Tag Archives: Cryopreservation

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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Freezing improves DNA integrity

Gamete cryopreservation could help improve the fertility of men whose spermatozoa show a high level of prefreeze DNA fragmentation, study findings indicate.

 Laura Thomson (Fertility First, Hurstville, Australia) and co-authors note potential cryoinjury of sperm from subfertile men is an issue of primary concern “considering that subfertile men form a very large proportion of the men requiring semen cryopreservation.”

The findings were observed during a study comparing different cryoprotectants used to store spermatozoa for fertility treatment. The study involved 320 men who presented for fertility investigations and provided semen samples.

Post-thaw sperm DNA integrity was unaffected by the type of cryoprotectant used during freezing, but showed a significant, negative correlation with the prefreeze level of DNA fragmentation. Among men with prefreeze sperm DNA fragmentation levels within the normal range, 89 percent showed an increase in fragmentation post-thaw. Conversely, 64 percent of those with very high levels of prefreeze fragmentation showed a decrease in fragmentation post-thaw.

The authors suggest that the result “gives rise to a possible novel method of reducing fragmentation in sperm used for assisted reproductive technology treatment cycles, in some cases without the need for invasive and expensive testicular sperm retrievals.”

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Breast cancer recurrence not increased with controlled ovarian stimulation

Ovarian stimulation using gonadotropins and letrozole to preserve fertility in patients with breast cancer undergoing chemotherapy is unlikely to increase their risk for recurrence, say US researchers.

Kutluk Oktay, from the Center for Human Reproduction in New York, and colleagues evaluated 215 women with breast cancer for fertility preservation before adjuvant chemotherapy. Overall, 79 of the women underwent controlled ovarian stimulation (COS) for embryo or oocyte preservation, while the remainder served as controls.

An average of 10.3 oocytes were retrieved from COS patients, with 5.97 embryos or oocytes cryopreserved per patient.

The time between surgery and chemotherapy was significantly longer for patients who underwent IVF than control patients, at 45.08 versus 33.46 days. In patients who had COS, peak estradiol levels ranged from 58.4 to 1,166 pg/ml.

In the COS group, median follow-up after chemotherapy was 23.4 months, compared with 33.05 months in the control group. Recurrence occurred in 3.8 and 8.1 percent of COS and control patients, respectively, at a nonsignificant hazard ratio of 0.56.

The team concludes: “COS before embryo or oocyte cryopreservation is unlikely to result in a significant increase in recurrence of breast cancer compared with those who did not undergo ovarian stimulation, at least in the short term.”

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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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Intracytoplasmic sperm injection (ICSI)

Intracytoplasmic sperm injection (ICSI) is an in vitro fertilization procedure in which a single sperm is injected directly into an egg; this procedure is most commonly used to overcome male infertility problems. With conventional IVF, one can expect approximately 75% of the oocytes (eggs) to fertilize if they are healthy and the sperm parameters are not severely abnormal.

 

However, when there are severe sperm defects in count, motility or morphology, prolonged unexplained infertility, or abnormalities of the oocyte membrane , there may be severely reduced fertilization or none at all.

Fertilization involves a complex series of physical and biochemical events, which take place between the egg and sperm. Conventional IVF overcomes the need for the sperm to swim long distances to reach the egg. However, the sperm must still be able to secrete enzymes to enable it to move through the cumulus mass, which surrounds the egg, be able to attach to and drill through the protein coat (zona pellucida) which protects the egg, and to attach to the egg membrane. The egg must be able to engulf the sperm and to cause the sperm head to swell and become a male pronucleus. This will then combine with the female pronucleus to complete the process of fertilization. Failure of any of these steps will result in no fertilization.

Intracytoplasmic Sperm Injection (ICSI) is used to treat severe male infertility. Male infertility is a lowering of a male’s sperm count or sperm quality sufficient to reduce a patient’s chance of pregnancy. Male infertility is often classified as mild, moderate or severe based on the number of motile sperm and the number of normally-shaped sperm in a man’s semen. Men with fewer than five million sperm or fewer than 10 percent normally-shaped sperm are classified as having severe male infertility.

During IVF, patients who do not have male infertility have their eggs inseminated by a standard insemination procedure that involves placing a small volume of specially prepared motile sperm with the eggs in a dish. The process of fertilization is complex and requires sperm to function normally. The sperm from men with male infertility often do not possess normal sperm functions. Therefore, when sperm and eggs are simply placed together in a dish, as is done in the standard insemination procedure, the eggs often do not fertilize.

The ICSI technique was developed to assist the fertilization process in patients with severe male infertility. ICSI is a highly successful procedure that involves injecting one sperm directly into the egg using a microscope equipped with specialized micromanipulation equipment. The first step in ICSI involves selecting a normal-appearing sperm for injection into the egg. The sperm is then inserted into the egg using a micropipet.

The ICSI procedure can be used successfully to treat a wide array of male infertility disorders, such as low sperm counts, low sperm motility, or abnormally-shaped sperm. ICSI may also be used to treat a condition called azoospermia, which is the complete absence of sperm in the man’s ejaculate. When no sperm are present in the ejaculate, the sperm aspiration techniques, Epididymal sperm aspiration and Testicular Sperm Extraction (TESE) may be used to obtain sperm from the male’s reproductive tract. These sperm may then be used in conjunction with IVF and ICSI, donor egg IVF and ICSI, surrogacy.

Posted by : Goral Gandhi, MSc,

                   Laboratory Director,

                   Rotunda – Center for Human Reproduction (Pvt) Ltd

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