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  Speakers Overviews Print
Amir Arav, Israel
CRYOTECHNOLOGY - WHAT IS NEXT?

This year we celebrate 75 years since the first vitrification of sperm done by Luyet in 1938.
Since then many techniques in cryobiology have been developed and the preservation of many cells, tissues and even organs has been made possible. What unifies the tissue and cell cryobanking is that they store the samples in liquid nitrogen. Storage of cryopreserved samples, under liquid nitrogen, is very demanding in terms of maintenance, storage space, storage equipment and costs. An alternative that would minimize costs, storage and maintenance has been gaining a foothold in the field of cell preservation in recent years - the dry storage. Drying of cells can be achieved by either convective-drying or freeze-drying. Freeze-drying is achieved by sublimation of the ice after freezing the sample to subzero temperatures. The process is damaging to the cellular membrane and some degree of chromosomal damage may also take place due to endogenous nucleases.
To date, embryonic development after intracytoplasmic sperm injection (ICSI) with freeze-dried sperm heads has been reported in humans, hamsters, cattle, pigs, rhesus macaque and cats, and live offspring were reported in mice, rabbits, rats and fish . We have recently demonstrated the use of sheep freeze-dried somatic cells for somatic cell nuclear transfer. We utilized the directional freezing technology to freeze-dry somatic cells which were kept at room temperature for 3 years. These cells were rehydrated and then used to direct embryonic development following nuclear transfer into in vitro matured enucleated oocytes. Finally, human hematopoietic stem cells that were lyophilized and rehydrated with water were viable and have maintained their clonogenic capacity showing that they were able to develop into all blood lineages. This was the first report to show cells that have undergone complete lyophilization and following rehydration have maintained not only their viability but also their functionality. Recently we have shown that bovine mature oocytes can be lyophilized and maintain the viability after rehydration.
I believe that the future of fertility preservation will be a simple "on shelf preservation" of gametes, embryos, gonad's tissue and stem cells.

Uzi Beller, Israel
HOW TO SELECT THE PATIENTS FOR FERTILITY PRESERVATION

Identifying and selecting the female patient, with gynecological or breast cancer, to whom we can offer Fertility Preservation (FP) depends upon a variety of factors each with significant as well as unique importance.
We should address the following issues prior to recommending any FP modality to our patient:
1.Oncologic prognosis and quality of life
2.Fertility and obstetrical history and potential.
3.Availability and reliability of the FP technologies
4.Surgical modalities and expertise
5.Medical and laboratory modalities
6.Explaining the meaning of success
7.The psycho-social perspective
8.The role of couple counseling
9.Obtaining informed consent

Fertility preservation is by definition a personalized medical therapy; the "one size fits all” approach does not exist in these situations. Therefore, for every woman, a multidisciplinary group of experts should convene and analyze all the related issues prior to recommending the best option for the particular patient and spouse.
One should not provide information or data with exaggerated reassurance of obstetrical success rates. We should definitely not compromise on oncologic proven treatments and outcome on account of possible FP.
We emphasize the need for a carefully obtained informed consent.
Galli Cesare, Italy
HORSE EMBRYO SLOW FREEZING

Assisted reproductive technology in the horse lagged behind other species. Several reasons include difference in ovarian physiology with fewer follicles present at any time on the ovaries, the difficulty in inducing superovulation, the extreme individual variability to the success of semen cryopreservation, the relative large size of the pre-implantation embryo and last but not least studbook regulations. Embryos crypopreservation allows more flexibility on the breeding programmes, the possibility of banking embryos and the marketing of the embryos on a world scale. Horse embryos to be successfully cryopreserved should be less than 300µm in diameter. These embryos can be recovered from inseminated mare 6-7 days after insemination, however at this stage the recovery rate is lower than normally obtained on day 8-9 because not all the embryos have migrated into the uterus yet. Embryos can also be produced in vitro through oocyte maturation, intracytoplasmic sperm injection (ICSI) and in vitro culture to the blastocyst stage. In this way the embryo can be frozen at the right stage and size thus maximizing its survival. Embryos are frozen using 10% glycerol as cryoprotectant and loaded into straws. The programmable freezer is set at -6°C , embryos are allowed to equilibrate at this temperature for 5 min. then after seeding are cooled at 0.5 °C per minute to -32°C and finally plunged in liquid nitrogen.
After thawing the cryprotectant is removed prior to transfer to synchronized recipients by non surgical transfer. Under these conditions pregnancy rates are in the range of 50 to 60% thus making cryopreservation a viable tool for the horse industry.
Ana Cobo, Spain
THE POSSIBILITY TO VITRIFY EGG, EMBRYO AND CREATING A SUIRABLE BANK.

Nowadays we can say that the challenge of successful oocyte cryopreservation has been reached by means of vitrification, making possible its introduction into the clinical practice. We are currently successfully storing gametes of many women due to different reasons. Fertile women are currently taking advantage of this technology to electively delay childbearing. Additionally, women at risk of losing their reproductive function caused by oncologycal treatments or premature ovarian failure could benefit greatly from this practice. In addition, oocyte vitrification increases the flexibility of the options offered by current assisted reproduction technology strategies, especially in cases where the delaying in embryo transfer is mandatory due to the risk of ovarian hyperstimulation syndrome; failure to obtain the sperm sample the day of ovum pick up; or low response to gonadotropins. One of the most interesting advantages of oocyte cryo-storage is the greater flexibility granted to ovum donation programs, since no synchronization would be needed, becoming safer and easier procedures since a quarantine period could be attained prior to the donation of the oocytes. The availability of donors is one of the greatest difficulties of oocyte donation programs generating long waiting lists. This issue has been overcome by oocyte cryo-banking as the stored oocytes are donated as soon as the recipient's endometrial preparation is completed. The use of stored oocytes has also eliminated the need for precise coordination of the donor and recipient cycles, which in some cases leads to the cycle cancellation due to unexpected inconveniences with consequent emotional and financial cost for the couple. One more remarkable advantage is the ability to quarantine the oocytes in the same way we do for sperm samples, greatly contributing to diminish the potential risk of infectious disease transmission. In this way the oocyte donation practice could be equate to the donation of any other cryopreserved tissue. In addition the use of donor's cryopreserved oocytes alleviates logistic aspects related to travel issues, setting the date for embryo replacement so that patients feel more confident and comfortable. In IVI-Valencia egg banking is currently an integral part of our clinical routine with over 30,000 vitrified oocytes donated, bringing great flexibility to our program. On the other hand, vitrification has proven to be very useful for embryo cryopreservation programs yielding excellent results at all embryo developmental stages. In addition to the clinical evidence, the safety of vitrification has been demonstrated by other studies assessing the subcellular damage and the physiology of embryos developed from vitrified oocytes. Indeed, it would seem a breakthrough has been reached in assisted reproductive technology.
Pierre Comizzoli, USA
COMPARATIVE AND TRANSLATIONAL CRYOBIOLOGY OF GAMETES AND GONADAL TISSUES FROM WILDLIFE SPECIES

One of the greatest challenges to retaining viability of frozen gametes and reproductive tissues is understanding and overcoming species-specificities. This also is important because there is significant diversity in the cryobiological properties and requirements among cell types and tissues themselves. Systematic studies can lead to successful post-thaw recovery, especially after determining (1) membrane permeability to water and cryoprotectant, (2) cryoprotectant toxicity, (3) tolerance to osmotic changes and (4) resistance to cooling and freezing temperatures. While species-dependency ultimately dictates the ability of specific cells and tissues to survive freeze-thawing, there are commonalities between taxa that allow a protocol developed for one species to provide useful information for another. This is the primary reason for performing comparative cryopreservation studies among diverse species. Characterizing the biophysical traits of gametes and tissues is the most efficient way to develop successful storage and recovery protocols. Such data are only available for a few laboratory, livestock and fish species, with virtually all others (wild mammals, birds, reptiles and amphibians) having gone unstudied in this arena. Nonetheless, when a rare animal unexpectedly dies, there is no time to understand the fundamentals of biophysics. In these emergencies, it is necessary to rely on experience and the best data from taxonomically-related species. At last, new preservation strategies will offer knowledge and tools to better manage species that serve as valuable biomedical models or require assistance to reverse endangerment.
John Crowe, USA
PRESERVATION OF DRY CELLS: LESSONS FROM NATURE

There are many organisms in nature that are capable of surviving nearly complete dehydration, a phenomenon known as anhydrobiosis. Using what we and others have learned about adaptations of such organisms to existence in the absence of water, we have for some years been drying mammalian cells, with considerable success. I will talk about the underlying mechanisms that make this possible and about work on human platelets and stem cells.
Andras Dinnyes, Hungary
CRYOPRE SERVATION OF ANIMAL STEM CELLS 

Stem cells offer biomedical research and therapeutic applications in various disorders and trauma.There is a major variability among different kind of stem cells (embryonic origin, somatic origin of various tissues, induced pluripotent stem cells). There is even a potential to turn somatic cells into pluripotent stem cells, then create chimeric embryos (with germline transmission, currently in mice only) or to use stem cells for gamete production. This would allow gene banking from almost any tissues. The most frequently used cell freezing technology offer a simple and moderately efficient way to preserve stem cells. However, in light of the speedy development of the stem cell field, more advanced and efficient cryopreservation methods (including directional freezing, vitrification, freeze dryingetc) would allow to bank precious stem cell material without genetic damage or loss of cell populations. Stem cell derived cells, cell conglomerates (f.ex. embryoid bodies) or tissues as commercially valuable products might need to be cryopreserved to extend their use without time or geographical distance limitations. This would allow specialized laboratories with appropriate conditions (GMP, GLP etc) to apply professionalstem cell culture, expansion and/or differentiation technologies then distribute their stem cell based products internationally. The lecture will discuss the cryopreservation aspects of advanced animal and human stem cell based applications, including current trends and additional needs. We acknowledge the support of EU FP7 AnistemPIAP-GA-2011-286264, RabPstem PERG07-GA-2010-268422, Resolve Health-F4-2008-202047 projects.
Marie-Madeleine Dolmans, Belgium
REIMPLANTATION OF CRYOPRESERVED OVARIAN TISSUE TAKEN FROM PATIENTS WITH VARIOUS MALIGNANT DISEASES: IS IT SAFE?!

Reversing treatment-related premature ovarian failure by means of autotransplantation of frozen-thawed ovarian tissue harvested before chemo-radiotherapy is becoming an increasingly realistic prospect for clinical application, with 22 live births already reported using this technique. Our objective is to offer young patients at risk of post-treatment premature ovarian failure, safe fertility preservation options.
One major concern raised by transplantation of ovarian cortical fragments in cancer patients is the potential risk that the cryopreserved ovarian tissue might harbor malignant cells that could induce a recurrence of the disease after reimplantation.
Hematological malignancies and breast cancer are the most frequent indications for ovarian tissue cryopreservation. Both carry the risk of ovarian metastasis.
We therefore decided to conduct a study to evaluate the presence of leukemic cells and breast cancer cells in cryopreserved human ovarian tissue from patients with chronic myeloid leukemia or acute lymphoblastic leukemia and advanced breast cancer.
In each case, histology, polymerase chain reaction for disease-specific markers, and long-term xenografting were used to test the frozen-thawed ovarian tissue.
Results showed that malignant cells may be present in ovarian tissue from acute leukemia patients and give rise to tumor development in mice after xenografting (n=5/12, acute leukemia). For mice grafted with ovarian tissue from patients with advanced breast cancer, PCR and MGB2 gene sequencing were positive in ovarian tissue in 5 out of 10 patients, but none of the xenografted mice developed tumor masses during the 6-month grafting period.
Although the malignant potential of these cells is not yet known, the current study demonstrates that conventional histology and IHC need to be associated with more sensitive screening methods, like PCR and sequencing, before ovarian tissue transplantation can be contemplated.
Jacques Donnez, Belgium
PREGNANCY FROM FROZEN SLICES OF OVARY

The different cryopreservation options available for fertility preservation in cancer patients are embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation.
Embryo cryopreservation requires the patient to be of pubertal age, have a partner, and be able to undergo a cycle of ovarian stimulation. Oocyte cryopreservation is also effective, but the quality of eggs in women suffering from cancer is so far unproven, and requires also tissue for ovarian stimulation.
Cryopreservation of ovarian tissue is the only option available for prepubertal girls, and for woman who cannot delay the start of chemotherapy.
Around 50-60 cases of orthotopicreimplantation of cryopreserved ovarian tissue have been reported to date and 22 live births have been achieved, yielding a pregnancy rate of more than 25%. In our department, ten women have undergone orthotopicreimplantation of cryopreserved tissue either once or twice. Restoration of ovarian function, proved by follicular development and estradiol secretion, occurred in all cases. A time interval of 3.5 to 5 months was observed. In the literature, pregnancies were naturally obtained in 50% of cases. Graft activity was found to persist for 2.5 to 4 years. In non-pregnant patients, IVF was performed, but the quality of oocytes and embryos was not optimal.
Prognostic factors (age, previous chemotherapy) are therefore discussed.
In conclusion, fertility preservationis now a real possibility for patients whose gonadal function is threatened by radiotherapy or chemotherapy.
Fulvio Gandolfi, Italy
DIRECTIONAL FREEZING OF OVARIAN TISSUE

Ovarian tissue cryobanking is proposed as an effective option for preserving female fertility in cancer patients. At present two options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. The use of whole ovary reduces ischemic insult. However, the larger the sample volume, the more difficult is to introduce the cryoprotective agents and to ensure an adequate cooling rate that minimizes tissue damage. For this reason we used the Multi-Thermal Gradient (MTG) method, based on running the sample through a temperature gradient. This allows an homogeneous cooling rate through the whole sample independently from its volume.
Using sheep as an experimental model we compared the viability of whole ovaries cryopreserved with conventional slow freezing (SF) versus directional freezing (DF) techniques. The exam vessel integrity, follicle development and cell vitality indicated that directional freezing provides a better preservation of the ovarian tissue. In addition we also investigated whether DF could preserve whole organs with the same efficiency of cortical fragments. Our results indicate that DF preserve ovarian tissue with high efficiency irrespectively of the sample size. Thus overriding the limitations usually associated with whole organ banking.
Gedis Grudzinskas, UK

Announcements of more than 20 babies born following first birth after human successful orthoptic transplantation of cryopreserved thawed ovarian tissue by Donnez et al. (2004), the news of the birth of healthy babies following orthotopic reimplantation appears to be increasing greatly . Although it may be a little too early to say that this rise is exponential, this cautious position may soon be cast aside. Moreover, Andersen and co-workers (2012) suggest that it might also be possible to postpone the normal time of the menopause or to alleviate menopausal symptoms using auto transplanted ovarian tissue. Since the pioneers in this field have generously given free access to their successful technology why is this technique not yet available to all suitable cancer survivors?
Mary Hagedorn, USA
CORRAL GAMETES AND STEM CELLS CRYO
Over the next 30 to 40 years, rising ocean temperatures and increased ocean acidification have the potential to overpower the vast majority of current in situ coral conservation efforts, resulting in severe loss of reef biodiversity. If this occurs, ex situ coral conservation incorporating cryopreservation may provide an important means for successful reef diversification. To build new tools for the continued protection and propagation of coral, an international group of coral and cryopreservation scientists known as the Reef Recovery Initiative joined forces at the Australian Institute of Marine Science during the November 2011 and 2012 mass-spawning event. The outcome was the creation of the first frozen bank for Australian coral from two important reef-building species, Acroporatenuis and Acroporamillepora. Sperm cells were successfully cryopreserved, and after thawing, samples were used to fertilize eggs, resulting in functioning larvae that settled, resulting in thousands of young coral that are now growing in captivity. Frozen and thawed coral sperm can now be used to create new coral, and in the near future, we may have a blueprint to move our work from the laboratory to the reefs to develop collaborative, practical conservation management tools to secure reef biodiversity. The Reef Recovery Initiative is seeking new partners to help train professionals, create new frozen repositories in countries with important coral ecosystems to help expand the scope of its important conservation mission.
Samir Hamamah, France
VITRIFICATION AND SLOW FREEZING EFFECTS ON GENE EXPRESSION OF HUMAN MII OOCYTES

The emergence of DNA chips offers the possibility to exhaustively catalogue the genes that are expressed in a given tissue at a given time. A number of studies suggest that changes in gene expression, such as GDF9 or Bone Morphogenic Protein-15 (BMP15) in oocytes can be monitored for healthy oocyte and selecting oocytes for fertilization. Therefore, gene expression studies in human oocyte could contribute not only to identify factors involved in the oocyte maturation pathway, but could also provide valuables molecular markers of abnormal gene expression in oocytes with reduced competence. Cryopreservation via vitrification is now considered as an efficient way to store human oocytes to preserve fertility. However, little is known about the effects of this procedure on oocyte gene expression. The aim of my lecture is to evaluate the effect of the two cryopreservation procedures, slow freezing and vitrification, on the gene expression profile of human metaphase II (MII) oocytes by using DNA micro array ships. Both cryopreservation procedures negatively affected the gene expression profile of human MII oocytes in comparison with controls. However, slowly frozen and vitrified MI oocytes displayed specific gene expression signatures. Slow freezing was associated with down-regulation of genes involved in chromosomal structure maintenance (KIF2C and KIF3A) and cell cycle regulation (CHEK2 and CDKN1B) that may lead to a reduction in the oocyte developmental competence. In vitrified oocytes, many genes of the ubiquitination pathway were down-regulated, including members of the ubiquitin-specific peptidase family and subunits of the 26S proteasome. Such inhibition of the degradation machinery might stabilize the maternal protein content that is necessary for oocyte developmental competence. The low pregnancy rates commonly observed when using human MII oocytes after slow freezing–thawing may be explained by the alterations of the oocyte gene expression profile.
Katsuhiko Hayashi, Japan
GENERATION OF EGGS AND SPERMATOZOA FROM PLURIPOTENT STEM CELLS IN MICE

Generation of gametes from pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), in culture is a key goal in developmental/reproductive biology. More practically, gametes from ESCs/iPSCs can be an indefinite source for reproduction and preservation of animals. In mice, a model of mammals, generation of gametes from ESCs/iPSCs in culture has been attempted for a decade. However, despite a lot of effort, a culture system that reconstitutes the entire process of germ cell development has not been established yet.
Toward establishing such culture system, we recently succeeded in producing a robust number of cells whose potential is equivalent to nascent primordial germ cells (PGCs), origin of eggs and spermatozoa. In the culture system, ESCs/iPSCs first differentiate into epiblast-like cells (EpiLCs) and then induce PGC-like cells (PGCLCs). The manner of the step-wise differentiation from ESCs/iPSCs to PGCLCs reproduces that of PGC specification in vivo. Importantly, PGCLCs produced from ESCs/iPSCs are capable of differentiating into eggs and spermatozoa, when transplanted into ovary and testis, respectively. Furthermore, PGCLC-derived eggs and spermatozoa are fully potent, as they give rise to healthy and fertile offspring.
Successful production of functional PGCLCs from ESCs/iPSCs may lead to an idea of applications to other mammalian species including human. There are, however, technical obstacles to apply immediately our culture system to other species. It would be necessary to understand similarity/difference between mice and other species, especially in nature of pluripotent stem cells and PGC development. Nevertheless, our study opens a possibility that pluripotent stem cells are a practical source for reproduction and preservation of animals.

Vladimir Kostal, Slovania
PRODUCTION OF FREEZE TOLERANCE ORGANISMS: LESSONS FROM FRUIT FLIES

Insect cold tolerance represents a complexofimpressivestrategies. A wholearrayofbiochemical and physiologicaladjustmentscounteractdamagingeffectsofsubzerotemperatures, e.g.accumulationoflowmolecularweightcryoprotectants, synthesisofantifreezeproteins, remodelingofcellularmembranes, and accumulationofheatshockproteins. Someinsect species display thehighestknownlevelsofcold tolerance amongallanimals. For instance, larvaeofthe malt fly, Chymomyzacostata (Drosophilidae) surviveaftersubmersion in liquidnitrogen (-196C) in a fullyhydratedstate. Wefoundthattheprinciplesunderlyingfreeze tolerance in malt flylarvae are transferable, at least partially, onto a chillsusceptiblelarvaeoffruitfly, Drosophilamelanogaster. Giventhefactthattheevolutionaryadaptationforfreeze tolerance isgenerallyconsidered as highlycomplex and uneasy to mimic in laboratory, ourrecentresults are encouraging and may open a newdirectionofresearch on long-term cryogenicstorageofbiologicalmaterial. So far,cryogenicprotocolshavebeendevelopedforembryosofseveralinsect species, includingD. melanogaster. Despiteindisputableprogress, methodsforcrypreservationofinsectembryoswere not widelyimplementedintopractice. The long term cryogenicstorageofcomplex animal tissuesorwholeorganismsstillremains a greatchallenge. In these respect, detailedanalysisofC. costata model appears as greatlypromising.

Sergio Ledda, Italy
STRATEGIES FOR CRYOPRESERVATION OF GENETIC RESOURCES OF WILD AND DOMESTIC UNGULATES (RUMINANTS): PROCESSES ANALYSIS AND IMPROVING TECHNIQUES

Cryopreservation systems of germplasma (spermatozoa, oocytes and preimplantation embryos) has been developed rapidly in the last 50 years . Succefull banking of genetic resources by freezing gametes and embryos have progressed in domestic specie as well in endangered animals- However, while the cryopreservation of sperm and embryos can be considered an applied technology, cryopreservation of the oocytes have not reached the same level of standardized procedures. The main difficulties are the preservation of the morphology of this large cell and its complex biochemical machinery. For these reasons, using sheep as large animals model, we have performed an series of studies in order to analyze and follow the major chances induces by the hypothermic exposition focusing our attention to the modification of the cythoscheletal cell organization and localization of major ooplasma constituents. In the same time analysis of gene expression have been performed by microarray experiments and RT validation. Our data showed, using confocal microscopy and confocal Raman microscopy, that during vitrification , significant chances are induces in the cythosckeletal proteins and in the composition and localization of the major constituents of the oocyte. Similarly the cryopreservation procedures affected several classes of stored mRNA that involved in meiotic resumption and early development. Moreover these changes are closely related to the quality of the oocytes and this unique cell respond to the thermal stress according to the intrinsic capacity.

Juergen Liebermann, USA
ASEPTIC VITRIFICATION OF BLASTOCYSTS: DID WE ACHIEVE ANY IMPROVEMENTS?

It has been more than two decades since the first publication of successful mouse embryo vitrification in 1985. Since then the experience with human blastocyst vitrification has progressed close to perfection and nonetheless has become an essential adjunct to improve clinical outcomes. In addition, by mastering successful vitrification at the blastocyst stage, the IVF lab has gained a power tool to encourage physicians and patients in considering the reduction of the number of fresh embryos to be transferred through cryo-storage of sibling embryos. The blastocyst vitrification technology involved has changed little in principle in this time other than swinging to the preference of closed systems instead of open systems.
In general, data suggest that expanded blastocysts showed no significant differences in viability, implantation potential or pregnancy outcome when frozen on day 5 versus day 6. However, our "body of data” refutes the comparable implantation of blastocysts cryopreserved on day 5 or 6. In awareness of published papers in the past in regards to artificial collapsing (AC) of blastocyst prior to cryopreservation, we were looking into an opportunity, which could potentially help us to improve the outcome of day 6 blastocysts by AC of the blastocoele prior to the vitrification steps. If this concept shows any improvement, then we would extend AC to day 5 blastocysts. Artificial collapse of the blastocoele can be performed using different approaches such as micro-needles, sucrose solutions or laser. Comparing no artificial collapse with artificial collapse in day 5 as well as day 6 blastocysts, an average increase for all ages in regards to clinical (40% vs. 58%), ongoing pregnancy rates (30% vs. 53%), and implantation rates (28% vs. 41.5%) were observed. In regards to the day of embryo development, comparing no artificial collapse with AC, an average increase for day 5 and day 6 were established for the following:
• Clinical (Day 5: 48% vs. 69%; Day 6: 36% vs. 58.5%),
• Ongoing pregnancy rates (Day 5: 39.5% vs. 64%; Day 6: 28.6% vs. 53%), and
• Implantation rates (Day 5: 36.5% vs. 50.2%; Day 6: 25.7 vs. 41.1%).
To conclude, AC in day 5 and day 6 blastocysts prior to the steps of vitrification is beneficial for all outcome parameters observed.

Pasqualino Loi, Italy
LATE EMBRYOGENESIS ABUNDANT PROTEINS AND DESICCATION TOLERANCE IN MAMMALIAN OOCYTES

Late Abundant proteins (LEA) are a large family of hydrophilic proteins expressed during seeds maturation. LEA proteins confer desiccation tolerance in seeds via a protection of structural proteins and lipids, and their expression has been reported not only is seeds, but also in anidrobiontes, small vertebrates that can survive in absence of environmental water. We have generated three LEA proteins (one targeting the membranes, one the mitochondria, and one which permeates the cyto/nucleoplasm) by DNA recombination.
In my talk I shall present our data on the sub cellular localization of recombinant LE A proteins injected into metaphase II sheep oocytes.

Peter Mazur, USA
RELATIVE IMPORTANCE OF COOLING AND WARMING RATE IN OBTAINING HIGH SURVIVALS OF MOUSE OOCYTES AND EMBRYOS AFTER VITRIFICATION.

There is accelerating interest in the cryopreservation of human oocytes and preimplantation embryos, and this has prompted parallel research with mouse oocytes and embryos by us and others. The survival of a cell requires that no more than minimal amounts of small crystals of ice form inside the cell. One way to avoid intracellular ice is to vitrify cells; i.e., to convert cell water to a glass rather than to ice. The belief has been that this demands that both the cooling rate and the concentration of glass-inducing solutes be very high. But high solute concentrations can themselves be damaging. Our studies have shown that the first belief (that cooling needs to be extremely rapid) is not correct with respect to the vitrification of mouse oocytes. The important requirement is that the warming rate be extremely high. We subjected mouse oocytes in the vitrification solution EAFS 10/10 to vitrification procedures using a broad matrix of cooling and warming rates. Morphological survivals exceeded 80% when they were warmed at the highest rate (117,000°C/min) even when the prior cooling rate was as low as 880°C/min. Functional survival was > 81% and 54% with the highest warming rate after cooling at 69,000 and 880°C/min, respectively. We have also found the second belief to be erroneous. We show that a high percentage of mouse oocytes survive vitrification in EAFS media that contain only half the usual concentration of solutes, provided they are warmed extremely rapidly; i.e., > 100,000°C/min. Again, the cooling rate is of less consequence. This suggests that the recrystallization of intracellular ice is playing a dominant role in survival. Whether the external EAFS medium freezes or vitrifies depends on both on the cooling rate and the relative concentration of the EAFS. Full strength EAFS vitrifies when cooled at 522 °C/min or faster. A third of samples of 0.5X EAFS freeze when cooled at 69,000°C/min, and all of them freeze when cooled at 1827°C/min or slower; yet, survivals are equally high with both concentrations. What we do not yet know is whether the cell interior freezes or not. These findings have implications for both fundamental and applied cryobiology.

Laura Rienzi, Italy
OPEN SYSTEM EFFICIENCY AND SAFETY

Recent studies suggest that oocyte vitrification significantly improves oocyte survival, embryo development and pregnancy rates compared to slow freezing procedure (Oktay et al., 2006, Gook et al., 2007, Smith et al., 2010). The most efficient and reproducible protocol being today the "minimum volume open system approach”, in which oocytes are directly exposed to liquid nitrogen in a very small volume to maximize ultra-rapid cooling and minimize ice crystal formation. However, there is a theoretical concern regarding such direct contact, that potentially expose oocytes to infectious organisms present in the liquid nitrogen (although infectious transmission has never been observed in ART). To avoid this theoretical cross-contamination, methods have been developed to sterilize liquid nitrogen by microfiltration or ultraviolet irradiation (Parmegiani et al., 2011).
To evaluate the success rate of oocytes vitrification different randomized controlled trials comparing outcomes with vitrified and fresh oocytes in ICSI cycles have been conduced (Cobo et al., 2008; 2010; Rienzi et al., 2010, Parmegiani et al., 2011). All these studies have used a similar minimum volume open system protocol. Overall, oocyte survival after vitrification and warming was >90%, fertilization rate was similar to the one obtained with fresh oocytes, and implantation rates ranged between 17%–41%. These studies and a recent meta-analysis (Cobo et al., 2011) suggest that the reproductive outcome of vitrified/warmed and fresh oocytes are similar. Moreover, to assess the efficacy and reproducibility of oocyte open system vitrificaiton a longitudinal cohort multicentre study has been conducted (Rienzi et al., 2012). The number of oocytes vitrified, the women age and blastocyst culture were significantly correlated to delivery rate.
In conclusion, there is good evidence that excellent fertilization and pregnancy rates can be obtained with open system oocyte vitrification protocol (similar to those obtained with fresh oocyte in expert centres). Although data are limited, no increase in chromosomal abnormalities, birth defects, and developmental deficits has been reported in the offspring born from vitrified oocytes. As recently stated by "The Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology” the evidence indicates that oocyte vitrification and warming should no longer be considered experimental (ASRM pages, in press).

Joseph Saragusty, Germany
The current decade was declared by the United Nations as the Decade of Biodiversity. The alarming rate at which biodiversity is lost in recent years has been likened to the 6th mass extinction in Earth's history, mass extinction that is largely anthropogenic in nature. To preserve the world's biodiversity, the establishment of genome resource banks is desirable. These institutions can preserve gametes, embryos and tissues that, when needed, can be used to replenish a genetically dwindling population, species that are extinct in the wild or, at least theoretically, resurrect extinct species. Currently cryopreservation (freezing and vitrification) is the leading long-term preservation technique. This technique, however, is not optimal, as it often requires species-specific customization and carries high costs in terms of liquid nitrogen, dedicated space and professional maintenance team. The consequence of all these is high carbon footprint. It ought to be our goal to overcome each of these shortcomings. To do that, we need to strive for accurate modeling of the cryopreservation process, development of techniques that will be truly universal, and alternatives that will help overcoming issues of maintenance costs and carbon footprint. These efforts should (ideally) be part of a global network so that data and knowledge can be shared, efforts can be concerted and collections can be truly dynamic.
Gerald Schatten, USA
SCIENCE FICTION OR FUTURE REALITY? PRODUCING GAMETES FROM STEM CELLS AND SOMATIC CELLS

The 2012 Nobel Prizes to Sir John Gurdon and Shinya Yamanaka for their seminal work in nuclear transfer and induced pluripotency, coupled with the 2010 Nobel Prize to Bob Edwards for pioneering In Vitro Fertilization in humans, attests to the contemporary importance of stem cells and regenerative medicine for ART. This lecture will consider pluripotent stem cells, their differentiation into both sperm and oocytes, as well as their role of PSCs in cancers and epigenetics.

Pluripotent stem cells from humans (hPSCs), including human embryonic stem cells (hESCs) and by induced pluripotency (hiPSCs) captivate medical attention due to their unique properties of unlimited self-renewal and differentiation. ESC lines have only been established robustly and investigated intensively in mice (mESCs) and more recently in humans after derivations from fertilized-blastocysts and after induced pluripotency (iPSCs). Scientists around the World are now asking whether these cells might treat or even cure juvenile diabetes with insulin secreting β-islet cells responsive to circulating glucose; cerebral palsy treatments with neuroprogenitors to repair white matter injuries due to premature births; heart muscle repair with cardiomyocytes; spinal cord regeneration with peripheral motor neurons; multiple sclerosis with neuroprogenitor cells or astrocytes for Schwann cell; Parkinson's disease using dopaminergic neurons; amyotrophic lateral sclerosis with neuronal lineages; reduction or replacement of whole organ transplantation by single cell transplantation of hepatocytes for diseased livers; renal cells in place of kidney transplants, and many others.

Our report entitled: Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells ((Easley et al., Cell Reports 2, 440–446, September 27, 2012; http://x.doi.org/10.1016/j.celrep.2012.07.015 ) notes that: ‘Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.' Understanding gametogenesis is vital for improving infertility therapies and contraceptives. Patient-specific stem cells undergoing gametogenesis in vitro represent models for mechanistic investigations and potential therapies. Here, we show that human embryonic and induced pluripotent stem cells differentiate into advanced germ cell lineages including spermatogonia, spermatocytes, and haploid spermatids with parent-of-origin genomic imprints similar to fertile human sperm. Developing an in vitro spermatogenesis model may prove critical for understanding the mechanistic causes of male infertility.

FERTILE OFFSPRING GENERATED FROM OOCYTES DERIVED FROM iPSCs. In their paper, Drs. Hayashi et al. from the Prof Saitou lab have reported in SCIENCE that: "Reconstitution of female germ-cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in mice are induced into primordial germ cell–like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, undergo X-reactivation, imprint erasure, cyst formation, and exhibit meiotic potential. Upon transplantation under mouse ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which then contribute to fertile offspring after in vitro maturation and fertilization. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ-cell development in vitro.” Please recognize though that extrapolations to ART clinic are highly speculative. This work teaches us features about oogenesis IN MICE. So unless your patients are rodents, please do not let ‘fertile' imaginations run wild yet…

Cancer Stem Cell Hypothesis: Notwithstanding decades of remarkably powerful chemotherapeutic and radiotherapeutic treatments for various cancers, combined with skillful removal of the tumors, cancer remains a devastating disease with largely unfavorable long-term outcomes. The scientific discoveries on stem cells, and especially the teratoma assay used to demonstrate pluripotency to stem cell scientists, has influenced thinking among oncologists and cancer biologists. The ‘Cancer Stem Cell' Hypothesis proposes that most treatments used today to destroy cancers are actually only targeting the differentiated cells, which comprise the bulk of the tumor. They miss however, the earliest precursor cells, which is the root cause of the disease – the cancer stem cell – since these cells are rare, are highly resistance to drugs, and mutate rapidly to evolve tolerance. This lecture will consider the strengths and weaknesses of the hypothesis as well as how emerging new therapeutic strategies are being developed for the next generation of cancer treatments.
Sherman Silber, USA
FRESH AND FROZEN OVARY TRANSPLANT RESULTS


The aim of this lecture is to summarize the state-of-the-art of ovarian tissue cryopreservation and transplantation. This clinical experience will then be applied to understanding 1) the mechanism for initiation of primordial follicle development, 2) controversially why cancer cells metastases do not severely limit the transplantation of frozen ovary tissue back to cancer patients, and 3) what we can learn fro comparing ovarian embryology to testis embryology.
First, all nine fresh ovary transplants were between identical twins all ovulating and menstruating, resulting in eleven healthy babies in seven of the nine recipients. Recipients always reinitiated ovulatory menstrual cycles and normal Day 3 serum FSH levels by 4-1/2 months. Most conceived naturally (three of them twice or three times from the same graft). Duration of function of fresh ovarian grafts, contrary to initial expectation, indicated a very acceptable or minimal oocyte loss from ischemia time. Grafts of just modest portions of ovarian tissue have lasted more than 7 years.
The same surgical techniques were then applied to 4 frozen ovary tissue transplants, up to 14 years after the ovary had been frozen, all resulting in normal ovulation and in 3 more healthy babies. Around the world, the number of healthy babies from ovary grafts has now risen to over 30, with more than 20 from frozen grafts. Although ovary freezing and transplantation has been referred to as "experimental” for preserving fertility in cancer patients, it should be noted that virtually all of the babies born from fertility preservation for cancer patients, have resulted thus far from ovary tissue freezing, rather than from egg freezing.
As to the efficiency of ovarian tissue freezing compared to the use of fresh tissue, slow freeze has resulted in only a 60% loss of oocytes, and vitrification has resulted in no observable loss. For practical purposes therefore both techniques are reliable. However, in vitro studies in humans, and in vivo studies in bovine, show that vitrification of ovarian tissue, may nonetheless be an improvement over slow freeze.
The basic science concept of vitrification is to completely avoid any ice crystal formation by using a very high concentration of cryoprotectant and a very rapid rate (virtually "instant”) of cooling. This is quite different from classic slow freeze cooling which relies on a partial and very gradual removal of water from the cell by encouraging ice crystal formation preferentially on the outside of the cell, drawing the water out.
A comparison has to be made between vitrification for ovarian tissue versus vitrificationofr mature eggs and embryos. For mature eggs and embryos, there is first an equilibration in 7.5% EG and 7.5% DMSO followed by a final solution of 15% EG and 15% DMSO with 0.5 molar sucrose. It is very important to allow enough time for full absorption of this more concentrated cryoprotectant solution usually more than one minute, as these solutions, contrary to myth, are not toxic. However, for ovarian tissue, there must be a longer incubation in 7.5% EG and 7.5% DMSO, and then a later incubation in a denser 20% EG and 20% DMSO with 0.5% sucrose, to make certain there is full absorption of cryoprotectant. In all cases, the eggs or embryos or ovarian tissue must not be frozen in a droplet (even a tiny microdroplet) as this would slow the rate of freeze and thaw. The ovarian tissue must have fully absorbed the cryoprotectant, but be "dry” on the outside. Just as thawed ovarian tissue works as well as fresh, thawed embryos have just as good a success rate as fresh embryos. With vitrification, embryos can be frozen with impunity, whereas with slow freeze (like with ovarian tissue) there is some viability loss.
A comparison of the embryology and anatomy of the ovary and the testis is very instructive for two issues, 1) the initiation of follicle development, and 2) the low risk of neoplastic metastasis to the ovary cortex and, therefore, the safety of ovarian cortical cryopreservation and transplantation in cancer patients. The ovarian cortex is actually identical to the tunica albuginea of the testis, except that in the male the germ cell cords to not invade the testes of boys and men with leukemia, but uncommonly the tunica albuginea of the testis. This phenomenon explains both of these issues.
It is clearly possible to preserve and restore fertility, using ovary and egg or embryo freezing in young women with cancer who are undergoing otherwise sterilizing chemotherapy and radiation. But this approach can also be used for any woman who wishes to prolong her reproductive lifespan. It may thus eventually obviate the growing worldwide epidemic of female age-related decline in fertility, and even could eliminate menopause.

 

 


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