Genetic analysis of patients with deep vein thrombosis during pregnancy and postpartum.

Deep vein thrombosis (DVT) is a serious pregnancy-related complication. Recent studies indicate that the genetic background for DVT differs with ethnicity. In our study, we enrolled 18 consecutive Japanese patients who had developed DVT during pregnancy and postpartum. We performed a genetic analysis of three candidate genes for DVT, protein S, protein C and antithrombin, in these patients. We found that four patients had missense mutations in the protein S gene, including the K196E mutation in two patients, the L446P mutation in one patient, and the D79Y and T630I mutations in one patient, as well as one patient with the C147Y mutation in the protein C gene. All five patients with genetic mutations had DVT in their first two trimesters. Nine of the patients without genetic mutations developed DVT in the first two trimesters, and four in the postpartum period. Thus, genetic mutations in the protein S gene were predominant in pregnant Japanese DVT women, and DVT in pregnant women with genetic mutations occurred more frequently at the early stage of pregnancy than postpartum. Considering the rapid decrease in protein S activity during pregnancy, we may need to assess thrombophilia in women before pregnancy.

Placental features of chorioamnionitis colonized with Ureaplasma species in preterm delivery.

Ureaplasma spp. is detected in the urogenital tract, including the vagina, cervix, chorioamnion, and placenta. Their colonization is associated with histologic chorioamnionitis (CAM), often observed in placentas from preterm delivery. We isolated Ureaplasma spp. from 63 preterm placentas among 151 specimens, which were delivered at <32 wk of gestation. Of the 63 placentas, 52 (83%) revealed CAM in cultures positive for Ureaplasma spp., however, CAM was observed only in 30% (26/88) of cultures negative for Ureaplasma spp. (p < 0.01). Colonization by Ureaplasma spp. was an independent risk factor for CAM (OR, 11.27; 95% CI, 5.09-24.98). Characteristic neutrophil infiltration was observed in the amnion and subchorion (bistratified pattern) in cultures positive for Ureaplasma spp. FISH analysis of CAM placenta with male infant pregnancy indicated that bistratified infiltrated neutrophils showed the XX karyotype and umbilical vein infiltrated neutrophils showed XY karyotype. The distribution of sulfoglycolipid, the receptor of Ureaplasma spp., was mainly detected in the amnion. Ureaplasmal urease D protein and ureB gene were both detected in the amnion, indicating direct colonization by Ureaplasma spp.

Up-regulation of alpha5-integrin by E-cadherin loss in hypoxia and its key role in the migration of extravillous trophoblast cells during early implantation.

During early pregnancy, cytotrophoblast cells differentiate into extravillous trophoblast (EVT) cells and invade the uterine spiral arteries. This physiological process is essential for the development of maternal-fetal circulation. Because EVT cells are exposed to a low-oxygen environment during this process, we investigated the role of hypoxia in the mechanism that regulates the invasive behavior of EVT cells. Real-time PCR and immunofluorescent analysis were performed to investigate how hypoxia influences the expression of E-cadherin in villous explants cultures and in trophoblast-derived BeWo cells. We determined that hypoxia induced E-cadherin down-regulation through Snail up-regulation in villous explant cultures. The influence of E-cadherin loss was examined by analyzing the expression of alpha(5)-integrin and phosphorylated focal adhesion kinase (FAK) by Western blot and evaluating trophoblast invasion using a matrigel invasion assay. E-cadherin loss induced the up-regulation of alpha(5)-integrin, which leads to the tyrosine phosphorylation of FAK, resulting in an increase in the invasive activity of EVT cells. An alpha(5)-integrin neutralizing antibody inhibited the invasion of EVT cells by attenuating FAK tyrosine phosphorylation. Immunohistochemical analysis using clinical placental bed biopsies revealed that alpha(5)-integrin was up-regulated and FAK tyrosine phosphorylated (Try(861)) as EVT cells invade the uterine myometrium, whereas E-cadherin expression was down-regulated. These results suggest that alpha(5)-integrin up-regulation induced by E-cadherin loss under hypoxia has a crucial role in regulating the migration of EVT cells. This finding should help us reach a better understanding of the pathogenesis of critical gestational diseases, such as preeclampsia.

Change in morphology and oxytocin receptor expression in the uterine blood vessels during the involution process.

BACKGROUND: The histological changes in uterine blood vessels during pregnancy have been well investigated, but there have been few reports focusing on the changes in blood vessels during the involution process, especially within the first 24 h. We observed the process of uterine involution, focusing on the vessels of the resected uterus. METHODS: Paraffin-embedded uterine samples from 15 patients who underwent hysterectomy because of severe cervical laceration and uterine rupture were examined. The time between delivery and hysterectomy ranged from 15 min to 456 h. The specimens were stained with hematoxylin-eosin, elastica-van Gieson and an antioxytocin receptor antibody. RESULTS: Changes in the uterine vessels varied substantially based on their location. The intima in arteries of the endometrial side thickened within 5 h after delivery. On the serosal side, phlebosclerosis was demonstrated 6 weeks postpartum. Immunoreactivity for the oxytocin receptor (OTR) appeared in the muscular medias of arteries 5 h after delivery although it was not expressed before this period. CONCLUSION: Remodeling of uterine vessels involved thickening of the arterial intima and OTR expression in vessel walls during the first 5 h postpartum; the parameters normalized within 6 weeks. However, phlebosclerosis persisted for a long time on the serosal side.

Anti-annexin A2 IgM antibody in preterm infants: its association with chorioamnionitis.

Intrauterine infection is associated with chorioamnionitis (CAM), which can lead to preterm delivery. We previously reported that the levels of IgM and the incidence of CAM were elevated in preterm infants with neonatal pulmonary emphysema. The pathogen and target of this IgM remain unclear. By using Western blot and amino acid sequences, we have determined one of the target proteins: annexin A2. Immunohistochemical analysis showed that annexin A2 was expressed at fetal chorion and amnion membranes. Among very low birth weight (VLBW) infants with hyper-IgM (> or = 30 mg/dL), 58.8% showed a high titer against annexin A2 (more than x 16), which accounted for about 20%-40% of the total IgM. Anti-annexin A2 IgM antibody inhibited plasmin generation. Furthermore, the median of anti-annexin A2 IgM titer from preterm infants who were delivered with high-grade (grade III) CAM was significantly higher than those from preterm infants without CAM (p = 0.011) and with low-grade CAM (grade I and II) (p = 0.010). Here, we indicate the fetal autoimmunoreactivity against the fetomaternal interface in preterm infants.

Adiponectin concentration in umbilical cord serum is positively associated with the weight ratio of fetus to placenta.

CONTEXT: Adiponectin (APN) concentration in umbilical cord serum is higher than that in adult serum. Except for the positive association between birth weight and cord APN concentration, little is known about the pathophysiological function of APN in fetal development. OBJECTIVE: The objective of this study was to evaluate the relationship of cord serum APN and IGF-I concentrations with the development of the fetoplacental unit. DESIGN AND METHODS: Umbilical cord serum APN and IGF-I concentrations were measured in term singleton deliveries (n = 94). The association of cord APN and IGF-I concentrations was evaluated in relation to fetal weight, placental weight, and fetoplacental (F/P) weight ratio. RESULTS: Mean concentrations and sd of APN and IGF-I were 36.1 +/- 14.0 microg/ml and 58.6 +/- 27.0 ng/ml, respectively. Cord APN concentration was positively associated with F/P weight ratio (r = 0.375, P < 0.001) as well as fetal weight (r = 0.389, P < 0.001) but not placental weight. Cord IGF-I concentration was positively associated with fetal weight (r = 0.405, P < 0.001) and placental weight (r = 0.400, P < 0.001) but not F/P weight ratio. In multiregression analysis, only APN concentration resulted in a significant determinant of F/P weight ratio among variables (beta = 0.376, P < 0.001). CONCLUSIONS: In cord hyperadiponectinemia, fetuses tend to be disproportionately larger for their placental weight and vice versa in cord hypoadiponectinemia. APN is shown to be the first biomarker positively associated with F/P weight ratio.

Huge pericardial rhabdomyoma with hypoplastic left ventricle and lung.

We describe the clinical course and autopsy findings of a female fetus with hydrops fetalis due to a huge pericardial rhabdomyoma. Fetal echocardiography at 21 weeks gestation demonstrated a huge tumor in the left ventricle. The fetus died of hydrops fetalis due to cardiac dysfunction at 24 weeks gestation. Autopsy demonstrated that the tumor protruded from the epicardial region of the apex into the pericardial cavity and induced a hypoplastic left ventricle and lung. Microscopically, the cardiac tumor showed characteristics of rhabdomyoma. This localization of cardiac rhabdomyoma is rare, but we remain aware of the possibility of an unusual and rapid progression of cardiac rhabdomyoma.

Impact of prepregnant body mass index and maternal weight gain on the risk of pregnancy complications in Japanese women.

BACKGROUND: To analyze the association of pregnancy complications with prepregnant body mass index and weight gain during pregnancy in Japanese women. METHODS: A retrospective cohort study was conducted with 21,718 Japanese women with a singleton pregnancy. Pregnant women were grouped by prepregnant body mass index and evaluated for association with pregnancy complications using multivariate logistic regression analysis. The women in each body mass index group were then divided into groups by weight gain during pregnancy using intervals of 0.05 kg/week to analyze the relationship between the weight gain and pregnancy complications by multivariate logistic regression association analysis. RESULTS: In both nulliparous and parous women, the least pregnancy complications were found among women with medium prepregnant body mass indexes (18-23.9). Significant risks of pregnancy complications were associated with low (< 18) and high (> or = 24) prepregnant body mass indexes, particularly high prepregnant body mass indexes. In nulliparous women, the optimal weight gain was 0.25-0.4 kg/week for low (< 18) prepregnant body mass index, 0.20-0.30 kg/week for medium (18-23.9) prepregnant body mass index, and > or = 0.05 kg/week for high (> or = 24) prepregnant body mass index. In parous women, the corresponding values were > or = 0.20, 0.20-0.30, and 0.05-0.30 kg/week. CONCLUSIONS: Japanese women with prepregnant body mass indexes from 18 to 23.9 are least associated with pregnancy complications, although there is a broad range of prepregnant body mass indexes associated with few pregnancy complications. Optimal weight gain is roughly inversely related to prepregnant body mass index.

Prenatal diagnosis of trisomy 9.

Fetal trisomy 9, especially its nonmosaic form, is a rare chromosomal abnormality and there are only 8 cases reported to have been sonographically detected in the prenatal period. We report a case of nonmosaic fetal trisomy 9, mimicking trisomy 13 on sonographic findings at 32 weeks' gestation. Although the incidence of trisomy 9 is rare, diagnosing trisomy 9 is important because the sonographic features are similar to those of trisomies 13 and 18, and cannot to be identified by routine fluorescencein situ hybridization. Because nonmosaic trisomy 9 is universally lethal, correct diagnosis and appropriate counseling is essential in patient care and clinical management.

Congenital cervical rhabdomyosarcoma arising in one fetus of a twin pregnancy.

OBJECTIVE: To describe a huge congenital cervical rhabdomyosarcoma. METHODS: We were recently confronted with a case of fetal solid neck mass arising in one fetus of a twin pregnancy. Prenatally, the cervical tumor was consistent with teratoma, but it was diagnosed histologically as a rhabdomyosarcoma. Genetic amniocentesis showed a mosaic pattern consisting of 46,XY/46,XY,t(2;8)(q35;q21.2). RESULTS: EXIT procedure was proposed to the parents but declined. The twin with huge cervical tumor died in utero at 35 weeks' gestation due to hydrops fetalis. CONCLUSION: Fetal cervical rhabdomyosarcoma is an extremely rare condition that has not been previously reported, but should be considered in the presentation of fetal solid neck mass.

Changed expression of heat shock proteins in various pathological findings in placentas with intrauterine fetal growth restriction.

Heat shock proteins (HSPs) are activated in the cells of most organisms in response to sublethal heat shock and other stressors. It has been reported that HSP27, HSP60, HSP70, and HSP90 are expressed in normal human placenta, and it was thought that these HSPs play a role in the demonstration of cell viability and function. In this study, we performed an immunohistochemical (IHC) study of these HSPs for 27 placentas that had complicated intrauterine fetal growth restriction (IUGR) and compared the IHC findings with the pathological findings. To quantify HSP27, HSP60, HSP70, and HSP90, immunoreacted cells in the chorionic villi, syncytiotrophoblasts (ST), and cytotrophoblasts (CT) were counted. In thrombus, excessive syncytial knots, and avascular villi, the expression of HSPs was higher in the pathological sections compared to control in both ST and CT. In contrast, all HSPs decreased in both ST and CT around the infarction region. The data suggested that chorionic villi cells locally responded to some stresses, e.g., hypoxia and increase or decrease in the expression of HSPs. Although the villous cells around the infarction histologically appear viable, they may have received lethal damage, and as a result the expression of HSPs was decreased. These results are expected to improve our understanding of the pathological findings of IUGR in placentas, including the quality, damage, and function of the chorionic villi.

Immunohistochemical distribution of Toll-like receptor 4 in term and preterm human placentas from normal and complicated pregnancy including chorioamnionitis.

Recently discovered Toll-like receptors (TLRs) are essential for the induction of innate immune responses. One member of the TLR family, TLR4 mediates lipopolysaccharide (bacterial endotoxin)-induced inflammatory responses. Although the innate immune system appears to be important in the pathogenesis of infection-induced preterm delivery, the distribution of TLRs in human placenta is poorly understood. Here we investigated the expression of TLR4 protein in 43 human placentas obtained from normal and complicated pregnancies delivered in the second and third trimesters, using immunohistochemistry. TLR4 was localized to the extravillous trophoblasts, intermediate trophoblasts/X cells in the degenerative villi, and villous Hofbauer cells of all preterm and term placentas examined and to the inflammatory cells in placentas with chorioamnionitis (CAM). The villous Hofbauer cells of preterm CAM placentas demonstrated increased TLR4 immunoreactivity compared with those of preterm placentas without CAM or those of term placentas with or without CAM. These results suggest an important role of the villous Hofbauer cells in the activation of innate immune system in response to infectious pathogens in preterm placentas. Elucidation of biological functions of placental TLR4 under physiological conditions requires further investigation.

Successful pregnancy with low molecular weight heparin in two women with recurrent miscarriage of unknown etiology.

We report here two cases of recurrent miscarriages that were successfully treated with continuous intravenous administration of low molecular weight heparin (LMWH). One patient experienced 11 spontaneous abortions, and the other eight abortions. Previous treatments including prednisone, aspirin and mononuclear-cell immunization were all unsuccessful. They were negative for anticardiolipin antibodies and lupus anticoagulant, and had no inherited thrombophilic disorder. Intravenous administration of LMWH, 4800 units of dalteparin, was started as soon as the conception was confirmed, and was continued until 34 weeks of gestation. They were delivered of live born infants.

Expression of vascular endothelial growth factor, placental growth factor, and their receptors Flt-1 and KDR in human placenta under pathologic conditions.

The vascular endothelial growth factor (VEGF) family and its receptors have multifunctional activities besides angiogenesis, and some of these molecules are induced by hypoxia/ischemia. They are known to be expressed in human placenta, but little is known about their involvement in pathologic conditions. We have investigated the expression patterns of VEGF, placental growth factor (PlGF), and their receptors fms-like tyrosine kinase (Flt-1) and kinase insert domain-containing region (KDR) in placentas with histopathological changes. Forty-two placentas from normal and complicated pregnancies delivered in the second and third trimesters were fixed with paraformaldehyde and embedded in paraffin. In situ hybridization and immunohistochemistry were performed on serial sections. In the villi with characteristic hypoxic/ischemic changes (HIC), including increased syncytial knots, infarction, or hypercapillarization, intense immunostaining for VEGF was detected in the media of blood vessels, and increased staining for KDR was demonstrated in the endothelial cells. Strong PlGF immunoreactivity was localized to the degenerative trophoblasts around the infarctions. Marked Flt-1 mRNA expression in the syncytiotrophoblast layers of HIC villi was identified, but some samples did not show ligand expression in these regions. Positive immunostaining for VEGF, PlGF, and Flt-1 was observed in infiltrated neutrophils and macrophages in the placentas with chorioamnionitis (CAM). These findings suggested that in the hypoxic/ischemic regions, VEGF and KDR expression is increased within the villous vessels by paracrine regulation, whereas the expression of PlGF and Flt-1 is enhanced in villous trophoblasts by autocrine regulation. The Flt-1 gene may also be up-regulated directly by hypoxia/ischemia independently of ligand mediation. Furthermore, the results indicated that VEGF and PlGF stimulate inflammatory cell migration by autocrine regulation via the Flt-1 receptor in the CAM placenta. Thus, various functions of VEGF family members participate in the development of pathologic changes in the placenta.

Detection of cytomegalovirus DNA in human placenta.

Although human cytomegalovirus (CMV) is one of the most common causes of viral intrauterine and perinatal infection, its distribution in the placenta is poorly understood. The purpose of this study was to determine the frequency of CMV DNA positivity in placentas, to demonstrate the localization of the viral genome, and to identify the clinical features related to placental CMV. A total of 254 placentas from 231 mothers were investigated, and the maternal serum CMV immunoglobulin antibodies were measured. Specimens from both the placental parenchyma and the placental membrane close to the ruptured site in each placenta were examined for the presence of CMV DNA using dot blot hybridization after PCR amplification. None of 57 placentas from seronegative mothers was positive for CMV DNA. Of 197 placentas from seropositive mothers, 60 (30.5%) had CMV DNA in either the parenchyma or the membrane by dot blot analysis. In situ hybridization was carried out on these 60 placentas, and the localization of the viral genome was established in 19; CMV DNA was localized mostly to the villi, including the mesenchyme and trophoblasts, extravillous trophoblasts, and decidual cells. The mean gestational age at delivery was significantly later in the CMV DNA-positive placentas than in the negative placentas (36.9 +/- 5.1 vs 34.7 +/- 6.2 weeks, P = 0.0059). CMV DNA was detected in only 6 of 33 placentas delivered in the second trimester, and all six were associated with either severe maternal nephritis or severe chorioamnionitis. These results suggest that the CMV genome is common in placentas at later gestational ages and in those of earlier gestational ages with certain maternal complications.

Hyperreactio luteinalis associated with severe twin-to-twin transfusion syndrome.

Twin-to-twin transfusion syndrome (TTTS) is a severe complication of monochorionic twin pregnancies, with high perinatal loss and significant morbidity. The etiology of this syndrome remains unclear. Hyperreactio luteinalis is a rare benign condition characterized by maternal ovarian enlargement due to theca lutein cysts. We present 4 cases of hyperreactio luteinalis associated with severe TTTS. We detected maternal ovarian enlargement by prenatal ultrasonography in 2 cases, at the cesarean section in 1 case, and postpartum in 1 case. Three cases showed hydrops fetalis and all cases showed congestive cardiac failure. Serial amnioreduction was performed in all cases, and regression of hydrops fetalis and maternal ovarian enlargement occurred in 1 case. These cases suggest that the pathophysiology of TTTS is closely related to the etiology of hyperreactio luteinalis.

Placental features in preterm infants with periventricular leukomalacia.

OBJECTIVE: Evaluation of the placenta provides some important insights into pathophysiologic changes that take place during the prenatal and intrapartum process. We investigated the relationship between placental findings and periventricular leukomalacia (PVL) to obtain a better understanding of its cause. METHODS: Thirty-two preterm infants with PVL delivered before 34 weeks' gestation, between 1990 and 1999, were classified into 4 groups according to the onset of brain injury assumed from ultrasonographic presentation and clinical course: 2 Antenatal, 22 Peripartum, 5 Postnatal, and 3 in an unknown time of onset group. We evaluated the gross and histopathologic features of the placentas of each group and compared them with those of a control group matched by birth weight and gestational age in terms of the frequency of major placental findings. Potential confounding factors were controlled in logistic regression analyses. RESULTS: Gross lesions with disturbance of uteroplacental circulation, including massive retroplacental hematoma, extensive infarction or thrombosis, and marked basal or perivillous fibrin deposition, were observed more frequently in the Antenatal + Peripartum combined subgroup than in the controls (41.7% vs 13.7%). Placentas from the Antenatal + Peripartum subgroup also demonstrated a significantly higher frequency of ischemic changes in villi, based on histopathologic examination, as compared with the control group (54.2% vs 13.7%). These associations remained after adjustment for confounding factors in logistic regression analyses (odds ratio: 4.04, 95% confidence interval: 1.40-11.67; and odds ratio: 7.28, 95% confidence interval: 2.50-21.20; respectively). Frequencies of chorioamnionitis and twin placentation tended to be higher in PVL cases than in the controls, although the differences were not statistically significant (46.9% vs 37.9%, 37.5% vs 20.0%, respectively). CONCLUSIONS: These results suggest that disturbed placental circulation underlies the development of PVL in the majority of cases with prenatal and peripartum brain injury. In chorioamnionitis cases, certain additional factors were suggested in the genesis of PVL. Thus, placental examination is essential for elucidating the pathophysiologic changes leading to PVL in the perinatal process.