Hormonal Stimulation

Limited initially by the single preovulatory oocyte generated with each natural menstrual cycle, the introduction of follicular hormonal stimulation has significantly increased the probability of a live birth through the retrieval of multiple oocytes per cycle. Hormonal regimens typically initiate a cycle with a gonadotropin-releasing hormone agonist to induce pituitary and ovarian suppression, followed by follicle-stimulating hormone and human menopausal gonadotropin to stimulate the development and growth of multiple ovarian follicles. Human chorionic gonadotropin (hCG) is later added to induce maturation and demargination of the oocyte from the follicular wall before retrieval. Although the goal of these regimens is the generation of 10 to 15 oocytes, the physiologic responses to hormonal manipulation can be unpredictable, particularly with primary cycles, resulting in either failed or superovulation. All visible ovarian follicles are aspirated (see later discussion), with each follicle usually containing a single oocyte.

After oocyte retrieval, pituitary function is usually insufficient to provide adequate hormonal support for the growing corpus luteum. For this reason, parenteral progesterone is administered until either the first pregnancy test results are known or the first trimester of pregnancy is completed.

Oocyte Retrieval

Originally conducted with direct visualization of the ovarian follicles through pelvic laparoscopy, the majority of oocyte retrievals are currently performed through a transvaginal approach with ultrasonographic guidance ( Fig. 15.3 ). Laparoscopic oocyte retrieval is typically reserved for situations in which tubal transfer is planned (i.e., gamete intrafallopian transfer [GIFT] or zygote intrafallopian transfer [ZIFT]; see later discussions).

Transvaginal ultrasound-guided oocyte retrieval. The ultrasonographic probe is placed in the vagina and advanced into the posterior fornix. The needle, previously inserted through the needle guide, is advanced through the vaginal wall and ovarian capsule.

(Redrawn from Steinbrook R. Egg donation and human embryonic stem-cell research. N Engl J Med 2006;354:324–326. Copyright © 2006 Massachusetts Medical Society. All rights reserved.)

Oocyte retrieval is performed promptly between 34 to 36 hours after hCG administration to prevent spontaneous ovulation from occurring. With the use of a needle introduced through the vaginal fornix and affixed to a channel on the side of a transvaginal ultrasound probe, the ovary is punctured and follicular fluid is aspirated. Oocytes are identified in the aspirated fluid, immediately washed in culture media, and microscopically examined to determine their stage of meiosis. Oocytes are classified as postmature metaphase II, mature metaphase II, metaphase I, or prophase I based on their nuclear, cytoplasmic, and extracellular composition.

In vitro Fertilization

Although the term in vitro fertilization is often used synonymously with any aspect of ART, technically it applies only to the process of oocyte fertilization with spermatozoa in culture media. After a microscopic examination, oocytes are incubated for 4 to 6 hours in culture media that resembles human fallopian tube fluid and are then inseminated. The insemination process is sometimes delayed with immature oocytes (e.g., metaphase I) to increase the probability of normal (i.e., monospermic) fertilization.

At 16 to 20 hours after insemination, the oocytes are examined for evidence of fertilization (i.e., the presence of two pronuclei and two polar bodies in the perivitelline space) ( Fig. 15.4 ). The advantages of IVF include the ability to document the process of fertilization and to enhance sperm motility or penetration (e.g., intracytoplasmic sperm injection). IVF followed by ET represents approximately 99% of the ART procedures used in the United States ; less than 1% occur via GIFT or ZIFT procedures (see later discussions). Male factor infertility is present in approximately 35% of the couples seeking ART procedures, with intracytoplasmic sperm injection being the most frequently used therapeutic intervention.

Pronuclear stage prezygote. (A) At 8 to 10 hours after insemination, pronuclei are barely visible and may be spaced slightly apart. (B) After 12 hours, pronuclei have migrated to the center of the cell and are clearly seen. (C) At 20 to 22 hours, nuclear envelopes break down and pronuclei begin to fade from view. (D) The one-cell zygote before the first cleavage.

(Redrawn from Veeck LL. Atlas of the Human Oocyte and Early Conceptus . Baltimore, MD: Williams & Wilkins; 1991:43.)

Embryo Transfer

Following incubation for 3 to 5 days, embryos resulting from IVF may be transferred via a catheter into the fallopian tubes (i.e., ZIFT) or, more commonly, into the uterine cavity (IVF-ET). The advantages of transcervical ET are (1) simplicity—it does not require laparoscopy or anesthesia; (2) low cost, especially compared with laparoscopic intrafallopian transfer procedures; and (3) the ability to proceed without patent fallopian tube(s). The primary disadvantage of transcervical ET is that the probability of successful pregnancy is slightly less than that with ZIFT. Embryos in excess of those required for transfer may be frozen in 1,2-propanediol or glycerol and stored for possible later transfer.

Gamete Intrafallopian Transfer

GIFT procedures consist of the transabdominal or transvaginal collection of oocytes followed by a microscopic inspection of the oocytes’ quality and maturation in a laboratory adjacent to the operating room. Mature oocytes are aspirated into a transfer catheter with washed partner or donor sperm, and the contents (gametes) are injected into the distal 3 to 6 cm of one or both fallopian tubes. The catheter is subsequently inspected microscopically to verify that oocytes have not been retained. The GIFT procedure does not involve IVF, because fertilization occurs in vivo in the natural milieu of the fallopian tube.

Specific advantages of the GIFT procedure include (1) the convenience of oocyte retrieval and ET occurring within a single operative event, (2) the elimination of IVF, and (3) the embryos reaching the uterine cavity at a potentially more appropriate (i.e., later) stage of development than with IVF-ET. The primary disadvantage is that fertilization cannot be documented, a critical factor when this capacity is in question (e.g., couples with male or immunologic factors). Normally, 50% to 70% of inseminated oocytes become fertilized ; however, lower fertilization rates are often observed in couples with severe male factor infertility or in women with antisperm antibodies. Other limitations are the required presence of at least one patent fallopian tube and the requirement for laparoscopic surgery.

Zygote Intrafallopian Transfer

ZIFT (also known as pronuclear stage transfer [PROST]) consists of oocyte retrieval followed by IVF. At 16 to 20 hours after insemination, the oocytes are examined for the presence of two distinct pronuclei (i.e., the pronuclear stage; see Fig. 15.4 ), which indicates that fertilization has occurred. The patient is anesthetized for laparoscopy, and pronuclear stage embryos (usually no more than four) are transferred through a catheter into the distal portion of a fallopian tube (as described for GIFT). Advantages of ZIFT include (1) the documentation of fertilization, (2) the avoidance of laparoscopy if fertilization is not successful (approximately 13% of inseminations), (3) a shorter exposure to the laboratory environment than with IVF-ET, and (4) the potential for embryos to reach the uterine cavity at a more appropriate stage of development than with IVF-ET (i.e., approximately the fifth day after insemination). Its disadvantages and limitations include (1) the added inconvenience and cost of a two-stage procedure, (2) the requirement for laparoscopic surgery, and (3) the requirement for at least one patent fallopian tube.