Liquid semen storage-induced alteration in the protein composition of turkey (Meleagris gallopavo) spermatozoa.

Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca2+-binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology.

Low acrosin activity is associated with decreased Spam1/acrosin expression and GSH deficiency-caused premature acrosome release of human sperm cells.

Low acrosin activity (LAA) is associated with sperm function anomaly and poor outcomes of in vitro fertilization. In this study, we confirm that 993 semen samples with LAA had a reduced sperm motility and low in vitro fertilization rate in comparison with 1332 normal controls (NC). Proteomic comparison between 11 LAA and 11 NC sperm samples identified 35 upregulated and 99 downregulated proteins in the LAA group. Indeed, proteomic data showed that acrosome enzymes Spam1 and Acrosin were among the downregulated proteins in the LAA group, which was validated by quantitative PCR and immunefluorescent staining of sperm cells. The KEEG pathway analysis revealed a deficiency of GSH and Gln biosynthesis in LAA sperm cells. Immunofluorescent staining of sperms and quantitative PCR verified downregulation of GLUL and GCLC, the key enzymes for GSH and Gln biosynthesis. Moreover, the results of ELISA assay confirmed low levels of GSH and Gln in LAA sperm cells. Mechanistic studies showed that addition of 10 mM H2O2 to semen samples led to a significant reduction of acrosin activity and sperm motility, most possibly by triggering premature acrosome release. In contrast, the presence of 20 mM GSH blocked the oxidative effects of H2O2. Since GSH counteracts the oxidative stress and Gln participates in TCA cycling, their deficiency may affect the redox balance as well as energy production of sperm cells. These findings shed new light on the pathological mechanisms of infertility associated with LAA. Male infertility patients could benefit from GSH supplement by improvement of acrosin activity and other sperm functions.

[Value of sperm acrosin activity detection in selecting the method of assisted reproduction for patients with unexplained infertility].

OBJECTIVE: To investigate the clinical significance of sperm acrosin activity detection in selecting the method of assisted reproduction for patients with unexplained infertility (UI). METHODS: This retrospective study included 49 UI couples treated by IVFET (49 cycles) after three failures in intrauterine insemination (IUI) and another 95 couples with uterine tube obstruction (UTO) treated by IVF (131 cycles). We analyzed the laboratory data, clinical outcomes and sperm acrosin activity in the two groups of patients. According to the level of sperm acrosin activity of the males, we further divided the UI patients into two subgroups, a < 36 IU/106 sperm group (20 cycles) and a ≥36 IU/106 sperm group (29 cycles), and compared the fertilization rates between the two groups. RESULTS: Compared with UI couples treated by IVFET, the UTO couples treated by IVF had a significantly lower rate of fertilization (67.0% vs 76.4%, P < 0.05) and a higher rate of remedial intracytoplasmic sperm injection (ICSI) (20.4% vs 6.1%, P < 0.05), but showed no statistically significant differences in the rates of MII oocytes, available embryos, highquality embryos, implantation, and clinical pregnancy from the latter group (P >0.05). The sperm acrosin activity was remarkably lower in the UI than in the UTO patients (36.03 vs 61.98 IU/106, P < 0.01), and so was the fertilization rate in the < 36 IU/106 than in the ≥36 IU/106 sperm subgroup (47.7% vs 80.3%, P < 0.01). CONCLUSIONS: The low fertilization rate caused by decreased sperm acrosin activity may be the main cause of infertility and the potential factor of UI. When sperm acrosin activity is < 36 IU/106 sperm, IVF plus shortterm fertilization by remedial ICSI should be preferred to IUI.

Effect of transient scrotal hyperthermia on sperm parameters, seminal plasma biochemical markers, and oxidative stress in men.

In this experimental prospective study, we aimed to analyze the effect of transient scrotal hyperthermia on the male reproductive organs, from the perspective of sperm parameters, semen plasma biochemical markers, and oxidative stress, to evaluate whether different frequencies of heat exposure cause different degrees of damage to spermatogenesis. Two groups of volunteers (10 per group) received testicular warming in a 43°C water bath 10 times, for 30 min each time: group 1: 10 consecutive days; group 2: once every 3 days. Sperm parameters, epididymis and accessory sex gland function, semen plasma oxidative stress and serum sex hormones were tested before treatment and in the 16-week recovery period after treatment. At last, we found an obvious reversible decrease in sperm concentration (P = 0.005 for Group 1 and P= 0.008 for Group 2 when the minimums were compared with baseline levels, the same below), motility (P = 0.009 and 0.021, respectively), the hypoosmotic swelling test score (P = 0.007 and 0.008, respectively), total acrosin activity (P = 0.018 and 0.009, respectively), and an increase in the seminal plasma malondialdehyde concentration (P = 0.005 and 0.017, respectively). The decrease of sperm concentration was greater for Group 2 than for Group 1 (P = 0.031). We concluded that transient scrotal hyperthermia seriously, but reversibly, negatively affected the spermatogenesis, oxidative stress may be involved in this process. In addition, intermittent heat exposure more seriously suppresses the spermatogenesis compared to consecutive heat exposure. This may be indicative for clinical infertility etiology analysis and the design of contraceptive methods based on heat stress.

Failed fertilization after ICSI and spermiogenic defects.

OBJECTIVE: To evaluate the relationship between late spermiogenic events, including histone-protamine replacement, acrosome integrity, and sperm morphology, with fertilization rate after intracytoplasmic sperm injection (ICSI). DESIGN: Prospective study. SETTING: Isfahan Fertility and Infertility Center, Royan Institute, Tehran, Iran. PATIENT(S): Semen samples from 68 infertile couples undergoing ICSI at Isfahan Fertility and Infertility center were assessed during this study. INTERVENTION(S): Semen analysis was carried out according to World Health Organization criteria. Protamine deficiency, acrosin activity, sperm morphology, and acrosome size were assessed by chromomycin A3 (CMA3) staining, gelatinolysis test, and Papanicolaou staining (strict criteria), respectively. MAIN OUTCOME MEASURE(S): The correlation between protamine deficiency, sperm morphology, acrosin activity, and acrosome size with each other and fertilization rate were assessed. RESULT(S): Percentage CMA3 positivity and mean halo diameter show a significant correlation with fertilization rate. However, no correlation was found between sperm normal morphology and fertilization rate. The mean values of acrosome size and fertilization rate were significantly different when patients were grouped for CMA3 positivity of 40%. Multiple linear regression analysis revealed that only protamine deficiency has direct effect on fertilization rate. CONCLUSION(S): Protamine deficiency appears to have a more significant effect on fertilization after ICSI than acrosin activity and semen parameters.

Effects of dietary vitamin A intake on acrosin- and plasminogen-activator activity of ram spermatozoa.

Acrosin and plasminogen activators are proteolytic enzymes of ram spermatozoa that play an essential role in the induction of the acrosome reaction, as well as the binding of spermatozoa to the oocyte and their penetration through the layers that surround the oocyte. Since vitamin A can alter gene expression in various tissues, testis included, this study was undertaken to evaluate the possible effect of vitamin A intake on acrosin- and plasminogen-activator activity. During a 20-week experiment, 15 rams of the Greek breed Karagouniki, divided to three groups, received different amounts of vitamin A per os in retinyl acetate capsules (group A, controls, 12,500 iu/animal per day; group B, 50,000 iu/animal per day; group C, 0 iu/animal per day up to the 13th week, then 150,000 iu/animal per day until the end of the experiment). Acrosin- and plasminogen-activator activity were determined by spectrophotometric methods. Vitamin A was determined in blood plasma by HPLC. No statistical differences were detected regarding the body weight of the rams or the qualitative and quantitative parameters of their ejaculate throughout the whole experiment. No statistically significant alterations of enzyme activity were detected in group B. In group C, both enzyme activities started declining in week 9. Compared with controls, maximum reduction for acrosin was 49% on week 11 and for plasminogen activators 51% in week 14. Activities returned to normal rates after vitamin A re-supplementation. To date, the main result of vitamin A deficiency was known to be arrest of spermatogenesis and testicular degeneration. A new role for vitamin A may be suggested, since it can influence factors related to male reproductive ability before spermatogenesis is affected.

Acrosin activity as a potential marker for sperm membrane characteristics in unexplained male infertility.

OBJECTIVE: To evaluate sperm membrane system integrity in unexplained infertile male subjects with three consecutive conception failures on IUI even though semen clinical parameters were normal. DESIGN: Prospective study. SETTING: Medical biotechnology laboratory, School of Medical Science and Technology IIT Kharagpur, India. PATIENT(S): Twenty-nine patients with unexplained infertility, 17 normal proven-fertile healthy donors, and 21 infertile males with low motility but with other semen parameters remaining normal. INTERVENTION(S): Semen samples were collected from unexplained infertile patients as well as from healthy fertile donors after abstinence of 3-5 days and were analyzed according to World Health Organization guidelines. MAIN OUTCOME MEASURE(S): Release of 5'-nucleotidase (plasma membrane marker), lactate dehydrogenase (mitochondrial marker) and free acrosin, proacrosin, and total acrosin (acrosomal membrane marker). RESULT(S): Plasma membrane integrity and respiratory activity of sperm cells were comparable in all three groups. The proacrosin-acrosin system was adversely affected in unexplained infertile subjects despite high sperm motility. CONCLUSION(S): Total acrosin activity may be considered as a sensitive biochemical marker for clinical evaluation of unexplained infertility in males.

[Proacrosin, an acrosomal marker for the detection of spermatogenic cells in ejaculates from azoospermic men]. / La proacrosine, un marqueur acrosomal pour la détection des cellules spermatiques dans le sperme éjaculé de patients azoospermes.

Between January 2003 and April 2004, a prospective study was performed on ejaculates from non-obstructive azoospermic men (n = 95), for the identification of spermatogenic cells using an immunohistochemical labeling for proacrosin. 48.4% of ejaculates (46/95) displayed labeled spermatogenic cells. A 38/95 (40%) of men had testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (i.c.s.i.). Testicular spermatozoa were extracted from 21 men (52.5%). The sensitivity of detecting spermatogenic cells is 66.7% in predicting the presence of testicular spermatozoa, and its specificity is 76.5%. Compared to histopathological diagnostic testicular biopsy, the detection of spermatogenic cells using proacrosin immunohistochemical method offers a predictive parameter for successful TESE. The immunohistochemical method for proacrosin has the advantages of simplicity and low cost. It could be used to predict spermatogenesis from non-obstructive azoospermic men. Further evaluation is required with extensive results to improve sensitivity and specificity.

Efecto del medio de fecundación in vitro sobre el patron de reacción acrosómica en el espermatozoide bovino / In vitro fertilization medium effect on the bovine acrosome reaction pattern

En este trabajo se ha analizado el proceso de reacción acrosómica de muestras congeladas de semen bovino cultivados en tres medios de cultivo utilizados para la fecundación in vitro TALP, TCM-199 y BO. El estado del acrosoma y la viabilidad espermática fue evaluada mediante una doble tinción fluorescente con lectinas unidas a fluoresceina y ioduro de propidio. El medio utilizado y el tiempo de cultivo ejercen un efecto significativo sobre los parámetros estudiados. Así el porcentaje de espermatozoides con acrosomas intactos es superior en el medio TCM-199 (valor medio 30.93ñ1.18) que en TALP (28.79ñ1.40) o BO (26.53ñ1.31). El medio TALP es el que induce un mayor porcentaje de espermatozoides con reacción acrosómica (valor medio TALP 9.33ñ0.81, TCM-199 6.76ñ0.64 BO 7.33ñ0.54), mientras que los espermatozoides cultivados en el medio TCM-199 presentan un retraso en el patrón de reacción acrosómica (AU)

Comparison, characterization, and identification of proteases and protease inhibitors in epididymal fluids of domestic mammals. Matrix metalloproteinases are major fluid gelatinases.

The testicular and epididymal fluids of ram, boar, and stallion were analyzed by means of one-dimensional and two-dimensional gelatin gel zymography. Five main gelatinolytic bands were revealed in the ram and at least seven were observed in the boar and stallion. These proteolytic bands showed regionalized distribution throughout the organs. The two main proteolytic activities at around 54-66 kDa retrieved in all three species were inhibited by EDTA and phenanthroline, indicating that they were metallo-dependent enzymes. The activity of some of the low-molecular-weight gelatinases was also decreased by EDTA, whereas others were inhibited by serine protease inhibitors. One of the main proteases at 60-62 kDa from the caput fluid of the stallion and the ram was N-terminal sequenced; in both cases, high sequence homology was found with the N-terminal of the matrix-metalloproteinase-2 pro-form (pro-MMP-2). Antibodies against MMP-2, MMP-3, and MMP-9 gelatinases confirmed the regional distribution in the fluids of pre -, pro-, active, or degraded forms of these metalloproteases in all three species. We also observed the presence of acrosin in epididymal fluids, which was probably released by dead spermatozoa, but this enzyme did not explain all the serine protease activity. Moreover, the majority of this enzyme is bound to the protease inhibitor alpha(2)-macroglobulin, which is present in the fluids of all three species. TIMP-2, a potent inhibitor of MMPs, was present in the fluid of the caput regions in the ram and boar, and in the caput and caudal fluids of the stallion. This study demonstrated that similar types of proteases and inhibitors are regionally distributed in the epididymal fluids of three domestic species, suggesting an identical role in the sperm maturation process, the plasticity of this organ, or both.

Differential release of guinea pig sperm acrosomal components during exocytosis.

The contents of the sperm acrosome are compartmentalized at the biochemical and morphological levels. Biochemically, the acrosome can be considered to be comprised of two compartments: one consisting of readily soluble proteins and one containing a particulate acrosomal matrix. To test the hypothesis that compartmentalization affects the release of acrosomal components during the course of secretion in guinea pig sperm, we examined the relationship between the presence of specific proteins and acrosomal status and monitored the recovery of acrosomal constituents in the medium surrounding sperm induced to undergo exocytosis with the ionophore A23187. Cysteine-rich secretory protein 2 (CRISP-2), a soluble component of the acrosome, was rapidly lost from the acrosome soon after ionophore treatment. However, acrosomal matrix components remained associated with the sperm for longer periods. AM67, a matrix component and the guinea pig orthologue of the mouse sperm zona pellucida-binding protein sp56, was released at a slower rate than was CRISP-2 but at a faster rate than were two other matrix proteins, AM50 and proacrosin. Coincident with their release from the sperm, AM50 and proacrosin were posttranslationally modified, probably by proteolysis. The release of proacrosin from the matrix appears associated with the conversion of this protein to the enzymatically active acrosin protease. These results provide strong support for the hypothesis that compartmentalization plays a significant role in regulating the release of proteins during the course of acrosomal exocytosis. Acrosomal matrix proteins remain associated with the sperm for prolonged periods of time following the induction of acrosomal exocytosis, suggesting that transitional acrosomal intermediates may have significant functions in the fertilization process.

Efecto del protocolo de preparación de los espermatozoides bovinos sobre el patrón de reacción acrosómica / Effect of sperm preparation protocol on the bovine acrosome reaction pattern

En este trabajo se ha analizado el proceso de reacción acrosómica mediante la tinción con lectinas de muestras de semen bovino congelado que han sido sometidos a diferentes tratamientos de preparación. La adición de heparina y cafeína supone un aumento significativo del número de espermatozoides con los acrosomas reaccionados. Al seleccionar los espermatozoides en un columna de Percoll se acelera el proceso de reacción acrosómica. Estos datos confirman que la preparación de los espermatozoides para la fecundación in vitro afecta al patrón de capacitación y reacción acrosómica, de manera que pueden ser determinantes para el proceso de fecundación (AU)

Effects of season and breed on sperm acrosin activity and semen quality of boars.

Acrosin activity and semen quality (sperm concentration, ejaculate volume and number of spermatozoa) were assessed from March 1997 to March 1998 in semen of Large White, Pietrain and Duroc x Pietrain boars. Semen quality varied with season, including high production of spermatozoa in autumn and winter and low production in summer. Semen quality also differed across breeds. Acrosin activity of boar spermatozoa was not affected by breed (range 3.16-3.32 mU/10(6) spermatozoa), but exhibited distinct seasonal changes. Monthly changes in acrosin activity were parallel to changes in number of sperm in the ejaculate from November to March. On the other hand, dramatic changes in acrosin activity between July and October (range 1.85-4.59 mU/10(6) spermatozoa) were not paralleled by similar changes in number of ejaculated sperm. These fluctuations in acrosin activity may reflect either changes in sperm acrosin production or disturbances to sperm membranes, probably related to effects of high summer temperatures during spermatogenesis. Results confirmed seasonal and breed-related differences in boar semen quality characteristics.

Immunolocalization of proacrosin/acrosin in bovine sperm and sperm penetration through the zona pellucida.

The aim of the present work was to immunolocalize acrosin in bull spermatozoa incubated for up to 6 h in capacitating culture medium (TALP-heparin), in order to study the kinetics of its release during the acrosome reaction and in vitro sperm penetration. Six replicates from semen of one bull were used. Acrosin was localized by the silver-enhanced immunogold technique using anti-bovine acrosin monoclonal antibody ACRO-C2E5. Spermatozoa thus showed the presence of acrosin only at the acrosomal region. Four different patterns were seen: (1) no labeling: (2) intense labeling on the rim of the portion of the acrosome; (3) diffuse label over the entire acrosomal region; and (4) intense label over the entire acrosomal region. Spermatozoa incubated in capacitating medium for 4 h showed that unlabeled (pattern 1) spermatozoa decreased from 72% to 28% difference that was found to be significant (p<0.05). Patterns 3 and 4 increased from about 10% to 20-29%, (p<0.05). With further incubation (4-6 h), pattern 1 increased while patterns 3 and 4 decreased differences were not significant (p0.05). The incidence of pattern 2 did not change through the whole incubation period. Sperm penetration through the zona pellucida of in vitro matured bovine oocytes (57%) or empty zonae pellucida (70.5%) increased (p<0.05) as a function of sperm incubation time in capacitating medium. The presence of acrosin, as determined by the silver-enhanced immunogold technique, was highly correlated with sperm penetration of in vitro mature bovine oocyte (r=0.98) and cryopreserved zonae pellucidae (r=0.93) (p<0.01).

Determination of sperm acrosin activity for evaluation of male fertility.

AIM: To investigate a simple method for assaying acrosin activity for the evaluation of male fertility. METHODS: The acrosin activity of 7.5 x 10(6) sperm without seminal plasma and acrosin activity inhibitors was assayed using N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and detergent (Triton X-100) as substrate. RESULTS: The acrosin activity of 60 normal fertile men (35 +/- 10 microIU/10(6) sperm ) was higher than that of 168 infertile men (16 +/- 8 microIU/10(6) sperm) (P < 0. 01). It was indicated that there was a significant positive correlation between the acrosin activity and the sperm motility (r > or = 0.6534, P < 0.01) and a significant negative correlation between the sperm malformed rate and the WBC number (r < or = -0.5426, P < 0.01). The temperature and time of incubation and the sperm concentration could influence the assay results. CONCLUSION: Acrosin activity is an important index for the evaluation of male fertility. The approach developed by the authors is a simple method for the determination of acrosin activity.

Immunolocalization of CRES (Cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa.

The CRES (cystatin-related epididymal spermatogenic) protein is a member of the cystatin superfamily of cysteine protease inhibitors and exhibits highly restricted expression in the reproductive tract. We have previously shown that CRES protein is present in elongating spermatids in the testis and is synthesized and secreted by the proximal caput epididymal epithelium. The presence of CRES protein in developing germ cells and in the luminal fluid surrounding maturing spermatozoa prompted us to examine whether CRES protein is associated with spermatozoa. In the studies presented, indirect immunofluorescence, immunogold electron microscopy, and Western blot analysis demonstrated that CRES protein is localized in sperm acrosomes and is released during the acrosome reaction. Interestingly, while the 19- and 14-kDa CRES proteins were present in testicular and proximal caput epididymal spermatozoa, the 14-kDa CRES protein was the predominant form present in mid-caput to cauda epididymal spermatozoa. Furthermore, following the ionophore-induced acrosome reaction, CRES protein localization was similar to that of proacrosin/acrosin in that it was detected in the soluble fraction as well as associated with the acrosome-reacted spermatozoa. The presence of CRES protein in the sperm acrosome, a site of high hydrolytic and proteolytic activity, suggests that CRES may play a role in the regulation of intraacrosomal protein processing or may be involved in fertilization.

Monoclonal antibodies to canine intra-acrosomal sperm proteins recognizing acrosomal status during capacitation and acrosome reaction.

Monoclonal antibodies Ds-1 and Ds-2 specifically labelling dog sperm acrosome were prepared by immunization of mice with acetic acid extracts of dog spermatozoa. Electron microscopy and indirect immunofluorescence localized the site of Ds-1 and Ds-2 proteins inside the acrosomal vesicle. Ds-1 antibody detected 55, 76, 115, 120 and 190 kDa proteins under non-reducing conditions, and 73 kDa and 54 kDa proteins after reduction (p73/Ds-1 and p54/Ds-1). 92 kDa and 40 kDa proteins recognized by Ds-2 (p92/Ds-2 and p40/Ds-2) migrated at > 200 kDa in the absence of reducing agent. In vivo, p73/Ds-1 and p54/Ds-1 are therefore likely to be present both in free and complexed form, while all of p92/Ds-2 and p40/Ds-2 form disulfide-bonded complexes. Decrease in the rate of acrosomes stained with Ds-1 and Ds-2 was correlated with the progress of capacitation resulting in the increased rate of spontaneous acrosome reactions, as suggested by a dramatic effect of A23187. Monoclonal antibody to boar acrosin (ACR-2) recognized dog sperm acrosin homologue. A higher rate of ACR-2-negative spermatozoa was observed after capacitation and A23187 treatment compared to Ds-1 and Ds-2, indicating that proteins recognized by Ds-1 and Ds-2 are localized in a specific compartment of acrosome, distinct from acrosin and possibly representing fraction of acrosomal matrix.

A novel method for evaluating the acrosomal status of mammalian spermatozoa.

A novel method was developed to evaluate the acrosomal status of mammalian spermatozoa. The method is based on the ability of the lectin Pisum sativum agglutinin (PSA) to bind specifically to glycoproteins of the acrosomal matrix released during the acrosome reaction. The amount of released acrosomal content is proportional to the fraction of spermatozoa that underwent acrosome reaction. The released glycoproteins present in the supernatant separated from the cells were detected via an ELISA-like assay. The authors suggest that one of these glycoproteins might be the acrosin as identified by anti-acrosin antibodies, using Western blot analysis. The new method (demonstrated here with ram and bull spermatozoa) correlates well with the results obtained by conventional methods. Its advantages are simplicity, objectiveness, rapidity, and low cost. In addition, many samples can be processed in parallel. The method can be used in experimental as well as clinical applications.

Prognostic value of objective semen parameters in an in vitro fertilization program.

PURPOSE: The basic semen parameters seem to have a limited predictive value in male fertility. Could other objective sperm analyses be helpful in the choice of the most adapted assisted procreation technique? METHODS: This study concerns 78 infertile couples with insemination failures. For each semen, 21 objective parameters are analyzed in fresh semen and after sperm selection procedure. The 78 couples are then included in an IVF protocol and classified into two groups: fertile (at least one cleaved embryo is obtained) and infertile. RESULTS: Using multiple variant discriminant factorial analysis, we have found nine nonconventional parameters which induce us to define two classes of semen. These two classes fit with the classification into fertile and infertile groups in 74.4% of the cases. CONCLUSIONS: So these parameters allow us to predict the chance of obtaining embryos during an IVF trial and to choose for each couple the most appropriate technique: IVF or ICSI.