Using autoimmune premature ovarian failure as a model condition, we investigate genetic, immunological, and molecular aspects of menstrual cycle disorders and disorders of human reproduction in order to develop diagnostic tools and therapeutic approaches for such disorders. Premature ovarian failure (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 50 percent of POF patients have follicles remaining in the ovary, even though the follicles fail to function normally. In 90 percent of cases, the mechanism of the ovarian insufficiency remains a mystery. Work in a mouse model of autoimmune POF, neonatal thymectomy of B6A mice, which induces autoimmune oophoritis and ovarian failure similar to human autoimmune 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 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.

MATER, a Human Gene Critical to Female Fertility
Tong, Nelson
During the past year, we defined the human homolog of the mouse Mater, a gene critical to female fertility. We previously reported that Mater is a maternal effect gene required for early embryonic development in mice. We cloned human MATER cDNA with PCR techniques, used hybridization and immunodetection to determine, respectively, the mRNA and protein, and relied on bioinformatics software to analyze the cDNA and protein sequences. We found that the human MATER gene spans an approximately 63 kbp DNA at chromosome 19 and is composed of 15 exons and 14 introns. Expression of its mRNA (about 4.2 kb) is restricted to the oocyte. Human MATER cDNA (3885 nt) shows an open reading frame (3600 nt) encoding a polypeptide chain composed of 1,200 residues with a predicted molecular mass of 134,236 Da. We detected MATER protein in human oocytes. The human and mouse cDNA share 67 percent homology while their deduced polypeptide chains exhibit 53 percent sequence identity. Further, their protein structures have a number of similar features. We have concluded that the human MATER and mouse Mater genes and their encoded proteins are conserved. Characterization of the human MATER and its protein provides a basis for us to investigate their clinical implications in autoimmune pre-mature ovarian failure and infertility in women.

We have generated a mouse line lacking Mater and are investigating the Mater mouse as a model for human idiopathic infertility. Some couples experience infertility despite the fact that no abnormalities can be detected in the man or the woman. A portion of these cases might be explained by poor egg quality related to genetic or functional deficiencies in heretofore unidentified human maternal effect genes. Maternal effect genes produce mRNA or proteins that accumulate in the egg during oogenesis, and these maternal products control the developmental program until the embryonic genome is activated. The factors governing the transition from the maternal to the embryonic genome are unknown. In mice, embryonic transcription is first detected in the late one-cell zygote stage and is required for development beyond the two-cell stage. Embryos from Mater female mice have a block in development at the two-cell stage.

Needs of Young Women with 46,XX Spontaneous Premature Ovarian Failure
Pastor, Groff, Vanderhoof, Nelson; in collabora-tion with Al-Zubaidi, Calis
The infertility associated with premature ovarian failure has been the primary focus of our research. An in-depth understanding of the mechanisms of follicle dysfunction in young women with 46,XX spontaneous premature ovarian failure might lead to treatments that could restore fertility. However, young women with the condition have other needs that must be met by their health care providers. We are conducting clinical research to define these other needs more fully and to determine how clinicians might best respond to them.

During the past year, we investigated the experiences of young women with spontaneous premature ovarian failure with regard to their initial presenting symptom, promptness of diagnosis, and patient education. We asked 50 patients previously diagnosed with spontaneous premature ovarian failure to participate in a structured interview survey. Disturbance in menstrual pattern was the most common initial symptom. Over half of the women who presented with this complaint reported visiting a clinician’s office three or more times before laboratory testing was performed to determine the diagnosis. For 25 percent of the women, more than five years elapsed before the diagnosis of premature ovarian failure was established. A majority of the women were dissatisfied with the manner in which they had been informed of the diagnosis and the amount of information they had received about their condition. Our findings demonstrate that women with spontaneous premature ovarian failure perceive a need for more aggressive evaluation of secondary amenorrhea and oligomenorrhea. Loss of menstrual regularity 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 premature ovarian failure had osteopenia of the femoral neck.

Autoimmune Adrenal Insufficiency
Vanderhoof, Nelson; in collaboration with Bakalov, Bondy
It is well established that patients with spontaneous premature ovarian failure 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 in these young women. During the past year, we completed studies testing the hypothesis that anti-steroid cell antibodies detected by indirect immuno-fluorescence may be an effective screening test. We comprehensively evaluated the adrenal axis of 123 women with spontaneous premature ovarian failure. We uncovered a new diagnosis of asymptomatic adrenal insufficiency in four women (3.2 percent). All four 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 presence of positive adrenal antibodies increased the pretest probability of adrenal insufficiency from 3.2 to 67 percent. As a screening method, the cortisol response during a standard adrenocorticotrophic hormone (ACTH) stimulation test gave two false positive results. Our findings demonstrate that measuring adrenal antibodies is an effective screening method with which to detect asymptomatic autoimmune adrenal insufficiency in young women with spontaneous premature ovarian failure. The standard ACTH stimulation test should be reserved to confirm adrenal insufficiency in women who test positive for adrenal antibodies or in those with signs and symptoms of adrenal insufficiency.

Mechanisms of Follicle Dysfunction in 46,XX Spontaneous Premature Ovarian Failure
Anasti, Shibanuma, Tong, Udofa, Emeche, Nelson; in collaboration with Bakalov
Autoimmune lymphocytic oophoritis is a clearly established mechanism of follicle dysfunction in some patients with 46,XX spontaneous premature ovarian failure. Due to ascertainment bias, the proportion of cases of premature ovarian failure that are caused by this mechanism is unclear. Evidence is accumulating to suggest that autoimmune oophoritis and adrenal autoimmun-ity may represent a continuum of one pathophysiologic process. To determine the true prevalence of steroid cell autoimmunity as a mechanism of premature ovarian failure, it is necessary to evaluate prospectively a group of young women who are representative of the population at large. To this end, we have been recruiting young women with 46,XX spon-taneous premature ovarian failure who meet two primary criteria: they are primarily concerned with infertility and amenorrhea, and they generally consider themselves to be in good health otherwise. In a prospective series, we found evidence for steroid cell autoimmunity in six of 123 women. In ongoing work, we are investigating the accuracy of steroid cell antibodies in predicting the presence of autoimmune oophoritis. We are collating the results of the antibody test with a gold standard: autoimmune oophoritis confirmed by ovarian biopsy.

We previously demonstrated that a low number of follicles is associated with follicle dysfunction in young women with 46,XX spontaneous premature ovarian failure and that the follicle dysfunction is mediated by inappropriate luteinization; the mechanism is related 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 premature ovarian failure. During the past year, we tested the hypothesis that mutations in the c-kit receptor may be the cause of the reduction in germ cell number in these women. Mice with such mutations have a reduced number of germ cells. The c-kit system, including c-kit receptor and its ligand, is known to play important regulatory roles in ovarian folliculogenesis and oogenesis. We found that mutations in the coding region of the kit receptor gene are uncommon in women with premature ovarian failure. In ongoing work, we are evaluating the kit ligand gene.

Antigenic Role of MATER in Autoimmune Premature Ovarian Failure
Vanevski, Tong, Nelson
Although autoantibodies develop in most autoimmune diseases, specific ovarian antigens involved in human autoimmune premature ovarian failure have yet to be confirmed. Autoimmune premature ovarian failure causes young women to develop amenorrhea, menopausal symptoms, and infertility. A similar syndrome appears in mice with post-thymectomy autoimmune oophoritis. We have been developing both a serum marker that will identify women with premature ovarian failure attributable to autoimmune oophoritis 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 premature ovarian failure in mice.