Tag Archives: Vitrification

ESHRE Endorses Egg Freezing : ASRM Lifts Experimental Label From Egg Freezing

The American Society of Reproductive Medicine (ASRM) and the European Society of Human Reproduction and Embryology (ESHRE) have endorsed oocyte freezing as a standard and safe procedure in  2012. ASRM  issued a new report on 22 October, 2012 stating that in young patients egg freezing techniques have been shown to produce pregnancy rates, leading to the birth of healthy babies, comparable to IVF cycles using fresh eggs. After much work and deliberation by fertility experts, who reviewed the world literature on the effectiveness and safety of egg freezing and, most importantly, on the desired outcome: healthy babies, egg freezing is can now be used in routine practice. More studies are being published regarding this age range, and all is reassuring.

 

APPLICATION:

Egg freezing could provide a viable alternative source for couples needing donor eggs to build their families. In addition, among the medical indications for its use are fertility preservation for patients who may be left infertile following medical treatments for other diseases  (viz., cancer),  some genetic conditions, or IVF treatment interrupted by the unexpected inability to obtain sperm.Cryotec

 

REASON FOR CAUTION:

The Committee points out that the age of the woman at the time of egg freezing is a very important factor. “Success rates with oocyte cryopreservation appear to decline with maternal age consistent with the clinical experience with fresh oocytes.”  ASRM did not encourage egg freezing for “social reasons,” such as a delay in childbearing as, although the technical procedure of egg freezing is safe, we do not have enough long-term data about babies born to women using eggs frozen when they are older than 35. Cryotec VitrificationCiting a lack of data on safety, efficacy, cost-effectiveness, and potential emotional risks, the report states, “Marketing this technology for the purpose of deferring childbearing may give women false hope and encourage women to delay childbearing. Patients who wish to pursue this technology should be carefully counseled.”

 

ROTUNDA EGG FREEZING PROGRAM:

Rotunda is now offering oocyte cryopreservation as part of its ART services  using the latest cutting edge Cryotec vitrification technique. We also have initiated donor egg bank. We have achieved comparable success rates with frozen donor oocytes  to fresh donor oocytes.

Rotunda Egg Freezing ProgramThe excellent survival rates, embryo development and pregnancy rates have given a tremendous new hope to young cancer women. These young cancer patients can now dream of becoming a mother one day in future when they are cured of their disease.

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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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Vitrified Embryos Seem To Produce Healthier IVF Babies

picture-121picture-111picture-10Three new studies have provided further evidence that vitrified embryos may be better than fresh for IVF. The studies were presented at the American Society for Reproductive Medicine conference in San Francisco, US, last week. The studies indicate that using vitrified embryos rather than fresh embryos reduces the risk of stillbirth and premature delivery. The technique allows more embryos to survive the thawing process than the older and more widely used slow-freezing method. It is unclear why this is the case; there are several theories. Some experts have suggested that when fresh embryos are used women may still be
suffering from the effects of the powerful drugs that are used to stimulate the ovaries, temporarily disrupting any IVF attempt shortly afterwards. Dr Allan Pacey, from the University of Sheffield and secretary of the British Fertility Society (BFS), said: ‘These findings are really quite interesting.
It kind of defies logic to a certain extent, because the stimulation drugs and anaesthetics that are used in egg collection have worn off by the time fresh embryo transfers are done. It seems to be an issue with the formation of the placenta, but how it has an effect isn’t known.’ It has also been suggested that only the best embryos survive the cryopreservation process. 
The three large, independent studies took place in Finland, Australia and the US. The Finnish study found that babies born from fresh embryos were 35 per cent more likely to be premature and 64 per cent more likely to have a low birth weight when compared to those born from vitrified embryos. The research that took place in Melbourne, Australia, showed that 11 per cent of babies born from fresh embryos had a low birth weight, compared to 6.5 per cent of those born from vitrified embryos. They also found that 12.3 per
cent of babies born from fresh embryos were premature, compared with 9.4 per cent of those born from vitrified embryos. Also, 1.9 per cent of babies from fresh embryos died a few days after birth, compared to 1.2 per cent from vitrified embryos. Similar findings were reported in June this year from a Danish study.
Typical IVF treatment involves stimulating a woman’s ovaries with hormones to produce eggs which are then collected and fertilised in the laboratory, with one or two embryos being transplanted into the womb two
days later. The remaining embryos can be slow-frozen or vitrified, then stored, to be used later if the initial cycle fails.
The new data may provide a dilemma for IVF clinics, as although vitrified embryos seem to result in a healthier pregnancy, the actual rate is less successful. Commenting on this, Dr Pacey said: ‘Frozen embryo
transfers are not as successful as fresh ones in terms of getting a pregnancy. So it may be that we have to balance the health of children against chance of success.’

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Blastocyst Embryo Transfer

Blastocyst transfer achieved the first IVF human pregnancy. Blastocyst transfer is claimed to be more physiological than pronucleate or cleaved-embryo transfer is as it mimics nature more closely. As the embryo advances in the development, after 5-6 days it becomes a blastocyst(see picture). This has an outer thin layer of cells, which will later form the placenta, and an inner cell mass, which will develop into the fetus. A blastocyst has about 120 cells. A blastocyst gives a better idea of the competence of an embryo and has a higher chance of implantation than a cleaved embryo. In conventional culture medium, about 20% of embryos will develop into blastocysts. Recently, the use of sequential culture medium (the embryos are cultured in different media according to their stage of growth) has enabled a larger number of embryos to develop into blastocysts. However, up to 40% of patients will not grow blastocysts and will not have blastocyst embryo transfer. The rationale behind a blastocyst transfer is that an embryo, which has failed to reach the blastocyst stage, would be unlikely to have resulted in a pregnancy. However, if it reaches the blastocyst stage it has about 50% chance of implanting. So the improved implantation rates following blastocyst transfer is due to selection of the best embryos.
Why then do 50% of the blastocysts fail to implant? A defective blastocyst (e.g. chromosomal abnormalities) is a possible cause; a non-receptive endometrium is another cause. Blastocyst embryo transfer into the uterine cavity is performed about 5-6 days after egg collection. Transfer of one or two blastocysts is recommended to avoid high-order multiple pregnancies. Supernumerary blastocysts can be frozen for future use.
Blastocyst transfer is recommended for patients who had repeatedly failed to achieve a pregnancy following the transfer of good quality cleaved embryos (If the embryo arrests and did not develop to blastocyst, this may indicate a potential egg problem). Patients who wish to achieve a pregnancy without the risk of multiple pregnancies will benefit from a single blastocyst transfer. Patients who do not wish to have their spare embryos frozen for whatever reasons may be advised to have blastocyst transfer. About 10% of the embryos that fail to develop to blastocyst in vitro may have done so if replaced inside the womb on day 2 or 3. Up to 40% of patients will not have blastocysts available for transfer. Freezing spare blastocysts was not as good as freezing cleaved embryos in the past. But, with the advent of Vitrification, high pregnancy rates have been reported. We, at Rotunda have a highly successful vitrification program for cleavage stage embryos as well as blastocysts.

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