Compensatory Increase in Ovarian Aromatase in Older Regularly Cycling Women.

CONTEXT: Serum estradiol (E2) levels are preserved in older reproductive-aged women with regular menstrual cycles despite declining ovarian function. OBJECTIVE: The objective of the study was to determine whether increased granulosa cell aromatase expression and activity account for preservation of E2 levels in older, regularly cycling women. DESIGN: The protocol included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. SUBJECTS: Healthy, regularly cycling older (36-45 y; n = 13) and younger (22-34 y; n = 14) women participated in the study. MAIN OUTCOME MEASURES: Hormone levels were measured in peripheral blood and follicular fluid aspirates and granulosa cell CYP19A1 (aromatase) and FSH-R mRNA expression were determined. RESULTS: Older women had higher FSH levels than younger women during the early follicular phase with similar E2 but lower inhibin B and antimullerian hormone levels. Late follicular phase serum E2 did not differ between the two groups. Follicular fluid E2 [older (O) = 960.0 [interquartile range (IQR) 765.0-1419.0]; younger (Y) = 994.5 [647.3-1426.5] ng/mL, P = 1.0], estrone (O = 39.6 [29.5-54.1]; Y = 28.8 [22.5-42.1] ng/mL, P = 0.3), and the E2 to testosterone (T) ratio (O = 109.0 ± 41.9; Y = 83.0 ± 18.6, P = .50) were preserved in older women. Granulosa cell CYP19A1 expression was increased 3-fold in older compared with younger women (P < .001), with no difference in FSH-R expression. CONCLUSIONS: Ovarian aromatase expression increases with age in regularly cycling women. Thus, up-regulation of aromatase activity appears to compensate for the known age-related decrease in granulosa cell number in the dominant follicle to maintain ovarian estrogen production in older premenopausal women.

Accumulated deep sleep is a powerful predictor of LH pulse onset in pubertal children.

CONTEXT: During puberty, reactivation of the reproductive axis occurs during sleep, with LH pulses specifically tied to deep sleep. This association suggests that deep sleep may stimulate LH secretion, but there have been no interventional studies to determine the characteristics of deep sleep required for LH pulse initiation. OBJECTIVE: The objective of this study was to determine the effect of deep sleep fragmentation on LH secretion in pubertal children. DESIGN AND SETTING: Studies were performed in a clinical research center. SUBJECTS: Fourteen healthy pubertal children (11.3-14.1 y) participated in the study. INTERVENTIONS: Subjects were randomized to two overnight studies with polysomnography and frequent blood sampling, with or without deep sleep disruption via auditory stimuli. RESULTS: An average of 68.1 ±10.7 (± SE) auditory stimuli were delivered to interrupt deep sleep during the disruption night, limiting deep sleep to only brief episodes (average length disrupted 1.3 ± 0.2 min vs normal 7.1 ± 0.8 min, P < .001), and increasing the number of transitions between non-rapid eye movement (NREM), REM, and wake (disrupted 274.5 ± 33.4 vs normal 131.2 ± 8.1, P = .001). There were no differences in mean LH (normal: 3.2 ± 0.4 vs disrupted: 3.2 ± 0.5 IU/L), LH pulse frequency (0.6 ± 0.06 vs 0.6 ± 0.07 pulses/h), or LH pulse amplitude (2.8 ± 0.4 vs 2.8 ± 0.4 IU/L) between the two nights. Poisson process modeling demonstrated that the accumulation of deep sleep in the 20 minutes before an LH pulse, whether consolidated or fragmented, was a significant predictor of LH pulse onset (P < .001). CONCLUSION: In pubertal children, nocturnal LH augmentation and pulse patterning are resistant to deep sleep fragmentation. These data suggest that, even when fragmented, deep sleep is strongly related to activation of the GnRH pulse generator.

Evidence that increased ovarian aromatase activity and expression account for higher estradiol levels in African American compared with Caucasian women.

CONTEXT: Serum estradiol levels are significantly higher across the menstrual cycle in African American (AAW) compared with Caucasian women (CW) in the presence of similar FSH levels, yet the mechanism underlying this disparity is unknown. OBJECTIVE: The objective of the study was to determine whether higher estradiol levels in AAW are due to increased granulosa cell aromatase mRNA expression and activity. DESIGN: The design of the study included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. SUBJECTS: Healthy, normal cycling AAW (n = 15) and CW (n = 14) aged 19-34 years participated in the study. MAIN OUTCOME MEASURES: Hormone levels in peripheral blood and follicular fluid (FF) aspirates and aromatase and FSH receptor mRNA expression in granulosa cells were measured. RESULTS: AAW had higher FF estradiol [1713.0 (1144.5-2032.5) vs 994.5 (647.3-1426.5) ng/mL; median (interquartile range); P < .001] and estrone [76.9 (36.6-173.4) vs 28.8 (22.5-42.1) ng/mL; P < .001] levels than CW, independent of follicle size. AAW also had lower FF androstenedione to estrone (7 ± 1.8 vs 15.8 ± 4.1; mean ± SE; P = .04) and T to estradiol (0.01 ± 0.002 vs 0.02 ± 0.005; P = .03) ratios, indicating enhanced ovarian aromatase activity. There was a 5-fold increase in granulosa cell aromatase mRNA expression in AAW compared with CW (P < .001) with no difference in expression of FSH receptor. FSH, inhibin A, inhibin B, and AMH levels were not different in AAW and CW. CONCLUSIONS: Increased ovarian aromatase mRNA expression, higher FF estradiol levels, and decreased FF androgen to estrogen ratios in AAW compared with CW provide compelling evidence that racial differences in ovarian aromatase activity contribute to higher levels of estradiol in AAW across the menstrual cycle. The absence of differences in FSH, FSH receptor expression, and AMH suggest that population-specific genetic variation in CYP19, the gene encoding aromatase, or in factors affecting its expression should be sought.

Insights into puberty: the relationship between sleep stages and pulsatile LH secretion.

CONTEXT: During the pubertal transition, LH secretion initially increases only during sleep; however, its relationship to sleep stage is unknown. OBJECTIVES: Our objective was to determine whether the initiation of LH pulses is related to a specific sleep stage in pubertal children. DESIGN AND SETTING: Frequent blood sampling and polysomnographic studies were performed in a Clinical Research Center. SUBJECTS: Fourteen studies were performed in nine healthy pubertal children, ages 9.9-15.6 yr. INTERVENTIONS: Subjects underwent one to two overnight studies with polysomnography and blood sampling for LH at 10-min intervals. RESULTS: Alignment of polysomnographic records and LH pulses demonstrated that LH pulses (n = 58) occurred most frequently during slow-wave sleep (SWS) (1.1 pulse/h, n = 30) compared with all other sleep stages or periods of wake after sleep onset (P < 0.001). There was also a significant increase in the amount of SWS in the 15 min preceding and the 5 min following each pulse compared with the amount of SWS seen across the study night (P < 0.01). CONCLUSIONS: During puberty, the majority of LH pulses that occur after sleep onset are preceded by SWS, suggesting that SWS is intimately involved in the complex control of pubertal onset. These studies raise concerns about the potential hormonal repercussions of the increasing prevalence of sleep disturbances in adolescents.

Gonadotropin responses to estrogen-positive and -negative feedback are identical in African-American and Caucasian women.

CONTEXT: Gonadotropin levels are similar in African-American women (AAW) and Caucasian women (CW), despite higher preovulatory estradiol (E2) levels in AAW, suggesting that AAW may be less sensitive to E2 feedback than CW. OBJECTIVE: The aim of the study was to determine whether responsivity to estrogen feedback differs in AAW and CW. DESIGN AND SETTING: Subjects were studied in the early follicular phase using a 5-d, graded E2 and progesterone infusion. SUBJECTS: Healthy, normal-cycling AAW (n = 10) and CW (n = 13) aged 23-30 yr participated in the study. MAIN OUTCOME MEASURES: Blood samples were collected every 4 h and assayed for LH, FSH, E2, and progesterone. RESULTS: There was no difference in E2-negative feedback on LH (nadir, 3.8 ± 0.4 vs. 5.4 ± 0.9 IU/liter; time of nadir, 33.2 ± 3.3 vs. 32.3 ± 2.7 h) or FSH (nadir, 3.1 ± 0.4 vs. 3.1 ± 0.3 IU/liter; time of nadir, 48.8 ± 2.7 vs. 50.5 ± 3.1 h) in AAW compared to CW. The two groups also demonstrated similar positive feedback responses of E2 on LH (peak, 80.3 ± 13.3 vs. 73.1 ± 11.6 IU/liter; time of peak, 80.4 ± 4.3 vs. 86.5 ± 3.1 h) and FSH (peak, 13.4 ± 1.4 vs. 10.2 ± 1.0 IU/liter; time of peak, 82.2 ± 4.0 vs. 97.2 ± 4.9 h). CONCLUSIONS: LH and FSH feedback responses to a controlled steroid infusion do not differ between AAW and CW, indicating that AAW do not have diminished hypothalamic-pituitary responsivity to E2. These studies support the concept of a threshold effect of E2 in generating LH-positive feedback, suggest pituitary insensitivity to differences in E2 of the magnitude observed in prior studies, and account for similarities in gonadotropins despite E2 differences in AAW compared with CW.

Estrogen levels are higher across the menstrual cycle in African-American women compared with Caucasian women.

CONTEXT: Previous studies have suggested that estrogen levels may be higher in African-American women (AAW) compared with Caucasian women (CW), but none have systematically examined estrogen secretion across the menstrual cycle or in relation to other reproductive hormones. OBJECTIVE: The objective of the study was to compare estradiol (E2), progesterone (P), gonadotropins, androstenedione (a'dione), inhibins, and SHBG levels between AAW and CW across the menstrual cycle. DESIGN, SETTING, AND SUBJECTS: Daily blood samples were collected from regularly cycling AAW (n = 27) and CW (n = 27) for a full menstrual cycle, and serial ultrasounds were performed. MAIN OUTCOME MEASURES: Comparison of E2, P, LH, FSH, SHBG, inhibin A, inhibin B, and a'dione levels. RESULTS: AAW and CW were of similar age (27.2 ± 0.6 yr, mean ± sem) and body mass index (22.7 ± 0.4 kg/m(2)). All subjects grew a single dominant follicle and had comparable cycle (25-35 d) and follicular phase (11-24 d) lengths. E2 levels were significantly higher in AAW compared with CW (P = 0.02) with the most pronounced differences in the late follicular phase (225.2 ± 14.4 vs. 191.5 ± 10.2 pg/ml; P = 0.02), midluteal phase (211.9 ± 22.2 vs.150.8 ± 9.9, P < 0.001), and late luteal phase (144.4 ± 13.2 vs. 103.5 ± 8.5, P = 0.01). Although LH, FSH, inhibins A and B, P, a'dione, and SHBG were not different between the two groups, the a'dione to E2 ratio was lower in AAW (P < 0.001). CONCLUSIONS: Estradiol is higher in AAW compared with CW across the menstrual cycle. Higher estradiol in the face of similar androstenedione and FSH levels suggests enhanced aromatase activity in AAW. Such differences may contribute to racial disparities in bone mineral density, breast cancer, and uterine leiomyomas.

Differential effects of aging on estrogen negative and positive feedback.

Recent studies have demonstrated an age-related decline in gonadotropins and a decrease in pituitary responsiveness to GnRH, indicating that aging influences the neuroendocrine components of the female reproductive axis independently of changes in ovarian function. To determine whether aging might also affect the luteinizing hormone (LH) negative and positive feedback responses to gonadal steroids, we administered a controlled, graded sex steroid infusion to 11 younger (45-56 yr) and nine older (70-80 yr) postmenopausal women (PMW) in whom endogenous ovarian steroids and peptides are uniformly low. The doses of estradiol (E(2)) and progesterone (P) were chosen to mimic levels across the normal follicular phase and have been shown previously to induce negative followed by positive feedback on LH. Similar E(2) and P levels were achieved in younger and older PMW (P = 0.4 and 0.3, respectively) and produced a biphasic LH response in all subjects. The early decline in LH to 53% of baseline was not different in older vs. younger PMW. However, the positive feedback effect was attenuated in older compared with younger PMW (peak LH 144.4 ± 19.5 vs. 226.8 ± 22.3 IU/l, respectively, P = 0.01). In conclusion, these studies in PMW demonstrate preservation of short-term steroid negative and positive feedback in response to exogenous E(2) and P with aging. Attenuation of positive feedback in older compared with younger PMW is consistent with previous reports of declining GnRH responsiveness with aging.

Estrogen negative feedback on gonadotropin secretion: evidence for a direct pituitary effect in women.

CONTEXT: Studies in humans and animals indicate that estrogen negative feedback occurs at the level of the hypothalamus, but it is unclear whether estrogen also exerts an inhibitory effect directly at the pituitary. OBJECTIVES: The aim of the study was to determine whether estrogen has a direct negative feedback effect at the pituitary and whether this varies with aging. DESIGN AND SETTING: A GnRH antagonist and graded doses of GnRH were used to isolate pituitary responsiveness before and after estrogen administration in Clinical Research Center studies at an academic medical center. SUBJECTS: Subjects were healthy postmenopausal women aged 48-56 yr (n = 8) or 70-75 yr (n= 8). INTERVENTIONS: A suppressive dose of the NAL-GLU GnRH antagonist was administered, followed by graded doses of GnRH before and after 1 month of estrogen administration. RESULTS: LH and FSH responses to GnRH decreased after estrogen administration (P = 0.01 and P = 0.0001, respectively). The ratio of FSH to LH amplitudes decreased in response to estrogen (P = 0.04) indicating a greater sensitivity of FSH than LH to inhibition by estrogen. The inhibitory effect of estrogen on FSH was attenuated with aging (P = 0.02), but was maintained for LH (P = 0.4). CONCLUSIONS: Studies that control for endogenous GnRH and estradiol demonstrate a direct pituitary site of estrogen negative feedback on LH and FSH responsiveness to GnRH in women. The effect of estrogen on FSH responsiveness is greater than on LH and is attenuated with aging. These studies indicate that estrogen negative feedback occurs directly at the pituitary and contributes to the differential regulation of FSH and LH secretion.

Potent inhibition of werner and bloom helicases by DNA minor groove binding drugs.

Maintenance of genomic integrity is vital to all organisms. A number of human genetic disorders, including Werner Syndrome, Bloom Syndrome and Rothmund-Thomson Syndrome, exhibit genomic instability with some phenotypic characteristics of premature aging and cancer predisposition. Presumably the aberrant cellular and clinical phenotypes in these disorders arise from defects in important DNA metabolic pathways such as replication, recombination or repair. These syndromes are all characterized by defects in a member of the RecQ family of DNA helicases. To obtain a better understanding of how these enzymes function in DNA metabolic pathways that directly influence chromosomal integrity, we have examined the effects of non-covalent DNA modifications on the catalytic activities of purified Werner (WRN) and Bloom (BLM) DNA helicases. A panel of DNA-binding ligands displaying unique properties for interacting with double helical DNA was tested for their effects on the unwinding activity of WRN and BLM helicases on a partial duplex DNA substrate. The levels of inhibition by a number of these compounds were distinct from previously reported values for viral, prokaryotic and eukaryotic helicases. The results demonstrate that BLM and WRN proteins exhibit similar sensitivity profiles to these DNA-binding ligands and are most potently inhibited by the structurally related minor groove binders distamycin A and netropsin (K(i)