The use of cryoprotective agents for the conventional cryopreservation of human spermatozoa, oocytes, zygotes, early cleavage stage embryos and blastocysts is an integral part of almost every human IVF programme. Moreover, the cryopreservation of these types of cells by direct plunging into liquid nitrogen usually requires high cryoprotectant concentrations with consequent cytotoxic effects. This review covers the history of this problem, and in this light offers an explanation, through physico-chemical concepts, for one of the most recent developments in this area: the recovery of motile and potent spermatozoa after cryoprotectant-free vitrification.

As early as 1985, ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification was reported in an attempted alternative approach to cryostorage. Since then, vitrification techniques have entered more and more the mainstream of animal reproduction as an alternative cryopreservation method to traditional slow-cooling/rapid-thaw protocols. In addition, the last few years have seen a significant resurgence of interest in the potential benefits of vitrification protocols and techniques in human-assisted reproductive technologies. The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human-assisted reproduction, and human preimplantation embryo vitrification is still considered to be largely experimental. Besides the inconsistent survival rates that have been reported, another problem is the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all preimplantation stages is encouraging for further research and clinical implementation. Clearly, however, attention needs to be paid to the inconsistent survival rates following vitrification.

PurposeFertility preservation methods are playing an increasing role in women up to the age of 40 years because of rising survival rates in those affected by cancer. However, balanced practical recommendations concerning all relevant fertility preservation, to support doctors in counselling and treating patients, are still rare.MethodsThese recommendations were prepared by the network FertiPROTEKT (http://www.fertiprotect.eu), a collaboration of around 70 centres in Germany, Switzerland and Austria. The recommendations were developed by specialists in reproductive medicine, reproductive biology and oncology, which gave a comprehensive overview of all named techniques as well as their benefits and risks. Furthermore, practice-orientated recommendations for the individual use of fertility preservation methods for various indications such as breast cancer, Hodgkin’s lymphoma and borderline ovarian tumours are given.ResultsVarious options such as ovarian stimulation and cryopreservation of unfertilised or fertilised oocytes, cryopreservation and transplantation of ovarian tissue, GnRH-agonist administration and transposition of the ovaries can be offered. All the techniques can be performed alone or in combination within a maximum of 2 weeks with low risk and different success rates.ConclusionsFertility preservation in women has become an option with realistic chances to become pregnant after cytotoxic therapies. The information provided allows a well balanced and realistic counselling and treatment.

Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 x 10(5) degrees C/min). This is achieved by directly plunging a copper cryoloop loaded with a sperm suspension into liquid nitrogen. After storage, vitrified spermatozoa are instantly thawed by melting in an agitated, warm medium. The goal of the present study was to compare the quality of spermatozoa cryopreserved using this rapid vitrification method with that of spermatozoa cooled relatively slowly by preexposure of the loaded cryoloop to liquid nitrogen vapor (-160 degrees C) with speed in the range 150-250 degrees C/min) before immersion into liquid nitrogen. Both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa. In both cases, instant thawing by melting in a warm medium was essential for successful cryopreservation. Our findings suggest that optimal regimes for the cryoprotectant-free cryopreservation of spermatozoa need not be restricted to very fast cooling before storage in liquid nitrogen, a wide range of cooling rates being acceptable. Herein, we discuss the implications of this finding in the light of the physics of extra- and intracellular vitrification.

Purpose Official guideline of the German Society of Gynecology and Obstetrics (DGGG), the Austrian Society of Gynecology and Obstetrics (ÖGGG) and the Swiss Society of Gynecology and Obstetrics (SGGG). The aim of this guideline was to standardize the diagnosis and treatment of couples with recurrent miscarriage (RM). Recommendations were based on the current literature and the views of the involved committee members. Methods Based on the current literature, the committee members developed the statements and recommendations of this guideline in a formalized process which included DELPHI rounds and a formal consensus meeting. Recommendations Recommendations for the diagnosis and treatment of patients with RM were compiled based on the international literature. Specific established risk factors such as chromosomal, anatomical, endocrine, hemostatic, psychological, infectious and immunological disorders were taken into consideration.

PurposeIn addition to guidelines focusing on scientific evidence, practical recommendations on fertility preservation are also needed.MethodsA selective literature search was performed based on the clinical and scientific experience of the authors. This article (Part II) focuses on fertility preservation techniques. Part I, also published in this journal, provides information on disease prognosis, disease-specific therapy, and risks for loss of fertility.ResultsOvarian stimulation including double stimulation and freezing of oocytes is the best-established therapy providing live birth chances in women < 35 years with high ovarian reserve of around 30–40%. Ovarian tissue freezing is especially useful in young women with good ovarian, if spontaneous conception is favoured and if < 1 week until chemotherapy is provided. Data on success rates are still limited, but this further evolving technique will possibly reach similar success rates as ovarian stimulation. GnRH agonists seem to reduce the risk of premature ovarian failure up to 50%; however, the effect is possibly not long-lasting. Ovarian transposition can easily be combined with freezing of ovarian tissue and is the preferred technique before pelvic radiotherapy. Other techniques, such as in vitro maturation, are limited to women with high ovarian reserve and remain less effective. In addition, procedures such as in vitro growth of follicles, etc. are still experimental.ConclusionsFertility preservation in women provides realistic chances of becoming pregnant. The choice of technique needs to be based on the time required, the woman’s age, its risks and efficacy, and the individual preference of the patient.

Human spermatozoa can be successfully cryopreserved without the use of cryoprotectants through vitrification at very high warming rates. This is achieved by plunging a small amount of frozen sperm suspension into a warming medium, or a large amount of sperm suspension into an agitated warming medium. The aim of the present study was to compare the motility of human spermatozoa cryopreserved using four different methodologies of cooling and warming: cryoloops, droplets, open-pulled straws and standard open straws. Evaluation of two parameters, motility and viability rate of spermatozoa, suggests that all four methods are suitable for use in assisted reproductive technology. However, only the use of open-pulled straws as well as standard open straws allows the isolation of spermatozoa from liquid nitrogen with low potential risk of microbial contamination during freezing and storage, and is thereby a clean method of vitrification.

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