L. Xanthopoulou

The nature and origin of binucleate cells in human preimplantation embryos: relevance to placental mesenchymal dysplasia

Cleavage-stage embryos often have nuclear abnormalities, one of the most common being binucleate blastomeres, which may contain two diploid or two haploid nuclei. Biopsied cells from preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) cycles were studied to determine the relative frequency of binucleate cells with two haploid versus two diploid nuclei. The frequency of mononucleate haploid biopsied blastomeres was also recorded. In the chromosomal PGD cycles 45.2% of the biopsied binucleate cells were overall diploid and 38.7% were overall tetraploid, compared with 50.0% and 29.2% for the PGS group, respectively. Placental mesenchymal dysplasia is a rare condition associated with intrauterine growth restriction, prematurity and intrauterine death. Recent work suggests that androgenetic diploid/haploid mosaicism may be a causal mechanism. There are two possible origins of haploid nuclei, either the cell contained only one parental genome initially or they may be derived from the cytokinesis of binucleate cells with two haploid nuclei. Binucleate formation therefore may be a way of doubling up the haploid genome, to produce diploid cells of androgenetic origin as seen in placental mesenchymal dysplasia.

Chromosome Breakage in Human Preimplantation Embryos from Carriers of Structural Chromosomal Abnormalities in Relation to Fragile Sites, Maternal Age, and Poor Sperm Factors

Chromosome breakage is a fairly widespread phenomenon in preimplantation embryos affecting at least 10% of day 3 cleavage stage embryos. It may be detected during preimplantation genetic diagnosis (PGD). For carriers of structural chromosomal abnormalities, PGD involves the removal and testing of single blastomeres from cleavage stage embryos, aiming towards an unaffected pregnancy. Twenty-two such couples were referred for PGD, and biopsied blastomeres on day 3 and untransferred embryos (day 5/6) were tested using fluorescence in situ hybridisation (FISH) with appropriate probes. This study investigated whether chromosome breakage (a) was detected more frequently in cases where the breakpoint of the aberration was in the same chromosomal band as a fragile site and (b) was influenced by maternal age, sperm parameters, reproductive history, or the sex of the carrier parent. The frequency of breakage seemed to be independent of fragile sites, maternal age, reproductive history, and sex of the carrier parent. However, chromosome breakage was very significantly higher in embryos from male carriers with poor sperm parameters versus embryos from male carriers with normal sperm parameters. Consequently, embryos from certain couples were more prone to chromosome breakage, fragment loss, and hence chromosomally unbalanced embryos, independently of meiotic segregation.

Polymorphisms in the MTHFR gene influence embryo viability and the incidence of aneuploidy.

MTHFR is an important enzyme in the metabolism of folic acid and is crucial for reproductive function. Variation in the sequence of MTHFR has been implicated in subfertility, but definitive data are lacking. In the present study, a detailed analysis of two common MTHFR polymorphisms (c.677C>T and c.1298A>C) was performed. Additionally, for the first time, the frequencies of different MTHFR alleles were assessed in preimplantation embryos. Several striking discoveries were made. Firstly, results demonstrated that maternal MTHFR c.1298A>C genotype strongly influences the likelihood of a pregnancy occurring, with the 1298C allele being significantly overrepresented amongst women who have undergone several unsuccessful assisted reproductive treatments. Secondly, parental MTHFR genotypes were shown to affect the production of aneuploid embryos, indicating that MTHFR is one of the few known human genes with the capacity to modulate rates of chromosome abnormality. Thirdly, an unusual deviation from Hardy–Weinberg equilibrium was noted for the c.677C>T polymorphism in subfertile patients, especially those who had experienced recurrent failure of embryo implantation or miscarriage, potentially explained by a rare case of heterozygote disadvantage. Finally, a dramatic impact of the MTHFR 677T allele on the capacity of chromosomally normal embryos to implant is described. Not only do these findings raise a series of interesting biological questions, but they also argue that testing of MTHFR could be of great clinical value, identifying patients at high risk of implantation failure and revealing the most viable embryos during in vitro fertilisation (IVF) cycles.

When is old too old for preimplantation genetic diagnosis for reciprocal translocations

Preimplantation genetic diagnosis (PGD) was developed as an alternative to prenatal diagnosis and involves the removal and testing of one or two blastomeres from in vitro fertilisation (IVF) generated embryos to select those embryos that are unaffected by a single gene or a chromosomal abnormality (Fragouli, 2007). Couples who are at risk of having a child with a specific single gene or chromosomal abnormality may opt to have PGD to avoid an affected pregnancy and to avoid having prenatal diagnosis that is associated with a 1% risk of miscarriage; moreover, their decision to have PGD is often linked to the couple’s view on pregnancy termination. At the same time, other couples who seek PGD have difficulty in achieving a pregnancy because of infertility or subfertility or are victims of repeated pregnancy loss. Fluorescence in situ hybridization (FISH) is used in PGD for the investigation of numerical and structural chromosomal abnormalities. It involves using a fluorescently labelled DNA probe, which will bind to its complementary sequence in the genome and as a result detect a chromosome or a part of a chromosome, thereby allowing determination of the copy number of that region in a particular sample (Hopman et al., 1988). Reciprocal translocations are the most common type of structural chromosomal abnormality in humans, seen in about 1 in 500 live births (Jacobs et al., 1992) and involve the exchange of genetic material between different chromosomes. Carriers are usually phenotypically normal because there is no loss of genetic material but are faced with unfavourable meiotic segregation when the translocation chromosomes pair up at meiosis. Therefore, carriers are at a high risk of producing unbalanced gametes and hence, genetically abnormal embryos are associated with recurrent miscarriage, infertility and abnormal offspring.

Male and female meiotic behaviour of an intrachromosomal insertion determined by preimplantation genetic diagnosis

BackgroundTwo related family members, a female and a male balanced carrier of an intrachromosomal insertion on chromosome 7 were referred to our centre for preimplantation genetic diagnosis. This presented a rare opportunity to investigate the behaviour of the insertion chromosome during meiosis in two related carriers. The aim of this study was to carry out a detailed genetic analysis of the preimplantation embryos that were generated from the three treatment cycles for the male and two for the female carrier.Patients underwent in vitro fertilization and on day 3, 22 embryos from the female carrier and 19 embryos from the male carrier were biopsied and cells analysed by fluorescent in situ hybridization. Follow up analysis of 29 untransferred embryos was also performed for confirmation of the diagnosis and to obtain information on meiotic and mitotic outcome.ResultsIn this study, the female carrier produced more than twice as many chromosomally balanced embryos as the male (76.5% vs. 36%), and two pregnancies were achieved for her. Follow up analysis showed that the male carrier had produced more highly abnormal embryos than the female (25% and 15% respectively) and no pregnancies occurred for the male carrier and his partner.ConclusionThis study compares how an intrachromosomal insertion has behaved in the meiotic and preimplantation stages of development in sibling male and female carriers. It confirms that PGD is an appropriate treatment in such cases. Reasons for the differing outcome for the two carriers are discussed.

P2 Sex chromosome abnormalities in embryos from women undergoing PGS: Focus on meiotic errors involving the X-chromosome

P1 Preimplantation genetic diagnosis and maternal age: the effect of embryo cohort size N. Ercelen1, E. Tutar1, M. Gultomruk1, H. Comert1, B. Yazar1, B. Balaban2, B. Urman2. 1American Hospital Genetic Disease Diagnostics Center, Guzelbahce Sok. No: 20, 34365 Nisantasi, Istanbul, Turkey, 2American Hospital Ob-Gyn and Reproductive Medicine Unit, Guzelbahce Sok. No: 20, 34365 Nisantasi, Istanbul, Turkey