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 150 participants over 2 years with a target for study
completion of 25 subjects. Eligible participants will be females greater than or equal to 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. Blood samples will be collected for eventual genotyping.

- INCLUSION CRITERIA:

1. Female

2. Between 18 and 28 years of age (inclusive)

3. Reported menarche between the ages of 11 and 14 years

4. Gynecological age of less than or equal to 14 years

5. 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

6. A BMI of 18.5 to 27 kg (Summation)m(2) and a weight >= 93 lbs.

7. Agrees to use barrier contraception method for the duration of the study

8. Agrees to abstain from alcohol consumption during both 5-day diet/exercise study
interventions

9. Agrees to abstain from donating blood during the study and within 30 days of
completing the study

10. Is willing and able to fulfill the requirements of the protocol and to provide
informed consent

11. Able to speak and read English

12. Lives within 50 miles of the Clinical Research Unit

EXCLUSION CRITERIA:

1. Currently lactating or pregnant or planning on becoming pregnant for the duration of
the study

2. Has ever given birth

3. History in the past 3 months of dieting or weight loss amounting to greater than 2 kg
(4.4 lbs)

4. > 4 hours per week of aerobic exercise for the past 3 months

5. 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

6. Currently using hormone-based contraception, including those administered orally,
vaginally, via injection, sub-dermally, or transdermally

7. 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

8. Current use of recreational drugs (alcohol intake will be monitored and excluded
during the two intervention periods)

9. Unable to consume food containing dairy or nuts

10. 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. History of thyroid disorder is permissible if
the patient is biochemically euthyroid on replacement.

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 less than or equal to 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