Santo Stefano del Sole, Italy
Santo Stefano del Sole, Italy

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Menezo Y.,Laboratoire dEylau | Dale B.,Center for Assisted Fertilization | Cohen M.,Clinique Natecia
Zygote | Year: 2010

The genome of all cells is protected at all times by mechanisms collectively known as DNA repair activity (DRA). Such activity is particularly important at the beginning of human life, i.e. at fertilization, immediately after and at the very onset of embryonic development. DRA in early development is, by definition, of maternal origin: the transcripts stored during maturation, need to control the integrity of chromatin, at least until the maternal/zygotic transition at the 4- to 8-cell stage in the human embryo. Tolerance towards DNA damage must be low during this critical stage of development. The majority of DNA damage is due to either apoptosis or reactive oxygen species (ROS). Apoptosis, abortive or not, is a common feature in human sperm, especially in oligoasthenospermic patients and FAS ligand has been reported on the surface of human spermatozoa. The susceptibility of human sperm to DNA damage is well documented, particularly the negative effect of ROS (Kodama et al., 1997; Lopes et al., 1998a, b) and DNA modifying agents (Zenzes et al., 1999; Badouard et al., 2007). DNA damage in sperm is one of the major causes of male infertility and is of much concern in relation to the paternal transmission of mutations and cancer (Zenzes, 2000; Aitken et al., 2003; Fernández-Gonzalez, 2008). It is now clear that DNA damaged spermatozoa are able to reach the fertilization site in vivo (Zenzes et al., 1999), fertilize oocytes and generate early embryos both in vivo and in vitro. The effect of ROS on human oocytes is not as easy to study or quantify. It is a common consensus that the maternal genome is relatively well protected while in the maturing follicle; however damage may occur during the long quiescent period before meiotic re-activation (Zenzes et al., 1998). In fact, during the final stages of follicular growth, the oocyte may be susceptible to damage by ROS. With regards to the embryo there is active protection against ROS in the surrounding environment i.e. in follicular and tubal fluid (El Mouatassim et al., 2000; Guerin et al., 2001). DNA repair activity in the zygote is mandatory in order to avoid mutation in the germ line (Derijck et al., 2008). In this review we focus on the expression of mRNAs that regulate DNA repair capacity in the human oocyte and the mechanisms that protect the embryo against de novo damage. © 2010 Cambridge University Press.


Menezo Y.,London Fertility Associates | Menezo Y.,Laboratoire CLEMENT | Evenson D.,South Dakota State University | Cohen M.,Procrelys | And 2 more authors.
Advances in Experimental Medicine and Biology | Year: 2014

According to worldwide statistics, between one in four and one in five couples have fertility problems. These problems are equally distributed between males and females. Modern lifestyle has obviously increased these problems: endocrine-disrupting chemicals, such as plastic polymer catalysts, alkylphenols, phthalates and so on, and cosmetic additives seem to be strongly involved in this fertility problem. Many of these compounds increase oxidative stress (OS) and thus impair spermatogenesis. The oocyte has only a finite capacity, decreasing with maternal age, to repair sperm-borne decays. To decrease this DNA repair burden, reducing the sperm DNA damages linked to OS is tempting. Antioxidant vitamins are often given haphazardly; they are not very efficient and potentially detrimental. A detailed analysis of the sperm nucleus is mandatory (DNA fragmentation or lack of nuclear condensation) prior to any treatment. Here we discuss new concepts in OS and the corresponding therapeutic approaches. © 2014 Springer Science+Business Media New York.


PubMed | Center for Assisted Fertilization, Clinique Natecia, Bourn Hall Clinic and Laboratoire Clement
Type: Review | Journal: Reproductive biomedicine online | Year: 2016

The negative effect of oxidative stress on the human reproductive process is no longer a matter for debate. Oxidative stress affects female and male gametes and the developmental capacity of embryos. Its effect can continue through late stages of pregnancy. Metabolic disorders and psychiatric problems can also be caued by DNA methylation and epigenetic errors. Age has a negative effect on oxidative stress and DNA methylation, and recent observations suggest that older men are at risk of transmitting epigenetic disorders to their offspring. Environmental endocrine disruptors can also increase oxidative stress and methylation errors. Oxidative stress and DNA methylation feature a common denominator: the one carbon cycle. This important metabolic pathway stimulates glutathione synthesis and recycles homocysteine, a molecule that interferes with the process of methylation. Glutathione plays a pivotal role during oocyte activation, protecting against reactive oxygen species. Assisted reproductive techniques may exacerbate defects in methylation and epigenesis. Antioxidant supplements are proposed to reduce the risk of potentially harmful effects, but their use has failed to prevent problems and may sometimes be detrimental. New concepts reveal a significant correlation between oxidative stress, methylation processes and epigenesis, and have led to changes in media composition with positive preliminary clinical consequences.


Santella L.,Stazione Zoologica Anton Dohrn | Dale B.,Center for Assisted Fertilization
Reproductive BioMedicine Online | Year: 2015

In a recent report in Reproductive Biomedicine Online by Ebner et al., a comprehensive multi-centre study was presented on the use of a calcium ionophore, A23187, to artificially activate oocytes from patients who had poor fertilization rates in previous cycles. Under physiological conditions, the calcium increase in oocytes at activation is caused by influx and release from specific stores and ion channels, and has precise temporal, quantitative and spatial patterns. Calcium ionophores may release Ca2+ in an uncontrolled fashion from intracellular stores that would not normally be involved in the activation process. Ionophores, including A23187, have a multitude of effects on cell homeostasis, not yet defined in oocytes, that may have long-term effects, for example on gene expression. We suspect that the successful births reported by Ebner et al. are a result of the overriding influence of the injected spermatozoa, rather than the effect of the ionophore; nevertheless, such an invasive non-physiological approach to assisted reproduction techniques is worrying, especially as epigenetic effects may result in future generations. © 2015 Reproductive Healthcare Ltd.


Di Caprio G.,National Research Council Italy | Di Caprio G.,Harvard University | El Mallahi A.,Free University of Colombia | Ferraro P.,National Research Council Italy | And 5 more authors.
Biomedical Optics Express | Year: 2014

In this paper we investigate the use of a digital holographic microscope, with partial spatial coherent illumination, for the automated detection and tracking of spermatozoa. This in vitro technique for the analysis of quantitative parameters is useful for assessment of semen quality. In fact, thanks to the capabilities of digital holography, the developed algorithm allows us to resolve in-focus amplitude and phase maps of the cells under study, independently of focal plane of the sample image. We have characterized cell motility on clinical samples of seminal fluid. In particular, anomalous sperm cells were characterized and the quantitative motility parameters were compared to those of normal sperm. © 2014 Optical Society of America.


Ferrara M.A.,National Research Council Italy | De Angelis A.,National Research Council Italy | De Luca A.C.,National Research Council Italy | Coppola G.,National Research Council Italy | And 2 more authors.
IEEE Journal on Selected Topics in Quantum Electronics | Year: 2016

Coupling digital holography with high-specific Raman spectroscopy is an attractive means of identifying biochemical changes in cells by both physical topography and spectroscopy. We have used this approach to simultaneously study biochemical and morphological characteristics of human sperm cells irradiated with green laser radiation. Severe spermatozoa variations associated with a topological redistribution of the sample and a gradual decrease in the Raman signal intensity were detected in a label-free configuration. Importantly, at laser fluences where no morphological alterations were detected (30 MJ/cm2), high specific spectral variations were monitored to evaluate the cell photodegradation. © 2015 IEEE.


PubMed | Stazione Zoologica Anton Dohrn and Center for Assisted Fertilization
Type: Journal Article | Journal: Reproductive biomedicine online | Year: 2015

In a recent report in Reproductive Biomedicine Online by Ebner etal., a comprehensive multi-centre study was presented on the use of a calcium ionophore, A23187, to artificially activate oocytes from patients who had poor fertilization rates in previous cycles. Under physiological conditions, the calcium increase in oocytes at activation is caused by influx and release from specific stores and ion channels, and has precise temporal, quantitative and spatial patterns. Calcium ionophores may release Ca(2+) in an uncontrolled fashion from intracellular stores that would not normally be involved in the activation process. Ionophores, including A23187, have a multitude of effects on cell homeostasis, not yet defined in oocytes, that may have long-term effects, for example on gene expression. We suspect that the successful births reported by Ebner etal. are a result of the overriding influence of the injected spermatozoa, rather than the effect of the ionophore; nevertheless, such an invasive non-physiological approach to assisted reproduction techniques is worrying, especially as epigenetic effects may result in future generations.


Dale B.,Center for Assisted Fertilization | Menezo Y.,London Fertility Associates | Coppola G.,Center for Assisted Fertilization
Journal of Assisted Reproduction and Genetics | Year: 2015

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. © 2014, Springer Science+Business Media New York.


Dale B.,Center for Assisted Fertilization | Defelice L.,Virginia Commonwealth University
Journal of Assisted Reproduction and Genetics | Year: 2011

The purpose of this review is to open a debate as to whether or not oocytes actively repel supernumerary sperm or in nature final sperm : oocyte ratios are so low that polyspermy preventing mechanisms are not necessary. Before encountering the oocyte, spermatozoa need to be primed, either by environmental factors as in animals exhibiting external fertilization, or by factors from the female reproductive tract, as in mammals. The spermatozoon must then recognize and interact with the outer layers of the oocyte and progression of the fertilizing spermatozoon through these layers is further controlled and modulated by a precise sequence of signals in situ. Removal of these outer coats may not inhibit fertilization, however does interfere with the dynamics of sperm-oocyte interaction. We propose that monospermy in mammals and sea urchins, under natural conditions, is ensured by the controlled and gradual encounter of a minimum number of spermatozoa with the oocyte and that fine tuning is ensured by the structural and molecular organization of the oocyte and its surrounding coats. We suggest that laboratory experiments using oocytes deprived of their investments and exposed to unnaturally high concentrations of spermatozoa are artifactual and argue that the conclusions leading to the hypothesis of a fast electrical block to polyspermy are unfounded. Under laboratory conditions the majority of spermatozoa, although motile and capable of attaching to the oocyte surface, are either physiologically incompetent or attach to areas of the oocyte surface that do not support entry. © 2010 Springer Science+Business Media, LLC.


Dale B.,Center for Assisted Fertilization
Biochemical and Biophysical Research Communications | Year: 2014

This purpose of this review is to look at the experimental evidence, both kinetic and electrophysiological, that led to the hypothesis of a fast electrical block to polyspermy in sea urchin eggs. The idea of a fast partial block, forwarded in the 1950's, that would reduce the receptivity of the egg surface by 1/20th following its interaction with the fertilizing spermatozoon, was based on experiments that treated fertilization as a first order chemical reaction. Here, I outline the criticisms of the Rothschild theory and demonstrate that the hypothesis of a fast partial block to polyspermy is unfounded. Notwithstanding, it was suggested in the 1970's that the membrane depolarization, induced by the fertilizing spermatozoon, prevented the interaction of supernumerary spermatozoa, the fast electrical block to polyspermy. While trans-membrane voltage recording has permitted a better understanding of the sequence of events occurring at fertilization, there is no evidence that depolarization prevents the interaction of supernumerary spermatozoa. Sperm entry is prevented at positive and negative potentials, in the voltage clamp configuration, however this is an artifact caused by the currents injected into the egg employed to hold the voltage constant in a non-physiological range. At permissive voltages, around -20 mV, where the current required to hold the voltage is minimal, only one spermatozoon normally enters the egg. Thus, irrespective of the egg voltage, the fertilizing spermatozoon is, in any case, attached to a privileged interaction site that permits entry and distinguishes it from supernumerary spermatozoa. Competence for monospermy is acquired during oocyte maturation and data on cortical organization in echinoderm eggs points to the actin filament system for regulating sperm entry. © 2014 Elsevier Inc. All rights reserved.

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