Weill Medical College
Cornell Institute for Reproductive Medicine
Center for Male Reproductive Medicine and Microsurgery
"State-of-the-Art Compassionate Care for the Infertile Couple"
Indications for ICSI
Until recently, the clinical application of direct injection of a single sperm into the cytoplasm of an oocyte during IVF was not shown to be feasible. The demonstration of fertilization and live births by Palermo et al. in 1993 was the first successful application of ICSI. Since that time, ICSI has been performed extensively in multiple centers to treat patients with severe male factor infertility. To date, the success of ICSI procedures has been related to several factors: (1) the viability of the spermatozoon, (2) the quality of the oocyte, (3) effective activation of the oocyte, and (4) ability of the oocyte to tolerate intracytoplasmic manipulation. Application of this treatment is described below. To date, rigorous indications for ICSI have not yet been defined. Most clinical series report on using ICSI in cases where standard IVF is highly unlikely to succeed, that is, in patients with less than 500,000 motile sperm present in the ejaculate, or less than 4% normal forms with strict criteria evaluation. In addition, couples who have failed to fertilize any oocytes in a prior IVF cycle are considered appropriate candidates for IVF-ICSI. We have proposed the following indications for ICSI:a) sperm concentration < 2 x 106
b) sperm motility < 5 %
c) strict criteria normal morphology < 4 %
d) use of surgically retrieved spermatozoa
e) failure of fertilization in a previous IVF cycle
Although fertilization and pregnancy rates with ICSI are similar or better than those achieved with normal sperm in other couples undergoing IVF concurrently at the same center, couples with only minor semen abnormalities have not been routinely treated with IVF-ICSI. Given the relatively brief history of ICSI, and its potential effects on progeny, it would seem prudent to avoid over-application of this new technology. Therefore, ICSI should not be recommended to couples for whom there is no documented benefit, since unknown risk may exist.
Technique of ICSI
1. Oocyte processing: Oocytes are prepared by removing the cumulus mass and corona radiata with hyaluronidase. The oocytes are then examined under the inverted microscope to assess the maturation stage by observing the presence of a germinal vesicle, germinal vesicle breakdown, and the extruded first polar body. Metaphase II oocytes are identified by the presence of the extruded first polar body. Intracytoplasmic sperm injection is performed on all metaphase II oocytes. Metaphase II oocytes have their diploid complement of chromosomes delicately arranged on the metaphase plate near the polar body. Mechanical disruption of the metaphase plate can occur by injury from the injection pipette or the presence of a motile sperm in the oocyte cytoplasm. Each oocyte is placed in a droplet of medium surrounding the central droplet which contains the spermatozoa. The droplets are covered with lightweight paraffin oil and the petri dish is placed on a heated stage of the microscope. The microscope is equipped with two hydraulic micromanipulators which are fitted to two tool holders for the micropipettes. During the injection procedure oocytes are stabilized with a holding micropipette, and injected with an injection pipette.
2. Microinjection: Details of the preparation of microtools and protocols for ICSI are described in detail elsewhere. The holding and injection pipettes are made by drawing glass capillary tubes with a pipette puller and further processed on microgrinder and microforges. The outer and inner diameters of the holding and injection pipettes are, respectively, 60 and 20 Ám, and 7 and 5 Ám. The injection pipette has a bevel angle of 50║ and a sharp spike to assist penetration through the oolemma. Washed sperm are prepared on a discontinuous mini-Percoll gradient. The sperm h-action is washed with T6 medium containing 5 mM CaCl2 just prior to the injection procedure. The sperm pellet is resuspended and transferred with a polyvinylpyrrolidone solution in HEPESbuffered Earle's medium. From a 3-5 Ál sperm-PVP droplet covered with lightweight paraffm oil, a single sperm is aspirated into an injection micropipette. A metaphase II oocyte is immobilized with slight negative pressure on the holding pipette. The polar body is held at the 12 or 6 o'clock position and the injection micropipette containing the single sperm is pushed through the zona pellucida and oolemma into the cytoplasm of the oocyte at the 3 o'clock position. A single sperm is injected head first into the ooplasm with 1-2 pl of medium. The injection pipette is withdrawn gently and the oocyte is released from the holding pipette. Further handling of injected oocytes is similar to that for oocytes in standard IVF.
Results of ICSI
One of the largest series reporting results using IVF/ICSI was from Van Steirteghem et al. at The Brussels Free University in Brussels, Belgium. In their preliminary report on 150 couples who underwent 150 consecutive treatment cycles, 1409 oocytes were injected and 830 were successfully fertilized for a fertilization rate of 59 percent. A total clinical pregnancy rate of 35 percent was achieved. The fertilization rate in this study was not influenced by the standard semen characteristics of concentration, motility, and strict criteria morphology. In another largest case serie on ICSI in the United States, Palermo et al. at Cornell reported successful fertilization in 1,142/1,923 (59 %) metaphase II oocytes injected, and ongoing pregnancies in 84/227 (37%) couples. Neither semen quality nor the source of sperm (ejaculated, surgically retrieved or electroejaculated) affected fertilization rates. They concluded that IVF/ICSI offers fertilization and pregnancy rates comparable to that achieved with normal sperm quality for couples who have failed to achieve fertilization on repeated IVF cycles or have severe impairments in semen quality. In addition, the success of IVF/ICSI was independent of standard semen parameters (density, motility, and morphology).
Factors Affecting Results Of ICSI
1. Spermatozoal factors: Nagy et al. evaluated the effect of spermatozoal factors on results of ICSI in 966 microinjection cycles. Despite no normal forms in a semen preparation, virtual azoospermia or essentially no motile sperm in the ejaculate, pregnancy could still be achieved. Nagy et al. found that the only absolute criterion for successful ICSI is the presence of at least one viable spermatozoon to inject per oocyte in the prepared pellet of the washed semen sample. The only category in which semen parameters had a significantly impaired fertilization and pregnancy rate was when there was no motility of sperm'o. If no motility is present, then viability is often impaired as well.
2. Female factors: Oehninger et al. investigated the role of matemal factors in a total of 92 couples, where 1163 oocytes were injected with an overall fertilization rate of 61 percent. Fertilization rates were unaffected by matemal age, but pregnancy rates were significantly lower with increased matemal age. Pregnancy rates were 49, 23 and 6 percent for couples in whom matemal age was <34, 35-39, and < 40 years. Similar results were found by Sherins et al., with a 30% pregnancy rate for the youngest couples and 13 % pregnancy rate for the couples with the oldest female partners. The rate of aneuploidy increases dramatically for embryos derived from the oocytes of women over 40 compared to those from women less than age 35. Therefore, it is likely that the chance of fertilization is unrelated to female factors, but that the chance of pregnancy occurring after ICSI is related primarily to oocyte factors, if a viable sperm is injected.
3. Oocyte activation: Since oocyte activation normally occurs in association with sperm binding, fusion and penetration of the oocyte, oocyte activation during intracytoplasmic may not necessarily occur. The importance of intentional induction of oocyte activation was demonstrated by Tesarik and Sousa who increased fertilization and pregnancy rates during ICSI with aggressive aspiration and injection of the oocyte cytoplasm. Direct comparison of gentle and vigorous cytoplasm aspiration resulted in an increase in fertilization rates per oocyte from 38% to 80% with increased pregnancy rates up to 52 % with aggressive aspiration/injection. Evaluation of calcium fluxes in oocytes during injections demonstrated an additional peak of intracellular calcium levels for aggressive aspiration, when compared with gentle aspiration. Intracellular calcium changes have long been thought to have a role in oocyte activation. An additional sperm factor may have a role in cytoplasmic activation. Aggressive immobilization of spermatozoa has been used to effect increased sperm membrane permeability. Gerris et al. evaluated the effects of sperm tail breakage on ICSI success by directly comparing fertilization rates achieved using sperm with intact tails compared to sperm with damaged tails. Aggressive immobilization of spermatozoa resulted in an increase in the percentage of normally fertilized oocytes from 36 to 60%. These authors and others" suggested that tail damage induces sperm membrane changes which facilitate biochemical events necessary for sperm nuclear decondensation and pronuclear formation. Palermo et al. have also investigated the effect of aggressive sperm immobilization on fertilization and pregnancy rates. Although there was little improvement in fertilization rates for ejaculated sperm, a dramatic improvement in epididymally-retrieved sperm fertilization rates was seen, from 51 to 84% per oocyte, with an associated increase in pregnancy rates from 5 1 to 82 %. The biochemical basis for the effect of increased sperm membrane permeability to improve fertilization rates is unclear. It is possible that increased permeability of the manipulated sperm resulted in better penetration of ooplasmic factors into the spermatozoon to induce male pronuclear formation. Alternatively, it is possible that increased permeability results in enhanced leakage of toxic factors out of the cytoplasmic droplet of immature epididymal spermatozoa.
4. Cytoplasmic Injection/oocyte injury: Disruption of the oocyte sufficient to cause oocyte demise may occur during ICSI. Results from some of the major centers performing ICSI show rates of oocyte loss after injection of 7 to 14 percent. Although the precise reasons for oocyte injury are not known, it is though to occur as a result of plasma membrane and ultrastructural disturbances associated with injection, damage to the meiotic spindle during injection, and/or extrusion of the oocyte cytoplasm following injection. In addition, other factors such as changes in temperature have been reported to cause irreversible changes in the meiotic spindle of the human oocyte. Clearly, there is a learning curve for embryologists performing the ICSI procedure. As greater expertise is gained, the oocyte injury rate decreases. Palemo et al. have recently described oocyte characteristics that may predispose to oocyte injury. These investigators described an oocyte membrane response of "sudden breakage" during attempted ICSI. The oocytes with this response did not form a normal oocyte membrane funnel around the injection pipette. Instead, the oocyte membrane separated, spilling the oocyte cytoplasm and resulting in a 14 % injury rate, compared to a 4 % injury rate for other oocytes. Oocytes demonstrating sudden breakage were more likely to be retrieved from women who received higher gonadotropin treatment doses, with lower serum estradiol levels at retrieval, yielding immature oocytes, including those requiring maturation in vitro. These observations suggest that ovarian stimulation characteristics may affect the ability of oocytes to successfully undergo ICSI.
Risks Of ICSI
Risks of IVF-ICSI include general risks of IVF as well as the specific risks related to the micromanipulation procedure of ICSI. One of the most significant risks associated with stimulation of the ovaries is the ovarian hyperstimulation syndrome (OHSS). This can manifest as massive ovarian enlargement, peritoneal irritation due to follicular rupture or hemorrhage, ovarian torsion, ascites, pleural effusion, oliguria, electrolyte imbalance, hypercoagulability3l I and sometimes death36 . The syndrome occurs in a moderate form for 3-4% percent of initiated cycles, and in a severe form for 0. 1-0.2 % of the populatioe undergoing controlled ovarian hyperstimulation. Other reported complications of ovarian hyperstimulation are pituitary hemorrhage, endometriotic bloody ascites, and genital cancer. Complications of ovarian retrieval have been reported for transvaginal aspiration as well as laparoscopic aspiration. Complications associated with wmsvaginal aspiration have been reported to occur in 0.3-3 percent of cases and include bleeding, pelvic infections, and abdominal viscera perforation . Laparoscopic complications include hemorrhage, intestinal perforation, infection, and carbon dioxide embolism. The laparoscopic risks are no higher in ovarian retrieval procedures than in other laparoscopic applications.
Finally, pregnancies resulting from ovarian stimulation are at risk for spontaneous abortion, ectopic pregnancy, and multiple gestational. The rate of spontaneous abortion after achieving a biochemical pregnancy with assisted reproduction is approximately 25 percent. These losses are attributed to advanced maternal age and the associated increased prevalence of chromosomal abnormalities, a higher rate of pregnancy loss due to multiple gestations, and early recognition of these pregnancies due to close monitoring. After achieving a clinical pregnancy, the chance of a spontaneous abortion occurring for IVF-ICSI cycles ranges from 10-16%. Fctopic pregnancies occur in up to 3-5.5 % of gestational cycles and can be life threatening. The etiology is usually pelvic adhesions and tubal damage from pelvic inflammatory disease or previous surgery. Multifetal pregnancies occur in 22 percent of cases of IVF with embryo transfer, and 44 to 46 percent of IVF/ICSI cases. Multifetal pregnancies are considered a complication of assisted reproductive techniques because of the associated increased incidence of preeclampsia, placenta previa, placental abruption, premature rupture of membranes, and postpartum hemorrhage. Most importantly, multiple gestations are almost universally associated with prematurity and the associated complications to offspring, including cerebral palsy and intracranial hemorrhage with mental retardation or blindness. To prevent multifetal pregnancies and their attendant complications, it would be preferable to avoid assisted reproduction unless it is specifically indicated, and limit the number of embryos transferred. Where there is government regulation of IVF, including England, Australia and France, transfer of only 3 embryos is allowed and multifetal pregnancies are less commoe. Unfortunately, there is significant pressure to transfer more than three embryos by couples in the United States who are desperate to conceive. In general, for women less than 35 years of age, only 3 embryos should be transferred.
Birth Defects After Assisted Reproduction
The bypass of natural barriers to fertilization, possible genetic defects in men with severe male infertility, and the use of severely abnormal sperm for intracytoplasmic sperm injection has engendered concern over the impact of ICSI on the genetic complement of the offspring . Previous studies have suggested no increase in birth defect rates when IV-F alone was used to induce conception. Van Steirteghem reported no increase in the congenital malformation rate in their center after ICSI when compared with the general population. Of 877 children born after ICSI procedures, 23 (2.6 percent) had major congenital malformations compared to 2.0 to 2.8 percent in the general population and 1.9 to 2.9 percent of children resulting from assisted reproductive techniques.
Sex chromosomal abnormalities have also been reported in ICSI cases. In't Veld et al. reported on 12 patients with ICSI pregnancies who underwent prenatal diagnosis for advanced matemal age. Three of the 12 women had twin pregnancies for a total of 15 diagnostic procedures by amniocentesis or chorionic villus sampling. A total of five chromosomal abnormalities were detected: two cases of XXY, one complex mosaic 45,X/46,X.dic(Y)(q11)/46.X.del(Y)(qll), and two cases of 45 XO. This high rate of sex chromosome abnormalities has not been corroborated by other studies. The Brussels group reported on a total of 585 prenatal diagnoses performed in pregnancies established by ICSI. A total of six sex chromosome abnormalities (1.0 percent) were detected compared to 0.2 percent in the general population. This difference did not achieve statistical significance. Govaerts et al. reported on 55 karyotypes obtained by amniocentesis or chorionic villus sampling in pregnancies from ICSI and found no sex chromosome abnormalities. When sex chromosome abnormalities have been identified it is unclear whether they are related to the ICSI procedure itself or can be ascribed to advanced matemal age. What is reassuring is that the rates of nonsex chromosomal abnormalities in the ICSI population published to date do not exceed the rates seen in the general population. The relationship of ICSI to sex chromosomal abnormalities in offspring may be related to the association between Y chromosomal abnormalities and severe male factor infertility. Several investigators have reported that up to- 13% of men with azoospermia or severe oligospermia may have deletions of 15,000 to 200,000 base pair lengths of Y chromosome. At least one gene (DAZ; deleted in azoospermia) is deleted in 13 percent of patients with non-obstructive azoospermia.
Although chromosomal abnormality rates in offspring after these procedures have not exceeded those in the general population, experience with these techniques is brief. Genetic counseling, preimplantation genetic diagnosis, and state of the art prenatal diagnosis must also be available to couples enrolled in assisted reproductive programs. Genetic counseling should be available to all couples. All couples undergoing micromanipulation procedures are strongly urged.to have prenatal diagnosis with amniocentesis or chorionic villus sampling. The need for prenatal diagnosis is dependent on whether the couple would consider terminating the pregnancy if the results are abnormal. If the couple would carry a pregnancy to term regardless of the results of prenatal diagnosis, then the procedure of prenatal intervention would carry risks to the fetus without benefit and therefore cannot be required.
In certain cases, manipulation of the embryo can enhance implantation and subsequent pregnancy. Common practice is to use acidified Tyrode's solution (pH 2.35) to thin the zona pellucida. An increase in implantation from 18 % to 25 % is achievable for oocytes with poor prognosis with this intervention, refeffed to as assisted hatching. Assisted hatching is not applicable only for male factor infertility, in fact, it is a treatment for specific defects in embryo development or oocyte abnormalities. However, it is a micromanipulation technique that is routinely performed during IVF at many centers.
The application of micromanipulation techniques in the IVF laboratory has allowed the development of analysis and selection of embryos with specific genetic, chromosomal or biochemical characteristics prior to transfer of those embryos. An embryo at the four or eight cell stage is isolated and an individual cell is extracted for evaluation. Chromosome-specific sequences can be identified using fluorescent hybridization probes or, alternatively, polymerase chain reaction (PCR) amplification of individual alleles on the chromosomes themselves may be applied to identify the genotype of the "biopsied" embryo. These techniques can allow identification of embryos at high risk of carrying X-linked diseases such as hemophilia A or von Willebrand's disease. In addition, specific genetic defects such as the homozygous delta-F508 mutation of the CFTR gene, associated with the development of a severe form of cystic fibrosis, can also be identified. These techniques have been applied for couples known to be at high risk of having children with specific genetic diseases. "Biopsied" embryos have been successfully transferred resulting in pregnancies and live births'. These micromanipulation techniques are highly labor intensive and carry some potential pitfalls. For example, if both parents in a couple are heterozygous for the delta-F508 CFTR mutation, then an individual embryo has a one-in-four chance of being homozygous for that gene mutation. PCR has the potential for identifying the homozygous condition since the delta-F508 mutation is a deletion of 3 base pairs in the normal allele. The normal CFTR gene is 3 base pairs longer than the delta-F508 mutated allele, and a heterozygous carrier would be expected to have both alleles detectable with PCR. However, PCR involves magnification of the single signal present on both chromosomal alleles; if one allele is preferentially bound, read and amplified with PCR, then the results of PCR analysis are significantly confounded.
For sex chromosomal analysis, this evaluation is more accurately performed (up to 95.5 % efficiency) using different colored fluorescent probes". A test for both X and Y-specific sequences is possible and provides further confirmation of the results of these tests. Given the extensive manpower needed for single-day biopsy and evaluation of the results of embryo biopsy, this technique is limited to those cases where life threatening genetic defects can be reliably detected prior to embryo transfer to prevent the potential termination of a fetus later in development.
Since the first U.S. report of a successful delivery from in vitro fertilization in 1983 the advances in the field of assisted reproduction and micromanipulation have been truly dramatic. Perhaps the most exciting advances have been in the area of male factor infertility. Couples who previously would have been offered donor insemination or adoption are now achieving . pregnancies despite severe impairments in semen quality, the presence of only single numbers of sperm in the ejaculate or unreconstructable reproductive tract obstruction. Techniques of micromanipulation that were revolutionary less than five years ago are now obsolete, replaced by even more successful methods. Even non-obstructive azoospermia due to maturation arrest or other impairments in germ cell maturation have been added to the list of treatable factors in male infertility since sperm can frequently be extracted directly from testicular parenchyma that is surgically biopsied. For patients without sperm in the testicular parenchyma, round spermatid or secondary spermatocyte injections are possible.
Several important questions remain with regard to IVF-ICSI:
(1) What should be the specific indications for IVF and IVF-ICSI'? Should IVF alone ever be used for male factor infertility? (2) What are the reasons for failure to achieve pregnancy after IVF-ICSI that still represent over half of our attempts at achieving ongoing pregnancies? (3) Can we be certain that using severely impaired or less mature sperm will not result in significant birth defects or in genetic abnormalities that could affect the offspring in adolescence or adulthood) and (4) What is the most cost effective approach for the infertile couple with impaired semen parameters? Contemporary application of IVF-ICSI for severe male factor infertility can allow pregnancy rates up to 52% , with ongoing pregnancy and live delivery rates as high as 37 % per IVF cycle attempt. As long as viable sperm are present in the ejaculate or retrievable from the male reproductive tract, then IVF-ICSI procedures can be applied.