greater width of the underlying perivitelline space. Hyperosmolar sucrose solution to extend the perivitelline space was not used. Depending on the thickness of ...
Human Reproduction vol.12 no.10 pp.2242–2245, 1997
Ultrastructural observations in human oocytes and preimplantation embryos after zona opening using an erbium–yttrium–aluminium–garnet (Er:YAG) laser
Andreas Obruca1,3, Heinz Strohmer1,3, Astrid Blaschitz2, Elisabeth Scho¨nickle2, Gottfried Dohr2 and Wilfried Feichtinger1,4 1Institute
for Sterility Treatment, Trauttmansdorffgasse 3a, A-1130 Vienna, Austria, 2Institute of Histology and Embryology, Karl-Franzens-University Graz, A-8010 Graz, Austria
3Present
address: Department of Obstetrics and Gynaecology, University of Vienna Medical School, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria
4To
whom correspondence should be addressed
For more than 3 years we have performed laser-assisted hatching prior to embryo transfer in patients with recurrent implantation failure using an erbium–yttrium–aluminium– garnet (Er:YAG) laser system that operates in the infrared region of the light spectrum. The laser beam is guided through a quartz fibre and is brought into direct contact with the zona pellucida. This study was undertaken to evaluate the ultrastructural effects of this laser on the zona pellucida and underlying cell membrane of unfertilized human oocytes and pathologically fertilized preimplantation embryos using light and scanning electron microscopy. The Er:YAG laser produces an almost circular zona opening in the shape of a truncated cone tapering off towards the inside, with a mean diameter of 18 mm. The exact diameter of the drilled site depends on the diameter of the fibre tip and the total number of pulses applied. After laser interaction, the zona matrix and the surface of the underlying ooplasm membrane showed no degenerative alterations. We conclude that the Er:YAG laser is an effective microsurgical tool for achieving reproducible, precise zona openings particularly suitable for purposes of assisted hatching because of their characteristic shape. Key words: assisted hatching/scanning electron microscopy/ infrared laser/micromanipulation/zona pellucida
Introduction A variety of laser systems have been considered for microsurgical procedures on human gametes. Thus, attention has focused on the possibility of using infrared laser optical traps to capture or transport spermatozoa (Tadir et al., 1989; Schu¨tze et al., 1994) or to measure their velocity (Colon et al., 1992). Moreover, numerous recent experiments have attempted to use laser and light delivery systems, instead of the chemical or mechanical methods currently in use, to achieve partial zona dissection by localized thinning or to open the zona pellucida for purposes of microsurgical fertilization (Cohen et al., 1988) 2242
and as a means of preimplantation-assisted hatching following in-vitro fertilization (IVF) (Cohen et al., 1990). The majority of previous experimental studies, most of which have been performed on animal cells, have used ultraviolet laser systems at 193–532 nm wavelengths (Tadir et al., 1991; Blanchet et al., 1992; Laufer et al., 1993; Simon et al., 1993) or, less frequently, infrared lasers (Coddington et al., 1992; Germond et al., 1995). Taking into account the prerequisites for the application of lasers in human embryology (Strohmer and Feichtinger, 1992a), we have designed an erbium–yttrium–aluminium–garnet (Er:YAG) laser system in our laboratory using a 2.9 µm wavelength, thus operating in the infrared part of the spectrum (Strohmer and Feichtinger, 1992b; Feichtinger et al., 1992). Because the safety and efficacy of this laser system have already been demonstrated in a clinical environment (Obruca et al., 1994; Antinori et al., 1996a), it was the aim of the current investigation to evaluate the ultrastructural effects of lasers on the zona pellucida and the underlying cell membrane of unfertilized human oocytes and preimplantation embryos using light and scanning electron microscopy.
Materials and methods The micromanipulation procedure was performed using the same laser equipment as reported previously (Strohmer and Feichtinger, 1992a; Obruca et al., 1994). Briefly, the system consists of a 2940 nm Er:YAG laser (Fertilaser; LISA laser products, Katlenburg, Germany) fed into a laser fibre with a tip diameter of 20 µm. We used pulses of ~10 µJ energy, causing a penetration depth of 3 µm in biological tissue. Six human oocytes showing fertilization failure 2 days after invitro insemination and two triploid embryos were obtained from our IVF programme. The oocytes or embryos were placed together in a microdrop of HEPES-buffered Earle’s balanced salt solution (EBSS) medium (Medicult, Copenhagen, Denmark) on the same microscope slide and covered with 37°C warm paraffin oil (Medicult). The procedure was carried out on an inverted microscope (Diaphot, Nikon, Tokyo, Japan) equipped with a heated stage and two hydraulic micromanipulators (Narishige, Tokyo, Japan). The oocyte or embryo was held by negative pressure using a holding pipette (Humagen, Virginia, USA) of 130 µm outer and 20 µm inner diameter. The glass fibre, fitted to the manipulator by a pipette holder, was brought into direct contact with the zona but the tip was not forced into it to avoid contact with the surface of the ooplasm or blastomere upon penetration of the zona. The preferred site of laser application was the region of the indentation between two adjacent blastomeres because of the greater width of the underlying perivitelline space. Hyperosmolar sucrose solution to extend the perivitelline space was not used. Depending on the thickness of the zona, which was between 15 and 18 µm, five to eight pulses were necessary to penetrate the zona. Because each laser pulse removes only small portions of the zona, © European Society for Human Reproduction and Embryology
Electron microscopy of laser assisted hatching
Figure 1. The unfertilized oocyte is attached to the holding pipette by negative pressure (left). The laser fibre is brought into direct contact with the zona pellucida at the 4 o’clock position. Application of three to five laser pulses leads to ablation of the surface layer of the zona pellucida while leaving the inner layer intact (zona thinning). (Original magnification 3400)
Figure 2. An additional three to four laser pulses are required to achieve complete penetration of the zona pellucida. The wellcircumscribed, funnel-shaped hole had a mean diameter of 18 µm. The preferred site of laser application was where the underlying perivitelline space was wide enough, e.g. at the conjunction between two adjacent blastomeres. (Original magnification 3400)
the fibre tip was readjusted continuously so as to guarantee close contact with the remaining zona. All oocytes and embryos were drilled at one site only. The drilling procedure itself took ~15–25 s per oocyte. Immediately after laser drilling, the oocytes and embryos were fixed in 2% glutaraldehyde and 2% paraformaldehyde freshly prepared in 0.1 M cacodylate buffer, washed thoroughly in 0.1 M cacodylate buffer (pH 7.2), and post-fixed in 2% osmium tetroxide in cacodylate buffer. Oocytes were mounted on alcian blue-coated cover slips, dehydrated in ethanol, incubated in acetone, critical point-dried, sputter-coated with gold and palladium, and examined in a Zeiss DSM 950 (Zeiss, Germany) scanning electron microscope.
Results Effect of zona drilling on the cytoplasm as seen by light microscopy Ablation of the zona pellucida could be followed easily by light microscopy. In a very few instances the laser pulse caused a weak mechanical vibration of the oocyte or embryo. None of the laser-treated oocytes of embryos exhibited discoloration or morphological changes in the underlying cytoplasm during or after laser interaction. Complete opening of the zona pellucida was evident by the ability of the fibre tip to pass freely through the hole into the perivitelline space without deforming the surrounding zona pellucida and by free influx and outflux of perivitelline granular material. The first three to five laser pulses removed only the surface layer of the zona while leaving the inner layer intact. This enabled precise control of the drilling process, resulting in reproducible zona thinning in a well-circumscribed area (Figure 1). As demonstrated in Figure 2, application of an additional three to four pulses resulted in localized opening of the zona pellucida, with light microscopy demonstrating a funnelshaped, clearly delimited hole with a mean diameter of 18 µm. Generally, the drilled holes exhibited a slightly wider outside and a narrower inside diameter. In the entire series of holes,
Figure 3. Scanning electron microscopy (original magnification 31000) of a well-circumscribed opening in the zona pellucida of an unfertilized oocyte exposed to Er:YAG laser light. The outer and inner diameters of the hole are 20317 and 14310 µm, respectively.
the diameter of the drilled site depended on the diameter of the fibre tip and the total number of pulses applied. Characterization of the drilled holes using scanning electron microscopy Scanning electron micrographs (SEM) of the holes generated in the zona pellucida are shown in Figures 3–6. The low magnification micrograph in Figure 3 shows a laser-generated hole in an unfertilized oocyte with outside and inside diameters of 20317 and 14310 µm, respectively. As already demonstrated under light microscopy, the SEM study also showed an almost circular hole of the shape of a truncated cone narrowing towards the perivitelline space. Figure 6 provides a particularly clear image of this special type of opening generated in the zona pellucida of a 4-cell embryo. Higher magnification details of the impact region show the 2243
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Figure 4. The high magnification micrograph (original magnification 35000) shows the ultrastructure of the zona pellucida previously shown in Figure 3. Significant features include the wellpreserved porous zona matrix, with no signs of damage to the zona surrounding the impact area.
Figure 5. A higher magnification detail (original magnification 310 000) of the opening shown in Figure 4 provides a clear image of the underlying cell membrane. The characteristic and highly sensitive microvilli were not found to be destroyed or broken, suggesting that laser drilling did not damage the surface of the ooplasm membrane.
well-preserved dense matrix of the zona pellucida adjacent to the ablation area (Figure 4). Also, the surface of the underlying ooplasm membrane appeared intact (Figure 5), because the characteristic microvilli on the surface of the membrane were found not to be destroyed or broken. Discussion The ultrastructural effects of various laser systems on the zona pellucida and the underlying cell membrane depend on whether laser light is delivered to the target in the non-contact mode through microscope objectives, hitting the zona pellucida tangentially with a given spot size, or whether the laser is applied in the contact mode through an optic fibre that is brought into direct contact with the zona pellucida, delivering the beam perpendicularly. However, on the one hand, contact laser systems need the use of micromanipulators, as used for intracytoplasmic sperm injection (ICSI), for moving and 2244
Figure 6. Scanning electron microscopy of a 4-cell embryo (original magnification 31000). As already demonstrated under light microscopy (cf. Figure 2), the electron micrograph shows an almost circular hole of the shape of a truncated cone narrowing towards the inside, with the diameter of the drilled site depending on the diameter of the fibre tip and the total number of pulses applied.
adjusting the optical fibre and training is necessary, while in contrast non-contact systems need a precision microscope stage for moving the oocytes or embryos. Nevertheless the routine handling of a contact laser offers great similarities to the ICSI procedure. Another important determinant is the wavelength, with most laser systems making use of either the ultraviolet or the infrared region of the spectrum. In addition, the laser pulse energy, pulse duration, and pulse repetition rate all play a role (Tadir et al., 1994). Non-contact laser systems use wavelengths which are transmitted well by microscope lenses and water but require increased pulse energy levels to perform zona ablation (Neev et al., 1993; Antinori et al., 1996b). The shape of the almost circular hole produced by the Er:YAG laser, resembling a truncated cone tapering off towards the inside, can be accounted for by several factors. The 2.9 µm wavelength used in our Er:YAG laser is strongly absorbed by water. Therefore, the laser beam cannot be focused directly through the culture medium but must be delivered to the zona surface by means of a quartz fibre, thus hitting the target perpendicularly. The size of the fibre tip determines the spot size of the laser and, consequently, the size of the hole it produces. Moreover, because of the relatively low penetration depth per pulse, several pulses are needed to achieve gradual ablation and opening of the zona pellucida, with the energy characteristics right at the tip of the fibre causing the diameter of the hole to decrease towards the inside. This 10–20 µm aperture differs in shape and size from the openings generated by microscope-delivered lasers directed tangential to the oocyte sphere. Tangential laser irradiation results in trench or tunnellike holes, depending on whether the laser beam is aimed at the zona pellucida tangentially on the equatorial plane or whether the beam is directed in a more poleward direction. The maximum attainable hole size is 10 µm, with the diameter of the drilled site depending on the pulse energy applied (Neev et al., 1992).
Electron microscopy of laser assisted hatching
In a study by Cohen and Feldberg (1991) it was demonstrated that complete hatching and normal trophoblast outgrowth depend on the size, shape, and number of zona pellucida openings. Thus, mechanical partial zona dissection, producing narrow incisions ø5 µm in diameter similar to those obtained by lasers delivered tangentially, led to complete hatching in only 16% of embryos, whereas the remainder were trapped in a typical figure-of-eight shape. In contrast, zona drilling by acidified Tyrode’s solution, creating larger, round holes 11– 25 µm in diameter comparable with the openings produced by our Er:YAG laser, allowed for normal hatching in 92% of embryos (Cohen and Feldberg, 1991). A subsequent study (Neev et al., 1993) performing assisted hatching in mouse embryos zona-drilled with acidified Tyrode’s solution or lased by means of a 308 nm non-contact excimer laser showed that significantly more embryos were hatching in the chemically drilled group (50%) than in the laser-treated group (24%). Therefore, although direct comparisons between contact and non-contact laser systems are lacking, it would appear from these results that the large, round holes produced by our contact laser are more effective at promoting complete embryonic hatching than the openings created by the non-contact approach, thus reducing the incidence of blighted ova (Malter and Cohen, 1989) or monozygotic twins (Cohen et al., 1990). Moreover, because it has been suggested that embryonic cells are highly vulnerable to acidified Tyrode’s solution (Gordon et al., 1988; Malter and Cohen, 1989), an equally effective but less detrimental laser system would be a welcome alternative to replace chemical zona drilling (Schiewe et al., 1995). Previous studies in mouse embryos (Strohmer and Feichtinger, 1992b) and in a clinical setting (Obruca et al., 1994) have confirmed the utility and safety of the Er:YAG laser system for zona pellucida drilling. Unlike ultraviolet radiation, the 2.940 µm wavelength of the Er:YAG laser does not have any detrimental effects on the genetic material. Also, in the current study, the ooplasm and its surface did not exhibit detectable alterations during or after laser interaction. In contrast, Germond et al. (1995) reported that laser irradiation using a 1.48 µm diode laser induced local changes in the appearance of the cytoplasmic material. Similarly, potential adverse effects on embryonic development (Li et al., 1993) have also been described after manipulation with 308 nm excimer lasers (Neev et al., 1993). In conclusion, the results of this study examining the ultrastructural effects of an Er:YAG laser on the zona pellucida and the underlying cell membrane of unfertilized oocytes and preimplantation embryos suggest that this laser system is particularly suitable for achieving partial zona dissection and facilitating assisted hatching.
micromanipulation in the mouse embryo: a novel approach to zona drilling. Fertil. Steril., 57, 1337–1341. Coddington, C.C., Veeck, L.L., Swanson, R.J. et al. (1992) The YAG laser used in micromanipulation to transect the zona pellucida of hamster oocytes. J. Assist. Reprod. Genet., 9, 557–563. Cohen, J. and Feldberg, D. (1991) Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo. Mol. Reprod. Dev., 30, 70–78. Cohen, J., Malter, H., Fehilly, C. et al. (1988) Implantation of embryos after partial opening of oocyte zona pellucida to facilitate sperm penetration. Lancet, ii, 162. Cohen, J., Elsner, C., Kort, H. et al. (1990) Impairment of the hatching process following IVF in the human and improvement of implantation by assisted hatching using micromanipulation. Hum. Reprod., 5, 7–13. Colon, J.M., Sarosi, P., McGovern, P.G. et al. (1992) Controlled micromanipulation of human sperm in three dimensions with an infrared laser optical trap: effect on sperm velocity. Fertil. Steril., 57, 695–698. Feichtinger, W., Strohmer, H., Fuhrberg, P. et al. (1992) Photoablation of oocyte zona pellucida by erbium–yag laser for in-vitro fertilisation in severe male infertility. Lancet, 339, 811. Germond, M., Nocera, D., Senn, A. et al. (1995) Microdissection of mouse and human zona pellucida using a 1.48-µm diode laser beam: efficacy and safety of the procedure. Fertil. Steril., 64, 604–611. Gordon, J.W., Grundfeld, L., Garrisi, G. et al. (1988) Fertilization of human oocytes by sperm from infertile male after zona pellucida drilling. Fertil. Steril., 50, 68–73. Laufer, N., Palanker, D., Shufaro, Y. et al. (1993) The efficacy and safety of zona pellucida drilling by a 193-nm excimer laser. Fertil. Steril., 59, 889–895. Li, L., Munne, S., Licciardi, F. et al. (1993) Microinjection of FITC-dextran into mouse blastomeres to assess topical effects of zona photoablation. Zygote, 1, 43–48. Malter, H.E. and Cohen, J. (1989) Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration. Fertil. Steril., 51, 139–148. Neev, J., Tadir, Y., Ho, P. et al. (1992) Microscope-delivered ultraviolet laser zona dissection: principles and practices. J. Assist. Reprod. Genet., 9, 513–523. Neev, J., Gonzalez, A., Licciardi, F. et al. (1993) Opening of the mouse zona pellucida by laser without a micromanipulator. Hum. Reprod., 8, 939–944. Obruca, A., Strohmer, H., Sakkas, D. et al. (1994) Use of lasers in assisted fertilization and hatching. Hum. Reprod., 9, 1723–1726. Schiewe, M.C., Neev, J., Hazeleger, N.L. et al. (1995) Developmental competence of mouse embryos following zona drilling using a non-contact holmium:Yttrium scandian gallium garnet (Ho:YSGG) laser system. Hum. Reprod., 10, 1821–1824. Schu¨tze, K., Clement-Sengewald, A. and Ashkin, A. (1994) Zona drilling and sperm insertion with combined laser microbeam and optical tweezers. Fertil. Steril., 61, 783–786. Simon, A., Palanker, D., Harpaz-Eisenberg, V. et al. (1993) Interaction between human sperm cells and hamster oocytes after argon fluoride excimer laser drilling of the zona pellucida. Fertil. Steril., 60, 159–164. Strohmer, H. and Feichtinger, W. (1992a) Application of laser for micromanipulation: relevance of biophysical criteria. Fertil. Steril., 48, 540. Strohmer, H. and Feichtinger, W. (1992b) Successful clinical application of laser for micromanipulation in an in vitro fertilization program. Fertil. Steril., 58, 212–214. Tadir, Y., Neev, J. and Berns, M.W. (1994) Lasers in micro-manipulation of preimplantation embryos and gametes. Semin. Reprod. Endocrinol., 12, 169–176. Tadir, Y., Wright, W.R., Vafa, O. et al. (1989) Micromanipulation of sperm by a laser generated optical trap. Fertil. Steril., 52, 870–873. Tadir, Y., Wright, W.H., Vafa, O. et al. (1991) Micromanipulation of gametes using laser microbeams. Hum. Reprod., 6, 1011–1016. Received on October 25, 1996; accepted on July 25, 1997
References Antinori, S., Panci, C., Selman, H.A. et al. (1996a) Zona thinning with the use of laser: a new approach to assisted hatching in humans. Hum. Reprod., 11, 590–594. Antinori, S., Selman, H.A., Caffa, B. et al. (1996b) Zona opening of human embryos using a non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy. Hum. Reprod., 11, 2488–2492. Blanchet, G.B., Russell, J.B., Fincher, C.R. and Portmann, M. (1992) Laser
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