Aquaporins in Fetal Development.

Water homeostasis is essential for fetal growth, and it depends on the successful development of the placenta. Many aquaporins (AQPs) were identified from blastocyst stages to term placenta. In the last years, cytokines, hormones, second messengers, intracellular pH, and membrane proteins were found to regulate their expression and function in the human placenta and fetal membranes. Accumulated data suggest that these proteins may be involved not only in the maintenance of the amniotic fluid volume homeostasis but also in the development of the placenta and fetal organs. In this sense, dysregulation of placental AQPs is associated with gestational disorders. Thus, current evidence shows that AQPs may collaborate in cellular events including trophoblast migration and apoptosis. In addition, aquaglyceroporins are involved in energy metabolism as well as urea elimination across the placenta. In the last year, the presence of AQP9 in trophoblast mitochondria opened new hypotheses about its role in pregnancy. However, much further work is needed to understand the importance of these proteins in human pregnancies.

The Placental Function Beyond Pregnancy: Insights from Latin America.

Currently, more than 100,000 papers had been published studying the placenta in both physiological and pathological contexts. However, relevant health conditions affecting placental function, mostly found in low-income countries, should be evaluated deeper. This review will raise some - of what we think necessary - points of discussion regarding challenging topics not fully understood, including the paternal versus maternal contribution on placental genes imprinting, placenta-brain communication, and some environmental conditions affecting the placenta. The discussions are parts of an international effort to fulfil some gaps observed in this area, and Latin-American research groups currently evaluate that.

Contribution of aquaporins in the transamniotic water flux.

We explored the contribution of each aquaporin (AQP) expressed in human amnion in the transcellular water flux across the human amnion. Human amnion was placed between two lucite chambers and net water transport (Jw) was recorded by applying a hydrostatic (7 cm H2O) and an osmotic (40 mOsm PEG 8000) pressure gradients. The hydrostatic (Phydr) and osmotic (POsm) permeabilities were calculated before and after the blocking of AQPs. Phdr showed no significant difference after the blocking of AQPs, while POsm was dramatically reduced. Interestingly, we also found that the blocking of AQP1 produced the highest decrease of POsm (80 ± 1%). Our results strongly suggested that AQP1 seems to contribute more to the maintenance of AF volume homeostasis.

Intact caveolae are required for proper extravillous trophoblast migration and differentiation.

Caveolae constitute membrane domains critical for the organization and synchronization of different signaling molecules related to numerous cell processes such as cell migration, invasion, and differentiation. Caveolin-1 (Cav-1) is the main integral membrane protein of these domains. Recently, it was found that a normal expression of aquaporin-3 (AQP3) is required for extravillous trophoblast (EVT) cell migration. Our aim was to investigate the role of caveolae in the migration, invasion, and endovascular differentiation of human EVT cells during placentation and its interaction with AQP3. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's modified Eagle's medium-nutrient mixture F12 and treated with 5 mM methyl-ß-cyclodextrin (MßCD) to disrupt caveolae. We found that after MßCD treatment, Cav-1 protein was undetectable. In this condition, the ability of the cells to migrate was significantly decreased compared with the control cells, while no differences were observed in the number of invading cells and the metalloproteinases activity between control and MßCD-treated cells. Surprisingly, the disruption of caveolae significantly enhanced EVT endovascular differentiation. On the contrary, the silencing of AQP3, negatively affected tube-like formation. The theoretical analysis of the primary sequence of AQP3 protein revealed a putative Cav-1-binding site. In addition, immunoprecipitation and double immunofluorescence assays showed that AQP3 colocalized with Cav-1. These results showed that during placentation an intact caveola in EVT cells may be necessary for AQP3 and Cav-1 interaction and any perturbations might result in serious pregnancy disorders.

Osmotic stress induces apoptosis in extravillous trophoblast cells. Role of TRPV-1.

In different tissues hyperosmolarity induces cell differentiation. Nevertheless an exacerbated hyperosmolar stress alters the normal cellular development. The transient receptor potential vanilloid 1 (TRPV-1) is a non-selective cation channel that is activated by hyperosmolarity and participates in many cellular processes. TPRV-1 is expressed in human placenta at term. Here, we evaluated the expression of TRPV-1 in first trimester extravillous trophoblast cells and its participation in the survival of these cells exposed to hyperosmolar stress. Our results showed that hyperosmolar stress up-regulates the expression of TRPV-1 and induces the apoptosis in Swan 71 cells. In addition, the inhibition of TRPV-1 abrogates the apoptotic events.

Cytotoxic effects of Shiga toxin-2 on human extravillous trophoblast cell lines.

Shiga toxin (Stx2) producing Escherichia coli infections during early gestation may impair placentation through a Stx2 damage of extravillous trophoblast (EVT) cells. We have previously demonstrated that Stx2 injected in rats in the early stage of pregnancy causes spontaneous abortion by a direct cytotoxic effect in the highly perfused feto-uteroplacental unit. The main aim was to evaluate the effects of Stx2 on EVT in order to understand the possible adverse effects that the toxin may have on trophoblast cells during early pregnancy. Swan 71 and HTR-8 cell lines were used as human EVT models. The presence of Stx2 receptor, globotriaosylceramide (Gb3), on Swan 71 and HTR-8 cells was evaluated by thin layer chromatography. The effects of Stx2 on cell viability were evaluated by neutral red uptake, migration by wound-healing assay and invasion was determined by the 'transwell chamber' assay. Metalloproteinase activity (MMP-2) was evaluated by zymography and tubulogenesis was analyzed by counting the total tube length and the number of branch formation. We have demonstrated that Swan 71 expresses high levels of Gb3 compared to HTR-8 cells. Stx2 decreased significantly Swan 71 viability in a dose-dependent manner after 72 h of toxin exposure. Furthermore, Stx2 impaired migration, invasion and tube-like formation of Swan 71 cells and decreased the MMP-2 activity. These cytotoxic effects were partially prevented by aminoguanidine, an inducible nitric oxide synthase inhibitor. These studies demonstrate that the function and viability of EVT cells may be altered by Stx2 and suggest that NO overexpression may be involved in the detrimental effects.

Oxygen regulation of aquaporin-4 in human placenta.

RESEARCH QUESTION: We recently reported that blocking of placental aquaporins (AQP) abrogates the apoptotic response of the trophoblast. As trophoblast apoptosis is exacerbated in pre-eclampsia, we hypothesized that changes in AQP in these placentae may trigger programmed cell death. We analysed AQP4 expression in pre-eclamptic placentae and its regulation by oxygen tension. DESIGN: AQP4 expression was studied in placentae from non-pathological and pre-eclamptic pregnancies by reverse transcription polymerase chain reaction (RT-PCR), Western blot, immunofluorescence and immunohistochemistry. Explants from non-pathological placentae were cultured in normoxia, hypoxia, hypoxia-reoxygenation and CoCl2. AQP4 expression was investigated by RT-PCR and Western blot. Hypoxia responsive elements sites on AQP4 promotor were investigated by in-silico analysis. AQP4 degradation was studied in the presence of proteosomal and lysosomal inhibitors. RESULTS: AQP4 protein expression was weakly detectable in pre-eclamptic placentae, but its mRNA was elevated compared with non-pathological placentae. In non-pathological explants cultured in hypoxia, AQP4 mRNA and protein were increased compared with placentae cultured in ambient oxygen but decreased after reoxygenation. Incubation with CoCl2, that stabilizes hypoxia inducible factor (HIF)-1α, also increased AQP4 levels. In-silico analysis showed three putative binding sites for HIF-1α in AQP4 promotor. CONCLUSIONS: Oxygen may regulate AQP4 expression in human placenta, possibly through HIF-1α. Therefore, the decrease in AQP4 throughout pregnancy, previously reported, is consistent with changes in HIF-1α, and suggests that AQP4 might have a crucial role during placentation. Therefore, the abnormal expression of AQP4 may be involved in the cause of pre-eclampsia, but it does not seem to take part in the apoptotic events.

Leptin upregulates aquaporin 9 expression in human placenta in vitro.

Aquaporins are integral membrane proteins that have permeability functions in many tissues. Aquaporin 9 may transport not only water but also small molecules, such as glycerol, monocarboxylates, purines and pyrimidines. Aquaporin 9 is expressed in syncytiotrophoblast of human term placenta, and it may contribute to the embryonic/fetal growth and survival. We have previously found that Aquaporin 9 expression levels seem to be increased in placenta from gestational diabetes. Since leptin plasma levels and leptin expression are increased in placenta from gestational diabetes, we aimed to study the possible role of leptin on Aquaporin 9 expression in human placenta in vitro. The present work shows that leptin produces a dose-dependent increase of Aquaporin 9 expression, resulting in an increase in Aquaporin-9 protein in human trophoblast explants.

Vascular Dysfunction in Mother and Offspring During Preeclampsia: Contributions from Latin-American Countries.

Pregnancy is a physiologically stressful condition that generates a series of functional adaptations by the cardiovascular system. The impact of pregnancy on this system persists from conception beyond birth. Recent evidence suggests that vascular changes associated with pregnancy complications, such as preeclampsia, affect the function of the maternal and offspring vascular systems, after delivery and into adult life. Since the vascular system contributes to systemic homeostasis, defective development or function of blood vessels predisposes both mother and infant to future risk for chronic disease. These alterations in later life range from fertility problems to alterations in the central nervous system or immune system, among others. It is important to note that rates of morbi-mortality due to pregnancy complications including preeclampsia, as well as cardiovascular diseases, have a higher incidence in Latin-American countries than in more developed countries. Nonetheless, there is a lack both in the amount and impact of research conducted in Latin America. An impact, although smaller, can be seen when research in vascular disorders related to problems during pregnancy is analyzed. Therefore, in this review, information about preeclampsia and endothelial dysfunction generated from research groups based in Latin-American countries will be highlighted. We relate the need, as present in many other countries in the world, for increased effective regional and international collaboration to generate new data specific to our region on this topic.

Aquaporins in Fetal Development.

Water homeostasis during fetal development is of crucial physiologic importance. The successful formation and development of the placenta is critical to maintain normal fetal growth and homeostasis. The expression of several aquaporins (AQPs ) was found from blastocyst stages to term placenta and fetal membranes. Therefore, AQPs are proposed to play important roles in normal pregnancy, fetal growth, and homeostasis of amniotic fluid volume, and water handling in other organs. However, the functional importance of AQPs in fetal development remains to be elucidated.

[Serum uric acid levels and risk of developing preeclampsia]. / Niveles de ácido úrico sérico y riesgo de desarrollar preeclampsia.

It is well known that preeclampsia is associated to high uric acid levels, but the clinical assessment of this relationship is still under consideration. Our research was to evaluate if periodic doses of uric acid during pregnancy might help to identify a high risk group prior to the onset of preeclampsia. We conducted a retrospective investigation in 79 primary gestates with normal blood pressure and 79 women with preeclampsia who were assisted at Hospital Nacional Posadas during 2010. Serum uric acid levels, creatininemia, uremia, and proteinuria data from the clinical records of the pregnant women were considered. Uric acid levels were similar in both groups during the first half of gestation. However, as of the 20th week, uric acid increased 1.5-times in preeclamptic women with no changes in creatinine and urea, confirming that these patients had no renal complications. Furthermore, we noted that higher levels of uric acid correlated with low birth weight. We also observed that pregnant women with a family history of hypertension were more likely to develop this condition. Moreover, we did not find a direct relationship with the fetal sex or the appearance of clinical symptoms. The analytical evidence suggests that changes in uric acid concentrations may be due to metabolic alterations at the initial stages of preeclampsia. Therefore, we propose that monitoring levels of uric acid during pregnancy might contribute to the early control of this condition.

Machine learning applied in maternal and fetal health: a narrative review focused on pregnancy diseases and complications.

Introduction: Machine learning (ML) corresponds to a wide variety of methods that use mathematics, statistics and computational science to learn from multiple variables simultaneously. By means of pattern recognition, ML methods are able to find hidden correlations and accomplish accurate predictions regarding different conditions. ML has been successfully used to solve varied problems in different areas of science, such as psychology, economics, biology and chemistry. Therefore, we wondered how far it has penetrated into the field of obstetrics and gynecology. Aim: To describe the state of art regarding the use of ML in the context of pregnancy diseases and complications. Methodology: Publications were searched in PubMed, Web of Science and Google Scholar. Seven subjects of interest were considered: gestational diabetes mellitus, preeclampsia, perinatal death, spontaneous abortion, preterm birth, cesarean section, and fetal malformations. Current state: ML has been widely applied in all the included subjects. Its uses are varied, the most common being the prediction of perinatal disorders. Other ML applications include (but are not restricted to) biomarker discovery, risk estimation, correlation assessment, pharmacological treatment prediction, drug screening, data acquisition and data extraction. Most of the reviewed articles were published in the last five years. The most employed ML methods in the field are non-linear. Except for logistic regression, linear methods are rarely used. Future challenges: To improve data recording, storage and update in medical and research settings from different realities. To develop more accurate and understandable ML models using data from cutting-edge instruments. To carry out validation and impact analysis studies of currently existing high-accuracy ML models. Conclusion: The use of ML in pregnancy diseases and complications is quite recent, and has increased over the last few years. The applications are varied and point not only to the diagnosis, but also to the management, treatment, and pathophysiological understanding of perinatal alterations. Facing the challenges that come with working with different types of data, the handling of increasingly large amounts of information, the development of emerging technologies, and the need of translational studies, it is expected that the use of ML continue growing in the field of obstetrics and gynecology.

Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Pregnancy. A Non-systematic Review of Clinical Presentation, Potential Effects of Physiological Adaptations in Pregnancy, and Placental Vascular Alterations.

In December 2019, the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) rapidly spread to become a pandemic. To date, increasing evidence has described the potential negative impact of SARS-CoV-2 infection on pregnant women. Although the pathophysiology of coronavirus disease 2019 (COVID-19) is not entirely understood, there is emerging evidence that it causes a severe systemic inflammatory response associated with vascular alterations that could be of special interest considering some physiological changes in pregnancy. Additionally, these alterations may affect the physiology of the placenta and are associated with pregnancy complications and abnormal histologic findings. On the other hand, data about the vaccine against SARS-CoV-2 are limited, but the risks of administering COVID-19 vaccines during pregnancy appear to be minimal. This review summarizes the current literature on SARSCoV2 virus infection, the development of COVID-19 and its relationship with physiological changes, and angiotensin-converting enzyme 2 (ACE2) function during pregnancy. We have particularly emphasized evidence coming from Latin American countries.

Lactic Acid Transport Mediated by Aquaporin-9: Implications on the Pathophysiology of Preeclampsia.

Aquaporin-9 (AQP9) expression is significantly increased in preeclamptic placentas. Since feto-maternal water transfer is not altered in preeclampsia, the main role of AQP9 in human placenta is unclear. Given that AQP9 is also a metabolite channel, we aimed to evaluate the participation of AQP9 in lactate transfer across the human placenta. Explants from normal term placentas were cultured in low glucose medium with or without L-lactic acid and in the presence and absence of AQP9 blockers (0.3 mM HgCl2 or 0.5 mM Phloretin). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase release. Apoptotic indexes were analyzed by Bax/Bcl-2 ratio and Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick-End Labeling assay. Heavy/large and light/small mitochondrial subpopulations were obtained by differential centrifugation, and AQP9 expression was detected by Western blot. We found that apoptosis was induced when placental explants were cultured in low glucose medium while the addition of L-lactic acid prevented cell death. In this condition, AQP9 blocking increased the apoptotic indexes. We also confirmed the presence of two mitochondrial subpopulations which exhibit different morphologic and metabolic states. Western blot revealed AQP9 expression only in the heavy/large mitochondrial subpopulation. This is the first report that shows that AQP9 is expressed in the heavy/large mitochondrial subpopulation of trophoblasts. Thus, AQP9 may mediate not only the lactic acid entrance into the cytosol but also into the mitochondria. Consequently, its lack of functionality in preeclamptic placentas may impair lactic acid utilization by the placenta, adversely affecting the survival of the trophoblast cells and enhancing the systemic endothelial dysfunction.

Hyperosmolarity Impairs Human Extravillous Trophoblast Differentiation by Caveolae Internalization.

We recently reported that an intact caveolar structure is necessary for adequate cell migration and tubulogenesis of the human extravillous trophoblast (EVT) cells. Emerging evidence supports that hyperosmolarity induces the internalization of caveolae into the cytoplasm and accelerates their turnover. Furthermore, signaling pathways associated with the regulation of trophoblast differentiation are localized in caveolae. We hypothesized that hyperosmolarity impairs EVT differentiation and caveolae/caveolin-1 (Cav-1) participates in this process. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 and exposed to hyperosmolar condition (generated by the addition of 100 mM sucrose). Hyperosmolarity altered the EVT cell migration and the formation of tube-like structures. In addition, cell invasion was decreased along with a reduction in the latent and active forms of matrix metalloproteinase-2 (MMP-2) secreted by these cells. With respect to Cav-1 protein abundance, we found that hyperosmolarity enhanced its degradation by the lysosomal pathway. Accordingly, in the hyperosmolar condition, we also observed a significant increase in the number of vacuoles and the internalization of the caveolae into the cytoplasm. Taken together, our findings suggest that hyperosmolarity may induce caveolae internalization and increase their turnover, compromising the normal differentiation of EVT cells.

Assessing the Role of Uric Acid as a Predictor of Preeclampsia.

We assessed the diagnostic utility of uric acid for the prediction of preeclampsia. An observational prospective approach was carried out during 2014. Preeclamptic women were classified into 4 groups accordingly to the onset of preeclampsia and the presence of intrauterine growth restriction (IUGR). Serum uric acid levels, urea, and creatinine were measured. Receiver operating curves (ROC) of the uric acid levels ratio (UAr) between a dosage before and after the 20th week of gestation were performed. One thousand two hundred and ninety-third pregnant women were enrolled in this study. Eight hundred ten had non-complicated pregnancies, 40 preeclampsia, 33 gestational hypertension, and 20 IUGR without preeclampsia. Uric acid significantly raised after 20 weeks of gestation in women who develop preeclampsia before 34 weeks (Group A) or in those who develop preeclampsia after 37 weeks associated with IUGR (Group C). In women who develop preeclampsia after 34 weeks without IUGR (Groups B and D), uric acid increased after the 30th week of gestation. In all groups, UAr was greater than 1.5. In gestational hypertension, UAr was superior to 1.5 toward the end of gestation, while in IUGR without preeclampsia, the behavior of serum uric acid was similar to non-complicated pregnancies. In all cases, urea and creatinine showed normal values, confirming that patients had no renal compromise. ROC area was 0.918 [95% confidence interval (CI): 0.858-0.979) for the preeclampsia group and 0.955 (95% CI: 0.908-1.000) for Group A. UAr at a cut-off point ≥1.5 had a very low positive predictive value, but a high negative predictive value of 99.5% for preeclampsia and it reached 100% for Group A. Thus, a UAr less than 1.5 may be a helpful parameter with a strong exclusion value and high sensitivity for those women who are not expected to develop preeclampsia. Additionally, this low-cost test would allow for better use of resources in developing countries.

High Levels of Tumor Necrosis Factor-Alpha Reduce Placental Aquaporin 3 Expression and Impair in vitro Trophoblastic Cell Migration.

Placentas from preeclamptic women display augmented tumor necrosis factor-alpha (TNF-α) levels with reduced expression of aquaporin 3 (AQP3). However, whether TNF-α modulates AQP3 expression remains to be elucidated. We hypothesize that elevated levels of TNF-α reduce AQP3 expression and negatively impact trophoblastic cell migration. Spontaneously hypertensive rats (SHRs) and Wistar rats (14-16 weeks) were divided into hypertensive and normotensive groups, respectively. Systolic blood pressure (SBP) was measured, and animals mated. In a third group, pregnant SHRs were treated with a TNF-α antagonist, etanercept (0.8 mg/kg, subcutaneously) on days 0, 6, 12, and 18 of pregnancy. Placentas were collected on the 20th day of pregnancy. Human placental explants, from normotensive pregnancies, were incubated with TNF-α (5, 10, and 20 ng/ml) and/or etanercept (1 µg/ml). Swan 71 cells were incubated with TNF-α (10 ng/ml) and/or etanercept (1 µg/ml) and subjected to the wound healing assay. AQP3 expression was assessed by Western blot and TNF-α levels by ELISA. SBP (mmHg) was elevated in the hypertensive group, and etanercept treatment reduced this parameter. Placental TNF-α levels (pg/ml) were higher in the hypertensive group. AQP3 expression was reduced in the hypertensive group, and etanercept treatment reversed this parameter. Explants submitted to TNF-α exposition displayed reduced expression of AQP3, and etanercept incubation reversed it. Trophoblastic cells incubated with TNF-α showed decreased cell migration and reduced AQP3 expression, and etanercept incubation ameliorated it. Altogether, these data demonstrate that high TNF-α levels negatively modulate AQP3 in placental tissue, impairing cell migration, and its relationship in a pregnancy affected by hypertension.

Aquaporins during pregnancy.

Aquaporins (AQPs) are water channels proteins that facilitate water flux across cell membranes in response to osmotic gradients. Despite of the differences in the mammalian placentas, the conserved combination of AQPs expressed in placental and fetal membranes throughout gestation suggests that these proteins may be important in the regulation of fetal water homeostasis. Thus, AQPs may regulate the amniotic fluid volume and participate in the trans-placental transfer of water. Apart from their classical roles, recent studies have revealed that placental AQPs may also cooperate in cellular processes such as the migration and the apoptosis of the trophoblasts. Aquaglyceroporins can also participate in the energy metabolism and in the urea elimination across the placenta. Many factors including oxygen, hormones, acid-basis homeostasis, maternal dietary status, interaction with other transport proteins and osmotic stress are proposed to regulate their expression and function during gestation and alterations result in pathological pregnancies.

AQP1 expression in the proximal tubule of diabetic rat kidney.

Polyuria is a hallmark symptom and the first clinical manifestation of diabetes mellitus (DM). The glucose that remains in renal tubules was proposed to produce an osmotic effect resulting in polyuria. Although water is reabsorbed in proximal tubules through an aquaporin-1 (AQP1) dependent mechanism, AQP1 role in the genesis of polyuria is unknown. AQP1 expression was studied in a rat model of Type-1 DM at 15-days and 5-months of evolution. A different AQP1 expression pattern was found in both experimental groups, with no changes in AQP1 localization, suggesting that changes in AQP1 may be involved in the development of polyuria.