Abstract
Today, about 10–15 percent of couples at reproductive age worldwide are infertile and they are unable to conceive naturally without medical assistance. Infertility can be caused by male-only factors, female-only factors or a combination of both. However, the cause of infertility is currently unidentifiable in about 20–30 percent of patients, who are classified as “unexplained infertility”. Currently, there is a lack of effective medical treatment for most infertile couples to achieve natural pregnancy. Although assisted reproductive technology (ART) such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are popular and effective procedures to treat both female and male factor infertility, they are very expensive and not always successful. Some patients require several attempts of treatment cycles to achieve a pregnancy and bear huge financial and emotional costs in the process.
11.1 Introduction/Background
Today, about 10–15 percent of couples at reproductive age worldwide are infertile and they are unable to conceive naturally without medical assistance. Infertility can be caused by male-only factors, female-only factors or a combination of both. However, the cause of infertility is currently unidentifiable in about 20–30 percent of patients, who are classified as “unexplained infertility”. Currently, there is a lack of effective medical treatment for most infertile couples to achieve natural pregnancy. Although assisted reproductive technology (ART) such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are popular and effective procedures to treat both female and male factor infertility, they are very expensive and not always successful. Some patients require several attempts of treatment cycles to achieve a pregnancy and bear huge financial and emotional costs in the process.
In clinical ART, the two major treatment procedures – conventional IVF and ICSI – are applied according to the cause of infertility. In general, IVF is effective in treating female factor infertility, whereas ICSI is effective for male factor infertility. Accurate diagnosis of sperm function is therefore critical in determining which treatment option is most suitable, as well as assisting couples to achieve optimal ART outcomes from each oocyte collection cycle. In most ART clinics, assignment of patients to IVF or ICSI treatment is mostly based on routine semen analysis results according to the World Health Organization (WHO) manual [1]. Patients with normal or mildly abnormal results are treated by IVF and those with moderate to severe sperm defects are treated by ICSI. Unfortunately, conventional semen analysis provides limited diagnostic information for the sperm fertilizing ability, and many patients with normal semen analysis but with subtle sperm defects impairing sperm-oocyte interaction go undiagnosed [2, 3].
In conventional IVF, failure of fertilization is highly associated with defective sperm-zona pellucida (ZP) binding and penetration, which is mostly due to sperm and not oocyte abnormalities [4, 5]. Before ICSI became available, patients with failure of fertilization in IVF were commonly associated with abnormal semen such as oligo-, astheno- and terato-zoospermia. Now, these patients with abnormal semen analysis are routinely treated by ICSI. However, patients with unexplained infertility and normal semen analysis are usually treated by conventional IVF in the initial cycle. If fertilization is low or completely fails, they are then treated by ICSI in the subsequent cycle. In order to minimize this risk of failure of fertilization in IVF, it is very important to diagnose subtle sperm defects for patients with unexplained infertility before starting ART treatment.
We have developed the bioassays using human oocytes that failed to fertilize in IVF/ICSI to examine the ability of sperm-ZP binding and ZP-induced acrosome reaction (AR) [6, 7]. The ZP-induced AR is a very good marker for the ability of sperm-ZP penetration [8]. The clinical predicative value of these tests for IVF fertilization rate has been extensively studied [9, 10]. Our large set of clinical data shows that defective sperm-ZP interaction is a major cause of male infertility and failure of fertilization in conventional IVF [11–13]. We identified two major defects of sperm-ZP interaction in unexplained infertile men: 1) defective sperm-ZP binding (DSZPB) in which sperm have little to no ability to bind to the ZP [13]; and 2) disordered ZP-induced AR (DZPIAR) in which sperm have normal capacity to bind to the ZP but are unable to undergo the AR induced by the ZP [10, 12]. Both defects severely impair the ability of sperm to penetrate the ZP of human oocytes which then leads to failure of fertilization in conventional IVF. We found that about 25–30 percent of patients with unexplained infertility and normal semen analysis have DSZPB or DZPIAR [11, 13]. These subtle defects are unable to be diagnosed by routine semen analysis and therefore require sperm-ZP binding and ZP-induced AR tests using human oocytes.
11.2 Principle/Mechanism
During the process of human fertilization in vivo or under IVF condition, fertile sperm must be able to bind to the ZP, undergo the AR, penetrate the ZP and finally fuse with the egg plasma membrane [14]. In the literature, the mechanism of human sperm-ZP interaction has been well established. The ZP of the human oocyte consists of four glycoproteins; ZP1, ZP2, ZP3 and ZP4. The ZP3 is regarded as the primary receptor responsible for binding acrosome intact sperm and inducing the AR. If there are defects impairing either sperm-ZP binding or the ZP-induced AR, the sperm will be unable to penetrate the ZP and fertilize oocytes under IVF condition.
In the clinical IVF program, we have observed a large variation in the number of sperm bound to the ZP between individual oocytes in IVF. Due to the variability of ZP quality, the sperm-ZP binding test is designed based on the competitive binding of patient and control (fertile donor) sperm labeled with different fluorochromes (green and red colors) to the same oocyte [6]. Under this experimental condition, if patient sperm have similar ZP binding capacity as fertile control sperm, the number of sperm bound to the same oocyte should be very similar. On the other hand, if patient sperm has DSZPB, the number of sperm bound to the ZP will be significantly lower or may be none.
The ZP-induced AR test is designed to predict the ability of sperm-ZP penetration in infertile men with normal semen analysis and normal sperm-ZP binding [7, 8]. For this test, patient sperm are incubated with a group of four oocytes. If the sperm have normal ZP binding capacity, then all sperm bound to the ZP will be removed from the surface to examine the ZP-induced AR. Our previous study showed that there is a highly significant correlation between the ZP-induced AR and the proportion of ZP penetrated and IVF fertilization rate [8]. Development of the ZP-induced AR test leads us to discover a subtle sperm defect called DZPIAR which causes failure of fertilization in IVF for patients with normal semen analysis and normal sperm-ZP binding [12].
11.3 Brief Methods/Protocol
11.3.1 Sources of Human Oocytes
For clinical tests of sperm-ZP binding and the ZP-induced AR, it is essential to use the ZP of human oocytes as there is currently no artificial human ZP or active recombinant human ZP available. In clinical IVF/ICSI, the average fertilization rate is about 70–80 percent. The remaining 20–30 percent of unfertilized oocytes, as well as immature oocytes (GV and MI) unsuitable for ICSI, can be used for clinical tests of sperm-ZP binding and ZP-induced AR. Our previous study confirmed that the ZP of immature oocytes has similar biological activity to those of mature oocytes for binding sperm and inducing the AR [13]. The oocytes can be used either fresh or after storage in a high concentration of salt solution (1M ammonium sulphate). Salt stored oocytes must be washed with culture medium to remove salt before being used for the test [6, 9].
11.3.2 Competitive Sperm-Zona Pellucida Binding Test
For this test, patient sperm (test) are labeled with the green fluorescence fluorescein isothiocyanate (FITC), and fertile donor (control) sperm are labeled with the red fluorescence tetramethylrhodamine isothiocyanate (TRITC). A mixture of equal numbers (1 × 105/mL) of labeled motile test and control sperm is incubated with a group of four oocytes for two hours. After incubation, the oocytes are washed with a large bore (250–300 µm) pipette to dislodge loosely attached sperm; then the number of green and red sperm tightly bound to the ZP are counted under a fluorescence microscope, and the ratio of test to control sperm is calculated and used as an endpoint [6, 9]. A sample of labeled test and control sperm bound to a salt stored human ZP is shown in Figure 11.1.
Figure 11.1 These photos are an example of the competitive sperm-ZP binding test using two different colored fluorescent sperm: A shows patient (test) sperm labeled with the green fluorescence (FITC); B is fertile donor (control) sperm labeled with the red fluorescence (TRITC). In image C, there are 14 green sperm and 12 red sperm bound to a salt-stored human ZP.
11.3.3 Zona Pellucida-Induced Acrosome Reaction Test
The ZP-induced AR test examines the ability of sperm undergoing physiological AR induced by the ZP after binding to the ZP [8]. Defective sperm-ZP induced AR will lead to a failure of sperm-ZP penetration and fertilization in vitro. Thus, the ZP-induced AR is an excellent marker for the ability of sperm-ZP penetration [3, 8]. This test is very simple (see Figure 11.2): a group of four oocytes are incubated with motile sperm selected by swim-up or discontinue gradients centrifugation for two hours then transferred to fresh medium to dislodge or remove all sperm loosely bound to the surface of the ZP. The number of tightly bound sperm remaining are counted under an inverted phase contrast microscope with 200× magnification. Then, all the ZP-bound sperm are removed to a glass slide for examination of the AR by staining with Pisum sativum agglutinin (PSA-FITC) conjugated with fluorescein isothiocyanate. For each sample, 200 sperm will be scored under fluorescent microscope using excitation wavelengths of 450–490 nm and a magnification of 400×. When more than half the head of a sperm is brightly and uniformly fluorescing, the acrosome is considered intact. Sperm with a fluorescing band at the equatorial segment or without fluorescence are considered acrosome reacted (Figure 11.2) and the percentage of ZP-induced AR is used as an endpoint [3, 11].
Figure 11.2 This is a diagram of the ZP-induced AR test. First, motile sperm are incubated with a group of four oocytes for two hours. The sperm which bind tightly to the ZP are then assessed after washing the oocytes to dislodge loosely adherent sperm. All sperm which bind to the surface of the ZP are removed by aspiration of the oocyte in and out of a small pipette. The removed ZP-bound sperm are then stained with fluorescein labeled Pisum sativum agglutinin for assessment of AR. As illustrated, the two acrosome reacted sperm (1 and 2) show a florescent band in the equatorial region and the two acrosome intact sperm (3 and 4) show uniform florescence of the anterior two-thirds of the head.
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