Triplet ‘waiting in freezer’ born 11 years after sisters

One of three triplets conceived on the same day through IVF was born last month in Britain — 11 years after her twin sisters who say she was waiting "in the freezer" to be born.The girls, daughters of Lisa and Adrian Shepherd, all come from the same round of fertility treatment and it is believed there is no other known case where such an age gap exists.

The British couple began treatment in 1998 after Ms Shepherd was diagnosed with medical conditions that made her chances of her becoming pregnant naturally slim. During the treatment at the Midland Fertility Clinic near Birmingham, doctors successfully fertilised 14 eggs with Mr Shepherd’s sperm. Two embryos were implanted with the remaining 12 placed into freezer storage.

Soon afterwards scans showed Ms Shepherd was expecting twins. The girls, Megan and Bethany, arrived six weeks early and weighed less than 8lbs between them but made a great recovery.

When the girls were nine, the couple started to consider another baby. "We had been so busy raising the twins that it wasn’t until then that we stopped to think about having another one," Ms Shepherd said.

"We asked the girls what they thought about having another addition to the family and they really wanted it."

So last year, they returned to the clinic for another cycle of IVF, using the embryos stored 10 years previously. "We didn’t know if it would work… It was one last chance, and if it was meant to be, then it would happen."

And so it was meant to be with Ryleigh was born last month, weighing 7lb 10oz.

"The girls are thrilled to have a sister — and they know that she was conceived at the same time that they were, but has been in the freezer," Ms Shepherd said.

"She’s a really happy baby and has got a really good appetite — it’s as though she’s making up for lost time."

Duration of Embryo Cryopreservation Does Not Affect Pregnancy Outcome

A study in the latest online issue of Fertility and Sterility has reported that the length of embryo cryostorage does not adversely affect the post thaw survival rate or pregnancy outcome, suggesting it to be a safe and valuable adjunct to assisted reproduction technology (ART).
Sergio Oehninger, Director of the Division of Reproductive Endocrinology and Infertility, Jones Institute for Reproductive Medicine, Virginia, and coworkers, conducted the retrospective study to investigate the effect of cryostorage duration on embryo survival, implantation competence, and pregnancy outcome. The cryopreserved embryos were isolated from in vitro fertilization (IVF) patients, and recipients of oocyte donation cycles. Researchers analyzed a total of 11,768 cryopreserved embryos which had undergone at least one thaw cycle during 1986 to 2007. Post thaw survival proportion and implantation, miscarriage, clinical pregnancy, and live birth rates were considered as the primary outcomes of the study.
The study findings showed that the extent of cryostorage duration for IVF or oocyte donation cycles did not significantly affect the post thaw survival of embryos (frozen at pronuclear or cleavage stages), implantation competence, or pregnancy outcomes. Logistic regression analysis, however, confirmed that factors, such as stage of oocyte, number of embryos transferred and survival proportion, positively predict pregnancy outcomes.
Earlier, Machtinger, et al. (Gynecological Endocrinology, 2002) conducted a case-control study to evaluate the impact of long-term cryopreservation of embryos on its survival, and implantation rate. The study compared 101 women, implanted with their embryos which were cryopreserved for 2–9 years, and the same number of women (control) implanted with embryos stored for 6 months or less. The implantation rate was found to be 4.5% and 5.5% in the study and control groups, respectively. Based on the similar implantation rate observed in both the groups, the researchers concluded that prolonged cryopreservation, do not have an adverse impact on embryo survival and could thereby be an effective option for embryo storage in ARTs.
Embryo cryopreservation plays a major role in the overall outcome achieved by assisted reproduction programs. The procedure also facilitates an improved cumulative success rate for IVF, preventing ovarian hyperstimulation syndrome (OHSS) and reducing the rate of multiple pregnancies. Although several researches suggest that long time preservation of embryos does not affect its survival rate and pregnancy outcome, the maximum duration up to which the embryo can remain in the frozen state for successful implantation, needs further investigation.
References
1. Riggs R, Mayer J, Dowling-Lacey D, et al. Does storage time influence postthaw survival and pregnancy outcome? An analysis of 11,768 cryopreserved human embryos. Fertil Steril. 2008 Nov 20. [Epub ahead of print]
2. Machtinger R, Dor J, Levron J, et al. The effect of prolonged cryopreservation on embryo survival. Gynecol Endocrinol. 2002 Aug;16(4):293-8.

Frozen Embryo Transfer

Embryo freezing is a well-established form of assisted conception treatment. An increasing number of IVF clinics worldwide are now able to freeze spare embryos for later transfer. The first frozen embryo baby was born in 1984. Embryo cryopreservation allows multiple embryo transfers from a single egg collection and improves the chances of livebirth. Amongst the advantages of embryo cryopreservation are maximizing the potential for conception for IVF and prevent wastage of viable normal spare embryos. Perhaps this is the most important advantage of cryopreservation. Approximately 50% of women may have spare embryos available for freezing. In some clinics, the pregnancy and live birth rates with frozen-thawed embryo transfer is as high as those achieved with fresh embryo transfer. Freezing all embryos for subsequent transfer may be advised for women who are at a high risk of developing severe ovarian hyperstimulation syndrome following ovarian stimulation for in-vitro fertilization (IVF).

Embryo Freezing is also resorted to when embryo implantation may be compromised in cases such as the presence of endometrial polyps, poor endometrial development, break through bleeding near the time of embryo transfer or illness as also difficulty encountered at fresh embryo transfer e.g. cervical stenosis (inability to pass through the cervical canal because the cervix is narrowed or scarred, etc). Cryopreservation of embryos is very important to be incorporated in the egg donation programs. It is not always possible to synchronize the recipient’s cycle with that of the egg donor. In some countries, it is mandatory to freeze all embryos created from donated eggs, quarantined for a period of six months and until the donor have a repeat negative screening tests. As a result of successful cryopreservation programs, frozen embryos have also become available for donation to infertile couples.

Embryos can be frozen at any stage (pronucleate, early cleaved and blastocyst) if they are of good quality. Embryos are stored in batches of one or more embryos depending on the number of embryos that are likely to be transferred into the uterus at a later date. Embryos are mixed with a cryoprotectant fluid (to protect embryos from damage during freezing process). Then, the mixture is put either in a plastic straw or a glass ampoule and stored in liquid nitrogen at a very low temperature –196°C using a specialized programmable machine. Thawing of embryo involves removing the embryos from the liquid nitrogen, thaw at room temperature, remove the cryoprotectant fluid and mix the embryo in a special culture media. The mixture is then kept in the incubator ready for transfer.

If the embryos were frozen at cleaved stage or blastocyst, they can be thawed and replaced in the same day. However, if they were frozen at the two-pronucleate stage, then they are thawed on the day before and cultured overnight to allow them to divide and are replaced when they become 2-4 cell embryo (s). Different countries have different regulation concerning the length of time that embryos can be frozen. In the United Kingdom, embryos can be stored for a maximum of 10 years. India is yet to define the upper limit of storage.

The first step to transfer embryos from one centre to another is to write to the centre which has the couple's embryos stored in order to release the embryos. Consent from both partners is required. The couple will need to take responsibility of the embryos once they leave the center. The embryos are transferred using a small liquid nitrogen container specially designed.
Not all embryos survive the freezing and thawing process. In a good freezing program, a survival rate of 75-80% should be expected. Therefore, it may be necessary to thaw out several embryos to get two or three good embryos to replace. Damage of embryos does occur as a result of freezing, not during the storage but during the cooling and thawing process. It is important that both the couple and the clinic to keep in contact with regard their frozen embryos. Most IVF clinics will send an annual reminder letter to patients who have frozen embryos stored.

The letter usually offer a couple five choices:

Continue storage for a further period of time.
Arrange for embryo transfer treatment cycle before the expiry date.
Allow embryos to perish by interfering with the thawing process.
Donate embryos to ethically approved research.
Donate embryos to infertile couple.

Frozen/thawed embryos may be transferred into the uterus in a natural cycle, a hormone replacement cycle or a stimulated cycle. In general, the three methods have similar pregnancy and live birth rates. A Natural cycle is usually recommended in young women with regular menstrual cycles and ovulation. It involves serial ultrasound scans to check the development of the follicle and endometrium, blood tests to check the levels of hormone LH, estrogen and progesterone. Embryo transfer is usually performed about 3-4 days after the LH surge (2-3 days after ovulation). The woman is given no drugs until the day of embryo transfer. On the day of embryo transfer, the woman may start a course of progesterone pessaries or tablets to support the luteal phase. Natural cycles have the advantages of a naturally prepared endometrium and reduced cost. The disadvantages of natural cycle frozen embryo transfer is the risk of failure of ovulation. Also, the date of ovulation can not be predicted.

Hormone replacement cycle with or without GnRh agonist is usually recommended for older women, woman without ovaries or non-functioning ovaries, women with irregular infrequent menstrual cycles or ovulation. It involves giving estrogen in the form of tablets or skin batches and later adds progesterone in the form of tablets, pessaries, gel or injection. Different IVF clinics have different protocols for giving these medications and in some women GnRh agonists may be given in addition to hormone replacement to "switch off" any hormone production by the ovaries which may interfere with the treatment. After embryo transfer, both estrogen and progesterone are continued until the pregnancy test. In the test is positive, the woman should continue the medication for a further 8-10 weeks. Hormone replacement cycle allows accurate programng the date of embryo transfer and ensures that the endometrium is adequately prepared to receive the embryos.

Stimulated cycle is where fertility drugs such as clomifene tablets or FSH injection is given aiming to produce one or two follicles. When the follicle is mature and the endometrium developed satisfactorily, hCG injection is given to induce ovulation. Embryo transfer is usually performed 2-3 days after the ovulation. This regimen is usually recommended for women do not ovulate regularly and did not respond to hormone replacement treatment in a previous cycle.

The success rates depend on many factors; mainly the woman’s age and number of embryos transferred. The outcome of pregnancies resulted from frozen embryo transfer is similar to fresh embryo transfer in the incidence of biochemical pregnancy, blighted ovum, early and late miscarriage, ectopic pregnancy, preterm deliveries and term deliveries. To date there is no evidence that babies born after frozen embryo transfer have any increased incidence of congenital abnormality.

There are several ethical and moral issues surrounding the embryo freezing process. These include the following:

Fate of the stored embryos on the death of couple - 'orphaned' embryos.
Ownership of the embryos if the couple divorce.
Safety of embryo freezing.
Concern that the length of time embryos have been kept in storage might have a detrimental effect on the outcome of frozen embryo transfer and possible increase in fetal abnormalities. However, no long-term studies have been carried out since the age of the oldest child born as a result of frozen embryo transfer is only 14 years. In addition, there is no evidence that extended storage is detrimental to the outcome of treatment.