Long-Term Use of Progestogens – Getting the Balance Right: Molecular Biology and the Endometrium

By Druckmann, Rene

Abstract The role of progestins in long-term treatment has recendy been under scrutiny, in particular as their effects on organs other than the endometrium, such as the breast or the cardiovascular system, could give rise to undesirable reactions. The use of progestins in hormone replacement therapy has highlighted the importance of choice of progestin. Insights into the molecular biology of normal processes in the endometrium can help to find accurate markers for progestin effects and help select progestins with a better benefit-risk profile. Such insight can also lead to a better understanding of the etiology of menstrual and fertility disorders and ultimately to new dierapeutic approaches.

Keywords: Progestogens, endometrium, molecular biology, long- term treatment, menstrual cycle

Introduction

Progesterone plays a pivotal role in female reproduction. It modifies the effects of estrogen on the endometrium, and the balance between the two regulates the menstrual cycle, ovulation, implantation and pregnancy. As a consequence, progesterone and synthetic progestins are used in hormone replacement therapy (HRT) and contraception, as well as to treat menstrual disorders and certain types of infertility.

An important area of use of progesterone and progestins is HRT for postmenopausal women with an intact uterus, in which they counteract the proliferating effect of estrogen in order to prevent endometrial hyperplasia and endometrial cancer. There are two main treatment regimens: estrogen is administered continuously or cyclically, with either sequential or continuous combined administration of a progestogen. A number of different progestational agents are used, such as progesterone itself, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), norethisterone acetate (NETA) or dydrogesterone (DYD).

Therapeutic use of progestins for HRT, in particular continuous combined HRT, but also progestinonly contraception, is often associated with irregular and unwanted bleeding episodes. These side- effects seem more pronounced with some progestins than with others.

In addition, recent studies have implicated some progestins with an increased risk of cardiovascular disease or breast cancer when combined with estrogen in HRT.

As the progestational agent in HRT is added in order to prevent endometrial hyperplasia and neoplasia, efficacy or dose-finding studies usually investigate the ability of the progestogen in question to evoke withdrawal bleeding and histological changes in appropriately estrogen-primed endometrium. Studying endometrial histology and the bleeding pattern induced by a progestin in estrogen-primed endometrium at a certain dosage can determine whether endometrial hyperplasia has been avoided. However, the bleeding pattern and histology do not provide sufficient information for comparing different progestins, nor to select the most suitable progestogen with a reduced risk of unwanted bleeding and other side- effects.

Molecular biology, endocrinology and immunology are contributing valuable information in the area of gynecology. Insights from all of these fields have given us glimpses of a new picture of the events in the endometrium during the menstrual cycle, implantation or pregnancy. This information, although still fragmentary, has led to new hypotheses on the background of menstrual and fertility disorders. It could also provide us with more precise markers for progestin actions and thus help avoid side-effects and, eventually, lead to new therapeutic options.

The following short overview does not aim to be complete. It tries to give an impression of the intricate and multidimensional way in which events during the menstrual cycle are regulated at the molecular level. Ovarian steroids are in overall control of these processes. Their intracellular effects are brought about by binding as a ligand to their specific receptors, thereby modulating gene expression. The specific reaction associated with the ligand binding to its receptor depends on the target cell; different cells can respond differently to the same ligand. Fine tuning of the events during the menstrual cycle, however, does not seem to be under the direct control of the ovarian steroids. An important part is played by local interactions in the endometrium between a multitude of factors, including cytokine production or differential expression of receptors. Every actor in this play seems to have a counter actor, and tissue proliferation or differentiation is the result of the balance between all players involved.

Angiogenesis

Angiogenesis is one of the key events during the menstrual cycle. Studies on angiogenic activity have shown three peaks. The first peak occurs with vascular repair during menstruation. Regrowth starts from the stumps of ruptured blood vessels and glands in the basal layer of the endometrium, which act as a germinal compartment for the regeneration of all cell types, i.e. epithelial, stromal and endothelial. Blood vessel elongation seems to be the mechanism particular to endometrial angiogenesis. A second peak is found during the proliferative phase of rapid endometrial growth, which is dominated by estrogen. The third peak is detected in the second half of the menstrual cycle, during the progesterone-dominated secretory phase when angiogenesis is characterized by differentiation rather than rapid growth [1-3].

During this latter phase, the characteristic spiral arterioles with their thick layer of vascular smooth muscle cells develop. Stromal fibroblasts surrounding the developing spiral arterioles begin to differentiate, or decidualize, by enlarging and depositing a decidua-specific extracellular matrix (ECM). Perivascular stromal cells and vascular smooth muscle cells retain steroid receptors throughout the cycle (in contrast to glandular and surface epithelium, which lose theirs during the secretory phase) and are thought to play an important part in vascular control [4,5]. Finally, in the absence of pregnancy, the withdrawal of estrogen and progesterone leads to shrinkage of the endometrium, vasoconstriction of spiral arterioles and extensive destruction of the ECM, resulting in the shedding of the whole functional layer of the endometrium.

It has been shown that human endometrial endothelial cells express both estrogen receptors and progesterone receptors, thus estrogen and progesterone may direcdy affect endometrial blood vessels [6]. However, stimulators of angiogenesis in general are also present in the endometrium: vascular epithelial growth factors (VEGFs), particularly VEGF-A, fibroblast growth factors (FGFs) and epidermal growth factors. The actions of these angiogenic factors are opposed by angiostatic factors, such as thrombospondin 1 (TSP- 1) [3,7].

Serum levels of angiogenic and angiostatic factors show a recurrent pattern during the menstrual cycle, though not necessarily parallel to the levels of the ovarian steroids. At the first peak of angiogenesis, during menstruation, hypoxia seems to play an important part in stimulating VEGF-A and FGF synthesis, as it does in angiogenesis in other tissues [8]. However, estradiol has been shown to stimulate the expression of VEGF-A and FGF, and it has been suggested that estradiol maintains VEGF expression through the cycle after the first peak of angiogenesis. Progesterone, on the other hand, inhibits the production of some angiogenic factors and stimulates the production of angiostatic factor TSP-1. The expression of TSP-1 is highest during the progesterone-dominated mid- secretory phase [3,9].

The process of angiogenesis is thought to be the result of the balance between the various stimulators and inhibitors of angiogenesis. When the stimulators are in excess, angiogenesis is promoted; when the inhibitors are predominant, angiogenesis stops. However, intermediate states of this balance are also possible, where rapid growth is slowed down and differentiation of blood vessels sets in. The overall modulators of cyclic endometrial changes are the ovarian steroids, but a host of angiogenic and angiostatic factors, only some of which have been mentioned here, provide the fine tuning of these processes. Estrogen seems to mainly stimulate angiogenesis, while progesterone shows a more complex pattern of both angiostatic and angiogenic actions [10]. Whereas estrogen seems to be responsible for rapid growth of capillaries during the first part of the cycle, progesterone shifts the processes of angiogenesis in the direction of differentiation during the second half of the cycle [3].

Breakdown of endometrial tissue and menstruation

In the absence of pregnancy, the withdrawal of progesterone and estrogen leads to menstruation. One of the first effects of hormone withdrawal is episodic spiral arteriole constriction under the influence of prostaglandin (PG) PGF^sub 2alpha^ and endothelin-1. Vasodilators, such as PGE^sub 2^, naturally counteract vasoconstrictive factors. Both vasoconstrictive and vasodilating factors are present in the endometrium, but at menstruation, the balance is shifted towards dominance of the vasoconstrictive factors [11]. As a result of arteriole constrictions, the endometrium suffers ischemia and reperfusion damage, which leads to the release of a range of substances such as cytokines and other signaling molecules. All of these probably play a part during the shedding of the functional layer of the endometrium [12,13]. The ECM plays an important role in the structural and functional organization of every tissue and organ. ECM constituents, in particular fibrillar collagens, are the most abundant proteins in the human body. Thus, the degradation and rebuilding of the ECM is an important part of every tissue remodeling process, such as during the menstrual cycle.

While various members of the transforming growth factor (TGF) family of proteins promote processes like collagen synthesis, which lead to ECM growth, the matrix metalloproteinases (MMPs) play a central role in the degradation of the ECM (see Figure 1). This group of proteinases hydrolyses components of the ECM, including collagen, fibronectin, gelatin, elastin and laminin. Some MMPs are also known under other names, according to their substrate: for instance, collagenase 1 (MMP-1) or gelatinase B (MMP-9). An overview of the MMPs and their substrates is given in Table I [14,15]. MMPs are secreted as inactive pro-enzymes. Under normal physiological conditions, the activities of MMPs are controlled at three levels: transcription, activation of the pro-enzymes, and inhibition by their particular endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). For an overview of the TTMPs see Table II [14-16].

Figure 1. Tissue remodeling of the human endometrium during the secretory phase. Tissue remodeling and breakdown characterizes the secretory endometrium. Whereas a small-scale localized breakdown of the extracellular matrix is necessary to facilitate embryo implantation during the receptive phase, complete tissue breakdown takes place during menstruation. Both events are probably mediated by matrix metalloproteinase (MMPs).

The MMPs and TIMPs involved in endometrial remodeling during the menstrual cycle seem to be globally controlled by ovarian steroids, although not necessarily directly. This was demonstrated in several studies on the expression of mRNA for various MMPs and TIMPs throughout the menstrual cycle and by experiments using explant models of endometrial tissue. The expression of several MMPs was shown to be inhibited by progesterone and, in agreement with these findings, most MMPs had their maximum activity at menstruation, after progesterone withdrawal [17-19].

However, progesterone does not direcdy inhibit MMP expression, but uses at least one mediator. An endometrial factor has been discovered which regulates ECM breakdown by both inhibiting collagen synthesis and enhancing its degradation via stimulation of MMP production. This factor was initially called ‘endometrial bleeding- associated factor’ (EBAF), as it is found in greatest amounts during the late secretory and menstrual phases of the cycle [20]. It is now more commonly called ‘LEFTY-A’ or just ‘LEFTY’, as it is similar to a member of the TGFbeta family called ‘lefty’. The expression of LEFTY-A is inhibited by ovarian steroids, including progesterone, and the withdrawal of these hormones results in an increase of LEFTY- A production [21,22]. The main place of LEFTY-A synthesis seems to be the endometrial stromal cells, which have been found to retain their progesterone receptors throughout the cycle. Whereas other members of the TGFbeta family promote collagen synthesis and inhibit MMP production, LEFTY-A stimulates the production of MMPs and opposes the effects of other TGFbeta proteins. Figure 2 presents a model of the regulation of MMP activity by LEFTY [15,21-23].

Table I. Classification of human matrix metalloproteinases (MMPs) [14,15].

Table II. Human tissue inhibitors of metalloproteinases (TIMPs) [14-16].

There is increasing evidence that leukocytes also play an important role in the remodeling of the endometrium. The endometrial leukocytes include T and B cells, eosinophils, neutrophils, macrophages, mast cells and the characteristic uterine natural killer (NK) cells. Cyclical alterations in the number and type of leukocytes in the endometrial stroma throughout the menstrual cycle have led to the suggestion that they are closely involved with the remodeling processes in the endometrium. At the initiation of menstruation, leukocytes can comprise 40% of the total cellular content of the endometrium and most leukocytes peak at this stage. Uterine NK cells, on the other hand, form the majority of the leukocyte population in the late secretory phase and pregnancy. Mast cell distribution and numbers are not altered during the cycle, but the cells seem to be activated prior to menstruation [24,25].

Figure 2. Model of the regulation of matrix metalloproteinase (MMPs) during the menstrual phase of the endometrium [15-18]. During the secretory phase, progesterone stimulates the production of transforming growth factor-beta (TGFbeta) and MMPs are suppressed. The balance of these signals favors collagen synthesis. Upon progesterone withdrawal during the late secretory phase, or in abnormal bleeding conditions, LEFTY expression is upregulated. LEFTY suppresses TGFbeta production and induces MMP production and/or activity. This leads to collagen and tissue breakdown in the endometrium.

There is no doubt that leukocytes are somehow involved in the processes of proliferation and tissue breakdown in the endometrium described above, although it is not known exacdy where. Uterine NK cells express VEGF and other angiogenic factors, in addition to being found in close spatial association with spiral arterioles. Macrophages are also a source of VEGF, as well as expressing MMPs and producing a variety of regulatory molecules that could stimulate MMP production or activation in adjacent cells. Mast cells have also been implicated in the upregulation of MMPs. The number of endometrial leukocytes seems to be negatively regulated by progesterone, but there is no evidence for the expression of progesterone receptors, which suggests an indirect regulation of the endometrial leukocyte population by progesterone [24,25].

Based on the available information (Figure 2), Dong and colleagues suggested how MMPs, TIMPs and leukocytes may interact during different phases of the menstrual cycle. The remodeling of the endometrium is brought about by shifts in the balance between MMPS and TIMPs. A host of odier factors are most probably also involved in regulating menstruation, but little is known so far about their actions [22,24].

A complicated web of interactions such as that involved in the regulation of the menstrual cycle could easily be unbalanced by the lack, or overproduction, of even just one of its factors. Most of the information on the biochemical parameters briefly described above has indeed come from studies comparing normal endometrial tissue with that from women with menstrual disorders. LEFTY-A, which is normally transiendy expressed during menstruation, was found to be expressed much more strongly in women with abnormal bleeding [20,23]. It has been suggested that abnormal bleeding is caused by an overproduction of LEFTY-A, which leads to an overproduction of MMPs and ultimately to an untimely breakdown of ECM and blood vessel basement membranes. Another set of biochemical factors involved in menstruation has also been implicated in bleeding disorders; an imbalance between PGs with vasoconstrictive and vasodilating effects has been shown in some women with menorrhagia, as well as an increase in PG receptors [11,26]. An increase in the number of leukocytes compared with that prior to menstruation has also been observed in cases of abnormal bleeding and in women with breakthrough bleeding while using progestogen-only contraception [25].

Bleeding disorders associated with long-term progestin treatment

Irregular bleeding episodes are very common in women receiving long-term progestin treatment as part of HRT or progestin-only contraception, and they result in a high percentage of treatment discontinuations.

Histological and hysteroscopic studies showed dilated, thin- walled and fragile vessels in the superficial endometrial stroma in users of LNG or progesterone [27,28]. The cause of this increased vascular fragility is unclear, but there is evidence of disturbances in molecular function in the endometrium. A number of studies have analyzed molecular patterns in association with irregular bleeding episodes in women receiving long-term progestin treatment. Irregular dysfunctional bleeding episodes in users of LNG-only contraceptives were associated with focal breakdown of ECM, linked with locally restricted uncontrolled activity of several MMPs, combined with a decreased expression of their TIMPs [29,30]. An upregulation of MMP activity and a decrease in TIMPs has also been observed with MPA, although to a lesser extent than with LNG [31,32]. Intrauterine LNG- releasing systems have been reported to alter endometrial VEGF expression in association with aberrant arteriole differentiation. The number of bleeding episodes was correlated with VEGF levels [3].

Irregular endometrial bleeding is also a common problem in women receiving combined HRT. Studies by Hickey and associates [33,34] in women suffering from HRT-related irregular bleeding showed that, in this population, as well the structural integrity of endometrial blood vessels, MMP and TIMP expression were also altered in comparison with controls.

Abnormalities in the ultrastructure of endometrial blood vessels were also observed in women suffering from spontaneous abnormal uterine bleeding [13,28], as well as an increased expression of several MMPs and LEFTY-A [20,23]. Thus, an imbalance in angiogenesis- modulating factors, causing aberrant endometrial vessel differentiation, could be the common background to irregular bleeding.

There is evidence indicating differences in the effect of progesterone and various synthetic progestogens on endometrial blood vessels [32,35]. In order to test this hypothesis, the effect of several progestins (MPA, NETA and LNG), progesterone and estradiol on the expression of angiogenesispromoting VEGF and angiogenesis- inhibiting TSP-1 was studied in explants of endometrial epithelium. Estradiol, as expected, caused an increase in VEGF levels in endometrial glandular epithelium in vitro. Progesterone had no effect on VEGF expression. The three synthetic progestins, however, were found to increase VEGF expression to a greater extent than estradiol. Progesterone increased TSP-1 mRNA, estradiol had no effect on TSP-1 expression and the effect of the synthetic progestins was below that found with progesterone [36,37]. Mirkin and co-workers suggested that the progestins used in HRT may alter the balance between angiogenic promoters and inhibitors. These alterations could induce proangiogenic activity, with consequent aberrant vasculogenesis and irregular endometrial bleeding. Synthetic progestins are supposed to mimic the effect of the natural ovarian steroid progesterone, while showing more favorable pharmacokinetics. However, in addition to their progestational activity, they have a wide range of other biological effects. Litde is known about the molecular mechanisms involved, but it has been shown that some progestins, like NETA or MPA, not only bind to the progesterone receptor but also to other steroid receptors, while others, like DYD, do so to a lesser degree [38]. The findings of Mirkin’s group [37] are therefore not too surprising and they add interesting information to our currently limited knowledge of the non-progestational effects of synthetic progestins.

Progestogen treatment for reproduction

The reason for the remarkable endometrial differentiation is to create a favorable environment for pregnancy if the egg is fertilized. One of the crucial first steps in pregnancy is implantation of the blastocyst. There appears to be only a limited phase of endometrial receptivity, the ‘implantation window’, during which blastocyst implantation is possible. During a normal, spontaneous menstrual cycle in humans, early blastocyst implantation takes place at day 6 to 8 after ovulation. Several studies comparing human mid-secretory phase endometrium with non-receptive stages have failed to show a consistent pattern of ‘receptive state’ gene expression which could be used to characterize this phase. The implantation window ends shortly before menstruation, probably when the sinking levels of progesterone lead to irreversible preparations for menstruation, such as the LEFTY-A-mediated increase in MMP production [23,39].

The blastocyst ‘invades’ the endometrium by producing a number of different proteinases, among them MMPs. The same basic mechanisms that play an important part in menstruation and other tissue remodeling processes take place here as a dialogue between embryo and maternal cells. The action of the proteinases produced by the embryo seems to be opposed by maternal production of proteinase inhibitors, like TIMPs, which keep the dissolution of endometrial components under control. Also involved in this process are a number of cytokines, such as several members of the TGF family, but their roles are unclear. In general, cytokines active during this stage of pregnancy at the site of implantation are predominantly pro- inflammatory [39,40].

A successful pregnancy depends not just on the invasion of the blastocyst, but also on tolerance of the genetically incompatible embryo by the maternal immune system. Although pregnant women have antifetal, antiplacental and antipaternal antibodies, maternal recognition of fetal antigens does not appear to compromise pregnancy. Tolerance is thought to depend on the interactions of various cytokines produced by the endometrial and embryonic cells.

Cytokines can be grouped into those with proinflammatory effects (e.g. tumor necrosis factor-alpha, interleukin (IL)-1, interferon- gamma) and diose with antiinflammatory effects (e.g. IL-4, IL-10, TGFbeta). The lymphocytes producing these cytokines are accordingly grouped into T helper (Th) 1 cells, producing pro-inflammatory cytokines, and Th2 cells, producing anti-inflammatory cytokines. Pro- inflammatory cytokines have embryotoxic effects, whereas antiinflammatory cytokines permit tolerance of the embryo. The original dominance of pro-inflammatory cytokines at the site of blastocyst invasion is soon shifted toward more anti-inflammatory (Th2) cytokines produced by endometrial cells under the influence of progesterone. The term ‘Th1/Th2 shift’ is an oversimplification, as not only lymphocytes produce cytokines, but it is commonly used for this event. As in other regulating processes, progesterone works indirectly via at least one mediator. Lymphocytes carry receptors for progesterone, which are much increased in number during pregnancy. By binding to these receptors, progesterone stimulates the lymphocytes to produce progesterone-induced blocking factor (PIBF), which in turn promotes the production of anti-inflammatory cytokines [41,42].

Ovarian steroids, in this case mainly progesterone, are responsible for the overall control of these events but again, the finely balanced interaction of a great number of factors is necessary for a successful pregnancy. Most cases of infertility are attributable to some known pathology, but in about 10% of the cases the etiology remains unknown. It has been suggested that in these cases, an imbalance of factors involved in the intricate signaling processes during or after implantation might be the cause.

In a comparison of LEFTY-A mRNA expression in the endometrium of normal fertile women with that in infertile women, Tabibzadeh [23] demonstrated that LEFTY-A expression was low during the secretory phase of the cycle and increased only during menstruation in fertile women. In 50% of the infertile women, on the other hand, LEFTY-A was already upregulated well before menstruation, during the ‘implantation window’. Based on diese findings, Tabibzadeh suggested mat progesterone greatly suppresses the expression of MMPs during die implantation window in normal women. Progesterone withdrawal leads to LEFTY-A expression, which in turn promotes MMP secretion, thus initiating menstruation. If LEFTY-A is already highly expressed early during the secretory phase, the receptive phase of the endometrium is shortened or totally absent. Interestingly, the women with poorly regulated LEFTY-A expression were suffering from various forms of infertility, such as endometriosis, polycystic ovary, anovulatory cycle or luteal-phase defect, habitual abortion or unknown causes. Thus, overexpression of LEFTY-A seems to be a common event in infertility.

In another scenario, infertility could be due to early abortion caused by a lack of PIBF. Without PIBF, the shift towards anti- inflammatory cytokines, which is necessary if the embryo is to be tolerated by the maternal system, does not take place. Studies supporting this hypothesis showed that PIBF is expressed in the lymphocytes of pregnant women after nidation of the blastocyst and that PIBF levels are higher in fertile women than in women suffering from habitual abortion. Pregnant women with clinical signs of threatened abortion also had fewer PIBF-positive lymphocytes than healdiy pregnant women [42,42].

As progesterone is the dominating hormone during pregnancy, progestins have been used to treat infertility for a long time, with varying success. In an interesting ongoing study of DYD in habitual abortion, the cytokines characterizing the Thl/Th2 shift are actually used as an early marker for successful treatment [43].

Outlook

Recent clinical studies have raised concern about possible unexpected side-effects, in addition to irregular bleeding, of some progestins used in HRT, thereby highlighting the importance of choice of synthetic progestin. The two most frequently used progestins, MPA and NETA, seem to increase the risk of breast cancer and cardiovascular disorders when given in combination with estrogen during long-term HRT [44]. Clearly, it is not sufficient to study whether a progestin used in HRT is able to prevent endometrial hyperplasia; ideally, its effects should be restricted to a progestational action only.

Insights from molecular biology can provide more sophisticated markers for the effects, wanted or unwanted, of progestins in clinical studies, which could show abnormalities long before clinical unwanted events become evident. The results reported by Mirkin and colleagues [37] concerning the different effects of synthetic progestins on angiogenic and angiostatic factors point to one possibility. On a molecular level, a progestin that has exclusively progestational effects is one that binds highly selectively to the progesterone receptor. A number of studies have shown considerable differences in this respect between the various synthetic progestins, depending on which parent compound they are derived from and how they are metabolized. MPA and NETA, for instance, were found to bind not only to the progesterone receptor but, unlike progesterone itself, also exhibit agonistic activity on the androgen and glucocorticoid receptors. DYD, in contrast, appeared much more specific, showing little or no effect, very similar to progesterone. It is this androgenic effect that has been associated with the side-effects of some synthetic progestins [37,44].

Molecular markers could aid in the choice of the right progestin, i.e. the one that is most selective and has the least risk of side- effects. In the long run, a better understanding of the molecular biology behind processes in the endometrium will also lead to the discovery of new, more selective progesterone ligands, with fewer side-effects overall and, in particular, fewer effects outside the target organ or tissue. An interesting new class of substances in development, which may fit this description, is the selective progesterone-receptor modulators (SPRMs), which exert progesterone agonist, antagonist or mixed agonist/antagonist effects on various progesterone target tissues in vivo. Importantly, SPRMs show tissue- specific actions [45]. These substances, which do not necessarily have a steroidal structure, may well be the next generation of therapeutic progesterone receptor ligands. However, for some time to come we will continue using synthetic progestin as therapeutic agents. We would be well advised to make use of all the available information when choosing a progestin, in particular for long-term treatment. References

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RENE DRUCKMANN

12 rue de France, Nice, France

(Received 17 May 2007; accepted 5 September 2007)

Correspondence: R. Druckmann, 12 rue de France, F-06000 Nice, France. Tel: 33 49 3820608. Fax: 33 49 3162743. E-mail: renedruckman@aol.com

Copyright Taylor & Francis Ltd. Oct 2007

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