Sergio C. Oehninger, MD, PhD

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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. 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

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 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).


         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: [1] 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%); [2] failure of IUI therapy in cases presenting with  abnormal sperm parameters including presence of anti-sperm antibodies);  [3] previous failed fertilization in IVF; and [4] 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.

1. Barroso G, Morshedi M, Oehninger S. (2000) Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod, 15, 1338-1345. 

2. Bonduelle M, Camus M, De Vos A., et al., (1999) Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum Reprod,, 1999, 14, 243-264. 

3. Burkman LJ, Coddington CC, et al., (1988) The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril, 49, 688-697. 

4. Center for Disease Control and Prevention. Reproduction Technology Success Rates (1998) National summary and Fertility Clinic reports. U.S. Department of Health and Human Services, 9-32. 

5. Centers for Disease Control and Prevention. (1997) Fertility, Family Planning and Women's Health: New Data from the 1995 National Survey of Family Growth. US Department of Health and Human Services, USA, 1-11. 

6. Dong KW, Chi TF, Juan YW, et al., (2001) Characterization of the biological activities of a recombinant human zona pellucida protein 3 (ZP3) expressed in human ovarian (PA-1) cells. Am J Obstet Gynecol, 184, 835-844.

7. ESHRE Andrology Special Interest Group (1996) Consensus Workshop on Advanced Diagnostic Andrology Techniques. Hum Reprod, 11, 1463-1479. 

8. Evenson DP, Jost LK, Marshall D, et al., (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod, 14, 1039-1049. 

9. Franken DR, Bastiaan HS, Oehninger S. (2000) Physiological induction of the acrosome reaction in human sperm: Validation of a microassay using minimal volumes of solubilized, homologous zona pellucida. J Assist Reprod Genet, 17, 156-160. 

10. Hinsch E, Oehninger S, Hinsch K. (April, 1998) ZP3-6 peptide and acrosome reaction. Semen evaluation, testing and selection: a new look in the 2000s. In Andrology in the Nineties Precongress Training Course. Genk, Belgium, 25-30. 

11. Irvine DS. (1998) Epidemiology and etiology of male infertility. Hum Reprod, 13, 33-44. 

12. 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. 

13. Liu DY, Lopata A, et al., (1988) A human sperm-zona pellucida binding test using oocytes that failed to fertilize in vitro. Fertil Steril, 50, 782-788. 

14. Oehninger S. (2001) Place of intracytoplasmic sperm injection in clinical management of male infertility. Lancet, 357, 2068-2069.

15. Oehninger S, Franken DR, et al., (2000) Sperm function assays and their predictive value for fertilization outcome in IVF therapy: a meta-analysis. Hum Reprod Update, 6, 160-168.

16. Oehninger S. (2000) Clinical and laboratory management of male infertility: An opinion on its current status. J Androl, 21, 814-821.

17. Oehninger S, Franken D, Kruger T. (1997) Approaching the next millennium: how should we manage andrology diagnosis in the intracytoplasmic sperm injection era? Fertil Steril, 67, 434-436. 

18. Oehninger S, Toner JP, et al., (1992a) Prediction of fertilization in vitro with human gametes: Is there a litmus test? Am J Obstet Gynecol, 167, 1760-1767.

19. Oehninger S, Franken D, et al., (1992b) Hemizona assay and its impact on the identification and treatment of human sperm dysfunctions. Andrologia, 24, 307-321.

20. Ombelet W, Bosmans E, Janssen M, et al., (1997) Semen parameters in a fertile versus subfertile population: a need for change in interpretation of semen testing. Hum Reprod, 12, 987-993. 

21. Sakkas D, Mariechoz E, Manicardi G, et al., (1999) Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod, 4, 31-37. 

22. St John JC. (1999) Incorporating molecular screening techniques into the modern andrology laboratory. J. Androl, 20, 692-701.

23. Tesarik J. (1989) Appropriate timing of the acrosome reaction is a major requirement for the fertilizing spermatozoon. Hum Reprod, 4, 957-961. 

24. van Duin M, Polman J, De Breet IT. (1994) Recombinant human zona pellucida protein ZP3 produced by Chinese Hamster Ovary cells induces the human sperm acrosome reaction and promotes sperm-egg fusion. Biol Reprod, 51, 607-617

25. World Health Organization. (1999) WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge, United Kingdom: Cambridge University Press, 4-33.

26. Zinaman MJ, Brown CC, Selevan SG, Clegg ED. (2000) Semen quality and human fertility: A prospective study with healthy couples. J. Androl, 21, 145-153 

Articles

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.

Author | Sergio C. Oehninger, MD, PhD

Articles

Is A Simple Sperm Analysis All We Need?