Polar body array CGH for prediction of the status of the corresponding oocyte. Part II: technical aspects

The purpose of this study was to assess the technical aspects related to polar body (PB) biopsy, which might have an influence on the results of the microarray comparative genomic hybridization analysis. Furthermore, a comparison was made between two biopsy methods (mechanical and laser). Biopsy of the first and second PB (PB1 and PB2) was performed by mechanical- or laser-assisted biopsy in two different IVF centres. PBs were separately amplified by whole genome amplification. The method of biopsy, mechanical or laser had no influence on the proportion of successfully biopsied oocytes. Especially, for the PB2, the timing of biopsy after ICSI was directly correlated to amplification efficiency. Special care has to be taken with respect to the timing of biopsy of the PB2. Mechanical- and laser-assisted biopsy give the same performance in terms of diagnostic efficienc

Methods for comprehensive chromosome screening of oocytes and embryos: capabilities, limitations, and evidence of validity

Preimplantation aneuploidy screening of cleavage stage embryos using fluorescence in situ hybridization (FISH) may no longer be considered the standard of care in reproductive medicine. Over the last few years, there has been considerable development of novel technologies for comprehensive chromosome screening (CCS) of the human genome. Among the notable methodologies that have been incorporated are whole genome amplification, metaphase and array based comparative genomic hybridization, single nucleotide polymorphism microarrays, and quantitative real-time PCR. As these methods become more integral to treating patients with infertility, it is critical that clinicians and scientists obtain a better understanding of their capabilities and limitations. This article will focus on reviewing these technologies and the evidence of their validity

What next for preimplantation genetic screening? A polar body approach!

Screening of human preimplantation embryos for numerical chromosome abnormalities has been conducted mostly at the preimplantation stage using fluorescence in situ hybridization. However, it is clear that preimplantation genetic screening (PGS) as it is currently practiced does not improve live birth rates. Therefore the ESHRE PGS Task Force has decided to start a proof of principle study with the aim of determining whether biopsy of the first and second polar body followed by subsequent analysis of the complete chromosome complement of these polar bodies using an array based technique enables a timely identification of the chromosomal status of an oocyte. If the principle of this approach can be proven, it is obvious that a multicentre randomized controlled trial should then be started to determine the clinical value of this technique. In this way the ESHRE PGS Task Force hopes to redirect preimplantation screening from the blind alley to the main road of assisted reproduction

Activation of ethylene-responsive p-hydroxyphenylpyruvate dioxygenase leads to increased tocopherol levels during ripening in mango

Mango is characterized by high tocopherol and carotenoid content during ripening. From a cDNA screen of differentially expressing genes during mango ripening, a full-length p-hydroxyphenylpyruvate dioxygenase (MiHPPD) gene homologue was isolated that encodes a key enzyme in the biosynthesis of tocopherols. The gene encoded a 432-amino-acid protein. Transcript analysis during different stages of ripening revealed that the gene is ripening related and rapidly induced by ethylene. The increase in MiHPPD transcript accumulation was followed by an increase in tocopherol levels during ripening. The ripening-related increase in MiHPPD expression was also seen in response to abscisic acid and to alesser extent to indole-3-acetic acid. The expression of MiHPPD was not restricted to fruits but was also seen in other tissues such as leaves particularly during senescence. The strong ethylene induction of MiHPPD was also seen in young leaves indicating that ethylene induction of MiHPPD is tissue independent. Promoter analysis of MiHPPD gene in tomato discs and leaves of stable transgenic lines of Arabidopsis showed that the cis elements for ripening-related, ethylene-responsive, and senescence-related expression resided within the 1590 nt region upstream of the ATG codon. Functionality of the gene was demonstrated by the ability of the expressed protein in bacteria to convert p-hydroxyphenylpyruvate to homogentisate. These results provide the first evidence for HPPD expression during ripening of a climacteric fruit

Hepsin regulates TGF beta signaling via fibronectin proteolysis

Transforming growth factor-beta (TGF beta) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGF beta activity by acting as a storage compartment of latent-TGF beta, but how TGF beta is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGF beta signaling through the release of latent-TGF beta from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGF beta signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGF beta 1, while overexpression of hepsin in mammary tumors increased TGF beta signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGF beta and, importantly, that the ability of hepsin to activate TGF beta signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGF beta pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.Peer reviewe

Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results

Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis. In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1-15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant. This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PB

In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1,2,3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages

Influence of soil water content and atmospheric conditions on leaf water potential in cv. "Touriga Nacional" deep-rooted vineyards

Abstract In this study, the influence of soil and atmosphere conditions on noon and basal leaf water potential of vines ‘‘Touriga Nacional’’ in the Da˜o region submitted to different irrigation treatments is analysed. Both indicators showed to be dependent on environmental conditions at the time of measurement. Leaf water potential at noon of fully watered plants was linearly related with atmospheric conditions, with values registered when vapour pressure deficit (VPD) was higher than approximately 3 kPa being no different from the values registered in stressed plants. Therefore, this indicator cannot be reliably used to distinguish different plant water stress levels when atmospheric conditions induce high evaporative demands. The basal leaf water potential (wb) was also influenced by VPD at the time of measurement for all soil water conditions. In well irrigated plants, it was even possible to establish a baseline that can therefore be used to identify nonwater stressed conditions (wb (MPa) = -0.062–0.0972 VPD (kPa), r2 = 0.78). A good correlation was found between soil humidity and wb. However, more than the average value of the whole thickness of soil monitored, the wb values were dependent on the distribution of soil humidity, with the plants responding to the presence of wet layers