Cryosurvival of human oocytes

 

Patients undergoing cancer treatment or patients with genetic conditions associated with premature ovarian failure now have options to help preserve fertility by freezing their unfertilized eggs.  This assisted reproductive technology is also available to healthy women who are unable or unwilling to freeze their fertilized eggs or choose to defer childbearing.  MGH bioengineers and clinicians are working to refine cooling and storing techniques to improve oocyte survival, fertilization, and pregnancy rates so that human oocyte preservation becomes a standard clinical laboratory procedure.  Read More

Why preserve and bank oocytes?

Cryopreservation of oocytes provides a lifeline for patients to preserve their reproductive potential before undergoing medical treatments that increase the risk of, or cause infertility, such as chemotherapy or radiotherapy for cancer.  For those patients with genetic conditions associated with premature ovarian failure, oocyte cryopreservation eliminates the reliance on donor oocytes and coordination of donor-recipient hormonal cycles.  For healthy women, oocyte cryopreservation has become an important alternative for women who are unable or unwilling to cryopreserve embryos (fertilized eggs) for ethical or legal reasons. Elective cryopreservation also offers the opportunity for women to postpone childbearing for social, career, or personal reasons.   Oocyte cryopreservation has become one of the most sought after advances in assisted reproductive technologies.

Oocyte cryopreservation

Cryopreservation techniques involve the cooling of cells or tissues to a frozen state to stop all biological activity, followed by storage and subsequent thawing for future use.  Over several decades, animal models using mammalian oocytes from mice, sheep, cows, and pigs have greatly contributed to the development of current protocols for human tissue preservation.  Cryopreserved human cells can include semen, blood (stem cells and umbilical cord blood), embryos, ovarian tissue, and oocytes (unfertilized eggs).

The ideal cryopreservation protocol for oocytes

There are two primary approaches to achieving cryopreservation of oocytes.  The conventional, slow-freezing approach uses a low cooling rate of less than 2°C per minute while the vitrification approach uses a rapid cooling approach that uses cooling rates of over 1,000°C per minute.  With either technique, use of one or more cryoprotective agents (CPAs) prevents freezing damage to cells.  With slow freezing, however, ice crystals can form inside the cells and damage the oocytes, which are sensitive to chilling injury.  Commonly used CPAs like ethylene glycol or 1,2-propanediol (PROH) in low concentration are used with the slow freeze method to penetrate the cell membrane, form hydrogen bonds with water molecules and prevent the formation of ice crystals.  Vitrification, the glass-like solidification of the cells, uses rapid cooling and warming together with high concentrations of CPAs to prevent intracellular ice formation.  Due to the toxicity and osmotic shock that can result from these high concentrations, CPAs must be added and removed (in a process known as CPA ‘loading’) from the cells in very short exposure times, which makes the process complicated and difficult to control.

Cryobiologists know that oocyte survival after cryopreservation is dependent on many factors including oocyte-cell characteristics, cell permeability to CPAs, and CPA toxicity, temperature, and duration of exposure.  Efficient vitrification protocols for cryopreservation would (1) yield high survival of oocytes; (2) achieve vitrification while preserving the cell’s integrity and structure; and (3) have no effect on the fertilization and embryonic development of oocytes.

“Thinking outside the box” with sugar-based CPAs

MGH bioengineers and clinicians since the early 2000’s have been developing techniques that combine the best features of slow freezing and vitrification – reduced toxicity secondary to low CPA concentrations and the absence of intracellular ice crystal formation.

Trehalose, a disaccharide with important functional properties

The investigators are inspired by nature’s survival scheme of a diversity of animals, bacteria, and yeast being able to use sugars to survive extreme environmental conditions of freezing and extreme drying.  The investigators are experimenting with using intracellular and extracellular trehalose, a natural sugar, as a penetrating CPA.  In an in vitro study with discarded human oocytes that failed to mature or to fertilize, investigators were able to obtain high cryosurvival using only a small amount of microinjected extracellular trehalose in the absence of any other CPA.  This provided a significant protection against freeze-associated stresses.  In follow-up studies, investigators using mouse fertilized eggs as a model explored the long-term effect of trehalose on embryonic and fetal development as a function of its intracellular concentrations.  Microinjection of a small amount of trehalose did not impair development of mouse zygotes to the blastocyte stage while transfer of two-cell embryos to foster mothers resulted in normal implantation and development rates, and birth of healthy mouse pups. In a study using low concentrations of a combination of intracellular and extracellular trehalose with small amounts of a conventional penetrating cryoprotectant (dimethylsulfoxide), mouse oocytes showed high survival, fertilization, and embryonic development rates statistically similar to untreated controls.  These studies demonstrated the successful use of trehalose as a CPA.

Low CPA concentrations + slush nitrogen in quartz capillaries

Quartz capillary vitrification device

Scientists working on vitrification techniques recognize the relationship between cell sample volume, cooling rate, and the critical CPA concentration needed to vitrify: the smaller the sample volume and the higher the cooling rate, the lower the concentration needed to vitrify.  MGH investigators developed an ultra-rapid vitrification technique that uses quartz capillary “straws” filled with a combination of trehalose and PROH cryoprotectant solution and plunged into slush nitrogen (a combination of liquid and solid nitrogen).  This represented the first report of oocyte vitrification using slow-freeze CPA concentrations.

Single oocyte on a microfluidic platform

Single oocyte passing under valve and captured in the oocyte analysis chamber

By combining microfluidic and biophysical modeling tools, MGH investigators designed a microfluidic device that can securely hold a single oocyte and expose it to varying concentrations of CPAs as a function of time.  In this microfluidic oocyte analysis chamber, the investigators were able to measure oocyte response to CPA concentration changes in a continuous manner.  Using a biophysical model together with these new measurements, the investigators were able to design optimized protocols for three distinct CPA loading protocols to achieve CPA loading in less than 15 minutes with less than 10% oocyte volume changes throughout the process.

The investigators believe this microfluidic device offers many advantages for the measurement of oocyte volumetric changes compared to conventional techniques.  And, it combines the advantages of precisely controlled CPA loading and gentle handling of oocytes.  This microfluidic technology for single oocyte handling has broad applicability for future advances in assisted reproductive technologies and fertility preservation especially for protocols that involve oocyte handling and manipulation.

Experimental procedure or clinical practice?

In their 2013 practice guidelines, the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology indicated that “mature oocyte vitrification and warming should no longer be considered experimental.”   The authors’ guidelines cite the dramatic improvements in oocyte cryopreservation techniques and the fact that this procedure has become the recommended treatment for fertility preservation in patients who will receive radiation or a chemotherapeutic agent.

There are several encouraging, albeit preliminary studies on post thaw oocyte outcomes that show that mature oocyte survival, oocyte fertilization, implantation, and pregnancy outcomes appear to be comparable to those of women with spontaneous births and those who become pregnant with in vitro fertilization (IVF) treatment.  Many of these early studies reporting live births were from egg donor programs and not from clinic practices of assisted reproduction so the reported results should be viewed with caution.  Well-designed, randomized,  and controlled clinical trials are required to assess the clinical safety issues with vitrified oocytes and any risks of adverse obstetrical, perinatal, or developmental outcomes from the vitrification process.  To this end, MGH investigators and other scientists are now studying the bioenergetics, metabolic, and transcriptional responses to cryopreservation.

Relevant publications

Practice Committees of American Society for Reproductive Medicine; Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. Fertil Steril. 2013 Jan;99(1):37-43. Epub  2012 Oct 22. PubMed PMID: 23083924

Heo YS, Lee HJ, Hassell BA, Irimia D, Toth TL, Elmoazzen H, Toner M. Controlled loading of cryoprotectants (CPAs) to oocyte with linear and complex CPA profiles on a microfluidic platform. Lab Chip. 2011 Oct 21;11(20):3530-7. PubMed PMID: 21887438; PubMed Central PMCID: PMC3755277

Lee HJ, Elmoazzen H, Wright D, Biggers J, Rueda BR, Heo YS, Toner M, Toth TL. Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations.  Reprod Biomed Online. 2010 Feb;20(2):201-8. PubMed PMID: 20113958; PubMed CEntral PMCID: PMC2818866

Eroglu A, Bailey SE, Toner M, Toth TL. Successful cryopreservation of mouse oocytes by using low concentrations of trehalose and dimethylsulfoxide. Biol Reprod. 2009 Jan;80(1):70-8.  PubMed PMID: 18815355; PubMed Central PMCID: PMC2804808

Eroglu A, Lawitts JA, Toner M, Toth TL. Quantitative microinjection of trehalose into mouse oocytes and zygotes, and its effect on development. Cryobiology. 2003 Apr;46(2):121-34. PubMed PMID: 12686202

Eroglu A, Toner M, Toth TL. Beneficial effect of microinjected trehalose on the cryosurvival of human oocytes. Fertil Steril. 2002 Jan;77(1):152-8. PubMed PMID: 11779606

Eroglu A, Russo MJ, Bieganski R, Fowler A, Cheley S, Bayley H, Toner M. Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nat Biotechnol. 2000 Feb;18(2):163-7. PubMed PMID: 10657121

Contact

Mehmet Toner, PhD Thomas L. Toth, MD
mtoner@hms.harvard.edu tltoth@mgh.harvard.edu
617-724-5336 617-726-8868

Read the MGH news report about this revolutionary technique

See Less

Grace McDonald-SmithCryosurvival of human oocytes