“Infertile couples from all over the world come to St. Louis, Missouri, to chase their dream, because Dr. Sherman Silber and his team are simply the best there is.” – Discovery Health Channel June 2004 Documentary

Video: Dr. Silber explains all the modern Assisted Reproductive Technologies (ART), including IVF

Video: Dr. Silber discusses Egg and Ovary Banking to Preserve Fertility

IVF (in vitro fertilization) is the most common form of ART (Assisted Reproductive Technology) today. If the fallopian tubes are damaged or the sperm is poor, it is the only acceptable approach. It is also usually the most effective treatment for most other types of infertility as well. The eggs must first be fertilized in our laboratory, and the resulting embryos then are placed into the uterus 2 to 5 days later. This procedure achieves remarkable pregnancies even in women with hopelessly damaged fallopian tubes and even "unexplained" infertility. When there is any problem at all regarding the sperm, we can fertilize the eggs with ICSI. In fact, in our program we routinely use ICSI in all cases to guarantee against the risk of failed fertilization. Our IVF pregnancy rate is over 50% per attempt, regardless of diagnosis, and we accept all of the most difficult cases.
    See: ART Pregnancy Rates

Extra embryos are frozen and saved for a later, much less expensive future pregnancy. The success rate for frozen embryo transfers in our program is just as high as for "fresh" transfers.

With IVF, the eggs are fertilized in the laboratory, as opposed to GIFT, which simply leaves fertilization up to nature in the fallopian tubes.

How Does IVF Work?

Eggs are retrieved by ultrasound-guided needle aspiration under light sedation (in the operating room). This involves no surgical incision, and virtually no pain afterward. You just leave the hospital directly from the operating room, with no pain, and come back three days later to have the embryo (or embryos) placed very simply into the uterus through the cervix with a tiny catheter. No incision and no anesthetic are needed. An hour later you are able to go home. There is no pain from the procedure.

Intra-Cytoplasmic Sperm Injection (ICSI)

If there is a question of the sperm's ability to fertilize the egg, due to either a low sperm count or poor quality of the sperm, that poses no problem whatsoever. Intra-Cytoplasmic Sperm Injection (ICSI) would be performed instead of regular IVF. With ICSI, the eggs are retrieved the same as if you were doing conventional IVF. However, the eggs and the sperm are then fertilized in the laboratory, by direct injection of a single sperm into each egg. Three days later the resulting embryos are simply placed into your uterus with no surgery, just as with IVF. Extra embryos are frozen for later attempts at pregnancy.

The availability of this Intra-Cytoplasmic Sperm Injection, "ICSI" technique (which was developed and perfected by the Brussels University and our institution in St. Louis) means that men whose sperm previously were too weak or too few to fertilize in vitro (IVF), now have no problem fertilizing their wife's eggs. The fertilization rate per egg using ICSI is about 70% despite the sperm being terrible, the fertilization rate per infertile couple is over 99% if the wife has adequate eggs, and the pregnancy rate per treatment cycle is over 50%. This is not significantly different from regular IVF with normal sperm. This technique is very cost-effective, and will give you the same high chance for getting pregnant as any couple with normal sperm.

How Does ICSI Work?

We can take a man who would otherwise have to resort to donor sperm, and if we can find just a few weak sperm in his otherwise sterile appearing ejaculate, it is more than enough to microsurgically inject these few sperm into his wife's eggs, fertilize them normally, and get her pregnant.

If there is absolutely no sperm in the ejaculate, we can perform a testicle biopsy, remove the few non-moving sperm that we find through a highly refined ultra micropipette, inject it into the wife's egg and still get her pregnant. Even in testicles where allegedly there is no sperm production, we can usually (but not always) find a few sperm, which is enough for successful ICSI.

	Figure 1: Immobilizing the sperm's tail before picking it up.
	Figure 2: Injection of sperm into the egg.
	Figure 3: Fertilized egg demonstrating the two nuclei – one from the father, one from the mother.

Mini-IVF

Mini-IVF - Fox News St. Louis

When patients contemplate IVF, their first reaction is often the fear of daily injections of hormones for months, the incredibly high cost of the drugs, the risk of multiple pregnancy and consequent prematurity, side effects related to high levels of estrogen resulting from large numbers of eggs, hyperstimulation syndrome, and the prospect of painful daily progesterone injections for a full ten weeks even after the IVF procedure. Mini-IVF is a very unique approach developed by our colleagues in Japan to circumvent these problems and to simplify IVF for patients, reducing the cost while maintaining comparable success rates.

Mini-IVF is designed to recruit only a few (but high quality) eggs, thus avoiding the risks of hyperstimulation, reducing the cost of drugs from an average of $4,000 to closer to $400, reducing the number of injections, and completely avoiding the painful progesterone injections. This approach is not just a simple-minded reduction in hormonal stimulation. It is an ingeniously conceived and completely different approach to IVF, that saves the patient much of the complexity and cost associated with more conventional IVF protocols. Here is how it works.

On Day 3 of the menstrual cycle, you start on a low dose of Clomid (50mg), but you don’t stop the Clomid in five days as is usually the custom. You just keep taking the Clomid until ultrasound monitoring shows the follicles to be ready for ovulation. A very low “booster” dose of gonadotropin (just 150 iu of FSH), is added on Days 8, 10, and 12. Clomid not only stimulates your own pituitary to release FSH naturally (by blocking estrogen’s suppressing effect), but also staying on the Clomid (a unique new approach) blocks estrogen’s stimulation of LH release, and so also usually prevents premature ovulation. Thus, with this simple change in protocol, the old-fashioned, cheap Clomid is able to stimulate the development of great quality eggs for IVF.

Another advantage of this protocol is that you did not have to go on Lupron first to suppress the pituitary. Staying on Clomid blocks estrogen from stimulating your pituitary to release LH, and this prevents premature ovulation without your having to be suppressed. This means that you can be induced to ovulate with just a simple injection or nasal sniff of Lupron. This causes a more natural LH surge, and avoids the luteal phase defect caused by HCG that would otherwise require months of progesterone injections.

The next step is to recognize that Clomid has a negative effect on the uterine lining (because it prevents estrogen from stimulating the endometrium). That is one reason why results in the past have been so poor with the use of Clomid for ovarian stimulation. The embryos are less likely to implant in such endometrium. But that problem is solved by using the Japanese protocol for embryo freezing, “vitrification,” which I discuss elsewhere. We can now freeze the embryos almost with impunity using this approach, with only a 1% risk of loss. Then these embryos are transferred the next month in a “natural cycle” with no need for taking any hormones at all.

The frozen embryo transfers can then all be performed in a later natural cycle (without hormones). Even if you don’t normally ovulate predictably, you can be given one injection of Lupron in the follicular phase (once your follicle reaches 1.5cm) to induce natural luteinization, and still have a natural cycle embryo transfer with no hormones. The Day 3 frozen embryo would then be transferred five days later, and there is no need for your taking any hormones at all.

Even for poor prognosis cases of older women with low remaining ovarian reserve, there is an advantage to mini-IVF over high dose stimulation. Such patients normally yield very few eggs anyway even with huge megadoses of gonadotropin. If they have any quality eggs remaining, mini-IVF is just as likely to yield as many eggs (very few, of course) as giving huge megadoses of gonadotropin. Even in the worst case scenario, if there are no good eggs left at all, at least they can discover this with only $400 spent on drugs instead of $7,100 (cost of maximum dosage).

Think of this simple parable: If you are sitting under an apple tree, and wish to eat the most ripe and ready apples, you have a choice. You can chop down the tree, and look at every apple on the fallen tree to see which ones were ready. Or you can simply try to shake the lower branches and eat the one or two that have fallen. That is the idea of mini-IVF. It may not work for everyone, but for many patients, it will remove much of the aggravation and complexity associated with IVF, and also dramatically reduce the cost.

Improvements in Embryo Culture

A major improvement in embryo culture was realizing that the oxygen content in the air we breathe is much too high for eggs and embryos. In fact, most cells in the body are exposed to a much lower concentration than the air we breathe. Too much oxygen delivered to these cells can, in a sense, overheat the cell. So it is much better to culture the embryos, not only in 5% CO₂, but also in only 5% oxygen (not the 20% that is in air). This is difficult to do. Large amounts of pure nitrogen gas have to be blown constantly through the incubator at a carefully controlled rate to lower the oxygen concentration in the incubator. But it is worth that extra effort to get higher pregnancy rates.

Classically, most IVF labs have cultured embryos at a pH of 7.4 (the normal acid-base of blood concentration), and at an oxygen concentration of 20% (the same as in the air we breathe). However, these are not the acid or oxygen concentrations that are most favorable for embryo growth and development. In fact, the acid concentration inside the embryo is normally much greater than that, and the oxygen concentration is much lower. Conventional IVF culturing conditions, therefore, are too alkaline and too oxygen-rich. In fact, oxygen concentration in the Fallopian tube is only about 8% (not 20% as in air), and in the uterus, it is as low as 2%.

This type of optimal culturing of embryos requires a lot of extra attention. To reduce the oxygen concentration in the incubator from 20% to 5% requires blowing through a huge amount of nitrogen (95%), and to keep the pH acid at 7.2 (but not too acid below 7.2), requires careful monitoring of the acidity of the media. This represents a lot of extra work, but it is well worth the effort.

Freezing Embryos by Vitrification

Video Showing the Vitrification Process

This new technique of freezing called “vitrification” avoids the damage caused by ice forming inside the cell by not trying to pull every last molecule of water out, because it is impossible to do this 100%. In fact, 70% of the cell is water, and at best you can reduce that to 30%. So with the conventional controlled rate slow-freezing technique, there is always going to be some intra-cellular ice crystal formation, causing some damage to embryos, and severely damaging most eggs. Vitrification uses a super high concentration of antifreeze (DMSO and ethylene glycol), and drops the temperature so rapidly that the water inside the cell never becomes ice. It just instantaneously super-cools into a solid with no ice crystal formation at all.

We can now freeze and thaw, and even refreeze and rethaw, with impunity, using this new protocol from Dr. Masashige Kuwayama from the Kato Clinic in Tokyo. With conventional “slow freezing,” the temperature of the embryo goes down at precisely 0.3°C per minute. With vitrification (using four times the concentration of antifreeze, or cryoprotectant), the temperature is dropped at 23,000 degrees C° per minute, that is 70,000 times faster. At that speed of cooling, and at that concentration of antifreeze, ice crystals simply cannot form.

Of course, it is not quite as simple as it might sound. Such high concentrations of antifreeze, in a few minutes, could be toxic to cells. Therefore, the embryos (or eggs) must first be placed in lower concentrations of antifreeze (and sucrose to draw some water out), and then left in high concentrations only for less than a minute before instantaneous freezing. Then when the time comes to thaw the embryo, it must be instantaneously warmed, immediately taken out of the high concentration of antifreeze, and then placed into a solution with lower concentration, in order to avoid antifreeze toxicity. This requires more skill than conventional freezing, but it is faster, cheaper, and most importantly, avoids almost all freezing damage to either eggs or embryos. Such a reliable method of embryo freezing gives the IVF program much greater ability to avoid dangerous multiple pregnancy, and makes scheduling for procedures like egg donation simpler for the patient.

Using this vitrification technique for freezing, we can now also preserve eggs as well as embryos and sperm. This allows us to preserve the fertility of young women for the future in egg banks if they wish to delay childbearing.

Blastocyst Culture — Dr. Silber’s comments

Whenever IVF or ICSI is performed, embryos may be cultured for either one day, two days, or even five days, before transferring them back into the woman. In order to culture the embryos for five days, i.e. to what we call the "blastocyst" stage, you need to use "sequential" culture media systems. We have used such a system since the summer of 1997 because it gives us the option of culturing the embryos for as long as is clinically appropriate for each particular patient's situation.

However, there is a great deal of debate, and some considerable commercial hype, centering around whether to culture for two days, three days, or five or six days. The media we use, and which many other of the best programs use, allows us to culture the embryos to whatever number of days is appropriate for the particular patient. Since this media is commonly available, it should not be used to hype any particular program.

For some patients with poor quality embryo development (a condition which is programmed into the genome of many infertile women), even with the best culture media, the embryos may be better off going directly into the fallopian tube immediately. For the average patient, day two or day three transfer either to the fallopian tube or to the uterus may be best. For some patients, day 5 transfer to the uterus may be a good option. The problem with extended culture to day 5 is that there may be a loss of some embryos that might have "made it" if they had been transferred earlier.

The major advantage (despite the already mentioned disadvantage) of day 5 transfer is embryo selection. The implantation rate per day 5 blastocyst transfer is greater than for transfer of day 2 or 3 embryos. But only 20 percent to 50 percent of day 2 embryos can develop in vitro to day five no matter how perfect the in vitro culture system. There is a potential loss therefore of what could have been viable embryos. So selection is the only advantage of blastocyst culture, and this selection has nothing to do with the "quality" of the baby, but rather just whether the embryo "makes it" or not to becoming a baby.

Either way, because our system of embryo freezing is so good, we do not lose anything if we transfer less embryos to reduce your risk of triplets or quadruplets, and just save the extra embryos for a later pregnancy. We can even transfer just one embryo at a time (if that is your wish) without at all reducing your chance of pregnancy.

Therefore, hype should be removed from this issue, and decisions should be made that are in the best interest of the particular patient's situation.

Nonetheless we believe that use of sequential culture media, as we routinely do, is an advantage, in that patients can have the option of prolonged embryo culture if that is in their best interest.

Costs For IVF

Since infertility treatment is expensive, we try to soften its impact by achieving the highest possible pregnancy rates, and thus reduce the number of treatment attempts and the overall expense. We also make sure not to charge extra for unnecessary testing or ineffective preliminary treatment approaches. We only recommend what we feel is necessary to help you achieve a pregnancy as soon as possible.

Once you are scheduled, there is no additional charge for office visits, semen analysis and/or sperm freezing performed at the time of your procedure, embryo freezing, assisted hatching, blastocyst culture, or ICSI, all of which are available to give you the greatest chance for pregnancy. The procedure that we normally recommend is ICSI with assisted hatching with an embryo transfer directly into the uterus (IVF). This treatment will increase your odds of fertilization and implantation.

In addition to IVF with conventional stimulation, we also offer IVF with minimal stimulation (mini-IVF), which is a new, lower cost option for patients. This approach would reduce the cost of your overall treatment (including medications) dramatically.

Assisted Hatching: Excerpt from "How to Get Pregnant With the New Technology, Updated and Revised"

Another attempt at improving the pregnancy rate is the idea of "assisted hatching". The concept of this procedure is that based on the fact that the embryo normally sits around in the uterus without any effort to implant until around day six. Until day six, the embryo keeps growing within its very tough zona pellucida (outer shell). But on day six, that zona pellucida begins to thin out, and the embryo then eventually "hatches," just like a chicken out of an egg. It is at this moment of hatching that the embryo, now called a "blastocyst," actually implants into the uterine lining, the endometrium. It is at this moment, around day seven after fertilization, that pregnancy actually occurs, and this free floating ball of cells finally becomes a part of the mother. One theory to explain the perplexing phenomenon that many IVF embryos do not result in a pregnancy has been that this thinning of the zona and hatching of the embryo may be defective and in some way impeded in embryos that have been cultured in vitro.

The solution to this problem would be to microsurgically thin out the wall of the zona pellucida of these embryos on day three, just prior to transfer. It was hoped that this micromanipulative process to the embryos might provide a necessary extra bit of help to improve the implantation rate. This proposition is very difficult to prove with certainty, but in many centers, including ours, assisted hatching is performed on most embryos, and the results have thereby improved. It is a beautiful procedure and a wonderful rationale, and in women who have failed to get pregnant through the transfer of perfectly good embryos, the standard approach at the present should be to give them the benefit of the doubt, and to do assisted hatching.

A healthy blastocyst hatching.

PGD

	Pre-implantation genetic diagnosis discussed on St. Louis network television. December, 2002.			Pre-implantation genetic diagnosis discussed on St. Louis network television. July, 2002.
	Embryo biopsy for genetic diagnosis. [technical video]			Marfan's Syndrome Report on St. Louis network Television. March 23, 2003.

WHAT IS PREIMPLANTATION GENETIC DIAGNOSIS (PGD)?

Most of the twenty-five thousand or so genes in the human genome have now been identified and their DNA sequenced. Molecular analysis of genes is becoming simpler and more efficient. As a consequence, PGD with IVF, can now prevent couples from having to face the horror of giving birth to children with almost any of the genetic defects such as Down syndrome, cystic fibrosis, muscular dystrophy, sickle cell anemia, Tay-Sachs, Gaucher’s disease, mental retardation, etc., that terrify every woman who ever gets pregnant. With PGD, we can also better understand the problem of recurrent, early miscarriage and the genetic errors that arise in pregnancies of older mothers.

PGD should not be construed as creating “designer babies,” an incorrect term used only by the press and not by physicians. We could not manipulate (even if we wanted to) the features or characteristics of an offspring. That is just pure fiction. All we can do is eliminate heartbreaking and devastating genetic disease.

Figure 1: 8 Cell Embryo Ready for Biopsy (i.e. Remove One Cell)

WHAT IS EMBRYO BIOPSY?

When an embryo reaches the third day of development, it normally has eight cells. One or two of these cells, called “blastomeres,” can be removed from the embryo with micromanipulation technique. The embryo is usually unharmed, and can go on to develop just as though this one cell were never removed. You can then subject those one or two cells to genetic analysis, and know the chromosomal composition of the embryos, and if they carry a specific disease-producing mutation.

Figure 2: Removing One Cell for DNA Analysis

BASIC GENETIC LESSONS

DNA Testing and Single Gene Disease

Everything we are physically is determined by approximately 25,000 genes located in our DNA. Your entire body is made up of many thousands of different proteins whose structure and orientation determine the incredible machinery of your body and brain. However, these many thousands of different proteins are in turn composed of just twenty amino acids. The variety of amino acids that theoretically could be expected to exist is unlimited. Chemists can synthesize many thousands of them. However, living nature is composed of only twenty specific amino acids, which are absolutely the same for all living things on earth. The differences between living creatures lie in the differences in the sequences of these twenty amino acids, which make up all the different proteins of which they are composed. In turn, this sequence of amino acids has been determined by the sequence of the 4 basic letters that comprise our DNA. The entire human genome represents 3 billion base pairs, or letters, of DNA. A specific set of combinations of three DNA letters are codes for each one of the twenty amino acids. Therefore, the order in which the DNA letters occurs on the chromosome determines the sequence of amino acids, and this, in turn, determines the proteins, which determine everything in our body. A single error in an amino acid sequence in any protein can cause a dramatic change in its structure, and a severe genetic disease like cystic fibrosis or Tay Sachs. These errors in DNA sequence can be diagnosed by a process (carried out in a test tube) called PCR, and that is the basis of PGD for single gene genetic disease.

Chromosome Testing and Aneuploidy Screening

All of our 6 billion DNA “letters” are located on forty-six chromosomes, which are coiled up inside the nucleus of every single cell in your body and are divided into twenty-three pairs, twenty-two “autosomal” pairs and one “sex” chromosome pair, the “X” and the “Y”. The child who has two X chromosomes will be a girl, and one with one Y chromosome and one X chromosome will be a boy.

For conception to occur, twenty-three chromosomes from the husband’s set of forty-six, and twenty-three chromosomes from the wife’s set of forty-six, must meet at the moment of fertilization and become an embryo with a new normal set of forty-six chromosomes. The process whereby primitive sperm cells and primitive eggs lose half of their chromosome number as they become sperm and mature eggs ready for fertilization is called “meiosis.” The aging process of eggs makes it harder for them to undergo the meiosis process than sperm. That is why the eggs from older women are less likely to result in a viable embryo, and that is also why older women are more infertile than younger women, and why older women have higher rates of miscarriage and of babies with abnormalities such as Down’s syndrome.

If there is an error in division of the egg’s chromosomes and one of the pairs of chromosomes fails to separate, then the egg will have twenty-four chromosomes instead of twenty-three. In Down’s syndrome, for example, the embryo has a total of forty-seven chromosomes instead of forty-six because it has three sets of chromosome 21 instead of the normal two sets. This kind of a chromosome error, in which one of the chromosomes has three copies instead of the proper two copies, is called “trisomy.” All of these chromosomal errors, including trisomies, monosomies, and various combinations can occur in virtually any of the chromosomes, and these errors, as a group, are called “aneuploidy.” These numerical chromosomal erros in a cell can be diagnosed by counting color signals on a slide by a process called FISH.

Most of these chromosomal defects that occur in human embryos are lethal and result in either failure of the embryo to implant, or result in a miscarriage. Only occasional chromosomal defects such as trisomy 21, or Down syndrome result in the actual birth of an abnormal baby, and even in that event (trisomy 21), only 20% of the time. Nothing about in vitro fertilization (IVF) or ICSI, increases the risk of these chromosomal abnormalities.

Criticisms and Limitations of PGD

Single gene defects are the rare genetic diseases like cystic fibrosis or Tay Sachs (there are thousands of them), which occur in the offspring of otherwise fertile couples who are carriers, and which are diagnosed by PCR. PGD for such couples is extremely accurate today because for verification, multiple DNA sequences near the defective gene (linked markers) can be tested for, as well as the mutation itself. PGD is also very accurate for recurrent miscarriage caused by chromosomal translocations for which about 1.5% of infertile couples are carriers.

However, PGD (referred to as PGS in Europe) for routine aneuploidy screening during IVF for infertile couples has recently come under severe criticism in Europe owing to several prospective, randomized control studies in Holland and Belgium. PGS has failed to show any improvement in IVF clinical outcome per initiated cycle for advanced maternal age, nor for recurrent implantation failure.

In European studies of even younger, infertile couples, only 36% of embryos subject to PGD were found to be chromosomally normal (where two embryos are removed and tested rather than one). When embryos diagnosed as chromosomally abnormal on Day 3 (and, therefore, not transferred to the patient) were re-examined on Day 5, only 54% turned out to have that abnormality. The error rate for the FISH technique has been clearly shown to be as low as 5%. Therefore, this 50% discordance appears to be mostly due to the wide prevalence of embryo mosaicism. This means that some of the cells in many embryos are normal and some are abnormal. About 50% of embryos in the European studies have thus been found to be mosaic. Therefore, the use of PGS may result in good embryos being discarded (diagnosed as abnormal) and abnormal embryos (diagnosed as normal) being transferred.

However, there is still some benefit to PGS in certain cases. Firstly, if two cells are tested instead of one, and both reveal chromosomal errors, the diagnosis of abnormal embryo is very likely to be correct. Likewise, the diagnosis of normal embryo using two cells also is likely to be correct. (However, the problem is that removing two cells is more likely to hurt the embryo than removing one cell.)

Secondly, for recurrent miscarriage in younger women, even with removing and testing only one cell, PGS is likely to reduce the miscarriage rate. (However, unfortunately, with PGS there are certain to be some normal embryos that are discarded as abnormal.) PGS can also vastly reduce the risk of a Downs Syndrome conception.

Thirdly, it may be useful in counseling for cases who consistently produce all chromosomally abnormal embryos in order to understand their problem better.

Our experience with PGS and PGD is extensive, and we can counsel you on whether it is a good idea or not for your particular IVF cycle. In general it is best reserved for carriers of single gene disease who do not want an offspring to have such disease, and for recurrent miscarriage.

Related information:
"Genetic screening for disorder offers hope: Woman with Marfan’s didn’t want to pass it on"

See also:

Also listen to Dr. Silber's radio commentaries:

Listen to Dr. Silber's internet radio chat on msnbc.com. (47:26 min.)

Listen to Dr. Silber and Joan Hamburg discuss the exciting new procedure for determining exactly how long a woman has left on her biological clock. (25:52 min.)

If you have any questions, you may call us at  (314) 576-1400.