Article

Mice generated by in vitro fertilization exhibit vascular dysfunction and shortened life span

The Journal of clinical investigation (Impact Factor: 13.22). 11/2013; 123(12). DOI: 10.1172/JCI68943
Source: PubMed

ABSTRACT

Children conceived by assisted reproductive technologies (ART) display a level of vascular dysfunction similar to that seen in children of mothers with preeclamspia. The long-term consequences of ART-associated vascular disorders are unknown and difficult to investigate in healthy children. Here, we found that vasculature from mice generated by ART display endothelial dysfunction and increased stiffness, which translated into arterial hypertension in vivo. Progeny of male ART mice also exhibited vascular dysfunction, suggesting underlying epigenetic modifications. ART mice had altered methylation at the promoter of the gene encoding eNOS in the aorta, which correlated with decreased vascular eNOS expression and NO synthesis. Administration of a deacetylase inhibitor to ART mice normalized vascular gene methylation and function and resulted in progeny without vascular dysfunction. The induction of ART-associated vascular and epigenetic alterations appeared to be related to the embryo environment; these alterations were possibly facilitated by the hormonally stimulated ovulation accompanying ART. Finally, ART mice challenged with a high-fat diet had roughly a 25% shorter life span compared with control animals. This study highlights the potential of ART to induce vascular dysfunction and shorten life span and suggests that epigenetic alterations contribute to these problems.

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Available from: Daniel Fuster, Nov 10, 2015
Research article
The Journal of Clinical Investigation http://www.jci.org 1
Mice generated by in vitro fertilization
exhibit vascular dysfunction
and shortened life span
Emrush Rexhaj,
1
Ariane Paoloni-Giacobino,
2
Stefano F. Rimoldi,
1
Daniel G. Fuster,
3
Manuel Anderegg,
3
Emmanuel Somm,
4
Elisa Bouillet,
1
Yves Allemann,
1
Claudio Sartori,
1,5
and Urs Scherrer
1,6
1
Swiss Cardiovascular Center Bern and Department of Clinical Research, University Hospital, Bern, Switzerland.
2
Department of Genetic and Laboratory Medicine and Swiss Center for Applied Human Toxicology, Geneva University Hospital, Geneva, Switzerland.
3
Division of Nephrology, Hypertension and Clinical Pharmacology, University Hospital, Bern, Switzerland.
4
Division of Development and Growth,
Department of Paediatrics, University of Geneva Medical School, Geneva, Switzerland.
5
Department of Internal Medicine,
CHUV, Lausanne, Switzerland.
6
Facultad de Ciencias, Departamento de Biología, Universidad de Tarapacá, Arica, Chile.
Children conceived by assisted reproductive technologies (ART) display a level of vascular dysfunction simi-
lar to that seen in children of mothers with preeclamspia. The long-term consequences of ART-associated
vascular disorders are unknown and difficult to investigate in healthy children. Here, we found that vascula-
ture from mice generated by ART display endothelial dysfunction and increased stiffness, which translated
into arterial hypertension in vivo. Progeny of male ART mice also exhibited vascular dysfunction, suggest-
ing underlying epigenetic modifications. ART mice had altered methylation at the promoter of the gene
encoding eNOS in the aorta, which correlated with decreased vascular eNOS expression and NO synthesis.
Administration of a deacetylase inhibitor to ART mice normalized vascular gene methylation and func-
tion and resulted in progeny without vascular dysfunction. The induction of ART-associated vascular and
epigenetic alterations appeared to be related to the embryo environment; these alterations were possibly
facilitated by the hormonally stimulated ovulation accompanying ART. Finally, ART mice challenged with
a high-fat diet had roughly a 25% shorter life span compared with control animals. This study highlights the
potential of ART to induce vascular dysfunction and shorten life span and suggests that epigenetic altera-
tions contribute to these problems.
Introduction
Epidemiological studies showing an association between patho-
logic events during early life and the development of cardiovas-
cular and metabolic disease in adulthood have led to the “fetal
programming of adult disease hypothesis” (1, 2). In line with
this hypothesis, experimental studies show that short-term
hypoxemia after birth predisposes to exaggerated hypoxic pul-
monary vasoconstriction later in life in rats (3) and humans (4)
and preeclampsia predisposes the offspring to pulmonary and
systemic endothelial dysfunction (5). Recent data suggest that
assisted reproductive technologies (ART) represent another
example for this hypothesis, since arterial pressure is increased
in rats and children generated by ART (6, 7) and young children
conceived by ART display generalized vascular dysfunction and
signs of early arteriosclerosis (8).
While these findings demonstrate vascular dysfunction in ART
children that is of magnitude similar to that observed in offspring
of mothers with preeclampsia (5) or preadolescents with type I dia-
betes (9), conditions known to be associated with an increased risk
for premature cardiovascular morbidity (10–12), several important
issues remain unclear. First, in humans, it is difficult to determine
whether this problem is related to the ART procedure itself or
to parental factors. Second, the long-term cardiovascular conse-
quences of ART are unknown because this population has not yet
reached the age at which premature cardiovascular diseases typi-
cally start to occur. Finally, the underlying mechanisms of ART-
induced vascular dysfunction are not known and are difficult to
study in healthy children.
We, therefore, developed a mouse model of ART to first test
the hypothesis that ART induces vascular dysfunction in the
offspring of normal mice. We found that ART mice display
endothelial dysfunction, increased arterial stiffness, and arterial
hypertension. To study whether ART predisposes to premature
mortality, we compared the life span of ART and control mice
fed with normal chow or challenged with a high-fat diet (HFD).
We then started to test for underlying mechanisms. Hormonal
stimulation of ovulation is inherently needed for ART and may
have effects on vascular function in the offspring. We, therefore,
compared vascular function between mice born after hormonal
stimulation in the mother and control mice. The time passed in
culture medium may be another factor involved in ART-induced
vascular dysfunction. To test this hypothesis, we compared vas-
cular function between mice born after implantation of 2 cell
embryos and blastocysts. Finally, in mice, epigenetic mechanism
are involved in the fetal programming of vascular dysfunction
(13), and in humans, the prevalence of very rare diseases caused
by epigenetic mechanisms is increased in ART (14, 15). Epigen-
etic alterations may be transmitted to the next generation (13, 16,
17). Therefore, we examined whether male ART mice transmitted
the vascular dysfunction to their progeny. In a next step, we then
Authorship note: Claudio Sartori and Urs Scherrer contributed equally to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. doi:10.1172/JCI68943.
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2 The Journal of Clinical Investigation http://www.jci.org
studied the methylation of imprinted genes and genes involved
in cardiovascular regulation in ART mice, offspring of ART mice,
and control mice. Finally, we examined the effects of administra-
tion of a deacetylase inhibitor on the methylation of genes and
vascular function in these 3 groups of animals.
Results
Endothelial dysfunction, increased arterial stiffness, and arterial
hypertension in ART mice
To assess the putative effects of ART on vascular function, we assessed
acetylcholine-mediated and nitroprusside-induced mesenteric artery
vasodilation in vitro in ART and control mice. We found that acetyl-
choline-induced vasodilation was impaired in ART mice compared
with control mice (Figure 1A). In contrast, nitroprusside-induced
vasodilation was normal in ART mice (Figure 1B), demonstrating
mesenteric artery endothelial dysfunction. To assess for premature
vascular senescence, we examined carotid artery vascular stiffness.
We found that vascular stiffness was increased in ART mice (Figure
1, C and D). To examine whether endothelial dysfunction in vitro had
functional consequences in vivo, we measured arterial blood pressure
and found that it was higher in ART mice than in control mice, both
during short-term measurements and during chronic measurements
by telemetry over 48 hours (Figure 1, E and F).
Decreased life span in ART mice
To evaluate whether ART had consequences on the life span in
mice, we compared the survival of ART and control mice. To
facilitate the detection of potential differences, in addition to
groups of mice fed with normal chow, groups of ART and con-
trol mice were challenged with a HFD. We found that in mice
fed with normal chow, life span was similar in the 2 groups. In
contrast, a HFD, while not having a detectable effect on life
span in control mice, significantly reduced survival in ART
mice (P < 0.0001, Figure 2).
Underlying mechanisms
Effects of stimulation of the ovulation in the mother on vascular function in
the offspring. To examine whether hormonal stimulation of ovula-
tion in the mother alters vascular function in the offspring, we
Figure 1
Mesenteric artery responsiveness, carotid artery pressure diameter relationship, and arterial blood pressure in mice generated by ART and con-
trol mice. (A) Acetylcholine-induced and (B) sodium nitroprusside–induced mesenteric artery vasodilation in vitro (data represent mean ± SEM).
(C and D) Carotid artery pressure diameter relationship in vitro. C shows outer diameter (OD) and D shows inner diameter (ID). (E) Mean arterial
blood pressure in vivo obtained by short-term measurements and (F) 48-hour long-term measurements by telemetry. Horizontal lines represent
the median; boxes, 25
th
to 75
th
percentiles; and T bars, 5th and 95th percentiles. Data are for at least 10 mice per group except for C and D
(n = 5 per group) and F (n = 4 per group).
Figure 2
Kaplan-Meier survival curves of ART and control mice fed with normal
chow (NC) or challenged with a HFD. Median survival (days): Control
NC = 853; control HFD = 787; ART normal chow = 736; ART HFD = 582.
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compared mesenteric artery responses to acetylcholine in off-
spring of superovulated and control mice. Overall, acetylcholine-
induced mesenteric vasodilation was comparable in the 2 groups
(Figure 3), but at lower doses of acetylcholine, curves tended to
diverge, and the responses to the 1 × 10
–8
dose were statistically
different between the 2 groups (P = 0.04).
Vascular dysfunction is similar in ART mice generated by implantation of
2 cell embryos and blastocysts. To examine whether a shorter duration
of embryo culture attenuates/prevents ART-induced vascular dys-
function in the offspring, we compared mesenteric artery respons-
es to acetylcholine and arterial blood pressure between mice gen-
erated by implantation of 2 cell embryos (30 hours of incubation)
and blastocysts (96 hours of incubation). ART-induced vascular
dysfunction and arterial hypertension were comparable in the 2
groups of mice (Figure 4).
Male ART mice transmit the vascular dysfunction to their progeny.
There is evidence that ART may alter epigenetic mechanisms
(14, 15). These alterations may be transmitted to the next gen-
eration (13, 16, 17). To test for this possibility, we examined
vascular function and arterial blood pressure in male offspring
generated by mating male ART mice with control females.
Endothelial dysfunction and arterial hypertension in offspring
of male ART mice were comparable to those observed in their
fathers (Figure 5, A and B).
Methylation of imprinted genes in the aorta is altered in ART mice
and their progeny
Normalization by administration of the deacetylase inhibitor butyrate.
To evaluate whether ART altered the methylation of genes in the
vasculature, we studied the methylation of imprinted genes in
the aorta. We found that the methylation of the imprinted genes
H19, Gtl2, and Peg3, but not of Peg1 and Snrpn, was altered in the
aorta of ART mice (Figure 6). To determine whether these altera-
tions were specific for vascular tissue or generalized, we examined
the methylation of these genes in the liver and found that it was
similar in the 2 groups (data not shown). Finally, to study whether
altered methylation was transmitted to the next generation, we
examined the methylation of imprinted genes in the aorta of the
progeny of male ART mice. The alterations of the methylation of
imprinted genes in the aorta of the offspring were comparable to
those observed in their fathers (Figure 6).
Deacetylase inhibitors are capable of normalizing altered meth-
ylation (13, 18). We therefore examined the effects of butyrate
administration to male ART mice on the methylation of imprinted
genes. We found that butyrate normalized the methylation of the
imprinted genes H19, Gtl2, and Peg3 (but had no detectable effect on
the methylation of the normally methylated imprinted genes Peg1
and Snrpn) in the aorta of ART mice, whereas it had no detectable
effect on the methylation of these genes in control mice (all P > 0.22,
control + vehicle vs. control + butyrate; Figure 6). Finally, butyr-
ate administration to male ART mice before mating prevented the
transmission of altered methylation to the progeny (Figure 6).
Altered DNA methylation of the promoter of the eNOS gene,
altered eNOS and eNOS mRNA expression in vascular tissue,
and decreased nitric oxide plasma concentration in ART mice
Normalization by administration of the deacetylase inhibitor butyrate. While
the above findings show altered methylation of imprinted genes in
ART mice, the consequences of these alterations are not known,
since the physiologic function of these genes is unknown. We, there-
fore examined DNA methylation of genes known to be important
in cardiovascular regulation. DNA methylation of the promoter of
the eNOS gene was increased in the aorta of ART mice (Figure 7A),
whereas DNA methylation of the promoter of the endothelin-1 and
the angiotensin converting enzyme (ACE) gene was not different
between ART and control mice (endothelin-1: 15.9% ± 1.0% vs. 16.4%
± 1.0%, control vs. ART, P = 0.27; ACE: 4.0% ± 0.5% vs. 3.9% ± 0.3%,
Figure 3
Acetylcholine-induced mesenteric artery vasodilation in vitro in off-
spring of superovulated and control mice. Data represent mean ± SEM
for at least 7 mice per group.
Figure 4
Effects of embryo culture time on mesenteric artery responsiveness and blood pressure in ART mice. Mesenteric artery responsiveness to acetylcho-
line (A) and arterial blood pressure (B) in ART-mice generated by implantation of 2 cell embryos and blastocysts. In A, data represent mean ± SE. In
B, horizontal lines represent the median; boxes, 25th to 75th percentiles; and T bars, 5th and 95th percentiles. Data are for at least 9 mice per group.
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control vs. ART, P = 0.33). Increased promoter DNA methylation
downregulates eNOS expression in endothelial cells in vitro (19).
In line with this concept, eNOS expression (Figure 7B) and eNOS
mRNA expression (0.56% ± 0.08% vs. 1.0% ± 1.21%, P = 0.047, ART
vs. control) in the carotid artery were decreased in ART mice com-
pared with control animals. Decreased eNOS expression appeared
to be of functional importance, because nitric oxide (NO
x
) plasma
concentration was lower in ART than in control mice (Figure 7C).
Butyrate administration to ART mice normalized DNA
methylation of the promoter of the eNOS gene (Figure 7A) and
eNOS expression (Figure 7B) and restored NO
x
plasma concen-
tration (Figure 7C).
Butyrate administration to ART mice normalizes endothelial function and
prevents transmission of vascular dysfunction to the offspring. To evaluate
whether normalization of the DNA methylation and vascular NO
synthesis by butyrate had favorable effects on vascular function in
ART mice,we examined vascular responsiveness to acetylcholine. We
found that butyrate administration to ART mice normalized mes-
enteric artery function, whereas it had no detectable effect on this
function in control mice (Figure 8A). Moreover, butyrate adminis-
tration to male ART mice before mating prevented the transmission
of vascular dysfunction to the next generation (Figure 8B).
Discussion
We recently showed that children conceived by ART display
vascular dysfunction (8). The long-term consequences of this
problem are unknown, and its underlying mechanisms are
difficult to investigate in healthy children. Here, we show for
what we believe is the first time that in mice generated by ART,
endothelial function of the systemic circulation in vitro is defec-
tive and translates into arterial hypertension in vivo. Moreover,
ART also had consequences on the life span; when mice were
challenged with a HFD, life span was roughly 25% shorter in
ART than in control mice.
Consistent with findings in children conceived by ART show-
ing endothelial dysfunction (8), in ART mice, acetylcholine-
induced mesenteric artery dilation was impaired while endothe-
lium-independent dilation was preserved. Moreover, as in ART
Figure 5
Transmission of vascular dysfunction and arterial hypertension by mice generated by ART to their offspring. Mesenteric artery responsiveness to
acetylcholine in vitro (A) and arterial blood pressure in vivo (B) in mice generated by ART, offspring of mice generated by ART, and control mice.
In A, data represent mean ± SEM. In B horizontal lines represent the median; boxes, 25th to 75th percentiles; and T bars, 5th and 95th percentiles.
P values by 1-way ANOVA and Dunnett’s post hoc test. Data are for at least 10 mice per group.
Figure 6
Methylation of imprinted genes H19, Gtl2, Peg3, Peg1, and Snrpn in the aorta of control mice, mice generated by ART, and their progeny. Effects
of administration of the deacetylase inhibitor butyrate to control and ART mice on the methylation of these genes and on the transmission of the
dysmethylation to the progeny. *P < 0.001, **P < 0.001, ***P = 0.02, ART + vehicle vs. control + vehicle;
#
P = 0.03,
##
P = 0.03,
###
P < 0.01, ART
+ butyrate vs. ART + vehicle;
P < 0.01,
††
P < 0.001,
†††
P = 0.014, offspring of ART + vehicle vs. control + vehicle;
P = 0.04,
‡‡
P = 0.03, offspring
of ART + butyrate vs. ART + vehicle. Data represent mean ± SEM for at least 7 mice per group.
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children, arterial stiffness was increased in ART mice. Whereas in
young ART children, endothelial dysfunction was not associated
with arterial hypertension, in adult ART mice, endothelial dys-
function in the systemic circulation was associated with arterial
hypertension. In humans, it has been speculated that parental
factors (e.g., older age of mothers necessitating to resort to ART,
sterility-associated vascular dysfunction that is transmitted to
the offspring) contribute to vascular dysfunction in offspring
of ART. The present findings showing that in normal mice ART
induces vascular dysfunction in the offspring indicate that ART
per se is the main culprit. Data in humans showing no relation-
ship between the age of the mother and vascular function in the
offspring and demonstrating normal vascular function in sterile
parents are in line with this concept (8).
Due to the young age of the ART population in humans (the
first ART child was born in 1978), it is not known yet whether ART
will affect life expectancy in humans. Here we found that when
fed normal chow, life span was not different in ART and control
mice. However, when mice were challenged with a Western style
HFD, life span in ART mice was roughly 25% shorter than in con-
trol mice. Premature vascular senescence and exaggerated HFD-
induced glucose intolerance and insulin resistance (20) may con-
tribute to this problem. These effects of ART were observed in FVB
mice, a mouse strain less susceptible to atherosclerosis than, for
example, the C57BL/6 strain (21, 22), suggesting that the effects of
IVF may have a greater impact in genetically susceptible individu-
als. These findings suggest that it may be worthwhile to provide
dietary advice to persons born with the help of ART.
Figure 7
Butyrate administration to ART mice normalizes DNA promoter methylation and expression of the eNOS gene in vascular tissue and NO
x
plasma
concentration. DNA methylation of the promoter of the eNOS gene in the aorta (A), eNOS expression in the carotid artery (B), and NO
x
plasma
concentration (C), in control + vehicle, ART + vehicle, ART + butyrate, and control + butyrate mice. Data represent mean ± SEM for at least 13
mice per group. *P = 0.004,
#
P < 0.0001,
P = 0.03 vs. control + vehicle by 1-way ANOVA and Dunnett’s post hoc test. In B, the lanes were run
on the same gel but were noncontiguous.
Figure 8
Butyrate administration to ART mice normalizes vascular function and prevents transmission of dysfunction to offspring. (A) Effects of butyrate
administration to ART and to control mice on acetylcholine-induced mesenteric artery dilation. (B) Effects of butyrate or vehicle administration to ART
mice before mating on acetylcholine-induced mesenteric artery dilation in the progeny. Data represent mean ± SEM for at least 10 mice per group.
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In mice, fetal programming of pulmonary vascular dysfunction
is associated with altered lung DNA methylation (13) and there
is evidence for transmission of epigenetic alterations to the next
generation. Here, we found that vascular dysfunction and arte-
rial hypertension of the progeny of male ART mice mated with
normal females was similar to what was found in their fathers.
In line with this observation, the methylation of imprinted
genes was altered similarly in the aorta of ART mice and their
progeny. While these findings provide proof of principle for
epigenetic alterations in the vasculature of ART mice, the con-
sequences of these alterations for vascular regulation, if any, are
unknown. In a next step, we, therefore, examined the methyla-
tion of genes known to be important for vascular regulation. We
found that DNA methylation of the promoter of the eNOS gene
was increased in arterial tissue of ART mice and associated with
decreased eNOS expression, decreased eNOS mRNA expression,
and lower NO
x
plasma concentration in ART than in control
mice. Taken together, these findings indicate that in mice, ART
alters the entire chain of events starting from altered eNOS meth-
ylation in the vasculature over endothelial dysfunction and pre-
mature vascular senescence to arterial hypertension and possibly
premature mortality. Finally, the methylation of the promoter of
the endothelin-1 and the ACE gene was not altered in ART mice,
suggesting that there exist no global changes of DNA methylation
of genes involved in vascular regulation.
There is increasing evidence for an interplay between histone
modifications and DNA methylation in gene silencing (23).
Changes of methylation can be reversed by histone deacetylase
inhibitors that are known to induce replication-independent
demethylation of ectopically methylated genes (13, 24, 25).
Histone deacetylase inhibitors have been shown to reverse epigen-
etic and phenotypic changes induced by pathologic events during
early life (26) and to prevent transmission of these changes to the
progeny (13). In line with these observations, we found that butyr-
ate administration to male ART mice normalized methylation and
expression of the eNOS gene in vascular tissue together with vas-
cular responsiveness to acetylcholine. Moreover, it prevented the
transmission of vascular dysfunction to the progeny. Collectively,
these data suggest that in mice, epigenetic mechanisms contribute
to ART-induced vascular dysfunction.
The present studies also provide insight regarding the etiology
of ART-induced cardiovascular and epigenetic alterations that may
have consequences for ART in humans. Culture time and hormon-
al effects related to stimulation of ovulation and embryo culture
per se (independent of time) are candidate mechanisms. Here, we
found that the culture time needed to obtain 2-cell embryos was
sufficient to cause these changes, since endothelial dysfunction
and arterial hypertension were comparable in ART mice generated
by implantation of 2-cell embryos and blastocysts. In humans,
embryos are generally implanted at the blastocyst stage. The pres-
ent findings in mice suggest that in humans, attempts to shorten
the time lag between fertilization and implantation are unlikely to
prevent ART-induced vascular dysfunction.
ART implies stimulation of the ovulation in the mother, and it
has been speculated that this procedure may have adverse effects
in the offspring (27). In line with recent findings in humans dem-
onstrating normal vascular function in children conceived during
ovarian stimulation in the mother (8), we found that endothelium-
dependent mesenteric artery vasodilation was normal in offspring
of superovulated mice. However, while we did not see a significant
overall effect of hormonal stimulation alone on endothelial func-
tion in mice, there was a significant (P = 0.04) difference at a lower
dose of acetylcholine between hormonally stimulated and control
mice, suggesting the possibility of an additive effect between hor-
monal stimulation and cell culture on ART-induced epigenetic
and vascular alterations in mice. In line with this speculation,
epigenetic alterations in mouse embryos induced by hormonal
stimulation and culture were reported to be greater than those
induced by embryo culture alone (28). Current culture media used
for in vitro fertilization are suboptimal, as evidenced by reduced
pregnancy rates, viability, and growth of cultured compared with
in vivo embryos. For example, culture media may lack or contain
at different concentration key metabolites and/or growth factors
present in oviductal fluid and are not capable of reproducing the
dynamic changes of oviductal fluid naturally occurring along the
female reproductive tract. Suboptimal in vitro culture conditions
result in compromised ability to maintain genomic imprinting of
in vitro compared with in vivo embryos (28) and may contribute
to the observed epigenetic and cardiovascular alterations in ART
mice in the present study. In line with this speculation, modifica-
tion of the culture medium may allow ART-induced cardiovascu-
lar dysfunction in mice to be attenuated (29). Taken together, the
present findings suggest that embryo culture per se is the main
culprit underpinning ART-induced epigenetic and vascular altera-
tions in mice. These alterations may be facilitated by hormonal
stimulation of ovulation in the mother. We speculate that similar
mechanisms may play a role in the rapidly growing population of
humans conceived by ART, who now make up 2% to 4% of births
in developed countries.
Methods
Chemicals and reagents. Pregnant mare serum gonadotropin (PMSG),
human chorionic gonadotropin (hCG), and ketamin were from Intervet.
Human tubal fluid medium (HTF), human serum albumin (HSA), and
paraffin oil were from Irvine Scientific. G1 and G2 media were from Vitro-
life. Sodium butyrate, acetylcholine and sodium nitroprusside were from
Sigma-Aldrich. Xylazine was from Bayer HealthCare. Temgesic (buprenor-
phine) was from Essex Chemie. Bactrim was from Roche. Biokema Flunix-
ine was from Biokema SA.
Animals. Wild-type (FVB and NMRI) mice were from the Charles-River
Laboratory. Animals were fed a standard chow diet (Safe). Throughout the
study, the mice were housed with lights on from 7:00 am to 7:00 pm, and
access to food and water was ad libitum.
In vitro fertilization and embryo culture. Eight- to twelve-week-old female
FVB mice were superovulated by i.p. injection of 5 IU (0.1 ml) PMSG, fol-
lowed 50 hours later by an i.p. injection of 5 IU (0.1 ml) of hCG.
Fourteen hours after hCG injection, cumulus-oocyte complexes were
recovered from oviducts in HTF supplemented with 5 mg/ml of HSA. Sper-
matozoa were collected from the cauda epididymis of 10- to 14-week-old
FVB mice and capacitated for 60 minutes in HTF/HSA medium at 37°C
under a humidified atmosphere of 6% CO
2
in air. Oocytes were insemi-
nated 14 hours after hCG injection with 10
6
spermatozoa in HTF/HSA
medium for 4 hours at 37°C in a CO
2
-enriched (6%) atmosphere. Eggs were
then transferred to 25 μl drops of G1 medium covered with paraffin oil.
The embryo culture was conducted up to the blastocyst stage in sequen-
tial G1 and G2 medium preequilibrated at 37°C under the CO
2
-enriched
atmosphere. Embryos were kept in the G2 medium for 48 hours before
the transfer to pseudopregnant females. The culture media used in these
studies are largely used during ART in humans. For these studies, the ART
procedure was repeated about 50 times, each time in a different animal.
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Embryo transfer. NMRI females of at least 6 weeks of age were placed with
vasectomized males to mate 2.5 days prior to embryo transfer. The morn-
ing after mating, females were checked for the presence of a vaginal plug.
On the transfer day, pseudopregnant females were anesthetized by i.p.
injection of xylazine (15 mg/kg) and ketamine (100 mg/kg). From 14 to 20
embryos were transferred to the uterus of each female.
Unless stated otherwise, 12- to 14-week-old male ART and control mice
were used for all the in vitro and in vivo studies described below.
Mesenteric artery function in vitro. ART and control mice were killed with
an overdose of pentobarbital sodium (200 mg/kg, i.p.). The mesenteric
artery was dissected free of parenchyma and cut into a ring. Each ring
was positioned between 2 stainless steel wires (diameter 25 μm) in a
5-ml organ bath of a small vessel myograph (DMT 610M; Danish Myo
Technology) (30). The organ bath was filled with modified Krebs-Ring-
er bicarbonate solution (composition: 118.3 mM NaCl, 4.7 mM KCl,
2.5 mM CaCl
2
, 1.2 mM Mg2SO
4
, 1.2 mM KH
2
PO
4
, 25.0 mM NaHCO
3
,
and 11.1 mM glucose), maintained at 37 ± 0.5°C and aerated with 95%
O
2
plus 5% CO
2
(pH = 7.4). At the beginning of the experiment, each
vessel ring was stretched to its optimal resting tension corresponding
to a transmural pressure of 100 mm Hg and allowed to equilibrate for
1 hour. MyoDaq software (MyoDaq 2.01 M610; Danish Myo Technol-
ogy) was used for data acquisition and display. Then, acetylcholine-
induced (ACh, 10
–9
to 10
–5
M) or sodium nitroprusside–induced (10
–10
to
10
–5
M) vasodilation was assessed in mesenteric arteries preconstricted
with phenylephrine (10
–5
mol/l) at a level corresponding at least to the
maximal response to potassium (100 mmol/l KCl). The ACh-induced
change in tension was expressed as the percentage of the initial contrac-
tion induced by phenylephrine.
To assess arterial stiffness, the left carotid artery was quickly excised,
placed in modified Krebs-Ringer solution (composition, see above) cleaned
of adhering tissue and fat. The artery was then mounted onto a pressure
myograph (Danish Myo Technology) in an organ bath filled with physi-
ological buffer, as described previously (31). After a 30-minute equilibra-
tion period, the carotid artery was transilluminated under an inverted
microscope (Zeiss Axio Vert A1) connected to a camera and a computer-
ized system (MyoVIEW II Software; Danish Myotechnology) allowing the
continuous recording of the artery diameter. Measurements of the inner
and outer artery diameter were obtained during stepwise increases (10 mm
Hg steps for 5 minutes from 0 to 180 mm Hg) of the intravascular pressure
and expressed as a percentage of the baseline value measured at 10 mm Hg
of intravascular pressure.
Arterial blood pressure. Arterial blood pressure was recorded continuously
in awake mice, as described previously (32). Briefly, a fluid-filled PE-10 tub-
ing connected to a pressure transducer was inserted into the carotid artery
under isoflurane anesthesia and tunneled subcutaneously to exit at the
back of the neck. Mice were allowed to recover for 4–5 hours before the
blood pressure measurement.
In additional ART and control mice, we measured arterial blood pres-
sure by telemetry, as described previously (33). Briefly, blood pressure
transmitters (TA11PA-C10 transmitter; DataSciences International) were
implanted under inhalational anesthesia with isoflurane on a heating
surface set at 36°C. Preoperatively, mice received 1 ml of prewarmed 0.9%
saline i.p. to support fluid homeostasis and trimethoprim (6 mg/kg)/sul-
fadoxin (30 mg/kg) (Bactrim) s.c. for anti-infective prophylaxis. The left
common carotid artery was exposed, and the tip of the telemetry catheter
was inserted, advanced into the aortic arch, and secured by silk sutures.
The body of the transmitter was positioned in an s.c. pocket on the right
flank. Postoperatively, mice received s.c. injections twice per day for 7 days
of flunixine (5 mg/kg body weight) for pain relief and 300 μl of glucose
(5%) and 300 μl of normal saline (0.9%) to prevent exsiccosis.
From 10–12 days after implantation, transmitters were activated mag-
netically and offset-corrected blood pressure was recorded at 4-minute
intervals for 48 consecutive hours. The recording room was maintained at
22–24°C with a 12-hour light/12-hour dark cycle, and mice had free access
to food and water. The telemeter signal was processed using RPC-1 receiv-
ers, an APR-1 ambient pressure monitor,and a Dataquest A.R.T. 4.31 Gold
acquisition system (DataSciences International).
Histone deacetylase inhibitor administration. To test for the potential patho-
genic role of epigenetic mechanisms, sodium butyrate (2 mg/kg body
weight/d in 200 μl of PBS, i.p.) was administered to 10-week-old male ART
and control mice for 14 days.
Methylation of imprinted genes in the aorta. The maternally expressed pater-
nally methylated H19 (34) and Gtl2 (35) genes and the paternally expressed
maternally methylated Peg3, (36) Peg1 (37), and Snrpn genes (38) were stud-
ied in the aorta. DNA was extracted using the QIAampDNA Microkit (Qia-
gen). Using the EZ Methylation Gold-Kit (Zymo Research), the extracted
DNA was treated with sodium bisulfite in order to convert unmethylated
cytosine residues to uracil. The converted DNA was eluted in 10 μl of TE
buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5). 2 μl of the bisulfite-treat-
ed DNA was used for subsequent PCR amplification.
The PCR amplifications were performed starting from 80–140 ng of
bisulfite-treated DNA. All reactions were performed with PCR reaction
mixtures (total volume 25 μl) containing oligonucleotides at 0.5 mM
concentration and 12.5 μl of HotStarTaq Master Mix (Qiagen). The PCR
conditions were the same for all 5 genes tested, i.e., 94°C for 15 minutes,
followed by 40 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C
for 30 seconds, and by a 72°C 10-minute final extension step. The char-
acteristics of the amplicons and the oligonucleotides chosen have been
described earlier (39).
The biotinylated PCR products were purified using streptavidin-
sepharose beads (Amersham) and sequenced using the PSQ 96 Gold
Reagent Kit (Biotage AB) with the following primers: H19: 5-GTGTA-
AAGATTAGGGTTGT-3; Gtl2: 5-GTTATGGATTGGTGTTAAG-3;
Peg1: 5-TCAATATCAACTAAATAATC-3; Snrpn: 5-GAATTGGAGTT-
GTGTGG-3; Peg3: 5-AATTGATAAGGTTGTAGATT-3. The degree of
methylation at each CpG site was determined using Pyro Q-CpG Software
(Biotage AB). All samples were analyzed in duplicate. Diagrams showing
the location of the DMDs and the DNA fragments of the 5 genes amplified
by PCR in the present study have been published previously (39).
Methylation of the eNOS gene promoter in the aorta. Using the same methods,
methylation analysis was performed in the eNOS (GenBank AF091262)
core promoter, where DNA methylation was shown to control gene
expression (19, 40). The following oligonucleotides were designed with
the PyroMark Assay Design 2.0 program (Qiagen): eNOS-F: 5-TGAGA-
TTTTTGTGGTTATAGGAATATGAT-3; eNOS-R: 5-biotin-7CAA-
CAAAATCCTAACCTTTTCCTTAA-3. The biotinylated PCR products
were purified using streptavidin-sepharose beads (Amersham) and
sequenced using the PSQ 96 Gold Reagent Kit (Biotage AB) with the fol-
lowing oligonucleotide: eNOS-S: 5-TTGGGTTTTTATTTATTAGTTT-
TA-3. This sequence analyzed by pyrosequencing was 151-bp long, encom-
passing 8 CpGs.
Protein extraction and eNOS Western blot. Four carotid arteries of the same
condition were pooled and grounded in liquid nitrogen. Each pool of
carotid tissue was suspended in SDS lysis buffer (62.5 mM Tris, pH 6.8, 5%
SDS, 10 mM EDTA). Following sonication and 5 minutes of full-speed cen-
trifugation, supernatant proteins were quantified by the Lowry method.
Samples were heated at 95°C for 3 minutes, loaded in duplicate (8 μg per
well) in a 8% acrylamid gel, and transferred to PVDF membrane (Millipore).
The membrane was blocked with 5% milk-TBS, 0.1% Tween (TBST) for 1
hour. Blots were probed with anti-eNOS (1:1000; BD Transduction Labo-
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8 The Journal of Clinical Investigation http://www.jci.org
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the PSQ 96 Gold Reagent Kit (Biotage AB) with the following oligonucle-
otide: ACE-S: CCTCCACACTCCAATTATAACTTTC-3. The sequence ana-
lyzed by pyrosequencing was 85-bp long, encompassing 10 CpGs.
NO plasma concentration. NO was measured in plasma samples obtained
by cardiac punctuation by chemiluminescence with a NO analyzer (Sievers
280 NOA) after reduction of NO
x
to NO with VCl
3
(44).
Survival. Eighty male FVB mice generated by ART and 80 control mice
born and raised in our animal facility were observed in these studies. Three
to four animals were placed in a cage in the clean conventional mouse facil-
ity. Six ART and seven control mice had to be sacrificed because of inju-
ries related to fighting. Throughout the study, the mice were housed with
lights on from 7:00 am to 7:00 pm, and access to food and water was ad
libitum. Thirty-two ART and twenty control mice were fed normal chow,
42 ART mice and 53 control mice were fed a HFD (49.5% fat, 31.5% pro-
tein, and 0% carbohydrates; Safe). One week each month, mice in the HFD
groups were fed a standard chow diet (pellets) to avoid dental problems.
Kaplan-Meier survival curves were constructed using known birth and
death dates, and the log-rank test was used to evaluate statistical differ-
ences between groups.
Statistics. Statistical analyses were made using JMP v. 7.0 software (SAS
Institute). Bivariate analyses were made using the unpaired 2-tailed Stu-
dent’s t test and 1- or 2-factor ANOVA. Post hoc comparisons were made
using the Tukey HSD test and Dunnett’s multiple comparison test. A
P value of less than 0.05 was considered to indicate statistical significance.
Unless otherwise indicated, data are given as mean ± SD.
Study approval. All animal protocols were approved by the CHUV Institu-
tional Animal Care Committee.
Acknowledgments
We are indebted to Françoise Urner for help with the initial studies
and to Caroline Mathieu, Christelle Stouder, Pierre Dessen, and
Arnaud Bichat for invaluable technical assistance. This work was
supported by the Swiss National Science Foundation, the Eagle
Foundation, the Leenaards Foundation, the FABER Foundation,
the Swiss Society of Hypertension, the Prof. Dr. Max Cloetta Foun-
dation, and the Placide Nicod Foundation.
Received for publication January 22, 2013, and accepted in revised
form September 4, 2013.
Address correspondence to: Urs Scherrer, Swiss Cardiovascular Center
Bern, University Hospital, Bern, Switzerland. Phone: 41.31.632.01.02;
Fax: 41.31.632.42.11; E-mail: Urs.Scherrer2@insel.ch.
ratories) in 5% milk/TBST overnight and were detected using peroxidase-
conjugated secondary antibody (1:10000) in 5% milk/TBST for 1 hour.
Products were visualized by chemiluminescence (GE Healthcare), and
band intensity was measured using ImageQuant 5.0 software (Molecu-
lar Dynamics). Equal protein loading was confirmed by β-actin (1:10000;
Thermo Scientific Pierce Products) hybridization on the same membrane.
RNA preparation and gene expression analysis. Total RNA from aorta was
extracted using the TRIzol RNA Isolation Reagents according to the manu-
facturer’s protocol (Life Technologies Europe B.V.). From 500 to 1000 ng
total RNA were reverse transcribed using 400 units of Moloney Murine
Leukemia Virus Reverse Transcriptase (Invitrogen) in the presence of 1
U/μl RNasin (Promega Corp.), 0.2 μg of random primers (oligo[dN]6)
(Promega Corp.), 2 mM dNTP, and 20 μM of DTT (Invitrogen). The expres-
sion of the cDNAs for mouse eNOS was determined by quantitative real-
time PCR using an ABI StepOne Plus Sequence Detection System (Applera
Europe) and normalized using the housekeeping gene Rps29. PCR prod-
ucts were quantified using Master SYBR Green Mix (Applera Europe),
and results are expressed in AU relative to the control group mean value.
Primers were designed using Primer Express software (Applera Europe)
and chosen when possible on both sides of an intron to avoid amplifica-
tion of eventual contaminating genomic DNA. Oligos were used at 217
nM each (Microsynth). The sequence of the primers used were as follows:
eNOS, forward: 5-AAGGCAGCGGTGGAAATTAA-3; eNOS, reverse:
5- TCACTTTGGCCAGCTGGTAAC-3
Methylation of the endothelin-1 gene promoter in the aorta. Methylation anal-
ysis was performed in the Edn-1 (GenBank NC_000079) promoter in a
region where DNA methylation was shown to control gene expression
(41, 42). The following oligonucleotides were designed with the Pyro-
Mark Assay Design 2.0 program (Qiagen): Edn-F: 5-GTGATTTTTTA-
AGGAGTTTTAGAAATAGG-3; Edn-R: 5-biotin-7AACCCTACAACCCTA-
AACACACTTATT-3. The biotinylated PCR products were purified using
streptavidin-sepharose beads (Amersham) and sequenced using the PSQ 96
Gold Reagent Kit (Biotage AB) with the following oligonucleotide: Edn-S:
5-GAGTTTTAGAAATAGGTAGG-3. The sequence analyzed by pyro-
sequencing was 239-bp long, encompassing 9 CpGs.
Methylation of the ACE gene promoter in the aorta. Methylation analysis was
performed in the ACE promoter, in a CpG island region where methyla-
tion was reported to be modified in the mouse fetus after maternal dietary
changes during pregnancy (19, 40, 43). The following oligonucleotides were
designed with the PyroMark Assay Design 2.0 program (Qiagen): ACE-F:
5-biotin-7GGTGGTGGTTGGGTTTTATA-3; ACE-R: 5-CCCAACTAAC-
CACCATACTCTAAAACAT-3. The biotinylated PCR products were puri-
fied using streptavidin-sepharose beads (Amersham) and sequenced using
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The Journal of Clinical Investigation http://www.jci.org 9
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  • Source
    • "The mouse model is particularly useful in studying the effects and underlying mechanisms of ART on the long-term health in later life [11, 30]. The findings of some investigations revealing the changes in the glucose metabolism and the vascular dysfunction in humans conceived by ART are consistent with those obtained in a mouse model of ART [10, 11, 29]. However, the mechanisms of ART-induced metabolic and cardiovascular disorders are still unknown. "
    [Show abstract] [Hide abstract] ABSTRACT: Environmental influences during early development increase the susceptibility to metabolism diseases in adulthood. Assisted reproductive techniques (ART) expose the gametes or preimplantation embryo to a non-physiological environment which increases the risk of metabolism diseases in later life. However, the precise underlying causes of ART-related metabolism disease remain unclear. In our previous study, by using a mouse model, we found that ART resulted in placental maldevelopment and dysfunction which led to a reduced fetal growth. The lipid metabolism and lipid transporters in the placenta were also affected by ART. Based on these findings, we hypothesized that ART may hamper fetal lipid metabolism, which could predispose to metabolic diseases in later life. In the present examination, by lipidomic analysis, we investigated for the first time the effect of ART on phospholipid profiles in the fetal liver in a mouse model and presented it in a detailed overview. We revealed that ART increased significantly the level of lysophosphatidylcholine (LPC), phosphatidic acid (PA), and lysophosphatidylethanolamine (LPE) in the livers of fetuses compared with those in the controls. LPC and PA acts as signaling molecules involved in the majority of cellular processes regulating many crucial physiological and pathophysiological processes. LPC has been evidenced to exert a crucial impact in the development of atherosclerosis and type 2 diabetes, and increase in PA can result in insulin resistance. We proposed that changes of LPC and PA may be one of the causes of the changes in glucose metabolism and vascular dysfunction in the mouse model of ART.
    Preview · Article · Mar 2016 · Biology of Reproduction
  • Source
    • "It seems that the duration of time that oocytes spending under in vitro conditions is an important factor determining success fertilization. Also, it has been reported that mice generated by IVF suffer from vascular dysfunction later in life and have a shortened lifespan (Rexhaj et al., 2013). As Ca 2+ is a signalling molecule regulating a range of cellular functions (Clapham, 2007), it is possible that even when conception occurs it may have consequences for later life. "
    [Show abstract] [Hide abstract] ABSTRACT: Study question: Could drugs targeting ATP-sensitive K(+) (KATP) channels prevent any spontaneous increase in intracellular Ca(2+) that may occur in human metaphase II (MII) oocytes under in vitro conditions? Summary answer: Pinacidil, a KATP channel opener, and glibenclamide, a KATP channel blocker, prevent a spontaneous increase in intracellular Ca(2+) in human MII oocytes. What is known already: The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca(2+) homeostasis is crucial for cell wellbeing and increased intracellular Ca(2+) levels is a well-established indicator of cell stress. Study design, size, duration: Supernumerary human oocytes (n = 102) collected during IVF/ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca(2+) levels were monitored in oocytes in the following experimental groups: (i) Control, (ii) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), (iii) Pinacidil, (iv) Glibenclamide, (v) DNP: an inhibitor of oxidative phosphorylation, (vi) Pinacidil and DNP and (vii) Glibenclamide and DNP. Participants/materials/settings/methods: Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean ± SD) age 34.1 ± 0.6 years, n = 41. Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca(2+)-sensitive dye, and monitored by laser confocal microscopy for 2 h at 10 min intervals. Time between oocyte collection and start of Ca(2+) monitoring was 80.4 ± 2.1 h. Main results and the role of chance: Intracellular levels of Ca(2+) increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0 ± 11.8 AU (AU = arbitrary units; n = 23) to 91.8 ± 14.0 AU (n = 19; P < 0.001) after 2 h of monitoring. Pinacidil (100 µM) inhibited this increase in Ca(2+) (85.3 ± 12.3 AU at the beginning of the experiment, 81.7 ± 11.0 AU at the end of the experiment; n = 13; P = 0.616). Glibenclamide (100 µM) also inhibited the increase in Ca(2+) (74.7 ± 10.6 AU at the beginning and 71.8 ± 10.9 AU at the end of the experiment; n = 13; P = 0.851. DNP (100 mM) induced an increase in intracellular Ca(2+) that was inhibited by glibenclamide (100 µM; n = 9) but not by pinacidil (100 µM; n = 5). Limitations, reasons for caution: Owing to clinical and ethical considerations, it was not possible to monitor Ca(2+) in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4 ± 2.1 h (n = 102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilized, it is possible that they may have been abnormal with impaired Ca(2+) homeostasis and, furthermore, the altered Ca(2+) homeostasis might have been associated solely with the protracted incubation. Wider implications of the findings: These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca(2+), which can be counteracted by drugs targeting KATP channels. As Ca(2+) homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that KATP channel openers and blockers should be tested as drugs for improving success rates of ART. Study funding/competing interests: University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside. Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. The authors declare no conflicts of interest.
    Full-text · Article · Dec 2015 · Human Reproduction
  • Source
    • "One example is the post-natal administration of leptin in rats, which was able to reverse the adverse effects of mother-undernutrition: the offspring phenotype as well as the expression and methylation of several hepatic genes were corrected [174] . One other example is the post-natal administration of butyrate (histone deacetylase inhibitor) in the mouse model, which normalized both DNA methylation of the promoter of the eNOS gene and vascular function [169]. Further studies in animals are needed to better understand tissue-specific epigenetic regulation in ART. "
    [Show abstract] [Hide abstract] ABSTRACT: Today, there is growing interest in the potential epigenetic risk related to assisted reproductive technologies (ART). Much evidence in the literature supports the hypothesis that adverse pregnancy outcomes linked to ART are associated with abnormal trophoblastic invasion. The aim of this review is to investigate the relationship between epigenetic dysregulation caused by ART and subsequent placental response. The dialogue between the endometrium and the embryo is a crucial step to achieve successful trophoblastic invasion, thus ensuring a non-complicated pregnancy and healthy offspring. However, as described in this review, ART could impair both actors involved in this dialogue. First, ART may induce epigenetic defects in the conceptus by modifying the embryo environment. Second, as a result of hormone treatments, ART may impair endometrial receptivity. In some cases, it results in embryonic growth arrest but, when the development of the embryo continues, the placenta could bring adaptive responses throughout pregnancy. Amongst the different mechanisms, epigenetics, especially thanks to a finely tuned network of imprinted genes stimulated by foetal signals, may modify nutrient transfer, placental growth and vascularization. If these coping mechanisms are overwhelmed, improper maternal-foetal exchanges occur, potentially leading to adverse pregnancy outcomes such as abortion, preeclampsia or intra-uterine growth restriction. But in most cases, successful placental adaptation enables normal progress of the pregnancy. Nevertheless, the risks induced by these modifications during pregnancy are not fully understood. Metabolic diseases later in life could be exacerbated through the memory of epigenetic adaptation mechanisms established during pregnancy. Thus, more research is still needed to better understand abnormal interactions between the embryo and the milieu in artificial conditions. As trophectoderm cells are in direct contact with the environment, they deserve to be studied in more detail. The ultimate goal of these studies will be to render ART protocols safer. Optimization of the environment will be the key to improving the dialogue between the endometrium and embryo, so as to ensure that placentation after ART is similar to that following natural conception.
    Full-text · Article · Aug 2015
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