Fertility preservation for non-medical reasons: controversial, but increasingly common.

BACKGROUND Fertility-preserving measures for women are increasingly being performed for non-medical reasons in Germany. This is now a controversial matter. METHODS The authors searched the PubMed database for pertinent publications on the basis of their clinical and scientific experience and evaluated relevant data from the registry of the German FertiPROTEKT network (www.fertiprotekt. com). The various fertility-preserving measures that are available are described and critically discussed. RESULTS In most cases, the creation of a fertility reserve currently involves the cryopreservation of unfertilized oocytes, rather than of ovarian tissue. Most of the women who decide to undergo this procedure are over 35 years old. According to data from the FertiPROTEKT registry, most such procedures carried out in the years 2012 and 2013 involved a single stimulation cycle. The theoretical probability of childbirth per stimulation is 40% in women under age 35 and 30% in women aged 35 to 39. If the oocytes are kept for use at a later date, rather than at once, the maternal risk is higher, because the mother is older during pregnancy. The risk to the child may be higher as well because of the need for in vitro fertilization (IVF). Pregnancy over age 40 often leads to complications such as gestational diabetes and pre-eclampsia. IVF may be associated with a higher risk of epigenetic abnormalities. Ethicists have upheld women's reproductive freedom while pointing out that so-called social freezing merely postpones social problems, rather than solving them. CONCLUSION Fertility preservation for non-medical reasons should be critically discussed, and decisions should be made on a case-by-case basis.

Sampling tissue for DNA analysis of trees: Trunk cambium as an alternative to canopy leaves

The number of studies of tropical tree species that use molecular tools is increasing, most of which collect leaf tissue for genomic DNA extraction. In tropical trees the canopy is not only frequently inaccessible, but also, once reached, the leaf tissue is often heavily defended against herbivory by high concentrations of anti-predation compounds, which may inhibit downstream applications, particularly PCR. Cambium tissue, accessed directly from the tree trunk at ground level, offers a readily accessible resource that is less hampered by the presence of defensive chemicals than leaf tissue. Here we describe a simple method for obtaining tissue from the cambial zone for DNA extraction and test the applicability of the method in a range of tropical tree species. The method was used successfully to extract DNA from 11 species in nine families. A subset of the DNA extracts was tested in more detail and proved to be highly suitable for AFLP analysis.

Preservation of nitric oxide availability as nitrite and nitrosothiols

Nitric Oxide (NO) has been known for long to regulate vessel tone. However, the close proximity of the site of NO production to "sinks" of NO such as hemoglobin (Hb) in blood suggest that blood will scavenge most of the NO produced. Therefore, it is unclear how NO is able to play its physiological roles. The current study deals with means by which this could be understood. Towards studying the role of nitrosothiols and nitrite in preserving NO availability, a study of the kinetics of glutathione (GSH) nitrosation by NO donors in aerated buffered solutions was undertaken first. Results suggest an increase in the rate of the corresponding nitrosothiol (GSNO) formation with an increase in GSH with a half-maximum constant EC50 that depends on NO concentration, thus indicating a significant contribution of NO2 mediated nitrosation in the production of GSNO. Next, the ability of nitrite to be reduced to NO in the smooth muscle cells was evaluated. The NO formed was inhibited by sGC inhibitors and accelerated by activators and was independent of O2 concentration. Nitrite transport mechanisms and effects of exogenous nitrate on transport and reduction of nitrite were examined. The results showed that sGC can mediate nitrite reduction to NO and nitrite is transported across the smooth muscle cell membrane via anion channels, both of which can be attenuated by nitrate. Finally, a 2-D axisymmetric diffusion model was constructed to test the accumulation of NO in the smooth muscle layer from reduction of nitrite. It was observed that at the end of the simulation period with physiological concentrations of nitrite in the smooth muscle cells (SMC), a low sustained NO generated from nitrite reduction could maintain significant sGC activity and might affect vessel tone. The major nitrosating mechanism in the circulation at reduced O2 levels was found to be anaerobic and a Cu+ dependent GSNO reduction activity was found to deliver minor amounts of NO from physiological GSNO levels in the tissue.

Cancer and fertility preservation: International recommendations from an expert meeting

In the last years, thanks to the improvement in the prognosis of cancer patients, a growing attention has been given to the fertility issues. International guidelines on fertility preservation in cancer patients recommend that physicians discuss, as early as possible, with all patients of reproductive age their risk of infertility from the disease and/or treatment and their interest in having children after cancer, and help with informed fertility preservation decisions. As recommended by the American Society of Clinical Oncology and the European Society for Medical Oncology, sperm cryopreservation and embryo/oocyte cryopreservation are standard strategies for fertility preservations in male and female patients, respectively; other strategies (e.g. pharmacological protection of the gonads and gonadal tissue cryopreservation) are considered experimental techniques. However, since then, new data have become available, and several issues in this field are still controversial and should be addressed by both patients and their treating physicians. In April 2015, physicians with expertise in the field of fertility preservation in cancer patients from several European countries were invited in Genova (Italy) to participate in a workshop on the topic of "cancer and fertility preservation". A total of ten controversial issues were discussed at the conference. Experts were asked to present an up-to-date review of the literature published on these topics and the presentation of own unpublished data was encouraged. On the basis of the data presented, as well as the expertise of the invited speakers, a total of ten recommendations were discussed and prepared with the aim to help physicians in counseling their young patients interested in fertility preservation. Although there is a great interest in this field, due to the lack of large prospective cohort studies and randomized trials on these topics, the level of evidence is not higher than 3 for most of the recommendations highlighting the need of further research efforts in many areas of this field. The participation to the ongoing registries and prospective studies is crucial to acquire more robust information in order to provide evidence-based recommendations.

Establishment of a protocol for the cryopreservation of cell sheets of adipose tissue stem cells

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Preservation of Nitric Oxide Availability as Nitrite and Nitrosothiols

Nitric Oxide (NO) has been known for long to regulate vessel tone. However, the close proximity of the site of NO production to “sinks” of NO such as hemoglobin (Hb) in blood suggest that blood will scavenge most of the NO produced. Therefore, it is unclear how NO is able to play its physiological roles. The current study deals with means by which this could be understood. Towards studying the role of nitrosothiols and nitrite in preserving NO availability, a study of the kinetics of glutathione (GSH) nitrosation by NO donors in aerated buffered solutions was undertaken first. Results suggest an increase in the rate of the corresponding nitrosothiol (GSNO) formation with an increase in GSH with a half-maximum constant EC50 that depends on NO concentration, thus indicating a significant contribution of ∙NO2 mediated nitrosation in the production of GSNO. Next, the ability of nitrite to be reduced to NO in the smooth muscle cells was evaluated. The NO formed was inhibited by sGC inhibitors and accelerated by activators and was independent of O2 concentration. Nitrite transport mechanisms and effects of exogenous nitrate on transport and reduction of nitrite were examined. The results showed that sGC can mediate nitrite reduction to NO and nitrite is transported across the smooth muscle cell membrane via anion channels, both of which can be attenuated by nitrate. Finally, a 2 – D axisymmetric diffusion model was constructed to test the accumulation of NO in the smooth muscle layer from reduction of nitrite. It was observed that at the end of the simulation period with physiological concentrations of nitrite in the smooth muscle cells (SMC), a low sustained NO generated from nitrite reduction could maintain significant sGC activity and might affect vessel tone. The major nitrosating mechanism in the circulation at reduced O2 levels was found to be anaerobic and a Cu+ dependent GSNO reduction activity was found to deliver minor amounts of NO from physiological GSNO levels in the tissue.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.

The legality of tissue transplants for the benefit of family members in the UK and Australia : implications for saviour siblings

The ethics of creating ‘saviour siblings’ for the benefit of another has received much attention, but little consideration has been given to the legal position of those saviours born who may be asked to provide tissue for transplantation to another during childhood. This article examines the ethical issues surrounding minor donation as well as the existing legal framework in the UK and Australia that regulates minors providing tissue for the benefit of another. Against this background the position of minor saviours, who are called upon to donate bone marrow or peripheral blood stem cells, is examined. This analysis suggests that the law does not provide sufficient protection for minor saviours who are called upon to donate to another. It is argued that specific ethical obligations are owed to saviours—that ought to be reflected in the law—in order to protect them from exploitation while they remain minors.

Commercialisation of regenerative human tissue : Regulation and reform in Australia and England, Wales and Northern Ireland

The commercialisation of therapeutic products containing regenerative human tissue is regulated by the common law, statute and ethical guidelines in Australia and England, Wales and Northern Ireland. This article examines the regulatory regimes in these jurisdictions and considers whether reform is required to both support scientific research and ensure conformity with modern social views on medical research and the use of human tissue. The authors consider the crucial role of informed consent in striking the balance between the interests of researchers and the interests of the public.

Translating tissue engineering technology platforms into cancer research

Technology platforms originally developed for tissue engineering applications produce valuable models that mimic three-dimensional (3D) tissue organization and function to enhance the understanding of cell/tissue function under normal and pathological situations. These models show that when replicating physiological and pathological conditions as closely as possible investigators are allowed to probe the basic mechanisms of morphogenesis, differentiation and cancer. Significant efforts investigating angiogenetic processes and factors in tumorigenesis are currently undertaken to establish ways of targeting angiogenesis in tumours. Anti-angiogenic agents have been accepted for clinical application as attractive targeted therapeutics for the treatment of cancer. Combining the areas of tumour angiogenesis, combination therapies and drug delivery systems is therefore closely related to the understanding of the basic principles that are applied in tissue engineering models. Studies with 3D model systems have repeatedly identified complex interacting roles of matrix stiffness and composition, integrins, growth factor receptors and signalling in development and cancer. These insights suggest that plasticity, regulation and suppression of these processes can provide strategies and therapeutic targets for future cancer therapies. The historical perspective of the fields of tissue engineering and controlled release of therapeutics, including inhibitors of angiogenesis in tumours is becoming clearly evident as a major future advance in merging these fields. New delivery systems are expected to greatly enhance the ability to deliver drugs locally and in therapeutic concentrations to relevant sites in living organisms. Investigating the phenomena of angiogenesis and anti-angiogenesis in 3D in vivo models such as the Arterio-Venous (AV) loop mode in a separated and isolated chamber within a living organism adds another significant horizon to this perspective and opens new modalities for translational research in this field.