In Vitro Fertilization for Male Factor Infertility

Peter N. Schlegel and Sarah K. Girardi

James Buchanan Brady Foundation, Department of Urology, The New York Hospital-Cornell Medical Center, and The Population Council, Center for Biomedical Research, New York, New York

Address correspondence and requests for reprints to: Peter N. Schlegel, M.D., Room F-905A, Department of Urology, The New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, New York 10021.


    Introduction
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
MALE FACTOR infertility is a general term that describes couples in whom an inability to conceive is associated with a problem identified in the male partner. This problem may be associated with low sperm production (oligospermia), poor sperm motility (asthenospermia), or abnormal morphology (teratospermia) (1). Abnormal sperm function may also be evaluated with sperm function tests that evaluate sperm interaction with cervical mucus (cervical mucus penetration test), the zona pellucida surrounding the oocyte (hemi-zona binding assay), or the oocyte itself (hamster-egg penetration assay) (2). Male factor infertility also describes men with normal sperm production but conditions that prevent sperm transport to the vagina during intercourse (e.g. reproductive tract obstruction or ejaculatory dysfunction). Obtaining serial semen analyses and questioning of the male partner should be part of the initial survey of an infertile couple. When a male factor is suspected during evaluation of a couple for infertility, complete evaluation of the man is warranted. If treatable conditions causing the male factor are found, they should be corrected. If treatment is unsuccessful, or if the couple still does not conceive, then assisted reproduction is indicated. Assisted reproductive techniques include intrauterine insemination (IUI), in vitro fertilization (IVF), and IVF with micromanipulation. Micromanipulation refers to a series of procedures that enhance the ability of sperm to fertilize an oocyte, in vitro. In this review we will emphasize recent advances in IVF, especially IVF with the advanced micromanipulation technique of intracytoplasmic sperm injection (ICSI), as tools for treatment of the infertile couple with male factor infertility.


    Evaluation of male factor infertility
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
The cornerstones of evaluation of a subfertile man include a comprehensive history, physical examination, multiple semen analyses, and an endocrine evaluation. In specific circumstances, additional testing may be indicated. For men with azoospermia or severe oligospermia (sperm concentration < 5 x 106/cc), consideration of karyotypic abnormalities such as Klinefelter’s syndrome is appropriate if clinically indicated. In addition, up to 13% of men with azoospermia may have microdeletions of the Y chromosome. Although routine evaluation for these microdeletions is available at only a few U.S. academic centers in 1996, evaluation at the SIMMY protocol referral center (Study of ICSI, Male Infertility and Microdeletions on the Y chromosome) can provide free testing of patients who are candidates for treatment with ICSI (3, 4, 5, 6). For men with unilateral or bilateral congenital absence of the vas deferens, cystic fibrosis transmembrane conductance regulator (CFTR) gene analysis is important, as 55–82% of men with congenital absence of the vas deferens will carry detectable CFTR mutations (7). In addition, patients with idiopathic epididymal obstruction have been estimated to have a 47% chance of carrying a detectable CFTR mutation (8). For couples with vasal or epididymal anomalies, testing of the female partner for CFTR mutations is even more important, as not all CFTR mutations are currently detectable in the man. In the case of any other genetic condition, including treatment of men with Klinefelter’s syndrome, and in the case of couples with a female partner over age 40 yr, genetic counselling is recommended before assisted reproduction treatments.


    Treatment of male infertility
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Up to 75% of men with a male factor will have identifiable or treatable conditions that affect their fertility (9, 10, 11). Nearly all men with male factor infertility are treatable with assisted reproductive techniques. Before applying more invasive techniques, however, avoidance of specific gonadotoxic factors such as exogenous heat, chemical gonadotoxins (e.g. sulfasalazine and cimetidine), or medications that can adversely affect fertilization (including calcium channel blockers) is appropriate. Treatment of varicoceles, endocrine disturbances, symptomatic infections, and obstructive azoospermia have all been demonstrated, using randomized or other appropriately designed studies, to have a role in the management of male infertility (12). Specific treatment of the man may be less invasive, more successful, and more cost effective (13, 14) with lower risk than IVF. In addition, it is worthwhile to remember that up to 1% of men with subfertility have a potentially life threatening condition associated with their fertility problem, (e.g. testis tumor) (1, 15). Suffice it to say that evaluation and treatment of a man with male factor is worthwhile, despite the recent advances in assisted reproduction.


    Background: in vitro fertilization
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Male factor infertility was initially considered a contraindication to IVF because abnormal sperm are less likely to fertilize oocytes than normal sperm (16). However, subsequent experience starting just over a decade ago indicated that fertilizations and subsequent live births were possible despite impaired sperm quality (17). IVF has had some success in the treatment of these men, however it has been recognized that even normal concentrations of sperm from oligozoospermic men, placed directly with oocytes in culture, do not fertilize at the same rates as sperm from otherwise normal men. In addition, adequate numbers of sperm cannot be obtained from all men to allow insemination of oocytes with the usual numbers of gametes (100,000 sperm/oocyte). Initial concerns, that assisted fertilization with apparently defective sperm might lead to the development of abnormal embryos and an increase in the number of birth defects, have not been founded. In fact, once fertilization has been achieved for male factor couples, implantation and subsequent pregnancy appear to be just as likely, if not more likely, to occur than in other cases of IVF (18).

Unless advanced age of the female partner is present, IVF is usually indicated after specific treatment of male and female factors affecting fertility has been unsuccessful and less invasive forms of assisted reproduction (intrauterine inseminations) have been attempted. If severe male factor infertility is present, direct treatment with IVF and micromanipulation may be indicated. The technique of IVF is described in greater detail elsewhere (19). Briefly, it involves down-regulation of the woman’s pituitary function with GnRH agonists given during the preceding luteal phase. This is followed by controlled ovarian hyperstimulation using FSH or FSH-stimulating agents, to increase the number of oocytes produced. Follicle development in the ovary is evaluated directly with transvaginal ultrasound imaging of follicular growth and by measurement of serial serum estrogen and progesterone levels. Final oocyte maturation is induced with an intramuscular dose of hCG (5–10,000 units) when optimal follicular development is obtained. Retrieval of oocytes is performed by transvaginal follicular aspiration using ultrasound guidance with intravenous sedation. The transvaginal approach has obviated the need for general anesthesia and laparoscopy to perform IVF. Many oocytes (mean of 12 oocytes) can be obtained from otherwise normal women with ovarian hyperstimulation. Morphologically mature, metaphase II oocytes may then be inseminated with sperm. Human oocytes survive freezing poorly since they are in metaphase; therefore, all retrieved and mature oocytes are inseminated. Immature oocytes may be matured in vitro and subsequently inseminated, although only anecdotal pregnancies have been achieved after in vitro oocyte maturation.

Sperm are washed free of seminal fluid and inseminated with oocytes at a concentration of 100,000 or more sperm per oocyte in simulated human (Fallopian) tubal fluid medium, and the oocytes that fertilize (embryos) are usually allowed to divide up to the 8-cell stage before embryo transfer. Embryo transfer back to the uterus is typically performed after 2–3 days of incubation in vitro. Up to four embryos may be transferred to the uterus, and excess embryos may be frozen. An implantation rate can be calculated by dividing the number of gestations (fetal heart on ultrasound) that result from embryo transfer by the number of total number of embryos transferred to the uterus in a population of treated patients. Implantation rates per transferred embryo range from 10–25% in most IVF programs.


    Micromanipulation
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Gamete micromanipulation has enabled the embryologist to circumvent inefficient steps in the fertilization process. Instead of simply bringing sperm and oocyte together in vitro (IVF), micromanipulation involves mechanical alteration of the oocyte in vitro to increase the chance of fertilization of the oocyte by sperm (Fig. 1Go.) The three categories of assisted fertilization by gamete micromanipulation that have been applied in humans are illustrated in Fig. 2Go. The first category involves the creation of an opening in the zona pellucida, an acellular layer surrounding the oocyte that serves as a major barrier to sperm penetration. Subsequently, the micromanipulated oocyte is inseminated according to standard IVF guidelines. These procedures have been broadly termed "zona drilling." One variant of zona drilling involving mechanical piercing of the zona pellucida has been successful in male factor patients (20). This method has been called partial zona dissection (PZD; Fig. 2AGo). A second category of micromanipulation techniques directed at facilitating sperm-oocyte interaction is the subzonal insertion of sperm (SuZI). SuZI involves direct placement of sperm into the perivitelline space between the zona pellucida and oocyte, completely bypassing the zona pellucida (Fig. 2BGo) (2, 21). The third and most invasive form of microsurgical fertilization is the microinjection of a single sperm into the cytoplasm of the oocyte, referred to as intracytoplasmic sperm injection (ICSI; Fig. 2CGo). This technique for manipulation has a higher risk of oocyte injury than SuZI or PZD, but overall higher fertilization and pregnancy rates (22). Most importantly, only very few sperm are necessary for ICSI. The tremendous superiority of fertilization and pregnancy rates after application of ICSI when compared with PZD and SuZI have relegated both PZD and SuZI to techniques of historical importance only. With micromanipulation, fertilization and pregnancy rates appear to be independent of sperm quality (23, 24), which is the opposite of what has been demonstrated for both IUI and IVF (16).



View larger version (79K):
[in this window]
[in a new window]
 
Figure 1. The structural components of an oocyte important for micromanipulation are schematically illustrated.

 


View larger version (80K):
[in this window]
[in a new window]
 
Figure 2. A, Schematic illustration of partial zona dissection technique. B, Schematic illustration of subzonal insertion procedure. C, The intracytoplasmic sperm injection technique is schematically presented.

 

    Intracytoplasmic sperm injection
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Until recently, the clinical application of direct injection of a single sperm into the cytoplasm of an oocyte during IVF had not been feasible. The demonstration of fertilization and live births by Palermo et al. (25) 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.


    Indications for ICSI
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
To date, rigorous indications for ICSI are not universally agreed upon. In general, any condition in which it is expected that oocyte fertilization might be impaired, ICSI should be considered. Early clinical series applied ICSI in cases where men had less than 500,000 motile sperm present in the ejaculate, less than 4% normal sperm forms (with strict criteria evaluation), or where couples have failed to fertilize any oocytes in an earlier IVF cycle. Sperm function tests (2) may provide additional insight into specific sperm-oocyte interaction defects that will define appropriate candidates for ICSI. We have proposed the following minimal indications for micromanipulation (26):

a. sperm concentration < 2 x 106 sperm/cc.

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 to or better than those achieved with normal sperm in other couples undergoing IVF at the same center (27), couples with only minor semen abnormalities have not been routinely treated with 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, as unknown risk to the embryo and resulting fetus may still exist (28).


    Technique of ICSI
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Oocyte processing. Oocytes are prepared by removing the cumulus mass and corona radiata with hyaluronidase. 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 by the presence of a motile sperm in the oocyte cytoplasm. The oocyte is stabilized with a holding micropipette and injected under an inverted microscope.


    Microinjection
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Details of the preparation of microtools and protocols for ICSI are described in detail elsewhere (27). Individual single sperm are aspirated from a prepared semen specimen and directly injected into an oocyte immobilized in a droplet of medium under paraffin oil. 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. Further handling of injected oocytes is similar to that for oocytes in standard IVF.


    Results of ICSI
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
One of the largest series reporting results using ICSI was from Van Steirteghem et al. (22) 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. Fertilization and pregnancy rates from their updated series (29) are shown in Table 1Go.


View this table:
[in this window]
[in a new window]
 
Table 1. Fertilization and clinical pregnancy rates from IVF/ICSI

 
In another large series, Palermo et al. (27) reported on 227 couples treated with ICSI for failed IVF cycles or for severe male factor infertility. Fertilization and pregnancy rates were evaluated relative to semen parameters and the origin of the semen samples. They reported successful fertilization in 1,142/1,923 (59 percent) metaphase II oocytes injected and ongoing pregnancies in 84/227 (37 percent) couples.


    Factors affecting results of ICSI
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Spermatozoal factors. Nagy et al. (29) reviewed 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 of semen parameters that had a significantly adverse effect on fertilization and pregnancy rates with ICSI was when there were no motile sperm (29). If no motility is present, then viability is often impaired as well.

Female factors. Oehninger et al. (30) investigated the role of female factors on ICSI results in a total of 92 couples, where 1163 oocytes were injected, with an overall fertilization rate of 61 percent. Fertilization rates were unaffected by maternal age, but pregnancy rates were significantly lower with increased maternal age. Pregnancy rates were 49, 23, and 6 percent for couples in whom maternal age was less than 34 yr, 35–39 yr, and 40 yr or over. Similar results were found by Sherins et al. (31), with a 30% pregnancy rate for the youngest couples and a 13% pregnancy rate for the couples with the oldest female partners. The rate of aneuploidy increased dramatically for embryos derived from the oocytes of women over 40 compared with those from women less than 35 yr (32). Implantation of an aneuploid embryo is highly unlikely. These observations suggest that the chance of a metaphase II oocyte being fertilized with ICSI is unrelated to female age, but the chance of a pregnancy occurring after transfer of ICSI embryos dramatically decreases with increased female age, especially female age over 40 yr.

Oocyte activation. Oocyte activation refers to the series of events that occur after sperm-oocyte fusion during natural fertilization, which result in the ability of the oocyte to complete its nuclear maturation, to synthesize proteins and DNA. Because sperm fusion with the oocyte is bypassed during ICSI, other approaches to induce oocyte activation have been attempted. Tesarik and Sousa (33) improved fertilization and pregnancy rates during ICSI by aggressive aspiration and injection of the oocyte cytoplasm during injection of sperm into the oocyte to induce oocyte activation. Vigorous cytoplasmic aspiration resulted in an increase in fertilization rates per oocyte from 38–80% when compared with results achieved using only gentle aspiration of the oocyte cytoplasm. Pregnancy rates increased up to 52% with aggressive aspiration/injection. Aggressive aspiration of cytoplasm caused additional peaks of oocyte intracellular calcium levels, when compared with gentle aspiration (33). Intracellular calcium changes have long been thought to have a role in oocyte activation, and these changes may constitute the mechanism by which aggressive cytoplasmic aspiration improves fertilization rates.

A sperm factor may also have a role in oocyte activation. This cytoplasmic sperm factor may need to diffuse through the sperm plasma membrane to induce post-ICSI events in the oocyte that facilitate pronuclear formation, including formation of the sperm aster by the paternally-derived centrosome and mitosis of the embryo. Aggressive immobilization of spermatozoa involves mechanical crushing of the sperm tail between the injection micropipette and the bottom of the petri dish containing the spermatozoon. Gerris et al. (34) reported an increase in the percentage of normally fertilized oocytes from 36–60% with aggressive immobilization. Palermo et al. (35) found the effect of aggressive sperm immobilization on fertilization rates was seen primarily in immature spermatozoa that were surgically retrieved from the epididymis and testis. For epididymal sperm, Palermo et al. demonstrated an increase in fertilization rates, from 51–84% per oocyte, with an associated improvement in pregnancy rates from 51–82% (35).

It appears that aggressive immobilization of immature spermatozoa may increase sperm membrane permeability, which enhances release of cytosolic sperm factors that facilitate oocyte activation (36). Alternatively, it is possible that the increased sperm membrane permeability results in leakage of toxic factors, such as reactive oxygen species, out of the cytoplasmic droplet of immature spermatozoa. Oocyte activation must be induced for optimal success with ICSI. Cytoplasmic sperm factors (37) as well as mechanical stimulation of the oocyte are helpful in inducing oocyte activation.

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 7–14%. Although the precise reasons for oocyte injury are not known, it is thought 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 over the first 50–100 oocyte injections, the oocyte injury rate decreases (38).

Palermo et al. (39) have described an oocyte membrane response of "sudden breakage" during attempted ICSI. The oocytes with this response did not form a normal funnel during attempted penetration of the injection pipette, but suddenly separated, spilling the oocyte cytoplasm. With sudden breakage, a 14% oocyte injury rate was seen, compared with a 4% injury rate for other oocytes. Oocytes demonstrating sudden breakage were more likely to be retrieved from women treated with higher gonadotropin treatment doses, with lower serum estradiol levels at retrieval, or where the oocytes were immature, requiring maturation in vitro. These observations suggest that ovarian hyperstimulation may affect the ability of oocytes to survive ICSI (39).


    Risks of ICSI
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Risks of 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 caused by follicular rupture or hemorrhage, ovarian torsion, ascites, pleural effusion, oliguria, electrolyte imbalance, hypercoagulability (40), and sometimes death (41). 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 population (42) undergoing controlled ovarian hyperstimulation. Other reported complications of ovarian hyperstimulation are pituitary hemorrhage, endometriotic bloody ascites, and genital cancer (43).

Complications of ovarian retrieval have been reported for transvaginal aspiration as well as laparoscopic aspiration. Complications associated with transvaginal aspiration have been reported in 0.3–3% of cases and include bleeding, pelvic infections, and abdominal viscera perforation (44). 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 (45), ectopic pregnancy (46), and multiple gestations (47, 48, 49). The rate of spontaneous abortion after achieving a biochemical pregnancy with assisted reproduction is approximately 25%. These losses are attributed to a) advanced maternal age and the associated increased prevalence of chromosomal abnormalities; b) a higher rate of pregnancy loss resulting from multiple gestations, and c) early recognition of these pregnancies because of close monitoring. After achieving a clinical pregnancy (the presence of at least one fetal heart beat on ultrasound), the chance of a spontaneous abortion occurring for ICSI cycles ranges from 10–16%. Ectopic 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 (46). Multifetal pregnancies occur in 22% of cases of IVF with embryo transfer (47), and 44–46% of ICSI cases (27, 30) in the United States. 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 (48). 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 to limit the number of embryos transferred. Where there is government regulation of IVF, including England, Australia, and France, transfer of only three or fewer embryos is allowed, and multifetal pregnancies are less common (49). 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 yr of age, only three embryos should be transferred.


    Birth defects after assisted reproduction
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
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 (28). Previous studies have suggested no increase in birth defect rates when IVF alone was used to induce conception (50). Van Steirteghem (51) 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 with 2.0–2.8% in the general population and 1.9–2.9% of children resulting from IVF without ICSI (51).

Sex chromosome abnormalities have also been reported in ICSI cases. In’t Veld et al. (52) reported on 12 patients with ICSI pregnancies who underwent prenatal diagnosis for advanced maternal 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 5 chromosomal abnormalities were detected: 2 cases of 47 XXY, 1 complex mosaic 45,X/46,X.dic(Y)(q11)/46,X.del(Y)(q11), and 2 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 6 sex chromosome abnormalities (1.0 percent) were detected compared with 0.2 percent in the general population (53). This difference did not achieve statistical significance. Govaerts et al. (54) 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 has been unclear whether they were related to the ICSI procedure, underlying paternal cytogenetic defects, or advanced maternal age. What is reassuring is that the rates of non-sex chromosomal abnormalities in the ICSI population published to date do not exceed the rates seen in the general population.

The source of sex chromosomal abnormalities in offspring after ICSI may be related to abnormalities in testicular germ cells. In addition to disomic sex chromosome abnormalities, several investigators have reported that up to 13% of men with azoospermia or severe oligospermia may have deletions of 15,000–200,000 base pair lengths of the Y chromosome (3, 4, 5, 6). At least one gene (DAZ; deleted in azoospermia) is deleted in 13% of patients with nonobstructive azoospermia and some men with severe oligospermia. Of greater concern is the possibility that additional unknown genetic problems may be present in infertile men who have never been able to conceive in the past, but can now become fathers with ICSI. Evaluation of 32 father-son pairs after ICSI showed that 3 (9%) ICSI-derived sons had microdeletions of the DAZ region in one study (6), possibly because of germ line mosaicism for Y chromosome microdeletions in the fathers of 2 of these boys. The third father had a Y chromosome microdeletion detected on peripheral leukocyte evaluation that was inherited by his son. ICSI-derived sons may be at increased risk of infertility or other abnormalities because of transmission of the genetic defects that are associated with male infertility.

Although chromosomal abnormality rates in offspring after assisted reproductive 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. 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.


    Application of ICSI: epididymal and testicular sperm
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
The application of ICSI has allowed treatment of couples who until very recently were considered sterile and untreatable. Men with bilateral congenital absence of the vas deferens and other unreconstructable obstructions of the male reproductive tract are good candidates for ICSI. In these men, microsurgical retrieval as well as cryopreservation of sperm is possible despite the fact that the sperm are immature (i.e. have not traversed most of the excurrent duct system.) Percutaneous aspiration of sperm from the epididymis or testis can also provide sperm for ICSI cycles, although the sperm are often not of adequate quality for cryopreservation; therefore a repeat sperm retrieval procedure mighty be needed with each ICSI attempt. Using ICSI and simultaneous open surgical sperm retrieval, clinical pregnancy rates per sperm and oocyte retrieval attempt range from 45–82% at established centers (35, 55, 56).

For men with nonobstructive azoospermia, sperm retrieval from the testis is often successful using an open biopsy technique. Even if a small diagnostic testis biopsy reveals a Sertoli cell-only pattern in a man with small volume testes and an elevated FSH level, sperm retrieval might still be possible with a more extensive biopsy. Taken together, 50–70% of men with nonobstructive azoospermia can have sperm retrieved surgically from the testis, with a biopsy performed simultaneous to an IVF-ICSI procedure (57, 58, 59). Up to 20% or more of attempts at sperm retrieval-ICSI for nonobstructive azoospermia will result in a clinical pregnancy (57, 58, 59).

Even for men who have no sperm production in their testes, immature germ cells (spermatids or spermatocytes) might have application in micromanipulation procedures with some chance of contributing to pregnancies. In an animal model, Kimura and Yanigamachi (60) have demonstrated induction of pregnancies by injecting the nucleus of a secondary spermatocyte from a normal animal into an oocyte and activating the egg with an electrical pulse. Transfer of resulting embryos to the uterus of a recipient animal resulted in normal offspring. In humans, round spermatids retrieved from the semen of seven men were microinjected into oocytes of their female partners, with ongoing pregnancies for two couples, including delivery of a normal child for one couple (61). The potential for future application of these techniques is difficult to predict. In our experience, we have rarely found spermatids in testicular biopsy specimens, unless more mature spermatozoa are present.


    Associated procedures
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Micromanipulation procedures can also be used to analyze and select embryos with specific genetic, chromosomal, or biochemical characteristics before the transfer of those embryos. Analysis of the embryo is performed at the four- or eight-cell stage by extracting an individual cell (blastomere) 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 sex selection to avoid transmission of 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 (62). These micromanipulation techniques are highly labor intensive and carry some potential pitfalls. For example, if both male and female partners 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. However, differential amplification of either the normal or mutated allele may result in a false positive or negative result by preimplantation diagnosis (63).

For sex chromosome analysis, this evaluation is more accurately performed (up to 95.5% efficiency) using different colored fluorescent probes (64). 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 before embryo transfer to prevent the potential termination of a fetus later in development. In addition, chromosomal mosaicism is common in embryos, limiting the accuracy of single blastomere biopsy to approximately 80%.


    Summary
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 
Since the first U.S. report of a successful delivery from in vitro fertilization in 1982 (65), progress in the field of assisted reproduction and micromanipulation has 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 yr ago are now obsolete, replaced by even more successful methods. Even nonobstructive azoospermia resulting from maturation arrest or other impairments in germ cell development have been added to the list of treatable factors in male infertility, as sperm can frequently be extracted directly from testicular parenchyma that is aspirated or surgically biopsied. For patients without sperm in the testicular parenchyma, round spermatid or secondary spermatocyte injections are at least theoretically 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 ICSI, which 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? 4) What is the most successful and cost effective approach for the infertile couple with impaired semen parameters?

For couples with male factor infertility, careful evaluation and treatment of the man should be considered before assisted reproduction, including ICSI. Contemporary application of ICSI for severe male factor infertility can allow pregnancy rates up to 52% (33), with ongoing pregnancy and live delivery rates as high as 37% per IVF cycle attempt (27). As long as viable sperm are present in the ejaculate or retrievable from the reproductive tract, then ICSI procedures can be applied.

Received March 1, 1996.

Revised July 11, 1996.

Revised October 18, 1996.

Accepted October 28, 1996.


    References
 Top
 Introduction
 Evaluation of male factor...
 Treatment of male infertility
 Background: in vitro...
 Micromanipulation
 Intracytoplasmic sperm injection
 Indications for ICSI
 Technique of ICSI
 Microinjection
 Results of ICSI
 Factors affecting results of...
 Risks of ICSI
 Birth defects after assisted...
 Application of ICSI: epididymal...
 Associated procedures
 Summary
 References
 

  1. World Health Organization. 1992 WHO Laboratory manual for the examination of human semen and sperm-cervical mucous interaction. Third Edition. Cambridge, UK: Cambridge Press.
  2. Liu DY, Baker HWG: 1992 Tests of human sperm function, and fertilization. in vitro. Fertil Steril. 58:465–483.[Medline]
  3. Reijo R, Tien-Yi L, Salo P, et al. 1995 Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nature Genet10 :383–393.
  4. Ma K, Sharkey A, Kirsch S, et al. 1992 Toward the molecular localization of the AZF locus: mapping of microdeletions in azoospermic men with 14 subintervals of interval 6 of the human Y chromosome. Hum Mol Gen 1:29–33.
  5. Kent-First MG, Kol S, Muallem A, Blazer S, Itskovitz-Eldor J. 1996 Infertility in intracytoplasmic-sperm-injection-derived sons. Lancet 348:332.
  6. Chandley AC, Hargreave TB. 1996 Genetic anomaly and ICSI. Hum Reprod. 11:930–931.[Medline]
  7. Schlegel PN, Shin D, Goldstein M. 1996 Urogenital anomalies in men with congenital absence of the vas deferens. J Urol. 155:1644–1648.[CrossRef][Medline]
  8. Jarvi K, Zielinski J, Wilschanski M, et al. 1995 Cystic fibrosis transmembrane conductance regulator and obstructive azoospermia. Lancet 345:1578.
  9. Johnson W. 1975 120 Infertile men. Br J Urol47 :230.
  10. Greenberg SH, Lipshultz LI, Wein AJ. 1978 Experience with 425 subfertile male patients. J Urol. 119:507–510.[Medline]
  11. Dubin L, Amelar RD. 1971 Etiologic factors in 1294 consecutive cases of male infertility. Fertil Steril 22:469–474.
  12. Sigman M, Howards SS. 1992 Male infertility. In Walsh PC, Retik AB, Stamey TA, Vaughan ED Jr., eds., Campbell’s urology, 6th ed. Philadelphia: W.B. Saunders; Chapter. 15:661–705
  13. Pavlovich CP, Schlegel PN. 1997 Fertility options after vasectomy: A cost effectiveness analysis. Fertil Steril. 67:133–141.[CrossRef][Medline]
  14. Schlegel PN. 1997 Is assisted reproduction the optimal treatment for varicocele-associated male infertility? A cost-effectiveness analysis. Urology. 49:83–90.[CrossRef][Medline]
  15. Jarow JP. 1994 Life-threatening conditions associated with male infertility. Urol Clin N Amer. 21:409–415.[Medline]
  16. Acosta A, Kruger T, Swanson RJ, et al. 1988 The role of in vitro fertilization in male infertility. Ann NY Acad Sci. 541:297–309.[Medline]
  17. Cohen J, Edwards RG, Fehilly CB, et al.1985 In vitro fertilization: a treatment for male infertility. Fertil Steril. 43:422–433.
  18. Van Uem JFHM, Acosta AA, Swanson RJ, et al. 1985 Male factor evaluation in in vitro fertilization: Norfolk experience. Fertil Steril. 44:375–383.[Medline]
  19. Davis OK, Rosenwaks Z. 1995 In vitro fertilization. In: Keyes Jr WR, Chang RJ, Rebar RW, Soules MR, eds. Infertility: evaluation and treatment. Philadelphia: W.B. Saunders; pp 759–771.
  20. Cohen J, Malter H, Wright G, et al. 1989 Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated. Hum Reprod. 4:435–442.[Abstract]
  21. Cohen J, Alikani M, Malter HE, et al. 1991 Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm, and embryo morphology. Fertil Steril. 56:696–706.[Medline]
  22. Van Steirteghem AC, Nagy Z, Joris H, et al. 1993 High fertilization, and implantation rates after intracytoplasmic sperm injection. Hum Reprod. 8:1061–1066.[Abstract]
  23. Cohen J, Alikani M, Adler A, et al. 1992 Microsurgical fertilization procedures: The absence of stringent criteria for patient selection. J Assist Reprod Genet. 9:197–206.[Medline]
  24. Van Steirteghem AC, Liu J, Joris H, et al. 1993 Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum Reprod. 8:1055–1060.[Abstract]
  25. Palermo G, Joris H, Devroey P, et al. 1992 Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 340:17.[Medline]
  26. Schlegel PN. 1994 Techniques of assisted reproduction. In: Bahnson RR, ed., Management of urologic disorders. Chicago: Mosby-Year Book; 28.
  27. Palermo GD, Cohen J, Alikani M, Adler A, Rosenwaks Z. 1995 Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril. 63:1231–1240.[Medline]
  28. Cummins JM, Jequier AM. 1995 Concerns and recommendations for intracytoplasmic sperm injection (ICSI) treatment. Hum Reprod. 10[Suppl 1]:138–143.
  29. Nagy ZP, Liu J, Joris H, et al. 1995 The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum Reprod. 10:1123–29.[Abstract]
  30. Oehninger S, Veeck L, Lanzendorf S, Maloney M, Toner J, Muasher S. 1995 Intracytoplasmic sperm injection: achievement of high pregnancy rates in couples with severe male factor infertility is dependent primarily upon female not male factors. Fertil Steril. 64:977–981.[Medline]
  31. Sherins RJ, Thorsell LP, Dorfmann A,et al. 1995 Intracytoplasmic sperm injection facilitates fertilization even in the most severe forms of male infertility: pregnancy outcome correlates with maternal age and number of eggs available. Fertil Steril.64:369–375.
  32. Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. 1995 Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril. 64:382–391.[Medline]
  33. Tesarik J, Sousa M. 1995 Key elements of a highly efficient intracytoplasmic sperm injection technique: Ca2+ fluxes and oocyte cytoplasmic dislocation. Fertil Steril. 64:770–776.[Medline]
  34. Gerris J, Mangelschots, Van Royen E, Joostens M, Eestermans W, Ryckaert G. 1995 ICSI and sever male-factor infertility: breaking the sperm tail prior to injection. Hum Reprod. 10:484–486.[Medline]
  35. Palermo GD, Schlegel PN, Colombero LT, Zaninovic N, Moy F, Rosenwaks Z. 1996 Aggressive sperm immobilization in ICSI using epididymal and testicular spermatozoa improves fertilization and pregnancy rates. Hum Reprod. 11:1023–1029.[Abstract]
  36. Dozortsev D, Rybouchkin A, De Sutter P, Dhont M. 1995 Sperm plasma membrane damage prior to intracytoplasmic injection: a necessary condition for sperm nucleus decondensation. Hum Reprod. 10:2960–2964.[Abstract]
  37. Parrington J, Swann K, Shevchenko VI, Sesay AK, Lai FA. 1996 Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature. 379:364–368.[CrossRef][Medline]
  38. Van den Bergh M, Bertrand E, Englert Y. 1994 Intracytoplasmic single sperm injection: preclinical training and first clinical results. J Assist Reprod Genet. 11:289–294.[Medline]
  39. Palermo GD, Alikani M, Bertoli M, Colombero LT, Moy F, Cohen J, Rosenwaks Z. 1996 Oolemma characteristics in relation to survival and fertilization patterns of oocytes treated by intracytoplasmic sperm injection. Human Reprod. 11:172–176.[Abstract]
  40. Schenker JG. 1993 Prevention and treatment of ovarian hyperstimulation. Hum Reprod. 8:653–659.[Medline]
  41. Mozes M, Bogowsky H, Anteby E, et al. 1965 Thromboembolic phenomena after ovarian stimulation with human menopausal gonadotrophin. Lancet. 2:1213.[CrossRef][Medline]
  42. Bergh T, Lundkvist O. 1992 Clinical complications during in vitro fertilization treatment. Hum Reprod. 7:625–626.[Abstract]
  43. Schenker J, and Ezra Y. 1994 Complications of assisted reproductive techniques. Fertil Steril. 61:411–422.[Medline]
  44. Bennett SJ, Waterstone JJ, Cheng WC, Parsons J. 1993 Complications of transvaginal ultrasound-directed follicle aspiration: A review. J Assist Reprod Genet. 10:72–77.[Medline]
  45. Wilcox LS, Peterson HB, Haseltine FP, Martin MC. 1993 Defining and interpreting pregnancy success rates for in vitro fertilization. Fertil Steril. 60:18–25.[Medline]
  46. Dubuisson JB, Aubriot FX, Mathieu I, et al. 1991 Risk factors for ectopic pregnancy in 556 pregnancies after in vitro fertilization: implications for preventive management. Fertil Steril. 56:686–690.[Medline]
  47. Seoud MAF, Toner JP, Kruithoff C, Muasher SJ. 1992 Outcome of twin, triplet, and quadruplet in vitro pregnancies: the Norfolk experience. Fertil Steril. 57:825–834.[Medline]
  48. Tan SL, Dolye P, Campbell S, et al. 1992 Obstetric outcome of the in vitro fertilization pregnancies compared with normally conceived pregnancies. Am J Obstet Gynecol. 167:778–784.[Medline]
  49. Australian In Vitro Fertilization Collaborative Group. 1988 In vitro fertilization pregnancies in Australia and New Zealand 1979–85. Med J Aust. 148:429–436.[Medline]
  50. Assisted reproductive technology in the United States and Canada. 1995 1993 results generated from the Amercian Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril. 64:13–21.[Medline]
  51. Van Steirteghem AC. Breaching the zona pellucida: Recent advances in micromanipulation. Presented at the Annual meeting of the American Society for Reproductive Medicine, 1995, Seattle, WA.
  52. Veld P, Brandenburg H, Verhoeff A, Dhont M, Los F. 1995 Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet. 346:773.[CrossRef][Medline]
  53. Liebaers I, Bonduelle M, Van Assche E, et al. 1995 Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet. 346:773.[CrossRef][Medline]
  54. Govaerts I, Englert Y, Vamos E, Rodesch F. 1995 Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet. 346:1095.[CrossRef]
  55. Schlegel PN, Palermo GD, Alikani M, et al. 1995 Micropuncture retrieval of epididymal sperm with IVF: importance of in vitro micromanipulation techniques. Urology. 46:238–241.
  56. Silber SJ, Nagy ZP, Liu J, Godoy H, Devroey, Van Steirteghem AC. 1994 Conventional in vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum Reprod9 :1705–1709.
  57. Tournaye H, Liu J, Nagy PZ, et al. 1996 Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum Reprod. 11:127–132.[Abstract]
  58. Devroey P, Liu J, Nagy Z, et al. 1995 Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in nonobstructive azoospermia. Hum Reprod. 10:1457–1460.[Abstract]
  59. Schlegel PN, Palermo GD, Goldstein M, Menendez S, Zaninovic N, Rosenwaks Z. Testicular sperm extraction with ICSI for non-obstructive azoospermia. Urology. In press.
  60. Kimura Y, Yanagimachi R. 1995 Development of normal mice from oocytes injected with secondary spermatocyte nuclei. Biol Reprod. 53:855–862.[Abstract]
  61. Tesarik J, Mendoza C, Testart J. 1995 Viable embryos from injection of round spermatids into oocytes. N Engl J Med. 333:525.[Free Full Text]
  62. Grifo JA, Tang YX, Cohen J, Gilbert F, Sanyal MK, Rosenwaks Z. 1992 Pregnancy after embryo biopsy and coamplification of DNA from x and Y chromosomes. JAMA. 268:727–729.[CrossRef][Medline]
  63. Liu J, Lissens W, Devroey P, et al. 1992 Efficiency and accuracy of polymerase-chain-reaction assay for cystic fibrosis allele delta F508 in single cell. Lancet. 339:1190–1192.[CrossRef][Medline]
  64. Munne S, Weier HU, Stein J, Grifo J, Cohen J. 1993 A fast and efficient method for simultaneous X and Y. in situ hybridization of human blastomeres. J Assist Reprod Genet. 10:82–90.[Medline]
  65. Jones Jr HW, Jones GS, Andrews MC, et al. 1982 The program for in vitro fertilization at Norfolk. Fertil Steril. 38:14–21.[Medline]
  66. Van Steirteghem AC, Joris H, Liu J, et al. 1994 Evolution of intracytoplasmic (ICSI) results. Fertil Steril. 63:S83.
  67. Harari O, Bourne H, McDonald M, et al. 1995 Intracytoplasmic sperm injection: a major advance in the management of severe male subfertility. Fertil Steril. 64:360–368.[Medline]
  68. Payne D, Flaherty SP, Jeffrey R, et al. 1994 Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection. Hum Reprod. 11:2051–2057.
  69. Tsirigotis M, Yang D, Redgment CJ, et al. 1994 Assisted fertilization with intracytoplasmic sperm injection. Fertil Steril. 62:781–785.[Medline]