Confirmation rates of array-CGH in day-3 embryo and blastocyst biopsies for preimplantation genetic screening.

PURPOSE: The purpose of this study was to compare the confirmation rate of day-3 embryo biopsy (blastomere) and trophectoderm biopsy using array-comparative genomic hybridization (array-CGH) technology. METHODS: A blinded study was conducted to re-analyse 109 embryos previously diagnosed as chromosomally abnormal by array-CGH. Preimplantation genetic screening (PGS) was performed using array-CGH on day 3 (n = 50) or day 5 (n = 59). Partial chromosome gains or losses were excluded (n=6), and only whole chromosome aneuploidies were considered. Re-analysis of whole blastocysts was carried out following the same array-CGH protocol used for PGS. RESULTS: The PGS result was confirmed in the whole blastocyst in (a) 49/50 (98 %) abnormal embryos after day-3 biopsy and (b) 57/59 (96.6 %) abnormal embryos after trophectoderm biopsy. One embryo (1/50; 2 %) was diagnosed as abnormal, with monosomy 18, on day 3, and software analysis of the whole blastocyst gave a euploid result; however, a mosaic pattern was observed for monosomy 18 in the whole blastocyst. Two trophectoderm biopsy cases (3.4 %) did not have the abnormalities (trisomy 7, and trisomy 1 and 4, respectively) verified in the whole embryo. Concordance rates for both biopsy strategies and for individual chromosomes were evaluated by Fisher's exact test and showed no significant differences. CONCLUSIONS: Both types of biopsies showed similar high concordance rates with whole blastocyst results. Therefore, regarding the confirmation rates shown in this work, day-3 embryo biopsies can be representative of the whole embryo and both types of biopsy can be used for clinical analysis in PGS following the described array-CGH protocol.

Impact of assisted reproduction treatments on Spanish newborns: report of 14,119 pregnancies.

PURPOSE: To investigate neonatal malformation, prematurity, and stillbirth in singleton and multiple pregnancies derived from different Assisted Reproductive Techniques (ART). METHODS: In this prospective cohort study data were collected, from private and public Spanish IVF units, during the years 2008 and 2009. During this period, 8,682 pregnancies were analysed from the initial 14,119 pregnancies reported. Pregnancies included in the study derived from IUI (n = 1,065), IVF (n = 838), ICSI (n = 5,080), FET (n = 1,404) and PGD (n = 295). This first analysis focuses primarily on neonatal malformation, prematurity, and stillbirth both in singleton and multiple pregnancies derived from different ART. Malformations were classified according to the WHO ICD 10 code. RESULTS: Malformations were found in 0.83 % of our newborns. No differences in malformations were observed between singletons or multiples independently of the ART used. There was a significant difference in prematurity rate among singletons depending on treatment but this association was not observed in multiple pregnancies. Stillbirth was significantly lower in singleton (0.72 %) than in multiple pregnancies (1.82 %). CONCLUSIONS: The percentage of malformations observed in ART newborns was similar to the rate observed in the normally-conceived Spanish population. Multiplicity seems to be the most important factor associated with an increased incidence of newborn complications such as prematurity or stillbirth.

Distribution of the HLA-DQA1 and polymarker alleles in the Basque population of Spain.

HLA-DQA1 and polymarker (LDLR, GYPA, HBGG, D7S8, and GC) genotypic and allelic frequencies are determined for a population sample of 102 unrelated Basque individuals using PCR-based methodology. All six loci met Hardy-Weinberg expectations in at least two of the three analyses performed (HLA-DQA1 failed to meet Hardy-Weinberg requirements in the heterozygote deficiency test). Three linkage analysis programs (GDA, GENEPOP and LINKDOS) detected possible linkage disequilibrium between LDLR and HBGG and results from one (GDA) indicated a possible non-random association between HBGG and HLA-DQA1 as well. Allelic data for the six loci are compared to that previously established for other populations (18 for polymarker alone, 16 for polymarker plus HLA-DQA1) to determine homogeneity between the Basque sample and these groups. According to the results of G-tests based on these loci, the Tadjik, a nomadic Caucasian group from western Asia, and the Basque residents are the only sample populations surveyed that are homogenous with the Basque sample. Phylogenetic analysis places the Basque sample correctly within the Caucasian cluster.

Embryonic regulation of endometrial epithelial apoptosis during human implantation.

Apoptosis is a mechanism of cell death in which cells undergo a genetically determined programme. The implanting blastocyst has to appose and adhere to the endometrial epithelium and subsequently invade it. Locally regulated uterine epithelial apoptosis induced by the embryo is a crucial step in epithelial invasion in rodents. To address the physiological relevance of this process in humans, we have investigated the effect of single human blastocysts on the regulation of apoptosis in cultured human endometrial epithelial cells (EEC) in the apposition and adhesion phases of implantation. We report a co-ordinated embryonic regulation of EEC apoptosis. In the apposition phase, the presence of a blastocyst rescues EEC from the apoptotic pathway. However, when the blastocyst adheres to the EEC monolayer it induces a paracrine apoptotic reaction.

An analysis of spontaneous hatching in a human endometrial epithelial coculture system: is assisted hatching justified?

PURPOSE: To evaluate spontaneous embryo hatching in an endometrial epithelial coculture system, and compare it with cases where coculture was performed because of maternal age, previous repeated implantation failures, or both. To clarify in which cases assisted hatching would be appropriate. METHODS: Individual human embryos were cocultured on an endometrial epithelial cell monolayer until Day 6. RESULTS: Blastocyst hatching rate at Day 6, depending on maternal age, was 9.1% (age <37 years) and 3.4% (age > or = 37 years). However, blastocyst hatching rates depending on number of previous IVF failures were similar. CONCLUSIONS: Maternal age and previous implantation failures are factors affecting the ability of human embryos to reach the blastocyst stage in coculture. However, assisted hatching is not justified in these populations because of the absence of hatching rate differences between blastocysts obtained from these two groups and the control group.

The human blastocyst regulates endometrial epithelial apoptosis in embryonic adhesion.

The implanting blastocyst must appose and adhere to the endometrial epithelium and, subsequently, invade it. Locally regulated uterine epithelial apoptosis induced by the embryo is a crucial step of the epithelial invasion in rodents. To address the physiological relevance of this process in humans, we investigated the effect of single human blastocysts on the regulation of apoptosis in cultured human endometrial epithelial cells (hEEC) in both apposition and adhesion phases of implantation. Here, we report a co-ordinated embryonic regulation of hEEC apoptosis. In the apposition phase, the presence of a blastocyst rescues hEEC from the apoptotic pathway. However, when the human blastocyst adheres to the hEEC monolayer, it induces a paracrine apoptotic reaction. Fas ligand (Fas-L) was present at the embryonic trophoectoderm. Fas was localized at the apical cell surface of hEEC, and flow cytometry revealed that 60% of hEEC express Fas. Neutralizing adhesion assays revealed that the Fas/Fas-L death system may be an important mechanism to cross the epithelial barrier, which is crucial for embryonic adhesion, and the manipulation of this system could have potential clinical implications as an interceptive mechanism.