Cell-free fetal DNA is present in the plasma of pregnant women. It consists of short DNA fragments among primarily maternally derived DNA fragments. We sequenced a maternal plasma DNA sample at up to 65-fold genomic coverage. We showed that the entire fetal and maternal genomes were represented in maternal plasma at a constant relative proportion. Plasma DNA molecules showed a predictable fragmentation pattern reminiscent of nuclease-cleaved nucleosomes, with the fetal DNA showing a reduction in a 166-base pair (bp) peak relative to a 143-bp peak, when compared with maternal DNA. We constructed a genome-wide genetic map and determined the mutational status of the fetus from the maternal plasma DNA sequences and from information about the paternal genotype and maternal haplotype. Our study suggests the feasibility of using genome-wide scanning to diagnose fetal genetic disorders prenatally in a noninvasive way.

Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus

Background: The discovery of circulating fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. MicroRNAs (miRNAs), a class of small RNAs, have been intensely investigated recently because of their important regulatory role in gene expression. Because nucleic acids of placental origin are released into maternal plasma, we hypothesized that miRNAs produced by the placenta would also be released into maternal plasma.

Methods: We systematically searched for placental miRNAs in maternal plasma to identify miRNAs that were at high concentrations in placentas compared with maternal blood cells and then investigated the stability and filterability of this novel class of pregnancy-associated markers in maternal plasma.

Results: In a panel of TaqMan MicroRNA Assays available for 157 well-established miRNAs, 17 occurred at concentrations >10-fold higher in the placentas than in maternal blood cells and were undetectable in postdelivery maternal plasma. The 4 most abundant of these placental miRNAs (miR-141, miR-149, miR-299-5p, and miR-135b) were detectable in maternal plasma during pregnancy and showed reduced detection rates in postdelivery plasma. The plasma concentration of miR-141 increased as pregnancy progressed into the third trimester. Compared with mRNA encoded by CSH1 [chorionic somatomammotropin hormone 1 (placental lactogen)], miR-141 was even more stable in maternal plasma, and its concentration did not decrease after filtration.

Conclusion: We have demonstrated the existence of placental miRNAs in maternal plasma and provide some information on their stability and physical nature. These findings open up a new class of molecular markers for pregnancy monitoring.

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Detection and characterization of placental microRNAs in maternal plasma.

Reproduction is required for the survival of all mammalian species, and thousands of essential 'sex' genes are conserved through evolution. Basic research helps to define these genes and the mechanisms responsible for the development, function and regulation of the male and female reproductive systems. However, many infertile couples continue to be labeled with the diagnosis of idiopathic infertility or given descriptive diagnoses that do not provide a cause for their defect. For other individuals with a known etiology, effective cures are lacking, although their infertility is often bypassed with assisted reproductive technologies (ART), some accompanied by safety or ethical concerns. Certainly, progress in the field of reproduction has been realized in the twenty-first century with advances in the understanding of the regulation of fertility, with the production of over 400 mutant mouse models with a reproductive phenotype and with the promise of regenerative gonadal stem cells. Indeed, the past six years have witnessed a virtual explosion in the identification of gene mutations or polymorphisms that cause or are linked to human infertility. Translation of these findings to the clinic remains slow, however, as do new methods to diagnose and treat infertile couples. Additionally, new approaches to contraception remain elusive. Nevertheless, the basic and clinical advances in the understanding of the molecular controls of reproduction are impressive and will ultimately improve patient care.

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The biology of infertility: research advances and clinical challenges

https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(14)63100-1/pdf

Real‐time PCR in the microbiology laboratory

Rapid progress in identifying biomarkers that are hallmarks of disease has increased demand for high-performance detection technologies. Implementation of electrochemical methods in clinical analysis may provide an effective answer to the growing need for rapid, specific, inexpensive, and fully automated means of biomarker analysis. This Review summarizes advances from the past 5 years in the development of electrochemical sensors for clinically relevant biomolecules, including small molecules, nucleic acids, and proteins. Various sensing strategies are assessed according to their potential for reaching relevant limits of sensitivity, specificity, and degrees of multiplexing. Furthermore, we address the remaining challenges and opportunities to integrate electrochemical sensing platforms into point-of-care solutions.

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Electrochemical Methods for the Analysis of Clinically Relevant Biomolecules

To the Editor: An invasive approach is still the gold standard for prenatal diagnosis of genetic disorders. Chorionic-villus sampling, the current procedure of choice, allows an early diagnosis, bu...

New strategy for prenatal diagnosis of X-linked disorders.

Fetal sex prediction can be achieved using PCR targeted at the SRY gene by analysing cell-free fetal DNA in maternal serum. Unfortunately, the results reported to date show a lack of sensitivity, especially during the first trimester of pregnancy. Therefore, determination of fetal sex by maternal serum analysis could not replace karyotype analysis following chorionic villus sampling. A new highly sensitive real-time PCR was developed to detect an SRY gene sequence in maternal serum. Analysis was performed on 121 pregnant women during the first trimester of pregnancy (mean gestational age: 11.8 weeks). Among them, 51 had at least one previous male-bearing pregnancy. Results were compared with fetal sex. SRY PCR analysis of maternal serum was in complete concordance with fetal sex. Among the 121 pregnant women, 61 were bearing a male fetus and 60 a female fetus. No false-negative results were observed. Furthermore, no false-positive results occurred, even though 27 women carrying a female fetus during the current pregnancy had at least one previous male-bearing pregnancy. This study demonstrates that a reliable, non-invasive sex determination can be achieved by PCR analysis of maternal serum during the first trimester of pregnancy. This non-invasive approach for fetal sex prediction should have great implications in the management of pregnant women who are carriers of an X-linked genetic disorder. Prenatal diagnosis might thus be performed for male fetuses only, avoiding invasive procedures and the risk of the loss of female fetuses.

First-trimester fetal sex determination in maternal serum using real-time PCR.

Circulating cell-free fetal DNA in maternal serum offers an early and non-invasive method for prenatal diagnosis, but the origin of this DNA is still unknown. We report the absence of the SRY gene in maternal serum of a pregnant woman despite male genitalia at ultrasound. The karyotype was 45,X after direct trophoblast analysis and 45,X/46,Xidic(Yp) after culture and in all fetal tissues studied. Due to the absence of the SRY sequence in maternal blood and in the cytotrophoblast, we presume that free fetal DNA in this case originates from trophoblastic cells. As the case presented here is exceptional, it only has a minor impact on the accuracy of fetal sex determination by maternal serum analysis, but highlights the importance of and the necessity for the complementary ultrasonographic control.

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Circulating cell‐free fetal DNA in maternal serum appears to originate from cyto‐ and syncytio‐trophoblastic cells. Case report

In the present study we investigated the results of cytogenetic analysis in male and female patients included in an intracytoplasmic sperm injection (ICSI) programme for severe male infertility as well as in conceptuses resulting from these ICSI treatments. In the 261 couples treated, 11 male (4.2%) and three female (1.2%) abnormal karyotypes were found, all consisting of structural chromosome anomalies. Chromosomal translocation exhibited the highest frequency (eight males and two females), and there were also three cases of chromosomal inversion (two males and one female) and one male with one additional marker chromosome. There was no difference in fertilization rates among couples with abnormal (n = 14) and normal (n = 147) cytogenetic results, and the rates of clinical pregnancy per ICSI attempt were 25.0% (5/20) and 20.6% (78/ 378) respectively. In pregnancies obtained in couples with normal karyotypes, all of the 108 fetuses were free of chromosomal abnormalities. Among the eight fetuses from couples with chromosome structural anomalies, three out of five and two out of three inherited from the cytogenetic defects found in their father or mother respectively. In this series of 83ICSI pregnancies there were no chromosomal abnormalities other than those inherited from the parents. These findings suggest that normal pregnancy rates can be obtained by ICSI in cases of chromosomal translocation in couples with severe male infertility. However, until further evaluations of available data can be performed, cytogenetic analysis must be conducted prior to ICSI in men with low sperm counts, and genetic counselling must include prenatal diagnosis for all growing conceptuses.

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Genetics: Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities

Fetal RHD genotype determination is useful in the management of sensitized RhD-negative pregnant women. It can be ascertained early during pregnancy by chorionic villus sampling (CVS) or amniocentesis. However, these procedures are invasive, resulting both in an increased risk of fetal loss and in an increased severity of immunization due to fetomaternal haemorrhage. A reliable determination of RHD genotype by fetal DNA analysis in maternal serum during the first trimester of pregnancy is reported in this study. One hundred and six sera from RhD-negative pregnant women were obtained during the first trimester of pregnancy. These sera were tested for the presence of RHD gene using a new real-time polymerase chain reaction assay and the results compared with those obtained later in pregnancy on amniotic fluid cells and by RHD serology of the new-born. All sera from women carrying a RhD-positive fetus (n = 62) gave positive results for RHD gene detection and sera from women carrying a RhD-negative fetus (n = 40) were negative. The high level of accuracy of fetal RHD genotyping obtained in this study could enable this technique to be offered on a routine basis for the management of RhD-negative patients during the first trimester of pregnancy.

Evelyne Gautier

Papers

New strategy for prenatal diagnosis of X-linked disorders.

First-trimester fetal sex determination in maternal serum using real-time PCR.

Circulating cell‐free fetal DNA in maternal serum appears to originate from cyto‐ and syncytio‐trophoblastic cells. Case report

Genetics: Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities

Fetal RHD genotyping in maternal serum during the first trimester of pregnancy