Oocyte and Embryo Cryopreservation docx

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Oocyte and Embryo Cryopreservation

Gary D. Smith

Reproductive Sciences Program, Departments of Obstetrics and Gynecology,

Urology, and Molecular and Integrated Physiology, University of Michigan,

Ann Arbor, Michigan, U.S.A.

Joyce Fioravanti

Huntington Center for Reproductive Medicine of Brazil, Sao Paulo, Brazil

HISTORY OF MAMMALIAN GAMETE/EMBRYO CRYOBIOLOGY

When one considers the history of gamete/embryo cryobiology, it is difficult

to select a specific event and point of origin. Early basic scientific advance￾ments in measurements of temperature and the chemistry of solutions and

gases are certainly sentinel events for cryobiology. It has been suggested that

original versions of a device to measure temperature were made by Galileo

Galilei in the early seventeenth century. The first accurate means of measur￾ing temperature were developed in the early 1700s by the German physicist

Gabriel Fahrenheit through application of mercury in glass. Since these

early days, modifications of instruments to assess temperature have become

significantly more accurate and easier to use. Equally important were early

advancements made in the nineteenth century involving understandings of

liquefaction of gases and potential use of such refrigerants to cool and store

specimens at extremely low temperatures.

When one traces the history of mammalian gamete cryopreservation,

numerous accounts reference the beginning of low-temperature biology to

1866, when an Italian military physician Mantegazza documented the obser￾vation that human spermatozoa became immotile when cooled in snow

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(1,2). He subsequently proposed that it might be possible for a soldier to

father a child, even after his death, by cooling and storing spermatozoa.

It is quite interesting that centuries later, as technological refinement of

cryopreservation has occurred, many of these same reproductive quandaries

exist and are still debated today (3–5). In the early 1930s, two papers were

published (6,7) that demonstrated the effects of numerous temperatures

ranging from 0 to 45
C on rabbit spermatozoa. Considering advances

that were being made at this time in basic understandings of reproduc￾tive biology, artificial insemination, and aeronautical engineering,

Dr. Hammond demonstrated his forward-thinking by stating ‘‘in these days

of rapid aeroplane transport, it might be possible to move entire herds of

animals around the world in the form of chilled samples of semen’’ (1).

Although there were numerous empirical studies on non-mammalian and

mammalian spermatozoa cryopreservation in the 1930s and 1940s, a major

breakthrough occurred with the serendipitous discovery that glycerol

imparts cryoprotective properties to spermatozoa during freezing and thaw￾ing (8). A tantalizing tale of this important moment in cryobiological history

is eloquently portrayed by Leibo (1). In the early 1950s, Bunge and Sherman

(9,10) extended Polge’s use of glycerol to cryopreservation of human sperm.

This ability to cryopreserve human sperm that upon thawing can fertilize ova

has subsequently resulted in thousands, and perhaps millions, of children

being born through intrauterine insemination (IUI) of cryopreserved semen.

Investigations in the late 1940s and early 1950s by Chang (11,12) on

low-temperature storage of rabbit oocytes, zygotes, and embryos paved

the way for studies on the cryopreservation of female gametes and embryos.

Subsequent experiments by Sherman and Lin (13–16) demonstrated that

mouse oocytes could also be cooled in glycerol, stored and subsequently fer￾tilized in recipients; furthermore, resulting embryos supported pregnancies.

In the 1960s and early 1970s, a merging of basic/theoretical cryobiology and

practical studies ultimately gave rise to increased success of embryo cryopre￾servation. Classical basic science investigations by Mazur (17–19) formed

the foundation for understanding cell-specific optimal cooling and warming

rates which today remain a pivotal key to successful mammalian gamete and

embryo cryopreservation. It was the combined strengths of Mazur, Leibo,

and Whittingham that resulted in successful cryopreservation of mouse

embryos (Fig. 1). These investigators used 1.5 M dimethylsulfoxide (DMSO)

as the cryoprotective agent combined with a slow cooling rate (0.3
C/min

to 80
C) and stored in liquid nitrogen (20). This procedure was based on

the appreciation that slow cooling rates would support dehydration during

cooling and avoid intracellular ice formation. In addition, it was established

that addition and removal of the cryoprotective agent (here DMSO) should

be performed in a stepwise manner to avoid osmotic shock or damage.

In the early 1980s, application of the same methodologies of cryopre￾servation led to the establishment of the first human pregnancies following

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freezing and thawing (21,22). The use of 1,2-propanediol (PROH) as a per￾meating cryoprotectant for pronuclear-stage zygotes was introduced by

Testart et al. (23). In addition, these investigators used sucrose in the

cryo-media as a non-permeating cryoprotectant to act as an osmotic buffer.

Slow cooling rates were used until 30
C was reached; samples were subse￾quently plunged into liquid nitrogen, and the warming rate was rapid. This

approach of cryopreserving pronuclear- and cleavage-stage mammalian

embryos has become an acceptable procedure in assisted reproductive tech￾nology (ART) laboratories across the United States and worldwide.

PRINCIPLES OF CRYOPRESERVATION

As mentioned previously, in the 1940s it was discovered that addition of

glycerol protected against cryo-damage and greatly enhanced survival

of cryopreserved living cells. This led to the investigational concept of cryo￾protectants. In hindsight, the use of cryoprotectants is logical considering

that, in insect biological systems, sugars and sugar-alcohols are used to with￾stand severe winter temperatures (24). Experience in cryopreservation of

various cell types led to the appreciation that as cell size increases, difficulty

in cryopreservation also increases (19). This concept is of particular impor￾tance in mammalian oocyte and embryo cryopreservation.

Currently, there are two methods used to cryopreserve mammalian

oocytes and embryos: slow-rate freezing and vitrification (25). 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

Figure 1 A photograph of Mazur (left), Leibo (center), and Whittingham (right)

taken in June 1972 on the occasion of the birth of the first mammals derived from

cryopreserved embryos.

Oocyte and Embryo Cryopreservation 333