Abstract
The incidence of infertility is up to 20–25 percent in men with poor semen quality with a contribution of the male factor in 30–50 percent of couples undergoing assisted reproduction, including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). In 17 studies sampling 6410 women, the proportion of couples seeking such medical care was, on average, 56.1 percent (range 42–76.3 percent) in more developed countries and 51.2 percent (range 27–74.1 percent) in less developed countries [1]. Investigation of male infertility or sub-fertility basically comprises of semen analysis [2]. However, a standard semen analysis cannot always assess the multifunctional events and biological properties that spermatozoa express following capacitation. In many cases, it is only when couples fail to achieve conception, the male factor is suspected and advanced laboratory tests are recommended to establish this reliably.
12.1 Introduction
The incidence of infertility is up to 20–25 percent in men with poor semen quality with a contribution of the male factor in 30–50 percent of couples undergoing assisted reproduction, including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). In 17 studies sampling 6410 women, the proportion of couples seeking such medical care was, on average, 56.1 percent (range 42–76.3 percent) in more developed countries and 51.2 percent (range 27–74.1 percent) in less developed countries [1]. Investigation of male infertility or sub-fertility basically comprises of semen analysis [2]. However, a standard semen analysis cannot always assess the multifunctional events and biological properties that spermatozoa express following capacitation. In many cases, it is only when couples fail to achieve conception, the male factor is suspected and advanced laboratory tests are recommended to establish this reliably.
The predictive value of semen analysis has been found to be limited, particularly in cases of oligoasthenoteratozoospermia, idiopathic or unexplained infertility. While considering the conditions of the partner, it is also imperative for the clinician to incorporate an effective and quick test of sperm function into the workup algorithm of such patients who may benefit from a variety of therapeutic options including assisted reproductive technology (ART) [3–5].These functional tests include zona binding, acrosome integrity, reactive oxygen species, chromatin decondensation and DNA integrity, among others.
As a clinically important test in the diagnosis and treatment of infertility [6], sperm-zona binding is one of the most powerful indicators of sperm fertilizing ability of oocytes in vitro [7, 8]. Since defective sperm-zona pellucida binding has been recognized to be a very common cause of fertilization failure in assisted reproduction [9, 10], this test is recommended in cases of poor fertilization, unexpected fertilization failure [5] and failed conventional IVF [11].
As of today, it is well known that fertilization starts when the male gamete binds to the antigenically/biochemically complex translucent extracellular glycoproteinaceous matrix coating of the oocyte, called the zona pellucida (ZP). The ZP is formed during the early stages of folliculogenesis and surrounds the embryo until the time of implantation [12]. The inner surface of the ZP is particulated and granular, whereas the outer surface resembles a fenestrated mesh or lattice [13]. In the human, the zona consists of four major component glycoproteins (ZP1, ZP2, ZP3 and ZP4), of which ZP3 acts as the primary sperm receptor and acrosome reaction inducer, whereas ZP2 is a secondary receptor [14]. During binding, any sperm first attach loosely to the ZP, which is closely followed by tight attachment to specific receptors. This ability of the sperm to bind tightly to the ZP is a critical and mandatory step in fertilization [15] and subsequent embryonic development [16]. Binding capacity depends heavily on the stage of maturity of the oocytes, the presence of complementary binding sites/receptors on the surface of the sperm as well as the surface structure of the ZP [17]. The structural and biochemical changes of the ZP that accompany the oocyte maturation process facilitate the highest binding when oocytes mature to the stage best for fertilization [18, 19].
For the first time, human sperm-oocyte interaction was described in an assay to record zona penetration. The methodology of this assay formed the basis of zona binding assays that developed subsequently [20]. Two tests have been designed to assess sperm capacity to bind to the ZP, namely the hemizona assay (HZA) [21] and the sperm-ZP binding ratio test [22].
12.2 Hemizona Assay
12.2.1 Clinical Utility
In andrology, the identification of specific gamete dysfunction is one of most significant steps prior to assisted reproduction [23]. The clinician usually opts for ART if at least one of the following conditions are fulfiled – i) failure of andrology treatment, ii) diagnosis of idiopathic infertility, iii) moderate to high level of sperm abnormalities revealed by standard semen analysis, and iv) functional abnormalities of sperm detected by advanced tests [24]. The HZA is a highly significant, internally controlled, homologous bioassay providing functional aspects of the sperm, which may assist the clinician in determining the management of men for whom conventional IUI and IVF therapy is likely to be unsuccessful and should rather be referred to ICSI [25, 26]. It has been highly predictive of IVF [27] and IUI fertilization and pregnancy outcomes [18]. Results of this test have also been useful in counseling couples before allocating them into controlled ovarian hyperstimulation (COH/IUI therapy) [26].
The percentage of normal spermatozoa bound to the ZP under HZA conditions reported for both normo- and teratozoospermic men also showed significant improvement when compared to the percentage of normal spermatozoa found after the swim-up procedure (Figure 12.1) [28]. Multiple regression analyses have demonstrated that sperm morphology is the most significant predictor of sperm-zona binding in the HZA, when compared to other sperm variables from the original semen sample (r=0.83, p=0.0001). However, curvilinear velocity (VCL) and hyperactivated motility (HA) have been the most significant predictors of successful zona binding, after separation of the motile sperm fraction (r=0.47 and r=0.46, respectively p=0.001) [28–30]. Sperm morphology and HZA data correlated with fertilization rates in a prospective study of a large number of infertile patients before IVF therapy [11].
The diagnostic utility of HZA has been confirmed based on a set of criteria such as the ability to produce few false-negative and false-positive values, as well as good positive (PPV) and negative (NPV) predictive values [31]. In terms of fertility and fertilization rates, a compilation of studies predicted high HZA sensitivity (75−100 percent), good specificity (57–100 percent), and high PPV (79–100 percent) and NPV (68–100 percent) [32].
12.2.2 Procedure and Evaluation
As shown in Figure 12.2, the ZP of freshly isolated or stored oocytes are divided into half either microsurgically (using a micromanipulator) or manually [25] followed by incubation of one half (called a hemizona) with fertile donor sperm (positive control) and the other half with patient sperm [33]. The 100 µL semen suspension drop containing 500,000 motile sperm/mL are kept under light white mineral oil and are co-incubated with each hemizona in a 35 × 10 mm Petri dish for four hours at 37°C with 5 percent CO2 in air. Both hemizonae, the control and the test one, are rinsed in a cold medium drop five times with a large drawn glass pipette to dislodge loosely attached sperm and then transferred to a fresh medium drop and positioned with the outer surface upward [21, 23, 34]. The numbers of tightly bound sperm to the respective hemizonae are counted with a 400× phase contrast microscopy [35, 36]. Binding capacity is expressed as the hemizona index (HZI) and calculated by expressing the number of tightly bound patient sperm as a percentage of the number of tightly bound control sperm [23, 35]. The peak number of control sperm bound to the hemizona ranges from 42–215 whereas that of the patient ranges from 27–142, respectively. Based on these findings, a cut-off binding value of 34 sperm was ascertained to be indicative of inferior ZP or subnormal control [23, 35].
Figure 12.2 The Hemizona Assay [37]. An oocyte is removed from the working droplet and placed in the middle of the microscopic field. The micromanipulation blade is lowered to touch the top surface of the oocyte and the oocyte is flattened by further lowering. The oocyte is bisected into two identical hemizonae using side-to-side excursions. Hemizona pairs are kept together in fresh drops of culture medium after removing the residual cytoplasm. Hemizona pairs are placed in the incubator overnight before transferring into sperm droplets. When sperm are ready for incubation with the hemizonae, one hemizona is transferred into the drop containing control (donor) sperm and the corresponding hemizona is transferred into the drop containing patient’s sperm.
12.2.3 Bisecting of Oocytes
Post-mortem oocytes obtained from donated ovarian tissue were used during the developing stages of the assay. A complete micromanipulation system is required for bisecting the oocytes. The assay is performed by separately incubating matching bisected halves of a ZP with sperm from a patient and proven fertile control, respectively. Oocytes are placed in 100 mm Petri dish, culture medium is poured into the dish to a depth of 3–4 mm. A holding pipette is used to stabilize the oocyte during cutting [5]. Using total magnification of 200× the cutting blade is lowered flattening the oocyte to initiate a midline cut and, with side-to-side excursions, two identical HZ are subsequently produced (Figure 12.3).
Figure 12.3 Bisecting an oocyte by side by side excursions of the micromanipulation blade [37]. The oocyte is held by a holding pipette and flattened by touching its surface by the micromanipulation blade.
Alternatively, a cost-effective manual hand-cutting method of oocytes recorded comparable recovery rate, diameter size of the hemizonae, sperm binding and HZI thereby advocating the possible elimination of an expensive micromanipulator making the assay more affordable to many fertility clinics across the globe [25, 38].
12.2.4 Oocyte Sources
During experimental studies, hemizonae were obtained from prophase I oocytes from post mortem ovarian tissue from different age groups namely, 7 months, 5 years, 7 years, 12 years and 30 years in the first experiment (Table 12.1) [39]. The age group studies indicated that ovarian age does not have any influence on the ZP’s capacity to bind spermatozoa.
Table 12.1 The Mean Number of Bound Sperm among the Different Age Groups [39]
| Age | Number sperm bound |
|---|---|
| 7 months | 38.9±17 |
| 5 years | 31.0±27 |
| 7 years | 49.3±21 |
| 12 years | 32.8±18 |
| 30 years | 39.5±17 |
| Pooled data | 37.7±7 |
| Donated prophase oocytes | 33.0±20. |
Hemizonae can also be recycled for at least a second binding experiment as metaphase II oocytes with previous exposure to sperm were found to retain their binding capacity. Zonae that had been exposed to sperm and that were subsequently stripped from bound sperm, revealed a mean number of bound sperm after re-insemination that were significantly higher than the prophase I oocytes; 115.0±2.8 versus 35.6±12 (P<0.0001) [2]. These results indicate that the upper limit for sperm binding in the presence of sperm populations with known zona binding defects and possibly poor zonae is 34 sperm per hemizona. The lower cut-off value of 34 bound sperm was used for sperm populations with normal (>14 percent normal forms) morphologic features and zona binding potential. Proven fertile sperm samples unavoidably include results with ZP showing inferior sperm binding capacity [40].
The problems of the availability of ZP from oocytes by surgical removal of ovarian tissue or by ovarian follicular aspiration has been largely overcome by utilizing surplus oocytes from IVF programs after gonadotropin stimulation, oocytes derived from post mortem tissue and various storage methods as alternatives of fresh oocytes [25, 39]. Under oocyte storage conditions, the ZP has been able to exhibit good sperm binding [23]. Fresh, long-term DMSO stored [41, 42] and short-term salt-stored oocytes [23, 43] have been used successfully during the initial stages of the assay. Even though the long-term technique of ultralow temperature liquid nitrogen storage could preserve oocytes for up to 12 months, the short-term method of up to seven days storage at 4ᵒC gained gradual widespread use in the HZA [39].
12.3 Perspective
The difficulty in obtaining the precious human ZP for the HZA has largely been overcome by the use of oocytes that fail to fertilize during ICSI or IVF [44]. Furthermore, an easy to perform bioluminescence-enhanced detection system employing a pool of solubilized ZP has been developed for easier routine use in the diagnosis of male infertility. This highly sensitive assay labels the ZP proteins with a luminescent probe and measures the light emission by the luciferin-luciferase system after almost every D-luciferin molecule is oxidized by the enzyme luciferase [45]. Yet, these approaches are still rather laborious and only very specialized laboratories are able to perform the test.
Recently, a 3D system has been developed to facilitate the ART investigations that recreated the spherical shape oocytes and the biochemical characteristics of the ZP [46]. As human sperm bind to the N-terminus of ZP2, thereby acting as a ligand for sperm binding [40, 47], the N-terminus of ZP2 attached agarose beads have been modeled to decoy sperm and prevent fertilization in vitro and in vivo [48]. Another in vitro model based on magnetic sepharose beads coated with single recombinant ZP glycoproteins that mimic the 3D oocyte’s shape has been proposed as a diagnostic predictor of sperm function in male infertility patients. The secreted recombinant ZP glycoproteins are capable of conjugating to beads thereby forming a 3D oocyte-like shape that supports sperm binding and reflects the event of capacitation successfully [49]. However, the problem of glycosylation remains to be addressed as the correct amino acid sequence alone is not sufficient as ZP binding with sperm is rather mediated by the glycosidic residues [50].
In conclusion, the HZA may be incorporated into the workup algorithm of patients as an effective test of sperm function, who may benefit from a variety of therapeutic options including ART. As a diagnostic test the HZA may be particularly beneficial in oligoasthenoteratozoospermic males as well as in men with idiopathic or unexplained infertility.
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