{"paper_id":"68c347ea-04a7-46b6-9a6c-61d78d9a5e8e","body_text":"Premature ovarian insufficiency (POI) during adolescence is rare, affecting less than 1 in 10,000 individuals ( 1 , 2 ), and is frequently associated with underlying genetic conditions or prior exposure to chemotherapy. In these situations, estrogen deficiency can be anticipated, and replacement initiated before consequences on bone health arise. Conversely, the diagnosis of idiopathic POI is often delayed due to the assumption that irregular menses are common among adolescents. This delay results in estrogen deficiency during a critical period for skeletal development and can compromise peak bone mass accrual ( 3 ). POI has also been associated with higher rates of depression and anxiety in adult women ( 4 ), but the impact of this diagnosis on the mental health of adolescents is unknown. Estrogen deficiency during menopause may negatively influence cognition and memory ( 5 ), but the impact on brain development during adolescence is unknown.\nOnce POI is diagnosed, treatment approaches vary as there is no consensus on optimal hormone replacement for adolescents ( 6 ). Physiologic estradiol may be more beneficial for bone density compared to combination oral contraceptives ( 7 , 8 ). In this longitudinal study, we compared female adolescents with idiopathic POI, treated with escalating doses of transdermal estrogen (TDE2), to normally menstruating female adolescents matched by age, race, and BMI. The purpose of this pilot study was to assess the clinical response of adolescent females newly diagnosed with POI to gradual titration of TDE2 relative to age-matched peers in terms of bone health, cognitive function, and mental health over a 24-month period.\n\nBetween November 2018 and January 2021, we recruited adolescent (ages 11.0–19.0 years) females diagnosed with idiopathic POI within the last three months from a pediatric referral center. Nine cases, 5 with primary amenorrhea and 4 with secondary amenorrhea, and 9 controls (recruited from an adolescent clinic and volunteer database) with typical age of menstrual onset and cycles occurring every 21–35 days, and matched for age, race, and BMI are included in this report. Exclusion criteria for all participants included a prior diagnosis of a chronic condition affecting bone health or use of medications that impact bone health. Additional exclusion criteria for controls were prior diagnoses of learning or developmental disabilities, anxiety or depression. Baseline characteristics of the study cohort have previously been reported ( 9 ).\nThis study protocol (2018–2375) was reviewed and approved by the Institutional Review Board at Cincinnati Children’s Hospital Medical Center. Written assent and consent were obtained from all participants and their legal guardian, respectively. This study was registered on  clinicaltrials.gov  ( NCT03568708 ).\nFive research visits were conducted (baseline and every 6 months over 2 years) between November 2018 and January 2023. For adolescents with idiopathic POI, weekly transdermal 17β-estradiol patch systems (Mylan Pharmaceuticals Inc. Canonsburg, Pennsylvania, USA) were used to deliver the following estrogen replacement: 0.025 mg/day for 3 months, 0.0375 mg/day for 3 months, 0.05 mg/day for 6 months, 0.075 mg/day for 6 months, 0.1 mg/day for 6 months. Progestin supplementation (10 mg of oral medroxyprogesterone acetate for 12 consecutive days each month) was initiated when participants reported spontaneous vaginal bleeding. Participants were asked to track use of estrogen patches and progestin on monthly calendars (provided) and bring completed calendars to subsequent study visits. Weeks with at least one reported estrogen patch change divided by weeks enrolled in the study was used to estimate compliance with the treatment regimen. As described previously in detail ( 9 ) at baseline and 12 and 24 months, we used validated surveys for adolescents to gather data on depression (Children’s Depression Index-II; CDI-II), anxiety (Screen for Anxiety Related Disorders; SCARED), dietary and lifestyle patterns, and quality of life (Child Health Questionnaire-9; CHQ-9). We also assessed memory (Child and Adolescent Memory Profile; ChAMP) and cognitive processing (NIH Toolbox Cognition Battery).  Table 1  provides an overview of assessments obtained at each study visit.\nAll participants provided a complete medical history. Pubertal staging using Tanner criteria was performed by a pediatric endocrinologist or pediatric and adolescent gynecologist.\nDual energy x-ray absorptiometry (DXA) scans (Hologic Horizon, Hologic Inc., Marlborough MA) were obtained of the lumbar spine (LS), hip and whole body for measurement of areal BMD and Z-scores calculated using age, race, and sex specific reference ranges ( 10 ). We obtained peripheral quantitative computed tomography (pQCT) scans (Stratec XCT 2000, Orthometrix, Inc., White Plains, NY) of the non-dominant radius (3% and 66% sites) and left tibia (3%, 38% and 66% sites). Measurements were acquired with voxel size of 0.4 mm, slice thickness of 2.3 mm, and scan speed of 25 mm/sec, and analyzed with manufacturer software version 6. The 3% sites provided measures of trabecular volumetric BMD (vBMD), whereas the 38% and 66% sites provided measures of cortical vBMD, cortical area and thickness and strength-strain index (SSI), the weighted section modulus to assess resistance to bending forces. A hand wrist radiograph was obtained at baseline on all adolescents with POI for bone age assessment with subsequent x-rays obtained until skeletal maturation was ≥15 years, as this coincides with >99.0% of final adult height.\nAdolescents with POI provided fasting serum samples for measurement of bone metabolism [osteocalcin, bone specific alkaline phosphatase (BSAP), C-telopeptide beta-cross-links (CTX), calcium, phosphorus, 25-hydroxyvitamin D (25-OHD)], cardiometabolic profile [lipid profile, aspartate aminotransferase (AST), alanine aminotransferase (ALT)], and hormonal activity [estradiol, insulin like growth factor 1 (IGF-1)]. Osteocalcin, BSAP, CTX, estradiol, and IGF-1 were obtained every 6 months whereas lipid profile, AST, ALT, 25-hydroxyvitamin D (25-OHD), calcium and phosphorus were obtained every 12 months. Osteocalcin was measured using a quantitative electro-chemiluminescent immunoassay, BSAP and CTX by enzyme-linked immunosorbent assay. 25-OHD, estradiol, and IGF-1 were measured by chemiluminescent assay (ARUP Laboratories, Salt Lake City, Utah, USA).\nDescriptive statistics (means, standard deviations and counts) were generated for each anthropometric and lifestyle variable, as well as mental health outcomes. For each group (POI and Control), initial comparisons between baseline and 24-months were conducted using paired t-tests while between group comparisons at each time point were compared using a two-sample t-test. A mixed model analysis was conducted where each outcome variable was fitted as a function of group, time and group-by-time interaction while controlling for age, race, BMI (BMI z-score for Z-score outcomes), vitamin D intake, calcium intake and reported hours of exercise per week. A first order autoregressive covariance structure was included in the model to adjust for the correlation of measurements on the same individual across time. Outcomes variables include those obtained from DXA, pQCT and behavioral questionnaires. SAS V9 was used for all analyses.\n\nTable 2  describes the anthropometric measures of cases and controls at each visit. All participants completed the 12-month visit and 89% completed the 24-month visit. Reported use of at least one TDE2 patch per week was 69%, although this includes one case who did not return any patch calendars, and thus had zero weeks of reported patch use. Within the POI group, laboratory data showed an expected rise in serum estradiol from baseline to study completion (7.49 ± 2.76 pg/mL vs. 98.63 ± 87.89, p=0.029), albeit with high variability. Bone formation markers trended up from baseline to 6 months (osteocalcin: +1.89 ± 2.76 ng/mL, p=0.68; BSAP: +9.13 ± 11.18 μg/L, p=0.05), but subsequently decreased at study completion (osteocalcin: −17.78 ± 20.60 ng/mL, p=0.025; BSAP: −17.92 ± 15.50 μg/L, p=0.010). Bone resorption markers (CTX) declined from baseline to 24 months (−305 ± 281 pg/mL, p=0.026). Hepatic enzymes, ALT and AST, decreased from baseline to 24 months (−4.89 ± 4.46 U/L p=0.038 and −3.33 ± 4.62 p=0.018, respectively). There were no significant changes in cardiometabolic markers (HDL, LDL, triglycerides, total cholesterol), IGF-1 or GGT. At baseline, the mean 25OHD level was 23.3 ± 7.6 ng/mL with insufficiency (20–30 ng/mL) in 4/9 (44%) and deficiency (<20 ng/mL) in 3/9 cases (33%). Vitamin D supplementation was prescribed as clinically indicated. However, on subsequent assessments, Vitamin D insufficiency (8/17) and deficiency (6/17) remained prevalent. Bone age was delayed an average of 2.2 years in all POI participants at baseline, but all had a bone age of at least 15 years by study completion.\nAt baseline, DXA BMD Z-scores at all skeletal sites were significantly lower in POI cases compared to controls ( Figure 1 ) and remained so throughout the study after controlling for BMI, reported calcium intake, vitamin D intake and exercise hours per week. Among POI cases, DXA BMD Z-scores significantly increased (p<0.05) from baseline to 24 months at all skeletal sites, whereas there was no change in DXA measures among controls except for an increase at the whole body less head (WBLH) (p=0.034). Among POI cases who started progestin compared to those who had not, BMD Z-scores were lower at the total hip and femoral neck in progestin users at 12 months (p= 0.038 and p=0.005 respectively); however, this difference was no longer significant at 24 months. BMD Z-scores at the LS and WBLH did not differ based on progestin exposure in the POI cases.\nResults of bone parameters measured by pQCT are shown in  Table 3 . At the 3% site of both the tibia and the radius, case participants had lower trabecular vBMD at all time points with no significant change over time for either group. Cortical vBMD did not differ between groups at any time nor at any skeletal site. Cortical thickness was initially lower in participants with POI compared to controls. However, this difference was no longer present at 6 months, and the groups remained similar throughout the rest of the study. There were no between group differences in cortical area nor stress strain index at any measurement site (data not shown).\nDepression scores (CDI-II) and quality of life scores (CHQ-87) did not differ between groups at baseline, and results were essentially unchanged over the two-year study period ( Table 4 ). Anxiety scores (SCARED) appeared higher in the POI group at each time point, but group differences were not statistically significant, and there was minimal variation across the 24-month study. While no between group differences in neurocognitive measures were identified, both POI participants (p=0.001) and controls (p=0.041) demonstrated increases in total memory index scores (ChAMP) from baseline to 24 months. There were no significant differences between POI participants and controls on the NIH Toolbox cognitive measures except for better performance by POI participants on Picture Sequence Memory at the 12-month visit (126.0 ± 18.2 vs 108.6 ± 15.0, p=0.042).\nPatch site irritation was the most common side effect in participants with POI, occurring in 30%. Additional symptoms reported during the study by participants with POI included weight loss, increase in underlying tic disorder symptoms, heavy menstrual bleeding, and benign fibroglandular breast tissue, with no individual reporting more than one of these concerns. One participant with POI developed a subclavian vein thrombosis during the study. Thrombophilia testing revealed factor V Leiden heterozygosity. ERT was continued and antithrombotic medication initiated though eventually discontinued after surgery for thoracic outlet syndrome was performed.\n\nIn this pilot study, we describe the impact of TDE2 treatment for POI on bone health, mental health, and cognitive functioning. At baseline, adolescents with POI had significantly lower height adjusted BMD Z-scores by DXA at all skeletal sites, as well as lower trabecular vBMD and cortical thickness at both weight-bearing (tibia) and non-weight-bearing (radius) sites than controls. There were statistically significant increases in height-adjusted BMD Z-scores at all skeletal sites in cases with POI. However, they remained below age, race and BMI matched peers after 24 months of TDE2 treatment. While no significant differences were noted in cognitive or mental health outcomes, our study had limited power to detect differences in these outcomes, and it is possible that differences may emerge as these young people mature into adulthood.\nFew studies have described the effect of TDE2 on bone density in young women with idiopathic POI ( 7 , 13 , 14 ). In one of the largest prior studies, women in their late twenties with POI had lower aBMD at the femoral neck (FN) and LS, compared to control participants at baseline ( 13 ). After three years of treatment (TDE2 0.1mg/day and cyclic oral medroxyprogesterone), women with POI had gained BMD at the FN (2.5%) and LS (7.7%) whereas controls had a 2.6% loss of BMD at the FN and little change at the LS removing between group differences in BMD at both skeletal sites. In our study, adolescents with POI experienced a similar net increase in raw aBMD of 3.7% and 6.5% at the FN and LS, respectively, whereas controls experienced subtle decreases in aBMD at the FN and LS. However, these changes were not sufficient to remove differences in BMD between groups. There are several reasons why differences in BMD resolved in adult women with POI compared to controls but persisted in adolescents. First, in the study of adult women, the onset of menstrual abnormalities occurred at age 24.9 ± 7.9 years. These women likely had normal peak bone mass accrual during adolescence. Second, adult women received full estrogen replacement for a mean of 2.2 ± 1.0 years whereas our participants were on full estrogen replacement dose for only 6 months. It is possible that further gains in BMD might be achieved with longer use of 0.1mg/day TDE2, representing a full estrogen replacement dose.\nAnother 24-month study of young women with POI (median age 19 years) found a +0.7 (95% CI 0.47–0.93) increase in LS BMD Z-score in those treated with TDE2 0.1mg/day compared to an increase of +0.41 (95% CI 0.25–0.58) in those treated with oral estradiol ( 14 ). While there were no between-group differences at baseline, the TDE2 group had a percentage increase in LS BMD nearly double that of the oral E2 group (16.87% vs 8.98%, p=0.003). The authors proposed that the higher gains in BMD in the TDE2 group may have been due to a lack of IGF-1 suppression. We also found that IGF-1 concentrations were not suppressed in our cohort, which likely reflects bypassing the hepatic first-pass effect with a transdermal system.\nFew studies have used pQCT to assess bone outcomes in women with ovarian insufficiency. In a study of young adult women who regularly exercised, those with estrogen deficiency (amenorrhea or <6 menstrual cycles per year) had lower total vBMD at the 4% distal radius site (p=0.007) and lower cortical vBMD at the 66% cortical site (p=0.001) compared to women with regular menses ( 15 ). Interestingly, total area was greater in the estrogen deficient group compared to those deemed estrogen replete, which the authors hypothesized was due to lack of inhibition of periosteal expansion. Bone strength index, a measure of bone’s ability to resist compressive forces at the distal end of bone, was lower in the estrogen deficient group (p=0.003) whereas at the 66% radius skeletal site, stress strain index was similar in each group. We did not see differences in bone area at any skeletal site although our sample size may have been insufficient to detect differences.\nAdult women with POI are 3.3 and 4.9 times more likely to have symptoms of depression and anxiety, respectively, compared with controls ( 17 ). However, mental health outcomes have not been reported among adolescents with POI. We did not find differences in self-reported depression between adolescents with POI and controls. Self-reported anxiety appears slightly elevated among those with POI, but differences were not statistically significant. We also did not find changes in depression or anxiety among either group over the 24-month study. Schmidt et al found that women with spontaneous POI were more likely to report a history of depression (55%) compared to those with Turner Syndrome (36%) ( 18 ). This may be due to the unexpected diagnosis and its consequences on fertility in women with spontaneous POI compared to those with Turner Syndrome who expected ovarian insufficiency as a component of their genetic condition. Adolescents in our cohort were not directly asked about their mental health in relation to fertility, although several asked for referrals to reproductive specialists.\nAdolescents in both groups reported similar overall health on the CHQ-87, and values were consistent across the 24-month study. Memory performance on the ChAMP was similar among both groups of adolescents, and both demonstrated improvements in memory tasks over the course of the study (likely due to increased familiarity with the tasks with repeated exposure). Likewise, performance on the NIH Toolbox cognitive assessments were similar among both groups of adolescents except for the significant difference in the Picture Sequence Memory task at the 12-month visit. This difference was not sustained at the 24-month visit, and performance variability was high among both groups of adolescents across the study (data not shown).\nLaboratory measurements in our cohort of adolescents with POI were reassuring and confirmed findings by Burgos et al. ( 19 ) that hormone replacement therapy did not alter the lipid profile. In addition, we confirmed that TDE2 use did not alter serum IGF-1 levels nor increase liver transaminases, likely reflecting the ability to bypass hepatic metabolism that occurs with oral estrogen ( 12 ).\nOur study is limited by small sample size. Nonetheless, it is one of the few protocols to phenotype extensively adolescents with POI and track characteristics in response to hormone replacement therapy. Furthermore, we included an age-matched comparator group. All analyses were performed based on intention to treat with TDE2, but there was significant variability in compliance with estrogen patch use. Our study was conducted during the COVID-19 pandemic, which had a significant impact on adolescents worldwide, and may have diminished our ability to find any differences in psychological measures between groups. We were unable to confirm vitamin D status of the control participants and estradiol levels may have varied even though these adolescents reported regularly occurring menses.\n\nAdolescents with POI have compromised bone health at baseline that improved but did not catch up to peers of similar age, race and BMI after 24 months of gradual TDE2 titration. Gains in bone density may continue into young adulthood with consistent physiologic estrogen replacement therapy. Future trials of a larger sample size are needed to confirm the current results and ask whether more rapid TDE2 dose titrations or higher doses of TDE2 are needed for adolescents than the transdermal doses typically prescribed for women experiencing natural menopause. The effects on the central vs. peripheral skeleton also merit further study. The impact of POI during adolescence and its treatment with TDE2 on mental health and neurocognitive outcomes remains uncertain. These data highlight the importance of improving the recognition of estrogen deficiency in adolescents, enabling timely initiation of hormone replacement therapy to optimize peak bone mass accrual, while also exploring the impact of this condition on adolescent mental health and cognition.","source_license":"CC0","license_restricted":false}