Lawrence M. Nelson, MD, Head, Unit on Gynecologic Endocrinology
Zhi-Bin Tong, MD, Staff Scientist
James Anasti, MD, Guest Researcher
Carmen Pastor, MD, Guest Researcher
Noriyuki Otsuka, MD, Postdoctoral Fellow
Sharon Covington, LCSW, Special Volunteer
Tamara Prodanov, MD, Special Volunteer
Vien Vanderhoof, RN, CRNP, Nurse Practitioner
Emily Corrigan, BS, Predoctoral Fellow
Jennifer Elizondo, BS, Predoctoral Fellow
Allison Groff, BA, Postbaccalaureate Fellow
Emily Hui, BA, Postbaccalaureate Fellow
Jackeline Soto, BS, Postbaccalaureate Fellow
June Ventura, BA, Postbaccalaureate Fellow
Ekemini Udofa, Summer Student

Using 46,XX spontaneous premature ovarian failure (POF) 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, POF causes young women to develop amenorrhea and infertility before age 40. Some women with the disorder can conceive, and 50 percent of such women have ovarian follicles. In 90 percent of cases, the mechanism of the ovarian insufficiency remains a mystery even after thorough investigation. We have been investigating clinical POF by recruiting patients to research protocols that are designed both to gain insight into the mechanisms of ovarian follicle dysfunction and to guide the development of appropriate treatments for such patients.

Genetic mechanisms of 46,XX spontaneous POF

Corrigan, Hui, Soto, Udofa, Vanderhoof, Tong, Nelson; in collaboration with Persani, Raygada, Reijo Pera, Zachman,

Genetic studies have identified several loci, at Xq22, Xq26-28, and Xp11.2-p22.1, whose disruption has been associated with the development of spontaneous POF. Fragile X syndrome, an X-linked disorder, is the most common hereditary cause of mental retardation and developmental delay. In nearly all cases, the disorder is caused by an expansion of CGG trinucleotide repeats in the 5′ untranslated region of FMR1 (Fragile Site Mental Retardation 1 Gene). Interestingly, premutations in FMR1, located at Xq27.3, have been associated with the development of 46,XX spontaneous POF. We reported a case of a young woman with established spontaneous POF who conceived subsequent to the diagnosis and had a child who manifests mental retardation due to fragile X syndrome. The case illustrates the need to inform patients with POF about the increased risk of carrying a premutation in FMR1, the options for testing, and the potential implications for family members with regard to diagnosis of menstrual irregularity, developmental delay, and neurological symptoms.

Members of the TGFβ superfamily play a role in supporting normal ovarian function. One member of this family, the gene encoding bone morphogenic protein 15 (PMB15), is located at Xp11.2. Based on evidence in animal models, as well as on a case report linking an A to G transition at position 704 of the BMP15 gene with familial POF, we participated in an international collaboration to perform BMP15 genetic analysis in a large series of women with spontaneous POF. The investigation revealed two novel heterozygous missense alterations (p.R68W in one case, p.A180T in five cases) affecting the proregion of BMP15. The alterations were not present in any of the controls. We found heterozygous gene BMP15 gene variants in 7 of 166 patients. Given that BMP15 is within the Xp locus linked to POF, the findings suggest that BMP15 represents one of the genes whose haploinsufficiency significantly contributes to the primary ovarian insufficiency in Turner's syndrome.

Deleted in AZoospermia-Like (DAZL), located at 3p24, is another candidate gene for POF. We participated in a large multicenter collaboration to determine if sequence variants in this gene affect the development of spontaneous POF. Rather than focusing on rare amino acid changes that severely impair protein function, the collaboration primarily tested the hypothesis that common variants, or SNPs (single nucleotide polymorphisms), might in aggregate have a measurable effect on the development of POF. Homozygosity for the 8145g SNP genotype (in the RNA binding domain in exon 2) was significantly associated with POF at a relatively early age. Furthermore, examination of haplotypes (groups of closely linked genetic markers that tend to be inherited together) showed that the combined effects of several SNPs, versus a reference haplotype, had a strong association with POF at an earlier age. Thus, SNPs in the DAZL gene may act singly or jointly to affect reproductive characteristics of women. In addition, sequencing of the entire coding region of DAZL, including all exons and flanking regions, identified four putative missense mutations. One mutation in the homozygous state was associated with POF, and three missense heterozygous mutations were associated with early menopause (age 40 to 45). Three mutations mapped to exon 2 and one to exon 5 of the DAZL gene; all were juxtaposed to or found within the RNA-binding domain. Further studies may shed light on the role of DAZL in the development of spontaneous POF.

Mice with mutations in the c-kit tyrosine kinase receptor Kit (W) and the c-kit ligand Kitlg (Steel) have impaired development of primordial germ cells. Both KIT and KITLG play critical roles in gametogenesis as well as in hematopoiesis and melanogenesis. Point mutations in the mouse Kit receptor tyrosine kinase can selectively impair fertility without inducing detectable abnormalities in hematopoiesis or pigmentation. The observations in mice suggest that mutations in either the KIT or KITLG gene might be a cause of POF in women. However, in previous experiments, we found that mutation of the KIT gene does not appear to be a common cause of 46,XX spontaneous POF in women. We recently demonstrated that mutations in the coding regions of the KITLG gene also do not appear to be a common cause of this condition.

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 may represent a continuum of one pathophysiologic process. Given that ascertainment bias has created uncertainty about the true prevalence of autoimmune lymphocytic oophoritis and steroidogenic cell autoimmunity as a mechanism of POF, we have been 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.

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 POF 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 the 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 to undergo ovarian biopsy and should facilitate clinical research directed to developing a therapy that could restore fertility for women with adrenal cortex antibodies.

The 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 young women. Particularly in the case of young women at genetic risk for developing POF, 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 with regular menstrual cycles experience an age-related decline in ovarian function as reflected in declining numbers of ovarian follicles.

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. We chose to employ, as a dynamic measure, a previously reported FSH stimulation test; in contrast to methods employing clomiphene citrate and gonadotropin releasing hormone agonist, the test involved both direct stimulation of the ovaries and direct measurement of ovarian follicle products (inhibin B and estradiol).

We recruited 42 healthy volunteer women age 18 to 50 years who had regular ovulatory menstrual cycles and a previous pregnancy. We administered a single dose of 300 IU human recombinant FSH on the third day of their 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. Total antral follicle count correlated most strongly with basal MIS level.

Interestingly, multiple regression analysis identified basal serum FSH, stimulated inhibin B, and stimulated estradiol levels as the parameters that most significantly correlated with age. From a physiological perspective, the regression results make sense as an integrated measure of ovarian function. Thus, from a theoretical perspective, the 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 our FSH stimulation test model of ovarian aging and its ability to detect asymptomatic ovarian endocrine insufficiency in young women.

Needs of young women with 46,XX spontaneous POF

Vanderhoof, Covington, Groff, Ventura, Godoy,1 Nelson; in collaboration with Bakalo, Calis, Corrigan, Fitzgerald, Kalantaridou, Koziol, Mazer, Troendle

We also conduct clinical research to define more fully other needs of young women with 46,XX spontaneous POF. The normal premenopausal ovary is an important source of androgen as well as of estrogen. In premenopausal women, daily testosterone production is approximately 300 µg, of which approximately half is derived from the ovaries and half from the adrenal glands. Thus, young women with spontaneous POF would be expected to have lower serum testosterone levels than normal women. However, there is considerable controversy as to whether female androgen deficiency syndrome exists and whether such a syndrome has clinical consequences, such as effects on bone, quality of life, or libido. Uncertainty also exists as to whether spontaneous POF is a cause of clinically significant testosterone deficiency. We thus undertook a study based on the rigorous method of equilibrium dialysis to measure levels of circulating free testosterone in women with 46,XX spontaneous POF. We evaluated 130 such women while they were off any estrogen replacement therapy and then again three months later while they were on a standardized estradiol regimen. We sampled regularly menstruating control women during the mid-follicular phase. While off estrogen therapy, POF patients had median serum free testosterone concentrations that were significantly lower than those in controls. While on physiologic transdermal estradiol therapy, their median serum free testosterone dropped significantly even though their sex hormone binding globulin levels did not change. While on estradiol replacement, 13 percent of the treated women had serum free testosterone levels below the lower limit of normal. The clinical implications of this testosterone deficiency may be significant, and we are currently investigation them further.

We have also been investigating women's psychological response to the diagnosis of spontaneous POF. In general, given that the inability to reproduce creates a profound sense of 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 POF, 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 POF by (1) collecting evidence on the manner in which 100 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 POF. We found that over two-thirds of women with POF were unsatisfied with the manner in which they were informed of the diagnosis. Nearly 90 percent reported that they experienced 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. They perceived that thorough and accurate medical information on POF, support of others, and spirituality were 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 POF.

Ninety percent of women with spontaneous POF reported to us that spirituality plays an important role in helping them cope with the emotional sequelae of their diagnosis. In a subsequent study, we demonstrated a statistically significant positive correlation between functional well-being and spiritual well-being by employing an instrument specifically designed and validated to measure spirituality apart from religiosity. Our findings suggest a need for a controlled interventional clinical trial to test the hypothesis that strategies to assist women in finding meaning and purpose in the diagnosis of spontaneous POF would improve their functional well-being and quality of life. A group of women with the disorder who receive standard management would serve as controls.

1 Heidi Godoy, BA, former Postbaccalaureate Fellow

Collaborators

Vladimir Bakalov, MD, Developmental Endocrinology Branch, NICHD, Bethesda, MD
Karim A. Calis, MD, Department of Pharmacy, NIH Clinical Research Center, Bethesda, MD
O. Ray Fitzgerald, PhD, Spiritual Ministry, NIH Clinical Research Center, Bethesda, MD
Jadwiga Furmaniak, MD, FIRS Laboratories, RSR Ltd., Cardiff, UK
Sophia Kalantaridou, MD, University of Ioannina, Ioannina, Greece
Deloris E. Koziol, PhD, Biostatistics Service, NIH Clinical Research Center, Bethesda, MD
Lony Chong-Leong Lim, PhD, Division of Immunology, Specialty Laboratories, Santa Monica, CA
Norman A. Mazer, PhD, Department of Pharmacy, NIH Clinical Research Center, Bethesda, MD
Maria J. Merino, MD, Laboratory of Pathology, NIH Clinical Research Center, Bethesda, MD
Luca Persani, MD, PhD, Università degli Studi di Milano, Milan, Italy
Ahalya Premkumar, MD, Imaging Sciences, NIH Clinical Research Center, Bethesda, MD
Margarita J. Raygada, PhD, Laboratory of Clinical Genomics, NICHD, Bethesda, MD
Renee A. Reijo Pera, PhD, University of California San Francisco, San Francisco, CA
James F. Troendle, PhD, Biometry and Mathematical Statistics Branch, NICHD, Bethesda, MD
Keith Zachman, MS, Reproductive Biology and Medicine Branch, NICHD, Bethesda, MD

For further information, contact lawrence_nelson@nih.gov.