Christina Vogt Isaksen

Serious foetal growth restriction is associated with reduced proportions of natural killer cells in decidua basalis

Extravillous trophoblasts are major participants in placental development and remodelling of spiral arteries. Trophoblast invasion is regulated by maternal immune cells, and abnormal leucocyte subpopulation composition has been reported in implantation failure. In pre-eclampsia (PE), with or without foetal growth restriction (FGR), superficial trophoblast invasion and insufficient remodelling of spiral arteries are common findings. In the present study, we have compared spiral artery remodelling and leucocyte composition in decidual tissue from 30 cases (PE=8, FGR=5, PE + FGR=17) and 31 controls. Six histological remodelling criteria were established, and each pregnancy obtained a remodelling score. Numbers of natural killer (NK) cells (CD56+), T cells (CD3+) and activated (CD25+ or CD69+) leucocytes were determined and related to total leucocyte (CD45+) numbers in serial sections. Cases demonstrated significantly impaired spiral artery remodelling, inappropriate placental growth and reduced NK cell proportions, as compared to controls (P=0.02, P<0.001 and P=0.01, respectively). Reduced NK cell proportion was primarily found in pregnancies complicated by FGR, with or without PE, and a significant positive correlation was observed between NK cell proportion, trophoblast infiltration and placental growth. Our in vivo observations support the hypothesized association between NK cells, impaired placental development and pathogenesis of PE/FGR.

Trophoblast Apoptosis in Human Placenta at Term as Detected by Expression of a Cytokeratin 18 Degradation Product of Caspase

CONTEXT Apoptosis occurs in the normal placenta. The monoclonal antibody M30 is directed against a novel epitope of cytokeratin 18 (CK18) that is formed by caspase cleavage early in the apoptotic cascade, and this antibody may therefore be useful for evaluating trophoblast apoptosis. OBJECTIVE We undertook the present study to evaluate the use of monoclonal antibody M30 to assess trophoblast apoptosis in placenta at term. METHODS We stained paraffin-embedded placental tissues from 15 deliveries at term with M30. We compared positive M30 staining and CK18 staining (as detected by a monoclonal antibody directed against CK18) of trophoblasts in serial slides. We also compared apoptotic rates as detected by M30 and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) in 7 of the placentas. RESULTS In fields of villous tissue, most M30-positive cells were CK18-positive syncytiotrophoblasts. Approximately half of M30-positive cells occurred as focal positive staining in the syncytial layer, and half occurred as abundant staining of syncytiotrophoblasts in areas with increased intervillous or perivillous fibrinoid. We found very few M30-positive cells in villous stroma. In decidual/basal plate tissues, most (two thirds) of the M30-positive cells were CK18-positive extravillous trophoblasts, whereas one third were syncytiotrophoblasts of anchoring villi. Since TUNEL detects apoptosis in both epithelial and nonepithelial cells, more cells were positively stained with TUNEL than with M30 in some tissue fields. However, our observations suggest that M30 was more sensitive than TUNEL in recognizing apoptotic trophoblasts and had less nonspecific staining than TUNEL. CONCLUSION We recommend the use of monoclonal antibody M30 for apoptosis studies in placental tissues. This antibody is easy to handle, the staining obtained seems specific, and the nonspecific staining seems negligible.

Decidual expression and maternal serum levels of heme oxygenase 1 are increased in pre-eclampsia.

Background. Pre‐eclampsia (PE) is associated with increased oxidative stress and excessive maternal inflammatory response. Heme oxygenase 1 (HMOX1) is an important stress response enzyme and a mediator of cytoprotection against a wide variety of tissue injuries. Methods. In the present study, microarray technology was used to compare the expression of HMOX1 and other genes involved in stress and inflammatory responses in decidua basalis from 16 pregnancies complicated by PE and 17 healthy controls. In addition, the presence of HMOX1 protein in decidua basalis was examined by means of immunohistochemistry, and ELISA was used to measure the maternal serum concentration of HMOX1. Results. Fifteen transcripts involved in stress response including HMOX1 were up‐regulated in cases, using a cut‐off value at p = 0.01. HMOX1 protein expression in decidua basalis was significantly increased in cases compared to controls reflected by more pronounced intensity of HMOX1 positive decidual cells (1.8±0.3 versus 1.5±0.4, p = 0.02) and an increased proportion of HMOX1 positive decidual leukocytes (31±29 versus 9±6%, p = 0.001). Finally, serum HMOX1 levels were significantly higher among cases compared to controls (3.1±1.3 versus 1.9±0.5 ng/ml, p = 0.008). Conclusions. Increased decidual and serum HMOX1 levels, together with altered decidual expression of some stress‐related genes in cases, support the role of oxidative stress and excessive maternal inflammatory response in the pathogenesis of PE.

Maternal Smoking, Intrauterine Growth Restriction, and Placental Apoptosis

AbstractPregnant women who smoke are at greater risk of delivering a growth-restricted infant than nonsmoking mothers. We wanted to see if apoptosis could be involved in the mechanisms behind smoke-induced growth restriction, and our aim was to compare apoptosis in the placenta of smoking mothers giving birth to growth-restricted infants and nonsmoking mothers with infants of appropriate weight. The project was conducted at the Magee—Womens Hospital and Magee—Womens Research Institute, University of Pittsburgh, PA. Histological sections from 20 placentas were selected from smoking mothers who had given birth to small-for-gestational-age infants (birth weight ≤ 2 SD). The controls were gestational-age matched nonsmoking mothers with infants having appropriate-for-gestational-age weight. The TUNEL method was used to demonstrate DNA fragmentation in nuclei, and a monoclonal antibody M30, specific for a neo-epitope on cytokeratin 18, was used to identify apoptotic epithelial cells. The positive nuclei (TUNEL) and positive cells (M30-positive cytoplasm) were counted blindly both in villous tissue and in decidual/basal plate tissue. M30-positive cells in villous tissues were significantly increased in placentas from smoking mothers compared to nonsmoking mothers. When evaluated by the TUNEL method, the difference between the two groups of women was not significant. Our study shows that apoptosis was increased in the placentas of smoking mothers with growth-restricted infants. The difference between the two groups was mainly in the syncytiotrophoblast layer and in connection with perivillous fibrin deposition. Cigarette smoke with reduction in blood flow has previously been shown to increase apoptosis, and it is possible that this could be one of the mechanisms playing a role in the growth restriction.

Sibs with anencephaly, anophthalmia, clefts, omphalocele, and polydactyly : Hydrolethalus or acrocallosal syndrome?

Major characteristics of the acrocallosal syndrome include severe mental retardation, agenesis or hypoplasia of the corpus callosum, and polydactyly of fingers and toes. In the past few years, anencephaly has also been noted, together with other midline defects. We report on a nonconsanguineous, Norwegian couple with a history of two pregnancies with a male and a female fetus, respectively, with anencephaly, median cleft lip and palate, omphalocele, and preaxial polydactyly, suggesting the diagnosis of the acrocallosal syndrome. Both fetuses also lacked eyes and nose, a finding not previously reported in the acrocallosal syndrome. Microphthalmia has been reported in the hydrolethalus syndrome, which may be caused by mutations in the same gene as the acrocallosal syndrome. The present report adds support to the hypothesis that the acrocallosal and hydrolethalus syndromes may be allelic conditions. The family history is consistent with autosomal recessive inheritance.

Cerebral nuclear magnetic resonance (MRI) in Kearns syndrome

Abstract. We describe an 11‐year‐old boy with external ophthalmoplegia, pigment retinopathy, hearing loss, elevated spinal protein and ragged‐red fibers on muscle biopsy. Cerebral nuclear magnetic resonance (MRI) demonstrated demyelinating lesions in the white matter of the cerebral hemispheres and the cerehellum. To our knowledge this is the first report on the cerebral MRI findings in Kearns syndrome.

Detection of Trisomy 18 on Formalin-fixed and Paraffin-embedded Material by Fluorescence In Situ Hybridization

Formalin-fixed and paraffin-embedded autopsy material from 10 fetuses and infants with unknown karyotype and anomalies suggestive of trisomy 18 were subjected to fluorescence in situ hybridization (FISH). Nuclei were extracted from the tissues and hybridized with a chromosome 18–specific centromere probe. The hybridization was successful in 9 of 10 cases. Two cases showed three hybridization signals in most of the nuclei (74% and 85%). These had anomalies frequently occurring with trisomy 18 (congenital heart defect, omphalocele, and horseshoe kidney). Two cases showed a mixture of two and three signals (47%/49% and 59%/36%), suggesting the possibility of mosaicism. One of these cases had anomalies consistent with a trisomy 18 phenotype. In the other case intrauterine growth retardation and syndactylies suggested triploidy. Hybridization with a chromosome 8–specific probe gave a distribution of two and three signals (34% and 62%, respectively). This result strengthened the suspicion of a possible triploid mosaicism. In five of the cases most of the nuclei showed two signals (85% to 88%). However, as only one type of tissue was examined for enumeration of chromosome 18, the possibility of organ mosaicism or other chromosome aberrations cannot be excluded. The FISH technique is applicable on macerated and autolysed formalin-fixed tissue, making it possible to retrospectively analyze autopsy material from aborted and stillborn fetuses and infants. This analysis contributes to a better quality of perinatal autopsies and is helpful in parental counseling.

Death of a 10-Month-Old Boy After Exposure to Ethylmorphine

Abstract:  Ethylmorphine, an opiate that is partially metabolized to morphine, is a common ingredient in antitussive preparations. We present a case where a 10‐month‐old boy was administered ethylmorphine in the evening and found dead in bed the following morning. Postmortem toxicological analyses of heart blood by gas chromatography‐mass spectrometry and liquid chromatography‐mass spectrometry revealed the presence of ethylmorphine and morphine at concentrations of 0.17 μM (0.054 mg/L) and 0.090 μM (0.026 mg/L), respectively. CYP2D6 genotyping showed that the deceased had an extensive metabolizer genotype, signifying a “normal” capacity for metabolizing ethylmorphine to morphine. The autopsy report concluded that death was caused by a combination of opiate‐induced sedation and weakening of respiratory drive, a respiratory infection, and a sleeping position that could have impeded breathing. This is the first case report where the death of an infant has been linked to ethylmorphine ingestion.

Sudden infant death syndrome not caused by Norwegian Jervell and Lange-Nielsen mutations

The long QT syndrome (LQTS) has been proposed as the cause of death in some cases of sudden infant death syndrome (SIDS) [Schwartz, 1976]. The most convincing evidence comes from a 19-year long prospective study involving ECG registration of approximately 34,000 infants, where 12 of 24 cases that died fromSIDSwere shown to have had prolongation of theQT interval [Schwartz et al., 1998]. The risk of SIDS in cases with prolonged QT interval was found to be 41.3, higher than for most factors known to increase the risk of SIDS. Several studies give support to this result [Maron et al., 1976; Sadeh et al., 1987], while others find no indication of prolonged QT interval in SIDS or amongSIDS families [Kukolich et al., 1977; Southall et al., 1986]. Recent studies have found LQTS gene mutations in SIDS victims [Christiansen et al., 1999; Priori et al., 2000;Ackerman et al., 2001; Piippo et al., 2001; Schwartz et al., 2001]. LQTS can be a direct cause of sudden infant death, as well as a possible predisposing factor of SIDS when combined with one or more environmental factors. The autosomal recessive form of LQTS, Jervell and LangeNielsen syndrome (JLNS), comprises deafness and prolonged QT interval and was first described in Norway in 1957 [Jervell and Lange-Nielsen, 1957]. JLNS has a highmortality rate, but is a rare disorder. However, JLNS has a high prevalence in the Norwegian population, especially in two counties in themiddle region ofNorway (Trønderlag) and in two counties in the south of Norway (Agder) [Tranebjaerg et al., 1999]. In Norwegian families with clinically diagnosed JLNS, only four different mutations in theKCNQ1 (KVLQT1) gene have been described; a frameshift at codon 191 (5-bp deletion) and the amino acid changes R518X, Q530X, and E261D [Tranebjaerg et al., 1999; Tyson et al., 2000]. The high prevalence of JLNS in Norway is intriguing considering the knowledge that SIDS has had a higher prevalence in Norway compared to other Nordic countries [Vege and Rognum, 1997; Tranebjaerg et al., 1999]. Tranebjaerg et al. [1999] suggest that the well-defined spectrum of JLNS mutations in Norway provides an excellent opportunity to investigate whether a proportion of SIDS cases in Norway might be explained by undiagnosed heterozygous mutation carriers who are asymptomatic or undiagnosed homozygous JLNS patients. In a previous case-control study with questionnaires to 16 SIDS families and 29 control families in southeast Norway, we hypothesized that some SIDS cases could be due to cardiac arrhythmias such as JLNS. We found that 19% of the SIDS families compared to 7%of the control families reportedhaving family members with hearing disabilities/deafness (OR 3.1 CI 0.5–21, P1⁄4 0.2). Although only 6% of SIDS family members reported having had syncopal attacks, this was found in as few as 3% of control families (OR 1.9 CI 0.1–32, P1⁄4 0.6). We therefore continued to look for the known Norwegian JLNS mutations in our SIDS cases. Twenty-six SIDS cases from the two middle counties (Trønderlag), 32 SIDS cases from the two southern counties (Agder), and 212 SIDS cases from the eastern region ofNorway were studied. Each case has been classified according to the Nordic criteria for the diagnosis of SIDS [Vege and Rognum, 1997]. The SIDS victims have a median age of 3.5 months (range, 0.5–36 months) and include 165 males (61%) and 105 females (39%). Two of the SIDS victims from the middle region of Norway and two of the SIDS victims from the southern region of Norway were twins dying simultaneously. DNA was extracted from frozen blood/spleen samples using standard methods (phenol/chloroform extraction and ethanol precipitation) or from formalin fixed, paraffin embedded tissue (spleen). For mutation analysis, polymerase chain reaction (PCR) was performed to amplify DNA products from the regions of interest in theKCNQ1 gene, using primer sequences and PCR temperature profiles described in Table I. The PCR products were then digested by restriction enzymes (Table I). The study was approved by the regional ethics committee. None of the Norwegian JLNS mutations was found in the Norwegian SIDS cases studied. Our results therefore indicate that JLNS is not hidden among SIDS victims. However, LQTS is a genetically heterogeneous condition [Tyson et al., 1997]. Therefore, the failure to demonstrate JLNS mutations in our material does not rule out involvement of long-QT syndrome as an explanation for some cases of SIDS inNorway. The study by Ackerman et al. [2001] demonstrated mutations in one of the other LQTS genes (SCN5A) in 2% of SIDS cases, making it worthwhile to screen SIDS cases for mutations in LQT genes. SIDS is still the main cause of death in infancy in Norway, but neglect, abuse, and homicide are more often mentioned as possible differential diagnoses. Much disputed is the so-called three-strike rule: One unexplained death is tragic but innocent, two is suspicious, and three is murder [Eastman, 2003]. However, it seems that the pendulum is about to move in the other direction: a recent report from a court case in the United Kingdom tells of a mother being acquitted of murder of three infants on the grounds of a possible genetic explanation for their deaths [Eastman, 2003]. It is stated that the outcome could lead to more extensive screening of babies for inherited disorders, as well as to genetic testing of mothers accused of killing their babies. The search for a genetic predisposition for *Correspondence to: Dr. Marianne Arnestad, Institute of Forensic Medicine, Rikshospitalet, 0027 Oslo, Norway. E-mail: marnesta@labmed.uio.no

P05.01: Prenatal diagnosis of characteristic CNS features in thanatophoric dysplasia (TD)

Objective: The objective of this study was to analyze the reasons of fetal cardiomegaly and prenatal and neonatal outcome. Material and methods: Between 2000 and 2008 over 10.000 pregnancies were referred to our department for fetal echocardiography and detailed anomaly scan. All examinations were performed with Voluson 730 Expert and Philips HDI 5500. We measured heart size dividing heart area by chest area (HA/CA). An inclusion criteria was HA/CA ratio 0,45 or more. There were 3 study groups in our series: 1. Cardiomegaly with normal heart anatomy (NHA), and no extracardiac malformations (ECM) 2. Cardiomegaly with congenital heart disease (CHD) without or with ECM, 3. Cardiomegaly and ECM with NHA. Results: Cardiomegaly was identified in 99 fetuses. Of the 99 fetuses we lost follow up in 21 fetuses. Of 78 fetuses 31 hearts were normal with no ECM (Group 1). In this group the most common reason of cardiomegaly were: intrauterine infection (n=10), 40% of demise, twin-twin transfusion syndrome (n=10), 67% of demise, intrauterine growth restriction (n=7) 43% of demise, and arrhythmias (n=5) 60% of demise. Total fetal and neonatal demises in group 1 was 52%. Group 2 (n=28): 12 fetuses had aortic stenosis (42% of demises), 8 fetuses had Ebstein anomaly (75% of demises), 2 fetuses had pulmonary stenosis (100% of demises), and 6 fetuses had complex CHD (67% of demises). The total demises in group 2 was 53%. Group 3: n= 19. There was renal agenesis in 8 fetuses (100% of demises), skeletal malformations in 5 fetuses (80% of demises), 6 fetuses with another fetal malformations including 4 fetuses with urinary tract disease (100% of demises). Conclusion: Cardiomegaly is a serious problem and it’s connected with high rate of demises, independently of the reason of cardiomegaly. All fetuses with cardiomegaly should be referred as soon as possible for fetal cardiology centre for haemodynamic assessment.