The cell-free DNA virome of 108,349 Dutch pregnant women

Objective

Viral infections during pregnancy are a major health concern to mother and fetus. By repurposing cell-free Non Invasive Prenatal Testing (NIPT) sequencing data, we investigated prevalence and abundance of viral DNA in a cohort of 108,349 pregnant women.

Method

Cell-free DNA (cfDNA) sequencing reads that did not map to any of the human chromosomes or mitochondrial DNA of the human reference genome build GRCh38 were aligned to 224 DNA viruses selected from the NCBI refseq viral database.

Results

In total 443,665 reads of viral origin were detected across 42,273 samples representing 165 viral species. Several are known to be potentially harmful during pregnancy and/or childbirth, including Cytomegalovirus, Parvovirus B19 and Hepatitis B. Viral sequences were mostly detected at very low abundance. However, several cases had exceptionally high viral loads for Parvovirus B19, Hepatitis B and others. We found statistically significant associations between presence of viral DNA and gestational age, maternal age, fetal fraction, cfDNA concentration and others.

Conclusion

We demonstrate the feasibility to detect viral DNA from typical genome-wide NIPT cfDNA sequencing and describe the main characteristics of the viral DNA in our cohort. Our dataset of detected viral sequence reads is made publicly available to guide future clinical implementations.     

Noninvasive detection of fetal sex: the laboratory diagnostician's view

Toward the end of the twentieth century it was discovered that cell-free fetal DNA sequences could be detected in maternal blood plasma. Initially, Y-chromosome sequences originating from male fetuses were targeted in cell-free DNA extracted from maternal plasma in order to demonstrate proof of this concept towards the development of noninvasive prenatal diagnosis methods. Clinical application of this approach is now possible. Fetal sex can be detected through a procedure that is noninvasive with respect to the fetus. Specifically, the presence of Y-chromosome sequences in maternal blood plasma indicates that the fetus is male, whereas lack of a signal will indicate that the fetus is female. Fetal sex can be detected very early, from at least the 7th week of pregnancy (and even earlier, according to several studies), about two months before this information is available through ultrasound scanning. Although the controversial issue of fetal sexing is not new, it is expected that with the availability of an accurate noninvasive test, public interest will rise. It is therefore imperative that an authorized committee of experts in each country generates an official policy regarding application of the test. Copyright © 2006 John Wiley & Sons, Ltd.     

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

An Erratum has been published for this article in Prenatal Diagnosis 22(13) 2002, 1241. 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 developped 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. Copyright © 2001 John Wiley & Sons, Ltd.     

An update on maternal medication-related embryopathies

There is a general perception that any exposure to medication during pregnancy poses a potential risk to the fetus. Most available data about teratogenic drugs is derived from animal studies, case reports, or cohort studies. As a result, counseling women and their partners about the safety of drugs during pregnancy can be difficult due to limited information about efficacy, pharmacokinetics, and teratogenicity of some drugs. However, this should always be done in the context of weighing up potential teratogenic risks with the perinatal risks of an untreated medical or psychiatric condition. Ideally, this counseling should occur prior to a planned pregnancy so that medications and treatment of chronic medical conditions can be optimized. It is important that clinicians providing antenatal care are able to confidently manage women including utilizing appropriate resources. This paper aims at reviewing a selected (non-exhaustive) list of the most commonly prescribed medications considered significant human teratogens and provides recommendations for pre-conception and antenatal counseling.     

Maternal thyroid disease and its effects on the fetus and perinatal outcomes

Thyroid disease is common in women of childbearing age and can have significant effects on the development of the fetus and perinatal outcomes. Maternal thyroid hormone is critical for proper fetal neurodevelopment, and the fetus relies on thyroid hormone from its mother for the first half of pregnancy. Both overt maternal hypothyroidism and overt maternal hyperthyroidism have been shown to be associated with adverse effects on central nervous system gray matter and neurocognitive development of offspring as well as increased obstetrical risks. Treatment of overt thyroid conditions improves outcomes. Subclinical maternal hypothyroidism may increase adverse neurocognitive and obstetrical outcomes although data are conflicting. To date, treatment of subclinical hypothyroidism has not shown benefit. Subclinical hyperthyroidism is well tolerated in pregnancy. Thyroid autoantibodies alone may also affect neurodevelopment and obstetrical outcomes; however, recent data have shown no improvement with levothyroxine treatment. Several rare maternal genetic thyroid conditions can affect the fetus including a thyroid-stimulating hormone receptor mutation leading to hypersensitivity to human chorionic gonadotropin and thyroid hormone resistance. The thyroid plays a crucial role in fetal health and understanding it is important for optimal care.     

Prenatal sonographic diagnosis of autosomal recessive polycystic kidney disease (arpkd) during the early second trimester

Autosomal recessive polycystic kidney disease (ARPKD) is a rare hereditary disease with a high neonatal mortality. Currently, prenatal diagnosis is possible only during the second half of pregnancy, when bilaterally enlarged, echogenic kidneys are visible by ultrasound. We describe a case in which a diagnosis of ARPKD was sought in the first half of pregnancy. High-resolution ultrasonography revealed echogenic, normal-sized kidneys at 15+4 weeks. Microsatellite DNA analysis of a chorionic villus sample, parental blood, and blood of an affected sibling showed that the fetus had the maternal haplotype and a recombination of the paternal haplotype. Thus, no distinction between homo- and heterozygosity for the ARPKD mutation in the fetus was possible. A further ultrasound examination at 19+4 weeks confirmed the previous results, indicating that the fetus was likely to be affected. After termination of the pregnancy, the diagnosis was confirmed on microscopic examination.     

The time of appearance and disappearance of fetal DNA from the maternal circulation

A single copy Y-chromosome DNA sequence was amplified using the polymerase chain reaction (PCR) from the peripheral blood of 30 women who had achieved a pregnancy through an in vitro fertilization (IVF) programme. The time of conception was known precisely and was confirmed by serial ultrasound scans. Conceptions were dated as the number of weeks after fertilization plus 2, to give a time equivalent to the obstetric menstrual dating of the pregnancy (LMP). Y-chromosome-specific DNA was detected in all pregnancies with a male fetus (18/30). The earliest detection was at 4 weeks and 5 days, and the latest at 7 weeks and 1 day. Y-chromosome-specific sequences were no longer detected in any of the male pregnancies 8 weeks after delivery. No Y-chromosome sequences were detected in any of the pregnancies where only female babies were delivered. This demonstrates that fetal DNA appears in the maternal circulation early in the first trimester, that it can be identified in all pregnancies tested by 7 weeks, that it continues to be present throughout pregnancy, and that it has been cleared from the maternal circulation 2 months after parturition. Early non-invasive prenatal diagnosis for aneuploidies and inherited disorders will be possible in all pregnancies if fetal cells can be isolated free from maternal contamination (or identified accurately in the presence of maternal cells) without problems of contamination from previous pregnancies.     

Autosomal dominant polycystic kidney disease: Prenatal diagnosis by DNA analysis and sonography at 14 weeks

A pregnant woman affected with autosomal dominant polycystic kidney disease (ADPKD) had a history of an affected fetus, diagnosed by sonography at 29 weeks of pregnancy. The proband's father was also affected. DNA analysis performed on chorionic villi at 11 weeks during a second pregnancy predicted an affected fetus, and sonographic examination at 14 weeks confirmed the diagnosis.     

Spontaneous cessation of umbilical blood flow in the acardiac fetus of a twin pregnancy

The acardiac fetus is a rare entity found only in monozygotic multiple pregnancy. Although the acardiac fetus is non-viable, the perinatal mortality rate for the normal fetus may be as high as 50 per cent, and is usually associated with fetal heart failure and hydrops fetalis, or as the result of prematurity. In this communication, we describe a case of spontaneous cessation of blood flow to an acardiac fetus and discuss the management of this condition with special reference to optimizing the outcome for the normal fetus.     

Microsatellite analysis provides efficient confirmation of fetal trophoblast isolation from maternal circulation

Fetal trophoblasts can be found in maternal circulation from an early stage of pregnancy and thus provide a potential source of DNA for non-invasive prenatal diagnosis. We have developed a two-step method for trophoblast isolation between the 8th and 12th week of pregnancy. Blood was sampled from 14 women undergoing termination of pregnancy or spontaneous abortion. Immunomagnetic beads precoated with HLA class I and II, and with anti-cytokeratin-18 monoclonal antibodies, were used to remove CD8+ and other maternal cells, and to select for fetal trophoblasts, respectively. Microsatellite analysis was performed on DNA extracted from the isolated, maternal, paternal and placental cells after PCR amplification. Recovery of the trophoblasts was confirmed in 13/14 cases (93%) by the identification of an identical microsatellite pattern for fetal and placental cells. Further evidence was the presence of heterozygous alleles of both maternal and paternal origin. The correct prediction of gender in all five male fetuses was an additional confirmation of trophoblast recovery. We conclude that trophoblasts can be effectively isolated from maternal blood in the first trimester, and by using polymorphic microsatellite markers to confirm sample purity, this method has potential future application in prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis of Apert syndrome: report of two cases

Two de novo cases with Apert Syndrome detected prenatally are presented herein. In the first, fetal ultrasound findings of syndactyly of the hands, craniosynostosis and proptosis resulted in a prenatal diagnosis in the nineteenth week of gestation. This is the earliest prenatal diagnosis of this syndrome in a not-at-risk case. Following counseling, this pregnancy was terminated and subsequent pathological examination and DNA analysis confirmed the diagnosis of Apert Syndrome and coarctation of the aorta. In the second case, fetal ultrasound at 21 weeks' gestation revealed a hypoplastic left heart and clover-leaf skull. Following counseling, this pregnancy was also terminated. Further examination of the fetus and DNA analysis led to a diagnosis of Apert Syndrome. These cases emphasize the need to complete a thorough fetal ultrasound in cases with potentially lethal cardiac abnormality and the importance of incorporating a fetal pathologist, as well as a medical geneticist, in the investigations performed after delivery or pregnancy termination when a fetal abnormality is detected on ultrasound. Copyright © 2003 John Wiley & Sons, Ltd.     

Oncogenic Papillomavirus and Polyomavirus in Water Environments: Is There a Potential for Waterborne Transmission?

Waterborne exposure to human viruses through contact with sewage-contaminated water environments can result in infections associated with a wide range of illnesses. Gastrointestinal symptoms are the most commonly encountered manifestations of waterborne viral illness. Respiratory diseases, neurological diseases and paralysis can also occur. Whether viral infections resulting in health outcomes like cancer might also be transmitted by the waterborne route is unknown. Recently, viruses belonging to two oncogenic groups—Human Papillomaviruses (HPVs) and Human Polyomaviruses (HPyVs)—have been detected in urban sewages worldwide. The latter have also been identified in other water environments. HPVs are epitheliotropic viruses responsible for several diseases of skin and mucosae, from common warts to squamous intraepithelial lesions that can either heal or progress to invasive carcinoma of the cervix, vulva, vagina, penis, anus or oropharynx. Human PyVs infect different tissues and organs, causing infections that are usually subclinical in immunocompetent individuals but can be serious in immunocompromised hosts. These pathogens belong to a family of DNA tumour viruses. Merkel cell polyomavirus, a HPyV identified in recent years, has attracted much attention due to its link with a rare and aggressive form of human cancer. Merkel cell carcinoma, the incidence of which has tripled over the past two decades. JC polyomavirus and BK polyomavirus are also potentially oncogenic. The observed abundance and wide dissemination of HPVs and HPyVs in water environments strongly suggest the need to shed light on the fate of these viruses in water environments and to elucidate their potential for waterborne transmission. Such information is essential for the improvement of wastewater management programs in terms of both sewage treatment and water quality surveillance.     

Prenatal evaluation and in utero platelet transfusion for thrombocytopenia absent radii syndrome

A fetus with absent radii in both forearms was discovered on routine ultrasound examination performed at 18 weeks of pregnancy. No other significant abnormalities were found, and no signs of haemorrhage were detected. Serial ultrasound examinations revealed no evidence of fetal internal bleeding. At 37 weeks of pregnancy, a CBC obtained by cordocentesis under ultrasound guidance confirmed the diagnosis of thrombocytopenia absent radii (TAR) syndrome. Apheresis platelets were transfused into the umbilical vein to correct the thrombocytopenia and was followed by an uncomplicated delivery. No bleeding was encountered during the remainder of the baby's neonatal course. We conclude that TAR syndrome can be readily identified prenatally on sonogram, and if severe thrombocytopenia is confirmed by cordocentesis, platelets should be transfused to diminish the risk of serious internal bleeding during and immediately after delivery.     

Mid-trimester fetal ultrasound: Diagnostic dilemmas

Two cases are reported in which an unusual ultrasound finding preceded diagnostic amniocentesis and led to further work-up. In both cases a decision was made to terminate the pregnancy. One fetus in which a neck mass was detected by ultrasound was shown to be normal on post-mortem examination. The second fetus was aborted because of Rh sensitization and had the abnormality seen by ultrasound. However, this lesion, calcified intrahepatic plaques, had no presumed pathological significance. These cases suggest caution in the interpretation of results obtained with the new technologies used for prenatal diagnosis.     

Monosomy 8q: Prenatal diagnosis and autopsy findings

The autopsy findings of a fetus with deletion of the long arm of chromosome 8 are described. Many of the features are similar to those of the tricho-rhino-phalangeal syndromes, types I and II, which are associated with deletions on chromosome 8q24. Other findings in this case, such as total absence of the corpus callosum and intestinal malrotation, have not been described in these syndromes. Genes involved in the development of the latter malformations may reside in adjacent regions on the long arm of chromosome 8. An elevated serum level of beta human chorionic gonadotropin (βhCG) was found during pregnancy. This aberration should be included with other chromosomal disorders which may be detected by this test.     

Detection of Potential Infectious Enteric Viruses in Fresh Produce by (RT)-qPCR Preceded by Nuclease Treatment

Foodborne illnesses associated with contaminated fresh produce are a common public health problem and there is an upward trend of outbreaks caused by enteric viruses, especially human noroviruses (HNoVs) and hepatitis A virus (HAV). This study aimed to assess the use of DNase and RNase coupled to qPCR and RT-qPCR, respectively, to detect intact particles of human adenoviruses (HAdVs), HNoV GI and GII and HAV in fresh produce. Different concentrations of DNase and RNase were tested to optimize the degradation of free DNA and RNA from inactivated HAdV and murine norovirus (MNV), respectively. Results indicated that 10 µg/ml of RNase was able to degrade more than 4 log₁₀ (99.99%) of free RNA, and 1 U of DNase degraded the range of 0.84–2.5 log₁₀ of free DNA depending on the fresh produce analysed. The treatment with nucleases coupled to (RT)-qPCR was applied to detect potential infectious virus in organic lettuce, green onions and strawberries collected in different seasons. As a result, no intact particles of HNoV GI and GII were detected in the 36 samples analysed, HAdV was found in one sample and HAV was present in 33.3% of the samples, without any reasonable distribution pattern among seasons. In conclusion, RT-qPCR preceded by RNase treatment of eluted samples from fresh produce is a good alternative to detect undamaged RNA viruses and therefore, potential infectious viruses. Moreover, this study provides data about the prevalence of enteric viruses in organic fresh produce from Brazil.     

Pericentric inversion of chromosome 19: prenatal diagnosis and genetic counselling

During prenatal diagnosis for advanced maternal age, a pericentric inversion of a chromosome 19 was detected in a male fetus. The inversion was familial, transmitted to the fetus by the phenotypically normal mother. The pregnancy resulted in a term birth of a phenotypically normal male infant. Inversion 19 appears to be a rare abnormality with only seven families reported thus far including ours. Infants with duplication deficiencies for chromosome 19 have not been reported in these families. This may suggest an apparent suppression of crossing over and recombination within the inverted segment of chromosome 19 during meiosis.     

Prenatal identification of i(YP) by molecular cytogenetic analysis

An i(Yp) is a rare marker chromosome. We present a case of de novo 46,X,i(Yp) detected prenatally in an amniotic fluid specimen. Fluorescence in situ hybridization (FISH) studies using a panel of Y-specific biotinylated DNA probes identified the marker chromosome as i(Yp). Comparative genomic hybridization (CGH) studies further confirmed the diagnosis. Upon pregnancy termination, external examination of the fetus revealed a generally well-developed male fetus with slight facial dysmorphism and prominent rocker-bottom feet. The molecular cytogenetic data in this case proved very useful in genetic counselling and served as a good example illustrating the important role of molecular techniques for accurate identification of marker chromosomes.     

First-trimester prenatal diagnosis of the nijmegen breakage syndrome and ataxia telangiectasia using an assay of radioresistant dna synthesis

Prenatal diagnosis was performed in two pregnancies at risk of the Nijmegen breakage syndrome. In one pregnancy, an affected fetus was diagnosed by demonstration of radioresistant DNA synthesis, using autoradiographic detection of incorporated tritiated thymidine in cultured chorionic villus cells. The diagnosis was confirmed in fetal skin fibroblasts. In the other case, the fetus appeared unaffected. Using the same procedure, unaffected fetuses were predicted from chorionic villus cells in two pregnancies at risk of ataxia telangiectasia, which is another genetic disorder showing the feature of radioresistant DNA synthesis. The present biochemical method for prenatal detection of Nijmegen breakage syndrome and ataxia telangiectasia can be used as a simplified alternative to the cytogenetic procedures reported earlier for ataxia telangiectasia.