Integrative Molecular and Functional Analysis of Human Sperm Subpopulations to Identify New Biomarkers of Fertilization Potential

IF 4.7 3区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Archives of Medical Research Pub Date : 2025-03-31 DOI:10.1016/j.arcmed.2025.103210

Pedro Caballero-Campo , Saúl Lira-Albarrán , Alexandra Amaral , Chibo Hong , Nakul Shah , Annaick Carles , Daofeng Li , David Barrera , Gabriela Hernández-Silva , Joao Ramalho-Santos , Ting Wang , Martin Hirst , Fernando Larrea , Joe Costello , Paolo Rinaudo , Mayel Chirinos

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引用次数: 0

Abstract

Background

Human ejaculates are composed of sperm subsets with heterogeneous characteristics. Comparative studies of sperm subpopulations with differences in motility may serve to investigate the functional and molecular features that are crucial for reaching the oocyte and fertilizing.

Objective

To identify functional and molecular markers that characterize sperm subpopulations with high and low motility.

Materials and Methods

Semen samples from 11 donors with proven fertility were processed by density gradient centrifugation to isolate high (F1) and low (F2) motility sperm subpopulations. Besides motility, we evaluated viability, chromatin integrity, mitochondrial membrane potential, capacitation, and acrosomal status. F1 and F2 were subjected to comparative methylome and transcriptome analyses by whole-genome bisulfite sequencing and RNA sequencing, respectively. Further validation of candidate biomarkers at the RNA and protein levels was performed with semen samples from six normozoospermic volunteers using quantitative PCR and Western blotting.

Results

Spermatozoa from the F1 fractions exhibited higher mitochondrial membrane potential and viability than F2. Comparative methylome and transcriptome analyses of F1 and F2 identified 271 differentially methylated genes and 82 differentially expressed genes. Notably, CEP128 and CSTPP1 were downregulated and differentially methylated in the F2 fraction. Quantitative PCR confirmed the downregulation of these two genes in F2, and the downregulation of CEP128 was further validated at the protein level by Western blotting.

Conclusion

F1 spermatozoa are characterized by elevated mitochondrial membrane potential, viability, and higher expression of CEP128 and CSTPP1. Future studies should evaluate the potential of these functional variables and genes as biomarkers of fertility, either individually or in combination.

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背景人类射出的精子由具有不同特征的精子亚群组成。材料与方法通过密度梯度离心法处理 11 位具有生育能力的供体的精子样本，分离出高（F1）和低（F2）活力精子亚群。除运动能力外，我们还评估了精子活力、染色质完整性、线粒体膜电位、获能能力和顶体状态。通过全基因组亚硫酸氢盐测序和 RNA 测序，分别对 F1 和 F2 进行了甲基组和转录组比较分析。利用定量 PCR 和 Western 印迹技术对六名正常无精子症志愿者的精液样本进行了 RNA 和蛋白质水平候选生物标记物的进一步验证。F1 和 F2 的甲基组和转录组比较分析确定了 271 个不同的甲基化基因和 82 个不同的表达基因。值得注意的是，在 F2 部分中，CEP128 和 CSTPP1 下调且甲基化程度不同。定量 PCR 证实了这两个基因在 F2 中的下调，Western 印迹进一步验证了 CEP128 在蛋白质水平上的下调。未来的研究应评估这些功能变量和基因作为生育力生物标志物的潜力，无论是单独使用还是结合使用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Archives of Medical Research 医学-医学：研究与实验

CiteScore

12.50

自引率

0.00%

发文量

审稿时长

28 days

期刊介绍： Archives of Medical Research serves as a platform for publishing original peer-reviewed medical research, aiming to bridge gaps created by medical specialization. The journal covers three main categories - biomedical, clinical, and epidemiological contributions, along with review articles and preliminary communications. With an international scope, it presents the study of diseases from diverse perspectives, offering the medical community original investigations ranging from molecular biology to clinical epidemiology in a single publication.