Article Meiotic spindle and zona pellucida characteristics as predictors of embryonic development: a preliminary study using PolScope imaging

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1 RBMOnline - Vol 14. No Reproductive BioMedicine Online; on web 8 Jnury 2007 Article Meiotic spindle nd zon pellucid chrcteristics s predictors of embryonic : preliminry study using PolScope imging GA Rm Rju obtined his medicl degree from Andhr Medicl College, Viskhptnm, Indi, nd DNB in obstetrics nd gynecology from NBE, Government of Indi, New Delhi. He underwent trining in ART t Kiel University, Bielefeld Institute of Reproduction, Germny nd in PGD t the Centre for Reproductive Medicine, Vrije Universiteit Brussel, Belgium. He hs been member of the visiting fculty in obstetrics nd gynecology t Justus Liebig University, Frnkfurt, Germny. He is currently the director of Krishn IVF Clinic, recognized centre for lproscopy trining. His reserch interests include reproductive endocrinology, vitrifiction of gmetes nd ovrin tissue nd PGD. Dr GA Rm Rju GA Rm Rju 1, GJ Prksh, KM Krishn, K Mdn Embryology Division, Krishn IVF Clinic, Zillprishd Junction, Viskhptnm, Pincode , Andhr Prdesh, Indi 1 Correspondence: Tel: +91 (0) ; Fx: ; e-mil: krishnivf@yhoo.com Abstrct This study ssesses meiotic spindle nd zon pellucid chrcteristics using the PolScope, nd nlyses their reltionship to embryonic potentil. A totl of 205 mtured oocytes retrieved from 25 ptients undergoing ovrin stimultion were imged for meiotic spindle nd zon pellucid chrcteristics using the PolScope. After intrcytoplsmic sperm injection, the oocytes were cultured nd ssessed for progression to blstocysts. Meiotic spindles were visulized in 78.0% of oocytes. Significntly more oocytes with visible spindles fertilized nd progressed to blstocysts compred with oocytes without visible spindles. Oocytes with spindle retrdnce of >3 nm showed greter progression to blstocysts compred with those with retrdnce of 2-3 nm or <2 nm. More blstocysts were obtined from oocytes with spindle lengths of >12 nm thn from oocytes with spindle lengths nm or <10 nm. A difference in progression to blstocyst ws observed in oocytes with zon inner lyer retrdnce of >3 nm compred with oocytes with retrdnce of 2-3 nm or <2 nm. Oocytes with n inner lyer zon of nm thickness showed better progression compred with those with thickness of 8 10 nm or <8 nm. Quntittive mesurement of length nd retrdnce of the meiotic spindle nd zon pellucid hs positive predictive vlue in reltion to embryonic. Keywords: embryonic, humn oocyte, meiotic spindle, PolScope, retrdnce Introduction 166 Rpid strides in of vrious spects of ssisted reproduction hve provided mny insights into humn embryonic. With the ultimte im of chieving pregnncy following single embryo trnsfer, vrious workers hve ttempted to identify oocyte prmeters which cn predict fvourble outcome (Serhl et l., 1997; Xi, 1997; Borini et l., 2005). With conventionl light microscope imging, oocyte chrcteristics such s cumulus, polr body, cytoplsm nd zon pellucid morphology hve been relted to embryonic nd pregnncy rtes. However, no single chrcteristic hs been infllible in predicting the bility of oocytes to chieve pregnncy. Hence, efforts to identify relible prmeters for the ssessment of oocyte qulity remin priority. One such prmeter tht plys mjor role in meiosis nd is the meiotic spindle. Until 1990, most of the work on spindles ws performed with the fluorescent microscope, which provided extensive informtion on the meiotic spindle nd its role in cell division. However, its routine use in ssisted reproduction procedures is precluded by its inbility to be pplied to live oocytes. With the dvent of the PolScope, it hs become possible to ssess meiotic spindle nd zon pellucid chrcteristics in live oocytes Published by Reproductive Helthcre Ltd, Duck End Frm, Dry Dryton, Cmbridge CB3 8DB, UK

2 Moreover, this procedure hs the dvntge of being totlly noninvsive nd preserves the oocyte vibility. The im of the present investigtion ws to evlute nd nlyse meiotic spindle nd zon pellucid chrcteristics of oocytes using the PolScope nd to ssess their influence on embryonic. Mterils nd methods Ptients The study group consisted of 205 metphse II (MII) oocytes obtined from 25 ptients. The ptient s ge rnged from 25 to 40 yers, with n verge of ± 6.2. Only femle infertility ws included; couples with mle infertility were excluded from this study protocol. Indictions for ssisted reproduction include polycystic ovry disese (n = 10), tubl fctor (n = 6), endometriosis (n = 4) nd idiopthic (n = 5). The study protocol nd the need to perform intrcytoplsmic sperm injection (ICSI) nd PolScope imging nlysis in spite of the bsence of mle fctor ws explined to the couples nd they were recruited into the study fter their pprovl. This study ws pproved by the Institutionl Review Bord (IRB), Krishn IVF Clinic. Stimultion protocol nd oocyte collection Ovrin stimultion ws chieved using gondotrophin-relesing hormone (GnRH) gonist nd recombinnt FSH. Downregultion ws initited using intrmusculr injection of triptorelin (Decpeptyl; Ferring Phrmceuticls, Mumbi, Indi) 3.75 mg on dy 21 of the cycle. Adequcy of down-regultion ws confirmed by mesuring oestrdiol (<50 pg/ml) nd LH concentrtions (<1 ng/ml). Ovrin stimultion ws chieved using recombinnt FSH (Recgon; Orgnon, USA) nd the dose ws djusted on the bsis of individul response. Humn chorionic gondotrophin (HCG) t dose of 10,000 IU ws given fter two follicles of 18 mm or more were visulized in the ultrsound scn. Oocyte retrievl ws scheduled 36 h lter by trnsvginl ultrsonogrphy (TVS) guided spirtion. Cumulus cells were removed using hyluronidse (Sigm, USA) nd mturtion of the oocytes ws confirmed by the presence of the first polr body. The oocytes were wshed twice in Gmete solution (VitroLife, Sweden) nd plced in n incubtor for 3 4 h before spindle imging. Visuliztion of spindle using the PolScope For spindle imging, ech oocyte ws plced in n equilibrted 5-μl medi droplet overlid with minerl oil in glss bottomed culture dish (Willco Wells, Netherlnds). Oocytes were imged using Zeiss Axiovert 200M microscope equipped with n LC PolScope (CRI, USA) controller nd CCD cmer. The imges were cquired t 200 mgnifiction nd the loction, length, men retrdnce of the meiotic spindle nd the men retrdnce of inner lyer of zon pellucid nd its thickness were nlysed using n LC computerized imge nlysis system. A temperture of 37 C ws mintined throughout the procedure using n incubtor (Incubtor XL; Crl Zeiss, Germny) fitted onto the microscope. In order to optimize the spindle nd polr body visuliztion, oocytes were rotted using the holding pipette. ICSI ws crried out by using one of the following methods. For oocytes with birefringent spindle, sperm injection ws performed fter plcing the spindle t the 6 o clock position. For oocytes with shift of meiotic spindle from the polr body, sperm injection ws performed ligning the imged spindle to the 6 o clock position. For oocytes without spindles, sperm injection ws performed fter plcing the polr body t the 6 o clock position. After ICSI, oocytes with nd without spindles were wshed twice nd cultured seprtely nd sequentilly in fertiliztion medi droplets (IVF; VitroLife) for further nlysis. Assessment of fertiliztion Oocytes were exmined under stereomicroscope for fertiliztion h fter ICSI. Oocytes with two pronuclei nd hving second polr body were considered s normlly fertilized. The fertilized oocytes were wshed twice nd cultured in fresh droplets of clevge medi (G1; VitroLife). After 3 dys post-icsi, embryos were trnsferred to blstocyst medi (G2; VitroLife). Sttisticl nlysis The results were compred using the chi-squred test nd one-wy nlysis of vrince (ANOVA). The vlues were expressed s ± SEM. A P-vlue of <0.05 ws considered sttisticlly significnt. Results A totl of 205 oocytes from 25 ptients were exmined with PolScope. The meiotic spindle ws visulized in 160 oocytes (78.0%), but could not be visulized in the remining 45 oocytes (22.0%). Significntly (P P < 0.05) more oocytes with visible spindles fertilized (82.5 versus 31.1%) nd progressed to the blstocyst stge on dy 5 (48.4 versus 14.2%) compred with the oocytes without visible spindles (Tble 1). Angle of spindle reltive to polr body versus embryonic Mture (MII) oocytes hd spindles t vrious ngles reltive to the first polr body (Figure 1). These oocytes were clssified into four groups on the bsis of the shift from the polr body. Oocytes in group A showed 0 5 shift, in group B showed 6 45 shift, in group C showed shift nd those in group D Tble 1. Influence of the presence of PolScope-visulised meiotic spindle on embryonic. PolScope No. of No. fertilized No. visulistion oocytes oocytes (%) proceeding (%) (%) to blstocyst (%) With spindle 160 (78.0) 132 (82.5) 64 b (48.5) Without spindle 45 (22.0) 14 (31.1) 2 b (14.3) 167

3 showed >90 shift. Among the 160 oocytes with visible spindle, 30.0% were ctegorized into group A, 38.8% into group B, 18.1% into group C nd 13.1% into group D (Tble 2). After ICSI, no significnt differences in the blstocyst progression rtes were observed mong the different groups. Spindle retrdnce versus embryonic The of n embryo to blstocyst ws tken s the criterion for good embryonic (Figure 2). Among 160 oocytes with visible spindle, 40% progressed to blstocysts. Of these, 60.9% showed retrdnce of >3 nm, 25.0% showed retrdnce between 2 nd 3 nm nd the remining 14.1% showed retrdnce of <2 nm (Tble 3). Significntly more oocytes with spindle retrdnce of >3 nm progressed to blstocysts when compred with oocytes hving retrdnce between 2 3 nd <2 nm respectively (P P < 0.05). Spindle length versus embryonic The length of the spindle in its long xis ws mesured using the PolScope softwre nd the results were nlysed with respect to blstocyst progression. Among the 64 oocytes which progressed to blstocysts, 45.3% hd spindle length between nm, 34.4% hd spindle length between nm nd the remining 20.3% hd spindle length of <10 nm (Tble 4). Oocytes with spindle lengths between 12 nd 15 nm progressed to significntly higher number of blstocysts (P P < 0.05) when compred with oocytes hving spindle lengths between nm nd <10 nm respectively. Inner zon retrdnce versus embryonic In the present study, the retrdnce of the inner lyer of the zon ws mesured prior to ICSI. Among 64 oocytes which progressed to blstocysts, 56.3% hd zon retrdnce of >3 nm, 26.6% showed retrdnce between 2 nd 3 nm nd the remining 17.2% showed retrdnce of <2 nm. Significntly (P P < 0.05) more oocytes hving zon retrdnce of >3 nm progressed to blstocysts on dy 5 when compred with oocytes hving zon retrdnce between 2 nd 3 nm nd <2 nm respectively (Tble 5 nd Figure 3). Inner zon thickness versus embryonic In humn oocytes, the innermost lyer (IL) of zon pellucid is the thickest mong the three zon lyers. In the present study, thickness of the IL of zon pellucid ws mesured before ICSI. Among 64 oocytes tht progressed to blstocysts, 53.1% hd n IL zon thickness of nm, 25.0% showed thickness between 8 nd 10 nm nd the remining 21.9% showed thickness of <8 nm. Oocytes with IL zon thickness between 10 nd 12 nm progressed significntly more frequently (P P < 0.05) to blstocysts compred with oocytes hving IL zon thicknesses between 8 10 nm or <8 nm (Tble 6). Spindle loction in vrious ge groups After nlysing the oocytes of ptients in different ge groups, no significnt reltionship ws observed between the degree of spindle shift nd ge of the ptient (Tble 7). Mternl ge versus spindle retrdnce nd length A significnt chnge in retrdnce nd lso the length of spindle with ge of ptients ws observed (Tble 8, P < 0.05). With increse in ge there ws decrese in retrdnce nd length of the spindle. b 168 Figure 1. Oocytes with meiotic spindle locted () djcent to polr body nd (b) with 90 shift from polr body. Mgnifiction 200.

4 Tble 2. Influence of meiotic spindle shift reltive to polr body on embryonic. Group Spindle ngle No. of No. proceeding to polr body oocytes to blstocysts (%) A (41.7) B (37.1) C (41.4) D > (42.9) There were no sttisticlly significnt differences in blstocyst progression rte between the groups. b Figure 2. PolScope imging of meiotic spindle. Oocytes exhibiting () wek retrdnce nd (b) norml retrdnce. Mgnifiction 200. Tble 3. Influence of men meiotic men spindle retrdnce on embryonic. Spindle retrdnce (nm) No of blstocysts (%) > (60.9) (25.0) <2.0 9 (14.1) b 169

5 Tble 4. Influence of meiotic spindle length on embryonic. Spindle No. of length blstocysts (nm) (%) (45.3) (34.4) <10 13 b (20.3) Tble 5. Influence of men retrdnce of the inner lyer of zon pellucid on embryonic. Retrdnce No of of inner lyer blstocysts of zon (nm) (%) > (56.3) (26.6) < b (17.2) b Figure 3. () An idel oocyte displying good spindle retrdnce nd length, trilminr structure of zon with good retrdnce of inner lyer. Mgnifiction 200. (b) Retrdnce profile of the three lyers of zon pellucid. Tble 6. Influence of thickness of the inner lyer of zon pellucid on embryonic. Thickness of No. of inner lyer blstocysts of zon (nm) (%) (53.1) (25.0) <8 14 b (21.9) Tble 7. Meiotic spindle loction in vrious ge groups. Spindle ngle Per cent of oocytes to polr body >35 yers yers old yers old b old c > oocytes; b 35 oocytes; c 17oocytes. There were no sttisticlly significnt differences between the ge groups. 170

6 Tble 8. Influence of mternl ge on meiotic spindle retrdnce nd length. Age (yers) b >35 c Retrdnce (nm) 3.01 ± 0.14 d,e 2.22 ± 0.07 d 1.84 ± 0.06 e Spindle length (nm) ± 0.36 f,g ± 0.30 f ± oocytes; b 35 oocytes; c 17 oocytes; d-g Vlues with the sme superscript letter re significntly different g Discussion The identifiction of relible criteri for ssessment of oocyte potentil prior to fertiliztion is one of the most investigted res in the field of ssisted reproduction. Attempts to estblish relible prmeters indictive of oocyte potentil hve involved ssessment of cumulus nd zon morphology, cytoplsmic fetures such s grnultion, vcuoles nd refrctile bodies, nd polr body fetures such s size nd frgmenttion. Conflicting reports hve been published by vrious uthors for the bove prmeters in reltion to embryonic (Desutter et l., 1996; Serhl et l., 1997; Blbn et l., 1998, 2001; Loutrdis et l., 1999; Rttnchiynon et l., 1999; Verlinsky et l., 2003; Ebner et l., 2006). For the pst decde, the meiotic spindle hs been extensively investigted s possible predictive feture for oocyte selection. Most of the erlier studies on meiotic spindle were performed using fluorescent microscopy (Liu et l., 1998). Although this technique gives detiled informtion bout chromosomes nd meiotic spindles, its clinicl use is limited by its invsive nture nd inbility to be used for studying live oocytes. Imging of meiotic spindle in live oocytes hs been mde possible by the of n orienttion-independent polrized light microscope, the polscope, which fcilittes study of the spindle s rchitecture non-invsively in live humn oocytes without ffecting vibility (Oldenbourg, 1996; Liu et l., 2000; Keefe et l., 2003). Unlike conventionl methods of imging spindles, the polscope does not require preprtive techniques such s fixtion nd stining nd hs unique potentil to visulize nd mesure birefringent structures dynmiclly nd non-invsively in living cells. The polscope opertes on the principle tht when two orthogonlly polrized light rys pss through orderly rrnged filmentous structures such s spindles composed of microtubules, they re differentilly slowed down. The resultnt chnge is mesured s retrdnce, nd cn be quntified (Sto et l., 1975; Oldenbourg, 1999). The mesured retrdnce is directly proportionl to the density of the microtubules. This reltionship between spindle retrdnce nd microtubule density ws first estblished by Sto et l. (1975). Liu et l. (2000b) observed tht oocytes with bsence of spindle when visulized with polscope exhibited disssembled spindles nd mislignment of chromosomes when visulized by confocl microscopy. Hence, the spindle retrdnce my be n importnt prmeter for the evlution of oocyte qulity prior to fertiliztion nd further embryonic (Wng et l., 2001; Trimrchi et l., 2004). The meiotic spindle, composed of cytoskeletl microtubules, plys mjor role in the successful completion of meiosis by controlling chromosoml movement throughout the vrious stges of meiosis. Severl uthors hve suggested tht disorgniztion of the spindle leds to disruption of orderly chromosoml segregtion, which results in neuploidy of zygotes (Pickering et l., 1988; Eichenlmb Ritter et l., 2002). The meiotic spindle morphology nd kinetics re influenced by severl vribles such s mternl ge, temperture nd in-vitro mnipultions of oocytes. Studies by Bttgli et l. (1996) indicted tht incidence of spindle bnormlity in oocytes ws significntly higher in older ptients. This is further supported by the work of Volrcik et l. (1999), who studied ge-relted effects on the meiotic process of in-vitro mtured humn oocytes from unstimulted ovries. This lso corresponds with the higher incidence of meiotic segregtion errors observed with incresed mternl ge. Temperture is n importnt prmeter ffecting the integrity of the spindle during hndling nd culturing of oocytes in invitro mnipultion. Meiotic spindles in humn oocytes re more sensitive to temperture fluctutions when compred with other nimls (Pickering et l., 1990; Amn, 1994; Almedi et l., 1995). Disssembled spindles my result from prolonged exposure to low temperture. This is further confirmed in report by Wng et l. (2001b, 2002). A recent study by Sun et l. (2004) on in-vitro mtured oocytes reported tht spindles in humn oocytes re lso sensitive to tempertures higher thn 37 C. Therefore, these results indicte tht when humn oocytes re mnipulted in vitro, rigorous control of 37 C temperture is essentil for mintennce of spindle integrity nd further embryonic. A further potentil for injury to the meiotic spindle during invitro mnipultion of oocytes is the sperm injection procedure. During ICSI, the loction of the spindle is ssumed to be in reltion to the polr body. In order to void dmge to the spindle, the polr body is positioned t 90 ngle reltive to the injection needle. Studies by Silv et l. (1999) nd Wng et l. (2001c) suggested tht the spindle is not lwys djcent to the polr body. In such circumstnces, where the displced spindle is in the pth of the injecting needle during ICSI, it my be subject to mechnicl dmge. 171

7 172 Developmentl potentil of oocytes with visulized spindle using the PolScope Wng nd Keefe (2002) reported tht oocytes without visible spindle led to high degree of neuploidy nd poor embryonic. Severl uthors hve suggested tht the bsence of spindle my be primrily becuse of compromised oocyte with disorgnized spindle or secondry to improper control of environmentl fctors such s temperture control during oocyte retrievl, nd micromnipultion procedures (Wng et l., 2001b; Cook et l., 2002). In the present study, it ws found tht the spindle ws bsent in 22.0% of mture (MII) oocytes. These findings re comprble with previous studies by Wng et l. (2001), Moon et l. (2003) nd Cohen et l. (2004), whose comprble figures were 18, 16.5 nd 24.5% respectively. Furthermore, the fertiliztion rte of 82.5% in oocytes with visulized spindles ws sttisticlly significntly greter compred with tht in oocytes with non-visulized spindles (31.1%, P <0.05) (Tble 1). These findings re in greement with previous study by Wng et l. (2001). A significnt ssocition between the presence of spindle nd blstocyst progression ws lso observed (Tble 1). These results re in ccordnce with Wng et l. (2001c) nd Moon et l. (2003). However, these results re contrry to those of Cohen et l. (2004), who reported no significnt difference in embryonic to dy 3 embryos between oocytes with nd without spindles. Role of shift of the spindle from the polr body in embryonic Studies on hmster nd rhesus monkey oocytes by Silv et l. (1999) nd Hewitson et l. (1999) suggested tht the meiotic spindle is not lwys locted djcent to the polr body. This is further supported by Wng et l. (2001c) for live humn oocytes. A possible explntion for the shift of the polr body my be due to physicl displcement during denudtion of cumulus nd coron prior to spindle imging. This explntion is in greement with previous studies by Rienzi et l. (2003, 2005) nd Moon et l. (2003). In the present study, out of 160 oocytes with visible spindle, 30% hd spindle djcent to the polr body nd the remining 70% hd spindles t different loctions, which is in greement with the bove uthors. No sttisticlly significnt difference ws observed between the ligned nd non-ligned oocytes in terms of embryonic. This my be due to the correction of the shift of the spindle during ICSI by ligning the spindle of the oocyte t the 6 o clock position insted of the polr body. Spindle retrdnce nd its role in embryonic Studies by Sto et l. suggested tht spindle birefringence ws contributed to by microtubules, nd lso proposed reltionship between spindle retrdnce nd microtubule density (Sto et l., 1975). Trimrchi et l. (2004) reported positive correltion between spindle retrdnce nd embryonic. A recent study by Shen et l. (2006) reported tht men retrdnce of the spindle is positively relted to pronucler score nd better conception rte. In the present investigtion, oocytes were ctegorized on the bsis of men retrdnce into three groups (Tble 3). Oocytes with men retrdnce of >3.0 progressed to significntly more blstocysts in comprison with other groups (P < 0.05). These findings re in greement with previous studies by Trimrchi et l. (2004) nd Shen et l. (2006). A trend ws lso observed between the low retrdnce, ge nd oocyte qulity in terms of further embryonic. Studies by De Sntis et l. (2005) lso observed the sme trend. Meiotic spindle length s predictor of embryonic Vrition in the meiotic spindle length of oocytes ws reported by Wng et l. (2001b). The proposed contributing fctors tht influence the length of spindle re meiotic ge (Eichenlub- Ritter et l., 1986, 1988, 1995) nd exposure to different culture conditions. Shen et l. (2006) recently reported tht the men spindle lengths of ll oocytes in different cohorts re similr. However, in the present study, oocytes with spindle length of >12 nm showed significntly higher blstocyst progression (Tble 4; P < 0.05). A decrese in spindle length ws lso observed with incresed mternl ge. Zon inner-lyer retrdnce, predictive mrker for embryonic Polscope study revels the zon pellucid s three-lyer structure, nd rrngements of filments within these lyers re different from ech other (Oldenbourg, 1997). Due to the different orienttion of these filments, these lyers exhibit different birefringence with the polscope. The filments in the inner lyer (IL) of zon re rrnged rdilly nd exhibit mximum birefringence, wheres filments of the outer lyer zon (OL) re oriented tngentilly nd retrd the light to lesser degree, yielding moderte birefringence (Oldenbourg, 1997). The inner nd outer lyers re seprted by the middle lyer (ML), which exhibits lest birefringence due to rndom orienttion of filments (Silv et l., 1997). For the pst few yers, mny uthors hve focused to estblish reltionship between these birefringent chrcteristics of zon pellucid of oocyte nd embryonic (Pelletier et l., 2004; Shen et l., 2005). Studies by Pelletier et l. (2004) suggested tht the retrdnce mgnitude of zon chnges with different mturtionl stges of the oocyte nd embryo. Shen et l. (2005) reported vrition in the men retrdnce of the IL zon of oocytes in conception nd non-conception cycles with higher men retrdnce of the IL zon in the oocytes of conception cycles compred with non conception cycles. They lso concluded tht the men retrdnce of middle nd outer lyers of zon were not sttisticlly significnt. Hence, the retrdnce mgnitude of the inner lyer of the zon pellucid hs been proposed to hve positive predictive vlue s mrker for the selection of oocytes with better l potentil. In the present study oocytes with zon inner lyer retrdnce of >3 nm progressed to significntly more blstocysts in comprison with oocytes with zon inner lyer retrdnce of <3 nm. These findings re in ccordnce with Shen et l. (2005), nd further strengthen the importnce of zon IL retrdnce s predictor of good embryonic.

8 Zon thickness versus embryonic The thickness of the zon pellucid increses with mturtion of the oocyte nd becomes thinner in the post-fertiliztion (Betrnd et l., 1996; Dirnfeld et l., 2003). Shen et l. (2005) reported significnt increse in thickness of the inner lyer of the zon pellucid in oocytes of conception cycles compred with non-conception group of oocytes, wheres the thickness of the middle nd outer lyers of the zon ws not significnt. In the present study, zon IL thickness ws mesured nd the present findings re in ccordnce with Shen et l., with oocytes hving zon IL thickness between 10 nd 12 nm exhibiting the highest blstocyst progression. Further lrger prospective studies re needed to vlidte the role of zon chrcteristics s possible positive predictor of good oocyte qulity. In conclusion, the present study shows tht the presence of visible meiotic spindle viewed with polscope, nd quntittive ssessment of its men retrdnce nd length, hve positive predictive vlue in reltion to progression to blstocysts. The mesurement of the thickness of the inner lyer of the zon pellucid nd its men retrdnce lso shows similr predictive vlue. The PolScope, by fcilitting the nlysis of the meiotic spindle nd zon pellucid chrcteristics non-invsively nd rpidly, hs proven to be revolutionry tool for ssessment of the meiotic spindle. However, further prospective studies with lrger ptient popultions re needed so tht the results cn be nlysed with the ptient s the sttisticl unit rther thn the oocyte. Such studies would llow further ssessment of the reltionship between chrcteristics of the meiotic spindle nd inner lyer of zon pellucid nd embryonic, nd the vrious fctors which my influence these chrcteristics. 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9 Article - Polscope imging of spindle nd zon chrcteristics - GA Rm Rju et l. correltion between oocyte coron cumulus complex morphology nd nucler mturity of oocytes collected in stimulted cycles for intrcytoplsmic sperm injection. Fertility nd Sterility 71, Rienzi L, Ubldi F, Mrtinez F et l Meiotic spindle visuliztion in living humn oocytes. Reproductive BioMedicine Online 10, Rienzi L, Ubldi F, Mrtinez F et l Reltionship between meiotic spindle loction with regrd to the polr body position nd oocyte potentil fter ICSI. Humn Reproduction 18, Sto H, Ellis GW, Inoue S 1975 Microtubulr origin of meiotic spindle from birefringence. Demonstrtion of the pplicbility of Wiener s eqution. Journl of Cell Biology 67, Serhl P, Riner D, Kinis A et l Oocyte morphology predicts outcome of intrcytoplsmic sperm injection. Humn Reproduction 12, Shen Y, Stlf T, Mehnert C et l Light retrdnce by humn oocyte spindle is positively relted to pronucler score fter ICSI. Reproductive BioMedicine Online 12, Shen Y, Stlf T, Mehnert C et l. 2006b High mgnitude of light retrdtion by the zon pellucid is ssocited with conception cycle. Humn Reproduction 20, Shen Y, Stlf T, Mehnert C et l High mgnitude of light retrdnce by the zon pellucid is ssocited with conception cycle. Humn Reproduction 20, Silv CS, Kpur K, Odenberg R, Keefe DL 1999 The first polr body does not predict ccurtely the loction of the metphse II meiotic spindle in mmmlin oocytes. Fertility nd Sterility 71, Silv CP, Silv V, Kommineni K, Keefe D 1997 Effect of in vitro culture of mmmlin embryos on the rchitecture of the zon pellucid. Biologicl Bulleten 193, Sun XF, Wng WH, Keefe DL Overheting is detrimentl to meiotic spindles within in vitro mtured humn oocytes. Zygote 12, Trimrchi JR, Krin RA, Keefe DL 2004 Averge spindle retrdnce observed using the polscope predicts cell number in dy 3 embryos. Fertility nd Sterility 82 (Suppl. 2), S268. Verlinsky Y, Lerner S, Illkevitch N et l Is there ny predictive vlue of first polr body morphology for embryo genotype or l potentil? Reproductive BioMedicine Online 7, Volrcik K, Sheen L, Goldfrb J et l The meiotic competence of in-vitro mtured humn oocyte is influenced by donor ge: evidence tht folliculogenesis is compromised in the reproductively ged ovry. Humn Reproduction 13, Wng W, Keefe DL 2002 Prediction of chromosome mislignment mong in vitro mtured humn oocytes by spindle imging with the polscope. Fertility nd Sterility 78, Wng W, Meng L, Hckett RJ et l Rigorous therml control during intrcytoplsmic sperm injection stbilizes the meiotic spindle nd improves fertiliztion nd pregnncy rtes. Fertility nd Sterility 77, Wng WH, Meng L, Hckett RJ, Keefe DL 2001 Developmentl bility of humn oocytes with or without birefringent spindles imged by PolScope before insemintion. Humn Reproduction 16, Wng WH, Meng L, Hckette RJ et l. 2001b Limited recovery of meiotic spindles in living humn oocytes fter cooling-rewrming observed using polrized light microscopy. Humn Reproduction 16, Wng W, Meng L, Hckett RJ et l. 2001c The spindle observtion nd its reltionship with fertiliztion fter intrcytoplsmic sperm injection in living humn oocytes. Fertility nd Sterility 75, Xi P 1997 Intrcytoplsmic sperm injection: correltion of oocyte grde bsed on polr body, perivitelline spce nd cytoplsmic inclusions with fertiliztion rte nd embryo qulity. Humn Reproduction 12, Received 10 October 2006; refereed 6 November 2006; ccepted 4 December