Sperm immobilization test and quantitative sperm immobilization test using frozen‐thawed sperm preparation applied with computer‐aided sperm analysis

Abstract Purpose In a previous study, a new method was described using the sperm immobilization test (SIT) with computer‐aided sperm analysis (CASA). However, obtaining high‐quality sperm as needed was a known issue. Here, we compared the results of using frozen‐thawed sperm and fresh sperm for the SIT using the CASA method. Methods For the frozen‐thawed preparation, 500 μL of condensed semen and 500 μL of Sperm Freeze were mixed in a cryovial and cryopreserved in liquid nitrogen. Density gradient centrifugation was used for the collection of motile sperm in both the fresh and frozen‐thawed sperm preparations. A total of 50 serum samples were prepared for both the fresh and frozen‐thawed sperm with each sample tested containing 10 μL of serum, 1 μL of either fresh or frozen motile sperm suspension, and 2 μL of complement. Sperm motilities were measured using CASA after a 1‐hour incubation period for both fresh and frozen‐thawed sperm. Results Both fresh and frozen‐thawed sperm reacted similarly when exposed to serum containing sperm‐immobilizing antibodies asserting the use of frozen‐thawed sperm for the diagnosis of immunological infertility. Conclusion These results suggest the possibility of using cryopreserved sperm for the SIT when fresh sperm is unavailable.

an anatomic, physiologic, or immunological barrier. In this case, sperm motility is also impaired. 2,3 The sperm-immobilizing (SI) antibodies in female serum, which is an ASAs, are secreted into the cervical mucus, follicle fluid, and fallopian tube. SI antibodies prevent the sperm from penetrating progressively into the cervical mucus and do not allow sperm to pass into the uterus. 6 SI antibodies are also known to inhibit fertilization even if sperm are able to enter the uterus through the cervix and reach the fallopian tubes. 7,8 The sperm immobilization test (SIT) and quantitative SIT have been developed as an useful tool for detecting SI antibodies and determining the SI antibody titers (SI 50 : 50% sperm immobilization unit), respectively. 9,10 These tests have been traditionally calculated by eye estimation using a microscope to determine the proportion of motile sperm, after the number of motile and immotile sperm were counted manually by a highly trained laboratory embryologist. 9 Recently, we outlined a new method for SIT and quantitative SIT using computer-aided sperm analysis (CASA), which aimed to make the SIT and quantitative SIT more convenient and objective. 11 However, in our previous study describing the new method using CASA, we only used fresh sperm when validating its effectiveness. In a practical setting, obtaining high-quality fresh sperm from a donor is not always readily available. Therefore, in this study, we compared frozen-thawed sperm with fresh sperm by using the new CASA method for SIT and quantitative SIT.

| Subjects
The study has been approved by the ethical committee (Hyogo College of Medicine College Hospital, Nishinomiya, Japan. No. 2865).

| Test serum
Fifty serum samples were collected from Japanese infertile women who received infertility treatment in our hospital from the year 2000 to 2020 and stored at −80°C. An opt-out consent method was provided. The serum samples were prepared by centrifugation at 1500 g for 5 minutes, and inactivation of endogenous complement activities was treated by heating the sera at 56°C for 30 minutes.

| Complement
The complement (Low-Tox guinea pig complement) was purchased from CADARLANE Lab. Lit. In some experiments, inactivation of the complement was treated at 56°C for 30 minutes to obtain a negative control.

Sperm samples
Sixteen freshly ejaculated normal semen samples from five healthy men were obtained by masturbation from volunteers under informed consent. The motility assay was evaluated using CASA. Sperm samples with motility above 68% were included in this study. The mean ± SD of the sperm concentration and sperm motility was 88.4 ± 55.9 × 10 6 /mL (range: 34.3-215.9) and 83.5 ± 7.3% (range: 68.1-95.3), respectively.
In both the fresh and the frozen-thawed sperm, motile sperm for experimentation was collected using density gradient centrifugation.
Sperm samples at a concentration of 100 × 10 6 /mL were prepared for experimentation.

Sperm cryopreservation
After liquefaction, the fresh semen samples were condensed by centrifugation at 540g for 5 minutes. Subsequently, 500 μL of condensed semen and 500 μL of Sperm Freeze ® (Kitazato Co.) were mixed well in a cryovial. Subsequently, the cryovial with the mixture was frozen in liquid nitrogen (LN 2 ) vapor for 5 minutes and submerged into LN 2 for cryopreservation. Afterward, the cryovial was immediately transferred to a storage tank with the LN 2 .

Sperm thawing
For thawing, the cryovial was taken out of the storage tank with the LN 2 . After warming for 1 minute at room temperature, the frozen sperm in the cryovials were thawed by placing them in a warming solution at 37°C for 5 minutes.

| Sperm immobilization test
Two wells in one Terasaki microplate were used for the SIT. Mixtures The mixtures were then incubated for 1 hour at room temperature.
The same steps were followed using both the fresh and the frozenthawed sperm. Sperm motilities were measured using CASA (Ditect Co.) after the 1-hour incubation for both fresh and frozen-thawed sperm. The percentages of motile sperm with active and inactivated complement were calculated for T% and C%. C/T was designated as the sperm immobilization value (SIV). A SIV-positive result for SI antibodies was defined as a value of 2 or more.

| Quantitative sperm immobilization test
Ten wells in one Terasaki microplate were used for quantitative SIT. Sperm motility in the SIT-positive serum and each of the diluted SIT-positive and control sera was determined using T% and C%, respectively. Sperm immobilization activity was defined as the value of (C − T)/C × 100. Sigmoid curve was obtained by plotting the value of the sperm immobilization activity against the serum dilution. The serum dilution with 50% sperm immobilization activity was defined as the quantitative titer of SI antibody (SI 50 ), indicating that the dilution time needed to recover 50% sperm motilities is defined as SI 50 .
The mixtures of the fresh and the frozen-thawed sperm were incubated simultaneously, and motile sperm in both mixtures were counted at the same time using the same serum sample and complement.

| Statistical analysis
The correlation between the values of SI 50 obtained by both fresh and frozen-thawed sperm was analyzed using JMP Pro 14 software (SAS Institute Inc). The correlation analysis was performed using Pearson's correlation coefficient. The differences were considered significant with a probability value of <.05. Table 1 shows the results of the SIT. The SIV between fresh and frozen sperm was compared. In the SIT-negative category, the SIV of fresh sperm had a mean value of 1.02 ± 0.14 (range: 0.76-1.58) and frozen-thawed sperm had a mean value of 1.03 ± 0.14 (range:

| D ISCUSS I ON
Sperm are foreign antigens for women, and therefore, women might produce ASAs impairing sperm motility. 4 Our results suggest that IVF is suitable as a treatment choice in patients with a high titer of SI antibodies. Patients with a high titer of SI antibodies have similar fertilization rate as patients with non-SI antibodies if IVF is used. 14 Therefore, it is important that the presence of SI antibodies is determined along with other standard tests for a clinical diagnosis of subfertility in women. It has been proposed that infertile patients should receive earlier examinations for SIT as a means of reducing treatment time so as not to consume their reproductive time using methods with a low chance of success such as TI or intrauterine IUI. 11 However, routine screening of female serum for SI antibodies is performed in only few fertility clinics although SIT can be performed conveniently by obtaining the complement and sperm. One reason for this is that the screening of all patients undergoing assisted reproductive technology (ART) for sperm-immobilizing antibodies in the patient's serum is ineffective because it may not change the management and prognosis of the patient undergoing ART. 14  Note: Table 1 shows that the results were identical, and 21 of 50 samples tested were positive, and 29 samples were negative for spermimmobilizing (SI) antibodies based on both fresh and frozen-thawed sperm.

TA B L E 1 (Continued)
subfertility with TI or IUI without knowing the presence of ASAs.
Thus, SIT should be performed early during medical treatments for infertility in order to reduce treatment time. Another reason SIT is not routinely used in clinical diagnosis is that the test lacks standardization and objectivity because the method of counting sperm is done by eye estimation by a highly skilled embryologist. 9 Although generally accurate enough, this method is still prone to human error. To manage this, we recently outlined a new method using CASA to make SIT evaluation more convenient and objective as compared to the traditional method. 11 We found the assay results for detecting the SI antibodies and estimating the quantitative titer of SI antibodies to be significantly correlated between the traditional method of manually counting sperm by eye estimation by a highly skilled embryologist and the new method of counting sperm through the chamber slides using CASA. 11 Moreover, this study provided evidence that a highly skilled embryologist is not necessarily required in order to perform this test. Another reason this test is not commonly done is that it is sometimes difficult to obtain high-quality fresh semen from a donor when required. For this reason, in this study, we examined whether cryopreserved sperm could be used for SIT using the CASA method.
The antigens involved in agglutination responses are thought to be surface antigen. 15 In case sperm antigens are recognized by a female patient's serum antisperm antibodies, this could affect the frozen-thawing process. The frozen-thawing process might affect the measurement of the SIT. Freezing and thawing have been reported to cause substantial protein changes. 16 The study conducted by Alexander et al 15  immobilization of frozen-thawed sperm has been reported to be unchanged and remained intact during the frozen-thawing process.
Using SIT, the immunoperoxidase assay, and separation of proteins by gel electrophoreses, they found there was no difference in the membrane antigens before and after frozen-thawing process. 12 Our results showed a significant correlation between fresh and frozenthawed sperm when detecting the presence of ASAs, thus supporting the findings that frozen-thawed sperm remain unchanged throughout the freezing and thawing process. Moreover, a significant correlation was found in SI 50 titer between fresh and frozenthawed sperm. Taken together, these results indicate that SIT and quantitative SIT can be performed using frozen-thawed sperm instead of using fresh sperm.
Cryopreservation of human spermatozoa is widely used for infertility therapy, the improvement of ART, and male fertility preservation before cancer therapy. 18,19 Ice crystallization during the cooling process causes increased toxicity and deleterious effects to the sperm plasma membrane covering the sperm head. 19 As such, the process of thawing sperm after cryopreservation leads to a significant decrease in spermatozoa viability, which decreases the number of motile normal sperm. 20 However, high-quality sperm must be obtained for SIT, and therefore, motile spermatozoa were collected using a Percoll density gradient method for SIT in our study. 21 Based on our findings, the possibility of using cryopreserved sperm for detecting SI antibodies using the CASA method when fresh sperm is unavailable is both practical and recommended for clinical practice.
However, there are possible limitations when using this test clinically. In particular, it is important to acknowledge the comparative sensitivity of the test in relation to the availability of high-quality sperm. High-quality sperm with good motility (in our case, above 68%) may be required to see clinically relevant results. Also, finding similar quality sperm in an amount necessary for clinical testing could be difficult.
In conclusion, we found that the method using CASA with frozen-thawed sperm enables SIT to be more a more convenient and practical test as a clinical indicator.