SHORT COMMUNICATION

Protective effect of fallopian tubal fluid against activatedleucocyte-induced sperm DNA fragmentation: preliminaryresultsP. Navarrete Gomez1,6, J. Espinoza Ruiz1,5, J. Parodi Rivera1, J. G. Alvarez2,3 & R. Sanchez Gutierrez4

1 Centro de Excelencia de Biotecnologıa de la Reproduccion (CEBIOR), Universidad de La Frontera, Temuco, Chile;

2 Instituto Marques, Barcelona, Barcelona, Spain;

3 Fundacion Leonardo Marques, Barcelona, Spain;

4 Departamento de Ciencias Preclınicas, Universidad de La Frontera, Temuco, Chile;

5 Tesista y;

6 Estudiante del Programa de Doctorado en Ciencias Mencion Biologıa Celular y Molecular Aplicada, Becarios CONICYT

Introduction

The integrity of the paternal genome is vital to the onset

and maintenance of a viable pregnancy to term

(Angelopoulou et al., 2007). The previous studies have

shown that exposure of human spermatozoa to tubal/

oviductal epithelial cells protects them from ROS-induced

damage, thus increasing sperm survival (El Mouatassim

et al., 2000). Despite all these studies, however, it is still

not known whether the oviduct exerts a protective effect

on sperm DNA fragmentation. The primary aim of this

study was to determine if human tubal explants or tubal

fluid exert a protective effect against ROS-induced sperm

DNA damage.

Materials and methods

Tubal explants were obtained from the fallopian tubes of

four healthy women subjected to hysterectomy. Tubal

fluid was obtained from the explants and incubated at

37 �C for 24 h in supplemented DMEM medium

(sDMEM). Semen samples were obtained from healthy

donors, processed for standard swim-up and incubated

with tubal fluid during 12 h.

Keywords:

Activated leucocytes—DNA

fragmentation—human sperm—ROS—

tubal fluid

Correspondence

Dr Raul Sanchez Gutierrez, Facultad de

Medicina, Universidad de La Frontera, Manuel

Montt 112, Temuco, Chile.

Tel.: +56 45 744248;

Fax: +56 45 325600;

E-mail: rsanchez@ufro.cl

Accepted: January 13, 2009

Summary

The integrity of the paternal genome is of paramount importance in the initia-

tion and maintenance of a viable pregnancy. Oxygen radicals (ROS) have been

identified as one of the main factors responsible for the induction of sperm

DNA damage. Spermatozoa are mainly protected against ROS-induced damage

by seminal plasma. However, this protective effect disappears once spermatozoa

enter the female genital tract. The fallopian tube mucosa may play a protective

role against ROS-induced sperm damage. The main objective of this study was

to determine whether human tubal explants and tubal fluid exert a protective

effect on ROS-induced sperm DNA damage. Spermatozoa were exposed to

tubal explants and/or tubal fluid in the presence of phorbol myristate acetate

(PMA)-activated polymorphonuclear leucocytes or control medium and sperm

DNA fragmentation was measured using the TdT-mediated dUTP-biotin nick

end labelling (TUNEL) test. Exposure of human spermatozoa to PMA-activated

leucocytes resulted in a 2-fold increase in sperm DNA fragmentation. Co-incu-

bation of spermatozoa with tubal explants did not reduce this damage.

However, pre-incubation of spermatozoa with tubal fluid resulted in a statis-

tically significant reduction in sperm DNA fragmentation levels, comparable to

those observed in control. In conclusion, tubal fluid appears to protect against

activated leucocyte-induced sperm DNA fragmentation, thus preserving the

integrity of the paternal genome.

196 ª 2009 Blackwell Verlag GmbH Æ Andrologia 41, 196–198

Polymorphonuclear (PMN) leucocytes were activated

with phorbol-12-miristate-13-acetate (PMA). Sperm DNA

fragmentation was determined using the TUNEL test

according to the manufacturer’s instructions (Roche,

Grenzach-Wyhlen, Germany). Following TUNEL labelling,

propidium iodide was added and the samples analysed by

flow cytometry. TUNEL test data were analysed using the

graphpad prism 5.0 program (GraphPad Software, La

Jolla, CA, USA).

Differences between the groups were analysed using the

Kruskal–Wallis and Dunn’s post-tests. A P-value less than

0.05 (P < 0.05) was considered as statistically significant.

Results and discussion

Incubation of human spermatozoa in sDMEM did not

result in any significant increase in DNA fragmentation

compared with the control with HTF medium (P > 0.05)

(Fig. 1a). Treatment with DNAse I produced a fragmenta-

tion level of 95 ± 3.06% (P < 0.05). Incubation of human

spermatozoa with activated leucocytes resulted in an

increase in DNA fragmentation levels, although these dif-

ferences were not statistically significant (P > 0.05).

Moreover, incubation of the spermatozoa with PMN,

PMA and DMSO did not produce any significant effect

(P > 0.05) on sperm DNA fragmentation. Incubation of

human spermatozoa with tubal explants produced a sig-

nificant decrease in sperm DNA fragmentation levels dur-

ing incubation at 37 �C (P < 0.05) (Fig. 1b), whereas

incubation of human spermatozoa with tubal medium

did not result in any significant effect on sperm DNA

fragmentation (P > 0.05). Incubation with activated leu-

cocytes did not produce any significant decrease

(P > 0.05) in activated leucocyte-induced sperm DNA

fragmentation levels, whereas incubation with tubal fluid,

after incubation with activated leucocytes, produced a sig-

nificant decrease in sperm DNA fragmentation levels

(P < 0.05) comparable to those observed in the control

(Fig. 1c).

The main findings of this study are that (i) tubal ex-

plants decrease sperm DNA fragmentation during incuba-

tion at 37 �C, whereas tubal fluid had no effect; and (ii)

tubal fluid prevents sperm DNA fragmentation induced

by PMA-activated leucocytes, whereas tubal explants had

no effect. The previous studies have shown that human

spermatozoa can establish direct contact with the tubal

epithelium and that this contact increases motility, pre-

vents the premature acrosome reaction and increases the

fertility capacity of spermatozoa (Morales et al., 1996;

Green et al., 2001). This could be related to a possible

antioxidant effect of the tubal cells that would neutralise

the ROS produced by spermatozoa in the extracellular

medium. In contrast, the tubal explants’ lack of protective

effect on activated leucocyte-induced sperm DNA frag-

mentation might be related to tubal cell damage induced

by the specific ROS produced by activated leucocytes,

which could inactivate enzymatic antioxidant defences of

these cells. In the case of tubal fluid, the antioxidant

defences would be mainly nonenzymatic soluble factors

that are not susceptible to inactivation by the high levels

(a)

(b)

(c)

Fig. 1 Effect of incubation in sDMEM, tubal explants (TU) or tubal

fluid (CM) on sperm DNA fragmentation. (a) Percentage of TUNEL-

positive spermatozoa after incubation in sDMEM (*P < 0.05); (b) Per-

centage of TUNEL-positive spermatozoa after incubation with TU or

CM; (c) Percentage of TUNEL-positive spermatozoa after incubation

with tubal TU or CM prior to incubation with activated leucocytes and

their respective controls.

P. Navarrete Gomez et al. Tubal fluid protects against sperm DNA damage

ª 2009 Blackwell Verlag GmbH Æ Andrologia 41, 196–198 197

of ROS produced by the activated leucocytes. Studies are

underway to try to answer the question of why the tubal

fluid protects against activated leucocyte-induced DNA

damage and not that produced during incubation with

nonactivated leucocytes.

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Tubal fluid protects against sperm DNA damage P. Navarrete Gomez et al.

198 ª 2009 Blackwell Verlag GmbH Æ Andrologia 41, 196–198