Cryoprotents are used in the solution that the embryos are frozen in. There are 2 basic types - permeating (e.g. propanediol) and extracellular, such as sucrose and lipoprotein (egg yolk). Cryoprotents are useful because they:
Lower freezing point and may prevent intracellular ice formation until temp very low.
May protect cells by interacting with membranes as they change from a pliable to a rigid state.
Embryos can be frozen at the pronuclear stage (one cell), or at any stage after that up to and including the blastocyst stage (5-7 days after fertilization).
Different cryoprotents are used for different stages of embryo development.
Embryo survival rates after thawing and pregnancy rates in most IVF programs are highest for embryos that were frozen at the pronuclear stage, or at the 2-cell to 4-cell stage.
How are frozen-thawed embryo transfer cycles managed?
There are many different protocols for both "natural cycle" and for "hormone replacement cycle" thawed embryo transfers. Following are examples for each. There is nothing magic about these protocols - these are just examples for educational purposes. At the Advanced Fertility Center of Chicago we usually use hormonally replaced cycles.
Ultrasound until the dominant follicle is greater than 14 mm in mean diameter.
Then, daily urine LH testing.
Once ovulation confirmed by LH surge, thawed embryo transfer is planned for 3 days after LH surge.
Pronuclear embryos are thawed the day before transfer, cleaved embryos are thawed on the day of transfer or the day before.
Luteal support: 200 mg vaginal suppositories can be used twice daily.
Pregnancy test 12-14 days after transfer.
Hormone replacement cycle
GnRH agonist (e.g. Lupron or Synarel) is given, either midluteal (such as day 21) or very early follicular phase, such as day 2.
Down-regulation is confirmed by ultrasound and blood tests about 10 days later
Estradiol valerate 2 mg twice daily is started once endometrium <4 mm and no follicular activity seen on ultrasound. This dose may need to be increased.
When the endometrium is satisfactory in thickness and reflectivity, progesterone is started.
Embryo transfer is planned for 3-6 days later - depending on the stage of development of the embryos to be replaced.
The same estrogen and progesterone doses are continued in the luteal phase.
Pregnancy testing is done 8-14 days after transfer - depending on the stage of development of the embryos replaced.
If pregnant, estrogen and progesterone are continued until at least 12 weeks and then weaned off gradually.
There are other protocols that use transdermal estrogen patches or various other methods of progesterone support, etc.
"Window of implantation"
Implantation in some other mammals
There are some very interesting variations among different mammalian species.
" Delayed implantation", also called embryonic diapause has been described in about 100 species of mammals.
Ovulation - mating - fertilization - and subsequent development to the blastocyst stage occurs. The blastocyst then remain in uterus without implanting or developing further. In some species, the corpus luteum in the ovary is later reactivated at which time the embryo implants and continues development.
The swamp wallaby, a marsupial, is a great example:
This animal mates during pregnancy: About 4-6 days before giving birth.
The sperm enter the non-pregnant uterus (this animal has a double uterus), and the egg is fertilized.
The resulting embryo develops to the blastocyst stage and then goes into "diapause" (like hibernation).
The mother gives birth to the pregnancy that was near the end, and, after the young are finished suckling, the blastocyst that is in diapause in the other uterus "wakes-up" and implants, develops, etc.
Implantation in humans
The concept of a "window" of implantation:
After sufficient estrogenic exposure, initiation of progesterone initiates a "clock" that results in the uterine lining passing through a receptive "window" of time when implantation can occur. Before, or after the window - implantation will not occur.
Rosenwaks et al, in 1987 published an excellent article that looked at donor embryo transfers done in natural cycles. They got good results when transferring 4-6 cell embryos on day 17-19 endometrium (day of LH surge was called day14).
Formigli et al, in 1987 reported uterine lavage of embryos from uteri of donors at 5 days post-ovulation. The embryos were then transferred to recipient women. They had pregnancies when the recipient's cycle was from 4 days in front of to 3 days behind the donor's at ovulation. This suggests a window of implantation of up to 7 days.
Navot et al, in 1991 reported on donor embryo transfers done with 2-3 day old embryos on recipient "cycle days" 15-20 (artificial cycles). Pregnancies resulted from transfers on all days. This suggests (at least) a 6 day transfer window.
The "window of transfer" is a little different from the window of implantation. A 2-3 day old embryo takes 3-4 days to become a blastocyst. Blastocysts can hatch and implant. Therefore, from all of the above information, the inferred window of implantation may extend from days 18-19 to 23-24 of the "idealized cycle".
In vitro fertilization (IVF) is a remarkable diagnostic and treatment tool for infertile couples. Since the birth of the first IVF baby in July 1978, IVF has become the ultimate treatment for almost every cause of infertility. Nevertheless, any single embryo transfer has a finite probability for success. Consequently, one of the important strategies of IVF treatment, similar to natural attempts at conception, is to keep trying. “Most of the important things in the world have been accomplished by people who have kept on trying when there seemed to be no help at all.” - Dale Carnegie (1888–1955), US author of How to Make Friends & Influence People.
With many IVF cycles there is help to keep trying and it comes in the form of frozen excess embryos. Embryo cryopreservation has its roots in the accidental successful cryopreservation of fowl sperm in 1948. Scientists apparently mislabeled the experimental freezing solutions and used glycerol instead of another compound. The glycerol solution was highly effective and its use led to a new branch of science. Subsequently, several solutions have been identified that protect the cells or tissues during the freezing process. Interestingly, cryopreservation of biological material has had its greatest practical impact in the field of reproduction. Cells and tissues freeze successfully and for very long periods of time. The process of freezing cells and tissues involves cryoprotents that prevent the build up of salts as water crystallizes during freezing. High concentrations of salts and perhaps the ice crystals themselves can mortally wound cells either during freezing or thawing. The cells (embryos in this case) are stored in liquid nitrogen after a controlled freeze by special machinery. Embryo freezing takes several hours while the thaw process takes about 30-45 minutes. Embryos have been successfully thawed after cryopreservation for as many as 13 years. Clinical pregnancies have been reported from embryos stored for 9 years.
Meticulous attention to detail and careful adherence to guidelines are required to achieve embryo survival and viability that results in live born babies. “It has long been an axiom of mine that the little things are infinitely the most important.” - Sir Arthur Conan Doyle, writer of Sherlock Holmes mysteries.
At Genetics & IVF Institute, our policy has been to freeze each embryo in its own straw. Many clinics put 3-4 embryos in the same cryopreservation vial. While our strategy is more labor intensive, it has important advantages for patients. First, we know the quality of the embryo at the time it was frozen. We can use that information to decide the order to thaw the embryos. Secondly, we can immediately judge the viability of each embryo at the time of thawing so that we thaw only the number of embryos that we want to transfer, avoiding embryo wastage.
Our strategy of individual freezing and thawing is critical to achieve the exact number of embryos desired for transfer. The alternative strategy often results in too many embryos transferred with increased multiple gestations or too few embryos transferred and lower pregnancy rates. Since we know the pre-freeze characteristics of each embryo frozen, and the embryo score is related to the probability of implantation, we can purposely thaw and transfer more embryos when the embryo quality is lower without concern for high multiple gestation rates while maximizing each embryo’s potential for implantation. We believe this strategy provides the greatest efficiency and use of embryos in a frozen transfer cycle.
Embryo cryopreservation also has enormous potential to avoid ethical dilemmas for many couples. We have three basic options for handling excess embryos. The embryos may be discarded, donated anonymously to other infertile couples or donated to scientific research. IVF couples choose one of these three options at the time of egg retrieval but their choice may be changed any time in the future. Embryo cryopreservation provides an opportunity to use every embryo produced without discarding them. Some infertile couples are uncomfortable with any of these choices and they choose to inseminate only a few eggs and then transfer all resulting embryos. Other couples continue to transfer their cryopreserved embryos in subsequent frozen embryo transfer cycles with the possibility of producing additional children. Overall, embryo cryopreservation avoids many of the ethical dilemmas inherent in producing a large number of embryos.
An uncommon but serious complication of IVF is ovarian hyperstimulation syndrome (OHSS). The high hormone levels cause water to leak from the ovarian blood vessels into the abdomen. If the woman becomes pregnant, the syndrome often is much more severe and lasts longer. Cryopreservation of all embryos in an IVF cycle to avoid conception in a woman at risk for OHSS also can help avoid a very serious medical illness.
Successful cryopreservation of excess embryos is an increasingly important tool to reduce the number of multiple gestations resulting from infertility treatments. Over time, infertility treatments have become more successful and efficient in producing pregnancies. As such, the potential for individual embryos to become babies (implantation rate) has increased. The worldwide trend is to transfer fewer embryos, which results in more embryos for freezing. The question on most couples minds when they contemplate a frozen embryo transfer (FET) cycle is how many embryos will survive the thaw process and what are my chances for pregnancy?
The factors that predict pregnancy are complex and interrelated. We recently evaluated our data and found that, when about 20 factors were introduced into a mathematical equation, the predictive value was approximately 10%. Thus 90% of the factors predictive of pregnancy are not observable by the medical staff. Nevertheless, we rely on some clinical factors to help us decide which embryos to thaw and to estimate the likelihood of embryo survival. We define embryo survival based on the number of viable cells in an embryo after thawing. An embryo has “survived” if >50% of the cells are viable. We consider an embryo to “partially survive” if <50% of its cells are viable and to be “atretic” if all the cells are dead at thaw. Approximately, 65-70% of embryos survive thaw, 10% partially survive and 20-25% are atretic. Our data suggests that embryos with 100% cell survival are almost as good as embryos never frozen but only about 30-35% survive in this fashion.
Embryo morphology (appearance of the cells / percentage of fragmentation) is one of the most influential factors for embryo survival. Interestingly, embryos produced from intracytoplasmic sperm injection (ICSI) also seem to survive somewhat better than embryos produced from conventional insemination. The following graph illustrates these points. The embryo grade in the graph goes from worst (3.2) to best (1.0).
Embryos that are 2, 4 or 8 cells when frozen have about 5-10% greater survival than embryos with an odd number of cells. Donor egg embryos have a 2-5% greater survival rate than embryos from infertile women when compared by morphology score.
Pregnancy rates are similarly affected by complex relationships and like embryo survival only 7-10% of the predictive value can be observed and measured. Age is not a significant factor with frozen embryos but many fewer older women have frozen embryos. From the approximately 20 factors reviewed, the most important factors predicting pregnancy rates are the number of surviving embryos transferred, the number of 100% surviving embryos transferred and the morphology scores of the transferred embryos. The delivered pregnancy rates ranged from 5% (a single poor quality embryo) to 36% (4 high quality embryos) when the cycles from 1987 to 2001 were combined.
Blastocysts (embryos cultured for 5 days rather than 2-3) are a special case. The embryos are much larger and have special needs with regard to freezing without damage. Many centers have had trouble with blastocyst cryo-survival and pregnancy rates. A new protocol developed in our laboratory and implemented in December 2000 led to a transfer rate of 62% and a 35% pregnancy rate per transfer. This important change now makes blastocyst transfer more appealing since excess blastocysts can be expected to yield pregnancy rates comparable to embryos frozen two to three days after retrieval.
A happy note to couples that have the opportunity to use frozen embryos is the children are healthy and normal. Many studies have evaluated the children born from frozen embryos (“frosties”). The result has uniformly been positive with no increase in birth defects or development abnormalities.
In summary, embryo cryopreservation adds an important dimension to assisted reproduction. It extends the possibility for pregnancy when fresh cycles fail or when couples want additional children after a successful embryo transfer. Cryopreservation helps avoid many ethical dilemmas by eliminating the need to dispose of embryos for couples unwilling to donate them to other couples or to scientific investigation. It also offers an alternative to couples that might transfer too many embryos and risk a multiple gestation pregnancy. Our policy to freeze embryos individually provides a critical feature for efficient use of the embryos. Embryo cryopreservation adds about 10-30% more pregnancies per retrieval cycle and the outcomes of the children are normal.