|
Items in cart [1] |
Questions? Call 888-624-8373 |
| Copyright © 2009. National Academy of Sciences. All rights reserved. Terms of Use and Privacy Statement |
Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 234
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
-
OCR for page 235
one approach to an investigation of =;`rly devel~al fa; flare
hen r—Lion 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 -
OCR for page 236
~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
. ,
OCR for page 237
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 -
OCR for page 238
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 cx—feted 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 ~
OCR for page 239
prior to fertilization. Cl - ply, the extent to which an ideas
~ of genetic ~ur`tion that can Ruse prep or Arty
Hi—lactation 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
OCR for page 240
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
aE—ar 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 cc—nags 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
OCR for page 241
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
OCR for page 242
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
OCR for page 243
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 -
OCR for page 244
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
-
OCR for page 245
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 P—r
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 -
OCR for page 246
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 —
OCR for page 247
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
call—of infertility: ache role of electron me n the
evaluation of senen duality. Fertil. Steril. 48: 711-734.
— 247 —
OCR for page 248
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
OCR for page 249
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
Representative terms from entire chapter:
van blerkam
