Efficiency of laser-assisted intracytoplasmic sperm injection in a human assisted reproductive techniques program

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1 ORIGINAL ARTICLE pissn eissn Clin Exp Reprod Med 2011;38(3): Efficiency of lser-ssisted intrcytoplsmic sperm injection in humn ssisted reproductive techniques progrm Kyoung Hee Choi, Jung Hyun Lee, Yun Hee Yng, Te Ki Yoon, Dong Ryul Lee, Woo Sik Lee Fertility Center of CHA Gngnm Medicl Center, CHA University College of Medicine, Seoul, Kore Objective: Lser-ssisted intrcytoplsmic sperm injection (), lso known s micro-opening or thinning of the zon pellucid (ZP) prior to ICSI, my help to reduce mechnicl dmge to the oocyte during the procedure. The im of the present study ws to evlute nd nlyze the efficcy of our institutionl progrm, which fetures lser-ssisted ZP thinning prior to ICSI, in comprison with conventionl ICSI (C- ICSI), performed on ptients with different clinicl chrcteristics. Methods: Ptients undergoing totl of 212 ICSI cycles were rndomly divided into n group (106 cycles) nd conventionl ICSI group (106 cycles). To reduce tissue dmge, we thinned the ZP by pproximtely 70%, using lser, before ICSI. Ptients thus treted formed the LA- ICSI group. Comprisons included the morphologicl qulity of trnsferred embryos, blstocyst development of the remining embryos, nd clinicl pregnncy, in terms of ICSI method nd ptient chrcteristics. Results: Fertiliztion, development of remining embryos, nd pregnncy rte were significntly higher in the group compred with the group. Fertiliztion, embryonic development, nd the pregnncy rte were ll improved in younger ptients ( < 38 yers of ge) nd in those who underwent low number of IVF-ET ttempts ( < 3 trils). In ddition, the pregnncy rte ws incresed in older ptients. Conclusion: my be useful in improving the chnce of pregnncy in ll ICSI ptients. Keywords: Intrcytoplsmic Sperm Injection; Lser-ssisted Intrcytoplsmic Sperm Injection; In Vitro Fertiliztion-embryo Trnsfer; Zon Pellucid Thinning; Clinicl Outcome; Humn Introduction In the time since the first successful pregnncy fter ICSI ws reported in 1992 [1], ICSI hs become routine method of fertiliztion if mle fctor infertility is evident, nd the tretment is lso used in Received: My 31, 2011 Revised: Aug 2, 2011 Accepted: Aug 16, 2011 Corresponding uthors: Dong Ryul Lee nd Woo Sik Lee Fertility Center of CHA Gngnm Medicl Center, CHA University College of Medicine, Yeoksm-dong, Gngnm-gu, Seoul , Kore Tel: Fx: E-mil: drleedr@ch.c.kr; wooslee@ch.c.kr *This work ws supported by grnt from the Kore Helthcre Technology R&D Project, Ministry for Helth, Welfre & Fmily ffirs, Republic of Kore (A084923). This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Non-Commercil License ( which permits unrestricted non-commercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. instnces of non-mle fctor infertility if repeted ttempts t IVF-ET fil [2-5]. However, ICSI hs severl drwbcks. These include mechnicl dmge to the cytoplsmic membrne, the cytoskeleton, nd the meiotic spindle tht occurs during membrne penetrtion nd sperm deposition [6,7]. In fct, oocyte degenertion nd bnorml fertiliztion constitute the principl resons for the cncelltion of ssisted reproductive techniques (ART) cycles. Additionlly, some oocytes re very frgile, nd the zon pellucid cn be very resistnt to penetrtion, resulting in sudden brekge of the membrne during ICSI [7-9]. To overcome these problems, severl groups hve developed piezo- or lser-ssisted ICSI techniques (). Piezo-ICSI, which uses blunt injection needle operted by piezo-electric device, efficiently prevents dmge to both mouse nd humn oocytes [10,11]. However, this technique hs become unpopulr owing to risk of mercury contmintion nd prcticl difficulties. Lsers hve Copyright THE KOREAN SOCIETY FOR REPRODUCTIVE MEDICINE

2 KH Choi et l. Efficiency of lser ICSI in humn ART lredy been used s convenient nd sfe tools in ssisted htching nd pre-implnttion genetic dignosis (PGD). Rienzi et l. [12] reported tht, feturing micro-opening or drilling of the ZP prior to ICSI, reduced the degenertion rte of humn oocytes nd incresed embryo development rtes in ptients who hd experienced prior ICSI filures cused by poor oocyte survivl [13]. Severl groups hve reported similr results in selected ptients with histories of poor ICSI outcomes, for whom only limited numbers of oocytes were vilble [13,14]. In ddition, when used to overcome fertiliztion problems, lser-ssisted ZP thinning prior to routine ICSI both improved oocyte survivl nd incresed the htching rte in vitro [15]. However, the clinicl utility of remins controversil becuse comprisons of fertiliztion, embryo qulity, nd clinicl outcomes hve not been performed in the generl ptient popultions eligible for routine ICSI. In ddition, the study groups hve hd included few cses. Therefore, the objective of the present work ws to evlute the possible benefits of, thus thinning of the ZP prior to ICSI, s prt of routine ICSI progrm in non-rndomized ptients. We compred the dt with those collected from conventionl ICSI (C- ICSI) ptients. The clinicl outcomes of both groups were re-nlyzed in terms of the ge nd history of femle ptients, to identify ny possible clinicl significnce of lser use. Methods This study ws pproved by the Institutionl Review Bord of the CHA Gngnm Medicl Center (Seoul, Kore) nd ws conducted between Februry nd September of Ptients experiencing totl of 212 ICSI cycles were rndomly divided into two groups (106 cycles of nd 106 cycles of ). 1. LA- nd procedures Strting on the third dy of the cycle, controlled ovrin hyperstimultion ws chieved using recombinnt FSH (rfsh; Gonl-F, Merck- Serono, Modugno Bri, Itly). To prevent ny premture LH surge, we employed GnRH gonist (Lucrin, Abbott, Seoul, Kore) using the long protocol, or GnRH ntgonist (Cetorelix, Cetrotide, Merck-Serono) given fter 5-6 dys of stimultion. Ovultion ws triggered by the injection of 10,000 IU hcg (Profsi, Merck-Serono) when t lest two follicles were over 18 mm in dimeter. Trnsvginl oocyte retrievl ws performed hours fter hcg dministrtion. For, n oocyte ws held with the polr body in the 12-o clock position. The ZP ws thinned by pproximtely 70% t the 3-o clock position (where the injection needle ws to penetrte) using lser pulse 100 µ sec in durtion (ZILOS-tk clss I lser, Hmilton Thorne Reserch, Beverly, MA, USA). An ICSI needle ws introduced into the oocyte through the pre-thinned region of the ZP, nd the overll process ws termed. The control group received conventionl ICSI nd ws termed the group. After both nd, oocytes injected with spermtozoon were incubted (under 5% CO2 nd 5% O2, both v/v, nd t 37 C) in drops of Quinn s Advntge Clevge medium (Sge, Trumbull, CT, USA) under oil. 2. Evlution of oocyte integrity, fertiliztion, embryo development, nd pregnncy Fertiliztion nd degenertion of injected oocytes were monitored hours fter ICSI. Fertilized embryos were trnsferred to drops of fresh clevge medium. At dy 3, embryo qulity ws grded into five levels bsed on the percentge of frgmenttion nd the number nd size of blstomeres [14]; two or three embryos were selected for embryo trnsfer. After trnsfer to the uterus, the remining embryos were cultured to dy 7 for evlution of blstocyst formtion. To rtificilly prepre the endometrium, E2 vlerte (4-6 mg/dy; Progynov, Schering, Byer, New Zelnd) ws dministrted nd progesterone (100 mg/dy; Smil Phrm. Co., Ltd., Seoul, Kore) ws injected when the endometrium ttined 8 mm in thickness. Pregnncy ws evluted by mesurement of the serum β-hcg level 12 dys fter embryo trnsfer nd clinicl pregnncy ws defined s thepresence of gesttionl sc with positive hert ctivity t 5-7 weeks. 3. Anlysis of clinicl outcomes by ptient profiles All clinicl outcomes, including fertiliztion nd degenertion rtes, development of remining embryos into blstocysts, clinicl pregnncy rte nd bortion rte were evluted. First, clinicl outcomes in the nd groups were compred mong ll ptients. Second, the dt were re-nlyzed with respect to femle ge ( < 38 yers nd 38 yers). The ge of ptients ws distinguished bsed on reserch results of study tht used lser for zon thining [16]. Lst, the dt were re-nlyzed in terms of the number of prior IVF- ET ttempts ( < 3 nd 3). 4. Sttisticl nlysis Unless otherwise specified, dt re expressed s mens±se. Clinicl outcomes were nlyzed using the chi-squre test nd Student s t-test. A p <5 ws considered to be sttisticlly significnt. Results 1. Comprisons of the clinicl outcomes of the nd groups The clinicl profiles of ptients in both groups re summrized in Tble 1. The verge ge, sperm strict morphology using Kruger s strict criteri nd the cycle dte of hcg injection were similr in both groups. Also, neither the E2 concentrtion on the dy of hcg injection 149

3 Clin Exp Reprod Med 2011;38(3): Tble 1. Clinicl profiles of the lser-ssisted intrcytoplsmic sperm injection () nd conventionl ICSI () groups p-vlue Number of cycles NS Averge of sperm strict morphology NS Averge ge of femles (yr) 35.8± ±0.4 NS E2 level (pg/ml) on the dy of hcg injection 1,823.0± ,823.1±131.9 NS Totl number of oocytes (oocytes/cse) 1,165 (11±0.8) 1,162 (11.0±0.7) NS Cycle durtion (dy) 12.8± ±0.2 NS Number of MII oocytes (oocytes/cse) 941 (8.9±0.6) 953 (9.0±0.5) NS Score of trnsferred embryos 3.5± ±0.1 NS Number of embryos trnsferred 2.6± ±0.1 NS Vlues re presented s mens ± SE. NS, not significnt ± 1.9 Fertiliztion 81.5± 1.8 Degenertion 29.1± 4.3± ±0.7 to blstocyst Clinicl Implnttion nor the totl number of retrieved oocytes differed between groups. In ptients, 941 MII oocytes retrieved from 106 cycles were injected with sperm; 714 (75.9%) becme fertilized nd 40 (4.3%) degenerted. A totl of 953 MII oocytes retrieved from 106 cycles were injected in the group; 777 (81.5%) becme fertilized nd 27 (3.0%) degenerted. Figure 1 shows tht, lthough the degenertion rte did not differ between groups, the fertiliztion rte in the group ws higher thn tht in the group (81.5% vs. 75.9%; p <5). Also, the blstocyst formtion rte from embryos remining fter trnsfer nd the clinicl pregnncy rte were significntly higher in the group (36.2% nd 5% vs. 29.1% nd 30.2%, respectively; p <5), even though the number of trnsferred embryos nd the scores of such embryos t dy 3 were similr in both groups. Use of slightly incresed the implnttion rte, but the increse ws not sttisticlly significnt. 2. Comprisons of clinicl outcomes of the nd groups with respect to femle ge 36.2± 3.3 To investigte the specific efficcy of, the dt from both groups were re-nlyzed in terms of femle ge. As shown in Figure 30.2 (32/106) 5 (53/106) (57/287) (39/275) 83.0 (44/53) 71.9 (23/32) Figure 1. Comprison of clinicl outcomes between the lser-ssisted intrcytoplsmic sperm injection () nd conventionl ICSI () groups. p < 5. 2, showed improved pregnncy rtes in both ge groups (58.2% vs. 39.7% in those < 38 yers old; 35.9% vs. 13.2% in those 38 yers old, p<5) when compred with. In ddition, LA- ICSI incresed the fertiliztion (81.9% vs. 74.4%, respectively; p<5) nd developmentl rtes of the remining embryos (38.4% vs. 30.8%, respectively; p < 5) nd lso reduced the degenertion rte (2.1% vs. 4.1%) in younger femles ( < 38 yers of ge) (Figure 2A) when compred to the group. LA-ISCI did not ffect these prmeters in older ptients ( 38 yers of ge) (Figure 2B). 3. Comprisons of clinicl outcomes of the nd groups in terms of repeted IVF filure In the present study, 42 IVF-ICSI ptients who hd experienced repeted IVF filure ( 3 IVF-ET ttempts) were divided into two groups nd the clinicl outcomes fter nd were nlyzed. As shown in Figure 3A, ll clinicl outcomes, including embryonic development, clinicl pregnncy, nd implnttion rtes, were slightly higher in the group thn in the group (13.3% vs. 25.9%, 3% vs. 5%, nd 14.0% vs. 21.8%, respectively), but these differences were not significnt. However, when the 170 ICSI cycles of ptients with low number of prior IVF-ET ttempts were nlyzed, fertiliztion, embryonic development, nd clinicl pregnncy rtes were significntly improved in the group compred with the group (82.4% vs. 75.6%, 37.6% vs. 30.7%, nd 5% vs. 30.2%; ll p<5, respectively) (Figure 3B). Discussion To overcome low fertiliztion rte cused by oocyte degenertion or bnorml embryonic development [4,6], the ICSI procedure hs been modified over the pst 20 yers to reduce mechnicl oocyte dmge. either cretes hole in or thins the ZP [9], nd this my permit esy entrnce of the needle into oocytes tht re bnorml in terms of thickness or ZP resistnce. Hence, mny reserchers hve suggested tht cn reduce the dmge suffered by oo- 150

4 KH Choi et l. Efficiency of lser ICSI in humn ART A B ± 2.4 Fertiliztion 81.9± ± ±1.1 Degenertion 30.8± ± 4.0 to blstocyst 58.2 (39/67) (27/68) 18.5 (43/189) (33/178) Clinicl Implnttion (31/39) (19/27) ± ± 3.6 Fertiliztion 4.8± ± ± 2.9 Degenertion 27.1± 5.6 to blstocyst 13.2 (5/38) 35.9 (14/39) Clinicl 6.2 (6/97) 14.3 (14/98) Implnttion (4/5) 92.9 (13/14) Figure 2. Comprison of clinicl outcomes between the lser-ssisted intrcytoplsmic sperm injection () nd conventionl ICSI () groups in femles of different ge groups (A: < 38 yers old, B: 38 yers old). p<5. A B ± 4.2 Fertiliztion 76.3± ± ±1.7 Degenertion 13.3± (6/20) 25.9± 8.9 to blstocyst 5 (11/22) Clinicl (12/55) (7/50) Implnttion 5 (3/6) 90.9 (10/11) Figure 3. Comprison of clinicl outcomes between the lser-ssisted intrcytoplsmic sperm injection () nd conventionl ICSI () groups ccording to the number of prior IVF-ET ttempts (A: 3 ttempts, B: < 3 ttempts). p< ± 2.1 Fertiliztion 82.4± ± ±0.9 Degenertion 30.7± ± 3.5 to blstocyst 30.2 (26/86) 5 (42/84) Clinicl (45/232) (32/725) Implnttion (34/42) (20/26) cytes during ICSI, with consequent benefits for embryonic development [14,17]. However, most prior studies used ZP penetrtion methods or nlyzed only smll numbers of specific ptients with poor ICSI histories. To dte, the efficcy of in ptients of vrying chrcteristics hs not been well studied. In the present work, we conducted prospective comprison ( vs. ) on ptients undergoing our routine ICSI procedure, which fetures conjugted IVF-ET progrm, nd next re-nlyzed clinicl outcomes in terms of ptient ge nd the number of prior IVF-ET ttempts. The results show tht my not mitigte dmge to injected oocytes, but enhnced both the fertiliztion rte nd blstocyst formtion by residul embryos. Although the scores of trnsferred embryos did not differ between groups, we hypothesized tht the qulity of embryos selected for trnsfer in the group my hve been better thn tht of group embryos, resulting in stisfctory clinicl pregnncy rte (Figure 1). In fct, the implnttion rte ws not incresed significntly nd ws only slightly improved in the group. Lser drilling of the ZP is used principlly for lser-ssisted htching (LAH). Although LAH my enhnce embryo implnttion by mechniclly fcilitting the htching process nd permitting embryo-endometrium contct, LAH is ssocited with the risks ssocited with ll invsive pproches. Mntoudis et l. [18] developed prtil LAH technique involving ZP thinning without bleching of the inner membrne; the pregnncy rte improved significntly. LAH hs lso been offered to selected ptients who were older, who hd experienced prior IVF-ET filures, or who responded poorly to gondotropins. As with LAH, hs been used to tret selected ptients who filed previous ICSI cycles or who produced limited numbers of MII oocytes [12-14]. By re-nlyzing ptients with specific indictions for, we found tht the technique significntly improved clinicl outcomes in both older nd younger femles. However, my not improve clinicl outcomes of ptients with histories of prior IVF-ET filures ( 3 ttempts); this ws in contrst to the results in ptients with fewer prior ttempts (Figure 3). Severl reports hve indicted tht opening of the ZP prior to ICSI, nd ssisted htching before embryo trnsfer re ssocited with twin [12,16,19-21]. Howev

5 Clin Exp Reprod Med 2011;38(3): er, in the present study, we did not fully penetrte the ZP before ICSI to void this problem. Our pproch my thus not ffect the multiple pregnncy rte. The results indicte tht is vluble in routine prctice. In conclusion, my reduce dmge to oocytes during fertiliztion, in turn leding to improved embryonic development. Enhnced clinicl outcomes fter use of were evident in selected ptients who hd experienced repeted IVF filure, or who were older, s well s when ll ptients undergoing routine ICSI were evluted s two unselected groups. Also, is simple procedure. Therefore, we suggest tht the procedure be used for ll ICSI ptients to improve clinicl outcomes. Conflict of interest No potentil conflict of interest relevnt to this rticle ws reported. References 1. Plermo G, Joris H, Devroey P, Vn Steirteghem AC. Pregnncies fter intrcytoplsmic injection of single spermtozoon into n oocyte. Lncet 1992;340: Ebner T, Moser M, Sommergruber M, Jescher K, Tews G. Complete oocyte ctivtion filure fter ICSI cn be overcome by modified injection technique. Hum Reprod 2004;19: Ol B, Afnn M, Shrif K, Ppionnou S, Hmmdieh N, Brrtt CL. Should ICSI be the tretment of choice for ll cses of in-vitro conception? Considertions of fertiliztion nd embryo development, cost effectiveness nd sfety. Hum Reprod 2001;16: Oehninger S, Gosden RG. Should ICSI be the tretment of choice for ll cses of in-vitro conception? No, not in light of the scientific dt. Hum Reprod 2002;17: Bhttchry S, Hmilton MP, Shbn M, Khlf Y, Seddler M, Ghobr T, et l. Conventionl in-vitro fertilistion versus intrcytoplsmic sperm injection for the tretment of non-mle-fctor infertility: rndomised controlled tril. Lncet 2001;357: Ebner T, Ymn C, Moser M, Sommergruber M, Jescher K, Tews G. A prospective study on oocyte survivl rte fter ICSI: influence of injection technique nd morphologicl fetures. J Assist Reprod Genet 2001;18: Liu J, Ngy Z, Joris H, Tournye H, Smitz J, Cmus M, et l. Anlysis of 76 totl fertiliztion filure cycles out of 2732 intrcytoplsmic sperm injection cycles. Hum Reprod 1995;10: Ngy ZP, Liu J, Joris H, Bocken G, Desmet B, Vn Rnst H, et l. The influence of the site of sperm deposition nd mode of oolemm brekge t intrcytoplsmic sperm injection on fertiliztion nd embryo development rtes. Hum Reprod 1995;10: Plermo GD, Alikni M, Bertoli M, Colombero LT, Moy F, Cohen J, et l. Oolemm chrcteristics in reltion to survivl nd fertiliztion ptterns of oocytes treted by intrcytoplsmic sperm injection. Hum Reprod 1996;11: Hung T, Kimur Y, Yngimchi R. The use of piezo micromnipultion for intrcytoplsmic sperm injection of humn oocytes. J Assist Reprod Genet 1996;13: Yngid K, Ktyose H, Yzw H, Kimur Y, Konni K, Sto A. The usefulness of piezo-micromnipultor in intrcytoplsmic sperm injection in humns. Hum Reprod 1999;14: Rienzi L, Greco E, Ubldi F, Icobelli M, Mrtinez F, Tesrik J. Lserssisted intrcytoplsmic sperm injection. Fertil Steril 2001;76: Demirol A, Benkhlif M, Sri T, Gurgn T. Use of lser-ssisted intrcytoplsmic sperm injection (ICSI) in ptients with history of poor ICSI outcome nd limited metphse II oocytes. Fertil Steril 2006;86: Abdelmssih S, Crdoso J, Abdelmssih V, Dis JA, Abdelmssih R, Ngy ZP. Lser-ssisted ICSI: novel pproch to obtin higher oocyte survivl nd embryo qulity rtes. Hum Reprod 2002;17: Moser M, Ebner T, Sommergruber M, Gisswinkler U, Jescher K, Puchner M, et l. Lser-ssisted zon pellucid thinning prior to routine ICSI. Hum Reprod 2004;19: Blkier H, Mndel R, Sojecki A, Motmedi G, Zver S, Librch C. Lser zon thinning in women ged <or=37 yers: rndomized study. Fertil Steril 2009;91: Vlojerdi MR, Eftekhri-Yzdi P, Krimin L, Ashtini SK. Effect of lser zon pellucid opening on clinicl outcome of ssisted reproduction technology in ptients with dvnced femle ge, recurrent implnttion filure, or frozen-thwed embryos. Fertil Steril 2008;90: Mntoudis E, Podsidly BT, Gorgy A, Venkt G, Crft IL. A comprison between qurter, prtil nd totl lser ssisted htching in selected infertility ptients. Hum Reprod 2001;16: Alikni M, Noyes N, Cohen J, Rosenwks Z. Monozygotic twinning in the humn is ssocited with the zon pellucid rchitecture. Hum Reprod 1994;9: d Cost AA, Abdelmssih S, de Oliveir FG, Abdelmssih V, Abdelmssih R, Ngy ZP, et l. Monozygotic twins nd trnsfer t the blstocyst stge fter ICSI. Hum Reprod 2001;16: Schieve LA, Meikle SF, Peterson HB, Jeng G, Burnett NM, Wilcox LS. Does ssisted htching pose risk for monozygotic twinning in conceived through in vitro fertiliztion? Fertil Steril 2000;74: