Novel isolation strategy to deliver pure fetal-origin and maternal-origin mesenchymal stem cell (MSC) populations from human term placenta.

The placenta is an abundant source of mesenchymal stem/stromal cells (MSC). Although presumed of translationally-advantageous fetal origin, the literature instead suggests a high incidence of either contaminating or pure maternal MSC. Despite definitional criteria that MSC are CD34-, increasing evidence suggests that fetal MSC may be CD34 positive in vivo. We flow sorted term placental digests based on CD34+ expression and exploited differential culture media to isolate separately pure fetal and maternal MSC populations. This method has considerable translational implications, in particular to clinical trials underway with "placental" MSC of uncertain or decidual origin.

Pregnancy-acquired fetal progenitor cells.

The transfer and persistence of fetal progenitor cells into the mother throughout pregnancy has sparked considerable interest as a trafficking stem cell and immunological phenomenon. Indeed, the intriguing longevity of semi-allogeneic fetal microchimeric cells (FMC) in parous women raises questions over their potential clinical implications. FMC have been associated with both immune-modulatory roles and participation in maternal tissue repair. Although their influence on maternal health is as yet unresolved, FMC selectively home to damaged maternal tissues and often integrate, adopting site-appropriate phenotypes. FMC features, such as plasticity and persistence in their maternal host, suggest that they likely include pluripotent, or various multipotent and committed stem and progenitor cells. Recent efforts to determine what cell types are involved have established that FMC include cells of ectodermal, endodermal, mesodermal, and perhaps trophectodermal lineages. This review details FMC phenotypes and discusses how FMC themselves may be considered a naturally occurring stem cell therapy.

Systemic delivery of scAAV9 in fetal macaques facilitates neuronal transduction of the central and peripheral nervous systems.

Correction of perinatally lethal neurogenetic diseases requires efficient transduction of several cell types within the relatively inaccessible CNS. Intravenous AAV9 delivery in mouse has achieved development stage-specific transduction of neuronal cell types, with superior neuron-targeting efficiency demonstrated in prenatal compared with postnatal recipients. Because of the clinical relevance of the non-human primate (NHP) model, we investigated the ability of AAV9 to transduce the NHP CNS following intrauterine gene therapy (IUGT). We injected two macaque fetuses at 0.9 G with 1 × 10(13) vg scAAV9-CMV-eGFP through the intrahepatic continuation of the umbilical vein. Robust green fluorescent protein (GFP) expression was observed for up to 14 weeks in the majority of neurons (including nestin-positive cells), motor neurons and oligodendrocytes throughout the CNS, with a significantly lower rate of transduction in astrocytes. Photoreceptors and neuronal cell bodies in the plexiform and ganglionic retinal layers were also transduced. In the peripheral nervous system (PNS), widespread transduction of neurons was observed. Tissues harvested at 14 weeks showed substantially lower vector copy number and GFP levels, although the percentage of GFP-expressing cells remained stable. Thus, AAV9-IUGT in late gestation efficiently transduces both the CNS and PNS with neuronal predilection, of translational relevance to hereditary disorders characterized by perinatal onset of neuropathology.

Identification of candidate surface antigens for non-invasive prenatal diagnosis by comparative global gene expression on human fetal mesenchymal stem cells.

Transplacental passage of circulating first-trimester fetal mesenchymal stem cells (fMSC) raises the prospect of harvesting fetal cells in maternal blood. Despite high sensitivity in model systems, negative selection and culture strategies yield fMSC only rarely in post-termination maternal blood. The different adhesion molecule profile of fMSC to competitor maternal cell types suggests that improved positive selection strategies may facilitate non-invasive prenatal diagnosis. We aimed to identify surface antigens specific to fMSC and not maternal peripheral blood lymphocytes (PBL), using genome-wide analysis of actively expressed transcripts. Maternal PBL and fMSC cultured from first-trimester blood, liver and bone marrow were assessed for global gene expression by Affymetrix U133Plus2.0 arrays. Data were analysed using Affymetrix GCOS01.2. Transcripts present in all fMSC (n = 9) but absent in all PBL samples (n = 3) were selected for further analysis of cell-surface membrane molecules by RT-PCR and immunocytochemistry. Of 1544 genes expressed in fMSC and not maternal PBL, filtering for cell-surface molecules yielded 159 genes. Of these, 29 had a mean expression ratio of >300 (P < 0.001), which represented 18 unique genes, and their positive expression in all fMSC samples was confirmed by RT-PCR. Candidates for non-invasive prenatal diagnosis were chosen for further analysis by immunocytochemistry. Surface expression of OSMR and COL1 proteins on all fMSC, but no maternal PBL was confirmed. Identification of novel surface antigens on circulating human fMSC and not maternal PBL facilitates positive selection strategies for isolating fMSC for non-invasive prenatal diagnosis.

Transfusional fetal complications after single intrauterine death in monochorionic multiple pregnancy are reduced but not prevented by vascular occlusion.

OBJECTIVE: To document co-twin death/pregnancy loss and brain injury after single intrauterine death (sIUD) in monochorionic pregnancies. DESIGN: A total of 135 pregnancies with sIUD were reviewed for co-twin IUD, miscarriage and abnormal antenatal and postnatal neuro-imaging. SETTING: A tertiary referral fetal medicine unit from 2000 to 2007. POPULATION OR SAMPLE: All cases referred with a single fetal death in monochorionic pregnancy, including those where sIUD was spontaneous or occurred after fetoscopic laser treatment, or resulted from selective termination by cord occlusion with bipolar diathermy or intrafetal vascular ablation with interstitial laser. METHODS: Clinical details and ultrasound findings of the study population were retrieved from ultrasound and institutional databases. Delivery and neonatal outcome data were obtained from discharge summaries supplemented by individual chart review. MAIN OUTCOME MEASURES: Co-twin death or pregnancy loss and neurologic injury assessed on antenatal ultrasound and MR-imaging. RESULTS: A total of 81 sIUDs resulted from vascular occlusive feticide (diathermy or interstitial laser), 22 followed placental laser and 32 were spontaneous. In 22 pregnancies (16.8%), the co-twin died in utero and eight pregnancies miscarried (6.1%). Antenatal magnetic resonance (MR) imaging in 76/91 (83.5%) continuing pregnancies detected antenatal brain injury in five (6.6%). Three infants (two not scanned antenatally) had abnormalities detected postnatally. Brain abnormality was detected less often after procedure related (2.6%, 2/77) than spontaneous sIUD (22.2%, 6/27, P = 0.003) and after early compared with late gestation sIUD (3.6%, 4/111 versus 20.0%, 4/20; P = 0.02). CONCLUSIONS: We confirm substantial co-twin loss (22.9%) after monochorionic sIUD, but a low risk of antenatally acquired MRI-identified brain injury, suggesting this risk has been overestimated. Procedures restricting inter-twin transfusion reduce, but do not negate risk of brain injury.

The basic and clinical science of twin-twin transfusion syndrome.

Twin-twin transfusion syndrome (TTTS) is a fascinating condition in which fetuses of identical genotype adopt discordant cardiovascular phenotypes, secondary to unbalanced placental inter-twin transfusion. Flow along the primary units of inter-twin transfusion, unidirectional arteriovenous anastomoses, can be as high as litres/day each, and TTTS develops when the placenta has insufficient compensatory counter-transfusional anastomoses. The initial phenotype reflects dysvolaemia, with added contributions from uteroplacental insufficiency in the donor, and raised afterload with diastolic dysfunction secondary to discordant endothelin and placentally derived renin-angiotensin system effectors in the recipient. Endoscopic laser ablation of placental anastomoses has become the primary treatment modality, supported by a randomised trial showing improved survival and short but not long-term neurological morbidity. Its uptake has facilitated comparative pre- and post-laser studies, which provide considerable insight into the pathophysiology. Despite the therapeutic advance, placental laser remains associated with a 25% incidence of fetal death within a week, and a 10% risk each of recurrence and twin anaemia/polycythaemia sequence due to residual anastomoses. In Stage I, high rates of non-progression with more conservative management have resulted in therapeutic equipoise as to whether laser is indicated primarily or only for progressive disease. The challenge ahead lies in improving double intact survival rates, which in addition to randomised trials will require technical advances, better understanding of the circulatory pathophysiology and more sophisticated surveillance tools.

Systematic analysis of research underfunding in maternal and perinatal health.

BACKGROUND: Little published evidence supports the widely held contention that research in pregnancy is underfunded compared with other disease areas. OBJECTIVES: To assess absolute and relative government and charitable funding for maternal and perinatal research in the UK and internationally. SEARCH STRATEGY, SELECTION CRITERIA, DATA COLLECTION, AND ANALYSIS: Major research funding bodies and alliances were identified from an Internet search and discussions with opinion leaders/senior investigators. Websites and annual reports were reviewed for details of strategy, research spend, grants awarded, and allocation to maternal and/or perinatal disease using generic and disease-specific search terms. MAIN RESULTS: Within the imprecision in the data sets, < or =1% of health research spend in the UK was on maternal/perinatal health. Other countries fared better with 1-4% investment, although nonexclusive categorisation may render this an overestimate. In low-resource settings, government funders focused on infectious disease but not maternal and perinatal health despite high relative disease burden, while global philanthropy concentrated on service provision rather than research. Although research expenditure has been deemed as appropriate for 'reproductive health' disease burden in the UK, there are no data on the equity of maternal/perinatal research spend against disease burden, which globally may justify a manyfold increase. AUTHOR'S CONCLUSIONS: This systematic review of research expenditure and priorities from national and international funding bodies suggests relative underinvestment in maternal/perinatal health. Contributing factors include the low political priority given to women's health, the challenging nature of clinical research in pregnancy, and research capacity dearth as a consequence of chronic underinvestment.

The natural caesarean: a woman-centred technique.

Although much effort has gone into promoting early skin-to-skin contact and parental involvement at vaginal birth, caesarean birth remains entrenched in surgical and resuscitative rituals, which delay parental contact, impair maternal satisfaction and reduce breastfeeding. We describe a 'natural' approach that mimics the situation at vaginal birth by allowing (i) the parents to watch the birth of their child as active participants (ii) slow delivery with physiological autoresuscitation and (iii) the baby to be transferred directly onto the mother's chest for early skin-to-skin. Studies are required into methods of reforming caesarean section, the most common operation worldwide.

The placenta contributes to activation of the renin angiotensin system in twin-twin transfusion syndrome.

The renin-angiotensin system (RAS) in twin-twin transfusion syndrome (TTTS) is up-regulated in the donor fetus's kidneys, but down-regulated in the recipient's. Ultrasonographic and echocardiographic features suggest that the recipient is also exposed to RAS components. In this study we investigated the role and origin of RAS components in the recipient fetus. Monochorionic diamniotic (MCDA) pregnancies were recruited from a tertiary fetal medicine service. Cord blood was collected from MCDA twins (TTTS and control non-TTTS) at delivery for renin and angiotensin II immunoassays. Placental tissue was flash-frozen for mRNA and protein expression or formalin-fixed for immunohistochemistry. Archival placenta and kidney samples were used for immunohistochemistry and in-situ hybridization. Plasma renin levels were elevated (p<0.05) in recipients (median 201 pg/ml, range 54-315 pg/ml) and donors (125 pg/ml, 25-296) with TTTS compared to controls (2.5 pg/ml, 1.1-1.5 pg/ml). The same was found with angiotensin II with high levels in both recipients (300.5 pg/ml, 86.1-488 pg/ml) and donors (239 pg/ml, 76.6-422) compared to controls (169.5 pg/ml, 89-220 pg/ml, p<0.05). Renin mRNA expression, and protein appeared qualitatively higher in the placental territory of the recipient compared to that of the donor and non-TTTS controls. We conclude that both fetuses in TTTS are exposed to high levels of RAS components; these appear to be produced from different sites, namely the kidney of the donor, and the placenta of the recipient. Given the markedly different phenotypes in the genetically identical fetuses with TTTS, we suggest that the source of RAS components may influence their clinical manifestations.

Transplantation of human fetal mesenchymal stem cells improves glomerulopathy in a collagen type I alpha 2-deficient mouse.

Fetal mesenchymal stem cell (fetal MSC) therapy has potential to treat genetic diseases with early onset, including those affecting the kidney and urinary tract. A collagen type I alpha 2-deficient mouse has a deletion in the alpha2 chain of the procollagen type I gene, resulting in the synthesis of abnormal alpha1(I)(3) homotrimers, which replace normal alpha 1(I)2 alpha 2(I)1 heterotrimers and a glomerulopathy. We first confirmed that col1 alpha 2-deficient homozygous mice show abnormal collagen deposition in the glomeruli, which increases in frequency and severity with postnatal age. Intrauterine transplantation of human MSCs from first trimester fetal blood led postnatally to a reduction of abnormal homotrimeric collagen type I deposition in the glomeruli of 4-12 week-old col1 alpha 2-deficient mice. Using bioluminescence imaging, in situ hybridization and immunohistochemistry in transplanted col1 alpha 2-deficient mice, we showed that the damaged kidneys preferentially recruited donor cells in glomeruli, around mesangial cells. Real-time RT-PCR demonstrated that this effect was seen at an engraftment level of 1% of total cells in the kidney, albeit higher in glomeruli. We conclude that intrauterine transplantation of human fetal MSCs improves renal glomerulopathy in a collagen type I-deficient mouse model. These data support the feasibility of prenatal treatment for hereditary renal diseases.

Exploring cortical subplate evolution using magnetic resonance imaging of the fetal brain.

The subplate is a transient structure essential for normal development of the cortex. We used magnetic resonance imaging of the fetal brain to assess cortical subplate evolution between 20 and 35 weeks gestation. Two-dimensional measures of diameter were obtained for the cortex, subplate and fetal white matter. The subplate was originally seen as a continuous band at early gestations measuring up to 4.5 mm. It became magnetic resonance invisible from approximately 28 weeks initially from the depths of the sulci and then from the tops of the gyri. The disappearance of the subplate was regional, involuting most rapidly in the parietal lobe and remaining prominent in the anterior temporal lobe up to 35 weeks. x

Stage I twin-twin transfusion syndrome: rates of progression and regression in relation to outcome.

OBJECTIVES: Twin-twin transfusion syndrome (TTTS) results in high rates of perinatal mortality and neurological morbidity. Fetoscopic laser ablation of placental anastomoses is now established as the treatment of choice for advanced disease. However, there remains controversy about its use in early-stage TTTS, in which laser-related fetal losses need to be balanced against relatively favorable outcomes with more conservative approaches. We investigated rates of progression and regression in Stage I TTTS and determined factors influencing the course of the disease. METHODS: We undertook a retrospective observational study of all TTTS cases referred to our tertiary referral fetal medicine service from 2000 to 2006. In patients presenting with Stage I TTTS, the following variables were evaluated for their ability to predict the course and progression of the disease: gestational age (GA) at presentation, amniotic fluid index, recipient and donor deepest vertical pool, presence of artery-artery anastomoses, small-sized bladder compared to normal donor bladder and fetal size discordance. Study end-points were disease regression or progression, and neonatal survival at 28 days. RESULTS: Among 132 consecutive cases of TTTS, 46 women presented with Stage I disease. In the majority (69.6%), disease remained stable (28.3%) or regressed (41.3%). Of cases that progressed, 79% did so within 2 weeks and 93% progressed to at least Stage III. No factor was significantly linked with progression or regression, although there was a trend towards the absence of an artery-artery anastomosis (P = 0.10) and the presence of a small rather than normal donor bladder (P = 0.10) influencing progression, and later GA at presentation (P = 0.07) influencing regression. At least one infant survived in 83% of cases and there was double survival in 59%. Perinatal outcome was significantly better in cases that regressed (the rates of at least one survivor and double survival being 89% and 89%, respectively) or remained Stage I (77% and 61%, respectively), compared with those cases that progressed (79% and 14%, respectively). Treatment with amnioreduction at first presentation did not influence progression or regression. CONCLUSIONS: This study demonstrates that a high percentage of Stage I TTTS cases regress or remain early stage. Identification of factors predicting progression would facilitate the selection of patients for definitive therapy, while avoiding treatment-related morbidities in mild or transient disease.

Amniotic fluid testosterone: relationship with cortisol and gestational age.

INTRODUCTION: Foetal exposure to testosterone is increasingly implicated in the programming of future reproductive and non-reproductive behaviour. Some outcomes associated with prenatal exposure to testosterone may be predicted from exposure to prenatal stress, suggesting a link between them. The peak serum levels of testosterone in the foetus are thought to be around 14-18 weeks' gestation, and we explored testosterone levels at different gestations. Although best investigated in foetal plasma, this is now difficult because of the decline in frequency of foetal blood sampling; in this study, we used amniotic fluid as a biomarker to investigate foetal exposure. AIMS: To investigate the relationship between amniotic fluid testosterone, amniotic fluid cortisol, foetal gender, and gestational age. METHODS: Paired amniotic fluid and maternal plasma samples were collected from 264 pregnant women undergoing amniocentesis between 15 and 37 weeks' gestation (median 17 weeks [119 days]). Total testosterone and cortisol in amniotic fluid, and total plasma testosterone (maternal) were measured by radioimmunoassay. RESULTS: Amniotic fluid testosterone levels were higher in male than in female foetuses, with a median (interquartile range) of 0.85 nmol/l (0.60-1.17 nmol/l) and 0.28 nmol/l (0.175-0.45 nmol/l), respectively. No relationship between amniotic fluid testosterone and gestational age was detected in either sex. Amniotic fluid testosterone correlated positively with amniotic fluid cortisol in both sexes (r = 0.30 male foetuses, r = 0.33 female foetuses, P < 0.001 for both), and remained significant in multivariate analysis. CONCLUSION: Testosterone in amniotic fluid did not change with gestation in the second and third trimester, raising questions about the timing of the reported early peak in the male foetus. The positive correlation between cortisol and testosterone in amniotic fluid suggests that increased foetal exposure to cortisol may also be associated with increased exposure to testosterone.

Ontogeny of foetal exposure to maternal cortisol using midtrimester amniotic fluid as a biomarker.

OBJECTIVE: There is increasing evidence that antenatal stress has long-lasting effects on child development, but there is less accord on the mechanisms and the gestational window of susceptibility. One possible mechanism is by foetal exposure to maternal cortisol. To explore this, we investigated the relationship between cortisol in maternal plasma and amniotic fluid, and any moderating influence of gestational age. PATIENTS AND MEASUREMENTS: Two hundred and sixty-seven women awaiting amniocentesis for karyotyping were studied. Samples were collected between 0900 and 1730 h. Gestational age was determined to the nearest day by ultrasound biometry and time of collection noted to the nearest 15 min. Total cortisol was measured by radioimmunoassay in paired amniotic fluid and maternal blood samples (n = 267) [gestation range 15-37 weeks, median 17 weeks (119 days)]. RESULTS: Both maternal and amniotic fluid cortisol levels increased with gestation (r = 0.25, P < 0.001; r = 0.33 P < 0.001, respectively). Amniotic fluid cortisol was positively correlated with time of collection (r = 0.22, P < 0.001) and negatively with maternal age (r =-0.24, P < 0.001). There was a positive correlation between amniotic fluid cortisol with maternal plasma levels (r = 0.32, P < 0.001), which persisted after multivariate analysis controlling for gestation, time of collection and maternal age. The association appeared to be dependent on gestational age, being nonsignificant at 15-16 weeks' gestation and increasing in strength thereafter. CONCLUSION: This study shows a positive correlation between maternal and amniotic fluid cortisol levels, which becomes robust from 17 to 18 weeks onwards. The results provide support for the hypothesis that alterations in maternal cortisol may be reflected in amniotic fluid levels from this gestation.

FastFISH: technique for ultrarapid fluorescence in situ hybridization on uncultured amniocytes yielding results within 2 h of amniocentesis.

Rapid aneuploidy detection methods allow prenatal diagnosis results to be released within 48 h, but not on the same day as the invasive test. We aimed to develop a rapid fluorescence in situ hybridization (FISH) method (FastFISH) that releases accurate results on the same day as amniocentesis. FastFISH was optimized to be completed within 2 h of sample collection using CEP and LSI probes for chromosomes 13, 18, 21, X, Y and DiGeorge syndrome (DGS). The technique was tested on 100 consecutive amniotic fluid samples in a blinded study. It was also validated as a 1-day molecular genetic test on three representative fetal tissue samples: chorionic villus, amniotic fluid and fetal blood. In the blinded study, FastFISH results were ready within 2 h of sample collection. Of the 100 amniotic fluid samples, 49 male and 50 female fetuses were identified. One fetus was 47, XXY (Klinefelter syndrome). Three fetuses had trisomy 21. One fetus suspected of DGS by ultrasound was identified as normal. Results of FastFISH analyses in all 100 cases were concordant with their karyotypes (100% accuracy; lower 95% CI, 97.05%). In the 1-day test validation, all results were released on the same day and were concordant with their respective karyotypes. FastFISH allows results to be released on the same day as amniocentesis. It represents the necessary development for a 1-day prenatal diagnosis service.

Longitudinal blood flow in shared (arteriovenous anastomoses) and non-shared cotyledons in monochorionic placentae.

OBJECTIVES: In this study we aimed to quantitate monochorionic twin placental blood flow in vivo through arterio-venous anastomoses (AVA) and corresponding vessels within normal cotyledons. METHODS: The topography of chorionic plate vasculature was mapped using colour Doppler in ten monochorionic diamniotic twin (MCDA) pregnancies. Cotyledonary flow was derived by insonation of chorionic veins draining normal (n=10) and paired control shared cotyledons (n=10). Venous volume flow was calculated from five determinations of vessel diameter and three of time average mean velocity (TAMV). Measurements were repeated every 2-4 weeks from 18 until 32 weeks' gestation. RESULTS: Blood flow through non-shared and shared cotyledons increased with gestation (p<0.0001). Median flow at 28 weeks through shared cotyledons was 16 ml/min (15-21) (median, interquartile range), lower than in shared cotyledons (31, 25-35) (p<0.001), as was median volume flow across gestation calculated as area under the curve (shared cotyledons 126 (122-167), control cotyledons 269 (214-274), p=0.01). However, velocity was similar, with the difference due to smaller vein diameters draining shared compared to normal cotyledons (mean 3.6mm (SD 0.8) vs. 4.5mm (0.8), p=0.004). Ex vivo quantitation of insonated cotyledons and of all cotyledons confirmed the difference in vein diameter in the placentae studied. CONCLUSIONS: Blood flow through shared cotyledons was lower across gestation than through paired normal cotyledons in the placenta studied due to the smaller diameter of the AVA vessels. The size of AVAs rather than simply their presence and direction may contribute to determining transfusional imbalance in monochorionic twins.

High perinatal survival in monoamniotic twins managed by prophylactic sulindac, intensive ultrasound surveillance, and Cesarean delivery at 32 weeks' gestation.

OBJECTIVES: Increased perinatal mortality in monoamniotic twin pregnancies is attributed to cord accidents in utero and at delivery. We evaluated the following parameters in monoamniotic pregnancies: (1) the incidence of cord entanglement; (2) the effect of sulindac on amniotic fluid volume and stability of fetal lie; and (3) the perinatal outcome with our current management paradigm. METHODS: This is a retrospective review of monoamniotic pregnancies of >or=20 weeks' gestation managed with serial ultrasound surveillance, medical amnioreduction and elective Cesarean delivery at 32 weeks' gestation. Mean amniotic fluid index (AFI) and change in AFI in monoamniotic pregnancies managed with oral sulindac was compared with 40 gestation-matched monochorionic-diamniotic controls. RESULTS: Among 44 monoamniotic pregnancies, 20 with two live structurally normal twins at 20 weeks' gestation satisfied the inclusion criteria. All fetuses survived to 28 days postnatally despite early prenatal cord entanglement in all but one case. Whereas AFI remained stable throughout gestation in the controls, the AFI fell in those patients on sulindac from a mean value of 21.0 cm (95% CI, 18.5-23.6 cm) at 20 weeks to a mean of 12.4 cm (95% CI, 10.1-14.6 cm) at 32 weeks (ANOVA P across gestation = 0.001) but mainly remained within normal limits. Fetal lie was stabilized in 11/20 cases in the monoamniotic group compared with 13/40 in the control group (P < 0.0001). CONCLUSIONS: Cord entanglement appears unpreventable, as it typically occurs in early pregnancy. Sulindac therapy reduces AFI, leads to more stable fetal lie, and may prevent intrauterine death by diminishing the risk of constricting cords that are already entangled. Perinatal survival in monoamniotic pregnancies managed by a regime of sulindac from 20 weeks' gestation, close ultrasound surveillance and elective abdominal delivery at 32 weeks' gestation seems empirically higher than that in the literature.

Maternal anxiety at amniocentesis and plasma cortisol.

OBJECTIVES: To assess whether anticipation of amniocentesis is linked with maternal anxiety, and whether this anxiety is associated with increased maternal plasma cortisol. METHODS: Two hundred and fifty-four women awaiting a morning amniocentesis for karyotyping (gestation range 15-37 weeks, median 17 weeks) completed Spielberger state and trait anxiety inventory (STAI) questionnaires, and provided blood samples immediately before the procedure for cortisol assay. Six hundred and five women at mean gestation of 20 weeks, attending the same hospital for routine ultrasound but not for amniocentesis, also completed Spielberger STAI questionnaires and served as a comparison group for the anxiety ratings. RESULTS: Mean state and trait anxiety scores (+/- SD) in the comparison group of 605 women at mean gestation of 20 weeks were 36.1 +/- 10.2 (range 20-70) and 35.6 +/- 8.9 (range 20-73), respectively. The mean state anxiety score (+/-SD) of 49.8 +/- 14.0 (range 20-77) of the amniocentesis group was considerably higher than the comparison group (p < 0.001), although the mean trait anxiety score in the amniocentesis group was similar at 36.4 +/- 8.6 (range 21-60). The state, but not trait, anxiety correlated with plasma cortisol (r = 0.176, p = 0.005). Maternal cortisol in the amniocentesis group increased with gestational age (r = 0.310, p < 0.001), whereas state anxiety scores showed no significant change with increase in gestational age (r = - 0.042, ns). Multivariate analysis demonstrated that maternal state anxiety was positively correlated with plasma cortisol independent of gestation and time of collection. CONCLUSION: Women awaiting amniocentesis experience a high state anxiety associated with modestly increased plasma cortisol.

Histomorphometric characterisation of shared and non-shared cotyledonary villus territories of monochorionic placentae in relation to pregnancy complications.

OBJECTIVE: Theoretical estimates and physiological inferences suggest that the structure of a shared cotyledon differs from a non-shared cotyledon. The aim of this study was to characterise the histomorphometry of terminal villi in shared and non-shared cotyledons in monochorionic placentae, both from uncomplicated twins and from those with twin-twin transfusion syndrome (TTTS) or discordant growth restriction (DeltaIUGR). METHODS: Forty-one monochorionic placentae from Caucasian non-smokers were obtained at caesarean section. Their vascular anatomy and placental territories were ascertained by dye injection. After fixation, full thickness histological blocks were obtained by systematic random sampling from each twin's territory and the shared cotyledons. Fifty randomly selected terminal villi were assessed for: (i) median villus diameter (ii) median villus capillary diameter (iii) median fetomaternal diffusion distance (iv) median no. of capillaries/villus (v) degree of vascularization (median percentage cross-sectional area of terminal villi occupied by capillaries) using a stage micrometer and image analysis programme. The histomorphometric findings were then correlated with birthweight discordance, placental territory discordance and DeltaAVAs (no. of AVAs from smaller twin (donor) to larger twin (recipient) minus no. of AVAs from larger to smaller twin). RESULTS: Histomorphometric variables were similar in shared and non-shared cotyledons of uncomplicated MCDA twins. However, the median diameter of terminal villi in shared cotyledons in DeltaIUGR and TTTS placentae was significantly smaller [51.2 microm (48.2-58.3), p<0.001 and 52.6 microm (53.1-50.4), p<0.001], and had a similar number of smaller capillaries, larger fetomaternal diffusion distance and reduced vascularization compared to non-shared IUGR and TTTS placentae. However, Deltadiameter (defined as the difference between median diameters of terminal villi in large minus small twins' territories) rose with increasing birthweight discordance (Pearson correlation coefficient=0.82, p<0.001). Multiple linear regression analysis revealed that Deltadiameter was influenced by placental territory discordance (p<0.001) and birthweight discordance (p<0.01): log10 Deltadiameter=1.38+(0.01 x birthweight discordance)+(0.56 x log10 placental territory discordance) (R2=0.82, p<0.001), but there was no significant relationship with DeltaAVA and AAA. In the TTTS group, Deltadiameter correlated significantly with DeltaAVA only: log10Deltadiameter=1.44+(0.02 x DeltaAVA) (R2=0.3, p<0.001). CONCLUSIONS: This is the first study to characterise the histomorphometry of shared and non-shared cotyledons in MC twins. The findings suggest that abnormal placentation, rather than placental vascular anatomy may be responsible for DeltaIUGR in MC twins, whereas TTTS arises from imbalance in interfetal transfusion with resultant differing terminal villus histomorphometric features in donor, recipient and shared cotyledons.