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Aim: This study aimed to clarify the genetic and epigenetic features of recurrent hydatidiform mole (RHM) in Japanese patients., Methods: Four Japanese isolated RHM cases were analyzed using whole-exome sequencing. Villi from RHMs were collected by laser microdissection for genotyping and DNA methylation assay of differentially methylated regions (DMRs). Single nucleotide polymorphisms of PEG3 and H19 DMRs were used to confirm the parental origin of the variants., Results: A novel homozygous nonsense mutation in NLRP7 (c.584G>A; p.W195X) was identified in 1 patient. Genotyping of one of her molar tissue revealed that it was biparental but not androgenetic in origin. Despite the fact that the RHM is biparental, maternally methylated DMRs of PEG3, SNRPN and PEG10 showed complete loss of DNA methylation. A paternally methylated DMR of H19 retained normal methylation., Conclusions: This is the first Japanese case of RHM with a novel homozygous nonsense NLRP7 mutation and a specific loss of maternal DNA methylation of DMRs. Notably, the mutation was identified in an isolated case of an ethnic background that has not previously been studied in this context. Our data underscore the involvement of NLRP7 in RHM pathophysiology and confirm that DNA methylation of specific regions is critical., (© 2015 S. Karger AG, Basel.)

The aim of this research was to study the concentration of urinary trypsin inhibitor in amniotic fluid (AF) and its effect on myometrial contraction in term and preterm deliveries. Urinary trypsin inhibitor was measured in AF of term and preterm labor. Immunohistochemical staining of amnion and myometrium was carried out. Isometric uterine contraction was studied to elucidate the effect of AF and urinary trypsin inhibitor on the contractile activity of term and preterm myometrium. The effect of urinary trypsin inhibitor on the production of prostaglandin E2 (PGE2) from myometrial cultures stimulated by IL-8, IL-1 and LPS was verified. Urinary trypsin inhibitor was significantly increased in AF of cases of preterm delivery (p < 0.0001). Amnion and myometrium of preterm deliveries were faintly stained for urinary trypsin inhibitor compared to term delivery. Amniotic fluid and urinary trypsin inhibitor could successfully inhibit-myometrial contraction. Also, urinary trypsin inhibitor could significantly inhibit the production of PGE2 in the myometrial cell cultures stimulated by IL-1 and LPS (p < 0.001 and 0.0005). IL-8 has no significant effect on PGE2 production from myometrial cell culture. Urinary trypsin inhibitor suppresses myometrial contraction in term and preterm deliveries. It may play a role in maintaining normal pregnancy and preventing preterm delivery.

Dehydroepiandrosterone sulfate (DHA-S) is one of the successful drugs used to induce cervical ripening. There is growing evidence that interleukin-8 (IL-8) plays an essential role during cervical ripening. IL-8 was found to be hormonally controlled in the cervix. The aim of this research was to study the effect of DHA-S on the function of IL-8 on the cervix. Sixteen pregnant white New Zealand rabbits where tested either by placebo (adeps solidus, n = 4), DHA-S (10 mg, n = 4), IL-8 (100 ng, n = 4) or combination of DHA-S and IL-8 (n = 4). Collagen contents in the cervices were assessed histologically after staining with picrosirius red. The number of neutrophils in 5 random fields of the cervical tissue was counted in all groups. Collagenase, elastase and gelatinase were measured in those groups. The collagen content of the cervices was significantly decreased after treatment with DHA-S, IL-8 or a combination of both drugs compared to the control group (p < 0.004, 0.001 and 0.001, respectively). A maximal decrease in collagen content was found in the DHA-S + IL-8-treated group (71.5%). Neutrophil infiltration was significantly increased in the IL-8 and DHA-S + IL-8-treated groups (p < 0.0001). Neutrophil numbers in the DHA-S + IL-8-treated group were significantly higher than in the IL-8 group (p < 0.006). Collagenase activity was increased in the DHA-S, IL-8 and DHA-S + IL-8 groups compared to the control group (p < 0.02, 0.008 and 0.001, respectively). Elastase activity was also significantly increased in the same groups (p < 0.002, 0.005 and 0.001, respectively). A similar increment was found in gelatinase activity (p < 0.004, 0.0001 and 0.0001, respectively). DHA-S has a synergistic effect with IL-8 during cervical ripening.

We experienced a case of congenital afibrinogenemia and successfully performed cesarean section with administration of fibrinogen. The patient was administered fibrinogen every week to sustain a fibrinogen level above 60 mg/ dl according to our previously reported first case. Pregnancy course was uneventful, and fetal growth was normal, but unfortunately placental abruption occurred after the spontaneous onset of labor at 37 weeks gestation. The fibrinogen level before labor was 96 mg/dl, but decreased to 33 mg/dl when placental abruption was diagnosed. During and after the operation, it was increased to 147 and 199 mg/dl, respectively, through infusion of 10 g of fibrinogen, and massive bleeding was stopped. Two grams of fibrinogen were infused daily after cesarean section, and postpartum hemorrhage was normal. It is obvious that fibrinogen is an extremely important factor in maintaining pregnancy, and we conclude that fibrinogen level must be at least 60 mg/dl during pregnancy, 120 mg/dl during surgery and 150 mg/dl during labor, if possible as high as 200 mg/dl under the continuous infusion of fibrinogen to prevent placental abruption.

We immunohistologically studied the hepatic tissue sections in cases with the syndrome of hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome; n = 2) and acute fatty liver of pregnancy (AFLP; n = 2) compared to necropsy controls. Unlike in the AFLP cases, a marked infiltration of neutrophils in liver tissues was found in both cases of the HELLP syndrome. Immunostaining with the antihuman (polyclonal) TNF-alpha, IL-1 beta, IL-8 and antihuman neutrophil elastase (monoclonal antibody) was performed in paraffin-embedded hepatic tissue sections. Liver tissues in HELLP syndrome patients were stained strongly with TNF-alpha and neutrophil elastase antibody. The strongest staining pattern was observed in the eclamptic case, whereas in the AFLP cases, as in the necropsy controls, a very weak staining for anti-TNF-alpha and elastase antibody was found. The liver sections of the HELLP syndrome cases were moderately stained with polyclonal IL-1 beta and IL-8 antibodies whereas AFLP and controls had a very faint staining. Significant correlations were found between the numbers of necrotic hepatocytes and elastase dots in the same microscopic fields (randomly selected) of liver sections from two cases of HELLP syndrome (r2 = 0.63; p < 0.0001), which might suggest a neutrophil-mediated tissue damage in such a disease. This study suggests that a cytokine- and neutrophil-mediated liver injury occurs in the HELLP syndrome but not in AFLP.

We studied the impact of blood coagulation and fibrinolysis on the clinical features of eclamptic patients (n = 20) in Bangladesh. The variables used were edema, proteinuria, blood pressure, number of convulsions, level of consciousness at the time of admission, thrombin antithrombin complexes (TAT), antithrombin (AT) III (%) activity and antigen, D dimer fibrin degradation product and alpha 2-plasmin inhibitor-plasmin complex (PIC) in plasma. Canonical correlation analysis was made to obtain clinical index, eclampsia index and two coagulation indices. On admission, the mean values of coagulation parameters were AT III activity: 83.2% (range 57-108), TAT complex: 47.6 ng/ml (range 11.5-60), D dimer: 1,693 ng/ml (range 417-8,276) and PIC 1.4 mg/ml (range 0.4-3.3). We found a significant correlation between the eclampsia index and clinical index (r = 0.601; p = 0.01). Gestosis index, clinical index, and eclampsia index have also a strong correlation with the coagulation index (r = 0.695, p < 0.005; r = 0.871, p < 0.0001 and r = 0.805, p < 0.0001, respectively). Coagulation and fibrinolysis were markedly activated in eclampsia. The correlation between the clinical status and coagulation status in this study suggested a close relation between the coagulation and the development and progression of the disease.

To examine the possible role of endothelin and vasospasm in eclamptic seizures, we studied and analyzed the electroencephalograms (EEG) of endothelin-1 (ET-1)-treated pregnant, nonpregnant and sham control (dextrose-treated) rabbits. After multiple intravenous bolus injections of ET-1 (500 pmol/kg) or 5% dextrose in the rabbits, we recorded EEG directly from the brain cortex and analyzed by Fast Fourier Transform (FFT). Water content was measured in the brain of all groups (n = 7). Repeated seizures occurred in all of the pregnant and 2 of the nonpregnant rabbits by variable doses of ET-1. FFT analysis showed remarkable changes in frequency and power arrays characterized by mild to severe form of dysrhythmia, high-voltage spikes, high-voltage fast and slow waves after ET-1 injections. Water content was increased in brain mass in ET-1-treated rabbits (p = 0.001) suggesting an ET-1-induced edema. Histologically we confirmed that ET-1 caused ischemic changes in brain tissues. However, ET-1 induced more pronounced changes in behavior, EEG, brain edema or ischemia in pregnant than in nonpregnant groups. The injections of exogenous ET-1 into the brain substances were strongly suggested by immunohistochemical study with polyclonal antiendothelin antibody in brain tissue sections. Therefore, we assume that endothelin along with other vasoactive substances causes acute cerebral vasospasm and ischemia inducing EEG changes leading to ultimate clinical convulsions in eclampsia.

The early pathogenesis of amniotic fluid embolism is not completely understood. The entrance of amniotic fluid (AF) into the systemic circulation leads to an initial phase of pulmonary vasospasm, pulmonary hypertension and cor pulmonale. We studied the effect of AF on endothelin (ET) production in vivo and in vitro. Injection of rabbits (pregnant and nonpregnant) with meconium-stained AF, raw AF and supernatant AF led to a significant increase in serum ET. This result was confirmed by using human umbilical vein endothelial cell cultures incubated with the same types of AFs. After infusion of rabbits with AFs, we observed that the lung, heart and kidney were positively stained for ET and von Willebrand factor. AF was found to have an injurious effect on endothelial cells measured by using fura-2. The maximal injurious effect of AFs was observed for the meconium-stained AF. We hypothesized that the early pathological changes in AF embolism may be mediated by ET.

Urinary trypsin inhibitor-related substance (UTI-R) was measured in the amniotic fluid (n = 30), first neonatal urine (n = 10), meconium (n = 10), and adult urine (n = 10). The concentration of UTI-R excreted in the neonatal urine was significantly higher than in adult urine and meconium. Positive immunostaining for UTI was observed in amnion, umbilical cord and placenta. The effect of UTI was studied on the amnion using fura-2-acetoxymethyl ester. Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-beta (TNF-beta) led to a significant increase in relative fluorescence release (RFR) of fura-2 (p < 0.05 and p < 0.02, respectively) while endotoxin did not show significant changes of RFR of fura-2. The effect of IL-1 beta and TNF-beta was almost abolished after incubation of UTI with the amnion cells (p < 0.03). This suggests that UTI may have a protective effect on the amnion especially against IL-1 beta and TNF-beta.

We experienced a typical case of eclampsia. After the onset of eclampsia, we performed magnetic resonance imaging angiography (MRI angiography) to estimate the function of the cerebral artery. MRI angiography showed that spasm occurred in many cerebral arteries. The spasm was still observed in some arteries 13 day after the onset of eclampsia. Vasospasm of eclampsia was clearly, easily and noninvasively confirmed in this case by MRI angiography. This observation suggests that MRI angiography is very useful and informative for diagnosis and in treatment of eclampsia.

We studied the effects of bolus injections and infusion of endothelin-1 (ET-1) in female rabbits by measuring serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), lactic dehydrogenase (LDH), antithrombin III (AT-III), thrombin antithrombin (TAT) complexes, platelet counts and indirect bilirubin. Two successive bolus doses of 0.125 and 0.25 nmol/kg of ET-1 with an interval of 30 min were given to conscious non-pregnant female rabbits (n = 8). GOT, GPT and LDH were found to be significantly increased after injections of ET-1 (p < 0.02, p < 0.04 and p < 0.05, respectively). The percent AT-III activity decreased significantly (p < 0.005). Vasospasm of the hepatic artery was demonstrated by angiography with the same bolus doses in rabbits. There was also an increase in GOT (p < 0.003), GTP (p < 0.05), LDH (p < 0.007), indirect bilirubin (p > 0.07) and TAT complexes (p < 0.02) and a decrease in AT-III (p < 0.03) and platelet counts (p < 0.02) in rabbits (n = 10) after 24 h of continuous infusion of ET-1 (0.6 nmol/kg/h). Histological examination of rabbit liver tissues showed varying degrees of ischemic necrosis of hepatocytes. Thus this study suggests that exogenously administered ET-1 causes vasospasm and liver ischemia resulting in HELLP syndrome-like blood parameters in rabbits.