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翻译文献 | 女性高雄激素状:目前对女性高雄激素状态实验室诊断的建议

2018-10-11 17:10 来源: 中国妇产科网 作者: 中国妇产科网 浏览量: 176

目前对总睾酮测定的建议

目前的立场主张适当使用免疫分析法和质谱分析法测量总睾酮以诊断女性的高雄激素状态。泰勒等人捍卫了这种现实的方法(40)。呼应了卡夫卡的哲学:“从正确的开始,而不是从可接受的开始”。考虑到这一目标,LC-MS /MS是一种可以提高特异性的非常精确的检测方法。然而,缺陷必须被识别和解决,它们同样呼应了伏尔泰(Voltaire)的格言:“最好是好的敌人”,认为用MS技术取代所有的免疫分析将是“一个不切实际和不必要的目标”,因为MS的可用性受到成本和技术需求的限制。对于睾酮检测方法的专家建议也应该是基于这种态度建立的。当世界各地都能够使用质谱分析设备时,最终的共识才可能达成(3,4,13,18)。

游离激素假说

游离激素假说(41,42)指出,未与蛋白结合的和循环中游离的激素部分才是有生物活性的。有充分的证据表明,游离激素的浓度比总的血浆激素水平能更准确地反映临床状况。这一概念已广泛应用于甲状腺功能的常规检查来避免甲状腺激素结合球蛋白(TBG)水平的变化对检测结果的影响,特别是在怀孕期间和口服避孕药的情况下。

同样的情况也适用于性激素结合球蛋白,性激素结合球蛋白是睾酮和雌二醇的主要转运系统,可以通过限制睾酮和雌二醇扩散到靶组织来调节它们的生物活性。游离激素假说已经在表达hSHBG的小鼠模型上得到了再次验证(43)。在这个模型中,循环中性激素结合球蛋白的显著增加使得与其结合的睾酮半衰期延长,从而使睾酮浓度升高。

相比之下,游离睾酮基本上没有变化,这可能是适应反馈调节使黄体生成素水平显著增加导致的。然而,雄性转基因小鼠表现出一种轻微的性腺功能低下的表型,这可能是由于靶细胞对性类固醇激素的生物利用度低导致的,可通过使用氚标记探针证明。这些结果有力地证实了性激素结合蛋白的一个重要生理功能,可维持睾酮水平并调节细胞对睾酮的生物利用度。这个模型可以转移为人类男性生理学,在男性中,性腺激素的反馈调节使得游离睾酮水平维持在正常范围内。

然而,在女性中,任何引起黄体生成素分泌的负反馈调节对雄性激素的影响都是微弱的(44)。相比之下,在晚卵泡期所观察到的,雌二醇升高的诱发可以使黄体生成素脉动分泌正反馈调节,也可以导致黄体生成素水平升高,并维持雄激素过量。在性激素结合蛋白水平较低的患者中尤其如此,这本身就可能导致临床高雄激素血症。有充分的证据表明,胰岛素抵抗(45)、肝脏的脂肪变和/或炎症(46,47)均可通过降调性激素结合球蛋白基因的肝脏表达可导致其水平降低。此外,也有文献记载,过量雄激素可降低正常作用下的黄体酮对促性腺激素释放激素脉冲频率的抑制,导致黄体生成素脉冲分泌加速,进一步维持或增加卵巢雄性激素的产生(48)。因此,黄体生成素的增加可促进卵巢生成更多的雄性激素,这是一个恶性循环,这种现象早期已经被Samuel Yen描述过(49)。目前的概念是雄激素过多会促进异常的中枢性黄体生成素分泌,从而使高雄激素状态持续下去(48)。值得注意的是,使用氟他胺(一种非甾类雄激素受体阻滞剂)对正常女性的促性腺激素调节基本上没有影响(50),但在青春期PCOS受试者中,氟他胺使得孕酮反馈表调节的敏感性部分恢复,使LH脉冲分泌减少(51)。

测量或计算游离睾酮浓度

        根据游离激素假说,测量未与蛋白结合的睾酮含量,可以对睾酮的总体产量和生物活性提供更好的指标(41,42)。技术上的挑战是一种开发方法,不干扰类固醇与结合蛋白之间的结合平衡,并可以实现比总睾酮浓度要低得的多的精确度和高敏感度游离睾酮浓度的测定。

平衡渗透法长期以来一直是定量前将结合蛋白从游离睾酮中分离出来的标准方法(52),这种方法认为可提供游离睾酮血浆浓度的最佳估计值,尽管在技术上难以实施(53)。在37度环境下选择适当的孵化时间,向血清标本中添加微量的3 H-睾酮,与性激素结合球蛋白及白蛋白相结合的部分睾酮可以透过小透析管被分离为游离睾酮。这种方法可以测量游离3H-睾酮的百分比,通常占血液循环中总睾酮的2-3%。这种方法还可以可靠的测量透析液中游离睾酮的实际浓度。此方法需要高纯度的3H -睾酮作为示踪剂(54,55)。

超滤离心透析已经被发现并且证实比平衡透析更快速,且技术要求更少(56)。将超滤法与GC-MS检测联用作为一种参照方法(57),近期改进为超滤法与LC-MS /MS相结合后进一步提高了分析灵敏度、方便性和样品要求(58)。

在等待这些有前景技术的常规应用时,临床医生必须记住,目前市面上可用于检测游离睾酮的免疫分析法的准确性和精密度都不够(59)。

用来计算游离睾酮浓度的替代方法

通过测定睾酮和SHBG浓度,应用精确的分析方法计算游离睾酮浓度,被认为是一种可以常规临床应用的可靠方法(60)。根据质量作用定律,它提供了直接测定游离睾酮的替代方法,但其依赖于所使用的方程式(61,62)。该计算方法假设性激素结合球蛋白的亲合力没有个体差异,且白蛋白浓度及白蛋白与睾酮的结合力是恒定的。它还假定,可以明显的从性激素结合球蛋白结合位点将睾酮置换的内源性配体产生的干扰是可以忽略不计的。

为了估计游离睾酮的含量,人们提出了各种各样的游离睾酮计算方法,包括游离睾酮指数(FTI),它是指总睾酮与SHBG之比,以百分比表示。理论上,当总睾酮与SHBG的摩尔比值较低时,游离睾酮指数与游离睾酮一致,这在女性中是如此,但在成年男性中并非如此(61,62)。一般而言,游离睾酮的计算值与平衡透析结果相关(60,63)。

我们应该认识到计算游离睾酮的陷阱。研究已经证明SHBG基因多态性导致了SHBG亲和力的不同(64),另一项研究报道了SHBG浓度对其亲合力的影响(65)。更重要的是,天然的内源性配体结合的邓恩平衡模型(19)证据表明,潜在的SHBG结合物如药物或内分泌干扰物可能干扰SHBG-结合位点,进而干扰游离激素比例(66)。

生物可利用的睾酮包括与白蛋白结合和与性激素结合球蛋白结合,这些被认为是循环中可被靶细胞生物利用的睾酮成分(67)。使用饱和硫酸铵可以沉淀球蛋白,包括SHBG,可以在离心前将SHBG结合的3H -睾酮与白蛋白结合的3H -睾酮和游离睾酮分离,这已被一些实验室报道是有效的方法(68)。饱和硫酸铵沉淀法的一个主要限制是球蛋白(包括SHBG)的不完全沉淀,这可能会增加测定内部和测定间的变异性。通过一些技术预防措施,未与性激素结合球蛋白结合的睾酮可以作为日常评估雄性激素过量的可靠工具(69,70)。

高雄激素患者的游离睾酮和总睾酮

根据Vermeulen等的研究,文献中所报道的大多数实验室是通过总睾酮水平、SHBG或白蛋白水平来计算游离睾酮水平(53)。Azziz等的研究报告称,在连续的临床高雄激素状态人群中,38%的病例总睾酮水平升高,而55.5%的病例出现游离睾酮升高;在这项研究中,有五分之一的患者雄性激素水平正常。Carmina等(72)的研究报告称,在连续的多毛症患者人群中,有三分之二的患者符合鹿特丹PCOS诊断标准,总睾酮是最常见的升高的雄性激素;游离睾酮对PCOS的诊断也有类似的提示,但并不优越。有趣的是,一项研究表明,血清SHBG水平对PCOS的诊断具有良好的敏感性和特异性(87%)(73)。据报道,游离睾酮浓度对PCOS的诊断很有用,但雌激素浓度单独或联合游离睾酮升高对PCOS更有鉴别性(74)。

替代方法:测量雄烯二酮

O ' reilly 等报告称(75),在鹿特丹标准诊断的PCOS患者中,睾酮水平升高与高雄烯二酮和游离雄激素指数相关;然而,高水平雄烯二酮是pcos相关的雄激素过量的一个更敏感的标志物。他们还报道了血清雄烯二酮和胰岛素敏感性之间的强烈负相关性,以及与雄激素表型的严重程度相关的糖耐量异常的高发生率。有趣的是,Lerchbaum(76)等的报道游离睾酮的升高与不良的代谢有关,而不像雄烯二酮升高是独立的。

联合评估总睾酮、雄烯二酮和游离睾酮的水平,为多囊卵巢综合征及其高雄激素亚型的诊断提供了良好的准确性(76,77,78)。然而,其在预测PCOS患者代谢异常风险中的应用需要进一步的研究(79)。

肾上腺来源的雄激素过量的研究

肾上腺皮质的网状带在促肾上腺皮质激素(ACTH)控制下的产生DHEA,并没有证据显示DHEA对垂体ACTH分泌存在负反馈调节。DHEA半衰期非常短,只有50分钟,其清除率高达2040升/天(80)。而有一部分DHEA通过DHEA磺基转移酶的作用被硫酸化为硫酸脱氢表雄酮(81)。硫酸脱氢表雄酮与白蛋白紧密结合,并以非常缓慢的速度(12.8 L/天)从循环中清除,其半衰期约为17 h(80)。因此,在女性中,DHEAS的浓度全天相对稳定(82)。硫酸脱氢表雄酮是活性雄激素的前体物质,可以被卵巢中的卵泡摄取,合成睾酮(83),甚至可以在外周组织直接转化为雄烯二酮,而不需要睾酮的前体形式(84)。

DHEA产生量随年龄增长而变化,30岁后其生成量开始下降(85),而ACTH的分泌并没有变化。这种雄激素分泌的老化过程可能与某些酶活性的下降有关,例如存在于肾上腺网状层的细胞色素-b5依赖性的(17,20)裂解酶(CYP17) (85),但仍有待进一步研究。胆盐磺基转移酶是在肾上腺和肝脏中负责DHEA硫酸化的主要的酶。硫酸盐供体磷酸硫酸(PAPS)是磺基转移酶维持活性所必需的。在人体中,酸合成酶合成的,即PAPSS1和PAPSS2。DHEA硫酸化为DHEAS是DHEA代谢的主要途径,这表明DHEA硫化物的增加可能限制了雄性激素合成所需的可利用的DHEA量。相比之下,已经有报道称在DHEAS水平很低但雄性激素水平增加的患者中发现了编码人类PAPS合成酶2 (PAPSS2)的基因突变,在DHEAS水平很低但雄激素水平增加的患者中被报道。这是一个单基因源性、肾上腺皮质原因导致的雄激素过量(86)。

免疫分析法测定硫酸脱氢表雄酮

因为硫酸脱氢表雄酮是一种水溶性抗原,大量存在于人类血浆中(年轻受试者中约6µmol / L),可以根据不同的方法(放射免疫法、带有非放射性标记的自动免疫法等)对经过或未经稀释的血浆样本进行免疫分析。使用特定浓度的高特异性抗体可以使得其他类固醇的干扰非常小(12)。

高雄激素状态中的硫酸脱氢表雄酮

当硫酸脱氢表雄酮被用作肾上腺雄激素(AAP)的标志物时,大约20-30%的高雄激素状态和/或PCOS女性表现出过量的肾上腺雄激素(AAP)(87)。高DHEAS水平的多毛症患者可能表现出类似PCOS的表型。在DHEA/DHEAS增加的患者中,导致类固醇生成异常的酶的遗传缺陷只占很小的一部分。相比之下,许多PCOS患者表现为在促肾上腺皮质激素刺激下的肾上腺源类固醇产量普遍增加,而没有证据显示下丘脑-垂体轴功能障碍(88)。

体内实验表明胰岛素可以促进PCOS患者肾上腺类固醇生成对促肾上腺皮质激素的反应(88,89),这与体外数据(90,91)一致。与此相反,二甲双胍(92)和噻唑烷二酮治疗降低了PCOS患者的DHEAS水平,因为这些药物降低了胰岛素抵抗和高胰岛素血症(93,94)。在猴子身上的研究与这些结果一致,尽管在这些领域也有一些相反的结果(96)。

关于卵巢雄激素对肾上腺雄激素分泌的影响及睾酮的作用,在体内和体外研究中都存在争议(97);在PCOS患者中得出的结论是卵巢雄激素,尤其睾酮,对肾上腺皮质功能的影响非常有限(87)。来自医学基因检测的初步结果支持了一个观点,即与胰岛素抵抗有关的一些基因可能与过量的肾上腺雄激素相关(98)。

非典型先天性肾上腺增生(NCAH)在高雄激素状态中的筛查

绝大多数的指南建议,NCAH诊断应依据晨间血浆17-羟孕酮(17OHP)的水平,且在周期的第一个阶段进行取样最佳,并排除多毛症和PCOS患者。对于基础17OHP水平的评估应在没有进行糖皮质激素治疗或已治疗后,否则可能导致假阴性结果出现。莫兰等进行了一项最大的多中心研究(99),结果显示基础17OHP水平高于10ng /mL是NCAH诊断的敏感标准,这一结论与最近的一项单中心研究一致(100)。而在已从基因学证实存在非典型先天性肾上腺增生的患者中,晨间基础17OHP < 2ng /mL与8%的假阴性相关。

为了提高筛选效率,人们设计开发了ACTH激发试验,使用250µg 促皮质素激发。Morel和Tardy(101)的研究表明,当以>10ng /mL为界线时,几乎100%的经过基因验证的NCAH患者,无论突变类型如何,都可以被识别。这种非常精确的界限值常规应用于高雄激素状态女性的NCAH筛查(102、103、104、105、106)。在不久的将来,测序平台的发展将使CYP21A2基因的常规测序成为可能。

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翻译:周瑶

单位:广州市南方医院


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