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Considerable correlative evidence suggests an important role for matrix metalloproteinases (MMPs) in menstruation, a process which occurs naturally in very few species. In this study, MMP expression was examined in a mouse model of endometrial breakdown and repair and the functional importance of MMPs determined. In the model, progesterone support was withdrawn from mice in which endometrial decidualization had been induced; 24 h later, endometrial breakdown was complete, and the entire decidual zone had been shed. Re-epithelialization had occurred by 36 h, and the endometrium had undergone extensive restoration toward a predecidualized state by 48 h. Immunoreactive MMP9 and MMP7 colocalized with leukocyte subsets, particularly neutrophils, whereas MMP13 staining was always extracellular. MMP3 and MMP7 were abundant during re-epithelialization in close proximity to newly reforming epithelium. The functional importance of MMPs in these processes was examined using two MMP inhibitors, doxycycline and batimistat. Both inhibitors effectively reduced MMP activity, as assessed by in situ zymography, but did not have significant effects on endometrial breakdown or repair. This study demonstrates that although MMPs are present in abundance during endometrial breakdown and repair in this mouse model, they are not the key mediators of these processes.

Human endometrium possesses a unique immunological environment enabling implantation of the semiallogeneic embryo. Large populations of macrophages and uterine-specific natural killer cells infiltrate the implantation site, believed to be important modulators of trophoblast invasion and decidualization. In the absence of pregnancy, there is a dramatic influx of neutrophils, eosinophils, and macrophages, likely to be critical for focal inflammatory endometrial destruction. However, little is known regarding selective recruitment of leukocyte subtypes. We employed a gene array approach to analyze the expression of 21 chemokines in endometrium. Real-time RT-PCR and immunohistochemistry was conducted to verify expression patterns and determine cellular source. Nine chemokines were highly abundant in human endometrium: monocyte chemotactic protein-3, eotaxin, fractalkine, macrophage inflammatory protein-1, 6Ckine, IL-8, hemofiltrate CC chemokine-1 and -4, and macrophage-derived chemokine. Chemokine mRNA was generally up-regulated during endometrial receptivity and early pregnancy, particularly of macrophage and natural killer chemoattractants. Chemokine protein was predominantly localized to epithelial glands, whereas differentiated stromal cells were a major source of chemokines after decidualization. This is the first study to use an unbiased approach to screen for endometrial chemokines, and we report the selective regulation of chemokines, corresponding to the recruitment of distinct leukocyte subpopulations required for pregnancy and menstruation. (J Clin Endocrinol Metab 89: 6155–6167, 2004)

A mouse model of menstruation1 was modified to evaluate the effects of long-term progestagens on the endometrium. Many women using long-acting progestin (P)-only contraceptives experience break-through bleeding (BTB) and this is the main reason for discontinuation of their use by 30% of users. To understand the mechanisms involved in BTB, ovariectomised mice treated with a strict steroid hormone regime, had etonogestrel- or levonorgestrel-releasing implants inserted subcutaneously. A decidualizing stimulus (oil) was administered into one uterine horn; decidualisation was maximal 48 h later, designated day 0 (d0). The implant was left in place for a further 45 days. Uteri were collected at 5-day intervals and analysed for morphological changes, leukocyte infiltration, and matrix metalloproteinase (MMP) expression. In uteri from mice treated with etonogestrel and examined at d0, decidualisation was maximal and very few leukocytes were present (neutrophils, NEU; Macrophages, Mac; uterine natural killer cells, uNK). Some loss of tissue integrity was seen at 5d, associated with some MMP-9 positive leukocytes. However, substantial stromal breakdown was observed at 10d and this progressed until 25d. A large infiltrate of NEU and uNKs, as well as increased MMP-9 expression, was observed at these times. At 25d there was evidence of re-epithelialisation and by 45d the tissue had fully repaired back to its pre-decidualised state. Re-epithelialisation was associated with a decline in NEU and uNKs, as well as MMP-9 positive immunostaining. The morphology of uteri from mice treated with levonorgestrel implants was compared to mice treated with etonogestrel. The changes observed were very similar with a loss of tissue integrity at 5d and tissue destruction at 10d. This mouse model mimics the changes in leukocyte populations and MMP expression observed in endometria of some women using P-only contraceptives. (1)Brasted M, White CA, Kennedy TG, Salamonsen LA. (2003) Biololgy of Reproduction 69(4), 1273–1280.

Strong correlative evidence supports a role for matrix metalloproteinases (MMPs) in the tissue breakdown at menstruation. As menstruation occurs in very few species besides women, there is a lack of suitable and easily accessible animal models available to examine the functional significance of potential key mediators of this process. A mouse model of endometrial breakdown and repair has been developed,1 which morphologically resembles that of human endometrium at menstruation. Previous studies in our laboratory showed that the expression patterns of various MMPs in the mouse model closely resembled those seen in the human.2 Administration of doxycycline, a broad spectrum MMP inhibitor, decreased gelatinase activity, but had no effect on tissue breakdown in this model. The aim of the present study was to further examine the importance of MMPs in endometrial breakdown and repair via administration of batimistat, a highly potent and specific MMP inhibitor. Batimistat was administered I.P to mice 24 h prior to the expected time of endometrial breakdown. The efficacy of batimistat within the uterus was proven using in situ zymography, which identifies MMP activity (rather than latent forms). This demonstrated that batimistat was reaching its target organ and effectively inhibiting MMP activities (both gelatinase and collagenase). Examination of gross uterine morphology revealed no apparent difference between groups, with batimistat treated uteri displaying a similar extent of tissue breakdown and repair to their control counterparts. Measurement of the breaking down area compared to total endometrial area revealed no difference between control and batimistat treatment, with the breaking down areas being 69 ±13% and 72 ± 9.8% of total endometrial cross-sectional area respectively. There was likewise no effect on endometrial repair. The results of this study together with our previous study using doxycycline, indicate that MMPs are not the key mediators of endometrial breakdown in this model. (1)Shen et al. (2004) Reprod. Fert. Dev. 16(Suppl), A265, p. 97.(2)Brasted et al. (2003) Biol. Reprod. 69, 1273.

Activin and inhibin subunits are co-expressed by human endometrial epithelial and decidualised stromal cells. Activin A is a potent stimulator of decidualisation in vitro, but the mechanisms are unknown. Matrix metalloproteases (MMPs) are known to be important during decidualisation, as administration of a broad spectrum MMP inhibitor in the rat results in reduced decidualisation. Transforming Growth Factor(TGF)-βs are closely related to activins and inhibit MMP production in endometrial epithelial cells. We hypothesised that activins regulate MMP production during decidualisation and/or trophoblast invasion. Epithelial and stromal cells were isolated from human endometrium and treated for 24 h with activin, inhibin, activin/inhibin, and follistatin. Media were collected and subjected to gelatin and caesin zymography. In epithelial cells, activin A stimulated the expression of latent forms of MMPs-1, -2, -7 and -9, and increased formation of active forms of MMPs-2 and -7. Cotreatment with inhibin prevented this stimulation, whilst inhibin alone completely inhibited MMP production. Treatment with follistatin treatment reduced MMP levels. Similar regulation was seen in stromal cells for MMPs-1, -2 and -9. These data show that activin stimulates the production and activation of MMPs in both endometrial cells, and that inhibin is a potent inhibitor. It is interesting that activin is acting in an opposing manner to TGF-β, indicating that these two closely related proteins have divergent signalling pathways in endometrial cells. Further, this is the first demonstration of a role for inhibin in regulating MMPs and indeed for inhibin action in the endometrium. These findings are of potential importance in understanding regulation of MMPs in the peri-implantation endometrium. Activin is the predominant dimer produced by decidual and epithelial cells, where it may be promoting decidualisation though enhancing MMP production and activation. Furthermore, activin secretion by invasive cytotrophoblasts may stimulate focal decidual MMP production promoting their invasion during embryo implantation.

Strong correlative evidence supports a role for matrix metalloproteinases (MMP) in the tissue breakdown at menstruation. Because menstruation occurs only in women and a few old-world primates, it has not been possible to examine the functional significance of potentially key mediators of this process. To this end, we developed a mouse model for menstruation (1), in which ovariectomised mice are subjected to a decidualising stimulus: injection of oil into the uterine lumen following appropriate hormone-priming. During the 24 h following withdrawal of progesterone (P), the decidualised tissue progressively breaks down, in a manner that morphologically resembles that of human endometrium at menstruation. The aims of the present study were to examine the pattern of MMP expression during the time from progesterone withdrawal until complete tissue breakdown, and to determine whether administration of doxycycline, (a known MMP modulator), 3 h prior to P withdrawal, affected the expression or activity of the MMPs or restrained the tissue destruction. MMP-3 was present at foci in the decidual zone: these were initially associated with the restructuring at decidualisation and subsequently with the tissue destruction. MMP-7 was detected both in epithelium and in leukocytes, predominantly neutrophils. These were first apparent in the basal zone during the earliest stages of tissue instability, and dramatically increased in numbers as breakdown progressed. MMP-9 was found only in leukocytes, predominantly neutrophils and some macrophages, with greatly increased numbers with time. Zymography revealed a dramatic increase in both latent and active MMP-9 as tissue breakdown proceeded. Doxycycline reduced immunoreactive MMP-3 but not MMP-7 or MMP-9 in the tissue, and also decreased gelatinase activity. However, no apparent effect on tissue breakdown was observed. Further studies with a more potent MMP inhibitor are required to fully establish the importance of MMPs in these processes. (1) Brasted et al. (2003) Biol. Reprod. 69, 1273.

At the time of implantation the endometrium becomes a specialised immune environment to allow implantation of the semi-allogenic embryo. A large population of leukocytes, predominantly uterine NK cells and macrophages, infiltrate the endometrium and are believed to modulate trophoblast invasion and facilitate decidualisation. Despite their importance, the factors responsible for recruiting these cells to the endometrium and inducing their activation/differentiation to the ‘uterine phenotype’ are unknown. In this study, a gene array approach was utilised to identify which chemokines (family of chemotactic factors for leukocytes) are expressed by the endometrium at the time of embryo implantation. We identified eight chemokines that were expressed at this time: monocyte chemoattractant protein (MCP)-3, eotaxin, macrophage inflammatory protein (MIP)-1β, fractalkine, 6Ckine, macrophage-derived chemokine (MDC), hemofiltrate CC chemokine (HCC)-1, and HCC-4. Real time RT-PCR was utilised to analyse their expression across the menstrual cycle and in early pregnancy. mRNA expression of MCP-3, 6Ckine, HCC-1, HCC-4, MDC and MIP-1β was upregulated in the mid-secretory phase, with high expression maintained by MCP-3, 6Ckine, HCC-4 and MDC in early pregnancy. Immunohistochemistry was conducted to examine protein production and cellular source. All chemokines were predominantly localised to the glandular and luminal epithelial cells and decidualised stromal cells. HCC-1, MDC and 6Ckine were upregulated in the secretory phase and early pregnancy whilst MCP-3 and MIP-1β were high throughout the cycle and early pregnancy. Immunostaining was also detected in infiltrating leukocytes for all chemokines, with maximal numbers of MDC-, HCC-1-, and MCP-3-positive leukocytes in early pregnancy. Furthermore staining for HCC1, 6Ckine and MDC was prominent in vascular endothelium. All of the identified chemokines possess chemotactic activity for monocytes/macrophages (HCC-1 and -4, MCP-3, MIP-1β) or NK cells (MCP3, MDC, MIP-1β), with the exception of 6Ckine which is a potent T cell chemoattractant. Chemokines expressed by the endothelium are likely to be involved in the initial recruitment of leukocytes from vessels, whilst chemokines produced by epithelial and stromal cells could be more important for the positioning and activation of leukocytes within the endometrium. This data supports a role for these chemokines in the establishment of pregnancy through the recruitment of pregnancy-associated leukocytes.