Unable to recruit adequate number of participants

Excessive exercise, disordered attitudes toward eating, physical and psychological stress, and/or hormonal imbalances may result in cessation of menstruation (secondary amenorrhea). The accompanying lack of estrogen may impair muscle power and oxygen recovery after exercise by reducing nitric oxide levels. The purpose of this study is to determine whether ingestion of beetroot juice containing nitrate, an alternative source of nitric oxide, can help reverse these changes.

Due to excessive exercise, disordered attitudes toward eating, physical and psychological stress, and/or hormonal imbalances, young women may be predisposed to developing secondary amenorrhea. Secondary amenorrhea is defined as the absence of menses for at least 3 months in regularly menstruating women or at least 6 months in irregularly menstruating women [1]. This disorder of the hypothalamic-pituitary axis (HPO) is associated with a short-term lack of endogenous estradiol (E2), which may have negative health consequences [2-7]. For example, E2 plays an important role in regulating skeletal muscle mass and contractile function, including both strength and power [4]. In part, this is because E2 increases phosphorylation of the regulatory light chain of myosin, thereby enhancing muscle contractility [6, 8]. Lack of E2 also alters lipid and carbohydrate metabolism [9, 10], which may be due to the important role E2 plays in regulating mitochondrial function, more specifically respiration and efficiency. This impairment in metabolism and mitochondrial respiration could increase CVD risk and alter plasma levels of lipids, cortisol, and insulin [2, 9, 10]. Indeed, Ronkainen et al. [11] found that postmenopausal women not on hormone replacement therapy (HRT) have reduced skeletal muscle strength and power compared to those on HRT, whereas Kleis-Olsen et al. [12] reported similar findings with respect to fat mass and mitochondrial respiratory capacity. Younger women with secondary amenorrhea may also suffer from similar impairments due to E2 deficiency.

It has also been shown that short term lack of E2 compromises nitric oxide synthase (NOS) activity, which plays a critical role in cardiovascular health, exercise capacity, and vasomodulation and mitochondrial function within skeletal muscle [7, 13, 14]. Conversely, dietary nitrate supplementation can increase nitric oxide bioavailailbity, via an enterosalivary pathway in which nitrate is reduced to nitrite that in turn is further reduced to nitric oxide [15]. It has been demonstrated that dietary nitrate improves muscle contractility [16], exercise capacity and performance [15, 17], vascular function [18], and mitochondrial efficiency during exercise [5, 19, 20]. Indeed, we have found nitrate supplementation to be particularly effective in increasing muscular speed and power in postmenopausal women [21]. Increasing NO bioavailability via ingestion of nitrate therefore may have beneficial effects on both muscle function and mitochondrial function in women with secondary amenorrhea. To date, however, this hypothesis has not been tested.

Although the aforementioned research has highlighted the beneficial effects of dietary nitrate supplementation on muscle contractile function and mitochondrial respiration in various populations, no studies have examined the effects of this supplementation on these parameters in women with secondary amenorrhea. The proposed study will therefore provide a better understanding of the effects of dietary nitrate supplementation on muscle function and mitochondrial respiration in women with secondary amenorrhea, potentially improving health, recovery, and performance outcomes within this vulnerable population.

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Inclusion Criteria:

• Women age 18-44 years old
• Missed >3 consecutive periods in the last 12 months if previously regularly menstruating (average cycle 21-35 d) OR
• Missed >6 consecutive periods in the last 12 month if previously irregularly menstruating (spontaneous menstruation, average cycle <21 or > 35 d)

Exclusion Criteria:

• Unable to provide informed consent
• Currently diagnosed with primary amenorrhea (no history of menstruation by age 15) or oligomenorrhea (menstrual cycle >35 d or <8 cycles per year)
• Currently diagnosed with a chronic illness, including thyroid disease, hyperprolactinemia, Cushing syndrome, and/or poly-cystic ovarian syndrome (PCOS)
• History or current clinical diagnosis of an eating disorder
• Currently dieting
• Weight instablility for the past 3 months
• Previously undergone a revascularization procedure involving a vascular graft or stenting of the femoral or popliteal arteries
• Currently at the time of screening, or in the past year, using hormonal contraceptives
• Pregnancy
• Epilepsy
• Current antibiotic use
• Current nicotine user
• Stage II hypertension (resting blood pressure >140/>90)
• Currently taking proton pump inhibitors, antacids, xanthine oxidase inhibitors, or on hormone replacement therapy
• An answer of yes to any of the seven questions on the first page of the Physical Activity Readiness Questionnaire (PAR-Q) indicating that the participant is not physically ready for exercise without a medical exam. These exclusions include the following:
• If participant's doctor has ever said that he/she has a heart condition and that he/she should only do physical activity recommended by a doctor
• Pain in chest when doing physical activity
• In past month, chest pain when not doing physical activity
• If participant has ever lost balance because of dizziness or has ever lost consciousness
• Muscle, bone, or joint problem that could be made worse by physical activity
• Currently on prescribed drugs for blood pressure or heart condition.
• If the participant knows of any other reason he/she should not do physical activity.

All non-PHI individual participant data will be shared upon reasonable request.

Data will be made available upon completion of the study and publication of the final results.