Original Research ajog.

org

OBSTETRICS
Metformin as a prevention and treatment for preeclampsia:
effects on soluble fms-like tyrosine kinase 1 and soluble
endoglin secretion and endothelial dysfunction
Fiona C. Brownfoot, MBBS; Roxanne Hastie, BBiomed Sc; Natalie J. Hannan, B Sci, PhD;
Ping Cannon, B Sci; Laura Tuohey, BSci; Laura J. Parry, B Sci, PhD; Sevvandi Senadheera, B Sci PhD;
Sebastian E. Illanes, MBBS, MSc; Tuuhevaha J. Kaituu-Lino, PhD1; Stephen Tong, MBBS, PhD1

BACKGROUND: Preeclampsia is associated with placental ischemia/
hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble
endoglin into the maternal circulation. This causes widespread endothelial
dysfunction that manifests clinically as hypertension and multisystem organ
injury. Recently, small molecule inhibitors of hypoxic inducible factor 1a have
been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin
secretion. However, their safety profile in pregnancy is unknown. Metformin
is safe in pregnancy and is also reported to inhibit hypoxic inducible factor
1a by reducing mitochondrial electron transport chain activity.
OBJECTIVE: The purposes of this study were to determine (1) the
effects of metformin on placental soluble fms-like tyrosine kinase 1 and
soluble endoglin secretion, (2) to investigate whether the effects of met-
formin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion
are regulated through the mitochondrial electron transport chain, and
(3) to examine its effects on endothelial dysfunction, maternal blood
vessel vasodilation, and angiogenesis.
STUDY DESIGN: We performed functional (in vitro and ex vivo) ex-
periments using primary human tissues to examine the effects of met-
formin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion
from placenta, endothelial cells, and placental villous explants. We used
succinate, mitochondrial complex II substrate, to examine whether the
effects of metformin on soluble fms-like tyrosine kinase 1 and soluble
endoglin secretion were mediated through the mitochondria. We also
isolated mitochondria from preterm preeclamptic placentas and gesta-
tionally matched control subjects and measured mitochondrial electron
transport chain activity using kinetic spectrophotometric assays.
Endothelial cells or whole maternal vessels were incubated with metformin
to determine whether it rescued endothelial dysfunction induced by either
tumor necrosis factor-a (to endothelial cells) or placenta villous
explanteconditioned media (to whole vessels). Finally, we examined the
effects of metformin on angiogenesis on maternal omental vessel explants.
RESULTS: Metformin reduced soluble fms-like tyrosine kinase 1 and
soluble endoglin secretion from primary endothelial cells, villous cyto-
trophoblast cells, and preterm preeclamptic placental villous explants. The
reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin
secretion was rescued by coadministration of succinate, which suggests
that the effects of metformin on soluble fms-like tyrosine kinase 1 and
soluble endoglin were likely to be regulated at the level of the mito-
chondria. In addition, the mitochondrial electron transport chain inhibitors
rotenone and antimycin reduced soluble fms-like tyrosine kinase 1
secretion, which further suggests that soluble fms-like tyrosine kinase 1
secretion is regulated through the mitochondria. Mitochondrial electron
transport chain activity in preterm preeclamptic placentas was increased
compared with gestation-matched control subjects.
Metformin improved features of endothelial dysfunction relevant to pre-
eclampsia. It reduced endothelial cell messenger RNA expression of vascular
cell adhesion molecule 1 that was induced by tumor necrosis factorea
(vascular cell adhesion molecule 1 is an inflammatory adhesion molecule
up-regulated with endothelial dysfunction and is increased in preeclampsia).
Placental conditioned media impaired bradykinin-induced vasodilation;
this effect was reversed by metformin. Metformin also improved whole
blood vessel angiogenesis impaired by fms-like tyrosine kinase 1.
CONCLUSION: Metformin reduced soluble fms-like tyrosine kinase 1
and soluble endoglin secretion from primary human tissues, possibly by
inhibiting the mitochondrial electron transport chain. The activity of the
mitochondrial electron transport chain was increased in preterm pre-
eclamptic placenta. Metformin reduced endothelial dysfunction, enhanced
vasodilation in omental arteries, and induced angiogenesis. Metformin has
potential to prevent or treat preeclampsia.
Key words: electron transport chain, metformin, preeclampsia, soluble
endoglin, soluble fms-like tyrosine kinase 1

organ injury.23-35 There are no treatments

to arrest disease progression; expectant

Cite this article as: Brownfoot FC, Hastie R, Hannan NJ,

et al. Metformin as a prevention and treatment for pre-
eclampsia: effects on soluble fms-like tyrosine kinase 1
and soluble endoglin secretion and endothelial dysfunc-
tion. Am J Obstet Gynecol 2016;214:356.e1-15.
0002-9378/free
Crown Copyright 2016 Published by Elsevier Inc. All rights
reserved.
http://dx.doi.org/10.1016/j.ajog.2015.12.019
P reeclampsia is a serious pregnancy
complication that globally is
responsible for >100 maternal and 400
perinatal deaths each day.1-7 An important
step in the pathophysiologic condition
may be placental ischemia/hypoxia,8-15
which leads to the release of soluble fms-
like tyrosine kinase 1 (sFlt-1)16-18 and sol-
uble endoglin (sENG)19 into the maternal
circulation.17,20-22 These cause endothelial
dysfunction that leads to multisystem
management and delivery remain the only
treatment options.1,25,36-39 A medication
that is safe in pregnancy, that reduces
placental sFlt-1 and sENG secretion, that
rescues endothelial dysfunction, and that is
angiogenic may be effective in the treat-
ment or prevention of preeclampsia.
There is interest in the use of drugs
that inhibit hypoxic inducible factor 1a
(HIF1a) to treat preeclampsia.9,40,41
HIF1a is up-regulated with ischemia/

356.e1 American Journal of Obstetrics & Gynecology MARCH 2016

ajog.org
hypoxia42 and facilitates sFlt-1 secre-
tion.43,44 Therefore, drugs that block
HIF1a activity may decrease sFlt-1
secretion. Indeed, the HIF1a inhibitors
YC-140 and ouabain41 have been shown
to reduce sFlt-1 secretion from placental
tissues. However, the safety prole of
YC-1 and ouabain in pregnancy are not
known, and they are still in clinical trials
for use in pulmonary hyperplasia45,46
and cancer,47,48 respectively.
This prompted us to explore repur-
posing a medication that is thought to be
safe in pregnancy that inhibits HIF1a
and lead us to investigate metformin.49
Metformin is an oral hypoglycemic
agent that is used to treat gestational
diabetes mellitus.49-51 Recently, metfor-
min has been reported to reduce
breast52,53 and prostate54 cancer metas-
tasis and prolong survival. This discov-
ery renewed interest in the further
exploration of its mechanism of action; it
recently was shown to inhibit HIF1a by
blocking complex I of the mitochondrial
electron transport chain.53,55,56 There-
fore, we hypothesized that metformin
may reduce sFlt-1 secretion in women
with preeclampsia.
Metformin has been reported to have
vasoprotective properties; epidemiologic
studies have shown that it reduces
cardiovascular morbidity in patients
with polycystic ovarian syndrome26 and
diabetes mellitus.57,58 This has been
attributed to its ability to reduce vascular
cell adhesion molecule 1 (VCAM-1),59,60
which is a molecule that is expressed on
the luminal surface of blood vessels in
the presence of inammation and is
increased in preeclampsia.61 Metformin
has also been reported to induce vaso-
dilation of diabetic rat vessels.62
Objective
This study had 3 objectives: (1) to assess
the effects of metformin on sFlt-1 and
sENG secretion from primary placental
and endothelial cells/tissues and to
investigate whether these effects are
mediated through mitochondrial elec-
tron transport chain inhibition; (2) to
assess whether mitochondrial electron
transport chain activity positively regu-
lates sFlt-1 secretion and if preterm
preeclamptic placenta have increased
OBSTETRICS
mitochondrial electron transport chain
activity; and (3) to assess whether met-
formin can reduce endothelial dysfunc-
tion, induce vasodilation, and stimulate
angiogenesis in human omental arteries.
Materials and Methods
Patient population
We performed functional experiments in
which we administered metformin
to human tissues and assessed its effects
on sFlt-1 and sENG secretion, mito-
chondrial electron transport chain func-
tion, and endothelial dysfunction. To
perform our experiments, we examined
tissues from placenta and blood vessels.
We collected several types of placental
tissues. We isolated human umbilical
vein endothelial cells (HUVECs)63 and
primary villous cytotrophoblast cells64
from the placenta and umbilical cord
that had been collected from patients at
term. We also collected placental villous
explants from patients with severe early
onset proteinuric preeclampsia (deliv-
ered at
34 weeks gestation by cesarean
delivery) as dened by the American
College of Obstetricians and Gynecolo-
gists (ACOG) guidelines.65 Placental
biopsy specimens were taken from 4
random placental sites as recommended
by the Co-Lab consortium.66 There
was hypertension and proteinuria (>300
mg of protein in a 24-hour urine
collection) in all cases. The placental
villous explants were prepared, as
previously described.63,67
To assess mitochondrial electron
transport chain activity, we obtained a
placental biopsy specimen from 23
women with severe preterm proteinuric
preeclampsia (dened by ACOG 2013
guidelines65) and from 25 gestationally
matched normotensive control subjects
who had a preterm delivery without ev-
idence of signicant chorioamnionitis or
maternal comorbidities (Supplementary
Table). All the women who were diag-
nosed with preeclampsia had protein-
uria. All placental samples were collected
from women who underwent a cesarean
delivery. Mitochondrial electron trans-
port chain activity studies were per-
formed, as previously described.68,69
We also collected omental tissue from
patients who had elective term cesarean
MARCH 2016 American Journal of Obstetrics & Gynecology
Original Research
delivery and dissected omental arteries,
as previously described.70 These omental
arteries were used to assess the effects of
metformin on vasodilation (by pressure
myography) and angiogenesis (omental
artery explant assay), as described later.
This study was approved by The
Mercy Health Human Research Ethics
Committee (Institutional review board
number R11/34; approved on the
November 12, 2014); all women gave
written informed consent.
In vitro experiments that
assessed metformin and
mitochondrial electron transport
chain activity inhibitors and
substrates on sFlt-1 and sENG
production and endothelial
dysfunction
HUVECs were plated at 24,000 cells/cm2
between passages 2 and 4 and cultured
at 37 C in 20% O2 and treated for 24
hours. Primary villous cytotrophoblast
cells were plated at 24,000 cells/cm2,
incubated overnight to ensure larger
viable villous cytotrophoblasts had
adhered, washed to remove apoptotic
mononuclear syncytiotrophoblast frag-
ments, then treated for 24 hours at
500,000 cells/cm2 and treated for 48 hours
at 37 C in 8% O2 to assess sFlt-1 and
sENG secretion, respectively. Placental
villous explants of approximately 20-mg
tissue per well (dried weight) were
treated for 72 hours at 37 C in 8% O2.
HUVECs, primary villous cyto-
trophoblast cells, and placental villous
explants were treated with 0, 1, 2, and 5
mmol/L metformin (Sigma Chemical
Company, St. Louis, MO); HUVECs and
primary villous cytotrophoblast cells
were treated with 0, 0.5, and 1 mmol/L
metformin  25 mmol/L succinate
(mitochondrial electron transport chain
substrate; Sigma Chemical Company).
Primary villous cytotrophoblast cells
were also treated with mitochondrial
electron transport chain inhibitors
rotenone (Sigma Chemical Company) at
0, 0.625, 1.25, 2.5, and 5 mmol/L or
antimycin (Sigma Chemical Company)
at 0, 0.156, 0.31, 0.63, and 1.25 mmol/L.
Endothelial dysfunction was induced
in (1) HUVECs that used a constant dose
of 10ng/mL tumor necrosis factor a
356.e2
Original Research OBSTETRICS
(TNFa; Sigma Chemical Company),
(2) whole omental arteries with the use
of 25% conditioned placental villous
explant media (this media is collected
24 hours after being cultured with
placental villous explants that were ob-
tained from normal pregnancies at
term), and (3) omental artery explants
that used 250 ng/mL sFlt-1 at 37 C at
20% O2. Metformin was administered
simultaneously to HUVECs at 0, 1, 2,
or 5 mmol/L for 24 hours, to whole
omental arteries at 0 and 5 mmol/L for
3 hours, and omental artery explants at
1 mmol/L for 120 hours.
Measurement of sFlt-1, sENG, and
VCAM-1, sFlt-1 e15a and i13
Conditioned media were collected, and
RNA was extracted with the RNeasy mini
kit (Qiagen, Valencia, CA) from func-
tional experiments and HUVEC endo-
thelial dysfunction assay. Enzyme-linked
immunosorbent assay for sFlt-1 and
sENG was performed with the DuoSet
VEGF R1/Flt-1 kit (R&D Systems by
Bioscience, Waterloo, Australia) and a
DuoSet Human Endoglin CD/105 ELISA
kit (R&D Systems), respectively. RNA
was quantied with the Nanodrop ND
1000 spectrophotometer (NanoDrop
Technologies Inc, Wilmington, DE).
RNA (0.2 mg) was converted to com-
plementary DNA with the use of Applied
Biosystems high capacity cDNA reverse
transcriptase kit (Life Technologies,
Mulgrave, Australia). Sybr gene expres-
sion assay for sFlt-1 e15a and sFlt-1 i13
(Geneworks, South Australia, Australia)
was performed,71 and a taqman gene
expression assay was used for VCAM-1
(Life Technologies).
Assessment of the effect of
metformin on whole omental
artery vasodilation
Treated whole omental arteries were
mounted on a pressure myograph organ
bath (Living Systems Instrumentation,
Burlington, VT). Incremental doses
of 0.01 nmol/L to 1mmol/L bradykinin
(Auspep, West Melbourne, Australia)
were infused, and vasodilation
was assessed with video microscopy
(Diamtrak Software, Adelaide, SA,
Australia).72
356.e3 American Journal of Obstetrics & Gynecology MARCH 2016
Assessment of the effect of
metformin on angiogenesis with
the use of omental artery explants
Omental artery explants (0.5-mm rings)
were stained with calcein AM (Merck
Millipore, Darmstadt, Germany) and
imaged at 40 magnication with the
EVOS FL microscope (Life Technolo-
gies); outgrowth was assessed with image
J (http://imagej.nih.gov/ij/).73
Statistical analysis
Technical triplicates were performed
for each experiment, with a minimum of
3 biologic replicates for each in vitro
study (samples from different patients
were used for each biologic replicate).
When 2 groups were analyzed, a t-test
(parametric) or a Mann-Whitney test
(nonparametric data) was used. When
3 groups were compared, a 1-way
analysis of variance test (parametric) or
a Kruskal-Wallis test (non-parametric)
was used. Statistical analysis was done
with GraphPad Prism 6 software
(GraphPad Software, La Jolla, CA). All
data are expressed as mean  SEM;
probability values of <.05 were consid-
ered signicant.
Detailed methods are included in the
supplementary Methods section.
Results
Metformin reduces sFlt-1 secretion
from primary endothelial cells and
placental tissues
We assessed the effects of metformin on
sFlt-1 secretion from endothelial and
placental tissues because they are its main
tissue source. Administering metformin
dose-dependently reduced sFlt-1 secretion
from endothelial cells (HUVECs; Figure 1,
A) and primary cells that were isolated
from placenta (villous cytotrophoblast
cells; Figure 1, B). At the highest doses,
metformin reduced endothelial and
placental cell secretion by 53% and 63%,
respectively. Metformin also reduced sFlt-
1 secretion from placental villous explants
that were obtained from 4 women who
had been diagnosed with preterm pre-
eclampsia (delivery required <34 weeks
gestation; Figure 1, C).
We investigated the effect of metfor-
min on messenger RNA (mRNA)
expression of different sFlt-1 variants in
ajog.org
the cells, or placental villous explant
tissues. sFlt-1 i13 is the most abundant
sFlt-1 variant in endothelial cells.74
Metformin dose-dependently reduced
sFlt-1 i13 mRNA expression in endo-
thelial cells (Figure 1, D). sFlt-1 e15a is
the predominant variant expressed in
human placenta.74 Metformin reduced
sFlt-1 e15a mRNA expression in primary
villous cytotrophoblasts cells (Figure 1,
E) and placental villous explants
(Figure 1, F) that were obtained from
women with preterm preeclampsia.
Thus, we conclude that metformin re-
duces sFlt-1 isoform expression and sFlt-
1 secretion in endothelial and placental
cells/tissues, including placental villous
explants from patients diagnosed with
preterm preeclampsia.
Metformin reduces sENG secretion
from primary endothelial and
placental tissues
We next investigated the effects of met-
formin on sENG secretion from primary
endothelial cells and placental cells/
tissues. Metformin dose-dependently
reduced sENG secretion from HUVECs
(Figure 2, A) and primary villous cyto-
trophoblast cells (Figure 2, B). Metfor-
min induced a trend towards a reduction
in sENG secretion from preterm pre-
eclamptic placental villous explants at
3 doses, but none of these decreases were
signicant (Figure 2, C).
Metformin reduces sFlt-1 and sENG
secretion by inhibiting the
mitochondrial electron transport
chain
Given that metformin inhibits mito-
chondrial electron transport chain activity
by blocking complex I,53,55,56 we exam-
ined whether the decrease in sFlt-1
secretion was mediated through mito-
chondrial electron transport chain inhi-
bition. Succinate is a substrate for complex
II of the mitochondria, downstream of the
effect of metformin blockade. Thus, if
metformin was reducing sFlt-1 secretion
by directly blocking complex I, then suc-
cinate should restore electron ow and
rescue this effect. Indeed, succinate
rescued a reduction in sFlt-1 secretion that
was induced by endothelial cells (Figure 3,
A) and primary villous cytotrophoblasts
ajog.org OBSTETRICS Original Research

FIGURE 1 FIGURE 2
Effect of metformin on soluble fms-like tyrosine kinase 1 secretion and Effect of metformin on soluble
isoforms e15a and i13 expression in endothelial cells and placental tissue endoglin secretion from endothelial
cells and placental tissue

Metformin (0, 1, 2, and 5 mmol/L) reduced

soluble endoglin secretion from A, endothelial
cells and B, villous cytotrophoblast cells. Met-
Metformin (0, 1, 2, 5 mmol/L) dose-dependently reduced soluble fms-like tyrosine kinase 1 secretion formin did not change soluble endoglin secre-
from A, endothelial cells, B, villous cytotrophoblast cells, and C, preterm preeclamptic placental tion from C, preterm preeclamptic placental
villous explants. Metformin reduced endothelial cell expression of D, sFlt-1 i13 isoform, E, villous villous explants. The single asterisk indicates
cytotrophoblast cells, and F, preterm preeclamptic placental villous explant messenger P < .05; the double asterisks indicate P < .01;
RNA expression of sFlt-1 e15a. The single asterisk indicates P < .05; the double asterisks indicate the quadruple asterisks indicate P < .00001.
P < .01; the triple asterisks indicate P < .0001; the quadruple asterisks indicate P < .00001. sENG, soluble endoglin.
sFlt-1, soluble fms-like tyrosine kinase 1.
Brownfoot et al. Metformin decreases sFlt-1 and sENG,
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol improves endothelial function, and is angiogenic. Am
2016. J Obstet Gynecol 2016.

cells (Figure 3, B). Succinate also rescued
a decrease in sENG secretion that
was induced by metformin in primary
HUVECs (Figure 3, C) and villous cyto-
trophoblast cells (Figure 3, D). These data
raise the possibility that the effects of
metformin on sFlt-1 and sENG secretion
are mediated through its effects on the
mitochondria.
Inhibition of the mitochondrial
electron transport chain reduces
sFlt-1 secretion from primary
villous cytotrophoblasts cells
To our knowledge, the concept that the
mitochondria regulate sFlt-1 secretion is
novel. To obtain further evidence that
this is the case, we examined whether
other mitochondrial electron transport
chain inhibitors reduce sFlt-1 secretion.
Administering rotenone, another com-
plex I inhibitor, to primary villous
cytotrophoblast cells reduced sFlt-1
secretion by 65% (Figure 4, A). Anti-
mycin, a complex III inhibitor, also
reduced st-1 secretion by 75%
(Figure 4, B). These doses of rotenone
and antimycin did not induce cell death

MARCH 2016 American Journal of Obstetrics & Gynecology 356.e4

Original Research OBSTETRICS ajog.org

FIGURE 3 FIGURE 4
Effect of metformin and mitochondrial electron transport chain complex I Mitochondrial electron transport
activity on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion chain inhibitors and sFlt-1
from endothelial cells and villous cytotrophoblast cells secretion

A, Rotenone (0, 0.62, 1.25, 2.5, and 5 mmol/L),

a complex 1 mitochondrial electron transport
chain inhibitor, and B, antimycin (0, 0.16, 0.31,
0.63, and 1.25 mmol/L), a complex III mito-
chondrial electron transport chain inhibitor, both
significantly reduce soluble fms-like tyrosine
The effect of metformin (0.5 and 1 mmol/L) on soluble fms-like tyrosine kinase 1 secretion from kinase 1 secretion from villous cytotrophoblast
A, endothelial cells and B, villous cytotrophoblast cells is likely mediated through mitochondrial cells. The single asterisk indicates P < .05; the
electron transport chain complex 1 inhibition because the effect is rescued in the presence of the double asterisks indicate P < .01; the triple
mitochondrial electron transport chain substrate, succinate (25 mmol/L). Furthermore, metformin asterisks indicate P < .0001.
also reduced soluble endoglin secretion likely through mitochondrial electron transport chain sFlt-1, soluble fms-like tyrosine kinase 1.
complex 1 inhibition from C, endothelial cells (0.5, 1 mmol/L) and D, villous cytotrophoblast cells Brownfoot et al. Metformin decreases sFlt-1 and sENG,
(1 mmol/L) because succinate (25 mmol/L) rescued its effect on secretion. The single asterisk in- improves endothelial function, and is angiogenic. Am
dicates P < .05; the double asterisks indicate P < .01; the triple asterisks indicate P < .0001; the J Obstet Gynecol 2016.

quadruple asterisks indicate P < .00001.

sENG, soluble endoglin; sFlt-1, soluble fms-like tyrosine kinase 1.
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol the preeclamptic placentas for all 4
2016. complexes, and this was signicant
for complex II (Figure 5). Therefore,
(MTS assay and calcein stain; data not we hypothesized that preeclamptic pla- mitochondrial electron transport chain
shown). These studies provide further centas might have increased mitochon- activity may be increased in preterm
evidence that the mitochondria is drial electron transport chain activity. We preeclamptic placenta.
involved in the regulation of sFlt-1 therefore compared mitochondrial elec-
secretion. tron transport chain activity in preterm Metformin reduces VCAM-1
preeclamptic placentas (n 23) and expression on endothelial cells
Mitochondrial electron transport normotensive gestation matched preterm Endothelial dysfunction is associated
chain activity is up-regulated in control placentas (n 25; Supplementary with increased VCAM-1 expression in
preterm preeclamptic placenta Table contains baseline characteristics). the endothelium.61,75 VCAM-1 is an
Given the mitochondria appears to posi- We observed an increase in mitochon- adhesion molecule that is expressed
tively regulate sFlt-1 and sENG secretion, drial electron transport chain activity in on the luminal surface of blood vessels

356.e5 American Journal of Obstetrics & Gynecology MARCH 2016

ajog.org OBSTETRICS Original Research

FIGURE 5 FIGURE 6
Mitochondrial electron transport chain activity in preterm preeclamptic Effect of metformin on endothelial
placenta compared with gestationally matched controls cell vascular cell adhesion
molecule 1 expression

Inflammatory cytokine tumor necrosis factor a

increased endothelial cell expression of vascular
cell adhesion molecule 1 and was significantly
reduced with increasing doses of metformin
(0, 1, 2, and 5 mmol/L). The single asterisk
indicates P < .05; the triple asterisks indicate
P < .0001.
TNFa, tumor necrosis factor a; VCAM 1, vascular cell adhesion
molecule 1.
Brownfoot et al. Metformin decreases sFlt-1 and sENG,
improves endothelial function, and is angiogenic. Am
J Obstet Gynecol 2016.

differ from the normal culture media

Activity of all (A-D) complexes in the mitochondrial electron transport chain was increased in
control (Figure 7).
preeclamptic placenta (n 23), compared with preterm placental controls (n 25), and was
significant for complex II (B). The single asterisk indicates P < .05.
CS, citrate synthase.
Metformin enhances angiogenic
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol sprouting from omental vessel
2016. explants
Reduced angiogenesis is thought to
and can cause an inammatory mesh into the maternal circulation have a contribute to placental hypoxia and
and snare circulating blood cells. role in inducing whole blood vessel contribute to the development of pre-
Preeclampsia is also associated with dysfunction. We incubated human eclampsia.23,77 We explored whether
increased circulating TNFa,76 which is omental arteries in either conditioned metformin could rescue the inhibition of
a proinammatory cytokine that upre- placental culture media or normal cul- angiogenesis that is caused by sFlt-1. We
gulates VCAM-1.61,75 We therefore per- ture media (not incubated with placental devised a human angiogenesis omental
formed an in vitro assay for which tissue). The vessels that were incubated ring explant assay that was based on
we added TNFa to HUVECs, which in normal culture media dilated 100% a mouse aorta explant model.73 We
signicantly up-regulated VCAM-1 in response to bradykinin (endogenous obtained omental biopsy specimens at
expression in endothelial cells (Figure 6). vasodilator), but vessels that were cesarean delivery, isolated the omental
The administration of metformin incubated in placental culture media vessels, dissected them into small
signicantly reduced TNFa-induced exhibited a signicant impairment in explants, and cultured them with or
VCAM-1 expression, which suggests that vasorelaxation (40% less; Figure 7, A). without sFlt-1. We found a signicant
it may have effects to decrease endothe- This suggests that placental factors reduction in angiogenic sprouting from
lial dysfunction. seem to impair vasorelaxation in our the vessels in the presence of sFlt-1
assay. (Figure 8). However, metformin res-
Metformin induces vasodilation in When metformin was coadministered cued sFlt-1-induced inhibition of
maternal vessels that are isolated to vessels that were cultured with the angiogenic sprouting (Figure 8).
from the omentum placental culture media, this impairment A PowerPoint presentation that
We next set up an assay to mimic the fact of vasodilation was reversed, and the summarizes these data is included in
that placental factors that are released bradykinin-mediated relaxation did not supplementary materials.

MARCH 2016 American Journal of Obstetrics & Gynecology 356.e6

Original Research OBSTETRICS ajog.org

Comment FIGURE 7
Primary findings of the study Effect of metformin on whole blood vessel dilation
Metformin reduces sFlt-1 and sENG
secretion from primary human tissues,
possibly by inhibiting the mitochondrial
electron transport chain. Second,
mitochondrial electron transport chain
activity positivity regulates sFlt-1 secre-
tion, and mitochondrial electron trans-
port chain activity is increased in
preterm preeclamptic placenta. By
using assays to replicate the endothelial
and vascular dysfunction that may be
occurring in preeclampsia, we found
that metformin reduces endothelial
dysfunction, improves vasodilation, and
is angiogenic. Collectively, our results
suggest that metformin has potential to
prevent or treat preeclampsia.

sFlt-1 secretion is regulated

through the mitochondrial
electron transport chain; metformin
possibly blocks this pathway
This is the rst report to show that
sFlt-1 secretion is regulated through the
mitochondrial electron transport chain.
Using 2 inhibitors, we showed that
blocking complex I or III signicantly
reduced sFlt-1 secretion.
We have generated circumstantial
evidence that suggests that metformin
inhibits sFlt-1 and sENG secretion by
inhibiting complex I of the mitochon-
drial electron transport chain. The
administration of succinate (a substrate
for complex II of the mitochondrial
electron transport chain) rescued the
metformin-induced reduction in sFlt-1
secretion but had no effect on sFlt-1
secretion when administered alone.
However, a limitation of this experiment
is that succinate is also thought to stabi-
lize HIF1a directly.78 Furthermore,
metformin is known to have other
intracellular targets, such as activating
AMP-activated protein kinase.79 Another
consideration is whether metformin may
be inhibiting syncytialization to reduce
sFlt-1 secretion. We do not believe this is
likely for the following reasons: (1)
metformin had the same effect on sFlt-1
secretion from endothelial cells, which
do not fuse; (2) we observed a reduction
in sFlt-1 secretion from intact placental
villous explants, and (3) there was a
Whole omental blood vessels that were cultured with placental villous explant media and were
compared with those blood vessels that were cultured with normal media have impaired relaxation to
the vasodilator bradykinin (1 mmol/L). Metformin (5 mmol/L) reversed the effect of placental villous
explant media on vasodilation and improved relaxation to levels that were seen in those vessels
cultured in normal media. The single asterisk indicates P < .05.
pEC50, half maximal effective concentration.
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol
2016.
reduction in sFlt-1 mRNA from primary activity in preeclamptic placenta,80 while
trophoblasts that indicated a direct effect the other concluded mitochondrial elec-
on the transcriptional machinery of the tron transport chain activity was
cell. We note that we have not demon- decreased.81 Both reports examined only
strated conclusively that metformin is 6 preeclamptic placentas that were ob-
decreasing sFlt-1 secretion via its effects tained predominately from term preg-
on the mitochondria. nancies (10 of the 12 placentas were
at term).80,81 Our study is the rst to
Mitochondrial electron transport examine preterm preeclampsia specif-
chain activity is increased in ically, and we investigated a considerably
preterm preeclamptic placenta larger cohort of samples compared with
We found an increase in activity in all those previous reports. 80,81
electron transport chain complexes in Given that our data suggests that
preterm preeclamptic placentas, which preeclamptic placentas have increased
was signicant for the activity of complex electron transport chain activity, we
II. There are 2 other reports that speculate that enhanced mitochondrial
have examined mitochondrial electron electron transport chain activity in
transport chain activity. One report preeclampsia may have a role in the
was concordant with our results, also increased sFlt-1 and sENG secretion seen
reporting an increase in complex II in this disease.82

356.e7 American Journal of Obstetrics & Gynecology MARCH 2016

ajog.org OBSTETRICS Original Research

FIGURE 8
Effect of metformin on angiogenesis

Omental whole vessel rings cultured in the presence of soluble fms-like tyrosine kinase 1 have reduced vessel outgrowth, which is restored with
the addition of metformin (1 mmol/L). The white arrows point at vessel outgrowth. Representative micrographs are shown by the asterisk that indicates
P < .05
sFlt-1, soluble fms-like tyrosine kinase 1.
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol 2016.

The therapeutic implication of our and associated with endothelial are affected by insulin resistance85 and
ndings that the mitochondria regulates dysfunction.61 Metformin previously diabetes mellitus,62 respectively. We
sFlt-1 and sENG secretion is that has been shown to reduce endothelial developed an assay to assess whether
screening other mitochondrial electron cell inammatory gene and protein metformin vasodilates maternal vessels
transport chain inhibitors may identify expression83,84 and serum VCAM-1 that are incubated in placental-
new therapeutic candidates for concentrations in patients with diabetes conditioned media. Indeed, metformin
preeclampsia. mellitus59 and impaired glucose toler- appeared to enhance the vasodilatory
ance.60 We demonstrated that, in the response to bradykinin. We propose
Metformin rescues endothelial presence of TNFa that is an inamma- that this technique could be used
dysfunction and impaired tory cytokine that is up-regulated in more widely to assess the effect of
vasodilation specific to preeclampsia,76 metformin reduced small molecules on vasodilation in the
preeclampsia VCAM-1 expression in endothelial cells. context of pregnancy. Experiments to
VCAM-1 is an inammatory protein Metformin has been shown to determine whether this effect was
that is up-regulated in preeclampsia vasodilate mouse and rat vessels that dependent on an intact endothelium

MARCH 2016 American Journal of Obstetrics & Gynecology 356.e8

Original Research OBSTETRICS
were not performed and merit investi-
gation in the future.
Metformin improves angiogenesis
There is an imbalance towards an
antiangiogenic state in preeclamp-
sia.16,19,20,23,82 We devised an assay to
examine the effects of metformin on
angiogenesis in maternal vessels. There
are a number of other angiogenesis
assays that could have been performed.
These include tube-forming assays
that use endothelial cells alone or are
cocultured with broblasts and mouse
or rat aortic ring assays.73 We believe
our assay more closely represents the
dysfunction that is present in pre-
eclampsia because it examines human
vessels, examines angiogenesis in
heterogeneous tissues that includes
both endothelial and vascular smooth
muscle; we induced dysfunction with
sFlt-1, the antiangiogenic molecule
that is of direct relevance to pre-
eclampsia. We demonstrated that sFlt-1
reduced human omental vessel
outgrowth and that metformin rescued
this effect. These data suggest that
metformin has angiogenic effects on
maternal vessels.
Metformin has potential to prevent
and treat preeclampsia
Metformin is safe in pregnancy and
currently is used to treat gestational
diabetes mellitus. Interestingly, a ran-
domized trial that compared metformin
and insulin to treat gestational diabetes
mellitus49 showed a nonsignicant
reduction in the incidence of gestational
hypertension (3.9% metformin arm,
compared with 6.2% insulin treatment)
and preeclampsia (5.5% metformin arm,
7% insulin treatment) among those
treated with metformin. A randomized
trial86 that assessed the effect of metfor-
min on reducing perinatal morbidity in
obese women did not identify a differ-
ence in preeclampsia risk. However, it is
possible that under-dosing and poor
compliance may be an explanation for
the reason that there was no effect on
the incidence of preeclampsia. Further-
more, a metaanalysis of 2 randomized
control trials that assessed the effect of
metformin on pregnancy outcome in
356.e9 American Journal of Obstetrics & Gynecology MARCH 2016
patients with polycystic ovarian syn-
drome also did not show a reduction in
preeclampsia.87
It is important to note that incidence
of hypertensive disorders of pregnancy
and preeclampsia were not the primary
outcome of these previous trials.49,86 In
light of our work, we propose that a
clinical trial that will examine whether
metformin can prevent or treat pre-
eclampsia is justied. Given our ndings
of reduced sFlt-1 and sENG placental
secretion and improved endothelial
dysfunction, we believe metformin
may be able to reduce both placental
and maternal vascular aspects of
preeclampsia
Conclusion
We have performed preclinical studies
using primary human tissues to show
that mitochondrial electron transport
chain activity is up-regulated in preterm
preeclamptic placenta and that the
mitochondrial electron transport chain
regulate villous cytotrophoblast cells and
endothelial cell sFlt-1 and sENG secre-
tion. Metformin reduces sFlt-1 and
sENG secretion by inhibiting complex 1
of the mitochondria. Furthermore,
metformin reduces key features of
endothelial dysfunction that are specic
to preeclampsia and enhances angio-
genesis. Metformin may be a novel
preventative or be therapeutic for pre-
eclampsia and has the potential to reduce
the burden of this major pregnancy
complication. n placental soluble fms-like tyrosine kinase 1
Acknowledgments
We thank the research midwives, Gabrielle Pell,
Genevieve Christophers, Rachel Murdoch, and
Debra Jinks, and the patients at Mercy Hospital
for Women for participating in this research.
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Author and article information
From the Translational Obstetrics Group, Department of
Obstetrics and Gynaecology, University of Melbourne,
Mercy Hospital for Women (Drs Brownfoot, Hannan,
Kaituu-Lino, and Tong and Ms Hastie, Cannon, and
Tuohey) Heidelberg, Victoria, Australia; Biosciences,
University of Melbourne, (Drs Parry and Senadheera),
Parkville, Victoria, Australia; and Department of Obstet-
rics and Gynecology, Laboratory of Reproductive Biology,
Faculty of Medicine, Universidad de Los Andes, Santiago,
Chile (Dr Illanes).
1
These authors have contributed equally to this article.
Received Nov. 30, 2015; revised Dec. 15, 2015;
accepted Dec. 15, 2015.
Supported by The National Health and Medical
Research Council of Australia (NHMRC; #1048707,
#1046484, #1101871) and an Arthur Wilson RANZCOG
scholarship; by an Australian Postgraduate Award and an
AVANT scholarship (F.B); by a CR Roper Research
Fellowship (N.J.R.); the NHMRC provided salary support
(#1050765 [S.T.]; #1062418 [T.K.L.]; #628549 [S.S.]).
The funders had no role in study design, data collection,
analysis, decision to publish or the preparation of the
manuscript.
The authors report no conflict of interest.
Corresponding author: Stephen Tong, MBBS, PhD.
s.tong@unimelb.edu.au
ajog.org
Supplementary:
Methods
Cell culture media
Placental tissues, endothelial cells,
and omental vessels were cultured in a
variety of media. Human umbilical
vein endothelial cells (HUVECs) were
cultured in M199 media (Life Technolo-
gies, Mulgrave, Australia) that contained
10% fetal calf serum, 1% antibiotic-
antimicotic (Life Technologies), 1%
endothelial cell growth factor (Sigma
Chemical Company, St. Louis, MO), and
1% heparin; primary trophoblasts and
placental explants were cultured in
DMEM high Glutamax (Life Technolo-
gies) that contained 10% fetal calf serum
(Sigma Chemical Company) and 1%
antibiotic-antimicotic (Life Technologies).
Isolation of HUVECs and
trophoblasts from human placenta
Placentas were obtained from women who
had undergone elective cesarean delivery
at term. HUVECs were isolated by ob-
taining the umbilical cord, infusing it with
10 mL (1 mg/mL) of collagenase (Wor-
thington, Lakewood, NJ), and ushing the
cells through. as previously described.1
Primary trophoblasts were isolated by
scraping 50 g of cotyledon tissue from the
placenta, subjecting it to enzyme digestion
buffer, and separating cells with the use of
a discontinuous Percoll gradient then
subjecting them to negative selection with
CD9 to remove contaminating cells, as
previously described.2
Cell viability assays (MTS assay
and calcein stain)
Cell viability assays were performed for
all HUVEC and primary trophoblast
functional experiments with the use of
either CellTiter 96-Aquesous One solu-
tion (Promega, Madison, WI) or calcein
stain (Merck Millipore, Darmstadt,
Germany) and were quantitated with a
Fluostar omega uorescent plate reader
(BMG Labtech, Victoria, Australia).
Whole omental vessel collection,
pressure myography, and omental
vessel explant outgrowth
Omental vessel tissue collection
Omental tissue was obtained from
women with normal pregnancies
OBSTETRICS
who had undergone elective cesarean
delivery at term. The omental biopsy
specimens were transferred to cold
Krebs physiologic solution (PSS) that
contained 112 mmol/L NaCl, 25 mmol/
L NaHCO3, 4.7 mmol/L KCl, 1.2 mmol/
L MgSO4.7H2O, 0.7 mmol/L KH2PO4,
10 mmol/L HEPES, 11.6 mmol/L D-
glucose, and 2.5 mmol/L CaCl2.2H2O;
pH 7.4; Sigma Chemical Company) and
washed 3 times to remove the anesthetic.
The average inner lumen diameters of
arteries that were used were 280-320 mm
at 60 mm Hg in zero calcium. All ex-
periments were performed at <20 hours
after surgery.
Whole vessel pressure
myography
Whole omental arteries were cleaned
carefully of connective and adipose tis-
sue. Arteries were cultured for 3 hours at
4 C in treatment groups (control group,
M199 media [Sigma Chemical Com-
pany] or treatment groups, M199 with
25% conditioned media that had been
exposed to placental explants for 24
hours and M199 with 25% conditioned
media with 5 mmol/L metformin
[Sigma Chemical Company]) for incu-
bation studies. All arteries were moun-
ted in a pressure myograph organ bath
(Living Systems Instrumentation, Bur-
lington, VT) and continuously super-
fused with PSS (37 C) at a rate of 4 mL/
min. In the absence of intraluminal ow,
arteries were pressurized gradually to
60 mm Hg, warmed to 37 C, and
checked for pressure leaks over a
50-minute equilibration period. The
outer diameters were measured through
video microscopy (Diamtrak Software,
Adelaide, SA, Australia). Before the start
of each experiment, the viability of the
smooth muscle was tested with potas-
sium physiologic saline solution
(KPSS, 100 mmol/L) or thromboxane
agonist 9,11-dideoxy-9a,11a-meth-
anoepoxy prostaglandin F2a (U46619, 1
mmol/L; Sigma Chemical Company)
washed with PSS to regain basal diam-
eter. Arteries were preconstricted with
thromboxane agonist U46619; endo-
thelial function was tested by applying
bradykinin (10 mmol/L; Auspep, West
Melbourne, Australia), and washed to
MARCH 2016 American Journal of Obstetrics & Gynecology
Original Research
regain basal diameter. Arteries were then
submaximally preconstricted (approxi-
mately 70% of maximal response to
KPSS or U46619) with U46619 (Sigma
Chemical Company); vascular reactivity
to drugs were assessed in the following
manner. In incubation studies, the effect
of treatments on vascular reactivity was
assessed with bradykinin (0.01-1000
nmol/L). At the end of the experiment,
smooth muscle integrity was tested
in response to the endothelium-
independent, nitric oxide donor, so-
dium nitroprusside (10 mmol/L). The
organ bath was then washed out with
PSS, and Ca2-free Krebs solution was
added for 20 minutes to determine the
maximum diameter. Ca2-free Krebs so-
lution contained no added CaCl2.2H2O
but contained EGTA (2 mmol/L).
Human omental vessel explants
To assess changes in angiogenesis
potential, a human omental vessel
outgrowth assay was conceived and
performed. Omental samples were
collected from patients who had had an
elective cesarean delivery at term. An
arteriole was dissected carefully out of
the fat, and the vessel was ushed with
a 26G needle with OptiMEM (Life
Technologies). The vessel was cut into
0.5-mm rings and serum-starved in
OptiMEM at 20% O2, 5% CO2 at 37 C
overnight. The ring was embedded in a
collagen matrix (1 mg/mL in DMEM,
pH adjusted so slightly basic using
NaOH) in a 96-well plate with 1 omental
ring per well. Rings were treated in
media that contained OptiMEM with
2.5% fetal calf serum (Sigma Chemical
Company) and 1% antibiotic anti-
mycotic (Life Technologies)  250 ng/
mL soluble fms-like tyrosine kinase 1
(sFlt-1) and 0 or 1 mmol/L metformin
(Sigma Chemical Company). For each
patient sample (n 4), we used 5 rings
(technical replicates) per treatment
group. Treatments were changed every
48 hours, and the experiment was
continued for 120 hours. Calcein AM
(Merck Millipore, Darmstadt, Ger-
many) was added to the wells for 45
minutes at 37 C, and images were ob-
tained at the same magnication (40)
with the EVOS FL microscope (Life
356.e12
Original Research OBSTETRICS
Technologies). Vessel outgrowth at the
completion of the experiment was
determined by a calculation of the area
of growth with the computer program
Image J (http://imagej.nih.gov/ij/).
Assessment of mitochondrial
electron transport chain activity
in preterm preeclamptic placenta
and preterm gestationally
matched placenta
Placental collection for
mitochondrial electron transport
chain activity assay
Placental tissue was obtained from 23
women with severe preterm pre-
eclampsia that was dened with the
American College of Obstetricians and
Gynecologists 2013 guidelines3 and
from 25 gestationally matched preterm
control subjects who delivered either
because of spontaneous preterm
labor without evidence of infection
(conrmed on histopathologic evalua-
tion), hypertensive disease, or maternal
comorbidities. Baseline patient clinical
characteristics are detailed in the Sup-
plementary Table.
Placental tissue was processed within
30 minutes of delivery. Placental tissue,
excluding fetal membranes, was washed
in sterile phosphate buffered saline,
frozen within 15 minutes, and stored
at e80 C.
Isolation of mitochondria from
preeclamptic and preterm
placental samples
Intact mitochondria were isolated, as
previously described.4 Briey, to isolate
intact mitochondria, the placental tissue
was homogenized in ice-cold Zheng
buffer (210 mmol/L mannitol, 70
mmol/L sucrose, 5 mmol/L HEPES, and
1 mmol/L EGTA, pH 7.2) and centri-
fuged at 600g for 10 minutes at 4 C; the
supernatant was transferred to an ice-
cold microcentrifuge tube and freeze
thawed 3 times in a methanol bath
(1-minute freeze followed by 4-minute
thaw at room temperature).4
Electron transport chain activity
All mitochondrial respiratory complex
activity was performed by spectro-
photometric assays, as previously
356.e13 American Journal of Obstetrics & Gynecology MARCH 2016
described.4 The Fluostar Omega Fluo-
rescent Plate Reader (BMG Labtech,
Victoria, Australia) was used to measure
absorbance. First, the activity of the
mitochondrial matrix enzyme citrate
synthase was measured by the rate of free
sulfhydryl group production. This was
determined by the administration of thiol
reagent 5,5-dithio-bis-(2-nitrobenzoic
acid) to placenta, which reacts with free
sulfhydryl to produce thio nitrobenzoate
acid. Placental samples were sonicated on
ice for 1 minute to disrupt the mito-
chondrial membrane and 1 mmol/L 5,5-
dithio-bis-(2-nitrobenzoic acid) and 12.5
mmol/L acetyl CoA were added to 10 mL
of placental sample or blank, and the
absorbance was read every 15 seconds for
2 minutes at 412 nmol/L. To begin the
reaction, 5 mmol/L of oxaloacetic acid
was then added, and absorbance was read
every 15 seconds at 412 nmol/L for 4
minutes.
The activity of complex I was
measured by the determination of
the transfer of electrons from b-nicotin-
amide adenine dinucleotide to coenzyme
Q. This was achieved by the activation of
complex 1 by adding 5mmol/L b-nico-
tinamide adenine dinucleotide, 50
mmol/L potassium cyanide, 0.5 mmol/L
antimycin A, 10% bovine serum albu-
min, and 5.0 mmol/L oxidized coenzyme
Q to 10 mL of placental sample or blank
and the inhibiting of the complex by the
addition of rotenone 0.125 mmol/L and
reading the absorbance every 15 seconds
for 3 minutes at 340 nmol/L.
Complex II activity was next assessed
by the measurement of the reduction of
coenzyme Q on oxidation of succinate
to fumarate. Baseline activity was
assessed initially by the addition of 50
mmol/L potassium cyanide, 0.5 mmol/L
antimycin A, 100 mmol/L succinate,
and 0.125 mmol/L rotenone to 10 mL
of placental sample or blank; the
absorbance was read every 15 seconds
for 2 minutes at 280 nmol/L; then 5
mmol/L of coenzyme Q was added, and
the rate of reduction was assessed by the
reading of absorbance every 15 seconds
for 3 minutes at 280 nmol/L.
The activity of complex III was
assessed next by the measurement of the
reduced form of decyl benzoquinone,
ajog.org
which is a short chain of analogue of
endogenous ubiquinone that donates
electrons to cytochrome c. Baseline ac-
tivity was assessed by the addition of 50
mmol/L potassium cyanide, 0.125
mmol/L rotenone, 10% bovine serum
albumin, 25 mmol/L n-dodecyl-b-D-
Maltoside, 10 mmol/L reduced dur-
oquinone, and 1 mmol/L oxidized cy-
tochrome c to 10 mL of placental sample
or blank; absorbance read every 12 sec-
onds for 4 minutes at 550 nmol/L; 25
mmol/L of L-ascorbate was added to
complete the reaction, and the absor-
bance was read every 15 seconds for 5
minutes at 550 nmol/L.
The activity of complex IV, the ter-
minal enzyme in the electron transport
chain, was measured by the assessment
of the loss of reduced cytochrome c as it
is oxidized. Baseline activity was deter-
mined by the addition of reduced cyto-
chrome c to 10 mL of placental sample or
blank; the absorbance was read every 15
seconds for 5 minutes at 550 nmol/L;
then 50 mmol/L of potassium ferricya-
nide was added to stop the oxidation of
cytochrome c, and the absorbance was
read every 15 seconds for 2 minutes at
550 nmol/L.
Electron transport chain activity was
calculated as rst-rate constants (rst
rate constant per minute per milligram)
that were determined by the subtraction
of the nal absorbance reading after the
addition of inhibiting substance from
the absorbance before and the data
plotted as a slope against time. The slope
is denoted the rst-order rate constant.
Activity was normalized to milligrams of
tissue or citrate synthase (maker of
mitochondrial content).
Enzyme-linked immunosorbent
assay (ELISA) analysis
Concentrations of sFlt-1 and soluble
endoglin were measured in conditioned
cell/tissue culture media with the use
of the DuoSet VEGF R1/Flt-1 kit
(R&D Systems by Bioscience, Waterloo,
Australia) and a DuoSet Human Endo-
glin CD/105 ELISA kit (R&D Systems)
according to the manufacturers in-
structions. The coefcient of variation
for our ELISA sFlt-1 was 3.4% and for
sENG was 2.4%.
ajog.org
Real timeepolymerase chain
reaction (PCR)
RNA was extracted from placental ex-
plants and HUVECs using an RNeasy
mini kit (Qiagen, Valencia, CA) and
quantied using the Nanodrop ND 1000
spectrophotometer (NanoDrop tech-
nologies Inc, Wilmington, DE). 0.2 mg
of RNA was converted to complemen-
tary DNA with the use of Applied Bio-
systems high capacity complementary
DNA reverse transcriptase kit (Life
Technologies), as per manufacturer
guidelines. The coefcient of variation
for the PCR for vascular cell adhesion
molecule 1 was 5.6%; the coefcient of
variation for the PCR sFlt-1-i13 and
e15a was 3.24%.
A taqman gene expression assay was
performed for VCAM-1 (Life Technol-
ogies). Real timeePCR was performed
on the CFX 384 (Bio-Rad, Hercules, CA)
with the use of FAM-labeled Taqman
universal PCR mastermix (Life Tech-
nologies) with the following run condi-
tions: 50 C for 2 minutes; 95 C for 10
minutes; 95 C for 15 seconds, and 60 C
for 1 minute (40 cycles). Sybr gene
expression assay for sFlt-1 e15a and sFlt-
1 i13 was used. Primers were designed as
previously described (Geneworks, South
Australia, Australia).5 RT-PCR was per-
formed with the following run condi-
tions: 95 C for 20 minutes; 95 C for
0.01 minutes, 60 C for 20 minutes, 95 C
for 1 minute (39 cycles), melt curve
OBSTETRICS
65 C to 95 C at 0.05 C increments at
0.05 seconds.
All data were normalized to GAPDH
as an internal control and calibrated
against the average Ct of the control
samples. Results were expressed as fold
change from control.
Statistical analysis
Technical triplicates were performed for
each experiment, with a minimum of 3
biologic replicates (different patient
samples) for each in vitro study. Data
were tested for normal distribution and
analyzed with the use of the appropriate
parametric or nonparametric test. The
statistical software we used was Graph-
Pad Prism 6 (GraphPad Software, La
Jolla, CA). When 3 groups were
compared, a 1-way analysis of variance
(for parametric data) or Kruskal-Wallis
test (for nonparametric data) was used.
Post-hoc analysis was carried out with
either the Tukey (parametric) or Dunns
test (nonparametric). When 2 groups
were analyzed, either an unpaired t-test
(parametric) or a Mann-Whitney test
(nonparametric) was used. Concentra-
tion response curves from omental ar-
teries were tted to a sigmoidal curve
with nonlinear regression to calculate
the sensitivity of each agonist (pEC50) or
maximum relaxation (Emax). All data
were expressed as mean  SEM; prob-
ability values of <.05 were considered
signicant.
MARCH 2016 American Journal of Obstetrics & Gynecology
Original Research
References
1. Brownfoot FC, Hannan N, Onda K, Tong S,
Kaituu-Lino T. Soluble endoglin production is
upregulated by oxysterols but not quenched by
pravastatin in primary placental and endothelial
cells. Placenta 2014;35:724-31.
2. Kaituu-Lino TJ, Tong S, Beard S, et al.
Characterization of protocols for primary
trophoblast purication, optimized for functional
investigation of sFlt-1 and soluble endoglin.
Pregnancy Hypertens 2014;4:287-95.
3. American College of Obstetricians and Gy-
necologists. Report of the American College of
Obstetricians and Gynecologists task force on
hypertension in pregnancy. Obstet Gynecol
2013;122:1122-31.
4. Frazier AE, Thorburn DR. Biochemical ana-
lyses of the electron transport chain complexes
by spectrophotometry. Methods Mol Biol
2012;837:49-62.
5. Whitehead CL, Palmer KR, Nilsson U, et al.
Placental expression of a novel primate-specic
splice variant of sFlt-1 is upregulated in preg-
nancies complicated by severe early onset pre-
eclampsia. BJOG 2011;118:1268-71.
356.e14
Original Research OBSTETRICS ajog.org

SUPPLEMENTARY TABLE
Baseline characteristics of placenta collected that compares mitochondrial electron transport chain activity
in placentas that were complicated by preterm preeclampsia vs controls (normotensive, preterm delivery)
Characteristic Preeclampsia (n 23) Preterm (n 25) P value
Maternal age, y a
30.6  5.9 31.1  6.6 .78
Gestation, wka 29.6  2.3 29.4  2.4 .75
Body mass index, kg/m 2a
28.3  5.5 27.6  7.9 .58
Systolic blood pressure, mm Hg a
169.9  15.4 119.8  14.7 < .0001
Diastolic blood pressure, mm Hg a
103.6  9.9 70.4  14.1 < .0001
Birthweight, g a
1190  448.5 1393  475.7 .14
Nulliparity, n (%) 15 (65) 9 (36) .08
Intrauterine growth restriction (birthweight, <10%), n (%) 11 (48) 10 (40) .77
a
Data are shown is mean  standard deviation.
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol 2016.

356.e15 American Journal of Obstetrics & Gynecology MARCH 2016