Assisted Reproductive Technology

Marwan M. Shaykh, M.D.
Marwan Shaykh, M.D. is a Reproductive Endocrinologist with
North Florida Assisted Fertility Program in Jacksonville.

Definition

Assisted reproductive technologies, also called ART procedures, are defined primarily by the first procedure that was developed, which was In Vitro Fertilization and Embryo Transfer (Figure 1). IVF, as it is called, is the removal of the human oocyte (egg) from the ovary, fertilization by the husband's partner or donor sperm in the laboratory, and then transfer of the fertilized egg (embryo) back into the uterus. GIFT (gamete intrafallopian transfer) and ZIFT (zygote intrafallopian transfer) are derivatives of IVF.

GIFT (Figure 2) is again the removal of the oocyte from the ovary, but instead of fertilization outside the body (in vitro), the egg and sperm are mixed together, placed into the fallopian tube, where fertilization and embryo development take place (this is where the sperm and egg normally meet). In ZIFT, the egg is removed and fertilized in the laboratory. The fertilized egg at the pronuclear stage (prior to the first cell division - Figure 3) is then placed into the fallopian tube hoping that the tubal environment, just as in GIFT, will aid in embryo development, growth and pregnancy.

Figure 1. In Vitro Fertilization (IVP) with transcervical embryo transfer (ET) in cases of fallopian tube abnormalities.	Figure 2. In Gamete Intra-Fallopian Transfer (GIFT) retrieved oocytes (eggs) and sperm are placed directly into the fallopian tube for fertilization in vivo.

History

Louise Brown, born in 1978, was the first reported live birth conceived by in vitro fertilization.¹ Steptoe and Edwards achieved this success by suctioning a single egg using refined laparoscopic equipment. Today we use the fertility hormone (FSH) to stimulate the development of several eggs. Those eggs are easily retrieved transvaginally under ultrasound guidance (Figure 1) without having to resort to surgery (laparoscopy). The first baby conceived by in vitro fertilization in the United States was in 1981, and in Jacksonville in 1986. Today over 100,000 babies in the U.S. have been conceived through ART.

Indication

Although the original indication for in vitro fertilization was tubal disease, this indication has now been expanded to include male factor, endometriosis, ovulatory dysfunction, unexplained infertility and uterine factors (Figure 4).

Success Rates Of ART

Early success rates using in vitro fertilization were 10%-15%, however improved techniques have more than doubled that rate (Table 1).

Figure 4. Primary diagnoses among couples who had fresh, nondonor ART Cycles, 1997.	Table 1. Pregnancy Rates Of ART

IVF, GIFT Or ZIFT?

In the past, higher success rates used to occur with GIFT and ZIFT, because the fallopian tube was thought to provide a more natural environment for conception and early embryo development. Over the years, in our North Florida Assisted Fertility Program, we have performed large numbers of GIFT procedures with success rates in the 25%-40% range. However because of the improved success rates with IVF, we are doing fewer GIFT procedures. IVF does not require general anesthesia or laparoscopic intervention.

Currently, the only patients that we're doing GIFT on are patients that may require diagnostic laparoscopy or laparoscopy for treatment of endometriosis, in which case we are combining the procedures as a cost saving measure. Because there is no demonstrable advantage for ZIFT over IVF, we no longer perform this procedure. In 1997, only 1.6% of the ART procedures done in the United States were ZIFT and only 3.2% were GIFT.²

Ovarian Stimulation

Most centers today utilize a long regimen of GnRh agonist such as LupronŽ which is initiated in the luteal phase of the cycle. By doing this, the FSH and LH pituitary reserves are depleted, and the preselected group of follicles for the next month have not initiated any growth. When the menstrual period starts we have a group of follicles that are essentially at the starting gate. By adding gonadotropins in the form of FSH or FSH and HMG combinations we can grow the eggs in a synchronous fashion, leading to increased number of eggs at the same stage of maturity. GnRh antagonists will soon change our stimulation protocols.

ART And The Age Factor

A woman's age is the most important factor affecting the chance of pregnancy with ART. Figure 5 shows both the pregnancy and live birth rates for women of different ages who had ART procedures in 1997. Among women in their twenties, both rates are relatively stable, however, both rates declined sharply from the mid-thirties onward.

Ovarian egg response can be an issue at any age group. The marker we use for ovarian response is cycle day 2 or day 3 FSH-Estradiol ratio, which correlates fairly well with advancing age. Younger women with an abnormal ratio have poor egg production and decreased chance of pregnancy. In our laboratory, we want FSH levels to be below 12 miu/ml and estradiol to be below 70pg/ml. This is related to the feedback between the ovary and the pituitary. If the ovary doesn't produce estrogens and inhibins, there is less negative feedback and FSH begins to rise.

ART And Donor Eggs

Because the ovarian response is a very important determinant of success in ART, using eggs from a young donor will improve the pregnancy rates of older or poor response patients (Figure 6). The live birth per transfer rate for cycles using embryos from donor eggs varies only slightly across all age groups.

Figure 5. Pregnancy and live births rates for fresh, nondonor ART cycles, by age of woman, 1997.	Figure 6. Live births per transfer for fresh embryos from own and donor eggs, by age of recipient, 1997.

ART In Male Fertility

Intrauterine insemination, where high concentration of washed sperm is injected into the uterus, may help some couples with male infertility, however when we have a severe abnormality especially in low sperm motility (less than 20% by the WHO criteria) the chance of success is so low, that we prefer to go directly to ART. We use 5 million total motile sperm as our cutoff before we go to ART. Of course sperm shape (morphology) is also important, and we go by Kruger's strict morphology criteria. If we have <4% normal looking sperm we go directly to ART.

If we cannot recover 500,000 to a million motile sperm from a given ejaculate, the chance that a man's sperm could fertilize his wife's eggs in in vitro fertilization is very low. In such cases we proceed to Intracytoplasmic sperm injection (ICSI - Figure 8). ICSI was first reported by Gianpiero Palermo in 1992³ where a single sperm is injected into the egg. This procedure allowed the treatment of almost any man that has any sperm with a good success rate. Sperm can be extracted from the vas deferens (Figure 9). Even though some literature is reassuring⁴ one of the continued concerns about ICSI is the potential of transmitting genetic abnormalities. Men who have congenital absence of the vas deferens carry cystic fibrosis gene mutations in up to 80% of cases. Men with severe oligospermia or azospermia have Y chromosome microdeletions in up to 15% of cases.

These microdeletions are passed to the male offspring, which doesn't appear to be a major problem to couples who consider themselves normal. Other chromosomal problems such as autosomal translocations as well as sex-chromosome abnormalities are seen in up to 20% of men with nonobstructive azospermia.

Blastocyst Culture In ART

After an egg is fertilized it divides and cleaves into several smaller cells called blastomeres. The mass of these cells is enclosed in the egg shell (Zona Pellucida). This eventually forms a solid mass called a "morula". Later the morula develops a fluid cavity and is called a blastocyst (Figure 3).

Until recently we did not have a good blastocyst culture system. Transferring embryos at the blastocyst stage seems to give better implantation and pregnancy rates than transferring at the 8-12 cell stage (day 3 embryos).^5,6 Because of the higher implantation rates of blastocyst embryos, the number of embryos transferred can be decreased leading to decrease in the incidence of multiple pregnancy which is currently a major problem in ART.⁷

In our program, we transfer 3 embryos in women less than 30 years old, 4 embryos in women between 30 and 40, and more than 4 in women older than 40. There doesn't seem to be increased incidence of malformation in babies conceived through ART.

The Future And ART

The future holds promise for rapid advances in ART. Advances in molecular medicine will help in Y chromosome mapping. Men with testicular failure and germ cell arrest will be able to father children. Future research with maturation, culture and endometrial synchronization may allow immature oocyte retrieval with in vitro oocyte maturation to replace in vitro fertilization in its present form. Storing frozen oocytes (eggs) will provide an alternative to embryo cryopreservation with it's ethical implications.

Advances in preimplantation genetics will help screen out abnormal embryos. New drugs such as GnRh antagonists hold promise of leading to better ovarian stimulation, egg quality and possibly implantation rates. Transfer of cytoplasm from younger donor oocytes into older oocytes may improve the viability of these gametes and give a chance for older patients to carry their own genetic pregnancy.

Discussion

While ART can help a large number of infertile couples, it is estimated that only 5% of ART candidates utilize this medical service, the reason being cost or lack of accessibility in certain geographic areas. Few states have mandated insurance coverage for ART. These technologies raise several ethical issues, such as what to do with leftover frozen embryos, egg donation, embryo donation, compensation for egg or embryo donation, donor sperm for single women or gay couples, number of embryos transferred into the uterus leading to high order multiple pregnancies with the possible need for selective reduction. Sorting out genetic factors, genetic counseling and possible preimplantation diagnosis should be used more often in ART programs.

Reported success rates of different clinics have been used to rank and allow direct comparison between practices, which can be misleading to couples. Because success rates depend on several factors, and selection criterion as well as cancellation rates, these comparisons have been found by the Society For Assisted Reproductive Technology as unacceptable and not permitted, yet is still practiced.

Certain ART practices such as assisted hatching are widely used even though there is no comprehensive, well controlled, prospective study that has been conducted conclusively to define which clinical circumstances are aided by its use.

Conclusion

Whereas ART procedures may be fraught with possible deleterious side-effects for offspring that arise from these procedures, our initial fears have so far been largely allayed with respect to these high technology form of human reproduction. Although well-researched caution is always advisable, ultimately with the use of more and more complete forms of embryo quality assessment through the use of preimplantation biopsy, we will be able to monitor the normality or otherwise of all techniques we apply before the transfer of any embryos generated by such techniques.

REFERENCES

1. Steptoe PC, Edwards RC. Birth after reimplantation of human embryo. Lancet. 1978; 2:366.
2. U.S. Department of Health and Human Services Centers for Disease Control and Prevention. The 1997 assisted reproductive technology success rates. December1999.
3. Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of a single spermatozoon into an oocyte. Lancet. 1992; 340;17.
4. Palermo GD, Cohen J, Rosenwaks Z. Intracytoplasmic sperm injection: a powerful tool to overcome fertilization failure. Fertil Steril. 1996; 65(5); 899-908.
5. Gardner DK, Lane M, Calderon I, Leeton J. Environment of the preimplantation human embryo in vitro: metabolic analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil Steril. 1996; 65: 349-53.
6. Cruz JR, Dubey AK, et al. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril. 1999; 72; 218-220.
7. Milki AA, Fisch JD, Behr B. Two-blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared with three blastocyst transfer. Fertil Steril. 1999; 72: 225-8.

Jacksonville Medicine / May, 2000

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