Vladimir Isachenko

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.

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.

Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryoinduction of capacitation, and acrosome reaction. Spermatozoa were isolated using the swim-up procedure performed using three different media: (a) human tubal fluid (HTF, control) medium; (b) HTF with 1% human serum albumin (HSA); and (c) HTF with 1% HSA and 0.25 M sucrose. From each group, 30 mul suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by quickly submerging the spheres in HTF with 1% HSA at 37 degrees C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction were investigated. Sperm viability, acrosome reaction, and capacitation were detected using the double fluorescence chlortetracycline-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye, 5,5,6,6-tetrachloro-1-1,3,3-tetraethyl-benzamidazolocarbocyanin iodide, commonly known as JC-1. The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1+/-3.2%, P<0.05) when compared with controls (19.4+/-1.9%). The combination of HSA and sucrose (65.2+/-2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compared with HSA alone (32.6+/-4.7%). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as HSA and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.

Effective method for in-vitro culture of cryopreserved human ovarian tissue.

It is widely accepted that it is possible to successfully cryopreserve human ovarian tissue by direct plunging into liquid nitrogen using permeable cryoprotectants only, without disaccharides. This study aimed to search for and test a new method for in-vitro culture of vitrified tissue. Ovarian biopsies were obtained during operative laparoscopy. Pieces of ovarian tissue were vitrified and warmed. After warming, tissue pieces were randomly distributed into three groups for further culture: in 2 ml of culture medium which was regularly renewed (group 1), in 30 ml of culture medium without agitation (group 2) and in 30 ml of culture medium with agitation (group 3). During the 2-week and 6-week culture, the growth of follicles within the vitrified-warmed ovarian tissue pieces was investigated. After 2 weeks of culture, mean numbers of non-degenerated follicles per mm(2) of tissue were 1.5, 1.7 and 4.5 for groups 1, 2 and 3 respectively (groups 1 and 2 versus group 3, P < 0.05). Agitation during culture of ovarian tissue is beneficial, and can be used as a prognostic tool for future warming and autotransplantation of ovarian tissue.

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.

Cryopreservation of human ovarian tissue: effect of spontaneous and initiated ice formation

This investigation compared conventional freezing of human ovarian tissue using either spontaneous or initiated (seeded) ice formation. Biopsies of ovarian tissue were obtained from women with indications for chemotherapy or radiotherapy. Small pieces of experimental tissue were randomly distributed into three groups that were then subjected to different treatments prior to culture in vitro for 16 days: the control group, no treatment, cultured immediately after biopsy (group 1); cryopreservation/thawing with spontaneous ice formation (group 2); and cryopreservation/thawing with initiated ice formation (group 3). Follicle viability and hormonal activity were then evaluated. There was no significant difference between groups regarding the concentration of oestradiol 17-beta in the culture supernatant, whereas progesterone concentration was significantly (P < 0.05) higher in group 1 compared with group 2 or 3. There was a significant (P < 0.05) difference in primordial and primary follicle density between all of the groups (group 1 having the highest and group 2 having the lowest) and group 2 had significantly (P < 0.05) fewer normal grade follicles than the other two groups. For optimal cryopreservation of human ovarian tissue, the protocol of conventional freezing should therefore include a step of initiated ice formation.

Vitrification Technique - New Possibilities for Male Gamete Low-Temperature Storage

The method is cheap and quick. Due to lowering the temperature of solutions glass transition the permeable cryoprotectants prevents the actual freezing of solution and allows to maintain its some flexibility in a glassy phase. Vitrification without permeable cryoprotectants allows to avoid the cryoprotectants toxicity and osmotic stress; the damage of plasmatic and mitochondrial membrane during equilibration with cryoprotectants; protects of plasmatic and mitochondrial membrane against lipid peroxidation and formation of reactive oxygen species and DNA damage.

Stellenwert der Vitrifikation in der Reproduktionsmedizin

ZusammenfassungDie Vitrifikation ist ein noch recht junges Verfahren zur Kryokonservierung von Zellen und Geweben. Bei der Vitrifikation werden die Zellen aus einem flüssigen Zustand direkt in einen amorphen, vitrifizierten Zustand überführt, und die Bildung von Eiskristallen wird unterbunden. Insbesondere im reproduktionsmedizinischen Bereich zeigen sich vermehrt Einsatzmöglichkeiten für die Vitrifikation. Im Vergleich zu der bereits etablierten Methode der langsamen Kryokonservierung, scheint die Vitrifikation für die als kryobiologisch problematisch einzufrierenden Zellen, Gewebe und Entwicklungsstadien von Vorteil zu sein. Unbefruchtete Eizellen im Metaphase-II-Stadium sowie Blastozysten zeigen nach Vitrifikation sehr gute Überlebens- und Entwicklungsraten. Neue Entwicklungen erlauben die Vitrifikation unter aseptischen Bedingungen und unter Vermeidung des direkten Kontakts zwischen der Probe und flüssigem Stickstoff. Wie bei jeder neuen Technik, müssen bei der Durchführung der Vitrifikation einige wichtige Details beachtet werden, um das volle Potenzial dieser relativ einfachen Technik auszuschöpfen.AbstractVitrification is a relatively new technique for the cryopreservation of cells and tissues. A key element of vitrification is the direct transformation of cells from a fluid state into an amorphous, vitrified state without the formation of ice crystals. Vitrification may be especially useful in the field of reproductive medicine. Compared to the more established method of slow freezing, vitrification seems to be more suitable for cryobiologically difficult to freeze cells, tissues and developmental stages. Unfertilized metaphase II oocytes and blastocysts show very high survival and developmental rates after vitrification. Recent improvements allow for aseptic vitrification by avoiding direct contact between the specimen and liquid nitrogen. As with every new laboratory technique, the introduction of vitrification into the the laboratory requires the careful application of all technical details in order to achieve its full potential.

Generierung und Konservierung von Keimzellen

ZusammenfassungDie In-vitro-Maturation (IVM) ist eine Technik, welche die Reifung von Eizellen im Germinalvesikelstadium bis hin zur befruchtungsfähigen Metaphase-II-Eizelle beinhaltet. Die Eizellen werden in der Regel gezielt aus kleinen antralen Follikeln gewonnen. Der Erfolg nach 24- bis 36-stündiger Maturation wird derzeit mit der Bildung des 1. Polkörpers als Anzeichen für eine abgeschlossene erste meiotische Reifeteilung belegt. Die Qualität einer in vitro gereiften Eizelle kann durch ein neuartiges polarisationsmikroskopisches Verfahren noch besser eingeschätzt werden. Dieses Verfahren ermöglicht eine deutlich bessere Beurteilung und ist somit ein einzigartiges Instrument, um bestehende IVM-Protokolle zu optimieren. Zusammen mit der inzwischen realistischen Option der erfolgreichen Kryokonservierung von unreifen und reifen Eizellen durch die Vitrifikation eröffnen sich künftig für die IVM neue Einsatzgebiete.AbstractIn vitro maturation (IVM) is a technique which allows the maturation of oocytes from the germinal vesicle stage up to the stage of the fertilization-competent metaphase-II oocytes. Immature oocytes are primarily retrieved from small antral follicles. Their successful maturation is usually documented by formation of the first polar body as an indicator of completion of the first meiotic division. The quality of in vitro matured oocytes can now be judged by polarisation microscopy. This technique allows better quality assessment and is hence a unique instrument for optimising existing IVM protocols. In combination with the now realistic option of successful cryopreservation of mature and immature oocytes by the technique of vitrification, IVM will soon enter new fields of application.

In vitro-Kultur von Embryonen mit Mikrovibration erhöht die take-Baby-home Raten: Daten von 4303 Patienten in einem Zeitraum von vier Jahren

Es wurde gezeigt, dass mechanische Stimulation von Zellen eine Welle von steigendem Ca2+ induziert. Diese verbreitet sich von den stimulierten zu benachbarten Zellen. Die in-vitro-Kultur von menschlichen Embryonen, die regelmasiger kurzer Vibration unterworfen ist, fuhrt zu erhohten Entwicklungsgeschwindigkeiten. Diese Art der Behandlung imitiert die Vibration in der Natur, wobei Eileiterflussigkeit mechanisch durch die epithelialen Zilien in Bewegung gebracht wird. Unsere Forschungsfrage lautet: Kann in-vitro-Kultur von Embryonen mit Mikrovibration die take-Baby-home Rate erhohen? Diese Arbeit wurde von August 2010 bis Dezember 2014 durchgefuhrt. Eine schriftliche Einwilligung wurde von allen teilnehmenden Paaren fur die Kultur der pronuklearen Eizellen und Embryonen eingeholt. Patientinnen mit idiopathischer Infertilitat wurden in IVF- oder ICSI-Zyklen stimuliert und in vier Altersgruppen eingeteilt: 40 Jahre. Einwilligung zur Verwendung der pronuklearen Oozyten (zwei pro Patient) wurde von 4303 Patientinnen (Durchschnittsalter 34 + 4,6) eingeholt. Die in vitro Kultur wurde unter zwei verschiedenen Bedingungen durchgefuhrt: mit mechanischer Bewegung (2152 Patienten (20 Hz 5 Sekunden einmal pro Stunde geliefert), n = 4304) sowie ohne Vibration des Kulturmediums (2151 Patienten, n = 4302). Embryotransfer (zwei Embryonen pro Patient) wurde am 3ten oder 5ten Tag nach der Follikelpunktion durchgefuhrt. Take-Baby-home Raten wurden mittels ANOVA fur kategoriale Variablen mit dem CATMOD Verfahren von SAS (SAS Institute Inc., 2011) analysiert. Vergleiche zwischen den Altersgruppen wurden durch paarweise Gegensatze und Bonferroni-Holm Einstellung fur multiple Vergleiche mit dem MULTTEST Verfahren von SAS durchgefuhrt. Folgende Take-Baby-home Rate wurde fur Gruppen 40 Jahre, bzw. (statisch gegen Vibration) festgestellt: 29,3% vs. 32,9% (P > 0,1, die Erhohung von der Hohe der Neigung), 27,1% gegenuber 38,7% (P < 0,05), 23,8% gegenuber 28,9% (P < 0,05), 9,1% vs. 14,5% (P < 0,05). In-vitro-Kultur von Embryonen unter Mikro-Vibrationsbedingungen die take-Baby-home Rate fur Patientinnen von 30 Jahren und alter deutlich erhoht.