Basic research to establish a treatment for asthenozoospermic patients.

目的：(1)探讨弱精子症患者精子中活性氧(ROS)的形成情况及其与精子运动参数的关系。(2)评价己酮可可碱对精子产生高ROS的弱精子症患者的体内外治疗效果。材料与方法：(1)采用化学发光法测定弱精子症患者和正常生育志愿者精子ROS的生成。在无刺激(稳态)和N-甲酰-甲硫基亮氨酸苯丙氨酸(f-MLP)或佛波酯-12-肉豆蔻酸酯-13-醋酸酯(PMA)刺激后，观察精子标本的ROS形成情况。(2)用己酮可可碱对15例弱精子症患者的精子进行体外处理，观察其对ROS生成和精子运动参数的影响。(3)相同的15例…患者和18例弱精子症患者以不同剂量(300 mg/d和1200 mg/d)的己酮可可碱处理更多稳态不产生ROS的精子，观察其对体内ROS生成、精子运动参数和精子受精能力的影响。结果：(1)在无刺激和f-MLP不刺激的情况下，精子产生ROS的来源可能是精子本身。(2)在71例弱精子症患者中，有15例的精子在稳态状态下产生ROS。这15例患者被分成A组。(3)己酮可可碱可减少A组患者精子产生ROS，并使曲线速度和搏动交叉频率的降低维持6h。(4)A组患者口服己酮可可碱不能减少精子产生ROS。A组口服己酮可可碱小剂量(300 mg/d)对精子活力、精子运动参数和精子受精能力无影响。大剂量(1200 mg/d)可提高精子活动率和搏动频率，但对精子受精能力无明显影响。结论：f-MLP刺激精子制备物可明确稳态产生ROS的来源。在弱精子症患者中，有一组患者(A组)的精子产生可检测到的稳定水平的ROS。在这组患者中，己酮可可碱似乎有效地减少了ROS的形成，并在体外保留了精子的运动参数。口服己酮可可碱在低剂量时无效，但在高剂量时可提高精子活力和某些精子运动参数，但不改变精子受精能力。未来：目前尚无有效治疗特发性弱精子症等精子功能缺陷患者的方法。要开发新的治疗策略，重要的是要更多地了解这些缺陷的性质。本研究结果可能为特发性男性因素不育症的治疗提供线索。为了证实这些结果，有必要进行前瞻性的双盲临床试验，将生殖力作为关键的结果衡量标准。较少

Purpose : (1) To determine the incidence of reactive oxygen species (ROS) formation by spermatozoa from asthenozoospermic patients and the relationship between ROS formation and sperm motion parameters. (2) To assess the efficacy of in vitro and in vivo pentoxifylline treatment of asthenozoospermic patients whose spermatozoa generated high ROS levels.Materials and Methods : (1) ROS formation by spermatozoa from asthenozoo-spermic patients and fertile volunteers was measured by chemiluminescence. ROS formation by the sperm preparations was investigated without stimulation (steady state) or after stimulation with N-formyl-methionyleucyl-phenylalanine (f-MLP) or phorbol-12-myristate-13-acetate (PMA). (2) Spermatozoa from 15 asthenozoospermic patients, whose spermatozoa produced high levels of ROS at steady state, were treated in vitro with pentoxifylline to determine its effect on ROS generation and sperm motion parameters. (3) These same 15 patients and 18 asthenozoospermic patients whos … More e spermatozoa did not produce ROS at steady state were treated with pentoxifylline at two different dosages (300 mg and 1200 mg/day) to determine its effect on ROS generation, sperm motion parameters and sperm fertilizing ability in vivo.Results : (1) When the steady state ROS formation was detected without stimulation and that was not stimulated by f-MLP,the source of ROS could be attributed to the spermatozoa themselves. (2) Spermatozoa from 15 out of 71 asthenozoospermic patients generated ROS at steady state. These 15 patients were assigned to group A patients. (3) Pentoxifylline decreased ROS generation by spermatozoa from group A patients and preserved the decrease of curvilinear velocity and beat cross frequency for 6 hrs in vitro. (4) For group A patients orally administered pentoxifylline failed to decrease ROS generation by spermatozoa. For group A patients orally administered pentoxifylline had no effect on sperm motility, sperm motion parameters and sperm fertilizing ability at low dosage (300mg/day). It increased motility and beat cross frequency at high dosage (1200mg/day), but had no effect on sperm fertilizing ability in group A patients.Conclusions : Stimulation of sperm preparations with f-MLP can identify the source of ROS generated at steady state. Among asthenozoospermic patients, there was a group of patients (group A) whose spermatozoa produces detectable steady state levels of ROS.For this group, pentoxifylline appeared to be effective for reducing ROS formation and preserving sperm motion parameters in vitro. Orally administered pentoxifylline had no effect at low dosage, however, it increased sperm motility and some sperm motion parameters without altering sperm fertilizing ability at high dosage.Future : Currently, there is no effective treatment for patients with deficient sperm functions, such as idiopathic asthenozoospermia. To develop new treatment strategies, it is important to learn more about the nature of these deficiencies. The present results may provide clues to a therapeutic approach for the treatment of idiopathic male-factor infertility. For the confirmation of these results, a prospective and double-blind clinical trial, dealing with fecundity as the crucial outcome measure, is necessary. Less