Monoclonal antibodies identify Fc gamma receptors on unfertilized human oocytes but not spermatozoa.

Oolemmal Fc receptors have previously been shown to play a role in the promotion of adhesion by antibody labeled human spermatozoa to zona-free hamster eggs. In this work, we demonstrated the presence of Fc gamma RI, Fc gamma RII and Fc gamma RIII on the oolemma of unfertilized human oocytes by means of monoclonal antibodies directed against these receptors, detected both by immunobead rosetting and indirect immunofluorescence. These receptors were also functionally active in that they were able to bind human aggregated IgG, human IgG-Fc, mouse IgG1 and IgG2a. While the presence of oolemmal IgG-Fc receptors might play a role in reproductive failure, by their promotion of polyspermic fertilization, in cases where antisperm antibodies bound to the spermatozoan surface, their role in the normal physiology of fertilization or in other events unrelated to sperm incorporation remains to be determined. In contrast, Fc gamma receptors were not present on human spermatozoa, irrespective of their functional state (fresh ejaculated, capacitated or acrosome reacted).

Inhibition of human sperm-zona pellucida tight binding in the presence of antisperm antibody positive polyclonal patient sera.

Patient sera previously characterized as containing high levels of IgG and IgA antisperm antibodies that bound to the sperm surface, most specifically the head region, were evaluated for their effect on sperm-zona pellucida tight binding as assessed by the hemizona assay (HZA). Of the ten patient sera tested, 7 reduced zona binding by approximately half. Two of the most strongly inhibitory (greater than 70% inhibition) were examined for their effect on the prefertilization maturation of sperm. The patient sera did not affect sperm motion characteristics, or development of hyperactivated motility. However, in the presence of these sera some impedance was noted in calcium uptake after stimulation with human follicular fluid and in the acrosome reaction after calcium ionophore induction. Whether these two sera specifically affect sperm-zona pellucida binding or non-specifically affect the normal progression of capacitation remains to be eludicated.

Reproductive problems in the DES-exposed female.

The records of 25 diethylstilbestrol-exposed women under treatment for reproductive dysfunction were reviewed. A tendency toward pregnancy wastage (5 of 10 patients), ectopic pregnancy (2 of 8 patients), cervical factor infertility (15 of 20 patients), hysterographic abnormalities (15 of 19 patients), and ovulatory dysfunction (10 of 25 patients) was noted. With the exceptions of ovulation induction and cervical cerclage, no means of treatment can be recommended.

Diagnosis of luteal phase inadequacy.

Plasma progesterone concentrations drawn at the time of endometrial biopsy in 26 infertility patients with histologically documented luteal phase inadequacy were compared with those of 26 infertility patients with normal biopsies. Although as a group the former patients had lower progesterone values and shorter cycles, there was considerable overlap. Therefore, although plasma progesterone determinations and temperature charts are useful in the detection of ovulation and in the interpretation of the biopsy results, a properly obtained endometrial biopsy is essential for the diagnosis of luteal phase inadequacy.

Detection in human sera of antibodies directed against the hamster egg oolemma.

Heteroantibodies were demonstrated by indirect immunofluorescence in human sera, which reacted with unfertilized and fertilized hamster eggs. Oolemmal antigens to which these antibodies were directed were distinct from antigen present on the surface of living human spermatozoa. Both species-specific and tissue-specific heteroantibodies were demonstrated by absorption with hamster liver and ovary. An increased degree of heteroantibody binding was noted following penetration of zona-free hamster eggs by human spermatozoa, indicating that an alteration in oolemmal antigen distribution had occurred. No evidence was found, however, that antisperm antibodies in these sera reacted with zona-free hamster eggs following their fertilization.

Lysis of periadnexal adhesions for correction of infertility.

A series of patients is presented in whom lysis of periadnexal adhesions was carried out for correction of infertility. These 35 couples had been infertile for at least 1 year prior to surgery. Seventy-seven per cent had been trying to conceive for more than 18 months. Following diagnostic evaluation, periadnexal adhesions were found to be the sole cause of infertility in 83% of cases. Subsequent to surgery, 63% of the patients conceived, 82% within 18 months, and 57% gave birth to at least one viable child. There can be no doubt that periadnexal adhesions represent true pathology. Although often seemingly insignificant in character, at the macroscopic level, they appear to play a major role at the microscopic level in impairing ovum pickup by the fallopian tube. Gynecologists should recognize the importance of these structures as mediators of a condition of relative sterility. Thus, despite the demonstration of tubal patency, the function of the rather delicate fimbria may be compromised by periadnexal adhesions, conglutinations, and hydatids so that only a fraction of those eggs that leave the ovary at ovulation reach the interior of the fallopian tube.

PIP: A series of patients is presented in whom lysis of periadnexal adhesions was carried out for correction of infertility. These 35 couples had been infertile for at least 1 year prior to surgery. 77% had been trying to conceive for more than 18 months. Periadnexal adhesions were found to be the sole cause of infertility in 83% of those who eventually conceived. Subsequent to surgery, 63% of the patients conceived, 82% within 18 months, and 57% gave birth to at least 1 viable child. There is no doubt that periadnexal adhesions represent true pathology. Although often seemingly insignificant in character at the macroscopic level, they appear to play a major role at the microscopic level in impairing ovum pickup by the fallopian tube. Gynecologists should recognize the importance of these structures as mediators of a condition of relative sterility. Thus, despite the demonstation of tubal patency, the function of the rather delicate fimbria may be compromised by periadnexal adhesions, conglutinations, and hydatids so that only a fraction of those eggs that leave the ovary at ovulation reach the interior of the fallopian tube.

Measurement of vitronectin content of human spermatozoa and vitronectin concentration within seminal fluid.

OBJECTIVE: Vitronectin previously has been extracted from human spermatozoa and messenger RNA (mRNA) encoding vitronectin localized by reverse transcriptase in situ polymerase chain reaction (PCR) to spermatocytes of human testis. In the present experiments, we have established ranges for the content of vitronectin in living human spermatozoa and vitronectin concentration within seminal fluid of human ejaculates. DESIGN: Seminal fluid was obtained by centrifugation and motile sperm selected by swim-up from men with normal and abnormal ejaculates, according to World Health Organization criteria, for vitronectin determinations. SETTING: Academic research environment. MAIN OUTCOME MEASURE(S): Seminal fluid vitronectin concentrations were measured by ELISA and sperm vitronectin content by polyacrylamide gel electrophoresis and semiquantitative Western blots. RESULT(S): Vitronectin seminal fluid concentration was 1.35 +/- 1.0 mg/mL (mean +/- SD) for normospermic samples (n = 26) and 0.78 +/- 0.4 mg/mL for azoospermic specimens (n = 6). Vitronectin sperm content ranged from 1 to 15 ng/10(6) motile cells (n = 20). Both high- and low-molecular-weight material was observed. Sperm content of vitronectin did not vary with sperm morphology. CONCLUSION(S): These results suggest that spermatozoa represent a major source of seminal fluid vitronectin, but that a secondary source exists, perhaps through transudation from serum.

Characterization of humoral antibodies reactive with spermatozoa, N-acetyl galactosamine, and a putative blood group antigen in seminal plasma.

Antisperm antibodies in sera of infertile women may react differently with spermatozoa of different men. We studied the reactivity of these antibodies with spermatozoa from men of varying blood group status. Increased immunoglobulin binding to sperm of group A or AB men was noted when compared with group O men. A diminution in binding of immunoglobulins to spermatozoa after absorption of these sera with human group A or AB red blood cells was noted as well as after coincubation of sera and sperm with N-acetyl galactosamine, the terminal sugar of blood group antigen A. These observations suggest that antibodies directed against blood group antigens adsorbed to sperm of secretor males may account in part for variations in immunobead binding levels between sperm of different men.

Effects of incubation time in serum and capacitation on spermatozoal reactivity with antisperm antibodies.

Sperm reside within the female reproductive tract before the occurrence of fertilization. During this time they undergo surface modifications associated with changes in their functional state. To study their antigenic expression, capacitated and acrosome-reacted sperm were incubated with sera that had previously been tested for antisperm antibodies against fresh washed sperm, as detected by indirect immunobead binding. Forty-eight percent of previously positive and 20% of previously negative sera reacted differently with sperm after an extended time (18 hours) of incubation in serum or after sperm capacitation. These results suggest that current techniques of antisperm antibodies detection be modified to include testing sera after prolonged incubation times with both capacitated as well as fresh sperm.

Sperm-oolemmal interaction: role of the Arg-Gly-Asp (RGD) adhesion peptide.

The Arg-Gly-Asp tripeptide (RGD) plays a widespread role in cell-to-cell and cell-to-matrix recognition. We demonstrated that an RGD-containing peptide (Arg-Gly-Asp-Val, RGDV) inhibits both oolemmal binding and penetration of zona-free hamster eggs by human spermatozoa in vitro when added to the incubation medium. These results suggest that RGD-containing proteins may play a role in sperm-oolemmal interactions required for fertilization.

A microwell sperm penetration assay.

Egg penetration rates in a modified SPA using microwells in tissue typing plates were comparable with those in a standard assay. This technique allows sperm penetrating ability to be determined using single zona-free hamster eggs and as few as 10,000 spermatozoa.

Progesterone-evoked increases in sperm [Ca2+]i correlate with the egg penetrating ability of sperm from fertile but not infertile men.

OBJECTIVES: To investigate the relationship between sperm capacitation and intracellular calcium ([Ca2+]i) and to correlate these findings with routine semen parameters and sperm fertilizing ability. DESIGN: Baseline and P-evoked increases in [Ca2+]i of fresh versus capacitated human sperm were measured for known fertile donors and infertile men and compared with the results of semen analysis and in vitro penetration of zona-free hamster eggs. SETTING: Andrology laboratory in a university hospital. PATIENTS: Infertile men undergoing semen analysis. INTERVENTIONS: Capacitation of spermatozoa and exposure of sperm to P (1 microgram/mL). MAIN OUTCOME MEASURES: [Ca2+]i as measured using fura-2, percent zone-free hamster eggs penetrated, and number of penetrating sperm per egg. RESULTS: Steady state [Ca2+]i increased from 74 +/- 32 nM to 166 +/- 97 nM after capacitation, as did P-evoked peak and plateau [Ca2+]i. Deletion of calcium from the assay buffer with ethylene-bis (oxy-ethylenenitriolo) tetraacetic acid abrogated the P-evoked increments. RU486, a P receptor antagonist; reduced the P-evoked response in a dose-dependent manner. Progesterone-evoked calcium responses of sperm varied between different ejaculates of the same fertile donor and correlated with their egg penetrating ability. Sperm from infertile men with abnormal morphology exhibited lower egg penetrating ability and lower mean peak P-evoked [Ca2+]i than morphologically normal sperm. However, free intracellular calcium parameters correlated only weakly with penetrating ability for individual infertile men. CONCLUSION: Progesterone-evoked increases in [Ca2+]i in motile capacitated spermatozoa cannot be used to discriminate between dysfunctional spermatozoa and those capable of penetrating eggs.

Complement-mediated effects of sperm head-directed human antibodies on the ability of human spermatozoa to penetrate zona-free hamster eggs.

The ability of immunoglobulins of IgA, IgG, and IgM classes to mediate complement-dependent membrane damage varies. Sera containing antisperm antibodies of differing immunoglobulin classes were studied, in association with complement, for their ability to alter human sperm penetration of zona-free hamster eggs. Sera that contained immunoglobulins of IgG, IgA, or IgM classes directed primarily against the sperm head (as determined by immunobead binding) were selected from men and women judged to be at risk for immune causes of infertility. Spermatozoa were incubated in these sera in the presence and absence of complement. Following an additional incubation in a modified Biggers, Whitten and Whittingham medium, zona-free hamster eggs were inseminated with these spermatozoa. Antibodies known to fix complement (IgG and IgM) diminished the percentage of eggs penetrated and the number of penetrating sperm per egg without impairing the ability of sperm to contact the egg surface, as judged by comparable numbers of spermatozoa adherent to the oolemma. IgA, which cannot fix the first component of complement, did not alter the ability of sperm to penetrate eggs.

Seminal fluid antisperm antibodies do not reflect those present on the sperm surface.

Given the increasing evidence that head-directed antibodies can impair fertilization in vitro, as well as play a role in the impaired entry of sperm into cervical mucus, our findings provide strong support for the direct analysis of immunoglobulins bound to the sperm surface, rather than by indirect analysis through the study of seminal fluid.

Autoimmunity to spermatozoa: effect on sperm penetration of cervical mucus as reflected by postcoital testing.

In couples with abnormal postcoital tests, where husbands exhibited autoimmunity to spermatozoa, the degree of impairment of sperm penetration into cervical mucus correlated with the proportion of sperm in ejaculates exhibiting surface-bound immunoglobulins. Residual sperm-directed antibodies detected within seminal fluid were not representative of the cell-bound immunoglobulins present on the sperm surfaces. When all sperm were antibody-bound, spermatozoa were rarely seen in cervical mucus. Conversely, the number of motile sperm seen at postcoital testing was normal, that is, no different from that of antibody-negative couples, when less than 50% of sperm were antibody-bound in the ejaculate. In this group, other causes of infertility should be explored. The extent of autoimmunity to spermatozoa as reflected in the proportion of sperm exhibiting immunobead binding, then, provides guidelines for treatment of these men.

Detection using antisperm monoclonal antibodies of shared epitopes expressed by human spermatozoa and oocytes.

OBJECTIVE: To determine whether human spermatozoa and oocytes share common antigenic epitopes, supporting the hypothesis that their cross-linking by antisperm antibodies present in the clinical sera of infertile couples could promote sperm adhesion to the oolemma. DESIGN: Human and hamster eggs were studied for the presence of antigens recognized by a panel of World Health Organization Task Force monoclonal antibodies (mAbs) originally raised against human spermatozoa. A new technique was devised, using frozen sections of paraformaldehyde-fixed individual human and hamster eggs, to screen rapidly antisperm mAbs for egg reactivity. Living zona-free human and hamster eggs then were exposed to Covaspheres (Duke Scientific, Palo Alto, CA) coupled with these mAbs to document the presence of reactive epitopes on the oolemma. SETTING: Academic research environment. MAIN OUTCOME MEASURE(S): Indirect immunofluorescence and Covasphere rosetting. RESULT(S): Eleven of 37 antisperm mAbs tested reacted with fixed hamster eggs and 10 reacted with human eggs. Five of 6 mAbs reactive with both fixed eggs also reacted with the oolemma of living, zona-free eggs. CONCLUSION(S): Common antigenic epitopes, some of which are shared with somatic tissues, exist on the oolemma of human eggs and on the plasma membrane of human spermatozoa.

Echistatin, a disintegrin, inhibits sperm-oolemmal adhesion but not oocyte penetration.

OBJECTIVE: To study the effects of echistatin, a disintegrin known to block the binding of fibronectin (FN) and vitronectin to their respective integrin receptors, alpha 5 beta 1 and alpha v beta 3, on the adhesion of human spermatozoa to the oolemma of zona-free hamster eggs and their subsequent penetration. DESIGN: Motile capacitated human spermatozoa and zona-free hamster eggs were coincubated in the presence of echistatin or in its absence and observed at short serial intervals. Whole mounts of these eggs, washed out of sperm suspension and stained with acridine orange, were scored for numbers of oolemmal adherent and penetrating sperm. SETTING: University Hospital laboratories. PATIENTS: Known fertile semen donors. MAIN OUTCOME MEASURES: Numbers of spermatozoa adherent to the oolemma and those penetrating the oocyte. RESULTS: Sperm adherence to the oolemma was reduced significantly at micromolar concentrations of echistatin, in a concentration-dependent manner. In contrast, echistatin did not inhibit the penetration of oocytes by sperm that had become adherent to the oolemma despite the presence of echistatin. CONCLUSION: We propose that two processes occur in the binding of sperm to the oolemma, one that is echistatin sensitive and possibly involving the integrin receptors that recognize FN and vitronectin, and a second process, resistant to echistatin, that leads to gamete membrane fusion.

Sperm-specific isoantibodies and autoantibodies inhibit the binding of human sperm to the human zona pellucida.

Heterologous antisperm antibodies induced by immunization with Freund's adjuvants are known to inhibit sperm-zona binding. We have tested whether spontaneously occurring sperm-directed isoantibodies and autoantibodies inhibit the binding of human sperm to the human zona pellucida. Zonae pellucidae were obtained from the ovaries of a woman of reproductive age undergoing surgery for uterine malignancy. Couples at risk for immunity to spermatozoa were screened for humoral antisperm antibodies by immunobead binding. Sera containing head-directed antibodies of IgA and/or IgG classes were selected for passive antibody transfer. Populations of spermatozoa exhibiting differing proportions of antibody-bound sperm were obtained by exposure of antibody-negative sperm of a fertile donor to untreated sera or sera depleted of head-directed antisperm antibodies by prior absorption. Each zona pellucida was challenged sequentially with two populations of sperm. an inverse relationship was found between the number of spermatozoa adherent to the zona and the proportion of sperm exhibiting binding of IgA or IgG immunoglobulins over the head, indicating that the presence of these antibodies in the acrosomal region of the sperm head inhibited their ability to attach to the zona pellucida.

Intrafollicular pressure within preovulatory follicles of the pig.

Antral pressure was measured within the follicles of unstimulated ovaries in prepubertal pigs and following an ovulatory stimulus with exogenous gonadotropins. No increase in intrafollicular pressure (IFP) was observed as the time of ovulation approached. A wide range of IFP was noted within follicles of the unstimulated ovary. In many follicles IFP was greater than 30 cm H2O, suggesting that the antral fluid was not in hydrostatic equilibrium with the surrounding thecal capillaries. IFP of unstimulated follicles could be increased to more than 400 cm H2O by antral injection of mineral oil without follicular rupture--a demonstration of the need for stigma formation in the release of the ovum from the follicle. Stimulated follicles were found to be more distensible than unstimulated follicles. The follicles also fell into two groups--those in which sustained versus transient elevation in IFP occurred following oil injection. It is postulated that the follicle wall develops the ability to undergo stress relaxation during follicular maturation and that this process plays a role in regulating IFP.

The detection in human sera of antisperm antibodies reactive with FA-1, an evolutionarily conserved antigen, and with murine spermatozoa.

Evolutionarily conserved antigens are present on spermatozoa of several mammalian species. We tested sera from infertile men and women containing antisperm antibodies (ASAs) for their reactivity with FA-1, an antigen known to be present on murine and human spermatozoa. Fifty percent of male sera and 63% of female sera contained anti-FA-1 antibodies, as judged by enzyme linked immunosorbent assay (ELISA). Fourteen percent of male sera and 50% of female sera were also shown to possess ASAs reactive with living mouse spermatozoa, and murine in vitro fertilization was inhibited by human antibodies. These results suggest that the transfer of immunoglobulins from human sera to spermatozoa of other species may provide a model to study how ASAs effect sperm function.