Karina Richani

A role of the anti-angiogenic factor sVEGFR-1 in the ‘mirror syndrome’ (Ballantyne's syndrome)

Background. ‘Mirror syndrome’ (Ballantynes syndrome) refers to the association of fetal hydrops with placentomegaly and severe maternal edema. Preeclampsia occurs in approximately 50% of these cases. Soluble vascular endothelial growth factor receptor-1 (sVEGFR-1), an anti-angiogenic factor, has been implicated in the pathophysiology of preeclampsia (PE). Objective. The objective of this study was to determine if the maternal plasma concentration of sVEGFR-1 is elevated in patients with mirror syndrome. Study design. This case-control study included patients with uncomplicated pregnancies (n = 40) and those with mirror syndrome (n = 4) matched for gestational age. Mirror syndrome was defined as fetal hydrops and severe maternal edema. Maternal plasma sVEGFR-1 concentrations were determined using specific enzyme-linked immunosorbent assays. Immunohistochemistry of sVEGFR-1 on villous trophoblasts was also performed in samples from one patient with mirror syndrome and compared with those from a patient with spontaneous preterm delivery matched for gestational age. Non-parametric statistics were used for analysis (p < 0.05). Results. (1) The median maternal plasma concentration of sVEGFR-1 was significantly higher in patients with mirror syndrome than in the control group (median: 3974 pg/mL, range: 3083–10 780 vs. median: 824 pg/mL, range: 260–4712, respectively; p < 0.001). (2) All patients with mirror syndrome had sVEGFR-1 concentrations above the 95th percentile for gestational age. Syncytiotrophoblast, especially syncytial knots, showed strong staining with antibodies against sVEGFR-1 in placental samples from the patient with mirror syndrome, but not in those from the patient with spontaneous preterm delivery. Conclusion. High maternal plasma concentrations of sVEGFR-1 were observed in mirror syndrome. We propose that this anti-angiogenic factor may participate in the pathophysiology of this syndrome. Thus, maternal plasma determination of sVEGFR-1 may help to identify the hydropic fetus that places the mother at risk for preeclampsia.

Resistin: a hormone which induces insulin resistance is increased in normal pregnancy.

Abstract Aims: Resistin, a newly discovered adipokine, is thought to play a key role in the regulation of insulin resistance. The objectives of this study were to develop a nomogram of maternal plasma concentrations of resistin from 11 weeks of gestation to term and to determine whether resistin concentrations differ between normal and overweight pregnant women. Methods: In this cross-sectional study, plasma concentrations of resistin were determined in normal pregnant women of normal body mass index (BMI 18.5–24.9; n=261), overweight pregnant women (BMI ≥25; n=140), and non-pregnant women of normal BMI (n=40). Blood samples were collected once from each woman between the first trimester and term. Percentiles for resistin concentration were determined for five pre-specified windows of gestational age. Plasma resistin concentration was determined by immunoassay. Non-parametric statistics were used for analysis. Results: The median maternal plasma concentration of resistin between 11 to 14 weeks of gestation in women of normal weight was significantly higher than non-pregnant women; the plasma concentration of resistin increased with gestational age. Conclusions: Normal pregnant women have a higher median plasma concentration of resistin than non-pregnant women and the concentration of this adipokine increases with advancing gestation. Alterations in the maternal plasma concentration of resistin during pregnancy could contribute to metabolic changes of pregnancy.

Human β-defensin-2: A natural antimicrobial peptide present in amniotic fluid participates in the host response to microbial invasion of the amniotic cavity

Objective. Human β-defensin-2 (HBD-2) is a potent antimicrobial peptide that is part of the innate immune response. The purpose of this study was to determine whether HBD-2 is present in amniotic fluid and if its concentration changes with microbial invasion of the amniotic cavity (MIAC) and labor. Study design. Amniotic fluid was retrieved by amniocentesis from 318 patients in the following groups: (1) mid-trimester (n = 75); (2) term not in labor (n = 28) and in labor (n = 51); (3) preterm labor and intact membranes without MIAC who delivered at term (n = 36), who delivered preterm without MIAC (n = 52), and preterm labor with MIAC who delivered preterm (n = 25); and (4) preterm premature rupture of membranes (preterm PROM) with (n = 25) and without MIAC (n = 26). MIAC was defined as a positive amniotic fluid culture for microorganisms. Amniotic fluid HBD-2 concentrations were determined using a sensitive and specific ELISA. Non-parametric statistics were used for analysis. Results. (1) HBD-2 was detected in all amniotic fluid samples; (2) the concentration of HBD-2 did not change with gestational age from mid-trimester to term (p = 0.8); (3) intra-amniotic infection was associated with a significant increase in amniotic fluid concentrations of HBD-2 in both women with preterm labor and intact membranes, and women with preterm PROM (p < 0.05 for each comparison); (4) patients with preterm labor and a negative amniotic fluid culture who delivered preterm had a higher median amniotic fluid HBD-2 concentration than those with preterm labor who delivered at term (p=0.001); and (5) among patients with preterm labor without MIAC, those who had intra-amniotic inflammation (amniotic fluid white blood cell count >100 cells per mL) had a higher median amniotic fluid concentration of HBD-2 than those without this condition (p < 0.002). Conclusion. (1) Amniotic fluid contains HBD-2, a natural antimicrobial peptide, and this may account for some of the antimicrobial activity of amniotic fluid; (2) amniotic fluid HBD-2 concentrations are increased in women with MIAC, regardless of the membrane status (intact membranes or PROM); and (3) we propose that amniotic fluid HBD-2 is part of the innate immune system within the amniotic cavity.

Evidence for complement activation in the amniotic fluid of women with spontaneous preterm labor and intra-amniotic infection

Objective. The complement system plays an important role in host defense against infection. Concentrations of complement split products or anaphylatoxins (C3a, C4a, and C5a) in biological fluids are considered to reflect complement activation. The purpose of this study was to determine if term and preterm parturition are associated with evidence of complement activation in the amniotic fluid. Study design. Amniotic fluid (AF) samples were collected from 270 women in the following groups: (1) normal pregnant women in midtrimester (n = 70), (2) term not in labor (n = 23), (3) term in labor (n = 48), and (4) preterm labor (PTL) (n = 129). PTL was categorized into: (a) PTL without microbial invasion of the amniotic cavity (MIAC) who delivered at term (n = 42), (b) PTL who delivered preterm without MIAC (n = 57), and (c) PTL with MIAC (n = 30). C5a, C4a, and C3a concentrations in amniotic fluid were determined by ELISA. Nonparametric tests were used for statistical analysis. Results. (1) The median AF C5a concentration was higher in women at term than that of those in the midtrimester (p = 0.02); (2) Spontaneous labor at term was not associated with changes in AF concentrations of anaphylatoxins C3a, C4a, and C5a (all p > 0.05); (3) Among patients with PTL who delivered preterm, those with MIAC had higher AF C4a and C5a concentrations than those without infection (p < 0.01); and (4) AF C3a, C4a, and C5a concentrations were higher in patients with PTL with MIAC than in those with PTL without MIAC who delivered at term. Conclusion. Patients with spontaneous preterm labor and intact membranes with microbial invasion of the amniotic cavity had higher median amniotic fluid concentration of complement split products C3a, C4a, and C5a than patients without intra-amniotic infection. These findings suggest that preterm labor in the context of infection is associated with activation of the complement system.

Anaphylatoxins in preterm and term labor.

Abstract Objective: The complement system plays a central role in the first line of defense against invading pathogens, and its activation involves the release of potent pro-inflammatory mediators such as anaphylatoxins C3a, C4a and C5a. The aim of this study was to determine whether differences existed in maternal plasma anaphylatoxin concentrations between patients with term and preterm parturition. Study design: A cross-sectional study was designed to determine the plasma anaphylatoxin concentrations in 296 pregnant women in the following groups: 1) normal pregnancy between 20–36 6/7 weeks (n=64); 2) term not in labor (n=70); 3) term in labor (n=60); and 4) preterm labor with intact membranes (n=102). Women with preterm labor were classified into: a) term delivery (n=24); b) preterm delivery without intra-amniotic infection (IAI) (n=62); and c) preterm delivery with IAI (n=16). Concentrations of C3a, C4a and C5a were determined by ELISAs. Statistical analysis was conducted with non-parametric methods. Results: 1) The median plasma C5a concentration was lower in women at term in labor than in those not in labor (P<0.001). In contrast, there were no differences in plasma C3a and C4a concentrations between the two groups (P>0.05). 2) Among patients with preterm labor, those with IAI had a higher median plasma C5a concentration than those without IAI and those who delivered at term (post-hoc tests P<0.001 and P=0.01, respectively). When comparing the preterm labor subgroups with normal pregnancy, only women with preterm delivery and IAI had a median plasma C5a concentration higher than that of normal pregnant women (Kruskal-Wallis P<0.001, post hoc test P<0.001). There was no difference in the plasma C4a concentration among patients with preterm labor. The median plasma C3a concentration in patients with preterm labor with IAI was higher than in those without IAI (Kruskal-Wallis P=0.01, and post-hoc test P=0.005). There was no difference in the plasma C3a concentrations between women with preterm labor who delivered at term and those with preterm delivery, with or without IAI. In addition, no differences were observed in the median plasma C3a concentration between women with normal pregnancy and those in each of the preterm labor subgroups. Conclusions: The maternal plasma concentration of anaphylatoxin C5a is increased in women with preterm labor and IAI, but not in spontaneous labor at term.

PREECLAMPSIA AND PREGNANCIES WITH SMALL-FOR-GESTATIONAL AGE NEONATES HAVE DIFFERENT PROFILES OF COMPLEMENT SPLIT PRODUCTS

Objective. The activation of the complement system results in the generation of split products with pro-inflammatory properties. The objective of this study was to determine whether preeclampsia and small-for-gestational age (SGA) are associated with changes in the maternal plasma concentrations of anaphylatoxins C3a, C4a and C5a. Methods. A cross-sectional study was conducted in the following groups: (a) normal pregnant women (n = 134); (b) women who delivered an SGA neonate (n = 53); (c) preeclampsia with (n = 52) and without SGA (n = 54). Maternal plasma anaphylatoxin concentrations were determined by enzyme-linked immunoassay. Results. (1) Women with preeclampsia with or without SGA had a significantly higher median plasma C5a concentration than that of normal pregnant women and those with SGA alone (all P < 0.01); (2) women with SGA alone did not have an increase in plasma C5a concentration; (3) in contrast, the median maternal plasma concentration of C4a was lower in women with preeclampsia and SGA than that of those with a normal pregnancy (P = 0.001); (4) no changes in C3a were observed among the study groups. Conclusion. Preeclampsia is associated with increased plasma concentration of C5a, regardless of the presence or absence of an SGA fetus. In contrast, there was no difference in the plasma C3a, C4a and C5a concentration in patients with SGA.

Increased concentration of the complement split product C5a in acute pyelonephritis during pregnancy

Objective. Pregnant women with acute pyelonephritis develop acute respiratory distress syndrome (ARDS) more frequently than non-pregnant women. The reasons for this remain unknown. The complement system is a complex set of self-assembling proteins that have been implicated in the pathophysiology of ARDS and sepsis. The purpose of this study was to determine if activation of the complement system occurs in pregnant women with acute pyelonephritis. Methods. A cross-sectional study was conducted to determine the plasma concentrations of C3a, C4a and C5a (i.e., complement split products) in pregnant patients with acute pyelonephritis (n = 38) and normal pregnant women (n = 38). The complement split products C3a, C4a and C5a were measured using ELISA. Data were analyzed using non-parametric statistics. Results. 1) The median plasma concentration of C5a in pregnant patients with acute pyelonephritis was significantly higher than that in normal pregnant women (p < 0.001); 2) there was no statistical difference in the median plasma concentration of C3a and C4a between the two groups (p > 0.05); and 3) concentrations of C3a, C4a and C5a were not different among patients with acute pyelonephritis with and without bacteremia. Conclusions. 1) Pyelonephritis in pregnant women is associated with an increased plasma concentration of C5a, but not C3a and C4a; and 2) an excess of C5a can predispose pregnant women to develop ARDS and multi-organ failure in pyelonephritis. This finding may have clinical implications since blocking C5a improves ARDS in experimental sepsis.

Tissue microarray: An effective high-throughput method to study the placenta for clinical and research purposes

Objective. Tissue microarray (TMA) technology allows simultaneous examination of the expression of many molecular markers (protein, mRNA, DNA, etc.) with high-throughput. The application of this technology, to date, has been largely confined to the study of cancer. Placental pathology poses unique challenges because of the size of the organ, its complex anatomy, as well as its histological heterogeneity. The objective of this study was to assess the feasibility and efficiency of TMAs for immunohistochemistry and in situ hybridization of placental tissues. Study design. TMAs were constructed using an automated tissue arrayer. Standard 0.6-mm or 1-mm microarray needles were used. Villous parenchyma, basal plate, and chorioamniotic membranes were targeted in each block. Five μm-thick TMA sections underwent immunohistochemical analysis of both cytoplasmic and nuclear antigens using a panel of antibodies against a variety of cytoplasmic [cytokeratin-7, vascular endothelial growth factor (VEGF), and protein Z], membranous (endoglin), and nuclear (c-fos and c-jun) antigens. mRNA in situ hybridization for surfactant protein A (SP-A) and chromogenic in situ hybridization for the Y chromosome (DYZ1) were also performed. Results. Validation of TMA immunoreactivity demonstrated comparable results with corresponding whole sections. When a two-tiered scoring system (positive/negative) was employed, there was agreement between two and three cores and whole tissue sections (kappa>0.7). When a three-tiered scoring system (negative, weak-positive, or strong-positive) was used, the data from three cores showed the highest agreement with whole tissue sections (kappa >0.7). In situ hybridization experiments for mRNA and DNA were also successful in that the signals were readily detectable. Successful transfer from the donor block to the recipient block differed according to the anatomical compartment. The transfer efficiency of villous parenchyma, basal plate, and chorioamniotic membranes were 96.9% (875/903), 76.7% (115/150), and 75.4% (224/297), respectively. Conclusion. TMA is a practical and effective tool for high-throughput molecular analysis of the human placenta. Duplicate and triplicate cores offer agreement with whole tissue sections for two-category distinction immunostaining. TMA also affords relevant results from in situ hybridization experiments for mRNA and DNA. The major advantages are the conservation of tissues and reagents, simultaneous comparison of molecular markers in different anatomical compartments of the placenta, and reduction of experimental error.

Three‐dimensional ultrasound in the prenatal diagnosis of cleidocranial dysplasia associated with B‐cell immunodeficiency

A patient with a singleton pregnancy was referred for three‐dimensional ultrasonography (3DUS) at 18 + 3 weeks for suspected hypomineralization of the skull bones and absence of the nasal bones. Three‐dimensional rendered images of the fetal skull revealed widening of the coronal sutures, absence of the squamous portion of the temporal bone, and absence of the occipital bone, except for two areas of ossification. In addition, a fractured right clavicle was identified. The remainder of the fetal anatomy was normal and biometry was appropriate for gestational age. Genetic amniocentesis revealed a 46,XX fetal karyotype. Family history was positive for a 5‐year‐old sibling with an open anterior fontanelle. Cleidocranial dysplasia was suspected. A female neonate was delivered by elective repeat Cesarean section at 40 + 3 weeks of gestation without complications and discharged home 3 days after delivery. Prenatal diagnosis was confirmed by physical and radiological evaluation.

OC2.05: 3D ultrasound evaluation of the fetal optic chiasm: a potential parameter for the differential diagnosis of developmental midline brain anomalies

Objective: Visualization of the fetal optic chiasm by ultrasound has not previously been reported. MRI is often used to image the fetal optic chiasm for the differential diagnosis of septo-optic dysplasia and absent cavum septum pellucidum. We describe herein a new sonographic technique to visualize the optic chiasm using multiplanar three-dimensional ultrasound and to evaluate its dimensions during gestation. Methods: This cross-sectional study included 76 singleton fetuses with no congenital or anatomical abnormalities. The optic chiasm was identified as a hypoechoic structure in the shape of an ‘‘X’’, located anterior to the cerebral peduncles and below the circle of Willis. The optic chiasm diameter was measured in the axial plane at its midline. Intraand inter-observer agreement was analyzed by the Bland–Altman plot. Regression analysis was used to determine the relationship between the optic chiasm and gestational age. Results: Seventy-two of 76 (94.7%) normal fetuses had a visible optic chiasm. The mean optic chiasm diameter at 12 and 40 weeks of gestation measured 1 mm and 4.27 mm, respectively. Intraand inter-observer mean differences between paired measurements were 0.007 mm (95% limits of agreement −0.30 to 0.32 mm) and 0.19 mm (95% limits of agreement −0.85 to 1.24 mm), respectively. The optic chiasm diameter and gestational age fit a linear regression model with a multiple R = 0.84, and a R2 = 0.70 (chiasm diameter = 0.129 + 0.119 * gestational age). Conclusions: 1) The fetal optic chiasm can be reproducibly imaged and measured from 12.5 weeks of gestation to term; 2) the diameter of the optic chiasm increases with gestational age; 3) this novel parameter may be useful in the differential diagnosis of septo-optic dysplasia and absent cavum septum pellucidum, which currently requires MRI. OC2.06 ‘‘Intraventricular fused fornices’’, a marker for complex midline anomalies