The aim of this pilot study was to assess if array comparative genomic hybridization (aCGH), non-invasive preimplantation genetic screening (PGS) on blastocyst culture media is feasible. Therefore, aCGH analysis was carried out on 22 spent blastocyst culture media samples after polar body PGS because of advanced maternal age. All oocytes were fertilized by intracytoplasmic sperm injection and all embryos underwent assisted hatching. Concordance of polar body analysis and culture media genetic results was assessed. Thirteen out of 18 samples (72.2%) revealed general concordance of ploidy status (euploid or aneuploid). At least one chromosomal aberration was found concordant in 10 out of 15 embryos found to be aneuploid by both polar body and culture media analysis. Overall, 17 out of 35 (48.6%) single chromosomal aneuploidies were concordant between the culture media and polar body analysis. By analysing negative controls (oocytes with fertilization failure), notable maternal contamination was observed. Therefore, non-invasive PGS could serve as a second matrix after polar body or cleavage stage PGS; however, in euploid results, maternal contamination needs to be considered and results interpreted with caution.

Meiotic errors during oocyte maturation are considered the major contributors to embryonic aneuploidy and failures in human IVF treatment. Various technologies have been developed to screen polar bodies, blastomeres and trophectoderm cells for chromosomal aberrations. Array-CGH analysis using bacterial artificial chromosome (BAC) arrays is widely applied for preimplantation genetic diagnosis (PGD) using single cells. Recently, an increase in the pregnancy rate has been demonstrated using array-CGH to evaluate trophectoderm cells. However, in some countries, the analysis of embryonic cells is restricted by law. Therefore, we used BAC array-CGH to assess the impact of polar body analysis on the live birth rate. A disadvantage of polar body aneuploidy screening is the necessity of the analysis of both the first and second polar bodies, resulting in increases in costs for the patient and complex data interpretation. Aneuploidy screening results may sometimes be ambiguous if the first and second polar bodies show reciprocal chromosomal aberrations. To overcome this disadvantage, we tested a strategy involving the pooling of DNA from both polar bodies before DNA amplification. We retrospectively studied 351 patients, of whom 111 underwent polar body array-CGH before embryo transfer. In the group receiving pooled polar body array-CGH (aCGH) analysis, 110 embryos were transferred, and 29 babies were born, corresponding to live birth rates of 26.4% per embryo and 35.7% per patient. In contrast, in the control group, the IVF treatment was performed without preimplantation genetic screening (PGS). For this group, 403 embryos were transferred, and 60 babies were born, resulting in live birth rates of 14.9% per embryo and 22.7% per patient. In conclusion, our data show that in the aCGH group, the use of aneuploidy screening resulted in a significantly higher live birth rate compared with the control group, supporting the benefit of PGS for IVF couples in addition to the suitability and effectiveness of our polar body pooling strategy.

The members of the Olig gene family encode for basic helix-loop-helix (bHLH) transcription factors involved in neural cell type specification. Three Olig genes (Olig1, Olig2 and Olig3) have been identified in all known vertebrate models and a fourth one in anamniotes (olig4). Here we have performed a global analysis of olig genes during zebrafish embryonic development and determined which signaling pathways control their induction and regionalization in the CNS. Interestingly, zebrafish olig3 and olig4 together establish most of the expression domains corresponding to mouse Olig3. According to our data, olig1 is specifically confined to the oligodendrocyte lineage, whereas the other members display stratified expression in diencephalon, hindbrain, and spinal cord. We observed differential expression of olig genes within specific motoneuron and interneuron domains of the spinal cord. olig2, olig3, and olig4 expression appears to be regulated by nodal and FGF signaling during gastrulation and early somitogenesis, by RA signaling in the hindbrain, and by BMP and Hh signals along the dorsoventral axis of the embryonic CNS. Our findings suggest a role for olig genes in CNS patterning as well as in multiple cell fate decisions during neural differentiation.

ObjectiveThe aim of this study was to evaluate the efficacy of a non-expensive, easy and fast technique (direct micro swim-up) for sperm preparation in intracytoplasmic sperm injection (ICSI) treatments without the use of centrifuge.MethodsWe carried out a multicentric study in which a total of 140 ICSI-cycles were included. Sibling oocytes were divided into two groups according to semen preparation procedures: group A, discontinuous gradients (DG) (oocytes n=668), and group B, direct micro swim-up (MSU) (oocytes n=660). We analyzed differences in some key performance indicators.ResultsFertilization rates were not statistically different between the DG and MSU groups (76.0% vs. 81.8%, respectively, p=0.248); while significant differences were found in blastulation rates per fertilized oocytes (41.7% vs. 58.5%, p=0.009), blastulation rates per D3 embryos (46.1% vs. 63.7%, p=0.045), and pregnancy rates (25.8% vs. 41.9%, p=0.045). The abortion rate was reduced in the MSU group as compared to DG, but not in a significant manner (12.9% vs. 29.4%, p=0.161).ConclusionThe MSU procedure has the advantage of reducing costs, time and mismatches, while ensuring comparable, and in some cases, better results than DG treatments. This technique can therefore be used as an alternative method to other conventional semen treatments.

Segmentation is a key step in embryonic development. Acting in all germ layers, it is responsible for the generation of antero-posterior asymmetries. Hox genes, with their diverse expression in individual segments, are fundamental players in the determination of different segmental fates. In vertebrates, Hox gene products gain specificity for DNA sequences by interacting with Pbx, Prep and Meis homeodomain transcription factors. In this work we cloned and analysed prep1.2 in zebrafish. In-situ hybridization experiments show that prep1.2 is maternally and ubiquitously expressed up to early somitogenesis when its expression pattern becomes more restricted to the head and trunk mesenchyme. Experiments of loss of function with prep1.2 morpholinos change the shape of the hyoid and third pharyngeal cartilages while arches 4-7 and pectoral fins are absent, a phenotype strikingly similar to that caused by loss of retinoic acid (RA). In fact, we show that prep1.2 is positively regulated by RA and required for the normal expression of aldh1a2 at later stages, particularly in tissues involved in the development of the branchial arches and pectoral fins. Thus, prep1.2 and aldh1a2 are members of an indirect positive feedback loop required for pharyngeal endoderm and posterior branchial arches development. As the paralogue gene prep1.1 is more important in hindbrain patterning and neural crest chondrogenesis, we provide evidence of a functional specialization of prep genes in zebrafish head segmentation and morphogenesis.