Assisted reproductive technology

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1 Assisted reprductive technlgy FERTILITY AND STERILITY Vl. 66, N., August 996 Cpyright 996 American Sciety fr Reprductive Medicine Printed n acid-free paper in U. S. A. Aneuplidy 6 in human embrys increases significantly with maternal age* Claudi A. Benadiva, M.D.t Lmac Kligman, M.D. Santiag Munne, Ph.D.:!: The Center fr Reprductive Medicine and Infertility, Department f Obstetrics and Gyneclgy, The New Yrk Hspital Crnell Medical Center, New Yrk, New Yrk Objectives: T determine aneuplidy fr chrmsrne 6 by recycling nuclei f cells already analyzed fr chrmsmes X,Y, 8, 3, and using multiple-prbe flurescence in situ hybridizatin in preimplantatin human embrys in a time frame cmpatible with clinical IVF and t assess the incidence f chrmsme 6 aneuplidy in embrys related t maternal age. Design: Prspective experimental study. Setting: In vitr fertilizatin prgram in a tertiary center. Patients: One hundred fur cnsenting patients underging IVF. Main Outcme Measures: Chrmsme 6 plidy was analyzed in a ttal f 95 embrys. In 89 embrys, a standard multiple-prbe flurescence in situ hybridizatin was used fr chrmsmes X, Y, 8, and 6 (series ). The remaining 06 embrys (series ) were reanalyzed with a new prcedure fr chrmsme 6, which invlves rehybridizatin with a digxigenin-iabeled a satellite prbe after the standard analysis fr chrmsmes X, Y, 8, 3, and was cmpleted. The embrys were assigned t ne f three grups accrding t the wmen's age; grup : :::;3 years (n = 3), grup : 35 t 39 years (n = 7); grup 3: ~0 years (n = 3). Results: Successful analysis, including bipsy, fixatin, and flurescence in situ hybridizatin was achieved in 86% f the blastmeres within apprximately 0 hurs. A significant relatinship was fund between the rate f aneuplidy fr chrmsme 6 and increasing maternal age: grup : 0%, grup : 6.3%, and grup 3:.7%. Mnsmy fr chrmsme 6 was fund in 7.7% f the embrys carrying chrmsme 6 anmalies, with the remaining three embrys having tw trismies and ne tetrasmy. This new prtcl was applied clinically t five patients underging preimplantatin aneuplidy assessment. Aneuplidy fr chrmsme 6 was fund in five embrys frm three f thse patients. Cnclusins: This study demnstrates that preimplantatin genetic diagnsis f the majr human aneuplidies is achievable within a time frame cmpatible with IVF. In additin, this study cnfirms, fr embrys, the existing data frm spntaneus abrtins suggesting that chrmsme 6 aneuplidy increases with maternal age. The high prevalence f embrynic mnsmy, which is rarely fund in spntaneus abrtins, suggests that mnsmy 6 culd be a factr assciated with failure f implantatin, as well as pinting t a different mechanism invlved in the generatin f chrmsme 6 aneuplidy. Fertil Steril 996;66:8-55 Key Wrds: Preimplantatin genetic diagnsis, in vitr fertilizatin, mnsmy, embry bipsy Received December 7, 995; revised and accepted February 9, 996. * Presented at the 5st Annual Meeting f The American Sciety fr Reprductive Medicine, Seattle, Washingtn, Octber 7 t, 995, where it was awarded the prize paper f the Sciety fr Assisted Reprductive Technlgy. 8 Benadiva et al. Chrmsme 6 assessment A shrt flurescence in situ hybridizatin prcedure using flurchrme and digxigenin-labeled DNA prbes has been applied t preimplantatin human embrys t analyze the five chrmsmes mst invlved in aneuplidies fund in livebrn infants t Present address: Center fr Fertility and Reprductive Endcrinlgy, New Britain General Hspital, New Britain, Cnnecticut. t Reprint requests and present address: Santiag Munne, Ph.D., The Institute fr Reprductive Medicine and Science, Saint Barnabas Medical Center, 0 Old Shrts Hills Rad, Suite 50, West Orange, New Jersey 0705 (FAX: ). Fertility and Sterility

2 (X, Y, 8, 3, and ) befre replacement int the uterus (). This methdlgy can be used in a time frame cmpatible with regular IVF prcedures, thereby reducing the risk f cmmn aneuplidies in newbrns. In additin, it may prevent the transfer f embrys carrying chrmsmal cmplements assciated with spntaneus abrtins r even cmplete failure f implantatin. One imprtant limitatin f the prcedure, hwever, is the reduced number f chrmsmes that can be analyzed. Flurescence in situ hybridizatin analysis f multiple chrmsmes requires the simultaneus use f DNA prbes labeled each with different flurchrmes and/r mixtures f tw r three flurchrmes per chrmsme (). With ur previus methd, we were able t detect fur different chrmsme-specific prbes simultaneusly. Trismy 6 is the mst cmmnly identified trismy in man, ccurring in 7.5% f all spntaneus abrtins and.5% fkarytyped preimplantatin embrys (3, ). Despite its imprtance, chrmsme 6 culd nt be detected with ur previus prtcl (). The purpse f this study was twfld: t develp a new methd f ascertaining aneuplidy fr chrmsme 6 by blastmere rehybridizatin with a digxigeninlabeled a satellite prbe after the analysis f chrmsmes X,Y, 8, 3, and was cmpleted and t assess the incidence f chrmsme 6 aneuplidy in preimplantatin human embrys related t maternal age. The apprach described in the present study prvides infrmatin n specific chrmsmes within 0 hurs frm the embry bipsy, which is cmpatible with embry replacement n day 3 after retrieval, at the 8- t -cell stage. Embrys MATERIALS AND METHODS Embrys were btained frm patients underging IVF at The New Yrk Hspital-Crnell Medical Center. The number f prnuclei per zygte was scred after inseminatin, and thse with tw prnuclei (mnspermic) were allwed t cleave fr 3 days. Mrphlgically nrmal embrys fr this study came frm tw surces. The first cnsisted f embrys frm patients underging preimplantatin genetic diagnsis. The embrys at risk were nt transferred and were used fr research. The secnd surce f pssibly nrmally develping embrys were thse that divided asynchrnusly and therefre were nt frzen because they were nt captured at interphase. In additin, in patients 0 years r lder, the fur embrys f best mrphlgical and develpmental characteristics were replaced, and any thers were discarded r dnated fr research, as freezing f these Vl. 66, N., August 996 embrys rarely results in a successful pregnancy. Embrys may be investigated by flurescence in situ hybridizatin accrding t prtcls apprved by the Human Investigatin Cmmittee f the New Yrk Hspital-Crnell University Medical Cllege (prtcls n , , and ). In additin, arrested r mrphlgically abnrmal embrys als were analyzed. Patients frm whm the embrys were btained had cnsented t these prcedures. Tw ffive patients in which this new prtcl was applied fr preimplantatin genetic diagnsis f aneuplidy underwent the prcedure at Saint Barnabas Medical Center (Institutinal Research Bard prtcl title: "Genetic diagnsis f eggs and embrys"). Mnspermic embrys were classified in three main grups: arrested, slw and/r fragmented, and develpmentally nrmal, accrding t develpmental fitness. Arrested embrys were cnsidered thse that did nt develp beynd the eight-cell stage n their th day and had nt cleaved fr '0: hurs by days 3 and. Slw embrys were thse that had nt reached the eight-cell stage n their th day f develpment but had cleaved during day 3 r ; fragmented embrys were slw r develpmentally nrmal but had mre than 5% extracellular fragmentatin. Develpmentally nrmal embrys were thse at the eight-cell stage by day 3 with less than 5% fragmentatin. Extensive diplid msaicism pssibly inhibiting nrmal embrynic develpment was defined as the ccurrence f (38%) f the cells in a diplid embry being chrmsmally abnrmal r multinucleated. This figure is derived frm ur center's data n crypreservatin, where embrys that have lst up t three f eight cells are capable f nrmal develpment, as well as frm experience derived frm embry bipsy experiments (5). Chrmsme 6 plidy was analyzed in a ttal f 95 embrys (develpmentally nrmal, n = 9; slw and/r fragmented, n = 68; and arrested, n = 35) btained frm 0 patients, with a mean maternal age f36.3 ±.7 years (mean ± 3D). In 89 embrys (series ), a standard multiple-prbe flurescence in situ hybridizatin was used fr chrmsmes X, Y, 8, and 6 (6). The remaining 06 embrys (series ) were reanalyzed with the new prcedure described in the present study. Results f bth series were cmbined t determine the incidence f chrmsme 6 aneuplidy in a larger number f patients. The embrys were assigned t ne f three grups accrding t the wmen's age; grup : :=;3 years (mean 3.0 ±.0; n = 3), grup : 35 t 39 years (mean 36.6 ±.; n = 7); grup 3: '0:0 years (mean.0 ±.6; n = 3). Differences between age grups were cmpared by X using Fisher's-type adjustment Benadiva et a. Chrmsme 6 assessment 9

3 fr small samples and the crrelatin between aneuplidy rates and maternal age was examined by linear regressin analysis using mving averages (7). Embry Bipsy and Flurescence In Situ Hybridizatin Embrys crrespnding t the five patients underging preimplantatin genetic diagnsis (n = 37) were bipsied in the mrning f the 3rd day after retrieval, and all their nnreplaced embrys were reanalyzed the next day t cnfirm the initial diagnsis. All ther embrys (n = 58) were bipsied n day and mst r all their cells were analyzed. Embry bipsy and blastmere fixatin were perfrmed as described previusly withut mdificatin (8,9). Tw sequential assays f flurescence in situ hybridizatin were perfrmed t analyze chrmsmes X, Y, 3, 6, 8, and in the same blastmere. The first flurescence in situ hybridizatin invlved the use f prbes fr chrmsmes X, Y, 8, and 3/. Mter washing ffthese prbes, a secnd flurescence in situ hybridizatin was perfrmed using a prbe fr chrmsme 6. Fr the first flurescence in situ hybridizatin, a mdificatin f a previus prtcl () was used. The prbes fr chrmsmes Y and 8 were labeled with blue (CEP Spectrum aqua; Vysis, Naperville, IL) and green (CEP Spectrum green; Vysis) flurescent haptens, respectively. Fr chrmsme X, a : mixture f prbes labeled with red and green flurescent haptens (CEP Spectrum green and range; Vysis) was applied t btain a yellw appearance when bserved with a dual green-red band pass filter. The prbe fr chrmsme 3/ was labeled with digxigenin (Oncr, Gaithersburg, MD) and demnstrated by using rhdamine-labeled antidigxigenin antibdies. An additin f. ILL f each prbe (0 t 50 ng/ill fr CEP prbes and 0 ng/ill fr the 3/ prbe) was made t 8. ILL f Spectrum CEP hybridizatin buffer (Vysis). The resulting 3 ILL f hybridizatin slutin were applied t a glass slide cntaining the fixed blastmeres and cvered with an 8 X 8 mm cverslip. The slide was placed n a slide warmer at 80 C fr 3 minutes, sealed with rubber cement, and placed t hybridize at 37 C in a dark mist chamber fr 3 hurs minimum. Mter the hybridizatin, the slides were washed and the digxigenin-iabeled prbe was demnstrated with rhdamine-labeled antibdies as described previusly withut mdificatin (). The slides then were munted with 0 ILL f DAPI cunterstain in antifade slutin (',6-diamin--phenyl indle; Vysis) and bserved under a flurescence. micrscpe (Olympus BX50) equipped with a triple-band pass 50 Benadiva et al. Chrmsme 6 assessment filter set fr simultaneus bservatin f Spectrum Orange, Spectrum Green, and Spectrum Aqua (Vysis). In additin, a DAPI filter (Olympus, Lake Success, NY) was used t find the nuclei, and a PI/ FITC (Olympus) filter was applied t distinguish red (invisible with this filter) and green signals frm autrefringent debris (yellw with this filter). Digital cmputer imaging was nt used during the analysis. After the analysis fr chrmsmes X, Y, 8, and 3/ was cmpleted, the slides cntaining the fixed blastmeres were washed in X phsphate-buffered detergent (Oncr) fr 30 minutes, dehydrated in ethanl (70%, 85%, and 00%, minutes each), and reanalyzed immediately. An aliqut ( ILL) f a digxigenin-iabeled ll' satellite chrmsme-6 prbe (Oncr) was added t 7 ILL f Spectrum CEP hybridizatin buffer (Vysis) and ILL f blcking human COT-I DNA (GIBCO BRL, Gaithersburg, MD). The resulting hybridizatin slutin was applied (0 ILL per 8 X 8 mm cverslip) t the slides and denatured at 80 C fr 3 minutes n a slide warmer. Several test experiments perfrmed during the develpment f this technique demnstrated that, after such treatment, the prir flurescent signals fr chrmsmes X, Y, 8, and 3/ becme undetectable (Fig. C). The slide then was placed t rehybridize at 37 C in a humidified chamber fr 3 hurs, fllwed by a wash in O.x saline-sdium citrate buffer, ph 7.5 at 7 C fr minutes and minutes in phsphate-buffered detergent at rm temperature. Next, 5 ILL f rhdamine-labeled antidigxigenin sheep antibdy (Oncr) was applied t the slide and incubated fr 30 minutes at 37 C. Tw additinal layers f anti-sheep antibdy frm rabbit (5 ILL; Oncr) and rhdamine-labeled anti-rabbit antibdy (5 ILL, Oncr) were applied t the slide and incubated fr 5 minutes each at 37 C, as described abve. Finally, 5 ILL f DAPI in antifade slutin was used as a cunterstain, befre analysis under flurescence micrscpy with a rhdamine DAPI filter (Vysis). The scring criteria and flurescence in situ hybridizatin failure criteria used in this study were described previusly (0). Fr preimplantatin genetic diagnsis f aneuplidy, when the cell was clearly abnrmal, the embry was nt transferred. Nevertheless, in the fllwing instances the replacement f embrys with abnrmal cells culd be cntemplated if enugh nrmal embrys are nt available fr transfer. The first case is when the bipsied cell is binucleated with each nuclei shwing a nrmal number f chrmsmes. This is based n previus flurescence in situ hybridizatin studies n multinucleated blastmeres, which shwed thse embrys usually are nrmal (). The secnd instance is when the cell is mnsmic fr an auts- Fertility and Sterility

4 Figure In situ hybridizatin with X, Y, 8, 3/, and 6 chrmsme-specific DNA prbes. The chrmsme X-specific prbe is shwn labeled in yellw, chrmsme Y is labeled in blue, chrmsme 8 is labeled in green, and the prbe specific fr chrmsmes 3/ and 6 is labeled in red. (A), Blastmere with tw X chrmsmes, tw 8, and fur signals fr chrmsmes 3 and. (B), Blastmere with ne X and Y chrmsmes, tw 8, and fur signals fr chrmsmes 3 and. (C), Blastmere nuclei after the prbes fr the first five chrmsmes were washed ff and befre reanalysis with a chrmsme 6 prbe. (D), Blastmere after reanalysis, shwing tw signals fr chrmsme 6. mal chrmsme and n nrmal embrys are available fr transfer. This criterin is based n the fact that the test has a % prbability f misdiagnsis, whereas mnsmies fr autsmal chrmsmes are lethal and very seldm implant (). The third circumstance in which embry replacement culd be cnsidered is when a tetraplid cell is fund in a mrphlgically and develpmentally nrmal embry. We have fund tetraplid cells in 7.% (6/ 3) f mrphlgically and develpmentally nrmal embrys (unpublished data). Flurescence in situ hybridizatin analysis f all the blastmeres revealed that such embrys were nt plyplid, and a nin msaic was fund in all the cases. This criterin is supprted further by the fact that nly eight nenates with cmplete tetraplidy have been reprted in the wrld (3). Vl. 66, N., August 996 RESULTS Flurescence In Situ Hybridizatin Efficiency Hybridizatin signals were fund in 99.3% (38/ ) f the blastmeres fixed and analyzed with the new prtcl (Table ). Errrs f flurescence in situ Table Efficiency f the New Flurescence In Situ Hybridizatin Prtcl Invlving a Secnd Hybridizatin fr Chrmsme 6* Ttal n. f blastmers analyzed Blastmeres with hybridizatin signals Blastmeres nt classified Flurescence in situ hybridizatin errrs Ttal flurescence in situ hybridizatin failure * Values in parentheses are percentages. 38/ (99.3) 3/ (0.7) 59/38 (3.) 6/ (.05) Benadiva et al. Chrmsme 6 assessment 5

5 Table Incidence f Chrmsme 6 Aneuplidy in Relatin t Maternal Age* Ttal embrys analyzed by flurescence in situ hybridizatin Chrmsme 6 aneuplidy Age Series t Series :: Ttal Mnsmy Trismy Ttal % y :53 (n = 3) 35 t 39 (n = 7) :0 (n = 3) Ttal (n = 0) % 6.3%.8% 5.6% * Grups,, and 3 are significantly different, P = 0.0. t Prbes X, Y, 8, and 6. :I: Prbes X, Y, 8, 3/, and 6. One tetrasmic. hybridizatin were detected in 59 cells (3.%); three false-negatives were detected fr gnsmes; fur were detected fr chrmsme 8; 3 were detected fr chrmsme 6, and 5 were detected fr chrmsmes 3/. In additin, tw false-psitives fr chrmsmes 3/ and ne fr chrmsme 8 were fund. The ttal flurescence in situ hybridizatin failure rate was.05% when cmbined with the 0.7% (3/) f bias tme res nt classified. Embrys With Chrmsme Abnrmalities Results fr chrmsme 6 (95 embrys) analyzed with the previus (series ) and the new (series ) prtcls are shwn in Table. A significant increase in the incidence f aneuplidy fr chrmsme 6 was bserved with increasing maternal age: grup (age :5 3 years): 0%, grup (age 35 t 39 years): 6.3%; and grup 3 (age ~ 0 years):.5% (P = 0.0). In grup, tw thirds fthe aneuplidies fund were trismies and the remainder were mnsmic. All the anmalies fund in grup 3 crrespnded t mnsmies. A significant linear trend als was established between maternal age and the incidence f chrmsme 6 aneuplidy (R: 0.879; P < 0.00). The incidence f chrmsme 6 aneuplidy can be estimated frm the abve analysis by using the fllwing equatin: percent aneuplidy = (age). Other chrmsmal anmalies fund after flurescence in situ hybridizatin analysis in series and cmbined are shwn in Table 3. In brief, 5.% f the develpmentally nrmal, 5.3% f the slw and/r fragmented, and 59.9% f the arrested embrys shwed chrmsmal abnrmalities. The mst cmmn anmaly fund in develpmentally nrmal embrys was extensive (~38% f cells) msaicism (.9%), fllwed by aneuplidy (.%). In cntrast, plyplidy-haplidy was the mst frequent abnrmality fund in arrested embrys (37.%), fllwed by msaicism (7.%) and aneuplidy. (5.7%). Slw and/r fragmented embrys shwed msaicism as 5 Benadiva et ai. Chrmsme 6 assessment their mst frequent anmaly (%), fllwed by plyplidy-haplidy (6.%) and aneuplidy (3.%). Preimplantatin Genetic Diagnsis f Aneuplidy The new flurescence in situ hybridizatin prtcl used in series was applied t five patients underging IVF wh cnsented t preimplantatin aneuplidy assessment. Findings in these five cases are summarized in Table. Of 37 embrys analyzed, results were btained in 36 (97.%). Twenty-five embrys (67.5%) revealed a presumed abnrmal chrmsmal cntent, and 8 were transferred t the patients. Amng the transferred embrys, ne bipsied cell analyzed fr preimplantatin genetic diagnsis was tetraplid, and five ther embrys, all frm the same patient with n ther nrmal embrys available fr transfer, were mnsmies 3 r. The patient with nly mnsmy 3/ embrys replaced did nt becme pregnant. Of the ther fur cases, ne has an nging singletn pregnancy. DISCUSSION Chrmsmes X, Y, 8, and 3/ can be assessed by flurescence in situ hybridizatin in a reasnable time frame cmpatible with regular IVF prcedures (). Chrmsme 6, hwever, culd nt be detected with the previus prtcl because f the limited number f flurchrmes available. This prblem was slved by "recycling" previusly analyzed cells fr five chrmsmes, washing-ff thse prbes, and reanalyzing the cells with a prbe fr chrmsme 6. The technique f cell recycling with chrmsmespecific prbes was applied previusly t a small grup f blastmeres that were reanalyzed fr chrmsme 8 (9). Mre recently, Thrnhill et al. () have applied the same cncept t cmbine flurescence in situ hybridizatin fr chrmsme analysis and plymerase chain reactin fr detectin f specific mutatins in muse blastmeres. Anther apprach that has been prpsed fr preimplantatin Fertility and Sterility

6 Table 3 Summary f the X, Y, 3/, 8, and 6 Chrmsme Abnrmalities Fund in Mrphlgically and Develpmentally Nrmal, Slw and/r Fragmented and Arrested Mnspermic Human Embrys' Flurescence in situ hybridizatin results Develpmentally nrmal (n = 9)* Slw and/r fragmented (n = 68) Arrested (n = 35) A Nrmal* B Msaics and/r multinucleated blastmeres «38% f cells): C Aneuplidy (%) Trismy 3/ Mnsmy 3/ Trismy 8 TrismyXXY Trismyltetrasmy 6 Mnsmy 6 Mnsmy 6 and 3/ D Aneuplidy and msaicism (%) Trismy 3/ and msaic (:38% f cells) Trismy 8 and msaic «38% f cells) Mnsmy Y/O and msaic (:38% f cells) Mnsmy 8 and msaic «38% f cells) Mnsmy 6 and msaic (:38% f cells) Mnsmy X and msaic (:38% f cells) Mnsmy 6, Trismy 3/ and msaic «38% f cells) E Plyplidy-haplidy (%) Plyplid and trismy 8 Plyplid and trismy 6 Plyplid Plyplid msaics Haplid Haplid msaic F Msaics and/r multinucleated blastmeres (:38% f cells) Ttal aneuplidy (C + D) (%) Ttal extensive msaicism (D :38% cells + F) (%) Ttal detrimental abnrmalities (C + D + E + F) (%) 3 (36.9) 3 (.) (0.6) (6.) 5 () () (3.) (5.7) (7.) * Values in parentheses are percentages. genetic diagnsis f aneuplidy is the analysis f the cyte's plar bdy befre fertilizatin (5,6). Such technique has the advantage f nt being susceptible t misdiagnsis prduced by msaicism. One imprtant limitatin f the methd is, hwever, that it cannt identify paternal aneuplidy r ther chr- Table Summary f Findings in Five Patients Underging Preimplantatin Genetic Diagnsis Age y N. f embrys bipsied N. f embrys transferred 3 * 5t * One tetraplid embry. t All five were mnsmy 3/ embrys. Vl. 66, N., August 996 Reanalysis f nntransferred embrys = Trismy 3/, mnsmy 6 = Mnsmy 3/ 3 = Msaics (;;,,66% abnrmal cells) = Duble Mnsmy 3/ and 6 = Triple Mnsmy 3, and 6 = Trismy 8 = Cmplex msaic = Mnsmy 6 = Tetrasmy 6 = Trismy 3/ 3 = Msaics r mnnuclear blastcysts (;;,,75% abnrmal cells) = Undiagnsed (nt reanalyzed) = Nrmal msmal abnrmalities (i.e., plyplidy-haplidy) that in the present study accunt fr 3.3% (6/95) f the abnrmal embrys detected. By applying this new prtcl, we are able t detect the six mst cmmn chrmsmes knwn t be invlved in aneuplid ffspring and spntaneus abrtin. Thus, preimplantatin genetic diagnsis can nw be applied t avid transfer f aneuplid embrys, thereby decreasing the risk f affected ffspring, particularly in the ppulatin f wmen ver age 35. In additin, preimplantatin genetic diagnsis may cntribute t bviate the issue f abrtin f clinically viable pregnancies, with the assciated psychlgical, mral, and ethical issues that the prcedure entails. Applicatin f this technlgy als is relevant because aneuplidy f chrmsmes X, Y, 8, 3, and in early embrys is manyfld higher than that detected in clinically recgnized pregnancies (7). In the present study, aneuplidy fr gnsmes and chrmsmes 3, 6, 8, and ccurred in 6.9% f all embrys, with 55% f thse being mnsmic. Cmpared with aneuplidy rates detected in spntaneus abrtins, ur results are strikingly different fr tw reasns. One, mnsmy is as frequent as trismy, which wuld be expected if indeed nndisjunctin is the main mechanism f Benadiva et al. Chrmsme 6 assessment 53

7 aneuplidy. Hwever, with the exceptin f mnsmy, with an incidence f per,000 karytyped abrtins (), the rest f autsmal mnsmies are practically undetected in clinically recgnized pregnancies. Because nndisjunctin prduces as many dismic as nullismic gametes, mnsmic embrys must be eliminated during the first days r weeks f pregnancy, as was demnstrated in the muse (8). Mnsmic muse embrys begin t die apprximately day 3 t day (late blastcyst stage) depending n the mnsmy and very rarely arrive t day 6 (pstimplantatin) stage f develpment (9). The ther difference between preimplantatin embrys and spntaneus abrtins is the higher rate f trismy in cleaving embrys (3, ). Because the rate f chrmsmal abnrmalities in newbrns after IVF is similar t the ne fund in the general ppulatin (0), these findings suggest that mst trismies als are eliminated befre clinical recgnitin. This cnclusin is supprted by previus wrks (8, ) in the muse mdel, which fund that mnsmic muse embrys disappear very rapidly during early embrynic stages, whereas trismic embrys survive lnger, althugh they t becme eliminated befre the end f the pregnancy. Indeed, althugh trismy 6 is the mst cmmn autsmal trismy nted in prducts f human cnceptins, there are nt reprted cases f live brn human ffspring with trismy 6. Thus, if there shuld exist a limitatin t the number f embrys that can be replaced safely, preimplantatin genetic diagnsis f aneuplidy may increase the prbability f achieving a viable pregnancy by aviding the transfer f embrys with a lw r null prbability f arriving t term. The flurescence in situ hybridizatin prtcl described in this study was applied successfully t five patients underging IVF wh cnsented fr preimplantatin aneuplidy assessment. Of 37 embrys analyzed, 3.5% revealed chrmsme 6 aneuplidy, suggesting that the additin f chrmsme 6 assessment prvides valuable infrmatin, especially in the lder age grup f wmen. The secnd gal f this study was t determine the maternal age effect n chrmsme 6 aneuplidy at the embrynic level. Althugh trismy 6 has been shwn t increase with maternal age in clinically recgnized pregnancies (3, ), this increase culd nt be shwn in cleavage-stage human embrys in a previus study because f the small sample size (6). Hence, by cmbining thse results with the present nes, we were able t demnstrate a significant increase f chrmsme 6 aneuplidy with maternal age. Of particular interest is the bservatin that trismy 6 had an incidence f.53% in preimplantatin embrys (3/95), which is very similar t the estimated incidence (.3%) in all clinically recg- 5 Benadiva et ai. Chrmsme 6 assessment nized pregnancies (3). This cntrasts with aneuplidy rates fr trismies X, Y, 8, 3, and, which are much higher in preimplantatin embrys than in clinically recgnized pregnancies (7). It is cnceivable that a large prprtin f human trismies may nt implant r they abrt in the very early stages f gestatin, whereas thers, such as trismy 6, survive lng enugh t be clinically recgnized. Nevertheless, apprximately 80% f the aneuplidies fr chrmsme 6 were mnsmies, which never are detected clinically. These results in embrys cnfirm data frm spntaneus abrtins suggesting that, in cntrast with ther chrmsmes, chrmsme 6 aneuplidy increases linearly, and nt expnentially, with maternal age (). The different rati f mnsmy t trismy fr chrmsme 6 when cmpared with ther chrmsmes supprts the existence f a different mechanism invlved in the rigin f aneuplidy 6 (3). In additin, there is nw cnsiderable evidence that a significant prprtin f trismy 6 cnceptins can autcrrect and prduce a karytypically nrmal cell lineage. Frequently, the nrmal cell line will frm the fetus, leading t a discrepancy between the chrmsmal cmplement f the fetus and its placenta, knwn as cnfined placental msaicism (, ). In ne third f cases, the diplid cell line is expected t have uniparental dismy (the derivatin f a pair f hmlgues frm ne parent) fr the chrmsme pair invlved in the riginal trismy (5). The ccurrence f this event has been assciated with pregnancy lss, delivery f severely grwth-retarded infants, and ccasinal late fetal lss (). In cnclusin, this study demnstrates that preimplantatin genetic diagnsis fthe majr human aneuplidies (X, Y, 3, 6, 8, and ) is achievable within a time frame cmpatible with IVF. The high prevalence f mn s my 6 (80%), which is nt fund in spntaneus abrtins, suggests that mnsmy 6 culd be a factr assciated with failure f embry implantatin. A larger grup f patients shuld be evaluated t test the hypthesis that the pregnancy rates and/r the miscarriage rates can be imprved by applying preimplantatin genetic diagnsis rutinely fr embry selectin, especially in lder wmen. Acknwledgments. We gratefully acknwledge the assistance f Fred My, Ph.D., fr his cllabratin in the statistical analysis. We als acknwledge Jacques Chen, Ph.D.; Zev Rsenwaks, M.D.; Jamie Grif, M.D., Ph.D.; and William Ledger, M.D., fr their supprt f this wrk. REFERENCES. Munne S, Lee A, Rsenwaks Z, Grif J, Chen J. Diagnsis f majr chrmsme aneuplidies in human preimplantatin embrys. Hum Reprd 993;8:85-9. Fertility and Sterility

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