Original Paper Fetal Diagn Ther 2014;35:280–288 DOI: 10.1159/000358516

Received: July 1, 2013 Accepted after revision: December 27, 2013 Published online: April 30, 2014

Maternal Plasma and Amniotic Fluid Cytokines in Monochorionic, Diamniotic Twin Pregnancies Complicated by Twin-to-Twin Transfusion Syndrome Caroline E. Fox a, b Gendie E. Lash d Samantha J. Pretlove a Ben C. Chan a Roger Holder c Mark D. Kilby a, b a

Fetal Medicine Centre, Birmingham Women’s Foundation Trust, b School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, c Department of Primary Care and General Practice, School of Population Studies, College of Medicine and Dentistry, University of Birmingham, Birmingham, and d Reproductive and Vascular Biology Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK

Key Words Amniotic fluid · Angiogenesis · Angiogenic factors · Biomarkers · Cytokines · Fetal therapy · Fetoscopic laser ablation · Laser therapy · Monochorionic twins · Plasma · Twin-to-twin transfusion syndrome

Abstract Introduction: Cytokine imbalance has been implicated in placental-related pathologies, i.e. recurrent miscarriage and pre-eclampsia. Such conditions are more prevalent in multiple pregnancies. Twin-to-twin transfusion syndrome (TTTS) is associated with asymmetric placental blood flow and intra-cardiac pressures. We hypothesised that cytokine expression may be aberrant in this condition and that fetoscopic laser ablation (FLA) may cause local cytokine release. Material and Methods: A prospective cohort of monochorionic, diamniotic twins with TTTS (n = 23) was studied. Circulating T helper cell type 1 (TH1)/TH2 maternal cytokines and cytokine-related and angiogenic factors were measured in plasma and amniotic fluid before and after FLA by human FASTQuant or ELISA. Basal comparisons were made with uncomplicated monochorionic and dichorionic (DC) twins. Results: Median maternal plasma platelet-derived growth factor-BB was highest in uncomplicated DC twins (p = 0.049), whereas tissue inhibitor of metalloproteinases (TIMP)-1 was

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highest in TTTS twins (p = 0.003). In TTTS amniotic fluid, interleukin (IL)-6, IL-1β, tumour necrosis factor-α, IL-10, IL-4, IL-8, interferon-γ, TIMP-1 and intercellular adhesion molecule-1 were significantly higher than maternal plasma concentrations. There were no significant differences in plasma or amniotic fluid cytokines after FLA, with the exception of amniotic fluid keratinocyte growth factor, which was significantly reduced. Discussion: TTTS is associated with minimal changes in cytokine levels when compared to uncomplicated twins, although the majority of cytokine levels were higher in amniotic fluid than maternal blood. It does not appear that FLA evokes a significant change in cytokines. © 2014 S. Karger AG, Basel

Introduction

Approximately 10–15% of monochorionic (MC) twin pregnancies are complicated by twin-to-twin transfusion syndrome (TTTS) [1, 2]. This condition, if untreated, carries a high fetal mortality rate of 80–90% [3]. The pregnancy losses are secondary to the risk of pre-term labour and intrauterine fetal demise of one or both twins [4], and surviving infants may develop significant neurodevelopmental morbidity [5]. In TTTS, the ‘donor’ is underperfused, with associated poor urine output, whilst its coProf. Mark D. Kilby Fetal Medicine Centre, Birmingham Women’s Hospital Foundation Trust School of Clinical and Experimental Medicine, College of Medicine and Dentistry University of Birmingham, Edgbaston, Birmingham B15 2TG (UK) E-Mail m.d.kilby @ bham.ac.uk

balance. We hypothesised that cytokine levels in amniotic fluid and maternal plasma may be aberrant in TTTS. FLA causes occlusion by coagulation of the arteriovenous anastomoses [19], and there is some evidence of transient feto-maternal haemorrhage and trophoblast destruction [11]. It is possible that such a ‘reaction’ leads to the local release of cytokines into the maternal blood and amniotic fluid, and these may have an effect both on decidualmyometrial interaction (affecting the risks of iatrogenic preterm delivery) [14] and potentially affect the risk of fetal morbidity. Neurodevelopmental morbidity is associated with survival in up to 19% of cases [20], and cytokines, i.e. TNF-α, IL-6 and IL-1β, have been implicated in neonatal white matter lesions and, by extension, cerebral palsy [21]. In this study, we examined maternal circulating plasma and amniotic fluid concentrations of cytokines in a cohort of TTTS pregnancies prior to treatment and compared these to a small cohort of uncomplicated MC and dichorionic (DC) twin pregnancies to aid understanding of the pathophysiology of the condition. However, we also measured changes in circulating plasma levels and amniotic fluid concentrations of cytokines in MC twin pregnancies complicated by severe TTTS after FLA. This is of vital importance to examine the role this now widely accepted treatment may have in the neurodevelopmental morbidity evident in survivors.

twin, the recipient, has a hyperdynamic circulation, with cardiac dysfunction (increased ventricular afterload and preload) [6]. This leads to polyuria, polyhydramnios, eventually cardiac failure and, if untreated, the development of fetal hydrops. Several treatment modalities have been advocated, but fetoscopic laser ablation (FLA) of the vascular anastomoses appears to be the optimal treatment [7, 8], as it has the potential to modify the underlying disease and significantly improves perinatal survival with a reduction in long-term morbidity. Placental vascular anastomoses are an anatomical prerequisite for the development of TTTS, but other pathologic mechanisms appear to be involved, including endocrine, autocrine and paracrine placental components [9–11]. Cytokines are the soluble glycoprotein messengers of the immune system, released predominantly by T lymphocytes [12]. More specifically, it is the T lymphocytes expressing CD4+, which may be further classified into T helper cell type 1 (TH1) and TH2 sub-types, that are the richest source of cytokines [12]. TH1 cytokines are involved in pro-inflammatory responses that include the release of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukins (IL-2, IL-6, IL-12, IL-1β) [12]. TH2 cytokines are released by lymphocytes involved in anti-inflammatory and allergic immune responses and include IL sub-types IL-4, IL-5, IL-10 and IL-13 [12]. Cytokines are expressed in the human placenta, decidua and fetal membranes during normal pregnancy and are considered of homeostatic importance to the establishment and function of the placental-maternal interface [13]. Disruption to the local cytokine balance has been implicated in conditions associated with failure of trophoblast invasion as well as placental hypoxia and vascular changes within the placenta, such as those associated with recurrent miscarriage, intrauterine growth restriction and pre-eclampsia [13, 14]. There is evidence that inflammatory cytokines (i.e. IL-6, IL-1β) expressed and produced by the decidua and fetal membranes are associated with spontaneous preterm labour and the associated reaction when infection is also implicated in this process [14]. IL-6 is one such cytokine, and this has also been investigated in relation to TTTS [15, 16]. However, circulating cytokine concentrations in maternal plasma in MC twins in relation to TTTS have not been described. It is known that in multiple pregnancies, there is an increased risk of abnormal placentation, predisposing to complications such as pre-eclampsia and fetal growth restriction [17]. In addition, in TTTS there are asymmetric arteriovenous anastomoses [18] within the placental ‘inter-twin circulations’ that predispose to the characteristic haemodynamic im-

Study Design A consecutive cohort of MC twins complicated by severe TTTS (n = 23) were prospectively studied at a single fetal medicine unit between October 2006 and December 2007. Twins were eligible for inclusion if they were confirmed by ultrasound scan (in the first trimester) to be MC [there was a single placental mass and only a thin dividing membrane (T sign)] and subsequently presented with a diagnosis of TTTS. The diagnosis was made prospectively according to internationally accepted ultrasound criteria, i.e. an MC twin pregnancy with polyhydramnios of ≥8 cm (deepest vertical pocket in the recipient), or ≥10 cm from 20 weeks onwards, and oligohydramnios of 34 weeks in time for analysis of the TTTS cohort. This study received ethical approval from Birmingham Black Country Local Research Ethics Committee (No. 06/Q2702/71), and all participants gave informed consent. Sample Collection and Assay Maternal peripheral venepuncture was performed under basal conditions (several hours prior to any fetal therapy in the MC cohort complicated by TTTS and at an antenatal visit in the uncomplicated twins). Venous blood was collected into a Sarstedt Lithium Heparin 7.5-ml tube and placed immediately on ice. Within 10 min, the sample was centrifuged at 3,000 rpm for 10 min at 4 ° C (Heraeus Labofuge 400). A sample of amniotic fluid was collected from the recipient twin sac just prior to FLA or at amniodrainage and again at the end of the procedure. Sample collection has been described previously [10, 11]. The two pregnancies treated by amniodrainage are included in the basal analysis and then reported separately. Briefly, the procedures were performed as described below. FLA was performed percutaneously under local anaesthesia by a single operator (M.D.K.) [25]. A 3.3-mm cannula with trochar was inserted under continuous ultrasound guidance, and a 2-mm fetoscope (STORZ, Germany) was introduced. Primarily, coagulation selectively targeted superficial and deep anastomoses on the chorionic plate using a diode laser system (30–50 W) [25]. The total numbers of vascular anastomoses on the chorionic plate crossing the inter-twin membrane were counted prior to the coagulation. At the end of the procedure, excess amniotic fluid was drained to an MPD of 5–6 cm (median 1,500 ml, range 900– 4,300 ml). This was the primary treatment if stage II–IV TTTS was diagnosed. For amniodrainage, an 18-gauge needle was inserted percutaneously into the recipient amniotic sac under continual ultrasound assessment. The initial 5-ml sample was discarded, and then 5 ml of amniotic fluid were taken for assay. Amniodrainage was performed until the MPD was measured to be 5–6 cm (median 2,500 ml, range 1,700–5,100 ml). This was the primary treatment if stage I TTTS was diagnosed.  

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Fetal Diagn Ther 2014;35:280–288 DOI: 10.1159/000358516

 

All treatment was performed within 24 h of diagnosis. Both maternal plasma and amniotic fluid samples were divided into 1,000-μl aliquots and stored at –80 ° C until assay (all within 16 months of collection, range 1.2–15.3 months). In order to determine the optimal concentration for plasma and amniotic fluid for each cytokine, a range of concentrations were assayed against the appropriate standard and if necessary diluted in accordance with the manufacturer’s instructions. All samples for any given analyte were assayed in duplicate at the same time using the same standard curve to minimise inter-assay variation. The intra-assay coefficients of variation using these methods ranged from 2.7 to 16.5%. Plasma and amniotic fluid samples were analysed for the TH1/ TH2 cytokines IFN-γ, TNF-α, IL-2, IL-6, IL-1β, IL-4, IL-5, IL-10 and IL-13, as well as keratinocyte growth factor (KGF), plateletderived growth factor (PDGF)-BB, fibroblast growth factor (FGF)basic, tissue inhibitor of metalloproteinases (TIMP)-1 and intercellular adhesion molecule (ICAM)-1, by Human TH1/TH2 or Human Angiogenesis FASTQuant kits (Kerafast, N.C., USA). IL-8 was measured by ELISA (R&D Systems, Abingdon, UK). The sensitivity of all assays ranged between 3.04 and 15.2 pg/ml, and these values were utilised if the assay result was outside the reference range. The methodology has previously been described in detail elsewhere [26–28]. All assays were performed by C.E.F. and G.E.L.  

 

Statistical Analysis Statistical analysis was performed using Graphpad Instat®, and graphs were generated using Graphpad Prism version 4 (Graphpad Software Inc., Calif., USA, 2004). As the data did not fit a normal distribution, values are expressed as medians and interquartile ranges (IQRs) and non-parametric statistical tests were used. The unpaired Mann-Whitney U test or paired Wilcoxon signed rank test were used to compare numerical variables where there were two groups, and for comparison of three or more groups, the Kruskal-Wallis test with Dunn’s post hoc testing was used. Spearman correlation was used to compare blood and amniotic fluid. A p value of