Single-cell analysis of [Ca2+]i signalling in sub-fertile men: characteristics and relation to fertilization outcome.
Single-cell analysis of [Ca2+]i signalling in sub-fertile men: characteristics and relation to fertilization outcome.
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DOI：
10.1093/humrep/dey096
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发表时间：
2018-06-01
期刊：
影响因子：
--
通讯作者：
Martins Da Silva S
中科院分区：
文献类型：
--
作者：
Kelly MC;Brown SG;Costello SM;Ramalingam M;Drew E;Publicover SJ;Barratt CLR;Martins Da Silva S
生育力低下的男性精子中黄体酮诱导（CatSper 介导）的单细胞 [Ca2+]i 信号有何特征？它们与受精能力有何关系？黄体酮诱导（CatSper 介导）[Ca2+]i 的单细胞分析表明，黄体酮敏感性降低是生育力低下患者精子的一个共同特征，并且与受精率相关。黄体酮刺激是一种广泛使用的方法，通过激活人类精子中的 CatSper 来评估 [Ca2+]i 动员。尽管数据有限，但精子群体研究表明，[Ca2+]i 对黄体酮的反应较差与受精能力降低有关。这是一项队列研究，使用了 21 名捐献者和 101 名在邓迪尼尼威尔斯医院辅助受孕科接受 ART 治疗的患者的精液样本。患者于 2016 年 1 月至 2017 年 6 月期间招募。精液捐献者和患者是根据苏格兰东部研究伦理服务 (EoSRES) REC1 的当地伦理批准 (13/ES/0091) 招募的。通过单细胞成像检查 [Ca2+]i 反应，并通过计算机辅助精子分析 (CASA) 评估运动参数。为了进行分析，患者样本分为三组：IVF(+ve)（受精成功；62 个样本）、IVF-FF（受精失败；8 个样本）和 ICSI（21 个样本）。另外 10 个 IVF 样本显示出较大的自发 [Ca2+]i 振荡，并且无法分析对黄体酮的反应。所有装载有[Ca2+]i-指示剂fluo4的患者样本对黄体酮刺激作出反应，荧光双相增加（短暂然后稳定），这与黄体酮刺激的供体样本中看到的相似。 IVF-FF 和 ICSI 患者组的平均标准化反应（黄体酮诱导的荧光强度标准化至静息水平）显着小于供体。通过绘制每个细胞的静息荧光强度和孕酮诱导的荧光增量之间的关系来进一步分析所有样品。在供体样本中，这些图紧密重叠，梯度为 ≈ 2，并且大多数 IVF(+ve) 样本的图与供体分布非常相似。然而，在 IVF(+ve) 样本的子集（约 10%）、3/8 IVF-FF 样本和三分之一的 ICSI 样本中，图的梯度明显较低，表明这些样本中的细胞对孕酮的反应异常小。对 IVF 样本的梯度（图的回归系数）与受精率之间关系的检查显示出正相关。在 IVF-FF 和 ICSI 组中，可检测到黄体酮反应的细胞比例显着低于供体和 IVF (+ve) 患者。供体、IVF(+ve) 和 ICSI 样品中大约 20% 的细胞在受到黄体酮刺激时产生 [Ca2+]i 振荡，但在 IVF-FF 样品中只有约 10% 的细胞产生振荡，并且没有观察到振荡的样品比例明显更高。与对照组相比，IVF(+ve) 和 IVF-FF 组的过度活跃水平较低，IVF-FF 的水平也低于 IVF(+ve)。这是一项体外研究，在体内推断这些结果时必须小心。这项研究揭示了生育力低下男性精子中 [Ca2+]i 信号传导受损的重要细节，而这些细节在群体研究中无法检测到。这项研究由 MRC 项目拨款 (MR/M012492/1; MR/K013343/1) 资助。首席科学家办公室/NHS 苏格兰研究中心提供了额外的资金。
What are the characteristics of progesterone-induced (CatSper-mediated) single cell [Ca2+]i signals in spermatozoa from sub-fertile men and how do they relate to fertilizing ability? Single cell analysis of progesterone-induced (CatSper-mediated) [Ca2+]i showed that reduced progesterone-sensitivity is a common feature of sperm from sub-fertile patients and is correlated with fertilization rate. Stimulation with progesterone is a widely used method for assessing [Ca2+]i mobilization by activation of CatSper in human spermatozoa. Although data are limited, sperm population studies have indicated an association of poor [Ca2+]i response to progesterone with reduced fertilization ability. This was a cohort study using semen samples from 21 donors and 101 patients attending the assisted conception unit at Ninewells Hospital Dundee who were undergoing ART treatment. Patients were recruited from January 2016 to June 2017. Semen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service (EoSRES) REC1. [Ca2+]i responses were examined by single cell imaging and motility parameters assessed by computer-assisted sperm analysis (CASA). For analysis, patient samples were divided into three groups IVF(+ve) (successful fertilization; 62 samples), IVF-FF (failed fertilization; eight samples) and ICSI (21 samples). A further 10 IVF samples showed large, spontaneous [Ca2+]i oscillations and responses to progesterone could not be analysed. All patient samples loaded with the [Ca2+]i-indicator fluo4 responded to progesterone stimulation with a biphasic increase in fluorescence (transient followed by plateau) which resembled that seen in progesterone-stimulated donor samples. The mean normalized response (progesterone-induced increase in fluorescence normalized to resting level) was significantly smaller in IVF-FF and ICSI patient groups than in donors. All samples were further analysed by plotting, for each cell, the relationship between resting fluorescence intensity and the progesterone-induced fluorescence increment. In donor samples these plots overlaid closely and had a gradient of ≈ 2 and plots for most IVF(+ve) samples closely resembled the donor distribution. However, in a subset (≈ 10%) of IVF(+ve) samples, 3/8 IVF-FF samples and one-third of ICSI samples the gradient of the plot was significantly lower, indicating that the response to progesterone of the cells in these samples was abnormally small. Examination of the relationship between gradient (regression coefficient of the plot) in IVF samples and fertilization rate showed a positive correlation. In IVF-FF and ICSI groups, the proportion of cells in which a response to progesterone could be detected was significantly lower than in donors and IVF (+ve) patients. Approximately 20% of cells in donor, IVF(+ve) and ICSI samples generated [Ca2+]i oscillations when challenged with progesterone but in IVF-FF samples only ≈ 10% of cells generated oscillations and there was a significantly greater proportion of samples where no oscillations were observed. Levels of hyperactivated motility were lower in IVF(+ve) and IVF-FF groups compared to controls, IVF-FF also having lower levels than IVF(+ve). This is an in vitro study and caution must be taken when extrapolating these results in vivo. This study reveals important details of impaired [Ca2+]i signalling in sperm from sub-fertile men that cannot be detected in population studies. This study was funded by a MRC project grant (MR/M012492/1; MR/K013343/1). Additional funding was provided by Chief Scientist Office/NHS research Scotland.
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DOI：
10.1093/humrep/dex055
发表时间：
2017-05-01
期刊：
Human reproduction (Oxford, England)
影响因子：
--
作者：
Martins da Silva SJ;Brown SG;Sutton K;King LV;Ruso H;Gray DW;Wyatt PG;Kelly MC;Barratt CLR;Hope AG
通讯作者：
Hope AG
影响因子：
2.7
作者：
Bedu-Addo, K.;Barratt, C. L. R.;Publicover, S. J.
通讯作者：
Publicover, S. J.
影响因子：
11.4
作者：
Brenker, Christoph;Goodwin, Normann;Struenker, Timo
通讯作者：
Struenker, Timo
DOI：
10.1073/pnas.0610286104
发表时间：
2007-01-23
影响因子：
11.1
作者：
Qi, Huayu;Moran, Magdalene M.;Clapham, David E.
通讯作者：
Clapham, David E.
DOI：
10.1093/humrep/dew056
发表时间：
2016-06
期刊：
Human reproduction (Oxford, England)
影响因子：
--
作者：
Brown SG;Publicover SJ;Mansell SA;Lishko PV;Williams HL;Ramalingam M;Wilson SM;Barratt CL;Sutton KA;Da Silva SM
通讯作者：
Da Silva SM