E. Isachenko

Comparison of necrosis in human ovarian tissue after conventional slow freezing or vitrification and transplantation in ovariectomized SCID mice.

This paper examines and compares necrosis in human ovarian tissue after conventional slow freezing or vitrification and ensuing xenotranplantation. Slow cryoconserved or vitrified ovarian tissue samples and fresh controls from nine patients were subcutaneously transplanted into SCID mice. The tissue samples were explanted after 6 weeks and the necrotic areas were examined by staining with Lucifer yellow SV. The size of the necrotic areas in parallel cultivated ovarian tissue samples was compared, as was necrosis in cultivated prostate tumour spheroids where the emergence of necrosis and its pathophysiological correlation have been described. Examinations showed no significant rise in the proportion of necrotic areas after slow cryoconservation/transplantation and in the controls (transplanted fresh tissue, not transplanted fresh tissue, long-term culture). The proportion of necrotic areas in the tumour spheroids was significantly higher than in the ovarian tissue. Vitrification could, after these results, be presented as an alternative to conventional slow cryoconservation.

Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation

OBJECTIVE In ovarian tissue grafts there is a massive loss of follicles during the ischaemic period until re-vascularisation is established. The aim of our study was to investigate the influence of different cryopreservation techniques on the ability for the re-vascularisation of ovarian tissue transplanted to SCID mice. STUDY DESIGN Ovarian fragments from five patients were cut into pieces (approximately 0.5 mm x 1.0 mm x 1.0 mm) and randomly distributed into three groups: fresh non-treated tissue (group A); tissue conventionally frozen in standard 0.5 ml insemination straws with 1.5 M ethylene glycol+0.1 M sucrose, with thawing in a 40 degrees C water bath and step-wise removal of cryoprotectants at room temperature in 0.5 M, 0.25 M and 0.15 M sucrose with gentle agitation (group B); tissue vitrified in 2.62 M dimethylsulphoxide+2.6 M acetamide+1.31 M propylene glycol+0.0075 M polyethylene glycol, with warming by direct plunging of solid specimens with ovarian pieces into 20 ml of 50% vitrification solution pre-warmed to 40 degrees C and dilution of cryoprotectants in a decreasing concentration of vitrification solution (25%, 12.5%) at room temperature (group C). We used a xenograft model in which ovarian tissue pieces of all three groups were subcutaneously transplanted in SCID mice. The animals were sacrificed on the third day after ovarian tissue transplantation and then weekly during 1 month to obtain the ovarian tissue grafts. These samples were examined by immunohistochemical staining with the endothelial cell-specific marker platelet endothelial cell adhesion molecule-1 (PECAM-1) to determine angiogenesis. Histological observation of tissue after explantation was performed and quality and quantity of follicles were assessed. RESULTS No PECAM-1 staining was observed in all treatment groups prior to grafting. After warming and in vivo culture of ovarian tissue, the beginning of angiogenesis in pieces from all treatment groups on the third day was detected by PECAM-1 staining. After 4 weeks of in vivo culture the overall area of PECAM-1-positive blood vessels significantly increased (P<0.05), independent of the type of cryopreservation (groups B and C vs. group A). It was found that transplantation technique had negative influence on the integrity of follicles independent of the type of treatment during in vivo culture. The duration of in vivo culture has a negative, but not statistically significant, influence on follicle quality in long-cultured transplants inside each treatment group (P>0.5). CONCLUSION The process of re-vascularisation of transplanted ovarian tissue is independent of the type of treatment and does not influence follicle quality.

Effect of long-term exposure at suprazero temperatures on activity and viability of human ovarian cortex

The experiment was designed to determine the optimal time and temperature for long-distance transport. Prolonged suprazero temperature exposure of ovarian tissue for 26 hours has no negative influence on follicle quality.

In-vitro culture of human embryos with mechanical micro-vibration increases implantation rates

The in-vitro culture of human embryos in a medium subjected to regular short intervals of mechanical agitation leads to increased development rates. This type of treatment tries to mimic conditions in nature whereby oviductal fluid is mechanically agitated by the epithelial cilia. This phenomenon can be explained by the fact that an embryo developing in vivo is naturally exposed to constant vibrations of around 6Hz with the periodically repeating increase to 20Hz. This review covers the history of this question and in this light offers an explanation through biological concept for one of the most recent developments in this area: in-vitro culture of human embryos with mechanical micro-vibration. The effect of mechanical micro-vibration on embryos during their in-vitro culture was examined. Pregnancy rates after the transfer of embryos in the group with in-vitro culture under mechanical vibration were increased.

Human ovarian tissue cryopreservation: quality of follicles as a criteria of effectiveness

Experiments comparing vitrification and conventional freezing of mammalian ovarian tissue show that vitrification can also guarantee the storage of viable follicles after warming, but conventional freezing is more effective. The central goal of cryotechnology is the preservation of intact follicles. This article presents a critical opinion about the normality of follicles after vitrification of human ovarian tissue and microbial contamination as a result of direct contact of this tissue with liquid nitrogen at vitrification.

Human ovarian tissue: vitrification versus conventional freezing

Sir, In the challenging paper by Wang et al. (2008), the authors have reported about an effective method of vitrification of human ovarian tissue with direct contact of cells in liquid nitrogen, named needle immersed vitrification. Childbirth after cryopreservation of ovarian tissue is now a reality (Donnez et al., 2004; Demeestere et al., 2007; Meirow et al., 2007; Andersen et al., 2008) and to study the new modifications of cryopreservation protocols is very interesting. In fact, vitrification is technologically promising, it is simpler and one cryocycle is less time-consuming and cheaper than the conventional freezing method. However, the central goal of the cryotechnology is the preservation of intact follicles, and not the guarantee of simplicity and availability of technology for operator to the detriment of the post-warming quality of follicles. However, results of the above investigations (Wang et al., 2008, Fig. 2) have shown that proposed vitrification protocol cannot guarantee the storage of viable follicles after warming in contrast to conventional freezing. Analysis of histological preparation evidence that post-warming follicles are far from normality (Paynter et al., 1999): in both ‘postwarming’ follicles presented in Fig. 2B the vacuolization of cytoplasm, especially in the top follicle as well as the chromatin condensation in both follicles, can be noted; the right follicle in Fig. 2C, which is also denoted by authors as normal, has partly damaged cytoplasm. In our opinion, in the evaluation of normality of cryopreservation protocol it would be better (i) to demonstrate a bigger sector of tissue (see Isachenko et al., 2008a) and (ii) to evaluate the quality of follicles after long-term culture (see Fig. 1 and Isachenko et al., 2007, 2008a). Besides, in our opinion, the above-mentioned method of vitrification cannot be recommended for use in the medical practice because these protocols presuppose a direct contact with liquid nitrogen, which is a potential source of microbial contamination. In fact, any technology in reproductive biology and especially in a medical approach must ensure and guarantee the full protection of biological objects from micro-organisms. Liquid nitrogen, which is used for the storage of frozen material, can be a source of contamination by these micro-organisms (Tedder et al., 1995; Bielanski et al., 2003). Filtration or ultra-violet treatment of liquid nitrogen cannot guarantee the absence of contamination of biological material by viruses. Different types of viruses, which are simple and very cryostable structures, may increase their virulence after a direct plunging and storage in liquid nitrogen, such as hepatitis virus, papova virus, vesicular stomatitis virus and herpes virus. The above vitrification methodology is based on the direct cooling of cells in liquid nitrogen. In contrast, conventional freezing completely avoids the direct contact between the liquid nitrogen and the tissue.

Epidermal growth factor effects on marmoset monkey (Callithrix jacchus) oocyte in vitro maturation, IVF and embryo development are altered by gonadotrophin concentration during oocyte maturation

BACKGROUND This is the first study of the effect of epidermal growth factor (EGF) on marmoset monkey oocytes matured in vitro. METHODS We have evaluated the effects of 10 ng/ml EGF in combination with 1 or 10 IU/ml of gonadotrophins (FSH/hCG 1:1 ratio) during in vitro maturation (IVM) of marmoset oocytes. Immature cumulus-oocyte complexes (COCs) were retrieved from ovarian antral follicles of unprimed monkeys. COCs from six animals (n= 268) used in this study were randomly distributed among four experimental groups: (A) 1 FSH +1 hCG; (B) 10 FSH +10 hCG; (C) 1 FSH +1 hCG + EGF; and (D) 10 FSH +10 hCG + EGF (where 1 and 10 are concentrations, IU/ml). After IVM, oocytes were fertilized in vitro and embryos were allowed to progress up to 87-88 h. RESULTS the highest rate of total and radial cumulus expansion was observed in Group A, with the lowest in Group B (P < 0.05). Neither maturation nor fertilization rate were affected by gonadotrophin concentration or presence of EGF. Addition of EGF increased degeneration and decreased first cleavage rate, which was significantly lower in Group C than Group A (P < 0.005). Interestingly, in the EGF groups some embryos cleaved faster than without EGF. CONCLUSIONS The effects of EGF are highly dependent on concentration of gonadotrophins present in IVM medium. EGF has a negative effect on oocytes in the presence of low gonadotrophins, but contrastingly partially protects oocytes from the negative effects of high gonadotrophins. We propose that these observed negative effects of EGF may suggest use of an inappropriate dose of growth factor.

Pregnancy after the calcium ionophore correction of pronuclei position in oocytes after intracytoplasmic sperm injection.

OBJECTIVE To report a successful pregnancy after transfer of embryos derived from oocytes after calcium ionophore correction of the pronuclei localization after intracytoplasmic sperm injection (ICSI). DESIGN Case report. SETTING University hospital. PATIENT(S) A 30-year-old patient and her 30-year-old husband, diagnosed with asthenoteratozoopermia, underwent ICSI because of three unsuccessful IUI attempts. INTERVENTION(S) Thirteen metaphase II oocytes were injected with morphologically normal spermatozoa and immediately divided into two groups. Group 1 (n = 7) was the untreated control and in group 2 (n = 6), the oocytes were treated with 10 μM calcium ionophore solution for 20 minutes at 37°C in 6% CO(2). The fertilization was checked 18 hours later and location of pronuclei in cytoplasm was assessed. Transfer of two embryos was performed on the third day after oocyte retrieval. MAIN OUTCOME MEASURE(S) Pregnancy after transfer of embryos after calcium ionophore correction of pronuclei localization. RESULT(S) Fertilized zygotes from calcium ionophore-treated oocytes with physiologically normal central localization of pronuclei were chosen for ET. Clinical pregnancy was confirmed at 7 weeks of gestation. CONCLUSION(S) Post-ICSI calcium ionophore activation of oocytes can be used for correction of the pronuclei localization, which can increase developmental rate of embryos.

Prevention of ovarian damage and infertility in young female cancer patients awaiting chemotherapy—clinical approach and unsolved issues

Great advances in the oncological therapy of childhood and adolescent cancer patients lead to an increase of young cancer survivors with a normal expectancy of life. The aggressive chemotherapy and/or radiation often compromises endocrine function with consecutive menopausal symptoms and sterility. Recently, new approaches were developed to preserve fertility with different methods to restore the ovarian function. The present review gives an overview of the current possibilities, which may be offered to these young cancer patients, as well as the chances of success and risks and the unsolved issues in special situations.