Micromanipulation as a Clinical Tool ppt

It is used in biopsy for preimplantation genetic diagnosisPGD, intra-cytoplasmic sperm injection ICSI, assisted hatching, and inother more controversial areas such as egg freezing via zy

Micromanipulation as a Clinical Tool

Jacques Cohen

Tyho-Galileo Research Laboratories and Reprogenetics, West Orange,

New Jersey, U.S.A

INTRODUCTION

Micromanipulation involves a well-integrated set of technologies in assistedreproductive technology (ART) Its applications are diagnostic as well astherapeutic, and it is practiced in mature gametes and all stages of preimplan-tation embryos It is used in biopsy for preimplantation genetic diagnosis(PGD), intra-cytoplasmic sperm injection (ICSI), assisted hatching, and inother more controversial areas such as egg freezing via zygote reconstitution,cryopreservation of isolated testicular spermatozoa, and cytoplasmic ormitochondrial transfer for reversal of cytoplasmic and potentially nuclearincompetence (1–6) In this context, it becomes increasingly difficult to dis-cuss micromanipulation as a separate subject In fact, the need for a separateassessment appears almost artificial

When different fields merge in science, exciting developments can beexpected This has occurred numerous times in ART, first in the integration

of biochemistry and reproductive endocrinology, and later when ogy and applied genetics emerged as tools to improve efficiency and safety

cryobiol-No reproductive specialist could have predicted that the field of mental micromanipulation would have such an enormous impact on assistedreproduction less than two decades after the first relatively simple butelegant applications appeared (1–3) Since then, hundreds of thousands of

experi-283

babies have been born worldwide from micromanipulative methods aimed

at alleviating male infertility and enhancing implantation and the exclusion

of chromosomal and single gene disorders Some laboratories now havethree or more complete stations for micromanipulation Rather than havingsome embryologists sub-specialize in the area of micromanipulation, thepractice in some laboratories, most embryologists now aim to become pro-ficient in one or more micromanipulation techniques

The main emphasis here will not be already integrated procedures such

as ICSI and in part embryo and polar body biopsy, which are covered indepth by other chapters of this book, but on other innovative techniques.The different procedures and concepts will be discussed in two sections;the first will focus on gamete micromanipulation and the second will dealwith the manipulation of embryos It is also important to note that thetopics discussed in this chapter are often considered controversial or unpro-ven and are seen by some as hazardous to clinical care and even the humanspecies at large No review would be complete if it does not refer to the alter-native opinion A recent 2004 opinion by Cummins gives a very differentperspective of some of the technologies described below (7)

GAMETE MICROMANIPULATION

Cryopreservation of Spermatozoa Under Zonae

Men who are azoospermic can be successfully treated through surgical lation of spermatozoa from their testicles or reproductive tract (8–10).Repeated surgical procedures, however, may not only be costly but invasive,especially in the case of testicular sperm extraction (11) Repetition can, insome cases, be avoided by normal cryopreservation of spermatozoa, butonly when sufficient numbers of functional cells are isolated (12,13) Freezingmethods are now available that can freeze and recover very few or even sin-gle spermatozoa, and avoid the need for repeated surgical sperm extraction(4,14,15) Single sperm can be frozen by insertion of spermatozoa into ani-mal or human evacuated zonae pellucidae or through variations of thismethod such as freezing in cryoloops

iso-Recovery rates of spermatozoa frozen and thawed in evacuatedhuman or animal zonae are high, with motility recovery rates in excess of75% (4,15) In standard freezing protocols, centrifugation is essential But

in this protocol, washing can be accomplished by individually pipettingand removing the cryoprotectant

In addition to reducing the need for repeated sperm retrievals andperhaps donor sperm, this approach has the advantage of avoiding theuncertain outcome of surgical extraction by freezing spermatozoa andretrieving eggs at different times (4,16) The time-consuming search for sper-matozoa can be conducted independent of an egg retrieval

Details of Procedure and Choice

of Technical Details

Micromanipulation can be performed using polyvinyl pyrrolidone (PVP) as

a tool to slow the insertion of spermatozoa into the zonae and to withdrawspermatozoa from the zonae after thawing Two different solutions of PVPare recommended: (i) an 8 to 10% solution for sperm capture and insertioninto empty zonae (this is produced by a number of manufacturers with vary-ing results) and (ii) a 10 to 12% solution for sperm recovery from the thawedzonae The ICSI procedures using thawed spermatozoa should be per-formed at 37
C, but all other micromanipulations can be performed at roomtemperature in order to reduce sperm velocity and perhaps prolong survival.The microtools needed for zona opening, avoidance of zona collapse, cellextraction, and ICSI have been described (3,4)

The pilot experiments were conducted using spermatozoa from cal retrievals and spermatozoa not cryopreserved from men with normalsemen analysis to test the fertilizing ability of donated research oocytes byICSI Human-evacuated zonae can be obtained from multiple sources:immature eggs, unfertilized ICSI eggs, and abnormal embryos that werenot exposed to sperm suspensions

surgi-It was shown that two small incisions in the zonae improved extraction

of the egg ooplasm and insertion of the sperm cells into the evacuated zonae

by preventing the collapse of the zona during suction and excessive inflation.With gained experience, a single-hole technique may be effective as well Atfirst, holes were made chemically in some pre-fertilization zonae by releasingacidified Tyrode’s solution from a 10 mm open microneedle However, pro-gressively motile spermatozoa escaped, resulting in poor recovery rates Thiscan be avoided by cutting a hole in the zona mechanically using partial zonadissection with a spear-shaped closed microneedle Alternatively, one canuse a laser-mediated opening of the zona pellucida, but the efficiency of thisneeds to be shown in clinical trials (17)

Cytoplasm can be extracted using a larger micropipette, connected inturn to a suction device The zona is positioned so that one of the two inci-sions is at the 3 o’clock position The beveled microtool is inserted throughthe aperture using the sharp edge on the lower end The tool is movedthrough the oolemma, and the cytoplasm is fully aspirated until the zona

is empty The pipette is occasionally emptied outside the zonae as moremedium is sucked up to remove any sticky cytoplasm from the pipette tip.Mouse and hamster zonae as well as human eggs and embryos can also

be prepared in the same fashion

Spermatozoa can be released into the 10% PVP solution prior to tion into empty zonae They are individually taken from small 2- to 5-mLdroplets of sperm suspension using an ICSI microtool Spermatozoa can

inser-be injected into the empty zona while motile or can inser-be immobilized inser-before

injection and freezing (17) In the latter case, the spermatozoa remain viable

at a high frequency, but fertilization and pregnancy have not yet beendemonstrated Considering the absence of clinical data, motile spermatozoaare recommended for this purpose The use of human zonae, while appro-priate for creating strict xeno-free conditions, is problematic as spermatozoaattach to the inside of the zona pellucida and may immobilize before freez-ing Recently, we developed and tested a combinatorially derived ligandpieczenik peptide sequence 2 from an artificial target that specifically binds

to human zonae pellucidae (18) When injected inside evacuated humanzonae, this ligand efficiently prevented sperm attachment by possibly inter-fering with sperm/zona pellucida (ZP3) interaction Whether this ligandcould be used for the purpose of freezing spermatozoa in evacuated zonaeand maintaining high motility requires further evaluation

Injected zonae can be frozen in a simple 8% glycerol solution using

a phosphate-buffered solution supplemented with 3% human serum albumin.Alternatively, one can use buffer containing TES (N-tris[hydroxymethy]-methyl-2-aminoethanesufonic acid) and Tris (Tris[hydroxymethyl]amino-methane) yolk buffer, but the recovery rate is not superior compared to earlierstudies (16) The zonae can be frozen singly in standard plastic straws betweentwo small air bubbles to indicate their position One end of the straw is closedusing sealant polyvinyl alcohol powder, whereas the other end is heat sealed.The freezing procedure is based on a simple standard semen cryopreservationprotocol (4)

Thawed straws can be inserted into a medium droplet as the vation medium containing the zona slowly releases The zona is washed toremove cryoprotectant and moved into an ICSI dish containing droplets ofN-2-Hydroxyethylpiperazine-N0-2-Ethanesulfonic acid (HEPES) bufferedmedium and a central droplet of 10 to 12% PVP in supplemented intra-cellular solution Some sperm cells may show considerable motility withinthe zona The zona is positioned using the holding pipette and a PVP-filledICSI microtool so one incision and a sperm cell line up This allows for thepenetration of the ICSI needle and aspiration of the sperm cell (mechanicalrecovery method) Minimum suction is used for this process These cells can

cryopreser-be aspirated by positioning the needle at the contralateral side and applyingsuction when the sperm cell passes the needle aperture All spermatozoa areremoved and released gently in the PVP solution Any still motile can then beimmobilized for ICSI The mechanical sperm recovery method is preferred,and other methods involving zona digestion have proved less successful.Recovered sperm are washed and immediately injected into eggs; thaws should

depen-through the narrow mechanical PZD incisions, but some may be lost whenusing acidified Tyrode’s solution or laser opening Loss after thawing throughnarrow incisions may also occur because of inadvertent excess suction appliedthrough the holding pipette This can be avoided by visualizing both holesprior to micromanipulation and sperm recovery The rate of sperm lossthrough the incisions diminishes markedly with increased operator experience.

Motility Recovery and General Efficiency

Motility recovery rates are defined as the percentage of motile cells seen, andvary between 73 and 100% Rates of recovery over 80% are found in experi-ments involving rodent zonae and human zonae that are embryonic inorigin, or spermatozoa that are immobilized prior to freezing Some cellsthat are motile without marked velocity become progressively more motileafter aspiration and exposure to medium Some spermatozoa inserted intohuman zonae may become caught up in cytoplasmic remnants or crevicesinside the glycoprotein matrix, but this occurs less frequently when animalzonae are used Aggregation of spermatozoa also appears to inhibit individ-ual motility, but is avoided when three or less spermatozoa are inserted perzona The use of only one to three sperm cells per zona pellucida appearsoptimal, yet insertion of up to 15 spermatozoa has been reported (16).General Considerations of Single Sperm Freezing

Although single sperm freezing is performed with spermatozoa from menwith extreme oligospermia, average motility recovery rates are considerablyhigher than those generally reported for moderately abnormal semen and arecomparable to results of donor semen or moderately oligospermic patientstreated with a combination of cryoseeds and dithiothreitol (DTT) (19–21).The use of the zona pellucida or perhaps cryoloops as a vehicle avoids theknown loss in motility associated with post-thaw dilution and sperm washingseen in frozen donor semen It is possible that the presence of multiple sperm

in a small volume exerts an internally deleterious effect during freezing, ing, and centrifugation, which may be avoided by freezing sperm singly or insmall groups This method makes it feasible to perform surgical extractionsindependently from the time and place of egg retrieval

thaw-Another distinct advantage is that animal zonae, such as those frommice and hamsters, can be used for storage This application has beenrejected by some because of the desire to culture under xeno-free conditions.Although it is immunologically unlikely that there will be any cross-specieshazards or reactive compounds, it is possible that compounds may adhere tothe sperm cell and become incorporated in the oocyte Whether these haveany metabolic effects on the embryo has yet to be clarified Nevertheless, theabsence of reverse transcriptase appears evident, thereby rigorously reduc-ing the possibility of any process resembling transgenesis

The use of combinatorially derived ligands as described above for biting sperm–egg binding in the homologous application may be preferred,but should be tested in pre-clinical models first.

inhi-Clinical Application

The experimental procedure was first tested in an azoospermic patient whorequired testicular biopsy and had scant motile cells (22) Extensive searchingfor sperm yielded enough spermatozoa for ICSI Other motile spermatozoa(n¼ 23) were isolated and trapped into two evacuated zonae derived from thepatient’s immature eggs The patient did not become pregnant and the couplereturned for another ICSI attempt, but this time without sperm retrieval bytesticular biopsy One zona with 12 trapped spermatozoa was thawed first,washed, and prepared for sperm extraction All but one sperm could berecovered Ten were motile, and five were injected into the patient’s matureeggs four hours after egg retrieval The patient delivered healthy twins

A total of 104 procedures have been performed, and 52 of themreturned for transfer Of these, 49 had transfers and 26 became clinicallypregnant The implantation rate was nearly 25% There were three miscar-riages and two biochemical pregnancies Although a small data set, the work

is encouraging but awaits confirmation by other teams

Ooplasmic Transplantation

Transplantation of ooplasm for clinical application involves a set of imental techniques designed to address oocyte-specific deficits in a definedand limited group of infertile patients who previously failed ART (5,23).The first clinical application was based on oocyte donation, although theuse of homologous ooplasm or mitochondria from the patient’s cumuluscells has also been suggested (24–26) The latter proposal was first made

exper-by Tzeng’s team at Taipei Medical University in Taiwan and this has led

to the birth of more than 20 babies There are a number of ways to plant ooplasm, but the one initially used was a minor modification of thestandard clinical ICSI technique A small portion of ooplasm was trans-ferred from a donor to the patient’s oocytes Other groups have reported

trans-on the experimental applicatitrans-on of modified versitrans-ons of ooplasmic transfer,including the use of cryopreserved donor oocytes, polyspermic zygotes, andcumulus cell mitochondrial suspensions as the source of the donor infusion(24,27,28) In the work performed from 1997 to 2001 at Saint BarnabasMedical Center’s Institute for Reproductive Medicine and Science, twoinstances of abnormal karyotypes were reported following the application

of ooplasmic transfer (both 45, XO) One resulted in an early spontaneousmiscarriage and the second in an elective reduction at 15 weeks following anabnormal ultrasonography (29) A single case of pervasive developmentaldisorder (a spectrum of autism-related diagnoses which has an incidence

of one in 250 children) was also diagnosed at 18 months in a ooplasmic

transfer infant, a boy from a mixed sex twin Other centers applying mic transplantation have not reported any potential side effects This may

ooplas-be ooplas-because there were no side effects or the studies were incomplete Due

to the very small sample size, any direct connection between the ties and the technique itself has yet to be established (30)

abnormali-In 2001, we voluntarily agreed to cease clinical application of mic transfer pending the application and review of an investigative new drug(IND) protocol with the U.S Food and Drug administration The pre-INDprocess was concluded in 2003, but we did not pursue the application furtherfor non-clinical reasons The procedure was not prohibited as Schultz andWilliams (31) and others have suggested It is currently unknown whetherother groups have applied for a permit

ooplas-The clinical application and science of ooplasmic transplantation havebeen extensively criticized by ethicists and scientists (7,31–33) Some carelessarticles of the lay press, fueled by some scientists, have likened the technique

to gene transfer and described a detrimental artificial scenario likely to changethe genetics of mankind Several recent publications have presented viewpointsbased on a poor understanding of the issues The debate should be based on afactual understanding of the procedure and the clinical and scientific realitiesinvolved The technique’s underlying experimental nature must be stressed andthe many scientific and clinical ‘‘unknowns’’ that surround it must be presented.Ooplasmic Transfer Procedures

Preliminary clinical investigations applying the technique to a defined group

of patients with ‘‘normal’’ ovarian reserve who exhibit consistent mental problems and implantation failure have been published (29,34).The final 27 couples treated had exhibited a record of failure in over 95 priorassisted reproduction treatment cycles Unlike what some critics have sug-gested, this is an unavoidable confounding factor in attempting to designand conduct true controlled trials (33,35) In this case, the ‘‘control’’ treat-ment is already consistently known to result in failure and the cause is notunknown since embryo morphology was clearly marginal in all these cases.The 43% pregnancy rate and the delivery of 17 babies following ooplasmictransfer was characterized solely on this prior failure criteria The issue ofcontrolled trials is obviously complex, and to suggest that this is a simpledeficit that we have failed to address is incorrect There is a considerableethical issue in forcing patients to engage in treatment modalities that offerthem no hope of success for the sake of these trials In a subsequent study,patients with diminished ovarian reserve were treated unsuccessfully withcytoplasmic transfer (36) It is known that in these patients most eggs areaneuploid, a condition that is irreversible at the MII stage (37)

develop-Ooplasmic transfer was conceived as a simple but critical extension

of the standard egg donation protocol that is a clinical option availablefor patients It theoretically provides for a beneficial donor egg while

maintaining the patient’s genetic contribution The technique is suitable foreggs ‘‘having a normal nuclear genome, but ooplasm that is abnormal or defi-cient due to maternally mediated factors’’ (38) Recently, several authors haveincorrectly represented our publications as suggesting that ooplasmic transfer isbased on a correction of deficits in mitochondrial function or ATP content(32,33) One publication goes so far as to raise this purported ATP deficit as

a ‘‘straw man’’ argument and, in rejecting it, raises the question that ooplasmictransfer might not be ‘‘biologically plausible.’’ We have clearly stated in ourpublications that there are a multitude of causative factors potentially underly-ing ooplasmic-related developmental deficits Energy metabolism is certainlyone of these factors However, the entire concept of ooplasmic transfer is toinfuse the potentially compromised patient oocyte with a whole source of healthydonor ooplasm We have never suggested that a manipulation of ooplasmicATP or any specific factor underlies any positive effect of ooplasmic transfer.Others have suggested that mitochondria transfer from isolated cumu-lus cells would be a safer alternative, but we reject the direct comparison based

on the arguments outlined here (24,25) Mitochondria transfer has been sidered sub-optimal compared to ooplasmic transfer in one artificial mousemodel (39) It is likely that a subset of infertile patients exhibit reproductivedysfunction derived from oocyte-related deficits and that replacing compro-mised oocytes with donor substitutes is not only ‘‘biologically plausible’’but also a technique compatible with early development and pregnancy (40).Mitochondrial Issues

con-An area of controversy concerns the transfer and persistence of donor chondria following ooplasmic transfer The transfer of heterologousmitochondria was not detectable in the first ooplasmic transfer cases, andtherefore initial reports and discussions reflected this (38) The oosplasmictransfer protocol has included an analysis of mitochondrial DNA and, insubsequent treatment cycles, donor mitochondrial DNA was identified inpre- and postnatal samples derived from ooplasmic transfer offspring Todate, donor mitochondrial DNA has been positively identified in three ofthe 13 tested ooplasmic transfer babies (34) This suggests a heteroplasmic con-dition with two populations of mitochondria (donor and recipient) present.However, current scientific evidence does not support the concept thatooplasmic transfer-related heteroplasmy constitutes a potentially deleteriouscondition The only heteroplasmy that has been observed in a small subset ofooplasmic transfer patients is in the form of benign polymorphisms in thenon-coding hyper-variable region of the otherwise highly conserved mitochon-drial genome (34) This form of heteroplasmy is now known to be a commonphenomenon in the normal human population and may not have an associ-ation with mitochondrial disease or dysfunction (41) Indeed, this form ofbenign heteroplasmy may have little significance Many mammalian speciesare routinely heteroplasmic in the replication control region, and this

mito-heteroplasmy may be conserved and transmitted from generation to generation(42,43) This benign form of heteroplasmy is different from that associated withdeleterious coding region mutations related to ‘‘aging and in inherited mitochon-drial disease’’ as incorrectly suggested in one critical opinion (33) Selection ofyoung and healthy oocyte donors providing gametes for ooplasmic transfer isbased on the same criteria as standard oocyte donation Ooplasmic transferpatients have no unique risk of the transmission of such rare deleterious mito-chondrial DNA mutations as whole oocyte donation is the only other clinicaloption available for them Suggestions that ooplasmic transfer donors should

be uniquely screened for mitochondrial mutations are more logically directed

at oocyte donation where 100% of the mitochondrial genome is derived fromthe donor (32) No incidence of mitochondrial disease transmission has beenreported over 10 of 1000 of oocyte donation cycles, although it is expected that

1 of 8000 children will develop mitochondrial disease de novo

A substantial body of animal research has been concerned with themanipulative creation of heteroplasmy in mice and large animals [reviewed

by Malter and Cohen (29)] Although the opinion piece by St John (32) tions this research, it fails to point out the underlying fact that much of thisresearch is based on the efficient generation of hundreds of healthy hetero-plasmic animals that have been produced through cytoplasmic transfer andmaintained over 15 generations with no obvious developmental or physio-logical problems From a genetic standpoint, many of these experimentsare also based on a much more drastic heteroplasmic scenario as the mixedmitochondrial populations are essentially derived from two ancestrallydifferent species While one should not place tremendous confidence inmodeling a complex phenomenon through research animals, in this casesuch research has demonstrated that a much more extreme heteroplasmiccondition than would be possible through clinical ooplasmic transfer is com-patible with normal mammalian development Other basic research suggestsconsiderable flexibility in nuclear/mitochondrial interaction, particularlyamong primates In the type of cellular hybrid experiments also discussed

men-by St John (32), chimp and gorilla mitochondria could readily replace humanmitochondria (a cross-genus ‘‘mismatch’’), and create fully functional cellswith human nuclear genomes and non-human primate mitochondria exhibit-ing unremarkable mitochondrial protein synthesis and function (44).Although the specific nature of the heteroplasmy observed in a small fraction

of ooplasmic transfer offspring is not fully understood, an honest review ofcurrent research in this area does not suggest a potential for negative devel-opmental or physiological outcomes This obviously does not mean thatfuture patients should not be informed of the uncertainties that remain.Epigenetic Aspects and Animal Models

Ooplasmic transfer, by design, generates a recipient oocyte that containsdonor-derived components such as proteins, messenger RNAs, mitochondria,

and other cytoplasmic constituents In theory, this infusion of healthy donorcomponents can have a positive effect on important ooplasmic functionsduring early development However, a recent opinion piece by Hawes et al.suggests a potential for adverse developmental outcomes resulting from thesimple creation of a mixed ooplasmic state (35) This argument is based onabnormal developmental syndromes that have been identified in uniqueinbred mouse strains [reviewed by Malter and Cohen (29)] These syndromesresult from genetic incompatibilities between inbred strains apparentlymanifested via unique epigenetic events in the cytoplasm of their oocytesand early embryos.

Studies with these inbred strains have been critical to understandingthe early cytoplasmic genome modification events that create the maturefunctional embryonic genome (29) The epigenetic processing that occursduring this period is necessary for proper development This supports theconcept that positive developmental effects could be obtained by a moderateinfusion of healthy ooplasm, although other outcomes, including negativeeffects, are plausible as well The scenarios and experimental manipulationsinvolved with the manifestation of these aberrant developmental outcomesare also essentially compatible with normal development in the mouse(and other species) outside of these experiments In our own research, simi-lar manipulation to the early cytoplasm of F1 hybrid mouse embryosresulted in a significant improvement in certain developmental parameterscompared to non-manipulated controls [Levron et al (6)] It has been sug-gested that similar alleleic combinations for these unique incompatiblemurine gene products could be present in the human, yet there is no proof

of this

The aberrant developmental outcomes observed in the inbred mousestrains are unique epigenetic anomalies with clear genetic causes (45,46).These specific deleterious anomalies are unknown outside of the uniqueinbred combinations used and any other animal model system Inbred miceare genetically anomalous strains considered to be homozygous at all loci.They manifest a great variety of adverse developmental and physiologicalconditions including reduced fertility (29) Even between related inbredstrains, drastic differences in morphology, physiology, and behavior makedrawing cross-strain conclusions questionable Such strains do not constitutevalid models for complex processes (particularly those related to fertility anddevelopment) in other mammalian species Furthermore, many differencesbetween the developmental processes in humans and other mammalianspecies have been well characterized, including a recent finding which demon-strates clear differences in the methylation-based imprinting system (47) Infact, the authors of this study go so far as to suggest that concerns aboutthe aberrant epigenetic-processing (and resulting developmental effects)observed after in vitro manipulation in other mammalian species and modelsystems may simply not apply to the human

Therefore, to suggest, based on artefacts and tenuous evidence, thatdeleterious epigenetic combinations could likely be created by clinicalooplasmic transfer—where the small volume of infused ooplasm is derivedfrom another highly outbred, healthy, and fertile human donor—seemsspeculative at best Unfortunately, this is an example of a growing trend

in the use of questionable animal model systems to judge the efficacy andsafety of human procedures

Despite these points, considerable effort and financial support fromthe governments are routinely expended on animal-based studies involvinghighly artificial conditions that assert some kind of relevance to the human.This type of research is now frequently being used to attack and questionclinical procedures Procedures such as in vitro fertilization (IVF) and ICSIhave long established histories of safety and efficacy in humans proven over

10 of 1000 of successful treatment cycles Complex medical follow-up of IVFoutcomes is performed by obtaining data from comparable age groups Todiscount direct data based on the minor results of contrived animal modelsystems with tenuous physical, physiological, and developmental connec-tions to the human is questionable science

Obviously, assisted reproduction is not safe beyond any doubt.Conclusive data has been published regarding reduced birth weight ofIVF singletons and congenital malformation rates that suggest there may

be inherent problems associated with treating infertile patients (48,49).These problems may be associated with follicular stimulation, in vitromanipulation, or an altered reproductive condition Some extensive fol-low-up studies have corrected prior (and current) suggestions that pre-and post-natal development may be compromised by procedures (50) Con-trol groups of the general population are not really appropriate because they

do not suffer from the same condition(s)

One recent study distinguishes between the inherent etiology of thestudy population (the infertile and possibly prenatal and peri-natalconditions that are associated with this) and consequences of hormonalalteration of the ovary and in vitro culture (51)

How Should Human Infertility Treatment Advance?

The first experimental clinical application of a new technique in ductive medicine always raises the question of the possible negative effects

repro-on the patient, offspring, and subsequent generatirepro-ons; this is certainly asober and critical issue in assessing the potential risks and benefits of theapplication The question has been raised throughout the history of humaninfertility treatment, occasionally in a panic-stricken fashion that often pre-vents progress in this area The current state-of-the-art in the field has arisenthrough a complex process involving patients, physicians, scientists, ethicistsand, with increasing frequency, government and social entities It has slowlyadvanced through research, and the development and clinical application of

experimental techniques like ooplasmic donation that address deficits in ourcapabilities derived from needs in the patient population Patients haveacknowledged and accepted risks to future generations on their behalf,although this remains a valid ethical question.

We feel strongly that ooplasmic transfer was developed and mentally applied in a reasonable and responsible fashion Others beg todiffer [for full opposing review see Cummins (7)] The decision to proceedwith experimental clinical trials of ooplasmic transfer was based on a carefulreview of mid-1990s scientific knowledge in this area (including animal mod-els, mitochondrial issues, and epigenetic aspects) as well as considerabledirect experience with successfully incorporating advanced manipulativetechniques into the human clinical environment Suggestions that experi-mental application of ooplasmic transfer in humans proceeded in theabsence of prior research in this area are incorrect as any unbiased review

experi-of the pertinent literature will demonstrate (29)

Appropriate ooplasmic transfer patients (n¼ 33) were selected from amuch larger group of potential patients (several thousand) who expressed

a strong interest in participating in such a trial These patients were carefullyinformed of all potential negative aspects of the technique includingphysical, developmental, mitochondrial, and epigenetic aspects They parti-cipated in an informed consent process that was supervised by our hospitalinternal review board and constantly updated to include all pertinent infor-mation on ooplasmic transfer results This included the chromosomalabnormalities observed, the incidence of heteroplasmy, and criticism frombio-ethicists and basic scientists Internal review board supervision ofinformed consent is an ethical and responsible process that works well toprotect patients and allow for clinical advancement Patient follow-up isalso a critical component of an experimental trial

hatching is not widely practiced based on the fact that there are only about

250 scientific papers regarding this topic in the literature, in sharp contrastwith, for instance, the ICSI literature Whereas there is a broad consensusabout efficacy regarding the latter, this is clearly not the case with assistedhatching A large proportion of studies have evaluated minor changes in thetechnical protocol without emphasis on clinical efficacy Five meta-analysisstudies have been published to date about assisted hatching, but three werebiannual updates from the same team (52–56) The consensus in these stud-ies is that assisted hatching improves outcomes in poor prognosis patients,particularly in the case of maternal aging, not all that different from ourconclusions reported in 1993 after conducting four randomized trials (57).Proof of efficacy was only attainable when patients were selected based

on maternal age or prior failed attempt The research team that has lished three consecutive meta-analysis studies fails to explain the difficultywhen randomizing unselected or partially selected populations In our workwith over 10,000 patients, the effect of assisted hatching is not noticeablebecause we are unable to select against suitable control patients, so onceone has a track record from randomized studies, a data dilution effect islikely to result When controlled studies are compared, there are no goodways to compensate for study design differences such as those caused byage cut-off, opening, thinning, and subtle variation in methodologies andnumber of attempts Hence, the comparisons become blurred, similar toattempting to evaluate the parameters of a single large IVF database withspecific groups represented in both experimental and control arms Inaddition, embryo culture technology and follicular stimulation, which areboth the most likely factors affecting embryonic health and associated zonachanges, have improved spectacularly during the past ten years This haspossibly reduced the need for assisted hatching in sub-groups of patients,yet the exact contribution of this change in technology on hatching behaviorneeds to be determined One factor that appears missing in all review papers

pub-of assisted hatching is the lack pub-of understanding pub-of nuances pub-of techniqueswithin each class of technologies used It does not matter whether one useslaser, acidified Tyrode’s solution, enzymes, mechanical opening, or someother derivative technology—there are only few appropriate ways of doingeach technique, but simply no guidelines for this in the literature Indeed,this does not only apply to assisted hatching but also to any medical tech-nology Most practitioners realize that there are ways of doing poor IVFand good IVF Likewise, there is also good assisted hatching technique andpoor assisted hatching technique Meta-analysis evaluations are not able toweigh these important aspects Indeed, nor can this reviewer provide a moreexact evaluation of the problem, but nevertheless, as the one who intro-duced assisted hatching, I would like to take this opportunity to share someobservations with the reader without evaluating each of the assisted hatch-ing papers one by one Other reviews have done this already and the more

recent ones will illustrate the difficulty in determining the efficacy of assistedhatching and the lack of consensus among practitioners (58,59).

The most common technology used is that of opening the zona cida with acidified Tyrode’s solution (57) The pH of zona breaching isabout 2.3; below this the zona will disappear as a coherent structure, andabove it the zona will remain intact In this respect it is important to con-sider the buffer solution that the embryos are kept in during the procedure.Some buffers, such as HEPES, are excellent in locally maintaining pH andhave high buffering capacity These solutions are more forgiving than bicar-bonate buffered systems which have reduced buffering capacity Anotherconsideration is that technologists, while aware of the aggressiveness ofacidified solution and therefore often like to perform the procedure gentlyand carefully, are not necessarily considering the physics of the system.Also, a hierarchy of teaching from developer to practitioner, as was the casewith ICSI, is largely absent as embryologists have interpreted zona opening

pellu-as a very simple procedure and have applied the technique directly by ing the original publications without communicating with the experiencedgroups While the first descriptions had some depth, it has become apparentover time that subtle aspects were not described in detail Hence, there havebeen a number of interpretations of the procedure and perceptions of thisbeing an easy technique have largely prevailed An example of this is thatmany embryologists will not deposit the acidified solution directly on thezona pellucida while keeping the microneedle pressed on the zona pellucida.This to many embryologists would seem aggressive, yet it is the only way toreduce release of very limited amounts of acidified solution While the zona

read-is dread-issolving, the microneedle should be moved into the thinning area ing the microneedle at distance and releasing acidified solution will cause abroad stream of acidified medium insufficiently lowering the pH below 2.3,the result being that larger quantities of acidified solution are being releasedbecause of perceived carefulness This is likely to affect cells adjacent to themanipulated area Similar considerations can be applied to the use of laser.The lack of referring to possibly detrimental effects in this respect in theoriginal clinical applications is worrisome

Keep-Less than half of all the embryos created after assisted reproductionappear genetically normal (60), but implantation rates are generally lowerthan that, indicating that a number of other factors must be involved.Assisted hatching promotes earlier implantation and may therefore elevatethe chance of implantation by optimizing the implantation window (61).There is additional proof that superficially thinning of the zona pellucida

is advantageous in certain patients (62)

Complete removal of the zona pellucida prior to compaction may lead

to loss of cells due to the absence of structural junctions Only a proportion ofhuman expanded blastocysts growing in vitro will hatch and this frequencymay be dependent on the quality of the culture system The incidence of

hatching in vitro is enhanced by zona opening, at least in a proportion of invitro studies Assisted hatching can potentially be applied to any embryo,but its application by clinics has been slow or it has been abandoned in theabsence of consistent results because of technical variations and subtlechanges to the originally published protocols Common problems identi-fied by us have been (i) the lack of immunosuppression (63) in patientswhose embryos are zona-drilled, (ii) the excessive use of acidified Tyrode’ssolution by keeping the micro-pipette more than 5 mm away from the zonapellucida, thereby decreasing the pH of a greater area than is needed forzona piercing as described above, (iii) the use of hand-controlled suctiondevices for release of acidified solution, which does not allow controlledrelease due to insufficient visualization of the fluid, (iv) the creation of holessmaller than 10 mm which may trap the embryos during hatching, (v) the cre-ation of holes larger than 25 mm which may lead to cell loss during or afterembryo replacement, and (vi) the inability to change the transcervical embryotransfer method in such a way that excess zona pressure is avoided duringreplacement.

At least five methods have been used with varying amounts of success:(i) partial zona dissection on day-two, three, or day 5, (ii) zona drilling withacidified Tyrode’s solution on day-three, (iii) laser-assisted drilling on day-three using an infrared non-contact laser, (iv) zona drilling with eithermethod at the blastocyst stage and (v) piezo-mediated drilling was alsoachieved in animal and human models and might in future be considered

an alternative for clinical application (64), yet results in our laboratory haveshown that excessive use of piezo devices is detrimental for embryo develop-ment in the mouse

We abandoned the use of the first method of partial zona dissection as

it may produce gaps which are too small, possibly resulting in cell separationduring the escape from the zona pellucida (65) The creation of a second per-pendicular gap may be beneficial (66) Larger mechanical openings may behelpful and indeed have been studied to some extent (67) Trapping of theblastocyst may occur if artificial gaps are too small A link between gap sizeand monozygotic twinning is illustrated by recent findings after embryobiopsy The rate of monozygotic twinning was not increased compared tothe frequency in the natural population (68) Gaps produced for biopsyare a factor 2 or 3 times larger than those produced for assisted hatching.Similarly, no monozygotic twins have been found after transferring blasto-cysts from which the zona pellucida was removed (Coughlin Wagner andMaravilla, Highland Park, Chicago, personal communication)

Though our practice indicates that assisted hatching improves tation, controversy continues to impede its widespread and dependableapplication with as many studies finding no effect as studies that do Inthe first meta-analysis (69), 20 studies were considered, but only 14 wereincluded in a meta-analysis as their information came from prospective