Gregory Duncombe

The Basic and Clinical Science of Twin–Twin Transfusion Syndrome

Twin-twin transfusion syndrome (TTTS) is a fascinating condition in which fetuses of identical genotype adopt discordant cardiovascular phenotypes, secondary to unbalanced placental inter-twin transfusion. Flow along the primary units of inter-twin transfusion, unidirectional arteriovenous anastomoses, can be as high as litres/day each, and TTTS develops when the placenta has insufficient compensatory counter-transfusional anastomoses. The initial phenotype reflects dysvolaemia, with added contributions from uteroplacental insufficiency in the donor, and raised afterload with diastolic dysfunction secondary to discordant endothelin and placentally derived renin-angiotensin system effectors in the recipient. Endoscopic laser ablation of placental anastomoses has become the primary treatment modality, supported by a randomised trial showing improved survival and short but not long-term neurological morbidity. Its uptake has facilitated comparative pre- and post-laser studies, which provide considerable insight into the pathophysiology. Despite the therapeutic advance, placental laser remains associated with a 25% incidence of fetal death within a week, and a 10% risk each of recurrence and twin anaemia/polycythaemia sequence due to residual anastomoses. In Stage I, high rates of non-progression with more conservative management have resulted in therapeutic equipoise as to whether laser is indicated primarily or only for progressive disease. The challenge ahead lies in improving double intact survival rates, which in addition to randomised trials will require technical advances, better understanding of the circulatory pathophysiology and more sophisticated surveillance tools.

The long term neurologic outcome of children from pregnancies complicated by twin-to-twin transfusion syndrome

Objective  To assess long term outcomes of children from pregnancies complicated by twin‐to‐twin transfusion syndrome.

The Effect of Glucose on the Release and Bioactivity of Exosomes From First Trimester Trophoblast Cells

CONTEXT Hyperglycemia and hypoxia are risk factors of metabolic complication during pregnancy. The interactions between oxygen and glucose-sensing pathways that regulate exosome bioactivity from placental cells, however, have not been established. OBJECTIVE The aim of this study was to test the hypothesis that exosomal signaling by placental cells (defined as the number of exosomes released per unit time and their bioactivity) is responsive to extracellular glucose concentration. METHODS First-trimester primary trophoblast cells were incubated with D-glucose (5 mM or 25 mM) under 1%, 3%, or 8% O2 for 48 hours. Exosomes were isolated from cell-conditioned media by differential and buoyant density centrifugation. The total number of exosome vesicles was determined by quantifying immunoreactive exosomal CD63. The effect of exosomes on cytokine (granulocyte macrophage colony-stimulating factor, IL-2, IL-4, IL-6. IL-8, IL-10, interferon-γ, and TNF-α) release from endothelial cells was established by a protein solution array analysis. RESULTS Glucose (25 mM) significantly increased the release of exosomes from trophoblast cells at all oxygen tensions tested (by approximately 2-fold when compared with controls, P < .001). Exosomes (100 μg/mL exosomal protein) released from trophoblast cells significantly increased (P < .05) the release of all cytokines from human umbilical vein endothelial cells when compared with the control (ie, cells without exosomes), with the exception of IL-2 and IL-10 (P > .05). CONCLUSIONS The effects of high glucose on exosomes bioactivity may be recapitulated in vivo and is of clinical relevance in association with maternal insulin resistance (resulting in hyperglycemia) and preeclampsia (associated with placental insufficiency and hypoxia).

Role of Extracellular Vesicles and microRNAs on Dysfunctional Angiogenesis during Preeclamptic Pregnancies

Preeclampsia is a syndrome characterized by hypertension during pregnancy, which is a leading cause of morbidity and mortality in both mother and newborn in developing countries. Some advances have increased the understanding of pathophysiology of this disease. For example, reduced utero-placental blood flow associated with impaired trophoblast invasion may lead to a hypoxic placenta that releases harmful materials into the maternal and feto-placental circulation and impairs endothelial function. Identification of these harmful materials is one of the hot topics in the literature, since these provide potential biomarkers. Certainty, such knowledge will help us to understand the miscommunication between mother and fetus. In this review we highlight how placental extracellular vesicles and their cargo, such as small RNAs (i.e., microRNAs), might be involved in endothelial dysfunction, and then in the angiogenesis process, during preeclampsia. Currently only a few reports have addressed the potential role of endothelial regulatory miRNA in the impaired angiogenesis in preeclampsia. One of the main limitations in this area is the variability of the analyses performed in the current literature. This includes variability in the size of the particles analyzed, and broad variation in the exosomes considered. The quantity of microRNA targets genes suggest that practically all endothelial cell metabolic functions might be impaired. More studies are required to investigate mechanisms underlying miRNA released from placenta upon endothelial function involved in the angiogenenic process.

Vasa Previa Diagnosis, Clinical Practice, and Outcomes in Australia.

OBJECTIVE To estimate the incidence of women with vasa previa in Australia and to describe risk factors, timing of diagnosis, clinical practice, and perinatal outcomes. METHODS A prospective population-based cohort study was undertaken using the Australasian Maternity Outcomes Surveillance System between May 1, 2013, and April 30, 2014, in hospitals in Australia with greater than 50 births per year. Women were included if they were diagnosed with vasa previa during pregnancy or childbirth, confirmed by clinical examination or placental pathology. The main outcome measures included stillbirth, neonatal death, cesarean delivery, and preterm birth. RESULTS Sixty-three women had a confirmed diagnosis of vasa previa. The estimated incidence was 2.1 per 10,000 women giving birth (95% CI 1.7-2.7). Fifty-eight women were diagnosed prenatally and all had a cesarean delivery. Fifty-five (95%) of the 58 women had at least one risk factor for vasa previa with velamentous cord insertion (62%) and low-lying placenta (60%) the most prevalent. There were no perinatal deaths in women diagnosed prenatally. For the five women with vasa previa not diagnosed prenatally, there were two perinatal deaths with a case fatality rate of 40%. One woman had an antepartum stillbirth and delivered vaginally and the other four women had cesarean deliveries categorized as urgent threat to the life of a fetus with one neonatal death. The overall perinatal case fatality rate was 3.1% (95% CI 0.8-10.5). Two thirds (68%) of the 65 neonates were preterm and 29% were low birth weight. CONCLUSION The outcomes for neonates in which vasa previa was not diagnosed prenatally were inferior with higher rates of perinatal morbidity and mortality. Our study shows a high rate of prenatal diagnosis of vasa previa in Australia and associated good outcomes.

Uptake of invasive prenatal tests in pregnancies conceived via assisted reproductive technologies: the experience in Queensland, Australia

Prenatal diagnosis of a chromosomal abnormality currently involves the use of an invasive procedure, which has a risk of fetal loss. The aim of this study was to identify whether pregnancies conceived via assisted reproductive technologies were more or less likely to be subjected to an invasive procedure compared with pregnancies that were conceived spontaneously.

Placenta-derived exosomes promote trophoblast invasion and spiral arterial remodeling - A possible role in the physiopathology of preeclampsia

Figures will be available only online at UNIV OF PITTSBURGH on June 19, 2015 rsx.sagepub.com Downloaded from at UNIV OF PITTSBURGH on June 19, 2015 rsx.sagepub.com Downloaded from Thrsday O als Scientific Abstracts Reproductive Sciences Vol. 22, Supplement 1, March 2015 57AINTRODUCTION: Statin use inadvertently during pregnancy and proposed use of statins for the treatment of preeclampsia, led us to question the evidence behind their current contraindicated status. Several studies have evaluated the relationship between statin use in pregnancy with fetal outcome but their results have not been quantitatively assessed by meta-analysis. Our objective was to undertake a systematic review of all published clinical evidence to assess the effects of statin use in pregnancy on subsequent fetal wellbeing. METHODS: A comprehensive search strategy was performed of all electronic databases and the Merck reporting database for studies published from 1966 to 2014. Two reviewers independently screened citations and undertook study quality assessment and data extraction. We obtained summary estimates of adverse fetal events that were classified as potentially fatal, clinically significant morbidity or minor adverse event. We identified 602 titles and reviewed 30 articles for inclusion and exclusion criteria. Meta-analysis was performed on seven studies (3 cohort, 3 case-series and 1 case-control). RESULTS: Of the 922 cases of statin exposure in pregnancy, 27 exposures were associated with lethal or clinically significant fetal morbidity and 10 with minor adverse events. Statin exposure was limited to the first trimester in all but two cases. The pooled rate of lethal or clinically significant fetal abnormalities in pregnant women exposed to statins was 0.01 (95% CI 0.00-0.04), less than the European rate of 0.026 (95% CI 2.54- 2.57)EUROCAT. The rate of fetal abnormality for simvastatin was 0.03 (95% CI 0.00-0.08), atorvostatin 0.11 (95% CI 0.00-0.52), pravastatin 0.01 (95% CI 0.00-0.2) and lovastatin use 0.04 (95% CI 0.00-0.28). Systems based anomalies were also calculated, congenital heart disease was 0.8 (95% CI 0.02-0.12) compared with the background rate of 0.79 (95% CI 0.78- 0.80). CONCLUSIONS: The published data suggests that statins may not be teratogenic when given inadvertently during pregnancy and prospective studies such as The StAmP Trial may provide more dataIntroduction. Being born small increases cardio-renal disease risk, with males exhibiting more se vere phenotypes than females. These disease risks are not limited to the first generation (F1) but may be transmitted to subsequent generations (F2 and F3). The F3 maternal line represents the first generation that is not directly exposed to the initial insults. There is limited evidence of paternal line transmission. W e characterized nephron number and cardio-renal phenotype of F3 of fspring born to normally grown and growth restricted (F1) mothers or fathers. Methods. Late gestation rat uteroplacental insuf ficiency was induced (Restricted) or sham (Control) surgery in F0. Rats were anaesthetized with 4% isoflurane and 650ml.min -1 oxygen flow (reduced to 3.2% isoflurane and 250ml.min -1 oxygen flow when suturing). T o generate F3 paternal line offspring, F1 Control and Restricted males were mated with normal females and the F2 Control and Restricted males were then mated with normal females. F3 maternal line of fspring were similarily generated. F3 body weights were measured from birth to 12 months. Nephron number w as quantified using unbiased sterology at postnatal day 35. 24h renal excretions, creatinine clearance and tail cuf f blood pressure were measured at 6 (maternal and paternal lines) and 12 (maternal line only) months of age. All data were analysed by t-test within a gender and line. Results. Although F1 offspring were born small, F2 and F3 offspring (both maternal and paternal lines) had normal birth weights. F3 body weight was not dif ferent from birth to 12 months of age with no dif ferences in kidney, heart or adipose weights at 6 months between groups or lines. F3 male and female nephron endowment and blood pressure w as not different between groups for paternal and maternal lines. F3 maternal line renal function was normal at 6 months of age. Ho wever, at 12 months, although creatinine clearance (eGFR) was normal, renal dysfunction emerged in F3 maternal line males (proteinuria) and females (increased urinary creatinine excretion). F3 offspring from fathers born small had e vidence of impaired eGFR (reduced creatinine clearance). Conclusions. F3 offspring, born to F1 growth restricted mothers or f athers are not programmed to be born of low birth weight but de veloped renal dysfunction in the absence of obesity . The proteinuria that emerged with aging in the F3 maternal line male of fspring in the absence of nephron deficits, hypertension and reduced eGFR, suggests tubulointerstitial injury mediated either through podocyte dysfunction/depletion or solely via proteinuria. In contrast, F3 paternal line of fspring had glomerular dysfunction (reduced eGFR), indicati ve of glomerular filtration deficits. Our paternal line results highlight sustained transgenerational inheritance of renal dysfunction. Since nephron number was preserved our results propose the progression of renal dysfunction via the “fibrosis hypothesis” rather than the “Brenner h ypothesis”. Our findings provide no vel evidence of transgenerational transmission of renal dysfunction to F3 offspring from both maternal and paternal lines.INTRODUCTION: Preeclampsia is a vascular disorder in pregnancyand is biochemical characterization by high soluble Flt-1 and lowplacenta growth factor as well as an imbalance in redox homeostasis.During conditions of high oxidative stress, cysteine residues on keyproteins are reversibly altered by S-glutathionylation, modifying theirfunction. Glutaredoxin-1 (Glrx) enzymatically catalyzes the removal of S-glutathione adducts, conferring reversible signaling dynamics toproteins with redox-sensitive cysteines. The role of Glrx in preeclampsiais unknown.METHODS: Immunohistochemistry and Western blot analysis for Glrx orglutathione were conducted on human placenta samples collected pre-termfrom early onset preeclamptic patients (n=10) or non-preeclamptic induceddeliveries (n=9). Human endothelial cells were infected with adenovirusencoding Glrx or LacZ prior to the cells being exposed to hypoxia (0.1%O2, 24h) to measure changes in soluble Flt-1 (sFlt-1). Quantitative PCRand ELISA were used to measure sFlt-1 at mRNA and protein level.RESULTS: Immunohistochemical staining for GSH revealed lowerS-glutathionylation adducts in preeclampsia placenta in comparison tocontrols. Glrx expression, which catalyses de-glutathionylation wasenhanced in early onset preeclampsia compared to pre-term controlsamples. In contrast, no change was observed in preeclamptic and IUGRplacentas at full term. In endothelial cells overexpressing Glrx, sFlt-1expression was dramatically enhanced at mRNA (3-fold P 0.01, n=4) after hypoxia andoverexpressing Glrxin mice enhanced levels of circulating sFlt-1 during in vivo ischemia.CONCLUSIONS: Enhanced Glrx expression in preeclamptic placentain line with an apparent decrease in S-glutathionylation may leavekey proteins susceptible to irreversible oxidation in conditions of highoxidative stress.

Hypoxia regulates the response of trophoblast-derived exosomes to hyperglycemia and displays a difference placental exosome profile in plasma from patients with gestational diabetes mellitus

Figures will be available only online at UNIV OF PITTSBURGH on June 19, 2015 rsx.sagepub.com Downloaded from at UNIV OF PITTSBURGH on June 19, 2015 rsx.sagepub.com Downloaded from Thrsday O als Scientific Abstracts Reproductive Sciences Vol. 22, Supplement 1, March 2015 57AINTRODUCTION: Statin use inadvertently during pregnancy and proposed use of statins for the treatment of preeclampsia, led us to question the evidence behind their current contraindicated status. Several studies have evaluated the relationship between statin use in pregnancy with fetal outcome but their results have not been quantitatively assessed by meta-analysis. Our objective was to undertake a systematic review of all published clinical evidence to assess the effects of statin use in pregnancy on subsequent fetal wellbeing. METHODS: A comprehensive search strategy was performed of all electronic databases and the Merck reporting database for studies published from 1966 to 2014. Two reviewers independently screened citations and undertook study quality assessment and data extraction. We obtained summary estimates of adverse fetal events that were classified as potentially fatal, clinically significant morbidity or minor adverse event. We identified 602 titles and reviewed 30 articles for inclusion and exclusion criteria. Meta-analysis was performed on seven studies (3 cohort, 3 case-series and 1 case-control). RESULTS: Of the 922 cases of statin exposure in pregnancy, 27 exposures were associated with lethal or clinically significant fetal morbidity and 10 with minor adverse events. Statin exposure was limited to the first trimester in all but two cases. The pooled rate of lethal or clinically significant fetal abnormalities in pregnant women exposed to statins was 0.01 (95% CI 0.00-0.04), less than the European rate of 0.026 (95% CI 2.54- 2.57)EUROCAT. The rate of fetal abnormality for simvastatin was 0.03 (95% CI 0.00-0.08), atorvostatin 0.11 (95% CI 0.00-0.52), pravastatin 0.01 (95% CI 0.00-0.2) and lovastatin use 0.04 (95% CI 0.00-0.28). Systems based anomalies were also calculated, congenital heart disease was 0.8 (95% CI 0.02-0.12) compared with the background rate of 0.79 (95% CI 0.78- 0.80). CONCLUSIONS: The published data suggests that statins may not be teratogenic when given inadvertently during pregnancy and prospective studies such as The StAmP Trial may provide more dataIntroduction. Being born small increases cardio-renal disease risk, with males exhibiting more se vere phenotypes than females. These disease risks are not limited to the first generation (F1) but may be transmitted to subsequent generations (F2 and F3). The F3 maternal line represents the first generation that is not directly exposed to the initial insults. There is limited evidence of paternal line transmission. W e characterized nephron number and cardio-renal phenotype of F3 of fspring born to normally grown and growth restricted (F1) mothers or fathers. Methods. Late gestation rat uteroplacental insuf ficiency was induced (Restricted) or sham (Control) surgery in F0. Rats were anaesthetized with 4% isoflurane and 650ml.min -1 oxygen flow (reduced to 3.2% isoflurane and 250ml.min -1 oxygen flow when suturing). T o generate F3 paternal line offspring, F1 Control and Restricted males were mated with normal females and the F2 Control and Restricted males were then mated with normal females. F3 maternal line of fspring were similarily generated. F3 body weights were measured from birth to 12 months. Nephron number w as quantified using unbiased sterology at postnatal day 35. 24h renal excretions, creatinine clearance and tail cuf f blood pressure were measured at 6 (maternal and paternal lines) and 12 (maternal line only) months of age. All data were analysed by t-test within a gender and line. Results. Although F1 offspring were born small, F2 and F3 offspring (both maternal and paternal lines) had normal birth weights. F3 body weight was not dif ferent from birth to 12 months of age with no dif ferences in kidney, heart or adipose weights at 6 months between groups or lines. F3 male and female nephron endowment and blood pressure w as not different between groups for paternal and maternal lines. F3 maternal line renal function was normal at 6 months of age. Ho wever, at 12 months, although creatinine clearance (eGFR) was normal, renal dysfunction emerged in F3 maternal line males (proteinuria) and females (increased urinary creatinine excretion). F3 offspring from fathers born small had e vidence of impaired eGFR (reduced creatinine clearance). Conclusions. F3 offspring, born to F1 growth restricted mothers or f athers are not programmed to be born of low birth weight but de veloped renal dysfunction in the absence of obesity . The proteinuria that emerged with aging in the F3 maternal line male of fspring in the absence of nephron deficits, hypertension and reduced eGFR, suggests tubulointerstitial injury mediated either through podocyte dysfunction/depletion or solely via proteinuria. In contrast, F3 paternal line of fspring had glomerular dysfunction (reduced eGFR), indicati ve of glomerular filtration deficits. Our paternal line results highlight sustained transgenerational inheritance of renal dysfunction. Since nephron number was preserved our results propose the progression of renal dysfunction via the “fibrosis hypothesis” rather than the “Brenner h ypothesis”. Our findings provide no vel evidence of transgenerational transmission of renal dysfunction to F3 offspring from both maternal and paternal lines.INTRODUCTION: Preeclampsia is a vascular disorder in pregnancyand is biochemical characterization by high soluble Flt-1 and lowplacenta growth factor as well as an imbalance in redox homeostasis.During conditions of high oxidative stress, cysteine residues on keyproteins are reversibly altered by S-glutathionylation, modifying theirfunction. Glutaredoxin-1 (Glrx) enzymatically catalyzes the removal of S-glutathione adducts, conferring reversible signaling dynamics toproteins with redox-sensitive cysteines. The role of Glrx in preeclampsiais unknown.METHODS: Immunohistochemistry and Western blot analysis for Glrx orglutathione were conducted on human placenta samples collected pre-termfrom early onset preeclamptic patients (n=10) or non-preeclamptic induceddeliveries (n=9). Human endothelial cells were infected with adenovirusencoding Glrx or LacZ prior to the cells being exposed to hypoxia (0.1%O2, 24h) to measure changes in soluble Flt-1 (sFlt-1). Quantitative PCRand ELISA were used to measure sFlt-1 at mRNA and protein level.RESULTS: Immunohistochemical staining for GSH revealed lowerS-glutathionylation adducts in preeclampsia placenta in comparison tocontrols. Glrx expression, which catalyses de-glutathionylation wasenhanced in early onset preeclampsia compared to pre-term controlsamples. In contrast, no change was observed in preeclamptic and IUGRplacentas at full term. In endothelial cells overexpressing Glrx, sFlt-1expression was dramatically enhanced at mRNA (3-fold P 0.01, n=4) after hypoxia andoverexpressing Glrxin mice enhanced levels of circulating sFlt-1 during in vivo ischemia.CONCLUSIONS: Enhanced Glrx expression in preeclamptic placentain line with an apparent decrease in S-glutathionylation may leavekey proteins susceptible to irreversible oxidation in conditions of highoxidative stress.

Hysterosalpingo-foam sonography (HyFoSy): Tolerability, safety and the occurrence of pregnancy post-procedure

Fallopian tube patency testing is an essential part of infertility evaluation. Hysterosalpingo‐contrast sonography (HyCoSy) has been described as reliable, well tolerated and safe compared to other modalities such as laparoscopy and a dye test or hysterosalpingography. Limited availability of the previously used contrast has led to the introduction of a foam contrast agent as an alternative.

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).