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Page 127 Wang et al. J Transl Genet Genom 2023;7:126-40 https://dx.doi.org/10.20517/jtgg.2023.07

Methods: A transcriptomic analysis of human placentas derived from various pregnancy outcomes was performed
as a discovery study. This was followed by a quantitative confirmation to validate the differential transcription of
lncADAM9, the lncRNA overlapping with the ADAM9 gene locus, and of lncRNA-overlapped mRNA of ADAM9
(mRNA-ADAM9). In vitro examination of lncADAM9 transgenic (TG) HTR8 cells were used to perform functional
assessment to address the role of lncADAM9-mediated epigenetic regulation of extracellular matrix-adhesion
(ECM-A) associated molecules. This assessment was then expanded to studies of human fetal membranes.

Results: We observed that expression of lncADAM9 was increased in sPTB, and this increase was further
associated with the down-regulation of mRNA-ADAM9 in human placentas. In vitro, overexpression of lncADAM9
in lncADAM9-transgenic HRT8 cells led to DEPs relevant to ECM-A molecules, particularly at the loci of CNTN1,
NRXN2, SPN, ICAM2, and HLA-DPB1. This was also true in fetal membranes from abnormal versus normal fetal
membranes.

Conclusion: We have studied the epigenetic impact of differentially expressed lncADAM9 on the ECM-A pathway
that is associated with sPTB and documented that this impact may be mediated through the down-regulation of
mRNA-ADAM9. Our results of demonstrating the epigenetic regulation of lncADAM9 on the ECM-A pathway may
help provide greater insight into critical pathogenic mechanisms underlying sPTB.

Keywords: Spontaneous preterm birth (sPTB), long non-coding RNA (lncRNA), epigenetic regulation, ADAM9,
extracellular matrix (ECM)

INTRODUCTION
Spontaneous preterm birth (sPTB), parturition before 37 gestational weeks (GW), is a leading cause of
neonatal morbidity and mortality worldwide. Clinically, sPTB may present as spontaneous preterm labor
(sPTL), defined as premature parturition with no prior rupture of fetal membrane, or as preterm premature
rupture of fetal membrane (pPROM) that presents as rupture of membrane before uterine contraction.
sPTB is a highly heterogeneous syndrome , resulting from gene-environment interaction that
[1,2]
[3]
consequently causes biological changes of (epi)genetic variation and functions as a predisposing factor .
Many studies of gene-environment interaction in human sPTB have focused on the determination of a
correlation between a monogenic factor, such as a specific genetic locus that is derived from single-omic
data generated from genome-wide variation in blood or placenta with the outcome of pregnancy while
associated with a specific environmental exposure [1,4-8] .

Long non-coding RNA (lncRNA) is a group of transcripts that are more than 200 nucleotides without
coding potential, but act as epigenetic expression regulators through several molecular mechanisms.
[9]
LncRNAs have been determined to play a role in human reproduction and development . It has been
reported that lncRNA is a critical player in regulating epigenetic modification and is thus an important
mediator of gene-environment interaction relevant to placental development including trophoblast
differentiation [10,11] . In addition to miscarriage (MC), intrauterine grown restriction (IUGR), preeclampsia
(PE), and gestational diabetes mellitus (GDM) [12,13] , a pathogenic role of lncRNA is linked to human
reproductive disorders, including sPTB . LncRNA was found to have an epigenetic regulatory function in
[2,3]
the relationship between social and environmental exposures and sPTB [14-18] .

Studies on the pathogenic mechanisms underlying sPTB have applied an in vitro cell culture model to probe
the interaction between genes and the intrauterine environment [19,20] . Currently, a collective, quantitative
framework is lacking to relate the complex, nonlinear mechanical behavior of the placenta to changes in the
pathogenic pathway, which could contribute to the clinical complex but overlap as a causal factor leading to