ICSI outcomes using testicular spermatozoa in non-azoospermic couples with recurrent ICSI failure and no previous live births.

BACKGROUND: The use of testicular over ejaculated spermatozoa for ICSI has been presented as an alternative to overcome infertility in men with poor semen parameters or high levels of sperm DNA fragmentation. OBJECTIVE: To evaluate the efficacy of testicular ICSI outcomes in couples with no previous live birth and recurrent ICSI failure using ejaculated spermatozoa by comparison to the outcomes of couples with similar history of recurrent ICSI using ejaculated spermatozoa only. MATERIALS AND METHODS: A total of 145 couples undergoing ejaculated or testicular ICSI cycles with no previous live births and with at least two previous failed ICSI cycles with ejaculated spermatozoa were evaluated retrospectively. ICSI was performed either with ejaculated (E-ICSI) or with testicular (T-ICSI) spermatozoa. Semen parameters and sperm DNA quality were assessed prior to the oocyte collection day. Primary outcomes included cumulative live birth and pregnancy rates. Secondary analysis included percentage of DNA fragmentation in ejaculated spermatozoa (SCSA® and TUNEL). RESULTS: Patients undergoing T-ICSI (n = 77) had a significantly higher clinical pregnancy rate/fresh embryo transfer (ET) (27.9%; 17/61) and cumulative live birth rate (23.4%; 15/64) compared to patients using E-ICSI (n = 68) (clinical pregnancy rate/fresh ET: 10%; 6/60 and cumulative live birth rate: 11.4%; 7/61). Further, T-ICSI yield significantly better cumulative live birth rates than E-ICSI for men with high TUNEL (≥36%) (T-ICSI: 20%; 3/15 vs. E-ICSI: 0%; 0/7, p < 0.025), high SCSA® (≥25%) scores (T-ICSI: 21.7%; 5/23 vs. E-ICSI: 9.1%; 1/11, p < 0.01), or abnormal semen parameters (T-ICSI: 28%; 7/25 vs. E-ICSI: 6.7%; 1/15, p < 0.01). CONCLUSIONS: The use of testicular spermatozoa for ICSI in non-azoospermic couples with no previous live births, recurrent ICSI failure, and high sperm DNA fragmentation yields significantly better live birth outcomes than a separate cohort of couples with similar history of ICSI failure entering a new ICSI cycle with ejaculated spermatozoa.

Quebec public funding facilitates fertility preservation for male cancer patients.

BACKGROUND: Sperm cryopreservation remains the only clinically feasible option to preserve male fertility. The quality of counselling provided by the treating physicians and the cost of sperm cryopreservation can both influence a patient's decision about whether to preserve sperm. On 5 August 2010, the Quebec government introduced provincial coverage of assisted reproductive technologies, with sperm cryopreservation included as a covered service. The aim of the present study was to evaluate whether and how such a program affects the behaviour of cancer patients with respect to sperm cryopreservation. METHODS: We analyzed the database derived from male patients undergoing sperm cryopreservation from August 2008 to August 2012 at our centre. The retrieved data included patient age, male infertility or oncologic diagnosis, sperm quality parameters, and details about the number of visits for sperm cryopreservation. RESULTS: The number of cancer patients who cryopreserved sperm before and after the policy change did not differ significantly, but a marked increase in the number of non-cancer patients was observed. Further analysis revealed that, after implementation of the public funding program, the total number of sperm cryopreservation sessions per patient increased significantly in cancer patients but not in non-cancer patients. CONCLUSIONS: It appears that cancer patients who are willing to freeze sperm are keen to return for more sessions of sperm banking when no fees are associated with the service. Those findings suggest that cost reduction is an important factor for improving delivery of fertility preservation services to male cancer patients.

[Lung cancer in Avila province, Spain. Incidence rates, epidemiolgy of the year 2012 and trends in the last 20 years]. / Cáncer de pulmón en la provincia de Ávila. Tasas de incidencia, epidemiología del año 2012 y tendencias en los últimos 20 años.

BACKGROUND AND OBJECTIVES: To determine the extent of lung cancer in Alvila. Its incidence rates and significant epidemiological aspects of the year 2012 were recorded, and the results of each 5-year period (up to 20 years) were compared with those of known studies conducted using the same methodology. PATIENTS AND METHODS: A prospective study was conducted on all patients diagnosed with lung cancer in the Province of Avila throughout the year 2012. RESULTS: A total of 81 patients were diagnosed, of whom 70 were males and 11 females, with a mean age of 72.1 years (range: 44-91), and was higher than that found in previous studies. This gave gross, and adjusted to the standard world population, incidence rates in 2012 of 80.99 and 31.23 per 100,000, respectively, in males, and 12.97 and 5.68 per 100,000, respectively in females. These rates are lower in both sexes than those found in Alvila in 2002. In 2012, 80.25% had been smokers (90% of males and 18.18% of the women), although, on diagnosis, 68.75% had quit smoking. A clinical-radiological diagnosis was made in 9 (11.1%), with a histocytological diagnosis in 72 (88.9%). The histological types were: adenocarcinomas in 37.5%; squamous in 33.3%; microcytic in 13.8%; undifferentiated non-small cell in 11.1%; large cell in 2.77%, and carcinoid in 1.38%. The most frequent treatments were chemotherapy (50.6%), symptomatic (23.4%), and surgery (12.3%). CONCLUSIONS: The incidence of lung cancer in Avila has decreased in both sexes in the last 10 years. In 2012, the patients have been older, the majority with adenocarcinoma histology, and receiving chemotherapy.

The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting.

Sperm chromatin integrity may affect the outcomes of assisted reproductive technology (ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting (MACS) technology coupled with differential density gradient centrifugation (DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic (n = 13), asthenoteratozoospermic (n = 17) and teratozoospermic (n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical value of MACS as a mean to enhance sperm quality that may improve assisted reproductive outcomes.

Tyrosine nitration in human spermatozoa: a physiological function of peroxynitrite, the reaction product of nitric oxide and superoxide.

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO-) produced from the reaction of nitric oxide (NO.) with superoxide (O2(.-)). Since human spermatozoa are able to produce both NO. and O2(.-) during capacitation in vitro, we investigated whether spontaneous tyrosine nitration of proteins occurs in human spermatozoa and evaluated the physiological effects of peroxynitrite on sperm function. We report here that human spermatozoa, incubated for 8 h under conditions conducive to capacitation, display a reproducible pattern of protein tyrosine nitration. Several proteins with mol. wt of 105-14 kDa become increasingly tyrosine-nitrated after 15 min incubation and then minimal changes are observed. Treatment of capacitated spermatozoa with human follicular fluid or calcium ionophore causes an increase of the nitrotyrosine content of proteins at the mol. wt of 85 kDa. Moreover, exposure of spermatozoa to ONOO- (2.5-50 micromol/l) increases motility and primes spermatozoa to respond earlier to human follicular fluid. ONOO- also increases protein tyrosine nitration and phosphorylation in a concentration-dependent manner. Taken together, these results demonstrate that tyrosine nitration of sperm proteins occurs in capacitated human spermatozoa, and that low concentrations of ONOO- modulate sperm functions, emphasizing the concept that capacitation is part of an oxidative process.

Effect of nitric oxide synthase inhibitors on ovum transport and oviductal smooth muscle activity in the rat oviduct.

The effect of the inhibition of nitric oxide synthase (NOS) on ovum transport and oviductal motility in rats was investigated. Three different NOS inhibitors were injected into the ovarian bursa at oestrus or day 3 of pregnancy. Oviducts and uteri were flushed 24 h later and the presence of ova was recorded. In oestrous and pregnant rats, treatment resulted in accelerated egg transport, as shown by a decrease in the number of ova present in the oviducts. In cyclic rats, intrabursal injection of 1 mg kg-1 of either N-monomethyl-L-arginine (L-NMMA) or N omega nitro-L-arginine methyl ester (L-NAME) elicited a 30% reduction in the number of ova present in the oviducts, whereas in pregnant animals, the same dose of L-NMMA produced a reduction of 40%. Simultaneous administration of the NO donor spermine NONOate (5 mg kg-1) completely reversed the effect of L-NMMA. Tubal motility was assessed by microsphere displacement analysis within the oviduct. Surrogate ova were transferred to the oviductal lumen at oestrus and 24 h later the effect of intraoviductal injection of 1 microgram L-NMMA or vehicle was assessed. The microspheres in the isthmus showed an oscillating motion, and periods in which movement was not detectable. However, L-NMMA treatment produced a 3.6-fold increase in the maximum instant velocities and a significant reduction in the resting periods of the microspheres compared with the control group (P < 0.001). These results provide evidence that NO inhibition increases tubal motility that results in accelerated ovum transport, and indicate that NO could act as a paracrine signal between different layers of the oviductal wall, providing a role for endogenous NO in regulation of tubal function.

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.

Progesterone enhances prostaglandin E2 production via interaction with nitric oxide in the mouse acrosome reaction.

In the present study we evidenced that progesterone could directly stimulate sperm nitric oxide synthase (NOS) in capacitated mouse spermatozoa. This stimulation led to an increase in sperm prostaglandin E2 (PGE2) production and subsequent acrosomal exocytosis. However, cGMP levels were not modified during the progesterone-induced acrosome reaction under our experimental conditions. These results suggest a functional role for nitric oxide as an intracellular messenger through, at least, stimulation of PGE2 production during the acrosome reaction triggered by progesterone.

Effect of prostaglandin F2 alpha (PGF2 alpha) on oviductal nitric oxide synthase (NOS) activity: possible role of endogenous NO on PGF2 alpha-induced contractions in rat oviduct.

In this paper, we evaluated the hypothesis that prostaglandin F2 alpha (PGF2 alpha) regulates NOS activity and we also investigated, by means of nitric oxide inhibitors (N-monomethyl-L-arginine monoacetate, L-NMMA) the role of endogenous NO on PGF2 alpha-induced contractions in rat oviduct. NOS activity was determined by measuring the conversion of 14[C]-L-arginine to 14[C]-L-citrulline on oviductal homogenates from estrogenized rats (1 microgram/rat). The presence of PGF2 alpha (10(-8) M) in the incubation medium produced an increase in NOS activity (p < or = 0.05). The effect of the prostanoid was blocked completely by the presence of two NOS inhibitors: N-nitro-L-arginine methyl ester (L-NAME, 0.6 mM) and aminoguanidine (Ag, 0.5 mM). These results suggested that PGF2 alpha could be modulating the Ca(2+)-independent NOS activity. We determined NOS activity using 1 mM EGTA, a chelator of Ca2+, in a free Ca2+ medium. These results indicated that PGF2 alpha produces an increase in Ca(2+)-independent NOS activity (p < or = 0.05). PGF2 alpha induces contraction of the oviductal smooth muscle in a concentration dependent manner. L-NMMA enhanced PGF2 alpha induced contraction of the oviduct, providing indirect evidence that there is a basal release of NO in the oviduct, which may reduce and/or modulate the contractile effects of PGF2 alpha.

Prostaglandin modulation of mouse and human sperm capacitation.

To determine whether prostaglandins produce a capacitation and/or acrosome reaction, the effect of prostaglandins on capacitated mouse spermatozoa and the effect of prostaglandin pre-incubation on human and mouse spermatozoa were studied. Prostaglandins did not induce an acrosome reaction in capacitated mouse sperm. PGE1 pre-incubation in a protein-free medium enhanced acrosome loss of mouse sperm challenged with A-23187 or solubilized mouse zona pellucida. Human sperm were pre-incubated in media containing prostaglandins, and an acrosome reaction was induced with calcium ionophore or human follicular fluid. PGE1 pre-incubation enhanced acrosome loss by human sperm when the action was induced with calcium ionophore, but had no effect on follicular fluid induction. We conclude that PGE1 acts as a capacitating factor in vitro for mouse spermatozoa, and enhances acrosome-reaction induction with calcium ionophore in human spermatozoa.

The nitric oxide synthase of mouse spermatozoa.

Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

Evidence that nitric oxide synthase is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa.

In a recent work, we detected nitric oxide synthase (NO synthase) in the acrosome and tail of mouse and human spermatozoa by an immunofluorescence technique. Also, NO-synthase inhibitors added during sperm capacitation in vitro reduced the percentage of oocytes fertilized in vitro, suggesting a role for NO synthase in sperm function. Therefore, in the present study the effect of three NO-synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-D-arginine methyl ester (D-NAME) and L-NG-nitro-arginine (NO2-arg), and of a nitric oxide donor, spermine-NONOate, on the progesterone-induced acrosome reaction of mouse sperm was examined. NO-synthase inhibitors were added at 0, 60 or 90 min during capacitation; at 120 min, mouse epididymal spermatozoa were exposed to 15 microM progesterone for another 15 min. In another set of experiments, different concentrations of spermine-NONOate were added to capacitated spermatozoa for 15 min; in these experiments, progesterone was not included. NO2-arg and L-NAME blocked progesterone-induced exocytosis regardless of the time at which these inhibitors were added. Moreover, D-NAME did not inhibit exocytosis. In contrast, spermine-NONOate stimulated the acrosomal exocytosis in vitro directly. These results provide evidence that mouse sperm NO synthase participates in the progesterone-induced acrosome reaction in vitro and that nitric oxide induces this event.

Analysis of the effect of nitric oxide synthase inhibition on mouse sperm employing a modified staining method for assessment of the acrosome reaction.

A commercially available staining kit (Spermac) was combined with swelling in a hypoosmotic medium (HOS) for simultaneous assessment of viability and acrosome reaction in mouse spermatozoa. We compared the results obtained with the combined technique (HOS-Spermac) with those obtained with currently used techniques: the chlortetracycline fluorescence assay and eosin exclusion. The results obtained with HOS-Spermac were the same as those obtained with the chlortetracycline fluorescence assay. Viability assessment with HOS-Spermac showed a good correlation with the percentage of spermatozoa showing eosin dye exclusion. Using this novel technique, we studied the effect of a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) on the acrosome reaction. L-NAME produced a dose-dependent inhibition of spontaneous acrosome reaction and its inhibitory effect was specifically counteracted by L-arginine. We conclude that HOS-Spermac provides a simple and reliable tool for assessment of the acrosome reaction and that nitric oxide synthase participates in this important function of the spermatozoon.

Influence of embryos on PGF2 alpha production by uterine tissue in pregnant mice.

The aims of the present study are to find out if preimplantation mice embryos can synthesize prostaglandins and if the presence of the embryos in the uteri modifies the uterine prostaglandin synthesis. Both uteri of adult pregnant mice and embryos were collected at day two, three and four of pregnancy and the synthesis and release of PGE and PGF2 alpha was measured by radioimmunoassay. At day five of pregnancy, embryos are tightly adherent to epithelium, so tied uterine horns (without embryos) were compared to control uteri (with embryos). We found that embryos synthesize PGE and PGF2 alpha and that PGE is significantly greater on the fourth day of pregnancy than on the second and third day (P < 0.001). In the uteri, during the four days of pregnancy, there is a significant increase in PGE (P < 0.05) and a significant decrease in PGF2 alpha (P < 0.01). On the fifth day, the synthesis of PGF2 alpha was significantly greater in tied uteri than in controls (P < 0.05). We conclude that mice embryos can synthesize prostaglandins and their presence in uteri significantly decreased the synthesis of PGF2 alpha during the peri-implantation period without modifying the production of PGE.

Localization by indirect immunofluorescence of nitric oxide synthase in mouse and human spermatozoa.

The localization of nitric oxide synthase was studied in mouse epididymal spermatozoa and freshly ejaculated human sperm. A rabbit antiserum against the neuronal isoform of the enzyme was used, and antibody binding was detected with a fluorescein isothiocyanate-conjugated polyclonal antibody specific for rabbit IgG. In mouse spermatozoa, the percentage of cells staining specifically ranged from 88% to 98%. Samples were examined after 0-, 90- and 150-min incubations in vitro. Three different patterns of staining were observed: (a) Pattern I, intense fluorescent staining localized in the acrosome and in a segment of the tail; (b) Pattern II, fluorescent staining localized only in the tail; and (c) Pattern III, faint fluorescent staining localized in the acrosomal cap and in the tail. The potential physiological significance of these patterns is discussed. Nitric oxide synthase was also localized in the acrosome of freshly ejaculated human sperm.

Effects of nitric oxide synthase inhibitors on the outcome of in vitro fertilization in the mouse.

The effect of three nitric oxide (NO) synthase inhibitors, L-NG-nitro-arginine (NO2Arg), NG-Nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, on in vitro fertilization in the mouse was examined. Mouse epididymal spermatozoa were capacitated in a medium with or without NO synthase inhibitors. Oocytes were inseminated and the percentage of oocytes with two pronuclei was scored after an 8-h incubation. NO2Arg and L-NAME, but not aminoguanidine, inhibited fertilization. L-NAME inhibited fertilization in a dose-dependent manner, and its effects were stereospecific. The inhibitory effect was neutralized by L-arginine but not by D-arginine. Moreover, D-NAME did not inhibit fertilization. The results suggest that NO synthase activity (presumably of the constitutive type is necessary for spermatozoa to display their full fertilizing ability.

Mouse spermatozoa can synthesize PGE2 and 5-HETE in vitro: stimulatory action of nitric oxide.

Cauda epididymal spermatozoa were examined for their capacity to synthesize prostaglandins (PGs) and hydroxy acids and the role of nitric oxide (NO) on cyclooxygenase and lipoxygenase enzyme activity was determined. Sperm suspensions were incubated in the presence or absence of 300 microM sodium nitroprusside (NP) and 20 micrograms/ml hemoglobin (Hb) and then incubated with [14C]arachidonic acid (AA) for 60 min. The cyclooxygenase and lipoxygenase products of AA metabolism were measured for their radioactivity. It was found that mouse spermatozoa could synthesize PGE2 and 5-hydroxyeicosatetraenoic acid (5-HETE) under these experimental conditions. The synthesis of these two metabolites was 100% stimulated by NP, the releaser of NO, and the response to NP was completely prevented by Hb, a scavenger of NO. These data provide evidence that mouse spermatozoa can synthesize PGE2 and 5-HETE in the presence of AA in vitro and that NO is involved in the production of AA metabolites in the male gamete.

Prostaglandin synthesis by cumulus-oocyte complexes: effects on in vitro fertilization in mice.

Cumulus-oocyte complexes, obtained from superovulated Balb/C virgin female mice, released to the incubation media significant amounts of PGE1, PGE2 and PGF2 alpha, as estimated by bioassay. Fertilization rates in vitro decreased sharply when cumulus-oocyte complexes were treated with indomethacin (10(-6) M) and then inseminated with 5000 sperm per oocyte. In order to explore if the reduced prostaglandin (PG) concentration was responsible for diminished fertilization rates, PGE1, PGE2 and PGF2 alpha (10(-9) M) were added to the fertilization media of treated oocytes. PGE1 and PGE2 but not PGF2 alpha returned fertilization rates to control levels. Besides, PGE1 (10(-9) M) enhanced fertilization rates with reduced sperm numbers (1000 sperm per oocyte) of untreated cumulus-oocyte complexes. In conclusion, PG synthesis and release of mouse cumulus-oocyte complexes affects fertilization in vitro, and it is suggested that PGs of the E series modulate sperm function at the moment of fertilization.

Effect of nitric oxide on mouse sperm hyperactivation.

Nitric oxide (NO) has recently been found to function as an intra and extra cellular messenger by activating guanylate cyclase. Its role in sperm hyperactivation was examined by adding to the capacitating medium a classical donor of NO (Sodium nitroprusside, NP) in two different concentrations (150 microM and 300 microM). In both treatments, the percentage of motile cells was evaluated, showing a significant decrease on motility and viability at 90 and 120 minutes when sodium nitroprusside was used in a concentration of 300 microM; no modifications were observed with 150 microM. The effect obtained with 300 microM of sodium nitroprusside was avoided by hemoglobin (20 micrograms/ml), a scavenger of the NO. The percentage of hyperactivated spermatozoa in the presence of 300 microM sodium nitroprusside increased significantly more than the control during the first 30 and 60 minutes of capacitation; but with 150 microM sodium nitroprusside a significant increase was observed at 60 and 90 minutes of incubation. Thus, the data strongly suggests that nitric oxide plays an important role in sperm hyperactivation "in vitro".