Frank Nawroth

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.

Cryoprotectant-Free Cryopreservation of Human Spermatozoa by Vitrification and Freezing in Vapor: Effect on Motility, DNA Integrity, and Fertilization Ability

Abstract Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 × 105 °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°C) with speed in the range 150–250°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.

Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success

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.

Clean technique for cryoprotectant-free vitrification of human spermatozoa

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.

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.

New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos

This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.

Conservative treatment of ectopic pregnancy in a cesarean section scar with methotrexate: a case report

Ectopic pregnancies sited in dehiscent cesarean section scars have a high risk of rupture and bleeding. Attempts at operative therapy frequently end in loss of the uterus. A connection with the cavum uteri justifies an attempt at dilatation and curettage. We describes a patient with combined systemic and local intra-amniotic methotrexate (MTX). The uterus was preserved.

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.

Lipolysis and Ultrastructural Changes of Intracellular Lipid Vesicles after Cooling of Bovine and Porcine GV‐oocytes

The aim of our investigation was to compare the ultrastructure of lipid droplets, and the effect of cooling on intracellular lipid vesicles of bovine and porcine GV oocytes. The lipid droplets in bovine GV oocytes have a homogeneous structure. The utilization of lipids takes place directly from these vesicles without formation of interim lipid compounds. In contrast, there are two kinds of lipid droplets in porcine GV oocytes: ‘dark’, homogeneous vesicles next to ‘grey’ vesicles with electron‐lucent streaks. Vesicles of each specific group are connected to each other. After a 12‐h culture, the formation of the cisternal smooth endoplasmic reticulum layer was always associated with ‘grey’ lipid vesicles. This is evidence that during oogenesis lipolysis takes place only in ‘grey’ vesicles. It is supposed that cytoplasmic lipolysis has two stages: ‘dark’ vesicles change into a ‘grey’ form followed by a utilization of these ‘grey’ lipids. Furthermore, both types of lipid droplets in porcine oocytes changed morphologically during cooling: they changed into a spherical form with lucent streaks. Lipid droplets in bovine GV oocytes revealed no visible morphological changes after cooling.