Fetal Genotype Research Articles

Sequencing Cell-free Fetal DNA in Pregnant Women With GCK-MODY.

Individuals with monogenic diabetes due to inactivating glucokinase (GCK) variants typically do not require treatment, except potentially during pregnancy. In pregnancy, fetal GCK genotype determines whether treatment is indicated, but noninvasive methods are not clinically available. This work aims to develop a method to determine fetal GCK genotype noninvasively using maternal cell-free fetal DNA. This was a proof-of-concept study involving 3 pregnant women with a causal GCK variant that used information from 1) massive parallel sequencing of maternal plasma cell-free DNA, 2) direct haplotype sequences of maternal genomic DNA, and 3) the paternal genotypes to estimate relative haplotype dosage of the pathogenic variant-linked haplotype. Statistical testing of variant inheritance was performed using a sequential probability ratio test (SPRT). In each of the 3 cases, plasma cell-free DNA was extracted once between gestational weeks 24 and 36. The fetal fraction of cell-free DNA ranged from 21.8% to 23.0%. Paternal homozygous alleles that were identical to the maternal GCK variant-linked allele were not overrepresented in the cell-free DNA. Paternal homozygous alleles that were identical to the maternal wild-type-linked allele were significantly overrepresented. Based on the SPRT, we predicted that all 3 cases did not inherit the GCK variant. Postnatal infant genotyping confirmed our prediction in each case. We have successfully implemented a noninvasive method to predict fetal GCK genotype using cell-free DNA in 3 pregnant women carrying an inactivating GCK variant. This method could guide tailoring of hyperglycemia treatment in pregnancies of women with GCK monogenic diabetes.

Non-invasive prenatal detection of ocutaneous albinism type I based on cfDNA barcode-enabled single-molecule test

To assess the value of non-invasive prenatal testing based on cfDNA barcode-enabled single-molecule test (cfBEST) for the prenatal diagnosis of oculocutaneous albinism type I in a family. Prenatal genetic diagnosis was carried out by using the cfBEST-based method as well as invasive prenatal diagnosis through amniocentesis. The outcome of the pregnancy was followed up. Non-invasive prenatal testing based on cfBEST showed a fetal DNA concentration of 6.6%, with the proportion of c.929_930insC (p.Arg311Lysfs*7) and c.1037-7T>A mutations being 45.7% and 0%, respectively. The posterior frequency of the negative results was 1, suggesting that the fetus carried neither of the two mutations. The result was consistent with that of invasive prenatal diagnosis, and the follow-up found that the fetus was normal. Non-invasive prenatal testing based on cfBEST can be used to detect maternal and fetal genotypes in maternal cell-free DNA, which is clinically feasible.

Prenatal Genetic Diagnosis in Three Fetuses With Left Heart Hypoplasia (LHH) From Three Unrelated Families.

Background: Congenital heart defects (CHDs) are the most common birth defects, and left heart hypoplasia (LHH) is a severe form of CHD and responsible for more than 20% cardiac deaths during the first week of life, however, its genetic causes remain largely elusive.Methods: Three families with fetal LHH were recruited. Genomic DNA from amniotic fluid or peripheral blood, and trio whole exome sequencing (trio-WES) and copy number variation sequencing (CNV-seq) were performed.Results: All the three couples had no family history, and mid-gestation ultrasound revealed LHH and other variable cardiovascular defects in the fetuses. Trio-WES revealed de novo pathogenic variations in KMT2D (p.Gly3465Aspfs*37) (NM_003482) and WDFY3 (p.Ser117Xfs*) (NM_014991), and CNV-seq identified a deletion of 150 kb encompassing NOTCH1. KMT2D and NOTCH1 previously have been reported to be associated with CHDs, however, WDFY3 is reported for the first time to be possibly related to CHD in human.Conclusion: Our study suggested that genetic component is an important risk factor for the development of LHH, and next generation sequencing is a powerful tool for genetic diagnosis in fetuses with CHDs and genetic counseling, however, more studies and data are need to establish the correlation of fetal phenotypes and genotypes.

The Norwegian experience with nationwide implementation of fetal RHD genotyping and targeted routine antenatal anti-D prophylaxis.

To reduce the risk of RhD alloimmunization during the last trimester of pregnancy, a targeted routine antenatal anti-D prophylaxis (RAADP) programme was implemented in Norway in 2016. Here, we present and discuss our experience with the nationwide implementation of the programme, and report sample uptake and preliminary data of de novo anti-D in pregnancy. The targeted RAADP was advised by the academic community and evaluated by the health authorities. A National Working Group has conducted the implementation in the transfusion services and contributed to organise the administration of the antenatal anti-D prophylaxis. Fetal RhD type is determined by non-invasive prenatal testing at gestational week 24, and anti-D prophylaxis is administrated at gestational week 28 only to women with RhD positive fetuses. We describe the implementation process of targeted RAADP in Norway. The sample uptake is calculated by comparing the number of fetal RHD screens with the expected number of samples. The sample uptake shows regional variations: 88%-100% after 3 years. Promising decrease in de novo anti-D detected during pregnancy is observed. Nationwide targeted RAADP is implemented and included in the Norwegian maternity care programme. Compliance to sample uptake should further improve in some regions. A remaining issue to fulfil is the documentation of the accuracy of the fetal RHD-typing at all sites. Post-natal prophylaxis will then be guided by the fetal RHD result. Dedicated registries will ensure data to evaluate the expected reduction in pregnancy-related RhD immunisations, which is the final success criterion of the programme.

Maternal one carbon metabolism and interleukin-10 &-17 synergistically influence the mode of delivery in women with Early Onset Pre-Eclampsia.

Studies on One Carbon Metabolism (OCM), Interleukins-10 &-17 (IL-10/-17) & βhCG in pre-eclampsia and its delivery outcome (preterm birth) reveal contradictory results, attributed to clinical heterogeneity (early/late onset pre-eclampsia) or preterm/term birth. Disturbed OCM also influences IL-10 &-17 during pregnancy. We sought to investigate the synergism between OCM and IL-10/-17 mediated immune-regulation through βhCG in Early onset pre-eclampsia (EO-PE) patients, delivering preterm, among North Indian women. Case-control study with a total of 399 pregnant women (EO-PE delivering preterm=199; Normotensives delivering at term=200). Maternal genotypes & biochemical estimations along with fetal genotypes on subset (n=72) pertaining to OCM and IL-10/-17 regulation were assessed. Association of 1) maternal plasma levels with EO-PE 2) maternal and fetal genotypes with EO-PE. 3) Effect of Hyper-homocysteinemia (surrogate of disturbed OCM) on differential immune regulation (IL10,-17, βhCG) in EO-PE and mode of delivery. Hyper-homocysteinemia posed an increased risk of three folds for EO-PE. Both, folate and B12 deficiencies were associated with elevated homocysteine in EO-PE. Further, MTHFR 677TT homozygotes was present only in EO-PE indicating its detrimental role. However, maternal IL17-197AA genotype showed decreased risk for EO-PE. Furthermore, elevated maternal plasma IL-17 along with elevated IL-10 & βhCG were observed in EO-PE. Taken together, altered homocysteine metabolism was associated with high IL10 in EO-PE; and was more pronounced in spontaneous vaginal deliveries as compared to induced/caesarean section deliveries. We report homocysteine mediated IL-10 &17 dysregulation and its influence on mode of delivery in EO-PE, possibly through initiation of cervical ripening. Further, these could serve potential biomarkers of EO-PE & its delivery outcome among vulnerable populations with similar nutritional & genetic predispositions.

Non-Invasive Prenatal Fetal Blood Group Genotype and Its Application in the Management of Hemolytic Disease of Fetus and Newborn: Systematic Review and Meta-Analysis.

Hemolytic disease of fetus and newborn (HDFN) imposes great healthcare burden being associated with maternal alloimmunization against parental-inherited fetal red blood cell antigens causing fetal anemia or death. Noninvasive prenatal analysis (NIPT) provides safe fetal RHD genotyping for early identification of risk pregnancies and proper management guidance. We aimed to conduct systematic review and meta-analysis on NIPT's beneficial application, in conjunction with quantitative maternal alloantibody analysis, for early diagnosis of pregnancies at risk. Search for relevant articles was done in; PubMed/Medline, Scopus, and Ovid (January 2006April 2020), including only English-written articles reporting reference tests and accuracy data. Nineteen eligible studies were critically appraised. NIPT was estimated highly sensitive/specific for fetal RHD genotyping beyond 11-week gestation. Amplifications from ≥2 exons are optimum to increase accuracy. NIPT permits cost-effectiveness, precious resources sparing, and low emotional stress. Knowledge of parental ethnicity is important for correct NIPT result interpretations and quantitative screening. Cut-off titer ≥8-up-to-32 is relevant for anti-D alloantibodies, while, lower titer is for anti-K. Alloimmunization is influenced by maternal RHD status, gravida status, and history of adverse obstetrics. In conclusion, NIPT allows evidence-based provision of routine anti-D immunoprophylaxis and estimates potential fetal risks for guiding further interventions. Future large-scale studies investigating NIPT's non-RHD genotyping within different ethnic groups and in presence of clinically significant alloantibodies are needed.

Pregnancy Serum DLK1 Concentrations Are Associated With Indices of Insulin Resistance and Secretion.

ContextDelta like noncanonical notch ligand 1 (DLK1) is a paternally expressed imprinted gene that encodes an epidermal growth factor repeat-containing transmembrane protein. A bioactive, truncated DLK1 protein is present in the circulation and has roles in development and metabolism.ObjectiveWe sought to investigate links between maternal pregnancy circulating DLK1 concentrations and: (1) maternal and fetal DLK1 genotypes, (2) maternal insulin resistance and secretion, and (3) offspring size at birth.Patients, design, and settingWe measured third-trimester maternal serum DLK1 concentrations and examined their associations with parentally transmitted fetal and maternal DLK1 genotypes, indices of maternal insulin resistance and secretion derived from 75-g oral glucose tolerance tests performed around week 28 of pregnancy, and offspring size at birth in 613 pregnancies from the Cambridge Baby Growth Study.ResultsMaternal DLK1 concentrations were associated with the paternally transmitted fetal DLK1 rs12147008 allele (P = 7.8 × 10-3) but not with maternal rs12147008 genotype (P = 0.4). Maternal DLK1 concentrations were positively associated with maternal prepregnancy body mass index (P = 3.5 × 10-6), and (after adjustment for maternal body mass index) with both maternal fasting insulin resistance (Homeostatic Model Assessment of Insulin Resistance: P = 0.01) and measures of maternal insulin secretion in response to oral glucose (insulinogenic index: P = 1.2 × 10-3; insulin disposition index: P = 0.049). Further positive associations were found with offspring weight (P = 0.02) and head circumference at birth (P = 0.04).ConclusionThese results are consistent with a partial paternal or placental origin for the maternal circulating DLK1 which may lead to increased maternal circulating DLK1 concentrations, stimulation of maternal insulin resistance and compensatory hyperinsulinemia during pregnancy, and the promotion of fetal growth.

1110 Predictive value of elevated MCA doppler in patients with alloimmunization

To evaluate pregnancy outcomes after abnormal middle cerebral artery peak systolic value (MCA PSV) measurement managed with percutaneous umbilical blood sampling (PUBS) or delivery as compared to repeat MCA PSV within 7 days. We performed a descriptive study including all patients in a single tertiary care center between 2006 and 2016 with a MCA PSV > 1.5 multiples of the median (MoM) in the setting of maternal red cell alloimmunization. The index visit was the first exam with MCA PSV > 1.5 MoM. We excluded cases with fetal hydrops prior to the index visit or abnormal MCA PSV at > 35 weeks. Patients undergoing immediate intervention (PUBS or delivery) (group 1) were compared to patients with repeat MCA PSV performed within 7 days (group 2). All scans were interpreted by board-certified Maternal-Fetal Medicine physicians. Fifty patients were identified. 19 underwent intervention based on the first abnormal MCA PSV (group 1) and 31 underwent repeat MCA PSV (group 2). The gestational ages (GA) or MCA PSV at index exam did not vary between the groups (table 1). Fetal red blood cell genotypes were infrequently known in this population. MCA PSV values at index exam were not different regardless of anemia severity (none, mild, moderate /severe). In group 1, 14/19 (74%) had anemia, with 8/19 (42%) having moderate/severe anemia (Figure 1). In group 2, 11/31 patients underwent PUBS for a persistently abnormal MCA PSV, all of whom had anemia which was moderate/severe in 7/11 (64%). Of the remaining 20 patients (65%) in group 2, 19/20 (95%) never underwent PUBS or developed moderate/severe anemia. One fetus had an abnormal MCA PSV 13 days after the index and was transfused for severe anemia. Of patients who underwent repeat MCA PSV testing rather than immediate intervention, 65% had normal repeat MCA PSVs and safely avoided PUBS. Persistently elevated MCA PSVs were more predictive of anemia than single elevated values preceding intervention. Other factors contributing to the decision to immediately pursue intervention should be further evaluated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Male fetal sex affects uteroplacental angiogenesis in growth restriction mouse model†.

Abnormally increased angiotensin II activity related to maternal angiotensinogen (AGT) genetic variants, or aberrant receptor activation, is associated with small-for-gestational-age babies and abnormal uterine spiral artery remodeling in humans. Our group studies a murine AGT gene titration transgenic (TG; 3-copies of the AGT gene) model, which has a 20% increase in AGT expression mimicking a common human AGT genetic variant (A[-6]G) associated with intrauterine growth restriction (IUGR) and spiral artery pathology. We hypothesized that aberrant maternal AGT expression impacts pregnancy-induced uterine spiral artery angiogenesis in this mouse model leading to IUGR. We controlled for fetal sex and fetal genotype (e.g., only 2-copy wild-type [WT] progeny from WT and TG dams were included). Uteroplacental samples from WT and TG dams from early (days 6.5 and 8.5), mid (d12.5), and late (d16.5) gestation were studied to assess uterine natural killer (uNK) cell phenotypes, decidual metrial triangle angiogenic factors, placental growth and capillary density, placental transcriptomics, and placental nutrient transport. Spiral artery architecture was evaluated at day 16.5 by contrast-perfused three-dimensional microcomputed tomography (3D microCT). Our results suggest that uteroplacental angiogenesis is significantly reduced in TG dams at day 16.5. Males from TG dams are associated with significantly reduced uteroplacental angiogenesis from early to late gestation compared with their female littermates and WT controls. Angiogenesis was not different between fetal sexes from WT dams. We conclude that male fetal sex compounds the pathologic impact of maternal genotype in this mouse model of growth restriction.

Evaluation of pregnancy outcomes in women with GCK-MODY.

To determine the fetal and maternal outcomes in pregnant women with Glucokinase-Maturity onset diabetes of the young (GCK-MODY). We studied the obstetric and perinatal outcomes in 99 pregnancies of 34 women with GCK-MODY. The mutation status of the offspring was known in 29 and presumed in 33. Clinical outcomes were determined and compared between affected (n=39) and unaffected (n=23) offspring. 59% of pregnancies were treated with diet alone and 41% received insulin. Birthweight, percentage of large for gestational age (LGA) and caesarean section (CS) in GCK-unaffected offspring was significantly higher than in GCK-affected offspring (4.0±0.7 vs. 3.4±0.4kg, p=0.001), 15 (65%) vs. 5(13%) (p=0.00006) and 17 (74%) vs. 11 (28%) (p=0.001), respectively. We observed an earlier gestational age at delivery on insulin in unaffected offspring (38.3±1.0 vs. 39.5±1.5 weeks, p=0.03) with no significant change in LGA (9 (82%) vs. 6 (50%); p=0.12), and a higher rate of CS (8 [73%] vs. 3 [11%]; p<0.001), and no change in small for gestational age (0 [0%] vs. 4 [14%]; p=0.30) in affected offspring. Insulin therapy in unaffected offspring did not reduce LGA and was associated with earlier gestational age at delivery. Insulin treatment in GCK-affected offspring was associated with an increased incidence of CS, but did not adversely affect fetal outcome. Fetal genotype determines birthweight rather than treatment. Pre-pregnancy diagnosis of GCK-MODY, use of continuous glucose monitoring and non-invasive fetal genotyping may enable further investigation of targeted therapy in this condition.

Rh disease prevention: the European Perspective

Background and ObjectivesIn Europe, postnatal and subsequently antenatal administration of anti‐RH1 (D) immunoglobulins (Ig) has reduced the number of maternal anti‐RH1 immunizations and the incidence of haemolytic disease of the foetus and newborn since the 1960s. Non‐invasive foetal RHD genotyping now enables antenatal prophylaxis to be targeted only to women carrying RHD‐positive foetus. We aimed at describing how Rh disease prevention is currently managed in different European countries.Materials and MethodsWe prepared an online survey on guidelines and biological tests performed for Rh disease prevention. The link was sent to 15 expert laboratories among Europe, selected by their publications in the field.ResultsExperts from thirteen countries responded. Guidelines on anti‐RH1 prophylaxis are similar regarding the major aspects of RH disease prevention, including indication and timing of anti‐RH1 Ig administration, as well as indication of foetal RHD genotyping. Different anti‐RH1 Ig preparations are used, and the dosing may differ depending on gestational age. Other controversial issues include (1) timing for foetal RHD genotyping, (2) indication of tests performed to quantitate feto‐maternal haemorrhage prior to anti‐RH1 Ig administration, (3) if there is a remaining indication for newborn RH1 phenotyping. Procedures for monitoring the prophylaxis efficiency and evaluating the national prevention programme also differ among countries.ConclusionDespite some differences among countries, the Rh disease prevention policies are very efficient in Europe, but HDFN cases due to maternal anti‐RH1 immunization have not completely disappeared. It therefore remains important to share best practices for continuous improvement in reducing anti‐RH1 alloimmunization.

Genome-Wide Noninvasive Prenatal Diagnosis of SNPs and Indels.

Noninvasive prenatal diagnosis (NIPD) is an emerging field, that enables testing for diseases in the fetus with no risk to the pregnancy, compared to invasive methods (e.g., amniocentesis). The procedure is based on the presence of fetal DNA within the mother's plasma cell-free DNA (cfDNA). Today, NIPD is performed for chromosomal abnormalities (e.g., Down syndrome) and some large deletions/duplications. It is also available for point mutations but is limited for one mutation or up to several genes simultaneously. Genome-wide detection of fetal point mutations was presented in a few studies, and the first software tool for this task, Hoobari, has recently become available. Here we describe the necessary steps in genome-wide noninvasive fetal genotyping, including examples using the Hoobari software. We discuss the various materials, software, computational infrastructure, and samples required for this analysis. Genome-wide analysis of point mutations in the fetus is not widely studied, albeit much space for algorithmic improvements exists. Here we suggest practical solutions for challenges along the process. Our work assists bioinformaticians in accessing NIPD data analysis and can eventually be utilized for other cfDNA-related fields.

Improved noninvasive fetal variant calling using standardized benchmarking approaches

The technology of noninvasive prenatal testing (NIPT) enables risk-free detection of genetic conditions in the fetus, by analysis of cell-free DNA (cfDNA) in maternal blood. For chromosomal abnormalities, NIPT often effectively replaces invasive tests (e.g. amniocentesis), although it is considered as screening rather than diagnostics. Most recently, the NIPT has been applied to genome-wide, comprehensive genotyping of the fetus using cfDNA, i.e. identifying all its genetic variants and mutations. Previously, we suggested that NIPD should be treated as a special case of variant calling, and presented Hoobari, the first software tool for noninvasive fetal variant calling. Using a unique pipeline, we were able to comprehensively decipher the inheritance of SNPs and indels. A few caveats still exist in this pipeline. Performance was lower for indels and biparental loci (i.e. where both parents carry the same mutation), and performance was not uniform across the genome. Here we utilized standardized methods for benchmarking of variant calling pipelines and applied them to noninvasive fetal variant calling. By using the best performing pipeline and by focusing on coding regions, we showed that noninvasive fetal genotyping greatly improves performance, particularly in indels and biparental loci. These results emphasize the importance of using widely accepted concepts to describe the challenge of genome-wide NIPT of point mutations; and demonstrate a benchmarking process for the first time in this field. This study brings genome-wide and complete NIPD closer to the clinic; while potentially alleviating uncertainty and anxiety during pregnancy, and promoting informed choices among families and physicians.

Evaluation of a Microhaplotype-Based Noninvasive Prenatal Test in Twin Gestations: Determination of Paternity, Zygosity, and Fetal Fraction.

As a novel type of genetic marker, the microhaplotype has shown promising potential in forensic research. In the present study, we analyzed maternal plasma cell-free DNA (cfDNA) samples from twin pregnancies to validate microhaplotype-based noninvasive prenatal testing (NIPT) for paternity, zygosity, and fetal fraction (FF). Paternity was determined with the combined use of the relMix package, zygosity was evaluated by examining the presence of informative loci with two fetal genome complements, and FF was assessed through fetal allele ratios. Paternity was determined in 19 twin cases, among which 13 cases were considered dizygotic (DZ) twins based on the presence of 3~10 informative loci and the remaining 6 cases were considered monozygotic (MZ) twins because no informative locus was observed. With the fetal genomic genotypes as a reference, the accuracy of paternity and zygosity determination were confirmed by standard short tandem repeat (STR) analysis. Moreover, the lower FF, higher FF, and combined FF in each DZ plasma sample were closely related to the estimated value. This present preliminary study proposes that microhaplotype-based NIPT is applicable for paternity, zygosity, and FF determination in twin pregnancies, which are expected to be advantageous for both forensic and clinical settings.

Prenatal RHD genotyping in Croatia: preliminary results.

Croatian Institute of Transfusion Medicine (CITM) implemented non-invasive fetal RHD genotyping as a request for targeted antenatal anti-D prophylaxis. The diagnostic performance of in-house RT-PCR method for fetal RHD genotyping and preliminary results are analyzed. Evaluation included results of RHD genotyping for 205 RhD negative pregnant women, 12-36th week of gestation, whose samples were received in period between 2015 and 2020. QIAsymphony SP DSP Virus Midi Kit was used for cffDNA extraction on QIAsymphony SP platform (Qiagen, Germany). Fragments of RHD exons 7 and 10 and later exon 5 were RT-PCR amplified. As internal controls, amplification of SRY gene or RASSF1A fragment and β-actin genes digested with BsTUI were used. We identified 70.72% (145/205) positive and 28.78% (59/205) negative fetal RHD genotypes. We had one inconclusive result (0.50%) due to the interference of maternal DNA with variant genotype RHD*09.02.00/01/*01N.01. When compared to newborns RhD phenotypes, no false negative and three false positive results (3/199, 1.50%) were observed. The test yielded 100% sensitivity and 95.08% specificity, while diagnostic accuracy was 98.48%. We were able to determine one case of fetal variant genotype RHD*04.04/*01N.01 inherited from the father. The negative and positive predictive test values were 100% and 97.86%, respectively. Automated cffDNA extraction and RT-PCR amplification of fetal RHD exons 5,7,10 and fragments of SRY, RASSF1A genes represents highly reliable system for determining fetal RHD status which enables targeted antenatal anti-D prophylaxis. To obtain high specificity of cffDNA extraction, strict and thoroughly cleaning procedures are required.

USING DROPLET DIGITAL PCR TO SCREEN FOR RARE BLOOD DONORS: PROOF OF PRINCIPLE

Abstract Background Digital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). Methods Nucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1–5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. Results DI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. Conclusion The proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

Fetal Rhesus D Genotyping and Sex Determination from Maternal Plasma of Rhesus D-Negative Antenatal Population: The Usefulness of Conventional Polymerase Chain Reaction in Resource-limited Settings.

Background This prospective cohort study evaluated the usefulness of conventional PCR in genotyping fetal Rhesus D (RhD) and sex from the maternal plasma of RhD-negative (RhD−) antenatal population in resource-limited settings. Methods Thirty apparently healthy RhD− pregnant women with RhD positive (RhD+) partners were included. Blood samples were collected from each participant (in the third trimester of pregnancy) for DNA extraction/purification and fetal RhD genotyping. Results Out of the 30 samples, 26 (86.7%) were found to be RhD+ while 4 (13.3%) were RhD−. The RhD+ comprised 24 (80.0%) RhD+ based on exons 5, 7, and 10 combined. Exons 5 and 7 were detected in two additional samples but not exon 10. Serological phenotyping of neonatal blood confirmed 26 RhD+ and 4 RhD−. There was a perfect agreement between the fetal RhD genotype and neonatal RhD phenotyping after delivery for exons 5 and 7 (concordance = 100%, κ = 100.0%, diagnostic accuracy = 100%, p < 0.0001) while exon 10 presented with an almost perfect agreement (concordance = 93.3%, κ = 76.2%, diagnostic accuracy = 93.3%, p < 0.0001). Regarding the prenatal test for the SRY gene, 9 (30.0%) were predicted to be males and the remaining 21 (60.0%) were females. All the 9 and 21 anticipated males and females, respectively, were confirmed after delivery (concordance = 100%, κ = 100.0%, diagnostic accuracy = 100%). Conclusion Our study suggests that conventional PCR using the SRY, RhD exons 5 and 7 could be useful for predicting fetal sex and RhD from maternal peripheral blood in resource-limited settings.

A common deletion in the growth hormone receptor gene (d3-GHR) in the offspring is related to maternal placental GH levels during pregnancy

BackgroundA common growth hormone receptor polymorphism with deletion of exon 3 (d3-GHR) has previously been linked to increased postnatal growth on the one hand and decreased fetal growth on the other. Regulation of fetal growth is positively dependent on secretion of placental GH (hGH-V). ObjectiveWe explored the effect of the fetal d3-GHR genotype on maternal serum levels of hGH-V and fetal growth. The cellular localization of hGH-V synthesis and the GH receptors were determined in normal placentas. Methods43 healthy mother-child pairs were examined during pregnancy with measurements of hGH-V during third trimester, and serial ultrasound measurements determined fetal growth rate. Birth anthropometrics were obtained. The GHR genotype of the child was analysed postnatally. Immunohistochemical (IHC) analysis was conducted on four placentas. ResultsThe presence of the d3-GHR genotype was associated with a markedly reduced concentration of hGH-V in maternal serum (β −0.52, SE 0.24, p = 0.04) compared to those who had a fl/fl genotype. Accordingly, a tendency towards reduced fetal growth rate during third trimester (β −25.8, SE 12.7, p = 0.05) and a lower birth weight were found among carriers of the d3-GHR allele, but these associations did not reach statistical significance (p = 0.08). IHC analysis showed expression of placental GH and GHR in the villous syncytiotrophoblast, the extravillous trophoblast, and the decidual cells and smooth muscle cells in chorionic vessels. ConclusionsThe presence of the d3-GHR polymorphism in the fetus was associated with lower maternal serum levels of hGH-V, decreased fetal growth rate in third trimester and lower birth weight compared to the wildtype.

Maternal-fetal genetic interactions, imprinting, and risk of placental abruption

Background: Maternal genetic variations, including variations in mitochondrial biogenesis (MB) and oxidative phosphorylation (OP), are associated with placental abruption (PA). However, the role of maternal-fetal genetic interactions (MFGI) and parent-of-origin (imprinting) effects in PA remain unknown. Objective: To investigate MFGI in MB-OP, and imprinting effects in relation to risk of PA. Methods: Among Peruvian mother-infant pairs (503 PA cases and 1052 controls), independent single nucleotide polymorphisms (SNPs), with linkage-disequilibrium coefficient <0.80, were selected to characterize genetic variations in MB-OP (78 SNPs in 24 genes) and imprinted genes (2713 SNPs in 73 genes). For each MB-OP SNP, four multinomial models corresponding to fetal allele effect, maternal allele effect, maternal and fetal allele additive effect, and maternal-fetal allele interaction effect were fit under Hardy-Weinberg equilibrium, random mating, and rare disease assumptions. The Bayesian information criterion (BIC) was used for model selection. For each SNP in imprinted genes, imprinting effect was tested using a likelihood ratio test. Bonferroni corrections were used to determine statistical significance (p-value < 6.4e-4 for MFGI and p-value < 1.8e-5 for imprinting). Results Abruption cases were more likely to experience preeclampsia, have shorter gestational age, and deliver infants with lower birthweight compared with controls. Models with MFGI effects provided improved fit than models with only maternal and fetal genotype main effects for SNP rs12530904 (p-value = 1.2e-04) in calcium/calmodulin-dependent protein kinase [CaM kinase] II beta (CAMK2B), and, SNP rs73136795 (p-value = 1.9e-04) in peroxisome proliferator-activated receptor-gamma (PPARG), both MB genes. We identified 320 SNPs in 45 maternally-imprinted genes (including potassium voltage-gated channel subfamily Q member 1 [KCNQ1], neurotrimin [NTM], and, ATPase phospholipid transporting 10 A [ATP10A]) associated with abruption. Top hits included rs2012323 (p-value = 1.6E-16) and rs12221520 (p-value1.3e-13) in KCNQ1, rs8036892 (p-value = 9.3E-17) and rs188497582 in ATP10A, rs12589854 (p-value = 2.9E-11) and rs80203467 (p-value = 4.6e-11) in maternally expressed 8, small nucleolar RNA host (MEG8), and rs138281088 in solute carrier family 22 member 2 (SLC22A2) (p-value = 6.8e-9). Conclusions We identified novel PA-related maternal-fetal MB gene interactions and imprinting effects that highlight the role of the fetus in PA risk development. Findings can inform mechanistic investigations to understand the pathogenesis of PA.

Effects of fetal and maternal genotype on placentome morphology in sheep

Both maternal and fetal genotypes contribute to conceptus development. The objective was to determine how placentome number, size, and type and fetal weight was influenced after reciprocal embryo transfer in Columbia and Romanov sheep. Reciprocal embryo transfer was conducted between Columbia and Romanov ewes where a single embryo was transferred into Romanov and Columbia recipients [Romanov embryo in a Romanov uterus (RinR, n = 9); Romanov embryo in a Columbia uterus (RinC, n = 7); Columbia embryo in a Columbia uterus (CinC, n = 8); Columbia embryo in a Romanov uterus (CinR, n = 4)]. On day 130 of gestation, fetuses were weighed and placentomes were morphologically typed, weighed, and measured.Regardless of maternal genotype, Romanov fetuses were smaller (P < 0.05) compared to Columbia fetuses. Moreover, CinC fetuses were larger (P < 0.05) than CinR fetuses. There was a tendency (P = 0.12) for a fetal by maternal genotype interaction on total placentome weight, but main effects were significant for fetal genotype (P = 0.04) and maternal genotype (P < 0.01). The number of Type A placentomes was greater than any other types. Type A placentomes had a greater (P < 0.05) contribution to total placentome weight within the Romanov uterus, or when associated with a Romanov fetus, than within the Columbia breed, in which placentome type was evenly distributed. The hypothesis that the Romanov uterus would limit the growth of a Columbia conceptus is accepted; however, the Romanov conceptus did not experience augmented growth when transferred into a Columbia uterus as predicted.