An alternative to PVP for slowing sperm prior to ICSI

B. Balaban1, K. Lundin2, J.M. Morrell*,3, H. Tjellström*,3, B. Urman1 and P.V. Holmes*,3,4

1 American Hospital, Istanbul, Turkey, 2 Reproductive Medicine, SU/Sahlgrenska, Gothenburg and 3 NidaCon International, Gothenburg, Sweden

4 To whom correspondence should be addressed. e-mail: paul{at}nidacon.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: There is a growing awareness of potential problems in exposing sperm to polyvinylpyrrolidone (PVP) to slow their motility, a procedure commonly used prior to ICSI. The study presented here evaluates an alternative product for slowing sperm motility, which contains hyaluronate, a substance found naturally in the reproductive tract. METHODS: Computerized sperm motility analysis was used to compare the motilities of sperm exposed to either a PVP-containing product (ICSI-100), or a hyaluronate-containing product (SpermCatchTM), or control sperm resuspended in a sperm maintenance medium. A subjective assessment was made of the ease with which sperm could be isolated and be drawn into, and expelled from, an injection pipette after having their tails nicked. Sperm exposed to either ICSI-100 or SpermCatch were used for ICSI. Fertilization rate, zygote development, grading, and outcome of transfer were recorded for the two treatment groups. RESULTS: The hyaluronate-containing product slowed sperm motility sufficiently for the sperm to be captured in an injection pipette, was easy to draw into and expel from the pipette, prevented sperm sticking to plastic or glassware, and did not affect post-injection zygote development. Clinical pregnancy rates were similar for the two groups. CONCLUSIONS: This product represents an alternative to PVP for slowing sperm motility prior to ICSI.

Key words: hyaluronate/ICSI/PVP/slowing sperm motility


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intracytoplasmic sperm injection (ICSI) requires the capture of an individual sperm in a glass pipette for injection into the oocyte. This procedure is facilitated by first immobilizing the sperm, which is accomplished by a variety of means, for example by laser (Montag et al., 2000Go; Ebner et al., 2001Go; 2002), or by sonication, freezing, detergent treatment and piezo-actuated pulses (reviewed by Mizuno et al., 2002Go). Probably the most widely practised method, since it does not require special equipment, is to reduce sperm motility by placing the sperm in a viscous medium prior to nicking the tail to immobilize the sperm completely (Van Steirteghem et al., 1993Go).

Previously, the only products commercially available for slowing sperm motility contained a synthetic plastic, polyvinylpyrrolidone (PVP). However, some PVP is injected into the oocyte along with the sperm and, as the PVP cannot diffuse out and is not digestible by lysosomal enzymes, it will remain in the oocyte for a prolonged period (Jean et al., 2001Go). To avoid any potential damaging effects of PVP, techniques have been developed which do not necessitate slowing sperm motility (Harari et al., 1995Go; Jean et al., 1996Go; 1997; Butler and Masson, 1997Go; Hlinka et al, 1998Go; Tsai et al., 2000Go). However, not everybody is able to crush the tails of sperm moving at their normal velocity.

Therefore, a physiological alternative to PVP has been sought for reducing sperm motility to facilitate capturing sperm for ICSI. Ideally the product should possess the following characteristics: (i) be sufficiently viscous, to slow sperm motility enough to aspirate them into an ICSI pipette; (ii) be sufficiently fluid, to facilitate ease of aspirating and dispensing the sperm and a little of the liquid into, and from, the pipette; (iii) be able to prevent sperm sticking to either the plastic culture dish or the glass ICSI pipette; and (iv) have no deleterious effects on post-ICSI zygote development.

This study was designed to evaluate SpermCatchTM (NidaCon International, Gothenburg, Sweden), a viscous liquid containing hyaluronate and human serum albumin, as a potential substitute for PVP. Since hyaluronate and human serum albumin are found naturally in the mammalian reproductive tract, this product may prove to be a physiological alternative to PVP which can be used in the clinic to reduce sperm motility. SpermCatch is manufactured according to current Good Manufacturing Practice (cGMP) by NidaCon International AB, an ISO-registered company, and is approved by medical authorities.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ejaculates from 11 healthy volunteers, whose semen parameters were considered ‘normal’ (World Health Organization, 1999Go) and whose fertility status was unknown, were prepared on PureSperm® (NidaCon) density gradients by centrifugation at 300 g. After washing the resulting sperm pellet in PureSperm Wash by centrifugation at 500 g, and resuspending in a sperm maintenance medium, SpermAssistTM, aliquots of the sperm preparations were added to equivalent volumes of SpermCatch, ICSI-100, or SpermAssist as a control (all materials were obtained from NidaCon International AB with the exception of ICSI-100 from VitroLife, Gothenburg, Sweden). Computer assisted sperm motility analysis (CASA) was performed using a Hobson Sperm Tracker (Hobson Vision Ltd, Derbyshire, UK). A total of 500 sperm from each donor was analysed per treatment group according to standard procedures (Holt et al, 1985Go; 1989).

The sperm were slowed by exposure to SpermCatch, immobilized by nicking their tail and drawn into an ICSI pipette. The ease with which the sperm could be caught, and drawn into, or expelled from, the pipette was assessed subjectively by workers accustomed to using PVP.

In a separate study, oocytes were injected with sperm exposed to either the hyaluronate or the PVP-containing product. The oocytes were from different patients, and were randomly assigned to the media that was used to slow sperm motility. Post-ICSI rates of fertilization and cleavage were compared between the two groups, and embryos were graded according to the following classification; G1 embryos: embryos with no fragmentation and equal sized homogeneous blastomeres; G2 embryos: embryos with <20% fragmentation and/or equal sized blastomeres; G3 embryos: embryos with 20–50% fragmentation and/or unequal sized or granular necrotic blastomeres; G4 embryos: embryos with >50% fragmentation.

All the sperm samples in the ICSI study came from patients with male factor infertility.

Mean velocities for each group were compared using ANOVA (Campbell, 1974Go). Significance was accepted where P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Motility was modified immediately for sperm exposed to the hyaluronate–containing medium (SpermCatch) compared with sperm in gamete-holding medium alone. Progressive forward motility was decreased without any noticeable change in the lateral movement of the sperm head. Although the spermatozoa did not cease moving completely, it was easy to immobilize and catch them individually and draw them into an injection pipette. The hyaluronate-containing medium was sufficiently viscous to allow fine control during sperm aspiration into, and expulsion from, the injection pipette. Sperm did not stick to the plastic culture dish or to the glass pipette.

CASA was performed on sperm exposed to either the hyaluronate-containing medium or the PVP product (ICSI-100). These sperm had mean curvilinear velocities, mean velocity of the average path and mean straight line velocities which were significantly slower (P < 0.001 for all parameters) than those of control sperm diluted in sperm maintenance medium (Table I).


View this table:
[in this window]
[in a new window]
 
Table I. Mean velocities of sperm exposed to either hyaluronate-containing or PVP-containing products, compared with control sperm in sperm maintenance medium (n = 11 sperm donors)
 
The rates of zygote cleavage and development after injection of the oocyte with sperm exposed to either the hyaluronate-containing medium or the PVP product are shown in Table II, together with the number of grade 1 and 2 embryos developing and transferred. The rates were not different between the two treatment groups. Apart from one pregnancy loss in each group, the remaining pregnancies proceeded to term. No genetic abnormality was observed in any of the offspring.


View this table:
[in this window]
[in a new window]
 
Table II. Proportion of oocytes cleaving and zygotes developing after ICSI with sperm exposed to hyaluronate-containing or PVP-containing products
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinicians have become increasingly concerned about the use of PVP-containing products to slow sperm motility prior to ICSI (Jean et al., 2001Go). Dozortsev et al. (1995Go) reported that PVP may interfere with sperm nucleus decondensation while Feichtinger et al. (1995Go) stated that they have always avoided the use of PVP. Detrimental effects on mouse zygote development were reported following exposure of the sperm to PVP (Mizuno et al., 2002Go). In contrast, bull zygotes were not affected by PVP (Motoshi et al., 1996Go), possibly reflecting an effect of either the source or concentration of PVP used for the procedure. Moreover, differential species susceptibility to the effects of PVP cannot be ruled out.

Some embryologists, concerned about the potential adverse effects of PVP, have developed modifed techniques to capture sperm without slowing sperm motility (Harari et al., 1995Go; Hlinka et al., 1998Go; Tsai et al., 2000Go). Hlinka et al. (1998Go) attached the sperm to the end of the injection needle by suction before crushing the tail. They claim that even fast-moving sperm can be caught by this method, with the added advantage that since the sperm is never aspirated into the needle, a minimal volume of fluid is expelled during sperm deposition in the oocyte (Hlinka et al., 1998Go). Others have reported crushing the sperm tail before aspirating the sperm into the tip of the ICSI pipette (Harari et al., 1995Go; Tsai et al., 2000Go). Jean et al. (2001Go) noted that training was sufficient to overcome any initial difficulties experienced in catching the sperm in the absence of PVP.

Although avoiding the use of exogenous substances altogether is probably the most desirable situation, sperm are not easy to tail-nick or catch without slowing their motility. Therefore, a physiological alternative to PVP is required. The new product, SpermCatch, which contains sodium hyaluronate, slowed sperm sufficiently for them to be caught, was easy to handle in the ICSI pipette and prevented sperm from sticking to the culture dish or to the pipette. Although sperm motility was somewhat faster in the hyaluronate-containing product than in the PVP-containing product, which necessitated some slight adaptations to the handling techniques on the part of the operator, it was not difficult to learn to use this product. Furthermore, since there was no difference in zygote development after ICSI, it would appear that the hyaluronate-containing product could be used in preference to PVP-containing products without detriment to the patient’s chances of becoming pregnant.

The observation that hyaluronic acid can modulate sperm motility is not new; for example, Zimmerman et al. (1994Go) reported that exposure of sperm to hyaluronic acid at 1 mg/ml caused a decrease in straight line velocity, amplitude of lateral head deviation and mean angular deviation compared with non-exposed sperm. Moreover, the suggestion that hyaluronic acid could be used to slow sperm motility prior to ICSI was advocated by Barak et al. (1999; 2001Go). These authors reported similar fertilization rates and pregnancy rates after ICSI with sperm exposed to either hyaluronic acid or PVP and recommended the use of hyaluronic acid as a physiological replacement for PVP.

The results reported here concur with those of Barak et al. (1999; 2001Go), in that sperm motility was effectively slowed by the hyaluronate-containing product, and zygote development after ICSI was not impaired. Furthermore, the hyaluronate-containing product was easy to handle in the ICSI pipette and prevented sperm from sticking to the culture dish or to the pipette. Therefore, it is concluded that SpermCatch represents an effective, physiological alternative to PVP for modulating sperm motility prior to aspirating a single sperm into an ICSI pipette.


    FOOTNOTES
 
* J.M.Morrell, H.Tjellström. and P.V.Holmes are employees of NidaCon International, the manufacturer of SpermCatch medium. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Barak, Y., Ménézo, Y., Veiga, A., Kogosowski, A. and Nevo, Z. (1999) Hyaluronic acid–a physiological replacement for polyvinylpyrrolidone (PVP) in Intracytoplasmic Sperm Injection (ICSI) Procedure. Fertil. Steril. (Suppl.), O-231.

Barak, Y., Ménézo, Y., Veia, A. and Elder, K. (2001) A physiological replacement for polyvinylpyrrolidone (PVP) in assisted reproductive technology. Hum. Fertil., 4, 99–103.

Campbell, R.C. (1974) Statistics for Biologists. 2nd edn. Cambridge University Press, Cambridge, UK.

Butler, E.A. and Masson, G.M. (1997) Development of a successful ICSI programme without the use of PVP. Hum. Reprod., 12, 1115.

Dozortsev, D., Rybouchkin, A., De Sutter, P. and Dhont, M. (1995) Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation. Hum. Reprod., 10, 2960–2964.[Abstract]

Ebner, T., Yaman, C., Moser, M., Sommergruber, M., Hartl, J. and Tews, G. (2001) Laser assisted immobilization of spermatozoa prior to inracytoplasmic sperm injection in humans. Hum. Reprod., 16, 2628–2631.[Abstract/Free Full Text]

Ebner, T., Moser, M., Yaman, C., Sommergruber, M. and Tews, G. (2002) Successful birth after laser assisted immobilization of spermatozoa before intracytoplasmic injection. Fertil. Steril., 78, 417–418.[CrossRef][ISI][Medline]

Feichtinger, W., Obruca, A. and Brunner, M. (1995) Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet, 346, 1566.

Harari, O., Bourne, H., Baker, G., Gronow, M. and Johnston, I. (1995) High fertilization rate with intracytoplasmic sperm injection in mosaic Klinefelter’s syndrome. Fertil. Steril., 63, 182–184.[ISI][Medline]

Hlinka, D., Herman, M., Vesela, J., Hredzak, R., Horvath, S. and Pacin, J. (1998) A modified method of intracytoplasmic sperm injection without the use of polyvinylpyrrolidone. Hum. Reprod., 13, 1922–1927.[Abstract]

Holt, W.V., Moore, H.D.M. and Hillier, S.G. (1985) Computer-assisted measurement of sperm swimming speed in human semen: correlation of results with in vitro fertilisation assays. Fertil. Steril., 44, 112–119.[ISI][Medline]

Holt, W.V., Shenfield, F., Leonrad, T. Hartman, T.D., North, D., and Moore, H.D.M. (1989) The value of sperm swimming speed measurements in asessing the fertility of human frozen semen. Hum. Reprod., 4, 292–297.[Abstract]

Jean, M., Barriere, P. and Mirallie, S. (1996) Intracytoplasmic sperm injection without polyvinylpyrrolidone: an essential precaution? Hum. Reprod., 11, 2332.

Jean, M., Mirallie, S., Bourdineau, M., Tatin, C. and Barriere, P. (2001) Intracytoplasmic sperm injection with polyvinylpyrrolidone: a potential risk. Fertil. Steril., 76, 419–420.[CrossRef][ISI][Medline]

Mizuno, K., Hoshi, K. and Huang, T. (2002) Fertilization and embryo development in a mouse ICSI model using human and mouse sperm after immobilisation in polyvinlypyrrolidone. Hum. Reprod., 17, 2350–2355.[Abstract/Free Full Text]

Montag, M., Rink, K., Delacretaz, G. and van der Ven, H. (2000) Laser-induced immobilization and plasma membrane permeabilization in human spermatozoa. Hum. Reprod., 15, 846–852.[Abstract/Free Full Text]

Motoishi, M., Goto, K., Tomita, K., Ookutsu, S. and Nakanushi, Y. (1996) Examination of the safety of intracytoplasmic injection procedures by using bovine oocytes. Hum. Reprod., 11, 618–620.[Abstract]

Tsai, M.-Y., Huang, F.-J., Kung, F.-T., Lin, Y.-C., Chang, S.-Y., Wu, J.-F. and Chang, H.-W. (2000) Influence of polyvinylpyrrolidone on the outcome of intracytoplasmic sperm injection. J. Reprod. Med., 45, 115–120.[ISI][Medline]

Van Steirteghem, A., Nagy, Z., Joris, H., Liu, J., Staesson, C., Smitz, J. Wisanto, A. and Devroey, P. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 1061–1066.[Abstract]

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.

Zimmerman, E., Drobnis, E.Z., Robertson, K.R., Nakajima, S.T. and Kim, H. (1994) Semen preparation with the Sperm Select system versus a washing technique. Fertil. Steril., 61, 269–275.[ISI][Medline]





This Article
Abstract
FREE Full Text (PDF )
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Request Permissions
Google Scholar
Articles by Balaban, B.
Articles by Holmes, P.V.
PubMed
PubMed Citation
Articles by Balaban, B.
Articles by Holmes, P.V.