Accepted Manuscript
Bio-compartmentalization of microRNAs in exosomes during gestational diabetes mellitus
Juvita Delancy Iljas, Dominic Guanzon, Omar Elfeky, Gregory E. Rice, Carlos Salomon
PII: S0143-4004(16)30648-8 DOI: 10.1016/j.placenta.2016.12.002 Reference: YPLAC 3515
To appear in: Placenta
Received Date: 7 November 2016 Revised Date: 27 November 2016 Accepted Date: 2 December 2016
Please cite this article as: Iljas JD, Guanzon D, Elfeky O, Rice GE, Salomon C,
Bio-compartmentalization of microRNAs in exosomes during gestational diabetes mellitus, Placenta (2017), doi: 10.1016/j.placenta.2016.12.002.
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Bio-compartmentalization of microRNAs in exosomes during gestational diabetes mellitus
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Juvita Delancy Iljas, 1Dominic Guanzon, 1Omar Elfeky, 1,2Gregory E. Rice, 1,2Carlos
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Salomon
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6 1
Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland
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Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of
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Queensland, Brisbane QLD 4029, Australia. 2 Maternal-Fetal Medicine, Department of
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Obstetrics and Gynecology, Ochsner Clinic Foundation, New Orleans, USA.
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*Correspondence: Dr Carlos Salomon PhD, DMedSc, MSc, BSc
Head of the Exosome Biology Laboratory | Centre for Clinical
Diagnostics | UQ Centre for Clinical Research | The University of
Queensland | Building 71/918 | Royal Brisbane Hospital | Herston
QLD 4029 | Faculty of Health Sciences | University of Queensland
Phone: +61 7 33465500 | Fax: +61 7 3346 5509 |
Email:[email protected] | Web: www.uqccr.uq.edu.au/
.
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Abstract 12 13Analysis of the human genome revealed that only 1.2% encoded for proteins, which raised
14
questions regarding the biological significance of the remaining genome. We now know that
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approximately 80% of the genome serves at least one biochemical function within the cell. A
16
portion of this 80% consists of a family of non-coding regulatory RNAs, one important
17
member being microRNAs (miRNAs). miRNAs can be detected in tissues and biofluids,
18
where miRNAs in the latter can be bound to proteins or encapsulated within lipid vesicles
19
such as exosomes. Gestational diabetes mellitus (GDM) is a complication of pregnancy,
20
which has harmful health impacts on both the fetus as well as the mother. The incidence of
21
GDM worldwide varies, but reached 18% in the HAPO cohort using the new International
22
Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria. Not only has GDM
23
been associated with increased risks of further complications during pregnancy, but also pose
24
long-term risks for both the mother and the baby. Thus, understanding the pathophysiology of
25
GDM is important from a public health perspective. Literature has demonstrated that GDM is
26
associated with elevated levels of circulating exosomes in maternal circulation. However,
27
there is a paucity of data defining the expression, role, and diagnostic utility of miRNAs
28
in GDM. This review briefly summarizes recent advances in the function and quantification
29
of intracellular and extracellular miRNAs in GDM.
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32
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Introduction 34 35During normal pregnancy, glucose intolerance gradually increases during the second and third
36
trimesters of pregnancy to ensure adequate nutrient supply for the fetus [1]. A pregnant
37
mother with acquired or chronic insulin resistance is not able to compensate for the increased
38
circulating glucose concentrations due to β-cell dysfunction. The resulting maternal
39
hyperglycaemia increases the risk of disease for both the mother and her offspring. Therefore,
40
any degree of abnormal glucose metabolism diagnosed during pregnancy is termed
41
gestational diabetes mellitus (GDM).
42
GDM accounts for 90% of hyperglycaemic complications of pregnancy, affects 3-8%
43
of all pregnancies and has a recurrence rate of 35-80% [1]. GDM can lead to morbidity and
44
mortality in both the mother and the infant, which includes pre-eclampsia, subsequent
45
development of diabetes mellitus type 2 (DM2) in the mother, fetal macrosomia, congenital
46
malformation, perinatal mortality and obesity [2].
47
Due to the comorbidities and risks associated with GDM pregnancies, it has become
48
apparent that screening in the first trimester or early-second trimester of pregnancy is critical
49
in metabolic disorders such as GDM [3]. Early diagnosis is essential in reducing GDM
50
associated complications for both the mother and the fetus, by implementing treatments which
51
normalize blood glucose levels. However, the current two-step approach screening method
52
based on plasma glucose measurement identify women at a later stage of GDM (24-28 weeks
53
of gestation) [4]. This means that treatment cannot start until 32 weeks of gestation, which
54
already presents a high risk of fetal morbidity and mortality. Consequently, many researchers
55
are investigating potential biomarkers present in the blood to accurately diagnose GDM
56
earlier than 24-28 weeks (e.g. 16-19 weeks of gestation) [5]. microRNAs (miRNAs) show
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great potential as early-trimester biomarkers for GDM, as they are highly stable in body fluids
58
and are accessible from maternal fluids throughout gestation [6].
59
Dysregulation of miRNA expression has been associated with metabolic disorders in
60
insulin secreted as well as insulin-targeted tissues and cells [7]. Furthermore, miRNAs have
61
been associated with GDM and its resulting health complications [8, 9]. Research in miRNA
62
profiling will provide valuable information in identifying unique and sensitive biomarkers in
63
body fluids to utilize as diagnostics or therapeutics in GDM. This can prevent adverse
64
pregnancy outcomes and reduce healthcare costs associated with GDM. This review aims to
65
provide an overview of recent advances in extracellular and intracellular miRNAs involved in
66
GDM pathogenesis.
67
68
miRNA biogenesis and function
69
70
Biogenesis:
71
miRNAs are small non-coding RNA fragments approximately 22 nucleotides in length [10].
72
In the initial stage of miRNA biogenesis, intergenic regions and introns where miRNAs
73
reside, are transcribed by RNA polymerase II and III [11, 12]. This produces a long transcript
74
called primary miRNA (pri-miRNA) that is greater than 70 nucleotides in length [13]. Within
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the nucleus, this pri-miRNA folds into a structure containing a stem-loop with single stranded
76
5’ and 3’ RNA ends. The proteins Drosha and its cofactor DGRC8 bind and cleave the
pri-77
miRNA at the stem-loop base, discarding the single stranded RNA ends [14, 15].
78
Subsequently, this stem loop structure (termed a pre-miRNA) is exported into the cytoplasm
79
by exportin 5 [16].
80
Within the cytoplasm, the pre-miRNA is further processed by protein Dicer, cleaving
81
the terminal loop [17]. The remaining double stranded RNA is loaded into a multi-protein
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complex called RNA induced silencing complex (RISC). At the heart of RISC are the
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argonaute proteins, that unwind the double stranded RNA [18]. During this process one RNA
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strand is removed (passenger strand), while the other RNA strand (guide strand) remains
85
within the RISC [19]. This completes the biogenesis pathway for miRNAs, as this guide
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strand is now the mature miRNA. This miRNA-RISC complex now has the capacity to
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epigenetically regulate gene expression.
88
89
Function:
90
The miRNA-RISC regulates gene expression by binding to the 3’ end of the messenger RNA
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(mRNA) and regulates its translation. Although not fully understood, this miRNA-RISC is
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hypothesized to regulate translation by two processes [20]: (1) miRNA-RISC obstructs the
93
binding of ribosomes to the mRNA [21] and (2) deadenylation and decapping of the mRNA
94
rendering the mRNA unstable, promoting its decay [22]. Ultimately, these two approaches
95
suppress translation resulting in a decrease of protein output. In rare situations, there are some
96
miRNAs that enhance translation. An example of this is miRNA miR-10a that promotes the
97
translation of ribosomal mRNA [23]. Orom et al. (2008) speculated that this miRNA
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competes with a negative regulator that suppresses translation of ribosomal mRNA.
99
The ability of miRNAs to recognize and bind to mRNA is primarily determined by its
100
seed region, located at nucleotide positions 2 to 7 from the 5’ end of the miRNA strand [24].
101
It has been predicted that a single miRNA can regulate more than 200 different mRNA
102
species [25]. Furthermore, it has been hypothesized that greater than 60% of genes are
103
regulated by miRNAs. Consequently, miRNAs are critically involved in regulating many
104
biological processes [26]. This makes miRNAs attractive as biomarkers or therapeutic targets,
105
as dysregulation of miRNA expression has been linked to several diseases such as cancer and
106
diabetes mellitus (DM) [27].
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108 Quantifying miRNAs 109 110There are limitations regarding miRNA detection due to inefficient RNA isolation, serum or
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plasma contamination by haemolysis, variable reverse transcription (RT) and polymerase
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chain reaction (PCR) efficiencies, inconsistent reference genes and the existence of different
113
qPCR platforms [28]. It is still a challenge to collect and quantify extracellular miRNAs. This
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is due to the low concentration present in body fluids, the difficulty distinguishing between
115
mature and immature miRNA and the presence of miRNA isoforms.
116
Currently, the most sensitive, reproducible and gold standard method to quantify
117
miRNAs is quantitative real-time PCR (qRT-PCR) [29]. qRT-PCR involves RT of miRNA
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into cDNA, before amplification and detection with specific probes. Advances have been
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made in detecting low amounts of miRNA in the range of approximately 7 fM [30], which is
120
superior to other qRT-PCR based technologies. Subsequently, high-throughput qRT-PCR
121
platforms were developed to allow rapid miRNA profiling of a large number of samples [31].
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The most commonly used qRT-PCR miRNA expression profiling method is TaqMan
low-123
density array (TLDA), which is both cost-effective and high-throughput. This TLDA
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technology has been utilized to profile miRNAs within serum isolated from GDM patients
125
(Table 1) [32].
126
Microarrays are a technology based on the hybridization of cDNA to DNA probes
127
[28]. The approach is limited by low sensitivity, high RNA input requirement (100 ng to 1µg),
128
and suffers from background and cross-hybridization when compared to qRT-PCR.
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Therefore, microarrays are not ideal for miRNA profiling, since they cannot detect highly
130
expressed miRNAs (due to signal over saturation) or distinguish between mature and
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immature miRNAs. Nevertheless, microarrays have been used as a discovery tool to identify
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miRNAs that are differentially expressed between GDM and normal controls [33, 34].
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Next generation sequencing technology (NGS) has the advantage over other
134
technologies, as it can identify novel miRNAs and other small RNA species [35]. NGS is
135
more laborious than qRT-PCR and microarrays and requires a larger amount of RNA input
136
(500 ng to 5 µg). It is possible to barcode multiple samples and sequence them simultaneously
137
thereby reducing the costs and time. miRNA databases and bioinformatics tools are available
138
that provide information on miRNA sequences, miRNA-mRNA target interactions, and the
139
involvement of miRNAs in biological as well as pathological processes. NGS was utilized to
140
identify differentially expressed miRNAs in GDM compared to normal pregnancies (Table 1)
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[36].
142
In summary, qRT-PCR is ideal for low input RNA, NGS technologies for
143
identification of novel miRNAs, and microarrays (low sensitivity) along with NGS (high
144
sensitivity) is utilised to screen for differentially expressed miRNAs in a large set of samples.
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Several researchers have compared these three technologies for miRNA quantification and
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concluded that each technology has its own strengths and weaknesses [37]. Deciding on
147
which technology to use for miRNA quantification and profiling depends on the experimental
148
design and its goals.
149
150
Extracellular and intracellular miRNAs during normal and GDM pregnancies
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152
Extracellular miRNAs present in body fluids can be located within extracellular vesicles
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(EVs) (exosomes, microvesicles and apoptotic bodies [38]), bound to protein complexes (such
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as argonaute2 complexes [39]) or within high-density lipoproteins (HDL) [40]. These
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structures protect extracellular miRNAs from degradation, rendering them very stable in
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biological fluids. Therefore, miRNAs are exciting candidates for biomarkers [41, 42]. Recent
157
studies have produced promising results on the potential of extracellular miRNAs in blood
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(termed circulating miRNAs) as biomarkers that may be useful in the diagnosis of GDM (see
159
Figure 1) [43].
160
The literature showed extensive studies of miRNAs in DM1 and DM2, but studies into
161
their role in GDM is lacking. There have only been 6 studies investigating miRNAs in GDM,
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only 2 of which specifically investigate extracellular miRNAs within the blood (see Table 1)
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[32, 36]. A study by Zhao et al. (2011) reported that miR-132, miR-29a and miR-222 are
164
decreased in serum of women with GDM [32]. A second study by Zhu et al. (2015) reported
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that hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-19a-3p, hsa-miR-19b-3p and hsa-miR-20a-5p
166
are increased in the plasma of women with GDM [36]. There is no apparent correlation
167
between these two studies with respect to the miRNAs detected.
168
Four other studies investigated intracellular miRNAs in GDM (see Table 1) [33, 34,
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44, 45]. Interestingly, the studies by Zhao et al. (2011) and Shi et al. (2014) share one miRNA
170
in common, which is miR-222 [32, 33]. However, the expression of miR-222 is not consistent
171
between the two studies. Zhao et al. (2011) reported that miR-222 is supressed in GDM,
172
while Shi et al. (2014) demonstrated that miR-222 was upregulated in GDM. One potential
173
reason for this inconsistency is that different tissue types were analysed, namely serum and
174
omental adipose tissue [32, 33].
175
Clearly, there is a lack of data regarding the expression of miRNAs in pregnancies
176
complicated by GDM. However, parallels can be drawn between GDM and DM. Collares et
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al. (2013) investigated the differences and similarities between DM1, DM2 and GDM in 178
peripheral blood mononuclear cells [46]. These investigators found 9 miRNAs: hsa-miR-126,
179
1307, 142-3p, 142-5p, 144, 199a-5p,
hsa-miR-180
27a, hsa-miR-29b and hsa-miR-342-3p that were shared in DM1, DM2 and GDM.
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Interestingly, miR-27a and miR-29b (a miRNA sharing sequence composition to
hsa-182
miR-29a) was also detected in GDM as observed by Li et al. (2015) and Zhao et al. (2011)
183
[32, 34]. Furthermore, Collares et al. (2013) reported that miR-222 is specific to DM2. This
184
miRNA has also been identified in GDM studies [32, 33], contradicting this observation by
185
Collares et al. (2013) [46]. Nevertheless, miR-222 seems to be closely linked to DM2 and
186
GDM pathology.
187
In addition, research detecting an increase of this miRNA within plasma of patients
188
with DM2 further supports the previous studies investigating miR-222 [47, 48]. However, a
189
study investigating miR-222 in plasma microparticles concluded that there was no difference
190
between DM2 and normal controls [49]. A majority of studies have found miR-222
191
differentially expressed in plasma between normal and GDM/DM2 patients [32, 46-48].
192
Consequently, this suggests that miR-222 is not located within microparticles, but within
193
other lipid vesicle subtypes or bound to protein complexes within blood. This miRNA
miR-194
222 has been heavily investigated in cancer and has been demonstrated to regulate Kip1, a
195
protein required for cell cycle entry and growth factor stimulation [50].
196
With regards to GDM, miR-222 has been shown to directly target the 3’ untranslated
197
region of ERα, a protein involved in regulating GLUT4 expression [33, 51]. Shi et al. (2011)
198
further demonstrated that silencing of miR-222 caused an increase of ERα and GLUT4
199
expression. Finally, insulin stimulation in miR-222 silenced cells caused an increase in
200
GLUT4 translocation from the cytoplasm to the plasma membrane, suggesting enhanced
201
glucose uptake [33]. This is very interesting as it means that cells with accumulated miR-222
202
would be less receptive to insulin stimulation. Perhaps this increase in intracellular miR-222
203
can be detected in blood, as demonstrated in the previous studies in DM [46-48]. However,
204
Zhao et al. (2011) found that miR-222 is decreased in serum of patients with GDM [32].
205
Further investigations are needed to understand the role of miR-222 in GDM.
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A study performing a meta-analysis on the current literature for miRNAs and DM has
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identified a range of circulating and tissue specific miRNAs [52]. Interestingly, miR-29a,
208
miR-19b, miR-132, miR-16 and miR-20b were differentially expressed in DM, corroborating
209
with studies in GDM (see Table 1) [32, 52]. Another study identifies 29, 19a,
miR-210
33a being involved in the insulin signalling pathway [53]. These miRNAs are also
211
differentially expressed in GDM, thus are attractive therapeutic targets [34, 36, 45]. Finally,
212
miR-103 and miR-107 have been closely connected to insulin sensitivity [54]. This previous
213
study led to the development of a GalNac-conjugated anti-miR, targeting 103 and
miR-214
107 (called RG-125) by the company Regulus™. RG-125 is currently undergoing phase 1
215
study in humans for the treatment of non-alcoholic steatohepatitis in patients with DM2. This
216
study shows the potential for miRNAs to be used as therapeutics for metabolic disorders, such
217
as GDM.
218
219
What are exosomes?
220
221
Exosomes are a member of the EVs and originate from the endosomal compartment by the
222
fusion of multivesicular bodies with the plasma membrane [55]. Exosomes are bi-layered
223
lipid vesicles between 40 to 120 nm in diameter, are secreted by multiple cell types, and are
224
key players in many biological functions. Furthermore, exosomes contain both RNA
225
(primarily miRNA) and protein. These secreted exosomes are involved in cell-to-cell
226
communication by delivering their cargo into recipient cells. Furthermore, these secreted
227
exosomes can be isolated from conditioned medium and body fluids (e.g. plasma, saliva,
228
urine). Exosome isolation involves several steps including ultracentrifugation, ultrafiltration
229
and separation by density gradient [56]. Centrifugation coupled with ultrafiltration can further
230
enrich for exosomes by separating vesicles based on their size. Specifically, larger vesicles
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such as apoptotic bodies and microvesicles can be separated from smaller vesicles such as
232
exosomes using a 0.22µm filter [57]. Additionally, exosomes can be further purified by
233
density using sucrose and iodixanol gradients. Another exosome isolation method mentioned
234
in the literature is immunoisolation using magnetic beads coated with specific antibodies
235
against exosomal proteins [58]. However, capturing exosomes with a specific exosomal
236
antibody can be limiting when an exosome does not possess the certain marker. Furthermore,
237
a microfluidic device has also been used for separation of exosomes from serum using
micro-238
channels [59]. Different exosome isolation techniques were also applied to obtain exosomes
239
in GDM patients (Table 2).
240
It is important to note that EVs are a heterogeneous group. Its nomenclature is
241
constantly being redefined by the scientific community [60]. A strict separation between the
242
different vesicle types by size or biogenesis has not been established. There is still no
243
agreement on markers (e.g. CD63, Hsp70, CD9 for exosomes) that can distinguish the
244
different type of vesicles by its origin once they leave the cell. Furthermore, Thery et al.
245
(2006) has investigated various exosome isolation techniques and concluded that each
246
isolation technique lead to different yields and purities of the sample [58]. Therefore, these
247
differences in methodologies can result in inconsistencies within downstream protein and
248
RNA analyses. Consequently, in the growing interest of EVs as a diagnostic tool,
249
standardization in EV isolation and analysis methodologies are required.
250
251
Exosomal miRNAs during GDM pregnancies
252
253
Dysregulation of miRNA expression has been linked with metabolic disorders, such as DM2
254
and dyslipidaemia [61]. Consequently, the miRNA population within exosomes can be
255
profiled and used as biomarkers for diseases such as GDM [62]. Consistent with this proposal,
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trophoblast/cytotrophoblast derived exosomes have been used to define fetal-maternal
257
interaction [63, 64] as well as placental dysfunction [65], which is important in GDM
258
pathogenesis.
259
Research into GDM and exosomes is still formative, with only 6 studies specifically
260
focused on exosomes and GDM pathogenesis (see Table 2). Sarker et al. (2014), Salomon et
261
al. (2016) and Nardi et al. (2016) observed an increase in exosome concentration in the 262
maternal plasma of first trimester pregnant women. Furthermore, exosome concentrations
263
correlated with gestational age [66-69], while the release of trophoblast-derived exosomes
264
decreased dramatically in the third trimester [67]. Rice et al. (2015) established a link
265
between increased exosome concentration and GDM by defining the effects of glucose
266
concentration on exosome release and bioactivity [70]. They reported that high glucose
267
concentrations increased the release of exosomes from first-trimester trophoblast cells and the
268
capacity of exosomes to induce the release of cytokine mediators from target cells.
269
A subsequent study by Salomon et al. (2016), reported the differential release of
pro-270
inflammatory cytokines (GM-CSF, IL-4, IL-6, IL-8, IFN-y and TNF-α) from endothelial cells
271
when treated with placental exosomes derived from women with GDM [68]. These data
272
warrant further investigation to elucidate the intracellular mechanisms by which glucose
273
regulates exosome biogenesis, packaging and bioactivity, and how this contributes to the
274
aetiology and progression of GDM.
275
Further research is necessary to identify miRNAs specific for GDM in placental or
276
maternal derived exosomes in blood. It is known that the chromosome 19 miRNA cluster
277
(C19MC) is exclusively expressed in the placenta [64]. Recently, Almohammadi et al. (2016)
278
identified that the abundance of miR-518a-5p, miR-518b, miR-518c, miR-518e, miR-520c-3p
279
and miR-525-5p (members of the C19MC region) in placental exosomes isolated from
280
women with GDM is increased when compared to women with normal pregnancies [71].
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Furthermore, Donker et al. (2012) also investigated the expression C19MC region under
282
placental hypoxia, which is implicated in fetal growth restriction [64]. Donker et al. (2012)
283
found that the C19MC miRNA region is not differentially expressed in placenta hypoxia
284
compared to control, but did observe a downregulation of miR-520c-3p. Furthermore, this
285
miRNA was found to be elevated within maternal plasma of GDM patients. These
286
aforementioned results suggests a role of miR-520c-3p in placental oxygen supply [71].
287
However, the role of the entire C19MC region in GDM pathogenesis remains to be
288 elucidated. 289 290 Conclusion 291 292
The early diagnosis of GDM during the first trimester of pregnancy is critical in preventing
293
complications for both the mother and the fetus. Current diagnostic methods fail to identify
294
GDM early, which leads to the urgent need of research and development for new diagnostic
295
tools. The measurement of extracellular miRNAs encapsulated in exosomes within blood
296
holds great promise as a reliable and accurate diagnostic tool for GDM. Using methods such
297
as qRT-PCR and NGS, these extracellular miRNAs have been demonstrated to be
298
differentially expressed in women with GDM. Currently, research into exosomal miRNAs in
299
GDM has not been sufficient. In addition, there is no apparent correlation between studies
300
investigating extracellular miRNAs and GDM. Therefore, additional research is required to
301
understand the role of exosomes and extracellular miRNA in GDM, how these contribute to
302
the aetiology of GDM, and the potential for using exosomes and extracellular miRNAs as
303
interventions for GDM.
304
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Acknowledgements 306 307This review was generated as part of the Queensland Perinatal Consortium Inaugural
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Conference held on July 15th 2016 in Brisbane, Queensland Australia. The conference was
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supported by an Intra-Faculty Collaborative Workshop grant from the Faculty of Medicine
310
and Biomedical Sciences, The University of Queensland. CS holds a Lions Medical Research
311
Foundation Fellowship.
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525
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Table 1: Tabulation of miRNAs in GDM compared to normal controls.
527 Publication Sample miRNA upregulated in GDM miRNA downregulated in GDM Detection technique
[32] Serum 132,
miR-29a, miR-222
Discovery = TaqMan low density array Validation = Real-time PCR [36] Plasma 16-5p, miR-17-5p, hsa-miR-19a-3p, 19b-3p, hsa-miR-20a-5p Discovery = Ion Torrent (Sequencing) Validation = Real-time PCR
[34] Placental tissue miR-508-3p
27a, miR-9, miR-137, 92a, miR-33a, miR-30d, miR-362-5p and miR-502-5p Discovery = Agilent Human miRNA Microarray Validation = Real-time PCR
[45] Placental tissue miR-518d Real-time PCR
[33] Omental
adipose tissue miR-222
Discovery = AFFX miRNA expression chips Validation = Real-time PCR [44] Primary
HUVEC miR-101 Real-time PCR
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Table 2: Exosomes and exosomal miRNAs in GDM pathogenesis
529 Publicati on Sample GDM linked pathogene sis Exosome isolation method Number of exosomes miRNA Detectio n techniq ue [71] Plasma (trophobl ast cells) To be determined Differential and buoyant density centrifugati on ↑ 518a-5p, 518b, 518c, 518e, miR-520c-3p and miR-525-5p (GDM patients vs normal pregnancy) qRT-PCR [66, 67, 69] Maternal or fetal plasma exosomes Exosomes might play a role in feto-maternal interaction and placenta developme nt [66, 67] Differential and buoyant density centrifugati on using a sucrose continuous gradient ultrafiltratio n. [69] ExoQuick™ or ultracentrifu gation Total exosome Increased 50-fold during pregnancy (pregnancy vs non-pregnant). Placental exosome number decline during pregnancy - - [68] Maternal periphera l plasma exosomes Bioactive (cytokine release) and promote cell migration. Differential and buoyant density centrifugati on using a sucrose continuous ultrafiltratio - - -
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530 531 [70] First trimester trophobla st cells Extracellul ar milieu regulate the release and bioactivity of exosomes Differential and buoyant density centrifugati on Release of exosomes increased with higher glucose and low oxygen tension. High oxygen tension decreased exosome release - - [64] Primary human trophobla st Placenta hypoxia (fetal growth restriction) Ultracentrif ugation and ultrafiltratio n No effect on other CM19MC miRNAs ↓ miR-520c-3p qRT-PCRM
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Figure Legend 532 533Figure 1: Overall summary of extracellular and intracellular miRNAs as biomarkers for
534
GDM. The placenta provides the exchange of nutrients between maternal and fetal blood
535
supply during pregnancy. The mother and fetus release exosomes containing miRNA and
536
proteins, which allows cell-to-cell communication via the delivery of their cargo into recipient
537
cells. In addition, miRNAs can be bound to protein and HDL complexes. Extracellular and
538
intracellular miRNAs can be isolated from plasma or tissue/cells, respectively. After isolation,
539
miRNAs can be quantified by methods including: qRT-PCR, NGS and microarrays. Using
540
these methods, the differential expression of candidate miRNAs between GDM and normal
541
pregnancies can be established and putative biomarkers of GDM (e.g. miR-222 and C19MC
542
miRNA region) identified and validated.
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Conflict of Interest Statement
