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.

Trans-rectal Electro-ejaculation (TREE)

Trans-rectal electroejaculation (TREE) technique is used to collect semen from men who are unable to ejaculate. The technique has been used in animals since 1930s and in man since 1948 onwards. The aim of trans-rectal electroejaculation (TREE) is to directly stimulate the seminal vesicles. There are selected groups of patients for whom trans-rectal electroejaculation (TREE) is recommended. The majority of spinal cord injuries are among young men. About 5% of these men are likely to achieve pregnancies with their partners without assistance. The loss of fertility in these men is due to a decline of sperm quality, impotence and ejaculatory failure. About 50% of them may able to achieve ejaculation with the use of penile vibrators. Men who are unable to ejaculate by other means, should be offered rectal electroejaculation. Sexual dysfunction is common in insulin dependent diabetics. Trans-rectal electroejaculation (TREE) may be advised for men who are unable to achieve erection and ejaculation. Men with multiple sclerosis who are unable to achieve erection and ejaculation will also benefit from TREE. Men who are impotent and or unable to ejaculate because of psychosexual problems may be offered rectal electroejaculation after failed psychosexual treatment.

The procedure is performed as a day case surgery usually under a short general anesthesia. However, in men with spinal cord injury (SCI) it may be performed without anesthetics (although about 5% of these men may require a general anesthesia. The procedure takes about 15 minutes. The man usually lies on his right side. A metal or plastic tube (proctoscope) is lubricated and inserted into his rectum to inspect the lining of the rectum (see picture). A special probe is inserted into the rectum, the probe is attached to an electric stimulation power unit through which electric stimulation is delivered(see picture). The power is increased gradually until ejaculation occurs. At the same time an assistant will apply massage to the prostate gland and milk the urethra to obtain the ejaculate. At the end of the procedure, another proctoscope is inserted to check for any burn injury in the rectum caused by the hot rectal probe. Like any surgical procedure there are a few potential problems. Autonomic hypereflexia (very high blood pressure) is a potentially severe complication to any spinal cord injured man. Because the normal defenses which protect against a sustained high blood pressure during orgasm are lost. This can have serious consequences such as brain hemorrhage. For this reason, SCI men are usually given pills to lower their blood pressure before they undergo trans-rectal electroejaculation (TREE). Other problems that may occur include burn injury and complications associated with the general anesthesia.

After sperm preparation the fresh sperm is then used for treatment such as insemination, IVF or ICSI depending upon the sperm count and quality. Alternatively the semen sample is frozen for future use. Treatment options will depend upon the sperm count and quality. Other factors such as the fertility of the female partner and the duration of infertility, etc. will also influence the treatment options available. The options include the following:

Intrauterine insemination (IUI)

Intrauterine insemination (IUI) with washed and prepared sperm. This will be advised if the sperm problem is mild and the female partner is young, has regular menstrual cycles and ovulation, and has healthy patent Fallopian tubes. A pregnancy rate of 5-10% per treatment cycle is expected, depending on many factors such as the woman’s age and treatment cycle number, etc.

IVF (In Vitro Fertilization)

In-vitro fertilization (IVF) is usually advised if the female partner is 35 years or older, has blocked or damaged Fallopian tubes or severe endometriosis and the male partners sperm count or quality is not suitable for IUI.

ICSI (Intra-cytoplasmic Sperm Injection)

Intracytoplasmic sperm injection (ICSI) is advised when there is a severe sperm problem, i.e. sperm is not suitable for standard IVF treatment.

Recent advances in assisted conception such as IVF and ICSI have made it possible to achieve pregnancies and live births with ejaculate containing just a few sperm.

All about hCG testing

The American College of Obstetricians and Gynecologists (ACOG) has issued advice on recognizing and avoiding false-positive human chorionic gonadotropin (hCG) test results. In its latest Committee Opinion report, the college acknowledges that "clinically significant false positive hCG test results are rare," citing one study in which five out of 162 women studied had evidence of serum assay interference sufficient to provide misleading results. But the college points out that such results, if unrecognized, can lead to significant and unnecessary clinical intervention.

Reviewing the causes of false-positive hCG results, the ACOG reports that some individuals have circulating factors in their serum that interact with the hCG antibody. The most common of these are heterophilic antibodies—human antibodies that can target animal-derived antigens used in immunoassays. People who have worked as animal laboratory technicians or in veterinary facilities, or who have grown up on farms, are more likely than others to have developed heterophilic antibodies, the college says.

What is a misleading serum test result? The ACOG states: "If results are misleading, they usually are seen with values below 1,000 mIU/mL." To rule out the presence of heterophilic antibodies or other interfering substances, it lists four methods:
1.A urine test: heterophilic antibodies are not present in urine.
2.Rerunning the assay with serial dilutions of serum: lack of linearity confirms assay interference.
3.Preabsorbing serum: some laboratories can pre-absorb serum to remove heterophilic antibodies before the assay is performed.
4.Using another assay: some assays may recognize the aberrant, non-active forms of hCG that some individuals naturally produce. "Repeating the hCG measurement in a different assay system can best detect this problem," says the college.

Concluding, it states: "Caution should be exercised whenever clinical findings and laboratory results are discordant. Although false-positive hCG results are rare, if unrecognized they may lead to unwarranted clinical interventions for conditions such as persistent trophoblastic disease. The physician must judge whether the risks of waiting for confirmation of results outweighs the risks of failing to take immediate action."

Venter Sci-Fi

Craig Venter, the scientist who controversially commercialized the efforts of the Human Genome Project, is claiming to have constructed a synthetic chromosome with his research team, giving rise to an artificial life form. Mr. Venter's institute is expected to make an announcement concerning the development in the coming weeks. If true, the potential applications are simply awe-inspiring. Organisms could be "created" for a plethora of uses. Potential applications include, but are not limited to; medical treatments, environment protection, energy production and, unfortunately, biological weapons of vast sophistication. Speaking about the research Mr. Venter said:

"[this is] a very important philosophical step in the history of our species. We are going from reading our genetic code to the ability to write it. That gives us the hypothetical ability to do things never contemplated before".

The team was able to construct an artificial chromosome of 381 genes and the DNA sequence they have pieced together is based upon the bacterium Mycoplasma genitalium (see picture). The original bacterium had a fifth of its DNA removed and was able to live successfully with the synthetic chromosome in place. The man-made organism, dubbed Mycoplasma laboratorium, still relied upon the intracellular machinery already present to carry out tasks such as replication and metabolism, and in this respect the entity is not an entirely new life form. Though the potential may be phenomenal, at the moment all there is to go on is a scientist's word, and in science facts are the only language of proof. We will be waiting for an official announcement with baited breath.

Assisted Reproductive Techniques (ART)

I am asked by many a patient & at times by friends about ART. Everyone wants to know what exactly is Assisted Conception in layman's terms. Today's blog will attempt to clear the basics of IVF or "test-tube baby" as is known in non-medical language. IVF was the first procedure used to fertilize eggs outside a woman's body. In 1978, the first "test tube baby," was conceived through IVF. Most assisted reproductive technologies (ART) are derived from the IVF procedure.
In an IVF Procedure, the doctor: prescribes hormonal medications to stimulate the ovaries, gathers the eggs with the help of transvaginal ultrasound (see picture), fertilizes the eggs in vitro (outside the body) and transfers the resulting embryos through the cervix into the uterus(see picture).

Follicular Stimulation and Monitoring
At the start of the woman's cycle, the physician prescribes hormones or other medications. These control the timing of the cycle and stimulate the development of multiple follicles, sacs of fluid in the ovary that may contain an egg. Usually, the patient can take these medications at home. After several days, the patient returns to the clinic for a vaginal ultrasound and blood tests that help determine the cycle's progress and the number of follicles. More than one visit may be required.
When the doctor is satisfied with the progress of the cycle and the number of follicles, the patient is instructed to administer an additional injectable medication. This ensures that the eggs will mature on schedule for the retrieval. A minimum number of follicles must develop to make the retrieval worthwhile.

Oocyte (Egg) Retrieval
Egg retrieval is normally performed under anesthesia or intravenous sedation. Guided by vaginal ultrasound monitoring, the doctor inserts a needle through the vagina and into the ovaries. The doctor then draws back the needle to remove the eggs from the follicles. Not all follicles contain eggs. Following the procedure, patients recuperate in a recovery room.

Oocyte (Egg) Retrieval
Patients usually receive intravenous antibiotic therapy during the egg retrieval process. Certain patients will be asked to take additional antibiotics by mouth for 3 to 4 days following retrieval.

Fertilization and Incubation
Once the eggs have been retrieved, an embryologist prepares the eggs and sperm from the partner or a donor. The eggs and sperm are mixed in the laboratory to promote fertilization. In cases of severe male infertility, the embryologist can inject a single sperm directly into an egg - this technique is called Intracytoplasmic Sperm Injection (ICSI). The fertilized eggs, called embryos, are incubated for several days.

Embryo Transfer Procedure
If the embryos develop normally, the embryologist selects the best candidates to transfer into the woman's uterus. Based on the individual situation, the physician and the patient determine the number of embryos to transfer. The physician uses a small catheter to pass the embryos through the cervix and into the uterus. After the transfer, which requires no anesthesia, the patient must rest in a recovery room.

Embryo Transfer
If more good embryos than are needed for the transfer, they can, in some cases, be frozen for use in another treatment cycle.
The physician will prescribe hormonal therapy after the transfer to improve the chances for conception. If one or more of the embryos implant in the uterus, the woman may become pregnant. However, as in the natural reproduction process, there is a risk that pregnancy will not occur.

Variations on the IVF Procedure

Transfer of cryopreserved embryos
This procedure uses embryos that were salvaged from a previous IVF cycle and frozen. The embryos are thawed and transferred into the uterus.

Gamete intrafallopian transfer (GIFT)
Based on IVF, but this procedure does not involve fertilization outside the body. The eggs are mixed with sperm, then surgically placed into the fallopian tubes to allow natural fertilization. GIFT requires anesthesia as well as time in the recovery room.

Intracytoplasmic Sperm Injection (ICSI)
Based on IVF, but the eggs are not merely mixed with sperm in a dish. Instead, a single sperm is directly injected into each egg. ICSI is particularly useful when the man has an extremely low sperm count.

Assisted hatching
A microsurgical procedure in which the embryologist chemically dissolves a small area of the zona pellucida, the shell surrounding the embryo, to facilitate hatching of the embryo and implantation.

Donor Procedures
Any of these procedures, but using sperm, eggs, or embryos from a donor.

Unique "Twin" Conference in Tehran!

A friend sent me these unusual pictures of identical & non-identical twins gathered for a unique Congress celebrating "Twins" in Iran. Have a lazy Sunday & enjoy these pictures. Cheers!

Preimplantation Genetic Diagnosis (PGD)

Although Rotunda does not presently offer any PGD services, I thought it worthwhile to educate our patients & readers about Preimplantation Genetic Diagnosis. Couples with a family history of a genetic disorder and older mothers are relatively more likely to have a baby with genetic birth defects. Preimplantation genetic testing and diagnosis (PGD) can help these parents dramatically improve their odds of giving birth to a healthy child. Embryos that have certain genetic defects develop improperly. Used with in vitro fertilization (IVF), PGD can help us select the best embryos and avoid specific birth defects.
In PGD, a embryologist removes one or two cells from each embryo created in the IVF cycle(see picture). The cells are tested for abnormal genes. Only the embryos that have normal cells are transferred into the woman. Since PGD is not 100% reliable and only tests for specific defects, parents should presently still use other prenatal genetic tests, such as amniocentesis or chorionic villus sampling. PGD is expensive and still considered an experimental procedure by the majority of IVF laboratories.
Presently, only couples with family history of genetic disease and women over age 35 are advised PGD. PGD can detect genetic disorders when the defect is understood. As we learn more about genes and gene defects, PGD will become useful for more patients. PGD can tell whether an embryo will become a boy or girl, and reveal certain genetic conditions, including Cystic fibrosis, Down Syndrome, Duchenne muscular dystrophy, Hemophilia A, Tay-Sachs disease, and Turner Syndrome.
Prior to PGD, many couples with a family history of severe genetic disorder may have decided against having children. PGD dramatically improves the odds of having a baby without the disorder. In some cases, biologists can see whether the embryo has the defect. Some disorders only affect male offspring, so that female embryos may be selected to avoid the condition even if the exact defect isn't understood.
PGD helps prevent the stress and trauma after an abnormal result from an amniocentesis or chorionic villus sampling. These prenatal genetic tests can only be performed after 10-12 weeks into the pregnancy. Patients who use PGD should follow up with one of these tests, but the odds of an abnormal result will be dramatically reduced.
PGD requires the removal of one or two cells from each embryo. The embryo development is slowed slightly, but is otherwise normal. Most embryos are not adversely affected by the procedure. Some embryos may be damaged during the removal. An embryo that the PGD detects as abnormal may be normal in a small percentage of cases. This embryo would not be transferred, even though it could have become a healthy baby. An embryo that the PGD detects as normal may be abnormal similarly. This embryo would be transferred, and would result in a miscarriage or child with birth defects. Because of this risk, other genetic tests, amniocentesis or chorionic villus sampling, should be performed. To decrease the false positive & false negative results, some IVF teams employ a combination of tests using more than one cell biopsies. Since PGD is performed using embryos from an IVF cycle, the patient should be aware of the risks of IVF. There may be too few or no embryos without the defect. The embryos may not implant and develop even if they do not have the defect. If you decide to pursue PGD, you will prepare for an IVF procedure. The woman will take hormones to stimulate the ovaries to produce many eggs. Just as in a normal IVF procedure, the doctor retrieves the mature eggs from the woman. The eggs are fertilized with sperm. After two days, the embryos grow to four to six cells. The biologist forms a small opening in the outer membrane of the embryo, the zona pellucida. This is a similar process to assisted hatching. The technician gently sucks one or two cells out of the embryo through the hole. These cells are then tested for genetic abnormalities. In most cases, all the cells of an embryo will have the identical genetic makeup. Therefore, the tested cells will show the genetics of the remaining, viable embryo. The remaining cells of the embryo are young enough that they will form a complete, normal fetus. The test itself is a standard genetic test. It takes less than 24 hours to perform, so that the patient follows essentially the same schedule as a standard IVF cycle.
Women over 35 are more likely to have eggs with an extra or missing chromosome (aneuploidy). In these cases, the laboratory will examine the cells to count the chromosomes that usually lead to severe birth defects. Each human chromosome has a number, except the X and Y chromosomes that determine gender. The biologist uses a technique called fluorescence in- situ hybridization (FISH) to attach a particular color to each 13, 16, 18, 21, X, and Y chromosome. The biologist counts the spots of each color for each cell (see picture). Normal cells will have two of each color for the numbered chromosomes, as well as two X chromosomes (female cells) or an X and a Y chromosome (male cells). For a family history of a disorder, the laboratory will test for the specific defect. The laboratory must first test cells from the parent who has the disorder or may be a carrier to determine the exact defect. The embryo cells are then tested in a process that uses FISH to see if they contain that exact defect. The test doesn't reveal other genetic defects. After the tests are completed, the best embryos without the defect are transferred into the woman's uterus as in a standard IVF cycle. Follow the recovery procedures for an IVF cycle. If you become pregnant, be sure to follow up the PGD with another prenatal genetic test.