1H Magnetic Resonance Spectroscopy of live human sperm.
1H Magnetic Resonance Spectroscopy of live human sperm.
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DOI：
10.1093/molehr/gax025
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发表时间：
2017-07-01
影响因子：
4
通讯作者：
Pacey AA
中科院分区：
文献类型：
--
作者：
Reynolds S;Calvert SJ;Paley MN;Pacey AA
1H磁共振波谱（MRS）可以用来获得有关活的人类精子中的分子和代谢物的信息吗？Percoll-based密度梯度离心（DGC），然后再进行两次洗涤步骤，产生了足够的精子，精液中的污染最小（<0.01%），以允许有效的MRS，检测到颗粒中的精子和40%/80%界面的精子之间的1H MRS谱的胆碱/甘油磷酸胆碱（GPC）、脂质和乳酸盐区域存在显著差异（P < 0.05）。目前检查精子的方法要么价值有限（例如精液分析），要么具有破坏性（例如免疫组织化学，精子DNA检测）。以前有一些研究使用MRS检查精子，但这些研究要么观察了具有不同射精质量的男性的精浆，要么观察了冻干精子混合样本中的分子。在通过三种不同方法之一从新鲜精液中回收后，通过1H MRS扫描使用1H激发雕刻溶剂抑制序列检查37°C下悬浮在磷酸盐缓冲盐水（PBS）中的精子：（i）简单离心;（ii）DGC一次洗涤;或（iii）DGC两次洗涤。在DGC的情况下，从颗粒（“80%”精子）和40/80界面（“40%”精子）收集精子。使用自定义Matlab脚本进行光谱处理，以确定精浆/Percoll污染的程度、1H MRS检测的最小精子浓度以及“40%”和“80%”精子的1H MRS光谱之间的差异。DGC用两次洗涤使精浆和Percoll/PBS溶液污染的1H MRS峰强度最小化，同时保留精子特异性峰。对于本研究中使用的MRS扫描仪，产生大于3：1信噪比（SNR）的胆碱/GPC 1H MRS峰所需的最低精子浓度估计为约3 × 106/ml。双因素方差分析表明，1H谱的胆碱/GPC和乳酸/脂质区域存在显著差异（P < 0.0001; n = 20）。这些区域的ROC曲线分析显示了区分两种精子群体的显著能力：胆碱/GPC ROC AUC = 0.65-0.67，乳酸/脂质ROC AUC = 0.86-0.87。仅使用3-4个精液样本来评估所检查的每个精子洗涤方案的有效性。MRS所需的估计最低精子浓度特定于我们研究中使用的硬件，并且在其他光谱仪中可能不同。光谱分箱是一种低分辨率分析方法，其对化学位移范围内的MRS峰进行求和。这可能会模糊哪些代谢物是造成精子群体之间差异的原因。需要进一步的工作，以确定相对贡献的体细胞的MRS频谱从“40%”和“80%”的精子。 1H MRS可以提供有关活精子中存在的分子的信息，因此可以允许研究活精子的潜在功能生物学或代谢组学。由于获得合适的MRS信号所需的精子浓度相对较低（约3 × 106/ml），因此可以对少精子症、弱精子症或畸形精子症男性的精子进行检测。这可能会导致新的诊断测试或最终的男性因素不孕症的新疗法的发展。这项工作得到了医学研究理事会拨款MR/M010473/1的支持。作者声明无利益冲突。
Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa? Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface. Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm. Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet (‘80%’ sperm) and the 40/80 interface (‘40%’ sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of ‘40%’ and ‘80%’ sperm. DGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65–0.67, lactate/lipid ROC AUC = 0.86–0.87. Only 3–4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the ‘40%’ and ‘80%’ sperm. 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility. This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.
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影响因子：
2.8
作者：
Huser, Thomas;Orme, Christine A.;Balhorn, Rod
通讯作者：
Balhorn, Rod
DOI：
10.1016/j.plefa.2007.07.001
发表时间：
2007-07-01
影响因子：
3
作者：
Tavilani, Heidar;Doosti, Mahmoud;Hosseinipanah, Seyed Mohammad
通讯作者：
Hosseinipanah, Seyed Mohammad
DOI：
10.1016/0731-7085(94)80004-9
发表时间：
1994-01-01
影响因子：
3.4
作者：
LYNCH, MJ;MASTERS, J;NICHOLSON, JK
通讯作者：
NICHOLSON, JK
DOI：
10.1016/b978-0-12-372568-4.00015-x
发表时间：
2009-01-01
期刊：
SPERM BIOLOGY: AN EVOLUTIONARY PERSPECTIVE
影响因子：
--
作者：
Pacey, Allan A.
通讯作者：
Pacey, Allan A.
影响因子：
6.7
作者：
Amaral, Alexandra;Paiva, Carla;Ramalho-Santos, Joao
通讯作者：
Ramalho-Santos, Joao