Type I interferon alters invasive extravillous trophoblast function.

Inappropriate type I interferon (IFN) signaling during embryo implantation and placentation is linked to poor pregnancy outcomes. Here, we evaluated the consequence of elevated type I IFN exposure on implantation using a biomimetic model of human implantation in an organ-on-a-chip device. We found that type I IFN reduced extravillous trophoblast (EVT) invasion capacity. Analyzing single-cell transcriptomes, we uncovered that IFN truncated endovascular EVT emergence in the implantation-on-a-chip device by stunting EVT epithelial-to-mesenchymal transition. Disruptions to the epithelial-to-mesenchymal transition is associated with the pathogenesis of preeclampsia, a life-threatening hypertensive disorder of pregnancy. Strikingly, unwarranted IFN stimulation induced genes associated with increased preeclampsia risk and a preeclamptic gene-like signature in EVTs. These dysregulated EVT phenotypes ultimately reduced EVT-mediated endothelial cell vascular remodeling in the implantation-on-a-chip device. Overall, our work indicates IFN signaling can alter EVT epithelial-to-mesenchymal transition progression which results in diminished EVT-mediated spiral artery remodeling and a preeclampsia gene signature upon sustained stimulation. Our work implicates unwarranted type I IFN as a maternal disturbance that can result in abnormal EVT function that could trigger preeclampsia.

Uterine macrophages and NK cells exhibit population and gene-level changes after implantation but maintain pro-invasive properties.

Introduction: Prior to pregnancy, hormonal changes lead to cellular adaptations in the endometrium allowing for embryo implantation. Critical for successful pregnancy establishment, innate immune cells constitute a significant proportion of uterine cells prior to arrival of the embryo and throughout the first trimester in humans and animal models. Abnormal uterine immune cell function during implantation is believed to play a role in multiple adverse pregnancy outcomes. Current work in humans has focused on uterine immune cells present after pregnancy establishment, and limited in vitro models exist to explore unique functions of these cells. Methods: With single-cell RNA-sequencing (scRNAseq), we comprehensively compared the human uterine immune landscape of the endometrium during the window of implantation and the decidua during the first trimester of pregnancy. Results: We uncovered global and cell-type-specific gene signatures for each timepoint. Immune cells in the endometrium prior to implantation expressed genes associated with immune metabolism, division, and activation. In contrast, we observed widespread interferon signaling during the first trimester of pregnancy. We also provide evidence of specific inflammatory pathways enriched in pre- and post-implantation macrophages and natural killer (NK) cells in the uterine lining. Using our novel implantation-on-a-chip (IOC) to model human implantation ex vivo, we demonstrate for the first time that uterine macrophages strongly promote invasion of extravillous trophoblasts (EVTs), a process essential for pregnancy establishment. Pre- and post-implantation uterine macrophages promoted EVT invasion to a similar degree as pre- and post-implantation NK cells on the IOC. Conclusions: This work provides a foundation for further investigation of the individual roles of uterine immune cell subtypes present prior to embryo implantation and during early pregnancy, which will be critical for our understanding of pregnancy complications associated with abnormal trophoblast invasion and placentation.

COVID-19 during pregnancy alters circulating extracellular vesicle cargo and their effects on trophoblast.

SARS-CoV-2 infection and the resulting coronavirus disease (COVID-19) complicate pregnancies as the result of placental dysfunction which increases the risk of adverse pregnancy outcomes. While abnormal placental pathology resulting from COVID-19 is common, direct infection of the placenta is rare. This suggests maternal response to infection is responsible for placental dysfunction. We hypothesized that maternal circulating extracellular vesicles (EVs) are altered by COVID-19 during pregnancy and contribute to placental dysfunction. To examine this, we characterized maternal circulating EVs from pregnancies complicated by COVID-19 and tested their functional effect on trophoblast cells in vitro. We found the timing of infection is a major determinant of the effect of COVID-19 on circulating EVs. Additionally, we found differentially expressed EV mRNA cargo in COVID-19 groups compared to Controls that regulates the differential gene expression induced by COVID-19 in the placenta. In vitro exposure of trophoblasts to EVs isolated from patients with an active infection, but not EVs isolated from Controls, reduced key trophoblast functions including hormone production and invasion. This demonstrates circulating EVs from subjects with an active infection disrupt vital trophoblast function. This study determined that COVID-19 has a long-lasting effect on circulating EVs and circulating EVs are likely to participate in the placental dysfunction induced by COVID-19.

The impact of the COVID-19 pandemic on inequalities in preventive health screenings: Trends and implications for U.S. population health.

The COVID-19 pandemic has profoundly impacted population well-being in the United States, exacerbating existing racial and socioeconomic inequalities in health and mortality. Importantly, as the pandemic disrupted the provision of vital preventive health screenings for cardiometabolic diseases and cancers, more research is needed to understand whether this disruption had an unequal impact across racialized and socioeconomic lines. We draw on the 2019 and 2021 National Health Interview Survey to explore whether the COVID-19 pandemic contributed to racialized and schooling inequalities in the reception of preventive screenings for cardiometabolic diseases and cancers. We find striking evidence that Asian Americans, and to a lesser extent Hispanic and Black Americans, reported decreased reception of many types of cardiometabolic and cancer screenings in 2021 relative to 2019. Moreover, we find that across schooling groups, those with a bachelor's degree or higher experienced the greatest decline in screening reception for most cardiometabolic diseases and cancers, and those with less than a high school degree experienced the greatest decline in screening reception for diabetes. Findings have important implications for health inequalities and U.S. population health in the coming decades. Research and health policy attention should be directed toward ensuring that preventive health care is a key priority for public health, particularly among socially marginalized groups who may be at increased risk of delayed diagnosis for screenable diseases.

Changing socioeconomic and geographic gradients in cardiovascular disease risk factors among Indians aged 15-49 years - evidence from nationally representative household surveys.

Background: Cardiovascular diseases (CVDs) are the leading cause of death in most low- and middle-income countries (LMICs). CVDs and their metabolic risk factors have historically been concentrated among urban residents with higher socioeconomic status (SES) in LMICs such as India. However, as India develops, it is unclear whether these socioeconomic and geographic gradients will persist or change. Understanding these social dynamics in CVD risk is essential for mitigating the rising burden of CVDs and to reach those with the greatest needs. Methods: Using nationally representative data with biomarker measurements from the fourth (2015-16) and fifth (2019-21) Indian National Family and Health Surveys, we investigated trends in the prevalence of four CVD risk factors: smoking (self-reported), unhealthy weight (BMI ≥25 kgm2), diabetes (random plasma glucose concentration ≥200 mg/dL or self-reported diabetes), and hypertension (one of: average systolic blood pressure ≥140 mmHg, average diastolic blood pressure ≥90 mmHg, self-reported past diagnosis, or self-reported current antihypertensive medication use) among adults aged 15-49 years. We first described changes at the national level and then trends stratified by place of residence (urban versus rural), geographic region (northern, northeastern, central, eastern, western, southern), regional level of development (Empowered Action Group member state or not), and two measures of socioeconomic status: level of education (no education, primary incomplete, primary complete, secondary incomplete, secondary complete, higher) and wealth (quintiles). Findings: Unhealthy weight increased among all social and geographic groups but both the absolute and the relative changes were substantially higher among people with low SES (as measured by education or wealth) and in rural areas. For diabetes and hypertension, the prevalence increased for those from disadvantaged groups while staying constant or even decreasing among the wealthier and more educated. In contrast, smoking consumption declined for all social and geographic groups. Interpretation: In 2015-16, CVD risk factors were higher among more advantaged subpopulations in India. However, between 2015-16 and 2019-21, the prevalence of these risk factors grew more rapidly for less wealthy and less educated subpopulations and those living in rural areas. These trends have resulted in CVD risk becoming far more widespread throughout the population; CVD can no longer be characterized as a wealthy urban phenomenon. Funding: This work was supported by the Alexander von Humboldt Foundation (grant received by NS); the Stanford Diabetes Research Center [grant received by PG] and the Chan Zuckerberg Biohub [grant received by PG].

Embryo cryopreservation leads to sex-specific DNA methylation perturbations in both human and mouse placentas.

In vitro fertilization (IVF) is associated with DNA methylation abnormalities and a higher incidence of adverse pregnancy outcomes. However, which exposure(s), among the many IVF interventions, contributes to these outcomes remains unknown. Frozen embryo transfer (ET) is increasingly utilized as an alternative to fresh ET, but reports suggest a higher incidence of pre-eclampsia and large for gestational age infants. This study examines DNA methylation in human placentas using the 850K Infinium MethylationEPIC BeadChip array obtained after 65 programmed frozen ET cycles, 82 fresh ET cycles and 45 unassisted conceptions. Nine patients provided placentas following frozen and fresh ET from consecutive pregnancies for a paired subgroup analysis. In parallel, eight mouse placentas from fresh and frozen ET were analyzed using the Infinium Mouse Methylation BeadChip array. Human and mouse placentas were significantly hypermethylated after frozen ET compared with fresh. Paired analysis showed similar trends. Sex-specific analysis revealed that these changes were driven by male placentas in humans and mice. Frozen and fresh ET placentas were significantly different from controls, with frozen samples hypermethylated compared with controls driven by males and fresh samples being hypomethylated compared with controls, driven by females. Sexually dimorphic epigenetic changes could indicate differential susceptibility to IVF-associated perturbations, which highlights the importance of sex-specific evaluation of adverse outcomes. Similarities between changes in mice and humans underscore the suitability of the mouse model in evaluating how IVF impacts the epigenetic landscape, which is valuable given limited access to human tissue and the ability to isolate specific interventions in mice.

A microphysiological model of human trophoblast invasion during implantation.

Successful establishment of pregnancy requires adhesion of an embryo to the endometrium and subsequent invasion into the maternal tissue. Abnormalities in this critical process of implantation and placentation lead to many pregnancy complications. Here we present a microenigneered system to model a complex sequence of orchestrated multicellular events that plays an essential role in early pregnancy. Our implantation-on-a-chip is capable of reconstructing the three-dimensional structural organization of the maternal-fetal interface to model the invasion of specialized fetal extravillous trophoblasts into the maternal uterus. Using primary human cells isolated from clinical specimens, we demonstrate in vivo-like directional migration of extravillous trophoblasts towards a microengineered maternal vessel and their interactions with the endothelium necessary for vascular remodeling. Through parametric variation of the cellular microenvironment and proteomic analysis of microengineered tissues, we show the important role of decidualized stromal cells as a regulator of extravillous trophoblast migration. Furthermore, our study reveals previously unknown effects of pre-implantation maternal immune cells on extravillous trophoblast invasion. This work represents a significant advance in our ability to model early human pregnancy, and may enable the development of advanced in vitro platforms for basic and clinical research of human reproduction.

Assessing mortality registration in Kerala: the MARANAM study.

Complete or improving civil registration systems in sub-national areas in low- and middle-income countries provide several opportunities to better understand population health and its determinants. In this article, we provide an assessment of vital statistics in Kerala, India. Kerala is home to more than 33 million people and is a comparatively low-mortality context. We use individual-level vital registration data on more than 2.8 million deaths between 2006 and 2017 from the Kerala MARANAM (Mortality and Registration Assessment and Monitoring) Study. Comparing age-specific mortality rates from the Civil Registration System (CRS) to those from the Sample Registration System (SRS), we do not find evidence that the CRS underestimates mortality. Instead, CRS rates are smoother across ages and less variable across periods. In particular, the CRS records higher death rates than the SRS for ages, where mortality is usually low and for women. Using these data, we provide the first set of annual sex-specific life tables for any state in India. We find that life expectancy at birth was 77.9 years for women in 2017 and 71.4 years for men. Although Kerala is unique in many ways, our findings strengthen the case for more careful attention to mortality records within low- and middle-income countries, and for their better dissemination by government agencies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s41118-021-00149-z.

Uterine natural killer cell biology and role in early pregnancy establishment and outcomes.

Objective: While immune cells were originally thought to only play a role in maternal tolerance of the semiallogenic fetus, an active role in pregnancy establishment is becoming increasingly apparent. Uterine natural killer (uNK) cells are of specific interest because of their cyclic increase in number during the window of implantation. As a distinct entity from their peripheral blood counterparts, understanding the biology and function of uNK cells will provide the framework for understanding their role in early pregnancy establishment and adverse pregnancy outcomes. Evidence Review: This review discusses unique uNK cell characteristics and presents clinical implications resulting from their dysfunction. We also systematically present existing knowledge about uNK cell function in three processes critical for successful human embryo implantation and placentation: stromal cell decidualization, spiral artery remodeling, and extravillous trophoblast invasion. Finally, we review the features of uNK cells that could help guide future investigations. Results: It is clear the uNK cells are intimately involved in multiple facets of early pregnancy. This is accomplished directly, through the secretion of factors that regulate stromal cells and trophoblast function; and indirectly, via interaction with other maternal cell types present at the maternal-fetal interface. Current work also suggests that uNK cells are a heterogenous population, with subsets that potentially accomplish different functions. Conclusion: Establishment of pregnancy through successful embryo implantation and placentation requires crosstalk between multiple maternal cell types and invading fetal trophoblast cells. Defects in this process have been associated with multiple adverse perinatal outcomes including hypertensive disorders of pregnancy, placenta accreta, and recurrent miscarriage though the mechanism underlying development of these defects remain unclear. Abnormalities in NK cell number and function which would disrupt physiological maternal-fetal crosstalk, could play a critical role in abnormal implantation and placentation. It is therefore imperative to dissect the unique physiological role of uNK cells in pregnancy and use this knowledge to inform clinical practice by determining how uNK cell dysfunction could lead to reproductive failure.

Mind-Mindedness and Stress in Parents of Children with Developmental Disorders.

Relations between mind-mindedness (assessed using the describe-your-child interview) and stress were investigated in parents of children with developmental disorders (ADHD, n = 51, ASD, n = 23, Down's Syndrome, n = 38, and 22q11.2 Deletion Syndrome, 22q11.2DS, n = 32) and typically-developing children (n = 89). Mind-mindedness did not differ across diagnostic groups, and mind-mindedness predicted parenting stress across groups. Parenting stress was lowest in the typically-developing and Down's Syndrome groups. Across all groups, mind-minded and positive descriptions predicted lower parenting stress, and negative descriptions predicted higher stress. In the developmental disorder groups, describing the children with reference to their disorder was negatively correlated with mind-mindedness. Results are discussed with regard to interventions for families where children have developmental disorders.

IFNs Drive Development of Novel IL-15-Responsive Macrophages.

Disruption in homeostasis of IL-15 is linked to poor maternal and fetal outcomes during pregnancy. The only cells described to respond to IL-15 at the early maternal-fetal interface have been NK cells. We now show a novel population of macrophages, evident in several organs but enriched in the uterus of mice and humans, expressing the ß-chain of the IL-15R complex (CD122) and responding to IL-15. CD122+ macrophages (CD122+Macs) are morphologic, phenotypic, and transcriptomic macrophages that can derive from bone marrow monocytes. CD122+Macs develop in the uterus and placenta with kinetics that mirror IFN activity at the maternal-fetal interface. M-CSF permits macrophages to express CD122, and IFNs are sufficient to drive expression of CD122 on macrophages. Neither type I nor type II IFNs are required to generate CD122+Macs, however. In response to IL-15, CD122+Macs activate the ERK signaling cascade and enhance production of proinflammatory cytokines after stimulation with the TLR9 agonist CpG. Finally, we provide evidence of human cells that phenocopy murine CD122+Macs in secretory phase endometrium during the implantation window and in first-trimester uterine decidua. Our data support a model wherein IFNs local to the maternal-fetal interface direct novel IL-15-responsive macrophages with the potential to mediate IL-15 signals critical for optimal outcomes of pregnancy.

Timing of exposure to gonadotropins has differential effects on the conceptus: evidence from a mouse model†.

Superovulation with gonadotropins alters the hormonal milieu during early embryo development and placentation, and may be responsible for fetal and placental changes observed after in vitro fertilization (IVF). We hypothesized that superovulation has differential effects depending on timing of exposure. To test our hypothesis, we isolated the effect of superovulation on pre- and peri-implantation mouse embryos. Blastocysts were obtained from either natural mating or following superovulation and mating, and were transferred into naturally mated or superovulated pseudopregnant recipient mice. Fetal weight was significantly lower after peri-implantation exposure to superovulation, regardless of preimplantation exposure (p = 0.006). Placentas derived from blastocysts exposed to superovulation pre- and peri-implantation were larger than placentas derived from natural blastocysts that are transferred into a natural or superovulated environment (p < 0.05). Fetal-to-placental weight ratio decreased following superovulation during the pre- or peri-implantation period (p = 0.05, 0.01, respectively) and these effects were additive. Peg3 DNA methylation levels were decreased in placentas derived from exposure to superovulation both pre- and peri-implantation compared with unexposed embryos and exposure of the preimplantation embryo only. Through RNA sequencing on placental tissue, changes were identified in genes involved in immune system regulation, specifically interferon signaling, which has been previously implicated in implantation and maintenance of early pregnancy in mice. Overall, we found that the timing of exposure to gonadotropin stimulation can have differential effects on fetal and placental growth. These findings could impact clinical practice and underscores the importance of dissecting the role of procedures utilized during IVF on pregnancy complications.

Epigenetic changes and assisted reproductive technologies.

Children conceived by Assisted Reproductive Technologies (ART) are at moderately increased risk for a number of undesirable outcomes, including low birth weight. Whether the additional risk is associated with specific procedures used in ART or biological factors that are intrinsic to infertility has been the subject of much debate, as has the mechanism by which ART or infertility might influence this risk. The potential effect of ART clinical and laboratory procedures on the gamete and embryo epigenomes heads the list of mechanistic candidates that might explain the association between ART and undesirable clinical outcomes. The reason for this focus is that the developmental time points at which ART clinical and laboratory procedures are implemented are precisely the time points at which large-scale reorganization of the epigenome takes place during normal development. In this manuscript, we review the many human studies comparing the epigenomes of ART children with children conceived in vivo, as well as assess the potential of individual ART clinical and laboratory procedures to alter the epigenome.

Effects of an mHealth intervention for community health workers on maternal and child nutrition and health service delivery in India: protocol for a quasi-experimental mixed-methods evaluation.

INTRODUCTION: Millions of children in India still suffer from poor health and under-nutrition, despite substantial improvement over decades of public health programmes. The Anganwadi centres under the Integrated Child Development Scheme (ICDS) provide a range of health and nutrition services to pregnant women, children <6 years and their mothers. However, major gaps exist in ICDS service delivery. The government is currently strengthening ICDS through an mHealth intervention called Common Application Software (ICDS-CAS) installed on smart phones, with accompanying multilevel data dashboards. This system is intended to be a job aid for frontline workers, supervisors and managers, aims to ensure better service delivery and supervision, and enable real-time monitoring and data-based decision-making. However, there is little to no evidence on the effectiveness of such large-scale mHealth interventions integrated with public health programmes in resource-constrained settings on the service delivery and subsequent health and nutrition outcomes. METHODS AND ANALYSIS: This study uses a village-matched controlled design with repeated cross-sectional surveys to evaluate whether ICDS-CAS can enable more timely and appropriate services to pregnant women, children <12 months and their mothers, compared with the standard ICDS programme. The study will recruit approximately 1500 Anganwadi workers and 6000+ mother-child dyads from 400+ matched-pair villages in Bihar and Madhya Pradesh. The primary outcomes are the proportion of beneficiaries receiving (a) adequate number of home visits and (b) appropriate level of counselling by the Anganwadi workers. Secondary outcomes are related to improvements in other ICDS services, and knowledge and practices of the Anganwadi workers and beneficiaries. ETHICS AND DISSEMINATION: Ethical oversight is provided by the Committee for the Protection of Human Subjects at the University of California at Berkeley, and the Suraksha Independent Ethics Committee in India. The results will be published in peer-reviewed journals and analysis data will be made public. TRIAL REGISTRATION NUMBER: ISRCTN83902145.

Epigenetic changes in preterm birth placenta suggest a role for ADAMTS genes in spontaneous preterm birth.

Preterm birth (PTB) affects approximately 1 in 10 pregnancies and contributes to approximately 50% of neonatal mortality. However, despite decades of research, little is understood about the etiology of PTB, likely due to the multifactorial nature of the disease. In this study, we examined preterm and term placentas, from unassisted conceptions and those conceived using in vitro fertilization (IVF). IVF increases the risk of PTB and causes epigenetic change in the placenta and fetus; therefore, we utilized these patients as a unique population with a potential common etiology. We investigated genome-wide DNA methylation in placentas from term IVF, preterm IVF, term control (unassisted conception) and preterm control pregnancies and discovered epigenetic dysregulation of multiple genes involved in cell migration, including members of the ADAMTS family, ADAMTS12 and ADAMTS16. These genes function in extracellular matrix regulation and tumor cell invasion, processes replicated by invasive trophoblasts (extravillous trophoblasts (EVTs)) during early placentation. Though expression was similar between term and preterm placentas, we found that both genes demonstrate high expression in first- and second-trimester placenta, specifically in EVTs and syncytiotrophoblasts. When we knocked down ADAMTS12 or ADAMTS16in vitro, there was poor EVT invasion and reduced matrix metalloproteinase activity, reinforcing their critical role in placentation. In conclusion, utilizing a population at high risk for PTB, we have identified a role for ADAMTS gene methylation in regulating early placentation and susceptibility to PTB.

Embryo Culture Conditions and the Epigenome.

Assisted reproductive technologies (ARTs) lead to an increased risk for pregnancy complications, congenital abnormalities, and specific imprinting disorders. Epigenetic dysfunction is thought to be one common mechanism which may be affecting these outcomes. The timing of multiple ART interventions overlaps with developmental time periods that are particularly vulnerable to epigenetic change. In vitro embryo culture is known to impact blastocyst development, in vitro fertilization (IVF) success rates, as well as neonatal outcomes. Embryo culture, in contrast to other procedures involved in ART, is obligatory, and has the highest potential for causing alterations in epigenetic reprograming. In this review, we summarize progress that has been made in exploring the effects of embryo culture, culture media, and oxygen tension on epigenetic regulation in the developing embryo. In humans, it is difficult to isolate the role of embryo culture on epigenetic perturbations. Therefore, additional well-controlled animal studies isolating individual exposures are necessary to minimize the epigenetic effects of modifiable factors utilized during ART. Findings from these studies will likely not only improve IVF success rates but also reduce the risk of adverse perinatal outcomes.

PIWI proteins are essential for early Drosophila embryogenesis.

PIWI proteins, a subfamily of the ARGONAUTE/PIWI protein family, have been implicated in transcriptional and posttranscriptional gene regulation and transposon silencing mediated by small non-coding RNAs, especially piRNAs. Although these proteins are known to be required for germline development, their somatic function remains elusive. Here, we examine the maternal function of all three PIWI proteins in Drosophila; Piwi, Aubergine (Aub) and Argonaute3 (Ago3) during early embryogenesis. In syncytial embryos, Piwi displays an embryonic stage-dependent localization pattern. Piwi is localized in the cytoplasm during mitotic cycles 1-10. Between cycles 11 and 14, Piwi remains in the cytoplasm during mitosis but moves into the somatic nucleus during interphase. Beyond cycle 14, it stays in the nucleus. Aub and Ago3 are diffusely cytoplasmic from cycle 1 to 14. Embryos maternally depleted of any one of the three PIWI proteins display severe mitotic defects, including abnormal chromosome and nuclear morphology, cell cycle arrest, asynchronous nuclear division and aberrant nuclear migration. Furthermore, all three PIWI proteins are required for the assembly of mitotic machinery and progression through mitosis. Embryos depleted of maternal PIWI proteins also exhibit chromatin organization abnormalities. These observations indicate that maternal Piwi, Aub and Ago3 play a critical role in the maintenance of chromatin structure and cell cycle progression during early embryogenesis, with compromised chromatin integrity as a possible cause of the observed mitotic defects. Our study demonstrates the essential function of PIWI proteins in the first phase of somatic development.

Untangling the web: the diverse functions of the PIWI/piRNA pathway.

Small RNAs impact several cellular processes through gene regulation. Argonaute proteins bind small RNAs to form effector complexes that control transcriptional and post-transcriptional gene expression. PIWI proteins belong to the Argonaute protein family, and bind PIWI-interacting RNAs (piRNAs). They are highly abundant in the germline, but are also expressed in some somatic tissues. The PIWI/piRNA pathway has a role in transposon repression in Drosophila, which occurs both by epigenetic regulation and post-transcriptional degradation of transposon mRNAs. These functions are conserved, but clear differences in the extent and mechanism of transposon repression exist between species. Mutations in piwi genes lead to the upregulation of transposon mRNAs. It is hypothesized that this increased transposon mobilization leads to genomic instability and thus sterility, although no causal link has been established between transposon upregulation and genome instability. An alternative scenario could be that piwi mutations directly affect genomic instability, and thus lead to increased transposon expression. We propose that the PIWI/piRNA pathway controls genome stability in several ways: suppression of transposons, direct regulation of chromatin architecture and regulation of genes that control important biological processes related to genome stability. The PIWI/piRNA pathway also regulates at least some, if not many, protein-coding genes, which further lends support to the idea that piwi genes may have broader functions beyond transposon repression. An intriguing possibility is that the PIWI/piRNA pathway is using transposon sequences to coordinate the expression of large groups of genes to regulate cellular function.