Fetal tissue sampling. The San Francisco experience with 190 pregnancies.

Prenatal diagnosis of genetic defects was done using fetal blood sampling in 167 at-risk pregnancies, by fetal skin biopsy in 15 pregnancies, and by fetal liver biopsy in 8 pregnancies. Fetal blood sampling was done by fetoscopy through January 1985 and by sonographically directed percutaneous umbilical blood sampling since then. In our series, cytogenetics has become the major indication for fetal blood sampling, increasing from 6% of the cases with fetoscopy to 48% with umbilical blood sampling. Fetoscopy provided pure fetal blood in 61% of cases while umbilical blood sampling provided pure fetal blood 97% of the time. The corrected risk of fetal demise after percutaneous umbilical fetal blood sampling was 2% and after fetoscopy was 4%.     

Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats: comparative effects on selected behavioural and blood variables

We compared the behaviours of rats, and measured various blood parameters, after three blood sampling techniques: orbital puncture while they were under diethyl-ether anaesthesia, blood collection by tail vein puncture under O2-N2O-halothane anaesthesia and puncture of the saphenous vein without anaesthesia. Twelve rats were subjected to the three treatments according to a Latin square design. After each treatment, the behaviour of the rats was automatically monitored using the so-called LABORAS method, which discriminates between grooming, locomotion and inactivity in rats. Based on excitation scores and urine production, it was found that induction of diethyl-ether anaesthesia combined with orbital puncture caused more distress than did the other two blood sampling techniques. The three techniques had no differential effects on the behaviours of grooming, locomotion and inactivity. Collecting 0.5 ml of blood by orbital puncture was +/-7 times faster than doing so by saphenous vein puncture and +/- 15 times faster than collecting blood by tail vein puncture while the rats were under O2-N2O-halothane anaesthesia. The levels of some haematological and plasma variables differed significantly between the three blood collection techniques. These observations may help to select the most appropriate technique of blood sampling with respect to anticipated discomfort in the animals.     

Effect of cooling and heating on the regional distribution of blood flow in fetal sheep

This is a study on the effect of cooling and heating amniotic fluid on blood flow to fetal tissues and organs. In 8 unanaesthetized, chronically-catheterised fetal sheep (129-137 days gestation) cold or warm water was passed through tubing encircling the fetus in utero and blood flow was measured using the radionuclide-labelled 15 mu spheres. Following cooling for 30 min, amniotic fluid temperature fell 9.6 degrees C to 29.9 +/- 2.1 degrees C (SEM) fetal arterial temperature fell 2.37 degrees C to 37.30 +/- 0.36, and maternal arterial temperature fell 0.53 degrees C to 38.58 +/- 0.16. Blood flow through the fetal skin fell 60% (P less than 0.01) to 13.6 ml/min per 100 g tissue. Blood flow to the brown fat increased 186% (P less than 0.05) to 99.6 ml/min per 100 g. Following warming for 20 min, fetal temperature rose to 40.43 +/- 0.19 degrees C, and skin blood flow did not change significantly relative to initial control period but rose 200% above that during cooling (P less than 0.01). During both cooling and heating, blood flow to the adrenals rose significantly (P less than 0.05) whereas flow to the carcass, brain, kidneys, and placenta was not altered detectably. Continuous sampling of blood from the inferior vena cava during microsphere injection failed to detect any evidence of arterio-venous shunting through the skin at any temperature studied. Overall, the blood flow responses are consistent with a thermoregulatory role for the skin and brown fat in the near-term fetal sheep.     

Effects of reducing uterine blood flow on fetal blood flow distribution and oxygen delivery.

We examined the effect of graded reduction in uterine blood flow on distribution of cardiac output and oxygen delivery to fetal organs and venous blood flow patterns in 9 fetal sheep using the radionuclide-labeled microsphere technique. We reduced uterine blood flow in two steps, decreasing fetal oxygen delivery to 70% and 50% of normal, and compared the results with those from a similar study from our laboratory on graded umbilical cord compression. With 50% reduction in fetal oxygen delivery, blood flow and the fraction of the cardiac output distributed to the brain, heart, and adrenal gland increased and that to the lungs, carcass, skin, and scalp decreased. Oxygen delivery to the brain and myocardium was maintained, while that to the adrenal doubled, and that to the brain stem increased transiently. The decrease in oxygen delivery to both carcass and lower body segment correlated linearly with oxygen consumption (P less than 0.001). The proportion of umbilical venous blood passing through the ductus venosus increased from 44.6% to 53% (P less than 0.05). The preferential distribution of ductus venosus blood flow through the foramen ovale to the heart and brain increased, but that to the upper carcass decreased so that ductus venosus-derived blood flow to the upper body did not change. Hence, the oxygen delivered to the brain from the ductus venosus was maintained, and that to the heart increased 54% even though ductus venosus-derived oxygen delivery to the upper body fell 34%. Abdominal inferior vena caval blood flow and its contribution to cardiac output decreased, but the proportion of the abdominal inferior vena caval blood distributed through the foramen ovale also increased from 23.0 to 30.9%. However, the actual amount of inferior vena caval blood passing through the foramen ovale did not change. There was a 70% fall in oxygen delivery to the upper body segment from the inferior vena cava. A greater portion of superior vena caval blood was also shunted through the foramen ovale to the upper body, but the actual amounts of blood and oxygen delivered to the upper body from this source were small. Thus, graded reduction of uterine blood flow causes a redistribution of fetal oxygen delivery and of venous flow patterns, which is clearly different from that observed previously during graded umbilical cord occlusion.     

Evaluation of bidirectional transfer of plasma DNA through placenta

To clarify the origin of cell-free fetal DNA in maternal plasma, we analyzed bidirectional transfer of plasma DNA between fetus and mother. We analyzed maternal and fetal plasma DNA obtained from 15 pregnant women at the time of Cesarean section. The subjects were five patients with preeclampsia and 10 gestational-age-matched normal controls. DNA was extracted from 1.5-ml plasma samples and the cellular fraction of maternal and umbilical blood. Seven polymorphic marker genes were analyzed. The relative concentration of fetal DNA in maternal plasma and maternal DNA in cord blood were evaluated. The relative concentration of maternal DNA in fetal circulation (median, 0.9%; range, 0.2–8.4%) was significantly lower than that of fetal DNA in maternal blood (14.3%, 2.3–64%), with P=0.007. The relative concentration of maternal DNA in fetal blood was not affected by preeclampsia. These findings indicate that cell-free DNA is unequally transferred through the placenta. The structural characteristics of the placenta suggest that the majority of cell-free fetal DNA in maternal plasma is derived from villous trophoblasts.     

The use of fetal bovine serum: ethical or scientific problem?

Fetal bovine serum (FBS) is a common component of animal cell culture media. It is harvested from bovine fetuses taken from pregnant cows during slaughter. FBS is commonly harvested by means of a cardiac puncture without any form of anaesthesia. Fetuses are probably exposed to pain and/or discomfort, so the current practice of fetal blood harvesting is inhumane. Apart from moral concerns, several scientific and technical problems exist with regard to the use of FBS in cell culture. Efforts should be made to reduce the use of FBS or, preferably, to replace it with synthetic alternatives.     

The impact of different blood sampling methods on laboratory rats under different types of anaesthesia

Rats with implanted telemetry transponders were blood sampled by jugular puncture, periorbital puncture or tail vein puncture, or sampled by jugular puncture in carbon dioxide (CO2), isoflurane or without anaesthesia in a crossover design. Heart rate, blood pressure and body temperature were registered for three days after sampling. Initially blood pressure increased, but shortly after sampling it decreased, which led to increased heart rate. Sampling induced rapid fluctuations in body temperature, and an increase in body temperature. Generally, rats recovered from sampling within 2-3 h, except for rats sampled from the tail vein, which showed fluctuations in body temperature in excess of 30 h after sampling. Increases in heart rate and blood pressure within the first hours after sampling indicated that periorbital puncture was the method that had the largest acute impact on the rats and that it might take an extra hour to recover from it. CO2 anaesthesia seemed unable to prevent the increase in blood pressure and the fluctuations in body temperature induced by blood sampling, and up to 10 h after sampling, the rats were still affected by CO2 anaesthesia. Rats anaesthetized with isoflurane showed lower increases in blood pressure after, and fewer fluctuations in body temperature during sampling, and the post-anaesthetic effects of isoflurane, if any, seemed to disappear immediately after sampling. It is, therefore, concluded that blood sampling in rats by jugular puncture seems to be the method from which rats most rapidly recover when compared with periorbital puncture and tail vein puncture, and that for anaesthesia, isoflurane is recommended in preference to CO2.     

Blood collection from the tail of a rat

Techniques for blood collection from the rat include puncture of the heart, retro-orbital plexus, jugular vein, saphenous vein, tail blood vessels, carotid artery, abdominal aorta, and vena cava. Most techniques (except saphenous vein and tail blood vessel puncture) require anesthesia. The following discussion focuses on two methods of blood collection - ventral tail artery puncture and dorsal or lateral tail vein puncture.     

Blood circulation and oxygen transport in the fetal guinea pig

Anaesthetized fetal guinea pigs near term were studied under conditions, where maternal placental flow of haemoglobin was maintained within the normal range. The rate of maternal fetal equilibration of intravenously injected 3H2O was found to be similar as in unanaesthetized animals (half time 4 min) indicating that fetal circulation was undisturbed under the present experimental conditions. Umbilical blood flow as determined by a modified 3H2O method was 0.13 ml . min-1 . g-1 of fetal body mass. Radioactive microspheres, injected into the fetal saphenous (jugular) vein, were distributed to the placenta, the lower body, the upper body and the lungs at a ratio of 31(47):27(39):30(6):12(8). From these data, cardiac output was calculated (0.38 ml . min-1 . g-1) and found to be almost equally distributed between the placenta, the lower body and the upper body. There was preferential streaming of the inferior vena caval blood to the upper body. There was no evidence for flow through a ductus venosus. The O2-saturation in the fetal carotid arterial blood was 59 +/- 4%. The O2-supply to the fetal tissues was estimated to be 3 times the oxygen consumption.     

Pulmonary vascular response to prostacyclin in fetal lambs

Eighteen prostacyclin injections (19.4±1.5 μg/kg) were performed in five chronically instrumented, intact fetal lambs in order to study the effects on pulmonary blood flow. These resulted in a brief period of bradycardia followed by a more prolonged period of increased pulmonary blood flow. In this latter phase, pulmonary blood flow increased from a baseline value of 49±4 ml/(kg min) to 122±10 ml/(kg min). Systolic/diastolic pulmonary arterial pressure simultaneously fell from to mm Hg. Flow through the ductus arteriosus was unchanged and right ventricular output increased to account for the increased pulmonary blood flow. Thus, prostacyclin causes pulmonary vasodilation in intact fetal lambs and may participate in the control of fetal pulmonary blood flow and the circulatory adjustments to extra-uterine life.     

ITP in pregnancy and the newborn: introduction

Idiopathic thrombocytopenic purpura (ITP) occurs more commonly in young women and is one of the commonest immune mediated disorders in pregnancy. It may exist as an incidental finding in an otherwise healthy pregnant woman or may be associated with symptomatic reduction in the platelet count and varying degrees of clinical hemorrhage. The condition termed incidental thrombocytopenia of pregnancy is invariably associated with a platelet count of greater than 100 x 10(9)/L and a very low incidence of fetal thrombocytopenia. Symptomatic thrombocytopenia is more commonly associated with low platelet counts in the fetus (estimated between 20%-40%). It has recently been suggested that the incidence of fetal thrombocytopenia is substantially lower than this figure. The management of ITP in pregnancy is complicated by the fact that fetal thrombocytopenia is difficult to diagnose and carries substantial risks during the delivery process with rare cases of fetal hemorrhage occurring spontaneously in utero. Unfortunately there are no laboratory studies that can be performed precisely in the mother that may predict the occurrence of fetal thrombocytopenia. Maternal management is usually directed towards treatment of maternal symptoms. Maternal treatment or response to treatment is inconsistently associated with predictable changes in the fetal platelet count. Obstetric management is aimed at reducing the risks of life threatening fetal hemorrhage occurring at the time of delivery, and fetal management is directed towards the obtaining of fetal platelet samples in order to plan an appropriate strategy for obstetrical delivery. Fetal blood samples are obtained either by a scalp vein puncture at the time of delivery or earlier in gestation by the use of the newer technique termed percutaneous umbilical blood sampling. Fetuses with platelet counts of less then 50 x 10(9)/L are generally delivered by cesarean section whereas those with counts greater than 50 x 10(9)/L are allowed to proceed with vaginal delivery assuming no obstetrical contraindications exist. The use of IVIgG therapy during pregnancy has theoretical implications on improving platelet counts in the mother in situations of severe hemorrhage, however cannot be considered to be appropriate treatment for the prevention of fetal thrombocytopenia, since the exogenous transport of IVIgG across the placenta appears to be inconsistent and unpredictable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cardiovascular responses to acute, severe haemorrhage in fetal sheep

In adults, the responses to acute haemorrhage vary greatly depending on the amount of blood lost. While many studies have documented fetal responses to mild haemorrhage, fetal responses to severe haemorrhage are not known. In this study we examined the effect of acute, severe haemorrhage in fetal lambs. Despite the severity of haemorrhage, we found that mean arterial blood pressure was restored within 2 min, and heart rate was restored within 30 min. This restoration of blood pressure and heart rate was facilitated by an increase in peripheral vascular resistance mediated in part by secretion of catecholamines and plasma renin. In addition, about 40% of the shed blood volume was restored within 30 min by fluid from either the fetal interstitium or placenta. The PO2 of umbilical venous blood increased from 33 +/- 9 mmHg to 49 +/- 17 mmHg 2 min post-haemorrhage, and to 47 +/- 15 mmHg 30 min post-haemorrhage. However, this increase was not sufficient to offset the fall in both haemoglobin concentration and umbilical-placental blood flow, so that oxygen delivery decreased from 21.1 +/- 5.5 ml/min per kg to 9.1 +/- 5.2 ml/min per kg 2 min post-haemorrhage, and 14.1 +/- 9.2 ml/min per kg 30 min post-haemorrhage. Because of this decrease in oxygen delivery, oxygen consumption fell and a metabolic acidemia ensued. Nevertheless, oxygen delivery to the heart and brain was maintained because hepatic vasoconstriction diverted more of the well oxygenated umbilical venous return through the ductus venosus. Although the fetus was able to tolerate acute loss of 40% of blood volume, larger volumes of haemorrhage resulted in fetal death.     

Fetal progenitor cells naturally transferred through pregnancy participate in inflammation and angiogenesis during wound healing

The phenotype and fate of fetal microchimeric cells transfered into the maternal circulation during pregnancy are not well described. Since progenitors from distal sites mobilize during wound healing, we analyzed the recruitment and plasticity of fetal progenitors into maternal wounds. Wounds were generated on normal and bleomycin-induced fibrotic skin of parous or pregnant wild-type females with fluorescent GFP(+) fetuses. Analyses were performed on skin and blood specimens through PCR, immunohistochemistry, and flow cytometry. Controls consisted of parous and pregnant females without wounds and virgin females with wounds. Fetal cells were detected in all skin specimens of parous mice as long as healing was not achieved. During early stages of wound healing, fetal cells expressed mainly leukocyte markers, while in later phases endothelial markers prevailed. Fetally derived vessels connected to maternal circulation were also found, demonstrating the transfer of fetal endothelial progenitor cells. Wounding mobilized fetal CD34(+)ckit(-) cells into the blood during pregnancy. Most of this population was CD11b(-)VEGFR2(-). Another part was CD11b(+) with a fraction expressing VEGFR2. VEGFa-spiked Matrigel plugs partially mimicked this fetal progenitor recruitment and mobilization into the blood. In summary, fetal cells that mobilize in response to wounding are mainly progenitor cells and participate in angiogenesis and inflammation.     

Improvement of cardiac puncture in mice

Cardiac puncture through the anterior thoracic aperture, which was originally reported by Frankeberg, was improved. The improved method involved the use of a restraining board instead of manual holding. A mouse anesthetized with ether was fixed in a dorsal position on the restraining board, and then a needle of 26G X 1/2 was inserted through the skin just in front of the anterior thoracic aperture, keeping the syringe parallel to the long axis of the animal. A blood volume of 0.6-0.9 ml could be obtained even by an inexperienced collector, and the blood collection was usually completed within 10-20 s. The potential for repeated blood collection of 0.4-0.6 ml was facilitated by use of the present method at intervals of 7 days or more.     

Haemodynamic and catecholamine responses to hypothermia in the fetal sheep in utero

Chronically catheterised fetal sheep (117-134 days) were cooled in utero via a tubing coil placed around the fetal trunk through which cold water was circulated for one hour. The fetal core temperature was reduced by 5.51 +/- 0.61 degrees C. This hypothermia was associated with tachycardia (P less than 0.001) and hypertension (P less than 0.001) (n = 12). The tachycardia was abolished by treatment with propranolol (n = 4) and the hypertension by treatment with phentolamine (n = 5). Blood flow in the left umbilical artery was measured by an electromagnetic flow probe in 4 fetuses and rose (P less than 0.001) with fetal cooling. The increase in blood flow was abolished by treatment with either phentolamine or propranolol. These observations are consistent with a redistribution of fetal blood flow from peripheral tissues to placental and thermogenic tissues during cooling. Fetal plasma adrenaline and noradrenaline concentrations rose (P less than 0.01) during fetal cooling (n = 5). These studies demonstrate that catecholamine and cardiovascular responses to environmental hypothermia have differentiated prior to birth in the sheep fetus.     

Studies on the growth of the fetal sheep. Effects of surgical reduction in placental size, or experimental manipulation of uterine blood flow on plasma sulphation promoting activity and on the concentration of insulin-like growth factors I and II

The effect of long- and short-term manipulations of uterine blood flow on fetal plasma levels of IGF-I and -II have been studied in sheep at days 125-139 of pregnancy and compared with those in near term rats and guinea pig. The primary objective is to show that both long- and short-term reduction of uterine blood flow is associated with increase in the fetal plasma concentration of IGF-II while that of IGF-I falls. In the pregnant sheep long-term depression of utero-placental blood flow was caused by surgical reduction in placental mass (carunclectomy) prior to conception. This reduced fetal weight to 2.42 +/- 0.49 kg (SD) compared with 3.41 +/- 0.46 in controls; the respective values for uterine blood flow being 1694 +/- 558 and 913 +/- 324 ml/min respectively. This was associated with a fall in fetal plasma IGF-I concentration from 22.6 +/- 3.4 ng/ml to 14.9 +/- 1.31 ng/ml and a rise in IGF-II from 1952 +/- 284 ng/ml to 3360 +/- 914 ng/ml respectively. Similar changes in the plasma concentrations of IGF peptides were observed in fetal rats and guinea pigs in response to uterine artery ligation. Short-term reduction (60 min) of the uterine blood flow was caused either by compression of the common uterine artery to depress flow from 1491 +/- 375 to 648 +/- 216 ml/min or through intraarterial infusion of adrenaline at 35 ug/min to lower flow from 1628 +/- 339 to 1195 +/- 128 ml/min. Such falls in uterine blood flow had no significant effect on fetal plasma IGF-I levels but increased IGF-II levels by 30 to 60%.     

Effect of the method of blood extraction on plasma levels of renin in the Wistar rat

To investigate the influence of blood extraction conditions on the renin-angiotensin system in rats, plasma renin activity (PRA) and plasma renin concentration (PRC) were measured in blood samples obtained by different methods. PRA and PRC in samples obtained by chronic catheterization, cardiac puncture without anesthesia, and decapitation immediately following light ether anesthesia were not significantly different from those obtained by simple decapitation (control group). In contrast, PRA and PRC in samples obtained by cardiac puncture and cavernous sinus puncture after light ether anesthesia were significantly (p less than 0.01) higher than those obtained in the control group. There was a significant direct correlationship between PRA and PRC in all samples studied (r = 0.87, p less than 0.001). The present results suggest that light ether anesthesia increases renin levels, except when blood samples are taken by decapitation, and that chronic catheterization and cardiac puncture are the choice blood extraction methods to evaluate the renin-angiotensin system in rats.     

Pulmonary vascular response to prostacyclin in fetal lambs.

Eighteen prostacyclin injections (19.4 +/- 1.5 micrograms/kg) were performed in five chronically instrumented, intact fetal lambs in order to study the effects on pulmonary blood flow. These resulted in a brief period of bradycardia followed by a more prolonged period of increased pulmonary blood flow. In this latter phase, pulmonary blood flow increased from a baseline value of 49 +/- 4 ml/(kg min) to 122 +/- 10 ml/(kg min). Systolic/diastolic pulmonary arterial pressure simultaneously fell from 73 +/- 2/48 +/- 1 to 68 +/- 2/42 +/- 1 mm Hg. Flow through the ductus arteriosus was unchanged and right ventricular output increased to account for the increased pulmonary blood flow. Thus, prostacyclin causes pulmonary vasodilation in intact fetal lambs and may participate in the control of fetal pulmonary blood flow and the circulatory adjustments to extra-uterine life.     

Uterine trauma and limb defects

The temporary clamping of the uterine blood vessels on one side of the uterus during late pregnancy in the rat (days 14-16) results in hemorrhage and tissue necrosis in the extremities of the fetuses from the experimental side and occasionally from the control side. A further series of experiments showed that similar fetal hemorrhage followed the temporary clamping (45 minutes) of the uterine wall or uterine fat, excluding major uterine vessels; handling the uterus for 5 minutes; and stretching of the uterine blood vessels. A low incidence of fetal hemorrhage was also associated with laparotomy alone, but the fetuses were unaffected by extensive handling of the uterus through the abdominal wall or by intraperitoneal anesthesia. Fetal hemorrhage was also induced by a short episode of severe maternal hyperthermia but not by a high dose of ethanol given by gavage. These results suggest that a range of uterine trauma may result in fetal hemorrhage, perhaps through a common mechanism.     

Blood puncture as a nondestructive sampling tool to obtain DNA in frogs: comparison of protocols and survival analysis

In molecular biology studies of Anura, nondestructive methods to obtain genetic material are needed as alternatives to toe clipping. This work evaluates a nondestructive method for sampling DNA from blood puncture, comparing the performance of three different extraction protocols (Qiagen Kit, Salting-out and Chelex). We collected 134 individuals of Eleutherodactylus johnstonei, extracting blood via puncture of the medial vein using commercial-grade glucometer lancets. We extracted 100-1880 ng DNA, finding no differences between the extraction protocols. We compared the quality of the resulting DNA through amplification and sequencing of the 16S mitochondrial gene. Amplification was successful for the three extraction protocols, although Chelex showed better performance, making it the most recommendable protocol for extraction of DNA from blood. The resulting sequences corresponded to those registered in the GenBank for this species. Additionally, we found no significant differences in survival or weight change between the individuals that were manipulated and a control group (mean survival 66.7% treated, 62.9% untreated). Data reveal that blood samples obtained by puncture are a convenient alternative to other tissues (phalange, buccal swab, liver) that have traditionally been used as DNA sources for anurans. The technique is applicable to small and large species, covering most anuran diversity, provides enough DNA for many genetic applications and produces no noticeable effect on the survival or performance, given that it does not affect the motor parts or the dexterity of the animals.