Showing papers by "Jan Deprest published in 2013"

Predictors of neonatal morbidity in fetuses with severe isolated congenital diaphragmatic hernia undergoing fetoscopic tracheal occlusion

Abstract: Objectives To investigate neonatal morbidity in fetuses with severe congenital diaphragmatic hernia (CDH) treated with fetoscopic endoluminal tracheal occlusion (FETO) and compare it with historical controls with less severe forms of CDH that were managed expectantly Methods This was a prospective, multicenter study on neonatal outcomes and prenatal predictors in 90 FETO survivors (78 left-sided, 12 right) and 41 controls from the antenatal CDH registry with either severe or moderate hypoplasia who were managed expectantly We also investigated early neonatal morbidity indicators, including the need for patch repair, duration of mechanical ventilation and supplemental oxygen, age at full enteral feeding and incidence of pulmonary hypertension Results Gestational age at delivery was predictive of duration of assisted ventilation (P = 0046), days on supplemental oxygen (P = 0019) and age at full enteral feeding (P = 0020) When delivery took place after 34 weeks' gestation, neonatal morbidity of FETO cases was comparable with that of expectantly managed cases with moderate hypoplasia Conclusions Fetal intervention for severe CDH is associated with neonatal morbidity that is comparable with that of an expectantly managed group with less severe disease Copyright © 2013 ISUOG Published by John Wiley & Sons Ltd

Placenta as a reservoir of stem cells: an underutilized resource?

Abstract: Introduction Both embryonic and adult tissues are sources of stem cells with therapeutic potential but with some limitations in the clinical practice such as ethical considerations, difficulty in obtaining and tumorigenicity. As an alternative, the placenta is a foetal tissue that can be obtained during gestation and at term, and it represents a reservoir of stem cells with various potential. Sources of data We reviewed the relevant literature concerning the main stem cells that populate the placenta. Areas of agreement Recently, the placenta has become useful source of stem cells that offer advantages in terms of proliferation and plasticity when compared with adult cells and permit to overcome the ethical and safety concern inherent in embryonic stem cells. In addition, the placenta has the advantage of containing epithelia, haematopoietic and mesenchymal stem cells. These stem cells possess immunosuppressive properties and have the capacity of suppress in vivo inflammatory responses. Areas of controversy Some studies describe a subpopulation of placenta stem cells expressing pluripotency markers, but for other studies, it is not clear whether pluripotent stem cells are present during gestation beyond the first few weeks. Particularly, the expression of some pluripotency markers such as SSEA-3, TRA-1-60 and TRA-1-81 has been reported by us, but not by others. Growing points Placenta stem cells could be of great importance after delivery for banking for autologous and allogeneic applications. The beneficial effects of these cells may be due to secretion of bioactive molecules that act through paracrine actions promoting beneficial effects. Areas timely for developing research Understanding the role of placenta stem cells during pregnancy and their paracrine actions could help in the study of some diseases that affect the placenta during pregnancy.

Routine clonal expansion of mesenchymal stem cells derived from amniotic fluid for perinatal applications

Abstract: Introduction Stem cells (SCs) isolated from amniotic fluid (AF) are a promising source for autologous perinatal cell therapy. The aim of this study was to develop a routine isolation, selection, and expansion protocol of clonal SC lines from redundant clinical amniocentesis samples. Materials and methods Amniotic fluids were collected between 15 and 22 weeks of gestation, and SCs were isolated by CD117-based and mechanical selection protocols. SCs were characterized by mesenchymal SC marker expression and differentiation protocols. Cells were manipulated with a lentiviral vector system expressing the β-galactosidase reporter gene and were injected into immunodeficient newborn mouse pups. Qualitative assessment was performed to detect the infused cells after 1 week. Results A total of 78 clonal AF SC populations were successfully isolated by mechanical selection from 21 consecutive amniocentesis samples. They were positive for mesenchymal SC cluster of differentiation markers and could be differentiated into the different lineages. SCs were stably labeled using β-galactosidase and were detected in the lungs and hearts of the neonatal mice. Conclusion We demonstrate that mesenchymal SCs can be routinely isolated and clonally expanded from mid-gestation human AF using mechanical isolation. They can easily be transduced and be tested for perinatal treatment in animal models. © 2013 John Wiley & Sons, Ltd.

Identification of dosage‐sensitive genes in fetuses referred with severe isolated congenital diaphragmatic hernia

Abstract: Objective Congenital diaphragmatic hernia (CDH) is a fetal abnormality affecting diaphragm and lung development with a high mortality rate despite advances in fetal and neonatal therapy. CDH may occur either as an isolated defect or in syndromic form for which the prognosis is worse. Although conventional karyotyping and, more recently, chromosomal microarrays support a substantial role for genetic factors, causal genes responsible for isolated CDH remain elusive. We propose that chromosomal microarray analysis will identify copy number variations (CNVs) associated with isolated CDH. Methods We perform a prospective genome-wide screen for CNVs using chromosomal microarrays on 75 fetuses referred with apparently isolated CDH, six of which were later reclassified as non-isolated CDH. Results The results pinpoint haploinsufficiency of NR2F2 as a cause of CDH and cardiovascular malformations. In addition, the 15q25.2 and 16p11.2 recurrent microdeletions are associated with isolated CDH. By using gene prioritisation and network analysis, we provide strong evidence for several novel dosage-sensitive candidate genes associated with CDH. Conclusions Chromosomal microarray analysis detects submicroscopic CNVs associated with isolated CDH or CDH with cardiovascular malformations. © 2013 John Wiley & Sons, Ltd.

Is general anaesthesia for caesarean section associated with postpartum haemorrhage? Systematic review and meta-analysis.

Abstract: Background: Postpartum haemorrhage (PPH) remains a leading cause of maternal morbidity and mortality. The odds for PPH were about eight times higher following general anaesthesia compared with neuraxial anaesthesia in a recent retrospective study. We aimed to conduct a systematic review and metaanalysis of the effect of type of anaesthesia (general vs. neuraxial) on estimated blood loss and transfusion requirements after caesarean section. Methods: A systematic literature search was performed. The quality of eligible reports was assessed using the Oxford Quality Scale. Data were subjected to meta-analysis using the random effects model. Results: The search identified 18 articles including 12,330 parturients. Meta-analysis of randomised controlled trials (RCTs) found a significant difference in blood loss, favouring neuraxial anaesthesia (spinal and epidural) when comparing with general anaesthesia {weighted mean difference −106.11 ml [95% confidence interval (CI) −209.80, −2.42 ml], P = 0.04}. In further analyses, we found a significant difference after epidural compared with general anaesthesia but not after spinal compared with general anaesthesia. Based on RCTs, there was no significantly increased risk of blood transfusions with general anaesthesia.Analysis of non-randomised studies found a significantly higher transfusion requirement after general anaesthesia (risk ratio 5.06, 95% CI 2.47‐10.36, P < 0.00001). The incidence of hypotension and the amount of fluid given were higher in the neuraxial anaesthesia groups. Conclusion: General anaesthesia is associated with a higher blood loss than neuraxial anaesthesia. However, based on highquality studies, the need for blood transfusion was not greater. The higher blood loss with general anaesthesia is therefore of uncertain clinical relevance.

Amniopatch procedure after previable iatrogenic rupture of the membranes: a two-center review

Abstract: Objective This study aimed to analyze success rates and pregnancy outcomes of amniopatch procedures for previable iatrogenic preterm prelabour rupture of the membranes (PPROM) with associated oligohydramnios. Methods Retrospective analysis of amniopatch procedures performed at the University Hospitals Leuven, Belgium, and the Mount Sinai Hospital Toronto, Canada. Cases were analyzed overall and in two sub-groups: PPROM after a needle-based procedure (NP group, n = 13) or after fetoscopic intervention (FI group, n = 11). Complete technical success was defined as cessation of leakage and normalization of amniotic fluid volume, partial success as cessation of leakage, or re-establishment of volume. Further outcomes were pregnancy duration and outcome, fetal/neonatal morbidity and mortality, and maternal morbidity. Results Gestational age at amniopatch was comparable in both groups (NP: 20.1, FI: 21.0 weeks). Amniopatch was completely and partially successful in 29% (NP: 31%; FI: 27%) and 29% (NP: 15%; FI: 45%), respectively. Mean gestational age at delivery was 27.5 weeks (NP: 25.5; FI: 29.4 weeks). Overall neonatal survival was 17/31 (55%) (NP: 4/13 (31%), FI: 13/18 (72%); p = .02). Chorioamnionitis occurred in three cases, two associated with maternal sepsis. Severe neonatal morbidity occurred in two survivors. Conclusion Amniopatch for iPPROM was successful in 58%, with an overall live birth rate of 68% and survival to discharge of 55%. © 2013 John Wiley & Sons, Ltd.

Antireflux surgery after congenital diaphragmatic hernia repair: a plea for a tailored approach.

Abstract: OBJECTIVES: Preventive antireflux surgery (ARS) at the moment of congenital diaphragmatic hernia (CDH) repair has been suggestedby some authors, particularly in subgroups with a liver herniated in the chest or patch requirement. We evaluated the incidence andassociated factors of gastro-oesophageal reflux disease (GERD) and the need for subsequent ARS in our CDH patients.METHODS: We retrospectively reviewed our CDH database. Demographics, prenatal assessment of severity, prenatal treatment, type ofrepair, intraoperative findings and incidences of gastro-oesophageal reflux and ARS were recorded.RESULTS: CDH repair was performed in 77 infants between July 1993 and November 2009. Eight died after repair. Seven were lost tofollow-up. The median follow-up was 4.0 (0.16–14.88) years. Fourteen of these 62 patients were prenatally treated with fetoscopicendoluminal tracheal occlusion (FETO) because of severe pulmonary hypoplasia. After CDH repair, GERD was diagnosed in 31 patients.In all of them, medical antireflux treatment was started. Thirteen (42%) patients needed ARS at a median age of 64 (37–264) days. Oneyear after starting medical treatment, 14 (45%) patients were completely off antireflux medication. In CDH subgroups with patch repair,liver herniated in the chest or previous FETO, the incidences of gastro-oesophageal reflux and ARS were 61 and 32%, 73 and 38% and71 and 43%, respectively. Univariable analysis of associated potentially predisposing factors shows that patch repair, liver herniated inthe chest, pulmonary hypertension, high-frequency oscillatory ventilation and FETO are associated with subsequent ARS. On multivari-able analysis, liver herniated in the chest was the only independent predictor for both gastro-oesophageal reflux and ARS.CONCLUSIONS: Of all CDH patients, 50% developed gastro-oesophageal reflux and 21% required ARS. For both, liver in the chest wasthe only independent predictor. Routine ARS in certain subgroups at the time of CDH repair seems not to be justified. Foetal endolum-inal tracheal occlusion creates a new cohort of survivors with an increased risk for undergoing ARS. The surgical group, in particular,reflects a more complex gastro-oesophageal reflux physiopathology.Keywords: Gastro-oesophageal reflux † Congenital diaphragmatic hernia † Fundoplication

Preoperative prediction of the individualized risk of early fetal death after laser therapy in twin-to-twin transfusion syndrome.

Abstract: The aim of this study is to evaluate the independent and combined value of gestational age, fetal weight, fetoplacental Doppler, and myocardial performance index for the prediction of individual risk of early ( 1.5 MoM (OR = 22, p = 0.015), but this event was present in only 3% of recipients. In donors, reverse end diastolic flow in the umbilical artery (OR = 14.748, p = 0.033), estimated fetal weight discordance (OR = 1.054, p = 0.036), and gestational age (OR = 0.757, p = 0.046) were independent predictors.In TTTS, preoperative fetal assessment can identify independent risk factors for early post-operative IUFD, particularly in donors. © 2013 John Wiley& Sons, Ltd.

Gestational age-specific reference ranges for amniotic fluid assessment in monochorionic diamniotic twin pregnancies

Abstract: Objectives To establish gestational age-specific reference ranges for amniotic fluid measurements in monochorionic diamniotic twin pregnancies, to compare them with previously reported singleton and twin reference ranges and to examine the rationale for using a gestational age-dependent cut-off to define polyhydramnios in twin–twin transfusion syndrome, as is the practice in most European centers. Methods We retrospectively evaluated amniotic fluid volume in 32 monochorionic diamniotic twin pregnancies that were followed longitudinally at 2-week intervals from the first trimester until birth. Amniotic fluid volume was assessed by measuring the deepest vertical pocket in both amniotic sacs. We used multilevel modeling to estimate the gestational age-specific reference ranges for deepest vertical pocket measurements. Results Based on 429 observations in 64 fetuses, we constructed gestational age-specific reference ranges from 11 weeks until term. The deepest pocket increased from the first trimester to reach a maximum at 26 weeks, followed by a gradual decrease towards term. Measurements between 18 and 28 weeks were comparable to those in singleton pregnancies. However, before 18 weeks values were higher, whereas after 28 weeks they were lower, as compared to singleton references. Conclusion In monochorionic twin pregnancies, the deepest vertical pocket is a gestational age-dependent measurement. Therefore, a gestational age-dependent definition of polyhydramnios in twin–twin transfusion syndrome, as used by most European centers, seems a logical approach. Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.

A morphometric study of the human fetal heart on post-mortem 3-tesla magnetic resonance imaging

Abstract: Objective To report on the feasibility of assessing cardiac structures on post-mortem 3-tesla MRI (pmMRI) and to provide morphometric data in fetuses without cardiac abnormalities. Methods Retrospective single center study on 3T pmMRI of 39 consecutive fetuses without cardiac abnormalities (13–38weeks of gestation). Fetal cardiac anatomy was assessed and measurements of cardiac structures were performed on T2-weighted 3D multiplanar reconstructed images. Linear regression analysis was performed to examine changes of cardiac dimensions during gestation. Results The four-chamber view of the fetal heart could be obtained and the measurements of cardiac chambers and ventricular walls could be performed in all 39 cases. The aorta and the pulmonary artery were visualized and their diameters were measured in 38 (97.4%) fetuses, ductus arteriosus in 32 (82%). All measurements showed strong linear correlation with gestational age. The relationship of the diameters of the pulmonary artery, aorta, and ductus arteriosus remained constant over pregnancy. All these observations are consistent with what is known from prenatal ultrasound. Conclusions The present study proves the feasibility of visualizing normal cardiac structures on 3-tesla pmMRI in fetuses beyond 14weeks. We provide morphometric data that may enable diagnostic evaluation of cardiac abnormalities on pmMRI. © 2013 John Wiley & Sons, Ltd.

Concerning the timing of antibiotic administration in women undergoing caesarean section: a systematic review and meta-analysis.

Abstract: Objective To assess the effects on maternal infectious morbidity and neonatal outcomes of the timing of antibiotic prophylaxis in women undergoing caesarean section. A recent National Institute for Health and Clinical Excellence (NICE) guideline reported that antibiotic administration before skin incision reduces the risk of maternal infection; this recommendation was based on a meta-analysis, however one including trials that were not double blind and not including a trial published recently. Design Systematic review and meta-analysis. Data sources Searches of PubMed and EMBASE and reference lists of the retrieved articles. Inclusion criteria Randomised double-blind controlled trials comparing the administration of antibiotics before skin incision with administration after cord clamping. Data extraction and analysis Data on maternal total infectious morbidity, endometritis and wound infection, as well as neonatal intensive care unit admission, neonatal infection and neonatal sepsis were extracted and combined using random effects meta-analysis. Results Five studies reporting on 1777 parturients were included in our systematic review. The relative risk (RR) for maternal total infectious morbidity for antibiotic administration before incision compared with antibiotic administration after cord clamping was 0.64 (95% CI 0.36 to 1.15). Likewise, there was no difference in the risk of wound infection (RR 0.72, 95% CI 0.41 to 1.27). Parturients receiving the antibiotic preoperatively had a significantly reduced risk of endometritis (RR 0.48, 95% CI 0.27 to 0.87; number needed to treat 41, 95% CI 23 to 165). Analyses of the neonatal outcome parameters revealed no differences between the regimens of antibiotic administration, but were based on few studies. Conclusions In contrast to a recent NICE guideline, we did not find a reduction in total infectious morbidity with antibiotic administration before skin incision; we confirmed a reduction in the risk of endometritis and a lack of effect on the risk for wound infection.

Fetal echocardiography and pulsed-wave Doppler ultrasound in a rabbit model of intrauterine growth restriction.

Abstract: Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn The other horn in the rabbit bicornuate uterus serves as internal control fetuses Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform The ductus venosus and middle cerebral artery Doppler is then examined Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting

Does website-based information add any value in counseling mothers expecting a baby with severe congenital diaphragmatic hernia?

Abstract: Objective We aimed to measure whether website-provided information about congenital diaphragmatic hernia (CDH) and fetal therapy for severe cases provides added value compared with clinical counseling of parents. Methods This is a single center study in 102 couples who earlier opted for fetoscopic endoluminal tracheal occlusion (FETO) because of isolated severe CDH. They were asked to fill out an anonymized web-based survey of 12 questions. Then, they were offered access to information on the web pages of the randomized Tracheal Occlusion to Accelerate Lung Growth (TOTAL) trial. One week later, their appreciation was measured again by a second questionnaire. Results Eighty-two (80%) parents completed the first questionnaire, and 48 (47%) completed the entire survey. Several items became more clear to the parents after reading the website, such as the length of hospital stay (23.2% prior to web information, 60.4% after; P = 0.004), maternal risk, or the requirement of fetal anesthesia for FETO (43.9% resp. 79.2%; P = <0.001). Conclusion Complementing prenatal counseling on CDH and FETO by standardized information via website is perceived by parents as of added value. Maternal risks and the need for fetal medication need more clarification during the verbal counseling prior to prenatal interventions. © 2013 John Wiley & Sons, Ltd.

Management of twin pregnancies: where do we go from here?

Abstract: Twins have a more complicated intrauterine journey than do singletons. Also, their management is complicated because the interests of both twins have to be taken into account at all times. Moreover, in monochorionic pairs, there is the added risk of the shared circulation that can cause transfusion imbalances and makes their wellbeing interrelated. Due to artificial reproductive technologies, we now encounter more twin pregnancies than ever before. Most twin pregnancies resulting from assisted conception are dizygotic and thus dichorionic. Nevertheless, the risk of monozygotic cleavage is increased after assisted conception and these monozygotic twins are more likely to have a monochorionic placenta1. As such, about 1 in 20 twin pregnancies following assisted conception will be monochorionic. Ultrasound plays a key role in the surveillance of twin pregnancies. The first-trimester scan is of paramount importance for determination of chorionicity, risk assessment for chromosomal anomalies and detection of major structural anomalies. Simply diagnosing a twin pregnancy is insufficient; chorionicity should always be specified and it is of paramount importance to identify the high-risk monochorionic twin pregnancy early. There is general consensus that monochorionic twin pregnancies should benefit from fortnightly scans to detect twin–twin transfusion syndrome (TTTS); this complicates 1 in 10 monochorionic pairs, mostly in the previable period between 16 and 26 weeks, and is diagnosed by measuring the deepest vertical amniotic fluid pockets. Timely detection of TTTS is necessary because invasive fetal therapy may salvage one or both twins2. Additionally, expert opinion recommends the inclusion of middle cerebral artery (MCA) peak systolic velocities (PSV) in the routine follow-up of monochorionic twins, in order to detect twin anemiapolycythemia sequence (TAPS). This condition occurs in 1 in 25 monochorionic twin pairs, mostly in the viable period after 26 weeks, and is diagnosed by measuring the MCA-PSV (< 0.8 in the recipient and > 1.5 multiples of the median in the donor). Timely detection of TAPS and intrauterine treatment or elective preterm birth may prevent a number of late intrauterine fetal deaths which presumably would have remained unexplained3,4. In addition to identifying complications of the shared circulation, the purpose of sonographic follow-up is to detect poor growth and structural anomalies, both of which occur more commonly in monochorionic twin pregnancies. Dichorionic twin pregnancies are usually scanned every 4 weeks, primarily to detect poor growth and structural anomalies. The convenient aspect of scanning twin pregnancies is that one essentially looks for discordance (in growth, Doppler recordings, amniotic fluid and anatomy) and the presence of two fetuses means that each one can serve as the other’s control. The downsides of scanning twins are that it is time-consuming and, more often than not, visualization is hampered by intrauterine crowding. As in singleton pregnancies, cervical length measurement is the most important predictor of preterm birth in twins and a cervical length < 25 mm is useful to decide whether or not a symptomatic patient should receive steroids and tocolysis5. Since there is no proven useful intervention that prolongs pregnancy or alters outcome, routine cervical length measurement at mid-gestation is not as yet recommended. From 32 weeks onward, biophysical profile score and fortnightly growth checks may help to confirm fetal wellbeing and determine the optimal time of birth in monochorionic as well as dichorionic pregnancies. Evidence as well as expert opinion suggest that uncomplicated monochorionic twin pregnancies are best delivered at 36–37 weeks and uncomplicated dichorionic twins at 37–38 weeks because, at these time points, intrauterine mortality of twin pregnancies equals that of post-term singleton pregnancies6–8. Finally, during the second stage of labor and after delivery of the first twin, the availability of a bedside scanning machine is practical to determine the position of the second twin and to assist with internal or external version if necessary. So, from the first trimester up to the time of birth itself, ultrasound examination plays an indispensable role in fetal surveillance and in optimizing the outcome of twin pregnancies. Just as twin pregnancies keep many obstetricians busy, so it is fitting that a substantial proportion of this month’s journal should be dedicated to papers dealing with their management. Three papers are from the Southwest Thames Obstetric Research Collaborative (STORK) multiple pregnancy cohort, which comprises more than 2000 twin pregnancies that have been followed from the first trimester over the past 10 years9–11. The first paper9 demonstrates that, in the absence of structural or chromosomal anomalies, the crown–rump length (CRL) difference is only a weak predictor of mortality; the main predictor of adverse pregnancy outcome is whether the twins share a single placenta, thereby underlining the

Psychosocial aspects of invasive fetal therapy as compared to prenatal diagnosis and risk assessment.

Abstract: Objective To explore the psychosocial impact of invasive fetal therapy (FT). Methods We studied 100 consecutive patients scheduled for invasive FT. Contemporary controls were women undergoing (1) invasive prenatal diagnosis (AC/CVS) and (2) first trimester risk assessment of aneuploidy (NT), and (3) women who declined the latter (CTR). Prior to the procedure, participants completed the Beck Depression Inventory II, the State-Trait Anxiety Inventory, the Dyadic Adjustment Scale and a questionnaire specifically designed to evaluate the process preceding the intervention. Results Thirty-five percent of women in the FT group had mild to severe depressive symptoms, and 30% showed high levels of state anxiety. Mean state anxiety was significantly higher in women facing invasive as compared to non-invasive procedures. Trait anxiety levels and relationship scores were comparable across all groups. FT patients were more satisfied with the information and support given, whereas women in the NT group felt a greater degree of self-determination and contentedness with the choices they made. Conclusion Pregnant women awaiting invasive prenatal diagnosis and FT face higher levels of state anxiety than women undergoing non-invasive procedures. Traits of depression and high state anxiety are found in at least one third of women undergoing FT. © 2013 John Wiley & Sons, Ltd.

A Comparative Study on Culture Conditions and Routine Expansion of Amniotic Fluid-Derived Mesenchymal Progenitor Cells

Abstract: Background: Amniotic fluid (AF) cell populations will be applied in perinatology. We aimed to test the feasibility of large-scale cell expansion. Study Methods: We determined the best out of three published expansion protocols for mesenchymal progenitors (AF samples, n = 4) in terms of self-renewal ability. Characterization was performed based on morphology, surface marker analysis, cytogenetic stability, and differentiation potential. The conditions for the best self-renewal ability were further determined in a consecutive series (n = 159). Results: The medium containing fetal bovine serum (FBS), epidermal growth factor, insulin, transferrin, and tri-iodothyronine, combined with seeding on gelatin-coated wells, best stimulated the growth of cells with mesenchymal features, as demonstrated by flow cytometry; however, only osteogenic differentiation was possible. Large-scale testing (n = 44) failed to confirm a robust self-renewal ability. Better results were obtained (n = 88) using optimized FBS or an increased initial cell density. Eventually over 81% of cultures continued growing after the initial medium change and had mesenchymal features but failed differentiation assays. Discussion: Routine in vitro expansion of AF-derived mesenchymal cells remains problematic. Despite an increase in successful cell cultures from 40 up to 80% using optimized serum and an increased cell density, eventually cells failed to demonstrate differentiation abilities. Routine isolation and expansion from unselected AF samples remains a challenge.

In vitro sealing of iatrogenic fetal membrane defects by a collagen plug imbued with fibrinogen and plasma

Abstract: Objectives We aimed to demonstrate local thrombin generation by fetal membranes, as well as its ability to generate fibrin from fibrinogen concentrate Furthermore, we aimed to investigate the efficacy of collagen plugs, soaked with plasma and fibrinogen, to seal iatrogenic fetal membrane defects Methods Thrombin generation by homogenized fetal membranes was measured by calibrated automated thrombography To identify the coagulation caused by an iatrogenic membrane defect, we analyzed fibrin formation by optical densitometry, upon various concentrations of fibrinogen The ability of a collagen plug soaked with fibrinogen and plasma was tested in an ex vivo model for its ability to seal an iatrogenic fetal membrane defect Results Fetal membrane homogenates potently induced thrombin generation in amniotic fluid and diluted plasma Upon the addition of fibrinogen concentrate, potent fibrin formation was triggered Measured by densiometry, fibrin formation was optimal at 1250 µg/mL fibrinogen in combination with 4% plasma A collagen plug soaked with fibrinogen and plasma sealed an iatrogenic membrane defect about 35% better than collagen plugs without these additives (P = 0037) Conclusions These in vitro experiments suggest that the addition of fibrinogen and plasma may enhance the sealing efficacy of collagen plugs in closing iatrogenic fetal membrane defects © 2013 John Wiley & Sons, Ltd

A Surgical Technique for Homogenous Renal Distribution of Substances in Rats

Abstract: Intra-arterial injection of mesenchymal stem cells has been proven to result in a superior nephroprotection compared to intravenous injection. This avoids initial passage through filter organs such as the lung, liver and spleen. The aim of the present study was to investigate whether suprarenal aortic delivery results in a homogenous distribution to both kidneys. Chinese ink was used to evaluate the renal distribution pattern for the comparison of two retrograde intra-aortic injection methods. In the first, the aorta caudal to the renal branches was temporarily clamped and Chinese ink was injected at the level of the renal arteries. In the second, a distal aortic clamp was combined with alternated clamping of the contralateral arteries. Immediately after injection, kidneys were harvested for histological analysis. Amniotic fluid stem cells labeled with LacZ were injected in the aorta by alternated clamping of the renal arteries in order to track the cells in a rat ischemia/reperfusion model. Without renal artery clamping, intra-aortic administration resulted in a delivery of the ink into the right kidney, whereas administration with alternated clamping of the contralateral renal artery, together with distal aortic artery clamping, resulted in a more homogenous distribution of the ink in both kidneys. Moreover, LacZ-positive cells were found in both kidneys after 6 h of injection. In conclusion, the retrograde administration of Chinese ink in two steps is a fast and reproducible technique, which results in a more homogenous distribution of the stain in both kidneys than a single administration combined by only clamping the aorta.