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Although diagnostic problems make it difficult to establish the extent of the male partner's contribution with certainty, a number of studies suggest that male problems represent the most common single defined cause of infertility.
Although diagnostic problems make it difficult to establish the extent of the male partner's contribution with certainty, a number of studies suggest that male problems represent the most common single defined cause of infertility. Male-related disorders are probably present in up to 40-50% of childless couples, alone or in combination with female factors (Centers for Disease Control, 1997: Irvine, 1998).
Treatment options for male infertility include a large number of urological procedures (surgical and non-surgical), medical-pharmacological interventions, low complexity assisted reproductive procedures (such as intrauterine insemination therapy or IUI), and the more advanced and complex assisted reproductive technologies (ARTs). Among the latter, in vitro fertilization (IVF) and embryo transfer, augmented with intracytoplasmic sperm injection (ICSI) in moderate and severe cases, constitute validated and successful ways to assist fertilization and conception. National statistics from the U.S.A. (Centers for Disease Control, 1998) reported a 40% incidence of ICSI in 61,650 IVF cycles performed in a one-year period, a figure that highlights the importance of this technique. Although ICSI has become a real "boom" in the treatment of men with various degrees of sperm anomalies, it may carry a risk of transmission of chromosomal/genetic disease (Bonduelle, et al., 1999).
In spite of the fact that contemporary therapies have enhanced the opportunities to conceive in couples suffering from male infertility, often these solutions are brought up in the absence of a defined etiological or pathophysiological diagnosis. Male infertility, unfortunately, is still considered "idiopathic" in a large proportion of cases. Within this clinical scenario, a simple practical question arises: what are the diagnostic steps that we should take to direct the infertile man to an optimized and cost-efficient therapeutic modality?
The Basic Semen Analysis
The cornerstone of the andrological evaluation in all cases is an exhaustive history and a physical examination followed by repeated semen analyses. A urological, endocrine, and/or imaging work-up should be implemented as appropriate. A comprehensive semen analysis following the World Health Organization guidelines (WHO, 1999) is fundamental at the primary care level to make a rational initial diagnosis and to select the appropriate clinical management. The collection and analysis of the semen must be undertaken by properly standardized procedures in appropriately qualified and accredited laboratories.
The "basic" semen evaluation should include: (1) assessment of physical semen characteristics (volume, liquefaction, appearance, consistency, pH and agglutination); (2) evaluation of sperm concentration, grading of motility and analysis of morphological characteristics (using strict criteria) (Kruger, et al., 1986); (3) determination of sperm vitality (viability), testing for sperm auto-antibodies (using the mixed antiglobulin reaction and/or the direct immunobead tests), presence of leukospermia and immature sperm cells; and (4) bacteriological studies. The identification and separation of the motile sperm fraction is also an integral part of the initial semen evaluation (Oehninger, 2000).
Sperm Function Tests
Other categories of assays that are usually considered include: (1) tests that examine defective sperm functions indirectly through the use of biochemical tests (i.e., measurement of the generation of reactive oxygen species or evidence of peroxidative damage, measurement of enzyme activities such as creatine phosphokinase and others); (2) bioassays of gamete interaction (i.e., the heterologous zona-free hamster oocyte test and the homologous sperm-zona pellucida binding assays) and induced-acrosome reaction scoring; and (3) computer-aided sperm motion analysis (CASA) for the evaluation of sperm motion characteristics (ESHRE, 1996; WHO, 1999).
We recently published the results of a meta-analysis that aimed to determine the diagnostic test accuracy and predictive value of various sperm function assays for IVF outcome (Oehninger, et al., 2000). The scrutinized tests were CASA, acrosome reaction testing, the zona-free hamster egg penetration test or sperm-penetration assay (SPA) and sperm-zona pellucida binding assays. The validity of such assays was objectively assessed through results obtained from 2,906 subjects evaluated in 34 prospectively designed, controlled studies. Results demonstrated a high predictive power of the sperm-zona pellucida binding and the induced-acrosome reaction assays for fertilization outcome. On the other hand, the findings indicated a poor clinical value of the SPA as predictor of fertilization and a real need for standardization and further investigation of the potential clinical utility of CASA systems.
The interaction between spermatozoa and the zona pellucida is a critical event leading to fertilization and reflects multiple sperm functions (i.e., completion of capacitation as manifested by the ability to bind to the zona pellucida and to undergo ligand-induced acrosome reaction) (Oehninger, et al., 1992a and b). The two most common sperm-zona pellucida binding tests currently utilized are the hemizona assay (or HZA) (Burkman, et al., 1988) and a competitive intact-zona binding assay (Liu, et al., 1992).
The induced-acrosome reaction assays appear to be equally predictive of fertilization outcome and are simpler in their methodologies. The use of a calcium ionophore to induce acrosome reaction is at the present time the most widely used methodology (Tesarik, 1989). Nevertheless, the implementation of assays using small volumes of human solubilized zonae pellucidae (micro assay) (Franken, et al., 2000), biologically active recombinant human ZP3 (van Duin, et al., 1994; Dong, et al., 2001) or active, synthetic ZP3 peptides (Hinsch, et al., 1998), will probably allow for the design of improved, physiologically oriented acrosome reaction assays.
It is now well established that a variety of genetic disorders are associated with male infertility, some of them potentially transmissible to the offspring. Chromosomal anomalies (structural and numerical, involving autosomal and sexual chromosomes) can be diagnosed by peripheral karyotyping and should be studied in men with severe oligozoospermia and non-obstructive azoospermia. At the gene level, reproductive failure may be associated with cystic fibrosis mutations (in men presenting with obstructive azoospermia due to congenital absence of the vas deferens) and with Y-microdeletions (in men with severe oligozoospermia and non-obstructive azoospermia due to spermatogenic failure). Such abnormalities can also be detected by peripheral blood screening using PCR methodologies (conventional, nested, multiplex, fluorescent or quantitative PCR) (St. John, 1999).
Spermatozoa of infertile men have also been shown to contain various nuclear alterations. Some of them include an abnormal chromatin structure, aneuploidy, chromosomal microdeletions and DNA strand breaks (Sakkas, et al., 1999). Presently, various tests are available for detection of some of those anomalies, including the aniline blue staining, acridine orange, sperm chromatin structure assay (SCSA) and assessment of DNA damage or fragmentation (Evenson, et al., 1999; Barroso, et al., 2000).
Clinicians and scientists are still searching for semen parameter thresholds in the so-called "normal fertile populations" in order to be able to more accurately define fertility, sub-fertility and infertility. Recent publications have appropriately re-addressed those issues as part as both European and American studies (Ombelet, et al., 1997; Zinaman, et al.,).
Notwithstanding the lack of uniform criteria, if sperm abnormalities are observed in the "basic" semen analysis or if the couple is diagnosed as "unexplained" infertility, the work-up should proceed to the analysis of sperm function/biochemical tests. The diagnosis of sub-fertility or infertility, based upon the first-tier (initial "basic" evaluation) and the "expanded" (functional/biochemical) screenings, will direct management toward a variety of therapeutic options (Oehninger et al, 1997). We have previously proposed that laboratory evaluation of sperm quality/quantity for assisted reproduction should be approached using such sequential, multi-step diagnostic scheme (Oehninger, et al., 1992 a and b; Oehninger et al, 1997).
Currently recommended ART options include: "low complexity" intra-uterine insemination (IUI) therapy, "standard" IVF and embryo transfer, and IVF augmented with ICSI. If the female partner is <35 years, typically 4-6 cycles of intrauterine inseminations (IUI) using husband's sperm are recommended as a simple (low complexity) ART approach. Although there are no established sperm cut-off levels, it is preferable to perform IUI if >5 million total motile spermatozoa can be used per insemination (particularly if sperm morphology is normal or only slightly abnormal). Patients with a motile sperm fraction >1.5 million motile spermatozoa following swim-up or gradient centrifugation, but with mild to moderate teratozoospermia (in the range of 4-14% normal forms by strict criteria) should be offered "standard" IVF therapy. In those cases, good fertilization and pregnancy rates are achieved with an increase in the sperm insemination concentration (Oehninger, 2000).
In our program, patients are selected for IVF augmented with ICSI according to the following indications:  poor sperm parameters (i.e., <1.5 x 106 total spermatozoa with adequate progressive motility after separation, severe teratozoospermia with <4% normal forms in the presence of a borderline to low total motile fraction, and/or poor sperm-zona pellucida binding capacity with a hemizona assay index <30%);  failure of IUI therapy in cases presenting with abnormal sperm parameters including presence of anti-sperm antibodies);  previous failed fertilization in IVF; and  presence of obstructive or non-obstructive azoospermia, where ICSI is combined with sperm extraction from the testes or the epididymis (Oehninger, 2001).
On the basis of current evidence, the use of ICSI should be restricted to male-factor infertility, for which it seems to be cost-effective and relatively safe. However, as in other areas of the ever-expanding specialty of ART, vigilance is required to identify any potential negative impact on the long-term health of children conceived after ICSI. Although ICSI constitutes a validated, formidable therapy that can help most cases of male infertility, the identification of specific sperm defects should allow the development of simpler, directed therapies. The basic semen analysis remains the cornerstone in the evaluation of the male partner. Validated sperm functional tests should expand the initial work up as indicated. The urologist and reproductive endocrinologist should work as a team to offer the best option to each couple suffering from male infertility.
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