IFPA meeting 2015 workshop report II: mechanistic role of the placenta in fetal programming; biomarkers of placental function and complications of pregnancy.

Workshops are an integral component of the annual International Federation of Placenta Association (IFPA) meeting, allowing for networking and focused discussion related to specialized topics on the placenta. At the 2015 IFPA meeting (Brisbane, Australia) twelve themed workshops were held, three of which are summarized in this report. These workshops focused on various aspects of placental function, particularly in cases of placenta-mediated disease. Collectively, these inter-connected workshops highlighted the role of the placenta in fetal programming, the use of various biomarkers to monitor placental function across pregnancy, and the clinical impact of novel diagnostic and surveillance modalities in instances of late onset fetal growth restriction (FGR).

The expression of placental soluble fms-like tyrosine kinase 1 in mouse placenta varies significantly across different litters from normal pregnant mice.

OBJECTIVE: Gene expression studies often pool tissues from multiple placentas when using animal models of preeclampsia without accounting for the potential confounders of litter origin or pup sex. We aimed to determine whether placental gene expression differs based on sex or litter. METHODS: We examined the differential expression of soluble fms-like tyrosine kinase 1 (Flt-1) using 35 pups from six normal pregnant mice. RESULTS: Expression of sFlt-1 (p = 0.003) was significantly different between litters but not between sexes (p = 0.17). CONCLUSIONS: These findings highlight the importance of adequate sampling from multiple litters in expression studies when using animal models in clinical research.

OS044. Morphological differences in murine placenta detected by magneticresonance imaging measurements of T2 relaxation times in mouse models ofpreeclampsia.

INTRODUCTION: We have demonstrated that morphologically distinct regions of the murine placenta can be detected by magnetic resonance imaging (MRI), with image contrast arising from the variation in T2 relaxation times between regions and dependent upon blood flow. Previous studies of human placenta by other groups have shown a homogeneous tissue with correlation of relaxation times with gestational age and a trend for shorter relaxation times in pregnancies complicated by preeclampsia and fetal growth restriction. The ability to detect morphological changes and alterations in blood flow in experimental models of preeclampsia would be a significant boost in understanding the relationship between abnormal placental implantation, reduced placental perfusion, inflammatory cytokines, angiogenic molecules and other factors that may play a role in the syndrome. OBJECTIVES: The aim of this study was to investigate whether morphological changes or abnormalities can be detected by T2 mapping in the placenta of mice subject to two experimental models of preeclampsia (reduced uterine perfusion pressure (RUPP) model and TNF-α induced model). METHODS: Pregnant C57BL/6JArc mice were, on day 13.5 of gestation, either subject to a unilateral ligation of the right uterine artery (RUPP) (n=2) or given an infusion of TNF-α by subcutaneous insertion of a mini-osmotic pump primed to deliver 500ng/kg/day for 4days (n=2). Controls were normal pregnant (n=2), sham-operated (n=1) or saline infused animals(n=1). MRI images were acquired on anaesthetised mice on day 17.5 of gestation using a Bruker Avance 11.7 Tesla wide-bore spectrometer with micro-imaging probe capable of generating gradients of 0.45T/m. T2 measurements were acquired using an MSME sequence protocol (Bruker MSME-T2-map) with an in-plane resolution of 0.1-0.2mm. Matlab was used to generate R2 (i.e.,1/T2) maps from the acquired data with the T2 values being calculated from selected regions of interest from 2-6 individual placenta from each mouse. RESULTS: Differences in the pattern of the regions of T2 contrast in the placenta were observed between normal, TNF-α treated and RUPP mice. The ratio of T2 values from the inner two regions was also significantly altered in TNF-α treated 1.98±0.09 (p=0.007); RUPP 1.94±0.11 (p=0.006) compared to normal animals 2.5±0.1 . CONCLUSION: These results demonstrate that morphological differences or abnormalities can be detected by T2 mapping in the placenta of mice subject to experimental models of preeclampsia and may be used to analyse changes quantitatively. This technology has the potential to be used when studying the dynamic changes in the placenta of pregnancies complicated by preeclampsia.

OS055. Sex-dependent differences in expression of FLT-1 variants andJMJD6 in mouse placenta.

INTRODUCTION: There is evidence for fetal sex-dependent differences in the way in which preeclamptic pregnancies proceed, and in maternal and neonatal outcomes. Mouse models are common in the study of preeclampsia and pooled tissue from multiple placentae is often used to obtain samples for expression studies. Potential concerns regarding this practice are the sex-dependent differences in placental expression of candidate factors. One biomolecule of interest is soluble fms-like tyrosine kinase 1 (sFLT-1) which is a known marker of preeclampsia and commonly used to determine the severity of the induced preeclampsia-like syndrome in rodent models. OBJECTIVES: It was the aim of this preliminary study to determine whether variation exists in the expression of different genes in murine placenta based on pup sex in C57BL/6JArc mice. A novel gene, Jumonji domain-containing protein 6 (Jmjd6) that may prove to have a role in the pathogenesis of preeclampsia, mFLT-1 and sFLT-1 were selected as targets. METHODS: Seventeen pups were retrieved from three normal pregnant female mice euthanized via cervical dislocation (CD) on day 17.5 or 18.5. Tails and corresponding placentas were collected from the pups, snap frozen in liquid nitrogen and stored at -80°C. Tails were used to accurately determine pup sex via PCR amplification of sex chromosome-specific sequences and revealed the presence of 3 females and 14 males. Quantitative PCR was used to determine the relative expression of the FLT-1 and Jmjd6 transcripts in each placenta. The placenta collected from the first pup of the first pregnant female served as the reference sample and transcript expression in the remaining samples was expressed relative to this sample. General linear modelling using linear regression with categorical variables was used to evaluate the difference in transcript expression between the sexes and Pearson's correlation coefficient used to examine relationships between variables. RESULTS: Pup sex was found to have a significant effect on the relative expression of sFLT-1 after controlling for litter, pup weight and gestational age (p=0.013), with 1.5 times more expression in the placentas of female pups. The expression of sFLT-1 was highly correlated with mFLT-1 (r=0.690,p=0.002). The relative expression of Jmjd6 was not significantly different in male and female placentas and sFlt-1 was not correlated with Jmjd6. CONCLUSION: This is the first study to demonstrate a link between fetal sex and placental sFLT-1 expression in mice, finding increased levels of this gene in the placentas of female pups. It is possible that in normal pregnancies, female placentas produce more sFLT-1 which acts to condition them and offer some protection during the sFLT-1 spike seen in preeclampsia. The findings of this study also highlight a possible need to consider sex as a variable in placental expression studies using mice to ensure the accuracy of results.

PP001. Variability in MRNA expression of Jmjd6 and FLT-1 variants in normal and preeclamptic human placenta.

INTRODUCTION: Biomolecules such as soluble fms-like tyrosine kinase 1 (sFLT-1) have been implicated in the pathogenesis of preeclampsia with many studies reporting on their expression in human placenta. OBJECTIVES: This study aimed to determine whether variation exists in the expression of different genes in human placenta based on collection site. Expression of different FLT-1 variants including the primate-specific sFLT-1e15a and a novel gene, Jumonji domain-containing protein 6 (Jmjd6) that may prove to have a role in the pathogenesis of preeclampsia, was selected as targets. METHODS: Placental tissue was collected from one normotensive and one preeclamptic woman following caesarean section at 38 weeks. Twelve 1.5cm diameter×2mm thick samples were excised from various sites around the decidual surface. Quantitative PCR was used to determine the relative expression of the FLT-1 and Jmjd6 transcripts in the separate samples. Within a placenta, the first sample collected served as the reference and transcript expression in the remaining 11 samples was expressed relative to this sample. Between placentas, a pooled normal sample was used as a reference to determine the relative expression in preeclamptic compared to normal placental samples. One sample t -tests and coefficients of variation (CV) were used to explore the variation and Pearson's correlation coefficient was used to examine relationships. RESULTS: Within the normal placenta, significant variation was seen in the 12 collection sites for sFLT-1 e15a (CV=45.1% p=0.008) and Jmjd6 (CV=30.4% p=0.019). The CVs for sFLT-1 i13 and mFLT-1 were 25.6% and 23.7% respectively. Within the preeclamptic placenta, significant variation was seen in the expression of all FLT-1 variants; mFLT-1 (CV=66.9% p=0.023), sFLT-1 i13 (CV=64.8% p=0.033) and sFLT-1 e15a (CV=61.1% p=0.001) across different collection sites. Significant variation was also seen between preeclamptic placenta sites and a normotensive pool; mFLT-1 (CV=66.9% p=0.012), sFLT-1 e15a (CV=61.1% p=0.005) and Jmjd6(CV=65.2% p=0.029). Using cumulative moving means, the minimum number of samples required to obtain a zero difference in means for all transcripts in a data subset was 8 for the normal placenta and 6 for the preeclamptic placenta. Overall, the expression of Jmjd6 and all FLT-1 variants was increased in the samples from the preeclamptic placenta compared to normal. Expression of mFLT-1 was highly correlated with sFLT-1 i13 and sFLT-1 e15ain preeclamptic (r=0.808 p=0.001; r=0.841p=0.001) but not normal placenta, and Jmjd6 was not correlated with any transcript in either placenta. CONCLUSION: This study demonstrates significant variation in expression levels of several new and commonly investigated genes across sites in both normal and preeclamptic human placenta. These data show samples should be obtained from no less than 8 separate sites when pooling samples for expression analysis. Further, given that many studies examine relationships between different colocalised molecules, it may also be prudent to examine expression levels in each site separately to ensure that no relationships are missed.

PP084. Magnetic resonance imaging measurements of T2 relaxation times within contrasting regions of murine placenta is dependent upon blood flow.

INTRODUCTION: It has been postulated that reduced placental perfusion as a result of abnormal placental implantation is the initiating event that leads to the maternal symptoms of preeclampsia. To be able to directly measure blood flow and perfusion in the placenta in experimental models of preeclampsia would provide valuable insight into the structural abnormalities of this syndrome. Magnetic resonance imaging (MRI) offers visualization of anatomy and analysis of changes in tissue morphology and function including blood flow and perfusion. The major source of image contrast in MRI comes from the variation in relaxation times between tissues. Previously, human placenta has appeared as fairly homogeneous in studies of T1 and T2 relaxation times, with no internal morphology apparent. OBJECTIVES: The aim of this study was to investigate, using much higher field strengths (11.7Tesla) and much higher resolution than have been used previously, whether structural inhomogeneities in the placenta can be discerned by T2 mapping and whether T2 mapping is capable of detecting structural abnormalities that may affect blood flow in a preeclamptic placenta. METHODS: Magnetic resonance images were acquired on an anaesthetised C57BL/6JArc mouse placed in a vertical animal probe using a Bruker Avance 11.7Tesla wide-bore spectrometer with micro-imaging probe capable of generating gradients of 0.45T/m. T2 measurements were acquired using an MSME sequence protocol (Bruker MSME-T2-map) with an in-plane resolution of 0.1-0.2mm. Matlab was used to generate R2 (i.e., 1/T2) maps from the acquired data with the T2 values being calculated from selected regions of interest within 5 individual placenta. Additional T2 measurements were acquired on the same slices immediately after blood flow was reduced to zero. RESULTS: Three distinct regions of T2 contrast were discerned in the mouse placenta, likely correlating to the labrynthine, junctional and decidual zones. The contrast between the inner two regions was substantially abrogated when blood flow ceased upon euthanasia of the animal by anaesthetic overdose (p<0.005), whereas the decidual region remained unchanged (p=0.13). CONCLUSION: These results demonstrate that morphologically distinct regions of the mouse placenta can be detected through the mapping of T2 relaxation times. The contrast between regions is lost when blood flow ceases and the placenta becomes homogeneous in appearance, suggesting that differences in T2 relaxation times across regions of the placenta may be used to non-invasively examine structural abnormalities and blood flow in the placenta of experimental animal models of preeclampsia.

PP162. sFlt-1 controlled by CPAP in a pregnant patient with chronic hypertension.

INTRODUCTION: Preeclampsia is recognised as the leading cause of perinatal mortality and morbidity worldwide. Interest has been increasing recently as to the possible impact of sleep disordered breathing (SDB) on the mechanisms of preeclampsia, possibly by augmenting placental hypoxia during sleep. A biomarker of preeclampsia, sFlt-1, has also come to prominence in recent years and is postulated to be a good predictor of preeclampsia as well as a strong indicator of the severity of the disease. OBJECTIVES: The aim of this study was to investigate the effect of CPAP treatment for SDB on sFlt-1 concentrations during pregnancy. METHODS: Patients were recruited from the outpatients' clinic in Campbelltown Hospital, Campbelltown, NSW in the first half of 2011. The levels of sFlt-1 in four pregnant women, with or without hypertensive disorders of pregnancy, and with or without SDB were measured using ELISA. RESULTS: Women with SDB and chronic hypertension or preeclampsia had higher levels of sFlt-1 and a greater percentage increase of this marker, and CPAP treatment appeared to attenuate the rise of sFlt-1 as shown in the table below. CONCLUSION: Elevated sFlt-1 was associated with untreated SDB in those with chronic hypertension. This study provides feasibility for a larger scale study to occur, to further examine the validity of the hypothesis that CPAP treatment has a beneficial effect on sFLt-1 levels and therefore, may lower the risk and severity of preeclampsia.

Responses of insulin-like growth factor (IGF)-I and IGF-binding proteins to nutritional status in peroxisome proliferator-activated receptor-alpha knockout mice.

Peroxisome proliferator-activated receptor alpha (PPARalpha) plays a central role in glucose and lipid homeostasis. Mice lacking PPARalpha(-/-) have a sexually dimorphic phenotype. We have characterized the IGF system in wild type and PPARalpha-/- mice. In normal mice fasting IGF-I and the IGFBP-3 ternary complex were 2-fold higher in males than in females. PPARalpha influenced the IGF/IGFBP response to feeding, particularly in males. Compared to wild type, male PPARalpha-/- mice had 40% lower total fasting IGF-I concentrations, decreased ALS and less IGFBP-3 ternary complex formation, but within 4 h of refeeding there was an increase in IGF-I and IGFBP-3 ternary complex to values similar to controls. Circulating IGFBP protease activity was induced in male PPARalpha-/- mice during refeeding. IGFBP-1 and insulin concentrations were higher in males than females, and were increased by PPARalpha knockout, suggesting significant hepatic insulin resistance. We speculate that gender differences in the IGF system contribute to the PPARalpha-/- phenotype.

In the absence of the invariant chain, HLA-DR molecules display a distinct array of peptides which is influenced by the presence or absence of HLA-DM.

The independent influences of invariant chain (Ii) and HLA-DM molecules on the array of naturally processed peptides displayed by HLA-DR molecules were studied using transfected cell lines. The absence of Ii led to an altered set of HLA-DR-bound peptides as judged by the discriminating responses of alloreactive T cell clones. While most T cell clones raised against DR+Ii+DM+ peripheral blood mononuclear cells (PBMC) failed to respond to DR+Ii-DM- cells, T cell clones raised against DR+Ii-DM- transfectants were not stimulated by DR+Ii+DM+ cells. Furthermore, coexpression of HLA-DM with HLA-DR1 in the absence of Ii augmented responses of anti-PBMC T cell clones but inhibited allorecognition by T cell clones raised against DR+Ii-DM- transfectants. The conformational integrity of the class II molecules, as judged by serology, suggests that the patterns of reactivity of the T cell clones reflect specificity for different alloantigen-bound peptides. Hence, discordant regulation of expression of major histocompatibility complex class II, Ii, and HLA-DM molecules in vivo may lead to the display of novel self-peptides and possible interruption of self-tolerance.

Radioimmunoassay of soluble insulin-like growth factor-II/mannose 6-phosphate receptor: developmental regulation of receptor release by rat tissues in culture.

The soluble form of the insulin-like growth factor-II/mannose 6-phosphate receptor, which has been detected in serum from a variety of species, is synthesized and released by rat tissue explants in culture. Investigations into its regulation and function, however, have been hampered by inadequate means of quantification. In this paper a RIA that enables sensitive and specific quantification of soluble receptor from serum and conditioned medium is described. In order to further clarify the source of soluble receptor in serum and to determine whether release of receptor from rat tissues is under developmental regulation, receptor release from rat explants was examined and compared to serum levels of receptor. Heart, skeletal muscle, kidney, and liver explants from fetal, neonatal, weanling, and adult rats were cultured in serum-free medium, and the conditioned medium was analyzed by RIA for the presence of receptor. Soluble receptor release was highest for fetal and neonatal tissues, with weanling and adult tissues showing dramatically decreased levels. Release of soluble receptor varied between tissues, with the tissue-specific pattern altering during development, such that while heart and muscle appeared as major tissues of release in fetal animals, the liver was the tissue of highest release in adult animals. It is concluded that release of soluble receptor by rat tissues is developmentally regulated, with levels of receptor in serum reflecting the pattern of release from tissues. The RIA will prove a useful tool for further examination of the regulation and function of the soluble insulin-like growth factor-II/mannose 6-phosphate receptor.

Secretion of soluble insulin-like growth factor-II/mannose 6-phosphate receptor by rat tissues in culture.

The insulin-like growth factor-II/mannose 6 phosphate receptor, which binds both IGF-II and mannose 6-phosphate containing proteins, such as lysosomal enzymes, has been detected in the serum of several species. Neither the source nor the role of this soluble, truncated form of the membrane receptor has been determined. This study has examined the ability of a variety of rat tissues in culture to synthesize and release soluble receptor. Explants (1 mm3) of 21-day fetal, neonatal and adult rat tissues were cultured in serum-free medium and the conditioned medium analyzed for the presence of receptor. Using IGF-II binding and immunochemical techniques, receptor was detected in media from heart, kidney, liver, lung and muscle explants. [35S]cysteine labeling indicated that de novo synthesis of the soluble receptor occurs in the cultured explants and can be inhibited by cycloheximide. This rat tissue explant culture system demonstrates that soluble receptor is released by a number of tissues, and should provide a useful model for further investigations into its function and regulation.

Purification and characterisation of proteins with cardiac stimulatory and haemolytic activity from the anemone Actinia tenebrosa.

Three new proteins with cardiac stimulatory and haemolytic activity, designated tenebrosins-A, -B and -C, have been purified from the Australian sea anemone Actinia tenebrosa. These proteins are basic (pI greater than or equal to 9.4), have mol. wt of about 20,000, and have very similar amino acid compositions and N-terminal amino acid sequences. None of the proteins contains cysteine or cystine residues. On isolated, spontaneously beating guinea pig atria they exhibit at 1-2 nM strong positive inotropic and slight to moderate chronotropic effects. In some cases a transient negative inotropic effect occurs prior to onset of the positive inotropic response. The proteins are also haemolytic, producing 50% haemolysis of guinea pig erythrocytes at concentrations similar to those showing positive inotropic effects.