PROJECT SUMMARY/ABSTRACT: Miscarriage is the most frequent pregnancy complication, and around
750,000 to 1,000,000 miscarriages occur annually in the United States. Up to five percent of couples will
experience two or more miscarriages and have recurrent pregnancy loss (RPL). RPL is a significant clinical
problem as even after a diagnostic workup, 50% of couples will not find a cause for their recurrent losses and
will be diagnosed with unexplained RPL (uRPL). Therefore, investigating new causes for RPL is crucial to help
couples with RPL to identify a cause and, therefore, possible therapeutic options. Male factor causes of uRPL
are understudied, and clinically, the only diagnostic testing performed on men with uRPL is a karyotype to
exclude chromosomal translocation/inversion. Our proposal is highly significant as it addresses this major gap
in our knowledge regarding paternal causes of RPL. Several lines of evidence show that abnormal sperm
quality as objectively measured by high DNA fragmentation and aberrant DNA methylation in imprinted genes
is associated with uRPL. This project will shift the present clinical diagnostic paradigm for RPL couples from a
mainly female-centric approach to a balanced approach, including molecular analysis of sperm. Our project will
involve a comprehensive analysis of sperm from a large cohort of uRPL couples to study DNA fragmentation
(Aim 1), DNA methylation (Aim 2), and histone patterns (Aim 3). In Aim 1, our experiments will investigate the
molecular mechanisms by which increased sperm DNA fragmentation in humans can lead to RPL by
investigating the type of DNA damage occurring and the genomic loci affected. One major innovation in our
approach is to uniquely study in parallel the genome of the miscarriages resulting from high DNA fragmented
sperm to elucidate the underlying defect. Although sperm genomic integrity is one crucial aspect of ensuring a
healthy pregnancy, another aspect is the sperm epigenome, which also plays a crucial role in pregnancy
viability. Prior studies show that sperm DNA methylation at imprinted loci is perturbed in men with uRPL, and
increased retention of histones in sperm from uRPL men has been observed. Building upon these
observations, in aim 2, we will pursue both a candidate (imprinted genes) and an unbiased genome-wide
approach to studying the sperm methylome using the latest comprehensive DNA methylation array technology.
In addition, we will also leverage a prior predictive model using DNA methylation as a marker of paternal aging
to assess if there is premature aging in the sperm from uRPL men. This work will identify epimutations in
sperm that could play a mechanistic role in RPL. In aim 3, we will expand our epigenetic analysis to perform a
genome-wide analysis of sperm histone patterns in uRPL men. Finally, we will combine our multi-omics
datasets to compare and contrast the genomic loci affected. In summary, this project will contribute
significantly to identifying novel male factor causes of RPL, which will ultimately lead to significant
advancements in the diagnosis and treatment of couples with RPL.