Paternal obesity-associated DNA methylation: an investigation into its reproducibility, reversibility and association with fetal growth restriction

SUMMARY
BACKGROUND:
The number of overweight and obese people is rising all over the world and in the UK. Obesity now affects children, adolescents and adults. Younger people are particularly prone to the lifelong consequences of obesity with type-2 diabetes and high blood pressure. Once obesity is acquired, weight loss is difficult to achieve and in particular to maintain. Therefore, preventing overweight and obesity in the first place, is an attractive ambition to improve public health.
The origins of obesity can be traced back to the time we spent in our mother's womb and even before conception. During pregnancy, the developing baby grows in response to the health and weight of its mother. However, the father's weight at the time of conception is also influential, but in the opposite way to the mother's influence. Whereas an overweight or obese mother is predisposed to have a large baby, an overweight or obese man is predisposed to father a small baby. How well a baby grows in the womb is important, as it influences the health of that baby in adult life. Babies that grow poorly in the womb have a low birth weight and are at increased risk of type-2 diabetes, high blood pressure and obesity. Very large babies are also at risk of future type-2 diabetes.
AIMS:
We wish to discover if humans behave in the same way as animals by passing on acquired characteristics such as obesity, from father to unborn baby. We have designed a multi-staged, integrated study to discover how an obese father might inhibit the growth of his unborn baby and whether paternal weight loss has the potential to improve fetal growth and provide the ultimate primary prevention of life-long disease in the next generation.
METHODS:
We will discover whether obesity, type-2 diabetes and low birth weight are associated with consistent changes in the chemistry around genes, known as epigenetic change. We have linked with a Norwegian group who have collected DNA from over 11,000 families (mother, father baby). We plan to investigate whether DNA from 1000 obese fathers shows consistent obesity-associated epigenetic changes, compared with 1000 normal-weight fathers. We will determine if obesity-associated epigenetic marks identified by others in published reports are also evident in the 1000 obese fathers. This will generate a validated list of 'obesity-associated' epigenetic changes.
We will next determine the relative contribution of validated obesity-associated epigenetic marks from the obese father on the birth weight of their offspring in a study of newly pregnant women. We will study 250 pregnancies fathered by obese men compared with 250 pregnancies fathered by normal weight men. We will determine whether obese fathers are more likely to father low birth weight babies. We will take into account the influence of mothers' size and genes from both parents.
We will determine whether placentae (genetically half paternal) from pregnancies fathered by obese men function differently to placentae from pregnancies fathered by normal weight men.
Finally, in a separate study of 15 obese men, we will determine whether dramatic weight loss through bariatric surgery (by-pass of the stomach), is associated with reversal of obesity-associated epigenetic marks in DNA from their blood and sperm.
EXPECTED RESULTS:
The results of this project are of both scientific and public health interest. If obesity-associated epigenetic marks are evident in sperm of obese fathers and their low birth weight babies, we would have discovered a potential mechanism through which acquired paternal obesity perpetuates a vulnerability to obesity in a future generation. If weight loss eliminates these epigenetic marks, then we will have established a scientific rationale for a future study to investigate whether pre-conception paternal weight loss improves fetal growth and potentially improves the long-term health of the next generation.

Acquired parental obesity can be transmitted across generations via epigenetic mechanisms in animals. We propose a series of human studies to establish if validated DNA methylation profiles (DMPs) are associated with paternal obesity, insulin resistance and offspring birth weight.
Study 1: In collaboration with a Norwegian mother and child cohort (MOBA), DNA from 1000 lean fathers and 1000 obese fathers will establish a validated set of obesity-associated DNA methylation marks. We will profile ~300 regions identified from a comprehensive review of published epigenome-wide association studies of obesity. Throughout, methylation will be measured using a targeted sequencing approach (Bis-PCR-seq).
To identify potentially reversible, genetically-independent, DNA methylation profiles, we will investigate those SNPs previously identified as being associated with obesity and related traits. Custom multiplex PCR will be used to profile validated obesity associated SNPs.
Study 2: We will prospectively recruit a cohort of pregnant women who have conceived with either an obese (n=250) or lean (n=250) partner. We will investigate whether obesity-associated DMPs are evident in blood and sperm of fathers and in cord blood of their offspring. We will determine relative parental contributions and validate our results using mother-offspring pairs from the MOBA cohort.
In study 3 we will investigate whether paternal weight loss is associated with reversal of obesity-associated DMPs or small RNAs in blood and sperm of 15 morbidly obese men before and after bariatric surgery, using methods described above.
In study 4 we will assess placental function, structure and metabolism of offspring of our obese fathers. Histology and quantitative PCR will characterise nutrient expression and Western blotting - metabolic signaling pathways. Analyses will establish whether small babies of obese fathers are a consequence of poor placental function, through obesity-epigenetic marks in sperm

The results of this project are of both scientific and public health interest. Discovering robustly validated, epigenetic changes associated with obesity and weight loss, with detailed knowledge of the surrounding genetic environment, will provide insight into novel metabolic pathways to disease that could be new targets for therapeutic intervention. These observations would be of interest to those involved in the development of weight-loss drugs and to those who manage and research the causes of obesity.
If obesity-associated epigenetic marks are evident in sperm of obese fathers and their growth restricted offspring, we would have discovered a potential mechanism through which acquired paternal obesity perpetuates a vulnerability to obesity in a future generation. This observation would be of great scientific interest as it supports the intergenerational inheritance of acquired epigenetic marks in humans, which would be of particular interest to geneticists and evolutionary biologists.
It would also be of public health interest, as our results would also provide a scientific rationale for a future study to investigate whether pre-conceptual paternal weight loss would eliminate or reduce transmissible obesity-associated epigenetic marks to his offspring. This could be a novel approach to the primary prevention of obesity and its co-morbidities that has an impact in future generations.
These observations would be of particular interest to public health doctors, diabetologists, primary care clinicians, epidemiologists, and not least couples planning a healthy family.