Background aimsHuman umbilical cord–derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods... Show moreBackground aimsHuman umbilical cord–derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods are applied. Here, we report on a robust and standardized method for hUC-MSC isolation and expansion.MethodsUsing 90 hUC donors, we compared and optimized critical variables during each phase of the multi-step procedure involving UC collection, processing, MSC isolation, expansion and characterization. Furthermore, we assessed the effect of donor-to-donor variability regarding UC morphology and donor attributes on hUC-MSC characteristics.ResultsWe demonstrated robustness of our method across 90 UC donors at each step of the procedure. With our method, UCs can be collected up to 6 h after birth, and UC-processing can be initiated up to 48 h after collection without impacting on hUC-MSC characteristics. The removal of blood vessels before explant cultures improved hUC-MSC purity. Expansion in Minimum essential medium α supplemented with human platelet lysate increased reproducibility of the expansion rate and MSC characteristics as compared with Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum. The isolated hUC-MSCs showed a purity of ∼98.9%, a viability of >97% and a high proliferative capacity. Trilineage differentiation capacity of hUC-MSCs was reduced as compared with bone marrow-derived MSCs. Functional assays indicated that the hUC-MSCs were able to inhibit T-cell proliferation demonstrating their immune-modulatory capacity.ConclusionsWe present a robust and standardized method to isolate and expand hUC-MSCs, minimizing technical variability and thereby lay a foundation to advance reliability and comparability of results obtained from different donors and different studies. Show less
BackgroundResearch in singletons identified fetal growth restriction (FGR) as a risk factor for retinopathy of prematurity (ROP), but is generally subject to confounding by genetic, obstetric, and... Show moreBackgroundResearch in singletons identified fetal growth restriction (FGR) as a risk factor for retinopathy of prematurity (ROP), but is generally subject to confounding by genetic, obstetric, and maternal factors. We investigated the effect of FGR on ROP in growth-discordant identical twins, thereby controlling for confounding factors.MethodsAll data of monochorionic (MC) twin pairs with a birth weight discordance >= 20% born in our center between 2010 and 2021 were retrospectively reviewed for the presence of ROP. Potential risk factors for ROP were analyzed. Outcomes were compared between the smaller and larger twin.ResultsWe included 88 MC twin pairs with growth discordance. In 34% (30/88), both neonates were at risk of ROP. Prevalence of ROP was higher among the smaller twin compared to the larger twin, 30% (9/30) versus 13% (4/30), respectively (OR 2.8, 95% CI: 1.2-6.6). The smaller twin had a longer duration of mechanical ventilation (8 (1-20) versus 2 (1-4) days) and received their first red blood cell transfusion at an earlier postmenstrual age (29.6 (28.1-31.6) versus 30.4 (29.7-32.6) weeks).ConclusionsIn this identical twin model, FGR is associated with almost tripled odds of ROP development, suggesting that both unfavorable antenatal growth conditions and adverse neonatal outcomes affect postnatal retinal vascular proliferation.ImpactFetal growth restriction in growth-discordant identical twins is associated with almost tripled odds of developing retinopathy of prematurity in the smaller twin.Since these twins do not only differ in birth weight but also duration of mechanical ventilation and timing of the first red blood cell transfusion, both unfavorable antenatal growth conditions and adverse neonatal outcomes can affect postnatal retinal vascular proliferation.More attention for preventing retinopathy of prematurity is needed in those with fetal growth restriction who received prolonged duration of mechanical ventilation, oxygen supplementation, or a first red blood cell transfusion Show less
Emrani, S. el; Groene, S.G.; Spekman, J.A.; Slaghekke, F.; Meeren, L.E. van der; Schalij-Delfos, N.E.; Lopriore, E. 2023
Introduction: The purpose of this study was to evaluate the within-pair difference in retinopathy of prematurity (ROP) between donors and recipients with twin-to-twin transfusion syndrome (TTTS)... Show moreIntroduction: The purpose of this study was to evaluate the within-pair difference in retinopathy of prematurity (ROP) between donors and recipients with twin-to-twin transfusion syndrome (TTTS) and to identify risk factors for ROP development.Methods: This retrospective cohort study included 147 TTTS twin pairs managed between 2002-2022 and eligible for ROP screening. Primary outcomes were any stage ROP and severe ROP. Secondary outcomes were hemoglobin at birth, red blood cell transfusions, mechanical ventilation days, postnatal steroids and neonatal morbidity.Results: Rates of any stage ROP (23% vs. 14%) and severe ROP (8% vs. 3%) were significantly higher in donors compared to recipients. Donors received a higher number of blood transfusions (1 (+/- 1.9) vs. 0.7 (+/- 1.5)). Five factors were univariately associated with any stage ROP: donor status (OR 1.9; 95% CI 1.3-2.9), lower GA at birth (OR 1.7; 95% CI 1.4-2.1), small for GA (OR 2.1; 95% CI 1.3-3.5), mechanical ventilation days (OR 1.1; 95% CI 1.1-1.2) and blood transfusions in phase 1 (OR 2.3; 95% CI 1.2-4.3). Three factors were independently associated with any stage ROP: donor status (OR 1.8; 95% CI 1.1-2.9), lower GA at birth (OR 1.6; 95% CI 1.2-2.1) and mechanical ventilation days (OR 1.1, 95% CI 1.0-1.1). Donor status was univariately associated with severe ROP (OR 2.3, 95% CI 1.1-5.0). Conclusion: Any stage ROP and severe ROP are detected twice as frequently in donors compared to recipients. Increased awareness for ROP is needed in donors, especially those with lower GA at birth and longer duration of mechanical ventilation. Show less
In 70% of identical twin pregnancies, the twins share a single placenta in the womb that provides them with nutrients. This shared placenta can be unequaly shared. This means that one of the twins... Show moreIn 70% of identical twin pregnancies, the twins share a single placenta in the womb that provides them with nutrients. This shared placenta can be unequaly shared. This means that one of the twins has a much smaller share of the placenta compared to the other twin, resulting in a large growth discrepancy during pregnancy, also known as 'selective fetal growth restriction'. At birth, a large difference in birth weight ensues. Sometimes one twin can be almost twice the size of the other twin.These twins are considered a vulnerable patient group in which perinatal complications are prevalent. Nevertheless, a large gap in knowledge persists, impeding proper parent counseling and risk assessment by health care providers. Simultaneously, these twins can also be considered a unique natural 'experiment' to uncover the early origins of health and disease following an adverse environment in the womb. The growth-restricted twin can be compared to a genetically identical, normally-grown twin who has been in the same womb of the same mother at the same time.In this thesis, Sophie Groene has investigated the placental mechanisms and the short- and long-term outcomes of this special group of twins. Show less
Background: Psychosocial development in monochorionic (MC) twins born after selective fetal growth restriction (sFGR) has been unreported to date, despite its importance for daily functioning and... Show moreBackground: Psychosocial development in monochorionic (MC) twins born after selective fetal growth restriction (sFGR) has been unreported to date, despite its importance for daily functioning and future relationships. Aims: To investigate psychosocial development, attachment and school functioning in MC twins with sFGR and compare outcomes with the general population and between smaller and larger twins. Study design: Observational cohort study. Subjects: MC twins with sFGR (defined as a birth weight discordance >= 20 %) born between 2002 and 2017 and aged 3-17 years. Outcome measures: Multiple parent report questionnaires: the Child Behavior Checklist (social-emotional devel-opment and behavior), the (Early) Childhood Behavior Questionnaire Very Short Form (temperament), the Attachment Insecurity Screening Inventory (attachment) and a school functioning questionnaire. Results: Median age for the 48 twin pairs was 11 (interquartile range (IQR) 8-13) years. Attachment insecurity for both twins was higher than in the general population for ambivalence/resistance (34 % (21/62) vs. 16 %, p = 0.024) and total attachment insecurity (35 % (22/62) vs. 16 %, p = 0.016). Smaller twins had more internalizing behavioral problems, i.e. negative emotions and behaviors turned inwards (22 % (10/46) vs. 11 % (5/46), p = 0.021) and a higher negative affect, i.e. more likely to experience negative emotions (3.2 (2.9-3.7) vs. 2.9 (2.2-3.2), p = 0.009) than larger twins, as well as a lower secondary school level (p = 0.031). Conclusion: MC twins with sFGR have more ambivalent/resistant attachment insecurity following the compli-cated pregnancy course. Smaller twins have a tendency towards negative emotions and internalizing behaviors compared to larger twins, indicating an increased sensitivity for depression and anxiety. Show less
Groene, S.G.; Tollenaar, L.S.A.; Middeldorp, J.M.; Lopriore, E. 2022
Monochorionic (MC) twin pregnancies are at increased risk of neonatal morbidity and mortality due to the shared placenta with vascular connections that can give rise to various complications,... Show moreMonochorionic (MC) twin pregnancies are at increased risk of neonatal morbidity and mortality due to the shared placenta with vascular connections that can give rise to various complications, including twin-twin transfusion syndrome, twin anemia polycythemia sequence (TAPS), selective fetal growth restriction, and other hematological imbalances at birth. Each complication presents its own challenges and considerations in the neonatal period. Measurement of hemoglobin levels and reticulocyte count is required to establish a correct diagnosis. Placenta dye injection is needed to properly distinguish between the various conditions. Risk factors for adverse outcome in MC twins include prematurity, severe cerebral injury, and the type of MC pregnancy complication. We, therefore, recommend cerebral ultrasound examinations in all complicated MC twins at birth to rule out a severe brain injury. Lastly, we strongly encourage screening for hearing loss using automated auditory brainstem response in all spontaneous TAPS donors to prevent permanent speech development delay. Show less
Background: Singletons born after fetal growth restriction (FGR) are at increased risk of poor neurodevelopmental outcomes. Studies of singletons with FGR usually compare outcomes with those... Show moreBackground: Singletons born after fetal growth restriction (FGR) are at increased risk of poor neurodevelopmental outcomes. Studies of singletons with FGR usually compare outcomes with those without FGR, a comparison that is inherently biased by obstetrical, parental, and genetic factors. We aim to compare neurodevelopmental outcomes between the smaller and larger twin in a population of discordant identical twins who shared a single placenta (monochorionic diamniotic), naturally eliminating these confounders. Methods: This study is part of the cohort study LEMON of monochorionic diamniotic twins with selective FGR. All monochorionic diamniotic twins with selective FGR who were born in Leiden University Medical Center (Leiden, Netherlands) between March 1, 2002, and Dec 31, 2017, were eligible for inclusion. Twin pregnancies that were complicated by twin-twin transfusion syndrome, twin anaemia polycythaemia sequence, or monoamnionicity were excluded. Cognitive performance was evaluated with two standardised psychometric age-appropriate tests, producing a full-scale intelligence quotient (FSIQ). Motor functioning was assessed with a standardised neurological examination. A composite outcome of neurodevelopmental impairment (NDI) was used, subdivided into mild NDI (defined as FSIQ <85, minor neurological dysfunction or cerebral palsy grade 1, or mild visual or hearing impairment) and severe NDI (defined as FSIQ <70, severe neurological dysfunction, or severe visual or hearing impairment). Findings: Between Jan 25, 2021, and March 15, 2022, 47 twin pairs were enrolled in the study and underwent neurodevelopmental assessment. The median gestational age at birth was 33middot9 weeks (IQR 31middot3-36middot0) for the 47 included twin pairs, with median birthweights of 1400 g (1111-1875) in the smaller twin and 2003 g (1600-2680) in the larger twin. The median age at neurodevelopmental assessment was 11 years (8-13). Median FSIQ was 94 (86-101) for the smaller twin and 100 (92-108) for the larger twin (p<0middot0001). More smaller twins had mild NDI (17 [36%] of 47) than did the larger twins (five [11%] of 47; odds ratio 4middot8 [95% CI 1middot6-14middot1]; p=0middot0049). There was no difference in the proportion of children with severe NDI (two [4%] of 47 in both groups, p=1middot0). Interpretation: As mild NDI can impede children in their daily functioning, we recommend standardised long-term follow-up, including neurodevelopmental testing, for monochorionic diamniotic twins with selective FGR to facilitate early identification of children at risk. Show less
Emrani, S. el; Groene, S.G.; Verweij, E.J.; Slaghekke, F.; Khalil, A.; Klink, J.M.M. van; ... ; Lopriore, E. 2022
This systematic review aims to assess the gestational age at birth and perinatal outcome [intrauterine demise (IUD), neonatal mortality and severe cerebral injury] in monochorionic twins with... Show moreThis systematic review aims to assess the gestational age at birth and perinatal outcome [intrauterine demise (IUD), neonatal mortality and severe cerebral injury] in monochorionic twins with selective fetal growth restriction (sFGR), according to Gratacos classification based on umbilical artery Doppler flow patterns in the smaller twin. Seventeen articles were included. Gestational age at birth varied from 33.0 to 36.0 weeks in type I, 27.6-32.4 weeks in type II, and 28.3-33.8 weeks in type III. IUD rate differed from 0%-4% in type I to 0%-40% in type II and 0%-23% in type III. Neonatal mortality rate was between 0%-10% in type I, 0%-38% in type II, and 0%-17% in type III. Cerebral injury was present in 0%-2% of type I, 2%-30% of type II and 0%-33% of type III cases. The timing of delivery in sFGR varied substantially among studies, particularly in type II and III. The quality of evidence was moderate due to heterogenous study populations with varying definitions of sFGR and perinatal outcome parameters, as well as a lack of consensus on the use of the Gratacos classification, leading to substantial incomparability. Our review identifies the urgent need for uniform antenatal diagnostic criteria and definitions of outcome parameters. Show less
Objectives: Fetal growth restriction (FGR) may alter brain development permanently, resulting in lifelong structural and functional changes. However, in studies addressing this research question,... Show moreObjectives: Fetal growth restriction (FGR) may alter brain development permanently, resulting in lifelong structural and functional changes. However, in studies addressing this research question, FGR singletons have been compared primarily to matched appropriately grown singletons, a design which is inherently biased by differences in genetic and maternal factors. To overcome these limitations, we conducted a within-pair comparison of neonatal structural cerebral ultrasound measurements in monochorionic twin pairs with selective FGR (sFGR). Methods: Structural cerebral measurements on neonatal cerebral ultrasound were compared between the smaller and larger twins of monochorionic twin pairs with sFGR, defined as a birth-weight discordance (BWD) >= 20%, born in our center between 2010 and 2020. Measurements from each twin pair were also compared with those of an appropriately grown singleton, matched according to sex and gestational age at birth. Results: Included were 58 twin pairs with sFGR, with a median gestational age at birth of 31.7 (interquartile range, 29.9-33.8) weeks and a median birth weight of 1155 g for the smaller twin and 1725 g for the larger twin (median BWD, 32%). Compared with both the larger twin and the singleton, the smaller twin had significantly smaller cerebral structures (corpus callosum, vermis, cerebellum), less white/deep gray matter and smaller intracranial surface area and volume. Intracranial-volume discordance and BWD correlated significantly (R-2 = 0.228, P < 0.0001). The median intracranial-volume discordance was smaller than the median BWD (19% vs 32%, P < 0.0001). After correction for intracranial volume, only one of the observed differences (biparietal diameter) remained significant for the smaller twin vs both the larger twin and the singleton. Conclusions: In monochorionic twins with sFGR, neonatal cerebral ultrasound reveals an overall, proportional restriction in brain growth, with smaller cerebral structures, less white/deep gray matter and smaller overall brain-size parameters in the smaller twin. There was a positive linear relationship between BWD and intracranial-volume discordance, with intracranial-volume discordance being smaller than BWD. (c) 2021 International Society of Ultrasound in Obstetrics and Gynecology. Show less
Groene, S.G.; Tollenaar, L.S.A.; Meeren, L.E. van der; Slaghekke, F.; Verweij, E.J.; Hooper, S.B.; ... ; Lopriore, E. 2021
We report a case of a monochorionic diamniotic twin with an uncomplicated pregnancy, but with an unexpected large intertwin hemoglobin (Hb) difference at birth. Twin 1 was delivered vaginally and... Show moreWe report a case of a monochorionic diamniotic twin with an uncomplicated pregnancy, but with an unexpected large intertwin hemoglobin (Hb) difference at birth. Twin 1 was delivered vaginally and had an uneventful neonatal course. The umbilical cord of Twin 1 was clamped approximately 5 min after birth. After the birth of Twin 1, Twin 2 developed severe bradycardia and showed limited cardiac output on ultrasound, for which an emergency cesarean section was performed. A full blood count revealed an Hb of 20.1 g/dL for Twin 1 and 10.2 g/dL for Twin 2 (intertwin difference 9.9 g/dL). Reticulocyte counts were similar, 40 parts per thousand and 38 parts per thousand, respectively. Placental examination revealed 10 vascular anastomoses, including one arterio-arterial anastomosis with a diameter of 1.4 mm. Additionally, a large chorangioma was present on the placental surface of Twin 2. There was no color difference on the maternal side of the placenta. Based on the reticulocyte count ratio and the placental characteristics, twin anemia polycythemia sequence was ruled out as the cause of the large intertwin Hb difference. In this report, we discuss the various potential causes that could explain the large intertwin Hb difference including the role of delayed cord clamping in Twin 1, and the role of a large chorangioma, which may have attracted blood from the fetal circulation of Twin 2. Show less
Background: Fetal growth restriction (FGR) is thought to negatively affect lung development resulting in increased respiratory morbidity. However, research performed in singletons is often limited... Show moreBackground: Fetal growth restriction (FGR) is thought to negatively affect lung development resulting in increased respiratory morbidity. However, research performed in singletons is often limited by a certain level of bias caused by individual differences in genetic constitution, obstetrical and maternal factors.Methods: Respiratory morbidity was compared between the smaller and the larger twin in monochorionic twins with selective fetal growth restriction (sFGR), defined as a birth weight discordance >= 20%, born in our center between 2010 and 2019 in this retrospective study. Respiratory distress syndrome (RDS) was diagnosed based on the clinical picture of a neonate with respiratory failure requiring mechanical ventilation and/or surfactant, confirmed by a chest X-ray. Bronchopulmonary dysplasia (BPD) was diagnosed when the neonate required treatment with >21% oxygen for at least 28 days.Findings: Median gestational age at birth for the 94 included pregnancies was 32.4 (IQR 30.4-34.3) weeks. Within-pair analyses showed that the prevalence of RDS was lower in the smaller twin compared to the larger twin, 19.1% (18/94) vs 34.0% (32/94), respectively (p = 0.004). The odds of RDS for the larger twin was doubled (OR 2.1 (CI95% 1.3-3.5). In contrast, the rate of BPD in the smaller twin was higher as opposed to the larger twin, 16.7% (15/90) vs 6.7% (6/89), respectively (p = 0.008), with a more than doubled odds (OR 2.5 (CI95% 1.3-4.9)).Interpretation: Despite being genetically identical, sFGR twins have different respiratory outcomes. Adverse growth condition in utero in the smaller twin is associated with a reduced odds of RDS at birth but a more than doubled odds of BPD, reflecting the pathophysiologic adverse effect of growth restriction on lung development. (C) 2021 The Authors. Published by Elsevier Ltd. Show less
Lifelong health is thought to be partially set during intrauterine life by persistent epigenetic changes induced by the prenatal environment. To evaluate this hypothesis, we initiated a prospective... Show moreLifelong health is thought to be partially set during intrauterine life by persistent epigenetic changes induced by the prenatal environment. To evaluate this hypothesis, we initiated a prospective longitudinal study in monochorionic (MC) twins: the TwinLIFE study. MC twins are monozygotic, thus in origin genetically identical, and share a single placenta. Although MC twins have many environmental factors in common, in one-third of the MC twin pairs, one fetus has significantly less access to nutrients and resources during pregnancy than its co-twin often resulting in a significant discordance in prenatal growth. Hence, MC twins constitute a unique natural experiment to study the influence of the prenatal environment on health. In TwinLIFE, we will chart intrapair differences in DNA methylation focusing on mesenchymal stromal cells isolated from cord as an advanced proxy of epigenetic dysregulation relevant for long-term health consequences. Next, we will follow up the MC twins for growth, cardiovascular and neurodevelopmental outcomes during childhood and evaluate the impact of an epigenetic signature at birth on future health. The current target is to include 100 MC twin pairs, but we aim to continue enrollment after procuring additional funding. TwinLIFE will not only address an unmet clinical need in the high-risk group of MC twins, but may also advance early-life strategies to prevent adverse growth, cardiovascular and neurodevelopmental outcomes in the general population. Show less
Groene, S.G.; Tollenaar, L.S.A.; Slaghekke, F.; Middeldorp, J.M.; Haak, M.; Oepkes, D.; Lopriore, E. 2018
