification of the normalized fluorescence intensity of E-cadherin. *** p < 0.001, ns p > 0.05. Each experiment was performed in triplicate. Full article ">Figure 2 Cont.
Effect of HtrA4 on cell fusion. (A) BeWo WT, BeWo KO, and BeWo KO-HtrA4 rescue cells were subjected to two conditions: they were treated with 50 μM of forskolin or equal amounts of DMSO to induce cell fusion. After 48 h, an immunocytochemistry analysis was performed to examine the expression of E-cadherin and β-hCG. nuclei were stained blue with DAPI, while E-cadherin and β-hCG were labelled with red and green, respectively. (B) Quantification of the normalized fluorescence intensity of E-cadherin. *** p < 0.001, ns p > 0.05. Each experiment was performed in triplicate. Full article ">Figure 3
Transwell invasion and scratch wound healing assays in BeWo and JEG3 cells. (A) Representative images of the invasion assay in BeWo WT, BeWo KO, and BeWo KO-HtrA4 rescue cells were obtained, and cell counts were compared among the groups. (B) Representative images of the wound healing assay in BeWo WT, BeWo KO, and BeWo KO-HtrA4 rescue cells were obtained after treatment with mitomycin C, and migration distances were compared. (C) Western blot analysis was performed with 30 μg of protein from whole cell lysate to determine the expressions of MMP-2, MMP-9, p-FAK (pY397), and FAK. (D) Representative images of the invasion assay in JEG3 control and shRNA-HtrA4 JEG3 cells were obtained, and cell counts were compared between the groups. (E) Representative images of the wound healing assay in JEG3 control and shRNA-HtrA4 JEG3 cells were obtained, and migration distances were compared. (F) Western blot analysis was performed with 30 μg of protein from whole cell lysate to determine the expressions of MMP-2, MMP-9, p-FAK (pY397), and FAK. β-actin was used as an internal control, and the statistical analysis of protein quantification using western blotting was performed using t-test. Data are presented as the means ± standard error (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05. Each experiment was performed in triplicate. Full article ">Figure 3 Cont.
Transwell invasion and scratch wound healing assays in BeWo and JEG3 cells. (A) Representative images of the invasion assay in BeWo WT, BeWo KO, and BeWo KO-HtrA4 rescue cells were obtained, and cell counts were compared among the groups. (B) Representative images of the wound healing assay in BeWo WT, BeWo KO, and BeWo KO-HtrA4 rescue cells were obtained after treatment with mitomycin C, and migration distances were compared. (C) Western blot analysis was performed with 30 μg of protein from whole cell lysate to determine the expressions of MMP-2, MMP-9, p-FAK (pY397), and FAK. (D) Representative images of the invasion assay in JEG3 control and shRNA-HtrA4 JEG3 cells were obtained, and cell counts were compared between the groups. (E) Representative images of the wound healing assay in JEG3 control and shRNA-HtrA4 JEG3 cells were obtained, and migration distances were compared. (F) Western blot analysis was performed with 30 μg of protein from whole cell lysate to determine the expressions of MMP-2, MMP-9, p-FAK (pY397), and FAK. β-actin was used as an internal control, and the statistical analysis of protein quantification using western blotting was performed using t-test. Data are presented as the means ± standard error (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05. Each experiment was performed in triplicate. Full article ">Figure 4
Effect of HtrA4 on cell proliferation. (A) Cell viability was evaluated using CCK-8 assay in BeWo WT, BeWo KO, and BeWo KO-HtrA4 rescue cells, and optical density (OD) values were measured at 0, 1, 2, 3, 4, and 5 days. (B) The colony formation assay was conducted in BeWo WT and BeWo KO cells, and representative images were captured. The number of cell colonies was compared between the two groups. (C) CCK-8 assay was conducted in control and shRNA-HtrA4 JEG3 cells, and OD values were measured at 0, 2, 4, 6, and 8 days. (D) The colony formation assay was conducted in JEG3 cells, and representative images were captured. The number of cell colonies was compared between control and shRNA-HtrA4 JEG3 cells. (E) Western blot analysis was conducted using 30 μg of proteins from whole cell lysate to determine the expressions of ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, Ras, Raf-1, MKK3, and MKK6. β-actin was used as an internal control. Statistical analysis of protein quantification in western blotting was performed using a t-test. Data are presented as the means ± standard error (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05. Each experiment was conducted in triplicate. Full article ">Figure 4 Cont.
Effect of HtrA4 on cell proliferation. (A) Cell viability was evaluated using CCK-8 assay in BeWo WT, BeWo KO, and BeWo KO-HtrA4 rescue cells, and optical density (OD) values were measured at 0, 1, 2, 3, 4, and 5 days. (B) The colony formation assay was conducted in BeWo WT and BeWo KO cells, and representative images were captured. The number of cell colonies was compared between the two groups. (C) CCK-8 assay was conducted in control and shRNA-HtrA4 JEG3 cells, and OD values were measured at 0, 2, 4, 6, and 8 days. (D) The colony formation assay was conducted in JEG3 cells, and representative images were captured. The number of cell colonies was compared between control and shRNA-HtrA4 JEG3 cells. (E) Western blot analysis was conducted using 30 μg of proteins from whole cell lysate to determine the expressions of ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, Ras, Raf-1, MKK3, and MKK6. β-actin was used as an internal control. Statistical analysis of protein quantification in western blotting was performed using a t-test. Data are presented as the means ± standard error (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05. Each experiment was conducted in triplicate. Full article ">Figure 5
Effect of HtrA4 on cell cycle. (A) Cell cycle distribution and quantitative measurement of cell cycle phases in BeWo WT cells over a 3 h period. (B) Cell cycle distribution and quantitative measurement of cell cycle phases in BeWo KO cells over a 3 h period. (C) Western blot analysis was performed with 30 μg of proteins to determine the expression levels of Cyclin D, Cyclin A, and Cyclin E. β-actin was used as an internal control. Statistical analysis of protein quantification in western blotting was performed using a t-test. Data are presented as the means ± standard error (SD). * p < 0.05, ** p < 0.01, *** p < 0.001. Each experiment was performed in triplicate. Full article ">Figure 6
Detection of serum HtrA4 level. The HtrA4 protein was determined using ELISA in sera obtained from normal controls and RPL patients with regular menstrual cycles on the 5th to 9th days after ovulation in the menstrual cycle. *** p < 0.001. The experiment was performed for 32 controls and 60 RPL patients at the same time. Full article ">

16 pages, 2941 KiB

Open AccessArticle

Maternal and Intrauterine Influences on Feto-Placental Growth Are Accompanied by Sexually Dimorphic Changes in Placental Mitochondrial Respiration, and Metabolic Signalling Pathways

by Esteban Salazar-Petres, Daniela Pereira-Carvalho, Jorge Lopez-Tello and Amanda N. Sferruzzi-Perri

Cells 2023, 12(5), 797; https://doi.org/10.3390/cells12050797 - 3 Mar 2023

Cited by 2 | Viewed by 2446

Abstract

Adverse maternal environments such as small size, malnutrition, and metabolic conditions are known to influence fetal growth outcomes. Similarly, fetal growth and metabolic alterations may alter the intrauterine environment and affect all fetuses in multiple gestation/litter-bearing species. The placenta is the site of [...] Read more.

Adverse maternal environments such as small size, malnutrition, and metabolic conditions are known to influence fetal growth outcomes. Similarly, fetal growth and metabolic alterations may alter the intrauterine environment and affect all fetuses in multiple gestation/litter-bearing species. The placenta is the site of convergence between signals derived from the mother and the developing fetus/es. Its functions are fuelled by energy generated by mitochondrial oxidative phosphorylation (OXPHOS). The aim of this study was to delineate the role of an altered maternal and/or fetal/intrauterine environment in feto-placental growth and placental mitochondrial energetic capacity. To address this, in mice, we used disruptions of the gene encoding phosphoinositol 3-kinase (PI3K) p110α, a growth and metabolic regulator to perturb the maternal and/or fetal/intrauterine environment and study the impact on wildtype conceptuses. We found that feto-placental growth was modified by a perturbed maternal and intrauterine environment, and effects were most evident for wildtype males compared to females. However, placental mitochondrial complex I+II OXPHOS and total electron transport system (ETS) capacity were similarly reduced for both fetal sexes, yet reserve capacity was additionally decreased in males in response to the maternal and intrauterine perturbations. These were also sex-dependent differences in the placental abundance of mitochondrial-related proteins (e.g., citrate synthase and ETS complexes), and activity of growth/metabolic signalling pathways (AKT and MAPK) with maternal and intrauterine alterations. Our findings thus identify that the mother and the intrauterine environment provided by littermates modulate feto-placental growth, placental bioenergetics, and metabolic signalling in a manner dependent on fetal sex. This may have relevance for understanding the pathways leading to reduced fetal growth, particularly in the context of suboptimal maternal environments and multiple gestation/litter-bearing species. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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Figure 1
Illustrative figure representing the crosses used in the study. WT = wild-type; α/+ = Heterozygous Pik3ca-D933A mice; F = female fetus; M = male fetus. Full article ">Figure 2
Fetal and placental growth of WT conceptuses in response to littermate and/or maternal p110α deficiency. Fetal weight (A), placenta weight (B), LZ weight (C), and fetal weight/LZ weight (D) in females and males on day 18 of pregnancy. Data are from WT fetuses generated by WT x WT, WT x α/+, and α/+ x WT parental crosses (n = 1–2 fetuses/sex/dam with 5–12 dams/group) and are displayed as individual data points with mean ± S.E.M. Data were analysed by one-way ANOVA with Tukey post hoc pairwise comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, pairwise comparison). LZ: labyrinthine zone. Full article ">Figure 3
Placental mitochondrial bioenergetics of WT conceptuses in response to littermate and/or maternal p110α deficiency. Oxygen consumption in the placental LZ associated with CILEAK (A), CIOXPHOS (B), CI + CIIOXPHOS (C), CII (D), total ETS (E), reserve capacity (F), CILEAK/total ETS (G), and CIOXPHOS/total ETS (H) for females and males on day 18 of pregnancy. Data are from WT fetuses generated by WT x WT, WT x α/+, and α/+ x WT parental crosses (n = 1–2 fetuses/sex/dam with 5–12 dams/group) and are displayed as individual data points with mean ± S.E.M. Data were analysed by one-way ANOVA with Tukey post hoc pairwise comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, pairwise comparison). Full article ">Figure 4
Protein abundance of mitochondrial complexes and key mitochondrial regulatory proteins in placental labyrinth of WT conceptuses in response to littermate and/or maternal p110α deficiency. Relative protein abundance of mitochondrial complexes in females (A) and males (B), as well as citrate synthase (C), PGC1α (D), PPARγ (E), and UCP2 (F) in females and males. Representative images from each antibody and Ponceau staining are included. Data are from 1 WT fetus per dam generated by WT x WT (n = 4), WT x α/+ (n = 5), and α/+ x WT (n = 5) parental crosses and are displayed as individual data points with mean ± S.E.M. Data were analysed by one-way ANOVA and Tukey post hoc pairwise comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, pairwise comparison). Full article ">Figure 5
Abundance of growth and metabolic signalling proteins in placental labyrinth of WT conceptuses in response to littermate and/or maternal p110α deficiency. Female (A) and male (B) representative images from each antibody immunodetection and Ponceau staining for phosphorylated and total AKT, AMPKα, MAPK 44/42, and P38 MAPK. Total AKT, AMPKα, MAPK 44/42, and P38 MAPK protein levels in females and males (C), and AKT, AMPKα, MAPK 44/42, and P38 MAPK phosphorylation levels as a ratio to total protein in females and males (D). Data are from 1 WT fetus per dam generated by WT x WT (n = 4), WT x α/+ (n = 5), and α/+ x WT (n = 5) parental crosses and are displayed as individual data points with mean ± S.E.M. Data were analysed by one-way ANOVA with Tukey post hoc pairwise comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, pairwise comparison). p = phosphorylated, t = total. Full article ">

9 pages, 1022 KiB

Open AccessCommunication

Enoxaparin Increases D6 Receptor Expression and Restores Cytoskeleton Organization in Trophoblast Cells from Preeclampsia

by Chiara Tersigni, Giuseppe Maulucci, Roberta Castellani, Giada Bianchetti, Marianna Onori, Rita Franco, Greta Barbaro, Marco De Spirito, Antonio Lanzone, Giovanni Scambia and Nicoletta Di Simone

Cells 2022, 11(13), 2036; https://doi.org/10.3390/cells11132036 - 27 Jun 2022

Cited by 1 | Viewed by 1782

Abstract

D6 is a scavenger receptor for CC chemokines expressed in the human placenta. It prevents excessive leukocyte tissue infiltration by internalizing chemokines through cytoskeleton-dependent intracellular transport. In preeclampsia (PE), the D6 receptor is overexpressed in trophoblast cells, but functionally impaired, due to cytoskeleton [...] Read more.

D6 is a scavenger receptor for CC chemokines expressed in the human placenta. It prevents excessive leukocyte tissue infiltration by internalizing chemokines through cytoskeleton-dependent intracellular transport. In preeclampsia (PE), the D6 receptor is overexpressed in trophoblast cells, but functionally impaired, due to cytoskeleton destructuring. Low molecular weight heparin (LMWH) represents a potential treatment for PE based on its anti-thrombotic and anti-inflammatory properties. Here, we investigated the effect of enoxaparin on D6 expression, and cytoskeleton organization primary cytotrophoblast cell cultures were obtained from the placentae of women with PE (n = 9) or uncomplicated pregnancy (n = 9). We demonstrated that enoxaparin is able to (i) increase D6 expression, and (ii) improve cytoskeletal fiber alignment in trophoblast cells from PE patients. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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Figure 1

Figure 1
(A) Representative immunoblotting showing expression of D6 receptor in trophoblast cell lysates from PE or controls in basal conditions (0) and after incubation with LMWH for 24 h (1 and 10 UI/mL). (B) Quantitative analysis of Western blot results shows higher expression of D6 receptor in trophoblast cells from preeclamptic women (n = 3) after incubation with enoxaparin for 24 h (1 UI/mL) compared to basal conditions (0). No significant differences in terms of D6 expression were observed in trophoblast cells from normal pregnancy (CTR, n = 3) with or without LMWH treatment. (C–E) Confocal analysis of trophoblast cells showed a basal higher expression of D6 in preeclamptic women (n = 9) compared to controls (n = 9) and a significant increase of D6 expression in PE after cells incubation with LMWH (10 UI/mL) for 24 h. Data are expressed as the mean ± standard error (SE). PE: preeclampsia; CTR: control; LMWH: low molecular weight heparin; NS: not significant. * p < 0.05; **** p < 0.0001. Full article ">Figure 2
(A1–B2) F-actin staining of trophoblast cells from normal pregnant (A1,A2) and PE (B1,B2) women before (A1,B1) and after (A2,B2) LMWH incubation (10 UI/mL) for 24 h. (C) F-actin polymerization analyzed as eccentricity is significantly reduced in PE compared to controls. Incubation of trophoblast cells with LMWH significantly improved F-actin polymerization and cytoskeleton spatial organization. No significant difference was found in cells obtained from normal pregnant women after treatment with LMWH. Data are expressed as median with related confidence interval. ** p < 0.01. Full article ">

11 pages, 1793 KiB

Open AccessArticle

Lung Inflammation Is Associated with Preeclampsia Development in the Rat

by Katrina Curtis, Derek Clarke, Makayla Hanegan, Brendan Stapley, Ryan Wendt, Nathan Beckett, Cade Litchfield, Kennedy Campbell, Paul Reynolds and Juan Arroyo

Cells 2022, 11(12), 1884; https://doi.org/10.3390/cells11121884 - 10 Jun 2022

Viewed by 2331

Abstract

Preeclampsia (PE) is an obstetric complication associated with significant health implications for the fetus and mother. Studies have shown a correlation between lung disease development and PE. Gas6 protein is expressed in the lung and placenta, and binds to the AXL Tyrosine kinase [...] Read more.

Preeclampsia (PE) is an obstetric complication associated with significant health implications for the fetus and mother. Studies have shown a correlation between lung disease development and PE. Gas6 protein is expressed in the lung and placenta, and binds to the AXL Tyrosine kinase receptor. Recently, our laboratory utilized Gas6 to induce preeclamptic-like conditions in rats. Our objective was to determine the role of Gas6/AXL signaling in the maternal lung during PE development. Briefly, pregnant rats were divided into control, Gas6, or Gas6 + R428 (an AXL inhibitor). Immunofluorescence was performed to determine AXL expression. Bronchoalveolar lavage fluid (BALF) was procured for the assessment of inflammatory cell secretion. Western blot was performed to detect signaling molecules and ELISA determined inflammatory cytokines. We observed increased proteinuria and increased blood pressure in Gas6-treated animals. AXL was increased in the lungs of the treated animals and BALF fluid revealed elevated total protein abundance in Gas6 animals. Extracellular-signal regulated kinase (ERK) and protein kinase B (AKT) signaling in the lung appeared to be mediated by Gas6 as well as the secretion of inflammatory cytokines. We conclude that Gas6 signaling is capable of inducing PE and that this is associated with increased lung inflammation. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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Figure 1
Blood Pressure and proteinuria during Gas6 treatment in the pregnant rat. There was an increase in blood systolic (A) and diastolic pressure (B) and urine proteinuria (+3 and +4) (C) in treated animals as compared to controls (n = 10). Systolic (A) and Diastolic (B) blood pressures and proteinuria (C) returned to basal levels in animals treated with Gas6 and the Axl inhibitor as compared to those treated with Gas6 alone. Representative data are shown with p ≤ 0.05. Full article ">Figure 2
Lung AXL expression during Gas6 treatment in the pregnant rat. Hematoxylin staining was performed for lung structure determination (A). There was an increase in AXL mRNA in the treated animals as compared to controls (B). This increase was also observed in AXL protein levels in the lung of treated animals as compared to controls (C). Full article ">Figure 3
BALF analysis in control and treated animals. Cell count (A) and protein (B) were increased in the BALF of Gas6-treated animals as compared to controls. Levels were decreased in animals co-treated with R428 and Gas6 (n = 10). Representative data are shown with p ≤ 0.05. Full article ">Figure 4
Lung-signaling molecules in control and treated animals. A representative Western blot for ERK and Akt is shown in (A,C). Lung levels of ERK (B), and AKT (D) were increased in Gas6-treated animals compared to controls (n = 6 per group). Molecules were deceased in animals co-treated with R428. Representative data are shown with p ≤ 0.05. Full article ">Figure 5
Inflammatory cytokines in the serum of control and treated animals. Serum levels of IL-1???? (A), IL-2 (B), and TNF???? (C) were increased in Gas6-treated animals compared to controls (n = 10). Cytokines were deceased in animals co-treated with R428. Coefficients of variations for the experiments are found in (D). Representative data are shown with p≤ 0.05. Full article ">

Review

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22 pages, 1446 KiB

Open AccessReview

The Role of the NLRP3 Inflammasome in the Molecular and Biochemical Mechanisms of Cervical Ripening: A Comprehensive Review

by Wojciech Flis and Maciej W. Socha

Cells 2024, 13(7), 600; https://doi.org/10.3390/cells13070600 - 29 Mar 2024

Cited by 2 | Viewed by 1394

Abstract

The uterine cervix is one of the key factors involved in ensuring a proper track of gestation and labor. At the end of the gestational period, the cervix undergoes extensive changes, which can be summarized as a transformation from a non-favorable cervix to [...] Read more.

The uterine cervix is one of the key factors involved in ensuring a proper track of gestation and labor. At the end of the gestational period, the cervix undergoes extensive changes, which can be summarized as a transformation from a non-favorable cervix to one that is soft and prone to dilation. During a process called cervical ripening, fundamental remodeling of the cervical extracellular matrix (ECM) occurs. The cervical ripening process is a derivative of many interlocking and mutually driving biochemical and molecular pathways under the strict control of mediators such as inflammatory cytokines, nitric oxide, prostaglandins, and reactive oxygen species. A thorough understanding of all these pathways and learning about possible triggering factors will allow us to develop new, better treatment algorithms and therapeutic goals that could protect women from both dysfunctional childbirth and premature birth. This review aims to present the possible role of the NLRP3 inflammasome in the cervical ripening process, emphasizing possible mechanisms of action and regulatory factors. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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19 pages, 1259 KiB

Open AccessReview

Modulation of NRF2/KEAP1 Signaling in Preeclampsia

by Giovanni Tossetta, Sonia Fantone, Federica Piani, Caterina Crescimanno, Andrea Ciavattini, Stefano Raffaele Giannubilo and Daniela Marzioni

Cells 2023, 12(11), 1545; https://doi.org/10.3390/cells12111545 - 4 Jun 2023

Cited by 56 | Viewed by 3277

Abstract

Placentation is a key and tightly regulated process that ensures the normal development of the placenta and fetal growth. Preeclampsia (PE) is a hypertensive pregnancy-related disorder involving about 5–8% of all pregnancies and clinically characterized by de novo maternal hypertension and proteinuria. In [...] Read more.

Placentation is a key and tightly regulated process that ensures the normal development of the placenta and fetal growth. Preeclampsia (PE) is a hypertensive pregnancy-related disorder involving about 5–8% of all pregnancies and clinically characterized by de novo maternal hypertension and proteinuria. In addition, PE pregnancies are also characterized by increased oxidative stress and inflammation. The NRF2/KEAP1 signaling pathway plays an important role in protecting cells against oxidative damage due to increased reactive oxygen species (ROS) levels. ROS activate NRF2, allowing its binding to the antioxidant response element (ARE) region present in the promoter of several antioxidant genes such as heme oxygenase, catalase, glutathione peroxidase and superoxide dismutase that neutralize ROS, protecting cells against oxidative stress damages. In this review, we analyze the current literature regarding the role of the NRF2/KEAP1 pathway in preeclamptic pregnancies, discussing the main cellular modulators of this pathway. Moreover, we also discuss the main natural and synthetic compounds that can regulate this pathway in in vivo and in vitro models. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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16 pages, 1177 KiB

Open AccessReview

Cross-Generational Impact of Innate Immune Memory Following Pregnancy Complications

by Nakeisha A. Lodge-Tulloch, Alexa J. Toews, Aline Atallah, Tiziana Cotechini, Sylvie Girard and Charles H. Graham

Cells 2022, 11(23), 3935; https://doi.org/10.3390/cells11233935 - 6 Dec 2022

Cited by 7 | Viewed by 3064

Abstract

Pregnancy complications can have long-term negative effects on the health of the affected mothers and their children. In this review, we highlight the underlying inflammatory etiologies of common pregnancy complications and discuss how aberrant inflammation may lead to the acquisition of innate immune [...] Read more.

Pregnancy complications can have long-term negative effects on the health of the affected mothers and their children. In this review, we highlight the underlying inflammatory etiologies of common pregnancy complications and discuss how aberrant inflammation may lead to the acquisition of innate immune memory. The latter can be described as a functional epigenetic reprogramming of innate immune cells following an initial exposure to an inflammatory stimulus, ultimately resulting in an altered response following re-exposure to a similar inflammatory stimulus. We propose that aberrant maternal inflammation associated with complications of pregnancy increases the cross-generational risk of developing noncommunicable diseases (i.e., pregnancy complications, cardiovascular disease, and metabolic disease) through a process mediated by innate immune memory. Elucidating a role for innate immune memory in the cross-generational health consequences of pregnancy complications may lead to the development of novel strategies aimed at reducing the long-term risk of disease. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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25 pages, 866 KiB

Open AccessReview

Signaling Pathways Regulating Human Cervical Ripening in Preterm and Term Delivery

by Maciej W. Socha, Wojciech Flis, Miłosz Pietrus, Mateusz Wartęga and Martyna Stankiewicz

Cells 2022, 11(22), 3690; https://doi.org/10.3390/cells11223690 - 21 Nov 2022

Cited by 11 | Viewed by 6721

Abstract

At the end of gestation, the cervical tissue changes profoundly. As a result of these changes, the uterine cervix becomes soft and vulnerable to dilation. The process occurring in the cervical tissue can be described as cervical ripening. The ripening is a process [...] Read more.

At the end of gestation, the cervical tissue changes profoundly. As a result of these changes, the uterine cervix becomes soft and vulnerable to dilation. The process occurring in the cervical tissue can be described as cervical ripening. The ripening is a process derivative of enzymatic breakdown and inflammatory response. Therefore, it is apparent that cervical remodeling is a derivative of the reactions mediated by multiple factors such as hormones, prostaglandins, nitric oxide, and inflammatory cytokines. However, despite the research carried out over the years, the cellular pathways responsible for regulating this process are still poorly understood. A comprehensive understanding of the entire process of cervical ripening seems crucial in the context of labor induction. Greater knowledge could provide us with the means to help women who suffer from dysfunctional labor. The overall objective of this review is to present the current understanding of cervical ripening in terms of molecular regulation and cell signaling. Full article

(This article belongs to the Special Issue Signaling Pathways in Pregnancy)

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