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Abnormal Offspring Syndrome
Abstract
Large offspring syndrome (LOS) refers to an overgrowth phenotype sometimes observed in cattle fetuses and newborns produced by the use of assisted reproductive technologies (ART). This chapter discusses the current knowledge on ART‐induced abnormal offspring syndrome (AOS). It also discusses findings from sheep as AOS has also been documented in this species, and from Beckwith–Wiedemann syndrome (BWS), a human congenital overgrowth condition which phenocopies AOS. In addition, the chapter presents evidence of spontaneous AOS in cattle and provides examples of financial loss incurred by the cattle industry as a result of this syndrome. As the causes of BWS at the molecular level have been largely attributed to epigenetic defects, which include alterations of DNA methylation and loss‐of‐imprinting, it discusses these epigenetic modifications/mechanisms. Early identification of AOS would allow early elective termination of the pregnancy in order to prevent the monetary losses and animal welfare concerns associated with AOS.
... For the purposes of this review, the condition will be referred to as LOS. Although LOS was first observed among offspring produced from IVP or somatic cell nuclear transfer (SCNT), we have recently reported that LOS is not exclusively a consequence of these technologies as spontaneous cases can occur (spontaneous LOS [SLOS]) (Li et al., 2019a andRivera et al., 2021). ...
... The human condition of BWS exhibits phenotypic similarities to LOS (Brioude et al., 2018;Li et al., 2019a;Rivera et al., 2021), with the main epigenetic dysregulations including loss of methylation at KvDMR1 (referred to as imprinting control region 2, ICR2), gain of methylation at H19/IGF2 ICR (ICR1), paternal uniparental disomy of 11p15, and mutation of the maternal CDKN1C gene on chromosome 11 (Brioude et al., 2018). Due to the phenotypic similarities to LOS, BWS is often used as a reference for characterizing LOS (Hori et al., 2010;Chen et al., 2013). ...
... The etiology of LOS is complex, and factors such as coculture with somatic cells, high levels of rumen-degradable nitrogen in the maternal diet, high levels of progesterone during early embryo development, asynchrony with the uterine environment, IVP, and cloning have been reported to promote its occurrence (Young et al., 1998;Rivera et al., 2021). Supplementation with serum or bovine serum albumin (BSA) during in vitro culture is also associated with LOS (Lazzari et al., 2002). ...
Large/abnormal Offspring Syndrome (LOS/AOS) is a congenital overgrowth condition of cattle and sheep, characterized by macrosomia, abdominal wall defects, organomegaly, difficulty to stand and suckle at parturition. The condition was first described as an exclusive consequence of assisted reproductive technologies, such as in vitro production and somatic cell nuclear transfer (cloning). However, we recently reported the spontaneous occurrence of this syndrome in cattle. The etiology of LOS is unclear, although the syndrome is an epigenetic condition characterized by multi-locus loss-of-imprinting, global dysregulation of small and long RNAs, changes in DNA methylation, and altered chromosomal architecture. These molecular and epigenetic changes affect biological pathways implicated in organ size, cell proliferation, cell survival, resulting in the phenotypes which characterize LOS. The lack of accurate tools for the prediction and diagnosis of LOS and the prevention of dystocia resulting from fetal overgrowth is a major concern for the dairy and beef industries. Furthermore, death of the calf and/or dam during calving adds animal welfare issues and affects the net income of the industry. An early diagnosis of LOS/AOS during gestation is critical to facilitate the decision-making process on whether to allow the pregnancy to continue or not in order to prevent harm to the dam as well as to provide producers with the timely necessary information to prepare for a difficult birth. The present review summarizes the definition, traits, incidence, and molecular characteristics of LOS to provide information and serve as a guide for future investigations regarding the early identification of LOS during pregnancy in cattle.
... Serum has been experimentally determined to cause LOS in sheep [16][17][18] and bovine offspring derived from embryos cultured in serum containing medium can develop LOS 2 . In addition, the syndrome can also occur in fetuses and calves derived from embryos cultured without serum supplementation 19,20 and, more recently, we have documented that this syndrome occurs spontaneously in cattle produced by natural or artificial insemination [21][22][23] . The latter is of interest as there is a similar loss-of-imprinting overgrowth syndrome in humans, namely Beckwith-Wiedemann syndrome, which occurs naturally, and its incidence is increased in children conceived by assisted reproduction 24 . ...
... Even if the newborn calf survives the difficult birth, the enlarged tongue or extreme body weight make suckling difficult, thus increasing the chances of postnatal death 26 . In addition to the possible death of calves and cows, other financial losses are incurred due to veterinary costs 22 and the associated negative economic impact in terms of losses in milk, fat, and protein yields in the subsequent lactation 27,28 . For example, two independent LOS cases have been recently reported with total estimated losses of approximately $30,000 each 22 . ...
... In addition to the possible death of calves and cows, other financial losses are incurred due to veterinary costs 22 and the associated negative economic impact in terms of losses in milk, fat, and protein yields in the subsequent lactation 27,28 . For example, two independent LOS cases have been recently reported with total estimated losses of approximately $30,000 each 22 . These monetary losses could have been minimized if the early identification of LOS was possible. ...
In vitro production (IVP) of embryos in cattle can result in large/abnormal offspring syndrome (LOS/AOS) which is characterized by macrosomia. LOS can cause dystocia and lead to the death of dam and calf. Currently, no test exists to identify LOS pregnancies. We hypothesized that fetal ultrasonography and/or maternal blood markers are useful to identify LOS. Bovine fetuses were generated by artificial insemination (control) or IVP. Fetal ultrasonographies were taken on gestation D55 (D55) and fetal collections performed on D56 or D105 (gestation in cattle ≈ D280). IVP fetuses weighing ≥ 97 percentile of the control weight were considered LOS. Ultrasonography results show that the product of six D55 measurements can be used to identify extreme cases of LOS. To determine whether maternal blood can be used to identify LOS, leukocyte mRNA from 23 females was sequenced. Unsupervised hierarchical clustering grouped the transcriptomes of the two females carrying the two largest LOS fetuses. Comparison of the leukocyte transcriptomes of these two females to the transcriptome of all other females identified several misregulated transcripts on gestation D55 and D105 with LOC783838 and PCDH1 being misregulated at both time-points. Together our data suggest that LOS is identifiable during pregnancy in cattle.
... Large/abnormal offspring syndrome (LOS/AOS) is a congenital overgrowth syndrome that has been reported in ruminants [1,2]. Frequently observed features include macrosomia, macroglossia, umbilical hernia, organomegaly, placentomegaly, hydrallantois, increased gestation length, and increased dystocia rate [3][4][5][6][7][8][9][10][11][12][13]. ...
... ART is known to induce errors in the epigenome including DNA methylation and genomic imprinting to offspring in humans and ruminants [14,15]. We recently reported that LOS can occur spontaneously [2,16], a phenomenon that in some cases may have been incorrectly ascribed to the sire's genetics [17,18]. Currently, there is a lack of documented incidence for both spontaneous LOS (SLOS) and ART associated LOS (ART-LOS) from the industry, although those experiencing them in their farm or practice incur steep financial losses [2]. ...
... We recently reported that LOS can occur spontaneously [2,16], a phenomenon that in some cases may have been incorrectly ascribed to the sire's genetics [17,18]. Currently, there is a lack of documented incidence for both spontaneous LOS (SLOS) and ART associated LOS (ART-LOS) from the industry, although those experiencing them in their farm or practice incur steep financial losses [2]. ...
Large/abnormal offspring syndrome (LOS/AOS) is a congenital overgrowth syndrome reported in ruminants produced by assisted reproduction (ART-LOS) which exhibit global disruption of the epigenome and transcriptome. LOS/AOS shares phenotypes and epigenotypes with the human congenital overgrowth condition Beckwith-Wiedemann syndrome. We have reported that LOS occurs spontaneously (SLOS), however, to date, no study has been conducted to determine if SLOS has the same methylome epimutations as ART-LOS. In this study, we performed whole genome bisulfite sequencing to examine global DNA methylation in SLOS and ART-LOS tissues. We observed unique patterns of global distribution of differentially methylated regions (DMRs) over different genomic contexts, such as promoters, CpG islands, shores and shelves, as well as at repetitive sequences. In addition, we included data from two previous LOS studies to identify shared vulnerable genomic loci in LOS. Overall, we identified 320 genomic loci in LOS that have alterations in DNA methylation when compared to controls. Specifically, there are 25 highly vulnerable loci that could potentially serve as molecular markers for the diagnosis of LOS, including at the promoters of DMRT2 and TBX18, at the imprinted gene bodies of IGF2R, PRDM8, and BLCAP/NNAT, and at multiple CpG islands. We also observed tissue-specific DNA methylation patterns between muscle and blood, and conservation of ART-induced DNA methylation changes between muscle and blood. We conclude that as ART-LOS, SLOS is an epigenetic condition. In addition, SLOS and ART-LOS share similarities in methylome epimutations.
... Large/abnormal offspring syndrome (LOS/AOS) is a naturally occurring congenital overgrowth syndrome in ruminants and its incidence increases with the use of assisted reproductive technologies (ART) (Rivera et al., 2021). Frequently observed abnormal phenotypes in LOS include macrosomia, omphalocele, and abnormal organ development (Rivera et al., 2021). ...
... Large/abnormal offspring syndrome (LOS/AOS) is a naturally occurring congenital overgrowth syndrome in ruminants and its incidence increases with the use of assisted reproductive technologies (ART) (Rivera et al., 2021). Frequently observed abnormal phenotypes in LOS include macrosomia, omphalocele, and abnormal organ development (Rivera et al., 2021). Gestational problems associated with the dam include dystocia and in some cases fetal and/or maternal death (Rivera et al., 2021). ...
... Frequently observed abnormal phenotypes in LOS include macrosomia, omphalocele, and abnormal organ development (Rivera et al., 2021). Gestational problems associated with the dam include dystocia and in some cases fetal and/or maternal death (Rivera et al., 2021). In humans, a similar congenital overgrowth syndrome, Beckwith-Wiedemann syndrome (BWS, OMIM #130650), shares phenotypes and molecular aberrations with LOS (Brioude et al., 2018;Li et al., 2019a). ...
Large offspring syndrome (LOS) and Beckwith-Wiedemann syndrome are a similar epigenetic congenital overgrowth conditions in ruminants and humans, respectively. We have reported global loss-of-imprinting, methylome epimutations, and gene misregulation in LOS. However, less than 4% of gene misregulation can be explained with short range (<20kb) alterations in DNA methylation. Therefore, we hypothesized that methylome epimutations in LOS affect chromosome architecture which results in misregulation of genes located at distances >20kb in cis and in trans (other chromosomes). Our analyses focused on two imprinted domains that frequently show misregulation in these syndromes, namely KvDMR1 and IGF2R. Using bovine fetal fibroblasts, we identified CTCF binding at IGF2R imprinting control region but not KvDMR1, and allele-specific chromosome architecture of these domains in controls. In LOS, analyses identified erroneous long-range contacts and clustering tendency in the direction of expression of misregulated genes. In conclusion, altered chromosome architecture is associated with LOS.
Genomic imprinting is an epigenetically-regulated process of central importance in mammalian development and evolution. It involves multiple levels of regulation, with spatio-temporal heterogeneity, leading to the context-dependent and parent-of-origin specific expression of a small fraction of the genome. Genomic imprinting studies have therefore been essential to increase basic knowledge in functional genomics, evolution biology and developmental biology, as well as with regard to potential clinical and agrigenomic perspectives. Here we offer an overview on the contribution of livestock research, which features attractive resources in several respects, for better understanding genomic imprinting and its functional impacts. Given the related broad implications and complexity, we promote the use of such resources for studying genomic imprinting in a holistic and integrative view. We hope this mini-review will draw attention to the relevance of livestock genomic imprinting studies and stimulate research in this area.
In cattle, vitrified/warmed (V/W) and frozen/thawed (F/T), in vitro-produced (IVP) embryos, differ in their physiology and survival from fresh embryos. In this study, we analyzed the effects of embryo cryopreservation techniques on the offspring. IVP embryos cultured with albumin and with or without 0.1% serum until Day 6, and thereafter in single culture without protein, were transferred to recipients on Day 7 as F/T, V/W, or fresh, resulting in N = 24, 14, and 13 calves, respectively. Calves were clinically examined at birth, and blood was analyzed before and after colostrum intake (Day 0), and subsequently on Day 15 and Day 30. On Day 0, calves from V/W and F/T embryos showed increased creatinine and capillary refill time (CRT) and reduced heartbeats. Calves from F/T embryos showed lower PCO2, hemoglobin, and packed cell volume than calves from V/W embryos while V/W embryos led to calves with increased Na⁺ levels. Colostrum effects did not differ between calves from fresh and cryopreserved embryos, indicating similar adaptive ability among calves. However, PCO2 did not decrease in calves from V/W embryos after colostrum intake. Serum in culture led to calves with affected (P < 0.05) temperature, CRT, HCO3-, base excess (BE), TCO2, creatinine, urea, and anion gap. On Day 15, the effects of embryo cryopreservation disappeared among calves. In contrast, Day 30 values were influenced by diarrhea appearance, mainly in calves from V/W embryos (i.e., lower values of TCO2, HCO3-, and BE; and increased glucose, anion gap, and lactate), although with no more clinical compromise than calves from fresh and F/T embryos. Diarrhea affected PCO2 and Na⁺ in all groups. Embryo cryopreservation, and/or culture, yield metabolically different calves, including effects on protein and acid–base metabolism.
This review is intended to draw attention to the importance of the culture media composition on the health of the embryos, fetuses, newborns, and adults derived from assisted reproductive technologies (ART). Although current research and industry trends are to use chemically defined media because of their suitability for manufacturing, commercialization, and regulatory purposes, compelling evidence indicates that those media fail to adequately account for the biological demands of early embryogenesis. Here, we list the main undesirable consequences of the ART described in the literature and results we and others have obtained over the past decade exploring an alternative and more natural way to support embryo growth in vitro: inclusion of endogenous reproductive fluids as additives in the ART culture media for pigs, cows, and humans. This review systematically assesses the pros and cons of using reproductive fluid additives, as well as the requirements to implement this approach in the future.
The use of assisted reproductive technologies (ART) in cattle can result in large/abnormal offspring syndrome (LOS/AOS) which is characterized by macrosomia. LOS can cause dystocia and lead to the death of dam and calf. Currently, no test exists to identify LOS pregnancies. We hypothesized that fetal ultrasonography and/or maternal blood markers are useful to identify LOS. Bovine fetuses were generated by artificial insemination (control) or ART. Fetal ultrasonographies were taken on gestation day 55 (D55) and fetal collections performed on D56 or D105 (gestation in cattle ≈280 days). ART fetuses weighing ≥97 percentile of the control weight were considered LOS. Ultrasonography results show that the product of six D55 measurements can be used to identify extreme cases of LOS. To determine whether maternal blood can be used to identify LOS, leukocyte mRNA from 23 females was sequenced. Unsupervised hierarchical clustering grouped the transcriptomes of the two females carrying the two largest LOS fetuses. Comparison of the leukocyte transcriptomes of these two females to the transcriptome of all other females identified several misregulated transcripts on gestation D55 and D105 with LOC783838 and PCDH1 being misregulated at both time-points. Together our data suggest that LOS is identifiable during pregnancy in cattle.
The use of assisted reproductive technologies (ART) in cattle can result in large/abnormal offspring syndrome (LOS/AOS) which is characterized by macrosomia. LOS can cause dystocia and lead to the death of dam and calf. Currently, no test exists to identify LOS pregnancies. We hypothesized that fetal ultrasonography and/or maternal blood markers are useful to identify LOS. Bovine fetuses were generated by artificial insemination (control) or IVP. Fetal ultrasonographies were taken on gestation day 55 (D55) and fetal collections performed on D56 or D105 (gestation in cattle ~280 days). ART fetuses weighing ~97 percentile of the control weight were considered LOS. Ultrasonography results show that the product of six D55 measurements can be used to identify extreme cases of LOS. To determine whether maternal blood can be used to identify LOS, leukocyte mRNA from 23 females was sequenced. Unsupervised hierarchical clustering grouped the transcriptomes of the two females carrying the two largest LOS fetuses. Comparison of the leukocyte transcriptomes of these two females to the transcriptome of all other females identified several misregulated transcripts on gestation D55 and D105 with LOC783838 and PCDH1 being misregulated at both time-points. Together our data suggest that LOS is identifiable during pregnancy in cattle.
The increasing use of in vitro embryo production (IVP) followed by embryo transfer (ET), alongside with cryopreservation of embryos, has risen concerns regarding the possible altered pregnancy rates, calving or even neonatal mortality. One of the hypotheses for these alterations is the current culture conditions of the IVP. In an attempt to better mimic the physiological milieu, embryos were produced with female reproductive fluids (RF) as supplements to culture medium, and another group of embryos were supplemented with bovine serum albumin (BSA) as in vitro control. Embryos were cryopreserved and transferred while, in parallel, an in vivo control (artificial insemination, AI) with the same bull used for IVP was included. An overview on pregnancy rates, recipients’ hormonal levels, parturition, and resulting calves were recorded. Results show much similarity between groups in terms of pregnancy rates, gestation length and calves’ weight. Nonetheless, several differences on hormonal levels were noted between recipients carrying AI embryos especially when compared to BSA. Some calving issues and neonatal mortality were observed in both IVP groups. In conclusion, most of the parameters studied were similar between both types of IVP derived embryos and the in vivo-derived embryos, suggesting that the IVP technology used was efficient enough for the safe production of calves.
Imprinted genes mediate fetal and childhood growth and development, and early growth patterns drive fetal programming effects. However, predictions and evidence from the kinship theory of imprinting have yet to be directly integrated with data on fetal programming and risks of metabolic disease. I first define paternal-gene and maternal-gene optima with regard to early human growth and development. Next, I review salient evidence with regard to imprinted gene effects on birth weight, body composition, trajectories of feeding and growth, and timing of developmental stages, to evaluate why and how imprinted gene expression influences risks of metabolic disease in later life. I find that metabolic disease risks derive primarily from maternal gene biases that lead to reduced placental efficacy, low birth weight, low relative muscle mass, high relative white fat, increased abdominal adiposity, reduced pancreatic β-cell mass that promotes insulin resistance, reduced appetite and infant sucking efficacy, catch-up fat deposition from family foods after weaning, and early puberty. Paternal gene biases, by contrast, may contribute to metabolic disease via lower rates of brown fat thermiogenesis, and through favoring more rapid postnatal catch-up growth after intrauterine growth restriction from environmental causes. These disease risks can be alleviated through dietary and pharmacological alterations that selectively target imprinted gene expression and relevant metabolic pathways. The kinship theory of imprinting, and mother-offspring conflict more generally, provide a clear predictive framework for guiding future research on fetal programming and metabolic disease.
The use of assisted reproductive technologies (ART) can induce a congenital overgrowth condition in humans and ruminants, namely Beckwith-Wiedemann syndrome (BWS) and large offspring syndrome (LOS), respectively. Shared phenotypes and epigenotypes have been found between BWS and LOS. We have observed global misregulation of transcripts in bovine foetuses with LOS. microRNAs (miRNAs) are important post-transcriptional gene expression regulators. We hypothesize that there is miRNA misregulation in LOS and that this misregulation is shared with BWS. In this study, small RNA sequencing was conducted to investigate miRNA expression profiles in bovine and human samples. We detected 407 abundant known miRNAs and predicted 196 putative miRNAs from the bovine sequencing results and identified 505 abundant miRNAs in human tongue. Differentially expressed miRNAs (DE-miRNAs) were identified between control and LOS groups in all tissues analysed as well as between BWS and control human samples. DE-miRNAs were detected from several miRNA clusters including DLK1-DIO3 genomic imprinted cluster in LOS and BWS. DNA hypermethylation was associated with downregulation of miRNAs in the DLK1-DIO3. mRNA targets of the DE-miRNAs were predicted and signalling pathways associated with control of organ size (including the Hippo signalling pathway), cell proliferation, apoptosis, cell survival, cell cycle, and cell adhesion were found to be enriched with these genes. Yes associated protein 1 (YAP1) is the core effector of the Hippo signalling pathway, and increased level of active (non-phosphorylated) YAP1 protein was detected in skeletal muscle of LOS foetuses. Overall, our data provide evidence of miRNA misregulation in LOS and BWS.
Large offspring syndrome (LOS) is a fetal overgrowth condition in bovines most often observed in offspring conceived with the use of assisted reproductive technologies (ART). Phenotypes observed in LOS include, overgrowth, enlarged tongues, umbilical hernias, muscle and skeleton malformations, abnormal organ growth and placental development. Although LOS cases have only been reported to be associated with ART, fetal overgrowth can occur spontaneously in cattle (S-LOS). S-LOS refers to oversized calves that are born at normal gestation lengths. ART-induced LOS has been characterized as an epigenetic syndrome, more specifically, a loss-of-imprinting condition. We propose that S-LOS is also a loss-of-imprinting condition.
Beckwith-Wiedemann syndrome (BWS) is caused due to the disturbance of imprinted genes at chromosome 11p15. The molecular confirmation of this syndrome is possible in approximately 85% of the cases, whereas in the remaining 15% of the cases, the underlying defect remains unclear. The goal of our research was to identify new epigenetic loci related to BWS. We studied a group of 25 patients clinically diagnosed with BWS but without molecular conformation after DNA diagnostics and performed a whole genome methylation analysis using the HumanMethylation450 Array (Illumina).
We found hypermethylation throughout the methylome in two BWS patients. The hypermethylated sites in these patients overlapped and included both non-imprinted and imprinted regions. This finding was not previously described in any BWS-diagnosed patient.
Furthermore, one BWS patient exhibited aberrant methylation in four maternally methylated regions—IGF1R, NHP2L1, L3MBTL, and ZDBF2—that overlapped with the differentially methylated regions found in BWS patients with multi-locus imprinting disturbance (MLID). This finding suggests that the BWS phenotype can result from MLID without detectable methylation defects in the primarily disease-associated loci (11p15). Another patient manifested small but significant aberrant methylation in disease-associated loci at 11p near H19, possibly confirming the diagnosis in this patient.
DNA methylation is an important epigenetic modification that undergoes dynamic changes in mammalian embryogenesis, during which both parental genomes are reprogrammed. Despite the many immunostaining studies that have assessed global methylation, the gene-specific DNA methylation patterns in bovine preimplantation embryos are unknown. Using reduced representation bisulfite sequencing, we determined genome-scale DNA methylation of bovine sperm and individual in vivo developed oocytes and preimplantation embryos. We show that: 1) the major wave of genome-wide demethylation was completed by the 8-cell stage; 2) promoter methylation was significantly and inversely correlated with gene expression at the 8-cell and blastocyst stages; 3) sperm and oocytes have numerous differentially methylated regions (DMRs)—DMRs specific for sperm were strongly enriched in long terminal repeats (LTRs) and rapidly lost methylation in embryos; while, the oocyte-specific DMRs were more frequently localized in exons and CpG islands (CGIs) and demethylated gradually across cleavage stages; 4) DMRs were also found between in vivo and in vitro matured oocytes; and 5) differential methylation between bovine gametes was confirmed in some but not all known imprinted genes. Our data provide insights into the complex epigenetic reprogramming of bovine early embryos, which serve as an important model for human preimplantation development.
Background
Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. ResultsOf the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. Conclusion
By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.
Beckwith–Wiedemann syndrome (BWS), a human genomic imprinting disorder, is characterized by phenotypic variability that might include overgrowth, macroglossia, abdominal wall defects, neonatal hypoglycaemia, lateralized overgrowth and predisposition to embryonal tumours. Delineation of the molecular defects within the imprinted 11p15.5 region can predict familial recurrence risks and the risk (and type) of embryonal tumour. Despite recent advances in knowledge, there is marked heterogeneity in clinical diagnostic criteria and care. As detailed in this Consensus Statement, an international consensus group agreed upon 72 recommendations for the clinical and molecular diagnosis and management of BWS, including comprehensive protocols for the molecular investigation, care and treatment of patients from the prenatal period to adulthood. The consensus recommendations apply to patients with Beckwith–Wiedemann spectrum (BWSp), covering classical BWS without a molecular diagnosis and BWS-related phenotypes with an 11p15.5 molecular anomaly. Although the consensus group recommends a tumour surveillance programme targeted by molecular subgroups, surveillance might differ according to the local health-care system (for example, in the United States), and the results of targeted and universal surveillance should be evaluated prospectively. International collaboration, including a prospective audit of the results of implementing these consensus recommendations, is required to expand the evidence base for the design of optimum care pathways.
Embryo culture and assisted reproductive technologies have been associated with a disproportionately high number of epigenetic abnormalities in the resulting offspring. However, the mechanisms by which these techniques influence the epigenome remain poorly defined. In this study, we evaluated the capacity of oxygen concentration to influence the transcriptional control of a selection of key enzymes regulating chromatin structure. In mouse embryonic stem cells, oxygen concentrations modulated the transcriptional regulation of the TET family of enzymes, as well as the de novo methyltransferase Dnmt3a. These transcriptional changes were associated with alterations in the control of multiple imprinted genes, including H19, Igf2, Igf2r, and Peg3. Similarly, exposure of in vitro produced bovine embryos to atmospheric oxygen concentrations was associated with disruptions in the transcriptional regulation of TET1, TET3 and DNMT3a, along with the DNA methyltransferase co-factor HELLS. In addition, exposure to high oxygen was associated with alterations in the abundance of transcripts encoding members of the Polycomb repressor complex (EED and EZH2), the histone methyltransferase SETDB1 and multiple histone demethylases (KDM1A, KDM4B and KDM4C). These disruptions were accompanied by a reduction in embryo viability and suppression of the pluripotency genes NANOG and SOX2. These experiments demonstrate that oxygen has the capacity to modulate the transcriptional control of chromatin modifying genes involved in the establishment and maintenance of both pluripotency and genomic imprinting.
Assisted reproductive therapies (ART) have become increasingly common worldwide and numerous retrospective studies have indicated that ART-conceived children are more likely to develop the overgrowth syndrome Beckwith-Wiedemann (BWS). In bovine, the use of ART can induce a similar overgrowth condition, which is referred to as large offspring syndrome (LOS). Both BWS and LOS involve misregulation of imprinted genes. However, it remains unknown whether molecular alterations at non-imprinted loci contribute to these syndromes. Here we examined the transcriptome of skeletal muscle, liver, kidney, and brain of control and LOS bovine fetuses and found that different tissues within LOS fetuses have perturbations of distinct gene pathways. Notably, in skeletal muscle, multiple pathways involved in myoblast proliferation and fusion into myotubes are misregulated in LOS fetuses. Further, characterization of the DNA methylome of skeletal muscle demonstrates numerous local methylation differences between LOS and controls; however, only a small percent of differentially expressed genes (DEGs), including the imprinted gene IGF2R, could be associated with the neighboring differentially methylated regions. In summary, we not only show that misregulation of non-imprinted genes and loss-of-imprinting characterize the ART-induced overgrowth syndrome but also demonstrate that most of the DEGs is not directly associated with DNA methylome epimutations.
Background
Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts. ResultsBovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological (“BASAL”) concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological (“HIGH COMBI”) concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. Conclusions
Overall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.
DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner.
Reproductive health problems are one of the most important problems that affect the production and productivity of dairy cows. Dystocia is also one of the reproductive health problems that cause considerable economic loss to the dairy industry. The main aims of this manuscript were to review risk factors and economic impact of dystocia in dairy cows. Dystocia is also defined as prolonged or difficult parturition and it is a condition in which the first or, especially the second stage of parturition was markedly prolonged for more than 6 hr and the cow required assistance. There are different causes and risk factors associated with dystocia in dairy cattle which can result from both maternal and foetal factors. Breed parity, weight and condition of cow at calving, sex and birth weight of calf, malpresentation, multiple calving can be a risk factors for dystocia. Foeto-pelvic incompatibility is the major reason leading to difficulty at calving. However, dystocia can result from other causes that interfere with the expulsive forces needed to expel the calf. This includes: lack of uterine contractions (weak labour), incomplete dilation of the cervix and vagina due to stenosis and uterine torsion. Dystocia has negative impacts on the farm, the cow and the calf. It is an undesirable reproductive event resulting in increased risk of calf morbidity and mortality, reduced fertility and milk production, as well as cow survival and consequently reduces farm profitability on dairy farms. Moreover, dystocia may have negative effects on reproductive performance, causing stillbirth, cow death, retained placenta, uterine infections, or increased involuntary culling, which have negative consequences for farm economics as well as for cow welfare. Thus to avoid its adverse effect every dairy should implement a dystocia monitoring program and employ management practices that limit the occurrence and impact of dystocia.
Genomic imprinting is an epigenetic mechanism that leads to parental-allele-specific gene expression. Approximately 150 imprinted genes have been identified in humans and mice but less than 30 have been described as imprinted in cattle. For the purpose of de novo identification of imprinted genes in bovine, we determined global monoallelic gene expression in brain, skeletal muscle, liver, kidney and placenta of day ∼105 Bos taurus indicus × Bos taurus taurus F1 conceptuses using RNA sequencing. To accomplish this, we developed a bioinformatics pipeline to identify parent-specific single nucleotide polymorphism alleles after filtering adenosine to inosine (A-to-I) RNA editing sites. We identified 53 genes subject to monoallelic expression. Twenty three are genes known to be imprinted in the cow and an additional 7 have previously been characterized as imprinted in human and/or mouse that have not been reported as imprinted in cattle. Of the remaining 23 genes, we found that 10 are uncharacterized or unannotated transcripts located in known imprinted clusters, whereas the other 13 genes are distributed throughout the bovine genome and are not close to any known imprinted clusters. To exclude potential cis-eQTL effects on allele expression, we corroborated the parental specificity of monoallelic expression in day 86 Bos taurus taurus × Bos taurus taurus conceptuses and identified eight novel bovine imprinted genes. Further, we identified 671 candidate A-to-I RNA editing sites and describe random X-inactivation in day 15 bovine extraembryonic membranes. Our results expand the imprinted gene list in bovine and demonstrate that monoallelic gene expression can be the result of cis-eQTL effects.
Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.
Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.
In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use Piwi-interacting RNA (piRNA) guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here, we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2 deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 is, suggesting that the proteins have independent roles in establishing DNA methylation patterns.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
We have previously shown that, while the intrinsic quality of the oocyte is the main factor affecting blastocyst yield during bovine embryo development in vitro, the main factor affecting the quality of the blastocyst is the postfertilization culture conditions. Therefore, any improvement in the quality of blastocysts produced in vitro is likely to derive from the modification of the postfertilization culture conditions. The objective of this study was to examine the effect of the presence or absence of serum and the concentration of BSA during the period of embryo culture in vitro on 1) cleavage rate, 2) the kinetics of embryo development, 3) blastocyst yield, and 4) blastocyst quality, as assessed by cryotolerance and gene expression patterns. The quantification of all gene transcripts was carried out by real-time quantitative reverse transcription-polymerase chain reaction. Bovine blastocysts from four sources were used: 1) in vitro culture in synthetic oviduct fluid (SOF) supplemented with 3 ...
The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.
Simple Summary
High calf loss rates are an international welfare problem though this is often not recognised. These loss rates have increased in recent years. Improvement in calf survival rates is dependent upon re-prioritization of this problem relative to other animal health and welfare issues and creation of awareness of this prioritization. Once the problem is recognised action needs to be taken at national and at farm levels, specifically on problem farms. Data recording, research, breeding, veterinary, extension and farmer organisations all have a role to play in improving bovine neonatal survival and hence improving animal welfare in the future.
Abstract
Poor bovine neonatal survival rates are an international animal welfare issue. The key modifiable risk factors associated with such loss are age at first calving in primiparae, calf breed, gender and gestation length and calving management. The primary causes of mortality in the perinatal period are calving problems, in particular dystocia, defined as both difficult and abnormal calvings. Calf loss rates are rising on modern dairy farms in many countries internationally. High calf loss rates are often not recognised at national or at farm-level; recording needs to be improved. Improving bovine neonatal survival requires re-prioritization of this issue. Stakeholders need to be made cognisant of this prioritization. Actions to effect change need to occur at both national and farm-levels. National-level actions need firstly to address raising awareness of the issue. Farm-level actions need to focus on identifiable problem farms through targeted surveillance. Application of existing knowledge to alter modifiable risk factors is the key to improving calf welfare in the future. Research also has a role to play in filling knowledge gaps in particular about the ‘unexplained stillbirth’.
Beckwith-Wiedemann syndrome (BWS) is characterized by cancer predisposition, overgrowth and highly variable association of macroglossia, abdominal wall defects, nephrourological anomalies, nevus flammeus, ear malformations, hypoglycemia, hemihyperplasia, and organomegaly. BWS molecular defects, causing alteration of expression or activity of the genes regulated by two imprinting centres (IC) in the 11p15 chromosomal region, are also heterogeneous. In this paper we define (epi)genotype-phenotype correlations in molecularly confirmed BWS patients. The characteristics of 318 BWS patients with proven molecular defect were compared among the main four molecular subclasses: IC2 loss of methylation (IC2-LoM, n=190), IC1 gain of methylation (IC1-GoM, n=31), chromosome 11p15 paternal uniparental disomy (UPD, n=87), and cyclin-dependent kinase inhibitor 1C gene (CDKN1C) variants (n=10). A characteristic growth pattern was found in each group; neonatal macrosomia was almost constant in IC1-GoM, postnatal overgrowth in IC2-LoM, and hemihyperplasia more common in UPD (P<0.001). Exomphalos was more common in IC2/CDKN1C patients (P<0.001). Renal defects were typical of UPD/IC1 patients, uretheral malformations of IC1-GoM cases (P<0.001). Ear anomalies and nevus flammeus were associated with IC2/CDKN1C genotype (P<0.001). Macroglossia was less common among UPD patients (P<0.001). Wilms' tumor was associated with IC1-GoM or UPD and never observed in IC2-LoM patients (P<0.001). Hepatoblastoma occurred only in UPD cases. Cancer risk was lower in IC2/CDKN1C, intermediate in UPD, and very high in IC1 cases (P=0.009). In conclusion, (epi)genotype-phenotype correlations define four different phenotypic BWS profiles with some degree of clinical overlap. These observations impact clinical care allowing to move toward (epi) genotype-based follow-up and cancer screening.European Journal of Human Genetics advance online publication, 22 April 2015; doi:10.1038/ejhg.2015.88.
Significance
Large offspring syndrome (LOS) is a fetal overgrowth condition that mimics the human syndrome Beckwith–Wiedemann. These conditions have been observed with higher incidence in offspring conceived with the use of assisted reproductive technologies and are believed to be the result of misregulation of a set of genes that are expressed only from the maternally or paternally inherited chromosomes. These genes are known as imprinted genes. In our study, we demonstrate that the kidney, brain, muscle, and liver of LOS fetuses show misregulation of multiple imprinted genes when compared with controls. Furthermore, we show that the magnitude of overgrowth in LOS fetuses correlates with the number of misregulated imprinted genes. Our results may help create diagnostics for these fetal syndromes.
Animal breeders have made widespread use of assisted reproductive technologies to accelerate genetic improvement programs aimed at obtaining more, better and cheaper food products. Selection approaches have traditionally focused on Mendel’s laws of inheritance using parental phenotypic characteristics and quantitative genetics approaches to choose the best parents for the next generation, regardless of their gender. However, apart from contributing DNA sequence variants, male and female gametes carry parental-specific epigenetic marks that play key roles during pre- and post-natal development and growth of the offspring. We herein review the epigenetic anomalies that are associated with artificial reproductive technologies in current use in animal breeding programs. For instance, we demonstrate that bovine embryos and fetuses derived by in vitro culture and somatic cell nuclear transfer show epigenetic anomalies in the differentially methylated regions controlling the expression of some imprinted genes. Although these genomic imprinting errors are undetected in the somatic tissues after birth, further research is warranted to examine potential germ cell transmission of epimutations and the potential risks of reproducing cattle using artificial reproductive technologies.
Genomic selection (GS) approaches, in combination with reproductive technologies, are revolutionizing the design and implementation of breeding programs in livestock species, particularly in cattle. GS leverages genomic readouts to provide estimates of breeding value early in the life of animals. However, the capacity of these approaches for improving genetic gain in breeding programs is limited by generation interval, the average age of an animal when replacement progeny are born. Here, we present a cost-effective approach that combines GS with reproductive technologies to reduce generation interval by rapidly producing high genetic merit calves.
In animals produced by assisted reproductive technologies, two abnormal phenotypes have been characterized. Large offspring syndrome (LOS) occurs in offspring derived from in vitro cultured embryos, and the abnormal clone phenotype includes placental and fetal changes. LOS is readily apparent in ruminants, where a large calf or lamb derived from in vitro embryo production or cloning may weigh up to twice the expected body weight. The incidence of LOS varies widely between species. When similar embryo culture conditions are applied to nonruminant species, LOS either is not as dramatic or may even be unapparent. Coculture with serum and somatic cells was identified in the 1990s as a risk factor for abnormal development of ruminant pregnancies. Animals cloned from somatic cells may display a combination of fetal and placental abnormalities that are manifested at different stages of pregnancy and postnatally. In highly interventional technologies, such as nuclear transfer (cloning), the incidence of abnormal offspring continues to be a limiting factor to broader application of the technique. This review details the breadth of phenotypes found in nonviable pregnancies, together with the phenotypes of animals that survive the transition to extrauterine life. The focus is on animals produced using in vitro embryo culture and nuclear transfer in comparison to naturally occurring phenotypes.
Background
Beckwith-Wiedemann syndrome (BWS), a congenital overgrowth disorder with variable expressivity and a predisposition to tumorigenesis, results from disordered expression and/or function of imprinted genes at chromosome 11p15.5. There are no generally agreed clinical diagnostic criteria, with molecular studies commonly performed to confirm diagnosis. In particular, methylation status analysis at two 11p15.5 imprinting control centres (IC1 and IC2) detects up to 80% of BWS cases (though low-level mosaicism may not be detected). In order to evaluate the relationship between the clinical presentation of suspected BWS and IC1/2 methylation abnormalities we reviewed the results of >1,000 referrals for molecular diagnostic testing.
Results
Out of 1,091 referrals, 507 (46.5%) had a positive diagnostic test for BWS. The frequency of tumours was 3.4% in those with a molecular diagnosis of BWS. Previously reported genotype-phenotype associations with paternal uniparental disomy, IC1, and IC2 epimutation groups were confirmed and potential novel associations detected. Predictive values of previously described clinical diagnostic criteria were compared and, although there were differences in their sensitivity and specificity, receiver operating characteristic (ROC) analysis demonstrated that these were not optimal in predicting 11p15.5 methylation abnormalities. Using logistic regression, we identified clinical features with the best predictive value for a positive methylation abnormality. Furthermore, we developed a weighted scoring system (sensitivity 75.9%, and specificity 81.8%) to prioritise patients presenting with the most common features of BWS, and ROC analysis demonstrated superior performance (area under the curve 0.85, 95% CI 0.83 to 0.87) compared to previous criteria.
Conclusions
We suggest that this novel tool will facilitate selection of patients with suspected BWS for routine diagnostic testing and so improve the diagnosis of the disorder.
Background
Macrosomia has been defined in various ways by obstetricians and researchers. The purpose of the present study was to search for a definition of macrosomia through an outcome-based approach.
Methods
In a study of 30,831,694 singleton term live births and 38,053 stillbirths in the U.S. Linked Birth-Infant Death Cohort datasets (1995–2004), we compared the occurrence of stillbirth, neonatal death, and 5-min Apgar score less than four in subgroups of birthweight (4000–4099 g, 4100–4199 g, 4200–4299 g, 4300–4399 g, 4400–4499 g, 4500–4999 g vs. reference group 3500–4000 g) and birthweight percentile for gestational age (90th–94th percentile, 95th-96th, and ≥97th percentile, vs. reference group 75th–90th percentile).
Results
There was no significant increase in adverse perinatal outcomes until birthweight exceeded the 97th percentile. Weight-specific odds ratios (ORs) elevated substantially to 2 when birthweight exceeded 4500 g in Whites. In Blacks and Hispanics, the aORs exceeded 2 for 5-min Apgar less than four when birthweight exceeded 4300 g. For vaginal deliveries, the aORs of perinatal morbidity and mortality were larger for most of the subgroups, but the patterns remained the same.
Conclusions
A birthweight greater than 4500 g in Whites, or 4300 g in Blacks and Hispanics regardless of gestational age is the optimal threshold to define macrosomia. A birthweight greater than the 97th percentile for a given gestational age, irrespective of race is also reasonable to define macrosomia. The former may be more clinically useful and simpler to apply.
Methylation of DNA is an essential epigenetic control mechanism in mammals. During embryonic development, cells are directed toward their future lineages, and DNA methylation poses a fundamental epigenetic barrier that guides and restricts differentiation and prevents regression into an undifferentiated state. DNA methylation also plays an important role in sex chromosome dosage compensation, the repression of retrotransposons that threaten genome integrity, the maintenance of genome stability, and the coordinated expression of imprinted genes. However, DNA methylation marks must be globally removed to allow for sexual reproduction and the adoption of the specialized, hypomethylated epigenome of the primordial germ cell and the preimplantation embryo. Recent technological advances in genome-wide DNA methylation analysis and the functional description of novel enzymatic DNA demethylation pathways have provided significant insights into the molecular processes that prepare the mammalian embryo for normal development.
Direct transfer (DT) of cryopreserved embryos to recipients facilitates on-farm application. We analyzed a new freezing/thawing (F/T) procedure for in vitro produced (IVP) embryos, integrating: 1) an ethylene-glycol based system; 2) a culture step without protein; and 3) a synthetic protein substitute (CRYO3) in cryopreservation medium. IVP embryos from abattoir ovaries were cultured in groups in BSA-containing synthetic oviduct fluid with or without 0.1% fetal calf serum (FCS) until Day-6. Morulae and early blastocysts were subsequently cultured without protein from Day-6 onwards. Day 7 and Day 8 expanded blastocysts (EXB) were subjected to F/T or vitrification/warming (V/W). Thawed and warmed EXB were cultured in vitro, and development rates, cell counts and dead cells were analyzed in surviving embryos. V/W improved survival over F/T (live and hatching rates at 2 h, 24 h and 48 h) (P < 0.0001), and FCS before Day 6 did not affect in vitro survival. After F/T, embryos had lower cell counts in the ICM, TE and total cells than after V/W. Day-7 embryos after F/T showed % apoptotic, % pycnotic and % total dead cells higher (p < 0.05) than their Day-8 counterparts, probably because F/T reduced the numbers of ICM cells within Day-8 embryos. Thereafter, Day-7 blastocysts were transferred to heifers in an experimental herd. There were no differences in birth rates with frozen (-FCS [n = 40]: 45%; +FCS [n = 14]: 28%), vitrified (-FCS [n = 47]: 53%; +FCS [n = 11]: 36%) and fresh (-FCS [n = 30]: 47%; +FCS [n = 17]: 53%) embryos. However, frozen embryos produced with FCS showed 5/9 miscarriages after Day-40. Calves born from frozen (n = 22), vitrified (n = 29) and fresh (n = 22) transfers did not differ in birth weight, gestation length and daily gain weight (P > 0.10). Subsequently, transfer of frozen embryos (n = 29) derived from oocytes collected from live, hormonally stimulated cows in experimental herd, led to pregnancy rates of 57% (heifers) and 40% (dry cows). with EXB on Day-62 Finally, embryos produced with BSA were transferred to cows in an on-field trial (frozen [n = 80]; fresh [n = 58]), with no differences in pregnancy rates (days 30-40). Pregnancy and birth rates could not be predicted from in vitro approaches. The new F/T system yields pregnancy and birth rates comparable to vitrified and fresh embryos without birth overweight. The absence of products of animal origin, defined chemical composition, and direct transfer entail sanitary, manufacturing and application advantages.
A protocol for production of bovine embryos from oocytes collected from ovaries obtained from an abattoir is described. The protocol includes methods for in vitro maturation of oocytes, capacitation of sperm, fertilization, and development of the resultant embryos to the blastocyst stage. The protocol can be easily modified to use oocytes collected by ultrasound-guided follicular aspiration.
Complex multicellular organisms, such as mammals, express two complete sets of chromosomes per nucleus, combining the genetic material of both parents. However, epigenetic studies have demonstrated violations to this rule that are necessary for mammalian physiology; the most notable parental allele expression phenomenon is genomic imprinting. With the identification of endogenous imprinted genes, genomic imprinting became well-established as an epigenetic mechanism in which the expression pattern of a parental allele influences phenotypic expression. The expanding study of genomic imprinting is revealing a significant impact on brain functions and associated diseases. Here, we review key milestones in the field of imprinting and discuss mechanisms and systems in which imprinted genes exert a significant role.
Objectives—This report presents 2016 data on U.S. births according to a wide variety of characteristics. Trends in fertility patterns and maternal and infant characteristics are described and interpreted. Methods—Descriptive tabulations of data reported on the birth certificates of the 3.95 million births that occurred in 2016 are presented. Data are presented for maternal age, live-birth order, race and Hispanic origin, marital status, tobacco use, prenatal care, source of payment for the delivery, method of delivery, gestational age, birthweight, and plurality. Selected data by mother’s state of residence and birth rates by age also are shown. Trend data for 2010–2016 are presented for selected items. Results—A total of 3,945,875 births were registered in the United States in 2016, down 1% from 2015. Compared with rates in 2015, the general fertility rate declined to 62.0 per 1,000 women aged 15–44. The birth rate for females aged 15–19 fell 9% in 2016. Birth rates declined for women in their 20s but increased for women in their 30s and early 40s. The total fertility rate declined to 1,820.5 births per 1,000 women in 2016. The birth rate for unmarried women declined, while the rate for married women increased. More than three-quarters of women began prenatal care in the first trimester of pregnancy (77.1%) in 2016, while 7.2% of all women smoked during pregnancy. The cesarean delivery rate declined for the fourth year in a row. Medicaid was the source of payment for 42.6% of all 2016 births. The preterm birth rate rose for the second straight year, and the rate of low birthweight increased 1%. Twin and triplet and higher-order multiple birth rates declined, although the changes were not statistically significant. © 2018, National Center for Health Statistics. All rights reserved.
Background and objectives:
The emerging association of assisted reproductive techniques (ART) with imprinting disorders represents a major issue in the scientific debate on infertility treatment and human procreation. We studied the prevalence of Beckwith-Wiedemann syndrome (BWS) in children conceived through ART to define the specific associated relative risk.
Methods:
Patients with BWS born in Piemonte, Italy, were identified and matched with the general demographic data and corresponding regional ART registry.
Results:
Between 2005 and 2014, live births in Piemonte were 379 872, including 7884 from ART. Thirty-eight patients with BWS were born, 7 from ART and 31 naturally conceived. BWS birth prevalence in the ART group was significantly higher than that of the naturally conceived group (1:1126 vs 1:12 254, P < .001). The absolute live birth risk in the ART group was 887.9 per 1 000 000 vs 83.3 per 1 000 000 in the naturally conceived group, providing a relative risk of 10.7 (95% confidence interval 4.7-24.2). During the 1997-2014 period, 67 patients were diagnosed with BWS out of 663 834 newborns (1:9908 live births). Nine out of the 67 BWS patients were conceived through ART (13.4%), and 8 were molecularly tested, with 4 having an imprinting center 2 loss of methylation, 2 with 11p15.5 paternal uniparental disomy, and 2 negative results.
Conclusions:
ART entails a 10-fold increased risk of BWS and could be implicated in the pathogenesis of genomic events besides methylation anomalies. These data highlight the need for awareness of ART-associated health risk.
p>Numerous variables can torpedo attempts to replicate cell experiments, from the batch of serum to the shape of growth plates. But there are ways to ensure reliability.</p
Commercial embryo transfer has evolved as an art and as a science since the early 1970s. Today's multiple ovulation embryo transfer is a widely used reproductive tool on many farms and is performed by veterinarians throughout the world. Propagation of the female genomes of select donors, through embryo transfer, has allowed a rapid progression of genetic gain in many breeds, much like what happened with artificial insemination since the 1940s. Advancement of this technology is migrating to in vitro fertilization technology today, allowing a higher volume of offspring to be produced with sex selection in the laboratory.
Beckwith-Wiedemann syndrome (BWS; OMIM #130650) is an overgrowth syndrome caused by different genetic or epigenetic alterations affecting imprinted regions on chromosome 11p15.5. Here we report a family with multiple offspring affected with BWS including giant omphalocoeles in which maternal transmission of a chromosomal rearrangement including an inversion and two deletions leads to hypomethylation of the imprint control region 2 (ICR2). As the deletion includes the promoter and 5' part of the KCNQ1 gene, we suggest that transcription of this gene may be involved in establishing the maternal methylation imprint of the ICR2, which is located in intron 10 of KCNQ1.European Journal of Human Genetics advance online publication, 3 February 2016; doi:10.1038/ejhg.2016.3.
Objective To determine the independent association of post-term pregnancy with neonatal outcome in low-risk newborns.
Design Retrospective cohort.
Setting Tertiary university-affiliated medical centre.
Patients All newborns of low-risk singleton pregnancies born at 39+0 to 44+0 weeks’ gestation over a 5-year period. Exclusion criteria: multiple gestation, maternal hypertensive disorder, diabetes or cholestasis, placental abruption or intrapartum fever (>38°C), small for gestational age (<10th centile) and major congenital or chromosomal anomalies.
Interventions None.
Outcome measures Admission to the neonatal intensive care unit (NICU), hospital length of stay, 5-min Apgar score, birth trauma, respiratory, neurological, metabolic and infectious morbidities and neonatal mortality. The adverse outcome rate was compared among three groups based on gestational age at birth: post-term (≥42+0 weeks), late term (41+0 to 41+6 weeks) and full term (39+0 to 40+6 weeks).
Results Of the 23 524 eligible neonates, 747 (3.2%) were born post-term, 4632 (19.7%) late term and 18 145 (77.1%) full term. Women in the post-term group versus the late-term group had a significantly higher rate of caesarean section (8.9% vs 5.6%, p<0.001) and operative vaginal delivery (9.6% vs 7.4%, p=0.024). Post-term pregnancy versus full-term pregnancy was associated with an increased risk of NICU admission (OR 2.0, 95% CI 1.4 to 2.8), respiratory morbidity (OR 2.2, 95% CI 1.3 to 3.8) and infectious morbidity (OR 1.88, 95% CI 1.32 to 2.69). Post-term pregnancy versus late-term pregnancy was similarly associated with an increased risk of NICU admission (OR 2.0, 95% CI 1.4 to 2.9), respiratory morbidity (OR 2.7, 95% CI 1.5 to 5.0) and infectious morbidity (OR 1.8, 95% CI 1.2 to 2.7) and with hypoglycaemia (OR 2.6, 95% CI 1.2 to 5.4). Post-term delivery was not associated with neonatal mortality.
Conclusions Post-term pregnancy is an independent risk factor for neonatal morbidity even in low-risk singleton pregnancies.
DNA and histone lysine methylation are dynamic chemical modifications that play a crucial role in the establishment of gene expression patterns during development. Both types of genomic methylation patterns are enzymatically regulated by the opposing activities of enzymes that introduce and remove these marks, known as methylation 'writers' and 'erasers', respectively. The appropriate localization and activity of these enzymes on chromatin is, in part, regulated by chromatin 'readers', protein modules that recognize histone and DNA modifications. Such reading modules are either encoded within the same polypeptide as the catalytic domains of writers and erasers, or present in protein partners that associate with them. Here, we review recent structural, biochemical and biological studies that demonstrate that there are multiple mechanisms by which reader domains can regulate the writers and erasers of histone and DNA methylation.
Abnormal placental development is frequent in nuclear transfer (NT) pregnancies and is likely to be associated with altered epigenetic reprogramming. In the present study, fetal and placental measurements were taken on Day 60 of gestation in cows with pregnancies produced by AI, IVF and NT. Placentas were collected and subjected to histological evaluation, the expression of genes important in trophoblast differentiation and expression of the placental imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2), as well as chromatin immunoprecipitation (ChIP) for histone marks within the promoter of PHLDA2. Fewer binucleated cells were observed in NT cotyledons, followed by IVF and AI cotyledons (P < 0.05). Expression of heart and neural crest derivatives expressed 1 (HAND1), placental lactogen (PL), pregnancy-associated glycoprotein 9 (PAG-9) and PHLDA2 was elevated in NT cotyledons compared with AI cotyledons. Expression of PHLDA2 was higher in IVF than AI samples (P < 0.05). ChIP revealed an increase in the permissive mark dimethylation of lysine 4 on histone H3 (H3K4me2), surprisingly associated with the silent allele of PHLDA2, and a decrease in the inhibitory mark H3K9me2 in NT samples. Thus, genes critical for placental development were altered in NT placentas, including an imprinted gene. Allele-specific changes in the permissive histone mark in the PHLDA2 promoter indicate misregulation of imprinting in clones. Abnormal trophoblast differentiation could have resulted in lower numbers of binucleated cells following NT. These results suggest that the altered expression of imprinted genes associated with NT are also caused by changes in histone modifications.
This chapter shows images of the various causes, anatomic relationships, and procedures. The most common cause of dystocia is fetopelvic disproportion, when the fetus is too large and/or the maternal pelvis is too small. Conceptus development following transfer of embryos from in vitro production (IVP) or somatic cell nucleus transfer (SCNT) in cattle may lead to early embryonic death, fetal death, or overgrown calves. Hybrids produced by the mating of an American bison bull with a domestic cow or heifer have an increased incidence of hydrops amnii. The primary predisposition to cervico-vaginal prolapse in cattle is elevated estrogen concentrations during the late third trimester. The prepubic tendon is the tendon of the rectus abdominis muscle that attaches to the cranial border of the pubis. Prolonged gestation is a syndrome in cattle that is characterized by failure of the cow to calve at the normal time.
Gamete and embryo manipulations can result in alterations to the epigenome, and are associated with altered gene expression. The initial objective of this study was to determine the transcript level of several epigenetic modifiers in embryos that had been cultured from the 2-cell stage until the late-blastocyst stage in four culture conditions. Cultured embryos were compared to control, in vivo-produced late blastocysts to ascertain if differences in gene expression existed among the culture conditions; none were observed. As all of the embryos used were produced in females that had undergone superovulation, we next compared the transcript level of the same epigenetic modifiers between superovulated, in vivo-produced embryos and embryos produced from natural ovulation. Following in vitro culturing, expression of the genes analyzed was increased in all superovulation groups. We therefore hypothesized that the superovulation procedure-used to increase the number of embryos obtained for experimentation-may have caused an inappropriate acquisition of epigenetic modifications in the maternal genome prior to ovulation, which in turn caused misexpression of genes at the blastocyst stage. To test this hypothesis, we compared the level of global DNA methylation and histone 3 lysine-9 or -14 acetylation in zygotes obtained by natural- or superovulation. Indeed, superovulation decreased global DNA methylation on the maternal pronucleus of zygotes, which inversely correlated with H3K9/14 acetylation. In conclusion, superovulation alters the epigenome of the oocyte, resulting in the dysregulation of gene expression at the blastocyst stage. Mol. Reprod. Dev. 2015. © 2015 Wiley Periodicals, Inc.
© 2015 Wiley Periodicals, Inc.
The word ‘epimutation’ is often used in a manner that can be misinterpreted. The strict definition of epimutation is a heritable change in gene activity that is not associated with a DNA mutation but rather with gain or loss of DNA methylation or other heritable modifications of chromatin. Unfortunately, there is a growing tendency in the cancer field to use the word in situations in which underlying DNA sequence changes have occurred.
Enhancers establish spatial or temporal patterns of gene expression that are critical for development, yet our understanding of how these DNA cis-regulatory elements function from a distance to increase transcription of their target genes and shape the cellular transcriptome has been gleaned primarily from studies of individual genes or gene families. High-throughput sequencing studies place enhancer-gene interactions within the 3D context of chromosome folding, inviting a new look at enhancer function and stimulating provocative new questions. Here, we integrate these whole-genome studies with recent mechanistic studies to illuminate how enhancers physically interact with target genes, how enhancer activity is regulated during development, and the role of noncoding RNAs transcribed from enhancers in their function.
Genes that are subject to genomic imprinting in mammals are preferentially expressed from a single parental allele. This imprinted expression of a small number of genes is crucial for normal development, as these genes often directly regulate fetal growth. Recent work has also demonstrated intricate roles for imprinted genes in the brain, with important consequences on behavior and neuronal function. Finally, new studies have revealed the importance of proper expression of specific imprinted genes in induced pluripotent stem cells and in adult stem cells. As we review here, these findings highlight the complex nature and developmental importance of imprinted genes.
The eukaryotic genome is organized in the three-dimensional nuclear space in a specific manner that is both a cause and a consequence of its function. This organization is partly established by a special class of architectural proteins, of which CCCTC-binding factor (CTCF) is the best characterized. Although CTCF has been assigned various roles that are often contradictory, new results now help to draw a unifying model to explain the many functions of this protein. CTCF creates boundaries between topologically associating domains in chromosomes and, within these domains, facilitates interactions between transcription regulatory sequences. Thus, CTCF links the architecture of the genome to its function.