Using oxytocin receptor gene methylation to pursue an agenda

A pair of 2019 Virginia studies involved human mother/infant subjects:

“We show that OXTRm [oxytocin receptor gene DNA methylation] in infancy and its change is predicted by maternal engagement and reflective of behavioral temperament.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795517 “Epigenetic dynamics in infancy and the impact of maternal engagement”

“Infants with higher OXTRm show enhanced responses to anger and fear and attenuated responses to happiness in right inferior frontal cortex, a region implicated in emotion processing through action-perception coupling.

Infant fNIRS [functional near-infrared spectroscopy] is limited to measuring responses from cerebral cortex..it is unknown whether OXTR is expressed in the cerebral cortex during prenatal and early postnatal human brain development.”

https://www.sciencedirect.com/science/article/pii/S187892931830207X “Epigenetic modification of the oxytocin receptor gene is associated with emotion processing in the infant brain”


Both studies had weak disclosures of limitations on their findings’ relevance and significance. The largest non-disclosed contrary finding was from the 2015 Early-life epigenetic regulation of the oxytocin receptor gene:

These results suggest that:

  • Blood Oxtr DNA methylation may reflect early experience of maternal care, and
  • Oxtr methylation across tissues is highly concordant for specific CpGs, but
  • Inferences across tissues are not supported for individual variation in Oxtr methylation.

This rat study found that blood OXTR methylation of 25 CpG sites couldn’t accurately predict the same 25 CpG sites’ OXTR methylation in each subject’s hippocampus, hypothalamus, and striatum (which includes the nucleus accumbens) brain areas. Without significant effects in these limbic system structures, there couldn’t be any associated behavioral effects.

But CpG site associations and correlations were deemed good in the two current studies because they cited:

“Recent work in prairie voles has found that both brain- and blood-derived OXTRm levels at these sites are negatively associated with gene expression in the brain and highly correlated with each other.”

https://www.sciencedirect.com/science/article/pii/S0306453018306103 “Early nurture epigenetically tunes the oxytocin receptor”

The 2018 prairie vole study – which included several of the same researchers as the two current studies – found four nucleus accumbens CpG sites that had high correlations to humans. Discarding one of these CpG sites allowed their statistics package to make a four-decimal place finding:

“The methylation state of the blood was also associated with the level of transcription in the brain at three of the four CpG sites..whole blood was capable of explaining 94.92% of the variance in Oxtr DNA methylation and 18.20% of the variance in Oxtr expression.”

Few limitations on the prairie vole study findings were disclosed. Like the two current studies, there wasn’t a limitation section that placed research findings into suitable contexts. So readers didn’t know researcher viewpoints on items such as:

  • What additional information showed that 3 of the 30+ million human CpGs accurately predicted specific brain OXTR methylation and expression from saliva OXTR methylation?
  • What additional information demonstrated how “measuring responses from cerebral cortex” although “it is unknown whether OXTR is expressed in the cerebral cortex” provided detailed and dependable estimates of limbic system CpG site OXTR methylation and expression?
  • Was the above 25-CpG study evidence considered?

Further contrast these three studies with a typical, four-point, 285-word limitation section of a study like Prenatal stress heightened adult chronic pain. The word “limit” appeared 6 times in that pain study, 3 times in the current fNIRS study, and 0 times in the current maternal engagement and cited prairie vole studies.

Frank interpretations of one’s own study findings to acknowledge limitations is one way researchers can address items upfront that will be questioned anyway. Such analyses also indicate a goal to advance science.

Prenatal stress heightened adult chronic pain

This 2019 McGill rodent study found:

Prenatal stress exacerbates pain after injury. Analysis of mRNA expression of genes related to epigenetic regulation and stress responses in the frontal cortex and hippocampus, brain structures implicated in chronic pain, showed distinct sex and region-specific patterns of dysregulation.

In general, mRNA expression was most frequently altered in the male hippocampus and effects of prenatal stress were more prevalent than effects of nerve injury. Recent studies investigating chronic pain-related pathology in the hippocampus in humans and in rodent models demonstrate functional abnormalities in the hippocampus, changes in associated behavior, and decreases in adult hippocampal neurogenesis.

The change in expression of epigenetic- and stress-related genes is not a consequence of nerve injury but rather precedes nerve injury, consistent with the hypothesis that it might play a causal role in modulating the phenotypic response to nerve injury. These findings demonstrate the impact of prenatal stress on behavioral sensitivity to a painful injury.

Decreased frontal mRNA expression of BDNF and BDNF IV in male offspring following neuropathic pain or prenatal stress respectively. Relative mRNA expression of other stress-related genes (GR17, FKBP5) and epigenetic-related genes (DNMTs, TETs, HDACs, MBDs, MeCP2) in male offspring.

A drastic decrease in expression of HDAC1 was observed in all groups compared to sham-control animals. CCI: chronic constriction injury.”


The study’s design was similar to the PRS (prenatal restraint stress) model, except that the PRS procedure covered gestational days 11 to 21 (birth):

“Prenatal stress was induced on Embryonic days 13 to 17 by restraining the pregnant dams in transparent cylinder with 5 mm water, under bright light exposure, 3 times per day for 45 min.”

None of the French, Italian, and Swiss PRS studies were cited.

The limitation section included:

  1. “Although our study shows significant changes in expression of epigenetic enzymes, it didn’t examine the impact of these changes on genes that are epigenetically regulated by this machinery or their involvement in intensifying pain responses.
  2. The current study is limited by the focus on changes in gene expression which do not necessarily correlate with changes in protein expression.
  3. Another limitation of this study is the inability to distinguish the direct effects of stress in utero vs. changes in the dam’s maternal behavior due to stress during pregnancy; cross-fostering studies are needed to address this issue.
  4. Functional experiments that involve up and down regulation of epigenetic enzymes in specific brain regions are required to establish a causal role for these processes in chronic pain.”

What do you think about possible human applicability of this study’s “effects of prenatal stress were more prevalent than effects of nerve injury” finding?

Are there any professional frameworks that instruct trainees to recognize that if a person’s mother was stressed while pregnant, their prenatal experiences could cause more prevalent biological and behavioral effects than a recent injury?

https://www.sciencedirect.com/science/article/pii/S0166432819315219 “Prenatal maternal stress is associated with increased sensitivity to neuropathic pain and sex-specific changes in supraspinal mRNA expression of epigenetic- and stress-related genes in adulthood” (not freely available)

A review of fetal adverse events

This 2019 Australian review subject was fetal adversities:

“Adversity during the perinatal period is a significant risk factor for the development of neurodevelopmental disorders long after the causative event. Despite stemming from a variety of causes, perinatal compromise appears to have similar effects on the developing brain, thereby resulting in behavioural disorders of a similar nature.

These behavioural disorders occur in a sex‐dependent manner, with males affected more by externalizing behaviours such as attention deficit hyperactivity disorder (ADHD) and females by internalizing behaviours such as anxiety. The term ‘perinatal compromise’ serves as an umbrella term for intrauterine growth restriction, maternal immune activation, prenatal stress, early life stress, premature birth, placental dysfunction, and perinatal hypoxia.

The above conditions are associated with imbalanced excitatory-inhibitory pathways resulting from reduced GABAergic signalling. Methylation of the GAD1/GAD67 gene, which encodes the key glutamate‐to‐GABA synthesizing enzyme Glutamate Decarboxylase 1, resulting in increased levels of glutamate is one epigenetic mechanism that may account for a tendency towards excitation in disorders such as ADHD.

The posterior cerebellum’s role in higher executive functioning is becoming well established due to its connections with the prefrontal cortex, association cortices, and limbic system. It is now suggested that disruptions to cerebellar development, which can occur due to late gestation compromises such as preterm birth, can have a major impact on the region of the brain to which it projects.

Activation of the maternal hypothalamic-pituitary adrenal (HPA) axis and placental protection. Psychological stress is perceived by the maternal HPA axis, which stimulates cortisol release from the maternal adrenal gland.

High levels of maternal cortisol are normally prevented from reaching the fetus by the 11β-hydroxysteroid dehydrogenase 2 (HSD11B2) enzyme, which converts cortisol to the much less active cortisone. Under conditions of high maternal stress, this protective mechanism can be overwhelmed, with the gene encoding the enzyme becoming methylated, which reduces its expression allowing cortisol to cross the placenta and reach the fetus.”


The reviewers extrapolated many animal study findings to humans, although most of their own work was with guinea pigs. The “suggest” and “may” qualifiers were used often – 22 and 37 times, respectively. More frequent use of the “appears,” “hypothesize,” “propose,” and “possible” terms was justified.

As a result, many reviewed items such as the above graphic and caption should be viewed as hypothetical for humans rather than reflecting solid evidence from quality human studies.

The reviewers focused on the prenatal (before birth) period more than the perinatal (last trimester of pregnancy to one month after birth) period. There were fewer mentions of birth and early infancy adversities.

https://onlinelibrary.wiley.com/doi/abs/10.1111/jne.12814 “Perinatal compromise contributes to programming of GABAergic and Glutamatergic systems leading to long-term effects on offspring behaviour” (not freely available)

Organismal aging and cellular senescence

I’ll curate this 2019 German review through its figures:

“With the discovery of beneficial aspects of cellular senescence and evidence of senescence being not limited to replicative cellular states, a redefinition of our comprehension of aging and senescence appears scientifically overdue.

Figure 1. Current determinants and relevant open questions, marking the processes of aging and senescence as discussed in the text. Aspects represented in green are considered as broadly accepted or scientifically consolidated. Novel aspects that are yet unproven, or are under debate, are highlighted in red.

SASP = senescence-associated secretory phenotype. AASP = putative aging-associated secretory phenotype as suggested in the text.

Figure 2. Theories on the causality and purpose of aging. Graphically summarized are four contrasting concepts crystallized from current evidence addressing the inductive driving force of aging. Apart from a stochastic deleteriome, there are arguments for a pseudo-programmed, programmed or at least partially programmed nature of aging.

Figure 3. Comparative representation of the aging and senescence processes highlighting different levels of interaction and putative sites of interventions.

(1) As discussed in the text, causative mechanisms of aging are still not well understood, however, multiple factors including genetic, epigenetic and stress-related effects seem to have an orchestrated role in the progression of aging. Senescence on the other hand, is seen as a programmed response to different kinds of stressors, which proceed in defined stages. Whether, in analogy, aging also follows a defined program or sequential stages is not known.

(2) Senescence involves autocrine and paracrine factors, which are responsible for a ‘seno-infection’ or bystander effect in neighboring cells. There is currently no direct evidence for a similar factor composition propagating the aging process via a kind of ‘gero-infection’.

(3) Accumulation of senescent cells has been described as a hallmark of aging; however, whether they are a causative factor or a consequence of tissue and organismal aging is still unknown. As discussed in the text, it appears possible that aging and senescence mutually influence each other through positive feedback at this level, leading to accelerated tissue damage and aging.

(4,5) Clearance of senescent or aging cells might constitute putative targets for interventional approaches aimed to reduce or reverse the impact of aging and improve cell and tissue homeostasis by inducing a ‘rejuvenation’ process.

Figure 4. Pathological and beneficial functions of aging and senescence, according to current knowledge. In red are represented pathological consequences and in green beneficial functions of aging and senescence.

The impact of aging has mainly been described at the organismal level, since a complete cellular functional profile has not yet been established. Accordingly, whether beneficial consequences of the aging process exist at the cellular level is unclear.”


The reviewers’ position on Figure 2 was:

“In our view, recent evidence that

  • Senescence is based on an unterminated developmental growth program and the finding that
  • The concept of post-mitotic senescence requires the activation of expansion, or ‘growth’ factors as a second hit,

favor the assumption that aging underlies a grating of genetic determination similarly to what is summarized above under the pseudo-programmed causative approach.”

Their position on Figure 4’s beneficial effects of aging began with the sentence:

“If we assume that aging already starts before birth, it can be considered simply a developmental stage, required to complete the evolutionary program associated with species-intrinsic biological functions such as reproduction, survival, and selection.”

Cited studies included:

https://www.mdpi.com/2073-4409/8/11/1446 “Dissecting Aging and Senescence-Current Concepts and Open Lessons”

Do genes or maternal environments shape fetal brains?

This 2019 Singapore human study used Diffusion Tensor Imaging on 5-to-17-day old infants to find:

“Our findings showed evidence for region-specific effects of genotype and GxE on individual differences in human fetal development of the hippocampus and amygdala. Gene x Environment models outcompeted models containing genotype or environment only, to best explain the majority of measures but some, especially of the amygdaloid microstructure, were best explained by genotype only.

Models including DNA methylation measured in the neonate umbilical cords outcompeted the Gene and Gene x Environment models for the majority of amygdaloid measures and minority of hippocampal measures. The fact that methylation models outcompeted gene x environment models in many instances is compatible with the idea that DNA methylation is a product of GxE.

A genome-wide association study of SNP [single nucleotide polymorphism] interactions with the prenatal environments (GxE) yielded genome wide significance for 13 gene x environment models. The majority (10) explained hippocampal measures in interaction with prenatal maternal mental health and SES [socioeconomic status]. The three genome-wide significant models predicting amygdaloid measures, explained right amygdala volume in interaction with maternal depression.

The transcription factor CUX1 was implicated in the genotypic variation interaction with prenatal maternal health to shape the amygdala. It was also a central node in the subnetworks formed by genes mapping to the CpGs in neonatal umbilical cord DNA methylation data associating with both amygdala and hippocampus structure and substructure.

Our results implicated the glucocorticoid receptor (NR3C1) in population variance of neonatal amygdala structure and microstructure.

Estrogen in the hippocampus affects learning, memory, neurogenesis, synapse density and plasticity. In the brain testosterone is commonly aromatized to estradiol and thus the estrogen receptor mediates not only the effects of estrogen, but also that of testosterone.”

https://onlinelibrary.wiley.com/doi/full/10.1111/gbb.12576 “Neonatal amygdalae and hippocampi are influenced by genotype and prenatal environment, and reflected in the neonatal DNA methylome” (not freely available)

Transgenerational epigenetic inheritance of thyroid hormone sensitivity

My 500th curation is a 2019 Portuguese human study of Azorean islanders:

“This study demonstrates a transgenerational epigenetic inheritance in humans produced by exposure to high TH [thyroid hormone] in fetal life, in the absence of maternal influences secondary to thyrotoxicosis. The inheritance is along the male line.

The present work took advantage of the relatively frequent occurrence of fetal exposure to high TH levels in the Azorean island of São Miguel. This is the consequence of a missense mutation in the THRB gene causing the amino-acid replacement R243Q, resulting in reduced affinity of the TH receptor beta (TRβ) for TH and thus RTHβ.

Its origin has been traced to a couple who lived at the end of the 19th century. F0 represented the third generation and F3 the sixth and seventh generation descendant.”


These researchers provided the first adequately evidenced human transgenerational epigenetic inheritance study! However, the lead sentence in its Abstract wasn’t correct:

“Evidence for transgenerational epigenetic inheritance in humans is still controversial, given the requirement to demonstrate persistence of the phenotype across three generations.”

Although found in this study, there is no “requirement to demonstrate persistence of the phenotype.” Observing the same phenotype in each generation is NOT required for human transgenerational epigenetic inheritance to exist!

Animal transgenerational studies have shown that epigenetic inheritance mechanisms may both express different phenotypes for each generation:

and entirely skip a phenotype in one or more generations!

  • Transgenerational pathological traits induced by prenatal immune activation found a F2 and F3 generation phenotype of impaired sociability, abnormal fear expression and behavioral despair – effects that weren’t present in the F1 offspring;
  • The transgenerational impact of Roundup exposure “Found negligible impacts of glyphosate on the directly exposed F0 generation, or F1 generation offspring pathology. In contrast, dramatic increases in pathologies in the F2 generation grand-offspring, and F3 transgenerational great-grand-offspring were observed.” (a disease phenotype similarly skipped the first offspring generation);
  • Epigenetic transgenerational inheritance mechanisms that lead to prostate disease “There was also no increase in prostate histopathology in the directly exposed F1 or F2 generation.” (a prostate disease phenotype skipped the first two male offspring generations before it was observed in the F3 male offspring); and
  • Epigenetic transgenerational inheritance of ovarian disease “There was no increase in ovarian disease in direct fetal exposed F1 or germline exposed F2 generation. The F3 generation can have disease while the F1 and F2 generations do not, due to this difference in the molecular mechanisms involved.” (an ovarian disease phenotype similarly skipped the first two female offspring generations before it was observed in the F3 female offspring).

Details of epigenetic inheritance mechanisms were provided in Another important transgenerational epigenetic inheritance study. Mechanisms from fetal exposure to the fungicide vinclozolin were compared with mechanisms from fetal DDT exposure, and summarized as:

The fetal exposure initiates a developmental cascade of aberrant epigenetic programming, and does NOT simply induce a specific number of DMRs [DNA methylation regions] that are maintained throughout development.

I emailed references to the studies in the first five above curations to the current study’s corresponding coauthor. They replied “What is the mechanism for the transgenerational inheritance you describe?” and my reply included a link to the sixth curation’s study.

Are there still other transgenerational epigenetically inherited effects due to fetal exposure to high thyroid hormone levels?

https://www.liebertpub.com/doi/full/10.1089/thy.2019.0080 “Reduced Sensitivity to Thyroid Hormone as a Transgenerational Epigenetic Marker Transmitted Along the Human Male Line”

PNAS politics in the name of science

This 2019 Germany/Canada human fetal cell study was a Proceedings of the National Academy of Sciences of the United States of America direct submission:

“In a human hippocampal progenitor cell line, we assessed the short- and long-term effects of GC [glucocorticoid] exposure during neurogenesis on messenger RNA expression and DNA methylation profiles. Our data suggest that early exposure to GCs can change the set point of future transcriptional responses to stress by inducing lasting DNAm changes.”


The study’s basic finding was that cells had initial responses to stressors that primed them for subsequent stressors. Since this finding wasn’t new, the researchers tried to make it exciting by applying it to novel contexts that were yet circumscribed by official paradigms.

Hypothesis-seeking associations of human fetal hippocampal cell behaviors with human behaviors were flimsy stretches, as were correlations to placental measurements. These appeared to have been efforts to find headline-making effects.

There wasn’t even a hint of the principle described in Epigenetic variations in metabolism:

“Because of the extreme interconnectivity of cell regulatory networks, even at the cellular level, predicting the impact of a sequence variant is difficult as the resultant variation acts:

  • In the context of all other variants and
  • Their potential additive, synergistic and antagonistic interactions.

This phenomenon is known as epistasis.”

It would have condemned pet models of reality to admit that a cell exists in multiple contexts of other cells with potential additive, synergistic, and antagonistic interactions.

A research proposal to trace a specific cell type’s behaviors – while isolated from their extremely interconnected networks – to trillion-celled human behaviors would be rejected in less-politicized organizations.

Sanctioned speculations manifested in this paper with phrases such as “although not significant..” and “although not directly tested..” The study’s title was probably a disappointment in that it conformed to the study’s evidence.

Involvements of psychiatry departments at the pictured Kings College, Harvard, etc., as part of PNAS entrenched politics, retard advancements of science past approved paradigms.

This is my final curation of PNAS papers.

https://www.pnas.org/content/pnas/early/2019/08/08/1820842116.full.pdf “Glucocorticoid exposure during hippocampal neurogenesis primes future stress response by inducing changes in DNA methylation”