The ex vivo expansion and maintenance of long-term hematopoietic stem cells (LT-HSC) is crucial for stem cell-based gene therapy. A combination of stem cell factor (SCF), thrombopoietin (TPO), FLT3... Show moreThe ex vivo expansion and maintenance of long-term hematopoietic stem cells (LT-HSC) is crucial for stem cell-based gene therapy. A combination of stem cell factor (SCF), thrombopoietin (TPO), FLT3 ligand (FLT3) and interleukin 3 (IL3) cytokines has been commonly used in clinical settings for the expansion of CD34(+) from different sources, prior to transplantation. To assess the effect of IL3 on repopulating capacity of cultured CD34(+) cells, we employed the commonly used combination of STF, TPO and FILT3 with or without IL3. Expanded cells were transplanted into NSG mice, followed by secondary transplantation. Overall, this study shows that IL3 leads to lower human cell engraftment and repopulating capacity in NSG mice, suggesting a negative effect of IL3 on HSC self-renewal. We, therefore, recommend omitting IL3 from HSC-based gene therapy protocols. Show less
Youssef, A.; Hoorn, M.L. van der; Eekelen, R. van; Geloven, N. van; Wely, M. van; Smits, M.A.J.; ... ; Lashley, E. 2022
Introduction: Recurrent pregnancy loss (RPL) is defined as the loss of two or more conceptions before 24 weeks gestation. Despite extensive diagnostic workup, in only 25%-40% an underlying cause is... Show moreIntroduction: Recurrent pregnancy loss (RPL) is defined as the loss of two or more conceptions before 24 weeks gestation. Despite extensive diagnostic workup, in only 25%-40% an underlying cause is identified. Several factors may increase the risk for miscarriage, but the chance of a successful pregnancy is still high. Prognostic counselling plays a significant role in supportive care. The main limitation in current prediction models is the lack of a sufficiently large cohort, adjustment for relevant risk factors, and separation between cumulative live birth rate and the success chance in the next conception. In this project, we aim to make an individualised prognosis for the future chance of pregnancy success, which could lead to improved well-being and the ability managing reproductive choices. Methods and analysis: In this multicentre study, we will include both a prospective and a retrospective cohort of at least 931 and 1000 couples with RPL, respectively. Couples who have visited one of the three participating university hospitals in the Netherlands for intake are eligible for the study participation, with a follow-up duration of 5 years. General medical and obstetric history and reports of pregnancies after the initial consultation will be collected. Multiple imputation will be performed to cope for missing data. A Cox proportional hazards model for time to pregnancy will be developed to estimate the cumulative chance of a live birth within 3 years after intake. To dynamically estimate the chance of an ongoing pregnancy, given the outcome of earlier pregnancies after intake, a logistic regression model will be developed. Ethics and dissemination: The Medical Ethical Research Committee of the Leiden University Medical Center approved this study protocol (N22.025). There are no risks or burden associated with this study. Participant written informed consent is required for both cohorts. Findings will be published in peer-reviewed journals and presentations at international conferences. Show less
Eikmans, M.; Morales-Prieto, D.M.M.; Hoorn, M.L. van der; Markert, U.R.R. 2022
Introduction The assisted reproductive technique of oocyte donation (OD) is comparable to in vitro fertilisation (IVF), with the distinction of using a donated oocyte and thus involving two women.... Show moreIntroduction The assisted reproductive technique of oocyte donation (OD) is comparable to in vitro fertilisation (IVF), with the distinction of using a donated oocyte and thus involving two women. Compared with IVF and naturally conceived (NC) pregnancies, OD pregnancies have a higher risk for pregnancy complications as pregnancy-induced hypertension (PIH) and pre-eclampsia (PE). Various covariates among women pregnant by OD, however, also contribute to an increased risk for developing hypertensive complications. Therefore, we will conduct the DONation of Oocytes in Reproduction individual participant data (DONOR IPD) meta-analysis to determine the risk for the development of hypertensive complications in OD pregnancy, in comparison to autologous oocyte pregnancy (non-donor IVF/intracytoplasmic sperm injection (ICSI) and NC pregnancy). The DONOR IPD meta-analysis will provide an opportunity to adjust for confounders and perform subgroup analyses. Furthermore, IPD will be used to externally validate a prediction model for the development of PE in OD pregnancy. Methods and analysis A systematic literature search will be performed to search for studies that included women pregnant by OD, and documented on hypertensive complications in OD pregnancy. The authors from each study will be asked to collaborate and share IPD. Using the pseudoanonymised combined IPD, we will perform statistical analyses with one-stage and two-stage approaches, subgroup analyses and possibly time-to-event analyses to investigate the risk of developing hypertensive complications in OD pregnancy. Furthermore, we will formally assess a prediction model on its performance in an external validation with the use of IPD. Ethics and dissemination Ethical approval and individual patient consent will not be required in most cases since this IPD meta-analysis will use existing pseudoanonymised data from cohort studies. Results will be disseminated through peer-reviewed journals and international conferences. PROSPERO registration number CRD42021267908. Show less
Eikmans, M.; Keur, C. van der; Anholts, J.D.H.; Drabbels, J.J.M.; Beelen, E. van; Lopes, S.M.C.D.; Hoorn, M.L. van der 2022
IntroductionTrophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their... Show moreIntroductionTrophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their usefulness in studying interaction with immune cells. MethodsAfter enzymatic digestion of first-trimester placental tissue from seven donors (6-9 weeks gestation) and trophoblast enrichment we cultured cytotrophoblasts (CTB) in stem cell medium. CTB were differentiated into EVT in a Matrigel-containing medium. A subset of CTB/EVT was profiled for microRNA levels. Expression of classical HLA molecules and of HLA-G was studied by flow cytometry, qPCR, and ELISA. Secondary trophoblast cell lines JAR and JEG-3 were studied as controls. Lymphocytes were investigated during co-culturing with EVT. ResultsThe trophoblasts could be easily maintained for several passages, upregulated classical trophoblast markers (GATA3, TFAP2C, chromosome-19 microRNAs), and upon differentiation to EVT they were selective in expressing HLA-C. EVT showed increasing expression of total HLA-G, an increasing proportion of HLA-G1 over G2- and G3 isoforms, and elevated excretion of soluble HLA-G. These features were distinct from those of the secondary trophoblast cell lines. TNF-alpha and IL-8 represented the most abundantly secreted cytokines by CTB, but their levels were minimal in EVT cultures. As proof of principle, we showed that EVT affect lymphocytes in three-day co-cultures (n=4) by decreasing activation marker HLA-DR. ConclusionWe verified the possibility culturing trophoblasts from first-term placentas, and their capability of differentiating to HLA-G expressing EVT. This culture model better represents the in-vivo situation than previously studied secondary trophoblast cell lines and enables mechanistic studies of fetal-maternal interactions. Show less
This special issue of Reproductive Sciences is focusing on ethnic health disparity and its impact on (fe)male reproduction. Indeed, studies regarding underlying mechanisms, interventions and... Show moreThis special issue of Reproductive Sciences is focusing on ethnic health disparity and its impact on (fe)male reproduction. Indeed, studies regarding underlying mechanisms, interventions and prognosis in reproduction are underexposed for the non-White male and female. Here, we call for documentation of race and ethnicity in the analysis and management of couples with recurrent pregnancy loss. Show less
Bentem, K. van; Bos, M.; Keur, C. van der; Kapsenberg, H.; Lashley, E.; Eikmans, M.; Hoorn, M.L. van der 2022
Oocyte donation (OD) pregnancies are characterized by more fetal-maternal human leukocyte antigen (HLA) mismatches compared with naturally conceived (NC) and in vitro fertilization (IVF)... Show moreOocyte donation (OD) pregnancies are characterized by more fetal-maternal human leukocyte antigen (HLA) mismatches compared with naturally conceived (NC) and in vitro fertilization (IVF) pregnancies. The maternal immune system has to cope with greater immunogenetic dissimilarity, but involved immunoregulation remains poorly understood. We examined whether the amount of regulatory T cells (Tregs) and immunoregulatory cytokines in decidua basalis of OD pregnancies differs from NC and IVF pregnancies. The cohort included 25 OD, 11 IVF and 16 NC placentas, maternal peripheral blood, and umbilical cord blood of uncomplicated pregnancies. Placenta slides were stained for FOXP3, IL-10, IL-6, gal-1, TGF-0 and Flt-1. Semi-quantitative (FOXP3+ Tregs) and computerized analysis (cytokines) were executed. The blood samples were typed for HLA class I and II to calculate fetal-maternal HLA mismatches. The percentage of Tregs was significantly higher in pregnancies with 4-6 HLA class I mismatches (n = 17), compared to 0-3 mismatches (n = 35; p = 0.04). Cytokine analysis showed significant differences between OD, IVF and NC pregnancies. Flt-1 was significantly lower in pregnancies with 4-6 HLA class I mismatches (p = 0.004), and in pregnancies with 6-10 HLA mismatches in total (p = 0.024). This study suggests that immunoregulation at the fetal-maternal interface in OD pregnancies with more fetal-maternal HLA mismatches is altered.(c) 2021 The Authors. Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/). Show less
Hoorn, M.L. van der; Bentem, K. van; Lashley, E. 2022
It is well known that oocyte donation (OD) pregnancies are associated with higher complication rates compared to autologous pregnancies. However, evidence-based information for pre-pregnancy... Show moreIt is well known that oocyte donation (OD) pregnancies are associated with higher complication rates compared to autologous pregnancies. However, evidence-based information for pre-pregnancy counseling designed for health care workers is scarce. Therefore, a systematic literature search was performed to find articles that address pre-pregnancy counseling before OD. A systematic search was conducted in September 2020 in various databases, including PubMed and Embase. Nine (systematic) reviews and meta-analyses were included that reported on pre-pregnancy advice in OD pregnancies. Studies are consistent in documenting a higher risk for hypertensive disorders, cesarean section, preterm birth, postpartum hemorrhage, and low birth weight. Based on these complications, pre-pregnancy advice is mentioned in all included systematic reviews to prevent complications in the next pregnancy. All studies recommend counseling women on the increased risk of complications during OD pregnancy. Other recommendations include the prophylactic use of aspirin in pregnancy and restriction to single embryo transfer. Individualized appropriate surveillance and management strategies should be considered for every patient achieving pregnancy by OD. Show less
Tian, X.Z.; Aiyer, K.T.S.; Kapsenberg, J.M.; Roelen, D.L.; Hoorn, M.L. van der; Eikmans, M. 2021
Problem The embryo of an oocyte donation (OD) pregnancy is completely allogeneic to the mother, which may challenge the maternal immune system to tolerize the fetus. Decidual macrophages are... Show moreProblem The embryo of an oocyte donation (OD) pregnancy is completely allogeneic to the mother, which may challenge the maternal immune system to tolerize the fetus. Decidual macrophages are essential in maintaining a healthy pregnancy, and type 2 macrophages may exhibit immune suppressive activity. We hypothesized that the composition of decidual macrophages is different between uncomplicated OD pregnancies and non-OD in vitro fertilization (IVF) pregnancies, and is related to fetal-maternal incompatibility. Method of study Women with uncomplicated pregnancy were enrolled: 25 singleton OD pregnancies and 17 non-OD IVF pregnancies. The extent of immunohistochemical staining of CD14 (pan-macrophage marker) and CD163 (type 2 macrophage marker) in both decidua basalis and parietalis was quantitated by digital image analysis. Maternal and fetal DNA was typed for human leukocyte antigen (HLA)-A, -B, C, -DRB1, and -DQB1, and fetal-maternal HLA mismatches were calculated. Results OD pregnancies showed a higher percentage of CD163+ staining (P = .040) and higher CD163/CD14 ratio (P = .032) in the parietalis than non-OD IVF. The OD group was separated into a semi-allogeneic group (<= 5 fetal maternal HLA mismatches) and a fully allogeneic group (> 5 mismatches). The HLA-fully-allogeneic OD group, but not the HLA-semi-allogeneic OD group, showed significantly elevated CD163/CD14 ratio in the parietalis compared with the non-OD IVF group (P < .05). Conclusions Uncomplicated OD pregnancies display a higher CD163-positive cell fraction in the total decidual macrophage population compared to autologous pregnancies, which may suggest that a local type 2 macrophage-related mechanism is needed to compensate for the higher fetal-maternal HLA mismatch load. Show less
Bentem, K. van; Bos, M.; Keur, C. van der; Kapsenberg, H.; Lashley, E.; Eikmans, M.; Hoorn, M.L. van der 2021
Research question: The definition of recurrent pregnancy loss (RPL) differs internationally. The European Society of Human Reproduction and Embryology (ESHRE) defines RPL as two or more pregnancy... Show moreResearch question: The definition of recurrent pregnancy loss (RPL) differs internationally. The European Society of Human Reproduction and Embryology (ESHRE) defines RPL as two or more pregnancy losses. Different definitions lead, however, to different approaches to care for couples with RPL. This study aimed to determine whether the distribution of RPL-associated factors was different in couples with two versus three or more pregnancy losses. If a similar distribution were found, couples with two pregnancy losses should be eligible for the same care pathway as couples with three pregnancy losses.Design: This single-centre, retrospective cohort study investigated 383 couples included from 2012 to 2016 at the Leiden University Medical Center RPL clinic. Details on age, body mass index, smoking status, number of pregnancy losses, mean time to pregnancy loss and performed investigations were collected. The prevalence of uterine anomalies, antiphospholipid syndrome, hereditary thrombophilia, hyperhomocysteinaemia, chromosomal abnormalities and positive thyroid peroxidase antibodies were compared in couples with two versus three or more pregnancy losses.Results: No associated factor was found in 71.5% of couples with RPL. This did not differ statistically between couples with two versus three or more pregnancy losses (73.6% versus 70.6%; P = 0.569). The distribution of investigated causes did not differ between the two groups.Conclusions: As the distribution of associated factors in couples with two versus three or more pregnancy losses is equal, couples with two pregnancy losses should be eligible for the same care pathway as couples with three. This study supports ESH RE's suggestion of including two pregnancy losses in the definition of RPL. Show less
Eikmans, M.; Zwan, A. van der; Claas, F.H.J.; Hoorn, M.L. van der; Heidt, S. 2020
Appropriate development of the placenta is required for healthy pregnancy to occur. After implantation of the fertilized blastocyst, fetal trophoblasts invade the endometrium and myometrium of the... Show moreAppropriate development of the placenta is required for healthy pregnancy to occur. After implantation of the fertilized blastocyst, fetal trophoblasts invade the endometrium and myometrium of the mother's uterus to establish placentation. In this process, fetal trophoblasts encounter maternal immune cells. In this review, we focus on the role of maternal T cells and myeloid cells (macrophages, dendritic cells) in pregnancy and their interaction with trophoblasts. To retain immunologic tolerization, trophoblasts evade immune recognition by T cells and produce factors that modulate their phenotype and function. On top of that, the local environment at the maternal-fetal interface favors expansion of regulatory T cells. Macrophages and dendritic cells are essential in maintaining a healthy pregnancy. They produce soluble factors and act as antigen-presenting cells, thereby interacting with T cells. Herein, M2 macrophages, immature dendritic cells, CD4(+)Th2 cells, and regulatory T cells represent an axis that maintains a local immune tolerant environment. We consider outstanding issues concerning these cell types and their pathways, which need to be addressed in future investigations. Data from recent single-cell sequencing experiments of the placental bed, to study heterogeneity of maternal immune cells and to predict cell-cell interactions, are discussed. Novel ways for long-term culturing of primary trophoblasts allow for cell-cell interaction studies in a functional way. Future directions should include study of the functionality of currently known and newly identified decidual immune cell subsets in healthy and complicated pregnancies, and their interaction with and modulation by trophoblast cells. Show less
The embryo of an oocyte donation (OD) pregnancy is completely allogeneic to the mother, which leads to a more serious challenge for the maternal immune system to tolerize the fetus. It is thought... Show moreThe embryo of an oocyte donation (OD) pregnancy is completely allogeneic to the mother, which leads to a more serious challenge for the maternal immune system to tolerize the fetus. It is thought that macrophages are essential in maintaining a healthy pregnancy, by acting in immunomodulation and spiral arterial remodeling. OD pregnancies represent an interesting model to study complex immunologic interactions between the fetus and the pregnant woman since the embryo is totally allogeneic compared to the mother. Here, we describe a narrative review on the role of macrophages and pregnancy and a systematic review was performed on the role of macrophages in OD pregnancies. Searches were made in different databases and the titles and abstracts were evaluated by three independent authors. In total, four articles were included on OD pregnancies and macrophages. Among these articles, some findings are conflicting between studies, indicating that more research is needed in this area. From current research, we could identify that there are multiple subtypes of macrophages, having diverse biological effects, and that the ratio between subtypes is altered during gestation and in aberrant pregnancy. The study of macrophages' phenotypes and their functions in OD pregnancies might be beneficial to better understand the maternal-fetal tolerance system. Show less
Hasaart, K.A.L.; Manders, F.; Hoorn, M.L. van der; Verheul, M.; Poplonski, T.; Kuijk, E.; ... ; Boxtel, R. van 2020
