Reprograming of murine blastocoele formation

The present study shows that there is communication between reaggregated asynchronous cleavage stage blastomeres that regulates blastocoele formation. Individual blastomeres from eight-cell murine embryos were transferred to empty zonae pellucidae, intact two-cell embryos, or enucleated two-cell embryos, and were examined over a period of 75 hours for development of cavitation. It was found that the isolated blastomeres cavitated concurrently with intact control eight-cell embryos, while intact control two-cell embryos cavitated 24 hours later. However, the embryos resulting from combining a two-cell embryo and a blastomere from an eight-cell embryo cavitated at a time in between the eight- and two-cell controls.     

Viability of nuclei of two-cell mouse embryos stored at 4 degrees C and fused with blastomeres of fresh two-cell embryos.

This study compares the resistance of the nuclei and the cytoplasm of two-cell mouse embryos to short-term storage at low temperature above 0 degrees C. Two-cell embryos were stored at 4 degrees C for 24-96 h in PB1 containing 0.25, 0.5, 0.75, and 1.0 M sucrose. The development to blastocysts in culture was highest in the presence of 0.5 M sucrose. However, only 3% of the embryos developed into blastocysts after 96 h of storage. On the other hand, the viability of the nuclei of two-cell embryos stored at 4 degrees C was significantly prolonged when they were transplanted into a blastomere of enucleated fresh F1 (C57BL/6JXCBA) two-cell embryos. The proportions of chimeric embryos that developed to blastocysts were 88, 67, 76, 71, 64, 45, 32, and 20% following storage for 0, 48, 72, 96, 120, 144, 168, and 192 h, respectively. In addition, there was no difference in the coat color of the young derived from nuclei stored at 4 degrees C or fresh nuclei, although the proportions of chimeric embryos that developed into live young after transfer tended to decrease with increased storage time. Moreover, the viability of nuclei stored at 4 degrees C for 192 h was confirmed in the germ cell population of chimeric mice mated with albino mice. These results demonstrated that the nuclei in the two-cell mouse embryos were more resistant to storage at low temperature than the cytoplasm.     

Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.     

Fusion and development rates of single blastomere pairs of mouse two- and four-cell embryos using the electrofusion method

The present study was undertaken to find suitable conditions for blastomere fusion of mouse two- and four-cell embryos using the electrofusion method to simplify the nuclear transfer procedure. Single blastomeres of ICR and F1 (C57BL/6J x CBA/N) two-cell embryos or ICR four-cell embryos and F1 two-cell embryos were paired and treated with electric stimulus under different fusion conditions. Two hours after electrofusion treatment, the fused blastomere pairs were encapsulated in alginate gel and cultured for 96 hours to observe their developmental potential. When the single blastomere pairs of two-cell embryos were exposed to electric pulses of 1.0, 1.5 and 2.0 kV/cm for 30, 60 and 90 mu sec, high fusion rates were obtained (84.6 to 100%). However, when two-cell blastomere were paired with four-cell blastomere and then treated under the same conditions, the fusion rates (27.5 to 87.5%) were lower than that of single blastomere pairs of two-cell embryos regardless of the duration and strength of the d.c. pulses. The blastocyst developmental rate after in vitro culture of the fused blastomere pairs of two-cell embryos using the above electrofusion conditions was high (81.8 to 100%). Lower blastocyst developmental rates were obtained on the fused blastomere pairs of two- and four-cell embryos (46.4 to 76.2%). Based on the results of this study, a pulse duration of 60 mu sec and a pulse strength of 1.0kV/cm were the most suitable conditions for single blastomere pair fusion of two-cell or two- and four-cell embryos. The study further showed that alginate gel is a good substitute for zonae pellucidae for encapsulating zona-free embryos.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.     

Production of identical twin and triplet mice by nuclear transplantation

Transplantation of a single nucleus from two- or four-cell embryos into one of the enucleated blastomeres of a two-cell embryo resulted in successful production of identical triplet and twin mice. The proportion of reconstituted embryos that developed in blastocysts was 71% (84/118) when four-cell embryos were used as donors of nuclei; 10 sets of quadruplet and nine sets each of triplet and twin blastocysts were obtained by this technique. After transfer to recipients, 30% (18/61) developed to term, and one set of identical triplet and four sets of identical twin mice were obtained. When two-cell embryos were used as donors of nuclei, 79 (95%) sets of twin embryos developed to blastocysts. Of 38 twin blastocysts transferred to recipients, 21 sets (55%) developed to term as identical twin mice. These results demonstrate that the enucleated two-cell embryo develops in vitro after transfer of a nucleus from a two- or four-cell embryo and the resultant blastocyst has high potential for development to term after transfer to a recipient.     

Cloning of bovine embryos by multiple nuclear transfer

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.     

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.     

Mouse cloned from embryonic stem (ES) cells synchronized in metaphase with nocodazole

Full-term development occurred when nuclei from mouse embryonic stem (ES) cells, synchronized in metaphase with nocodazole, were fused with enucleated oocytes or nuclei of reconstituted eggs and again fused with the enucleated blastomeres of fertilized two-cell embryos using inactivated Sendai virus. Two surviving male mice were derived from undifferentiated ES cell nuclei, one from single nuclear transfer and another from serial nuclear transfer. Both were noticeably small and died within 24 hr of birth for unknown reasons. These findings demonstrate that nuclear transfer of ES cells using the fusion method produces young, as does the piezoelectric-actuated nuclear transfer. J. Exp. Zool. 289:139-145, 2001.     

Full-term development of rat after transfer of nuclei from two-cell stage embryos

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.     

How many blastomeres of the 4-cell embryo contribute cells to the mouse body?

The aim of this study was to estimate how many blastomeres of the 4-cell mouse embryo contribute cells to the embryo proper and finally to the animal. To this end, 4-cell embryos of pigmented and albino genotypes were disaggregated and single blastomeres (henceforth called '1/4' or 'quarter' blastomeres) were reaggregated in the following combinations: one 'pigmented' blastomere + three 'albino' blastomeres or vice versa (henceforth called '1+3') and two pigmented blastomeres + two albino blastomeres (henceforth called '2+2'). The aggregations were cultured in vitro and transferred as blastocysts either to the oviduct or uterus of pseudopregnant females. Recipients were allowed to litter naturally, or the foetuses were removed by Caesarian section and raised by lactating foster mothers. Chimaerism was assessed on the basis of coat (adults) or eye pigmentation (dead neonates). Among 28 '1+3' animals, there were 13 chimaeric and 15 non-chimaeric individuals. The pigmentation of non-chimaeras was always concordant with the genotype of the three 1/4 blastomeres and not with the genotype of the single blastomere in the given aggregation. These results make rather unlikely the possibility that the mouse is built of cells derived either from one or all four 1/4 blastomeres. Both two remaining options (2 or 3 1/4 blastomeres) are conceivable but the observed ratio of chimaeras to non-chimaeras among '1+3' animals (13:15) fits better the assumption of two 1/4 blastomeres contributing cells to the animal body. This assumption finds additional support in the observation that among '2+2' animals there were non-chimaeras (5 out of 7) and these would not have been expected should three 1/4 blastomeres contribute cells to the mouse body.     

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.     

Nuclear transfer of mouse follicular epithelial cells pretreated with spermine, protamine, or putrescine

The in vitro and in vivo developmental potential of nuclear transferred embryos receiving follicular epithelial cells pretreated with spermine (5 and 20 mM), protamine (0.25 and 25 mg/ml), or putrescine (1 and 100 microg/ml) at room and reduced temperatures was examined in the mouse. The pretreated donor cells were first fused with enucleated oocytes, and then nuclei from reconstituted eggs at the two-cell stage were fused with the enucleated fertilized two-cell embryos. The proportion of reconstituted embryos that developed into blastocysts was not significantly different among groups. After transfer to recipients, implantation rates were not different between groups and fetuses were obtained in protamine- and spermine-treated groups as well as in control groups. These results demonstrate that pretreatment of nuclear donor cells with spermine, protamine, or putrescine does not enhance the developmental potential in vitro or in vivo in the mouse. J. Exp. Zool. 289:208-212, 2001.     

Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.     

Efficient production of sex-identified and cryosurvived bovine in-vitro produced blastocysts

To establish a protocol for production of bovine in-vitro produced (IVP) blastocysts that were sex-identified and cryopreserved, we examined the sexing efficiency and accuracy of Day-3 and Day-4 embryos by polymerase chain reaction (PCR), the development of the biopsied embryos into Day-7 blastocysts and the freezability of these blastocysts by vitrification in gel-loading tips. One or two blastomeres were isolated from IVP embryos at either the 8-cell or 16-cell stage (Days 3 and 4, respectively) by a pressing-out method, and were then subjected to primer extension preamplification (PEP)-PCR. The successful sex-identification rate of biopsied samples amplified, purified and analyzed for sex by a second PCR (88.9%) was higher than that of those amplified and analyzed without purification (32.0%). Developmental rates into Day-7 blastocysts of biopsied embryos (Day-3, 65.5%; Day-4, 70.8%) were similar to those of non-biopsied control embryos (Day-3, 74.5%; Day-4, 65.1%). Total cell numbers and the inner cell mass (ICM) ratio of blastocysts derived from biopsied embryos were also comparable with those of control embryos. Blastocysts were vitrified-warmed in the presence of 20% DMSO, 20% ethylene glycol and 0.6M sucrose using gel-loading tips as containers. The proportions of biopsied blastocysts that were hatched or hatching rates after warming were high, regardless of the biopsy time (Day-3, 94.1%; Day-4, 91.9%), similar to the rates for control blastocysts (Day-3, 97.5%; Day-4, 96.9%). In conclusion, a protocol that allows sexing of Day-3 and Day-4 bovine embryos without compromising either the developmental ability to the blastocyst stage or freezability of Day-7 blastocysts was developed.     

Allocation of cells in mouse blastocyst is not determined by the order of cleavage of the first two blastomeres

The second cleavage of the mouse embryo is asynchronous. Some recent investigators have proposed that the sequence of division of blastomeres in two-cell embryos may predict the ultimate location of the descendants of these blastomeres within the blastocyst. To verify this model, we tracked the cells derived from two-cell stage blastomeres using tetramethylrhodamine-conjugated dextran as a lineage tracer. In the first variant of the experiment, we labeled one of two blastomeres in two-cell embryos and subsequently recorded which blastomere cleaved first. In the second variant of the experiment, fluorescent dextran was injected at the three-cell stage into the blastomere that had not yet cleaved. Subsequently, the fate of the progeny of labeled and unlabeled blastomeres was followed up to the blastocyst stage. Our results suggest that allocation of cells into the embryonic and abembryonic parts of the blastocyst is not determined by the order of cleavage of the first two blastomeres.     

Development of single mouse blastomeres enlarged to zygote size in conditions of nucleo-cytoplasmic synchrony

The following blastomeres were enlarged to the size of the zygote by one, two or three rounds of blastomere enucleation and electrofusion: (1) from the 2-cell stage (referred to as 2/1 embryos), (2) from the 4-cell stage (referred to as 4/1 embryos), (3) from the 8-cell stage (referred to as 8/1 embryos). Such single enlarged blastomeres developed into blastocysts in vivo in 55.5% (2/1), 28% (4/1) and 6.6% (8/1) of cases. Their mean cell numbers were 45.3, 24.5 and 13.0 in 2/1, 4/1 and 8/1 embryos, respectively. When a blastomere nucleus from another mouse strain (heterologous nucleus) was substituted for a blastomere's own (homologous) one, then fewer blastocysts were formed from 2/1 embryos (34.6%), but not from 4/1 and 8/1 embryos. Five young (10.4%) were born from 2/1 embryos with a homologous nucleus, and nine (8.3%) from 2/1 embryos with heterologous nuclei. Four young (7.1%) were born from 4/1 embryos with heterologous nuclei. No young were obtained from 8/1 embryos. Incorrect cavitation resulting in trophoblastic vesicles and false blastocyst formation was common in 4/1 embryos (18.7% of those with homologous nuclei and 41.3% with heterologous nuclei) and in 8/1 embryos (53.3% and 43.7%, respectively). The results show that neither enlargement to zygote size nor nucleo-cytoplasmic synchrony improve postimplantation development of 4- and 8-cell stage blastomeres when compared with less enlarged non-synchronous ones; therefore, it appears that an insufficient number of inner cell mass cells in blastocysts and not too small a size of isolated blastomeres precludes their postimplantation development.     

Comparison of the validity of preimplantation genetic diagnosis for embryo chromosomal anomalies by fluorescence in situ hybridization on one or two blastomeres

Is it necessary to analyze two blastomeres in preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridization (FISH) or is one blastomere enough, as suggested by some teams? We analyzed the sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), false positives (FP), false negatives (FN), and the efficiency (Eff) of FISH performed on one (Group I) or two (Group II) blastomeres. Ninety embryos were analyzed (day 3), 19 blastocysts were replaced (day 5), 64 embryos were reanalyzed (day 5), (Group I = 23; Group II = 41). No differences were observed between the two groups for all of the parameters considered, but one false negative was observed in Group I. Furthermore, two embryos from Group II, which had a discordant diagnosis at PGD (one blastomere being normal and one abnormal), were read as abnormal after reanalysis. The accidental biopsy of the normal blastomere could have lead to the selection of these 2 embryos for transfer, causing a misdiagnosis rate of 4.8%. We conclude that embryo reanalysis is a useful tool to test the reliability of PGD in each laboratory: that PGD on two blastomeres is safer because the practice of PGD on one blastomere can result in a false-negative misdiagnosis.