Gary D. Hodgen

The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential * † ‡

The authors present their initial results with the hemizona assay (HZA), which was developed to predict the fertilizing potential of spermatozoa. The HZA uses the matching halves of a human zona pellucida from a nonfertilizable and nonliving oocyte, providing an internal control on zona-to-zona variability. Maximal binding of human sperm to the hemizona usually occurred after 4 to 5hours of coincubation. Sperm from fertile men exhibited significantly higher binding capacity to hemizonae compared with sperm from men who had fertilization failure during in vitro fertilization (IVF) treatment. The HZA index is calculated as follows: (bound sperm from subfertile male) ÷ (bound sperm from fertile male)×100. These findings demonstrate that the HZA may be a useful diagnostic tool in male infertility evaluations.

A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes

In vitro fertilization (IVF) is recognized as an accepted treatment for male infertility. However, the fertilization rate is significantly lower than the fertilization rate of other IVF patient groups. Some male factor infertility patients still have a basic semen quality too poor for treatment by IVF. Microinjection of a spermatozoon directly into ooplasm has been recommended to assist fertilization in this subfertile population. This study found that oocytes from 5 of 11 patients microinjected with human spermatozoa demonstrated successful pronuclear formation and correlated with the incidence of pregnancy in these patients transferred with same-source oocytes inseminated by standard protocols. This initial evidence promotes the supposition of clinical feasibility of assisted fertilization by sperm microinjection.

Factors affecting fertilization: endometrial placental protein 14 reduces the capacity of human spermatozoa to bind to the human zona pellucida*†

OBJECTIVE To examine whether placental protein 14 (PP14) may affect directly those sperm functions crucial to fertilization and early embryo development. DESIGN In these prospective studies, we evaluated semen samples of fertile men incubated under capacitating conditions with and without PP14. SETTING Academic tertiary institution. INTERVENTIONS Biologically active PP14 was purified from human midtrimester amniotic fluid by anion exchange and immunoaffinity chromatography. After separation of the motile fraction, spermatozoa were incubated for 30 minutes with or without PP14 (concentration range of 0.01 to 100 micrograms/mL), washed, and then aliquots were prepared for use in the different assays. Human sperm-zona pellucida (ZP) binding was assessed using the hemizona assay (HZA) in a 4-hour gametes coincubation period. Sperm motility parameters were evaluated using a computerized semen analyzer. The acrosome reaction (AR) was determined by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and indirect immunofluorescence. MAIN OUTCOME MEASURES Sperm-ZP binding, sperm motility patterns, and AR. RESULTS Preincubation of sperm (and not the hemizonae) with PP14 produced a significant and dose-dependent inhibition of binding in the HZA. Monoclonal antibodies generated against PP14 showed no direct effect in the HZA and partially neutralized the inhibitory activity of PP14 in the HZA. Insulin-like growth factor binding protein-1 (IGFBP-1), an endometrial stromal cell product, showed no effect in the HZA. Neither PP14 nor IGFBP-1 interfered with sperm motility parameters or the AR. CONCLUSIONS Placental protein 14 produced a potent, fast, and dose-dependent inhibition of binding of human spermatozoa to the human ZP without affecting other prefertilization events (i.e., hyperactivated motility or AR). The detrimental effect on sperm-zona interaction seems to be specific for this endometrial epithelial protein (not observed with an endometrial stromal product) and may have fundamental bearance to the fertilization process thus providing a mechanism for endometriosis-related infertility.

Induction of menstruation by an antiprogesterone steroid (RU 486) in primates: site of action, dose-response relationships, and hormonal effects

The antiprogesterone compound RU 486 (17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-[ 1-propynyl]estra-4,9-dien-3-one; Roussel-Uclaf, Paris, France) was used to induce menstruation in a randomized controlled trial. Castrated adult female cynomolgus monkeys (n = 17) received sequential subcutaneous Silastic capsules of estradiol or progesterone over a 49-day regimen which stimulated the steroidal milieu of the fertile ovarian/menstrual cycle. RU 486 was injected at 10.0, 1.0 to 3.0, or 0.1 mg/kg from days 31 to 34 of this protocol. At all concentrations tested, menstruation followed RU 486 injection within 48 hours and persisted through 72 hours. No bleeding followed placebo injections. All monkeys that menstruated after RU 486 administration also manifested vaginal bleeding after removal of exogenous steroid treatments. No changes in daily serum follicle-stimulating hormone, luteinizing hormone, prolactin, or serum and urinary free cortisol accompanied RU 486 treatment. We conclude that (1) RU 486 promptly induces menstruation by a local action upon the endometrium; (2) familiar endometrial proliferation and withdrawal bleeding can redevelop after exposure to this compound; (3) no effects of RU 486 on serum cortisol or pituitary gonadotropins were noted; and (4) extensive combination therapy with estrogen and progesterone causes mild hyperprolactinemia.

Noncompetitive antiestrogenic effect of RU 486 in blocking the estrogen-stimulated luteinizing hormone surge and the proliferative action of estradiol on endometrium in castrate monkeys

The noncompetitive antiestrogenic effects of RU 486 were examined using estradiol (E2)-treated ovariectomized monkeys given RU 486, progesterone (P), or both. The E2-induced luteinizing-hormone (LH) surge of control animals was abrogated by P and/or RU 486. Secretory transformation by P was inhibited by RU 486 coadministration. RU 486 alone (1 mg/kg) induced endometrial secretory transformation, but higher doses (5 mg/kg) inhibited proliferation and secretory activity. Thus in the presence of P, RU 486 is antagonistic but, in absence of P, exhibits endometrial progestational effects at low doses and an antiproliferative (antiestrogenic) effect at higher doses. These data encourage continued evaluation of RU 486 as a potential contraceptive agent acting at the pituitary and/or endometrial level.

Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome

The hemizona assay (HZA) was used in a prospective, blinded study to assess the relationship between tight sperm binding in the HZA and sperm fertilizing ability in in vitro fertilization (IVF). In each controlled assay, the authors compared sperm binding of proven fertile men with that of patients undergoing IVF. Human oocytes stored in a salt solution were used in the study, and binding results were correlated with the fertilization rate of preovulatory oocytes during IVF. Patients with poor fertilization rates in IVF had significantly lower binding than those cases with successful fertilization (7.3 +/- 1.4 versus 62.1 +/- 10.9, respectively; mean +/- standard error, P less than 0.02). Based on current standards, the HZA was able to predict fertilization accurately in 26 of 28 cases (sensitivity of 83%, specificity of 95%, positive predictive value of 83%). The authors conclude that the HZA is a valuable tool for evaluating dysfunctional sperm-zona pellucida binding, with good predictive value for fertilization in vitro.

The hemizona assay (HZA): A predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment

The hemizona assay (HZA) was developed to assess human sperm fertilizing potential. This blinded study investigated the relationship between sperm binding to the hemizona and in vitro fertilization (IVF) success (36 patients). Nonliving human oocytes were recovered from excised ovaries and stored. Each zona pellucida was cut into equal hemispheres by micromanipulation. For the HZA, one droplet exposed a hemizona to abnormal spermatozoa, while the control droplet contained the matching hemizona and spermatozoa from normal semen. After 4 hr, the number of tightly bound spermatozoa was counted. Binding to the hemizona was significantly higher for those having IVF success (mean of 36.1±7, versus 10.4±4 from the failure group;P<0.05). Fewer sperm from the failure group had a strictly normal morphology (3,2 versus 12.7%;P<0.05, Kruger method). Tight zona binding was significantly correlated with the percentage motile sperm, percentage normal morphology, and seminal sperm concentration. These results enhanced our confidence that the HZA is diagnostic for identification of patients at high risk of failing to achieve fertilization in vitro.

Composite pattern of circulating LH, FSH, estradiol, and progesterone during the menstrual cycle in cynomolgus monkeys.

Summary Circulating levels of LH, FSH, estradiol and progesterone were measured by radioimmunoassays in daily serum samples throughout the menstrual cycle of seven adult cynomolgus monkeys. The composite pattern of these hormones in cynomolgus monkeys strongly resembles that reported for rhesus monkeys, and is similar to that in the menstrual cycle of women. Thus, in the face of diminishing supplies and higher costs of rhesus monkeys, the cynomolgus monkey can also serve effectively as a model for studies in primate reproduction. The expert technical assistance and animal care by D. L. Barber, C. K. Turner, and J. Lewis, Jr., are gratefully acknowledged.

Menopause in rhesus monkeys: Model for study of disorders in the human climacteric

Hormonal and menstrual patterns were studied in rhesus monkeys 22 years of age or older. Sustained elevations of serum gonodotropins, low circulating levels of estradiol and progesterone, associated with oligomenorrhea or amneorrhea, were similar to changes reported for peri- and postmenopausal women. During the menopausal transition, pituitary FSH appears to be modulated independently of LH. These observations suggest that the rhesus monkey may be a suitable model for study of disorders afflicting women in the climacteric.

Effects of hydrogen peroxide on human spermatozoa

PurposeReactive oxygen species (ROS) have been reported widely to cause deleterious effects on sperm viability and function due to peroxidation of membrane lipids. However, their action appears more selective at low concentrations; recent evidence indicates that the superoxide anion can promote capacitation and induce hyperactivated motility (HA) in human spermatozoa and that hydrogen peroxide (H2O2)may participate in capacitation of hamster spermatozoa. The objective of these studies was to investigate the direct effects of H2O2on functions crucial to fertilization in human spermatozoa.MethodsIn these prospective studies, we examined the dose-and time-dependent effects of H2O2on sperm membrane-mediated events (binding to the zona pellucida and changes in intracelłular calcium concentration [Ca2+]i,motility patterns, and acrosome reaction). Sperm from fertile donors were used in the experiments under capacitating conditions after separation of the motile fraction by wash/swim-up. [Ca2+]iwas measured by the fluorescent fura-2 indicator, and sperm-zona pellucida binding was assessed with the hemizona assay (HZA). Hyperactivated motility was evaluated by computerized analysis, and the percentage of acrosomereacted sperm was detected by FITC-Pisum sativumlectin and indirect immunofluorescence.ResultsIn the HZA, H2O2did not influence sperm-zona pellucida binding at low concentrations (0.05 mMand 0.1 mM),but significantly reduced binding at 0.2 mM (P<0.004 vs controls). H2O2significantly decreased HA in a dose-dependent manner (P<0.0001) and had a significant effect (P<0.01) on acrosome reaction (stimulatory effect at 0.01 mM).H2O2did not affect basal[Ca2+]i;however, H2O2 (0.1mMthrough 10 mM)decreased the initial phase of progesterone-induced (P4: 1 μM)enhancement of [Ca2+]iin a dose-and time-dependent fashion. Preincubation of sperm with catalase (20 μg/ml) potentiated the P4-induced increase of [Ca2+]i.H2O2did not significantly modify [Ca2+]iincrease in response to inomycin (10 μM ).ConclusionsThese experiments show that H2O2directly affects sperm functions crucial to fertilization in a dose-and time-dependent fashion. Low concentrations maintain capacitation, whereas higher concentrations have deleterious effects, as determined by the end points of the capacitation process. The latter effects are probably dependent on modifications of plasma membrane and intraceliular homeostasis by the oxidative process.