Novel approaches to manipulating foetal cells in the maternal circulation for non-invasive prenatal diagnosis of the unborn child

Due to the risks to the foetus with invasive prenatal diagnosis, non-invasive prenatal diagnosis (NIPD) is gaining tremendous interest but no reliable method that can be widely used has been developed to date. Manipulation of foetal cells and foetal cell-free genetic material in the maternal blood are two promising approaches being researched. The manipulation of foetal cells in the maternal circulation is more popular as it can provide complete genetic information of the foetus particularly the diagnosis of aneuploidies. However, the foetal cell numbers in the maternal circulation are small and their enrichment and ex vivo culture remain two major challenges for NIPD. Primitive foetal erythroblasts (pFEs) have been considered as a good potential candidate for early first trimester NIPD but their nature, properties and manipulation to provide adequate cell numbers remain a challenging task and several approaches need to be meticulously evaluated. In this review we describe the current status of NIPD and suggest some novel approaches in manipulating pFEs for future clinical application of NIPD. These novel approaches include (1) understanding the pFE enucleation process, (2) enriching pFE numbers by individual pick-up of pFEs from maternal blood using micromanipulation and microdroplet culture, (3) expansion of pFEs using mitogens and (4) decondensation of the pFE nucleus with histone deacetylase (HDAC) inhibitors followed by reprogramming using gene delivery protocols with/without small reprogramming molecules to improve reprogrammed pFE proliferation rates for successful NIPD.     

Non-invasive optical monitoring of cerebral blood oxygenation in the foetus and newborn: preliminary investigation

Near infra-red spectroscopy was applied as a non-invasive and continuous technique for the in vivo monitoring of blood and tissue oxygenation in human neonates. Monitoring of cerebral blood oxygenation in the wavelength range 775–904 nm was carried out on preterm infants after inducing a transient mild hypoxic change; the measurements were performed either by the transmission or reflection (backscattering) mode of monitoring. The results of these investigations were used to assess the application of the technique to foetal monitoring. A series of foetal monitoring studies was performed to investigate the influence of maternal contractions on foetal cerebral blood oxygenation. Although only changes in haemoglobin concentration can be monitored at present, the results suggest that near infra-red monitoring could provide a non-invasive, real-time monitoring method in intensive neonatal and intrapartum care.     

Placentation and associated aspects of gestation in the bonnethead shark, Sphyrna tiburo

The left ovary of the bonnethead shark, Sphyrna tiburo, is rudimentary, and the right ovary supplies both oviducts which share a common ostium situated in the falciform ligament. Preceding ovulation the nidamental gland of each oviduct hypertrophies and the caudal two-thirds of each oviduct is modified to form a uterus. In the Florida-Caribbean area Sphyrna tiburo probably mates in March and 3–7 eggs are fertilized in the vicinity of the nidamental gland of each oviduct. The developing embryo is nourished during the first 3–4 months of gestation by yolk stored in its extensive yolk sac. Approximately three and one-half months after fertilization, the distal portion of the yolk sac becomes convoluted and interdigitates with deep folds in the uterine wall to form a yolk-sac placenta. As the placenta develops, the maternal uterine epithelium is reduced from columnar cells to squamous cells, and the foetal yolk-sac epithelium is reduced from columnar and cuboidal cells to squamous cells. Exchange between the maternal and foetal blood systems takes place through maternal endothelium, reduced maternal epithelium, egg-case membrane, reduced foetal epithelium, and foetal endothelium.     

Non-invasive prenatal diagnosis by massively parallel sequencing of maternal plasma DNA

The presence of foetal DNA in the plasma of pregnant women has opened up new possibilities for non-invasive prenatal diagnosis. The use of circulating foetal DNA for the non-invasive prenatal detection of foetal chromosomal aneuploidies is challenging as foetal DNA represents a minor fraction of maternal plasma DNA. In 2007, it was shown that single molecule counting methods would allow the detection of the presence of a trisomic foetus, as long as enough molecules were counted. With the advent of massively parallel sequencing, millions or billions of DNA molecules can be readily counted. Using massively parallel sequencing, foetal trisomies 21, 13 and 18 have been detected from maternal plasma. Recently, large-scale clinical studies have validated the robustness of this approach for the prenatal detection of foetal chromosomal aneuploidies. A proof-of-concept study has also shown that a genome-wide genetic and mutational map of a foetus can be constructed from the maternal plasma DNA sequencing data. These developments suggest that the analysis of foetal DNA in maternal plasma would play an increasingly important role in future obstetrics practice. It is thus a priority that the ethical, social and legal issues regarding this technology be systematically studied.     

Effects of a beta-sympathomimetic drug on the foetal and maternal cardiovascular system in a chronic sheep model

The effects of the beta-sympathomimetic agent terbutalin (1-/3.5 dihydroxyphenyl/-allilaminoethane sulphonate) on maternal and foetal circulation were studied in 11 ewe at 110-130 days of pregnancy. None of them exhibited uterine contractions. Catheters were implanted in the carotid artery and jugular vein, together with four stainless steel electrodes on the limbs. Terbutalin was added intravenously in Ringer-lactate (5%) infusion (1 ml/min). The following maternal and foetal parameters were measured simultaneously: heart rate, central arterial pressure, blood glucose levels, ECG. Intravenous administration of 100 micrograms terbutalin resulted in a 19% increase of maternal and 10% enhancement of foetal heart rate. Maternal systolic blood pressure rose by 9%, whereas diastolic blood pressure fell by 7%. Maternal and foetal blood glucose levels was fairly constant during the entire experiment. In further six experiments terbutalin was administered into the foetal circulation (1 ml/3 min). The same effects on foetal heart rate and pulse pressure were observed as after injecting into the maternal circulation, however but the time necessary for the maximum action to develop was significantly shorter.     

Refined quantitative fluorescent PCR of Y-chromosome DNA sequences mosaics in Turner's syndrome patients--alternative to real-time PCR

BACKGROUND: Real-time polymerase chain reactions (PCRs) are the most frequently used techniques for gonosomal mosaics quantification. The primary aim of this work is to assess and optimize the refined technique of quantitative fluorescent polymerase chain reaction (RQF PCR) in the quantification of Y-chromosome sequences in gonosomal mosaics. The method was applied to the analysis of Y-chromosome sequences (amelogenin gene, AMELX/Y-loci) in peripheral lymphocytes and gonadal tissues in Y-positive Turner's syndrome (TS) patients. METHODS: RQF PCR was used for molecular quantification, and fluorescent in situ hybridization (FISH) technique was used for comparison. RESULTS: Based on a formulated calibration curve, DNA mosaics from six Y-positive patients and gonads from one patient were deducted. For calculation of rare mosaics, it is possible to take advantage of a new empirical formula. FISH results were comparable to RQF PCR. CONCLUSION: The sensitivity of RQF PCR brings significant progress in the analysis of gonosomal aberrations. RQF PCR also finds applications in prenatal diagnostics of maternal contaminations of amniotic fluid and foetal DNA in maternal blood and analysis of chimerism in patients after bone marrow transplantation. The method is very convenient for determining the number of testis-specific protein, Y-linked (TSPY) gene repetitions.     

Human type 2 17beta-hydroxysteroid dehydrogenase mRNA and protein distribution in placental villi at mid and term pregnancy

Background  

During human pregnancy, the placental villi produces high amounts of estradiol. This steroid is secreted by the syncytium, which is directly in contact with maternal blood. Estradiol has to cross placental foetal vessels to reach foetal circulation. The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) was detected in placental endothelial cells of foetal vessels inside the villi. This enzyme catalyzes the conversion of estradiol to estrone, and of testosterone to androstenedione. It was proposed that estradiol level into foetal circulation could be regulated by 17beta-HSD2.     

Microdensitometric and autoradiographic comparison of the DNA contents of foetal and adult rat liver nuclei

Summary Glare-corrected, scanning Feulgen microdensitometry and ³H-thymidine autoradiography were applied to squash preparations of rat 18-day foetal and maternal liver cells, and to smears of maternal blood. No significant differences were found between the mean Feulgen-DNA contents of autoradiographically unlabelled diploid foetal and maternal hepatocytes. The Feulgen-DNA contents of other unlabelled foetal and maternal hepatocytes were also as predicted by the DNA-constancy hypothesis, i.e. were twice or four times that of diploid cells. Small (less than about 4%) but statistically significant discrepancies in the mean Feulgen-DNA contents of foetal haemopoietic cells and adult leucocytes were attributable to uncorrected residual distribution and chromatic errors in the microdensitometry.None of the 371 maternal nuclei measured had Feulgen-DNA contents substantially (i.e. more than ±10%) different from a modal value. About 12% of these nuclei were classified as labelled. Evidence was found suggesting a significantly non-random distribution of background grains in the autoradiographs, which would materially affect the proportion of cells incorrectly classified. After taking this factor into account there seems no reason to suppose that the apparently labelled adult nuclei were in fact synthesising DNA.Of 376 foetal cells measured, 107 had inter-modal Feulgen-DNA contents. Eleven of these were classified as unlabelled. All the inter-modal cells were however probably in the S-phase of the cell cycle, statistical variation in autoradiographic grain distribution accounting for those appearing to be unlabelled.Our results are consistent with the DNA-constancy hypothesis, and are at variance with previous claims for the existence of metabolic DNA in adult and/or foetal rat hepatocytes.     

Doppler evaluation of maternal and foetal vessels during normal gestation in queen

The aim of this work is to evaluate the haemodynamic characteristics of maternal and foetal vessels during normal pregnancy in queens, using colour Doppler and pulsed wave Doppler ultrasonography, in order to obtain information about maternal and foetal circulation. The blood waveforms of the uteroplacental arteries, aorta, caudal cava vein and umbilical cord of the fetuses were recorded weekly in seven healthy pregnant queens. Also, the measurements of peak systolic, end diastolic velocities, resistance and pulsatility indices were carried out. Uteroplacental blood flow was biphasic while the ones of the umbilical artery and aorta were first systolic and then diastolic. The caudal cava vein showed a typical waveform of venous vessels. During gestation the EDV and PSV of foetal vessels increased ( < 0.05) while the PI and RI of all vessels examined decreased ( < 0.05) except for the IP of the aorta. The Doppler ultrasonography, also in queens, can be used to evaluate the characteristics of maternal and foetal vessel flow and their progressive changes during pregnancy. This study can be considered the basis for further contribution in diagnosing and monitoring high-risk pregnancies in Veterinary Medicine.     

A method for obtaining mitotic figures from blood leucocyte cultures of rainbow trout, Salmo gairdneri

A method for obtaining dividing cells for chromosome analysis from blood cultures of the rainbow trout, Salmo gairdneri , is described. The effects of different phytohaemagglutinin and foetal calf serum concentrations, oxygenation of the cultures, and varying the initial volume of cell inoculum on the number of mitoses obtained are described and discussed.     

Prostaglandin production in the umbilical and uterine circulations in pregnant sheep at 129-136 days gestation.

Prostaglandins circulating in the maternal and foetal blood have been implicated in important physiological systems. These functions include foetal adrenal function, maintenance of patency of the ductus arteriosus, regulation of uterine and umbilical circulations, and labor and delivery type myometrial contractions. The placenta is a major site of prostaglandin production in pregnancy. Limited data are available which combine measurements of veno-arterial differences across the uterine and umbilical circulations with blood flow in these circulations to enable calculation of umbilical-placental and utero-placental production rates for the prostaglandins. In chronically instrumented pregnant ewes, between 129 and 136 days of gestation, prostaglandin F2 alpha(PGF2 alpha), 13, 14 dihydro-15-keto prostaglandin F2 alpha (PGFM), prostaglandin E2 (PGE2) were measured in the maternal carotid artery and uterine vein. Foetal PGE2, and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) (the major metabolite of prostacyclin) were measured in umbilical venous and foetal descending aorta arterial plasma. Umbilical and uterine blood flow were measured using the diffusion-equilibrium technique. Uterine blood flow was 1693 +/- 137 ml.min-1 (mean +/- SEM); uterine production rates were 480 +/- 88 ng.min-1 for PGF2 alpha, 517 +/- 144 ng.min-1 for PGFM, and 165 +/- 27 ng.min-1 for PGE2. Umbilical blood flow was 147 +/- 17 ml.min-1.kg-1 foetal body weight. Umbilical production rates into the foetal circulation were 11 +/- 2 ng.min-1.kg-1 for PGE2 and 6 +/- 2 ng. ng.min-1.kg-1 foetal body weight for PGI2.     

Variations in the distribution of calcium, magnesium and inorganic phosphorus within chronically catheterized sheep conceptuses during the last eight weeks of pregnancy

The concentrations of calcium, magnesium and inorganic phosphorus were higher in foetal arterial plasma than in maternal jugular plasma in sheep examined between 90 and 145 days of gestation. During the same period the calcium and magnesium concentrations of foetal urine were usually less than amniotic fluid values which in turn were less than maternal plasma concentrations. In allantoic fluid, calcium concentrations were usually less and magnesium concentrations greater than maternal and foetal plasma values. A 2-5 fold increase in the calcium concentrations of allantoic fluid after superfical uterine surgery and in amniotic fluid from a group of foetuses that were exposed during operation, were considered to be artefacts of technique. Inorganic phosphorus concentrations in foetal urine, amniotic fluid and allantoic fluid were variable.     

Reduction of biliverdin and placental transfer of bilirubin and biliverdin in the pregnant guinea pig.

Biliverdin was reduced to bilirubin in pregnant and foetal guinea pigs, and the 100000 g supernatant from homogenates of foetal liver, placenta and maternal liver showed high biliverdin reductase activity. The placental transport of unconjugated bilirubin and biliverdin was compared by injecting unlabelled and radiolabelled pigments into the foetal or maternal circulation and analysing blood collected from the opposite side of the placenta. Injected bilirubin crossed the placenta from foetus to mother and vice versa, but injected biliverdin did not appear to cross without prior reduction to bilirubin. The guinea-pig placenta is apparently more permeable to bilirubin than biliverdin. Reduction of biliverdin to bilirubin in the foetus may, therefore, be essential for efficient elimination of haem catabolites from the foetus in placental mammals.     

Localisation and synthesis of α-fetoprotein in the rabbit

Summary The cellular location and sites of synthesis of -fetoprotein (AFP) in the foetal, neonatal and maternal rabbit, were studied by the fluorescent antibody technique and by culturing tissuesin vitro with labelled amino acids. AFP was found to be localised intracellularly within liver hepatocytes and yolk sac endoderm of the foetus, and within the maternal uterine epithelium. Analysis of extracts of the cultured tissues for incorporation of radioactivity into serum proteins separated by polyacrylamide gel electrophoresis or analysed by autoradiography of immuno-precipitation lines, confirmed that the foetal liver and yolk sac splanchnopleur were the principal sites of primary synthesis of AFP. Localisation of AFP in the uterine epithelium and other foetal organs was consistent with a secondary derivation from the uterine fluid or from the blood circulation. These findings are discussed in relationship to findings in man and other mammals.Supported by an award from the Medical Research Council to whom grateful acknowledgement is made.     

Up regulation of the maternal immune response in the placenta of cattle naturally infected with Neospora caninum

Neospora caninum is an intracellular protozoan parasite which is a major cause of abortion in cattle worldwide. It forms persistent infections which recrudesce during pregnancy leading to foetal infection and in a proportion of cases, abortion. The mechanisms underlying abortion are not understood. In this study, recrudescence of a persistent infection in eight naturally infected cows occurred between 20 and 33 weeks of gestation. Animals were killed at the time of recrudescence and parasites were detected in the placentae and foetuses. An active maternal immune response consisting of an infiltration of CD4+ and CD8+ T cells and a 46-49 fold increase in interferon-γ and interleukin-4 mRNA was detected. Other cytokines, notably interleukin-12 p40, interleukin-10 and tumour necrosis factor-α were also significantly increased and Major Histocompatibility Class II antigen was expressed on maternal and foetal epithelial and stromal fibroblastoid cells. Significantly, despite the presence of an active maternal immune response in the placenta, all the foetuses were alive at the time of maternal euthanasia. There was evidence of parasites within foetal tissues; their distribution was restricted to the central nervous system and skeletal muscle and their presence was associated with tissue necrosis and a non-suppurative inflammatory response involving lymphocytes and macrophages, irrespective of the gestational age of the foetus. Whilst an active maternal immune response to a pathogen in the placenta is generally considered to be damaging to the foetal trophoblast, our findings suggest that the presence of a parasite-induced maternal immune response in the placenta is not detrimental to foetal survival but may contribute to the control of placental parasitosis.     

Regeneration of renal proximal tubules after mercuric chloride injury is accompanied by increased binding of aminoacyl-transfer ribonucleic acid.

The liver of the foetal guinea pig accumulates a large quantity of triacyglycerol late in gestation at the same time that adipose-tissue mass grows at its maximum rate and foetal adipose-tissue lipoprotein lipase activity and sensitivity to lipolytic hormones has substantially declined. The fatty acid for triacyglycerol synthesis is not synthesized in the foetal liver and it is unlikely that it originates from any of the foetal tissues. Before the accumulation of hepatic triacyglycerol the concentration of free fatty acids increases in both the umbilical vein and the maternal inferior vena cava. This occurs at a time when the triacyglycerol lipase activity in maternal adipose tissue is elevated and the rate of lipolysis, but not of fatty acid esterification, is higher than earlier in gestation or than in the non-pregnant state. It is proposed that the increase in lipolysis in maternal adipose tissue, brought about by an increase in circulating lipolytic hormones, mobilizes fatty acid, which passes to the foetus and is partly stored as hepatic triacylglycerol. The foetal liver effectively removes both long-and short-chain fatty acids from umbilical-vein blood. The rate of placental fatty acid transfer is more than adequate to account for the triacylglycerol accumulation.     

从实验感染OPPV的山羊外周血单核细胞中分离病毒的研究

用绵羊胎肺细胞与接种绵羊进行性肺炎病毒（OPPV）的山羊外周血单核细胞共同培养的方法可以分离到病毒,这说明OPPV可以感染山羊。用细胞病变观察、间接荧光抗体试验、电镜切片观察和聚合酶链式反应对分离毒进行了鉴定,进一步证实了分离毒为OPPV。分离结果表明这是一种较为敏感的分离方法。绵羊胎肺细胞可传到40多代,且每一代次的细胞都可用于病毒的分离,因此这是一种非常实用的分离OPPV的方法。     

The passage of orally fed proteins from mother to foetus in the rat

Pregnant rats were orally fed with a mixture of bovine IgG, bovine albumin, ovalbumin and beta-lactoglobulin. Using immunoprecipitation methods, these proteins were detected in the maternal blood serum, urine and uterine fluids, and also in the foetal blood serum and the amniotic fluid. The results imply that a variety of dietary proteins, still immunoprecipitable, are able to cross the materno-foetal barriers to be detected in the foetal circulation, where they may influence the maturation of the immune system of the foetus.     

A cross-sectional study of umbilical cord blood donor profiles and their influence on umbilical cord blood collection in a Brazilian hospital

Background aimsUmbilical cord blood (UCB) cells are a new alternative to bone marrow source for hematopoietic stem cell transplantation and their use has increased in the last decade. Thus efforts are being made to improve the umbilical cord blood unit's quality. Besides compatibility, other factors, such as the total nucleated cell (TNC) count and the percentage of CD34⁺ cells in the product, are very important for a successful transplant outcome. Our aim was to describe our donor population and assess the best cord blood collection technique at Hospital Israelita Albert Einstein's cord blood bank (São Paulo, Brazil).MethodsThis was a retrospective study in which all analyses were performed simultaneously. A Student's t-test was used for qualitative variables for non-matched samples. For qualitative analyses, we used either the chi-square test or the exact Fisher's test.ResultsThe stratification of the population characteristics allowed us to determine which ones had an impact on unit volume, TNC count and percentage of CD34⁺ cells. A significant correlation was observed between donor characteristics and the quality of UCB units as related to maternal and gestational age, type of pregnancy, route of delivery, cord blood collection technique and birth weight.ConclusionsWe found that cord blood collection technique and newborn weight were significantly correlated with the TNC content. The collection technique used at our institution significantly improved the UCB unit volume and consequently the TNC count. Some findings, such as the impact of maternal age and newborn weight, have led us to re-evaluate our protocol in order to achieve better results.     

Cellular and molecular atlas of the placenta from a COVID‐19 pregnant woman infected at midgestation highlights the defective impacts on foetal health

ObjectivesThe impacts of the current COVID‐19 pandemic on maternal and foetal health are enormous and of serious concern. However, the influence of SARS‐CoV‐2 infection at early‐to‐mid gestation on maternal and foetal health remains unclear.Materials and methodsHere, we report the follow‐up study of a pregnant woman of her whole infective course of SARS‐CoV‐2, from asymptomatic infection at gestational week 20 to mild and then severe illness state, and finally cured at Week 24. Following caesarean section due to incomplete uterine rupture at Week 28, histological examinations on the placenta and foetal tissues as well as single‐cell RNA sequencing (scRNA‐seq) for the placenta were performed.ResultsCompared with the gestational age‐matched control placentas, the placenta from this COVID‐19 case exhibited more syncytial knots and lowered expression of syncytiotrophoblast‐related genes. The scRNA‐seq analysis demonstrated impaired trophoblast differentiation, activation of antiviral and inflammatory CD8 T cells, as well as the tight association of increased inflammatory responses in the placenta with complement over‐activation in macrophages. In addition, levels of several inflammatory factors increased in the placenta and foetal blood.ConclusionThese findings illustrate a systematic cellular and molecular signature of placental insufficiency and immune activation at the maternal–foetal interface that may be attributed to SARS‐CoV‐2 infection at the midgestation stage, which highly suggests the extensive care for maternal and foetal outcomes in pregnant women suffering from COVID‐19.