Minoru Shigeta

Direct Production of the Fab Fragment Derived From the Sperm Immobilizing Antibody Using Polymerase Chain Reaction and cDNA Expression Vectors

PROBLEM: Sperm immobilizing antibodies cause infertility mainly through complement dependent sperm immobilization. To analyze any effect of sperm immobilizing antibody on fertilization, we had already established cell lines that secrete IgM monoclonal antibody (MAb H6‐3C4) and IgG monoclonal antibody (MAb EnBCMGS). The latter was a class‐switched recombinant IgG antibody that shares the same variable region as MAb H6‐3C4. The biological effects of the IgG antibody were also reported previously to eliminate sperm immobilizing or sperm agglutinating activities. However, the method of chemical digestion of IgG had some disadvantage to prepare the purified Fab fragment stably and in large quantities. This time we report a unique method to obtain the recombinant Fab fragments (Fab EnBCMGS) using polymerase chain reaction (PCR) and cDNA expression vectors.

Establishment and characterization of a human hybridoma secreting monoclonal antibody with high titers of sperm immobilizing and agglutinating activities against human seminal plasma.

Peripheral blood lymphocytes isolated from an infertile woman possessing strong sperm immobilizing and agglutinating antibodies were stimulated by culturing with poke-weed mitogen (PWM) and spermatozoa from a healthy donor for 5 days. The stimulated lymphocytes were fused with mouse myeloma NS-1 by PEG-1000. Fused growing hybrid cells were observed in 58 of 96 wells, and 22 of these showed the production of human immunoglobulin. Among the 22, one hybridoma clone (H6-3C4) was found to produce human IgM (lambda) with strong sperm immobilizing and agglutinating activities. The supernatant from the culture medium contained approximately 1.5 microgram IgM/ml and the antibody titers were 5000 SI50 units on sperm immobilization and 1:1600 dilutions on sperm agglutination. The hybridoma H6-3C4 has continuously produced high titers of antibody exhibiting sperm immobilizing and agglutinating activities over 8 months and contains chromosomes of acrocentric type from mouse and metacentric type from human. The monoclonal antibody (Mab) H6-3C4 reacted specifically to human seminal plasma, ejaculated spermatozoa and male accessory gland but not to testis, any other somatic tissues, or secreted fluids tested. Immunofluorescence staining indicated that the antigen corresponding to Mab H6-3C4 was present over the surface of ejaculated spermatozoa. The binding of Mab H6-3C4 to human spermatozoa was blocked by the serum of the patient from whom the lymphocytes were obtained for cell fusion.

Sperm Immobilizing Antibodies Interfere With Sperm Migration From the Uterine Cavity through the Fallopian Tubes

PROBLEM: It is well known that sperm migration in cervical mucus is impaired by sperm immobilizing antibodies secreted in the mucus. However, it is not clear yet whether sperm migration from the uterine cavity through the fallopian tubes to the peritoneal cavity is impaired by sperm immobilizing antibodies. To test the possible impairment of sperm migration in the tubes, laparoscopic examinations were carried out and the presence of motile sperm in the peritoneal fluid after intra‐uterine insemination was investigated.

Contraceptive potential of synthetic peptides of zona pellucida protein (ZPA)

Previously, we produced a fertilization-blocking monoclonal antibody (MAb-5H4) to find a candidate peptide for a contraceptive vaccine. MAb-5H4 recognized a linear amino acid sequence of ZPA (No. 50-67) in pigs, humans and rabbits. In the present study, 18mer peptides corresponding to the sequence were conjugated with diphtheria toxoid as a carrier protein before immunization in rabbits. All three antisera recognized human zona pellucida on testing by immunofluorescent staining method. The two produced against human and rabbit peptides effectively inhibited human sperm binding to the zona pellucida, but the antiserum against the pig peptide did not. The former two peptides include an identical sequence (LDPEKLTL) of the minimum binding motif for MAb-5H4, but the latter peptide includes one amino acid replacement (K to N) in the sequence. It is thus concluded that a synthetic peptide including the sequence of LDPEKLTL could be a feasible candidate for developing a contraceptive vaccine for humans.

Correlation between quantitative antibody titers of sperm immobilizing antibodies and pregnancy rates by treatments

Immunological infertility in women who possessed sperm immobilizing (SI) antibodies made it very difficult to conceive using the usual treatments. We examined SI antibodies by the quantitative Sperm Immobilization Test and found the antibody titers (50% sperm immobilization unit: SI50 unit) associated with pregnancy rates. Patients with high SI50 titers (greater than 10 units) did not conceive by ordinary or repeated artificial inseminations with husbands semen (AIH) except when treated with in vitro fertilization (IVF) and embryo replacement. Patients with relatively low SI50 titers (less than 10 units) could conceive either by repeated or ordinary AIH, though the success rates were lower than by IVF-embryo replacement. It is important to assess the SI50 titers by the quantitative method to select treatments for infertile women with SI antibodies. In follow-up studies of the patients who conceived successfully, it was found that SI50 titers tended to decline as pregnancy proceeded.

Effects of Sperm Immobilizing Antibodies on Pregnancy Outcome in Infertile Women Treated With IVF‐ET

PROBLEM: Since it was found that anti‐sperm antibodies could impair in vitro development of fertilized eggs in the presence of complement in rats, the effects of sperm immobilizing antibodies on human pregnancy were examined in infertile women treated with IVF‐ET.

Relationship Between the Sperm Motility Index Assessed by the Sperm Quality Analyzer and the Outcome of Intracytoplasmic Sperm Injection

Purpose:Intracytoplasmic sperm injection (ICSI) has been validated as a useful treatment in severe male-factor patients who could not achieve fertilization and live births by conventional in vitro fertilization treatment. To examine the impact of male factors on ICSI outcome, clinical laboratory data were retrospectively analyzed.Methods:One hundred two cycles of ICSI treatment indicated by severe male-factor infertility were entered into this study. Sperm parameters including sperm motility, sperm concentration, and sperm motility index assessed by the Sperm Quality Analyzer were evaluated.Results:Five hundred seventy-six metaphase II oocytes retrieved were manipulated. The normal fertilization (2 PN) rate per oocyte was 64.9 ± 26.0% (mean ± SD). Of the 99 transfers, 31 clinical pregnancies were obtained, yielding an average pregnancy rate of 31.3% per transfer. The mean sperm motility, sperm concentration, and sperm motility index were 20.3 ± 16.1% (range, 0 to 50%), 18.2 ± 25.1 × 106/ml (range, <1 to 150 × 106/ml), and 31.2 ± 45.0 (range, 0 to 220), respectively. Sperm concentration did not have a significant impact on fertilization rate by ICSI. In four cases, ICSI was performed using totally immotile sperm and the fertilization rate was 43.5%, which was significantly lower than that of some of the other sperm motility groups, and no pregnancy could be achieved. In 14 cases in which the sperm motility index assessed by the Sperm Quality Analyzer was 0, the fertilization rate (50.0%) was significantly lower than in most of the other sperm motility index groups.Conclusions:These findings suggest that in severe malefactor cases with totally immotile sperm or a sperm motility index of 0, the selection of good-quality sperm should be verified before injection.

Analysis of B cell epitopes of a glycoprotein porcine zona pellucida (pZP1)

The zona pellucida (ZP) of mammalian oocytes forms an extracellular matrix composed of three major glycoproteins and plays an important role in sperm-zona interactions. As ZP had a strong organ-specific but species-cross-reactive antigenicity and passive or active immunization with ZP antigens could impair fertilization, the possibility of developing a immunocontraceptive vaccine has been extensively studied. Studies on active immunization with porcine ZP (pZP) that contain B cell epitopes and T cell epitopes demonstrated that a temporary infertility could be induced along with the elevation of antibody titers, but it was always associated with ovarian failure. This could be due to the oophoritis by activation of pathogenic T cell immunity. It is the general consideration that any adverse effects by vaccination should be avoided for an immunocontraception. From this point of view, the analysis of B cell epitopes of pZP protein would be helpful for construction of a safe immunocontraceptive vaccine with zona antigens. We determined the amino acid sequence of the B epitope in the pZP1 protein by using a monoclonal antibody (MAb-5H4) that possesses a fertilization blocking ability. In addition, antiserum raised to the epitope sequence was revealed to block in vitro fertilization of homologous animal species.

Prevention of multiple pregnancies by restricting the number of transferred embryos: randomized control study

IntroductionThe high incidence of multiple pregnancies is a major concern in the treatment of infertility by in vitro fertilization (IVF). Risks and costs are associated with multiple pregnancies and it is important to find a way to decrease multiple pregnancies. The standard protocol for IVF includes the transfer of two or three embryos.MethodsIn this study, we compared the rate of single and multiple pregnancies between patients who received two and three embryos. One hundred and sixty-nine patients were randomly divided into two groups, with 106 cycles of IVF each. Group 1 received two embryos, and group 2 three.ResultsGroup 1 had total 40 (37.8%) pregnancies, while group 2 had 29 (27.4%). The number of ongoing pregnancies was 33 (31.1%) in group 1 and 26 (24.5%) in group 2. The number of baby-take-home was 32 (30.1%) in group 1 and 26 (24.5%) in group 2. These results show that there was no significant difference between the two groups in both the total and ongoing pregnancies. On the other hand, the number of twin pregnancies was 6 (15.0%) in group 1 and 12 (41.4%) in group 2, showing a significant difference. Group 1 had no triplet pregnancies, while Group 2 had two.ConclusionWe conclude that the transfer of two embryos reduces the incidence of multiple pregnancies while maintaining an acceptable rate of pregnancy in IVF.

Diagnostic Laparoscopy in Infertility: A Retrospective Study

STUDY OBJECTIVE To evaluate the role of laparoscopy in the diagnosis and therapy of infertility. DESIGN Retrospective study. SETTING Academically affiliated reproductive endocrinology practice. PATIENTS One hundred seventy patients. INTERVENTION Diagnostic/therapeutic laparoscopy. MEASUREMENTS AND MAIN RESULTS One hundred seventy infertile patients underwent diagnostic laparoscopy between 1996 and 2000 in our clinic, and 109 of them were seen at follow-up more than 1 year after laparoscopy. Of the 109 patients, 77 (70.6%) were treated with assisted reproductive technology, such as in vitro fertilization-embryo transfer, and 32 (29.4%) were treated with conventional procedures. Of the 109 patients, 68 (62.4%), including 39 (50.6%) of the 77 treated with assisted reproductive technology and 29 (90.6%) of the 32 treated with conventional procedures, became pregnant. Of the 68 patients who became pregnant, 49 (72.1%) of them conceived within 1 year after laparoscopy. CONCLUSION Laparoscopy is an important procedure in the treatment of infertility.