Age as only predictive factor for successful sperm recovery in patients with Klinefelter's syndrome.

The study was performed to determine factors affecting successful sperm retrieval by testicular sperm extraction in patients with nonmosaic Klinefelter's syndrome (KS). From May 2001 to February 2007, 27 azoospermic patients were diagnosed as having nonmosaic KS. All patients underwent sperm testicular extraction. Patient's age, testicular volume, serum follicle-stimulating hormone (FSH) and inhibin B were assessed as predictive factors for successful sperm recovery. Of the 27 Klinefelter's patients examined, eight (29.6%) had successful sperm recovery. The comparisons of serum FSH, inhibin B and testicular volume between patients with and without successful sperm retrieval did not show any statistical significance. The patients with successful sperm recovery were significantly younger (28.6 +/- 3.11 years) than those with failed attempts (33.9 +/- 4.5 years, P = 0.002). The rate of positive sperm retrieval was significantly higher in patients younger than 32 years compared with patients older than 32 years (P = 0.01, chi-squared test). The study showed that clinical parameters such as FSH, inhibin B and testicular volume do not have predictive value for sperm recovery in patients with KS. The mean age of our patients with successful sperm recovery was significantly lower than that of men with unsuccessful results. Testicular sperm extraction or testicular sperm aspiration should be performed before the critical age of 32 years.

[Single-embryo transfer: Rennes' Hospital experience]. / Transfert monoembryonnaire : expérience du CHU de Rennes.

In 2005-2006, 905 punctures of oocytes were realised in the Assisted Reproductive Technology Centre of Rennes' Hospital in France, the source of 173 pregnancies after fresh embryos transfers and 185 pregnancies after frozen embryos transfers. The single-embryo transfer (SET) was proposed in all patients aged less than 38 years with at least two embryos type I or II on the first two cycles. Sixty-three percent of patients (n=293 cycles) chose the SET with 21.5% pregnancies through fresh embryo transfer. The SET with frozen embryos has been completed on 708 cycles with 16.8% of pregnancies per transfer. The cumulative rate of pregnancies by puncture is 39% and the overall risk of multiple pregnancies has dropped to 12%. The SET associated with an effective embryo cryopreservation therefore allows to reduce the risk of multiple births while maintaining a satisfactory pregnancy cumulative rate.

[Vitrification: Principles and results]. / La vitrification: principes et résultats.

Sperm and embryos cryopreservation is a commonly applied technique for several years. Recently authorized in France, vitrification tends to replace gradually the conventional technique of slow freezing, so upsetting the practices in the management of patients. It allows from now on the cryopreservation of oocytes and opens new perspectives in egg donation either still in fertility preservation. This review thus attempted to examine the contribution of vitrification in the freezing of oocytes and human embryos at various stages of development. If obviously vitrification appears as the current method of choice for the cryopreservation of oocytes as well as blastocysts, the results are less cut as regards embryos to early stages. No increase in adverse obstetric and perinatal outcomes in children conceived from vitrified oocytes or embryos is noted in the literature.

Assessment of acrosome-reacted boar spermatozoa using monoclonal antibody GB 24 and propidium iodide.

Fluorescein-labeled GB 24, a mouse monoclonal antibody, was evaluated as an acrosomal dye for boar spermatozoa that had previously been stained with propidium iodide (PI) to assess sperm viability. A specific sperm-staining pattern with fluorescein-labeled GB 24 was shown to be associated with acrosome reaction on freshly ejaculated sperm when fixed with acetone or induced with ionophore A 23187, whereas the presence of PI staining was typical of dying spermatozoa. The GB 24-PI procedure was as accurate as the glutaraldehyde method in assessing acrosomal presence or absence on freshly ejaculated spermatozoa when spontaneous or A 23187-induced acrosomal reactions were considered. Approximately half of A 23187-induced spermatozoa with acrosomal loss did not exhibit a PI fluorescence; these were potentially viable acrosome-reacted spermatozoa. On semen diluted in a boar sperm-specific diluent (BTS-A) and stored, percentages of spermatozoa with nonintact acrosome from glutaraldehyde and GB 24-PI were not significantly different. Conversely, data from GB 24-PI was significantly lower than those from glutaraldehyde when semen were undiluted. This suggested that spermatozoa with reacted acrosome gradually lost their ability to bind with GB 24. Providing unequivocal and rapid scoring of acrosome-reacted spermatozoa, the GB 24-PI procedure may be a valuable tool in the evaluation of the acrosomal status of porcine fresh spermatozoa.

[Microsurgical epididymal sperm aspiration (MESA), testicular biopsy and intracytoplasmic sperm injection (ICSI) in the treatment of male infertility]. / Aspiration microchirurgicale des spermatozoïdes épididymaires (MESA), biopsie testiculaire et injection intra-cytoplasmique du spermatozoïde (ICSI) dans le traitement de l'infertilité masculine.

OBJECTIVE: To assess if ICSI using epididymal or testicular spermatozoa is effective in the treatment of couples with male factor infertility. MATERIAL AND METHODS: 56 couples suffering from male infertility underwent a total of 88 treatment cycles of ICSI, in combination with microsurgical epididymal sperm aspiration (MESA): 62 cycles, or testicular sperm retrieval: 26 cycles. RESULTS: Overall, fertilization rate was 63% per injected metaphase II oocyte. In MESA group, fertilization rate was 64% compared to 61% embryos in testicular sperm aspiration. The overall pregnancy rate was 26% per started ICSI cycle. Pregnancy rates were also similar in both group: 24% for MESA and 32% for testicular sperm extraction. 9 pregnancy (41%) were obtained using cryopreserved epididymal sperm (6 cases) or testicular sperm (3 cases). CONCLUSION: ICSI combined with epididymal or testicular sperm achieved a high fertilization and pregnancy rate. It constitutes an efficient alternative in the treatment of men suffering of testicular failure or azoospermia not amenable to surgical reconstruction.

[Oocyte donation as in France]. / Pour un don d'ovocytes à la française.

Oocyte donation, initially proposed in agonadal women, saw indications expand to ovarian deficiencies and failures of in vitro fertilization (IVF), resulting in a significant increasing demand. The recruitment of oocyte donors is a critical issue for all countries that have allowed this practice. The French legislation, with the laws of bioethics, is clearly the most restrictive of European countries, imposing an absolute free gift from mother. The different solutions in the neighboring countries are analysed and in particular the interpretations made in respect of gratuity and compensation. Motivating donors (spontaneous, relational, or by reciprocity), but also motivating the medical teams can organize a program of oocyte donation in France. The authors present their results of three years experience, demonstrating that this system is possible in the current legislative framework.

Effects of antioxidants on human sperm preparation techniques.

The effect of two different sperm preparation techniques, Percoll gradient centrifugation and swim-up from a washed pellet were tested on the functional competence of the selected spermatozoa. Percoll gradient centrifugation brought about an improvement in sperm motility parameters such as curvilinear velocity and straight-line velocity, an increase in the rates of hyperactivation and the acrosome reaction and an increase in the percentage of motile spermatozoa after 24 h of incubation compared to the centrifugation, swim-up procedure. The effects of antioxidants such as dithiothreitol (DTT) or reduced glutathione (GSH), and reactive oxygen species-scavenging enzymes such as catalase or superoxide dismutase (SOD) during the stage of centrifugation before the swim-up procedure were also studied. Though all of these agents prevented the fall in sperm motility after 24 h incubation, only DTT and SOD improved the rates of both hyperactivation and the acrosome reaction. GSH also improved the acrosome reaction, whereas catalase was without significant effect on the rates of hyperactivation or the acrosome reaction. These results indicate that Percoll gradient centrifugation selects spermatozoa with better functional competence than does centrifugation swim-up. The damage caused by the centrifugation can be prevented by the addition of antioxidants, suggesting that the differences noted with the Percoll gradient method was due to better protection against peroxidative damage due to the centrifugation of unselected spermatozoa. However, the use of DTT is limited by virtue of the fact that this sulphydryl reducing agent leads to destabilization of the sperm chromatin. In contrast, GSH and SOD could have therapeutic potential.

Reactive oxygen species and human spermatozoa: physiology and pathology.

The role of reactive oxygen species (ROS) in the pathophysiology of human sperm function has been emphasized in recent years. ROS production in semen has been associated with loss of sperm motility, decreased capacity for sperm-oocyte fusion and loss of fertility. There is a current presumption that the most prolific source of ROS in sperm suspensions is an NADPH oxidase located in leukocytes or in spermatozoa which produces superoxide which is further converted to peroxide by the action of superoxide dismutase. Hydrogen peroxide has been recognized as the most toxic oxidizing species for human spermatozoa, which are very sensitive to lipid peroxidation owing to the high content of polyunsaturated fatty acids in their plasma membrane, though this is not the sole mechanism by which sperm function might be impaired by ROS. Although the excessive production of ROS is detrimental to human spermatozoa, there is a growing body of evidence which suggests that ROS are also involved in the physiological control of some sperm functions. This review focuses on the nature and source of the ROS generated by human spermataozoa as well as their operational mechanisms and their effects, which may be detrimental or beneficial.

Influence of oxygen tension on reactive oxygen species production and human sperm function.

Human spermatozoa were exposed to the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to stimulate endogenous production of reactive oxygen species (ROS). They were incubated under a gas phase of 5% CO2/90% N2/5% O2, or 5% CO2/95% air (20% O2) to investigate whether a lower than atmospheric oxygen tension in the gas phase of the incubator limited the endogenous production of ROS by human spermatozoa and thus was able to reduce the cytotoxic effects of ROS on sperm function. Exposure of human spermatozoa or exogenous NADPH induced an 8-fold higher production of superoxide anion under ambient vs. low oxygen tension. This marked difference in the stimulation of superoxide anion generation was associated with significantly different sperm motility parameters, according to the oxygen tension in the gas phase of the incubator. Whereas under 5% oxygen the percentage of motile spermatozoa was unaffected by the presence of NADPH, all of the motility parameters recorded under an atmosphere of 5% CO2 in air were dramatically affected, not only compared to their respective controls, but also compared to the motility parameters observed under low oxygen tension. The presence of superoxide dismutase plus catalase protected spermatozoa against the toxic effects of NADPH, confirming a cause/effect relationship between the increased superoxide production and reduced sperm function. Even at a concentration of NADPH which did not alter the percentage of motile spermatozoa, hyperactivated motility and acrosome reaction were significantly lower under an atmosphere of 5% CO2 in air compared to a gas phase of 5% CO2/90% N2/5% O2. These results suggest that there is an advantage in using 5% O2 rather than 20% O2 in the gas phase of the incubator to prevent the excessive production of ROS by spermatozoa and related alterations of sperm functions. This may be of clinical value in fertilization programmes.

An in vitro promoting role for hydrogen peroxide in human sperm capacitation.

A complex process of maturation called capacitation is an essential step for spermatozoa to fertilize oocytes. Recent studies have shown that reactive oxygen species (ROS) can enhance the capacitation of human spermatozoa and sperm-zona interaction. We have investigated whether hydrogen peroxide (H2O2) could trigger capacitation of human spermatozoa and the acrosome reaction. The addition of catalase, a specific H2O2 scavenger, at the beginning of the capacitation process decreased the levels of both hyperactivation and induced-acrosome reaction whereas catalase added 15 min before the induction of the acrosome reaction by the calcium ionophore had no effect. Supplementation of the medium with H2O2 resulted in increased levels of hyperactivation and the acrosome reaction, whereas H2O2 added 15 min before induction of the acrosome reaction did not have any stimulatory effect. These results suggest that H2O2 may be involved in the capacitation process of human spermatozoa but not in the acrosome reaction.

Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process.

The involvement of superoxide anion (O2o-) in human sperm capacitation and/or acrosome reaction was investigated. Addition of superoxide dismutase (SOD) to the medium at the beginning of the capacitation process or 15 min before induction of the acrosome reaction, decreased the level of ionophore-induced acrosome reaction. Hyperactivation was unaffected by the presence of SOD during the capacitation process. Addition of calcium ionophore to the sperm suspension increased production of O2o- by the spermatozoa by four to five-fold and induced the acrosome reaction. In the presence of SOD, superoxide anion could not be detected in the medium and the rate of induced-acrosome reaction was decreased greatly. The presence of an inhibitor of protein kinase C inhibited the production of O2o- in the medium and reduced the induced-acrosome reaction. The production of O2o- and the acrosome reaction were also increased by exposure of spermatozoa to 12-myristate 13-acetate phorbol ester, a specific activator of protein kinase C. While the level of spontaneous acrosome reaction was not increased by the direct addition of O2o- to the medium, its presence induced the release of unesterified fatty acids from membrane phospholipids. These findings suggest that the production of O2o- by spermatozoa could be involved in the ionophore-induced acrosome reaction, possibly through the de-esterification of membrane phospholipids. However, this production of superoxide anion is not sufficient on its own to induce the acrosome reaction.

Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa.

The reactive oxygen species, hydrogen peroxide (H2O2) and superoxide anion (O2o-), were generated with a xanthine-xanthine oxidase system and their effect on human sperm function was studied. The action of reactive oxygen species on selected human spermatozoa resulted in a decreased capacity for ionophore-induced acrosome reaction, a decrease in sperm motility, an increase in the concentration of lipid hydroperoxides and a loss of membrane polyunsaturated fatty acids. H2O2 was the key intermediate of the deleterious effects exerted by the xanthine and xanthine oxidase. Among these parameters, the acrosome reaction appeared most susceptible to the reactive oxygen species generated by the xanthine-xanthine oxidase system, and was decreased without sperm motility being affected. Treatment with H2O2 was shown to inactivate several enzymatic activities involved in the antioxidant defence of spermatozoa: glutathione peroxidase, superoxide dismutase and glucose-6-phosphate dehydrogenase. H2O2 and O2o- were shown to be involved in the lipid alterations triggered by the xanthine-xanthine oxidase system. Singlet oxygen is proposed to intervene in the lipoperoxidation process. The inefficacy of mannitol in protecting spermatozoa suggests that hydroxyl radicals were not produced in the extracellular medium.

Effects of chamber depth on the motion pattern of human spermatozoa in semen or in capacitating medium.

The computer-aided sperm analysis system (CASA) permits precise calculation of the trajectory characteristics of human spermatozoa. Comparison between different chamber depths (10, 20 and 100 microns) revealed variations in the results, which were more evident as the magnitude of the spermatozoon flagellar beat increased. In seminal spermatozoa, the reduced amplitude of movement, linked to the relatively short flagellum and high viscosity of seminal plasma, indicates that the 10 microns-deep chamber can be used for motion analysis without involving extensive modifications. On the other hand, analysis of quicker movement, such as in capacitated spermatozoa, revealed large variations; in particular the proportion of non-progressive hyperactivated spermatozoa was higher in the 20 microns than in the 10 microns chamber (17.9 +/- 14% and 6.9 +/- 4.5% respectively, P < 0.01). In fact the distribution of non-progressive and progressive hyperactivated spermatozoa is depth-dependent. It is therefore necessary to use a chamber of at least 20 microns in depth for sperm analysis in capacitated medium.

Decondensation of human sperm nuclei and HP1 protamine degradation from normospermia and asthenospermia in Xenopus egg extracts.

The process of human sperm decondensation has been studied in vitro in cytoplasmic extracts prepared from unfertilized Xenopus laevis eggs. The chromatin decondensation-recondensation cycle was divided into four stages according to chromatin appearance. Spermatozoa from normospermia and asthenospermia were evaluated according to their capacity to reach these stages, and their DNA integrity was assessed by acridine orange (AO) staining. We observed a significant difference between normospermia and asthenozoospermia in the ability to achieve the cycle of chromatin decondensation-recondensation. These results correlated with AO staining. The role of human protamine 1 degradation in the decondensation process was evaluated by immunostaining. It was found not to be a prerequisite for the earlier stage of chromatin decondensation and it was not implied in the latest stages of pronuclear development.

Influence of oxygen tension on function of isolated spermatozoa from ejaculates of oligozoospermic patients and normozoospermic fertile donors.

Oxygen radical generation is known to be detrimental to sperm function. An example of a reactive oxygen species-associated male pathology is oligozoospermia in which fertilization and pregnancy rates are low in in-vitro fertilization (IVF) programmes. As the extent of the modifications induced by reactive oxygen species (ROS) depends on several factors, notably from oxygen tension in the incubation medium, the aim of this study was to examine the influence of a low (5%) rather than atmospheric (20%) oxygen tension in the incubator gas phase on the function of Percoll-selected spermatozoa from ejaculates of oligozoospermic patients and normozoospermic fertile donors. After incubation for several hours in a gas phase of either 5% CO2/90% N2/5% O2 or 5% CO2/95% air (20% O2), none of the parameters investigated, e.g. movement characteristics, potential of spermatozoa to acquire hyperactivated motility, to undergo the acrosome reaction when challenged with a calcium ionophore and to fuse with zona-free hamster oocytes, was significantly different between the two oxygen tensions in fertile donors. In contrast, among oligozoospermic patients, the motility parameters, the percentage of hyperactivated motility and of induced-acrosome reaction were significantly improved under a gas phase of 5% O2 compared with those observed under an atmosphere of 20% O2 (P < 0.05). Exposure to 5% rather than 20% oxygen tension also induced a significant increase in the percentage of penetration of zona-free hamster eggs after capacitation for 17 h, but no difference was found in the mean number of bound spermatozoa per oocyte. After incubation for 24 h, a significantly higher survival rate was observed under 5% compared with 20% oxygen tension. These results show that the use of a low oxygen tension rather than air might improve spermatozoan competence of oligozoospermic patients during IVF programmes.

Influence of seminal plasma on cryopreservation of human spermatozoa in a biological material-free medium: study of normal and low-quality semen.

The objective was to evaluate the efficiency of a biological material-free medium and the role of seminal plasma (SP) in the cryopreservation of human spermatozoa. Normal semen samples and low-quality semen samples were used for this study. After centrifugation of 300 microL fractions of whole semen, pellets were resuspended either in autologous SP or in a chemically defined medium (BM) supplemented or not with 3% bovine serum albumin (BSA); after 15 min at 37 degrees C, the samples were diluted (V/V) with cryoprotective medium (30 mM NaCl; 22 mM sodium citrate, 19.4 mM fructose; 80 mM glutamine; 14%, V/V, glycerol) and maintained for 15 min at room temperature before freezing. Assessment of viability and motility was performed using fresh semen (T0), after centrifugation and resuspension prior to adding the cryoprotectant (T15), after adding the cryoprotectant (T30) and after freezing and thawing (Tpost). In all three resuspending media used, sperm viability and motility (forward and total) decreased (p < 0.05) during both the equilibration period especially before addition of the cryoprotective medium (between T0 and T15) and during the freeze-thaw process comparison between T30 and Tpost. The recovery of viable and motile spermatozoa (post-thaw values/values of fresh samples) was higher (p < 0.05) in normal semen than in low-quality semen. In both groups, the recovery was slightly, but significantly, higher with SP than with BM and the presence of BSA has no beneficial effect. To conclude, these data suggest that SP may reduce the deleterious effects of cryopreservation. Nevertheless cryopreservation of spermatozoa in a medium containing neither SP nor biological substances could offer an acceptable cryoprotection of spermatozoa to be used in assisted fertilization procedures, especially for intracytoplasmic sperm injection.

Improvement of motility and fertilization potential of postthaw human sperm using glutamine.

The effectiveness of three amino acids, glutamine, proline, and histidine, and one amino acid-related compound, betaine, in preserving human sperm diluted v/v in a basal medium (BM) containing 14% glycerol during the freeze-thaw (FT) process was studied. At 80 mM in BM, only glutamine improved the 5- to 60-min postthaw total and progressive motilities and velocity. The presence of glutamine at 80 mM is not sufficient to achieve lower concentrations of the toxic agent glycerol in FT medium. Glutamine may therefore have a mechanism of protection on human spermatozoa that is independent from that of glycerol. The zona-free hamster egg penetration test showed that the percentage of eggs penetrated was significantly greater when spermatozoa were frozen-thawed with 80 mM glutamine in BM. Consequently, the presence of glutamine at 80 mM in a glycerol-FT medium maintains human sperm motility and fertilizing ability during the FT process.

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.