This cross-sectional comparison and prospective cohort design study will investigate differences in calcium metabolism, biochemical markers of bone and reproductive health, musculoskeletal health, and iron status between women using different hormonal contraceptives (combined oral contraceptive pill (COCP), hormonal implant, hormonal intra-uterine system (IUS), hormonal contraceptive injection, and eumenorrheic non-hormonal contraceptive users). The same outcomes will also be examined across a menstrual cycle in the eumenorrheic non-hormonal contraceptive users.

The study will test the following hypotheses:

Hormonal contraceptive use

1. Biochemical markers of bone resorption and formation and ratio of urinary 44Ca:42Ca will be higher in the implant and injection groups compared with IUS (which exerts localised effects) and non-HC users (ovulatory phase), and lower in COCP compared with non-HC users;
2. Oestradiol and progesterone will be lower in hormonal contraceptive users compared with non-HC users during the ovulatory phase;
3. Bone macro- and microstructure, muscle strength, and tissue properties are different in hormonal contraceptive users compared with non-HC users;
4. Calcium and bone metabolism, reproductive hormones and musculoskeletal function are different between the pill phase and non-pill phase of COCP use.

Menstrual cycle phase

1. Calcium and bone metabolism are lower during the ovulatory phase compared with menses, mid follicular and mid luteal phases.
2. Muscle strength and tissue properties are different across the menstrual cycle in non-HC users.

Women of reproductive age experience cyclical variation during the menstrual cycle in the female sex steroid hormones, oestrogens and progesterone. Oestrogens performs a primary function in sexual development and reproduction; but, non-reproductive effects on bone, muscle, sinew tissue (e.g. ligaments and tendons) and metabolism may influence injury risk and physical performance. Hormonal contraceptive use, which is common in athletes and military service women, disrupts the reproductive axis and suppresses endogenous hormone production. The purpose of this study is to compare calcium metabolism, biochemical markers of bone and reproductive health, iron status and musculoskeletal health between women using one of four methods of hormonal contraceptives-combined oral contraceptive pill (COCP), hormonal implant, hormonal intra-uterine system (IUS) and hormonal contraceptive injection-and eumenorrheic, non-hormonal contraceptive users (non-HC).

The study will involve a pre-screening visit followed by the main study visits. During the pre-screen visit a venous blood sample will be taken to assess vitamin D status alongside several questionnaires to evaluate health and lifestyle and determine eligibility. Eumenorrheic women will be given a commercially available fertility tracking wearable bracelet (Ava Science Inc.) which will be worn throughout at least two menstrual cycles in the non-HC group for prediction and detection of ovulation. Following pre-screen, the non-HC group will attend the laboratory on four occasions corresponding to the start of the menstrual bleed, the mid-follicular phase, ovulatory phase and mid-luteal phase; the COCP users will attend on two occasions corresponding to the end of the pill phase and the end of the pill-free phase; and the Long Acting Reversible Contraceptives (LARC) users (hormonal injection, hormonal implant, IUS) will attend for a single study visit. On each study visit, participants will provide a urine and venous blood sample, undertake muscle function tests (isokinetic dynamometry and single-leg drop) and have tendon, muscle and ligament characteristic measurements taken (digital palpation). Bone measurements (DXA, HRpQCT, ultrasound) will be taken on one occasion, (day 14 for the non-HC group; day 21 for the COCP group (i.e. end of pill-using weeks); and the single testing day for other LARC groups. The final measurement of impact microindentation (IMI) will be performed using the Osteoprobe within 4 weeks after the skeletal imaging; the IMI is a specialised procedure and will be scheduled in set sessions each month.

Primary Outcomes: Bone calcium balance (44Ca:42Ca) measured in urine.

Secondary Outcomes: Markers of bone turnover, reproductive function, iron status and musculoskeletal health.

Inclusion Criteria:

1. Aged 18-40 years old
2. No hormonal contraceptive use (with regular menstrual cycles 24-35 days in length, and have not been taking any hormonal contraceptives for at least 12 months), or;
3. Use of the combined oral contraceptive pill containing ≥25 mg ethinyl-oestradiol (EE) and an anti/low androgenic progestin for at least 12 months, or;
4. Use of the hormonal coil (IUS) continuously for at least 2 years, or;
5. Use of the hormonal implant continuously for at least 2 years, or;
6. Use of the hormonalDMPA injection continuously for at least 2 years;
7. Weight stable (no change in self-reported body mass ≥ 5% over the previous 3 months);
8. BMI between 18 and 30 kg·m2.

Exclusion Criteria:

1. Taking a hormonal contraceptive other than the combined oral contraceptive pill containing ≥25 mg EE and an anti/low androgenic progestin, hormonal coil (IUS), the hormonal contraceptive implant or the DMPA hormonal injection;
2. History of DMPA hormonal injection use in those women not currently using the DMPA hormonal injection;
3. Diagnosed Premature Ovarian Insufficiency;
4. Pregnancy;
5. Less than 2 years postpartum;
6. Given birth to more than 2 children;
7. Evidence of disordered eating (≥ 20 on the EAT-26);
8. Any self-diagnosed eating disorder;
9. Self-reported change in body mass of ≥ 5% over the previous 3 months;
10. Body mass index of < 18 or > 30 kg·m2;
11. Evidence of menstrual disturbance (oligomenorrhea: < 9 menstrual cycles in previous 12 months or amenorrhoea: ≤ 3 menstrual cycles in the previous 12 months);
12. Habitual smoking (regularly smoking more than 10 cigarettes per day);
13. Taking any medications known to affect bone or calcium metabolism (e.g., treatment for thyroid disorders).
14. Total 25-hydroxyvitamin D (25(OH)D)level < 30 nmol/l at baseline, confirmed with a venous blood sample;
15. Self-declared history of heart, liver or kidney disease, diabetes, or thyroid disorder;
16. Self-reported bone fracture in the previous 12 months.

Exclusion Criteria for Reference Point Indentation Only:

1. Local oedema;
2. Local skin infection or cellulitis;
3. Prior clinical or stress fracture in the tibial diaphysis;
4. Dermatological lesions around the measurement site;
5. Focal tibial lesions like in primary or metastatic tumour, Paget's disease, Gaucher;
6. Osteomyelitis of the tibia;
7. Systemic infection or fever (unless unrelated to infection);
8. Allergy to lidocaine.

Healthy women of reproductive age.

Data will be shared pending approval from the UK Ministry of Defence.