1 Research Laboratories of Schering AG, Müllerstr 170178, 13342 Berlin, 2 Institute of Anatomy, Medical School, University of Essen, Germany, 3 University of Texas Medical Branch, Galveston, USA
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Abstract |
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Key words: antiprogestin/embryo development/implantation/nitric oxide/progesterone
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Introduction |
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The exact factors which invoke decidualization of the endometrial stroma are still poorly understood. Cytotrophoblast invasion, encompassing dynamic changes in cellcell and cell-matrix 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 cytokine-inducible 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 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 uterofetoplacental 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 cytokine-dependent 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-nitro-L-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 evaluate whether NO is involved in the preparation of the endometrium for implantation during the pre-receptive phase (days 14 post coitum); (ii) to determine whether it plays a role during the peri-implantation phase (days 68 p.c.); and (iii) to examine the interaction of NO with progesterone during implantation.
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Materials and methods |
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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; 11ß-[4-(dimethylamino)phenyl]-17-hydroxy-17ß-(3-hydroxypropyl)-13
-estra-4,9-dien-3-one) was formulated in 0.2 ml benzylbenzoate + castor oil (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 of the synergistic effects of onapristone on fertility in rats (data not shown).
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Experiment 2: the effects of aminoguanidine in the presence and absence of onapristone on implantation after treatment on days 14 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 14 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 2122 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 14 p.c. The rats (n = 8/group) were treated with a combination of aminoguanidine and low-dose onapristone or the vehicles (controls) at 9 a.m. on days 14 p.c. The autopsy was performed between 12 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 58 p.c.; Figure 1B)
Experiment 5: the effects of L-NAME alone or in combination with low-dose onapristone after treatment on days 58 p.c. Pregnant rats (n = 8/group) were treated on days 58 p.c. using the same groups as described for experiment 1.
Experiment 6: the effects of aminoguanidine alone and in combination with low-dose onapristone after treatment on days 58 p.c. Rats were randomly allocated to four groups (n = 7/group) and were treated on day 58 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 58 p.c. The animals were treated on days 58 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.
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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 = 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.
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Results |
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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 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.
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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 14 p.c.
Treatment with L-NAME plus onapristone Dramatic changes 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 14 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.
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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 58 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 plus onapristone were more pronounced than those of aminoguanidine plus onapristone (Table II).
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Discussion |
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The results of experiments employing oviductal flushing indicate that this treatment inhibited embryonic development thus causing the implantation defect. However, during the peri-implantation 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 Ca2+-independent and Ca2+-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 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-11R
) (Robb et al., 1998
) remain infertile because of implantation failure. Interestingly, both LIF and IL-11R
knock out mice also exhibit a decidualization defect.
iNOS can produce high levels of NO for prolonged periods of time (typically 46 h) in response to pro-inflammatory cytokines, including IL-1 and tumour necrosis factor (TNF-
) (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-1ß 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, 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 68 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 peri-implantation 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 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.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed
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References |
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Submitted on July 23, 1998; accepted on November 5, 1998.