Fetal gender determination in early first trimester pregnancies of rhesus monkeys (Macaca mulatta) by fluorescent PCR analysis of maternal serum

Non-human primate fetal gender determination can be a powerful tool for research study design and colony management purposes. The recent discovery of the presence of fetal DNA in maternal serum has offered a new non-invasive approach for identification of fetal gender. We present a rapid and simple method for the sexing of developing rhesus monkeys in the first trimester by polymerase chain reaction (PCR) analysis of maternal serum. Serum samples were obtained from 72 gravid rhesus monkeys during 20-32 days of gestation (term 165 +/- 10 days). Fetal gender and the quantity of circulating fetal DNA were determined by real-time PCR analysis of the rhesus Y-chromosomal DNA sequences. The sensitivity for identifying a male fetus was 100% by 30 days gestation, and no false-positive results were observed. This study demonstrates that fetal gender can be reliably determined in the early first trimester from maternal serum samples, a non-invasive method for routine gender screening.     

Fetal cells in the maternal circulation during first trimester in pregnancies

To investigate the presence of fetal cells in the maternal circulation during early pregnancy, the polymerase chain reaction was used to test the presence of human Y chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood specimens from 19 women carrying male fetuses and 12 women carrying female fetuses. The presence of fetal cells was suggested as early as 6 weeks gestation in 1 of the 19 women bearing male fetuses. Fetal cells were present in the maternal circulation of 15 of the 19 women by 9 weeks gestation, and in only 1 of the 19 were fetal cells not detected until the 12th week after conception. These results suggest that identification of fetal cells in the maternal circulation is possible with a properly designed and executed polymerase chain reaction. However, there was considerable variation with respect to when these fetal cells first became detectable during pregnancy. These fetal cells are potentially a valuable source of material for biochemical and genetic studies of the fetuses.     

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.     

Application of fetal DNA detection in maternal plasma: a prenatal diagnosis unit experience.

Non-invasive prenatal diagnosis tests based on the analysis of fetal DNA in maternal plasma have potential to be a safer alternative to invasive methods. So far, different studies have shown mainly fetal sex, fetal RhD, and quantitative variations of fetal DNA during gestation with fetal chromosomal anomalies or gestations at risk for preeclampsia. The objective of our research was to evaluate the use of fetal DNA in maternal plasma for clinical application. In our study, we have established the methodology needed for the analysis of fetal DNA. Different methods were used, according to the requirements of the assay. We have used quantitative fluorescent polymerase chain reaction (QF-PCR) to perform fetal sex detection with 90% sensitivity. The same technique permitted the detection of fetal DNA from the 10th week of gestation to hours after delivery. We have successfully carried out the diagnosis of two inherited disorders, cystic fibrosis (conventional PCR and restriction analysis) and Huntington disease (QF-PCR). Ninety percent of the cases studied for fetal RhD by real-time PCR were correctly diagnosed. The detection of fetal DNA sequences is a reality and could reduce the risk of invasive techniques for certain fetal disorders in the near future.     

Quantification of fetal and total circulatory DNA in maternal plasma samples before and after size fractionation by agarose gel electrophoresis

Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal (SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100-300, 300-500, 500-700, 700-900, and >900 bp) after size fractionation by agarose gel electrophoresis. DNA was extracted from maternal plasma samples from 11 pregnant women carrying male foetuses at the 16th week of gestation. Fetal circulatory DNA was mainly detected in the 100-300 bp fraction with the median concentration being 14.4 GE/ml. A lower median amount of 4.9 GE/ml was also found in the 300-500 bp fraction. Circulatory DNA extracted from the 100-300 bp fraction contained 4.2 times enriched fetal DNA when compared with unseparated DNA sample. Fetal DNA within the 300-500 bp fraction was 2.5 times enriched. Circulatory fetal DNA is predominantly present in a fraction with molecular size <500 bp, which can be used for the detection of paternally inherited alleles. However, the usage of size-separated DNA is not suitable for routine clinical applications because of risk of contamination.     

Fetal DNA in maternal serum: does it persist after pregnancy?

Fetal DNA and cells present in maternal blood have previously been used for non-invasive prenatal diagnosis. However, some fetal cells can persist in maternal blood after a previous pregnancy. Fetal rhesus status and sex determination have been performed by using amplification by real-time polymerase chain reaction (PCR) of fetal DNA sequences present in maternal circulation; no false-positive results related to persistent fetal DNA from a previous pregnancy have been reported. This idea has recently been challenged. An SRY real-time PCR assay was performed on the serum of 67 pregnant women carrying a female fetus but having previously given birth to at least one boy and on the serum of 30 healthy non-pregnant women with a past male pregnancy. In all cases, serum was negative for the SRY gene. These data suggest that fetal DNA from a previous pregnancy cannot be detected in maternal serum, even by using a highly sensitive technique. Therefore, non-invasive prenatal diagnosis by fetal sex determination for women at risk of producing children with X-linked disorders, and fetal RHD genotyping is reliable and secure as previously demonstrated.     

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     

Analysis of cell-free fetal DNA in plasma and serum of pregnant women.

Sixty blood samples from pregnant women during gestational weeks 9-28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.     

Noninvasive Fetal Sex Determination by Real-Time PCR and TaqMan Probes

Background:Noninvasive fetal sex determination by analyzing Y chromosome-specific sequences is very useful in the management of cases related to sex-linked genetic diseases. The aim of this study was to establish a non-invasive fetal sex determination test using Real-Time PCR and specific probes.Methods:The study was a prospective observational cohort study conducted from August 2018 to September 2019. Venous blood samples were collected from 25 Iranian pregnant women at weeks 7 to 25 of gestation. Cell-free DNA (cfDNA) was isolated from the plasma of samples and fetal sex was determined by SRY gene analysis using the Real-Time PCR technique. In the absence of SRY detection, the presence of fetal DNA was investigated using cfDNA treated with BstUI enzyme and PCR for the epigenetic marker RASSF1A.Results:Of the total samples analyzed, 48% were male and 52% female. The RASSF1A assay performed on SRY negative cases also confirmed the presence of cell-free fetal DNA. Genotype results were in full agreement with neonate gender, and the accuracy of noninvasive fetal sex determination was 100%.Conclusion:Fetal sex determination using the strategy applied in this study is noninvasive and highly accurate and can be exploited in the management of sex-linked genetic diseases.Key Words: Cell-free fetal DNA, Fetal sex determination, Noninvasive prenatal diagnosis, Sex-linked genetic diseases, SRY     

Zinc, copper, and iron metabolism during porcine fetal development

Zinc, copper, and iron levels in maternal and fetal pig tissues and fluids were measured starting on d 30 of gestation and continuing to term (d 114) at 10-d intervals. Fetal hematocrit increased from a low of 19% on d 30 to 32% by d 50, after which it remained above 30% to term. Amniotic fluid zinc, copper, and iron all reached maximal levels by d 60 of gestation. Maternal serum zinc levels fluctuated little during gestation, but fetal serum zinc concentration was significantly elevated above maternal levels during the second trimester. Fetal serum copper levels were significantly lower than maternal values throughout gestation and this was also the case for ceruloplasmin oxidase activity. Maternal serum iron reached its lowest level by d 80 of gestation when rate of transfer of iron to the developing fetuses was high. Fetal serum iron declined throughout gestation, reaching its lowest level on d 100. In general, fetal liver concentrations of zinc, copper, and iron were higher than the corresponding maternal values throughout gestation. Distinct increases were noted for fetal hepatic zinc and copper concentrations during the second trimester of pregnancy and these were accompanied by increases in cytosolic and metallothionein-bound zinc and copper levels. Maternal hepatic iron declined during the second trimester, reaching its lowest point on d 80, indicative of the shunting of maternal iron reserves to fetal tissues. Fetal kidney metal levels did not demonstrate any distinctive developmental patterns with respect to zinc, copper, or iron concentrations, but a general accumulation of each metal was observed as gestation progressed. The results of this study highlight some of the distinct changes occurring in the metabolism of zinc, copper, and iron in both maternal and fetal tissues and fluids during gestation in the pig. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other suitable products.     

The effects of maternal undernutrition on maternal and fetal serum insulin-like growth factors, thyroid hormones and cortisol in the guinea pig.

The insulin-like growth factors (IGF-I and -II) are potential mediators of the effects of maternal undernutrition on fetal growth and muscle development. The effects of a 40% reduction in maternal feed intake on serum levels of the IGFs, the thyroid hormones and cortisol, were investigated for the last two trimesters (day 25 to birth). This level of undernutrition is known to cause a 35% reduction in fetal and placental weights, and a 20-25% reduction in muscle fibre number. Maternal IGF-I level was greater than non-pregnant levels on day 25 gestation, in both control and restricted dams, and declined with gestational age. The increase in IGF-I level in the 40% restricted group was approximately two-thirds that of control animals. Fetal serum IGF-I was also reduced in undernourished fetuses throughout gestation. Maternal IGF-II did not change with gestational age and was unaffected by undernutrition. Fetal IGF-II reached a peak at day 55 of gestation, this peak was greatly diminished by maternal feed restriction. Both IGF-I and IGF-II tended to be related to fetal, placental and muscle weights at day 65 of gestation. Thyroid hormone concentration declined in maternal serum and increased in fetal serum with increasing gestational age. Levels were not significantly affected by undernutrition. Both triiodothyronine (T3) and thyroxine (T4) were correlated with IGF-I in maternal serum (P < 0.05), but not in fetal serum. Cortisol levels were elevated by undernutrition in both maternal and fetal serum, and increased with gestational age. Cortisol was inversely correlated with serum IGF-I in both maternal and fetal serum. Maternal serum IGF-I may mediate the effects of undernutrition on fetal growth by affecting the growth and establishment of the feto-placental unit in mid-gestation. Fetal IGF-I may mediate the effects on muscle growth, whereas IGF-II seems to be related to hepatic glycogen deposition. Cortisol may play a role via its effect on the IGFs, but the thyroid hormones are unlikely to be important until the late gestation/early postnatal period.     

SRY sequence in maternal plasma: Implications for non-invasive prenatal diagnosis: First report from India

AIM:

The presence of circulatory cell-free fetal DNA in maternal plasma has found new applications in non-invasive risk-free prenatal diagnosis.

MATERIALS AND METHODS:

We made use of a size separation approach along with real time polymerase chain reaction (PCR) to evaluate the use of fetal DNA in the detection of the sex of the fetus. Cell-free fetal DNA was isolated from the plasma of 30 women (10–20 weeks gestation) using a size separation approach. We made use of Taq Man Chemistry and real time PCR using primers and probes for GAPDH and SRY.

RESULTS:

Only 24 cases could be studied as there was no amplification in six cases. Fetal sex was accurately determined in all of the 24 cases wherein 19 women were carrying male fetuses and five women were carrying female fetuses. An increase in the amount of fetal DNA was observed with an increase in the gestational age.

CONCLUSIONS:

Real time PCR analysis is a highly sensitive and accurate tool for non-invasive prenatal diagnosis, allowing detection of the sex of the fetus as early as 10 weeks of gestation. Non-invasive prenatal diagnosis eliminates the risk of fetal loss associated with the invasive procedure.     

Magnesium levels in the body fluids of dam and fetal rabbits during the reproductive phase

Gestation- and lactation-dependent variations of magnesium concentrations were measured in the maternal and fetal body fluids and tissues of rabbits. Samples of serum, milk, and mammary gland were investigated. A decrease in magnesium in the maternal serum during the early gestation period from 0.80 mmol/l in the first week to 0.73 mmol/l in the third week was observed. In the last week of gestation a rapid increase of magnesium concentrations both in maternal (0.83 mmol/l) and fetal (2.84 mmol/l) serum was determined. An assumed active diaplacental transfer of magnesium from the doe to the fetus was indirectly established. An active transport mechanism for magnesium from the maternal serum into the mammary glands was enhanced by strong concentration gradients of approximately 1:33 between serum and milk.     

Quantitation of fetal DNA in maternal serum in normal and aneuploid pregnancies

We investigated whether the amount of circulating cell-free fetal DNA in maternal serum is influenced by fetal karyotype, using real-time quantitative polymerase chain reaction assay. Serum samples were obtained from pregnant women at gestational ages ranging from 15 to 17 weeks, prior to their undergoing amniocentesis. In total, we examined 70 samples consisting of 55 cases of pregnancy with 46,XY, 5 cases with 47,XY,+21, 3 cases with 47,XY,+18, a single case with 46,XY,dup(1) and 2 cases with twins of 46,XY, and 4 cases with 46,XX which were used as negative controls. We measured the concentration of the SRY sequence as a molecular marker for fetal DNA. The SRY sequence was detectable and measurable when the fetuses were male except for one case with 47,XY,+18. This case showed fetal growth retardation and bradycardia. No amplification signals of the SRY sequence were detected when the fetuses were female. The mean concentration of fetal DNA in maternal serum was 31.5 copies/ml in the pregnancy with 46,XY, 23.5 copies/ml in the pregnancies with 47,XY,+21 and 21.5 copies/ml in the pregnancies with 46,XY,+18. There were no significant differences in the concentration of fetal DNA between pregnancies with fetuses of normal karyotype and those with fetuses of abnormal karyotype.     

Heterogeneous Distribution of Fetal Microchimerism in Local Breast Cancer Environment

Fetal cells enter maternal circulation during pregnancy and persist in the woman’s body for decades, achieving a form of physiological microchimerism. These cells were also evidenced in tumors. We investigated the frequency and concentration of fetal microchimerism in the local breast cancer environment. From 19 patients with confirmed breast neoplasia, after breast surgical resection, we collected three fresh specimens from the tumor core, breast tissue at tumor periphery, and adjacent normal breast tissue. The presence of male DNA was analyzed with a quantitative PCR assay for the sex determining region gene (SRY) gene. In the group of women who had given birth to at least one son, we detected fetal microchimerism in 100% of samples from tumors and their periphery and in 64% (9 of 14) of those from normal breast tissue. The tissues from the tumor and its periphery carry a significantly increased number of SRY copies compared to its neighboring common breast tissue (p = 0.005). The median of the normalized SRY-signal was about 77 (range, 3.2–21467) and 14-fold (range, 1.3–2690) greater in the tumor and respectively in the periphery than in the normal breast tissue. In addition, the relative expression of the SRY gene had a median 5.5 times larger in the tumor than in its periphery (range, 1.1–389.4). We found a heterogeneous distribution of fetal microchimerism in breast cancer environment. In women with sons, breast neoplasia harbors male cells at significantly higher levels than in peripheral and normal breast tissue.     

Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13 but not trisomy 18

Cell-free fetal DNA in the maternal circulation is a potential noninvasive marker for fetal aneuploidies. In previous studies with Y DNA as a fetal-specific marker, levels of circulating fetal DNA were shown to be elevated in women carrying trisomy 21 fetuses. The goal of this study was to determine whether cell-free fetal DNA levels in the serum of pregnant women carrying fetuses with trisomies 13 or 18 are also elevated. Archived maternal serum samples from five cases of male trisomy 13 and five cases of male trisomy 18 were studied. Each case was matched for fetal gender, gestational age, and duration of freezer storage to four or five control serum samples presumed to be euploid after newborn medical record review. Real-time quantitative polymerase chain reaction amplification of DYS1 was performed to measure the amount of male fetal DNA present. Unadjusted median serum fetal DNA concentrations were 97.5 GE/ml (genomic equivalents per milliliter; 29.2-187.0) for the trisomy 13 cases, 31.5 GE/ml (18.6-77.6) for the trisomy 18 cases, and 40.3 GE/ml (3.7-127.4) for the controls. Fetal DNA levels in trisomy 13 cases were significantly elevated ( P=0.016) by analysis of variance of the ranks of values within each matched set. In contrast, fetal DNA levels in trisomy 18 cases were no different from the controls ( P=0.244). Second trimester maternal serum analytes currently used in screening do not identify fetuses at high risk for trisomy 13. Fetal DNA may facilitate noninvasive screening for trisomy 13 provided that a gender-independent fetal DNA marker can be developed.     

Bovine fetal microchimerism in normal and embryo transfer pregnancies and its implications for biotechnology applications in cattle

Fetal cells and DNA have been detected in the maternal circulation during and after pregnancy in a few mammalian species. The incidence of similar microchimerism in cattle could have repercussion for the application of modern biotechnologies such as the transfer of transgenic embryos. To determine if feto-maternal leakage can occur in pregnant cows, we have analyzed maternal blood samples for the presence of fetal DNA during gestation and post-partum periods. Y chromosome-specific DNA was detected in up to 73% of blood samples from naturally mated heifers carrying conventional bull calves and a transgene-specific sequence in up to 50% of recipient cows carrying transgenic fetuses. These findings document for the first time that transplacental leakage of fetal DNA into the maternal circulation can occur in cattle despite the epitheliochorial placenta of ruminants, with potential implications for the utilization of recipient cows in the food chain.     

Presence of fetal DNA in maternal plasma decades after pregnancy

Cells of fetal origin and cell-free fetal DNA can be detected in the maternal circulation during pregnancy, and it has recently been shown that fetal cells can persist long after delivery. Given the various biological and clinical implications of this fact, we tested the hypothesis that cell-free fetal DNA can be present in maternal plasma decades after pregnancy. We extracted DNA from plasma samples and nucleated blood cells of 160 healthy women with male offspring at different time intervals after delivery (range 1-60 years). All of the samples were tested by means of a real-time quantitative PCR assay for a specific Y chromosome sequence (the SRY gene). Y chromosome-specific DNA was detected in 16 peripheral blood cell samples (10%) and 35 plasma samples (22%). The women with male sequences in the cell fraction had significantly greater total parity ( P=0.018). The proportion of women with detectable Y sequences in the plasma or cell samples was not related to the time since delivery. The fetal DNA concentrations in the genomic material extracted from plasma samples were significantly higher than those extracted from the Y-positive cell samples (149+/-140 vs 20+/-13 genome-equivalents/ml; P<0.001). There was no relationship between the concentration of fetal DNA and the time since delivery. Not only fetal cells, but also fragments of fetal DNA can be present in the maternal circulation indefinitely after pregnancy. This finding has practical implications for non-invasive prenatal diagnoses based on maternal blood, and may be considered for possible pathophysiological correlations.     

利用孕妇血浆DNA检测胎儿性别的研究

本文探讨应用孕妇血浆中游离DNA进行无创性产前性别诊断的可行性。用柱分离法提取73例孕妇血浆中DNA,用巢式PCR技术检测其胎儿SRY基因。 结果73位孕妇血浆DNA含量为0.0062~0.3399μg/μL。巢式PCR检测胎儿SRY基因的灵敏度为97.37%（37/38）,假阴性率2.86%(1/35),特异度85.71%（30/35）,假阳性率13.16%(5/38),总符合率91.78%（67/73）。采用孕妇血浆胎儿DNA和巢式PCR技术可以快速简便的进行无创性产前性别诊断,诊断结果的准确率为91.8%,对性连锁遗传病的预防具有重要意义。 Abstract:To investigate the feasibility and possibility of application of fetal DNA from maternal plasma for noninvasive prenatal diagnosis of fetal sex,plasma DNAs in blood samples of 73 pregnant women at the gestational period of 26 to 41 weeks were extracted by column separation and nested polymerase chain reaction were employed to amplify the SRY gene.A comparison was made between the amplification results and the real sex of the fetus after their delivery.The concordance rate of SRY gene amplification results of plasma free DNA with real fetal sex was 91.78% (67/73),the sensitivity rate was 97.37% (37/38),and the specific rate was 85.71% (30/35).The cell-free fetal DNA in maternal blood can be one of the valuable material sources for noninvasive prenatal diagnosis and the method of nested PCR could be useful for fetal sex determination.The specific rate of the test was 91.78%.It is of significance to prevent sex-linked inheritant diseases.     

Determination of bovine fetal sex by PCR using fetal fluid aspirated by transvaginal ultrasound-guided amniocentesis

Fetal sex can be determined by the polymerase chain reaction (PCR) using cells from fetal fluid collected by transvaginal ultrasound-guided amniocentesis. A total of 35 aspirates from 30 cows, 15 Holsteins and 15 Japanese Blacks at 59 to 250 d of pregnancy were used. Five cows were aspirated twice at a 10-d interval. A 5.0 MHz convex array transducer connected to a scanner was inserted into the vagina under caudal epidural anesthesia. The transducer was equipped with a 65-cm long, 21-g needle within the probe carrier. A bovine male-specific primer and a bovine gender-neutral primer were used. Fetal fluid was obtained from all except 2 cows in early pregnancy. Five animals aborted within 1 wk following aspiration. A total of 33 samples, 29 of amniotic fluid and 4 of allantoic fluid, was subjected to PCR analysis. Fetal gender was verified in 31 33 samples (18 females and 13 males). Gender was also determined by gross examination of external genitalia of offspring after calving or abortion. Fetal gender was correctly identified by PCR analysis of aspirated fetal fluid in 16 16 females and in 13 15 males. Transvaginal ultrasound-guided amniocentesis followed by PCR analysis of aspirated cell DNA can be used accurately to determine fetal sex in cows at 70 to 100 d of gestation. The procedure requires considerable skill and is not without some risk to fetal viability.