Embryo freezing after IVF: Human blastocyst and embryo cryopreservation and vitrification

Frozen embryo transfer cycle - FET- issues and explanation of details

What is cryopreservation?

Cryopreservation is freezing tissue or cells in order to preserve it for the future. This is what Darth Vader did to Han Solo in the Star Wars movie. Jabba later used the frozen guy as a wall decoration.

With current technology, we can freeze some cells (like sperm and embryos) and small tissue fragments fairly well. I think we could all think of a few people that we would prefer as frozen wall decorations. However, we can't freeze and thaw people yet...

Cryopreservation is used in infertility programs mainly to freeze and store sperm or to freeze "leftover" embryos from an in vitro fertilization cycle.

What is vitrification for IVF?

To embryologists, vitrification is ultra-rapid IVF embryo freezing instead of the traditional slow freezing process. To a science dictionary, vitrification is the process of converting something into a glass-like solid - free of any crystal formation. For example, by adding a cryoprotectant, water can be cooled until it hardens like glass without any ice crystals forming. This is important in the embryology world because ice crystal formation can be very damaging to frozen embryos (or other frozen cells). Vitrification in IVF can allow freezing of spare embryos with better post-thaw survival rates and higher pregnancy and live birth rates from frozen embryo transfer cycles. We started vitrification of blastocysts in our IVF lab in early 2008 and have seen excellent post-thaw embryo survival and substantially higher pregnancy rates after frozen transfer procedures.

A short history lesson:

In 1972 preimplantation mammalian embryos were successfully cryopreserved. The method was very time consuming. Slow cooling (1 degree/min or less) to about -80 degrees Centigrade. The embryos were then placed in liquid nitrogen. Also, the embryos needed to be thawed slowly and cryoprotectant added and removed in many small steps. This was a lot of work. The first reported human pregnancy from frozen embryos was reported in 1983 by Trounson and Mohr.

Most of the research has been done on mouse embryos. Development of frozen thawed mouse embryos, in vitro and in vivo, is not statistically reduced as compared to their nonfrozen controls. This is not yet true for human embryos. Research continues in this area and human embryo freezing and thawing protocols have improved tremendously over the past 20 years. Hopefully, the freeze-thaw technology and culture techniques will soon allow equivalent IVF success rates with human blastocyst embryos transferred fresh or after freeze-thaw.

Techniques

Cryoprotectants 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).

Cryoprotectants are useful because they:

1. Lower freezing point and may prevent intracellular ice formation until temp very low.
2. 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 cryoprotectants and freezing solutions and protocols are used for different stages of embryo development.

Many programs, including ours are currently doing all of their embryo freezing at the blastocyst stage. There has been a recent trend in the IVF world away from the traditional slow freezing method that has been used since the 1980's. Vitrification for IVF embryo freezing is becoming a more widely used technology.

How are frozen-thawed embryo transfer cycles managed?

There are many different protocols for both "natural cycle" and for "hormone replacement cycle" frozen-thawed embryo transfers, often referred to as FET cycles. Below are examples for both types of FET. 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, but some patients with regular menstrual cycles prefer the natural cycle FET approach. Success rates seem to be a little higher with the hormone replacement "controlled" FET cycles.

Natural FET cycle

1. Ultrasound until the dominant follicle is greater than 14 mm in mean diameter.
2. Then, daily urine LH testing.
3. Once ovulation confirmed by LH surge, thawed embryo transfer is planned for 3 days after LH surge.
4. Pronuclear embryos are thawed the day before transfer, cleaved embryos are thawed on the day of transfer or the day before. Blastocyst FET cycles can also be done by thawing the afternoon prior, or thawing the morning of the FET.
5. Luteal support: 200 mg vaginal suppositories can be used twice daily (this can be optional).
6. Pregnancy test 8-14 days after transfer, depending on the stage of embryos at FET and the preferences of the IVF clinic.

Blastocyst implantation in an FET cycle

Implantation after frozen blastocyst transfers (FET) can be slightly delayed compared to that seen with fresh blastocyst transfer. However, it is not different enough to warrant changing the timing of the blood pregnancy test. Frozen blastocyst transfers should have hatching and implantation by about 1-2 days after the FET and early pregnancy detection following blastocyst transfer is possible with a sensitive blood assay for HCG hormone by about 9 days after fresh or frozen blastocyst transfer. A urine HPT (home pregnancy test) can be done by 12 days after blastocyst transfer (fresh or frozen), if it is a sensitive, high quality test kit.

Medicines in Frozen Embryo Transfer FET cycle

Hormone replacement cycle:

1. GnRH agonist (e.g. Lupron or Synarel) is given, either midluteal (such as day 21) or very early follicular phase, such as day 2.
2. Down-regulation is confirmed by ultrasound and blood tests about 10 days later
3. 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.
4. When the endometrium is satisfactory in thickness and reflectivity, progesterone is started.
5. Embryo transfer is planned for 3-6 days later - depending on the stage of development of the embryos to be replaced.
6. The same estrogen and progesterone doses are continued in the luteal phase.
7. Pregnancy testing is done 8-14 days after transfer - depending on the stage of development of the embryos replaced and the preferences of the fertility specialist doctor.
8. 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".

When does blastocyst implantation actually occur in IVF or normal cycles?

A very good study of implantation was published in 1992 by Bergh & Navot. They studied 33 pregnancies from egg donation or frozen-thawed cycles (FET) with serial HCG levels on the mothers to find the time of "first embryonic signal". The HCG assay used can detect very low levels. Average first detection was at an embryonic age of 7.1 +/- 0.28 days (range 6.6-7.4 days). This correlates with the studies of Hertig and Rock in the 1950's (hysterectomy studies) that showed the day of implantation to be day 6. They did not find any evidence to support the concept of an embryonic diapause in humans.

So human blastocysts hatch from the shell and begin to implant 1-2 days after IVF blastocyst transfer (day 5 transfer). In a natural situation (not IVF), the blastocyst should hatch and implant at the same time - which is about 7 days after ovulation and fertilization.

Pregnancy success rates with FET - frozen embryo transfers:

Success rates for frozen embryo transfer cycles vary considerably by the program handling the case. Some programs have low pregnancy and live birth from their frozen embryo transfer cycles while other IVF programs have live birth rates of over 50% per transfer procedure in women under 35 for frozen-thawed embryo cycles. As more IVF programs vitrify rather than slow freeze the embryos the FET success rates across the US should improve significantly.

There is no good way to know about the pregnancy success results in the IVF program that you are considering being treated at other than:

1. Checking the CDC or SART results for that program for frozen embryo transfers.
2. Asking very specific questions of your physician.

Ask for:

1. The percentage of embryo thaw cycles that resulted in a transfer. Some thaws might result in no potentially viable embryos - therefore, no transfer is done.
2. The live birth rate per transfer procedure for frozen-thawed embryos.

Cost:

We charge $700 for cryopreservation (embryo freezing). We are currently using the vitrification technique for embryo freezing (see above).

Our IVF pricing page lists our current charges for storage of frozen embryos and also the cost for a frozen embryo transfer cycle.

Cryopreservation of oocytes, eggs:

There are currently (as of 2007) about 200 babies (worldwide) that have been born after frozen eggs were thawed, fertilized and the embryos then transferred to the mother.

The technology to successfully freeze and thaw human eggs is new and is not close to being perfected and disseminated. Although it is still experimental, it is a very exciting and promising development in the field of reproductive medicine. Vitrification of eggs seems to have some promise for the future.

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