Clinical and in Vitro Evidence against Placenta Infection at Term by Severe Acute Respiratory Syndrome Coronavirus 2.

Despite occasional reports of vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during pregnancy, the question of placental infection and its consequences for the newborn remain unanswered. Herein, we analyzed the placentas of 31 coronavirus disease 2019-positive mothers by reverse transcriptase PCR, immunohistochemistry, and in situ hybridization. Only one case of placental infection was detected, which was associated with intrauterine demise of the fetus. Differentiated primary trophoblasts were then isolated from nonpathologic human placentas at term, differentiated, and exposed to SARS-CoV-2 virions. Unlike for positive control cells Vero E6, the virus inside cytotrophoblasts and syncytiotrophoblasts or in the supernatant 4 days after infection was undetectable. As a mechanism of defense, we hypothesized that trophoblasts at term do not express angiotensin-converting enzyme 2 and transmembrane protease serine 2 (TMPRSS2), the two main host membrane receptors for SARS-CoV-2 entry. The quantification of these proteins in the placenta during pregnancy confirmed the absence of TMPRSS2 at the surface of the syncytium. Surprisingly, a transiently induced experimental expression of TMPRSS2 did not allow the entry or replication of the virus in differentiated trophoblasts. Altogether, these results underline that trophoblasts are not likely to be infected by SARS-CoV-2 at term, but raise concern about preterm infection.

Haem oxygenases play a pivotal role in placental physiology and pathology.

BACKGROUND: Haem oxygenases (HO) catabolise haem, which is the prosthetic group of numerous haemoproteins. Thus, multiple primary cellular pathways and functions rely on haem availability. HO exists in two isoforms, both expressed in the placenta, namely HO-1 and HO-2, the first being inducible. Haem oxygenases, particularly HO-1, have garnered specific interest in the field of physiological and pathological placental function. These enzymes mediate haem degradation by cleaving the alpha methene bridge to produce biliverdin, which is subsequently converted to bilirubin, carbon monoxide and iron. HO-1 has anti-inflammatory and antioxidant activities. SEARCH METHODS: An initial literature analysis was performed using PubMed on 3 October 2018 using key terms such as 'haem oxygenase and pregnancy', 'haem oxygenase and placenta', 'HO-1 and pregnancy', 'HO-1 and placenta', 'HO and placenta', 'HO and pregnancy', 'genetic variant and HO', 'CO and pregnancy', 'CO and placenta', 'Bilirubin and pregnancy', 'Iron and pregnancy' and 'PPAR and Haem', selecting consensus conferences, recommendations, meta-analyses, practical recommendations and reviews. A second literature analysis was performed, including notable miscarriages, foetal loss and diabetes mellitus, on 20 December 2019. The three authors studied the publications independently to decipher whether they should be included in the manuscript. OBJECTIVE AND RATIONALE: This review aimed to summarise current pieces of knowledge of haem oxygenase location, function and regulation in the placenta, either in healthy pregnancies or those associated with miscarriages and foetal loss, pre-eclampsia, foetal growth restriction and diabetes mellitus. OUTCOMES: HO-1 exerts some protective effects on the placentation, probably by a combination of factors, including its interrelation with the PGC-1α/PPAR pathway and the sFlt1/PlGF balance, and through its primary metabolites, notably carbon monoxide and bilirubin. Its protective role has been highlighted in numerous pregnancy conditions, including pre-eclampsia, foetal growth restriction, gestational diabetes mellitus and miscarriages. WIDER IMPLICATIONS: HO-1 is a crucial enzyme in physiological and pathological placentation. This protective enzyme is currently considered a potential therapeutic target in various pregnancy diseases.

Does soluble fms-like tyrosine kinase-1 regulate placental invasion? Insight from the invasive placenta.

OBJECTIVE: Soluble fms-like tyrosine kinase (sFLT-1) is a potent antiangiogenic growth factor that has been found to be markedly elevated in preeclampsia. In healthy pregnancy, serum sFLT-1 concentrations are 50-fold higher than in the nonpregnant state. The functional significance of this physiologic elevation in serum sFLT-1 in normal pregnancy is unknown. We hypothesized that sFLT-1 regulates placental cytotrophoblast invasion and that lower levels of sFLT-1 would be observed locally in invasive placentas (accreta/increta/percreta). STUDY DESIGN: We performed a retrospective case-control study comparing placental sFLT-1 expression in hysterectomy specimens from 3 groups: group 1, focally invasive placenta; group 2, normal invasion from the same specimen; and group 3, normal invasion associated with placenta previa. Immunohistochemistry for sFLT-1 was performed, and staining intensity was graded on a scale from 1+ (weak) to 5+ (strong). RESULTS: We identified 10 hysterectomy specimens from women with invasive placentation and 3 with placenta previa. The median sFLT-1 staining score for group 1 was 1.75 compared to 4.0 for group 2 (P = .01). A significant difference was also found between group 1 and group 3 (P = .01). When comparing depth of invasion, there was a trend toward lower staining score as depth of invasion increased (P = .11). Interobserver agreement for immunohistochemistry scoring was 87%. CONCLUSION: Lower levels of sFLT-1 protein expression were associated with invasive placentation suggesting a critical functional role for sFLT-1 in regulation of placental invasion.

Phosphorylation and carbonylation of placental proteins in normal pregnancy and gestosis.

Study of posttranslational changes in placental proteins revealed disorders in the intensity of their phosphorylation and carbonylation in patients with placental failure. Phosphorylation was reduced for the majority of endogenous placental proteins, substrates for cAMP- and cGMP-dependent protein kinases. An opposite dynamics was noted for oxidative modification of proteins. The content of carbonyl derivatives evaluated in spontaneous and metal-catalyzed oxidation of placental proteins was elevated in gestosis in comparison with the normal level.

A fetal cyclooxygenase-2 gene polymorphism is associated with placental malperfusion.

Prostaglandin levels vary during pregnancy, mostly under the control of the inducible enzyme cyclooxygenase-2 (COX-2). The expression of COX-2 has been associated with ischemic events in the heart and brain, but its direct effect on human placental perfusion has not been previously examined. The purpose of this study was to investigate whether a functional polymorphism in the COX-2 gene that controls enzyme expression levels is associated with placental histopathologic lesions. Maternal and neonatal DNA from twin gestations were analyzed by a polymerase chain reaction-based assay for a single G to C nucleotide polymorphism at position -765 in the COX-2 gene promoter. Placental histopathology was evaluated in 6 major categories: meconium, malperfusion, inflammation, umbilical cord problems, villitis, and thrombosis. There was no significant association between placental histopathologic findings and polymorphisms of the COX-2 gene in the mother. In the fetus, carriage of the COX-2 C allele, which is correlated with decreased COX-2 gene expression, was negatively associated with lesions of placental ischemia/malperfusion (P = 0.02). Placental ischemic lesions were positively associated with intrauterine growth restriction (IUGR; P < 0.001). No other group of histopathologic lesions was associated with fetal polymorphisms in the COX-2 gene or with IUGR. Thus, a fetal polymorphism in the COX-2 gene influences the occurrence of placental malperfusion and ischemia, which may be of sufficient severity to promote or allow the development of IUGR.

Mice deficient in heparan sulfate 6-O-sulfotransferase-1 exhibit defective heparan sulfate biosynthesis, abnormal placentation, and late embryonic lethality.

Heparan sulfate (HS) plays critical roles in a variety of developmental, physiological, and pathogenic processes due to its ability to interact in a structure-dependent manner with numerous growth factors that participate in cellular signaling. The divergent structures of HS glycosaminoglycans are the result of the coordinate actions of several N- and O-sulfotransferases, C5-epimerase, and 6-O-endosulfatases. We have shown that 6-O-sulfation of the glucosamine residues in HS are catalyzed by the sulfotransferases HS6ST-1, -2, and -3. To determine the biological and physiological importance of HS6ST-1, we now describe the creation of transgenic mice that lack this sulfotransferase. Most of our HS6ST-1-null mice died between embryonic day 15.5 and the perinatal stage, and those mice that survived were considerably smaller than their wild-type littermates. Some of these HS6ST-1-null mice exhibited development abnormalities, and histochemical and molecular analyses of these mice revealed an approximately 50% reduction in the number of fetal microvessels in the labyrinthine zone of the placenta relative to that in the wild-type mice. Because we observed a modest reduction in VEGF-A mRNA and protein in the tissues of HS6ST-1-null mice, an HS-dependent defect in cytokine signaling probably contributes to increased embryonic lethality and decreased growth. Biochemical studies of the HS chains isolated from various organs of our HS6ST-1-null mice revealed a marked reduction of GlcNAc(6SO(4)) and HexA-GlcNSO(3)(6SO(4)) levels and a reduced ability to bind Wnt2. Thus, despite the presence of three closely related 6-O-sulfotransferase genes in the mouse genome, HS6ST-1 is the primary one used in HS biosynthesis in most tissues.

Gene expression of nitric oxide synthase by human umbilical vein endothelial cells: the effect of fetal plasma from pregnancy with umbilical placental vascular disease.

OBJECTIVE: To determine whether endothelial cell injury would be produced by factor(s) released into the fetal circulation, manifested by altered messenger RNA expression of nitric oxide synthase. DESIGN: Case-control study. SETTING: University teaching hospital. SAMPLES: Fetal plasma was collected from 34 normal pregnancies, 44 pregnancies with umbilical placental vascular disease identified by an abnormal umbilical Doppler and 11 pregnancies with maternal pre-eclampsia but with normal umbilical Doppler studies. METHODS: Aliquots from a common culture of human umbilical vein endothelial cells (HUVECs) were incubated with fetal plasma from the members of the three patient groups. The total RNA was extracted from the endothelial cells and mRNA for nitric oxide synthase was measured by reverse transcription and semi-quantitative polymerase chain reaction (RT-PCR). This was standardised by comparison of the amplified inducible nitric oxide synthase (iNOS) or endothelial constitutive nitric oxide synthase (ecNOS) to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). MAIN OUTCOME MEASURE: Endothelial cell gene expression of iNOS and ecNOS. RESULTS: The mRNA expression of iNOS and ecNOS were significantly higher (P < 0.05) in HUVECs stimulated by fetal plasma from pregnancies with umbilical placental vascular disease [iNOS 1.12 (0.16); ecNOS 1.78 (0.18)] when compared with normal pregnancies [iNOS 0.56 (0.06); ecNOS 1.06 (0.10)]. In the maternal pre-eclampsia group, the NOS expression [iNOS 0.76 (0.11); ecNOS 1.39 (0.26)] did not differ from normal pregnancy. In the vascular disease group, there was no difference in NOS expression between the subgroups with and without maternal pre-eclampsia. CONCLUSIONS: Our study demonstrates that umbilical placental vascular disease is associated with a factor(s) in fetal plasma that produces an increase in the expression of iNOS and ecNOS mRNA by endothelial cells. Our findings raise the possibility that the release of factors causing an up-regulation of iNOS and ecNOS in the endothelium in the fetal placenta may occur as part of an inflammatory response of the vascular endothelium to injury.

Possible involvement of PKC isoforms in signalling placental apoptosis in intrauterine growth retardation.

Apoptosis contributes to uteroplacental dsyfunction in intrauterine growth retardation (IUGR). Specific protein kinase C (PKC) isoforms regulate apoptosis in other cell types. PKC isoforms thought to be anti-apoptotic include the conventional PKC isoforms (cPKC-alpha, -beta I and -beta II), whereas the novel PKC isoforms nPKC-delta and nPKC-epsilon may be apoptotic. Dexamethasone administration during the last third of pregnancy in the rat leads to IUGR. We used the dexamethasone model to test the hypothesis that adverse changes in fetal growth might be associated with a modified placental PKC isoform profile. Dexamethasone administered from day 15 of gestation via subcutaneous infusion (osmotic minipump; 100 or 200 microg dexamethasone/kg maternal body wt. per day) induced a dose-dependent decline in placental and fetal body weights at day 21 of gestation. Placental protein expression of all three cPKC isoforms was downregulated by maternal dexamethasone exposure, whereas placental nPKC-epsilon protein expression and activity was significantly upregulated in a dose-dependent manner. These data indicate that IUGR induced by excessive glucocorticoid exposure late in pregnancy leads to changes in the placental PKC isoform profile consistent with the concept that members of the PKC family participate in apoptosis signalling in the placenta.

Relationship between granulocyte elastase levels and perinatal infections.

This study was conducted in order to investigate the usefulness of granulocyte elastase levels as predictive factors in the onset of perinatal infections. The subjects were 41 patients who delivered within 48 h after amniocentesis after giving their informed consent. The relationship between cervical granulocyte elastase (Cx-E), serum C-reactive protein (CRP) and amniotic fluid granulocyte elastase (Af-E), and placental infections and neonatal infections was comparatively investigated. In some cases, gastric juice granulocyte elastase in neonates (Gj-E) was measured, and the correlation by site was investigated. Elastase levels were not used as a management protocol. In predicting neonatal infections, diagnostic efficacy (sensitivity x specificity) of placental infections (0.97) and abnormal Af-E (0.79) were superior to those of abnormal Cx-E (0.40) and abnormal CRP (0.49). There was no correlation between Cx-E and Af-E or between Cx-E and Gj-E; however, a very close correlation was noted between Af-E and Gj-E. In predicting abnormal amniotic fluids, Cx-E (> or = 1.2 micrograms/ml) + CRP (> or = 1.0 mg/dl) had the highest diagnostic efficacy with 0.58. These findings demonstrate that Af-E is a good index for predicting the onset of neonatal infections. In predicting abnormal amniotic fluid, it might be advisable to consider amniocentesis in order to diagnose intrauterine infections, when both Cx-E and CRP show abnormal levels.

Aryl hydrocarbon hydroxylase activity in human placenta and threatened preterm delivery.

Induction of aryl hydrocarbon hydroxylase (AHH) activity in the placenta has been well documented. This enzyme may be induced by a variety of Polycyclic Aromatic Hydrocarbons (PAHs) and the AHH inducibility is associated with harmful effects of environmental chemicals. Toxic effects of PAHs in tissues such as placenta have been demonstrated to be due to their metabolites, epoxides, which interact with DNA. Thus, environmental PAHs may be related to its alterations in fetal development. Founded on these findings the PAH metabolites could interfere with the normal course of the pregnancy and may be an aborticide, a teratogen or a carcinogen. We hypothesize that low increased activity of placental Aryl Hydrocarbon Hydroxylase (AHH) may be an important determinant of human fetotoxicity. The present investigation was designed to examine the possible implications of PAH exposure at environmental exposure levels on the normal course of the pregnancy using AHH induction as an indicator of PAH exposure. Threatened Preterm Delivery (TPD) was used as an index of problems in the normal course of pregnancy. A group of forty pregnancies at term with TPD was compared with eighty controls for placental AHH induction. Macroscopic placental examination was also performed. A significant increase in prevalence of placental AHH induction with TPD was shown (Odds-Ratio = 2.8; 95% confidence bounds [1.3-6.2]; chi 2 = 6.7 p < 0.01). No such increases were found associated with placental pathology. When taking into account the group of placenta without basal plate calcifications, the significant increase in prevalence of placental AHH induction with TPD above mentioned was greatly increased (Odds-Ratio = 8.9; 95% confidence bounds [2.4-32.9]; chi 2 = 11.1 p < 0.001) controlling for gestational age. The increase in prevalence of placental AHH induction with TPD disappeared when taking into account the subgroup with basal plate or parenchyma calcifications. It is hypothesized that the high estrogen and progesterone at term may explain these associations.

UDP galactose:ceramide galactosyltransferase, CDP choline:1,2-diacyl-sn-glycerol phosphocholine transferase, and microsomal reductases in major regions of the developing rat brain in nutritional stress.

The influence of nutritional inadequacy during the active growth phase of the developing brain, coinciding with myelinogenesis, was examined in rat pups. Developmental profiles of enzyme activities involved in biosynthesis of myelin membrane lipids, and those of associated pathways which generate precursor compounds for such biosynthetic reactions, were followed as functions of age in major anatomical regions of the brain. It was observed that while galactosyltransferase and choline phosphotransferase activities were significantly diminished, the microsomal cytochrome reductases, which contribute to the process of fatty acid elongation and desaturation, were also lowered. An attempt at adaptative increase of enzyme activity, in response to the stress, was apparent in the case of glycerol-3-phosphate dehydrogenase, which could possibly explain, in part, the lower susceptibility of phospholipid biosynthesis to impairments induced by nutritional insufficiency.

Vascular placental insufficiency in the rabbit. Changes in creatine kinase and adenylate kinase activities in fetal tissues.

Vascular placental insufficiency is considered a common pathogenic factor in human intrauterine growth retardation. To mimic this condition, the rabbit, a 'perinatal brain developer' was utilized as an experimental model. Ischemic conditions were achieved by total ligation of approximately 30% of the uteroplacental vessels of half of the fetuses in each pregnant rabbit in the last third of gestation. The change in activity of the brain type isozyme of creatine kinase (CKBB), involved in energy regeneration and regulation, was assessed as a response marker to tissue ischemia in rabbit tissues: cerebellum, cerebrum, kidney, liver and placenta. A significant transient increase in CK-specific activity was found in the kidney and the cerebellum but not in the other organs tested, at 24 and 48 h after ligation. This increase was not seen with adenylate kinase, another enzyme involved in energy regeneration and regulation. It is proposed that an increase in CK-specific activity could serve as a metabolic marker of vascular insufficiency in rapidly developing tissues, representing part of a compensatory mechanism to overcome an energetic gap induced by ischemia.

Peripartal changes in serum alkaline phosphatase activity and lactate dehydrogenase activity in dairy cows.

Peripartal serum alkaline phosphatase activity and lactate dehydrogenase activity were measured in 30 dairy cows in order to examine the association between retained fetal membranes and enzyme activity. Daily blood samples were obtained from pregnant cows, starting 15 days before the expected day of calving until eight days after parturition. Sera from 15 cows which retained fetal membranes longer than 24 hours and 15 cows which shed fetal membranes within six hours after parturition were analyzed for alkaline phosphatase and lactate dehydrogenase enzyme activities. Mean alkaline phosphatase enzyme activities ranged from 15.93 to 32.6 U/L in retained and nonretained placenta cows. There was a trend towards higher serum alkaline phosphatase activities in retained placenta cows but the differences were not significant among the groups (P greater than 0.05). Mean lactate dehydrogenase activities ranged from 307.2 to 438.86 U/L in nonretained and retained placenta cows. Lactate dehydrogenase enzyme activities in nonretained and retained placenta cows were similar (P greater than 0.05). The alkaline phosphatase and lactate dehydrogenase enzyme activities peaked at the time of parturition in both groups. However, the differences in alkaline phosphatase and lactate dehydrogenase activities on different days within non-retained and retained placenta cows were significant (P less than 0.05). Results indicate that prepartal changes in alkaline phosphatase and lactate dehydrogenase enzyme activities are not predictive of placental retention postpartum.

Cord blood serum creatine kinase isoenzymes with placental dysfunction.

It has been suggested that perinatal asphyxia is not generally followed by neurological impairment unless there is preexisting chronic fetal distress. In cases of brain damage one can observe elevated levels of CK-BB. The purpose of our study was to evaluate CK isoenzymes in umbilical cord blood sera of newborns affected by chronic fetal distress. Fetal distress reflected by placental dysfunction was characterized by a diminished HPL level and decreased activity of CAP. We estimated CK isoenzymes with the use of DEAE-sepharose CL-6B column chromatography. Total CK activity was measured using kits supplied by Boehringer-Mannheim (Monotest CK-NAC aktiviert). The clinical state of examined newborns was estimated. Investigations were carried out in the group of 57 infants delivered after 37 weeks of gestation. Total CK activity in cord sera ranged from 40 to 400 U/l. Our results showed a significant rise of CK-BB activity in cord sera of newborns delivered from pregnancies with placental dysfunction (figure 2) as well as in cases of asphyxiated infants (figure 3). We were unable to demonstrate differences in total CK, CK-MM and CK-MB activities in all examined groups of newborns. Other authors have confirmed that severe asphyxia results in increase in CK-BB activity in cord blood. Infants with ominous fetal heart rate patterns have higher CK-BB activity. There are several possible sources for CK-BB activity in umbilical cord blood sera, i.e. fetal brain, lung, gastrointestinal tract, placenta and uterus. It appears that the brain is most likely the source of elevated CK-BB activity found in cord blood in cases of placental dysfunction.

Placental sulfatase deficiency: maternal and fetal expression of steroid sulfatase deficiency and X-linked ichthyosis.

PSD-X-linked ichthyosis are manifestations of a similar disorder of an inborn error of metabolism characterized by a deficiency of steroid sulfatase. The decreased enzyme activity is due to the absence of the expression of enzyme (steroid sulfatase) protein. Affected individuals with this disorder are males (X-linked inheritance) with a frequency of 1/2000 to 1/6000 births. Homozygous females from cosanguineous marriages have been reported with this disorder. The diagnosis is suspected and confirmed by: Low estriol excretion; Negative DHEAS loading test Increased DHEAS in amnionic fluid; Normal DHEAS in cord plasma; Possible delayed or abnormal labor patterns; Decreased sulfatase activity in the placenta, fibroblast, erythrocytes, lymphocytes or leukocytes of affected individuals; Development of ichthyosis in male infants at 2 to 3 months of age.

[Endocrinological and histochemical studies of placental sulfatase deficiency].

A pregnancy with placental sulfatase deficiency was suspected when a 31-year-old patient at 36 weeks of gestation was found to have extremely low urinary estriol excretion but an otherwise normal prenatal course. The maternal plasma level of estrogens was extremely low, whereas dehydroepiandrosterone sulfate (DHA-S) was normal. Following intravenous infusion of 100mg DHA-S, no rise in urinary or serum estrogens was noted. At 40 weeks of gestation, a male infant was delivered vaginally. In vitro incubation study confirmed the diagnosis of placental sulfatase deficiency. Arylsulfatase C activity was not demonstrated in sulfatase deficient placenta, whereas arylsulfatase C activity was detected in the cytoplasma of syncytiotrophoblast cells of all normal placentas, by light and electron microscopy.

[Structuro-functional changes in the placenta as a result of exposure to atmospheric pollutants]. / Strukturno-funktsional'nye izmeneniia platsenty pod vliianiem atmosfernykh zagriaznenii.

Under the influence of atmospheric pollutions certain structural-functional changes take place in placenta: terminal villi per stipulated square unite, villi with desquamated epithelium, with dilated vessels, with deposition of fibrinoid masses, with plasmodial buds increase in number; section area occupied by epithelial layer decreases; RNA concentration and histoenzymatic activity change in the latter.

[Effect of protein-calorie restriction during pregnancy in rats, on the activity of glycolytic enzymes in the placenta]. / Efecto de la restricción calórico-proteínica durante el embarazo de la rata, en la actividad de algunas enzimas glicolíticas en la placenta.

The specific activities and changes of four placental enzymes: pyruvate kinase (PK), glucose-6-phosphate-dehydrogenase (G6PD), 6-phosphogluconic-dehydrogenase (6PGD), and NADP malate dehydrogenase (NADP-MD) occurring during the course of gestation and during maternal restricted food intake, were studied in rats. Enzymes activities are expressed in relation to DNA. With the progress of pregnancy, a significant increase in the activity of all enzymes, except NADP-MD, was observed in both groups. A 50% food restriction during pregnancy markedly decreased cell placental enzymes in different stages of development. PK was lower during early and late pregnancy, but NADP-MD was reduced only in the early developmental stage. The activities of G6PD and 6PGD were significantly lower only in the near-term stage in the malnourished group in comparison with the control group. Our findings suggest that this kind of nutritional insult markedly reduces glycolytic capacity and NADPH2 generation enzymes, a key factor in the placental steroids metabolism.