X-Message-Number: 28082
From: "Basie" <>
Subject: New Egg Freezing Technique 
Date: Thu, 22 Jun 2006 13:31:56 -0400

New Egg Freezing Technique Offers Hope To Hundreds Of Women
A new and highly successful method of freezing human eggs will help to even 
out the current inequality between men and women whereby, until now, men 
have been able to use their previously frozen sperm for IVF treatment but 
women have not been able to do the same with their eggs.
The research, presented today (Monday 19 June) at the 22nd annual meeting of 
the European Society of Human Reproduction and Embryology, gives hope to 
hundreds of women who want to preserve their future fertility but who, for 
whatever reason, only have eggs, not embryos, available for freezing.
Dr Masashige Kuwayama told a news briefing that although sperm could be 
frozen, thawed and used for in vitro fertilisation with high levels of 
success, the freeze-thawing process could damage eggs and, until now, it had 
been very difficult to perform successful IVF using frozen-thawed eggs. 
Research by Professor Stefania Nottola and Dr Sandrine Chamayou, presented 
at the conference, gave examples of the current problems of conventional 
freeze-thawing and the need for more effective techniques. It is thought 
that worldwide less than 150 babies have been born using eggs that have been 
frozen.
Dr Kuwayama, scientific director of the Kato Ladies Clinic in Tokyo, Japan, 
has developed a new method of freezing eggs (oocytes) called the Cryotop 
method, which he first used for artificial insemination in sheep and cattle.
He said: "The Cryotop method is a highly efficient freezing procedure that 
opens a new way to resolve the various aspects of the problem of human 
oocyte cryopreservation. Using this method, we achieved a more than 90% 
survival rate for the freeze-thawed oocytes and a high pregnancy rate of 
nearly 42% after the oocytes had been fertilised and implanted in the women. 
This pregnancy rate is practically the same as the rate we can achieve in 
our clinic using fresh oocytes."
The Cryotop method involves very rapid freezing in a tiny amount (less than 
0.1 microlitres) of a special vitrification solution, before storing in 
liquid nitrogen. This process prevents ice crystals forming, which do so 
much damage to the structure of the egg. Dr Kuwayama froze 111 eggs, of 
which 94.5% survived freeze-thawing, 90.5% were fertilised using ICSI 
(intra-cytoplasmic sperm injection), and 50% of the resulting zygotes were 
successfully developed for embryo transfer. Twelve pregnancies resulted from 
29 embryo transfers (a pregnancy rate of 41.9%, compared with 42.5% using 
fresh eggs), with an average of 2.3 embryos transferred each time (compared 
with an average of 1.1 embryos formed from fresh eggs). Eleven healthy 
babies were born (nine singletons and one pair of twins), while two 
pregnancies miscarried. The women were aged between 25 and 37.
Dr Kuwayama said: "The conventional slow freeze-thawing method has been used 
successfully for human embryos in assisted reproduction for two decades, but 
has been far less successful for oocytes. The post-thaw survival rate for 
human embryos is about 85-90%. Now, using the Cryotop method, we can achieve 
post-thaw survival rates for oocytes of 90-95%, making oocyte 
cryopreservation a real option for women.
"This technology opens up new horizons for medically assisted reproduction 
in women, enabling them to have the option of having children at a later 
date by freezing eggs rather than embryos. Moreover, it will help to 
eliminate the existing time differences in fertility between men and women, 
whereby women's supplies of eggs decline at a faster rate than men's 
supplies of sperm."
Dr Chamayou, scientific director of the Unita di Medicina Della 
Riproduzione, Fondazione HERA, Catania, Italy, told the conference that 
Italy had banned embryo and zygote freezing in 2004, and any embryos created 
for IVF, up to a maximum of three, had to be implanted in one transfer. As a 
consequence of this new law, interest in egg freezing had grown. "Even 
although oocyte freezing is still considered experimental by the scientific 
community, nowadays in our centre it is routinely proposed to patients as an 
alternative to disposing of any surplus oocytes. Surplus oocytes become 
available in 78.5% of IVF cycles."
Her research into the potential of freeze-thawing eggs, using conventional 
methods of slow freezing and rapid thawing, revealed the consequences of 
freezing on fertilisation, subsequent initial cell division (cleavage) and 
embryo quality. Out of 337 thawed eggs, 263 survived (78%), 221 were 
fertilised using ICSI (67.9%), 116 underwent cleavage up to the second day 
(77.3%), 92 embryos were implanted in 37 embryo transfer procedures and two 
pregnancies resulted with two babies born (13.5%, although this figure was 
not significant due to the small numbers involved).
Dr Chamayou also found that the quality of embryos differed significantly 
depending on whether fresh or frozen eggs had been used; out of the total 
number of embryos, 36.7% were grade 1 embryos after IVF using fresh eggs, 
19.5% were grade 1 after ICSI using fresh eggs (giving an overall percentage 
after IVF and ICSI of 24.7% grade 1 embryos), but only 12.1% were grade 1 
using frozen-thawed eggs.
"We concluded that oocyte preservation decreased the proportion of cleavage 
and grade 1 embryos, but did not influence fertilisation rates," she said. 
One reason for the high percentage of failure after fertilisation could be 
the damage done to the structure of the egg during freeze-thawing, in 
particular to a part called the meiotic spindle which is involved in cell 
division. The meiotic spindle is a bundle of microtubules, some of which 
become attached to chromosomes, providing the mechanism for chromosomal 
movement.
"Before freezing we observed meiotic spindles in 62.5% of oocytes, but in 
only 43.4% after thawing. This is statistically significant and I deduced 
that cryopreservation may induce irreversible damage to the bonding of the 
microtubules," she said.
Professor Nottola, an associate professor of anatomy in the Department of 
Anatomy, University La Sapienza, Rome, Italy, told the conference that a 
variety of abnormalities became apparent when frozen-thawed eggs were 
inspected more closely, even in those that appeared undamaged after thawing. 
In addition, the type and/or concentration of agent that was used to protect 
the egg during freezing (cryoprotectant agent or CPA) might affect the eggs 
as well.
Six eggs were frozen using a multi-step procedure that involved increasing 
concentrations of ethylene glycol (EG) as a CPA. Twelve were frozen using 
propane-1,2-diol (PrOH) and two different amounts of sucrose as CPAs. After 
thawing, the eggs were studied using light and transmission electron 
microscopy (LM and TEM).
The eggs appeared normal when observed with LM, although some eggs showed 
the development of vacuoles (small cavities that sometimes contain water, 
food or metabolic waste) in their cytoplasm. When TEM was used, more 
differences became apparent. These included:
changes to the linkages between mitochondria (microscopic respiratory 
structures in the cytoplasm) and intracellular membranes amongst the 
EG-treated eggs
changes to the amount and density of cortical granules (particles that 
harden the exterior of the egg once it has been fertilised to prevent other 
sperm penetrating it)
changes to the structure of the egg exterior (the zona pellucida).
TEM also confirmed that some eggs, particularly those treated with EG and 
with the PrOH agent containing more sucrose, had vacuoles developing - a 
process known as vacuolisation.
Professor Nottola said: "These data suggest that frozen-thawed oocytes may 
look similar to fresh oocytes, but when they are examined more closely it 
becomes apparent that there are a number of very small but important 
alterations to specific parts of numerous oocytes, which presumably are 
responsible for their reduced developmental potential. The type and/or 
concentration of the CPA used may play a role, at least in part, in 
producing these alterations. In particular, EG appears less suitable than 
PrOH.
"In my opinion, the most important alteration found in frozen-thawed oocytes 
could be the presence of vacuolisation. This is a quite non-specific feature 
commonly found in cells that are responding to an injury and, even in 
absence of other alterations, might lead to an impairment of the 
developmental potential of the frozen-thawed oocytes.
"As far as the other possible alterations are concerned, the reduction in 
amount and density of the cortical granules and the consequent hardening of 
the zona pellucida in frozen-thawed oocytes may be an expected phenomenon 
that possibly could be by-passed by the application of ICSI technique at 
insemination; other zona pellucida damage may greatly affect oocyte survival 
but it depends on the type and amount of the damage; alterations in the 
mitochondrial organisation can be very subtle and deserve to be further 
investigated by transmission electron microscopy."
Professor Arne Sunde, former chairman of ESHRE, said: "Cryopreservation of 
human semen and embryos has been a routine procedure since the early 1980s, 
while cryopreservation of mature oocytes has proved to be very difficult. 
Current techniques for freezing oocytes have a very low success rates in 
terms of viable pregnancies per frozen oocyte. This is probably due to basic 
biological differences between oocytes and sperm cells and embryos.
"For decades men have had the opportunity to freeze sperm prior to treatment 
for malignant diseases, and thousand of babies have been born to couples 
where the male is an infertile survivor of cancer treatment. With the 
current techniques, women would need to freeze hundreds of oocytes in order 
to have a reasonable chance of obtaining a child, but by using the technique 
of Dr Kuwayama and his colleagues, it will be possible to achieve the same 
rates of success with 10-20 frozen-thawed oocytes as with fresh oocytes. 
This is a major improvement, and for the first time, cryopreservation of 
oocytes represent a realistic option for the preservation of fertility in 
women who are in need of aggressive treatment for malignant diseases."

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