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.     

Hunter syndrome: prenatal diagnosis in maternal serum.

Iduronate sulfate sulfatase (ISS), the deficient hydrolase in Hunter syndrome, consistently increases in the serum of pregnant women, reaching a three- to fourfold increase from pre-pregnancy levels toward the end of pregnancy. In Hunter carriers, a correlation occurs between the status of the fetus with regard to Hunter syndrome and the ISS increase in maternal serum. Thus, in pregnancies with Hunter-affected fetuses, enzyme levels did not change in the serum of heterozygous mothers until abortion was performed, while in nonaffected fetuses, ISS increased usually very early in pregnancy--as early as the 6th-12th week. In heterozygote female fetuses, this increase might be delayed. These data imply that a prenatal diagnosis of Hunter syndrome might be accomplished in maternal serum at early conventional procedures for the prenatal diagnosis of Hunter syndrome.     

Prenatal diagnosis using fetal cells from the maternal circulation.

All current methods of fetal karyotyping are invasive and carry a definite, albeit small, procedure-related risk. Because of this and testing costs, only women older than 35 years who have a greater risk for fetal aneuploidy are currently offered prenatal testing. But this detects only 20% to 25% of fetuses with Down syndrome. It would be a tremendous advance to find a noninvasive technique for prenatal diagnosis that carries no procedure-related risk and could be offered to all pregnant women. We describe a possible technique for noninvasive prenatal diagnosis that aims to identify fetal cells in the peripheral maternal circulation and successfully garner them for prenatal testing. Early attempts at fetal karyotyping were hampered by inaccurate diagnostic methods and cumbersome cell-counting techniques. Today, improved capabilities of identifying and enriching for fetal cells, coupled with sensitive methods of analysis such as the polymerase chain reaction, bring renewed enthusiasm to this task. Many technical issues, as well as serious questions regarding the test''s utility, still exist, however, and must be explored and answered before the capture of fetal cells in the maternal circulation translates into reality for noninvasive prenatal diagnosis.     

Probing the fetal genome: progress in non-invasive prenatal diagnosis

Progress in our understanding of the molecular basis of heritable diseases, through identification of specific mutations, has provided a foundation for the development of DNA-based prenatal diagnosis. Genetic analysis of fetal DNA is now routinely performed from chorionic villus samples obtained as early as the tenth week of gestation or by amniocentesis from week 15 onwards. However, both of these approaches involve invasive procedures with increased risk of fetal loss. To avoid such complications, attempts have been made to develop non-invasive tests through the identification, characterization and isolation of fetal cells or free fetal DNA from the maternal circulation. Recently, progress has been made towards the development of novel strategies that are expected to provide non-invasive means for early prenatal diagnosis in pregnancy.     

A monoclonal antibody with potential for aiding non-invasive prenatal diagnosis: utility in screening of pregnant women at risk of preeclampsia.

The development of a non-invasive method of prenatal diagnosis in maternal blood has been the goal of our investigations during the last years. We have developed several anti-CD71 monoclonal antibodies and optimized a protocol for the isolation of nucleated red blood cells (NRBC) from peripheral maternal blood. The enhanced traffic of fetal erythroblasts into the maternal circulation in preeclampsia has been investigated by several groups. In this study, we compared one of our antibodies, 2F6.3, with a commercial anti-CD71 antibody in blood samples from pregnant women suffering pregnancy-induced hypertension (PIH) and in a control group of pregnant women without clinical features suggestive of an increased risk of developing preeclampsia. The mAb 2F6.3, developed by our group, has succeeded in isolating a significantly higher number of erythroblasts with less maternal cell contamination than the commercial antibody in both women with PIH and in the control group (p<0.01; Wilcoxon Signed Ranks Test). Fluorescence in situ hybridization analysis also demonstrated that 2F6.3 is a better antibody for the isolation of fetal NRBC in maternal blood than the commercial anti-CD71 antibody.     

The fetal erythroblast is not the optimal target for non-invasive prenatal diagnosis: preliminary results.

Fetal cells, present in the blood of pregnant women, are potential targets for non-invasive prenatal diagnosis. The fetal erythroblast has been the favorite target cell type. We investigated four methods of enrichment for fetal erythroblasts, identifying only three fetal erythroblasts in 573 ml of maternal blood. This is much less than the expected two to six fetal cells per ml of maternal blood. Hamada and Krabchi used a cell type-independent marker, i.e., the Y chromosome in maternal blood from male pregnancies after Carnoy fixation, leaving the nuclei for hybridization with X-and Y-chromosome-specific probes. We found with a similar technique 28 fetal cells in 15 ml of maternal blood. The fetal origin of cells was confirmed by hybridizing the nuclei with X- and Y-chromosome-specific probes, using two consecutive hybridizations with the two probes in opposite colors (reverse FISH). Candidate fetal cells were inspected after each hybridization. Only cells that were found to change the color of both probe signals from first to second hybridization were diagnosed as fetal. To reduce the labor-intensive slide screening load, we used semiautomated scanning microscopy to search for candidate cells. We conclude that erythroblasts form only a small fraction of fetal cells present in maternal blood.     

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.     

The molecular basis of beta-thalassemia in Thailand: application to prenatal diagnosis.

To enable the prenatal diagnosis of beta-thalassemia by direct detection of the mutant beta-globin genes, we have determined the spectrum of mutations causing this disease in Thailand. The techniques employed included a combination of synthetic oligonucleotide probe hybridization, direct sequencing of genomic DNA enzymatically amplified by the polymerase chain reaction, and cloning and sequencing of the beta-globin genes. A total of 116 beta-thalassemia genes from 78 Hb E/beta-thalassemia patients and from 19 homozygous beta-thalassemia patients were analyzed, and the mutation was characterized in 112/116 (97%) of them. Eleven mutations were found, of which four (-CTTT in codon 41/42, AAG----TAG in codon 17, C----T in position 654 of the IVS-2 region, and A----G in position -28 upstream of the beta-globin gene) accounted for 83%; two previously undescribed mutations have been identified. The spectrum of beta-thalassemia mutations is similar to that reported among the Chinese. However, within the Thai population itself, patients with homozygous beta-thalassemia show a wider spread of mutations in comparison with the Hb E/beta-thalassemia group, in whom the frameshift 41/42 mutation predominates at a frequency of 62%. This difference in distribution may reflect the difference in ethnic origin of the two groups. Characterization of these mutations should aid the planning of a prenatal diagnosis program for beta-thalassemia in Thailand.     

Mitosis of maternal lymphocytes in the presence of fetal cells: Possible implication in prenatal diagnosis from fetal blood samples

Summary The suppression of proliferation of maternal lymphocytes by the lymphocytes of their own male newborns have been tested in a PHA-induced two-way stimulation system. The mixed lymphocyte cultures of 6 out of 12 such mother/son pairs had 23–50% metaphases with 46,XX karyotype. In 2 more cases 10% maternal metaphases have been observed. Hence, it appears that fetal lymphocytes are unable to suppress the proliferation of maternal cells completely.     

Cryopreservation and in vitro expansion of chondroprogenitor cells isolated from the superficial zone of articular cartilage

Understanding the proliferation mechanisms of chondroprogenitor cells and their influence on cell differentiation is crucial in order to develop large-scale expansion processes for tissue engineering applications. Proliferation control mechanisms were mainly attributed to substrate limitation and cell-cell contact inhibition. The limiting substrates were found to be components of the FCS, with an optimal proliferation rate achieved in the presence of 40% FCS. In addition, the medium supply rate was found to be essential in reducing substrate limitation. In terms of FCS, 10 microL FCS cm(-2) h(-1) was the threshold feed rate required to prevent substrate limitation. Above this rate, maximum cell densities of 5.3 x 10(5) cells/cm2 were achieved, representing a 53-fold expansion. To reduce the need for high supply rates, the effect of specific growth factors was also investigated. Cell densities of 3.3 x 10(5) cells/cm2 were achieved in batch cultures using 40% FCS and 1 ng/mL TGF-beta1. Chondroprogenitor cells were expanded in this medium up to three passages without compromising their ability to differentiate and produce cartilage-like matrix in pellet cultures. In addition to substrate limitation, cell-cell contact, even at very sparse subconfluent densities, appeared capable of exerting some degree of growth inhibition. The cells exhibited deceleratory growth kinetics, characterized by a decrease of specific growth rates over time.     

Untargeted metabolomics: an overview of its usefulness and future potential in prenatal diagnosis

ABSTRACT

Introduction: Metabolomics opens up new avenues for biomarker discovery in different branches of medicine, including perinatology. Chromosomal aberration, preterm delivery (PTD), congenital heart defects, spina bifida, chorioamnionitis, and low birth weight are the main perinatal pathologies. Investigations using untargeted metabolomics have found the candidate metabolites for diagnostic biomarkers.

Areas covered: This review describes areas of prenatal diagnosis in which untargeted metabolomics has been used. Data on the disease, type of sample, techniques used, number of samples used in the study, and metabolites obtained including the sign of their regulation are summarized.

Expert commentary: Untargeted metabolomics is a powerful tool which can shed a new light on prenatal diagnostics. It helps to discover affected metabolic pathways what may help to reveal disease pathogenesis and propose potential biomarkers. Among others, glycerol and 2- and 3-hydroxybutyrate were proposed as markers of chromosomal aberration. Serum metabolic signature of PTD was characterized by increased lipids and decreased levels of hypoxanthine, tryptophane, and pyroglutamic acid. Lower level lipids and vitamin D3 metabolites together with increased bilirubin level in maternal serum were associated with macrosomia. However, to give a real value to those assays and allow their clinical application multicenter, large cohort validation studies are necessary.     

In vitro activity of potential probiotic Lactobacillus murinus isolated from the dog

Aims:  The aim of this study was to isolate and identify Lactobacillus spp. isolates from faeces of a healthy dog, and to characterize their potential as probiotics in order to evaluate their possible use as probiotics for dogs.
Methods and Results:  An in vitro approach was used to characterize the isolates as potential probiotics including the evaluation of pH and bile salts tolerance, production of antimicrobial substances, biofilm formation on glass and polystyrene surfaces, aggregation ability and adhesion to canine intestinal mucus. The isolates survived to different pH and bile salts conditions, inhibited the in vitro growth of Escherichia coli and Clostridium perfringens , and adhered to glass and intestinal mucus.
Conclusions:  The properties shown by these isolates may indicate that they could colonize and persist in the gastrointestinal tract and induce beneficial effects to the host.
Significance and Impact of Study:  The evaluation of native canine isolates and future exprimental feeding assays may be useful tools to develop probiotics to improve animal health and reduce the risk of gastrointestinal disorders.     

Linkage analysis in X-linked adrenoleukodystrophy and application in post-and prenatal diagnosis

Summary We have performed linkage analysis with the DNA markers DXS52 and the clotting factor VIII gene (F8C), in several large families with X-linked adrenoleukodystrophy (ALD). The tight linkage to DXS52 could be extended giving a maximal LOD score of 22.5 at 1 cM. F8C was also tightly linked to ALD with a maximal LOD score of 7.8 without recombination. Multipoint linkage analysis with the markers DXS304, DXS52, and F8C indicated that both the gene for ALD and for F8C are distal to DXS52. In four patients with ALD, no major structural rearrangement in the Xqter region was observed; in particular, there were no abnormalities in the vision blindness genes. DNA analysis appeared to be of use in determination of the carrier status of females at risk, for the determination of the origin of the mutation in a particular family, and for prenatal diagnosis.     

Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro

Isolation of a true self-renewing stem cell from the human brain would be of great interest as a reliable source of neural tissue. Here, we report that human fetal cortical cells grown in epidermal growth factor expressed low levels of telomerase and telomeres in these cultures shortened over time leading to growth arrest after 30 weeks. Following leukemia inhibitory factor (LIF) supplementation, growth rates and telomerase expression increased. This was best demonstrated following cell cycle synchronization and staining for telomerase using immunocytochemistry. This increase in activity resulted in the maintenance of telomeres at approximately 7 kb for more than 60 weeks in vitro. However, all cultures displayed a lack of oligodendrotye production, decreases in neurogenesis over time and underwent replicative senescence associated with increased expression of p21 before 70 weeks in vitro. Thus, under our culture conditions, these cells are not stable, multipotent, telomerase expressing self-renewing stem cells. They may be more accurately described as human neural progenitor cells (hNPC) with limited lifespan and bi-potent potential (neurons/astrocytes). Interestingly, hNPC follow a course of proliferation, neuronal production and growth arrest similar to that seen during expansion and development of the human cortex, thus providing a possible model neural system. Furthermore, due to their high expansion potential and lack of tumorogenicity, these cells remain a unique and safe source of tissue for clinical transplantation.     

Genetic prenatal maternal effects on organ size in mice and their potential contribution to evolution

Two embryo transfer experiments were carried out in order to estimate the magnitude of prenatal maternal effects, independent of postnatal maternal factors, on the growth of internal organs and fat pads in mice. Reciprocal embryo transfers between the inbred mouse strains C3HeB/FeJ and SWR/J yielded three significant findings. First, all traits were not equally influenced by prenatal maternal factors. Genetic prenatal maternal factors, stemming from the genotype of the uterine mother, had a significant effect on testis weight, subcutaneous fat pad weight and epididymal fat pad weight in 21 day old progeny, but they had no effect on cranial capacity, an index of brain size, kidney weight, or liver weight. Prenatal litter size, defined as the sum of live and dead pups at birth, had a significant negative relationship with 21 day testis weight and kidney weight, and a significant positive association with subcutaneous and epididymal fat pad weights. Cranial capacity and liver weight at 21 days postnatally were not influenced by prenatal litter size. Second, the experiments demonstrated that there was ontogenetic variability in the strength of prenatal maternal effects. At 70 days of age, only subcutaneous fat pad weight was significantly influenced by genetic prenatal effects, and prenatal litter size had a significant negative relationship only with subcutaneous fat pad weight and body weight. Third, genetic prenatal effects had a significant influence on the among-trait covariances at 21 days postnatally, but not at 70 days. Because multivariate evolution involves covariances among characters, the latter results suggest that prenatal effects due to the mother's genotype can affect phenotypic evolution of mammals, especially for selection imposed early in life.     

Pyruvate carboxylase activity in chorionic villi: possibility of application to prenatal diagnosis

Pyruvate carboxylase (PC) activity was assayed in 27 chorionic villi samples (CVS) obtained at 9-12 weeks of gestation. The kinetic properties of the CVS enzyme were similar to those of liver PC; more than 75% of PC activity was recovered in the mitochondrial fraction of CVS. Apparent Km for pyruvate, ATP, acetyl CoA and HCO3- in the presence of saturation concentrations of the other reactants, were 0.3, 0.44, 0.015 and 6.0 mmol/l, respectively. The optimum pH was 7.5-8.0. The activity of PC in CVS was 3.2 +/- 0.3 nmol/min/mg protein, which is severalfold higher than that of amniotic fluid fibroblasts.