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Session 5: The altered epigenomes of sperm from infertile and aging men
And the possible consequences of these alternations to a pregnancy.

Summary of Session 4: The epigenome of fertile human sperm

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SUMMARY of Hammoud et al.

Nucleosomes in human sperm are associated with genes that regulate development
and/or transcription.

Many of those genes are bound by histones with transcriptionally permissive

modifications.

Others (e.g. SOX2 and FOXD3) are bound by histones with both transcriptionally
permissive and transcriptionally repressive modifications. The promoters of those
genes do not contain 5-methy cytosine residues.
Thus, they are poised for transcription in the embryo.

Conclusion: The sperm epigenome potentially affects embryonic development.

Studies of the effects on embryonic development of manipulating the

epigenomes of mouse sperm support this conclusion.

H3K4me3 in genomic areas that overlap the transcription start sites of 1570 genes.

Each row = 1 gene.

Data are clustered
for similar histone
methylation
densities.

Near homogeneous H3K4Me3

across a broad region that
overlaps the TSS of 1570 genes

TSS= transcription start site

Nature Communications 11: 349, 2020.

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Quantitative Analysis of the Distribution of Histone-Bound DNA in Human Sperm

Which Gene TSSs Are Associated with Peaks of H3K4me3 or H3K27me3?
What are the Densities of H3K4me3 and H3K27me3 at Those TSSs?
What fraction of sperm have those modified histones at That TSS?

H3K27me3 peak
H3K4me3

Transcription Start Site

H3K27me3
130 gene TSSs are associated with a peak of H3K4me3

320 genes TSSs are associated with a peak of H3K27me3

DNA of those 320 genes is not methylated. These
genes are poised for transcription.
Many regulate development.
Nature Communications 11: 349, 2020

An important conclusion of M. Oikawa et. al.

In almost every fertile human sperm, regulatory regions of few genes are bound by
histone 3 lysine4 trimethyl and/or histone 3 lysine 27 trimethyl. A significant percentage
of these genes encode intrinsic regulators of development.

The epigenome determines which of a sperm’s genes are the first to be expressed in
an embryo. The proper, early expression of these genes may be essential for normal
embryonic development.

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Session 5: The epigenome of sperm from infertile and aging men.

Part A: Imprinting of Maternal and Paternal Genes

Definitions:
Imprinting: Epigenetic chromatin modifications that result in specific genes being expressed
in a parent-of-origin (Mom or Dad) manner.

Maternal allele: The copy of the gene we inherit from our mother.
Paternal allele: The copy of the gene we inherit from our father.

Maternally imprinted allele: The maternal allele is not expressed; the paternal allele
is expressed
Paternally imprinted allele: The paternal allele is not expressed; the maternal allele
is expressed

Currently, 128 human genes are known to be imprinted.

104 additional genes are predicted to be imprinted *

* www.geneimprint.com/site/genes-by-species.

During Early Embryonic Development, The Epigenome of Imprinted Genes is Not erased .

Erasure of the entire epigenome

Mouse embryo
> eDay 17
Erasure of epi-
genome of all
but imprinted The mouse
genes
blastocyst
forms on eDay
3. PGCs are
Formed on
eDay 7.5 in
the epiblast.

Is imprinting normal in infertile sperm?

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Imprinting of Maternal and Paternal Genes

More Definitions:

DMD: Differentially methylated domain. Also called differentially methylated region.

Methylation of DMDs determine whether a maternal or paternal allele of an imprinted gene
is expressed.

Locus: A specific location in a chromosome.

CTCF (CCCTC binding factor): This insulator protein binds to an unmethylated DMD that
separates two genes within a locus. This binding insulates one of the genes from important
transcriptional regulatory elements.

The confusing part: Your somatic cells contain one paternal allele and one maternal allele.
However, in sperm all imprints are paternal, even though each sperm inherits chromosomes
from the Dad and the Mom.

Likewise, in eggs all imprints are maternal, even though each egg inherits chromosomes
from the Dad and the Mom.

Imprinting of the Igf2 - H19 locus (human chromosome 11)

These 2 genes constitute a single genetic locus and their expressions are linked
Maternal allele: The
binding of CTCF to the
H19 DMD unmethylated
DMRs/DMDs insulates
the IGF2 promoter from
downstream enhancers.
Maternal allele H19 is expressed.
(DMDs not
methylated Paternal allele: CTCF
does not bind to meth-
ylated CTCF. IGF2
Paternal allele promoter interacts with
(DMDs downstream enhancers.
methylated) IGF2 is expressed.

Loebel and Tam (2004) Nature 428: 809-811

IGF2 promoter H19 promoter Transcription from the IGF2-H19 locus

requires interaction of the down-
stream enhancer with a promoter.

• H19 – Long non-coding RNA; maternal allele is expressed.

• Igf2 – Insulin-like growth factor 2; DMD is methylated; paternal allele is expressed. Correct
expression of the paternal allele of IGF2 is critical for growth of the embryo.
In normal sperm, the maternal and paternal H19 and IGF2 alleles are methylated.

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Are gene imprints abnormal in sperm of infertile men?

Boissonnas et al. Specific epigenetic alterations of IGF2-H19 locus in

spermatozoa from infertile men. European Journal of Human Genetics 18: 73, 2010.

Hypothesis: Sperm from infertile men have (DNA) imprinting errors at the H19-IGF2 locus.

Approach: Compare DNA methylation of the IGF-1 - H19 locus in human normal sperm and
and in infertile human sperm. Pyrosequencing* quantified methylation of each
CpG dinucleotide. Samples of 0.5-1µg of DNA were analyzed.

* Investigators sequenced DNA from a pool of 1000s of sperm. Pyrosequencing quantifies

the relative numbers of C’s and U’s at a specific position in the DNA sequence.

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Two different infertile sperm phenotypes.

Teratozoospermia: Numbers of sperm in ejaculate are within the normal range but
96% of sperm in an ejaculate have severe morphological abnormalities

Oligo-asthenoteratozoospermia: Both numbers and motility of sperm are

abnormally low and most are morphologically abnormal.

Fertile human sperm Teratozoospermia

Sources: sciencephotolibrary & PNAS 111:7054, 2014

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Normal Methylation of the DMRs in the IGF2-H19 Locus of Sperm

Normally, both alleles (one per sperm) have paternal epigenetic marks resulting in expression of IGF2.
IGF2 H19

DMR = differentially
Methylated region
27 CpG’s in the H19 DMR.

Location
of CpG’s

Normally, most
DMRs in the Sperm from
sperm IGF2- 17 fertile men.
H9 locus are Each sample
methylated analyzed
separately.
Data; Mean +
In diploid cells SD.
IGF2 is express-
ed only from the
paternal allele.

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Analysis of
Teratozoospermia
Tetratozoospermia =
= % of a specific severe
CpG methylated in morphological
1 pool of sperm abnormalities
from one patient.

Methylation of
DMRs in a pool
of sperm from CTCF6
each patient.

DNA methylation within normal range. DNA methylation significantly below normal range.

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Oligo-Asthenoteratozoospermia

Oligo-astheno-
tratospermia =
sperm morphology,
concentration and
motility defects

Different
Subgroups CTCF6
Of patients

DNA methylation within normal range. DNA methylation below normal range.

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Sperm concentration in ejaculate correlates with the extent of methylation of H19 CTCF6

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Epimutations in sperm of oligospermic men may not be inherited by the child.

Analysis of the imprinting of CTCF6 of H19 DMR.
Hypothesis: Sperm epimutations are inherited by the child.

Cytosine is not
methylated

Cytosine is methylated

Sperm and blood were collected one oligospermic man. Umbilical blood was collected at the
birth of his child. DNA was isolated and treated with sodium metabisulfite or not (control)
DNA was cloned, and 5 clones per sample sequenced.

*Oligospermia is a condition of infertile men characterized by low sperm count (<15 million sperm/
ml ejaculate) but normal morphology. This is a less severe infertility phenotype than
tetratozoospermia or astheno-teratozoospermia.

There are no follow-up

Koyayashi et al. Aberrant DNA methylation of imprinted loci
in sperm from oligospermic patients. studies of men with more severe
Human Molecular Genetics 16: 2542, 2007 sperm abnormalities.

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Is there any clinical evidence that an abnormal sperm epigenome

has a deleterious effect on fetal development or the progress of a
pregnancy?

Consider the effects of paternal age on risk for birth defects,

on the the sperm epigenome and on pregnancy complications

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Children Born to Older Fathers Are at a Higher Risk of Major Birth Defects
This risk is independent of the age of the mother.
Mothers < 25 years

National Birth Defects Mothers 25-34 years Birth defects include:

Septal heart defects
Prevention Study
Constriction of aorta

Pulmonary Vein returns to

right rather than
left atrium
Mothers > 34 years

Diaphragmatic hernia

Source: National Birth Defects

Prevention Study. Ann Epidemiol
20: 241, 2020 Paternal Age (< 25 to > 55)

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What is the effect of paternal age on the human sperm epigenome?

M. Cao et al. High-resolution analysis of human sperm dynamic methylome

Reveal thousands of age-related epigenetic alterations (2020). Clin. Epigenet.
12:192.

Hypothesis advancing paternal age is associated with increased numbers

of sperm epimutations as defined by altered DNA methylation.

Subjects:
48 men 18-38 years old
46 men 46-71 years old

Analyzed ~ 2.6 million CpG’s in sperm genome analyzed.

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Effect of Paternal Age on CpG Methylation

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606
420

Where are these clusters relative to the genes?

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Distribution of age-associated hypo- and hypermethylated CpGs in relation to gene region

Hypomethylation Hypermethylation

Hypomethylated sites are proximal to the transcription start of a gene

Hypermethylated sites are distal to the transcription start site of a gene

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GO Terms for Genes that Show Age-Associated Changes in Methylation

Hypomethylation

15 of 40 GO terms
associated with
Hypermethylation development

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Conclusion: The sperm epigenome changes with human aging.

Some genes linked to these changes regulate development.

Mouse studies demonstrate that the placenta is more prone to

epimutations than an embryo. Are the epimutations of sperm of older
men passed on to the placenta?

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S. Choufani et al. Impact of assisted reproduction, infertility, sex and

paternal factors on the placental DNA methylome. Human Molecular Genetics
(2019) 28: 372-385.

Hypothesis: The epigenome of the human placenta is sensitive to conception by

Assisted Reproductive Technologies and to male-factor infertility and to paternal age.

Placental sources:
44 control couples did not present for infertility and conceived spontaneously
44 couples had presented for infertility.
Children conceived by ICSI, IVF, IUI.

Extent of methylation at 450,000 different CpG sites in each of the 88 placentas was quantified.

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PCA Analysis Identifies A Subset of Placentas from ART Conceptions

With DNA Abnormal DNA Methylation

Abnormal
DNA Methylation.
The “Outliers.”

PCA analysis gives an overview of CpG methylation in the 88 samples.

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Placental DNA methylation is significantly increased in a subset of ART cases.

Heat Map PCA Analysis: Normal vs. Outliers
Normal conception ART conception
436 CpG dinucleotides

Methylation level

From the publication:

Further analysis of the clinical attributes of
these groups identified an association/enrichment
of male factor etiology for infertility and increased
paternal age in the separate cluster of the in vitro
group compared to the in vivo and the remaining in
vitro group.

ART Outliers

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Which Pathways Might Be Affected in the Placentas in the Outlier Group?

5 of 11
Go Terms
associated with
development.

Note that age-associated changes in sperm methylation were associated with genes that regulate development.

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Both age and infertility are associated with abnormal sperm DNA methylation.

Older infertile fathers are associated with abnormal placental DNA methylation.

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Does an altered placental epigenome matter? Possibly because:

1. Placental wrights increase with age of the father1.

2. IVF is associated with a significantly increased risk factor for

pathological invasion of the placenta towards or into the uterine
myometrium2.

Thus, both paternal age and IVF may affect placentation and thus,
the success of a pregnancy.

1 Human Reproduction 28: 3126, 2013.

2 PLoS One. 2012;7(12):e52893. doi: 10.1371/journal.pone.0052893. Epub 2012 Dec 27

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Summary:

1. Sperm from infertile men may have abnormal gene imprints.

2. Aberrant imprints are higher in sperm with the most severe morphological
abnormalities.
3. Paternal age is associated with abnormal sperm DNA methylation. About 40% of the
linked genes regulate development.
4. Placental DNA methylation is abnormal in a subset of placentas.
They are associated with older fathers and use of IVF for conception.
5. Both paternal age and IVF are associated with formation of abnormal placentas.

Conclusion: Abnormal sperm CpG methylation may have deleterious

effects on placentation and thus on pregnancy.

Next session: Studies with mice demonstrate that an abnormal sperm

epigenome can have dire consequences for embryo development.

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