Embryo Development and Assessment of Viability potx

hyper-In other words, the actual implantation potential may be overestimatedalthough oocyte morphology, fertilization, and cleavage rate may appear in-conspicuous at first glance.. On the

Embryo Development and Assessment

of Viability

Thomas EbnerIVF-Unit, Women’s General Hospital, Linz, Austria

Germ cell wastage is a universal phenomenon throughout reproductive life

in mammals, including humans Before puberty and adult life, the vastmajority of oocytes become atretic at various stages of follicular develop-ment and, of those actually managing to ovulate, only a limited numberare capable of repeating the life cycle

Compared to the natural cycle, the situation in controlled ovarian stimulation is substantially aggravated because accidental maturation andovulation of germ cells of reduced developmental potential may occur (1)

hyper-In other words, the actual implantation potential may be overestimatedalthough oocyte morphology, fertilization, and cleavage rate may appear in-conspicuous at first glance On the other hand, even embryos of worst qualitymay sometimes turn out to be viable, e.g., giving birth to healthy babies.Taken together, viability of individual embryos is strongly correlated

to optimal maturational steps in the ovary, adequate fertilization, ive development through all pre-implantation stages, as well as subsequentimplantation in the endometrium Combining cytogenetical analysis—morphological evaluation throughout preimplantation development (2),and embryo metabolism (3)—the ability to select the most competentembryo out of a pool of concepti will further improve and definitely help

progress-to reach the ultimate goal in assisted reproduction, namely a healthy ton delivery

single-199

THE FOLLICLE

It is well accepted that the developmental fate of an embryo is largely tated by the quality of the oocyte, which in turn reflects the follicular milieu.Most likely, affected gametes are derived from follicles with reducedblood supply since various reports suggest a close relationship between fol-licular blood flow and developmental competence of the correspondingoocyte or embryo (4,5) If vascularization in ovaries is underdeveloped,some follicles will be confronted with hypoxia which in turn causes a change

dic-in energy metabolism by switchdic-ing from oxidative phosphorylation to colysis As a consequence, adenosine triphosphate (ATP) production inthe affected follicle will decrease dramatically, since glycolysis generatesonly two molecules of ATP compared with oxidative phosphorylation (38molecules) In addition, ATP depletion is increased since the vast majority

gly-of ATP is used for remodeling the vascular network via angiogenesis which

is triggered by chronical underoxygenation (6) Since vascular endothelialgrowth factor (VEGF) is a potent mediator of angiogenesis, it can beexpected that it is produced by granulosa and theca cells in response tohypoxia In fact, a significant correlation between elevated levels of VEGF

in follicles and a reduced viability of the corresponding embryo has beendescribed (7)

Since conventional parameters, such as follicle size or fluid volume, arenot considered to be adequate predictors of developmental potential of har-vested oocytes and arising embryos, pulsed color Doppler ultrasound may

be the first-line indirect technique for screening for competent oocytes whichmight serve as a basis for viable embryos or blastocysts, followed by follicu-lar fluid analysis for oxygen, ATP, and/or VEGF

THE OOCYTE

It is still unknown how follicular underoxygenation affects normal cellularand genetic development of the human oocyte; however, there is evidencethat gametes with a reduced internal cytoplasmic pH and ATP contentmay arise if oxygen saturation falls below a certain threshold of less than

by a complete spindle absence More interestingly, the rate of unaligned

chromosomes increased dramatically from 13% in the 20% oxygen group to35% in the low oxygen cohort Similarly, Van Blerkom et al (11) reportedthat 92% of the oocytes exhibiting chromosome displacement or abnormalchromosomal alignment originated from follicles with dissolved oxygen con-tents of less than 3%.

Considering the importance of the follicular and in vitro milieu and itsclose relationship to the health of the gamete, it is not surprising that up to38% of analyzed MII oocytes lack a spindle apparatus as shown using apolarized light microscope (12) Though detectability improved withincreasing PolScope experience (Table 1), one characteristic remained con-sistent, namely a reduction in developmental competence in spindle negativemature oocytes as assessed by fertilization rate (13,15,16) Even in spindlepositive gametes, grading in terms of fertilizability is suggested (16) withthose oocytes of worst quality showing a spindle deviation of more than

90 degrees from the first polar body

However, absence of a birefringent spindle does not predict tion failure and developmental arrest In fact, it has been found that humanoocytes with a polar body but without birefringent spindle may still be attelophase I or prometaphase I stage (18) Thus, precocious intracytoplasmicsperm injection (ICSI) in human prometaphase I oocytes with unalignedchromosomes may be one reason why oocytes without a birefringent meta-phase II spindle have a significantly worse prognosis

fertiliza-Knowing the actual position of the birefringent spindle during ICSI canimprove embryo quality (14) If no spindle at all has been detected, the prob-ability of a good quality embryo decreases dramatically (13,15), though thissuspected correlation is still subject to controversial discussion (16,17)

Table 1 Visualization of Metaphase II Spindle by Means of Polscope and itsInfluence on Fertilization Rate

Spindle inproximity toPb1

Fertilization rateSpindle No spindleWang et al (12) 327/533 (61.4) 61 (18.7) 202 (61.8)a 91 (44.2)a

Moon et al (15) 523/626 (83.6) 252 (48.2) 444 (84.9)c 78 (75.7)cRienzi et al (16) 484/532 (91.0) 254 (52.5) 362 (74.8)d 16 (33.3)d

The only paper correlating spindle detection with further tion development to the blastocyst stage (13) reports increased rates ofblastocyst development by day five arising from spindle-positive oocytes(51.1%) compared with the spindle-negative counterparts (30.3%), thus sup-porting the hypothesis that spindle detection may be used as indicator of theoocyte’s capacity to form a viable, chromosomally balanced embryo.

preimplanta-In addition, oocytes rather tend to show a visible spindle apparatus ifpostovulatory age exceeds 38 hours (17), making spindle imaging a newmarker for optimal timing of the ICSI procedure and thus increasing thechance to generate viable embryos

First polar body morphology takes the same line, since the mostnotable characteristic of postovulatory aging is the spontaneous division

or fragmentation of the first polar body (19) Bearing this in mind, it isnot surprising that a close correlation between the first polar body appear-ance and the further fate of the oocyte was observed (20–23) In detail,heavily fragmented first polar bodies were negative predictors of embryoquality, blastocyst formation rate as well as rates of implantation and clini-cal pregnancy Apparently this benefit is somewhat reduced with increasingtime span between ovulation induction and injection, since a retrospectivestudy applying a different schedule could not find any relationship betweenconstitution of the first polar body and subsequent ICSI outcome (24)

In contrast to postovulatory age, chromosomal status of the oocyte isnot reflected by the morphology of the first polar body as suggested fromdata of polar body biopsy Regardless of the grade of the first polar body,more than two-thirds of the oocytes were found to be aneuploid (25), but,unfortunately, the most interesting grade consisting of large polar bodieswas not analyzed in this highly selected patient cohort

It has been summarized that MII oocytes of good morphology should

be of regular size and show a clear, moderately granulate cytoplasm, a smallperivitelline space, and a colorless zona pellucida (2) As a precaution, eggswith an observed deviation in size should not be kept in culture since, e.g.,giant oocytes will mostly result in trigynic triploidy (26,27) On the otherhand, any reduction in diameter might reflect a certain cytoplasmic loss dur-ing manipulation of the oocyte (28)

Cytoplasmic Component

The degree to which cytoplasmic abnormalities, probably being the result of

an impaired cytoplasmic maturation, influence fertilizability and furtherdevelopmental potential is still a matter of debate (29–33) According toVan Blerkom and Henry (34), the further fate of female gametes is dependent

on the first occurrence of certain ooplasmic anomalies, e.g., those developingearly in maturation may be associated with failed fertilization and aneu-ploidy while those occurring later in maturation may express developmental

failure despite normal fertilization However, summarizing the relevantliterature dealing with cytoplasmic abnormalities, one may conclude thatonly few cytoplasmic dysmorphisms actually impair viability of the resultantembryo (29,31,33).

On the one hand, aggregation of the smooth endoplasmic reticulum(sER) was shown to significantly reduce rates of implantation and clinicalpregnancy (34), even if transferred embryos did not derive from sER aggre-gation positive ova, which is presumed to be the result of an underlyingadverse factor that might have affected the entire follicular cohort (34) Onlyone pregnancy went to term after transfer of an embryo developed from anaffected gamete (34), and to make matters worse, this baby was diagnosedwith Beckwith–Wiedemann syndrome

On the other hand, vacuolization is the most apparent and dynamiccytoplasmic anomaly in human oocytes Vacuoles are membrane-bound cyto-plasmic inclusions filled with fluid virtually identical with perivitelline fluid andthey vary in size as well as in number It is assumed that vacuoles arise eitherspontaneously (35) or by fusion of preexisting vesicles derived from thesmooth endoplasmic reticulum and/or Golgi apparatus (36)

Recently, a prospective analysis revealed that larger vacuoles above acut-off value (e.g., 14 mm) affect adequate fertilization and severely impairblastocyst development (37) Two hypotheses could explain these phenom-ena First, it is likely that a larger vacuole or multiple vacuoles will cause

a much more detrimental effect to the oocyte than a small vacuole since alarger portion of the cytoskeleton (e.g., microtubuli) cannot function as sup-posed to Secondly, large vacuoles are thought to displace the MII spindlefrom its polar position which may result in fertilization failure (35).Regardless of the different types of cytoplasmic inclusions, it has beenobserved that a deficiency in ooplasmic texture can also reduce reproductivesuccess Thus, oocytes with impaired fluidity of the cytoplasm, as assessed

by the persistence of the injection funnel after ICSI, had a developmentaldisadvantage compared to MII gametes with regular viscosity (38) How-ever, extensive cytoplasmic granularity is recognized as the most severe form

of cytoplasmic texture anomaly since more than half of affected gametesshow chromosomal abnormalities (39), which led to minimal rates ofimplantation (4.2%) and clinical pregnancy (12.8%)

THE ZYGOTE

Normal fertilization follows a defined course of events, although the timing

of these events may vary considerably (for more details, refer to Chapter 11).Either direct deposition (ICSI) or active propulsion [conventional in vitrofertilization (IVF)] ensures presence of a spermatozoon in the cytoplasm.Its head decondenses in the ooplasm prior to the extrusion of the secondpolar body The male pronucleus appears in the center of the oocyte

and the female one in close proximity to the meta phase spindle at the iphery of the gamete Microtubuli growing from the paternal centrosomeorganize central apposition of both pronuclei (40) This phase isaccompanied by final pronuclear growth, nucleolar movement, and coalesc-ence as well as a certain withdrawal of ooplasmic components to theperinuclear region (41).

per-Abnormal Findings

At least in terms of oocyte polarity, a good quality two-pronuclear zygote ischaracterized by two polar bodies being located near the pronuclear axis(42) Any deviation from this presumed optimal arrangement that cannot

be corrected by microtubuli-driven rotation of the pronuclei (43) could lead

to embryos of reduced morphology (42) This drawback is in line with a highrate of complex genetic abnormalities found in embryos derived fromzygotes with impaired polarity (44)

However, it may happen that an intrinsic defect of the cytoskeleton orthe parental centrosome causes peripheral apposition of both pronuclei (42)

or a complete failure in alignment (45), which can result in chromosomalaberrations (44)

The first scenario is more frequent in conventional IVF than in ICSI(3.3 vs 11.8%), probably due to varying sites of sperm entrance in IVF(42), e.g., spindle-near penetration of the zona could force eccentric forma-tion of pronuclei (46) According to Garello et al (42), zygotes with eccen-tric pronuclei show a limited capacity to cleave regularly (47.4%)

The second phenomenon is less frequent in assisted reproduction nologies (approximately 1%) but much more detrimental since the vastmajority of zygotes with unaligned pronuclei fail to cleave or show develop-mental arrest at early stages (47)

tech-Though the female pronucleus usually is smaller than its malecounterpart (41), more extensive differences in size (7–10 mm) may occur.This divergence most likely is the result of problems arising during male pro-nucleus formation (48) and severely affects viability of the correspondingembryos since more than 80% were found to be aneuploid (49,50)

Normal Fertilization

However, the vast majority of zygotes will present with two centrally alignedpronuclei 18 to 20 hours post-insemination Within these pronuclei, nucleolitend to align at the pronuclear junction, but since this condition is time-dependent (51), embryologists may be confronted with various pronuclearpatterns at the time of fertilization check

Scott and Smith (45) were the first to report a prognostic value of azygote score involving pronuclear appearance on implantation and deliveryrate This rather complex score was simplified by focusing exclusively on

pronuclear morphology (47) Thus, it could be shown that interpronuclearsynchronicity is a strong predictor of embryo viability (47,52–55) In fact,there is only one report critically questioning this suspected correlation(56), but since it is based on single embryo transfers, it may reflect the actualimplantation potential more accurately than studies dealing with double ortriple embryo transfers.

In addition to pronuclear pattern, cytoplasmic appearance at zygotestage was part of the original Scott score (45) As demonstrated by time-lapse video cinematography (41), ooplasm withdraws mitochondria andother cell organelles to the perinuclear region during fertilization, leaving

a clear halo around the cortex

Initial studies (56–58) analyzing a suggested relationship between haloformation and outcome (45) were characterized by a lack of standardization

in terms of halo scoring, since they either pooled all variations of haloes(e.g., concentric haloes and polar ones) or excluded certain subtypes fromanalysis Despite this fact, halo-positivity was found to influence embryoquality (56,59) and blastocyst formation rate (58) Recently, it could beproven that any halo effect, irrespective of its grade and dimension, is ofpositive predictive power in terms of blastocyst quality and, consequently,clinical pregnancy rate (55)

During evaluation of zygote morphology, it has to be considered thatboth halo and pronuclear formation follow a fixed schedule Since directooplasmic placement of a viable spermatozoon is performed in ICSI, thusbypassing most steps of fertilization (including acrosome reaction and zonabinding), the further course of development will be somewhat accelerated ascompared to conventional IVF (60) Consequently, more optimal zygoteswere observed in ICSI than in IVF at the time of analysis and pronuclearpattern was performed (54) Therefore, different observation times formicroinjected and conventionally inseminated oocytes are recommended

To summarize, though pronuclear morphology turned out to be anunstable factor within the dynamic process of fertilization, optimal pronu-clear patterns, e.g., those with alignment of fused nucleoli, may characterize

a subgroup of oocytes showing a developmental advantage compared withzygotes developing more slowly (those showing pronuclear asynchrony).This is in line with recent findings indicating that during syngamythose zygotes with an accelerated breakdown of the pronuclear membranes(PMB) 22 to 25 hours post-insemination or injection implanted significantlymore frequently than those with delayed dissolution (61)

THE CLEAVING EMBRYO

However, just like pronuclear appearance, dissolution of the pronuclei is not

a static event and, using it for selection purposes, embryologists may befaced with undocumented zygotes in terms of pronuclear location, size,

and number Thus, it may happen that, unintentionally, chromosomallyimbalanced embryos may be kept in culture if pronuclear morphology couldnot be checked due to abnormal developmental speed or intense ooplasmicgranulation (62,63).

Interestingly, slow cleaving embryos at day 1 were shown to have lessblastomeres at later stages of preimplantation development (67), whichcould be the reason for the observed decrease in blastocyst formation(69), implantation, and clinical pregnancy

Several reasons may account for this phenomenon Apart from thefact that, at least in conventional IVF, embryos dividing early may be asso-ciated with earlier fertilization, oocyte intrinsic factors are considered topromote early cleavage after fertilization (65) Though currently unknown,

Table 2 Prognostic Relevance of Early Cleavage Behavior on Pregnancy Rate

Authors Method

Hours post IVF/ICSI

Day of transfer

Clinical pregnancy rate Early cleavage

No cleavage Shoukir et al (64) IVF 25 2 33.3 a 14.7 a

Sakkas et al (65) ICSI 27 2 25.9b 3.2bSakkas et al (66) IVF/ICSI 23–27 2 45.0c 23.8cLundin et al (67) IVF/ICSI 25–27 2, 3 40.5 d 31,3 d

Bos-Mikich et al (68) IVF/ICSI 25–29 3 54.8e 25.0eFenwick et al (69) IVF 25 2 31.3f 10.5fSalumets et al (70) IVF/ICSI 25–27 2 50.0 g 26.4 g

Windt et al (71) ICSI 26 2, 3 37.5 h 11.1 h

Van Montfoort et al (72) IVF/ICSI 23–28 2 37.1i 10.3i

Abbreviations: IVF, in-vitro fertilization; ICSI, intracytoplasmic sperm injection.

Soruce: From Ref 72a.

such factors could be related to the expression of human leukocyte antigen

G (73), a candidate human functional homolog to the mouse Qa-2 antigen,which, as a product of the preimplantation embryo development (Ped) gene,promotes rapid mitotic divisions An alternative explanation would be thatslow cleaving human embryos have an early lag phase in cell cycle, whichwas found to be detrimental to blastocyst rate of bovine embryos (74).Whether a genetic predisposition influences viability of the developingembryo or to what extent metabolic disturbances (e.g., mitochondrial con-tent, ATP production, mRNA, cytoplasmic maturation) cause late cleavage

is still matter of discussion However, several data from embryo culturerather support a genetic reason On the one hand, it could be documentedthat early dividing embryos show a lower rate of multinucleated blastomeres(70), and on the other hand, tripronuclear zygotes show a limited capacity tocleave early as compared to their binucleated counterparts (67)

Further Cleavages

As cleavage continues, the first blastomeres of the two-cell embryo dividesmeridionally followed by approximately equatorial cleavage of the other cell(75) This lesson from mouse embryos may explain the typical crosswiseappearance of the 4-cell human conceptus on day two of development.Although a regular tetrahedral configuration of blastomeres with six inter-cellular contacts is the most common outcome of second cleavage, boththe distribution and the relationship between blastomeres may vary, includ-ing specimens that are essentially planar This arrangement involves areduced number of cell–cell contacts which could impair compaction anddelay blastulation of the embryo (28,76)

Apart from the number of blastomeres, routine assessment of embryoquality from day two onward also includes the degree of fragmentation.There is a considerable lack of objective and standardized methods forassessing embryonic fragmentation In fact, a cell size of 45 mm on daytwo has been suggested which allowed distinguishing between anuclear frag-ments and blastomeres Below this cut-off value, only 3% of the cellscontained DNA compared to 67% with a diameter above this cut-off Simi-lar results were published for day three embryos with the exception that, due

to ongoing cleavage, a threshold of 40 mm was indicative in terms of entiation (77)

differ-It is generally accepted that minor fragmentation does not impairviability of the embryo (78,79) and may disappear during in vitro culture,either by lysis or resorption (80,81) Larger amounts of fragments, however,significantly reduce the chance to achieve pregnancy (82) and, even moreimportantly, perinatal outcome of babies derived from heavily fragmentedembryos (greater than equal to 50% fragmentation) was found to be worsecompared with that after transfer of more or less fragment-free embryos (83)

As fragments are structures of blastomeric origin, the actual amount ofcytoplasmic fragmentation during cleavage stage can be estimated by thedifference between the previous zygote volume and the overall blastomerevolume (84) In cases of moderate fragmentation, it appears that differentspatial patterns of fragmentation are of more severe developmental conse-quences than fragmentation per se (81,82) In detail, smaller, more localizedfragments did not impair viability, whereas larger and more scattered frag-ments had a disastrous effect on implantation (82) Theoretically, thedetrimental effect of such patterns may be explained by the fact that anuclearfragments lying in close proximity to an assumed cleavage axis may impairfurther cleavage and/or reduce the number of cell-to-cell contacts requiredfor regular compaction and blastocyst formation Viability of bad qualityembryos may be improved in certain instances if spatial relationship of blas-tomeres is restored by cosmetical removal of the acellular remnants (82).The higher the degree of blastomeric decay, the higher the risk ofchromosomal imbalances, such as mosaicism (63) In these cases, selectivefragmentation could function as a means to completely exclude affectedblastomeres from further cell aggregation (85) This process is most likelyrelated to programmed cell death (85,86), e.g., the ratio of the two apoptosis-related gene families bcl-2 and bax (85) Others (87) question a directrelationship between apoptotic phenomena and fragmentation and muchrather speculate that apoptosis may be triggered if the degree of mitochon-drial and proteinic loss due to fragmentation reaches a certain level.

It is an undisputed fact that two morphological phenomena, namelymultinucleation and inequality of cleavage, reduce viability of the cleavingembryos to a minimum Frequently, both anomalies coincide (80), whichmay be explained by the larger cell size of multinucleated blastomeres(84) In general, it can be expected that about one-third of all day twoand three embryos show at least one multinucleated blastomere (88) Theoverall incidence, however, will generally be underestimated since nucleiare only visible at interphase Most previous studies report a disastrousimplantation rate of less than 6% after exclusive transfer of bi-or multinu-cleated embryos (88,89) This reduced outcome seems to be a reflection ofthe chromosomal constitution of the embryos since the vast majority ofthem (approximately 75%) were chromosomally abnormal (80,90) In detail,cytokinesis may fail during any mitotic division (91) with the worst outcome

to be expected if problems arise during the first cleavage ending up withboth cells being multinucleated (92)

Even if the embryo is composed of a stage-appropriate number ofequally divided mononuclear cells, this does not mean that the texture

of the blastomeres will correspond to the assumed normal condition, which

is a translucent cytoplasm with moderate granulation As a result of cultureconditions (e.g., media composition), cytoplasm of a day 3 embryo maychange to a more mottled appearance showing numerous small (1.5 mm) pits

on the surface (93,94) Pitting is physically different from excessive lation and mostly affects embryos after embryonic genome activation (93).

granu-In humans, this switch from maternal genome control is considered to takeplace around the 8-cell stage (95) Definitely, it is an important hallmark ofpreimplantation development prior to which it is an assessment of oocytequality rather than embryo viability

However, this temporal coincidence of cytoplasmic pitting is not apositive predictor of outcome (96); but much rather it seems to be com-pletely unrelated to implantation and pregnancy Recent findings, however,suggest a certain influence on viability since some 30% of implantations van-ished after exclusive transfer of pitted embryos compared to only 16% ofearly pregnancy loss in a nonaffected control group (97)

Compacting

While at earlier cleavage stages embryos resemble an accumulation of tary blastomeres with a rudimentary level of biosynthesis, compaction phase(beginning on day 3) is characterized by increased biosynthetic rates and thecapacity to metabolize glucose more efficiently In addition, the compactingembryo is capable of actively regulating ionic gradients, thus controlling itsinternal environment (98)

soli-Compaction is due to the formation and the number of tight cellular junctions (e.g., desmosomes, gap, and tight junctions) causingblastomeres to become closely apposed (76,99) Due to this highly interac-tive cell mass, blastomeres loose their totipotent characteristics

inter-In humans, compaction begins around eight-cell stage probably lowing an intrinsic developmental clock Precocious compacting at day 2could result in formation of trophoblastic vesicles leaving no predecessorcells of inner cell mass (6) On the other hand, 16-cell embryos withoutthe slightest evidence of compaction are of reduced capacity and will hardlyreach blastocyst stage (99)

fol-Tao and co-workers (100) successfully tried to predict implantationscoring embryos at the compaction stage These authors showed that theimplantation potential is positively related to the proportion of blastomeresundergoing compaction Consequently, embryos had the worst prognosis ifless than half of the blastomeres were involved in the compaction process.Blastomeres and fragments that are unable to form appropriate contactsare generally excluded from the compaction process and remain withinthe empty zona pellucida after hatching (99)

EMBRYO METABOLISM

Despite the fact that numerous morphological criteria have been publishedwhich could add to predictive power on further developmental potential of