Trophoblastic mitochondrial DNA induces endothelial dysfunction and NLRP3 inflammasome activation: Implications for preeclampsia.

AIMS: Preeclampsia (PE) is characterised by systemic vascular endothelium dysfunction. Circulating trophoblastic secretions contribute to endothelial dysfunction, resulting in PE; however, the underlying mechanisms remain unclear. Herein, we aimed to determine the potential correlation between the release of trophoblastic mitochondrial deoxyribonucleic acid (DNA) (mtDNA) and endothelium damage in PE. MATERIALS AND METHODS: Umbilical cord sera and tissues from patients with PE were investigated for inflammasome activation. Following this, trophoblastic mitochondria were isolated from HTR-8/SVneo trophoblasts under 21 % oxygen (O2) or hypoxic conditions (1 % O2 for 48 h) for subsequent treatments. Primary human umbilical veinendothelial cells (HUVECs) were isolated from the human umbilical cord and then exposed to a vehicle (phosphate-buffered saline [PBS]), mtDNA, hypo-mtDNA, or hypo-mtDNA with INF39 (nucleotide oligomerisation domain-like receptor family pyrin domain containing 3 [NLRP3]-specific inhibitor) for 12 h before flow cytometry and immunoblotting. The effects of trophoblastic mtDNA on the endothelium were further analysed in vivo using enzyme-linked immunosorbent assay (ELISA) and vascular reactivity assay. The effects of mtDNA on vascular phenotypes were also tested on NLRP3 knockout mice. RESULTS: Elevated interleukin (IL)-1ß in PE sera was accompanied by NLRP3 inflammasome activation in cord tissues. In vitro and in vivo experiments revealed that the release of trophoblastic mtDNA could damage the endothelium via NLRP3 activation, resulting in the overexpression of NLRP3, caspase-1 p20, IL-1ß p17, and gasdermin D (GSDMD); reduced endothelial nitric oxide synthase (eNOS) levels; and impaired vascular relaxation. Flow cytometric analysis confirmed that extensive cell death was induced by mtDNA, and simultaneously, a more pronounced pro-apoptotic effect was caused by hypoxia-treated trophoblastic mtDNA. The NLRP3 knockout or pharmacologic NLRP3 inhibition partially reversed tumour necrosis factor-α (TNF-α) and IL-1ß levels and endothelium-dependent vasodilation in mice. CONCLUSION: These findings demonstrate that trophoblastic mtDNA induced NLRP3/caspase-1/IL-1ß signalling activation, eNOS-related endothelial injury, and vasodilation dysfunction in PE.

Low-dose aspirin inhibits trophoblast cell apoptosis by activating the CREB/Bcl-2 pathway in pre-eclampsia.

Excessive apoptosis of placental trophoblast cells is considered a major cause of pre-eclampsia (PE) pathogenesis. Phosphorylation of the widely expressed cAMP response element binding protein (CREB) regulates apoptosis and may be involved in PE incidence. Low-dose aspirin (LDA) is an effective approach for preventing PE with unclear mechanisms. Thus we examined whether LDA protects against PE by inhibiting trophoblast cell apoptosis through CREB. The effects of LDA on human PE placenta, PE model rat placenta, and hydrogen peroxide (H2O2)-induced HTR-8/SVneo cell apoptosis were analyzed. TUNEL assay, immunohistochemistry, Cell Counting Assay Kit-8 (CCK-8) assay, western blot, and flow cytometry assay were performed. In the placenta of human PE and rat PE models, the TUNEL index increased and was partially corrected with LDA pre-treatment. Meanwhile, decreased Bcl-2 and increased Bax expression were significantly reversed by LDA pre-treatment. In HTR-8/SVneo cells, H2O2 decreased cell viability, promoted apoptosis, reduced the Bcl-2/Bax ratio, aggravated loss of mitochondrial membrane potential (MMP), increased cytoplasmic cytochrome c release, and simultaneously activated caspase-9 and caspase-3. These effects were effectively restored by LDA pre-treatment in the cells. Moreover, LDA promoted CREB phosphorylation in trophoblast cells. CREB interference further promoted apoptosis, reduced the Bcl-2/Bax ratio, and increased MMP loss. CREB interference also reversed the inhibitory effect of LDA on H2O2-induced apoptosis in HTR-8/SVneo cells. Thus, LDA was shown to inhibit trophoblast cell mitochondrial apoptosis by activating the CREB/Bcl-2 pathway, providing novel evidence for the protective mechanism of LDA in PE.Abbreviations; PE: Pre-eclampsia; LDA: low-dose aspirin; CREB: cAMP response element binding protein; ROS: reactive oxygen species; H2O2: hydrogen peroxide; PBS: Phosphate-buffered saline; Bcl-2: B-cell lymphoma-2; MMP: Mitochondrial membrane potential; Cyt-c: CytochromeC.

Surface electromyography of the pelvic floor at 6-8 weeks following delivery: a comparison of different modes of delivery.

INTRODUCTION AND HYPOTHESIS: The objective of this study was to compare the impact of different modes of delivery, especially forceps delivery (FD), on pelvic floor muscles (PFMs) through vaginal surface electromyography (sEMG) in primiparous women at early (6-8 weeks) postpartum. METHODS: A total of 1259 primiparous women with full-term singleton births were included in this cross-sectional study. Of these, 98 were delivered by forceps, 865 underwent spontaneous vaginal delivery (SD) and 296 underwent elective cesarean delivery (CD). Clinical demographic characteristics and vaginal sEMG variables of parturients 6-8 weeks after birth were collected and analyzed using SPSS software. One-way ANOVA with Bonferroni correction, Chi-square test or Student's t-test was used according to the variable type. Spearman correlation and binary logistic regression analyses were also used. P/α ≤ 0.05 was considered statistically significant. RESULTS: Amplitude of fast and sustained contractions on sEMG in the FD group was significantly lower compared with the CD and SD groups. The sEMG amplitude of all contractions was significantly higher in the CD group compared with the FD and SD groups (P < 0.01). According to binary logistic regression analysis, mode of delivery was a major influencing factor in sEMG. CONCLUSIONS: An early postpartum sEMG test appears to be helpful for the assessment of PFM activity. Mode of delivery was a major influencing factor on sEMG. Forceps delivery significantly inversely influenced PFM activity.

Osmotic water permeability diversification in primary trophoblast cultures from aquaporin 1-deficient pregnant mice.

AIM: Aquaporins (AQP) are water channel proteins, and some play an important role in maternal-fetal fluid exchange. The present study aimed to measure the osmotic water permeability in primary cultures of trophoblast cells from AQP1-deficient (AQP1(-/-) ) pregnant mice and to define the quantitative role of AQP1 in water transport across the trophoblast plasma membrane. MATERIAL AND METHODS: Trophoblast cells were obtained from placental tissue cell culture of AQP1(-/-) pregnant mice and were characterized by cytokeratin 7 immunostaining. The expression of the AQP1 gene in trophoblast cells of wild-type (AQP1(+/+) ) mice was confirmed by immunofluorescence. The osmotic water permeability of trophoblast plasma membranes was measured by a calcein fluorescence quenching method in response to osmotic gradients. RESULTS: A primary cell culture system for trophoblasts was successfully established. Immunofluorescence showed the expression of AQP1 in the trophoblast cell membrane of AQP1(+/+) mice. The osmotic water permeability of AQP1(-/-) trophoblast cells was significantly lower than that in AQP1(+/+) trophoblast cells, in response to both hypotonic and hypertonic challenges. CONCLUSION: The results suggest an important role of AQP1-mediated plasma membrane water permeability in maternal-fetal fluid balance and also provide a potential direction for the identification of therapeutic targets for the treatment of abnormalities in amniotic fluid volume.

Maternal-fetal fluid balance and aquaporins: from molecule to physiology.

Maternal-fetal fluid balance is critical during pregnancy, and amniotic fluid is essential for fetal growth and development. The placenta plays a key role in a successful pregnancy as the interface between the mother and her fetus. Aquaporins (AQPs) form specific water channels that allow the rapid transcellular movement of water in response to osmotic/hydrostatic pressure gradients. AQPs expression in the placenta and fetal membranes may play important roles in the maternal-fetal fluid balance.

Pregnant phenotype in aquaporin 8-deficient mice.

AIM: Aquaporin 8 (AQP8) is expressed within the female reproductive system but its physiological function reminds to be elucidated. This study investigates the role of AQP8 during pregnancy using AQP8-knockout (AQP8-KO) mice. METHODS: Homozygous AQP8-KO mice were mated, and the conception rate was recorded. AQP8-KO pregnant mice or their offspring were divided into 5 subgroups according to fetal gestational day (7, 13, 16, 18 GD) and newborn. Wild type C57 pregnant mice served as the control group. The number of pregnant mice, total embryos and atrophic embryos, as well as fetal weight, placental weight and placental area were recorded for each subgroup. The amount of amniotic fluid in each sac at 13, 16, and 18 GD was calculated. Statistical significance was determined by analysis of variance of factorial design and chi-square tests. RESULTS: Conception rates did not differ significantly between AQP8-KO and wild type mice. AQP8-KO pregnant mice had a significantly higher number of embryos compared to wild type controls. Fetal/neonatal weight was also significantly greater in the AQP8-KO group compared to age-matched wild type controls. The amount of amniotic fluid was greater in AQP8-KO pregnant mice than wild type controls, although the FM/AFA (fetal weight/amniotic fluid amount) did not differ. While AQP8-KO placental weight was significantly larger than wild type controls, there was no evidence of placental pathology in either group. CONCLUSION: The results suggest that AQP8 deficiency plays an important role in pregnancy outcome.

[Analysis of the cause and clinical characteristics of maternal cardiac arrest].

OBJECTIVE: To analyze the cause and clinical characteristics of maternal cardiac arrest. METHODS: The data of all cases of maternal cardiac arrest from January 2005 to December 2009 in Third Affiliated Hospital of Guangzhou Medical College was retrospectively studied. RESULTS: (1) A total of 41 maternal cardiac arrests (6 in prenatal period, 2 in the first stage of labor, 7 in the third stage of labor, 26 in postpartum period) were included. All patients regained spontaneous circulation after basic life support. Twelve (29%) mothers survived. Twelve cardiac arrests occurred in the hospital, and the total delivery number from January 2005 to December 2009 was 17 101, with occurrence rate of 1:1425. (2) The causes of arrest were hemorrhagic shock (12, 29%), amniotic fluid embolism (7, 17%), severe preeclampsia/eclampsia (7, 17%), septic shock (6, 15%), cardiac disease (2, 5%), unidentified cause (2, 5%) and other occasional causes. (3) Thirty-seven (90%) in-hospital maternal cardiac arrest occurred in operation room (16, 39%), ICU (7, 17%), maternity wards (6, 15%), delivery room (5, 12%) and the emergency room (3, 7%). Three (7%) arrest occurred out of hospital and one in the ambulance. Maternal survival rate was 2/3 in the emergency room, 8/16 in the operation room, 1/5 in the maternity wards, and 1/6 in the delivery room. No mother survived in ICU, ambulance or out of hospital. (4) Five of the 12 survived women showed ischemic encephalopathy after cardiac arrest and one of them developed cerebral infarction in the right corona radiate. (5) In 4 of the 8 cases of cardiac arrest in pregnancy, perimortem caesarean section (PMCS) was performed. In the four PMCS, 2 mothers and 2 children survived. In the 4 cases that PMCS was not carried out, no infant survived. CONCLUSIONS: Hemorrhagic shock, severe preeclampsia and eclampsia, amniotic fluid embolism are the major obstetric causes of maternal cardiac arrest. Septic shock and cardiac diseases are the major non-obstetric causes. Cardiac arrests occurred in emergency room and operation room has a higher maternal survival rate than those occurred in the delivery room and maternity wards. Timely PMCS may ensure the optimal outcome for mothers and fetuses.

[Clinical outcomes and characteristics of concurrent eclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome].

OBJECTIVE: the purpose was to describe the outcomes and characteristics of the obstetric patients with concurrent eclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome (HELLP) syndrome. METHODS: we retrospectively collected the materials between December 1999 and December 2008 in Obstetric Critical Care Center of Guangzhou. There were 76 patients in rolled then they were divided into two groups according to with or without HELLP syndrome. All the patients were injected Magnesium Sulfate to control seizure and to prevent the recurring of seizure. We analyzed the characteristics (such as age, gestational weeks, blood pressure after seizure), complications, biochemistry markers, the rate for intensive care unit (ICU) admittion, the need for mechanical ventilation, the Glasgow coma score (GCS) when admitted into ICU, computed tomography scan (CT) or magnetic resonance imaging (MRI), death rate of maternal and others, then compared between the two groups. RESULTS: (1) general data: There were 17 patients admitted with both eclampsia and HELLP syndrome, and 59 patients admitted eclampsia without HELLP syndrome. The incidence of eclampsia with HELLP syndrome was 22% (17/76). In eclampsia with HELLP syndrome group, the systolic blood pressure was higher and the rate of preterm also was higher [(182 ± 20) mm Hg (1 mm Hg = 0.133 kPa) vs. (159 ± 21) mm Hg, P < 0.05]. But in regard to the age, gestational weeks, the rate of regular prenatal care and diastolic blood pressure, there were no differences between the two groups. (2) Biochemistry markers: the aspartate transaminase (AST), lanine transaminase (ALT), blood urea nitrogen and creatinine were significantly increased in eclampsia with HELLP syndrome group than eclampsia without HELLP syndrome group [(879 ± 337) U/L vs. (90 ± 27) U/L, (344 ± 83) U/L vs. (43 ± 11)U/L, (2245 ± 294) U/L vs. (485 ± 61) U/L, (14 ± 9) mmol/L vs. (7 ± 3) mmol/L, (140 ± 92) µmol/L vs. (83 ± 28) µmol/L, P < 0.01, P < 0.05], and the platelet was lower in eclampsia with HELLP syndrome group [(38 ± 13) × 10(9)/L vs (172 ± 46) × 10(9)/L, P < 0.01]. (3) Clinical outcomes: The maternal death rate was 35% (6/17) in eclampsia with HELLP syndrome patients, and significantly higher than the rate in eclampsia without HELLP syndrome group (3%, 2/59) (P < 0.05). There were more patients admitted to ICU and more patients who need mechanical ventilation in eclampsia with HELLP syndrome (13/17 vs. 34%, 9/17 vs. 24/, P < 0.05), also more patients with GCS ≤ 8 in eclampsia with HELLP syndrome when admitted to ICU (8/17 vs. 7/59, P < 0.05), compared to the eclampsia without HELLP syndrome group. There were more patients complicated with cerebral venous thrombosis and cerebral hemorrhage in eclampsia with HELLP syndrome group than other group (8/17 vs. 7%, P < 0.05). Five of six patients died of cerebral hemorrhage in eclampsia with HELLP syndrome group, while other two missing cases in eclampsia without HELLP syndrome group all died of cerebral hemorrhage. The all missing cases were performed CT or MRI and seven (7/8) of them showed cerebral hemorrhage. CONCLUSION: the incidence of concurrent eclampsia and HELLP syndrome was not rare, it happened seriously and with more mortalities, such as cerebral hemorrhage, and also the maternal mortality rate was significantly higher. It should be warning that the obstetrician should take great attention for these women, and consider life support treatment for them.

[Correlation between sleep apnea hypopnea syndrome and preeclampsia and the possible pathogenesis of preeclampsia].

OBJECTIVE: To investigate the relationship between sleep apnea-hypopnea syndrome (SAHS) and preeclampsia and the possible pathogenesis of the latter. METHODS: Twenty-five healthy pregnant women, 43 pregnant women with preeclampsia, and 27 with preeclampsia complicated by SAHS were enrolled in this study. Apnea-hypopnea index (AHI) and the lowest arterial oxygen saturation (LSaO2) were measured through a 7-hour polysomnography (PSG), and the maternal age, gestational age, body mass index and 24-hour urine protein were recorded. RESULTS: All the indexes except for the maternal age and gestational age showed significant differences between the 3 groups. The two groups of preeclampsia patients showed a significant difference in BMI from the control cases. Significant positive correlations of AHI to BMI, MAP and 24-hour urine protein were noted; LSaO2 was found to inversely correlate to BMI, MAP, and 24-hour urine protein. In spite of the significant correlation of BMI to the other indexes, we found that BMI was less important than AHI and LSaO2. CONCLUSION: SAHS may induce or aggravate preeclampsia. Greater attention should be given to the presence of SAHS in pregnant women with obesity, but obesity is not the predominant predisposing factor for preeclampsia.

[Expression of aquaporin-1 in human oligohydramnios placenta and fetal membranes].

OBJECTIVE: To detect aquaporin-1 mRNA (AQP1) expression in human oligohydramnios placenta and fetal membranes. METHODS: Placenta and fetal membranes samples were obtained from 5 women with oligohydramnios and 5 with normal amniotic fluid volume. AQP-1 mRNA expression in the tissue samples was detected by semi-quantitative RT-PCR. RESULTS: The expression of AQP1 mRNA was significantly lower in oligohydramnios placenta than in normal pregnancy placenta at term (P<0.05), and also significantly lower in oligohydramnios fetal membranes than in normal fetal membranes at term (P<0.05). CONCLUSION: Alterations in AQP1 mRNA expressions in human placenta and fetal membranes may play an important role in the disorder of maternal-fetal fluid exchange and amniotic fluid volume.

[Clinical application of the Epworth sleepiness score in pregnant women with obstructive sleep apnea-hypopnea syndrome].

OBJECTIVE: To provide a convenient method for screening obstructive sleep apnea-hypopnea syndrome (OSAHS) in pregnant women. METHODS: Seventy-eight pregnant women with suspected OSAHS were calculated for the EP index using Epworth sleepiness score (ESS) with also measurement of the neck circumference (NC) and body mass index (BMI). The apnea/hypopnea index (AHI) was calculated and the lowest SaO(2) (LSaO(2)) measured through a 7-h polysomnography (PSG). The women were then divided into 4 groups according to the AHI and LSaO(2). The ESS was compared with the PSG-AHI and the receiver operating characteristic curve (ROC) was generated. RESULTS: All the clinical indexes (NC, BMI, EP, AHI, and LSaO(2)) showed significant differences between the 4 groups (P<0.05). EP and PSG were found to have greater correlations to AHI (r=0.759, P=0.000) than NC (r=0.668) and BMI (r=0.663). The area under the ROC of the EP (0.825) was greater than that of NC (0.772) and BMI (0.784). The index of EP showed greater clinical diagnostic value of OSAHS in pregnancy. Base on the ROC, EP at the optimal operating point of 7.5 had a sensitivity of 76.8% and specificity of 68.2% for diagnosis of OSAHS in pregnant women. CONCLUSION: The ESS is an economic and convenient method for screening OSAHS in pregnant women with high diagnostic sensitivity and specificity.

[Relationship between changes of endogenous nitric oxide synthase inhibitor and hydrolase and initiation of pre-eclampsia].

OBJECTIVE: To investigate the role of dimethylarginine dimethylaminohydrolase-2 (DDAH-2)/asymmetric dimethylarginine (ADMA) in pathophiology of preeclampsia by detecting expression of DDAH-2 in placenta and serum plasma ADMA. METHODS: From Jan. 2004 to Jan. 2005, 30 preeclampsia patients (PE group) were chosen in the Third Affiliated Hospital, Guangzhou Medical College matched with 10 normal third trimester women as control (control group). The placental DDAH-2 mRNA expression was detected by fluorescence quantitative polymerase chain reaction (FQ-PCR) and the plasma concentration of ADMA was determined by high performance liquid chromatography (HPLC). RESULTS: (1) The level of ADMA in PE group was significantly higher that than of control group [(18.0 +/- 7.2) mg/L vs. (10.3 +/- 1.7) mg/L, P < 0.01]. The expression level of ADMA in preeclampsia occurring before 34 gestatinal weeks was significantly higher than that of preeclampsia occurring after 34 gestational weeks [(22.0 +/- 7.0) mg/L vs. (12.7 +/- 2.8) mg/L, P < 0.01]. (2) The Placental DDAH-2 mRNA expression in preeclampsia patients was remarkably lower than that of control group [1 x 10((5.23 +/- 0.45)) copy/microl vs. 1 x 10((5.65 +/- 0.08)) copy/microl, P < 0.01]. The Placental DDAH-2 mRNA in preeclampsia occurring before 34 gestatinal weeks was significantly lower than that of preeclampsia occurring after 34 gestational weeks [1 x 10((5.02 +/- 0.46)) copy/microl vs. 1 x 10((5.61 +/- 0.19)) copy/microl, P < 0.01]. CONCLUSION: Our results suggested that low expression of DDAH-2 in placenta and increased serum ADMA level might confer the susceptibility to preeclampsia.

[Expression of aquaporin 1 in human placenta and fetal membranes].

OBJECTIVE: To detect aquaporin1 (AQP1) expression in normal human placenta and fetal membranes. METHODS: Human placenta and fetal membrane specimens were collected from 5 pregnant women with intact membranes undergoing elective cesarean sections at term. Expression and localization of AQP1 was detected by RT-PCR, Western blotting and immunohistochemistry. RESULTS: AQP1 mRNA was detected in the placenta and fetal membranes by RT-PCR, and Western blotting also yielded positive results for the specimens, showing a specific band around 28 kD. Immunohistochemical staining showed AQP1 expression in the vascular endothelial cells and syncytiotrophoblasts of the placenta, epithelial cells of the amnion, and cytotrophoblasts of the chorion. CONCLUSION: The presence of AQP1 expression in the placenta and fetal membranes suggest that AQP1 may play an important role in maternal-fetal fluid exchange and amniotic fluid balance.