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Contraceptive Research and Development: Looking to the Future (1996)

Chapter: Appendix A: Female Methods

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Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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Appendixes

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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A: Female Methods

Horacio Croxatto, M.D.

Instituto Chileno de Medicina Reproductiva, Santiago

Michael Harper, Ph.D., Sc.D.

Center for Reproductive Medicine, Baylor College of Medicine

Donald McDonnell, Ph.D.

Department of Pharmacology, Duke University Medical Center

Wylie Vale, Ph.D.

The Clayton Foundation Laboratories for Peptide Biology,

The Salk Institute, La Jolla, Calif.

Introduction

The advent of modern molecular and cell biology has permitted a detailed look at the regulation of ovarian secretory function, follicular and oocyte maturation, and ovulation. This, in turn, has disclosed targets within the ovary which, at the current time, appear to have the greatest potential for leading to the development of a deliverable contraceptive within the next 10 to 15 years.

Interference With Prefertilization Events

General

Oocyte maturation and ovulation are coordinated by a series of cascading signals involving the brain, pituitary, and gonads. Fertility in mammals is modulated by multiple factors including length of day, availability of food, exposure to stressors, illness, presence or evidence of potential mates and competitors, and breastfeeding. The final common pathway for the effects of this external and internal sensory information on fertility is the collection of GnRH (gonadotropin-releasing hormone)-producing neurons in the hypothalamus that provide this neuropeptide to the anterior pituitary gland via a local vascular connection. The

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×

pituitary gonadotropes respond to GnRH by secreting the gonadotropins, LH (luteinizing hormone) and FSH (follicle-stimulating hormone); these act in concert with each other and with many other local ovarian factors to control steroidogenesis and release of mature oocytes. Each regulatory component is a potential target for interfering with oocyte maturation and ovulation and hence fertility. Among possible contraceptive targets are receptors and transcriptional regulators for GnRH, activin, inhibin, gonadotropins, and intragonadal paracrine/autocrine factors; however, much basic and applied work is still needed to generate specific pharmacologic tools for controlling these molecules.

Brain

Contraception can be achieved by preventing production of GnRH, a strategy that would only require steroid replacement at a physiologic level with potential health benefits. The maintenance of GnRH expression may be dependent upon tissue-specific transcription factors which, if sufficiently restricted, could be blocked pharmacologically. GnRH action can be prevented by synthetic analogues or specific antibodies. The GnRH-producing cells in the hypothalamus secrete this peptide in a rhythmic fashion that is critical for normal gonadotropin secretion and work is under way to determine the cellular and molecular basis of the ''GnRH pulse generator." Pharmacologic disruption of pulse parameters can have differential effects on the secretion of the two pituitary hormones, FSH and LH; thus, it may be possible to selectively disorganize gametogenesis. Superimposed upon the GnRH pulse generator are numerous neural and hormonal inputs mediated by monoamines, neuropeptides, prostaglandins, nitric oxide, sex and adrenal steroids, thyroid hormones, cytokines, and peptide growth factors that stimulate or inhibit GnRH production. Some of these could be targets for contraception, provided that cell-type-specific drugs could be developed.

Hypothalamus and Pituitary

The production of both LH and FSH is dependent upon receipt of periodic pulses of GnRH from the hypothalamus. GnRH binds to the GnRH receptor, a serpentine, G protein-coupled receptor, and induces its second messengers. The exposure of the pituitary to persistently high levels of GnRH or to potent longacting agonist analogues (superagonists) results in an initial stimulation that is followed by desensitization secondary to receptor down-regulation and attenuation of receptor signal transduction.

Prolonged superagonist administration suppresses both LH and FSH secretion leading to hypogonadism. Superagonists are now on the market for several indications-including precocious puberty and the treatment of hormone-dependent neoplasias and dysplasias. These agents inhibit fertility in females and males, reduce steroid production, and induce postmenopausal symptoms. Thus,

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×

any continuous use of superagonists as contraceptives will necessitate steroid replacement therapy.

An alternative approach, involving agents that bind to the GnRH receptor, is to inhibit gonadotrope functions with GnRH receptor antagonists. These compounds have the advantage of producing an immediate suppression of gonadotropin secretion, thereby avoiding transient stimulation of the gonadotropes, gonads, and steroid-dependent tissues. However, much higher doses of the presently available antagonists must be delivered than in the case of the agonists. The development of potent, orally active antagonists would provide a means of reversibly suppressing gonadal functions. Screening of chemical and microbiological libraries with high throughput GnRH receptor assays may provide leads for further optimization. These antagonists, given with steroid replacement, should be very effective contraceptives in both women and men. Furthermore, because GnRH antagonists do not transiently stimulate sex hormone production, they would be more appropriate than agonists for the treatment of steroid-dependent neoplasia and dysplasia. The recent development of tissue-specific steroids raises the possibility that new steroids might be found that are specific for GnRH cells of the hypothalamus or pituitary gonadotropes.

The selective regulation of pituitary FSH is achieved physiologically by the interplay of GnRH, gonadal steroids, and peptide hormones/growth factors. There are reports in the literature of the existence of a small peptide, FSH-releasing factor (FRF), that acts at the pituitary level to stimulate FSH but not LH. If such a putative factor were identified and found to be physiologically necessary for normal FSH production, then blockers of this peptide would suppress fertility. Activin, a dimeric peptide growth factor produced locally within the pituitary, is probably the key trophic factor maintaining expression of FSH. Activin has little effect on LH production in most systems. Two inhibitors of activin have been identified, inhibin and follistatin. Inhibin, a heterodimeric protein structurally related to activin (they share a common subunit) blocks the responses of some (but not all) cells to activin; follistatin binds to activin and bioneutralizes it. Both proteins reduce the production of FSH in animals and suppress follicular development. Inhibin is secreted by the ovary under FSH stimulation and provides a negative feedback signal that shuts off further secretion of that pituitary hormone; follistatin is produced locally and serves to limit all effects of activin. The binding and signaling receptors for activin have been cloned and it may be possible to develop small molecules that would interfere with these functions. The way inhibin blocks activin is currently unknown, but studies of this process may provide insight for developing inhibin-mimetics. Because inhibin only suppresses a subset of activin effects, such drugs may be relatively specific to suppression of reproduction (DePaolo et al. 1991; Vale et al. 1994, 1990).

The finding that activin and inhibin can uncouple the transcription of FSHß from that of LHß provides evidence for the existence of distinct intracellular regulatory pathways for the two proteins that may be exploitable.

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×
Follicular Development

A unique organ, the ovary is made up of hundreds of thousands of primary follicles that die during the lifetime of a female. Of the 400,000 follicles found in the human female at puberty, only about 400 will ever make it to ovulation.

From before birth, and throughout the reproductive years, small cohorts of follicles start growing continuously, one after the other, out of the pool of primary follicles. The vast majority undergo atresia after reaching different stages of growth. Beginning with puberty, growing follicles are periodically subjected to a process of recruitment and selection, from which a dominant follicle emerges in each menstrual cycle. This follicle proceeds to grow to maturity, a condition that makes it responsive to the ovulatory stimulus. The fact that a single follicle is selected to complete the process of maturation in each menstrual cycle is remarkable. This dominant follicle survives the other partners of its cohort, which enter atresia, and it proceeds to ovulation. It has been suggested that this process is regulated in part both by hormonal gradients within the ovary and by the ability of the developing follicle to respond to these signals by virtue of its physical location. However, the fidelity of the system and the usual outcome, that is, the production of one ovum, suggests that follicle selection is a tightly controlled process, the molecular basis for which has yet to be determined.

It has been established that most ovarian cell turnover occurs as a consequence of programmed cell death or apoptosis, an important cellular process by which superfluous or unwanted cells are deleted from an organism during tissue remodeling and differentiation. Although not identical in all cells, it has received much attention recently as a consequence of the identification of several transcription factors involved in regulating the process and the definition of the external stimuli that modulate the function of these proteins. Within the ovary, several substances have been shown to modulate the rate of cellular apoptosis by acting as follicular survival factors or mediators of cell death. From among the factors identified thus far, gonadotropins, steroid hormones, cytokines such as IGF-1, and interleukins seem likely to be important (Artini et al. 1994; Erickson and Danforth 1995).

Modulation of Ovarian Follicle Apoptosis as a Potential Contraceptive Approach

Follicular atresia is a well-regulated apoptotic event and not the result of cell necrosis. The ovary is a unique tissue with massive cell death throughout reproductive life. As suggested above, more than 99 percent of ovarian follicles endowed at early life are destined to undergo apoptosis. Based on extensive literature dealing with follicle selection and ovulation, as well as recent analysis of follicle apoptosis and follicle recruitment, one can propose a multistep model for the life cycle of ovarian follicles (Figure A-1).

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×

Figure A-1

Life cycle of ovarian follicles. FSH = follicle-stimulating hormone; FSHR = follicle-stimulating hormone receptor; LH = luteinizing hormone; and LHR = luteinizing hormone receptor. Source: Prepared for this report by Aaron Hsueh.

At early stages of ovarian development, a fixed number of primordial follicles are endowed in the ovary. Later on, no mitosis of germ cells can be detected in the ovary, and gradual depletion of the follicle pool begins as subpopulations of follicles initiate growth. As they reach the early antral stage, all follicles undergo degeneration through apoptosis. However, after the activation of the hypothalamic-pituitary axis at puberty, circulating gonadotropins (mainly FSH) act as survival factors to prevent the demise of a small subgroup of early antral follicles (Hsueh et al. 1994). From these, a dominant follicle is selected for final maturation and ovulation.

Throughout reproductive life, the cyclic process of follicle recruitment, atresia selection, and ovulation continues until the follicle pool is exhausted around the time of reproductive senescence (or menopause in women). Ovarian cell apoptosis in early antral follicles, before their final selection into preovulatory ones, represents a unique stage for contraceptive intervention. Recent studies have demonstrated that gonadotropins, estrogens, growth hormone, growth factors (IGF-I, EGF/TGFß, basic FGF), a cytokine (interleukin-1), and nitric oxide act in concert to ensure the survival of preovulatory follicles. In contrast, androgens, interleukin-6, and gonadal GnRH-like peptide are apoptotic factors. The selection of these follicles and their continuing maturation can be blocked by treatment with antagonists for the follicle survival factors or by increasing the levels of apoptotic factors. Promising candidates include the FSH blockers, such as deglycosylated FSH antagonists, and the extracellular fragments of FSH re-

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×

ceptors that may serve as a neutralizing binding protein. These agents would act in an ovary-specific manner. It should be realized, however, that this approach must be complemented with physiologic replacement therapy, since preventing the emergence of the dominant follicle would probably lead to hypoestrogenism. The fact that all but one follicle within a selected cohort degenerates indicates that selective regulation of atresia within the ovary actually occurs. Understanding the molecular events that control follicle selection could have a major impact on contraceptive research.

Disruption of the Ovulatory Process

The ovulatory process comprises several coordinated changes that occur in mature follicles, triggered by the ovulatory stimulus (the gonadotropin surge). They include resumption of meiosis, cumulus expansion with detachment from the mural granulosa, onset of luteinization expressed as a new steroidogenic pattern, and the collagenolitic cascade that leads to rupture of the follicle wall and release of follicle contents. Although all these events are triggered by the same stimulus, they can be dissociated by specific pharmacologic interventions. It is therefore possible to prevent follicular rupture without interfering with luteinization and steroidogenesis. In other words, although the oocyte is prevented from leaving the follicle to be fertilized, at the same time the normal hormonal oscillations of the menstrual cycle are preserved. Recent data indicate that LH uses at least three signal transduction pathways to produce follicular rupture: CAMP-dependent protein kinase, the protein kinase C, and the calcium/calmodulin-dependent protein kinase II pathway (Kugu et al. 1995). In addition, the LH surge induces the expression of the prostaglandin synthase 2 gene (PGS-2), which codes for an enzyme whose activity is essential for follicular rupture. This enzyme could be selectively inhibited, eliminating ovulation without blocking luteinization and the synthesis of steroid hormones (Morris and Richards 1993, 1995). Luteinizing hormone induces prostaglandin endoperoxide synthase-2 and luteinization in vitro by A-kinase and C-kinase pathways (Sirois 1995).

It should also be possible to intervene with the onset of meiosis, advancing it to such a point that, by the time the oocyte is released, it is no longer fertilizable. Meiotic cell division is a process, unique to the gonads, which is designed to permit the exchange of genetic material between maternal and paternal DNA. While this process of what is essentially the creation of genetic diversity is tightly regulated, because of its unique mode and site of action it has potential as a realistic target for contraceptive intervention. Based on work in mammalian cells, as well as in species as diverse as Drosophila and Xenopus, it has become clear that meiotic and mitotic cell division and the way they are regulated are sufficiently distinct to assume that process-specific regulatable targets can be identified. One point of intervention might be the regulation of progression of primary oocytes from prophase I to metaphase II (Grigorescu et al. 1994). The

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×

oocyte contained in each follicle rests in an animated state, arrested in the prophase I of the first meiotic division, until the follicle matures and receives the ovulatory stimulus. The ovulatory stimulus has the peculiar property of stimulating resumption of meiosis only of the oocyte contained in the mature follicle. The processes that maintain the oocyte in a quiescent stage for between 12 and 50 years remain largely unknown. Fully grown oocytes resume meiosis when they are separated from granulosa cells or when isolated cumulus-oocyte complexes are stimulated with FSH. This has led to the concept that the cell layer surrounding the oocyte produces a substance—oocyte maturation inhibitor (OMI)—which is inhibitory, and that the ovulatory stimulus releases this inhibition and adds a stimulatory factor for germinal vesicle breakdown (GVB), a prerequisite for completion of meiosis. Purines produced by follicular cells and transmitted to the oocyte via gap junctions are known to be involved in preventing resumption of meiosis (Downs 1993).

Unusual sterols present in human follicular fluid and bovine testis have been shown recently to activate mouse oocyte meiosis in vitro and are believed to play a crucial role in the resumption of meiosis in mammals (Byskov et al. 1995). It is likely that synthetic analogues may be efficient both as agonists and as antagonists for pharmacologic manipulation of the onset of meiosis. An intracellular factor that regulates the transition from first meiotic prophase to metaphase II—maturation promoting factor (MPF)—has been identified and determined to be a heterodimer comprised of the regulatory subunit cyclin B and the catalytic subunit, the cyclin-dependent protein kinase p34cdc2 (Dorce 1990).

These concepts introduce two specific areas that must be investigated further, one to define the precise chemical messenger(s) responsible for keeping the oocyte in the dormant state, the second to define the events responsible for the termination of dormancy and the resumption of meiosis. There are, of course, specific concerns about intervention in the process of meiosis. Most of these deal with the possibility of consequences from interfering with the process of genetic exchange and any effects on an ovum or resulting fetus were an oocyte to escape regulation by a pharmaceutical agent. Although these are valid considerations, they are premature until such time as the events that regulate the meiotic process are defined.

Granulosa Cell-specific Targets

The granulosa cell layer is the innermost lining of the follicle wall and surrounds the oocyte. Under the influence of FSH, granulosa cells proliferate and produce estradiol. It is likely that targeting the FSH receptor (different ways to achieve this are discussed in Appendix B) would be useful in interrupting the process of follicular development. However, the ensuing decrease in circulating estrogens on a long-term basis would be contraindicated, owing to the important role of estrogen in regulating the processes responsible for maintenance of bone

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×

mass and cardiovascular tone, and in regulating vasomotor function (Barzel 1988), so that FSH receptor blockers would have to be given in conjunction with steroid replacement therapy. The new generation of tissue-selective steroid receptor agonists, for example, raloxifene, may be available for use in contraception.

It is not clear that blocking FSH actions in the granulosa cell has to result only from a pharmacological action at the receptor. It is possible that an understanding of the intermediate steps from the cell surface to the nucleus, and of the genes within the ovary that respond to this hormonal stimulus, may permit the development of specific agents that uncouple the synthesis of estrogen from other gene products required for follicular development.

Ovary-specific Gene Transcription

One of the most exciting areas of reproductive research has to do with whatever is involved in determining the expression and role of novel hormone-dependent transcription factors within the ovary, including follicular and corpus luteum cells and the oocyte. In the last 10 years, the cloning and characterization of the steroid hormone receptors has been accomplished and has assisted in our understanding of the mechanism of action of the reproductive hormones estrogen and progesterone (Evans 1988). The specific impact of these observations will be discussed below. Emanating from this research was the unexpected finding that the family of proteins to which the steroid hormone belongs comprises up to 50 members that are related structurally but, for many of which, no ligands have been identified (O'Malley 1991).

These "orphan receptors" are so structurally related to those of the steroid receptors that it is considered likely that some, if not all, will be found to have an endogenous ligand. This contention has received some support from the discovery in 1992 of a ligand for the orphan nuclear receptor RXR (Heyman et al. 1992). This ligand is 9-cis retinoic acid, a metabolite of retinoic acid. Since then, progress has been slow in defining specific ligands for other such receptors, although agents that regulate their biological activity indirectly have been identified. In this category, the identification of a nuclear receptor that responds to the addition of farnesyl pyrophosphate (FAR) (Forman et al. 1995) and a family of related receptors (peroxisome proliferator-activated receptors [PPAR]) that are transcriptionally regulated by fatty acid derivatives, are intriguing examples of an emerging new endocrinology (Kliewer et al. 1992). Of particular relevance to reproduction is the recent discovery that the transcriptional effects of melatonin, a hormone intimately involved in reproduction, are manifested through the orphan receptor RZR (Becker-Andre et al. 1994). A combination of melatonin and a synthetic progestin is being studied as a novel type of oral contraceptive preparation.

It is possible that the slow pace of the discovery of ligands for additional orphan receptor members of this subfamily may indicate that they do not, in fact,

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×

have ligands but are regulated in other ways. This contention is supported by a series of independent findings that indicate that some orphan receptors can be activated by membrane depolarization, as well as by agents such as dopamine and nerve growth factor that act at the cell surface (Power et al. 1991; Lydon et al. 1992). Nevertheless, the recent discovery that the orphan receptor PPARy is the target for the biological efficacy of the thiazolidinediones, a class of drugs used for the treatment of non-insulin-dependent diabetes, indicates that these receptors are realistic pharmaceutical targets (Lehmann et al. 1995). The discovery of this compound, which binds to PPARy with high affinity and permits it to activate target gene transcription, is an important result from a pharmaceutical perspective. It demonstrates that, without knowing whether or not an orphan receptor has an endogenous ligand, it can be regulated by a synthetic pharmaceutical in a manner that impacts on a relevant biological process. This paradigm is likely to be reiterated with other orphan receptors, some of which may be relevant to oocyte maturation.

More research into the role of nuclear hormone receptors in the regulation of the transcriptional program within the oocyte and in the ovary is needed. The recent finding that the nuclear receptor germ cell nuclear factor (GCNF) is restricted in expression to developing gametes and is detectable in all stages of oocyte development justifies this contention (Chen et al. 1994). Although a direct link between its expression and function has not yet been established, GCNF's unique pattern of expression would imply a critical function. Efforts to identify the target genes that are responsive to GCNF, as well as identification of an endogenous ligand or a synthetic molecule that could regulate its transcriptional activity, will be required at a minimum to evaluate its potential role as a target for contraception. In addition to GCNF, the nuclear receptor SF-1 (steroidogenic factor1) may also play a key regulatory role in gametogenesis (Ikeda et al. 1994; Lala et al. 1992; Luo et al. 1994). This receptor, identified initially as a positive transcriptional regulator of steroidogenic enzymes, has now been determined to have a much more central effect on gonadal development.

Although a ligand has not yet been identified, there have been significant advances in our understanding of the biology of SF- 1. In recent published experiments in which the SF-1 protein has been genetically disrupted in mice, it was determined that the ensuing progeny failed to develop gonads and their pituitary lacked gonadotropes. The mechanism by which SF-1 may exert its regulatory activities is just beginning to become clearer. The gene-encoding Mullerian inhibiting substance (MIS), a cytokine required for male reproductive tract development, is positively regulated by SF-1 (Shen et al. 1994). All these examples illustrate the impact of basic nuclear factor research on our understanding of the molecular events underlying the development of reproductive capacity.

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×
Steroid Hormone Receptors

In addition to more attention to new targets in the ovary, much progress will be made by taking a fresh look at targets that are already established. In particular, the actions of steroid hormones at the gonadal/pituitary/hypothalamic axis, a well-defined series of targets acted upon by existing oral contraceptives, could advance to a new level by developments in our understanding of the steroid hormone receptor signal transduction pathways. The cloning of the estrogen and progesterone receptors and the ability to use these tools to dissect the progesterone- and estrogen-mediated signal transduction pathways indicate that, in addition to the receptor, its environment impacts on the biological efficacy of the hormone.

A recent and unexpected finding is that different ligands induce distinct alterations in receptor structure, the consequence of which is that these ligands can then promote unique sets of protein-protein interactions and subsequently regulate gene transcription in a differential manner (Allan et al. 1992). One of the most striking examples of this process is the observation that estradiol and tamoxifen, ligands for the estrogen receptor, induce distinct conformational changes within the receptor, with distinct biological consequences in vivo (McDonnell et al. 1995; Tzukerman et al. 1994). In this instance it has been shown that tamoxifen functions as an antagonist of the estrogen receptor in the breast and as an agonist in bone and in the cardiovascular system. The splitting by tamoxifen of the desirable and undesirable effects of estrogen receptor activation reinforces the notion that tissue- or process-specific steroid receptor modulators can be developed. Exploitation of these observations should lead to the generation of novel compounds with improved therapeutic efficacy.

Support is needed for basic studies of the hormones and growth factors, their receptors, and intracellular signaling mechanisms mediating the neuroendocrine and gonadal regulation of reproduction. Strategies for interfering with this regulation can target critical molecules for blockade or inappropriate expression through development of orally-active or very long-acting small molecules. In the long term, gene therapy approaches may permit the utilization of biosynthetic molecules, including larger proteins produced by biosynthetic organoids with extended but finite life spans.

Recommendations

Based on our current understanding of the state of research in the regulation of ovarian function and oocyte development, we make the following recommendations:

  1. The mechanisms underlying the pulsatile release of GnRH and the differential regulation of FSH and LH synthesis and secretion should be elucidated.
Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×
  • Specific molecular events associated with maintenance of oocytes in prophase I, and release of this block by the ovulatory stimulus only in mature follicles, should be defined. This should be accompanied by a determination of the suitability of these targets for pharmacological intervention.
  • The horizons of apoptosis research should be expanded to include the developing follicle as a target. Apoptosis research in diverse areas, including this one, could "cross-fertilize" these complementary fields of research and perhaps produce benefit to each other. One very important research objective is to understand the mechanism by which the dominant follicle progresses while other developing follicles undergo atresia.
  • Examination of molecular and cellular aspects of follicular development, definition of key "players" and their specific targets, and identification of endogenous and synthetic ligands should be major research objectives.
  • Studies should be undertaken to elucidate the factors that control follicular rupture, since inhibition of this process would be an ideal way to prevent fertilization through simulation of a normal nonconception cycle with unaltered steroid patterns, steroid levels, and cycle length.
  • Established targets, for instance, the steroid hormone receptors, should be reexamined, the rationale being the recent observation that tissue-selective compounds can be developed to control specific subsets of genes that are regulated by the natural hormone.

Interference with Postfertilization Events

After ovulation, the oocyte enters the oviduct (fallopian tube) and progresses into the ampullary segment, where it may be fertilized if it is met by capacitated spermatozoa that retain their fertilizing potential. Fertilization could be prevented by interventions that hinder the sperm from reaching the ampullary segment, by causing the sperm to undergo a premature acrosome reaction, by blocking sperm binding to the zona pellucida, by changing the consistency of the zona to prevent sperm penetration, or by premature aging of the oocyte. Once fertilization has occurred, the fertilized egg is retained in the ampullary segment of the oviduct for the next 72 hours. During this period, it undergoes several mitotic cycles so that, after traversing the isthmic segment, which takes only hours, it reaches the uterus at the 8- to 12-cell stage. It then lies free in the uterus for another three to four days while undergoing continued mitotic cycles, reaching first the morular stage and then undergoing cavitation to form a blastocyst (Croxatto 1995). Even before attachment to the endometrial epithelium, the blastocyst commences to cause changes in the endometrium, for example, increased blood flow at the future site of attachment. After attachment, the trophoblastic cells of the blastocyst start to invade through the endometrial epithelial lining into the stromal compartment, where the stromal cells begin a process of differentiation known as decidualization. Implantation is considered the process

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×

that starts with attachment and ends with decidualization. Concomitant with implantation in primates, the trophoblastic cells of the embryo start to secrete chorionic gonadotropin (CG), which is necessary for maintenance of the corpus luteum and continued progesterone production. Progesterone is an essential requirement for the establishment and maintenance of early pregnancy.

Oviductal and/or Myometrial Function

Based on the fact that agents that accelerate oviductal transport of eggs in polyovular animals with cornuate uteri also decrease the number of embryos that implant successfully, it has been felt for many years that, if the developing embryos could be expelled from the oviduct prematurely, then pregnancy would not take place. While this effect has been demonstrated in animals, at the moment there is no good proof that this is the case in humans. On the contrary, placement of human in-vitro-fertilized embryos in the uterus at a very early developmental stage (equivalent to what would happen if the embryo were expelled prematurely from the oviduct) has produced viable human offspring. It has been established that the main reason why embryos arriving prematurely in the uterus do not implant in some animals is because they are rapidly expelled from the uterus (Adams 1980; Ortiz et al. 1991). Close to the time when embryos normally enter the uterus, the activity of the myometrium changes so that, instead of being expelled, the embryos are retained. The human may differ in that the shift from expulsion to retention may occur much earlier than in it does in rodents; it is also possible that the different shape of the uterus makes the expulsive mechanism less efficient. In any event, it would appear that, for effective contraception, a drug that would accelerate tubal transport should also cause expulsion of the embryo from the uterus by itself or, in combination with an oxytocic agent, stimulate uterine contractility. On the other hand, an agent that stimulated only enough uterine contractility to cause embryonic expulsion, even when the embryo entered the uterus at the normal time, would also provide effective contraception. In the human female there is a three-day window from the moment the egg enters the uterus until blastocyst adhesion to the endometrium begins. By accelerating tubal transport, this window could be widened to six days, during which time expulsion of the egg could be achieved.

Recent data indicate that the time at which the early-stage embryos pass from the oviduct to the uterus is dictated in some species predominantly by ovarian sex steroids; in others, this is due to substances secreted by the embryo during its journey through the oviduct. Two of the substances involved appear to be prostaglandin E2 (PGE2) (Weber et al. 1991 [mare]) and platelet-activating factor (PAF) (Velásquez et al. 1995 [hamster]). Since the human oviduct does not respond with accelerated transport to acute increases in estrogen or progesterone levels (Croxatto 1995), it is likely to belong to that class of mammals in which the embryo itself, rather than ovarian steroids, control its passage to the uterus. Until

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
×

this is confirmed and the substance involved is identified, it will be difficult to assess the feasibility of using this natural mechanism to put this process out of phase.

Hormonal treatments that cause retention of embryos in the oviducts of animals also reduce their fertility, but this approach does not appear applicable to human females because of the likelihood that it would cause an intratubal (ectopic) pregnancy. This complication, which does not occur in nonprimates, can be life-threatening in human females. New understanding of the regulation of embryo transport may result in new approaches to contraception that would act at the tubal and/or myometrial level.

Postcoital Contraception
Estrogens , Progestins , and Estrogen/progestin Combinations

As indicated earlier, administration of estrogens soon after coitus to nonprimates causes a notable decrease or suppression of fertility; it was for this reason that estrogens were first tried as a method of emergency contraception in women. Large-scale trials of various estrogens showed that, in human females, estrogens were moderately effective in preventing pregnancy when taken within 72 hours after a single mid-cycle act of intercourse (Blye 1973; Haspels 1994). The exact mode of action of this treatment is unknown. For the reasons given above, it is not likely that embryo transport through the oviduct is accelerated; what may be involved is either expulsion of the embryo from the uterus, disruption of the synchronous development of the endometrium with that of the embryo that is necessary for implantation, or ovulation blockade.

One drawback of this treatment is the extent of side effects, especially nausea and vomiting. To alleviate these consequences, the therapy has been modified to a combined estrogen/progestin regimen. This reduces side effects, although it does not eliminate them, at the same time that the window of efficacy remains unaltered (Yuzpe et al. 1982; Yuzpe and Lancee 1977). Another emergency contraceptive approach is vaginal administration of steroids, the objective of which is to increase efficacy and reduce gastrointestinal side effects. One piece of research that should be implemented is formulation of pills for postcoital contraception for vaginal administration, with performance to be comparable to that of orally administered pills. Progestins alone (''minipills") have also been used and seem to be effective (Farkas et al. 1981; Ho and Kwan 1993).

A major advantage of combination therapy is that, although in the United States no postcoital contraceptive preparation is marketed as such, administration of four marketed oral contraceptive pills taken two and two, 12 hours apart, provides effective postcoital contraception. This is not widely known among either providers or users who might need such treatment. However, because of the probable disruption of the menstrual cycle produced by such therapy, it is not

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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suitable for use cycle after cycle or where there are multiple acts of unprotected intercourse in any single cycle.

Antiprogestins

Current antiprogestins are synthetic steroids that bind to the progesterone receptor and prevent the transcription activation that is normally initiated by the ligand-receptor complex. Since the antiprogestin mifepristone has been shown to terminate pregnancy when given after a missed menses, it has also been tried as a postcoital contraceptive. In two trials in which mifepristone was given at the dose of 600 mg orally within 72 hours after unprotected intercourse to a total of 597 women, there was not a single pregnancy; there were nine pregnancies among 589 women similarly treated with estrogen/progestin or progestin-only formulations (Glasier et al. 1992; Webb et al. 1992). This low failure rate indicates that this particular antiprogestin is an effective postcoital contraceptive. In both studies, the incidence of nausea and vomiting was significantly lower than has been the case with other presently available compounds. Nevertheless, menstrual delay was more common in the group receiving mifepristone.

Postcoital emergency use of mifepristone is being studied further by the Special Programme of Research, Development, and Research Training in Human Reproduction of the World Health Organization (WHO/HRP 1995). An ongoing clinical trial comparing single doses of 10, 50, and 600 mg taken within five days of unprotected intercourse is expected to be completed in 1996.

Mifepristone can delay ovulation due to temporary arrest of the growth of the dominant follicle, can offset the positive feedback of estrogen on the discharge of gonadotropin from the pituitary gland, and can disrupt the required secretory changes of the endometrium. These features make an antiprogestin effective for preventing either fertilization or implantation, depending on the stage of the menstrual cycle at which it is taken. Conceivably, a combination of estrogen and an antiprogestin could be more effective than either compound alone and would permit reduction in the amount of each ingredient, with a concomitant reduction in side effects.

Anti-implantation Agents
Targeting Hormones

Antiestrogens given in small single doses during the 24 hours prior to implantation can specifically inhibit implantation in species such as the rat and the mouse, which require a surge of endogenous estrogen to initiate the implantation process. However, these compounds have not been shown to be effective in species such as the rabbit and the hamster, which require only progesterone and need no maternal estrogen to initiate implantation (Harper 1972).

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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The situation in primates is more complex. Since there is a rise in plasma estrogen levels in primates around the time of implantation, it was thought for some time that they, too, would require estrogen for implantation. However, a series of experiments in which monkeys were ovariectomized after ovulation and mating and given only progesterone replacement indicated that pregnancy could occur normally. Furthermore, recent studies in women with premature ovarian failure who used in vitro fertilization and embryo transfer to overcome their infertility showed that a combination of estrogen and progesterone during the first half of the artificial cycle, followed by progesterone alone starting several days before the transfer and continuing through the implantation period, was perfectly adequate to ensure pregnancy (see Harper 1992 for discussion). Similar results have been demonstrated in monkeys (Ghosh et al. 1994). Thus, it is clear that maternal estrogen is not required at the time of implantation to initiate that process in primates. However, there have been several studies in primates where antiestrogens were given after ovulation and before implantation and produced an antifertility effect (Harper 1992).

In any event, a lack of progesterone action on the uterus always leads to pregnancy failure. There are three ways to prevent progesterone from acting on its target organs and each has its limitations. One way is to prevent synthesis of progesterone by enzyme inhibitors, for example, azastene or epostane (Crooij 1988; Schane et al. 1978; Snyder and Schane 1985). Epostane (200 mg orally every 6 hours for 7 days) terminated pregnancy in about 80 percent of women up to the eighth week of pregnancy, but nausea was a common side effect (Bigerson and Odlind 1987; Crooij et al. 1988). The main problem with this approach is that it may affect synthesis of adrenal steroids.

A second approach is to intercept progesterone in the circulation by specific antibodies. This could be achieved on a long-term basis by active immunization (Hillier et al. 1975; Nieschlag and Wickings 1978; Rider et al. 1985). A possible drawback would be exposure of the endometrium to the action of unopposed estrogen. However, administration of a synthetic progestin, not crossreactive with the antiprogesterone antibody, during the last quarter of the cycle would produce withdrawal bleeding regardless of other events in the cycle. This is a potentially interesting target.

The third approach is to block progesterone action at the receptor level using antiprogestins. Mifepristone has been administered to primates and to human females within the time frame between ovulation and implantation to assess effects on the endometrium and on fertility. A single dose of 10 mg administered 5 to 8 days after the LH surge produced marked asynchrony between glandular and stromal elements of the endometrium (Greene et al. 1992), an effect which presumably would be incompatible with implantation. Single doses of 2 or 10 mg/kg given 2 days after ovulation to rhesus monkeys were highly effective for preventing pregnancy (Ghosh and Sengupta 1993). Similarly, administration of 200 mg on the second day after the LH surge caused retarded endometrial devel-

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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opment in human females (Swahn et al. 1990), as well as reduced fertility (Gemzell-Danielsson et al. 1993).

It is also plausible that combinations of progesterone synthesis inhibitors with progesterone receptor blockers might be more effective than either alone (Birgerson and Odlind 1987; Birgerson et al. 1987; Crooij et al. 1988; Selinger et al. 1987). Since most of these hormonal manipulations result in making endometrial development out of synchrony with or hostile to the embryo, a combination of an anti-estrogen and an antiprogestin might also be effective. Until recently, except for the WHO/HRP research team, no one has been able to conduct the required human trials, even though there has been extensive clinical experience with such candidate compounds for other indications. India and the People's Republic of China have recently supported work in this neglected field.

Cell-adhesion Molecules

One of the first events in the implantation process is the attachment of the blastocyst to the uterine epithelial surface, an effect possibly accomplished by cell-adhesion molecules. Under appropriate conditions, functional and structural changes occur at the apical surfaces of epithelial cells lining the endometrial cavity which permit blastocyst attachment. Carbohydrates appear to be intimately involved in cell-cell adhesion. Synthesis of the histo-blood group Htype-1 antigen is hormonally regulated and is expressed in the endometrial epithelium at the peri-implantation period in several species, including the human. It is thought that endometrial carbohydrates carrying the H-type-1 antigen may be involved in the initial blastocyst adhesion (Kimber 1994; Kimber et al. 1995).

The integrins are a family of cell-adhesion molecules. The distribution of several a and ß integrin subunits has been studied in the human endometrium during the menstrual cycle. It was noted that the collagen/laminin receptors (a2, a3, and a6) were expressed mainly on the epithelial cells, whereas the fibronectin receptor (a5) was found mainly in the stroma. The two subunits of the vitronectin receptor (avß3) and integrin a4ß1 varied throughout the cycle, with av increasing throughout the cycle and ß3 only appearing abruptly on day 20, and a4 decreasing about day 24. Changes in the expression of these integrins may define the putative period of uterine receptivity for blastocyst attachment (Ilesanmi et al. 1993; Lessey 1994; Lessey et al. 1992, 1994; Schultz and Armant 1995). Blockade or disruption of this expression of integrins might also provide a specific means of preventing implantation.

Another cell surface glycoprotein with potential importance in blastocyst adhesion is a high molecular weight glycoprotein identified by mouse ascites golgi antibodies. This glycoprotein involves N-acetyl-galactosamine and other determinants, is secreted from the endometrial glands in the human during the period of uterine sensitivity for implantation (Kliman et al. 1995), and is thought to be involved in the initial adhesion phase of implantation.

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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Mucins are O-linked glycoproteins present on the apical surface of epithelial cells (Strous and Dekker 1992). Mucin domains can extend for 200-500 nm from the cell surface (Jentoft 1990). A high density of such glycoproteins on apical cell surfaces could block access to the cell membrane. Muc- 1 (also known as episalin) is a member of this family of mucin glycoproteins. Muc- 1 expression (protein and message) is found only in the endometrial epithelial cells of mouse uterus. It is high at proestrus and estrus and is barely detectable on day 4 of pregnancy (just prior to implantation). Using steroid-treated ovariectomized mice, it was shown that Muc-1 was stimulated by estrogen and down-regulated by progesterone and antiestrogens owing to inhibition of estrogen action (Braga and Gendler 1993; Surveyor et al. 1995). It seems reasonable, therefore, to argue that loss of Muc-1 is necessary, though not sufficient, to generate the receptive state for implantation. Muc-1 is also found in granulated metrial cells of the mouse decidua by day 8 of pregnancy. This is unusual since Muc- 1 is considered to be an epithelial differentiation marker (Braga and Gendler 1993). If manipulation of Muc-1 expression is to serve as a basis for a contraceptive approach, ways of decreasing it prematurely or delaying its down-regulation would have to be found. It may prove difficult to obtain a sufficient degree of asynchrony between the development of uterine receptivity and the embryo to constitute a reliable method of contraception.

Heparin-binding epidermal growth factor (HB-EGF) binds to the EGF receptor and is a mitogen for keratinocytes and fibroblasts. Its binding to the EGF receptor and its biological activity are potentiated by heparin sulfate proteoglycans, which are present in high amount in the basal lamina of the luminal epithelium. At least in the rat, HB-EGF is regulated by progesterone with expression in the uterine stromal cells and suppression in epithelial cells. It appears that it is involved in the development and maintenance of the decidual cells that are required for subsequent stages of implantation after attachment. The decidualizing stimulus first causes differentiation in the stromal cells underlying the epithelium and then is propagated to other stromal cells. Thus, HBEGF may be important for establishing uterine receptivity for implantation and causing stromal cell proliferation (Zhang et al. 1994a). Its appearance can be blocked by antiprogestins and thus may be a good contraceptive target (Zhang et al. 1994b).

Two new adhesion molecules have recently been described, trophinin and tastin. Trophinin is an intrinsic membrane protein which mediates self-binding, while tastin is a cytoplasmic protein whose role is to permit trophinin to act as a cell-adhesion molecule. These molecules are associated with the cytoskeleton and have been found on monkey blastocyst trophectoderm and human endometrial luminal epithelium at the beginning of the appearance of the period of receptivity (Fukuda et al. 1995). The functional significance of these factors in implantation remains to be determined, but they may also be useful new leads for a contraceptive acting to prevent blastocyst attachment.

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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Clearly, prevention of attachment of the blastocyst to the endometrial lining could be an attractive possibility for developing a novel method of contraception. Much basic work still remains to be done to establish which of the cell-adhesion molecules are crucial for this process. Unless agents that act in this manner have a long biological half-life, there will also be practical difficulties in determining the optimal time during the cycle for administration.

Cytokines/Growth Factors

Within the last 10 years there has been a veritable explosion of research and interest in a variety of endogenous glycoproteins that are known as growth factors and/or cytokines. The importance of many of these agents, especially members of the interleukin family, for regulation of the immune system is now well established. However, the study of mutant mouse strains or production of transgenic mice with the gene for a particular cytokine disabled (the so-called "knockout" mouse) has recently revealed that some of these cytokines also regulate endometrial functions (Harvey et al. 1995). Several candidates have been proposed for an essential role in implantation.

Interleukin- 1 is used to designate two structurally related proteins, IL- la and IL- 1ß , with a molecular mass of about 17,000 daltons. Both proteins bind to the same receptor (IL-1R) and mediate similar actions. The receptor is also in two forms, IL- 1 type I and type II, of which the type I receptor binds both IL- 1a and ß and the function of the type II receptor is unknown. IL- 1 a is present in mouse (message and protein) uterus during the preimplantation period and appears to be hormonally regulated since it peaks just before implantation (De et al. 1993; Tackacs et al. 1988). IL-1ß mRNA has been found in human endometrium, as has the IL-1R which peaks in the luteal phase (Sim6n et al. 1993a, 1993b; Tabibzadeh et al. 1990). Furthermore, IL-1 ß, which is increased during endometrial decidualization, also acts as a modulator of the degree of decidualization (Frank et al. 1995). The action of IL-1 on its receptor can be inhibited by an endogenous inhibitor, the IL-1a receptor antagonist. Treatment of mice with the IL-1R antagonist during the preimplantation period prevented pregnancy (Sim6n et al. 1994). Experiments to repeat these results have been only partially successful and the critical importance of IL-1 in implantation per se remains to be established.

In a mutant inbred mouse strain that has undetectable levels of colonystimulating factor-1 (CSF- 1), breeding of homozygous males and females results in a failure of implantation. This effect is not specific to the maternal reproductive tract, because breeding the homozygous females with heterozygous males results in a certain degree of rescue of the pregnancy, which may indicate an embryonic function in this rescue (Pollard et al. 1991). This may mean that CSF1 might also be a critical factor for implantation. It regulates gene expression and induces synthesis of another cytokine, tumor necrosis factor-a (TNF-a). In these

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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homozygous mutant mice, TNF-a expression in the reproductive tract was not different from that in mice with normal CSF-1 levels (Hunt et al. 1993). When defective early development of mouse embryos is induced by exogenous CSF- 1, preimplantation development can be restored to normal by TNF-a or granulocyte macrophage colony-stimulating factor but not by transforming growth factor ß 1 (Tartakovsky and Ben-Yair 1991). Interestingly, TNF-a inhibits decidualization of human endometrial stromal cells in vitro (Inoue et al. 1994), so that the balance between CSF- 1 and TNF-a may be critical for normal progression of the implantation process. In human endometrial stromal cells in culture, secretion of macrophage CSF is progesterone dependent and should therefore be increased during the luteal phase of the cycle (Hatayama et al. 1994; Kariya et al. 1994). Short-term blockade of CSF-1 action to prevent implantation may be free of the side effects such as osteopetrosis that are seen in the mutant animals.

Leukemia inhibitory factor (LIF) is a 38- to 67-kDA glycoprotein, which can be secreted or remain cell-associated. LIF exerts actions on a variety of different cell types. The results of experiments in mice with the gene for LIF mutated are very promising. Homozygous mice deficient in LIF appeared normal except for being approximately 25 percent smaller than wild-type animals. Homozygous males were fertile and produced offspring from both normal and heterozygous females. In contrast, homozygous females mated to wild-type heterozygous or homozygous males did not become pregnant. These females were not sterile because normal blastocysts were found in the uterus on both day 4 (the day of implantation) and day 7, which indicates that implantation was delayed. Such blastocysts when transplanted to wild-type females implanted normally but when wild-type blastocysts were transplanted to the homozygous females, implantation did not occur. Replacement therapy with recombinant LIF (without the glycosylation) induced implantation of some blastocysts in the homozygous mice. Expression of LIF mRNA in the uteri of pseudopregnant mice was similar to that seen in pregnant mice, suggesting that the embryo was not involved in this expression (Bhatt et al. 1991; Stewart et al. 1992).

These results clearly indicate that, in the mouse, LIF production by and action on the uterus is a critical factor for initiation of implantation. In the mouse both LIF message and protein is regulated by estrogen (Bhatt et al. 1991; Yang et al. 1996), and the mouse is a species requiring estrogen for blastocyst implantation. The importance of LIF in the implantation process in other species, especially those not requiring estrogen, has not been established; this is of crucial importance for relevance to the human.

At the same time, there is some supporting evidence from other species. LIF protein, LIF receptor, and gp130 have been shown to peak in the uteri of both pregnant and pseudopregnant rabbits before implantation, in a similar fashion to mice (Yang et al. 1994, 1995a, 1995b). Since the rabbit does not require estrogen for induction of implantation, it would seem likely that LIF in this species is regulated differently from the mouse. Indeed, this has proved to be the case. LIF

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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protein uterine levels are increased by progesterone and unaltered by estrogen. In endometrial specimens from women during nonconception cycles, both LIF message and protein are low or absent during the proliferative phase and high during the secretory phase, remaining high to the end of the cycle (Charnock-Jones et al. 1994; Kojima et al. 1994). LIF secretion from human endometrial cell cultures, however, shows a peak at the mid-luteal phase (around the time of implantation in a conception cycle) (Chen et al. 1995). LIF and IL-6 are both found in pig uterine secretions just prior to embryo attachment (Anegon et al. 1994). The reason for the discrepancy between message and protein levels and secretion in the human may be due to changes between cell-associated and secretory forms or between conception and nonconception cycles. In mice and rabbits, no examination was done for LIF expression in pseudopregnant animals after the equivalent time of implantation in pregnant animals. In sum, the data in hand suggest that LIF may be an important lead in the development of specific anti-implantation agents.

There are, nonetheless, some possible problems. There are significant similarities in primary amino acid sequences and predicted secondary structures of LIF with other cytokines, e.g., oncostatin M (OSM), IL-6, ciliary neurotrophic factor (CNTF), and granulocyte colony-stimulating factor (G-CSF) (Horseman and Yu-Lee 1994; Rose and Bruce 1991). These structurally related cytokines modulate differentiation in a variety of cells so that there is a possibility that, in some cells at least, they could substitute for each other, although the present understanding is that LIF and related cytokines are not functionally equivalent (Piquet-Pellorce et al. 1994) despite similarities in receptor structure (see below). The importance of LIF is underscored by the high degree of conservation in the coding regions and high degree of similarity of the protein across species (Willson et al. 1992).

Actions of these cytokines on cells are mediated through membrane-bound receptors. Binding of these cytokines to their receptor complexes activates a signal transduction pathway, resulting in rapid tyrosine phosphorylations, followed by activation of a protein kinase cascade and early gene responses. These appear to be nonreceptor tyrosine kinases. Specificity of response may be ensured by intracellular structure of the cytokine receptor dictating which signaling molecules are activated and expression levels of signaling molecules in the different cell types (Taniguchi 1995). Similarities between the receptor signal transducing units of CNTF, G-CSF, IL-6, IL-11, and LIF have been reported (Horseman and Yu-Lee 1994). There are two LIF receptor types, a high- and a low-affinity type (Hilton et al. 1988; Yamamoto-Yamaguchi et al. 1986). The cloned LIF receptor (LIFR) only binds LIF with low affinity, but after the binding of a common receptor component gp 130 (the signal transduction unit for IL-6R), LIFR is converted to the high-affinity type (Gearing et al. 1991, 1992). The receptors for all the above cytokines involve gp130 binding, acting as a signal transduction unit. The IL-6R comprises a homodimer of gp130, but OSMR,

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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LIFR, and CNTFR involve only one gp 130 subunit plus the LIFR ß subunit, all complexed with a third element, a specific a subunit, which may be membrane-bound or act as a soluble factor, and regulate the binding of factors to the ß subunits (Davis et al. 1993; Hirano et al. 1994; Ip et al. 1992; Murakami et al. 1993). Receptor activation results from binding of gp130 which converts the a:ß complexes to the high-affinity form. These receptor components constitutively associate with Jak-Tyk kinases, which are activated by receptor dimerization (Lütticken et al. 1994; Murakami et al. 1993; Stahl et al. 1994). A family of proteins known as STATs (signal transduction and activation of transcription) are involved in the actions of many polypeptide ligands on cells. STAT3 is activated through phosphorylation on tyrosine as a DNA binding protein by the LIF-IL-6CNTF family of ligands (Zhong et al. 1994). Selection of the particular substrate STAT3 is specified not by the particular Jak activated but by the tyrosine-based motifs in the receptor components gp130 and LIFR (Stahl et al. 1995). Blockade of the tyrosine kinases prevents phosphorylation of the receptor subunits and gene induction.

Since gp130 is involved in signal transduction for all members of this receptor family, inactivation of the gp130 signaling function may not provide a good contraceptive target owing to lack of specificity. Nevertheless, inhibition of tyrosine kinase activity itself has been suggested as a potential drug target (Levitzki and Gazit 1995), although whether sufficient specificity can be ensured is again an unresolved question. However, specific blockade of the formation of the LIFR a:ß complex, given the essential need of LIF for implantation, appears a much more appealing target, if it can be established that LIF plays the same role in other species that it does in the mouse. Such inhibition targeted at the essential LIFR a subunit should provide the specificity needed to avoid interference with function of OSM, CNTF, and IL-6. Although LIF has actions at sites other than the uterus, the fact that the mutant mice deficient in LIF were apparently normal gives hope that short-term inhibition of LIF action in the uterus will be without other adverse consequences.

Studies in nonhuman primates have identified a variety of other growth factors whose secretion by the endometrium is hormonally regulated. There are cell-specific changes in gene expression of the receptors for insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF), and for the secreted proteins IGF-binding protein 1 and retinol-binding protein (Fazleabas et al. 1994). In human endometrial cell cultures, IGF-I, its receptor, and the IGF-binding proteins 1-4 are all localized to the epithelial cells and highest at the early- to midsecretory phase of the cycle (Tang et al. 1994). It is postulated that these changes are modulated by the embryo and are essential for implantation. However, at present the data are purely correlative. Fibroblast growth factors (FGFs) are involved in angiogenesis, cell growth, and cell differentiation, and disruption of the gene for FGF-4 in mice causes severe inhibition of the growth of the blastocyst inner cell mass (the cells that form the embryo) and failure of pregnancy just

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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after implantation (Feldman et al. 1995). FGFs have a dual receptor system; one component is the FGF receptor with an extracellular ligand-binding domain and an intracellular tyrosine kinase domain, and the other is a series of heparin or heparin sulfate proteoglycans required for FGF binding. There are several binding sites for these proteoglycans on FGF, and thus design of compounds able to modulate FGF action should be possible (Ornitz et al. 1995).

In summary, there are several new and promising approaches that could form the basis for a once-a-month contraceptive pill. Yet, all this research must still be brought to proof of concept stage and, even when an LIFR blocker that blocks implantation is developed, many questions will remain. There will be a need to determine specific dosages and frequencies, all of which will depend on the half-life of the compound being used. Will it be necessary to deliver the agent locally, or will an oral preparation suffice? If the agent is given too late, will the effect be simply a normal pregnancy and is there a risk of a teratogenic effect? Is there likely to be a shortening of the cycle that causes difficulties in determining the time for optimal dosing in the next cycle? Obviously, there are difficulties; still, those may be fewer than those produced through hormonal manipulation, making the approach worthy of further investigation.

Inhibition of hCG Production

As discussed above, peptide agonists and antagonists of GnRH have been developed and their actions in blocking the central hypothalamic-pituitary axis have been extensively investigated as a method of contraception in both men and women, as discussed above. Investigations are under way to develop nonpeptide antagonists since they are likely to be simpler and cheaper. Whether such agents could block the action of trophoblast GnRH in stimulating the hCG secretion necessary for luteal support and pregnancy maintenance is unknown. It may also be that other factors are involved in regulation of early hCG production that might be amenable to attack. Inhibition of the early hCG production necessary for luteal maintenance would cause early pregnancy failure without disrupting menstrual cyclicity.

Induction of Luteolysis

In nonprimates, it has been relatively easy to develop agents that cause luteolysis, that is, premature demise of the corpus luteum with interference in progesterone secretion and consequent failure of pregnancy. In primates, in whom the mechanisms of luteal support appear to differ significantly from those of nonprimates, this has proven difficult. At this time, induction of luteolysis does not appear to be a promising avenue, but basic research on regulation of the corpus luteum may provide new leads. One alternative means to achieve the same effect is by blockade of progesterone synthesis in the corpus luteum. Such

Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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a functional luteolysis has been demonstrated following administration of either azastene or epostane during the luteal phase of the cycle to rhesus monkeys (Schane et al. 1978; Snyder and Schane 1985); in pregnant monkeys, pregnancy was terminated (Schane et al. 1978). Even with a luteolytic agent in hand, it might prove to be effective only during the period before the trophoblast provides luteal support, although this was not the case with the progesterone synthesis inhibitors (Asch et al. 1982; Snyder and Schane 1985). In such cases, the agent would not be luteolytic at the end of the cycle but only until the peri-implantation period, after which cycle disruption might ensue.

Expected Menses Induction

Another possible once-a-month contraceptive modality aims at insuring the occurrence of endometrial sloughing at the time of expected menses, regardless of whether or not an embryo has implanted. Since progesterone is essential to maintain the integrity of the endometrium, it is theoretically possible that a single high dose of an antiprogestin given at, or shortly before, the time of expected menses might achieve this purpose. Mifepristone has been given as a single dose of 600 or 400 mg, or 100 mg for four consecutive days in the late luteal phase to women who had detectable levels of the ß subunit of hCG; actual failure rates, expressed as percentages of subjects continuing to be pregnant, were from 17 to 19 percent. This level of efficacy is similar to that observed after use of mifepristone to interrupt pregnancy when given up to 11 days after a missed menses. In order to improve effectiveness, WHO/HRP is conducting a two-center study in which mifepristone is followed by administration of misoprostol, an orally active prostaglandin that has been shown to increase significantly the rate of pregnancy termination when both are combined after missed menses. Data from this study may be available during 1996.

Recommendations

Available evidence suggests that there are several promising targets that can be pursued now and we therefore make the following recommendations:

  1. There should be expeditious examination of combinations of antiprogestins and other hormonal or antihormonal drugs, given orally or vaginally, as a method of emergency or once-a-month contraception. A more effective combination could be developed in a relatively short time frame.
  2. Studies in nonhuman primates should be conducted to develop the concept and test the safety of immunization against progesterone as a simple and easily reversible contraceptive method.
  3. The most promising of the adhesion molecules should be studied to deter-
Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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  1. mine how essential they are for initial blastocyst attachment to the endometrial epithelium.
  2. Experiments using active or passive immunization should be conducted to determine whether, for example, LIF is essential for implantation in species other than the mouse. If such experiments prove positive, then means to disrupt uterine LIF function for a short period should be sought; the most specific approach would appear to be through interference with binding of the specific LIFRa subunit. Examination of the obligatory requirement for other growth factors/cytokines should be carried out.
  3. Nonpeptide GnRH antagonists, which would be useful for applications both before and after fertilization, should be developed.

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Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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Suggested Citation:"Appendix A: Female Methods." Institute of Medicine. 1996. Contraceptive Research and Development: Looking to the Future. Washington, DC: The National Academies Press. doi: 10.17226/5156.
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The "contraceptive revolution" of the 1960s and 1970s introduced totally new contraceptive options and launched an era of research and product development. Yet by the late 1980s, conditions had changed and improvements in contraceptive products, while very important in relation to improved oral contraceptives, IUDs, implants, and injectables, had become primarily incremental. Is it time for a second contraceptive revolution and how might it happen?

Contraceptive Research and Development explores the frontiers of science where the contraceptives of the future are likely to be found and lays out criteria for deciding where to make the next R&D investments.

The book comprehensively examines today's contraceptive needs, identifies "niches" in those needs that seem most readily translatable into market terms, and scrutinizes issues that shape the market: method side effects and contraceptive failure, the challenge of HIV/AIDS and other sexually transmitted diseases, and the implications of the "women's agenda."

Contraceptive Research and Development analyzes the response of the pharmaceutical industry to current dynamics in regulation, liability, public opinion, and the economics of the health sector and offers an integrated set of recommendations for public- and private-sector action to meet a whole new generation of demand.

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