Maternal DNA Research Articles

Non-invasive prenatal testing (NIPT): Combination of copy number variant and gene analyses using an "in-house" target enrichment next generation sequencing-Solution for non-centralized NIPT laboratory?

Recent studies have integrated copy number variant (CNV) and gene analysis using target enrichment. Here, we transferred this concept to our routine genetics laboratory, which is not linked to centralized non-invasive prenatal testing (NIPT) facilities. From a cohort of 100 pregnant women, 22 were selected for the analysis of maternal genomic DNA (gDNA) along with fetal cell-free DNA. Using targeted enrichment, 135 genes were analyzed, combined with aberrations of chromosomes 21, 18, 13, X, and Y. The data were subjected to specificity and sensitivity analyses, and correlated with the results from invasive testing methods. The sensitivity/specificity was determined for the CNV analysis of chromosomes: 21 (80%/75%), 18 (-/82%), 13 (100%/67%), and Y (100%/100%). The gene detection was valid for maternal gDNA. However, for cell-free fetal DNA, it was not possible to determine the boundary between an artifact and a real sequence variant. The target enrichment method combining CNV and gene detection seems feasible in a regular laboratory. However, this method can only be responsibly optimized with a sufficient number of controls and further validation on a strong bioinformatic background. The present results showed that NIPT should be performed in specialized centers, and that its introduction to isolated laboratories may not provide valid data.

A Study on the Causes of Apomixis in Malus shizongensis

Apomixis is a unique reproductive process that produces fertile offspring without the combination of sperm and egg cells. This process perfectly reproduces maternal DNA, making it possible to fix heterosis during reproduction. Malus shizongensis is a newly discovered species that is closely related to Malus hupehensis Rehd. After de-male bagging, it was found that the fruit set rate reached 78.7%. Preliminary analysis indicated that M. shizongensis have apomictic reproductive characteristics. In this work, we employed paraffin sectioning and electron scanning microscopy to explore apomixis in M. shizongensis during the development of male–female gametes and embryo sacs. Stigma fluorescence assays showed that pollen germination was normal, but less pollen entered the ovaries. Additionally, analysis of anthers indicated the presence of dysplasia and paraffin sectioning revealed that the pollen mother cells were aborted due to abnormal disintegration of the tapetum layer. Taken together, our results indicate that the primary causes of apomixis in M. shizongensis are anther dysplasia and male gamete development failure, resulting in reduced pollen tube entry into ovaries and reduced reproduction of female gametes. In conclusion, this study provide a theoretical basis and technical supports for apple stock breeding and apple industry development.

Influence of genetic polymorphisms on arsenic methylation efficiency during pregnancy: Evidence from a Spanish birth cohort

BackgroundInorganic arsenic (iAs) is a widespread toxic metalloid. It is well-known that iAs metabolism and its toxicity are mediated by polymorphisms in AS3MT and other genes. However, studies during pregnancy are scarce. We aimed to examine the role of genetic polymorphisms in AS3MT, GSTO2, N6AMT1, MTHFR, MTR, FTCD, CBS, and FOLH1 in iAs methylation efficiency during pregnancy. MethodsThe study included 541 pregnant participants from the INMA (Environment and Childhood) Spanish cohort. Using high-performance liquid chromatography coupled to inductively coupled plasma-tandem mass, we measured arsenic (iAs and the metabolites monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) in urine samples collected during the first trimester. iAs methylation efficiency was determined based on relative concentrations of the As metabolites in urine (%MMA, %DMA, and %iAs). Thirty-two single nucleotide polymorphisms (SNPs) in nine genes were determined in maternal DNA; AS3MT haplotypes were inferred. We assessed the association between genotypes/haplotypes and maternal As methylation efficiency using multivariate linear regression models. ResultsThe median %MMA and %DMA were 5.3 %, and 89 %, respectively. Ancestral alleles of AS3MT SNPs (rs3740393, rs3740390, rs11191453, and rs11191454) were significantly associated with higher %MMA, %iAs, and lower %DMA. Pregnant participants with zero copies of the GGCTTCAC AS3MT haplotype presented a higher %MMA. Statistically significant associations were also found for the FOLH1 SNP rs202676 (β 0.89 95%CI: 0.24, 1.55 for carriers of the G allele vs. the A allele). ConclusionsOur study shows that ancestral alleles in AS3MT polymorphisms were associated with lower As methylation efficiency in early pregnancy and suggests that FOLH1 also plays a role in As methylation efficiency. These results support the hypothesis that As metabolism is multigenic, being a key element for identifying susceptible populations.

Overview of Noninvasive Prenatal Testing (NIPT) for the Detection of Fetal Chromosome Abnormalities; Differences in Laboratory Methods and Scope of Testing.

Although nearly all noninvasive prenatal testing is currently based on analyzing circulating maternal cell-free DNA, the technical methods usedvary considerably. We review the different methods. Based on validation trials and clinical experience, there are mostly relatively small differences in screening performance for trisomies 21, 18, and 13 in singleton pregnancies. Recent reports show low no-call rates for all methods, diminishing its importance when choosing a laboratory. However, method can be an important consideration for twin pregnancies, screening for sex chromosome abnormalities, microdeletion syndromes, triploidy, molar pregnancies, rare autosomal trisomies, and segmental imbalances, and detecting maternal chromosome abnormalities.

Noninvasive prenatal diagnosis of Mendelian disorders for consanguineous couples by relative genotype dosage.

Noninvasive prenatal diagnosis relies on the presence in maternal blood of circulating cell-free fetal DNA released by apoptotic trophoblast cells. Widely used for aneuploidy screening, it can also be applied to monogenic diseases (NIPD-M) in case of known parental mutations. Due to the confounding effect of maternal DNA, detection of maternal or biparental mutations requires relative haplotype dosage (RHDO), a method relying on the presence of SNPs that are heterozygous in one parent and homozygous in the other. Unavoidably, there is a risk of test failure by lack of such informative SNPs, an event particularly likely for consanguineous couples who often share common haplotypes in regions of identity-by-descent. Here we present a novel approach, relative genotype dosage (RGDO) that bypasses this predicament by directly assessing fetal genotype with SNPs that are heterozygous in both parents (frequent in regions of identity-by-descent). We show that RGDO is as sensitive as RHDO and that it performs well over a large range of fetal fractions and DNA amounts, thereby opening NIPD-M to most consanguineous couples. We also report examples of couples, consanguineous or not, where combining RGDO and RHDO allowed a diagnosis that would not have been possible with only one approach.

Integrative profiling of extrachromosomal circular DNA in placenta and maternal plasma provides insights into the biology of fetal growth restriction and reveals potential biomarkers.

Introduction: Fetal growth restriction (FGR) is a placenta-mediated pregnancy complication that predisposes fetuses to perinatal complications. Maternal plasma cell-free DNA harbors DNA originating from placental trophoblasts, which is promising for the prenatal diagnosis and prediction of pregnancy complications. Extrachromosomal circular DNA (eccDNA) is emerging as an ideal biomarker and target for several diseases. Methods: We utilized eccDNA sequencing and bioinformatic pipeline to investigate the characteristics and associations of eccDNA in placenta and maternal plasma, the role of placental eccDNA in the pathogenesis of FGR, and potential plasma eccDNA biomarkers of FGR. Results: Using our bioinformatics pipelines, we identified multi-chromosomal-fragment and single-fragment eccDNA in placenta, but almost exclusively single-fragment eccDNA in maternal plasma. Relative to that in plasma, eccDNA in placenta was larger and substantially more abundant in exons, untranslated regions, promoters, repetitive elements [short interspersed nuclear elements (SINEs)/Alu, SINEs/mammalian-wide interspersed repeats, long terminal repeats/endogenous retrovirus-like elements, and single recognition particle RNA], and transcription factor binding motifs. Placental multi-chromosomal-fragment eccDNA was enriched in confident enhancer regions predicted to pertain to genes in apoptosis, energy, cell growth, and autophagy pathways. Placental eccDNA-associated genes whose abundance differed between the FGR and control groups were associated with immunity-related gene ontology (GO) terms. The combined analysis of plasma and placental eccDNA-associated genes in the FGR and control groups led to the identification of potential biomarkers that were assigned to the GO terms of the epigenetic regulation of gene expression and nutrient-related processes, respectively. Conclusion: Together, our results highlight links between placenta functions and multi-chromosomal-fragment and single-fragment eccDNA. The integrative analysis of placental and plasma eccDNA confirmed the potential of these molecules as disease-specific biomarkers of FGR.

O-197 Vertical transmission of maternal mitochondrial DNA via extracellular vesicles (EVs) modulates embryo bioenergetics during the periconceptional period

Abstract Study question Does maternal endometrial mitochondrial (mt) DNA cargo of EVs modulate embryo bioenergetics during embryo implantation? Summary answer We demonstrate the vertical transmission of maternal mtDNA within endometrial-derived EVs and their uptake by the trophoblast which reduces mitochondrial respiration and ATP production. What is known already The release and uptake of membrane-enclosed compartments with specific cargos, commonly known as EVs, represents a novel cell-to-cell communication mechanism in physiological and pathogenic conditions. EVs are generally classified into three populations based on their biogenetic pathways, composition, and physical characteristics: apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs). Among other contents, EVs contain single- and double-stranded DNA, with their relative abundance varying depending on the cell and vesicle type. The vertical transmission of EV-associated DNA has been proposed as a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. Study design, size, duration Prospective observational multicenter analysis in which EVs were obtained from endometrial fluid from healthy donors aged 18–35 years (n = 10) during the receptive phase of their natural cycle and under hormonal replacement therapy in pre-receptive (P + 2), receptive (P + 5), post-receptive (P + 8) stages (n = 13). Participants/materials, setting, methods Endometrial EVs isolated using ultracentrifugation and classified according to `parameters obtained from electron microscopy, Western blotting, and size distribution analysis. DNA copy number identified using high throughput sequencing. EV-associated DNA tagged with 5-ethynyl-2’-deoxyuridine was followed by confocal imaging after co-incubation of EVs with murine embryos (n = 200). ATP levels assessed using the FLASC luciferase reporter system, and the Seahorse XFE96 extracellular flux analyzer used to measure embryo oxygen consumption rate (OCR) (n = 400). Main results and the role of chance The human endometrium secretes all three EV types - ABs, MVs, and EXOs - into the human endometrial fluid. Deep sequencing revealed that EVs encapsulated nuclear and mtDNA. When analyzing endometrial biopsies, we observed the reduced mtDNA content of endometrial cells and the activation of mitochondrial clearance mechanisms, which coincided with the time of embryo implantation together with specific enrichment in endometrial MVs secreted during the periconceptional period. EVs were internalized and DNA was transferred to the cytoplasm and nuclei of trophectoderm of murine embryos. We analyzed ATP concentrations in murine embryos and found a significant reduction in ATP levels following the coculture of embryos with a combination of all EVs types compared to control embryos cultured without endometrial EVs (p < 0.001). Finally, we demonstrated a reduction in the OCR in embryos treated with endometrial EVs obtained during the receptive phase compared with the pre-receptive phase. In conclusion, maternal EVs modulate the bioenergetics of the preimplantation embryo by increasing the embryo's metabolic rate and oxygen consumption during the periconceptional period. Limitations, reasons for caution These results were obtained using a combination of a human endometrial model and a murine embryo model. Wider implications of the findings Our results suggest that the vertical transmission of maternal mtDNA encapsulated within EVs to the trophectoderm might energetically assist the preimplantation embryo through the implantation process. Trial registration number N/A

P-731 The accuracy of truly non-invasive PGT using spent culture media is insufficient to justify routine clinical use

Abstract Study question Can non-invasive PGT (niPGT), using a protocol that avoids manipulation of the embryo before spent culture medium is collected, accurately determine embryo chromosomal status? Summary answer Accuracy using a truly non-invasive PGT method was low, suggesting this strategy is not appropriate for clinical use and should be restricted to research studies. What is known already PGT requires embryo biopsy to obtain small numbers of cells for genetic testing. The procedure requires skilled personnel and specialist equipment, significantly adding to the cost of PGT while also creating training and logistical bottlenecks that reduce patient access to PGT. Additionally, there have been concerns that biopsy could damage embryos. These considerations have stimulated interest in potential non-invasive PGT strategies. Thus far, niPGT methods have mostly focused on cell-free embryonic DNA found in spent culture media, but studies reporting highest accuracy have tended to involve embryo manipulations likely to promote DNA release and therefore cannot be considered truly non-invasive. Study design, size, duration Samples of spent culture medium (SCM) associated with 128 embryos were collected. Oocytes had been denuded of cumulus cells, fertilised using ICSI and the resulting embryos thoroughly washed on day-4 before transfer to a fresh drop of medium. SCM samples were collected on day-5 or day-6 of culture. Embryos had not previously been cryopreserved and underwent minimal manipulation prior to SCM collection. Results of SCM analysis were compared to those obtained following conventional ‘invasive’ PGT. Participants/materials, setting, methods Media samples were subjected to whole genome amplification using the PG-Seq Rapid Non-Invasive PGT kit. Concentrations of sequencing libraries were measured, and next-generation sequencing (NGS) was undertaken. The resulting NGS data was analysed with PG-Find Software to predict chromosomal status. In parallel, trophectoderm biopsies from the corresponding embryos were analysed using a well-established and highly validated PGT method, based upon amplification of thousands of sites across the genome, followed by NGS. Main results and the role of chance Despite the implementation of rigorous measures to prevent contamination of SCM samples, extraneous female DNA, likely of cumulus cell origin, was frequently observed. Depending on the clinic, 16.7% to 38.1% of media samples associated with male embryos gave a discordant (female) result. This highlights the difficulty of avoiding inadvertent sampling of maternal DNA. There were no instances of male DNA contamination affecting SCM samples from female embryos. We assessed whether clinic of origin, ploidy status of the embryo, and day of SCM collection influenced the quality of the sequencing libraries (ANOVA). Higher concentrations of libraries and superior sequencing quality scores were observed in SCM collected on day-6 compared to day-5 (p = 0.011 and p = 0.002, respectively). Chromosomal assessments obtained from SCM were compared to those from standard, trophectoderm-based PGT in a blinded fashion. 50% of SCM samples gave an identical predicted karyotype to their corresponding trophectoderm biopsy (concordant copy number for all chromosomes). When applying a simple aneuploid/euploid classification, 80% of embryos classified chromosomally abnormal using the validated PGT method were also aneuploid according to analysis of the associated SCM sample, but without necessarily having identical karyotypes, while 62% of embryos classified euploid by PGT received the same designation after SCM analysis. Limitations, reasons for caution SCM samples were designated ‘contaminated’ when embryos of one sex (determined using invasive PGT) were incorrectly classified as the opposite sex using niPGT. However, our study was unable to determine when SCM samples were contaminated with DNA of the same sex as the embryo. Consequently, contamination rates could be higher. Wider implications of the findings Most niPGT methods are not truly non-invasive, as they include embryo manipulations that increase likelihood of cell death and DNA release. Our results show that obtaining reliable niPGT results is difficult without deviating significantly from optimal embryological protocols. Currently, the accuracy of niPGT seems insufficient to justify routine clinical use. Trial registration number Not applicable

Hemoglobinopathies, merozoite surface protein-2 gene polymorphisms, and acquisition of Epstein Barr virus among infants in Western Kenya

BackgroundEpstein Barr virus (EBV)-associated endemic Burkitt’s Lymphoma pediatric cancer is associated with morbidity and mortality among children resident in holoendemic Plasmodium falciparum regions in western Kenya. P. falciparum exerts strong selection pressure on sickle cell trait (SCT), alpha thalassemia (-α3.7/αα), glucose-6-phosphate dehydrogenase (G6PD), and merozoite surface protein 2 (MSP-2) variants (FC27, 3D7) that confer reduced malarial disease severity. The current study tested the hypothesis that SCT, (-α3.7/αα), G6PD mutation and (MSP-2) variants (FC27, 3D7) are associated with an early age of EBV acquisition.MethodsData on infant EBV infection status (< 6 and ≥ 6–12 months of age) was abstracted from a previous longitudinal study. Archived infant DNA (n = 81) and mothers DNA (n = 70) samples were used for genotyping hemoglobinopathies and MSP-2. The presence of MSP-2 genotypes in maternal DNA samples was used to indicate infant in-utero malarial exposure. Genetic variants were determined by TaqMan assays or standard PCR. Group differences were determined by Chi-square or Fisher’s analysis. Bivariate regression modeling was used to determine the relationship between the carriage of genetic variants and EBV acquisition.ResultsEBV acquisition for infants < 6 months was not associated with -α3.7/αα (OR = 1.824, P = 0.354), SCT (OR = 0.897, P = 0.881), or G6PD [Viangchan (871G > A)/Chinese (1024 C > T) (OR = 2.614, P = 0.212)] and [Union (1360 C > T)/Kaiping (1388G > A) (OR = 0.321, P = 0.295)]. There was no relationship between EBV acquisition and in-utero exposure to either FC27 (OR = 0.922, P = 0.914) or 3D7 (OR = 0.933, P = 0.921). In addition, EBV acquisition in infants ≥ 6–12 months also showed no association with -α3.7/αα (OR = 0.681, P = 0.442), SCT (OR = 0.513, P = 0.305), G6PD [(Viangchan (871G > A)/Chinese (1024 C > T) (OR = 0.640, P = 0.677)], [Mahidol (487G > A)/Coimbra (592 C > T) (OR = 0.948, P = 0.940)], [(Union (1360 C > T)/Kaiping (1388G > A) (OR = 1.221, P = 0.768)], African A (OR = 0.278, P = 0.257)], or in utero exposure to either FC27 (OR = 0.780, P = 0.662) or 3D7 (OR = 0.549, P = 0.241).ConclusionAlthough hemoglobinopathies (-α3.7/αα, SCT, and G6PD mutations) and in-utero exposure to MSP-2 were not associated with EBV acquisition in infants 0–12 months, novel G6PD variants were discovered in the population from western Kenya. To establish that the known and novel hemoglobinopathies, and in utero MSP-2 exposure do not confer susceptibility to EBV, future studies with larger sample sizes from multiple sites adopting genome-wide analysis are required.

Prenatal maternal stress is associated with site-specific and age acceleration changes in maternal and newborn DNA methylation

ABSTRACT Prenatal maternal stress has a negative impact on child health but the mechanisms through which maternal stress affects child health are unclear. Epigenetic variation, such as DNA methylation, is a likely mechanistic candidate as DNA methylation is sensitive to environmental insults and can regulate long-term changes in gene expression. We recruited 155 mother-newborn dyads in the Democratic Republic of Congo to investigate the effects of maternal stress on DNA methylation in mothers and newborns. We used four measures of maternal stress to capture a range of stressful experiences: general trauma, sexual trauma, war trauma, and chronic stress. We identified differentially methylated positions (DMPs) associated with general trauma, sexual trauma, and war trauma in both mothers and newborns. No DMPs were associated with chronic stress. Sexual trauma was positively associated with epigenetic age acceleration across several epigenetic clocks in mothers. General trauma and war trauma were positively associated with newborn epigenetic age acceleration using the extrinsic epigenetic age clock. We tested the top DMPs for enrichment of DNase I hypersensitive sites (DHS) and found no enrichment in mothers. In newborns, top DMPs associated with war trauma were enriched for DHS in embryonic and foetal cell types. Finally, one of the top DMPs associated with war trauma in newborns also predicted birthweight, completing the cycle from maternal stress to DNA methylation to newborn health outcome. Our results indicate that maternal stress is associated with site-specific changes in DNAm and epigenetic age acceleration in both mothers and newborns.

Early biomarkers of neurodevelopmental disorders in preterm infants: protocol for a longitudinal cohort study

IntroductionPreterm (PT) infants are at high likelihood for poor neurodevelopmental outcomes, including autism spectrum disorder (ASD), attention-deficit hyperactivity disorder (ADHD) and other neurodevelopmental disorders (NDDs), which could considerably impair the...

Polygenic prediction of preeclampsia and gestational hypertension.

Preeclampsia and gestational hypertension are common pregnancy complications associated with adverse maternal and child outcomes. Current tools for prediction, prevention and treatment are limited. Here we tested the association of maternal DNA sequence variants with preeclampsia in 20,064 cases and 703,117 control individuals and with gestational hypertension in 11,027 cases and 412,788 control individuals across discovery and follow-up cohorts using multi-ancestry meta-analysis. Altogether, we identified 18 independent loci associated with preeclampsia/eclampsia and/or gestational hypertension, 12 of which are new (for example, MTHFR-CLCN6, WNT3A, NPR3, PGR and RGL3), including two loci (PLCE1 and FURIN) identified in the multitrait analysis. Identified loci highlight the role of natriuretic peptide signaling, angiogenesis, renal glomerular function, trophoblast development and immune dysregulation. We derived genome-wide polygenic risk scores that predicted preeclampsia/eclampsia and gestational hypertension in external cohorts, independent of clinical risk factors, and reclassified eligibility for low-dose aspirin to prevent preeclampsia. Collectively, these findings provide mechanistic insights into the hypertensive disorders of pregnancy and have the potential to advance pregnancy risk stratification.

Association of vitamin D and polymorphisms of its receptor with antiviral therapy in pregnant women with hepatitis B.

The interruption of mother-to-child transmission (MTCT) is considered important to decrease the individual and population morbidity of hepatitis B virus (HBV) infection as well as the global burden of hepatitis B. Serum vitamin D (VD) is associated with hepatitis B. To assess whether baseline VD levels and single nucleotide polymorphisms of the VD receptor gene (VDR SNPs) are associated with the efficacy of tenofovir disoproxil fumarate (TDF) in the prevention of MTCT in pregnant women with high HBV viral loads. Thirty-eight pregnant women who were at high risk for MTCT of HBV (those with an HBV DNA level ≥ 2 × 105 IU/mL during 12-24 wk of gestation) receiving antiviral therapy of TDF between June 1, 2019 and June 30, 2021 in Mianyang were included in this retrospective study. The women received 300 mg TDF once daily from gestational weeks 24-28 until 3 mo after delivery. To further characterize the clinical relevance of maternal serum HBV DNA levels, we stratified patients according to HBV DNA level as follows: Those with levels < 2 × 105 (full responder group) vs those levels ≥ 2 × 105 IU/mL (partial responder group) at delivery. Serum levels of 25-hydroxyvitamin D [25(OH)D], liver function markers, virological parameters, VDR SNPs and other clinical parameters were collected to analyze their association with the efficacy of TDF. The Mann-Whitney U test or t test was used to analyze the serum levels of 25(OH)D in different groups. Multiple linear regressions were utilized to analyze the determinants of the maternal HBV DNA level at delivery. Univariate and multivariate logistic regression analyses were employed to explore the association of targeted antiviral effects with various characteristics at baseline and delivery. A total of 38 pregnant women in Mianyang City at high risk for MTCT of HBV were enrolled in the study. The MTCT rate was 0%. No mother achieved hepatitis B e antigen or hepatitis B surface antigen (HBsAg) clearance at delivery. Twenty-three (60.5%) participants were full responders, and 15 (39.5%) participants were partial responders according to antiviral efficacy. The present study showed that a high percentage (76.3%) of pregnant women with high HBV viral loads had deficient (< 20 ng/mL) or insufficient (≥ 20 but < 31 ng/mL) VD levels. Serum 25(OH)D levels in partial responders appeared to be significantly lower than those in full responders both at baseline (25.44 ± 9.42 vs 17.66 ± 5.34 ng/mL, P = 0.006) and delivery (26.76 ± 8.59 vs 21.24 ± 6.88 ng/mL, P = 0.044). Serum 25(OH)D levels were negatively correlated with maternal HBV DNA levels [log(10) IU/mL] at delivery after TDF therapy (r = -0.345, P = 0.034). In a multiple linear regression analysis, maternal HBV DNA levels were associated with baseline maternal serum 25(OH)D levels (P < 0.0001, β = -0.446), BMI (P = 0.03, β = -0.245), baseline maternal log10 HBsAg levels (P = 0.05, β = 0.285) and cholesterol levels at delivery (P = 0.015, β = 0.341). Multivariate logistic regression analysis showed that baseline serum 25(OH)D levels (OR = 1.23, 95%CI: 1.04-1.44), maternal VDR Cdx2 TT (OR = 0.09, 95%CI: 0.01-0.88) and cholesterol levels at delivery (OR = 0.39, 95%CI: 0.17-0.87) were associated with targeted antiviral effects (maternal HBV DNA levels < 2 × 105 at delivery). Maternal VD levels and VDR SNPs may be associated with the efficacy of antiviral therapy in pregnant women with high HBV viral loads. Future studies to evaluate the therapeutic value of VD and its analogs in reducing the MTCT of HBV may be justified.

The origin and possible mechanism of embryonic cell-free DNA release in spent embryo culture media: a review.

The presence of cell-free DNA in spent embryo culture media (SECM) has unveiled its possible utilization for embryonic ploidy determination, opening new frontiers for the development of a non-invasive pre-implantation genetic screening technique. While a growing number of studies have shown a high concordance between genetic screening using cell-free DNA (cfDNA) and trophectoderm (TE), the mechanism pertaining to the release of cfDNA in SECM is largely unknown. This review aims to evaluate research evidence on the origin and possible mechanisms for the liberations of embryonic DNA in SECM, including findings on the self-correction abilities of embryos which might contribute to the presence of cfDNA. Several databases including EMBASE, PUBMED, and SCOPUS were used to retrieve original articles, reviews, and opinion papers. The keywords used for the search were related to the origins and release mechanism of cfDNA. cfDNA in SECM originates from embryonic cells and, at some levels, non-embryonic cells such as maternal DNA and exogenous foreign DNA. The apoptotic pathway has been demonstrated to eliminate aneuploid cells in developing mosaic embryos which might culminate to the release of cfDNA in SECM. Nonetheless, there is a recognized need for exploring other pathways such as cross-talk molecules called extracellular vesicles (EVs) made of small, round bi-layer membranes. During in vitro development, embryos physiologically and actively expel EVs containing not only protein and microRNA but also embryonic DNA, hence, potentially releasing cfDNA of embryonic origin into SECM through EVs.

Intermediate hepatitis B virus infection prevalence among 1622 pregnant women in rural Burkina Faso and implications for mother-to-child transmission

In highly endemic countries for hepatitis B virus (HBV) infection, childhood infection, including mother-to-child transmission (MTCT), represents the primary transmission route. High maternal DNA level (viral load ≥ 200,000 IU/mL) is a significant factor for MTCT. We investigated the prevalence of HBsAg, HBeAg, and high HBV DNA among pregnant women in three hospitals in Burkina Faso and assessed the performance of HBeAg to predict high viral load. Consenting pregnant women were interviewed on their sociodemographic characteristics and tested for HBsAg by a rapid diagnostic test, and dried blood spot (DBS) samples were collected for laboratory analyses. Of the 1622 participants, HBsAg prevalence was 6.5% (95% CI, 5.4–7.8%). Among 102 HBsAg-positive pregnant women in DBS samples, HBeAg was positive in 22.6% (95% CI, 14.9–31.9%), and viral load was quantified in 94 cases, with 19.1% having HBV DNA ≥ 200,000 IU/mL. HBV genotypes were identified in 63 samples and predominant genotypes were E (58.7%) and A (36.5%). The sensitivity of HBeAg by using DBS samples to identify high viral load in the 94 cases was 55.6%, and the specificity was 86.8%. These findings highlight the need to implement routine HBV screening and effective MTCT risk assessment for all pregnant women in Burkina Faso to enable early interventions that can effectively reduce MTCT.

Sex differences in the intergenerational link between maternal and neonatal whole blood DNA methylation: a genome-wide analysis in 2 birth cohorts

BackgroundThe mother–child inheritance of DNA methylation (DNAm) variations could contribute to the inheritance of disease susceptibility across generations. However, no study has investigated patterns of mother–child associations in DNAm at the genome-wide scale. It remains unknown whether there are sex differences in mother–child DNAm associations.ResultsUsing genome-wide DNAm profiling data (721,331 DNAm sites, including 704,552 on autosomes and 16,779 on the X chromosome) of 396 mother–newborn pairs (54.5% male) from the Boston Birth Cohort, we found significant sex differences in mother–newborn correlations in genome-wide DNAm patterns (Spearman’s rho = 0.91–0.98; p = 4.0 × 10–8), with female newborns having stronger correlations. Sex differences in correlations were attenuated but remained significant after excluding X-chromosomal DNAm sites (Spearman’s rho = 0.91–0.98; p = 0.035). Moreover, 89,267 DNAm sites (12.4% of all analyzed, including 88,051 [12.5% of analyzed] autosomal and 1,216 [7.2% of analyzed] X-chromosomal sites) showed significant mother–newborn associations in methylation levels, and the top autosomal DNAm sites had high heritability than the genome-wide background (e.g., the top 100 autosomal DNAm sites had a medium h2 of 0.92). Additionally, significant interactions between newborn sex and methylation levels were observed for 11 X-chromosomal and 4 autosomal DNAm sites that were mapped to genes that have been associated with sex-specific disease/traits or early development (e.g., EFHC2, NXY, ADCYAP1R1, and BMP4). Finally, 18,769 DNAm sites (14,482 [77.2%] on the X chromosome) showed mother–newborn differences in methylation levels that were significantly associated with newborn sex, and the top autosomal DNAm sites had relatively small heritability (e.g., the top 100 autosomal DNAm sites had a medium h2 of 0.23). These DNAm sites were mapped to 2,532 autosomal genes and 978 X-chromosomal genes with significant enrichment in pathways involved in neurodegenerative and psychological diseases, development, neurophysiological process, immune response, and sex-specific cancers. Replication analysis in the Isle of Wight birth cohort yielded consistent results.ConclusionIn two independent birth cohorts, we demonstrated strong mother–newborn correlations in whole blood DNAm on both autosomes and ChrX, and such correlations vary substantially by sex. Future studies are needed to examine to what extent our findings contribute to developmental origins of pediatric and adult diseases with well-observed sex differences.

Haplotype-based non-invasive prenatal diagnosis of recessive dystrophic epidermolysis bullosa via targeted capture sequencing of maternal plasma.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe hereditary skin disease, caused by mutations in the COL7A1. However, whether non-invasive prenatal testing (NIPT) can be used for this monogenic genodermatosis remains unknown. Accordingly, we conducted a study in which one couple at high risk of having a fetus with RDEB were recruited and tested by haplotyping-based NIPT. Next-generation sequencing-based multi-gene panel testing was carried out in this couple and their first child as proband who was affected with RDEB. We deduced parental haplotypes via single nucleotide polymorphism (SNP)-based haplotype linkage analysis. Then the maternal plasma cell-free DNA was also sequenced to determine the fetal haplotypes using a parental haplotype-assisted hidden Markov model (HMM) analysis. Results show that the fetus was only a heterozygous mutation carrier in COL7A1 and the identical results were obtained after birth. These results demonstrate that haplotyping-based NIPT is a feasible method for NIPT of RDEB.

Diagnosis of hydatidiform moles using circulating gestational trophoblasts isolated from maternal blood

IntroductionIdentifying hydatidiform moles (HMs) is crucial due to the risk of gestational trophoblastic neoplasia. When a HM is suspected on clinical findings, surgical termination is recommended. However, in a substantial fraction of the cases, the conceptus is actually a non-molar miscarriage. If distinction between molar and non-molar gestations could be obtained before termination, surgical intervention could be minimized. MethodsCirculating gestational trophoblasts (cGTs) were isolated from blood from 15 consecutive women suspected of molar pregnancies in gestational week 6–13. The trophoblasts were individually sorted using fluorescence activated cell sorting. STR analysis targeting 24 loci was performed on DNA isolated from maternal and paternal leukocytes, chorionic villi, cGTs, and cfDNA. ResultsWith a gestational age above 10 weeks, cGTs were isolated in 87% of the cases. Two androgenetic HMs, three triploid diandric HMs, and six conceptuses with diploid biparental genome were diagnosed using cGTs. The STR profiles in cGTs were identical to the profiles in DNA from chorionic villi. Eight of the 15 women suspected to have a HM prior to termination had a conceptus with a diploid biparental genome, and thus most likely a non-molar miscarriage. DiscussionGenetic analysis of cGTs is superior to identify HMs, compared to analysis of cfDNA, as it is not hampered by the presence of maternal DNA. cGTs provide information about the full genome in single cells, facilitating estimation of ploidy. This may be a step towards differentiating HMs from non-HMs before termination.

Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods: This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results: Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy. Conclusion: This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.

Genome-wide analysis facilitates estimation of the amount of male contribution in meiotic gynogenetic three-spined stickleback (Gasterosteus aculeatus).

Gynogenetic embryos - those inheriting only maternal DNA - can be experimentally created by fertilizing eggs with radiation-treated sperm containing inactivated paternal chromosomes. Diploidy in the zygotes can be maintained through prevention of the second meiosis or restored by preventing the first mitosis after the maternal chromosome complement has been replicated. These gynogenetic organisms are useful in many fields including aquaculture, evolutionary biology and genomics. Although gynogenetic organisms have been created in numerous species, the completeness of uni-parental inheritance has often been assumed rather than thoroughly quantified across the genome. Instead, when tests of uni-parental inheritance occur, they typically rely on well-studied genetically determined phenotypes that represent a very small sub-set of the genome. Only assessing small genomic regions for paternal inheritance leaves the question of whether some paternal contributions to offspring might still have occurred. In this study, the authors quantify the efficacy of creating gynogenetic diploid three-spined stickleback fish (Gasterosteus aculeatus). To this end, the authors mirrored previous assessments of paternal contribution using well-studied genetically determined phenotypes including sex and genetically dominant morphological traits but expanded on previous studies using dense restriction site-associated DNA sequencing (RAD-seq) markers in parents and offspring to assess paternal inheritance genome-wide. In the gynogenetic diploids, the authors found no male genotypes underlying their phenotypes of interest - sex and dominant phenotypic traits. Using genome-wide assessments of paternal contribution, nevertheless, the authors found evidence of a small, yet potentially important, amount of paternally "leaked" genetic material. The application of this genome-wide approach identifies the need for more widespread assessment of paternal contributions to gynogenetic animals and promises benefits for many aspects of aquaculture, evolutionary biology and genomics.