Human Reproduction vol.14 no.2 pp.542–552, 1999
Synergistic role of nitric oxide and progesterone during
the establishment of pregnancy in the rat*
Kristof Chwalisz1,4, Elke Winterhager2,
Thomas Thienel2 and Robert E.Garfield3
1Research Laboratories of Schering AG, Müllerstr 170–178, 13342
Berlin, 2Institute of Anatomy, Medical School, University of Essen,
Germany, 3University of Texas Medical Branch, Galveston, USA
4To
whom correspondence should be addressed
Successful pregnancy is strictly dependent on the trophoblast-decidual interaction and on an adequate blood supply
to the implantation sites. Nitric oxide (NO) has been shown
to play an important role during advanced gestation,
although its role during early pregnancy is unclear. The
aim of the present study in rats was to evaluate whether
NO plays a role during the preimplantation [days 1–4 post
coitum (p.c.)] and peri-implantation (days 6–8 p.c.) phases
of pregnancy. The rats were treated with the non-specific
nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine
methyl ester (L-NAME), and the iNOS inhibitor aminoguanidine in the presence and absence of low-dose antiprogestin, onapristone, and evaluated on days 9 p.c. and 19 p.c.,
respectively. Before implantation, the treatments alone
(L-NAME, aminoguanidine, onapristone) had little effect
on pregnancy outcome. Conversely, aminoguanidine plus
onapristone treatment completely prevented pregnancy,
whereas L-NAME plus onapristone reduced the pregnancy
rate to approximately 50%. In addition, both treatments
drastically reduced decidualization. Oviductal flushing
experiments revealed arrest of embryo development at
around the 8-cell stage after aminoguanidine plus onapristone treatment on days 1–4 p.c. Similarly, treatment during
the peri-implantation period with L-NAME, aminoguanidine, and onapristone each had only marginal effects on
pregnancy. However, a combination of L-NAME and onapristone, and aminoguanidine plus onapristone prevented
pregnancy in 71% and 42% of dams, respectively, as
determined on day 19 p.c. These treatments also markedly
inhibited the decidualization process. This study demonstrates synergistic effects of NOS inhibitors and an antiprogestin in preventing pregnancy. NOS, particularly the
cytokine- and progesterone-inducible iNOS, may represent
a new target for novel therapeutic agents capable of
promoting or inhibiting pregnancy.
Key words: antiprogestin/embryo development/implantation/
nitric oxide/progesterone
*This work was presented in part at the 44th and 45th Annual
Meetings of the Society for Gynecological Investigation in San Diego,
California (March, 1997), and in Atlanta, Georgia, USA (March,
1998), respectively.
542
Introduction
Implantation of the embryo is a critical event in pregnancy. In
humans, peri-implantation pregnancy loss may contribute to
more than 20% of unexplained infertility. Approximately onethird of human pregnancies end in spontaneous abortions, twothirds of them occurring prior to the clinical detection of
pregnancy (Wilcox et al., 1988). Successful implantation of
blastocysts is strictly dependent on the synchrony between
embryo development and the cascade of cellular and molecular
events that occur in the endometrium during the peri-implantation period of the fertile cycle (Beier et al., 1994). In rodents
during early pregnancy, the surrounding endometrial stroma
undergoes a dramatic differentiation into the decidua, a specialized, well-vascularized tissue that encapsulates the developing
embryo. Decidual cells are believed to play a key role in
providing nutrients to the embryo, and in controlling trophoblast invasion (Parr and Parr, 1989; Loke and King, 1995). In
rodents, the decidual cell reaction occurs in response to either
blastocysts or artificial stimuli and is always preceded by an
increase in endometrial vascular permeability which is normally initiated at the antimesometrial sites where blastocysts
implant (Psychoyos, 1973).
The exact factors which invoke decidualization of the
endometrial stroma are still poorly understood. Cytotrophoblast
invasion, encompassing dynamic changes in cell–cell and cellmatrix interactions, can be viewed as an inflammatory reaction.
There is ample evidence indicating that prostaglandins mediate,
at least in part, the changes in endometrial vascular permeability
and subsequent decidualization in pregnant as well as in
pseudo-pregnant animals. Hence, prostaglandin concentrations
are elevated at implantation sites in areas of increased permeability (Kennedy, 1980; Evans and Kennedy, 1978), and prostaglandin inhibitors, such as indomethacin, delay or inhibit (i)
implantation, (ii) the localized increase in endometrial vascular
permeability, and (iii) artificially-induced decidualization in
several non-primate mammals (Kennedy, 1977; Psychoyos
et al., 1995). In addition, the targeted disruption of the cytokineinducible cyclooxygenase (COX) isoform COX-2, but not
COX-1 (a constitutive isoform), impairs fertilization, implantation and decidualization in mice (Lim et al., 1997). In rhesus
monkeys, however, post-ovulatory treatment with high-dose
diclofenac, a non-specific COX inhibitor, had only a partial
effect on pregnancy establishment (Nayak et al., 1997) suggesting that prostaglandins are not essential in primate
implantation. It is, therefore, likely that other vasoactive
agents are able to replace prostaglandins in order to support
vascularization and the decidual reaction during implantation.
Nitric oxide (NO) has come to prominence recently as a
© European Society of Human Reproduction and Embryology
Nitric oxide and implantation
major mediator of numerous biological processes, including
vascular functions and inflammation (Ignarro, 1989; Moncada
and Higgs, 1993). NO is produced by nitric oxide synthesizing
enzymes (NOS enzymes) which exist as three isoforms: ecNOS
or endothelial NOS; iNOS or cytokine-inducible NOS, and
nNOS or neural NOS (Nathan and Xie, 1994). NO was recently
implicated as an important regulatory agent in various steps
of female reproduction (reviewed by Chwalisz et al., 1996;
Rosseli, 1997). During pregnancy NO plays a pivotal role in
controlling uterine contractility, cervical ripening, and utero–
feto–placental blood flow (Izumi et al., 1993; Sladek et al.,
1993; Buhimschi et al., 1996; Liao et al., 1997). Our previous
studies have shown that in rats the iNOS is the dominant NOS
isoform in the pregnant uterus, cervix and placenta (Buhimschi
et al, 1996; Purcell et al., 1997; Ali et al., 1997). In rats, the
uterine, cervical and placental iNOS expression is gestationally
regulated at protein and mRNA levels, with progesterone as
the key physiological regulating agent (Buhimschi et al., 1996;
Chwalisz et al., 1996; Ali et. al., 1997). The effects of
progesterone on iNOS expression are tissue-dependent. In the
uterus and placenta progesterone up-regulates iNOS expression
and NO production, whilst exerting opposite effects in the
cervix. The L-arginine-NO-cGMP system (NO system) is also
present in the decidua in rats (Purcell et al., 1997; Sladek
et al., 1998) and rabbits (Sladek et al., 1993). An increased
expression of both iNOS and ecNOS was recently found in
the mouse uterus during implantation (Purcell et al., 1998). In
addition, increased expression of iNOS was described in human
decidualized stromal cells (Telfer et al., 1997) and in the
secretory endometrium (Tschugguel et al., 1998). Overall,
these data suggest that (i) iNOS is the dominant NOS isoform
in the endometrium, (ii) endometrial iNOS is directly or
indirectly controlled by progesterone, and (iii) NO may play
a role in the local control of endometrial functions. However,
the precise function of NO during implantation has not yet
been defined.
Meanwhile, it is well established that iNOS-derived NO
may invoke pro-inflammatory effects, including vasodilatation,
oedema, tissue remodelling, and the mediation of cytokinedependent processes at the inflammation site (Evans et al.,
1995; Salvemini et al., 1995). It is also well known that the
proinflammatory cytokines are up-regulated in the uterus during
implantation (Simón et al., 1993; Chard, 1995; Tazuke and
Giudice, 1996). Studies with antiprogestins clearly show that
progesterone is essential for endometrial receptivity and for
the entire implantation process in all mammals investigated to
date (reviewed by Chwalisz et al., 1997). However, it still
remains unclear how exactly progesterone acts in the uterus
during the pre- and peri-implantation stages of pregnancy.
Because NO mediates certain progesterone effects in the
myometrium, placenta and cervix, we hypothesize that NO and
progesterone play a synergistic role during the establishment of
pregnancy. To test this hypothesis, we performed a series of
functional studies in rats using the NOS inhibitors NG-nitroL-arginine methyl ester (L-NAME, a non-specific NOS inhibitor) and aminoguanidine [a 20- to 30-fold selective inhibitor
of iNOS (Misko et al., 1993)], and an antiprogestin (onapristone) prior to and during implantation. Our aims were: (i) to
Figure 1. Experimental design.
evaluate whether NO is involved in the preparation of the
endometrium for implantation during the pre-receptive phase
(days 1–4 post coitum); (ii) to determine whether it plays a
role during the peri-implantation phase (days 6–8 p.c.); and
(iii) to examine the interaction of NO with progesterone during
implantation.
Materials and methods
Animals
Early pregnant female Wistar rats (Schering, TZH, Berlin, Germany)
were used for all experiments. The animals were kept under standard
conditions. The light/dark-cycle was 14/10 h (light 6:30–20:30). The
presence of sperm in the vaginal smear on the morning following
mating was defined as day 1 post coitum (day 1 p.c.).
Compounds and formulations
L-NAME (Sigma-Aldrich Chemie, Munich, Germany) was dissolved
in sterile 0.9% saline solution. Osmotic minipumps (Alza, Palo Alto,
CA, USA; model 2ML1 with a pumping rate of 10 µl/h) were filled
with vehicle or with L-NAME solution and implanted s.c. during
ether anaesthesia. The drug-release was verified by opening the
minipumps during autopsy to determine the remaining volume.
Aminoguanidine (Sigma-Aldrich Chemie) was dissolved in water at
pH 6.0 and administered orally (p.o.) in 1 ml. The specific progesterone
antagonist onapristone (ZK 98 299; 11β-[4-(dimethylamino)phenyl]17α-hydroxy-17β-(3-hydroxypropyl)-13 α-estra-4,9-dien-3-one) was
formulated in 0.2 ml benzylbenzoate 1 castor oil (1 1 4 vol/vol)
and administered s.c. Onapristone was synthesized at Schering AG,
Berlin, Germany.
Experimental design
Treatments with L-NAME (50 mg/rat/day s.c.) and aminoguanidine
(120 mg/rat/day) alone as well as in combination with low-dose
onapristone (0.3 mg/rat/day s.c.) were performed after randomization
in accordance with two experimental protocols (Figure 1A, B).
L-NAME at this dose produced hypertension and fetal growth
restriction in late pregnant rats (Liao et al., 1997). The aminoguanidine
dose used was based on the results of pilot dose-finding studies
543
K.Chwalisz et al.
of the synergistic effects of onapristone on fertility in rats (data
not shown).
The effects of NO inhibition and low-dose onapristone on implantation
after treatment during the preimplantation phase (days 1–4 p.c;
Figure 1A)
Experiment 1: the effects of L-NAME in the presence and absence of
onapristone on implantation after treatment on days 1–4 p.c. Osmotic
minipumps containing L-NAME or 0.9% saline (vehicle) were
implanted s.c. on day 1 p.c. and removed on day 4 p.c. Onapristone
or the respective vehicle (controls) was administered (s.c.) once daily
on days 1–4 p.c. (n 5 7/group). Group 1 (vehicle control) received
the respective vehicles (saline-containing mini-pump and onapristone
vehicle). Group 2 was treated with L-NAME and the onapristone
vehicle. Group 3 received onapristone and the L-NAME vehicle (i.e.
saline-containing mini-pump was implanted). Group 4 received a
combination of L-NAME plus onapristone. Peripheral blood for
progesterone and oestradiol radioimmunoassays was collected from
the retro-orbital plexus during CO2 anaesthesia at 9–10 a.m. on days
1, 5, 7 and 9 p.c. During autopsy on day 9 p.c. the uteri were opened,
photographed and macroscopically examined for implantation sites
and resorptions. They were then removed and processed for histology.
Experiment 2: the effects of aminoguanidine in the presence and
absence of onapristone on implantation after treatment on days
1–4 p.c. This experiment was performed in accordance with the
protocol described for experiment 1, using the same animal numbers
per group. Group 1 (controls) was treated orally and s.c. with both
vehicles. Group 2 received aminoguanidine orally and onapristone
vehicle s.c. Group 3 was treated s.c. with onapristone and with the
aminoguanidine vehicle orally. Group 4 was treated orally with
aminoguanidine and s.c. with onapristone at the respective dose.
Peripheral blood collections and autopsy (day 9 p.c.) were performed
as described for experiment 1.
Experiment 3: pregnancy outcome (on day 19 p.c). after treatment
with L-NAME and aminoguanidine in the presence and absence of
onapristone on days 1–4 p.c. Treatments were performed as described
for experiments 1 and 2. However, in order to assess the effects of
treatment on pregnancy outcome, the autopsy was performed on day
19 p.c., i.e. approximately 3 days before birth (term: days 21–22
p.c.). During autopsy, the number and condition of pups, fetal weight,
placental weight and the number of resorptions per uterus were
recorded. In addition, the empty uteri were stained with 10%
ammonium sulphide in order to identify early resorptions.
Experiment 4: the effects of combined aminoguanidine and onapristone treatment on tubal transport, preimplantation embryo development and endometrial receptivity after treatment on days 1–4 p.c. The
rats (n 5 8/group) were treated with a combination of aminoguanidine
and low-dose onapristone or the vehicles (controls) at 9 a.m. on days
1–4 p.c. The autopsy was performed between 1–2 p.m. on days 4
p.c. [groups 1 and 2 (controls)], 5 p.c. [groups 3 and 4 (controls)],
and 6 p.c. [groups 5 and 6 (controls)]. During autopsy the uteri and
oviducts were dissected and flushed with 0.9% saline which was
subsequently examined for the presence or absence of preimplantation
embryos using inverted phase contrast microscopy.
The effects of NO inhibition and low-dose onapristone on implantation
after treatment during the peri-implantation phase (days 5–8 p.c.;
Figure 1B)
Experiment 5: the effects of L-NAME alone or in combination with
low-dose onapristone after treatment on days 5–8 p.c. Pregnant rats
(n 5 8/group) were treated on days 5–8 p.c. using the same groups
as described for experiment 1.
Experiment 6: the effects of aminoguanidine alone and in combination
544
with low-dose onapristone after treatment on days 5–8 p.c. Rats were
randomly allocated to four groups (n 5 7/group) and were treated
on day 5–8 p.c. The same treatment groups as described for experiment
2 were used.
Experiment 7: pregnancy outcome (on day 19 p.c). after treatment
with L-NAME and aminoguanidine alone and in combination with
low-dose onapristone on days 5–8 p.c. The animals were treated on
days 5–8 p.c. analogously with experiment 3. The following groups
were used (see Table II for numbers): group 1: vehicle control;
group 2: onapristone alone; group 3: L-NAME alone; group 4:
aminoguanidine alone; group 5: L-NAME plus onapristone; group 6:
aminoguanidine plus onapristone.
Morphology
In experiments 2, 5 and 6, the uteri were fixed in 4% paraformaldehyde
overnight and routinely embedded in paraffin. The uteri were then
cut into serial sections, stained with haematoxylin/eosin and viewed
on a Zeiss Axiophot® microscope.
Radioimmunoassays
Progesterone and oestradiol concentrations were measured in serum
samples after ether extraction with a specific radioimmunoassay as
previously described (Hasan and Caldwell, 1971).
Statistical analysis
For the analysis of hormonal data, the differences between experimental days 5, 7 and 9 in each individual group were calculated. The
logarithmic ratios of the treatment groups were then compared with
those of the control group on each day using Dunnett’s test at the
significance level of α 5 0.05. The treatment effects on pregnancy
rates were analysed using one-sided Fisher’s exact test. For the
statistical analysis of treatment effects on implantation numbers, the
Wilcoxon test for comparison between the groups was applied.
Results
The effects of NO inhibition and low-dose onapristone after
treatment during the preimplantation phase (days 1–4 p.c.)
The effects on pregnancy rates and implantation numbers after
treatment on days 1–4 p.c (experiments 1 and 2)
Neither L-NAME nor aminoguanidine alone significantly affected pregnancy rates (Figure 2A and B) nor implantation
numbers (Figure 2C and D) when administered before
implantation. The implantation sites were normal in these
groups (Figure 2; black bars). However, treatment with onapristone alone reduced the total number of implantation sites in
both experiments by approximately 50–60% compared to
controls (Figure 2C and D), the majority of them being
macroscopically changed (smaller and in part haemorrhagic;
presented as striped bars). In addition, the effects of each NOS
inhibitor in combination with low-dose onapristone on both
pregnancy rates and implantation numbers were quite dramatic.
After L-NAME plus onapristone treatment, implantation sites
were detectable in only two animals: six normal implantation
sites in one animal and one very small implantation site in the
second animal (Figure 2A and C). After aminoguanidine
plus onapristone treatment, only two highly retarded and
haemorrhagic implantation sites were observed in 1/7 animals
(Figure 2B, D). Thus, the combination of onapristone and
aminoguanidine completely prevented pregnancy.
Nitric oxide and implantation
Figure 2. The effects of the NOS inhibitors L-NAME (LN) and
aminoguanidine (A) in the presence or absence of low-dose
onapristone (ONA) on pregnancy rates (upper panel; A, B) and
implantation number (lower panel; B, C) established on day 9 p.c.
after treatment at preimplantation (day 1–4 p.c.). There were seven
animals per group. The autopsy was performed on day 9 p.c.
Upper panel (A, B) presents the percentage of pregnant animals per
experimental group. Black bars demonstrate the percentage of
animals exhibiting macroscopically normal implantation sites.
Striped bars present the percentage of animals showing
macroscopically changed implantation sites (haemorrhagic and
growth retarded). Lower panel (C, D) shows the total number of
implantation sites per group. Black bars present the number of
normal implantation sites, whereas striped bars demonstrate the
number of macroscopically changed implantation sites. Asterisks*
mark the statistically different values (P , 0.05) of normal
implantation sites (black bars) from the respective control group.
The effects on serum progesterone and oestradiol concentrations after treatment on days 1–4 p.c. (experiments 1 and 2)
Figure 3 presents the results of progesterone measurements.
Neither treatment before implantation (experiments 1 and 2)
significantly influenced the serum progesterone (Figure 3) and
oestradiol concentrations (data not shown) compared to the
control animals.
The effects on implantation chamber morphology after treatment on days 1–4 p.c. (experiments 1 and 2)
For morphological evaluation, cross-sectional slides of the
middle part of the implantation chambers of each experimental
group were selected.
Vehicle control groups The cross-sections of an implantation
chamber of all controls (Figure 4A, B) on day 9 p. c. revealed
a high degree of stromal decidualization surrounding the
developing embryo. Decidualization was most developed at
the antimesometrial side, whilst the decidual cells formed a
special wall-like region surrounding the embryo, the primary
Figure 3. The effect of the NOS inhibitors L-NAME (LN)
and aminoguanidine (A) in the presence or absence of
low-dose onapristone (ONA) on serum progesterone
concentrations after treatment at preimplantation
(day 1–4 p.c.). Upper panel: L-NAME 6 onapristone treatment.
Lower panel: Aminoguanidine 6 onapristone treatment. Blood
samples were taken on days 1, 5, 7 and 9 p.c. C 5 vehicle
control. The results are expressed as means 6 SD. For statistical
analysis Dunnett’s test at the significance level of α 5 0.05 was
used. There were no significant differences between the treatment
groups and the control.
decidual zone. Higher magnification demonstrates the typical
morphological features of the decidual cells (Figure 4B) with
closely packed polygonal, partly multinuclear cells, separated
only by the small extracellular matrix.
L-NAME and onapristone alone Compared to controls, treatment with either L-NAME or onapristone alone before
implantation produced relatively subtle changes in endometrial
morphology. Each drug exerted virtually identical effects on
decidual differentiation, thus both are described together. The
differentiation process seemed retarded after L-NAME or
onapristone alone. The decidual cells were more loosely
connected (Figure 4D). Typically, the closure reaction in the
mesometrial part remained incomplete, despite the degeneration of the epithelium leading to a kind of cleft connecting the
antimesometrial chamber to the uterine lumen (Figure 4E
and F).
Aminoguanidine only treatment Aminoguanidine alone had
virtually no effect on the implantation reaction which was
indistinguishable from those treated with the vehicle (data not
shown) on days 1–4 p.c.
Treatment with L-NAME plus onapristone Dramatic changes
545
K.Chwalisz et al.
occurred after combined treatment with L-NAME and onapristone. The main morphological disturbances were: incomplete
decidualization, incomplete closure of the uterine lumen, and
hyperplasia of the uterine epithelium (Figure 4G). Only in
one case could signs of stromal decidualization be observed
(Figure 4H). In this implantation chamber, merely a compact
primary decidual zone was formed without enclosing any
remnants of embryonic tissues (Figure 4G).
Treatment with aminoguanidine plus onapristone Surprisingly,
no implantation reaction was found in any animal, the morphological appearance of the uteri being comparable to those of
non-pregnant animals. The serial uterine sections of animals
treated with this combination revealed neither the presence of
decidualization nor any evidence of early resorptions.
The effects on pregnancy outcome (on day 19 p.c.) of treatment
on days 1–4 p.c. (experiment 3)
The results are summarized in Table I. No pregnancies were
found after aminoguanidine plus onapristone treatment. After
a combined L-NAME plus low-dose onapristone treatment
there was a 50% inhibition of fertility and an increase in early
resorptions. Only seven live, but extremely growth-retarded,
fetuses were found in this group. There were no inhibitory
effects on pregnancy rates after either treatment alone; however,
onapristone treatment alone significantly reduced the number
and weight of living fetuses.
Figure 4A–H. Cross sections of an implantation chamber on day 9
p.c. after treatment with L-NAME (50 mg/rat/day) in the presence
and absence of low-dose onapristone (0.3 mg/rat/day) before
implantation (days 1–4 p.c.). Vehicle control group (A): the
embryo proper (EM) is surrounded by compact, well-developed
decidual tissue up to the myometrial layers. The decidua is
subdivided into different regions: the primary decidual zone (PD),
the mesometrial (M) and antimesometrial (AM) decidua. B: the
polygonal decidual cells of the antimesometrial region are densely
packed and the extracellular matrix is virtually missing. Some of
the decidual cells contain several nuclei (arrow). L-NAME alone
(C): overview of an implantation chamber treated with L-NAME
during preimplantation. The decidua is well developed enclosing an
appropriate differentiated embryo (EM). D: higher magnification of
the antimesometrial side reveals more loosely-packed decidual cells
compared to controls. Some spindle shaped cells can be observed
(arrow). Onapristone alone (E): decidualization is reduced. Closure
reaction is incomplete, the uterine lumen being still preserved
(arrow). F: antimesometrial decidual tissue is less compact
compared to both control and L-NAME-treated animals, and the
cells are separated by matrix. L-NAME plus onapristone (G): a
dramatic reduction in decidualization without embryonic tissue. H:
a compact primary decidual zone (PD) at higher magnification.
Stromal cells are not completely differentiated into decidual cells,
as shown by small cells and large amounts of intracellular matrix.
(AM, antimesometrial side; M, mesometrial side. Slides A, C, E, G
are at the same magnification, i.e. represented by 400 µm bar in G.
Slides B, D, F are at the same magnification and shown with a
30 µm bar in F. Bar in H represents 100 µm).
546
The effects of combined aminoguanidine and onapristone
treatment on preimplantation embryo development, tubal transport and nidation after treatment on days 1–4 p.c.
(experiment 4)
The results of oviductal and uterine flushing on days 4 and 6
p.c. (first day after attachment) are presented in Figure 5.
On day 4 p.c, the total number of 74 embryos at various
developmental stages were recovered from the oviducts of the
control animals. In contrast, only 25 pathologically changed
embryos (small size, irregular plasma membranes and necrotic
changes), mostly at the 8-cell stage, could be recovered from
the oviducts of animals treated with aminoguanidine plus
onapristone. On day 6 p.c., 77 well-developed implantation
sites were recorded in control animals, whereas in animals
treated with aminoguanidine plus onapristone, only small
points of increased vascular reaction could be observed. There
was no evidence of tubal transport time change (e.g. accelerated
tubal transport) after treatment with aminoguanidine plus
onapristone.
The effects of NO inhibition and low-dose onapristone after
treatment during the peri-implantation phase (days 5–8 p.c.)
The effects on pregnancy rates and implantation numbers after
treatments on days 5–8 p.c. (experiments 5 and 6)
Neither treatment alone had any significant effect on pregnancy
rate (Figure 6A and B), nor on the number of implantation
sites (Figure 6C and D) on day 9 p.c. Almost all of the
implantation sites were macroscopically unchanged (presented
as black bars in Figure 6). However, in combination with
low-dose onapristone, both L-NAME and aminoguanidine
significantly reduced pregnancy rates and the total number of
implantation sites. Moreover, in those animals which remained
Nitric oxide and implantation
Figure 5. The effects of combined aminoguanidine (A) plus onapristone (ONA) treatment on preimplantation embryo development on
day 4 p.c. (left panel) and implantation rate on day 6 p.c. (right panel). There were eight animals per group. During autopsy the uteri and
oviducts were dissected, flushed with 0.9% saline and examined for the presence or absence of preimplantation embryos using inverted
phase contrast microscopy. White bars: normal preimplantation embryos, striped bars: abnormal preimplantation embryos, black bars:
nidation points (N). Numbers 1, 2, 4, 8, 16 on the x-axis 5 cell stage of the preimplantation embryos, M: morula, B 5 blastocyst.
Table I. Outcome of pregnancy on day 19 p.c. after treatment with the NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (A)
alone and in the presence and absence of low-dose onapristone (ONA) during the pre-receptive phase (days 1–4 p.c.; experiment 3). Asterisks mark the values
which are significantly different from the vehicle control group (P , 0.05)
Group
Treatment
Total dams
(n)
Dams pregnant
(n)
Implantation sites
(n)
Fetal weight (g)
(mean 6 SD)
1
2
3
4
5
6
Vehicle
ONA
L-NAME
A
L-NAME 1 ONA
A 1 ONA
6
7
7
7
6
7
6
7
7
7
3*
0*
64
53
67
63
25*
0*
1.43
1.06
1.43
1.54
0.76
-
pregnant, most implantation sites were macroscopically abnormal (necrotic and haemorrhagic areas, and small size;
presented as striped bars in Figure 6). In animals treated with
L-NAME plus onapristone, 23 normal versus 62 macroscopically abnormal implantation sites were recorded
(Figure 6A and C). Similarly, after aminoguanidine plus
onapristone treatment, only eight macroscopically normal
implantation sites were discovered (Figure 6B and D).
The effects on serum concentrations of progesterone and
oestradiol after treatment on days 5–8 p.c.
Neither treatment in either experiment had any significant
effect on serum progesterone concentrations on days 1, 5 and
7 p.c. (Figure 7). A slight, but statistically significant reduction
in serum progesterone levels was observed only after L-NAME
plus onapristone treatment on day 9 p.c. No statistically
significant changes in serum oestradiol concentrations were
observed with either treatment (data not shown).
The effects on implantation chamber morphology after treatment on days 5–8 p.c.
Onapristone alone A reduction in the extent of decidualization
was observed in the implantation chambers. Except for the
primary decidual zone, the decidua was less compact and the
mesometrial decidualization was virtually missing. The tissue
was composed of a mixture of either undifferentiated or
less differentiated decidual cells that were separated by the
extracellular matrix (Figure 8D).
L-NAME and aminoguanidine alone The decidualization process was comparable to that of onapristone treatment although
6
6
6
6
6
0.16
0.22*
0.19
0.15
0.07*
Living fetuses
(n)
Resorptions
(n)
60
44
62
57
7*
0*
4
9
5
6
18*
0
less in extent (Figure 8A, B). Typical decidual cells dominated
the antimesometrial decidua and substantial amounts of extracellular matrix were only observed in the mesometrial part of
the implantation chamber. However, the embryos did not
appear to be significantly altered, as also observed for those
animals treated with onapristone alone.
L-NAME plus onapristone This treatment produced pronounced
changes in decidualization as well as in embryonic development. In the majority of implantation chambers examined no
embryonic tissue could be found, except for some necrotic
cells of embryonic origin. Decidualization was profoundly
disturbed (Figure 8E, F).
Aminoguanidine plus onapristone The effects were comparable
to those exerted by L-NAME plus onapristone treatment, but
slightly less pronounced (Figure 8G, H). As in the case of
L-NAME plus onapristone there was a gross reduction in the
extent of decidualization.
The effects on pregnancy outcome on day 19 p.c. after treatment
on days 5–8 p.c. (experiment 7)
Treatment with each NOS inhibitor alone did not produce any
significant effects on the pregnancy rate nor on the number of
living fetuses. A slight reduction in the number of living
fetuses was observed after onapristone alone. Interestingly,
both L-NAME and onapristone significantly reduced fetal
weights when administered alone. However, both combination
regimens exerted significant inhibitory effects on pregnancy
rates and the number of living fetuses. The effects of L-NAME
547
K.Chwalisz et al.
Figure 6. The effects of the NOS inhibitors L-NAME (LN) and
aminoguanidine (A) in the presence or absence of low-dose
onapristone (ONA) on pregnancy rates (upper panel; A, B) and
implantation number (lower panel; B, C) established on day 9 p.c.
after treatment at peri-implantation (days 5–8 p.c.). There were
seven animals per group. Upper panel (A, B) presents the
percentage of pregnant animals per experimental group. Black bars
demonstrate the percentage of animals exhibiting macroscopically
normal implantation sites. Striped bars present the percentage of
animals showing macroscopically changed implantation sites
(haemorrhagic and growth retarded). Lower panel (C, D) shows the
total number of implantation sites per group. Black bars present the
number of normal implantation sites, whereas striped bars
demonstrate the number of macroscopically changed implantation
sites. Asterisks* mark the statistically different values (P , 0.05) of
normal implantation sites (black bars) from the respective control
group.
plus onapristone were more pronounced than those of aminoguanidine plus onapristone (Table II).
Discussion
The results of our study in rats clearly demonstrate that NO
inhibition with aminoguanidine and L-NAME substantially
increased the inhibitory effects of the antiprogestin onapristone
(i) on the establishment of pregnancy after treatment before
implantation, (ii) on implantation and decidualization, and (iii)
on the pregnancy outcome after treatment during the periimplantation phase. These effects were synergistic, since either
treatment alone only marginally affected implantation and
pregnancy outcome. Treatment with onapristone alone particularly before implantation induced macroscopic changes in
implantation sites such as haemorrhage and size reduction
during autopsy at day 9 p.c. (Figure 2). Interestingly, the
majority of these embryos apparently recovered during the
later stages of pregnancy except after combined treatments
(Table I). Similar synergistic effects of NOS inhibitors and
548
Figure 7. The effect of the NOS inhibitors L-NAME (LN) and
aminoguanidine (A) in the presence or absence of low-dose
onapristone (ONA) on serum progesterone concentrations
after treatment at peri-implantation (day 5–8 p.c.).
Upper panel: L-NAME 6 onapristone treatment.
Lower panel: aminoguanidine 6 treatment. Blood samples were
collected on days 1, 5, 7 and 9 p.c. C 5 vehicle control. The
results are expressed as means 6 SD. Asterisks* mark the
statistically different values (P , 0.05) from respective control
group on the respective day.
antiprogestins on pregnancy maintenance were observed in
late pregnant rats (Yallampalli et al., 1996) and in guinea
pigs (Chwalisz et al., 1996). Before implantation, each NOS
inhibitor in combination with low-dose onapristone exerted a
dramatic inhibitory effect on the establishment of pregnancy,
aminoguanidine being more effective than L-NAME.
The results of experiments employing oviductal flushing
indicate that this treatment inhibited embryonic development
thus causing the implantation defect. However, during the periimplantation phase, inhibition of the decidual reaction was the
main treatment effect subsequently leading to the degeneration
of the implantation sites. In both phases of pregnancy, neither
treatment produced any significant changes in serum progesterone or oestradiol concentrations until day 7 p.c. indicating that
the effects observed were direct, and not mediated by changes
in ovarian hormone secretion.
The results of our study are consistent with recent molecular
studies on the expression of NOS isoforms in mice and rats.
Western blot analysis of iNOS and ecNOS in conjunction with
trace optical density reading revealed a significant elevation
of iNOS and ecNOS expression in implantation sites versus
intersite tissues on days 6, 7 and 8 of mouse pregnancy (Purcell
et al., 1998). An increase in Ca21-independent and Ca21-
Nitric oxide and implantation
Figure 8A–H. Cross-sections of an implantation chamber on day 9
p. c. after peri-implantation (days 5–8 p.c.) treatment with
L-NAME and onapristone each, and the combination of onapristone
with either L-NAME or aminoguanidine. L-NAME alone (A):
compared to controls, the decidualization process is reduced. The
tissue shows, however, less compaction than after onapristone
treatment alone. B: most of the decidual cells in the
antimesometrial (Am) region seem to be well differentiated and
intermingled with some spindle-shaped cells (arrows). Onapristone
alone (C): this treatment reduced the extent of decidualization, the
cells being loosely packed. D: higher magnification of the
antimesometrial region shows small cells (arrow) separated by the
extracellular matrix. L-NAME plus onapristone (E): a dramatic
reduction in the extent of decidualization. Some necrotic embryo
rudiments are detectable (arrow). Decidualization process seems to
be incomplete. F: a higher magnification reveals spindle-like
stromal cells with large intercellular spaces, some of the cells being
necrotic (arrow). Aminoguanidine plus onapristone (G): an
extensive retardation of decidualization, especially in the
mesometrial (M) region, although the embryo is still present
(arrow). H: at higher magnification, the decidual cells appear welldifferentiated and closely packed, virtually without any intracellular
space. Slides A, C, E, G are at the same magnification, represented
by 400 µm bar in G. Slides B, D, F, H are at the same
magnification, represented by 30 µm bar in H.
dependent NOS activity was also described in the rat uterus
on day 5 of pregnancy (Novaro et al., 1997).
Our present study might also indicate that NO is not essential
for the establishment of pregnancy, since NOS inhibitors
did not prevent implantation nor terminate pregnancy when
administered alone. The results of knock-out experiments in
mice indicate that the disruption of individual genes encoding
iNOS (MacMicking et al., 1995), ecNOS (Huang et al., 1995),
and bcNOS (Huang et al., 1993) did not significantly alter
fertility or litter size. However, uterine NO production may be
maintained by the remaining isoforms in animals lacking a
single NOS isoform. The most likely explanation for the
absence of any significant effects of NOS inhibitors on the
establishment of pregnancy is the existence of redundant
mediators such as prostaglandins (Kennedy, 1977, 1980) or
histamine (Day and Hubbard, 1981) which may compensate
for NO deficiency in both the preimplantation embryo and
decidua. Another possible explanation is that the increased
local NO production in both the preimplantation embryo and
the decidua is not entirely inhibited by NOS inhibitors around
the time of implantation, even when high doses are administered.
Our findings from experiments employing oviduct flushing
indicate that the inhibitory effects of aminoguanidine plus
onapristone were due to the impairment of early embryo
development, resulting in the inhibition of cleavage between
the 8-cell and morula stages. Our data are consistent with the
results of recent studies in mice indicating that preimplantation
embryos express both iNOS and ecNOS, and can produce NO
under in-vitro conditions (Gouge et al., 1998). In addition,
this study shows that the non-specific NOS inhibitor L-NAME
impairs embryogenesis. Overall, these data suggest that NO is
required for normal embryonic development.
Whether progesterone has a direct effect on preimplantation
embryo development is still a matter of dispute. The growth
rate and the implantation ability of preimplantation monkey
embryos were not affected by treatment with RU 486 in vitro
(Wolf et al., 1990). Similarly, blastocysts transferred from rats
exposed to RU 486 displayed delayed implantation in the
untreated recipients (Roblero and Croxatto, 1991). In addition,
it was clearly demonstrated in rabbits that short-term treatment
with various antiprogestins during the luteal phase leads to the
transposition of the implantation window by antiprogestins
acting primarily on the endometrium without any effects on
the embryo (Beier et al., 1994). However, there are also studies
suggesting that progesterone does play a role in preimplantation
embryo development. Impairment of embryonic growth and
development was reported after pre-ovulatory or early luteal
phase treatment with RU 486 in rats and mice (Psychoyos and
Prapas 1987; Roh et al., 1988; McRae, 1994), guinea pigs
(Chwalisz et al., 1997), and monkeys (Ghosh et al., 1997).
Implantation is subject to the interaction of the trophoblast
cells with the decidua. The results of the present study strongly
suggest that NO is involved in the decidual reaction since
NOS inhibitors, in particular L-NAME, partially reduced the
extent of decidualization even in the absence of onapristone
after peri-implantation treatment. It is well established that
cytokines (some of which are indispensable) play an important
role during implantation (Chard, 1995; Tazuke and Giudice,
1996). IL-1 and its receptor are up-regulated in the human
endometrium during the peri-implantation period (Simón et al.,
1993), and are also present in preimplantation mouse embryos
reaching maximal expression in hatching blastocysts (Cresol
et al., 1997). The female osteopetrotic (op/op) mice, which
549
K.Chwalisz et al.
Table II. Outcome of pregnancy on day 19 p.c. after treatment with the NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (A)
alone and in the presence and absence of low-dose onapristone (ONA) during the peri-implantation phase on days (days 5–8 p.c.; experiment 7). Asterisks
mark the values which are significantly different from the vehicle control group (P , 0.05)
Group
Treatment
Total dams
(n)
Dams pregnant
(n)
Implantation sites
(n)
Fetal weight (g)
(mean 6 SD)
Living fetuses
(n)
1
2
3
4
5
6
Vehicle
ONA
L-NAME
A
L-NAME 1 ONA
A 1 ONA
7
7
6
7
7
7
7
6
6
7
2*
4*
70
64
61
67
22*
54
62
52
54
58
19*
44
1.37
1.25
1.30
1.55
1.34
1.33
exhibit mutation of the colony-stimulating factor (CSF-1) gene,
have markedly impaired fertility (Pollard et al., 1991). Also,
female mice with a null mutation of the genes encoding
leukaemia inhibitory factor (LIF) (Stewart et al., 1992), and
interleukin-11 receptor alpha chain (IL-11Rα) (Robb et al.,
1998) remain infertile because of implantation failure. Interestingly, both LIF and IL-11Rα knock out mice also exhibit a
decidualization defect.
iNOS can produce high levels of NO for prolonged periods
of time (typically 4–6 h) in response to pro-inflammatory
cytokines, including IL-1 and tumour necrosis factor α (TNFα) (Moncada and Higgs, 1993; Nathan and Xia-wen, 1994).
Similarly, several types of CSF have been shown to up-regulate
NO via iNOS induction in macrophages (Jorens et al., 1993).
On the other hand, pro-inflammatory cytokines are known to
up-regulate COX-2 at the inflammation site. Furthermore, it
was recently found in various models of inflammation that
NO is also a powerful inducer of COX-2 and elevates local
PGE2 concentrations (Salvemini et al., 1995). The results of
the present study suggest that NO acts as a local mediator of
inflammatory changes during various stages of implantation.
It may act on endometrial blood vessels and contribute to the
vascular reaction. Since NO may directly regulate the activity
of matrix metalloproteinases (MMP) (Trachtman et al., 1996),
it may play a role in extracellular matrix changes occurring
during the trophoblast invasion. Recent in-vitro (Behrendtsen
et al., 1992) and in-vivo (Alexander et al., 1996) studies in
mice show that embryo-activated MMPs and their inhibitors
(TIMP-1, TIMP-2, and TIMP-3) play an important role during
implantation. In addition, the treatment of early pregnant mice
with a MMP inhibitor reduced the length and overall size of
implantation sites and disturbed embryo orientation (Alexander
et al., 1996). These effects are similar to those observed in
the present study.
To date an up-regulation of the IL-1 system during mice
and human implantation has been linked to the attachment
process in the early stages of implantation (Simón et al., 1993;
Kruessel et al., 1997). Since IL-1β is produced by the embryo
itself in humans (Simón et al., 1993; Kruessel et al., 1997),
this cytokine may also act as an embryonic signal promoting
implantation. It could in fact, via iNOS and COX-2 induction,
initiate the vascular reaction, decidulization, and the remodelling of the extracellular matrix during trophoblast invasion.
In all mammals during early pregnancy, an adequate uterine
blood supply is essential for embryo development. In women,
550
6
6
6
6
6
6
0.09
0.01*
0.11*
0.19
0.01
0.10
Resorptions
(n)
8
12
5
9
3
10
impaired blood flow to the uterus can jeopardize the establishment of pregnancy (Edwards, 1995). Furthermore, uterine
blood flow, as measured by colour Doppler, was proposed as
the physiological parameter to assess endometrial receptivity
to blastocysts implantation following assisted reproductive
treatments (Achiron et al., 1995; Friedler et al., 1996). In
guinea pigs dilatation of uteroplacental arteries was observed
when invading trophoblast cells coexpressing ecNOS and
iNOS are present in the exravillous trophoblast (Nanaev et al.,
1995) suggesting that NO mediates spiral arterial changes
occurring during pregnancy. The present study demonstrates
that both L-NAME and onapristone alone induced fetal growth
retardation after treatment on days 6–8 p.c. (Table II). Interestingly, in rats during advanced pregnancy, the fetal growth
retardation induced by L-NAME can be partially reversed by
progesterone and some synthetic progestins, but not by oestrogen (Liao et al., 1997). The present results further support the
concept of NO and progesterone being the key factors regulating the adaptation-related changes in uterine and placental
blood vessels during pregnancy.
The exact mechanism of the synergistic effects of onapristone
and NOS inhibitors before implantation and in the periimplantation period remains, however, unclear. Hence, further
studies are needed to determine whether these effects are simply
due to a more powerful combined blockade of embryonic and
decidual NO, or to more complex, perhaps independent effects
of NOS inhibitors and onapristone. It is well established that
the endometrium is generally very sensitive to antiprogestins
that inhibit or modulate a number of progesterone-dependent
genes even at very low doses (reviewed by Chwalisz et al.,
1997). Hence, onapristone may inhibit other (redundant) pathways involved in embryogenesis, vascularization and decidualization, thereby contributing to the synergistic effect with NOS
inhibitors.
In conclusion, this study clearly demonstrates the synergistic
inhibitory effects of NOS inhibitors and an antiprogestin in
preventing pregnancy by inhibiting preimplantation embryo
development and impairing decidualization. Furthermore, these
findings suggest that NOS, in particular iNOS, may represent
a new target for novel therapeutic agents capable of promoting
or inhibiting implantation. Up-regulating uterine NO production with either the NO substrate L-arginine and NO donors
alone or in combination with progesterone might have beneficial effects on pregnancy outcome during assisted conception.
Such schedules may also have implications for the management
Nitric oxide and implantation
of early pregnancy disorders, including recurrent abortions.
On the other hand, the effects of NOS inhibitors in combination
with antiprogestins point to a novel method for controlling
fertility, particularly by enhancing the efficacy of antiprogestins
used for endometrial contraception, menstrual induction and
post-coital contraception.
Acknowledgements
We are grateful to Mrs B. Bragulla and Mrs P. Maulwurf for expert
technical assistance in animal studies. We also thank Dr N. Benda
for the statistical analyses, Mrs R. Jäger for reading the manuscript,
and T. Purcell for discussions on studies of NOS expression in rats
and mice.
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Received on July 23, 1998; accepted on November 5, 1998
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