menstrual dysfunction in girls from the treatment options ... · conclusions: menstrual dysfunction...

Menstrual Dysfunction in Girls from the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) Study

Megan M. Kelsey, MD, MS, Barbara H. Braffett, PhD, Mitchell E. Geffner, M.D., Lynne L Levitsky, MD, Sonia Caprio, MD, Siripoom V. McKay, MD, Rachana Shah, MD, Jennifer E. Sprague, MD, PhD, Silva A. Arslanian, for the TODAY Study Group

The Journal of Clinical Endocrinology & Metabolism Endocrine Society Submitted: January 17, 2018 Accepted: April 02, 2018 First Online: April 24, 2018

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The Journal of Clinical Endocrinology & Metabolism; Copyright 2018 DOI: 10.1210/jc.2018-00132

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Menstrual Dysfunction in Girls with T2D

Menstrual Dysfunction in Girls from the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) Study

Megan M. Kelsey, MD, MS, Barbara H. Braffett, PhD, Mitchell E. Geffner, M.D., Lynne L Levitsky, MD, Sonia Caprio, MD, Siripoom V. McKay, MD, Rachana Shah, MD, Jennifer E. Sprague, MD, PhD, Silva A. Arslanian, for the TODAY Study Group

Department of Pediatrics, University of Colorado School of Medicine

Biostatistics Center, George Washington University

The Saban Research Center, Children’s Hospital Los Angeles, Keck School of Medicine of USC

Division of Pediatric Endocrinology, Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School

Department of Pediatric Endocrinology, Yale School of Medicine

Division of Pediatric Endocrinology, Baylor College of Medicine, Texas Children's Hospital

Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia; Philadelphia, PA

Department of Pediatrics, Washington University in St. Louis

University of Pittsburgh, Children’s Hospital of Pittsburgh of UPMC Received 17 January 2018. Accepted 02 April 2018.

Context: Little is known about reproductive function in girls with youth-onset type 2 diabetes. Objectives: To characterize girls with irregular menses, and effects of glycemic treatments on menses and sex steroids in the Treatment Options for Type 2 Diabetes in Youth (TODAY) study. Design: Differences in demographic, metabolic, and hormonal characteristics between regular vs. irregular menses groups were tested; treatment group (metformin +/- rosiglitazone, metformin + lifestyle) effect on menses and sex steroids over time in the study was assessed. This is a secondary analysis of TODAY data. Setting: Multi-center study in an academic setting. Patients: TODAY girls not on hormonal contraception and those > 1-year post-menarche were included. Irregular menses was defined as < 3 periods in the prior 6 months. Results: Eligible participants with serum measurement of sex steroids (n=190, mean age 14 years), 21% had irregular menses. Those with irregular vs. regular menses had higher body mass index (BMI) (p=0.001), AST (p=0.001), free androgen index (p=0.0003), total testosterone (p=0.01); and lower sex-hormone binding globulin (SHBG) (p=0.004) and estradiol (p=0.01). Differences remained after adjusting for BMI. There was no treatment group effect on menses or sex steroids at 12 or 24 months, and no association of sex steroids with measures of insulin sensitivity or secretion. Conclusions: Menstrual dysfunction is common in girls with recently diagnosed T2D and associated with alterations in sex steroids, SHBG, and AST, but not with alteration in insulin sensitivity or β-cell function, and did not improve with 2 years of anti-hyperglycemic treatment.

We examined reproductive function in girls with youth-onset T2D. These data suggest that menstrual dysfunction is common in girls with diagnosed T2D and associated with sex steroids, SHBG, and AST.

Introduction:

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Obesity is associated with irregular menses and decreased fertility (1), partly due to its co-occurrence with polycystic ovarian syndrome (PCOS) (2). In adolescents, PCOS is characterized by clinical and/or biochemical evidence of elevated testosterone and oligomenorrhea (2; 3). PCOS is also associated with insulin resistance, particularly in women who are obese, and increased risk for type 2 diabetes in adults (4; 5). Systemic insulin resistance is presumed to result in elevated testosterone through preserved ovarian responsiveness to compensatory hyperinsulinemia, which excessively stimulates ovarian androgen production (6) and suppresses hepatic sex-hormone binding globulin (SHBG) production by the liver (7). A similar pathophysiological schema has been proposed for adolescents with PCOS who have decreased insulin sensitivity and compensatory hyperinsulinemia compared to obese controls (8). Obesity is also thought to be associated with functional hypogonadotropic hypogonadism in some women (9), but this disorder has not been well-characterized. The frequency of menstrual dysfunction among adolescent girls with type 2 diabetes has not been reported. Furthermore, although metformin treatment has been demonstrated to improve glucose tolerance in adolescent girls with PCOS and impaired glucose tolerance (10), little is known about treatment effects in girls with type 2 diabetes and PCOS or other menstrual disorders.

The Treatment Options for Type 2 Diabetes in Youth (TODAY) Study was designed to assess the addition of lifestyle improvement or rosiglitazone to standard metformin therapy on durability of glycemic control. This secondary analysis takes advantage of the largest reported adolescent type 2 diabetes cohort in the world to: (1) assess the frequency of self-reported menstrual dysfunction in girls with recently diagnosed type 2 diabetes, (2) compare metabolic and hormonal characteristics in TODAY girls with and without menstrual dysfunction, (3) assess the effect of treatment on menstrual function, and (4) evaluate associations of sex steroids with estimates of insulin sensitivity, insulin secretion, and disposition index (oDI, β-cell function in relation to insulin sensitivity).

Methods:

Study Cohort The TODAY study was designed to evaluate the effects of three treatment arms (metformin alone, metformin + rosiglitazone, and metformin + lifestyle) on time to failure to maintain glycemic control (HbA1c > 8% for 6 months or inability to wean from temporary insulin started for acute metabolic decompensation). Detailed methods are published elsewhere (11; 12). Briefly, TODAY study participants were identified from 15 participating diabetes centers. Eligibility criteria included: negative diabetes autoantibodies (glutamic acid decarboxylase-65 and tyrosine phosphatase), measurable c-peptide, BMI > 85th percentile, age at entry 10-17 years, and < 2 years’ duration of type 2 diabetes. After an initial screening visit, those who met eligibility criteria participated in a 2-6-month run-in period, which consisted of: mastery of standardized diabetes education, titration of metformin to a maximum dose of 1000 mg twice daily (minimum of 500 mg twice daily), discontinuation of all other oral diabetes medications, and discontinuation of insulin. In order to successfully complete the run-in period, participants were required to maintain an HbA1c of < 8%. Participants were followed for an average of 3.86 years. For the following analyses, we present data from baseline (randomization) and months 12 and 24. Measures performed at the baseline and through follow-up included: anthropometrics (height, weight, and waist circumference), blood pressure, fasting laboratory studies (collected before 10 AM after a 10-14 hour fast), and a 2-hour oral glucose

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tolerance test (OGTT). All participants provided both informed parental consent and minor child assent.

Assessment of menstrual dysfunction Menstrual history was assessed retrospectively by self-report. At baseline (after run-in, and just prior to randomization), the following questions were asked: (1) “Have you had your first period?; (2) If yes, how old were you when your periods began?; and (3) How many periods have you had in the past 6 months?” Regular periods were defined as: YES to ever having a period at baseline, having 5 periods or more in the past 6 months, AND having had the first period at least one year prior to baseline. One year was chosen based on evidence that most girls achieve a regular menstrual cycle within one year after menarche (13). Irregular menses was defined as YES to having a period at least one year prior to baseline AND having 3 periods or fewer in the past 6 months.

At every follow-up visit, the following questions were asked: (1) “Have you had a period since the last visit?” and (2) “If yes, how many have you had?” This information was collected every 2 months during the first year of TODAY and every 3 months thereafter. Therefore, to determine the total number of periods in the 6-month period before the year 1 and year 2 visits, we combined information from visits at months 8, 10, and 12 for year 1 and from visits at months 21 and 24 for year 2.

Assays and calculations Measurements of lipids (total cholesterol, low-density lipoprotein [LDL] cholesterol, high-density lipoprotein [HDL] cholesterol, and triglycerides), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, insulin, c-peptide, HbA1c, and sex steroids/SHBG were performed at a centralized laboratory (Northwest Lipid Research Laboratory, University of Washington [Seattle, WA]) in stored morning serum samples using chemiluminescent immunoassays. The analytical range for testosterone is 10-1600 ng/dL with intra- and inter-assay coefficients of variation (CVs) of 4.4-9.4% and 3.2-5.3%, respectively. The analytical range for SHBG is 1-200 nmol/L with intra- and inter-assay CVs of 3.1-4.9% and 6.6-7.5%, respectively. The analytical range for estradiol is 20-4800 pg/mL with intra- and inter-assay CVs of 7.9-19.9% and 4.7-8.9%, respectively.

Free androgen index was used as an estimate of free testosterone and calculated as follows: 100 * (total testosterone [nmol/L]/SHBG [nmol/L]). Free testosterone was also calculated, assuming normal albumin, using the Vermeulen equation(14). Insulin sensitivity was estimated using 1/fasting insulin (1/IF) (15). Insulin secretion was estimated from 30-minute glucose (G), insulin (I), and C-peptide (C) samples collected during the OGTT using the insulinogenic index (∆I30/∆G30) and C-peptide index (∆C30/∆G30) (16). Oral disposition index (oDI) was used as an estimate of β-cell function in relation to insulin sensitivity from 30-minute samples obtained during an OGTT. Two methods were used to calculate oDI: 1/IF x ∆I30/∆G30

and 1/IF x ∆C30/∆G30 (17).

Statistical analyses Baseline demographic and metabolic characteristics were compared between girls with regular and irregular menses using the Student t-test or Wilcoxon rank-sum test for quantitative variables, and the chi-square test for categorical variables. Additional adjustments were made for waist circumference as a surrogate for adiposity. Separate generalized linear mixed models were used to evaluate the effects of original treatment group assignment on the mean of each sex steroid over repeated time points. The models assumed an unstructured covariance structure. The

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TODAY study visits (baseline and months 12 and 24) were included as a class effect and the interaction between treatment group and visit was evaluated. Separate linear regression models were used to evaluate the association between each of the sex steroid measurements at baseline with markers of insulin resistance and β-cell function. Of note, those who were started on hormonal contraception (n=26 between baseline and month 12 and n=28 between months 12 and 24) during the study were removed from analysis at subsequent visits. This is a secondary exploratory analysis; thus, adjustment for multiple comparisons were not made.

Results:

Frequency of menstrual dysfunction A total of 432 girls from the TODAY study who were not on hormonal contraception in the 6 months prior to randomization; of those, 278 met the remaining inclusion criteria and had adequate menstrual data (see CONSORT diagram, and 190 of those girls with adequate stored serum for measurement of sex steroids and SHBG were included for the remainder of the analyses. At months 12 and 24 a total of 123 and 101 girls, respectively, had adequate stored samples and were not on hormonal contraception. In the final cohort of 190 participants, 151 (79.5%) reported having at least 5 periods in the previous 6 months (regular menses) and 39 (20.5%) had < 3 periods in the previous 6 months (irregular menses). When compared with the girls (n=262) who were not included in the analysis, the final cohort was older (14.2 ± 1.7 vs. 13.4 ± 2.2 years, p<0.0001) and had lower insulin secretion, as measured by insulinogenic index and C-peptide index, but similar disposition index, and slightly lower AST (21.3 ± 9.6 vs. 24.0 ± 11.0, p=0002). They also had higher total and free testosterone and estradiol (data not shown). When compared to those included in the study, those who were excluded due to hormonal contraceptive use were, on average, older (baseline: 14.2±1.7 vs. 15.2±1.6 years, p=0.0308; month 12: 15.0±1.6 vs. 16.2±1.2 years, p=0.0033; month 24: 16.1±1.6 vs. 16.9±1.5 years, p=0.0138). Those who were on hormonal contraceptives were not different than the included cohort in terms of BMI, HbA1c or treatment group at any time point.

Clinical, metabolic, and hormonal characteristics associated with menstrual dysfunction Demographic and metabolic characteristics at baseline are presented in Table 1. TODAY girls with menstrual dysfunction were similar to those with regular menses in terms of age, race/ethnicity, diabetes duration, and diabetes control. However, those with irregular menses showed greater clinical evidence of metabolic dysfunction, as indicated by higher BMI, waist circumference, and liver transaminases. The difference in mean AST remained significant after adjusting for BMI. There were no significant differences in mean insulin sensitivity (1/IF) or oDI between the two groups. The mean insulinogenic index was higher in those with irregular menses, but this difference was not statistically significant after adjusting for BMI. Mean total testosterone and free androgen index were higher and mean SHBG and estradiol lower in girls with menstrual dysfunction; differences remained significant after adjusting for BMI. The majority of girls with irregular menses (79%) and a significant proportion of those with regular menses (58%) had a free androgen index in a range (cut point >6) consistent with previous reports for girls with clinical evidence of hyperandrogenism and women with PCOS (18; 19). The distribution of free androgen index in those with and without irregular menses is shown in the Figure 2. Of note, there was no difference in the frequency of irregular menses at baseline between girls who did not reach primary outcome by the end of TODAY (20% irregular menses) and those who had failure of glycemic control on oral therapy (21% irregular menses), p=0.75.

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There were no significant associations between sex steroids or SHBG and insulin sensitivity, insulin secretion, or oDI estimates at baseline (data not shown).

Effect of treatment group on menstrual irregularity and sex steroids/SHBG There was no significant effect of treatment group on menstrual irregularity between baseline and at 12 and 24 months, data not shown), The frequency of menstrual irregularity did not significantly improve over time (Figure 3). Menstrual frequency was also assessed at 12 and 24 months including the girls who did not have adequate stored samples and results were similar (data not shown). In the overall cohort, there was a significant increase in testosterone (p=0.0095), SHBG (p=0.0017), and estradiol (p=0.001) across treatment visits, but there were no effects of treatment group on sex steroids or SHBG over time (Figure 4).

Discussion:

This is the first report of the frequency of menstrual dysfunction and associated metabolic defects in adolescent girls with type 2 diabetes. The 20.5% frequency of irregular menses in the TODAY girls who are >1 year post-menarche appears to be higher than in the general adolescent population, although reported prevalence of irregular menses in normal adolescents varies widely. One study suggests that 75% of healthy girls achieve a normal adolescent cycle length (21-45 days) within 1 year of menarche and that the frequency increases to at least 80% in the second post-menarchal year (20); however, the proportion of these girls with more strictly defined oligomenorrhea is unknown. Data from two large studies that include a total of 3,358 girls and 307,592 menstrual cycles suggest that <5% of girls have an average cycle length of 3 months or more in the first year after menarche (20; 21). The prevalence rate of menstrual dysfunction in obese non-diabetic girls is not well-studied, though a study of 835 Serbian girls suggests that BMI is higher in those with irregular menses (22). In one smaller study of girls randomly selected from an adolescent medicine clinic, the frequency of irregular periods, defined as <9 menses per year, in obese girls was 46% (19) and another very small study of severely obese girls undergoing bariatric surgery reported a 32% frequency of oligomenorrhea (23). While the frequency of menstrual dysfunction in the TODAY study is much lower than in these other obese populations, there are significant differences in the definitions of irregular menses in these studies and in the populations themselves that make exact comparisons with TODAY difficult. Moreover, 80% of the TODAY girls were already on metformin at the time of screening (Table 1) and all were on a maximized metformin dose at baseline, so that the girls with PCOS would be considered treated at that time. This may have masked some of the menstrual dysfunction that would have been present if the girls had been untreated.

In the TODAY cohort, girls with irregular menses showed some differences in concentrations of circulating sex steroids compared to those with regular menses. Notably, they had significantly higher total testosterone (despite significantly lower SHBG) and free androgen index, and lower estradiol. These differences persisted after adjustment for BMI. Elevated testosterone is a hallmark of the diagnosis of PCOS, a condition that affects 10-20% of the normal adult female population and is commonly associated with obesity and increased risk for type 2 diabetes. However, PCOS can only be diagnosed 2 years after menarche and youth-onset type 2 diabetes is often apparent before a diagnosis of PCOS can be made. As pointed out above, because the standard first-line treatment for type 2 diabetes is also a treatment for PCOS, underlying PCOS may be masked in these youth. These factors make the relationship between PCOS and type 2 diabetes in adolescents particularly challenging to elucidate. Characterization of PCOS in the TODAY cohort is further complicated by the fact that clinical hyperandrogenism

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was not assessed. However, studies in adult (18) and adolescent (19) women suggest that indices of free testosterone, including free androgen index, are excellent markers of clinical hyperandrogenism and irregular menses. Receiver-operating characteristic (ROC) analysis in a study of 120 lean and obese adolescent girls was used to define a free androgen index cut-point of 6.0 to indicate high risk for clinical hyperandrogenism. The proportion of TODAY girls with free androgen index > 6.0 is high both in the regular menses group (58%) and in those with menstrual dysfunction (79%), indicating that many girls with treated diabetes still show biochemical, and, therefore, likely clinical, hyperandrogenism. Thus, while PCOS cannot be clearly defined in the TODAY sample, key features of PCOS are common. It is important to note that a subset of TODAY girls with menstrual dysfunction did not have biochemical hyperandrogenism (20.5%), though this could potentially reflect previous treatment with metformin. Obesity itself has been demonstrated to be associated with menstrual disruption through suppression of gonadotropins (24; 25), which may explain some of menstrual dysfunction seen in TODAY (although gonadotropins were not measured).

The differences in sex steroid levels between those who were included in the analysis vs. those who were excluded are likely reflective of the relative reproductive immaturity of those excluded, as a significant proportion of them were premenarchal or had only recently experienced menarche (n=112), and they were significantly younger than those included. Earlier pubertal status could also explain the higher insulin secretion in the excluded cohort, as insulin secretion is known to increase significantly during puberty in lean and obese youth without diabetes (26; 27).

Based on known effects of obesity on reproductive factors in adults and animal evidence regarding the role of sex steroids in metabolism, we hypothesized that there may be some interaction between sex steroids during adolescence and metabolic function in obese girls with diabetes. However, we did not find any interactions among sex steroids, insulin sensitivity, and β-cell function in our cohort. Evidence from ovariectomized rats (28; 29) and post-menopausal women (30-35) suggests that estrogen may improve insulin sensitivity. However, when estrogen concentrations are physiologically elevated, as in the mid-luteal phase of the menstrual cycle (36), insulin sensitivity worsens. Even less is known about estrogen effects on β-cell function; however, animal evidence suggests that estrogen promotes β-cell survival, insulin synthesis, and insulin secretion (37). Elevated androgens as seen in girls with PCOS may also attenuate the favorable effects of estradiol in obese females during puberty. This body of evidence suggests a complex role for sex hormones in the regulation of insulin action and secretion that needs further study. However, our study is limited by the fact that it does not include measurements prior to puberty that all girls were on metformin at the time of sex steroid measurement, and that sex steroid measurements were not performed by the most sensitive methodology (tandem mass spectrometry/mass spectroscopy). Further longitudinal studies are needed to better characterize the impact of obesity on reproductive hormones and β-cell function during the pubertal transition period and, thus, to better understand potential sex differences in the pathophysiology of youth-onset type 2 diabetes.

Although the TODAY participants with irregular menses were similar to those with regular menses in terms of insulin sensitivity and most metabolic markers, they did have a higher BMI and liver transaminases. Even after adjusting for BMI, AST remained significantly higher in the TODAY girls with menstrual dysfunction. This may specifically represent a link between PCOS and fatty liver disease. One recent study of non-diabetic obese adolescent girls with and without PCOS demonstrated a high frequency of hepatic steatosis, as measured by MRI, in girls with

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PCOS (49%) compared to those without (14%) (38). As new treatments for non-alcoholic fatty liver disease emerge, developing better ways to clinically identify patients at the greatest risk will be important, with menstrual dysfunction being one such marker that can be used in combination with others, such as liver transaminases.

We did not find any added treatment effects of lifestyle or rosiglitazone on the biochemical hyperandrogenism in TODAY participants, who were previously treated with metformin. Studies of adult women with PCOS treated with metformin or thiazolidinediones demonstrate improvements in insulin sensitivity and reductions in total or free testosterone, and improvement in fertility (39-41). Fewer studies have compared the addition of thiazoladinediones to metformin treatment in women with PCOS. One study that compared metformin and rosiglitazone monotherapy reported improvement in androgens, insulin sensitivity, and menstruation in both groups; however, improvements in menstruation were greater in the rosiglitazone group (42). A Chinese study showed a better therapeutic effect of metformin + rosiglitazone compared with metformin alone on menstrual function/fertility, and also demonstrated a reduction in triglycerides, testosterone, and insulin resistance in the metformin + rosiglitazone group (40). In the TODAY cohort, there was no significant improvement in menstrual regularity or free androgen index over time. Furthermore, although metformin + rosiglitazone showed beneficial effects compared to metformin monotherapy in terms of glycemic control, the combination did not have added benefit for menstrual regularity or serum testosterone concentrations. It is important to note that the TODAY participants were already treated with metformin for a minimum of 2 months when the baseline sex steroids and SHBG were measured, which may have somewhat masked the true treatment effects on these measures.

In summary, in girls with recent-onset type 2 diabetes, we found a higher frequency of irregular menses than in previous reports of healthy adolescents; however, it is difficult to quantify how much of this menstrual irregularity is related to obesity alone due to the lack of an obese non-diabetic comparison group. Irregular menses were associated with higher testosterone and AST, and lower estradiol concentrations. This is the first study to examine sex steroids and menstrual health in a large cohort of girls with obesity and recent youth-onset type 2 diabetes treated with metformin +/- rosiglitazone. Further studies are needed to better understand the role of reproductive hormones on sex differences in pathophysiology of type 2 diabetes in youth.

Grant support:

This work was completed with funding from NIDDK/NIH grant numbers U01-DK61212, U01-DK61230, U01-DK61239, U01-DK61242, and U01-DK61254; from the National Center for Research Resources General Clinical Research Centers Program grant numbers M01-RR00036 (Washington University School of Medicine), M01-RR00043-45 (Childrens Hospital Los Angeles), M01-RR00069 (University of Colorado Denver), M01-RR00084 (Children’s Hospital of Pittsburgh), M01-RR01066 (Massachusetts General Hospital), M01-RR00125 (Yale University), and M01-RR14467 (University of Oklahoma Health Sciences Center); and from the NCRR Clinical and Translational Science Awards grant numbers UL1-RR024134 (Children’s Hospital of Philadelphia), UL1-RR024139 (Yale University), UL1-RR024153 (Children’s Hospital of Pittsburgh), UL1-RR024989 (Case Western Reserve University), UL1-RR024992 (Washington University), UL1-RR025758 (Massachusetts General Hospital), and UL1-RR025780 (University of Colorado Denver).

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National Institute of Diabetes and Digestive and Kidney Diseases http://dx.doi.org/10.13039/100000062, U01-DK61212, U01-DK61230, U01-DK61239, U01-DK61242, and U01-DK61254, Not Applicable

Corresponding Author: Barbara H. Braffett, PhD, 6110 Executive Boulevard Suite 750, Rockville, MD 20852, Phone 301-881-9260, Fax 301-881-3742, [email protected]

Reprint requests: (corresponding author)

ClinicalTrials.gov Identifier: NCT00081328

Disclosure Summary MK is a site principal investigator for Merck and Daiichi-Sankyo. LL is a consultant for Eli Lilly. MG is a clinical trial steering committee member for Daiichi-Sankyo. None of the other authors have anything to disclose.

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12. Zeitler P, Epstein L, Grey M, Hirst K, Kaufman F, Tamborlane W, Wilfley D: Treatment options for type 2 diabetes in adolescents and youth: a study of the comparative efficacy of metformin alone or in combination with rosiglitazone or lifestyle intervention in adolescents with type 2 diabetes. Pediatr Diabetes 2007;8:74-87 13. Legro RS, Lin HM, Demers LM, Lloyd T: Rapid maturation of the reproductive axis during perimenarche independent of body composition. The Journal of clinical endocrinology and metabolism 2000;85:1021-1025 14. Vermeulen A, Verdonck L, Kaufman JM: A critical evaluation of simple methods for the estimation of free testosterone in serum. The Journal of clinical endocrinology and metabolism 1999;84:3666-3672 15. George L, Bacha F, Lee S, Tfayli H, Andreatta E, Arslanian S: Surrogate estimates of insulin sensitivity in obese youth along the spectrum of glucose tolerance from normal to prediabetes to diabetes. The Journal of clinical endocrinology and metabolism 2011;96:2136-2145 16. Uwaifo GI, Fallon EM, Chin J, Elberg J, Parikh SJ, Yanovski JA: Indices of insulin action, disposal, and secretion derived from fasting samples and clamps in normal glucose-tolerant black and white children. Diabetes Care 2002;25:2081-2087 17. Sjaarda LG, Bacha F, Lee S, Tfayli H, Andreatta E, Arslanian S: Oral disposition index in obese youth from normal to prediabetes to diabetes: relationship to clamp disposition index. J Pediatr 2012;161:51-57 18. Bui HN, Sluss PM, Hayes FJ, Blincko S, Knol DL, Blankenstein MA, Heijboer AC: Testosterone, free testosterone, and free androgen index in women: Reference intervals, biological variation, and diagnostic value in polycystic ovary syndrome. Clinica chimica acta; international journal of clinical chemistry 2015;450:227-232 19. Rieder J, Santoro N, Cohen HW, Marantz P, Coupey SM: Body shape and size and insulin resistance as early clinical predictors of hyperandrogenic anovulation in ethnic minority adolescent girls. The Journal of adolescent health : official publication of the Society for Adolescent Medicine 2008;43:115-124 20. Treloar AE, Behn BG, Cowan DW: Analysis of gestational interval. American journal of obstetrics and gynecology 1967;99:34-45 21. Vollman RF: The menstrual cycle. Major Probl Obstet Gynecol 1977;7:1-193 22. Radivojevic T, Anselmi J, Scalas E: Ergodic transition in a simple model of the continuous double auction. PloS one 2014;9:e88095 23. Hillman JB, Miller RJ, Inge TH: Menstrual concerns and intrauterine contraception among adolescent bariatric surgery patients. J Womens Health (Larchmt) 2011;20:533-538 24. Rosenfield RL, Bordini B: Evidence that obesity and androgens have independent and opposing effects on gonadotropin production from puberty to maturity. Brain research 2010;1364:186-197 25. van Hooff MH, Voorhorst FJ, Kaptein MB, Hirasing RA, Koppenaal C, Schoemaker J: Predictive value of menstrual cycle pattern, body mass index, hormone levels and polycystic ovaries at age 15 years for oligo-amenorrhoea at age 18 years. Hum Reprod 2004;19:383-392 26. Goran MI, Gower BA: Longitudinal study on pubertal insulin resistance. Diabetes 2001;50:2444-2450 27. Kelly LA, Lane CJ, Weigensberg MJ, Toledo-Corral CM, Goran MI: Pubertal changes of insulin sensitivity, acute insulin response, and beta-cell function in overweight Latino youth. J Pediatr 2011;158:442-446

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28. Campbell SE, Febraio MA: Effect of the ovarian hormones on GLUT4 expression and contraction-stimulated glucose uptake. American Journal of Physiology: Endocrinology and Metabolism 2002;282:1139-1146 29. Kumagai S, Holmang A, Björntorp P: The effects of oestrogen and progesterone on insulin sensitivity in female rats. Acta Physiologica Scandinavica 1993;149:91-97 30. Cucinelli F, Paparella P, Soranna L, Barini A, Cinque B, Mancuso S, Lanzone A: Differential effect of transdermal estrogen plus progestagen replacement therapy on insulin metabolism in postmenopausal women: relation to their insulinemic secretion. European Journal of Endocrinology 1999;140:215-223 31. Espeland MA, Hogan PE, Fineberg SE, Howard G, Schrott H, Waclawiw MA, Bush TL: Effect of postmenopausal hormone therapy on glucose and insulin concentrations. PEPI Investigators. Postmenopausal Estrogen/Progestin Interventions. Diabetes Care 1998;21:1589-1595 32. Evans EM, Van Pelt RE, Binder EF, Williams DB, Ehsani AA, Kohrt WM: Effects of HRT and exercise training on insulin action, glucose tolerance, and body composition in older women. Journal of Applied Physiology 2001;90:2033-2040 33. Jensen MD, Martin ML, Cryer PE, Roust LR: Effects of estrogen on free fatty acid metabolism in humans. American Journal of Physiology 1994;266:E914-920 34. O'Sullivan AJ, Ho KKY: A comparison of the effects of oral and transdermal estrogen replacement on insulin sensitivity in postmenopausal women. J Clin Endocinol Metab 1995;80:1783-1788 35. Van Pelt RE, Gozansky WS, Schwartz RS, Kohrt WM: Intravenous estrogens increase insulin clearance and action in postmenopausal women. American journal of physiology Endocrinology and metabolism 2003;285:E311-317 36. Pulido JME, Salazar MA: Changes in insulin sensitivity, secretion and glucose effectiveness during menstrual cycle. Archives of Medical Research 1999;30:19-22 37. Mauvais-Jarvis F: Role of Sex Steroids in beta Cell Function, Growth, and Survival. Trends Endocrinol Metab 2016;27:844-855 38. Ohman-Hanson RA, Cree-Green M, Kelsey MM, Bessesen DH, Sharp TA, Pyle L, Pereira RI, Nadeau KJ: Ethnic and Sex Differences in Adiponectin: From Childhood to Adulthood. The Journal of clinical endocrinology and metabolism 2016;101:4808-4815 39. Baillargeon JP, Jakubowicz DJ, Iuorno MJ, Jakubowicz S, Nestler JE: Effects of metformin and rosiglitazone, alone and in combination, in nonobese women with polycystic ovary syndrome and normal indices of insulin sensitivity. Fertil Steril 2004;82:893-902 40. Liao L, Tian YJ, Zhao JJ, Xin Y, Xing HY, Dong JJ: Metformin versus metformin plus rosiglitazone in women with polycystic ovary syndrome. Chinese medical journal 2011;124:714-718 41. Steiner CA, Janez A, Jensterle M, Reisinger K, Forst T, Pfutzner A: Impact of treatment with rosiglitazone or metformin on biomarkers for insulin resistance and metabolic syndrome in patients with polycystic ovary syndrome. Journal of diabetes science and technology 2007;1:211-217 42. Yilmaz M, Biri A, Karakoc A, Toruner F, Bingol B, Cakir N, Tiras B, Ayvaz G, Arslan M: The effects of rosiglitazone and metformin on insulin resistance and serum androgen levels in obese and lean patients with polycystic ovary syndrome. Journal of endocrinological investigation 2005;28:1003-1008

Figure 1: Consort diagram for inclusion in analysis.

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Figure 2: Free Androgen Index at Randomization by Menstrual Status in Female Youth with Type 2 Diabetes in the TODAY study. Legend: Distribution of free androgen index among 39 girls with irregular menses and 151 girls with regular menses at randomization in the TODAY study. There was a high frequency of elevated free androgen index (>6) in both groups, despite treatment with metformin for at least 2 months during the run-in period.

Figure 3: Menstrual Irregularity at Annual Visits. Legend: Proportion of TODAY girls not on hormonal contraception with irregular menses at baseline, 12 months, and 24 months. There was no significant difference in prevalence of irregular menses over time (p=0.10). There was also no significant treatment effect on menstrual irregularity (data not shown, p=0.60)

Figure 4: Mean Sex Steroid Levels at Annual Visits Overall and by Treatment Group (Metformin Only=blue, Metformin + Rosiglitazone =red, Metformin + Lifestyle=green, and Overall=black. Legend: In the overall cohort, there was a significant increase in testosterone (p=0.0095), SHBG (p=0.0017), and estradiol (p=0.001) across treatment visits, but there were no effects of treatment group on sex steroids or SHBG over time

Table 1. Baseline Characteristics of female TODAY study participants (n=190) by menstrual status

Characteristics Regular periods* (n=151) Irregular periods†

(n=39) p-value Adjusted p-

value Age (years) 14.1 ± 1.7 14.3 ± 1.7 0.7150 - Gynecological age (years) 2 (1, 4) 2 (1, 3) 0.3704 Diabetes duration (months) 6 (4, 11) 5 (4, 9) 0.3155 - Race/ethnicity (%) Black non-Hispanic 39.1 25.6 0.1793 - Hispanic 42.4 41.0 White non-Hispanic 15.2 25.6 Other 3.3 7.7 Ever smoked (%) 13.9 12.8 0.8603 - Treatment group (%) Metformin only 36.4 38.5 0.9375 - Metformin + rosiglitazone 29.8 30.8 Metformin + lifestyle 33.8 30.8 Metformin use at screening (%) 80.1 79.5 0.9284 - Physical examination BMI (kg/m2) 34.2 ± 6.8 37.6 ± 7.9 0.0097 - Waist circumference (cm) 106.6 ± 14.6 112.4 ± 15.7 0.0374 - Systolic BP (mm Hg) 111.4 ± 9.9 112.9 ± 9.5 0.4063 - Diastolic BP (mm Hg) 67.2 ± 8.3 67.1 ± 6.5 0.9773 - Lipid/Liver Total cholesterol (mg/dL) 146.2 ± 29.9 144.6 ± 27.2 0.7742 - LDL cholesterol (mg/dL) 83.3 ± 26.3 82.9 ± 23.2 0.9219 - HDL cholesterol (mg/dL) 40.7 ± 9.7 37.9 ± 8.6 0.1064 - Triglycerides (mg/dL) 113.2 ± 81.6 118.9 ± 55.5 0.1757 - ALT (U/L) 25.5 ± 18.1 33.1 ± 21.7 0.0066 0.0695 AST (U/L) 20.3 ± 8.8 25.1 ± 11.6 0.0012 0.0034 Metabolic HbA1c (%) 6.1 ± 0.7 6.1 ± 0.7 0.9132 0.8941 Fasting glucose (mg/dL) 113.2 ± 23.2 109.8 ± 21.1 0.4078 0.4085 Fasting insulin (µU/mL)‡ 28.7 ± 18.9 36.7 ± 28.2 0.1474 0.1870 Fasting c-peptide (ng/mL)‡ 3.6 ± 1.4 4.1 ± 1.5 0.0415 0.3372 Insulin sensitivity [1/IF] (mL/µU)‡ 0.052 ± 0.049 0.043 ± 0.028 0.1263 0.4593 Insulinogenic index [∆I30/∆G30] (µU/mL per mg/dL)‡

1.140 ± 1.018 1.507 ± 1.289 0.0226 0.0777

C-peptide index [∆C30/∆G30] (ng/mL per mg/dL)‡ 0.061 ± 0.052 0.077 ± 0.067 0.0977 0.1871

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oDI [1/IF x ∆I30/∆G30]‡ 0.049 ± 0.055 0.061 ± 0.073 0.2336 0.2579 oDI [1/IF x ∆C30/∆G30]‡ 0.003 ± 0.003 0.003 ± 0.004 0.7976 0.5934 Sex Steroids Free androgen index (free androgen index, no units)‡

10.6 ± 12.1 16.9 ± 14.5 0.0003 0.0019

Free testosterone (nmol/L)‡ 0.035 ± 0.038 0.045 ± 0.023 0.0011 0.0062 Testosterone (ng/mL)‡ 0.39 ± 0.38 0.44 ± 0.20 0.0129 0.0422 SHBG (nmol/L)‡ 20.1 ± 23.1 13.6 ± 10.0 0.0042 0.0124 Estradiol (pg/mL)‡ 70.5 ± 56.4 50.5 ± 34.8 0.0114 0.0021

Gynecological age represents years since menarche. Data are mean ± SD, median (interquartile range), or percent. The p-values are calculated from Student’s t-test or Wilcoxon rank-sum test for continuous variables and from the chi-square test for categorical variables. P-values were also adjusted for waist circumference. P-values <0.05 are bolded. * Defined as YES to ever having a period at baseline and, if yes, having 5 periods or more in the past 6 months. † Defined as YES to ever having a period at baseline and, if yes, having 3 periods or less in the past 6 months. ‡ Variables were log-transformed prior to testing.

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1

452 Girls randomized to the TODAY Study

424 Girls

342 Reported having a

period

278 Girls

54 Reported 0-3 periods

IRREGULAR PERIODS

BASELINE

39 with stored blood samples

MONTH 12


(excluding 3 using contraceptives)

MONTH 24



224 Reported 5+ periods

REGULAR PERIODS

BASELINE

151 With stored blood samples

MONTH 12


(exlcuding 16 using contraceptives)

MONTH 24



64 Excluded from analyses

12 Reported 4 periods

31 Reported menarche had occurred within the past year

21 Incomplete menstrual frequency data

81 Reported NOT ever having a period1 Reported unknown status

28 Excluded from analyses

20 Were using hormonal contraceptives

8 Missing menstrual questionnaire

Figure 1: Consort diagram for inclusion in analysis.

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Figure 2: Free Androgen Index at Randomization by Menstrual Status in Female Youth with Type 2 Diabetes in the TODAY study

Legend: Distribution of free androgen index among 39 girls with irregular menses and 151 girls with regular menses at randomization in the TODAY study. There was a high frequency of elevated free androgen index (>6) in both groups, despite treatment with metformin for at least 2 months during the run-in period.

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Figure 3: Menstrual Irregularity at Annual Visits

79% 85% 89%

21% 15% 11%

0%

20%

40%

60%

80%

100%

0 12 24

Time in TODAY in Months

Irregular

Regular

Legend: Proportion of TODAY girls not on hormonal contraception with irregular menses at baseline, 12 months, and 24 months. There was no significant difference in prevalence of irregular menses over time (p=0.10). There was also no significant treatment effect on menstrual irregularity (data not shown, p=0.60)

n=190 n=101 n=123

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Figure 4: Mean Sex Steroid Levels at Annual Visits Overall and by Treatment Group (Metformin Only=blue, Metformin + Rosiglitazone =red, Metformin + Lifestyle=green, and Overall=black.

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Legend: In the overall cohort, there was a significant increase in testosterone (p=0.0095), SHBG (p=0.0017), and estradiol (p=0.001) across treatment visits, but there were no effects of treatment group on sex steroids or SHBG over time

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menstrual dysfunction in girls from the treatment options ... · conclusions: menstrual dysfunction...

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