A new multiparameter flow cytometric method for human semen analysis

S. Perticarari, G. Ricci, M. Granzotto, R. Boscolo, C. Pozzobon, S. Guarnieri, A. Sartore, G. Presani

Research output: Contribution to journalArticle

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Original languageEnglish
Pages (from-to)485-494
Number of pages10
JournalHuman Reproduction
Volume22
Issue number2
DOIs
Publication statusPublished - Feb 2007

Fingerprint

Semen Analysis
Spermatozoa
Semen
Caspases
Apoptosis
Leukocytes
Flow Cytometry
Caspase Inhibitors
Fluorescein
Microspheres
Microscopy
Fluorescence
Staining and Labeling

Keywords

  • Apoptosis
  • Flow cytometry
  • Fluorospheres
  • Sperm
  • Syto 16

ASJC Scopus subject areas

  • Physiology
  • Developmental Biology
  • Obstetrics and Gynaecology
  • Reproductive Medicine

Cite this

Perticarari, S., Ricci, G., Granzotto, M., Boscolo, R., Pozzobon, C., Guarnieri, S., ... Presani, G. (2007). A new multiparameter flow cytometric method for human semen analysis. Human Reproduction, 22(2), 485-494. https://doi.org/10.1093/humrep/del415

A new multiparameter flow cytometric method for human semen analysis. / Perticarari, S.; Ricci, G.; Granzotto, M.; Boscolo, R.; Pozzobon, C.; Guarnieri, S.; Sartore, A.; Presani, G.

In: Human Reproduction, Vol. 22, No. 2, 02.2007, p. 485-494.

Research output: Contribution to journalArticle

Perticarari, S, Ricci, G, Granzotto, M, Boscolo, R, Pozzobon, C, Guarnieri, S, Sartore, A & Presani, G 2007, 'A new multiparameter flow cytometric method for human semen analysis', Human Reproduction, vol. 22, no. 2, pp. 485-494. https://doi.org/10.1093/humrep/del415
Perticarari S, Ricci G, Granzotto M, Boscolo R, Pozzobon C, Guarnieri S et al. A new multiparameter flow cytometric method for human semen analysis. Human Reproduction. 2007 Feb;22(2):485-494. https://doi.org/10.1093/humrep/del415
Perticarari, S. ; Ricci, G. ; Granzotto, M. ; Boscolo, R. ; Pozzobon, C. ; Guarnieri, S. ; Sartore, A. ; Presani, G. / A new multiparameter flow cytometric method for human semen analysis. In: Human Reproduction. 2007 ; Vol. 22, No. 2. pp. 485-494.
@article{069d8674c5ac42f18366e3c758149822,
title = "A new multiparameter flow cytometric method for human semen analysis",
abstract = "Background: 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. Methods: 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. Results: 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. Conclusions: 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.",
keywords = "Apoptosis, Flow cytometry, Fluorospheres, Sperm, Syto 16",
author = "S. Perticarari and G. Ricci and M. Granzotto and R. Boscolo and C. Pozzobon and S. Guarnieri and A. Sartore and G. Presani",
year = "2007",
month = "2",
doi = "10.1093/humrep/del415",
language = "English",
volume = "22",
pages = "485--494",
journal = "Human Reproduction",
issn = "0268-1161",
publisher = "Oxford University Press",
number = "2",

}

TY - JOUR

T1 - A new multiparameter flow cytometric method for human semen analysis

AU - Perticarari, S.

AU - Ricci, G.

AU - Granzotto, M.

AU - Boscolo, R.

AU - Pozzobon, C.

AU - Guarnieri, S.

AU - Sartore, A.

AU - Presani, G.

PY - 2007/2

Y1 - 2007/2

N2 - Background: 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. Methods: 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. Results: 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. Conclusions: 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.

AB - Background: 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. Methods: 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. Results: 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. Conclusions: 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.

KW - Apoptosis

KW - Flow cytometry

KW - Fluorospheres

KW - Sperm

KW - Syto 16

UR - http://www.scopus.com/inward/record.url?scp=33846491446&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33846491446&partnerID=8YFLogxK

U2 - 10.1093/humrep/del415

DO - 10.1093/humrep/del415

M3 - Article

C2 - 17079246

AN - SCOPUS:33846491446

VL - 22

SP - 485

EP - 494

JO - Human Reproduction

JF - Human Reproduction

SN - 0268-1161

IS - 2

ER -