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Related Experiment Video

Updated: Oct 16, 2025

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Published on: July 12, 2017

Optimization of the sperm processing protocol for subsequent molecular cytogenetic studies.

Anastasiya Aleksandrovna Tarlycheva¹, Zh G Markova¹, D A Yurchenko¹

¹Research Centre for Medical Genetics.

Klinicheskaia Laboratornaia Diagnostika

|October 19, 2021

Summary

Chromosomal abnormalities cause pregnancy loss and infertility. A new FISH protocol using TCEP decondensation agent significantly improves sperm analysis for assisted reproductive technologies (ART).

Keywords:

DTT TCEP aneuploidy sperm FISH

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Last Updated: Oct 16, 2025

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

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Published on: July 12, 2017

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Published on: September 1, 2009

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

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Published on: December 28, 2021

Area of Science:

Reproductive biology
Genetics
Cytogenetics

Background:

Chromosomal abnormalities (CA) and aneuploidies in gametes contribute to pregnancy termination, infertility, and developmental issues.
Couples facing reproductive challenges or carrying CA often utilize assisted reproductive technologies (ART) for preimplantation genetic diagnosis.
Sperm analysis, typically via Fluorescent In Situ Hybridization (FISH), is crucial for assessing gamete aneuploidy but faces challenges due to sperm chromatin condensation.

Purpose of the Study:

To optimize the pre-hybridization processing of ejaculate samples for improved FISH analysis of male gametes.
To identify the most effective decondensation agent and protocol for FISH analysis of sperm chromatin.

Main Methods:

Five distinct pre-hybridization protocols were tested on ejaculate samples from nine phenotypically normal men.
Comparative analysis of hybridization efficiency was performed for each protocol.
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was evaluated as a decondensation agent.

Main Results:

The protocol utilizing TCEP as a decondensation agent demonstrated the highest efficiency for subsequent molecular cytogenetic studies.
A hybrid protocol combining proteolytic pretreatment, TCEP, and thermal decondensation proved effective when other methods failed.

Conclusions:

TCEP is a highly effective decondensation agent for optimizing FISH analysis of sperm.
The developed hybrid protocol offers a valuable solution for challenging ejaculate samples in ART programs, improving diagnostic accuracy for chromosomal abnormalities.