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Objective: To test the effect of sperm specimen volume in the freezing-thawing process on specimen quality., Design: Experimental prospective study., Setting: Tertiary academic medical center., Patient(s): Fifty high-quality sperm donors donated ∼3 times each. Sperm samples were split into two aliquots and frozen in volumes of 0.25 mL and 0.5 mL., Intervention(s): Semen analyses., Main Outcome Measure(s): Eight sperm quality parameters of thawed specimens., Result(s): Thawed 0.5-mL specimens had a higher percentage of motility and viability, progressive motility concentration, percentage of cells with high mitochondrial membrane potential, and intact chromatin compared with 0.25-mL specimens. Although there were fewer cells with intact acrosomes in the 0.5-mL thawed samples, they had a similar ability to respond to ionophore by acrosome reaction as the 0.25-mL specimens. Both groups had similar percentages of cells with oxidative stress and numbers of cells that bound to the zona pellucida. The remaining air volume in the straw and freezing medium composition had a minimal effect on tested parameters., Conclusion(s): Better quality thawed human sperm was achieved after cryopreservation of high volumes compared with low volumes of specimens. Air volume in the straw had no influence on specimen quality., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate perfringolysin O, a cholesterol-dependent pore-forming cytolysin, as a tool to study several aspects of human sperm physiology., Design: Prospective study., Setting: Basic research laboratory., Patient(s): Human semen samples with normal parameters obtained from healthy donors., Intervention(s): Interaction of recombinant perfringolysin O with human spermatozoa., Main Outcome Measure(s): Assessment of perfringolysin O binding to spermatozoa, tests for acrosome and plasma membrane integrity, and acrosomal exocytosis assays., Result(s): Perfringolysin O associated with human spermatozoa at 4°C. The binding was sensitive to changes in cholesterol concentrations and distribution occurring in the plasma membrane of these cells during capacitation. When perfringolysin O-treated sperm were incubated at 37°C, the plasma membrane became permeable, whereas the acrosome membrane remained intact. Permeabilized spermatozoa were able to respond to exocytic stimuli. The process was inhibited by proteins that interfere with membrane fusion, indicating that large molecules, including antibodies, were able to permeate into the spermatozoa., Conclusion(s): PFO is a useful probe to assess changes in the amount and distribution of the active sterol fraction present in the sperm plasma membrane. The toxin can be used for the efficient and selective permeabilization of this membrane, rendering a flexible experimental model suitable for studying molecular processes occurring in the sperm cytoplasm., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the effects of thawing temperature on sperm function after cryopreservation. The technical aspects of sperm cryopreservation have significantly improved over the last few decades. However, a standard protocol designed to optimize sperm motility recovery after thawing has not yet been established., Design: Prospective study., Setting: Private infertility institute and university-based research laboratory., Patient(s): Eighty consenting normozoospermic patients consulting for infertility., Intervention(s): Spermatozoa from donor semen samples were thawed at different temperatures., Main Outcome Measure(s): Sperm motility, viability, adenosine-5'-triphosphate (ATP) content, acrosomal status, and DNA integrity were evaluated as a function of thawing temperature in cryopreserved human sperm samples., Result(s): Thawing at 40 degrees C resulted in a statistically significant increase in sperm motility recovery compared with thawing at temperatures between 20 degrees C and 37 degrees C. There were no statistically significant differences in sperm viability, acrosomal status, ATP content, and DNA integrity after thawing at 40 degrees C compared with thawing at temperatures between 20 degrees C and 37 degrees C., Conclusion(s): Sperm thawing at 40 degrees C could be safely used to improve motility recovery after sperm cryopreservation., (Copyright 2010 American Society for Reproductive Medicine. All rights reserved.)

We analyzed the appearance and localization of the sub-acrosomal perinuclear theca (PT) during human spermiogenesis. The PT is tightly associated with acrosomal biogenesis.

Objective: To investigate the role of the cumulus cells and the cumulus matrix in affecting the penetrability, morphology, acrosome reaction, and motility of human spermatozoa penetrating the cumulus oophorus., Design: Controlled experimental laboratory study., Setting: University gynecology unit., Patient(s): Women undergoing assisted reproduction treatment and men visiting the subfertility clinics., Intervention(s): Human spermatozoa were allowed to penetrate through the cumulus oophorus and cell-depleted cumulus matrix in a capillary, and were treated with cumulus cell extract or hyaluronic acid., Main Outcome Measure(s): The morphology, acrosomal status, and motility of human spermatozoa were determined., Result(s): Fewer spermatozoa could penetrate the fresh cell-depleted matrix compared with intact cumulus oophorus. Spermatozoa that penetrated through the cumulus oophorus had higher percentages of normal morphology and acrosome reaction and had specific motility pattern. These effects were lost or reduced in the cell-depleted matrix that had been stored overnight. Hyaluronic acid, a main component of the cumulus matrix at concentration found in the cumulus oophorus, modulated sperm motility but did not affect spontaneous acrosome reaction. Cumulus cell extract did not affect sperm motility, but induced acrosome reaction., Conclusion(s): Both the cumulus matrix and the cumulus cells contribute to the effect of cumulus oophorus on spermatozoa penetrating through it.

Objective: To asses the effects of seminal plasma on sperm function., Design: Retrospective case-control study., Setting: University hospital., Patient(s): One hundred fourteen infertile men., Intervention(s): Acrobeads Test scores (0-4) and measurement of interleukin (IL)-6, soluble IL-6 receptor, epidermal growth factor, insulin-like growth factor-I (IGF-I), transforming growth factor-beta I, superoxide dismutase, calcitonin, and macrophage migration inhibitory factor (MIF) levels in seminal plasma., Main Outcome Measure(s): Kruskal-Wallis test to compare the concentrations of substances as a nonparametric test for differences among Acrobeads Test scores and a multivariable logistic regression model to find independent risk factors associated with abnormal Acrobeads Test results., Result(s): The Acrobeads Test score was 0 for 7 samples, 1 for 20 samples, 2 for 18 samples, 3 for 28 samples, and 4 for 41 samples. Age, abstinence period, and semen parameters, except for sperm motility and percentage of sperm with abnormal morphology, had no effect on the Acrobeads Test results. Concentrations of IGF-I and MIF were significantly higher in patients with abnormal Acrobeads Test results. Multivariate analysis indicated that MIF and IGF-I were significantly associated with abnormal Acrobeads Test results (scores 0 to 1)., Conclusion(s): Although further studies are needed, IGF-I and MIF in seminal plasma may have negative effects on sperm function.

Objective: To test, both in semen and washed-sperm fractions, whether hyaluronic acid (HA) binding is restricted to sperm that have completed cellular maturation., Design: Comparisons of sperm in semen and in HA-bound sperm fractions., Settings: University-based diagnostic and research andrology laboratory., Patient(s): Semen samples originated in men being tested for infertility., Intervention(s): The attributes of sperm maturity were tested by immunocytochemistry with creatine kinase and HspA2 antisera (highlights cytoplasmic retention in diminished-maturity sperm), aniline blue chromatin staining (detects persistent histones), pisum sativum lectin staining (reveals acrosomal integrity), and the FertiLight viability kit (highlights viable and nonviable sperm)., Result(s): All markers of sperm maturity and immaturity supported the hypothesis that HA-bound sperm are mature. Nonbinding sperm exhibited cytoplasmic and nuclear properties of diminished maturity. The acrosomal status of HA-bound sperm was either unreacted or slightly capacitated, but not acrosome reacted. Only viable sperm exhibited HA binding., Conclusion(s): Sperm that are able to bind to HA are mature and have completed the spermiogenetic processes of sperm plasma membrane remodeling, cytoplasmic extrusion, and nuclear histone-protamine replacement. Hyaluronic acid-bound sperm show unreacted acrosomes. These studies provide further insights into the relationship between spermiogenesis and sperm function.

Objective: To determine extracellular calcium (Ca(2+)) requirements for the maintenance of human sperm function in vitro., Design: Prospective study., Setting: Basic research laboratory., Patient(s): Normozoospermic volunteers provided fresh semen samples; follicular fluid (human FF) and oocytes were collected from women undergoing IVF-ET., Intervention(s): Spermatozoa were incubated for /=0.1 mM of Ca(2+) were able to undergo the AR when exposed to human FF in the presence of 2.5 mM of Ca(2+). Calcium concentrations of >/=0.22 mM were sufficient to reach protein tyrosine phosphorylation levels and hyperactivated motility values similar to those of controls. Higher Ca(2+) concentrations (>/=0.58 mM) were required to produce maximum human FF-induced AR in previously capacitated cells and to obtain an adequate sperm-ZP binding., Conclusion(s): Different steps of the fertilization process have distinctive Ca(2+) requirements. Whereas 0.22 mM of Ca(2+) is sufficient for the development of some capacitation-related events, human FF-induced AR and sperm-ZP interaction require 0.58 mM of this cation.

Objective: To determine the effect of phorbol myristate acetate (PMA) on human acrosome morphology and the acrosome reaction., Design: Controlled experiments on sperm and unfertilized oocytes from volunteers., Setting: Academic research and teaching tertiary hospital., Patient(s): Sperm samples were from normospermic men and unfertilized oocytes from IVF patients., Main Outcome Measure(s): Acrosome morphology was assessed by using transmission and scanning electron microscopy. The acrosome reaction was assessed by using fluorescein-labeled Pisum sativum agglutinin., Result(s): PMA induced acrosome ruffling, indicated by a marked wavy appearance. A significant correlation was found between PMA-induced ruffling and PMA enhancement of the zona pellucida-induced acrosome reaction. Protein kinase C inhibitors bisindolylmalemide I and sangivamycin had no effect on PMA-induced acrosomal ruffling, but actin polymerization inhibitors cytochalasin B and cytochalasin D significantly decreased PMA-induced acrosomal ruffling. In contrast, bisindolylmalemide I, sangivamycin, cytochalasin B, and cytochalasin D significantly decreased both the zona pellucida-induced acrosome reaction and the PMA enhancement of the zona pellucida-induced acrosome reaction., Conclusion(s): PMA-induced acrosomal ruffling involves actin polymerization, possibly independent of conventional protein kinase C. Acrosomal ruffling is involved in the PMA augmentation of the zona pellucida-induced acrosome reaction.

Objective: To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears., Design: The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined., Setting: Clinical and academic research environment., Patient(s): 59 patients undergoing fertility treatment., Intervention(s): Sperm samples leftover from IVF procedures were fixed and processed for the comet assay., Main Outcome Measure(s): Sperm head DNA density and sperm variables., Result(s): A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology., Conclusion(s): The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.

Objective: To investigate the effects of the phosphodiesterase inhibitor pentoxifylline on hyperactivated motility and acrosome reaction in human sperm and to determine whether its stimulatory effects occur via increased intracellular calcium levels., Design: Prospective study., Setting: Academic tertiary care facility., Participant(s): Healthy male donors., Intervention(s): The effects of pentoxifylline on hyperactivated motility, acrosome reaction, and intracellular calcium were studied and compared with the effects of progesterone. Thapsigargin, a known mobilizer of intracellular calcium, also was used as positive control., Main Outcome Measure(s): Hyperactivated motility was assessed by computer-assisted sperm motion analysis using the HTM-IVOS, acrosome reaction was evaluated with the fluorescent probe fluorescein isothiocyanate-labeled Pisum sativum agglutinin, and intracellular calcium was determined by fura-2 using spectrofluorometry., Result(s): Pentoxifylline significantly increased both hyperactivated motility and acrosome reaction. Enhancement of hyperactivated motility by pentoxifylline in the capacitation medium persisted for up to 5 hours after pentoxifylline was washed from the medium. It also enhanced the percentage of acrosome-reacted spermatozoa after 4 hours of incubation. These effects occurred in the presence of a marginally significant decrease in intracellular calcium., Conclusion(s): Pentoxifylline stimulates hyperactivated motility and acrosome reaction in spermatozoa from fertile men. Its stimulatory effects occur through mechanism(s) other than increase in intracellular calcium.

Objective: To investigate the direct effects of sodium nitroprusside, a nitric oxide donor, on the fertilizing ability of human spermatozoa in vitro., Design: Prospective, controlled in vitro study., Setting: IVF Unit, Medical College Hospital., Patient(s): Women undergoing conventional IVF., Intervention(s): Human spermatozoa samples were incubated with a nitric oxide donor and a nitric oxide quencher, carboxy-imidazolineoxyl N-oxides., Main Outcome Measure(s): The capacitation and the acrosome reaction rates were determined by chlortetracycline assay. Sperm zona pellucida binding and sperm penetration into oocytes were determined using the hemizona assay and the human aged zona-free oocyte sperm penetration assay., Result(s): High concentrations of sodium nitroprusside (10(-3) and 10(-5) M) inhibited sperm motility and viability. In contrast, low concentrations of sodium nitroprusside (10(-7) and 10(-8) M) did not affect motility and resulted in increased capacitation (47 +/- 6% at 10(-7) M, 42 +/- 6% at 10(-8) M, and 24 +/- 4% in controls, respectively, P < 0.01). A twofold increase in the hemizona index occurred compared to the matched control. However, low levels of sodium nitroprusside treatment did not affect the acrosome reaction and human zona-free oocyte sperm penetration rates., Conclusion(s): Low concentrations of nitric oxide may have some physiologic role in fertilization through the enhancement of capacitation and zona pellucida binding but not by the induction of the acrosome reaction or the facilitation of penetration into oocytes.

Objective: To evaluate the influence of human granulosa cell (GC) cultures and follicular fluid (FF) on sperm kinetic parameters, hyperactivation, and the acrosome reaction compared with the influence of human tubal fluid (HTF) and Ham's F-10 medium., Design: Sperm kinetic parameters, hyperactivation, and the acrosome reaction were evaluated after 6 hours of incubation in HTF, Ham's F-10 medium, FF, and GC cultures., Setting: Infertility and In Vitro Fertilization Centre, Reproductive Endocrinology Unit, Institute of Obstetrics and Gynaecology., Patient(s): Sixteen normal semen samples., Intervention(s): Sperm kinetic parameters and hyperactivation were analyzed using an automated videomicrography system, the acrosome reaction was performed using a triple-stain technique, and progesterone and 17OH-progesterone levels were measured with the use of commercially available kits., Main Outcome Measure(s): Sperm kinetic parameters, hyperactivation, acrosome reaction., Result(s): The percentage of motile sperm, the mean curvilinear velocity, and the mean of the maximum amplitude of lateral head movement were increased significantly after 6 hours of incubation in FF or GC cultures compared with incubation in HTF or Ham's F-10 medium, whereas the mean linearity was decreased significantly. Follicular fluid and GC cultures significantly increased hyperactivation and the acrosome reaction compared with the values obtained using HTF and Ham's F-10 medium. Progesterone and 17OH-progesterone levels were increased significantly after incubation in FF and GC cultures compared with HTF and Ham's F-10 medium., Conclusion(s): Follicular fluid and GC cultures increase sperm motility parameters, hyperactivation, and the acrosome reaction. This effect may be related to GC detoxification of the microenvironement or GC secretion of peptides, glycoproteins, growth factors (insulin-like growth factors 1 and 2), or steroids (progesterone and 17OH-progesterone).

Objective: To understand the effect of sperm contact with the apical plasma membrane of tubal epithelial cells on sperm motility, velocity, and capacitation., Design: Prospective, controlled in vitro study., Setting: University medical center., Patient(s): Women of reproductive age undergoing hysterectomy for benign gynecologic indications and normozoospermic donors of proved fertility., Main Outcome Measure(s): Sperm motility as measured manually, velocity as measured by computer-assisted sperm motility analysis, and capacitation status as measured by the chlortetracycline fluorescence assay., Result(s): Sperm incubated with apical membrane vesicles had a significantly higher motility at 12 (87.4% +/- 3.4% versus 69.2% +/- 4.8% [mean +/- SEM]), 24 (85.2% +/- 3.1% versus 60.5% +/- 7.2%) and 48 hours (78.9% +/- 5.3% versus 42.4% +/- 11.3%) compared with control (sperm incubated with human tubal fluid media in the absence of apical membrane vesicles) (n = 4). Progressive velocity was significantly higher at 12 (78.2 +/- 1.4 versus 61.7 +/- 16.1 microns/s) and 24 (66.2 +/- 3.9 versus 34.4 +/- 9.8 microns/s) hours (n = 4). Incubation with apical membrane vesicles significantly slowed the transition from uncapacitated to capacitated chlortetracycline fluorescence pattern (n = 5)., Conclusion(s): Contact with the apical plasma membrane of tubal epithelial cells enhances sperm motility and delays sperm capacitation in vitro.

Objective: To determine whether antisperm antibodies can interfere with the induction of the acrosome reaction (AR) by the zona pellucida (ZP) and whether this interference also can occur in the absence of an inhibitory effect on ZP binding., Design: Prospective in vitro study., Setting: A tertiary care center, the Andrologic Clinic, University of L'Aquila., Patient(s): Sera from 12 infertile patients with high titers of circulating antibodies directed against the sperm head were studied., Intervention: None, Main Outcome Measure(s): The effect of antisperm antibodies on ZP binding was evaluated by matching antibody-exposed and nonexposed donor sperm suspensions labeled with fluorescein or rhodamine, respectively, and incubated with the same salt-stored human ZPs. The effect of antibodies on ZP-induced AR was determined by challenging antibody-exposed and nonexposed donor sperm suspensions with human ZPs disaggregated with acidic NaH2PO4. Acrosomal status was evaluated using fluorescein-labeled Pisum sativum agglutinin and supravital stain Hoechst 33258. In some selected cases, the effect of antisperm antibodies on the acrosomal status of sperm bound to intact ZP also was evaluated using transmission electron microscopy., Result(s): Five of 12 sera exhibited an inhibitory effect on ZP binding. An inhibition of AR induction by disaggregated ZPs (ranging from 64% to 98%) was produced by all 5 sera with an inhibitory effect on ZP binding and by 2 of 7 sera without an inhibitory effect on ZP binding. The different effects of antisperm antibodies on AR induction by disaggregated ZP were confirmed by comparing with ultrastructural evaluations on the acrosomal status of sperm bound to intact ZP., Conclusion(s): Antisperm antibodies can interfere with the induction of AR by ZP. This inhibition can occur even in the absence of an inhibitory effect on ZP binding. Neither effect may occur.

Objective: To determine whether intracytoplasmic sperm injection (ICSI) is effective treatment for patients with disordered zona pellucida (ZP)-induced acrosome reaction (AR) who have failure of standard IVF-ET., Design: Tests of sperm-ZP binding and penetration and the ZP-induced AR were used to diagnose patients with disordered ZP-induced AR who then were treated with ICSI., Setting: Academic reproductive medicine research laboratory associated with a tertiary referral infertility clinic., Main Outcome Measure(s): Clinical features and results of previous IVF-ET, semen analysis and sperm-ZP interaction tests, and ICSI were analyzed., Result(s): The majority of patients had consistently normal sperm characteristics and all patients had normal sperm-ZP binding but failure of sperm-ZP penetration. The AR of sperm bound to the ZP was significantly lower in the patients (mean 6%, range 0% to 16%) than in the fertile men (mean 61%, range 27% to 96%). Intracytoplasmic sperm injection was performed on 34 patients and the normal fertilization rate averaged 73% per injected oocyte. Five ongoing and eight term pregnancies including two with twins were achieved after an average of two ET procedures per patient were performed. Two couples have second ongoing pregnancies from transfer of frozen embryos after successful first pregnancies. Overall, the implantation rate was 10.5% per embryo and the pregnancy rate 19% per ET and 38% per patient., Conclusion(s): Disordered ZP-induced AR is a cause of severe infertility and persistent failure of IVF-ET. Intracytoplasmic sperm injection is effective treatment for this condition. Patients with idiopathic infertility should be tested for this condition before commencing IVF-ET treatment.

Objective: To determine whether mannose ligand receptor and acrosome reaction deficits in sperm from men with varicocele are related to the transition metal content of their semen., Design: Cadmium and zinc in semen and blood plasma were assayed for fertile males, men without varicocele who required intracytoplasmic sperm injection to achieve fertilization, and men evaluated for potential varicocele-associated infertility. The relationship between actin cytoskeletal distributions and acrosome status was determined for fertile donor sperm in the presence and absence of exogenous cadmium., Setting: University hospital-based molecular biology research laboratory., Patient(s): Patients from two university hospital-based IVF-assisted reproductive technology programs and two male urology private practices., Intervention(s): Fertile donor sperm were exposed to exogenous cadmium during capacitating incubations followed by culture at temperatures up to 41 degrees C., Main Outcome Measure(s): Metal ion levels in semen and blood plasma were determined by graphite furnace atomic absorption spectroscopy. Motile sperm were examined for mannose ligand binding and the ability to undergo spontaneous and induced acrosome reactions. Unfixed, Triton-permeabilized sperm were probed with antiactin and antimyosin antibodies., Result(s): Cadmium was elevated and zinc was decreased in the seminal plasma of men with varicocele. The content of these metals in semen and blood was not correlated. Cadmium exposure in vitro reduced mannose receptor expression, acrosome exocytosis, and cytoskeletal formation by fertile donor sperm., Conclusion(s): Defects in transition metal regulation or excessive cadmium exposure are involved in varicocele-associated infertility.

Objective: To examine the ability of a citrate-yolk buffer extender to preserve human semen samples at ambient temperatures over a 25- to 30-hour period., Design: Human semen samples were diluted 1:1 with citrate-yolk buffer or homologous seminal plasma and transported at ambient temperature between two distant locations (London to Edinburgh, United Kingdom). Various criteria of semen quality then were assessed before and after 25 to 30 hours storage and transportation in these diluents., Setting: An institutional research laboratory and a private fertility clinic., Patient(s): Samples were provided by 21 donors of unknown fertility and 7 asthenozoospermic patients., Intervention(s): The diluent used to preserve human semen comprised an egg yolk buffer supplemented with fructose and citrate., Main Outcome Measure(s): Aspects of semen quality assessed included movement, hyaluronate penetration, viability, acrosome reaction, and reactive oxygen species generation., Result(s): The deterioration of semen quality at ambient temperatures could be prevented by the presence of citrate-yolk buffer, permitting the accurate analysis of oxidative stress and human sperm function, 25 to 30 hours postejaculation., Conclusion(s): Citrate-yolk buffer offers considerable promise as a medium for the ambient temperature storage and transportation of human semen.

Objective: To obtain quantitative measures of morphometric selectivity of the human spermzona pellucida binding process as determined by light microscopy., Design: Fully automated sperm head morphometric based on a 32-dimensional parameterization of images of Shorr-stained sperm. Zona pellucida selected sperm removed from reinseminated oocytes that previously failed IVF., Setting: Academic research group associated with a tertiary infertility service., Patient(s): Semen samples from 51 infertile patients., Intervention(s): None., Main Outcome Measure(s): Differences in morphometric parameter means observed before and after swim-up and binding to zonae pellucidae., Result(s): Significant differences between insemination and bound sperm were observed in 17 parameter means and 21 standard deviations. The sperm-zona pellucida binding process preferentially selects sperm heads with a large anterior region with relatively low optical density, as well as high axial symmetry and minimal neck anomalies. Bias against sperm with pyriform morphology was not observed., Conclusion(s): A causal link has been established between sperm head morphometry, particularly within the acrosomal region, and the ability of sperm to bind to the human zona pellucida. As sperm-zona binding is necessary for fertility, it is possible to derive a physiologically based assessment for clinical diagnosis of male infertility using the "zona-preferred" morphometry results.

Objective: To assess the relationship between the incidence of acrosome reaction (AR) and the timing of pronucleus (PN) formation after intracytoplasmic sperm injection (ICSI)., Design: Prospective study., Setting: Infertility Research Center, Jeil Women's Hospital., Main Outcome Measure(s): Human semen obtained from fertile donors was prepared by one of the following methods: washing only (washed control); Percoll gradient; pentoxifylline; human follicular fluid (FF); pentoxifylline + FF; or platelet-activating factor (PAF) treatment. The AR of each group was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin or Arachis hypogea agglutinin. Spermatozoa of washed control, pentoxifylline + FF, and PAF treated groups, with significantly higher AR rate than others, were injected into mature hamster oocytes. Spermatozoon-injected oocytes were cultured for 6, 9, 12, or 15 hours. Then they were stained with Toluidine blue for PN formation examination under a light microscope., Result(s): Acrosome reaction rates of washed control, Percoll gradient, pentoxifylline, FF, pentoxifylline + FF, and PAF treated groups were 10.5% +/- 2.6%, 10.3% +/- 1.7%, 16.4% +/- 1.8%, 24.8% +/- 5.6%, 28.4% +/- 3.8%, and 33.3% +/- 5.2%, respectively. Pronuclear formation rate in washed control, pentoxifylline + FF, and PAF treated groups were 5.6% (3/54), 19.0% (11/58), and 18.9% (10/53) at 6 hours; 32.7% (18/55), 51.8% (29/56), and 57.4% (31/54) at 9 hours; 36.1% (22/61), 53.6% (30/56), and 50.0% (27/54) at 12 hours; and 47.2% (25/53), 64.8% (35/54), 53.6% (30/56) at 15 hours after ICSI. Pronuclear formation rate was significantly higher in pentoxifylline + FF, and PAF treated groups than that in the washed control group at 6 and 9 hours after ICSI., Conclusion(s): Pronuclear formation of oocytes takes place faster on those that were injected with acrosome-reacted spermatozoon than those injected with acrosome-intact spermatozoon. It could be concluded that induction of the AR of spermatozoa accelerates the time of PN formation and early development of the embryo in ICSI.

Objective: To evaluate human sperm acrosomal status, zona pellucida (ZP)-binding capacity, and sperm motion characteristics after treatment with pertussis toxin followed by exposure to increasing concentrations of solubilized human ZP., Design: Prospective analytical study., Setting: Normal human sperm donors in an academic research environment., Intervention: Sperm were prepared with a wash and swim-up method and treated with a final concentration of 100 ng/mL pertussis toxin. Acrosomal status were determined using a Pisum sativum agglutinin-fluorescien-isothiocyanate method after exposure of sperm to 0.25, 0.5, 0.75, and 1.00 ZP/microL solutions of human ZP. Zona binding potential was recorded using intact zona-binding assays. Motion characteristics were recorded with a semen analyzer., Main Outcome Measure: Percentage acrosome-reacted sperm, number of zona-bound sperm, and sperm motion parameters., Results: Spermatozoa treated with 100 ng/mL pertussis toxin, followed by ZP-mediated acrosome reaction induction, showed a significant decrease in the percentage of acrosome-reacted sperm compared with untreated controls. Motion characteristics of 3-hour capacitated sperm after treatment with either phosphate-buffered saline (PBS) or pertussis toxin were not different. Pertussis toxin-treated sperm populations bound significantly more sperm to the ZP after 4 hours incubation compared with the PBS-control groups: 137.1 +/- 8.0 compared with 96.3 +/- 7.0 (mean +/- SEM)., Conclusions: The data support the concept of the controlling mechanism and importance of G proteins during the ZP-mediated acrosome reaction. Intact acrosome correlate with and are needed to ensure tight zona binding.

Objective: To determine whether the proportions of sperm undergoing the acrosome reaction (AR) on the zona pellucida (ZP) or on exposure to the calcium ionophore A23187 correlate with sperm-ZP penetration in men with normal sperm-ZP binding., Design: Sperm samples from 14 fertile, 50 normozoospermic, and 21 teratozoospermic men with normal sperm-ZP binding were used to study ZP and ionophore-induced AR and sperm-ZP penetration., Setting: University research unit associated with a clinical infertility and IVF program., Main Outcome Measures: The proportion of sperm undergoing the AR induced by ZP and A23187, the number of sperm bound to and penetrating into the ZP, the proportion of ZP penetrated by sperm, and semen characteristics were analyzed., Results: There were highly significant correlations between the percentage AR of sperm bound to the ZP and both the proportion of ZP penetrated and average number of sperm penetrating per ZP (Spearman r = 0.697 and 0.703). In contrast, A23187-induced AR did not correlate with either ZP-induced AR or sperm-ZP penetration. Although sperm concentration in semen correlated with both ZP-induced AR and sperm-ZP penetration by Spearman test, it was not significant in multivariate regression analyses., Conclusion: Ionophore A23187-induced AR does not correlate with either ZP-induced AR or sperm-ZP penetration. The ZP-induced AR is correlated highly with sperm-ZP penetration. Testing ZP-induced AR may be useful for assessing infertile men with normal sperm-ZP binding. In particular, it will identify failure of ZP penetration because of specific defects of the AR on the ZP.

Objective: To evaluate the binding of three fluorescein (FITC)-labeled fucose-specific lectins, Anguilla anguilla agglutinin (AAA), Tetragonolobus purpureas agglutinin (TPA), and Ulex europaeus-1 agglutinin (UEA-1), to unfixed, acrosome-intact and acrosome-reacted (AR) human sperm by flow cytometry and to compare the results with those found using FITC-labeled Pisum sativum agglutinin (PSA) and Arachis hypogaea agglutinin (PNA)., Design: The binding of five FITC-labeled lectins (PSA, PNA, AAA, TPA, and UEA-1) to capacitated, calcium ionophore A23187 (CaI)-treated, or solvent-treated human sperm was quantitated by fluorescence flow cytometry. The binding of FITC-labeled lectins was compared with the binding of anti-CD46 monoclonal antibody (mAb), a marker for the human sperm AR. The effect of fucose alpha-1->2-, alpha-1->3-, and alpha-1->4-linked oligosaccharides to inhibit the binding of FITC-fucolectins to AR sperm also was tested., Setting: University of Oklahoma Health Sciences Center, a tertiary care referral center., Results: The average percentage of fluorescing, solvent-treated sperm labeled with PSA, PNA, AAA, TPA, or UEA-1 was 98 percent, 97 percent, 15 percent, 13 percent, and 17 percent, respectively. The corresponding values for CaI-treated, lectin-labeled sperm were 98 percent, 98 percent, 89 percent, 91 percent, and 92 percent, respectively. The increase in mean fluorescence intensity for the binding of the five lectins to CaI-treated versus solvent-treated sperm was 2.9-, 6.4-, 34.5-, 22-, and 36.7-fold, respectively. The binding site of the fucolectins was confined to the equatorial segment of the AR sperm. High positive correlations were observed between the percentage of AR sperm detected using three FITC-labeled fucolectins (AAA, TPA, and UEA-1) and anti-CD46 mAb (r2 = 0.87, 0.99, and 0.99, respectively). Fucolectin binding to AR sperm was sensitive to competitive inhibition by fucose alpha-1->2-linked lacto-N-fucopentaose I and fucoidan., Conclusions: Fucosylated glycans are expressed on AR human sperm. Fluorescence-labeled fucolectins markedly improved the signal:noise ratio in the detection of acrosomal loss of human sperm when compared with FITC-PSA or FITC-PNA. Fluorescence-labeled fucolectins can be used as specific markers for flow cytometric quantitation of unfixed AR sperm in suspension.

Objective: To study the time course of capacitation, spontaneous, and A23187-induced acrosome reaction of human spermatozoa during 8 hours incubation in vitro using the chlortetracycline (CTC) assay with a revised fluorescent pattern classification., Design: Fertile donor spermatozoa were isolated by direct swim-up and incubated in Earle's balanced salt solution for up to 8 hours. At hourly intervals, spermatozoa were stained with CTC before and after the addition of A23187 to induce the acrosome reaction., Setting: The University Clinic, Jessop Hospital for Women, Sheffield, United Kingdom., Patients: Donors participating in the Donor Insemination Program., Main Outcome Measures: Eight fluorescent patterns identified by the CTC assay and acrosome-reacted spermatozoa detected by indirect immunofluorescence using 18.6 monoclonal antibody., Results: Using a statistical model defined by analysis of deviance allowed rationalization of the CTC pattern classification by grouping together patterns that showed a similar and significant change over time. In addition, spontaneous and A23187-induced acrosome-reacted spermatozoa identified by the CTC assay were shown to be correlated significantly to those identified by indirect immunofluorescence., Conclusion: The CTC assay using a revised pattern classification offers a more precise description of human spermatozoa capacitation in vitro. Also, CTC-identified acrosome reaction (both spontaneous and A23187 induced) was confirmed independently by indirect immunofluorescence.

Objective: To determine the relationships between sperm function tests and fertilization of human oocytes in vitro., Design: Analysis of infertile patients undergoing IVF therapy., Setting: Diagnostic Andrology Laboratory and Assisted Conception Service., Patients: Forty-one couples who underwent IVF-ET therapy were studied., Interventions: None., Main Outcome Measures: The ability of human spermatozoa to achieve fertilization in vitro was examined in relation to numerous criteria of semen quality, including the conventional semen profile, the computer-aided assessment of sperm movement, ionophore-induced acrosome reaction, acridine orange staining, sperm morphology, and chemiluminescent signals induced by phorbol ester and N-formyl-methionyl-leucyl-phenylalanine (FMLP)., Results: Significant correlations were observed between fertilization rates and several attributes of the sperm preparations, including elements of sperm function (acrosome reaction), movement (percentage motile, hyperactivation, the amplitude of lateral sperm head displacement), morphology (normal morphology, midpiece defects, multiple anomalies index), nuclear normality (acridine orange staining), and reactive oxygen species generation (chemiluminescence induced by phorbol ester and FMLP). In a stepwise multiple regression analysis, an accurate prediction of fertilization rates was obtained using a multiple regression equation incorporating six variables of which sperm morphology and FMLP-induced chemiluminescence were the most informative., Conclusions: A set of criteria have been identified that accurately predict the fertilizing potential of human sperm suspensions in vitro and that place particular emphasis on sperm morphology and the degree of leukocyte contamination.

Objective: To determine the relationship between acrosome reactions and hamster egg penetration after ionophore challenge in nonteratozoospermic semen samples., Setting: A tertiary care center, the Andrologic Clinic, Department of Internal Medicine, University of L'Aquila., Patients: Twenty-five nonteratozoospermic patients with infertile marriages and nine fertile men., Interventions: The hamster egg penetration assay and the acrosome reaction assessment were performed on capacitated sperm suspensions in basal conditions and after ionophore challenge with ionomycin., Main Outcome Measure: The relationship between the acrosome reactions and the hamster egg penetration was analyzed., Results: Although in basal conditions the spontaneous acrosome reaction rate was not correlated with the hamster egg penetration, after ionophore challenge both the induced acrosome reaction rate and the acrosome reaction increase (induced minus spontaneous acrosome reaction rate) correlated with the hamster egg penetration. The highest correlation was observed between the induced acrosome reaction rate and the penetration index. Considering a penetration index = 2 as the threshold of a good response of the hamster egg penetration assay to the ionophore challenge, significantly lower acrosome reaction rates and increases were associated with poor hamster egg penetration. However, different penetration indexes occurred at the same level of induced acrosome reaction rate., Conclusions: The capability of sperm to react after ionophore challenge reflects to some extent the capability of the acrosome-reacted sperm to undergo the changes of the plasma membrane that are necessary for sperm-oocyte fusion. The simple evaluation of the acrosome reactions may represent a useful complement rather than a substitute for the hamster egg penetration assay in monitoring the responses of human sperm to the ionophore challenge. An impaired inducibility of the acrosome reactions may account for poor hamster egg penetrations exhibited by nonteratozoospermic semen samples.

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.

Objective: To assess different parameters of TEST (TES and Tris) yolk buffer (TYB) treatment of human sperm that may contribute to the biologic effects of TYB on sperm function., Design: The influence of TYB treatment on occurrence of acrosome reactions was studied, as was the influence of the concentration of acrosome-reacted sperm reacted by TYB or Biggers-Whitten-Whittingham medium (BWW) incubation on penetration levels in the sperm penetration assay (SPA). The necessity for the TYB to achieve enhanced SPA performance as well as the effect of heat shock on sperm also were studied., Setting: Andrology laboratory of a university hospital., Patients: Sperm donors., Main Outcome Measures: Sperm penetration levels in the SPA and acrosomal loss as evaluated by a fluorescent lectin staining technique., Results: Sperm incubated in TYB for 42 to 46 hours at 4 degrees C demonstrated a higher rate of acrosomal loss than did sperm capacitated in BWW media for 20 to 22 hours. The difference was not significant. When insemination concentrations were normalized to identical concentrations of acrosome-reacted sperm, TYB treated specimens demonstrated much higher penetration levels compared with BWW specimens. Samples incubated in BWW versus TYB for 42 to 46 hours at 4 degrees C before heat shock had identical penetration levels. Samples washed with 37 degrees C BWW (positive heat shock) had significantly higher penetration levels than did samples washed with 4 degrees C BWW (negative heat shock)., Conclusion: Although TYB treatment does increase the occurrence of acrosome reactions, this alone does not account for the dramatic increase in penetration levels in SPA seen with these samples. TEST yolk buffer is not required for enhancement of penetration, and the heat shock step of the procedure seems to be most important for enhancement of sperm fusion ability in the SPA.

Objectives: To determine whether or not acrosome evaluation can enhance the prediction of IVF results when associated to conventional semen parameters., Design: Acrosome reaction, sperm concentration, motility, and morphology were recorded in 131 semen samples from patients undergoing an IVF attempt., Main Outcome Measures: Spontaneous acrosome loss after a 24-hour incubation in B2 medium and after induction by calcium ionophore A23187 and phorbol ester, phorbol 12-myristate 13-acetate 4-O-methyl ether (TPA)., Results: Statistically significant differences between fertilization failures and successes were found for concentration, viability, spontaneous and induced acrosome reaction, and most parameters of motility and morphology. However, none of the parameters could predict > 64% of IVF results when studied alone. A progressive discriminant analysis allowed to predict up to 83% of IVF results, by classifying sperms through their normal forms, rapid motility, spontaneous acrosome loss, enlarged heads, multiflagellar forms, vitality, linear motility, and acrosome response to TPA. The other parameters, including concentration and response to calcium ionophore, had no additive value., Conclusion: The study of acrosome function, through spontaneous acrosome loss and response to TPA, is of great interest in clinical practice when associated to some parameters of motility and morphology. However, it appears that response to calcium ionophore, one of the most studied parameters, is of poor practical interest.

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.

Objective: To examine the effects of egg yolk on the spontaneous and ionophore-induced acrosome reaction (AR) and sperm-zona pellucida (ZP) binding in spermatozoa from patients with AR prematurity., Design: Prospective analysis., Setting: Private hospital, public research center, and a university-based laboratory., Patients: In vitro fertilization patients selected on the basis of previous AR testing., Interventions: None., Main Outcome Measures: Frequency of the AR, number of ZP-bound spermatozoa., Results: The pretreatment of spermatozoa with egg yolk decreased the frequency of AR occurring spontaneously during subsequent sperm incubation in capacitating medium but did not modify the sperm response to ionophore. The egg yolk-treated spermatozoa also showed a greater capacity of binding the ZP than their untreated counterparts., Conclusions: The pretreatment of spermatozoa with egg yolk alleviates the sperm-ZP binding deficiency resulting from premature initiation of the AR and may be helpful in IVF with patients showing this anomaly.

Objective: To determine the effects of peritoneal and follicular fluids (PFs, FFs) on sperm acrosomal reaction (AR)., Design: Prospective, randomized study., Setting: Three hospital-based infertility units., Patients: Twenty-three women participating in GIFT programs; 23 men participating in AIH programs., Interventions: Hormonal stimulation after buserelin desensitation; laparoscopy 36 hrs after hCG injection., Main Outcome Measure: Percentage of acrosomally-reacted sperm., Results: Compared with Earle's medium (control), moderate but significant increases of ARs were observed as function 1) of the relative content of FF in the incubation medium and 2) as function of time (these increases were constantly lower than those registered for the respective positive control, i.e. 2x frozen/thawed sperm). In contrast, when PF alone was present in the incubation medium, no such effects on AR were registered., Conclusions: FF and mixtures of PF and FF--but not PF alone--seem to induce some rapid and time-dependent processes which finally lead to an AR. Therefore, and independently on the infertility cause (tubal, male-dependent, unexplained infertility), PF seems able to exert effects on sperm motility (Revelli et al., Fertil. Steril 57, 654-60 [1992]) while maintaining an unreacted sperm status.

Objective: To determine if the acrosome reaction of human spermatozoa can occur without prior incubation to induce capacitation or in calcium-free medium., Design: Noncapacitated (washed ejaculated) or capacitated (incubated for 3 hours in the presence of albumin) human spermatozoa were treated with either solubilized zonae pellucidae (ZP); a calcium ionophore (A23187); or activators of protein kinases A, G, and C, and the acrosomal status was monitored by the double stain technique. During agonist treatment, the capacitated spermatozoa were in medium either with or without calcium ions (Ca2+). The noncapacitated spermatozoa were always in Ca(2+)-containing medium. Sperm motility was monitored throughout the experiments., Results: Solubilized ZP and kinase activators induced acrosomal exocytosis of capacitated spermatozoa (both in Ca(2+)-containing and Ca(2+)-free medium) and of noncapacitated spermatozoa. The lack of added Ca2+ or capacitation reduced the percentage of spermatozoa that reacted in response to solubilized ZP but not to the kinase activators. Ionophore A23187 stimulated acrosomal loss from noncapacitated spermatozoa to the same extent as capacitated spermatozoa in Ca(2+)-containing medium but had no effect on capacitated spermatozoa in Ca(2+)-free medium., Conclusions: Human spermatozoa do not require incubation under capacitating conditions or the presence of extracellular Ca2+ before they can undergo the acrosome reaction in response to certain agonists. Therefore, Ca2+ influx and/or preincubation to induce capacitation are not absolute requirements for the in vitro agonist-induced acrosome reaction. However, these conditions can optimize the acrosome reaction response to zona proteins. The intracellular mechanisms leading to the acrosome reaction appear to be functional in noncapacitated spermatozoa but membrane changes probably are required before certain extracellular ligands such as zona proteins can exert their maximal effect.

Objective: To develop a method to detect acrosome-reacted spermatozoa on human zonae pellucidae using only commercially available reagents and without need for sperm fixation., Design: Sperm head labeling with biotinylated soybean trypsin inhibitor (SBTI-biotin) was compared with results of a known method using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin. The SBTI-biotin method was applied to sperm bound to human zonae pellucidae., Setting and Subjects: Healthy sperm donors with normal semen characteristics were recruited by the Laboratory for Reproductive Medicine in a university medical center., Main Outcome Measures: Soybean trypsin inhibitor-biotin binding patterns on nonfixed spermatozoa were visualized with avidin-Texas Red. The development in time of various patterns upon induction of acrosome reaction (AR) in suspension with Ca(2+)-ionophore A23187 was noted and compared with P. sativum agglutinin FITC labeling patterns. Soybean trypsin inhibitor-biotin labeling patterns of spermatozoa bound to human zonae pellucidae were determined., Results: Soybean trypsin inhibitor-biotin bound specifically, via the SBTI-moiety, to an acrosomal factor as soon as AR started. Sperm-head labeling patterns could be assigned to defined stages of the AR process. The results were highly correlated to those obtained with P. sativum agglutinin FITC. The end point of the AR in suspension and on zonae pellucidae was SBTI-biotin binding confined to the equatorial segment., Conclusion: The SBTI-biotin method can be used to detect nonfixed acrosome-reacted spermatozoa both in suspension and on zonae pellucidae.

Objective: To evaluate the effects of calcium ion (Ca2+) channel blockers on male fertility potential., Design: A case comparison of the surface expression of mannose-ligand receptors on motile spermatozoa from 10 known fertile males and from 10 normospermic men taking Ca2+ channel blockers who were seeking infertility treatment. Examination of the effects of in vitro exposure of sperm from fertile donors (n = 14) to antihypertensive medications., Setting: Patients from a successful university hospital-based IVF-assisted reproductive technology program and from a male urology private practice., Interventions: Prescription of alternate hypotensive medications for four male patients; cholesterol loading and unloading in vitro of fertile donor sperm., Main Outcome Measures: Motile sperm were tested for their ability to bind fluorescein isothiocyanate-labeled, mannosylated bovine serum albumin as an index of the surface expression of mannose-ligand receptors associated with fertility potential. Acrosome status was simultaneously evaluated by fluorescence microscopy with rhodamine-labeled Pisum sativum lectin. Sperm were assayed before and after an 18-hour or 3-day incubation under capacitating conditions in vitro., Results: Motile spermatozoa of normospermic men taking calcium antagonists for hypertension control do not express head-directed mannose-ligand receptors at high frequency, nor do they undergo spontaneous acrosome loss. Unexpectedly, mannose-ligand receptor translocation from the subplasmalemmal space over the acrosome to the sperm surface and aggregation over the equatorial-postacrosomal regions occurred in acrosome-intact sperm. This differs from fertile controls in whom receptor translocation to the equatorial-postacrosomal segment is coupled with the acrosome reaction (AR). Discontinuation of calcium antagonists results in complete recovery of parameters associated with sperm fertilizing potential: time-dependent increases in the percentages of spermatozoa exhibiting surface mannose-ligand binding and spontaneous ARs in vitro. The effects of in vivo administration of calcium antagonists is mimicked in control fertile donor sperm by inclusion of a Ca2+ channel blocker in the media employed during capacitating incubations., Conclusions: Therapeutic administrations of calcium antagonists for hypertension control cause reversible male infertility associated with an IVF failure. A mechanism of inhibition of sperm fertilizing potential through insertion of lipophilic calcium ion antagonists into the lipid bilayer of the sperm plasma membrane is consistent with our in vitro studies.

Objective: To compare the sequential effects of human cervical mucus (CM), oviductal fluid, and follicular fluid (FF) on sperm motility, hyperactivation, and the acrosome reaction during 9 hours of incubation in vitro., Design: Spermatozoa from a fertile donor were allowed to penetrate human CM in vitro and then were recovered and incubated in either 30% human oviductal fluid or a simple salt solution for 3 hours before challenge with 20% FF. Thus, the sequential effects of the fluids collected from the female reproductive tract on sperm hyperactivation and acrosome reaction were examined., Setting: The donor insemination program at the University Clinic within the Jessop Hospital for Women, Sheffield, United Kingdom., Patients: All donors used in this study were involved in the donor insemination program., Main Outcome Measures: Sperm motility, hyperactivation, and percentage of acrosome-reacted spermatozoa were measured., Results: This study used an in vitro technique to mimic sperm transport in vivo. Spermatozoa sequentially incubated with female reproductive tract secretions in vitro showed a maintenance in the level of FF-induced hyperactivated motility and a decreased response to the FF-induced acrosome reaction when compared with sperm challenged with FF in the absence of prior incubation with human oviductal fluid., Conclusion: It is suggested that human oviductal fluid may serve to delay the FF-induced acrosome reaction.

Objective: To assess the utility of the acrosome reaction (AR) to ionophore challenge test in determining the sperm treatment protocols for patients undergoing assisted reproduction., Design, Setting, Patients: One hundred twenty-one couples undergoing an IVF-ET or GIFT procedure from January to July 1992 were included in this prospective study. All cases had a preliminary semen analysis within the previous 3 months and an AR to ionophore challenge test was carried out unless an acceptable fertilization rate occurred on previous IVF. For those patients whose AR to ionophore challenge score was below the accepted fertile range of > or = 10%, a second AR to ionophore challenge test was performed after exposure of sperm to the stimulant pentoxifylline. Couples then were managed by assisted reproduction with randomized allocation of oocytes for fertilization with a standard sperm preparation or with added sperm stimulants, either 3.6 mM pentoxifylline alone or combined with 3.0 mM 2-deoxyadenosine. The study was double-blind with neither the patients nor the embryologist knowing the AR to ionophore challenge result at the time of the IVF procedure., Main Outcome Measures: Data from the preliminary semen analyses and AR to ionophore challenge scores were correlated with the fertilization rates achieved using control and treated sperm preparations. The rates of total fertilization failure and the numbers of clinical pregnancies occurring in each subgroup were also recorded., Results: All AR to ionophore challenge groups showed normal sperm counts except the groups with poor AR to ionophore challenge, which demonstrated reduced sperm counts. The group with normal AR to ionophore challenge scores or previous normal fertilization showed satisfactory fertilization rates with either control or treated sperm, although some individual cases showed reduced fertilization with treated sperm. The fertilization rate for the group with low AR to ionophore challenge scores improved significantly with pentoxifylline, and the benefit was greatest when this had been predicted from the AR to ionophore challenge studies. Cases with persisting poor AR to ionophore challenge despite pentoxifylline showed no significant improvement in fertilization rates with sperm exposed to either sperm stimulant regimens. Poor AR to ionophore challenge scores were also predictive of total fertilization failure, but this problem was reduced by sperm stimulation. The AR to ionophore challenge score at 10% cutoff level showed optimal levels of sensitivity (82.1%), highest negative predictive value (82.1%), and lowest false negative rate (17.9%)., Conclusions: The AR to ionophore challenge test is useful in the assessment and management of the male factor in assisted reproduction. It can be used to identify the majority of cases who will benefit from the use of sperm stimulants.

In an attempt to determine whether human spermatozoa contain steroid hormones, in this study we have measured the immunoreactive levels of P, 17 alpha-OHP, and F in the extracts of sonicated specimens of sperms obtained as ejaculates from husbands of infertile couples. The results of this study demonstrate for the first time that human spermatozoa contain definite amounts of the measured steroids and suggests an association between steroid levels and sperm count. These steroids identified intracellularly in the haploid germ cells may play a role in the molecular events associated with fertilization.

Objective: To determine if a low acrosin activity in otherwise normal ejaculate from infertile patients is associated with an impairment of sperm functions involved in the hamster egg penetration., Setting: A tertiary care center, the Andrology Clinic, Department of Internal Medicine, University of L'Aquila., Patients: Nine infertile patients with low acrosin activity in otherwise normal ejaculate (including normal immunoreactivity for acrosin) were studied; nine fertile men served as a control group. The two groups were homogeneous for seminal parameters., Interventions: The hamster egg penetration assay and the acrosome reaction rate assessment were performed on capacitated sperm suspensions in basal conditions and after ionophore challenge., Main Outcome Measures: The penetration rate, the penetration index, and the acrosome reaction rate were measured and compared between the groups., Results: No penetration was achieved in seven patients and a low penetration was achieved in two cases. The difference with the control group was significant. The ionophore challenge was associated to penetration of hamster eggs in seven of nine patients, but the penetration index was significantly lower than the controls. Acrosin activity was correlated to hamster egg penetration. Both spontaneous and induced acrosome reaction rate were similar in the two groups., Conclusions: A low sperm acrosin activity in otherwise normal ejaculate is associated with an impaired hamster egg penetration. This impairment does not seem to be due to altered dynamics of acrosome reaction.

Objective: To determine if the solubilized human zona pellucida (ZP)-induced acrosome reaction is mediated by protein kinases., Design: Capacitated spermatozoa were incubated with inhibitors of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent kinase (KT5720), Ca(2+)- and phospholipid-dependent kinase (Calphostin C), and cyclic guanosine 3':5'-monophosphate (cGMP)-dependent kinase (KT5823) and then treated with a corresponding kinase stimulator (dibutyryl cAMP, phorbol 12-myristate 13-acetate and dibutyryl cGMP, respectively) to determine the effect on the acrosome reaction. Appropriate controls were performed. Zonae obtained from the unfertilized oocytes of women attending an IVF program were solubilized using acidic NaH2PO4, and the effect of solubilized ZP on the acrosome reaction was tested in dose-response fashion. Comparative studies with solubilized, zona-free oocyte-treated spermatozoa were performed. The effect of the kinase inhibitors on the solubilized ZP-induced acrosome reaction was then determined., Results: No significant stimulation of the acrosome reaction by kinase stimulators occurred when spermatozoa were pretreated with inhibitors of the kinases, in contrast to the controls. Capacitated spermatozoa incubated with 2, 4, and 6 solubilized ZP showed a dose-dependent increase in the acrosome reaction. Solubilized oocytes had no effect on the acrosome reaction. Pretreatment of spermatozoa with kinase inhibitors significantly lowered the acrosome reaction induced by solubilized ZP but not completely. When a "cocktail" of the three inhibitors was used, a significant reduction in the acrosome reaction occurred in comparison with single inhibitor treatment., Conclusion: The present data indicate a role for human ZP-induced activation of multiple second messenger pathways, involving kinases A, C, and G in the human sperm acrosome reaction.

Objective: To evaluate the frequency with which acrosome-reacted spermatozoa fuse with the oocyte after subzonal insemination (SUZI)., Design: The acrosome reaction frequency determined in sperm suspensions used for SUZI was related with the frequency of sperm-oocyte fusion after the microinsemination procedure. In some experiments, the acrosome reaction frequency was compared in sperm suspensions aspirated into the microinjection needle and in those leaving the needle., Setting: Private hospital, public research center, and university-based laboratory., Patients, Participants: Patients involved in a program of micromanipulation-assisted fertilization and healthy sperm donors., Interventions: IVF-ET., Main Outcome Measures: Frequency of the acrosome reaction, frequency of sperm-oocyte fusion., Results: The procedure of sperm aspiration to the microinjection needle followed by active swim-out of motile spermatozoa did not change the overall percentage of acrosome-reacted spermatozoa even though the subpopulation of acrosome-reacted spermatozoa leaving the needle was enriched in recently reacted cells. The frequency of sperm-oocyte fusion was considerably lower than the frequency of the acrosome reaction in both patient and donor sperm samples, irrespective of the number of spermatozoa inserted per egg., Conclusions: A spontaneous acrosome reaction does not necessarily predispose a spermatozoon to fuse with the oocyte after SUZI. It remains to be elucidated whether this relative inefficiency of acrosome-reacted spermatozoa is related to technical aspects of SUZI or whether, in a more general sense, the acrosomal exocytosis and the development of sperm-oocyte fusion capacity are distinctly regulated, though closely related, phenomena.

Objective: To evaluate the acrosome reaction and its prerequisite, a calcium influx, in spermatozoa of infertile men with a high incidence of abnormal sperm forms., Design: Prospective, controlled study., Setting: Academic tertiary assisted reproduction center., Patients: Patients (n = 14) were allocated in the study after semen evaluation showed teratozoospermia (< 14% normal sperm forms) as diagnosed by strict criteria., Interventions: After swim-up separation of the motile fraction, acrosome reactions were evaluated using Pisum sativum agglutinin (both spontaneously and exogenously induced with P and the calcium ionophore A23187, both at 10 microM); the intracellular-free [Ca2+]i was assessed by the fluorescent fura-2 indicator (basal and after P)., Results: Patients did not show the typical P-induced wave of [Ca2+]i that was observed in controls but rather a blunted response, no response at all, or abnormal basal [Ca2+]i levels. The percent of basal acrosome reaction was significantly lower for patients versus controls postswim-up, and at 1 hour and 3 hours. Furthermore, there was a significant difference in the response of acrosome reaction to P both at 1 hour and 3 hours, with patients showing almost no response at all. However, patients' acrosome reaction response to the calcium ionophore was similar to those of fertile men., Conclusion: Infertile patients with a high incidence of abnormal sperm forms as diagnosed by strict criteria have a low incidence of spontaneous acrosome reaction and a diminished P-stimulated acrosome reaction, whereas the nonspecific response to a calcium ionophore is conserved. Parallel abnormalities of [Ca2+]i were observed in patients, suggesting that these sperm populations may have a defective nongenomic P sperm receptor and/or abnormalities of other membrane transduction systems.

Objective: To evaluate the effect on the function on human sperm of overnight incubation with mycoplasma species., Design: Prospective controlled in vitro study., Setting: Community-based sperm bank and andrology laboratory., Interventions: Percoll-separated sperm were incubated overnight with a low densities of Myco-plasma hominis and Ureaplasma urealyticum serotypes., Main Outcome Measures: Motility, midpiece and tail morphology, hyperactivation after overnight capacitation, and acrosome status after incubation with calcium ionophore., Results: Overnight incubation with mycoplasma species resulted in small but highly statistically significant differences in motility, morphology, hyperactivation after overnight capacitation, and proportion acrosome reacted after incubation with calcium ionophore compared with controls., Conclusions: Incubation with mycoplasma species impairs sperm physiology. Identification and treatment of mycoplasma may augment a physiology-based infertility evaluation.

Objective: To evaluate the effects of a coculture with human endometrial cells on the function of spermatozoa from samples obtained from infertile couples., Design: In a prospective study, human spermatozoa selected by swim-up from fresh samples were cultured on human endometrial feeder layers. Thereafter, their viability, motility, acrosome integrity, and ability to penetrate zona-free hamster oocytes were evaluated. Spermatozoa from the same samples incubated under the same conditions but in the absence of endometrial cells, as well as in the medium previously spent for cell culture, were used as controls., Setting: Andrology Laboratory of the Infertility Center of San Raffaele Hospital., Patients: Spermatozoa were obtained from 17 infertile men attending the Infertility Center at our hospital., Results: Spermatozoa incubated in the presence of endometrial cell feeder layers did not differ from controls with regard to their viability or motility. Conversely, the percent spontaneous acrosome reactions after 18 hours of incubation was significantly higher for spermatozoa cocultured (19.7 +/- 2.2 versus 11.2 +/- 1.9; mean +/- SE). The mean number of spermatozoa penetrating hamster oocytes was also significantly improved (1.24 +/- 0.3 versus 0.68 +/- 0.24). This effect did not seem to be solely due to the secretion of soluble factors by endometrial cells in the medium, in that spermatozoa incubated in the medium spent for endometrial cell culture had a similar acrosome reaction percentage but a lower rate of hamster egg penetration., Conclusions: The coculture with human endometrial cells appeared to be beneficial for improving the sperm function. This effect partially may be due to the secretion of steroids in the medium, which increases the quota of spontaneous acrosome reaction and in part due to the direct contact of cells with spermatozoa, maybe for the detoxification of the medium or the release of trophic factors. Coculture might be a promising approach to preparing spermatozoa for assisted fertilization in cases of subfertile males.

Objective: To determine the applicability of flow cytometry to assess human sperm acrosome reaction., Design: Prospective evaluation of semen samples incubated overnight for the development of spontaneous acrosome reaction or exposed to the calcium ionophore A23187 (5 microM) for 3 hours for induction of the acrosome reaction., Setting: University-affiliated tertiary care center., Patients: Normal healthy volunteers., Interventions: The spermatozoa were stained with fluorescein isothiocyanate (FITC)-labeled pea agglutinin. The labeled samples were assessed visually and also subjected to analytic flow cytometry and fluorescence-activated cell sorting., Main Outcome Measures: Acrosome reaction assessed visually and by flow cytometry., Results: Flow cytometric analysis showed a single peak of FITC fluorescence in the washed semen samples. A second peak of lower FITC fluorescence intensity was noted after overnight incubation or exposure to A23187, suggesting loss of fluorescence, which indicated the occurrence of the acrosome reaction. There was a statistically significant correlation between the assessment by the two methods (n = 41). However, although the mean difference between the methods was small (2.49%), the difference between the two methods for each individual sample can vary between -24% to +29%. When the sperm cells were subjected to cell sorting based on green fluorescence intensity, reanalysis and visual scoring verified that the low intensity peak contained a majority of acrosome-reacted spermatozoa (77.52% +/- 2.39%)., Conclusion: These results validated the flow cytometric method for assessment of acrosome-reacted spermatozoa. Although flow cytometry is more objective and less time consuming when many samples are assessed at the same time, the visual method remains a useful and practical procedure in the clinical andrology laboratory.

A case of an isolated impairment of the ability of acrosome-reacted spermatozoa to fuse with the oocyte is reported. This impairment was associated with fertilization failure after subzonal sperm insertion even though elevated numbers of spermatozoa were inserted per oocyte. This observation shows the need for further studies into the relationship between the acrosome reaction and the sperm-oocyte fusion to better understand the advantages and the limitations of subzonal sperm insertion in different types of sperm pathology.

Objectives: To investigate the induction of the acrosome reaction in human spermatozoa by the zona pellucida (ZP), cumulus oophorus, and cervical mucus (CM), and to examine the effect of cumulus oophorus and CM on the zona-induced acrosome reaction. DESIGN, PATIENTS, MAIN OUTCOME MEASURES: The acrosome status of spermatozoa from healthy donors that were cultured with salt-stored human ZP, cumulus oophorus, and/or cervical mucus was assessed using fluoresceinated pisum sativum agglutinin., Results: The acrosome reaction rate after sperm attachment to the zona for 6 hours was 35.7% +/- 17.7%, which was higher than controls (2.8% +/- 1.9%). The acrosome reaction was not observed after passage through cumulus oophorus or CM; however, the acrosome reaction rate of spermatozoa passed through CM after the attachment to zona was 51.6% +/- 6.8%, a higher value in comparison with spermatozoa that had not passed through CM (25.6% +/- 9.4%)., Conclusion: These data indicate that human ZP are capable of inducing the acrosome reaction in human spermatozoa; however, cumulus oophorus and CM are unable to do so. The data further provide evidence that CM promotes the zona-induced acrosome reaction in human spermatozoa.