Non-invasive epigenetic detection of fetal trisomy 21 in first trimester maternal plasma

Background

Down syndrome (DS) is the most common known aneuploidy, caused by an extra copy of all or part of chromosome 21. Fetal-specific epigenetic markers have been investigated for non-invasive prenatal detection of fetal DS. The phosphodiesterases gene, PDE9A, located on chromosome 21q22.3, is completely methylated in blood (M-PDE9A) and unmethylated in the placenta (U-PDE9A). Therefore, we estimated the accuracy of non-invasive fetal DS detection during the first trimester of pregnancy using this tissue-specific epigenetic characteristic of PDE9A.

Methodology/Principal Findings

A nested, case-control study was conducted using maternal plasma samples collected from 108 pregnant women carrying 18 DS and 90 normal fetuses (each case was matched with 5 controls according to gestational weeks at blood sampling). All pregnancies were singletons at or before 12 weeks of gestation between October 2008 and May 2009. The maternal plasma levels of M-PDE9A and U-PDE9A were measured by quantitative methylation-specific polymerase chain reaction. M-PDE9A and U-PDE9A levels were obtained in all samples and did not differ between male and female fetuses. M-PDE9A levels did not differ between the DS cases and controls (1854.3 vs 2004.5 copies/mL; P = 0.928). U-PDE9A levels were significantly elevated in women with DS fetuses compared with controls (356.8 vs 194.7 copies/mL, P<0.001). The sensitivities of U-PDE9A level and the unmethylation index of PDE9A for non-invasive fetal DS detection were 77.8% and 83.3%, respectively, with a 5% false-positive rate. In the risk assessment for fetal DS, the adjusted odds ratios of U-PDE9A level and UI were 46.2 [95% confidence interval: 7.8–151.6] and 63.7 [95% confidence interval: 23.2–206.7], respectively.

Conclusions

Our findings suggest that U-PDE9A level and the unmethylation index of PDE9A may be useful biomarkers for non-invasive fetal DS detection during the first trimester of pregnancy, regardless of fetal gender.     

Multiple testing in fetal gender determination from maternal blood by polymerase chain reaction

Fetal male DNA can be identified in maternal blood by polymerase chain reaction (PCR) amplification of Y-specific sequences. This technology has not reached a satisfactory accuracy and reproducibility in fetal gender determination because of the very low concentration of fetal cells. Our purpose was to evaluate the possibility of improving the reliability of this test by setting up a repeated amplification system. We amplified, by nested PCR of the Y-specific sequence DYS14, 137 DNA samples extracted from maternal peripheral blood (93 from male-bearing and 44 from female-bearing pregnancies ranging from the 6th to the 36th gestational week). Each maternal DNA sample was tested doubly, in two different PCR sessions, with a total of four amplifications. We obtained discordant results in the four amplifications in 82/137 (60%) samples. The best interpretation of these discordant results was obtained by applying a positivity cutoff of at least two positive amplifications for considering a DNA sample as belonging to a male-bearing pregnancy. We obtained a sensitivity of 83%, a specificity of 93%, a positive predictive value of 96% and a negative predictive value of 72% in fetal male gender diagnosis. By applying this quadruple testing system, we significantly improved PCR accuracy and predictive values compared with single and double testing of the same samples. We conclude that, for future investigations of fetal DNA retrieved from maternal blood, the application of a quadruple testing system is better than the single PCR test. Received: 18 August 1997 / Accepted: 12 January 1998     

Plasma catecholamines in the hypoxaemic fetal rhesus monkey

To assess the response of the sympathoadrenal system of the primate fetus to oxygen deprivation, we measured plasma catecholamines in 8 chronically catheterized fetal rhesus monkeys. A range of fetal hypoxaemia was produced by having the mother inspire 15, 10, or 9% oxygen mixtures while tranquilized with ketamine. Catecholamines from fetal carotid and maternal femoral arteries were measured by radioenzymatic assay. Fetal plasma norepinephrine and epinephrine concentrations increased significantly at all levels of hypoxaemia, but dopamine increased only at very low fetal oxygen tensions. Norepinephrine levels exceeded those of epinephrine and dopamine under all conditions. Relatively more severe hypoxaemia was necessary to elevate concentrations of epinephrine above baseline as compared with norepinephrine. A negative exponential correlation (P less than 0.001) was found between both fetal arterial PO2 and oxygen content and plasma norepinephrine and epinephrine, which was qualitatively similar to that observed previously in the sheep fetus. Maternal catecholamines were found to increase during hypoxaemia as well, but to a lesser degree than in the fetus.     

Effects of fetal hypoinsulinemia on fetal hepatic insulin binding in the rat

Fetal hepatic insulin binding was studied in term fetal rats born to control mothers, mothers fasted for 48 h and mothers made hyperinsulinemic by the chronic, exogenous administration of insulin for 5 days prior to term. Maternal hyperinsulinemia was associated with fetal hypoglycemia and an approx. 70% reduction in fetal plasma insulin. Fetuses from these mothers exhibited an increase in hepatic insulin binding as indicated by a significant change in Scatchard analyses. No significant effect on fetal hepatic insulin binding by Scatchard analysis was seen with maternal fasting, despite a modest decrease in fetal plasma insulin. However, analysis of all animals showed that high-affinity fetal hepatic insulin binding and specific 125I-insulin binding were inversely correlated with fetal plasma insulin concentration. These results indicate that fetal rat liver, similar to adult rat liver, responds to a decrease in circulating insulin to below normal concentrations with an increase in insulin receptor binding.     

Fetal DNA detection in maternal plasma throughout gestation

The presence of fetal DNA in maternal plasma may represent a source of genetic material which can be obtained noninvasively. We wanted to assess whether fetal DNA is detectable in all pregnant women, to define the range and distribution of fetal DNA concentration at different gestational ages, to identify the optimal period to obtain a maternal blood sample yielding an adequate amount of fetal DNA for prenatal diagnosis, and to evaluate accuracy and predictive values of this approach. This information is crucial to develop safe and reliable non-invasive genetic testing in early pregnancy and monitoring of pregnancy complications in late gestation. Fetal DNA quantification in maternal plasma was carried out by real-time PCR on the SRY gene in male-bearing pregnancies to distinguish between maternal and fetal DNA. A cohort of 1,837 pregnant women was investigated. Fetal DNA could be detected from the sixth week and could be retrieved at any gestational week. No false-positive results were obtained in 163 women with previous embryo loss or previous male babies. Fetal DNA analysis performed blindly on a subset of 464 women displayed 99.4, 97.8 and 100% accuracy in fetal gender determination during the first, second, and third trimester of pregnancy, respectively. No SRY amplification was obtained in seven out of the 246 (2.8%) male-bearing pregnancies. Fetal DNA from maternal plasma seems to be an adequate and reliable source of genetic material for a noninvasive prenatal diagnostic approach.     

Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA)

In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.     

Fetal gender determination in early pregnancy through qualitative and quantitative analysis of fetal DNA in maternal serum

Fetal DNA in maternal plasma and serum has been shown to be a useful material for fetal gender determination and for screening tests for abnormal pregnancies except during early gestational ages. Maternal serum samples were obtained from 81 pregnant women during the 5th-10th weeks of gestation. Fetal gender was determined by conventional polymerase chain reaction (PCR) to detect a Y-chromosomal sequence (DYS14) in maternal serum during early gestation and confirmed by examination of the newborns after delivery. Real-time quantitative analyses of the SRY and beta-globin genes were also performed in order to determine fetal gender and to quantify fetal DNA concentration in maternal serum during early gestation. When using conventional PCR, the total sensitivity of identifying a male fetus was 95%, but its sensitivity after the 7th week was 100%, whereas in real-time quantitative PCR, the total sensitivity after the 5th week was 100%. Quantitative analyses of the SRY gene revealed that the mean concentration of fetal DNA in maternal serum was 30.55 copies/ml, that fetal DNA concentration showed a tendency to increase with the progression of pregnancy, and that it had a wide normal range. Thus, we could confidently determine fetal gender by using maternal serum samples taken as early as the 7th week.     

Detection of Y STR markers of male fetal dna in maternal circulation

BACKGROUND:

Circulating fetal cells and cell free DNA in the maternal blood has been shown to help in prenatal diagnosis of genetic disorders without relying on invasive procedures leading to significant risk of pregnancy loss.

AIM:

The current study was undertaken to detect the male fetal population using Y STR markers DYS 19, DYS 385 and DYS 392 and also to study the extent of persistence of fetal DNA in the mother following delivery.

MATERIALS AND METHODS:

Blinded study was conducted on 50 mothers delivering male and female babies. Cellular and cell free DNA was extracted from maternal and fetal cord blood and amplified for Y STR markers by PCR.

RESULTS:

The amplification sensitivity of Y specific STR, DYS19 was 100% (22/22) in the male fetal DNA samples. The incidence of other STRs, i.e., DYS385 and DYS392 were 91% (20/22) each. Analysis of results revealed that thirteen of the twenty six women had detectable male fetal DNA at the time of delivery. However fetal DNA was not detectable twenty four hours after delivery.

CONCLUSION:

Preliminary results show that the separation of fetal cell-free DNA in the maternal circulation is a good low-cost approach for the future development of novel strategies to provide non-invasive techniques for early prenatal diagnosis.     

Detection and Quantification of Male-Specific Fetal DNA in the Serum of Pregnant Cynomolgus Monkeys (Macaca fascicularis)

Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research. We have developed a specific, highly sensitive PCR system for detecting and quantifying male-specific fetal DNA in pregnant cynomolgus monkeys. We used multiplex quantitative real-time PCR to analyze cell-free DNA in maternal blood serum obtained from 46 pregnant monkeys at gestational weeks 5, 12, and 22. The presence of SRY gene and DYS14 Y chromosomal sequences was determined in 28 monkeys with male-bearing pregnancies. According to confirmation of fetal sex at birth, the probe and primers for detecting the Y chromosomal regions at each time point revealed 100% specificity of the PCR test and no false-positive or false-negative results. Increased levels of the SRY-specific sequences (mean, 4706 copies/mL serum DNA; range, 1731 to 12,625) and DYS14-specific sequences (mean, 54,814 copies/mL serum DNA; range, 4175–131,250 copies) were detected at week 22. The SRY- and DYS14-specific probes appear to be an effective combination of markers in a multiplex PCR system. To our knowledge, this report is the first to describe the detection of cell-free DNA in cynomolgus monkeys.Abbreviations: C_t, threshold cycleAnalysis of cell-free circulating nucleic acids in human maternal plasma or serum has led to the development of risk-free methods for prenatal genetic diagnosis and the assessment of several fetal and maternal conditions, for example, sex determination for paternally inherited diseases, pregnancy-associated complications, sex-linked disorders for ambiguous genitalia, and embryo tracking.^{1,4,12,14,18,19} Technical challenges associated with detecting fetal DNA arise due to the low concentration of fetal DNA in maternal plasma during pregnancy and the difficulty of differentiating the genetic material of the fetus from that of the mother.^5,13,20 Fetal sex determination using sequences derived from the Y chromosome only is relatively simple and has a reported accuracy rate in humans of approximately 99.0% at 7 wk of gestation and 100% after 20 wk, depending on the protocol and methods used.^3,5,17,20 In other species, researchers have used real-time PCR assays during pregnancy to predict fetal sex from cell-free DNA at an accuracy of 100%.^9,10,11 Cell-free fetal DNA in the maternal circulation represents only 3% to 6% of the total free DNA obtained from plasma throughout pregnancy; however, this percentage is variable between pregnancies.^5,13,20In clinical biomedical research, it is essential to develop animal models for human diseases to reveal their mechanisms.^16,22 Continued progress in surgical intervention and molecular medicine suggests that it may soon be possible to develop potential treatments or even cures for several fetal genetic diseases at an early stage of pregnancy.¹⁵ Fetal developmental research during early pregnancy might be facilitated by using cell-free fetal DNA in the maternal blood rather than other methods, such as serum screening and ultrasonography. Nonhuman primates, especially macaques, are useful model animals for studying fetal development because of the similarity of the reproductive characteristics, placental structure, and developmental events between these animals and humans.^9,10 These developmental similarities highlight the importance of the study of cell-free fetal DNA in nonhuman primates and its usefulness as a marker to obtain genetic information about the fetus.In the current study, we investigated the presence of cell-free fetal DNA in the maternal plasma of cynomolgus monkeys by developing and using a standardized PCR system. To this end, we selected the SRY (sex-determining region Y) gene and DYS14 sequences of the cynomolgus monkey to use as sex-associated markers. The Y chromosome-specific sequences in the single-copy sex determination region of SRY and the multicopy (thus yielding increased sensitivity) sequences of DYS14 in the TSPY (testis-specific protein, Y-linked) gene have had wide clinical use in humans as molecular markers for detecting and quantifying cell-free fetal DNA.^3,7 In addition, TSPY has been used in bovines for detecting cell-free fetal DNA² and in rhesus macaques for long-term evaluation of microchimerism.⁸ Given the reports of fetal sex determination in rhesus macaques^9,10 and sheep¹¹ by analyzing Y chromosome-specific sequences from cell-free DNA, we hypothesized that we could predict the fetal sex of cynomolgus monkeys at different stages of gestation. This information has been extremely useful in optimizing the design of experimental studies in biomedical research and in managing a nonhuman primate breeding colony.¹⁰ Because cynomolgus and rhesus macaques are closely related members of the same genus, the current experiments are similar to a previous study.⁹We developed an efficient 2-color multiplex PCR system to detect and quantify fetal DNA in the maternal serum of cynomolgus monkeys during pregnancy. We used 2 loci on the Y chromosome in a single PCR test to minimize the likelihood of false-positive signals. Here we report the results of detection and analysis of fetal DNA at various weeks of gestation and evaluate our PCR system for its ability to determine fetal sex from pregnant monkeys’ cell-free DNA.     

Cattle fetal sex determination by polymerase chain reaction using DNA isolated from maternal plasma

The objective of this study was to evaluate the use of polymerase chain reaction analysis (PCR) of fetal cells/DNA in the maternal plasma of pregnant cows to determine the sex of the fetus. Plasma was harvested from 35 cows of mixed genotype at different stages of pregnancy ranging from 5 to 35 weeks. A male calf and a heifer calf provided the control samples. Fetal sex was determined by amplification of Y-specific sequences. For the 35 cows, the fetal sex predicted by this technique was in accordance with the sex of the calf at birth in 88.6% of cases. The agreement between predicted and observed fetal sex was less for cows with a gestational length of 35-48 days (63.6%). Regression analysis showed that there was a strong relationship between the probability of correctly predicting fetal sex and the stage of gestation. It was estimated that the test performed at 43.8 days post fertilization would have 95% accuracy, increasing to 99% accuracy for testing at 48.4 days and 99.9% accuracy for tests at 55.0 days or later. It was concluded that PCR analysis of fetal cells in maternal plasma can be used to predict successfully the sex of the fetus in cattle.     

Effect of maternal hypercholesterolemia on fetal sterol metabolism in the Golden Syrian hamster.

The fetus obtains a significant amount of cholesterol from de novo synthesis. Studies have suggested that maternal cholesterol may also contribute to the cholesterol accrued in the fetus. Thus, the present studies were completed to determine whether diet-induced maternal hypercholesterolemia would affect fetal sterol metabolism. To accomplish this, maternal plasma cholesterol concentrations were increased sequentially by feeding hamsters 0.0%, 0.12%, 0.5%, and 2.0% cholesterol. At 11 days into a gestational period of 15.5 days, cholesterol concentrations and sterol synthesis rates were measured in the three fetal tissues: the placenta, yolk sac, and fetus. In the placenta and yolk sac, the cholesterol concentration increased significantly when dams were fed as little as 0.12% cholesterol (P < 0.0167), and sterol synthesis rates decreased in dams fed at least 0.5% or 2% cholesterol, respectively (P < 0.0167). In the fetus, changes in fetal cholesterol concentration and sterol synthesis rates occurred only when dams were fed at least 0.5% cholesterol, which corresponded to a greater than 2-fold increase in maternal plasma cholesterol concentrations. When the cholesterol concentration in the fetal tissues in each animal was plotted as a function of maternal plasma cholesterol concentration, a linear relationship was found (P < 0.001).These studies demonstrate that sterol homeostasis in fetal tissues, including the fetus, is affected by maternal plasma cholesterol concentration in a gradient fashion and that sterol metabolism in the fetus is dependent on sterol homeostasis in the yolk sac and/or placenta.     

Rapid clearance of fetal DNA from maternal plasma.

Fetal DNA has been detected in maternal plasma during pregnancy. We investigated the clearance of circulating fetal DNA after delivery, using quantitative PCR analysis of the sex-determining region Y gene as a marker for male fetuses. We analyzed plasma samples from 12 women 1-42 d after delivery of male babies and found that circulating fetal DNA was undetectable by day 1 after delivery. To obtain a higher time-resolution picture of fetal DNA clearance, we performed serial sampling of eight women, which indicated that most women (seven) had undetectable levels of circulating fetal DNA by 2 h postpartum. The mean half-life for circulating fetal DNA was 16.3 min (range 4-30 min). Plasma nucleases were found to account for only part of the clearance of plasma fetal DNA. The rapid turnover of circulating DNA suggests that plasma DNA analysis may be less susceptible to false-positive results, which result from carryover from previous pregnancies, than is the detection of fetal cells in maternal blood; also, rapid turnover may be useful for the monitoring of feto-maternal events with rapid dynamics. These results also may have implications for the study of other types of nonhost DNA in plasma, such as circulating tumor-derived and graft-derived DNA in oncology and transplant patients, respectively.     

Effects on maternal and fetal steroid concentrations of induction of parturition in the sheep by inhibition of 3 beta-hydroxysteroid dehydrogenase

Changes in circulating steroid hormones, the incidence of myometrial contractions, and the onset of labour were all monitored after administration of the 3 beta-hydroxysteroid dehydrogenase inhibitor, epostane, to chronically catheterized ewes and fetuses near term. In all animals the drug induced delivery 33-36 h after injection or infusion into the ewe with the birth of live healthy lambs which showed normal subsequent development. Epostane induced immediate, permanent falls in both maternal and fetal plasma progesterone concentrations, accompanied by increased PGF metabolite concentrations in the uterine vein beginning 15 min after treatment. Of the other hormonal changes observed, the most striking was the pronounced drop in both maternal and fetal plasma cortisol. In the fetus this fall was followed by increasing concentrations of circulating ACTH which eventually restored the cortisol levels. By 12-24 h after epostane a substantial overshoot had occurred and at 27-30 h the fetal plasma cortisol concentrations were as high as those seen during normal parturition at term. No significant changes in maternal plasma oestradiol-17 beta could be detected after epostane treatment or during labour. The incidence of slow myometrial contractions increased significantly during the second 3-h period after epostane, although their duration did not change. Contraction patterns typical of first stage labour were seen from 20 to 24 h. These results show that epostane may be used as a safe, predictable inducing agent in sheep if given 6-10 days before term; the lambs showed no signs of prematurity despite their lowered plasma cortisol concentrations which persisted for some hours before labour was induced.     

Changes in plasma adenosine during simulated birth of fetal sheep

Adenosine is known to inhibit nonshivering thermogenesis in adult brown fat. These experiments were undertaken to test whether fetal adenosine, normally present in high concentrations, suppresses lipolysis in utero and then falls after birth, permitting thermogenesis to begin. To test this hypothesis, we measured fetal plasma adenosine concentration [ADO] using high-performance liquid chromatography in 11 fetal sheep at 135-140 days gestation during simulated birth. During an initial control period, fetal [ADO] averaged 1.9 +/- 0.3 microM, about four times maternal [ADO] (0.4 +/- 0.1 microM, P less than 0.001). The fetus was then cooled by circulating cold water through a plastic coil encircled about the fetal torso. One hour later, when fetal core temperature had decreased 2.3 degrees C, fetal [ADO] averaged 2.8 +/- 0.5 microM, a 50% increase (P less than 0.05), while thermogenesis remained inactive. Next the fetal lungs were ventilated with O2 to raise arterial Po2 to greater than or equal to 150 Torr. Fetal [ADO] decreased only slightly, and thermogenic responses were modest. Finally, the umbilical cord was occluded. Fetal [ADO] decreased rapidly and 60 min later averaged 1.1 +/- 0.2 microM, 40% below initial control (P less than 0.05) and 57% below the previous period (P less than 0.001). As [ADO] fell, strong thermogenic responses became apparent, as indicated by seven- to eightfold increases in plasma glycerol (P less than 0.001) and a doubling in fetal O2 consumption (P less than 0.001). These results are consistent with the hypothesis that high fetal [ADO] inhibits thermogenesis before birth but then decreases after cord occlusion, allowing thermogenesis to begin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect on maternal and fetal renal function and plasma renin activity of a high salt intake by the ewe

The effect on renal function of replacing maternal drinking water with a solution containing 0.17 M NaCl was studied in 9 ewes and their chronically catheterised fetuses over a period of 9 days. Maternal sodium intake increased from control values of 2.19 +/- 0.09 mmol/h to 44.3 +/- 7.4 (P less than 0.001) and 46.3 +/- 6.5 mmol/h (P less than 0.001) on the 3rd and 6th days of salt ingestion. Maternal plasma sodium levels were not affected, but the urinary sodium/potassium ratio increased from 0.15 +/- 0.07 to 2.26 +/- 0.34 (P less than 0.001) after 6 days and plasma renin activity fell from 2.87 +/- 0.76 to 1.00 +/- 0.25 ng/ml per h (P less than 0.05). The changes in maternal sodium intake had no effect on fetal plasma sodium levels nor on fetal plasma renin activity. Sodium excretion and fetal urinary sodium/potassium ratio did not change. However, 3 days after the ewes returned to drinking water fetal plasma renin activity was significantly higher than it was prior to maternal ingestion of 0.17 M NaCl. Fetal plasma renin activity was inversely related to fetal plasma sodium levels (P less than 0.01). The results show that changes in maternal sodium intake had no long term effect on fetal plasma sodium levels nor on fetal renal sodium excretion. The fall in maternal plasma renin activity in the absence of any change in the fetal renin activity, indicates that the fetal renin angiotensin system is controlled by factors other than those influencing the maternal renin angiotensin system. Since fetal urinary sodium/potassium ratios remained unchanged it would suggest that fetal sodium excretion is not influenced by maternal levels of aldosterone.     

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.     

Prenatal sex determination from maternal peripheral blood using the polymerase chain reaction

We have investigated the use of a nested polymerase chain reaction assay for the detection of a fetal-specific Y-chromosomal sequence (DYS14) from DNA extracted from unsorted maternal peripheral blood. Serial dilutions of male DNA into female cord blood DNA indicated that the assay could detect an equivalent of a single male cell in 300000 female cells. The assay exhibited absolute specificity for male DNA with no amplification from a DNA panel obtained from 10 female cord blood samples. When used on DNA extracted from unsorted peripheral blood from a series of pregnant women, the predictive values of a positive test for a male fetus were 86%, 67% and 87% in the first, second and third trimesters, respectively. We have also demonstrated that retesting the samples allows the detection of a proportion of male-bearing pregnancies with a high degree of accuracy, in that all 15 women who gave positive signals in two consecutive amplifications had male fetuses. We have also applied the test at 8 weeks post-partum to eight women who had previously delivered male babies; no Y-specific signal could be detected in any of them, suggesting that most women have cleared their circulation of fetal cells by 8 weeks after parturition.     

Metabolic and hormonal responses to cooling the fetal sheep in utero

The metabolic and hormonal effects of cooling 10 fetal sheep in utero (115-142 days of gestation) for 2h were studied. The fetal core temperature fell by 2.81 +/- 0.14 degrees C while the maternal temperature fell 0.86 +/- 0.15 degrees C. This hypothermia caused a significant rise in the fetal and maternal plasma glucose concentrations (P less than 0.001) and a fall in the fetal insulin concentrations (P less than 0.01). The fetal plasma lactate and cortisol concentrations rose rapidly (P less than 0.01) while the growth hormone fell (P less than 0.01) and remained low until cooling ceased when a rapid rebound occurred. There was no significant change in any of the fetal iodothyronines and no elevation of nonesterified free fatty acid concentrations, in contrast to the rapid rise (P less than 0.01) which occurred when newborn lambs were cooled. These observations demonstrate that appropriate glucose, insulin, lactate and cortisol responses to hypothermia have differentiated by 120 days of gestation. However, neither a thyroid hormone response nor an elevation in free fatty acid levels was observed. Thus not all components of the thermogenic response to hypothermia can be demonstrated in the late gestation fetail sheep in utero.     

Effects of hyperthermia on fetal and maternal plasma prostaglandin concentrations and uterine activity in sheep

The exposure of pregnant sheep to high ambient temperatures (43 degrees C) for 8 hours, sufficient to significantly elevate maternal and fetal body temperature +2.0 degrees C (p less than 0.001) and +1.9 degrees C (p less than 0.001) respectively, resulted in significant increases in PGE2 plasma concentrations in both the maternal and fetal circulations. Plasma PGF2 alpha concentrations were significantly raised in the fetal circulation but not the maternal during hyperthermia. The increase in prostaglandin concentrations were correlated with the magnitude of the increase in maternal and fetal body temperature. Uterine activity also increased during hyperthermia, probably as a result of the increase in prostaglandin concentrations. We propose that increased synthesis and release of prostaglandins from the uterus and/or placenta is an adaptive response to hyperthermia, and may protect the fetus from the consequences of heat stress.     

Insulin-like growth factors and binding proteins in the fetal rat: Alterations during maternal starvation and effects in fetal brain cell culture

Maternal malnutrition adversely affects fetal body and brain growth during late gestation. We utilized a fetal brain cell culture model to examine whether alternations in circulating factors may contribute to reduce brain growth during maternal starvation; we then used specific immunoassay and western blotting techniques, and purified peptides to investigate the potential role that altered levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) may play in impaired growth during maternal nutritional restriction.Fetal, body, liver, and brain weight were reduced after 72 hr maternal starvation, and plasma from starved fetuses were less potent than fed fetal plasma in stimulating brain cell growth. Circulating levels of IGF-I were reduced in starved compared to fed fetuses, while levels of IGF-II were similar in both groups. In contrast, [¹²⁵I]-IGF-I binding assay demonstrated an increase in the availability of plasma IGFBPs following starvation. Western ligand blotting and densitometry indicated that levels of 32 Kd IGFBPs were 2-fold higher in starved compared to fed fetal plasma. Immunoblotting and immunoprecipitation with antiserum against rat IGFBP-1 confirmed that heightened levels of immunoreactive IGFBP-1 accounted for the increase in 32 Kd IGFBPs in starved plasma. Levels of 34 Kd BPs, representing IGFBP-2, were unaffected by starvation. Reconstitution experiments in cell culture showed that IGF-I promoted fetal brain cell growth, and that when they were supplemented with IGF-I, the growth promoting activity of starved fetal plasma was restored to fed levels. These changes were measured using MTT to assess mitochondrial reductase activity. Conversely, addition of physiological amounts of rat IGFBP-1 inhibited the effects of fed fetal plasma on brain cell growth, and bioactivity was reduced even further with higher concentrations of IGFBP-1. Based on these results, we conclude that reciprocal changes in circulating levels of IGFBP-1 (increased) and IGF-I (decreased) may combine to reduce the availability of IGF-I to this tissue and limit fetal brain cell growth when maternal nutrition is impaired.