The introduction of intracytoplasmic sperm injection. (ICSI) in 1992 has ... available for most patients with testicular or idiopathic causes of male infertility. These .... (gonorrhea, chlamydia). Drugs (e.g. cytotoxic drugs, alkylating agents, alcohol,.
0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society
Vol. 86, No. 6 Printed in U.S.A.
COMMENTARY Reproductive Technologies for Male Infertility O. KHORRAM, P. PATRIZIO, C. WANG,
AND
R. SWERDLOFF
Departments of Obstetrics and Gynecology (O.K.) and Internal Medicine (C.W., R.S.), Harbor–UCLA Medical Center, Torrance, California 90509; and Department of Obstetrics and Gynecology (P.P.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
The introduction of intracytoplasmic sperm injection (ICSI) in 1992 has revolutionized the treatment of male infertility (1) and has allowed couples whose only prior options were donor insemination to achieve pregnancies or adoption. Although in vitro fertilization (IVF) and related procedures have been used in the past for the treatment of male infertility, most assisted reproductive technology centers are using ICSI as the primary treatment for male infertility. Table 1 lists the cause of male factor infertility. Only a small minority of such patients are amenable to specific hormonal or pharmacologic therapy. Of these, the most probable candidates are the 1–2% of men with male infertility secondary to hypothalamic-pituitary (gonadotropin) insufficiency. These patients respond well to gonadotropin or GnRH therapy. Specific medical treatment is not available for most patients with testicular or idiopathic causes of male infertility. These patients are candidates for assisted reproductive technologies using oocytes from their spouses or partners. This article reviews the ICSI procedure, its indications, and techniques used to retrieve sperm and the risks and concerns that have been raised in regard to its safety, including the genetic risks to the fetus. Hormonal testing, semen analysis, and sperm function test
In the assessment of a patient with male infertility, a medical history, complete physical examination, and serum testosterone measurement are performed to exclude androgen deficiency and other specific etiologies; a serum FSH level is also measured as a marker of the severity of spermatogenic dysfunction. An elevated FSH generally indicates severe seminiferous epithelium damage and is a poor prognostic sign. Examination of the semen remains the cornerstone for the diagnosis of male infertility. In most laboratories, semen analyses are done with manual methods. The reference procedures are described in the World Health Organization Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction (2). These methods are recommended for use for most andrology laboratories. Computer-assisted semen analyses, when used carefully and with Received February 16, 2001. Accepted March 16, 2001. Address correspondence and requests for reprints to: Ronald Swerdloff, M.D., Division of Endocrinology, Department of Internal Medicine, Harbor–UCLA Medical Center, 1000 West Carson Street, Torrance, California 90509.
adequate quality control, provide a useful alternative to manual methods for analyzing the sperm concentration and motility and will yield additional sperm movement parameters (2). Studies have shown that some of these motility characteristics have predictive value on sperm’s ability to fertilize human oocytes. The development and change in the method of assessment of sperm morphology by the “strict criteria” of Kruger et al. (3) has been helpful in standardization of the morphology parameter. Morphology based on the strict criteria has been reported to have the advantage of predicting the outcome of IVF, with ejaculates showing 4% or less normal forms being associated with decreased rates of fertilization and pregnancy (3). In contrast to the experience with IVF, sperm morphology is not predictive of ICSI outcome, although the characteristics of sperm morphology may be important. When the percentage of strict normal morphology is 4% or less, the fertilization rate in ICSI is lower in the case of severely tapered heads, compared with other deformities (4). Chromosomal aberrations in different types of sperm may account for these differences in fertilization, because smallor large-headed sperm may not be associated with chromosomal abnormalities, whereas the incidence of structural abnormalities is higher in spermatozoa with amorphous and elongated heads (5). There has been a significant effort in developing sperm function tests, which could predict fertilization in vitro. This information may be useful particularly in cases where it is unclear whether IVF or ICSI should be performed. The majority of these tests evaluate the ability of sperm to undergo the acrosome reaction (spontaneously or after stimulation) (2), to penetrate and fuse with a zona-free hamster egg (2), and to bind the zona pellucida either directly (hemizona bioassay) (6) or indirectly using cytochemical probes to detect sperm surface receptors for zona ligands (7). Since the introduction of the ICSI procedure for the treatment of male infertility, the value of these zona penetration and binding tests in the clinical management of a patient with male infertility has been greatly reduced. ICSI procedure
For ICSI, oocytes (obtained from the ovarian follicles through ultrasound-guided transvaginal needle aspiration) in the metaphase-II stage are first prepared by removing the
2373
2374
KHORRAM ET AL.
TABLE 1. Causes of infertility in men Hypothalamic-pituitary disorders (GnRH, LH and FSH deficiency) Congenital Congenital GnRH deficiency (Kallmann’s syndrome) Hemochromatosis Multiorgan genetic disorders (e.g. Prader-Willi syndrome, Laurence-Moon-Beidl syndrome, familial cerebral ataxia) Acquired disorders Pituitary and hypothalamic tumors (macroadenoma, craniopharyngioma) Infiltrative disorders (sarcoidosis, histiocytosis, tuberculosis, fungal infections) Trauma, postsurgical, postirradiation Vascular (infarction, aneurysm) Hormonal (hyperprolactinemia, androgen excess, estrogen excess, cortisol excess) Drugs (opioids and psychotropic drugs, GnRH agonists or antagonists) Systemic disorders Chronic illnesses Nutritional deficiencies Obesity Primary gonadal disorders Congenital disorders Klinefelter’s syndrome (XXY) and its variants (XXY/XY; XXXY) Cryptorchidism Myotonic dystrophy Functional prepubertal castrate syndrome (congenital anorchia) Varicocele Androgen insensitivity syndromes 5 ␣ reductase (type 2) deficiency Y chromosome deletions Acquired disorders Viral orchitis (mumps, echovirus, arbovirus) Granulomatous orchitis (leprosy, tuberculosis) Epididymo-orchitis (gonorrhea, chlamydia) Drugs (e.g. cytotoxic drugs, alkylating agents, alcohol, marijuana, antiandrogens, ketoconazole, spironolactone, histamine receptor antagonists) Ionizing radiation Environmental toxins (e.g. dibromochloropropane, carbon disulfide, cadmium, lead, mercury, environmental estrogens, phytoestrogens) Hyperthermia Immunological, including polyglandular autoimmune disease Trauma Torsion Castration Systemic illness (e.g. renal failure, hepatic cirrhosis, cancer, sickle cell disease, amyloidosis, vasculitis, coeliac disease) Disorders of sperm transport (posttesticular) Epididymal dysfunction (drugs, infection) Abnormalities of the vas deferens (congenital absence, Young’s syndrome, infection, vasectomy) Ejaculatory dysfunction (spinal cord disease, autonomic dysfunction, premature ejaculation) Unexplained male factor infertility
cumulus mass and corona radiata with hyaluronidase. A single sperm obtained from the ejaculate, or epididymis or testis, is then directly injected via a micropipette (inner diameter of 6 –7 m) through the zona pellucida and oolema at the equatorial level into the cytoplasm of an oocyte that has been immobilized in a droplet of medium under oil. During the injection the oocyte cytoplasm is aggressively aspirated and injected to cause oocyte activation. Such cytoplasmic aspiration was found to improve fertilization and pregnancy
JCE & M • 2001 Vol. 86 • No. 6
rates (8). Because the spermatozoa also contribute to oocyte activation, immobilization of the spermatozoa is induced by mechanical crushing of the sperm tail between the injection micropipette and the bottom of a Petri dish. This maneuver, which increases the fertilization and pregnancy rates (9), is particularly useful in the cases of epididymal and testicularderived sperm (10). In experienced centers, ICSI results in oocyte damage and/or oocyte death in no more than 10% of cases. This damage could be a result of injury of the meiotic spindle or extrusion of the oocyte cytoplasm following injection (11). Indications for ICSI
Different groups have published different criteria for patients who would benefit from ICSI. These criteria include abnormalities detected on semen analysis, such as severe oligozoospermia (⬍2–10 ⫻ 106 sperm/mL), severe asthenozoospermia (⬍5–10% motile spermatozoa), poor sperm morphology (⬍4% normal oval forms), use of surgically retrieved spermatozoa, and failed fertilization in a previous IVF cycle. Relative indications include antisperm antibodies or poor fertilization in a prior IVF cycle (12, 13). In addition, a recently proposed indication independent of severe male infertility is when a low number of oocytes are retrieved for other assisted reproduction techniques (14). Many reports indicate that fertilization and pregnancy rates in ICSI are comparable with IVF for tubal obstruction. The Belgian group who developed ICSI in the human reported a normal fertilization rate of 65.6% in ICSI; this outcome rate was similar to that in IVF. Abnormal fertilization occurred as one pronuclear oocyte in 2.8% and three pronuclei in 3.7% of injected metaphase II oocytes (15). A survey of the world literature, in 1995, reported an average fertilization rate of 64% with ejaculated sperm, 62% with epididymal sperm, and 52% with testicular sperm (11). Clinical pregnancy rates from the latest database (1998) published by the Society of Assisted Reproductive Technology showed live birth rates per embryo transfer of 33.2% for IVF and 32.2% for ICSI (16). Use of spermatozoa precursors for ICSI
Initial animal studies showed that injection of round spermatid nuclei into hamster oocytes formed pronuclei that could participate in syngamy (17), and normal offspring were reported after injection of cryopreserved round spermatid in mice (18). These encouraging results led to use of sperm precursors either collected from the ejaculate or testicular tissue for human ICSI. Elongating and elongated mature spermatids were found to produce better fertilization rates (54%) as compared with round spermatids (17%) (19). Based on this observation, investigators have cultured round spermatids until they develop a short tail before use in ICSI (20). In general, pregnancy rates are dramatically lower using spermatids compared with mature sperm. Despite this fact, a number of human births using either round or elongated spermatids have been reported (21–23). A number of concerns have been raised with the use of sperm precursors: 1) accurate identification and classification of the precursor cells are difficult, and wide differences
COMMENTARY
in fertilization ability are dependent on the phases of development of the germ cells; 2) the identification and isolation of live round spermatids from the other types of cells present in the ejaculate or in testicular tissue may be difficult; and 3) the issue of viability or genomic normality of these sperm precursors may limit outcome and encourage dysmorphic fetuses (19). In a recent case report on four pregnancies resulting from the use of spermatids for ICSI, two cases of major malformations occurred with one of the fetuses having trisomy 9. The authors raised caution on the use of spermatids for ICSI and emphasized the need for extensive counseling of the couple (24). Sperm retrieval techniques
Epididymal spermatozoa retrieval. The main indication for epididymal sperm retrieval is obstructive azoospermia, caused by different disorders (Table 2). In cases of nonobstructive azoospermia, epididymal aspiration should not be attempted because the epididymal lumen is collapsed and spermatozoa cannot be retrieved. Currently, there are two methods to obtain epididymal spermatozoa: microsurgical epididymal sperm aspiration (MESA) (25) and percutaneous epididymal sperm aspiration (PESA) (26). Most of the literature on epididymal sperm has focused on the technique of MESA based on the claim that the amount of sperm retrieved with PESA may not be enough for cryopreservation, thus limiting the overall efficacy of this method. However, as experience with PESA accumulates and the method is much less invasive and expensive, it is now considered the preferred method to obtain epididymal spermatozoa. MESA: surgical technique. The patient may be under general or regional anesthesia. A scrotal longitudinal incision of approximately 2.5 cm is carried down to the underlying tunica vaginalis. After exposing the epididymis, at a magnification varying between ⫻6 and ⫻40, a tubule of the caput epididymis is longitudinally incised. The outflowing fluid is collected in a 22 Medicut attached to a tuberculin syringe, prefilled with small aliquots of medium to avoid the risk of drying out the sample. Each aspirate is handed to the biologist to assess for quantity and quality of sperm. This information will aid the surgeon on whether to continue the aspiration in the same place or move to a different segment. At times, it is necessary to perform aspirations from the vasa efferentia. If no sperm are found in the vasa efferentia, testicular sperm extraction should follow [see testicular sperm extraction (TESE)]. Epididymal sperm retrieval is completed once motile (even twitching) spermatozoa are found. PESA: surgical technique. The technique of PESA is extremely simple, and many centers have now adopted it, replacing the more invasive MESA. The patient requires conscious sedaTABLE 2. Indications for MESA and PESA Bilateral congenital CAVD Cystic fibrosis Vasectomy or failed vasectomy reversal Inoperable ejaculatory ducts or distal vasal obstruction Postinflammatory obstructions (Tbc, gonorrhea, chlamydia, etc.) Radical cysto-prostatectomy
2375
tion and/or only spermatic cord block (0.5% Marcaine). The testis is immobilized with one hand while the aspiration is carried out with a butterfly needle connected to a 20-cc plastic syringe, inserted through the scrotum directly into the proximal caput of the epididymis. An assistant is required to pull the syringe plunger to create a negative pressure. If the tip of the needle is properly positioned, epididymal fluid will be seen flowing within the plastic tubing of the butterfly needle. In the laboratory, the needle is flushed four to five times in a Petri dish and a drop of this solution is examined under the microscope to check for motile sperm. The epididymal fluid may contain very few or many spermatozoa (sperm counts can fluctuate from few thousands up to 200 millions). Generally, two to three aspirates is all that is needed, but, if no sperm are found, six to eight aspirates from each side should be carried out before switching to testicular sperm aspiration (TESA) or TESE. Testicular spermatozoa retrieval (TESA and TESE). The indications to obtain testicular spermatozoa are listed in Table 3. There are two techniques to harvest testicular spermatozoa: TESA and TESE (27). The first is carried out with the use of a needle, usually 21 gauge or 19 gauge, whereas the second uses a sample of testicular biopsy from which sperm are extracted in vitro and used for ICSI. Currently, no clinical tests are able to predict reliably for the presence of sperm in the testes of patients with azoospermia in their ejaculate before resorting to the aspiration or biopsy. This creates difficult clinical decisions: should a testicular biopsy precede an IVF-ICSI cycle or should it be performed in conjunction with ICSI? It is possible to have conditions in which the majority of the seminiferous tubules have a histological pattern of Sertoli cell only, and yet few tubules may show severely reduced spermatogenesis. In these instances, the testes are small, the FSH level is high, the patient is azoospermic, but there is still a 30 – 40% chance that TESA or TESE will be successful in retrieving sperm. In other instances, azoospermic patients with normal or borderline elevated FSH and normal size testis may have complete maturation arrest or true Sertoli cell only, in which case TESA or TESE will be ineffective. The use of molecular probes for meiosis provide a screening tool to identify differentiated germ cells. Absence of these markers indicates that no sperm can be retrieved from the testis (28). Testicular sperm can be retrieved by open or excisional biopsy, TESE, or by fine-needle TESA. For the open biopsy, a 1-cm transversal incision is carried through the tunica vaginalis down to the tunica albuginea, trying to obtain tissue from the midanterior surface of the testis. Gentle pressure is used to extrude testicular seminiferous tissue. The protruding seminiferous tubules are excised with scissors TABLE 3. Indications for TESA and TESE Nonobstructive azoospermia (maturation arrest, severe hypospermatogenesis, incomplete sertoli cell only) Obstructive azoospermia (rete testis blockage, no sperm in the epididymis, absent epididymis, extensive scarring) Anejaculation (not responding to electroejaculation or vibrostimulation) Complete terato/necrozoospermia Complete sperm immobility
2376
KHORRAM ET AL.
and transferred to a Petri dish containing 1 mL human tubal fluid (HTF)-buffered medium. A single biopsy is generally sufficient for the ICSI procedure and for freezing any excess testicular spermatozoa. However, in cases of incomplete Sertoli cells only or incomplete maturation arrest, up to three to four biopsies may be required. Another approach, recently used in conjunction with the open biopsy, is the use of a microscope for selecting the seminiferous tubules to be excised (micro TESE). The assumption is that the seminiferous tubules containing spermatogenetic activity will appear more dilated (29). The closed technique (TESA) uses a transcutaneous aspiration by inserting a needle directly in the testicular parenchyma and using negative pressure. The number of passes through the testicular tissue may vary from 1 to 8 or 10. In the laboratory, the testicular tissue is finely minced in HTFHEPES-buffered medium, and after centrifugation the pellet is suspended in culture media and examined for free testicular sperm. Testicular sperm show initially very low motility (flagellar twitching), which improves over time (10 –12 h). Other investigators have reported that testicular sperm motility improves with extended time (up to 3 days) in culture (30). Seminal tract washout (STW)
Some forms of male infertility are due to incomplete voiding of the distal seminal tract, and spermatozoa can be retained anywhere downstream of the epididymis. The most common of these are those resulting from ejaculatory duct incomplete obstructions, either secondary to the presence of intraprostatic mu¨llerian cysts between the two ejaculatory ducts, narrowing of the ejaculatory ducts after inflammation, or secondary to functional emptying disturbances of the ampullo-vesicular tract due to diabetes, spinal cord injury, extended retroperitoneal lymph node dissection or idiopathic origin (see Table 4). In these instances the technique of STW (31, 32) may be useful particularly when either electroejaculation or vibro-stimulation may fail. The technique involves the cannulation of the vas deferens and the subsequent antegrade washing of the vas and collection of sperm from the bladder. The operating time is about 20 min, and the patient can go home in 1 h. At times, it is difficult to cannulate the vas and, thus, a hemi-vasotomy is required. The vas is then reapproximated by using microsurgical suture. Cryopreservation and thawing of retrieved spermatozoa
Because the post-thaw recovery of motile epididymal sperm is optimized when the specimen is processed before cryopreservation, either washing or gradients for filtration are recommended. After sufficient sperm for ICSI have been set aside, the remainder of the specimen is pooled and proTABLE 4. Indications for STW Spinal cord injury (paraplegia, spina bifida) Ejaculatory duct subobstruction (mu¨llerian cysts, postinflammatory) S/P retroperitoneal lymph node dissection Anejaculation (absence of retrograde ejaculation) Postmortem collection
JCE & M • 2001 Vol. 86 • No. 6
cessed. Washed specimens are first concentrated and then diluted 1:1 with freezing medium (TEST-yolk buffer with glycerol). Buffer media containing glycerol provide the most effective recovery of motile sperm after cryopreservation (33, 34). For the thawing the cryo-vial is brought to room temperature or to 37 C, diluted with HTF-HEPES-buffered medium (Irvine Scientific, Santa Ana, CA), and washed once to remove the cryoprotectant. The pellet is then resuspended in a small aliquot of medium from which the motile or twitching epididymal sperm are isolated for the ICSI procedure. Similar procedures are used for testicular tissue, except the tissue must first be macerated and minced (33, 34). Testicular tissue can be frozen either in stepwise fashion manually or by using the Planer Kryo lll apparatus (T. S. Scientific, Perkasie, PA; Ref. 34). When the number of testicular spermatozoa is extremely small, the procedure of single sperm freezing has been advocated (35). This technique requires cell-free human zona pellucida where sperm (up to five) and cryoprotectant (8% glycerol solution in phosphate-buffered saline supplemented with 3% human serum albumin) are inserted into liquid nitrogen. The cell-free zona are loaded separately in 0.25-mL straws, exposed to nitrogen vapor overnight, and plunged the next day. Genetics and male infertility: implications for ICSI
A significant proportion of infertile males with azoospermia and severe oligozoospermia have a genetic etiology for their reproductive failure. The three most common genetic factors related to male infertility are cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations leading to congenital absence of the vas deferens (36 –38), Y-chromosome microdeletions in the azoospermia factor locus (39, 40) leading to spermatogenic impairment, and karyotype abnormalities (e.g. Klinefelter’s syndrome; Ref. 41). Currently, among men with bilateral congenital absence of the vas deferens (CAVD), 80% have at least one allele mutated in the CFTR gene. Before resorting to assisted reproduction, both partners of the infertile couple need to have CF testing, including a particular CFTR variant, the IVS8-5T, which is common among men with CAVD as opposed to patients with the classical form of CF. About 20% of men with CAVD do not have identifiable CFTR mutations, and they may have either unidentified mutations or a different etiology. Among all azoospermic men, the frequency of chromosomal abnormalities is estimated to be around 16% (41), of which 13% are represented by Klinefelter’s syndrome (47 XXY, XY/XXY mosaics and other sex chromosome aneuploidies) and the remaining 3% by other chromosomal aberrations, such as ring Y chromosome and translocations. Among men with oligozoospermia (sperm count ⬍20 million/mL), 5– 6% have karyotype anomalies, mostly autosomal Robertsonian and reciprocal translocations. Even when the somatic karyotype seems normal, men with impaired spermatogenesis may produce sperm with a high frequency of aneuploidy. In these instances, the use of ICSI with disomic sperm will lead to the production of trisomic embryos. Testicular sperm and sperm from men with extremely severe
COMMENTARY
oligoasthenoteratozoospermia were recently found to have significantly higher frequency of aneuploidy and diploidy (42). The future availability and applicability of DNA probes for the entire set of chromosomes would limit the risk of paternal origin of karyotype anomalies in ICSI offspring. The significance of the Y chromosome in regulating spermatogenesis has recently been recognized. Several groups have reported that up to 5– 6% of men with severe oligozoospermia and 10 –20% with azoospermia have microdeletions within a region of the Y chromosome known as DAZ (deleted in azoospermia) gene (43, 44). This deletion can be passed on to their male offspring (45). Therefore, these couples should receive counseling before proceeding with ICSI, as testing for Y chromosome deletion is now available at many centers and a test kit has been developed by Promega Corp. (Madison, WI) for this purpose. In counseling such couples, it is important to specify that it is uncertain as to what extent a son who inherits a microdeletion will have a fertility problem, and that there are no other known health consequences of Y microdeletions. Androgens, mainly testosterone and 5-␣-dihydrotestosterone, are essential regulators of human spermatogenesis. Their action is mediated by the androgen receptor (AR), a DNA-binding transcription factor protein encoded by a gene located on chromosome Xq11–12. Two highly polymorphic CAG and GGN microsatellite repeats are present in exon 1 of the AR gene. An expansion of the CAG microsatellite repeat to greater than 40 repeats is the causative AR mutation in patients with Kennedy’s disease, an X-linked form of spinobulbar muscular atrophy, with onset in the third decade of life. These patients also become infertile due to testicular atrophy resulting in marked oligozoospermia. Previous studies examining the number of CAG repeats in the AR gene of infertile males with unexplained oligozoospermia have reported conflicting results, with some (46, 47) showing no expansions or gross deletions of trinucleotide repeats within exon 1 of the AR gene, and others (48, 49) reporting increased trinucleotide repeat size. The study by Dadze et al. (47) pointed out ethnic differences as a possible explanation for the contradictory findings. A very recent study (50) assessed the length of the AR CAG repeats in North American Caucasian infertile males with severely disturbed spermatogenesis compared with proven fertile controls. Overall, the mean number of CAG repeats was found to be significantly greater in men with extremely severe oligozoospermia (sperm count ⱕ1 million/mL) than controls. These minor, but statistically significant, deviations from the norm in the number of the CAG repeats, could represent one genetic alteration in a multifactorial set of genetic polymorphisms or mutations that may lead to male infertility. This finding is of great interest because the expansion of the same trinucleotide repeat (in excess of 38 – 40 repeats) is responsible for a neuromuscular disease, spino-bulbar muscular atrophy, or Kennedy’s disease, which appears later in life and is associated with a severe reduction in sperm count. Because the AR gene is on the X chromosome, using ICSI to treat patients with extremely severe oligozoospermia and intermediate CAG trinucleotide repeats has the potential risk for transmitting Kennedy’s disease in two generations. The mechanism of transmission of this fatal neuromuscular disease will
2377
involve first an expansion of the CAG repeat, with transmission to a daughter who will be a carrier (first generation), and then the subsequent risk (50%) of transmission of the expanded CAG repeat to a son (second generation) who will be affected (50). ICSI concerns
There has been controversy concerning the risks associated with ICSI. The question raised is whether the risks associated with ICSI are due to the procedure itself or a consequence of using severely defective sperm that may be genetically abnormal. Karyotypic analysis of children born by ICSI has shown increased incidence of sex chromosome aneuploidy, specifically the absence of an X chromosome or the presence of an extra X or Y chromosome (51, 52). The prevalence of sex chromosome aneuploidy was reported to be 0.83% in children conceived by ICSI based on 1082 prenatal chromosome studies from Belgium compared with a prevalence of 0.2% in the general population (52). Loft et al. (53) did not find an increase in sex chromosome abnormalities in 730 children born after ICSI but found a 3.3% incidence of autosomal abnormalities. An increase in the incidence of de novo chromosomal structural abnormalities (0.36% compared with 0.07% general population), especially when the sperm used showed extreme oligo-astheno-teratozoospermia, was also found in ICSI offspring (52). Van Opstal et al. (54) reported that in all cases of sex chromosome anomalies, the abnormality was of paternal origin. These chromosomal anomalies in the offspring may be secondary to increased incidence of sex chromosome abnormalities in sperm of men needing ICSI (55). However, the presence of abnormal paternal karyotype does not necessarily lead to karyotypically abnormal offspring, and several groups have reported on healthy offspring born after ICSI with sperm from men with nonmosaic Klinefelter’s syndrome (56, 57). Most reports, to date, have not shown an increase in major congenital malformations in children born by ICSI (2–3%) when compared with children born by IVF or the general population (15, 58, 59). Most of the malformations found in ICSI offspring have been attributed to prematurity secondary to multiple births. One specific defect found more frequently is hypospadias, which may be a result of the association between paternal subfertility and hypospadias (59). Kurinczuk and Bower (60) recently reclassified birth defects reported in infants born by ICSI (in Belgium) and compared it with the prevalence of malformation in children born in Western Australia during the same period. They reported that infants born by ICSI were twice as likely to have a major birth defect and 50% more likely to have a minor defect. They found an excess of major cardiovascular defects, genitourinary defects, and gastrointestinal defects in these children (60). Additional studies to address the discrepancy between these reports are needed. Several studies have addressed differences in cognitive development of children born after ICSI. Bowen et al. (61), comparing 1-yr-old children conceived through ICSI, IVF, and natural conception, reported decreased average standardized mental developmental index scores in children conceived by ICSI compared with other groups. They reported
2378
KHORRAM ET AL.
that 17% of these children had significantly delayed development (61). In contrast, two other studies (62, 63) have not shown a significant developmental difference in children 1–2 yr old born by ICSI as compared with children born by IVF. The reason for this discrepancy is unknown but warrants further studies. The ICSI procedure has been reported to influence embryonic development. These studies showed that the chances for ICSI-derived embryos developing to the blastocyst stage are lower than those derived from IVF (64, 65). The reasons for this are unknown because ICSI-derived embryos do not have higher incidence of numerical chromosomal abnormalities than embryos from IVF (66). One explanation put forth has been that the timing and pattern of calcium transients may be altered in ICSI embryos (65). Because the implantation and pregnancy rates of ICSI embryos is the same as IVF (11, 16), the significance of ICSI on blastulation rates remains to be determined. Summary
ICSI has helped many couples with severe male factor infertility to achieve pregnancies. Even in cases of azoospermia, sperm retrieval techniques from the epididymis or testis have provided mature spermatozoa or spermatids for use in ICSI. Some concerns and unresolved issues still remain with this procedure. These include the need for development of universal criteria for who would benefit from ICSI, and the realization that men with severe defects in semen parameters often have sperm with genetic abnormalities that could potentially be transmitted to their offspring. Such couples must be provided with updated information and counseling regarding the prognosis and advised about the availability of prenatal fetal diagnosis (67). In addition, the long-term consequences of ICSI such as effects on later development are far from being resolved; the use of sperm precursor cells may result in potential birth defects and genetic abnormalities; and ICSI is not available or accessible for all couples who need the procedure for their infertility because of social, religious or financial reasons. Couples undergoing this technique should be informed about these issues before proceeding. These concerns also indicate that this procedure should not be used indiscriminately. Although ICSI is a very effective treatment of male infertility, the causes of idiopathic infertility need to be further investigated and noninvasive, and more specific treatments should be developed that may not carry the same potential risks and costs as ICSI. References 1. Palermo G, Joris H, De P, et al. 1992 Pregnancies after intracytoplasmic injection of single spermatozoa into an oocyte. Lancet. 340:17–18. 2. World Health Organization Laboratory. 1999 Manual for the examination of human semen and sperm-cervial mucus interaction, ed 4. Cambridge: Cambridge University Press. 3. Kruger TF, Menkveld R, Stander FSH, et al. 1986 Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril. 46:1118 –1123. 4. Osawa Y, Sueoka K, Iwata S, et al. 1999 Assessment of the dominant abnormal form is useful for predicting the outcome of intracytoplasmic sperm injection in the case of severe teratrozoospermia. J Assist Reprod Genet. 16:436 – 442. 5. Lee JD, Kamiguchi Y, Yan Agimachi R. 1996 Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection in mouse oocytes. Hum Reprod. 11:1942–1946. 6. Liu DY, Baker HWG. 1992 Tests of human sperm function and fertilization in vitro. Fertil Steril. 58:465– 483.
JCE & M • 2001 Vol. 86 • No. 6
7. Benoff S, Cooper GW, Hurley I, et al. 1993 Human sperm fertilization potential in vitro is correlated with differential expression of a head specific mannose-ligand and receptor. Fertil Steril. 59:854 – 862. 8. Tesarik J, Sousa M. 1995 Key elements of a highly efficient intra cytoplasmic sperm technique and oocyte cytoplasmic dislocation. Fertil Steril. 64:770 –776. 9. Gerris J, Mangelschots K, Van Royen E, Joostens M, Eastermans W, Ryckaert G. 1995 ICSI and severe male-factor infertility: breaking the sperm tail prior to injection. Hum Reprod. 10:484 – 486. 10. Palermo GD, Schlegl PN, Colombero LT, Zaninovic N, Moy F, Rosenwaks Z. 1996 Aggressive sperm immobilization in ICSI using epid testicular spermatozoa improves fertilization and pregnancy rates. Hum Reprod. 1:1023–1029. 11. Tarlatzis BC, Bili H. 2000 Intracytoplasmic sperm injection. Survey of world results. Ann N Y Acad Sci. 900:336 –344. 12. Thornton K. 2000 Advances in assisted reproductive technologies. Obstet Gynecol Clin North Am. 27:517–527. 13. Schlegel PN, Girardi SK. 1997 Clinical review 87: in vitro fertilization for male factor infertility. J Clin Endocrinol Metab. 82:709 –716. 14. Ludwig M, Al-Hasani S, Kupker W, Bauer O, Diedrich K. 1997 A new indication for an intracytoplasmic sperm injection procedure outside the cases of severe male factor infertility. Eur J Obstet Gynecol. 75:207–210. 15. Bondeuelle M, Camus M, De Vos A, et al. 1999 Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum Reprod. 14:243–264. 16. Centers for Disease Control and Prevention. 1998 Society for Assisted Reproductive Technology success rates. National summary and fertility clinic reports; 29. 17. Ogura A, Yanagimachi R. 1993 Round spermatid nuclei injected into hamster oocytes form pronuclei and participate in syngamy. Biol Reprod. 48:219 –225. 18. Ogura A, Matsuda R, Yanagimachi R. 1994 Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc Natl Acad Sci USA. 91:7460 –7462. 19. Schoysman R, Vanderzwalmen P, Berlin G, Nijs M, Van Damme B. 1998 Oocyte insemination with spermatozoa precursors. Curr Opin Urol. 9:541–545. 20. Aslam I, Fishel S. 1998 Short term in vitro culture and cryopresentation of spermatogenic cells used for human in vitro conception. Hum Reprod. 13:634 – 638. 21. Fishel S, Green S, Bishop M, et al. 1995 Pregnancy after intracytoplasmic injection of spermatid. Lancet. 245:1641–1642. 22. Kahraman S, Polat G, Samli M. 1998 Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection. Hum Reprod. 13:104 –110. 23. Tesarik J, Rolet F, Brami C, et al. 1996 Spermatid injection into human oocytes. II. Clinical application in the treatment of infertility due to non-obstructive azoospermia. Hum Reprod. 11:780 –783. 24. Zech H, Vanderzwalmen P, Prapas Y, et al. 2000 Congenital malformations after intracytoplasmic injection of spermatids. Hum Reprod. 15:969 –971. 25. Patrizio P, Silber S, Ord T, Balmaceda JP, Asch RM. 1988 Two births after microsurgical epididymal sperm aspiration. Lancet. 2:1364. 26. Craft I, Tsirigotis M, Bennett V, et al. 1995 Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia. Fertil Steril. 63:1038 –1042. 27. Schlegel P, Su LM, Li PS. 1998 Gonadal sperm retrieval: potential for testicular damage in non-obstructive azoospermia. In: Filicori M, Flamingi C, eds. Princeton Junction, NJ: Communications Media for Education; 383–392. 28. Patrizio P, Ricci MS, Tomaszewski JE, Hecht NB. 2000 Identification of meiotic and post-meiotic gene expression in testicular tissue of patients histologically classified as Sertoli cell only. Fertil Steril. 74:785–790. 29. Bachtell NE, Conaghan J, Turek PJ. 1999 The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation. Hum Reprod. 14:3048 –3051. 30. De Croo I, Van der Elst J, Everaert K, De Sutter P, Dhont M. 1998 Fertilization, pregnancy and embryo implantation rates after ICSI with fresh or frozenthawed testicular spermatozoa. Hum Reprod. 13:1893–1897. 31. Colpi GM, Negri L, Scroppo FI, Grugnetti C, Patrizio P. 1994 Seminal tract wash-out: a new diagnostic tool in complicated cases of male infertility. J Androl Suppl. 15:17S–22S. 32. Colpi GM, Negri L, Patrizio P, Pardi G. 1995 Fertility restoration by seminal tract washout in ejaculatory duct obstruction. J Urol. 153:1948 –1950. 33. Al Hasani S, Demirel LC, Schopper B, et al. 1999 Pregnancies achieved after frozen-thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen-thawed testicular tissues from non-obstructive azoospermic men. Hum Reprod. 14:2031–2035. 34. Kupker W, Schlegel P, Al-Hassani S, et al. 2000 Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection. Fertil Steril. 73:46 –58. 35. Cohen J, Garrisi JG, Congedo-Ferrara TA, et al. 1997 Cryopreservation of single human spermatozoa. Hum Reprod. 12:994 –1001. 36. Anguiano A, Oates RD, Amos JA, et al. 1992 Congenital bilateral absence of the vas deferens. A primarily genital form of cystic fibrosis. J Am Med Assoc. 267:1794 –1797. 37. Patrizio P, Asch RH, Handelin B, Silber SJ. 1993 Etiology of congenital
COMMENTARY
38. 39. 40. 41. 42. 43. 44. 45. 46. 47.
48. 49. 50. 51. 52.
absence of the vas deferens: genetic study of three generations. Hum Reprod. 8:215–220. Patrizio P, Zielenski J. 1996 Congenital absence of vas deferens: a mild form of cystic fibrosis. Mol Med Today. 2:24 –31. Reijo R, Alagappan RK, Patrizio P, Page DC. 1996 Severe oligospermia resulting from deletions of the azoospermia factor gene on the Y-chromosome. Lancet. 347:1290 –1293. Reijo R, Lee TY, Salo P, et al. 1995 Diverse spermatogenic defects in humans caused by Y-chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet. 10:383–393. Chandley AC. 1997 Karyotype and oligozoospermia. In: Barratt C, De jonge C, Mortimer D, Parinaud J, eds. Genetics of human male fertility. Paris: Editions E.D.K.; 111–122. Bernardini L, Gianoroli L, Fortini D, et al. 2000 Frequency of hyper-hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients. Hum Reprod. 15:2165–2172. Hargreave TB. 2000 Genetics and male infertility. Curr Opin Obstet Gynecol. 12:207–219. Reijo R, Alagappian PK, Patrizio P, et al. 1996 Severe oligospermia resulting from deletion of azoospermia factor gene on Y chromosome. Lancet. 347:1290 –1293. Page DC, Silber S, Brown LG. 1999 Men with infertility caused by AZFc deletion can produce sons by intracytoplasmic sperm injection, but are likely to transmit the deletion and infertility. Hum Reprod. 14:1722–1726. Komori S, Kasumi H, Kanazawa R, et al. 1999 CAG repeat length in the androgen receptor gene of infertile Japanese males with oligozoospermia. Mol Hum Reprod. 5:14 –16. Dadze S, Wieland C, Jakubiczka S, et al. 2000 The size of the CAG repeat in exon 1 of the androgen receptor gene shows no significant relationship to impaired spermatogenesis in an infertile Caucasoid sample of German origin. Mol Hum Reprod. 6:207–214. Yoshida KI, Masataka Y, Chiba K, Honda M, Kitahara S. 1999 CAG repeat length in the androgen receptor gene is enhanced in patients with idiopathic azoospermia. Urology. 54:1078 –1081. Dowsing AT, Yong EL, Clark M, McLachlan R, De Kretser DM, Trounson AO. 1999 Linkage between male infertility and trinucleotide repeat expansion in the androgen receptor gene. Lancet. 354:640 – 643. Patrizio P, Leonard DGB, Chen KL, Hernandez-Ayup S, Trounson AO. 2001 Larger trinucleotide repeat size in the androgen receptor gene of infertile males with extremely severe oligozoospermia. J Androl. In press. Int Veld P, Brandenburg H, Verhoeff A, et al. 1995 Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet. 346:773. Bonduelle M, Wilikens A, Buyssie A, et al. 1998 A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and tes-
53. 54. 55. 56. 57.
58. 59. 60. 61. 62. 63. 64. 65. 66. 67.
2379
ticular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod. 13:196 –207. Loft A, Petersen K, Erb K, et al. 1999 A Danish national cohort of 730 infants born after intracytoplasmic sperm injection (ICSI) 1994 –1997. Hum Reprod. 14:2143–2148. Van Opstal D, Los FJ, Ramlakhan S, et al. 1997 Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection. Hum Reprod. 12:682– 686. Storeng RT, Plachot M, Theophile D, et al. 1998 Incidence of sex chromosome abnormalities in spermatozoa from patients entering an IVF or ICSI protocol. Obstet Gynecol Scand. 77:191–197. Palermo GD, Schlegel PN, Sills ES, et al. 1998 Birth after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter syndrome. N Engl J Med. 338:588 –590. Nodan F, De Vincentis S, Olmedo SB, et al. 1998 Birth of twin males with normal karyotype after intracytoplasmic sperm injection with the use of testicular spermatozoa from a nonmosaic patient with Klinefelter syndrome. Fertil Steril. 71:1149 –1159. Palermo G, Colombero L, Schattman G, et al. 1996 Evolution of pregnancies and initial follow up of newborns delivered after intracytoplasmic sperm injection. J Am Med Assoc. 276:1893–1857. Wennerholm V-B, Bergh C, Hamsberger L, et al. 1996 Obstetric and perinatal outcome of pregnancies following intracytoplasmic sperm injection. Hum Reprod. 11:1113–1119. Kurinczuk JJ, Bower C. 1997 Birth defects in infants conceived by ICSI: an alternative explanation. Br Med J. 315:1260 –1265. Bowen JR, Gibson FL, Leslie GL, Saunders DM. 1998 Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet. 351:1529 –1534. Bonduelle M, Joris M, Hofmans K, GL, Saunders DM. 1998 Mental development of 201 ICSI children at 2 years of age (Letter). Lancet. 351:1553. Sutcliffe AG, Taylor B, Li J, et al. 1999 Children born after intracytoplasmic sperm injection: population control study. Br Med J. 318:704 –705. Shoukir Y, Chardonnens D, Campana A, Sakkas D. 1998 Blastocyst development from super embryos after intracytoplasmic sperm injection: a paternal influence? Hum Reprod. 13:1632–1637. Griffith TA, Murdoch AP, Herbert M. 2000 Embryonic development in vitro is compromised by the ICSI procedure. Hum Reprod. 15:1592–1596. Munne S, Marquez C, Reing A, et al. 1998 Chromosomal abnormalities in embryos obtained after conventional in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril. 69:5. 904 –908. Johnson MD. 1998 Genetic risks of intracytoplasmic sperm injection in the treatment of male infertility: recommendation for genetic counseling and screening. Fertil Steril. 70:397– 411.
