Evaluation and assessment of semen for IVF/ICSI *

D.Y. Liu, H.W.G. Baker

Asian J Androl 2002 Dec; 4: 281-285            

Keywords: male infertility; sperm function tests; sperm-oocyte interactions; IVF /ICSI

Abstract

Evaluation and assessment of semen is very important for both diagnosis of male infertility and selection of patients for treatment with IVF or ICSI. In standard IVF, sperm function is essential for normal fertilization: sperm must be able to bind to zona pellucida (ZP), undergo the acrosome reaction and penetrate the ZP and fuse with the oolemma before fertilization takes place. In contrast, most sperm functions are not required for fertilization in ICSI since sperm bypass the ZP and oolemma by injection of a single sperm directly into cytoplasm of oocyte. Therefore, the clinical decision on treatment of patients with either IVF or ICSI is mostly dependent on results of sperm tests. However, conventional semen analyses do not provide accurate information about sperm fertilizing ability since many patients with subtle sperm defects can not be detected. More advanced sperm function tests are required to detect sperm defects that may lead to failure of fertilization in standard IVF. In the last 15 years we performed extensive studies on relationship between sperm functions and fertilization rates by logistic regression analysis in large numbers of IVF patients including 370 patients with zero fertilization rate by IVF. We confirmed sperm morphology assessed strictly was strongly related to fertilisation rate with standard IVF. Thus sperm morphology assessment is very useful for selection of patients for ICSI. We also developed a number of new tests including sperm-ZP binding, sperm-ZP penetration and the ZP-induced AR and evaluated the clinical value of these tests. Sperm-ZP binding and sperm-ZP penetration tests are the most powerful indicators for sperm fertilizing ability in vitro. The ZP-induced AR is highly correlated with sperm-ZP penetration. We discovered a condition we call disordered ZP-induced AR which causes serve infertility in up to 25% men with otherwise idiopathic infertility In conclusion, the combination of semen analysis with advanced sperm function tests provide important diagnostic and prognostic information for male infertility and is crucial for selection of patients for treatment with IVF or ICSI.

Conventional semen analysis including sperm count, motility, and morphology is widely used as a fundamental indicator of male fertility [1]. However, the results do not provide accurate diagnosis or prognosis for human fertility in vivo or in vitro, except for those with ejaculates persistently containing no sperm, or all immotile sperm, or all sperm with uniform structural morphological defect such as round-headed acrosomeless sperm [1, 2]. But these are uncommon causes of severe male infertility. Most men seen for infertility have either reduced sperm count, motility and morphology alone, or in combination. Other (about 25 %) infertile men even have consistently normal semen analysis results and they are usually classified as having unexplained infertility. Semen analysis has limited clinical value for predicting fertility and other sperm function tests are needed to improved the clinical management of patients and allocation to IVF or ICSI.

Although the mechanisms of human sperm-oocyte interaction are not completely understood, they are crucial. During the process of human fertilization, sperm must bind to the zona pellucida (ZP), undergo the acrosome reaction (AR), penetrate the ZP and then fuse with the oolemma [3]. The sperm receptor on the ZP called ZP3 is probably responsible for binding sperm and induction of the AR [4]. Human sperm binding to the ZP is more strictly species-specific than it is for other mammals and human sperm only bind to the ZP of human oocytes [5]. In 1976, Overstreet et al first studied sperm-ZP binding and penetration using non-viable human oocytes from cancer patients [6]. They suggest that ability of sperm-ZP interactions may predict subtle sperm defects causing infertility that can not be diagnosed by standard semen analysis. Because there were limited numbers of human oocytes available at that time, the clinical value of sperm-ZP interaction tests was not widely investigated. In the late 1980s, we and Burkman et al developed the sperm-ZP binding ratio test and the hemizona assay using unfertilized human oocytes from clinical IVF for prediction of fertilization rate in standard IVF [7, 8]. Since then other sperm-ZP interaction tests have also been developed including ZP-induced AR, sperm-ZP penetration and sperm-oolemma binding. Over the last 15 years, many studies showed that the sperm-ZP interaction tests provide significant information about sperm fertilizing ability which is of clinical value for predicting fertilization rates in standard IVF. The aim of this presentation is to summarise the most important sperm function tests for prediction of sperm fertilizing ability in vitro and selection of patients for IVF or ICSI treatment.

2 Relationship between sperm defects and failure of fertilization in IVF

To determine which sperm defects were most significantly related to failure of fertilization in IVF, we performed a number of serial studies on various sperm function tests [2, 8, 9-19]. All these tests were determined for sperm in the insemination medium and semen remaining after preparation for IVF. The tests included sperm concentration, motility, velocity (straight line velocity, VSL, Hamilton-Thorn) viability (eosin Y exclusion), hypo-osmotic swelling, strict sperm morphology (Shorr stain), acrosome status (Pisum sativum agglutinin labelled with fluorescein isothiocyanate, PSA-FITC), total acrosin activity, nuclear maturity (acidic aniline blue stain) and DNA abnormality (acridine orange florescence stain). The number of sperm bound to the ZP and the proportion of ZP penetrated in oocytes that failed to fertilize in IVF were also analysed [19-20]. At the same time, oocyte characteristics such as maturity (presence of germinal vesicle, number of polar bodies) and morphological quality (good, fair and poor) were also included [12]. All these tests were performed without knowledge of the results of IVF and the data analyzed by logistic regression to determine which groups of sperm tests were independently sisignificant. Usually over 100 IVF treatments were investigated in each study. The statistical analysis showed the proportion of ZP penetrated, number of sperm bound to the ZP and percentage normal sperm morphology were most strongly related to fertilization rates in vitro and others sperm characteristic were less significant.

To further determine if defective sperm-ZP binding and penetration are major causes of failure of fertilization in vitro, we examined sperm-ZP binding and penetration of all unfertilized oocytes from 369 IVF patients who had zero fertilization rates of 3 mature oocytes inseminated [20]. We found that over 70 % of patients had low or zero sperm-ZP binding (an average of < 5 sperm bound/ZP) and 43 % had no sperm penetrating the ZP of any oocyte. Furthermore, 35 % of patients had low (<5 %) normal sperm morphology in the insemination medium. The latter mostly had low sperm-ZP binding however, 12 % (10 of 82) of patients with normal sperm-ZP binding (10 sperm bound/ZP) had no sperm penetrating into any oocyte. Over 50 % of patients with poor sperm-ZP binding and penetration had defects of sperm motility and morphology revealed by semen analysis. Another 50 % of the patients had normal semen analysis results. Therefore, sperm-ZP binding and penetration are important markers for sperm fertilizing ability.

3 Effect of sperm and oocyte abnormalities on sperm-ZP interaction

3.1 Ooctye

Sperm-ZP interaction is related to the functional integrity of both sperm and oocytes. Abnormalities from either sperm or oocytes will affect sperm-ZP interaction. However, the maturity of human oocytes does not often affect either sperm-ZP binding or penetration. Human sperm penetrate the ZP of oocytes at all stages of maturity from germinal vesicle to metaphase II either fresh or after storage in 1 mol/L ammonium sulphate solution [21, 22]. There is no relationship between the number of sperm bound to the ZP and oocyte quality assessed morphologically as discussed the above [2, 12]. To further confirm that oocyte quality was not a common cause for poor sperm-ZP binding in IVF, oocytes from patients with zero fertilisation rate and <5 sperm bound/ZP were re-incubated with fertile donor sperm. This resulted in large numbers of sperm binding to the ZP and penetrating into the ZP (>90 % had one or more sperm in the ZP) [20]. Thus patients with complete failure of fertilisation, low sperm-ZP binding usually results from sperm defects not oocyte defects. However, oocyte defects can still to be responsible for defective sperm-ZP binding particularly in patients with only one or a few oocytes from the batch which fail to fertilise. Therefore, as a general rule when most or all oocytes from a patient fail to fertilise, there is a sperm defect. Furthermore, defective sperm-ZP binding is mostly due to obvious sperm defects of motility or strict morphology. Thus tests of sperm-ZP binding provide a powerful measure of sperm fertilizing ability.

3.2 Sperm

Several aspects of sperm function affect sperm-ZP interaction. Normal sperm morphology is one of the most significant variables of semen analysis related to sperm-ZP binding [8, 13, 23]. This may explain why normal morphology is so significantly related to fertilization rates in vitro. Sperm motility and concentration in the insemination medium are also correlated with sperm-ZP binding as expected since both factors directly influence the chance of sperm contacting the oocyte [8, 13]. The proportion of sperm with a normal intact acrosome in the insemination medium is also correlated with the number of sperm bound to the ZP [11-15, 24]. This suggests that intact acrosomes are important for sperm binding to the ZP. Therefore, the sperm-ZP binding test reflects multiple sperm functions [2,25] and the results are the most significantly correlated with fertilization rates in standard IVF or all sperm tests investigated.

The average human ejaculate contains high proportions of morphologically abnormal sperm but both the ZP and oolemma are selective for binding sperm with normal morphology [23, 26, 27]. Importantly, sperm bound to the ZP tend to have normal acrosome areas occupying more than half of sperm head, suggesting that human acrosome region is very important for sperm binding to the ZP. Thus a normal appearing acrosome area should be considered as one of the most important criteria for assessment of sperm morphology. Sperm with gross head abnormalities such as amorphous or small head with a tiny acrosome do not bind to the ZP or oolemma. Round-headed sperm without acrosomes do not bind or penetrate the ZP, and fertilise oocytes either in vivo or in vitro by standard IVF [28].

The AR is very important for sperm-ZP penetration in the human. Although many agents such as the calcium ionophore A23187, follicular fluid and progesterone may stimulate the AR, the native human ZP is the most efficient inducer of the AR [29-32]. There is no relationship between A23187-induced AR and ZP-induced AR and sperm-ZP penetration [29, 30]. In contrast, there is a high correlation between human ZP-induced AR and sperm-ZP penetration [29]. The ZP-induced AR is limited to the subpopulation of sperm which are able to bind to the ZP and therefore have been selected for better morphology, motility and acrosome intactness. In contrast other inducers such as A23187 and progesterone potentially involve all the sperm present in the medium. Inhibition of acrosin activity with soybean trypsin inhibitor does not affect sperm-ZP binding but significantly blocks both the AR of sperm bound on the ZP and sperm-ZP penetration [33, 34]. As a result of these results we predicted that defective ZP-induced AR could cause failure of sperm-ZP penetration and infertility.

4 Disordered ZP-induced AR in unexplained male infertility

In 1994, we first reported this sperm defect called disordered ZP-induced AR (DZPIAR) causes failure of sperm-ZP penetration in group of IVF patients with a long duration of infertility and persistent zero or low (< 25 %) fertilisation rates with standard IVF [35]. These patients were previously classified as having idiopathic infertility since they have normal sperm characteristics including sperm concentration, motility/velocity, morphology, nuclear maturity or DNA normality and ultrastructure. No antisperm antibodies have been detected by the immunobead test. Sperm from the men have normal acrosomes and normal sperm-ZP binding capacity but no sperm penetrate into the ZP during IVF treatments. When tested with oocytes which failed to fertilise from other patients, the ZP-induced AR was severely reduced: average 6 % compared with 50 % for fertile men. Patients with this condition can not be treated with standard IVF because their sperm can not penetrate the ZP. Therefore DZPIAR patients need to be diagnosed by the ZP-induced AR test and treated by ICSI. Results of ICSI showed that DZPIAR had significantly higher fertilization, implantation and live birth pregnancy rates than patients with other diagnoses [36-38]. The frequency of DZPIAR in patients with unexplained infertility with normal semen and normal sperm-ZP binding is about 25 % [38, 39]. Therefore it is very important to diagnose patients with this condition before they commence ART treatment to avoid failure of fertilization by IVF which is usually recommended for idiopathic infertility.

5 Clinical application of sperm function tests in IVF/ICSI

Allocation of patients to IVF or ICSI is mainly dependent on adequate sperm function since it is required for fertilization in IVF but is less needed in ICSI. Although ICSI can be used for treatment of patients with and without sperm defects, most clinics only use ICSI to treat patients with severe sperm defects who would do less well with standard IVF. Therefore, diagnosis of cause of infertility and logical selection of the optimal procedure for treatment are necessary.

Sperm morphology is one of the most important routine semen analysis variables for selection of patients for IVF or ICSI [40]. As discussed the above, sperm morphology is strongly related to sperm-ZP binding. With strict morphology assessment [41], patients with <5 % normal sperm morphology should be recommended for ICSI since our previous study showed that they had an average fertilization rate 30 % by standard IVF [40] and many had complete failure of fertilization Although patients with normal sperm morphology between 6 to 15 % can be treated by either IVF or ICSI depending on other sperm tests results such as sperm count and motility and also clinical factors. In uncertain situations the ZP-binding test can be helpful. Patients with normal ZP binding should do well with IVF. On the other hand those with abnormal ZP-binding need ICSI. Patients with normal sperm morphology >15 %, mostly will be treated with standard IVF in the initial cycle. However, patients with more than two years unexplained infertility and also consistent normal semen analysis, the ZP-induced AR should be performed to determine if they have DZPIAR which will cause failure of sperm-ZP penetration poor results with standard IVF [35, 38, 39, 42]. Patients with DZPIAR should be treated with ICSI rather than standard IVF.

Details of all technical aspects of sperm-ZP binding and the ZP-induced AR tests were described elsewhere [2, 7, 8, 29, 30, 35]. About 20 to 30 % of oocytes inseminated in clinical IVF fail to be fertilized and these oocytes are valuable for these tests. Degenerated oocytes and morphologically abnormal oocytes should not be used for sperm-ZP interaction tests since they have high incidence of abnormalities or cortical granule reaction occurred which may affect on the activity of ZP receptors. In the future, development of diagnostic kits using recombinant human ZP for the testing sperm binding and the ZP-induced AR would have great potential for routine clinical testing [43-45].

Defective sperm-ZP binding and penetration are the major causes for failure of fertilization in standard IVF. Sperm-ZP binding and the ZP-induced AR tests are useful before patients commence ART treatment. In addition of sperm-ZP interaction tests to routine semen analysis could improve both clinical diagnosis and management of patients in IVF/ICSI. Over 25 % of unexplained infertility with normal semen may have DZPIAR who should be identified and treated with ICSI rather then standard IVF.

References

[1] World Health Organisation (WHO) Laboratory Manual for Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge: Cambridge University Press; 1992.

[2] Liu DY, Baker HWG. Test of human sperm function and fertilisation in vitro. Fertil Steril 1992; 58: 465-83.

[3] Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill J, editors. The physiology of Reproduction. 2nd ed. New York: Raven Press; 1994. p 189-317.

[4] Wassarman PM. Mammalian fertilization molecular aspects of gametes adhesion, exocytosis, and fusion. Cell 1999; 96: 175-83.

[5] Liu DY, Lopata A, Pantke P, Baker HWG. Horse and marmoset sperm bind to the zona pellucida of salt stored human oocytes. Fertil Steril 1991; 56: 764-7.

[6] Overstreet JW, Hembree WC. Penetration of the zona pellucida of nonliving human oocytes by human spermatozoa in vitro. Fertil Steril 1976; 17: 815-31.

[7] Burkman LJ, Coddington CC, Franken DR et al. 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 1988; 49: 688-97.

[8] Liu DY, Lopata A, Johnston WIH, Baker HWG. A human sperm-zona pellucida binding test using oocytes that failed to fertilize in vitro. Fertil Steril 1988; 50: 782-8.

[9] Liu DY, Elton RA, Johnston WIH and Baker HWG. Spermatozoal nuclear chromatin decondensation in vitro. A test for sperm immaturity. Comparison with results of human in vitro fertilisation. Clin Reprod and Fertil 1987; 5: 191-201.

[10] Liu DY, Du Plessis YP, Nayudu PL, Johnston WIH, Baker HWG. The use of in vitro fertilization to evaluate putative tests of human sperm function. Fertil Steril 1988; 49: 272-7.

[11] Liu DY, Baker HWG. The proportion of sperm with poor morphology but normal intact acrosomes detected with Pisum Sativum agglutinin correlates with fertilization in vitro. Fertil Steril 1988; 50: 288-93.

[12] Liu DY, Lopata A, Johnston WIH, Baker HWG. Human sperm-zona pellucida binding, sperm characteristics and in vitro fertilization. Hum Reprod 1989; 4: 696-701.

[13] Liu DY, Clarke GN, Lopata A, Johnston WIH, Baker HWG. A sperm-zona pellucida binding test and in vitro fertilization. Fertil Steril 1989; 52: 281-7.

[14] Baker HWG, Liu DY. New tests of human sperm function and in vitro fertilization. Reprod Fertil Dev 1989; 1: 179-82.

[15] Liu DY, Baker HWG. Relationships between human sperm acrosin, acrosomes, morphology and in vitro fertilization. Hum Reprod 1990; 5: 298-303.

[16] Liu DY, Lopata A, Baker HWG. Use of oocytes that failed to be fertilized in vitro to study sperm-oocyte interaction: comparison of sperm-oolemma and sperm-zona pellucida binding, and relationship with results of IVF. Reprod Fertil Dev 1990; 2: 641-50.

[17] Liu DY, Clarke GN, Baker HWG. Relationship between sperm motility assessed with the Hamilton-Thorn Motility Analyzer and fertilization rates in vitro. J Androl 1991; 12: 231-9.

[18] Liu DY, Baker HWG. Sperm nuclear chromatin normality: relationship with sperm morphology, sperm-zona pellucida binding and fertilization rates in vitro. Fertil Steril 1992; 58: 1178-84.

[19] Liu DY, Baker HWG. A new test for the assessment of sperm-zona pellucida penetration: relationship with results of other sperm tests and fertilisation in vitro. Human Reprod 1994; 9:489-96.

[20] Liu DY, Baker HWG. Defective sperm-zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization. Hum Reprod 2000; 15: 702-8.

[21] Lopata A, Leung PC. The fertilizability of human oocytes at different stages of meiotic maturation. In: Jones HW Jr, Schrader C, editors. In Vitro Fertilization and Other Assisted Reproduction. New York: New York Academy of Sciences; 1988. p 324-36.

[22] Yanagimachi R, Lopata A, Odom CB. Retention of biological charactristics of zona pellucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil Steril 1979; 31: 562-74.

[23] Liu DY, Baker HWG. Morphology of human spermatozoa bound to human zona pellucida of the oocytes that failed to fertilise in vitro. J Reprod Fertil 1992; 94: 71-84.

[24] Liu DY, Baker HWG. Inducing the human acrosome reaction with a calcium ionophore A23187 decreases sperm-zona pellucida binding with oocytes that failed to fertilize in vitro. J Reprod Fertil 1990; 89: 127-34.

[25] Liu DY, Baker HWG. Tests for human sperm-zona pellucida binding and penetration. In: Tesarik J,editors. Male Factor in Human Infertility, Ares-Serono Symposia Publications, Frontiers in Endocrinology; v 8. Italy: Rome; 1994. p 169-85.

[26] Liu DY, Baker HWG. Acrosome status and morphology of human sperm bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro. Hum Reprod 1994; 9: 673-9.

[27] Garrett C, Liu DY, Baker HWG. Selectivity of the human sperm-zona pellucida bining process to sperm head morphometry. Fertil Steril 1997; 67: 362-71.

[28] Bourne H, Liu DY, Clarke GN, Baker HWG. Normal fertilization and embryo development with an round headed acro-someless sperm. Fertil Steril 1995; 63: 1329-32.

[29] Liu DY, Baker HWG. A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona pellucida: lack of relatioship with ionophore A23187-induced acrosome reaction. Hum Reprod 1996; 11: 551-7.

[30] Liu DY, Baker HWG. Relationship between the zona pellucida (ZP) and ionophore A23187 induced acrosome reaction and the ability of sperm to penetrate the ZP in men with normal sperm-ZP binding. Fertil Steril 1996; 66: 312-5.

[31] Liu DY, Baker HWG. Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen. Hum Reprod 1998; 13: 905-10.

[32] Baker HWG, Liu DY, Garrett C, MarticM. The human acrosome reaction. Asian J Androl 2000; 2: 172-8.

[33] Liu DY, Baker HWG. Inhibition of acrosin activity with a Trypsin inhibitor blocks human sperm penetration of the zona pellucida. Biol Reprod 1993; 48: 340-8.

[34] Llanos M, Vigil P, Salgado AM, Morales P. Inhibition of the acrosome reaction by trypsin inhibitors and prevention of penetration of spermatozoa through the human zona pellucida. J Reprod Fertil 1993; 97: 173-8.

[35] Liu DY, Baker HWG. Disordered acrosome reaction of sperm bound to the ZP: a newly discovered sperm defect causing infertility with reduced sperm-ZP penetration and reduced fertilisation in vitro. Human Reprod 1994; 9: 1694-700.

[36] Liu DY, Bourne H, Baker HWG. Fertilization and pregnancy with acrosome intact sperm by intracytoplasmic sperm injection in patients with disordered zona pellucida induced acrosome reaction. Fertil Steril 1995; 64: 116-21.

[37] Liu DY, Bourne H, Baker HWG. High fertilization and pregnancy rates after intracytoplasmic sperm injection in patients with disordered zona pellucida-induced acrosome reaction. Fertil Steril 1997; 67: 955-8.

[38] Liu DY, Baker HWG. Disordered zona pellucida-induced acrosome reaction and failure of fertilization in patients with unexplained infertility. Fertil Steril: in press.

[39] Liu DY, Clarke GN, Martic M, Garrett C, Baker HWG. Frequency of disordered zona pellucida (ZP)-induced acrosome reaction in infertile men with normal semen analysis and normal sperm-ZP binding. Hum Reprod 2001; 6: 1185-90.

[40] Baker HWG, Liu DY, Bourne H, Lopata A. Diagnosis of sperm defects in selecting patients for assisted fertilization. Hum Reprod 1993; 8: 1779-80.

[41] Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilisation. Fertil Steril 1988, 49: 112-7.

[42] Esterhuizen AD, Franken DR, Lourens JDH. Van Rooyen LH. Clinical importance of zona pellucida induced acrosome reaction (ZIAR): predictive value of the ZIAR test for in vitro fertilization. Hum. Reprod 2001; 16: 138-44.

[43] Brewis IA, Clayton R, Barratt CLR, Hornby DP, Moore HDM. Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa. Mol Hum Reprod 1996; 2: 583-9.

[44] Whitmarsh AJ, Woolnough MJ, Moore HDM, Hornby DP, Barratt CLR. Biological activity of recombinant human ZP3 produced in vitro: potential for a sperm function test. Mol Hum Reprod 1996; 2: 911-9.

[45] Dong KW, Chi TF, Juan YW, Chen CW, Lin Z, Xiang XQ et al. Characterization of the biologic activities of a recombinant human zona pellucida protein 3 expressed in human ovarian teratocarcinoma (PA-1) cells. Am J Obstet Gynecol 2001; 184: 835-43.

home

Correspondence to: DY Liu, Ph D, Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's  Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia.

Tel: +61-3-9344 2042,   Fax: +61-3-347 1761

E-mail: dyl@unimelb.edu.au

* Presented at the First Asia-Pacific Forum on Andrology, 17-21 Oct 2002, Shanghai, China

Received 2002-09-26    Accepted 2002-10-05