Steps of an IVF Cycle

Steps of an IVF Cycle
Micromanipulation Techniques

Superovulation Stimulates Egg Development

The controlled "superovulation" techniques used in IVF are designed to stimulate the ovaries to produce several eggs (oocytes) rather than the usual single egg as in a natural cycle. Multiple eggs increase the potential availability of multiple embryos (fertilized eggs) for transfer and ultimately increase the probability of conception.

The medications required to boost egg production may include, but are not limited to the following: Lupron (gonadotropin releasing hormone-agonist), Antagon or Cetrotide (gonadotropin releasing hormone-antagonist), Follistim, Bravelle or Gonal-F (FSH, follicle stimulating hormone), Repronex (combination of FSH and LH, luteinizing hormone), and Pregnyl or Novarel (hCG, human chorionic gonadotropin). Each is administered by injection only. Most medications are given subcutaneously (beneath the skin), though some are intramuscular injections (into the muscle). Risks associated with injectable fertility medications may include but are not limited to, tenderness, infection, hematoma, and swelling or bruising at the injection site.

Retrieving the Oocytes (egg retrieval)

For IVF, collection of eggs is usually performed under transvaginal ultrasound guidance. To accomplish this, a needle is inserted (under IV sedation) through the vaginal wall into the ovaries using ultrasound to locate each follicle. The follicular fluid is drawn up into a test tube to obtain the eggs. Although patients are given pain medications intravenously and are carefully monitored by an anesthesiology staff, some women may experience some discomfort during the procedure. Generally, the oocyte (egg) retrieval takes 20-30 minutes. Patients are usually discharged home within hours after the retrieval. Risks of oocyte (egg) retrieval may include, but are not limited to, the following:

1.- Potential reactions from the drugs and procedures used in the administration of anesthesia.
2.- Risks associated with the passage of the needle through the vagina into the ovaries (including infection, bleeding, inadvertent damage to adjacent structures including, but not limited to, the bowel, bladder, blood vessels, ureter, uterus or ovary(ies), and adhesion formation (internal scarring) following the procedure. Although uncommon, significant bleeding or damage to the bowel may occur, and surgery may be required to repair such damage; this is a very uncommon event. Rarely, infection may become severe enough to require hysterectomy and/or removal of one or both ovaries.

Collecting and Preparing the Sperm

A semen sample will be obtained from the partner by masturbation on the day of the oocyte (egg) retrieval. This is usually obtained while the retrieval is being performed. Abstinence from ejaculation for two to five days prior to providing this semen specimen is recommended. After the specimen is produced, the sperm will be prepared for inseminating the collected eggs in our laboratory. Because this can be a stressful time period for men, the man/partner may be unable to produce a specimen when needed. Men who feel that they may have difficulty producing a semen specimen have the opportunity to have their specimens frozen by our laboratory ahead of time for use in this situation. Testicular biopsy can also be performed as a method to extract sperm for IVF.

Insemination of Eggs and Embryo Culture

Following egg retrieval, the follicular fluid is immediately transferred to the adjacent laboratory for identification of eggs, evaluation, and preparation for insemination. In the process of collecting the follicular fluid, it is possible that a large number of eggs may be retrieved. It is strongly recommended that all of these eggs be inseminated to maximize the number of embryos available for subsequent transfer. Any objection(s) to this policy should be stated in writing and attached to the IVF-ET consent form with the understanding that pregnancy success may be reduced. Otherwise, the prepared sperm will be added to each egg and they will be allowed to incubate overnight under controlled laboratory conditions. The next day, each egg is evaluated for evidence of fertilization. However, it is possible that no eggs are fertilized. If this happens, the laboratory staff will re-inseminate the eggs or perform intracytoplasmic sperm injection (ICSI) in hopes of obtaining embryos for transfer. If fertilization still does not occur, the eggs will be discarded and the remainder of the procedure will be cancelled. In the case of severe male factor, the couple may be asked to consider the option of using anonymous donor sperm (obtained through a licensed sperm bank for use as a "backup" or secondary sperm source) if it is not possible to obtain sufficient sperm from the partner at the time of fertilization.

The eggs that have fertilized will be allowed to develop for two or more additional days under controlled laboratory conditions before they are placed inside the woman's uterus. Depending upon the couple's wishes, some fertilized eggs/ embryos may be frozen and stored for future use.

After the embryos are transferred to the womb, the woman will continue progesterone supplementation that begins on the evening of your egg retrieval procedure. Progesterone can be taken as a combination of oral troches and rectal/vaginal suppositories or by injections. Administration of these medications after egg collection has been shown to create a more favorable uterine environment for the embryos, which therefore increases pregnancy rates. Side effects of progesterone may include, but are not limited to the following:

1.- Vaginal dryness;
2.- Bloating, breast tenderness;
3.- Depression, mood swings;
4.- Delay of menses.

Synthetic progesterone-like medications have been associated with certain birth defects. By using only natural progesterone, the risk of drug-induced birth defects is significantly reduced. It is important to note, however, that birth defects occur in approximately 3% of spontaneously-conceived pregnancies in the USA. Therefore, use of natural progesterone does not guarantee a child without a birth defect.

Transferring Embryos to the Uterus

Embryos are transferred on either day three or day five of development. The embryologists at SEMERT are highly-skilled in identifying "healthy" embryos and in some cases will recommend that a patient extend embryo development to day five, known as the blastocyst stage. Blastocyst transfer has become quite common in IVF cycles as it can increase chances for success while decreasing the likelihood of multiples. Your physician will work closely with the embryologists to determine if a day three or day five transfer would be ideal for your cycle.

Embryos are transferred to the uterus through a small tube (catheter). This procedure is much like a pap smear and does not require any anesthesia and is usually painless. The embryos are placed in a small amount of fluid inside the catheter, which is passed through the cervix at the time of a speculum examination. The embryos are placed in a manner so they reach the top part of the uterus. The number of embryos transferred depends on individual circumstances of the couple, and this decision will be made collectively by you, your physicians and the embryologist. Typically, two to four embryos are be transferred in one treatment cycle.

Embryo transfer can cause mild cramping. Although unlikely, during the embryo transfer the embryo(s) may be displaced through the cervix (causing loss of embryos) or into the fallopian tubes (causing possible tubal pregnancy). There is a small risk of bleeding or infection as a result of the transfer procedure.

After transfer, the woman may get dressed and leave after a brief recovery period. A pregnancy test will be done twelve to fourteen days after the transfer, regardless of the occurrence of any uterine bleeding.

The transfer of several embryos increases the probability of success. A multiple embryo transfer also increases the risk of a multiple pregnancy.Any multiple pregnancies carries an increased risk of miscarriage(s), premature labor and premature birth as well as an increased financial and emotional cost. Pregnancy-induced high blood pressure and diabetes are more common in women pregnant with more than one fetus. Prolonged hospitalization may be necessary for these pregnant women and for the mother and babies after delivery. Tubal (ectopic) pregnancy is also possible, and a combination of normal pregnancy and ectopic pregnancy may occur. A tubal pregnancy is a condition that may require laparoscopy or major surgery for treatment. Like spontaneous (natural) conceptions, pregnancies that arise through IVF may result in miscarriage. In the event of a miscarriage, a dilatation and curettage (D&C) may be necessary.

Couples going through therapy must choose and formalize their choice in the appropriate SEMERT consent form by indicating one of the following options for handling of any remaining embryos:

1.- Freezing (cryopreservation) of remaining embryos for use by the couple in future treatment cycles
2.- Anonymously donating the embryos for use by another infertile couple(s), if the donating couple and the donated embryos meet the screening criteria (You will not receive any money for this donation, nor will SEMERT "sell" them. SEMERT reserves the right to cryopreserve (freeze) any donated embryos as well as the right to discard any donated embryos if a suitable woman cannot be identified to receive the embryos)
3.- Allowing the embryos to develop in the laboratory until they perish, at which time they would be disposed of in a manner consistent with professional ethical standards and applicable legal requirements (This usually occurs within six to eight days after egg collection).
4.- Alternatives to IVF-ET:
Depending upon the individual and unique cause(s) of infertility for each couple, the chance of conception through alternative means, including intrauterine insemination (IUI) and medicinal therapy, other than IVF-ET may or may not exist. Possible success rates of these alternatives may vary depending upon the type and severity of the cause of the infertility. For some couples, it may even be possible to conceive spontaneously without a physician's help. You should discuss these alternative treatment methods with your physician before you proceed with IVF-ET therapy.

Micromanipulation

INTRODUCTION

Since the birth of the first baby achieved through conception outside of the human body in 1978, the principles of "in vitro" (literally "in glass") fertilization and culture have remained the same - careful establishment and maintenance of a well-controlled, sterile environment in which the normal physiology of fertilization and early development can be played out relatively undisturbed to provide healthy embryos for transfer back into the body. During the ensuing two decades, much has been learned, however, about the tolerances of such a system and how this technique can be exploited to treat a widening range of infertility cases. There have been great strides made in development of more appropriate culture media that has enabled embryos to be grown for extended periods of time in culture. Surplus embryos and possibly eggs may now routinely be cryopreserved in liquid nitrogen for use in subsequent attempts at pregnancy. Fertilization itself is no longer a hit-and-miss affair with the advent of assisted fertilization through micromanipulation. Embryos can be micro-manipulated for cell biopsy to determine their genetic status as well as aid in their ability to implant through drilling into their outer shell (assisted hatching).

CONVENTIONAL IN VITRO FERTILIZATION (IVF)

Through the controlled application of ovarian hyperstimulation, it is current practice to time the retrieval of mature oocytes (eggs) from a woman's ovary. The yield may vary anywhere from one to 30 or more eggs that may be retrieved depending on the responsiveness of the ovaries to the gonadotropins used to stimulate them. These eggs are gathered by the embryologist into an appropriately balanced salt solution and maintained at body temperature (37°C) until such time as they are ready to be inseminated. Meanwhile, a sample of semen containing the sperm destined to be used for each specific set of eggs is collected and processed by cell separation techniques to provide as clean and active a sample of sperm as possible. A major emphasis of the IVF laboratory is directed toward guaranteeing that the correct sperm go with the right eggs through good labeling and check systems. Ultimately, following several hours in culture, eggs and sperm can be mixed and allowed to bind and fertilize in a relatively natural fashion. Depending on the quality and maturity of both eggs and sperm, it is common for fertilization rates to vary considerably relative to the original number of eggs collected. Twenty eggs retrieved in no way guarantees 20 embryos. Likewise, 20 fertilized eggs in no way guarantees that there will be 20 embryos of sufficient quality for both cryopreservation and fresh transfer to the woman's body.

Central to the question of how many embryos are actually utilized in any IVF treatment cycle is the period during which the embryos are cultured in vitro. This can be as little as one day, or up to seven in the case of blastocyst growth and transfer. Assuming that culture conditions are relatively optimal, there is less and less reason not to culture embryos throughout their pre-implantation stages to allow the embryos to "select" themselves for transfer or cryopreservation. The blastocyst is the term given to the very last stage of an embryo prior to it implanting into the endometrial lining of the uterus. The poorer the rates of blastocyst growth are, the more restricted the choice of embryo is at this stage of development. In any event, growth of any embryos to the blastocyst stage improves the level of discrimination of embryo viability available to the embryologist, and is a key to reducing the numbers of embryos used for uterine transfer. The more confidence a clinic has in the viability of the embryos it transfers, the less need there is for multiple transfers of three or more embryos. Thus with the transfer of three or less embryos, the risk of multiple pregnancies is significantly reduced, in turn minimizing risks of pregnancy loss or fetal abnormalities common in multi-fetal pregnancies.

Micromanipulation Techniques

MICROMANIPULATION IN IVF THERAPY

Micromanipulation is the technique whereby sperm, eggs and embryos can be handled on an inverted microscope stage, performing minute procedures at the microscopic level via joysticks that hydraulically operate glass micro tools.

MALE FACTOR INFERTILITY

Micromanipulation first saw clinical use in IVF for purposes of assisted fertilization in the treatment of male factor infertility, where fertilization potential was low in cases of poor sperm quality. The ultimate evolution of this approach has been the development of the single sperm injection procedure referred to as Intracytoplasmic Sperm Injection, or ICSI. Sperm of virtually any quality and from any level of the male reproductive tract may be used with the only criterion for use being that the sperm is alive even if it is not moving (motile). Dead sperm may be able to achieve fertilization; however, the DNA or genetic material from such sperm is too degenerate to form a viable embryo. Immature sperm from the testicle or the epididymis can be retrieved for use with ICSI for men who possess no sperm in their ejaculated semen (azoospermia). This azoospermia is either due to an obstruction in the tract (obstructive), or to extremely low production of sperm in the testicle itself (non-obstructive).

INTRACYTOPLASMIC SPERM INJECTION (ICSI)

With the almost unlimited potential to achieve some level of fertilization with ICSI regardless of sperm quality, it would seem that male factor infertility would no longer be of concern. It must be noted, however, that sub-fertility in men can be related to certain numerical and structural defects of the chromosomes and, therefore, there is a strong recommendation for all couples that achieve pregnancies from ICSI to undergo prenatal screening. In certain cases of obstructive azoospermia, there is a higher incidence of cystic fibrosis in the male. Hence, before embarking upon treatment of the more extreme forms of male factor infertility, it is advisable to have some cytogenetic screening performed. Incidentally, very subtle compromise in sperm quality may well be responsible for a marginally lower embryonic viability rate and a slightly higher early miscarriage rate even if such embryos implant. Such observations have led to the suggestion that the technique ICSI itself is at fault; but this misses the point that ICSI per se is not causing the problem, merely facilitating the use of sperm, which under other circumstances would never have even achieved fertilization.

ICSI FOR NON-MALE FACTOR INFERTILITY

The use of ICSI is now routinely applied to a range of clinical situations wherever there is a possibility that conventional in vitro fertilization may be suppressed or not occur. Such situations include the following: idiopathic or unexplained fertility; hyper-responsive ovarian stimulation cases where egg quality may be reduced; post-thaw sperm samples that survive poorly; post-thaw egg insemination; generation of embryos for pre-implantation genetic screening where embryos "clean" from any extraneous contaminating sperm is needed; or, indeed, any case where there is an extreme need to maximize normal fertilization, for example, when a woman has only a few eggs retrieved. It is possible to "rescue" cases following complete failed conventional fertilization with ICSI. The viability potential of these "late-fertilized" embryos is approximately half of timely fertilized embryos; nevertheless, they do generate successful live births. ICSI has become such a common feature of IVF therapy that it is fast becoming the insemination technique of choice.

ASSISTED HATCHING

It has been proposed that a certain number of otherwise viable embryos do not implant simply because they are unable to break free from the surrounding "jelly coat" (zona pellucida) when they reach the blastocyst stage of development. Around an unfertilized egg there exists a transparent glyco-protein coat that acts to protect the egg and regulate normal fertilization by any penetrating sperm. This jelly-like coat continues to protect the early preimplantation embryo until, as a blastocyst, the embryo fills itself up with fluid like a water-filled balloon, pumping itself larger and larger until it ruptures and "hatches" from the zona pellucida. The embryo is now ready to make contact in its naked form with the endometrium and implant. Inappropriate ovarian environment due to advanced maternal age or other factors that may compromise the follicular environment may in certain cases render the zona pellucida thicker or tougher. Such IVF cases may benefit from the application of a form of micromanipulation referred to as "assisted hatching" In this process, the embryo has a hole made in the surrounding zona pellucida prior to transfer to enable it to "hatch" free from the zona pellucida more easily when it expands as a blastocyst in the uterus. This technique has never been unconditionally proven to be effective in any well-defined group of IVF patients, and as such remains essentially an experimental procedure. Holes in the zona pellucida may be made mechanically, chemically, or by laser. With the advent of more routine transfer of blastocyst stage embryos, the future of this technique, usually carried out on day three of development, may seem in question. Indeed, at the blastocyst stage in vitro, it may be most appropriate to dissolve off the entire zona pellucida prior to transferring naked embryos into the uterus. This could be considered the ultimate form of assisted hatching without the need for micromanipulation. Currently, however, assisted hatching can be easily performed using an infrared laser to create a hole in the zona pellucida that allows the embryo an easy means of escape when it is time to try and implant into the uterine wall.

EMBRYO BIOPSY

Briefly, it is of relevance in any discussion of micromanipulation techniques to mention the potential to biopsy both eggs and embryos. This approach is known as preimplantation genetic diagnosis (PGD) and enables the screening of both the unfertilized egg by removal of the first polar body, or the fertilized multi-cellular embryo by removal of one or more cells either at the 6-12 cell stage or from the trophectoderm of the blastocyst. This material can be probed for either genetic mutations or gross chromosomal errors. This technology remains in its infancy and can be of profound importance clinically, but at this time only for cases with very clear medically-defined needs. The biopsy procedure requires very exacting skills of the IVF laboratory, and the egg or embryo is not entirely free of risk during the procedure. Hence, couples whose offspring have a high chance of inheriting a genetic disorder may have their embryos screened. Women who are at risk of generating eggs with a high risk of chromosomal anomalies can benefit from having their eggs or embryos screened for chromosomal normality. While embryos can have their sex determined through this procedure, the SEMERT team considers it inappropriate to do so except in cases of sex chromosome-linked disorders.

CONCLUSION

It is a privilege for any scientist or clinician to have access to the earliest stages of human development through culturing gametes and embryos in vitro. And, as such, it requires a high degree of ethical responsibility to provide as safe and optimal an environment as possible for these microscopic changes. While much has been done to maximize IVF pregnancy rates over the last two decades, it nevertheless remains to improve individual embryo selection to the point where we can routinely transfer only one embryo at a time, while being able to successfully and consistently freeze all surplus embryos of sufficient quality for later use in attempting pregnancy.

Human Embryo Cryopreservation

CURRENT STATUS

The first pregnancy from a frozen/thawed human embryo was reported in 1983, and a birth from this source occurred the following year. Of 99,629 cases of Assisted Reproductive Technology (ART)* in the United States in 2000, about 16% of cases (16,194) used frozen/thawed embryos. In 2000, live birth rates per thaw cycle were 18.3% versus 26.6% from fresh embryo transfer. At SEMERT, the ongoing pregnancy rate for IVF using frozen/thawed embryos is currently 52%.

CRYOPRESERVATION OF HUMAN EMBRYOS

Egg retrieval under ultrasound guidance and subsequent fertilization and embryo culture are carried out according to our current procedures. If there happens to be a surplus of embryos following selection for fresh transfer (usually between one to four embryos are transferred to the uterus), then embryos of sufficient quality may be considered for cryostorage. While embryos can be frozen at any preimplantation stage between one-cell (one day old) to the blastocyst stage (5-6 days old), in an attempt to minimize the freezing of excessive numbers of "spare" embryos and to help pre-select the most potentially viable embryos, we generally choose to cryopreserve only at the blastocyst stage. In certain cases where all embryos need to be frozen without a fresh transfer (e.g., when a woman may be at risk from ovarian hyperstimulation that might be complicated by pregnancy), we generally freeze all embryos the day after egg collection at the one-cell stage.

Techniques of controlled-rate freezing are utilized that slowly cool embryos in cryoprotectant fluid ("anti-freeze" solution) from body temperature down to -196°C, at which temperature they are stored in containers of liquid nitrogen called dewars. The embryos are actually contained within special indelibly labeled plastic vials, or straws, that are sealed prior to freezing. Once frozen, they are placed inside labeled tubes attached to aluminum canes and stored in numbered canisters within the liquid nitrogen dewar. Site and label designations are stored in three separate file systems to avoid confusion and misidentification of cryopreserved embryos. When it comes time to thaw the embryos, all available identifiers of the stored specimen must match and be confirmed before thawing commences. The embryos are thawed out at room temperature, which takes about one to two minutes. However, the most critical element of the thaw procedure is not the timing but the careful dilution of the cryoprotectant fluid to return the embryo to its favored culture medium. This permits resumed growth and development in vitro. Once this is done, the embryo is assessed for cryodamage to determine if it is suitable for transfer. Experience has shown that if the embryo survives 50% or more intact, it is worthwhile to replace it. Embryos can accommodate such levels of cellular damage and still establish healthy pregnancies. All thawed embryos routinely undergo assisted hatching prior to transfer. The zona pellucida, which surrounds the embryo, has been shown to suffer a certain amount of hardening during cryopreservation. This can be overcome by artificially making an opening in the outer embryo shell.

Varying strategies may be applied according to how many and which embryos are thawed prior to transfer. It should be noted that not every couple undergoing IVF will need to worry about embryo freezing/thawing, since not every couple will have sufficiently large number of "surplus" or non-transferred embryos available for freezing. Indeed, most couples have only one or two embryos frozen, so that all are thawed and any surviving are replaced. In the event that there are more than two or three embryos frozen, thawing is usually undertaken until two to three healthy appearing embryos are recovered. In some cases, this may mean that all the cryopreserved embryos are thawed, in others just two or three. There always remains a possibility that there may be no embryo survival after thaw occurs, and no transfer is possible. If many early embryos are frozen, it is possible to thaw all of them and culture them for several days to allow selection of the best for transfer. When too many embryos are available for transfer in this circumstance, then extra embryos of sufficient quality may be refrozen for later use. This course of action has produced healthy offspring, proving the efficacy of double freezing of embryos.
During a medication-prepared frozen/thawed embryo transfer cycle as a patient, you will follow a treatment schedule using Synarel or Lupron, estrogen (pills, lozenge or patch) and progesterone (lozenge and/or suppository) in order to achieve appropriate endometrium (uterine wall lining) for embryo transfer. Following embryo transfer, estrogen and progesterone will be administered daily until the 7th to 8th week of pregnancy or until a negative pregnancy test.

CONSIDERATIONS AND RISKS

The Ethics Committee of the American Society of Reproductive Medicine (ASRM) has published guidelines for ethical consideration of human embryo cryopreservation. Possible advantages of cryopreservation of embryos suggested by the Committee include:

1.- Reduction of the risk of triplets or quadruplets by cryopreservation of embryos exceeding an optimal number for transfer to an individual patient
2.- Possibly increasing pregnancy rates by replacing thawed embryos during spontaneous ovulatory cycles or cycles in which the estrogen and progesterone hormone levels do not exceed that which occurs naturally
3.- Possibly decreasing the number of stimulated ovary drug treatment cycles needed for the attainment of pregnancy

The primary concern with the use of cryopreservation techniques is the possible loss of embryos to cryoinjury, meaning some healthy embryos may not survive the stress of freezing & thawing. The exact number of embryos lost to cryoinjury varies, but it is very likely that freezing will cause loss of some embryos, perhaps as many as 25-50% of those cryostored. One interpretation of this is that cryopreservation may even act as a "selection gate" for the more viable embryos, though this has never been proven.

Another concern with cryopreservation is the potential risk of birth defects in children produced from frozen/thawed embryos. In the domestic animal industry, large-scale freezing and transfer of embryos has not resulted in increased birth defects. Studies to date on those human offspring arising from thawed embryos have not shown any significant increase in abnormalities when compared to pregnancy outcomes in the rest of the population.
To optimize the likelihood of successful embryo cryopreservation at SEMERT, the mechanical processes of human embryo cryopreservation will be strictly controlled to minimize the chances of technical failure. A back-up freezing system is always available to decrease the risk of interruption in the freezing process, as well as generator back-up power in the event of a power outage. Individual embryos are placed in permanently labeled storage containers and identified according to origin, developmental stage, and date frozen. Permanent records are kept at Servicios Medicos Reproductivos de Tijuana for each individual's embryos. Liquid nitrogen dewars are connected to alarm systems to monitor the liquid nitrogen levels and prevent premature thawing. However, even with all these safeguards, the possibility of technical failure leading to loss of stored embryos following natural disaster cannot be totally and completely eliminated.

PERIOD OF CRYOSTORAGE

The disposition of any frozen embryos that are not transferred must be arranged in writing before cryopreservation (see Cryopreservation Consent Form). In the event that a successful pregnancy is established following a fresh or subsequent embryo thaw cycle, it will be at the discretion of the couple as to whether the remaining frozen embryos should continue to be cryostored, or appropriately discarded or donated. This may include the option to donate the embryos for research or to other infertile couples for transfer. Cryopreservation records, along with all records of medical care and procedures, will be maintained in the strictest confidence at Servicios Medicos Reproductivos de Tijuana.