Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study

BACKGROUND: Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in PGD cycles for beta-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts. METHODS: This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis. RESULTS: In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born. CONCLUSIONS: This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.     

Integrins and adhesion molecules: Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage-stage and blastocyst-stage human embryos

Human embryos have been biopsied at either the cleavage or theblastocyst stage of development. One to two blastomeres wereremoved from cleavage-stage embryos and 2–6 cells fromblastocysts. The biopsy specimens were subjected to gene amplificationby the polymerase chain reaction (PCR) and a comparison madeof amplification efficiencies of two unique target sequences,one located within the -globin gene and containing the sickle-celllocus and the other a polymorphic dinucleotide repeat. Whenthe cleavage-stage biopsy sample consisted of an intact blastomerewith a clearly discernible nucleus, an amplification efficiencyof 89% was achieved for each target locus. This was similarto that achieved with cleavage-stage biopsy samples consistingof two blastomeres or with blastocyst biopsy samples consistingof 2–3 trophectoderm cells. When biopsy samples consistedof four or more trophectoderm cells, both target loci were amplifiedin all samples tested. When the biopsy sample was heterozygousat the dinucleotide repeat locus and the biopsy consisted ofone or more intact cells with a clearly discernible nucleus,both alleles were amplified in >80% of biopsy samples. Whenfour or more trophectoderm cells were used for the PCR, bothalleles were amplified in all heterozygous samples. Target sequenceswere never amplified from biopsy samples which lysed prior totransfer into the reaction tube. Analysis of DNA fragments amplifiedfrom the dinucleotide repeat locus indicated that in most casesfaithful amplification of biopsy DNA template had taken place.However, in one case, fragments were identified which couldnot have resulted from the amplification of embryonic sequencesalone, indicating that contamination with extraneous DNA mayhave taken place. The significance of this finding for therapeuticpreimplantation genetic diagnosis is discussed.     

Cryopreservation of biopsied embryos at the blastocyst stage

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.     

Pregnancies following blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.

BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.     

Trophectoderm biopsy in human blastocysts

Trophectoderm biopsy was carried out on 47 human blastocysts. A slit was made in the zona pellucida opposite the inner cell mass by micromanipulative techniques. The human blastocyst zona offered more resistance to slitting compared to that of the mouse. After 18-24 h, controlled herniation of the trophectoderm cells was observed. These cells were biopsied when the diameter of the herniation was approximately equal to that of the blastocyst. The size of the slit and the stage of embryonic development at which slitting was performed were important for successful herniation to occur. After slitting, 76% of day 5-6 blastocysts showed herniation whilst only 42% of day 7-8 blastocysts herniated. Further development of the manipulated embryos was not apparently impaired, as hatching occurred in 44% of the former and 20% of the latter, compared with 18.1% in non-manipulated controls. The biopsied cells (approximately 10-30) usually remained in a clump but 14% formed vesicles on the day after biopsy. There was, however, no evidence of adherence to the dish or formation of monolayers. These results demonstrate the feasibility of trophectoderm biopsy in human blastocysts and that sufficient extra-embryonic material can be obtained by this technique for preimplantation diagnosis of genetic disorders.     

Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage

BACKGROUND: A correlation between morphology, developmental competence and chromosome abnormalities is established. However, since absolute correlations are rare, embryo selection remains one of the most arduous tasks in assisted reproduction. This study was undertaken in order to determine which chromosomal abnormalities are compatible with development to the blastocyst stage. METHODS: Embryos diagnosed by preimplantation genetic diagnosis (PGD) as chromosomally abnormal or unsuitable for transfer were cultured to day 5 or 6. Morphology and development were observed daily. After extended culture, embryos were fixed and analysed by two rounds of FISH with the same probes used for PGD. RESULTS: Some types of numerical chromosome abnormalities do not preclude full differentiation in vitro. For instance, extensive mosaicism was detected in blastocysts and trisomic embryos reached the blastocyst stage with a frequency of 37%. Interestingly, only those monosomies compatible with first trimester development (monosomy X and 21) were detected at blastocyst stage. CONCLUSION: Even though there is a strong selection against chromosomally abnormal embryos, extended culture to day 5 or 6 cannot be used as a reliable tool to select against clinically relevant chromosome abnormalities such as trisomies.     

Genetic diagnosis using polymerase chain reaction and fluorescent in-situ hybridization analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos

Cultured human preimplantation embryos have been used to developmethods which allow preimplantation genetic diagnosis (PGD)analyses by polymerase chain reaction (PCR) and fluorescentin-situ hybridization (FISH) on biopsied blastomeres and trophectodermcells from the same embryo. An experimental design is describedand experiments undertaken, which demonstrate the feasibilityof extending biopsy and PGD procedures currently in use. Wehave shown that dual-stage biopsies are possible, and that thePCR and FISH analyses of the biopsied cell samples are effective.One to two blastomeres were biopsied from an 8- to 10-cell embryoand processed for the simultaneous PCR amplification of a -globinand a cytosine adenine (CA) repeat sequence, or a Y chromosomesequence. FISH procedures were also used to detect the presenceof Y chromosome markers. The biopsied cleavage-stage embryocan be cultured to the blastocyst stage, where the serial biopsyof three to five mural trophectoderm cells provides two furthercell samples. These can be used to repeat and/or undertake additionalPGD analyses. The biopsied blastocyst is either used to confirmearlier diagnoses, or placed in culture for a further 4–24h. Maintenance of a blastocoele cavity, hatching and formationof an outgrowth demonstrates continuing viability followingthe dual-stage biopsy procedures. The PCR DNA amplificationprocedures are effective at the cellular level for both biopsiedblastomeres and mural trophectoderm cells. The FISH techniqueshave shown a definitive Y signal in 50% (one out of two) and100% (two out of two) of the biopsied blastomeres and 72% (twoout of three, four out of five and 7 out of 10) for the trophectodermcell nuclei. Preliminary experiments have demonstrated thatthe FISH preparations can be re-amplified to improve the signal,and dual fluorescent procedures using the X and Y probes areeffective. A retrospective PCR analysis has also been undertakenon preparations of biopsied cells which were previously usedfor PGD analysis by FISH.     

Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis

BACKGROUND: Preimplantation genetic diagnosis or screening (PGD, PGS) involves embryo biopsy on Day 3. Opting for one- or two-cell biopsy is a balance between the lowest risk for misdiagnosis on the one hand and the highest chance for a pregnancy on the other hand. METHODS: A prospective controlled trial was designed and 592 ICSI cycles were randomly assigned to the one-cell (group I) or the two-cell group (group II). Primary outcomes were diagnostic efficiency and embryonic development to delivery with live birth (analysed by cycle). The false-positive rate for the PCR cycles is presented as a secondary outcome (analysed by embryo). RESULTS: A strong significant correlation was observed between embryonic developmental stage on Day 3 and post-biopsy in vitro development on Day 5 (P < 0.0001). The influence of the intervention on Day 3 was less significant (P = 0.007): the biopsy of one cell is less invasive than the biopsy of two cells. PCR diagnostic efficiency was 88.6% in group I and 96.4% in group II (P = 0.008). For the fluorescence in situ hybridization (FISH) PGD cycles no significant difference in efficiency was obtained (98.2 and 97.5% in group I and II, respectively). Similar delivery rates with live birth per started cycle were obtained [58/287 or 20.2% in group I versus 52/303 or 17.2% in group II, P = 0.358; the absolute risk reduction = 3.05%; 95% confidence interval (CI): -3.24, 9.34]. Post-PGD PCR reanalysis showed six false positives in 97 embryos (6.2%) in group II and none in group I (91 embryos reanalysed). No false negatives were found. CONCLUSIONS: While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, Day 3 in vitro developmental stage is a stronger predictor for Day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. Delivery rates with live birth per started cycle were not significantly different.     

Preimplantation diagnosis of a human beta-globin transgene in biopsied trophectoderm cells and blastomeres of the mouse embryo.

The preimplantation diagnosis of a HbSA-globin transgene in biopsied trophectoderm cells and blastomeres in embryos using a transgenic mouse model for the trait of human sickle-cell anaemia has been undertaken. A sensitive procedure was developed for the amplification of the human beta-globin gene sequence flanking the sickle mutation. Polymerase chain reaction (PCR) assays were undertaken on one to five biopsied trophectoderm cells and isolated blastomeres of the preimplantation mouse embryo. After biopsy the blastocysts were cultured whilst the cells were analysed for the presence of the transgene, and a high proportion (82-91%) were viable as assessed by the presence of a blastocoele cavity within a 5-h period. The majority of the biopsied cultured blastocysts were frozen and used to confirm the diagnosis; 90 biopsied cultured blastocysts were transferred to pseudopregnant recipients and 34% established pregnancy. Material from day 13.5 post-coitum fetuses was also used to confirm the original diagnosis. The time (4-5 h) required to carry out the analysis obviates a need for extended culture or cryopreservation of the biopsied embryo. In individual experiments under optimal conditions, the presence of the transgene in biopsied cells was detected with 100% accuracy, and the PCR analysis was sensitive at the 1-cell level. The overall success rate of diagnosis and confirmation of the presence or absence of the human beta-globin sequence in the biopsied embryo was 70%. Over the entire experimental period (14 months) DNA contamination from a variety of sources did occasionally occur; the methods used to overcome this problem are discussed.     

Fertilization and Empryology: The effect of epidermal growth factor on growth and differentiation of mouse preimplantation embryos in vitro

The effect of epidermal growth factor (EGF) on embryonic growth,development, attachment and spreading in vitro was studied.EGF was added to 130 embryos at the 4-cell stage; to 128 embryosat the blastocyst stage; and to 147 embryos 24 h following spreading.Development of embryos from the 4-cell to the blastocyst stage,differentiation of the inner cell mass (ICM) and trophectoderm,and the occurrence of attachment and spreading were evaluated.Embryo development was significantly inhibited in cultures supplementedwith 100 ng/ml EGF compared to the controls (P < 0.001).Development of 4-cell embryos to blastocysts occurred in 25%of the EGF group compared to 85% of controls. Spreading occurredin 20% of 4-cell embryos and 30% of blastocysts treated withEGF, compared to 80 and 90% of corresponding controls. In embryosdeveloping from the 4-cell stage, massive growth of the ICMand inhibition of the trophectoderm occurred, whereas both ICMand trophectoderm were inhibited by EGF in embryos developingfrom the blastocyst stage. Following spreading, EGF caused massivegrowth of the ICM and regression of the trophectoderm. Our preliminaryresults show that EGF may be involved in the modulation andcontrol of early embryonic growth and differentiation.     

Epithelial cadherin distribution in abnormal human pre-implantation embryos

BACKGROUND: E(epithelial)-cadherin is a vital cell adhesion protein that plays a critical role in morphogenesis. Previous studies of E-cadherin distribution in human embryos have produced equivocal results. METHODS: Immunocytochemistry in conjunction with laser scanning confocal microscopy was used to detect E-cadherin in 97 human cleavage stage embryos and 35 blastocysts from normal and abnormal fertilization. An antibody against human placental E-cadherin was used to locate the protein. RESULTS: In blastomeres of cleaving embryos on the second and third days following insemination, E-cadherin was located in the cytoplasm--mostly concentrated in the cell margins. On the fourth day of development, the protein was relocated in compacting embryos to membranes in areas of cell-cell contact. In other abnormally compacted or non-compacted embryos with extensive cytoplasmic fragmentation, cell arrest or blastomere multi-nucleation, E-cadherin relocalization was either absent or erratic. In apparently normal blastocysts, E-cadherin in the inner cells was diffuse and cytoplasmic while properly organized trophectoderm cells were surrounded by a band of membrane E-cadherin. Disorganization of trophectoderm was associated with disruption of the regular E-cadherin banding pattern. CONCLUSION: As in other mammalian species examined, E-cadherin distribution in human embryos is stage-dependent. Disturbances in the distribution of E-cadherin occur in embryos with cleavage abnormalities and suggest one path to abortive or abnormal blastulation and loss of embryonic viability. The implications of similar changes in the blastocyst are well worth investigating since they could threaten blastocyst integrity.     

Flow of cells from polar to mural trophectoderm is polarized in the mouse blastocyst

During growth of the blastocyst there is a net flow of cells from the polar to the mural trophectoderm which is presumed to be radially symmetrical. However, such a pattern of cell movement is inconsistent with findings from a recent clonal analysis. To visualize the overall flow of cells directly, the polar trophectoderm of expanding blastocysts was labelled globally with fluorescent microspheres. Following further growth, the great majority of blastocysts that remained labelled throughout the polar trophectoderm exhibited a polarized rather than radial spread of label into the mural region. This was the case regardless of the labelling technique, whether the blastocysts were grown in utero or in vitro, or had the zona pellucida removed or left on. Intriguingly, where there were two foci of spread of label into the mural trophectoderm rather than one, these were diametrically opposite each other. In further experiments, fluorescent lineage labels were used to distinguish junctional trophectoderm cells with and without an extension onto the blastocoelic surface of the inner cell mass. The location of clones formed following further blastocyst growth provided no evidence that egress of cells from the polar trophectoderm is restricted circumferentially by the presence of junctional cells having an extension.     

Preimplantation development of mouse and human embryos biopsied at cleavage stages using a modified displacement technique

A modified embryo biopsy method was tested on four- and eight-cell stage mouse embryos and used on human embryos to obtain blastomeres for preimplantation genetic diagnosis. The biopsy method tested combines zona drilling and fluid displacement to force one or two cells through an opening in the zona pellucida of the cleavage-stage embryo. Rates of cell division and the percentage of mouse embryos forming blastocysts following biopsy at the eight-cell stage were not significantly different from those observed in unoperated control embryos. The percentage blastocyst formation was not significantly different in embryos biopsied at the four-cell stage and in control embryos, although cell division was significantly retarded following biopsy. 96% of the mouse blastomeres isolated at the eight-cell stage were recovered intact and 96% of those placed in culture underwent cell division. Survival and division of cells isolated at the four-cell stage were 92 and 84% respectively. Most of the cultured blastomeres cleaved several times and formed small trophoblast vesicles. Chromosomes were observed in 59% of blastomeres incubated in the presence of colcemid. In the initial use of this biopsy technique for human preimplantation genetic diagnosis, blastocyst formation was observed in 9 of 13 human embryos biopsied at the 7- to 10-cell stage. These findings support the use of this biopsy method as an alternative to aspiration techniques.      

Reduced survival after human embryo biopsy and subsequent cryopreservation.

Preimplantation genetic diagnosis (PGD) is performed in couples at risk of genetic disease, so as to avoid transfer of embryos which are affected by a monogenic disease or which carry chromosomal aberrations. As in all in-vitro fertilization (IVF) cycles, supernumerary non-affected good-quality embryos may be available after PGD. These embryos can be cryopreserved. So far, limited data on survival after cryopreservation of biopsied human embryos are available. In this study, human embryos of good morphological quality derived from abnormal fertilization were used to evaluate the influence of the embryo biopsy procedure on survival after cryopreservation. Embryos were allocated to three different groups: control (n = 20), drilling-only (n = 16), and biopsy (n = 29). After freezing and thawing, a significantly lower number of blastomeres was intact in the drilling-only group (46/118, i.e. 39.0%, P < 0.01) and in the embryo biopsy group (46/156, i.e. 29.5%, P < 0.0001) than in the control group (85/151, i.e. 56.3%). This difference was reflected in survival rates of embryos. Fifty-five per cent of the control embryos, 37.5% of the drilling-only group, and 33.3% of the biopsy group had at least 50% of their blastomeres intact. After further in-vitro culture, four blastocysts, three from the drilling-only group and one from the biopsy group, developed from the surviving embryos. From this study it can be concluded that current cryopreservation procedures are less successful when biopsied human embryos are cryopreserved, but that surviving embryos can develop to the blastocyst stage and thus may have the potential to develop to term.     

Use of co-culture of human embryos on Vero cells to improve clinical implantation rate

Co-culture of human embryos (n = 384 cycles) to the blastocyst stage using Vero cell monolayers was carried out between August 1995 and December 1997. A total of 2868 zygotes were co-cultured and 1027 embryos reached the blastocyst stage (blastocyst formation rate 35.8%). The blastocysts were frozen in 43.7% of patients. A mean of 1.8 blastocysts was transferred per patient and 95 pregnancies were obtained (pregnancy rate/cycle 24.7%). The blastocyst implantation rate was 23.6%. Miscarriage occurred in 15 patients (15.7%) and ectopic pregnancy in three (3.1%) patients. The multiple pregnancy rate was 32.6%. No differences were observed in the blastocyst rate between poor, normal or high response patients. Blastocyst formation was significantly lower when frozen donor spermatozoa were used. Significantly higher pregnancy rates per transfer and blastocyst implantation rates were attained when embryos were transferred on days 5 or 6 compared with day 7. No advantage was observed when co-culture was used in first cycle IVF patients, in comparison with conventional day 2 replacements. The use of blastocysts for preimplantation genetic diagnosis (PGD) increases the diagnostic reliability and widens diagnostic possibilities. A total of 215 cycles with frozen-thawed co-cultured blastocysts were carried out, with a pregnancy rate of 22.7% per replacement.     

Expression of fibronectin and a fibronectin-binding molecule during preimplantation development in the mouse

Using an indirect immunofluorescent technique, expression ofcell surface fibronectin and a cell surface fibronectin-bindingmolecule was studied during mouse embryo preimplantation development.We also studied the expression of fibronectin on immunosurgicallyisolated inner cell masses (ICMs) and regenerated mouse blastocysts.Fibronectin and the fibronectin-binding molecule were not detectedat the morula stage. From the early to late blastocyst stage,fibronectin expression increased on the trophectoderm. Expressionof the fibronectin-binding molecule was found only in the polartrophectoderm region of the early blastocyst, then in the polarand mural trophectoderm regions of the middle blastocyst. Inthe late blastocyst stage, this fibronectin-binding moleculewas only present in the mural trophectoderm. Fibronectin expressionby ICMs of early blastocysts was more intense than that of lateblastocysts. After 24 h of culture, 10% of ICMs isolated fromearly blastocysts regenerated a trophectoderm which stainedintensively for fibronectin in the mural and polar trophectodermregions. After 48 h of culture, regenerated blastocyst-likestructures closely resembled the normally obtained late blastocystsand stained for fibronectin in the mural and polar trophectodermregions. The significance of the results is discussed in relationto mouse embryo development, trophectoderm formation and blastocystimplantation.     

In-vitro studies on 'spare' human preimplantation embryos in culture

Methods previously used for the biopsy of preimplantation mouse embryos have been applied to individual 'spare' human embryos. Early cleavage-stage human embryos have been cultured and individual blastomeres removed following zonae thinning or drilling. Embryos have also been cultured to the blastocyst stage for the biopsy of three to five trophectoderm cells. Both the biopsied embryo and the biopsied cells have been allowed to develop and/or grow in vitro.     

Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage

Normally fertilized human embryos biopsied 3 days after in-vitro fertilization (IVF) have been examined for effects on viability and development in vitro after removal of one or two cells at the 8-cell stage (1/8 and 2/8) from each embryo. A high proportion of 7/8 and 6/8 biopsied and unmanipulated embryos developed to the blastocyst stage between days 5 and 6 (79, 71 and 59%, respectively), and many biopsied embryos (56%) hatched from the zona pellucida in vitro. The viability of biopsied embryos which developed to the blastocyst stage was assessed by daily non-invasive measurement of the uptake of two energy substrates, glucose and pyruvate. Uptake of both substrates was generally lower in 7/8 and 6/8 biopsied embryos but only in proportion to the reduced cellular mass. The total cell number and the numbers of both trophectoderm (TE) and inner cell mass (ICM) cells in biopsied embryos at the blastocyst stage, counted by differential labelling of their nuclei, were also reduced in proportion but the ratio of ICM to TE cells was maintained in both 7/8 and 6/8 biopsied embryos. We conclude that removal of one or two cells at the 8-cell stage, while reducing the cellular mass, does not adversely affect the preimplantation/development of biopsied embryos in vitro and suggest that this approach could be used for preimplantation diagnosis of genetic defects.     

Progression to the blastocyst stage of embryos derived from testicular round spermatids

Progression to the blastocyst stage of embryos derived from testicular round spermatids in men with non-obstructive azoospermia was studied. A total of 56 men were studied in whom partial spermatogenesis failure had occurred where only very few spermatozoa (fewer than the number of oocytes retrieved) were extracted from multiple testicular biopsy specimens. Oocytes remaining after intracytoplasmic injection of testicular spermatozoa (group 1) were injected with round spermatids (ROSI, group 2). Only embryos derived from group 1 were transferred. Remaining embryos were observed under culture for 8 days and their progression to the blastocyst stage was recorded. Of the 546 oocytes injected with testicular spermatozoa, 404 (73.9%) showed evidence of 2-pronuclear (2PN) fertilization. Injection of testicular round spermatids resulted in 2PN fertilization rate of 50% (P < 0.05). Using a four-point grading system, 53% of the good quality embryos (grade 1 or 2) in group 1 reached the blastocyst stage compared with 25% in group 2 (P < 0.05). The rate of progression to the blastocyst stage of grade 3 and grade 4 embryos was 46 and 8.5% in the two groups respectively (P < 0.05). Using a different three-point grading system for the blastocysts, 75.3% of the blastocysts in group 1 were either grade 1 or grade 2 and 24.7% were grade 3. However, in group 2 all blastocysts were grade 3. All embryos observed in group 1 reached the blastocyst stage by day 5 or 6 compared with 25% of the embryos reaching the blastocyst stage by this time in group 2. While 31.2% of the blastocysts in group 1 showed evidence of spontaneous hatching in vitro, none of the blastocysts in group 2 hatched. In conclusion, progression to the blastocyst stage occurred at a much lower and slower rate in embryos derived from testicular round spermatids. Furthermore, all blastocysts resulting from ROSI were of poor quality and none showed spontaneous hatching. These results may explain the dismal outcome associated with ROSI.     

Preimplantation sexing and diagnosis of hypoxanthine phosphoribosyl transferase deficiency in mice by biochemical microassay

Hypoxanthine phosphoribosyl transferase (HPRT)-deficient male embryos derived from heterozygous (carrier) female mice were diagnosed by biochemical microassay of X-chromosome-coded HPRT activity in a single cell taken from the 8-cell embryo or in 5-10 cells sampled from the blastocyst. In the latter procedure, carrier female blastocysts could also be distinguished from affected males, and normal males and females, as having intermediate HPRT activity in the sampled trophectoderm cells. During the assay procedures, the operated preimplantation embryos were cultured. They were then transferred, in batches as diagnosed, to recipient females. The resulting fetuses were sexed by gonad morphology and assayed for HPRT activity. All those identified as HPRT-negative embryos by biopsy at the 8-cell or blastocyst stages were indeed HPRT-negative males. The heterozygous females were also correctly identified by the trophectoderm biopsy procedure. The sex of an embryo can also be diagnosed by HPRT activity dosage in a single blastomere taken from 8-cell embryos from a normal mating and cultured for 12 hours before assay. Both X chromosomes are active in female morulae and the blastomeres sampled from female preimplantation embryos have twice the X-coded HPRT activity compared to those from the male embryos. The accuracy of this procedure for sexing was again verified by transfer of the putative male and putative female embryos into recipient females.