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Antiprogestins as a model for progesterone withdrawal
Steroids 68 (2003) 1061–1068
Antiprogestins as a model for progesterone withdrawal Hilary O.D. Critchley a,∗ , Rodney W. Kelly b , Robert M. Brenner c , David T. Baird d a
b
Obstetrics and Gynaecology, Centre for Reproductive Biology, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK c Oregon National Primate Research Centre, 505 NW 185th Avenue, Beaverton, OR 97006, USA d Obstetrics and Gynaecology, Centre for Reproductive Biology, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK
Abstract The key physiological function of the endometrium is preparation for implantation; and in the absence of pregnancy, menstruation and repair. The withdrawal of progesterone is the initiating factor for breakdown of the endometrium. The modulation of sex steroid expression and function with pharmacological agents has provided an invaluable tool for studying the functional responses of the endometrium to sex steroids and their withdrawal. By administration of the antiprogestin mifepristone, it is possible to mimic progesterone withdrawal and study local events in early pregnancy decidua that may play a role in the process of early pregnancy failure. Our data indicate that antagonism of progesterone action at the receptor level results in an up-regulation of key local inflammatory mediators, including NF-B, interleukin-8 (IL-8), monocyte chemotactic peptide-1 (MCP-1), cyclooxygenase 2 (COX-2) and others in decidua. Bleeding induced by mifepristone in the mid-luteal phase of the cycle is associated with changes in the endometrium similar to those that precede spontaneous menstruation including up-regulation of COX-2 and down-regulation of PGDH. Administration of antagonists of progesterone provide an excellent model to study the mechanisms involved in spontaneous and induced abortion as well as providing information which may help devise strategies for treating breakthrough bleeding associated with hormonal contraception. © 2003 Elsevier Inc. All rights reserved. Keywords: Endometrium; Progesterone; Steroid receptors; Antiprogestins
1. Introduction The key physiological function of the endometrium is preparation for implantation; and in the absence of pregnancy, menstruation and repair. The endometrium undergoes marked morphological and functional changes during each menstrual cycle. The cyclical features of endometrial proliferation and differentiation are the consequence of sequential exposure to estradiol and progesterone from the developing ovarian follicle and corpus luteum, respectively. The withdrawal of progesterone that occurs with demise of the corpus luteum is the initiating factor for breakdown of the endometrium at menstruation. The molecular mechanisms by which sex steroids induce these events within the endometrium, involve complex interactions between
∗ Corresponding author. Tel.: +44-131-242-6439; fax: +44-131-242-6440. E-mail address: [email protected] (H.O.D. Critchley).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2003.07.001
the endocrine and immune system and have recently been reviewed [1]. Steroids interact with their target organs via specific nuclear receptors. The expression of endometrial sex steroid receptors (progesterone receptor, PR; estrogen receptor, ER; androgen receptor, AR) varies temporally and spatially across the menstrual cycle [2–6]. The modulation of sex steroid expression and function with pharmacological agents has provided an invaluable tool for studying the functional responses of the endometrium to sex steroids. Progesterone is essential for preparation of the endometrium for implantation. Specifically, study of the action of antiprogestins on endometrial morphology and function, has provided invaluable information about the roles of progesterone in reproductive tissues. The following is a review of known roles for progesterone in the endometrium and early pregnancy decidua, and the response of the endometrium (and decidua) to both physiological and pharmacological progesterone withdrawal.
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2. Progesterone action in human endometrium and early pregnancy decidua Progesterone is essential for the transformation of an estrogen-primed endometrium in preparation for implantation. Much remains poorly understood about the molecular and cellular mechanisms involved by which the sex steroid hormones promote uterine receptivity. It is, however, recognised that sex steroids, acting via their cognate receptors, initiate a pattern of gene expression important for implantation and the early stages of pregnancy. Consequently, if specific steroid-induced molecules are identified, there is the potential for their use as markers of uterine receptivity or targets for early pregnancy interruption. Multiple studies have examined the temporal and spatial expression of presumed progesterone regulated genes across the menstrual cycle. For example, expression of calcitonin mRNA has been demonstrated to be temporally restricted to the mid-secretory phase of the cycle (maximal expression during days 19–21), a period that coincides with the putative window of implantation [7]. The site of post-ovulatory synthesis of calcitonin mRNA and protein is the glandular epithelium. Evidence for regulation of this gene by progesterone was derived from examination of endometrium collected from women treated with a progesterone antagonist, mifepristone. Calcitonin expression was dramatically reduced in women exposed to acute administration of mifepristone in the early luteal phase (administered day LH + 2; Fig. 1). Other examples of endometrial enzymes that are up-regulated by progesterone include, glandular secretion of glycodelin (PP14; [8]), 15-hydroxyprostaglandin dehydrogenase (PGDH; [9–11]) and 17-hydroxysteroid dehydrogenase (17-HSD type2; [12]). Decidualisation is a crucial step in the initiation and establishment of pregnancy and is a feature of those species that have an invasive haemochorial placentation. In the hu-
Fig. 1. Calcitonin immunostaining intensity in human endometrium. The relative signal intensity for calcitonin protein staining in glandular epithelial cells declines after treatment with the antiprogestin, mifepristone (mean ± S.E.M.; ∗ P < 0.001). Reproduced with permission from Kumar et al. [7]. Published by The Endocrine Society.
man, decidualisation is independent of the blastocyst and early signs of predecidual changes are first observed in stromal cells close to vascular structures in the mid-late secretory phase (Fig. 2). These same stromal cells express progesterone receptor throughout the luteal phase. In vivo, decidualisation is controlled most effectively by progesterone action on an estrogen-primed uterus. This process can, however, be induced as a consequence of administration of exogenous progestogens [13]. In vitro, a central role for cAMP has also been demonstrated as a decidualisation stimulus [14,15]. Studies of mouse mutants where decidualisation does not occur have identified a requirement for genes in the endometrial epithelium and/or stromal cells [16]. Key genes include cyclooxygenase 2 (COX-2, the rate-limiting enzyme in prostaglandin synthesis; [17]), Hoxa 10 and 11 [18,19]. Progesterone dependence of Hoxa 11 has been demonstrated [20]. The cellular interactions and progesterone target genes involved in decidualisation are complex. Multiple growth factors, cytokines and protein hormones have been recognised as important signals for initiation and maintenance of decidualisation [21,22]. Prolactin is a key cytokine secreted by decidualised endometrium. Prolactin expression is controlled by progesterone [23]. In vivo administration of the antiprogestin RU486 significantly reduces prolactin expression in decidualised stromal cells [24]. The evidence available indicates that progesterone is important for inducing and maintaining decidualisation but it does not induce the prolactin gene directly. Prolactin mediates its effect on target cells via single pass transmembrane spanning receptors. Prolactin receptor expression in the human endometrium is temporally regulated throughout the menstrual cycle. There is minimal expression during the proliferative phase and expression is up-regulated during the mid-late secretory phase. The prolactin receptor in non-pregnant endometrium is localised predominantly to the glandular epithelial cells. Prolactin receptor expression is maintained in pregnancy and localised to the decidualised stromal cells [25,26]. Prolactin receptor is also expressed by the phenotypically unique endometrial population of CD56+, CD16−, CD3− uterine natural killer (NK) cells that do not express the progesterone receptor [27]. Valuable insights are gained from gene ablation studies. In the above context, the prolactin receptor knock-out mouse displays an implantation defect [28] that may be a reflection of a central pituitary defect. Ablation of the progesterone receptor leads to inappropriate inflammation in the uteri of mice [29]. Different roles for the progesterone receptor isoforms, PRB and shorter PRA [30], have recently been elucidated by the production of PRA and PRB null mice [31]. For example in the PRA null mouse, estrogen induces uterine epithelial hyperplasia which progesterone treatment cannot suppress. This observation implies that progesterone-mediated suppression of epithelial growth stimulated by estrogen is dependent upon PRA, not PRB. Furthermore, in PRA + PRB null mice, there is a marked influx of inflammatory leukocytes, that cannot be inhibited
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Fig. 2. Photomicrograph of perivascular progesterone receptor immunostaining in secretory phase endometrium. Arrow indicates positive nuclear immunoreactivity.
by progesterone [29]. This observation supports a role for progesterone in suppression of the traffic of inflammatory cells into the uterus in wild type animals. The selective ablation of PRA in mice [32], has demonstrated that in mice the PRB isoform modulates a subset of progesterone-responsive target genes. Hence, PRA and PRB are likely to act as functionally distinct mediators of progesterone action in vivo. It is not known whether these observations can be extrapolated to the functions of the human uterus. 3. Physiological progesterone withdrawal Withdrawal of progesterone prevents implantation and converts the refractory pregnant uterus once again into a spontaneously steroid responsive organ [33,34]. The physiological withdrawal of progesterone from an estrogen– progesterone primed endometrium (that occurs with demise of the corpus luteum due to the absence of pregnancy) is also the triggering event for the cascade of molecular and cellular interactions that result in menstrual bleeding. A current hypothesis for menstruation is based on lines of evidence derived from studies on local endometrial response to progesterone withdrawal ([1]; Fig. 3).
(IL-8), monocyte chemotactic peptide-1 (MCP-1), and COX-2, the inducible enzyme responsible for synthesis of prostaglandins. COX-2 is markedly expressed in human menstrual phase endometrium at a time when prostaglandin levels have been demonstrated to rise [35]. Interestingly, these important local mediators display a perivascular location for their site of expression. IL-8, an ␣-chemokine that is chemotactic for neutrophils and MCP-1, a -chemokine that is chemotactic for monocytes both have a perivascular localisation [35–37]. PGDH, the enzyme responsible for metabolism of prostaglandins (PGs) to inactive metabolites, is a progesteronedependent enzyme [9]. Antagonism of progesterone action results in an inhibition of PGDH expression. Taken together these early local events in response to progesterone withdrawal result in an elevation of prostaglandin concentrations (PGE and PGF2␣) and potential synergism with the chemokine, IL-8 [38,39]. Consequently, there is a peri-menstrual influx of leukocytes consisting of neutrophils, macrophages and other haemopoietic cells. The endometrial population of leukocytes is a source of cytokines that further augment leukocyte traffic. 3.2. Up-regulation of matrix metalloproteinases (MMPs)
3.1. Progesterone withdrawal and up-regulation of inflammatory mediators and leukocyte influx The withdrawal of progesterone up-regulates key inflammatory mediators, including chemokines—interleukin-8
The endometrial population of leukocytic cells release enzymes, for example matrix metalloproteinases (MMPs) that act on the extracellular matrix along with the MMPs produced by the endometrial stromal cells following
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Fig. 3. Progesterone withdrawal results in up-regulation of inflammatory mediators, production of MMPs, a leukocyte influx and expression of stromal KDR in the upper endometrial zones. Menstrual sloughing takes place from the superficial regions of the endometrium. Reproduced with permission from Critchley et al. [1]. Published by Blackwell Science Ltd., Oxford, UK.
progesterone withdrawal at this time [40–42]. An important observation in this context is the secretion of MMPs by stromal cells in the upper endometrial zones after 48 h following progesterone withdrawal [43]. 3.3. Effects on vasoconstriction, hypoxia and up-regulation of vascular endothelial growth factor (VEGF) receptor type 2 (KDR) An increased production of PGF2␣, consequent upon progesterone withdrawal, produces myometrial contractions and vasoconstriction [44]. There is coincident vascoconstriction of the endometrial spiral arteries [45], and thus the uppermost endometrial zones are presumed to become hypoxic with resultant distal ischemia. There is some current controversy about the timing and role for hypoxia in the endometrium as Zhang and Salamonsen found no correlation between hypoxia inducing factors and MMPs in human endometrial tissues [46]. Local mediators that may be stimulated by hypoxia or indeed progesterone withdrawal from endometrial stromal cells include the potent angiogenic factor, vascular endothelial growth factor (VEGF; [47,48]). Progesterone withdrawal has been reported by our group to up-regulate the endometrial stromal expression of VEGF receptor type 2 (KDR) in women and non-human primates [49]. The stromal, but not vascular endothelial expression of KDR, can be blocked by adding back progesterone 24 h after progesterone withdrawal. Pro-MMP-1 was also observed to be up-regulated in a coordinate manner in the same stromal cell population by withdrawal of progesterone. In further support of these observations Nayak and Brenner have recently
described the up-regulation of VEGF mRNA in the glands and stroma of the superficial endometrial zones [48]. Thus, as VEGF, KDR and MMPs are co-ordinately expressed by stromal cells of the upper zones of pre-menstrual stage endometrium at the time of progesterone withdrawal the conclusion is that a VEGF–KDR–MMP link is a component of the pre-menstrual/menstrual process ([1,49]; Fig. 3). The end result is breakdown of the endometrium and initiation of menstrual bleeding. It is fully accepted that many other factors also operate during the pre-menstrual phase. 3.4. Progesterone withdrawal and two phases of menstrual bleeding The withdrawal of progesterone will initially affect cells expressing the progesterone receptor. It has recently been proposed that there are two phases of menstruation [50]. The early events in menstruation involve vasoconstriction and cytokine changes and are initiated by progesterone withdrawal and are likely to be reversible. Subsequent events are likely to be inevitable and will include the activation of lytic mechanisms that are presumably the consequence of hypoxia. Thus, the latter phase of menstruation is progesterone independent and will involve cells that may not express the progesterone receptor, for example luteal phase epithelial cells and uterine leukocytes [51–53]. Consistent with the view that early events occurring in PR-positive cells may be inhibited by add back of progesterone, are observations from a study in the rhesus macaque monkey [54]. The “adding back” of progesterone before 36 h following progesterone withdrawal prevented menstrual bleeding, whereas add back of progesterone after 36 h failed to inhibit the onset of menstruation.
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4. Pharmacological progesterone withdrawal: antiprogestin administration Much has been learned about endometrial function from the observations of physiological withdrawal of progesterone from the endometrium and the role for steroid withdrawal in the menstrual induction cascade. In a similar manner, studies of the actions of antiprogesterones has informed understanding about the local mechanisms that are targeted in order to maximise the contragestive and abortifacient properties of these compounds. The antiprogestin, mifepristone (RU486), is known to exert its inhibitory effects by impairing the gene regulatory activity of the progesterone receptor [55,56]. 4.1. Antiprogestin administration in early pregnancy By using a model of progesterone receptor antagonism in vivo, that is, administration of the antiprogestin mifepristone, it is possible to study local events in early pregnancy decidua that may play an important role in the process of early pregnancy failure. In a study examining the role of progesterone in the recruitment of selected leukocyte populations in the decidua of the first trimester of pregnancy, decidual tissue was examined 6, 12, 24 and 36 h after administration of a single dose of mifepristone (200 mg) in women with a pregnancy of less than 63 days gestation [57]. There was a significant increase in numbers of macrophages in the decidua parietalis 12 h following mifepristone administration in vivo (Fig. 4; [57]). In an earlier report by Wang et al. where first trimester decidua was examined 48 h after a 200 mg dose of mifepristone no such increase was described [58]. It is possible that the changes
Fig. 4. Oral administration of the antiprogestin mifepristone to women in early pregnancy (6–8 weeks gestation) results in an influx of monocytes (CD68 positive cells) into the decidua and an up-regulation of IL-8 production. The percentage of leukocytes that are monocytes increases significantly by 12 h. IL-8 was measured in supernatants derived from culture of decidual explants for 24 h. Within 6 h there was an increase in the amount of IL-8 released into the medium. Redrawn from Critchley et al. [57].
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in macrophage number observed by Critchley et al. (Fig. 4; [57]) in a time course earlier than 48 h post-antiprogestin administration play an important role in the local cellular responses which lead in due course to abortion. Antagonism of progesterone action at the level of its receptor results in an up-regulation of key local inflammatory mediators. Decidual IL-8 concentrations are significantly increased 6 h after mifepristone administration [57]. Expression of the prostaglandin metabolising enzyme, PGDH, was also inhibited in decidua by systemic administration of mifepristone [59]. Prostaglandin concentrations in decidua will thus be elevated and as indicated above there is an opportunity for synergism with IL-8 that should facilitate leukocyte entry into the decidua [38,39].
4.2. Acute antiprogestin administration in the luteal phase In vivo studies where antiprogestins have been administered acutely in the luteal phase of the cycle provide evidence for those functions in the non-pregnant endometrium regulated by progesterone. Several studies have described the increased expression of sex steroid receptors, estrogen receptor, progesterone receptor and androgen receptor in both the glandular and stromal compartments in mid-luteal phase endometrium after early luteal phase (usually day LH + 2) administration of antiprogestins [5,12,60,61]. Administration of an antiprogestin in the early luteal phase (LH + 2) will adversely affect local factors of potential importance to implantation. For example, treatment with antiprogestins will retard the development of usual luteal secretory changes and progesterone regulated endometrial expression of PGDH, leukaemia inhibitory factor (LIF) and glycodelin [62–64]. The endometrial changes (including marked alterations in the endometrial vasculature; [65]) associated with withdrawal of progesterone and menstrual bleeding indicate an involvement of vasoactive local mediators. Endometrial prostaglandin activity is modulated by progesterone, and hence prostaglandins, with widely recognised vasoactive properties, are prime candidates for local modulation of progesterone action [66] and the following observations [67] are entirely in keeping with our hypothesis described above [1]. Women administered 200 mg of mifepristone on day 8 after the onset of the urinary luteinising surge (LH + 8) had an endometrial sample collected between 6 and 48 h after ingestion of the antiprogestin. All women reported vaginal bleeding 36–48 h after taking mifepristone. After mifepristone, a significant increase in endometrial COX-2 immunoreactivity was evident at 36–48 h. There was a coincidental decrease in PGDH immunostaining in both glands and stromal tissue by 36–48 h (Fig. 5; [67]). In sum, the acute administration of an antiprogestin in the mid-luteal phase induced endometrial bleeding by a mechanism that involves a local increase in prostaglandin levels in endometrium. It is interesting that PGDH decline has been postulated as a
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Fig. 5. (a) PGDH immunostaining in mid-luteal phase endometrium, demonstrating positive immunoreactivity in the glands and stroma. (b) Endometrium, biopsied 36 h after administration of mifepristone, on day LH + 8 of cycle. Note the decrease in immunoreactivity in the glandular and stromal compartments. (c) COX-2 immunostaining in an endometrial biopsy collected in the mid-luteal phase. Negligible immunostaining in glands and stroma. (d) COX-2 immunostaining in endometrium, collected 36 h after administration of mifepristone, on day LH + 8. Note the increase in immunoreactivity in the glandular cytoplasm. Reproduced with permission from Hapangama et al. [67]. Published by The Endocrine Society.
potential marker of the closure of the implantation window, and hence local effects of mid-luteal administration of antiprogestins on such markers may contribute to current understanding of the action of mifepristone as a contraceptive. The chronic low dose oral administration of mifepristone has demonstrated the sensitivity of the endometrial morphology to antiprogestin exposure. Administration of mifepristone in low daily doses will inhibit ovulation and induce amenorrhoea or a marked reduction in endometrial bleeding [68]. The threshold dose for procuring a delay in endometrial maturation is 0.5 mg daily. Chronic antiprogestin administration inhibits both endometrial secretion and proliferation (anti-estrogen effects). This effect has been described as a “functional non-competitive anti-estrogenic action” of an antiprogestin [69]. This aspect of antiprogestin action in the endometrium has recently been addressed in a detailed review from Brenner et al. [70] and will not be considered further here.
5. Summary The molecular and cellular mechanism by which the withdrawal of sex steroids induces breakdown of the endometrium involves complex interactions between the endocrine and immune system. The modulation of sex steroid expression and function with pharmacological agents has provided an invaluable tool for studying the functional responses of the endometrium to sex steroids and their withdrawal. Specifically, study of the action of antiprogestins on endometrial morphology and function, has provided information about the roles for progesterone in reproductive tissues. The data derived from such studies to date (examples described above) support our thesis that administration of antagonists of progesterone provide an excellent model for the study of the mechanisms involved in spontaneous and induced abortion and can provide information that may help devise new strategies for
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treating breakthrough bleeding associated with hormonal contraception.
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We are grateful to Ted Pinner for expert assistance with the illustrations and Natasha Mallion for her secretarial help. Some of the data described herein are derived from studies supported by the Medical Research Council (UK; Grant nos.: G9620138; G0000066 to HODC, DTB); Wellbeing/RCOG (UK; Grant no. C2/99 to HODC) and support to RMB (Grants nos. DAMDIS-6096C and NIH-RR-00163). References [1] Critchley HOD, Kelly RW, Brenner RM, Baird DT. The endocrinology of menstruation—a role for the immune system. Clin Endocrinol 2001;55:701–10. [2] Garcia E, Bouchard P, DeBrux J, Berdah J, Frydman R, Schaison G, et al. Use of immunocytochemistry of progesterone and estrogen receptors for endometrial dating. J Clin Endocrinol Metab 1988;67:80–7. [3] Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL, McCarty KS. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metab 1988;67:334–40. [4] Snijders MP, de Goeij AFPM, Debets-Te Baerts MJC, Rousch MJ, Koudstaal J, Bosman FT. Immunocytochemical analysis of oestrogen receptors and progesterone receptors in the human uterus throughout the menstrual cycle and after the menopause. J Reprod Fertil 1992;94:363–71. [5] Slayden OD, Nayak NR, Chwalisz K, Cameron ST, Critchley HOD, Baird DT, et al. Progesterone antagonists increase the androgen receptor expression in the rhesus macaque and human endometrium. J Clin Endocrinol Metab 2001;86:2668–79. [6] Critchley HOD, Brenner RM, Drudy TA, Williams K, Nayak NR, Slayden OD, et al. Estrogen receptor beta, but not estrogen receptor alpha, is present in vascular endothelium of the human and nonhuman primate endometrium. J Clin Endocrinol Metab 2001;86:1370–8. [7] Kumar S, Zhu L-J, Polihronis M, Cameron ST, Baird DT, Schatz F, et al. Progesterone induces calcitonin gene expression in human endometrium within the putative window of implantation. J Clin Endocrinol Metab 1998;83:4444–50. [8] Chard T, Olajide F. Endometrial protein PP14: a new test of endometrial function. Reprod Med Rev 1994;3:43–52. [9] Casey ML, Hemsell DL, MacDonald PC, Johnston JM. NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity in human endometrium. Prostaglandins 1980;19:115–22. [10] Cameron ST, Critchley HOD, Buckley CH, Chard T, Baird DT. The effects of post ovulatory administration of onapristone on the development of a secretory endometrium. Hum Reprod 1996;11:40– 9. [11] Greenland KJ, Jantke I, Jennatschke S, Bracken KE, Vinson C, Gellerson B. The human NAD+ dependent 15-hydroxyprostaglandin dehydrogenase gene promoter is controlled by Ets and activating protein-1 transcription factors and progesterone. Endocrinology 2000;141:581–97. [12] Maentausta O, Svalander P, Gemzell-Danielsson K, Bygdeman M, Vihko R. The effects of an antiprogestin, mifepristone, and an anti-estrogen, tamoxifen, on endometrial 17 hydroxysteroid dehydrogenase and progestin and estrogen receptors during the luteal
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[33] Csapo AI, Resch BA. Prevention of implantation by antiprogesterone. J Steroid Biochem 1979;11:963–9. [34] Csapo AI, Pulkkinen M. Indispensibility of the human corpus luteum in the maintenance of early pregnancy. Lutectomy evidence. Obstet Gynecol Surv 1978;33:69–81. [35] Jones RL, Kelly RW, Critchley HOD. Chemokine and cyclooxygenase-2 expression in human endometrium coincides with leukocyte accumulation. Hum Reprod 1997;12:1300–6. [36] Critchley HOD, Kelly RW, Kooy J. Perivascular expression of chemokine interleukin-8 in human endometrium. Hum Reprod 1994;9:1406–9. [37] Milne SA, Critchley HOD, Drudy TA, Kelly RW, Baird DT. Perivascular interleukin-8 messenger ribonucleic acid expression in human endometrium varies across the menstrual cycle and in early pregnancy decidua. J Clin Endocrinol Metab 1999;84:2563–7. [38] Colditz IG. Effect of exogenous prostaglandin E2 and actinomycin D on plasma leakage induced by neutrophil activating peptide-1/ interleukin-8. Immunol Cell Biol 1990;68:397–403. [39] Rampart M, Van Damme J, Zonnekyn L, Herman AG. Granulocyte chemotactic protein/interleukin-8 induces plasma leakage and neutrophil accumulation in rabbit skin. Am J Pathol 1989;135: 21–5. [40] Schatz F, Papp C, Aigner S, Krikun G, Hausknecht V, Lockwood CJ. Biological mechanisms underlying the clinical effects of RU486: modulation of cultured endometrial stromal cell stromelysin-1 and prolactin expression. J Clin Endocrinol Metab 1997;82:188–93. [41] Lockwood CJ, Krikun G, Hausknecht VA, Papp C, Schatz F. Matrix metalloproteinase and matrix metalloproteinase inhibitor expression in endometrial stromal cells during progestin-initiated decidualisation and menstruation-related progestin withdrawal. Endocrinology 1998;139:4607–13. [42] Salamonsen LA, Woolley DE. Menstruation: induction by matrix metalloproteinases and inflammatory cells. J Reprod Immunol 1999;44:1–27. [43] Rudolph-Owen LA, Slayden O, Matrisan LM, Brenner RM. Matrix metalloproteinase expression in Macaca mulatta endometrium: evidence for zone-specific regulatory tissue gradients. Biol Reprod 1998;59:1349–59. [44] Ylikorkala O, Makila UM. Prostacyclin and thromboxane in gynecology and obstetrics. Am J Obstet Gynecol 1985;152:318–29. [45] Markee JE. Menstruation in intraocular transplants in the rhesus monkey. Contrib Embryol Carnegie Inst 1940;177:211–308. [46] Zhang J, Salamonsen LA. Expression of hypoxia-inducible factors in human endometrium and suppression of matrix metalloproteinases under hypoxic conditions do not support a major role for hypoxia in regulating tissue breakdown at menstruation. Hum Reprod 2002;17:265–74. [47] Sharkey AM, Day K, McPherson A, Malik S, Licence D, Smith SK, et al. Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia. J Clin Endocrinol Metab 2000;85:402–9. [48] Nayak NR, Brenner RM. Vascular proliferation and vascular endothelial growth factor expression in the rhesus macaque endometrium. J Clin Endocrinol Metab 2002;87:1845–55. [49] Nayak NR, Critchley HOD, Slayden O, Menrad A, Chwalisz K, Baird DT, et al. Progesterone withdrawal up-regulates vascular endothelial growth factor receptor type 2 in the superficial zone stroma of the human and macaque endometrium: potential relevance to menstruation. J Clin Endocrinol Metab 2000;85:3442–52. [50] Kelly RW, King AE, Critchley HOD. Cytokine control in human endometrium. Reproduction 2001;121:3–19. [51] King A, Gardner L, Loke YW. Evaluation of oestrogen and progesterone receptor expression in uterine mucosal lymphocytes. Hum Reprod 1996;11:1079–82.
[52] Henderson TA, Saunders PT, Moffet-King A, Groome NP, Critchley HOD. Steroid receptor expression in uterine natural killer cells. J Clin Endocrinol Metab 2003;88:440–9. [53] Stewart J, Bulmer JN, Murdoch AP. Endometrial leucocytes: expression of steroid hormone receptors. J Clin Pathol 1998;51:121– 6. [54] Slayden OD, Mah K, Brenner RM. A critical period of progesterone withdrawal exists for endometrial MMPs and menstruation in macaques. Program. Society for the Study of Reproduction. Abstract 579. Biol Reprod 1999;60(Suppl 1):273. [55] Baulieu EE. Contragestion and other clinical applications of RU486, an antiprogesterone at the receptor. Science 1989;245:1351–7. [56] Baulieu EE. The antisteroid RU486. Its cellular and molecular mode of action. Trends Endocrinol Metab 1991;2:233–9. [57] Critchley HOD, Kelly RW, Lea RG, Drudy TA, Jones RL, Baird DT. Sex steroid regulation of leukocyte traffic in human decidua. Hum Reprod 1996;11:2257–62. [58] Wang JD, Fu Y, Zhu JB. Lymphocyte population in human decidua after RU486 treatment. Biol Reprod 1995;52(Suppl 11):184. [59] Cheng L, Kelly RW, Thong KJ, Hume R, Baird DT. The effects of mifepristone (RU486) on prostaglandin dehydrogenase in decidual and chorionic tissue in early pregnancy. Hum Reprod 1993;8:705–9. [60] Cameron ST, Critchley HOD, Thong KJ, Buckley CH, Williams AR, Baird DT. Effects of daily low dose mifepristone on endometrial maturation and proliferation. Hum Reprod 1996;11:2518–26. [61] Berthois Y, Brux JD, Salat-Baroux J, Kopp F, Cornet D, Martin PM. A multiparametric analysis of endometrial estrogen and progesterone receptors after the postovulatory administration of mifepristone. Fertil Steril 1991;55:547–54. [62] Cameron ST, Critchley HOD, Buckley CH, Kelly RW, Baird DT. Effect of two antiprogestins (mifepristone and onapristone) on endometrial factors of potential importance for implantation. Fertil Steril 1997;67:1046–53. [63] Gemzell-Danielsson K, Wetlund P, Johannisson E, Swahn ML, Bygdeman M, Seppala M. Effect of low weekly doses of mifepristone on ovarian function and endometrial development. Hum Reprod 1996;11:256–64. [64] Gemzell-Danielsson K, Swahn ML, Wetlund P, Johannisson E, Seppala M, Bygdeman M. Effects of daily low doses of mifepristone on ovarian function and endometrial development. Hum Reprod 1997;12:124–31. [65] Johannisson E, Oberholzer M, Swahn M-L, Bygdeman M. Vascular changes in the human endometrium following the administration of the progesterone antagonist RU 486. Contraception 1988;39:103–17. [66] Baird DT, Cameron ST, Critchley HOD, Drudy TA, Howe A, Jones RL, et al. Prostaglandins and menstruation. Eur J Obstet Gynecol Reprod Biol 1996;70:15–7. [67] Hapangama DK, Critchley HOD, Henderson TA, Baird DT. Mifepristone-induced vaginal bleeding is associated with increased immunostaining for cyclooxygenase 2 and decrease in prostaglandin dehydrogenase in luteal phase endometrium. J Clin Endocrinol Metab 2002;87:5229–34. [68] Brown A, Cheng L, Lin S, Baird DT. Daily low-dose mifepristone has contraceptive potential by suppressing ovulation and menstruation: a double-blind randomised control trial of 2 and 5 mg per day for 120 days. J Clin Endocrinol Metab 2002;87:63–70. [69] Wolf JP, Hsiu JG, Anderson TL, Ulmann A, Baulieu EE, Hodgen GD. Noncompetitive antiestrogenic effect of RU486 in blocking estrogen-stimulated luteinising hormone surge and the proliferative action of estradiol on endometrium in castrate monkeys. Fertil Steril 1989;52:1055–60. [70] Brenner RM, Slayden OD, Critchley HOD. Antiproliferative effects of progesterone antagonists in the primate endometrium: a potential role for the androgen receptor. Reproduction 2002;124:167–72.
Antiprogestins as a model for progesterone withdrawal Hilary O.D. Critchley a,∗ , Rodney W. Kelly b , Robert M. Brenner c , David T. Baird d a
b
Obstetrics and Gynaecology, Centre for Reproductive Biology, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK c Oregon National Primate Research Centre, 505 NW 185th Avenue, Beaverton, OR 97006, USA d Obstetrics and Gynaecology, Centre for Reproductive Biology, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK
Abstract The key physiological function of the endometrium is preparation for implantation; and in the absence of pregnancy, menstruation and repair. The withdrawal of progesterone is the initiating factor for breakdown of the endometrium. The modulation of sex steroid expression and function with pharmacological agents has provided an invaluable tool for studying the functional responses of the endometrium to sex steroids and their withdrawal. By administration of the antiprogestin mifepristone, it is possible to mimic progesterone withdrawal and study local events in early pregnancy decidua that may play a role in the process of early pregnancy failure. Our data indicate that antagonism of progesterone action at the receptor level results in an up-regulation of key local inflammatory mediators, including NF-B, interleukin-8 (IL-8), monocyte chemotactic peptide-1 (MCP-1), cyclooxygenase 2 (COX-2) and others in decidua. Bleeding induced by mifepristone in the mid-luteal phase of the cycle is associated with changes in the endometrium similar to those that precede spontaneous menstruation including up-regulation of COX-2 and down-regulation of PGDH. Administration of antagonists of progesterone provide an excellent model to study the mechanisms involved in spontaneous and induced abortion as well as providing information which may help devise strategies for treating breakthrough bleeding associated with hormonal contraception. © 2003 Elsevier Inc. All rights reserved. Keywords: Endometrium; Progesterone; Steroid receptors; Antiprogestins
1. Introduction The key physiological function of the endometrium is preparation for implantation; and in the absence of pregnancy, menstruation and repair. The endometrium undergoes marked morphological and functional changes during each menstrual cycle. The cyclical features of endometrial proliferation and differentiation are the consequence of sequential exposure to estradiol and progesterone from the developing ovarian follicle and corpus luteum, respectively. The withdrawal of progesterone that occurs with demise of the corpus luteum is the initiating factor for breakdown of the endometrium at menstruation. The molecular mechanisms by which sex steroids induce these events within the endometrium, involve complex interactions between
∗ Corresponding author. Tel.: +44-131-242-6439; fax: +44-131-242-6440. E-mail address: [email protected] (H.O.D. Critchley).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2003.07.001
the endocrine and immune system and have recently been reviewed [1]. Steroids interact with their target organs via specific nuclear receptors. The expression of endometrial sex steroid receptors (progesterone receptor, PR; estrogen receptor, ER; androgen receptor, AR) varies temporally and spatially across the menstrual cycle [2–6]. The modulation of sex steroid expression and function with pharmacological agents has provided an invaluable tool for studying the functional responses of the endometrium to sex steroids. Progesterone is essential for preparation of the endometrium for implantation. Specifically, study of the action of antiprogestins on endometrial morphology and function, has provided invaluable information about the roles of progesterone in reproductive tissues. The following is a review of known roles for progesterone in the endometrium and early pregnancy decidua, and the response of the endometrium (and decidua) to both physiological and pharmacological progesterone withdrawal.
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2. Progesterone action in human endometrium and early pregnancy decidua Progesterone is essential for the transformation of an estrogen-primed endometrium in preparation for implantation. Much remains poorly understood about the molecular and cellular mechanisms involved by which the sex steroid hormones promote uterine receptivity. It is, however, recognised that sex steroids, acting via their cognate receptors, initiate a pattern of gene expression important for implantation and the early stages of pregnancy. Consequently, if specific steroid-induced molecules are identified, there is the potential for their use as markers of uterine receptivity or targets for early pregnancy interruption. Multiple studies have examined the temporal and spatial expression of presumed progesterone regulated genes across the menstrual cycle. For example, expression of calcitonin mRNA has been demonstrated to be temporally restricted to the mid-secretory phase of the cycle (maximal expression during days 19–21), a period that coincides with the putative window of implantation [7]. The site of post-ovulatory synthesis of calcitonin mRNA and protein is the glandular epithelium. Evidence for regulation of this gene by progesterone was derived from examination of endometrium collected from women treated with a progesterone antagonist, mifepristone. Calcitonin expression was dramatically reduced in women exposed to acute administration of mifepristone in the early luteal phase (administered day LH + 2; Fig. 1). Other examples of endometrial enzymes that are up-regulated by progesterone include, glandular secretion of glycodelin (PP14; [8]), 15-hydroxyprostaglandin dehydrogenase (PGDH; [9–11]) and 17-hydroxysteroid dehydrogenase (17-HSD type2; [12]). Decidualisation is a crucial step in the initiation and establishment of pregnancy and is a feature of those species that have an invasive haemochorial placentation. In the hu-
Fig. 1. Calcitonin immunostaining intensity in human endometrium. The relative signal intensity for calcitonin protein staining in glandular epithelial cells declines after treatment with the antiprogestin, mifepristone (mean ± S.E.M.; ∗ P < 0.001). Reproduced with permission from Kumar et al. [7]. Published by The Endocrine Society.
man, decidualisation is independent of the blastocyst and early signs of predecidual changes are first observed in stromal cells close to vascular structures in the mid-late secretory phase (Fig. 2). These same stromal cells express progesterone receptor throughout the luteal phase. In vivo, decidualisation is controlled most effectively by progesterone action on an estrogen-primed uterus. This process can, however, be induced as a consequence of administration of exogenous progestogens [13]. In vitro, a central role for cAMP has also been demonstrated as a decidualisation stimulus [14,15]. Studies of mouse mutants where decidualisation does not occur have identified a requirement for genes in the endometrial epithelium and/or stromal cells [16]. Key genes include cyclooxygenase 2 (COX-2, the rate-limiting enzyme in prostaglandin synthesis; [17]), Hoxa 10 and 11 [18,19]. Progesterone dependence of Hoxa 11 has been demonstrated [20]. The cellular interactions and progesterone target genes involved in decidualisation are complex. Multiple growth factors, cytokines and protein hormones have been recognised as important signals for initiation and maintenance of decidualisation [21,22]. Prolactin is a key cytokine secreted by decidualised endometrium. Prolactin expression is controlled by progesterone [23]. In vivo administration of the antiprogestin RU486 significantly reduces prolactin expression in decidualised stromal cells [24]. The evidence available indicates that progesterone is important for inducing and maintaining decidualisation but it does not induce the prolactin gene directly. Prolactin mediates its effect on target cells via single pass transmembrane spanning receptors. Prolactin receptor expression in the human endometrium is temporally regulated throughout the menstrual cycle. There is minimal expression during the proliferative phase and expression is up-regulated during the mid-late secretory phase. The prolactin receptor in non-pregnant endometrium is localised predominantly to the glandular epithelial cells. Prolactin receptor expression is maintained in pregnancy and localised to the decidualised stromal cells [25,26]. Prolactin receptor is also expressed by the phenotypically unique endometrial population of CD56+, CD16−, CD3− uterine natural killer (NK) cells that do not express the progesterone receptor [27]. Valuable insights are gained from gene ablation studies. In the above context, the prolactin receptor knock-out mouse displays an implantation defect [28] that may be a reflection of a central pituitary defect. Ablation of the progesterone receptor leads to inappropriate inflammation in the uteri of mice [29]. Different roles for the progesterone receptor isoforms, PRB and shorter PRA [30], have recently been elucidated by the production of PRA and PRB null mice [31]. For example in the PRA null mouse, estrogen induces uterine epithelial hyperplasia which progesterone treatment cannot suppress. This observation implies that progesterone-mediated suppression of epithelial growth stimulated by estrogen is dependent upon PRA, not PRB. Furthermore, in PRA + PRB null mice, there is a marked influx of inflammatory leukocytes, that cannot be inhibited
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Fig. 2. Photomicrograph of perivascular progesterone receptor immunostaining in secretory phase endometrium. Arrow indicates positive nuclear immunoreactivity.
by progesterone [29]. This observation supports a role for progesterone in suppression of the traffic of inflammatory cells into the uterus in wild type animals. The selective ablation of PRA in mice [32], has demonstrated that in mice the PRB isoform modulates a subset of progesterone-responsive target genes. Hence, PRA and PRB are likely to act as functionally distinct mediators of progesterone action in vivo. It is not known whether these observations can be extrapolated to the functions of the human uterus. 3. Physiological progesterone withdrawal Withdrawal of progesterone prevents implantation and converts the refractory pregnant uterus once again into a spontaneously steroid responsive organ [33,34]. The physiological withdrawal of progesterone from an estrogen– progesterone primed endometrium (that occurs with demise of the corpus luteum due to the absence of pregnancy) is also the triggering event for the cascade of molecular and cellular interactions that result in menstrual bleeding. A current hypothesis for menstruation is based on lines of evidence derived from studies on local endometrial response to progesterone withdrawal ([1]; Fig. 3).
(IL-8), monocyte chemotactic peptide-1 (MCP-1), and COX-2, the inducible enzyme responsible for synthesis of prostaglandins. COX-2 is markedly expressed in human menstrual phase endometrium at a time when prostaglandin levels have been demonstrated to rise [35]. Interestingly, these important local mediators display a perivascular location for their site of expression. IL-8, an ␣-chemokine that is chemotactic for neutrophils and MCP-1, a -chemokine that is chemotactic for monocytes both have a perivascular localisation [35–37]. PGDH, the enzyme responsible for metabolism of prostaglandins (PGs) to inactive metabolites, is a progesteronedependent enzyme [9]. Antagonism of progesterone action results in an inhibition of PGDH expression. Taken together these early local events in response to progesterone withdrawal result in an elevation of prostaglandin concentrations (PGE and PGF2␣) and potential synergism with the chemokine, IL-8 [38,39]. Consequently, there is a peri-menstrual influx of leukocytes consisting of neutrophils, macrophages and other haemopoietic cells. The endometrial population of leukocytes is a source of cytokines that further augment leukocyte traffic. 3.2. Up-regulation of matrix metalloproteinases (MMPs)
3.1. Progesterone withdrawal and up-regulation of inflammatory mediators and leukocyte influx The withdrawal of progesterone up-regulates key inflammatory mediators, including chemokines—interleukin-8
The endometrial population of leukocytic cells release enzymes, for example matrix metalloproteinases (MMPs) that act on the extracellular matrix along with the MMPs produced by the endometrial stromal cells following
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Fig. 3. Progesterone withdrawal results in up-regulation of inflammatory mediators, production of MMPs, a leukocyte influx and expression of stromal KDR in the upper endometrial zones. Menstrual sloughing takes place from the superficial regions of the endometrium. Reproduced with permission from Critchley et al. [1]. Published by Blackwell Science Ltd., Oxford, UK.
progesterone withdrawal at this time [40–42]. An important observation in this context is the secretion of MMPs by stromal cells in the upper endometrial zones after 48 h following progesterone withdrawal [43]. 3.3. Effects on vasoconstriction, hypoxia and up-regulation of vascular endothelial growth factor (VEGF) receptor type 2 (KDR) An increased production of PGF2␣, consequent upon progesterone withdrawal, produces myometrial contractions and vasoconstriction [44]. There is coincident vascoconstriction of the endometrial spiral arteries [45], and thus the uppermost endometrial zones are presumed to become hypoxic with resultant distal ischemia. There is some current controversy about the timing and role for hypoxia in the endometrium as Zhang and Salamonsen found no correlation between hypoxia inducing factors and MMPs in human endometrial tissues [46]. Local mediators that may be stimulated by hypoxia or indeed progesterone withdrawal from endometrial stromal cells include the potent angiogenic factor, vascular endothelial growth factor (VEGF; [47,48]). Progesterone withdrawal has been reported by our group to up-regulate the endometrial stromal expression of VEGF receptor type 2 (KDR) in women and non-human primates [49]. The stromal, but not vascular endothelial expression of KDR, can be blocked by adding back progesterone 24 h after progesterone withdrawal. Pro-MMP-1 was also observed to be up-regulated in a coordinate manner in the same stromal cell population by withdrawal of progesterone. In further support of these observations Nayak and Brenner have recently
described the up-regulation of VEGF mRNA in the glands and stroma of the superficial endometrial zones [48]. Thus, as VEGF, KDR and MMPs are co-ordinately expressed by stromal cells of the upper zones of pre-menstrual stage endometrium at the time of progesterone withdrawal the conclusion is that a VEGF–KDR–MMP link is a component of the pre-menstrual/menstrual process ([1,49]; Fig. 3). The end result is breakdown of the endometrium and initiation of menstrual bleeding. It is fully accepted that many other factors also operate during the pre-menstrual phase. 3.4. Progesterone withdrawal and two phases of menstrual bleeding The withdrawal of progesterone will initially affect cells expressing the progesterone receptor. It has recently been proposed that there are two phases of menstruation [50]. The early events in menstruation involve vasoconstriction and cytokine changes and are initiated by progesterone withdrawal and are likely to be reversible. Subsequent events are likely to be inevitable and will include the activation of lytic mechanisms that are presumably the consequence of hypoxia. Thus, the latter phase of menstruation is progesterone independent and will involve cells that may not express the progesterone receptor, for example luteal phase epithelial cells and uterine leukocytes [51–53]. Consistent with the view that early events occurring in PR-positive cells may be inhibited by add back of progesterone, are observations from a study in the rhesus macaque monkey [54]. The “adding back” of progesterone before 36 h following progesterone withdrawal prevented menstrual bleeding, whereas add back of progesterone after 36 h failed to inhibit the onset of menstruation.
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4. Pharmacological progesterone withdrawal: antiprogestin administration Much has been learned about endometrial function from the observations of physiological withdrawal of progesterone from the endometrium and the role for steroid withdrawal in the menstrual induction cascade. In a similar manner, studies of the actions of antiprogesterones has informed understanding about the local mechanisms that are targeted in order to maximise the contragestive and abortifacient properties of these compounds. The antiprogestin, mifepristone (RU486), is known to exert its inhibitory effects by impairing the gene regulatory activity of the progesterone receptor [55,56]. 4.1. Antiprogestin administration in early pregnancy By using a model of progesterone receptor antagonism in vivo, that is, administration of the antiprogestin mifepristone, it is possible to study local events in early pregnancy decidua that may play an important role in the process of early pregnancy failure. In a study examining the role of progesterone in the recruitment of selected leukocyte populations in the decidua of the first trimester of pregnancy, decidual tissue was examined 6, 12, 24 and 36 h after administration of a single dose of mifepristone (200 mg) in women with a pregnancy of less than 63 days gestation [57]. There was a significant increase in numbers of macrophages in the decidua parietalis 12 h following mifepristone administration in vivo (Fig. 4; [57]). In an earlier report by Wang et al. where first trimester decidua was examined 48 h after a 200 mg dose of mifepristone no such increase was described [58]. It is possible that the changes
Fig. 4. Oral administration of the antiprogestin mifepristone to women in early pregnancy (6–8 weeks gestation) results in an influx of monocytes (CD68 positive cells) into the decidua and an up-regulation of IL-8 production. The percentage of leukocytes that are monocytes increases significantly by 12 h. IL-8 was measured in supernatants derived from culture of decidual explants for 24 h. Within 6 h there was an increase in the amount of IL-8 released into the medium. Redrawn from Critchley et al. [57].
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in macrophage number observed by Critchley et al. (Fig. 4; [57]) in a time course earlier than 48 h post-antiprogestin administration play an important role in the local cellular responses which lead in due course to abortion. Antagonism of progesterone action at the level of its receptor results in an up-regulation of key local inflammatory mediators. Decidual IL-8 concentrations are significantly increased 6 h after mifepristone administration [57]. Expression of the prostaglandin metabolising enzyme, PGDH, was also inhibited in decidua by systemic administration of mifepristone [59]. Prostaglandin concentrations in decidua will thus be elevated and as indicated above there is an opportunity for synergism with IL-8 that should facilitate leukocyte entry into the decidua [38,39].
4.2. Acute antiprogestin administration in the luteal phase In vivo studies where antiprogestins have been administered acutely in the luteal phase of the cycle provide evidence for those functions in the non-pregnant endometrium regulated by progesterone. Several studies have described the increased expression of sex steroid receptors, estrogen receptor, progesterone receptor and androgen receptor in both the glandular and stromal compartments in mid-luteal phase endometrium after early luteal phase (usually day LH + 2) administration of antiprogestins [5,12,60,61]. Administration of an antiprogestin in the early luteal phase (LH + 2) will adversely affect local factors of potential importance to implantation. For example, treatment with antiprogestins will retard the development of usual luteal secretory changes and progesterone regulated endometrial expression of PGDH, leukaemia inhibitory factor (LIF) and glycodelin [62–64]. The endometrial changes (including marked alterations in the endometrial vasculature; [65]) associated with withdrawal of progesterone and menstrual bleeding indicate an involvement of vasoactive local mediators. Endometrial prostaglandin activity is modulated by progesterone, and hence prostaglandins, with widely recognised vasoactive properties, are prime candidates for local modulation of progesterone action [66] and the following observations [67] are entirely in keeping with our hypothesis described above [1]. Women administered 200 mg of mifepristone on day 8 after the onset of the urinary luteinising surge (LH + 8) had an endometrial sample collected between 6 and 48 h after ingestion of the antiprogestin. All women reported vaginal bleeding 36–48 h after taking mifepristone. After mifepristone, a significant increase in endometrial COX-2 immunoreactivity was evident at 36–48 h. There was a coincidental decrease in PGDH immunostaining in both glands and stromal tissue by 36–48 h (Fig. 5; [67]). In sum, the acute administration of an antiprogestin in the mid-luteal phase induced endometrial bleeding by a mechanism that involves a local increase in prostaglandin levels in endometrium. It is interesting that PGDH decline has been postulated as a
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Fig. 5. (a) PGDH immunostaining in mid-luteal phase endometrium, demonstrating positive immunoreactivity in the glands and stroma. (b) Endometrium, biopsied 36 h after administration of mifepristone, on day LH + 8 of cycle. Note the decrease in immunoreactivity in the glandular and stromal compartments. (c) COX-2 immunostaining in an endometrial biopsy collected in the mid-luteal phase. Negligible immunostaining in glands and stroma. (d) COX-2 immunostaining in endometrium, collected 36 h after administration of mifepristone, on day LH + 8. Note the increase in immunoreactivity in the glandular cytoplasm. Reproduced with permission from Hapangama et al. [67]. Published by The Endocrine Society.
potential marker of the closure of the implantation window, and hence local effects of mid-luteal administration of antiprogestins on such markers may contribute to current understanding of the action of mifepristone as a contraceptive. The chronic low dose oral administration of mifepristone has demonstrated the sensitivity of the endometrial morphology to antiprogestin exposure. Administration of mifepristone in low daily doses will inhibit ovulation and induce amenorrhoea or a marked reduction in endometrial bleeding [68]. The threshold dose for procuring a delay in endometrial maturation is 0.5 mg daily. Chronic antiprogestin administration inhibits both endometrial secretion and proliferation (anti-estrogen effects). This effect has been described as a “functional non-competitive anti-estrogenic action” of an antiprogestin [69]. This aspect of antiprogestin action in the endometrium has recently been addressed in a detailed review from Brenner et al. [70] and will not be considered further here.
5. Summary The molecular and cellular mechanism by which the withdrawal of sex steroids induces breakdown of the endometrium involves complex interactions between the endocrine and immune system. The modulation of sex steroid expression and function with pharmacological agents has provided an invaluable tool for studying the functional responses of the endometrium to sex steroids and their withdrawal. Specifically, study of the action of antiprogestins on endometrial morphology and function, has provided information about the roles for progesterone in reproductive tissues. The data derived from such studies to date (examples described above) support our thesis that administration of antagonists of progesterone provide an excellent model for the study of the mechanisms involved in spontaneous and induced abortion and can provide information that may help devise new strategies for
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treating breakthrough bleeding associated with hormonal contraception.
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We are grateful to Ted Pinner for expert assistance with the illustrations and Natasha Mallion for her secretarial help. Some of the data described herein are derived from studies supported by the Medical Research Council (UK; Grant nos.: G9620138; G0000066 to HODC, DTB); Wellbeing/RCOG (UK; Grant no. C2/99 to HODC) and support to RMB (Grants nos. DAMDIS-6096C and NIH-RR-00163). References [1] Critchley HOD, Kelly RW, Brenner RM, Baird DT. The endocrinology of menstruation—a role for the immune system. Clin Endocrinol 2001;55:701–10. [2] Garcia E, Bouchard P, DeBrux J, Berdah J, Frydman R, Schaison G, et al. Use of immunocytochemistry of progesterone and estrogen receptors for endometrial dating. J Clin Endocrinol Metab 1988;67:80–7. [3] Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL, McCarty KS. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metab 1988;67:334–40. [4] Snijders MP, de Goeij AFPM, Debets-Te Baerts MJC, Rousch MJ, Koudstaal J, Bosman FT. Immunocytochemical analysis of oestrogen receptors and progesterone receptors in the human uterus throughout the menstrual cycle and after the menopause. J Reprod Fertil 1992;94:363–71. [5] Slayden OD, Nayak NR, Chwalisz K, Cameron ST, Critchley HOD, Baird DT, et al. Progesterone antagonists increase the androgen receptor expression in the rhesus macaque and human endometrium. J Clin Endocrinol Metab 2001;86:2668–79. [6] Critchley HOD, Brenner RM, Drudy TA, Williams K, Nayak NR, Slayden OD, et al. Estrogen receptor beta, but not estrogen receptor alpha, is present in vascular endothelium of the human and nonhuman primate endometrium. J Clin Endocrinol Metab 2001;86:1370–8. [7] Kumar S, Zhu L-J, Polihronis M, Cameron ST, Baird DT, Schatz F, et al. Progesterone induces calcitonin gene expression in human endometrium within the putative window of implantation. J Clin Endocrinol Metab 1998;83:4444–50. [8] Chard T, Olajide F. Endometrial protein PP14: a new test of endometrial function. Reprod Med Rev 1994;3:43–52. [9] Casey ML, Hemsell DL, MacDonald PC, Johnston JM. NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity in human endometrium. Prostaglandins 1980;19:115–22. [10] Cameron ST, Critchley HOD, Buckley CH, Chard T, Baird DT. The effects of post ovulatory administration of onapristone on the development of a secretory endometrium. Hum Reprod 1996;11:40– 9. [11] Greenland KJ, Jantke I, Jennatschke S, Bracken KE, Vinson C, Gellerson B. The human NAD+ dependent 15-hydroxyprostaglandin dehydrogenase gene promoter is controlled by Ets and activating protein-1 transcription factors and progesterone. Endocrinology 2000;141:581–97. [12] Maentausta O, Svalander P, Gemzell-Danielsson K, Bygdeman M, Vihko R. The effects of an antiprogestin, mifepristone, and an anti-estrogen, tamoxifen, on endometrial 17 hydroxysteroid dehydrogenase and progestin and estrogen receptors during the luteal
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