Gary N. Clarke

A sperm-zona pellucida binding test and in vitro fertilization.

Sperm binding to the zona pellucida was studied in 106 in vitro fertilization (IVF) patients. Oocytes that failed to fertilize in vitro were inseminated with a mixture of equal numbers of test and fertile donor sperm differentially labeled with fluorescein or rhodamine to control for variability in the sperm-zona pellucida binding capacity of oocytes. The ratio of the number of test and control sperm bound to four to six zonae pellucidae was significantly correlated with sperm morphology, viability, motility, motility index, and normal intact acrosomes in semen. The sperm-zona pellucida binding ratio was the most significant factor related to IVF rates by logistic regression analysis. But the proportions of sperm with normal morphology and intact acrosomes in semen also were significant. In patients with

Antisperm antibodies and conception

Sperm have been known to be antigenic for more than a century. There is a strong body of evidence that in humans and in other species at least some antibodies that bind to sperm antigens can cause infertility. Therefore, these antibodies are of interest today for two practical reasons. Firstly, the association of the antibodies with infertility means that they must be detected and then the couples treated appropriately. Secondly, because these antibodies can induce infertility they have the potential to be developed for contraceptive purposes in humans and also for the control of feral animal populations.

Intracytoplasmic sperm injection for treating infertility associated with sperm autoimmunity

Objective: To determine whether intracytoplasmic sperm injection (ICSI) can be used to achieve normal fertilization, embryo cleavage, and pregnancies in cases of sperm autoimmunity. Design: A retrospective analysis of ICSI results in sperm antibody-positive and randomly selected antibody-negative groups. Setting: University- and hospital-based reproductive research laboratory and tertiary referral IVF program. Patient(s): Thirty-nine couples selected on the basis of a strongly positive result for sperm antibodies of immunoglobulin (Ig) G and/or IgA immunoglobulin class in the male partner and a control group of 140 antibody-negative couples. Intervention(s): Human menopausal gonadotropin, hCG and Lucrin (Abbott Australasia, Kurnell, NSW, Australia) were given by injection. Oocyte collection was by transvaginal ovarian puncture. Blood was collected for β-hCG measurement. Main Outcome Measure(s): Normal fertilization, embryo cleavage, establishment of clinical pregnancy, and delivery. Result(s): There were no significant differences in fertilization rates (62% versus 58%) or clinical pregnancy rates (19% versus 12%) between sperm antibody-positive and sperm antibody-negative patient groups. Conclusion: Intracytoplasmic sperm injection is an effective treatment for patients with severe sperm autoimmunity.

Inhibition of human sperm-zona pellucida and sperm-oolemma binding by antisperm antibodies*

The results of this preliminary investigation suggest that antisperm antibodies interfere predominantly with sperm-zona pellucida binding. The observation of similar numbers of control (Ab-) and test (Ab+) sperm bound to the oolemma implies that the antibody-mediated inhibition of capacitation, acrosome reaction, or oolemma binding may not be major causes of failed fertilization with sperm autoimmunity. However, only seven patients were studied, and further investigation with larger numbers of subjects are required.

Sperm Antibodies, Immunoglobulins, and Complement in Human Follicular Fluid

ABSTRACT: The levels of immunoglobulins (IgG, IgA, IgM), complement (C3, C4, C1EI) and sperm antibodies were determined in plasma and follicular fluid samples from 26 patients undergoing in vitro fertilization (IVF) treatment. The results show that IgG, IgA, C3, C4, and C1EI concentrations in follicular fluid are similar to plasma concentrations (63.1–96.1% of plasma levels). The follicular fluid concentration of IgM was severely reduced, however, being only approximately 10% of plasma concentrations. Sperm antibody titres were compared in three patients using sperm agglutination, immobilization, and immunobead binding. The titres in plasma and follicular fluid were similar, apart from antibodies of IgM class, which were undetectable in follicular fluid.

Sperm antibodies and human in vitro fertilization.

In order to directly evaluate the effects of sperm antibodies in human in vitro fertilization (IVF), the authors preincubated donor sperm in female sera containing sperm antibodies and then inseminated supernumerary human oocytes from a gamete intrafallopian transfer (GIFT) program. The sperm were incubated for 30 minutes in medium containing 20% serum with antisperm activity (Test); or no antisperm activity (Control) as assessed by the immunobead test (IBT). Each oocyte was inseminated with 1 to 2 X 10(5)/ml of the preincubated motile sperm with Control or Test treatments allocated on a random basis. Six positive sera were tested in 17 experiments, resulting in a fertilization rate of 41% (25/61) versus 84% (36/43) for controls (P less than 0.001). When considered individually, three of six positive sera caused significant inhibition. The only serum that gave complete inhibition had the highest titer for IgG (10,000) and lower IgA (100). Absorption with protein A reduced the IgG titer to less than 10 and removed the fertilization inhibitory activity. These results confirm that sperm antibodies from female sera can inhibit human IVF.

Detection of Antispermatozoal Antibodies of IgA Class in Cervical Mucus

ABSTRACT: A simple procedure for detection of antisperm antibodies of IgA class in human cervical mucus is described and the results of its application to samples from 102 patients are presented. The results suggest that the IgA immunobead test (IgA‐IBT) is a specific and clinically useful test for sperm antibodies. There was a strong correlation between the IgA‐IBT and the presence of complement‐dependent sperm immobilization in serum (Spearmans, r = 0.92, p < 0.001). Positive IgA‐IBT results occurred only in mucus samples that showed poor penetration by normal sperm. An added advantage of the IgA‐IBT is that both the immunoglobulin class and the site of binding to the sperm surface can be determined simultaneously.

Normal fertilization and embryo development by intracytoplasmic sperm injection of round-headed acrosomeless sperm

OBJECTIVE To investigate the ability of round-headed acrosomeless sperm to bind to the human zona pellucida (ZP) and oolemma and to fertilize human oocytes by intracytoplasmic sperm injection. DESIGN Oocytes that had failed to fertilize in IVF were used for sperm-ZP and spermoolemma binding tests. Sperm from a fertile donor was used as a control for oocyte variability. Intracytoplasmic sperm injection was used for assisted fertilization. SETTING University- and hospital-based reproductive research laboratory and tertiary referral IVF program. PATIENTS Case study of a couple in which the man has 100% round-headed acrosomeless sperm in the ejaculate. MAIN OUTCOME MEASURE Fertilization and embryo development and the ability of sperm to bind to the ZP and oolemma. RESULTS No ZP or oolemma binding was achieved, but normal fertilization and embryo development was obtained after intracytoplasmic injection of round-headed acrosomeless sperm. However, no pregnancy was achieved after the transfer of two cleaving embryos. CONCLUSIONS Normal fertilization and embryo development from round-headed acrosomeless sperm is possible with intracytoplasmic sperm injection. However, it remains to be reported whether pregnancy can result from fertilization with this type of sperm defect.

Effect of sperm antibodies in females on human in vitro fertilization

The effect of sperm antibodies derived from the female partners serum on fertilization and embryo cleavage was evaluated by analyzing the Royal Womens Hospital in vitro fertilization (IVF) data. The results suggest that antispermatozoal isoantibodies detected by the immunobead test (IBT) can interfere with IVF. Thus, in a group of patients with IBT-IgG and IBT-IgA sperm antibody titers of greater than or equal to 10 in serum, a low fertilization rate (15%) was obtained when the wifes serum was used as serum supplement in the IVF culture medium. Where replacement (antibody-negative donor or cord) serum was used in the culture medium, a higher fertilization rate (69%) was obtained (P less than 0.01). These results underline the importance of using replacement serum in cases where the wife has significant sperm antibody levels in her serum. Six pregnancies were obtained in the antibody-positive group (n = 20), five of which occurred in patients with IBT-IgG and IBT-IgA-titers less than 10, for a pregnancy rate of 5/9 in this subgroup. Four of these patients delivered (4/9). Analysis of larger groups of antibody-positive patients is required for further evaluation of these results and ascertainment of the likelihood of occurrence of posttransfer effects of sperm antibodies on the embryo.

Analysis of HIV-1 viral load in seminal plasma samples

BACKGROUND With HIV-1-infected individuals now facing the prospect of relatively long and healthy lives, many discordant couples (where the male is HIV-1 seropositive) are seeking to have children. To assist reducing the risks of heterosexual and subsequent vertical transmission in this situation, quantification of HIV-1 viral load in seminal plasma may be effective as one of several measures to reduce the risk of infecting the mother during insemination, potentially providing a better indication of infectivity than blood plasma analysis. OBJECTIVE(S) To modify existing molecular methods for the purpose of analysing HIV-1 viral load in seminal plasma. METHODS Two commercial assays for HIV-1 RNA quantification were used to assess their sensitivity, specificity and precision for quantification of seminal plasma samples. Seminal plasma samples were prepared with an additional centrifugation step to aid removal of inhibitors to molecular assays. RESULTS Seminal plasma samples exhibited specificity of >95%, equivalent to that reported by the manufacturers of the commercial assays. With additional centrifugation, complete inhibition of 2/19 (10%) seminal plasma samples was observed using the RT-PCR assay, and inhibition was not apparent in the bDNA assay. Quantification of HIV-1 RNA in seminal plasma samples in both assays was equivalent to that observed in plasma samples and did not appear to be affected by the additional centrifugation step. CONCLUSION Minor modification of the RT-PCR assay procedure by additional centrifugation of seminal plasma improved the sensitivity of the assay. Inhibition was not apparent with the bDNA assay.