Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

pronuclear and day 3 features in their retrospective

study, which defined embryos derived from Z-1

zygotes (based on Scott et al

) that became grade 1

embryos as ‘top quality.’ These ‘top quality’ embryos

had a 92% day 5 survival rate and were associated

with significantly greater implantation and preg-

nancy rates.

De Placido et al also used a combined

pronuclear and day 3 assessment, but included a

weighting based on embryonic growth rates/cell

number. The weighted day 3 embryo score had the

highest correlation with pregnancy rates, and top

scoring embryos achieved a pregnancy rate of 71%.

Several multiday assessments have used the com-

bination of pronuclear morphology and day 3 mor-

phology, and some have included early cleavage as

part of the weighted score. A recent prospective trial

randomized embryos to two groups according to

the manner in which they were scored.

Group 1

embryos were given a score based on a combination

of pronuclear morphology and day 3 morphology;

pronuclear morphology was scored as follows: sub-

group A, nucleoli large or medium sized and nucle-

olar alignment seen; subgroup B, nucleoli large or

medium with no alignment; and subgroup C, nucle-

oli small or pinpoint with no nucleolar alignment.

Group 2 embryos were assessed at 26 h for early cleav-

age combined with day 3 morphology.

Embryos at

early cleavage were segregated into those that had

cleaved to 2 cells (subgroup A), those in which pro-

nuclear breakdown had occurred but cleavage had

not occurred (subgroup B), and those still exhibit-

ing two distinct PNs (subgroup C). These authors

found no difference in implantation rates between

group 1 and group 2, although they did find that sub-

group A (in both groups) had a higher implantation

and pregnancy rate than subgroup C. However, when

the corresponding subgroups were compared, there

was no difference in pregnancy rates.

This study

raises questions as to whether or not it is necessary

(or even beneficial) to evaluate embryos for both

pronuclear morphology and for early cleavage. In

addition, these authors found that pronuclear mor-

phology and early cleavage were interrelated. For

example, those embryos with subgroup A pronu-

clear morphology were more likely to undergo early

cleavage, and likewise the embryos that underwent



early cleavage were more likely to arise from sub-

group A. Therefore, evaluation of only one of these

markers of developmental competence may achieve

results similar to those following evaluation of both

markers.

Along these lines, another combined scoring

system has been recently reported, which used the

relatively novel combination of early cleavage, day 2

mononucleation and day 3 morphology. In this paper,

the implantation and pregnancy rates were highest

in the group displaying both early cleavage and

mononucleation.

Available evidence suggests that multiday assess-

ment provides a more accurate picture of develop-

mental progression as opposed to a single static

observation. However, the ultimate combination of

morphological features required for optimum eval-

uation of developmental competence has yet to be

resolved. When the critical time points have been

identified, we can then determine the ideal timing

for embryo transfer.

TIMING OF EMBRYO TRANSFER:

OPTIMIZING PREGNANCY RATES

There has been much debate surrounding the ideal

timing of embryo transfer in human IVF. Compared

with animal models, the human uterus is far more

forgiving in terms of receptivity to embryo transfer,

and this has allowed pregnancies to be successfully

established not only with transfers from day 1 to

day 6, but also, remarkably on day 0 with eggs and

sperm being introduced into the uterus.

However,

as implantation rates and pregnancy rates continue

to improve, the optimal embryo transfer window

for each patient needs to be determined.

Until recently, the vast majority of embryo trans-

fers were carried out on day 2; since culture condi-

tions did not permit extended culture, it was felt that

the embryos should be transferred to the maternal

environment as quickly as possible. In the late 1990s,

when improved culture media became available,

several groups began to question this timing, partic-

ularly since the embryo typically enters the uterus

late on day 4 in vivo. Extending embryo culture may

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DEVELOPMENT RATE, CUMULATIVE SCORING, AND EMBRYONIC VIABILITY



also serve to improve selection of embryos with

greater degrees of developmental competence.

In 1995, Dawson et al retrospectively analyzed

day 2 versus day 3 transfers, and although there was

no difference in implantation or pregnancy rates,

they found that day 3 transfers were associated with

a significant difference in development to the fetal

heart stage, with fewer miscarriages.

These authors

also concluded that the additional day in culture

allowed them to select embryos that had passed an

additional test of development, in that they had sur-

vived the stage of embryonic gene activation with-

out arresting at the 4- to 8-cell stage. Carillo et al also

compared day 2 and day 3 transfers retrospectively,

and found that delaying the transfer by 24 hours did

result in an improved implantation rate and ongoing

pregnancy rate.

However, prospective studies have not substanti-

ated these findings. Laverge et al designed a prospec-

tive randomized study to compare day 2 and day 3

transfers in terms of implantation and pregnancy

rates. They found no differences in implantation or

pregnancy rates, but did observe a significant decrease

in embryo quality when embryos were kept in cul-

ture until day 3.

These results were consistent with

two recent additional prospective studies that also

found no difference in pregnancy rates between day

2 and day 3 transfers.

68,69

Finally, a meta-analysis

carried out in 2004 that reviewed the timing of

embryo transfer found a slightly improved clinical

pregnancy rate for day 2 versus day 3 transfer, but

no difference in live birth rate.

Based on the principle that additional observation

in culture would allow embryos that did not arrest

to be identified, several authors have suggested that

extended culture to the blastocyst stage may further

improve implantation rates, thus allowing transfer

of fewer embryos and decreasing multiple gesta-

tions. Indeed, blastocyst transfer became a viable

option following development of sequential media

systems,

although there is currently debate regard-

ing the need to use a sequential two-step system,

rather than a single one-step system.

72,73

A retrospective pilot study by Gardner et al

showed that blastocyst transfer resulted in a signifi-

cantly higher implantation rate, when compared with

day 3 transfer.

A follow-up prospective, randomized

study confirmed increased implantation rates from

day 5 transfers, but showed equivalent clinical preg-

nancy rates with day 3 transfer.

These observations

have led some authors to postulate that blastocyst

transfer may provide a strategy to optimize preg-

nancy rates while decreasing multiple gestations.

However, blastocyst transfer may not be ideal in

all cases, and may compromise a successful outcome

that might otherwise have been achieved following

a day 3 transfer.

This suggests that the uterus may

provide a superior environment compared with

that of the in vitro system, and that day 3 embryo

transfer may have ‘rescued’ embryos that otherwise

may not have developed to blastocysts in culture.

A Cochrane review of blastocyst transfer versus day

3 embryo transfer identified 16 trials for meta-

analysis and concluded that there was no significant

difference in live birth rate, pregnancy rate, inci-

dence of multiple gestations, or miscarriage rates. In

addition, they found that those patients who under-

went a day 3 transfer were more likely to have addi-

tional embryos available for cryopreservation, and

patients undergoing a day 5 transfer were more likely

to have no embryos available for transfer.

In con-

trast, a recent prospective randomized trial compar-

ing single blastocyst transfer versus single cleavage

stage transfer concluded that the delivery rate from

fresh transfers was significantly higher after single

blastocyst transfer.

However, fewer embryos were

frozen on day 5 (2.2 vs 4.2, p ⫽ 0.001) and the live-

birth rate was higher following transfer of thawed

day 3 embryos (15% vs 11%). Given these findings,

and depending on whether pregnancy rates are cal-

culated from solely fresh transfers versus fresh and

frozen transfers (i.e. the cumulative pregnancy rate),

debate remains as to whether single transfer at the

blastocyst or the cleavage stage is the optimal proce-

dure. Furthermore, many programs have difficulty

with successful blastocyst freezing and therefore have

reverted to performing day 3 transfer.

The optimum day for embryo transfer has yet to

fully be elucidated,and is also influenced by the success

of cryopreservation on specific days, within an indi-

vidual laboratory. It is clear that a variety of patient

and laboratory factors play a role in determining the

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HUMAN PREIMPLANTATION EMBRYO SELECTION

ideal timing for each individual patient. Moreover,

while the majority of studies use pregnancy rates

after fresh transfer as the index of success, cumula-

tive pregnancy rates derived from both fresh and

frozen transfers for each stimulated cycle provide a

more accurate reflection of cycle efficacy and suc-

cess. A recent study proposing single blastocyst

transfer reported high pregnancy rates of cryopre-

served blastocysts in either a subsequent natural

cycle or hormonally prepared endometrium, sug-

gesting that perhaps the transfer of only one blasto-

cyst at first attempt may lead to a higher cumulative

pregnancy rate, by allowing transfer of cryopre-

served blastocysts to a more favorable uterine

environment.

NUMBER OF EMBRYOS TO TRANSFER

Single embryo transfer provides the potential oppor-

tunity to change the landscape of IVF. Currently, the

incidence of multiple gestations remains high. In

2003, the percentage of live births that had multiple

infants resulting from ART in the United States

ranged from 17.4% to 38.4%,depending on maternal

age.

Although there have been efforts to decrease

high-order multiple gestations (triplets and higher-

order multiples), until recently, both patients and

providers have accepted the consequence of twins,

in return for an acceptable pregnancy rate. This

implied agreement between patients and doctors

has recently come under scrutiny, as twin preg-

nancies are associated with increased maternal

and fetal complications compared with singleton

gestations.

In the early days of IVF, a high number of

embryos was typically transferred so as to achieve a

reasonable pregnancy rate.

Indeed, in early studies,

the total number of embryos transferred was highly

correlated with pregnancy.

2–4,79

In the late 1990s,

investigators began questioning the practice of trans-

ferring three or more embryos in routine IVF.

5,7

The

logical next step in the attempt to reduce multiple

gestations is to provide appropriate patients with the

option of elective single embryo transfer. However,

selecting the appropriate patient population and the



best embryo from any given cohort remains the clin-

ical challenge. Van Royen et al defined a ‘top quality

embryo’ by identifying the embryonic features asso-

ciated with ongoing twin pregnancies following

double embryo transfer. They found that the pres-

ence of top quality embryos was highly associated

with both ongoing pregnancies and multiple gesta-

tions, and that twin pregnancies after double embryo

transfer only occurred in patients ⬍38 years of age.

Hellberg et al used similar methods (analysis of twin

pregnancies after a double transfer) to identify the

following characteristics: maternal age ⱕ38 years,

infertility cause other than male factor (male factor

was associated with a decreased risk of twinning),

two high quality embryos, ability to freeze at least

one embryo, and ⱖ5 fertilized oocytes, and concluded

that all of the above were associated with increased

twinning.

Building on the definition of a top quality

embryo, De Neubourg et al

used Van Royen’s defi-

nition of a top quality embryo (see above) to select

embryos for single embryo transfer on day 3. Using

these selection criteria, they were able to demon-

strate a 50% pregnancy rate with transfers to women

⬍38 years of age. Thurin et al

randomized women

⬍36 years of age with at least two good quality

embryos to undergo single or double embryo transfer

and then analyzed cycles with 0% or 100% implan-

tation to identify variables associated with success.

Multivariate analysis revealed that first IVF cycle,

IVF as method of fertilization (as opposed to

ICSI), 4-cell embryos (day 2), and number of IU

of FSH per oocyte retrieval were predictive of

implantation.

Recently, randomized trials have shown that single

embryo transfer shows increasing promise. A ran-

domized multicenter trial of single embryo transfer

(with subsequent transfer of frozen embryos in the

event of no live birth) versus double embryo transfer

(in women ⬍36 years of age with at least two good

quality embryos) demonstrated roughly equivalent

cumulative pregnancy rates (39% versus 43%) with

a substantial reduction in multiple pregnancy rates

(0.8% compared with 33%).

Single blastocyst

transfer followed by cryostored blastocyst transfer

has also showed equivalent pregnancy rates with

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DEVELOPMENT RATE, CUMULATIVE SCORING, AND EMBRYONIC VIABILITY



decreased rates of multiple gestations. In addition,

there were five perinatal deaths in the double embryo

transfer group, all involving twin pregnancies.

CONCLUSION

Although IVF has made remarkable strides in the

optimization of culture media, stimulation regimens,

and, ultimately, pregnancy rates, the challenge to

reduce the number of twin pregnancies remains.

Clearly, single embryo transfer is the answer. However,

in order for this practice to become acceptable to

both doctors and patients, both the patient and

embryo must be appropriately selected. To accom-

plish optimal embryo selection, investigators have

evaluated several features and time points for

embryo morphologic assessment including pronu-

clear morphology, early cleavage, cell number, the

extent of fragmentation, the degree of asymmetry,

the presence of mononucleation on day 2, and stan-

dard morphological features on day 3, including

early compaction. Compared with exclusive day 3

morphological assessment, evidence is accumulat-

ing that multiday scoring systems provide improved

predictability as to which embryos are likely to

implant.

61–63

Nevertheless, it remains to be deter-

mined whether evaluation must be performed on

each day of culture, to what extent repeated obser-

vations may compromise embryo development, and

which combination of features provides greatest

selection sensitivity. In addition, the incremental

improvements in implantation potential must be

weighed against the time and labor of the embryol-

ogy staff; indeed, multiday assessments may not be

possible in every IVF program. As a randomized

trial recently demonstrated, it may not be necessary

to perform both pronuclear and early cleavage scor-

ing.

Going forward, we must demand more rigor-

ous studies, comprised of prospective, randomized

trials, to evaluate not only the scoring systems

themselves, but also the differential weighting of all

the different parameters and time points. The ulti-

mate result of such trials will hopefully improve

standardization of embryo scoring systems, and pro-

vide improved efficacy and safety for our patients.

ACKNOWLEDGMENTS

We thank the entire embryology team at Brigham

and Women’s Hospital for their expertise in embryo

grading. We would particularly like to thank Kerry

Kelleher for photographing the embryo images in

this chapter and Paulette Nippet for her assistance

in acquiring copyright permissions.

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following in vitro fertilization or intracytoplasmic sperm injection. The

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71. Gardner D, Schoolcraft W, Wagley L et al. A prospective randomized

trial of blastocyst culture and transfer in in vitro fertilization. Hum

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72. Biggers J, Racowsky C. The development of fertilized human ova to

the blastocyst stage in KSOM

medium: is a two-step protocol neces-

sary? Reprod Biomed Online 2002; 5: 133–40.

73. Biggers J, McGinnis L, Lawitts J. One-step versus two-step culture of

mouse pre-implantation embryos: is there a difference? Hum Reprod

2005; 20: 3376–84.

74. Gardner D, Vella P, Lane M et al. Culture and transfer of human blas-

tocyst increases implantation rates and reduces the need for multiple

embryo transfers. Fertil Steril 1998; 69: 84–8.

75. Blake D, Proctor M, Johnson N et al. Cleavage stage versus blastocyst

stage embryo transfer in assisted conception. Cochrane Database Syst

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76. Papanikolaou E, Camus M, Kolibianakis E et al. In vitro fertilization

with single blastocyst-stage versus single cleavage-stage embryos.

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reduces twin rates without compromising pregnancy rates. Fertil Steril

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78. Centers for Disease Control and Prevention. 2003 Assisted reproduc-

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versus double embryo transfer in in vitro fertilization. N Engl J Med

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10. Human embryo cryopreservation and its

effects on embryo morphology

James J Stachecki and Klaus Wiemer



CRYOPRESERVATION AND ITS EFFECTS ON

EMBRYO MORPHOLOGY

Cryopreservation may have a variety of effects on

embryo morphology, ranging from subtle damage on

intracellular organelles, cytoplasm, or processes that

can negatively affect normal cell development, to

overtly blatant effects such as the lysis of one or more

blastomeres. Many underlying factors can influence

the outcome of cryopreservation, e.g. the quality of the

oocyte or embryo itself will often determine the effects

of the process on subsequent morphology. The type of

freezing protocol used (slow-cooling or vitrification)

and the concentration and type of cryoprotectant(s)

will also have an effect on subsequent morphology.

The goal of this chapter is to offer an insight into the

question: how does cryopreservation affect subse-

quent embryo morphology? We address this question

in three major sections: background, types of cryo-

preservation damage, and causes of damage.

BACKGROUND

Cells have been successfully cryopreserved for over

100 years. Glycerol was discovered to have protective

effects on sperm cells in 1949, and since that time

both gametes and embryos have been successfully

frozen from a number of mammalian species includ-

ing mice, sheep, cows, pigs, horses, hamsters, rats,

cats, and humans. The foundation of modern cryobi-

ology was established throughout the 1940s, 1950s,

and 1960s by Lovelock, Meryman, Polge, Smith,

Levitt, Luyet, Mazur, and others.

1–4

Further advances

in animal oocyte and embryo cryopreservation

were achieved during the 1970s by Whittingham,

Willadsen, Leibo, Mazur, and Wilmut,

5–7

and these

prepared the way for human oocyte and embryo storage

in the 1980s. Protocols for human embryo storage

were refined during the subsequent years up to 2000,

so that they have now become a routine procedure in

IVF clinics throughout the world. Cryopreservation

exposes embryos and oocytes to numerous types of

stress, and the fact that these cells can survive and go

on to form a viable fetus after freezing and thawing

is truly remarkable. However, freeze–thawing proto-

cols are not perfect, and many cells do not survive

and/or go on to develop normally.

CONSEQUENCES OF CRYOPRESERVATION-

INDUCED DAMAGE

Damage can occur at any time throughout the cryo-

preservation process, and may be manifested in dif-

ferent ways and at different times. Extreme types of

damage such as intracellular ice formation (IIF) and

cell fracture will lead to immediate cell lysis and death,

and these are easily documented through routine

microscopic observation of morphology. Damage can

also occur on a cellular structural/functional level,

involving intracellular organelles, and this is more

difficult to diagnose.

There is very little ultrastruc-

tural data documenting morphological damage in

cryopreserved oocytes and embryos. However, dif-

ferences in appearance between fresh and cryopre-

served embryos are sometimes mentioned in the

discussion section of manuscripts that focus on var-

ious freezing techniques. From these studies we can

gather at least some information about the effect of

cryopreservation on embryo morphology. However,

reported differences in morphology postcryopreser-

vation may not be universal, and are likely to be due to

the specific protocol and overall set of circumstances

used in the study. Cryopreservation procedures may

not inevitably result in specific observable morpho-

logical aberrations, but the embryo might still be

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HUMAN PREIMPLANTATION EMBRYO SELECTION

affected. In other words, the fact that one investigator

using propanediol finds 80% embryo mortality,

does not necessarily mean that propanediol is a toxic

or inferior cryoprotectant. Likewise, if the same inves-

tigator shows 95% embryo survival with dimethylsul-

foxide (DMSO), this does not mean that DMSO is the

best or the safest cryoprotectant to use. Ultimately,

regardless of morphological appearance, the most

important feature is that an embryo should remain

viable after cryopreservation.

CRYOPRESERVATION THEORY

A basic understanding of the cryopreservation pro-

cedure is important in order to better comprehend

common morphological characteristics observed

following cryopreservation. The first theoretical basis

for cryopreservation of cells was proposed by Mazur

and later applied to mouse embryos

5,9,10

and other

species including cows and sheep.

7,11

Conventional cryopreservation methods consist

of several steps:

(1) Pre-equilibration: embryos are exposed to a

simple salt solution containing a permeable

cryoprotectant (1,2-propanediol, DMSO, glyc-

erol, ethylene glycol, etc.) and usually a low

concentration of non-permeable cryoprotectant

(sucrose).

(2) Cooling: after a brief time of exposure to allow

uptake of cryoprotectant and initial dehydra-

tion, the cells are cooled rapidly to a tempera-

ture slightly below the melting point of the

solution (usually around ⫺7⬚C).

(3) Seeding: at this point the container with the cells

is super-cooled in a process known as ‘seeding’

so that ice forms in the extracellular solution.

(4) Slow cooling: upon ice formation and further

cooling at a slow rate (usually ⬍1⬚C/min to

below ⫺30⬚C), the osmolarity of the extracellular

solution increases as water freezes to ice, causing

the cells to dehydrate with the increasing tonicity.

(5) Plunging/vitrification: dehydration continues

during slow-cooling until the cells are plunged

into liquid nitrogen, usually at a temperature

below ⫺30⬚C. At this point the intracellular

cryoprotectant concentration is high enough

so that the remaining intracellular water will

vitrify, preventing IIF.

(6) Thawing and rehydration: during thawing, the

dehydrated cells are exposed to hypotonic con-

ditions and rehydrate as the cryoprotectant is

removed.

TYPES OF DAMAGE

INTRACELLULAR COMPONENTS

The effects of cryopreservation are not always evi-

dent; embryos may be adversely affected by cryo-

preservation at the intracellular level, with the

potential of altering the function of the intracellular

organelles and cytoplasm. Protein structure and

function, as well as metabolism can also be affected.

It is likely that embryos require a period of ‘recovery’

following cryopreservation, before they are able to

continue normal intracellular function. Therefore,

those embryos that are able to compensate for lost or

decreased function are the ones most likely subse-

quently to implant and develop further. We have

noted that human embryos following thawing often

begin to develop initially slightly slower than their

fresh in vitro counterparts. Following extended

culture, frozen embryos may resume normal rates of

development prior to transfer.

EMBRYO ORIGIN

The origin of the embryo itself may have a profound

impact on the survival of embryos following cryo-

preservation. In cattle, we have noted that in vivo

oocytes have a large perivitelline space; cleaved

embryos have uniform blastomeres, and compaction

at the morula stage is not irregular. In most cases,

expanded blastocysts contain distinct inner cell

masses that are not dark in appearance. In contrast,

in vitro 1-cell embryos have a very small perivitelline

space, blastomeres are irregular in early cleavage-

stage embryos, and morulae tend to exhibit irregular

patterns of compaction. Resulting blastocysts retain

a dark appearance and a slightly more irregular shape.



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HUMAN EMBRYO CRYOPRESERVATION AND ITS EFFECTS ON EMBRYO MORPHOLOGY

Evidence suggests that in vitro developed bovine

embryos may be more sensitive (to cryopreservation)

than their in vivo counterparts.

13,14

A reduced

implantation rate for in vitro produced blastocysts

following cryopreservation was noted. Reduced sur-

vival and pregnancy rates after transfer of in vitro

produced bovine embryos when compared with in

vivo embryos has also been reported by others.

15,16

The reason for this is unclear, other than the fact

that in vivo produced embryos are inherently differ-

ent to in vitro embryos. Some of the inherent differ-

ences may be due to suboptimal maturation and

culture systems, inability to adapt cryopreservation

methodologies to suit the kinetics of in vitro embryos,

and inabilities to manipulate energy substrates or

biochemical pathways following thawing.

INTRACELLULAR LIPIDS

It is well known that embryos from certain species

including goat, pig, and cow are substantially more

sensitive to injury from a reduction in temperature

than are mouse or human embryos. This chill sensi-

tivity or direct chilling injury is defined as an irre-

versible damage from exposure to low temperatures.

Martino et al

describe the effect of embryo stage,

cooling temperature, and cooling duration on sub-

sequent fertilization and development in vitro of

bovine oocytes. Leibo and Loskutoff

noted that in

vitro and in vivo cattle embryos have different buoy-

ant densities. In vivo embryos will sink in 2.35 M

sucrose solutions, while their in vitro counterparts

will float in solutions containing more than 1.6 M

sucrose. This may be due to altered ratios of lipids to

proteins found in cell membranes. In vitro produced

bovine embryos have a higher lipid content, making

them less buoyant, and this lower buoyancy renders

the embryo more sensitive to chilling and freezing

when compared with its in vivo counterpart.

Horvath and Seidel

pointed out that membranes

with higher cholesterol concentrations are more

fluid at lower temperatures, and are thus more chill-

resistant. They demonstrated this effect by loading

cumulus–oocyte complexes with cholesterol-loaded

methyl-beta-cyclodextrin; these cholesterol-loaded

oocytes showed a higher survival after vitrification

than did the non-loaded controls. However, no

data on further development and birth were given.

Arav et al

also showed that there is a difference in

lipid phase transition, and the temperature at which

this phase transition took place was directly related

to the effect of temperature on the plasma membrane.

Phase transition is simply the progression from one

phase or physical state to another. For example from

liquid to solid or from liquid to gas. During cooling,

‘liquid’ water will progress to ‘solid’ ice at a warmer

temperature than would oil or lipids, or the cryopro-

tectants propanediol and DMSO. Differences in lipid

phase transition were observed between in vivo and

in vitro matured oocytes. This suggests that alteration

of membrane composition affects chill sensitivity, and

subsequently survival following freezing. Nagashima

et al

showed that chilling sensitivity of porcine

embryos was directly related to their lipid content.

Embryos that were partially or fully delipidated sur-

vived cooling better than control non-delipidated

embryos, and delipidated non-cooled embryos did

not differ in their development compared with

control embryos. In a more recent manuscript,

Beeb et al

noticed that early stage porcine blasto-

cysts could survive vitrification and produce piglets

following centrifugation of lipids within the embryos

prior to cooling. This was the first report of piglets

from frozen early-stage blastocysts with lipid removal.

It may be possible that the variation found in in vitro

human embryos is in part due to the amount and

distribution of lipids found in the cell membranes.

In accordance with this hypothesis, Ghetler et al

showed that human zygotes had a higher resistance

to chilling injury compared with oocytes of differ-

ent stages of maturation. Since many species that

have been studied to date exhibit variable, and more

specifically, higher lipid content in the cell mem-

branes of in vitro derived embryos compared to their

in vivo counterparts, it is reasonable to suppose that

human in vitro derived embryos may also exhibit

this variation.

INTRA- AND INTERSPECIES DIFFERENCES

The effects of cryopreservation on intracellular organ-

elles may not be limited to in vitro produced embryos.



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