CaREFREE Study (Calorie Restriction, Environment and Fitness: Reproductive Effects Evaluation Study)

Functional hypothalamic amenorrhea (HA) is a reversible form of hypogonadotropic hypogonadism (HH) that can be triggered by stressors such as exercise, nutritional deficits, and psychological stress. Dysfunction of the hypothalamic component of the reproductive axis plays a key role in functional HA and is manifest by an altered pattern of luteinizing hormone (LH) pulses detectable in peripheral blood. There is ample evidence supporting the use of LH as a surrogate marker of hypothalamic gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. There is also significant evidence that women vary in their susceptibility to such stress-induced amenorrhea, pointing to a role for both environmental and genetic factors in the etiology of functional HA. However, the variation in changes in GnRH pulse frequency in response to stressors in healthy women has not been defined. Data from previous work in our lab has suggested that rare variants in genes associated with other forms of HH may also contribute to the variability seen in susceptibility to functional HA. The long-term goal of our research is to examine the interaction of environment and genes in HA. In this pilot study we propose to examine the inter-individual variability in pulsatile LH secretion in response to standardized neutral and deficient energy availability (NEA and DEA, respectively) in normal women. We will then relate this primary end-point to proposed predictive factors including past reproductive and family history and markers of current metabolic status and their response to energy availability. Our initial analyses will help to determine simplified biomarkers that can be translated to larger studies examining the potential combined effect of energy availability and genotype.

The proposed pilot study is a single-site, 2-period study in healthy female volunteers. The study will enroll approximately 300 participants with a target for study completion of 36 subjects. Eligible participants will be females >= 18 years of age. Eligible participants will have had menarche at or before 14 years of age and no earlier than age 11. Eligible participants will have a gynecological age (years after menarche) of 14 years or less. The upper age limit will vary based on each subject s age of menarche and fall between 25 and 28 among participants. Eligible participants will confirm at the pre-screening call having normal menstrual cycles (self-reported) for at least the previous 2 months and ovulation will be confirmed during the menstrual cycle before the start of intervention.

The primary outcome will be changes in daytime LH pulse frequency, when comparing NEA vs DEA. Secondary measures will evaluate past reproductive history, family history, and current metabolic status using medical history interviews, lifestyle questionnaires and maximum oxygen uptake (as a measure of fitness). Resting energy expenditure, body composition as well as metabolic and stress hormones will be measured at baseline and in association with the interventions.

• INCLUSION CRITERIA:

Participants will be enrolled with distribution across race. Each potential participant must meet all of the following inclusion criteria in order to be eligible to enroll in the study:

• Female
• Between 18 and 28 years of age (inclusive)
• Reported menarche between the ages of 11 and 14 years
• Gynecological age of <= 14 years
• A history of self-reported regular menstrual cycles when not on contraceptive medication of between 25 and 35 days (inclusive) at prescreen and knowledge of date of onset of menses before the screening visit
• A BMI of 18.5 to 27 kg x m^2 and a weight >= 93 lbs.
• Agrees to use barrier contraception method for the duration of the study and the follow up period
• Agrees to abstain from alcohol consumption during both 5-day diet/exercise study interventions
• Agrees to abstain from donating blood during the study and within 30 days of completing the study
• Agrees to abstain from biotin supplements for the duration of the study
• Is willing and able to fulfill the requirements of the protocol and to provide informed consent
• Able to speak and read English
• Lives within 50 miles of the Clinical Research Unit

Exclusion criteria

A potential participant meeting any of the following exclusion criteria is not eligible to enroll in the study:

• Currently lactating or pregnant or planning on becoming pregnant for the duration of the study
• Has ever given birth
• History in the past 3 months of dieting or weight loss amounting to greater than 2 kg
• > 4 hours per week of aerobic exercise for the past 3 months
• Has initiated training for an athletic sport or event in the past 3 months that, in the opinion of the investigator, may interfere with the results of the study
• Currently using hormone-based contraception, including those administered orally, vaginally, via injection, sub-dermally, or transdermally
• Current use of medications or supplements that may interfere with the results of the study, including:

i.Steroids

ii.Hormone-based contraception

iii.Sleeping pills

iv.Homeopathic substances (e.g. Chinese herbs, protein or other powders, and other-the-counter extracts)

v.Stimulants (e.g. Ritalin)

vi.Antidepressants or anti-epileptic medications or centrally acting anti-hypertensive medications

• Current use of recreational drugs (alcohol intake will be monitored and excluded during the two intervention periods)
• Unable to consume food containing dairy or nuts
• Has regular overnight awake periods, for example overnight or rotating shift work, that will continue during the study, at the discretion of the investigator
• Has currently or has a history of any of the following: autoimmune, heart, liver, renal disease, diabetes, or another health condition deemed by the PI to be a contraindication to study participation.

Additional Eligibility Criteria to be Met Prior to Start of Intervention(s):

Criteria 1 Habitual energy intake between 35-55 kcal/kg LBM*day

Criteria 2 VO2max <= 40 ml/kg/min with the option to increase this at the discretion of the PI, depending on the current and past exercise level of the participant.

Criteria 3 Hemoglobin, prolactin and TSH within normal female range for testing laboratory.

Criteria 4 Ovulation confirmed in the cycle before each study intervention by self-reported positive urine test, ultrasound and/or progesterone blood-levels