The erbium-yttrium-aluminium-garnet (Er: YAG) laser has been applied to micromanipulation in humans. It was used in the fertilization process for both subzonal insemination (SUZI) and for partial zona dissection (PZD). Laser-assisted micromanipulation achieved significantly higher fertilization rates (34.8%) when compared to mechanical SUZI (16.1%), but use of the laser did not improve the PZD results (laser 14.8% versus mechanical 14%). The Er: YAG laser was used to assist hatching. In the mouse it significantly improved the hatching rate (80 versus 29.3%) 110 h after administration of human chorionic gonadotrophin. This technique was applied in two different centres to patients with previous in-vitro fertilization (IVF) failures. The implantation rate per embryo (14.4% laser-assisted hatching versus 6% control group) and the pregnancy rate per transfer (40 versus 16.2%) were improve

Barak, Y.

Feichtinger, W.

Kogosowski, A.

Menezo, Y.

Obruca, A.

Sakkas, D.

Strohmer, H.

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Human Reproduction vol.9 no.9 pp. 1723-1726, 1994Use of lasers in assisted fertilization and hatchingA.Obruca1, H.Strohmer1, D.Sakkas2, Y.Menezo23,A.Kogosowski4, Y.Barak4 and W.Feichtinger1'5'Institute of Sterility Treatment, Trauttmansdorffgasse 3A,A-1130 Vienna, Austria, 2H6pital Cantonal Universitaire deGeneve, Switzerland, 3Institut Rhonalpin, Lyon, France and4Herzliya Medical Centre, Herzliya-on-Sea, Israel5To whom correspondence should be addressedThe erbium-yttrium-aluminium-garnet (Er:YAG) laserhas been applied to micromanipulation in humans. It was usedin the fertilization process for both subzonal insemination(SUZI) and for partial zona dissection (PZD). Laser-assistedmicromanipulation achieved significantly higher fertilizationrates (34.8%) when compared to mechanical SUZI (16.1%),but use of the laser did not improve the PZD results (laser14.8% versus mechanical 14%). The Er:YAG laser was usedto assist hatching. In the mouse it significantly improved thehatching rate (80 versus 29.3%) 110 h after administrationof human chorionic gonadotrophin. This technique wasapplied in two different centres to patients with previous in-vitro fertilization (IVF) failures. The implantation rate perembryo (14.4% laser-assisted hatching versus 6% controlgroup) and the pregnancy rate per transfer (40 versus 16.2%)were improved.Key words: assisted hatching/Er:YAG laser/in-vitro fertilization/laser micromanipulation/zona pellucidaIntroductionSince Cohen et al. (1988) reported the first pregnancy aftermechanical partial zona dissection (PZD), this micromanipulationtechnique has been used routinely to enhance the chance offertilizing the ovum in instances of poor sperm quality (Tuckeret al., 1991). Recently, micromanipulation was performed tofacilitate embryonic hatching (Cohen, 1991; Cohen et al., 1992).The increasing importance of lasers in medicine has encouragedus to consider the application of this technology for zona drilling,subzonal insemination (SUZI) and assisted hatching in our in-vitro fertilization (IVF) programme.However, it is recommended that the following four criteriaare taken into account when using lasers in human embryology(Welch et al., 1991): (i) absolute avoidance of thermic effects;(ii) prevention of genetic damage by a wavelength sufficientlydistant from the absorption maximum of DNA; (iii) low ablationthreshold to ensure precision and to minimize mechanicalvibration; (iv) easy handling using existing micromanipulationequipment as briefer manipulations are always more efficient.© Oxford University PressSpecial laser equipment fulfilling all these criteria has beendeveloped in our laboratory (Feichtinger et al., 1992; Strohmerand Feichtinger, 1992a)In this paper, we present the use of the erbium-yttrium-aluminium-garnet (Er:YAG) laser in two types ofmicromanipulation in human IVF technology, i.e. SUZI andPZD. For assisted hatching, a control experiment was tested inthe mouse and then applied in humans, particularly in patientswith repeated failure of embryo transfer.Materials and methodsLaser technologyThe laser system used consisted of an EnYAG laser (Lisa LaserProducts, Katlenburg, Germany) and a laser fibre of 20 /xm tipdiameter. The application of low energy resulted inphotoablation—a non-thermic effect of the laser on biologicaltissue. A wavelength of 2.9 pm was selected to avoid damagingthe genetic structure of the embryo (Strohmer and Feichtinger,1992b), yet offering a high absorption in water. At 2.9 /*mwavelength, penetration depth in biological tissue is about 3 /im,creating an ablation of the zona pellucida in layers by laser pulsesof — 10 ixi energy. Additionally, the specially developed glassfibre allowed a procedure similar to conventional mechanicalPZD.MicromanipulationTwo hydraulic fine-control micromanipulators (Narishige, Tokyo,Japan) mounted on a Leitz Labovert inverted microscope (Leitz,Wetzlar, Germany) were used in conjunction with a HoffmannModulation Contrast System (Modulation Optics, Greenvale, NY,USA).All oocytes or embryos to be treated were placedsimultaneously on a microscope slide and covered with paraffinoil. During the procedure, the oocyte or embryo was held bynegative pressure using a holding pipette of 130 /*m outer and20 /im inner diameter. The glass fibre, fitted to the manipulatorby a pipette holder, was brought in direct contact with the zona,but the tip was not forced further into it. This prevented anycontact of the fibre with the surface of the ooplasm or blastomereonce it penetrated the zona. Five to eight pulses were necessaryto penetrate the zona, creating a 20-30 jtm opening. Theefficiency of the system was easily controlled and visualized byinserting the fibre tip into the hole.Laser SUZIThese techniques were performed in cases of male subfertilitythat had been defined according to World Health Organization1723A.Obruca el at.criteria (WHO, 1987). In most of the cases, there had been atleast one preceding IVF attempt, with failure of fertilization. Forlaser SUZI, a double micro-instrument holder was used with thelaser fibre and one thin, but not sharpened, glass pipette. Themain advantage of this system was that after a hole had beencreated by the laser, SUZI was performed easily withoutmechanical manipulations and uncontrolled sheering forces.Mechanical SUZI was carried out in a number of control cases.Laser zona drillingOne hole (20 fim diameter) was made in the zona beforeconventional in-vitro insemination. The protocol was performedon patients with poor semen parameters, similar to the patientsselected for SUZI.Mouse experiments with laser-assisted hatchingThe embryo culture media used in this study were M16 mediumcontaining 4 mg/ml of type V bovine serum albumin (BSA; SigmaPharmaceuticals, Buchs, Switzerland) and M2 HEPES bufferedmedium (Whittingham, 1971). One-cell embryos were obtainedfrom 3- to 4-week-old C57/bl (IFFA-CREDO, L'Arbresle,France) female mice. Ovulation was induced as previouslydescribed (Sakkas and Trounson, 1990). One-cell embryos wereobtained 21 h after administration of human chorionicgonadotrophin (HCG) by tearing the ampulla and releasing thecumulus cell complex plug in M2 medium containing 1 mg/mlof hyaluronidase type IV (Sigma Pharmaceuticals). The embryoswere incubated at 37°C in 5% CO2 in air. Between 66 and 68h after HCG at the 8-cell stage, the assisted hatching procedurewas performed according to the following protocol. Groups of10—15 embryos were placed under paraffin oil in M2 mediumon two slides. One slide was left on a warming stage while onthe other slide the embryos were subjected to the laser treatment:one hole of the size of the probe was made in the zona pellucida.Once the procedure was finished (10 min per group), the embryosfrom the two slides were washed in M16 medium and placedin culture droplets in their respective groups. Embryos weresubsequently assessed for the number reaching the blastocyst stageand for their ability to expand and hatch at 110 h after HCG.In a short series, blastocysts were transferred to the uteri of day3 pseudopregnant recipients. To each uterine horn of eachrecipient, six embryos from the control and treatment groups weretransferred. Recipient females were examined on day 15 to countthe number of implantation sites and the developing fetuses.Laser-assisted hatchingAssisted hatching was performed in two different IVF centres(Institut fur Sterilitatsbetreuung, Vienna, Austria, and HerzliyaMedical Centre, Herzliya-on-Sea, Israel) on 129 patients whohad had repeated IVF failures. In both centres, the results werecompared to a matched control group which consisted of IVFpatients with repeated failures of transfer, treated in both centresbefore the laser was introduced. The mean number of repeatedfailures of transfer (3.6 assisted hatching versus 3.1 control group)and the mean age were not significantly different between thegroups (33.8 versus 34.3 years). There were also no differencesin ovulation induction and sperm treatment. The number ofprevious embryos transferred was 11.8 in the laser-treated groupTable I. Micromanipulation in cases of male factor infertilityNo. oftreatedoocytesNo. offertilizedoocytes (%)No. ofembryotransfersNo. ofpregnancies(% per embryotransfer)Mechanical PZD 57Laser PZD 149Mechanical SUZI 143Laser SUZI 238 (14.0)22 (14.8)23 (16.1)8 (34.8)*31413501 (7.1)3(23.1)I (20)***Significantly different from other values in the same column. P < 0.05.**Not significantly different from mechanical SUZI value.PZD = partial zona dissection; SUZI = subzonal insemination.Table II. Assisted hatching of mouse embryos by EnYAG laser (AHL). 110 hpost-HCGNumber (%) reaching each stageStage Control AHLDegenerated blastocysts 20 (34.5)Expanded blastocysts 21 (36.2)Hatched (day 4.5 post-HCG) 17 (29.3)Total 585 (9.0)6 (10.9)44 (80.0)*SS*Significantly different from control value. P = 0.0001.HCG = human chorionic gonadotrophin.versus 9 in the matched control group. The laser technologyassisted in making one hole of the size of the fibre tip, at the2- to 4-cell stage 48 h post-insemination. The embryos weretransferred immediately after the laser-assisted hatching.Statistical analysisThe results were analysed using the x2 test and unpaired Mest.A standardized computer program (StatView; Abacus Concepts,Berkeley, CA, USA) was applied.ResultsSUZI and zona drillingThe results of micromanipulation procedures in male factorinfertility patients are summarized in Table I. The best resultswere obtained with laser SUZI (34.8% fertilization rate), whichwas significantly better (P < 0.05) than mechanical SUZI(16.1 %). However, the pregnancy rate per transfer was similarfor both groups (20 versus 23.1 %). The results of laser-assistedPZD were as poor as those of the mechanical method: fertilizationrate 14.8 versus 14%; pregnancy rate 7.1 versus 0%.Assisted hatching in the mouseLaser-assisted hatching shortened the hatching time bycomparison with the control group. At day 4.5 post-HCG, 80%of the mouse embryos had already started the hatching processin the laser-treated group versus 29.3% in the control group(Table II).In the short series of embryo transfers in day 3 synchronizedpseudopregnant recipients, no differences were observed betweenthe laser-treated embryos and the control group (control group:1724Laser application in the IVF programmeTable III. Results from assisted hatching by ErYAG laser (AHL) in patients with repeated IVF failure in two centres (Institut fur Sterilitatsbetreuung, Austria,and Herzliya Medical Centre, Israel)No.embryostransferredTotalembryostransferredimplantationrate/embryoClinicalpregnanciesLostpregnanciesOngoingpregnancyrate (%)Assistedhatching"(n = 129)Controlgroupb(n = 167)1234Mean/total1234Mean/total81247621633675112.516.713.514.914.4*12.54.564.97.01 (12.5)4 (33.3)16 (34)30 (48.4)51 (40)*2 (12.5)3 (9.1)12 (17.9)10 (19.6)27 (16.2)004(25)4 (13.3)81 (50)01 (8.3)3(30)51 (12.5)4 (33.3)12 (25.5)26 (41.9)43 (33.3)*1 (6.3)3(9.1)11 (16.4)7(13.7)22 (13.2)"Patients with repeated IVF failures from 1992/1993bPatients with repeated IVF failures before the laser was introduced.•Significantly different from control values, P < 0.05.two fetuses and four resorptions out of 12 embryos transferred;laser group: three fetuses and four resorptions out of 12 embryostransferred).Assisted hatching in humansLaser-assisted hatching in 129 patients who had had repeated IVFfailures was compared with 167 controls who were treated beforethe laser was introduced. The number of previously failedattempts for the two groups was 3.6 ± 3 and 3.1 ± 2.6respectively, and the corresponding number of transferredembryos was 11.8 ± 12.1 and 9 ± 9.2 respectively. The meanages of the two groups were similar: 33.8 ± 5.4 and 34.3 ±5. Laser treatment increased the implantation rate per embryo(14.4 versus 7%) as well as the clinical pregnancy rate perembryo transfer (40 versus 16.2%) and the ongoing pregnancyrate per embryo transfer (33.3 versus 13.2%) (Table in). Theincrease was not related to differences of age, stimulation, spermtreatment or number of embryos transferred.So far (November 1993), 44 children have been born from37 patients who had laser-assisted micromanipulation.DiscussionApplication of ultraviolet (UV) lasers in non-contact mode hasbeen proposed by Tadir et al. (1993) for differentmicromanipulation procedures. These systems have the advantagethat there is no need for disposable fibres and for sterilization;also, there is the possibility of intracellular application. However,certain wavelengths in the UV range bear potential hazards forthe genetic structure, the question of heat deposition by thesesystems has yet to be determined, and the spot size is limitedby the wavelength and optics. The creation of large gaps in thezona requires longer exposure and higher laser energies. Lastbut not least, expensive optics are needed for the microscope.Basic studies in laser-gamete interaction have to be carried outin order to determine the potential hazards of UV lasers.Therefore, these systems are not ready to be applied to humansyet (Neev et al, 1993; Li et al., 1993).We intended to avoid such problems by using a totally differentsystem: an infrared laser in contact mode. Although disposableor sterilizable fibres are needed, this system, which uses existingmicromanipulation equipment, is simple, and the micromanipu-lation process can be quickly carried out. The spot size can bemodulated by the fibre size (3-30 /mi); very low energies andfew pulses are needed to achieve appropriate zona piercing.However, for assisted fertilization (SUZI and zona drilling)the results are as disappointing as for mechanical processes. Thisconfirms that these technologies are not really improving thefertilization process for the treatment of male subfertility (Sakkaset al., 1992; Ord et al., 1993).Er:YAG laser-assisted hatching shortens the hatching time inthe mouse; in this case, our data are comparable to thosedescribed by Neev et al. (1993) for acidic cutting of the zona,but not for laser drilling. UV and Er:YAG laser give totallydifferent results in this case, as confirmed by the degenerationrate observed by the authors. EnYAG laser treatment does notprevent further implantation when compared to controls.Used in human FVF, laser-assisted hatching improves both theimplantation rate per embryo and the pregnancy rate per transfer.The increases were effective for the two different centres involvedin the protocol. So far, 37 patients have delivered 44 healthychildren using these laser experiments, and these are the firstcases reported in the literature.The Er:YAG laser offers an alternative method of treatmentfor repeated failures of embryo transfer. It can be consideredas safe and efficient as the assisted hatching using acidic Tyrodeproposed by Cohen (1991). However, further evaluations in-atotally randomized protocol are necessary to confirm theefficiency of this technology and to define for which indicationsit is especially useful and can be recommended, as alreadyproposed by Cohen et al. (1992).ReferencesCohen.J. (1991) Assisted hatching of human embryos. J. In Vitro Fertil.Embryo Transfer, 8, 179-190.Cohen,J., Malter,H., Fehilly.C, Wright.G., Elsner,C, Kort.H. andMassey,J. (1988) Implantation of embryos after partial opening ofoocyte zona pellucida to facilitate sperm penetration. Lancet, ii, 162.Cohen,J., Alikani,M., Trowbridge,J. and Rosenwaks.Z. (1992)1725A.Obruca el al.Implantation enhancement by selective assisted hatching using zonadrilling of human embryos with poor prognosis. Hum. Reprod., 7,685-691.Feichtinger,W., Strohmer,H., Fuhrberg.P., Radivojevic,K., Antinori,S.,Pepe.G. and Versaci,C. (1992) Photoablation of oocyte zona pellucidaby erbium-yag laser for in-vitro fertilisation in severe male infertility.Lancet, 339, 811.Li,L., Munne.S., Licciardi,F., Neev,J., Tadir.Y., Berns,M., Godke.R.and Cohen,J. (1993) Microinjection of FITC-dextran into mouseblastomeres to assess topical effects of zona photoablation. Zygote,1, 43-48.Neev.J., Gonzalez,A., Licciardi.F., Alikani,M., Tadir.Y., Berns,M.and Cohen.J. (1993) Opening of the mouse zona pellucida by laserwithout a micromanipulator. Hum. Reprod., 8, 939-944.Ord.T., Patrizio.P., Balmaceda,J.P. and Asch,R.H. (1993) Can severemale factor infertility be treated without micromanipulation? Fertil.Steril., 60, 110-115.Sakkas,D. and Trounson.A.O. (1990) Co-culture of mouse embryos withoviduct and uterine cells prepared from mice at different days ofpseudopregnancy. J. Reprod. Fertil., 90, 109-118.Sakkas.D., Lacham.O., Gianaroli,L. and Trounson,A. (1992) Subzonalsperm microinjection in cases of severe male factor infertility andrepeated in vitro fertilization failure. Fertil. Steril., 57, 1279- 1288.Strohmer,H. and Feichtinger.W. (1992a) Successful clinical applicationof laser for micromanipulation in an in vitro fertilization program.Fertil. Steril., 58, 212-214.Strohmer,H.and Feichtinger,W. (1992b) Application of laser formicromanipulation: relevance of biophysical criteria. Fertil. Steril.,Abstr. Suppl. 48, S40.Tadir,Y., Neev,J., Ho,P. and Berns,M.W. (1993) Laser for gametemicromanipulation: basic concepts. J. Assist. Reprod. Genet., 10,121-125.Tucker,M.J., Bishop.F.M., Cohen,J., Wiker,S.R. and Wright,G. (1991)Routine application of partial zona dissection for male factor infertility.Hum. Reprod., 6, 676-681.Welch,A.J., Motamedi,M., Rastegar.S., Le Carpentier.G.L. andJansen.D. (1991) Laser thermal ablation. Photochem. Photobiol., 53,815-823.Whittingham,D.G. (1971) Culture of mouse ova. J. Reprod. Fertil.,14 (Suppl.), 7 -21 .World Health Organization (1987) WHO Laboratory Manual for theExamination of Human Semen and Semen —Cervical MucusInteraction. 2nd edn. The Press Syndicate of the University ofCambridge, Cambridge.Received on December 7, 1993; accepted on May 10, 19941726

Fertilization and early embryology: Use of lasers in assisted fertilization and hatching

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