Predictive value of hypo-osmotic swelling test to identify viable non-motile sperm

Keywords: azoospermia; sperm spermmotility; hypo-osmotic swelling (HOS) test; sperm viability; predictive value

Abstract

Aim: To determine the predictive value of the hypo-osmotic swelling (HOS) test to identify viable, non-motile sperm. Methods: Semen samples from 20 men with severe asthenozoospermia underwent traditional seminal analysis, eosin-nigrosin (EN) staining and the HOS test. A further EN stain was then performed on a HOS pre-treated aliquot and a total of 2000 further sperm examined. Results: The median sperm density was 5.1 million/mL (IQR 4.3~13.1) and the median motility was 3.0 % (IQR 0~7). Seven samples showed complete asthenozoospermia. Initial EN staining showed 59 % viability (range 48~69) despite the poor standard parameters and 47 % (range 33~61) in the complete asthenozoospermia subgroup. The HOS test showed 49.9 % reacted overall (range 40~59) and 41.7 % (range 22~61) in the complete asthenozoospermia subgroup. The combined HOS/EN stain showed the positive predictive value of the HOS test to identify viable sperm was 84.2 % overall and 79.7 % in the complete asthenozoospermia subgroup. Conclusion: The HOS test can effectively predict sperm viability in patients with severe and complete asthenozoospermia.

1 Introduction

The prognosis for male factor infertility has dramatically improved with the advent of intracytoplasmic sperm injection (ICSI). Men with severe or complete astheno-zoospermia have very poor spontaneous pregnancy rates and poor fertilization rates with standard in vitro fertilization (IVF) [1] and, although medical treatments can improve motility, ICSI is often the treatment of choice for this group [2].

In order to achieve the optimum fertilization rate it is necessary to select the most suitable sperm for oocyte injection. This is normally achieved by studying the motility characteristics of the sperm [3]. However, when there are very few or no motile sperm, it then becomes a matter of chance as to whether the sperm selected is viable. Our experience and that reported from other centres have shown that the viability of sperm is <50 % in samples showing complete asthenozoospermia [4].

The standard tests to assess sperm viability involve the exclusion of vital dyes, usually eosin-nigrosin, and air-drying which then render the sperm unsuitable for further use. This problem could be overcome using the hypo-osmotic swelling (HOS) test, which identifies sperm with a functionally intact plasma membrane without further damaging the sperm [5]. In cases of severe asthenozoopsermia, this would allow selection of viable sperm for oocyte injection. Although cases of successful pregnancies have been reported using the HOS test to identify viable non-motile sperm for oocyte injection [4, 6] and although the HOS test has been shown to accurately assess the viability of non-motile sperm in sperm donors [7], no study has measured the predictive value of the HOS test in identifying viable sperm in severe or complete asthenozoospermia.

The objective of this study was to perform combined HOS-viability staining on sperm from patients with severe or complete asthenozoospermia to determine the positive predictive value of the HOS test to identify viable sperm.

2 Materials and methods

Patients were identified from the regional andrology clinic, which is a tertiary referral and teaching center. All had previously diagnosed asthenozoospermia. The Hospital Ethical Committee approval had been granted prior to the study.

Fresh samples were collected by masturbation into sterile plastic containers after 4 days sexual abstinence from 20 men presenting with primary infertility and asthenozoospermia, which was defined as a total motility of < 10 %. Routine seminal analysis [8] was performed within one hour of production after liquefaction. HOS test and eosin-nigrosin (EN) viability studies were also performed, followed by a further EN stain on a HOS-pretreated aliquot.

The HOS test was performed by mixing 0.1 mL aliquots of semen with 1.0 mL of a 150 mmol/kg hypo-osmotic solution prepared by mixing 7.35 g sodium citrate and 13.5 g fructose in 1000 mL distilled water. The mixture was incubated for 60 min at 37 oC in 5 % CO2 and 95 % air. Then 0.2 mL of the mixture was placed on a slide and mounted with a coverslip and immediately examined at a magnification of ?400 under a phase contrast microscope. The percentage of reacted sperm (curled tails) and non-reacted sperm (non-curled tails) were assessed by counting a minimum of 100 sperm.

The EN stain was performed by dissolving 0.5 g eosin Y and 3 g nigrosin in 30 mL distilled water. Two drops of the prepared solution were added to a drop of the fresh semen sample on a clean microscope slide. A smear of this mixture was then made on another microscope slide and allowed to air dry. This slide was then examined under a phase contrast microscope at ?00. The percentage of viable sperm (sperm head unstained) and non-viable sperm (sperm head stained) was assessed by counting a minimum of 100 sperm.

The EN viability test was performed in exactly the same manner as described above, but using 0.5 mL sperm/HOS mixture in the place of fresh semen. All slides were examined by the same two people, the pre-study inter-observer error being <5 %.

All data were assessed for normality of distribution using the Shapiro-Wilk test. Normally distributed values are quoted as means with standard deviations (SD) or total ranges. Non-normally distributed values are quoted as medians with interquartile ranges (IQR) or total ranges when the data were small. Frequency distributions are compared using c² tests. The positive predictive value was determined directly.

3 Results

Overall the median sperm density was 5.1 million/mL (IQR 4.3~13.1) and the median total motility was 3.0 % (IQR 0~7). Seven samples showed complete asthenozoospermia. The EN stain demonstrated a mean viability of 59.0 % (range 48~69) despite the poor parameters. The HOS test demonstrated a mean of 49.9 % reacted sperm (range 40~59).

Table 1 shows the results of the combined HOS-EN test of the 2000 sperm examined. The positive predictive value of the HOS test for sperm viability is 84.2 %. The differences of EN viability (59.0 % vs. 46.8 %) and HOS reacted sperm (49.9 % vs. 37.3 %) between the raw sample and the combined slides are not statistically significant (P=0.12 for EN viability and P=0.25 for HOS).

Table 1. Results of combined HOS-EN stain on semen demonstrating severe asthenozoospermia. HOS: hypo-osmotic swelling test; EN: eosin-nigrosin. *Positive predictive value of a HOS reacted sperm is calculated at 84.2 %.

In the complete asthenozoospermia subgroup (n=7), the median sperm density was 6.1 million/mL (IQR 4.7~13.1), the mean viability 47.3 % (range 33~61) and the mean HOS reacted sperm 41.7 % (range 22~61). The positive predictive value of the HOS test in this subgroup was 79.7 %.

Although previous studies have assessed the role of the HOS test and demonstrated an association between HOS reacted sperm and viable sperm using viability/vitality stains [7, 9, 10], these have been performed on semen samples from fertile and infertile men and the higher numbers of healthy and motile sperm skew the data in favor of a positive association. This study demonstrates that the HOS test can reliably identify viable non-motile sperm with a positive predictive value of about 80 % in semen samples with severe or complete asthenozoospermia, the very group that would benefit from HOS test selection of non-motile sperm for oocyte injection [4, 6].

The HOS test assesses the functional integrity of the sperm plasma membrane. The value of the HOS test has been extensively studied, particularly with relevance to more elaborate sperm function tests, fertilization potential in IVF-ET treatment and pregnancy rates. Some authors have demonstrated a positive association with these outcomes [11, 12, 13] whereas others have reported no association [14, 15]. Despite these controversies, the HOS test remains an easy and reliably repeatable test. This study provides the confirming evidence for the continued clinical application of the HOS test.

The EN stain viability test was first described over 50 years ago [16] and remains the current standard. Viable sperm repel the vital stain, whereas dead sperm have lost the structural integrity of the plasma membrane and therefore will absorb the dye. The use of the EN stain and subsequent air drying obviously result in the inability to use these sperm for treatment.

The combined HOS-EN staining results showed that 5.9 % spermatozoa were non-viable but still HOS reacted. This discrepancy could be for two reasons. Firstly, prolonged hypo-osmotic stress may lead to irreversible effects on the cell membrane and cell death [7] or secondly, non-viable sperm may exhibit spontaneous swelling before exposure to the HOS solution and therefore be counted as HOS reacted when in fact they were already non-viable [17].

In couples undergoing infertility treatment for severe male factor with complete asthenozoospermia, fertilisation rates with ICSI are higher with motile sperm compared with randomly selected immotile sperm [18, 19]. However, oocyte fertilization and pregnancies have been reported following ICSI with HOS selected sperm in men with complete asthenozoospermia [4, 6]. Nevertheless in our experience, technical difficulties can be encountered when washing HOS reacted sperm prior to oocyte injection and other authors have used modified HOS tests to try to overcome this [20, 21]. Although, some studies have suggested that the use of HOS reacted sperm is safe [22, 23], further clinical studies need to compare the fertilization, pregnancy and live birth rates following treatment of severe/complete asthenozoosper-mia with HOS selected and non-HOS selected sperm for oocyte injection.

In conclusion, these results confirm that the HOS test can reliably predict sperm viability in cases of severe and complete asthenozoospermia.

References

[1] Kasai T, Ogawa K, Mizuno K, Nagai S, Uchida Y, Ohta S, et al. Relationship between sperm mitochondrial membrane potential, sperm motility, and fertility potential. Asian J Androl 2002; 4: 97-103.
[2] Haidl G, Kohn FM, Schill WB. Drug treatment of male fertility disorders. Asian J Androl 2000; 2: 81-5.
[3] Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of a single spermatozoon into an oocyte. Lancet 1992; 340: 17-8.
[4] Casper RF, Meriano JS, Jarvi KA, Cowan L, Lucato ML. The hypo-osmotic swelling test for selection of viable sperm for intracytoplasmic sperm injection in men with complete asthenospermia. Fertil Steril 1996; 65: 972-6.
[5] Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld LJ. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984; 70: 219-28.
[6] Ved S, Montag M, Schmutzler A, Prietl G, Haidl G, Van der Ven H. Pregnancy following intracytoplasmic sperm injection of immotile spermatozoa selected by the hypo-osmotic swelling-test: a case report. Andrologia 1997; 29: 241-2.
[7] Smikle CB, Turek PJ. Hypo-osmotic swelling can accurately assess the viability of nonmotile sperm. Mol Reprod Dev 1997; 47: 200-3.
[8] World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Semen-Mucus Interaction. Cambridge: Cambridge University Press, 1987.
[9] Chan PJ, Tredway DR, Corselli J, Pang SC, Su BC. Combined supravital staining and hypoosmotic swelling test. Hum Reprod 1991; 6: 1115-8.
[10] Ramirez JP, Carreras A, Mendoza C. Sperm plasma membrane integrity in fertile and infertile men. Andrologia 1992; 24: 141-4.
[11] Van der Ven HH, Jeyendran RS, Al-Hasani S, Perez-Pelaez M, Diedrich K, Zaneveld LJ. Correlation between human sperm swelling in hypoosmotic medium (hypoosmotic swelling test) and in vitro fertilization. J Androl 1986; 7: 190-6.
[12] Check JH, Stumpo L, Lurie D, Benfer K, Callan C. A comparative prospective study using matched samples to determine the influence of subnormal hypo-osmotic swelling test scores of spermatozoa on subsequent fertilization and pregnancy rates following in vitro fertilization. Hum Reprod 1995; 10: 1197-200.
[13] Oosterhuis GJ, Hampsink RM, Michgelsen HW, Vermes I. Hypo-osmotic swelling test: a reliable screening assay for routine semen specimen quality screening. J Clin Lab Anal 1996; 10: 209-12.
[14] Barratt CL, Osborn JC, Harrison PE, Monks N, Dunphy BC, Lenton EA, et al. The hypo-osmotic swelling test and sperm mucus penetration test in determining fertilization of the human oocyte. Hum Reprod 1989; 4: 430-4.
[15] Biljan MM, Taylor CT, Manasse PR, Joughin EC, Kingsland CR, Lewis-Jones DI. Evaluation of different sperm function tests as screening methods for male fertilization potential - the value of the sperm migration test. Fertil Steril 1994; 62: 591-8.
[16] Blom E. A one-minute live-dead sperm stain by means of eosin-nigrosin stain. Fertil Steril 1950; 1: 176-81.
[17] Correa-Perez JR. Testing for 'life'- how predictable? Fertil Steril 2002; 77: 426-7.
[18] Nagy ZP, Verheyen G, Tournaye H, Van Steirteghem AC. Special applications of intracytoplasmic sperm injection: the influence of sperm count, motility, morphology, source and sperm antibody on the outcome of ICSI. Hum Reprod 1998; 13 (suppl 1): s143-54.
[19] Shulman A, Feldman B, Madgar I, Levron J, Mashiach S, Dor J. In-vitro fertilization treatment for severe male factor: the fertilization potential of immotile spermatozoa obtained by testicular extraction. Hum Reprod 1999; 14: 749-52.
[20] Verheyen G, Joris H, Crits K, Nagy Z, Tournaye H, Van Stierteghem A. Comparison of different hypo-osmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection. Hum Reprod Update 1997; 3: 195-203.
[21] Sallam N, Farrag A, Agameya A, Ezzeldin F, Eid A, Sallam A. The use of a modified hypo-osmotic swelling test for the selection of viable ejaculated and testicular immotile spermatozoa in ICSI. Hum Reprod 2001; 16: 272-6.
[22] El-Nour AM, Al Mayman HA, Jaroudi KA, Coskun S. Effects of the hypo-osmotic swelling test on the outcome of intracytoplasmic sperm injection for patients with only nonmotile spermatozoa available for injection: a prospective randomized trial. Fertil Steril 2001; 75: 480-4.
[23] Zeyneloglu HB, Baltaci V, Ege S, Haberal A, Batioglu S. Detection of chromosomal abnormalities by flourescent in-situ hybridization in immotile viable spermatozoa determined by hypo-osmotic sperm swelling test. Hum Reprod 2000; 15: 853-6.

home

Correspondence to: Dr William M. Buckett, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, McGill University, Royal Victoria Hospital, 687, avenue des Pins Ouest, montral, (Qubec), Canada H3A 1A1.
Tel: +1-514-842 1231, Fax: +1-514-843 1496
E-mail: william.buckett@muhc.mcgill.ca
Received 2003-04-15  Accepted 2003-08-04

This article has been cited by other articles:

• Bjorndahl L, Soderlund I, Johansson S, et al.Why the WHO recommendations for eosin-nigrosin staining techniques for human sperm vitality assessment must change JOURNAL OF ANDROLOGY 25 (5): 671-678 SEP-OCT 2004. [Abstract] [Full Text]