Establishing the MC Twin Cohort

The Leiden University Medical Center (LUMC) is the national referral center for complicated MC twin pregnancies in the Netherlands. The LUMC provides top-referent care that includes close monitoring of development and health of MC twins from early pregnancy to childhood. Therefore, a large proportion of clinical data available for TwinLIFE is routinely collected as part of standard care. In the LUMC, approximately 1 MC twin pair is born per week ensuring a steady enrollment of MC twins into TwinLIFE. We aim to enroll at least 100 MC twin pairs over a period of 2–3 years. Provided that we successfully acquire additional funding, we will increase our sample size beyond 100 pairs. Twins will be followed throughout childhood. This includes the time from inclusion (from week 14 of gestation to birth) to the completion of the follow-up assessments at 2, 5 and 8 years (Figure 1).

Fig. 1. An overview of the study design and the main procedures that subjects will undergo over the course of the study.

Monochorionicity is diagnosed with ultrasound in the first trimester of gestation, a procedure undergone by all pregnant women in the Netherlands. Women carrying an MC twin are referred to a hospital, either a general hospital or a university medical center, for close monitoring of the pregnancy which consists of an ultrasound every other week for the early detection of amniotic fluid discordance, differences in growth or other complications. If complications are detected, women are referred to a university medical center for specialist care. MC twin pregnancies are at risk of developing numerous complications, regularly in combination, among which selective fetal growth restriction (sFGR), twin–twin transfusion syndrome (TTTS) and twin anemia-polycythemia sequence (TAPS) represent the large majority. sFGR (occurring in up to 25% of MC twins) is characterized by unequal placenta sharing and fetal growth discordance as illustrated in Figure 2 (Inklaar et al., Reference Inklaar, van Klink, Stolk, van Zwet, Oepkes and Lopriore2014). TTTS (occurring in up to 10% of MC twins) results from severe intertwin blood flow imbalances and is diagnosed based on discordancy in amniotic fluid volume (twin oligo-polyhydramnios sequence) (Lewi et al., Reference Lewi, Jani, Blickstein, Huber, Gucciardo, Van Mieghem and Deprest2008). Lastly, TAPS (occurring up to 5% of MC twins) is caused by chronic blood flow imbalances through minuscule vascular anastomoses and diagnosed based on large hemoglobin discordance before or at birth (Slaghekke et al., Reference Slaghekke, Kist, Oepkes, Pasman, Middeldorp, Klumper and Lopriore2010).

Fig. 2. Selective fetal growth restriction: (a) an injected placenta demonstrating an unequal placental share division (79% vs. 21%) and a laser demarcation line following fetoscopic laser surgery for coexistent TTTS; (b) an MC twin pair affected by selective fetal growth restriction treated at the LUMC (gestational age at birth 32 weeks, birth weights 2300 and 1200 g). The larger placental share (a, left) belonged to the larger co-twin (b, left).

Written informed consent from both parents (≥18 years and able to consent) is a prerequisite before entering TwinLIFE. The following cases will be excluded from the study: twins with major anatomical abnormalities (interference with outcomes), triplet or higher order multiple pregnancies (different population, cannot be compared to MC twins), cases with Twin Reversed Arterial Perfusion (TRAP) sequence (acardiac twin) and/or cases with single or double fetal demise (no intertwin comparison possible after birth).

We will conduct a comprehensive series of pre- and perinatal assessments (Tables 1 and 2), which consist of a series of assessments part of standard care supplemented with a fetal cardiac functioning measurement, a postnatal cardiac ultrasound and questionnaires specific for the study. Prenatally, fetal growth and cardiac functioning will be recorded using detailed ultrasound scans every 2 weeks or more frequently on clinical indication. Furthermore, maternal and paternal characteristics will be recorded prenatally using a questionnaire, which collects information on demographics, medication and nutritional supplement use, intoxications and medical history. Also, parental wellbeing will be assessed using the Brief Symptom Inventory (BSI) (Lang, Reference Lang2003) for quantification of somatization, depression and anxiety. After birth, we will assess anthropometric measurements (birth weight, length in supine position and head circumference), brain and cardiac development. Placental characteristics will be recorded after color dye injection by trained staff according to our routine clinical program for placenta analysis in MC twins for in-depth analysis of the placental angioarchitecture (Lopriore et al., Reference Lopriore, Slaghekke, Middeldorp, Klumper, van Lith, Walther and Oepkes2011). Common neonatal morbidities will be recorded from birth and admission to the neonatal intensive care unit until discharge to home by the treating neonatologist. At 6 months (corrected for prematurity), parental wellbeing will be reevaluated using the BSI and child nutrition will be recorded using a standardized questionnaire.

Table 1. Timing and goal of assessments of TwinLIFE

Note: trim = trimester, wks = weeks, MSCs = mesenchymal stromal cells, CBCL = Child Behavior Checklist, PedsQoL = Pediatric Quality of Life Inventory, BRIEF = Behavior Rating Inventory of Executive Functioning, Infant Behavior Questionnaire, ECBQ = Early Childhood Behavior Questionnaire, CBQ = Children’s Behavior Questionnaire, AISI = Attachment Insecurity Screening Inventory, aPWV = aortic pulse wave velocity, cIMT = carotid intima–media thickness, CVD = cardiovascular disease.

Defining Epigenetic Changes at Birth

At birth, we will collect umbilical cord tissue, cord blood and buccal swabs for the TwinLIFE Biobank. Umbilical cords will be collected in a sterile PBS-antibiotic/antimycotic solution on birth as a source of MSCs. MSCs are cells of mesodermal origin with the capacity to differentiate into multiple cell types including osteoblasts, adipocytes and chondroblasts using specific culture conditions (Swamynathan et al., Reference Swamynathan, Venugopal, Kannan, Thej, Kolkundar, Bhagwat and Balasubramanian2014; van der Garde et al., Reference van der Garde, van Pel, Millan Rivero, de Graaf-Dijkstra, Slot, Kleinveld and Zwaginga2015). MSCs can be obtained from umbilical cord without any risk for the donor and expanded to high numbers for experiments. For MSC isolation, a modified explant method operational at the LUMC will be adapted and standardized (van der Garde et al., Reference van der Garde, van Pel, Millan Rivero, de Graaf-Dijkstra, Slot, Kleinveld and Zwaginga2015). After expansion for approximately 10 days, the explants are removed and the MSCs are transferred into culture flasks. The transferred cells will be grown to >70% confluence, frozen and stored until further expansion is required. Subsequent MSC expansions of MSCs obtained from co-twins will be performed simultaneously to minimize batch effects. The uniform classification system for MSCs, introduced by the International Society of Cellular Therapy, will be adopted to ensure unambiguous identification and separation of MSCs (Dominici et al., Reference Dominici, Le Blanc, Mueller, Slaper-Cortenbach, Marini, Krause and Horwitz2006). The number of cells after expansion will be sufficient for the epigenetic profiling, RNA expression analysis, cellular phenotyping, and functional differentiation studies.

Cord blood will be collected in sterile blood collection tubes, containing a citrate phosphate dextrose adenine (CPDA) solution. Subsequently, mononuclear cells and plasma will be isolated using a Ficoll density gradient. The isolated cells and plasma will be stored for further experiments, such as DNA methylation analysis of specific cells types. Buccal swabs will be collected, postnatally for DNA extraction and DNA methylation analysis.

We will perform genomewide DNA methylation profiling of MSCs from the twins, using the Illumina EPIC methylation array (Zhou et al., Reference Zhou, Laird and Shen2017). The platform provides a cost-effective coverage of ~850,000 potentially methylated CG dinucleotides across the genome and is enriched for regions known to be involved in genomic regulation. The EPIC array is the successor of the 450k array that has been widely used to successfully identify epigenetic changes after adverse prenatal exposures, including by ourselves in the context of the Dutch Hunger Winter (Tobi et al., Reference Tobi, Slieker, Stein, Suchiman, Slagboom, van Zwet and Lumey2015). We will use our established work flow for quality control, normalization and analysis methylation array data, which is publicly available (https://github.com/molepi/DNAmArray) and combines our own BioConductor packages with the best practices in the field (van Iterson et al., Reference van Iterson, Tobi, Slieker, den Hollander, Luijk, Slagboom and Heijmans2014, Reference van Iterson, van Zwet, Consortium and Heijmans2017, Reference van Iterson, Cats, Hop, Consortium and Heijmans2018). Co-twins will be measured on the same array and array row and will be randomized with respect to array column.

Uncovering Epigenetic Influences on Growth, CVD and NDI Risk

To study the associations of epigenetic changes at birth with precursors of CVD and NDI risk in childhood, we will initiate a follow-up examination of MC twins at least 2 years of age aimed at a focused growth, cardiac, vascular and neurodevelopmental characterization. Parents and their MC twins will be invited to the LUMC for follow-up examinations at the age of 2, 5 and 8 years (at term equivalent age). Transthoracic echocardiography will be used to assess differences in structural and functional cardiac measures (Toemen et al., Reference Toemen, Gaillard, van Osch-Gevers, Helbing, Hofman and Jaddoe2017). Furthermore, aortic pulse-wave velocity (PWV) reflecting vascular stiffness will be assessed using aortic length regression equation based on the subject’s height and two pulsed Doppler recordings of the aorta (Jo et al., Reference Jo, Lande, Meagher, Wang and Vermilion2010). Measurement of the intima–media thickness (IMT) of the carotid artery, demonstrating the remodeling of the arterial wall, is feasible in small children and will be measured with ultrasonography with a linear array probe of 7.5 MHz (Dilli et al., Reference Dilli, Ozkan, Ozkan, Aydin, Ozyazici, Fettah and Okumus2017).

Long-term follow-up of complicated MC twin pregnancies is part of our standard clinical care at the LUMC and includes a formal neurological examination (Touwen et al., Reference Touwen, Hempel and Westra1992), measurement of relevant growth parameters, measurement of blood pressure and an assessment of cognitive and motor development using standardized psychometric tests (van Klink et al., Reference van Klink, Koopman, Middeldorp, Klumper, Rijken, Oepkes and Lopriore2015, Reference van Klink, Slaghekke, Balestriero, Scelsa, Introvini, Rustico and Lopriore2016). At the (term equivalent) age of 24 months, cognitive and motor development will be assessed using Bayley scales (Bayley, Reference Bayley2006). At 5 and 8 years of follow-up, cognitive development will be assessed using Wechsler scales (Wechsler, Reference Wechsler2002, Reference Wechsler2014). During the visit, we will also collect anthropometric data through our national system of primary care physicians who monitor growth in every Dutch child throughout the first 2 years of life.

To assess quality of life, language and behavioral development of the twins, online questionnaires will be filled in by the parents using a web-based application (Baron, Reference Baron2000; Varni et al., Reference Varni, Seid and Kurtin2001). These questionnaires are part of our standard clinical care. In addition, we will evaluate temperament and attachment using validated questionnaires (Putnam et al., Reference Putnam, Gartstein and Rothbart2006; Spruit et al., Reference Spruit, Wissink, Noom, Colonnesi, Polderman, Willems and Stams2018) (Table 2).

Table 2. Overview of evaluations and measurements within TwinLIFE

Note: MC = monochorionic, MSCs = mesenchymal stromal cells, qPCR = quantitative polymerase chain reaction, CVD = cardiovascular disease, NDI = neurodevelopmental impairment, cIMT = carotid intima–media thickness, aPWV = aortic pulse-wave velocity