Konstantinos G. Michalakis, MD, PhD
Alan H. DeCherney, MD
Alan S. Penzias, MD
In vitro fertilization (IVF) is a process by which egg cells are fertilized in vitro, that is, by sperm outside of the womb. IVF is a major treatment in infertility when other methods of assisted reproductive technology have failed. Assisted reproductive technologies (ART) include multiple techniques that allow gamete manipulation outside the body and have evolved greatly over the past 2 decades.
IN VITRO FERTILIZATION
IVF involves egg retrieval from the ovary, fertilization in the laboratory (fluid medium), and replacement of the zygote in the patient’s uterus. The first live birth resulting from this technique occurred in June 1978. Since then, over 1 million children have been born throughout the world with the use of assisted reproduction.
Assisted reproductive techniques have been used for more than 20 years, reporting an increasing number of cycles treated, an increasing pregnancy rate, and an increase in live births per cycle (from 6.6% in 1985 to 27% in 2006) for IVF. In 2003 there were 122,872 ART cycles (99.4% were IVF cycles), whereas in 2006, 41,343 live-birth deliveries were reported; approximately <1.0% were gamete intrafallopian transfer for fertilization (GIFT) and <1.0 % accounted for zygote intrafallopian transfer (ZIFT) cycles. In approximately half of the ART cycles (53%), intracytoplasmic sperm injection (ICSI) is used.
One of the most important prognostic predictors for pregnancy is the age of the female partner. Whereas for women younger than 35 years, live birth rate/cycle varies from 30 to 35%, women older than 40 years face live birth rates <6%, down to 2.4%. Table 57–1 presents data according to the National Summary and fertility reports of the US Department of Health and Human Services.
Table 57–1. In vitro fertilization.
Approximately 39% of patients who undergo egg retrieval will become pregnant with sonographic documentation of an intrauterine pregnancy (clinical pregnancy); 82% of these patients will carry to term. Many “biochemical pregnancies” occur, but these should not be included in pregnancy statistics. A biochemical pregnancy is one in which serum levels of human chorionic gonadotropin (hCG) rise and then fall before sonographic detection of pregnancy is possible. Eggs are almost always obtained by aspiration, and under ordinary circumstances, approximately 75% of eggs will fertilize and cleave. The clinical pregnancy rate of approximately 34% per embryo transfer per IVF cycle (women <35 years old) is > 20–25% pregnancy rate per cycle observed in spontaneous conceptions in the general population.
The success rate with ART has been augmented by replacing more than 1 embryo, but doing so results in one of the major complications of ART treatment: the development of multiple gestations. In 2002, the European Society of Human Reproduction and Embryology (ESHRE) reported a multiple gestation incidence between 26.3 and 29.1%, whereas in the United States, among pregnancies from fresh donor cycles, 57.3% were singletons, 37.1% were twins, and 5.6% were triplets or more. Although multiple gestations are often welcomed by the infertility couples, they are riskier pregnancies that may result in preterm births.
Indications
The basic concept of IVF–embryo transfer (IVF-ET) initially was to bypass the potential mechanical obstacles of the female reproductive tract. It was first developed for patients with severe tubal disease, for patients with bilateral salpingectomy, or for women whose tubes are so badly damaged that they cannot function. As expertise increased, the variations of IVF and ICSI applied to a wider spectrum of other infertility problems. Indications for ART now include the following:
1. Male factor infertility
2. Tubal disease (tubal and pelvic adhesions)
3. Absent or damaged fallopian tubes
4. Endometriosis
5. Preimplantation genetic diagnosis (PGD)
6. Need for third-party reproduction/donor eggs or gestational surrogate
7. Unexplained infertility
8. Age-related infertility
9. Decreased ovarian reserve
10. Recurrent intrauterine insemination failure
When the probability of conception by ART exceeds that of conception by conventional therapy, ART appears to be the procedure of choice. Because of an increased incidence of infertility in our modern society, the timing of reproduction tends to move to the right of the female reproductive curve, as career-women work earlier and conceive later. Thus there is an increased awareness and availability of ART, and the application of such alternatives has expanded.
Although IVF is successful in treating many infertility problems, its success hinges on entry of sperm into the egg. It was initially hoped that routine IVF could be used to compensate for severe oligospermia (<5 million sperm/mL). However, early results were often poor. Modern microsurgical techniques with ICSI are now used in several cases, attempting placement of sperm directly into the cytoplasm of the oocyte. This is discussed in detail later. In addition to male factor issues, another barrier to success with IVF is hydrosalpinx (fluid collection in the fallopian tube). This condition may interfere with implantation, and additional surgery may be needed so that implantation and pregnancy rates improve.
Technique
IVF consists of the following steps:
1. Ovarian stimulation
2. Oocyte retrieval
3. Fertilization with capacitated sperm and ICSI
4. Embryo culture
5. Embryo transfer
A. Ovarian Stimulation—Superovulation
Multiple eggs increase the possibilities of producing multiple embryos, which adds to the likelihood of successful conception. Apart from that, multiple eggs are desired because some eggs will not develop or fertilize after retrieval. By using fertility medication, the ovaries are stimulated to produce several high-quality eggs, and timing for aspiration is better controlled.
Almost all ART programs use superovulation. The type of ovulation-induction therapy varies from group to group. The following methods are used alone or in combination:
1. Combination of gonadotropins and gonadotropin-releasing hormone (GnRH) analogues
2. Combination of gonadotropins and GnRH antagonist
3. Follicle-stimulating hormone (FSH) products—urinary or recombinant
4. Human menopausal gonadotropins—urinary or recombinant
5. Luteinizing hormone (LH) agonists
6. Clomiphene citrate (rarely)
In order to monitor the number and growth of follicles, as well as the uterine lining, superovulation is carefully monitored with ultrasound. To assess the function of the follicles, serial serum estradiol levels are drawn. At least 2 or 3 follicles should be developing before proceeding with egg aspiration; otherwise, the cycle is usually abandoned, and an alternative stimulation regimen is selected for a subsequent cycle. Serum estradiol levels are complementary to ultrasonography in evaluating the maturation and growth of the developing follicles (200 pg/mL per mature follicle is expected). There is evidence that the pattern of serum estradiol may predict the cycles most likely to result in pregnancy. When the mature follicles have reached at least 17 mm in diameter and the amount of estradiol reaches approximately 500 pg/mL, 10,000 IU of hCG (either urinary or recombinant) are usually administered to induce ovulation. Ovulation and subsequent retrieval usually occurs 36 hours after hCG injection.
The introduction of GnRH agonists or antagonists to superovulation regimens has drastically reduced the likelihood of a premature LH surge; consequently, they are used in the majority of IVF patients in the United States. There are many ways to add an agonist in the whole procedure: commonly the GnRH agonists are administered on day 21 or the previous cycle (long protocol) or at the beginning of menses, along with the addition of gonadotropins (short protocol), and they are continued until the day of hCG. When GnRH antagonists are used, treatment with antagonists begins after 5–6 days of gonadotropins or when the lead follicle is 13 mm, whereas recent data allow the antagonist initiation even with a 16–17 mm leading follicle. The antagonists have the advantage of requiring fewer injections; however, there may not be a difference in pregnancy rates between the agonists or the antagonists.
B. Oocyte Retrieval
Aspiration of the preovulatory follicles is performed approximately 34–36 hours after the hCG injection. Egg aspiration is performed using either of 2 methods. Laparoscopy was the first method to be used and is rarely used today. The current method uses ultrasonography to direct transvaginal aspiration (occasionally, egg collection is performed through the abdominal wall under ultrasound guidance, in cases in which the ovaries are abnormally placed). In transvaginal aspiration, a needle is passed through the posterior vaginal fornix using a vaginal ultrasound probe and directed into the ovary. Fluid from the follicles is drawn into a test tube to retrieve the eggs. The advantage of ultrasound aspiration is that it can be performed on an outpatient basis (approximately 30-minute procedure), it is simpler, less invasive, and less expensive.
C. Fertilization With Capacitated Sperm and ICSI
Freshly ejaculated sperm cannot fertilize an egg; the sperm must be capacitated. Fortunately, capacitation is a very simple process in humans and involves only a short incubation period in a culture medium, soon after the collection procedure.
Because of the nature of the superovulatory process, eggs will be in different stages of maturation. Once the eggs have been identified, the embryologist classifies them as either mature (preovulatory) or immature. Mature eggs have an expanded cumulus oophorus, have undergone the first meiotic division (and so the first polar body is visible), and are usually fertilized 5 hours after aspiration, whereas immature eggs have a very compact cumulus, have not undergone the first meiotic division. and can be incubated in the laboratory for up to 36 hours before fertilization. If sperm and eggs are mixed too early, fertilization and cleavage will not take place. Between 50,000 and 150,000 motile sperm are placed with each egg.
Male infertility has been considered a major contributory factor to infertility. In order to face and eventually bypass male factor, microassisted fertilization techniques, principally ICSI, were developed. In the context of assisted conception, they seem to have revolutionized the management of couples with so-called male factor infertility. The causes of spermatogenetic failure found in most cases of male infertility remain largely idiopathic, however, and unfortunately, there is no effective treatment to improve spermatogenesis for idiopathic male infertility patients. For male factor infertility (<5 million total normal motile sperm/mL), ICSI has resulted in higher fertilization rates and expanded possibilities for cryopreservation. In this procedure, 1 normal motile sperm is selected per oocyte and injected through the intact zona pellucida directly into the cytoplasm away from the polar body. Other indications for ICSI include surgically retrieved sperm (for men with azoospermia who need testicular or epididymal biopsy), cryopreserved oocytes, or cases in which PGD is performed for single gene disorders. For couples with borderline quality semen, ICSI results in higher fertilization rates than IVF, and couples with very poor semen will have better fertilization outcomes with ICSI than with subzonal insemination or additional IVF. In 2005, approximately 60% of all ART cycles in the United States involved intracytoplasmic sperm injection, whereas in 2006, it was used in 1 in 2 cycles.
D. Embryo Culture
Embryos are incubated in an atmosphere of ≤5% carbon dioxide and 37°C temperature, close to the temperature of the fallopian tubes. Various culture media are used and are often supplemented with either the patient’s serum or synthetic albumin as well as essential and nonessential amino acids and sugars. At various intervals after the attempted fertilization, the embryos are examined in order to identify pronuclei, which confirm fertilization (genetic material from both partners), as well as the stage of cleavage.
After pronuclei identification, the embryos will develop for another 24 hours. At this point, embryos are usually monitored for cell division and should have evolved to 2- or 4-cell embryos.
Embryos can be cultured for various days, mostly relevant to the reproductive obstacles the parents were facing. Embryos can be cultured for:
• 2 Days—This type of culture is used for couples who have a low number of embryos available for transfer or who have embryos that are slowly developing. Those embryos are transferred at the 2- or 4-cell stage.
• 3 Days—Embryos cultured for 3 days are checked for gene activations and cleavage, thus increasing the potential of transferring a viable embryo. These embryos are usually transferred at the 6- to 8-cell stage.
• 5 Days—These embryos reach the blastocyst stage. Blastocysts consist of 12 to 16 cells and are ready for implantation into the uterus.
E. Embryo Transfer
After 3–5 days of laboratory culture, the embryos are replaced into the patient’s uterus, a procedure termed embryo transfer. Before transfer, the embryos are graded from A to D depending on their appearance and on the degree of fragmentation. Embryos that are not transferred at this time can be cryopreserved and stored in liquid nitrogen use in later IVF cycles, if necessary. If day 5 or 6 transfers are performed, the embryos are at the blastocyst stage, as mentioned earlier. There are 2 types of embryo transfers:
• Day 3 embryo transfer, which is performed 72 hours after egg retrieval
• Blastocyst transfer, which is the transfer of blastocysts and, as mentioned, raises the possibilities of transferring a healthy embryo
The decision of how many embryos to transfer is made by the patient in conjunction with the physician and the embryologist in accord with the American Society for Reproductive Medicine (ASRM) recommendations based on the patient’s age (Table 57–2). The exact number of embryos transferred depends on the number of embryos produced, the health of the embryos, the risk level for multiple pregnancy, and the woman’s age.
Table 57–2. Recommended number of embryos to transfer.
Most embryo transfers are performed under direct visualization with 2-dimensional (2-D) or 3-dimensional (3-D) ultrasounds. Before the embryo transfer is performed, the patient is usually asked to drink water to fill the bladder. A full bladder helps straighten the uterus as well as improve visualization by ultrasound during the transfer. The embryologist prepares the best embryos by aspirating them into a small catheter with some media, and after the physician cleans the cervix with culture media and aspirates the extra cervical mucous, the catheter is passed transcervically into the uterus, and the embryos are injected into the uterine cavity under direct visualization, usually in the space at the top of the uterus. The probability of pregnancy after embryo transfer can be affected by the patient’s age, the cause of infertility, the endometrial thickness, and the average embryo grade.
In some patients, assisted hatching or an opening in the zona is performed in order to improve implantation. This is thought to be beneficial in older patients (age 38 years and older) who have harder zonae; however, it is not routinely performed in all IVF centers.
Retrospectively, the decision to establish such recommendations indeed helped the number of multiple births decrease substantially, although the absolute number did not ultimately increase because of the increase in total IVF births.
F. Luteal Phase Support
In order to avoid a short period of luteal phase, after embryo transfer is performed, progesterone supplementation is usually recommended by most physicians until approximately 7 weeks’ gestation. Progesterone administration tends to correct the ratio of estradiol to progesterone and as a result provide a secretory endometrium, which is needed for the implantation. Progesterone is usually administered by an intramuscular injection or by a vaginal suppository or gel.
Complications
Few risks are associated with ART. The risks of ART can be considered in 5 major areas:
A. Risks Associated with Drugs Used to Stimulate Egg Production
1. Ovarian hyperstimulation syndrome—This syndrome is characterized by ovarian enlargement, ascites, and hemoconcentration, whereas the clinical manifestations are abdominal distention, abdominal discomfort, and nausea. Its incidence reaches 5%. Risk factors include polycystic ovary syndrome, multiple follicles, and high estradiol levels. The prognosis is usually worse in patients who get pregnant and have this syndrome. Patients with this syndrome may be at risk for blood clots. In 0.5–1.0% of all IVF cycles, admission is required, with fluid drainage and replacement of albumin. This situation resolves in 1–2 weeks.
2. Cancer—Two studies suggested that the use of the drug clomiphene increases the risk of ovarian cancer, although this has not been reported in other studies. Uterine, cervical, or breast cancer incidence does not increase with IVF.
B. Surgical Risks Associated with IVF
• General anesthetic and intravenous sedation: similar risk to any other surgery.
• Damage to other structures: 1 in 2500 retrievals.
• Pelvic infection: This could occur as a result of the needle insertion and manipulations and requires antibiotic treatment and, rarely, abscess drainage.
C. Risks Associated with Pregnancy
1. Multiple gestations—The likelihood of a twin pregnancy is 10% (0.5% for triplets) with the use of clomiphene, 20–30% after IVF with 2 embryos (increased incidence of triplets in 3 embryo replacement), and 10–20% after intrauterine implantation treatment (1–2%). The complications of multiple pregnancy are increased risk of miscarriage, increased risk of premature labor, increased risk for hemorrhage and high blood pressure, increased requirement for caesarian section, increased loss of an infant, and increased risk of an abnormal infant with a physical or learning disability. Transferring more embryos does not necessarily lead to a greater IVF success rate.
2. Ectopic and heterotopic pregnancies—Patients who undergo an ART procedure are at twice the risk for having an ectopic pregnancy as the general population (1–3% of all pregnancies from embryo transfer). Heterotopic pregnancies, which are rare but seen more commonly with ART, involve cases in which there is an intrauterine pregnancy and an ectopic pregnancy (usually in the fallopian tube) in the same patient.
3. Miscarriage—No difference has been reported in relation to naturally conceived pregnancies.
4. Preterm birth and low-birth-weight infants—These are higher in patients undergoing IVF.
D. Risk of an Abnormal Baby
The risk of congenital abnormalities may be slightly higher in patients who use ART; however, this concept is still controversial (2.6% risk of an abnormal baby with IVF, 2.0% with natural conception). In patients who use ICSI, the risk of imprinting disorders, such as Angelman’s syndrome and Beckwith-Wiedemann syndrome, may be increased.
Intellectual impairment seems to occur more often in offsprings of fathers who had to go through ICSI or surgical extraction of sperm.
Babies born after replacement of thawed embryos do not show any increased incidence of abnormalities.
E. Cost
Currently only a few states allow health insurance to cover infertility treatment, which leaves many couples with tremendous expenses (the estimated cost per delivery is $66,667).
OTHER TECHNIQUES RELATED TO IVF-ET
Ovum Donation
Embryos have been donated from one woman to another with many resultant live births. Women who receive donated embryos include those with ovarian failure (premature, autoimmune) or absence (eg, gonadal dysgenesis), diminished ovarian reserve, or genetically transmitted disorders.
Ovum donation can occur under either of 2 circumstances. One circumstance is the infertile patient who produces a large number of oocytes during her own IVF or GIFT cycle and elects to donate some of them to another woman who is otherwise incapable of producing eggs. The other, more common circumstance involves the recruitment of a woman who undergoes superovulation and oocyte retrieval purely for the purpose of donating her oocytes. The donor may be known to the patient (a family member or friend) or, more commonly, may be anonymous. Although the genetics of the resulting pregnancy are derived from the husband and the donor, the infertile woman incapable of producing her own eggs goes through the pregnancy. In these cases, the endometrium of the recipient must be primed with estrogen and progesterone before transfer of the donated embryos, and progesterone and estrogen supplementation must be maintained for at least 10 weeks. The number of embryos transferred is decided based on the age of the donor, not the age of the recipient.
Gestational Surrogacy
A surrogate mother is a woman who is pregnant with a child but who does not intend to raise it after birth. The intended parent(s) is an individual or couple who intends to raise the child after its birth. In traditional surrogacy the surrogate is pregnant with her own biological child, but this child was conceived with the intention of relinquishing the child to be raised by others.
In gestational surrogacy the surrogate becomes pregnant via embryo transfer with a child of which she is not the biological mother. In altruistic surrogacy, the surrogate receives no financial reward for her pregnancy, whereas in commercial surrogacy, the gestational carrier is paid to carry out the pregnancy by the infertile couple. This procedure is legal in several countries.
Gamete Intrafallopian Tube Transfer (GIFT)
GIFT is an alternative to IVF but is used infrequently, typically for women with unexplained infertility or with normal tubal function plus endometriosis. Live birth rates per cycle are approximately 25–35% at most infertility centers. However, with the improved pregnancy rates in IVF, GIFT procedures are rarely done now. Usual indications for GIFT nowadays include patients who have moral or religious objections to IVF and want to have fertilization in vivo rather than in vitro. As with IVF, superovulation is induced, and the follicles are aspirated vaginally under ultrasound guidance. The eggs are then identified in the laboratory. Thereafter, sperm is collected and capacitated, and laparoscopy is performed. Sperm are then mixed with the eggs and drawn up into a catheter. The sperm and eggs can also be separated by an air bubble in the catheter, after which they are transferred into one of the fallopian tubes, permitting in vivo fertilization and cleavage.
Obviously, GIFT is useful only in patients who have normal tube function and are not of advanced age. It has been argued that the requirement of normal tubal function renders the direct comparison of IVF-ET and GIFT results impossible. Among proponents of each technique, there is vigorous ongoing debate regarding the advantages of GIFT over IVF-ET.
In unexplained infertility, IVF-ET will differentiate the etiology of fertilization problems between egg and sperm; GIFT will not. Additionally, GIFT exposes patients to the risks of general anesthesia and laparoscopy. GIFT is now rarely used.
Zygote Intrafallopian Transfer (ZIFT)
Zygote intrafallopian transfer (ZIFT) is used to treat infertility that is caused by a blockage in the fallopian tubes that prevents the normal binding of sperm to the egg. ZIFT is a procedure that combines IVF and GIFT. Ovulation is induced and the oocytes are removed and fertilized in vitro. Soon thereafter, the zygotes are placed into the fallopian tubes by laparoscopy, similar to GIFT, and the embryo travels to the uterine cavity. ZIFT has a success rate of 64.8% in all cases, but is now rarely used.
Preimplantation Genetic Diagnosis (PGD)
PGD is a technology that has been around since early 1990. It allows many genetically heritable diseases to be identified using a variety of molecular biologic techniques. These techniques include but are not limited to polymerase chain reaction and fluorescent in situ hybridization. Currently, there are mainly 2 groups of patients for which PGD is indicated.
1. Couples with a high risk of transmitting an inherited condition that is either a monogenic disorder (autosomal recessive, autosomal dominant, or X-linked disorders) or a chromosomal structural abnormality/translocation.
2. Couples whose embryos are screened for chromosome aneuploidies in the context of IVF procedures. The technique mostly used for screening is actually referred to as preimplantation genetic screening (PGS) and is used to increase the chances of an ongoing pregnancy. The main reasons for this procedure are advanced maternal age or history of recurrent miscarriages. Patients with nonobstructive azoospermia are also candidates for PGD.
Recent advances in embryo manipulation have made possible the removal of 1 or 2 cells, or blastomeres, from a developing 8-cell human embryo without harm to the embryo. Biopsy of the first and/or second polar bodies can also be done for several single-gene defects. In patients at risk of passing along a heritable genetic disease, PGD has made possible the identification of normal embryos (those with no risk of passing the heritable disease). PGD is available for a large number of monogenic disorders; the most frequently diagnosed autosomal recessive disorders are β-thalassemia, sickle cell disease, cystic fibrosis, and spinal muscular atrophy type 1. These normal embryos are then transferred back to the patient. More than 1000 live births have been reported after application of these techniques. PGD is also performed for patients with recurrent miscarriages, previous failed IVF cycles, aneuploidy diagnosis for patients with advanced maternal age, and for sex selection, but these indications are still controversial.
Cryopreservation
Cryopreservation is a process by which cells or whole tissues are preserved by cooling to low subzero temperatures, such as (typically) 77 K or −196° C (the boiling point of liquid nitrogen). At these low temperatures, all biologic activities, including cell death, are stopped.
As expected, the combination of cryopreservation and IVF means that embryos or eggs are frozen to be used at a later time after thawing (unfreezing). Cryopreservation of embryos is very successful and has greatly improved since the first case in 1983. Survival rates of frozen embryos have been reported to be between 50 and 90%. Before implanting thawed embryos, the patient’s cycle is usually synchronized so that embryo transfer occurs during the implantation window of the uterus. Consequently, pretreatment with estrogen and progesterone is recommended. In 2003, the live birth rate per transfer of frozen embryos was 27%.
Cryopreservation of oocytes has been gaining attention and has improved over the past few years. In the fall of 2004, the American Society for Reproductive Medicine (ASRM) issued an opinion on oocyte cryopreservation concluding that the science was “promising” because recent laboratory modifications have resulted in improved oocyte survival, fertilization, and pregnancy rates from frozen-thawed oocytes in IVF. The ASRM noted that from the limited research performed to date, there does not appear to be an increase in chromosomal abnormalities, birth defects, or developmental deficits in the children born from cryopreserved oocyes. Pending further research, oocyte cryopreservation should be introduced into clinical practice only on an investigational basis and under the guidance of an institutional review board.
Cryopreservation of ovarian tissue is of interest to women who want to preserve their reproductive function beyond the natural limit or whose reproductive potential is threatened by cancer therapy. Research on this issue is promising; autologous transplantation is the process by which the ovary is removed and transferred to a different location, such as the forearm or abdomen.
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