Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5～6.2, 4.6,5.1,5.5～5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

Effect of GABA on sperm acrosome reation in antisperm antibody positive patients

To investigate the effect of gamma-aminobutyric acid (GABA) on the rate of sperm acrosome reaction both in normal and antisperm antibody (AsAb) positive men. Methods: The sperm acrosome reaction was tested with triplestain technique in two groups of 18 men each. Results: (1) GABA increased the rate of sperm acrosome reaction both in normal and AsAb positive subjects (P<0.01); (2) GABA increased the Na^+-K^+-ATPase activity of sperm (P<0.01); (3)GABA increased the Ca^(2+)-ATPase activity of sperm (P<0.05); (4) GABA decreased the production of MDA and oxygen free radicals of sperm. Conclusion: GABA could regulate the rate of sperm acrosome reaction. (Chin J Andro12002; 16: 355)

Studies on relationship between semen viscosity and other semen parameters, Ureaplasma urealyticum infection and seminal plasma antisperm antibody

Objective: To study the relationship between semen viscosity and other semen parameters, Ureaplasma urealyticum (UU) infection and seminal plasma antisperm antibody (AsAb) in male infertiles. Methods: Semen parameters, Ureaplasma urealyticum (UU) infection and antisperm antibody (AsAb) were measured and analyzed in 4337 infertile men. Results: The seminal viscosity was higherr than normal in 65.02 % of 4337 male infertiles. The sperm motility and grade (a, b) motile sperm were significantly lower in the high viscosity group than in the normal viscosity group (P<0.05-0.01). The rate of abnormal morphology sperm was higher and duration of semen liquefaction was longer in the high viscosity than in the normal viscosity group (P<0.01). The seminal volume, sperm concentration and semen pH were not significantly different between the two groups. The semen viscosity is significantly higher in subjects with higher seminal WBC (>5/ HP) than in those with lower WBC (<5/HP). The positive AsAb and UU infection rates were significantly higher in the high viscosity group (P<0.01). Conclusion: The semen viscosity is related to other seminal parameters, as well as to UU infection and seminal AsAb.

Analysis of sperm membrane antigens relevant to antisperm antibody using Western blot

To identify the sperm membrane antigens associated with antisperm antibody. Methods: The antisperm antibody in serum was tested by ELISA. Antisperm antibody positive sera from 18 infertile men and 15 infertile women were used. The molecular weight (MW) of sperm membrane antigens associated with the antisperm antibody was analyzed with antisperm antibody positive serum using Western blot. Results: Eight kinds of MW of sperm membrane antigens were identified. The ratio of identification on the 78 KD(60.7 %), 60KD (71.4 %), 51 KD (14.9 %) and 23 KD (14.29 %) sperm antigen was higher than others. Conclusion: Sperm membrane antigens with MW of 78 KD, 60 KD, 51 KD and 23 KD were associated with antisperm antibody and immunological infertility. (Chin J Andro12002; 16: 345)

Analysis of Antisperm Antibody in Cervix Mucus for Azoospermic Men’s Wives

ELISA method was used to detect antisperm antibodies in cervix mucus. Out of 400 women whose husbands had sperms, 92 were antisperm antibody positive (23% ). 42 of 200 women with azoospermic partners were antisperm antibody positive (21% ). No difference was found between the two groups. Hence, antisperm antibodies in azoospermic men's wives may be caused by sperm'sadhering antigens in semina.

Clinical Observation on Effect of Combination of Zhuanyindan (转阴丹) and Hormone in Treating MaleInfertility with Positive Antisperm Antibody

Objective:To explore the therapeutic effect of combination of Zhuanyindan (ZYD, 转阴丹, a self-made Chinese herbal preparation) and hormone in treating male infertility with positive antisperm antibody and its influence on nitric oxide (NO) level. Methods:Eighty-two patients were randomly divided (according to the digital list) into the WM group (n = 20, treated with prednisone), the TCM group (n=28, treated with ZYD) and the ICWM group (n=34, treated with prednisone plus ZYD). The clinical effect, negative converting rate of antisperm antibody, changes of NO level in semen and various parameters of sperm motion before and after treatment were observed. Results: The total effective rate in the ICWM group was 88.2%, that in theTCM group 75. 0% and in the WM group 65. 0%. Significant difference was seen in the ICWM and TCM group before and after treatment in NO level, sperm motion parameters, including linear motion speed, linearity, propulsion, whip frequency, sperm vitality and mean moving angle, and quality of semen ( P < 0. 05 or P < 0. 01). In the WM group, significant difference in comparison before and after treatment was seen in NO level, propulsion, whip frequency, mean moving angle and quality of semen, including vitality and survival rate (P<0. 01). Conclusion: Combination of Chinese herbs and hormone could lower the NO level in semen and improve the quality of sperm.Original article on CJITWM (Chin) 2004 ;23 (3): 223

Changes in soluble interleukin-2 receptor level in serum and Na^+ -K^+ -exchanging ATPase activity in semen of infertile men caused by antisperm antibody

Aim: To explore the possible mechanisms of male infertility caused by antisperm antibody (AsAb). Methods: Thesoluble interleukin-2 receptor (sIL-2R) level in serum was analyzed by ELISA and Na^+ -K^+ -exchanging ATPase activi-ty in semen by phosphorus (Pi) assay. Results: The slL-2R level in serum was significantly higher and the Na^+ -K^+ -exchanging ATPase activity in semen significantly lower in AsAb positive infertile men when compared with thecontrols. Conclusion: The AsAb titer varies with the slL-2R level in serum. A decrease in Na^+ -K^+ -exchangingATPase activity in semen may play a role in male infertility caused by AsAb.

Circulating Antisperm Antibody (ASA) in Women is not Associated with Missed Abortions at the First-trimester Pregnancy

Objective To determine the role of the circulating antisperm antibody （ASA） in the pathogenesis of missed abortion at the first-trimester pregnancy. Methods Sixty-two patients with a history of missed abortion at the first-trimester pregnancy were enrolled into this study. Indirect immunobead test （IBT） was used to measure the circulating ASA levels. Fifty healthy women with the first-trimester pregnancy set as the control. Results No case had the positive level of ASA according to the World Health Organization criteria （50% or more of the motile sperm with immunobead binding）. Only 1 case in patient group and 1 case in the control had 10%-20% of the motile spermatozoa with ASA-IgG bead binding. In both patient and control groups, ASA-IgA was found to be completely negative binding. Conclusion The circulating ASA is not associated with the pathogenesis of missed abortions at the first-trimester pregnancy.

Circulating Antisperm Antibody is not Related to the Pathogenesis of Infertility in Women with Endometriosis

Objective To investigate the role of circulating antisperm antibody （ASA） in the pathogenesis of endometriosis-associated infertility. Methods Serum samples were collected from 75 infertile women with minimal or mild endometriosis. Indirect immunobead test reporting on the percentage of motile sperm with adherent immunobeads was used to detect the circulating ASA levels. Results No infertile cases enrolled in the present study developed significant ASA level in the serum samples. That is to say, no one could achieve the diagnosis of ASAmediated infertility according to the World Health Organization criteria （50% or more of the motile sperm with immunobead binding）. There were only 5 cases （6.7%） who had 20%-40% of the motile sperm that were found to have adherent particles of ASA- IgG. All the cases were found to be completely absent of ASA-IgA. Conclusion Endometriosis seems to have little impact on the production of circulating ASA. Clearly, ASA is not the key factor implicated in the pathogenesis of endometriosis-associated infertility. The presence of a low titer of ASA in a small proportion of the infertile women with endometriosis may exist by chance and, at least in part, explain the impaired fecundity in those patients.

Evaluation of Circulating Antisperm Antibody in Infertile Women with Polycystic Ovary Syndrome

Objective To evaluate levels of the circulating antisperm antibody （ASA） in infertile women with polycystic ovary syndrome （PCOS）.Methods Forty-six infertile women with PCOS were enrolled into this study. The serum was screened by the indirect immunobead test for ASA type IgG and IgA according to the WHO laboratory manual.Results Of the 46 patients assessed for ASA-IgG, 3 cases had immunobead binding which were 20%, 27% and 35%, respectively, and the sub-positive rate was 6.5%. No case had the clinical positive level according to the WHO criteria （50% or more of the motile sperm with immunobead binding）. ASA-IgA was not detected in all cases. Conclusion The circulating ASA is not associated with the pathogenesis of infertile women with PCOS.

Evaluation on Antisperm Antibody in Infertile Men with Oligoasthenoteratozoospermia

To evaluate the level of antisperm antibody （ASA） in infertile men with oligoasthenoteratozoospermia （OAT）. Methods Forty-six infertile men with OAT were enrolled into this study. Sperm samples were screened by the direct immunobead test for ASA type IgG and lgA according to the WHO laboratory manual. Results Of the 46 patients with OAT assessed for ASA-IgG, 2 had immunobead binding which were 22% and 27%, respectively, and the sub-positive rate was 4.3%. No case had the clinical positive level according to the WHO criteria （ ≥ 50% of the motile sperm with immunobead binding）. ASA-IgA was not detected in all cases. Conclusion A significant incidence or high level of ASA could not be found in infertile men with OAT, which suggests that ASA is not associated with the pathogenesis of infertile men with OAT.

Hemospermia is not associated with the production of antisperm antibody

Objective To investigate whether hemospermia is involved in the production of antisperm antibodies （ASA）. Methods A total of 40 males with hemospermia were enrolled in this study. These males were divided into 3 groups according to their fertile status： 18 cases were patients who visited the infertility clinic （group A）, 8 had fertility （group B）, and 14 did not know their fertile status （group C）. Semen samples were collected and ASA levels were detected by the direct immunobead test （D-IBT）. Leukocytes in semen were assessed by the peroxi- dase assay. Results None of these males with hemospermia were found to display significant levels of ASA in the 3 groups. The number of cases who had increased levels of leukocytes in the semen samples of groups A, B and C were 15 （83.3%）, 6 （75.0%） and 12 （85.7%）, respectively. Conclusion Hemospermia is not associated with the production of ASA. It is worth noting that the leukocyte levels are increased in semen of males with hemospermia.

Evaluation on Antisperm Antibody in Infertile Women with Chronic Salpingitis

Objective To evaluate the level of antisperm antibody （ASA） in infertile women with chronic salpingtis. Methods Forty-eight infertile women with chronic salpingtis were enrolled into this study. Serum samples were screened by the direct immunobead test for ASA type IgG and IgA according to the WHO laboratory manual. About 50% or more of the motile sperm attaching to one or more immunobeads were regarded as clinical positivity according to the WHO criteria. About 20% 50% motile sperm that had adherent particles were deemed to be sub-positive. Results Of the 48 patients with chronic salpingtis assessed for ASA-IgG, 4 had immunobead bindings ranged from 50% to 60%, and the positive rate was 8.3%. In addition, 5 cases had 20%-40% of immunobead bindings, and the sub-positive rate was 10.4%. For ASA-IgA detecting, 2 cases with ASA-IgG positivity also had ASA-IgA immunobead bindings, and the positive rates were 22%, and 28%, respectively. Conclusion The incidence of ASA could be found in infertile women with chronic salpingtis, which suggests that an increased risk for the production of ASA would exist in the inflammatory status o f fallopian tube.

Antisperm Antibodies on the Surface of Spermatozoa before Ejaculation from Vasectomized Men

The antisperm antibodies (AsAbs) coated on spermatoZoa of the proximal vas deferens (sperm before ejaculation, SBE) from 48 fertile men who were volunteers of vasectomy and 24 vasectomized men who asked for vasovasotomy,were determined by immunobead test (IBT) and sperm-cervical mucus contact test (SCMC). The results showed that in fertile men there were no positive samples of SBE in IBT and SCMC. In vasectomized men positive samples of SBE were found in 79.4% for IgG, 38.2% for IgA and 35.5% for SCMC. The AsAbs on SBE could be found at the time of less than one year to more than 3 years after vasectomy. The AsAbs were still found on the semen samples at 1~3months after vasovasotomy. Our results also indicated that the incidence of AsAbs on SBE from vasectomized men could not predict the levels of AsAbs on their ejaculated sperm after vasovasotomy. There was no significant correlation between the levels of AsAbs in serium before vasovasotomy and those on SBE from vasectomized men.

The Presence of Antisperm Antibodies in Semen of Subfertile Men—Positive ASA in Subfertile Men

Background: In more than 50% of male subfertility, the aetiology remains unknown. Antisperm antibodies (ASA) might be involved, however the exact role of ASA in unexplained male subfertility is not clear, yet. Objective: The aim of this study was to examine 1) the prevalence of ASA in subfertile men, 2) the possible causes of the presence of ASA, and 3) the influence of ASA on sperm parameters and fertilization including assisted reproductive technologies (ART) and pregnancy outcomes. Study Design, Size, Duration: In this retrospective single center study, all men with semen analyses between January 2003 and December 2017 were included as well as all subfertile couples getting treatment if at least one sperm analysis showed a spermMar test ≥50%. Methods: Collected parameters were: intoxications, medication and professions that could have an adverse effect on fertility, sperm parameters, the type and amount of ART, mode of conception, pregnancy rates and outcomes. Results: 3098 semen analyses were performed. In total, 233 ASA positive men were observed, including 175 subfertile couples with an ASA positive man in the additional analyses. The prevalence of ASA in the subfertile population was 8.2%. The presence of ASA was significantly associated with the presence of oligoasthenoteratozoospermia (OAT), asthenoteratozoospermia and asthenozoospermia (p = 0.008, p = 0.004, and p = 0.02 respectively). However, 50% of the couples with an ASA positive man became pregnant without ART. Conclusions: The presence of ASA did not seem to have a negative effect on spontaneously pregnancy rates or pregnancy rates after ART. Therefore, it might be justified to advice 6 - 12 months expectant management, before starting ART in ASA positive men.

Traditional Chinese Medicine Compound Mianbu Mixfang Is Effective in Treating Immunological Infertility: a Female Mice

Objective To study the therapeutic effectivess of Traditional Chinese Medicine compound mixture Mianbu Fang (Immunological infertility therapy) on immunological infertility caused by antisperm antibody (AsAb) in female mice. Materials & Methods Forty-two female Kunming mice were evenly divided into 7 groups by weight. Group A was control group; Group B was model of infertility. Group C, D and E were fed with normal, half and double dosage of Mianbu I respectively. Group F and G were fed with Mianbu II and prednisone Acetates respectively. Animal model of immunological infertility were set up by injecting mice sperm to the other 36 Kunming female mice except Group A. The AsAb levels in serum, cervical mucus were measured, the histological and immunohistochemistry changes in ovary and endometrium were observed, and the pregnancy indexes were compared in different groups. Results Compared with the infertility model group, the AsAb level in serum and cervical mucus in treatment group was lower. Less immune compounds in ovary and endometrium and atretic follicle of ovary was found in treatment group than in model and control group. The immune compounds in ovary and endometrium were less in the treatment group than that in the model and control group. Conclusion By regulating immunological system, Traditional Chinese Medicine compound mixfang Mianbu Fang lowers AsAb in the circulation system and special organs, eliminates immunological compound, repairs tissue impairment and increases pregnancy of female mice.

Histology and Immunohistochemistry Study on Mianbu Fang Healing Immunological Infertility of Male Mice

To observe the histological and immunohistochemical changes caused by of Traditional Chinese Medicine——Mianbu Fang (Immunological Infertility Healing Mixture) in testis and epididymus of male mice with immunological infertility induced by antisperm antibody (AsAb). Materials & Methods Models of mice with immunological infertility were used by injection of mice sperm. These mice were treated with Mianbu Ⅰ, Mianbu Ⅱ or Prednisone Acetates. The characteristics on histology and immunohistochemistry of mice were analyzed. Results High level of AsAb was detected in serum and seminal vesicle plasma of model mice after treatment. Immune compounds mostly deposited in limiting membrane of seminiferous tubules, spermatogonium and epithelia of epididymis ducts. The number of spermatogenic cells per convoluted seminiferous tubule decreased. However, the Chinese medicine could significantly reduce the level of AsAb, or even eliminate the antibodies and clear out the immune compounds and increase the number of spermatogenic cells of seminiferous tubules. Conclusion Chinese Medicine Mianbu Ⅰand Ⅱ can regulate, absorb and clear out circulation and partial AsAb, as well as immunological compound. Hence, they can increase the number of spermatogenic cells of seminiferous tubules and increase the pregnancy of mice.

Effect of NO on spontaneous acrosome reaction in AsAb positive rat spermatozoa

Objective: To explore the effect of NO on the spontaneous acrosome reaction in antisperm antibody (AsAb) positive rat spermatozoa. Methods: The rat model of AsAb was set up by artificial immunization. The level of AsAb in blood serum was determined by TAT and ELISA. Rat spermatozoa was visualized by staining the acrosome with Coomassie brilliant blue. The NO concentration in rat spermatozoa was assayed by HPLC. Results: The percentage of acrosome reaction, NO concentration, superoxide dismutase (SOD) and Na^+-K^+ATPase activity in AsAb positive rat spermatozoa were significantly decreased compared with the control group. Low dose of NO (SNP 10^(-9)～10^(-8) mol/L) increased the percentage of acrosome reaction and SOD activity, but had no effect on Na^+-K^+ATPase activity. High dose of NO (SNP 10^(-6)～10^(-4) mol/L) decreased the three items. Conclusion: The decrease in acrosome reaction in positive AsAb rat spermatozoa might be related to a decrease in NO and increase in O_2. (the SOD activity was decreased) in sperm. A low dose of NO might increase the percentage of acrosome reaction in AsAb positive rat by inhibiting the superoxide, while a high dose of NO was harmful to sperm function. [Reprod Contracep (in Chinese) 2002; 22: 203]

Relationship between serum antisperm antibodies and anticardiolipin antibodies and clinical pregnancy outcome in an in vitro fertilization and embryo transfer program

Objective To study the influence of maternal immunological factors on clinical pregnancy outcome in an in vitro fertilization and embryo transfer (IVF ET) program. Methods One hundred and fifty IVF ET treatment cycles from November 1995 to November 1996 were studied. The indication for IVF ET treatment was bilateral blocked tubes. Serum antisperm antibodies and anticardiolipin antibodies were measured using enzyme linked immunosorbent assay (ELISA). Cleavage rate and successful pregnancy rate in relation to antibody status of infertile women after IVF ET treatment were assessed. Results Lower cleavage rate (64.2%±32.1%) was found in 44 cycles of antisperm antibody seropositive women, compared with 84.8%±18.7% in 106 cycles of seronegative women (P<0.05). The clinical pregnancy rate was 31.8% in antisperm antibody positive cycles and 20.8% in negative cycles (P> 0.05). The abortion rates of the two groups were similar (P>0.05). Lower pregnancy rate (9.5%) was found in 21 cycles of serum anticardiolipin antibody positive group, compared with 26.3% in 129 cycles of seronegative women (P<0.05). Of patients with bio chemical pregnancy and no pregnancy, 20.0% and 16.2%, respectively, had seropositive anticardiolipin antibody, compared with 5.6% of patients with clinical pregnancy (P<0.05). Conclusion Serum immunological factors may play a part in clinical pregnancy outcome in IVF ET.

Expression and function of HSP110 family in mouse testis after vasectomy

HSP110 functions to protect cells, tissues, and organs from noxious conditions. Vasectomy induces apoptosis in the testis; however, little is known about the reason leading to this outcome. The aim of the present study was to evaluate the expression and function of HSP110 in mouse testis after vasectomy. Following bilateral vasectomy, we used fluorescent Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） to detect apoptosis, Western blotting and immunohistochemistry to examine HSP110 expression and localization. Serum antisperm antibody （AsAb） and testosterone were measured by Enzyme-linked immunosorbent assay （ELISA） and radioimmunoassay, respectively. Expression of endoplasmic reticulum stress （ERS） sensors and downstream signaling components was measured by Reverse Transcription-Polymerase Chain Reaction （RT-PCR）, and the phosphorylation of elF2a and JNK was detected by Western blotting. Vasectomy induced morphologic changes, increased apoptosis in the testis, increased serum AsAb, and decreased testosterone levels. After vasectomy, ORP150 mRNA level was increased first and then decreased, Bcl-2 was decreased, and the expression of HSPA41, GRP78, GADD153, PERK, ATF6, IRE-l, XBP-ls, Bax, Bak, and caspases and the phosphorylation of elF2a and JNK were increased. We present that an ER stress-mediated pathway is activated and involved in apoptosis in the testis after vasectomy. HSPA41 and ORP150 may play important roles in maintaining the normal structure and function of testis.