Using 46,XX spontaneous premature ovarian failure as a model condition, we investigate genetic, immunological, and molecular aspects of disorders of the menstrual cycle and human reproduction. Once considered an irreversible condition similar to normal menopause, premature ovarian failure causes young women to develop amenorrhea and infertility before age 40. We now know that some women with this disorder can conceive and that ovarian follicles are present in 50 percent of patients with 46,XX spontaneous premature ovarian failure. In 90 percent of cases, the mechanism of the ovarian insufficiency remains a mystery even after thorough investigation. Work in neonatally thymectomized B6A mice, a model of autoimmune premature ovarian failure, 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 Materis a maternal effect gene essential for normal female fertility. In addition, we have been investigating clinical premature ovarian failure 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 premature ovarian failure.
MATER and autoimmune premature ovarian failure
Vanevski,1 Tong, Nelson; in collaboration with Bondy, de Pol
Although autoantibodies develop in most autoimmune diseases, the specific ovarian antigens involved in human autoimmune premature ovarian failure have yet to be defined. In certain strains of mice, neonatal thymectomy induces an autoimmune oophoritis that leads to premature ovarian failure. We demonstrated that mice with autoimmune oophoritis develop antibodies against MATER and defined the human homologue of the mouse Mater. The human MATER gene spans 63 kbp at chromosome 19 and, as in the mouse, comprises 15 exons and 14 introns, with expression restricted to the oocytes. The human and mouse cDNA share 67 percent homology while their deduced polypeptide chains have 53 percent identity of amino acids. Further, the human and mouse protein structures exhibit several similar features. We concluded that the human MATER and mouse Mater genes and proteins are conserved. Characterization of the human MATER and its protein provides a basis for investigating their clinical implications in autoimmune premature ovarian failure and infertility in women.
We analyzed the expression profile of Mater and its protein during mouse oogenesis and embryogenesis as well as their subcellular localization in oocytes. Mater mRNA was detectable earliest in oocytes of type 2 follicles, whereas MATER protein appeared earliest in oocytes of type 3a primary follicles. Both mRNA and protein accumulated during oocyte growth. In situ hybridization showed that Mater mRNA was progressively less abundant in oocytes beyond type 5a primary follicles. As assessed by ribonuclease protection assay, Mater mRNA was abundant in germinal vesicle oocytes but undetectable in all stages of pre-implantation embryos. In contrast, the protein persisted throughout pre-implantation development. Immunogold electron microscopic analysis revealed that MATER was located in oocyte mitochondria and nucleoli and close to nuclear pores. Taken together, our data indicate that Mater gene transcription and protein translation are active during oogenesis but appear inactive during early embryogenesis. Thus, Mater and its protein are expressed in a manner typical of maternal effect genes. The presence of MATER protein in mitochondria and nucleoli suggests that it may participate in both cytoplasmic and nuclear events during early development.
Tong ZB, Gold L, De Pol A, Vanevski K, Dorward H, Sena P, Palumbo C, Bondy CA, Nelson LM. Develop-mental expression and subcellular localization of mouse MATER, an oocyte-specific protein essential for early development. Endocrinology 2004;145:1427-1434.
Autoimmune oophoritis and steroidogenic cell autoimmunity
Anasti, Vanderhoof, Nelson; in collaboration with Bakalov, Calis, Furmaniak, Merino, Premkumar
Evidence is accumulating to suggest that autoimmune oophoritis and adrenal autoimmunity represent a continuum of one pathophysiologic process in women. Given that ascertainment bias has created uncertainty about the true prevalence of autoimmune lymphocytic oophoritis and steroidogenic cell autoimmunity as a mechanism of premature ovarian failure, we have been studying the prevalence of steroidogenic cell autoantibodies in young women with 46,XX spontaneous premature ovarian failure who meet two primary criteria: (1) infertility and amenorrhea as a primary concern and (2) otherwise general good health.
Over the last year, we completed a controlled prospective evaluation to assess the association between serum adrenal cortex autoantibodies and histologically confirmed autoimmune lymphocytic oophoritis. We tested 266 women with 46,XX spontaneous premature ovarian failure for the presence of adrenal cortex antibodies as assessed by indirect immunofluoresence. For 10 women, we used immunohistochemical lymphocyte markers to test for the presence of autoimmune oophoritis in ovarian biopsy specimens. We obtained a histological diagnosis of autoimmune oophoritis in four women who tested positive for adrenal cortex autoantibodies and excluded this diagnosis in ovarian biopsies from six women who tested negative for these autoantibodies. Women with histologically confirmed autoimmune oophoritis had a greater total ovarian volume as assessed by transvaginal sonography. They were also more likely to have subclinical adrenal insufficiency and clinical signs of androgen deficiency. Overall, 10 of the 266 women tested positive for adrenal cortex autoantibodies. The availability of a validated serum marker with which to detect autoimmune lymphocytic oophoritis will spare women the need for ovarian biopsy and should facilitate clinical research toward developing a therapy that could restore fertility for women with autoimmune oophoritis.
Bakalov VK, Anasti JN, Calis KA, Vanderhoof VH, Premkumar A, Chen S, Furmaniak J, Smith BR, Merino MJ, Nelson LM. Autoimmune oophoritis as a mechanism of follicular dysfunction in 46,XX spontaneous premature ovarian failure. Fertil Steril 2005;84:958-965.
Normal age-related decline in ovarian function
Pastor, Vanderhoof, Nelson; in collaboration with Calis, Premkumar
Greater insight into the physiology of the normal ovarian aging process might provide a basis from which to assess more accurately ovarian endocrine function in a young woman. Particularly in the case of young women at genetic risk for developing premature ovarian failure, an accurate method to assess ovarian function might assist them in making family planning decisions. The literature has reported several measures of ovarian endocrine function for evaluating infertile women, but we know little about how these measures relate to the normal ovarian aging process in women without fertility problems. Even younger women who are experiencing regular menstrual cycles experience an age-related decline in ovarian function reflected in declining ovarian follicle numbers.
During the past year, we completed a pilot study in which we assessed measures of ovarian function reflecting the ovarian aging process in normal young women. The purpose of the study was to examine the feasibility of developing a research test that could eventually define an endocrine functional ovarian age. As a dynamic measure, we chose to employ a previously reported FSH stimulation test; in contrast to methods employing clomiphene citrate and gonadotropin releasing hormone agonist, the FSH test involves both direct stimulation of the ovaries and direct measurement of ovarian follicle products (inhibin B and estradiol).
We recruited 42 healthy volunteer women aged 18 to 50 years who had regular ovulatory menstrual cycles and a previous pregnancy. We administered a single 300-IU dose of human recombinant FSH on the third day of the menstrual cycle. The main outcome measures were ovarian antral follicle count as determined by transvaginal ultrasound and basal and FSH-stimulated serum markers. We found that age correlated most strongly with FSH-stimulated serum inhibin B levels, followed by antral follicle count, basal FSH, basal Müllerian Inhibiting Substance (MIS), and basal inhibin B. Total antral follicle count correlated most strongly with basal MIS level.
Interestingly, when taking into consideration the available factors, multiple regression analysis identified basal serum FSH, stimulated inhibin B, and stimulated estradiol levels as the parameters that most significantly contributed to a correlation with age. From a physiological perspective, the outcome makes sense as an integrated measure of ovarian function. Thus, from a theoretical perspective, this three-parameter FSH stimulation test model of the age-related decline in ovarian function seems worth pursuing. Further investigation is required to determine the cycle-to-cycle reproducibility of this FSH stimulation test model of ovarian aging and its ability to detect asymptomatic ovarian endocrine insufficiency in young women.
Pastor CL, Vanderhoof VH, Lim LCL, Calis KA, Premkumar A, Guerro NT, Nelson LM. A pilot study investigating the age-related decline in ovarian function of regularly menstruating normal women. Fertil Steril 2005;84:1462-1469.
Needs of young women with 46,XX spontaneous premature ovarian failure
Vanderhoof, Covington, Groff, Corrigan, Nelson; in collaboration with Bakalov, Calis, Raygada, Fitzgerald, Smith
We conduct clinical research to define more fully other needs of young women with 46,XX spontaneous premature ovarian failure. To investigate dry eye syndrome, we used neonatally thymectomized B6A mice, which develop lacrimal gland autoimmunity as well as autoimmune ovarian failure and thus provide 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 premature ovarian failure, we collaborated with investigators at NEI and found an association between ocular surface disease and premature ovarian failure that has not been reported before. We found that young women with 46,XX spontaneous premature ovarian failure have greater prevalence and severity of both signs and symptoms of ocular surface disease than age-matched control women. Given that not all patients had dry eye, it is possible that the dry eye phenotype may signal a particular mechanism of premature ovarian failure, such as autoimmunity. It is also possible that endocrine factors, such as the androgen deficiency associated with premature ovarian failure, might explain the association. Additional studies are planned to characterize further this pathology and determine its etiology.
We have also been investigating women’s psychological response to the diagnosis of spontaneous premature ovarian failure. In general, given that the inability to reproduce creates a profound loss for most women and affects their self-esteem and relationships with others, the literature has thoroughly documented the psychological distress of women with infertility. Most commonly, women discover that they are infertile in a gradual manner after many failed attempts at conception. However, in cases such as premature ovarian failure, medical conditions that preclude normal fertility can be uncovered during the course of investigation of other presenting complaints. Thus, the clinician is confronted with communicating information about a sudden, unexpected diagnosis that is life-altering but not life-threatening.
The manner in which bad news is communicated can have a profound effect on patient satisfaction, treatment compliance, quality of life, and other health outcomes. We sought to learn more about the emotional processes associated with premature ovarian failure by (1) collecting evidence regarding the manner in which women were informed of the diagnosis; (2) assessing the initial emotional impact; (3) identifying areas of support used in coping; and (4) identifying factors that might improve care for women with premature ovarian failure. We conducted two focus groups and then administered structured telephone interviews to 100 women. We found that over two-thirds of women with premature ovarian failure were unsatisfied with the manner in which their clinician informed them of the diagnosis. Nearly 90 percent reported experiencing moderate to severe emotional distress at the time, the degree of which was positively correlated with the degree of dissatisfaction with the manner in which they had been informed of the diagnosis. Thorough and accurate medical information on premature ovarian failure, support of others, and spirituality were perceived as helpful in coping. The findings suggest that the manner in which patients are informed of the diagnosis can significantly affect their level of distress. Patients perceive a need for clinicians to spend more time with them and provide more information about premature ovarian failure.
Corrigan EC, Raygada MJ, Vanderhoof VH, Nelson LM. A woman with spontaneous premature ovarian failure gives birth to a child with fragile X syndrome. Fertil Steril 2005;84:1508.
Folgi A, Gauthier-Barichard F, Schiffmann R, Vanderhoof VH, Bakalov VK, Nelson LM, Boespflug-Tanguy O. Screening for known mutations in EIF2B genes in a large panel of patients with premature ovarian failure. BMC Women’s Health 2004;4:8.
Groff AA, Covington SN, Halverson LR, Fitzgerald OR, Vanderhoof VH, Calis KA, Nelson LM. Assessing the emotional needs of women with spontaneous premature ovarian failure. Fertil Steril 2005;83:1734-1741.
Nelson LM, Covington SN, Rebar RW. An update: spontaneous premature ovarian failure is not an early menopause. Fertil Steril 2005;83:1327-1332.
Smith JA, Vitale S, Reed GF, Grieshaber SA, Goodman LA, Vanderhoof VH, Calis KA, Nelson LM. Dry eye signs and symptoms in women with premature ovarian failure. Arch Ophthalmol 2004;122:151-156.
1Konstantina Vanevski, MD, former Postdoctoral Fellow
Collaborators
Vladimir Bakalov, MD, Developmental Endocrinology Branch, NICHD, Bethesda, MD
Carolyn Bondy, MD, Developmental Endocrinology Branch, NICHD, Bethesda, MD
Karim A. Calis, DPharm, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
Anto de Pol, PhD, Università degli Studi di Modena e Reggio Emilia, Modena, Italy
O. Ray Fitzgerald, PhD, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
Anne Fogli, PhD, INSERM, Clermont-Ferrand, France
Jadwiga Furmaniak, MD, FIRS Laboratories, RSR Ltd., Cardiff, UK
Lony Chong-Leong Lim, PhD, Specialty Laboratories, Santa Monica, CA
Maria J. Merino, MD, Laboratory of Pathology, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
Ahalya Premkumar, MD, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
Margarita Raygada, PhD, Laboratory of Clinical Genomics, NICHD, Bethesda, MD
Raphael Schiffmann, MD, Developmental and Metabolic Neurology Branch, NINDS, Bethesda, MD
Janine A. Smith, MD, Division of Epidemiology and Clinical Research, NEI, Bethesda, MD
For further information, contact Lawrence_Nelson@nih.gov.