Trophectoderm biopsy of blastocysts following IVF and embryo culture increases epigenetic dysregulation in a mouse model.

STUDY QUESTION: Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? SUMMARY ANSWER: TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. WHAT IS KNOWN ALREADY: Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. STUDY DESIGN, SIZE, DURATION: We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). PARTICIPANTS/MATERIALS, SETTING, METHODS: Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. LARGE SCALE DATA: Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. LIMITATIONS, REASONS FOR CAUTION: This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

CHK1-CDC25A-CDK1 regulate cell cycle progression and protect genome integrity in early mouse embryos.

After fertilization, remodeling of the oocyte and sperm genomes is essential to convert these highly differentiated and transcriptionally quiescent cells into early cleavage-stage blastomeres that are transcriptionally active and totipotent. This developmental transition is accompanied by cell cycle adaptation, such as lengthening or shortening of the gap phases G1 and G2. However, regulation of these cell cycle changes is poorly understood, especially in mammals. Checkpoint kinase 1 (CHK1) is a protein kinase that regulates cell cycle progression in somatic cells. Here, we show that CHK1 regulates cell cycle progression in early mouse embryos by restraining CDK1 kinase activity due to CDC25A phosphatase degradation. CHK1 kinase also ensures the long G2 phase needed for genome activation and reprogramming gene expression in two-cell stage mouse embryos. Finally, Chk1 depletion leads to DNA damage and chromosome segregation errors that result in aneuploidy and infertility.

KRAB-zinc-finger proteins regulate endogenous retroviruses to sculpt germline transcriptomes and genome evolution.

As transposable elements (TEs) coevolved with the host genome, the host genome exploited TEs as functional regulatory elements. What remains largely unknown are how the activity of TEs, namely, endogenous retroviruses (ERVs), are regulated and how TEs evolved in the germline. Here we show that KRAB domain-containing zinc-finger proteins (KZFPs), which are highly expressed in mitotically dividing spermatogonia, bind to suppressed ERVs that function following entry into meiosis as active enhancers. These features are observed for independently evolved KZFPs and ERVs in mice and humans, i.e., are evolutionarily conserved in mammals. Further, we show that meiotic sex chromosome inactivation (MSCI) antagonizes the coevolution of KZFPs and ERVs in mammals. Our study uncovers a mechanism by which KZFPs regulate ERVs to sculpt germline transcriptomes. We propose that epigenetic programming in the mammalian germline during the mitosis-to-meiosis transition facilitates coevolution of KZFPs and TEs on autosomes and is antagonized by MSCI.

New twists of a TAIL: novel insights into the histone binding properties of a highly conserved PHD finger cluster within the MLR family of H3K4 mono-methyltransferases.

Enhancer activation by the MLR family of H3K4 mono-methyltransferases requires proper recognition of histones for the deposition of the mono-methyl mark. MLR proteins contain two clusters of PHD zinc finger domains implicated in chromatin regulation. The second cluster is the most highly conserved, preserved as an ancient three finger functional unit throughout evolution. Studies of the isolated 3rd PHD finger within this cluster suggested specificity for the H4 [aa16-20] tail region. We determined the histone binding properties of the full three PHD finger cluster b module (PHDb) from the Drosophila Cmi protein which revealed unexpected recognition of an extended region of H3. Importantly, the zinc finger spacer separating the first two PHDb fingers from the third is critical for proper alignment and coordination among fingers for maximal histone engagement. Human homologs, MLL3 and MLL4, also show conservation of H3 binding, expanding current views of histone recognition for this class of proteins. We further implicate chromatin remodeling by the SWI/SNF complex as a possible mechanism for the accessibility of PHDb to globular regions of histone H3 beyond the tail region. Our results suggest a two-tail histone recognition mechanism by the conserved PHDb domain involving a flexible hinge to promote interdomain coordination.

OBOX regulates mouse zygotic genome activation and early development.

Zygotic genome activation (ZGA) activates the quiescent genome to enable the maternal-to-zygotic transition1,2. However, the identity of transcription factors that underlie mammalian ZGA in vivo remains elusive. Here we show that OBOX, a PRD-like homeobox domain transcription factor family (OBOX1-OBOX8)3-5, are key regulators of mouse ZGA. Mice deficient for maternally transcribed Obox1/2/5/7 and zygotically expressed Obox3/4 had a two-cell to four-cell arrest, accompanied by impaired ZGA. The Obox knockout defects could be rescued by restoring either maternal and zygotic OBOX, which suggests that maternal and zygotic OBOX redundantly support embryonic development. Chromatin-binding analysis showed that Obox knockout preferentially affected OBOX-binding targets. Mechanistically, OBOX facilitated the 'preconfiguration' of RNA polymerase II, as the polymerase relocated from the initial one-cell binding targets to ZGA gene promoters and distal enhancers. Impaired polymerase II preconfiguration in Obox mutants was accompanied by defective ZGA and chromatin accessibility transition, as well as aberrant activation of one-cell polymerase II targets. Finally, ectopic expression of OBOX activated ZGA genes and MERVL repeats in mouse embryonic stem cells. These data thus demonstrate that OBOX regulates mouse ZGA and early embryogenesis.

Epigenetic programming in the ovarian reserve.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades. The ovarian reserve consists of oocytes residing in primordial follicles arrested in meiotic prophase I and is maintained independent of DNA replication and cell proliferation, thereby lacking stem cell-based maintenance. Largely unknown is how cellular states of the ovarian reserve are established and maintained for decades. Our recent study revealed that a distinct chromatin state is established during ovarian reserve formation in mice, uncovering a novel window of epigenetic programming in female germline development. We showed that an epigenetic regulator, Polycomb Repressive Complex 1 (PRC1), establishes a repressive chromatin state in perinatal mouse oocytes that is essential for prophase I-arrested oocytes to form the ovarian reserve. Here we discuss the biological roles and mechanisms underlying epigenetic programming in ovarian reserve formation, highlighting current knowledge gaps and emerging research areas in female reproductive biology.

Histone remodeling reflects conserved mechanisms of bovine and human preimplantation development.

How histone modifications regulate changes in gene expression during preimplantation development in any species remains poorly understood. Using CUT&Tag to overcome limiting amounts of biological material, we profiled two activating (H3K4me3 and H3K27ac) and two repressive (H3K9me3 and H3K27me3) marks in bovine oocytes, 2-, 4-, and 8-cell embryos, morula, blastocysts, inner cell mass, and trophectoderm. In oocytes, broad bivalent domains mark developmental genes, and prior to embryonic genome activation (EGA), H3K9me3 and H3K27me3 co-occupy gene bodies, suggesting a global mechanism for transcription repression. During EGA, chromatin accessibility is established before canonical H3K4me3 and H3K27ac signatures. Embryonic transcription is required for this remodeling, indicating that maternally provided products alone are insufficient for reprogramming. Last, H3K27me3 plays a major role in restriction of cellular potency, as blastocyst lineages are defined by differential polycomb repression and transcription factor activity. Notably, inferred regulators of EGA and blastocyst formation strongly resemble those described in humans, as opposed to mice. These similarities suggest that cattle are a better model than rodents to investigate the molecular basis of human preimplantation development.

Embryonic microRNAs are essential for bovine preimplantation embryo development.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression after transcription. miRNAs are present in transcriptionally quiescent full-grown oocytes and preimplantation embryos that display a low level of transcription prior to embryonic genome activation. The role of miRNAs, if any, in preimplantation development is not known. The temporal pattern of expression of miRNAs during bovine preimplantation development was determined by small RNA-sequencing using eggs and preimplantation embryos (1-cell, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst). Embryos cultured in the presence of α-amanitin, which permitted the distinguishing of maternal miRNAs from embryonic miRNAs, indicated that embryonic miRNA expression was first detected at the two-cell stage but dramatically increased during the morula and blastocyst stages. Targeting DGCR8 by a small-interfering RNA/morpholino approach revealed a role for miRNAs in the morula-to-blastocyst transition. Knockdown of DGCR8 not only inhibited expression of embryonically expressed miRNAs but also inhibited the morula-to-blastocyst transition. In addition, RNA-sequencing identified an increased relative abundance of messenger RNAs potentially targeted by embryonic miRNAs in DGCR8-knockdown embryos when compared with controls. Results from these experiments implicate an essential role for miRNAs in bovine preimplantation embryo development.

PRC1-mediated epigenetic programming is required to generate the ovarian reserve.

The ovarian reserve defines the female reproductive lifespan, which in humans spans decades due to robust maintenance of meiotic arrest in oocytes residing in primordial follicles. Epigenetic reprogramming, including DNA demethylation, accompanies meiotic entry, but the chromatin changes that underpin the generation and preservation of ovarian reserves are poorly defined. We report that the Polycomb Repressive Complex 1 (PRC1) establishes repressive chromatin states in perinatal mouse oocytes that directly suppress the gene expression program of meiotic prophase-I and thereby enable the transition to dictyate arrest. PRC1 dysfuction causes depletion of the ovarian reserve and leads to premature ovarian failure. Our study demonstrates a fundamental role for PRC1-mediated gene silencing in female reproductive lifespan, and reveals a critical window of epigenetic programming required to establish ovarian reserve.

Placental Abnormalities are Associated With Specific Windows of Embryo Culture in a Mouse Model.

Assisted Reproductive Technologies (ART) employ gamete/embryo handling and culture in vitro to produce offspring. ART pregnancies have an increased risk of low birth weight, abnormal placentation, pregnancy complications, and imprinting disorders. Embryo culture induces low birth weight, abnormal placental morphology, and lower levels of DNA methylation in placentas in a mouse model of ART. Whether preimplantation embryos at specific stages of development are more susceptible to these perturbations remains unresolved. Accordingly, we performed embryo culture for several discrete periods of preimplantation development and following embryo transfer, assessed fetal and placental outcomes at term. We observed a reduction in fetal:placental ratio associated with two distinct windows of preimplantation embryo development, one prior to the morula stage and the other from the morula to blastocyst stage, whereas placental morphological abnormalities and reduced imprinting control region methylation were only associated with culture prior to the morula stage. Extended culture to the blastocyst stage also induces additional placental DNA methylation changes compared to embryos transferred at the morula stage, and female concepti exhibited a higher loss of DNA methylation than males. By identifying specific developmental windows of susceptibility, this study provides a framework to optimize further culture conditions to minimize risks associated with ART pregnancies.

CDC25B is required for the metaphase I-metaphase II transition in mouse oocytes.

Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.

Understanding stream bank erosion and deposition in Iowa, USA: A seven year study along streams in different regions with different riparian land-uses.

Agricultural activities such as row-cropping and grazing, have accelerated stream bank erosion. Accelerated stream bank erosion increases nonpoint source pollutants in aquatic ecosystems, significantly degrading them. Mitigating stream bank erosion is a priority worldwide, especially in agricultural watersheds. The objective of this study was to analyze the impacts of riparian land-use management on stream bank erosion and deposition, along with analyzing its temporal and spatial patterns. The study was conducted in three regions of Iowa (central, northeast and southeast) along 30 stream reaches adjacent to seven different riparian land-uses. The riparian land-uses were riparian forest buffers, grass filters, pastures with the cattle excluded from the stream, intensive rotational grazing, rotational grazing, continuous grazing and row crop fields. Seasonal erosion and deposition data (Spring, Summer and Autumn) were collected along these reaches for 5 years and yearly for the following two years. To analyze the data, conventional statistical methods (ANOVA and Tukey's test) along with innovative techniques (percentile plots, cumulative erosion curves and bubble charts) were utilized. Based on the analysis, of this extensive in time (seven years) and large in size (1500 pins measured 17 times in three regions) field dataset, major results were obtained in regard to stream bank erosion in Iowa, USA. Stream banks exhibited high year-to-year variation in erosion and deposition showcasing the need for long-term datasets to better understand stream bank erosion and deposition. Seasonal erosion, also had high variability with Spring recording the most erosion followed by Summer and Autumn. Certain seasons exhibited high stream bank erosion indicating that managers need to focus on these seasons, to reduce erosion effectively. In addition, seasonal measurements can highlight depositional events that might be masked with annual measurements. Riparian land-uses significantly impacted stream bank erosion. Riparian forest buffers and grass filters significantly mitigated stream bank erosion while traditional agricultural practices like continuous grazing and row-crop agriculture had accelerated stream bank erosion. Finally, the percentile plots, cumulative erosion curves and bubble charts captured some stream bank responses that would have been unnoticed using conventional statistical methods, allowing decision makers, stakeholders and the general public, to support and approve measures to mitigate this environmental problem. Nature-based solutions utilizing riparian perennial vegetation can sustainably mitigate stream bank erosion.

Sex-specific effects of in vitro fertilization on adult metabolic outcomes and hepatic transcriptome and proteome in mouse.

Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, there is increasing concern about the long-term and sex-specific health implications. Augmenting our IVF mouse model to longitudinally investigate metabolic outcomes in offspring from optimal neonatal litter sizes, we found sex-specific metabolic outcomes in IVF offspring. IVF-conceived females had higher body weight and cholesterol levels compared to naturally conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through adult liver transcriptomics and proteomics, we identified sexually dimorphic dysregulation of the sterol regulatory element-binding protein (SREBP) pathways that are associated with the sex-specific phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.

Unscrambling the oocyte and the egg: clarifying terminology of the female gamete in mammals.

Most reproductive biologists who study female gametes will agree with the 16th century anatomist William Harvey's doctrine: 'Ex Ovo Omnia'. This phrase, which literally translates to 'everything from the egg', recognizes the centrality of the egg in animal development. Eggs are most impressive cells, capable of supporting development of an entirely new organism following fertilization or parthenogenetic activation. Not so uniformly embraced in the field of reproductive biology is the nomenclature used to refer to the female germ cell. What is an oocyte? What is an egg? Are these terms the same, different, interchangeable? Here we provide functional definitions of the oocyte and egg, and how they can be used in the context of mammalian gamete biology and beyond.

Chromatin remodeling in bovine embryos indicates species-specific regulation of genome activation.

The shift from maternal to embryonic control is a critical developmental milestone in preimplantation development. Widespread transcriptomic and epigenetic remodeling facilitate this transition from terminally differentiated gametes to totipotent blastomeres, but the identity of transcription factors (TF) and genomic elements regulating embryonic genome activation (EGA) are poorly defined. The timing of EGA is species-specific, e.g., the timing of murine and human EGA differ significantly. To deepen our understanding of mammalian EGA, here we profile changes in open chromatin during bovine preimplantation development. Before EGA, open chromatin is enriched for maternal TF binding, similar to that observed in humans and mice. During EGA, homeobox factor binding becomes more prevalent and requires embryonic transcription. A cross-species comparison of open chromatin during preimplantation development reveals strong similarity in the regulatory circuitry underlying bovine and human EGA compared to mouse. Moreover, TFs associated with murine EGA are not enriched in cattle or humans, indicating that cattle may be a more informative model for human preimplantation development than mice.

Long-term nitrate removal in three riparian buffers: 21 years of data from the Bear Creek watershed in central Iowa, USA.

Riparian buffers are a conservation practice that increases vegetation diversity on the agricultural landscape while providing environmental benefits. This study specifically focused on the ability of riparian buffers to remove nitrate from shallow groundwater. There are many studies that assessed nitrate removal within buffers, but not many have a long-term, continuous data set that can analyze for variation in nitrate removal rates over time. Here we report on 21 years of nitrate well data, from 1996 through 2017, for three buffers in the Bear Creek watershed in central Iowa. These buffers are named using abbreviations to help keep landowners anonymous (e.g. RN, RS, and ST). Studied buffers RS and ST showed greater nitrate reduction (or removal) after 10 and 6 years of its establishment, respectively. Buffer RN did not experience a significant nitrate removal increase with time, but instead had higher nitrate removal rates when compared to buffers RS and ST of 10.3 g NO3--N m-1 day-1 from the start of this study. From this data, we suggest that past land management played a major role in the responses observed. RN had previously been established in cool-season grasses for grazing before being converted to a buffer, while RS and ST had been managed in a corn and soybean rotation. RN was thought to have higher denitrification immediately with increased labile soil carbon input and enhanced soil aggregation due to the grassland perennials, while buffer vegetation establishment increased soil carbon inputs and soil aggregation over time for RS and ST. These nitrate removal trends would not have been observed without access to long-term, continuous data. This study highlighted the importance of long-term data sets and the need to assess conservation practices over time to determine their longevity and efficiency with time.

Assisted reproductive technologies induce temporally specific placental defects and the preeclampsia risk marker sFLT1 in mouse.

Although widely used, assisted reproductive technologies (ARTs) are associated with adverse perinatal outcomes. To elucidate their underlying causes, we have conducted a longitudinal analysis of placental development and fetal growth using a mouse model to investigate the effects of individual ART procedures: hormone stimulation, in vitro fertilization (IVF), embryo culture and embryo transfer. We demonstrate that transfer of blastocysts naturally conceived without hormone stimulation and developed in vivo prior to transfer can impair early placentation and fetal growth, but this effect normalizes by term. In contrast, embryos cultured in vitro before transfer do not exhibit this compensation but rather display placental overgrowth, reduced fetal weight, reduced placental DNA methylation and increased levels of sFLT1, an anti-angiogenic protein implicated in causing the maternal symptoms of preeclampsia in humans. Increases in sFLT1 observed in this study suggest that IVF procedures could increase the risk for preeclampsia. Moreover, our results indicate that embryo culture is the major factor contributing to most placental abnormalities and should therefore be targeted for optimization.

Characterization of Historical Methane Occurrence Frequencies from U.S. Underground Natural Gas Storage Facilities with Implications for Risk Management, Operations, and Regulatory Policy.

Defining a baseline for the frequency of occurrences at underground natural gas storage facilities is critical to maintaining safe operation and to the development of appropriate risk management plans and regulatory approaches. Currently used frequency-estimation methods are reviewed and broadened in this article to include critical factors of cause, severity, and uncertainty that contribute to risk. A Bayesian probabilistic analysis characterizes the aleatoric historical occurrence frequencies given imperfect sampling. Frequencies for the three main storage facility types in the United States (depleted oil-and-gas field storage, aquifer storage, solution-mined salt cavern storage) are generally on the order of 3 to 9 × 10-2 occurrences, of all causes (surface, well integrity, subsurface integrity) and severities (nuisance, serious, catastrophic), per facility-year. Loss of well integrity is associated with many, but not all, occurrences either within the subsurface or from there up to the surface. The probability of one serious or catastrophic leakage occurrence to the ground surface within the next 10 years, assuming constant number of facilities, is approximately 0.1-0.3% for any facility type. Storage operators and industry regulators can use occurrence frequencies, their associated probabilities and uncertainties, and forecasts of severity magnitudes to better prioritize resources, establish a baseline against which progress toward achieving a reduction target could be measured, and develop more effective mitigation/monitoring/reduction programs in a risk management plan.