Perticarari S, Ricci G, Granzotto M, Boscolo R, Pozzobon C, Guarnieri S, Sartore A, Presani G
Clinical Analysis Unit, Department of Laboratory Medicine, Institute of Child Health IRCCS Burlo Garofolo, Italy.
Hum Reprod. 2007 Feb;22(2):485-94. doi: 10.1093/humrep/del415. Epub 2006 Nov 1.
The objectives of this study were (i) to evaluate whether the combined use of Syto 16 and 7-amino-actinomycin-D (7-AAD) allows the detection of sperm apoptosis and (ii) to describe a new multiparameter flow cytometric method to assess simultaneously sperm concentration (SC), viability and apoptosis as well as leukocyte concentration.
Semen samples from 68 patients were evaluated according to World Health Organization (WHO) criteria (normal, n=26; abnormal, n=42). The detection of activated caspases before and after betulinic acid (BA) incubation was carried out in 13 semen samples by flow cytometry using fluorescein-labelled inhibitors of caspases (FLICA). A multiparameter flow cytometric analysis was performed in 55 semen samples. Fluorescent microspheres were used to assess SC. Sperm apoptosis was detected by staining sperm with Syto 16 and 7-AAD. Leukocytes were counted using monoclonal anti-CD45.
A significant correlation between the percentage of the spermatozoa with low Syto 16 fluorescence and the percentage of spermatozoa containing activated caspases was found (r=0.68, P=0.0106; n=13). After incubation with BA, an increase of the percentage of apoptotic cells was observed in all samples, using both the Syto 16/7-AAD and the caspase activation methods. There was a good correlation between flow cytometry and optical microscopy for sperm (r=0.98, P < 0.0001) and leukocyte counting (r=0.64, P <0.0001). The percentage of apoptotic sperm was inversely correlated with both SC (r=-0.303, P=0.0246) and morphology (r=-0.384, P=0.0050) but not with motility.
The combination of Syto 16/7-AAD provides a sensitive assay to detect sperm apoptosis. The multiparameter flow cytometric method described offers the possibility of a simultaneous, simple, rapid and accurate assessment of several semen parameters.
本研究的目的是：（i）评估联合使用Syto 16和7-氨基放线菌素D（7-AAD）能否检测精子凋亡；（ii）描述一种新的多参数流式细胞术方法，以同时评估精子浓度（SC）、活力、凋亡以及白细胞浓度。
根据世界卫生组织（WHO）标准对68例患者的精液样本进行评估（正常，n = 26；异常，n = 42）。使用荧光素标记的半胱天冬酶抑制剂（FLICA）通过流式细胞术在13份精液样本中检测桦木酸（BA）孵育前后活化半胱天冬酶的情况。对55份精液样本进行多参数流式细胞术分析。使用荧光微球评估精子浓度。通过用Syto 16和7-AAD对精子进行染色来检测精子凋亡。使用单克隆抗CD45对白细胞进行计数。
发现Syto 16荧光低的精子百分比与含有活化半胱天冬酶的精子百分比之间存在显著相关性（r = 0.68，P = 0.0106；n = 13）。用BA孵育后，使用Syto 16/7-AAD和半胱天冬酶激活方法，在所有样本中均观察到凋亡细胞百分比增加。流式细胞术与光学显微镜在精子计数（r = 0.98，P < 0.0001）和白细胞计数（r = 0.64，P <0.0001）方面具有良好的相关性。凋亡精子的百分比与精子浓度（r = -0.303，P = 0.0246）和形态（r = -0.384，P = 0.0050）均呈负相关，但与活力无关。
Syto 16/7-AAD组合提供了一种检测精子凋亡的灵敏方法。所描述的多参数流式细胞术方法为同时、简单、快速且准确地评估多个精液参数提供了可能性。
