The Menstrual Cycle and Human Reproduction

Lawrence M. Nelson, MD, Head, Unit on Integrative Reproductive Medicine
Zhi-Bin Tong, MD, Staff Scientist
Vaishali Popat, MD, MPH, Clinical Fellow
Sophia Kalantaridou, MD, Guest Researcher
Susan Orshan, PhD, RN, Guest Researcher
Vien Vanderhoof, RN, CRNP, Nurse Practitioner
Sharon Covington, LCSW, Special Volunteer
Tamara Prodanov, MD, Special Volunteer
Emily Corrigan, BS, Predoctoral Fellow
June Ventura, BA, Postbaccalaureate Fellow

Using spontaneous 46,XX primary ovarian insufficiency 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 and previously referred to as “premature menopause,” “premature ovarian failure,” or “PO F,” primary ovarian insufficiency (POI) causes young women to develop amenorrhea and infertility before age 40. In 90 percent of cases, the mechanism of the ovarian insufficiency remains a mystery even after thorough investigation. We investigate POI with the use of mouse models of the condition and by recruiting patients to research protocols that are designed to gain insight into the mechanisms of ovarian follicle dysfunction and to guide the development of appropriate treatments for such patients.

Genetic mechanisms of spontaneous 46,XX POI

Tong, Vanderhoof, Corrigan, Nelson; in collaboration with Wittenberger, Reijo Pera, Castrillon

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 POI. 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 CG trinucleotide repeats in the 5′ untranslated region of FMR1 (Fragile Site Mental Retardation 1 gene). Interestingly, pre-mutations in the FMR1 gene, located at Xq27.3, have been associated with the development of spontaneous 46,XX POI. We previously reported a case of a young woman with established spontaneous POI 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 POI about their increased risk of carrying a pre-mutation in FMR1, their options for testing, and the potential implications for family members with regard to diagnosis of menstrual irregularity, developmental delay, and neurological symptoms.

The forkhead transcription factor Foxo3 is a master regulator and potent suppressor of primordial follicle activation. Loss of Foxo3 function in the mouse leads to primary ovarian insufficiency due to global follicle activation. The mouse Foxo3 locus is haploinsufficient, and Foxo3^−/+ females undergo early reproductive senescence consistent with an increased rate of primordial follicle utilization. As part of an international collaboration, we sought to determine if heterozygous or homozygous polymorphisms or mutations of the human orthologue FOXO3 contribute to primary ovarian insufficiency. We sequenced the exons and flanking splice sequences of the gene in a large number (302) of women with idiopathic primary ovarian insufficiency. We identified a total of eight single-nucleotide polymorphisms (SNPs), revealing a substantial amount of genetic variation at the FOXO3 locus. Allelic frequencies in control samples excluded several of the variants as causal. For the remaining variants, we performed site-directed mutagenesis to assess their functional impact but observed that these rare sequence variants were not associated with significant decreases in FOXO3 activity. Taken together, our findings suggested that, despite the potential for FOXO3 haploinsufficiency to cause POI, FOXO3 mutations are not a common cause of primary ovarian insufficiency.

Gallardo TD, John GB, Bradshaw K, Welt C, Reijo-Pera R, Vogt PH , Touraine P, Bione S, Toniolo D, Nelson LM, Zinn AR, Castrillon DH. Sequence variation at the human FOXO3 locus: a study of premature ovarian failure and primary amenorrhea. Hum Reprod 2008;23:216-221.
McConkie-Rosell A, Abrams L, Finucane B, Cronister A, Gane LW, Coffey SM, Sherman S, Nelson LM, Berry-Kravis E, Hessl D, Chiu S, Street N, Vatave A, Hagerman RJ. Recommendations from multidisciplinary focus groups on cascade testing and genetic counseling for fragile X-associated disorders. J Genet Couns 2007;16:593-606.
Wittenberger MD, Hagerman RJ, Sherman SL, McConkie-Rosell A, Welt CK, Rebar RW, Corrigan EC, Simpson JL, Nelson LM. The FMR1 premutation and reproduction. Fertil Steril 2007;87:456-465.

Testosterone deficiency and sexual function in young women with spontaneous 46,XX POI

Kalantaridou, Vanderhoof, Corrigan, Nelson; in collaboration with Calis, Troendle

The normal premenopausal ovary is an important source of androgen as well as of estrogen production. 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 POI would be expected to have lower serum testosterone levels than normal women. However, there is considerable controversy as to whether female androgen deficiency syndrome in fact exists and whether such a syndrome has clinical consequences for women, with effects on bone, quality of life, or libido. We sampled regularly menstruating control women during the mid-follicular phase. While off estrogen therapy, patients with POI had median serum free testosterone concentrations that were significantly lower than those in controls. While on physiologic transdermal estradiol therapy, the patients’ median serum free testosterone dropped significantly lower even though their sex hormone binding globulin levels did not change. While on estradiol replacement, 13 percent of the women had serum free testosterone levels below the lower limit of normal.

No controlled studies to date have specifically evaluated sexual function in women with spontaneous 46,XX POI. We sought to determine whether women with this condition who have been given estradiol replacement experience sexual dysfunction as compared with young women of similar age with normal ovarian function. Sexual dysfunction can have harmful effects on relationships, self-esteem, and quality of life. Normal sexual function involves the interaction of emotional, cultural, personal, interpersonal, contextual, and medical factors. Young women find the diagnosis of 46,XX spontaneous POI particularly traumatic. Disorders of mental health, especially depression, frequently underlie the presentation of sexual dysfunction. The traditional separatist notion that sexual dysfunction has either psychological or organic origins has given way to an understanding that the two are closely linked and must be taken into consideration together.

We assessed sexual function in 143 women with spontaneous 46,XX POI after at least three months of a standardized hormone replacement regimen (100 μg/day estradiol patch and oral medroxyprogesterone acetate 10 mg for 12 days each month) and compared the findings with those of 70 control women with normal ovarian function and regular menstrual periods. We employed the Derogatis Interview for Sexual Function Self Report (DISF-SR), a validated self-administered questionnaire. Women with POI had significantly lower DISF-SR composite scores than control women. Their serum total testosterone levels were significantly correlated with their DISF-SR composite scores, but accounted for only 4 percent of the variance in the score. Patients with testosterone levels below normal tended to have lower DISF-SR composite scores. As assessed by the DIS F-SR, sexual function is in the normal range for most young women with 46,XX spontaneous POI who are receiving physiologic estradiol replacement. However, as a group, these women score significantly lower on this sexual function scale than control women. We are currently investigating the relative contributions of psychosocial factors and testosterone deficiency to the sexual function of these patients.

Kalantaridou SN , Vanderhoof VH, Calis KA, Corrigan EC , Troendle JF, Nelson LM . Sexual function in young women with spontaneous 46,XX primary ovarian insufficiency. Fertil Steril 2008;90:1805-1811.

Needs of young women with spontaneous 46, XX primary ovarian insufficiency

Popat, Ventura, Fitzgerald, Covington, Vanderhoof, Nelson; in collaboration with Orshan, Calis, Koziol, Troendle

We investigate women’s psychological response to the diagnosis of spontaneous POI. In general, given that the inability to reproduce creates a profound loss for most women and affects their selfesteem and relationships, 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 POI, medical conditions that preclude normal fertility may 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 previously demonstrated that over two-thirds of women with POI 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 POI, support of others, and spirituality were all helpful in coping. Patients perceived a need for clinicians to spend more time with them and provide more information about premature ovarian failure.

In structured interviews, 90 percent of women with spontaneous POI reported to us that spirituality plays an important role in how they cope with the emotional sequelae of the diagnosis. In a follow-up 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 POI 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.

We tested the hypothesis that women with spontaneous POI differ from control women with regard to perceived social support. Using validated self-reporting instruments (Personal Resource Questionnaire-85 [PRQ85], Rosenberg Self-Esteem Scale), we investigated the relationship between perceived social support and self-esteem by comparing 154 women diagnosed with spontaneous POI at a mean age of 27 years with 63 healthy control women. We found that women with POI had significantly lower scores than controls on the perceived social support scale and self-esteem scale. The findings remained significant after modeling with multivariate regression for differences in age, marital status, and having children. The modeling showed a significant positive correlation between self-esteem scores and perceived social support and no significant differences in perceived social support or self-esteem related to marital status, number of children, or time since diagnosis. The evidence supports the need for prospective controlled studies. Strategies to improve social support and self-esteem might provide a therapeutic approach for reducing the emotional suffering that accompanies the life-altering diagnosis of spontaneous POI.

We also investigated methods that might improve fertility in women with POI. Normally, ovarian follicles grow in response to FSH stimulation; then, the mid-cycle LH surge induces follicle rupture, ovulation, terminal differentiation of granulosa cells into luteal cells, and formation of the corpus luteum. In up to 78 percent of women with POI, the normal process of ovulation usually fails despite the presence of antral follicles. We demonstrated previously that many of these follicles fail to function normally; instead, they become prematurely luteinized because of associated chronically elevated serum LH levels. The process is akin to “luteinized unruptured follicle syndrome.” Estrogen replacement therapy might improve ovulation rates in women with spontaneous POI by lowering serum LH levels to normal. We found that a regimen of 100 μg per day of transdermal estradiol replacement achieves normal serum LH levels in approximately half of women with spontaneous POI. Theoretically, by avoiding inappropriate luteinization, physiologic estradiol replacement therapy might improve follicle function in women with POI. Accordingly, we are undertaking controlled studies to assess the effect of estrogen replacement on follicle function in such women.

Covington SN, Martinez PE, Popat V, Nandagopol R, Ryan M, Nelson LM. The psychology of antecedents to adult reproductive disorders in adolescent girls. Ann NY Acad Sci 2008;1135:155-162.
Orshan SA, Ventura JL, Covington SN, Vanderhoof VH, Troendle JF, Nelson LM. Women with spontaneous 46,XX primary ovarian insufficiency (hypergonadotropic hypogonadism) have lower perceived social support than control women. Fertil Steril 2008 [E-pub ahead of print].
Popat VB, Prodanov T, Calis KA, Nelson LM. The menstrual cycle: a biological marker of general health in adolescents. Ann NY Acad Sci 2008;1135:43-51.
Popat VB, Vanderhoof VH, Calis KA, Troendle JF, Nelson LM. Normalization of serum LH levels in women with 46,XX spontaneous primary ovarian insufficiency. Fertil Steril 2008;89:429-433.
Ventura JL, Fitzgerald OR, Koziol DE, Covington SN, Vanderhoof VH, Calis KA, Nelson LM. Functional well-being is positively correlated with spiritual well-being in women with spontaneous premature ovarian failure. Fertil Steril 2007;87:584-590.

Collaborators

Karim A. Calis, MD, Pharmacy, NIH Clinical Research Center, Bethesda, MD
Diego H. Castrillon, MD, PhD, University of Texas Southwestern, Dallas, TX
O. Ray Fitzgerald, PhD, Spiritual Ministry, NIH Clinical Research Center, Bethesda, MD
Deloris E. Koziol, PhD, Biostatistics Service, NIH Clinical Research Center, 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
Michael Wittenberger, MD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD

For further information, contact lawrence_nelson@nih.gov or visit http://poi.nichd.nih.gov.