Gianfranco Coppola

Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa

PurposeWe used computer assisted sperm selection (MSOME) during cycles of intracytoplasmic sperm injection to test whether this technique improves results over traditional ICSI protocols. We also used the TUNEL assay to test whether MSOME could deselect physiologically abnormal spermatozoa.MethodsIndividual spermatozoa were examined with MSOME. Normal and abnormal spermatozoa were tested for the level of DNA fragmentation using TUNEL assay. In a prospective, randomized trial, patients were selected for standard ICSI, or IMSI techniques. We tested the two groups for biological and clinical parameters.Results64.8% of spermatozoa, otherwise selectable for ICSI, were characterized by abnormalities after computer-assisted sperm analysis. These sperm were also characterized by an increase in the level of DNA fragmentation. We noted an increase in embryo quality, pregnancy and implantation rates after computerized sperm selection during ICSI procedures.ConclusionsComputerised selection of spermatozoa during ICSI procedures deselects physiological abnormal spermatozoa and improves clinical results.

Mitochondria and human preimplantation embryo development

Human reproduction, like all biological systems, is characterised by a large level of variability. In this field, the variability is observed as a large difference in implantation potential of human embryos developing in vitro, despite similarities in observable parameters such as rate of development and morphology of these embryos. One of the underlying factors that determines developmental potential in these embryos is the availability of energy in the form of ATP for development. Here, we suggest that, despite the evidence suggesting that mitochondrial metabolism is relatively inactive during preimplantation embryo development, aerobic (mitochondrial) metabolism contributes a major role in the supply of ATP. A second pathway, anaerobic respiration, is also active and the two pathways work in synchrony to supply all the ATP necessary. We discuss the differences in the two forms of energy production and suggest that, although anaerobic respiration can supplement deficiencies in the energy supply in the short term, this is not sufficient to substitute for aerobic respiration over long periods. Therefore, we suggest that deficiencies in the levels of aerobic respiration can explain variability in the implantation potential of apparently equivalent embryos.

S-adenosylhomocysteine treatment of adult female fibroblasts alters X-chromosome inactivation and improves in vitro embryo development after somatic cell nuclear transfer

The poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell. The objective of this study was to investigate the effects of treatment with S-adenosylhomocysteine (SAH) a DNA demethylation agent, on DNA methylation levels and X-chromosome inactivation status of bovine female fibroblast donor cells and the subsequent impact on developmental potential after SCNT. Compared with non-treated controls, the cells treated with SAH revealed (i) significantly (P<0.05) reduced global DNA methylation, (ii) significantly (approximately 1.5-fold) increased telomerase activity, (iii) diminished distribution signals of methylated histones H3-3mK9 and H3-3mK27 on the presumptive inactive X-chromosome (Xi), (iv) alteration in the replication pattern of the Xi, and (v) elevation of transcript levels for X-chromosome linked genes, ANT3, MECP2, XIAP, XIST, and HPRT. SCNT embryos produced with SAH-treated donor cells compared with those derived from untreated donor cells revealed (i) similar cleavage frequencies, (ii) significant elevation in the frequencies of development of cleaved embryos to hatched blastocyst stage, and (iii) 1.5-fold increase in telomerase activity. We concluded that SAH induces global DNA demethylation that partially reactivates the Xi, and that a hypomethylated genome may facilitate the nuclear reprogramming process.

How do spermatozoa activate oocytes

Although the spermatozoon is 500,000 times smaller in volume than the oocyte, it induces rapid and dramatic changes in oocyte physiology that lead to meiosis re-initiation. These oocyte activation events are described here, as is the evidence for a soluble activating factor in the spermatozoon. Since changes in plasma membrane conductance, calcium ion release and maturation-promoting factor inactivation are common to all animal oocytes at activation, it is expected that the sperm-borne trigger is also ubiquitous. One likely candidate, phospholipase C (PLC) zeta 1, induces calcium release in mammalian oocytes; however, work on other deuterostomes suggests that the sperm factor is non-specific and multifactorial, regulating several activation events. Human, sea urchin and ascidian gametes are remarkably similar and comparative studies across the deuterostomes may help in elucidating basic principles in fertilization. Questions to be answered include the identification of PLC zeta 1 in invertebrate spermatozoa and the characterization of other targets in mammalian oocytes, such as the adenosine diphosphate ribose/nitric oxide pathway.

Spatial distribution of histone isoforms on the bovine active and inactive X chromosomes.

The inactive X chromosome (Xi) in female mammals serves as an important model for studying the role of histone isoforms in directing specific nuclear processes leading to inherited differences in transcription. In the present study, we investigated the distribution of some histone isoforms known to be involved in the process of human X inactivation on their bovine counterparts. To ascertain the identity of active and inactive X chromosome, their distribution was investigated on the X chromosomes in a cell line derived from a bovine female carrying an X;autosome translocation rcp(Xp+;23q–) which allowed the recognition of the maternal (translocated) and paternal (normal) X chromosome. The distribution patterns of histone H3 trimethylated at lysine 9 (H3K9me3) and trimethylated at lysine 27 (H3K27me3), and histone macroH2A1 and macroH2A2 (isoforms specific to heterochromatin) were determined by immunocytochemistry and compared to the temporal pattern of replication using BrdU pulse labeling prior to staining. Immunostaining revealed that H3K9me3, H3K27me3, and macroH2A1 are preferentially concentrated on the Xi, whereas the histone variant macroH2A2 is not a marker for this chromosome. H3K9me3, H3K27me3, and macroH2A1 were consistently located in bands along the Xi, while H3K9me3, macroH2A1 and macroH2A2 localized in the pericentromeric regions of the autosomes. H3K27me3 identified two intense bands on the Xi at Xp22 and Xq31, representing the early replication regions of the chromosome. H3K27me3 and macroH2A1 overlapped in the Xq31 region. It was concluded that different heterochromatin regions on the bovine inactive X chromosome can be identified by their histone isoform composition.

Identification of the homologue of the bovine Rob(1;29) in a captive gaur (Bos gaurus).

Robertsonian translocations have been well documented in domestic cattle, with the most commonly occurring fusion involving chromosomes 1 and 29. The widespread nature of this translocation is indicative of its ancient origin. The gaur (Bos gaurus) is one of many wild cattle species currently listed as vulnerable or endangered. Due to the small founder stock and 50 years of restricted breeding, the captive herd is showing signs of inbreeding and reduced fertility. Recent cytogenetic analysis of a female gaur at Toronto Zoo found that the individual contained 2n=57 chromosomes instead of the normal 2n=58, with an extra submetacentric and the loss of two acrocentric chromosomes being observed. This study was undertaken to identify the translocation in this individual and to examine the karyotype of immediate family members. Chromosome analysis of fibroblast cell cultures was carried out using GTG-banding, C-banding and FISH (bovine 1 and 29 paints) techniques to characterize the translocation. Results from the GTG-banding and FISH analyses confirm that the two autosomes involved in the translocation are the bovine homologues 1 and 29. A monocentric centromere was observed by C-banding. Chromosome abnormalities have not been detected in other gaur tested to date. This study demonstrates the importance of cytogenetic analysis for the establishment of screening protocols for the assessment of reproductive potential in this and other exotic bovinae.

Maternal non-Mendelian inheritance of a reduced lifespan? A hypothesis

PurposeA negative correlation exists between advanced maternal age and reproduction. Current data suggest that this correlation is due to a decline in oocyte quality with respect to female age. Since a new individual is derived from the fusion of a single sperm and egg, we tested whether the quality of this material could influence the long-term physiological health of offspring, by examining whether a link between parental age and lifespan of offspring exists.MethodsWe requested a search from the Swedish demographic database POPUM 3 maintained by the University of Umeå, Sweden between years 1700 and 1900. Parameters requested included mothers’ and fathers’ age at gestation, the lifespan of the children, cause of death of children and the region of birth.ResultsComplete data was obtained for 30,512 children born to 12,725 mothers and fathers. Kaplan-Meier estimators demonstrated a strong relationship between mother’s age at gestation and the longevity of offspring. Extrinsic factors such as century of birth also had an effect on the data. The forward stepwise procedure on Cox’s model of proportional hazards suggested that most significant intrinsic factors were mother’s lifespan and mother’s age at gestation.ConclusionsThese data demonstrate that intrinsic and extrinsic factors influence the lifespan of children. Among intrinsic factors, mother’s lifespan and age at gestation had a significant influence on the data. The influence of intrinsic factors remained significant despite a strong extrinsic influence. We suggest that the influence of the mother on the lifespan of offspring is due to extra-genomic factors.

Trends, Fads and ART!

Morphological selection techniques of gametes and embryos are of current interest to clinical practice in ART. Although intracytoplasmic morphologically selected sperm injection (IMSI), time lapse imaging morphometry (TLIM) or quantification of chromosome numbers (PGS) are potentially useful in research, they have not been shown to be of statistically predictive value and, thus, have only limited clinical usefulness. We make the point that morphological markers alone cannot predict the success of the early embryo, which depends on the correct orchestration of a myriad of physiological and biochemical activation events that progress independently of the maternal or zygotic genome. Since previous attempts to identify metabolic markers for embryo quality have failed and there is no evidence that the intrinsic nature of gametes and embryos can be improved in the laboratory, embryologists can only minimize environmental or operator induced damage while these cells are manipulated ex vivo.