Fertility preservation and preimplantation genetic assessment for women with breast cancer

Breast cancer is the most common cancer diagnosed among reproductive aged women, and its treatment can compromise future fertility. Options for fertility preservation include oocyte or embryo cryopreservation after ovarian stimulation (OS), which are the most established choices and are applicable for adult women with cancer. Ovarian tissue freezing may also be appropriate, as it offers potentially the least delay. The recognisation of the role of BRCA1 and BRCA2 mutations in some women has led to the involvement of preimplantation genetic diagnosis (PGD), recently renamed preimplantation genetic testing for monogenic disorder (PGT-M), whereby embryos are created by IVF and cell(s) are removed and genetically analyzed for specific disease-related mutations. PGT-M offers a valid option for women wishing to avoid transmission of the predisposition for hereditary breast cancer to their offspring. The aim of this paper is to provide an overview of the factors that influence fertility preservation in newly diagnosed breast cancer patients, and to illustrate the option of PGT-M to enable conception of an unaffected child.     

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution

Cryopreservation is the only long-term storage option for the storage of vessels and vascular constructs. However, endothelial barrier function is almost completely lost after cryopreservation in most established cryopreservation solutions. We here aimed to improve endothelial function after cryopreservation using the 2D-model of porcine aortic endothelial cell monolayers.?The monolayers were cryopreserved in cell culture medium or cold storage solutions based on the 4°C vascular preservation solution TiProtec^®, all supplemented with 10% DMSO, using different temperature gradients. After short-term storage at ?80°C, monolayers were rapidly thawed and re-cultured in cell culture medium.?Thawing after cryopreservation in cell culture medium caused both immediate and delayed cell death, resulting in 11 ± 5% living cells after 24 h of re-culture. After cryopreservation in TiProtec and chloride-poor modifications thereof, the proportion of adherent viable cells was markedly increased compared to cryopreservation in cell culture medium (TiProtec: 38 ± 11%, modified TiProtec solutions ≥ 50%). Using these solutions, cells cryopreserved in a sub-confluent state were able to proliferate during re-culture. Mitochondrial fragmentation was observed in all solutions, but was partially reversible after cryopreservation in TiProtec and almost completely reversible in modified solutions within 3 h of re-culture. The superior protection of TiProtec and its modifications was apparent at all temperature gradients; however, best results were achieved with a cooling rate of ?1°C/min.?In conclusion, the use of TiProtec or modifications thereof as base solution for cryopreservation greatly improved cryopreservation results for endothelial monolayers in terms of survival and of monolayer and mitochondrial integrity.     

Mechanisms underlying insect freeze tolerance

Freeze tolerance – the ability to survive internal ice formation – has evolved repeatedly in insects, facilitating survival in environments with low temperatures and/or high risk of freezing. Surviving internal ice formation poses several challenges because freezing can cause cellular dehydration and mechanical damage, and restricts the opportunity to metabolise and respond to environmental challenges. While freeze‐tolerant insects accumulate many potentially protective molecules, there is no apparent ‘magic bullet’ – a molecule or class of molecules that appears to be necessary or sufficient to support this cold‐tolerance strategy. In addition, the mechanisms underlying freeze tolerance have been minimally explored. Herein, we frame freeze tolerance as the ability to survive a process: freeze‐tolerant insects must withstand the challenges associated with cooling (low temperatures), freezing (internal ice formation), and thawing. To do so, we hypothesise that freeze‐tolerant insects control the quality and quantity of ice, prevent or repair damage to cells and macromolecules, manage biochemical processes while frozen/thawing, and restore physiological processes post‐thaw. Many of the molecules that can facilitate freeze tolerance are also accumulated by other cold‐ and desiccation‐tolerant insects. We suggest that, when freezing offered a physiological advantage, freeze tolerance evolved in insects that were already adapted to low temperatures or desiccation, or in insects that could withstand small amounts of internal ice formation. Although freeze tolerance is a complex cold‐tolerance strategy that has evolved multiple times, we suggest that a process‐focused approach (in combination with appropriate techniques and model organisms) will facilitate hypothesis‐driven research to understand better how insects survive internal ice formation.     

Functional impairment of MSC induced by transient warming events: Correlation with loss of adhesion and altered cell size

Background

We recently showed that transient warming effects decreased the functional and adhesion properties of mesenchymal stromal cells (MSC) while post-thaw viability remained high. In an attempt to better predict functional impairment of cryopreserved MSC, we further analysed the correlation between viability, immunosuppressive activity and adhesion of cells exposed or not to warming events.

杏愈伤组织的超低温保存

杏茎段在附加１．０ｍｇ／Ｌ ２,４－Ｄ和０．１ｍｇ／Ｌ ＢＡ的改良ＭＳ培养基中诱导出愈伤组织,放入１０％ＤＭＳＯ＋０．５ｍｏｌ／Ｌ山梨醇或葡萄糖的冰冻保护剂中,以１Ｃ／ｍｉｎ的降温速度降至－４０℃,停留２ｈ后投入液氮保存,在４０℃水浴中化冻。继代培养８次生长１５－２０ｄ的愈伤组织保存后的相对存活率最高,不同品种基因型保存效果差异很大,保存后的愈伤组织生长量比对照减少,但经过２次继代培养后不再表现差     

Cryopreservation of Unfertilized Mouse Oocytes: The Effect of Replacing Sodium with Choline in the Freezing Medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stagein vitro.Specifically, the effects of cooling to subzero temperatures, thawing rate, LN₂plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or −7.0°C in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN₂from −10, −20, or −33°C at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, −0.3 C/min, plunging at −33°C) rapid thawing by direct submersion in 30°C water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.     

In vitro culture and conservation of microalgae: Applications for aquaculture, biotechnology and environmental research

Summary Microalgae are a highly diverse group of unicellular organisms comprising the eukaryotic protists and the prokaryotic cyanobacteria or blue-green algae. The microalgae have a unique environmental status; being virtually ubiquitous in euphotic aquatic niches, they can occupy extreme habitats ranging from tropical coral reefs to the polar regions, and they contribute to half of the globe’s photosynthetic activity. Furthermore, they form the basis of the food chain for more than 70% of the world’s biomass. Microalgae are a valuable environmental and biotechnological resource, and the aim of this review is to explore the use of in vitro technologies in the conservation and sustainable exploitation of this remarkable group of organisms. The first part of the review evaluates the importance of in vitro methods in the maintenance and conservation of microalgae and describes the central role of culture collections in applied algal research. The second part explores the application of microalgal in vitro technologies, particularly in the context of the aquaculture and biotechnology industries. Emphasis is placed upon the exploitation of economically important algal products including aquaculture feed, biomass production for the health care sector, green fertilizers, pigments, vitamins, antioxidants, and antimicrobial agents. The contribution that microalgae can make to environmental research is also appraised; for example, they have an important role as indicator organisms in environmental impact assessments. Similarly, designated culture collection strains of microalgae are used for ecotoxicity testing. Throughout the review, emphasis is placed on the application of in vitro techniques for the continued advancement of microalgal research. The paper concludes by assessing future perspectives for the novel application of microalgae and their products.     

COMMERCIAL DEVELOPMENTS IN MICROALGAL BIOTECHNOLOGY

A number of important advances have occurred in microalgal biotechnology in recent years that are slowly moving the field into new areas. New products are being developed for use in the mass commercial markets as opposed to the "health food" markets. These include algal-derived long-chained polyunsaturated fatty acids, mainly docosahexaenoic acid, for use as supplements in human nutrition and animals. Large-scale production of algal fatty acids is possible through the use of heterotrophic algae and the adaptation of classical fermentation systems providing consistent biomass under highly controlled conditions that result in a very high quality product. New products have also been developed for use in the development of pharmaceutical and research products. These include stable-isotope biochemicals produced by algae in closed-system photobioreactors and extremely bright fluorescent pigments. Cryopreservation has also had a tremendous impact on the ability of strains to be maintained for long periods of time at low cost and maintenance while preserving genetic stability.     

Birth of a Western Lowland gorilla (Gorilla gorilla gorilla) following in vitro fertilization and embryo transfer

A 21-year-old multiparous female exhibiting 31–41 day menstrual cycles was given hFSH (225 IU/day, Metrodin 75, from cycle day 3 through 9 (menses = day 1) and hCG (10,000 IU, Profasi, on day 10 to stimulate follicular development. At 35 h after hCG, under isoflurane (AErrane) anesthesia, follicles were aspirated by controlled suction under transvaginal ultrasound guidance. Metaphase II oocyctes (n = 11) were placed in modified human tubal fluid (mHTF, 100 μl) medium under oil at 37°C in humidified 5% CO₂. Frozen semen, collected by voluntary ejaculation, was thawed (70°C H₂O bath, 6 sec), diluted slowly, centrifuged, and resuspended in mHTF, and 160,000 motile spermatozoa/ml were added at 6 h after oocyte recovery. At 21 h postinsemination (p.i.) eight oocytes were at the two-cell stage, five were cryopreserved, and three were cultured to the six- to eight-cell stage in mHTF with granulosa cells before transcervical uterine transfer at 47 h p.i. using a Teflon catheter. Micronized progesterone (400 mg/d) was orally administered for 10 weeks posttransfer (p.t.). Ultrasound examination revealed a single fetus at 15 weeks p.t., and unassisted delivery of a live 1.37 kg female infant occurred at 29 weeks. Am. J. Primatol. 41:247–260, 1997. © 1997 Wiley-Liss, Inc.