Results
A total of 553 donors were included in this study, including 276 new donors ( i.e., the control group) and 277 repeat donors ( i.e., the donor group), respectively. There were statistically significant differences in age, annual household income, exercise intensity, protein intake and calcium supplementation between the two groups ( P < 0.05), as shown in Table 1 . There were significant differences in the levels of 25OHD, P1NP, β -CTX and PTH between the two groups ( P < 0.05). The inadequacy rate of vitamin D in the repeat donor group (82.31%) was significantly higher than that in the new donor group (68.48%) ( P < 0.001).

In this part of the study, the statistically different indicators related to bone metabolism biomarkers in Table 1 was grouped according to three donation behavior indicators ( i.e., total number of donations, the recent frequency of donation, and interval between the last donation). The results showed that the level of 25OHD and β -CTX was lower while PTH and PINP were higher in the repeat donor group. High total number of donations, high recent frequency and short interval have significant influence on these biomarkers. After 100 times of plasmapheresis donation in Figure 1 , it has an impact on the bone formation indicators (P1NP). When the frequency of plasmapheresis donation increases to more than 17 times recently, both bone resorption ( β -CTX) and bone formation (P1NP) indicators undergo significant changes. When the interval between plasmapheresis donation exceeds 14 days, it has no effect on the levels of bone resorption ( β -CTX) and bone formation (P1NP) indicators. When the interval between plasmapheresis donation exceeds 28 days, the levels of 25OHD and PTH return to before plasmapheresis donation. In summary, the main influencing factors of bone metabolism related indicators are recent plasmapheresis donation frequency. When it increases to a certain level ( i.e., more than 17 times/nearly 12 months, note: the current upper limit in China is 24 times/nearly 12 months), both bone resorption and bone formation indicators undergo significant changes. When the interval between plasmapheresis donation exceeds 14 days (the current minimum interval requirement in China), the levels of bone resorption and bone formation indicators return to before plasmapheresis donation. When the interval between plasmapheresis donation exceeds 28 days, the levels of 25OHD and PTH return to before plasmapheresis donation. As the total number of plasmapheresis donation increases, it first affects bone resorption indicators, and after 100 plasmapheresis donations, it will further affect bone formation indicators.

Multiple linear regression analysis was performed for bone metabolism biomarkers (25OHD, P1NP, β -CTX, PTH) with significant differences in Table 1 , and adjusted for a large number of possible confounding factors (age, body mass index (BMI), annual household income, physical activity, protein intake, calcium supplementation and vitamin D supplementation). Only indicators of plasmapheresis donation behaviors that were clearly risk factors were shown ( Table 2 ). The results showed that the level of 25OHD was positively correlated with the total number of plasmapheresis donations, and negatively correlated with the frequency of plasmapheresis donation. The level of P1NP was positively correlated with the total number of donations, the higher the number of donations, the higher the level of P1NP. PTH level was positively correlated with the frequency of donation, while β -CTX level was negatively correlated with the frequency of donation. The interval from the last plasma donation has no correlation with changes in the indicators after adjustment.

From Figure 2 , it can be seen that non-linear relationships existed between biomarkers and behavior indicators in Table 2 . The 25OHD level decreased rapidly at first with the increase of the total numbers, then began to rise towards 100 times, and then remained stable after 200 times. However, it decreased rapidly with the increase of recent frequency. It may be noteworthy that even new donors have vitamin D inadequacies, which may be exacerbated by plasma donation. Both PTH and P1NP levels showed an upward trend as the total number and recent frequency increased. β -CTX decreased slowly as the recent frequency decreased, but values remained within the normal range.

The results indicate that there is a statistical difference between the two groups at the SF level. Compared with the control group, the donor group had lower SF levels. The iron accumulation rate in the group of women who repeatedly donate plasma is significantly lower than that in the group of new donors ( P < 0.001). There is no statistically significant difference in iron accumulation rate between male repeat donors and new donors ( Table S1 ).

Total number of donations, the recent frequency of donation, interval between the last donation, SF, β -CTX and P1NP were included in the Pearsoncorrelation analysis to analyze the correlation between the two pairs. There is a correlation between total numbers and the levels of SF and P1NP, with correlation coefficients of −0.306 and 0.148, respectively. The level of SF is correlated with total numbers and P1NP, with correlation coefficients of −0.306 and −0.187, respectively. P1NP is correlated with total numbers and SF ( Table S2 ). The above results indicate that there is a correlation between total numbers, SF, and P1NP pairwise. Therefore, total numbers, SF, and P1NP were selected for the next analysis. Use Bootstrap method for mediating effect analysis. According to the research purpose of this article, the variable details are as follows: total numbers is the independent variable, SF level is the mediator variable, and P1NP level is the dependent variable, as shown in Figure 3 . According to the results in Table 3 , it can be concluded that SF partially mediates the relationship between the total number of plasma donations and the level of P1NP. The total effect is c = 0.156, the direct effect is c ′ = 0.122, and the indirect effect is a * b = 0.034. The mediating effect percentage is a ∗ b / c = 21.8%.

There was no significant difference in BMD between the two groups ( P > 0.05). There was no difference in the prevalence of osteoporosis and low bone mass rate between the two groups, indicating that there was no significant difference in BMD between the two groups ( Table 4 ). Meanwhile, we conducted multiple linear regression analysis on BMD in different parts and found that plasmapheresis donation related behaviors were not independent factors affecting BMD ( Table S3 ), which is consistent with the single factor results. Therefore, this indicates that plasmapheresis donation does not affect the donor’s BMD.

Notes:

Model 1, Predicts the SF level based on the total number of donations; Model 2, Prediction of P1NP level based on the total number of donations; Model 3, Predicting P1NP level together with the total number of donations and SF level.