Tag Archives: cryopreserved embryos

Vitrification of Cleavage Stage Embryos

Freezing Cleavage-stage Embryos by Vitrification Improves Outcome July 9, 2009 Embryo cryopreservation is known to offer several advantages during ART cycles, including enhancing cumulative pregnancy rates, preventing ovarian hyperstimulation syndrome, reducing multiple pregnancy rates, and lowering treatment costs. After the vitrification technology for cryopreservation was developed, several studies have compared the slow freezing technique and vitrification method in relation to post-thaw survival, implantation, and live-birth rates. Now, a new retrospective study published in the Journal of Assisted Reproduction and Genetics highlights the efficacy of cleavage-stage embryo vitrification in improving the survival rate, post-thaw embryo morphology, and pregnancy outcomes, compared to the slow-freezing technique. Mojtaba Rezazadeh Valojerdi and colleagues, from the Embryology Department, Royan Institute, Iran, compared the effect of vitrification against slow-freezing of cleavage-stage embryos with regard to post-thaw survival rate, embryo morphology, and clinical outcomes. Cleavage-stage embryos of 305 patients were either subjected to vitrification (n=153) or slow-freezing (n=152) procedures. The following results observed during the study demonstrated that vitrification is a better cryopreservation technique compared to the slow-freezing method. Variables Vitrification (%) Slow-freezing (%) Odds Ratio Survival rate 96.9 82.8 6.607 Morphology with intact blastomeres 91.8 56.2 8.769 Clinical pregnancy rate 40.5 21.4 2.427 Implantation rate 16.6 6.8 2.726 Previously, Loutradi et al (Fertility and Sterility, 2008) conducted a systemic review and meta-analysis to compare post-thaw survival rates following vitrification and slow-freezing of human embryos. The investigators analyzed four studies, including three randomized controlled trials, comprising of 7,482 vitrified and 1,342 slow-frozen human blastocysts/cleavage stage embryos. A substantially higher cleavage stage embryo survival rate was observed in the vitrification group as compared to the slow-freezing group (OR=15.57; random effects model). Post-thaw survival rate of blastocysts was also found to be considerably greater in the vitrification group than the slow-freezing group (OR=2.20; fixed effects model). The conventional cryopreservation, by means of the slow-rate freezing protocol is associated with disadvantages such as osmotic shock, cryoprotectant toxicity, and mainly intracellular ice formation that can damage the cell wall and structure. In contrast, vitrification, the ultra-rapid cryopreservation method, eliminates the formation of ice crystals, thereby reducing the chances of cellular damage. The superiority of vitrification over slow-freezing for embryo preservation has been documented by several authors. Balaban et al (Human Reproduction, 2008) demonstrated that vitrification has a lower effect on embryo metabolic rate, compared to slow-freezing; as evident by the higher survival rate and subsequent in vitro development. Apart from the potential advantages of embryo vitrification, the ultra-rapid technique of cryopreservation has also shown its superiority in oocyte and sperm cryopreservation, and is hence becoming a more favorable procedure in comparison to the slow-freezing technique. In a more recent review study, Kolibianakis and colleagues (Current Opinion in Obstetrics and Gynecology, 2009) noted that vitrification was significantly better than slow-freezing with regard to post-thaw survival rates and embryo development of cleavage-stage embryos and blastocysts. However, the clinical pregnancy rates per transfer were comparable between the two groups. Although there seems to be ample evidence from retrospective studies and meta-analyses on the potential benefits of vitrification compared to the conventional freezing techniques, further prospective, randomized controlled trials are mandated for validating these findings and also to assuage the concerns of embryo toxicity due to the cryoprotectants used for vitrification.

References:
1. Rezazadeh Valojerdi M, Eftekhari-Yazdi P, Karimian L, Hassani F, Movaghar B. Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet. 2009 Jun 10. [Epub ahead of print]
2. Loutradi KE, Kolibianakis EM, Venetis CA, et al. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril. 2008 Jul;90(1):186-93.
3. Balaban B, Urman B, Ata B, et al. A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod. 2008 Sep;23(9):1976-82.
4. Kolibianakis EM, Venetis CA, Tarlatzis BC. Cryopreservation of human embryos by vitrification or slow freezing: which one is better? Curr Opin Obstet Gynecol. 2009 Jun;21(3):270-4.

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Surrogate Pregnancy after transfer of Cryoshipped, Vitrified Human Blastocysts

Rotunda achieved its first pregnancy with Cryoshipped Vitrified embryos from USA and transferring them into a surrogate mother.

Till now, we have received frozen embryos from many countries and successfully transferred them into surrogate mothers at Rotunda. Most of these embryos were frozen by the slow freezing process. As vitrification is becoming popular as a method of choice for freezing gametes, we have started receiving vitrified embryos from world over. Our first case of cryoshipped, vitrified blastocyst transfer has resulted in a pregnancy.

A short history lesson:

In 1972 preimplantation mammalian embryos were first successfully cryopreserved. The method was very time consuming. Slow cooling was used (1 degree/min or less) to about -80 degrees Centigrade. Then the embryos were placed in liquid nitrogen.

The embryos also needed to be thawed slowly and a cryoprotectant added and removed in many gradual steps. This was a lot of work.

The first reported pregnancy in humans from frozen embryos was in 1983.

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 counterparts.

Research continues in this area and human embryo freezing and thawing protocols have improved tremendously over the past 25 years. Hopefully, the newer vitrification technique will prove to have equivalent success rates with human blastocyst embryos transferred fresh or after freezing and thawing.

What is the difference between slow freezing and vitrification?

Patients who undergo IVF may have several eggs collected. The eggs are then fertilized with a sperm and checked for fertilization. Fertilized eggs are called embryos. A patient may have multiple high quality embryos eligible for embryo transfer back to the uterus. A certain number of embryos are chosen for embryo transfer, and the surplus of high quality embryos can be cryopreserved for future use.

Previously, embryos were cryopreserved using a slow freeze method. Embryos were run through different solutions of media toStorage of Cryopreserved embryosdehydrate the cells of water and replace it with cryoprotectant. Then the cryoprotected embryos were individually labeled and stored in cryopreservation straws, which were put in special freezers. These freezers slowly (-0.3 degrees Celsius per minute), cooled the embryos to -35 degrees Celsius using liquid nitrogen. They were then stored in liquid nitrogen (-196 degrees Celsius). At that extremely cold temperature, cellular activity is essentially brought to a halt, allowing the embryos to remain viable indefinitely.

When patients decide to use their cryopreserved embryos to try for a pregnancy, the embryos are removed from the liquid nitrogen, warmed and run through solutions of media to remove the cryoprotectant and rehydrate the cells with water. During cryopreservation, the formation of intracellular ice crystals can damage the cells of the embryo, decreasing future viability. Therefore, new methods were developed to improve cryopreservation techniques.

vitrification-hook 1Recent technical advancement in the field of cryobiology has opened up various options for freezing gametes and embryos at different developmental stages. The tendency of the IVF world to switch over to natural cycle IVF and to elective single-embryo transfer has put cryotechnology in the forefront of IVF. Vitrification method is gaining popularity as the method of choice for gamete/embryo cryopreservation.

Vitrification is a new process for cryopreserving embryos. Through vitrification, the water molecules in an embryo are removed and replaced with a higher concentration of cryoprotectant than in the slow freeze method. The embryos are then plunged directly into liquid nitrogen. This drastic (-12,000 degrees Celsius per minute) freezing creates a glass transition temperature, commonly called a “glass” state, and the embryos are vitrified. This quick freezing reduces the chance for intercellular ice crystals to be formed, thus decreasing the degeneration of cells upon thawing for embryo transfer.

In 1998, it was shown that vitrification using an EG-based vitrification solution (EFS40) (Kasai et al., 1990) with conventional cryo-straws was effective for human embryos at the 4- to 8-cell stage (Mukaida et al., 1998). The effectiveness of vitrification was confirmed for human embryos at the 8- to 16-cell stage (Saito et al., 2000) and the morula stage (Yokota et al., 2001b), also using EG-based solutions.

Many studies show survival rates of vitrified embryos to be far higher than survival rates of slow freeze embryos. Thus far at Rotunda, vitrification results are very encouraging, and we are excited to offer this cutting edge technology to our patients.

For more information about vitrification, ask to speak to the embryologist at your center.

Vitrification, a cutting edge technology for cryopreservation of embryos, is now available at Rotunda – Center for Human Reproduction.

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The Frozen Embryo Transfer

Frozen Embryo Transfer (FET)

If you have recently gone through infertility treatments or if you are considering undertaking IVF, you may be wondering what will happen to any extra embryos that are created during the procedure. If you and your partner have extra embryos that are not used during initial IVF procedures, these embryos can be frozen and then transferred to your uterus at a later date. Known as frozen embryo transfer (FET), this procedure has helped many couples facing infertility achieve pregnancy.

What is Frozen Embryo Transfer?
This procedure takes embryos that have been frozen for a period of time and replaces them into your uterus after they have been thawed. FET is a relatively non-invasive procedure, which is why many couples choose to have it performed. It can be successfully performed on women who are experiencing either natural or controlled menstrual cycles.Why Choose Frozen Embryo Transfer?
Many couples choose to have FET performed if they have had extra embryos remaining from an initial IVF cycle. Some couples do not like the idea of destroying embryos simply because they are “left over” from an IVF cycle. Other couples know or suspect that they will need to do IVF again in the future and prefer to freeze their embryos in order to make future IVF cycles less stressful physically for the female. 

In order to perform IVF, numerous embryos are created in order to ensure that healthy and viable embryos are available for transfer. Many couples decide to freeze some of these embryos in order to allow them the opportunity to get pregnant again in the future or for use in a later IVF cycle. 

Embryo Freezing
The FET procedure involves having your embryos frozen, or cryopreserved. The freezing procedure is as follows:

 

  • Your embryos are placed inside of special glass vials, that look much like straws.
  • These embryos are then mixed with a special solution, called cryoprotectant. This cryoprotectant prevents ice from forming in between the cells of your embryo.
  • The glass vials containing the embryos are then inserted into a controlled freezer filled with liquid nitrogen.
  •  They are cooled slowly until they reach a final temperature of -196° C.

 

Embryo Thawing
Before FET can take place, your embryos must be thawed after the freezing process. When your reproductive endocrinologist decides it is time to begin the FET procedure, your embryos will be removed from the freezer and thawed.

 

  • The embryos are allowed to thaw naturally, until they come to room temperature.
  • The embryos are then steeped in four separate solutions to help remove any cryoprotectant used during the freezing process.
  •  Your embryos are then warmed to body temperature (37°C) and mixed with a small amount of culture medium.

The Frozen Embryo Transfer Procedure

The FET procedure is actually fairly straightforward. 

Before Embryo Transfer
Before your embryos can be thawed and transferred, you and your reproductive endocrinologist need to decide how many embryos to transfer into your uterus. The number of embryos transferred will directly impact the success rate of the FET procedure. Typically, between three and four embryos are transferred during each FET procedure.

Your health care provider will then monitor your body in order to determine the best time for the embryo transfer. We usually give oral estradiol tablets to prepare the uterine lining. The thickness is measured on ultrasound scan. Your embryos will be thawed the day before your FET procedure.

The Transfer
The actual transfer of the frozen embryos is painless and straightforward, and only takes about 15 minutes.

 

  • A catheter is inserted through your cervix and into your uterus.
  • The embryos are injected into the catheter and deposited in your uterus.

 

After the Transfer
After the transfer your reproductive endocrinologist will likely have you continue any fertility medications that you may be using. Twelve days after the FET procedure, you will return to your clinic for a pregnancy test. 

Success Rates of Frozen Embryo Transfer

The success rates of FET really depends upon a variety of factors, particularly maternal age and the number of embryos transferred. Typical success rates are around 20% per cycle. It is important to know that not all embryos will survive the freezing and thawing process though. About 70% of embryos survive cryopreservation, and this can sometimes impact the success rates of FET. This makes it important to freeze and thaw a number of embryos when performing the FET procedure.

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Frozen is better than fresh when it comes to transplanting embryos in IVF

Danish scientists found babies born after a frozen embryo was thawed and implanted had higher birth weights than those born from fresh embryos.  

The study of over 19,000 babies also found no added risk of birth defects.

A European fertility conference heard frozen embryo babies did better because only the most robust embryos survived the freezing and thawing process. Freezing embryos allows couples to have several cycles of treatment from one egg collection.

That means it cuts the amount of times women have to take ovarian stimulation drugs.

As doctors want to avoid multiple pregnancies, it is common for just one embryo – which has been fertilised in the lab – to be transferred into the womb, and the rest frozen.

In later cycles, a frozen embryo is thawed and implanted three to five days after ovulation, exactly the same way as fresh embryos are used.

While single embryo transfers are becoming increasingly common, the researchers said there was little data on the results of using frozen embryos.

But earlier mouse studies had shown a higher rate of behavioural and development problems in animals born from frozen embryos.

In this study, presented to the European Society for Human Reproduction and Embryology meeting in Barcelona, all 1,200 babies who had been born from frozen embryos between 1995 and 2006 in Denmark were compared to the 17,800 babies born from fresh embryos.

The data showed no increase in the rate of congenital malformations – which include conditions such as spina bifida and cleft palate.

Fewer frozen embryo babies were admitted to neonatal care units, but the researchers said this was probably because there was a higher rate of multiple births in the fresh embryo group.

In addition, pregnancies lasted slightly longer in the frozen embryo group, and babies were an average around 200 grams bigger.

There was also a lower proportion of low-birth weight babies weighing under 2,500 grams (5.5lbs) and fewer premature births, before 37 weeks.

Dr Anja Pinborg, who led the research, said: “We think the reason for the differences is probably positive selection of the embryos for frozen embryo replacement.

“Only the very top quality embryos survive the freezing and thawing process.

“And you only get pregnancies in patients with lots of good embryos to freeze.”

She added that by the ovarian stimulation patients have to go through in order to get fresh embryos could negatively influence a consequent pregnancy – something women using frozen embryos would not be affected by.

Dr Pinborg said: “The findings are reassuring.

“If our results continue to be positive, it can be accepted as a completely safe procedure, which can be used more frequently than it is currently.”

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