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THE MENSTRUAL CYCLE AND HUMAN REPRODUCTION
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Lawrence M. Nelson, MD, Head, Unit on Gynecologic Endocrinology Zhi-Bin Tong, MD, Staff Scientist |
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| To develop diagnostic tools and therapeutic approaches for menstrual cycle and human reproduction disorders, we investigate genetic, immunological, and molecular aspects of such disorders, using autoimmune premature ovarian failure (POF) as a model condition. POF causes young women to develop amenorrhea and infertility before age 40 and was once considered an irreversible condition similar to normal menopause. We now know that, even though they fail to function normally, follicles remain in the ovaries of 50 percent of patients with POF. In 90 percent of such cases, the mechanism of the ovarian insufficiency remains a mystery. Work in neonatally thymectomized B6A mice, which display autoimmune oophoritis and ovarian failure similar to human autoimmune ovarian failure and are thus a model of autoimmune POF, led us to discover Mater, a novel oocyte protein that serves as a major antigen in ovarian autoimmunity. Using transgenic technology, we also demonstrated that Mater is essential for normal female fertility. In addition, we have been investigating clinical POF by recruiting patients to research protocols that are designed to gain insight into the mechanisms of ovarian follicle dysfunction and to develop appropriate treatments for patients with 46,XX spontaneous POF. Premature ovarian failure, autoimmune oophoritis, and steroidogenic cell autoantibodies Anasti, Vanderhoof, Nelson; in collaboration with Bakalov, Bondy, Calis Evidence is accumulating to suggest that autoimmune oophoritis and adrenal autoimmunity may represent a continuum of one pathophysiologic process. We have been conducting an IRB-approved protocol investigating autoimmune oophoritis as a mechanism causing 46,XX spontaneous POF. We are studying the accuracy of steroidogenic cell antibodies in predicting the presence of autoimmune oophoritis and collating results of the antibody test with a gold standard: autoimmune oophoritis confirmed by ovarian biopsy. Given that ascertainment bias has created uncertainty about the true prevalence of autoimmune lymphocytic oophoritis as a mechanism of POF, we are studying the prevalence of steroidogenic cell autoantibodies in young women with 46,XX spontaneous POF who meet two primary criteria: (1) infertility and amenorrhea as a primary concern and (2) otherwise general good health. It is well established that patients with spontaneous POF are at increased risk of developing autoimmune adrenal insufficiency, a potentially fatal disorder. Adrenal insufficiency has an insidious onset, and patients with the condition often experience a delay in diagnosis. There has been ongoing controversy regarding the best strategy to detect adrenal insufficiency at an early stage, and we recently studied the effectiveness of antisteroid cell antibody detection by indirect immunofluorescence as a screening test. We comprehensively evaluated the adrenal axis of 123 women with spontaneous POF and diagnosed asymptomatic adrenal insufficiency in four women who tested positive for adrenal antibodies. A positive adrenal antibody test was highly associated with adrenal insufficiency while a negative test was associated with normal adrenal function in all cases; the cortisol response during a standard adrenocorticotrophic hormone (ACTH) stimulation test gave two false positive results. Thus, measuring adrenal antibodies is an effective screening method with which to detect asymptomatic autoimmune adrenal insufficiency in young women with spontaneous POF, whereas the standard ACTH stimulation test should be reserved to confirm adrenal insufficiency in women who test positive for adrenal antibodies or in women with signs and symptoms of adrenal insufficiency. Bakalov VK, Vanderhoof VH, Bondy CA, Nelson LM. Adrenal antibodies detect asymptomatic autoimmune adrenal insufficiency in young women with spontaneous premature ovarian failure. Human Reprod 2002;17:2096-2100. Antigenic role of MATER in autoimmune premature ovarian failure Vanevski, Soto, Tong, Nelson Although autoantibodies develop in most autoimmune diseases, the existence of specific ovarian antigens involved in human autoimmune POF has yet to be confirmed. Autoimmune POF causes young women to develop amenorrhea, menopausal symptoms, and infertility. We have been developing a serum marker that will identify women who have POF due to autoimmunity and a treatment that will restore fertility. We have demonstrated that mice with autoimmune oophoritis develop antibodies against MATER. Experiments are under way to test the antigenic role of MATER in the development of autoimmune POF in mice and in women. We recently defined the human homolog of the mouse Mater and have initiated efforts to produce human recombinant MATER and to develop an assay to determine if some women with 46,XX spontaneous POF exhibit autoantibodies to this oocyte-specific protein. We previously reported that the maternal effect gene Mater is required for early embryonic development in mice. We cloned human MATER cDNA by PCR techniques and found that the human MATER gene spans about 63 kbp at chromosome 19 and comprises 15 exons and 14 introns. Expression of its mRNA is restricted to the oocytes. Human MATER cDNA shows an open reading frame encoding a polypeptide chain of 1,200 residues with a predicted molecular mass of 134,236 Da, and MATER protein was detected in human oocytes. The human and mouse cDNA share 67 percent homology while their deduced polypeptide chains have 53 percent amino acid identity. In addition, their protein structures have a number of similar features. Characterization of human MATER and its protein provides a basis for us to investigate their clinical implications in autoimmune POF and infertility in women. During the last year, we demonstrated the unreliability of a commercially available ovarian antibody test that uses cynomologous monkey ovary as substrate in an indirect immunofluorescence procedure. Some clinicians use this test to make treatment decisions, but, to our knowledge, the test had never been validated. We performed the test in a blinded manner in young women with 46,XX spontaneous POF, in control women with regular menstrual cycles (matched for age, race, and parity), and in control men (matched for age and race). We also compared the frequency of other autoantibodies associated with ovarian autoimmunity and found that women with POF were more likely to exhibit thyroid peroxidase and parietal cell antibodies. Unexpectedly, use of the commercially available test revealed that nearly one-third of normal control women had ovarian antibodies. One half of young women with POF were found to have antibodies, but the frequency of a positive test was not significantly different from that in normal women. None of 26 men was found to have ovarian antibodies. We were unable to improve the specificity of the test and thus conclude that the method has poor specificity and is not a reliable tool for the diagnosis of autoimmune ovarian failure. Our findings underscore the need to demonstrate the specificity of any ovarian antibody test before it is used clinically. Novosad JA, Kalantaridou SN, Tong Z-B, Nelson LM. Ovarian antibodies as detected by indirect immunofluorescence are unreliable in the diagnosis of autoimmune premature ovarian failure: a controlled evaluation. BMC Womens Health 2003;3:2. Tong Z-B, Bondy CA, Zhou J, Nelson LM. A human homologue of mouse Mater, a maternal effect gene essential for early embryonic development. Human Reprod 2002;17:903-911. Low follicle number as a mechanism of 46,XX spontaneous premature ovarian failure Tong, Udofa, Emeche, Nelson We previously demonstrated that the mere existence of a low number of follicles creates an indirect mechanism causing follicle dysfunction in young women with 46,XX spontaneous POF. We demonstrated histologically that the follicle dysfunction is mediated by inappropriate luteinization and that the mechanism relates to inadequate negative feedback on gonadotropins due to a low follicle cohort size. For this reason, we are looking for gene mutations that might cause germ cell deficiency and low cohort size as a mechanism of POF. Given that mice with mutations in the c-kit receptor have a reduced number of germ cells, we recently tested the hypothesis that such mutations are a cause of reduced germ cell number. The c-kit system, including c-kit receptor and its ligand, is known to play important regulatory roles in ovarian folliculogenesis and oogenesis. However, we found that mutations in the coding region of the kit receptor gene are uncommon in women with POF. In ongoing work, we are evaluating the kit ligand gene and other candidate genes in which mutations are known to cause ovarian follicle deficiency in mice. Nelson LM, Bakalov VK. Mechanisms of follicle dysfunction in 46,XX spontaneous premature ovarian failure. Endocrinol Metab Clin North Am 2003;32:613-637. in women with 46,XX spontaneous premature ovarian failure. BMC Womens Health 2002;2:8. Needs of young women with 46,XX spontaneous premature ovarian failure Groff, Spitalnik, Pastor, Vanderhoof, Nelson; in collaboration with Calis, Smith We are conducting clinical research to define more fully other needs of young women with 46,XX spontaneous POF. In B6A mice, neonatal thymectomy induces lacrimal gland autoimmunity as well as autoimmune ovarian failure and provides the basis for an animal model of Sjögren's syndrome. To test the hypothesis that a similar association may be present in a subset of women with 46,XX spontaneous POF, we are collaborating with investigators at the National Eye Institute. We found an association between ocular surface disease and POF that has not been reported before. Keratoconjunctivitis sicca is defined as at least one symptom of dry eye occurring often or present all of the time. There are two major mechanisms of keratoconjunctivitis defined on the basis of either aqueous tear deficiency or evaporative tear deficiency. A decreased volume of tear production characterizes aqueous tear deficiency and may be the result of autoimmunity directed against the lacrimal glands. A qualitative disturbance in the tear film with resultant instability, leading to increased evaporation and dryness of the ocular surface, characterizes evaporative tear deficiency and is most frequently the result of meibomian gland disease. Meibomian gland dysfunction leads to decreased lipid production, which results in tear film instability. Interestingly, androgens, which may be deficient in women with POF, appear to play an important role in supporting normal meibomian gland function. Compared with age-matched control women, we found that young women with 46,XX spontaneous POF have an increased prevalence and severity of both signs and symptoms of ocular surface disease. Given that not all patients had dry eye, it is possible that the dry eye phenotype may signal a particular mechanism of POF, such as autoimmunity. It is also possible that endocrine factors, such as the androgen deficiency associated with POF, might explain the association. Additional studies are planned to characterize this pathology further and determine its etiology. We also recently completed an investigation of the experiences of young women with spontaneous POF with regard to their initial presenting symptom, promptness of diagnosis, and patient education. We asked 50 patients previously diagnosed with spontaneous POF to participate in a structured interview. Disturbance in menstrual pattern was the most common initial symptom. Over half of the women who presented with this complaint reported visiting three different clinicians' offices before laboratory testing was performed to determine the diagnosis. For 25 percent of the women, it took more than five years for the diagnosis of POF to be established. A majority of the women were unsatisfied with the manner in which they had been informed of the diagnosis and with the amount of information they had received about their condition. Our findings demonstrate that women with spontaneous POF perceive a need for more aggressive evaluation of secondary amenorrhea and oligomenorrhea. Menstrual irregularity can be a sign of ovarian insufficiency, and the associated estrogen deficiency is a well-established risk factor for osteoporosis. Indeed, in an earlier study, we found that two-thirds of young women with spontaneous POF had osteopenia of the femoral neck. Adams Hillard PJ, Nelson LM. Adolescent girls, the menstrual cycle, and bone health. J Pediatr Endocrinol Metab 2003;16:673-681. Alzubaidi NH, Chapin HL, Vanderhoof VH, Calis KA, Nelson LM. Meeting the needs of young women with secondary amenorrhea and spontaneous premature ovarian failure. Obstet Gynecol 2002;99:720-725. Gordon CM, Nelson LM. Amenorrhea and bone health in adolescents and young women. Curr Opin Smith JA, Vitale S, Reed GF, Grieshaber SA, Goodman LA, Vanderhoof VH, Calis KA, Nelson LM. Dry eye COLLABORATORS Vladimir Bakalov, MD, Developmental Endocrinology Branch, NICHD, Bethesda MD Carolyn Bondy, MD, Developmental Endocrinology Branch, NICHD, Bethesda MD Karim A. Calis, MD, Warren Grant Magnuson Clinical Center Pharmacy, Bethesda MD Janine A. Smith, MD, Division of Epidemiology and Clinical Research, NEI, Bethesda MD For further information, contact nelsonl@cc1.nichd.nih.gov
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