7.1 Physiological Aspects

The hypo-osmotic swelling (HOS) test, introduced in 1987 to assess the functional integrity of the human sperm membrane, appears to have withstood the test of time. Many articles have been published on the subject since its inclusion in the third edition of the World Health Organization (WHO) manual in 1992 [1, 2, 3, 4, 5].

The concept of the HOS test is that only sperm with intact membranes will swell (balloon) when exposed to hypo-osmotic conditions due to the influx of water, thus indicating that the membrane is morphologically intact and physiologically active [6]. A membrane, such as the one used for dialysis, will allow solutes of a certain size to passively pass through it to maintain osmotic equilibrium when exposed to hypo-osmotic conditions, a phenomenon called semi-permeability. A physiologically active membrane, however, will prevent such an osmotic equilibrium until it reaches a critical stage when the osmotic stress cannot be actively managed, resulting in rupture of the membrane. Indeed, physiologically, the osmolarity in the intracellular compartment is higher than that of the extracellular compartment, resulting in an osmotic pressure gradient. This would normally cause an influx of water into the cell, which would in turn cause swelling, if it was not for the presence of active ionic transport processes (physiologically active membrane). The maintenance of cell volume relies heavily on those systems. However, they are only efficient up to a certain point, and in very low osmolarity environment, such as during the HOS test, they can be overwhelmed, leading to cell swelling [7].

The HOS test evaluates different characteristics of the sperm membrane as compared to the dye exclusion test. The dye exclusion test is based on how the living cell membrane acts as a physical barrier to dye penetration, thus, not allowing nuclear staining. When the membrane is damaged, such as that found in non-vital (dead) cells, it allows the entry of dye into the cell. Treatment of sperm with the membrane fixative glutaraldehyde (2 percent) had no significant effect on Eosin Y (0.5 percent) uptake versus non-treated cells. In contrast, almost no swelling was observed in the HOS test after glutaraldehyde treatment [8], suggesting that membrane function, rather than a physically intact membrane, is of critical importance when sperm are subjected to a HOS test. In addition, electron microscopy indicated that the sperm membrane of the tail bulges and swells in response to the hypo-osmotic condition. Bollendorf et al. [9] emphasized this phenomenon, showing that the majority of patients with a subnormal HOS test have a normal vitality based on the dye exclusion test, and that only 12.5 percent of men with a low HOS test exhibit low vitality. It therefore appears that while the dye exclusion test explores the membrane in a structural way, the HOS test is based on its functional capacities, which explains the discrepancy between dye exclusion and HOS tests.

7.2 Technical Aspects

The HOS test is performed by combining 0.1 mL of ejaculate with 1.0 mL of a hypo-osmotic solution prepared by mixing 7.35 g sodium citrate and 13.51 g fructose in 1 L of distilled H₂O. After incubation of the solution for at least 30–60 minutes, but no more than 120 minutes at 37⁰C, 100 or 200 spermatozoa are observed with a phase contrast microscope, and the percentage of spermatozoa with tail changes typical of a reaction in the HOS test (swollen, HOS-reactive, or HOS-positive spermatozoa) is determined [6, 10]. No additional equipment is required. The spermatozoa can be fixed with formaldehyde (18.5 percent, 0.1 mL) or ethanol/acetic acid (3:1), which retains the shape of the spermatozoa, so that they can be stored and observed at a later date [11]. A longitudinal study evaluating the semen quality monthly over nine months from 45 men revealed a mean of 64.08 percent HOS-positive with a median fluctuating only 7 percent over that period, which highlights the precision and reproducibility of the HOS test as compared to sperm motility [12].

7.3 Hypo-Osmotic-Swelling-Positive Spermatozoa Evaluation as a Diagnostic Tool

Ejaculates are classified as normal if they contain 60 percent or more HOS-positive spermatozoa and are regarded as abnormal if fewer than 50 percent of the spermatozoa are HOS-positive. The 50–59 percent range is considered “equivocal” [11]. The large majority (96 percent) of the ejaculates from 1890 participants who requested a vasectomy gave more than 60 percent HOS-positive spermatozoa [13]. Additional studies have confirmed the validity of these cut-off values, based on outcomes such as pregnancy rates or embryo implantation rates [14, 15, 16, 17, 18].

A physiologically active membrane is a prerequisite for sperm motility, capacitation, acrosome reaction and binding of the spermatozoon to the oolemma. The HOS test may detect subtle injury or incompetence of the sperm plasma membrane, which may be responsible for the reduced fertilizing potential of the sperm that results in non-viable embryos, low pregnancy rates, or miscarriages. Thus, the assessment of the sperm membrane may prove valuable in routine semen analysis, especially when the value of conventional semen analysis is the subject of debate [19].

Although significant, the correlation between the dye exclusion test and the HOS test results is surprisingly low with a correlation coefficient of r = 0.52 [6], even though the HOS test had initially been thought to evaluate sperm vitality, similarly to the dye exclusion test. In addition, the correlation coefficients between the HOS test results and several standard sperm parameters are generally so low that the outcome of the HOS test cannot be accurately predicted from these other observations [20]. However, the correlation between the HOS test results and the zona-free hamster egg penetration (Sperm Penetration Assay; SPA) as well as in vitro fertilization outcome is very high [6, 11, 21, 22, 23, 24, 25, 26]. Multiple studies have reported on the ability of the HOS test to predict in vivo and in vitro pregnancies [2, 17, 26, 27, 28, 29] as well as recurrent pregnancy loss [30, 31, 32].

Using the HOS test as an independent variable for sperm function and the IVF outcome (fertile/infertile) as the dependent variable in a forward logistic regression analysis, the HOS test could correctly predict the IVF outcome in 88.4 percent of the cases with 12.0 percent false positive and 7.7 percent false negative rate [23](Jeyendran et al., 1989).

Check et al. [2, 15] concluded that the results of the HOS test are significantly more reliable than the results of the standard semen analysis as a predictor of conception. These investigators found that no couple, regardless of the standard semen analysis results, achieved a pregnancy when the HOS test was abnormal. By contrast, even if the semen analysis was abnormal, the large majority of men were able to fertilize their partners’ oocytes as long as the HOS test was normal or equivocal (i.e., ≥50 percent reactive spermatozoa). Comparing the HOS test with normal sperm morphology, SPA, and IVF, Coetzee et al. [22] concluded that an abnormal HOS test was the most reliable indicator of a male factor infertility.

Incidentally, the HOS test has also been effectively used to evaluate spermicidal effects of chemotherapeutic agents [33, 34], pesticides [35] and to screen potential contraceptive agents [36, 37]. Polystyrene and polypropylene specimen containers affect the HOS test response as compared to glass containers [15]. Moreover, common laboratory treatment procedures may also influence the hypo-osmotic swelling responses [38]. Finally, the HOS test also appears to be useful as a measure of cryosurvival [16, 39, 40].

7.4 A Further Look: The Hypo-Osmotic Swelling Classification

The different types of tail swelling that can be observed under hypo-osmotic condition are: no tail swelling (a), swelling present only at the tip of the tail (b and c), bending at the hairpin curvature of the tail (d and e), shortened and thickened tail (f), and swelling of the entire tail (g), as described by Jeyendran et al. [6] (Figure 7.1). With regard to this classification, sperm that were formerly classified as “HOS-positive” are sperm that exhibit a flagellar swelling, that is to say a swelling of the “b” to “g” categories. Continuous monitoring of tail swelling changes of individual sperm revealed several subsets of sperm populations within an ejaculate that respond differently to hypo-osmotic stress [41]. It has been proposed that the differential analysis of the various types of tail curling may aid in minimizing false positives [42]. For instance Bassiri et al. [43] compared different types of sperm swelling under hypo-osmotic conditions and concluded that the sperm with the tail tip swelling were the most competent based on analysis of head morphology, DNA fragmentation, protamine deficiency (nuclear immaturity marker) and Annexin V expression (apoptotic marker). Stranger et al. [44] had previously confirmed the DNA competency of the sperm with tail tip swelling.

Figure 7.1 Schematic representation of typical morphological changes in human spermatozoa subjected to hypo-osmotic stress. (A) No change, (B)–(G) various types of tail changes. Swelling in tail is indicated by the hatched area.

In that study, looking at the different HOS classes, the HOS test morphology exhibited a stronger association with DNA fragmentation than with the presence of head vacuoles, suggesting that membrane alteration is an earlier phenomenon than vacuole formation in sperm physiology. They showed that abnormal sperm samples (based on routine semen analysis) exhibited a higher proportion of HOS test-positive “a” spermatozoa, and a lower proportion of HOS test “d/e” and “f” spermatozoa, suggesting that the latter are associated with normal fertility potential. Similar results were obtained with regards to DNA fragmentation, with the “d/e” and “f” classes carrying the lowest DNA fragmentation rate. Bassiri et al. [43] showed similar results, this time suggesting that the “b” and “d” classes were associated with the most fertile sperm in terms of DNA fragmentation, protamine deficiency, and externalization of phosphatidylserine. These two studies were performed not at the sperm level, but at the global level, looking at the aforementioned quality parameters globally, and correlating them to the respective proportion of each HOST class.

7.5 Hypo-Osmotic Swelling Test-Based Sperm Selection

As opposed to those two studies, the following were performed “cell by cell”, looking at each cell individually based on their HOST class, suggesting the HOS test could be used to select spermatozoa during intra-cytoplasmic sperm injection (ICSI). Pang et al. [5] showed that HOS can allow for the selection of sperm with a lower rate of aneuploidy in patients with a normal karyotype, with 17 times less aneuploid sperm in the “b”, “c”, and “d” types as compared to the rate observed prior to the HOS test. Rouen et al. [45] evidenced that the HOS test could be used to select chromosomally balanced spermatozoa in chromosomal rearrangement carriers (reciprocal and Robertsonian translocations, pericentric inversions), describing a new HOS test class termed “b+”. These results on normal karyotype aneuploidy and abnormal segregation in chromosomal rearrangement carriers suggest that membrane integrity can be viewed as a reflection of nuclear balance and organization. Rouen et al. hypothesized that an abnormal chromosomal content would prevent full nuclear condensation, and, in turn, might initiate an early apoptosis process, altering the sperm membrane. This membrane alteration would consequently alter the sperm response to hypo-osmolarity, and affect the tail conformation when incubated in a hypo-osmotic solution. These studies conclude that the HOS test may aid in selecting chromosomally balanced spermatozoa in patients with high aneuploidy rates or chromosomal rearrangement carriers, and selecting spermatozoa with high nuclear and membrane quality. Recent studies by Rouen et al. (to be published) suggest that in patients with a normal karyotype, b+ spermatozoa might be associated with significantly lower rates of DNA fragmentation and DNA decondensation and a more normal nuclear architecture. This suggests that b+ spermatozoa would be the most competent.

Since the HOS test identifies sperm with intact functional membranes without altering them, it has been effectively utilized to select sperm for ICSI from a population of non-motile sperm [3, 4, 46, 47, 48, 49, 50, 51, 52]. Indeed, according to the WHO guidelines [53], it is possible and safe to perform HOS prior to ICSI, in order to select viable spermatozoa. Recent studies suggest that this could also be used to select highly competent spermatozoa in a way to optimize reproductive outcome.

7.6 Conclusion

The HOS test is of particular interest for routine semen analysis because it measures an entirely different entity than the already existing sperm parameters. Compilation of all the data that have been published so far, and reanalysis, where appropriate and possible, leads to the conclusion that the HOS test provides additional information not obtained by conventional semen analysis. An abnormal HOS test is often associated with poor results in the SPA and unsuccessful IVF, and in-vivo fertilization rarely occurs when the HOS test is abnormal and is independent of the other semen parameters. Thus, an abnormal HOS test provides reasonable certainty that the ejaculate is infertile [54]. Furthermore, it has been suggested that response to hypo-osmolarity not only reflects membrane integrity, but also spermatic nuclear organization. On this basis, it can be concluded that the HOS test is a useful adjunct to existing sperm assays and could be included as a standard test during routine semen analysis. In addition, the HOS test can be used to monitor other sperm procedures, such as sperm selection for ICSI, sperm processing, sperm cryosurvival, and the effect of toxins on sperm. Finally, HOS can also be used to inject highly competent spermatozoa prior to ICSI, in patients with high aneuploidy rates, in patients with chromosomal rearrangements, or even in the global infertile population.