Fetal Genotype Research Articles

Following targeted routine antenatal anti-D prophylaxis, almost half of the pregnant women had undetectable anti-D prophylaxis at delivery.

In September 2016, a nationwide targeted routine antenatal anti-D prophylaxis program was implemented in Norway. The prophylaxis (anti-D immunoglobulin) aims to cover the whole third trimester and is administered in gestational week 28 to RhD-negative women who carry RhD-positive fetuses. However, in many women, antibody screening at delivery does not detect anti-D immunoglobulin. The goal of this study was to investigate the presumable role of dose and timing of antenatal anti-D immunoglobulin administration in non-detectable prophylaxis at the time of delivery. In this retrospective observational study, RhD-negative pregnant women who gave birth at Oslo University Hospital and Akershus University Hospital between January 2017 and December 2019 were analyzed. Women who received antenatal anti-D immunoglobulin (1500IU at Oslo University Hospital and 1250IU at Akershus University Hospital) when fetal RHD genotyping at gestational week 24 predicted an RhD-positive fetus were included if an antibody screen at delivery was available. Data from the blood bank, maternity information systems, and electronic patient records were used. Analysis of the 984 RhD-negative women at the two hospitals revealed that 45.4% had non-detectable anti-D at delivery. A significant difference between the two hospitals was observed: 40.5% at Oslo University Hospital (n=509) and 50.7% at Akershus University Hospital (n=475) (p=0.001). The proportion with non-detectable anti-D increased to 56.0 and 75.3%, respectively (p=0.008) in the group of women who gave birth 12 weeks after routine antenatal anti-D prophylaxis. Significantly fewer women had detectable anti-D at delivery when the lower anti-D immunoglobulin dose (1250IU) was administered antenatally. Multiple logistic regression indicated that the time interval between routine antenatal anti-D prophylaxis and delivery, in addition to anti-D dose, were significantly associated with detectable anti-D at delivery (p<0.001). We do not know which RhD-negative pregnant women, despite antenatal anti-D prophylaxis, are at risk of RhD alloimmunization, when antibody screening is negative at delivery. Administration of antenatal prophylaxis should probably be moved closer to delivery, since the risk of fetomaternal hemorrhage is higher during the last weeks of the third trimester.

Maternal Metabolic State and Fetal Sex and Genotype Modulate Methylation of the Serotonin Receptor Type 2A Gene (HTR2A) in the Human Placenta.

The serotonin receptor 2A gene (HTR2A) is a strong candidate for the fetal programming of future behavior and metabolism. Maternal obesity and gestational diabetes mellitus (GDM) have been associated with an increased risk of metabolic and psychological problems in offspring. We tested the hypothesis that maternal metabolic status affects methylation of HTR2A in the placenta. The prospective study included 199 pairs of mothers and healthy full-term newborns. Genomic DNA was extracted from feto-placental samples and analyzed for genotypes of two polymorphisms (rs6311, rs6306) and methylation of four cytosine residues (−1665, −1439, −1421, −1224) in the HTR2A promoter region. Placental HTR2A promoter methylation was higher in male than female placentas and depended on both rs6311 and rs6306 genotypes. A higher maternal pre-gestational body mass index (pBMI) and, to a lesser extent, diagnosis of GDM were associated with reduced HTR2A promoter methylation in female but not male placentas. Higher pBMI was associated with reduced methylation both directly and indirectly through increased GDM incidence. Tobacco use during pregnancy was associated with reduced HTR2A promoter methylation in male but not female placentas. The obtained results suggest that HTR2A is a sexually dimorphic epigenetic target of intrauterine exposures. The findings may contribute to a better understanding of the early developmental origins of neurobehavioral and metabolic disorders associated with altered HTR2A function.

Cost-effectiveness of noninvasive fetal RhD blood group genotyping in nonalloimmunized and alloimmunized pregnancies.

We sought to determine the cost-effectiveness of noninvasive fetal RhD blood group genotyping in nonalloimmunized and alloimmunized pregnancies in Canada. We developed two probabilistic state-transition (Markov) microsimulation models to compare fetal genotyping followed by targeted management versus usual care (i.e., universal Rh immunoglobulin [RhIG] prophylaxis in nonalloimmunized RhD-negative pregnancies, or universal intensive monitoring in alloimmunized pregnancies). The reference case considered a healthcare payer perspective and a 10-year time horizon. Sensitivity analysis examined assumptions related to test cost, paternal screening, subsequent pregnancies, other alloantibodies (e.g., K, Rh c/C/E), societal perspective, and lifetime horizon. Fetal genotyping in nonalloimmunized pregnancies (at per-sample test cost of C$247/US$311) was associated with a slightly higher probability of maternal alloimmunization (22 vs. 21 per 10,000) and a reduced number of RhIG injections (1.427 vs. 1.795) than usual care. It was more expensive (C$154/US$194, 95% Credible Interval [CrI]: C$139/US$175-C$169/US$213) and had little impact on QALYs (0.0007, 95%CrI: -0.01-0.01). These results were sensitive to the test cost (threshold achieved at C$88/US$111), and inclusion of paternal screening. Fetal genotyping in alloimmunized pregnancies (at test cost of C$328/US$413) was less expensive (-C$6280/US$7903, 95% CrI: -C$6325/US$7959 to -C$6229/US$7838) and more effective (0.19 QALYs, 95% CrI 0.17-0.20) than usual care. These cost savings remained robust in sensitivity analyses. Noninvasive fetal RhD genotyping saves resources and represents good value for the management of alloimmunized pregnancies. If the cost of genotyping is substantially decreased, the targeted intervention can become a viable option for nonalloimmunized pregnancies.

Prediction of fetal blood group antigens from maternal plasma using Ion AmpliSeq HD technology.

Fetal blood group (BG) and platelet (HPA) antigens may trigger maternal immunization, causing a fetal disease. Noninvasive prenatal diagnostics (NIPT) predicts fetal genotype, identifying pregnancies with no risk. All current techniques detect fetal antigen alleles with unspecific background and without estimation of fetal fraction, thus new protocols for detection of fetal BG/HPA alleles with ultrahigh sensitivity still need to be tested to improve NIPT. To design NIPT of clinically important antigens using Ion AmpliSeq HD technology. Plasma DNA from 36 pregnant women (9-33 week of gestation, 24 immunized with anti-HPA-1a,-3b,-15a, -K, or -D+C+S), with known BG/HPA genotypes of their neonates/partners, was tested on Ion S5 System using the Ion AmpliSeq HD designer custom gene panel. NGS contained 25 rs-targets encoding relevant BG/HPA antigens and 10 markers. Using the NGS protocol, 76 out of 85 differences in fetal/maternal BG/HPA genotypes were determined in concentration above 2% fetal paternally inherited allele chimerism. The level of unspecific reads for BG/HPA alleles was below 0.87%. In 24 immunized women NGS revealed feto-maternal incompatibility in 11 cases (from 2.44% to 7.41%) and excluded in 10 (<0.05%), three cases had inconclusive results (1.79%, 0.19%, 0.11%). The presence of fetal DNA was confirmed in each case by detecting markers with at least 2% chimerism. The use of Ion AmpliSeq HD technology improves the prediction of feto-maternal incompatibility, increasing the sensitivity of BG/HPA NIPT and serving confirmation of the fetal DNA at the same workflow.

Noninvasive Prenatal Testing of Methylmalonic Acidemia cblC Type Using the cSMART Assay for MMACHC Gene Mutations.

Noninvasive prenatal testing (NIPT) for monogenic disorders has been developed in recent years; however, there are still significant technical and analytical challenges for clinical use. The clinical feasibility of NIPT for methylmalonic acidemia cblC type (cblC type MMA) was investigated using our circulating single-molecule amplification and re-sequencing technology (cSMART). Trios molecular diagnosis was performed in 29 cblC type MMA-affected children and their parents by traditional Sanger sequencing. In the second pregnancy, invasive prenatal diagnosis (IPD) of the pathogenic MMACHC gene was used to determine fetal genotypes, and NIPT was performed using a novel MMACHC gene–specific cSMART assay. Maternal–fetal genotypes were deduced based on the mutation ratio in maternal plasma DNA. Concordance of fetal genotypes between IPD and NIPT, and the sensitivity and specificity of NIPT were determined. After removing two cases with a low P value or reads, the concordance ratio for NIPT and IPD was 100.00% (27/27), and the sensitivity and specificity were 100.00% (54.07–100.00%) and 100.00% (83.89–100.00%), respectively. This study demonstrates that NIPT using the cSMART assay for cblC type MMA was accurate in detecting fetal genotypes. cSMART has a potential clinical application as a prenatal diagnosis and screening tool for carrier and low-risk genotypes of cblC type MMA and other monogenic diseases.

Pregnancy in Women With Monogenic Diabetes due to Pathogenic Variants of the Glucokinase Gene: Lessons and Challenges.

Heterozygous loss-of-function variants of the glucokinase (GCK) gene are responsible for a subtype of maturity-onset diabetes of the young (MODY). GCK-MODY is characterized by a mild hyperglycemia, mainly due to a higher blood glucose threshold for insulin secretion, and an up-regulated glucose counterregulation. GCK-MODY patients are asymptomatic, are not exposed to diabetes long-term complications, and do not require treatment. The diagnosis of GCK-MODY is made on the discovery of hyperglycemia by systematic screening, or by family screening. The situation is peculiar in GCK-MODY women during pregnancy for three reasons: 1. the degree of maternal hyperglycemia is sufficient to induce pregnancy adverse outcomes, as in pregestational or gestational diabetes; 2. the probability that a fetus inherits the maternal mutation is 50% and; 3. fetal insulin secretion is a major stimulus of fetal growth. Consequently, when the fetus has not inherited the maternal mutation, maternal hyperglycemia will trigger increased fetal insulin secretion and growth, with a high risk of macrosomia. By contrast, when the fetus has inherited the maternal mutation, its insulin secretion is set at the same threshold as the mother’s, and no fetal growth excess will occur. Thus, treatment of maternal hyperglycemia is necessary only in the former situation, and will lead to a risk of fetal growth restriction in the latter. It has been recommended that the management of diabetes in GCK-MODY pregnant women should be guided by assessment of fetal growth by serial ultrasounds, and institution of insulin therapy when the abdominal circumference is ≥ 75th percentile, considered as a surrogate for the fetal genotype. This strategy has not been validated in women with in GCK-MODY. Recently, the feasibility of non-invasive fetal genotyping has been demonstrated, that will improve the care of these women. Several challenges persist, including the identification of women with GCK-MODY before or early in pregnancy, and the modalities of insulin therapy. Yet, retrospective observational studies have shown that fetal genotype, not maternal treatment with insulin, is the main determinant of fetal growth and of the risk of macrosomia. Thus, further studies are needed to specify the management of GCK-MODY pregnant women during pregnancy.

Noninvasive Prenatal Diagnosis of Fetal RHD Status Using Cell-free Fetal DNA in Maternal Plasma.

Background:The main cause of hemolytic disease of the fetus and newborn (HDFN) is the incompatibility of the RHD antigen between mother and fetus. Following the discovery of cell-free fetal DNA (cffDNA), noninvasive fetal RHD genotyping also became possible, which will help in the better management of immunized RHD negative mothers and in the targeted prenatal injection of Rho(D) immune globulin (RhIG). The objective of this study was to establish a reliable method with high accuracy to determine the fetal RHD genotype.Methods:The project was a prospective observational cohort study. After cell-free DNA (cfDNA) extraction from maternal plasma, fetal RHD genotyping was performed by duplex real-time polymerase chain reaction (PCR) and exons 5, 7, and 10 of the RHD gene were examined. SRY and RASSF1A genes were used as internal controls to confirm the presence of cffDNA in maternal plasma.Results:Out of 40 samples, 33 were RhD positive heterozygous mothers and 7 cases were RHD negative. In three cases where both the fetal RHD and SRY genotypes were negative, RASSF1A was amplified in cell-free DNA sample treated with the BstUI enzyme, and the presence of cffDNA was confirmed.Conclusion:The findings reveal that the strategy used in this study is reliable and it is possible to determine the fetal RHD status with high accuracy. The strategy can help targeted injection of RhIG and prevent unnecessary injection in RhD negative mothers who carry an RhD negative fetus.

Haplotype-Based Noninvasive Prenatal Diagnosis of 21 Families With Duchenne Muscular Dystrophy: Real-World Clinical Data in China.

Noninvasive prenatal diagnosis (NIPD) of single-gene disorders has recently become the focus of clinical laboratories. However, reports on the clinical application of NIPD of Duchenne muscular dystrophy (DMD) are limited. This study aimed to evaluate the detection performance of haplotype-based NIPD of DMD in a real clinical environment. Twenty-one DMD families at 7–12 weeks of gestation were prospectively recruited. DNA libraries of cell-free DNA from the pregnant and genomic DNA from family members were captured using a custom assay for the enrichment of DMD gene exons and spanning single-nucleotide polymorphisms, followed by next-generation sequencing. Parental haplotype phasing was based on family linkage analysis, and fetal genotyping was inferred using the Bayes factor through target maternal plasma sequencing. Finally, the entire experimental process was promoted in the local clinical laboratory. We recruited 13 complete families, 6 families without paternal samples, and 2 families without probands in which daughter samples were collected. Two different maternal haplotypes were constructed based on family members in all 21 pedigrees at as early as 7 gestational weeks. Among the included families, the fetal genotypes of 20 families were identified at the first blood collection, and a second blood collection was performed for another family due to low fetal concentration. The NIPD result of each family was reported within 1 week. The fetal fraction in maternal cfDNA ranged from 1.87 to 11.68%. In addition, recombination events were assessed in two fetuses. All NIPD results were concordant with the findings of invasive prenatal diagnosis (chorionic villus sampling or amniocentesis). Exon capture and haplotype-based NIPD of DMD are regularly used for DMD genetic diagnosis, carrier screening, and noninvasive prenatal diagnosis in the clinic. Our method, haplotype-based early screening for DMD fetal genotyping via cfDNA sequencing, has high feasibility and accuracy, a short turnaround time, and is inexpensive in a real clinical environment.

Noninvasive Fetal RhD Blood Group Genotyping: A Systematic Review of Economic Evaluations.

Noninvasive fetal rhesus D (RhD) blood group genotyping may prevent unnecessary use of anti-D immunoglobulin (RhIG) in non-alloimmunized RhD-negative pregnancies and can guide management of alloimmunized pregnancies. We conducted a systematic review of the economic literature to determine the cost-effectiveness of this intervention over usual care. Systematic literature searches of bibliographic databases (Ovid MEDLINE, Embase, and Cochrane) until February 26, 2019, and auto-alerts until October 30, 2020, and of grey literature sources were performed to retrieve all English-language studies. We included studies done in serologically confirmed non-alloimmunized or alloimmunized RhD-negative pregnancies, comparing costs and effectiveness of the intervention versus usual care. Two reviewers extracted data from the eligible studies and assessed their methodological quality (risk of bias) using the Quality of Health Economic Studies (QHES) and Drummond tools. We narratively synthesized findings. Our review included 8 economic studies that evaluated non-invasive fetal RhD genotyping followed by targeted RhIG prophylaxis in non-alloimmunized pregnancies. Five studies further considered a subsequent alloimmunized pregnancy. The cost-effectiveness of the intervention versus usual care (e.g., universal RhIG or prophylaxis conditional on results of paternal testing) for non-alloiummunized pregnancies was inconsistent. Two studies indicated greater benefits and lower costs for the intervention, and another 2 suggested a trade-off. In 4 studies, the intervention was less effective and costlier than alternatives. Three studies were determined to be of high quality by both tools. Two of these studies favoured the intervention, and one assessed benefits in quality-adjusted life-years. No study clearly examined the cost-effectiveness of repetitive use of fetal genotyping in multiple non-alloimmunized or alloimmunized pregnancies. The cost of genotyping was the most influential parameter. The cost-effectiveness of noninvasive fetal RhD genotyping for non-alloimmunized pregnancies varies between studies. Potential savings from targeted management of alloimmunized pregnancies requires further research.

Clinical application of non-invasive prenatal diagnosis of phenylketonuria based on haplotypes via paired-end molecular tags and weighting algorithm

BackgroundPhenylketonuria (PKU) is a metabolic disease that can cause severe and irreversible brain damage without treatment.MethodsHere we developed a non-invasive prenatal diagnosis (NIPD) technique based on haplotypes via paired-end molecular tags and weighting algorithm and applied it to the NIPD of PKU to evaluate its accuracy and feasibility in the early pregnancy. A custom-designed hybridization probes containing regions in phenylalanine hydroxylase (PAH) gene and its 1 Mb flanking region were used for target sequencing on genomic and maternal plasma DNA (7–13 weeks of gestation) to construct the parental haplotypes and the proband’s haplotype. Fetal haplotype was then inferred combined with the parental haplotypes and the proband’s haplotype. The presence of haplotypes linked to both the maternal and paternal mutant alleles indicated affected fetuses. The fetal genotypes were further validated by invasive prenatal diagnosis in a blinded fashion.ResultsThis technique has been successfully applied in twenty-one cases. Six fetuses were diagnosed as patients carrying both of the mutated haplotypes inherited from their parents. Eleven fetuses were carriers of one heterozygous PAH variants, six of which were paternal and five of which were maternal. Four fetuses were absence of pathogenic alleles. All results were consistent with the prenatal diagnosis through amniotic fluid.ConclusionsThe results showed that our new technique applied to the genotyping of fetuses with high risk for PKU achieves an accurate detection at an early stage of pregnancy with low fetal fraction in cell free DNA.

Do Genetic Variants Modify the Effect of Smoking on Risk of Preeclampsia in Pregnancy?

Maternal smoking is associated with as much as a 50% reduced risk of preeclampsia, despite increasing risk of other poor pregnancy outcomes that often co-occur with preeclampsia, such as preterm birth and fetal growth restriction. Researchers have long sought to understand whether this perplexing association is biologically based, or a result of noncausal mechanisms. We examined whether smoking-response genes modify the smoking-preeclampsia association to investigate potential biological explanations. We conducted a nested case-control study within the Norwegian Mother, Father and Child Birth Cohort (1999-2008) of 2,596 mother-child dyads. We used family-based log-linear Poisson regression to examine modification of the maternal smoking-preeclampsia relationship by maternal and fetal single nucleotide polymorphisms involved in cellular processes related to components of cigarette smoke (n = 1,915 with minor allele frequency ≥10%). We further investigated the influence of smoking cessation during pregnancy. Three polymorphisms showed overall (p < 0.001) multiplicative interaction between smoking and maternal genotype. For rs3765692 (TP73) and rs10770343 (PIK3C2G), protection associated with smoking was reduced with two maternal copies of the risk allele and was stronger in continuers than quitters (interaction p = 0.02 for both loci, based on testing 3-level smoking by 3-level genotype). For rs2278361 (APAF1) the inverse smoking-preeclampsia association was eliminated by the presence of a single risk allele, and again the trend was stronger in continuers than in quitters (interaction p = 0.01). Evidence for gene-smoking interaction was limited, but differences by smoking cessation warrant further investigation. We demonstrate the potential utility of expanded dyad methods and gene-environment interaction analyses for outcomes with complex relationships between maternal and fetal genotypes and exposures. · Maternal and fetal genotype may differentially influence preeclampsia.. · Smoking-related genes did not strongly modify smoking-preeclampsia association.. · Smoking cessation reduced strength of gene by smoking interactions..

Noninvasive Prenatal Test for β-Thalassemia and Sickle Cell Disease Using Probe Capture Enrichment and Next-Generation Sequencing of DNA in Maternal Plasma.

Noninvasive prenatal testing (NIPT) of chromosomal aneuploidies based on next-generation sequencing (NGS) analysis of fetal DNA in maternal plasma is well established, but testing for autosomal recessive disorders remains challenging. NGS libraries prepared by probe capture facilitate the analysis of the short DNA fragments plasma. This system has been applied to the β-hemoglobinopathies to reduce the risk to the fetus. Our probe panel captures >4 kb of the HBB region and 435 single-nucleotide polymorphisms (SNPs) used to estimate fetal fraction. Contrived mixtures of DNA samples, plasma, and whole blood samples from 7 pregnant women with β-thalassemia or sickle cell anemia mutations and samples from the father, sibling, and baby or chorionic villus were analyzed. The fetal genotypes, including point mutations and deletions, were inferred by comparing the observed and expected plasma sequence read ratios, based on fetal fraction, at the mutation site and linked SNPs. Accuracy was increased by removing PCR duplicates and by in silico size selection of plasma sequence reads. A probability was assigned to each of the potential fetal genotypes using a statistical model for the experimental variation, and thresholds were established for assigning clinical status. Using in silico size selection of plasma sequence files, the predicted clinical fetal genotype assignments were correct in 9 of 10 plasma libraries with maternal point mutations, with 1 inconclusive result. For 2 additional plasmas with deletions, the most probable fetal genotype was correct. The β-globin haplotype determined from linked SNPs, when available, was used to infer the fetal genotype at the mutation site. This probe capture NGS assay demonstrates the potential of NIPT for β-hemoglobinopathies.

Precision medicine in diabetes: A non-invasive prenatal diagnostic test for the determination of fetal glucokinase mutations.

ABSTRACTHyperglycemia caused by mutations in the glucokinase gene, GCK, is the most common form of monogenic diabetes. Prenatal diagnosis is important, as it impacts on treatment. This study reports a monogenic non‐invasive prenatal diagnostic (NIPD‐M) test on cell‐free DNA in maternal plasma using the relative haplotype dosage. In three pregnancies of two families with known maternal GCK mutations, the fetal genotype was determined unambiguously already at 12 weeks of gestation. In summary, proof is provided of the feasibility for NIPD‐M in GCK diabetes.

Significance of inhibitory maternal killer-cell immunoglobulin-like receptor (KIR) and fetal KIR ligand genotype combinations in placenta related obstetric complications

Some maternal killer-cell immunoglobulin-like receptor (KIR) and fetal KIR ligand genotypes are associated with obstetric complications, such as recurrent miscarriage, fetal growth restriction, preeclampsia, and preterm birth. However, how KIR/KIR ligand genotypes affect these placenta-related obstetric complications has not been fully understood. We aimed to demonstrate the association of maternal KIR-fetal KIR ligand genotype combinations with immunological/metabolic risk factor associated placenta-related obstetric complications. This study consisted of three groups of pregnant women: 1) Miscarriage group (n = 30), 2) Complicated Pregnancy (CP) group (n = 30), and 3) Control group (n = 30). The observed maternal genotype frequencies of all inhibitory and activating KIRs were similar in all groups (p > 0.05). However, inhibitory 2DL3 was quite frequent in the miscarriage group (p = 0.052). There was no difference between groups in terms of centromeric and telomeric maternal haplotypes (p > 0.05). The fetal group 1 HLA-C genotype was frequently detected in the miscarriage and CP groups with rates of 83.3 % and 93.3 % respectively, while the observed frequency was 70 % in the control group. The fetal group 2 HLA-C genotype was the same in all groups. The results demonstrated significantly less fetal group 2 HLA-C homozygosity in the CP groups when compared to the control group (p = 0.020). The fetal HLA-Bw4 genotype was detected more frequently in the miscarriage and CP groups (p = 0.028 and p = 0.001, respectively). The inhibitory KIR/KIR ligand genotype combinations of 2DL3-C1 and 3DL1-Bw4 were more frequent in the miscarriage and CP groups (p = 0.045 and p = 0.002, respectively). Enhanced NK cell inhibition may be one of the mechanisms underlying placenta-related obstetric complications.

Higher maternal adiposity reduces offspring birthweight if associated with a metabolically favourable profile

Aims/hypothesisHigher maternal BMI during pregnancy is associated with higher offspring birthweight, but it is not known whether this is solely the result of adverse metabolic consequences of higher maternal adiposity, such as maternal insulin resistance and fetal exposure to higher glucose levels, or whether there is any effect of raised adiposity through non-metabolic (e.g. mechanical) factors. We aimed to use genetic variants known to predispose to higher adiposity, coupled with a favourable metabolic profile, in a Mendelian randomisation (MR) study comparing the effect of maternal ‘metabolically favourable adiposity’ on offspring birthweight with the effect of maternal general adiposity (as indexed by BMI).MethodsTo test the causal effects of maternal metabolically favourable adiposity or general adiposity on offspring birthweight, we performed two-sample MR. We used variants identified in large, published genetic-association studies as being associated with either higher adiposity and a favourable metabolic profile, or higher BMI (n = 442,278 and n = 322,154 for metabolically favourable adiposity and BMI, respectively). We then extracted data on the metabolically favourable adiposity and BMI variants from a large, published genetic-association study of maternal genotype and offspring birthweight controlling for fetal genetic effects (n = 406,063 with maternal and/or fetal genotype effect estimates). We used several sensitivity analyses to test the reliability of the results. As secondary analyses, we used data from four cohorts (total n = 9323 mother–child pairs) to test the effects of maternal metabolically favourable adiposity or BMI on maternal gestational glucose, anthropometric components of birthweight and cord-blood biomarkers.ResultsHigher maternal adiposity with a favourable metabolic profile was associated with lower offspring birthweight (−94 [95% CI −150, −38] g per 1 SD [6.5%] higher maternal metabolically favourable adiposity, p = 0.001). By contrast, higher maternal BMI was associated with higher offspring birthweight (35 [95% CI 16, 53] g per 1 SD [4 kg/m2] higher maternal BMI, p = 0.0002). Sensitivity analyses were broadly consistent with the main results. There was evidence of outlier SNPs for both exposures; their removal slightly strengthened the metabolically favourable adiposity estimate and made no difference to the BMI estimate. Our secondary analyses found evidence to suggest that a higher maternal metabolically favourable adiposity decreases pregnancy fasting glucose levels while a higher maternal BMI increases them. The effects on neonatal anthropometric traits were consistent with the overall effect on birthweight but the smaller sample sizes for these analyses meant that the effects were imprecisely estimated. We also found evidence to suggest that higher maternal metabolically favourable adiposity decreases cord-blood leptin while higher maternal BMI increases it.Conclusions/interpretationOur results show that higher adiposity in mothers does not necessarily lead to higher offspring birthweight. Higher maternal adiposity can lead to lower offspring birthweight if accompanied by a favourable metabolic profile.Data availabilityThe data for the genome-wide association studies (GWAS) of BMI are available at https://portals.broadinstitute.org/collaboration/giant/index.php/GIANT_consortium_data_files. The data for the GWAS of body fat percentage are available at https://walker05.u.hpc.mssm.edu.Graphical abstract

Fetal RhD Genotyping using Cell Free Fetal DNA from Maternal Plasma in RhD Negative Women in Pakistan

Background: The Rhesus (Rh) blood group system is one of the most hemogenic and polymorphic system in the body. RhD negative pregnant women carrying RhD positive fetus are always at the risk of hemolytic disease of the fetus and newborn. Prenatal fetal RhD genotyping leads to appropriate management and targeted anti-D immunoprophylaxis in RhD-positive pregnancies only. Objective: To determine the accuracy and diagnostic feasibility of non-invasive fetal RhD genotyping by analysis of cell free fetal DNA based on real time polymerase chain reaction (RT-PCR). Materials and Methods: A total of 100 EDTA-blood samples from pregnant women between 10-32 weeks of gestation were collected. The cell free fetal DNA was extracted from maternal plasma followed by PCR analysis for SRY, RhD (exons 5,7,10) and RASSF1A genes using specific primers. Fetus was considered as RhD-positive when amplification of all targeted RhD exons were observed and in case, where no amplification for RhD exons was detected but Beta-globin gene and SRY gene was amplified, fetus were characterized as RhD-negative. When one or two RhD exons were amplified, the results were predicted to be inconclusive and RT-PCR assay with new extracted DNA samples was repeated. Results: Out of 100 pregnancies, none of the sample was found to be inconclusive for fetal RhD status. No false- positive and falsenegative results were registered. The sensitivity, specificity and diagnostic concordance was predicted to be 100%. Conclusion: The protocol we applied was highly accurate for non-invasive fetal RhD determination. We therefore recommend implementation of non-invasive RhD genotyping in prenatal screening. Keywords: Cff DNA; Fetal RhD genotyping; Maternal plasma

Practices for RhD alloimmunization prevention: a vignette-based survey of midwives

Introduction Despite the availability guidelines to prevent RhD alloimmunization, severe hemolytic disease of fetus and newborn still occurs in high-income countries. The aim of the study was (1) To assess variations in practices for the prevention of RhD alloimmunization, and (2) to understand midwives' acceptance and appropriation of fetal RhD genotyping. Methods Descriptive cross-sectional survey of French midwives from September 2017 through January 2018. Participants were asked to complete an internet-based questionnaire that included three clinical vignettes. They were questioned about their practices concerning early pregnancy visit by RhD-negative women, prevention of RhD alloimmunization in women with second-trimester metrorrhagia, and RhD fetal genotyping. Results A total of 827 midwives completed the questionnaire. Only 21.1% reported that they practice all the preventive measures recommended in early pregnancy. In a situation at high risk of RhD alloimmunization during pregnancy, 97.2% of midwives would perform immunoprophylaxis. Nearly, all midwives reported providing information about RhD alloimmunization (92.4%) at the beginning of pregnancy, although only 11.3% offered both written and verbal information; at the time of systematic anti-D immunoprophylaxis (28 weeks), 78% provided information, but only 2.7% both verbally and in writing. Finally, only 50.8% of midwives preferred to include RhD fetal genotyping in routine prenatal prophylaxis. Discussion This study showed significant variations in French midwives' practices to prevent RhD alloimmunization. Better dissemination of guidelines is needed to improve both consistent use of these practices and the quality of information delivered to RhD-negative pregnant women.

Droplet-based digital PCR for non-invasive prenatal genetic diagnosis of α and β-thalassemia.

Non-invasive prenatal diagnosis (NIPD) of isolated cell-free DNA from maternal plasma has been applied to detect monogenic diseases in the fetus. Droplet digital PCR (ddPCR) is a sensitive and quantitative technique for NIPD. In the present study, the development and evaluation of ddPCR-based assays for common α and β-thalassemia variants amongst the Asian population was described; specifically, Southeast Asian (SEA) deletion, HbE, and 41/42 (-CTTT). SEA is caused by deletion of a 20 kb region surrounding the α-globin gene, whilst HbE and 41/42 (-CTTT) are caused by point mutations on the β-globin gene. Cell-free DNA samples from 46 singleton pregnant women who were carriers of these mutations were isolated and quantified using ddPCR with specially designed probes for each target allele. Allelic copy number calculation and likelihood ratio tests were used to classify fetal genotypes. Classification performances were evaluated against ground truth fetal genotypes obtained from conventional amniocentesis. Copy number variation analysis of SEA deletion accurately classified fetal genotypes in 20 out of 22 cases with an area under the receiver operating characteristic curve of 0.98 for detecting Hb Bart's hydrops fetalis. For HbE cases, 10 out of 16 samples were correctly classified, and three were inconclusive. For 41/42 (-CTTT) cases, 2 out of 8 were correctly classified, and four were inconclusive. The correct genotype was not rejected in any inconclusive case and may be resolved with additional ddPCR experiments. These results indicate that ddPCR-based analysis of maternal plasma can become an accurate and effective NIPD for SEA deletion α-(0) thalassemia. Although the performance of ddPCR on HbE and 41/42 (-CTTT) mutations were not sufficient for clinical application, these results may serve as a foundation for future works in this field.

Noninvasive prenatal exome sequencing diagnostic utility limited by sequencing depth and fetal fraction.

Sequencing cell-free DNA now allows detection of large chromosomal abnormalities and dominant Mendelian disorders in the prenatal period. Improving upon these methods would allow newborn screening programs to begin with prenatal genetics, ultimately improving the management of rare genetic disorders. As a pilot study, we performed exome sequencing on the cell-free DNA from three mothers with singleton pregnancies to assess the viability of broad sequencing modalities in a noninvasive prenatal setting. We found poor resolution of maternal and fetal genotypes due to both sampling and technical issues. We find broad sequencing modalities inefficient for noninvasive prenatal applications. Alternatively, we suggest a more targeted path forward for noninvasive prenatal genotyping.

Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.