Vit Kit - Thaw. Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage)

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1 Vit Kit - Thaw Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage) Catalog No DSO Includes: Thawing Solution - TS (yellow cap) 4 x 2 ml Vials Dilution Solution - DS (orange cap) 1 x 2 ml Vials Washing Solution - WS (red cap) 1 x 2 ml Vials For assisted reproductive procedures.

2 Caution: Federal law restricts this device to sale by or on the order of a physician or a practitioner trained in its use. Caution: The user should read and understand the Directions for Use, Warnings and Precautions, and be trained in the correct procedure before using the Irvine Kits for vitrification of human embryos. INTENDED USE Vit Kit - Thaw is intended for use in the warming of pronuclear (PN) zygotes through blastocyst stage embryos, which were vitrified using Irvine Scientific s Vitrification Freeze Kit (Vit Kit - Freeze, Catalog No DSO) for embryos. PRECAUTIONS AND WARNINGS This device is intended to be used by staff trained in assisted reproductive procedures that include the indicated application for which the device is intended. Do not use any vial of solution which shows evidence of damage, leaking, particulate matter, cloudiness or not red to reddish-orange in color. Discard the product in accordance with applicable regulations. To avoid problems with contamination, handle using aseptic techniques. Vitrification Thaw Kit Solutions contain the antibiotic gentamicin sulfate. Appropriate precautions should be taken to ensure that the patient is not sensitized to this antibiotic. The long term safety of embryo vitrification on children born following this method of embryo cryopreservation is unknown. *Human source materials used in the manufacture of this product have been tested by FDA licensed kits, and found to be non-reactive to the antibodies for Hepatitis B surface antigen (HbsAg), antibodies to Hepatitis C (HCV) and antibodies to Human Immunodeficiency Virus, (HIV). Donors of the source material have also been screened for CJD. However, no test method offers complete assurance that products derived from human sources are noninfectious. Handle all human source material as if it is capable of transmitting infection, using universal precautions. PRODUCT DESCRIPTION Thawing Solution-TS is a HEPES buffered solution of Medium-199 containing gentamicin sulfate (35 µg/ml), 1.0 M sucrose and 20% (v/v) Dextran Serum Supplement*. Dilution Solution-DS is a HEPES buffered solution of Medium-199 containing gentamicin sulfate (35 µg/ml), 0.5M sucrose and 20% (v/v) Dextran Serum Supplement*. 1/7

3 Washing Solution-WS is a HEPES buffered solution of Medium-199 containing gentamicin sulfate (35 µg/ml) and 20% (v/v) Dextran Serum Supplement*. These three solutions are to be used in sequence according to the step-wise microdrop warming protocol. QUALITY ASSURANCE The solutions in Vit Kit-Thaw are membrane filtered and aseptically processed according to manufacturing procedures which have been validated to meet a sterility assurance level (SAL) of Each lot of Vit Kit- Thaw receives the following tests: Solutions: Endotoxin by LAL methodology Biocompatibility by mouse embryo assay (one-cell) Sterility by the current USP Sterility Test <71> Albumin Test All results are reported on a lot-specific Certificate of Analysis, which is available upon request. FOR WARMING CRYOTIP AS THE STORAGE DEVICE: MATERIALS REQUIRED BUT NOT INCLUDED Irvine Scientific Connector (Catalog No ) or adaptor Sterile Petri Dishes (50 X 9 mm, Falcon or equivalent) Disposable gloves Hamilton GASTIGHT Syringe (50 µl) catalog #80901 Transfer pipettes (pulled glass pipettes or micropipette tips with an inner tip diameter of ~200 µm) Tweezers Stopwatch or timer Liquid Nitrogen Reservoir (Dewar or Styrofoam container with lid, 1-2 L volume) Liquid Nitrogen (sufficient volume to achieve 4 inch depth in reservoir) Sharp scissors (sterile) 37 C waterbath (>500 ml) Culture Medium (with 20% protein) appropriate for develop mental stage of specimen to be recovered. Prepare and pre-equilibrate dish with culture medium to 37 C in CO 2 incubator prior to thawing specimens. DIRECTIONS FOR USE Vit Kit-Thaw components (per application): 50 µl of Thawing Solution-TS 50 µl of Dilution Solution-DS 100 µl of Washing Solution-WS 1 Connector WARMING PROTOCOL (FOR EMBRYOS) NOTE: Procedures are to be done at room temperature (20-27º C). DO NOT use heated microscope stage for the following procedures. CAUTION: Minimize exposure of specimen to light during manipulations through warming solutions. 2/7 1. Bring the quantity to be used of TS, DS and WS to room temperature (20-27 C) prior to warming vitrified specimens. NOTE: Avoid bringing the entire vials of TS, DS and WS to room temperature repeatedly when a small quantity of the solution is needed each time. It is better to aliquot the quantity to be used and return the vials to 2-8 C right after aliquoting. 2. Fill the liquid nitrogen reservoir with liquid nitrogen (~80 % full) and place close to the LN 2 freezer containing the specimens to be warmed. 3. Remove the canes with goblets containing the CryoTips with vitrified specimens from liquid nitrogen storage and transfer them into the liquid nitrogen filled reservoir. Place the reservoir close to the microscope for rapid manipulation. CAUTION: Make sure that CryoTips remain submerged in LN 2 (in goblet) during transfer from storage to LN 2 reservoir to prevent uncontrolled thawing of specimens. 4. Label a sterile petri dish (or lid) with necessary information. 5. Gently invert each vial of TS, DS and WS twice to mix contents before use. 6. To set up warming dish, aseptically dispense: One (1) 50 μl drop of TS One (1) 50 μl drop of DS Two drops of WS will be set up later at Step 15 Figure 1. CryoTip Contents (~1 µl) TS DS WS1 (4 min each) Transfer to Culture medium with 20% (v/v) protein for Recovery 7. Place the 37 C waterbath close to the microscope. Have the following nearby: a transfer pipet and tips, sterile sharp scissors, Hamilton syringe and sterile wipes. 3/7 (1 min) (4 min) WS2 50 µl Drops

4 8. Using tweezers (or tongs), quickly remove CryoTip from LN 2 and within 1 second fully immerse the device in the 37 C waterbath (>500 ml) and swirl device gently for 3 seconds. Swirling the device is critical to ensure the most rapid warming rate (+24,000 C/min). Figure While visualizing under the microscope, dispense contents of CryoTip as a small drop directly adjacent to TS drop. Once you visualize the specimen(s) touch the CryoTip contents drop to TS drop with end of CryoTip to mix. Set timer for 1 minute and leave undisturbed. NOTE: AVOID BUBBLES WHILE DISPENSING THE CONTENTS. 37 C H C/min NOTE: The specimens will shrink and float to the top of the drop. LN 2 (3 sec) Waterbath >500mL NOTE: After each transfer of the specimen(s), blow out any remaining fluid in the transfer pipet and draw up some solution from the next drop prior to the next manipulation. Avoid creating bubbles during the transfers. 9. Remove CryoTip from the waterbath and promptly remove metal cover sleeve from device by firmly grasping the lower end of the cover sleeve and pulling away from the CryoTip. Gently wipe away any water with a sterile dry tissue ensuring the tip of the device is dry. 10. Using sterile medical grade sharp scissors make Cut #1 below seal at wide end of CryoTip. 11. Withdraw the plunger of the syringe (with connector attached) to the half way position. Gently attach CryoTip to connector and syringe (or pipette). 12. Place fine tip end over the prepared warming dish and quickly make Cut #2 above the seal at the fine end. Figure Transfer specimen(s) to DS, for 4 minutes. Gently pipette specimens once to ensure complete rinse in DS. NOTE: The specimen will remain shrunken during exposure to DS. 15. During the 4 minute exposure in DS aseptically dispense two (2) 50 μl drops of WS (WS1, WS2) as in Figure Transfer specimen(s) to WS1 then WS2 for 4 minutes each undisturbed. NOTE: The specimen(s) should re-expand to the original size within 2-3 minutes in WS. 17. There are two options for warmed EMBRYO(S): a) For immediate transfer to patient: transfer EMBRYO(S) to pre-equilibrated transfer medium containing 20% (v/v) protein supplement or 12 mg/ml. Mark #4 Seal #2 Attach Connector Cut #1 b) For further culture: transfer EMBRYO(S) to pre-equilibrated culture medium containing 20% (v/v) protein supplement or 12 mg/ml for a 4 hour recovery period. After recovery period transfer EMBRYO(S) to culture medium with 10% (v/v) protein and incubate accordingly until desired developmental stage has been reached for transfer to patient. 37 C Mark #3 After Warming Mark #2 Mark #1 Seal #1 Cut #2 4/7 5/7

5 FOR WARMING OTHER CRYOSTORAGE DEVICES: MATERIAL REQUIRED BUT NOT INCLUDED Sterile 4-well dish (Nunc , or equivalent), or organ culture dish (BD Falcon ) Disposable gloves Transfer pipettes Tweezers Stopwatch or timer Liquid nitrogen resevoir Liquid Nitrogen Scissors Culture Medium with 20% protein, pre-equilibrated to 37 C in CO 2 incubator prior to thawing procedure. 37 C incubator without CO 2, or heating stage DIRECTIONS FOR USE Vit Kit Thaw components (per application) 250 µl of Thawing Solution-TS 50µL of Dilution Solution-DS 50 µl of Washing Solution-WS WARMING PROTOCOL (FOR EMBRYOS) NOTE: The warming steps include plunging the device into the 37 C TS and subsequent diluting and washing in DS and WS at room temperature 1. Set up warming dishes (see Figure 4): At 37 C: Aseptically dispense a minimum of 250 µl of TS into a sterile 4-well dish or an organ culture dish and place it in a 37 C incubator without CO 2 or on a heating stage at least 30 minutes prior to thawing procedure Figure 4. TS 37 C 1 min 250 µl 2. Identify cryostorage device(s) to be thawed from LN 2 storage and quickly transfer to LN 2 filled reservoir in preparation for warming procedure. 3. Place LN 2 reservoir close to microscope for subsequent rapid manipulation. 4. Remove TS dish from 37 C incubator or heating stage and place it under focus on top of the microscope stage. Steps 7-10 must be performed at room temperature At room temperature: Aseptically dispense one (1) 50 µl drop of DS on a sterile Petri dish as in Figure 5. Figure 5. WS1 WS2 (4 min each) 7. Transfer specimen(s) to DS for 4 minutes. Gently pipette specimens once to ensure complete rinse in DS. NOTE: The specimen will remain shrunken during exposure to DS. 8. During the 4 minute exposure in DS, aseptically dispense two (2) 50 µl drops of WS (WS1, WS2) as shown in Figure Transfer specimen(s) to each WS1 then WS2 for 4 minutes each, undisturbed. NOTE: The specimen(s) should re-expand to the original size within 2-3 minutes in WS. 10. There are two options for warmed EMBRYO(S): a) For immediate transfer to patient: transfer EMBRYO(S) to pre-equilibrated transfer medium containing 20% (v/v) protein supplement or 12 mg/ml. b) For further culture: transfer EMBRYO(S) to pre-equilibrated culture medium containing 20% (v/v) protein supplement or 12 mg/ml for a 4 hour recovery period. After recovery period transfer EMBRYO(S) to culture medium with 10% (v/v) protein and incubate accordingly until desired developmental stage has been reached for transfer to patient. For additional details on the use of these products, each laboratory should consult its own laboratory procedures and protocols which have been specifically developed and optimized for your individual medical program. STORAGE INSTRUCTIONS AND STABILITY Store the unopened vials of solutions refrigerated at 2 C to 8 C. When stored as directed, Vitrification Thaw Kit solutions are stable until the expiration date shown on the vial label. Do not use media for more than eight (8) weeks once containers have been opened. As human source material is present in the product it may develop some particulate matter during storage. This type of particulate matter is not known to have an effect on product performance. 5. Warm the selected cryostorage device and dispense specimen(s) into 37 C TS solution according to manufacturer s instructions. 6. Leave specimen(s) in TS for 1 minute. GASTIGHT is a Registered Trademark of Hamilton Co. 6/7 7/7 DS (4 min) 50 µl Drops Transfer to Culture medium with 20% (v/v) protein for Recovery

6 REFERENCES 1. Kuwayama M, Vajta G, Kato O, Liebo SP. Highly efficient vitrification of human oocytes. RBM Online 11: , Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor V, Brohammer R, Kato O. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. RBM Online 11:53-57, Takahashi K, Mukaida T, Goto T, Oka C. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil Steril 84:88-92, Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. RBM Online 11: , Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. RBM Online 6: , Isanchenko V, Selman H, Isachenko E, Montag M, El-Danasouri I, and Nawroth F. Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure. Reproductive BioMedicine Online 7, , Van den Abbeel E, Camus M, Van Waesberghe L, Devroey P, Van Steirteghem AC. A randomized comparison of the cryopreservation of one-cell human embryos with a slow controlled-rate cooling procedure or a rapid cooling procedure by direct plunging into liquid nitrogen. Hum. Reprod. 12: , Shee-Uan Chen, M.D., Yih-Ron Lien, M.D., Kuang-Han Charo, M.D., Hsin-Fen Lu, M.D., Hong-Nerng Ho, M.D., an Yu-Shih Yang, M.D., Ph.D.,, Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws, Fertility and Sterility, vol. 74(4), , Michel Camus, M.D., Etienne Van den Abbeel, B.Sc., Linda Van Waesberghe, M. Sci., Arjoko Wisanto, M.D., Paul Devroey, M.D., and Andre C. Van Steirteghem, M.D., PhD., Human embryo viability after freezing with dimethylsulfoxide as a cryoprotectant, Fertility and Sterility, Vol. 51 (3), , Balaban, et. al., A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation, Human Reproduction, Vol. 23, No. 9, pp , Yoko Kumasako, Mami Kumon, Takafumi Utsunomiya, and Yasuhisa Araki, Successful Pregnancy after the Vitrification of Zygotes Using Commercial Vitrification Solutions and Conventional Straws to Protect Against Infections of Liquid Nitrogen, Journal of Assisted Reproduction and Genetics, Vol. 22, No. 1, January S. Hashimoto, Y. Murata, M. Kikkawa, M. Sonoda, H. Oku, T. Murata, K. Sugihara, F. Nagata, Y. Nakaoka, A. Fukuda, and Y. Morimoto, Successful delivery after the transfer of twice-vitrified embryos derived from in vitro matures oocytes: A Case Report, Human Reproduction, Vol. 22, No. 1, pp , Safaa Al-Hasani, Batuhan Ozmen, Nikoleta Koutlaki, Beate Schoepper, Klaus Diedrich, Askan Schultz-Mosgau, Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing?, RMB Online, Vol. 14, No. 3, pp , Meghan B. Oakes, M.D., Claudia Messias Gomes, M.D., Joyce Fioravanti, B.S., Paulo Serafini, M.D., Ph.D., Eduardo L.A. Motta, M.D., Ph.D., and Gary D. Smith, Ph.D., A case of oocyte and embryo vitrification resulting in clinical pregnancy, Fertility and sterility, Vol. 90, No. 5, November Yoko Kumasako, B. E., Eiko Otsu,.En., Takafumi Utsunomiya, M.D., and Yasuhisa Araki, Ph.D., The efficacy of transfer of twice frozen-thawed embryos with the vitrification method, Fertility and Sterility, Vol. 91, No. 2, pp , February Nina Desai, Heather Blackmon, Julia Szeptycki, James Goldfarb, Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births, RBM Online, Vol. 14, No. 2, pp , A-J Kartberg, F Hambiliki, T Arvidsson, A Stavreus-Evers, P Svalander., Vitrification with DMSO protects embryo membrane integrity better than solutions without DMSO. RBM online, Vol 17, No. 3, pp , 2008.

7 Symbols: Catalog Number Lot Number Sterilized using aseptic processing techniques (filtration) Expiration: Year - Month - Day Caution: See instructions for use 2 C 8 C Storage Temperature Manufacturer 2511 Daimler Street, Santa Ana, California USA Telephone: Fax: PN Rev.10

Vit Kit - Thaw. Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage)

Vit Kit - Thaw. Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage) Vit Kit - Thaw Vitrification Thaw Kit for Embryos (PN through Blastocyst Stage) Catalog No. 90137DSO Includes: Thawing Solution - TS (yellow cap) 4 x 1 ml Vials Dilution Solution - DS (orange cap) 1 x