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Purpose: Our purpose was to assess the effect of chromosomal mosaicism in cleavage-stage human embryos on the accuracy of single-cell analysis for preimplantation genetic diagnosis., Methods: Multicolor fluorescence in situ hybridization with X, Y, and 7 or X, Y, 7, and 18 chromosome-specific probes was used to detect aneuploidy in cleavage-stage human embryos., Results: Most nuclei were diploid for the chromosomes tested but there was extensive mosaicism including monosomic, double-monosomic, nullisomic, chaotic, and haploid nuclei., Conclusions: Identification of sex by analysis of a single cleavage-stage nucleus is accurate but 7% of females are not identified. One or both parental chromosomes 7 were absent in at least 6.5% of the nuclei. With autosomal recessive conditions such as cystic fibrosis, carriers would be misdiagnosed as normal or affected. With autosomal dominant conditions, failure to analyze the affected parents allele (1.6-2.5%) would cause a serious misdiagnosis and analysis of at least two nuclei is necessary to reduce errors.

Purpose: Our purpose was to achieve preimplantation genetic diagnosis (PGD) of the dominant cancer predisposition syndrome, familial adenomatous polyposis coli (FAPC), as an alternative to prenatal diagnosis., Methods: The affected patient was superovulated and oocytes were retrieved and fertilized by intracytoplasmic sperm injection (ICSI). Two cells were biopsied from each embryo and the whole genome was amplified by primer extension preamplification (PEP). Nested PCR was then used to amplify two APC fragments: one including the APC mutation site and the other an informative intragenic polymorphism. Both were detected by simultaneous single-strand conformation polymorphism and heteroduplex analysis., Results: Four normally fertilized embryos were biopsied on day 3 post ICSI, and two cells were successfully removed from each embryo. Following PEP the APC mutation was successfully amplified in 7 of 8 cells, and the polymorphism in 6 of 8 cells. The APC mutation was detected in three embryos. This result was confirmed by identification of the mutation associated polymorphism in two cases. A single embryo was diagnosed as homozygous normal for the mutation and the polymorphism in both cells sampled. This unaffected embryo was transferred to the mother, but no pregnancy resulted., Conclusions: We report here the first diagnosis of a cancer predisposition syndrome in human preimplantation embryos. Our results indicate that difficulties associated with single-cell PCR, allele-specific amplification failure in particular, need not prevent preimplantation diagnosis of diseases with a dominant mode of inheritance, provided appropriate strategies are applied.

Purpose: Our purpose was to investigate early biochemical and ultrasonic measurements of pregnancies resulting from embryos biopsied for preimplantation diagnosis of inherited disease., Results: Singleton pregnancies following biopsy had lower initial hCG levels [10 or 12 days after oocyte recovery (OR)], which rose steeply to match the controls by 16 days after OR. Twin biopsied pregnancies showed hCG levels lower than those of twin control pregnancies, which rose in parallel with the controls but remained lower for a longer period than the singletons. Progesterone levels showed a wide variation. Ultrasound measurements showed that overall the mean sac diameter and crown-rump length at 28 and 42 days after egg collection were similar in biopsied and control pregnancies., Conclusions: Pregnancies resulting from biopsied embryos behave similarly to control IVF pregnancies. However, the reduction in cell mass following embryo biopsy occasionally results in reduced levels of circulating serum hCG and smaller ultrasound measurements in early pregnancy.

Introduction: Preimplantation diagnosis involves detecting genetic defects in one or two blastomeres biopsied from cleavage stage embryos following a vitro fertilization (IVF). For X-linked recessive disease, identification of the sex of embryos allows transfer of only unaffected females. To examine how critical the preparation of the single blastomere is for amplification of a Y chromosome specific repeat sequence using the polymerase chain reaction (PCR), the incidence of amplification failure has been examined following two lysis protocols., Materials and Methods: Amplification of a Y alphoid repeat sequence from single blastomeres disaggregated from cleavage stage embryos was examined after either (1) lysis in distilled water and freeze-thawing twice or (2) a two-step lysis protocol involving an initial treatment in potassium hydroxide and dithiothreitol. Some of the embryos had been previously sexed by cleavage-stage biopsy and fluorescent in situ hybridization with X- and Y-specific probes., Results: Amplification failure occurred in 6 of 50 (12%) and 4 of 60 (7%) single blastomeres from male embryos following lysis in distilled water or using the two-step protocol, respectively. Conversely, amplification from contaminating DNA occurred in 5 of 63 (8%) single blastomeres from female embryos and 6 of 94 (6%) of control medium-blanks., Conclusions: The incidence of amplification failure was improved but not eliminated using the two-step lysis protocol. At least two cells, therefore, would be necessary for accurate identification of males by amplification of Y-specific repeat sequences alone. Nevertheless, this protocol for preparing cleavage-stage blastomeres is likely to give more consistent amplification of any unique or repeat sequences.

Background: For couples at risk of transmitting a known single-gene defect, preimplantation genetic diagnosis (PGD) allows the identification and transfer of only unaffected embryos following in vitro fertilisation (IVF), single-cell biopsy at about the eight-cell stage, and genetic analysis by PCR. This technique therefore avoids the risk of terminating an affected pregnancy diagnosed later in gestation., Methods and Results: Using nested PCR, the delta F508 mutation causing cystic fibrosis can be detected in single cells and we previously reported successful PGD in a couple in whom both partners carry the delta F508 mutation. To date we have treated 12 couples in a total of 18 cycles. This resulted in five singleton births confirmed to be homozygous normal. Single blastomeres from disaggregated embryos which had not been transferred were analysed to confirm the original diagnosis and assess reliability in clinical practice. Amplification efficiency and accuracy were high, with blastomeres from embryos diagnosed as homozygous normal or affected. In a proportion of blastomeres from presumed carrier embryos, one of the parental alleles failed to amplify, apparently at random (allele dropout, ADO). A possible explanation is the relative inaccessibility of one of the target allele early in the PCR. To test this we have used single lymphocytes from delta F508 carriers and investigated the effects of various denaturation temperatures in the early cycles of amplification., Conclusions: Increasing the denaturation temperature reduced the rate of ADO without affecting amplification efficiency.

Purpose: Our purpose was to assess the clinical application of dual fluorescent in situ hybridization (FISH) for the diagnosis of sex in the human preimplantation embryo., Results: Over a 2-year period, 18 couples at risk of transmitting X-linked recessive disorders underwent preimplantation diagnosis of embryo sex by dual FISH with X and Y chromosome-specific DNA probes. A total of 27 in vitro fertilization (IVF) treatment cycles led to nine pregnancies; 7 reached the stage of clinical recognition, of which 2 spontaneously aborted. There were five live births, three singleton and two twin: none in disagreement with the diagnosed sex. The diagnosis was corroborated in 51 of the 74 nontransferred embryos. The efficiency of the procedure improved throughout the four treatment cycles. This was reflected in the increased proportion of double embryo transfers (from 50% in series 1 and 2 to 100% in series 3 and 4), with a consequent improvement in pregnancy rate (from 28 to 71% per embryo transfer). The excess of male embryos (male:female, 60:40 overall) and the high proportion of biopsied embryos with abnormal numbers of X and Y chromosome signals (14.5%) effectively reduced the number of normal female embryos available for transfer., Conclusion: Dual FISH is an efficient technique for determination of the sex of human preimplantation embryos and the additional ability to detect abnormal chromosome copy numbers, which is not possible via the polymerase chain reaction, (PCR), makes FISH the preferred technique.

Purpose: Pyruvate uptake is higher in human embryos developing to the blastocyst stage than those arresting at cleavage stages. To investigate whether pyruvate uptake provides an improved criterion for selecting embryos for transfer, we have measured uptakes by individual embryos noninvasively over 24-hr periods between the first day (day 1) postinsemination and embryo transfer on day 2 to 3 and correlated the levels with implantation and pregnancy outcome., Results: The mean uptake was significantly lower for embryos that implanted than for those which failed to implant: 22.9 +/- 1.0 and 27.1 +/- 0.6 pmol/embryo/hr, respectively on day 2, and 22.4 +/- 1.5 and 26.9 +/- 0.8 pmol/embryo/hr, respectively, on day 3, but the wide range of uptakes by individual embryos was overlapping., Conclusion: We conclude that pyruvate uptake as the sole criterion for embryo selection cannot predict which embryos will implant after transfer. Assessment of embryos using morphological and developmental criteria, therefore, remains the most consistent, though inefficient, indicator of pregnancy potential.