Human Reproduction, Vol. 14, (Suppl. 1), pp. 186-193, 1999
Possible applications of lasers in assisted
reproductive technologies
B.Schopper1, M.Ludwig, J.Edenfeld, S.Al-Hasani and
K.Diedrich
Department of Obstetrics and Gynecology, Medical University of Liibeck,
Ratzeburger Allee 160, 23538 Liibeck, Germany
l
To whom correspondence should be addressed
Laser systems seem to be the most promising technical tools to be introduced
in assisted reproduction treatment in recent years. The 1.48 Jim diode laser
in particular is on course to supersede conventional mechanical and chemical
methods for opening the zona pellucida in assisted hatching, and for biopsy
of polar bodies or blastomeres. This is because it works without physically
touching the cells, has no traceable toxic effects on living cells and is easy
to handle. There will be manifold future possibilities for micromanipulation
with lasers, as already demonstrated by the immobilization of spermatozoa
prior to intracytoplasmic sperm injection, and the successful dissection of
the zona pellucida into two halves with equal diameter for the hemizona assay.
Key words: ART/embryo biopsy/hemizona assay/laser/preimplantation genetic
diagnosis
Introduction
Since the introduction of human in-vitro fertilization (IVF) and the establishment
of the first successful steps, micromanipulation of embryos has been an integral
part of assisted reproductive technologies. Techniques implemented include zona
rubbing, zona thinning, assisted hatching to improve embryonic implantation, as
well as partial zona dissection, subzonal insemination and, most recently,
intracytoplasmic sperm injection (ICSI) to increase the fertilization rate in cases
of severe male factor infertility. Another application of embryo micromanipulation
is preimplantation genetic diagnosis (PGD), with the biopsy of polar bodies or
single blastomeres after opening the zona pellucida (ZP).
All these techniques had to be validated for their impact on the further
preimplantation and post-implantation development of the embryos. Obviously,
the least invasive procedures are the most appropriate ones for manipulation of
gametes, zygotes and embryos.
The introduction of laser techniques into the field of assisted reproduction
technology has opened different possibilities for fast and efficient manipulation
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© European Society for Human Reproduction and Embryology
Lasers in assisted reproductive technologies
procedures and it will supersede many of the currently used mechanical and
chemical treatments. The introduction of a laser system is the result of the wish
to standardize procedures, as was the computer-controlled micromanipulator
(Al-Hasani et al, 1999).
The history of laser technology in assisted reproductive technologies
Laser microbeams were first applied to gametes 10 years ago (Tadir et al, 1989).
Tadir et al. used a neodynium:yttrium aluminium garnet (Nd:YAG) laser to
generate an optical trap for spermatozoa and were able to manipulate the
movements of spermatozoa. Most of the subsequent clinical and research studies
of laser application in assisted reproduction have concentrated on the treatment
of the ZP. The first laser drilling of ZP was performed with an ArF-excimer laser
emitting in the UV region at 193 nm (Palanker et al, 1991). Oocytes treated in
this way fertilized in vitro at low sperm concentrations and developed to the
blastocyst stage. The low penetration depth of this laser radiation in the saline
culture medium, together with high absorption by the microscope optics, made
it necessary to touch the ZP with a laser-delivering micropipette. The erbium: YAG
laser also works in this contact mode, emitting 2.9 (im radiation, and has been
applied to improve fertilization in cases of severe male infertility (Feichtinger
et al, 1992) and to perform assisted hatching of mouse and human embryos
(Strohmer and Feichtinger, 1992). It was proved (Obruca et al, 1997) with the
help of light and scanning electron microscopy that ZP drilling with the
erbium:YAG laser does not cause any detectable alterations of the ooplasm
membrane next to the laser target region of the ZP and underlined the safety of
this system in contrast to lasers emitting UV light. As mentioned in a review
about laser practice (Neev et al, 1992a), the difficulties with systems requiring
contact, such as energy delivery to the target, and the necessity for special laserdelivering instruments, such as micropipettes or optical fibres, defeat most of the
advantages of using laser light and essentially reduce the laser-based techniques
to a mechanical/contact mode, similar to conventional ZP drilling methods.
One of the first non-contact laser systems was the 248 nm KrF excimer, which
was used for drilling of the ZP of mouse embryos (Blanchet et al, 1992).
Radiation of this wavelength is well transmitted through the culture medium and
the optical components of the microscope and can therefore be used in a
microscope objective delivery mode. Possible mutagenic effects are an important
argument against the application of radiation with wavelengths close to the
absorption peak of DNA at 260 nm. With respect to higher wavelengths and a
non-contact laser application, it was found (Neev et al, 1992b) that the 308 nm
emission of the xenon chlorine laser was suitable for ZP drilling. This same
group was able to show that oocytes treated in this way had enhanced
in-vitro fertilization rates by spermatozoa from long-term vasectomized mice
(El-Danasouri et al, 1993). A Nd:YAG laser with 1.06 \xm wavelength was also
used to drill hamster oocytes (Coddington et al, 1992). The combined usage of
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B.Schopper et al.
an UV nitrogen laser and the Nd:YAG laser has been described (Schiitze et al,
1994), which functioned as an optical tweezer. With this system, Schiitze et al.
were able to drill holes in the ZP and to catch and transport a spermatozoon
through the drilled holes into the perivitelline space.
The 1.48 |xm diode laser: most suitable for assisted reproduction
techniques
A crucial step in the further development of laser applications in assisted
reproduction techniques was made in 1994 with the introduction of an infrared
diode laser emitting at 1.48 |im (Rink et al, 1994). This system allows noncontact, microscope objective-delivered accessibility of laser light to the target
with minimal absorption by the culture dish and the aqueous medium. In addition,
the emitted wavelength is far from the absorption peak of DNA.
Ultrastructural studies on mouse oocytes and zygotes revealed that the laser
effect on the ZP is greatly localized and produces cylindrical holes with sharp
edges and smooth walls.
Laser-drilled mouse embryos gave rise to normal and fertile offspring (Germond
et al, 1995), with improved fertilization and implantation rates (Germond et al,
1996). Veiga et al (1997) applied the diode laser system to human embryos,
dissecting ZP in order to biopsy blastocysts for PGD, and achieved good results.
It was shown that laser drilling of the ZP of mouse oocytes facilitates polar body
biopsy (Montag et al, 1998a), and recently one pregnancy was reported after
successful use of the diode laser for cleavage-stage biopsy of a human embryo
for PGD (Boada et al, 1998).
Sperm immobilization has also been done with the diode laser prior to ICSI
(Montag et al, 1998b) and before cryopreservation of spermatozoa in an empty
ZP (Montag et al, 1998c). Recognition of the advantages of the diode laser
system (commercially available as FERTILASE™, MTM Medical Technologies
Montreux, Clarens, Switzerland) has led to its extensive use in IVF laboratories,
not least because this laser is compact, affordable and easily adapted to all types
of inverted microscopes.
Here, we review and discuss the possible applications of a non-contact diode
laser, including ZP drillings for embryo biopsy, assisted hatching, and zona
dissection to perform a hemizona assay (HZA). We also present some new data
on two of these topics.
Applications of the diode laser
Embryo biopsy after ZP drilling
Different techniques have been described to dissect the zona for embryo and
polar body biopsy in PGD. Acid Tyrode's solution (Tarin and Handyside, 1993)
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Lasers in assisted reproductive technologies
Table I. Results of mouse embryo biopsy after zona perforation using either acid Tyrode's
solution or a laser. No statistically significant differences exist between the different groups
No. of 2-cell stage embryos (A)
No. of 8-cell stage (% of A)
No. biopsied/manipulated (B)
No. of totally hatched blastocysts
(% of B)
No. of not totally hatched
blastocysts (% of B)
No. of arrested embryos (% of B)
Control
Laser
(control*)
Laser
+ biopsy
Acid Tyrode's
(control*)
Acid Tyrode's
+ biopsy
132
112 (85)
112
87 (78)
95
83 (87)
83
59 (71)
123
105 (85)
105
68 (65)
91
74 (81)
74
53 (72)
116
102 (88)
102
68 (67)
15(13)
11 (13)
10(10)
9(12)
13(13)
10 (8.9)
13 (15.7)
27 (25.7)
12 (16.2)
21 (20.6)
*Only the zona was perforated; no blastomeres were biopsied.
and mechanical dissection using sharp pipettes (Thompson et al, 1995) were
used previously. These methods were combined with different approaches to
remove the blastomeres, including aspiration with a biopsy pipette forced through
the ZP like a needle (Krzyminska et al, 1990), or after previous opening of the
ZP by acid Tyrode's (Handyside et al, 1989, 1990; Hardy et al, 1990) or
mechanically (Grifo et al, 1990). Others have demonstrated the advantages of
using an extrusion technique, i.e. pulling out the blastomeres through a hole
without touching the cell. This can be done by displacement, i.e. the injection
of culture medium into the embryo after opening the zona at another site, which
helps blastomeres to protrude beyond the zona (Roudebush et al, 1990; Takeuchi
et al, 1992). Finally, blastomeres can be removed by squeezing the blastomere
out of the zona using pressure to a point somewhat distant to a hole in the ZP
made either mechanically or enzymatically (Gordon and Gang, 1990).
To confirm that no differences in in-vitro developmental potential exist between
embryos biopsied after opening of the ZP with either acid Tyrode's or the diode
laser system, we randomly allocated 557 fresh 2-cell stage mouse embryos (B6
CBA strain, F{ hybrids: C57B1/JXCBA) to five groups. The embryos of one
group were cultured up to the hatched blastocyst stage (control), two groups had
the zona perforated by either the 1.48 i^m diode laser (laser control) or acid
Tyrode's (Tyrode's control) without biopsy, and two groups were perforated by
one of the methods and biopsied. The methods of hormonal stimulation, in-vivo
conception, retrieval of embryos, embryo culture and biopsy have been described
elsewhere (Ludwig et al, 1998). As can be seen in Table I, the developmental
rate up to the 8-cell stage was comparably high in all groups, with a total
cleavage rate of 85% and no statistically significant differences between groups.
Also, the hatched blastocyst rate, with an overall value of 70%, was similar for
all groups and did not show statistical difference. This confirms, in a prospective,
controlled way, the observations made in other studies (Veiga et al, 1997; Boada
et al, 1998; Montag et al, 1998a). We did not transfer the treated embryos to
foster mothers, so no data are available regarding their implantation rates.
In most cases using standardized micromanipulators, performing a biopsy is a
time-consuming, two-step procedure. First, one embryo after another is fixed
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B.Schopper et al.
with the holding pipette, drilled with the perforation pipette and then released.
In the second step, the perforation has to be replaced by the biopsy pipette and
each embryo has to be positioned and fixed with the holding pipette again to
align the drilled hole with the biopsy pipette. Furthermore, the released acid
Tyrode's solution can harm the exposed blastomeres. Using the non-contact
1.48 (im diode laser system, there is no longer any need to replace pipettes.
After fixing and positioning the embryo, the laser is switched on and the biopsy
can be done directly thereafter through the laser-created hole in one step.
Therefore, the time to handle one embryo is significantly shorter when using the
laser compared to micromanipulation with acid Tyrode's solution.
Assisted hatching
Prior to their implantation in the endometrium, embryos at the blastocyst stage
have to leave the ZP. It is a widely believed that artificial generation of a hole
in the ZP supports hatching of the embryos. In the case of IVF treatments, the
so-called assisted hatching has been suggested to improve the pregnancy rate
under certain conditions (Magli et al, 1998). In-vitro culture or cryopreservation
of oocytes and embryos can cause structural alterations of the ZP, and these are
collectively known as zona hardening. However, there is an ongoing debate as
to whether assisted hatching really has advantages in defined subgroups of
patients (Schoolcraft et al, 1994; Antinori et al, 1996a,b; Tucker et al., 1996;
Bider et al, 1997; Lanzendorf et al, 1998).
Methods for creating holes in the ZP for assisted hatching are the same as
described above for the biopsy procedure. Mechanical and chemical opening of
the ZP are difficult to handle and the results are not reproducible. In particular,
the use of acid Tyrode's solution can have detrimental effects on embryos.
Within this setting, the use of a diode laser will have the advantage of giving
reproducible results, with a hole of a defined diameter, which can be made within
a few milliseconds with one or two pulses. By using a standardized procedure,
it will really be possible to answer the question of whether there is a beneficial
effect of assisted hatching on implantation and pregnancy rates in certain patients.
The hemizona assay: a new application for laser technique
Searching for other potential applications of the diode laser, we tried to perform
dissection of ZP into matching halves for the hemizona assay (HZA). The HZA
is a commonly used functional bioassay to test binding of spermatozoa to the
ZP. Cutting the ZP from a non-fertilizable oocyte into two matching hemizonae
provides an internal control for the great variability between the zonae of different
oocytes. The HZA is not only an important diagnostic tool for assessing the
fertilizing potential of human spermatozoa in IVF treatments (Coddington et al.,
1994; Oehninger et al, 1997), but is also used to test whether certain substances,
e.g. sera, follicular fluids or seminal plasma (Huyser et al, 1997) or xenobiotics
(Hinsch et al, 1997), affect the interaction between spermatozoa and ZP.
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Lasers in assisted reproductive technologies
Hemizonae are normally generated with the help of a micromanipulator
working with a holding pipette at one side to fix the oocyte and a microscalpel
at the other side to cut it (Burkman et al, 1988). Some results were achieved
by 'handcutting' with regard to recovery rate, equal diameter of the hemizonae
and sperm binding capacity (Sanchez et al, 1995). With the 1.48 |um diode laser,
we were able to bisect the ZP of oocytes into two halves with nearly equal
diameter. A radiation time of 20 ms per pulse caused an ablation line of -15 (im,
which was considered to be small compared with the diameter of the oocyte.
Approximately 20 pulses were necessary to dissect the ZP into two roughly
equal halves.
The production of hemizonae with the diode laser is fast and efficient, and
there is no need for special bent holding pipettes as in the case of scalpel cutting.
Once again, it was shown that the laser is a very quick and precise working tool.
Conclusion
To conclude, in all cases described so far, applications of the diode laser system
have been proven to be highly effective and time saving. No disadvantages
compared with the established methods were shown for blastomere biopsy and
preparing hemizonae for the HZA. However, since the laser is easy to handle
and allows precise working even for beginners in the field of assisted reproduction,
the introduction of laser technology seems to be one of the most promising ways
to simplify the various techniques of gamete and embryo manipulation.
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