Oocyte and Embryo Cryopreservation and vitrification

PRINCIPLES OF CRYOPRESERVATION

As mentioned previously, in the 1940s it was discovered that addition ofglycerol protected against cryo-damage and greatly enhanced survival of cryopreserved living cells. This led to the investigational concept of cryoprotectants In hindsight, the use of cryoprotectants is logical considering that, in insect biological systems, sugars and sugar-alcohols are used to withstand severe winter temperatures. Experience in cryopreservation of various cell types led to the appreciation that as cell size increases, difficulty in cryopreservation also increases. This concept is of particular importance in mammalian oocyte and embryo cryopreservation.
Currently, there are two methods used to cryopreserve mammalian oocytes and embryos: slow-rate freezing and vitrification . Independent of the methodology used for cryopreservation, effects on oocyte and embryonic cellular functions can compromise abilities to develop normally following the cryopreservation process. These compromised cellular events can be collectively termed oocyte and/or embryo "cryo-damage." Documented and/or theoretical-specific cellular structures and functions that are/may be compromised by cryopreservation, as well as subsequent effects on oocyte and embryonic developmental competence have been previously reviewed.
During cryopreservation, cells are exposed to numerous stresses including mechanical, thermal, and chemical, which can lead to compromised cell function and cell death. In general, it has been demonstrated that oocytes are more sensitive to cryo-damage than later embryonic stages. A detailed discussion of the biophysics of cryopreservation is beyond the scope of this review, yet such information is available. Slow-rate freezing attempts to control biophysical properties of freezing, such as cooling and warming rates, in conjunction with cryoprotectants to minimize adverse cellular events. This method allows cells to be cooled to very low temperatures while minimizing intracellular ice crystal formation, and at the same time attempting to minimize the detrimental influences of increased solute concentrations and osmotic stress. Thus one can appreciate that with slow-rate freezing extracellular ice formation drives cellular dehydration through an equilibrium process. Conversely, vitrification, a form of rapid cooling, utilizes very high concentrations of cryoprotectant that solidify without forming ice crystals. The term "vitrification" is derived from the Latin word vitreous, which means glassy or resembling glass. Vitrification can be considered a non-equilibrium approach to cryopreservation originally developed for cryopreservation of mammalian sperm and embryos. The vitrified solids therefore contain the normal molecular and ionic distributions of the original liquid state and can be considered an extremely viscous, supercooled liquid. In this technique, oocytes or embryos are dehydrated by brief exposure to a concentrated solution of cryoprotectant before plunging the samples directly into liquid nitrogen. Utilization of vitrification for both oocytes and embryos is an area of current focus for many clinical, rodent, and domestic animal production laboratories. Human oocytes , pronuclear zygotes, cleavage-stage embryos, and blastocysts have been successfully vitrified. Excellent reviews of vitrification history, utilization, and potential advantages are available.

PROTOCOLS

It is important to recognize that there are important fundamental aspects of gamete/embryo cryopreservation that must be followed for successful cell survival,normal cellular function, and subsequent development. It is important also to recognize that numerous slight permutations exist between cryopreservation protocols that do not appear to influence clinical outcomes. This generalizing statement, while non-scientific and potentially concerning, is a result of inabilities to perform well-controlled, randomized, prospective clinical trials with human oocytes/embryos. Thus, keys to successful gamete/embryo cryopreservation are basing protocols on a combined strong understanding of cell developmental biology, empirical and theoretical aspects of cryobiology, and practical experience. Below are protocols for both slow-rate freezing/thawing and vitrification/warming that are known to work well for oocytes, cleavage-stage embryos, and blastocysts. Obviously, there are numerous protocols that vary slightly from those described below, and are known to provide commendable results.

Slow-Rate Freezing

Oocytes

Numerous reports of successful human oocyte slow-rate freezing and thawing with subsequent healthy live births exist. The following is a human oocyte slow-rate freezing protocol based on work from Porcu et al.

Freezing:

1. Materials (expendables): Conical tubes, organ culture dish, 5-and 10-mL disposable pipettes, pulled-pipettes-inner diameter just larger than the oocytes being cryopreserved, 0.2 mM filter, cryo-straws, cryo-canes, cryo-goblets, and cryo-sleeves.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, labeling device, timer, heat-sealer, programmable biological freezer, liquid nitrogen storage canister with lock and alarm, safety goggles, and cryo-gloves.

3. Cryo-solutions: Freeze medium #1 ¼ phosphate-buffered saline (PBS) 18%(w/v) protein source (WASH) Freeze medium #2¼ freeze medium #1 1.5M 1,2-PROH Freeze medium #3 ¼ freeze medium #2 0.3M sucrose (PROH SUCR)

4. Procedure :
a. Prepare freeze media inside the hood, at least one day prior to use. Rinse all tubes, syringes, and filters with PBS prior to use. Filter all freeze media with a 0.2-mM syringe filter.

b. Label three organ culture dishes with patient identifiers and one each with WASH, PROH, PROH SUCR. After rinsing dishes, add 1mL of the appropriate medium to each
dish and keep at room temperature.

c. Two to three hours after egg retrieval, denude oocytes and
select MII oocytes for freezing.

d. Transfer oocytes for freezing into WASH.
_ When transferring oocytes to a dish, always aspirate a small volume of the medium into which you are moving the oocytes prior to picking them up.
_ Move the oocytes in as small a volume of medium as possible.
_ Place the oocytes in at least three different areas of the dish to fully rinse them of the last medium.

e. Transfer oocytes into PROH for 10 minutes.

f. Transfer oocytes in PROH SUCR for five minutes.
During that five minutes period:
_ Label the straw(s), canes(s), goblet(s), and or sleeve(s) with patient identifiers.
_ Rinse the straw(s) with PROH SUCR and expel (do not let medium touch the cotton-plug/sealant end).

_ Load the straw(s) with PROH SUCR and oocytes in the following order:

a. 1-inch medium

B. 1/4-inch air space

C. 1/2-inch medium

D. 1-inch medium containing oocytes

E. 1/4-inch air space

f. Fill the remainder of the straw with medium. Make sure the medium completely wets the strawsealant at the end of the straw to ensure a proper seal.
_ Seal the straw(s)
_ Place straw(s) into programmable biological freezer with the sealant plug toward the top.

g. Once straw(s) are loaded into the programmable freezer begin the program.

h. When the temperature changes to _7_C, hold for five minutes, then seed straw(s) by briefly touching a liquidnitrogen- soaked cotton-tipped applicator at the meniscus
Just below the first airspace.

I. Wait two minutes, then inspect the seeded straw(s) to confirm ice crystal formation/growth.

j. When the programmable biological freezer temperature reaches _30_C, it can initiate free-fall to _150_C. Samples can then be removed and plunged into liquid nitrogen after ten to twelve minutes of stabilization at this temperature. During transfer, use goggles, cryo-protective gloves, and liquid-nitrogen-cooled forceps. Place straw(s) into liquidnitrogen- Submerged goblet on cane, and rapidly transfer into liquid nitrogen storage canister.

Thawing:

1. Materials (expendables): Conical tubes, organ culture dish, 5 and 10mL disposable pipettes, pulled-pipettes-inner diameter just larger than the oocytes being cryopreserved, 0.2 mM filter, and paper tissues.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, Dewar, timer, scissors, straw plunger, safety goggles and cryo-gloves, incubators (CO2 and non- CO2, 37_C).

3. Thaw-solutions: Thaw medium #1¼PBS 18%(w/v) protein source (TM1 or WASH)
Thaw medium #2¼TM1 0.3M sucrose (SUCR)
Thaw medium #3¼ TM1 1.0M PROH 0.3M sucrose
(1.0M PROH SUCR)
Thaw medium #4¼ TM1  0.5M PROH 0.3M sucrose
(0.5M PROH SUCR).

4. Procedure:
a. Prepare thaw media inside the hood, at least one day prior to use. Rinse all tubes, syringes, and filters with PBS prior to use. Filter all thaw media with a 0.2 mM syringe filter.

b. Prepare oocyte culture dishes and place into CO2 incubator at least four hours before thaw.

c. Label five organ culture dishes with patient identifiers and one each with 1.0M PROH SUCR, 0.5M PROP SUCR, SUCR, WASH1, WASH2. After rinsing dishes, add 1.0mL of the appropriate medium to each dish and keep at room temperature (except for WASH2_37_C).

d. Transfer the cryo-straw(s) containing oocytes to be thawed from the liquid nitrogen storage canister to a filled liquidnitrogen- portable Dewar. Always wear protective goggles and gloves when handling liquid nitrogen. Check the cryo-straw out of the patient oocyte logbook and out of the patient's cryopreservation record. Transport the cryostraw(s) in liquid nitrogen to the site of thaw.

f. Gently wipe the straw to remove condensation.

g. Submerge the straw in 30_C water bath for 40 seconds.

h. Remove the straw from the water bath and wipe dry.

i. Expel the oocytes from the straw.
_ Hold the straw vertically over the organ culture dish containing 1.0M PROH SUCR with the sealed, cotton-plug end up.
_ Cut the sealed end of the straw, opposite to the cotton-plug end.
_ Cut the cotton-plug end in the middle of the seal.
_ Use a straw plunger to expel the contents of the straw into the organ culture dish containing 1.0MPROH SUCR.
_ Move the oocytes to three different areas of the dish and hold for five minutes.

j. Transfer oocytes to 0.5MPROH SUCR for five minutes.
_ When transferring oocytes to another dish always aspirate, a small volume of the medium into which you are moving oocytes prior to picking them up.
_ Move oocytes in as small of a volume of medium as possible.
_ Place oocytes in at least three different areas of the dish to fully rinse them of the last medium.

k. Transfer oocytes to SUCR and leave oocytes in this solution for 10 minutes.

l. Transfer oocytes to WASH1 and leave oocytes in this solution for 10 minutes.

m. Transfer oocytes to WASH2 (at 37_C) and leave oocytes in this solution for 10 minutes.

n. Transfer oocytes into oocyte-culture dish (bicarbonate-buffered media protein) and place into CO2 incubator for four hours before proceeding with intracytoplasmic sperm injection.

Cleavage-Stage Embryos

Cleavage-stage embryos can be frozen successfully on either day 2 or 3 (d0 ¼ day of insemination). It is important to note that embryos can be cryopreserved in straws or vials. The below protocol is based on use of straws as cryo-containers.

Freezing:

1. Materials (expendables): Conical tubes, organ culture dish, 5- and 10-mL disposable pipettes, pulled-pipettes-inner diameter just larger than the embryos being cryopreserved, 0.2 mm filter, cryo-straws, cryo-canes, cryo-goblets, and cryo-sleeves.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, labeling device, timer, heat-sealer, programmable biological freezer, liquid nitrogen storage canister with lock and alarm, safety goggles, and cryo-gloves.

3. Cryo-solutions: Freeze medium #1¼HEPES [4(2-hydroxyethyl)1-piperazineethane sulfonic acid)]-buffered medium 1215% (w/v) protein source (WASH) Freeze medium #2¼freeze medium #1 1.5M 1,2-PROH Freeze medium #3¼freeze medium #2 0.1M sucrose (PROH SUCR).

4. Procedure
a. Prepare freeze media inside the hood, at least one day prior to use. Rinse all tubes, syringes, and filters with HEPESbuffered media prior to use. Filter all freeze media with a 0.2 mM syringe filter.

b. Label four organ culture dishes with patient identifiers and one each with WASH1, WASH2, PROH, PROH SUCR. After rinsing dishes, add 1mL of the appropriate medium to each dish and keep at room temperature.

c. Select embryos to freeze that meet your specific freezing criteria.

_When transferring embryos to a dish, always aspirate a small volume of the medium into which you are moving the embryos prior to picking them up.
_ Move the embryos in as small of a volume of medium as possible.
_ Place the embryos in at least three different areas of the dish to fully rinse them of the last medium.

e. Transfer embryos into WASH2 dish.

f. Transfer embryos into PROH for 15 minutes.

g. Transfer embryos into PROH SUCR for 15 minutes.

During that 15-minute period:
_ Label the straw(s), canes(s), goblet(s), and or sleeve(s) with the patient identifiers.
_ Rinse the straw(s) with PROH SUCR and expel (do not let medium touch pre-sealed end).
_ Load the straw(s) with PROH SUCR and embryos in the following order:

a. 1-inch medium
b. 1/4-inch air space
c. 1/2-inch medium
d. 1-inch medium containing embryos
e. 1/4-in air space
f. Fill the remainder of the straw with medium.

Make sure the medium completely wets the presealed end of the straw to ensure a proper seal.
_ Seal the straw(s)
_Place straw(s) into programmable biological freeze.

h. Once straw(s) are loaded into the programmable freezer, begin the program. The program listed below is one of many that have been successfully used to cryopreserve embryos.

I. When the temperature changes to _5 to _7 (seeding temperature), hold for five minutes, then seed straw(s) by briefly touching a liquid-nitrogen-soaked cotton-tipped applicator at the meniscus just below the first airspace.

j. Wait two minutes; inspect the seeded straw(s) to confirm ice crystal formation/growth.

k. When the programmable biological freezer temperature reaches _32_C, the programmable biological freezer can initiate free-fall or samples can be removed and plunged into liquid nitrogen. During transfer, use goggles, cryo-protective gloves, and liquid-nitrogen-cooled forceps. Place straw(s) into liquid-nitrogen-submerged goblet on cane, and rapidly transfer into liquid nitrogen storage canister.

Thawing:

1. Materials (expendables): Conical tubes, organ culture dish, 5- and 10-mL disposable pipettes, puller-pipettes-inner diameter just larger then the embryos being cryopreserved, 0.2 mm filter, paper tissues.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, Dewar, timer, scissors, straw plunger, safety goggles and cryo-gloves.

3. Thaw-solutions: Thaw medium #1¼HEPES-buffered medium 1215% (w/ v) protein source (TM1 or WASH) Thaw medium #2 ¼ TM1 0.2M sucrose (SUCR) Thaw medium #3¼TM1 1.0M PROH 0.2M sucrose (1.0M PROH SUCR) Thaw medium #4¼TM1 0.5M PROH 0.2M sucrose (0.5M PROH SUCR).

4. Procedure
a. Prepare thaw media inside the hood at least one day prior to use. Rinse all tubes, syringes, and filters with HEPESbuffered medium prior to use. Filter all thaw media with a 0.2 mM syringe filter.

b. Prepare embryo culture dishes and place into CO2 incubator at least four hours before thaw.

c. Label five organ culture dishes with the patient identifiers and one each with 1.0M PROH SUCR, 0.5M PROH SUCR, SUCR, WASH1, WASH2. After rinsing dishes, add 1.0mL of the appropriate medium to each dish and keep at room temperature.

d. Transfer the cryo-straw(s) containing embryos to be thawed from the liquid nitrogen storage canister to a filled liquid-nitrogen-portable Dewar. Always wear protective goggles and gloves when handling liquid nitrogen. Check the cryo-straw out of the patient embryo logbook and out of the patient's cryopreservation record. Transport the cryo-straw(s) in liquid nitrogen to the site of thaw.

e. Remove the straw from the liquid-nitrogen-filled Dewar with forceps. Hold the straw horizontally at the sealed, cotton-plugged end and allow the straw to partially thaw at room temperature for 40 seconds.

f. Gently rub the straw between your fingers with a paper tissue until all the ice crystals have disappeared.

g. Expel the embryos from the straw.

_ Hold the straw vertically over the organ culture dish containing 1.0M PROH SUCR with the sealed, cotton-plug end up.
_Cut the sealed end of the straw, opposite to the cotton- plug end.
_Cut the cotton-plug end in the middle of the seal.
_ Use a straw plunger to expel the contents of the straw into the organ culture dish containing 1.0M PROH SUCR.
_ Move the embryos to three different areas of the dish and hold for five minutes.

h. Transfer embryos to 0.5M PROH SUCR for five minutes.
_When transferring embryos to another dish, always aspirate a small volume of the medium into which you are moving embryos prior to picking them up.
_ Move embryos in as small of a volume of medium as possible.
_Place embryos in at least three different areas of the dish to fully rinse them of the last medium.

I. Transfer embryos to SUCR for five minutes.

j. Transfer embryos to WASH1; rinse for _3060 seconds.

k. Transfer embryos to WASH2; rinse for _3060 seconds.

l. Check and record embryo development. Transfer embryos to a CO2-equilibrated medium embryo culture dish and replace the dish into the CO2 incubator.

Blastocyst

The cryopreservation of preimplantation human embryos at the blastocyststage has become an important part of many ART programs. The following protocol is one of many based on pioneering work by Menezo utilizing glycerol and sucrose as permeating and non-permeating cryoprotectants, respectively. This protocol utilizes cryo-straws as the cryo-container.

Freezing:

1. Materials (expendables): Conical tubes, organ culture dish, 5- and 10-mL disposable pipettes, pulled pipettes-inner diameter just larger then the blastocysts being cryopreserved, 0.2 mm filter, cryo-straws, cryo-canes, cryo-goblets, and cryo-sleeves.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, labeling device, timer, heat-sealer, programmable biological freezer, liquid nitrogen storage canister with lock and alarm, safety goggles, and cryo-gloves.

3. Cryo-solutions: Freeze medium#1¼HEPES-buffered medium 1215% (w/v) protein source (WASH) Freeze medium #2¼freeze medium #1 5% (v/v) glycerol (5GLYC) Freeze medium #3 ¼freeze medium #1 9% (v/v) glycerol 0.2M sucrose (9GLYC SUCR).

4. Procedure
a. Prepare freeze media inside the hood at least one day prior to use. Rinse all tubes, syringes, and filters prior to use. Filter all freeze media with a 0.2 mM syringe filter.

b. Label four organ culture dishes with the patient identifiers and one each with WASH1, WASH2, 5GLYC, 9GLYC SUCR. After rinsing dishes, add 1mL of the appropriate medium to each dish and keep at room temperature.

c. Select blastocysts that meet your specific freezing criteria to freeze.

d. Transfer blastocysts to freeze into WASH1.

_ When transferring blastocysts to a dish, always aspirate a small volume of the medium into which you are moving the blastocysts prior to picking them up.
_ Move the blastocyst(s) in as small of a volume of medium as possible.
_ Place the blastocyst(s) in at least three different areas of the dish to fully rinse them of the last medium.

e. Transfer blastocyst(s) into WASH2 dish.

f. Transfer blastocyst(s) into 5GLYC for 10 minutes.

g. Transfer blastocyst(s) into 9GLYC SUCR for 10 minutes.

During this 10 min period:
_ Label the straw(s), canes(s), goblet(s), and or sleeve(s) with the patient identifiers.
_ Rinse the straw(s) with 9GLYC SUCR and expel (do not let medium touch pre-sealed end).
_ Load the straw(s) with 9GLYC SUCR and blastocysts.
in the following order:

a. 1-inch 9GLYC SUCR
b. 1-inch air space
c. 1/2-inch 9GLYC SUCR
d. 1-inch 9GLYC SUCR containing blastocysts
e. 1/4-inch air space
f. Fill the remainder of the straw with 9GLYC SUCR. Make sure the medium completely wets
the presealed end of the straw to ensure a proper seal.
_ Seal the straw(s)
_ Place straw(s) into programmable biological freeze.

h. Once straw(s) are loaded into the programmable freezer, begin the program. The program listed below is just one of many that has been successfully used to cryopreserve
blastocyst(s).

i. When the temperature changes to _7_C, hold for five minutes, then seed straw(s) by briefly touching a LN2-soaked cotton-tipped applicator at the meniscus just below the first airspace.

j. Wait two minutes; inspect the seeded straw(s) to confirm ice crystal formation/growth.

k. When the programmable biological freezer temperature reaches _30_C, it can initiate free-fall too approximately _100_C. Subsequently samples can be removed and plunged into liquid nitrogen. During transfer, use goggles, cryoprotective gloves, and liquid-nitrogen-cooled forceps. Place straw(s) into liquid-nitrogen-submerged goblet on cane, and rapidly transfer into liquid nitrogen storage canister.

Thawing:

1. Materials (expendables): Conical tubes, organ culture dish, 5- and 10-mL disposable pipettes, pulled-pipettes-inner diameter just larger then the blastocysts being cryopreserved, 0.2 mm filter, paper tissues.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, Dewar, timer, scissors, straw plunger, safety goggles, and cryo-gloves.

4. Procedure
a. Prepare thaw media inside the hood at least one day prior to use. Rinse all tubes, syringes, and filters with HEPESbuffered medium prior to use. Filter all thaw media with a 0.2 mM syringe filter.

b. Prepare blastocyst culture dishes and place into CO2 incubator at least four hours before thaw.

c. Label three organ culture dishes with the patient identifiers and one each with 0.5SUCR, 0.2SUCR, WASH. After rinsing dishes, add 1.0mL of the appropriate medium to each dish and keep at room temperature.

d. Transfer the cryo-straw(s) containing blastocysts to be thawed from the liquid nitrogen storage canister to a filled liquid-nitrogen-portable Dewar. Always wear protective goggles and gloves when handling liquid nitrogen. Check the cryo-straw out of the patient embryo logbook and out of the patient's cryopreservation record. Transport the cryo-straw(s) in liquid nitrogen to the site of thaw.

e. Remove the straw from the liquid-nitrogen-filled Dewar with forceps. Hold the straw horizontally at the sealed, cotton-plugged end and allow the straw to partially thaw at room temperature for 30 seconds.

f. Gently wipe the condensation off the straw with a paper tissue.

g. Submerge the straw into a 30_C water bath for 45 seconds.

h. Remove the straw from the water bath and gently wipe dry with a paper tissue.

i. Expel the embryos from the straw.
_ Hold the strawvertically over the organ culture dish containing 0.5SUCR with the sealed, cotton-plug end up.
_ Cut the sealed end of the straw, opposite to the cotton- plug end.
_ Cut the cotton-plug end in the middle of the seal.
_ Use the straw plunger to expel the contents of the straw into the organ culture dish containing 0.5SUCR.
_ Move blastocysts to three different areas of the dish and hold for 10 minutes.

j. Transfer blastocysts into 0.2SUCR.
_ Use a pulled pipette with diameter just larger than the blastocysts
_ Before loading blastocysts into pipette, partially fill the pipette with 0.2SUCR.
_ Pick up the blastocysts with the least amount of 0.5SUCR.
_ place the blastocysts into 0.2SUCR, pick them up and move (release) them into three separate areas within the 0.2SUCR.

k. Leave the blastocysts in 0.2SUCR for 10 minutes.

l. Move blastocysts into WASH, in a similar manner as mentioned above, and leave for five minutes.

m. Check and record embryo development. Transfer embryos to a CO2-equilibrated medium embryo culture dish and replace the dish into the CO2 incubator.

Vitrification

It is important to recognize that while a recent surge in interest of vitrification has been experienced in the field of human ART, this is not a new technology. Pioneering work by the likes of Polge et al., Rall and Fahy , and Mazur et al. have provided the foundation for current
research and application of vitrification for human oocytes and embryos. In addition, the importance of extensive experience in vitrification of domestic animal oocytes and embryos is immeasurable.

Oocytes numerous reports of successful human oocyte vitrification, followed by fertilization, embryo development and transfer, and healthy live births exist. The following is ahuman oocyte vitrification protocol that has been successfully used for mouse, bovine, and human oocyte cryopreservation. Vitrifying:

1. Materials (expendables): Conical tubes, tissue culture dish, organ culture dish, 5- and 10-mL disposable pipettes, pulledpipettes- inner diameter just larger than the oocytes being
cryopreserved, 0.2 mm filter, 20 ml pipette tips, cryo-goblet, cryo-cane, cryo-sleeve, 1-cm3 syringe, "pulled-straw" with an inner diameter of approximately 200 mm, syringe"pulledstraw" connector.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, labeling device, timer, heat-sealer, forceps, liquid nitrogen Dewar, liquid nitrogen storage canister with lock and alarm, safety goggles, and cryo-gloves.

3. Cryo-solutions:
Wash solution¼HEPES-buffered medium 12% (w/v) protein source (WASH) Equilibration solution (ES)¼HEPES-buffered medium 7.5% (v/v) ethylene glycol 7.5% DMSO 12% (w/v) protein source Vitrification solution (VS)¼HEPES-buffered medium 15% (v/v) ethylene glycol 15% DMSO 0.5M sucrose 12% (w/v) protein source.

4. Procedure
a. Prepare vitrification media inside the hood at least one day prior to use. Rinse all tubes, syringes, and filters prior to use. Filter all cryo-media with a 0.2 mM syringe filter. Prepare oocyte culture dish at least four hours before use and allow equilibrating in CO2 incubator.

b. Warm WASH, ES, and VS to room temperature. Make certain the solutions are mixed and homogeneous.

c. For each straw of oocytes being vitrified, prepare a culture dish with patient identifiers and written indication of 20- mL drop placements. In addition, label the "pulled-straw" with proper patient identifiers.

d. Immediately before beginning the procedure, dispense 20 mL drops of WASH (one drop), ES (three drops), and VS (four drops) onto the culture dish. Alternatively, the VS drops can be dispensed when oocytes are in the ES3 drop.

E. With a pulled-pipette and the least amount of medium carry-over, transfer denuded metaphase II (MII) oocytes from culture medium into WASH drop for one minute.

F. Using the pulled-pipette tip, merge the ES1 drop with WASH drop in a dragging motion from ES1 to WASH. There is no need to move oocytes. Set timer for two minutes.

g. When timer sounds, merge ES2 drop with the WASH/ ES1 drop in a similar manner, dragging from ES2 toward WASH. Set timer for two minutes.

h. When timer sounds, transfer oocytes using a pulled-pipette from merged drops to ES3 drop. Leave in ES3 for three minutes. During this three minute period, prepare the pulled straw for loading, sealing, and plunging.

Figure 2 Photograph representation of solution drop orientation used for oocyte vitrification.
Drops are 20 mL each and at room temperature. Abbreviations: HTF-H, human tubal fluid-HEPES medium; ES, equilibration solution; VS, vitrification solution.

I. Using a pulled-pipette, transfer oocyte through the VS drops. This should be done with microscope visualization and manual counting.
_ Move oocytes into the VS1 drop for five seconds.
_ Move oocytes into the VS2 drop for five seconds.
_ Move oocytes into the VS3 drop for 10 seconds.
_ Move oocytes into the VS4 drop.

j. The goal at this point is to load the pulled-straw, seal the straw, and plunge the straw in liquid nitrogen within a 90-second interval.
_ To load the pulled straw, aspirate VS4 into straw to the first line (closest to the fine-pulled tip), aspirate VS4 and oocytes so that the fluid meniscus reaches the second line, aspirate additional VS4 so that the fluid meniscus reaches the third line.
_ Heat-seal the narrow end of the straw just below the first mark, and then above the fourth mark.
_ Hold the sealed straw with forceps and immerse, while swirling, directly into liquid nitrogen.

Figure 3 Photographs and micrograph demonstrating the size and line marks on the closed-pulled straw used to vitrify oocytes, zygotes, cleavage-stage embryos, and blastocysts. The bottom panel demonstrates the size of the fine-pulled end of the straw containing mouse MII oocytes.

Warning:

1. Materials (expendables): Conical tubes, tissue culture dish, organ culture dish, 5- and 10-mL disposable pipettes, pulledpipettes- inner diameter just larger than the oocytes being cryopreserved, 0.2 mm filter, 20 mL pipette tips, 1 cm3 syringe, syringe/pulled-straw connector, paper tissue, or sterile gauze.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, labeling device, timer, forceps, thermometer, water bath, scissors, liquid nitrogen Dewar, safety goggles, and cryo-gloves.

3. Warming-solutions:
Initial warming solution¼HEPES-buffered medium 1.0M sucrose 12% (w/v) protein source (IWS) Dilution solution¼HEPES-buffered medium 0.5M sucrose 12% (w/v) protein source (DS)
Wash solution¼HEPES-buffered medium 12% (w/v) protein source (WASH) (VS)

4. Procedure

a. Prepare warming solutions inside the hood at least one day prior to use. Rinse all tubes, syringes, and filters prior to use. Filter all warming media with a 0.2 mM syringe filter. Prepare oocyte culture dishes at least four hours before use and allow to equilibrate in the CO2 incubator.

b. Warm IWS, DS, and WASH to room temperature. Make certain the solutions are mixed and homogeneous.

c. For each straw of oocytes being warmed, prepare a culture dish with patient identifiers and written indication of 20 mL drop placements.

d. Immediately before beginning the procedure, dispense 20 mL drops of IWS (one drop), DS (two drops) onto the culture dish.

e. Before beginning the warming process, ensure that all equipment and expendables are accounted for and functioning. Select the straw of oocytes to be warmed and rapidly transfer (in goblet containing liquid nitrogen) from the liquid nitrogen storage tank to liquid-nitrogen-filled Dewar. Place the Dewar close to the 37_C water bath.

f. Begin the warming process.
_With forceps, remove the close-pulled straw with oocytes from the liquid nitrogen and immediately submerge completely into the 37_C water bath while swirling the straw for three to five seconds.
_ Remove from the water bath and wipe dry with paper tissue or sterile gauze.
_ Using scissors, cut the straw at the end near the fourth mark at the large end of the straw.
_ Attach to pipetting device.
_ Position the straw tip over the culture dish and using scissors, cut the straw between the first and second marks but closest to the first mark.
_ Dispense the pulled-straw contents as a small drop onto the culture dish; avoid bubbles.
_ Rinse the pulled-straw with contents from the IWS drop by aspiration up to the third mark. Dispense this rinse IWS as a small drop next to the initial pulled-straw contents and merge the two drops together with a dragging motion of the pulled-straw. Set the timer for one minute.

g. When timer sounds, transfer oocytes using a pulledpipette to the bottom of the 20 mL IWS drop. Set the timer for one minute.

h. When timer sounds, transfer oocytes using a pulledpipette to the bottom of the DS1 drop for two minutes, then transfer to DS2 drop for two minutes. During the
DS2 exposure, dispense three 20 mL drops of WS.

i. Using a pulled-pipette, transfer the oocyte through the WASH (1, 2, and 3) drops, with a two-minute exposure in each drop. In the final WASH, drop check and record survival/development.

j. Transfer oocytes into CO2-equilibrated culture media, place into CO2 incubator, and perform ICSI four hours after warming.

Zygotes, Cleavage-Stage Embryos, and Blastocysts the ability to vitrify human pronuclear-stage zygotes, cleavage-stage embryos, and blastocysts has been demonstrated by many laboratories.
The protocol listed below is just one of many that have been demonstrated to produce quite acceptable results for vitrification of all stages of preimplantation human embryos.

Vitrifying:

1. Materials (expendables): Conical tubes, tissue culture dish, organ culture dish, 5- and 10-mL disposable pipettes, pulled-pipettes- inner diameter just larger than the cells being cryopreserved, 0.2 mm filter, 20mL pipette tips, cryo-goblet, cryo-cane, cryosleeve,
1cm3 syringe, "pulled-straw" with an inner diameter of approximately 200230 mm, syringe"pulled-straw" connector.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, labeling device, timer, heat-sealer, forceps, liquid nitrogen Dewar, liquid nitrogen storage canister with lock and alarm, safety goggles, and cryo-gloves.

3. Cryo-solutions: Wash solution¼HEPES-buffered medium 12% (w/v) protein source (WASH) Equilibration solution¼HEPES-buffered medium 7.5% (v/v) ethylene glycol 7.5% DMSO 12% (w/v) protein source (ES) Vitrification solution ¼ HEPES-buffered medium 15% (v/v) ethylene glycol 15% DMSO 0.5M sucrose 12% (w/v) protein source (VS)

4. Procedure
a. Prepare vitrification media inside the hood at least one day prior to use. Rinse all tubes, syringes, and filters prior to use. Filter all cryo-media with a 0.2 mM syringe filter.
Prepare embryo culture dish at least four hours before use and allow to equilibrate in the CO2 incubator.

b. Warm WASH, ES, and VS to room temperature. Make certain the solutions are mixed and homogeneous.

c. For each straw of zygotes/embryos being vitrified, prepare a culture dish with patient identifiers and written indication of 20 mL drop placements. In addition, label the "pulled-straw" with proper patient identifiers.

d. Immediately before beginning the procedure, dispense 20 mL drops of WASH (one drop), ES (one drop) onto the culture dish.

e. Select zygotes/embryos for vitrification that meet your cryopreservation criteria. Using a pulled-pipette and the least amount of media carry-over, transfer cells (maximum two at a time) from culture media into WASH drop for one minute.

f. Using the pulled-pipette tip, transfer zygotes/embryos into the top of the ES drop. The cells will begin to shrink and sink to the bottom of the drop. Zygotes/embryos with gradually return to their original size. Leave the zygotes/embryos in this ES drop for five to 15 minutes or until they re-expand to original size. During this equilibration step, dispense four 20 mL drops of VS (VS1VS4).

g. Transfer zygotes/embryos from ES to the VS1 drop. Microscopically observe zygotes/embryos and count to five seconds.

h. Transfer zygotes/embryos to VS2 for five seconds.

i. Transfer zygotes/embryos to VS3 for 10 seconds.

j. Transfer zygotes/embryos to VS4. The goal at this point is to load the pulled-straw, seal the straw, and plunge the straw in liquid nitrogen within a 90 seconds interval.
_To load the pulled straw, aspirate VS4 into straw to the first line (closest to the fine-pulled tip), aspirate VS4 and zygotes/embryos with fluid meniscus reaching the second line, aspirate additional VS4 with fluid meniscus reaching the third line.
_ Heat-seal the narrow end of the straw just below the first mark, and then above the fourth mark.
_ Hold the sealed straw with forceps and immerse, while swirling, directly into liquid nitrogen.

Warming:

1. Materials (expendables): Conical tubes, tissue culture dish, organ culture dish, 5- and 10-mL disposable pipettes, pullerpipettes- inner diameter just larger than the cells being cryopreserved, 0.2 mM filter, 20 mL pipette tips, 1-cm3 syringe, syringe/ pulled-straw connector, paper tissue or sterile gauze.

2. Equipment: Pipetting devices, laminar-flow hood, dissecting microscope with lighted base, labeling device, timer, forceps, thermometer, water bath, scissors, liquid nitrogen Dewar, safety goggles, and cryo-gloves.

3. Warming-solutions:
Initial warming solution¼HEPES-buffered medium 1.0M sucrose 12% (w/v) protein source (IWS) Dilution solution HEPES-buffered medium 0.5M sucrose 12% (w/v) protein source (DS)
Wash solution¼HEPES-buffered medium 12% (w/v) protein source (WASH).

4. Procedure:
a. Prepare warming solutions inside the hood at least one day prior to use. Rinse all tubes, syringes, and filters prior to use. Filter all cryo-media with a 0.2 mM syringe filter.Prepare zygote/embryo culture dishes at least four hours before use and allow to equilibrate in the CO2 incubator.

b. Warm IWS, DS, and WASH to room temperature. Make certain the solutions are mixed and homogeneous.

c. For each straw of zygotes/embryos being warmed, prepare a culture dish with patient identifiers and written indication of 20 mL drop placements.

d. Immediately before beginning the procedure, dispense 20 mL drops of IWS (one drop) DS (two drops) on to the culture dish.

e. Before beginning the warming process, ensure that all equipment and expendables are accounted for and functioning. Select the straw of zygotes/embryos to be warmed and rapidly transfer (in goblet containing liquid nitrogen) from the liquid nitrogen storage tank to liquid-nitrogen filled Dewar. Place the Dewar close to the 37_C water bath.

f. Begin the warming process.
_With forceps remove the close-pulled straw with zygotes/embryos from the liquid nitrogen and immediately and completely submerge into the 37_C water bath while swirling the straw for three to five seconds.
_ Remove from the water bath and wipe dry with a paper tissue or sterile gauze.
_ Using scissors cut the straw at the end near the fourth mark at the large-end of the straw.
_ Attach to pipetting device.
_ Position the straw tip over the culture dish and using scissors cut the straw between the first and second marks but closest to the first mark.
_ Dispense the pulled-straw contents as a small drop onto the culture dish; avoid bubbles.
_ Rinse the pulled-straw with contents from the IWS drop by aspiration up to the third mark. Dispense this rinse IWS as a small drop next to the initial pulledstraw contents and merge the two drops together with a dragging motion of the pulled-straw. Set the timer
for one minute.

g. When timer sounds, transfer zygotes/embryos using a pulled-pipette to the bottom of the 20 mL IWS drop. Set the timer for one minute.

h. When timer sounds, transfer zygotes/embryos using a pulled-pipette to the bottom of DS1 drop for two minutes, then transfer to DS2 drop for two minutes. During the DS2 exposure, dispense three 20 mL drops of WS.

i. Using a pulled-pipette, transfer zygotes/embryos through the WASH (1, 2, and 3) drops, with two minutes exposure in each drop. In the final WASH drop, check and record survival/development.

j. Transfer zygotes/embryos into culture media, place into incubator.

TROUBLESHOOTING AND A SUCCESSFUL
CRYOPRESERVATION PROGRAM

As a preface, it is fundamental to recognize that clinical human oocyte / embryo cryopreservation programs are inherently difficult to troubleshoot and improve because of material used (usually not the best embryos) and passage of time between cryopreservation and oocyte/embryo utilization. All would likely agree that a successful embryo cryopreservation program is pivotal in maximizing the cumulative pregnancy rate from a single oocyte retrieval. However, it is also essential that one be realistic in expectations of a cryopreservation program. Even with embryos derived from donor oocytes, the pregnancy rate of recipients receiving thawed embryos has been reported to be approximately 60% of that of recipients receiving fresh embryos.In general terms, a key factor that dictates success of oocyte/embryo cryopreservation is the condition and quality of the starting material. This is likely true independent of the method of cryopreservation. Having ARTprogram- specific cryopreservation criteria are important. They can be determined by reviewing past success of pregnancies and live births of cryopreserved embryos of different grades. It is inherently obvious that "poor-quality in" and "poor-quality out" applies to embryo and likely oocyte cryopreservation. Rate of cell division and degree of fragmentation are important considerations in determining expectations of any cryopreservation protocol. Lastly, there are likely numerous intracellular functions and cell-cycle-dependent events that are susceptible to cryo-damage, influence cryopreservation success, and yet are currently underappreciated. Attempting to troubleshoot or improve success of cryo-protocols requires attention to specific steps within a cryo-protocol. This holds true for both slow-rate freezing/thawing and vitrification/warming. There is no doubt the "devil is in the details." The following are just a few of many possible practical points to consider when attempting to optimize cryopreservation protocols. In all cases, it is prudent to systematically implement single alterations and, whenever possible, experimentally test such alterations in an animal model system before implementing them into use within the clinical laboratory.
Proteins can act as osmotic regulators within cryo-solutions. Because "osmotic shock" can be a critical determinant of cell survival during freezing/thawing or vitrification/warming, the concentration of protein in cryo-solutions should be considered. Many successful cryopreservation protocols will utilize protein addition at 1215 mg/m Inclusion of a higher concentration of sucrose or similar non-permeating cryoprotectant in the initial thaw/warming solution compared to the final concentration of non-permeating cryoprotectant in the freezing/vitrifying solution is believed to be important to enhanced cryo-survival. Such elevations in non-permeating cryoprotectants in the thawing/warming solutions are seen in most of the protocols above, with the exception of the oocyte slow-rate freezing protocol which is based on work from the Italian group of Porcu et al.. These investigators demonstrated that cryo-survival of oocytes slow-rate cryopreserved and thawed in 0.3M sucrose was significantly better than 0.2 or 0.1M sucrose. However, these investigators did not test the comparison of lower sucrose in freeze medium in relation to thaw medium. Boldt et al.  reported a commendable cryo-survival rate of 74% using human MII oocytes and slow-rate freezing in sodium-depleted cryopreservation medium where sucrose concentrations were elevated in thaw solutions compared to freeze solutions. The replacement of sodium chloride with choline chloride was demonstrated previously to be beneficial for mouse oocyte slow-rate cryopreservation and likely holds great promise in improving cryo-survival of human oocytes. A critical aspect of sequential cryoprotectant exposure is attention to the means of cell movement, solution carry-over, solution dissociation, and fluid microenvironments. As one can note in the above protocols, movement from a solution of one concentration of cryoprotectant to another should use a pulled-pipette just larger than the cells being transferred. This reduces cryoprotectant carry-over. Before transfer, one should also aspirate a small amount of solution in which the cells will be moved prior to taking up the cells.

This allows better pipetting control and reduces carry-over of the previous solution into the subsequent solution. Once cells are moved into the next cryo-solution, it is recommended that they be moved through a few different areas of the solution. This minimizes formation of a fluid microenvironment of the previous solution surrounding the cells. The above-mentioned cautions can help ensure exposure of cells to the desired concentration of cryoprotectant for the proper amount of time. Slow-rate freezing and seeding is often an area of concern and discussion. When using vials, seeding at the cryo-solution meniscus is recommended. If straws are oriented in a vertical fashion and one ensures that cells are not near the top meniscus, one can seed at the top meniscus of this solution column. When seeding, ensure good ice nucleation. This can be done with liquid-nitrogen-supercooled forceps or cotton swabs of a wooden stick applicator. The device should be supercooled between each seeding. Lastly at approximately two minutes after seeding, one should rapidly visualize the seeded area to ensure that ice nucleation is still present and growing. Finally, with regards to vitrification of oocytes and/or embryos, it is essential that the technologist practice and demonstrate a high degree of proficiency in cell movement through solutions of very different viscosities and buoyancies. This should be done with cells obtained from experimental model systems (mouse, hamster, bovine, 3PN zygotes, etc.). For successful vitrification it is essential that one be extremely well prepared and has all solutions, expendables, and equipment at hand before starting the procedure. Once the vitrification or warming has been started, there is very little time for mistakes or misplacement of a procedural component.

FUTURE OF GAMETE/EMBRYO CRYOBIOLOGY

Although cryobiology has theoretical and experimental roots anchored in the nineteenth and early twentieth centuries, the major experimental and applicable advances of gamete and embryo cryobiology have been made in the last 60 years. The same might be said of numerous other areas of developmental biology, biochemistry, cell biology, genetics, and molecular biology. Since the 1950s, there have been astonishing shifts in scientific paradigms in all these areas of science. Advancements in cryopreservation of mammalian gametes and embryos have had an enormous impact on clinical ARTs in the last three decades. When one thinks back in history, it is remarkable to consider that these advancements are based, at least philosophically, on techniques and protocols pioneered by the likes of Polge, Audrey Smith, and James Lovelock in the 1950s. So the question arises: what might one expect in the future? It is quite likely that vitrification of gametes and embryos will gain a more widespread acceptance. Again, it is essential to appreciate that the practical advantages of vitrification have been appreciated for numerous decades. If one considers the views expressed by the prominent cryobiologist Luyet in his seminal work of Life and Death at a Low Temperature , it is easy to capture his contemporary view of vitrification:
Good vitrification is not injur[i]ous, there being no molecular disturbance, while an incomplete vitrification or devitrification and, a fortiori, crystallization, are injur[i]ous to the extent that they disrupt the living structure.
The advantages of vitrification are still acknowledged today, as expressed by Taylor and colleagues:
A vitrified liquid is essentially a liquid in molecular stasis. Vitrification does not have any of the biologically damaging effects associated with freezing because no degradation occurs over time in living matter trapped within a vitreous matrix. Vitrification is potentially applicable to all biological systems.
When one cryopreserves millions of cells, such as transformed cell lines, sperm, or even tissues, the significant lose of a percentage of cells may not be viewed as critical for subsequent usage. This is not the case when one considers cryopreservation of a small finite set of cells or group of cells, such as oocytes and embryos. These cells require the most efficient means of cryopreservation and retrieval of viability. While slow-rate freezing has been successful, one has to ask whether vitrification will have added benefit.
Advancements in the area of "freeze-drying" platelets and sperm are very exciting and beg the question of whether such techniquesmight have future application in long-termstorage of oocytes and embryos.The potential to provide such storage in a freeze-dried state has numerous advantages, one being the actual means of long-term storage without liquid nitrogen. Whether such advances will be made for oocytes and embryos remains to be seen.
The fields of infertility treatment and ARTs are indebted to the numerous cryobiologists, whose basic and translational research has contributed significantly to the current success of gamete and embryo cryopreservation. With this said, there is always room for improvement. Knowledge continues to accumulate regarding intricacies of cell biology, regulation of gene expression, factors influencing epigenetic modifications, and how all these cellular functions culminate into normal physiology. The last 50 year have been exciting times in cryobiology; one could venture an opinion that the next fifty years may provide currently inconceivable advancements, that once applied, will lead to betterment of health and increased happiness.

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