Effects of human follicular fluid and high-density lipoproteins on early spermatozoa hyperactivation and cholesterol efflux.

The preovulatory human follicular fluid contains only HDLs as a lipoprotein class with a typically high proportion of prebeta HDL. We first examined the role of follicular fluid and HDL subfractions on human spermatozoa capacitation, a process characterized by a hyperactivation of the flagellar movement and a depletion of plasma membrane cholesterol. Whole follicular fluid and isolated HDL, used at constant free cholesterol concentration, were both able to promote an early flagellar hyperactivation.

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).

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.

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.

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.

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.

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.

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.

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.

[Evaluation of fertilizing ability of sperm 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.

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.

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.

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.

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.

[Selection and characterization of human acrosome-reacted sperm: application to subzonal insemination].

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.

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).

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.

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.

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.

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.

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%).

Culture of human preovulatory granulosa cells: effect of extracellular matrix on steroidogenesis.

Human luteal granulosa cells, harvested from preovulatory follicles during in vitro fertilization attempts, were cultured in a serum-precoated substratum ('serum cells') or on a collagen matrix ('collagen cells'). Concerning the 'serum cell' model, E2 secretion was very low in the absence of androgen; when androstenedione was added to the culture medium, cells secreted 180 +/- 52 pmol/ml/24 h of estradiol, 440 +/- 78 pmol/ml/24 h of testosterone and lower quantities of estrone and estriol. Follicle stimulating hormone induced a significant increase in estradiol and estriol, while the secretion of the other steroids was not altered.

High progesterone concentrations induce acrosome reaction with a low cytotoxic effect.

Objective: To determine the optimal conditions to obtain live acrosome-reacted spermatozoa for micromanipulation.

Design: Experiments were performed to determine time and dose-dependent effects of calcium ionophore A23187 or steroids on acrosome reaction of fertile donor sperm. The percentages of total reacted and live reacted spermatozoa were assessed with the peanut agglutinin lectin procedure.

The effect of oxytocin on oestradiol-17 beta and testosterone secretion by cultured human granulosa cells.

The effect of oxytocin at different concentrations was tested on the secretion of oestradiol-17 beta and testosterone by cultured human granulosa cells obtained by follicular punctures during in-vitro fertilization (IVF) attempts. Oxytocin had no effect on testosterone secretion, either in the absence or the presence of follicle stimulating hormone (FSH). It had no effect on oestradiol-17 beta in the absence of FSH.

Effect of clomiphene citrate on oestrogen secretion by human granulosa cells in culture.

The effect of clomiphene citrate (CC) and 17 beta-oestradiol (E2) on oestrogen secretion was studied in human preovulatory granulosa cells in culture. CC stimulated E2 secretion at 1 and 10 ng/ml (respectively, +36 +/- 14% and +33 +/- 5%) in basal conditions but had no effect on FSH-stimulated E2 secretion. On the other hand, E2 had no effect at any of the tested concentrations (0.