Humphrey H.H. Kanhai

Isolation of Mesenchymal Stem Cells of Fetal or Maternal Origin from Human Placenta

Recently we reported that second‐trimester amniotic fluid (AF) is an abundant source of fetal mesenchymal stem cells (MSCs). In this study, we analyze the origin of these MSCs and the presence of MSCs in human‐term AF. In addition, different parts of the human placenta were studied for the presence of either fetal or maternal MSCs. We compared the phenotype and growth characteristics of MSCs derived from AF and placenta.

Nonexpanded primary lung and bone marrow-derived mesenchymal cells promote the engraftment of umbilical cord blood-derived CD34 + cells in NOD/SCID mice

OBJECTIVE Previously, we have found that human culture-expanded fetal lung-derived mesenchymal stem cells (MSC) promote the engraftment of umbilical cord blood (UCB)-derived CD34((+)) cells. The high frequency of MSC in fetal lung allowed us to study whether this represented a biological feature of these cells or a property that was acquired during expansion in culture. MATERIALS AND METHODS Irradiated NOD/SCID mice (n=80) were transplanted with 0.1x10(6) UCB CD34(+) cells in the presence or absence of 10(6) primary nonexpanded or culture-expanded fetal lung, liver, or BM CD45(-) cells, or with nonexpanded fetal lung liver or BM CD45(-) cells only. RESULTS In comparison with transplantation of UCB CD34(+) cells only, cotransplantation of UCB CD34(+) cells and primary fetal lung or BM CD45(-) cells resulted in a significantly higher level of engraftment (% hCD45(+) cells) in BM, PB, and spleen. In addition, primary mesenchymal cells derived from adult BM had a similar promoting effect. The engraftment-enhancing effect was similar to that of culture-expanded fetal lung and BM MSC. Primary mesenchymal cells, but not culture-expanded MSC, were detected in recipient mice, suggesting that the primary cells were able to home and that this capacity was lost after expansion. CONCLUSIONS These results show that primary mesenchymal cells from fetal lung and BM promote the engraftment of UCB-derived CD34(+) cells to a similar degree as culture-expanded MSC, indicating that it reflects a biological property of primary MSC that is preserved during expansion in culture.

Preeclampsia and genetic risk factors for thrombosis: A case-control study

OBJECTIVE Recently, it has been proposed that hereditary coagulation abnormalities leading to an increased venous thrombosis risk may play a role in the development of preeclampsia. We tested this hypothesis in women who have had preeclampsia compared with matched control subjects. STUDY DESIGN We conducted a case-control study of 163 women with preeclampsia during 1991-1996. Control subjects were matched for age and delivery date. Patients and control subjects were tested for the presence of factor V Leiden, prothrombin 20210A allele, protein C, protein S, and antithrombin deficiency. Logistic regression methods were used for data analysis. RESULTS The prevalence of these genetic risk factors was similar in the patient group (12.9%) and the control group (12.9%; odds ratio, 1.0; 95% confidence interval, 0.5-3.9). Unexpectedly, we found a high prevalence of factor V Leiden in the control group (9.2%). CONCLUSION We found no differences in the prevalence of genetic risk factors of thrombosis in women with preeclampsia compared with control subjects.

Fetoscopic Laser Surgery in 100 Pregnancies with Severe Twin-to-Twin Transfusion Syndrome in the Netherlands

Objective: In this prospective cohort study, we evaluated the initial results of fetoscopic laser surgery for severe second trimester twin-to-twin transfusion syndrome (TTTS) treated at our centre. Method: A total of 100 consecutive pregnancies with severe second trimester TTTS treated at our centre with selective fetoscopic laser coagulation of vascular anastomoses on the placental surface between August 2000 and November 2004 were included in the study. Perinatal survival was analysed in relation to Quintero stage. Results: Median gestational age was 20 weeks at fetoscopy (range: 16–26) and 33 weeks at delivery (range: 18–40). Perinatal survival rate was 70% (139/200). The treatment resulted in at least one survivor at the age of 4 weeks in 81% of pregnancies. Perinatal survival was significantly higher when treatment was performed in the early Quintero stages (95% in stage 1, 76% in stage 2, 70% in stage 3, 50% in stage 4) (p = 0.02). Conclusion: Results of fetoscopic laser surgery for TTTS in our centre are similar to those in specialised centres in other countries. Diagnosis and treatment in the early Quintero stages resulted in significantly higher perinatal survival.

Long-term neurodevelopmental outcome after intrauterine transfusion for hemolytic disease of the fetus/newborn: the LOTUS study

OBJECTIVE To determine the incidence and risk factors for neurodevelopmental impairment (NDI) in children with hemolytic disease of the fetus/newborn treated with intrauterine transfusion (IUT). STUDY DESIGN Neurodevelopmental outcome in children at least 2 years of age was assessed using standardized tests, including the Bayley Scales of Infant Development, the Wechsler Preschool and Primary Scale of Intelligence, and the Wechsler Intelligence Scale for Children, according to the childrens age. Primary outcome was the incidence of neurodevelopmental impairment defined as at least one of the following: cerebral palsy, severe developmental delay, bilateral deafness, and/or blindness. RESULTS A total of 291 children were evaluated at a median age of 8.2 years (range, 2-17 years). Cerebral palsy was detected in 6 (2.1%) children, severe developmental delay in 9 (3.1%) children, and bilateral deafness in 3 (1.0%) children. The overall incidence of neurodevelopmental impairment was 4.8% (14/291). In a multivariate regression analysis including only preoperative risk factors, severe hydrops was independently associated with neurodevelopmental impairment (odds ratio, 11.2; 95% confidence interval, 1.7-92.7). CONCLUSION Incidence of neurodevelopmental impairment in children treated with intrauterine transfusion for fetal alloimmune anemia is low (4.8%). Prevention of fetal hydrops, the strongest preoperative predictor for impaired neurodevelopment, by timely detection, referral and treatment may improve long-term outcome.

Differential immunomodulatory effects of fetal versus maternal multipotent stromal cells.

Protective mechanisms are likely to be present at the fetomaternal interface because fetus-specific alloreactive T cells present in the decidua do not harm the fetus. We tested the immunosuppressive capacity of maternal and fetal multipotent stromal cells (MSC). Single cell suspensions were made from second-trimester amnion, amniotic fluid, and decidua. Culture-expanded cells were identified as MSC based on phenotype and multilineage potential. Coculture of MSC in a primary mixed lymphocyte culture of unrelated responder-stimulator combinations resulted in a dose-dependent inhibition of proliferation. Fetal MSC demonstrated a significantly higher inhibition compared with maternal MSC. This stronger inhibition by fetal MSC was even more prominent in a secondary mixed lymphocyte reaction (MLR) with primed alloreactive T cells. Analysis of cytokine production revealed that fetal MSC produced significantly more interleukin (IL)-10 and vascular endothelial growth factor than maternal MSC. Cell-cell contact is needed for part of the inhibitory effects of MSC. In addition, soluble factors play a role because blocking experiments with anti-IL-10 revealed that the inhibition of the MLR response by fetal MSC is mainly mediated by IL-10. For maternal MSC, other soluble factors seem to be involved. Fetal MSC derived from the fetomaternal interface have a stronger inhibitory effect on naive and antigen-experienced T cells compared with maternal MSC, which is probably related to their higher IL-10 production.

Benefits and risks of fetal red-cell transfusion after 32 weeks gestation

OBJECTIVE To compare the outcome after intrauterine transfusion (IUT) between fetuses treated before and those treated after 32 weeks gestation. SETTING National referral center for intrauterine treatment of red-cell alloimmunization in The Netherlands. STUDY DESIGN Retrospective evaluation of an 11 year period, during which 209 fetuses were treated for alloimmune hemolytic disease with 609 red-cell IUTs. We compared fetal and neonatal outcome in three groups: fetuses only treated before 32 weeks gestation (group A, n=46), those treated both before and after 32 weeks (group B, n=117), and those where IUT was started at or after 32 weeks (group C, n=46). RESULTS Survival rate was 48% in group A, 100% in group B, and 91% in group C. Moreover, fetuses in group A were hydropic significantly more often. Short-term perinatal loss rate after IUT was 3.4% in the 409 procedures performed before 32 weeks and 1.0% in the 200 procedures performed after 32 weeks gestation. CONCLUSION Perinatal losses were much more common in fetuses only treated before 32 weeks gestation. Two procedure-related perinatal losses in 200 IUT after 32 weeks remain a matter of concern because of the good prospects of alternative extrauterine treatment.

Ultrasonographic fetal spleen measurements in red blood cell-alloimmunized pregnancies

OBJECTIVES This study was performed to evaluate the possible relationship between fetal spleen size and fetal hemoglobin levels and to assess the predictive value of ultrasonographically measured fetal spleen size as an estimate of the severity of fetal hemolytic anemia. STUDY DESIGN Before 85 consecutive fetal blood samples in 28 red blood cell-alloimmunized pregnancies ultrasonographic fetal spleen measurements were performed. Results were compared with our own longitudinally derived reference ranges and were correlated with fetal hemoglobin deficit. RESULTS A significant positive correlation was found between spleen perimeter and fetal hemoglobin deficit. The ultrasonographic finding of splenomegaly correctly predicted severe fetal anemia (hemoglobin deficit > 5 SD from normal mean) in 44 of 47 cases, a positive predictive value of 94%. At first transfusion all fetuses showing splenomegaly were severely anemic. CONCLUSION Fetal spleen measurements may be a useful adjunct to ultrasonographic evaluation in the management of severe red blood cell-alloimmunized pregnancies.

Fetal intracranial haemorrhages caused by fetal and neonatal alloimmune thrombocytopenia: an observational cohort study of 43 cases from an international multicentre registry

Objective To characterise pregnancies where the fetus or neonate was diagnosed with fetal and neonatal alloimmune thrombocytopenia (FNAIT) and suffered from intracranial haemorrhage (ICH), with special focus on time of bleeding onset. Design Observational cohort study of all recorded cases of ICH caused by FNAIT from the international No IntraCranial Haemorrhage (NOICH) registry during the period 2001–2010. Setting 13 tertiary referral centres from nine countries across the world. Participants 37 mothers and 43 children of FNAIT pregnancies complicated by fetal or neonatal ICH identified from the NOICH registry was included if FNAIT diagnosis and ICH was confirmed. Primary and secondary outcome measures Gestational age at onset of ICH, type of ICH and clinical outcome of ICH were the primary outcome measures. General maternal and neonatal characteristics of pregnancies complicated by fetal/neonatal ICH were secondary outcome measures. Results From a total of 592 FNAIT cases in the registry, 43 confirmed cases of ICH due to FNAIT were included in the study. The majority of bleedings (23/43, 54%) occurred before 28 gestational weeks and often affected the first born child (27/43, 63%). One-third (35%) of the children died within 4 days after delivery. 23 (53%) children survived with severe neurological disabilities and only 5 (12%) were alive and well at time of discharge. Antenatal treatment was not given in most (91%) cases of fetal/neonatal ICH. Conclusions ICH caused by FNAIT often occurs during second trimester and the clinical outcome is poor. In order to prevent ICH caused by FNAIT, at-risk pregnancies must be identified and prevention and/or interventions should start early in the second trimester.

Differential distribution of NK cells in decidua basalis compared with decidua parietalis after uncomplicated human term pregnancy

As pregnancy progresses, a characteristic decline in the percentage of CD56bright CD16- uterine natural killer (NK) cells occurs. Studies of term decidua, however, have focused only on leukocytes derived from decidua basalis, the site of implantation. The decidua parietalis, lining the remainder of the uterine cavity is another important region of the maternal-fetal interface that forms contact with fetal tissue at the end of the first trimester. The aim of this study was to evaluate possible differences in expression of CD16 and CD56 on leukocytes from normal term decidua basalis and decidua parietalis. Decidua basalis and parietalis samples were obtained from 30 placentas collected after elective cesarean section. Percentages of leukocyte subpopulations and NK cell subsets within the CD45+ cell fraction were determined by flow cytometry. In six decidual samples, concurrent immunohistochemical staining was performed. Higher percentages of CD56dim CD16+ NK cells and CD56- CD16+ cells were found in decidua basalis in comparison to decidua parietalis. In contrast, the percentage of CD56bright CD16- uterine NK cells was significantly higher in decidua parietalis. Immunohistochemical quantification supported flow cytometric results. We conclude that significant differences exist with respect to the distribution of NK cells in term decidua basalis and parietalis. Future functional studies may improve our understanding of their role at the maternal-fetal interface.