Subjects
Collegiate and high school track and field athletes competing at a national level were recruited. The athletes were mostly recruited from the Kanto, Kansai, and Tokai regions of Japan and were examined at a designated hospital and universities. Since the body composition and the prevalence of amenorrhea is different per discipline, we recruited athletes specializing in the following events; 100meters (m), 200m, 400m, 100mH (meter hurdles), 400mH, long jump, triple jump, heptathlon, 4x100mR (meter relay) and 4X400mR. Since hormones could get affected by some medications including oral contraceptive pills, for those taking medications that could affect the hormonal levels were excluded (Garnero et al., 1995; Trémollieres, 2013). Also, for those who are experiencing amenorrhea and oligomenorrhea were also excluded since they have different bone metabolism and low bone mineral density compared to eumenorrheic athletes (Yuka Tsukahara et al., 2021). The measurement was performed during off-season and the recruitment took place between April 2021 to February 2022. Written informed consent was obtained from both the subjects and their legal guardians after being informed of the benefits and risks of the investigation. The study protocol was approved by the Ethics Review Procedures Concerning Research with Human Subjects Group of the authors’ affiliated institutions (approval number 2020 − 416).
Laboratory data
Participants were instructed to refrain from training and consume no food for at least 2 hours prior to blood collection which was performed between the timeframe of 11AM to 2PM. In order to assess bone metabolism, the following bone turnover markers were measured.
1) bone resorption markers
tartrate-resistant acid phosphatase type 5b (TRACP-5b) and N-terminal telopeptide total (NTx)
2) bone formation markers
pro-collagen type 1 amino-terminal propeptide (T-P1NP), bone-specific alkaline phosphatase (BAP), undercarboxylated osteocalcin (OC), and intact parathyroid hormone (iPTH)
3) bone matrix related markers
osteocalcin undercarboxylated osteocalcin (ucOC), intact parathyroid hormone (iPTH)
In addition to the above, 25-OH vitamin D (25OHVD) were also measured since although the relationship between vitamin D and stress fractures in athletes are scarce (Myburgh et al., 1990), some studies have reported the link between vitamin D and BMD in non-athletes (Dawson-Hughes et al., 1997; Mezquita-Raya et al., 2001; Palacios, 2006; Tenforde et al., 2010). In addition, although it was a study on male athletes, free testosterone was also reported to be low in male endurance athletes and thus we also decided to measure free testosterone to seek the relationship between stress fractures along with prolactin (Boden et al., 2001).
Height weight and Bone mineral density
Height was measured with the athlete standing erect without shoes to the nearest 0.1 cm using a stadiometer. Bone mineral density was assessed by whole body modedual-energy X-ray absorptiometry using a Delphi Bone Densitometer (Hologic, Inc., Bedford, MA, USA). After calibration, as for the whole body mode, the subjects, wearing a t-shirt without objects that could interfere with the test, were instructed to lay on a table with the shoulders slightly abducted and elbows slightly flexed, so that the forearms were parallel to the body axis, while extending, but not spreading, the fingers with the palms facing downward. The feet were bound to a polystyrene block in the shape of an isosceles triangle with the vertex angle of 30º, so that the fibulae were not hidden by the tibiae. The same technician performed all scans at each site.
Training schedules and injuries
A questionnaire was distributed to the athletes via online inquiring about the amount of training hours per day on both weekdays and weekends, resting days per week, weight training days per week, any past medical history of bone injuries. We also interviewed all athletes regarding their injuries and those who answered that they had been diagnosed to have stress fracture after high school and included those who were properly diagnosed by a physician following an imaging study were marked as stress fracture (+) and those without a history of stress fracture were marked as stress fracture (-).
Statistical Analysis
All data were analyzed using the Stata 16.1 (Stata Corporation, TX, USA). The results are presented as the mean ± standard deviation. The Shapiro–Wilk test was applied to assess the distribution of the data. Continuous data were compared using the independent t-test. A probability (p) value of < 0.05 was considered statistically significant. Logistic regression analysis was performed to identify independent variables and having a stress fracture after high school was used as a dependent variable.