Meta-analyses consistently find a substantial possible association between neonatal jaundice (hyperbilirubinemia) and later autism risk. The obvious question this poses is "what is the source of this risk?" This review explores the complementary roles of jaundice severity and time, racial and geographic disparities, and early infant feeding regime change, and discusses potential implications of these findings. A range of factors appears to increase the risk of autism development following neonatal jaundice, all of which are associated with the "exclusive breastfeeding" paradigm. Severity presents an intuitive risk factor in the context of bilirubin neurotoxicity; jaundice from the modal root cause of insufficient milk intake progresses as that condition persists. Racial and geographic disparities present another intuitive set of risk factors, including a heightened risk of missed diagnosis for darker-skinned neonates and delayed care access in poorer settings. In addition to these intuitive factors, near- or full-term as opposed to preterm status and phototherapy treatment may also heighten risk. These counter-intuitive findings provide additional support for deprivation/starvation as a crucial antecedent or independent variable, and time as a mediator to progression in and subsequent risk from jaundice; heightened medical monitoring and supplementation seem to protect preterms, and phototherapy risks iatrogenesis, having replaced without sufficient safety evidence the prior standard treatment of switching jaundiced, breastfed babies to formula. Critically, jaundice associated with insufficient milk intake due to breastfeeding insufficiencies is fully preventable and trivially treatable with appropriate supplemental milk. Feeding neonates adequately may play an important role in preventing autism and other neurodevelopmental disorders including attention deficit hyperactivity disorder, cerebral palsy, epilepsy, hearing impairment, learning disorders, and mood disorders. Precautionary principle invocation is overdue. Recently, a third meta-analysis found neonatal jaundice is associated with substantial possible increased autism risk -3 - the First-time and more exhausted mothers tend to have more breastfeeding problems, and the risk of autism may be correspondingly greater in first-borns -14 and tRising rates of autism ,30 and oThe evidence on neonatal jaundice and autism suggests that rising autism rates may be part of that epidemic. As Amin et al. observe, if unconjugated hyperbilirubinemia is a significant cause of autism, it "is easily testable, implies prevention, and is of public health importance." The precautionary principle, acting to prevent harm when risk is uncertain and stakes are high, applies. Neonates should drink appropriate supplemental milk early, adequately, and often. This practice should be standard of care whenever insufficient breastmilk intake is suspected, particularly in cases of jaundiced neonates. This usually entails formula-feeding due to factors including limited banked donor breastmilk availability and infectious disease transmission concerns . MinimizA PubMed search  for the terms "jaundice," "autism," and "meta-analysis" returned four published systematic reviews and meta-analyses. Of these, the first three comprehensively analyzed the relationship between jaundice and autism in the selected centrally indexed, peer-reviewed literature; this review details, synthesizes, and builds chiefly on their results, which consistently showed a substantial possible association between neonatal jaundice and autism. The fourth search result, a meta-analysis by Lai et al., focused on something different - the association between bilirubin and kernicterus spectrum disorder (KSD) - and limited its analysis of results relating to autism as a dependent variable to one study, which also showed a substantial possible association . These aThis review first summarizes the previous meta-analytical findings, which all show substantial possible risk. Second, this review arbitrates disagreement between these previous meta-analyses on the results of publication bias testing, noting common misuse of funnel plot tests for publication bias and presenting results of novel publication bias testing with a more modern tool, p-curve analysis. This analysis shows that results are unaffected by selective reporting (publication bias) and the underlying literature has evidential value. Hence, the jaundice-autism effect may be quite large and the link seems real; but where does the risk come from?The link between jaundice severity and risk suggests that time matters; preventing jaundice progression may prevent harm. This is consistent with the evidence on global disparities relating to delayed healthcare access. The evidence on subgroup effects and global disparities further suggests that, in order to better understand where the risk comes from, we need to reconceptualize the dependent variable as a broader continuum of harm; existing estimates are likely underestimates, because other neurodevelopmental harms may occur alongside, or instead of, autism and patient-level mortality (survivorship bias) selects against severity. Other identifiable biases also likely bias existing estimates down: mild case inclusion dilutes the effect, study-level exclusion criteria select against severity, and study populations tend to over-represent wealthy, Western populations, under-representing populations likely to suffer disproportionate associated harm.This body of evidence raises the question: If jaundice increases autism risk, time matters, and autism prevalence has substantially increased in recent history, then why might have untreated jaundice substantially increased in roughly the same period? The resurgence of breastfeeding from historical lows in modern societies, with novel emphasis on exclusivity and in the absence of previously widespread safety infrastructure guarding against common breastfeeding insufficiencies, offers a plausible explanation: neonates accidentally deprived of sufficient nutrition/hydration may suffer permanent harm -28.Two puzzles in the jaundice-autism literature fit this story. First, meta-analyses report weaker associations in the preterm subgroup, a puzzle because preterms are more vulnerable. Different early infant feeding norms in preterms may make them the control group in the exclusive breastfeeding natural experiment. Second, phototherapy, the current standard treatment for neonatal jaundice, is associated with possible increased autism risk of greater magnitude than that associated with jaundice. The prior norm that phototherapy replaced was treating the root cause of modal neonatal jaundice, insufficient milk intake, through feeding neonates early, adequate, and often supplemental milk. Moreover, phototherapy appears to depend on excretion for its efficacy, and possibly safety, which insufficient milk intake compromises; many researchers have raised questions about possible risks associated with phototherapy, including increased autism risk. The evidence is consistent with the possibility of preventable neurodevelopmental harm from untreated insufficient milk intake in neonates treated with phototherapy for jaundice.Jaundice-autism meta-analyses: consistent findings of substantial possible riskAmin et al., Jenabi et al., and Kujabi et al. consistently found that jaundice in near and full-term neonates may risk substantial autism increase -3. TheirAmin et al. reported the results of a systematic review and meta-analysis on jaundice and autism including 13 studies retrieved from PubMed and MEDLINE databases published until 2009, mostly using retrospective matched case-control designs  prevalence of ASD globally" . This woRising autism prevalence: causes, estimates, and historical coincidence with the exclusive breastfeeding paradigmExclusive breastfeeding along the starvation jaundice pathway is only one of many possible contributors to rising autism rates. Future research should undertake a full exposition of all these factors with preventive emphasis, integrating available evidence on early insufficient milk intake and neurodevelopmental risk into available evidence on perinatal risk factors and autism. Reviewing the latter, Cheng et al. state that prolonged breastfeeding is reportedly linked to possible autism risk reduction . The autOther possible contributors to rising autism incidence also warrant full exploration with a harm prevention focus. Some relate to the breastfeeding starvation hypothesis directly (as in other complications of insufficient milk intake), others indirectly (as in other facets of the modern Western condition). Chief among the directly related possible contributors are hypoglycemia and hypernatremia, which can also cause brain damage and are also often associated in previously healthy near- and full-term neonates with insufficient milk intake, although jaundice appears to be the complication most commonly diagnosed. Chief among the indirectly related ones are greater cumulative exposure to other stressors for fetuses, neonates, and mothers that is likely to have raised their allostatic load - the cumulative burden of chronic stress across physiological and psychological systems - in turn raising the likelihood of occult or frank disease, which may contribute to breastfeeding insufficiencies . Modern The modern Western condition links these possible allostatic load-increasing factors with the conditions that gave rise to the modern notion of "exclusive breastfeeding." New mothers supported by more experienced mothers with direct knowledge of safe breastfeeding, as in most traditional and non-Western societies, would not risk accidentally starving their newborns in the same way that new mothers without that human capital habitually do under the exclusive breastfeeding paradigm. Thus, intergenerational knowledge loss, the marginalization of laywomen as experts in childrearing and other forms of care work, and increasing social isolation all play a role in neonatal starvation from exclusive breastfeeding, as well as in other modern changes that may contribute to increased autism prevalence along other pathways.Two additional possible contributing factors to rising autism prevalence do not quite so neatly fit this story, but also have to do with modernity in a broader sense. First, as Reser hypothesized, assortative mating may play a role in more severe autism, with solitary foraging conditions previously selecting for subclinical autistic traits and then modern systematizing thinkers (increasingly) selecting one another for mates . ResultsMTHFR C677T polymorphic live births due to fewer miscarriages from hyperhomocysteinemia and associated thrombotic events, and thus an increased incidence of infants requiring additional and/or more bioavailable folate for normal methylation during the sensitive postnatal developmental period, resulting in increased autism incidence from subpar folate status [Second, Rogers hypothesized that in the early 1990s in the US, increased folic acid intake among pregnant women that was intended to prevent neural tube defects resulted in an unintentional reduction of natural selection pressures against the polymorphic form of the 5-methylenetetrahydrofolate reductase (MTHFR) enzyme needed to activate folate for methylation in neurodevelopment, leading to an increase in e status . If trueMTHFR C677T TSB" returns no results ; searching for "MTHFR C677T jaundice" revealed a case report and a sickle cell study; and searching for "MTHFR C677T neonatal screening meta-analysis" returns one meta-analysis by Wu et al., who found preterm birth and low birth weight associated with the polymorphism [MTHFR C677T polymorphism compounds neurodevelopmental harm from starvation.Rogers' hypothesis, and its intersection with the breastfeeding starvation hypothesis, remains largely untested. A PubMed search for "morphism , and a smorphism . In a stmorphism ; this paElsabbagh et al. suggest that increased autism incidence is likely an artefact of social and administrative, rather than biological, changes . This viNassar et al. suggested rising autism prevalence in Western Australia from 1983-1999 appears related to social and administrative changes but noted that a true increase cannot be ruled out . In a syAdditional evidence for the social/administrative hypothesis of artefactual autism increase comes from two Scandinavian birth cohort studies, and this evidence is weak. First, in a Danish study of children born from 1980-1991, Hansen et al. found that diagnostic criteria changes and the inclusion of outpatient contacts  accounted for most of the increase . But agaOverall, the evidence suggesting no true rise in autism is weak; substantial autism prevalence increase cannot be dismissed as artefactual based on existing data. So how big is the increase?As part of Public Health Surveillance in the National Center on Birth Defects and Developmental Disabilities, Child Development and Disability Branch, the US Centers for Disease Control and Prevention (CDC) maintains an autism prevalence study estimates collection. It tables information from English-language, peer-reviewed studies from PubMed searches for autism/autistic and prevalence (current through July 2021) that produce at least one autism prevalence estimate and are population-based within a defined geographic area. A scatterplot of that data  beyond 72 hours postpartum ,10 and 1Pre-modern organized societies that left sufficient historical records for this type of investigation appear to have similarly maintained safety infrastructure to protect newborns from common breastfeeding insufficiencies. Wetnursing -41, co-nWhy are breastfeeding insufficiencies so common if they are an antecedent condition of so much neonatal harm? The fact that homo sapiens tend to be a slow-maturing species offers one plausible sort of explanation. The commonality of breastfeeding insufficiencies, particularly among first-time mothers, is likely due physiologically to lack of full breast tissue differentiation during adolescence and first pregnancy; human breast tissue continues differentiating as a woman continues maturing in her reproductive life through childbearing, with mammary gland differentiation typical of pregnancy . Slow mHinde and Milligan observed that little is known about changes in primate milk synthesis across lactation, information that seems relevant to answering this question . UrashimOne alternate, though not mutually exclusive, explanation is that lessened selection pressures on functioning lactation for infant survival in herding societies may have caused women in those societies, over generations, to disproportionately lose breastfeeding as a reliable function. Levin posits and Post builds on this hypothesis ,57. BothLess selection pressure might help explain more lactation problems in evolutionary time, but it is also possible that novel modern conditions cause marked increases in the frequency and severity of breastfeeding insufficiencies. Increasing exposure to environmental pollutants is linked with the increasing incidence of numerous fertility and other health problems in animals including humans - including ones associated with breastfeeding problems, such as PCOS, overweight/obesity, and diabetes. Such exposures might drive a rising incidence of breastfeeding insufficiencies, though again, data do not exist to establish base rates of such problems over time to measure changes in them.For instance, dioxin exposure during pregnancy impairs murine mammary gland differentiation, blocking lactation  by activA growing body of evidence shows that infants do ingest pollutants through breastmilk, and these exposures appear to adversely affect development; so it could be a protective mechanism that some toxins short-circuit lactation, as long as offspring have an alternate source of sustenance. Analyzing Faroese birth cohort serum evidence, Mogensen et al. showed breastfeeding duration and exclusivity are associated with concentration increases of most PFASs in children . GrandjeIf the transfer of environmental pollutants to infants through breastmilk may cause pediatric harm, then it should also benefit mothers by offloading those otherwise persistent toxins; and there is evidence that it may. Examining PFAS exposures and type 2 diabetes incidence in a case-control study of women from the US Nurses' Health Study II, Sun et al. found PFAS exposures in the late 1990s are associated with increased later diabetes risk, consistent with a potential diabetogenic effect of PFAS exposures . AnalyziSwan and Colino offer a framework to situate such concerns in the broader context of growing evidence that male and female reproductive development, behavior, and function are increasingly and substantially impaired by environmental pollution . So modeWhat does all this mean in terms of the evolutionary puzzle that common breastfeeding insufficiencies pose? When, why, and how did humans evolve as not only cooperative breeders, but also specifically as cooperative breastfeeders? Were breastfeeding insufficiencies primarily an accident of biology, e.g., of the costliness of reproduction in our slow-maturing species? Or were common breastfeeding insufficiencies adaptive in evolving humans as cooperative breeders? Pre-human and early human mothers, especially first-time mothers, likely to physically require the assistance of more experienced mothers in order to feed their babies initially may well have benefited in other ways from proximity to such expert help. So, too, might their offspring and alloparental support networks. Conversely, such advantages would imply possible disadvantages from current, relatively atomistic, modern Western early infant feeding and other care norms - norms that many criticize as contributing to postpartum mental health problems. Future research should review evidence relevant to this puzzle.Historically and culturally, the real puzzle is not why humanity before modern Western society generally shared certain infant feeding norms, but rather why modern Western reformers changed those norms radically and without evidence establishing their safety, running a global natural experiment in the absence of informed consent or data collection about its possible risks. Aware that they were missing generations of knowledge about early infant feeding, reformers constructed a new, historically anomalous set of breastfeeding practices that introduced a single point of failure (one mother's lactation) into what had previously been a cooperative enterprise. Future research should examine the question of why modernity atomizes in this context, assessing to what extent this story is sui generis. Where else have modern societies marginalized, devalued, and lost traditional care work knowledge, and with what consequences for health? The rapid global loss of linguistic diversity, traditional diets, and ecosystems comes to mind.Moving back down again from the evolutionary to the mechanistic level, it would be ideal to directly test the breastfeeding starvation hypothesis of increasing autism prevalence. But, because of a lack of systematic data collection on relevant variables such as neonatal starvation periods, breastfeeding problems including maternal perceptions of delayed and insufficient milk, and heterogeneity in implementation of exclusive breastfeeding policies, the data for these tests do not appear to exist. This heterogeneity is notable: Hofvander reports that Swedish Baby-Friendly hospitals gave healthy neonates prelacteal feedings  anywhere from under 10% to 90% of the time ; implemeNeonatal jaundice incidence presents another set of measurement difficulties in relation to direct tests of the breastfeeding starvation hypothesis of increasing autism prevalence. As previously noted, under-diagnosis despite heightened associated risks in darker-skinned infants may persist for several reasons; related error rates are likely to have changed over time as this issue has garnered greater attention and understanding. Diagnostic criteria changes present another challenge, as do differences in measurement, severity, duration, standard use of TSB instead of unconjugated bilirubin (discussed in greater detail later), and growing normalization of related problems as the exclusive breastfeeding paradigm gained dominance. Future research should note these sorts of limitations and synthesize published neonatal jaundice incidence data with the aim of seeing if the base rate appears to have increased.It is past time to put the burden of proof where it belongs: Existing evidence is insufficient to establish the safety of days-long neonatal deprivation/starvation with respect to children's neurodevelopmental outcomes. The burden of proof should be on the side of establishing the novel modern medical intervention, exclusive breastfeeding, as safe. Research and practice must honor the Hippocratic Oath by focusing on first preventing harm when this intervention results in lengthy periods of insufficient milk intake for neonates, which carry serious possible risks.If modern exclusive breastfeeding at least partly caused increased autism prevalence via neonatal neurological damage from deprivation, then we might think of the shift in early infant feeding norms that the new construction of breastfeeding introduced as a natural experiment. We would expect to see worse outcomes in the treatment than in the control group. The evidence fits: the apparent paradox of weakened preterm jaundice-autism (and related) associations makes sense in the context of different subgroup feeding norms among other factors. Preterms may be the control group in this natural experiment.The preterm paradox: a natural experiment in preventive nutrition?Preterm subgroup results from the jaundice-autism literature present an apparent paradox. While substantially more vulnerable to morbidity and mortality, including jaundice  and autiAmin et al. estimated a subgroup OR of .7 (95%CI: 0.38-1.02) and suggested it still "deserves further investigation" because it was "contrary to our hypothesis and biological plausibility." They hypothesized it might be due to lower protein concentration and altered binding affinity due to competitors with bilirubin for albumin binding causing high (neurotoxic) free unconjugated bilirubin concentration despite low TSB concentration in preterms. In a later review of the evidence on unconjugated hyperbilirubinemia's developmental influence on neurobehavioral disorders, Amin noted, "Limited data is available on the ontogeny of (bilirubin binding capacity) and affinity in premature infants" . SimilarThree factors may help explain this apparent paradox. First, preterm subgroup estimates are particularly vulnerable to downward bias. Underpowering caused by small sample size, narrow operationalization of the outcome of interest (focusing on autism to the exclusion of other risks), and survivorship bias are particularly likely to contribute to Type-II errors (finding no significant effect when there is one) in preterms, a minority of births with considerably higher morbidity and mortality risks. Walani notes preterm birth is the leading cause of death among children, with substantial variations in preterm birth rates and associated mortality between and within countries . Thus, hSecond, preterms have heightened exposure to cumulative perinatal risk factors for autism. This heightened exposure to other risk factors may make jaundice appear less impactful in this subgroup, because susceptible children's genes or other predisposing factors in prodromal syndromes could have already been activated in relevant ways. In a review of evidence on environmental influences on autism development, Mandy and Lai suggest gene-environment interplay influences the development of autism spectrum condition, with postnatal social factors influencing the likelihood of prodromal autism progressing . This loFor instance, Chien et al. reported that autistic children had higher numbers of prenatal/perinatal factors that may increase the risk of ASD as compared to both their unaffected siblings and typically developing controls; particularly severe autism symptoms were associated with preeclampsia, polyhydramnios, oligoamnios, placenta previa, umbilical cord knot, and gestational diabetes . In a twTogether, findings like these provide some preliminary support for the hypothesis of a threshold of cumulative adverse event exposure, perhaps acting on more susceptible children to increase autism risk such that not all exposures affect all children equally. This hypothesis warrants further investigation and may help explain the weakened jaundice-autism preterm effect. With power limitations, it may be testable with meta-analyses exploring subgroup effects within the preterm subgroup. For instance, we might expect a notably weakening male subgroup effect for progressively later- rather than earlier-occurring groupings of cumulative adverse event independent variables, because males tend to have a greater susceptibility to most relevant variables and so we might expect the effect of cumulative adverse event exposure to be greater on them, sooner.Third, the preterm paradox may also (simultaneously) reflect a true difference in jaundice-autism risk due to more intensive early intervention that improves nutrition, limiting jaundice progression and duration. Heightened monitoring likely results in faster diagnosis and treatment for preterms who develop jaundice, hypoglycemia, and/or hypernatremic dehydration - frequent complications of insufficient milk intake associated with breastfeeding that cause most of the approximately 80,000 annual US neonatal hospital readmissions ,5. DiffeIn this way, the preterm paradox may reflect results of a natural experiment in preventive nutrition. While healthy near- and full-term infants act as the experimental group in the modern "exclusive breastfeeding" experiment, preterms are the control group. The control group fares considerably better when it comes to increased autism risk associated with jaundice. This is consistent with analogous findings that neonatal hypoglycemia increases term infants' autism risk threefold but does not increase the risk in preterms  - althouSo the preterm puzzle fits the breastfeeding insufficiencies story, but what else do we know about how medical management influences jaundice outcomes with respect to autism risk? Does phototherapy, the current standard treatment for neonatal jaundice that is determined severe enough to require medical intervention, mitigate the risk? The body of evidence on this question presents an under-recognized puzzle, and a parable in the wisdom of scientific modesty.Phototherapy and autism: we don't know what we think we knowA few generations ago, the standard medical treatment for jaundice in breastfed neonates was switching them to formula. Then modern reformers brought back breastfeeding with a historically anomalous emphasis on exclusivity. Around that time, phototherapy replaced formula-feeding as the standard treatment for neonatal jaundice severe enough to require medical intervention but not severe enough to require the more invasive and riskier hospital treatment of exchange transfusion. But the evidence for this shift was limited. Safety concerns plague current practice. This matters because it underscores the wisdom of applying the precautionary principle by treating the root cause of modal neonatal jaundice, insufficient milk intake associated with breastfeeding, by returning to previously widespread early infant feeding norms centered on ensuring that newborns drink enough milk.Many researchers caution that phototherapy may cause harm  includinWe might see correlations between phototherapy and worse outcomes for four reasons, and they are not mutually exclusive. First, jaundice severity/duration could predict phototherapy treatment, with one or both of those facets of jaundice (in combination with other risk factors) doing the harm. More severely jaundiced neonates are probably more likely to both receive phototherapy treatment for hyperbilirubinemia and to develop later autism as a result of bilirubin-induced brain injury. Duration, an under-studied factor, offers a particularly promising explanation for why even studies that account for jaundice severity may show increased autism risk associated with phototherapy; bilirubin-induced brain injury, with time as a critical factor, could explain irreversible neurological harm. Second, other factors including prematurity and infection could drive jaundice severity/duration, phototherapy exposure, and associated risk. But while jaundice duration is not usually reported and so not included in relevant analyses, preterms are usually identified and analyzed separately due to widespread recognition of increased vulnerability, making this type of explanation less likely. Third, phototherapy could cause iatrogenic harm through posited mechanisms including pro-inflammatory gene expression, incurring additional risks on top of damage jaundice does by the time of diagnosis. This could be largely masked by jaundice and other comorbidities; if phototherapy heightens a spectrum of pro-inflammatory risks, but children already more vulnerable to such risks are disproportionately treated with phototherapy in the first place, then evidence of such harm could be wrongly dismissed on the basis of analyses that appear to correct for confounding variables. Fourth, phototherapy treats jaundice but not other complications of insufficient milk intake including hypoglycemia and hypernatremia; yet it replaced the prior standard treatment of switching jaundiced, breastfed neonates to formula. Thus phototherapy may proxy for untreated insufficient milk intake, other complications of which also risk possible permanent neurological damage and could also explain an apparent phototherapy-autism effect.There is some evidence that phototherapy depends on sufficient milk intake for its efficacy and possibly safety. McDonagh notes that the effect of phototherapy hinges on excretion: "The principal effect of the treatment is not photodegradation of bilirubin, but the conversion of the pigment to structural isomers that are more polar and more readily excreted than the normal, more toxic 'dark' form of the pigment. This, coupled with some photooxidation of bilirubin, diminishes the overall pool of bilirubin in the body and lowers plasma levels" . Thus, pThere appear to be no published meta-analyses on phototherapy and autism or other neurodevelopmental harm along the potentially related continuum of the BIND spectrum that Amin et al.'s review detailed . PubMed There are two clues in the jaundice-autism meta-analytical literature that phototherapy treatment for neonatal jaundice may risk increased autism. First, Amin et al. noted that, according to Sugie et al. , jaundicResults from the six studies included in the most recent jaundice-autism meta-analysis, Kujabi et al's , that alOverall, these sets of estimates substantially overlap, reflecting possibly crucial effects that warrant careful consideration and further research. This evidence troubles the notion that phototherapy protects infants from harm. Rather, it suggests that phototherapy for jaundice may be associated with even more preventable harm, in terms of later autism risk, than jaundice itself. This is a major unreported finding of reported data. Why might it have been missed?A benign possible explanation centers on the common misuse of statistical significance tests as tests of practical significance. A less benign one centers on hospital systems making more money treating neonatal jaundice with phototherapy than they would preventing and treating insufficient milk intake with formula supplementation - and many also standing to lose additional funding for promoting the exclusive breastfeeding paradigm, should they depart from it. These explanations are not mutually exclusive. This review focuses on statistics. Future research should investigate the financial stakes, and how they relate to other sources of possible bias including not only perverse incentives in the classical understanding, but also cognitive and emotional, social and political, and normative and institutional factors and forces.So how often do researchers in this field confuse statistical for practical significance, and what are the implications of this confusion? In these six phototherapy-autism studies, half (three) have confidence intervals overlapping zero, so making this mistake is possible; of those, most (two) make it. Wu et al. concluded "After adjustment for the effects of sociodemographic factors and birth weight, neither hyperbilirubinemia nor phototherapy was an independent risk factor for ASD," even though their confidence intervals estimated substantial possible risk increases . SimilaRecently, the commonality and gravity of such misinterpretation of statistical tests -245 motiBut while banning p-value thresholding offers one simple solution, statistical significance testing misuse is not merely a technical or linguistic problem; it also has cognitive, social, and epistemological roots and implications. Greenland suggests that a cognitive bias of "dichotomania" drives misinterpretation of uncertainty as certainty in dismissing potentially practically significant but statistically non-significant results; statistics education, he argues, should address such biases . Ziliak Such calls to epistemic reorientation have considerable social and political significance. They resonate with Oreskes and Conway's analysis of how wealthy, powerful industries from tobacco to oil have misused uncertainty to misrepresent evidence on serious health risks that threatened their financial interests . ScientiThe simplicity of the technical change belies its implementation’s cognitive and emotional complexity, as it entails adopting a different approach to uncertainty. In publications, it requires shifting substantive results reporting focus from binary statistical significance test results to the practical significance of findings, incorporating uncertainty as part of the actionable scientific picture. This may involve moving from narrow "normal science" puzzle-solving in the Kuhnian sense, to bigger-picture questions about what would make the world better. This work is worth doing, but it takes a lot of (unpaid) work; uncertainty can and should be used to invoke the precautionary principle in the public interest, not only to discredit scientific evidence that threatens the financial interests of the powerful. "Publish or perish" remains the most oft-cited reason why this work remains largely undone, but only naming the problem risks feeding a vicious cycle of alienation and bad science. The political implications of changing this system have yet to be fully articulated or harnessed for change. We should talk more about why critically reviewing statistical methodology is political, and embracing uncertainty is a way of resisting illegitimate authority. Could more institutional support for partnerships between methodology, subject-area, and strategic litigation experts help advance this shift? How might methods training and open-access publishing help experts who are outsiders, like grassroots activists and traditionally disadvantaged stakeholders, advance their causes? How do courts, legislatures, and the public react when "the little guy" uses (among the more reputable) industry tactics and empiricist tools to serve under-served communities? The social and political impact of the recommended shift from statistical to practical significance remains to be shaped by the scholars and institutions struggling to address the crisis in science, and more awareness in shaping it could generate more momentum to help address it. One thing is clear: implementing the indicated epistemic shift toward embracing uncertainty suggests a need for better safety data on phototherapy treatment for neonatal jaundice. Future research should better assess whether and how phototherapy might contribute to neurodevelopmental and other risks in formerly jaundiced neonates. Methodological considerations relevant to the implicit question of interaction suggest this research should be experimental. Greenland cautions that "even with huge effects and perfect data... identification of specific mechanisms of interaction will depend on biologic assumptions that are untestable (nonidentifiable) with epidemiologic observations alone" . We needAt the same time, available evidence on a possible phototherapy-autism link is already sufficient to support the application of the precautionary principle. Clinicians 50 years ago frequently advised mothers to switch their jaundiced, breastfed infants to formula. Then the pendulum swung in favor of the modern exclusive breastfeeding paradigm - a counter-intuitively medicalizing shift, backed by the naturalistic fallacy, that included phototherapy for the epidemic of jaundice associated with insufficient milk intake that it may have created. The evidence suggests it is past time it swung back.DiscussionMeta-analytical estimates of increased possible autism risk associated with neonatal jaundice are consistently substantial. Their notably overlapping confidence intervals, based on included studies’ pooled ORs, reiterate estimated possible risk increases for near- and full-terms up to 67%, 68%, and 76%. These are sizeable possible effects.Discrepant publication bias findings from previous meta-analyses reveal universal misuse of the funnel plot test, leaving open the question of whether selective results reporting may account for the jaundice-autism association. P-curve analysis finds that studies that estimate a statistically significant jaundice-autism link display no evidence of publication bias. We should think of this result as a quality control stamp supporting the validity of the widely-noted correlation.Future research might extend this form of publication bias testing to the available evidence on complications of insufficient milk intake, filling a hole in the meta-analytical literature on the risk of preventable harm associated with common breastfeeding insufficiencies by evaluating a matrix of associated independent variables including hypoglycemia and hypernatremia, and dependent variables with which they covary according to systematic literature review. For jaundice, Amin et al suggested reconceptualizing the dependent variable in terms of a jaundice-associated subtle kernicterus or BIND spectrum of disorders including cognitive delay, ADHD, autism, specific learning disorder, and language disorder. A reconceptualized continuum might also include other manifestations of potential bilirubin-induced neurological harm such as cerebral palsy, epilepsy, hearing impairment, kernicterus, mood disorders, and lower IQ. It should also account for survivorship bias, especially in the preterm subgroup, by including death on a reconceptualized jaundice-associated harm continuum. This measure should count death from acute bilirubin encephalopathy (ABE) as well as death with an uncertain relationship to jaundice, particularly in less well-resourced settings.Including death on the jaundice-associated harm continuum presents special challenges, because the cause of death in these settings may be difficult or impossible to establish. Acknowledging likely bias valence and magnitude, the ethical and practical significance of persistent racial and geographic disparities, and uncertainty may be the best we can do with available data; it is worth noting that here is uncertainty again, and writing it out of analyses leaves out the most vulnerable people concerned. This sort of limitation highlights again the importance of incorporating context, variety, and uncertainty into thinking about risk, and interpreting fuller findings rather than binary results. Synthesized results of the most current, correct, and comprehensive matrix of possible publication bias tests would advance knowledge about the evidential value of statistically significant links between complications of common breastfeeding insufficiencies and a spectrum of clinically significant harms. But that evidence is itself shaped by identified biases in practically significant ways that should at least be mentioned in its interpretation. Just because we cannot count sub-Saharan African infant deaths like we can WEIRD autism cases does not justify ignoring the former.This dilemma resonates with Feinstein's concern that his evidence-based medicine revolution had been hijacked by the "distraction" of quantitative models . He suggThus Feinstein's warning also resonates more broadly with pro-breastfeeding bias, particularly confirmation bias, in the medical and public health literature . Future Reapplying Feinstein's insight to the core finding of substantial possible jaundice-associated autism risk, this review notes that the meta-analytical literature may offer substantial underestimates. This article identifies numerous features in the jaundice-autism literature likely contributing to underestimation of associated harm. In addition to the features identified above - primarily effect dilution from mild case inclusion; selection against severe cases in study-level exclusion criteria and survivorship bias; underpowering from small sample size, low base rate, and over-narrow operationalization of the dependent variable as autism to the exclusion of other harms; and over-representation of WEIRD populations - there are two additional sources of possible underestimation bias that bear mentioning.Operationalizing jaundice as TSB concentration (at best) is another way in which the literature may tend to underestimate the jaundice-autism effect. TSB reportedly has high sensitivity but low specificity for associated brain injury in near- and full-term infants , so relyRelatedly, interaction presents an additional source of possible underestimation and overestimation bias; other predisposing factors and associated conditions may interact with jaundice to magnify risk in many groups, and this magnification may be part of what existing jaundice- and phototherapy-autism effect estimates are really capturing. Subgroup effects  might be difficult to estimate using existing datasets; as previously discussed, there are theoretical and empirical reasons for clinicians to have a lower threshold of suspicion for neonatal jaundice and associated risk in many subgroups. Such clinically relevant subgroup effects may include and interact with etiological subgroup effects. In particular, jaundice associated with insufficient milk intake from breastfeeding correlates in a non-negligible percentage of cases with other complications of insufficient milk intake, chiefly hypernatremic dehydration and hypoglycemia. These other complications could matter for neurodevelopmental risk. While future research might better quantify these conditions' overlap and relationships with risk, a harm prevention focus better applies fundamental medical and research ethics than an approach that focuses primarily on better measuring irreversible damage once it has been done.At the same time, this identification of numerous likely underestimation biases underscores the importance of taking seriously consistent meta-analytic findings of a substantial possible jaundice-autism link. That these findings are consistent is a novel insight: This review shows that the article by Kujabi et al. contains mistakes that, if corrected, would support different conclusions. Its authors misinterpreted statistical significance test results to claim no jaundice-autism association when their analysis showed a quite sizeable possible effect. They also misused the funnel-plot test for publication bias, claiming without merit that its results indicated a high risk of publication bias. Their claim that "there is no evidence to suggest jaundice should be treated more aggressively to prevent autism" was based on these mistakes, incorrect temporal sequencing, and apparent lack of consideration of evidence on jaundice severity, etiology, and different prevention, screening, and treatment approaches. Such mistaken conclusions are likely to contribute to practices that endanger neonates. Kujabi et al. did not substantively respond to attempts to communicate about these issues. The relevant journal, Pediatric Research, declined to retract the paper - despite a six-month lag between this author's first manuscript submission raising these concerns (May 2021) and the journal's print publication of Kujabi et al.'s meta-analysis (November 2021). The journal also declined to publish earlier versions of this review, which was revised and resubmitted at the journal's direction as an article and letter to the editor. As stated in correspondence with the journal, failing to address these issues may contravene relevant publishing ethics  and retrThe evidence supports a paradigm shift in early infant feeding that is radical from the contemporary perspective, but that only represents a long-overdue pendulum swing back toward previous norms. Modern early infant feeding emphasis on exclusive breastfeeding breaks with all known, prior such practices by introducing a days-long neonatal starvation period that compounds the risks associated with the historically anomalous structural vulnerability of a single point of failure, one mother as the exclusive source of recommended infant nutrition and hydration. This break corresponds with a substantial increase in reported autism prevalence that may be causally explained, at least in part, by a corresponding increase in brain damage from increased neonatal jaundice associated with insufficient milk intake due to common breastfeeding insufficiencies as the new exclusive breastfeeding paradigm achieved ascendance.If previously healthy full-term neonates are the experimental group in this natural experiment, preterms are the control group, as they are typically supplemented early and often. The preterm paradox of weakened jaundice-autism effect estimates in this subgroup is consistent with a protective effect of this supplementation. The three previous meta-analyses  agree that the evidence for a jaundice-autism link appears weaker among preterms, losing statistical significance as well as magnitude, and generating estimated possible risk increase values up to 2%, 13%, and 12%, respectively. This weakened effect might be explained in part by factors relating to the "experiment" and in part by other factors. Crucially, heightened monitoring combined with more supplementation likely reduces preterm starvation periods and associated risks  in comparison with full-terms, protecting their brains from bilirubin-induced neurodevelopmental harm. Unrelatedly, the effect could also appear (but not necessarily be) weaker due to smaller subgroup sample sizes, survivorship bias as more preterms die leaving fewer to develop autism, and relatively greater full-term remaining susceptibility with respect to a triggering threshold of cumulative adverse event exposure.Safety concerns about phototherapy are similarly consistent with an overarching story about under-recognized risks associated with the exclusive breastfeeding paradigm, and the crisis in science that they illustrate. Included studies that analyze a possible phototherapy-autism link tend to suggest a possible substantial increased risk of even greater magnitude than the possible jaundice-autism link. A common misuse of statistical significance testing again plays a role in the under-recognition of this risk. Until more definitive studies are available, medical practitioners should try to prevent autism and other possible long-term complications of jaundice and phototherapy treatment. This means that neonatal jaundice needs more aggressive - but less medicalizing - prevention, diagnosis, and treatment. Early, adequate, and often formula or other appropriate infant milk supplementation would prevent harm from insufficient milk intake associated with breastfeeding, the most frequent cause of jaundice in previously healthy near- and full-term neonates. At least until more evidence establishes phototherapy as safe, such supplementation should replace it as the standard of care, ideally before jaundice is even diagnosed in breastfed infants; and when phototherapy treatment is still deemed medically necessary, complementary supplementation should also be standard of care due to greater bilirubin clearance in formula-fed versus breastfed neonates.Future research should investigate how this shift affects neonates with respect to jaundice, later neurodevelopmental outcomes, other potentially associated outcomes such as allergic diseases, and mortality in a variety of settings. How might that research be structured? This question matters for meta-analysis; current methodological recommendations for systematic reviews and meta-analyses, in the form of the Cochrane Handbook chapter on assessing the risk-of-bias in a non-randomized study, suggest authors start by describing "a 'target trial', which is a hypothetical pragmatic randomized trial of the interventions compared in the study, conducted on the same participant group and without features putting it at risk of bias" . One sucIn line with the Cochrane Handbook guidance, this target trial suggests direction for future meta-analysis. Are formula-fed neonates less prone to phototherapy-associated risks, correcting for possible confounds including prematurity and jaundice severity/duration? Do available data let us see whether other complications commonly associated with insufficient milk intake, like hypoglycemia, seem to be associated with phototherapy risk, which might support the hypothesis that phototherapy proxies for starvation? What do data from jaundice complication prevention experiments show?In sum, meta-analyses consistently estimate a quite substantial possible neonatal jaundice-autism effect. The association seems real; there is no evidence of publication bias. Severity and time matter, so prevention is key. Racial and geographic disparities are troubling, and there are many likely sources of underestimation bias in existing estimates including such disparities. The introduction and rise of the historically anomalous exclusive breastfeeding paradigm coincide with and could causally explain increasing autism prevalence from brain damage from complications of insufficient milk intake including jaundice. Preterms' weaker jaundice-autism link is consistent with strong supplementation norms protecting that subgroup. An even more substantial possible phototherapy-autism link is consistent with untreated insufficient milk intake doing at least part of the damage. This review synthesizes the state of the literature, which contains clear and convincing evidence that jaundice may be associated with common and preventable harm to neonates. So what is the public health apparatus doing to protect them?Documents obtained recently through the Freedom of Information Act (FOIA) appear to show that US Centers for Disease Control and Prevention (CDC) awareness of exclusive breastfeeding-associated risks, including the possible jaundice-autism link, has outpaced related safety monitoring and harm prevention efforts . A FOIA The CDC's combination of inaction in public health surveillance on one hand, and continued action in exclusive breastfeeding funding and promotion on the other, contributes substantially to the ongoing dominance of the exclusive breastfeeding paradigm in the US, which may contribute to the paradigm's continued global dominance. Heightened democratic oversight might enhance CDC efforts to fulfill its functions including preventing harm to neonates. For instance, the apparent lack of exclusive breastfeeding safety monitoring and harm prevention efforts, despite agency knowledge of associated risks, might be an appropriate subject for a Congressional inquiry, Government Accountability Office (GAO) review, class action lawsuit, or other standard democratic action intended to bring actual public agency behavior into greater accord with stated public agency goals and obligations.Neonatal jaundice may substantially elevate the risk of preventable, irreversible neurological damage including autism. Publication bias testing with p-curve analysis finds that studies that estimate a statistically significant jaundice-autism link do not show evidence of selective results reporting; they have evidential value. Jaundice severity heightens risk, so preventing progression is key; in most cases, it is as easy as giving a bottle. Racial and geographic disparities seem to mean that traditionally disadvantaged groups are worst-affected by jaundice-associated harm, and global unrepresentativeness likely generates considerable underestimates of associated harm including death and permanent disability. Additional identified factors likely further bias available estimates down, particularly overly narrow operationalization of the outcome of interest, survivorship bias, and inclusion/exclusion criteria that tend to include mild cases and exclude severe ones. The preterm paradox, in which a more vulnerable subgroup appears less vulnerable to the jaundice-autism link, may be due largely to different early infant feeding norms that minimize preterm deprivation/starvation; preterms may be the control group in the exclusive breastfeeding natural experiment. Phototherapy, the current standard treatment for neonatal jaundice, may additionally increase autism risk but could proxy for untreated insufficient milk intake.This review synthesizes a large body of evidence that strongly supports precautionary principle invocation. Until more definitive studies are available, medical practitioners should try to prevent autism and other possible long-term complications of neonatal jaundice by treating its modal root cause, insufficient milk intake associated with breastfeeding. Breastfeeding need not confer risk of preventable neurodevelopmental harm. It is rather the modern construction of "exclusive breastfeeding" - a practice without historical precedent that was invented relatively recently by reformers lacking intergenerational knowledge about breastfeeding safety - that introduces this risk by omitting the safety infrastructure that characterized prior societies' early infant feeding practices; prelacteal feeding, shared nursing, and wetnursing guarded against harm from common breastfeeding insufficiencies. Mothers' milk usually takes days to come in, days in which newborns in previous advanced civilizations (and foraging societies today) would usually drink other mothers' milk or another substitute; ours may be the only society ever in human history in which experts routinely advise caretakers to deprive newborns of adequate nutrition and hydration for days. A return to previously widespread prelacteal and other supplementary feeding practices characterized by early, adequate, and often formula (or other appropriate milk) supplementation would prevent harm from insufficient milk intake associated with breastfeeding, the modal cause of jaundice in previously healthy near- and full-term neonates. Future research and practice should apply the precautionary principle to act to avoid harm when risks are uncertain and stakes are high. Early infant feeding and jaundice management guidelines urgently require change.


Mesenchymal stem cells (MSCs) have long been regarded as critical components of regenerative medicine strategies, given their multipotency and persistence in a variety of tissues. Recently, the specific role of MSCs in mediating regenerative outcomes has been attributed (in part) to secreted factors from transplanted cells, namely extracellular vesicles. This viewpoint manuscript highlights the promise of cell-derived extracellular vesicles as agents of regeneration, enhanced by synergy with appropriate biomaterials platforms. Extracellular vesicles are a potentially interesting regenerative tool to enhance the synergy between MSCs and biomaterials. As a result, we believe these technologies will improve patient outcomes through efficient therapeutic strategies resulting in predictable patient outcomes. Originally conceived to solve the shortage of organs for transplantation, the field of tissue engineering has evolved to encompass a broad clinical scope including regeneration of simple and complex tissues in a variety of clinical settings . At theiMesenchymal stromal cells (MSCs) have attracted significant attention as an ideal multipotent stem cell source since their discovery as fibroblast-colony forming cells . MSCs arConcurrent with advances in tissue engineering, advances in molecular and developmental biology have significantly informed innovative tissue engineering strategies . In thisin vitro administration and secretion in vivo, which increases efficiency and minimizes off-target effects  (PLGA) microsphere. Over time, the PLGA polymer is degraded to allow EV release to local cells. We demonstrated that this delivery system was sufficient to induce odontogenesis  as a novel pulp-capping strategy to protect vital tooth tissue, where EV or cell administration would be otherwise limited. In this way, EVs are locally released from a depot for up to 12 weeks . As a fuComparisons of MSC-based and EV-based regenerative technology consider that MSC sources are well-characterized and readily accessible . While cin vitro and in vivo, sufficient to catalyze wound healing without requiring the transplantation of exogenous cells  may be minimized as an instructive combination of EVs and biomaterial matrix provide sufficient selection criteria for regenerative cell populations, allowing more crude preparations.The potential implications of combined EV and biomaterial therapeutics allow for a tailored, predictable, tissue/patientspecific approach to regeneration, which is highly desirable by both patients and clinicians. EVs and biomaterial constructs are significantly easier to manufacture, store, and regulate compared to MSCs. These attributes represent significant cost savings, as well as increased likelihood of clinical adoption as these technologies would not require sophisticated technical expertise or equipment to implement into existing clinical workflows. As a result of the increased bio-instructive nature of optimized EV-biomaterial platforms, we believe that this may lead to simpler cell sourcing. EVs have been demonstrated to induce cell migration both us cells . In the Predictability of regenerative outcomes is ultimate goal of next generation tissue engineering technology. In this Viewpoint, we highlight the synergy for development of biomaterial platforms which contain EVs, rather than rely on transplantation of stem cells. EVs in conjunction with tuned biomaterials matrices represent an exciting avenue for discovery, translation, and commercialization. We believe that EV-based biomaterial technologies hold the potential to democratize access to regenerative medicine therapeutics across medical disciplines and care settings given their decreased cost, increased manufacturing throughput, advantageous storage character and potentially easier point of care use. Successful clinical translation of these technologies will continue to rely on an intimate understanding of the molecular cargo encapsulated by EVs, interactions at cell-biomaterial interface and means of efficient EV delivery. We believe that regenerative potential represents a significant benefit to patients for a variety of conditions; therapeutic approaches which circumvent challenges associated with, such as EV-based therapies, will allow for more expedient clinical trials, regulatory approval, and widespread clinical adoption, ultimately improving patient care outcomes and quality of life.


Echocardiographic evaluation is an essential part of the diagnostic work-up in patients with known or suspected cardiovascular disease. Transthoracic Doppler echocardiography (TTDE) enables straightforward and reliable visualization of flow in the left anterior descending artery. In the absence of obstructive coronary artery disease, low TTDE-derived coronary flow velocity reserve (CFVR) is considered a marker of coronary microvascular dysfunction (CMD). TTDE CFVR is free from ionizing radiation and widely available, utilizing high-frequency transducers, pharmacologic vasodilator stress, and pulsed-wave Doppler quantification of diastolic peak flow velocities. European Society of Cardiology guidelines recommend TTDE CFVR evaluation only following preceding anatomic invasive or non-invasive coronary imaging excluding obstructive CAD. Accordingly, clinical use of TTDE CFVR is limited and CMD frequently goes undiagnosed. An evolving body of evidence underlines that low CFVR is an important and robust predictor of adverse prognosis and continuing symptoms in angina patients both with and without obstructive CAD. The majority of angina patients have no obstructive CAD, particularly among women. This has led to the suggestion that there may be a gender-specific female atherosclerotic phenotype with less epicardial obstruction, and a low CFVR signifying CMD instead. Nevertheless, available evidence indicates low CFVR is an equally important prognostic marker in both men and women. In this review, TTDE CFVR was evaluated regarding indication, practical and technical aspects, and interpretation of results. Association with symptoms and prognosis, comparison with alternative invasive and non-invasive imaging modalities, and possible interventions in angina patients with low CFVR were discussed, and key research questions were proposed. Coronary artery disease remains one of the leading causes of morbidity and mortality in Western countries in both men and women, with male sex as a risk factor for the early development of coronary artery disease (CAD) , 2. The via transesophageal, and later transthoracic Doppler echocardiography (TTDE)  –12. TTDEy (TTDE) –15. Latey (TTDE) , 16, 17.y (TTDE) .The relevant body of evidence related to TTDE CFVR in angina patients with no obstructive CAD consists of a combination of (i) studies in selected patient cohorts with no obstructive CAD and (ii) studies performed in mixed patient populations both with and without obstructive CAD. We had chosen to include both study types, while clearly highlighting possible interpretation difficulties and limitations of the evidence related to the mixed nature of the patient populations in the latter group.In this review, we evaluated TTDE CFVR regarding indication, practical and technical aspects, and interpretation of results in patients with angina and no obstructive coronary artery disease (ANOCA). Moreover, association with symptoms and prognosis, comparison with alternative invasive and non-invasive imaging modalities, and possible interventions in angina patients with low CFVR were discussed. Finally, we proposed a possible translation of TTDE CFVR to the clinic and questions in relation to angina patients and CFVR that remain unanswered.via several regulatory mechanisms acting in conjunction and interdependent mutual stimulation  a reduced CFR is considered a marker of reduced vasodilatory capacity in the small coronary arteries and arterioles, i.e., coronary microvascular dysfunction (CMD)  , 19, 25.on (CMD) , 26–29.Different vasodilatory and adrenergic agents induce a near-maximal increase in coronary blood flow during CFR quantification, partly due to indirect concomitant activation of both hemodynamic, metabolic, and neural mechanisms. Direct quantification of coronary flow reserve of the entire myocardium can be measured by positron emission tomography (PET). Quantification of flow reserve in any epicardial vessel is obtainable during ICA. TTDE visualization of the main coronary arteries allows assessment of coronary flow velocity as an alternative to absolute flow , 25, 29.via identification of the mid-distal LAD due to its position near the chest wall, thus providing reliable and optimal images and flow curves. Nonetheless, if LAD visualization is difficult in selected patients, measurements may also be obtained in other larger coronary branches . Stress coronary flow velocity should be the highest value obtained during vasodilator infusion and three consecutive cardiac cycles should preferably be obtained, although this is sometimes challenging due to cardiac motion and hyperventilation during stress. The patient may be instructed to hold their breath to limit hyperventilation artifacts . Reading2A-receptors on vascular smooth muscles cells in the microvascular vessel wall eliciting relaxation and vasodilation. Dipyridamole stimulates the same pathway indirectly by inhibiting adenosine deaminase and phosphodiesterase, effectively increasing the adenosine concentration in the vessel wall  perfusion, and single-photon emission computed tomography , 50. Thesel wall , 19, 21.sel wall , 56. It sel wall , 58. How2B adenosine receptors in small airways, and cardiac conduction abnormalities, such as higher degree atrioventricular block, which may be aggravated via stimulation of A1 adenosine receptors  produces comparable values of CFVR although maximal stress coronary flow velocity values are possibly only achieved with adenosine . Due to eceptors , 59, 60.eceptors , 61.via adrenergic stimulation, it also dilates the epicardial coronary arteries via β-adrenergic activation, resulting in the more difficult interpretation of coronary flow velocity changes. The increase in contractility makes consistent measures of coronary flow more difficult. Accordingly, dobutamine stress in CFVR evaluation is mainly suggested as an alternative if adenosine/dipyridamole are contraindicated due to comorbidity  Guideline on Chronic Coronary Syndromes recommended non-invasive assessment of microcirculatory function with TTDE CFVR as a recommendation IIb and evidence level B. Furthermore, it is only recommended in patients with both (i) clear-cut angina, (ii) an abnormal non-invasive functional test , and (iii) epicardial coronary vessels that are normal or have only mild stenosis at coronary computed tomography angiography or ICA . These cIn contrast to the brief coverage of TTDE CFVR in guidelines, an increasing body of White Papers, reviews, and editorials are proposing a reappraisal of the clinical utilization of coronary microvascular assessment , 83–86. Symptom characteristics, together with age, gender, and risk factors, play a prominent role when determining the clinical likelihood of obstructive CAD in stable angina patients. The rationale for this approach is a relatively strong association between typical angina symptoms and obstructive CAD . The symRegarding acute angina symptoms, the recently updated ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation emphasize ICA remains the reference standard for any high-risk patient regardless of sex . HoweverObservational studies have shown a greater risk of major adverse cardiovascular events (MACE) in patients with ANOCA compared with asymptomatic peers, in some studies even similar to those with obstructive CAD , 96. Thep < 0.001]. Subgroup-specific ORs according to sex or presence of obstructive CAD were not reported, plausibly because neither obstructive CAD nor sex were individually related to the composite outcome. Another study from 2014 in the context of SE wall motion analysis also included both patients with suspected and previous obstructive CAD but excluded patients if they had any current regional wall motion abnormality (RWMA)  . The comp < 0.001) and RWMA on SE , while sex was not a significant predictor. The optimal prognostic CFVR cut-off value was comparable for men and women at 2.03 and 2.02, respectively, and was also comparable for patients with known CAD and suspected CAD at 2 and 2.03, respectively, as determined by receiver operating characteristics analyses. These results imply that low CFVR is likely a sex-independent significant predictor adding to the predictive accuracy offered by standard SE wall motion analysis, both in patients with and without known previous CAD.The study of Cortigiani et al. has authored several studies evaluating angina patients using a SE protocol involving both wall motion score index analysis and CFVR measurement , 98, 99.p < 0.001 and 1.6, p = 0.009, respectively). Patients were all-comers for SE and thus represented a wide clinical spectrum of patients from severe to no prior CV disease, and stress modalities including both dobutamine and exercise were used in addition to vasodilators.Another recent study from 2019 is a prospective kind which included a cohort of angina patients and reported preliminary ad-interim composite outcome analyses on 1,867 patients with suspected or known CAD . RWMA anp = 0.001) and with all-cause mortality in secondary exploratory analysis. In subgroup analyses, the presence of a low CFVR remained significantly related to the composite outcome in both patients with and without non-obstructive atherosclerosis on the invasive angiogram (interaction p = 0.91), but there was significant interaction with BMI. CFVR was not associated with the composite outcome in patients with BMI > 30, possibly due to stress CFV acquisition difficulties and underestimation of CFVR in obese subjects. In short, low CFVR was also a significant prognostic marker in this highly selected patient cohort of women with relatively low event rates.In contrast to the more heterogeneous patient cohorts described above, a study published in 2021 prospectively enrolled only women with stable or unstable angina and no prior CAD or other cardiac diseases, and an invasive angiogram with no obstructive CAD . A cutpoA meta-analysis conducted in 2018 about TTDE CFVR also included some of the studies referenced above and found that low CFVR was significantly associated with adverse outcomes in all four studies evaluating angina patients with no obstructive CAD  . There wRegarding the optimal prognostic CFVR cut-off value, the continuous nature of the inverse association between CFVR and the adverse outcome is generally acknowledged. Several studies had shown that subdivision of patients into >2 subgroups according to CFVR value yields a dose-response relationship , 101. NoMoreover, the optimal cut-off is not necessarily the one that maximizes the sensitivity + specificity sum. A decrease in cut-point will necessarily involve a trade-off between the benefit of fewer false positives on the one hand and the disadvantage of more false negatives on the other, and the opposite will be the case if the cut-off point is elevated. Inherently, the issue of optimal cut-point also depends on weighing the benefits and risks of misclassifications and cannot be decided based on receiver operating characteristic statistics alone .In summary, when reviewing the larger prognostic studies utilizing TTDE CFVR, only a few of them directly reported risk estimates according to sex or presence of known or current obstructive CAD. However, most of these studies found that sex and obstructive CAD status did not significantly modify the relation between low CFVR and adverse outcome, suggesting that low CFVR value is a significant and important marker of adverse prognosis in patients with no obstructive CAD regardless of sex.The conception of the relationship between the degree of stenosis of the coronary artery lumen and resting and hyperemic flow, i.e., coronary flow reserve, was initially established using invasive Doppler quantification of coronary flow , 13, 14.Positron emission tomography quantification of mutual and balanced force reduction (MBFR) has long been considered the non-invasive reference standard for CMD diagnosis , 25, 106Application of cMRI perfusion quantification at rest and during pharmacologic stress for the diagnosis of CMD is a research field in development with advantages similar to PET MBFR, e.g., evaluation of all coronary territories and high-quality assessment in obese individuals , 82, 109Cardiac CT perfusion imaging is a further emerging modality in the assessment of both the functional significance of epicardial stenosis and microvascular function, and several studies demonstrated a decrease in perfusion during pharmacologic stress in patients both with and without obstructive CAD , 113–115In summary, TTDE CFVR has shown a good correlation with invasive CFR measurements and ambiguous correlation with PET MBFR, while studies comparing TTDE with cMRI and cardiac CT perfusion were lacking.Given that low CFVR at TTDE is an important predictor of adverse prognosis in angina patients there have been surprisingly few larger interventional studies in this patient group targeting CMD , 117. Con < 100 in all), however, there was not even an insignificant tendency toward an increase in CFVR value in the intervention groups.Three placebo-controlled interventional studies from our group investigated the effect of liraglutide , enalaprThe recent CorMica trial randomized patients with ANOCA to either standard care or a comprehensive interventional diagnostic procedure evaluating both coronary flow reserve and acetylcholine vasoreactivity. Patients were subsequently stratified to specific medical treatment based on the findings, and the study found an improvement in angina severity and quality of life in the intervention group . To our In summary, larger intervention studies providing a stronger evidence base for treatment recommendations in ANOCA patients are needed, ideally investigating the effect on both improvement in microvascular function, e.g., CFVR, symptom amelioration, and prognostic benefit , 119.The main strengths of TTDE CFVR in the evaluation of possible CMD include its non-invasive and inexpensive nature, whereas it is readily available at the bedside with no radiation exposure. Since the echocardiographic examination is already a mainstay in cardiologic evaluation, TTDE CFVR could be readily introduced in clinical practice. Importantly, low TTDE CFVR has been consistently associated with an adverse prognosis in diverse angina patient populations and specifically in the subgroup with no obstructive CAD. Several studies have reported feasibility >90% in the experienced hands, and this number may be even higher with the use of intravenous contrast in challenging cases. Inter- and intra-observer reproducibility has been high , 12, 30.The most important limitation of TTDE CFVR is the need for extensive training even in the hands of otherwise experienced echocardiographers. Examination quality continues to improve even after the performance of more than a 100 examinations, underlining the importance of sustained diligence, supervision, and adjustment to obtain high-quality reliable results . TTDE CFAnother limitation is that only the LAD is accessible for evaluation in all patients, and consequently the obtained CFVR value is strictly the only representative of the LAD region. Even when it is ensured that any present LAD stenosis is non-significant (<50%) and FFR-negative (>0.8), it may be suspected maximal flow is still slightly attenuated, leading to possible underestimation of universal cardiac perfusion during stress. Furthermore, possible vasospasm and/or endothelium-dependent blunted vasodilation may only be assessed invasively using acetylcholine and is therefore not evaluated with TTDE. Comparisons with PET MBFR which is considered the present non-invasive reference standard have been ambiguous, and the use of TTDE CFVR may be challenged in future years by cMRI, PET MBFR, or cardiac CT perfusion in case these modalities become more accessible and are further developed in the decades to come.Transthoracic Doppler echocardiography CFVR is feasible in the large majority, inexpensive, accessible, and carries prognostic information in ANOCA patients of both sexes. A low CFVR identifies angina patients who are at significantly higher risk of adverse outcomes. Given the large number of angina patients with negative non-invasive diagnostic work-up or absence of significant epicardial stenosis at ICA, there is a need for diagnostic tools that are capable of large-scale screening of patients. Although PET MBFR and stress cMRI are becoming increasingly available in many countries the current capacity only allows evaluation of a small part of the large population of ANOCA patients. A suggested algorithm for diagnostic work-up of angina patients integrating CMD evaluation is shown in Ultimately, implementation of new diagnostic tools should be based on their potential to change treatment recommendations and patient outcomes, and current evidence on beneficial treatment interventions in patients with CMD is lacking. This is reflected in the latest guidelines on chronic coronary syndromes which only briefly address microvascular disease. Prior to the potential implementation of CFVR evaluation in guidelines and clinical practice, randomized interventional trials stratifying patients according to low or normal CFVR must clarify whether it is possible to improve patient outcomes, ameliorate symptoms and improve microvascular function in ANOCA patients. We proposed future large-scale studies recruiting ANOCA patients with either a negative non-invasive diagnostic test or an ICA with no significant obstruction and a low CFVR, randomizing them to pharmacological therapy and lifestyle interventions, with a primary outcome of symptom severity and prognosis, and secondary outcome of increased CFVR on follow-up . An equiRegarding the choice of modality for CMD assessment, it may be argued implementation of TTDE CFVR is more straightforward in countries and hospitals with a tradition for SE and wall motion analysis in the evaluation of patients with suspected CAD. Even so, with proper education and training, it is feasible for experienced echocardiographers to include TTDE CFVR in their diagnostic armamentarium. Invasive evaluation of microvascular function, the only modality with the capacity for identification of vasospasm, will most likely remain an option only in a smaller, selected subgroup of angina patients. Both TTDE CFVR, PET MBFR, stress cMRI, or perhaps cardiac CT perfusion may appear as the non-invasive diagnostic test of choice for microvascular assessment in the years to come, likely based on local expertise and availability, and future studies evaluating the benefit of CMD assessment.JS and EP were responsible for the drafting and final revision of the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cerebral venous sinus thrombosis (CVST) is a central nervous system disease characterised by thrombosis in cerebral venous or dural sinuses. Autoimmune diseases, a series of diseases caused by immune responses to autoantigens, are important causes of CVST. The most common diseases that lead to CVST are Behçet’s syndrome, systemic lupus erythematosus, antiphospholipid syndrome, and Sjögren’s syndrome. Each of these diseases have different clinical and imaging manifestations and treatment for CVST varies by aetiology. This review summarises the characteristics and the current management strategies for autoimmune disease-associated CVST and emphasises controversial therapeutic strategies to provide informative reference information for diagnosis and treatment. Risk factors of autoimmune antigens should not be neglected when unconventional CVST occurs, and both drugs and interventional therapy need further standardisation and discussion with more prospective clinical studies. Cerebral venous sinus thrombosis (CVST) is a cerebrovascular disease presenting with headache, changes in intracranial pressure, and disturbance of consciousness caused by hypercoagulability and vessel wall damage. In this review, we aim to summarise the pathological mechanisms, clinical manifestations, therapeutic strategies, and prognosis of CVST caused by each disease. Further studies should be conducted regarding standard therapy and outcomes of autoimmune disease-associated CVST.BS is a recurrent autoimmune disease characterised by systemic vasculitis. The main clinical manifestations are recrudescent oral ulcers and multi-system complications, including genital ulcers, ocular or skin lesions, nervous and haematological system syndromes, and arthritis . ComplicBS-related CVST is mostly identified 24 months after CNS complications appear. It is mainly characterised by intracranial hypertension and a progressive headache , followeFVL) gene mutation and methyltetrahydrofolate reductase (MTHFR) gene polymorphisms, may be related to BS thrombosis but are still controversial  CSF reduction caused by lumbar puncture resulted in venous dilatation and high venous blood viscosity, 2) cerebral ptosis caused by CSF reduction led to venous and sinus occlusion, or 3) the onset of aseptic meningitis and steroid therapy directly caused the CVST  deposition and immune-mediated vasculitis, is also the main cause of SLE-related CVST. LAC and antiphospholipid (APL) antibodies can be detected in 40% of CNS-affected SLE patients , which ppatients . Clusterpatients , 23. Thethe CVST . During the CVST .APS is characterised by recurrent arterial and venous thrombosis, spontaneous abortion, circulating APL antibodies, and thrombocytopaenia, with positive laboratory examinations for both LAC and APL. APS is commonly secondary to SLE but can also be idiopathic, and CVST is less common than transient ischaemic aneurysm or stroke . APS witSS manifests as lymphocyte infiltration in salivary and lacrimal glands, therefore leading to simultaneous xerostomia and xerophthalmia. CNS complications among SS patients include aseptic meningitis, myelitis, microcirculation vasculitis related to white matter lesions, and encephalopathy . The proIntravenous heparin or low-molecular weight heparin followed with oral vitamin K antagonists (VKA) for 3-12 months according to various risk factors is recommended in the current guidelines of different countries –36. HoweThere is a lack of high-level evidence of the safety and efficacy of DOAC treatment for autoimmune disease-associated CVST, and it has never been recommended in current guidelines as a major oral therapy. Nevertheless, several retrospective studies and case reports have used DOACs during APS treatment and revealed a higher risk of thrombosis recurrence than VKA. In addition, a recent meta-analysis of randomised controlled trials on DOACs in APS showed a higher risk for only arterial thrombosis recurrence rather than venous thrombosis, suggesting the therapeutic potential of DOACs for APS-associated CVST.9/L and the haemoglobin level is stable, while the dose is suggested to be halved when platelets <50×109/L; glucocorticoids (GCs) or intravenous immunoglobulin (IVIg) should be used to elevate platelets to a safe level (30-50 ×109/L)  guidelines since there are common characteristics between ITP and APS-associated thrombocytopaenia . Normal  ×109/L) , 41.GCs and immunosuppressive therapy are well-known anti-inflammatory treatments. Azathioprine (AZP) is the top immunosuppressants for BS, while methotrexate, cyclophosphamide, and mycophenolate mofetil (MMF) are also recommended . CertainET has been conventionally used in refractory CVST patients when large and extensive thrombi are unlikely to dissolve after anticoagulant treatment and has shown a reasonable safety profile . In a siAutoimmune diseases are important risk factors for CVST, of which pathological mechanisms can be explained based on the mechanism of thrombosis, such as hypercoagulability or inflammation caused by vascular endothelial injury, while the specific pathologies of autoimmune disease-associated CVST have yet to be studied. It is easy to diagnose CVST according to clinical symptoms, case history, and radiography. When clinical manifestations like intracranial hypertension cannot be explained by conventional aetiologies of CVST, serum autoimmune antibodies and thrombohaemophilia tests should be considered. There are no standard or official guidelines for autoimmune disease-associated CVST treatment. Immune and anticoagulant therapy should be carefully analysed before use since there are advantages and disadvantages according to various complications and severities. Generally, the prognosis of autoimmune disease-related CVST is favourable with prompt treatment.BZ and YL conceived the review, collected the data, wrote the paper, and contributed equally to this work. WZ helped analyse the data and revise the manuscript. LC and FD supervised the review and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a grant from the National Natural Science Foundation of China (82071293) and the National Natural Science Foundation of China (82071351).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Droughts negatively affect sorghum’s productivity and nutritional quality. Across its diversity centers, however, there exist resilient genotypes that function differently under drought stress at various levels, including molecular and physiological.Sorghum is an economically important and a staple food crop for over half a billion people in developing countries, mostly in arid and semi-arid regions where drought stress is a major limiting factor. Although sorghum is generally considered tolerant, drought stress still significantly hampers its productivity and nutritional quality across its major cultivation areas. Hence, understanding both the effects of the stress and plant response is indispensable for improving drought tolerance of the crop. This review aimed at enhancing our understanding and provide more insights on drought tolerance in sorghum as a contribution to the development of climate resilient sorghum cultivars. We summarized findings on the effects of drought on the growth and development of sorghum including osmotic potential that impedes germination process and embryonic structures, photosynthetic rates, and imbalance in source-sink relations that in turn affect seed filling often manifested in the form of substantial reduction in grain yield and quality. Mechanisms of sorghum response to drought-stress involving morphological, physiological, and molecular alterations are presented. We highlighted the current understanding about the genetic basis of drought tolerance in sorghum, which is important for maximizing utilization of its germplasm for development of improved cultivars. Furthermore, we discussed interactions of drought with other abiotic stresses and biotic factors, which may increase the vulnerability of the crop or enhance its tolerance to drought stress. Based on the research reviewed in this article, it appears possible to develop locally adapted cultivars of sorghum that are drought tolerant and nutrient rich using modern plant breeding techniques. Drought is one of the most prominent abiotic stresses limiting crop production and productivity. It occurs recurrently in large parts of the world and affects all major crops. Severe drought substantially reduces crop yield and quality, and it can lead to famine in food insecure areas. However, crops differ in their tolerance to drought stress, and variation exists within a crop species.Myriads of biotic and abiotic stresses persistently challenge crops growing in the field under various environmental conditions. Because of global climate change, temperature and atmospheric COSorghum is a major staple crop for over half a billion people, mostly in developing countries in the semi-arid and arid tropics. It provides protein, fiber rich, and gluten-free nutrition  significantly reduces rate and percentage of seed germination in sorghum  growth  and post-flowering stage (between flowering and grain development)  were found to contain significantly higher protein content than others , transpiration rate (E), A:E ratio and WUE under normal and drought stress conditions. Different studies reported that a rise in A:E and WUE improve drought tolerance during pre-flowering stage in sorghum . The identification and understanding of the QTLs associated with these traits are highly important for the development of drought-tolerant sorghum cultivars. A number of drought related traits have been identified and several QTLs associated with these traits were mapped in sorghum Table . However00MN7645 , has been used as a major source of stay green genes in sorghum breeding programs aimed at improving its drought tolerance, in the United States, Australia, India and other parts of the world  and B35 ,  associated with stay green were identified using 98 RILs (derived from B35 × Tx7000 cross) grown under post-flowering drought stress conditions  are indeed associated with the trait. Similarly, using NILs mapping population derived from BTx642 × RTx7000, alleles that contribute to stay green were mapped to the four major QTLs   and abscisic acid (ABA), which are important for drought and heat stress tolerance and grain yield in sorghum  explaining 20–30% of the phenotypic variability in chlorophyll content were identified through exposing 98 sorghum RILs to post-flowering drought stress  and accounted for 46% of phenotypic variance  and maturity (SPADM) were associated with stay green ratings in sorghum  and proteins associated with response to ABAs  proteins, Delta 1-pyrroline-5-carboxylate synthase (P5CS2), high-affinity Kn et al. , which wn et al. . Furthern et al. . The tran et al. . Two ethn et al. .Transcripts encoding for the mitochondrial Transcription tERmination Factor (mTERF) family, anion-transporting ATPase family proteins and LEA, hydroxyproline-rich glycoprotein family protein were highly up-regulated under mild stress whereas proteins with putative homology to ABscisic acid- Insensitive 2 (ABI2) and mannosyltransferase were among the most highly elevated during severe drought stress  are plant-specific transcriptional regulators characterized by one or more DNA binding AP2/ERF domains. AP2/ERFBP TFs perform diverse roles in plant biological processes, such as cell proliferation, vegetative and reproductive development, plant hormone and abiotic/biotic stress responses  metabolism. Eleven transcription-related proteins (six histone proteins and three nascent polypeptide-associated complex (NAC) subunit beta proteins were expressed in root proteome of only drought-tolerant genotype in response to water deficit condition , leaf relative water content (RWC), leaf water potential (Ψ), and leaf osmotic potential (π) in sorghum (Machado and Paulsen Domestication of crops has led to the loss of genetic diversity in plants and the microorganisms associated with them (Perez-Jaramillo et al. Drought is a key limiting factor in major sorghum growing areas, which substantially reduces the productivity and hence production of the crop. In the face of climate change and decreasing water availability, the effects of drought stress imposed at different growth and developmental stages on grain yield and quality of sorghum are well established. Although drought stress spanning across several plant developmental stages is a common occurrence in major sorghum growing areas, most studies have focused only on the effect of drought stress that occur during specific plant growth stages. Hence, in order to gain a more definitive understanding of the overall effect of drought on sorghum and the characteristics of plant responses to drought, it is imperative to conduct well-planned and detailed studies that cover the entire plant growth and development stages. Overall, developing drought-tolerant cultivars that suit a range of agro-climatic conditions, especially in the arid and semi-arid regions, is crucial to avert the negative impacts of drought stress. In this regard, the identification of sorghum genotypes with strong tolerance to drought stress is the first step towards the development of drought-tolerant cultivars bearing various desirable characteristics. However, identification of acceptable drought-tolerant genotypes requires well-planned studies that represent spatiotemporal patterns, variable intensity and duration of drought stress during crop growing season, and variation in edaphic conditions across major sorghum growing areas. Dissecting molecular mechanisms of drought tolerance using genetics, genomics, proteomics and metabolomics studies would lead to the identification of molecular signatures that can be targeted for improving the drought tolerance of desirable sorghum germplasm using molecular breeding techniques.KBA, ME, AC and MG contributed to conceptualization of the manuscript. KBA, ME and RV wrote the draft manuscript. DN and TM provided the sorghum nutritional content data. MG, AC, TF, DN and TM edited the manuscript and contributed to the revision process. All authors read and approved the submitted version.