Serum leptin levels and reproductive function during the menstrual cycle




Objective


The purpose of this study was to investigate the role of leptin on reproductive hormones and ovulation.


Study Design


The BioCycle Study (2005-2007) followed 259 healthy premenopausal women not using hormonal contraceptives for ≤2 menstrual cycles (n = 509 cycles). Serum leptin, estradiol, progesterone, luteinizing hormone (LH), follicle-stimulating hormone, and testosterone were measured ≤8 times per cycle. The association of time-varying leptin and reproductive hormones over the cycle was estimated with the use of linear mixed models that were adjusted for percent body fat and age with inverse probability weighting for time-varying physical activity, caloric intake, and other reproductive hormones. The odds ratio for sporadic anovulation (n = 42 cycles) was estimated with the use of generalized linear models that were adjusted for percent body fat and age.


Results


Geometric mean serum leptin levels increased from menses to the late luteal phase (16.7-20.4 ng/mL; P < .01), with a mid-cycle peak (21.7 ng/mL) at the time of the LH surge ( P < .01). A 10% higher leptin level across the menstrual cycle was associated with higher estradiol levels (2.2%; 95% CI, 1.5–3.0), luteal progesterone levels (2.1%; 95% CI, 0.5–3.7), ovulatory LH levels (1.2%; 95% CI, 0–2.3), testosterone levels (0.6%; 95% CI, 0.3–0.9), and lower follicle-stimulating hormone levels (–0.7%; 95% CI, –1.1 to –0.4). Leptin at the time of the expected LH surge was moderately inversely associated with sporadic anovulation (per log increase in leptin; adjusted odds ratio, 0.58; 95% CI, 0.28–1.22).


Conclusion


The association that was observed between leptin level and reproductive function points to a possible relationship between serum leptin level and enhanced fertility.


Leptin, a product of the LEP gene, is known widely to regulate appetite and energy expenditure. Its involvement in the reproductive system was first suspected in 1949 when leptin homozygous recessive female mice were observed to be not only obese but sterile. Future research that demonstrated that the administration of recombinant leptin to these mice restored fertility led researchers to theorize that leptin served as a signal of adequate fat deposition, allowing for the energy-intensive reproduction system to function appropriately. Recent studies on the administration of recombinant leptin to women with lipodystrophy (ie, leptin deficiency) have also demonstrated restored menstrual cycle regularity and fertility. Despite the clear involvement of leptin in the female reproductive system, its relationship to reproductive hormone production, menstrual cycle characteristics, and ovarian function remains unclear.


The role of leptin on menstrual cycle regulation was first suggested more than a decade ago by researchers who found that leptin levels varied across the menstrual cycle while remaining stable for men and postmenopausal women over a 28-day period. Subsequently, a number of studies have found that either serum leptin increases from the follicular to the luteal phase (in a cyclic fashion) or shows no trend across the menstrual cycle. Limitations of previous work included the small number of women studied, the limited number of serum samples that were collected over the cycle, and unverified menstrual cycle phase determination. Furthermore, associations between leptin and reproductive hormones have been identified primarily by statistical correlations, without further consideration for factors such as diet, physical activity, and other hormone levels, which may have resulted in bias. In addition, because adipose tissue is a source of both leptin and estradiol production, adjustment for adiposity is critical for understanding leptin’s effect on reproductive hormones outside of the influence of body fat and could help inform future clinical interventions.


The primary objective of our study was to describe leptin levels across the menstrual cycle among a cohort of premenopausal women. Our secondary objectives were to examine the associations between leptin and reproductive hormones (including estradiol, progesterone, luteinizing hormone [LH], follicle-stimulating hormone [FSH], and testosterone), menstrual cycle characteristics and the odds of sporadic anovulation. The results of our study are important for understanding the role of leptin on reproduction and fertility.


Methods


Study population


The BioCycle Study (2005-2007) was a prospective cohort study of 259 regularly menstruating, healthy premenopausal women from western New York who were observed over 1 (n = 9) or 2 (n = 250) menstrual cycles. Women were not eligible for the study if they were using oral contraceptives or medications for a chronic medical condition, had been pregnant or breastfeeding within the past 6 months, had been diagnosed with a menstrual or ovulatory disorder, or self-reported their body mass index (BMI) as <18 or >35 kg/m 2 at screening. Additional information about the study population is described in more detail elsewhere. The University at Buffalo Health Sciences Institutional Review Board approved the study and served as the institutional review board that was designated by the National Institutes of Health for this study under a reliance agreement. All participants provided written informed consent.


Measures


Leptin and reproductive hormones


Women provided morning fasting blood samples up to 8 times per cycle. Fertility monitors (Clearblue Easy Fertility Monitor; Inverness Medical, Waltham, MA) were used to time mid-cycle visits; the remaining visits were scheduled according to an algorithm that considered each woman’s typical cycle length. Consequently, blood samples were collected during the following expected phases of the menstrual cycle: menses, the middle and late follicular phase, LH surge, ovulation, and the early, mid, and late luteal phase. Most women adhered to the study protocol; 94% of them provided blood samples for at least 7 visits per cycle. Blood samples were processed according to standard protocols and frozen at –80°C within 90 minutes of phlebotomy. Frozen sera were later shipped on dry ice to analytical laboratories. Samples from each participant were measured within a single run to limit analytical variability.


Leptin concentration was measured in multiple batches by immunoassay with the use of the Mercodia Leptin ELISA (Mercodia AB, Uppsala, Sweden) at the Advanced Research and Diagnostics Laboratory, University of Minnesota, Minneapolis, MN. No values were below the lower limit of detection for this assay (0.05 ng/mL). Select batches of measurements were recalibrated post-assay by a calibration curve that was estimated from all the calibration data. The maximum interassay coefficient of variation was 10.2% after recalibration.


Estradiol, progesterone, LH, and FSH concentrations were measured by solid-phase competitive chemiluminescent enzymatic immunoassays on the Immulite 2000 analyzer (Siemens Medical Solutions Diagnostics, Deerfield, IL) at Kaleida Laboratories in Buffalo, NY. Total testosterone was measured by liquid chromatography/tandem mass spectrometry with the use of the Shimadzu Prominence Liquid Chromatogram (Shimadzu Corporation, Kyoto, Japan) with a tandem mass spectrometer (AB Sciex 5500; AB Sciex, Framingham, MA) at the Advanced Research and Diagnostics Laboratory in Minneapolis, MN. Increased sensitivity was achieved by using 100% acetonitrile mobile phase B as the solvent gradient elution and adding a low standard of 4 ng/dL. The maximum coefficient of variation for each assay was <10% for estradiol, <14% for progesterone, <4% for LH and FSH, and <7% for testosterone. Values falling below the lower limit of detection for each assay were rare (<3%) and were replaced with values equal to the lower limit of detection divided by the square root of 2. All hormone measurements, including leptin, were transformed logarithmically for the analysis.


Sporadic anovulation and menstrual cycle characteristics


Sporadic anovulatory cycles were defined as cycles with a peak progesterone concentration of ≤5 ng/mL and no observed serum LH peak among samples that were collected during the later cycle visits (n = 42 cycles). In a sensitivity analysis, a subgroup of anovulatory cycles with peak progesterone concentrations of ≤3 ng/mL (n = 28 cycles) were examined as an alternative definition of anovulation. Menstrual cycle characteristics (menstrual cycle length, menses length, and total blood loss during menses) were determined from daily diaries that documented bleeding days and blood loss with the use of validated pictograms. Follicular and luteal phase lengths were determined based on the expected date of ovulation with the use of information from the fertility monitors and serum hormone levels.


Hormone realignment


To correct for any residual errors in blood collection timing, hormone measurements were realigned within ovulatory cycles according to an algorithm based on the day of the serum LH peak. This realignment affected 70% of ovulatory cycles, with 42% of realignments because of an LH peak that was detected at the visits adjacent to the expected LH surge visit. All hormone measurements were realigned together for a given cycle. Realignment procedures sometimes resulted in missing hormone levels; therefore, longitudinal multiple imputation methods were applied to these missing data. Anovulatory cycles were excluded from the realignment and imputation procedures because no ovulation was presumed to have occurred.


Covariates


Self-administered questionnaires were used to assess demographics, smoking behavior, physical activity, and perceived stress (measured by the Cohen Perceived Stress Scale) during a baseline visit that was scheduled to occur 1-2 weeks before the participant’s next expected start of menses. Physical activity was assessed by the measurement of past-week metabolic equivalent of task (MET)-hours per week with the use of the long-form International Physical Activity Questionnaire. BMI was calculated with weight and height as measured by trained personnel. At the end of the follow-up period, body composition was determined with duel energy x-ray absorptiometry scans (software version 12.4.1; Hologic Discovery Elite, Waltham, MA), and total percent body fat was derived. Past-week MET-hours per week and caloric intake were assessed 4 times during each cycle with the short-form International Physical Activity Questionnaire and the 24-hour dietary recall, respectively. These within-cycle measures were realigned and imputed along with the hormone measurements.


Statistical analysis


Participant characteristics were compared by tertile of average leptin concentration across the study period with Fisher exact and analysis of variance tests used to examine differences. Unadjusted linear mixed models were used to estimate geometric mean leptin by menstrual cycle phase and to assess differences in leptin between phases. To evaluate the effect of our realignment and imputations procedures, leptin concentrations across the cycle were also examined in the original data before realignment. Unadjusted linear mixed models were used to estimate mean reproductive hormones and menstrual cycle characteristics by tertile of leptin within each cycle. Unadjusted generalized linear models were used to assess differences in the proportion of anovulatory cycles by tertile of leptin within each cycle.


Linear mixed models with random intercepts were used to evaluate the association between time-varying leptin and reproductive hormones (estradiol, luteal progesterone, ovulatory LH, FSH, and testosterone). In these models, hormone levels varied over the cycle within women, which allowed for the estimation of the average association between leptin and reproductive hormones rather than the association within women who were categorized by average leptin level. Models were adjusted for (1) age and (2) age and percent body fat. For the 11 women who had no duel energy x-ray absorptiometry scans, percent body fat was estimated with the following internally derived linear regression formula: percent body fat = 1.7 + BMI × 1.2. Results of the models were presented as average percent change in nontransformed reproductive hormone values per 10% increase in nontransformed leptin value with the following formula: ([{1.1 ˆ β} – 1] × 100%). Because leaner women may be more sensitized to the effects of leptin, we conducted a secondary analysis by limiting our regression models to records from women with a percent body fat <30% (50th percentile).


To account for the possibility that time-varying confounders could be both causes and consequences of leptin levels, we used marginal structural models with inverse probability weights to adjust for these confounders. These models were estimated with the use of weighted linear mixed models; stabilized inverse probability weights were constructed with concurrent MET-hours per week and caloric intake and previous reproductive hormone measurements (estradiol, progesterone, FSH, LH). Confounders that were included in the weight models were based on a directed acyclic graph approach. Model weights were the cumulative product of all previous time-points’ weights for each woman and had a mean value of 1.0 (range, 0.01–16.9).


Linear mixed models were used to estimate leptin levels across the menstrual cycle in ovulatory and anovulatory cycles, with adjustment for age and percent body fat. The odds of sporadic anovulation were estimated with the use of average leptin levels from only the first one-half of the menstrual cycle to preserve the temporal ordering of the exposure-outcome relationship. Leptin on the day of the expected LH surge was also examined in relation to sporadic anovulation. We used generalized linear models to estimate the odds ratio (OR), adjusting for age and percent body fat. In addition, we explored the relationship between leptin and sporadic anovulation nonparametrically with restricted cubic splines.


All analyses accounted for multiple imputations and were performed with SAS software (version 9.3; SAS Institute, Cary, NC).




Results


Study population


Study participants had a mean age of 27.3 ± 8.2 (SD) years, a BMI of 24.1 ± 3.9 kg/m 2 , and a percent body fat of 29.5 ± 6.0 ( Table 1 ). BMI and percent body fat increased with leptin tertile ( P < .01, respectively); lower educational attainment was associated with higher leptin level ( P = .01); however, no other significant differences were observed.



Table 1

Participant characteristics by tertile of average leptin measured across ≤2 menstrual cycles (n = 259 women)

















































































































































































































Characteristic All (n = 259 a ) Tertile of average leptin across ≤2 menstrual cycles P value b
First: <13.7 ng/mL (n = 85) Second: 13.7-28.2 ng/mL (n = 85) Third: ≥28.2 ng/mL (n = 89)
Age, y c 27.3 ± 8.2 (18, 44) 27.8 ± 8 (18, 43) 26.9 ± 8.8 (18, 44) 27.2 ± 8 (18, 44) .78
Body mass index, kg/m 2c 24.1 ± 3.9 (16.1, 35.0) 21.2 ± 2 (16.1, 27.3) 23.6 ± 2.6 (18.5, 30.2) 27.3 ± 3.9 (18.4, 35.0) < .0001
Percent body fat c , d 29.5 ± 6.0 (15.1, 45.2) 23.7 ± 3.4 (15.8, 33.7) 29.6 ± 3.6 (15.1, 37.6) 35.1 ± 4.1 (24.8, 45.2) < .0001
Race, n (%) .13
White 154 (59.5) 56 (65.9) 54 (63.5) 44 (49.4)
African American 51 (19.7) 13 (15.3) 13 (15.3) 25 (28.1)
Other race 54 (20.8) 16 (18.8) 18 (21.2) 20 (22.5)
Education: highest grade completed, n (%) .01
Post-secondary 226 (87.3) 79 (92.9) 77 (90.6) 70 (78.7)
≤High school 33 (12.7) 6 (7.1) 8 (9.4) 19 (21.3)
Marital status, n (%) .85
Single/divorced 193 (74.5) 62 (72.9) 65 (76.5) 66 (74.2)
Married/living as married 66 (25.5) 23 (27.1) 20 (23.5) 23 (25.8)
Nulliparous, n (%) 187 (74) 57 (67.1) 65 (76.5) 65 (73.0) .51
Physical activity at baseline, n (%) 1.0
Low 25 (9.7) 8 (9.4) 8 (9.4) 9 (10.1)
Moderate 92 (35.5) 30 (35.3) 31 (36.5) 31 (34.8)
High 142 (54.8) 47 (55.3) 46 (54.1) 49 (55.1)
Smoking, n (%) .44
Nonsmoker 249 (96.1) 80 (94.1) 82 (96.5) 87 (97.8)
Current smoker 10 (3.9) 5 (5.9) 3 (3.5) 2 (2.2)
≥1 anovulatory cycle, n (%) e 35 (13.5) 15 (17.6) 8 (9.4) 12 (13.5) .30
Perceived stress score c , f 20.2 ± 6.8 (3, 44) 19.8 ± 6.2 (6, 34) 19.7 ± 6.2 (7, 38) 21.0 ± 7.9 (3, 44) .34
Dietary intake c , g
Total caloric intake, kcal 1607± 354 (793, 2686) 1626 ± 364 (793, 2622) 1611 ± 359 (858, 2686) 1587 ± 344 (821, 2667) .76
Total fat, g 62.1 ± 18.5 (18.5, 133.9) 62.9 ± 16.4 (28.6, 113.4) 62.1 ± 19.8 (26.1, 124.1) 61.5 ± 19.4 (18.5, 133.9) .88
Total carbohydrate, g 201.1 ± 49.0 (75.7, 330.3) 202.9 ± 52.9 (92.5, 325.3) 200.6 ± 47.0 (94.2, 330.3) 199.9 ± 47.4 (75.7, 312.4) .91
Total protein, g 62.2 ± 15.6 (26.3, 132.1) 63.2 ± 17.0 (28.5, 132.1) 63.0 ± 14.7 (37.6, 108.8) 60.4 ± 15.1 (26.3, 100.1) .41

Ahrens. Serum leptin and the menstrual cycle. Am J Obstet Gynecol 2014 .

a Two hundred fifty women were observed for 2 cycles, and 9 women were observed for 1 cycle, for a total of 509 cycles


b Two-sided probability values for continuous variables were calculated with the use of analysis of variance, and categoric variables were assessed with the Fisher exact test


c Data are given as mean ± SD (minimum, maximum)


d 248 participants were assessed for percent body fat at end of study


e Twenty-eight women in the study had only 1 anovulatory cycle, and 7 women in the study had 2 anovulatory cycles


f Measured at baseline


g Averaged over ≤8 24-hour dietary recalls that were conducted during the study.



Leptin


The geometric mean of average leptin levels within-women over the study period was 19.7 ng/mL (95% confidence interval [CI], 18.1–21.4) and varied across the menstrual cycle ( P < .01). Leptin concentrations increased over the menstrual cycle (from 16.7 ng/mL [95% CI, 15.3–18.2] during menses to 20.4 ng/mL [95% CI, 18.7–22.3] during the late luteal phase [ P < .01]), with a mid-cycle peak at the time of the LH surge (21.7 ng/mL [95% CI, 19.9–23.7]; P < .01 for comparisons with late follicular and ovulation, respectively). In the original data before realignment, the mid-peak (20.5 ± 2.0 ng/mL) was discernible, but only statistically different from the late follicular phase ( P < .03 and .65 for comparisons with late follicular and ovulation, respectively).


Menstrual cycle characteristics


Menses length differed by tertile of average leptin within each cycle ( P = .01), with the shortest menses length observed for cycles within the highest leptin tertile ( Table 2 ). This relationship was attenuated after post-hoc adjustment for percent body fat ( P = .09; data not shown). No other differences in menstrual cycle characteristics were notable, including the proportion of cycles that were anovulatory (overall, 42 of 509; 8.3%).



Table 2

Menstrual cycle parameters by tertile of average leptin levels within each cycle a








































































































Characteristics Total cycles Tertile of average leptin within each cycle P value b
First: <13.6 ng/mL (n = 167) Second: 13.6-28.6 ng/mL (n = 168) Third: ≥28.6 ng/mL (n = 174)
Reproductive hormones c , d
Estradiol, pg/mL 87.3 (83.9–90.9) 83.5 (78.2–89.2) 89.3 (84.0–94.9) 89.3 (83.8–95.2) .25
Progesterone, ng/mL 1.4 (1.4–1.5) 1.4 (1.3–1.5) 1.5 (1.4–1.6) 1.4 (1.3–1.5) .33
Follicle-stimulating hormone, mIU/mL 5.2 (5.0–5.4) 5.7 (5.4–6) 5.1 (4.8–5.3) 4.9 (4.6–5.2) < .01
Luteinizing hormone, ng/mL 6.4 (6.2–6.7) 6.4 (6.0–6.9) 6.5 (6.1–6.9) 6.3 (5.9–6.7) .63
Testosterone, ng/dL 27.8 (26.7–29.0) 26.8 (25.3–28.3) 28.1 (26.8–29.5) 28.6 (27.1–30.2) .15
Menstrual cycle characteristics d
Menstrual cycle length, d e 28.8 (28.4–29.3) 28.4 (27.6–29.1) 28.9 (28.2–29.6) 29.3 (28.6–30.0) .24
Follicular phase length, d f 15.3 (14.9–15.8) 15.1 (14.4–15.9) 15.4 (14.6–16.1) 15.4 (14.7–16.1) .87
Luteal phase length, d f 13.9 (13.7–14.1) 13.6 (13.1–14) 13.9 (13.5–14.3) 14.3 (13.9–14.7) .09
Menses length, d g 5.4 (5.2–5.5) 5.5 (5.3–5.8) 5.6 (5.3–5.8) 5.0 (4.8–5.3) .01
Total blood loss, g h 43.8 (38.9–49.3) 46.1 (38.2–55.7) 46.9 (39.4–55.8) 38.8 (32.3–46.7) .24
Anovulatory cycles, n (%) I , j 42 (8.3%) 18 (10.8%) 10 (6.0%) 14 (8.1%) .34

Ahrens. Serum leptin and the menstrual cycle. Am J Obstet Gynecol 2014 .

a Two hundred fifty women were observed for 2 cycles; 9 women were observed for 1 cycle


b Continuous measures were assessed by type III effects from unadjusted linear mixed models; anovulatory cycle value was assessed by type III effects from unadjusted generalized linear regression


c Geometric mean was calculated within-women with the use of ≤8 measurements over the cycle; testing was performed on log hormone mean then transformed by exponentiation for Table display


d Data are given as mean (95% CI)


e Available for 476 cycles


f Among ovulatory cycles, follicular phase length available for 467 women; luteal phase length available for 443 women


g Available for 497 cycles


h Available for 467 cycles; geometric mean was calculated within-women with ≤2 measurements over the study; testing performed on log total blood loss mean then transformed by exponentiation for table display


i Defined as progesterone concentration <5 ng/mL and no luteinizing hormone peak detected in serum during the later cycle visits


j Percentage of the column.



Reproductive hormones


Average FSH concentrations differed by tertile of average leptin within each cycle ( P < .01), but the other reproductive hormones were similar among tertiles ( Table 2 ). In the time-varying analysis, a 10% increase in leptin across the menstrual cycle was associated with increased estradiol (2.2%; 95% CI, 1.5–3.0), luteal progesterone (2.1%; 95% CI, 0.5–3.7), ovulatory LH (1.2%; 95% CI, 0–2.3), and testosterone (0.6%; 95% CI, 0.3–0.9), and decreased FSH (–0.7%; 95% CI, –1.1 to –0.4; Table 3 ). Adjustment for time-varying physical activity, caloric intake, and other hormone concentrations had little impact on the effect estimates, which indicated that confounding by these factors was minimal. Estimates of the association between leptin and reproductive hormones in women with <30% body fat were similar (±20%) to those found overall, with the exception of ovulatory LH (which increased by 22% from 1.4% [95% CI, 0.5–2.3] to 1.7% [95% CI, 0.4–3.0]) and FSH (which decreased by 26% from –0.7% [95% CI, –1.0 to –0.3] to –0.5% [95% CI, –1.0 to 0]).


May 11, 2017 | Posted by in GYNECOLOGY | Comments Off on Serum leptin levels and reproductive function during the menstrual cycle

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