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EXPE:R:~ AN; TO An: She OF EN I~VEIO~L Fit ~ IN ~5CH . . . Jo VAN 11[= EM AL ~ ~ ]HE: AN . . . . Cane of the ~ unfortunate facts of hen repro is Bat Be majority of normally fertilized eggs will net develc~ into a vinier tire pro Retards, 1986~. quit occlusion is sty by the relatively law rate at ~id, an ongoing pro is achieved after in Vito fert;1 ization and Irn~ltiple echo transfer at exert IVF preys (between 1# are 25%: Liu et al, 1988; Gibbons, 1988). Ike values are derived fan a typical pcpulation of infertile couples that present with a variety of etiologies (male factor, female factor, or Achy. Not infrequently, the failure of fertilization ~ Nor or of <3evelc~xnent to persist can be attrih~ ~ abnorne~1 immunological or endocrine patterns, or to ~r~ologir~.1 Notions in the structure of the uterine ppithelium. Lo, c~lymnis" Berm, whim include sum factors as low count, aberrant or reduced matili by, and 2 ~ nra1 morphology that results from spermatogenic or maturational dysfunction, can influence both the frequency of fertilization and the prognosis for postfertilization em,bryogenesis ((Bacoetti, '983; Zamboni, 1987~. While certa ~ classes of reproductive dysfunction can often be oorrecbed by relatively simple means (e.g., intrauterine insem m ation, sperm antibody suppression) ~ more complex surging or endocrinological , or both, intention is often rosary. However, surgical and endocrinc~logi~1 approaches are fitly precluded because of severe Olivia, or as a result of blocked, damaged or agents fallopian to. In this "se In writ fertilization Is indicated. Hay Die vast majority of embryos created by in vitro fertilization fad] to establish a pry after transfer to ~ uterus is: ye of the Seal Dart questions in ye application of this trilogy to human infertility. miS is especially relevant when multiple early cleavage Encage errs that appear normal at the level of ye light m~c~ are transferred win t the detectable ~ of implar~ation. C'n sore occasions, endocrinologist, inn~ologi~1 and uterine Apologia factors can be identified as contributing to the failure of implantation. For m~stenibryos transfers, hammer, itis very difficult ~ ascribe a specific Season for the fails. - 234 -

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one approach to an investigation of =;`rly devel~al fa; flare hen rLion is to ask whether ache inability of an once to be fertilized, or of an `~Tibryo to oc~ntinue ~velc~ or to implarrt is: an Detent rather than an Anal ina~uapy or pysfur~tian, that is to say that ~ devel~al capacitor of an oracle or embryo is limit" from the Am. this Lion is of particular relevarx~e An, in He ~ of spermatozoa of known fertility, penetration of sam or all of the normal ~earir~, icily mature opiates curtains fen hy~ti~ated },uman ovaries fails to occur. If fertilization, or not <3evelc~: ~ ir~G*ly limit In the human specie=, then to ~t degree might this durir~ both In vivo am In criers> ads to achieve a Prague? ~ Sony of human Axes and early errd3~0s Squires clew' arm for Aid analyses Eat abbess questions which ll be Airy of clinical relevance In the breast or Nadirs of He causes of infertility. He TV; of aralysE~; did In this paper fads on, two such a ~ of Any human development. He fir ~ type of analysis addresses the question of the degree of chro~oEcrul abnormality ~ the preovulatory ~ e. Some investigators have indicated that more than half of all p ~ atory oocytes produced by ovarian hyperstim,lation are chrcrLson ally aberrant (see Table 1~. If this is indeed the prevailing situation, then attempts to achieve a pregnancy, and more importantly a normal pry, may be cruised even before sperm and cxx~:e nut. ye following two questions also arise face ache analysis of the Lhl~1 Stately: of ye pre7vulatory acute: (1) is the fry of Cal aberration higher in Glen whose ouches persist fail to fertilize in vitro, and (2) Is the o Our ~ e of chr ~ ally abnormal oocytes more patient specific than a ubiquitous and obligate car sequence of exagencus ovarian stimulation? With some ncted exceptions, the fi ~ related to the chr=moEcral status of human oocytes are derived frum the examination of occytes that fail to fertilize after Insemination in vitro (~e Table 1). The second type of analysis focuses on the failure of newly fealty ized eggs to progress in development. ~~ rly studies of embryonic development in viva by Hertig, et al (1954) noted that in humans an urexFectedly high pro portion of embryos arrested development prior to the blastccyst stage. Mbre recent studies by Euster et al (1985) described a very high fregwenoy of embryonic demise during the preimplantation stages of in viva development in we men undergoing ovarian hyperstimulation and intrauterine insemination for subsequent embryo transfer. Early developmental failure is clearly evident ~' ring in vitro culture, where approximately 10-15% of the normally fertilized human eggs actually develop to the blastocyst stage (Fisher et al, 1985: Lluden berg and Hytell, 1988 ). Even for blostocyst-stage embryos, however, the normality of develop is often Actionable owing to suspec teed quantitative (inappropriate ~11 number) and qualitative disorders (e.g. multinucleate Ills) in He Car cell my art tr~ph~erm (ester et al, 1985; Edwards, 1986; Lir~g art - 23o -

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~tell, 1988). what cleavage arrest concurs with similar fr~ies in IVF Cycles that involved differs Ovarian sti~ati~ propels are a,Jtuna n - Ha its that devel~tal failure is ~r~relat~ rather Ian a ~ of how cues are general or the nag of the Orient ~ which ibryos are fertilized are Grain. Collectively, a considerable body of evidence irxlica - ; gab even when a noun ferL;1ization has c~, whether in viva or ~ vitro, develc~t to the stage of implantation is by no mans assure. When, shy, are to what extent duly develc~nenta~ arrest is mediated with me In specie is one of the f~an~4cal issues in ur~tar~ir~ In Rice. Be of Ye erg, ethical are potential legal ramifications that al the analytical or ~peri~tal t~ of presumably normal zygote are preimplantation e~ib~os, stay of early human develc~t usually involves - 3ryos that (1) are derived fme anally fertilize ~; (polyspermic), (2) clearly develc~p in an art pattern, arx] (3) have unambigualsly ~ to press in vitro. In spite of the restricted nature of "analyzable specimens," a considerable ant of information relevant ~ normal are abnormal clevel~nt=1 pries has Yen gig. As discussed belay, scare of these firings not only indicate shy develc~nt fails but also provide insight into ff e Ire sale origins of infertility. E~CY OF HAL ~ ~ HEN Cx'C~ AfTER OVARIAN ION Table 1 prompts Current estimate; of the frequency of ar~ploidy (hyp~aploidy and hyperhaploidy) in meiatically mate human oc~te~; that were Oryx after failed fertilization. It is aunt that these values vary widely face a low of 11% to a high of 65%. In Imprison to other studies, the extraordinarily high rate of aneuploidy reports by W~by are Saga (1987) appears to be an arerestimate that may have resulted fr - ~` ache curatively law nurser of go; Omit, fig patient population included in the study are, for the hypc~aploid ==i~, perhaps freon artifactual 1~= during preparation of ~ oodles for cue ~antitatic~n (Pladh~ et al, 1988; ran Blends ax Henry, 1988~. me reports of Bongos et al (1988), Pell~tor and Sele (1988), Plachat et al. (1988), Van Blerlc~n arc] Henry (1988) and Van Bler)cam (1989a) provide a frequent of aneuploidy that is fairly axis ax is derived If the analysis of nearly 1000 occur; Hairs Dun hyp~im~atec! ovaries. At present, it ~1d appear that between 20 arc] 25 Esprit of the ~iot:i~lly mature cog; Hat are Driver once stimulated ovaries are which fail to fertilize in vie are Anally z~r~r~c. Ihis value appears to be a Extant that is irk of the pruta~ol of ovarian stimulation (Planet et al, 1988; Van Blerkan, 1989a). - 236 . ,

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A signified limitation ~ the ~nterpre=tion of Me above fire that the fregu~cy of ar~loidy is derive alI=St entry few ~ e~ninatic~n of I; Cat failed to fern ize. It may be reasonably argued that sigh ooc~ are already cat, art in such a It predation of c~; Cal armies may be cry one manifesta~cic~n of a devel~ntally There state. In Nation, Van f3;1erl~ (1989a) ~~ that the cation of Nether ~z~1 ~r=tic=; are an Avoid construe of the stimulation and ~re~q~ of multiple follicles is difficult to antler at present because We baseline I: - of ~ te ar ~ ploi~y that ooours in natural (unstimulat~) cycles has not been determined for a large and clinically varied population. However, one approach to obtain this bacigrcund Blue is to determine the fregoency of chrrmosomal abnormalities in oocytes that matured in vitro from the GV to MlI-stages. The most extensive analysis of in vitro matured human oocytes was reported by Jagiello et al. (1976~. These investigators detected only 6 chromoscmL1 anomalies (1.5%) in 411 MII-stage oocytes that had resumed meiosis spontaneously in vitro after collection at the GV stage. this finding tends to suggest that the current protocols for the remitment and cle~rel~al station of multiple follicl~/c~ces are mediated with an elevate frequency of aneuploi~y. Anc~h-' approach to the question of the beck grounl frequency of aneuploi~y ~ to determine the chrcncsomal status of meictica~ly mature oocytes obtained from the ovary and prepared for karyotyping immediately preceding the anticipated time of ovulation. W~ameby et al. (1987) noted that approximately 50% of the meictically mature oocytes obtained ~ this fashion were aneuploid. By contrast, Van Blerkom and Henry (1988) reported that 11% of normal-appearing preovulatory (un inseminated) oocytes were aneupoloid. As discussed previously, the extent of aneuploidy indicated ~ the studies of ~ y and his collaborators appears to be an overestimate. However, while the frequency of aneuploidy reported by Van Blerkom and Henry (1988) is approximately half of the frequency described for failed fertilizations (20-25~), it ~ nevertheless significantly higher than the 1.5% value reported by Jagiello et al (1976) for hen I; Stained frc~unstimulated ovaries, and~i~h ~pontar~sly z~ and depleted amidic maturation entirely in vitro. mile it sums likely that ovarian hyper:;ti~atic~n is mediated with an elevated frequency of aneuploi~, a reacher Eric question, that has not .~n fully a~r~ is whether such an is genetic risk ~ an unavoidable consequer~ce of attest to gestate multiple oomph, or whey Ply adamant I; are Are likely to be pati~ific. He notion that a rather Sal Ink of In account for a di~pr~ortionately large share of Ally art of was a Air ~ on a limited hasps (163 w ~ n) in the s ~ y of Van Bler ~ and Henry (1988~. these investigators reported that nearly 27% of the hypohaploid occytes and 33% of the oocytes that exhibited chroncEomes not associated with the metaphase II spindle (i.e., potential aneuploids) were - 237 -

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derived form too patients during single attests at IVF. Incus, the f~ with wilily ly abnolIIlal ocher; may Our, or may be anticipated after Ovarian hyper~;ti~lation, may be difficult to estimate with red ~ a papillar indivi~. she At anbigui~ associated with ~ rink of ar~loi~y after Ovarian Estimation ~d be resolved through a drawl, nulti~er analysis of axe;, both in the living state and by l~ry~pir~ after tn~ive fixation. i; from both felled fertilization arm under ci~tan~ He a truly emotive comber are available for fertilization, arm Here embryo cry~pr~vation is not an option, ~1d be USA for this purpose. Ibe report of Plait et al (1988) tic He rats of such a multiple - r effort. In this rat, Van BlerXr" arm Henry (1988) cles~ib~ a method for ode Genetic evaluation Hat cad be performs on living is which failed to fertilize or fertilization was ret urxiertak~n. lhis method entails the t~ of font, Oral INA stains. Not only were aneuploid Abates detente by this prone, but also of; that exhibited major disorders ~ or =m struck ark ass aviation with the mete spindle were Red. such Bytes pliably contribute significantly to the Avery frequency of anepploic~. A fused effort to examine the Genetics of ox; that wax otherwise be ctiscx~rded, first In the living state arm subtly by conver~tional lcazyotyping methods, wax go a long way to definitively establishing (1) ache freq~y of aneuploidy in stimulated cycles, (2) whether it was patient ardor age related, arm (3) whether specific protocols of stimulation Acid be Are appropriate in order to Deduce the risk of generating a genetically abnormal c~ytefs). me production of an anepploid opiate is not a trivial Horn because it cannot be assay, a priori, that such an octane Is either unfertilizable or, if fertilized, Is irritable of i~p~an~cation. Gel, en triploid eribryos that rat fun disc penetration are capable of apparently normal prei~plantatian develc~t (Van B1erkam et al, 1984) arc] can develop to term on Rae Abrasions (Wertel~ki et al, 1976~. In this regard, Van Blerkam awl Henry (1988) Semi the car =al state of botch ~~ arc] unfertilized mattes Stains fmrn Karen whose Go;, on multiple attests at IVF, per~;istently failed to fertilize in ache presence of ~pennatozoa of Lee fertility (both husband arc] donor). me fr~uen~y of stn~ral aberration arc] aneuploidy was no different from that cued ~ IVF cycles (for different patients) Hen only scam of the ~ioti~lly mature Go; failed ~ fertilize. This firmed suggests that failed fertilization may not have a direst Gal Radiation. py Gash, structurally aberrant ocean;, such as them in whim one or Are d~r~s have Gene detadh~ from the metaphase ~pirx31e, shallot be fertilizable, especially if all ether aspects of pr~vulatory maturation have been cxfeted su~=fully. In this Parr, Ar~ell et al (1983) Ported that a Other high pedant of cleavagff;tage embryc6 promos by in vitro fertilization are Galley abnormal. C>ne Interpretation of this ~t Is that sure of these genetic anomies preexist In an ooze 238 ~

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prior to fertilization. Cl - ply, the extent to which an ideas ~ of genetic ~ur`tion that can Ruse prep or Arty Hilactation develc~ Is associate with ovarian hy~ti~latic~n As to be determine with Ad; Cat are r~i~y available arm, for m}, Icy dot. GIL ~ 1X~G am: ~rZP=CN H:RrOD File fertilization in vita of icily Stud human Ax; Staid Frau hyperstimulated ovaries occurs at a relatively high ~ (typically by 60 and 80%), mast of these zygotes will argot develc~nt during the prei~plantation stages (yards, 1986; Van Blerkam, 1988b). she perifertilization period is a particularly cxiti=~1 stage of develc~rent bemuse a Aries of cellular and Rear charges rear on an ~:~eper~ent, progressive pattern in order for the zygc~e to initiate arm complete the fit medic or cleavage cliv~sic~n. For example, major cellular change= involve the cortical granule reaction, i~ration into the oopla~rn of the fertilizing spermatozoon, a}=triction of the sec~rx] polar body, arm formation arm translation of prcnucl=. some of the major molecular changes ~-=cn~-iated pith this stage of development are Recondensation of the sperm chromatin, replication of DNA, and differential modification of male and female genomes at the prc nuclei stage (generic imprinting; Surani, 1987~. Developmental failure at the 1-cell stage Is of particular interest owing to the importance of ache sulfur cc~letion of each of these prod for it ebbryogenesis. Contently, much of air analytic efforts have been did to human de~rel~rent=1 failure at the 1 cell stage (for Neigh Van Blerkan, 1988~. After closer inaction by high-resol~ion, differential i~rferer~e trash Ad, approximately 5-10% of the Ax; that were thought be unfertilized did index have one or more Term in tile perivit^1 fine apace (Van Blerkc~n, 1988b) . At the electron ~cr~pic level, a feature typical of these opiates is the absence of birding between the Germ and Axe m~crovilli- -ache first shy In the actual process of fertilization. Ibr such Axles, three fw ~ Cal a ~ s of s ~ m ~ te it erection may be abnormal. First, microvilli-ascrciated ~11 surface glycc proteins that recognize spermrassociated surface proteins (or vice versa) may either be absent, altered, or distributed abnormally. Therefore, although the juxtaposition of the gamete= appropriate for attachment can ~ or, the mol-~,lar interactions necessary for incorporation of the fertilizing spermatozoon may be at sent or inadequate. This particular hypothesis is Katie at present because information concerning the biochemical and spatial nature of the m-11 surface mulches involved in the fertilization process is lacking for the human oocyte. Clearly, an understanding of the temporal and spatial aspects of the first molecular interactions between human garret Is a Rudy prerequisite to det~rmini~ whether, for sax LOCI, subtle biochemical versions are responsible for velc~nta1 failure. - 239

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A so po~tial camp of Me failure of ~ Mae gauge to Irate ~ of may be associated with the organizatic~n of cortical Iota elf;, sum as ~crofilarents. Akin filaments nat fly Pride the stn~1 support for Me elaboration of m~crov;1li. ho also aEar to be clammy involved in the ~tili~, of the cortical ~topla~n during the pa; of Form penetration (for review, ~ Van Blerka~, 1988~. Gently, an armnaly ~ either stratum, organization or distri~ticn of these eler~nts may preclude ~ per~tratian of the fertilizing spermatozoon aft a probably norm atta~nt to the He surface. Although Hailed information Stairs Me Hal o~zation of ~ ~~ humn oogybe is labia, a few Pies have Then the i~ortar~e of Me spatial distribution of actin filaments for human octane devel~nt ark fertilizab; ~ if. Sate et al, 1985 described the presence of a portico bark of ~crof;1aments ~at, in the G7-~GVB (germinal v~-icle~og~ni~ vesicle }>real~awn)ustage human Gate, a~iea:c~d to provide a p~ysir=1 barrier that prevent the premature deposition of cortical gramales ~ the subplasmal~1 cytoplasm. these ir~restigators suggests that Me develc~rent of such a barrier preludes a prelature (preavulatory) cortical Faction that wood make an He refract to sperm penetrat~c~n rnr~ one zone pellucid ~zona reaction) after ovulation. Van Blerk~n ark Henry (1988) arx] Van Blerkam (1988) Gibed an armnaly in the stnlc~ organization of the ~rt~x that was Fiats winch the failure of fertilization in gently nor~l-a~aring, ioti~=lly mature Han odes. In this situation, nearly the entire call surface was devoid of ~crovilli, are, in the majority of the s~lasmaler~nal Guam, cortical granules, files arx] acorn filaments were agent. By contrast, all of these Cons were present at high density in nag region that }carried the portion of the cue frown which the first polar body had been apprised. Me o~rzerce of this ~er~type in meiatically mature pr~vulatory Bytes d~strated that it did not necessarily develop as a consequer~e of Ally or in intro Ire to spermatozoa. Nearly all of the locates that displayed is pattern of Cortex polarization were deserved in won Hat had a history of idiopathic infertility, arm ~~- axis failed ~ ferriage after multiple attempts by in vitro methods. While the precise origin of this Genie is not Con, indict eviderKx suggests that it arises *tiring the stage at Sigh the first polar body is Strict (Van Blerk~n arx] Henry, 1988~. the polarization of the cellular ark Liar ccnags ir~rolved ~ the fertilization process 1d be a significant factor ass aviated with per~;istent fertilizatian failure ~ this patient Plato. A clear indication of when baring Lenses this abnor~lit~r may develp d be Rived from He analysis Of - fully~n but meioti~1 ly imna~re cxx~;, Staid for fistic pus fmn wan in which this cor~ition has either been pr~ialsly identifier] or is oust. Under these ci~tarx~:, oo~ Maid be Emil at the germinal vesicle stage and at ti~ int~ls during Biotic maturation in vitro (Van Blends, 1989b). me defiled elongation 24Q

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of ~eE; fen wc~n witch persisted f~ilizatic~n failure, ad. es~i~lly whom a male factor has teem precluded (as far as Visible), id indicate the fry with which this tape of ~fertili~hle axle of; sixth ~ Estimated (in vitro caddy and stipulated cycles (whiff, arx] without He hangar induction of ovulation). the rearganizatic~n end polarization of ~ cortical Cytoplasm arm plasma membrane ~ also viewed as a P~*.= ~vel~ren~cal and ~1 1 biological pi. An upstairs of the mc~lea~ar and cellular ~i~ by whip this Rich By; amid provide f~1 insights into how Rectal shapes in He stubby arc] organization of He cortical pytopl~;m arm plasma membrane lead to the attainment of the fertilizable state ~ ache human species. ]0 cipher cellular conditions associate with pert-fertilization failure In ache Han Cat are often Perfect after imagination In vitro are (1) ant or in~lete recondensation of ~rmatozoal by, and (2) ir~lete formation, migration or juxtaposition of prunuclei that Is Reid for Myra (for details, ~ Van Blerkam, 1988~. me failure of sperms decondense Lobe ascribe ~ a stat defy Ante pa~ging or organization of During ~rmatogenesis. ~r-studies have attests to reduce this possibility by focusing the analysis on thee develc~nt~lly at 1~11 embryos that are derived fee grossly normal-a~arir~, ~iotin=]ly mature opiates in#lr~with~perm with rx~ l morphology, Utility and of established fertility. In many of the ~ that have been Gamin, routine light microscopic inspection suggested that fertilization had not~rr~. Asa st~ardpr~:ol, these putative ooc~tes were stained for =~ ~ arm Omit by floors m~cr~py (Van Blerkom et al, 1987~. For Proximately 5% Of the of, fertilization was indicated by the pr~enoe of a sperm head within the cytoplasm. Electron mid revealed Berm He in variants stages of dec:orxlensation (Figs. ~ and 2~. Hover, in rune of He Us Did was the state of deoondensation Arable to that typically observed in a rx)rma1 h ~ n egg (Van Blerk~n, 1988~. For hu m n eggs exhibiting this type of "ally develoFmenta1 failure, it ~ difficult to eliminate completely the possibly ity that the fertilizing spermatozoa contained suckle defects in DNA packaging. With this caveat noted, work f'"u animal species (for review of pronuclear development, Doe LDngo, 1985) suggests that for normal development to occur, the cytoplasm of m ~t mammalian species ac ~ s the ability to Recondense sperm DNk during the latter stages of preovulatory maturation. This able ity appears to involve the appearance or activation of cytoplasmic enzymes and/or other proposed "factors" that rapidly pro mote DNA decondensati~1 Masai and Clark, 1979; Iongo, 1985). The varying degrees of ONE decondensation Observed 1nsemunated human oocybes may be a manifestation of an absent or inadequate bioch~n;~l zbiJi~ of the mature An c - lawn to interact add - riat^1y with An A. He cellular and m'1eadar bioicgy of this interaction r~r~s Bather area of early Sian develc~nt where available experiment information is of a limit r~a~cure. For exile, the precise number and identity of the bioc:hemic~1 factor:; Dialed with d~ndensation have not been established definitively. It, weeder - 241

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Cart ~ He sty of ~ In in the region of Be pene~cra~ Berm are r~ui~ for, or Dial with ~ Nation or p~CI-:~~ formation, or Ah, have not been detcrmi~. A c~r~hensive urxi~st~ir~ of the cellular art Par pro involved in Berm IN decor~sation art prorn~cl~r formation am clergy a pre~isite to begin to ~q?~er~ why ~K? penetrated In Dies fall ~ prier;s in develc~nt. It is also Dot At Rhine light mimic lion may not realize the situation ~ an He is it penetrates] but aces not form pmr~lei. Be fray with whim axles are penetrated but fa;1 to initiate or Poe r~ensatia~e prowar formation is urchin for the Amen species. Ibe Dun of this type of Arty devel~tal failure may be especially relevant in ~ersta~i~ the subtle origins of infertility arm consequently its Dry Child be correlated bath With the prow of ovarian hyperstimlllat~on arm the prior fertility/infertilit~r (e.g., idippathic) history of the cope. If On d~nd~sation and male pronucl-=' formation have Pro, the next major devel~renta1 landmark is ache migration and jeans - ition of the Nuclei. Nuclear juxtaposition art ache subsequent brown of the pr~rucl-=r Manes is a necessary prerequisite for the association of the materTa1 and paternal ~ = s at Do. A representative six of normal pranucl~=' Yarns is On in Figures 3-6. Based on studies fmn other Annals, it is Ed that replication of Natal ~ occurs in the human Purina Me r~rarn~clear stnaes that _ ~ . ~ . ~ ~ . ~ _ ~ prime ~uxtapos~tlon. Alar me Telethon or stray arm the formation of the mitatic spindle, the first cleavage division, usually cxx~rs within hairs. A relatively small per ~ nt of fertilized human e ~ s (less than 5%) arrest develcpment during the pronuclear stage. While an occasional egg 11 display pranuclei that have formed but fail to mlgz-ate, the majority of ega,s with this p enotype arrest development with nuclei juxtaposed. Electron microscopic studies indicate that the changes in prone clear membrane geometry that typically precede dissolution do not never in eggs arrested with juxtaposed pranuclei pVan Blerkom et al, 1987; Van EDerbom, 1988). Clearly, the dissolution of the pronucl-=r membranes ~ of fundamental importanoe in the formation of the zygote, becalm=- ~ shout this event the maternal and paternal chroncsomes remain in separate ccr}artrents. Chile the causes of developmental failure at the pronucl~ar stage are not r- - ily apparent, the analysis of fertilized ~ that arrest at this stage of de~relc~nent ~d provide basic insight into the Anises arm differentiative pro that establish oorxlitio~ permissive for Be actual joint of parental genres. ~ the human, to investigate the Ear and ~1 lular factors; iTrvolvec] in pronucl-=r formation chairs normal develc~arent golly roared invasive analyses that weld destroy the rawly fertilized egg. by contrast, pronuclea~ arrests ego; may be - 242

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file Midair for analysis ~ it is apparent mat develc~ent has truly cad. Van Bleary et al (1987) ~;~ a pries of ~r~c events involvir~ tire Charles In nuclear geometry are r~leolar distribution that were correlated with ark predictive of it detrelc ~ nt arx] devel ~ tat potential (~e also Figs. ). With this basis, analysis of arrested embryos can be undertaken with reasonable assurance that a particular egg is indeed nonprogressive. one relatively simple question that we have asked with respect to prcnuclear-arres ted human embryos ~ whether DAN replication had occurred in one or both pronuclei prior to juxtaposition (Van Elerkaml 1988). At present, semiquantitative chrlnosccal fluorescence has been amp for 4 eggs to assess whether or not replication had oocurreS. While thi_ particular approach ~ useful for app Mismating ploidy, it is not of sufficient sensitivity to determine the absolute amount of DNA (e.g., whether a particular chrcnrec~e is absent). The demonstration of haploi~ An the male and female pronuclei of 3 of the 4 eggs indicated that `wlication had failed to Our or was incomplete. Apse of Be 1 Or of pr~nuclear As Amid, these results are preliminary. However, they Suggest the pcssibilit~r that the ability of the pramcl~ar membrane to undergo prc~ssive Cartes scat lead to fragrren~cation at pronucl-~r juxtaposition is associated with the cc~rrenx or camp~etion of INK replication. Perhaps such a Manic exists ~ ache human to prevent Lady when replication has failed to Or in one or both pr~rnaclei. ~ ethical questions attends to experimentation ~ normally fertilized human eggs preclude the types of analyses ark e~eri~al design that may provide a di ~ t ens ~ r to whether INA r ~ lication Is a prerequisite for pronucl-ar membrane dissolution. However, this question ~ one type of question that can be examined in model systems such as the mc use. Aphidicol~n is a potent and relatively specific inhibitor of the enzyme DNA polymerase ~ (Spardi et al, 1982~. If this inhibitor is present during the in vitro fertilization and subsequent Mature of 1-cell mouse eggs, pronuclPa' formation, migration and juxtapcsiticn occur on sale (Hawlett, 1986; van Blerkan, ur~ibliEhed d~;ervati~s). ~ever, in ~ Hence of ~ replication, ion of ~ pronucl~r membranes ark therefore Syrian do not char. she inhibiticn of replication ~ one or bath prunuclei with ultraviolet activates agents Bat cmsslir~c ~ is ark her e ~ ri ~ approach to determine whether an ass aviation exists between DNA synchs is and~prcnucl-a' membrane breakdown. Recent studies from my laboratory indicate that culture of newly forth ized Trace eggs in the presence of trioxsalen (4,5', 8'Trimethylpsoralen), a W -activated DAN crosslinker, develop to the stage of pronucl-a' j ~ sition, but neither fragmentation nor changes in the membrane geometry that typically pry pronuclF=' fragmentatian ~ lo. For the== studies, either male or female, or both, pronuclei were irradiated with a W micrcbeam. At this rather gross level of analysis, the aphidicol~n and trioKsalen findings suggest that inhibition of pronuclear DNA `~lication in the mouse produces a develcpmenta1 arrest that is phenotypir=1ly similar to the situation that is observed in newly fertilized human eggs. - 243 -

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Collectively, the above cations surest Mat the newly fertilized me egg may serve as an amp - riate Mel to examine sam of the ca,~(s) of specific devel~1 abnormalities awn to early In Irises. For example, what are We floral arc] Iliad arcs of organization are ~~atioaa with the p~v~ butane ~ normally relc~pir~ arm z~plicati~irhibit~ eggs? With such information available fmn anion system;, Prisons deco ache situation that axe; in Man eggs that arrest at this stage of development can be redly ad. S[~RY In this paper, the division of real and Martial devel~tal Formalities In oneness Fran Blercam, 1989b) arx] early human monogenesis was intentionally limited to the ~1 stain= of the pr~vulatory Forte arxl the possible origins of failure in the Indwell, presy~amic enbryo. these particular topics represent only a few of the chr~al and cellular Notions that are end In He preiTrtplantation human embryo (for a c ~ Prehensive Hi~ion, fee Van Elerkom, 1988;1989b). Other areas of human developmental failure that are currently receiving both analytical and experimental attention include (1) the association between the arrest of development at the 4-cell stage a the failure of the embryonic genome to become exuressionallY activated , _ _ , - ~ - ~ . (Braude et al' 1988; and Braudel this volume) ! (2) the process by which Idly human embryos became genetically mosaic, i-.e.-, develop a mixture of diploid and aneuploid mononucleate blasts meres (Angell et al, 1983; Van Elerkom et al, 1984), (3) the mechanism by which normally fertilized eggs, at surprisingly high frequency, develop mLltinucleated blastoneres during cleavage (Plachot. 1985: Tesarik et al. 19871. and (41 the etioloov of ~ ~ - , , #, ~ ~ - " abnormal inner cell mass and tr~ec~erm devel~: at the blasts stage (Linde~andE~tell, 1988). me areas of resorb discussed in this paper represent sane of the critical stages of early human devel~nt ~chat, be t~ subject ~ frequent and often anvils alteration, preclude the establi~t of a normal pry. me Inanition arx]-unde~ta~i~ of he sFe~:ific devel ~ tal st ~ in early h ~ n ontogeny be ~ perturbed are Cry for a realistic appreciation of the extent to which clinical protocols that arise frill heroic science findings (e.g., m~croinjection of spermatozoa, removal of awry pronuclei, twining, etc.) can be (or should be) applied in a meaningful and effective fashion to initiate a pregnancy that he= a reasonable potential of develcpment to birth. - 244 -

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Al This work was short by fats freon P~cive Genetic ~ vita, Ever, Oolor~do and by grams fma the National Entity; of Health (HO 21582) and the National Science Elation (RPS-8601231). REVEREND Orwell, R.R., Aitken, R.J., Van Elk, P.F.A., In, M.A. arx] ]~letan, A.A. (1983). an abnormalities in human crier - ; after in vitro fertilization. Nature, 303: 336-337. Ba ~ etti, B. (1984~. the human ~ matozoon, An Ultra ~ Lecture of Pr auction fJ. Van Blerkom and P. Motta, edit. pp. 110-~96. Martinus Nijhaff Publishers Boston and The Hague. Eongso, A., Soon Chye, N., Ratman, S., Sathananthanm, H. and Long, P.C. (1988~. Chromosome abnomalies in human oocybes failing to fealty ize after Onset nation in vitro. Human Repro. 3: 645-649. Claude, P., Bolton, V. and Moore, S. (1988~. Human gene expression between four- and eight-cell stages of preimplantation development. Nature 332. 459-461. Buster, J.E., Sills, M., Rcdi, I.A., Cohen, S.W., et.al. tI985). Biologic and morphoslogic development of donated human ova recovered by non-surgic~l1 uterine ravage. Am. J. Obstet. and Gyneco1. 153: 211-217. Edwards, R. G. (1986). Causes of pregnancy Len. Human repro. 185-198. Fishel, S.B., Cohen, J., Fehilly, C., Purdy, J.M., et al. (1985). Factors influencing human embryonic development An vitro. NY Academy of Science. 442: 342-356. Gibbons, J.H. (1988). Infertility: Medical and Social Choices. Office of Technology Assessment, U.S. Gov't Printing Office, Washington, D.C. Hertig, A.T., Rock, J., Adams, E.C., and Mulligan, W.J. (1954). On the preimplantation stages of the human ovum: A description of four normal and four abnormal specimens ranging from the second to the fifth day of development. Contrib. Embryol. 35: 201-220. Howlett, S.K. (1986~. The effect of inhibiting DNA replication in the one-cel1 mouse embryo. Rcux's Arch Dev. Biol. 195: 499-505. - 245 -

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Jagiello, G., I:ucayen, M., Few, J-. ant Graffeo, J. (1976). Acetic Ovations In Sian oozes: In is Stay (P.K. Pearson arxiK.R. Iewis, in). vol..5, pp43-63, ,Jcl~n Wiley arx]Sans, New York. Ill, S. arx] } - tel. P. (1988) . In vitro stoic of Are peri- i~pla~ation Erases of human embryos In Ult~ze of Human Game~genes~s arm E~bryagenes~s (J. Van Blerkc~ arm P. 2~a, a.) Seer Academic Publishers, Boston (m press). Liu, BLOC., Jones, G., Jones, H., ark Rosenwaks, Z. (1988). ~ an and factors of early pregnancy wastage in in vitro ~ fealty ization-embryo transfer patients. Fertil. Steril 50: - Longo, F.J. (1985). Pronuclear events during fertilization In Biology of Fertilization (C.B. Ritz and A. Mbnroy, Eric.). vol. 3, pp. 251-298. Academic Press, New York A Masui, Y. and Clack, H. (1979~. Oocyte maturation Int. Rev. Cyto1. 57: 185-282, Academic Press, New York - Pellestor, F. and Sele, B. (1988~. Assessment of ane~ploidy in the human female by using c~ger~etics of IVF failures. Am. J. Hum. Genet. 42: 274-283 . Pladhot, M. (1985). Contribution a l'ebude de la fecorx~ation et flu ~ development ~ vitro de l'oeuf h~na~n. Ih-~ de cloc~rat cl 'etat es sciences natur~les. Paris: Univert;ite Paris VI, A. 1-114. Pladhat, M., Veiga, A., ~ntagut, J., de Grouchy, J., Sideman, G., arm others (1988~. Are clinical and biological Parameters oorr`31ated with ~31 disorders An early life: a multi~xntric sty. Oman Ripped. 3: 627-635. : Sathananthan A.H., Hi, S.C., Aria, C.M., law, H.Y., ~~;risinghe, W.R. and Ratnan, S.S. (1985~. The origin an~distributionofoorticalgranules In human of; with refererxx 0~ Golgi, Nucleolar and =~vfila~c activity. Annal NY Acad. Sci. 442: 251-264.- Spielmann, H., drug en, C., Stabber, M., and V ~ el,- R. (1985). Abnormal chrc~osore behavior in human oocytes which remained unfertile ized Curing human in vitro fertilization. IVF 2: 138-142. i, S., Sala, F., -and Pedroli-Noy, G. (1982~. Aphidicol~n: A specific inhibitor of-nuclear DNA replication in eukaryotes. Trends in Biocil~mir=l Scierxxs. 7: 29-31. Surani, M.A.H. (1981~. Evidence arm considers of differerx~s between maternal and paternal geneses during embryogenes~s In ~ ~se. In EX~rimer~tal Approadhes to Mammalian Embryonic D~elc~;t. (J. Rcssant and R. Peder~;on, eds). UP 410-435. Academic P=ss, NO York. 246

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Tesarik, J., By, V., Plac~ot, arm ~elbamn, J. (1987) . Ultra- s~al arx] a~xtoradiographic c~;ervations on Antinuclear blasters of hewn cleaving embryos contain" by in vitro fertilization. ~ Bean Repr=. 2: 127-136. . . . . . . . . . . . . . Van Clerks, J. (1988). Develc~nt=1 failure In hen repression a.~iat~ with preovulatory oogenes~s arm preimplantatic~n ~zyogenes=. : In Ultrastruct~re of In Ga~tc~sis arm Folly Eabryogenesis (J. Van Blerkan arm P. Matte, Is). ~ 125-180. gluier Academic Polishers, In are London. Van Bler~rn, J. (1989a). the origin arx] detection of ~r~1 ~~ malities in meiotically mature hen odes cocaine from stimulated follicles are after failed fertilization ~ vitro. In- Develc~s in Ult~ctllre of P~pr~x3uction (P. Cotta, ed). Alan I, New York. ~ press. Van Blerkon, J. (1989b). me French arx] devel~enbal Is of abberant cellular organization in meiori~1ly mature human of; after exogenous Ovarian hyperstilrnulation. J=r (in pro;). Van Blerkan, J. and Henry, G. H., (1988). genetic analysis of living human ounces: cellular basis and devel~rent~ c~sequerxxs of Lotions an Sal organization and c~nplemnt. Oman Reprod-. 3: 777-79Q. - ~ tan Blerkam, J. and Henry, G. H., arm Pore, R.P. (1984). Preimplanta- tion hewn erdbryorac clevel~nt fray polyprorTuclear eggs after in vitro fertilization. Fertil. Steril. 41: 686096. Van Blerkam, J., null, H., arx] Henry, G.H. (1987). Be errs, recognition arm cievel~zren~1 fat of pseudo~p~vnucl~ar ~5 aft In vitro fertilization of human ~es. than Repro. 2: 217-225. Ski, W., Graham, J.M., art Sergavi~h, F.R. (1976) . The clinical Myrrh of ~iploidy. C - bet. ~1. 47: 69-73. by, H. art Alga, K. (1987). ~r~ analysis of hen of; failing to cleave after inspiration in vitro. Human Repel. 2: 137-142. Or, H., Saga, K. are 7;edholm, P. (1987). ~3~ analysis of human axe; revel friar preovulatory follicles In stimulated - :les. N. Errol. J. Ad. 316: 121-124. Zam~ni, L. (1987). The ult~cural pathology of the yltozocn as a callof infertility: ache role of electron me n the evaluation of senen duality. Fertil. Steril. 48: 711-734. 247

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ME 1 XE;~RI'1;D Fat OF AN=PIDIDY IN METOll=LI~ ON (X)CY]:ES ~ WIT in ~ ~ ~ ~~m AUI~RE; ,`;~.TD air AL (1976) SON alp AL (1985) VAN B[E~M AND HENRY (1988) E~I=IOR AND SE[E ( 1988 ) ~06 EI AL (1988) BIAS EI A.L (1988) MP,E~ EI! AL (1986) ~BY ET AL (1987) WRAMSBY AID ~ (1987) % ANE~ID* 1.5 ~1 5 19 21 26 34 50 65 * percent Fines both hypc~aploid arc] hyE=rhaploicl situations ** fern analysis of unin~ted yes that ma~ In vitro frill the GV to ache HI stage. (fray Van Blerkam, 1989a) - 248

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Fi _ 1 and 2: Failed (Fig. 1) and inoo~plete (Fig. 2) deco Sensation of sperm AND (S) after successful on of the human opplasm. M, itoch ad ia Fig. 1: 28000X; Fig. 2: 44000X. from Can B1 ^ (1988b]- Figures 3-~: The normal Leagues ion of changes in pecouclear (P~] _ geometry (arrows) and nocleolar (h) distribution during the peeisyr~c _ of human deve1qpment (14-22 bra post- insemination) as by differential interference _ ~_. ~, ~ m1~ ma. from Awn B1 ^ et al (1987), - 249