Introduction
Cryopreservation
of gametes and embryos is a highly desirable goal for numerous
reasons, primarily for the ability to store excess genetic
material and to control the timing of fertilization and embryo
transfer. Oocyte
storage is especially appealing because it would: 1) allow women
to delay reproduction until later in life, after establishing a
career, for instance thus giving them more reproductive choices;
2) alleviate religious or ethical concerns of embryo storage; 3)
make “egg banks” possible thus eliminating problems with
donor-recipient synchronization and thus standardize the number
of eggs for the recipient; and 4) provide a feasible opportunity
for women undergoing radio- or chemo-therapy for cancer to have
their own children.
Since
the discovery of glycerol’s protective effects on sperm cells
in 1949, both gametes and embryos have been successfully frozen
from a number of mammalian species including mice, sheep, cows,
pigs, horses, hamsters, rats, cats, and humans.
Throughout the 1950’s and 1960’s the foundations of
modern cryobiology theory were laid down by Lovelock, Meryman,
Polge, Smith, Levitt, Luyet, etc.
During the 1970’s, breakthroughs in mammalian embryo
and oocyte cryopreservation by Whittingham and Willadsen paved
the way for human oocyte and embryo storage in the 1980’s.
Over the past twenty-some years leading up to the year
2000, refinements of protocols have made human embryo and sperm
storage a routine procedure in IVF clinics throughout the world.
However, efficient techniques for oocyte cryopreservation
have eluded scientists and simple modifications to embryo
storage protocols have not allowed the mastery of oocyte
cryopreservation. Furthermore,
human embryo and sperm cryopreservation techniques, although
successful to a certain degree, have room for improvement.
Background
(Oocyte Cryopreservation)
In 1986 Chen (Chen,
1986) was the first to report a pregnancy originating
from a frozen-thawed human oocyte.
At the same time studies
in mice suggested that although eggs could survive freezing and
thawing, they might possess higher levels of chromosomal
anomalies following this procedure when compared to fresh eggs (Johnson
and Pickering, 1987; Pickering and Johnson, 1987; Sathananthan et al., 1988). This
led to a slow-down in oocyte cryopreservation work until the mid
90’s. During
the 9 years following the first pregnancy, only a few
investigators (Al-Hasani
et al., 1987; Chen,
1988; van Uem et al.,
1987) added to Chen’s success and collectively
established 5 successful births by 1995.
By this time embryo cryopreservation had become routine
and the need for oocyte freezing was not a high priority for
clinicians. Additional work in the mouse did not overcome the skepticism
of possible abnormalities that could occur when cryopreserving
human oocytes. Further
research on mouse and human oocytes had started to show that
cryopreservation was not as detrimental as prior studies
suggested (Gook
et al., 1994; Gook et al., 1993). Also,
ICSI was being used to ensure fertilization and prevent
polyspermia of frozen-thawed oocytes (Gook
et al., 1995; Kazem et al.,
1995). During
the next several years Tucker and Massey in the United States,
Fabbri and Porcu in Italy, and other laboratories around the
world had, through concentrated efforts, become successful in
producing numerous babies from frozen-thawed oocytes (Antinori
et al., 1998; Borini et
al., 1998; Nawroth and Kissing, 1998; Polak de Fried et
al., 1998; Porcu, 1999; Porcu et
al., 1999a; Porcu et
al., 1999b; Porcu et
al., 1997; Porcu et al., 1998; Tucker et al.,
1996; Tucker et al.,
1998; Vidali et al.,
1998; Yang et al., 1999; Yang et al.,
1998; Young et al.,
1998). By
2000 the number of children born from frozen oocytes was over
20. These reports
caused a resurgence in egg freezing research and clinical
studies (Boldt
et al., 2003; Cha et
al., 2000; Fabbri et
al., 2001; Fosas et al., 2003; Marina and Marina, 2003; Porcu, 2001; Porcu et
al., 2002; Porcu et al., 2000; Yang et al.,
2002). Some
of the work was done with conventional slow-cooling, however
rapid freezing and vitrification held promise of a quick fix to
the problem, and investigations in this area became numerous (Ali,
2001; Chen et al.,
2000; Chung et al., 2000; Hong et al.,
1999; Hunter et al.,
1995; Kuleshova et al.,
1999; Kuleshova and Lopata, 2002; Liebermann and Tucker, 2002;
Liebermann et al., 2003; Pensis et al.,
1989; Wininger and Kort, 2002; Wu et
al., 2001; Yoon et al.,
2003).
To
date there have been around 100 children born from oocyte
freezing. However,
in reviewing these studies the number of offspring that were
produced per number of oocytes frozen was seldom greater than 1
to 5 percent. In a
recent commentary by Marina and Marina (Marina
and Marina, 2003) they reported 4 births from 99 oocytes
frozen in their clinic (4%).
Porcu et al. (Porcu
et al., 1999a),
who has done the majority of work in clinical human oocyte
freezing, reported 16 pregnancies from 1502 thawed eggs (1%).
Yang et al. (Yang et al.,
1999) reported 7 pregnancies using 120 oocytes (5.8%).
Ahuja et al. (Ahuja et al.,
1998) a 2% average number of pregnancies in UK IVF
centers. Therefore,
simply using human embryo cryopreservation protocols for
freezing human oocytes did not work well, similar to results
obtained when mouse oocytes were frozen using mouse embryo
cryopreservation procedures.
Even with modifications to freezing protocols and an
improvement in survival rates, it was difficult to obtain a high
percentage of fertilized and normal cleaving embryos after
oocyte cryopreservation. Initial
survival rates for frozen-thawed human oocytes were around
50-70% of all oocytes frozen, but that number was significantly
reduced after pronuclear formation (fertilization), and cleavage
beyond the 2-cell stage. Although,
little progress has been made when standard embryo freezing
protocols have been used to store unfertilized oocytes
investigators have reported similar pregnancy rates for frozen
oocytes and frozen embryos (2002 ASRM Annual Meeting; (Marina
and Marina, 2003).
Cryopreservation
Theory
As
a general review, the basic principles of cryopreserving oocytes
or embryos are as follows. Cells are exposed to a simple isotonic salt solution
containing a permeable cryoprotectant (1,2-propanediol) and
usually, a low concentration of non-permeable cryoprotectant
(sucrose). After a
brief exposure time to allow uptake of cryoprotectant, the cells
are cooled rapidly to a temperature slightly below the melting
point of the solution (usually around -7°C).
At this point the container with the cells is
“seeded” so that ice forms in the extracellular solution.
Upon ice formation and with additional cooling (slow
cooling to below -30°C) the osmolarity of the extracellular
solution increases as water freezes out as ice.
With the increasing tonicity, the cells dehydrate.
Dehydration continues during slow-cooling until the cells
are plunged into liquid nitrogen, usually at a temperature below
-30°C. At this
point the intracellular cryoprotectant concentration is high
enough that the remaining intracellular water will vitrify,
preventing intracellular ice formation (IIF).
Upon rewarming, ice can once again form, as the vitrified
solution melts and refreezes in the process of devitrification. This
happens when the temperature is high enough that the molecular
mobility of water has increased to a point where the water
molecules can move and rearrange themselves from a disorderly
amorphous vitrified position to an orderly crystalline position.
This occurs well below the melting point, and is
therefore a potentially lethal problem during the thawing stage (Luyet,
1970; Mazur and Schmidt, 1968).
During thawing, the dehydrated cells are exposed to
hypotonic conditions and rehydrate along with cryoprotectant
removal (Mazur,
1977). The
cryoprotectants are usually removed gradually by dilution
through a series of media with decreasing concentrations of
cryoprotectants until the cells are returned to a culture medium
and allowed to continue development.
For
more information regarding cryopreservation or specifically
human oocyte cryopreservation see the special upcomming issue of
Reproductive BioMedicine Online (RBMO).
Due out late 2003 or early 2004.
