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In-vitro Fertilization Procedural Manual - MTG

1. _ P In vitro Fertilization _ Procedural Manual e SCIENTIFIC 2511 Daimler Street Santa Ana California 92705 Tel 800 437 5706 Irvine 949 261 7800 Grow With Us Fax 949 261 6522 www irvinesci com pn 3 Irvine Scientific Contents Sonnie r 1 1 DUG COMO u u am u UPC PS TERMS 2 1 Embryology Protocols iore eset qd qu ep es 3 1 Pr01000 52 u us u u u usuy E PER k zhez TS V A TAa A PURSE OPES am EUR UR 4 1 Micromanipulation ICSI Protocols ccce mr Rn 5 1 Cryopreservation Protocols o past eie tang Ead EU REIR Y RUE ICT beca qd P Paris 6 1 2 7 1 IrvineScientific www irvinesci com Rev 1 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific MISSION STATEMENT Irvine Scientific 5 mission is to set the standard for EXCELLENCE through Quality Products for the Cell Culture Diagnostics and Biopharmaceutical Markets Quality Service with respect courtesy and loyalty to our customers Quality People dedicated to complete customer satisfaction COMPANY BACKGROUND Irvine Scientific was established in May 1970 in Fountain Valley Cali
2. 9 During the equilibration time prepare the CryoTip carefully attach the larger end of the tip to the connector on the suction device then slide the metal cover sleeve toward the larger end to expose the fine tip end Avoid touching or bending the fine tip end while handling the CryoTip The following steps should be completed in 90 seconds exposure of the embryos to VS should be limited to prevent cytotoxicity 10 After the equilibration of embryos is complete draw up some ES into the transfer pipette and transfer the specimen with minimal volume from the drop of ES into the bottom of the first drop of VS VS1 for 5 seconds The embryos will rise to the surface of the drop immediately This step should be observed under a magnification level that allows the observer to follow this movement of the embryos to the top of the drop 11 Quickly transfer the specimen from the first drop of VS to the bottom of the second drop of VS VS2 for 5 seconds and follow their rise to the top of the drop 12 Then transfer the specimen to the bottom of the third drop of VS VS3 for 10 seconds 13 Finally transfer the embryos to the fourth drop of VS VS4 Immediately Load and heat seal the CryoTip as follows While observing under the microscope carefully aspirate a small volume of VS to the 1st mark on the CryoTip Continue observation under the microscope and gently aspirate the specimen with VS to the 2nd mark on the CryoTip Now obs
3. Warming Vitrified Oocytes and Embryos IRVINE SCIENTIFIC VITRIFICATION THAW MEDIA KIT Cat 90137 Vitrification Thaw Media Kit is intended for use in assisted reproductive procedures for thawing and recovery of human oocytes MII and embryos 2PN to blastocyst that have been vitrified using Irvine Scientific s Vitrification Freeze Media Kit The product includes Thawing Solution TS is a HEPES buffered solution of Medium 199 containg 1 0 M sucrose and 20 v v Serum Substitute Supplement This solution is packaged in the tubes with the yellow labels and tops Dilution Solution DS is a HEPES buffered solution of Medium 199 containg 0 5 M sucrose and 20 v v Serum Substitute Supplement This solution is packaged in the tubes with the orange labels and tops Washing Solution WS is a HEPES buffered solution of Medium 199 containg 20 v v Serum Substitute Supplement This solution is packaged in the tubes with the red labels and tops Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Cryopreservation Protocols Using the Vitrification Thaw Media Kit Critical Points Great caution should be exercised when handling liquid nitrogen during the vitrification process The CryoTip is very thin and the solution in it will thaw within 1 3 seconds Therefore do not expose the tip to room air until ready to thaw This means that all transport or transfer of the embryos from one vessel to another should be done under liquid nitrogen A variet
4. 15 mm IS 373 MAKLER COUNTING CHAMBER CASA IS 365 MAKLER COUNTING CHAMBER INVERTED IS 363 MAKLER COUNTING CHAMBER KIT IS 360 MAKLER INSEMINATION DEVICE 99175 MODIFIED HAM S F 10 BASAL MEDIUM IrvineScientific www irvinesci com Catalog Number Description MODIFIED HAM S F 10 BASAL MEDIUM HEPES 99168 99109 90126 9984 90139 9305 99242 9235 99219 99311 90119 90129 IS 363 CG IS 374 IS 366 IS 364 IS 360RSB IS 360RSH 99193 IS 361A 99176 9983 MEI816NST 18165 CE118A CE123A CE418A CE423A N176740 METT1816 METT1816N 99252 90133 MODIFIED HAM S F 10 WITH ALBUMIN MODIFIED HTF MEDIUM WITH GENTAMICIN HEPES MODIFIED SPERM WASHING MEDIUM MULTIBLAST MEDIUM OIL FOR EMBRYO CULTURE 1 MEDIUM WITH GENTAMICIN PBS 1X DULBECCO S PHOSPHATE BUFFERED SALINE SOLUTION POLYVINYLPYRROLIDONE PVP LYOPHILIZED POLYVINYLPYRROLIDONE PVP SOLUTION 10 POLYVINYLPYRROLIDONE PVP SOLUTION 7 REFRIGERATION MEDIUM TEST YOLK BUFFER TYB WITH GENTAMICIN REPLACEMENT CHAMBER GRIP REPLACEMENT COVER GLASS CASA REPLACEMENT COVER GLASS INVERTED REPLACEMENT COVER GLASS WITH GRID REPLACEMENT SLIDING BRACKET REPLACEMENT SYRINGE HOLDER SERUM SUBSTITUTE SUPPLEMENT SHORT INSEMINATION CANNULA 15 mm SPERM MAINTENANCE MEDIUM SPERM WASHING MEDIUM STYLET RIGID 23 cm STYLET RIGID 18 cm SUREVIEW EMBRYO TRANSFER CATHETER 18 cm SUREVIEW EMBRYO TRANSFER CATHETER 23 cm SUREV
5. 5 Normally fertilized pronuclear embryos may be cultured in groups or individually in P 1 Medium ECM of the Complete Media series through Day 2 or 3 before transferring into the uterus or to ongoing culture Media that is not purchased as the Complete formulation should have protein added to it 5 mg mL HSA 10 v v SSS or 10 v v DSS at the time the dishes are prepared EMBRYO CULTURE AND ASSESSMENT General Considerations Following fertilization determination embryos may be transferred or cultured for several days depending on clinic policy Procedure 1 Some programs choose to transfer embryos on Day 2 some on Day 3 and others delay transfer until the blastocyst stage Each laboratory should determine their criteria for assessment of development and how to determine which embryos to transfer at a given time 2 There appears to be no agreement among laboratories with regard to the best day to transfer or which embryos to pick for transfer Numerous embryo evaluation systems have been reported in the literature and utilize a variety of parameters for assigning some type of value to the embryo s appearance Such criteria may include combinations of one or more of the following pronuclear alignment the presence or absence of a cytoplasmic halo surrounding the pronuclei early division at 25 27 hours cell numbers and fragmentation rates early on Day 2 multinucleation during the multinuclear stage cell numbers and fragmentation rates on Da
6. The formation of ice crystals could damage the embryos Allow the programmable freezing system to continue the freezing process to 35 Remove straws from the freezing unit and plunge into liquid nitrogen then store in the liquid phase of liquid nitrogen for long term storage Procedure for Thawing Early Cleavage Embryos 1 Remove the straw from the liquid nitrogen tank and hold in ambient tem perature 20 25 C in air for 30 40 seconds Then immerse the straw into a 30 35 C water bath for 40 50 seconds until all the ice has been melted Remove straw from water bath and wipe it gently with a sterile wipe Cut the plug end first with a pair of sterile scissors and then attach it to 1mL syringe or other expulsion device Bring the straw with syringe attached to the top of a sterile dish and then cut the heat sealed end Gently expel the contents of the straw as a single drop into the sterile dish and quickly locate the embryos An alternative to this method is to cut the sealed plug at the division between the two plugs Cut the heat sealed end insert a red plastic plunger and depress the innermost portion of the plug to the end The contents should be expelled onto the dry surface of a Falcon dish Locate the embryos in the drop pick up them up in a minimal amount of solution with a pulled sterile Pasteur Pipette or a sterile Stripper tip and transfer them to 2 3 mL of Embryo Thaw Medium T1 Incubate the embryos in T1 f
7. Federal Regulations CFR Title 21 Part 610 12 or current USP lt 71 gt recommendations For sterility testing random samples of every lot are filtered through 0 45 u membranes The membranes are incubated at 30 35 in Fluid Thioglycollate and at 20 25 in Soybean Casein Digest media and observed regularly for 14 days Any lot which shows growth over the course of the 14 days is retested If the sample is positive a second time the entire lot is rejected and will not be released for sale Mouse Embryo Testing Mouse embryo testing also referred to as the Mouse Embryo Assay or MEA is performed on gamete and embryo culture media as a functional test The test utilizes fresh one cell mouse embryos Aliquots of every lot are used to grow one cell mouse embryos to the blastocyst stage Test results indicate the percentage of mouse embryos developing to fully expanded blastocyst after 96 hours in culture Other biocompatibility testing such as sperm survival sperm motility recovery or blastocyst recovery assay is routinely performed on products where appropriate Any lot which fails one of these tests is retested If it again fails the lot of media is not released for sale Endotoxin Testing Endotoxin is measured using the LAL gel clot or kinetic chromogenic methodology Endotoxin testing is considered a biological test An aliquot of media is taken at the time of manufacture for Endotoxin testing Any products containing protein are heat treated
8. In the rare event that the pilot and target do not line up and the microscope has not been adjusted double click on the target with the computer mouse This unlocks the target and it can then be picked up and moved with the mouse Once aligned with the pilot Figure 2 the target is locked by double clicking on it again Turn off the pilot light using the same button as before The laser has 3 settings and these are selected by going back and forth with the arrows on the control unit Select the largest of the 3 settings The laser is now ready to use Figure 2 Make sure that the target is aligned with the pilot light This must be checked every time the laser is used 4 Take the plate containing the patient s embryos out of the incubator and move the embryos to be transferred to the central well using the Drummond pipette 5 Place the dish on the stage of the inverted scope and locate these embryos under low power 4x lens Rotate the laser lens into position and focus on the first embryo 6 The laser target should placed over the zona in a place that has no blastomere directly underneath The safety circle around the target indicates the area through which heat from the laser will be dispersed Blastomeres should be outside this circle see Figure 3 Figure 3 Position the target on the zona making sure that the safety circle does not overlap a blastomere Outer Cercle a Safety Zone Inmet a Hola Sire IrvineS
9. available in three formulations 1 ISolate 99264 is kit which provides upper and lower layer concentrations that ready to used without further dilution The ISolate concentration of the upper Layer is 5090 while the concentration of the lower layer is 9090 2 ISolate Stock Solution Cat 99275 is a 90 density gradient medium for use in a one step procedure or for further dilution 3 ISolate Concentrate Cat 99306 is the undiluted ISolate solution 100 which can be diluted to any final desired concentration Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Andrology Protocols Two Layer Discontinuous Gradient Separation Procedure This is the preferred method for preparing normozoospermic specimens 1 Bring all of the media to 37 C 2 If using dilutions other than the standard 50 and 90 layers prepare appropriately 3 Using a sterile disposable pipette transfer 1 5 2 0 mL of the lower layer into a sterile disposable 15 0 mL conical centrifuge tube 4 Using a new sterile pipette transfer an equal volume of the upper layer on top of the Lower layer This is done by contacting the surface of the lower layer at the side of the tube with the tip of the pipette Carefully dispense the upper layer by spiraling the pipette tip around the circumference of the tube in an upward motion as the level of the upper layer rises 5 Mix the liquefied semen well with a 5 0 mL or 10 0 mL volumetric pipet
10. choice D If sperm has been removed directly from the testes or the epididymus then potentially there may be very few sperm present in the sample and a simple wash will be sufficient to remove contaminating elements but retain the majority of sperm Each laboratory should have a procedure to determine which method of sperm preparation is appropriate for a specific patient This procedure detail the decision making process to identify the appropriate method for processing General Considerations 1 The patient should be informed ahead of time to have 2 3 days of sexual abstinence before the day of collection He should also be informed in writing about what he will be expected to do on the day of collection This is particularly important for patients who speal a language other than that primarily spoken by the laboratory personnel 2 All semen specimens should be accompanied by a test request form or have some form of documentation from the physician indicating the type of test processing that is to be done for the patient Appropriate documentation from the physician will save significant problems later if there is a discrepancy between what test the laboratory performs and what test the physician thought was going to be performed 3 For legal and liability purposes it is important to have a procedure in place to document the chain of custody for handling specimens in order to track each step in the specimen s movements within the laborato
11. drop into a sterile dish and quickly locate the blastocysts 7 Pick the blastocysts up in a minimal amount of solution and transfer them into 2 3 mL of Blastocyst Thaw Medium T1 for 10 minutes 8 Transfer the embryos in minimal amount of T1 into 2 3 mL of the Blastocyst Thaw Medium 12 for another 10 minutes 9 Wash the blastocysts by transferring them in a minimal amount of medium into 2 3 mL of Modified HTF Medium supplemented with 12mg mL HSA for 10 minutes at ambient temperature 10 Transfer into a dish with culture media and 12 mg mL HAS or 20 SSS until transfer VITRIFICATION OF OOCYTES EMBRYOS BLASTOCYSTS Vitrification is an ultra rapid freezing method in which human oocytes early cleavage embryos or blastocysts suspended in a high concentration of cryoprotectant solution are plunged directly into liquid nitrogen The thawing protocol is also an ultra rapid thawing method by removing the frozen specimens from liquid nitrogen and plunging directly into 37 C waterbath FREEZING OF OOCYTES AND EMBRYOS IRVINE SCIENTIFIC VITRIFICATION FREEZE KIT Cat 90133 The Vitrification Freeze Kit is intended for use in assisted reproductive procedures for vitrification of human oocytes MII and embryos 2PN to blastocyst The CryoTip the specimen carrier device is designed to be a closed system to prevent potential viral contamination of the oocytes embryos from liquid nitrogen as both ends of the device are securely sealed b
12. embryo cryopreservation outcomes Some QI programs can be conceived executed and completed within a few days Others may take months or even years to complete A comprehensive program detailing multiples levels of outcomes will be necessary for a lab to continually evolve to meet the challenge of creating the best possible opportunity for a patient to achieve a pregnancy A lab should at the very least have a program in place to verify all aspect of incubator function including monitoring and verification of media temperature within the incubator media pH sterility decontamination and cleaning gas levels and tank replacement and humidity control There should be a program to monitor ICSI fertilization and survival outcomes conventional insemination fertilization rates pregnancy outcomes cryopreservation outcomes and physician and scientist impact on transfer IrvineScientific www irvinesci com Rev 1 2 4 Embruologu Protocols ogy Protocols OVERVIEW General Considerations 1 Media that is manufactured by Irvine Scientific for use in the Embryology Laboratory has been quality control tested for pH osmolality sterility endotoxin and mouse embryo development prior to its release After receipt each ART lab may choose to perform additional QC testing at their own discretion 2 All media that is shipped cold should be stored in the refrigerator at the recommended temperatures until the discard date indicated on the label 3 M
13. extended storage up to 2 years Once the beads have been reconstituted refrozen and then thawed they should be used with no more than one subsequent refreeze and rethaw Therefore it is advisable to freeze the reconstituted reagent beads in small enough volumes that repeated freezing and thawing is not necessary Aliquot into 12 x 75 mm tubes each with 2 mg pl of stock solution and freeze 2 On the day of the test thaw one tube of each of the stock bead solutions IgG and IgA Add the contents of the tube to 10 mL of Buffer A in a separate conical centrifuge tube Centrifuge at 1000 x g for 10 minutes Decant the supernatant blot the rim and resuspend the final pellet in 0 2 mL of Buffer B 3 Prepare the sperm sample to be tested for the presence of antibodies Place the semen in a conical centrifuge tube and add sufficient amounts of Buffer A to bring the final volume to 10 mL Centrifuge at 600 x g for 10 minutes Discard the supernatant Wash the 0 sperm a second time with 10 mL Buffer A centrifuge and discard the supernatant Add a sufficient volume of Buffer B to bring the final concentration of sperm to 50 million motile sperm per mL Ideally the final dilution should yield approximately 10 20 mil motile sperm per mL 4 Begin testing by adding 5 pL of washed sperm suspension to 5 pL of each washed beads IgA and Ig Observe at 400X to 500X well cover with a cover slip and leave at room temperature in a moist chamber for up t
14. in boiling water for 2 minutes to denature the enzymes that interfere with the enzyme cascade reaction for endotoxin detection and then tested Any sample that is positive for endotoxin is retested If the sample fails again the lot of media is not released for sale pH Testing The pH of each lot of media is measured at a 1X concentration in accordance with finished product specifications Due to the constraints imposed during manufacture 10X and powdered media may require some adjustment when diluted or dissolved for filtration The pH readings reported on the Certificate of Analysis are indicative of the pH of the product immediately before capping and sealing and reflect the pH of the product at room temperature and under room air conditions The user is reminded that a medium in free exchange with CO in an incubator may establish a final pH different from the initial pH Each user should determine pH of the media in their own laboratory With pH sensitive cells the user should always check aliquots of the final media with a device specifically designed to measure pH such as a pH meter as visual estimation can be deceptive The pH of any sample is very sensitive to temperature and environmental CO and should be tested in such a way as to control for these variables Irvine Scientific recommends that the pH of a sample be tested in every incubator that is used for human embryo culture Furthermore we recommend that the CO of each incubator be adjust
15. medium dropwise until a 1 1 sample medium ratio is achieved Note Samples displaying high viscosity may require the additional step of passage through an 18 gauge needle or repeated pipetting to ensure thorough mixing Place the sample medium mixture into a beaker or other suitable container of 37 C water 0 000 20 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Cryopreservation Protocols 5 Place the container into the refrigerator at 2 C to 5 C and store for a maximum of 96 hours to allow a slow cooling of the mixture 0 5 C minute Thawing of Frozen Sperm General Considerations For raw semen that was cryopreserved the specimen should be washed using a density gradient For sperm that was processed using density gradient centrifugation prior to adding it to the Sperm Freezing Medium or Sperm Maintenance Medium the specimen may be thawed and then simply washed Thawing Procedure for raw semen 1 Remove frozen specimen s from storage tank thaw as follows For cryovials allow the vial to warm for at least 15 minutes to room temperature For cryostraws allow the straws to thaw for 2 minutes in room air Most laboratories use vials when freezing raw specimens and straw when routinely freezing processed specimens 2 While waiting for the specimen to thaw label a 15 mL Falcon tube with the patient name 3 Layer 1 5 2 0 mL of ISolate lower layer and then and equal volume of ISo
16. minutes 6 Load blastocysts into the pre rinsed straws by following the diagram shown in the embryo freezing procedure 7 Place the straws into the freezing chamber and start the freezing program 8 Cool straws from room temperature to 6 C begin the freezing program by loading the straws into a chamber that has been pre cooled to 6 C 9 Manually induce ice nucleation seed at 6 C by touching each straw near cotton plug with cold forceps Hold them at 6 C for a total of 10 15 minutes 10 Allow the programmable freezing system to continue the freezing process to 35 C 11 Remove straws and plunge into liquid nitrogen then store in the liquid phase of liquid nitrogen for long term storage Procedure for Thawing Blastocysts 1 Remove the straw from the liquid nitrogen tank and thaw them in ambient temperature 20 25 C in air for 30 seconds 2 Immerse straw in a 30 35 C water bath for 45 seconds until all the ice has been melted 3 Remove straw from water bath and wipe it gently with a sterile wipe Cut the plug end first with a pair of sterile scissors and then attach it to a 1mL syringe Or cut the plug half way up between the two sections of the plug and insert the red plastic plunger 5 Bring the straw to the top of a sterile dish and cut the heat sealed end 6 11 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Cryopreservation Protocols 6 Gently expel the contents of the straw as a single
17. nontraumatic catheters such as the Wallace Classic Embryo Transfer Catheter Cat ME1816 or ME1816N or Wallace SureView Embryo Transfer Catheter Cat CE118A or CE123A are ideal for embryo transfers HTF with or without HEPES Cat 90126 or 90125 and without protein is suitable for washing out the vagina and the cervix 10 20 mL of media should be warmed overnight by placing in an incubator in a container with the lid tightly capped if using a HEPES buffered media or with the lid loose if using one that is buffered with bicarbonate All embryo transfer dishes should be prepared at least 4 hours prior to transfer preferably on the afternoon of the day before transfer Transfers may be performed in Modified HTF Cat 90126 with protein HSA SSS or DSS if embryos are loaded into the transfer catheter outside of an IVF Chamber or in HTF Cat 90125 with protein HSA SSS or DSS or in the Complete Media series if the catheter is loaded inside a chamber A variety of protein concentrations have been reported in the literature ranging from 10 50 Each laboratory should decide which concentration is appropriate for its own use Once patients arrive in the room and in position the cervix may be visualized by inserting a speculum into the vagina The vagina may be cleaned using gauze or swabs and Modified HTE The cervix may be washed several times by flushing with media and a small catheter attached to a syringe until as much of the mucus is re
18. pipette Orient the embryo on the holding pipette so that the area at 3 o clock where the hatching will be done falls between two blastomeres or is adja cent to an area of fragmentation in the embryo 8 Lower the AH microtool while applying a slight amount of pressure to prevent oil from entering the AH pipette Align the AH pipette with the space between the blastomeres or next to the area of fragmentation 9 Begin to expel the solution onto the zona The outermost layer of the zona usually dissipates most easily While expelling the solution move the tip of the pipette up and down slightly to assist the removal of the zona and move in through the zona as it dissolves Move more carefully as the zona thins to avoid blowing large amounts of acidified Tyrode s into the interior of the embryo once the integrity of the innermost layer of the zona is breached The hole will look like a triangle with the widest part of the triangle at the outermost edge of the zona and the point being at the interior of the zona 10 Some embryologists choose to remove fragments from the interior of the embryo at the time of hatching This requires a steady hand and patience to prevent sucking up intact blastomeres as well as fragments but can be done at this time if the laboratory chooses 11 Once the zona has been breached and the fragments have been removed pull the pipette out Aspirate the immediate area adjacent to the hole in an effort to reduce continued e
19. storage of human sperm at 2 to 5 C before IVF or ICSI Sperm may be stored in this medium for a few hours up to 4 days 96 hr if maintained at 2 to 5 C It contains 20 v v egg yolk heat inactivated at 56 C for 30 minutes and gentamicin sulfate but does not contain glycerol This medium can also be used to capacitate sperm during a 2 hour incubation at room temperature SEMEN COLLECTION General Considerations Semen should be collected by masturbation following two to three days of abstinence Collection instructions should be given to each patient prior to collection of the specimen At the time of collection the laboratory should verify that the patient understands the instructions Patient identification at the time of collection should be carefully verified and documented If the patient has a partner she should also be identified Chain of custody documentation should be maintained in order to trace all steps and individuals handling the sperm during processing Patients should be assigned a unique accession number at the time of collection and that number should be used to identify all tubes and vials used during processing and for storage After collection the sample should be allowed to liquefy at room temperature or 37 C for 15 30 minutes and a semen analysis should be performed and documented to include the sperm counts motility and morphology Prior to mixing with liquefied semen one vial of the sperm cryopreservation medi
20. the tail and stabilize its movement in the injection pipette Some laboratories choose to inject testicular sperm tail first rather than head first into oocytes Multiple sperm may be pulled up into the ICSI pipette provided sufficient space between is provided to pre vent injecting multiple sperm into one oocytes 12 Raise the pipette slightly off the bottom of the dish and move to the first i injection droplet or the trough containing the oocyte s Lower the holding pipette and position the oocyte on the holding pipette so that the polar body is between 11 and 1 o clock or 5 and 7 Make sure that the oocyte is sitting on the bottom of the dish Bring the injection ICSI ipee down to the edge of the oocyte and check the position of the sperm Advance the sperm to the very tip of the needle IrvineScientific www irvinesci com Rev 1 5 4 Micromanipulation Protocols 13 Change magnification to 400X and focus on the oolemma Bring the injection pipette into focus Insert the injection pipette at the 3 o clock position into the oocyte to approximately 3 4 of its diameter or until the membrane pops or gives indicating that it has been punctured Aspi rate a small portion of the cytoplasm to further ensure that the oocyte membrane has been broken Aspiration of the cytoplasm is critical for oocyte activation 14 Inject cytoplasm and sperm back into the oocyte Place the sperm head slightly outside the injection path this helps to hold the sperm i
21. then plunged into the liquid nitrogen Exposure to VS loading tip and plunging in LN2 should be completed within 90 seconds 3 SA Wm wi lk ES VSI VS2 V53 V54 5 5 fowd L deep FIGURE 4 IrvineScientific www irvinesci com Cryopreservation Protocols 6 17 5 Load and heat seal the CryoTip as follows While observing under the microscope carefully aspirate a small volume of VS to the Ist mark on the Cryolip Continue observation under the microscope and gently aspirate the specimen with VS to the 2nd mark on the CryoTip Now observe the CryoTip directly from the side and aspirate more VS to the 3rd mark Heat seal the CryoTip on the 1st mark with the heat sealer set on 2 3 carefully slide the metal cover sleeve toward the fine tip end to cover the fine tip completely remove the aspiration syringe and then heat seal at the other thick end of the CryoTip above the 4th mark with the heat sealer set at 5 6 6 Attach the 1 2 cc cryostraw at this time if using it Hold the covered CryoTip with tweezers and immerse it metal end first directly into liquid nitrogen 7 Place the covered tip into the submerged liquid nitrogen filled 3 6 mL cryotube or goblet on the cryocane 8 Repeat these steps for remaining oocytess to be vitrified 9 Move the liquid nitrogen reservoir close to the liquid nitrogen tank and transfer the cryocane and its contents into the liquid nitrogen tank for long term storage
22. to the Falcon tube or cut the cryostraw and allow contents to drain into the Falcon tube 3 Using another sterile pipette add 3 mL of Sperm Washing Medium 4 Centrifuge the tube at 300 x g for 10 minutes 5 Then remove the supernatant with a clean pipette leaving about 500 pL of media and the sperm pellet Discard supernatant and resuspend the pellet in 3 mL of Sperm Washing Medium Using a sterile pipette tip perform count and motility on specimen and record values 7 Place the specimen in a warming block or in a 377C incubator with the lid tightly closed until time for insemination EMBRYO CRYOPRESERVATION Successful embryo cryopreservation is an important tool for the IVF laboratories in order to maximize a patient s IVF cycle The storage of excess embryos for future transfer allows multiple transfers from one stimulation cycle thereby reducing the monetary cost to the patient and reducing the potential for complications associated with repeated drug stimulation cycles The embryo cryopreservation procedure includes exposure of the embryos to cryoprotectants cooling to sub zero temperatures storage in liquid nitrogen tanks and the later thawing procedures The resulting pregnancy rates from the transfer of cryopreserved embryos has often been shown to be comparable to that of fresh transfers IRVINE SCIENTIFIC CROPRESERVATION MEDIA FOR EMBRYOS Irvine Scientific s Cryopreservation Media has been specially formulated for the c
23. 0 3 An SAL of 10 3 allows for no more than one bottle in one thousand to fail sterility testing This level of SAL is determined by a filling validation test A test run of three thousand bottles of media are tested using Trypticase Soy Broth Samples are taken from each of the 3000 bottles and tested for microorganism growth If more than three samples show the presence of any organism capable of growing in TSB the filling process must be re evaluated and re validated internally Once a lot of media has been manufactured the finished product testing is performed including all of the standard quality control procedures required to complete the Certificate of Analysis that accompanies every lot of media manufactured in Irvine Scientific s facility All final product testing is standardized and performed in accordance with written Standard Operating Procedures Powdered Media Bulk powered media are prepared in accordance with the corresponding original formula for the liquid when feasible Anhydrous salts are substituted on an equimolar basis The final powdered formulation is packed in screw capped containers and should be stored in controlled conditions according to product labeling Each lot of powdered media is checked for particle size uniformity and complete solubility Irvine Scientific pays particular attention to maintaining rigorous quality standards to ensure thorough distribution of trace compounds in powdered media Chemicals All raw mate
24. BRYO FREEZE MEDIA POLYVINYLPYRROLIDONE PVP SOLUTION 7 EMBRYO THAW MEDIA HTF MEDIUM WITH GENTAMICIN MODIFIED HTF MEDIUM WITH GENTAMICIN HEPES FREEZING MEDIUM TEST YOLK BUFFER TYB WITH GLYCEROL amp GENTAMICIN REFRIGERATION MEDIUM TEST YOLK BUFFER TYB WITH GENTAMICIN VIT KIT FREEZE 90133 STARTER VIT KIT FREEZE STARTER 90137 VIT KIT THAW 90137 STARTER VIT KIT THAW STARTER 90138 90139 90142 90143 99109 99140 99141 99168 99175 99176 99193 99219 99242 99252 99264 7 3 EARLY CLEAVAGE MEDIUM ECM MULTIBLAST MEDIUM COMPLETE EARLY CLEAVAGE MEDIA with DSS COMPLETE MULTIBLAST with DSS MODIFIED HAM S F 10 WITH ALBUMIN COMPLETE EARLY CLEAVAGE MEDIA ECM with SSS COMPLETE MULTIBLAST with SSS MODIFIED HAM S F 10 BASAL MEDIUM HEPES MODIFIED HAM S F 10 BASAL MEDIUM SPERM MAINTENANCE MEDIUM SERUM SUBSTITUTE SUPPLEMENT POLYVINYLPYRROLIDONE PVP LYOPHILIZED P 1 MEDIUM WITH GENTAMICIN TYRODE S SOLUTION ACIDIFIED ISOLATE Catalog Number Description 99275 ISOLATE STOCK SOLUTION 99306 ISOLATE CONCENTRATE 99311 POLYVINYLPYRROLIDONE PVP SOLUTION 1096 CEIIBA SUREVIEW EMBRYO TRANSFER CATHETER 18 cm CEI23A SUREVIEW EMBRYO TRANSFER CATHETER 23 cm CF4I8A SUREVIEW TRIAL TRANSFER CATHETER 18 cm CE423A SUREVIEW TRIAL TRANSFER CATHETER 23 cm E 101 EPPENDORF TRANSFER TIP RP ICSI MICROCAPILLARIES E201
25. EPPENDORF VACUTIP HOLDING MICROCAPILLARIES 15 300 CENTRIFUGE IS 325A CENTRIFUGE TUBE IS 360 MAKLER INSEMINATION DEVICE IS 360RSB REPLACEMENT SLIDING BRACKET IS 360RSH REPLACEMENT SYRINGE HOLDER IS361A SHORT INSEMINATION CANNULA 15 mm IS 362A LONG INSEMINATION CANNULA 15 mm IS 363 MAKLER COUNTING CHAMBER KIT 19363 CG REPLACEMENT CHAMBER GRIP 15 364 REPLACEMENT COVER GLASS WITH GRID 15 365 MAKLER COUNTING CHAMBER INVERTED 15 366 REPLACEMENT COVER GLASS INVERTED 15 373 MAKLER COUNTING CASA 15 374 REPLACEMENT COVER GLASS CASA MEI816 EMBRYO TRANSFER CATHETER 18 cm MEI816N EMBRYO TRANSFER CATHETER 23 cm MEI816NST RIGID 23 cm MEI816ST STYLET RIGID 18 cm ME2316 EMBRYO TRANSFER CATHETER 23 cm 8 INTRAUTERINE INSEMINATION CATHETER 18 cm MEONS1633 16g SINGLE LUMEN NEEDLE 33 16335 16g SINGLE LUMEN NEEDLE 33 cm MEONSI733 175 SINGLE LUMEN NEEDLE 33 MEONSI7338 17g SINGLE LUMEN NEEDLE 33 cm METTI816 TRIAL TRANSFER CATHETER 18 cm METTI81GN TRIAL TRANSFER CATHETER 23 cm NI76740 TCMULTIDISH N366656 1 CRYOTUBE 1 800 577 6097 949 261 7800 IrvineScientific IrvineScientific Irvine Scientific 2006 Grow With Us P N40833 Rev 0
26. IEW TRIAL TRANSFER CATHETER 18 cm SUREVIEW TRIAL TRANSFER CATHETER 23 cm TC MULTIDISH TRIAL TRANSFER CATHETER 18 cm TRIAL TRANSFER CATHETER 23 cm TYRODE S SOLUTION ACIDIFIED VIT KIT FREEZE 90133 STARTER VIT KIT FREEZE STARTER 90137 VIT KIT THAW 90137 STARTER VIT KIT THAW STARTER 9307 WATER FOR ASSISTED REPRODUCTIVE TECHNOLOGIES ART USE Rev 1 NUMERICAL INDEX Catalo Numb 1092 9235 9301 9305 9307 9910 9922 9926 9983 9984 9988 9999 15375 15376 15377 15378 15380 15381 15391 40709 40736 90101 90103 90108 90110 90116 90119 90124 90125 90126 90128 90129 90133 er Description BOVINE ALBUMIN FRACIION V POWDER PBS IX DULBECCOS PHOSPHATE BUFFERED SALINE SOLUTION DEXTRAN SERUM SUBSTITUTE OIL FOR EMBRYO CULIURE WATER FOR ASSISTED REPRODUCTIVE TECHNOLOGIES A R T USE COMPLETE 1 MEDIA with DSS COMPLETE HTF MEDIA with SSS COMPLETE P 1 MEDIA with SSS SPERM WASHING MEDIUM MODIFIED SPERM WASHING MEDIUM HUMAN SERUM ALBUMIN SOLUTION BOVINE SERUM ALBUMIN SOLUTION IMMUNOBEAD RABBIT ANTI HUMAN IgG A IMMUNOBEAD RABBIT ANTI HUMAN IgA IMMUNOBEAD RABBIT ANTI HUMAN IgM IMMUNOBEAD RABBIT ANTI HUMAN Ig IMMUNOBEAD BUFFER A IMMUNOBEAD BUFFER B ACTIVATED IMMUNOBEAD MATRIX ACTIVATED IMMUNOBEAD CRYOTIP CONNECTOR HYALURONIDASE SOLUTION EMBRYO BIOPSY MEDIUM BLASTOCYST FREEZE MEDIA BLASTOCYST THAW MEDIA EM
27. L of antibody positive serum and 200 pL of Buffer B in a 12 x 75 mm tube Incubate for 1 2 hours at 37 C At the end of the incubation time wash the sperm with Buffer B twice and resuspend in 400 pL of Buffer B Combine 5 uL of this sperm with 5 uL of Immunobeads of each type as described above and assess the percent of motile sperm bearing beads A negative control should also be performed Frozen thawed sperm from a known negative donor may be used as described for the test sample Similarly if using co inubation with serum to create a positive control a negative control may be performed simultaneous by co incubating donor sperm with a known antibody negative serum in the same way as described for the positive control INDIRECT IMMUNOBEAD ASSAY The indirect assay is used when testing samples that do not contain sperm such as serum follicular fluid or seminal plasma li Sample Collection Processing a Serum should be collected from the patient in question in a sterile red top tube no anti coagulant allowed to clot and then centrifuge at 3000 rpm for 10 minutes Transfer serum to tubes and heat inactivate complement 56 C for one hour Store frozen until needed for test b Seminal plasma After routine sperm collection the sample should be centrifuged at 600 x g for 10 minutes to remove sperm and other cells and the supernatant collected It should then be filtered through a 0 4 um filter to remove any remaining sperm and debri
28. OCYST FREEZE MEDIA 90110 BLASTOCYST THAW MEDIA 1092 BOVINE ALBUMIN FRACTION V POWDER 9999 BOVINE SERUM ALBUMIN SOLUTION 15 300 CENTRIFUGE IS 325A CENTRIFUGE TUBE 99140 COMPLETE EARLY CLEAVAGE MEDIA ECM with SSS 90142 COMPLETE EARLY CLEAVAGE MEDIA ECM with DSS 9922 COMPLETE HTF MEDIA with 555 99141 COMPLETE MULTIBLAST with SSS 90143 COMPLETE MULTIBLAST with DSS 9926 COMPLETE P 1 MEDIA with SSS 9910 COMPLETE P 1 MEDIA with DSS 40736 CONNECTOR 40709 CRYOTIP N366656 l CRYOTUBE 9301 DEXTRAN SERUM SUBSTITUTE 90138 EARLY CLEAVAGE MEDIUM ECM 90103 EMBRYO BIOPSY MEDIUM 90116 EMBRYO FREEZE MEDIA 90124 EMBRYO THAW MEDIA MEI816 EMBRYO TRANSFER CATHETER 18 cm ME1816N EMBRYO TRANSFER CATHETER 23 cm ME2316 EMBRYO TRANSEER CATHETER 23 cm E 101 EPPENDORF TRANSFER TIP RP ICSI MICROCAPILLARIES E 201 EPPENDORF VACUTIP HOLDING MICROCAPILLARIES 90128 FREEZING MEDIUM TEST YOLK BUFFER TYB WITH GLYCEROL amp GENTAMICIN 90125 HTF MEDIUM WITH GENTAMICIN 9988 HUMAN SERUM ALBUMIN SOLUTION 90101 HYALURONIDASE SOLUTION 15380 IMMUNOBEAD BUFFER A 15381 IMMUNOBEAD BUFFER B 15378 IMMUNOBEAD RABBIT ANTI HUMAN Ig H L 15376 IMMUNOBEAD RABBIT ANTI HUMAN IgA 15375 IMMUNOBEAD RABBIT ANTI HUMAN IgG A 15377 IMMUNOBEAD RABBIT ANTI HUMAN IgM t MEAICI8 INTRAUTERINE INSEMINATION CATHETER 18 cm 99264 ISOLATE 99306 ISOLATE CONCENTRATE 99275 ISOLATE STOCK SOLUTION IS 362A LONG INSEMINATION CANNULA
29. a m to 5 00 p m Pacific Time Customer Service 800 577 6097 Direct Line Technical Service 800 437 5706 Corporate Offices 800 437 5706 Telephone 949 261 7800 Facsimile 949 261 6522 24 hours International Orders All international orders must be pre paid prior to shipment Payment must be made in U S Funds drawn on a 5 Bank or by direct wire transfer to MELLON BANK Pittsburgh PA Reference ABA 4043000261 and indicate for credit to MERRILL LYNCH Account 1011730 for further credit to Irvine Scientific Sales Co Inc Account 27224 04244 Swift MELNUS Please add 20 to your wire transfer amount to cover bank fees An additional export documentation fee will be added to orders shipped outside of the United States Rev 1 1 800 577 6097 949 261 7800 IrvineScientific General Information GENERAL INFORMATION Corporate Offices Irvine Scientific 2511 Daimler Street Santa Ana CA 92705 5588 Phone 800 437 5706 Customer Support Customer Service 800 577 6097 Technical Service 800 437 5706 Telephone 949 261 7800 Facsimile 949 261 6522 Lot Reservation Policy Samples will be provided at no charge for the purpose of testing a particular lot Reserved lots will be held for three weeks from the date the sample is shipped Upon completion of your testing a purchase order for the amount reserved is required to secure product The reserve will be automatically cancelled at the end of the three week
30. ased that is already supplemented Cat 9922 Complete HTF Medium with SSS Cat 9926 Complete P 1 with SSS Cat 9910 Complete P 1 with DSS Cat 90140 Complete ECM with SSS Cat 90142 Complete ECM with DSS If the media does not have protein already added Cat 90125 HTF Cat 99242 P 1 Medium Cat 90138 ECM Human Serum Albumin Cat 9988 HSA Synthetic Serum Substitute Cat 99193 SSS or Dextran Serum Substitute Cat 49301 DSS should be added Recommended levels are 5 mg mL v v of HSA 10 v v SSS or 10 v v DSS as the additives for oocyte culture or sperm washing Higher concentrations may be used for freezing specimens up to 12 HSA 20 SSS or 20 DSS This higher concentration of protein may improve cryosurvival of specimens 18 Once protein has been added to make a stock the stock should be kept in the refrigerator and is stable for up to 4 weeks or until expiration date 19 There is little agreement in the literature regarding universally recognized methods for performing IVE Each laboratory should develop their own procedures and document those procedures in a Procedure Manual that is kept current and reviewed by the Labortory Director on an annual basis The following is a general overview 3 3 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific 090 Protocols OOCYTE RETRIEVAL General Considerations In most IVF programs oocytes are collected from patients who have undergone ovarian stimulation
31. bes are numbered to receive the cells and loaded with 5uL lysis buffer tubes and buffer are supplied by Dr Hughes in advance of the case Each blastomere will need one tube and it is usual to create a blank tube buffer without a cell to be tested in parallel with the blastomere Label the tubes 1 1B 2 2B 3 3B and so on accounting for the number of embryos to be biopsied and also allow for extra blastomeres A patient with 12 embryos therefore might have 12 blastomere tubes 12 blank tubes and 4 6 extra tubes all labeled and containing bufter Figure 6 Insert the biopsy pipette through the zona hole and gently aspirate a blastomere either fully or partially into the pipette Drag the blastomere out of the embryo For cytogenetic aneuploidy cases skip to step 24 The blastomere should be washed twice in wash buffer and placed in the tube of lysis buffer The embryologist performing this part of the procedure should be careful to observe the cell clearly going into the buffer If the cell is not clearly seen going into the tube or if the cell is observed to have an abnormal number of nuclei a second cell should be biposied from the embryo Using the medium remaining in the pipette after depositing the blas tomere open the blank tube and deposit about the same volume of medium as the cell containing tube received Both tubes should then be tightly capped and placed in the freezer Complete the paperwork and tracking documentati
32. ble 15 0 mL conical centrifuge tube 4 Mix the liquefied semen well with a 5 0 mL or 10 0 mL volumetric pipette 5 Gently place 0 5 2 0 mL of the liquefied semen onto the lower layer using a new sterile pipette If the total volume of semen is more than 2 0 mL then use the appropriate number of tubes and distribute the final semen volume accordingly Each tube of lower layer can be used to process up to 2 mL of semen Additional volumes require additional tubes 6 Centrifuge for 20 minutes at 300 x g 7 At the completion of the spin remove the layer by inserting a clean 5 mL pipette tip just below the surface of the liquid Hold the tip in this position during aspiration Aspirate the layers without disturbing the sperm pellet at the bottom of the tube until approximately 0 5 mL of lower layer remains Even if a sperm pellet is not visible this volume should contain sperm If the sperm pellet occupies more than 0 5 mL at the bottom of the tube aspirate as much liquid from above the pellet as possible but leave the pellet intact 8 Using a new sterile pipette add 2 0 3 0 mL of Sperm Washing Medium Cat 49983 or Modified Sperm Washing Media Cat 9984 to the tube and resuspend the pellet Centrifuge at 300 x g for 10 minutes Remove the supernatant with a clean pipette add 2 3 mL of Sperm Washing Media again and centrifuge 9 After the second wash discard the supernatant and resuspend the pellet in 0 25 0 5 mL of Sperm Washing Medium if
33. cavity of the embryo before freezing also improves survival of the embryo Collapsing the cavity may be done by using a PZD needle and inserting it from right to left through the blastocyst in an area removed from the inner cell mass Another method that has been employed is to pull the blastocyst up and down in decreasing sizes of stripper tips during the incubation in the first freezing media solution Procedure for Freezing Blastocysts 1 Prepare and label sterile non toxic straws for freezing by rinsing them with the Blastocyst Freeze Medium F2 2 Bring the freezing media to room temperature 20 25 and aliquot 1mL of each medium F1 and F2 into pre labeled independent culture dishes or wells Some programs have reported that they begin blastocyst freezing with the first solution at 37 C once the blasts have been placed in this solution the temperature is then allowed to come to room temperature This may expedite absorption of the glycerol into the expanded blastocoel 3 Chose the fully expanded high quality blastocysts for freezing 4 Transfer blastocysts to be frozen recommend 2 blastocysts straw in minimal volume of culture medium into the F1 medium for 10 minutes The blastocysts will initially shrink and then return to the normal size af ter equilibration unless the blastocoel has been artificially collapsed In this case re expansion should not be observed 5 Transfer blastocysts to the Blastocyst Freeze Medium F2 for 10
34. ce there is some data to suggest that the intracellular pH of embryos is approximately 7 12 Measuring pH with the classic pH meter is difficult in an IVF setting as pH of embryo culture media changes rapidly following exposure to room air and room temperatures Therefore the most appropriate measurement of pH would be to determine the pH of the media in situ in the incubator Since most laboratories do not have the kind of equipment necessary for this kind of analysis it is recommended that pH be tested in way that accounts for the temperature and sensitivity of the media One method that has been used to measure the pH of the media is a blood gas analyzer Other methods are available and any method is suitable as long as it takes into account temperature and pH drift 7 Adjusting the pH of the media by the addition of acids or bases is not recommended Instead adjust pH by adjusting the CO setting of the incubator 8 The CO levels in the incubator should be adjusted to attain the pH that is determined to be appropriate by the laboratory Increasing the CO percentage in the incubator will decrease the pH of the media and decreasing the CO levels will raise the pH Some laboratories use reduced levels of oxygen for culture The lower levels of in the incubator will not affect pH of the media Each laboratory should determine the incubator setting that produces the desired pH for that incubator It is important to understand that ever
35. ced in a CO incubator 10 The chosen method to collect and fertilize oocytes may differ between labs Options include test tubes multi well dishes with or without an oil overlay or microdrops under oil Each laboratory should decide which system works best in their hands 11 Opinions vary regarding whether oocytes should be fertilized and cultured in groups or individually Both methods have been shown to work effectively The most commonly used methods appear to be IrvineScientific www irvinesci com Rev 1 3 4 Embryology Protocols a Group culture Group culture in 5 pL drops of media under 8 10 mL oil overlay in a dish b Group culture Group culture in 500 1000 pL of media under 300 pL of oil overlay in multiwell dish c Individual culture Individual oocytes cultured in 10 20 pL of media under an oil overlay in a dish Therefore each lab should determine which system works best in their facility OOCYTE INSEMINATION FERTILIZATION General Considerations There is debate regarding the ideal time to inseminate oocytes after retrieval for optimal fertilization rates Inseminating human oocytes efficiently and in a timely manner is fundamental to achieving successful fertilization Some claim that oocytes require at least 2 5 hours in a CO incubator at 37 C before performing conventional insemination Other laboratories have documented good fertilization rates with ICSI of mature eggs when retrieval cleaning and injection is pe
36. ch as prostaglandins which are capable of initiating intense uterine contractility if introduced directly into the uterine cavity during the course of an IUI Seminal plasma also contains factors that inhibit the final capacitation and activation of the sperm and appears to contain elements that may be embryotoxic Therefore washing of semen is required to remove seminal plasma and it s associated capacitation inhibiting factors and to remove dead sperm and any contaminating white cells present in the specimen prior to introducing the sperm into the uterus during IUI or exposing oocytes to the sperm during or ICSI Determining the method of sperm preparation prior to IUI or IVF depends on the quality and source of the specimen A If the specimen is normal in terms of sperm count and motility or if it has significant amounts of seminal debris and dead sperm then the density gradient preparation is recommended B A swim up preparation may also be done if the ejaculated specimen is normal in terms of counts and motility and if the specimen is not highly viscous or if significant agglutination is not present Recovery of sperm from swim up preparations is highly variable and more time intensive Hence swim up preparations tend to be used less often for IUI and more often for IVF where high numbers of sperm are not required C If the specimen has low sperm concentration and low sperm motility then a single or double wash should be the method of
37. cientific www irvinesci com Rev 1 5 8 Micromanipulation Protocols 7 lf it is not possible to place the target over an area of zona without the safety circle overlapping a blastomere consider the following alternatives a Change to a lower power setting on the laser and make 2 or 3 small holes in the zona instead of one b Using any laser setting thin the zona as much as can be achieved without going to close to a blastomere Thinning should be done by using one two or at most 3 pulses from the laser and the pulses should overlap c Discuss the issue with the physician and explain that it is not possible to hatch a particular embryo without risk of damage 8 Hatch each embryo in turn by breaching the zona with a single pulse from the laser If the zona is particularly thick 2 pulses might be needed to make a hole one hitting the inner zona and one outside the first hole on the outer zona 9 Hatching of each embryo in turn should be accomplished quickly and efficiently Find a target site on the zona fire the laser and move on immediately to the next embryo 10 After all embryos have been hatched they can be moved to the transfer dish C Embryo Biopsy General Considerations 1 Embryo biopsy is the procedure where one or two blastomeres are removed from a 6 8 cell embryo for Preimplantation Genetic Diagnosis PGD The procedure is performed just before compaction of the blastomeres 2 Patients having In Vitro Fertiliza
38. cycle Glass Iype 1 Borosilicate Not recommended for re use Not currently recyclable Plastic PETG Not recommended for re use Disinfected bottles can be recycled with Plastic PET consumer PET soda bottles Incineration results in CO 0 Plastic HDPE Not recommended for re use Disinfected bottles can be recycled with consumer HDPE Bubble wrap PE Re use as packaging material Contact Recycle Hot Line 800 944 8448 for the nearest collection site Polystyrene Foam EPS Deliver to commercial packaging Contact Recycle Hot Line 800 944 8448 CFC Free outlets i e Mail Boxes Etc for re use for the nearest collection site 1 4 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific General Information Irvine Scientifics Global Presence ARGENTINA AUSTRALIA AUSTRIA BAHRAIN BELGIUM BOLIVIA BRAZIL CANADA CHILE CHINA COLOMBIA COSTA RICA CYPRUS CZECH REPUBLIC DENMARK DOMINICAN REPUBLIC ECUADOR EGYPT FINLAND FRANCE GERMANY GREECE GUAM GUATEMALA GUYANA HONDURAS HONG KONG HUNGARY INDIA INDONESIA IRELAND ISRAEL ITALY JAMAICA JAPAN JORDAN KOREA KUWAIT LEBANON LUXEMBOURG MALAYSIA MEXICO NETHERLANDS NEW ZEALAND NIGERIA NORWAY PANAMA PARAGUAY PERU PHILIPPINES POLAND PORTUGAL PUERTO RICO RUSSIA SAUDI ARABIA SINGAPORE SOUTH AFRICA SPAIN SWEDEN SWITZERLAND TAIWAN THAILAND TURKEY U A E UKRAINE UNITED KINGDOM UNITED STATES URUGUAY VENEZUELA For the local distributor of Irvine Scientific products in your country please contact our International Custome
39. d as biohazardous and the appropriate precautions taken EMBRYO BIOPSY PROCEDURE 1 Prepare the embryo biopsy dishes with 4 x 25 pL drops of Embryo Biopsy Medium and overlay with oil The dishes should be clearly labeled with the patient s name and the type of medium in the dish Locate the drops centrally in the dish and place in the non gassed 37 C incubator to warm up for at least 1 hour The dishes can also be made the day prior to the biopsy procedure 2 Setup the microscope with the biopsy pipette diameter 35 um on the right manipulator Attach a holding pipette on the other manipulator such that it opposes the biopsy pipette exactly Align the pipettes such that they will both have good access to any embryo placed on the stage The pipettes should also be checked for range of motion i e don t have them at the end of their range while working on an embryo 3 Prepare the paperwork for the case i 4 Score the embryos in the normal way It is usual to do the biopsies starting with the embryo with the fewest intact cells working through to the best embryos last Also it is often a good idea to biopsy an abnormal or degenerating embryo first to ensure that the biopsy can be performed easily This allows for problems to be identified and solved before beginning work on the viable embryos 5 Set up the inverted microscope turn it on and check the temperature of the stage If not already on turn on the computer to which the laser and micro
40. drop is added so it equilibrates adeguately After the entire volume of freezing medium has been added to the semen specimen allow the mixture to equilibrate for 3 minutes Transfer aspirate the final mixture into the cryostraws or cryovials seal and start the freezing process The freezing process from room temperature 20 25 C to 80 C can be accomplished either by a programmable freezing system or by manually assisted vapor phase cooling For programmagle freezing systems use according to the manufacturers instruction manual For the manually assisted vapor phase cooling method the cryostraws cryovials should be attached to the cryocane and placed in the refrigerator 2 5 C for one hour then exposed to liquid nitrogen vapor for 1 5 2 0 hours by either suspending them in the liquid nitrogen stor age tank above the liquid level or by placing them in the vapor phase in a small temporary liquid nitrogen dewar The final step should be to transfer the cryostraws cryovials quickly onto a labeled cane and then into the liquid nitrogen tank for storage Sperm Maintenance Medium Cat 99176 General considerations Store Sperm Maintenance Medium with Glycerol at 10 C or colder Do not expose medium to repeated freeze thaw cycles If smaller aliquots are desired thaw the product aliquot working volumes into sterile labeled containers and freeze until needed When stored as directed Sperm Maintenance Medium is stable until the expirat
41. e Complete Media series the tubes bottle should be loosely capped The type of media selected will be determined by whether retrieved oocytes are placed in an IVF chamber or closed in room air If the aspiration tubes are constantly maintained in a CO environment then a media containing bicarbonate buffer may be used If the tubes are transported to the laboratory and examined under room air then a HEPES buffered medium mHIF should be used for oocyte collection The media used for oocyte retrieval does not require protein supplementation as follicular fluid contains significant levels of protein but a Complete Medium containing protein may be use if determined to be advantageous by the laboratory Heparin may be added to the media 2 5 10 0 Units mL that is used for follicular aspiration at the discretion of the lab 5 Verify patient name prior to beginning an oocytes retrieval procedure Rinse the aspiration needle lumen and tubing with media mH TF and protein and discard the rinse media 7 Examine follicular aspirates as quickly as possible following collection after pouring the aspirate into a pre warmed collection dish Identify and rinse oocytes 8 Once the oocytes have been aspirated located and rinsed they should be transferred to media containing a bicarbonate buffer HTE P 1 or ECM 9 Media should be supplemented with protein 5 mg mL HAS 1096 v v SSS or 1096 v v DSS Complete Media which is already supplemented may be pla
42. e aggressively inactivated rather than injected into the oocytes while they were still moving Although some laboratories have managed to perfect the art of catching and inactivating a sperm without the use of additives or viscous solutions the most common method used to immobilize the sperm and slow its motility down is by suspending the sperm in Polyvinylpyrrolidone PVP solution Semen should be processed in the usual manner either through swim up density gradient centrifugation or washing and the resulting motile sperm fraction should be resuspended in Sperm Washing Medium HTF P 1 or ECM supplemented with protein HSA SSS or DSS Sperm that has been collected by surgical means such as testicular biopsy or epididymal aspiration may be low enough in count and or motility that very little processing should be done prior to picking up the sperm for ICSI INTRACYTOPLAMIC SPERM INJECTION 1 When doing ICSI the microscope stage should be heated and main tained at 37 C 2 The ICSI micropipettes along with any other micro tools should be kept sterile Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Micromanipulation Protocols 3 Setup the scope the microinjectors and the micro tools in advance aligning the tips of the micro tools in the center of the field of view Raise the tools until ready to use them or place a holding dish on the scope with PVP in a droplet in the center of a Falcon 1006 lid covered with oil Lower th
43. e blastomere does not necessarily have to be aspirated completely into the pipette Often having just 1 3 of or 1 2 of the blastomere in the pipette gives enough of a hold to drag the blastomere out of the embryo see Figure 6 gt Figure 5 A triangular shaped hole made with 3 laser pulses is often useful for guiding the biopsy pipette into the embryo The first and central pulse is the only one to breach the inner zona Two additional pulses flank the first hole and breach only the outer zona 14 Drop the aspirated blastomere next to the embryo and examine If the blastomere has a clear nucleus and is not lysed it can be given to the embryologist preparing the slides tubes for the genetics lab If not repeat step 13 IrvineScientific www irvinesci com Rev 1 5 12 Micromanipulation Protocols 19 16 17 18 19 20 2 8 22 23 24 Continue to biopsy the embryos until each one has resulted in a blastomere with nucleus that is acceptable to the embryologist preparing the cells for analysis In some cases this individual may ask you to re biopsy an embryo if they are not happy with what they received For example the individual fixing the cells in a cytogenetics case may fail to get the nucleus fixed onto a slide and ask you for another cell from that embryo In cytogenetics cases 5 10 blastomeres will be fixed and numbered on individ ual glass slides In molecular genetics cases the PCR tu
44. e injection needle into the PVP and allow it to equilibrate The holding pipette may be equilibrated in the oil 4 It appears as though injecting immature oocytes those that have not completed the first round of metaphase and extruded the first polar body may result in embryos that are genetically abnormal if they fertilize at all Therefore it is advisable to only inject oocytes with a clearly visible polar body Metaphase II The timing of the procedure varies between laboratories throughout the world and no standard timing has been established for ICSI following oocytes retrieval Therefore each laboratory should standardize the timing of the injection within its own facility Ihe most common time for injection appears to be around 38 40 hours post hCG administration 5 Prepare the ICSI dish Most laboratories have used the lid to the Falcon 1006 dishes due to their low clearance and depth However extended culture in these lids should not be done for any reason as they have not been treated in the same manner as the bottoms of the dishes by the manufacturer and there has been some evidence that the lids to these dishes may be embryo toxic following extended exposure A time should be specified regarding what is considered extended 6 The most common method for setting up a dish is to place 5 10 pL drop of PVP into the center of the ICSI dish and then to surround the PVP drop with a ring of 5 10 pL droplets of HEPES buffered media with
45. eScientific www irvinesci com Rev 1 6 2 Cryopreservation Protocols IRVINE SCIENTIFIC SPERM CRYOPRESERVATION MEDIUM Media containing TEST yolk buffer TYB have been shown to be effective in preserving sperm for short term storage when refrigerated and for long term storage with the addition of glycerol as a cryoprotectant when frozen For long term storage Freezing Medium Test Yolk Buffer T YB with Glycerol amp Gentamicin Cat 90128 for long term freezing and storage of human sperm This medium contains 12 v v glycerol and 20 v v heat inactivated egg yolk inactivated at 56 C for 30 minutes as well as 10 pg mL gentamicin sulfate Sperm Maintenance Medium Cat 99176 for long term cryopreservation of human sperm This medium contains a higher concentration of glycerol than Sperm Freezing Medium with 28 v v glycerol and has 20 mg mL of Human Serum Albumin also added but does not contain egg yolk or antibiotics It may be used in place of Sperm Freezing Medium TYB The formulation of Sperm Maintenance Medium with Glycerol is a modified version of Human Sperm Preservation Medium HSPM that was first described by Mahadevan and Trounson in 1983 The major formula tion changes included lowering the osmolarity stabilizing the buffer system and increasing the albumin and glycerol concentrations For short term storage and transport Sperm Refrigeration Medium Test Yolk Buffer TYB with Gentamicin Cat 90129 for short term
46. ed individually based on the detected pH of the sample tested in that incubator Osmolality Osmolality of each lot of media is determined either by the freezing point or vapor pressure methodology Testing is performed on precalibrated instrumentation using specific Standard Operating Procedures to ensure accuracy and consistency Certificate of Analysis C of A A Certificate of Analysis reporting all of the QC results is provided with each shipment Results are reported for each lot Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Quality Control Program QUALITY CONTROL PROGRAM Each ART laboratory should have detailed Quality Control Quality Assurance and Quality Improvement programs in place A Quality Control program is a defined set of steps undertaken to ensure correct operation of laboratory equipment and processes A Quality Assurance program is a set of steps taken to ensure that the Quality Control program is working as expected Finally Quality Improvement is a series of steps taken to review all aspects of the QC QA program with the intent of improving outcomes A QC QA QI program is a cooperative effort between the Laboratory Director and his her staff There are no nationally accepted or required steps that a laboratory must have in place in order to demonstrate that they have an appropriate AC QA QI program Instead each lab may design their own program to examine those factors they believe to be most critical As an e
47. edia should be removed from the refrigerator and equilibrated at 37 C prior to use The time and method required for equilibration will depend on the volume needed and the presence of bicarbonate buffer If using a media that contains bicarbonate buffer equilibrate for at least 4 hours in a CO incubator with the lid of the container loose If using media that does not contain a bicarbonate buffer equilibrate in either a warming block in room air or in an incubator with the lid tightly closed for enough time to warm the media prior to use 4 For oocyte collection Irvine Scientific recommends using mHTF Cat 90126 Modified Human Tubal Fluid when working on the benchtop or HIF Cat 90125 if working in an IVF chamber 5 Media should not be kept in the incubator for a prolonged period of time Once media has been placed in the incubator it should be used within one to two days If media has been in the incubator for more than this length of time it should be discarded and fresh media equilibrated just prior to use 6 The pH of the bicarbonate buffered media after equilibration in a CO incubator should be checked on a routine basis Irvine Scientific recommends keeping the pH of all media that will be used to culture embryos within a range of 7 20 to 7 40 There is limited data and little agreement regarding the precise pH requirements of embryos in an in vitro setting however keeping the pH closer to 7 20 rather than 7 40 may be beneficial sin
48. efore plunging into liquid nitrogen The Kit includes Equilibration Solution ES a HEPES buffered solution of Medium 199 containing 7 5 v v of each DMSO and ethylene glycol and 20 v v Serum Substitute Supplement This solution is packaged in the tubes with the white labels and tops Vitrification Solution VS a HEPES buffered solution of Medium 199 containing 15 v v of each DMSO and ethylene glycol 20 v v Serum Substitute Supplement and 0 5 M sucrose This solution is packaged in the tubes with the blue labels and tops CryoTips with metal cover sleeve stores up to 2 specimens Connectors 2 connectors are provided that will allow attachment of the Cryotip to a number of aspiration tools Using the Vitrification Freeze Media Kit Critical Points The ES and VS solutions are to be used in sequence for the step wise microdrop vitrification protocol Incubation time in ES and VS solutions should be monitored closely The high concentration of the cryoprotectants will cause toxicity to the specimens if the incubation time is extended for too long The kits are designed to be used at room temperature and without oil Recommended drop size is 20 pL IrvineScientific www irvinesci com Rev 1 6 12 Cryopreservation Protocols It is strongly recommended to practice the sealing and expelling processes before starting the vitrification protocol Great caution should be exercised when handling liquid nitrogen during the vitrification p
49. en has not liquefied after 30 minutes mix the specimen 1 2 with Sperm Washing Medium Cat 9983 and gently pull the mixture up and down several times using a Pasteur pipette If the specimen remains viscous it may be forcibly dispersed by it in and out through a 19 or 22 gauge needle attached to a 10 20 cc syringe or equivalent If the sperm preparation will be used to inseminate oocytes during IVE do not use syringes with black rubber plunger tips as solutions that have come in contact with these tips have been shown to be embryotoxic 10 Sperm Washing Medium contains HEPES buffer It should be pre warmed in a 37 C water bath or in a CO incubator but not exposed to the CO atmosphere therefore store all aliquots of media to be used in tubes that are kept tightly capped 11 Sperm Washing Medium and gradient layers should be stored in the refrigerator at 2 to 8 C and should not be used beyond the expiration date printed on the label METHODOLOGY A DENSITY GRADIENT CENTRIFUGATION METHODS General Considerations ISolate is a colloidal suspension of silica particles stabilized with covalently bound hydrophilic silane in HEPES buffered HTE It is designed to separate the motile sperm from the debris and seminal plasma Since mature live sperm with an intact cell membrane have a higher density than the dead sperm or any other debris in the semen they migrate through the ISolate layers to the bottom of the tube in a pellet form ISolate is
50. erm parameters are within normal ranges as outlined by the World Health Organization reference needed or 2 when the number of available oocytes is low to optimize the chance for fertilization During an ICSI procedure metaphase II oocytes are prepared for injection of a sperm by removing the outer layers of cells that surround the oocyte the cumulus oophorus and the corona Once a clear view of the interior of the oocyte is obtained sperm may be injected into the oocytes in order to overcome a number of sperm and or oocyte defects leading to low fertilization rates At the time of ICSI sperm may be placed into a solution of PVP in order to slow its movements down although not all laboratories choose to use these solutions Sperm is inactivated prior to insertion by rubbing the sperm at the midpiece or tail hard enough to kink the tail using a small beveled ICSI needle The inactivated sperm is then picked up into the needle and injected into the ooctye D IrvineScientific www irvinesci com Rev 1 5 2 Micromanipulation Protocols DENUDATION 1 The ICSI procedure requires that the cells surrounding the oocytes be removed in order to visual the interior of the cell a process called denudation stripping or cleaning The time between oocyte retrieval and cumulus removal should be standardized by each laboratory Denuding of oocytes can be achieved by either of two methods Dissection of the surrounding cumulus complex by cu
51. erve the CryoTip directly from the side and aspirate more VS to the 3rd mark Heat seal the CryoTip on the 1st mark with the heat sealer set on 2 3 carefully slide the metal cover sleeve toward the fine tip end to cover the fine tip completely remove the aspiration syringe and then heat seal at the other thick end of the Cryo above the 4th mark with the heat sealer set at 5 6 14 Attach the 1 2 cc cryostraw at this time if using it Hold the covered Cryo with tweezers and immerse it metal end first directly into liquid nitrogen 15 Place the covered tip into the submerged liquid nitrogen filled 3 6 mL cryotube or goblet on the cryocane 16 Repeat these steps for remaining embryos to be vitrified 17 Move the liquid nitrogen reservoir close to the liquid nitrogen tank and transfer the cryocane and its contents into the liquid nitrogen tank for long term storage IrvineScientific www irvinesci com Rev 1 6 14 Cryopreservation Protocols Procedure for Vitrification of Oocytes 1 After warming the vials of ES and VS to room temperature set up the vitrification dish as shown in Figure 1 Add one drop of culture medium CM HEPES buffered media containing 20 SSS 12 mg mL HSA and three drops of ES 20 uL for each Place the CM drop and the first two drops of the ES solution solutions from the white top tubes in close proximity to each other The third ES drop may be placed a further distance away from the gro
52. f the freezing and thawing rates as well as on the quality of the embryos chosen for cryopreservation Only morphologically normal embryos should be cryopreserved Other factors affecting the success of the embryo cryopreservation are the type of cryoprotectant used the method of cryopreservation used and the developmental stage of the embryos This kit may be used for embryonic stages ranging from the pronuclear through the multicell stage Pronucleate embryos zygotes should be frozen 24 28 hours after insemination prior to syngamy or fusion of the pronuclei Multicell cryopreservation may be performed on Day 2 or 3 CAP inspection in the United States requires redundancy in the main tanence of cryo information for a patient In addition a program must document that they are capable of satisfactorily cryopreserving embryos Therefore each laboratory should have a QC QA program that monitors outcomes for freezing embryos to determine whether the program is successful All media should be brought to room temperature before using Verify patient name and identify prior to thawing Verify number of em bryos to be thawed Thawing timing will depend on stage of embryos frozen Procedure for Freezing Embryos through Day 3 l Prepare and label sterile non toxic straws Labeling may be performed by writing on the straws themselves adding colored plugs to the tops of the straws or by applying a label to the straw the plug or a larger straw that is a
53. fornia to produce serum products for the cell culture market In the fall of 1973 Irvine Scientific reorganized and emerged as a respected entity for cell culture products on the West Coast In the years to follow we extended our products services and operations to become multi disciplined and international in scope By the fall of 1977 Irvine Scientific had outgrown its Fountain Valley facility and relocated to our present site in Santa Ana In the late 1980 s we further expanded and acquired the serum free cell culture and diagnostic product lines from Hana Biologics Inc Most recently Irvine Scientific has emphasized its manufacturing capabilities for custom formulations and also custom packaging with Irvine Scientific s MEDIA MANAGER line of products In August 1987 a Japanese based firm Japan Energy Company JEC formerly named Nippon Mining Company acquired an 8096 interest in Irvine Scientific JEC brought new capital to Irvine Scientific and a stated goal to become a leader in the expanding bio technical field JEC became the sole shareholder in July 1996 Their solid support has enabled us to provide our products worldwide to manufacturers of diagnostic and pharmaceutical products as well as the cytogenetic reproductive and clinical laboratories Today Irvine Scientific is a diversified company We sell products in a variety of markets including media and other related cell culture systems used in a wide variety of application
54. ged For example if the lab determines that the desired pH of culture media in the incubator for 24 hours prior to testing should be 7 20 7 25 and the measured pH as recorded on the data sheets is actually 7 35 then the pH of the media is out of range If the CO level on the incubator is turned up the pH of the media should drop The CO levels can be adjusted until the pH falls within the accepted range These kinds of adjustments can take several days to make The next step would be to monitor whether the changes in the CO level affected outcomes Therefore an outcome measure should also be determined at the start as part of the overall Quality Improvement program Any measure that the lab chooses can be assessed The measure should be meaningful such as fertilization rate early cleavage rate number of eight cell embryos or pregnancies to give a few examples In this example setting the CO level to drive the pH and monitoring pH levels on a routine basis would be the QC procedure QA would require reviewing the QC records to ensure that pH is staying within the acceptable range and QI would be monitoring outcomes before and after to determine whether pH changes affected the outcome Any number of parameters can be examined in an effort to improve pregnancy rates For example blastocyst conversion rates may be monitored in an effort to improve blastocyst culture Embryo survival rates may be determined as an endpoint when working to improve
55. he largest of the 3 settings The laser is now ready to use Figure 2 Make sure that the target is aligned with the pilot light This must be checked every time the laser is used 8 For molecular genetics cases have PCR tubes each with 5 pL of lysis buffer ready to receive the cells Each embryo will require a tube for the cell and a tube for a media blank It is also useful to have a number of extra tubes ready in case any embryo needs to be biopsied twice or in case a tube is dropped For cytogenetics cases the hypotonic and fixative solutions should be freshly made and glass slides ready for the fixing procedure 9 the plate containing the patient s embryos out of the incubator and move the first embryos to be biopsied to the Ist drop of the Ist biopsy dish containing the Embryo Biopsy Medium using a Drummond pipette with a sterile 170 um tip 10 Once the above steps have been completed the biopsy can begin Two embryologists should be involved from this point forward one to do the biopsies and one to move embryos and blastomeres around The first and worst embryo should now be in the dish and placed on the microscope Lower the microtools into the oil and pick up the embryo on the holding pipette Make a hole in the zona that will be big enough for the biopsy pipette to squeeze through almost the size of an 8 cell blastomere Place the dish on the stage of the inverted scope and locate the embryo under low power 4x lens Ro
56. he zona Therefore the setup and use of laser hatching should be determined by each laboratory However the following is one option for laser hatching using a Research Instruments Laser This protocol is used with the permission of Joe Conaghan PhD Director Fertility Laboratories Pacific Fertility Center San Francisco CA USA 1 Setup the inverted microscope turn it on and check the temperature of the stage If not already on turn on the computer to which the laser and microscope camera are attached Double click on the coronus icon on the desktop to activate the laser software Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Micromanipulation Protocols 2 Activate the laser by pressing any button on the hand control Figure 1 Ignite the pilot light by holding down the circular red button in the cen ter of the control until it beeps Rotate the laser lens into position on the microscope and place an empty petri dish in the light path Focus the microscope until the pilot light can be seen in focus on the surface of the dish Now check that the pilot light is aligned with the laser target on the computer screen Figure 1 Hand control for the laser 3 If the pilot and target are not aligned first check that none of the microscope controls have been adjusted to alter the light path In particular if the control to send light to the camera has been moved even slightly it could alter the position of the pilot light
57. ime to be used for IUI IVF or ICSI procedure For HEPES buffered media such as Sperm Washing Medium the tube should be tightly capped For media with a bicarbonate buffering system the tube should be loosly capped IrvineScientific www irvinesci com Andrology Protocols 4 7 QUALITY ASSESSMENT CRITERIA Semen analysis is performed in order to assess the specific parameters of the semen specimen Normal semen values have been established by the World Health Organization and published in WHO Laboratory Manual for the Examination of Human Semen and Semen cervical Mucus Interaction 4th ed Cambridge University Press 1999 A semen specimen with the following characteristics is considered normal e Volume 2 0 mL and above e pH 7 2 8 0 pH levels greater than this may indicate the presence of an infection The physician requesting the test should be notified in order to address appropriate treatment for the infection If the pH is less than 7 0 and the sperm count is low an abnormality or potentially a blockage somewhere in the reproductive tract may be suspected and the patient should be referred for evaluation Sperm concentration 20 x 105 per mL e Total sperm count N 40 x 105 per mL e Motility gt 50 or more with forward progression ie rapid and linear progressive motility or slow sluggish linear or nonlinear motility If 25 of the sperm have rapid linear progression this is also considered normal e Progress
58. ing of human blastocysts uses a two step freezing and two step thawing protocol Basal medium consists of Modified HTF with HEPES 12 mg mL Human Serum Albumin HSA and 10 pg mL Gentanicin Sulfate Blastocyst Freeze Media Kit Cat 90108 Includes Blastocyst Freeze Medium F1 is the 1st step freeze medium contains 5 glycerol Blastocyst Freeze Medium F2 is the 2nd step freeze medium contains 996 glycerol and 0 2 M Sucrose Blastocyst Media Thaw Kit Cat 790110 Includes Blastocyst Thaw Medium T1 is the 1st step thaw medium contains 0 5 M Sucrose Blastocyst Thaw Medium 12 is the 2nd step thaw medium contains 0 2 M Sucrose Blastocyst Freezing Critical Points The procedures should be performed in ambient atmosphere The success of blastocyst cryopreservation depends on the accuracy of the freezing and thawing rates as well as the high quality of the blastocyst chosen for cryopreservation Only high quality of the blastocyst should undergo cryopreservation All media should be brought to room temperature before using Verify patient name and identify prior to thawing Verify number of em bryos to be thawed Some programs have reported improved survival rates when culturing blastocysts in media that contains 20 SSS or at least 12 mgs mL after thawing and before transfer IrvineScientific www irvinesci com Rev 1 6 10 Cryopreservation Protocols Some programs have also reported that artificially collapsing the blastocoel
59. ing sperm freezing and thawing slow freezing rapid thawing for embryos and blastocysts and vitrification for gametes and embryos The ultimate goals of cryopreservation regardless of cell type are to reduce intracellular ice crystal formation minimize solute effects and toxicity of cryoprotectants and curtail osmotic shock Cryoprotectants both permeating and non permeating are used to reduce Ice crystal formation by dehydrating the cells before the cooling process Any potential toxic effects of the cryoprotectants may in turn be minimized by limiting the exposure time or reducing the concentration of cryoprotectants used during the freezing and thawing process The choice of cryoprotectant and freezing protocol depends on the developmental stage of the gamete or embryo All cryopreservation media utilize a buffered basal medium typically with HEPES buffer permeating and non permeating cryoprotectants and macro molecules such as Plasmanate Human Serum Albumin HSA or Serum Substitute Supplement SSS The most common permeating cryoprotectants those that enter the cell are DMSO glycerol ethylene glycol and propanediol Sucrose is the most commonly utilized non permeating cryoprotectant which serves to increase the extracellular concentration of solute and regulate the rate and extent of dehydration and rehydration during cooling and warming respectively Irvine Scientific manufactures several types of cryopreservation media and kits for u
60. ion The rapid and linear progressive motility determines the sperm s ability to be used for IUI or IVE The most common scale used for sperm progressive motility is the 0 4 scale with 4 rating the rigorous rapid forward progression and 0 rating the absence of any forward progression e Morphology 30 or greater with normal morphology is considered acceptable e White Blood Cells Less than 1 x 10 per mL is normal If greater than this number of non sperm round cells are seen e A differential staining should be performed to distinguish immature sperm from white cells e The patient should also be referred for evaluation by a urologist D ANTISPERM ANTIBODY ASSESSMENT General considerations Spermatozoa are manufactured in a portion of the body that is immunologically isolated during development in most men When the immunological barrier is breached by such events as trauma or vasectomy a man s immune system may begin to recognize his own sperm as being foreign or not self Part of the immune response to cells that are not recognized as self is the production of antibodies against the invading cells by the immune system These antibodies then bind to the offending cell and are used by the systemic immune system to assist in the removal of non self cells A number of different kinds of antigens that are present on the sperm may be immunogenic and stimulate the production of antibodies directed against various portions of the
61. ion date shown on the bottle label The medium does not contain antibiotics For procedures requiring antibiotics Gentamicin Sulfate or Penicillin G and Streptomycin Sulfate may be optionally added prior to use In all cases antibiotic usage should be determined by appropriate medical personnel to ensure that the patient is not sensitized to these antibiotics IrvineScientific www irvinesci com Rev 1 6 4 Cryopreservation Protocols Freezing Procedure m Sperm should be collected by masturbation following 2 3 days of abstinence and the sample allowed to liquefy at room temperature or 37 C for 15 30 minutes Refer to section above Semen Collection General Considerations One vial of a previously prepared aliquot of Sperm Maintenance Medium with Glycerol should be thawed and brought to room temperature or 37 If antibiotics are desired they may be added at this step The liquefied semen sample is transferred to a sterile 15 mL conical centrifuge tube The specimen volume is determined and an appropriate volume of thawed Sperm Maintenance Medium is added drop wise until a 3 1 sample to medium ratio is reached For example for each 1 mL of specimen add 0 33 mL of medium Aliquot the sample medium mixture into labelled cryotubes or straws To allow for expansion do not overfill cryotubes Freeze specimens either directly or after optional slow cooling step see below using a programmable freezer or vapor freezing pr
62. late upper layer into the tube using a sterile pipette according to product insert instructions Cap tube and place in warming block while specimen thaws 4 When the specimen has thawed use a 1 cc pipette to layer the specimen from the cryovial onto the prepared two layer ISolate gradient 5 Centrifuge tube at 300 x g for 20 minutes 6 Then remove the supernatant with a clean pipet leaving about 500 pL of medium without disturbing to pellet 7 Using another sterile pipette add 2 mL of Sperm Washing Medium Cat 9983 8 Centrifuge the tube at 300 x g for 10 minutes 9 Then remove the supernatant with a clean pipet leaving about 500 pL of medium and the sperm pellet Discard supernatant and resuspend the pellet in 2 mL of Sperm Washing Medium 10 Using a sterile pipette tip perform count and motility on specimen and record values 11 Place the specimen in a warming block or in a 377C incubator with the lid tightly closed until time for insemination IrvineScientific WWW irvinesci com Rev 1 6 6 Cryopreservation Protocols Thawing Procedure for processed sperm 1 Remove the frozen specimen from storage tank thaw as follows For cryovials allow the vial to warm for at least 15 minutes to room temperature For cryostraws allow the straws to thaw for 2 minutes in room air 2 Label a 15mL Falcon tube with patient s name After the specimen has thawed use a sterile 1 cc pipette to transfer the specimen from the cryovial
63. ll now be impaled on PZD needle with the tip of PZD needle exiting from the zona on the left and the wider back end of the PZD needle entering the zona from the right 7 Release the pressure on the holding pipette Lower the PZD needle to the bottom half of the field of view Move the holding pipette up to the top of the embryo 8 Rub the holding pipette from left to right across the top of the zona squeezing the zona between the PZD needle and the holding pipette Rub the zona until the PZD needle has created a slit in the zona and the embryo falls off of the PZD needle 9 Occasionally the zona will be sticky enough that the embryo does not actually fall off of the PZD but may be rolled off with the holding pipette once the slit has been made 10 Transfer the embryo to the dish where it will be held until transfer 11 Repeat with the remaining embryos Assisted Hatching using a Laser The zona may be hatched using a series of holes created by a laser also However there appears to be little agreement among embryologists as to how many holes to place in the zona whether to simply thin the zona or to actually breach the zona what pattern to use when making the holes ie multiple holes along the inside of the zona but only one smaller opening on the outside multiple holes along the outside of the zona but only one on the inside or to created one aligned set of holes breaching the entire thickness of t
64. loat to the top of the drop 11 Draw up some DS into the transfer pipette and transfer the oocytes or embryos from the drop of TS with minimal volume to the bottom of the first drop of DS DS1 for two minutes 6 19 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Cryopreservation Protocols 12 Then transfer them to the bottom of the second drop of DS DS2 for two minutes Note During exposure to DS the cells will remain shrunken During this two minutes waiting period dispense a sequence of 3 micro drops 20 uL each of WS on the same dish 13 Transfer the oocytes embryos to the bottom of the first drop of WS WS1 for three minutes Note They should re expand to the original size within three minutes in WS 14 Then transfer the oocyes embryos to the top of the second drop of WS WS2 for three minutes 15 Transfer them to the top of the third drop of WS WS3 for three minutes 16 Finally transfer them to a prepared dish containing the appropriate culture medium Incubate the oocytes for 3 4 hours to let them recover prior to injection Embryos may also be held prior transfer or further culture procedures after warming Note This same protocol is applicable for use with oocytes or embryos However when using oocytes they must be allowed to recover for at least 3 hours prior to insemination to allow for reformation of the spindle apparatus Insemination must be performed by ICSI as zona hardening due to release
65. moved as possible A variety of devices have been utilized to load transfer catheters including AirTite syringes and gas tight Hamilton syringes Using standard 1 cc tuberculin syringes with the black rubber tip is not recommended as the rubber tips have been shown to be embryotoxic Catheter filling should be performed in an aseptic manner Some programs prefer to load the catheter using a continuous column of media other programs use small bubbles of air to isolate the embryos Both methods appear to be satisfactory Following transfer the Embryologist should rinse the catheter well to verify that no embryos remain in the catheter If embryos are retained some reports in the literature suggest that the chance of achieving a pregnancy is diminished for the patients Retained embryos at the time of transfer should be documented Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Protocols Andrology Protocols Andrology Protocols OVERVIEW Ejaculated semen is composed of two major components spermatozoa and seminal plasma In the body during intercourse only sperm but not the seminal plasma component of the ejaculate pass through the cervix to enter the uterus and then move on up into the fallopian tubes where fertilization takes place The seminal plasma portion of the ejaculate is filtered out and retained within the vagina and cervix Seminal plasma contains a number of small factors and compounds su
66. n over the wider section of the CryoTip and over the pinched upper end of the metal sleeve once it has been pulled down to cover the tip of the CryoTip Additional labels can then be attached to the 1 2 cc cryostraws or colored plugs may be added to the opposite open end 6 Mix one vial of ES content and one vial of VS content by gently inverting the vial twice Then dispense a series of five 20 pL micro drops on a dish as follows One x 20 pL drop of ES white topped tube see standard vitrification protocol diagram for embryos Four x 20 uL drops of VS blue topped tube 7 Remove the culture dishes with the embryos from the incubator and check the growth and quality of the embryos under the microscope Select the best embryos for vitrification 8 Draw up some ES solution from the ES drop with the transfer pipette Then move the embryos no more than the number to be placed in one CryoTip at a time with a minimal volume of medium from the culture dish to the top of the drop of the ES and start the timer Watch the embryos Once they have shrunken and then re expanded they may be moved to the VS drops Pronuclear embryos will take less time to shrink probably less than 5 minutes although this is patient and embryo dependent and re expand than blastocysts 10 15 minutes Blastocysts may not fully re expand 0 000 0 0 6 13 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Cryopreservation Protocols
67. n the cytoplasm and provides a better area of contact for pronuclear formation Slowly remove the injection pipette while expelling the sperm in the oocyte 15 If using the trough method move the oocyte to the other end of the trough and then return to the uninjected oocytes and continue as described in the previous steps until all oocytes are injected If using drops release the oocytes in its drop and move the dish to the next drop 16 When finished injecting move all oocytes through three rinses of cul ture medium and place them into a drops of media covered with oil in a culture dish Oocytes may be cultured in groups or individually Dishes for culture should have been made the previous day to allow for equilibration especially when using low oxygen incubators as it appears that oxygen diffusion through oil occurs much more slowly than CO diffusion If using group culture then drops may be in the 20 50 pL range When using single embryo culture drop size may be less in the 10 20 pL range 17 Check for two pronuclei at 16 20 hours post injection as the evidence of fertilization Separate out the fertilized from unfertilized zygotes and culture B ASSISTED HATCHING General Considerations Assisted Hatching is used as a method to assist the embryo in its final expul sion from the zona as it begins to implant or to open the zona sufficiently at the time of embryo biopsy for Preimplantation Genetic Diagnosis PGD Several method
68. ng the vitrified embryos Fill the liquid nitrogen reservoir with liquid nitrogen 80 full and place it close to the liquid nitrogen tank Remove the canes containing the cryovials with the vitrified specimens from the liquid nitrogen tank and transfer them into the liquid nitrogen reservoir and then place it close to the microscope for rapid manipulation Label a sterile culture dish with necessary information and prepare the dish with the thaw media Mix each vial of TS DS and WS by gently inverting twice then aseptically dispense a sequence of 3 micro drops on a sterile culture dish as follows See procedural diagram for thawing protocol One 20 pL drop of TS and Two 20 uL drops 40 uL of DS and place the dish under the microscope IrvineScientific www irvinesci com Rev 1 6 18 Cryopreservation Protocols 6 Place the 37 C water bath close to the microscope Have a transfer pipette a pair of scissors syringe with connector timer and sterile wipes ready 7 Using tweezers retrieve the covered CryoTip from the cane in liquid nitrogen reservoir quickly immerse it into the 37 C water bath and swirl gently for 3 seconds to thaw 8 Gently wipe the CryoTip with a sterile wipe and cut the seal on the thick end on the 4th mark on the tip Attach the thick end to the connector and the syringe or other expulsion device Slide the metal cover sleeve toward the thick end to expose the fine tip Gently wipe the fine tip with a s
69. o 10 minutes magnification under a phase contrast microscope 5 Score the percentage of motile sperm that have Immunobeads attached Assess each type of bead separately Count at least 100 200 motile sperm per preparation Do not count non moving sperm even if they appear to have beads attached Some moving sperm may appear to have beads attached but careful observation will show that the beads are not carried with the sperm as they move Therefore observe only moving sperm and record sperm as positive only if it can be observed that the sperm are carrying the beads with them as they move Record the site of binding of the beads to the sperm head midpiece tail head and tail 6 The test is positive if 20 or more of the motile sperm have beads attached to them and is clinically significant if 50 or more are coated with beads Binding of the beads to only the tail tip is not considered clinically significant 1 4 8 IrvineScientific www irvinesci com Andrology Protocols Tests should also be performed using positive and negative controls alongside the sperm being analyzed A positive control may be frozen thawed and washed sperm from a patient who has previously tested highly positive for both IgA and IgG binding or antibody free sperm may be bound with antibody by co incubating the sperm in the serum from a patient that has previously been shown to be anti sperm antibody positive Incubate 100 pL of washed sperm with 100 u
70. ocedure Optional directions for slow cooling prior to freezing Attach the filled cryovials to an aluminum cane Place the cane s sample end down into an ambient temperature 20 25 C water bath and then place the water bath in a refrigerator 2 to 8 C for 60 to 90 minutes before freezing Once the initial pre freezing has been performed the canes may be frozen using either a programmable freezing system or by manually assisted vapor phase freezing by suspending the canes in liquid nitrogen vapor Store in appropriately identified locations Sperm Refrigeration Medium Cat 90129 General Considerations Store the unopened bottles at 109C or colder Do not expose medium to repeated freeze thaw cycles If smaller aliquots are desired thaw the product aliquot working volumes into sterile labeled containers and freeze until time of use Refrigeration Medium is stable until the expiration date shown on the bottle or vial label when stored as directed Procedure l Semen should be collected by masturbation following 2 3 days of abstinence and the sample allowed to liquefy at room temperature for 37 C for 15 30 minutes Refer to section above Semen Collection General Considerations One vial of medium can be used per patient Ihaw one vial and bring to room temperature After the specimen has liquefied transferred it to a sterile 15 mL conical centrifuge tube Determine the volume and add the refrigeration
71. of the cortical granules occurs during vitrification just as it does during slow cooling procedures IrvineScientific www irvinesci com Rev 1 6 20 TRADEMARKS American Express is registered trademark of American Express Company is trademark of Irvine Scientific is a trademark of Irvine Scientific FedEx is a registered trademark of Federal Express Corp IMMUNOBEAD is a registered trademark of Bio Rad Laboratories Inc Isolate is a registered trademark of Irvine Scientific MasterCard is a registered trademark of MasterCard International Incorporated MultiBlast is a trademark of Irvine Scientific MYCOTRANS is a registered trademark of Irvine Scientific MYCOTRIM is a registered trademark of Irvine Scientific P 1 is a registered trademark of Irvine Scientific Percoll is a registered trademark of Pharmacia Biotech AB Sweden SSS is a trademark of Irvine Scientific SUREVIEW is a trademark of Smiths Group plc UPS is a trademark of United Parcel Service of America Vit Kit is a trademark of Irvine Scientific VISA is a registered trademark of VISA International Rev 1 1 800 577 6097 949 261 7800 IrvineScientific ALPHABETICAL INDEX Catalog Number Description MEONS1633 16g SINGLE LUMEN NEEDLE 33 cm 516335 16g SINGLE LUMEN NEEDLE 33 cm MEONS1733 17g SINGLE LUMEN NEEDLE 33 cm MEONS17338 17g SINGLE LUMEN NEEDLE 33 cm 15391 ACTIVATED IMMUNOBEAD MATRIX ACTIVATED IMMUNOBEAD 90108 BLAST
72. on for this embryo and blastomere Repeat steps 10 through 20 until all the embryos have been biopsied To conclude the procedure the frozen tubes should be packaged up with ice blocks to keep them cool and sent to the appropriate lab for analysis For molecular genetics cases skip to step 25 Place the biopsied cell into mHTF with protein and pass to the embryologist to do the fixing Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Micromanipulation Protocols 25 The biopsied embryo should be washed twice in mHIF and then placed back in culture medium It is important that each embryo should spend no more than 5 minutes in the biopsy medium The embryo should be placed in culture in such a way as there is no possibility of having it confused with subsequent biopsied embryos 26 Continue to culture the embryos until the results of the biopsy are available usually 2 more days Embryo transfer is performed in the normal way 27 The embryo transfer will happen on Day 5 2 days after the biopsy References Mark Hughes 2000 PGD and biopsy protocol Trounson A O and Gardner D K 2000 Handbook of in vitro fertilization CRC Press New York 9 0 0 00000 IrvineScientific www irvinesci com Rev 1 5 14 Cryopreservation Protocols Cryopreservation Protocols General Considerations There are many protocols that have been introduced for different cryopreserva tion applications includ
73. on time wash the mixture three times by adding one mL of Buffer B and centrifuging at 600 x g for 5 minutes Remove supernatant and wash again After the final wash resuspend the sperm in 400 pL of media Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Andrology Protocols 7 During the incubation period prepare the Immunobeads as described for the Direct Immunobead Assay a Thaw one tube of each of the stock bead solutions IgG and IgA b Add the contents of the tube to 10 mL of Buffer A in a separate conical centrifuge tube c Centrifuge at 1000 x g for 10 minutes d Decant the supernatant blot the rim and resuspend the final pellet in 0 2 mL of Buffer B 8 Begin testing by adding 5 uL of washed sperm suspension to 5 pL of each washed Immunobeads IgA and IgG on a microscope slide Mix well cover with a cover slip and leave at room temperature in a moist chamber for up to 10 minutes Observe at 400X to 500X magnification under a phase contrast microscope 9 Score the percentage of motile sperm that have attached Immunobeads Assess each type of bead separately Count at least 100 200 motile sperm per preparation Do not count non moving sperm even if they appear to have beads attached Some moving sperm may appear to have beads attached but careful observation will show that the beads are not carried with the sperm as they move Therefore observe only moving sperm and record sperm as positive only if it can be observed that
74. or 5 minutes Move the embryos in a minimal amount of medium into 2 3 mL of the Embryo Thaw Medium 12 and incubate for 5 minutes Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Cryopreservation Protocols 10 Transfer the embryos in a minimal amount of medium into 2 3 mL of the Embryo Thaw Medium T3 for 10 minutes 11 Wash the embryos by transferring them in a minimal amount medium into 2 3mL of mHTF Medium supplemented with 12mg mL HSA for 10 minutes at ambient temperature 12 Finally transfer the embryos to fresh culture medium HTE P 1 or ECM supplemented with 5 mg mL of HSA 10 v v SSS or 10 v v DSS Use HTF or MultiBlast Medium supplemented with protein instead if the embryos were cryopreserved on Day 3 The embryos at this step could be further cultured to blastocyst stage or transferred to the patient s uterus after thawing BLASTOCYST CRYOPRESERVATION The ability to reliably freeze and thaw blastocysts has become increasingly important in recent years As more programs are trying to reduce the twin rate by taking more and more patients and their embryos on to the blastocyst stage it has become critical that a laboratory have a healthy blastocyst freezing program The blastocyst cryopreservation requires different cryopreservation media and procedures as compared to freezing at the early cleavage stages IRVINE SCIENTIFIC BLASTOCYST FREEZING MEDIA Media specially formulated for the cryopreservation and thaw
75. ore variable than density gradient separation Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Andrology Protocols Procedure 1 Place 1 mL of warm Sperm Washing Media Cat 9983 into a clean 15 mL centrifuge tube 2 Gently layer 1 mL of semen under the media using a clean disposable 1 mL pipette 3 Place the tube into the incubator at a 30 angle and allow the sperm to swim up into the overlying media for at least one hour 4 After one hour gently remove the top 200 300 pL of media and place in a clean tube 5 Multiple aliquots of several tubes may be combined into one if multiple tubes were used Sperm is ready to use at this point after assessment of count and motility C SPERM WASHING METHOD FOR POOR SPECIMENS General Consideration Poor semen specimens are those with low sperm concentrations low motility and or low progression Density gradient recovery typically averages around 25 therefore for specimens with extremely low initial counts density gradient centrifugation may not be appropriate The following procedure may be used to wash sperm that has been obtained by ejaculation or by testicular biopsy or epididymal sperm aspiration Procedure 1 Bring Sperm Wash Media to 37 C 2 Testicular tissue may be minced in Sperm Washing Media dispersed in and out through an 18 guage needle and then washed twice with 2 3 mL of Sperm Washing Media The specimen should be centrifuged at 300 x g centrifugation bet
76. prematurely the embryo should be re biopsied 5 9 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Micromanipulation Protocols 7 Unacceptable specimen UNLABELED OR IMPROPERLY LABELED SPECIMENS ARE UNACCEPTABLE AS THEY CANNOT BE USED FOR A GENETIC DIAGNOSIS IT IS IMPORTANT THAT THE BIOPSIED CELL SPECIMEN IS LABELLED CORRECTLY WITH THE SAME IDENTIFICATION AS THE EMBRYO FROM WHICH IT WAS OBTAINED Cells without nuclei or those with nuclear abnormalities multiple nuclei fragmented nuclei cannot be considered reliable for the diagnosis 8 Compromising conditions If the cell is lysed or dead or if the nucleus is missing or abnormal the analysis may not succeed If any of these scenarios arise consultation with the individual placing the cells in the PCR tubes or fixing and analyzing the nuclei should be undertaken as soon as they have had a chance to examine the cell Such conditions would require the embryo to be re biopsied to obtain an acceptable cell for analysis 9 Timing considerations The embryos should be biopsied as early as possible on the morning of Day 3 to minimize complications arising from embryo compaction 10 Special equipment For the purpose of the biopsy a laser can be used to allow the embryologist to work quickly on the embryos Specifically the laser allows a hole to be made in the zona pellucida without the need for a double tool holder acid Tyrodes and a hatching pipette 11 Embryos should be viewe
77. protein Another method is to place 5 pL triangle of PVP in the center of the dish and then a 5 10 pL trough of media with protein above it 7 Cover the drops with oil 8 Place sperm into the PVP droplet Sperm may be placed gently into the center of the drop or the outside edge of the triangle The amount of sperm added to the PVP droplet should be determined by the count and motility of the processed specimen The final dilution in the PVP droplet should be high enough that it is easy to visually find motile sperm around the edges of the drop or the opposite side of the triangle but low enough that it is easy to pick up only the chosen sperm without pulling multiple sperm up into the ICSI needle 9 Transfer oocytes into each of the droplets surrounding the sperm or into the left side of the trough in the ICSI dish 10 Take the dish containing the oocytes and sperm to the scope Raise the micro tools remove the holding dish and position the ICSI dish on the stage so that the edge of the PVP droplet is in focus 11 At 200X magnification focus on the sperm at the edge of the PVP drop Search for sperm that are slightly motile at the bottom of drop or at the drop oil interface Assess the sperm for the quality of the motility and its morphology Lower the ICSI needle to the top of the chosen sperm and use the tip of the pipette to swipe or snap the tail of the sperm in order to immobilize it Pull the sperm up into the pipette by
78. r Service Department Phone 001 949 261 7800 Fax 001 949 261 6522 WwWw irvinesci com IrvineScientific www irvinesci com Rev 1 1 5 Quality Control Program Quality Control Program QUALITY ASSURANCE Products manufactured by Irvine Scientific are classified as medical devices by the Food and Drug Administration FDA They are produced in compliance with the FDA s current Good Manufacturing Practices CGMP s and ISO 13485 2003 regulations utilizing detailed production protocols and quality control test procedures Irvine Scientific is licensed by both Federal and State agencies and is inspected regularly for compliance Irvine Scientific is certified to the ISO 13485 2003 International Quality Standard and is certified to the European Norm 46001 by the National Standards Authority of Ireland MANUFACTURING Liquid Media Liquid media for all applications is manufactured filtered and bottled on site in our aseptic class 100 environment All steps in the process undergo rigorous testing to validate sterility repeatability and reliability Approved filtration and filling protocols include testing for filter integrity sterility and product quality Filter integrity is assessed using the bubble point test to determine the presence of holes in the filling system and is performed with every fill Sterility is achieved by passing the media through a series of filters the final being 0 1 u to achieve a Sterility Assurance Level SAL of 1
79. rformed immediately Therefore the timing of insemination is procedure dependent Most laboratories no longer inseminate immature oocytes by ICSI since it may lead to abnormal cleavage rates and embryo fragmentation Precise timing of the insemination should be determined and documented by each laboratory Notes 1 Insemination dishes should be prepared in advance preferably on the day prior to oocyte retrieval If conventional insemination is used the same dishes that were used at the conclusion of oocyte retrieval may be used If performing ICSI the oocytes should be stripped or cleaned to remove the surrounding cumulus cells and rinsed and maintained in fresh dishes with fresh media containing protein until injection Following injection oocytes should be rinsed at least three times and then placed into fresh dishes with fresh media for culture overnight 2 It is critical to monitor and control temperature pH and exposure to potential contaminants during all phases of insemination regardless of insemination method 3 However attention should be paid to temperature pH and exposure to potential contaminants in air during all phases of insemination whether by conventional insemination or ICSI 4 Following insemination by either conventional IVF or ICSI oocytes should be placed in the incubator overnight FERTILIZATION CHECK General Considerations If oocytes embryos will be moved into new dishes following determination of fertiliza
80. rial chemicals where possible conform to Multi compendial standars United States Pharmacopeia USP or National Formulary NF standards Certificates of Analysis are obtained from manufactures or vendors for all chemicals used Once the chemicals arrive at Irvine Scientific samples of each lot are tested for endotoxin identity by Fourier Transform Infrared Spectroscopy efficacy and toxicity as appropriate Human Source Raw Materials Human Source Raw Materials used by Irvine Scientific in the manufacturing of reproductive media are of therapeutic grade and obtained from CBER licensed facilities All human source material has been tested at the donor level with FDA licensed kits and found to be nonreactive for the antibodies to Hepatitis B Surface Antigen HbsAg antibodies to Hepatitis C HCV and antibodies to Human Immunodeficiency Virus HIV 1 amp 2 Source material is also screened by questionnaire at the donor level for risk factors associated with CJD Creutzfeldt Jakob Disease IrvineScientific www irvinesci com Rev 1 2 2 Quality Control Program Water The water system is routinely monitored for microbial levels endotoxin conductivity pH and TOC Total Organic Carbon The finished product water meets USP test requirements for Water for Injection QUALITY CONTROL TESTS Sterility Testing The Sterility test is considered a biological test Sterility testing of all media is performed in accordance with the Code of
81. rocess A variety of suction devices have proven effective at loading the CryoTip Each laboratory may find that they are comfortable with their own version of a suction device Some alternatives include 250 pL Hamilton gas tight syringes 1 cc Airtight syringes Eppendorf or Gilson repipettors or the Mushroom microinjectors Before using a CryoTip slide the metal sleeve up and down to make sure it moves freely along the CryoTip Once embryos or oocytes are vitrified they cannot be warmed without immediate thawing of the CryoTip Therefore care should be taken to ensure that the tips are never exposed to ambient air temperatures until the time of thawing Procedure for Vitrification of Embryos 2PN to Blastocyst 1 Bring one vial of each ES and VS to room temperature 20 25 C for at least 30 minutes prior to vitrification of specimens 2 the liquid nitrogen reservoir with liquid nitrogen at least 4 inches in depth and place it close to the microscope Attach 4 5 mL cryotube uncapped or a cryogoblet to the bottom clamp of a cryocane and submerge in the liquid nitrogen in preparation for storage of the vitrified specimens 3 Determine the number of embryos to be vitrified 4 NOTE 1 2 embryos may be loaded into each CryoTip 5 Label each sterile culture dish and CyroTip with the patient s One half cryostraws may also be labeled and used as a handle for the CryoTips as the larger cryostraw will slide easily dow
82. rrounding the oocytes prior to ICSI procedure 2 PVP Polyvinylpyrrolidone Available in three convenient configurations a Lyophilized PVP Cat 499219 10 lyophilized PVP to be reconstituted with mHTE designed to be used with normal sperm specimens b 10 PVP Solution Cat 99311 Ready to use 10 PVP reconstituted in mHTF medium designed to be used with normal sperm specimens c 7 PVP Solution Cat 90119 Ready to use 7 PVP reconstituted in mHTF medium designed to be used with poor sperm specimens low motility sperm 3 Tyrode s Solution Acidified Cat 99252 Ready to use solution for thinning of the zona pellucida assisting hatching or drilling a hole in the zona of the embryos 4 Embryo Biopsy Medium Cat 90103 Embryo Biopsy Medium is a ready to use HEPES buffered salt solution mHTF with HSA 5 mg mL lacking Calcium and Magnesium ions and containing EDTA as a chelating agent It is designed for use as a temporary short term culture medium while performing embryo biopsy on cleavage stage 6 10 cell embryos to remove blastomeres for Preimplantation Genetic Diagnosis PGD A ICSI Procedures General considerations ICSI is performed when sperm count motility or morphology is low such that En may not occur when performing conventional insemination incubation of oocytes and sperm in a dish ICSI is also performed when 1 there has been evidence of failed fertilization in a previous cycle even when sp
83. rvical mucins within the female partner However in rare instances women may also produce their own IgG and IgA anti sperm antibodies IgG and IgA on spermatozoa may be assessed directly in semen using purified antibodies covalently bound to micron sized hydrophilic polyacrylamide beads Antibodies directed against spermatozoa in samples that do not contain spermatozoa such as cervical mucous or serum may be indirectly assessed using antibody free sperm that is co incubated with the sample containing the anti sperm antibodies The antibodies will then bind to the normally antibody free sperm and can be detected when the sperm are in turn incubated with the polyacrylamide beads Procedure DIRECT IMMUNOBEAD ASSAY Covalently linked beads are available for detection of IgG Cat 15375 Immunobead Rabbit Anti Human IgG y Riga Cat 15376 Immunobead Rabbit Anti Human IgA a and IgM Cat 15377 Immunobead Rabbit Anti Human IgM u These anti antibodies have been raised in rabbits to detect the presence of specific human anti sperm antibodies present on the surface of the sperm 1 Immunobead reagents of any type may be reconstituted and divided into smaller volumes then refrozen for later use Upon receipt of the beads reconstitute the reagent with 5 mL deionized water to produce a working stock solution of 10 mg of beads mL of water This rehydrated stock solution of beads is stable for 6 months when stored at 4 C or may be frozen for
84. ry from receipt of the specimen to final disposition whether via IUI or IVE All personnel handling the specimen from receipt to insemination should be identified in writing and sign off on each step as it progresses a 1 4 2 IrvineScientific www irvinesci com Andrology Protocols 4 The patient should also be expected to present appropriate identification and there should be an established protocol for verifying the female partner s association with the sperm provider 5 All semen specimens should be collected in sterile non toxic containers no more than one hour before processing and be kept at room temperature or 37 C body temperature 6 A defined identification protocol should be followed for each patient and his partner to ensure that there is no confusion among multiple laboratory personnel regarding which sperm to use for insemination purposes 7 The specimen should be processed in a clean work place while using non toxic gloves sterile non toxic disposable tubes needles and pipettes All disposable items should be used one time and then discarded in an approved biohazard waste container 8 Laboratory personnel processing the specimen should use aseptic techniques and all specimens should be treated as potentially infectious Therefore Universal Precautions should be followed during the course of all handling and processing 9 The specimen should be allowed to liquefy for 20 30 minutes before processing If the specim
85. ryopreservation of human cleavage stage embryos using a two step freezing protocol and three step thawing protocol Basal medium consists of Modified HTF with HEPES 12 mg mL Human Serum Albumin HSA and 10 ug mL Gentamicin Sulfate Embryo Freeze Media Kit Cat 90116 Includes Embryo Freeze Medium F1 is the 1st step freeze medium contains 1 5 M Propanediol Embryo Freeze Medium F2 is the 2nd step freeze medium contains 1 5 M Propanediol and 0 1 M sucrose Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Cryopreservation Protocols Embryo Thaw Media Kit Cat 90124 This kit allows stepwise dilution of the permeating cryoprotectant propanediol and utilizes a sucrose solution that is twice that of the Freeze Media in order to prevent rapid re expansion of the embryo prior to removal of the propanediol Embryo Thaw Medium T1 is the Ist step thawing medium and contains 1 0 M Propanediol plus 0 2 M sucrose Embryo Thaw Medium 12 the 2nd step thaw medium contains 0 5 M Propanediol and 0 2 M sucrose Embryo Thaw Medium 13 the 3rd step thaw medium contains 0 2 M sucrose Pronuclear and Multicell Cryopreservation Critical Points The procedures should be performed at room temperature atmosphere and does not require the use of an oil overlay However if a lab chooses to perform the procedures under oil there is no evidence to suggest that this is harmful The success of embryo cryopreservation depends on the accuracy o
86. s Seminal plasma may be tested without inactivation of complement c Follicular fluid should be centrifuged at 600 x for 10 minutes and heat inactivated by incubating at 56 C for one hour Fresh donor sperm that has previously been shown to be antibody free should be washed three times with 10 mL Buffer A and centrifugation at 600 x g for 10 minutes on the day the indirect assay is to be performed At the end of the final spin adjust the final sperm concentration to 50 million sperm mL in Buffer B Donor sperm that has been washed is then incubated with the test solution If antibodies are present in the test solution they will bind to the sperm during this time Add 100 pL of patient test fluid to 100 pL of washed donor sperm suspension and 200 pL of Buffer B in a 12 x 75 mm tube Positive and Negative controls should be run alongside the test solution a The positive control should be the serum of a patient who has previously been shown to be antibody positive with an indirect assay Once a patient has been shown to be antibody positive serum may be collected heat inactivated and then allocated into small volumes and stored frozen until needed b The negative control should be the serum of a patient who has not tested positively for anti sperm antibodies in the past using the indirect assay Incubate the patient test solution the positive and the negative controls with the donor sperm for 1 2 hours at 377C At the end of the incubati
87. s including specialty bulk sera products as components for the Diagnostics market media for the Cytogenetics laboratory as well as a complete product line for the Reproductive laboratory Irvine Scientific recently expanded our manufacturing facilities In 2003 we received ISO 13485 2003 certification for our manufacturing process Irvine Scientific s efforts are directed toward continued growth of our products and the markets we serve Our full line of products may be viewed on our website www irvinesci com IrvineScientific www irvinesci com Rev 1 1 1 General Information ORDERING INFORMATION Ordering Options Online Ordering You may access our online ordering by visiting us at www irvinesci com Orders may be placed 24 hrs a day Orders may be tracked Check our website for promotions and specials Access contact numbers and customer support Obtain copies of all product inserts and specifications Customer Support Line Domestic Orders Telephone fax and mail orders are accepted Please include a purchase order number and your telephone number on all orders Confirming purchase orders are NOT required on phone orders If a confirming purchase order must be sent be sure it is clearly marked CONFIRMATION DO NOT DUPLICATE Place orders for items in this website with IRVINE SCIENTIFIC Attention Customer Service Department 2511 Daimler Street Santa Ana CA 92705 5588 U S A Customer Service Hours 7 00
88. s Solution 1 Setup the micromanipulation microscope with a holding pipette on the left and the AH pipette on the right Align the microtools in the center of the field of view 2 Remove the Acidifed Tyrode s Solution from the refrigerator and allow it to come to room temperature while setting up the scope 3 AH should be done at 37 C 4 Place 10 pL of Acidified Tyrode s solution in the center of low walled dish cover with oil and place it on the stage of the microscope Lower the hatching microtool into the Tyrode s solutions apply suction and allow the microtool to fill and equilibrate for a few minutes before using 5 While waiting for the tool to equilibrate place several 10 pL drops of Modified HIF with protein HSA SSS or DSS in the dish and cover with oil 6 Add one embryo to the first drop It is not advisable to let an embryo sit in a droplet with acid Tyrode s for an extended period of time Therefore once the embryo has been hatched it should be transferred out of that drop and rinsed immediately Then the next embryo may be placed in the second drop hatched removed rinsed and then the third embryo hatched if multiple embryos are being hatched and transferred 7 Take the dish to the micromanipulation scope Raise the hatching microtool while applying a slight amount of pressure to prevent oil from entering the tool as it is raised through the oil Locate the drop with the embryo and pull the embryo onto the holding
89. s may be used to put a hole in the zona such as etching or dissolving a hole in the zona with acidified Tyrode s solution using a spike or PZD needle to rub a hole in the zona or using a laser to remove portions of the zona Performing zona removal in an effort to improve implantation rates is still somewhat controversial and the outcomes reported in the literature seemed to be mixed However when a benefit is shown it seems to be when using embryos from older women and or frozen thawed embryos However some programs routinely perform assisted hatching on all embryos prior to transfer Therefore criteria for determining when and which embryos to hatch should be developed by each individual laboratory The technique for performing assisted hatching for implantation improvement and opening a hole in the zona for embryo biopsy is similar except for the final size of the opening When doing assisted hatching to improve implantation the size of the hole should be approximately one quarter to one third the size of a blastomere When doing assisted hatching for embryo biopsy the size of the hole will have to be large enough to allow introduction of the biopsy tool or approximately the size of blastomere Therefore the procedure should be modified to accommodate the conditions for its use Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Micromanipulation Protocols Procedure for assisted hatching for implantation improvement using Acid Tyrode
90. s unless otherwise notified Pricing and Terms Irvine Scientific reserves the right to change prices without notice Any price reduction will automatically apply to your invoice Io obtain a copy of our current price list please fill out our online Catalogue Request Form Invoices are due in net 30 days in U S dollars A finance charge of 1 5 percent per month annual percentage rate of 18 percent will be charged to past due accounts Special Quotations Quantity discounts are available on Standing Orders Products can be bulk packaged in custom sized containers at specially quoted prices Quotations can be obtained by contacting your Territory Manager e Shipping e Returns Cancellations Facility Tours Irvine Scientific would like to invite our customers to visit our manufacturing facilities We are located in Santa Ana California approximately one hour south of Los Angeles If you would like to visit our site please contact your sales representative and they can arrange a tour for you IrvineScientific www irvinesci com Rev 1 1 3 General Information REDUCE RE USE RECYCLE Irvine Scientific is concerned with the environmental impact of our packaging materials and we encourage our customers to dispose of these materials responsibly Effective re use and recycling of these components depends on you Please use the following guidelines to help us protect our environment for future generations Packaging Material Re use Re
91. scope camera are attached Double click on the coronus icon on the desktop to activate the laser software 6 Activate the laser by pressing any button on the hand control Figure 1 Ignite the pilot light by holding down the circular red button in the center of the control until it beeps Rotate the laser lens into position on the microscope and place an empty petri dish in the light path Focus the microscope until the pilot light can be seen in focus on the surface of the dish Now check that the pilot light is aligned with the laser target on the computer screen IrvineScientific www irvinesci com Rev 1 5 10 Micromanipulation Protocols Figure 1 Hand control for the laser 7 If the pilot and target are not aligned first check that none of the microscope controls have been adjusted to alter the light path In particular if the control to send light to the camera has been moved even slightly it could alter the position of the pilot light In the rare event that the pilot and target do not line up and the microscope has not been adjusted double click on the target with the computer mouse This unlocks the target and it can then be picked up and moved with the mouse Once aligned with the pilot see Figure 2 the target is locked by double clicking on it again Turn off the pilot light using the same button as before The laser has 3 settings and these are selected by going back and forth with the arrows on the control unit Select t
92. se with sperm oocytes and embryos 1 Sperm Freezing Medium Test Yolk Buffer TYB with glycerol amp gentamicin Cat 90128 2 Sperm Refrigeration Medium Test Yolk Buffer TYB with gentamicin Cat 90129 3 Sperm Maintenance Medium Cat 99276 4 Embryo Freeze amp Thaw Media Cat 90116 amp 90124 5 Blastocyst Freeze amp Thaw Media Cat 90108 amp 90110 6 Vitrification Freeze amp Thaw Kits Cat 90133 amp 90137 SPERM CRYOPRESERVATION Sperm freezing offers patients the opportunity to preserve sperm for later use or for sperm donors to provide specimens for use by others through sperm banks Patients undergoing cancer therapies or having prostate or testicular surgery may bank their sperm prior to treatment for subsequent use at a later date Freezing also allows stored sperm to be used for repeated inseminations via intrauterine insemination IUI or in vitro fertilization by conventional IVF or ICSI This allows couples to continue therapy for infertility and have sperm available for insemination even when the male partner may be unable to produce a semen specimen due to surgery illness or absence Cryopreservation of sperm has also been used to accumulate multiple samples from a patient with low counts and then combine them to increase the volume and concentration of viable sperm for insemination Irvine Scientific manufactures several types of media for sperm cryopreservation transport and storage Irvin
93. sperm Depending on the antigen and the concentration of antibodies that are produced the sperm may begin to agglutinate or form clumps in the semen sample Another consequence of antibody formation occurs when the antibody bound sperm are exposed to proteins in the serum and other white cells of the immune system and result in sperm death This cytotoxic effect can be observed by a decline in motility and vitality in a semen specimen However this latter is a fairly rare phenomenon The major consequence of the presence of antibodies bound particularly to the head of the sperm is that the sperm may have a reduced capacity to bind to the zona pellucida of the oocyte inhibiting fertilization Antibodies present on sperm are not always directed at sperm specific antigens but may also be directed against molecules that are loosely attached to the sperm Antibodies of several different families may be produced against antigens on or associated with sperm Antibodies of the IgG type can appear in the gential tract by transudation from the serum Antibodies of the IgA type can be produced by cells within the mucosal lining of the tract IgM is found occasionally but appears to have no clinical significance for fertility Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Andrology Protocols Once antibodies are bound to the sperm as they move through the male reproductive tract particularly those of the IgA type they can in turn bind to ce
94. tate the laser lens into position and focus on the first embryo 11 The laser target should be placed over the zona the target is fixed so this is accomplished by moving the microscope stage in a place that has no blastomere directly underneath The safety circle around the target indicates the area through which heat from the laser will be dispersed Blastomeres should be outside this circle see Figures 3 and 4 5 11 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Micromanipulation Protocols Figure 3 Position the target on the zona making sure that the safety circle does not overlap a blastomere 12 An alternative method for making a hole is to line up the embryo with a blastomere directly under the area where the hole is to be made In this case the blastomere will receive some heat from the laser but that particular blastomere is the one to be biopsied so no harm will come to the embryo This method best allows the operator to get a cell with a nucleus at the first attempt since a cell with a clear nucleus can be targeted before ever making a breach in the zona Ba Figure 4 Embryo before and after Ist laser pulse 13 Once a hole has been made slip the biopsy pipette through and very gently aspirate a single blastomere Figure 5 Target a blastomere that is accessible close to the hole and one in which a nucleus can be seen Avoid pushing the pipette too far into the opening or up against other blastomeres Th
95. te 6 Gently place 1 5 2 0 mL of the liquefied semen onto the upper layer of the gradient system using a new sterile pipette If the total volume of semen is more than 2 0 mL then use the appropriate number of tubes and distribute the final semen volume accordingly Each tube of upper and lower layers can be used to process 1 3 mL of semen Additional volumes require additional tubes 7 Centrifuge for 20 minutes at 300 x g 8 At the completion of the spin remove the layers by inserting a clean 5 mL pipette tip just below the surface of the liquid Hold the tip in this position during aspiration Aspirate the layers without disturbing the sperm pellet at the bottom of the tube until approximately 0 5 mL of lower layer remains Even if a sperm pellet is not visible this volume should contain sperm If the sperm pellet occupies more than 0 5 mL at the bottom of the tube aspirate as much liquid from above the pellet as possible but leave the pellet intact 9 Using a new sterile pipette add 2 0 3 0mL of Sperm Washing Medium Cat 9983 or Modified Sperm Washing Medium Cat 9984 to the tube and resuspend the pellet Centrifuge at 300 x g for 10 minutes Remove supernatant with a clean pipette and add 2 3 mL of Sperm Washing Media again and centrifuge 10 After the second wash discard the supernatant and resuspend the pellet in 0 25 0 5 mL of Sperm Washing Medium if the sperm will be used for or fertilization media HTE P 1 ECM or the Comple
96. te Media series if the sperm will be used for IVF ICSI Place the tube containing the washed sperm in a warming block or water bath if it is to be used for IUI or into a CO incubator if the ns will be used for IVF ICSI For IUI procedures the sperm specimen should be concentrated in 0 25 0 50 mL to accommodate the volume capacity of the uterus Tubes should be kept at 377C until insemination by placing the tube in a CO incubator with the lid tightly sealed or in a warming block or water bath For IVF procedures a final dilution of the sperm specimen in a bicarbonate buffered media such as P 1 or the Complete Media series can be made depending on the insemination concentration and volume that is desired 11 Medium that does not contain protein should be supplemented and added prior to adding to the sperm Protein supplementation products include HSA Cat 49988 555 Cat 799193 and DSS Cat 49301 Prepare 5 mg mL of HSA or 1096 v v 555 or 1096 v v DSS in the appropriate volume of fertilization media IrvineScientific www irvinesci com Andrology Protocols One Layer Discontinuous Gradient Separation Procedure This method may also be used for normozoospermic patients or frozen specimens 1 Bring all of the media to 377C 2 If using a dilution other than 90 prepare appropriate dilution of ISolate 3 Using a sterile disposable pipette transfer 1 5 2 0 mL of the lower layer into a sterile disposa
97. terile wipe With the fine tip positioned over the thawing dish cut the seal on the 2nd mark of the fine tip end and dispense the contents of the CryoTip as a small drop with the embryos onto a dry region of the dish near but not in the TS drop Note avoid bubbles while dispensing the contents Oocytes embryos should immediately be visible in this drop The following steps should be done using a timer After each embryo transfer blow out the contents of the transfer pipette prior to the next manipulation Avoid creating bubbles during the transfers 9 Using the CryoTip pull up an equivalent volume 1 pL of TS and place it immediately adjacent to the already expelled contents of the CryoTip that contains the oocytes or embryos without touching the drop Then connect the two drops with the pipette tip and wait for one minute If no oocytes embryos are visible in this time check the blades of the scissors If no solution could be expelled from the tip then the tip may not have been cut far enough back to completely remove the seal Examine the tip under the scope to make sure that the seal has been cut away 10 Draw up some TS into the transfer pipette and transfer the oocytes or embryos with minimal volume into the bottom of the drop of TS 20 pL drop for one minute Transferring the oocytes or embryos from this point on may be done using a pulled Pasteur Pipette or a stripper tip of the appropriate size Note The cells will shrink and f
98. the sperm are carrying the beads with them as they move Record the site of binding of the beads to the sperm head midpiece tail head and tail 10 The test is positive if 2096 or more of the motile sperm have beads attached to them and is clinically significant if 5096 or more are coated with beads Binding of the beads to only the tail tip is not considered clinically significant ln IrvineScientific www irvinesci com Micromanipulation Protocols Micromanipulation Protocols OVERVIEW Micromanipulation media are designed for procedures including ICSI Intra Cytoplasmic Sperm Injection Assisted Hatching AH and Embryo Biopsy All three procedures are performed on an inverted microscope usually with Hoffman optics and using one of a variety of micromanipulation and microinjection devices that allow fine control of the pulled glass needles and holding pipettes that are used for these procedures All micromanipulation procedures should be done using a heated stage that has been calibrated to maintain the temperature of the media at 37 C within the dish that is being used Irvine Scientific Micromanipulation Media 1 Hyaluronidase Solution Cat 90101 Hyaluronidase is an enzymatic solution containing 80 IU mL of bovine derived hyaluronidase in a HEPES buffered HIF medium supplemented with 5 0 mg mL or 0 5 therapeutic grade Human Serum Albumin HSA It is designed for removing denuding cumulus and corona cells su
99. the sperm will be used for or fertilization media P 1 ECM or the Complete Media series if the sperm will be used for IVF ICSI Place the tube containing the washed sperm in a warming block or water bath if it is to be used for IUI or into a CO incubator if the ec will be used for IVF ICSI For IUI procedures the sperm specimen should be concentrated in 0 25 0 50 mL to accommodate the volume capacity of the uterus Tubes should be kept at 377C until insemination by placing the tube in a CO incubator with the lid tightly sealed or in a warming block or water bath For IVF procedures a final dilution of the sperm specimen in a bicarbonate buffered media such as 1 or the Complete Media series can be made depending on the insemination concentration and volume that is desired 10 Medium that does not contain protein should be supplemented and added prior to adding to the sperm Protein supplementation products include HSA Cat 9988 SSS Cat 99193 and DSS Car 9301 Prepare a 5 mg mL of HSA or 10 v v SSS or 10 v v DSS in the appropriate volume of fertilization media B SWIM UP PREPARATION General Considerations Recommended for use with samples that have normal sperm counts and motility Particularly appropriate for samples with elevated white cell contamination or when the lab wishes to reduce the risk of potentially generating reduced oxygen radicals by centrifugation Recovery counts and motility are m
100. tion and a known risk for a genetic defect are eligible for this procedure It is strongly recommended that ICSI fertilization is used for patients requesting embryo biopsy as this prevents possible contamination from sperm attaching to the blastomere during biopsy 3 The embryos to be biopsied should be at day 3 of development 72 hours from oocyte retrieval and have 5 or more cells Embryos with fewer cells or those that have not cleaved for 24 hours may not give reliable results and the viability of the embryos may be adversely impacted following the biopsy 4 The requirement is to get a cell containing a nucleus from each embryo If the first biopsied cell has an abnormal fragmented or multiple nucleus or no visible nucleus the embryo should be biopsied again until a cell with a normal nuclear number morphology is obtained In some instances this may adversely impact the viability of the embryo if too many blastomeres are removed 5 The cell should be biopsied using a biopsy pipette with an inner diameter of not less than 30 um 6 Stability of specimen The biopsied cell should be kept at room temperature and should be placed in lysis buffer in a PCR tube or lysed and fixed on a glass slide for transport to the genetics laboratory within one hour after the procedure Occasionally cells will be seen to lyse or die following biopsy and although these can be sent to the genetics laboratory they may not yield a result If the cell lyses
101. tion on the morning after the retrieval the dishes should be prepared on the afternoon of the day of oocyte retrieval and allowed to equilibrate overnight in the CO incubator particularly when using low O concentrations and an oil overlay Procedure 1 If conventional insemination was performed oocytes should be removed from their cumulus masses and examined for fertilization after 15 18 hours Cumulus masses and the surrounding coronal cells may be removed by aspirating oocytes up and down into 26 gauge needles manufactured denuding pipettes or finely pulled pasteur pipettes If ICSI was performed evidence of fertilization may be present as early as 12 hours post ICSI 2 The presence or absence of pronuclei should be assessed in the cleaned oocytes Rev 1 1 800 577 6097 949 261 7800 IrvineScientific logy Protocols 3 Oocytes with 2 pronuclei are normally fertilized The two pronuclei should be touching each other and be approximately at the center of the embryo where syngamy the conjugation of the male and female gametes occurs Oocytes with single pronuclei multiple pronuclei or fragmented pronuclei are abnormally fertilized Each laboratory should have a written policy documenting disposition policies for abnormally fertilized oocytes 4 Following syngamy of the two pronuclei the embyo begins to divide Embryo cleavage into two cells can be observed as early as 22 hours after insemination more commonly at 25 27 hours
102. ttached to the end of the cryostraw that will contain the embryos Bring the freezing media to room temperature 20 25 and aliquot 1mL of each medium F1 and F2 into pre labeled independent culture dishes or wells Identify the morphologically normal embryos to be frozen Transfer embryos to be frozen recommend 2 embryos straw to the FI medium for 10 minutes Move the embryos about two three times to rinse them The embryos will initially shrink and then return to the original size after equilibration Transfer the embryos to F2 Medium Allow pronucleate embryos to equilibrate for 2 3 minutes before loading Load multicellular embros immediately Load embryos into the pre rinsed straws shown as following IrvineScientific www irvinesci com Rev 1 6 8 Cryopreservation Protocols 10 11 Place the straws into freezing chamber start the freezing program Cool straws from room temperature to 6 C Alternatively the freezing chamber may be pre cooled and held 6 C until the straws are ready Once the straws are placed in the 6 C chamber they may be held for 5 minutes and then seeded Manually induce ice nucleation seed at 6 C by touching each straw near cotton plug with cold forceps or with a cotton swab that has been chilled by placing it in liquid nitrogen Hold the embryos 6 C for a total of 10 15 minutes NOTE When seeding do not touch the area of the straw that contains the embryos
103. tting away the cells surrounding the oocyte using conventional small bore injection needles or Short term exposure to Hyaluronidase Solution 80 IU mL to loosen up the cumulus corona cells A brief 30 second exposure to Hyaluronidase 50 pL drops or larger under oil is usually sufficient to initiate breakdown of the matrix holding the cumulus cells around the oocytes 2 After the 30 second exposure of the oocytes to the Hyaluronidase the oocytes is aspirated into a finely pulled pipette and transferred into Hyaluronidase free media such as mHTE P 1 ECM with protein HSA SSS or DSS Once the oocytes are in the rinse media they may be pulled repeatedly up and down in 135 150 um diameter pipette tips to remove the cells from around the oocytes 3 Following cumulus corona cell removal the oocytes should be transferred through several rinses to remove any remnants of the Hyaluronidase Solution 4 After all oocytes have been denuded and rinsed the presence or absence of a polar body may be observed Most laboratories choose to only perform ICSI on mature Metaphase II oocytes since oocytes at earlier stages of development do not possess either mature nuclei or cytoplasm Oocytes that are not at the MII stage may be cultured iv vitro until expulsion of the polar body and then injected if the laboratory chooses SPERM IMMOBILIZATION It became obvious early in the development of ICSI that fertilization rates increased when sperm wer
104. uld be monitored on a regular basis using a device that is routinely calibrated Irvine Scientific recommends performing all micromanipulation procedures on a heated stage maintained at 37 C and keeping all dishes under CO even when out of the main incubators to prevent pH drift unless using a HEPES buffered media 14 Culturing oocytes and embryos under oil may slow temperature pH and osmolality fluctuations and provide a barrier against airborne dust and microbial agents 15 Traditionally many laboratories have pre washed the oil used to overlay culture media Irvine Scientific oil Cat 9305 Oil for Embryo Culture does not require washing or filtration An aliquot of the oil may be kept in the incubator to allow it to be prewarmed before use but is not required as a step before preparing the dishes for oocyte retrieval and embryo culture 16 Generally it is recommended that all culture dishes and media to be used for a patient be prepared the day before and equilibrated overnight in an incubator that is gassed with CO However in the event of an emergency a minimum of 2 to 3 hours of equilibration may suffice for media that requires CO equilibration to attain the appropriate pH especially when using microdrops of media under an oil overlay Shorter times may be used if only temperature is of concern 17 Media that is used for oocyte fertilization and embryo culture should be supplemented with protein before use Media may be purch
105. um should be brought to room temperature It is important to add the cryopreservation medium to the semen specimen drop wise over a 30 second time period and mix it thoroughly after each drop is added to allow for adequate equilibration Note Sperm counts motility and morphology should be determined according to standard lab procedures established at each facility 0 000 20 6 3 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Cryopreservation Protocols Sperm Freezing Medium Cat 90128 General Considerations Store Freezing Medium TYB at 10 C or colder Do not expose medium to repeated freeze thaw cycles If smaller aliquots are desired thaw the product aliquot working volumes into sterile labeled containers and freeze until needed When stored as directed Freezing Medium TYB is stable until the expira tion date shown on the bottle label Freezing Procedure l Take a 5 OmL vial of Freezing Medium TYB out of the freezer and allow it to come to room temperature 20 25 After collection and liquefaction the semen specimen may be used as a raw specimen or it can be processed with a density gradient and washed with Sperm Washing Medium Cat 9983 prior to mixing with freezing medium Add a comparable volume of freezing medium TYB to the semen processed specimen to make a 1 1 v v dilution Add the freezing medium drop wise over a 30 second period Mix it thoroughly after each
106. up FIGURE 1 2 Transfer the oocytes from main culture dish into the 20 pL drop of culture medium CM Connect the drop of ES1 to the CM drop See Figure 2 arrow 1 moving from the ES1 drop toward the CM drop with the tip of the transfer pipette and allow gradual mixing of the two solutions to occur for 2 minutes Then connect the drop of ES2 arrow 2 to the merged drop of CM and 51 arrow 2 Allow gradual mixing of the 3 solutions to occur for 2 more minutes Then transfer the oocytes from the combined drops to the bottom of the ES3 drop for 3 minutes using a pulled Pasteur Pipette or a stripper tip of the appropriate size FIGURE 2 6 15 Rev 1 1 800 577 6097 949 261 7800 IrvineScientific Cryopreservation Protocols 3 During the 3 minutes waiting time set up 4 drops of VS solution as shown on Figure 3 below FIGURE 3 4 Transfer the oocyte to the BOTTOM of the first drop of VS VS1 for 5 seconds counting mentally After 5 seconds transfer the oocytes to the second drop where it will remain for 5 seconds then to the third drop for 10 seconds and finally into the last drop As with embryos oocytes will also float to the top of the drop Therefore observe the moving of the oocytes through the drops from a low enough power to be able to locate the oocytes at the top of the drop after placing them at the bottom Once the oocytes are in the final drop they should immediately be loaded into the CryoTip the tip sealed and
107. ween washes The final pellet should be resuspended in fertilization media P 1 ECM or the Complete Media series containing protein HSA SSS or DSS and the tube placed in a CO incubator Alternatively sperm may be expelled from testicular tissue by dissecting out the seminiferous tubules from the surrounding cells then using the edge of a glass slide or the back of the bevel of a needle to apply pressure to the tubules to expel the sperm Samples may then be washed resuspended in a fertilization media of choice and allowed to stand in a incubator until use or frozen if being used at a later date f 3 Epididymal samples or ejaculates with extremely low count and or motility may be processed by gently pipetting 1 5 2 0 mL of into a sterile centrifuge tube adding 2 0 3 0 mL of Sperm Washing Medium and gently mixing the contents by pulling the solut in and out ofa 5 mL pipette If the total volume of semen is more than 2 0 mL use the appropriate number of tubes and the sperm sample volume accordingly Centrifuge at 300 g for 5 10 minutes Remove supernatant and gently resuspend the pellet with 2 0 3 0 mL of Sperm Washing Medium and centrifuge for a second time for another 5 10 minutes 4 After the final wash discard the supernatant and resuspend the pellet in 0 25 0 5 mL Sperm Washing Medium or the appropriate medium depending on the next procedure Place the tube containing the washed sperm in a CO incubator until it is t
108. with medications to increase the number of mature oocytes that can be collected and fertilized at one time Occasionally patients may undergo a natural cycle that is relatively drug free or uses significantly reduced levels of medications Oocyte retrieval is commonly performed by ultrasound guided visualization of the follicles within the ovary Aspirated follicular fluid is collected in tubes and then examined under a microscope for the presence of oocytes Oocytes are isolated from follicular fluid rinsed and placed in culture for insemination Procedure 1 Pre warm all the surfaces and materials to 37 C that may come in contact with the oocytes 2 All of the tubes pipettes and dishes should be labeled with the patient s ID before proceeding with the collection Color coding can be used to ensure patient s identification 3 Dishes that will be used for oocytes culture should be prepared at least four hours in advance However there is some data showing that the equilibration rate of media to CO is much faster than that to O Therefore when using a low oxygen culture environment and an oil overlay dishes should be prepared the day before oocyte retrieval 4 Pre equilibrate flushing medium at 37 C in an incubator without CO or in a CO incubator with the tubes bottle tightly capped if using a HEPES buffered media Cat 90126 mHTF If using an Chamber with bicarbonate buffered media P 1 MultiBlast or th
109. xample of the steps involved in a QC QA QI program a Laboratory Director may believe that controlling pH during all phases of the IVF program is an important step in improving pregnancy outcomes or improving fertilization rates or cleavage outcomes any or all of these may be determined to be the appropriate indicator Therefore it would be up to the director to plan an overall program to assess and control pH The first step to establishing would be to initially determine how to assess pH which instrument to use to measure pH when and where to measure it in the incubators in a Hoffman chamber on the benchtop etc and how often to measure it The Director should then create a series of tools ie data sheets that will help the lab staff collect the data that is necessary to assess the affect of pH The act of measuring pH on a regular basis and in a defined manner would be the QC portion of the overall QI program The lab staff would begin measuring pH in the prescribed manner At regular intervals the Director would review the data collected and determine whether the appropriate data is being collected and what if anything should be changed The Director also determines whether the pH determinations are being performed correctly and if the pH is falling within the range that is expected or identified as being the range that the Director is interested in If any observations are outside the expected range then the instrumentation should be chan
110. xposure of the embryo to the acidified Tyrode s 12 Remove dish from the field of view and return the dish with the drop of acid in it Lower hatching pipette into the Tyrode s and let it re equilibrate 13 Transfer the embryo from the hatching drop through at least three rinses in media with protein Place it in the dish that will be used for transfer 14 Repeat with remaining embryos IrvineScientific www irvinesci com Rev 1 5 6 Micromanipulation Protocols Procedure for assisted hatching for implantation improvement using Partial Zona Dissection 1 The use of Acidifed Tyrodes Solution is not necessary when using PZD 2 Place the holding pipette on the left Align the microtools in the center of the field of view then raise both tools 3 Prepare a dish for PZD using Modified HTF plus 5 mg mL HSA or 10 v v SSS or 10 v v DSS Place 2 4 x 10 pL drops in a circle and cover with oil 4 Transfer one embryo into the first drop Place the dish onto the micromanipulation microscope stage and lower the holding pipette 5 Suck the embryo onto the holding pipette aligning the embryo such that the biggest space within the embryo or the widest distance between blastomeres is oriented at the top of the embryo 6 Insert the PZD needle from right to left across the top of the embryo entering at about the one o clock position and exiting at the 11 o clock position without entering any blastomeres The embryo wi
111. y 3 blastocele expansion appearance of the inner cell mass and trophectoderm cellularity and continuity 3 Ifembryos are to be cultured to the blastocyst stage transfer embryos from P 1 or ECM into MultiBlast Medium Cat 90139 supplemented with 5 mg mL of HSA 10 v v SSS or 10 v v DSS Embryos may be transferred into this media on the afternoon of Day 2 or on the morning of Day 3 Culture dishes with MultiBlast Medium should be set up on the morning of Day 2 if the intent is to place the embryos into blastocyst culture on the afternoon of Day 2 or set up on the afternoon of Day 2 if embryos will be transferred into blastocyst media on the morning of Day 3 4 Blastocyst culture may be performed using slightly higher pH values pH 7 30 if so desired by the laboratory however the optimal in vitro media pH value for each stage of embryo development has yet to be determined EMBRYO TRANSFER General Considerations Following culture of the embryos they must be placed back into the uterus A great deal has been written about transfer technique and again there is little agreement as to the proper technique IrvineScientific www irvinesci com Rev 1 3 6 Embryology Protocols Procedure i Most programs elect to use ultrasound guidance for transfers as observation of catheter placement with ultrasound appears to improve pregnancy rates Patients preparing to have this procedure done will need to have full bladder Flexible
112. y incubator may be different Therefore pH needs to be assessed in each incubator and each incubator should be individually adjusted 9 Irvine Scientific manufactures its media to attain an osmolality of 282 290 Osmols 10 Each laboratory needs to determine the best method to use specify pass fail values and rejection procedures IrvineScientific www irvinesci com Rev 1 3 2 Embruologu Protocols 11 Plasticware may be off gassed by opening the sleeves of dishes or the packages of pipettes and allowing them to sit in room air for at least 24 hours prior to use 12 The use of filter systems for room air and or incubator gases is advocated but may not be reguired Ihe decision to use filter systems is laboratory dependent and may be driven by laboratory location Laboratories in busy metropolitan areas with high levels of pollution may choose to employ more filtration systems than laboratories located in suburban areas or those with lower levels of air pollutants 13 Oocytes and embryos appear to be exguisitely sensitive to temperature fluctuations Allowing the temperature of the media that contains oocytes and embryos to drift from 37 C may be detrimental to the developmental potential of the embryo Therefore once the oocytes leave the body all handling and manipulations of the oocytes and embryos should be performed under tightly controlled conditions Temperature measurements of media on the stages of microscopes or in incubators sho
113. y of pressure devices may be used to expel the embryos from the CryoTip These include but are not limited to 250 pL Gas tight Hamilton syringes 1cc Airtight syringes Eppendorf or Gilson re pipettors or Mushroom microinjectors Each lab should determine which device works best for them It is very important to cut the thin end where embryos located on top of the dish since the pressure inside the tip after thawing will push out the contents after cutting Cutting the tip should be done using very fine scissors and a quick cutting motion Check the blades of the scissors after cutting to make sure that no fluid from the tip has accumulated on the blades If no oocytes embryos are found in the expelled solution then the fluid on the blade may contain the oocytes embryos and should be washed off and collected in a small volume of TS Bubbles are detrimental to fragile thawed embryos Care should be taken to avoid the formation of bubbles while pushing out the contents from the tip All drops are 20 pL and thawing should be done with drops at room temperature and without an oil overlay Allow 3 hours for oocytes recovery after thawing before performing ICSI to inseminate the oocytes Procedure for Thawing of Oocytes and Embryos 2PN to Blastocyst 1 Bring one vial each of Thaw Solution TS yellow top Dilution Solution DS orange top and Washing Solution WS red top to room temperature 20 27 C for at least 30 minutes prior to thawi

In-vitro Fertilization Procedural Manual - MTG

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