This 14-month, phase IV, randomized controlled crossover trial aims to compare the effects of oral versus transdermal estrogen replacement therapy (ERT) in women with Turner syndrome (TS). The study's objectives are to clarify endocrine, metabolic, cardiovascular, and thromboembolic risk factors in TS after a wash-out period without estrogen (E2) treatment; compare the effects of oral versus transdermal (TD) ERT regimens; and examine the long-term effects of E2 administration via these two routes. The study involves 50 TS women aged 18-50 years and 50 control participants. TS participants are randomized to receive either oral or TD ERT for six months, followed by crossover to the alternate treatment for another six months. Prior to randomization, any existing ERT will be discontinued for a 1-month washout period. A second 1-month washout period will occur between the two 6-month treatment phases. Laboratory analyses and clinical investigations are performed after the first wash-out period, after the first six months of treatment, and after the last six months of treatment. We anticipate that this study may provide a basis for new and improved recommendations for sex hormone replacement therapy in TS.

OBJECTIVES

1. Clarify endocrine, metabolic, cardiovascular and thromboembolic risk factors in Turner syndrome (TS) after a wash out period
2. Compare the effects of oral versus transdermal (TD) estrogen replacement therapy (ERT) in women with Turner syndrome
3. Examine long term effects of ERT via the two routes on endocrine, metabolic, cardiovascular, physiologic and thromboembolic risk endpoints

BACKGROUND Turner syndrome (TS) is a rare genetic condition affecting approximately 1 in 2,000 female births. A hallmark of TS is ovarian dysgenesis, leading to hypogonadism, premature ovarian failure, and infertility. Consequently, estrogen replacement therapy (ERT) is typically initiated around age 11-12 to induce puberty and continued until the average age of menopause (50-55 years), aiming for at least 42 years of adequate estrogen exposure.

Hypogonadism in TS is associated with various health complications. Importantly, estradiol (E2) replacement may mitigate these risks. Estrogen deficiency in TS affects cardiovascular health (hypertension, congenital cardiac disease, altered lipid profiles), metabolic function (diabetes, thyroid dysfunction, hepatic disorders, kidney disease, skeletal abnormalities), and is linked to neurocognitive and social challenges.

E2 can be administered orally or transdermally (TD), but it remains unclear whether either route offers specific advantages. There is ongoing debate regarding a potential increased thromboembolic risk in TS patients treated with oral E2. Epidemiological studies in postmenopausal women have reported an elevated thromboembolic risk associated with oral estrogen treatment, but to a lesser extent with TD administration. However, extrapolating data from postmenopausal women to TS patients is inappropriate, as women with TS receive estrogen as replacement therapy due to inadequate endogenous production. Furthermore, limited knowledge exists regarding the side effects of oral versus TD estrogen replacement therapy for TS patients.

MATERIALS AND METHODS

Study group:

Women aged 18-50 years with TS recruited primarily from the Department of Endocrinology at Aarhus University Hospital (n=50); 300 patients with TS are currently followed in the outpatient clinic. The investigators also have the opportunity to recruit from the Turner Association in Denmark, and finally the investigators do have contact with other outpatient clinics with TS patients in Denmark from where the investigators have previously recruited TS patients.

An age-matched control group of healthy women is included by advertisement (n=50).

Inclusion criteria:

For participants with TS:

• Diagnosis of TS regardless of karyotype
• Age 18-50 years
• Already receiving estrogen treatment

For healthy controls:

• Female
• Age 18-50 years
• Previously healthy
• Not receiving any medication
• Not using any form of contraceptive pills
• No mental or psychiatric disorders

Exclusion criteria:

• Active systemic chronic diseases
• Known or suspected breast cancer
• Known or suspected estradiol-dependent tumors (endometrial cancer or similar)
• Untreated endometrial hyperplasia
• Current or previous venous thromboembolism
• Acute or previous liver disease where liver enzymes are still elevated by a factor 3 or more
• Known hypersensitivity to the medications used
• Pregnancy
• Menopause (for the control group only)

Design:

A 14-month, phase IV, randomized controlled crossover study involving women with Turner Syndrome (TS) (n=50) and healthy, age-matched controls (n=50). TS participants are randomized to receive either oral or TD ERT for six months, followed by crossover to the alternate treatment for another six months. Prior to randomization, any existing ERT will be discontinued for a 1-month washout period. A second 1-month washout period will occur between the two 6-month treatment phases. Healthy controls will not receive any treatment. They will undergo a single set of assessments for comparison.

Laboratory analyses and clinical investigations will be conducted at the Department of Endocrinology and the associated Medical Research Unit at Aarhus University Hospital. These will include blood and urine sample collection, as well as specific clinical investigations. At baseline, both TS patients and healthy controls will undergo these assessments. Only TS patients will undergo follow-up assesments.

Clinical investigations:

• Insulin sensitivity assessment
• 24-hour blood pressure monitoring
• DEXA scan (Dual-Energy X-ray Absorptiometry)
• Bioelectrical impedance analysis (Multiplate)
• Comprehensive clinical examination
• Quality of life assessments using questionnaires
• SphygmoCor analysis
• VO2 max test using a stationary bike
• MRI of the thigh muscles to measure muscle cross-sectional area (CSA) and intramuscular fat content
• Isometric muscle strength testing and functional testing

STATISTICS Data will be summarized by treatment group and assessment time point. The investigators will use a mixed model with repeated measures analysis of variance (ANOVA) to compare the mean changes in each of the study variables between treatments over time. When appropriate, transformations or nonparametric methods will be used. All tests are two-tailed with a 5% level of significance. Data are presented as mean ± SE or median with CI for metrics not normally distributed.

PERSPECTIVES Patients with TS undergo hormone replacement therapy from puberty to menopause, spanning more than 40 years of treatment. To date, only two experimental studies have compared oral and TD ERT in TS, focusing solely on metabolic parameters and finding no differences between the two regimens. If the investigators' hypotheses are correct and if the side effects, including increased thromboembolic risk, are higher with oral than TD ERT, this project could be crucial for optimizing treatment, improving quality of life, and reducing morbidity and mortality in TS. The investigators aim for this study to provide a basis for new and improved national and international recommendations for ERT in TS patients and to contribute new knowledge about hormonal treatment for the general population as well.

Inclusion criteria:

For participants with TS:

• Diagnosis of TS regardless of karyotype
• Age 18-50 years
• Already receiving estrogen treatment

For healthy controls:

• Female
• Age 18-50 years
• Previously healthy
• Not receiving any medication
• Not using any form of contraceptive pills
• No mental or psychiatric disorders

Exclusion criteria:

• Active systemic chronic diseases
• Known or suspected breast cancer
• Known or suspected estradiol-dependent tumors (endometrial cancer or similar)
• Untreated endometrial hyperplasia
• Current or previous venous thromboembolism
• Acute or previous liver disease where liver enzymes are still elevated by a factor 3 or more
• Known hypersensitivity to the medications used
• Pregnancy
• Menopause (for the control group only)

Only patients with Turner syndrome can participate and age mathed healthy controls (women)

The patients with TS will be randomized to receive either oral (tablet) or transdermal (gel) estrogen replacement therapy. The study is not blinded, as both investigator and study participants know which medication, they are receiving.