Lecture 12: Developmental Programming

What is Developmental Origins of Health and Disease (DOHaD)?

• It suggests that events during development increase the risk of disease

• These events do not cause disease but alter physiology and confer a greater risk of having certain disease with age

• It suggests that adverse in utero environment disrupt normal fetal growth and development, leading to FGR and a small birth weight, which in turn leads to an increased susceptibility to various diseases as an adult (e.g. cardiovascular, metabolic)

What is the Barker Hypothesis?

• Concept proposed by Professor David Barker (discovered mid–late 1980s; first published in the 1990s) → led to the DOHaD paradigm

• Based on studies of men and women born in Hertfordshire, SE England, where detailed birthweight records had been kept since 1911.

• Demonstrated a strong correlation between low birth weight and increased risk of coronary heart disease in adulthood (~70 years later).

• Suggests that adverse conditions in fetal life can “program” long-term health and disease risk.

• Also known as fetal or developmental programming.

What Did Barker’s Hertfordshire Cohort Study Show About Birthweight and Adult Coronary Disease?

• The standardised mortality ratio (SMR) for coronary heart disease was calculated, with higher SMR values indicating increased mortality from CHD.

• Individuals born with a low birth weight (<5.5 lb ≈ 2.5 kg) had a higher incidence of CHD in adulthood. → high risk of CHD with low birthweight

  • CHD risk decreases as birth weight increases, with a slight increase again at very high birth weights.

• This is more pronounced in men, but also present in women.

What Did Barker’s Hertfordshire Cohort Study Show About Birthweight and Type 2 Diabetes?

• 370 Males, aged 60-71 from Hertfordshire, were assessed

• Prevalence for type 2 diabetes

  • 40% in males with a birthweight of <5.5 lb (2.5 kg)

  • 14% in males with a birthweight of 9.5 lb (4.3kg)

• Low birthweight is not a cause; but increases the risk for Type 2 diabetes in later life

• Implications of DOHaD for public health → a poor message would suggest the sole cause of T2DM is low birthweight; not the case, lifestype factors play an important role

What did the Barker Hertfordshire cohort study show about birth weight and adult BMI?

• Assessed the effect of birthweight and BMI in adulthood

  • Least favourable condition: Low birthweight + highest adult BMI → combined effect → significantly increased risk of insulin resistance and type 2 diabetes.

• Most favourable: High birthweight + lowest adult BMI → normal metabolic risk.

What did the Barker Hertfordshire cohort study show about birth weight and adult obesity?

• Investigated effect of birthweight on adult obesity

  • Low birthweight (<5.5 lbs / 2.5 kg): 43% increased risk of adult obesity.

  • High birthweight (>10 lbs / 4.5 kg): 34% increased risk of adult obesity.

• Demonstrates that both low and high birthweights are associated with a higher risk of adult metabolic disease, demonstrating a U-shaped relationship between birthweight and adult obesity.

What are the recent developments in understanding the Developmental Origins of Health and Disease (DOHaD)?

• Not limited to low birthweight: Preterm birth and high birthweight also influence long-term health.

• Birthweight as a surrogate marker → Reflects the in-utero environment; low or high BW suggests a suboptimal environment affecting fetal health.

• Critical windows beyond the fetal period, with preconception, early pregnancy, throughout pregnancy, and early postnatal years (first 2–4 years), having lifelong health consequences if disrupted or impaired.

  • Pregnancy is a marker of long term health and risk association → used as a window for theraputic intervention

What Maternal and Environmental Factors Influence Fetal Growth and DOHaD Outcomes?

• Nutrition:

  • Low birthweight: indicator of fetal undernutrition;

  • May result from maternal undernutrition or placental dysfunction.

• Early development outside an ideal environment: Preterm birth, IVF/embryo culture.

• Stress, with Maternal glucocorticoid exposure reducing birthweight and leading to DOHaD-related abnormalities and phenotypes in the offspring.

How do physiological adaptations to a sub-optimal prenatal environment affect postnatal health?

• Mismatch between pre- and post-natal environment: Low prenatal nutrition (leading to low birthweight) followed by abundant postnatal nutrition leads to a rapid catch-up growth in offspring to match (though to be bad)

  • Mismatch between what the fetus has adapted to and is exposed to in pregnancy and what it is exposed to post-natally

  • Confirmed with animal models, with undernutrition during pregnancy + high-fat diet after birth leading to similar outcomes.

• This results in an altered HPA axis and altered metabolism (promotion of fat gain), increasing the risk of obesity, insulin resistance, high blood pressure, cardiovascular disease, and altered stress response → post natal enviroment does not match anticipated environment

• Adaptive prenatal changes may become maladaptive if the postnatal environment differs from the prenatal expectation.

How do critical developmental windows in pregnancy influence organ-specific outcomes in growth restriction?

• Different organs develop at specific gestational time points, known as developmental windows and are critical time points

• Acute stressors or insults during these windows can have different effects on organ growth and function:

  • Kidneys → involved in blood pressure regulation; insults can increase risk of hypertension.

  • Pancreas → important for blood glucose control through insulin; insults can increase risk of diabetes.

  • Brain → development occurs towards the end of pregnancy; psychological and behavioural insults are of concern.

• Placenta → can affect all fetal organs; insults affecting the placenta can impact multiple fetal organs simultaneously.

What Did Khashan et al’s Study Show About the Effect of Severe Stress on Pre-Term Birth

• Cohort study: investigated individuals suffering from severe stress , characterised as the death or severe illness of a close relative (form of acute or chronic stress)

  • Stress exposure 6 months prior to pregnancy → Increased rate of preterm birth (RR - 1.16; 95% CI)

  • Stress exposure during pregnancy (1^st/2^nd trimester) had little effect on the risk of pre-term birth

    • Other studies suggest there may be effects of stress within pregnancy

How does peri-conceptual undernutrition affect fetal HPA axis activation and preterm birth in animal models?

• Animal models allow findings to be replicated or investigated further in a controlled environment

• Model: Sheep model of protein restriction (Bloomfield et al., 2003)

  • Protein restriction 60 days before pregnancy and during the first 30 days of pregnancy (overlap) → undernutrition and stressor model.

• Results:

  • Increased rates of preterm delivery and neonatal death in response to undernutrition stress.

  • As labour is triggered prematurely → the fetal adrenal gland releases cortisol, which triggers uterine activation and the onset of labour.

    • Normally, the fetal adrenal is inactive until term.

  • Premature activation/maturation of the fetal HPA axis leads to fetal stress caused by undernutrition.

  • Fetal stress caused by undernutrition may generate a signal activating pre-term labour, causing preterm birth.

Does peri-conceptual undernutrition affect pregnancy outcomes in human?

• Yes as

• Low maternal BMI (an indicator of poor nutrition) is associated with:

  • Increased risk of preterm delivery

  • Insulin resistance and elevated blood pressure in the offspring

• Famine during preconception or early pregnancy increases the risk of early or preterm birth

• Example: Dutch Hunger Winter (WW2)

  • Severe calorie restriction (~50% reduction), due to Nazi invasion of Netherlands, led to rationing acted as a major stressor

  • Evidence of earlier deliveries linked to maternal undernutrition

How Does Paternal Undernutrition Prior to Conception Affect Offspring?

• Invesgitaged in an animal model: Male mice fed a low protein diet (9% casein, ~50% of normal protein level) for 8 weeks prior to mating (~2 spermatogenesis cycles)

• Effects on testes: Seminiferous tubule area increased (compensatory mechansism in response to stress)

• Effects on offspring (as adults):

  • Increased body weight

  • Increased adiposity

  • Altered glucose tolerance

• Suggests that male pre-conception nutrition is important → affects gametogenesis and has long-term metabolic consequences for the offspring beyond pregnancy

What happens during preimplantation development of the human embryo?

• Fertilisation occurs in the ampulla of the fallopian tube

• Early development occurs in the oviduct

  • 2-cell stage → morula → blastocyst

• In IVF, this pre-implantation development occurs outside the body

What is In Vitro Fertilisation

• Common procedure (>8 million babies born via IVF)

  • Louise brown first baby born via IVF (47 y/o)

• Fertilisation and pre-implantation development occur in a culture dish

  • Questioned as to whether this is an optimal environment for embryo development

• Associated with higher rates of pregnancy complications, e.g. Pre-Eclampsia, FGR, Preterm Birth

  • Suggests IVF plays a role, e.g.embryo culture medium

How Does IVF Affect Trophectoderm and Inner Cell Mass Cells of the Embryo at the Blastocyst Stage?

• Investigated in mouse embryos at the blastocyst cells

  • In IVF vs in vivo group: Fewer Trophectoderm and ICM cells in IVF

  • Overall there are less cell in IVF

• Suggests that the IVF procedure and embryo culutre conditions affect fundemental processes e.g. cell proliferation and/or allocation of cells to the ICM or TE as study shows there are fewer cells in the blastocyst

How Does The Time Spent in IVF Embryo Culture Affect Long-Term Health in the Offspring?

• Investigated using mice conceived naturally or via IVF; embryos transferred at either the 2-cell stage or the blastocyst stage; offspring followed between 9–21 weeks of age

  • Systolic blood pressure and glucose response (marker of diabetes risk) are measured as markers of long-term health

• Stepwise increase in systolic blood pressure with age; highest in offspring from IVF-blastocyst stage embryos

• Increased diabetes risk in IVF groups; 2-cell stage IVF embryos showed worse glucose handling than blastocyst-stage IVF embryos.

• Length of time spent in embryo culture and stage of embryo prior to transfer influences long-term health outcomes (BP and glucose metabolism).

  • IVF and preimplantation culture can have subtle but significant long-term physiological effects.

What is Genomic Impriniting?

• An epigenetic mechanism that inactivates either the maternal or paternal allele of a gene

• Normally, genes are biallelically expressed (both maternal and paternal copies).

• Imprinting leads to monoallelic expression → only one allele is active

• Embryo culture (e.g., in IVF) can alter the expression of imprinted genes, which are particularly susceptible to environmental changes.

How Was a Loss of Genomic Imprinting in Response to Embryo Culture Demonstrated?

• Suboptimal embryo culture conditions lead to loss of imprinting (shown in multiple studies).

• H19, normally monoallelic, becomes biallelic after mouse embryo culture.

• H19 is a negative regulator of IGF-II, which is important for fetal and placental growth.

  • IGF-II promotes placental development and transporter activity

• Biallelic H19 (double amount of typical H19 present) → reduced IGF-II expression → impaired placental development and nutrient transporter activity → downstream effects on fetal and placental growth.

What Are the Long-Term Effects of a Loss of Genomic Imprinting?

• Loss of imprinting linked to (human) conditions including:

  • Angelman Syndrome

  • Prader-Willi Syndrome

  • Beckwith-Wiedemann Syndrome

• It is also associated with behavioural abnormalities.

• Higher incidence of Angelman and Beckwith-Wiedemann syndromes in children conceived via ART.

• Raises the question of whether this is due to embryo culture conditions.

How are the long-term effects of embryo culture assessed?

• Assessed in mice using three groups of embryos:

  1. In vivo embryos (naturally conceived, transferred to control for transfer effects)

  2. IVF embryos cultured in vitro in culture medium 1

  3. IVF embryos cultured in vitro in culture medium 2

• All embryos transferred to recipient mothers.

• Comparison of in vivo vs. cultured embryos.

• Outcome measured: long-term behavioural effects in adult offspring e.g. using Elveated zero maze

How Does Embryo Culture Affect Behaviour in Mice as Assessed by the Elevated Zero Maze?

• Behaviour assessed using the Elevated Zero Maze, an elevated circular track with open and enclosed areas.

• Relies on mice’s natural aversion and apprehension to open/elevated spaces (evolutionary preference for enclosed/safe areas which conferred survival).

• Normal mice spend most time in enclosed sections.

• Culture-derived mice spent more time in open areas (p = 0.02).

• This suggests that the embryo culture may reduce anxiety and/or cause memory impairments, indicating long-term behavioural effects as mice fail to recognise or avoid open spaces compared to controls → consistent with DOHaD

How Does Maternal Undernutrition During Pregnancy Affect Offspring Health and Demonstrate DOHaD

• Demonstrated in a rodent model given a low-protein diet during pregnancy (9% vs 18%)

• Offspring outcomes:

  • Low birthweight

  • Increased obesity

  • Impaired glucose tolerance

  • Hypertension

• Effects are pronounced when offspring are given a high-fat diet after weaning (different from in utero diet → mismatch between pre- and post-natal environment), confirming mismatch theory (Vickers et al., 2000; Langley & Jackson, 1994).

Where Has Maternal Undernutrition During Pregnancy Been Observed in Humans and What Were the Effects?

• Dutch Hunger Winter (1944–45) → severe calorie reduction due to Nazi invasion of Netherlands .

• This led to reduced maternal calorie intake → undernutrition during pregnancy.

• Offspring outcomes:

  • Reduced birthweight

  • Reduced glucose tolerance in adulthood (pre-diabetes/diabetes)

  • Evidence of insulin resistance

• Provides evidence of cardiometabolic changes, demonstrating that maternal undernutrition during pregnancy affects long-term offspring health.

• Demonstrated that Imbalanced protein & carbohydrate intake is linked to low birthweight, and the micronutrients may be important

  • Micronutrient deficiencies (low folate & iron) are also associated with low birthweight.

How does maternal stress affect fetal growth?

• Acute or chronic stress during pregnancy due to natural disasters, wars can lead to sustained elevated levels of cortisol, which leads to reduced fetal growth.

• Clinical glucocorticoid (GC) use during threatened preterm labour to promote lung maturation (in pre-term births, lungs not sufficiently matured)

  • Side effect: reduced birthweight.

• The placenta protects the fetus against cortisol via 11β-HSD2, an enzyme which metabolises cortisol to cortisone (inactive), protecting the fetus.

  • In FGR, 11β-HSD2 levels reduced → fetus exposed to excess cortisol → impaired growth.

What are the effects of glucocorticoid exposure during pregnancy?

• Assessed in pregnant rats treated with dexamethasone (synthetic GC analogue)

• Offspring outcomes:

  • Reduced birthweight

  • Reduced placental weight

  • Long-term consequences: metabolic syndrome, obesity, and hypertension

• High-fat diet after weaning worsens outcomes → a “second mismatch” with uterine environment (makes things worse)

  • Similar effects seen in maternal stress models

• Demonstrates that GCs during pregnancy can have both short-term and long-term effects on offspring.

What are the effects of high liquorice consumption during pregnancy?

• Liquorice contains glycyrrhiza (11b-HSD2 inhibitor)

• High liquorice consumption (>500mg/week) is linked to:

  • Earlier labour

  • Cognitive behavioural problems in children (speech, memory, increased incidence of ADHD) → increased cortisol exposure affects brain development

    • Seen in Scandinavian Populations

• Glycyrrhiza present in various substances at low levels (chewing gum, herbal teas, alcoholic drinks, tobacco, and cough medicine)

• Outiside pregnancy, high consumption of these substances islinked to cardiac effects

What are the Consequences of Pre-Term Birth?

• Children aged 4-10 years, born prematurely (24–32 weeks’ gestation), show:

  • Reduced insulin sensitivity

  • Increased risk for type 2 diabetes in childhood and adulthood

    • Early T2DM is concerning

• The early 3rd trimester is a critical window for permanent metabolic programming

• Incidence of type 2 diabetes in adulthood (18–43 years) is ~1.5× higher in those born prematurely (preterm)

• Preterm birth has lasting negative implications for long-term health

• Preterm individuals may also experience fetal growth restriction and/or glucocorticoid exposure

How Does Pre-Term Birth Affected Neurological Development?

• Investigated in extreme preterm birth (~<26 weeks of gestation)

• High prevalence of disability observed at 6 years old

  • Cognitive impairment was the most common disability

  • Odds ratio: 56 (95% 13-250) → 56 times more likely to have a cognitive impairment postnatally

• This is because brain development occurs late in pregnancy

  • If development occurs outside the uterus (as in preterm delviery and NICU admission) it is detrimental

• *Cohort in study exposed to antenatal glucocorticoids to mature lungs = potential confounder

How May Omega-3 Mitigate The Programming Effects of Glucocorticoids

• Experimental set up:

  • Pregnant rats treated with dexamethasone (Dex) → reduced birthweight and a slight increase in offspring systolic blood pressure (threshold for hypertension)

  • Dex-treated rats fed a high omega-3 (fish oil) diet:

    • Prevented Dex-induced increase in blood pressure

    • Normalised BP in Dex-treated and control animals

• Suggests omega-3 may mitigate the programming effects of prenatal glucocorticoid exposure

How May Leptin Mitigate The Programming Effects of Glucocorticoids?

• Leptin: an adipokine secreted by adipose tissue that assists satiety

• Experimental setup

  • Rats are undernourished during pregnancy → offspring show programmed metabolic phenotype

  • Offspring treated with leptin in the neonatal period → growth assessed over time

• Effects of leptin treatment in programmed offspring

  • prevents key aspects of the induced metabolic phenotype

  • Offspring do not become heavier

  • Reduced body fat and adipose tissue

  • increased locomotor activity and reduced food intake

  • Rests pathways that regulate energy homeostasis

• No effect on normal rats

How Can Treatments in Pregnancy Lead to Long-Term Problems?

• Clinical interventions in pregnancy can have long-term consequences

• Investigated in Igf2 P0 knockout mice (placenta-specific IGF2 KO), Sildenafil citrate (Viagra) administered from E12.5–E18.5 (late pregnancy 3^rd trimester)

  • Enhances placental blood flow when fetal/placental blood flow is compromised

• Follow-up → offspring monitored postnatally

  • Assessed for long-term outcomes such as blood pressure and other health factors

What are the Key Features of the Igf2 P₀ Knockout Rodent Model

• Placenta specific IGF KO

• Exhibit fetal growth restriction (FGR)

• Reduced placental and fetal size

• Abnormal placental morphology and function

How Does Antenatal Treatment With Sildenafil Citrate Lead to Long-Term Health Problems?

• Follow-up study (Renshall et al, 2020) revealed mice (WT or FGR, male or female) treated with sildenafil for 6 days during pregnancy show increased blood pressure as adults.

• Drug crosses the placenta:

  • Evidence on improving fetal weight is variable from clinical trials and studies (controversial); some studies suggest a benefit, but at the cost of higher blood pressure in adults

  • Due to safety concerns, sildenafil was no longer carried forward as a therapy for FGR.

• Crucial to evaluate the long-term effects of any drug administered during pregnancy.

How Does a Suboptimal Environment Before and During Pregnancy Affect Long-Term Health?

• Causes / Contributors:

  • Maternal or paternal undernutrition (pre-conception and during pregnancy)

  • Stress (maternal or fetal)

  • Lack of growth factors (e.g., IGF-II)

  • Poor placental development or function

  • Premature birth

  • IVF / embryo culture outside the body

• Mechanisms / How It Affects the Fetus (Leads to)

  • Fetal undernutrition

  • Exposure to glucocorticoids (GCs)

  • Organ maldevelopment

  • Physiological adaptations (e.g., altered metabolism)

  • Epigenetic changes (altered gene expression)

• Short Term Consequnces (Results): low birthweight; high birthweight; preterm birth

• Long-Term Consequences: Cardiovascular disease (CVD); Hypertension; Stroke; Obesity and metabolic syndrome (including diabetes); Increased risk of cancers

What are the long-term health problems associated with Pre-Eclampsia

• ‘Females’ who experienced preterm pre-eclampsia (delivery <37 weeks) were identified as a high-risk cohort

• Associated with persistent cardiovascular morbidity (6 months after delivery):

  • 61% had diastolic dysfunction

  • 75% had raised total vascular resistance

  • Only 5% had a completely normal echocardiogram

    • Individuals with pre-eclampsia during pregnancy have evidence of cardiovascular problems for 6 months after

• Severe pre-eclampsia can have lasting cardiovascular effects beyond pregnancy

Why Might Pre-Eclampsia Occur During Pregnancy?

• Question as to whether individuals are predisposed to PE due to pre-existing cardiovascular vulnerability, or if PE develops in an otherwise healthy individual

• Pregnancy acts as a cardiovascular stress test, requiring profound vascular adaptations

• PE is thought to occur when the maternal cardiovascular system fails to adapt

How Does Pre-Pregnancy Cardiac Health Affect Pre-Eclampsia and Fetal Outcomes?

• Studies of individuals with pre-existing cardiac myopathy (cardiac issue prior to pregnancy):

  • No overall increase in PE incidence.

  • Modest increase in pre-term PE.

  • Increased risk of FGR.

• These findings do not necessarily support a causal relationship between pre-existing cardiac dysfunction and Pre-eclampsia → mechanism underpining this research requires further research (statement from paper)

  • PE itself may contribute to long-term cardiovascular problems.

• Beta-blockers may be used in pregnancy: linked to reduced fetal weight;

• Demonstrates that interventions used may improve short-term outcomes but have long-term consequences.

What Factors Influence Long-Term Disease Risk and Developmental Programming?

• Risk factors for disease:Genetic factors; Adult lifestyle and environmental factors; Developmental events during and prior to pregnancy

• Developmental events influencing long-term health: Pre-conception period; Preterm birth; Early life growth (lactation, diet); Transmission to future generations (female oocytes develop in mother and are effected the grandmother’s environment)

• Pregnancy complications (e.g., pre-eclampsia) can affect maternal post-natal health and offspring outcomes

• Future: Human population studies to confirm animal findings (e.g., effects following IVF)