Effects of different exercise levels on serum trace element concentrations

Abstract

The present study aims to evaluate the effect of training levels on serum concentrations of trace elements including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and selenium (Se). Three groups of male participants at different levels of physical exercise were involved in the survey, such as professional middle- and long-distance runners (n = 20), amateur (n = 22), and sedentary subjects (n = 25). The determination of trace elements was carried out by the kinetic energy discrimination (KED) collision mode of inductively coupled plasma mass spectrometry (ICP-MS) with minimal sample consumption. It was found that there were significant differences in the concentration of Zn, Fe, Cu (all, P < 0.001) and Se (P < 0.05) between the professional group and the sedentary subjects. Mn concentration in the amateur and professional group was significantly higher than that in sedentary subjects (P < 0.001), and inversely, the concentrations of Zn (P < 0.01), Fe (P < 0.05) and Cu (P < 0.05) in the professional group were significantly lower than that in amateur. The correlations analysis demonstrated that changes in trace elements were significantly correlated with training levels: Zn (r = − 0.589, P < 0.001), Fe (− 0.469, P < 0.001), Cu (− 0.442, P < 0.001) and Se (− 0.313, 0.01) presented significant negative correlations with the degree of training, while Mn (0.674, P < 0.001) revealed a positive correlation with the degree of exercise. In addition, the professional group demonstrated that the malondialdehyde (MDA) levels (P < 0.001) and Cu/Zn ratio (P < 0.05) increased significantly under high-intensity training.

Introduction

Zinc (Zn), Iron (Fe), Copper (Cu), Manganese (Mn), and Selenium (Se) are essential trace elements for constituting human tissues, participating in metabolic processes, and maintaining physiological functions^1–3. Although their concentrations are subject to homeostatic control, physical training will alter their levels in the body. Researchers believe that training, especially high intensity training, usually leads to the loss of trace elements, which makes athletes susceptible to abnormal trace elements and affects their enzyme functions, hormone states, and immune functions of the body^4,5. Such conditions will adversely affect athletic performance and may even cause health problems. Therefore, it is necessary to monitor the trace elements in athlete’s bodies and supplement them as needed, which is of great significance for improving athletes’ performance and relieving sports fatigue^6,7.

The effects of exercise on trace elements levels have been widely studied, but there are some contradictory reports due to the intensity, duration and type of exercise^8,9. For instance, Khaled et al. reported that footballers had a low serum Zn concentration with a high blood viscosity due to increased erythrocyte rigidity during exercise¹⁰. Brun et al. reported that regular exercise can reduce the serum Zn concentration in young gymnasts¹¹. Clarkson stated that athletes may have a Zn deficiency due to its loss from the body through sweat and urine¹². Chu et al. believed that serum Zn concentration was significantly increased after aerobic exercise, indicating acute perturbations in Zn homeostasis¹³. In terms of Fe, Öner et al. demonstrated that high-intensity interval training increased oxygen transport in the blood of athletes, leading to an increase in iron concentration¹⁴. Skarpanska-Stejnborn et al. reported that the serum Fe levels decreased significantly during the recovery compared with pre- and post-exercise levels¹⁵. With respect to Cu, there was also no consensus. It was reported that the plasma Cu of athletes was not change significantly after intense exercise, and gradually returned to initial values after resumed training¹⁶. Kikukawa et al. discovered that the serum Cu concentration decreased immediately following + Gz acceleration¹⁷. In the case of Se, Emre et al. stated that the decline in serum Se after intense exercise was related to the transfer of lactate from muscle to blood¹⁸. Sánchez et al. also found that active group had lower Se levels compared to control group¹⁹. Maynar et al. reported that the Se concentration of students who did not participate in daily sports increased significantly after the test²⁰. As for Mn, Maynar and colleagues found that the serum Mn levels in individuals who participated in aerobic exercise were significantly higher than those in sedentary individuals, whereas the anaerobic and aerobic-anaerobic athletes groups presented lower concentrations of Mn than those of sedentary groups²¹. Maynar’s group also reported that exercise increased the activity of Mn-superoxide dismutase (Mn-SOD), which may be related to the reduction of serum Mn concentration²². Park also showed that Fe deficiency increased blood Mn levels, which was associated with the levels of serum ferritin and hemoglobin²³.

On the whole, the precise levels of trace elements under different exercise intensities are still ambiguous, especially for athletes. The purpose of this study is to explore the variation of trace elements concentration among groups with different exercise levels. Thus, modern techniques such as inductively coupled plasma mass spectrometry (ICP-MS) is used to measure and determine the proportions of trace elements to promote the development of this field. ICP-MS owns many advantages including high precision, low limits of detection (LOD), wide linear range and multi-element determinations²⁴. However, a primary disadvantage of ICP-MS is the spectral interferences among multiple elements with the same mass/charge ratio in the sample²⁵. To eliminate interferences, a quadrupole collision/reaction cells is used for ICP-MS operated in kinetic energy discrimination (KED) mode. In KED collision mode, the interference of polyatomic ions in mass spectrometry can be removed by employing the differences in energy loss when different ions collide with inert gas (helium). Furthermore, the pretreatment of trace elements determination in serum samples mainly adopts microwave digestion and wet digestion, which are complicated, time-consuming, more sample consumption, and not conducive to the detection of a large number of samples^26–28. As a simple and efficient pretreatment method, direct dilution can measure numerous samples in a short time, restrain matrix effects, and reduce exogenous contaminations, which is a promising option for serum detection.

In this study, a direct dilution method was employed for the pretreatment of serum samples, and an ICP-MS was used to detect concentrations of Zn, Fe, Cu, Mn, and Se in professional middle- and long-distance runners (n = 20), amateur (n = 22), and sedentary subjects (n = 25), so as to determine the changes in trace elements and whether trace element supplements were needed in athletes’ nutrition. This method is simple and highly efficient with less sample needed, enabling the measurement of a large number of samples in a short time.

Experimental design, materials and methods

Participants and procedures

This study recruited 67 male volunteers who were divided into three groups: sedentary subjects (n = 25), amateur (n = 22), and professional middle- and long-distance runners from the track and field team (n = 20). All of them were healthy and non-smoking, and no vitamins, trace elements or other supplementation were added other than the normal diet during the test. Anthropometric characteristics of all participants were shown in Table 1. There were no significant differences in age, height, weight and BMI parameters among the three groups, and significant differences can be observed in VO_2max, heart rate and lactate threshold. Both professional and amateur groups underwent 4 weeks concentrated training before blood collection. The training schedule for the professional group was 6 days per week, while that for the amateur group was 3 times per week. The professional group was performed aerobic physical training with an average of 120 km per week, which consisted of 4 days of aerobic continuous running or aerobic fartlek and 2 days of intense series and interval training, forming the high-intensity training group. For the amateur group, the training protocol was 60 km per week including 2 days of aerobic continuous running and 1 days of intense series and interval training, forming the low-intensity training group. All tests were performed in the morning (between 8 and 12 a.m.). The sedentary group maintained normal daily activities during the whole experimental period without any specific physical training program. To further evaluate training effects, the professional group completed high-and low-intensity training tests in different two weeks, respectively.

Table 1.

Anthropometric and cardiorespiratory characteristics of the study groups.

	Sedentary (n = 25)	Amateur (n = 22)	Professional (n = 20)
Age (years)	20.62 ± 1.12	20.86 ± 1.53	20.37 ± 1.69
Height (m)	1.73 ± 0.06	1.75 ± 0.05	1.76 ± 0.04
Weight (kg)	66.52 ± 7.39	65.28 ± 8.31	63.79 ± 6.32
BMI (kg/m2)	21.79 ± 2.43	21.26 ± 1.75	20.43 ± 1.50
VO2max (ml/min/kg)	43.61 ± 7.23	51.23 ± 4.46■■■	66.54 ± 5.39†††***
Heart rate (beats/min)	69.12 ± 9.52	63.70 ± 5.18	56.29 ± 7.52†††**
Lactate threshold (mmol/L)	3.05 ± 0.57	3.89 ± 0.66■■■	4.73 ± 0.85†††***
Exercise experience (years)	—	5.36 ± 2.38	9.15 ± 2.05
Exercise Schedule	—	3 times per week	6 days per week

ANOVA and Bonferroni tests.

†Differences between the professional and sedentary (†p < 0.05; ††p < 0.01; †††p < 0.001).

+Differences between the amateur and sedentary (■p < 0.05; ■■p < 0.01; ■■■p < 0.001).

*Differences between the professional and amateur (*p < 0.05; **p < 0.01; ***p < 0.001).

Reagents, instrument and parameters settings

The multi-element standard solution (Zn, Fe, Cu, Mn, Se, Cd, Pb, 100 mg/L), and the internal standard solutions (Sc, Ge, Rh, In, Re, Bi, 100 mg/L) were purchased from Merck (Germany), whereas Triton X-100 and nitric acid (65%) were from Sinopharm (China). ICP-MS tuning solutions (Li, Mg, Y, Ce, Tl, Co, 1 µg/L) were from Agilent Technologies Inc. (USA). Ultra-pure water (~ 18 MΩ cm) was obtained from a Milli-Q^® Advantage A10 water purification system (Millipore, USA).

An Agilent 8900 ICP-MS (Agilent Technologies Inc., USA) with a collision/reaction cell was used for this study. The instrument was tuned to optimal conditions daily using tuning solutions. The main instrument settings and parameters were detailed in Table 2.

Table 2.

ICP-MS operating parameters.

Parameter	Value
Radio frequency power	1600 W
Plasma gas flow	15.0 L/min
Auxiliary gas flow	1.2 L/min
Nebulizer gas flow	0.9 L/min
Collision gas	He
Replicates	3
Dwell time	50 ms
Cones	Ni
Scan Mode	Peak Hopping
Universal Cell Technology	KED

Malondialdehyde (MDA) levels were used to analyze the metabolic stress levels of the subjects before and after physical training. MDA levels were measured by an Agilent 1200 Series, high-performance liquid chromatography (HPLC, Agilent Technologies Inc., Germany).

Samples preparation

Blood samples were collected from the antecubital vein of participants and stored in venous blood vacuum collection tubes. After standing for 30 min, the blood samples were centrifuged at 3000 rpm for 10 min to separate serum, which was transferred into polyethylene tubes and stored at − 30 ℃ for later analysis. All glassware and plastic ware were cleaned with 10% HNO₃ and rinsed with ultrapure water. In the process of ICP-MS experiment, serum samples were thawed at room temperature, and uniformly mixed with a vortex mixer, and then quantitatively diluted 10-fold with the diluent solutions.

Preparation of the mixed standard solution

Diluent solution

The diluent solution (0.01% Triton X-100, 0.1% HNO₃) was prepared using Triton X-100 (0.1 mL), concentrated nitric acid (1 mL), and ultra-pure water in volumetric flask (1000 mL).

Standard solution and internal standard solution

The multi-element standard solution (Zn, Fe, Cu, Mn, Se, 100 mg/L) was successively diluted with the diluent solution to prepare a series of standard solutions (0, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500 µg/L). The internal standard solution (Sc, Ge, Rh, In, Re, Bi, 100 mg/L) was successively diluted to 50 µg/L with the diluent solution.

Statistical evaluations

Statistical analysis was proceeded using the SPSS 27.0 software. The results were denoted as mean values ± standard deviation. The normal distribution was checked by the Shapiro–Wilk test. One-way Anova test was applied to find differences between control and sports men groups, and Bonferroni post hoc test was performed to locate the source of differences. A paired t-test was used to compare the differences between Pre and Post samples. The correlations between serum element levels and physical training were evaluated using the Pearson’s test. The p-value was less than 0.05, indicating statistical significance.

Results

The linear range, detection limit and correlation coefficient

The internal standard calibration curves were plotted with the mass concentration of each element as the x-axis and the response values of the elements calibrated by the internal standard element as the y-axis. The linear regression equation and correlation coefficient were calculated based on the standard curves. The blank sample solution was measured 10 times under the same experimental conditions to calculate the standard deviation. The limit of detection (LOD) was calculated as 3.3 times the ratio of the standard deviation to the slope of the calibration curve. As shown in Table 3, there was a good linear relationship between the element concentration and the signal response intensity within the selected linear range (R² > 0.999). The instrument detection limit for the internal standard method was 0.0193–0.621 µg/L, which can meet the actual detection requirements.

Table 3.

The determination of the linear range, correlation coefficient, and instrument detection limit of trace elements by internal standard method.

Element	Linear range (µg·L−1)	Correlation coefficient	LOD (µg·L−1)
Zn	0.5 ~ 500	0.9994	0.378
Cu	0.5 ~ 500	0.9998	0.0193
Fe	0.5 ~ 500	0.9993	0.621
Mn	0.5 ~ 150	0.9995	0.0853
Se	0.5 ~ 200	0.9992	0.0919

The changes in serum trace elements and their correlation

Table 4 presented the serum concentrations of each element in sedentary, amateur and professional groups. When comparing concentrations between sedentary and amateur group, the amateur group showed significant differences in both Zn (P < 0.05) and Mn (P < 0.001) concentrations, indicating their decreased and increased levels, respectively. When comparing concentrations between sedentary and professional groups, Zn (P < 0.001), Fe (P < 0.001), Cu (P < 0.001) and Se (P < 0.05) concentrations were significantly lower in professional group, and only the case of Mn (P < 0.001) revealed a significant increase in its concentrations. When comparing concentrations between amateur and professional groups, the Zn (P < 0.01), Fe (P < 0.05) and Cu (P < 0.05) concentrations in professional were significantly lower than that in amateur, and Mn (P < 0.05) concentration in professional was significantly higher than that in amateur. No significant statistical differences were observed in the rest of the values.

Table 4.

Serum trace elements concentrations of sedentary, amateur and professional group.

	Sedentary (n = 25)		Amateur (n = 22)		Professional (n = 20)
	Determined (µg·L−1)	Recovery/%	Determined (µg·L−1)	Recovery/%	Determined (µg·L−1)	Recovery/%
Zn	970.75 ± 112.58	92.6	893.23 ± 88.04■	97.9	795.88 ± 94.16†††**	94.5
Fe	1060.09 ± 105.57	98.9	1012.81 ± 91.34	89.7	922.65 ± 121.30†††*	89.7
Cu	797.85 ± 134.09	96.6	749.49 ± 125.66	87.8	656.53 ± 82.18†††*	91.7
Mn	1.22 ± 0.43	92.5	1.88 ± 0.57■■■	99.3	2.34 ± 0.54†††*	93.9
Se	111.49 ± 35.56	88.5	101.81 ± 32.81	87.7	86.26 ± 24.38†	97.8

ANOVA and Bonferroni tests.

†Differences between the professional and sedentary (†p < 0.05; ††p < 0.01; †††p < 0.001).

+Differences between the amateur and sedentary (■p < 0.05; ■■p < 0.01; ■■■p < 0.001).

*Differences between the professional and amateur (*p < 0.05; **p < 0.01; ***p < 0.001).

To further verify the accuracy of the method, the spiked recovery tests were performed on the serum samples. Standard solutions containing five elements with accurate masses were added to the samples. The serum sample was measured 3 times, and the average value was taken as the determined value. As shown in Table 4, the ranges of recovery rates for the internal standard method were 87.7% to 99.3%.

The correlations between the trace elements and training level were investigated and the results were listed in Table 5. Zn (r = − 0.589, P < 0.001), Fe (− 0.469, P < 0.001), Cu (− 0.442, P < 0.001) and Se (− 0.313, 0.01) presented significant negative correlations with the degree of training, while Mn (0.674, P < 0.001) revealed a positive correlation with the degree of exercise.

Table 5.

Correlation analysis between serum trace elements and the level of training.

Elements	Pearson correlation analysis
	r value	P value
Zn	−0.589	< 0.001
Fe	−0.469	< 0.001
Cu	−0.442	< 0.001
Mn	0.674	< 0.001
Se	−0.313	0.010

As shown in Table 6, the training for the professional group led to a significant increase in oxidative stress level due to the enhanced MDA level: from 0.76 ± 0.11 to 0.98 ± 0.15 under high-intensity training (p < 0.001) and from 0.78 ± 0.12 to 0.91 ± 0.14 under low-intensity training (p < 0.01). The increase in MDA levels under high-intensity training was higher than that under low-intensity training. After high-intensity training, the levels of Zn (P < 0.001), Fe (P < 0.001), Cu (P < 0.05) and Se (P < 0.05) decreased significantly, while the concentration of Mn (P < 0.05) increased significantly. There were no statistical differences in the initial concentrations of MDA and trace elements between high- and low-intensity training. The Cu/Zn ratio significantly increased from 0.78 ± 0.12 to 0.85 ± 0.08 (P < 0.05) after high-intensity training, but there was no statistically difference after low-intensity training (P = 0.517).

Table 6.

MDA and serum trace elements concentrations of professional group before and after a week training.

	High-intensity training			Low-intensity training
	Before training	After training	P value	Before training	After training	P value
MDA (µmol/L)	0.76 ± 0.11	0.98 ± 0.15	< 0.001	0.78 ± 0.12	0.91 ± 0.14	< 0.01
Zn (µg/L)	823.12 ± 103.5	702.96 ± 92.34	< 0.001	801.76 ± 83.22	743.89 ± 76.69	< 0.05
Fe (µg/L)	891.63 ± 84.59	783.52 ± 102.95	< 0.001	914.60 ± 146.35	832.51 ± 85.82	< 0.05
Cu (µg/L)	630.70 ± 65.28	582.25 ± 81.40	< 0.05	633.49 ± 70.82	609.11 ± 75.57	0.299
Mn (µg/L)	2.26 ± 0.53	2.57 ± 0.39	< 0.05	2.34 ± 0.54	2.55 ± 0.46	0.193
Se (µg/L)	85.38 ± 21.67	72.36 ± 16.34	< 0.05	88.60 ± 24.97	74.61 ± 16.85	< 0.05
Cu/Zn ratio	0.78 ± 0.12	0.85 ± 0.08	< 0.05	0.81 ± 0.14	0.83 ± 0.15	0.517

P value: paired t-test.

Discussion

Zn, as a cofactor of enzymes, is related to the activity of more than 200 enzymes^20,29. Zn-containing enzymes participate in carbohydrate, lipid, protein, and nucleic acid metabolism. In addition, some Zn-dependent enzymes, such as lactate dehydrogenase and carbonic anhydrase, play critical roles in removing lactate, facilitating gas exchange, regulating the body’s acid-base balance and energy metabolism¹³. After intense exercise, a large number of free radicals were produced in athletes’ bodies, which caused oxidative damage to cell membranes, leading to exercise fatigue and increasing the risk of sports injuries. Zn can inhibit the generation of free radicals, and the inhibitory effect is enhanced with the increase of its concentration. Besides, Zn is involved in the formation of the coenzyme of Cu, Zn-SOD, which converts superoxide anion radicals into hydrogen peroxide (H₂O₂) and oxygen (O₂), thus exerting antioxidant effects. Kara et al. believed that Zn supplementation for male wrestlers prevented production of free radicals by activating the antioxidant system, which increased levels of serum SOD, serum glutathione peroxidase (GPx) and serum glutathione (GSH). Accordingly, after exercise program, their serum Zn levels were higher than those before exercise³⁰. This study found that the serum Zn level was significantly lower in professional group, which was related to the following interactions. On the one hand, the large amount of fluid loss in the body caused by high-intensity exercise can decrease Zn levels. As oxygen consumption increased, more Zn was transported into cells forming Cu, Zn-SOD to resist the increased reactive oxygen species (ROS)¹⁶. On the other hand, Zn elements presented in muscles (60%) and bones (30%) were mobilized to neutralize their loss, thereby buffering the fluctuations of body³¹.

Fe is the most abundant trace element in the body which is related to the transport ability of oxygen/carbon dioxide and the body’s immune function^32,33. Fe also participates in the body’s energy metabolism, and the enzymes and cytokines in the tricarboxylic acid cycle need iron to effectively perform transport functions^34,35. Fe deficiency can hinder the production of red blood cells and reduce the amount of hemoglobin, which prevents the body from normal aerobic metabolism and directly leads to a decrease in the body’s aerobic performance, and the endurance of athletes will also be significantly reduced accordingly³⁶. In addition, iron deficiency can affect the activity of various enzymes in the human body, which causes metabolic disorders and reduces the body’s exercise ability³⁷. This study found a significant decrease in serum Fe content of professional group due to the reduced hemoglobin concentration and the Fe loss through excessive sweat. Slight Fe loss does not seem to affect athletic performance but excessive Fe deficiency does³⁸. Generally, athletes have a higher demand for Fe during exercise, so it is necessary to increase dietary Fe content or use Fe supplements.

Cu is a main component of antioxidant enzymes including ceruloplasmin and SOD, which can prevent or reduce the damage to cells caused by superoxide anion free radicals^22,39,40. The catalytic effect of Cu on hemoglobin synthesis ensures aerobic endurance exercise, and its elimination of free radicals can relieve fatigue and aid in muscle recovery⁴¹. Cu also participates in a variety of immune responses and energy metabolism in the body³¹. For athletes, Cu deficiency in athlete’s body can lead to malnutrition and a weakened physique. Although Cu can be lost through sweat and urine, moderate or low-intensity exercise and training generally do not cause significant Cu deficiency, and there is no need to supplement with Cu¹². If athletes undergo high-intensity training, their physiological functions will decline accordingly, and they can moderately supplement with Cu-rich foods. This research found that exercise intensity had a significant effect on serum Cu level, and the Cu concentration in the professional group was lower than that in other groups. The possible reason is that the body generates more free radicals, and Cu in the blood enters muscle cells to form Cu, Zn-SOD to resist the invasion of free radicals.

Mn is an integral part of several metabolic enzymes, such as arginase, pyruvate carboxylase and Mn-SOD. Among them, Mn-SOD is a mitochondrial antioxidant enzyme that neutralize superoxide radicals formed during the exercise. Maynar et al. found that the serum Mn level in aerobic-exercise group was higher than that in sedentary group²¹. Sánchez et al. reported that the blood Mn concentration in more active participants was higher than that of the sedentary population¹⁹. A number of studies mentioned that physical exercise increased the activity of Mn-SOD, which was related to the higher serum Mn concentrations in sportsmen⁴². In our study, the increased serum Mn concentration was associated with lower serum ferritin levels which caused excess absorption of Mn^43,44. This phenomenon was common among long-distance runners, which was in accordance with the previous report⁴⁵.

Se exists in selenoproteins in the form of selenocysteine and selenomethionine⁴⁶. Selenoproteins affect the free radical metabolism, antioxidant function, and immune function in the body^18,47,48. Among the different selenoproteins, the glutathione peroxidase (GPx) and glutathione reductase (GR) constitute the glutathione redox cycle, which is an essential antioxidant system in the body⁴⁹. This study found that the decline in serum Se levels is associated with the prolonged state of oxidative stress maintained by athletes during training, when a large amount of reactive oxygen species will consume selenium-containing antioxidants, such as selenoproteins, thioredoxin reductase, and methionine sulfoxide reductase B. The depletion of antioxidant proteins causes the Se element to be released from the proteins, existing in body fluids in a free form and being lost through sweat. According to the reports, the depletion of Se-containing antioxidants such as glutathione peroxidase (GPx) during training caused the transfer of selenium from the blood to tissues, thereby leading to a decrease in the concentration of Se in the blood^50,51.

It was reported that acute exercise induced oxidative stress increased the level of MDA in serum⁵². This study found that both high- and low-intensity training caused significant increases in MDA levels compared with pre-exercise, owing to the aggravated lipid peroxidation⁵³. However, their difference in increased MDA levels was attributed to the fact that high-intensity exercise decreased the free radical scavenging ability of SOD, thereby slowing the decrease in the MDA level⁵⁴. The increased MDA levels during high-intensity training played a significant role in raising levels of Cu/Zn ratio, which indicated an important inflammatory-nutritional biomarker in human health⁵⁵.

Conclusion

In this study, the serum samples were pretreatment by direct dilution method, and the contents of several trace elements in the serum were determined by ICP-MS. The effects of different exercise levels on serum trace element concentrations were investigated among professional, amateur and sedentary subjects. It can be concluded that regular physical exercise significantly increased the serum Mn concentration and decreased the concentrations of Zn, Fe, Cu and Se. The correlations between trace element concentrations and training levels were significantly correlated. This study established a practical and efficient analytical method for serum samples with less sample needed, which was suitable for the determination of large-scale samples. The results may provide guidance for athlete’s training and nutritional supplementation strategies, prevent sports injuries and ensure the best competitive status.

Author contributions

Yumeng Xue: Investigation, Formal analysis, Methodology, Writing – original draft, Data curation, Conceptualization. Lei Zhang: Validation, Supervision, Formal analysis. Fei Fei: Supervision, Formal analysis, Writing – review & editing. Jiagen Yang: Investigation, Methodology, Data curation.

Funding

Declaration.

The authors declare that the funding for this research was supported by Doctoral Research Foundation of Zunyi Normal College (BS[2019]12) and Academic New Seedling Project of Zunyi Normal College (XM[2023] No. 1–07).

Data availability

Data is provided within the manuscript. More data may be provided from corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethic statement

This study was approved by the Ethics Committee of Liaocheng Maternal and Child Health Hospital (Approval No.: 20250501). All data used in this research were solely for scientific purposes, and all methods were carried out in accordance with relevant guidelines and regulations. All the participants were informed about the purpose of the study and voluntarily signed the informed consent.