Jessicah S. Collins, Jennifer P. Beller, Christine Burt Solorzano, James T. Patrie, R. Jeffrey Chang, John C. Marshall, Christopher R.

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1 Supplemental Materials for manuscript entitled Blunted Day-Night Changes in Luteinizing Hormone Pulse Frequency in Girls with Obesity: the Potential Role of Hyperandrogenemia Jessicah S. Collins, Jennifer P. Beller, Christine Burt Solorzano, James T. Patrie, R. Jeffrey Chang, John C. Marshall, Christopher R. McCartney Table of Contents 1. Supplemental analyses: Day-to-night changes in mean LH and mean FSH pp. 2-5 Supplemental Figures 1 and 2 pp Supplemental analyses: Day-to-night changes in LH IPI as a function of menstrual status pp. 6-9 Supplemental Figures 3 and 4 pp

2 Supplemental analyses: Day-to-night changes in mean LH and mean FSH Previous reports have suggested that early pubertal girls with obesity or overweight demonstrate blunted nocturnal increases in gonadotropin secretion (1-3). To provide additional assessments of this phenomenon in an expanded data set, we analyzed day-to-night changes in mean LH and mean FSH. Subjects All subjects described in the main text were included in the following analyses. Statistical Analysis Mean LH and FSH concentrations during the h and 23-7 h time blocks were designated as daytime and nighttime values, respectively. ANOVA was used to assess the effects of menarcheal and obesity statuses on day-to-night changes in mean LH and mean FSH. We hypothesized that such changes are blunted in girls with obesity. Results are expressed as mean differences (nighttime values minus daytime values) with Bonferroni-corrected lower and upper 95% confidence limits. A twosided p-value.5 was utilized as the null hypothesis rejection rule. Correction for multiple comparisons was performed using the Bonferroni method; only corrected p-values are reported. Results Nonobese premenarcheal girls exhibited a significant overnight increase in mean LH (+1.72 IU/liter [+.81, +2.63]; p <.1) (Supplemental Figure 1). Obese counterparts demonstrated a nonsignificant trend in this regard (+.79 IU/liter [.23, +1.81]). Between-group differences were not statistically significant. Mean LH did not change overnight in nonobese girls (.4 IU/liter [.88, +.81]). Obese girls exhibited a non-significant overnight decrease in mean LH (.69 IU/liter [ 1.66, +.28]); the between-group comparison was not significant. Premenarcheal nonobese girls exhibited a significant overnight increase in mean FSH (+.83 IU/liter [+.39, +1.27], p <.1), but their obese counterparts did not (+.36 IU/liter [.12, +.85]) (Supplemental Figure 2). Between-group differences were not significant. Mean FSH did not change overnight in either nonobese girls (.8 IU/liter [.31, +.16]) or obese girls (.19 IU/liter [.46, +.9]); the between-group comparison was not significant. Discussion A recent study suggested that, compared to normal weight controls, overweight early pubertal girls had blunted overnight increases in both mean LH and mean FSH, although only the former was statistically demonstrable (3). Our current results support these findings. Taken together, we believe that these data provide robust evidence that early pubertal obese girls exhibit blunted sleep-related increases in serum gonadotropin concentrations. Although the recent study by Rosenfield and colleagues suggest that blunted overnight changes in mean LH relate primarily to blunted increases in LH pulse amplitude (3), our data suggest that blunted increases in LH pulse play a role as well (see primary text). We did not observe significant overnight changes in mean gonadotropin concentrations in either group. Although LH pulse decreases overnight in subjects, LH pulse amplitude increases at the same time (see primary text); this reciprocal relationship between LH pulse and LH pulse amplitude likely accounts for the absence of clear overnight changes in mean LH. Importantly, this reciprocal relationship is not a new finding. In a study of GnRH-deficient men receiving fixed doses of GnRH at varying intervals, LH pulse amplitude varied inversely with the preceding LH interpulse interval (4); this is compatible with the idea that longer intervals between GnRH pulses allow increased accumulation of LH to be released with the subsequent GnRH pulse. It is important to note that increased GnRH release per pulse may also contribute to overnight increases in LH pulse amplitude in subjects, although our data do not allow us to address this issue specifically. 2

3 References 1. McCartney CR, Prendergast KA, Blank SK, Helm KD, Chhabra S, Marshall JC 29 Maturation of luteinizing hormone (gonadotropin-releasing hormone) secretion across puberty: evidence for altered regulation in obese peripubertal girls. The Journal of Clinical Endocrinology and Metabolism 94: Bordini B, Littlejohn E, Rosenfield RL 29 Blunted sleep-related luteinizing hormone rise in healthy premenarcheal pubertal girls with elevated body mass index. The Journal of Clinical Endocrinology and Metabolism 94: Rosenfield RL, Bordini B, Yu C 213 Comparison of Detection of Normal Puberty in Girls by a Hormonal Sleep Test and a Gonadotropin-Releasing Hormone Agonist Test. The Journal of Clinical Endocrinology and Metabolism 98: O'Dea LS, Finkelstein JS, Schoenfeld DA, Butler JP, Crowley WF, Jr Interpulse interval of GnRH stimulation independently modulates LH secretion. The American Journal of Physiology 256:E

4 A 2 premenarcheal nonobese obese nonobese obese Mean LH (IU/liter) B Day-to-night change in mean LH (IU/liter) day night day night day night day night * a * -2 nonobese obese nonobese obese premenarcheal Supplemental Figure 1. Day-to-night changes in mean LH in peripubertal girls partitioned by menarcheal and obesity statuses. (A) Daytime (19-23 h) and nighttime (23-7 h) mean LH, with individual subject data shown as open circles and means shown as solid circles. (B) Absolute day-to-night changes in mean LH with Bonferroni-adjusted 95% confidence limits. a = Bonferroni-corrected p <.5 for day-to-night change within a group; * = Bonferroni-corrected p <.5 for comparison of day-to-night changes between groups. 4

5 A 12 premenarcheal nonobese obese nonobese obese 1 Mean FSH (IU/liter) day night day night day night day night B Day-to-night change in mean FSH (IU/liter) * a * -1. nonobese obese nonobese obese premenarcheal Supplemental Figure 2. Day-to-night changes in mean FSH in peripubertal girls partitioned by menarcheal and obesity statuses. (A) Daytime (19-23 h) and nighttime (23-7 h) mean FSH, with individual subject data shown as open circles and means shown as solid circles. (B) Absolute day-to-night changes in mean FSH with Bonferroni-adjusted 95% confidence limits. a = Bonferroni-corrected p <.5 for day-to-night change within a group; * = Bonferroni-corrected p <.5 for comparison of day-to-night changes between groups. 5

6 Supplemental analyses: Day-to-night changes in LH IPI as a function of time since most recent menses Sleep-related slowing of LH (GnRH) pulse during the early follicular phase is well described (1-7). Studies also suggest that late follicular LH pulse in women slows by 2-3% during sleep (8-1). Of interest, some studies suggest that the gradual increase in 24-h LH across the follicular phase is primarily related to a gradual increase in sleep-related but not waking LH pulse (i.e., a gradual reduction in nocturnal LH slowing) (2, 5). It is possible that an increase in sleep-associated LH pulse across the follicular phase and hence the less marked overnight slowing of LH pulse in the late follicular phase compared to the early follicular phase reflects a gradual waning of progesterone s effect to slow (nocturnal) LH pulse (11). Anovulatory women with PCOS do not appear to exhibit nocturnal decreases in LH pulse (12), and length of time since the last luteal phase may help explain this finding. Since some subjects in the current study were evaluated on cycle days 8-1 and others were evaluated at least 6 days after most recent menses, length of time since the most recent luteal phase (with cycle day being a surrogate marker) could be a potential confounder in our analysis. To assess this possibility, we performed additional analyses in subjects. Subjects The majority of subjects described in the main text were included in the following analyses. Specific cycle day was unavailable for two subjects with obesity and hyperandrogenemia (HA) studied at UCSD; these subjects were thus omitted from these analyses. Statistical Analysis Day-to-night change in LH IPI was calculated as nighttime (23-7 h) IPI minus daytime (19-23 h) IPI. Thus, positive and negative values represent day-to-night decreases and increases, respectively, in LH pulse. As an initial analysis, day-to-night IPI changes were compared between 39 girls with recent menses (cycle day 8-1) and 22 girls without recent menses (cycle day 6). Then, day-to-night IPI changes were compared between obese girls with (n = 8) and without (n = 17) recent menses. All pairwise comparisons were performed using the Wilcoxon rank sum test, a non-parametric test that is based on ranks of observations and require no assumptions about the underlying distribution of data. The method of normal approximation was used for pairwise comparisons involving at least 1 observations per group; otherwise, exact Wilcoxon tests were employed. Given that the above analyses suggested that cycle day could be a potential confounder regarding the impact of HA on day-night LH pulse changes in obese girls, day-to-night changes in LH IPI were more formally analyzed by way of two-way ANOVA. The dependent variable was day-to-night change in log e (LH IPI), and the two ANOVA factors were HA status and menstrual status (i.e., whether or not the subject had experienced recent menses). This allowed a comparison of daynight LH pulse changes among 4 obese subgroups: (1) non-ha with recent menses (n = 5), (2) HA with recent menses (n = 3), (3) non-ha without recent menses (n = 4), (4) HA without recent menses (n = 13). Lastly, multiple linear regression was performed in subjects to independently assess predictors of (a) daytime (19-23 h) LH IPI (model 1) and (b) nighttime (23-7 h) LH IPI (model 2). For these models, predictor variables included age, BMI z-score, free testosterone concentration, and cycle day. The regression models were estimated via ordinary least squares, and restricted-cubic-spline functions of the predictor variables were utilized to assess non-linear trends. Conventional F-tests were employed to test for predictor variable versus outcome variable association. For all tests, a two-sided p-value.5 was utilized as the null hypothesis rejection rule. Adjustment for multiple pairwise comparisons was performed as necessary using the Bonferroni method; only corrected p-values are reported. 6

7 Results Wilcoxon rank sum tests When considering all subjects, subjects with recent menses had more marked day-to-night decreases in LH pulse compared to subjects without recent menses (mean ± standard deviation [median] IPI increase of 42.6 ± 37.8 [31.] vs. 7.3 ± 21.9 [1.] minutes; p <.1) (Supplemental Figure 3A). However, those without recent menses were also more obese (BMI z-scores 2.1 ±.5 [2.2] vs..9 ±.9 [1.], p <.1) and had 2.7-fold higher free testosterone concentrations (42.1 ± 22.8 [43.] vs ± 13. [1.] pmol/liter; p <.1). When considering only obese subjects, those with and without recent menses did not demonstrate a statistically significant difference of day-to-night decrease in LH pulse (IPI increases of 26.4 ± 41. [11.] vs. 8.1 ± 23.3 [8.8] minutes, respectively; p = 1.) (Supplemental Figure 3A). Although BMI z-scores were similar (2.2 ±.3 [2.2] vs. 2.3 ±.3 [2.4]), free testosterone concentrations were 8% higher in those without recent menses (26.5 ± 17.8 [19.6] vs ± 22.6 [46.3] pmol/liter; p =.48). Although the above analysis suggested that cycle day may be an important predictor of day-tonight decrease in LH pulse, it is of interest that nearly identical results were obtained when comparing day-to-night changes in LH pulse between subjects with and without HA (p <.1 by the Wilcoxon rank sum test) (Supplemental Figure 3B). This is not particularly surprising given that oligomenorrhea and HA tended to coexist among our cohort of obese girls; among such girls, the Spearman rank (non-parametric) correlation between cycle day and free testosterone concentration was high (r =.61, p =.1). Two-way ANOVA The non-ha subgroup with recent menses exhibited a day-to-night decrease in LH pulse (IPI change: +33%, 95% CI [+7, +51%]; p =.18), but day-night LH pulse changes were not demonstrable in any other subgroup (Supplemental Figure 4). There was no association between day-to-night changes in LH pulse and either HA status (p =.492) or menstrual status (p =.663). Multiple linear regression analyses Multiple linear regression model 1 was not predictive of daytime LH IPI (p =.133). Free testosterone appeared to be a unique predictor of daytime LH IPI (p =.34), but none of the other variables were (BMI z-score, p =.42; age, p =.568; cycle day, p =.727). Multiple linear regression model 2 was predictive of nighttime LH IPI (p =.7, adjusted R 2 =.2), although none of the independent variables were unique predictors of nighttime LH IPI (free testosterone, p =.285; age, p =.33; BMI z-score, p =.499; cycle day, p =.56). Discussion The above supplemental analyses suggest that cycle day may be an important predictor of nocturnal LH pulse slowing among subjects. Thus, although our data suggest that day-to-night changes in LH pulse are altered in obese girls, firm conclusions regarding whether this finding relates to HA vs. menstrual status (possibly remoteness from progesterone exposure) vs. some other factor related to obesity are difficult because these factors frequently coexisted in our cohort of girls. However, these supplementary analyses do not provide evidence that the presence or absence of recent menses is a more important determinant of nocturnal LH pulse slowing compared to HA. For example, among obese subjects with HA, day-night changes appeared to be identical between those with and without recent menses (Supplemental Figure 4). Also of interest, among obese girls with recent menses, our data suggest the possibility of less overnight LH pulse slowing in girls with HA compared to those 7

8 without. A similar consideration pertains to girls without recent menses, although apparent differences were less prominent. Importantly, our analyses were likely inadequately powered in this regard, preventing us from making firm conclusions. One may hypothesize that the degree of nocturnal LH pulse slowing during the follicular phase is partly determined by the length of time since most recent exposure to luteal progesterone levels. This notion could help explain why anovulatory women with PCOS do not exhibit nocturnal decreases in LH pulse (12). A study by Taylor and colleagues (13) also provides data that are consistent with this idea: compared to anovulatory women with PCOS, recently post-ovulatory women with PCOS had significantly lower pool LH, lower pool LH-to-FSH ratio, and fewer LH pulses over 24-h (8 vs. 18 pulses). However, nocturnal changes in LH secretion were not assessed in that particular study. Importantly, when assessing the effects of ovulatory status on nocturnal LH pulse slowing, testosterone concentrations represent an important potential confounder. While normal women exhibit nocturnal slowing of LH pulse during the follicular phase and anovulatory women with PCOS may not, androgen concentrations are also different between normal follicular controls and women with PCOS. Similarly, in the aforementioned study by Taylor et al (13), post-ovulatory women with PCOS had significantly lower testosterone concentrations compared to anovulatory women with PCOS (.8 vs. 1.3 ng/ml). Lastly, among our subjects, cycle day (ovulatory status) correlated strongly with free testosterone concentrations (HA status). Thus, we were unable to separate the influence of these two characteristics in a reliable fashion. In conclusion, ovulatory status (recently post-ovulatory vs. anovulatory) may be an important predictor of nocturnal LH pulse slowing among obese subjects and is thus a potential confounder in our primary analysis. However, we are unaware of data that clearly confirms this hypothesis. We propose that this is an important topic for future research. References 1. Soules MR, Steiner RA, Cohen NL, Bremner WJ, Clifton DK 1985 Nocturnal slowing of pulsatile luteinizing hormone secretion in women during the follicular phase of the menstrual cycle. The Journal of Clinical Endocrinology and Metabolism 61: Filicori M, Santoro N, Merriam GR, Crowley WF, Jr Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle. The Journal of Clinical Endocrinology and Metabolism 62: Rossmanith WG, Yen SS 1987 Sleep-associated decrease in luteinizing hormone pulse during the early follicular phase of the menstrual cycle: evidence for an opioidergic mechanism. The Journal of Clinical Endocrinology and Metabolism 65: Loucks AB, Mortola JF, Girton L, Yen SS 1989 Alterations in the hypothalamic-pituitaryovarian and the hypothalamic-pituitary-adrenal axes in athletic women. The Journal of Clinical Endocrinology and Metabolism 68: Rossmanith WG, Liu CH, Laughlin GA, Mortola JF, Suh BY, Yen SS 199 Relative changes in LH pulsatility during the menstrual cycle: using data from hypogonadal women as a reference point. Clinical Endocrinology 32: Rossmanith WG, Lauritzen C 1991 The luteinizing hormone pulsatile secretion: diurnal excursions in normally cycling and postmenopausal women. Gynecological Endocrinology 5: Hall JE, Sullivan JP, Richardson GS 25 Brief wake episodes modulate sleep-inhibited luteinizing hormone secretion in the early follicular phase. The Journal of Clinical Endocrinology and Metabolism 9: Loucks AB, Heath EM 1994 Dietary restriction reduces luteinizing hormone (LH) pulse during waking hours and increases LH pulse amplitude during sleep in young menstruating women. The Journal of Clinical Endocrinology and Metabolism 78:

9 9. Loucks AB, Thuma JR 23 Luteinizing hormone pulsatility is disrupted at a threshold of energy availability in regularly menstruating women. The Journal of Clinical Endocrinology and Metabolism 88: McCartney CR, Blank SK, Marshall JC 27 Progesterone acutely increases LH pulse amplitude but does not acutely influence nocturnal LH pulse slowing during the late follicular phase in women. American Journal of Physiology Endocrinology and Metabolism 292:E McCartney CR, Gingrich MB, Hu Y, Evans WS, Marshall JC 22 Hypothalamic regulation of cyclic ovulation: evidence that the increase in gonadotropin-releasing hormone pulse during the follicular phase reflects the gradual loss of the restraining effects of progesterone. The Journal of Clinical Endocrinology and Metabolism 87: Waldstreicher J, Santoro NF, Hall JE, Filicori M, Crowley WF, Jr Hyperfunction of the hypothalamic-pituitary axis in women with polycystic ovarian disease: indirect evidence for partial gonadotroph desensitization. The Journal of Clinical Endocrinology and Metabolism 66: Taylor AE, McCourt B, Martin KA, Anderson EJ, Adams JM, Schoenfeld D, Hall JE 1997 Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. The Journal of Clinical Endocrinology and Metabolism 82:

10 A 16 all subjects only obese subjects all subjects * * B 16 only obese subjects Day-to-night change in IPI (minutes) decrease increase decrease increase -6 with recent menses without recent menses with recent menses without recent menses -6 with HA without HA with HA without HA Supplemental Figure 3. (A) Day-to-night changes in LH interpulse interval (IPI) in subjects stratified by menstrual status. (B) Day-to-night changes in LH IPI in subjects stratified by hyperandrogenemia (HA) status. Day-to-night IPI change was calculated as nighttime (23-7 h) IPI minus daytime (19-23 h) IPI. Thus, positive and negative values represent day-to-night decreases and increases, respectively, in LH pulse. * = Bonferroni-corrected p <.1 by Wilcoxon rank sum test. 1

11 75 Percent day-to-night change in GM LH IPI decrease increase a -5 non-ha HA non-ha HA obese with recent menses obese without recent menses Supplemental Figure 4. Day-to-night changes in LH pulse in girls partitioned by menstrual status (presence or absence of recent menses) and hyperandrogenemia (HA) status. Changes in LH pulse are expressed as percent change in geometric mean (GM) interpulse interval (IPI) with Bonferroni-adjusted 95% confidence limits. Day-to-night increases in GM IPI represent decreases in pulse and vice versa. a = Bonferroni-corrected p <.5 for day-to-night change within a group. 11

Progesterone administration does not acutely alter LH pulse secretion in the mid-follicular phase in women

ORIGINAL RESEARCH Physiological Reports ISSN 51-817X Progesterone administration does not acutely alter LH pulse secretion in the mid-follicular phase in women Su Hee Kim 1,, Christine M. Burt Solorzano,3