Effects of various combinations of cryoprotectants and cooling speed on the survival and further development of mouse oocytes after vitrification

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1 ORIGINAL ARTICLE doi: /erm pissn eissn Clin Exp Reprod Med 211;38(1):24-3 Effets of vrious omintions of ryoprotetnts nd ooling speed on the survivl nd further development of mouse ooytes fter vitrifition Soo Kyung Ch 1 *, Bo Yeun Kim 2 *, Mi Kyung Kim 1, You Shin Kim 1, Woo Sik Lee 1, Te Ki Yoon 1, Dong Ryul Lee 1,2 1 Fertility Center of CHA Gngnm Medil Center, CHA University College of Mediine; 2 Deprtment of Biomedil Siene, College of Life Siene, CHA University, Seoul, Kore Ojetive: The ojetives of this study were to nlyze effiy of immture nd mture mouse ooytes fter vitrifition nd wrming y pplying vrious omintions of ryoprotetnts (CPAs) nd/or super-rpid ooling using slush nitrogen (). Methods: Four-week old ICR femle mie were superovulted for GV- nd MII-stge ooytes. Experimentl groups were divided into two groups. Ethylene glyol (EG) only group: pre-equilirted with 1.5 M EG for 2.5 minutes nd then equilirted with 5.5 M EG nd 1. M surose for 2 seonds. EG+dime thylsulfoxide () group: pre-equilirted with 1.3 M EG M for 2.5 minutes nd equilirted with 2.7 M EG M +.5 M surose for 2 seonds. The ooytes were loded onto grids nd plunged into or liquid nitrogen (). Stored ooytes were wrmed y five-step me thod, nd then their survivl, mturtion, levge, nd developmentl rtes were oserved. Results: The EG only nd EG+ groups showed no signifint differene in survivl of immture ooytes vitrified fter wrming. However, mturtion nd levge rtes fter onventionl insemintion were greter in the EG only group thn in the EG + group. In mture ooytes, survivl, levge, nd lstoyst formtion rtes fter wrming showed no signifint differene when EG only or EG+ ws pplied. Furthermore, levge nd lstoyst formtion rtes of MII ooytes vitrified using were inresed in oth the EG only nd EG+ groups. Conlusion: A omintion of CPAs in ooyte ryopreservtion ould e formulted ording to the ooyte stge. In ddition, my improve the effiieny of vitrifition y reduing ryoinjury. Keywords: Vitrifition; Ethylene Glyol; Dimethyl Sulfoxide; Cooling Rte; Nitrogen; Mie, Inred ICR; Cryoprotetive Agents Introdution Reeived: Nov 23, 21 Revised: De 9, 21 Aepted: De 16, 21 Corresponding uthors: Dong Ryul Lee nd Te Ki Yoon Fertility Center of CHA Gngnm Medil Center, CHA University, 66-5 Yeoksm-dong, Gngnm-gu, Seoul , Kore Tel: Fx: E-mil: drleedr@h..kr, tkyoon@h..kr *Ch SK nd Kim BY ontriuted eqully to this work. * This work ws prtly supported y grnt from the Kore Helthre Tehnology R&D Projet, Ministry for Helth, Welfre & Fmily Affirs, Repuli of Kore (A84923) nd grnt ( ) from the Priority Reserh Centers Progrm funded y the Ministry of Edution, Siene nd Tehnology, Repuli of Kore. This is n Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Non-Commeril Liense ( whih permits unrestrited non-ommeril use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. Sine the first suessful pregnny derived from ryopreserved humn ooytes ws reported in 1986 [1,2], vrious freezing nd thwing protools hve een pplied to the ryostorge of ooytes. Ooyte ryopreservtion osts vrious dvntges over emryo ryopreservtion. Ooyte ryopreservtion would signifintly ontriute in ssisted reprodutive tehnology (ART) progrms. It llows ptients to ryopreserve their ooytes when they hve no prtner or re out to lose their ovrin funtion due to surgery, hemotherpy, or rdiotherpy or wnt to dely the delivery [3]. Also, it voids ethil issues nd legl restritions. Unfortuntely, despite remrkle progress, ooyte ryopreservtion remins demnding tsk, s there is no preise stndrdiztion of ryopreservtion nd wrming proedures. Hene, the tehnique is not yet widely used in linil prtie. The slow ooling method ws initilly used for ooyte ryopreservtion. However, only few studies hve demonstrted suessful outomes of ooytes tht were frozen using slow ooling method 24 Copyright 211. THE KOREAN SOCIETY FOR REPRODUCTIVE MEDICINE

2 SK Ch et l. Effets of CPAs nd ooling speed on mouse ooytes fter vitrifition [1,2]. This n e explined y zon pelluid hrdening from premture ortil grnule exoytosis, hromosoml nondisjuntion used y serious disturne of the mirotuules, disturne in pronuler (PN) formtion, nd polr ody relese from mirofilment dmge nd ytoskeleton ltertion fter ryopreservtion [4-1]. Severl studies hve reported effetive outomes of pplying vrious improved systems inluding reduing the onentrtion of sodium in ryoprotetnts (CPAs) [11,12] or using different types nd onentrtion of CPAs during ryopreservtion [13-17]. In ddition, some studies reported exellent survivl, emryoni development, nd pregnny rtes y pplying vitrifition method whih voids the formtion of ie rystls using high onentrtion of CPA nd ultr-rpid ooling speed [18]. Advntges of vitrifition inlude tht it is time-sving, esy to perform, nd does not require expensive equipment. Most importntly, it ould minimize the dmge to ooytes in tht it voids the forming of ie rystls. However, its drwk is tht it requires high onentrtion of CPA, whih my use toxiity nd osmoti dmge to the ooyte. Sine the development of vitrifition, the toxiity of CPA hs een the one of the mjor onerns in pplying this method s ryopreservtion method. Severl studies hve een onduted to void the drwk y using CPA with high speed permeility nd less toxiity or y pplying omintion of permele nd non permele CPAs to derese the solute onentrtion without derese in the reltive onentrtion. Also, in order to inrese the ooling rte for vitrifition, mny studies hve een ttempted for minimizing the CPA solution volume or improving the het ondutivity of ryo-equipment. Furthermore, using liquid nitrogen in slush stte (slush nitrogen, ) hs improved the survivl nd emryoni development rtes fter vitrifition of ooytes nd emryos nd this my e due to the inrese in the het trnsfer rte tht is ssoited with [19,2]. Ethylene glyol (EG) is widely used in the vitrifition method s it is one of the mjor permele CPAs with low moleulr weight, nd it is lso less toxi to mmml ooytes or emryos inluding humns [21-24]. In prtiulr, the strtegy for reduing ell injury y pplying short exposure to high onentrtion of CPA hs een widely used. In ontrst, Mukid et l. [25] hs reently reported high survivl rte of emryos tht were vitrified t lstoyst stge with omintion of EG nd dimethylsulfoxide (, slow permele CPA). The omintion of CPAs for the vitrifition proess my hve indued lower reltive onentrtion nd lso lower toxiity of CPAs. In ft, s penetrtes into the ell, it elertes its hrteristis of glss-forming nd it inreses the permeility rte s it omines with the other types of CPAs whih omplement eh other. Also, there hs een report tht when omintion of CPAs ws used, higher lstoyst development ws otined ompred to using only 4% EG in ovine ooytes [26]. These studies hve een onduted only with emryos or lstoyst stge emryos, ut not mny studies hve worked with germinl vesile (GV) stge or metphse II (MII) stge ooytes. In ddition, few studies hve een onduted regrding the ooling rte, whih is one of ftors tht ffet ooytes or emryos during vitrifition [27]. Therefore, in this study, we nlyzed the survivl nd susequent emryoni development rtes of immture nd mture mouse ooytes fter vitrifition nd wrming proess using omintions of CPAs nd/or using to develop n effiient vitrifition method for immture nd mture ooytes. Methods 1. Preprtion of immture nd mture ooytes ICR mie (Smtko, Seoul, Kore) were mintined in temperture-nd-humidity-ontrolled room under 12 hours: 12 hours light: drk yle. For immture ooyte olletion, four-week-old femle mie were superovulted vi n intrperitonel injetion of 5 IU pregnnt mre serum gondotropin (PMSG; De Sung Miroiologil Ls, Seoul, Kore). At hours post PMSG, mie were srified y ervil dislotion for the olletion of ovries. The ovries were then trnsferred to Quinn s dvntge medium with HEPES (Quinn s- HEPES; Sge, In Vitro Fertiliztion, Trumull, CT, USA) ontining 1% sustitute protein serum (SPS; Sge BioPhrm, In., Bedminster, NJ, USA). Immture ooytes were olleted y punturing of folliles with needle (29 G). Cumulus-enlosed immture ooytes were seleted for the experiment. For the olletion of mture ooytes, 4- week-old ICR femle mie were superovulted with 5 IU PMSG, followed y injetion with 5 IU humn horioni gondotropin fter 48 hours (hcg; Intervet, Boxmeer, the Netherlnd). Cumulus-enlosed mture ooytes were retrieved t hours post-hcg from the oviduts. 2. Vitrifition nd wrming of ooytes Quinn s-hepes with 2% (v/v) fetl ovine serum (FBS; Gio, Grnd Islnd, NY, USA) ws used s the se medium for preprtion of ll vitrifition nd wrming solutions. As CPA, EG (Sigm-Aldrih, St. Louis, MO, USA) only or omintion of EG nd (Sigm-Aldrih) were used for the vitrifition proedure. For group 1, ooytes were pre-equilirted with 1.5 M EG for 2.5 minutes, followed y equilirtion with 5.5 M EG nd 1. M surose for 2 seonds. For group 2, ooytes were pre-equilirted with 1.3 M (7.5%) EG nd 1.1 M (7.5%) for 2.5 minutes, followed y 2.7 M (15%) EG, 2.1 M (15%) nd.5 M surose for 2 seonds. CPA-equilirted ooytes were loded onto n eletron mirosopi (EM) opper grid nd plunged into liquid nitrogen () or. ws produed using Vit-mster (IMT, Ness Zion, Isrel). The onentrtion of CPA 25

3 Clin Exp Reprod Med 211;38(1):24-3 ws determined ording to the study of Mukid et l. [25]. Vitrified immture nd mture ooytes were stored for t lest 2 weeks nd then wrmed to ompre their survivl nd susequent emryoni development. The vitrified ooytes were wrmed y five-step method. The opper grids were sequentilly trnsferred to 1.,.5,.25,.125, nd M surose with n intervl of 2.5 minutes. The vitrified/wrmed ooytes were then wshed with fresh ulture medium for three times. The immture ooytes were trnsferred to the ulture medium nd oserved under mirosope for the survivl rte of ooytes 1 hour fter wrming. The surviving immture ooytes were then indued to develop into mture ooytes y ulturing them in the in vitro mturtion medium. The mture ooytes from the immture stge nd the survived mture ooytes otined erlier were fertilized in vitro for further experimenttion. 3. In vitro mturtion of ooytes Survived immture ooytes from the vitrifition/wrming proess were indued for mturtion in G2.3 medium (Vitrolife, Göteorg, Sweden) ontining 2% FBS,.75 IU/mL FSH (Gonl-F, Serono, Modugno Bri, Itly),.5 IU/mL hcg (Ovidrel, Serono) nd 1. µg/ml estrdiol (Sigm-Aldrih) for 14 hours. Cumulus ells from mture ooytes were removed with modified Duleo s phosphte-uffered sline (D-PBS, HyClone, South Logn, UT, USA) solution ontining.1% hylurodse (Sigm-Aldrih). Denuded ooytes were wshed with fresh ulture medium three times nd their mturtion rte ws then evluted y oservtion of the first polr ody under the mirosope. 4. In vitro fertiliztion nd ulture Epididyml spermtozo were otined from 8- to 1-week-old mle ICR mie. Sperm suspension ws pitted in the inutor t 37 C, in 5% CO2 in ir for 9 minutes. Cpitted spermtozo were mixed ( /ml) with umulus-ooyte omplex in Quinn s dvntge fertiliztion medium nd inuted for 6 hours. The ooytes were then wshed three times in modified simplex-optimized medium (KSOM; Millipore, Dnvers, MA, USA) supplemented with.3% ovine serum lumin (BSA; Sigm-Aldrih). The fertilized ooytes were ultured in KSOM under 37 C in 5% CO2 for 5 dys to nlyze emryoni development. 5. Sttistil nlyses Survivl nd mturtion rtes of ooytes, emryoni development, nd lstoyst formtion rtes were nlyzed for sttistil signifine with one-wy ANOVA (Dunn-test). p-vlues<.5 were onsidered sttistilly signifint. Results 1. Effet of CPA on the survivl, mturtion, nd emryoni development of immture ooytes fter vitrifition using or The survivl nd mturtion rtes of immture ooytes were ompred fter the vitrifition/wrming proess using EG only or omintion of EG + s CPAs (Tle 1). After vitrifition/wrming using, there ws no signifint differene in the survivl rtes of the EG only group nd the EG + group (79.8 ± 4.7% vs ± 1.1%; p >.5), nor ws there ny differene etween the groups when using (79.4 ± 3.2% vs ± 1.4%; p>.5). However, the EG + group showed higher mturtion rte thn the EG only group in oth nd (73.2 ± 6.1%, 73.9 ± 1.2% vs ± 9.%, 52.3 ± 3.4 %; p <.5). The leve rte of immture ooytes in the EG only group ws signifintly lower thn tht of the ontrol nd EG + groups fter vitrifition using (26.3±7.8% vs. 62.9±12.2% nd 57.7 ± 5.%; p<.5). After vitrifition using, the leve rte in the EG group ws lower thn in the EG + group, ut the differene ws not sttistilly signifint (37.4 ± 4.8% vs ± 3.7%; p>.5). All groups showed very low lstoyst formtion rte. Numerilly, higher rte of lstoyst formtion ws oserved in the EG+ group fter vitrifition using, thn the other groups ut it ws not sttistilly signifint (3.9±1.7% vs. ±.%, 1.3±.7%, 1.9 ± 1.2%; p >.5). Tle 1. The effet of different ryoprotetnts nd ooling speed generted y using or on the survivl nd mturtion of immture ooytes following vitrifition Ooyte stge Tretment (solution) No. (%) of ooytes / Pre-equilirtion Equilirtion Conentrtion Vitrified Survivl (%) Mturtion (%) GV ooyte Control (87.9 ± 1.1) 1.5 M EG 5.5 M EG + 1. M Su 5.5 M (79.8 ± 4.7) 56 (49.1 ± 9.) 1.5 M EG 5.5 M EG + 1. M Su 5.5 M (79.4 ± 3.2) 56 (52.3 ± 3.4) 1.3 M EG M 2.7 M EG M +.5 M Su 4.8 M (87.9 ± 1.1) 19 (73.2 ± 6.1) 1.3 M EG M 2.7 M EG M +.5 M Su 4.8 M (88.9 ± 1.4) 113 (73.9 ± 1.2), GV, germinl vesile; EG, ethylene glyol;, dimethylsulphoxide; Su, surose;, liquid nitrogen;, slush nitrogen.,, Different supersripts within the sme olumn indite signifint differenes (p<.5). 26 doi: /erm

4 SK Ch et l. Effets of CPAs nd ooling speed on mouse ooytes fter vitrifition 2. Effet of CPA on the survivl, mturtion, nd emryoni development of mture ooytes fter vitrifition using or Mture ooytes were vitrified nd wrmed s immture ooytes nd the survivl rte ws evluted (Tle 2). After vitrifition using or, there ws no signifint differene etween the survivl rtes of the EG only nd EG + groups (71. ± 2.4%, 75. ± 3.5% vs. 6. ±8.8%, 79. ±4.3%; p >.5). There were no signifint differenes in leve rtes in the EG only nd EG + groups fter vitrifition using or (49. ± 5.9% vs. 43. ± 4.5%, 59. ± 1.1% vs. 62. ± 2.6%; p >.5). However, the leve rte ws signifintly higher in thn in in the EG only nd EG + groups (59. ±1.1%, 62. ±2.3% vs. 49. ±5.9%, 43. ±4.5%; p <.5). Furthermore, higher lstoyst formtion rte ws oserved in thn in for the EG+ group. (21.±2.2% vs. 7.±.8%; p <.5). Disussion Cryopreservtion of immture ooytes t the GV stge ws onsidered n lterntive to void the drwks of ryopreservtion of mture ooytes suh s hromosoml nondisjuntion indued y dmge to the spindle nd premture ortil grnule exoytosis [28,29]. However, s umulus ells nd ytoplsm re tightly onneted together, immture ooyte ryopreservtion resulted in mjor dmge nd lso low mturtion rte; hene, its linil pplitions hve een limited. Reently, the vitrifition proess ws pplied to overome these drwks. In ft, in this study, high survivl rte ws otined y pplying vitrifition; in prtiulr, using omintions of CPAs, EG, nd in vitrifition showed n pproximtely 9% survivl rte (Tle 1). The fertiliztion nd leve rtes of ooytes were lso higher in the CPA omintion group. In mny studies, the pre-equilirium nd equilirium time were longer when ws used for ryopreservtion, s it hs lower permeility rte thn EG [3]. Hene, we hve onduted preliminry study nd ompred the lstoyst formtion rte of ooytes nd emryos y using different pre-equilirium nd equilirium times. As result, there ws no differene in the lstoyst formtion for vrious time nd, in ft, when ooytes or emryos were treted with with the sme exposure time s to EG, they showed even etter results [31]. Consequently, unlike other studies, we used the sme exposure time for the EG group nd EG + omintion group. Slush nitrogen ws used to nlyze the effet of the ooling rte. The Tle 2. The effet of different ryoprotetnts nd ooling speed generted y using or on the survivl nd mturtion of mture ooytes following vitrifition Ooyte stge Tretment (solution) No. (%) of ooytes / Pre-equilirtion Equilirtion Conentrtion Vitrified Survivl (%) MII ooyte 1.5 M EG 5.5 M EG + 1. M Su 5.5 M 1 71 (71. ± 2.4) 1.5 M EG 5.5 M EG + 1. M Su 5.5 M 1 75 (75. ± 3.5) 1.3 M EG M 2.7 M EG M +.5 M Su 4.8 M 1 6 (6. ± 8.8) 1.3 M EG M 2.7 M EG M +.5 M Su 4.8 M 1 79 (79. ± 4.3) MII, met phse II; EG, ethylene glyol;, dimethylsulphoxide; Su, surose;, liquid nitrogen;, slush nitrogen. The sme supersript within the olumn indites no signifint differenes. A 1 B % of 2 ell (%) ± 12.2 n = ± 7.8, 37.4 ± 4.8,, 58.8 ± ± 5. n = 114 n = 17 n = 149 n = 153 % of lstoyst (%) ± 3.7 n = 124 n = 114 n = 17 n = 149 n = 153,,,. ±. 1.9 ± ± ± 1.7 Figure 1. The effet of different ryoprotetnts nd ooling speed generted y using or on the first levge (A) nd lstoyst formtion (B) of immture ooytes following vitrifition.,, Different supersripts indite signifint differenes (p<.5)., liquid nitrogen;, slush nitrogen; EG, ethylene glyol;, dimethylsulphoxide. 27

5 Clin Exp Reprod Med 211;38(1):24-3 A ± 2.1 B 1 % of 2 ell (%) n = ± ± ± ± 2.6 n = 1 n = 1 n = 1 n = 1 % of lstoyst (%) ± 7.9 n = 14, 5. ±.7 9. ±.6 7. ± ± 2.2 n = 1 n = 1 n = 1 n = 1 Figure 2. The effet of different ryoprotetnts nd ooling speed generted y using or on the first levge (A) nd lstoyst formtion (B) of mture ooytes following vitrifition.,, Different supersripts indite signifint differenes (p<.5)., liquid nitrogen;, slush nitrogen; EG, ethylene glyol;, dimethylsulphoxide. results showed tht there ws no prtiulr effet on survivl, mturtion, or levge rtes fter wrming immture ooytes (Figure 1). The fertiliztion rte showing two PN ould not e identified euse the onventionl insemintion method ws used in this study nd lso the lstoyst formtion rte ws very low. These results my e due to the ineffiieny of the vitrifition/wrming proess or the possiility of side effets suh s prthenogenesis. Hene, this results in limittion of the study. For evlution of the effetiveness of immture ooyte ryopreservtion, it is neessry to rry out further reserh using method suh s intrytoplsmi sperm injetion, whih diretly injets sperm into n ooyte. Furthermore, in this study, only the survivl nd emryoni development rtes were oserved, ut not intrellulr hnges tht my our during the vitrifition/ wrming proess, nd so the diret effet of using omintion of CPAs ws not possile to evlute. Hene, further study is required. In mture ooytes, the results otined were different from those of immture ooytes. After the vitrifition/wrming proess, the survivl, levge, nd lstoyst rtes of the EG only nd EG + groups were not signifintly different (Tle 2, Figure 2). However, when ws used for vitrifition, there ws no signifint differene in the survivl rte, ut it did inrese the levge nd lstoyst formtion rtes. Also, the lstoyst formtion rte ws signifintly higher when omintion of CPAs, EG nd, nd were used. As stted previously, this result is my e due to the inrese in the effiieny of vitrifition y using, whih elertes its glss-forming hrteristis s it penetrtes into the ell nd lso due to, whih inreses the ooling rte [19,2,25]. From this study, we oserved tht djusting n existing vitrifition solution tht is generlized worldwide is required in order to inrese the effiieny of vitrifition, s the hrteristis of immture nd mture ooytes re different. Also, lthough pplying in vitrifition proess did not ffet immture ooytes, it did ffet the emryoni development of mture ooytes fter the wrming proess. These results re due to the hrteristis of mture ooytes whih inlude n exposed spindle tht is essentil for hromosoml division; however, further study is required to evlute the mehnism in more detil. In this study, lthough the survivl rte of immture ooytes ws s high s tht otined from mture ooytes, immture ooytes still showed low emryoni development rte fter vitrifition/ wrming proess. This result ws similr to studies of humn ooytes [32,33]. It is suggested tht this is not only used y prolems with the ooyte ryopreservtion proess ut lso the lk of reserh on the in vitro mturtion proess. Therefore, if studies on the proess of in vitro mturtion of ooytes nd development of ulture system move forwrd then the effiieny of immture ooyte ryopreservtion should e expeted to improve. In onlusion, the omintion of CPAs, EG, nd showed greter effetiveness in the vitrifition of immture ooytes while for tht of mture ooytes, it is possile to use either EG only or EG nd. In ddition, my improve effiieny y reduing ryoinjury during vitrifition. Conflit of interest No potentil onflit of interest relevnt to this rtile ws reported. Referenes 1. Chen C. Pregnny fter humn ooyte ryopreservtion. Lnet 1986;1: vn Uem JF, Siezehnrül ER, Shuh B, Koh R, Trotnow S, Lng N. Birth fter ryopreservtion of unfertilized ooytes. Lnet 1987; 1: Lee DR, Yoon TK. Effet of slush-nitrogen on the ryopreservtion 28 doi: /erm

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