Gohar Rahimi

Role of reactive oxygen species and phosphatidylinositol 3-kinase in cardiomyocyte differentiation of embryonic stem cells.

Cardiotypic development in embryonic stem cell‐derived embryoid bodies may be regulated by reactive oxygen species (ROS). ROS were generated by a NADPH oxidase‐like enzyme which was transiently expressed during the time course of embryoid body development. Incubation with either H2O2 or menadione enhanced cardiomyogenesis, whereas the radical scavengers trolox, pyrrolidinedithiocarbamate and N‐acetylcysteine exerted inhibitory effects. The phosphatidylinositol 3‐kinase (PI‐3‐kinase) inhibitors LY294002 and wortmannin abolished cardiac commitment and downregulated ROS in embryoid bodies. Coadministration of LY294002 with prooxidants resumed cardiomyocyte differentiation, indicating a role for PI‐3‐kinase in the regulation of the intracellular redox state.

Potential Importance of Vitrification in Reproductive Medicine

Abstract As early as 1985, ice-free cryopreservation of mouse embryos at −196°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.

Effects of electrical fields on cardiomyocyte differentiation of embryonic stem cells

The effects of electromagnetic fields (EMFs) on the differentiation of cardiomyocytes in embryoid bodies derived from pluripotent embryonic stem (ES) cells were investigated. A single direct current (DC) field pulse was applied to 4‐day‐old embryoid bodies. The electrical field induced a hyperpolarization of the anode‐facing side of embryoid bodies and a depolarization at the cathode‐facing side. Significant effects of a single electrical field pulse applied for 90 s on cardiomyocyte differentiation were achieved with field strengths of 250 and 500 V/m, which increased both the number of embryoid bodies differentiating beating foci of cardiomyocytes and the size of the beating foci. The 500‐V/m electrical field increased intracellular reactive oxygen species (ROS), but not [Ca2+]i and activated nuclear factor kappa B (NF‐κB). A comparable increase in the number of beating embryoid bodies was achieved by an incubation for 1 h with H2O2 (1–10 nM), indicating that the electrical field effect was transduced via the intracellular generation of ROS. Because the radical scavengers dehydroascorbate and pyrrolidinedithiocarbamate (APDC) and the NF‐κB antagonist N‐tosyl‐L‐phenylalanine chloromethyl ketone (TPCK) inhibited cardiac differentiation, we assume that ROS and NF‐κB may play a role in early cardiac development. J. Cell. Biochem. 75:710–723, 1999.

Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen.

AIM To establish a prospective direction for further development of the protocol for cryopreservation of ovarian tissue by direct plunging into liquid nitrogen. MATERIALS AND METHODS Human ovarian biopsies from 20 patients (cut in approximately 0.5mm(3) pieces) were exposed to: 40% ethylene glycol+0.35 M sucrose+5% egg yolk; 40% ethylene glycol+18% Ficoll-70+0.35 M sucrose; 20% ethylene glycol+20% dimethyl sulphoxide. Cryopreservation of pieces was accomplished by plunging 0.25 ml straws or copper grids into liquid nitrogen or 0.25 ml straws into precooled (-196 degrees C) metallic powder. Thawed pieces were transferred to sucrose solution for incremental dilution of cryoprotectants. Histological observation of the tissue was performed after cryopreservation and in vitro culture was done to study hormone production ability after cryopreservation. RESULTS Only ultrarapid cooling in ethylene glycol-sucrose-egg yolk solution protected both follicles and stroma from damage. CONCLUSION The following parameters were established as required for a protocol of human ovarian tissue cryopreservation by direct plunging into liquid nitrogen: the vitrification medium should include ethylene glycol, disaccharide and egg yolk; ultrarapid cooling/thawing should take place using standard 0.25 straws or copper grids.

Effect of different vitrification protocols for human ovarian tissue on reactive oxygen species and apoptosis

The aim of the present study was to evaluate the effect of different vitrification protocols on reactive oxygen species (ROS) and apoptosis in human ovarian tissue. Human ovarian tissue pieces were exposed to different vitrification solutions. The intracellular redox state level was measured using the fluorescent dye dichlorodihydrofluorescein diacetate. Imaging of apoptotic cells was monitored by anti-caspase-3 immunolabelling after vitrification and warming. Following equilibration in either 40% ethylene glycol (EG) (v/v), 0.35 M sucrose + 10% egg yolk extract (v/v) or 40% EG (v/v), 18% Ficoll-70 (w/v) + 0.35 M sucrose for 6 min, ovarian pieces were cooled to -196 degrees C using four different protocols. Tissue that was cooled very rapidly (plunged directly into liquid nitrogen in straws or on grids or plunged directly into metal filings precooled to -196 degrees C) showed no statistically significant increase in either tissue ROS levels or the number of apoptotic cells after warming. In contrast, cooling using a less rapid method (nitrogen vapour at -120 degrees C) resulted in significantly elevated ROS levels and apoptosis after warming. There were no significant differences between the two vitrification solutions. This indicates that human ovarian tissue pieces should be vitrified using very rapid cooling rates.

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.

Vitrification of Human ICSI/IVF Spermatozoa Without Cryoprotectants: New Capillary Technology

The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up-prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 μL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 μL of spermatozoa vitrified in 50-μL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P < .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P < .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P > .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.

Comparison of In Vitro- and Chorioallantoic Membrane (CAM)-Culture Systems for Cryopreserved Medulla-Contained Human Ovarian Tissue

At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hens eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5–2.0×1.0–1.2×0.8–1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips.

In-vitro maturation of germinal-vesicle oocytes and cryopreservation in metaphase I/II: a possible additional option to preserve fertility during ovarian tissue cryopreservation

This study describes the possibility of combining two options in order to preserve female fertility: cryopreservation of human ovarian tissue and in-vitro matured germinal vesicle (GV) oocytes retrieved during tissue dissection. In contrast to ovarian tissue cryopreservation, the cryostorage of in-vitro matured unfertilized metaphaseI/II oocytes could be a more realistic option. This concept of preserving fertility before chemotherapy and/or radiotherapy without a long time delay could be an additional reason for favouring ovarian tissue cryopreservation. This concept is discussed in regard to two cases.