Abstract
Background
Lifetime ovulatory years (LOY) is estimated by the difference between ages at menopause and menarche subtracting time for events interrupting ovulation. We tested whether LOY influences sex-hormone levels in postmenopausal women with at least one intact ovary not using hormones.
Methods
Estradiol, estrone, estrone sulfate, total testosterone, dehydroepiandrostendione sulfate (DHEAS), prolactin, and sex-hormone-binding globulin (SHBG) were measured in 1,976 postmenopausal women from the Nurses’ Health Study. Associations of age, body mass index (BMI), smoking, alcohol use, and other factors on hormones were assessed by t-tests and ANOVA. Linear regression was used to assess multivariable-adjusted associations between LOY and hormones and trends in hormone levels per 5-year increases in LOY were estimated.
Results
Women averaged 61.4 years old, 11.0 years since menopause, with BMI of 25.8 kg/m2. 13.6% had irregular cycles, 17.5% hysterectomy, 6.4% unilateral oophorectomy, and 13.8% were current smokers. Variables associated with one or more hormone levels were included as covariates. Each 5-year increase in LOY was significantly associated with a 5.2% increase in testosterone in women with BMI<25 kg/m2 and a 7.4% increase in testosterone and 7.3% increase in estradiol in women with above-average BMI.
Conclusions
This is the first study to show that greater LOY is associated with higher testosterone in postmenopausal women and higher estradiol in those with elevated BMI, suggesting accumulation of functioning stromal and thecal cells from repeated ovulations and peripheral conversion of testosterone.
Impact
A possible explanation for why greater LOY increases risk for breast, endometrial, and ovarian cancer is offered.
Keywords: Testosterone, Estradiol, Menopause, Ovulation
Introduction
Why more estimated ovulations accumulated before menopause increases risk for cancers of reproductive organs and to what extent the postmenopausal ovary retains potential for hormone production are two important questions to cancer research and reproductive biology. Lifetime ovulatory years (LOY) is estimated by the difference between ages at menopause and menarche subtracting time for events interrupting ovulation. Greater LOY, described by the phrase “incessant ovulation,” was first hypothesized to explain risk for ovarian cancer in 1971(1) and supported by subsequent studies.(2–7) Repetitive damage to the ovarian surface from ovulation was originally postulated; later, exposure of tubal-fimbria cells to growth-factor-enriched follicular fluid was suggested.(8,9) The hypothesis was extended to breast and endometrial cancers(10), using the term “sterile menstrual cycles” and also supported by subsequent studies.(11–14) Cumulative effects of cyclic hormones on proliferation of breast and endometrial tissue could explain associations between LOY and these cancers.
Concerning hormone production from the postmenopausal ovary, it has been argued that the adrenal glands may be the more important source of androgens after menopause.(15) However, more persuasive, we believe, are studies in which ovarian veins were cannulated in postmenopausal women having pelvic surgery. These showed higher levels of testosterone and androstenedione in the ovarian venous blood than peripheral blood.(16–18) Cross-sectional studies of hormone levels in postmenopausal women are also relevant. Thirteen such studies were pooled and reanalyzed to identify determinants of estrone, estradiol, androstenedione, dehydroepiandrostendione sulfate (DHEAS), testosterone, and sex-hormone-binding globulin (SHBG).(19) Older women had lower hormone levels except for SHBG. Androgens were lower in women who had bilateral oophorectomy compared to women with intact ovaries. All hormones were higher in obese women except for SHBG.
An association between estimated LOY and hormone levels was not examined in either the cross-sectional or cannulation studies cited above, bringing us to a third question addressed in this study. Do postmenopausal ovaries which underwent more estimated ovulations have greater potential for sex hormone production? We address this question with cross-sectional data from the Nurses’ Health Study (NHS).
Methods
Study population
NHS was established in 1976 among 121,700 US female registered nurses, ages 30 to 55 years. Women completed questionnaires at baseline and biennially on exposures and disease diagnoses. Blood samples were collected in 1989–1990 from 32,826 postmenopausal participants not currently using hormonal therapy (HT).(20) Subsequently, plasma hormone levels were measured in nested case-control studies of various diseases.(21–26) Data on hormone levels in women selected as controls in these studies were consolidated to assess the effect of oophorectomy on postmenopausal hormone levels.(27) Updating these data through 2021, we identified a total of 2,364 postmenopausal women with plasma samples who had not used HT in the 5 months prior to the blood draw. Women were excluded who had: estradiol levels above 30 pg/ml, suggesting recent HT use (n=17); bilateral oophorectomy prior to the blood draw (N=285); and missing data necessary to calculate ovulatory years (N=86). After these exclusions (n=388), the final sample included 1,976 women.
Exposures
Variables to estimate LOY included ages at menarche and menopause, oral contraceptive (OC) use, parity, and breastfeeding. Age at natural menopause was the age after which no menstrual cycles occurred during the subsequent 12 months. For women who had a hysterectomy (without removal of both ovaries) before natural menopause, age at menopause was imputed.(28) Parity counted pregnancies lasting longer than 6 months. Years of OC use and data on number of children breastfed and total months of breastfeeding was also collected. LOY was estimated as the difference between age at menopause and age at menarche, subtracting years of OC use, 1 year for each pregnancy, and total years of breastfeeding. The following covariates were considered: age, years since menopause, body mass index (BMI, weight(kg)/height(m)2) at blood draw, smoking status (never, former, current), alcohol use (no regular use, ≤10, >10–20, >20–30, >30 grams/day), hysterectomy, unilateral oophorectomy, and menstrual cycle irregularity between ages 20–35 (very or usually regular and usually or very irregular).
Laboratory Assays
We evaluated estradiol, estrone, estrone sulfate, total testosterone, DHEAS, prolactin, and SHBG. Original descriptions of the assays used are found in three papers from NHS (24,29,30) and briefly summarized here. Estradiol, estrone, and testosterone were measured at Quest Diagnostics (San Juan Capistrano, CA) by radioimmunoassay following extraction and celite column chromatography or at the Mayo Clinic (Rochester, MN) by liquid chromatography-tandem mass spectrometry. Estrone sulfate was assayed at the University of Massachusetts Medical Center’s Longcope Steroid Radioimmunoassay Laboratory (Worcester), Quest Diagnostics, or the Mayo Clinic, after estrone extraction, by enzymatically cleaving the estrone sulfate to release estrone, which was then measured as noted above. DHEAS was measured either at Quest Diagnostics by radioimmunoassay without a prior separation step or at the Mayo Clinic by a solid phase, competitive chemiluminescent enzyme immunoassay (Siemens Healthcare Diagnostics, Deerfield, IL). SHBG was assayed at the Longcope Laboratory or at the Clinical Laboratory Research Core at Massachusetts General Hospital (Boston MA) using a solid-phase two-site chemiluminescent enzyme immunometric assay (Immulite; DPC, Inc). Prolactin was measured by microparticle enzyme immunoassay at either the Clinical Laboratory Research Core at the Massachusetts General Hospital using the ARCHITECTR chemiluminescence immunoassay system (Abbott Diagnostics, Chicago, IL) or at the Longcope Laboratory (Worcester) using the IMx System (Abbott Laboratory, Abbott Park, IL). The correlation between assays of the same hormone measured in two different labs was ≥0.9. To assess laboratory precision, masked replicates of 10% of all samples assayed were randomly interspersed; the average coefficients of variation across batches were <12%.
Statistical Analysis
Hormone levels were log-transformed and outliers were identified using the generalized extreme studentized deviate many-outlier detection approach and recalibrated to have a comparable distribution to an average batch according to the methods described by Rosner and colleagues.(31,32) Women with missing or outlying hormone information were excluded for specific analyses related to that hormone. We calculated geometric means for each hormone by categories of age at blood draw, years since menopause, BMI, smoking, alcohol use, hysterectomy, irregular cycles, age at menarche, age at menopause, OC use and duration, parity, breastfeeding, and LOY. Distributions were compared with t-tests and ANOVA.
We then used linear regression to examine relationships between LOY and hormone levels, adjusted for age at blood draw, years since menopause, BMI, smoking, alcohol use, hysterectomy, unilateral oophorectomy, and regularity of cycles. Variables which are components of LOY were not included as adjustment variables. We examined regression coefficients from the multivariable models to assess associations between LOY and hormone levels and calculated adjusted geometric means for each log-transformed hormone by LOY quartile (<29.8, 29.8–33.0, 33.1–35.9, ≥36.0 years). We tested for linear trends using LOY as a continuous variable and estimated the percent difference in biomarker levels for a 5-year increase in LOY. Results for all women were adjusted for BMI and presented separately by BMI category (<25, ≥25 kg/m2). Tests for significant interaction between continuous LOY and dichotomous BMI were assessed by the Wald test. The linear regression analysis was repeated excluding women with imputed ages at menopause.
Ethics Statement
The formation of the Nurses’ Health Study, subsequent data and blood collection protocols, and hormonal studies were approved by the Committee on the Use of Human Subjects in Research at the Brigham and Women’s Hospital (Boston, MA) under guidelines of the U.S. Common Rule
Data Availability
The data underlying this article are available by application. Further information including the procedures to obtain and access data from the Nurses’ Health Studies is described at https://www.nurseshealthstudy.org/researchers
Results
Table 1 shows participants’ characteristics. On average, women were 61.4 years old at blood draw; 12.7 years old at menarche; 50.4 years old at menopause; and were 11.0 years past menopause. The following percentages of women reported: irregular cycles (13.6%), hysterectomy before menopause (17.5%), and unilateral oophorectomy (6.4%). 95.8% of the women were parous and their mean parity was 3.5 births. 64.5% of women had breastfed and their mean total years of breastfeeding was 0.7. 32.2% had used OCs and mean years used was 4.4. Mean estimated LOY was 32.4 years. The mean BMI for the sample was 25.8 kg/m2; and 13.8% and 41.5% were current or former smokers, respectively. Not all hormones were measured in the various studies leading to differences in numbers for the individual hormones measured. The median values were: 5.0 pg/mL for estradiol; 22.9 pg/mL for estrone; 230.4 pg/ml for estrone sulfate; 19.8 ng/dL for testosterone; 61.2 ug/dL for DHEAS; 8.8 ng/mL for prolactin; and 58.5 nmol/L for SHBG.
Table 1.
Characteristics of postmenopausal women not using hormone therapy whose plasma hormone levels were measured in the Nurses’ Health Study (n=1976).
| Characteristic | All participants (n=1976) |
|---|---|
| Age at blood draw, y (Mean, SD) | 61.4 (4.7) |
| Age at menarche, y (Mean, SD) | 12.7 (1.4) |
| Irregular cycles (n, %) | 252 (13.6%) |
| Parous (n, %) | 1892 (95.8%) |
| Parity (Mean, SD)* | 3.5 (1.7) |
| Ever breastfed (n, %) | 1275 (64.5%) |
| Duration of breastfeeding, y (Mean, SD)† | 0.7 (0.8) |
| Ever OC use (n, %) | 633 (32.2%) |
| Duration of OC use, y (Mean, SD)‡ | 4.4 (4.4) |
| Age at menopause, y (Mean, SD) | 50.4 (3.2) |
| Years since menopause, y (Mean, SD) | 11.0 (5.2) |
| Hysterectomy (n, %) | 346 (17.5%) |
| Unilateral oophorectomy (n, %) | 127 (6.4%) |
| Ovulatory years (Mean, SD) | 32.4 (5.2) |
| BMI at blood draw, kg/m2 (Mean, SD) | 25.8 (4.7) |
| Smoking status (n, %) | |
| Never | 885 (44.8%) |
| Former | 819 (41.5%) |
| Current | 272 (13.8%) |
| Alcohol (grams/day) | |
| No regular use | 787 (41.7%) |
| ≤20 | 964 (51.1%) |
| >20 | 135 (7.2%) |
| Plasma hormone levels (median, 10th - 90th percentile, n) | |
| Estradiol (pg/mL) | 5.0 (2.7–10.6), 1624 |
| Estrone (pg/mL) | 22.9 (12.8–41.8), 1536 |
| Estrone sulfate (pg/mL) | 230.4 (98.3–544.9), 1182 |
| Testosterone (ng/dL) | 19.8 (10.4–37.5), 1869 |
| DHEAS (ug/dL) | 61.2 (22.9–135.1), 1374 |
| Prolactin (ng/mL) | 8.8 (5.3–15.6), 1192 |
| SHBG (nmol/L) | 58.5 (28.1–103.1), 1779 |
Among parous women
Among women who ever breastfed
Among women who ever used OCs
Table 2 shows the distribution of participant characteristics across quartiles of LOY with the first row showing the corresponding means for those quartiles. Variables which are components of LOY affected LOY in predictable directions. More LOY occurred with: earlier menarche, lower parity, less breastfeeding, less OC use, and later menopause. Among variables which are not components of LOY, there were trends for women with more LOY to: be older, have had fewer years since menopause at blood draw, have a greater BMI, be non-smokers, and not regularly consume alcohol. The prevalence of hysterectomy was lowest among women with fewest LOY. No obvious trends in LOY were seen with irregular cycles and unilateral oophorectomy.
Table 2.
Characteristics of postmenopausal women not using hormone therapy at the time of blood draw in the Nurses’ Health Study by lifetime ovulatory years quartile (n=1976).
| Characteristic | Ovulatory years | |||
|---|---|---|---|---|
|
| ||||
| <29.8 (n=492) | 29.8–33.0 (n=507) | 33.1–35.9 (n=471) | ≥36 (n=506) | |
| Ovulatory years (Mean, SD) | 25.3 (3.7) | 31.6 (1.0) | 34.5 (0.7) | 38.2 (1.7) |
| Components of ovulatory years | ||||
| Age at menarche, y (Mean, SD) | 13.1 (1.5) | 12.9 (1.4) | 12.7 (1.2) | 11.9 (1.2) |
| Parous (n, %) | 482 (98.0%) | 499 (98.4%) | 459 (97.5%) | 452 (89.3%) |
| Parity (Mean, SD)* | 4.4 (2.2) | 3.9 (1.6) | 3.3 (1.2) | 2.6 (1.1) |
| Ever breastfed (n, %) | 362 (73.6%) | 326 (64.3%) | 333 (70.7%) | 254 (50.2%) |
| Duration of breastfeeding, y (Mean, SD)† | 1.0 (1.0) | 0.7 (0.8) | 0.6 (0.6) | 0.5 (0.5) |
| Ever OC use (n, %) | 319 (65.0%) | 150 (29.8%) | 102 (21.7%) | 62 (12.3%) |
| Duration of OC use, y (Mean, SD)‡ | 7.0 (4.6) | 2.5 (2.7) | 1.5 (1.9) | 0.7 (0.8) |
| Age at menopause, y (Mean, SD) | 48.0 (4.0) | 49.6 (2.3) | 51.1 (1.9) | 52.8 (2.0) |
| Other variables | ||||
| Age at blood draw, y (Mean, SD) | 60.1 (5.0) | 61.1 (4.8) | 61.8 (4.6) | 62.5 (4.2) |
| Years since menopause, y (Mean, SD) | 12.0 (6.0) | 11.5 (5.1) | 10.7 (4.6) | 9.8 (4.5) |
| Hysterectomy (n, %) | 59 (12.0%) | 94 (18.5%) | 107 (22.7%) | 86 (17.0%) |
| Unilateral oophorectomy (n, %) | 33 (6.7%) | 30 (5.9%) | 34 (7.2%) | 30 (5.9%) |
| Irregular cycles (n, %) | 79 (17.3%) | 59 (12.3%) | 52 (11.7%) | 62 (13.0%) |
| BMI at blood draw, kg/m2 (Mean, SD) | 25.4 (4.9) | 25.5 (4.4) | 25.8 (4.7) | 26.5 (4.8) |
| Smoking status (n, %) | ||||
| Never | 216 (43.9%) | 218 (43.0%) | 207 (44.0%) | 244 (48.2%) |
| Former | 195 (39.6%) | 213 (42.0%) | 201 (42.7%) | 210 (41.5%) |
| Current | 81 (16.5%) | 76 (15.0%) | 63 (13.4%) | 52 (10.3%) |
| Alcohol (grams/day) | ||||
| No regular use | 181 (38.4%) | 200 (41.9%) | 180 (39.6%) | 226 (46.8%) |
| ≤20 | 257 (54.6%) | 243 (50.9%) | 233 (51.2%) | 231 (47.8%) |
| >20 | 33 (7.0%) | 34 (7.1%) | 42 (9.2%) | 26 (5.4%) |
Among parous women
Among women who ever breastfed
Among women who ever used OCs
Table 3 shows unadjusted geometric mean hormone levels by participant characteristics. Older age at blood draw and greater years since menopause were associated with higher testosterone, lower DHEAS, and higher SHBG. Greater BMI was associated with higher estrogen levels and lower SHBG. Current smoking was associated with lower estrone sulfate and prolactin and higher testosterone, DHEAS, and SHBG. Alcohol use was associated with lower estradiol and estrone and higher estrone sulfate, DHEAS, and SHBG. Hysterectomy was associated with lower estrone sulfate, testosterone, and DHEAS. Women who had a unilateral oophorectomy had lower testosterone and women who with irregular cycles had lower SHBG. Greater LOY was associated with higher estradiol, estrone, testosterone, and lower SHBG.
Table 3.
Unadjusted geometric mean hormone levels by potential covariates in postmenopausal women not using hormone therapy at the time of blood draw in the Nurses’ Health Study (n=1976)
| Estradiol (pg/mL) | Estrone (pg/mL) | Estrone sulfate (pg/mL) | Testosterone (ng/dL) | DHEAS (ug/dL) | Prolactin (ng/mL) | SHBG (nmol/L) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | |
| Age at blood draw | ||||||||||||||
| <55 | 649 | 5.3 (5.1–5.6) | 612 | 22.7 (21.8–23.5) | 519 | 239.3 (225.2–254.3) | 781 | 19.1 (18.5–19.8) | 557 | 68.6 (64.6–72.8) | 506 | 9.1 (8.8–9.5) | 739 | 54.5 (52.5–56.6) |
| ≥55 | 975 | 5.1 (5.0–5.3) | 924 | 22.9 (22.2–23.6) | 663 | 226.4 (214.9–238.4) | 1088 | 20.3 (19.7–21.0) | 817 | 51.2 (48.7–53.8) | 686 | 8.8 (8.5–9.1) | 1040 | 57.2 (55.5–58.9) |
| t-test p-value | 0.20 | 0.65 | 0.17 | 0.009 | <0.0001 | 0.20 | 0.05 | |||||||
| Years since menopause | ||||||||||||||
| <10 | 632 | 5.4 (5.1–5.6) | 598 | 22.9 (22.1–23.8) | 501 | 242.2 (227.5–257.8) | 743 | 19.2 (18.6–19.9) | 530 | 68.0 (64.0–72.4) | 482 | 9.2 (8.9–9.6) | 707 | 53.5 (51.5–55.6) |
| ≥10 | 992 | 5.1 (4.9–5.3) | 938 | 22.7 (22.1–23.4) | 681 | 224.7 (213.6–236.4) | 1126 | 20.2 (19.6–20.9) | 844 | 51.9 (49.4–54.6) | 710 | 8.7 (8.5–9.0) | 1072 | 57.8 (56.1–59.5) |
| t-test p-value | 0.08 | 0.69 | 0.07 | 0.03 | <0.0001 | 0.05 | 0.002 | |||||||
| BMI | ||||||||||||||
| <25 | 830 | 4.1 (4.0–4.3) | 789 | 20.1 (19.5–20.7) | 591 | 189.9 (180.8–199.5) | 958 | 19.8 (19.2–20.5) | 708 | 58.3 (55.2–61.6) | 612 | 9.0 (8.7–9.3) | 923 | 68.9 (67.1–70.8) |
| 25–29.3 | 531 | 5.7 (5.5–6.0) | 499 | 23.6 (22.6–24.6) | 392 | 248.2 (231.7–265.8) | 622 | 19.5 (18.7–20.3) | 458 | 57.7 (54.0–61.7) | 392 | 9.0 (8.6–9.4) | 581 | 48.4 (46.6–50.3) |
| ≥30 | 263 | 8.9 (8.4–9.4) | 248 | 31.9 (30.2–33.8) | 199 | 367.6 (334.8–403.7) | 289 | 20.5 (19.4–21.8) | 208 | 55.1 (49.8–61.1) | 188 | 8.7 (8.1–9.3) | 275 | 38.3 (36.1–40.7) |
| t-test p-value | <0.0001 | <0.0001 | <0.0001 | 0.36 | 0.63 | 0.60 | <0.0001 | |||||||
| Smoking status | ||||||||||||||
| Never | 729 | 5.3 (5.1–5.5) | 691 | 22.8 (22.0–23.6) | 532 | 245.8 (231.7–260.7) | 832 | 19.5 (18.8–20.2) | 625 | 57.5 (54.4–60.8) | 528 | 9.3 (8.9–9.6) | 788 | 55.7 (53.8–57.7) |
| Former | 673 | 5.2 (5.0–5.4) | 636 | 22.6 (21.8–23.5) | 491 | 228.4 (214.8–242.8) | 778 | 19.5 (18.8–20.2) | 565 | 54.7 (51.3–58.3) | 498 | 9.0 (8.6–9.3) | 740 | 54.2 (52.3–56.3) |
| Current | 222 | 5.1 (4.7–5.4) | 209 | 23.5 (22.1–24.9) | 159 | 200.3 (180.3–222.5) | 259 | 21.8 (20.5–23.1) | 184 | 68.2 (61.2–76.0) | 166 | 7.9 (7.4–8.4) | 251 | 62.9 (59.3–66.8) |
| ANOVA p-value | 0.62 | 0.61 | 0.004 | 0.006 | 0.002 | 0.0002 | 0.0003 | |||||||
| Alcohol (grams/day) | ||||||||||||||
| No regular use | 646 | 5.6 (5.3–5.8) | 609 | 23.9 (23.0–24.8) | 463 | 230.8 (215.8–246.8) | 741 | 20.1 (19.4–20.9) | 559 | 52.8 (49.6–56.2) | 465 | 8.8 (8.4–9.1) | 703 | 54.0 (51.9–56.2) |
| ≤20 | 798 | 4.9 (4.7–5.1) | 759 | 22.0 (21.3–22.7) | 587 | 223.6 (212.1–235.7) | 913 | 19.3 (18.7–20.0) | 661 | 58.9 (55.8–62.2) | 595 | 9.1 (8.7–9.4) | 879 | 57.9 (56.0–59.7) |
| >20 | 110 | 5.3 (4.8–5.8) | 106 | 22.9 (20.9–25.0) | 85 | 275.0 (237.9–318.0) | 130 | 21.2 (19.4–23.3) | 91 | 82.1 (70.2–95.9) | 81 | 8.7 (7.8–9.7) | 117 | 54.9 (50.3–60.0) |
| ANOVA p-value | <0.0001 | 0.005 | 0.03 | 0.07 | <0.0001 | 0.46 | 0.02 | |||||||
| Hysterectomy | ||||||||||||||
| No | 1342 | 5.2 (5.1–5.4) | 1275 | 22.8 (22.2–23.4) | 992 | 237.4 (227.4–247.8) | 1545 | 20.4 (19.9–20.9) | 1128 | 60.4 (57.9–63.1) | 996 | 8.9 (8.6–9.1) | 1471 | 56.3 (54.9–57.8) |
| Yes | 282 | 5.1 (4.8–5.5) | 261 | 22.8 (21.5–24.1) | 190 | 205.5 (186.2–226.7) | 324 | 17.5 (16.4–18.5) | 246 | 46.4 (42.2–51.0) | 196 | 9.2 (8.7–9.8) | 308 | 54.8 (51.8–57.9) |
| t-test p-value | 0.70 | 0.99 | 0.008 | <0.0001 | <0.0001 | 0.26 | 0.38 | |||||||
| Unilateral oophorectomy | ||||||||||||||
| No | 1517 | 5.2 (5.1–5.4) | 1435 | 22.8 (22.2–23.3) | 1102 | 233.8 (224.5–243.5) | 1745 | 20.0 (19.5–20.5) | 1295 | 58.2 (55.9–60.6) | 1112 | 8.9 (8.7–9.2) | 1659 | 56.0 (54.7–57.4) |
| Yes | 107 | 5.0 (4.5–5.6) | 101 | 23.3 (21.3–25.6) | 80 | 207.6 (177.1–243.2) | 124 | 17.5 (16.0–19.1) | 79 | 49.0 (40.6–59.0) | 80 | 9.1 (8.2–10.1) | 120 | 56.7 (51.5–62.4) |
| t-test p-value | 0.49 | 0.61 | 0.14 | 0.004 | 0.04 | 0.74 | 0.81 | |||||||
| Irregular cycles | ||||||||||||||
| No | 1317 | 5.2 (5.0–5.3) | 1248 | 22.8 (22.2–23.4) | 970 | 234.3 (224.2–244.9) | 1513 | 19.7 (19.2–20.2) | 1110 | 58.1 (55.6–60.7) | 974 | 9.0 (8.8–9.3) | 1445 | 56.8 (55.3–58.3) |
| Yes | 206 | 5.5 (5.1–5.9) | 196 | 23.6 (22.2–25.2) | 140 | 239.5 (215.6–266.1) | 240 | 20.5 (19.2–21.8) | 181 | 54.6 (49.0–60.7) | 143 | 8.7 (8.1–9.4) | 223 | 52.3 (49.0–55.9) |
| t-test p-value | 0.15 | 0.32 | 0.73 | 0.29 | 0.29 | 0.39 | 0.02 | |||||||
Table 4 presents unadjusted results for the association of the components LOY with hormones. An early age at menarche was associated only with higher estradiol levels while menopause after age 51 was associated with higher estradiol and estrone sulfate, but lower DHEAS and SHBG levels. Women who had any use of OCs had lower testosterone and prolactin levels and higher DHEAS levels, but no clear associations with duration of OC use. Conversely, hormone levels did not differ between ever and never parous women, but women with more children had lower estrone sulfate, testosterone, and DHEAS levels. Breastfeeding appeared to have the fewest associations with hormone levels. The final entry in Table 4 indicates that greater LOY was associated with higher estradiol and testosterone levels and lower SHBG.
Table 4.
Unadjusted geometric mean hormone levels by components of lifetime ovulatory years quartile in postmenopausal women not using hormone therapy at the time of blood draw in the Nurses’ Health Study (n=1976)
| Estradiol (pg/mL) | Estrone (pg/mL) | Estrone sulfate (pg/mL) | Testosterone (ng/dL) | DHEAS (ug/dL) | Prolactin (ng/mL) | SHBG (nmol/L) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | N | Mean (95% CI) | |
| Age at menarche | ||||||||||||||
| <12 | 347 | 5.5 (5.2–5.8) | 323 | 23.6 (22.4–24.8) | 260 | 244.1 (224.1–265.8) | 402 | 19.7 (18.8–20.7) | 282 | 59.8 (54.9–65.1) | 264 | 8.9 (8.4–9.4) | 383 | 55.1 (52.4–58.0) |
| 12 | 408 | 5.3 (5.0–5.6) | 391 | 23.3 (22.2–24.5) | 303 | 233.9 (215.0–254.5) | 468 | 20.0 (19.1–20.9) | 334 | 59.8 (55.1–65.0) | 298 | 9.0 (8.6–9.4) | 447 | 55.4 (53.0–58.0) |
| 13 | 498 | 5.1 (4.9–5.4) | 468 | 22.5 (21.5–23.4) | 345 | 228.3 (212.6–245.1) | 554 | 19.9 (19.1–20.8) | 402 | 55.2 (51.4–59.4) | 344 | 9.0 (8.6–9.4) | 533 | 56.1 (53.7–58.6) |
| 14 | 227 | 5.1 (4.8–5.5) | 221 | 22.2 (20.9–23.6) | 169 | 221.1 (201.5–242.6) | 268 | 19.9 (18.7–21.1) | 214 | 58.1 (52.8–64.0) | 173 | 8.8 (8.2–9.5) | 250 | 56.3 (53.0–59.8) |
| >14 | 144 | 4.7 (4.3–5.1) | 133 | 21.7 (20.1–23.4) | 105 | 227.1 (198.8–259.4) | 177 | 19.3 (17.8–21.0) | 142 | 54.6 (48.4–61.6) | 113 | 8.9 (8.2–9.7) | 166 | 59.8 (55.0–65.0) |
| ANOVA p-value | 0.04 | 0.27 | 0.64 | 0.96 | 0.45 | 0.99 | 0.50 | |||||||
| Age at menopause | ||||||||||||||
| <48 | 240 | 5.3 (4.9–5.6) | 225 | 22.8 (21.5–24.3) | 173 | 250.5 (225.9–277.7) | 280 | 20.0 (18.9–21.2) | 200 | 66.6 (60.1–73.8) | 179 | 9.2 (8.5–9.9) | 269 | 57.0 (53.6–60.7) |
| 48–49 | 246 | 4.8 (4.5–5.2) | 236 | 21.9 (20.7–23.1) | 191 | 211.3 (192.1–232.5) | 287 | 19.9 (18.9–21.1) | 213 | 62.6 (56.8–69.1) | 187 | 8.7 (8.2–9.3) | 272 | 61.5 (58.0–65.3) |
| 50–51 | 564 | 5.1 (4.9–5.3) | 533 | 22.6 (21.7–23.5) | 403 | 215.0 (201.1–229.9) | 648 | 19.2 (18.4–20.0) | 484 | 54.1 (50.6–57.7) | 409 | 8.9 (8.5–9.3) | 619 | 57.5 (55.3–59.7) |
| >51 | 574 | 5.5 (5.2–5.7) | 542 | 23.4 (22.5–24.4) | 415 | 252.4 (235.9–270.0) | 654 | 20.3 (19.5–21.1) | 477 | 55.8 (52.1–59.7) | 417 | 9.0 (8.6–9.4) | 619 | 52.1 (50.0–54.3) |
| ANOVA p-value | 0.02 | 0.28 | 0.008 | 0.22 | 0.002 | 0.76 | <0.0001 | |||||||
| OC use | ||||||||||||||
| No | 1107 | 5.3 (5.1–5.4) | 1052 | 23.0 (22.3–23.6) | 779 | 229.0 (218.1–240.4) | 1257 | 20.3 (19.7–20.9) | 929 | 55.6 (53.1–58.3) | 784 | 9.1 (8.8–9.4) | 1200 | 56.3 (54.7–58.0) |
| Yes | 511 | 5.1 (4.9–5.4) | 479 | 22.5 (21.5–23.4) | 398 | 238.6 (223.0–255.3) | 605 | 18.9 (18.1–19.7) | 441 | 61.8 (57.5–66.3) | 403 | 8.6 (8.2–8.9) | 572 | 55.5 (53.3–57.8) |
| t-test p-value | 0.35 | 0.40 | 0.33 | 0.004 | 0.01 | 0.03 | 0.58 | |||||||
| Duration of OC use | ||||||||||||||
| ≤0.5 | 101 | 4.9 (4.4–5.5) | 97 | 21.9 (19.8–24.3) | 76 | 222.8 (189.4–262.0) | 121 | 19.4 (17.5–21.5) | 83 | 59.8 (50.1–71.4) | 81 | 8.6 (7.9–9.3) | 116 | 56.3 (51.5–61.5) |
| >0.5–3 | 140 | 5.2 (4.7–5.8) | 129 | 22.9 (21.0–25.0) | 118 | 244.0 (214.9–277.1) | 168 | 19.1 (17.5–20.8) | 125 | 58.0 (50.4–66.7) | 115 | 8.3 (7.6–9.0) | 160 | 54.6 (50.7–58.7) |
| >3–8 | 131 | 5.2 (4.8–5.7) | 122 | 22.3 (20.5–24.2) | 91 | 239.4 (210.7–272.1) | 154 | 18.9 (17.5–20.5) | 114 | 68.2 (60.4–77.1) | 97 | 8.5 (7.9–9.2) | 147 | 54.3 (49.9–59.1) |
| >8 | 117 | 5.1 (4.6–5.6) | 111 | 22.6 (20.6–24.8) | 99 | 244.6 (210.9–283.7) | 132 | 18.0 (16.7–19.5) | 96 | 60.1 (51.1–70.7) | 95 | 9.0 (8.2–9.9) | 124 | 55.8 (51.3–60.6) |
| ANOVA p-value | 0.84 | 0.92 | 0.80 | 0.69 | 0.39 | 0.60 | 0.92 | |||||||
| Parity | ||||||||||||||
| Nulliparous | 75 | 5.3 (4.6–6.0) | 74 | 23.0 (20.6–25.7) | 50 | 261.4 (211.9–322.4) | 81 | 21.0 (18.6–23.8) | 58 | 62.0 (51.6–74.4) | 50 | 8.7 (8.0–9.5) | 79 | 60.2 (53.8–67.3) |
| Parous | 1549 | 5.2 (5.1–5.3) | 1462 | 22.8 (22.3–23.4) | 1132 | 230.7 (221.6–240.2) | 1788 | 19.8 (19.3–20.2) | 1316 | 57.4 (55.2–59.8) | 1142 | 8.9 (8.7–9.2) | 1700 | 55.9 (54.6–57.2) |
| t-test p-value | 0.81 | 0.87 | 0.21 | 0.28 | 0.45 | 0.55 | 0.20 | |||||||
| Parity | ||||||||||||||
| 1–2 | 462 | 5.3 (5.0–5.6) | 439 | 23.3 (22.3–24.4) | 333 | 240.0 (221.7–259.9) | 513 | 20.9 (20.0–21.9) | 379 | 62.7 (58.0–67.8) | 331 | 9.4 (8.9–9.9) | 497 | 55.0 (52.5–57.5) |
| 3 | 394 | 5.2 (4.9–5.4) | 371 | 22.5 (21.5–23.6) | 292 | 243.0 (225.7–261.7) | 477 | 20.0 (19.1–20.9) | 352 | 56.1 (51.9–60.6) | 300 | 8.9 (8.4–9.3) | 448 | 55.4 (52.9–58.0) |
| 4 | 355 | 5.1 (4.8–5.5) | 330 | 22.6 (21.5–23.7) | 252 | 227.9 (209.1–248.4) | 410 | 19.0 (18.1–20.0) | 300 | 55.8 (51.4–60.5) | 265 | 8.9 (8.4–9.4) | 397 | 56.6 (53.9–59.5) |
| >4 | 338 | 5.2 (4.9–5.5) | 322 | 22.6 (21.5–23.8) | 255 | 209.0 (192.5–226.8) | 388 | 18.8 (17.9–19.8) | 285 | 54.4 (49.8–59.3) | 246 | 8.6 (8.1–9.1) | 358 | 56.9 (54.0–60.0) |
| ANOVA p-value | 0.83 | 0.66 | 0.04 | 0.007 | 0.05 | 0.10 | 0.71 | |||||||
| Breastfeeding | ||||||||||||||
| No | 563 | 5.3 (5.0–5.5) | 532 | 23.3 (22.4–24.2) | 411 | 231.9 (216.7–248.1) | 663 | 20.4 (19.6–21.2) | 488 | 56.5 (53.0–60.3) | 422 | 9.2 (8.8–9.6) | 630 | 56.3 (54.2–58.6) |
| Yes | 1061 | 5.2 (5.0–5.3) | 1004 | 22.5 (21.9–23.2) | 771 | 232.0 (221.0–243.5) | 1206 | 19.5 (19.0–20.1) | 886 | 58.2 (55.4–61.2) | 770 | 8.8 (8.5–9.1) | 1149 | 55.9 (54.3–57.6) |
| t-test p-value | 0.44 | 0.18 | 0.99 | 0.09 | 0.48 | 0.08 | 0.76 | |||||||
| Months breastfed | ||||||||||||||
| ≤3 | 450 | 5.2 (4.9–5.4) | 423 | 22.7 (21.7–23.8) | 326 | 225.0 (208.2–243.1) | 510 | 19.6 (18.8–20.6) | 362 | 59.8 (55.4–64.6) | 329 | 8.7 (8.3–9.1) | 492 | 56.1 (53.7–58.6) |
| 4–11 | 328 | 5.1 (4.8–5.4) | 311 | 22.3 (21.1–23.5) | 243 | 236.0 (217.6–256.1) | 374 | 19.1 (18.1–20.1) | 282 | 57.4 (52.6–62.6) | 244 | 8.5 (8.1–9.0) | 357 | 56.5 (53.6–59.6) |
| ≥12 | 283 | 5.2 (4.9–5.6) | 270 | 22.6 (21.3–23.9) | 202 | 238.8 (216.7–263.1) | 322 | 19.8 (18.7–21.0) | 242 | 56.9 (51.7–62.7) | 197 | 9.2 (8.6–9.9) | 300 | 54.9 (51.7–58.4) |
| ANOVA p-value | 0.86 | 0.86 | 0.56 | 0.60 | 0.68 | 0.17 | 0.77 | |||||||
| Ovulatory years | ||||||||||||||
| <29.8 | 403 | 5.0 (4.8–5.3) | 381 | 21.9 (20.9–23.0) | 313 | 230.3 (213.4–248.7) | 470 | 18.5 (17.7–19.4) | 347 | 59.7 (55.2–64.6) | 312 | 8.4 (8.0–8.9) | 445 | 57.7 (55.0–60.5) |
| 29.8–33.0 | 408 | 5.0 (4.7–5.2) | 392 | 22.4 (21.4–23.5) | 296 | 226.2 (210.2–243.3) | 478 | 19.6 (18.8–20.5) | 355 | 58.4 (54.1–63.0) | 301 | 9.2 (8.7–9.7) | 451 | 57.9 (55.4–60.5) |
| 33.1–35.9 | 392 | 5.3 (5.0–5.6) | 365 | 23.1 (22.0–24.3) | 269 | 223.5 (205.4–243.1) | 442 | 20.6 (19.6–21.6) | 323 | 55.4 (51.1–60.1) | 278 | 9.1 (8.6–9.5) | 422 | 56.7 (53.9–59.6) |
| ≥36.0 | 421 | 5.5 (5.2–5.8) | 398 | 23.8 (22.7–24.9) | 304 | 247.4 (227.8–268.7) | 479 | 20.6 (19.7–21.6) | 349 | 56.9 (52.7–61.5) | 301 | 9.1 (8.7–9.6) | 461 | 52.3 (50.0–54.8) |
| ANOVA p-value | 0.02 | 0.09 | 0.28 | 0.004 | 0.59 | 0.05 | 0.007 | |||||||
Table 5 shows hormone levels by quartiles of LOY and examines the trend in hormone levels for 5-year increments in LOY as a continuous variable. Means and trends are adjusted for participant characteristics in Table 3 but not by components of LOY. BMI was included either as an adjustment or a stratification variable. In all women, adjusting for BMI, a significant trend was seen for testosterone with more LOY associated with higher levels. For each 5-year increase in LOY, there was a 6.2% increase in testosterone (95% CI: 3.5%−9.0%) overall. Similar trends with estradiol and estrone were also significant. For women with BMI<25 kg/m2, each 5-year increase in LOY was associated with a 5.2% increase in testosterone (95% CI: 1.3%, 9.1%). In women with above-average BMI, each 5-year increase in LOY was associated with a 7.4% (95% CI: 3.6%, 11.4%) increase in testosterone, a 7.3% (95% CI: 3.1%,11.7%) increase in estradiol, and a 7.0% (95% CI: 3.1%,11.1%) increase in estrone. Differences between the higher and lower BMI groups in the association of LOY with hormone levels were significant for estrone (p=0.02) and estrone sulfate (p=0.04), and borderline for estradiol (p=0.07). Slightly stronger associations were seen after excluding women whose age at menopause was imputed. (Supplemental Table 1). This included greater increases in testosterone with increasing LOY in all women (6.5%, 95% CI: 3.6%, 9.5%), women with BMI<25 kg/m2 (5.3%, 95% CI: 1.2%, 9.5%), and women with BMI≥25 kg/m2 (7.9%, 95% CI: 3.8%, 12.1%). Similarly, estradiol increased more with increasing LOY in all women (3.9%, 95% CI: 1.0%, 6.8%) and in women with above-average BMI (8.7%, 95% CI: 4.1%, 13.5%).
Table 5.
Adjusted geometric mean hormone levels by lifetime ovulatory years quartile among postmenopausal women not using hormone therapy at the time of blood draw in the Nurses’ Health Study (n=1976)
| Hormone | N | Ovulatory years | Difference per 5 years* | ||||
|---|---|---|---|---|---|---|---|
|
|
|||||||
| <29.8 Mean (95% CI)* | 29.8–33.0 Mean (95% CI)* | 33.1–35.9 Mean (95% CI)* | ≥36.0 Mean (95% CI)* | % (95% CI) | p-trend | ||
| All women | |||||||
| Estradiol (pg/mL) | 1624 | 5.1 (4.8, 5.3) | 5.1 (4.9, 5.3) | 5.3 (5.0, 5.6) | 5.4 (5.1, 5.6) | 2.7 (0.1, 5.4) | 0.05 |
| Estrone (pg/mL) | 1536 | 21.8 (20.8, 22.8) | 22.8 (21.8, 23.8) | 23.1 (22.1, 24.2) | 23.6 (22.5, 24.7) | 2.6 (0.1, 5.1) | 0.04 |
| Estrone sulfate (pg/mL) | 1182 | 232.4 (215.5, 250.7) | 233.1 (216.7, 250.8) | 223.8 (207.2, 241.7) | 237.7 (220.1, 256.7) | 0.3 (−3.6, 4.3) | 0.89 |
| Testosterone (ng/dL) | 1869 | 18.0 (17.1, 18.9) | 19.5 (18.7, 20.5) | 20.9 (20.0, 22.0) | 21.0 (20.0, 22.1) | 6.2 (3.5, 9.0) | <0.0001 |
| DHEAS (ug/dL) | 1374 | 55.9 (51.5, 60.5) | 57.6 (53.5, 62.1) | 56.7 (52.4, 61.3) | 60.4 (55.8, 65.5) | 2.2 (−2.1, 6.7) | 0.32 |
| Prolactin (ng/mL) | 1192 | 8.3 (7.9, 8.7) | 9.1 (8.7, 9.6) | 9.1 (8.6, 9.6) | 9.3 (8.8, 9.8) | 1.3 (−1.5, 4.2) | 0.35 |
| SHBG (nmol/L) | 1779 | 55.5 (53.1, 58.1) | 56.6 (54.2, 58.9) | 57.6 (55.1, 60.1) | 54.9 (52.5, 57.3) | 0.7 (−1.7, 3.1) | 0.57 |
| BMI<25 | |||||||
| Estradiol (pg/mL) | 830 | 4.2 (3.9, 4.5) | 4.2 (3.9, 4.5) | 4.0 (3.8, 4.3) | 4.2 (3.9, 4.5) | 0.1 (−3.6, 3.9)† | 0.96 |
| Estrone (pg/mL) | 789 | 20.1 (18.9, 21.4) | 20.5 (19.4, 21.7) | 19.4 (18.2, 20.7) | 20.2 (18.9, 21.5) | −0.9 (−4.2, 2.5)† | 0.60 |
| Estrone sulfate (pg/mL) | 591 | 202.7 (183.3, 224.2) | 196.2 (178.5, 215.7) | 177.5 (159.9, 197.1) | 181.4 (163.1, 201.8) | −4.5 (−9.5, 0.8)† | 0.09 |
| Testosterone (ng/dL) | 958 | 18.3 (17.1, 19.6) | 19.6 (18.5, 20.9) | 20.7 (19.3, 22.2) | 21.2 (19.7, 22.8) | 5.2 (1.3, 9.1)† | 0.008 |
| DHEAS (ug/dL) | 708 | 57.1 (51.2, 63.6) | 60.0 (54.2, 66.4) | 53.1 (47.5, 59.4) | 63.9 (56.7, 72.0) | 2.5 (−3.7, 9.0)† | 0.44 |
| Prolactin (ng/mL) | 612 | 8.5 (7.9, 9.1) | 8.7 (8.1, 9.3) | 9.4 (8.8, 10.1) | 9.5 (8.8, 10.3) | 1.5 (−2.2, 5.4)† | 0.43 |
| SHBG (nmol/L) | 923 | 67.5 (63.9, 71.3) | 68.4 (64.9, 72.0) | 72.5 (68.5, 76.8) | 67.7 (63.8, 71.8) | 0.7 (−2.3, 3.8)† | 0.64 |
| BMI≥25 | |||||||
| Estradiol (pg/mL) | 794 | 6.1 (5.6, 6.6) | 6.2 (5.7, 6.7) | 6.9 (6.4, 7.5) | 7.2 (6.7, 7.8) | 7.3 (3.1, 11.7)† | 0.0006 |
| Estrone (pg/mL) | 747 | 23.6 (21.8, 25.4) | 24.9 (23.2, 26.7) | 27.5 (25.7, 29.4) | 28.2 (26.3, 30.2) | 7.0 (3.1, 11.1)† | 0.0004 |
| Estrone sulfate (pg/mL) | 591 | 264.9 (235.1, 298.5) | 274.7 (243.9, 309.3) | 279.6 (248.0, 315.1) | 313.3 (278.9, 352.0) | 6.0 (−0.3, 12.7)† | 0.06 |
| Testosterone (ng/dL) | 911 | 17.6 (16.4, 18.9) | 19.4 (18.2, 20.8) | 21.2 (19.9, 22.7) | 20.8 (19.5, 22.2) | 7.4 (3.6, 11.4)† | 0.0001 |
| DHEAS (ug/dL) | 666 | 54.4 (48.3, 61.4) | 55.6 (49.8, 62.1) | 60.5 (54.2, 67.6) | 57.2 (51.4, 63.6) | 1.7 (−4.2, 8.0)† | 0.58 |
| Prolactin (ng/mL) | 580 | 8.1 (7.4, 8.8) | 9.6 (8.9, 10.4) | 8.8 (8.1, 9.5) | 9.2 (8.5, 9.9) | 1.3 (−2.8, 5.6)† | 0.54 |
| SHBG (nmol/L) | 859 | 45.5 (42.2, 49.1) | 46.1 (43.0, 49.4) | 45.7 (42.7, 48.9) | 42.9 (40.1, 45.8) | −1.0 (−4.6, 2.5)† | 0.60 |
Adjusted for age at blood draw (continuous), smoking (never, former, current), alcohol (no regular use, ≤10, >10–20, >20–30, >30 grams/day), years since menopause (continuous), hysterectomy, unilateral oophorectomy, and irregular cycles. Models for all women additionally adjusted for BMI.
p-values for heterogeneity comparing difference in hormone levels per 5 ovulatory years among those with BMI<25 to those with BMI≥25, estradiol: p=0.07, estrone: p=0.02, estrone sulfate: p=0.04, testosterone: p=0.47, DHEAS: p=0.94, prolactin: p=0.37, SHBG: p=0.66.
Discussion
We used existing data on sex-hormone levels measured in NHS participants to address whether estimated LOY is associated with postmenopausal hormone levels in women not using exogenous hormones. In all women, adjusting for BMI and other factors, we found significant trends for women with greater LOY to have higher testosterone and estrogen levels. The trends with testosterone were also apparent among women stratified by BMI<25 kg/m2 and BMI≥25 kg/m2. The latter group also had significant trends between LOY and estrogen levels. That women with more LOY and above-average BMI had both elevated testosterone and estrogen is consistent with the known ability of fatty tissue to aromatize androgens into active estrogens.(33) Prospective studies of postmenopausal women have found that the combination of elevated serum testosterone and estradiol predict endometrial, breast, and ovarian cancer risks.(34–37)
While the link between elevated testosterone and estradiol is easily explained, a link between LOY and testosterone first requires showing that the ovaries are a major source of testosterone in postmenopausal women. We believe this is established by cannulation of ovarian veins during pelvic surgery showing higher levels of testosterone and androstenedione than peripheral blood.(16–18) Importantly, studies in women with ovarian and endometrial cancer at their surgeries revealed even higher levels of testosterone compared to women coming to oophorectomy for other indications.(38,39) Cannulation studies revealed that the level of estradiol in ovarian veins were not, or only modestly, elevated over peripheral levels indicating that the postmenopausal ovary is not a major source of estradiol. Although LOY was not examined in the cannulation studies, nor in the pooled study of hormones in postmenopausal women,(19) the latter study did show that oophorectomized women had substantially lower levels of testosterone. This has been confirmed in other populations, including NHS(27) indicating that the ovaries are, indeed, the key source of testosterone in postmenopausal women. This conclusion is further supported by the observation in Table 3 that lower testosterone was associated with unilateral oophorectomy compared to women with both ovaries intact.
A study of Dutch women did examine sex-hormone levels in relation to estimated lifetime ovulatory cycles (which would equate with LOYx13 for a cycle length of 28 days).(40) Women age 50–69 who participated in a regional breast cancer screening program were invited to complete questionnaires and provide blood. Out of 50,313 women invited, 17,357 (34.3%) agreed to be studied; and 97.5% of these provided a blood sample.(41) Hormones were measured in a 10% sample of the bloods and associations with number of cycles examined, which did not reveal any link between number of cycles and testosterone. This makes important a comparison of the features of this study with those of ours. The Dutch study had a higher percentage of women who were nulliparous and had fewer livebirths, greater use of birth control pills, and more current smokers. Out of 1400 eligible participants, 860 remained after excluding hysterectomized women, indicating that 540 (38.6%) had a hysterectomy before menopause. This is more than double the 17.5% in our study. This is important because of our observation in Table 3 that women who had a hysterectomy were more likely to have a greater number of ovulatory years. The exclusion of hysterectomized women in the Dutch study could have skewed the distribution of women in the Dutch Study towards those with fewer ovulatory cycles. Variables similar to ours were used to construct number of cycles, but information on incomplete pregnancies was not available in the NHS. As we did, the Dutch study included years of pill use in calculating ovulatory cycles, but also adjusted for OC use in their analysis of its effect on sex hormones. As pointed out by Yang et al., adjusting for variables which are components of LOY weakens estimates of its effect on disease risk (and vice versa).(6)
Despite a lack of confirmation from the Dutch study, we believe a good biologic explanation exists for the link between LOY and testosterone. This link likely involves the ovary’s germ cell reserve, the process of follicle maturation, and the fate of follicles recruited during a cycle but not selected for ovulation.(42,43) Primordial follicles are oocytes surrounded by a single layer of pre-granulosa cells. The maximum quota of primordial follicles, likely more than a million, is established at birth, of which about 300,000 remain when puberty begins. During each menstrual cycle, a cohort of follicles is selected to undergo maturation involving growth of the granulosa layer, formation of a thecal-cell layer, secretion of follicular fluid to form antral follicles, and selection of, usually, just one to become a Graffian follicle from which the oocyte will be released. Remnants of the dominant follicle form the corpus luteum which produces progesterone to support early pregnancy if fertilization occurs. During a cycle, some follicles that reach the early-antral stage will become quiescent and remain at that stage as a more dynamic reserve than primordial follicles. The remainder of follicles that developed further during a cycle will undergo apoptosis to become atretic follicles; and the corpus luteum regresses. The fate of developing follicles during an ovulatory cycle and the process of follicular atresia are likely most relevant to the potential consequences of repetitive ovulation.(44) Evidence that an antral follicle is on the path to atresia is apoptosis within the granulosa cell layer. The granulosa cell decrease leads to lower estradiol, both from reduced production and reduced aromatization of androgens secreted by thecal cells into estradiol. Thecal cells remain viable after disappearance of granulosa cells and retain their ability to secrete androgens, but their longer-term fate is unclear. In the ovarian vein cannulation studies, a significant excess of testosterone from the ovary over peripheral levels was found in women coming to surgery ten years after their menopause.(18)
Relevant to the fate of stromal and thecal remnants are two related conditions called ovarian stromal hyperplasia (OSH) and hyperthecosis (OHT). OSH refers to nodular or diffuse proliferation of the stromal cells of the ovary in varying degrees while OHT is distinguished by the presence of round to polyhedral theca-lutein-like cells occurring singly or in nests within a stroma that itself is proliferative.(45) For decades, these conditions have been of interest in the context of describing ovaries removed from women with endometrial cancer.(46) A link between OSH and breast cancer has also been described;(47) and OHT can be seen at the edge of epithelial ovarian malignancies.(48) Clearly needed are studies which quantify the degree of OSH/OHT in relation to number of estimated LOY. Elevated testosterone and estradiol, OSH and OHT, and predisposition to endometrial cancer are components of the disorder polycystic ovary syndrome (PCOS) affecting younger women.(49,50) This raises the issue of how elevated testosterone and estradiol in postmenopausal women with greater LOY relates to PCOS. Although speculative, we suggest that PCOS involves abnormal cycles with too many follicles and premature luteinization during each cycle while greater LOY involves too many “normal” ovulatory cycles.
In assessing the potential effect of LOY on hormone levels, we adjusted for “lifestyle” variables, including BMI, smoking, and alcohol use, previously associated with level of postmenopausal hormones in a pooled analysis of 13 cohort studies.(19) All three factors were found to have some effects on estradiol and testosterone levels. However, a limitation of our study was that we did not consider all potential lifestyle or medical factors (e.g., thyroid conditions), and, in our adjusted models, residual confounding may remain. The main limitation of our study is a “generic” one. Is any epidemiologic study able to accurately estimate the number of ovulations a woman experienced based on her reproductive history? Unless pregnancy ensued, proving ovulation occurred during a cycle requires biologic measures of ovulation in blood or urine. Such studies involve small numbers of women followed over a relatively brief period-of-time. These studies indicate that anovulatory cycles are more likely to occur around the time of menarche and menopause; but estimates vary on how long it takes for ovulatory cycles to be established after menarche and how soon before menopause they become anovulatory.(51–53) While it is easy to be skeptical that even the most detailed algorithm could accurately quantify the number of ovulations, the fact is that even the crudest algorithms with only ages at menarche and menopause, births, and OC use produce estimates that correlate with risk not only for ovarian cancer, but also breast and endometrial cancer.
In conclusion, we studied postmenopausal hormone levels in NHS participants in relation to estimated lifetime ovulations not interrupted by pregnancies, breastfeeding, or oral contraceptive use. We observed an association between greater LOY and testosterone levels and, in women with both greater LOY and BMI, higher estrogen levels. We have suggested that the association of LOY with testosterone reflects some degree of ovarian stromal hyperplasia or hyperthecosis which may be consequences of uninterrupted ovulation and persistence of stromal and thecal cells accumulated during repetitive cycles. Our findings need replication which could be readily accomplished by estimating LOY in existing cross-sectional studies of sex hormones in postmenopausal women, ideally in a more demographically diverse population than NHS. Future studies should focus on hormonal profiles, especially testosterone and estradiol, in postmenopausal women coming to oophorectomy whose LOY have been well-characterized to correlate with histologic evidence of stromal or thecal cell activity.
Supplementary Material
Acknowledgements
The authors assume full responsibility for analysis and interpretation of these data. This work was supported by the National Institutes of Health (A.H. Eliassen, grant numbers UM1CA186107 and P01CA87969; S.E. Hankinson, grant number R01CA49449; and D.W. Cramer, grant number R35CA197605)
Funding
The project was supported by the National Institutes of Health (UM1CA186107 (A.H. Eliassen), P01CA87969 (A.H. Eliassen), R01CA49449 (S.E. Hankinson), and R35CA197605 (D.W. Cramer)). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Footnotes
Potential Conflicts of Interest
The authors report no conflict of interests related to the topic of this study. Dr. Cramer reports being paid for expert testimony in litigation related to talc and ovarian cancer.
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Data Availability Statement
The data underlying this article are available by application. Further information including the procedures to obtain and access data from the Nurses’ Health Studies is described at https://www.nurseshealthstudy.org/researchers