Robert M. Brenner

The endocrinology of menstruation – a role for the immune system

The human endometrium displays characteristic features, both structural and functional, across the menstrual cycle. It is the sex steroid hormones, oestrogen and progesterone, that drive the endometrium through the different phases of the cycle. Oestrogen and progesterone act sequentially to regulate cellular concentrations of their respective receptors, this interaction initiates gene transcription. Thereafter a cascade of local events prepares the endometrium for implantation, but in the absence of pregnancy, progesterone withdrawal leads to menstruation and cyclic repair. Withdrawal of progesterone from an oestrogen-progesterone primed endometrium is the initiating event for the cascade of molecular and cellular interactions that result in menstruation. Progesterone withdrawal first affects cells with progesterone receptors. Early events in the menstrual process are vasoconstriction and cytokine up-regulation. The activation of lytic mechanisms is a later event and involves cells that may lack progesterone receptors, for example, uterine leucocytes and epithelial cells. Hence progesterone withdrawal results in a local increase of inflammatory mediators and the enzymes responsible for tissue breakdown. The total complex of local factors implicated in normal menstrual and aberrant menstrual bleeding are yet to be fully defined.

Antiproliferative effects of progesterone antagonists and progesterone receptor modulators on the endometrium

Progesterone antagonists (PAs, antiprogestins) can modulate estrogenic effects in various estrogen-dependent tissues. These modulatory effects are complex and depend on species, tissue, type of compound, dose, and duration of treatment. In non-human primates, PAs, including mifepristone, ZK 137 316 and ZK 230 211, inhibit endometrial proliferation and induce amenorrhea. When administered chronically at relatively low doses, these compounds block the mitotic activity of endometrial epithelium and induce stromal compaction in a dose-dependent manner in both spayed and intact monkeys at high estradiol concentrations. These effects were accompanied by an atrophy of spiral arteries. The antiproliferative effects were endometrium-specific, since the estrogenic effects in the oviduct and vagina were not inhibited by PAs. Similar endometrial antiproliferative effects were also found after treatment with the progesterone receptor modulator (PRM), mesoprogestin J1042. The endometrial antiproliferative effects of PAs, particularly within the endometrial glands, were also observed in spayed rabbits. In spayed rats, however, the PAs did not inhibit, but rather enhanced, various estrogen responses, including endometrial proliferation, pointing to species-specific differences. In conclusion, our studies indicate that both pure PAs and PRMs selectively inhibit estrogen-dependent endometrial proliferation in the primate endometrium without affecting estrogenic response in other estrogen-dependent tissues or inducing unscheduled bleeding. Our studies indicate that the spiral arteries, which are unique to the primate endometrium, are the primary targets that are damaged or inhibited by PAs and PRMs. The damage to these unique vessels may underlay the paradoxical, endometrium-specific, antiproliferative effects of these compounds. Hence, the properties of PAs and PRMs (mesoprogestins) open up new applications in gynecological therapy and hormone replacement therapy.

VEGF blockade inhibits angiogenesis and reepithelialization of endometrium

Despite extensive literature on vascular endothelial growth factor (VEGF) expression and regulation by steroid hormones, the lack of clear understanding of the mechanisms of angiogenesis in the endometrium is a major limitation for use of antiangiogenic therapy targeting endometrial vessels. In the current work, we used the rhesus macaque as a primate model and the decidualized mouse uterus as a murine model to examine angiogenesis during endometrial breakdown and regeneration. We found that blockade of VEGF action with VEGF Trap, a potent VEGF blocker, completely inhibited neovascularization during endometrial regeneration in both models but had no marked effect on preexisting or newly formed vessels, suggesting that VEGF is essential for neoangio‐genesis but not survival of mature vessels in this vascular bed. Blockade of VEGF also blocked reepithelialization in both the postmenstrual endometrium and the mouse uterus after decidual breakdown, evidence that VEGF has pleiotropic effects in the endometrium. In vitro studies with a scratch wound assay showed that the migration of luminal epithelial cells during repair involved signaling through VEGF receptor 2‐neuropilin 1 (VEGFR2‐NP1) receptors on endometrial stromal cells. The leading front of tissue growth during endo‐metrial repair was strongly hypoxic, and this hypoxia was the local stimulus for VEGF expression and angio‐genesis in this tissue. In summary, we provide novel experimental data indicating that VEGF is essential for endometrial neoangiogenesis during postmenstrual/postpartum repair.—Fan, X., Krieg, S., Kuo, C. J., Wiegand, S. J., Rabinovitch, M., Druzin, M. L., Brenner, R. M., Giudice, L. C., Nayak, N. R. VEGF blockade inhibits angiogenesis and reepithelialization of endometrium. FASEB J. 22, 3571–3580 (2008)

Selective Progesterone Receptor Modulators (SPRMs)

Abstract: Endometriosis, the presence of endometrial tissue outside the uterus, is a progressive, estrogen‐dependent disease and occurs nearly exclusively in menstruating women of reproductive age. Pain syndrome, however, represents the major clinical problem of this disease, manifested as dysmenorrhea, pelvic pain, lower abdominal pain, and dyspareunia. The manifestation of the disease, that is, the pain syndrome, rather than the disease itself currently represents the major indication for both the medical and surgical therapies of endometriosis. The major drawbacks of current medical therapies of endometriosis are sometimes severe side effects. In this review, selective progesterone receptor modulators (SPRMs, mesoprogestins) as a potential therapeutic concept in endometriosis are discussed. Due to endometrial selectivity and favorable pharmacological profile, SPRMs may have advantages over the current medical treatments of this disease. Other emerging therapeutic approaches for this disease are also mentioned.

Regulation of human endometrial function: mechanisms relevant to uterine bleeding

This review focuses on the complex events that occur in the endometrium after progesterone is withdrawn (or blocked) and menstrual bleeding ensues. A detailed understanding of these local mechanisms will enhance our knowledge of disturbed endometrial/uterine function – including problems with excessively heavy menstrual bleeding, endometriosis and breakthrough bleeding with progestin only contraception. The development of novel strategies to manage these clinically significant problems depends on such new understanding as does the development of new contraceptives which avoid the endometrial side effect of breakthrough bleeding.

Premenstrual and menstrual changes in the macaque and human endometrium: relevance to endometriosis.

Abstract: According to current theory, endometriosis is initiated during retrograde menstruation when menstrual fragments flow out of the fimbriated end of the fallopian tubes and become established on the ovarian surface or other sites in the peritoneal cavity. In recent years, new data have accumulated on the properties of menstruating tissue itself, and several laboratories agree that this tissue is rich in matrix metalloproteinases (MMPs) that may facilitate endometriotic implantation. Recently, we found that vascular endothelial growth factor (VEGF) and its receptor VEGFR‐2 (KDR) were dramatically upregulated in the stromal cells of the superficial endometrial zones by progesterone (P) withdrawal during the premenstrual phase. A unique role of VEGF at this stage of the cycle may be to stimulate MMP expression in stromal cells because VEGF, KDR, and MMPs were all coordinately induced in these cells in the superficial zone of the primate endometrium by P withdrawal. The rich content of MMPs and VEGF in the menstrual fragments could facilitate attachment and angiogenesis of menstrual fragments in ectopic sites. In addition, a variety of chemokines, cytokines, and cellular regulators are induced by P withdrawal in the premenstrual human endometrium. These include NFκB, prostaglandins, interleukin‐8 (IL‐8), cyclooxygenase‐2 (COX‐2), and monocyte chemotactic peptide‐1 (MCP‐1), among others. The perivascular expression of several of these factors may facilitate the rapid invasion of leukocytes into the endometrium, especially in the superficial zones. Consequently, menstrual fragments may be rich in IL‐8 and MCP‐1, both of which would add to the angiogenic potential of such fragments in ectopic sites. In sum, menstrual tissue is rich in VEGF, KDR, MMPs, leukocytes, chemokines, cytokines, and prostaglandins, all factors that may facilitate attachment and angiogenesis when menstrual fragments exit from the tubes and implant on pelvic sites. Additional research on these and other factors in premenstrual and menstrual endometrium may deepen our understanding of both the establishment and progression of this debilitating disease.

In vitro fertilization and embryo transfer in primates

This volume contains the proceedings of a symposium which provides an in-depth analysis of current IVF-ET technology as applied to nonhuman primates. Although IVF-ET is now considered standard treatment for several categories of human infertility, a nonhuman primate model remains highly desirable for the study of reproductive and developmental processes, as well as for the establishment of improved disease models for medical research and the preservation of endangered nonhuman primate species. A better understanding of the primate reproductive system is thus essential to facilitate a global effort to control human population and to aid childless families attempting to overcome their infertility.

Antiprogestins as a model for progesterone withdrawal

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-kappaB, 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.

Analysis of monomeric-dimeric states of the estrogen receptor with monoclonal antiestrophilins.

We studied the antibody-combining properties of 3 forms of the estrogen receptor found in buffers of high ionic strength. Shifts to a faster sedimenting peak on sucrose gradients or a faster eluting peak on a gel filtration column with antibody addition allowed us to determine whether a given form contained one, two or more antibody-binding sites. The monomeric cytosolic estrogen receptor, ERC, contained one antibody binding site for each of 2 monoclonal antiestrophilins (H222 and H165, provided by Abbott Laboratories). Both the heat-transformed cytosolic estrogen receptor, ERC*, and a major fraction of the estrogen receptor extracted from nuclei, ERN, contained two sites for H165, but only one for H222. A minor fraction of ERN had only one site for each antibody. The kinetics of transformation of ERC to a species with two H165 binding sites were appropriate to a dimerization of ERC*. Addition of H222, but not H165, before the onset of the heat-induced transformation blocked the formation of ERC to ERC. These data suggest that ERC* and a major form of ERN are comprised of two immunologically similar subunits identical to ERC. Also, the antigenic determinant for H222, but not H165, appears to be located close to the dimerization domain. The minor form of ERN appears to contain an altered or dissimilar subunit.

Progesterone-mediated suppression of estradiol receptors in cynomolgus macaque cervix, endometrium and oviduct during sequential estradiol-progesterone treatment

We used sequential treatment with implants of estradiol (E2) and progesterone (P) to create varied hormonal states in a group of spayed cynomolgus macaques. The reproductive tracts were removed, and nuclear and cytosolic estrogen receptors were analyzed in the cervical mucosa, endometrium, and oviducts. Nuclear receptor quantities were greater in tissues of E2-treated monkeys than in tissues of spayed animals. Sequential P treatment, even in the presence of continuous E2, decreased the amounts of nuclear and cytosolic E2 receptors. In the oviduct and endometrium, the P-mediated suppression of receptors occurred within 1 or 2 days. In the cervix, suppression occurred only if the serum P:E2 ratio was elevated to twice the amount (approximately 100:1) usually found during the luteal phase of the menstrual cycle (approximately 50:1) in this species. Of these three reproductive tract tissues, the cervix had the highest threshold for suppression by P of E2 receptors in the presence of E2.