背景：维生素D是指一组在调节人体内钙和磷的稳态方面具有明显作用的脂溶性类固醇。在人体中，维生素D主要以维生素D3(胆钙化醇)和维生素D2(麦角钙化醇)两种形式存在。维生素D在肝脏经历了第一次羟基化并产生了25羟基维生素D(25 hydroxyvitamin D，25(OH)D)，25(OH)D是维生素D的主要循环形式，是评估体内维生素D水平的最佳指标。接着，25(OH)D被运送到肾脏，经历另一步羟化过程，转变为具有生物学活性的1α，25二羟基维生素D(1α，25-dihydroxycholecalciferol，1,25(OH)₂D)。近年来，维生素D缺乏已成为全球性的健康问题，对维生素D与男性生殖系统的研究也逐渐成为热点。此前有研究分析了血清维生素D水平与男性生殖功能的关系，但尚无统一结论。 目的：本研究旨在综合探讨血清及精浆维生素D水平与男性生殖功能的关系，为研究维生素D对男性生殖的具体作用机制提供研究方向。并在体外以维生素D的活性物质—1，25(OH)₂D处理精子，研究其对精子的具体作用机制。 方法：临床试验：根据纳排标准纳入来我科男科门诊检查的不育患者及正常体检者，通过电化学发光免疫分析法(Electrochemiluminescence immunoassay， ECLIA)检测25(OH)D水平，并评估血清和精浆25(OH)D与精液质量，精子运动学参数，循环抑制素B(Inhibin B，INHB)，抗苗勒管激素(Anti-Mullerian Hormone，AMH)，及精浆生化指标之间的关系。体外实验：用密度梯度离心法获取精子，在不含或者含不同浓度1,25(OH)₂D的培养液中进行孵育，检测精子的运动参数，爬高能力及相关指标的变化。 结果：临床试验：血清25(OH)D与精液量，精浆果糖浓度，总果糖量和总锌量呈正相关。精浆25(OH)D水平与血清25(OH)D水平无关，而与精液量和精子运动参数呈正相关。体外实验：使用维生素D的活性形式1,25(OH)₂D孵育精子，精子的运动学参数显著增加，尤其是在用0.1nmol／L 1，25(OH)₂D处理30分钟时，增加最为显著。在这种孵育条件下，精子的爬高高度伴随三磷酸腺苷(Adenosine triphosphate，ATP)的增加而提升。精子胞内的环磷酸腺苷(Cyclic adenosine monophosphate，cAMP)的浓度升高，蛋白激酶A(Protein kinase A，PKA)的活性增加，同时PKA抑制剂(H89)阻碍了1,25(OH)₂D对ATP的促进作用。精子胞内钙离子和烟酰胺腺嘌呤二核苷酸(Nicotinamide adenine dinucleotide，NADH)的浓度都有增加，磷脂酶C(Phospholipase C，PLC)抑制剂U-73122阻碍了1,25(OH)2D对胞内钙离子及NADH的促进作用，而线粒体钙离子转运体(Mitochondrial calcium uniporter，MCU)抑制剂(Ru360)没有阻碍1,25(OH)₂D对NADH及ATP的促进作用。 结论：1.精浆中的维生素D可能参与了精子运动的调节。2.1,25(OH)₂D可通过促进cAMP／PKA通路及细胞内钙离子的增加来促进ATP合成，从而增强精子活力。 关键词：精浆维生素D，男性生殖功能，精子运动参数，钙离子，NADH，cAMP， PKA，ATP

 BACKGROUND: Vitamin D refers to a group of fat-soluble steroids that play a pronounced role in regulating the homeostasis of calcium and phosphorus in the body. In humans, the most important compounds in this group of steroids are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Both vitamin D2 and vitamin D3 undergo their first hydroxylation in the liver to yield 25 hydroxyvitamin D (25(OH)D) , which is the major form of vitamin D in circulation and is the best indicator for assessing vitamin D level. 25(OH)D is transported to the kidney where, through another hydroxylation process, it becomes the biologically active form 1α,25-dihydroxycholecalciferol ( l,25(OH)₂D ) . Vitamin D deficiency is regarded as a widespread health problem. In recent decades, researches on vitamin D have extended the spectrum to the male reproductive system. Previous studies on the relationship between the overall level of vitamin D and semen quality have presented conflicting results. OBJECTIVE : In this study, we aimed to evaluate the associations of male reproductive function with both serum and seminal plasma vitamin D levels and study the mechanisms underlying these by incubating spermatozoa with l,25(OH)₂D in vitro. METHODS: Clinical trial: Based on the enrollment and exclusion criteria, men for andrological evaluation owing to male infertility or normal physical examination were included in our study. 25(OH)D was detected using an electrochemiluminescence method. Then the relationships between serum and seminal plasma 25(OH)D with semen quality, sperm kinetic parameters, Anti-Mullerian Hormone (AMH ) , Inhibin B ( INHB ) and seminal plasma biochemical markers were evaluated. In vitro: Spermatozoa were isolated by density gradient centrifugation method and were incubated in the medium without or with different concentrations of l,25(OH)₂D. Then the kinectic parameters, upward migration and relative makers of spermatozoa were detected. RESULTS: Positive relationships of serum 25(OH)D with semen volume, seminal plasma fructose and zinc were identified. Seminal plasma 25(OH)D level showed no relationship with serum 25(OH)D level, while it had positive associations with semen volume and the sperm kinetic values. In vitro, sperm kinetic parameters increased after incubation with l,25(OH)₂D, especially upon incubation for 30 min with it at a concentration of 0.1 nmol / L. Under these incubation conditions, the upward migration of spermatozoa increased remarkably with increasing adenosine triphosphate (ATP) concentration. The concentration of cyclic adenosine monophosphate (cAMP) and the activity of protein kinase A (PKA) were both elevated, and the PKA inhibitor (H89) reversed the increase of ATP production. The concentrations of cytoplasmic calcium ions and nicotinamide adenine dinucleotde (NADH) were both enhanced, PLC (Phospholipase C) inhibitor U-73122 reversed the increase of cytoplasmic calcium ions and NADH, while mitochondrial calcium uniporter (MCU) inhibitor, Ruthenium 360 (Ru360) did not reverse the increase of NADH and ATP production. CONCLUSIONS: 1. Seminal plasma vitamin D may be involved in regulating sperm motility. 2. l,25(OH)2D may enhance sperm motility by promoting the synthesis of ATP both through the cAMP/PKA pathway and the increase in intracellular calcium ions. KEYWORDS: Seminal plasma vitamin D, male reproductive function, sperm kinetic parameter, calcium ion, NADH, cAMP, PKA, ATP