Effect of IL-1 in amniotic fluid on the fetal keratinocyte.

IL-1 alpha is predominantly increased in amniotic fluid of the patients with premature rupture of the membrane (PROM). In the present study we examined the effect of IL-1 on the fetal keratinocytes, one of the main tissue exposed to the increased IL-1. Exposure of IL-1 at over 0.1 pg/ml IL-1 significantly (p < 0.01) inhibited proliferation of the keratinocytes in low calcium keratinocyte growth medium obtained from the umbilical cord. Immunohistochemical analysis using antibodies to various cytokeratins revealed that the umbilical cord keratinocytes is more similar to the mucosal epithelium than the skin epithelium. These results suggest that IL-1 alpha increased in amniotic fluid in the PROM cases leads the immature proliferating mucosal epithelium to maturate for protection of the fetus against environment.

High implantation rate and consequently high pregnancy rate by in vitro fertilization-embryo transfer treatment in infertile women with antisperm antibody.

OBJECTIVE: To examine the effect of antisperm immunity on postfertilization steps, such as implantation of embryos and fetal growth in IVF-ET treatment of women with sperm-immobilizing antibodies. DESIGN: Retrospective analysis of clinical laboratory data. SETTING: The IVF-ET program of the Department of Obstetrics and Gynecology. The University of Tokushima, School of Medicine. PATIENTS: Eighteen women with sperm-immobilizing antibodies and 122 infertile patients with nonimmune etiology as controls. Infertile couples due to a male factor and with unknown etiology were excluded. INTERVENTIONS: All patients received the same IVF-ET program with GnRH agonist. MAIN OUTCOME MEASURES: Rates of fertilization and cleavage, implantation rate per embryo transferred and pregnancy rate (PR) in both test and comparison groups. RESULTS: The rate of fertilization in the antisperm group (61.3%) was significantly lower than that in the comparison group (76.8%). But the implantation rate per embryo transferred (23.5%) and consequently the modified PR per oocyte recovery procedure (34.4%) in immunologically infertile women were significantly higher than those in the comparison group (7.9% and 17.8%, respectively). CONCLUSIONS: Although sperm-immobilizing antibodies prevent sperm-egg interaction, they do not seem to have any adverse effects on achievement of pregnancy. Moreover, the existence of antisperm immunity in woman with antisperm antibodies is suggested to be favorable for successful pregnancy by the IVF-ET procedure.

Immunoglobulin binding factor: a new tumor marker for prostatic tumors.

Human seminal plasma contains an immunoglobulin gamma binding factor (IgBF) with an estimated molecular weight of 16 kD under reducing condition. IgBF was detected only in the prostate, including benign prostatic hypertrophy (BPH) and neoplasm. The present study was performed to determine whether IgBF is a useful prostatic marker. Serum IgBF levels were measured in patients with prostatic tumors and in control patients without tumor by radioimmunoassay. Serum prostatic-specific antigen (PSA), the standard prostatic marker, was also determined. Serum IgBF levels in patients with prostate cancer were significantly higher than those in age-matched controls (P < 0.05). Also, patients with BPH tended to have elevated IgBF levels than the controls, although the values were not statistically significant. In control patients, serum IgBF levels increased with advancing age. There was no correlation between serum levels of IgBF and PSA in patients with prostate cancer. Using cut-off level at 28.5 ng/ml (2 S.D. above the mean IgBF level of age-matched control), the sensitivities were 41.2% (7/17) for prostate cancer, 23.1% (6/26) for BPH, and 5.6% (1/18) for control patients. In conclusion, serum IgBF is a useful marker in the diagnosis of patients with prostatic tumor, and in evaluating the course of treatment.

Blocking of human fertilization by carbohydrates.

The zona penetration test and triple stain technique were used to elucidate the blocking effects of carbohydrates on human fertilization and their mechanisms. In the presence of D-mannose or D-fructose (final concentration, 50 mmol/l), sperm penetration through the human zona pellucida was completely blocked. The triple stain technique revealed that D-fructose (50 mmol/l) significantly (P < 0.01) suppressed the acrosome reaction of human spermatozoa, while D-mannose did not show a suppressive effect on the acrosome reaction. These results reinforce our hypothesis proposed previously, that a mandatory step in human fertilization is the binding of a D-mannose-binding constituent of the sperm surface to a D-mannose residue in the sperm receptor site on the zona pellucida. In addition, D-fructose may play an important role as an acrosome stabilizing factor in seminal fluid.

Immunosuppressive 30-kDa protein in urine of pregnant women and patients with trophoblastic diseases.

Urine samples obtained from normal pregnant women and patients with trophoblastic diseases contain 30-kDa protein that suppresses phytohemagglutinin-induced T cell proliferation. The immunosuppressive protein was measured by a newly developed radioimmunoassay. The 30-kDa protein was demonstrated in almost all urine samples examined, fluid from hydatid vesicles and chorionic extracts, but not in any serum samples except at low levels in some sera from patients with choriocarcinoma. During pregnancy, the level of urinary 30-kDa protein was higher in the first (1625.5 +/- 1212.0 ng/ml, mean +/- S.D.) and second (1457.4 +/- 1332.4 ng/ml) trimesters than in the third trimester (460.6 +/- 419.0 ng/ml). The urinary 30-kDa protein/hCG ratios in patients with choriocarcinoma (8.3 +/- 10.9) were significantly higher than those in patients with hydatidiform mole (0.67 +/- 1.00, P < 0.01) and in all trimesters than those of normal pregnant women (0.54 +/- 0.44 in first trimester, P < 0.05; 0.63 +/- 0.46 in the second trimester, P < 0.05; 0.24 +/- 0.17 in the third trimester, P < 0.01). There is no significant difference between the ratios in hydatidiform mole and normal pregnancy. These findings and the fast disappearance of the 30-kDa protein from the circulation suggest that the 30-kDa protein plays a part in proliferation of trophoblastic cells in, or their invasion into the host by locally suppressing the immune reaction of the host and that the increase in the urinary 30-kDa protein level, in cases of choriocarcinoma, may be due to the malignant transformation of trophoblastic cells resulting in their rapid invasion.

Immunoglobulin binding factor in human seminal plasma: immunological function.

Human seminal plasma contains a protein with an estimated molecular weight of 16 kd that binds serum immunoglobulin gamma (IgG) and is named IgG binding factor (IgBF). Purified IgBF specifically suppressed pokeweed mitogen-induced lymphocyte blastogenesis, having little or no effect on lymphocyte blastogenesis stimulated with phytohemagglutinin or Concanavalin A; antibody-dependent cell-mediated cytotoxicity; natural killer cell activity; or complement-dependent cytotoxicity of antibodies against sperm. It would appear that IgBF may suppress activation of B cells in the male and female genital tract.

Predominant production of amniotic interleukin-1 alpha in cases with premature rupture of the membranes.

PROBLEM: The physiological significances of the two known subtypes of interleukin-1 (IL-1) in amniotic fluid (AF) were examined by measurements of their concentrations and detection of their location. METHOD: Each IL-1 subtype in AF collected from 61 patients were examined by newly developed enzyme-linked immunosorbent assay with high sensitivity. Placenta and umbilical cord from 17 patients were examined immunohistochemically for localization of IL-1. RESULTS: Different types of IL-1 were predominant in AF; the mean (+/- SE) concentration of IL-1 beta (412.8 +/- 105.4 pg/ml) was significantly higher (P < 0.01) than that of IL-1 alpha (116.4 +/- 26.7 pg/ml) in cases with spontaneous vaginal delivery, whereas the concentration of IL-1 alpha (243.1 +/- 81.4 pg/ml) was significantly higher (P < 0.01) than that of IL-1 beta (139.7 +/- 59.4 pg/ml) in cases with premature rupture of the membranes (PROM) without intrauterine infection. In these cases, immunohistochemical studies revealed the presence of IL-1 alpha in the trophoblastic layer of the placenta and epithelium of the umbilical cord. CONCLUSION: Amniotic IL-1 alpha was detected predominantly in cases with PROM, and it is thought to be produced in placenta and fetal skin keratinocyte.

Heterophile antigens in serous cystadenocarcinoma of the human ovary.

As the aberrance of carbohydrate moieties, the appearance of heterophile antigens in human ovarian cancer tissue was studied using various heterophile antigens and antibodies together with antisera raised in rabbits by immunization with extracts of human ovarian cancer tissues. In the immunodiffusion test, rabbit antisera reacted with a heat-stable, perchloric-acid extractable antigen that was different from CEA and CA125 and possessed Hanganutziu-Deicher (HD)-type specificity in all 4 specimens of serous cystadenocarcinoma of the ovary. Furthermore, a pathologic serum with strong HD-antibody complex formed a precipitin line with one of them. In the cancer tissue, we also immunochemically demonstrated the presence of rat-erythrocyte antigens. However, other types of heterophile antigens, such as Paul-Bunnell and Forssman, were not detected. These results suggest that HD-type antigens appear in serous cystadenocarcinoma of the human ovary, one in broad distribution and some in specific cases.

Etiological implication of autoantibodies to zona pellucida in human female infertility.

PROBLEM: Autoantibodies to zona pellucida (ZP) have been implicated as a cause of infertility in woman by three lines of clinical and laboratory evidence. METHOD: First, positive anti-zona activities, as assessed by the passive hemagglutination reaction (PHAR) using bovine red blood cells sensitized with porcine zona antigen, were detected in 45 of 1,872 serum samples collected from infertile women, but in only three (two nonpregnant and one pregnant) of 592 serum samples from control subjects. Their incidence in 320 women with unexplained infertility (5.6%) was much higher than that in 1,552 women with infertility of known cause (1.7%, P < 0.01), which was comparable to the incidence observed in 193 age-matched fertile nonpregnant and pregnant women (1.5%, P < 0.05). None of the serum samples from 292 age-matched adult men and 100 children of between 5 and 10 years old gave a positive PHAR. Second, follow-up study for a minimum of 2 years with treatment revealed that no pregnancy occurred in 11 infertile women with a consistently positive PHAR, and only three pregnancies in 19 infertile women with a fluctuating positive PHAR. Third, three of seven serum samples from women who gave a consistently positive PHAR produced strong immunofluorescence reactions on human ZP even after adsorption with porcine and human AB blood cells. Pre-exposure of human ZP to the sera showing positive immunofluorescence, including that of one patient undergoing an in vitro fertilization program, greatly diminished the number of spermatozoa of normal quality that bound to and penetrated across human ZP. CONCLUSIONS: The observed high incidence of anti-zona activities and long-term resistancy to treatment in women with unexplained infertility may be closely correlated with inhibition of sperm-egg interaction by anti-zona autoantibodies produced in these women.

Effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa.

OBJECTIVE: To study by a triple stain technique the effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa. DESIGN: The spermatozoa were allowed to swim up and culture in a medium containing 7.5% (vol/vol) serum with sperm-immobilizing antibodies or control serum up to 6 hours. Sperm mobility was analyzed, and the percentage of live acrosome reacted spermatozoa was determined. SETTING: Samples were collected from patients referred to university hospital infertility clinics. MATERIALS: Serum samples were drawn from seven patients with sperm-immobilizing antibodies. All the sera were heat activated and stored at -40 degrees C until use. Semen samples were taken from two healthy donors. RESULTS: During culture for 6 hours, the percentage of live sperm showing the acrosome reaction increased significantly (P less than 0.01) in the control group but not in the sperm-immobilizing antibodies group. However, the inhibitory effect of sperm-immobilizing antibodies on the acrosome reaction was reversed when sperm was reincubated in medium with control serum (P less than 0.01). CONCLUSIONS: Sperm-immobilizing antibodies block fertilization at least in part by inhibiting the acrosome reaction of human spermatozoa.

Significance of D-mannose as a sperm receptor site on the zona pellucida in human fertilization.

The role of monosaccharides in human fertilization was studied by testing their effects on penetration of spermatozoa into mature human oocytes (zona penetration test). When oocytes were pretreated with concanavalin A, wheat germ agglutinin, or Ricinus communis agglutinin-I at a concentration of 100 micrograms/ml, no spermatozoa were found to bind to or penetrate through the zona pellucida. Penetration of spermatozoa was restored when the zona pellucida pretreated with wheat germ agglutinin and Ricinus communis agglutinin-I were rinsed with N-acetyl-D-glucosamine (wheat germ agglutinin inhibitor) and D-galactose (Ricinus communis agglutinin inhibitor), respectively. Conversely, the blocking effect of concanavalin A on sperm penetration was not reversed by treatment with D-mannose (concanavalin A inhibitor). Furthermore, pretreatment of spermatozoa with D-mannose (50 mmol/L) completely inhibited sperm penetration through the zona pellucida. However, sperm penetration was clearly demonstrated when the zona pellucida was pretreated with D-mannose. These data suggest that D-mannose residues are essential in, or sterically closely related to, the sperm receptor site on the human zona pellucida.

Treatment of malignant ascites with allogeneic and autologous lymphokine-activated killer cells.

Two cases of peritonitis carcinomatosa with gynecological cancer, which did not respond to conventional treatment, were treated by intraperitoneal adoptive immunotherapy with allogeneic or autologous lymphokine-activated killer (LAK) cells plus interleukin-2. In case 1, reduction of acute ascites and disappearance of malignant cells from the peritoneal fluid and decreased levels of tumor markers (CA12-5 and CA19-9) were demonstrated during the treatment. In case 2, which also received additional treatment for pleuritis carcinomatosa, reduction of ascites and pleural effusion and disappearance of malignant cells from the pleural fluid were noted. In these cases allogeneic LAK therapy was well tolerated. This therapy did not prolong the patients' lives, but the findings indicate that it had appreciable local antitumor effects on peritonitis and pleuritis carcinomatosa in these patients with gynecological cancer.

[Clinical implication of serum antibody to Thomsen-Friedenreich antigen in patients with gynecological malignancies].

Thomsen-Friedenreich antigen (T-antigen) is a carbohydrate antigen that is expressed in a variety of cancer tissues. T-antigen is thought to have an antigenicity because circulating T antibodies can be detected as natural antibodies in humans. In this study, we examined the serum T antibody titers in patients with gynecological cancer using the hemagglutination test, and studied the relationship between the expression of T-antigen in cancer tissues and serum TA-4 levels and serum T antibody titers. Serum T antibody titers in patients with gynecological cancer were lower than those in normal controls, especially in endometrial and ovarian cancer (p less than 0.05) in which T-antigen was strongly expressed. Furthermore, the low antibody titers correlated with the expression of T-antigen in cancer tissues. T antibody titers significantly increased (p less than 0.01) after operation and the inverse relationship was found with the levels of circulating TA-4 in cervical cancer patients. These findings suggests that patients with gynecological cancer immunologically responded to T-antigen and the measurement of circulating T antibodies may be useful as an indicator of the progression of cancer in tissues.

[Expression of Thomsen-Friedenreich antigen (T-Ag) in gynecologic cancer tissues].

The expression of Thomsen-Friedenreich antigen (T-Ag), the precursor of MN blood group antigen, was studied by the ABC method with Arachys Hypogaea lectin (PNA) and the relationship of the results to the histology and clinical behavior of primary gynecologic cancers were examined. The results were as follows: 1. In cervical squamous cell tumors, the incidence of the expression of T-Ag in invasive cancers (52.9%, 37/70) was significantly higher (p less than 0.05) than that in intraepithelial tumors (28.6%, 8/28). No cryptic T-Ag(-) tissue was found in intraepithelial tumors or microinvasive cancers. 2. In squamous cell carcinomas of the cervix, the incidence of cryptic T-Ag(-) tissue was high (36.4%, 4/11) in tissue of the small cell non-keratinizing type, the most undifferentiated type, whereas it was not found in 5 cases of well-differentiated keratinizing carcinoma examined. 3. In stage Ib and II cervical cancers, no relationship was recognized between the expression of T-Ag in primary lesion and extension to the parametrium or the pelvic lymph nodes. 4. The incidence of T-Ag(+) tissue in adenocarcinomas of the uterine cervix (62.5%, 5/8), the endometrium (91.7%, 11/12), and the ovarian (80%, 8/10) was higher than that in squamous cell carcinoma of the uterine cervix (52.5%, 32/61). 5. In endometrial cancers, no relationship was recognized between the expression of T-Ag and hormone receptor status.

Differences in clinical significance of blood group antigens A, B, and H in carcinoma tissue in the uterine cervix.

The losses of blood group antigens A, B, and H in carcinoma tissue of the uterine cervix were studied by the avidin-biotin-peroxidase complex (ABC) method and the relations of these losses to invasion and dedifferentiation of primary cancer were examined. The incidence of cases showing complete loss of A or B antigen increased in proportion to the progression of cancer, but in most cases even of invasive cancer, H antigen, the precursor of A and B antigens, was detected. Complete loss of H antigen was not demonstrated in well-differentiated keratinizing invasive carcinomas, but was seen in 15% (15/101) of the cases of large cell non-keratinizing type cancer and 50% (8/16) of those of small cell non-keratinizing type cancer. No relationship was found between losses of A, B, and H antigens and parametrial spread of carcinoma or metastasis to the pelvic lymph nodes, but the incidence of death within 2 years after hysterectomy was higher in H antigen-negative cases than in H antigen-positive cases. These results indicate that loss of A and B antigens depends on some activity of invasion of cancer, while loss of H antigen strongly indicates dedifferentiation of cancer cells and also may indicate a poor prognosis.

[Studies on biological actions of sperm immobilizing antibody on human fertilization in vitro].

Effects of sperm immobilizing antibodies on sperm penetration through human zonae pellucidae have been studied. Exposure of human spermatozoa obtained from fertile donors to seven serum samples with sperm immobilizing antibody impaired sperm penetration completely in six cases and incompletely in one case. During the course of treatment of a patient with circulating sperm immobilizing antibody by means of an in vitro fertilization and embryo transfer program, it was found that fertilization was completely blocked in the presence of the patient's serum, but three matured ova fertilized successfully when umbilical cord serum was used instead of autoserum from the patient. Furthermore, when spermatozoa were exposed to an IgG fraction of sera containing sperm immobilizing antibody, sperm binding and penetration were markedly inhibited. The spermatozoa, preincubated with sperm immobilizing antibody, showed penetrability across the zona pellucida. However, exposure of possibly capacitated sperm to the antibody completely blocked sperm binding to and penetration through the zona pellucida. These results suggest that sperm immobilizing antibodies cause infertility by preventina sperm binding to and penetration through the zona pellucida, possibly by interfering with the step of fertilization beyond sperm capacitation.

Blocking of human fertilization in vitro by sera with sperm-immobilizing antibodies.

Serum samples with sperm-immobilizing antibody activity from six women were examined for ability to block sperm-egg interaction by a zona penetration test where human follicular ova matured in vitro were used. Exposure of spermatozoa from a fertile healthy donor to the sera impaired binding to and penetration through the zona pellucida of the spermatozoa completely in five cases and incompletely in one case. Successful fertilization in vitro was achieved by using fetal cord serum instead of autoserum of the patient included in the in vitro fertilization and embryo transfer program. These results suggest that interference with sperm-egg interaction may be an additional mechanism of infertility that is caused by antisperm antibodies.