University of Padova, Department of Medical and Surgical Sciences, Center for Male Gamete Cryopreservation, Clinica Medica 3, Via Ospedale 105, 35128 Padova, Italy
1 To whom correspondence should be addressed. Email: marco.rossato{at}unipd.it
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: acrosome reaction/antisperm antibodies/calcium/human sperm/hypo-osmotic swelling test
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since its introduction, the results of the different published studies evaluating the clinical usefulness of the HOS test have shown that semen samples with low HOS test scores are associated with normal fertilization but low pregnancy rates during IVF techniques (Check et al., 1995; Katsoff et al., 2000
) although controversial data have been reported so far (Check et al., 1989
, 1995
, 2001b
; Enginsu et al., 1992
; Abu-Musa et al., 1993
; Biljan et al., 1996
; Katsoff et al., 2000
).
Previous reports have described the association between the presence of sperm antibodies and reduced HOS test score (Kiefer et al., 1996; Katsoff and Check, 1997
; Wen et al., 2000
). As reported recently by Check's group, it is not clear if sperm from subjects affected by antisperm autoimmunity have some plasma membrane alteration leading to low HOS test scores or if the antisperm antibodies directly affect the sperm plasma membrane functionality (Jaraj et al., 2000
). The fact that antisperm antibodies can influence plasma membrane functional integrity (as evaluated by HOS test) has been further confirmed by a study reporting that long-term corticosteroid therapy for antisperm autoimmunity improves HOS test scores as well as sperm motility and pregnancy rates in a group of men affected by autoimmune infertility (Omu et al., 1996
).
In the present study, we evaluated the correlation between HOS test score and standard sperm parameters in a large group of infertile subjects with autoimmune infertility. The effects of antisperm antibodies bound to the sperm plasma membrane on the hypo-osmolarity-induced calcium influx and acrosome reaction were examined.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HOS test
The HOS test was performed as originally reported by Jeyendran et al. (1984): 0.1 ml of semen sample was mixed with 1.0 ml of a hypo-osmotic solution (150 mOsm) containing fructose and sodium citrate. After incubation for 30 min at 37°C,
100 sperm were analysed by phase-contrast microscopy, evaluating the modifications of the sperm tail to score swollen sperm, which were reported as a percentage of all sperm observed.
Sperm selection and incubation
When performing functional studies, we isolated sperm using density gradients (Isolate; Irvine Scientific, USA) following the manufacturer's instructions to obtain a population of highly motile sperm. All experiments were performed utilizing sperm isolated with this technique. Sperm isolated by this method from ASA positive subjects and fertile controls were collected and resuspended in standard saline containing (in mmol/l): 125 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 KH2PO4, 5.6 glucose, 25 NaHCO3, 1.7 CaCl2, 20 HEPES (pH 7.4, 37°C). Sperm concentration was adjusted to 15 x 106/ml.
To evaluate the effects of sperm exposure to hypo-osmotic medium on intracellular calcium concentration ([Ca2 + ]i) and acrosome reaction, sperm aliquots from seven infertile subjects, each showing 100% of sperm with ASA bound to their plasma membrane, were processed as described (Rossato et al., 1996). As controls, we considered sperm samples from five normozoospermic fertile subjects that were treated as described for ASA positive sperm samples. To obtain a reduction of medium osmolarity, isolated motile sperm were diluted with distilled water to obtain a reduction of medium osmolarity of 30%.
[Ca2+ ]i measurement
[Ca2+ ]i was measured utilizing the fluorescent probe fura-2/AM as previously described (Rossato et al., 1996): isolated sperm were incubated for 30 min at 37°C in the presence of fura-2/AM (2 mmol/l). After loading, sperm were washed by centrifugation at 800 g for 10 min, resuspended in standard saline and maintained at room temperature until used. [Ca2 + ]i was measured before and after sperm medium dilution in distilled water as previously described (Rossato et al., 1996
).
[Ca2+]i was measured using a Perkin Elmer LS50B fluorimeter equipped with a thermostatic and magnetically stirred cuvette holder and utilizing 1.0 ml sperm aliquots. The excitation wavelength was alternated between 350 and 380 nm and emission fluorescence was continuously monitored at 505 nm.
Acrosome reaction evaluation
Sperm aliquots were retrieved before and after reduction of external medium osmolarity by 30%. After fixation with formaldehyde the percentage of acrosome reacted sperm was assayed using an indirect fluorescence technique with fluorescein isothiocyanate-conjugated lectin from Pisum sativum, which selectively binds to the acrosomal matrix of acrosome-intact sperm. Non-fluorescent sperm were then scored as acrosome-reacted while fluorescent sperm were scored as acrosome intact. Two hundred sperm were scored in each sample to evaluate the percentage of acrosome-reacted sperm.
Statistical analysis
For statistical analysis the mean percentage of swollen and live sperm from controls and ASA positive subjects were compared using Student's t-test. A correlation analysis between HOS test scores and percentages of live sperm was performed by simple linear regression and determination of the correlation coefficients.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
The location of antibody binding on sperm plasma membrane was mixed (head, middle piece and tail) and it was therefore not possible to correlate the effects of ASA location on changes in HOS test percentages, variation of [Ca2 + ]i and acrosome reaction percentages induced by reduction of external medium osmolarity.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous preliminary studies (Omu et al., 1996, 1997
; Jairaj et al., 2000
) have suggested that ASA may somewhat modify sperm plasma membrane integrity, leading to low HOS test score, but to date, no planned studies have confirmed these observations. The present data clearly demonstrate that sperm with ASA bound to their plasma membranes show low HOS test scores, and this non-specific alteration of the plasma membrane integrity may participate in the determination of infertility due to ASA. How ASA interfere with sperm plasma membrane functionality has been not elucidated yet but it is possible that ASA modify sperm plasma membrane permeability or fluidity, leading to low fertilizing potential. One possible explanation for low HOS test score in ASA positive sperm samples is that ASA modify water permeability. In this regard it has been demonstrated that water transport across cell plasma membranes utilizes specific water channels named aquaporins (Ishibashi et al., 1997
; Borgnia et al., 1999
; Calamita et al., 2001
). It is possible that ASA bound to sperm surface may non-specifically or specifically block these water channels, thus altering sperm water permeability. Furthermore, antibody cross-linking could prevent plasma membrane distensibility, thus reducing sperm swelling when exposed to hypo-osmotic medium. Further confirmation that ASA may alter sperm plasma membrane permeability derives from a recent study reporting that sperm from a subject who underwent vasectomy reversal show low HOS test scores that remain low also after 1 year from the vasectomy reversal (Wen et al., 2000
). It is well known that vasectomized subjects develop ASA (Gubin et al., 1998
) and thus it is possible that the low HOS test score reported by these authors in sperm from subjects after vasectomy reversal may be due to ASA bound to the sperm surface.
Jairaj et al. (2000) reported no significant reduction of HOS test score in sperm previously incubated with positive ASA serum. The different results obtained in that study compared to ours may be due to the fact that in our study ASA were present in semen and directly bound to sperm. Furthermore those authors incubated sperm with ASA positive serum for 60 min, and it cannot be excluded that longer incubation or higher ASA concentration may be necessary to induce sperm plasma membrane alteration. Our data suggest that subjects with ASA show a wide range of HOS scores irrespective of the percentage of ASA, thus favouring the association with some possible common aetiology other than a direct effect of ASA in determining the lower sperm plasma membrane functionality during incubation in hypo-osmotic medium. The nature of this phenomenon remains to be elucidated.
The role of sperm plasma membrane permeability to water in regulating important sperm functions has been shown in previous studies (Morisawa and Suzuki, 1980; Inoda and Morisawa, 1987
) and we have demonstrated that human sperm exposure to hypo-osmotic medium activates an influx of water within sperm cytoplasm. This water influx induces a sperm volume increase and plasma membrane stretching, leading to the opening of osmosensitive calcium channels, calcium influx within sperm cytoplasm and activation of acrosome reaction (Rossato et al., 1996
). On the other hand, the osmosensitivity of sperm acrosome reaction in man has been reported previously (Bielfeld et al., 1993
) and the role of external osmolarity in the regulation of mammalian sperm functions is well known (Liu and Foote, 1998
; Rossato et al., 2002
). The effects of ASA and of other still unknown mechanisms present in autoimmune infertility in reducing plasma membrane water permeability, as shown by reduced HOS test score, could induce also a reduction of sperm responsiveness to putative hypo-osmotic stimuli fundamental for sperm activation during the fertilization process, as suggested by the low osmolarity of female genital tract secretions with respect to that of semen (Polak and Daunter, 1984
; Rossato et al., 1996
). Indeed this hypothesis was demonstrated to be true since sperm with ASA bound to their surface show a marked reduction of [Ca2 + ]i rise and acrosome reaction percentage increase induced by sperm exposure to hypo-osmotic medium, as evidenced in sperm from normozoospermic subjects without ASA. These observations confirm that ASA alter sperm membrane functionality, impairing important transduction signalling pathways, leading ultimately to alteration of the sperm fertilizing ability.
The results of the present study, beyond the suggestion of performing the HOS test during routine semen analysis, could also have a practical aspect since, when a sperm sample shows a low HOS score associated with normal percentages of live sperm, ASA may be bound to those sperm. This may indicate evaluating the presence of ASA, a procedure that is not performed as a routine evaluation during semen sample analysis in a number of laboratories.
Furthermore these observations could also have important clinical implications. When a semen sample shows a low HOS test score, it could be positive for ASA, and when performing an assisted reproduction technique, ICSI should be preferred to IVF or intrauterine insemination as recently suggested (Check et al., 2001).
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Avery S, Bolton UM and Mason BA (2000) An evaluation of the hypo-osmotic sperm-swelling test as a predictor of fertilizing capacity in vitro. Int J Androl 13, 9399.
Bahamondes L, Faxano F, de Lucio MA, Neves PA, Bottcher LF and Lorenzetti GB (2001) Evaluation of human sperm membrane integrity using the water test and the hypo-osmotic test. Andrologia 33, 7577.[CrossRef][Medline]
Barratt CL, Osborn JC, Harrison PE, Monless N, Dumphy BC, Lenton EA and Cooke ID (1989) The hypo-osmotic swelling test and the sperm mucus penetration test in determining fertilization of the human oocyte. Hum Reprod 4, 430434.[Abstract]
Bielfeld P, Jeyendran RS and Zaneveld LJ (1993) Osmo-sensitivity of the human sperm acrosome reaction. Hum Reprod 8, 12351239.[Abstract]
Biljan MM, Buckett WM, Taylor CT, Luckas M, Aird I, Kingsland CR and Lewis-Jones DI (1996) Effect of abnormal hypo-osmotic swelling test on fertilization rate and pregnancy outcome in in vitro fertilization cycles. Fertil Steril 66, 412416.[ISI][Medline]
Borgnia M, Nielsen S, Engel A and Agre P (1999) Cellular and molecular biology of the aquaporin water channels. Annu Rev Biochem 68, 425458.[CrossRef][ISI][Medline]
Calamita G, Mazzone A, Cho YS, Valenti G and Svelto M (2001) Expression and localization of the aquaporin-8 water channel in rat testis. Biol Reprod 64, 16601666.
Chan SYW, Wang C, Chan STH and Ho PC (1990) Differential evaluation of human sperm hypo-osmotic swelling test and its relationship with the outcome of in vitro fertilization of human oocytes. Hum Reprod 5, 8488.[Abstract]
Check JH, Epstein R, Nowroozi K, Shanis BS, Wu CH and Bollendorf A (1989) The hypo-osmotic swelling test as a useful adjunct to the semen analysis to predict fertility potential. Fertil Steril 52, 159161.[Medline]
Check JH, Stumpo L, Lurie D, Benfer K and Callan C (1995) A comparative prospective study using matched samples to determine the influence of subnormal hypo-osmotic test scores of spermatozoa on subsequent fertilization and pregnancy rates following in-vitro fertilization. Hum Reprod 10, 11971200.[Abstract]
Check JH, Katsoff D and Check ML (2001a) Some semen abnormalities may cause infertility by impairing implantation rather then fertilization. Med Hypoth 56, 653657.
Check JH, Katsoff D, Check ML, Choe JK and Swenson K (2001b) In vitro fertilization with intracytoplasmic sperm injection is an effective therapy for male factor infertility related to subnormal hypo-osmotic swelling test scores. J Androl 22, 261265.
Check ML, Kiefer D, Check JH, Hourani W and Long R (2002) Treatment of sperm with subnormal host scores with chymotrypsin/viable pregnancy after IUI. Arch Androl 48, 155158.[CrossRef][Medline]
Enginsu ME, Dumoulin JC, Pieters MH, Bergers M, Evers JL and Geraedts JP (1992) Comparison between the hypoosmotic swelling test and morphology evaluation using strict criteria in predicting in vitro fertilization (IVF). J Assist Reprod Genet 9, 259264.[Medline]
Gubin DA, Dmochowski R and Kutteh WH (1998) Multivariant analysis of men from infertile couples with and without antisperm antibodies. Am J Reprod Immunol 39, 157160.[ISI][Medline]
Inoda T and Morisawa M (1987) Effect of osmolality on the initiation of sperm motility in Xenopus laevis. Comp Biochem Physiol A 88, 539542.[CrossRef][Medline]
Ishibashi K, Kuwahara M, Gu Y, Kageyama Y, Tohsaka A, Suzuki F, Marumo F and Sasaki S (1997) Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol and urea. J Biol Chem 272, 2078220786.
Jairaj S, Check JH and Bollendorf A (2000) Do antisperm antibodies cause functional impairment of the sperm membrane as manifested by a low hypoosmotic swelling test score? Arch Androl 44, 231235.[CrossRef][ISI][Medline]
Jeyendran RS, Van der Ven HH, Perez-Palaez BG, Crabo BG and Zaneveld LJD (1984) Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 70, 219228.[Abstract]
Katsoff D and Check JH (1997) Two methods of achieving pregnancies despite subnormal hypo-osmotic swelling test scores. Fertil Steril 68, 549551.[CrossRef][Medline]
Katsoff D, Check ML and Check JH (2000) Evidence that sperm with low hypo-osmotic swelling scores cause embryo implantation defects. Arch Androl 44, 227230.[CrossRef][Medline]
Kiefer D, Check JH and Katsoff D (1996) The value of motile density, strict morphology and the hypo-osmotic swelling test in in vitro fertilization-embryo transfer. Arch Androl 37, 5760.[Medline]
Liu Z and Foote RH (1998) Bull sperm motility and membrane integrity in media varying in osmolality. J Dairy Sci 81, 18681873.
Morisawa M and Suzuki K (1980) Osmolality and potassium ion: their roles in initiation of sperm motility in teleosts. Science 210, 11451147.[ISI][Medline]
Omu AE, al-Qattan F and Abdul-Hamada B (1996) Effect of low dose continuous corticosteroid therapy in men with antisperm antibodies on spermatozoa quality and conception rate. Eur J Obstet Gynecol Reprod Biol 69, 129134.[CrossRef][Medline]
Omu AE, Makhseed M, Mohammed AT and Munim RA (1997) Characteristics of men and women with circulating antisperm antibodies in a combined infertility clinic in Kuwait. Arch Androl 39, 5564.[Medline]
Polak B and Daunter B (1984) Osmolarity of human seminal plasma. Andrologia 16, 224227.[ISI][Medline]
Rossato M, Di Virgilio F and Foresta C (1996) Involvement osmo-sensitive calcium influx in human sperm activation. Mol Hum Reprod 2, 903909.[Abstract]
Rossato M, Balercia G, Lucarelli G, Foresta C and Mantero F (2002) Role of seminal osmolarity in the reduction of human sperm motility. Int J Androl 25, 230235.[CrossRef][ISI][Medline]
Sjoblum P and Coccia E (1989) On the diagnostic value of the hypo-osmotic sperm swelling test in an in vitro fertilization program. J In Vitro Fertil Embryo Transfer 6, 4143.[Medline]
Wen RQ, Lie MY, Tian P, Yang N, Jiang YJ and Chen AP (2000) Sperm function tests after vasovasostomy. Asian J Androl 2, 111114.[Medline]
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge, UK.
Submitted on January 16, 2004; accepted on April 22, 2004.
|