In this thesis the aim was to study immune cell interactions at the maternal-fetal interface to understand the role for immune cells during healthy pregnancy development an pregnancy complications.... Show moreIn this thesis the aim was to study immune cell interactions at the maternal-fetal interface to understand the role for immune cells during healthy pregnancy development an pregnancy complications. Specifically in cases of recurrent pregnancy loss and chronic histiocytic intervillositis. Show less
This thesis deals with various aspects of twin anemia polycythemia sequence (TAPS). TAPS is a condition that can develop due to unbalanced feto-fetal blood transfusion through minuscule vascular... Show moreThis thesis deals with various aspects of twin anemia polycythemia sequence (TAPS). TAPS is a condition that can develop due to unbalanced feto-fetal blood transfusion through minuscule vascular placental anastomoses in monochorionic twin pregnancies, causing the donor twin to become anemic and the recipient twin to become polycythemic. In this thesis we show that a difference in middle cerebral artery peak systolic velocity (MCA-PSV) > 0.5 Multiples of the Median (MoM) has a high diagnostic accuracy for the the antenatal diagnosis of TAPS. For postnatal diagnosis of the condition, inspection of the color of the maternal side of the placenta can be of great value. Furthermore, we present the results of a large international registry, and report on outcomes after different treatment options for TAPS. As the best treatment for TAPS is unclear, we propose the protocol of The TAPS Trial (a multicenter open-label international RCT) to investigate the potential beneficial effect of fetoscopic laser surgery for the outcome in TAPS twins . In the last chapters of this thesis we discuss short- and long-term outcome and show that TAPS donors show significantly higher rates of perinatal mortality and long-term neurodevelopmental impairment than their recipient co-twins. Show less
Pregnancy can be seen as an immunologic paradox. Even though the fetus expresses paternally inherited alloantigens it is protected from rejection by a proper regulation of the maternal immune... Show morePregnancy can be seen as an immunologic paradox. Even though the fetus expresses paternally inherited alloantigens it is protected from rejection by a proper regulation of the maternal immune system. With the studies described in this thesis, we want to get more insight in the immunologic mechanisms that play a role in pregnancy. The results of this research can help to identify underlying etiologies in patients with unexplained pregnancy complications, such as recurrent miscarriage. Identifying these causes is important for providing answers and taking away anxiety in these couples, and eventually for the development of effective therapies. Furthermore, elucidating the mechanism leading to survival or rejection of the fetal allograft is not only essential for our understanding of processes leading to normal and abnormal pregnancies, but may also result in important concepts in the field of transplantation and autoimmunity. Show less
During pregnancy a unique situation arises in which the mother's immune system accepts the fetus, which carries both maternal and paternal genes, and does not reject it as can occur in solid organ... Show moreDuring pregnancy a unique situation arises in which the mother's immune system accepts the fetus, which carries both maternal and paternal genes, and does not reject it as can occur in solid organ transplantation. The aim of this dissertation was to unravel the immunological mechanisms that ensure tolerance during a healthy pregnancy and uncover how alterations could contribute to the development of pregnancy complications, such as pre-eclampsia and preterm birth.We applied the new technique mass cytometry and the associated computational analyzes to map all immune cells of the mother during a healthy pregnancy. Furthermore, we demonstrated the presence of three types of functional regulatory CD4+ T cells, identified a phenotype of CD8+ T cells that can offer both tolerance and immunity against infections, and demonstrated potential cross-reactivity of T cells against fetal allo-antigens. The results described in this thesis have contributed to a better understanding of healthy pregnancies and form a basis on which further research can be built. Show less
In this thesis, Chapter 1 presents the state of the literature on early development in humans and mice. Chapter 2 describes the development of gonadal and extragonadal PGCs in humans. Chapter 3... Show moreIn this thesis, Chapter 1 presents the state of the literature on early development in humans and mice. Chapter 2 describes the development of gonadal and extragonadal PGCs in humans. Chapter 3 investigates the status of XCI in human pre-implantation embryos. One X chromosome is indicated to be inactive in E4-E6 cells and E7 trophectoderm cells, whereas it becomes reactivated in E7 epiblast and primitive endoderm cells. Chapter 4 describes the entering of EVTs in maternal circulation (via decidual veins and lymphatic vessels) starts since W5.5, much earlier than previously accepted W8 (via decidual spiral arteries). Chapter 5 studies the spatial imprinting pattern of IGF2/H19 in human first-trimester placental villi. A normal imprinting pattern of IGF2/H19 is revealed in multi-site villi collections as in the embryo. Chapter 6 explores different consequences of Turner syndrome in mouse placenta. The significantly larger area of glycogen cells in XpO placental outer zone and the significantly higher expression of Ldha in XpO labyrinth zone suggest a more severe placental phenotype in E18.5 XpO placentas than in XmO placentas. Finally, Chapter 7 provides general discussion about the findings and discusses the future perspectives on assisted reproduction and new treatment for infertility, pregnancy complications. Show less
Increasing body of evidence shows that perinatal outcomes in MC twins are strongly associated with the complications resulting from the unique angioarchitecture in MC placentas, in particular... Show moreIncreasing body of evidence shows that perinatal outcomes in MC twins are strongly associated with the complications resulting from the unique angioarchitecture in MC placentas, in particular the placental vascular anastomoses. Due to the extensive application of prenatal ultrasound examination, an increasing number and types of complication dedicated for MC twins are being diagnosed. Delineation of the placental characteristics classified by specific complications may shed light on the pathophysiology of various complications in MC twins. One of the great successes in fetal therapy is the introduction of fetoscopic laser coagulation of vascular anastomoses for the treatment of twin–twin transfusion syndrome (TTTS). The investigation on postoperative complications in TTTS placentas is crucial for the further improvement of fetoscopic laser surgery and improvement of perinatal outcome. Since 2002, all MC placentas delivered at the Leiden University Medical Center (LUMC) are consecutively being examined and injected with colored dye. This large database of MC placentas (n=940 in 2016) allows detailed investigation of the pathogenesis and clinical outcome of these rare diseases. Show less
Human pregnancy is an interesting immunological paradox. The fetus is a semi-allograft, carrying paternal and maternal genes but is not rejected by the maternal immune system. The placenta is a key... Show moreHuman pregnancy is an interesting immunological paradox. The fetus is a semi-allograft, carrying paternal and maternal genes but is not rejected by the maternal immune system. The placenta is a key player in maintaining the pregnancy, since this fetus-derived organ is in direct contact with the mother. This thesis describes the results of investigations on the immune regulation at the fetal-maternal interface with emphasis on two immunological challenges during pregnancy. First, preeclampsia, which might be immunologically related to host versus graft disease as seen in solid organ transplantation and second, egg donation (ED) pregnancies, which show that even complete allogeneic fetal allografts can be tolerated by the mother. The immunological mechanisms involved in acceptance of the totally allogeneic fetus in ED pregnancies are not well understood yet. It is possible that it leads to differential immunological regulation. This hypothesis is tested in this thesis. We found differential immunological interactions in successful ED and in preeclamptic pregnancies compared with naturally conceived pregnancies. These results indicate that preeclampsia and ED pregnancies are indeed immunological challenges during pregnancy. It is a scientific challenge to further reveal the immunological mechanisms, contributing to precious information for the fields of immunology, transplantation and obstetrics. Show less
The incidence of spontaneous twinning in the Netherlands is approximately 1%, of which are 70% dizygotic and 30% monozygotic twins. Dizygotic twinning occurs after fertilization of two eggs (non... Show moreThe incidence of spontaneous twinning in the Netherlands is approximately 1%, of which are 70% dizygotic and 30% monozygotic twins. Dizygotic twinning occurs after fertilization of two eggs (non-identical twins). Dizygotic twins almost invariably have two separate placentas (dichorionic) and two separate amnions (diamniotic). Monozygotic twinning occurs after fertilization of one egg that splits into two embryos (identical twins). In 70-75%, these twins share one common placenta (monochorionic) and have two separate amnions (diamniotic). The incidence of monochorionic twinning is 1 in every 400 pregnancies. During gestation, monochorionic twins compared to dichorionic twins are at increased risk of several complications, such as intrauterine fetal death, intrauterine growth restriction, discordant fetal anomalies, and, most severe, twin-to-twin transfusion syndrome (TTTS). TTTS complicates 10 to 15% of monochorionic twin pregnancies. With an annual birth rate of 188 000, between 47 and 67 cases of TTTS are expected in the Netherlands per year. In virtually all monochorionic twin placentas, vascular connections between the two twins are present, whereas these almost never occur in dichorionic placentas. Thus, intertwin transfusion is the norm in monochorionic pregnancies and a normal physiological phenomenon as long as blood flow between the fetuses is balanced. TTTS develops when blood flow gets unbalanced. Hypovolemia, oliguria and oligohydramnios develop in the donor twin. The recipient twin suffers from hypervolemia, polyuria and polyhydramnios, which may lead to circulatory volume overload, cardiac failure and, eventually, hydrops. TTTS is diagnosed sonographically by the detection of an oligo/polyhydramnios sequence. Quintero et al. developed a staging system for TTTS based on the oligo/polyhydramnios sequence (Stage 1), and also included absent bladder filling in the donor (Stage 2), pathological Doppler findings in donor or recipient (Stage 3), hydrops (Stage 4), and eventually fetal death (Stage 5). TTTS usually emerges in the second trimester of pregnancy, although first-trimester and early third-trimester cases have been described. Due to massive polyhydramnios, TTTS may lead to maternal discomfort and present with clinical symptoms, such as premature rupture of membranes or contractions. This may result in (extremely) premature birth and high mortality and morbidity rates. If left untreated, mortality rates exceed 80% and survivors are handicapped in 10 to 50%. Since the 1980__s, several forms of treatment have been available, of which fetoscopic laser coagulation of the vascular anastomoses on the monochorionic placenta has been proven to be superior compared to serial amniodrainage in terms of perinatal survival and absence of neurological disease in survivors. Moreover, treatment in the early Quintero stages resulted in better outcome. Since 2000, monochorionic twin pregnancies complicated by TTTS have been treated with fetoscopic laser coagulation of placental anastomoses in the Leiden University Medical Center (LUMC), which is a tertiary medical center in the Netherlands and serves as the national referral center for fetal therapy. Since then, several studies on monochorionic twins with and without TTTS were started. TULIPS, Twins and ULtrasound In Pregnancy Studies, was one of these projects. Between July 2003 and July 2005, 58 monochorionic twins with and without TTTS had an ultrasound examination performed at least biweekly. The aims of our study were to evaluate serial ultrasound examinations combined with patient instructions in achieving timely detection of TTTS in a cohort of monochorionic diamniotic twin pregnancies, and to study the effects of TTTS and fetoscopic laser coagulation of the placental anastomoses on fetal hemodynamics of monochorionic twins. Chapter 1 contains a review of the literature on ultrasound examination in monochorionic twins and twin-to-twin transfusion syndrome during gestation. In part 1 of this chapter, the importance of first-trimester ultrasound examination to diagnose chorionicity is discussed in detail. To assess chorionicity, the intertwin membrane should be imaged at its insertion site to the placental mass. A lambda (_)-, __Y__- or twin peak sign indicates dichorionicity, whereas a __T__ sign must be visualized in monochorionic diamniotic twin pregnancies. The observation of two separate placentas alone is not sufficient to diagnose dichorionicity. A single placental mass does not prove monochorionicity. Thickness of the intertwin membrane and fetal gender are not considered reliable indicators of chorionicity. The complications of monochorionic twinning, such as single intrauterine fetal death, intrauterine growth restriction, discordant fetal anomalies, and TTTS, are outlined in short. Part 2 is focused on ultrasound and TTTS. Current insights in the pathophysiology, diagnosis, treatment and outcome are reviewed. Sonographic markers early in pregnancy that could forecast the development of TTTS are described, such as increased nuchal translucency, abnormal Doppler studies of the ductus venosus, folding of the intertwin membrane, and the sonographic absence of arterioarterial anastomoses. Furthermore, an overview of the most important Doppler studies in TTTS is supplied. Pathological Doppler studies in the donor are consistent with decreased venous return due to hypovolemia and increased cardiac afterload due to increased placental resistance. Pathological Doppler studies in the recipient are caused by congestive heart failure due to hypervolemia. Fetoscopic laser ablation of the placental anastomoses in TTTS affects the fetal and fetoplacental circulation in various ways, such as transient volume overload in donors and improvement of cardiac function in recipients, resulting in changed Doppler studies after therapy. Finally, the fetal heart in TTTS is discussed. Particularly recipients may be affected by prenatal cardiac failure. Donors show no or little cardiac pathology. The exact cause of cardiac dysfunction is unclear, however, primary cardiac pathology, increased preload, or increased afterload are suggested to play a role. In conclusion, most twin pregnancies have an uneventful course, although twins are at greater risk than singletons, particularly those that are monochorionic. TTTS is the most severe complication during gestation. TTTS is diagnosed sonographically, and that is why ultrasound examination is an essential tool in prenatal care for monochorionic twins. In chapter 2 we undertook a study to report the occurrence of bipartite monochorionic twin placentas. Examination of 109 monochorionic placentas delivered at our institution between June 2002 and June 2005 was performed. Placental characteristics on prenatal ultrasound were studied, including single or double appearance and type of intertwin membrane-placental junction (__T__ sign or lambda sign). Monochorionicity was confirmed by postnatal histologic confirmation (diamniotic intertwin membrane without chorionic tissue within the dividing septum). Bipartition was diagnosed when two separate placental masses attached by membranes were identified. Of the 109 monochorionic placentas, three were composed of two separate placental masses. Prenatal ultrasound examination showed two separate placental masses in each case. Monochorionicity was suspected on prenatal ultrasound due to the presence of __T__ sign in two cases and TTTS in another case. Microscopic examination of the dividing septum was consistent with monochorionicity in each case. Vascular anastomoses were present in two of the three placentas, and led in both cases to the development of TTTS. We concluded that two separate placental masses in twin pregnancies are not per se dichorionic and may occur in almost 3% of monochorionic placentas. Second-trimester twin-to-twin transfusion is well known, but first-trimester cases have been rarely described. In chapter 3 we present the case of a monochorionic twin at 11+0 weeks of gestation with single increased nuchal translucency and normal karyotypes. At 12+5 weeks of gestation, double intrauterine death was diagnosed, followed by delivery of a strikingly red and white fetus. In conclusion, TTTS can be seen in various ways at different gestational ages. Besides the well-known risks of severe second-trimester TTTS, we believe that TTTS can cause fetal death or neurological damage, even in the first trimester of pregnancy. The only presenting symptom may be a single increased nuchal translucency. In chapter 4 we assessed the value of serial ultrasound examinations together with patient instructions to report the onset of symptoms in achieving timely detection of TTTS in a cohort of monochorionic diamniotic twin pregnancies, and to evaluate sonographic TTTS predictors. Timely detection of TTTS was defined as diagnosis before severe complications of TTTS occurred, such as preterm prelabor rupture of membranes, very preterm delivery (24-32 weeks of pregnancy), fetal hydrops, or intrauterine fetal death. During a two-year period, a prospective series of 23 monochorionic twin pregnancies was monitored from the first trimester until delivery. At least every two weeks we performed ultrasound and Doppler measurements (nuchal translucency thickness, presence of membrane folding, estimated fetal weight, deepest vertical pocket, bladder filling, and Doppler waveforms of the umbilical artery, ductus venosus, and umbilical vein). Measurements of TTTS cases were compared to those of non-TTTS cases matched for gestational age. Furthermore, patients were informed about the symptoms caused by TTTS, and instructed to consult us immediately in case of rapidly increasing abdominal size or premature contractions. In all four TTTS cases, the diagnosis was timely. At the time of diagnosis, one case was at Quintero Stage 1, two at Quintero Stage 2, and one at Quintero Stage 3. Two of the TTTS cases became apparent after the patients__ feeling of rapidly increasing girth. The identification of TTTS predictors was successful with respect to one parameter: isolated polyhydramnios in one sac, without oligohydramnios in the other, preceded the ultimate diagnosis of TTTS in two of the four TTTS cases. All other ultrasound measurements of TTTS cases, prior to the diagnosis of TTTS, were within the range of measurements of non-TTTS cases. We concluded that biweekly ultrasound examinations, with special attention to amniotic fluid compartments of both fetuses, combined with detailed patient instructions to report the onset of symptoms resulted in timely diagnosis of all TTTS cases and appears to be a safe program for monitoring monochorionic twin pregnancies. In chapter 5 we investigated fetal hemodynamics in monochorionic twins with TTTS before and after fetoscopic laser therapy, focusing on the renal and cerebral blood flow. In a prospective study, we performed Doppler studies in monochorionic twin pregnancies with TTTS. The pulsatility index (PI) and end-diastolic flow (EDF) of the umbilical artery (UA) (recorded as present, absent or reversed); the PI and the peak systolic velocity of the middle cerebral artery (MCA PSV); the maximum flow velocity (V max) and flow pattern of the intrahepatic part of the umbilical vein (UV) (classified as pulsatile or non-pulsatile); the pulsatility index for veins (PIV) and A-wave of the ductus venosus (DV) (recorded as present, absent or reversed); and the PI and PSV of the renal artery (RA) were measured within 24 h before, 12 to 24 h and 4 to 10 days after laser therapy. At each examination, the presence or absence of tricuspid regurgitation (TR) and of hydropic signs (pleural effusion, ascites, pericardial effusion, or skin edema) was recorded. Hemoglobin values and reticulocyte counts were determined at birth. Long-term follow-up was assessed at the age of 2 years. In donor twins (n=34), DV PIV increased significantly 12 to 24 h after laser therapy, however returned to pre-operative values within 4 to 10 days. A significant decrease in UA PI and increase in UV V max was detected after laser treatment. Twenty percent (6/30) showed signs of TR 12 to 24 h after laser therapy, which was resolved completely after 4 to 10 days. The MCA PI and RA PI were significantly decreased 12 to 24 h after laser treatment, however returned to pre-operative values within 4 to 10 days. MCA and RA PSV values were unchanged by fetoscopic laser therapy. In recipient twins (n=32), DV PIV decreased significantly 4 to 10 days after laser therapy. The RA PI increased non-significantly after laser treatment; RA PSV values were unchanged. MCA PI and MCA PSV values increased significantly after laser therapy. After birth, mean hemoglobin values of donors (17.3 _ 4.9 g_/dL) and recipients (16.1 _ 4.2 g_/dL) were comparable (p=0.43). At the age of 2 years, neurodevelopmental impairment was diagnosed in 15% (4/26) of donors and in 10% (2/21) of recipients and was not related to abnormal MCA flow. None of the children suffered from chronic renal failure. We concluded that fetoscopic laser ablation of the placental anastomoses in TTTS affects the fetal and fetoplacental circulation in various ways, such as transient volume overload in donors and improvement of cardiac function in recipients. Cerebral and renal flow changes occur after laser therapy. Whether these are permanent or temporarily fetal adaptations needs further investigation with prolonged follow-up. In our studies, the changes found were not associated with long-term neurological or renal sequelae. In chapter 6 the influence of fetoscopic laser therapy on fetal cardiac size in monochorionic twins complicated by TTTS was evaluated. In a longitudinal, prospective study, we assessed sonographically the fetal cardiac size in monochorionic diamniotic twins with TTTS treated with laser therapy and in monochorionic twins without TTTS. The fetal cardiothoracic ratio (cardiac circumference divided by thoracic circumference) of TTTS twins was determined within 24 h before, 12 to 24 h after and 1 week after laser treatment, and from then on every 2 to 4 weeks until birth. TTTS twins were classified at Quintero Stage 1-2 (n=18) and Stage 3-4 (n=16) and measurements were compared to biweekly measurements of non-TTTS monochorionic twins matched for gestational age (n=38). Cardiomegaly was defined as a cardiothoracic ratio above the 97.5th percentile. Before laser treatment, cardiomegaly was observed in 44% (8/18) and in 50% (8/16) of recipients at Quintero Stage 1-2 and Stage 3-4, respectively. Cardiomegaly occurred in none of the donors before treatment. After laser treatment, cardiomegaly was observed in 76% (13/17) and 50% (7/14) of recipients at Stage 1-2 and Stage 3-4, respectively. Cardiomegaly was found in 17% (3/18) and 13% (2/15) of donors at Stage 1-2 and Stage 3-4, respectively. Non-TTTS monochorionic twins and singletons showed cardiomegaly in 18% (7/38) and 8% (2/25). After laser therapy, cardiothoracic ratios of recipients at Stage 1-2 and Stage 3-4 were not significantly changed (p=0.34 and 0.67, respectively). Cardiothoracic ratios of donors at Stage 1-2 and Stage 3-4 were increased compared to their cardiothoracic ratios before laser therapy (p-values 0.0002 and 0.005, respectively). Cardiothoracic ratios of non-TTTS monochorionic twins were not significantly different from our reference range in singletons throughout gestation, and were smaller as compared to both recipients and donors after laser therapy. It was concluded that recipients show cardiomegaly both before as well as after fetoscopic laser therapy for TTTS. Donors develop cardiomegaly only after laser treatment for TTTS. Our findings emphasize the significant effect of TTTS and fetoscopic laser therapy on the fetal hearts of both recipient and donor twins. In chapter 7 we compared fetal cardiac output (CO) in donor and recipient twins of TTTS pregnancies after fetoscopic laser therapy to monochorionic twins without TTTS and to normal singletons. In a longitudinal, prospective study, we sonographically assessed fetal CO in donors (n=10) and recipients (n=10) with TTTS after fetoscopic laser therapy, in monochorionic twins without TTTS (n=20) and in 20 normal singleton pregnancies. The fetal CO of TTTS twins was determined 1 day and 1 week after laser treatment, and from then on every 2 to 4 weeks until birth. Twins without TTTS were examined biweekly until birth. Singletons were examined twice with an 8-week interval at different gestational ages between 17 and 35 weeks. Absolute CO increased exponentially with advancing gestational age (p<0.001), and was significantly related to fetal weight for all groups (p<0.0001). The median CO/kg in donors after laser therapy, recipients after laser therapy, and non-TTTS monochorionic twins was significantly higher compared to singletons (all p-values <0.001). Median CO/kg in donors after laser therapy, recipients after laser therapy, and non-TTTS monochorionic twins was not significantly different from each other. It was concluded that monochorionic twins with TTTS have an increased CO/kg after laser treatment as compared to normal singletons. These results may be of importance in view of the increasing awareness of fetal origins of adult disease. In conclusion, knowledge about monochorionic twinning and its complications such as TTTS is crucial for clinicians participating in the care of pregnant women and for children born as monochorionic twins. With the studies described in this thesis, we aimed at designing a framework that is helpful in providing high quality prenatal care for monochorionic twins. A first-trimester scan to establish chorionicity is vital and should be followed by biweekly ultrasound examinations and patient instructions. Specific __guidelines__ that may be used both before and after fetoscopic laser treatment for TTTS are provided in the recommendations for clinical practice. We hope that the studies presented in this thesis will contribute to increased awareness of the potential problems and optimization of management of this unique subset of pregnancies: the monochorionic twins. Show less
