1 Department of Endocrinology and Reproductive Medicine, University of Bonn, 53105 Bonn, Germany and 2 Institut d'Optique Appliquée, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland
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Abstract |
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Key words: ICSI/immobilization/human/laser/spermatozoa
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Introduction |
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Some investigators reported that immobilization and permeabilization improves fertilization rates (Palermo et al., 1993; Fishel et al., 1995
; Gerris et al., 1995
), presumably because this manipulation facilitates sperm nuclear decondensation after injection of the spermatozoon into the oocyte (Dozortsev et al., 1995
), whereas others have reached opposite conclusions (Lacham-Kaplan and Trounson, 1994
; Hoshi et al., 1995
). However, it was shown that aggressive sperm immobilization, leading to a permanent permeabilization of the sperm membrane, improved fertilization rates and in cases of immature spermatozoa led to higher pregnancy rates (Palermo et al., 1996
).
So far, most centres have performed sperm manipulation in a 10% solution of polyvinylpyrrolidone (PVP) in culture medium. The viscosity of PVP reduces sperm motility and facilitates subsequent sperm manipulation (Palermo et al., 1995). However, PVP is a potentially toxic substance and the use of PVP in human tissue culture or ICSI is in debate. Some groups reported successful ICSI without the use of PVP (Feichtinger et al., 1995
; Jean et al., 1996
; McDermott and Ray, 1996
; Butler and Masson, 1997); their results compare favourably to those of centres routinely using PVP (Van Steirteghem et al., 1995
). However, sperm handling for ICSI without PVP is more problematic regarding immobilization.
We recently applied a non-contact 1.48 µm wavelength diode laser system for immobilization of spermatozoa prior to cryopreservation (Montag et al., 1999). The ease of this procedure prompted us to investigate in detail the potential use of this laser system for a controlled immobilization of spermatozoa and for permeabilization of the sperm tail membrane in PVP as well as in tissue culture medium.
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Material and methods |
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Instrumentation
We used a non-contact 1.48 µm wavelength diode laser system (initially developed at the École Polytechnique Fédéral de Lausanne, EPFL, Lausanne, Switzerland). A description of the technical concept of this system and its precision in laser application is described elsewhere (Rink et al., 1994). In short, the InGaAsP laser diode emitting at a wavelength of 1.48 µm is coupled to an inverted microscope (DMIRB; Leica, Bensheim, Germany) and the laser beam is directed along the microscope optical axis. Specially attached lenses and mirrors within the laser unit allow focusing of the laser beam at the image plane of the microscope objective. The laser energy delivered to the image plane by a single laser shot can be easily adjusted by varying the length of the single laser pulse (Rink et al., 1996
). The microscope was equipped with a heated stage, a x40 objective and micromanipulation devices (Narishige, Tokyo, Japan) for sperm manipulation and for intracytoplasmic sperm injection (Montag et al., 1998
).
Laser-assisted immobilization and permeabilization
For laser treatment, spermatozoa were pipetted into 10 µl droplets of a solution of 10% PVP in culture medium (ICSI 1; Scandinavian IVF Sciences AB, Göteborg, Sweden) or pure tissue culture medium (Gamete 100; Scandinavian IVF Sciences AB) under mineral oil. Each droplet was placed next to another droplet of hypo-osmotic swelling (HOS) test medium (Hypo10; Scandinavian IVF Sciences AB).
We evaluated two strategies for immobilization of spermatozoa and permeabilization of the sperm membrane. In a first series of experiments a single laser irradiation was applied close to the middle of the sperm tail. We used a distance of 1015 µm from from the tail (see Figure 1A). This distance offered the best choice for the requirements of our field of view. Usually we did not chase spermatozoa but instead caught sperm cells, which, during their forward progression, moved within the laser beam interaction area (Figure 1A
). This strategy helped to avoid direct application of the laser pulse to the sperm head, although it is unknown if a direct hit to the sperm head would have biological consequences.
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Both experiments were performed at different energy values in PVP and in culture medium with spermatozoa showing different degrees of motility [World Health Organization (WHO) Type A, B, C; WHO, 1993]. Immediately after laser treatment, each individual spermatozoon was transferred into HOS medium to assess the integrity of the sperm tail membrane. Permeabilized spermatozoa show a negative reaction in the HOS test (Jeyendran et al., 1984; Ved et al., 1997
).
All these experiments were performed with one sperm sample. However, initial studies have shown that there were no variations in the susceptibility of spermatozoa to the laser treatment in different sperm samples (data not shown).
Injection of laser-treated spermatozoa into mouse oocytes
According to federal law of Germany, we obtained permission to perform the animal experimentation presented in this study (K32 147-2737/3203). Superovulation of female mice, isolation of oocytes, denudation by hyaluronidase and incubation was performed as described previously (Montag et al., 1998), except that females were not mated and isolation of oocytecumulus complexes occurred at 14 h after administration of human chorionic gonadotrophin (HCG).
For injection we used human spermatozoa which were immobilized in culture medium using a double laser shot technique with two successive laser irradiations, as described above. As a control we injected human spermatozoa which were immobilized in PVP using the common ICSI immobilization procedure using a glass capillary.
Injection of mouse oocytes was performed as prevously described (Rybouchkin et al., 1996) with the modification that we used HEPES-buffered culture medium (Gamete-100) throughout the manipulation procedure. Mouse oocytes were randomly allocated to the treatment or the control group. When we injected laser-immobilized spermatozoa, which were treated in culture medium, we rinsed the injection capillary prior to the first injection in a viscous medium (PureSperm; Nidacon, Gothenborg, Sweden), followed by several washes in culture medium. This pretreatment avoided sticking of the spermatozoa to the glass capillary. It was not applied for the conventional injection procedure where the use of PVP fulfilled the same purpose. Injected oocytes were cultured for another 12 h and were checked periodically for the extrusion of second polar body and formation of pronuclei.
Statistics
Differences in the effects of treatments were assessed using the 2-test.
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Results |
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Spermatozoa showing no or only a temporary motility arrest always retained membrane integrity and exhibited a positive response to the HOS test. Spermatozoa with damaged membranes (HOS-negative) were only detected among permanently arrested spermatozoa. This observation was consistent for PVP and for culture medium. The effect of laser treatment in PVP is summarized in Table I. Since no difference could be observed in PVP between the exposed spermatozoa of WHO types A and B, the data concerning these groups were combined. A 100% immobilization and permeabilization of WHO A/B spermatozoa was only achieved at an energy of 2 mJ. Spermatozoa of type WHO C showed a 100% immobilization at 0.5 mJ, but 100% permeabilization only occurred at 2 mJ.
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Discussion |
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As recently demonstrated for zona pellucida drilling (Germond et al., 1995), the use of a 1.48 µm wavelength laser for sperm immobilization provides an increased precision and added features. Sperm immobilization can be performed according to two simple procedures: in the first, the spermatozoon identified by the operator is placed within the area of laser action, which is typically 1015 µm wide; in the second, the operator waits until a spermatozoon having the appropriate features enters the laser interaction area. Both approaches allow for selection in real conditions prior to injection. Moreover, as permanent arrest is induced after laser treatment, any spermatozoon can be immobilized for further morphological inspection prior to injection. The laser immobilization procedure maintains the sperm tail aligned with the head, thus facilitating aspiration into the injection pipette. Using either the single or double shot immobilization techniques, permanent immobilization is accomplished within less than 1 min.
Although permanent sperm immobilization can be induced by a single 1.48 µm laser shot, we prefer a double shot technique which allows us to reduce the total laser energy dose delivered to the spermatozoon. The double shot technique allows us to reduce as far as possible the energy used for immobilization, while an even lower energy is sufficient to achieve membrane permeabilization with a 100% success rate due to precise aiming of the laser beam at the tail. The energy necessary for permeabilization of previously immobilized spermatozoa is as low as 0.25 mJ and not dependent on the viscosity of the medium. It should be noted that the dose for laser treatment of spermatozoa is five to 10 times lower than that required for laser zona drilling (Primi et al., 1999) and we can be reasonably confident that it is a safe approach.
The laser energy required for immobilization depends on the degree of progressive motility of the spermatozoa. Spermatozoa with a high progressive speed, which stay for only a short time within the laser interaction zone, require a higher immobilization energy. This is not the case in PVP, where no difference in energy was noticed due to the slow speed of spermatozoa of each category in the viscous medium.
Interestingly, two types of effects have been observed after irradiating the sperm tail. A low laser dose induces only a temporary immobilization, whereas above a certain threshold energy permanent immobilization is obtained. So far we have no detailed information on the type of cellular alteration associated with immobilization in laser-treated spermatozoa. However, we know from previous studies that the mode of action of the 1.48 µm diode laser is thermal (Rink et al., 1996; Hollis et al., 1997
). Laser irradiation induces a local temperature elevation within the sperm tail. At low laser doses, the temperature elevation perturbs the thermodynamic processes of the molecular motor driving the tail movements (Khan and Sheetz, 1997
): the spermatozoon stops. As a given time is needed to restore the activation mechanisms of the tail, a temporary motility arrest is observed. Above a given threshold temperature, protein denaturation is very likely and permeabilization of the sperm tail membrane is induced, leading to permanent motility arrest. However, the high absorption characteristics of the 1.48 µm wavelength in water, as well as the application of the laser near the end of the spermatozoon, guarantees that the local thermal effect does not affect the DNA within the sperm head. At present we do not know whether a direct application of the laser beam to the sperm head at a high energy could cause any damage to the DNA. This question is under investigation.
The new laser approach presented here has to be clearly distinguished from other laser sperm manipulations described previously. Laser tweezers have been proposed to catch and move spermatozoa in the context of laser subzonal insemination (SUZI) (Tadir et al., 1989; Schütze et al., 1994
). In this application the sperm head is trapped at the centre of a highly focused laser beam due to restoring forces induced by the optical field. The spermatozoon cannot escape, but the movements of the sperm tail are not affected. Although the wavelength of laser tweezers is chosen such that virtually no light is absorbed by the trapped objects, it has been demonstrated that too long an exposure to the optical trap may cause alteration of sperm motility (Tadir et al., 1989
) or even cell death (König et al., 1996
). In the other attempt, a pulsed UV-laser beam has been proposed to cut the sperm tail before injection or SUZI (Schütze et al., 1994
); however, ultraviolet light may bear mutagenic risks (Kochevar, 1989
), and for that reason is not recommended.
The benefit and the necessity of permeabilization of the sperm membrane is still a subject of controversy. However, Dozortsev et al. (1995) reported that permeabilization is beneficial for subsequent sperm nuclear decondensation. Permeabilization of the sperm plasma membrane may facilitate the influx of a sperm nucleus decondensing factor (SNDF) from the oocyte (Perreault and Zirkin, 1982; Dozortsev et al., 1997
). It was postulated that this factor releases a sperm-associated oocyte activation factor (SAOF), whose identity is uncertain at present (Stice and Robl, 1990
; Swann, 1990
; Kuretake et al., 1996
; Parrington et al., 1996
; Sette et al., 1997
). There might also be differences between the requirements for human ICSI and for ICSI in other species, where sperm pretreatment might be not so important (Yanagimachi, 1998
).
Laser immobilization without PVP
The laser technique developed here did not require the addition of a viscous substance to facilitate sperm capture. It offers the potential to avoid PVP, whose use is still controversial. It has been recently shown that PVP has a primary detrimental action on the plasma membrane of spermatozoa (Strehler et al., 1998). Moreover, it is obvious that a certain quantity of PVP is co-injected into the oocyte during the ICSI procedure. A significant increase of degeneration and a reduction of the fertilization rates has been observed, correlated to an increase of the volume of PVP injected during the ICSI procedure, although a direct effect of the volume injected could not be excluded (Payne et al., 1998
). It was suggested that chromosomal abnormalities after ICSI could be due to the injection of PVP into the oocyte (Feichtinger et al., 1995
). Finally, although cytogenetic mutagenicity tests did not reveal mutagenic effects of PVP (Ray et al., 1995
), it is commonly agreed that PVP might be a potentially harmful agent and as a precaution should be avoided (Jean et al., 1997
). Recently, Hlinka and coworkers proposed a modified method for ICSI without using PVP (Hlinka et al., 1998
). These authors observed a significantly higher rate of normally fertilized oocytes and concluded that this may be associated with the elimination of the potentially harmful effects of the conventional ICSI procedure.
The use of PVP is known to facilitate ICSI procedures by preventing sticking of spermatozoa to the inner wall of the pipette. By contrast, mechanical immobilization in culture medium frequently results in the formation of kinks in the sperm tail, which greatly complicates subsequent sperm pipetting, as kinked spermatozoa tend to stick inside the injection capillary. The 1.48 µm diode laser technique for immobilization and permeabilization bypasses these problems, as we never observed kinked sperm tails following laser treatment. Pipetting of laser-treated spermatozoa in culture medium is thus easy to perform.
During our initial trials with injection of spermatozoa laser-treated in culture medium, we noted that the inner wall became sticky after some injections although no ooplasmic remnants were detectable inside the injection capillaries. Sticking of spermatozoa usually does not occur with PVP (Palermo et al., 1995). We found that this problem is not related to the laser treatment but to the fact that we used culture medium. It could be prevented by an initial rinsing of the injection capillary in a viscous medium (e.g. a sperm preparation medium) followed by several washes with culture medium prior to starting the injection session.
Further experimentation in an animal model is required to confirm the safety of the laser procedure before its application in human ICSI. However we were able to achieve activation of mouse oocytes and pronuclear formation following injection of laser-treated spermatozoa. This shows that laser-immobilized and permeabilized spermatozoa are at least capable of initiating fertilization.
In conclusion, a novel laser-induced sperm immobilization and permeabilization technique is proposed. The technique is simple and quick and does not require the use of an additional medium to slow down the spermatozoa. As compared to the PVP technique, the laser approach offers the possibility of controlling the sperm motility and behaviour prior to immobilization in representative conditions and allows a final morphological check before injection due to complete arrest of the spermatozoa. However, prior to applying this technique in human IVF, the safety of our approach needs to be investigated in animal experiments with special emphasis on the evaluation of embryonic development, implantation potential and pregnancy outcome.
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Acknowledgments |
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Notes |
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References |
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Submitted on August 27, 1999; accepted on January 10, 2000.