Department of Biological Science, University of Newcastle, Callaghan, NSW, Australia.
Department of Pharmacology and Therapeutics, University of Melbourne, Parkville, VIC, Australia.
Hum Reprod. 2021 Jul 19;36(8):2073-2082. doi: 10.1093/humrep/deab134.
What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes.
The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells.
Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation.
STUDY DESIGN, SIZE, DURATION: We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells.
PARTICIPANTS/MATERIALS, SETTING, METHODS: We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa.
CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation.
LIMITATIONS, REASONS FOR CAUTION: The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique.
CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes.
STUDY FUNDING/COMPETING INTEREST(S): The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare.
N/A.
用于体外受精目的的高密度纯化人精子通常具有怎样的核异质性。
数据表明，虽然密度梯度分离已经改善了整体精子群体，但这些细胞内仍然存在很大程度的核异质性。
吖啶橙（CMA3）是评估男性生育能力的重要 DNA 结合荧光染料。它通常用于评估整个精子样本的体外受精结果，其接受者操作特征曲线非常高。在这里，我们使用 CMA3 来描述经过密度梯度分离后用于体外受精目的的典型人精子群体。
研究设计、规模、持续时间：我们比较了来自男性的高密精子群体中 CMA3 结合的强度。通过荧光显微镜和流式细胞术分析独立证实了结合的异质性。我们还在该精子群体中直接观察了 CMA3 染色与头部形态之间的关系。最后，我们观察了 CMA3 染色细胞经过密度梯度分离后的精子核异质性（空泡、染色质浓缩）的电子显微镜照片。
参与者/材料、设置、方法：我们使用已经生育了一个或多个孩子的捐精者。禁欲 2 天后采集精液，并在 Percoll 梯度上纯化。只有高质量的精子，即用于辅助受孕的精子，才被用于后续实验。细胞用 CMA3 染色，并用流式细胞术进行分选。之后，用电子显微镜观察 CMA3 依赖的分选精子的核异质性。
CMA3 染色发生在形态正常和形态异常的精子中。高强度 CMA3 染色的精子具有大的空泡，而低 CMA3 染色的精子中则没有。此外，高 CMA3 染色的细胞具有更高数量的核颗粒。
局限性、谨慎的原因：本研究仅描述了这些细胞染色质内的问题，并未提出替代选择技术。
CMA3 是预测妊娠结局的一种较好的报告预后检测方法，尤其是在男性有问题的情况下。然而，很明显，即使在人精子的分离群体中，也有形态正常但具有高强度 CMA3 染色和染色质颗粒的精子。这意味着，选择基于精子形态的胚胎学家可能会选择具有不良染色质的细胞，这可能导致胚胎结局不佳。
研究资金/利益冲突：该项目由澳大利亚国家健康与医学研究委员会资助，项目号为 APP1118943。作者没有利益冲突需要申报。
无。
