Departments of Obstetrics and Gynecology (O.K.) and Internal Medicine (C.W., R.S.), HarborUCLA Medical Center, Torrance, California 90509; and Department of Obstetrics and Gynecology (P.P.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
Address correspondence and requests for reprints to: Ronald Swerdloff, M.D., Division of Endocrinology, Department of Internal Medicine, HarborUCLA Medical Center, 1000 West Carson Street, Torrance, California 90509.
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 12% 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.
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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 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 sperms 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 small- or 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 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 67 µm) 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 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 testicular-derived 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 (<210 x 106 sperm/mL), severe asthenozoospermia (<510% 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, 22, 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 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.
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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 sedation 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.
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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 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 HTF-HEPES-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 (1012 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 müllerian 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.
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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 processed. 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, 37, 38), Y-chromosome microdeletions in the azoospermia factor locus (39, 40) leading to spermatogenic impairment, and karyotype abnormalities (e.g. Klinefelters 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 Klinefelters 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), 56% 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 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 56% of men with severe oligozoospermia and 1020% 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 Xq1112. 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
Kennedys 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
1 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 3840 repeats) is responsible for a neuromuscular disease,
spino-bulbar muscular atrophy, or Kennedys 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
Kennedys disease in two generations. The mechanism of transmission of
this fatal neuromuscular disease will 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-terato-zoospermia, 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 Klinefelters syndrome (56, 57).
Most reports, to date, have not shown an increase in major congenital malformations in children born by ICSI (23%) 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 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 12 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.
Received February 16, 2001.
Accepted March 16, 2001.
References