THE MENSTRUAL CYCLE AND HUMAN REPRODUCTION
, Head, Unit on Integrative Reproductive Medicine
Zhi-Bin Tong, MD, Staff Scientist
Sophia Kalantaridou, 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
Emily Hui,BA, Postbaccalaureate Fellow
Lindsey Lawless, BS, Postbaccalaureate Fellow
Jackeline Soto, BS, Postbaccalaureate Fellow
June Ventura, BA, Postbaccalaureate Fellow
Ekemini Udofa, Summer Student
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 or premature ovarian failure (POF), primary ovarian insufficiency (POI) causes young women to develop amenorrhea and infertility before age 40. In 90 percent of cases, the mechanism of ovarian insufficiency remains a mystery even after thorough investigation. We have been investigating POI by using mouse models of the condition and by recruiting patients to participate in 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
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 CGG trinucleotide repeats in the 5¢ untranslated region of the FMR1 (Fragile Site Mental Retardation 1) gene. Interestingly, premutations in the FMR1 gene, located at Xq27.3, have been associated with the development of spontaneous 46,XX POI. We reported a case of a young woman with established spontaneous POI who conceived following 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 premutation 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.
Members of the TGFb superfamily play a role in supporting normal ovarian function. One member of this family, the gene for bone morphogenic protein 15 (BMP15), is located at Xp11.2. Based on evidence in animal models as well as on a case report of familial POI linking an A-to-G transition at position 704 of the BMP15 gene, we participated in an international collaboration to perform BMP15 genetic analysis in a large series of women with spontaneous POI. The investigation revealed two novel heterozygous mis-sense alterations (p.R68W in one case and 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, significantly more than in controls. Given that BMP15 is within the Xp locus linked to POI, the findings support the concept that BMP15 represents one of the genes whose haploinsufficiency significantly contributes to POI in Turner syndrome.
Deleted in AZoospermia-Like (DAZL), located at 3p24, is another candidate gene for POI. We participated in a large multicenter collaboration to determine whether sequence variants in this gene cause spontaneous POI (Tung JY et al., Hum Genet 2006;118:730). Rather than focusing on rare amino acid changes that severely impair protein function, the effort primarily tested the hypothesis that common variants, or SNPs (single-nucleotide polymorphisms), might in aggregate have a measurable effect on the development of POI. We found that homozygosity for the 8145g SNP genotype (in the RNA-binding domain in exon 2) was significantly associated with POI. Furthermore, an 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 an earlier age of POI. Thus, SNPs in the DAZL gene may act singly or jointly to affect women’s reproductive characteristics. In addition, sequencing of the entire coding region of DAZL, including all exons and flanking regions, revealed four putative mis-sense mutations. One mutation in the homozygous state was associated with POI, and three mis-sense 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 POI.
Foxo3-null female mice exhibit the distinctive ovarian phenotype of global follicle activation leading to oocyte death, early depletion of functional follicles, and secondary infertility. Thus, Foxo3 functions at the earliest stages of follicle growth as a suppressor of follicle activation, raising the possibility that accelerated follicle initiation plays a role in the development of POI in women. We participated in an international collaboration to test the hypothesis that sequence variation at the human FOXO3 locus is a mechanism of POI and primary amenorrhea. We sequenced the entire coding region (two exons) in a large number of women with POI (N = 273) or primary amenorrhea (N = 29). We identified eight SNPs, revealing a substantial amount of genetic variation at the FOXO3 locus. We determined allelic frequencies and compared them with controls. All coding variants were single–amino acid substitutions that were either conservative or not predicted to have a major impact on protein structure or function. Using a luciferase reporter under the control of a FoxO-responsive promoter, we performed site-directed mutagenesis to assess further the potential functional impact of these coding variants. Taken together, our findings do not exclude the possibility that, in some individuals, FOXO3 mutations contribute to POI or primary amenorrhea, but they do argue that de novo FOXO3 mutations are not a common cause of either condition.
Mice with mutations in the c-kit tyrosine kinase receptor Kit (W) gene and the c-kit ligand Kitlg (Steel) gene exhibit 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. These observations suggest that mutations in the KIT or KITLG gene might be a cause of POI in women. However, we found in previous experiments that mutation of the KIT gene does not appear to be a common cause of 46,XX spontaneous POI in women. Furthermore, we recently demonstrated that mutations in the coding regions of the KITLG gene do not appear to be a common cause of this condition.
Di Pasquale E, Rossetti R, Marozzi A, Bodega B, Borgato S, Cavallo L, Einaudi S, Radetti G, Russo G, Sacco G, Sacco M, Wasniewska M, Cole T, Beck-Peccoz P, Nelson LM, Persani L. Identification of new variants of human BMP15 gene in a large cohort of women with premature ovarian failure. J Clin Endocrinol Metab 2006;91:1976-9.
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 [E-pub ahead of print].
Hui ES, Udofa EA, Soto J, Vanderhoof VH, Zachman K, Tong Z-B, Nelson LM. Investigation of the human stem cell factor KIT ligand gene, KITLG, in women with 46,XX spontaneous premature ovarian failure. Fertil Steril 2006;85:1502-6.
Tung JY, Rosen MP, Nelson LM, Turek PJ, Witte JS, Cramer DW, Cedars MI, Reijo Pera RA. Novel missense mutations of the Deleted in AZoospermia-Like (DAZL) gene in infertile women and men. Reprod Biol Med 2006;4:40.
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;87:456-65.
Testosterone deficiency in young women with spontaneous 46,XX POI
The normal premenopausal ovary is an important source of androgen as well as of estrogen production. Premenopausal women produce approximately 300 µg testosterone a day, 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, considerable controversy persists as to whether female androgen deficiency syndrome in fact exists and, if so, whether it has clinical consequences for women, such as effects on bone, quality of life, or libido. It is also unclear whether spontaneous POI is a cause of clinically significant testosterone deficiency. Using the rigorous method of equilibrium dialysis, we undertook a study to measure levels of circulating free testosterone in women with 46,XX spontaneous POI. We evaluated 130 such women while they were off any estrogen replacement therapy (ERT) and then again after months of a standardized estradiol regimen. We also sampled regularly menstruating control women during the midfollicular phase. While off ERT, patients with POI had median serum concentrations of free testosterone that were significantly lower than those in controls. While on physiologic transdermal estradiol therapy, the control women’s median free testosterone dropped significantly lower even though their sex hormone–binding globulin levels did not change. Among women on ERT, 13 percent had serum levels of free testosterone below the lower limit of normal. The clinical implications of this testosterone deficiency may be significant, and we are currently investigating them further.
Kalantaridou SN, Calis KA, Vanderhoof VH, Bakalov VK, Corrigan EC, Troendle JF, Nelson LM. Testosterone deficiency in young women with 46,XX spontaneous premature ovarian failure. Fertil Steril 2006;86:1475-82.
Sexual function in young women with spontaneous 46,XX POI
To date, no controlled studies have specifically evaluated sexual function in women with spontaneous 46,XX POI. We sought to determine whether women with this condition who had been given estradiol replacement have higher rates of sexual dysfunction than 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 a 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 been replaced by an understanding that the two are closely linked and must be considered together.
We assessed sexual function in 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). We compared our findings with those for 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 the DISF-SR composite score, but accounted for only 4 percent of the variance in DISF-SR composite score. Patients with testosterone levels below normal tended to have lower DISF-SR composite scores. Of patients with POI, 9 of 127 (7 percent) scored below the second centile on the composite sexual function score compared with 2 percent of control women, although the difference was not statistically significant. We conclude that, as assessed by the DISF-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 young women score significantly lower on the DISF-SR 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 [E-pub ahead of print].
Needs of young women with spontaneous 46,XX primary ovarian insufficiency
We have been investigating 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 self-esteem and relationships with others, the literature has thoroughly documented the psychological distress of women with infertility. Most commonly, women gradually discover that they are infertile 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 dissatisfied 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 learned of the diagnosis. They perceived that thorough and accurate medical information on POI, the support of others, and spirituality were all helpful in coping. The findings suggest that the manner in which patients are informed of a POI diagnosis can significantly influence their level of distress. Patients perceive a need for clinicians to spend more time with them and provide more information about POI.
Ninety percent of women with spontaneous POI reported to us in structured interviews that spirituality plays an important role in helping them cope with the emotional sequelae of a POI 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 a 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.
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;87:584-90.
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
Luca Persani, MD, PhD, Università degli Studi di Milano, Milan, Italy
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
Keith Zachman, MS, Reproductive Biology and Medicine Branch, NICHD, Bethesda, MD
For further information, contact Lawrence_Nelson@nih.gov.