1. Hepatitis C( None A,None B) L. Motamedzadeh Vitrification of Human ICSI/IVF Spermatozoa Without Cryoprotectants V. Isachenko, R. Maettner Department of Obsterics and Gynecology, Cologne University, Kerpener Str.Cologne,Germany Section of Gynecological Endocrinology and Reproductiv Medicine, University of Ulm, Prittwitzstr, Germany

2. Cryobiology is a rapidly evolving field which only relatively recently has found broad applications in reproductive medicine,However, as any emerging technology, it has both a great potential and a need for further developments Introduction

3. The use of programmable or non-programmable “slow” (conventional) freezing: To preserve large volumes of diluted ejaculate or sperm preparations from 0.25 to 1.0 mL with acceptable rates of motility of spermatozoa after thawing and adequate integrity of acrosomal and cytoplasmic membranes Instrumental in minimizing the cryopreservation- induced cell deterioration resulting in membrane destabilization processes, such as membrane phosphatidylserine translocation

4. This method is based on the rapid cooling of the cells by immersion into liquid nitrogen Reducing the chance of the formation of big ice crystals Much faster, simpler in application and more cost- effective than conventional freezing At the same time vitrification can effectively protect spermatozoa from cryo-injuries associated with cooling-warming The mutagenic effects of permeable cryoprotectants can be avoided Spermatozoa vitrification

5. 68 ejaculates were obtained from 68 patients with oligo- astheno-terato-azoostermia All specimens used for this study had fulfilled following quality criteria for spermatozoa concentration, motility and morphology: less than 20 millions spermatozoa/mL, 35 % progressive motile and minimum 3 % morphologically normal (WHO, 1999). All investigations were carried out on swim up-prepared spermatozoa, Human Tubal Fluid (HTF)+ 1 % human serum albumin (HSA) Material & Method Spermatozoa preparation

6. After swim up, each sample was centrifuged, resuspended: To achieve concentration of 5 x 106 spermatozoa/mL Alliquot into three equal sub-samples: Group 1 included “fresh”, noncryopreserved swim-up spermatozoa preparations (control) Group 2:conventionally frozen Group 3 were vitrified

7. Vitrification Spermatozoa were diluted 1:1 with 0.5 M sucrose at room temperature The final concentration of spermatozoa was approximately of 0.5 x 10 spermatozoa/ml Specifically for our purposes, 50 μL-plastic capillaries cell were used(Fig.1) The end of the straw was labeled on the top to mark the cutting-off position(Fig.1, arrows) The capillary was filled with 10μL of spermatozoa suspension by aspiration (Fig. 1a) Vitrification and Warming

8. Aspirating the volume of sperm cell suspension above the mark and correcting it by lowering the fluid level inside the capillary after aspiration is technologically wrong and would result in excess of portion’s volume after thawing After the aspiration was completed, the capillary was inserted into 0.25 ml straw One end of this straw was sealed in advance using heat-sealer System, After sealing the second end of the straw (Fig. 1b), the straw was plunged into liquid nitrogen and cooled at a cooling speed of 600°C/min Spermatozoa were stored in liquid nitrogen at least for 24 h before warming

9. Capillary was removed from isolating 0.25 mL straw The straw was disinfected with ethanol in the area where the marked end of capillary was (Fig. 1, arrows) The second end of capillary is fixed tightly on the inner surface of the straw and the part of straw containing spermatozoa is still half submerged in liquid nitrogen (Figs. 1c,d) The capillary was expelled with a conical bolt (Fig. 1d) (Capillary must be open from both sides) with vitrified spermatozoa into 1.8 mL centrifuged tube with 0.7 mL pre-warmed to 37 °C thawing medium for approximately 20 sec. (Fig. 1e) The suspension of spermatozoa was expelled from the capillary for immediate evaluation of spermatozoa quality and IVF or ICSI. Thawing

13. After swim up procedure, The sperm suspension was diluted 1:2 with freezing medium (12 % (v/v) glycerol and 20 % (v/v) egg yolk) Diluted sperm preparations were equilibrated at room temperature for 10 min 10 μL of spermatozoa suspension were portioned by aspiration into 50 μL plastic capillaries using aspirator These capillaries were placed into 0.25 ml- standard insemination straws Straws were put in the horizontal position into liquid nitrogen vapor (-80°C, 10 cm over liquid nitrogen surface) 30 min Finally placed into liquid nitrogen where they were stored minimum 24 h until evaluation Thawing of spermatozoa was performed in accordance with technique described above for vitrified spermatozoa. Conventional Freezing and Thawing

14. Motility of spermatozoa was assessed 1 h after liquefaction, swim up procedure, cryopreservation,warming of samples and after 24h and 48h in vitro culture for evaluation of long-term spermatozoa The Makler’s chamber was used for all the motility studies Spermatozoa with progressive motility (categories ‘a’ [rapid, regular forward progression] and ‘b’ [moderate, slow or sluggish forward progression]) according to the World Health Organization (WHO,1999) were assessed Motility

15. The integrity of the plasma membrane was assessed with LIVE / DEAD sperm viability kit (includes SYBR-14 dye and propidium iodide (PI) which stain nucleic acid Differentiate between intact and damaged spermatozoa Due to its properties the SYBR-14 accumulates inside of living cells and emit green fluorescence and can, therefore be used for identification of live spermatozoa and exclusion of non-DNA containing particles and debris and the dead cells’ membrane will be permeable for PI Plasma membrane integrity

16. Changes in the membrane status and permeability associated with the capacitation are traditionally evaluated by using the double fluorescence chlortetracycline (CTC)-Hoechst 33258 staining technique Acrosomal integrity and Capacitation-like changes

17. At least 200 spermatozoa were observed in each plate and three patterns were identified: A uniform fluorescence on the head of the spermatozoa (spermatozoa classified as ‘non-capacitated) A band of fluorescence diminished in the post-acrosomal region and a relatively shining fluorescence in the acrosomal region (spermatozoa identified as undergoing capacitation- like changes) A fluorescence in the complete head of the spermatozoa, except a tenuous band of fluorescence in the equatorial segment (identified as ‘acrosome-reacted’ spermatozoa) The dead spermatozoa displayed a pattern of blue fluorescence in the whole head.

18. Result Motility Motility of spermatozoa vitrification in small volume (10 μL) in absence of permeable cryoprotectants displayed statistically higher levels of motility as compared to slow conventional freezing (28.0 +6.0 %vs 18.0 + 9.2 %, respectively, P<0.05)( In fresh 35.0 + 9.5%) Motility of vitrified spermatozoa after 24 h in vitro culture was also statistically different from that of frozen spermatozoa (12.0 +2.8 % vs 5.0 + 3.1 %, respectively, P<0.05) (in fresh 20.0 + 3.9%) Motility rates of vitrified spermatozoa after 48 h in vitro culture was not statistically different from those of frozen spermatozoa (6.0 +1.0 % vs 0.5 + 0.02 %, respectively) (P>0.1) (in fresh 10.0 + 1.9%)

20. The higher rates of membrane integrity were achieved in vitrified spermatozoa as compared to slow conventional freezing (56.0 ± 5.1 % vs 22.0 ± 3.5 %, respectively) (P<0.05) However, as compared to non-treated control (fresh spermatozoa) the spermatozoa viability was significantly diminished in frozen-thawed spermatozoa in both cases (in fresh spermatozoa 96.0 ± 0.6 %) (P<0.05) Plasma membrane integrity

22. There was a statistically significant difference between percentages of spermatozoa identified as acrosome intact after vitrification as compared to conventional freezing The aseptic vitrification technique had not provided significantly stronger prevention against “cryo-capacitation” as compared to conventional freezing In both cases the percentage of cells with capacitation-like changes was higher than in fresh spermatozoa Acrosomal integrity and Capacitation-like changes

23. The effect of two procedures used for cryopreservation on acrosomal membrane status by CTC staining

24. The presence of capacitation-like changes after different modes of cryopreservation

25. Discussion

26. Vitrification method can achieve up to 60%-and 20% - motility levels after thawing in normospermic and oligo asthenoteratozoospermic (OAT) patients respectively Vitrified spermatozoa can be processed for further use immediately after warming No additional treatment such : gradient separation, removal of cryoprotectants etc is required It is worth to mention that the protocol for vitrification does include swim up treatment, therefore, after swim up, vitrification and warming spermatozoa are also free from seminal plasma with potential pathogens

27. slow, step-wise adding of freezing solution to the ejaculate cooling during 20 to 45 min warming in water bath preparation of spermatozoa by the density gradient or the swim-up procedure for removal of permeable cryoprotectants. “ Slow” freezing of human spermatozoa

28. This removal of permeable cryoprotectants is the ultimate target of the last manipulation : Dilution of semen suspension with culture medium in order to reduce the toxicity of permeable cryoprotectant In fact, sensitivity of spermatozoa to additional mechanical manipulation is increased after freezing-thawing, and the negative effects of cryopreservation on cell viability and functional competence can be aggravated by additional procedures

29. The diameter of the pulled part of straw is not uniform, the volumes of the portions packaged in that way can not be standardized One of the most important features of this novel method of vitrification in capillaries is its potential for standardization which can be instrumentalised for the clinical practice standardization of the portion volume

30. Motility plasma membrane integrity It is well known that spermatozoa ryopreservation is associated with a large decline in spermatozoa viability and other sperm functional parameters The results of the present study let suggest that cryopreservation by vitrification helps to preserveessential determinants of spermatozoa function

31. The outcomes indicated that the motility of spermatozoa and membrane integrity were better preserved after Vitrification in capillaries Pilot results have been obtained with respect to evaluating capacitation-like changes associated with cryopreservation, so called “cryo-capacitation” Some spermatozoa intracellular signaling pathways can be affected during cryopreservation, and after warming spermatozoa display features commonly observed in capacitating or capacitated spermatozoa Spermatozoa quality after vitrification and after conventional freezing with addition of permeable cryoprotectant