LINE-1 and Alu Methylation Levels Among Middle Aged Women With Low Cardiovascular Risk Profile in Respect of Menopausal Hot Flashes

Suspended

• Conditions: Menopause; Sleep; Epigenetics; DNA Methylation; Hot Flashes; Cardiovascular Diseases
• Interventions: Diagnostic Test: Polysomnography; Diagnostic Test: Skin conductance; Genetic: ALU and LINE-1 DNA methylation analysis; Diagnostic Test: The Menopause-Specific Quality of Life Questionnaire (MENQOL); Diagnostic Test: Pittsburgh Sleep Quality Index (PSQI)

• Registration Number: NCT05892211
• Lead Sponsor: Istanbul University - Cerrahpasa

Brief Summary

Vasomotor symptoms are the most common symptoms seen during climacterium. The hypoestrogenic state causes dysfunction of hypothalamic preoptic area, a thermoregulatory center. The sympathetic overactivation during the hot flashes is associated with awakening during sleep and have a negative impact on cardiac indexes and vascular reactivity. Therefore, hot flashes are accepted as subclinical cardiovascular risk factor.

The association between the severity of the hot flashes and cardiovascular risk may have an epigenetic background. Recently, methylation changes of DNA was found to be associated with clinical and subclinical cardiovascular disease risk (atherosclerosis and hypertension etc.). A transposable element in the DNA, Long interspersed nuclear elements (LINE-1), was found to be hypomethylated in cases with ischemic heart disease and stroke. Therefore, the expression of repeating elements in the DNA (LINE-1 and ALU) may be considered as a mediator in the ischemic heart disease. Until now, menopausal age, vasomotor symptoms and epigenetic and biological aging have been evaluated. However, the epigenetic impact of severe vasomotor symptoms in postmenopausal women with low cardiovascular disease risk profile has not been evaluated. In this study, we aimed to evaluate the epigenetic basis of cardiovascular disease risk for women with vasomotor symptoms which disturb sleep by assessing the methylation levels of ALU and LINE-1.

Detailed Description

Vasomotor symptoms are the most common symptoms seen during climacterium. The hypoestrogenic state causes dysfunction of hypothalamic preoptic area, a thermoregulatory center. The variations in the range of thermoneutral zone determine the severity of the vasomotor symptoms. Sympathetic overactivation during the hot flash and decreased rate of parasympathetic control on the heart rate are the main factors contributing to the cardiovascular disease risk. The hot flashes which occur and cause awakening during sleep have negative impact on cardiac indexes and vascular reactivity. Therefore, vasomotor symptoms are accepted as subclinical cardiovascular risk factor.

Hot flashes associated with nighttime awakenings were shown to increase systolic and diastolic blood pressure and decrease pre-ejection period. Objectively recorded hot flashes and nighttime awakenings were found to be correlated white matter hyperintensities in the brain which show poor brain health. In addition, white matter hyperintensities may be considered as a cerebral small vascular disease and is associated with greater odds of having stroke and dementia.

Among postmenopausal women, cardiovascular disease is the most prevalent cause for mortality and morbidity. It results from the interaction of environmental and genetic factors. The association between the severity of hot flashes and cardiovascular risk may have an epigenetic background. The global DNA methylations were found to be decreased in postmenopausal women with high cardiovascular disease risk. Recently, methylation changes of DNA was found to be associated with clinical and subclinical cardiovascular disease risk (atherosclerosis and hypertension etc.). A transposable element in the DNA, Long interspersed nuclear elements (LINE-1), was found to be hypomethylated in cases with ischemic heart disease and stroke. Therefore, the expression of repeating elements in the DNA (LINE-1 and ALU) may be considered a mediator in the ischemic heart disease. Until now, menopausal age, vasomotor symptoms and epigenetic and biological aging have been evaluated. However, the epigenetic impact of severe vasomotor symptoms in postmenopausal women with low cardiovascular disease risk profile has not been evaluated. In this study, we aimed to evaluate the epigenetic basis of cardiovascular disease risk for women who have low cardiovascular disease risk profile at baseline and have vasomotor symptoms which disturb sleep by assessing the methylation levels of ALU and LINE-1.

Study Timeline

• Study Start: 2020-12-01
• Primary Completion: 2022-12-01
• Study First Submitted: 2023-05-09
• Study First Submitted QC: 2023-05-27
• Study First Posted: 2023-06-07
• Status Verified: 2025-04
• Last Update Submitted: 2025-04-29
• Last Update Posted: 2025-05-02
• Study Completion: 2032-03-08

Recruitment & Eligibility

• Status: SUSPENDED
• Sex: Female
• Target Recruitment: 30

Inclusion Criteria

• 45-55 years of age
• Women with serum FSH levels >35 IU/L, serum estradiol levels <20 pg/mL
• Women with low cardiovascular disease risk profile (Framingham score for coronary heart disease <10%),
• Low high sensitive CRP levels (<5 mg/L),
• Fasting glucose <90 mg/dL,
• Fasting levels of insulin <37.06 µIU/mL,
• Blood pressure <140/90 mmHg (measured 2 times with 10 minutes interval),
• TSH <4.2 mIU/L and fT4 <1.7 ng/dL,
• Hemoglobin levels 12-16 g/dL, leucocyte count <10.3*10^3/µL and neutrophil count <4.9*10^3/µL; eosinophil count <0.5*10^3/µL, basophil count <0.2*10^3/µL

Exclusion Criteria

• Women with high cardiovascular disease risk profile (Framingham score for coronary heart disease >10%),
• Women who were diagnosed with a cardiovascular disease (coronary heart disease, stroke),
• Women with hypertension,
• Smokers,
• BMI >30 kg/m2,
• Women with diabetes
• Women who were surgically postmenopausal,
• Women who used hormone therapy in the last three months,
• Women with obstructive sleep apnea
• Women who are using medicine which may cause sleep disturbances.

Study & Design

• Study Type: OBSERVATIONAL
• Study Design: Not specified

Arm && Interventions

Group	Intervention	Description
Group 1 (vasomotor symptoms present)	The Menopause-Specific Quality of Life Questionnaire (MENQOL)	Participants who have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.
Group 2 (vasomotor symptoms absent)	ALU and LINE-1 DNA methylation analysis	Participants who don't have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.
Group 1 (vasomotor symptoms present)	Polysomnography	Participants who have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.
Group 1 (vasomotor symptoms present)	Skin conductance	Participants who have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.
Group 1 (vasomotor symptoms present)	Pittsburgh Sleep Quality Index (PSQI)	Participants who have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.
Group 1 (vasomotor symptoms present)	ALU and LINE-1 DNA methylation analysis	Participants who have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.
Group 2 (vasomotor symptoms absent)	The Menopause-Specific Quality of Life Questionnaire (MENQOL)	Participants who don't have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.
Group 2 (vasomotor symptoms absent)	Pittsburgh Sleep Quality Index (PSQI)	Participants who don't have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.
Group 2 (vasomotor symptoms absent)	Polysomnography	Participants who don't have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.
Group 2 (vasomotor symptoms absent)	Skin conductance	Participants who don't have vasomotor symptoms. Sleep is recorded by polysomnography for a night. Hot flashes during sleep are recorded by two electrodes measuring the skin conductance near sternum. Any hot flashes reported subjectively by the patient during the recording is noted. Their blood samples are obtained.

Primary Outcome Measures

Name	Time	Method
Number of objective hot flashes per night	1 night	Number of objective hot flashes per night measured by sternal skin conductance in women with vasomotor symptoms
Methylation levels of ALU and LINE-1	1 day	Methylation levels of ALU and LINE-1 in women with vasomotor symptoms
Polysomnographic findings	1 night	Sleep stages in women with vasomotor symptoms
Number of nighttime awakenings per night	1 night	Number of nighttime awakenings per night associated with hot flash episodes

Secondary Outcome Measures

Name	Time	Method
Subjective sleep findings (Pittsburgh Sleep Quality Index (PSQI))	1 night	Scores of PSQI questionnaire in women with vasomotor symptoms (\>5 can be considered as a significant sleep disturbance.)
Menopause-Specific Quality of Life Questionnaire (MENQOL) scores and its association with hot flashes per night	1 day	Scores of MENQOL and its association with hot flashes per night (There is no limit, higher scores on the MENQOL indicate a poorer quality of life.)

Trial Locations

Locations (1)

Istanbul University-Cerrahpasa; 🇹🇷 Istanbul, Turkey