Enhancement of zona binding using 2-hydroxypropyl- beta-cyclodextrin.

Since membrane cholesterol depletion is known to play an important role in sperm capacitation, we have investigated the effect of 2-hydroxypropyl-beta-cyclodextrin, a cyclic oligosaccharide that mediates cholesterol efflux, on sperm functions. Sperm treatment with cyclodextrin did not affect the motility patterns but induced an increase in sperm binding to zona pellucida (24 +/- 5 versus 13 +/- 4 in control, P < 0.01). Cyclodextrin treatment was associated with an increase in spontaneous acrosome reaction (32 +/- 8% versus 22 +/- 4% in controls after a 4 h incubation, P < 0.05; 61 +/- 10% versus 50 +/- 11% in controls after a 24 h incubation, NS) but with a decrease in acrosome response to ionophore challenge (44 +/- 5% versus 51 +/- 3% in controls, P < 0.05). Concerning cell sterols, cyclodextrin induced a rapid and dramatic fall in the cholesterol and desmosterol content of spermatozoa. We conclude that cyclodextrin is a powerful capacitating agent, but since it induces an increase in spontaneous acrosome loss, it needs to be further evaluated before routine use in assisted reproductive technology media.

Computer-assisted assessment of sperm morphology: comparison with conventional techniques.

The aim of the present study was to compare conventional and computer-assisted morphology assessment of spermatozoa. Sixty-two semen samples from patients undergoing in vitro fertilization (IVF) and 40 samples from patients undergoing an intracytoplasmic sperm injection (ICSI) were studied using both techniques. The percentage of normal spermatozoa found was closely correlated between the techniques (r=0.788, p < 0.0001). The intra-operator variation was low for both techniques but the inter-operator variation was much higher with the conventional than with the computer-assisted method (coefficient of variation = 0.43 vs. 0.08, respectively, for conventional and computer-assisted assessments). The percentage of spermatozoa with normal morphology, as well as sperm motility, was significantly enhanced after PureSperm preparation, whatever the method used for assessment. In the IVF study, fertilization rate was poorly correlated with sperm morphology using both methods. However, combined with motility, morphology assessed with the computer allowed discrimination of two groups of patients with significantly different fertilization rates (30.5 +/- 5.4% vs. 63.1 +/- 5.4%, p < 0.0001). In contrast, the fertilization rate in ICSI was influenced neither by sperm morphology nor by motility. In conclusion, computer-assisted assessment of sperm morphology has a slightly better predictive value for ART than conventional assessment, but above all is much more reproducible, allowing standardization.

Use of a medium devoid of any human or animal compound (SMART2) for embryo culture in intracytoplasmic sperm injection.

PURPOSE: Our purpose was to evaluate the efficiency of a medium, devoid of any human or animal compound and specially designed for early embryo development (from the zygote to the eight-cell stage), SMART2, in intracytoplasmic sperm injection (ICSI) and to compare it with a medium containing human serum albumin (EllioStep2). METHODS: Oocytes from 50 ICSI attempts were randomly placed, after sperm injection, into either SMART2 or EllioStep2. After a 48-hr incubation, the embryos were examined for quality scoring before transfer or freezing. RESULTS: The percentage of normally fertilized oocytes per intact oocytes was slightly higher using SMART2 (139/199 vs. 135/224, respectively, for SMART2 and EllioStep2; P < 0.05). The distribution of embryo scores and the percentage of embryos with a fair morphology (71/143 vs. 72/148, respectively, for SMART2 and EllioStep2; not significant) were identical in both media. CONCLUSIONS: These data show that SMART2 medium can be successfully used for early embryo growth and, because it is devoid of any human or animal compound, offers better safety for patients than conventional media.

Human sperm capacitation and in-vitro fertilization in a chemically defined and protein-free medium SMART1.

Media for sperm capacitation and in-vitro fertilization (IVF) are supplemented by proteins (albumin, globulins) extracted from human or animal sera, which raises the problem of potential contamination by pathogens. The present study aimed to evaluate the efficiency of a protein-free medium (SMART1, Bio-Media, Boussens, France) and to compare it with a human serum albumin (HSA) containing medium (FertiCult, FertiPro NV, Aalter, Belgium). In the first part of the study, media were compared for their ability to support human sperm functions. Total motility, progressive motility and rapid motility were no different between media after a 30 min and a 4 h incubation, but were significantly reduced using SMART1 after a 24 h incubation. However, the kinematic parameters (straight line velocity, mean path velocity, curvilinear velocity and mean amplitude of lateral head displacement) were significantly lower using SMART1, whatever the incubation time. The spontaneous acrosome reaction and the acrosome response to A23187 ionophore were similar in both media. In the second part of the study, media were compared in a randomized trial in 93 IVF attempts. No significant difference was found in the transfer per attempt rate (92 versus 87% respectively for SMART1 and FertiCult, NS) but the percentage of fertilized oocytes was significantly higher using SMART1 (65 versus 55% respectively for SMART1 and FertiCult, P < 0.01). The percentage of embryos with a fair morphology was identical in both media (30 versus 30% respectively for SMART1 and FertiCult, NS). In conclusion, despite a decrease in sperm kinematics, SMART1 medium allows an increase in fertilization rate and, since it is devoid of any human or animal compound, may be preferable for human use.

Use of a plant enzyme preparation (Coronase) instead of hyaluronidase for cumulus cell removal before intracytoplasmic sperm injection.

The aim of this study was to compare the efficiency of a plant enzyme preparation (Coronase) with animal extracted hyaluronidase to remove cumulus cells before intracytoplasmic sperm injection (ICSI). The first part of the study was performed on mouse oocytes and embryos. Coronase displayed a similar efficiency to that of hyaluronidase for removing cumulus cells and the same percentage of activated oocytes was obtained with both techniques. However, prolonged incubation in Coronase, 120 min, led to a degeneration of oocytes. Incubation of 2-cell mouse embryos for 10 min with Coronase did not affect their subsequent in-vitro development to blastocyst. Coronase was then compared to hyaluronidase in the treatment of human oocytes prior to ICSI. The time required for total denudation was slightly longer using Coronase (98 s +/- 25 s versus 84 s +/- 24 s respectively for Coronase and hyaluronidase; P < 0.01). However, the two pronuclear (2PN) fertilization rate (70/103 versus 63/107 respectively for Coronase and hyaluronidase, not significant) and the percentage of embryos with a good morphology (39/74 versus 32/67 respectively for Coronase and hyaluronidase, not significant) were identical with both treatments. In conclusion, Coronase displays an efficiency close to that of hyaluronidase, without any adverse effect on oocytes, and may be preferable for human use.

Randomized comparison of three media used for embryo culture after intracytoplasmic sperm injection.

Since the metabolic requirements of fertilization and early embryonic development are very different, we have tested a new culture medium (EllioStep2, Ellios Bio-Media, Paris, France) specially designed for the first cleavages and compared it with two conventional media: BM1 (Ellios Bio-Media, Paris, France) and IVF50 (Scandinavian IVF Science, Gothenburg, Sweden). In order to avoid any interference with fertilization, the test was performed as part of an intracytoplasmic sperm injection (ICSI) study. A total of 416 ICSI attempts were randomly performed using one or other of the media. After sperm injection, oocytes were incubated either in EllioStep2 or in BM1 or in IVF50. The embryo quality, pregnancy and implantation rates, number of frozen embryos were compared in the different media. The percentage of fair embryos (grades 4 and 3) was significantly higher when EllioStep2 was used than when oocytes was cultured in BM1 medium (54 versus 47%; P < 0.01) or in IVF50 (69 versus 61%; P < 0.01). The pregnancy rate per transfer and the implantation rate were not significantly higher with EllioStep2 than with BM1 or IVF50. However, the percentage of embryo freezings per attempt was significantly higher with EllioStep2 than with BM1 (47/105 versus 28/105; P < 0.01). In conclusion, the use of EllioStep2 is associated with an increase in embryo quality permitting a higher number of embryo freezings.

Structural and functional comparison of HDL from homologous human plasma and follicular fluid. A model for extravascular fluid.

In the preovulatory period, follicular fluid contains only HDL. Biochemical characterization of such lipoproteins showed that follicular fluid HDLs were cholesterol-poor particles compared with serum HDLs, whereas the amount of phospholipids, expressed as percent weight, was significantly higher in follicular fluid HDLs (28.5%) than in serum HDLs (25.0%, P < .05). The amount of apolipoprotein (apo) A-IV per apo A-I was significantly higher in follicular fluid than in serum (0.77 versus 0.58 mg/g apo A-I, P < .02). To explore the role of HDLs as cholesterol acceptors in physiological media, we compared the ability of either whole human follicular fluids or homologous sera to promote cellular cholesterol efflux using Fu5AH rat hepatoma cells. At equivalent concentrations of HDL cholesterol in follicular fluid and in serum, t1/2 values for cholesterol efflux were in the same range. In addition, estimated maximal efflux values were not significantly different in follicular fluid and serum (45.9% and 49.6%, respectively), as were K(m) values (0.064 and 0.071 mmol/L HDL cholesterol respectively). In addition, isolated HDLs displayed the same capacity to promote cellular cholesterol efflux in both media. Thus, the kinetics and dose-response data between these two physiological media showed that HDLs play the major role in cellular cholesterol efflux. The rate of cholesterol esterification, as measured in the presence of cells, was significantly higher in follicular fluid than in serum at constant HDL cholesterol concentrations, whereas the rate of esterified cholesterol transfer toward added LDL was lower. In contrast, in a cell-free system, lecithin:cholesterol acyltransferase activity represented only 26% of that in serum HDL, whereas cholesterol ester transfer protein activities were comparable. In summary, in this particular model, we confirmed the essential role of HDLs as physiological acceptors in the removal of cellular cholesterol.

[Evaluation of fertilizing ability of sperm in vitro]. / Evaluation du pouvoir fécondant du sperme in vitro.

For the study of 145 semen samples in an IVF program, a discriminant analysis allowed to calculate a score, including 8 parameters, able to predict up to 83% of IVF results.

Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit) following ejaculation.

Oxygen radical generation is known to be detrimental to sperm function, especially motility, through the lipid peroxidation of the membranes. Generation of reactive oxygen species can be induced by leukocyte contamination, sperm centrifugation and the presence of abnormal spermatozoa with excess residual cytoplasm. This study aims to evaluate the effect on sperm motility of incubation in an antioxidant-containing solution, during liquefaction and centrifugation. Thirty semen samples were each divided into two equal parts: one mixed with Tyrode's solution, the other with a salt solution containing antioxidants (Sperm-Fit; Ellios Bio-Media, Paris, France). All the procedures were identical in the two groups. The ratio of leukocytes to spermatozoa was significantly correlated with the motility after liquefaction and after a 24 h incubation in routine in-vitro fertilization (IVF) medium and with the number of motile spermatozoa recovered after Percoll preparation. Moreover, when this ratio was > or = 0.2, all motility parameters were lowered. Incubation with Sperm-Fit allowed a higher percentage of motility after Percoll preparation when the ratio was > or = 0.2 (48 +/- 5% versus 41 +/- 6% for Sperm-Fit and Tyrode's solution respectively; P < 0.05) and a greater number of motile spermatozoa recovered after Percoll preparation, whatever the ratio (3.2 +/- 1.0 x 10(6) versus 2.4 +/- 0.7 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio > or = 0.2; 18.1 +/- 3.4 x 10(6) versus 14.4 +/- 2.9 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio < 0.2; P < 0.05). These results show that incubation with antioxidants during liquefaction and centrifugation increases recovery of motile spermatozoa.

Relationships between motility parameters, morphology and acrosomal status of human spermatozoa.

A total of 130 semen samples were examined for motility (by computer-assisted sperm analysis), morphology and acrosomal status. A high positive correlation was found between percentages of normal forms and progressive motility in the whole semen (r = 0.539, P < 0. 0001) as well as in the Percoll fraction (r = 0.702, P < 0.0001). Among the specific abnormalities, acrosome defects were most highly correlated with progressive motility (r = -0.492, P < 0.0001, in the Percoll fraction). The percentage of total spontaneously acrosome-reacted spermatozoa in the Percoll fraction was negatively correlated with the progressive motility (r = -0.499, P < 0.0001) and with the percentage of normal forms (r = 0.430, P < 0.0001). Surprisingly, the percentage of total spontaneously acrosome-reacted spermatozoa was poorly linked with head abnormalities but displayed significant positive correlations with the percentages of bent tails (r = 0.359, P < 0.0001) and of coiled tails (r = 0.371, P < 0.0001). These data suggest that sperm defects are often linked together, reflecting spermiogenesis and/or epididymal dysfunctions.

Are the characteristics of spermatozoa in the insemination medium useful for predicting in-vitro fertilization results?

To determine whether the characteristics of Percoll-selected spermatozoa are more predictive of in-vitro fertilization (IVF) results than are those of native semen, 118 semen samples from patients undergoing an IVF attempt were studied. Motility, using computer-assisted sperm analysis, and morphology were recorded before and after sperm selection on a Percoll gradient. Percoll selection increased the percentage of morphologically normal spermatozoa (58.2 +/- 19.9% versus 47.9 +/- 17.9%; p < 0.0001). This increase concerned almost all abnormalities and especially abnormalities of the midpiece (cytoplasmic droplets and bent tails). However, morphology of spermatozoa in the Percoll fraction had the same predictive value for IVF as did that for whole semen. Concerning motility, all parameters, except linearity, were improved by Percoll preparation, and their predictive value for IVF results was significantly higher in the Percoll fraction than in native semen. Therefore, it is concluded that, even if sperm characteristics are improved dramatically after Percoll selection, only motility analysis then gave more practical information than did analysis of sperm characteristics in native semen. This suggests that impairment of spermiogenesis, which presumably induces sperm abnormalities, is more important than are the actual characteristics of the spermatozoa.

Validation of a scoring method predicting the in-vitro fertilizing ability of human spermatozoa.

The fertilizing ability of human spermatozoa depends upon numerous functions such as motility, normal morphology, ability to bind to the zona pellucida and to undergo the acrosome reaction. Hence a lot of tests have been developed to try and predict IVF results. In a previous study we had established a scoring method, based on parameters such as sperm morphology, vitality, motility and the acrosome reaction, which was able to predict up to 83% of in-vitro fertilization results. The present study aimed to validate this score on a separate set of sperm samples. The results confirmed those of the first series. The score allowed prediction of fertilization failures with a 56% sensitivity, a 91% specificity, a 56% positive predictive value and a 91% negative predictive value. Therefore, this score could be used routinely to choose between conventional IVF and ICSI.

Variations in spontaneous and induced acrosome reaction: correlations with semen parameters and in-vitro fertilization results.

To determine whether variations in spontaneous and induced acrosome reactions are correlated with semen quality, and to identify the inducers of clinical interest, the acrosome reaction, sperm concentration, motility and morphology were recorded in 117 semen samples from patients undergoing an in-vitro fertilization (IVF) attempt. The spontaneous acrosome loss after 24 h incubation in Ménézo B2 medium and after induction by calcium ionophore A23187, progesterone, human follicular fluid, cyclic adenosine 3'-5'-phosphate (cAMP) analogue and phorbol ester (TPA) were measured using the fluorescein isothiocyanate-GB24 antibody. The mean (range) spontaneous acrosome reaction was 3.4 +/- 0.6% (0.0-30.0). Response to the tested inducers was 23.3 +/- 1.6% (0.0-72.0) for calcium ionophore, 5.4 +/- 0.8% (0.0-60.0) for progesterone, 1.0 +/- 0.7% (0.0-24.0) for human follicular fluid, 2.6 +/- 0.7% (0.0-39.0) for the cAMP analogue and 2.3 +/- 0.7% (0.0-31.0) for TPA. The response to calcium ionophore was correlated significantly to sperm concentration, motility and morphology, while the responses to progesterone and TPA were correlated significantly to motility and acrosome morphology. The responses to other inducers were not linked to classic semen parameters. When studying acrosome reaction as a function of IVF results, the responses to calcium ionophore and TPA were discriminant. The results of this study show that the spontaneous acrosome loss and the responses to acrosome reaction inducers are highly variable and partially linked to semen quality. The responses to calcium ionophore and TPA could be of interest in predicting the fertilizing ability in vitro.

[Selection and characterization of human acrosome-reacted sperm: application to subzonal insemination]. / Sélection et caractérisation des spermatozoïdes humains ayant fait la réaction acrosomique: application au SUZI.

Acrosome reacted spermatozoa were selected with immunobeads coated with an antibody directed against the inner acrosomal membrane (GB24) and then detached immunologically using an anti-Fab'2 antibody. Using this method, we have shown that acrosome reaction occurs in the most morphologically normal spermatozoa and is followed by a loss in motility and a decrease in longevity. Moreover, when injected in the perivitelline space of hamster oocytes, the selected spermatozoa had a higher fertilization rate than unselected spermatozoa, confirming that acrosome reaction is necessary for the fusion with the oocyte plasma membrane and that the selection method does not alter the fertilizing ability of the spermatozoa. Therefore, this method represents a new way for understanding the sperm functions.

Relevance of acrosome function in the evaluation of semen in vitro fertilizing ability.

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.

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

Follicular fluid and progesterone, which are present in the natural environment of oocytes, have been reported to induce the acrosome reaction and we compared their use in pretreatment of spermatozoa for human sub-zonal insemination (SUZI). Pre-treatment with follicular fluid (20% v/v) was associated with a higher fertilization rate than pre-incubation with progesterone (1 mmol/l) 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/injected spermatozoon rate (10.5 +/- 3.3% versus 3.6 +/- 1.9%, respectively; P < 0.01). Since we have previously reported that pre-treatment with progesterone allowed a higher percentage of live-reacted spermatozoa to be obtained, these results suggest that either progesterone induces modifications of the plasma membrane, which prevent fusion with the oolema, or that follicular fluid not only induces the acrosome reaction but increases the fusion ability by compounds other than progesterone.

Selection and micro-injection of acrosome-reacted human spermatozoa.

A method of selection of acrosome-reacted human spermatozoa is described. Petri dishes were coated with GB24 antibody, specific to the inner acrosomal membrane. The acrosome-reacted spermatozoa were fixed on the antibody and could be removed by aspiration with a micro-pipette. They were then injected into the perivitelline space of hamster eggs in order to check their fertilizing ability. This selection allowed the fertilization rate to be significantly increased (24 versus 7% for control spermatozoa; P < 0.01). This method could enhance the results of human sub-zonal insemination.

Influence of sperm parameters on embryo quality.

OBJECTIVE: To evaluate the influence of sperm defects on embryo quality. DESIGN: Retrospective study. SETTING: In vitro fertilization center. PATIENTS: Embryo transfers (710) from IVF attempts for tubal disease (626) or male infertility (84). MAIN OUTCOME MEASURES: Embryo morphology as a function of causes of infertility, semen, and follicular growth parameters. Embryos were classified into three groups according to their morphology. RESULTS: Transfers of embryos with good morphology were associated to a higher pregnancy rate (34%) than those with intermediate (24%) and poor (10%) morphology. Transfers of embryos with a poor morphology were more frequent (26 of 84 versus 114 of 626) and those with a fair aspect were less frequent (24 of 84 versus 229 of 626) in male infertility than in tubal disease. Embryos with a poor morphology were associated with lower percentage of morphologically normal sperms (62% +/- 19% versus 67% +/- 18%; means +/- SD) and a higher percentage of abnormalities of the postacrosomial region (29% +/- 15% versus 18% +/- 7%). Moreover, sperms with counts < 10 x 10(6)/mL were associated with a lower percentage of embryos with good morphology (18% versus 37%) than sperms with counts > or = 10 x 10(6)/mL. CONCLUSION: Embryo quality is influenced by the semen quality and especially by sperm head abnormalities, suggesting an important role of the male gamete on the early stages of embryogenesis.

Comparison between fluorescent peanut agglutinin lectin and GB24 antibody techniques for the assessment of acrosomal status.

In order to compare fluorescent peanut (Arachis hypogaea) agglutinin lectin and GB24 antibody (specific for the inner acrosomal membrane) techniques for the assessment of acrosome reaction, both methods were applied on semen specimens obtained from patients undergoing in-vitro fertilization (IVF). The acrosome status was evaluated after a 4 h incubation in B2 medium with and without calcium ionophore A23187. Results obtained with both techniques were compared and studied as a function of IVF outcome. The percentage of spontaneous acrosome-reacted spermatozoa was higher when assessed by lectin than by GB24 (19 +/- 2% versus 11 +/- 1%; P < 0.001). The difference between the two methods (lectins minus GB24) was significantly higher in abnormal than in normal spermatozoa (10 +/- 2% versus 4 +/- 2%; P < 0.05), but did not significantly correlate with the percentage of acrosomes with abnormal morphology (r = 0.28; NS). When studied in relation to the IVF results, the response to A23187 was higher in successes than in failures (45 +/- 2% versus 34 +/- 4%; P < 0.05) but there was no significant difference between methods. Thus the assessment of acrosome reaction is strongly influenced by the method used, particularly in abnormal spermatozoa. Since the results obtained with lectins were higher in abnormal spermatozoa, GB24 seems to be more effective for assessment of true acrosome reaction.

Effect of clomiphene citrate on sperm cell function.

Experiments were performed to determine time and dose-dependent effects of clomiphene citrate on the function of fertile donor spermatozoa. The percentages of acrosome-reacted, motile and live spermatozoa were assessed. Incubation with clomiphene citrate (1-1000 mumol/l; 1-4 h) induced a dose- and time-dependent increase in the percentage of acrosome-reacted spermatozoa (up to 100%). Acrosome reaction was associated with an impairment of both motility and viability. The maximal value for percentage of live reacted spermatozoa (37 +/- 1%) was obtained when cells were incubated for 1 h with 100 mumol/l of clomiphene citrate. Oestradiol neither affected sperm function when used alone nor inhibited clomiphene action. These results show that clomiphene strongly triggers the acrosome reaction, but that this action is not related to its anti-oestrogenic properties.