Endometriosis: Effect of peritoneal fluid from infertile women with endometriosis on ionophore-stimulated acrosome loss

The effect of peritoneal fluid (PF) from endometriosis patientswas studied in spontaneous and stimulus-induced (Ca-ionophore;A23187) acrosome reactions. PF samples were obtained from 21infertile women with endometriosis and five normal women (controls).Sperm acrosomes were examined by staining with Pisum sativumagglutinin labelled with fluorescein isothiocyanate. The incidenceof spontaneous acrosome reaction after 1 and 6 h of incubation(6.7 ± 1.6 and 6.9 ± 1.4 respectively) was significantly(P < 0.001) lower when the incubation was performed withPF from endometriosis patients in comparison with spermatozoaincubated in PF from the control group (12.8 ± 1.1 and12.8 ± 0.8). Similarly, the incidence of A23187-inducedacrosome reaction after 1 and 6 h of incubation (19.8 ±2.7 and 20.0 ± 2.4) was significantly (P < 0.001)lower when spermatozoa were incubated with PF from endometriosispatients in comparison with spermatozoa incubated with PF fromthe control group (34.6 ± 9.8 and 34.4 ± 1.1).The incidence of A23187-inducible acrosome reaction was alsosignificantly (P < 0.001) lower when the incubation was performedwith PF from endometriosis patients (13.1 ± 2.8 and 13.1± 2.4) when compared with that from the control group(21.8 ± 2.6 and 21.6 ± 1.5). No relationship wasfound between the stage of endometriosis and the incidence ofacrosome loss. In conclusion, the PF from endometriosis patientsdecreased both spontaneous and stimulus-induced acrosome reaction.This may represent a mechanism for the detrimental effect ofthe PF from endometriosis patients on the spermatozoa-oocyteinteraction and partially explain the aetiology of infertilityin patients with endometriosis.     

Endometriosis: Radionuclide migration through the genital tract in infertile women with endometriosis

The migration of radionuclide through the genital tract wasobserved, comparing 20 patients with endometriosis and infertilitywith a control group of 23 infertile patients who had a normalpelvis. All patients had patent tubes at laparoscopy and chromopertubation,performed in both groups for the investigation of infertilityand to diagnose the presence and extent of endometriosis. Aradionuclide tubal test, using human albumin microspheres labelledwith 99m-technetium (^99mTc) was subsequently undertaken to observethe extent of genital tract migration of radionuclide to uterus,Fallopian tube and peritoneal cavity. The results show thatradionuclide migration to the peritoneal cavity was impairedin patients with endometriosis, compared with the control group(30 versus 83%, respectively; P < 0.001). There was no relationshipbetween migration and the severity of endometriosis. We concludethat impaired tubal function may be a cause of infertility insome patients with endometriosis.     

Endometriosis: Effect of endometriosis on white blood cell subpopulations in peripheral blood and peritoneal fluid of baboons

In women with endometriosis, changes in peripheral blood andpentoneal fluid white blood cell (WBC) populations have beenreported, but it is not known whether these alterations arecausally related to or a consequence of endometriosis. The purposeof this study was to test the hypothesis that peripheral bloodand peritoneal fluid WBC populations are altered in baboonswith spontaneous and induced endometriosis compared to animalswithout dis ease. Peripheral blood and peritoneal fluid sampleswere obtained at laparoscopy from 60 baboons with a normal pelvis(n=23), spontaneous endometriosis (n=19) and induced disease(n=18) during menses (n=9), folllcular phase (n=12), lutealphase (n=20), pregnancy or nursing (n=11) and in non-cyclinganimals (n=8). The WBC concentration was analysed with a Coultercounter and fluorescent antibody cell separation (FACS) analysiswas used to measure cluster designation (CD)2, CD4, CD8, interleukin(IL)2R and leuclne (Leu) M5 subsets. In peripheral blood, thepercentage of CD4⁺ and IL2R⁺ cells was increased in baboonswith stage II-IY spontaneous or induced endometriosis, suggestingthat alterations in peripheral blood WBC populations may bean effect of endometriosis. In peritoneal fluid the WBC concentrationand percentages of Leu M5⁺ macrophages and CD8⁺ lymphocyteswere only increased in baboons with spon taneous endometriosisand not in animals with induced disease, suggesting that alterationsin peritoneal fluid WBC populations may lead to the developmentof endometriosis. In summary, the results of this study suggestthat peripheral blood and pentoneal fluid immune cell populationsare affected in baboons with endometriosis.     

ORIGINAL ARTICLE: Leptin on Peritoneal Macrophages of Patients with Endometriosis

Citation Wu M‐H, Huang M‐F, Chang F‐M, Tsai S‐J. Leptin on peritoneal macrophages of patients with endometriosis. Am J Reprod Immunol 2010; 63: 214–221 Problem The expression of cyclooxygenase (COX)‐2 is considered as a marker of macrophage activation and has been implicated in the development of endometriosis. Leptin is an immunomodulator, which may also affect the development of endometriosis. However, how leptin contributes to these pathological processes has not been completely understood. The aim of this study was to investigate the effects of leptin on peritoneal macrophages and its relationship with endometriosis. Methods of study Peritoneal fluid from 60 women of reproductive age was obtained while they underwent laparoscopy. Forty patients had endometriosis and 20 patients did not have endometriosis. The concentration of leptin in the peritoneal fluid and prostaglandin F_2α levels was measured by ELISA, and the other protein expression using Western blot when peritoneal macrophages were stimulated with leptin. Results Concentration of leptin in peritoneal fluid was increased in patients with endometriosis compared with disease‐free normal control. Functional leptin receptor was present in peritoneal macrophages. Treatment of peritoneal macrophages with leptin induced COX‐2 expression. Production of prostaglandin F_2α by peritoneal macrophages was increased after leptin stimulation in women with endometriosis. Conclusion Elevated concentration of leptin in peritoneal fluid may contribute to the pathological process of endometriosis through activation of peritoneal macrophages.     

Endometriosis: Serum-soluble CD23 in patients with endometriosis and the effect of treatment with danazol and leuprolide acetate depot injection

Activated B cells have recently been shown to produce solubleCD23 from their membranes. The serumsoluble CD23 concentrationin 21 patients with pelvic pain diagnosed as having endometriosisand confirmed by histology, and in 18 patients without pelvicpain, who had a normal pelvis during laparoscopic sterilization,was studied by chemiluminescent enzyme-linked immunosorbentassay. The endometriosis patients were randomized to 3 monthsof either danazol or leuprolide acetate injection. Serum wastaken before and after 3 months of therapy. The serum-solubleCD23 concentration was significantly elevated in patients withendometriosis when compared with the controls (P < 0.0001).There was no correlation between soluble CD23 concentrationsand the severity of endometriosis (r = 0.48, P > 0.05). Theserum concentration of soluble CD23 decreased significantlyon treatment with danazol but not leuprolide acetate (P <0.05). We conclude that the elevation of soluble CD23 in patientswith endometriosis suggests that there is activation of B cells,which respond to danazol but not leuprolide acetate injection.     

Resistin Concentration is Increased in the Peritoneal Fluid of Women with Endometriosis

Citation Yi KW, Shin J‐H, Park HT, Kim T, Kim SH, Hur J‐Y. Resistin concentration is increased in the peritoneal fluid of women with endometriosis. Am J Reprod Immunol 2010 Problem The aim of this study was to investigate the concentration of resistin and adiponectin in the peritoneal fluid (PF) of patients with endometriosis. Method of study PF sampling was obtained from women with (n = 48) and without endometriosis (n = 36), and the anthropometric indices of the patients were measured. Resistin and adiponectin concentrations in the PF were determined using the enzyme‐linked immunosorbent assay. Results The mean concentration of PF resistin was significantly higher in women with endometriosis compared to the controls. PF resistin concentrations were not associated with any of the anthropometric indices. The PF adiponectin did not differ between the two groups, but showed a significant association with the weight, body mass index, and hip circumference. After adjusting for these factors, PF adiponectin expression was not associated with endometriosis. Conclusion The findings of this study suggest a potent role for resistin in endometriosis. Further studies are needed to elucidate the biological implications of resistin in endometriosis.     

Fertilization and early embryology: Determination of the acrosome reaction in human spermatozoa is predictive of fertilization in vitro

The acrosome reaction was determined in aliquots from ejaculatesof 74 patients undergoing in-vitro fertilization at the Universityof Giessen, Germany, by means of the triple-stain technique.The percentage of acrosome-reacted spermatozoa after low-temperatureinduction of the acrosome reaction was not significantly relatedwith the fertilization rate (H test, P = 0.693, SJ test, P =0.366). However, all patients showing < 13.0% acrosome-reactedspermatozoa had poor fertilization rates. Highly significantdifferences between patients could be detected by correlatingthe inducibility of the acrosome reaction with the fertilizationrate (H test, P = 0.018; SJ test, P = 0.004); patients withhigh fertilization rates showed a corresponding high inducibilityof acrosome reactions. From our results, it is evident thatpercentages of acrosome-reacted spermatozoa < 13.0% or aninducibility of the acrosome reaction of <7.5% are indicativeof subfertility.     

IL-1β TNF-α, and IL-2 in Peritoneal Fluid and Macrophage-Conditioned Media of Women With Endometriosis

PROBLEM : The presence of various cytokines in human peritoneal fluid has been incompletely evaluated. Changes in cytokine levels may be related to the development of endometriosis, infertility, and activation of peritoneal macrophages. This study assesses levels of IL-1β, IL-2, and TNF-α in peritoneal fluid and macrophage conditioned media of women with endometriosis. METHOD : Peritoneal fluid was collected from 51 women at the time of diagnostic or operative laparoscopy for benign gynecologic disease. Peritoneal macrophages were isolated, cultured for 24 h, and the culture media collected. IL-1β, IL-2, and TNF-α levels were determined by commercial ELISA kits. RESULTS : The mean concentration of IL-1β and TNF-α was significantly higher in macrophage conditioned media of patients with endometriosis (P < 0.02). However, there were no significant changes in peritoneal fluid cytokine levels. Peritoneal macrophage concentrations were also higher in patients with endometriosis. CONCLUSION : This study supports the concept that endometriosis is associated with activation of peritoneal macrophages, and a higher concentration of these cells. This activation is reflected by the increased levels of cytokines found in macrophage conditioned media. The absence of significant changes in peritoneal fluid cytokine levels would seem to indicate that the above derangements are not responsible for the development or progression of endometriosis.     

Andrology: High fertilization prediction by flow cytometric analysis of the CD46 antigen on the inner acrosomal membrane of spermatozoa

The study was set up to determine the relationship between thehuman sperm acrosome reaction and fertilization in couples undergoingroutine in-vitro fertilization (IVF) treatment. Prospectivedata analysis was carried out on all IVF patients during a 6month period. Exceptions were those patients having insufficientsperm concentration to allow both acrosome reaction determinationand insemination. The main outcome measures were the predictionof fertiliza tion in IVF patients using flow cytometric analysisof the spontaneous and ionophore-induced acrosome reaction [givingthe acrosomal response to ionophore challenge (ARIC) score]in the male partner's spermatozoa versus standard analyticalmethods of sperm motion parameters and morphology. Stepwiselogistic regression indicated only two independent factors predictiveof fertilization: ARIC score (x² = 109.6, P < 0.0001) andpost-Percoll % motility (x² 8.8, P < 0.003). Of patientswith an ARIC score of >10, 92% had >30% of oocytes fertilized;100% of patients with an ARIC score of <10 had <30% fertilizationof oocytes. The sensitivity and specificity of the assay systemwere 1.00 and 0.82 respectively. The results would indicatethat the ARIC test as measured by flow cytometric analysis ofCD46 binding is a sensitive and specific assay for use in theprediction of fertilization in IVF patients, thus enabling directchannelling of those patients with ARIC scores of <10 intothe more invasive micro-assisted fertilization schemes.     

Andrology: Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro

It is believed that during the process of human fertilization,acrosome-intact spermatozoa bind to the surface of the zonapellucida which triggers the acrosome reaction and the enzymesreleased facilitate sperm penetration through the zona pellucida.We describe here reduced frequency of the acrosome reactionon the zona pellucida as a cause of infertility in 10 coupleswith long durations of infertility (average 6 years) and low(<15%, n= 3) or zero (n= 7) fertilization rates in vitro.Sperm concentration, motility, velocity (Hamilton-Thorn), morphologyand DNA normality were within the normal range in all the patients.Electron microscopy of spermatozoa did not reveal any specificultrastructural defects. All couples were negative for antispermantibodies by immunobead tests. Oocytes from other patientswhich failed to fertilize in in-vitro fertilization and normaldonor spermatozoa were used as controls for sperm-zona pellucidabinding and penetration experiments. Acrosome status of spermatozoabound to the zona pellucida was assessed with a fluorescentlectin and electron microscopy. The mean number of spermatozoabound to the zona pellucida was not significantly differentbetween patients and controls. However, the acrosome reactionof spermatozoa bound to the zona pellucida after 2 h incubationwas significantly lower (P< 0.001) in the patients (mean5%, range 0–16) than in the controls (mean 68%, range44–96). No zona pellucida (out of 40) was penetrated bypatient spermatozoa whereas most (39/40) zonae were penetratedby control spermatozoa (average 27 spermatozoa/four zonae pellucidae).The spontaneous acrosome reaction of spermatozoa in inseminationmedium was not different between patients (4%) and controls(3%), the acrosome reaction induced by calcium ionophore waslow (21 and 43% respectively) in six of the eight patients examined.In conclusion, these patients have spermatozoa with a disorderof the zona pellucida-induced acrosome reaction that resultsin failure of sperm-zona pellucida penetration and explainstheir infertility.     

Endometriosis: Expression of the 60 kDa heat shock protein in peritoneal fluids from women with endometriosis: implications for endometriosis-associated infertility

Proinflammatory cytokines and activated macrophages and T lymphocyteshave been detected in peritoneal fluids of women with endometriosisand may impair fertility. Expression of the 60 kDa heat shockprotein (hsp60) is one mechanism leading to a localized activationof macrophages and T lymphocytes and cytokine release. Peritonealfluids, obtained from 68 women undergoing a diagnostic laparos-copy,were assayed for hsp60. As independent evidence of local immuneactivation, the fluids were analysed for interferon (IFN).Fluids were also tested for antibodies to Chlamydia trachomatisbecause a chronic asymptomatic infection by this organism mayalso release hsp60. At laparoscopy, 26 women were diagnosedwith pelvic adhesions, 19 had endometriosis, 16 had a visiblynormal pelvis, four had ovarian cysts while three had myomas.The prevalence of hsp60 was higher in peritoneal fluids fromthe women with endometriosis than in the other subjects (P =0.005). Hsp60 was detected in seven (36.8%) of the endometriosispatients and in only one each of the women with adhesions, anormal pelvis or an ovarian cyst; all women with myomas werenegative. Detection of IFN in peritoneal fluids was highly correlatedwith the presence of hsp60 (P = 0.0003). IFN was present inseven of nine (77.8%) women with hsp60 and in only five of 40(12.5%) women lacking hsp60. Women with pelvic adhesions hadan increased prevalence of immunoglobulin G antibodies to C.trachomatiscompared with the other women (P = 0.01). There was no relationshipbetween evidence of exposure to C.trachomatis and hsp60 in peritonealfluids. These data suggest that hsp60 may be released into theperitoneal fluid as a consequence of implanted ectopic endometrium.Hsp60-mediated immune activation may be one mechanism leadingto endometriosis-associated infertility.     

CA 125 in peritoneal fluid from patients with endometriosis.

This study was performed to evaluate CA 125 in peritoneal fluid as an indicator of endometriosis. Peritoneal fluid from patients with mostly minimal and mild endometriosis (n = 43) and normal controls (n = 17) was collected at laparoscopy or laparotomy. The median concentration of CA 125 in peritoneal fluid did not differ significantly between patients and controls (79 IU/ml versus 76 IU/ml). In patients with endometriosis, a significantly increasing concentration of CA 125 in peritoneal fluid was seen from the early follicular to the late luteal phase; a similar change was not observed in the controls. In 14 patients, peritoneal fluid was sampled again after treatment with danazol and a significant reduction in median CA 125 concentration (76.5 IU/ml versus 57 IU/ml), peritoneal fluid volume (17.5 ml versus 10.5 ml) as well as reduced endometriosis scores (4 versus 2) were found. In controls, the concentration of CA 125 was about 10 times higher in peritoneal fluid than in serum. As the peritoneal levels of CA 125 did not differ significantly between patients with endometriosis and controls and as the reduction seen after danazol treatment did not correlate with the decrease of endometriotic implants, it is concluded that the monitoring of CA 125 in peritoneal fluid will not be useful in the diagnosis or control of endometriosis.     

Immunological factors in endometriosis-associated reproductive failure: studies in fertile and infertile women with and without endometriosis

Immunopathophysiological mechanisms in endometriosis-associated reproductive failure were studied in appropriate populations: infertile and fertile women with and without endometriosis. The incidence of sera positive for any of the autoantibodies tested among infertile women with endometriosis (n = 25) was similar to that observed in the three control groups [unexplained infertility patients (n = 25) and fertile women with (n = 10) and without (n = 25) endometriosis]. The mean volume of peritoneal fluid was significantly elevated in women with endometriosis (both fertile and infertile) as compared with patients without endometriosis (fertile or infertile). The concentration of peritoneal fluid leukocytes and the percentage of cells positive for macrophage markers were significantly increased and the percentage of T lymphocytes significantly decreased in infertile women with endometriosis but not in patients with unexplained infertility and fertile women with endometriosis, as compared with fertile controls without endometriosis. Macrophages from infertile patients with endometriosis had higher sperm phagocytosis than did those from infertile women without endometriosis or fertile subjects with or without endometriosis. Incidences of serum and peritoneal fluid samples embryotoxic to the in-vitro development of 2-cell mouse embryos were significantly higher in infertile patients with endometriosis than in unexplained infertility patients and fertile women with or without endometriosis. It is concluded that immunological mechanisms of endometriosis-associated infertility exist but that these peritoneal immunological factors in infertile women with endometriosis are related to their subfertility rather than to the presence of ectopic endometrial implants. This is supported by the lack of immunological abnormalities observed among fertile women with endometriosis. These immunological abnormalities are lacking in patients with unexplained infertility.      

Endometriosis: Fluorescence of experimental endometriosis in rabbits, using tamoxifen--eosin association

A major problem with endometriosis is the detection of microscopicand atypical lesions. An incomplete surgical eradication maylead to recurrence of the disease. This study aimed to investigatethe diagnostic improvement of endometriosis by the use of tamoxifen—eosininduced fluorescence. The experimental study was performed onsurgically induced endometriosis in the rabbit. Endometriosiswas induced by grafting endometrium onto the broad ligamentin 10 rabbits. After 5 weeks, the fluorescence of excised endometriosiswas studied after systemic injection of tamoxifen and localapplication of eosin. Healthy peritoneal samples served as controls.The fluorescence of endometriotic foci was also compared with(n = 5) or without (n = 5) tamoxifen. Fluorescence excitationwas carried out using a 150 W filtered lamp connected to anoptical fibre. Fluorescence emission was measured using an opticalfibre connected to a spectrofluorometer. Spectral analysis showeda specific fluorescence of endometriosis 72 h after systemicinjection of tamoxifen and eosin application. This result isexplained by binding to oestrogen receptors of tamoxifen whichwas protonized to form an ionic pair with eosin. Histologicalstudy of samples from the graft of endometrial tissue showedthat experimental endometriosis had developed in eight out ofthe 10 rabbits. However, the fluorescence was not significantlydifferent among the 10 rabbits. This observation was in accordancewith previous studies in which endometriosis was confirmed byroutine histological techniques or electron microscopy in 70–80%of cases. Consequently, the fluorescence of the two sampleswhich did not present histological evidence of endometriosisindicates the presence of microscopic endometriotic foci. Thisobservation suggests that the diagnosis of endometriosis bythe use of tamoxifen—eosin induced fluorescence improvesthe sensitivity of detection. Identification of microscopicendometriosis will be carefully studied and the consequencesof an early identification, which could lead to excessive surgicaltreatment of this disease, will be evaluated.     

Transferrin Receptor (CD71) Expression in Peritoneal Macrophages from Fertile and Infertile Women With and Without Endometriosis

PROBLEM: Hyperactivated macrophages are implicated in the pathophysiology of endometriosis-associated infertility. This study investigates transferrin receptor expression (CD71) as a marker of hyperactivity in peritoneal macrophages of infertile patients with minimal to mild endometriosis (group 1, n = 25). METHOD OF STUDY: Expression of the activation antigen CD71 on peritoneal fluid macrophages was determined by a specific monoclonal anti-CD71 antibody using indirect immunofluorescence technique and was analyzed by flow cytometry. Three different control groups of women were used: women with unexplained infertility (group 2, n = 25), fertile women with endometriosis (group 3, n =10), and fertile women without endometriosis (group 4, n = 25). RESULTS: The percentage of CD71 positive cells was significantly increased in infertile women with endometriosis as compared with the three control groups. There were no differences among groups 2, 3, and 4 with respect to the percentage of CD71 positive macrophages. CONCLUSIONS: Our results favor the concept that hyperactivated macrophages play a role in the pathophysiology of endometriosis-associated subfertility, a feature which is lacking in patients with unexplained infertility.     

Lectin binding as a biological test in vitro for the prediction of functional activity of human spermatozoa

Adequate acrosome reaction is one of the essential events thathas to occur in successful mammalian fertilization. The purposeof the present study was to assess the acrosome reaction inhuman spermatozoa by means of iodine-labelled lectins concanavalinA and peanut agglutinin ([¹²⁵I]Con A and [¹²⁵I]PNA). Six spermsamples in the control (fertile) group were compared with 24samples obtained from infertile patients. The acrosome reactionin both groups was induced in vitro by adding follicular fluid.Iodine-labelled lectins were bound to the sperm surface, andalteration in the binding capacity for [¹²⁵I]PNA and [¹²⁵I]ConA after induced acrosomal reaction was the main parameter forthe prediction of acrosome reaction and fertilizing ability.It is hoped that with the availability of this test, in-vivodata may be accumulated.     

Effect of sperm pre-treatments on the results of sub-zonal insemination (SUZI)

Follicular fluid and progesterone, which are present in thenatural environment of oocytes, have been reported to inducethe acrosome reaction and we compared their use in pretreatmentof spermatozoa for human sub-zonal insemination)SUZI). Pre-treatmentwith follicular fluid (20% v/v) was associated with a higherfertilization rate than pre-incubation with progesterone (1mmol/I) as assessed by both the embryos/injected oocytes rate(31.7 ± 6.2% versus 13.5 ± 5.9%, respectively;P <0.01) and the male pronuclei/imjected spermatozoon rate(10.5 ± 3.3% versus 3.6 ± 1.9%, respectively;P <0.01). Since we have previously reported that pre-treatmentwith progesterone allowed a higher percentage of live-reactedspermatozoa to be obtained, these results suggest that eitherprogesterone induces modifications of the plasma membrane, whichprevent fusion with the oolema, or that follicular fluid notonly induces the asrosome reaction but increases the fusionability by compounds other than progesterone.     

Endometriosis and human infertility: a new investigation into the role of eutopic endometrium

BACKGROUND: Endometriosis is related to infertility even in the absenceof mechanical alterations of the reproductive tract. Even thoughthe pathogenesis of this phenomenon is still unclear, an impairedendometrial receptivity has been recently suggested. The aimof the present study was to investigate if endometriotic peritonealfluids (EPF) could interfere with endometrial stromal cell (ESC)decidualization and if tumor necrosis factor (TNF)- could beinvolved in the EPF effect. METHODS: Eutopic ESC were isolated from patients with or without endometriosis.ESC were treated with 17β-estradiol 10^–8 M and 6-methyl-17-hydroxyprogesteroneacetate2x10^–7 M for 16 days. In vitro decidualization was morphologicallyand biochemically assessed. We analysed whether ESC decidualizationcould be affected by EPF or peritoneal fluids from control patients(CPF), with or without soluble TNF- receptor 1 (sTNFR-1). RESULTS: Compared with ESC from control patients, eutopic ESC from patientswith endometriosis showed an impaired decidualization. Decidualizationof normal ESC was morphologically normal but biochemically abnormalin the presence of EPF, which was able to decrease the secretionof decidualization markers. sTNFR-1 was able to partially counteractthis effect. CONCLUSIONS: In endometriosis, the milieu surrounding the uterine cavitymay be involved in impaired eutopic ESC decidualization, partiallydue to increased peritoneal levels of TNF-.     

Fertilization and early embryology: Human follicular fluid inhibits the binding of human spermatozoa to zona pellucidain vitro

The effect of human follicular fluid on human zona pellucidabinding of spermatozoa was investigated using the hemizona bindingassay (HZA). This effect was compared to that of progesterone,a known component of human follicular fluid. Exposure of spermatozoato 25% pooled human follicular fluid for 1 h significantly reducedthe number of spermatozoa bound to zona pellucida when comparedto those without human follicular fluid treatment (149.1 ±30.7 versus 177.1 ± 33.8, P 0.01). The same phenomenonwas observed after 3 h of treatment The corresponding numbersof bound spermatozoa were 140.4 ± 19.1 and 200.2 ±23.4 (P 0.0001). Progesterone (1.0µg/ml) stimulated thezona pellucida-binding capacity of spermatozoa significantlyunder the same conditions (P 0.01). The numbers of bound spermatozoaafter 1 and 3 h progesterone treatment were 235.5 ± 44.7(control, 168.1 ± 32.9) and 204.3 ± 27.4 (control,162.3 ± 20.1) respectively. HZA comparing the effectsof human follicular fluid and progesterone at concentrationsequivalent to those found in human follicular fluid using matchinghemizonae confirmed the inhibitory effect of human follicularfluid on sperm binding to zona pellucida (80.4 ± 28.4versus 149.8 ± 35.2, P 0.05). This inhibitory effectwas also found in another eight individual human follicularfluid samples. Both human follicular fluid and progesteronedid not affect the motility and viability of the treated spermatozoawhen compared to the controls with the same incubation period.Although more spermatozoa underwent the acrosome reaction after1 and 3 h of human follicular fluid treatment than in the control,the extent was comparable to those after progesterone treatmentThese results suggested that human follicular fluid inhibitedthe zona pellucida-binding capacity of spermatozoa in vitro.This inhibitory effect of human follicular fluid was not mediatedby progesterone, and did not result from the effects of humanfollicular fluid on sperm motility, viability and acrosome reaction.