Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT03962699 |
| Other study ID # |
C18-55 |
| Secondary ID |
2019-A00833-54 |
| Status |
Not yet recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
July 2019 |
| Est. completion date |
June 2022 |
Study information
| Verified date |
May 2019 |
| Source |
Institut National de la Santé Et de la Recherche Médicale, France |
| Contact |
Valerie GRANDJEAN, Dr |
| Phone |
+33 4 89 06 42 56 |
| Email |
grandjea[@]unice.fr |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Increasing evidence suggests that non-communicable diseases such as in particular obesity and
its associated metabolic diseases are inherited from parents to children throughout several
generations by epigenetic mechanisms. Thus, this environmental stress would induce epigenetic
modification in the germ line that once transmitted and maintained in the progeny would
induce the development of the parental pathologies. Considering the increasing prevalence of
these pathologies worldwide, we urgently need to understand this process in human. Based on
published and unpublished data demonstrating that sperm RNAs are vectors of epigenetic
inheritance of obesity mouse model, the investigative team hypothesizes that epitranscriptome
of obese men play a central role in the paternal epigenetic inheritance of obesity and its
associated metabolic diseases as epigenetic vectors in this process.
To validate this hypothesis, the investigative team will use sperm from non-obese and obese
men taken before and after surgery weight loss. Thanks to these cohorts, they propose to: (i)
compare the epitranscriptome profiles of non-obese and obese men to identify the RNAs
molecules which will be either qualitatively or quantitatively epigenetically modulated by
obesity; (ii) compare the epitranscriptome profiles of obese men before and after
surgery-weight loss to assess the reversibility of the newly acquired RNA modifications.
Giving some answers to this central question will provide not only some clues about the
molecular mechanisms involved in this process, elements which might be crucial to stop the
spread of this disorder, but will also allow the identification of obese-susceptibility loci
which expression may be modulate by environmental factors and consequently able to transmit
the disease.
Description:
1. Background and present state of the art in the research field. Sperm non-coding RNA is
an important vector for epigenetic inheritance of diet-induced obesity Obesity is a
multifactorial pathology associated with a high heritability (50-75%). To date, multiple
Genome Wide Association Studies (GWAS) have attempted and failed to identify the genetic
factors that contribute to the etiopathogenesis of obesity. In fact, the variance
explained by a high Body Mass Index (BMI) at these GWAS mutations is very low, estimated
to be approximately 2%[1]. Recent epidemiological and experimental studies strongly
suggest that this high heritability of obesity might be associated with the transmission
of obesity-induced epigenetic modifications.
Non-genetic inheritance of a newly acquired phenotype is a concept in biology whereby
changes induced by a specific signal and/or environmental stress can be passed to the
next generation in the absence of a genetic change [2, 3]. The biological relevance of
this process is proven by its diversification in various organisms including plants,
insects, nematodes, and mammals [4, 5]. However, evidence is now emerging that a wide
range of dietary factors including fats and proteins lead to changes in gene expression
that are maintained through mitotic and meiotic divisions[6]. These changes can
significantly impact the health of the offspring.
Very recently, the investigator has gained some insight into the molecular mechanisms
involved in this process, in particular those that pertain to the epigenetic inheritance
of newly acquired paternal pathologies [7] However, much remains to be uncovered about
the functional and molecular characterization underlying the mode of action of the
specific factors responsible.
Changes in DNA methylation [8-10], chromatin modification [11] and expression levels of
non-coding small RNA, including miRNA, piRNA and transfer RNA fragments [12-14] occur in
germ cells upon changes in environmental cues. However, it is not clear whether these
changes would mediate epigenetic inheritance. While several studies have indicated that
an altered DNA methylation signature of spermatozoa from HFD-obese individuals could be
transmitted to progeny [8], a recent study indicated that the sperm methylome is shaped
by genetic and epigenetic variations, but not diet [15], thus disproving the idea that
DNA methylation is the vector of epigenetic inheritance in this HFD model.
Based on this first evidence showing that small non-coding RNAs (sncRNAs) act as
trans-generational vectors of epigenetic information in mice [16], the investigative
team and others extended this discovery by deep investigation of the role of sperm small
RNAs as determinants of the inheritance in an acquired metabolic disorder [12-14]. In
this regard, experimental approaches include the micro-injection of one-cell embryos
with well-defined sperm RNAs from individuals fed by high-fat (HFD) or control diet
(CD). They found that mice derived from the microinjection of HFD sperm RNAs into naive
one-cell embryos developed adult onset diet-induced pathologies, such as obesity or
signs of type 2 diabetes even though they had been fed a control diet [14].
Deep-sequencing analysis of small RNAs of testis from HFD-fed animals and control
revealed that several small-RNAs are indeed deregulated upon HFD. Among those small
RNAs, we found miRNAs, piRNAs and tRNA-derived small RNAs (tsRNA). Importantly,
microinjection into naive one-cell embryos of one of the deregulated microRNAs - namely
microRNA-19b, induced metabolic alterations that were similar to the diet-induced
phenotypes. Likewise, other groups found that tsRNA may also contribute to
intergenerational inheritance of metabolic disorders. Indeed, Chen et al. showed that
micro-injecting sperm tsRNAs from HFD-fed individuals into naive one-cell embryos caused
impaired glucose tolerance in the resulting offspring [12]. However, in this model,
synthetic microRNAs or synthetic tsRNAs did not induce metabolic disorders in the
offspring, suggesting a role of post-transcriptional RNA modifications in the transfer
of epigenetic information. This hypothesis is consistent with the elevated levels of m5C
and m2G modifications of tsRNAs in sperm from the HFD group [12]. Hence, sperm small
RNAs represent a type of paternal epigenetic vector involved in intergenerational
inheritance of diet-induced metabolic phenotypes.
The majority of data on RNA-mediated epigenetic inheritance are from mouse models
[12-14]. Whether this mechanism is conserved in human as yet unknown. Noteworthy, a
study performed in human by Donkin et al. indicated that obesity might modify the sperm
epigenetic landscape as well as sperm epitranscriptome. However, epigenetic and
transcriptomic analysis were performed in only a small number of men[17] Taken together
these observations from human and rodent studies suggest that obesity-induced small RNA
changes are transmitted to the next generation, and then result in heritable metabolic
changes. However, although in rodents the obesity-induced changes have been quite well
described, the impact of obesity on the sperm epitranscriptome/epigenome in human
remains an open question of urgency in the field.
Reversal of the obesity-induced epigenetic changes: a hope for the next generation?
Although epigenetic modifications are mitotically heritable, they are erased and
re-established twice during development [18]. This reprogramming takes place early in
embryogenesis and during gametogenesis in primordial germ cells that will give rise to
eggs and sperm. [18]. Furthermore, phenotypes conferred by environmental compromise can
also be reversed via active intervention, such as change to healthy diet or physic
exercise during the preconception period [19, 20]. It is not understood whether there is
a relationship between developmental reprogramming and reversibility of environmentally
induced phenotypic states. Although the erasure of these newly established modifications
has been clearly described in rodents [20, 21] only limited studies have been addressed
in human [17, 22, 23]. Those studies only focused on DNA methylation pattern modulated
by obesity in somatic and germ cells. To date, nothing is known about the reversibility
of trans-generational epigenetic RNA in human sperm.
Preliminary results Obesity negatively impacts on sperm epigenome and early embryonic
development in human
After having demonstrated the role of sperm RNA (small and long RNA) in paternal
heredity of obesity and its associated diseases, the investigative team is currently
investigating the relevance of sperm RNA-mediated epigenetic inheritance of metabolic
disorders in human. To achieve this goal, they have carried out two complementary sets
of experiments. First, they recently found that the development capacity of human
embryos derived from obese men was impaired and noted that pre-implantation embryo
morphokinetic parameters were altered when sperm derived from obese fathers was used to
fertilize oocytes (Raad et al. 2019), suggesting obesity induced epigenetic changes in
sperm. Then, to determine whether obesity induced sperm small-non coding RNA
dysregulation in human, they performed a small RNA-seq differential expression analysis
between obese and non-obese subjects. Briefly, upon multivariant analysis (Principal
Component Analysis, PCA), they identified distinct small RNA profiles between obese and
lean men. However, while sperm small RNA profiles among lean men (N1, N2, N3 and N4
samples) were homogeneous, small RNA profiles among obese men (O1, O2, O3 and O4
samples) were very heterogeneous, suggesting stochastic dysregulation of sperm small RNA
expression in obesity.
Together, our results highlight the negative impacts of excessive weight mass on the
sperm epi-transcriptome and human early embryo development and strongly suggest that
paternal obesity negatively affects the sperm epigenome and subsequently influences the
health of the descendants.
2. Descriptive of the objectives Based on the published and unpublished data described
above, the investigative team hypothesize that epi-transcriptomes are responsive to
transient environmental changes such as obesity, leading to modulation of the progeny's
epigenomes in human and subsequently to the development of the father's phenotypes.
To validate this hypothesis, the investigator will use sperm collected from 2 groups of men:
the first group will be composed of non-obese men and the second one will be composed of
obese men taken before and after weight loss induced by surgery. Thanks to these cohorts, we
propose to:
- Identify the sperm epitranscriptomic signature of obese men Team 1 has recently
demonstrated that sperm RNAs are vectors of epigenetic inheritance of obesity and its
associated pathologies in mice. In parallel, while confirming the role of these RNAs in
epigenetic inheritance, two other independent groups have shown the involvement of RNA
posttranscriptional modifications in this process in mice. However, it is important to
determine whether such process exists for human. By combining the complementary
expertise of three groups in either epigenetic inheritance in mice (Team1) and in the
handling and collection of human sperm samples (Teams 2 and 3), the investigative team
will have the opportunity to answer this unresolved question.
- Study the reversibility of the sperm epitranscriptomic signature of obese men The
question of reversibility of sperm epigenetic signature induced by obesity in human is
an important question in term of public health. Barrey's group has recently shown a
possible reversion of obesity-induced epigenetic upon surgically incurred-weight loss.
However, this study relies on sperm DNA methylation studies [17]. The role of DNA
methylation as vector of epigenetic inheritance of obesity remains to be established,
however, we and others have recently demonstrated the involvement of sperm
epitranscriptome in this process in mice, [12, 14]. The possible reversibility of these
obese-induced epitranscriptomic changes remains to be demonstrated both in mice and in
human.
The impact of bariatric surgery with weight loss on metabolic health in progeny of obese
fathers could be, in principle, studied directly on human cohorts. However, the few
longitudinal studies currently available from offspring born after parental weight loss are
based on maternal weight loss studies [22, 24, 25]. These results are not only inconsistent
but also biased by the fact that the surgery procedure leads to malnutrition risks in the
mother. Given the importance of nutrition during pregnancy, the potential impact of surgical
weight loss on gametic epigenomic reversibility and on metabolic health of offspring could
not be evaluated in this context. By performing comparison of epitranscriptomic profiles
between obese men before and after surgery, the investigative team will bring new insights
into this very important biological question.
The investigative team expect that this proposal will provide clues about the molecular
mechanisms involved in this process, and will allow the identification of
obese-susceptibility loci as well, which their expression may be modulated by environmental
factors and consequently affects the inheritance of the disease.
