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Fetal microchimeric cells (FMCs) enter the maternal circulation and persist in tissue for decades. They have capacity to home to injured maternal tissue and differentiate along that tissue's lineage. This raises the question of the origin(s) of cells transferred to the mother during pregnancy. FMCs with a mesenchymal phenotype have been documented in several studies, which makes mesenchymal stem cells an attractive explanation for their broad plasticity. Here we assessed the recruitment and mesenchymal lineage contribution of FMCs in response to acute kidney fibrosis induced by aristolochic acid injection. Serial in vivo bioluminescence imaging revealed a biphasic recruitment of active collagen-producing FMCs during the repair process of injured kidney in post-partum wild-type mothers that had delivered transgenic pups expressing luciferase under the collagen type I-promoter. The presence of FMCs long-term post injury (day 60) was associated with profibrotic molecules (TGF-β/CTGF), serum urea levels, and collagen deposition. Immunostaining confirmed FMCs at short term (day 15) using post-partum wild-type mothers that had delivered green fluorescent protein-positive pups and suggested a mainly hematopoietic phenotype. We conclude that there is biphasic recruitment to, and activity of, FMCs at the injury site. Moreover, we identified five types of FMC, implicating them all in the reparative process at different stages of induced renal interstitial fibrosis.

Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling. ispartof: Molecular Therapy vol:20 issue:10 pages:1953-1967 ispartof: location:United States status: published

Intrauterine gene transfer (IUGT) offers ontological advantages including immune naiveté mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0-1.95 × 10(13) vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2-22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.

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.

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.

Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.

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

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.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.Blood flow through non-shared and shared cotyledons increased with gestation (p0.0001). 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) (p0.001), 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.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.

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).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).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), p0.001 and 52.6 microm (53.1-50.4), p0.001], 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, p0.001). Multiple linear regression analysis revealed that Deltadiameter was influenced by placental territory discordance (p0.001) and birthweight discordance (p0.01): log10 Deltadiameter=1.38+(0.01 x birthweight discordance)+(0.56 x log10 placental territory discordance) (R2=0.82, p0.001), 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, p0.001).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.

Objective To characterise arterio-venous anastomoses (AVA) in monochorionic (MC) placentae and determine (i) whether shared cotyledons lie beneath the co-termination of an artery from one twin and a vein to the contralateral twin and (ii) whether all AVA can be detected by visual inspection of the chorionic plate. Methods Vascular casts were made of 15 MC placentae. The number of typical AVAs suspected visually before digestion was compared with the number of AVAs identified after acid digestion. Results Thirty-three of 67 (49%) suspected typical AVAs were confirmed as typical after casting. There were five false positives and no false negatives. The remainder were classified as atypical AVAs, found in ≥90% of MC placentae. Type I (small vascular connections between two apparently normal cotyledons not seen before casting) and Type II (shared cotyledons arising within larger apparently normal cotyledons) atypical AVAs were found in 53% and 73% of placentae, respectively. Conclusions Only half the shared cotyledons in MC placentae are characterised by co-termination of an artery and vein on the chorionic plate. We report the existence of deep anastomoses beneath the chorionic plate that cannot be visualised by chorionic plate inspection. These findings have implications for laser treatment of twin–twin transfusion syndrome.

Fetal cells enter maternal blood during pregnancy and persist in women with autoimmune disease. The frequency of subsequent fetomaternal microchimerism in healthy women and its cell type is unknown. To test the hypothesis that fetal mesenchymal stem cells persist in maternal organs, we studied female bone marrow and ribs. Male cells were identified by XY fluorescence in-situ hybridisation in marrow-derived mesenchymal stem cells and in rib sections from all women with male pregnancies, but not in controls (9/9 vs 0/5, p=0.0005). We conclude that fetal stem cells transferred into maternal blood engraft in marrow, where they remain throughout life. This finding has implications for normal pregnancy, for obstetric complications that increase fetomaternal trafficking, and for graft survival after transplantation.

Objective The purpose of this study was to determine whether physical stimulation is stressful to the ovine fetus, as judged from physiologic changes that are similar to those reported for other stressors (such as hypoxia); whether any stress response could be blocked by clinically used doses of fentanyl; and whether fentanyl alone had any potentially deleterious physiologic effects in the fetus. Study design We investigated the effect of fentanyl analgesia on the cardiovascular and endocrine response to cutaneous electrical stimulation in the late gestation (>125 days) ovine fetus (n = 7 fetuses). Chronically implanted catheters and blood flow probes were used to measure fetal arterial blood pressure, heart rate, carotid and femoral blood flow, pH, P o 2, P co 2, lactate, cortisol, and β-endorphin levels before, during, and for 1 hour after 5 minutes of cutaneous electrical stimulation to the lip, forelimb, and abdomen, in a crossover design. Clinically used 30 or 150 μg doses of fentanyl (which approximated 10 or 50 μg/kg estimated fetal weight) or saline solution were given intravenously to the fetus 2 minutes before stimulation. Results When compared with the control, stimulation caused a significant rise in fetal heart rate (P = .003; mean maximal rise, 48.6±14.0 beats/min, 0-10 minutes after the start of stimulation) but caused no change in any other parameters studied. Neither dose of fentanyl attenuated the changes in heart rate that were observed in response to stimulation alone. Fentanyl alone significantly increased fetal heart rate, carotid blood flow, and lactate and cortisol levels and significantly decreased pH and P o 2. Conclusion Cutaneous electrical stimulation in the fetal sheep causes an increase in heart rate, which fentanyl does not block. Fentanyl itself has significant effects on the cardiovascular and endocrine system, which might adversely affect the fetus.

Despite the sound experimental basis and initial promise of early animal models, the results of antenatal intervention have been disappointing, with high rates of misdiagnosis of urethral valves, complications from vesicoamniotic shunting, perinatal mortality, and long-term renal impairment and bladder dysfunction in survivors. The recent development of a cystoscopic approach might obviate some of these problems, but to date the procedure has been limited by technical difficulty in negotiating the urethrovesical angle. Overcoming these difficulties through equipment modifications might allow definitive testing of whether or not alleviating distal urinary obstruction in utero is beneficial.

Objectives: To characterize the phenomenon of retrograde transmission of arterio-arterial anastomosis (AAA) interference patterns on umbilical artery (UA) waveform by (a) documenting the periodicity, (b) correlation with in vivo and in vitro demonstration of AAAs and (c) reproducing these patterns by computer modelling. Methods: Monochorionic twins (MC) twins underwent placental and umbilical Doppler studies. AAAs were sought by pulse wave Doppler of their bi-directional interference pattern and confirmed by postnatal injection studies. The periodicity of transmitted patterns in the UA was determined. Determinants of the transmitted patterns were ascertained by computer modelling of physiological and fetal variables. Results: Among 83 prospectively studied MC twin pregnancies; a transmitted pattern was observed in 6 (7 per cent) patients for 15–114 days. This was found in 20 per cent (6/30) of smaller MC twins discordant for growth restriction but in no appropriately grown twins. It was only observed in association with AAAs validated both in vivo and in ex vivo. Computer modelling demonstrated that this pattern could be reproduced by summating end diastolic flow with a high pulsatility index in the UA in the presence of a large AAA. Consistent with this, MC twins with a transmitted pattern had larger AAAs (median diameter 4.3 mm interquartile range 4.1–5.2) compared to MC twins discordant for intrauterine growth restriction (2.1 mm interquartile range 1.5 to 2.8) (P

Multiple pregnancy rates remain high after assisted conception because of a misconceived assumption that transferring three or more embryos will maximize pregnancy rates. Maternal morbidity is sevenfold greater in multiple pregnancies than in singletons, perinatal mortality rates are fourfold higher for twins and sixfold higher for triplets, while cerebral palsy rates are 1-1.5% in twin and 7-8% in triplet pregnancies. Therefore, multiple pregnancies must be considered a serious adverse outcome of assisted reproductive techniques. Primary prevention of multiple pregnancies is the solution. The overwhelming evidence presented in this chapter demonstrates that limiting the embryo transfer in in vitro fertilization to two embryos would significantly reduce adverse maternal and perinatal outcomes by reducing the incidence of high order multiple pregnancies without reducing take-home-baby rates. Secondary prevention by multifetal pregnancy reduction is effective, but not acceptable to all patients. New developments in blastocyst culture, single embryo transfer, embryo cryopreservation and pre-implantation aneuploidy exclusion, should allow improvements in pregnancy rates without increasing multiple pregnancies.

Objectives: Our purpose was to describe the fetal loss rate and platelet dynamics in fetal alloimmune thrombocytopenia managed by serial platelet transfusions. Methods: Retrospective analysis over 10 years of consecutive pregnancies affected by fetal alloimmune thrombocytopenia requiring in utero platelet transfusions. Results: There were 2 perinatal losses in 12 pregnancies managed by 84 platelet transfusions. One was obviously procedure related from exsanguination despite platelet transfusion. The attributable procedurerelated fetal loss rate was 1.2% per procedure but 8.3% per pregnancy. The median rate of fall in fetal platelet count per day after transfusion was lower at the placental cord insertion (n = 54) 40.5 × 10 9 /L (range, 5.4-96.1 × 10 9 /L) compared with that at the intrahepatic vein (n = 30) 50.9 × 10 9 /L,(range, 29.5-91 × 10 9 /L) ( P = .0009). Conclusion: Pooling our results with those previously published yields a cumulative risk of serial weekly transfusions of approximately 6% per pregnancy, indicating the need for development of less invasive approaches. (Am J Obstet Gynecol 2002;186:826-31.)

Invasive diagnostic and therapeutic techniques are increasingly applied to the fetus. It is not known if the fetus feels pain during such procedures, but the fetus does mount significant stress hormonal and circulatory changes in response to these from 18-20 weeks. Perinatal stress may have long-term neurodevelopmental implications. During open fetal surgery, maternal general anaesthesia provides fetal anaesthesia. However, in closed procedures, fetal analgesia presents difficulties. The optimal drug, dose, and route of administration remain to be determined.

Fetal abnormality is more common in multiple than in singleton pregnancies. This, together with the requirement to consider the risks with at least two babies to sample correctly each fetus and to undertake accurately-targeted selective termination, amounts to a major challenge for obstetricians involved in prenatal diagnosis. Early determination of chorionicity should be routine, since this influences not only the genetic risks but also the invasive procedure chosen for karyotyping or genotyping. Assessment of nuchal translucency identifies individual fetuses at risk of trisomy. Contrary to expectation, invasive procedures in twins appear to have procedure-related miscarriage rates that are similar to those in singletons. Instead, contamination remains a concern at chorionic villus sampling. Elective late karyotyping of fetuses may have a role in some countries. Whereas management options for discordant fetal abnormality are relatively straightforward in dichorionic pregnancies, monochorionic pregnancies are at risk of co-twin sequelae after any single intrauterine death. Techniques have now been developed to occlude completely the cord vasculature by laser and/or ultrasound guided bipolar diathermy. Given the complexities associated with prenatal diagnosis, all invasive procedures in multiple pregnancies should be performed in tertiary referral centres.

Monochorionic (MC) placentation occurs in two-thirds of monozygous twins and approximately 0.3% of all spontaneous conceptions. Vascular anastomoses within the placenta allow intertwin transfusion to occur, which is in most cases a normal event. However, imbalance in this flow may lead to the clinical sequelae observed, with acute, chronic or reverse intertwin transfusion. Acute transfusion describes the events following the death of one of an MC twin pair in utero, with an approximately 1 in 4 chance of demise or neurological damage in the co-twin. Chronic intertwin transfusion, otherwise termed feto-fetal transfusion syndrome, arises following the gradual flux of blood from one twin (the donor) to its sibling fetus (the recipient). The ensuing polyhydramnios places the pregnancy at risk of preterm amniorrhexis and/or labour, notwithstanding the specific sequelae seen in these fetuses following chronic in utero insult. Reverse transfusion describes the most bizarre form of intertwin transfusion: acardiac twinning. Retrograde perfusion of one of the twins with deoxygenated blood leads to the formation of a non-viable acardiac parasite and a 'pump twin' struggling to maintain the cardiac output required to perfuse both twins.

Throughout history twins have been the subject of either wonder or condemnation by various religious and cultural groups. Indeed, in some parts of the world even today twins are believed to be non-human and are punished accordingly. There has long been scientific interest in twin pregnancies, from Hippocrates and Aristotle to Galton, who was the first to appreciate the unique value of twins in research.

We investigated whether reliable prenatal diagnosis is possible from fetal cells harvested transcervically in first-trimester pregnancies.Fetal cells were obtained transcervically from 87 women undergoing pregnancy termination. Fetal gender was determined in 51 pregnancies with three different polymerase chain reaction techniques and in 36 pregnancies with fluorescent in situ hybridization. In known male pregnancies the number of male fetal cells present was also determined.Polymerase chain reaction detected male deoxyribonucleic acid in up to 79% of cases in male pregnancies and up to 45% of cases in female pregnancies. Fetal gender was correctly predicted in up to 72% of cases with fluorescent in situ hybridization. However, fetal cells were identified in40% of informative male pregnancies and were present in low numbers-0.7% to 3.4% in swabs and 4.4% to 24.8% in flushes.The use of fetal cells obtained by minimally invasive first-trimester transcervical sampling is unreliable for prenatal diagnosis.

Our purpose was to determine whether the presence of heme pigments in amniotic fluid is associated with the ultrasonographic findings of increased fetal bowel echogenicity in the second trimester.Spectrophotometric analysis of amniotic fluid for optical density at 410 nm was prospectively performed to study the presence of heme pigments in (1) 104 pregnancies undergoing second-trimester amniocentesis for routine cytogenetic indications and (2) in 14 pregnancies undergoing amniocentesis for prenatal karyotyping because of fetal strongly echogenic bowel. In the routine amniocentesis group the fetal small bowel echogenicity was assessed immediately before amniocentesis and classified as nonechogenic (n = 64), mildly echogenic (n = 36), or hyperechogenic (n = 4) with the fetal iliac wing and liver used as references. Only amniotic fluid specimens that were obtained at the first attempt and that were not blood-stained were included in this study, with the first milliliter being discarded in all samples.In the routine amniocentesis group abnormal amniotic fluid optical density readings were significantly more frequent in fetuses with increased bowel echogenicity compared with those with nonechogenic bowel (8/40 [20%] vs 3/64 [5%], respectively; p0.001). In the hyperchogenic bowel group abnormal amniotic fluid optical density readings were found in four samples (29%). Overall, 12 of 54 fetuses (22%) with increased bowel echogenicity had a detectable peak at 410 nm. Three of the 12 (25%) fetuses with echogenic bowel and positive readings for hemoglobin were chromosomally abnormal.Fetal small bowel echogenicity is associated with the presence of heme pigments in amniotic fluid as determined by amniotic fluid optical density at 410 nm. Swallowing of amniotic fluid after intraamniotic bleeding seems implicated in the etiology of second-trimester echogenic bowel in both euploid and aneuploid fetuses.

OBJECTIVE: In spite of recent advances in the assessment and treatment of fetofetal transfusion syndrome, its underlying mechanism remains controversial. We aimed to determine whether the clinical features of fetofetal transfusion syndrome could be explained by unidirectional or bidirectional intertwin transfusion along placental vascular anastomoses. STUDY DESIGN: We constructed a dynamic computerized model of monochorial twin fetoplacental units on the basis of numerous interrelated hemodynamic, osmotic, and metabolic physiologic variables. The circulations were then linked by various combinations of direction and number of arteriovenous anastomoses. RESULTS: With unidirectional anastomoses disease severity, characterized by disparity in blood solids, depended on donor arterial pressure but not on the number of anastomoses. In the chronic state water movement resulting from raised osmotic pressure in the recipient and reduction in the donor produced hydroosmotic pressure equilibrium, reducing anastomotic flow to near zero. Atrial natriuretic peptide - driven urine production was markedly increased in the recipient because of the raised vascular hydrostatic pressure component. With bidirectional anastomoses recirculation between twins reduced discordancy in colloids and hematocrit, and the clinical picture was determined by the degree of asymmetry in the number of connections. CONCLUSIONS: Severe manifestations of fetofetal transfusion syndrome can be explained by unidirectional intertwin transfusion and lesser degrees by asymmetric bidirectional transfusion. (AM J OBSTET GYNECOL 1996;174:598-608.)

Expansion of amniotic fluid volume is usually achieved by instillation of sterile fluid into the amniotic cavity by either the transabdominal or transcervical route. However, it has recently been demonstrated that oral hydration of the mother can have the same effect. Amnioinfusion always has the same indication, which is expansion of reduced amniotic fluid volume, but has several different applications. Oligohydramnios hinders ultrasound diagnosis as the lack of acoustic window and degree of fetal flexion limit the view obtained. Diagnostic amnioinfusion circumvents these problems by restoring amniotic fluid volume. Midtrimester oligohydramnios is associated with a poor fetal prognosis, whatever the underlying aetiology. Part of this is attributable to the underlying pathology, but a significant proportion is mediated through the fetal effects of oligohydramnios. Thus, serial amnioinfusion has been proposed as a therapeutic technique to maintain amniotic fluid volume. Amnioinfusion can be used to introduce antibiotics into the uterine cavity either for infection prophylaxis or treatment. Intrapartum amnioinfusion has been used to dilute thick meconium to prevent meconium aspiration, and to alleviate fetal heart rate pattern abnormalities. This article will examine these different roles of expansion of amniotic fluid volume.

Our purpose was to examine the reproducibility of intertwin membrane thickness measurements used to predict chorionicity in twin pregnancies.Twenty-seven twin pregnancies were scanned with high-resolution ultrasonography on 52 occasions during the second and third trimesters. Two observers, blind to other criteria of chorionicity, measured the dividing membrane twice in five different sites (total measurements 1040). The data were log-transformed and the coefficient of repeatability calculated as a measure of intraobserver variability. Interobserver variability was estimated by the Bland and Altman 95% limits of agreement. Random variation was assessed with the restricted maximum likelihood procedure in Genstat.The overall estimate of the coefficient of repeatability was 2.14, indicating that 95% of repeated measurements would be expected to be within 114% of each other. Measurements taken close to the placenta (up to 3 cm) were the most repeatable and displayed no bias when repeated. Coefficients of repeatability at this site ranged from 1.42 to 1.91, with no evidence of consistent differences between monochorionic and dichorionic twins. Gestational age was not significantly associated with membrane thickness for any of the models. The pregnancy type x subject x observer and pregnancy type x subject x site interactions were statistically significant (p0.001 and p0.005, respectively), implying that interobserver variability depends on the subject being measured, the site of sampling, and chorionicity.Ultrasonographic measurement of membrane thickness has high intraobserver and interobserver variability in the second and third trimesters. Our findings provide an explanation for the suboptimal accuracy reported with this method in determining chorionicity in the second and third trimesters.

Our purpose was to determine whether acute maternal hydration in pregnancies with third-trimester oligohydramnios (1) increases amniotic fluid index and hourly fetal urine production rate and (2) alters uteroplacental perfusion and fetal blood flow.Ten women with third-trimester oligohydramnios (amniotic fluid indexor = 5 cm) and 10 controls with normal amniotic fluid volume (amniotic fluid index7 cm) were prospectively recruited for this study. Maternal plasma and urine osmolality, amniotic fluid index, hourly fetal urine production rate, and Doppler flow velocimetry of maternal uterine artery and fetal umbilical, descending aorta, middle cerebral, and renal arteries were determined before and after oral hydration by having the patient drink 2 L of water over 2 hours.There was a significant reduction in maternal plasma (p0.05) and urine osmolality (p0.0001) in both groups after short-term oral hydration. Hydration increased amniotic fluid volume in women with oligohydramnios (mean change in amniotic fluid index 3.2 cm, 95% confidence intervals 1.1 to 5.3; p0.02) but not in those with normal amniotic fluid volume (mean change in amniotic fluid index -2.0, 95% confidence intervals -4.1 to +0.2). The hourly fetal urine production rate, however, did not increase in either group (mean change in hourly fetal urine production rate 3.5 ml/hr, 95% confidence intervals -11.7 to +18.7 and -6.8 ml/hr, 95% confidence intervals -2.9 to -10.7, respectively). Hydration was associated with an increase in uterine artery mean velocity in the oligohydramnios group (mean change in mean velocity 16.7 cm/sec, 95% confidence intervals 8.0 to 25.3; p0.006) but not in controls (mean change in mean velocity 1.2 cm/sec, 95% confidence intervals -19.7 to +22.1). There was no change in pulsatility index or in velocity in any of the fetal vessels studied in either group.Short-term maternal oral hydration increases the amniotic fluid index in women with third-trimester oligohydramnios. Although the mechanism for this effect remains unclear, it could not be accounted for by fetal urination in this study but instead was associated with improved uteroplacental perfusion.

OBJECTIVE: Our purpose was to develop a molecular assay to determine the fetal RhD blood type on single diploid cells, including blastomeres. STUDY DESIGN: Polymerase chain reaction amplification of a 99 bp deoxyribonucleic acid fragment of the RhD gene or a 113 bp fragment from the RhCE gene was performed from 20 venous blood samples and 20 amniotic fluid samples and from 60 single-cultured lymphoblasts and 12 media blanks mixed in a blinded fashion. This reaction was similarly tested after whole-genome amplification on 10 lymphoblasts and seven human blastomeres. RESULTS: Deoxyribonucleic acid amplification was successful and correct from all genomic deoxyribonucleic acid samples. Ninety-seven percent of single cells amplified: correct diagnosis was made in 96%. Five blastomeres successfully amplified. No media blanks produced amplified, contaminating deoxyribonucleic acid. CONCLUSIONS: The RhD blood type can be determined reliably from single cells and can be used for preimplantation genetic diagnosis for the prevention of rhesus hemolytic disease.

OBJECTIVE: Our purpose was to describe the clinical utility of a deoxyribonucleic acid amplification method for determining fetal RhD status in alloimmunized pregnancies STUDY DESIGN: Six RhD-negative women with alloimmunized pregnancies and heterozygous partners underwent amniocentesis ( n = 5) or chorionic villus sampling ( n = 1). Fetal RhD type was determined by polymerase chain reaction and results disclosed to the attending physicians. RESULTS: Knowledge of the fetal RhD status avoided further invasive procedures in two pregnancies and facilitated the timing or performance of intrauterine transfusions in the remainder. CONCLUSIONS: In alloimmunized pregnancies the ability to RhD-type the fetus in amniotic fluid avoids the risks of fetomaternal. hemorrhage and increased sensitization associated with fetal blood sampling or chorionic biopsy. This allows more rational pregnancy management, avoiding invasive procedures in the presence of an RhD-negative fetus, or planning therapeutic interventions or offering termination of pregnancy in the presence of an RhD-positive fetus.

The purpose of this study was to investigate whether the fetus mounts a hormonal stress response to a potentially painful procedure, intrauterine needling. Cortisol and beta-endorphin concentrations in fetal plasma obtained during uncomplicated fetal blood sampling or intrauterine transfusions by needling the fetal intra-abdominal portion of the umbilical vein (intrahepatic vein) were compared to hormone concentrations in fetal plasma obtained by the conventional technique of needling the placental cord insertion, which is not innervated. Cortisol and beta-endorphin concentrations did not increase within 10 minutes of fetal abdominal needling (n = 15). However, more prolonged needling during transfusion at the intrahepatic vein was associated with an increase in fetal plasma cortisol (median increase 48 nmol/L; 95% Cl, 23-86) and beta-endorphin (207 pg/mL; 113-307) concentrations compared to transfusion at the placental cord insertion (p0.005 for both hormones). The magnitude of rise in hormone increased linearly with the duration of needling (cortisol, r = 0.80; beta-endorphin, r = 0.88, p0.05 for both). These data suggest that the fetus mounts a hormonal stress response to invasive procedures. They raise the possibility that the human fetus feels pain in utero, and may benefit from anaesthesia or analgesia for invasive procedures.

Our purpose was to study the effect of operator transducer pressure on amniotic fluid index and single deepest pool measurements and their intraobserver and interobserver variability.Forty subjects in the third trimester with intact membranes were studied by two observers at three predetermined operator pressures. Pressure was measured by interfacing a flexible water-filled reservoir connected to a manometer between the maternal abdomen and the ultrasonography transducer. The amniotic fluid index and single deepest pool were measured on screen by another operator, and data were analyzed by calculating components of variance.Compared with medium pressure, low pressure resulted in a 13% increase in amniotic fluid index (p0.001), and high pressure resulted in a 21% fall in amniotic fluid index (p0.001). The single deepest pool was less sensitive to pressure, with a 11% increase at low pressure (p0.001) and a 16% fall at high pressure (p0.001). Intraobserver error was 17% for amniotic fluid index and 18% for single deepest pool when operator pressure was controlled at medium pressure; this increased to 28% and 24%, respectively, when pressure was not controlled (p0.01). Interobserver error was consistently very low.Both techniques are highly sensitive to the pressure applied to the maternal abdomen during scanning. The reproducibility of amniotic fluid index and single deepest pool is comparable. The use of the same observer for serial measurements of amniotic fluid index would appear to be less important than careful attention to transducer pressure.

Chorioamnionitis is strictly defined as inflammation of the chorioamniotic membranes. The term has occasionally been used to indicate the clinical syndrome of infection within the amniotic cavity but this is more appropriately termed amniotic infection syndrome. Although chorioamnionitis has generally been associated with clinical amniotic infection and pre-term labour some authors have considered it to be a non-specific inflammation of minimal perinatat significance (Driscoll et al, 1973). This apparent contradiction is due, at least in part, to differences to histological techniques and pathological criteria. Chorioamnionitis begins with an initially patchy polymorphonuclear leukocyte infiltration into the chorion which becomes more diffuse to involve the fetal surface of the amnion and the placenta. Preparation of fetal membrane rolls for histological examination allows a large area to be examined and diffuse chorioamnionitis to be distinguished from small areas of inflammation, for example at the site of membrane rupture. Pre-term labour, defined as labour before 37 weeks of amenorrhoea, remains a significant problem in obstetric practice. It occurs in up to 10% of all births but is associated with 85% of perinatal deaths in otherwise normal babies (Rush et al, 1976) and accounts for an unknown proportion of physical and mental handicap. Multiple pregnancy, polyhydramnios or Miillerian anomalies account for only a minority of cases of pre-term labour; in most cases the cause is unknown. The most reliable predictor that a woman will deliver pre-term is that she had done so in a previous pregnancy. Pre-term labour is weakly associated with advanced maternal age, cigarette smoking, high parity, and poor socioeconomic status (Newcombe and Chalmers, 1981). Only in the past decade has the role of infection or abnormal colonization of the genital tract been recognized as a major factor in the aetiology of pre-term labour.

Objective: The etiology of oligohydramnios-related pulmonary hypoplasia is not understood but is known to involve chronic lung liquid loss. We tested the hypothesis that low amniotic pressure in oligohydramnios disturbs the normal tracheal-amniotic pressure gradient to increase lung liquid loss and impair lung development. Study Design: Chronic pharyngeal catheterization with drainage to the exterior was used in 15 fetal sheep to mimic reduced amniotic pressure at the upper airway in the presence of normal amniotic fluid volume. Results: Pharyngeal pressures relative to amniotic pressures were negative in all drained fetuses (mean ± SE −3.0 ± 0.6 mm Hg), in contrast to positive pressures in controls (0.7 ± 0.1 mm Hg). There was no significant difference in lung weight or deoxyribonucleic acid relative to body weight, or in lung morphometry, between 10 fetuses drained for 10 to 21 days and their control cotwins. Conclusion: Mimicking low amniotic pressure in the upper airway by chronic fetal pharyngeal drainage does not impair lung development in fetal sheep.

The mechanism of oligohydramnios-related pulmonary hypoplasia (PH) is not understood, but is known to involve chronic loss of lung liquid. In order to investigate the recent suggestion that fetal breathing movements are absent or diminished in oligohydramnios-related PH, we made continuous recordings of fetal breathing movements (FBM) during chronic lung drainage in tracheostomized fetal sheep. FBM remained present throughout the drainage period. There was no significant difference in the number or duration of FBM epochs in tracheostomized fetuses compared to controls, although there was a slight reduction in incidence between 126 and 133 days (P = 0.02). PH in the fetuses undergoing chronic lung liquid drainage was confirmed by significant reductions in lung weight, volume, DNA, and total airspace, and in altered maturation. This study is consistent with other data suggesting that inhibition of FBM is not the primary mechanism of oligohydramnios-related pulmonary hypoplasia.

In this pilot study, 50 patients underwent preinduction cervical assessment by digital Bishop Score (BS) and transvaginal ultrasonography. The BS was lower in 7 patients requiring caesarean section than in 43 delivered vaginally (P = 0.017). Of seven ultrasound parameters examined, six (cervical length, width, dilatation, application and position and lower segment thickness) were similar in both groups. Cervical angle, however, was more acute in those delivered abdominally than vaginally (median = 60 degrees and 90 degrees, respectively, P = 0.002). Posterior cervical angle was more accurate than BS in predicting vaginal delivery (Kappa = 0.48 versus 0.21). Patient discomfort was significantly less with transvaginal ultrasound than digital examination. Combining posterior cervical angle greater than 70 degrees and BS greater than 5 yielded the best accuracy in predicting successful induction of labour (sensitivity = 88%, specificity = 100%, Kappa = 0.68).

We measured the effects of 2 minutes of vibroacoustic stimulation on the activity of unanesthetized fetal sheep in utero. We were unable to detect any changes in activity after stimulation with a model 5C electrolarynx. With more powerful stimulation using a mechanical oscillator, fetal electrocortical activity desynchronized. Fetal breathing and eye movements changed with electrocortical activity and there was an increase in nuchal muscle activity. Stimulation caused a small decrease in fetal heart rate. Responses to stimulation were not seen in four of the five fetuses with bilateral cochlear ablation. These results indicate that in fetal sheep the auditory apparatus is necessary for the detection of vibroacoustic stimuli. The difference in responsiveness of the sheep and human fetus appears to be because of a difference in central processing after detection of the stimulus.

Fetal acid-base status was evaluated on 66 blood samples taken for rapid karyotyping from 58 growth-retarded fetuses. Before blood sampling, doppler blood flow studies of the umbilical artery showed end-diastolic frequencies to be absent in 32 fetuses (group 1) and present in 26 (group 2). Fetuses with chromosomal (n = 4) or structural (n = 8) abnormalities were excluded from subsequent analysis. Gestational age at blood sampling (27.8 [95% CI 26.5-29.1] vs 32.2 [30.4-34.1] weeks) and time from sampling to delivery (median 2 (range 0-35] vs 14 [0-77] days) were significantly lower in group 1 than group 2. There were no perinatal deaths in group 2 whereas mortality in group 1 was 65.4%. There were significant differences between the groups at blood sampling in pH, pO2, pCO2, base equivalents, and nucleated-red-cell count, but within group 1 these measurements were similar in surviving fetuses and those who died perinatally. Since acid-base determination does not predict perinatal outcome in growth-retarded fetuses, fetal blood sampling has a limited role in monitoring fetal wellbeing.

Analysis of archival samples from cohorts of pregnant women may be key to discovering prognosticators of stillbirth and pregnancy/perinatal complications. Growth hormone (GH) and its receptor (GHR) are pivotal in feto-placental development and pregnancy maintenance. We report a rapid, optimized method for genotyping the GHR full-length versus exon 3-deleted isoform (GHRd3). TaqMan single nucleotide polymorphism (SNP) genotyping proved superior to standard multiplex polymerase chain reaction (PCR) in allele detection and GHR genotyping from archived samples, including those with poor genomic deoxyribonucleic acid quality/quantity such as formalin fixed, paraffin embedded, blood, and serum. Furthermore, this assay is suitable for high through put 96 or 384-well plate quantitative PCR machines with automated genotype calling software. The TaqMan genotyping assay can increase the data obtained from precious archival human samples.

This letter comments on a study which provides new estimates for maternal mortality rates (MMR) in 181 countries. The authors of the letter state that unsafe abortion - an important factor in maternal deaths in many countries and one identified as explaining the observed reduction in MMR in several of the country case studies cited - was surprisingly not mentioned in the paper in addition to some unexpected country estimates.