Heteroparental blastocyst production from microsurgically corrected tripronucleated human embryos

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Heteroparental blastocyst production frommicrosurgically corrected tripronucleatedhuman embryosMaría-José Escribá, Ph.D.,a Julio Martín, Ph.D.,a Carmen Rubio, Ph.D.,a Diana Valbuena, M.D.,b

José Remohí, M.D.,a,c Antonio Pellicer, M.D.,a,c,d and Carlos Simón, M.D.a,b,c

a IVI Foundation, IVI University, Valencia; b Valencia Stem Cell Bank, Centro Investigación Príncipe Felipe, Valencia;c Department of Paediatrics, Obstetrics and Gynaecology, University School of Medicine, Valencia University, Valencia; andd Department of Obstetrics and Gynaecology, Hospital Dr. Peset, Valencia, Spain

Objective: To prove the efficiency of identification and removal of one of the surplus paternal pronuclei indispermic IVF zygotes to obtain heteroparental blastocysts.Design: Experimental.Setting: One hundred fourteen tripronucleated (3PN) embryos from conventional IVF.Patient(s): After informed and signed consent, the patients from Instituto Valenciano Infertilidad (IVI), Valencia,donated their abnormally fertilized embryos.Intervention(s): Seventy-two embryos were diploidized by microsurgical removal of the pronucleus located at thefarthest position to the second polar body. Forty-two 3PN embryos served as controls.Main Outcome Measure(s): Survival and correction rate; in vitro development up to the blastocyst stage; X, Y,and 18 chromosome determination by triple fluorescent in situ hybridization and, inheritance analysis for 10polymorphic repeat regions using polymerase chain reaction (PCR) amplification and sequencing.Result(s): Seventy-eight percent of 3PN zygotes (56/72) survived manipulation and eventually 51 zygotes hadtwo pronuclei (71%). Forty-one percent of manipulated embryos progressed in vitro to the blastocyst stage(21/51). Fluorescent in situ hybridization analysis performed on eight manipulated embryos confirmed theirdiploid state; all four controls were triploid. Heteroparental inheritances were also confirmed in four of sixmanipulated embryos.Conclusion(s): Heteroparental blastocysts can be derived from corrected dispermic zygotes. (Fertil Steril� 2006;86:1601–7. ©2006 by American Society for Reproductive Medicine.)

Key Words: Tripronucleated embryos, diploidization, heteroparental inheritance, FISH, human

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he alteration of ploidy and parental constitution of zygotesas proved to be efficient and valuable for embryologicaltudies in several mammalian species (1–5). However, in-estigation in human embryos is very limited due to ethicalnd legal constraints. Initial efforts in humans were focusedn microsurgical embryo diploidization (6–10) and hap-oidization (11) by pronuclear removal from tripronucleated3PN) zygotes.

In humans, after conventional IVF, 4% of inseminatedggs are 3PN zygotes (12). The extra chromosome set hashown to be of paternal origin in 86% of cases (12), andt may result from fertilization of normal (haploid) oo-ytes by diploid (nonreduced) spermatozoa, or fromispermic fertilization (13).

Morphologically, the paternal pronucleus can be identifiedy the near presence of sperm tail remnants and increasedronuclear diameter (6, 14–16). However, when fertilization

eceived January 17, 2006; revised and accepted April 28, 2006.upported by the IVI Foundation, Valencia University.eprint requests: María-José Escribá, Ph.D., Instituto Universitario IVI,Instituto Valenciano de Infertilidad, Plaza Policía Local, 3, Valencia

f46015, Spain (FAX: 34-963050999; E-mail: [email protected]).

015-0282/06/$32.00oi:10.1016/j.fertnstert.2006.04.047 Copyright ©2006 American Soc

s assessed (20–22 hours after insemination), pronuclei arevident, similar in size. At this stage, the sperm remaindersre undetectable under light microscopy (7–10, 15).

On the other hand, in the zygote, the maternal pronucleusas been suggested to correspond to that located at theearest distance from the second polar body (8, 9, 15, 17).he presence of the second polar body, larger than the firstolar body and always connected to the ooplasmic mem-rane, indicates that meiosis was resumed and successfullyulminated, with a haploid maternal pronucleus formed athe nearest location (18–21).

In humans, successful microsurgical removal of a singleronucleus is technically possible, rendering bipronucleated,iploid embryos (6–10). However, diploidization has noteen technically feasible as a reproductive technology be-ause of the resulting embryos showed limited in vitro de-elopmental potential and there were unconclusive preim-lantational data on their heteroparental inheritance (6–10).

In this study, we resumed early studies on human embryoiploidization, using cytoskeleton relaxants to investigatehether the selection and elimination of the pronucleus

arthest from the second polar body restores the normal

1601Fertility and Sterility� Vol. 86, No. 6, December 2006iety for Reproductive Medicine, Published by Elsevier Inc.

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eteroparental combination in 3PN human zygotes emergingrom IVF. In addition, we also assessed the in vitro potentialf such corrected embryos to achieve the blastocyst stage.

ATERIALS AND METHODSripronuclear human zygotes from 169 conventional IVFycles were obtained on day 1 (oocyte retrieval: day 0) fromnfertile couples at the Instituto Valenciano InfertilidadIVI), Valencia, Spain. The use of abnormally fertilizeduman zygotes for research was approved by the Spanishommittee of Assisted Reproduction and complies with thepanish Law of Assisted Reproductive Technologies (35/8). Abnormal fertilized eggs included in this study belongo patients who received detailed information of the projectnd signed a written statement of consent.

The average age of the women was 34.9 � 4.6 yearsrange 19–45 years) and the average age of the men was7.1 � 4.7 years (range 20–52 years). The majority of theatients had tubal infertility (30%), idiopathic infertility30%), endometriosis (14%), aging (17%), or polycysticvary syndrome (PCOS) (8%). There were no cases ofevere male factor infertility. Protocols for follicular stimu-ation, oocyte recovery, in vitro insemination (day 0), andmbryo culture (days 1–6) are described elsewhere (22, 23).riefly, ova were cultured for 4 hours before insemination inuman tubal fluid (HTF) medium supplemented with 10%uman albumin at 37°C in an atmosphere of 5% CO2 in air.emen obtained from patients’ partners was prepared bywim-up technique. A final concentration of 100,000 motilepermatozoa/mL was adjusted with HTF medium. Insemi-ation drops of 50 �L were dispensed and then, a singlevum per drop was added. After 18–22 hours of insemina-ion, 2,108 inseminated oocytes were examined for the pres-nce and number of pronuclei. A total of 114 3PN embryos5.4%) with the two polar bodies checked, were included inhis study.

pronucleation Procedure: Microtools andicromanipulationor micromanipulation, 3PN zygotes (cases) were placed insmall (25 �L) drop of IVF-20 (Scandinavian IVF, EMB,arcelona, Spain) medium containing 7.5 �g/mL cytocha-

asin B (C6762; Sigma, Madrid, Spain) and 0.1 M demecol-ine (D1925; Sigma) and covered with equilibrated mineralil.

Zygotes were incubated for 3 minutes under controlledulture conditions (discussed later) and placed on the stagef an inverted microscope (Diaphot 300, Nikon, Barcelona,pain) fitted with hydraulic micromanipulators (MM188,arishige, London, UK).

Two types of handmade microtools were used for micro-anipulation: a holding pipette (outer diameter, 100 �m;

nner diameter, 20 �m) and a 6-�m outer diameter opened
icroneedle. To accommodate the micromanipulation set- f
1602 Escribá et al. Correction of tripronucleated embryos

p, both microtools were bent to a 45° angle and used atandard syringe system (IM-26-2; Narishige). The teflonubing for the hydraulic systems was filled with light mineralil.

Before pronuclear removal (epronucleation), 3PN zygotesere clumped against the holding pipette, rotated, and repo-

itioned until we could identify all pronuclei and the secondolar body. The three pronuclei are at syngamy, usuallylaced in a typical pyramidal position, in the center of theoplasm. At this time, the supernumerary male pronucleus toe removed was present at the farthest position from theecond polar body.

For epronucleation, the tip of the micropipette was in-erted through the zona pellucida (ZP) into the vitellus andently positioned next to the targeted surface of the pronu-leus. Negative pressure was then applied until the pronu-leus was picked up. Pressure was maintained while theipette and pronucleus were gently withdrawn from theyngamy area to the oolema. Aspiration progressed untilhe total elimination of the pronucleus and some of theytoplasm was freely drawn into the epronucleation nee-le. Then the micropipette was removed and the zygoteeleased. The micromanipulation time for each zygote waspproximately 5 minutes.

After manipulation, zygotes were washed three times inVF-20 medium and cultured in a 5% CO2 atmosphere in airt 37.5°C for 3 hours. The zygotes were assessed for survivalnd number of pronuclei. Only embryos that survived withhe two desired pronuclei were cultured; all others wereemoved from culture. Tripronucleated zygotes (negativeontrols) were similarly handled, but not microsurgicallyanipulated, and served as the 3PN control group.

mbryo Developmental Abilityorrected bipronucleated (2PN) human zygotes and 42 3PNontrol zygotes were co-cultured until the blastocyst stage onuman endometrial epithelial cells (hEEC), as routinely usedn our clinic (22, 23). Forty-eight hours after insemination,mbryos at the 2- to 4-cell stage were individually co-ultured in 1 mL of IVF-20/CCM (1/1; v/v) on the hEEConolayer until they reached the 8-cell stage.

On the morning of day 3, the medium was replaced by 1L of CCM (Scandinavian IVF) and embryos were cultured

or 3 more days. Embryonic development was checked onays 2, 3, and 5. Multinucleation was not assessed duringulture of corrected or negative control embryos.

For this study, developmental data from normal fertilizedmbryos of ongoing treatments, obtained by in vitro insem-nation and embryo co-culture were compiled and served asositive controls. Developmental ability of 2PN and 3PNmbryos was statistically compared using a �2 test. When aingle degree of freedom was involved, the Yate’s correction
or continuity was used.
Vol. 86, No. 6, December 2006

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hromosome Analysisn day 6, eight expanded blastocysts, derived from cor-

ected zygotes and, having a well-defined inner cell mass andnlarged trophoectoderm, were selected for chromosometudies. Similarly, four expanded negative control blasto-ysts were selected for chromosome analysis.

Expanded blastocysts were fixed using the Tarkowskyrotocol (24) without hypotonic treatment. Briefly, blasto-ysts were incubated in a Ca2�- and Mg2�-free mediumEB-10, Scandinavian IVF) before removal of the zona pel-ucida (ZP) by brief incubation in acidified Tyrode’s solutionpH 2.4, ZD-10, Scandinavian IVF). Then, blastocysts werelaced on a grease-free slide with a minimum volume ofedium.

Chromosomal analysis was performed by fluorescent initu hybridization (FISH) using centromeric probes for chro-osomes X (Spectrum Green; Vysis Inc., Downers Grove,

L), Y (Spectrum Orange; Vysis Inc.), and 18 (Spectrumqua; Vysis Inc.). The FISH procedure was performed ac-

ording to the manufacturer’s instructions and samplesnalyzed using an Olympus AX-70 epifluorescence mi-roscope (Olympus Optical Co., Hamburg, Germany)quipped with a triple-band pass filter for DAPI/Texased/FITC and single band pass filters for FITC, Texased, and Aqua Blue. Images were recorded with an Olym-us DP-50 video camera.

arental Determinationo analyze the genetic inheritance of the micromanipulatedlastocysts, 10 polymorphic short tandem repeat (STR) re-ions were studied on both blastocysts and genomic DNA ofheir corresponding parents.

On day 6 of development, six corrected blastocysts, hav-ng a well-defined inner cell mass and trophoectoderm wereelected for genetic inheritance determination. All whole

TABLE 1In vitro developmental ability of experimentally e(negative controls), and normal fertilized embryo

Number of zygotes

Initial Cultured C

Experimental 72 51a

Negative controls 42 42Positive controls 947 947a Number of zygotes that survived to microsurgical removb Eighteen blastocysts were morphologically normal in ap

for FISH studies and six blastocysts for STR-DNA analc Four tripronucleated control blastocysts were used for Fd,e P�.038; d,f P�.25; e,f P�.00013.

Escribá. Correction of tripronucleated embryos. Fertil Steril 2006

ertility and Sterility�

ona-free blastocyst was placed in 0.2-mL polymerase chaineaction (PCR) tubes containing 5 �L of lysis buffer solution50 mM KCl, 10 mM Tris-HCl at pH 8.3, 0.5% Tween-20,nd 100 �g/mL of proteinase K). Every embryo was iden-ified and immediately frozen and stored at �80°C until theCR amplification.

For genomic DNA extraction, whole blood samples of thembryo’s parents were processed using standard techniques.or PCR, amplification of STR loci was performed using thempFlSTR Profiler Plus PCR amplification kit (Appliediosystems, Foster City, CA), following manufacturer indi-ations. Before PCR, frozen blastocysts were incubated at0°C for 30 minutes and then placed at 95°C for 10 minutes.

For fragment analysis on ABI Prism 310, 1.5-�L PCRroducts was evaluated. Data were analyzed using theeneScan Analysis Software 3.1.2 (Applied Biosystems).

ESULTSifty-one of 72 3PN human zygotes (71%) survived to theicrosurgical manipulation stage and finally contained

he desired two pronuclei (Table 1). All 51 diploidizedmbryos cleaved at 24 hours of culture and 96% of themeached the 6- to 8-cell stage on day 3 of development. Onay 5, 21 manipulated human zygotes (41%) progressed tohe blastocyst stage (Table 1). This progress in in vitroevelopment was comparable to that observed on positiveontrol embryos (8-cell stage: 85%; blastocyst: 50%;�.05; Table 1). Moreover, most of day 5 blastocysts,erived either from co-cultured positive controls or frompronucleated embryos, were morphologically character-zed by a well-defined inner cell mass and trophoectodermFig. 1A,B, respectively).

These results were clearly opposed to 3PN control em-ryos (negative controls, Table 1). They developed signifi-antly less efficiently in our co-culture system, only render-

ucleated zygotes, tripronucleated zygotesIVF treatments (positive controls).

No. of embryos (%) reaching the stage of

vage 8-Cell Morula Blastocyst

(100) 49 (96) 24 (47) 21 (41)b,d

(100) 40 (95) 15 (36) 8 (19)c,e

(99) 808 (85) 478 (50)f

f the targeted pronucleus.ance (35%). Eight blastocysts were whole fixed and used

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ng 19% blastocysts with a reduced or absent inner cell massnd linear trophoectoderm (Fig. 1C). The FISH results wereonsidered informative when more than 20 nuclei could bevaluated in each blastocyst. Using this criterion, 7 of 8anipulated embryos and 3 of 4 negative control blastocystsere informative (Fig. 2).

Four diploidized blastocysts were diploid (three 46,XY

FIGURE 1

Morphological appearance of human blastocysts dermicrosurgically diploidized (B), and tripronucleated (Cinner cell mass in A and B compared to C.


nd one 46,XX; Fig. 2A,B), two were mixoploid with similar s


ercentages of coexisting diploid and tetraploid cell lines46,XX/ 92,XXXX and 46,XY/ 92, XXYY), and the lastorrected embryo had a tetraploid complement (92, XXXX).n the negative control group, the FISH results from threelastocysts confirmed the expected triploid condition (69,XY; Fig. 2C).

Genomic DNA from all parents’ samples and from four of

from extended co-culture of normal fertilized (A),gotes. Note the feature of trophoectoderm and

ived) zy

ix analyzed blastocysts (67%) resulted in electropherograms


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ith expected amplification patterns for the analyzed STR loci.nheritance of the markers indicated that all four amplifiedmbryos were diploid and genetically heteroparental (Fig. 3).hree embryos were female and one was male. Seven remainedlastocysts, derived from corrected zygotes and normal in ap-earance were used for workup human embryonic stem cell

FIGURE 2

Triple fluorescent in situ hybridization for centromericchromosomes of day 5 corrected blastocysts (A anddouble signal for chromosomes 18 and X in correctetriple signal for the chromosome 18 and XXY inherita


erivation; however, no colony was established. c


ISCUSSIONeteroparental blastocysts, derived from abnormal, nonvia-le 3PN embryos, from IVF protocols have been obtainedollowing the epronucleation technique, as described herend based on the elective microsurgical removal of theronucleus located distal from the second polar body using

ion of X (green signal), Y (orange), and 18 (blue)nd tripronucleated control blastocysts (C). Note the

astocysts (arrowheads in A and B) compared to thein control blastocysts (arrowhead in C).

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The in vitro developmental ability of our corrected zy-otes contrasts with earlier reports where embryos either didot cleave (6), grew until the 8-cell stage (9), and rarelyeached the compactation stages (7, 8). These differencesould be due to the technical procedure itself or to theesultant ploidy and parental inheritance in the embryo afterpronucleation.

Almost all protocols of mammalian nuclear transfer in-lude cytoskeletal relaxing agents (25–27). In humans, theemoval of an extra male pronucleus has been performedith (5–7, 11) or without (7, 8, 11) intervention on the

ytoskeletal organization.

In spite of oocyte or embryo, exposure to cytochalasineduces mechanical damage due to micromanipulation, in-lusion of cytoskeletal relaxing agents in epronucleationrotocols has been suggested to obtain subsequent embryoevelopment (6, 7). Such detrimental effects are dose andime exposure dependent; thus, reducing cytoskeleton relax-nt time exposure from 30 minutes (6, 7) to 10 minutes oress (5; this work), epronucleated zygotes cleaved and de-eloped in vitro to the blastocyst stage (41%) as efficiently ashose nonmanipulated, correctly fertilized embryos (50%).

On the other hand, 3PN human embryos, dispermic inrigin, contain more than one functional centrosome thatarticipates in the formation of multipolar spindles, severelympairing the subsequent embryo development (9, 28). Thus,

FIGURE 3

Representative panel of electropherograms displayincorrespond to blood samples from one couple includof corrected blastocyst from these parents (at the boinheritance in some loci.


he removal of the extra paternal pronucleus and the centro- a


ome is needed. Because of the close association of theentrosome to the paternal pronucleus, aspiration of wholeronucleus plus some of the nearby cytoplasm (10 picoliter)ay restore the centrosome inheritance (10).

Embryo development is severely influenced by bothloidy and parental inheritance. Tripronucleated human zy-otes, dispermic in origin, progress poorly in in vitro devel-pment (13, 28, 29, present work), may derive into partialydatidiform moles in rare instances and they are mosaic (9,0, 31) or 69, XXY (this work).

Self-correction of 3PN human embryos was earlier re-orted by Kola and Trounson (12). Briefly, these investiga-ors observed that at 38–40 hours after insemination, 14% ofultured 2-cell-plus-extrusion cleaved embryos had an extrahromosome set, showing a diploid condition in their blas-omeres. However, subsequent development of those auto-iploidized embryos was not obtained (12).

Removal of one pronucleus could render diploid androge-etic 46, XX and 46, XY complete hydatidiform moles (32,3) or heteroparental 46, XX and 46, XY embryos (mice:34]; human: [10] and this work), depending on the maternalr paternal origin of the removed pronucleus, respectively.

Correction of 3PN human embryos has been used foreproductive applications in a case report (10). Because ofhe parental origin of the removed pronucleus could severely

polymorphic loci. Two analyses at the topin the study (Mother and Father). Electropherogram, BTCIII). Note the informative heteroparental

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hisms to identify the parental inheritance in the resultingmbryo are strongly recommended by embryo biopsy on day

of development, before embryo transfer. In countrieshere handling and manipulation of abnormal embryos haveo other ending than research (i.e., Spain) we propose to useonviable 3PN embryos, discarded from IVF cycles, as anlternative source of chromosomally normal embryos fortem cell research. This option could be of particular interestn countries where the use of normal embryos or the creationf human embryos for stem cell research is not allowed (i.e.,nited States). In any case, this is exciting for a number of

easons, both scientific and ethical.

Thus, handling and final use of chromosomally correctedmbryos originating from 3PN zygotes are mainly dependantn governmental bans rather than on biological or technicalonstrains.

We conclude that microsurgical removal of one pronu-leus located at the farthest position to the second polar bodyrom 3PN zygotes is feasible and can result, in vitro, into aorphologically normal, heteroparental diploid blastocysts.his technique of embryo recycling could be useful for

eproductive purposes or human embryonic stem cell re-earch.

cknowledgments: The authors thank the IVI staff for their help andupport.

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Heteroparental blastocyst production from microsurgically corrected tripronucleated human embryos