Slow Freezing of Oocytes

Abstract

Oocyte cryopreservation has several potential applications in human assisted reproduction technology, such as to maximize cycle cumulative outcome, limit the number of embryos generated for fresh embryo transfer, and support programs of fertility preservation and oocyte donation. Mature oocytes are intrinsically more susceptible to cryodamage compared with embryos. Fully grown immature oocytes are even less amenable to cryopreservation, due to the necessity to preserve morpho-functional relationship with companion cumulus cells. Consequently, not surprisingly, initial attempts aimed at cryostoring oocytes with conventional controlled rate slow-cooling (CRSC) protocols were frustrated by low survival rates and poor clinical outcomes. Several studies have confirmed that cryopreservation, if performed with suboptimal protocols, generates diverse types of cell damage, such as zona pellucida rupture, release of cortical granules, ultrastructural damage, and alterations in cell cycle regulation. Notwithstanding, perseverant and systematic research efforts have led to improved slow-cooling protocols that, although unable to increase survival rates above 75–80%, can achieve clinical outcomes that, if assessed in terms of number of implantations per number of thawed oocytes, are comparable with those obtained by vitrification. Younger patients seem to benefit particularly from oocyte CRSC, while results from older women are rather disappointing even beyond the expected effect of female age. The health of babies derived from oocytes stored by CRSC has been investigated. Relevant studies are few and numerically limited but do not suggest a health impact of cryopreservation. Overall, over the last decade, oocyte cryopreservation by CRSC has been replaced by vitirification. However, some intrinsic advantages of CRSC should not be overlooked, such as a higher reproducibility of protocols and automated monitoring of cooling phases.

Appendices

Review Questions

• 1. What are the limitations of oocyte slow-cooling approach?

• 2. Are all the protocols adopted equivalent in term of survival and outcomes?

• 3. Are data provided from invasive methods valuable to improve SC protocols?

• 4. Are there any advantages in the laboratory management of female fertility preservation via slow cooling?

• 5. Is it time to discontinue slow freezing?

Freezing

1.1 Freezing Solutions

Freezing Solution 1:	1.5 mol/l PrOH
	1.08 ml PROH
	6.92 ml PBS
	2.00 ml PPS

Freezing Solution 2:	1.5 mol/l PrOH, 0.2 mol/l sucrose
	1.08 ml PROH
	6.92 ml PBS
	2.00 ml PPS
	684 mg sucrose

Notes

• (a) Freezing solutions must be stored at 4 °C. Warmed to room temperature (24−25 °C) before use.

• (b) All dehydration steps must be performed at room temperature.

Dehydration

• (a) For each oocyte (or group of oocytes), dispense 0.5 ml of the freezing solutions in separate wells of a 4-well plate.

• (b) Incubate sequentially oocytes in the freezing solutions according to the times indicated below.

  Solution	Time
  Freezing Solution 1	10 min
  Freezing Solution 2	5 min

• (c) Load oocyte(s) into straw(s)

• (d) Seal straw(s)

Cooling to LN2 temperature

• (a) Place straws into the cryofreezer.

• (b) Run the controlled rate freezing program (see below).

Controlled rate freezing program

• 1. Decrease temperature from +20 °C to –7 °C at a rate of −2 °C/min.

• 2. “Hold” at –7 °C for 10 min.

• 3. Perform manual seeding at about 30% of the “hold” ramp.

• 4. Decrease temperature from −7 °C to –30 °C at a rate of −0.3 °C/min.

• 5. Decrease temperature from −30 °C to −150 °C at a rate of −50 °C/min.

• 6. Hold at –150 °C for 10 min.

• 7. Transfer into LN₂ for long-term storage.

Thawing

1.1 Thawing Solutions

Thawing Solution 1:	1.0 mol/l PrOH, 0.3 mol/l sucrose
	0.72 ml PROH
	7.28 ml PBS
	2.00 ml PPS
	1026 mg sucrose

Thawing Solution 2:	0.5 mol/l PrOH, 0.3 mol/l sucrose
	0.36 ml PROH
	7.64 ml PBS
	2.00 ml PPS
	1026 mg sucrose

Thawing Solution 3:	0.3 mol/l sucrose
	8.00 ml PBS
	2.00 ml PPS
	1026 mg sucrose

Thawing Solution 4:
	8.00 ml PBS
	2.00 ml PPS

Note

• (a) Freezing solutions must be stored at 4 °C. Warmed to room temperature (24−25 °C) before use.

• (b) All dehydration steps must be performed at room temperature.

Thawing

• (a) For each oocyte (or group of oocytes), dispense 0.5 ml of the thawing solutions in separate wells of a 4-well plate.

• (b) Remove the straw from LN₂ and keep at room temperature for 30 sec.

• (c) Transfer the straw in a + 30 °C water bath for 40 sec.

Rehydration

• (d) Release the oocyte(s) from straw(s).

• (e) Incubate sequentially the oocyte(s) in the freezing solutions according to the times indicated below.

  Solution	Time
  Thawing Solution 1	5 min
  Thawing Solution 2	5 min
  Thawing Solution 3	10 min
  Thawing Solution 4	10 min

• (f) Transfer the oocyte(s) to 37 °C for 10 min, while in thawing solution 4 and after the initial incubation at room temperature.

• (g) Transfer the oocyte(s) in medium for oocyte culture and incubate under standard conditions for 60−90 min before microinjection.