Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

maternal age, patient’s previous history, and basal

FSH levels. An evaluation of even the most recent

literature shows that a great deal of confusion still

exists about the usefulness of assisted hatching, and

to which patient population it should be applied.

There is no general consensus of agreement about

the appropriate technique, or stage of development.

In 2006 assisted hatching was reported to be of no

confirmed benefit in good prognosis patients;

however, this had already been acknowledged by

the team who developed the original technique in

1992.

Since that time there have been numerous

papers arguing that there is no benefit (but no detri-

ment either!) to the use of assisted hatching in

young and first time patients. Due to the complexity

of patient etiology and diversity in technique, meta-

analyses

11–15

have failed to demonstrate a gain from

artificially opening the zonae of fresh embryos. This

is hardly surprising, since few (if any) studies allow-

able for meta-analysis are comparable – many are

unsuitable for analyses because of different selec-

tion and technical criteria. Conclusions vary from

demonstrating significantly improved odds of in-

creasing the clinical pregnancy rate, to no evidence

of an increase in birth rates. One single independent

meta-analysis study did show a demonstrable effect.

Although the different hatching techniques seem

comparable, minor technical differences could

account for the contradictory results. Over the past

2 years alone, two prospective randomized studies

were carried out on the use of laser guided assisted

hatching in frozen–thawed embryos. Balaban et al.

showed a clear improvement in pregnancy and

implantation rates, whereas Ng et al.

in an appar-

ently similar study, showed no effect at all. Three

other recent comparable non-randomized studies

of frozen embryo assisted hatching also disagreed;

one argued that assisted hatching had a positive

effect,

but this was not confirmed by two other

groups.

19,20

A critical analysis suggests that these

studies were performed with substandard approaches

to assisted hatching. In the first study

a quarter of

the zona pellucida was removed before embryo

freezing. Zona opening before freezing at the cleavage

stage is known to significantly decrease implantation

after thawing.

The other group

used a proteolytic

enzyme to thin the zona pellucida – a technique that

is rarely applied and therefore not comparable to

assisted hatching involving a full breach of the zona,

since the toxicological consequences of enzyme

breaching are unknown in experimental models.

In contrast to the published literature for ICSI,

only approximately 275 scientific papers have been

published about assisted hatching; it is therefore

assumed that the technique is not widely practiced.

Whereas there is a general consensus about the

efficacy of ICSI, any review of the literature demon-

strates that this is clearly not the case with assisted

hatching. A large proportion of studies have eval-

uated minor changes in the technical protocol

without emphasis on clinical efficacy. Five meta-

analyses on assisted hatching have been reported:

four from the same group, published over a period

of 4 years (three in the same journal), to some extent

re-evaluating a small proportion of acceptable pros-

pective randomized studies.

22–26

The consensus of

earlier reviews

11–15

supports our conclusions from

four randomized trials carried out in 1993:

assisted

hatching may improve outcomes in poor prognosis

patients, particularly in the case of maternal aging.

Two further meta-analyses

11,12

conclude that there is

no clinical benefit to assisted hatching. Proof of effi-

cacy was only attainable when patients were selected

based on maternal age or prior failed attempt.

Conflicting opinions may be due to the problems

inherent in randomizing unselected or partially selec-

ted populations. In our experience with over 10 000

patients, the effect of selective assisted hatching in

certain low prognosis patients is not noticeable

because we are unable to select against suitable con-

trol patients; with experience gained from random-

ized studies, differing selection criteria and a drift in

technology may cause a deviation in overall results.

In addition, applying selective assisted hatching to a

much larger number of embryos and patients than

was indicated by the initially accepted criteria may

result in a dilution effect. Based on the supposition

that a detrimental effect of assisted hatching has

never been shown, practitioners may have loosened

their criteria over time. In comparing controlled

studies, there is no effective means of compensating

for study design differences such as those caused by

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age cut-off, opening, thinning, and subtle variation

in methodologies and number of attempts. Hence,

the comparisons become blurred, in the same

manner that results from attempting to evaluate the

parameters of a single large IVF database with spe-

cific groups represented in both experimental and

control arms. In addition, embryo culture technol-

ogy and follicular stimulation, which are both the

most likely factors affecting embryonic health and

associated zona changes, have improved spectacu-

larly during the past 15 years. This has possibly

reduced the need for assisted hatching in sub-groups

of patients; however, the exact contribution of this

change in technology on hatching behavior needs to

be determined. Papers reviewing assisted hatching

reveal that there is a lack of understanding regard-

ing the nuances of techniques within the different

methodologies used. There are only a certain num-

ber of appropriate ways of carrying out each tech-

nique, whether it involves the use of laser, acidified

Tyrode’s solution, enzymes, mechanical opening,

or some other derivative technology. However, no

guidelines for this exist in the literature. Indeed, this

is the case not only for assisted hatching, but for any

applied medical technology. Most practitioners real-

ize that there are ways of doing suboptimal IVF and

good IVF, and likewise, there is good assisted hatching

technique and suboptimal assisted hatching tech-

nique. Meta-analysis evaluations are not able to weigh

these important aspects. Indeed nor can this reviewer

provide a more exact evaluation of the problem, but

nevertheless as the one who introduced assisted

hatching, I would like to take this opportunity to

share some general observations, without evaluating

each of the assisted hatching papers one by one. Pre-

vious attempts to do so illustrate both the difficulty

in determining the efficacy of assisted hatching, and

the lack of consensus among practitioners.

22,23

The most common hatching method used is zona

drilling, opening the zona pellucida with acidified

Tyrode’s solution.

The zona reacts to a pH of about

2.3: at pH ⬍2.3 the zona will disappear as a coher-

ent structure, whilst it remains intact at pH ⬎2.3. In

this respect it is important to consider the buffer

solution that the embryos are maintained in during

the procedure. Buffers such as HEPES are excellent

in locally maintaining pH and have high buffering

capacity. These solutions are more forgiving than

bicarbonate buffered systems, which have reduced

buffering capacity. Another consideration is that in

being aware of the aggressive nature of acidified

solution, technologists may not necessarily consider

the physics of the system, and therefore often like to

perform the procedure gently and carefully. Also, a

hierarchy of teaching from developer to practi-

tioner, as was the case with ICSI is largely absent;

zona opening has unfortunately been interpreted

as a very simple procedure, and the technique was

applied directly by reading the original publica-

tions, without communicating with the experienced

groups. While the first descriptions had some depth,

it has become apparent over time that subtle aspects

were not described in detail. Hence a number of

interpretations of the procedure have been applied,

and its perception as an easy technique has largely

prevailed. One example is that many embryologists

will not deposit the acidified solution directly on the

zona pellucida by keeping the microneedle pressed

on the zona. This may seem aggressive, but it has the

specific purpose of limiting the release of acidified

solution further. While the zona is dissolving, the

microneedle should be moved into the thinning

area. Releasing acidified solution from a microneedle

that is kept at distance causes a broad stream of

acidified medium that will not sufficiently lower the

pH below 2.3, with the result that larger quantities

of acidified solution are released because of perceived

carefulness. This is likely to affect cells adjacent to

the manipulated area.

Similar considerations can be applied to the

use of a laser; original clinical applications do not

refer to possibly detrimental effects due to details

of methodology. The same arguments can be app-

lied to opening the zona pellucida for biopsy pur-

poses. Less than half of all the embryos created after

assisted reproduction appear genetically normal

(see Chapter 18), but implantation rates are gener-

ally lower than that, indicating that a number of

other factors must be involved. Assisted hatching

promotes earlier implantation and may therefore

elevate the chance of implantation by optimizing

the implantation window.

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Assisted hatching can potentially be applied to

any embryo, but in the absence of consistent results,

many clinics have been slow to introduce it, or have

abandoned its use. Although our own practice indi-

cates that assisted hatching improves implantation,

controversy continues to impede its widespread

and dependable application. Inconsistent results are

invariably due to technical variations and subtle

changes to the originally published protocols; the

initial learning curve with this technique must be

emphasized, because individual and team results

may improve considerably after experience. Complete

removal of the zona pellucida prior to compaction

may lead to loss of cells due to the absence of struc-

tural space. At the blastocyst stage, the proportion of

human expanded blastocysts growing in vitro that

will hatch may be related to the quality of the cul-

ture system, and the frequency of successful hatch-

ing in vitro is enhanced by zona opening in at least a

proportion of in vitro studies. Common technical

problems that we have identified include:

(1) Lack of immunosuppression

in patients whose

embryos are zona drilled at the cleavage stage;

(2) Excessive use of acidified Tyrode’s solution by

keeping the micropipette more than 5 ␮m away

from the zona pellucida, thereby lowering the

pH over a greater area than is required for zona

piercing as described above;

(3) Use of hand-controlled suction devices for

release of acidified solution – insufficient visual-

ization of the fluid impedes its controlled release;

(4) Creating holes smaller than 10 ␮m, which may

trap the embryos during hatching;

(5) Creating holes larger than 25 ␮m, which may

lead to cell loss during or after embryo transfer;

(6) Inappropriate trans-cervical embryo transfer

method, so that excess zona pressure is created

during replacement;

(7) Inappropriate or inconsistent use of laser.

At least five different assisted hatching methods

have been used, with varying degrees of success:

(1) Mechanical partial zona dissection on day 2, 3

or 5;

(2) Zona drilling with acidified Tyrode’s solution

on day 2 or 3;

(3) Laser assisted drilling on day 3 using an infrared

non-contact laser;

(4) Zona drilling with acid or laser at the blastocyst

stage.

(5) Piezo-mediated drilling has also been used in

animal and human models, and might in future

be considered an alternative for clinical applica-

tion.

However, results in our laboratory have

shown that excessive use of piezo devices is detri-

mental to embryo development in the mouse.

In our experience, the first method of mechanical

partial zona dissection tended to produce gaps

which were too small, possibly resulting in cell

separation during the escape from the zona pellucida;

this approach was therefore abandoned.

Mouse

experiments and clinical studies from polar body

biopsies suggest that creating a second perpendicu-

lar gap may be beneficial.

Larger mechanical open-

ings were also studied to some extent, and may be

helpful.

If artificial gaps are too small, the blasto-

cyst may become trapped during hatching; recent

findings following embryo biopsy demonstrated a

link between gap size and monozygotic twinning,

but the rate of monozygotic twinning was not

increased when the incidence was compared with

the frequency in the natural population.

Gaps

created for biopsy are usually 2 or 3 times larger

than those created for assisted hatching.

SELECTION FOR ASSISTED HATCHING

Our current policy is to apply assisted hatching to

a large proportion of both IVF and ICSI patients,

with the exception of those in whom traumatic

transfers are anticipated, and those who do not con-

sent to the procedure. A number of programs have

simplified their selection criteria by using a patient

age cut-off limit. Other selection criteria include all

patients who have previously failed IVF or ICSI,

or who are otherwise considered to have a poor

prognosis for achieving a pregnancy.

Guidelines for selecting individual embryos for

assisted hatching depend upon a number of variables.

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Hypothetical cut-off levels above or below which

the chance of implantation is considerably reduced

are related to maternal age, elevated FSH, zona

thickness, percentage fragmentation, and number

of blastomeres. However, ultimately the selection

process remains a clinical rather than a scientific

decision. One guideline suggests that embryos from

patients with elevated basal FSH levels should

always be manipulated, regardless of other evalua-

tions, but this argument is not supported by solid

data. Embryos from patients 38 years or older may

also benefit from zona drilling. A truly thick zona is

defined as having a mean zona pellucida greater

than 18 ␮m, but this number is dependent on the

duration of follicular stimulation and maternal age

as well as the instrument used for observation.

Assisted hatching can also be used for embryos with

a detrimental degree of fragmentation. Patients

with previous failed treatment after apparently nor-

mal embryo transfer without zona drilling are also

considered for assisted hatching.

TECHNIQUES FOR ZONA OPENING:

ACIDIFIED SOLUTION

Acidified Tyrode’s solution is used in embryo biopsy

procedures, and therefore it is also widely used

for zona drilling. The diameter of needles used

for directing the acidified solution ranges from 10 to

12 ␮m. The microneedle is front-loaded with acidi-

fied Tyrode’s solution before each hatching event,

precisely controlled with mouth suction. This precise

control is especially important, because the menis-

cus of the acidic fluid is difficult to manipulate.

Although heated stages may be used, the optimal

temperature for this or any other type of embryo

micromanipulation is not known.

The embryo should be pre-aligned with the

holding pipette only, so that there is an area directly

subjacent to the region to which the acidified solu-

tion will be directed (usually the 3 o’clock position)

that shows either an open area between blastomeres,

an area of unusually large perivitelline space, or an

area with a concentration of fragments. The likeli-

hood of a small amount of acidified solution

expelled into the perivitelline space coming into

immediate contact with the surface of a blastomere

before being aspirated back into the hatching needle

is therefore diminished. When the embryo is appro-

priately positioned with the use of the single tool,

the micropipette filled with acidified solution is

lowered into the medium and brought adjacent to

the target area as quickly as possible. This is

intended to avoid diluting the acidified solution

with medium of normal pH: releasing fluid that

is insufficiently acidic will jeopardize the embryo

without affecting the zona pellucida. The key to suc-

cessful assisted hatching is producing a gap in the

zona whilst minimizing the impact of exposure to

acidified solution on the embryo. Since the acidified

Tyrode’s solution is aspirated through a needle of

very small diameter, considerable residual suction

(lower pressure) remains in the hatching needle

even after it is removed from the reservoir drop of

acid solution. When the hatching needle enters the

droplet of medium containing the embryo to be

hatched, this residual suction will aspirate culture

medium, so that a column of neutral pH medium

will therefore be at the tip of the hatching needle,

diluting the upstream acidified Tyrode’s solution.

This technical problem can be prevented by prepar-

ing the system in advance, so that the hatching nee-

dle will be in precisely the correct position relative

to the embryo when it is lowered into the medium

droplet. There should be a delay of no more than

2 seconds between the time that the hatching needle

enters the drop until the fluid is expelled to initiate

hatching. A slight positive pressure in the hatching

needle, applied as soon as the needle breaks the

surface of the drop when it is lowered, will also serve

to counteract the residual suction. The procedure

should be ceased if thinning is not immediately

obvious. The acid solution should be expelled force-

fully, so that the hole is made as quickly as possible,

minimizing the time of acid exposure. The total

time required to breach the zona should not exceed

a couple of seconds; most zonae will yield in fewer

than 5 seconds. The needle should be applied directly

to the zona pellucida, massaging the area to be

opened while the hole is being made, with the nar-

rowest point created at the interior surface of the

zona. The massaging motion should create a hole

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that is nearly rectangular. The inside layer of the zona

pellucida is frequently more resistant to reduced pH

than the outer layers, and care should therefore be

taken to ensure that zona breakthrough occurs over

a sufficiently wide area of at least 20 ␮m, and not at

a single small point.

The flow through the assisted hatching needle

must be immediately reversed as soon as the zona is

breached. This is most effectively achieved via the

use of a mouth controlled suction tube, and for

this reason mouth pipetting may be preferable for

assisted hatching. All of the expelled acidified solu-

tion must be aspirated, paying particular attention

to any solution that may have entered the peri-

vitelline space. The embryo must be simultaneously

moved to another area of the droplet, away from the

area of reduced pH.

ASSISTED HATCHING WITH LASER

Non-contact infrared (IR) laser has emerged as the

methodology perhaps best suited to mammalian

zona cutting applications.

Several FDA approved

commercial systems are now available that use IR

diode lasers, and these have been used in human

clinical embryology procedures such as assisted

hatching and biopsy. However, some of the basic

models that have been used to investigate the

infrared delivery system are questionable, and local-

ized effects such as heat have been assessed only in

other laser systems (for review see Malter et al).

Early clinical studies have been generally hampered

by lack of appropriate controls. FDA studies have

been conducted, but the study designs have in some

cases been suboptimal, as comparisons were made

between laser manipulated embryos and non-

manipulated control embryos.

This confounds the

effect of zona opening per se with any effects of the

laser. A more appropriate study would be to com-

pare the laser technique with other methods for

mechanical or chemical zona opening.

Recent studies using laser applications have

focused only on the efficacy of the method, with

safety evaluated in terms of pregnancy rates. The

results reported appear promising, and apparently

demonstrate simple, repeatable, and appropriate

zona ablation with no obvious detrimental effects

reported.

33,34

Two recent randomized studies show

no real consensus;

16,35

one study suggests that laser

assisted thinning is of no benefit in patients older

than 36 years, whereas the other study shows a sig-

nificant improvement in patients whose embryos

have been thawed. Earlier studies did show a possible

benefit in terms of implantation and clinical preg-

nancy rate, and a relatively large group of healthy

babies have now been born following laser-mediated

assisted hatching. However, basic safety studies are

rare, and those that exist

mainly do not involve clin-

ical applications. Whether these tools are safe and can

improve pregnancy rates is yet to be determined.

REMOVAL OF FRAGMENTS AND LYSED

CELLS AFTER CRYOPRESERVATION

Small fragments or lysed cells can be aspirated with

the hatching needle after the gap in the zona pellu-

cida has been created. Using a mouse model, in 1993

Alikani et al were the first to explore the possibility

of removing degenerate blastomeres from embryos

before compaction.

They also described that clini-

cal pregnancies could be established after removing

lysed cells from human embryos, but the efficacy of

this procedure was not demonstrated. Data sets of

embryos transferred with lysed cells remaining inside

their zonae were recently compared with embryos

the lysed cells of which had been removed, and this

comparison showed a remarkable improvement in

implantation after fragment removal.

The clinical benefit of this procedure is still

debated,

36,38

but promising results have now been

reported after removing lysed cells from thawed

embryos before transfer.

37,39

The technique requires

a high level of skill in order to remove all or most of

the fragments and lysed material from an embryo

via a single hole without causing damage. Great cau-

tion must be exercised, since even the slightest touch

of the hatching needle on the membrane of a blas-

tomere can result in the loss of membrane integrity.

Since 1993, we have removed fragments and

lysed cells from the embryos of more than 3000

patients. A 12 ␮m diameter needle is preferred for

fragment removal, compared with the approximate

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10 ␮m of a regular assisted hatching needle. If the

membrane of a blastomere appears to be reacting

in any way during the procedure, suction must be

instantly ceased. Some fragments and lysed debris

may be firmly attached to blastomeres, and removal

may be counter-productive in such cases. The pro-

cedure can be time-consuming, and should be done

gently and patiently. Since fragments are not all in

the same focal plane as the pipette tip and artificial

opening, the microscope must be continually

adjusted in order to focus on target material and

adjacent blastomeres. The zona should be turned

after removing some of the fragments, as a different

angle of approach increases the likelihood that more

fragments can be removed. Fragments in between

blastomeres and those opposite the aperture should

be removed last. Removing fragments from areas

between cells is especially advantageous, because

this apparently improves cell–cell interaction during

compaction.

Fragment and debris removal should only be

practiced at the highest magnification, using a state-

of-the-art interference microscope. An awareness of

the distinct patterns of fragmentation is essential,

along with continuous re-focusing and moving the

zona around while determining the best approach.

Most of the benefits of assisted hatching and frag-

ment removal lie in creating the artificial gap in the

zona pellucida. The benefit derived from fragment

removal over and above that of assisted hatching

alone is real, but modest in comparison. It is proba-

bly unwise to spend a great deal of time attempting

to remove only a few fragments on the periphery of

an embryo, whereas it is more appropriate to take

time in removing larger numbers of fragments,

especially if they are present in an orientation that

may interfere with normal cell–cell contact and

impede compaction.

MONOZYGOTIC TWINNING AND

ASSISTED HATCHING

The incidence of monozygtic twinning is quadrupled

after follicular stimulation with or without assisted

reproduction,

and this has not been described in

relation to all aspects of ART. Follicular stimulation,

poor embryo development, assisted hatching, and

blastocyst development have all been described as

risk factors, but evidence of direct associations is

largely missing. In the case of blastocyst transfer, the

association may be related to changes in the zona

pellucida or the ability of the embryo to prepare for

hatching, but this may be due to subtle aspects of

embryo culture and could be remedied with improved

culture conditions. The incidence of identical twin-

ning after blastocyst transfer may also be reduced by

removing the zona mechanically or enzymatically,

or by performing a partial yet vigorous opening

procedure. The association between assisted hatching

and monozygotic twinning is complicated by con-

founding factors such as selection of patients and

embryos. Again, the association between mono-

zygotic twinning and micromanipulation may be due

to technical variations – a more constraining zona

opening is likely to lead to trapped or split embryos.

This is illustrated by the observation that only 1/140

(0.7%) embryos that were biopsied developed into

monozygotic twins.

Adapting the procedure tech-

nically so that a larger zona opening is created

might therefore reduce nearly all the factors that are

associated with trapping and splitting.

EMBRYO BIOPSY

PGD can now be performed at nearly any stage

between egg maturation and cavitation of the blas-

tocyst. The validity of PGD and its scope of applica-

tion in relation to embryo selection is a complex topic

that requires broad discussion, and is described

elsewhere in this book by Munné and by Kuliev and

Verlinsky. The general interest in the biopsy tech-

nique itself and in understanding aspects of the

culture system that may play a role during micro-

manipulation for PGD is rather limited. Few studies

have emphasized these aspects, in spite of a genuine

and broad interest in the genetic aspects of PGD.

Biopsy of the cleaved embryo on day 3 is the

method preferred by most clinics that are interested

in PGD, and this is discussed briefly here. The zona

can be opened for embryo biopsy at the cleavage

stage in three different ways, and at least two methods

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can be used to extract a blastomere through the

artificially created gap.

40–44

The zona can be opened

mechanically (but this is rarely applied) via the use

of acidified solution (see Assisted hatching section

above) or with the use of a laser. The targeted blas-

tomere can be extracted using a biopsy needle that

absorbs the cell partially or completely and releases

it elsewhere in the droplet, or it can be freed from

the embryo by applying pressure to the zona pellu-

cida with a thin tool that either enters, or does not

enter the zona pellucida. The latter is a single tool

technique, and is often used in combination with a

laser. Surprisingly, there are no randomized studies

that compare any of these techniques, although

serial observations have been described.

The cir-

cumstances of biopsy have not been studied in great

detail. Despite its widespread use, there is no infor-

mation available from the literature regarding the

optimal use of Ca- and Mg-free medium and any

safety aspects. There is a consensus of agreement

about its technical advantages, since blastomere sur-

vival is clearly enhanced. Other aspects of the tech-

nique which lack information include the use of

buffered culture systems, optimal temperature,

and appropriate guidelines for targeting a certain

blastomere, although it is agreed that the blas-

tomere should not be dividing and should have a

single clear nucleus. Whether polarity exists that

predisposes blastomeres to the inner cell mass and

trophoblast at the 8 cell stage is uncertain. Cryo-

preservation studies involving models of cell sur-

vival indicate that early allocation is either absent or

has little clinical effect after biopsy.

45,46

The most important question regarding embryo

biopsy is the possible effect on further development

and outcome if an associated genetic diagnosis was

not performed. In other words, does a single blas-

tomere biopsy affect the embryo and is this loss

compensated in one way or another by the subse-

quent genetic diagnosis? Certainly, the notion that

PGD would be successful and result in acceptably

high pregnancy rates after testing for single gene

disorders was assumed many years ago, but results

in clinical practice have been disappointing.Although

the effects of cell loss after biopsy can be tested

by performing prospective analysis of intact and

biopsied embryos without a genetic diagnosis,

such experiments present an ethical challenge. The

proposition that blastocysts obtained after single

cell biopsy have cell numbers proportional to their

initial cell count is often quoted as showing that the

embryo can lose a cell without detriment, but those

experiments were performed with embryos showing

good development obtained from young donors.

IVF specialists often defer that the number of cells

on day 3 is not 8 cells on average, but a lower number

that is dependent on patient selection and other fac-

tors. A discussion about this and the consequences

of losing one instead of two cells from embryos has

been discussed in the chapter by Munné in the

context of PGD efficiency.

OTHER CONSIDERATIONS

Reproductive medicine is singled out in most

countries as the only form of clinical practice that

requires legislation. Many apparently secular govern-

ments appear to introduce contemporary limita-

tions that are sanctioned by organized religion. The

field of reproductive cell surgery apparently causes

great concern among lawmakers and ethicists alike,

especially since it involves the tools that enable

scientists to perform nuclear transplantation.

However, there are advantages to be gained from the

use of similar technologies. Transferring a nucleus

into an oocyte, such as is the case with ICSI, is

cloning derived technology, as are the attempts by

reproductive specialists to transfer haploid sperm

precursor cells. Removing cells from embryos during

PGD (a technology now prohibited in several

European countries) is yet another application where

experience from cloning technology has been advan-

tageous. The opportunity to treat infertility using

micromanipulation for embryo selection should

not be confused with or mistaken for cloning or

alteration of the genome in an as yet undefined and

possibly eugenic direction (genetic engineering).

Nothing could be further from the truth. These pro-

cedures aim to enhance development and reduce

multiple pregnancy, consequently improving condi-

tions for the developing fetus and the mother.

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Just as the success of advanced surgery depends

upon the skill of the surgeon, successful duplication

of embryology procedures depends in great measure

on mastering the skills of micromanipulation, and

on the experience and expertise of the individual

embryologist. This appears to be especially the case

with procedures such as assisted hatching, fragment

removal, and embryo biopsy. The field of microma-

nipulation in embryology is just as exciting as that

of surgery, perhaps even more so – and may ultimately

lead to more widespread use of some of the tech-

niques described here. It is naturally to be expected

that new, and perhaps surprising, applications will

be discovered that may extend beyond infertility.

This timeline is not controlled by patients or their

doctors, and it is not dependent on scientists or their

lack of funding. It is almost entirely constrained by

the attitude of powerful religious extremists.

ACKNOWLEDGMENTS

I am grateful to Santiago Munné, John Garrisi,

Mina Alikani, Tim Schimmel, Steen Willadsen, and

Henry Malter for countless contributions. I am

thankful for editorial comments by Rashmi Dalai.

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INTRODUCTION

Implantation is one of the least well understood

aspects of assisted reproduction. Over the past 20

years embryologists have improved culture tech-

niques and physicians have refined stimulation

protocols and optimized transfer techniques. Still,

the fate of embryos after transfer remains largely a

matter of speculation and hope. ‘Implantation’ is

actually a complex, choreographed dialog between

the embryo and the waiting endometrium. Pros-

taglandins, cytokines, integrins, and growth factors

are all reported to play a role.

The suppression of the maternal immune system

is critical to successful implantation. The body

discriminates self from non-self by the human

leukocyte antigen (HLA) on the major histocom-

patability complex (MHC), encoded by a series of

grouped genes on chromosome 6.

The MHC elicits

T cell mediated immune responses to allogenic (non-

self antigens expressed by another individual of the

same species) MHC antigens. This phenomenon,

first recognized in tissue transplantation experiments

in mice, may also play a role in the natural state,

since the mammalian fetus is a semi-allograft in

the maternal uterus due to paternal MHC antigens.

During normal pregnancy, the maternal immune

system undergoes changes that lead to fetal tolerance.

A growing body of literature suggests that HLA-G,

produced predominantly by extravillous cytotro-

phoblasts (which are the only fetal cells in direct

contact with the maternal decidual cells) confers

immunotolerance through interaction with mater-

nal decidual lymphocytes.

Immunolocalization

studies of trophoblast invasion indicate that

invading trophoblasts express HLA-G in a highly

regulated process. The effects of growth factors and

cytokines on trophoblast invasion suggest that

molecules of uterine origin can modify the process

of implantation.

HLA-G is expressed on placental extravillous

cytotrophoblast cells in direct contact with maternal

tissues. Circumstantial evidence suggests HLA-G

protects the fetus from the maternal immune

response and appears to be the functional analog of

mouse Qa-2 protein.

Using reverse transcriptase

polymerase chain reaction (RT-PCR) on surplus

embryos and unfertilized oocytes from IVF patients,

HLA-G heavy chain mRNA was detected in 40% of

148 blastocysts tested. HLA-G was also detected in

unfertilized oocytes and early embryos, but not in

control cumulus oophorus cells. HLA-G levels in the

preimplantation period correlated with the cleavage

rate of embryos.

In contrast to classical HLA-A and -B class Ia

genes that are down regulated in human trophoblast

cells, HLA-G is expressed by the placenta through-

out gestation. In addition to extravillous cytotro-

phoblast, HLA-G expression was also observed in

endothelial cells in the chorionic villi, as well as in

amnion cells and amniotic fluid.

Both membrane-

bound and soluble HLA-G isoforms were identified,

raising the possibility that membrane-bound HLA-G

is involved in regulation of chorionic villi angiogene-

sis and that soluble HLA-G (sHLA-G) isoforms may

act as specific immunosuppressors during pregnancy

(Figure 12.1).

This chapter reviews the role of sHLA-G in

implantation and how assessment of sHLA-G secre-

tion from preimplantation embryos can influence

the outcome from assisted reproduction. We first

briefly review the identification and characteriza-

tion of HLA-G, examine the role of HLA-G on

implantation, and, finally, evaluate the effect of

12. Assessment of soluble human leukocyte

antigen G in human embryos

Jeffrey D Fisch, Levent Keskintepe, and Geoffrey Sher

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