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Hum. Reprod. Advance Access originally published online on October 3, 2008
Human Reproduction 2009 24(1):28-36; doi:10.1093/humrep/den365
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© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Dual use of Diff-Quik-like stains for the simultaneous evaluation of human sperm morphology and chromatin status

Ana Paula M. Sousa1,{dagger}, Renata S. Tavares1,{dagger}, Juan Felipe Velez de la Calle3, Helena Figueiredo4, Vasco Almeida5, Teresa Almeida-Santos2 and João Ramalho-Santos1,2,6

1 Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal 2 Department of Maternal-Fetal Medicine, Genetics and Human Reproduction, University Hospitals of Coimbra, 3000 Coimbra, Portugal 3 In Vitro Fertilization Unity, Pasteur Saint-Esprit Clinic, 29200 Brest, France 4 Laboratorio FIV, Centro Hospitalar V.N. Gaia, 4400-129 V.N. Gaia, Portugal 5 Department of Zoology and Anthropology, Faculty of Sciences, University of Oporto, and Centro de Estudos de Infertilidade e Esterilidade (CEIE), 4050-345 Oporto, Portugal

6 Correspondence address. Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal. Tel:+351-239-855-760; Fax: +351-239-855-789; E-mail: jramalho{at}ci.uc.pt

BACKGROUND: Sperm chromatin status and nuclear DNA damage can be detected using well-established assays. However, most techniques are time-consuming and/or involve elaborate protocols and equipment. We have recently developed a simple and fast method to monitor sperm chromatin status in field conditions using the Diff-Quik assay which is employed in fertility clinics to assess sperm morphology with standard bright field microscopy. In the present study, we demonstrate that any Diff-Quik-like stain can easily, reproducibly and routinely monitor human sperm chromatin status as well.

METHODS: Different Diff-Quik-like stains were used to assess sperm morphology and the presence of abnormal dark nuclear staining in human sperm from four ART centres. The TUNEL assay was performed in the same samples, and fertility outcomes were assessed.

RESULTS: A significant correlation was found between TUNEL-positive sperm and dark sperm nuclei. Moreover, associations were also found between the percentage of dark sperm nuclei and seminal parameters, embryo development rate, embryo quality and clinical pregnancy, as well as with cryptorchidism, and there was a tendency towards an association with age. A value of 32% abnormal staining is suggested as a predictive threshold for embryo development and pregnancy.

CONCLUSIONS: Our results show that any Diff-Quik-like stain, already implemented in most laboratories to assess sperm morphology, can be adapted as an indicator for chromatin status in human sperm.

Key words: human sperm/chromatin/nuclear DNA/Diff-Quik staining/sperm morphology

{dagger} These authors contributed equally to this work, and should be considered joint first authors.

Submitted on June 4, 2008; resubmitted on September 5, 2008; accepted on September 9, 2008.


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