- Email: [email protected]
Characterizing the reproductive hormone milieu in infertile women with diminished ovarian reserve
Characterizing the reproductive hormone milieu in infertile women with diminished ovarian reserve Lubna Pal, M.B.B.S.,a Katie Zhang, M.D.,b Gohar Zeitlian, M.D.,c and Nanette Santoro, M.D.c a Department of Obstetrics & Gynecology and Reproductive Sciences, Division of Reproductive Endocrinology, Yale University School of Medicine, New Haven, Connecticut; b Department of Obstetrics & Gynecology, University of Maryland Medical Center, Baltimore, Maryland; and c Department of Obstetrics and Gynecology & Women’s Health, Albert Einstein College of Medicine, Bronx, New York
Objective: To elucidate the reproductive hormone profiles in association with a diagnosis of diminished ovarian reserve (DOR). Design: Prospective observational study. Setting: Academic tertiary care infertility practice. Patient(s): Eight regularly cycling infertile women diagnosed with DOR as the underlying contributor to infertility and 14 age-comparable healthy controls. Intervention(s): Daily morning urine voids were collected during one menstrual cycle. Main Outcome Measure(s): Urinary excretion of gonadotropins (FSH, LH) and metabolites of estrogen (E; estrone conjugate) and P (pregnanediol 3 glucoronide) during an entire menstrual cycle in women with DOR and healthy controls. Result(s): Women with DOR demonstrated significantly higher urinary FSH levels in the early follicular phase, exaggerated amplitudes, significantly protracted durations of LH, concomitant FSH surges, and significantly impaired luteal phase urinary excretions of estrone conjugate and pregnanediol 3 glucoronide compared with the controls. Conclusion(s): Women with DOR demonstrate reproductive hormone profiles that are distinct from age-comparable controls, share similarities with profiles previously described during menopause transition, as well as exhibit unique features not previously recognized in the context of reproductive aging. (Fertil Steril 2010;93:1074–9. 2010 by American Society for Reproductive Medicine.) Key Words: Diminished ovarian reserve, Pdg, E1c, luteal, LH surge, urinary hormones, infertility
Peak reproductive capacity in women is attained before age 30 years (1, 2). Transgression into and beyond the fourth decade is accompanied by a progressive decline in fertility. The term ovarian reserve alludes to the available repertoire of gametes for procreative purpose, at any given age (3, 4). In addition to the rather dismal implications of diminished ovarian reserve (DOR) from the perspective of success of fertility treatments, limited data suggest that premenopausal women manifesting features of DOR may be destined for an early menopause (5–7). Equally concerning are suggestions that processes contributory to morbidities of postmenopausal years, specifically skeletal fragility and risk for cardiovascular disease, may be initiated conReceived August 31, 2008; revised October 12, 2008; accepted October 29, 2008; published online December 18, 2008. L.P. has nothing to disclose. K.Z. has nothing to disclose. G.Z. has nothing to disclose. N.S. has nothing to disclose. Supported in part by National Institutes of Health grants K12 (to L.P.) and (CD 41978) to N.S. Presented as an abstract at the 53rd annual meeting of the Society for Gynecologic Investigation, Toronto, Canada, March 22–27, 2006. Reprint requests: Lubna Pal, M.B.B.S., M.R.C.O.G., M.S., Department of Obstetrics & Gynecology and Reproductive Sciences, Division of Reproductive Endocrinology & Infertility, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208063, New Haven, CT 06520 (FAX: 203-785-7134; E-mail: [email protected]).
1074
comitantly with decline in ovarian reserve (8, 9), thus adding to the psychological burden (10) and potential health care-related cost attributable to premature decline in ovarian reserve. Quantitative and qualitative decline in the oocyte repertoire and a concomitant impaired granulosa cell (GC) efficiency are described in the context of DOR (11–13) and are suggested as contributors to the magnitude of subfertility in these women. Lower E2 levels during attempts at ovarian hyperstimulation are well described, as is evidence of luteal dysfunction in association with DOR (3). Our study was aimed at better characterizing the reproductive hormone profile and dynamics of both the pituitary gonadotropins and the ovarian hormones that are critical to reproductive physiology, in regularly menstruating infertile women diagnosed with DOR as the primary contributor to their infertility. Enrollees were asked to collect daily morning urine voids during the course of an entire menstrual cycle and urinary levels of FSH, LH, and of metabolites of circulating estrogens (estrone conjugates [E1c]) and P (pregnanediol- 3 diglucoronide [Pdg]) were measured using previously validated methodology (14–19) in infertile women diagnosed with DOR and in age-comparable healthy controls.
Fertility and Sterility Vol. 93, No. 4, March 1, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2008.10.069
FIGURE 1 Daily profile of urinary FSH levels in cases with diminished ovarian reserve (DOR) and controls presented across an entire menstrual cycle. Compared with the controls, the cases demonstrate significantly higher FSH levels in the early follicular phase (P¼ .03), and a significantly protracted increase in FSH concomitant with the LH surge (P¼ .01). Note: centering of cycles relative to the LH surge (day 0). Data are presented as mean SEM.
Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010.
MATERIALS AND METHODS Participants collected daily urine void (first specimen for the day) during the course of an entire menstrual cycle. The study was approved by the institutional review board (IRB) at Albert Einstein College of Medicine and Montefiore Medical Center and written consent was obtained. DOR Cases Infertile, yet otherwise healthy women attending the clinical practice in whom DOR was determined as the primary contributor to infertility were identified during a 24-month period. Patients were deemed to demonstrate DOR if early follicular phase serum FSH levels assessed within the first 3 days of onset of menses were more than 10 mIU/mL, as per clinical practice guidelines. In addition these participants must have demonstrated suboptimal ovarian response to previous attempts at ovarian hyperstimulation (i.e., utilization of gonadotropins in excess of 300 U/day with attainment of <3 dominant follicles). Additional inclusion criteria were regular menstrual cycles (21–35 days) normoprolactinemia, normal thyroid function, and absence of chronic diseases (e.g., lupus, rheumatoid arthritis, diabetes, hypertension, psychological or seizure disorders). Eight healthy, albeit infertile, women diagnosed with DOR based on the specified criteria were enrolled between April 2004 and April 2006. Controls Daily urinary samples available on 14 healthy, regularly cycling normoprolactinemic and euthyroid women, aged 18–38 Fertility and Sterility
years, represented the control group. Data on urinary reproductive hormones in the controls has been previously reported (16, 19). Quality control steps were undertaken to assure against assay drift on the available stored control samples (19). Specimen Collection Details on specimen collection procedures have been published previously (16). Briefly, the participants were asked to collect the first morning voided urine specimen in supplied containers and transfer a portion of the volume to the provided polypropylene tubes up to the specified mark. The tubes were prefilled with glycerol (final concentration of 7% glycerol was achieved in the collected urine sample) (20). Participants were instructed to store the tubes in a provided box within the home freezer. Specimens were transported to the laboratory at the end of the collection period and stored at -20 C. Subjects were provided with written instructions regarding the procedure of collection and storage of urine samples during an entire menstrual cycle (first day of menses to the first day of next menstrual flow). Hormone Assays Urinary LH and FSH were measured using a solid-phase, two-site specific fluoroimmunometric assay (DELFIA; PerkinElmer, Turku, Finland) and were validated in our laboratory using previously described methods (14–19). For urinary LH, interassay coefficient of variation (CV) and intra-assay CVs were 13.7% and 5.0%, respectively, and 16.4% and 7.6% for FSH, respectively. Estrone conjugates and Pdg were measured in duplicate by ELISA using antibodies and conjugate tracers. Interassay CV for E1c was 10.1% and intra-assay CV was 8.4%. Corresponding CVs for Pdg were 15.0% and 14.0%, respectively. Urinary hormone concentrations were adjusted for glycerol and normalized to urinary creatinine (21). Data Analysis Data presented reflects the urinary hormone profile during a single menstrual cycle in 8 patients with DOR and 14 healthy controls. Cycle length was normalized to 28 days, after determination of individual mean hormone levels for the entire cycle. Cycle days were aligned to the day of LH surge (set at day 0), as specified by predefined criteria (16, 17). The duration of the LH surge was defined as days of persistent elevation in LH levels above a threshold. Using a 5-day moving average, a 3 SD increase in LH levels was considered consistent with an LH surge. Presence of luteal activity was determined by a sustained increase in Pdg concentrations R3 mg/ mg creatinine for 3 consecutive days, as previously described (17). The duration of the follicular phase was determined to extend from cycle day 1 (first day of menses) to day -1 (with reference to the LH surge). Early follicular phase was specified as before and excluding day -6 (reference to day of LH surge set at 0). The luteal phase was determined to span from day þ2 (reference to LH surge) until and excluding the first day of next menses. 1075
TABLE 1 Cumulative hormone profiles (normalized to a 28-day cycle) in 8 infertile women with DOR and 14 controls. Urinary hormone levelsa FSH (mU/mg Cr) Early follicular Magnitude of surge Duration of surge LH (mU/mg Cr) Magnitude of surge Duration of surge E1Cb(ng/mg Cr) Presurge Lutealc Pdg(mg/mg Cr) Lutealc Luteal adequacyb,d
DOR (N [ 8)
Controls (N [ 14)
P value
7.66 0.63 28.93 10.43 2.30 0.56 63.90 45.5 64.33 18.55 3.29 0.39
6.27 0.32 17.00 3.47 1.14 0.10 34.10 25.90 40.85 7.87 1.93 0.24
.03* .20 .01* .31 .18 < .01*
64.26 7.44 28.39 2.10
83.26 13.71 38 1.70
.36 < .01*
4.41 0.42 6.86 1.39
7.58 0.66 10.21 1.11
< .01* .09
a
Mean SEM. Estrone conjugates. c One patient with diminished ovarian reserve (DOR) who failed to provide luteal phase urine samples was excluded from these analyses. d Pregnanediol 3 diglucoronide levels >3 mg/mg Cr. * Statistically significant. b
Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010.
The integrated urinary excretion of the individual urinary hormones was evaluated for participants with DOR and the healthy controls. Mean hormone levels for early follicular phase, the duration of follicular and luteal phases, and for peak LH and FSH surges in the two groups were compared by two-tailed Student’s t test. The levels of E1c on day -1 (i.e., the day before LH surge) and the duration of LH and FSH surges (in days) were similarly assessed in the two groups. STATA Intercooled 8.2 (StataCorp, College Station, TX) was used for statistical analyses. P value < .05 was considered statistically significant.
RESULTS Data presented reflects the urinary hormone profile during a single menstrual cycle in 8 patients with DOR and 14 healthy controls. The ages (34.12 1.36 vs. 32.55 6.15) and body mass index (BMI) (21.74 2.62 vs. 25.33 2.50) of the patients and controls, respectively, were comparable (mean SD). One of the eight patients with DOR failed to provide daily urine samples beyond the period of LH surge þ2 days. This patient was excluded from analyses evaluating the duration of LH and FSH surge and luteal Pdg levels. Evidence of an LH surge (as previously defined) was noted in all participants (cases and controls). Luteal activity (as previously defined) was observed in all 14 controls and in 7/8 patients with DOR. One patient with DOR did not exhibit any increase in Pdg excretion despite documentation of an LH surge. 1076
Pal et al.
Reproductive hormone milieu in DOR
Not surprisingly, those diagnosed with DOR demonstrated higher urinary levels of FSH through the phases of the menstrual cycle compared with the controls (Fig. 1). These differences were of statistical significance for the early follicular phase (P¼.03). Although the pre-LH surge E1c levels were lower in women with DOR compared with controls (Table 1), the difference was not of statistical significance (P¼.36). Patients with DOR demonstrated a robust gonadotropin surge (LH and FSH) as reflected by urinary levels of the respective pituitary gonadotropins. Although the amplitudes of urinary LH and FSH surges were higher in women with DOR compared with the controls (Figs. 1 and 2), these differences did not reach statistical significance (Table 1). The durations of the LH surge and the concomitant FSH surge, however, were significantly protracted in women with DOR (Table 1) compared with healthy controls. Luteal phase urinary E1c and Pdg excretions were noted to be significantly lower in patients with DOR compared with the controls (Figs. 3 and 4 and Table 1). The temporal span of ‘‘adequate’’ luteal activity as previously defined (i.e., duration of luteal phase demonstrating Pdg levels R3 mg/mL) was shorter in women with DOR compared with the controls. This association approached statistical significance (P¼.09). DISCUSSION Suboptimal ovarian responses to attempts at controlled ovarian hyperstimulation (COH) are well described in infertile Vol. 93, No. 4, March 1, 2010
FIGURE 2
FIGURE 3
Evidence of a robust and protracted LH surge is seen in patients with diminished ovarian reserve (DOR) compared with controls, as assessed by daily profile of urinary levels across an entire menstrual cycle. The differences between the two groups are of statistical significance for the duration of the LH surge (P< .01). Note: centering of cycles relative to the LH surge (day 0). Data are presented as mean SEM.
The daily profile of urinary estrone conjugate (E1c) levels across one menstrual cycle is suggestive of relative hypoestrogenism in premenopausal women with diminished ovarian reserve (DOR) compared with the controls. The differences are of statistical significance for the luteal phase of the cycle (P< .01). Note: centering of cycles relative to the LH surge (day 0). Data are presented as mean SEM.
Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010. Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010.
women diagnosed with DOR (4). Our findings of significantly lower urinary levels of estrogen (E) and P metabolites in a natural menstrual cycle in young infertile women with DOR are suggestive of ovarian GC dysfunction in these otherwise healthy and regularly cycling women. Although impairments in GC viability and proliferative indices are described in association with DOR (11–13, 22), in the present study we provide evidence suggestive of altered reproductive hormone profile in women with DOR. Of interest are findings reported by Leach et al. (23) using a similar paradigm of daily urinary hormone assessment, albeit in a different population of infertile women. Comparing urinary reproductive hormone levels in 12 women with unexplained infertility and normal baseline hormone profiles with 12 healthy controls, the investigators report significant elevations in urinary gonadotropins in the follicular phase and lower P levels in the midluteal phase of menstrual cycle in women with unexplained infertility. Therefore we identify certain similarities in the hormonal milieu of women with unexplained infertility and those with reduced ovarian reserve. Ovarian reserve compromise is suggested in cases of unexplained infertility (24) and consistency in our observations with those described by Leach et al. (23) underscores these concerns. Of interest are absence of differences in preovulatory or periovulatory hormone profiles in the two groups studied by the latter investigators. Our data, in contrast, demonstrate lower preovulatory E1c and higher magnitude and duration of gonadotropin surge in women with reduced ovarian reserve compared with healthy controls. Our observations add to the existing literature of distinctions and yet similarities between cases of unexplained infertility and reduced ovarian reserve. These Fertility and Sterility
observations identify a distinct hormonal milieu in the setting of DOR and merit substantiation by future studies. Impaired luteal function is a recognized accompaniment to chronological aging (16). Our findings of significantly impaired urinary Pdg excretion in women with DOR suggest similarities between DOR diagnosed in the mid-30s and features of reproductive aging that are recognized in the perimenopausal years (i.e., a decade later). In the context of infertility, we acknowledge that although our findings are suggestive of suboptimal luteal reproductive hormone profile (significantly lower urinary excretion of Pdg and E1c, and shorter duration of adequate Pdg excretion, as specified in the text), these observations per se are not confirmatory of luteal insufficiency. Of interest are observations reported by Soules et al. (25), who in a study of daily serum hormones in patients with endometrial biopsy proven luteal phase deficiency identified significantly lower luteal levels of E2 and P. Our data corroborate these observations. Although our observations allow us to propose that luteal dysfunction may contribute to the subfertility of DOR, this assumption needs to be substantiated by appropriately designed studies that incorporate endometrial sampling for the diagnosis of luteal phase deficiency (26). If indeed this assumption is confirmed, luteal support with exogenous P may hold potential for benefit for infertile women with DOR. A decrease in ovarian estrogen is suggested to underlie health morbidities of early menopause (i.e. risks for cardiovascular disease and osteoporosis) (27, 28). An atherogenic lipid profile has been previously described in infertile women demonstrating evidence of reduced ovarian reserve (9) as is enhanced bone turnover and evidence of low bone mass in 1077
FIGURE 4 Premenopausal women with diminished ovarian reserve (DOR) demonstrate impaired luteal excretion of P metabolite Pdg (P< .01) and shorter duration of luteal adequacy (i.e., days of Pdg R3 mg/mg creatinine [Cr]) (P¼ .09) compared with healthy controls. Note: centering of cycles relative to the LH surge (day 0). Data are presented as mean SEM.
Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010.
healthy infertile women with DOR (8). The relatively hypoestrogenic milieu suggested by the presented data may partly explain these observations of skeletal attrition and a proatherogenic milieu previously described in premenopausal women with reduced ovarian reserve. Our findings thus identify DOR as a state of relative estrogen deficiency. Our observations suggest an overlap in hormonal profiles of DOR and chronologically advanced reproductive aging (i.e., elevated FSH levels and impaired luteal excretion of Pdg) (16) is suggested. These findings corroborate the hypothesis that processes of reproductive aging may indeed be accelerated in women with DOR. Of note, however, our observations also provide glimpses of a distinct reproductive hormone milieu in the context of DOR. Significantly lower levels of E1c through the menstrual cycle, as well as robust LH and FSH surges in women with DOR, are aspects that may be unique to the DOR phenotype. In contrast to the hypoestrogenic milieu of DOR (as suggested by our data), relative hyperestrogenemia has been described in association with more advanced stages of reproductive aging that predate menopause (i.e in perimenopause) (16, 29). Furthermore, although subtle elevations in the amplitude of LH surge have previously been reported in the perimenopausal years (16), the protracted gonadotropin surges observed in young women with DOR appear unique to this population. Interestingly, a decline in the putative ovarian gonadotropin surge attenuating factor is suggested concomitant with ovarian reserve, as well as with aging (30). Although a presumable in the ovarian gonadotropin surge attenuating factor in women with DOR may underlie the noted robust and protracted LH surge, this latter conjecture remains unsubstantiated. An alternative explanation for the distinct gonadotropic profiles noted in patients with 1078
Pal et al.
Reproductive hormone milieu in DOR
DOR may be a possible central hypothalamoituitary dysregulation. These observations are of interest and merit further evaluation including attempts to identify the thus far elusive ovarian gonadotropin surge attenuating factor. The small sample size is an inherent limitation of our study, and this is reflected in the relatively large standard errors of some of the measurements (e.g., LH) and, hence identify instability of some of the estimates of group means. Although utilization of historic sample controls is not optimal, stability of urinary levels of reproductive hormones in appropriately collected and stored urine samples is well substantiated (19, 20). Quality control steps ensured integrity of the respective assays and absence of any assay drift in the historic control samples (data not shown). We are thus confident of the noted distinctions in the urinary hormone profiles between patients with DOR and the healthy controls. In summary, our data are supportive of an altered reproductive hormonal milieu in young and infertile women with DOR. In a natural menstrual cycle, the urinary hormone profiles identify DOR as a state of relative hypoestrogenism and of luteal insufficiency. Although our data suggest similarities in the reproductive hormone milieu of premenopausal infertile women with DOR with parameters described in an older population of perimenopausal women, certain aspects of the hormonal profile appear unique to the entity of DOR. Our data encourage us to propose that the known subfecundity in women with DOR may, at least in part, be explained by an altered reproductive hormonal profile. Although the hormone profiles in women with DOR are suggestive of ovarian GC dysfunction, the possibility of central functional alterations (i.e., at the level of the hypothalamic GnRH pulse generator or the pituitary gonadotrophs) cannot be excluded. Our findings (i.e., hypoestrogenism) provide insights into mechanisms that may underlie the previously identified osseo and cardio deleterious implications of DOR. The reported observations are thus of clinical relevance and merit prospective substantiation in a larger cohort. Acknowledgments: We thank Dr. Bill Lasley, University of California-Davis, Davis, California, for the provision of critical reagents for the conduct of urinary E1c and Pdg assays, and to Goli Adel for her invaluable contributions in the conduct of the urinary immunoassays.
REFERENCES 1. Gosden RG, Faddy MJ. Ovarian aging, follicular depletion, and steroidogenesis. Exp Gerontol 1994;29:265–74. 2. Faddy MJ, Gosden RG. A model confirming the decline in follicle numbers to the age of menopause in women. Hum Reprod 1996;11:1484–6. 3. Toner JP. Ovarian reserve, female age and the chance for successful pregnancy. Minerva Ginecol 2003;55:399–406. 4. Karande VC. Managing and predicting low response to standard in vitro fertilization therapy: a review of the options. Treat Endocrinol 2003;2:257–72. 5. de Boer EJ, den Tonkelaar L, te Velde ER, Burger CW, van Leeuwen FE, OMEGA-project group. Increased risk of early menopausal transition and natural menopause after poor response at first IVF treatment. Hum Reprod 2003;18:1544–52.
Vol. 93, No. 4, March 1, 2010
6. Nikolaou D, Templeton A. Early ovarian ageing: a hypothesis. Detection and clinical relevance. Hum Reprod 2003;18:1137–9. 7. Lawson R, El-Toukhy T, Kassab A, Taylor A, Braude P, Parsons J, et al. Poor response to ovulation induction is a stronger predictor of early menopause than elevated basal FSH: a life table analysis. Hum Reprod 2003;18:527–33. 8. Pal L, Bevilacqua K, Zeitlian G, Shu J, Santoro N. Implications of diminished ovarian reserve (DOR) extend well beyond reproductive concerns. Menopause 2008;15:1086–94. 9. Chu MC, Rath KM, Huie J, Taylor HS. Elevated basal FSH in normal cycling women is associated with unfavourable lipid levels and increased cardiovascular risk. Hum Reprod 2003;18:1570–3. 10. Friese C, Becker G, Nachtigall RD. Rethinking the biological clock: eleventh-hour moms, miracle moms and meanings of age-related infertility. Soc Sci Med 2006;63:1550–60. 11. Seifer DB, Gardiner AC, Ferreira KA, Peluso JJ. Apoptosis as a function of ovarian reserve in women undergoing in vitro fertilization. Fertil Steril 1996;66:593–8. 12. Seifer DB, Charland C, Berlinsky D, Penzias AS, Haning RV Jr, Naftolin F, et al. Proliferative index of human luteinized granulosa cells varies as a function of ovarian reserve. Am J Obstet Gynecol 1993;169: 1531–5. 13. Greenseid K, Jindal S, Hurwitz J, Zapantis, A, Nihsen M, Santoro N, et al. Declining ovarian reserve adversely influences granulosa cell viability. Fertil Steril. Published online June 17, 2008. 14. Saketos M, Sharma N, Adel T, Raghuwanshi M, Santoro N. Time-resolved immunofluorometric assay and specimen storage conditions for measuring urinary gonadotropins. Clin Chem 1994;40:749–53. 15. Saketos M, Sharma N, Santoro NF. Suppression of the hypothalamic-pituitary-ovarian axis in normal women by glucocorticoids. Biol Reprod 1993;49:1270–6. 16. Santoro N, Brown JR, Adel T, Skurnick JH. Characterization of reproductive hormonal dynamics in the perimenopause. J Clin Endocrinol Metab 1996;81:1495–501. 17. Santoro N, Crawford SL, Allsworth JE, Gold EB, Greendale GA, Korenman S, et al. Assessing menstrual cycles with urinary hormone assays. Am J Physiol Endocrinol Metab 2003;284:E521–30. 18. Brown JR, Skurnick JH, Sharma N, Adel T, Santoro N. Frequent intermittent ovarian function in women with premature menopause: a longitudinal study, 1993. Endocrinol J 1993;1:467–74.
Fertility and Sterility
19. Zhang K, Pollack S, Ghods A, Dicken C, Isaac B, Adel G, et al. Onset of ovulation after menarche in girls: a longitudinal study. J Clin Endocrinol Metab 2008;93:1186–94. 20. Livesey JH, Roud HK, Metcalf MG, Donald RA. Glycerol prevents loss of immunoreactive follicle-stimulating hormone and luteinizing hormone from frozen urine. J Endocrinol 1983;98:381–4. 21. Taussky HH. A procedure increasing the specificity of the Jaffe reaction for the determination of creatine and creatinine in urine and plasma. Clin Chim Acta 1956;1:210–24. 22. Seifer DB, Honig J, Penzias AS, Lavy G, Nadkarni PM, Jones EE, et al. Flow cytometric analysis of deoxyribonucleic acid in human granulosa cells as a function of chronological age and ovulation induction regimen. J Clin Endocrinol Metab 1992;75:636–40. 23. Leach RE, Moghissi KS, Randolph JF, Reame NE, Blacker CM, Ginsburg KA, et al. Intensive hormone monitoring in women with unexplained infertility: evidence for subtle abnormalities suggestive of diminished ovarian reserve. Fertil Steril 1997;68:413–20. 24. Randolph JF, Ginsburg KA, Leach RE, Blacker CM, Moghissi KS, Diamond MP, et al. Elevated early follicular gonadotropin levels in women with unexplained infertility do not provide evidence for disordered gonadotropin-releasing hormone secretion as assessed by luteinizing hormone pulse characteristics. Fertil Steril 2003;80:320–7. 25. Soules MR, McLachlan RI, Ek M, Dahl KD, Cohen NL, Bremner WJ. Luteal phase deficiency: characterization of reproductive hormones over the menstrual cycle. J Clin Endocrinol Metab 1989;69:804–12. 26. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol 1975;122:262–3. 27. Matthews KA, Wing RR, Kuller LH, Meilahn EN, Plantinga P. Influence of the perimenopause on cardiovascular risk factors and symptoms of middle-aged healthy women. Arch Intern Med 1994;154:2349–55. 28. Slemenda C, Hui SL, Longcope C, Johnston CC. Sex steroids and bone mass. A study of changes about the time of menopause. J Clin Invest 1987;80:1261–9. 29. Prior JC. Ovarian aging and the perimenopausal transition: the paradox of endogenous ovarian hyperstimulation. Endocrine 2005;26: 297–300. 30. Martinez F, Barri PN, Coroleu B, Tur R, Sorsa-Leslie T, Harris WJ, et al. Women with poor response to IVF have lowered circulating gonadotrophin surge-attenuating factor (GnSAF) bioactivity during spontaneous and stimulated cycles. Hum Reprod 2002;17:634–40.
1079
Objective: To elucidate the reproductive hormone profiles in association with a diagnosis of diminished ovarian reserve (DOR). Design: Prospective observational study. Setting: Academic tertiary care infertility practice. Patient(s): Eight regularly cycling infertile women diagnosed with DOR as the underlying contributor to infertility and 14 age-comparable healthy controls. Intervention(s): Daily morning urine voids were collected during one menstrual cycle. Main Outcome Measure(s): Urinary excretion of gonadotropins (FSH, LH) and metabolites of estrogen (E; estrone conjugate) and P (pregnanediol 3 glucoronide) during an entire menstrual cycle in women with DOR and healthy controls. Result(s): Women with DOR demonstrated significantly higher urinary FSH levels in the early follicular phase, exaggerated amplitudes, significantly protracted durations of LH, concomitant FSH surges, and significantly impaired luteal phase urinary excretions of estrone conjugate and pregnanediol 3 glucoronide compared with the controls. Conclusion(s): Women with DOR demonstrate reproductive hormone profiles that are distinct from age-comparable controls, share similarities with profiles previously described during menopause transition, as well as exhibit unique features not previously recognized in the context of reproductive aging. (Fertil Steril 2010;93:1074–9. 2010 by American Society for Reproductive Medicine.) Key Words: Diminished ovarian reserve, Pdg, E1c, luteal, LH surge, urinary hormones, infertility
Peak reproductive capacity in women is attained before age 30 years (1, 2). Transgression into and beyond the fourth decade is accompanied by a progressive decline in fertility. The term ovarian reserve alludes to the available repertoire of gametes for procreative purpose, at any given age (3, 4). In addition to the rather dismal implications of diminished ovarian reserve (DOR) from the perspective of success of fertility treatments, limited data suggest that premenopausal women manifesting features of DOR may be destined for an early menopause (5–7). Equally concerning are suggestions that processes contributory to morbidities of postmenopausal years, specifically skeletal fragility and risk for cardiovascular disease, may be initiated conReceived August 31, 2008; revised October 12, 2008; accepted October 29, 2008; published online December 18, 2008. L.P. has nothing to disclose. K.Z. has nothing to disclose. G.Z. has nothing to disclose. N.S. has nothing to disclose. Supported in part by National Institutes of Health grants K12 (to L.P.) and (CD 41978) to N.S. Presented as an abstract at the 53rd annual meeting of the Society for Gynecologic Investigation, Toronto, Canada, March 22–27, 2006. Reprint requests: Lubna Pal, M.B.B.S., M.R.C.O.G., M.S., Department of Obstetrics & Gynecology and Reproductive Sciences, Division of Reproductive Endocrinology & Infertility, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208063, New Haven, CT 06520 (FAX: 203-785-7134; E-mail: [email protected]).
1074
comitantly with decline in ovarian reserve (8, 9), thus adding to the psychological burden (10) and potential health care-related cost attributable to premature decline in ovarian reserve. Quantitative and qualitative decline in the oocyte repertoire and a concomitant impaired granulosa cell (GC) efficiency are described in the context of DOR (11–13) and are suggested as contributors to the magnitude of subfertility in these women. Lower E2 levels during attempts at ovarian hyperstimulation are well described, as is evidence of luteal dysfunction in association with DOR (3). Our study was aimed at better characterizing the reproductive hormone profile and dynamics of both the pituitary gonadotropins and the ovarian hormones that are critical to reproductive physiology, in regularly menstruating infertile women diagnosed with DOR as the primary contributor to their infertility. Enrollees were asked to collect daily morning urine voids during the course of an entire menstrual cycle and urinary levels of FSH, LH, and of metabolites of circulating estrogens (estrone conjugates [E1c]) and P (pregnanediol- 3 diglucoronide [Pdg]) were measured using previously validated methodology (14–19) in infertile women diagnosed with DOR and in age-comparable healthy controls.
Fertility and Sterility Vol. 93, No. 4, March 1, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2008.10.069
FIGURE 1 Daily profile of urinary FSH levels in cases with diminished ovarian reserve (DOR) and controls presented across an entire menstrual cycle. Compared with the controls, the cases demonstrate significantly higher FSH levels in the early follicular phase (P¼ .03), and a significantly protracted increase in FSH concomitant with the LH surge (P¼ .01). Note: centering of cycles relative to the LH surge (day 0). Data are presented as mean SEM.
Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010.
MATERIALS AND METHODS Participants collected daily urine void (first specimen for the day) during the course of an entire menstrual cycle. The study was approved by the institutional review board (IRB) at Albert Einstein College of Medicine and Montefiore Medical Center and written consent was obtained. DOR Cases Infertile, yet otherwise healthy women attending the clinical practice in whom DOR was determined as the primary contributor to infertility were identified during a 24-month period. Patients were deemed to demonstrate DOR if early follicular phase serum FSH levels assessed within the first 3 days of onset of menses were more than 10 mIU/mL, as per clinical practice guidelines. In addition these participants must have demonstrated suboptimal ovarian response to previous attempts at ovarian hyperstimulation (i.e., utilization of gonadotropins in excess of 300 U/day with attainment of <3 dominant follicles). Additional inclusion criteria were regular menstrual cycles (21–35 days) normoprolactinemia, normal thyroid function, and absence of chronic diseases (e.g., lupus, rheumatoid arthritis, diabetes, hypertension, psychological or seizure disorders). Eight healthy, albeit infertile, women diagnosed with DOR based on the specified criteria were enrolled between April 2004 and April 2006. Controls Daily urinary samples available on 14 healthy, regularly cycling normoprolactinemic and euthyroid women, aged 18–38 Fertility and Sterility
years, represented the control group. Data on urinary reproductive hormones in the controls has been previously reported (16, 19). Quality control steps were undertaken to assure against assay drift on the available stored control samples (19). Specimen Collection Details on specimen collection procedures have been published previously (16). Briefly, the participants were asked to collect the first morning voided urine specimen in supplied containers and transfer a portion of the volume to the provided polypropylene tubes up to the specified mark. The tubes were prefilled with glycerol (final concentration of 7% glycerol was achieved in the collected urine sample) (20). Participants were instructed to store the tubes in a provided box within the home freezer. Specimens were transported to the laboratory at the end of the collection period and stored at -20 C. Subjects were provided with written instructions regarding the procedure of collection and storage of urine samples during an entire menstrual cycle (first day of menses to the first day of next menstrual flow). Hormone Assays Urinary LH and FSH were measured using a solid-phase, two-site specific fluoroimmunometric assay (DELFIA; PerkinElmer, Turku, Finland) and were validated in our laboratory using previously described methods (14–19). For urinary LH, interassay coefficient of variation (CV) and intra-assay CVs were 13.7% and 5.0%, respectively, and 16.4% and 7.6% for FSH, respectively. Estrone conjugates and Pdg were measured in duplicate by ELISA using antibodies and conjugate tracers. Interassay CV for E1c was 10.1% and intra-assay CV was 8.4%. Corresponding CVs for Pdg were 15.0% and 14.0%, respectively. Urinary hormone concentrations were adjusted for glycerol and normalized to urinary creatinine (21). Data Analysis Data presented reflects the urinary hormone profile during a single menstrual cycle in 8 patients with DOR and 14 healthy controls. Cycle length was normalized to 28 days, after determination of individual mean hormone levels for the entire cycle. Cycle days were aligned to the day of LH surge (set at day 0), as specified by predefined criteria (16, 17). The duration of the LH surge was defined as days of persistent elevation in LH levels above a threshold. Using a 5-day moving average, a 3 SD increase in LH levels was considered consistent with an LH surge. Presence of luteal activity was determined by a sustained increase in Pdg concentrations R3 mg/ mg creatinine for 3 consecutive days, as previously described (17). The duration of the follicular phase was determined to extend from cycle day 1 (first day of menses) to day -1 (with reference to the LH surge). Early follicular phase was specified as before and excluding day -6 (reference to day of LH surge set at 0). The luteal phase was determined to span from day þ2 (reference to LH surge) until and excluding the first day of next menses. 1075
TABLE 1 Cumulative hormone profiles (normalized to a 28-day cycle) in 8 infertile women with DOR and 14 controls. Urinary hormone levelsa FSH (mU/mg Cr) Early follicular Magnitude of surge Duration of surge LH (mU/mg Cr) Magnitude of surge Duration of surge E1Cb(ng/mg Cr) Presurge Lutealc Pdg(mg/mg Cr) Lutealc Luteal adequacyb,d
DOR (N [ 8)
Controls (N [ 14)
P value
7.66 0.63 28.93 10.43 2.30 0.56 63.90 45.5 64.33 18.55 3.29 0.39
6.27 0.32 17.00 3.47 1.14 0.10 34.10 25.90 40.85 7.87 1.93 0.24
.03* .20 .01* .31 .18 < .01*
64.26 7.44 28.39 2.10
83.26 13.71 38 1.70
.36 < .01*
4.41 0.42 6.86 1.39
7.58 0.66 10.21 1.11
< .01* .09
a
Mean SEM. Estrone conjugates. c One patient with diminished ovarian reserve (DOR) who failed to provide luteal phase urine samples was excluded from these analyses. d Pregnanediol 3 diglucoronide levels >3 mg/mg Cr. * Statistically significant. b
Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010.
The integrated urinary excretion of the individual urinary hormones was evaluated for participants with DOR and the healthy controls. Mean hormone levels for early follicular phase, the duration of follicular and luteal phases, and for peak LH and FSH surges in the two groups were compared by two-tailed Student’s t test. The levels of E1c on day -1 (i.e., the day before LH surge) and the duration of LH and FSH surges (in days) were similarly assessed in the two groups. STATA Intercooled 8.2 (StataCorp, College Station, TX) was used for statistical analyses. P value < .05 was considered statistically significant.
RESULTS Data presented reflects the urinary hormone profile during a single menstrual cycle in 8 patients with DOR and 14 healthy controls. The ages (34.12 1.36 vs. 32.55 6.15) and body mass index (BMI) (21.74 2.62 vs. 25.33 2.50) of the patients and controls, respectively, were comparable (mean SD). One of the eight patients with DOR failed to provide daily urine samples beyond the period of LH surge þ2 days. This patient was excluded from analyses evaluating the duration of LH and FSH surge and luteal Pdg levels. Evidence of an LH surge (as previously defined) was noted in all participants (cases and controls). Luteal activity (as previously defined) was observed in all 14 controls and in 7/8 patients with DOR. One patient with DOR did not exhibit any increase in Pdg excretion despite documentation of an LH surge. 1076
Pal et al.
Reproductive hormone milieu in DOR
Not surprisingly, those diagnosed with DOR demonstrated higher urinary levels of FSH through the phases of the menstrual cycle compared with the controls (Fig. 1). These differences were of statistical significance for the early follicular phase (P¼.03). Although the pre-LH surge E1c levels were lower in women with DOR compared with controls (Table 1), the difference was not of statistical significance (P¼.36). Patients with DOR demonstrated a robust gonadotropin surge (LH and FSH) as reflected by urinary levels of the respective pituitary gonadotropins. Although the amplitudes of urinary LH and FSH surges were higher in women with DOR compared with the controls (Figs. 1 and 2), these differences did not reach statistical significance (Table 1). The durations of the LH surge and the concomitant FSH surge, however, were significantly protracted in women with DOR (Table 1) compared with healthy controls. Luteal phase urinary E1c and Pdg excretions were noted to be significantly lower in patients with DOR compared with the controls (Figs. 3 and 4 and Table 1). The temporal span of ‘‘adequate’’ luteal activity as previously defined (i.e., duration of luteal phase demonstrating Pdg levels R3 mg/mL) was shorter in women with DOR compared with the controls. This association approached statistical significance (P¼.09). DISCUSSION Suboptimal ovarian responses to attempts at controlled ovarian hyperstimulation (COH) are well described in infertile Vol. 93, No. 4, March 1, 2010
FIGURE 2
FIGURE 3
Evidence of a robust and protracted LH surge is seen in patients with diminished ovarian reserve (DOR) compared with controls, as assessed by daily profile of urinary levels across an entire menstrual cycle. The differences between the two groups are of statistical significance for the duration of the LH surge (P< .01). Note: centering of cycles relative to the LH surge (day 0). Data are presented as mean SEM.
The daily profile of urinary estrone conjugate (E1c) levels across one menstrual cycle is suggestive of relative hypoestrogenism in premenopausal women with diminished ovarian reserve (DOR) compared with the controls. The differences are of statistical significance for the luteal phase of the cycle (P< .01). Note: centering of cycles relative to the LH surge (day 0). Data are presented as mean SEM.
Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010. Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010.
women diagnosed with DOR (4). Our findings of significantly lower urinary levels of estrogen (E) and P metabolites in a natural menstrual cycle in young infertile women with DOR are suggestive of ovarian GC dysfunction in these otherwise healthy and regularly cycling women. Although impairments in GC viability and proliferative indices are described in association with DOR (11–13, 22), in the present study we provide evidence suggestive of altered reproductive hormone profile in women with DOR. Of interest are findings reported by Leach et al. (23) using a similar paradigm of daily urinary hormone assessment, albeit in a different population of infertile women. Comparing urinary reproductive hormone levels in 12 women with unexplained infertility and normal baseline hormone profiles with 12 healthy controls, the investigators report significant elevations in urinary gonadotropins in the follicular phase and lower P levels in the midluteal phase of menstrual cycle in women with unexplained infertility. Therefore we identify certain similarities in the hormonal milieu of women with unexplained infertility and those with reduced ovarian reserve. Ovarian reserve compromise is suggested in cases of unexplained infertility (24) and consistency in our observations with those described by Leach et al. (23) underscores these concerns. Of interest are absence of differences in preovulatory or periovulatory hormone profiles in the two groups studied by the latter investigators. Our data, in contrast, demonstrate lower preovulatory E1c and higher magnitude and duration of gonadotropin surge in women with reduced ovarian reserve compared with healthy controls. Our observations add to the existing literature of distinctions and yet similarities between cases of unexplained infertility and reduced ovarian reserve. These Fertility and Sterility
observations identify a distinct hormonal milieu in the setting of DOR and merit substantiation by future studies. Impaired luteal function is a recognized accompaniment to chronological aging (16). Our findings of significantly impaired urinary Pdg excretion in women with DOR suggest similarities between DOR diagnosed in the mid-30s and features of reproductive aging that are recognized in the perimenopausal years (i.e., a decade later). In the context of infertility, we acknowledge that although our findings are suggestive of suboptimal luteal reproductive hormone profile (significantly lower urinary excretion of Pdg and E1c, and shorter duration of adequate Pdg excretion, as specified in the text), these observations per se are not confirmatory of luteal insufficiency. Of interest are observations reported by Soules et al. (25), who in a study of daily serum hormones in patients with endometrial biopsy proven luteal phase deficiency identified significantly lower luteal levels of E2 and P. Our data corroborate these observations. Although our observations allow us to propose that luteal dysfunction may contribute to the subfertility of DOR, this assumption needs to be substantiated by appropriately designed studies that incorporate endometrial sampling for the diagnosis of luteal phase deficiency (26). If indeed this assumption is confirmed, luteal support with exogenous P may hold potential for benefit for infertile women with DOR. A decrease in ovarian estrogen is suggested to underlie health morbidities of early menopause (i.e. risks for cardiovascular disease and osteoporosis) (27, 28). An atherogenic lipid profile has been previously described in infertile women demonstrating evidence of reduced ovarian reserve (9) as is enhanced bone turnover and evidence of low bone mass in 1077
FIGURE 4 Premenopausal women with diminished ovarian reserve (DOR) demonstrate impaired luteal excretion of P metabolite Pdg (P< .01) and shorter duration of luteal adequacy (i.e., days of Pdg R3 mg/mg creatinine [Cr]) (P¼ .09) compared with healthy controls. Note: centering of cycles relative to the LH surge (day 0). Data are presented as mean SEM.
Pal. Reproductive hormone milieu in DOR. Fertil Steril 2010.
healthy infertile women with DOR (8). The relatively hypoestrogenic milieu suggested by the presented data may partly explain these observations of skeletal attrition and a proatherogenic milieu previously described in premenopausal women with reduced ovarian reserve. Our findings thus identify DOR as a state of relative estrogen deficiency. Our observations suggest an overlap in hormonal profiles of DOR and chronologically advanced reproductive aging (i.e., elevated FSH levels and impaired luteal excretion of Pdg) (16) is suggested. These findings corroborate the hypothesis that processes of reproductive aging may indeed be accelerated in women with DOR. Of note, however, our observations also provide glimpses of a distinct reproductive hormone milieu in the context of DOR. Significantly lower levels of E1c through the menstrual cycle, as well as robust LH and FSH surges in women with DOR, are aspects that may be unique to the DOR phenotype. In contrast to the hypoestrogenic milieu of DOR (as suggested by our data), relative hyperestrogenemia has been described in association with more advanced stages of reproductive aging that predate menopause (i.e in perimenopause) (16, 29). Furthermore, although subtle elevations in the amplitude of LH surge have previously been reported in the perimenopausal years (16), the protracted gonadotropin surges observed in young women with DOR appear unique to this population. Interestingly, a decline in the putative ovarian gonadotropin surge attenuating factor is suggested concomitant with ovarian reserve, as well as with aging (30). Although a presumable in the ovarian gonadotropin surge attenuating factor in women with DOR may underlie the noted robust and protracted LH surge, this latter conjecture remains unsubstantiated. An alternative explanation for the distinct gonadotropic profiles noted in patients with 1078
Pal et al.
Reproductive hormone milieu in DOR
DOR may be a possible central hypothalamoituitary dysregulation. These observations are of interest and merit further evaluation including attempts to identify the thus far elusive ovarian gonadotropin surge attenuating factor. The small sample size is an inherent limitation of our study, and this is reflected in the relatively large standard errors of some of the measurements (e.g., LH) and, hence identify instability of some of the estimates of group means. Although utilization of historic sample controls is not optimal, stability of urinary levels of reproductive hormones in appropriately collected and stored urine samples is well substantiated (19, 20). Quality control steps ensured integrity of the respective assays and absence of any assay drift in the historic control samples (data not shown). We are thus confident of the noted distinctions in the urinary hormone profiles between patients with DOR and the healthy controls. In summary, our data are supportive of an altered reproductive hormonal milieu in young and infertile women with DOR. In a natural menstrual cycle, the urinary hormone profiles identify DOR as a state of relative hypoestrogenism and of luteal insufficiency. Although our data suggest similarities in the reproductive hormone milieu of premenopausal infertile women with DOR with parameters described in an older population of perimenopausal women, certain aspects of the hormonal profile appear unique to the entity of DOR. Our data encourage us to propose that the known subfecundity in women with DOR may, at least in part, be explained by an altered reproductive hormonal profile. Although the hormone profiles in women with DOR are suggestive of ovarian GC dysfunction, the possibility of central functional alterations (i.e., at the level of the hypothalamic GnRH pulse generator or the pituitary gonadotrophs) cannot be excluded. Our findings (i.e., hypoestrogenism) provide insights into mechanisms that may underlie the previously identified osseo and cardio deleterious implications of DOR. The reported observations are thus of clinical relevance and merit prospective substantiation in a larger cohort. Acknowledgments: We thank Dr. Bill Lasley, University of California-Davis, Davis, California, for the provision of critical reagents for the conduct of urinary E1c and Pdg assays, and to Goli Adel for her invaluable contributions in the conduct of the urinary immunoassays.
REFERENCES 1. Gosden RG, Faddy MJ. Ovarian aging, follicular depletion, and steroidogenesis. Exp Gerontol 1994;29:265–74. 2. Faddy MJ, Gosden RG. A model confirming the decline in follicle numbers to the age of menopause in women. Hum Reprod 1996;11:1484–6. 3. Toner JP. Ovarian reserve, female age and the chance for successful pregnancy. Minerva Ginecol 2003;55:399–406. 4. Karande VC. Managing and predicting low response to standard in vitro fertilization therapy: a review of the options. Treat Endocrinol 2003;2:257–72. 5. de Boer EJ, den Tonkelaar L, te Velde ER, Burger CW, van Leeuwen FE, OMEGA-project group. Increased risk of early menopausal transition and natural menopause after poor response at first IVF treatment. Hum Reprod 2003;18:1544–52.
Vol. 93, No. 4, March 1, 2010
6. Nikolaou D, Templeton A. Early ovarian ageing: a hypothesis. Detection and clinical relevance. Hum Reprod 2003;18:1137–9. 7. Lawson R, El-Toukhy T, Kassab A, Taylor A, Braude P, Parsons J, et al. Poor response to ovulation induction is a stronger predictor of early menopause than elevated basal FSH: a life table analysis. Hum Reprod 2003;18:527–33. 8. Pal L, Bevilacqua K, Zeitlian G, Shu J, Santoro N. Implications of diminished ovarian reserve (DOR) extend well beyond reproductive concerns. Menopause 2008;15:1086–94. 9. Chu MC, Rath KM, Huie J, Taylor HS. Elevated basal FSH in normal cycling women is associated with unfavourable lipid levels and increased cardiovascular risk. Hum Reprod 2003;18:1570–3. 10. Friese C, Becker G, Nachtigall RD. Rethinking the biological clock: eleventh-hour moms, miracle moms and meanings of age-related infertility. Soc Sci Med 2006;63:1550–60. 11. Seifer DB, Gardiner AC, Ferreira KA, Peluso JJ. Apoptosis as a function of ovarian reserve in women undergoing in vitro fertilization. Fertil Steril 1996;66:593–8. 12. Seifer DB, Charland C, Berlinsky D, Penzias AS, Haning RV Jr, Naftolin F, et al. Proliferative index of human luteinized granulosa cells varies as a function of ovarian reserve. Am J Obstet Gynecol 1993;169: 1531–5. 13. Greenseid K, Jindal S, Hurwitz J, Zapantis, A, Nihsen M, Santoro N, et al. Declining ovarian reserve adversely influences granulosa cell viability. Fertil Steril. Published online June 17, 2008. 14. Saketos M, Sharma N, Adel T, Raghuwanshi M, Santoro N. Time-resolved immunofluorometric assay and specimen storage conditions for measuring urinary gonadotropins. Clin Chem 1994;40:749–53. 15. Saketos M, Sharma N, Santoro NF. Suppression of the hypothalamic-pituitary-ovarian axis in normal women by glucocorticoids. Biol Reprod 1993;49:1270–6. 16. Santoro N, Brown JR, Adel T, Skurnick JH. Characterization of reproductive hormonal dynamics in the perimenopause. J Clin Endocrinol Metab 1996;81:1495–501. 17. Santoro N, Crawford SL, Allsworth JE, Gold EB, Greendale GA, Korenman S, et al. Assessing menstrual cycles with urinary hormone assays. Am J Physiol Endocrinol Metab 2003;284:E521–30. 18. Brown JR, Skurnick JH, Sharma N, Adel T, Santoro N. Frequent intermittent ovarian function in women with premature menopause: a longitudinal study, 1993. Endocrinol J 1993;1:467–74.
Fertility and Sterility
19. Zhang K, Pollack S, Ghods A, Dicken C, Isaac B, Adel G, et al. Onset of ovulation after menarche in girls: a longitudinal study. J Clin Endocrinol Metab 2008;93:1186–94. 20. Livesey JH, Roud HK, Metcalf MG, Donald RA. Glycerol prevents loss of immunoreactive follicle-stimulating hormone and luteinizing hormone from frozen urine. J Endocrinol 1983;98:381–4. 21. Taussky HH. A procedure increasing the specificity of the Jaffe reaction for the determination of creatine and creatinine in urine and plasma. Clin Chim Acta 1956;1:210–24. 22. Seifer DB, Honig J, Penzias AS, Lavy G, Nadkarni PM, Jones EE, et al. Flow cytometric analysis of deoxyribonucleic acid in human granulosa cells as a function of chronological age and ovulation induction regimen. J Clin Endocrinol Metab 1992;75:636–40. 23. Leach RE, Moghissi KS, Randolph JF, Reame NE, Blacker CM, Ginsburg KA, et al. Intensive hormone monitoring in women with unexplained infertility: evidence for subtle abnormalities suggestive of diminished ovarian reserve. Fertil Steril 1997;68:413–20. 24. Randolph JF, Ginsburg KA, Leach RE, Blacker CM, Moghissi KS, Diamond MP, et al. Elevated early follicular gonadotropin levels in women with unexplained infertility do not provide evidence for disordered gonadotropin-releasing hormone secretion as assessed by luteinizing hormone pulse characteristics. Fertil Steril 2003;80:320–7. 25. Soules MR, McLachlan RI, Ek M, Dahl KD, Cohen NL, Bremner WJ. Luteal phase deficiency: characterization of reproductive hormones over the menstrual cycle. J Clin Endocrinol Metab 1989;69:804–12. 26. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol 1975;122:262–3. 27. Matthews KA, Wing RR, Kuller LH, Meilahn EN, Plantinga P. Influence of the perimenopause on cardiovascular risk factors and symptoms of middle-aged healthy women. Arch Intern Med 1994;154:2349–55. 28. Slemenda C, Hui SL, Longcope C, Johnston CC. Sex steroids and bone mass. A study of changes about the time of menopause. J Clin Invest 1987;80:1261–9. 29. Prior JC. Ovarian aging and the perimenopausal transition: the paradox of endogenous ovarian hyperstimulation. Endocrine 2005;26: 297–300. 30. Martinez F, Barri PN, Coroleu B, Tur R, Sorsa-Leslie T, Harris WJ, et al. Women with poor response to IVF have lowered circulating gonadotrophin surge-attenuating factor (GnSAF) bioactivity during spontaneous and stimulated cycles. Hum Reprod 2002;17:634–40.
1079