A RESEARCH AGENDA FOR DEPARTMENTS OF OBSTETRICS AND GYNECOLOGY
The previous chapters developed the themes that identifiable weaknesses exist in the OB/GYN research enterprise and that actions to strengthen and support investigation are needed. This chapter describes research topics, the accomplishment of which would significantly improve the health of women and the results of pregnancy. The breadth and depth of this research agenda underscore the importance of ensuring a thriving research enterprise in OB/GYN.
The criteria for inclusion of topics in the research agenda and the process used by the committee to develop the agenda axe described in Chapter 2. They are reiterated here because they emphasize the sense of the committee that OB/GYN has the potential to make important contributions to health. They also point to the committee's insistence that research topics included in the agenda should be directed toward the melioration of significant health problems and be particularly suited to the work of departments of OB/GYN. To ensure that the research agenda fulfills its purpose of highlighting the need for expanded research efforts in OB/GYN, the following criteria were applied:
The research should contribute to the resolution of an important health problem. Importance can be defined in terms of high prevalence or incidence of a problem, or the serious effect of the problem on individuals who experience it. Importance can also be defined in terms of impact on the health care system where the costs of caring for the problem axe incurred.
The research approach should be promising. That is to say, there should be reason to think that following the selected avenue of investigation will provide solutions or that answering the question posed by the research is an essential step in finding a solution.
The research should be done in a department of OB/GYN or in collaboration with members of such departments. The mere fact that patients with the problem are first seen by an OB/GYN professional is not sufficient justification. Rather, OB/GYN must be the discipline with the knowledge or skill needed to accomplish the research. If the research is interdisciplinary, OB/GYN should be a necessary element. Lack of interest by other specialties would also be sufficient justification (i.e., the work would not be accomplished if OB/GYN did not do it).
During the period of this study, NIH initiated three activities that will result m research agendas that overlap many areas of the committee's work. The first of these is the Pregnancy, Birth, and Infant Research Plan of the National Institute of Child Health and Human Development. The second is a research agenda being developed by the Task Force on Opportunities for Research on Women's Health. This group, which was assembled by the Office of Research on Women's Health, has been asked to identify the research needed to improve the health of women at all stages of their lives. Its deliberations therefore include such areas as reproductive science and early developmental biology. Discussion at a workshop held by the task force emphasized the need to stress the epidemiological and behavioral aspects of research on women's health. Finally, NIH is engaged in an effort to develop a strategic plan and to that end has drawn on the expertise of several panels, including one on reproductive biology and development and one on infant health and mortality. In their initial work, both panels emphasized the personal and social consequences of unsolved problems in these areas. The panel on reproductive biology and development highlighted seven areas, each of which in whole or in part covers topics that the committee included in its research agenda: the control of reproductive function, infertility, contraception, the molecular basis of embryonic development in animal and plant models, environmental factors affecting reproductive biology and development, and postnatal growth.
In light of these large-scale efforts, the committee felt that it would be duplicative to produce a comprehensive and detailed research agenda. Instead, individual committee members were asked to highlight areas of investigation that meet the criteria listed above and that exemplify the range of questions that might fruitfully be investigated. Because there were no committee members with expertise in behavioral sciences, technology assessment, and outcomes analysis, the agenda outlined in the following sections does not sufficiently emphasize those areas. The committee therefore wishes to stress its opinion that departments of OB/GYN, in conjunction with individuals with relevant
expertise, are well suited to undertake investigation of many topics related to behavior that affects reproductive health, the technologies used by the field of OB/GYN, and the outcomes of care provided by OB/GYNs. The large number of patients who receive care in the OB/GYN clinics of academic centers represents an opportunity for clinically relevant epidemiological research—including research on the efficacy of treatment, on the natural history of disease, and on the prevention of disease. Faculty of departments of OB/GYN, in collaboration with epidemiologists, sociologists, statisticians, and health services researchers, have the patient base and the discipline-specific interests needed to investigate questions that other disciplines are not likely to undertake. The committee also believes that the advantages of the patient base and the knowledge that resides in departments of OB/GYN suggest that these departments should organize and conduct clinical/epidemiological trials that are often now initiated by other departments.
The following sections were written by committee members, as acknowledged, and are in large part based on background papers prepared for the committee, whose contributions are gratefully recognized (see Appendix C for a list of background papers).
Oocyte and Follicular Development in the Ovary*
This section identifies areas of research within the broad field of ovarian function that are best and most appropriately pursued in departments of obstetrics and gynecology.
The ovary, an ever-changing tissue, is a multicompartmental tincture with different and variable biological properties. Responding to cyclic pituitary hormone secretion the various types of ovarian cells interact in a highly integrated manner to secrete sex steroids, elaborate a variety of regulatory proteins, and produce a fertilizable oocyte or egg. This section focuses on key unresolved areas in ovarian physiology. All axe viewed as important not only to the understanding of ovarian function but also to the promotion of fertility and fertility control.
The primordial germ cells that will become the ovarian eggs originate near the bottom of the embryo where they can be identified as early as the end of the third week of gestation. Migration of germ cells to their final location is accomplished prior to the fifth week of gestation. Whereas some chemotaxis is clearly operational, the precise cellular mechanism or mechanisms underlying the guidance of germ cells to the future ovary remain uncertain. Most importantly, germ cells appear unable to persist elsewhere, and thus the future ovary may be viewed as the only bodily region competent to sustain oocyte development. By the same token, these germ cells play an indispensable role in the induction of gonadal development. During the subsequent 2 weeks of intrauterine life (weeks 5–7 of gestation), often referred to as the "indifferent stage," the primordial ovarian structure constitutes no more than a bulge on the medial aspect of the urogenital ridge. By about 8 weeks of intrauterine life, the future eggs are subject to three simultaneous ongoing processes: mitosis, meiosis, and atresia. As a result of the combined impact of these processes, the number of germ cells peaks by 20 weeks of gestation only to be followed by relentless lifelong and irreversible attrition to a point when the oocytic complement is finally exhausted, thereby giving rise to the menopause.
The prophase of the first meiotic division occurs between week 8 and week 13 of fetal life. Once formed, these primary oocytes persist in prophase until ovulation decades later when meiosis is resumed and the first polar body is formed and extruded. Although the exact cellular mechanism or mechanisms responsible for this meiotic arrest remain uncertain, it is generally presumed that the granulosa cells surrounding the oocyte secrete a putative oocyte meiosis inhibitor (OMI) that arrests egg development. This hypothesis is predicated on the observation that denuded (granulosa cell-free) oocytes are capable of spontaneously completing meiotic maturation under in vitro circumstances. It is not until the first luteinizing hormone (LH) surge occurs, indicating ovulation at puberty, that the first meiotic division is in fact completed. Again, little consensus exists as to the cellular events at play. The first primordial ovarian follicle is noted by around 16 weeks of intrauterine life. It is generally accepted that primordial follicle formation ends no later than 6 months postpartum. There is little information regarding the morphogenic principles responsible for the follicular organization surrounding the oocytes, but it is certain that formation of primordial follicles, the first step in ovarian follicular development, is independent of pituitary hormonal secretion. This presumption is strongly supported by the recognition that gonadotropin resistance, such as that
encountered in the so-called "resistant ovary syndrome," does not preclude primordial follicle formation.
The mechanisms responsible for recruiting some, but not other, primordial follicles for further development are unknown. However, this phenomenon underlies the presumed waves of follicular growth responsible for follicular replenishment. Although other factors are undoubtedly at play, it is virtually certain that even the earliest phases of follicular development beyond the primordial follicle stage are dependent on the pituitary hormones, follicle-stimulating hormone (FSH) and LH. Support for this conclusion is again derived from studies of the so-called "resistant ovary syndrome," whereto the ovaries display no evidence of follicular maturation beyond the primordial follicle stage. As such, this experiment of nature demonstrates the virtually absolute gonadotropin dependence of early follicular growth beyond the primordial follicle stage.
Elucidation of the events responsible for the transformation of endodermal cells into germ cell elements. Have the cells in question been somehow imprinted so as to form a germ cell lineage? Or is the endodermal cell totipotential and thus in a position to form unique cellular elements such as a germ cell?
Understanding of the forces responsible for guiding the germ cell toward the proper location in the future ovary. Clearly, chemotaxis appears to be at work. However, tissue remodeling appears equally inevitable, thus implicating extracellular matrix in the genesis of the required path.
Clarification of the cellular origins of the somatic follicular cells—for example, the steroid hormone producing granulosa and theca-interstitial cells surrounding the egg. There is reason to believe that the germ cells may play a morphogenic role by inducing the appearance of their somatic counterparts. Similarly, new information is required as to why germ cells are unable to subsist in bodily regions other than the future ovary
Analysis of the cellular mechanism or mechanisms responsible for the initiation of meiosis and for its arrest at the prophase stage of the first division. Clearer understanding of the ability of the mid-cycle surge to reinitiate meiotic division is required. The apparent biological differences between the cumulus
granulosa cells around the egg and those cells incorporated into the surrounding follicle must play a role.
Improved understanding of the role of putative intraovarian paracrine and autocrine regulators. Understanding such cytokine-mediated mechanisms could eventually lead to improved therapeutic strategies.
Literally translated, atresia (a Greek term) means the closure or obliteration ("a," not) of a normal body orifice or passage ("tresos," perforated). In the context of ovarian physiology, follicular atresia denotes the still enigmatic process whereby oocytes are lost from the ovary by means other than ovulation. In fact, atretic follicles are rendered incapable of ovulation. First noted in utero around month 6 of human gestation, atresia continues uninterrupted throughout life, thereby resulting in relentless and irreversible attrition of the ovarian germ cell endowment. It is noteworthy that the newborn human female enters life having lost as much as 80 percent of her egg cell endowment. By the onset of puberty, virtually 95 percent of all follicles have been lost. Of the residual follicular mass, only 400 to 500 follicles (i.e., less than 1 percent of the total) will ovulate in the course of a reproductive life span. Although clear-cut conclusions cannot be drawn at this time, it is generally presumed that postpubertal follicular atresia comprises an underlying tonic component reflecting a lifelong process on which losses of a cyclic, ovulatory nature are superimposed.
The forces guiding most but not all follicles toward certain demise remain unknown. Since all follicles appear to have comparable pituitary stimulation, one is forced to invoke the existence of as yet unrecognized intraovarian principles, the highly regionalized and exquisitely timed expression of which may well determine the direction of follicular development. This reasoning provides compelling arguments in favor of the concept of putative intraovarian regulators, presumably to exert in situ paracrine or autocrine modulatory effects at the follicular level.
The precise identity of the inciting atretic signal notwithstanding, recent advances suggest that follicular atresia represents an example of apoptosis, or programmed cell death, a relatively well-defined process first described by Kerr and colleagues in 1972. An active, energy-requiring process, apoptosis propels affected cells toward selective deletion. Viewed in this light, regulation of the
cellular complement revolves the concurrent and reciprocal processes of mitosis and apoptosis.
Much of the evidence identifying ovarian follicular atresia as an apoptotic process is morphologic in nature. Many of the morphologic features of atresia are reminiscent of the apoptotic process, suggesting that the two may be identical. However, these findings suggest that gonadotropin-primed follicles may respond to an as yet undescribed atretic signal with apoptosis.
Understanding of the molecular events responsible for determining follicular fate, which is a central goal of reproductive physiology. Clearly, if we could pharmacologically control and perhaps arrest the process of atresia, premature ovarian failure might be successfully treated and the age of the menopause substantially delayed. Likewise, fertility objectives could be served by an improved and augmented germ cell endowment.
Development of a reliable, reproducible experimental model for improved understanding of the atretic process. None exist at this time.
Understanding of the apoptotic nature of the atretic process and, in particular, of the ionic events that appear to trigger the molecular enzymatic events.
Focused investigation of potential putative intraovarian regulators concerned with the atretic process. Although sex steroids have been extensively implicated in the genesis and prevention of atresia, the body of literature remains equivocal and insufficient.
Follicular Recruitment, Selection, and Dominance
The term recruitment is used to indicate that a follicle has entered the so-called growth trajectory, that is, the process whereto the follicle leaves the resting pool to begin a well-characterized pattern of growth and development leading to ovulation. Clearly, recruitment is a necessary but not sufficient condition for ovulation to occur.
Follicular selection implies the final winnowing of the maturing but not yet quite dominant follicular cohort to the size of the species-characteristic ovulatory quota, which for the human is a single follicle each month. In the human,
follicular selection is presumed to occur during the first 5 days of the cycle when the leading follicular diameter is 5–10 mm.
The term dominance refers to the status of the follicule destined to ovulate, given its presumed key role in regulating the size of the ovulatory quota. It is generally agreed that a selected follicle becomes dominant about a week before ovulation, that is, as early as days 5–7 of the cycle when the diameter is around 10 mm. The earliest hormonal index reflecting dominance, which is evident at times as early as days 3–5 of the cycle, is an increase in the circulating levels of estradiol in the vein draining the ovary later shown to bear the corpus luteum. According to one view, the follicle selected for ovulation is functionally (not merely morphologically) dominant in that it inhibits the development of other competing follicles on both ovaries. Presumably then, the dominant follicle takes on an active role in ensuring its preferred status. Inevitably, and for reasons not entirely clear, the dominant follicle continues to thrive under physiologic circumstances it has made inhospitable to others.
Development of more specific markers capable of predicting the general well-being of the follicle in question and most importantly the quality of the resident oocyte. Such parameters would be of clinical relevance to in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT).
Improved understanding of existing known cytokine and growth factor regulators and the elucidation of the potential role of as yet unrecognized peptides. Although the central role or roles of gonadal steroids m folliculogenesis are well accepted, the variable fate of follicles afforded comparable gonadotropic stimulation strongly suggests the existence of additional intraovarian modulatory systems. This kind of investigation should yield clues as to how a follicle is selected and spared from atresia.
Corpus Luteum Function
As interesting as cell growth during follicular development is the cessation of cell growth and/or cell death. Granulosa cells divide on average only once following the preovulatory LH surge and then differentiate into luteal cells.
• It would be interesting to determine if the block to cell proliferation involves known genes associated with suppression of cell growth (perhaps one or more of the recently discovered tumor-suppressor genes, such as the retinoblastoma, or RB) or new examples of similarly functional genes. Action of the RB gene product involves TGF-B. Relatively little is known about TGF-B in the ovary except for cent reports by Schomberg and co-workers that at least forms of this factor axe synthesized there and that the expression of TGF-B2 is hormonally regulated.
It is now recognized that the extracellular matrix and cell surface molecules that specifically bind to matrix components play an important role in the regulation of cell growth and function. These include various glycosaminoglycans, like heparinsulfate, which are known to be synthesized by granulosa cells and axe present in the ovarian follicle. Almost nothing is known about the extracellular matrix or adhesion molecules in the ovary during the differentiation process from granulosa to luteal cell and during the lifetime of the corpus luteum.
Not surprisingly, numerous growth factors have been reported to be synthesized in the ovary. Despite the common belief that these factors play important roles in follicle rupture and corpus luteum formation and function, very little hard evidence about their specific actions and receptors is available. The only exception may be the insulin-like growth factors (IGFs), which have been the focus of several groups. Most of the studies on growth factors in the ovary have involved the addition of these factors to granulosa cells or other ovarian cell types in vitro and an examination of their effect on gonadotropin binding, steroid synthesis, and other markers of differentiated cell function. In the absence of adequate information about the spatial and temporal production of these factors and their receptors in the ovary, however, such studies are of limited value; the findings may or may not be of physiological importance.
The availability of sensitive molecular techniques such as in sire hybridization and reverse transcription-polymerase chain reaction now makes it possible to accurately determine where and when the genes for growth factors and their receptors are expressed in the ovary.
Leukocytes, Cytokines, and Ovarian Function
It has long been recognized that various leukocytes are present in the ovary and that their numbers increase dramatically in the corpus luteum. However, almost nothing is known about their possible role in ovarian function. There have been numerous recent reports of the presence of various cytokines in the ovary and of the effects of cytokines on the differentiated function of ovarian cells. These include various interleukins, tumor necrosis factor (TNF), transforming growth factors, platelet activating factor (PAF), and heparin. Heparin, a product of mast cells, appears to play an important role in the actions of various growth factors.
Determine the physiological role of immune system—derived products on ovarian function.
We know surprisingly little about the specialized microenvironment of the follicle, and until we do, it will be impossible to understand completely how the follicle (1) synthesizes the hormones that integrate all reproductive function and (2) nurtures the gamete to maturity. This applies to the follicular fluid, which bathes the granulosa cells and developing oocyte. While follicular fluid has been readily available, we have only the most rudimentary knowledge of the biologically active materials that it contains. We know that human follicular fluid is a potent enhancer of serum-stimulated endothelial cell proliferation. Recent reports indicate that follicular fluid stimulates sperm motility and contains a chemoattractant for sperm. The level of chemoattractant activity correlates with fertilizability of the egg from the same follicle.
• The identification of this factor might lead to ways to identify healthy eggs and to promote the fertilization process with direct benefit for women using the new reproductive techniques of IVF and GIFT.
Although traditional wisdom ascribes the role of ''master gland'' to the pituitary, the ovary may, in fact, play an active rather than a passive role in the initiation and maintenance of reproductive cyclicity and function. Thus, the ovary is central to a woman's reproductive capacity. Our limited under-standing of these reproductive events mandate continued and focused research in the areas of ovarian function described above. Not only is there an abundance of basic research topics that have important implications for solving problems of human fertility and infertility, but a better understanding of ovarian function would illustrate other normal and pathologic processes involving the regulation of cell growth and differentiation in many other organ systems.
The basic questions of physiology, cell growth, and differentiation are relevant to many human disease states that result in infertility and clinical aberrations of ovarian function. Such clinical problems as premature ovarian failure and anovulation, polycystic ovarian disease, and luteinized unruptured follicle syndrome are the province of gynecologists and obstetricians. It is, therefore, most appropriate that research on the intricacies of ovarian function be performed in departments of gynecology and obstetrics. These physician-scientists have the critical ability to investigate basic biological questions with the full knowledge and understanding of their relevance to human disease states. Not only does the gynecologist's interest, by definition, lie in the realm of ovarian function, but his or her understanding of the myriad facets of clinical aberration and disease allows for the critical link between basic scientific investigation and its applications to human disease with the potential for cure.
Reproductive cell biology can be considered the science basic to obstetrics and gynecology, but the literature, cell biology, physiology, and biochemical information dealing with the fertilization process is primarily comparative. Most of the information is derived from invertebrates, nonmammalian vertebrates, mammals such as rats and mice, and nonhuman primates. Little is derived from studies of humans.
Ignorance about the basic science of the fertilization process in humans no doubt is revealed in the high failure rate of some human in vitro fertilization procedures. This failure rate underscores the point that conclusions drawn from mice, rats, rabbits, and hamsters may not wholly apply to humans. Animal models should not, however, be abandoned. Moreover, the high failure rate may be because certain aspects of the fertilization process are unique to humans. Therefore, there is some urgency about the acquisition of a fully detailed analysis of the cell biology, physiology, biochemistry, and molecular biology of the human fertilization process. Investigations of the basic science of the fertilization process in humans (and other animal models) by an obstetrician trained in cell biology, physiology, biochemistry, or molecular biology in a department of obstetrics and gynecology can and will make important contributions to our understanding of this important process.
What follows are general aspects of the fertilization process in mammals. For details, readers are directed to the references at the end of the chapter. This discussion also offers avenues of research that appear warranted to gain further insights into the fertilization process in humans.
Fertilization is a multistep phenomenon beginning with the interaction of the female and male gametes and the subsequent interaction of maternal and paternal chromosomes derived from the female and male pronuclei. During the process of fertilization each gamete becomes activated, which leads to biparental heredity and subsequent cleavage of the non-uninucleated zygote. Fertilization involves the following steps:
Sperm first become associated with the ovulated egg with its associated cumulus cells (granulosa cells). Sperm become associated with the cumulus cells, and some are attached to the extracellular coat or matrix known as the zona pellucida.
The associated sperm brads to the zona pellucide.
The bound sperm completes the important acrosomal reaction.
The acrosomal-reacted sperm then digests its way through the thick zona pellucida.
When the sperm reaches the perivitelline space (the space between the egg and the zona pellucida), it fuses with the plasma membrane of the egg.
The fusion of the sperm with the plasma membrane of the egg initiates the release of cortical granules, thereby musing the zona reaction and the release of the second polar body.
The decondensation of the sperm nucleus occurs.
The female and male pronucleus forms.
Cleavage of the zygote takes place.
As the sperm travels through the female reproductive system it becomes capacitated, that is, competent for fusing with the egg in the ampullary region of the oviduct. Over the head of the sperm is the acrosome, a lysosome-like organelle. In an effort to expose the inside hydrolytic enzymes to the zona pellucida, the acrosome undergoes the acrosomal reaction, which is Na+ and Ca2+ dependent. This reaction consists of the fusion of the acrosomal membrane with the sperm membrane, thereby producing a series of hybrid vesicles. The hydrolytic enzyme acrosin (a trypsin-like proteinase) permits the sperm to penetrate or enzymatically digest its way through the zona pellucida.
The zona pellucida consists of three different glycoproteins of different molecular weights known as ZP (zona pellucida) 1, 2, and 3. Of interest is the fact that the mouse sperm receptor is the ZP3 glycoprotein, which consists of a 44,000-dalton polypeptide chain that has a number of asparagine-linked and serine-threonine-linked oligosaccharides covalently linked. It is believed that ZP3 is the acrosome reaction inducer alluded to above.
When the acrosome-reacted sperm makes its way by proteolysis through the thick zona pellucida, the postnuclear cap region of the sperm fuses with the plasma membrane of the egg. The fusion of the sperm with the egg's plasma membrane induces a zona reaction. The zona reaction is a biochemical change of the zona pellucida induced by the contents of the cortical granules. This biochemical change in the zona pellucida prevents other sperm from penetrating and sets up the slow block to polyspermy, the results of which induce pathological conditions. On the other hand, when the sperm fuses with the egg, depolarization of the egg's plasma membrane occurs within seconds. This transient alteration of the egg plasma membrane presumably results from an altered membrane permeability to certain ions. This depolarization of the egg plasma membrane after sperm fusion provides a fast block to polyspermy, a situation that is not well understood in mammals. However, a fast block to polyspermy in mammals should not be ruled out and requires further detailed investigations.
Cortical granules are glycoprotein-rich organelles that are produced by the corporation of the rough endoplasmic reticulum and the Golgi complex. These membrane-bound organelles migrate to the cortical cytoplasm where each becomes associated with the inner aspects of the egg plasma membrane. There is evidence that some of the cortical granules are released prior to ovulation. However, after sperm fusion, the remaining cortical granules fuse with the egg
plasma membrane, thereby intercalating their membrane into that of the egg. The conical granules, as reported by investigators, contain various hydrolytic enzymes such as proteinases and peroxidases, which are deposited within the perivitelline space and diffuse into the zona pellucida, thereby inducing the zona reaction mentioned above.
The fusion of the egg with the sperm initiates the activation process that alters the metabolic activity of the egg and sperm. As indicated earlier, the fusion of the egg produces inter alia the release of the second polar body, which means that the meiotic process is now complete—a process reinitiated by the sperm. This fusion also permits the continuation of a cascade of events that produces a maturation-promoting factor.
Following the release of the second polar body, the chromosomes form the female pronucleus, which contains a nucleolus and associated chromatin. Along with the formation of the female pronucleus is the formation of the male pronucleus. The condensed, incorporated sperm nucleus commences to lose its nuclear envelope, and the chromatin becomes dispersed and eventually acquires another nuclear envelope. The dramatic morphological changes, and no doubt biochemical changes, leading up to the formation of the male pronucleus prompted a classical embryologist to say that it is almost as if the sperm needed fertilizing.
The male and female pronuclei are large, round structures and it is difficult to tell which is female and which is male. The male and female pronuclei become closely opposed. Two asters that are established will become the poles of the mitotic spindle for first cleavage. The chromatin of each pronucleus condenses into individual chromosomes. The nuclear envelopes break down. Ultimately, the chromosomes move to the metaphase plate, and cleavage takes place, thereby forming two-unit nucleate blastomeres containing the chromosomal number of the investigated mammalian species.
Continued investigation of the role of maturation-promoting factor(s) in the reinitiation of meiosis and the continuation of egg maturation.
Continued investigation of the molecular biology of sperm chromatin processes.
Continued investigation of the biochemical composition of cortical granules and the significance of cortical granule dehiscence prior to sperm-egg fusion, as well as their general role in the fertilization process.
Determination of the physiology and biochemistry of germinal vesicle breakdown.
Further investigation of the molecular events and physiology of the formation of maternal and paternal pronuclei.
Determination of the physiology and biochemistry of male and female pronuclei (envelope) breakdown and the re-condensation of their chromosomes.
Continued investigation of the molecular biology of the zona proteins and their significance to sperm binding. Particular questions include how are zona proteins related to the slow block to polyspermy, and how sperm receptors are inactivated?
Continued investigation of the fast block to polyspermy following the sperm-egg fusion.
Investigation of the biophysics of sperm-egg-cortical granule fusion.
Continued investigation of the molecular biology of sperm capacitation.
Continued investigation of the molecular biology of the acrosome reaction with an emphasis on understanding the significance of the hydrolytic enzymes and their role in the general process of fertilization.
Definition of the molecular events of the first cleavage, focusing on the involvement of cyclins. Continued focus on each of the fertilization events, keeping in mind a possible means of interruption as a contraceptive tactic.
Fetal Growth and Development*
Research in the area of embryonic and fetal development remains an important frontier of biomedical research. Because of their unique positions as the guardians of reproductive health, obstetricians and departments of obstetrics and gynecologists should have a central role in the development of research programs concerned with human developmental biology, including the study of early embryonic events and mechanisms underlying embryoinc and fetal growth. Such research draws on the techniques and methodologies of diverse disciplines including genetics, molecular and cell biology, and physiology. Leaders in departments of OB/GYN should take the initiative in promoting the study of fetal growth and differentiation using the array of methodologies made available through modern science. A broad approach to the study of fetal growth will not only provide an opportunity to examine the basis of growth and differentiation but will also stimulate investigation into fundamental aspects of placental
function and fetal nutrition, including the study of mechanisms by which nutrients are transferred to the fetus from the maternal compartment. Departments of OB/GYN should also take the lead in examining environmental and genetic influences on fetal growth, and participate in the development of techniques and methodologies to diagnose and treat the fetus with structural or genetic defects or abnormalities resulting from maternal disease states. While there has been progress in our ability to diagnose fetal disease, the options for intervention remain quite limited. Research to develop such interventions should be vigorously pursued by departments of obstetrics and gynecology. Specific areas of research opportunity are outlined below.
Embryology and Congenital Malformations
Abnormal fetal development imposes a great societal economic burden, and its emotional costs to families and patients are incalculable. Three percent of infants will be born with a major malformation, and a much higher percentage of pregnancies terminate because of major chromosomal and/or structural defects. Congenital defects have their origin in early gestation, which is characterized by rapid cell division and organ development. The speed of these events is illustrated by closure of the neural tube between 19 and 29 days of gestation and by development of the heart from the time of the first heartbeat at 21 days to its differentiation as a four-chambered pumping organ by 56 days. Major malformations that can profoundly influence subsequent fetal growth and development are already established by the end of the third fetal month, and it is during this period of time that major genetic and structural defects commonly result in pregnancy loss. Obstetric investigators should participate in the challenge of understanding the basis of abnormal development.
Investigation of the basis of genetic regulation of early embryogenic events, including the role of homeotic genes in both normal embryogenesis and in congenital malformations.
Characterization and study of embryologic mechanisms, including cell-cell interactions, cell migration, cell matrix interactions, and programmed cell death, all of which are important in normal and abnormal development. Development and exploitation of tissue and embryo culture techniques to examine
developmental mechanisms and teratogenic influences on development including a study of drug-induced malformations as well as those resulting from conditions such as maternal diabetes or abnormal immune states.
Investigation of endocrine and growth factor signaling that modulates fetal growth and organ maturation—for example, the basis of actions of muellerian inhibitory factor (MIF) and androgens in regulating sexdifferentiation.
Fetal Growth and Placental Transport
The second trimester of pregnancy is largely a period of growth and functional refinement of organ systems that must be mature by the time of delivery. For example, the brain undergoes the waves of migration and differentiation that are the basis for neural integration and the behaviors necessary for postnatal survival. During this time there is rapid functional differentiation and growth of such systems as the lungs and gut.
During the third trimester, there is rapid fetal growth and deposition of storage fuels such as fat and glycogen, and the fetus quadruples in weight. Fetal growth throughout pregnancy can be influenced by genetic or environmental factors that are important subjects for obstetric investigation. Genetic disorders such as trisomy 18, Down's syndrome, or Turner's syndrome are obvious causes of fetal growth restriction, but the mechanisms by which aneuploid chromosomal defects result in abnormal fetal growth remain largely unexplored. Growth restriction resulting from decreased uteroplacental blood flow in such conditions as pregnancy-induced hypertension also continues to be an important cause of newborn morbidity and is representative of a group of problems that should be pursued vigorously by obstetric investigators.
Placental transport during normal development and under conditions in which nutrient flow is compromised.
The mechanisms by which specific disease states alter transport processes and the basic signaling mechanisms that regulate fetal growth and organ maturation. For example, infants of diabetic mothers with excessive substrate delivery and of large fetal size show a delay in organ maturation, whereas those with intrauterine growth restriction secondary to fetal malnutrition
exhibit accelerated lung and brain maturation. The mechanisms underlying such changes are largely unknown.
Metabolic regulation during development.
Congenital Infection and Substance. Abuse
Congenital infections can also have profound influences on fetal growth and should be part of the domain of obstetric research. This area includes viral infections such as cytomegalovirus disease, which is the most common congenital infection, and conditions such as congenital syphilis and toxoplasmosis. Congenital HIV infection is a national tragedy with thousands of infected infants, and there is a growing incidence of other sexually transmitted diseases that can have an impact on fetal and newborn health. The obstetric investigator must also be concerned about our modern epidemic of substance abuse, including heroin, alcohol, cocaine, and cigarettes. Cigarette smoking remains the most important cause of preventable mortality and morbidity, yet women continue to smoke during pregnancy. Research needed in these areas includes biological investigations as well as opportunities for epidemiologic and behavioral research.
Studies of mechanisms of maternal to fetal transmission of viruses.
Development of strategies to alter high-risk behaviors.
Investigation of pathogenesis of defects resulting from congenital infection.
Development of drug surveillance and treatment programs.
There are opportunities for perinatal research in departments of obstetrics—as, for example, studies of mechanisms responsible for the transition from fetal to extrauterine life. Important developmental events include maturation of lung and the surfactant system, intestinal maturation, and the cardiopulmonary adaptations necessary for extrauterine survival. A major challenge will be to develop a more complete understanding of influences on
fetal growth and maturation so that new therapies and interventions can be developed that will ensure the most optimal fetal outcome. Since Liggins defined the role for glococorticoids in pulmonary lung maturation nearly 20 years ago, there have been a myriad of clinical studies and evaluations, but relatively few of these have been translated into clinical trials.
Investigation of what controls the signaling that induces lung maturation in preparation for the extrauterine environment.
Development of new therapies to induce maturation.
Investigation of the influences of maternal disease states and environmental insults on maturational events.
Refinement of techniques for fetal surveillance and the development of better indices for normal and abnormal function.
Development of new systems to deliver drugs, replacement hormone therapy, or nutrients to the fetus.
There are clinical epidemiology research opportunities for departments of OB/GYN in defining and following at-risk populations and also in clinical trials involving obstetric patients. Programs in clinical epidemiology can be carried out in many institutions including those that would not have the resources for expensive and highly technical laboratory research programs. Almost every aspect of obstetric practice can generate important questions.
How does prenatal care reduce perinatal morbidity?
How can we measure the effectiveness of social and behavioral interventions in changing high-risk behaviors that impair and limit fetal development?
How do specific obstetric interventions—for example, cesarian section and maternal nutritional supplementation—affect newborn outcomes?
There are many additional research opportunities for departments of OB/GYN that are not developed here. These include technologies for fetal surveillance and diagnosis, which have opened a new window on early developmental events; embryology and fetal physiology have been a major focus of the new biology, and departments of obstetrics should be full participants in the research being conducted.
Preterm birth is a major health hazard of humans worldwide, being the leading cause of newborn and infant mortality and the principal cause of significant, often severe, morbidity for infants who survive. And ironically, the successful salvage of very small neonates today is largely attributable to rapid advance in neonatal care, which makes it inevitable that even more premature infants are now destined to sustain life-long disabilities. The only apparent solution to this devastating health problem is the prevention of premature birth. There are a number of different causes of preterm birth, including maternal and fetal complications that mandate delivery independent of the onset of labor, and preterm rupture of the membranes. But regrettably, preterm labor remains a common problem not yet solved in spite of the widespread use of tocolytic agents to arrest myometrial contractions. Therefore, new approaches must be developed to enable us to arrest or prevent the parturitional process when preterm labor threatens a pregnancy.
Preterm, Premature Rupture of the Fetal Membrane
Preterm delivery is the natural outcome of preterm, premature rupture of the fetal membranes. After the integrity of the fetal membranes is compromised, by whatever cause, preterm labor commonly follows. If preterm labor does not begin soon after membrane rupture, infection by way of colonization by microorganisms arising in the vagina or endocervix will precipitate preterm labor or else threaten the fetus by way of fetal respiratory movemeats that carry the infected amniotic fluid into the fetal lungs. The incidence of preterm, premature rupture of the fetal membranes as the primary cause of preterm delivery varies somewhat among populations of pregnant
women, but generally accounts for 35 percent to 40 percent of preterm births. Despite the devastation wrought by this complication of pregnancy, our understanding of the regulation of synthesis and degradation of the extracellular matrix of the fetal membranes and contiguous decidua parietalis is primitive. The metalloproteinases have been isolated, the amino acid sequence is known, and the genes have been cloned. The same is true of the major metalloproteinase inhibitors. Much is known of the regulation of synthesis of these major determinants of extracellular matrix formation and degradation in other tissues, but little or no information is available concerning the fetal membranes.
Research must be directed to understanding the regulation of synthesis and degradation of the extracellular matrix of the fetal membranes and contiguous decidua parietalis.
It is suspected, but not established, that infection by way of the action of bacterial toxins (lipopolysaccharide, or LPS) may serve to initiate the formation of metalloproteinases that act upon the extracellular matrix of chorion laeve and amnion. We must ascertain if this is a mechanism by which fetal membrane rupture is commenced because if this is indeed the case, the condition is theoretically preventable.
Complications of Pregnancy That Compromise Fetal or Maternal Well-Being Independent of the Onset of Labor
Depending on the population of pregnant women studied, 25 percent to 30 percent of preterm deliveries are mandated by complications of pregnancy that beset the mother or fetus, or both, that are independent of the onset of labor. Among the major categories of complications are pregnancy-associated hypertension that sometimes is also associated with abruptio placenta, diabetes mellitus, intrauterine growth retardation, and multiple pregnancies. Pregnancy-induced hypertension (PIH) or preeclampsia/eclampsia remains a common problem of pregnancy, especially in a first pregnancy. It is difficult to generalize about the incidence of hypertensive disorders in pregnancy because of the striking variations, depending on parity, socioeconomic status, and race
of the population examined. Nonetheless, in all populations of carefully monitored women, the incidence is greater than 2.6 percent. The subject is reviewed in Williams' Obstetrics (18th ed., pp. 656–657). Commonly, the incidence for hypertension among all pregnant women in the United States is believed to be 5 to 6 percent. With limited family size, however, a larger proportion of the obstetric population now is accounted for by women having their first child; and in nulliparous women, the incidence of PIH is much greater than in multiparous women. And with respect to the major problems of pregnancy, for instance, maternal death, fetal growth retardation, abruptio placenta, and so forth, all forms of hypertension in pregnancy must be considered high-risk factors. In approximately 50,000 pregnancies at Parkland Memorial Hospital, the incidence of pregnancy-reduced or pregnancy-aggravated hypertension was 13 percent. Indeed, 20 percent of nulliparous women developed hypertension. Although a largely indigent population was studied, it is a population in which the rate of delivery of prenatal care is high.
There is some optimism that modifications in the formation of prostaglandins at the level of platelets may reduce the incidence or severity of pregnancy-induced hypertension. Such modifications are accomplished by daily administration of low-dose aspirin. At best, however, this appears to be a temporizing approach, and little insight has been gained into the cause or methods of preventing this disorder that threatens fetal well-being and in some cases maternal health. Moreover, many pregnancies are threatened by failure of fetal growth, with or without coexisteat mammal hypertension. Yet we have no knowledge of the basic pathophysiology of the causes of severe fetal growth retardation.
Research is needed on the pathogensis of pregnany-associated hypertension.
Research must be directed toward defining the pathophysiology of the processes that mandate delivery prematurety even though independent of labor. Commonly, the obstetrician is faced with choosing between a deteriorating intrauterine environment for the fetus and the neonatal intensive care nursery for a sick newborn.
Preterm Onset of Labor
Preterm labor in pregnancies with intact membranes accounts for 30–40 percent of preterm deliveries in most populations. The incidence of preterm birth in the past 10 years has not decreased despite the extensive use of B-mimetic tocolytic agents. Indeed, it is estimated that more than 100,000 pregnancies in the United States alone are treated each year with such agents; yet there is no evidence that such treatments have resulted in a significant reduction in preterm birth or perinatal mortality. The reasons for this are probably multiple, including severe side effects of such drugs in the mother, down regulation of beta-adrenergic receptors, and complicating factors of pregnancy that preclude the use of or the effectiveness of the tocolytic drugs.
For the past 25 years, investigators have sought to define the nature of a presumed uterotonin that is produced in incase amounts at or near term to cause the spontaneous onset of labor. The most thoroughly studied candidates have been oxytocin and prostaglandins. Yet it now seems likely that the generation of these uterine contractants, in increased amounts, occurs after the onset of parturition and not before. Perhaps such agents act in the normal parturitional process to facilitate and maintain labor once parturition is initiated by some other mechanism.
Possibly the most curious feature of normal human pregnancy is the remarkable tolerance of the uterus to its burden. The myometrial smooth muscle is, inherently, a contractile organ. Strips of human myometrium placed in an organ bath contract spontaneously. If a very small intrauterine device is placed into the uterine cavity of a nonpregnant woman, the uterine contractions commonly are so severe as to produce expulsion or to necessitate its removal because of the pain that occurs. But in normal pregnancy, the uterus remains quiescent, accepting intrauterine distention to accommodate an 8-pound baby, 1–2 pounds of placenta and fetal membranes, and 1 liter of amniotic fluid.
Information must be assembled to understand the fundamentals of the maintenance of pregnancy and the spontaneous initiation of parturition at term.
What are the physiological processes that effect such a stronghold on uterine contraction during human pregnancy?
How are these processes translated at the biomolecular level?
What is the role of the fetus in the maintenance of pregnancy and in the retreat from pregnancy maintenance at the end of normal gestation? It now seems very likely that retreat from pregnancy maintenance is the most likely choice of potential mechanisms for the initiation of spontaneous labor at term. Therefore, we must define in great detail the processes that bring this remarkable situation about.
An understanding of the contractile properties of the uterus before and during pregnancy must be gained.
The role of Ca2+ channels and Ca2+ sequestration must be understood as these apply to the uterus of pregnancy.
The contribution of the unusual hormonal milieu of human pregnancy to the maintenance of uterine quiescence must be investigated. Before we can realistically address the causes of preterm labor, an understanding of these processes operative in normal human parturition at term must be acquired.
Preterm Labor and Infection
The only major clue to the cause of preterm labor is that ''silent'' infection in some pregnancies appears to lead to the premature onset of labor. It is envisioned that colonization of fetal tissues (fetal membranes [amnion and chorion laeve]) or maternal tissues (uterine decidua parietalis), or both, with microorganisms arising by ascending spread from the vagina or cervix, may cause preterm labor. Specifically, the elaboration of bacterial toxins may cause the generation of cytokines, namely, IL-1 and TNF-a, which act in many tissues to provoke prostaglandin formation. This may be true; but it has not been established definitively whether evidence of infection (inflammation) is the cause or the consequence of labor. That is, did inflammation cause the onset of parturition, or did labor beget the inflammatory processes? This is an extremely important issue because if infection is a common cause of preterm labor in pregnancies with intact fetal membranes, there is reason to believe that preterm labor in those instances could be prevented. On the other hand, if preterm labor leads to inflammatory processes, we would have no success in preventing preterm labor by use of antimicrobial agents. At present, millions of dollars are being expended each year to treat women with antibiotics to evaluate this issue. If inflammation follows the onset of labor, such ventures are doomed to failure.
There are other considerations of great importance in defining the cause of preterm labor. Clinical data make it clear that there must be a heterogeneous group of associated disorders. One of the common antecedents of preterm labor
is preterm cervical effacement and dilatation. A large number of pregnancies that terminate in preterm labor will be known to have preterm cervical dilatation without apparent increased myometrial activity for days to weeks before the onset of active labor. And in some such cases, the preterm labor is very prolonged; that is, slow cervical dilatation may continue with or without contractions noted by the pregnant woman for days. During this time of cervical patency without vigorous contractions, the forebag is exposed to microorganisms and bioactive agents in the vagina. Thus, the superimposition of an inflammatory response may both confuse and facilitate the progression of labor.
Research must be conducted to establish the role, if any, of infection in the preterm onset of labor.
An understanding of the cause or cause, of preterm cervical dilatation is urgently needed. We must understand the role of this condition within the sequence of events that terminatej in the preterm onset of labor. In addition, it is commonly believed that extrauterine refections, such as pneumonia, appendicitis, and pyelonephritis are commonly associated with preterm labor.
The nature of the pathophysiology of the association with preterm labor and extrauterine infections also must be defined. Other conditions, including fetal abnormalities with or without hydramnios, also seem to be associated with the preterm onset of labor. Thus, there may be multiple causes of preterm labor or else a common cause mediated by way of several contributing factors.
Role of the Obstetrician in Research to Define Physiological and Pathophysiological Mechanisms of Spontaneous Parturition at Term and the Preterm Onset of Labor
The obstetrician is ideally suited by training and experience to conduct fundamental studies related to the problem of preterm delivery and preterm birth, provided he or she has acquired skills in basic science research. The anatomical relationships of fetal and maternal tissues are understood best by the obstetrician. It is appreciably easier for the obstetrician to place in perspective the role of trauma sustained during labor and the physiological or
pathophysiological sequelae thereof in the category of consequence, not muse, of parturition. The obstetrician can appreciate the potential role of infection, whether it occurs before or during labor, in the parturitional process. The obstetrician also is best suited to distinguish between presumed preterm labor and labor that does in fact eventuate in preterm delivery. And the obstetrician is best suited to deal with the complications of pregnancy, revolving either the fetus or mother, or both, that may mandate delivery independent of the onset of labor. Thus, there is an urgent need for obstetricians trained in the basic sciences to conduct research to define the sequence of biomolecular events that lead to the initiation of parturition and to seek to identify the muses of preterm labor.
There are numerous studies on the biological consequences of various contraceptives. Even though the focus of most research has been on the risks of contraceptives, the benefits continue to be elucidated. For example, it is now established that oral contraceptives (OCs) offer women protection against gonococcal pelvic inflammatory disease. (PID), anemia, ovarian cysts, and ovarian and endometrial cancer. There is also an array of evidence to suggest that women using OCs are also protected against osteoporosis and atherosclerotic cardiovascular disease. These are impressive beneficial effects for an agent that is taken primarily to prevent unwanted pregnancy. In spite of this, women are not happy with their contraceptive options. At best, 50 percent of current contraceptors are satisfied with their present method. Thus, there is an opportunity for research on contraceptive development with an emphasis on methods that will have beneficial effects for users.
Develop contraceptives that protect women against breast and cervical cancer.
Increase user satisfaction by offering contraceptors a wider array of choices.
Provide contraception for some underserved groups including men, lactating mothers, teenagers, and permenopausal women.
Develop contraceptives that protect women against sexually transmitted diseases (STDs).
If contraceptive research is to be successful, it must be conducted with the cooperation of women who wish to have new and better methods of family planning. Departments of obstetrics and gynecology are in an almost unique position to develop new contraceptives since many young women seek advice from gynecologists for reproductive health care, including contraception. Thus, the gynecologist can study the desires of women and conduct appropriate clinical research into new contraceptive methods.
In the past, almost any kind of research in the field of reproduction has been considered contraceptive research with the view that any new finding could conceivably lead to a new method of fertility control. However, since new ideas can take up to 20 years to be developed into a method that can be used in clinical practice, one must reasonably conclude that a major portion of contraceptive research should focus on studies that will advance the introduction of new clinical entities in the foreseeable future, preferably within the lifetime of the investigator. As a consequence, the agenda that follows is focused on methods that could be completed in the present century, were funds available to conduct the research.
The recent introduction of Norplant has offered women a new option for long-term, low-dose, progestin-only contraception. In view of the acceptability of this method, it is clear that improved implants using levonorgestrel and other progestins such as 3-ketodesogestrel, ST 1435, and 19 nor-medroxyprogesterone acetate (19 nor-MPA) would be highly desirable. These highly potent progestins are good candidates for a single or double implant system that will provide contraception for 1 to 3 years. The ability to provide effective contraception with one or two implants would be a major improvement over Norplant, the six-capsule system, which is the only approved contraceptive implant. ST 1435 and 19 nor-MPA are structurally similar to progesterone, distinguishing them from most other currently marketed progestins, which are related to androgens and estrogens. Like progesterone, and unlike most other progestins, ST 1435 and 19 nor-MPA have no apparent effect on serum
lipoprotein patterns. In addition, the fact that some of these progestins, like progesterone, are rapidly metabolized by the liver after oral administration, suggests that they may be suitable contraceptives for lactating women, since any steroid transferred to the infant through the milk would be inactivated.
The contraceptive action of progestins used in implants must occur at dosages of 50 to 100 µg/day so that inhibition of ovulation and alteration of cervical mucus will occur at doses that can be delivered by this method. At these doses, many of these progestins have not shown unexpected effects m humans. For example, in clinical trials, ST 1435 had no apparent effect on carbohydrate metabolism, serum lipoprotein patterns, and hepatic proteins such as hormone-binding globulins. The most common complaint associated with implant use, and with all other forms of progestin-only contraception, is bleeding irregularities. Other side effects have been minor medical conditions, such as headache or acne.
Develop new drug delivery systems for steroids that would improve the pharmacokinetic profile to eliminate long-term tail-off of drug release once implants were sufficiently depleted of steroid as to be ineffective.
Assess the carcinogenic and other long-term effects of progestins on the breast, cardiovascular system, and other organs.
Conduct and evaluate implants in clinical trials.
Conduct long-term studies on NORPLANT to determine the health benefits and risks of long-term, low-dose, progestin-only contraception compared with combined oral contraceptives.
Develop biodegradable implants that can be removed at any time and that do not have a long period of drug tail-off.
Conduct studies in lactating women with ST 1435.
Contraceptive rings (CRs) that deliver steroids by the vaginal route have several advantages over other methods of contraception. First, tings are convenient since, unlike oral contraception, they do not require daily administration or attention. Second, steady blood levels of steroid resulting from the ring's constant drug release allow for more efficient contraception, thus lowering
contraceptive dosage, which diminishes the likelihood of adverse effects. Third, if estrogen is included in the CR, the adverse reactions that may be associated with oral contraception are milder. Fourth, ring contraception is under user control, a feature desired by many women.
Contraceptive rings may be formulated in two ways. The first type consists of a progestin combined with an estrogen. Any progestin/estrogen combination used in contraceptive pills could, in theory, be used in CRs. A progestin/estrogen-containing ring is used for 3 weeks and is then removed for 1 week to allow menstrual bleeding. This 3 weeks-in/1 week-out schedule is continued for the lifetime of the ring. Such a formulation is designed to inhibit ovulation and to produce minimal menstrual disturbance. There are currently no combination rings on the market, but several are being developed.
The second type of ring is a progestin-only formulation. Once inserted, this CR remains in the vagina continuously, and its effectiveness depends on a combination of actions of the progestin, including inhibition of ovulation and thickening of cervical mucus. The only such CR available is one that delivers levonorgestrel (20 µg/day); it will soon be marketed in Europe. This CR has a failure rate of 3.0 percent but without a significant incidence of ectopic pregnancies. Since a large number of ectopic pregnancies were expected but not observed, it is possible that the vagina may be the optimal route for progestin-only contraception. The ring also offers a more convenient method of delivering continuous progesterone to women, and such rings have been shown to provide effective contraception in lactating women.
Determine the optimal steroid for use in different CRs.
Determine how much the hormone dose can be decreased without compromising effectiveness and safety.
Perform specialized phase 2 studies on CRs to determine whether vaginally administered steroids are different from orally administered steroids with respect to ovarian function; lipoprotein levels; metabolism; effects on cervical, uterine, and vaginal pathology; and carbohydrate metabolism.
Determine the long-term effects of CR use.
Transdermal delivery of drugs has received increasing attention during the past decade because of the potential advantages of controlled continuous drug administration. Transdermal delivery of steroids is also an expanding field, especially in its application to hormonal replacement therapy. Transdermal application of estrogen has been used in the treatment of postmenopausal women. Percutaneous absorption of progesterone has been demonstrated in women as has treatment of male hypogonadism with transdermal testosterone. There are currently no transdermal contraceptives, but the popularity of this route of drug administration suggests that such methods would be highly acceptable. Both progestin-only and progestin/estrogen methods are possible. These drugs could be delivered by patches applied once each week or by a cream applied daily.
Determine what type of transderrnal delivery will be most acceptable to women: high-tech patches vs. low-tech creams.
Conduct optimization studies to select appropriate contraceptive steroids and their proper doses.
Determine subject-to-subject variability in absorption using pharmacokinetic studies.
Conduct local dermal irritation and toxicity studies. Conduct clinical studies for effectiveness.
Intrauterine Devices (IUDs)
IUDs are the most widely used reversible methods of contraception in the world. Only sterilization is used by more couples as a method of birth control. The popularity of IUDs is due to their ease of use since they require no action by women once they are inserted. Furthermore, the improved designs of modern IUDs have increased their effectiveness and reduced side effects while providing contraception at a very low cost. In addition, IUDs can carry delivery systems that target drugs to a selected part of the reproductive tract. The prevalence of IUD use in Norway, Sweden, and Finland ranges between 20 and 30 percent of contraceptive users; in the United States, it is less than 14 percent.
These observations suggest that factors that are not operative in other developed countries influence IUD use in the United States.
Conduct behavioral studies to determine why women do not wish to use IUDs and why many health care workers will not insert them.
Develop effective methods to identify those women who are not good candidates for IUD use, that is, those who will have to discontinue IUD use because of bleeding and pain.
Develop IUDs that act as barriers to infection of the upper reproductive tract.
Develop hormone-releasing IUDs that will further reduce IUD side effects.
There is such a bewildering array of combined oral contraceptives available to women throughout the world that it may be impossible to distinguish the relative risks and benefits of individual formulations. Since the doses of contraceptive steroids used in such pills have been progressively lowered, it is likely that the risks and benefits of currently used OCs are different from those used years ago. Thus, there is a continuous need to monitor the long-term effects of combination and progestin-only oral contraceptives, particularly in women who begin use of these agents at an early age. Although it is not reasonable to argue for the development of new Ocs based on progestins or estrogen/progestin combinations, new drug combinations would be welcome, such as the recently recommended method which uses progestin plus mellatonin for 3 weeks followed by mellatonin for 1 week when menstruation occurs. Since mellatonin is a hormone of seasonally breeding animals, its action in humans warrants study.
Study the long-term consequences of Ocs, and determine the mechanism of action of mellatonin in women.
There is a need for convenient formulations that when introduced before coitus will protect against the transmission of HIV and chlamydia, as well as other STDs, and will, in addition, provide protection against pregnancy. With heterosexual transmission of HIV being the principal route of infection worldwide and the percentage of AIDS caused by heterosexual transmission growing in the United States, there is a pressing need to offer protection against this mode of sexual transmission of the HIV virus. Although the widespread distribution of chlamydia refections and their consequences are not well known by the public, it is currently estimated that chlamydia infections are the most prevalent sexually transmitted disease. The classical disease mused by chlamydia is lymphogranuloma venereum (LGV). Although rare in the United States, LGV is common in developing countries, especially in central Africa. In this country, about 4 million chlamydia infections occur each year. It is a particular threat because infections frequently go undetected and thus cause tubal damage that results in infertility and ectopic pregnancies. When symptoms axe present, they can include cervicitis and salpingitis in women and urethritis and, occasionally, epididymitis in men. Although chlamydia can be successfully treated with antibiotics, damage is frequently severe before this condition is detected.
The goal of identifying novel spermicides has several motivations. Spermicidal agents approved for use in the United States are limited to surface-active agents (with the minor exception of phenylmercuric borate), with nonoxynol-9 (N-9) being by far the most widely used. Considerable evidence from both clinical trials and animal studies indicates that N-9 causes microulceration of the vagina. This is of concern since epidemiological evidence suggests that microulcerations increase susceptibility to HIV infection. At least as important as the need for alternative spermicides is the expectation that antifertility action would add to the incentive to use a product giving protection against one or more sexually transmitted diseases. Any new product that has both antifertility and anti-STD activity should perform as well as N-9, which will be the basis of selecting and testing new products. Unlike the forms of contraception mentioned above, development of barrier methods against HIV and other STDs will be long-term research projects, and is likely to be quite expensive.
Select candidate compounds from results of previous screening tests on sperm and STDs.
Test candidate compounds for evidence of antifertility effects and effectiveness against selected STDs in vitro. Prepare formulations (suitable for human use) of individual multiple compounds for animal tests. Test formulations in vitro.
Test selected formulations for evidence of effectiveness in animal model systems.
Prepare selected candidates for tests of effectiveness in humans.
Conduct comparative trials in humans.
There are no contraceptive methods for men other than condoms and sterilization, and while there are compelling reasons related to STD transmission to argue for increased utilization of those procedures, there is also a desire to provide new methods for this underserved population. The two approaches currently under investigation use drugs that either act directly on male germ cells or withdraw their hormonal support. To date, all drugs that act directly on germ cells have toxic effects and are relatively nonreversible, while hormonal suppression is readily reversible but is not as effective. The major approach to new contraceptives has been to improve hormone suppression using analogs of luteinizing hormone-releasing hormone (LHRH). controls the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. However, large doses of LHRH analogs block the secretion of both of these hormones, which are necessary for the production of sperm. Administration of LHRH analogs in men has resulted in decreased serum testosterone, sperm counts, and sperm motility. If the LHRH analog treatment is supplemented with an androgen, there are no signs of androgen deficiency, but suppression of sperm production may be less effective depending on when the androgen is given in relation to the onset of analog treatment. Some studies suggest that an LHRH antagonist may be better than an agonist, but there are
no studies in which the actions of agonists and antagonists have been compared when both are delivered from an optimally formulated release system.
Over the past several years, injectable and implantable peptide delivery systems have been formulated that could be effective for months to years when used as a component of a male contraceptive. This second component will be used as an androgen injection or implant.
Determine whether LHRH agonists or antagonists are the optimal component of a male method.
Develop long-term delivery systems for LHRH analogs.
Select an appropriate androgen for long-term administration, and develop an appropriate delivery system.
Conduct phase I and 2 clinical studies of the androgen and the LHRH analog.
Investigate new approaches by developing methods that will interfere with the autocrine/paracrine control of germ cell maturation in the testis (a long-term objective).
The concept of antifertility vaccines was introduced with immunization against either human gonadotropin-beta (hCG-B) or fragments of this gonadotropin subunit. Currently, three clinical trials are under way to evaluate various forms of this contraceptive vaccine. In the future, a series of other antigens will be investigated.
Immunological approaches to contraception are likely to be very long-term approaches to contraception for a variety of reasons:
Antigens are not readily available and must be produced by direct synthesis or recombinant DNA technology.
It is unlikely that a single antigen will produce a uniform response in a heterogeneous population, such as humans.
There is a long delay from the onset of treatment while antibody titers rise.
For the vaccine to be used widely, the incidence of idiosyncratic reactions must be very low.
Sperm Antigens Since autoantibodies to sperm are associated with infertility, sperm antigens appear to be attractive candidates for antifertility vaccines. A major obstacle will be the lack of purified sperm protein in sufficient quantities owing to the scarcity of human sperm to perform the biological assay for antifertility activity. To avoid having to rely on human as a starting material for antigen isolation, it will be necessary to produce the sperm proteins by recombinant technology or to synthesize peptide segments of the sperm protein. Such an approach will require a team of scientists including molecular biologists (for cloning, DNA sequencing, and preparation of expression vectors), a protein chemist (for peptide synthesis), a reproductive biologist (for gamete and fertility testing), and an endocrinologist (for clinical trials). These researchers must accomplish the tasks below.
Isolate a full-length cDNA that encodes promising sperm proteins, and determine their nucleotide sequences.
Identify the nucleotide segment encoding the extracellular domain of membrane proteins and the entire sequence of secreted proteins; express such proteins in the baculovirus or similar expression system; and isolate expressed proteins for biological testing.
Study the effect of immunization with the recombinant proteins and/or synthetic polypeptides.
Produce a human dosage form, and test it in animals.
Perform trials in humans.
LHRH-Vaccine Used With or Without a Vaccine to the Luteinizing Hormone or FSH Receptor The rationale for tiffs approach is based on the principle of controlling LH and FSH actions by immunoneutralization. Although there are several immunological strategies to control LH and FSH activities, the one that has shown the most promise is LHRH coupled with a carrier protein. Animals given this antigen become infertile as titers rise. An attractive alternative approach is to interfere with hormone-receptor interaction on the surfaces of the selected target cells in the gonads (i.e., Leydig cells in the testis and luteal cells in the ovary for LH, and Sertoli cells and granulosa cells for FSH), by
immunoblocking the receptor. The achievement of cloning and sequencing of rat and porcine LH and rat FSH receptor cDNAs opened the path to produce the extracellular domain of the LH and FSH receptors by recombinant DNA technology, to synthesize specific peptide segments of the receptor, and to determine the receptor regions that participate in the binding of the hormone and in the stimulation of the signal-transducing pathway. Identification of these critical segments of the LH and FSH receptors offers an opportunity to interfere with hormone action by raising antibodies against these specific regions alone or in combination with the LHRH antigen. The objective is to demonstrate that immunization with specific LH and FSH receptor segments will result in alterations of reproductive functions and reduce a state of infertility in experimental animals. The same team approach will be used as for sperm antigens.
Conduct trials of the LHRH vaccine in animals and humans.
Prepare recombinant polypeptides of the LH and FSH receptors, and study their immunogenicity.
Prepare synthetic peptide segments of LH and FSH receptors corresponding to the hormone-binding and adenylate cyclase-stimulating domains, and conjugate the peptides with a carrier protein.
Establish immunogenicity of the LH and FSH receptor peptide segments by determining the interaction of antibodies developed against specific receptor peptide segments with the recombinant extracellular domain of the respective receptor, and with isolated ovarian and testicular membranes containing the LH and FSH receptors, respectively.
Immunize male and female rats with various combinations of LHRH-antigen and specific LH/FSH receptor peptide segments, and determine their effects on sex steroid production, gonadotropin secretion, spermatogenesis, ovulation, and fertility.
Since U.S. government funds, which support most biomedical research, may not be used for research on medical abortifacients, such research cannot be conducted without private support. Studies are vitally necessary to achieve
safer, more effective, and more acceptable methods for terminating pregnancy without surgery.
RU 486 is effective in terminating established pregnancies. When combined with a prostaglandin, RU 486 is 95 percent effective in inducing abortion in pregnancies of less than 7 weeks' duration; however, this combination reduces abdominal cramps and requires multiple trips to the clinic, which adversely affects acceptance and compliance. For medical abortion to be used widely in outpatient clinics, especially in inner-city clinics, effectiveness of the regimen ought to be increased to greater than 99 percent; it should be effective throughout the first trimester of pregnancy; and it should be administered in a way that will improve acceptance and compliance. There is also concern about the extremely limited availability of the RU 486 regimen. Since RU 486 is not and may not become widely available to women, and since, in any case, it should be seen as the beginning, not the end, of medical abortifacient development, work in this field should move forward vigorously. Thus, a better regimen beyond the one currently used (RU 486 plus prostaglandin) is still needed. Additionally, as noted, a separate and important issue is that RU 486 is not available to most women. Because RU 486's availability remains uncertain at best, the alternative drug regimen should not depend on RU 486. since it is probable that an antiprogestin will be a component of the new medical abortifacient, a substitute for RU 486 should be identified.
Identify an antiprogestin that can be used as a substitute for RU 486 in a new medical abortifacient.
Test combinations of an antiprogestin, anordrin analogs, progesterone synthesis inhibitors, and prostaglandins in pregnant animals to determine the lowest effective dosages in terminating pregnancy.
Determine the window of effectiveness during the postcoital period when the combined drugs could be most effectively administered.
Select the most promising combination of drugs for small-scale clinical trials, and perform the appropriate toxicology.
Develop an appropriate delivery system so that the drug combination could be administered in only one clinical visit.
Investigate the acceptability of new delivery systems to users and providers.
It has been said that infertility is reaching epidemic proportions in the United States. Certainly, the overall number of couples who are involuntarily infertile has increased, and today, as many as one in nine couples are unable to conceive a child. This increased incidence of infertility stems from a number of biological and sociological factors including an increased incidence of sexually transmitted diseases resulting in adhesions and tubal infertility, the increased number of women pursuing careers and delaying childbirth until their late 30s or 40s, and a wide variety of environmental toxins with putative effects on both male and female fertility Couples turn to the gynecologist, and to subspecialists in the gynecologic discipline of reproductive endocrinology, for clinical evaluation and therapy for infertility. This clinical need and the gynecologists' ongoing interest in the reproductive biological events of conception and implantation make it appropriate that the research issues of infertility be undertaken by departments of gynecology and obstetrics. These physicians care for infertile couples and are ultimately responsible for the therapeutic interventions resulting in successful pregnancy. Innovative and focused research in both the pathophysiology of infertility and its therapies will benefit the thousands of couples who are involuntarily infertile. Also, dissecting the basic processes that render both men and women incapable of conceiving children will certainly suggest new methodologies for fertility control and contraception.
In order to conceive a child, the following stringent requirements of both male and female physiology must be met. First, a sufficient number of motile sperm must be produced with the appropriate enzymatic apparatus to penetrate the zona pellucida of the oocyte. To achieve successful fertilization, the male gamete must be normal and functional, and the female oocyte must be properly matured by appropriate ovarian follicular development. Sperm motility is required to propel the sperm from the exocervix through the female reproductive tract to the ovarian follicle and the site of fertilization. An obligatory requirement for this transit is the presence of hospitable cervical mucus. At the time of ejaculation, sperm are deposited around the exocervix and require estrogenized, watery cervical mucus to maintain motility. We know almost nothing of the chemical changes of cervical mucus that allow sperm to pass into the uterine cavity or of the immunologic requirements and barriers that protect this open passageway into the female peritoneal cavity and prevent ascending
infection. The female requirements are even more complex, mandating cyclic ovarian function, including the attendant hormonal fluxes that affect and prepare all the reproductive organs, the fallopian tube, uterine lining, and cervix for conception.
Once successful fertilization is acheived, the slowly developing human embryo must journey from the distal fimbria through the various portions of the tube and into the uterus in order to implant in the endometrium. The fallopian tube is far more than a simple conduit; patency is not sufficient. The complex but poorly understood interaction of ciliated tubal epithelium and muscle contraction, which actually transport the developing embryo, as well as the mechanisms for nourishing it during its week-long transit, must be perfectly timed for conception to occur. If aberrations occur, a very early and often undiagnosed pregnancy loss may result or, more seriously, the life-threatening situation of an ectopic pregnancy implanted along the course of the tube.
After the developing embryo reaches the uterus, midway through the luteal phase, the proper sequence of implantation events must occur to maintain the growing pregnancy. Although implantation of animal embryos has been well studied, we know that the reproductive events in humans are, in many ways, vastly different from those in lower mammals. Increased research on primate models of implantation, as well as information gleaned from the new reproductive technologies such as in vitro fertilization, are needed to enhance our knowledge of the events of human implantation.
In order to have a child, all of the above processes must work properly and in the appropriate chronological sequence. Infertility can result from abnormal physiologic response at any of the described points along the continuum of gamete function, fertilization, transport, and implantation. This section will define six major areas in which a focused effort of research would both increase our understanding of the basic reproductive physiologic processes and lead to new therapies that will allow infertile couples to conceive and bear children.
A structured, comprehensive research program, including an epidemiologic description of the etiologies of infertility and basic research in cervical, tubal, and sperm development and function, would both expand our knowledge and the therapies available for infertile couples.
Specific disease processes associated with infertility, such as endometriosis and tubal adhesions, need investigation.
The new reproductive technologies of in vitro fertilization and gamete intrafallopian transfer (GIFT) offer a tremendous opportunity for understanding the specific cellular processes of human reproduction.
Many women's health issues are cloaked in emotional rhetoric without substantiating statistical support. An example is in vitro fertilization of which the public continues to be sometimes unaware, and at other times overly optimistic regarding the true success rates currently attainable. The entire area of infertility is dotted with suppositions and unsubstantiated estimates. It is ''common knowledge'' that infertility, is on the increase, perhaps in epidemic proportions, in this country. Yet recent epidemiological data have clarified that about 8.4 percent of reproductive-age women (15–44 years) had unresolved, impaired fecundity in 1982, and this percentage was virtually unchanged in 1988. However, there was a significant increase in the actual number of women reporting infertility, secondary to the trend of delayed childbearing and to the fact that more women were entering this age group (25–44 years), because of the "baby boom" generation. As an example, there was a 37 percent increase (from 454,000 to 620,000) in the number of women aged 35–44 who were without children from 1982 to 1988; however, expressed as a percentage for these years (21 percent), this figure was essentially unchanged.
Solid epidemiological data are lacking for relating many aspects of reproductive health and function to environmental toxins. Likewise, there is a dearth of solid data in the field of in vitro fertilization. Despite the attempts of a national registry in this area, the reporting is strictly voluntary, and there are no checks on the individual clinic's accuracy, and no punitive procedures for lack of reporting. Along these lines, even firm data on "normal" fecundity and fertility are difficult to obtain; monthly fertility (i.e., the chance of conceiving in any particular cycle) has been estimated from 9 percent to 25 percent, and estimations of chromosomal abnormalities in early pregnancy losses have ranged from 25 percent to 75 percent. If, indeed, nearly 50 percent of human oocytes and 10 percent of male sperm are karyotypically abnormal, substantial natural barriers to successful artificial reproduction are in place.
In the present climate of potentially unlimited achievement in reproduction, the concept that age is no barrier needs to be examined, both for practical as well as emotional reasons. Until very recently, it has generally been accepted that female fecundity is minimal after age 40 and virtually non-existent after age
45. Due to lavish publicity in recent in vitro fertilization cases, the public may now perceive age as no barrier to fertility. In fact, good studies clarifying the relationship of natural fertility and age currently do not exist. One simply cannot give a knowledgeable answer to the question from a 46-year-old woman (or even from a 40-year-old) regarding her chances of natural conception.
Sound epidemiological data on many aspects of women's health and reproductive function are lacking. The research areas discussed below are by no means an all-inclusive list but rather provide the starting point for a concerted effort to develop and maintain not only normative data on both male and female fecundity, but also the effect of both the environment and sociologic behaviors on fertility and childbirth.
Research is needed on the effect of chemical contaminants on sperm and oocyte function. In addition, more research on the effect of such substances as alcohol, tobacco, and drugs on gametogenesis and fertilization is necessary.
Firm, normative data on normal fecundity and fertility, and a multitude of other reproductive issues, are needed for comparative data as the newer reproductive technologies continue to expand.
There is a need to ascertain the relationship between age and human (both male and female) fertility.
Cervical Physiology and Function
The cervix is the gateway allowing sperm to enter the female genital tract. Concomitantly, it provides an entryway directly into the peritoneal cavity, potentially resulting in ascending refection. Thus, the cervix must not only allow entry of sperm but must protect against bacterial and vital infection. Remarkably little is known about the physiology of the secretions that facilitate sperm motility while providing an immunologic barrier.
The cervix is lined with a highly active secretory endothelium that produces mucus in a hormonally dependent fashion. Under the influence of circulating estrogen, the cervical mucus becomes thin, clear, and acellular allowing optimal sperm motility and passage into the female genital tract. After ovulation, when progesterone levels increase, the mucus changes to a thick viscous secretion that is virtually impenetrable to sperm. Not only is our knowledge of cervical
secretory physiology extremely limited, but there are no good diagnostic tests of mucus function that can be shown to correlate with conception or birth.
It is estimate that as many as 5 percent of infertility patients are unable to conceive because of a cervical factor: either inadequate or insufficient mucus or an antibody-mediated immunologic response to sperm.
It is hoped that the long-range outcome of studies described below will be improved therapeutic modalities to address questions of cervical infertility.
Research is needed to understand the physical and chemical properties of cervical mucus that facilitate sperm motility and to develop solid criteria for diagnostic tests of mucus function.
Study is needed of the relationship of cellular and antibody mediated immunologic function to normal sperm motility, as well as to the prevention of pelvic infection.
More research is needed to define normal cervical function and immunology with the goal of improved therapies for cervical factor infertility.
Fallopian Tube Function
The human fallopian tube is not simply a conduit for sperm and the newly fertilized embryo. It is a complicated structure of longitudinal and circular muscle surrounding a convoluted, ciliated epithelium, which is hormonally responsive to both steroids and proteinaceous growth factors. Little is known about tubal function and evaluation. Currently, the hysterosalpingogram provides radiologic information on tubal patency, but there are no tests to evaluate tubal function. Such research is of tremendous importance given the rapidly increasing incidence of ectopic pregnancy in the United States. In some populations, as many as 1 in 60 pregnancies occur in the tube, necessitating surgical intervention and often resulting in severe maternal morbidity and occasionally mortality.
New techniques must be developed to evaluate tubal function and to describe the specific etiologies of abnormal tubal function.
Studies are required to assess ciliary function and the role of muscular contractions in transporting the embryo into the uterus.
The area of steroid and growth factor interactions with tubal epithelium requires a major research commitment.
Normal implantation in the endometrium is modulated by a number of growth factors, and research into the role of growth factors in tubal function may provide important answers on the etiology and genesis of tubal ectopic pregnancies.
Despite more than 100 years of investigation and attempts at treatment, pelvic endometriosis remains a ubiquitous and enigmatic disease. Its incidence, etiology, and management consistently arouse controversy, and its relationship with infertility remains unclear. It has been estimated that 1–5 percent of reproductive-age women have endometriosis, whereas as many as 45–50 percent of women suffering infertility are so afflicted. In 1982, 2.4 million married couples in the United States were infertile; since that estimate was of married couples a decade ago, today's estimate yields a number in excess of 1.5 million women with endometriosis and infertility.
Although it is generally agreed that minimal, or stage 1, endometriosis is not a significant etiologic factor in infertility, the natural history of such early, untreated disease is completely unknown. Such mild disease may well be the cause of significant later morbidity.
The natural course of endometriosis and its etiology are also unclear. Sampson's theory of retrograde menstruation is widely accepted as the etiology. However, it does not explain why particular women are prone to develop the disease, whereas others are not. Recently it has been postulated that immunological factors are significantly involved in the causation of endometriosis; however, this concept, too, is under attack.
Thus, endometriosis is a major health problem in women, both because of its associated infertility and its resultant morbidity, and the reasons particular women develop the disease and others do not are obscure.
Research is needed on the relationship of endometriosis to infertility.
Basic and clinical research into questions of who needs treatment and what is the best modality could yield an excellent societal return on investment.
A male factor is listed as the Primary cause of infertility in 30 percent of couples, and is implicated as a contributory factor in an additional 20 percent of cases. Despite our awareness for decades of male factor infertility, there has been minimal progress in its diagnosis and treatment. Perhaps nothing more typifies the current state of affairs in this area than the two recent diametrically opposed statements regarding the postcoital (Simms-Huhner) test first described in 1888 and considered to be a mainstay of the initial infertility evaluation of a couple. On the one hand it is said that the postcoital test is an important and mandatory investigation in the workup of the infertile couple. On the other hand it is said that there is a problem of poor validity, and the test suffers from a lack of standard methodology, lack of a uniform definition of normal, and unknown reproducibility. Even worse, perhaps, is the recognition that the time-honored standard semen analysis may have minimal correlation with male infertility.
Controversies such as the above pervade the subject of male infertility and its management. Specific debates include the role of antibodies (male and female) and theft appropriate treatment, the value (if any) of hormonal treatment of various forms of male subfertility, the role of intrauterine and intracervical insemination in unexplained infertility, the necessity of, and the physiologic factors involved in, capacitation, and the acrosome reaction. The obvious corollary of a clear understanding of sperm function would be the ability to develop new methods of contraception based on interfering with the normal fertilization process.
Research at the basic science level must be initiated before a true understanding of the causes and possible treatments of male infertility can be proposed.
Research is needed into sperm-oocyte interaction, including details of fertilization and chromosome exchange, sperm acrosome reaction, sperm maturation, sperm metabolism, and detailed sperm morphology.
In Vitro Fertilization and New Reproductive Technologies
A decade after the first in vitro fertilization (IVF) birth in 1978, the number of stimulation cycles initiated in women for the purpose of embryo transfer (ET) exceeded 25,000, despite its high cost (approximately $5,000 per cycle) and relatively poor success rate (14 percent for live-born infants), which has barely changed in the past 5 years. Were success rates of even 25 percent possible, it would be of major benefit to many more couples; because a success rate of 50 percent could eliminate most currently practiced infertility surgery, research in this area could be rewarding. Although many small clinical studies have been conducted in this area, no dramatic breakthroughs have resulted. However, recent reports involving co-culture of oocytes with maternal tubal or endometrial epithelial cells have suggested a new avenue of approach to this problem.
It has long been known that assisted reproduction is more successful in animals than in humans. Specifically, bovine ET, in which fertilization occurs in vivo (as opposed to in vitro) and the embryos are flushed from the uterus and then transferred (often after freezing) to a recipient uterus, routinely carries success rates of 60 percent. This and other observations, have led to the concept of very early embryo-maternal cell signaling, and an understanding that interaction is much more complex than previously envisioned. As a result a new " science" in this area is taking shape revolving chemical signaling (autocrine and paracrine), growth factors, follicular regulation of oocyte maturation, activation of the embryonic genome, and energy substrates and metabolism of the very early embryo.
Though distinct improvement in human IVF-ET success rates has not yet been forthcoming, a focused research effort might find specific etiologies for reproductive failures and specific corrections that might be offered. Perhaps even more exciting than the potential advances and success rates in IVF is the realization that research information gathered from in vitro studies may be directly applicable to natural human reproduction and population control. Such knowledge portends hope for controlling human reproduction, and on a worldwide scale.
Clearly, questions of human reproductive efficiency and infertility require a carefully formulated, rigorous research agenda. Such research would most
appropriately be conducted in departments of obstetrics and gynecology in which infertility patients are seen and cared for on a daily basis. Research on all areas of infertility is needed, not only to further the possibility of conception for infertile couples but to understand this basic reproductive function so that appropriate interventions can be made for population control. Research in reproductive biology and infertility has been hampered by lack of federal support, which is needed to advance knowledge of the basic processes of human fertilization and conception.
Research using appropriate animal model systems in the primate and research utilizing human follicular fluid, corona, and cumulus cells should investigate the molecular biology of human fertilization and early cell division.
Research should be conducted on the involvement of growth factors, activation of the embryonic genome, and metabolism in the very early embryo.
The incidence of premenstrual syndrome (PMS) is difficult to evaluate precisely because of the great variability in symptoms and in the severity of these symptoms among women. Nonetheless, it is clear that a large number of ovulatory women are affected by significant aberrations in feelings of well-being and in some cases by severe distress during the luteal phase of the ovulatory cycle. Symptoms are sometimes so severe as to be disabling at some time after ovulation during each menstrual cycle. Therefore, this disorder constitutes a major health problem and may cause periodic loss of a large population from the work force. Yet despite the common occurrence of PMS among young women and despite our relatively advanced understanding of biomolecular events of the ovarian cycle, we have very little definitive knowledge of the cause of PMS.
In part this may be because of the reluctance of some segments of society. and science to accept PMS as a biological disorder that can be related to defined endocrinological events. But the lack of understanding is also attributable to an inability to identify meaningful differences in the hormonal changes of ovulatory women who do or do not suffer from PMS. In particular, there are no major
differences in the blood levels of estrogens or progesterone during the luteal phases of women with and without symptoms of PMS. No significant differences have been found in the rate of secretion of aldosterone among the two groups; and differences in blood levels of prolactin have not been detected. The same is true of plasma testosterone and androgen prohormones. Thus, it has not been possible to demonstrate a substantial difference in the hormonal milieu of women during the postovulatory phase of the ovarian cycle. There is a growing sense, however, that attempts to identify differences in the endocrinology of the luteal phase of the cycle may have been relatively superficial, considering the likelihood that steroid metabolites are bioactive and that the bioactions of these metabolites as well as the steroid hormone precursor may act by way of nongenomic processes.
For example, until recently, the metabolic fate of the majority (60–70 percent) of progesterone was not defined. In recent studies, it appears that this unaccounted-for metabolism of progesterone proceeds by way of initial 5a-reduction, which occurs in both hepatic and extrahepatic tissues. Extrahepatic metabolism may be especially important because this mechanism of progesterone clearance would avoid immediate conjugation of potentially bioactive metabolites, a process that occurs in the liver. It has also been demonstrated that metabolites of progesterone, especially those reduced in the 5a-position, are bioactive. In particular, such compounds act to cause anesthesia, analgesia, and anxiolysis in both humans and experimental animals. Therefore, it is highly possible that metabolites of progesterone affect biobehavioral events; moreover, differences in metabolism of progesterone among women may give rise to decided differences in the bioresponses of women when progesterone production rates are high. In addition, the withdrawal of progesterone metabolites, as occurs at the end of each nonfertile ovarian cycle and after pregnancy, may also contribute to modifications in well-being. Therefore, because of person-to-person variations in the metabolism of progesterone, the stereospecific nature of the bioactions of progesterone metabolites, and the potential for biobehavioral modifications with progesterone metabolite withdrawal, many aberrations in behavior and well-being could be the consequence of the recurrent production of progesterone in large amounts in otherwise normal young women.
It is also highly likely that bioactions of progesterone by way of classic progesterone receptor-mediated processes that may affect the well-being of women are yet to be defined. It was recently demonstrated, for example, that progesterone acts, at least in some tissues, to increase the activity of the enzyme enkephalinase, which degrades enkephalins and other highly active small peptides, such as atrial natriuretic factor, and substance P, as well as the endothelins -1, -2, and -3. If progesterone were to act to induce increased
enkephalinase activity in the brain, enkephalin withdrawal could obtain. If progesterone were to increase enkephalinase activity in the kidney, marked attenuation of the effect of atrial natriuretic peptide could occur, leading to sodium and water retention.
Proposed Research—The metabolism and bitactions of progesterone and its metabolites are fruitful areas for research to define the biological causes of symptoms referred to as the premenstrual syndrome.
Studies of the role of endorphins in the precipitation of symptoms of premenstrual syndrome already are in progress in some laboratories, and the findings of these studies may be complementary to those suggested.
The Brain and Reproduction*
Human reproduction, as in all mammals, is the consequence of a cascade of neuroendocrine events that originate in a central signal generator residing in the region of the arcuate nucleus of the mediobasal hypothalamus. The existence of this signal generator was first evidenced some 20 years ago by rhythmic, oscillatory time courses in the plasma concentrations of the pituitary luteinizing hormone (LH) in women1 and rhesus monkeys,2 leading to the conclusion that these hormonal patterns axe the consequence of the pulsatile release of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), into the pituitary portal circulation. This supposition has been amply verified by the direct measurement of this neuropeptide in pituitary portal blood of experimental animals lending the name "GnRH pulse generator" to the neural timing mechanism in the hypothalamus. Beyond the fact that in the unmodulated state, the pulse generator is activated approximately once per hour and that it is an intrinsic property of the mediobasal hypothalamus, little is known about the cellular basis of its operation. Evidence has been adduced in support of the view that the pulse generator may be resident within the GnRH-producing cells of the hypothalamus, but the integrating system that leads to the synchronous activation of a number of GnRH cells and to the rhythmic discharge of the neuropeptide remains to be elucidated. The hypothesis of a pacemaker outside
the GnRH system that signals the GnRH cells to secrete their product cannot be eliminated at present.3
During the follicular phase of the menstrual cycle, the period of the GnRH pulse generator, and that of the resultant gonadotropic hormone pulses, approximates one hour, but it is profoundly reduced during the luteal phase as a consequence of the action of progesterone on the hypothalamic system, an action mediated by endogenous opioid peptides.4 While a minimal frequency of LH stimulation is required to maintain the functional integrity of the corpus luteum,5 normal ovulatory menstrual cycles can be achieved in the absence of endogenous GnRH production by the administration of GnRH at an invariable frequency of one pulse per 680 or 90 minutes, leading to the conclusion that GnRH is a permissive component of the control system that governs the ovarian cycle in the higher primate, the regulation being achieved primarily by an interaction between the ovarian hormones and the gonadotrophs of the adenohypophysis.4,6 Thus, the preovulatory gonadotropin surge is initiated by a positive feedback action of estradiol acting on the pituitary that can proceed in the face of an unchanging ambient GnRH environment.
The pathophysiological consequences of alterations in the secretory patterns of GnRH have recently been extensively reviewed.4,7,8 The total absence of GnRH production, as in Kallmann's syndrome, is characterized by an absence of gonadotropic hormone secretion and gonadal infantilism. This syndrome is associated with anosmia, a phenomenon that has received recent explication with the finding that, in mice, the GnRH cells have their origin in the olfactory placode of the embryonic brain. They migrate caudally to their ultimate location in the mediobasal hypothalamus.9,10 Apparently, in Kallmann's syndrome, this migration is interrupted by a midline defect. Less extreme disturbances in GnRH production can also lead to failures in follicular development, anovulation, and infertility. It has been estimated that some 15 percent of all infertility cases are attributable to anovulation,11 but better data bases are needed before definitive quantitative judgments can be made in this regard. While isolated pituitary gonadotropin deficiency can be a cause of ovarian failure, a most infrequent phenomenon, anovulation is mainly attributable to hypothalamic dysfunction. This can be psychogenic or stress induced.12 In the rhesus monkey, even seemingly mild perturbations in the environment can lead to arrest of the GnRH pulse generator.13 A similar phenomenon can be reduce in such animals by the administration of the hypothalamic corticotropin-releasing factor, an action apparently mediated by endogenous opiates.14 Morphine itself is a potent inhibitor of GnRH pulse generator activity.15 The role of endogenous opiates in the mediation of the responses to actual stress, however, is still not entirely clear. Similarly, while hyperprolactemia can lead to derangements of
pulse generator activity and pituitary malfunction, the role of this peptide in the anovulatory states associated with lactation is quite uncertain.16
Severe exercise, as well as caloric deficits, leads to anovulation, undoubtedly occasioned by a reduction in GnRH pulse generator frequency or its complete arrest.17 The link between caloric balance and the functioning of the GnRH pulse generator is unknown. While GnRH stimulates the secretion of both LH and FSH by the gonadotrophs (hence its name), the physiological control of the production of these two hormones is not the same. In addition to the stimulating role of GnRH, FSH secretion is preferentially inhibited by a family of peptides, the inhibins, produced largely by the gonads. While the physiological role of inhibin in the control of FSH secretion in the male is unambiguous,18 its role in the control of the menstrual cycle remains to be fully elucidated. A related family of peptides, the activins, stimulate FSH secretion,19 but their physiological roles are not clear at present. The roles of autotrine and paracrine factors in the control of gonadotropic hormone secretion and their interactions with GnRH are similarly conjectural.
The mechanisms underlying the ontogeny of the GnRH pulse generator remain a mystery. It is fully functional at birth and is inexplicably inhibited some weeks later to reawaken in anticipation of puberty.20 In fact, puberty can be defined as the reactivation of the pulse generator following its quiescence during the long period of infancy. Elucidation of the nature of this normal inhibitory influence may provide clues to the muses of hypothalamic amenorrhea and the mechanisms of action of the negative energy balances that obtain in caloric deprivation and severe exercise, and vice versa.
The electrophysiological substrates of the GnRH pulse generator have now been identified and monitored in the rhesus monkey,21 the rat,22 and the goat.23 In these models, each LH pulse is immediately preceded by a burst of multiunit electrical activity recorded from electrodes implanted in the mediobasal hypothalamus. During the rhesus monkey menstrual cycle, these volleys of multiunit activity last for about 2 minutes, whereas following ovariectomy their duration is some 15–20 minutes. This prolongation of electrical activity is abruptly reversed by estrogen administration.24 While the marked increase in the duration of increased hypothalamic electrical activity observed in the absence of ovarian function is not accompanied by significant changes in the dynamics of the resultant LH pulses, it may well be related to the manifestation of other neural activities such as the autonomic discharges characteristic of ''hot flashes'' that have also been shown to be synchronous with LH pulses in postmenopausal women.25
Reproductive physiology can be considered the science basic to obstetrics and gynecology. The GnRH pulse generator and the remainder of the
hypothalamo-hypophysial unit are central to human reproduction as they are in all vertebrates studied to date. The malfunction of this apparatus probably accounts for a major portion of infertility, both persistent and transient. It may also play a role in one of the most vexing endocrine disorders of women, polycystic ovarian disease. It seems regrettable, therefore, that the neuroendocrinology of human reproduction has been a research area explored more by internists and pediatricians than by obstetricians and gynecologists. This exciting field should be attractive to physician-investigators bent on an academic career, especially those interested in the still neglected interface between the activity of higher brain centers and reproductive function.
The nature, specific localization, and mode of operation of the GnRH pulse generator must remain a critically important subject for intensive investigation at the systems, cellular, and subcellular levels.
While estradiol can initiate the preovulatory gonadotropin surge in the absence of changes in GnRH production, what actually happens during the normal menstrual cycle is not known and should be investigated.
The quantitative role of neuroendocrine deficits in the causation of infertility in women must be defined.
The mechanisms whereby "stress" inhibits the GnRH pulse generator and consequent ovarian function must be elucidated.
The mechanisms whereby lactation, severe exercise, and caloric deficits lead to amenorrhea and infertility must be characterized.
The mechanisms of action of a variety of modulators of GnRH pulse generator activity must be elucidated. The opiates, catechol amines, NPY, and other neurotransmitters are cases in point.
It is now clear that the control of LH and FSH secretion by the pituitary gland is not the same. The role of activins and inhibins and other factors in the control of FSH secretion must be investigated in a physiological context.
The mechanisms that cause the inhibition of the GnRH pulse generator shortly after birth and its reawakening at the time of puberty remain a complete mystery. The initiation of puberty continues to be a central, unsolved problem in human biology.
The functional relationship between the hourly activation of the GnRH pulse generator and "hot flashes," synchronous events in postmenopausal women, should be a subject of concerted study with the aim of discovering the
physiological basis of the phenomenon and its potential alleviation by alternatives to estrogen therapy.
It is estimated that there are more than 40 million women in the United States today who are postmenopausal; and it is likely that this number will continue to increase rapidly in the next few decades. At menopause, estrogen secretion by the ovaries ceases. As the adrenal cortex does not secrete estrogen, menopause is associated with severe estrogen withdrawal. Indeed, estrogen production in postmenopausal women is limited to the extraglandular formation of the biologically weak estrogen, estrone, principally in adipose tissue, from the aromatization of plasma androstenedione (derived primarily from adrenal secretion). Estrogen deprivation at menopause results in troublesome and distressing (sometimes disabling) symptoms such as vasomotor instability (hot flashes) and urogenital atrophy (vaginal dryness and shrinkage). In addition, estrogen deprivation facilitates the more rapid development of life-threatening disorders of aging, including osteoporosis and possibly atherosclerosis.
Estrogen treatment is very effective in preventing hot flashes and in promoting growth of the vaginal epithelium. It also is clearly established that estrogen treatment of postmenopausal women serves to retard the loss of bone density and reduce the risk of myocardial infarction. Severe osteoporosis in elderly women is life-threatening; the death rate from complications of bone fractures, especially those of the hip, in elderly persons is high. Yet relatively few postmenopausal women today are treated with estrogen for any extended time. This is a health issue of major concern today and one that must be addressed if we are to improve the quality of life for an enlarging population of elderly women in this country and around the world.
The risk of estrogen treatment of postmenopausal women, when estrogen is given in adequate but reasonable doses, is believed to be confined almost exclusively to the development of endometrial carcinoma. The risk of breast cancer in postmenopausal women treated with estrogen, if different from that m postmenopausal women not ingesting estrogen, is not clearly established; but the risk ratio must be small, if it is different from 1. Nonetheless, any potential risk of breast cancer, however small, cannot be dismissed because of the very
common occurrence of breast cancer in women. Namely, a very small increase in risk, if significant, would represent a large amber of affected women.
Presently, however, the major concern of estrogen treatment of most postmenopausal women is the risk of endometrial carcinoma. The risk ratio for the development of endometrial cancer in postmenopausal women ingesting estrogen is believed to be approximately 4 (for average duration of treatment and dose). This risk should be analyzed, however, in light of the belief that endometrial carcinoma that develops in estrogen-treated, postmenopausal women is ordinarily a highly differentiated neoplasm for which the cure rate is near 100 percent. In fact, the cure rate for endometrial carcinoma in general is very high, commonly stated as being greater than 85 percent when all cases axe included. But to avoid even this small risk, many physicians choose to add a progestin to estrogen treatment regimens. This choice is problematic.
There are a few studies dealing with the relatively short-term effects of such a regimen on the concentration of plasma lipoproteins; there are a few studies that address the effect of the addition of progestins on the maintenance of bone density; but there are no long-term studies to evaluate the potential for increased incidence of heart disease and stroke when progestins are added to estrogen treatment regimens. It has been argued that the progestin can be given in low doses and therefore should be safe. This is not necessarily correct if the effects of the progestin are intravascular at the level of platelets or vascular endothelial cells. Rather, this would constitute intravascular therapeutics of a nature similar to that upon which the use of low-dose aspirin is based. In addition, the problem of patient compliance with various estrogen treatment regimens when progestins are added also must be addressed.
Thus, there are many data supportive of the belief that estrogen treatment of postmenopausal women is salutary in the prevention of major disorders of the skeletal and cardiovascular system and in the relief of hot flashes and the cure of urogenital atrophy.
It is established that estrogen treatment alone is a risk factor only for the development of endometrial carcinoma, and that this risk is low and the disorder is highly curable. Therefore, is it necessary to add progestin to this low-risk, high-benefit therapeutic plan?
These are questions of vital importance to the health care of millions of postmenopausal women. The prospective, long-term studies that should be conducted are easily envisioned and clearly call for the participation of gynecologists who are expert in the management of problems of the postmenopause and who are expert in the follow-up examination and evaluation of the postmenopausal woman taking estrogen—including the sampling of the endometrium to identify the development of abnormal endometrial tissue. The
endocrinology of the postmenopause has been defined by gynecologists; and it is the gynecologist who is most familiar with the endocrinology of the postmenopausal woman. Thus, the gynecologist who is trained in the basic sciences can make important contributions to the study of the endocrinology and endocrine treatment of postmenopausal women. Indeed, it is difficult to envision the conduct of a study of postmenopausal women without the guidance of experienced gynecologists. Few other physicians are expert in the conduct of pelvic examinations w evaluate ovarian status or to monitor cervical, vaginal, or vulvar health or disease. Cancer screening for pelvic neoplasias is accomplished almost exclusively by gynecologists. Therefore, gynecologists should make a major effort to design and conduct research to define the optimum acceptable hormone treatment regimens for postmenopausal women.
Long-term, prospective studies to evaluate the effects and side effects of combinations of estrogen and progestins in the treatment of postmenopausal women should be conducted.
Studies are needed to explain why very few postmenopausal women are treated with estrogen.
Studies are needed to discover and assess the risks of adding progestin to estrogen treatment.
Gynecologic malignancies will account for approximately 71,700 cancer diagnoses in women during 1991 and 23,500 cancer-related deaths. The following section briefly describes the current state of knowledge regarding cancer of the reproductive system, emphasizing recent advances and promising avenues of future research.
Ovarian cancer is the most lethal gynecologic cancer. Of the 20,700 women diagnosed with this disease in 1991, 12,500 women will die. Most ovarian neoplasms are of epithelial origin, and these cancers occur mostly in women beyond the age of 45. Risk factors are not well understood, but it is believed that repetitive ovulation increases the risk. Thus, early menarche and late menopause are risk factors while oral contraceptive use appears to diminish the risk.
More than half of the cases of ovarian cancer are discovered in an advanced stage. Current techniques of potential diagnosis include pelvic examination by a health professional, vaginal ultrasound, and the measurement of an ovarian tumor marker, CA125. However, the latter is not specific, nor is it effective as a screening tool. Pelvic examination is unfortunately imprecise, and currently vaginal ultrasound is both expensive and not widely available nor proven as a cost-effective measure.
What are the factors that predispose the development of ovarian cancer?
What preventive measures can be identified that could be implemented on a wide scale?
Is there a cost-effective method for early detection, such as the development and refinement of sensitive vaginal ultrasound, that would greatly improve survival?
The genetic changes that accompany the neoplastic ovarian phenotype are being investigated. As mesothelial cells undergo transformation, synthesis of a unique 200-kilodalton protein has been detected. Although normal ovarian tissue and benign ovarian lesions do not express M-CSF and c-fms proto-oncogene expression is low, 78 percent of ovarian neoplasms express M-CSF and 89 percent express c-fms transcripts. TGF alpha is not expressed by normal ovarian tissue but is detectable in neoplastic ovarian tissue.
Neoplastic ovarian tissue also demonstrates a variety of genetic changes. Allelic loss of the retinoblastoma gene has been reported. Allelic deletion of the Ha-ras proto-oncogene is also common. The p53 tumor suppressor gene is frequently overexpressed and mutated in these neoplasms. Amplification of the c-myc proto-oncogene has also been reported.
Studies of this type on ovarian and other gynecologic tumors will provide information concerning tumors arising at nongynecologic anatomic sites and may provide answers to the issues of basic molecular and cell biology of neoplasia and the effect of growth factors on this process.
The treatment of ovarian cancer relies on aggressive operative debalking. The stage of the disease is the single most important prognostic factor. Currently, platinum based chemotherapeutic regimens are most effective and produce initial responses in 60 to 80 percent of patients with advanced-stage (stages 3 and 4) disease. Unfortunately, only about 15 to 20 percent of patients following these excellent responses become long-term survivors.
A dose-response relationship for ovarian cancer in chemotherapy treatment has been demonstrated and this provides a rationale for the intraperitoneal administration of therapeutic agents. It also offers a route for other cell-specific therapies. High-dose chemotherapy with autologous bone marrow transplantation has been investigated but has not proven thus far to be efficacious.
A promising new drag is Taxol, a plant-derived antineoplastic agent. The drug is currently under investigation through a National Cancer Institute (NCI)-directed trial. In spite of initial good responses, long-term survival in a large group has not been demonstrated.
While the intraperitoneal route is currently the topic of many trials and may prove to be effective, particularly in patients with minimal disease, experimental models need to be explored for tumor-specific, site-directed therapies. For example, tumor-specific antibodies, particularly those linked to cytotoxic radionuclides, offer a potentially efficacious method W improve current therapeutic results, particularly for chemotherapy-resistant tumors. In addition, site-directed chemotherapy may also provide improved therapeutic benefits. Currently, these strategies are limited by the lack of cell-specific agents. Although immunotherapy has not yet proved successful, this research is emerging as a potential and exciting new method of treatment. Advances in these areas offer the promise of markedly increasing survival in this usually lethal disease.
Which genetic alterations, if any, play a causative role in neoplastic transformation merits further investigation.
Are there methods, including human tumor clonogenic assay, which can provide useful information and important clues to guide therapists to the optimal form of chemotherapy for specific patients?
What new agents or new approaches can be developed to kill the cancer cell—for example, novel delivery systems or specialized treatment approaches such as the improved use of intraperitoneal therapy as well as the immunologic development of new biological response modifiers?
Uterine neoplasms constitute the most common site of lower genital tract cancers in women, of the 33,000 new cases diagnosed in 1991, it is estimated that 5,500 women will die of this disease. There is evidence that the age-standardized incidence rates for endometrial cancer are rising.
Although unopposed estrogen appears to play a role in the etiology of most endometrial adenocarcinomas, this model is not universally applicable. Comparisons of neoplastic, hyperplastic, and normal endometrium have revealed differences at the molecular level that will provide mights into the changes accompanying the initiation and promotion of endometrial neoplasia. As aa example, normal endometrium is uniformly diploid and expresses PDGF beta, IGF-I, IGF-II, and the EGF receptor. Hyperplastic endometrium is characterized by an increased likelihood of a nondiploid DNA index and increased proliferative activity. Neoplastic endometrium exhibits frequent aneuploidy, loss of hormonal responsiveness, and a variety of proto-oncogene abnormalities.
The diagnosis of endometrial cancer is typically prompted by the onset of postmenopausal vaginal bleeding. Transvaginal ultrasonography shows promise as a means to detect the presence of uterine pathology utilizing color flow doppler. This offers a potential way to screen women for unsuspected uterine pathology. Transvaginal ultrasonography is also a useful way to determine the depth of myometrial invasion in cases of diagnosed carcinoma.
The prognosis for women with endometrial cancer is based on tumor grade and depth of invasion. The histopathological evaluation of endometrial carcinoma for prognosis may be further refined by the determination of DNA content, proliferative index, steroid receptor expression, and the presence of specific oncogene abnormalities. Hormone receptor status appears to be an important prognostic indicator. Preliminary studies indicate that receptor negative lesions with a high S-phase fraction behave more aggressively than
lesions without these characteristics. The presence of HER-2/neu amplification may also identify neoplasms that are more likely to recur and metastasize.
Surgery remains the mainstay of treatment for endometrial carcinoma since most patients present with clinical stage 1 disease. Adjunctive radiation therapy is reserved for patients with high risk factors that predict local/regional recurrence. Although radiation therapy is effective in decreasing pelvic recurrence, a beneficial effect on long-term survival has been difficult to demonstrate.
Progestins have been used in the treatment of recurrent endometrial cancer with a response rate of 30 percent for a median duration of 10 to 12 months. Responses are more likely when used to treat progesterone receptor-positive neoplasms, but not all patients who express hormone receptors respond to hormonal therapy.
Recent evidence suggests that the combination of chemotherapy and hormone therapy may offer enhanced responses and survival in uterine cancer.
Although uterine cancers are often hormone dependent, preliminary data have suggested that estrogen replacement therapy utilized in this group of women may enhance their survival and quality of life by diminishing the frequency of the deleterious effects of estrogen deprivation.
Can transvaginal ultrasonography become a cost-effective tool for early uterine cancer detection, similar to the project described in the discussion on ovarian cancer?
Specialized treatment trials are needed to determine optimal methods for combining chemotherapy and radiation therapy, as well as chemotherapy and hormone manipulation, to enhance responses in survival.
What is the safety and risk of estrogen replacement therapy in those who have been successfully treated for uterine cancer?
Cervical cancers have fallen to third in frequency after endometrial and ovarian malignancies. In 1991, 13,000 cases will be diagnosed, and 4,500 cervical cancer-related deaths will occur. Five- and 10-year trends demonstrate a continuing decline in the number of cases of invasive disease with a
concomitant increase in the number of cases of dysplasia. These trends have been attributed to increasingly effective cytologic (pap smear) screening programs.
The etiology of cervical cancer, particularly squamous cell carcinoma, has historically been associated with multiple sexual partners, early age at the initiation of sexual activity, cigarette smoking, and a history of sexually transmitted diseases. The effect of certain dietary deficiencies as a risk factor for cervical dysplasia is debated. During the past decade, attention has been focused on the role of the human papillomavirus (HPV) in premalignant lesions of the cervix (dysplasia, carcinoma in situ, or CIN I, II, III). Although HPV has not been demonstrated as the causative agent of cervix dysplasia and neoplasia, its role as an important cofactor is generally accepted. More than 60 subtypes of HPV have been identified, but a much smaller number appear to be important in cervical pathology. HPV 6 and 11 are associated with benign condyloma or low-grade neoplasia and are retained in the episomal state, whereas HPV 16 and 18 are generally found in high-grade dysplasia and in invasive carcinoma and are usually integrated into the host genome.
Current evidence suggests that only 16 percent of cases of mild dysplasia will progress to higher grade premalignant lesions, while carcinoma in sire carries a major risk for progression to invasive cancers. Specific times for progression are unknown but in most cases are believed to take years. However, recently some patients have been diagnosed with cervical cancer in whom the rate of progression to malignancy appears to have been much more rapid.
The transforming activity of the HVP 18 LCR-E6-E7 region is approximately 10- to 50-fold more active in transforming activity than comparable regions of HVP 16. This may explain the biological aggressiveness of HVP 18 adenocarcinomas of the cervix diagnosed in young women.
Immunologic alterations also appear to play a role in susceptibility to HVP cervical infections and in aa increased susceptibility to cervical cancer. Chronically immunosuppressed women, such as renal transplant patients, are at increased risk of cervical cancer. Pregnant women, in a state of relative immunosuppression, demonstrate an increased rate of a variety of HPV subtypes compared with nonpregnant women.
A new reporting system for cytological abnormalities of the cervix, the Bethesda system, has been proposed to facilitate communication between the cytopathologist and the clinician as well as to replace the numerical Papanicolaou designation. This new classification proposes to combine the changes of CIN I and those resulting from an HPV infection into a single diagnostic group. To date, the Bethesda system has not been uniformly adopted by obstetrician-
gynecologists or by cytopathologists. There is concern among gynecologists that the adoption of the Bethesda system will exacerbate the already serious problem of overtreatment of HPV cervical lesions. Since premalignant cervical lesions are treated and followed predominantly by obstetrician-gynecologists, these problems offer an important are for research in departments of obstetrics and gynecology. Most current therapeutic approaches to premalignant conditions of the cervix are empirical, and analyses based on cost-effective approaches are needed. For example, in some European countries such as the Netherlands, patients with mild dysplasia are followed with Pap smears twice yearly while in the United States the condition is almost universally treated. As noted, the optimal approach to widely prevalent HPV cervical refections is not known.
The stage of invasive cervical cancer remains the single most important clinical prognostic indicator of 5-year survival. Approximately one-half of cervical cancer patients with stage 1 disease that recurs after a radical hysterectomy have negative surgical margins and retroperitoneal pelvic lymph nodes free of metastatic disease, indicating the need for better prognostic indicators. Amplification of the c-myc oncogene has been correlated in some, but not all, studies as a poor prognostic finding. Overexpression of the c-myc oncogene has also been associated with early recurrence and decreased 5-year survival. Cervical neoplasms that express the Ha-ras p21 protein are at increased risk for pelvic lymph node metastases.
Carcinomas confined to the cervix may be treated with radiation therapy or surgical therapy with comparable 5-year survival rates. Neoadjuvant chemotherapy with cisplatin shows promise as a means to reduce the size of the primary lesion and to decrease the incidence of metastatic nodal disease prior to surgery, particularly in advanced cases.
With the exception of women who develop central pelvic recurrence amenable to removal by exenterative surgery, recurrent cervical cancer following radiation therapy is fatal. Cisplatin is the most active agent for recurrent cervical cancer, but response rates are limited to 30 percent and cures are anecdotal.
What is the influence of human immunodeficiency virus (HIV)-related immunosuppression upon the risk of cervical HPV infection, cervical dysplasia, and cervical neoplasia? This requires population studies.
Do HPV infections require therapy and if so, which types are needed to reduce the frequency of cervical cancer?
Can a methodology be developed to identify which ''premalignant'' cervical neoplastic conditions are at risk for progression?
What are the optimal intervals for cervical cytologic screening?
What are the optimal methods of treating various degrees of cervical intraepithelialneoplasia, and which are most cost-effective?
What is the role of HPV virus in the genesis and progression of cervical neoplasia?
What characteristics (oncogene amplification, for example) can be identified that will reliably predict aggressive tumor behavior and thus provide the basis for improved initial treatment strategies?
How can the standard therapies of radiation or operation for cervical cancer be combined with newer modalities of chemotherapy or immunotherapy to improve survival?
What new strategies can be developed to improve the therapy of recurrent cervical cancer, which currently is almost uniformly fatal?
Vulvar malignancies account for 3 to 4 percent of gynecologic neoplasms, and the majority of these are squamous cell carcinomas. The etiologic role of the human papillomavirus (HPV) in vulva dysplasia and neoplasia has not been fully determined. Microscopic evidence of benign HVP-associated lesions is found in conjunction with vulvar carcinomas. Using molecular biological techniques, HPV has been found in 30 to 80 percent of vulvar carcinomas. The difference in age-adjusted incidence rates for invasive cervical versus vulvar carcinoma, in addition to the observation that HVP is found more often in premalignant lesions versus malignant lesions of the cervix and vulva, suggests that the pathogenic role of HPV may be different in lesions of the cervix and of the vulva.
Vulvar and cervical neoplasms may share a common etiologic factor in HPV. However, the biological behavior of preinvasive vulvar disease appears to be different from that of cervical disease. There is little evidence to suggest that dysplastic lesions of the vulva have a significant probability of progression to invasive disease in the absence of appropriate treatment, as is the case for premalignant lesions of the cervix.
Not only is the biological behavior of preinvasive vulvar disease different from that of cervical disease—it is also less predictable. It appears that many of the vulvar premalignant lesions spontaneously regress, although there is currently no method to differentiate these cases from those that progress.
Surgery is the primary method of therapy of invasive vulvar carcinoma and consists primarily of radical vulvectomy or hemivulvecromy and inguinal-femoral node dissection. Radiation is used as an adjunct for advanced-stage tumors and to treat pelvic nodes in case in which the inguinal-femoral nodes are involved. Recently, less extensive surgical procedures for vulvar cancers, such as modified radical vulvectomy and unilateral lymph node dissection, have been advocated to achieve a more cosmetic result with presumed comparable therapeutic effectiveness. The potential for future use of chemotherapy as an independent and combined modality of treatment is similar to the situation described with cervical carcinomas.
Clinical trials are needed to establish efficacy and safety of new treatments.
What is the optimal method of therapy of premalignant lesions of the vulva, and can one identify which of these lesions actually require therapy? This should include investigation of rates of progression and regression, identification of lesions that require therapy, and determination of optimal screening intervals. Understanding the molecular biology of premalignant vulvar disease should help in this area of research.
Breast cancer is the most common cancer in women. In 1991, 175,000 women will be diagnosed with this disease, and 44,500 will die. It has been estimated that 1 in 10 women in the United States will develop breast cancer during her lifetime, and some recent estimates indicate that the number may be close to 1 in 9.
Obstetrician-gynecologists, as care givers to women, are often primarily involved in the initial detection of breast cancer. Therapy is usually performed by surgeons, radiotherapists, and medical oncologists. Nonetheless obstetrician-gynecologists not only have a major role in the diagnosis of the disease but are also medically responsible for the major events in the life of a
woman that may bear on the causation or prevention of breast cancer such as pregnancy, lactation, oral contraceptive use, estrogen replacement, and the ordering of such screening tests as mammography. All of these provide areas of research that require the participation of departments of obstetrics and gynecology.
Most benign breast diseases are not considered premalignant, although the risk of cancer in women with benign breast disease may be increased. The use of oral contraceptives appears to decrease the incidence of benign breast disease, although their effect on the risk of breast cancer is unclear at present. Most studies have indicated no effect, although some have suggested an increased effect after long-term usage.
The NIH-sponsored cancer and steroid hormone (CASH) study suggests a decrease in the relative risk of breast cancer, diagnosed in the 40s for women who have used oral contraceptives. It appears that there is a strong interaction between parity and age, in which parity increases the risk of breast cancer in younger women and decreases the risk in older women. Some of the data suggest an acceleration phenomenon for cases that may have already been initiated, and such would be consistent with the effects of parity.
Future studies may be directed at classifying women into high- and low-risk groups. It is possible that measurement of proto-oncogenes may theoretically yield information concerning genetic risk. A 16-alphahydroxylation pathway for the degradation of estrogen has been observed in women with breast cancer. Suspected risk factors for breast cancer include obesity, a high-fat diet, hereditary (genetic) factors, and alcohol retake. These factors appear to be associated with an increase in the ratio of the urinary estrogen metabolite while those factors suspected to be associated with decreased risk (thinness, exercise, decrease in fish oil consumption, etc.) decrease the excretion.
Adjuvant therapy reduces relapse rates and prolongs survival among those treated for breast cancer. Endocrine therapy has emerged as a major treatment modality for early-stage disease. The anti-estrogen Tamoxifen improves survival in estrogen receptor-positive cases, particularly for women over the age of 50. Currently, a clinical trial is being conducted in Europe to ascertain the efficacy of long-term prophylactic Tamoxifen therapy on the prevention of breast cancer. However, long-term Tamoxifen therapy may increase the risk of liver cancer; it has also been suggested that it increases the risks of endometrial cancer and of cardiovascular disease.
The issue of hormone replacement therapy is assuming increased importance as larger numbers of younger women with early-stage breast cancer have been diagnosed. Currently, estrogen therapies are believed to be contraindicated m women who have been diagnosed with breast cancer. Yet there are no clear
data to substantiate the general belief that hormone replacement therapy increases the risk of recurrent breast cancer.
What is the potential effect of oral contraceptives on pre- and postmenopausal breast cancer?
Does prolonged oral contraceptive use or early initiation of use (prior to age 20) alter the risk of the development of breast cancer?
Does prolonged estrogen replacement therapy alter the risk of breast cancer?
Does the addition of a progestin (protective for endometrial carcinoma) alter breast cancer risks?
Can estrogen replacement therapy be safely used in patients who have been successfully treated for breast cancer to avoid the morbidity of estrogen deprivation?
Does tamoxifen therapy for breast cancer alter the risk of endometrial neoplasia?
Can groups of high-risk and low-risk women be identified through metabolic hormonal investigation or through molecular studies such as those involving proto-oncogenes?
Trophoblastic disease is usually associated with pregnancy and occurs in the uterus, although similar tumors can arise at other sites such as the ovary. The most common variant of trophoblastic disease, hydatidiform mole, occurs in about 1,000–2,000 pregnancies. Most of these cases require no further treatment after surgical evacuation of molar tissue from the uterus, but some cases progress and require chemotherapy. The fact that these tumors all secrete hCG allows the measurement t of hormone as a specific marker for therapy. The development of a sensitive assay using the beta subunit of hCG has provided a unique tool to follow these patients.
Risk factors include young age at pregnancy (less than 15 years of age) or older age (more than 45 years of age), but the precise mechanism of development is not known. These diseases were the first to be cured by chemotherapy using initially methotrexate. Currently, actinomycin D is used for
low-risk cases. High-risk cases are treated with multiple agent chemotherapy with a more recent combination involving etopiside (VP16)- platinum (EP) or etoposide methotrexate, actinomycin-D, cyclophosphamide, and vincristine (oncovin) (EMA-CO). Brain metastases usually receive radiation. Although outstanding cure rates in this disease have been obtained with chemotherapy, fatalities still occur. The best results are for patients treated at large centers specializing in these diseases, and these centers are primarily located in departments of obstetrics and gynecology.
What are the effects on future fertility of successful chemotherapy of trophoblastic diseases?
What are the effects of chemotherapy in the mother on future genetic abnormalities in the offspring?
What improved treatment strategies can be developed to help patients who currently succumb to the disease?
What are the genetic or other causes that lead to the development of gestational trophoblastic diseases?
Sexually Transmitted Diseases*
The recent dramatic increase in sexually transmitted diseases (STDs) in the United States has had a major impact on the reproductive health of women.
Untreated or inadequately treated gonococcal and chlamydial infections result in approximately 1 million cases of pelvic inflammatory disease (PID) each year. The acute and chronic sequelae, which include infertility, tubal pregnancy, and chronic pelvic pain, are devastating to many women. The number of reported cases of syphilis among women has also increased and last year was the highest in 40 years. As a result, congenital syphilis has increased over 200 percent. More than 100,000 infants die or suffer birth defects because of STDs transmitted during pregnancy or at birth. Vital STDs including human immunodeficiency virus (HIV), genital herpes simplex (HSV), and human papillomavirus (HPV) also have become major problems. Genital herpes simplex vital refection is a painful, incurable disorder that affects many women.
An estimated 20 million women are infected with human papillomavirus and thus are at increased risk for carcinoma of the cervix. In 1990, the largest proportional increase in AIDS cases was among women. HIV infection in pregnancy can result in increased abortion, stillbirth prematurity, low-birthweight infants, and neonatal mortality. The health care costs to deal with sexually transmitted diseases and consequent sequelae are in the billions of dollars.
Major efforts will have to be directed toward early diagnosis and treatment to reduce the magnitude of the problems associated with STDs.
Preventing sexually transmitted diseases by developing clinically effective and safe vaccines.
Developing cost-effective tests for early diagnosis.
Developing new therapies where needed and new cost-effective antibiotics that are easily administered and sufficiently acceptable to enhance compliance.
Clarifying the natural history of genital infections.
Defining behaviors associated with the acquisition and spread of sexually transmitted diseases.
Characterizing the role of STDs in adverse pregnancy outcomes.
Although an interdisciplinary approach involving microbiology, immunology, genetics, and molecular biology will be needed to meet some of these challenges, obstetrics and gynecology—because obstetricians and gynecologists are among the primary providers of health care for women—will have to play a major role in meeting these challenges, particularly in relation to epidemiologic studies and clinical trials.
Prevent Sexually Transmitted Diseases by Developing Clinically Effective and Safe Vaccines
The structural components of STD organisms have been intensively analyzed and dissected, providing information for a rational approach to vaccine development that can be utilized to prevent further infections. Vaccines have been developed and are in various stages of testing for gonorrhea, chlamydia, HSV, and HIV.
Basic research on the microbiology, immunology and pathogenesis of STDs is essential to the eventual design and development of effective vaccines against them.
Development of prototypes of vaccines for use in the prevention of N. gonorrhoeae, C. trachomatis, HIV, and HSV is under way and should be intensified with additional resources.
The mucosal immune response to organisms that cause STDs is critical for the development of successful vaccines, which may stimulate both B- and T-cell limbs of the immune response. Consequently, detailed mapping and analysis of the epitomes of the proteins associated with STD organisms in eliciting immune response are necessary.
The mucosal immune system of the human female genital tract and its role in the prevention of infection and/or susceptibility to infection should be studied more intensely.
The function of the mucosal immune system, specifically, antigen-processing, humoral, and cellular immune responses and the effects of hormones on these responses, should be studied.
Develop Cost-Effective Tests for Early Diagnosis of STDs
With the development of molecular probes and monoclonal antibodies, improved diagnostic methods have been developed for the early detection of STDs. With the recommendation that asymptomatic women undergo routine screening with these newer diagnostic tools, asymptomatic infections have been identified and treated, thus avoiding further development of complications and sequelae.
Develop simple, inexpensive, rapid STD detection methods that are accurate in both symptomatic and asymptomatic women. Highest priority in this area is the development of a test for chlamydial infections. Development of a similar test for vital STDs, such as HSV, HPV, and HIV, is also critical.
Investigate the safety, and efficacy of experimental antiviral drugs against HIV and treatment of opportunistic infections in both pregnant and nonpregnant women.
Evaluate the efficacy, of treatment regimens for pelvic inflammatory disease in relation to preservation of normal reproductive function. This will require a long-term multicenter trial to adequately assess long-term outcomes.
Develop improved methods to diagnose PID and to identify women at high risk for reproductive sequelae. Accurate, noninvasive approaches must be developed, particularly to address the challenges posed by atypical infections. Virulence factors and immunologic markers should be sought that are predictive of postinfectious infertility or ectopic pregnancy.
Develop New Therapies Where Needed and New Cost-Effective Antibiotics That Are Easily Administered and Sufficiently Acceptable to Maximize Compliance
Antiviral drugs have been developed specifically for the treatment of HSV and HIV. These drugs have significantly decreased morbidity and, in the case of herpes, have decreased occurrence rates. Molecular studies have also delineated the mechanisms of antimicrobial resistance, an area of growing importance with the increasing spread of antibiotic-resistant Neisseria gonorrhoeae, acyclovir-resistant HSV, and AZT-resistant HIV.
Develop curative antiviral agents for infections with HPV, HSV, and HIV. Studies are also needed to better define the effect of existing palliative therapies on transmission and progression of their infections.
Evaluate PID treatment regimens for efficacy in preserving normal reproductive function, as well as for ability to achieve clinical and microbiological resolution of acute infection. This will require a multicenter clinical trial, with support for a minimum of 7 to 10 years, to permit adequate assessment of relevant long-term outcomes. The role of adjunctive PID therapy using anti-inflammatory or immunomodulating agents to reduce long-term sequelae should also be examined.
Conduct further studies to document the safety and efficacy of STD/HIV regimens during pregnancy.
Evaluate the safety and efficacy of experimental antiviral drugs against HIV and treatment of opportunistic infections in both pregnant and nonpregnant women.
Expand community research programs for the treatment of HIV, and identify mechanisms to increase access to care, particularly among low-income women.
Develop an understanding of the nature of pathogen-cell interactions, especially virus attachment and entry, in order to formulate effective strategies for interruption of transmission. Natural history studies of HPV infection and the influence of the immune system are critically important in attempts to prevent the development of cervical cancer.
Encourage therapeutic studies of STDs that specifically address efficacy and safety as well as compliance and cost.
Develop inexpensive, accessible therapeutics that can be used reliably by women who must frequently manage multiple responsibilities (e.g., family, job) despite declining health.
Evaluate and develop clinical trial recruitment and retention procedures to facilitate enrollment and follow-up of women (e.g., access to primary medical care, child care, transportation to clinic sites, as well as other support services).
Review clinical trial eligibility criteria in ongoing studies, specifically, inclusion/exclusion criteria that may be too restrictive and thus prohibit the participation of women (e.g., definitions of active drug use, pregnancy, anemia, elevated liver enzymes, etc.).
Study and develop better barrier/contraceptive methods (e.g., condoms vs. female-controlled methods) and viricides that are effective, safe, and acceptable to women; especially needed are methods that can be controlled by women and that may be used without detection by their sexual partners.
Clarify the Natural History of Genital Infections
Studies elucidating the pathogenesis of the microbial agents responsible for these refections have provided us with a better understanding of the factors responsible for the reduction of the disease process. In some cases, studies have identified the molecular basis of microbial attachments to mucosal surfaces and the subsequent immune response resulting in beth inflammatory changes and resistance to further infection.
Describe the full spectrum of HIV-related illnesses and malignancies in women to fully evaluate current AIDS case definitions and standards of medical care for women.
Establish prospective cohorts of women to determine the natural history and clinical presentation of HIV infection in women. Factors that affect the progression to AIDS among HIV-infected women should be identified, and the types of opportunistic infections that occur in women should be studied more intensively. Clinical, virologic, and immunologic markers of disease progression should be evaluated to the female-specific endpoints of disease progression.
To better understand, prevent, and treat HIV infection in women, conduct studies to address the frequency and factors responsible for transmission of HIV to women with specific focus on STDs, stage of disease, hormonal influence, and age.
Continue studies on the frequency and factors responsible for transmission of HIV from mother to child, and evaluate the use of therapy that prevents transmission.
Initiate detailed studies on the impact of STD infections on HIV transmission and the impact of HIV on STD infections. For example, detailed studies on HPV infection in HIV-infected women should be conducted to determine the impact of HIV on HPV in the subsequent development of cervical cancer.
Define the factors and mechanisms that alter risk of disease progression, such as HPV infection and its association with premalignant and malignant lesions of the genital tract. Epidemiologic studies are necessary to further define the factors required for initiation versus potentiation of typical cell growth.
Conduct epidemiological and basic studies to better define the risk factors and biological mechanisms that influence progression of HPV infection to anogenital neoplasia. Urgently needed are HPV natural history studies that examine the roles of viral type and immune status.
Examine the mucosal immune system of the human female genital tract, its relationship to other mucosal immune systems, and its role in the prevention of STDs and HIV infection. Specifically, antigen-processing, humoral, and cellular immune responses and the effects of hormones on the responses should be studied.
Define the chronology and the host and pathogen factors involved in ascent of lower tract organisms into the endometrium and fallopian tubes, and
subsequent tubal scarring. Development of improved animal models for PID would greatly facilitate this research.
Determine the clinical and microbiological spectrum, the frequency, and the natural history of atypical PID. Seroepidemiological studies of infertile women and women with tubal pregnancies strongly suggest that atypical or subclinical PID is responsible for a substantial proportion of these disorders.
Define Behaviors Associated with the Acquisition and Spread of STDs
Major advances have been made in our knowledge of the epidemiology of STDs including HIV infections. Factors contributing to the recent epidemic of STDs among women are complex and appear to involve the interaction of a number of variables including socioeconomic status, exchange of sexual services for drugs, health care-seeking behavior, changes in population demographics, and residence in areas of high disease prevalence. The increasing STD rate has important implications:
rises in heterosexual adult STDs predict similar trends in congenital STDs;
community health education messages, generated by concerns about HIV, to reduce risky sexual behavior have not yet permeated minority heterosexual populations; and
because of the association of both genital ulcer disease and genital nonulcerative diseases with HIV transmission, control of STDs could further reduce HIV spread in this population.
Investigate determinants of health care-seeking behavior in women, including the role of social networks and support systems in facilitating women's access to services.
Develop a specific behavioral research agenda in STD prevention. Epidemiologic studies are needed to identify the type and prevalence of behaviors that put individuals at risk for transmission or progression of an STD.
Identify behavioral risk factors; this work would be facilitated by a national survey of sexual behavior.
Determine population rates for STDs, and conduct natural history studies for disease progression in specific, well-characterized populations.
Study the psychosocial needs of HIV-positive women and their family systems (traditional and nontraditional, including lesbian women) as they cope with the chronic, crisis-oriented, and usually fatal nature of HlV disease. Give special attention to adolescent psychosocial needs with emphasis on suicide prevention and support strategies.
Characterize the Role of STDs in Adverse Pregnancy Outcomes
Perinatal infections, specifically, Group B streptococcus, cytomegalovirus, and chlamydia, are being studied to determine their incidence and resulting mammal, fetal, and neonatal outcomes.
Study factors such as the infecting pathogen, the stage of gestation during which infection occurs, chronicity of infection, and behavioral patterns such as drug abuse. Organisms should be specifically examined for virulence factors and for other markers associated with specific patterns of fetal or neonatal morbidity.
Conduct further studies to demonstrate whether drugs such as acyclovir and zidovudine are safe and effective for use during pregnancy.
Direct immunologic studies toward the protective immune responses during breastfeeding to identify the components in breast milk that are primarily responsible for inhibition of specific pathogens.
Similarly, identify the role that breastfeeding plays in the transmission of certain infections such as HIV.
Examine such factors as chronicity of infection and stage of gestation during which infection occurs to identify specific pathogens. Improved understanding of the immunobiology of pregnancy and the use of both natural and artificial animal models of STDs in pregnancy are likely to be important to productive research in this area. In addition, organisms should be examined for virulence factors or other markers associated with specific patterns of fetal or neonatal morbidity.
Oocyte and Follicular Development in the Ovary
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Fetal Growth and Development Embryology and Congenital Malformations
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Congenital Infection and Substance Abuse
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The Brain and Reproduction
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Bergkvist L., Adami H-O, Persson I., Hoover R. Schairer C.: The risk of breast cancer after estrogen and estrogen-progestin replacement. N Engl J Med 321:293–297, 1989.
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Rubin G.L., Peterson H.B., Lee N.C., Maes E.F., Wingo P.A. Becker S.: Estrogen repalcement therapy and the risk of endometrial cancer: Remaining controversies. Am J Obstet Gynecol 162:148–154, 1990.
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Oncology Ovarian Neoplasms
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