Progesterone induces Ca++-dependent 3',5'-cyclic adenosine monophosphate increase in human sperm.

Progesterone (P) has been reported to modulate numerous sperm functions through the binding of P to plasma membrane. One of the effects is an increase in sperm hyperactivation, which is known to be cAMP-dependent. To evaluate the effect of P on cAMP levels, human spermatozoa were incubated 2 h with increasing P concentrations. P significantly induced cAMP increase in a dose-dependent manner, reaching a 3-fold increase at 100 micromol/L (P < 0.01). During the study of the kinetics of P effect, two cAMP peaks were observed: one occurring after a 30-min incubation, with a 1.5-fold increase (P < 0.05), and the second one after a 120-min incubation, with a 2.5-fold increase (P < 0.01). These effects of P on cAMP levels correlated with significant rises in the percentage of hyperactivated spermatozoa, occurring at the same times as those of cAMP. To evaluate the Ca++-dependence of these P effects, the experiments were performed in the presence of and in the absence of Ca++ in the incubation medium. The effects of P at the 30th min and the 120th min were completely abolished in the absence of Ca++. Moreover, calcium ionophore A23187, after a 30-min incubation, induced an increase in cAMP levels identical to that obtained with P. The effect of P was partially reproduced by gamma-amino-butiric acid (GABA) and inhibited by GABA antagonist picrotoxin. It was also inhibited by tyrosine kinase inhibitor genistein but not by RU486. Based on these findings, we conclude that P induces Ca++-dependent cAMP increase in human sperm, that this effect is likely caused by the influx of Ca++ (previously reported), and that the effect partially involves GABA(A)-like receptors.

[Plasma androgens and 17 beta-estradiol of the Pyrenean urodele Euproctus asper during the period of reproduction]. / Androgènes et 17 beta-Estradiol plasmatiques de l'Urodèle pyrénéen Euproctus asper durant la période de reproduction.

The concentration of plasma androgens (A) and 17 beta-estradiol (E) in both males and females of Euproctus asper from June to November was determined by radioimmunoassay. The levels of androgens in males varied from a maximum of 980 ng/100 ml in June to a minimum of 172.5 ng/100 ml in July. A small increase was observed in September and a decrease in October-November. The concentration of androgens in females was comparable to that of males at the end of the cycle: 320 ng/100 ml; the values were high (470 ng/100 ml) in July and decreased to a minimum (80 ng) in September. The values of 17 beta-estradiol in females showed a different pattern during the reproductive cycle. The concentration was at a maximum in September, at the same time of the low concentration of androgens. In the males, the concentration of estradiol was very low (less than 17 ng/100 ml). Measurements of adenohypophysial volume and gonadotrophic cell diameter are discussed with respect to levels of circulating steroids.

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.

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.

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.

[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.

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.

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.

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.