Andrology: An indiscriminate use of pentoxifylline does not improve in-vitro fertilization in poor fertilizers

In order to enhance fertilization in vitro, pentoxifylline (PTX)was used in couples showing low fertilization rates in previousin-vitro fertilization (IVF) attempts for the treatment of malefactorinfertility. Sibling oocytes were inseminated at random withspermatozoa prepared with or without PTX. After selection byPercoll gradient, sperm samples were divided into two equalaliquots. One aliquot was incubated in Earle's medium containing3.6 mM PTX (treatment group), the other aliquot was incubatedwith PTX-free Earle's medium (control group). After 30 min,both suspensions were washed twice. Sperm parameters after preparationdid not differ between treatment and control samples, and nordid the mean fertilization rates, which were 49.3 and 42.6%respectively. Cleavage characteristics and morphological qualityof the embryos were not significantly different between thetreatment and control groups. The results of this study demonstratethat indiscriminate use of PTX in an IVF programme increasesneither fertilization rate nor embryo transfer rate in poorfertilizers. More prospective research is needed to evaluatethe role of PTX in IVF in couples selected according to theeffect of the compound on sperm function, e.g. hyperactivationand acrosome reaction.     

DNA and chromatin structure: Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry

Flow cytometry analysis was used for the acurate and objectiveevaluation of sperm chromatin condensation and chromatin stabilityof sperm nuclei. It was also possible to determine the influenceof incubation on sperm chromatin. Different types of spermatozoawere studied: unprocessed spermatozoa at 1 and 45 min afterejaculation, after swim-up (migrated), spermatozoa incubatedfor 6 h in non-capacitating conditions (aged), or in B2 medium(capacitated) or B2 medium followed 1 h later with A23187 (reacted).All types of spermatozoa were analysed before and after treatmentwith various decondensation agents: sodium dodecyl sulphate(SDS), SDS plus EDTA and SDS plus disulphide-reducing agent[dithiotreitol (DTT)). Sperm nuclei were enzymatically isolatedand stained with propidium iodide. Three flow cytometric parameterswere then measured: forward light scatter (cellular size), sidelight scatter (cellular complexity) and fluorescence (uptakeof propidium iodide). Fluorescence was the most suitable parameterto study the degree of condensation and resistance to decondensationof DNA in the spermatozoa. Unprocessed spermatozoa 1 min afterejaculation underwent decondensation by all assessed treatments(anionic detergent, chelating or disulphidereducing agents).Unprocessed spermatozoa 45 min after ejaculation and migratedspermatozoa did not undergo decondensation with SDS treatment,but decondensation occurred after treatment with SDS + EDTAor SDS + DTT. Spermatozoa incubated for 6 h under both non-capacitating(aged spermatozoa) and capacitating conditions (capacitatedspermatozoa) and reacted spermatozoa were decondensed only aftertreatment with SDS + DTT. In conclusion, the post-ejaculationand incubation time have to be taken into account when clinicalinterpretation of the effect of different treatments on spermchromatin condensation is made.     

Andrology: Intracytoplasmic sperm injection does not require special treatment pf the spermatozoa

In order to assess whether specific treatment of spermatozoais required prior to intracytoplasmic sperm injection (ICSI),three methods of sperm preparation were compared in this study.These three methods were (A) incubation of spermatozoa withpentoxifylline (PTX) and 2-deoxyadenosine (DOA), (B) electroporationfollowed by incubation in medium with PTX, and (C) no furthertreatment with the Percoll gradient. Controlled comparisonswere carried out between method A and method B in 21 patients,and between method A and method C in 32 patients. There wasno difference in the rates of fertilization and embryo cleavagewhen ICSI was done with spermatozoa treated by procedures A,B or C. Furthermore, the sperm selection procedure prior toICSI was done in two different media: T6 medium containing 1.78mM CaCl₂·2H₂O and a final washing step after the Percollgradient in T6 medium containing 5.0 mM CaCl₂·2H₂O, andEarle's medium containing 1.78 mM CaCl₂·2H₂O. The resultsof ICSI on sibling oocytes from 12 patients revealed no differencein the fertilization and embryo cleavage rates between the twodifferent media used during the sperm selection procedures.In conclusion, it appears that high fertilization and pregnancyrates can be obtained in couples with severe male-factor infertilityby ICSI and that no special treatment of the spermatozoa priorto ICSI is required.     

Fertilization and early embryology: The effect of pentoxifylline on mouse in-vitro fertilization and early embryonic development

This study was designed to assess the effect of pentoxifylline(PTX) on fertilization and early embryonic development in themouse. Oocytes from superovulated B6CBA female mice were inseminatedin vitro with spermatozoa from B6CBA males incubated with PTXaccording to different protocols, i.e. (i) 3.6 and 7.2 mM PTXwashed out prior to insemination, (ii) 3.6 and 7.2 mM PTX dilutedsix times in the insemination medium and (iii) PTX present at3.6 and 7.2 mM in the insemination medium. After inseminationand washing, fertilization was assessed by the presence of 2-cellstage embryos. These were further cultured up to the blastocystor egg-cylinder stage to asses embryonic development. Parthenogeneticactivation was evaluated by exposing post-ovulatory oocytesto 3.6 and 7.2 mM PTX. If spermatozoa were washed free fromPTX before insemination, no effect on either fertilization orsubsequent development was found. If PTX was not washed out,fertilization was reduced significantly, yet development offertilized oocytes was unaffected. If insemination was performedin the presence of PTX both fertilization and development wereimpaired. Parthenogenetic activation was not increased by PTXexposure. We conclude that if used in in-vitro fertilization,exposure of oocytes and/or zygotes to PTX has to be avoidedby washing out the compound thoroughly to prevent adverse effectson early embryonic development.     

Andrology: The variable effects of 2'-deoxyadenosine on human sperm motility and hyperactivation in vitro

The response of human sperm motility and hyperactivation tothe stimulant 2'-deoxyadenosine (2'-DEA) was studied in vitrousing computer-assisted sperm motion analysis. A total of 20randomly selected individuals with normal sperm counts as definedby the World Health Organization were chosen and their migration-separatedspermatozoa exposed to a range (0.1–10.0 mM) of concentrationsof 2'DEA. The straight line velocity (VSL) was increased abovecontrol values only at 0.1 mM, while the curvilinear velocity(VCL) and lateral head displacement (ALH) were increased significantlyat all concentrations. Linearity of progression (LIN), on theother hand, declined with increasing concentration of 2'-DEA.These changes were related to a significant increase in thenumber of spermatozoa exhibiting hyperactive-like motion. Therewas, however, considerable intra-individual variability in theresponse to 2'-DEA. In some individuals VCL and ALH exhibitedlittle or no response to 2'-DEA, whilst in others an increaseabove the control of 50–55% occurred. The maximum responsefor VCL and ALH occurred at 2.5 mM 2'-DEA. Individuals showedgreater variability in the percentage of spermatozoa exhibitinghyperactivity in response to 2'-DEA, with increases rangingfrom 76 to 948% of the control value, although the maximum responsewas also most commonly seen at 2.5 mM 2'-DEA. The diversityof response to 2'-DEA emphasizes the importance of tailoringdoses to the individual rather than employing one concentrationfor all. Further tests on a subgroup of the individuals examinedthe longevity of spermatozoa in response to 24 h of continuedexposure to 2'-DEA. Prolonged exposure to 0.1 mM 2'-DEA continuedto enhance VSL, while higher concentrations produced detrimentaleffects on all other motion characteristics including hyperactivation.     

The effect of nitric oxide on sperm cell function and embryo development

PROBLEM: Nitric oxide (NO) has been known to have multifunctional roles both in the male and female reproductive systems. We investigated the effects of sodium nitroprusside (SNP)-dependent NO release on sperm cell function and embryo development to elucidate the mechanisms of action of NO. METHOD OF STUDY: Semen samples from 20 healthy men were processed by the swim-up method. Sperm motility, hyperactivation, and acrosome reaction were examined following incubation with various concentrations of SNP. The concentration of 10 nM to 1 mM was used for sperm motility and hyperactivation measurement and 1 microM to 1 mM for examining the effect on acrosome reaction. Embryo development to blastocyst stage was assessed using 100 nM to 1 mM of SNP added before transferring the mouse embryos into the culture medium. Finally, to understand the mechanism of action of NO, changes in embryo development were examined after zygotes were treated with various concentrations ranging up to 1 mM of 8-bromo-cGMP, an analog of cGMP. RESULTS: Both sperm motility and hyperactivation were significantly reduced at 100 microM and 1 mM concentrations of SNP after 6 hr of incubation. After 24 hr of incubation, they were greatly decreased with all, except the 10 nM concentration of SNP. The percentage of acrosomal-reacted spermatozoa was increased with the increasing concentration of SNP following incubation with 10 microM and 1 mM of SNP. Embryo development was arrested since the two-cell embryonic stage with all except the 100 nM concentration of SNP, and inhibited by 200 microM of SNP regardless of SNP treatment stage. However, embryo development was not influenced by 8-bromo-cGMP. CONCLUSIONS: We concluded that SNP-inhibited sperm cell function and embryo development in a dose- and time-dependent manner, and the inhibitory effect on embryo development, may not be a stage-specific treatment mediated via a cGMP-independent pathway. This result suggests that NO may be enough to affect the fecundity potential in vivo.     

Andrology: Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction

This study was designed to test the effects of pentoxifyllineand progesterone upon capacitation of fresh human spermatozoa.Capacitation and acrosomal integrity were assessed using thefluorescent probe chlortetracycline on spermatozoa co-stainedwith a supravital fluorescent dye, Hoechst 33258. Hyperactivatedmotility was measured using computer-assisted movement analysis.After exposure to pentoxifylline (1 mg/ml; 30 min), the fluorescent‘B’ pattern, characteristic of capacitated, acrosome-intactcells, increased significantly (P < 0.01), though no increasein ‘AR’ pattern, characteristic of acrosome-reactedcells, was detected. There was a significant increase in hyperactivemotility (P < 0.001). Exposure to progesterone (1µg/ml;60 min) resulted in a significant increase in ‘B’pattern (P < 0.05) and ‘AR’ pattern (P < 0.005),though no effect on the expression of hyperactivation was detected.No effect upon hyperactivation was detected on exposure of freshor cryopreserved spermatozoa to a physiological range of progesteroneconcentrations (0.1–1000 ng/ml). Sequential exposure topentoxifylline then progesterone resulted in a significant increasein ‘B’ pattern, acrosome loss and hyperactivation.Sperm viability was not affected in any treatment group. Theseobservations suggest that pentoxifylline and progesterone affectcapacitation through independent mechanisms. Stimulation ofboth capacitation and acrosome reaction resulted from sequentialexposure to pentoxifylline and progesterone. This may have implicationsfor sperm handling for assisted reproductive techniques.     

Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation

Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook Sydney IVF medium, Cook Sydney IVF sperm buffer, Earle's balanced salt medium (capacitating medium) or a modified Earle's balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37 degrees C and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA.     

Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry

Flow cytometry analysis was used for the acurate and objectiveevaluation of sperm chromatin condensation and chromatin stabilityof sperm nuclei. It was also possible to determine the influenceof incubation on sperm chromatin. Different types of spermatozoawere studied: unprocessed spermatozoa at 1 and 45 min afterejaculation, after swim-up (migrated), spermatozoa incubatedfor 6 h in non-capacitating conditions (aged), or in B2 medium(capacitated) or B2 medium followed 1 h later with A23187 [GenBank] (reacted).All types of spermatozoa were analysed before and after treatmentwith various decondensation agents: sodium dodecyl sulphate(SDS), SDS plus EDTA and SDS plus disulphide-reducing agent[drthiotrertol (DTT)]. Sperm nuclei were enzymatically isolatedand stained with propidium iodide. Three flow cytometric parameterswere then measured: forward light scatter (cellular size), sidelight scatter (cellular complexity) and fluorescence (uptakeof propidium iodide). Fluorescence was the most suitable parameterto study the degree of condensation and resistance to decondensationof DNA in the spermatozoa. Unprocessed spermatozoa 1 min afterejaculation underwent decondensation by all assessed treatments(anionic detergent, chelating or disulphide-reducing agents).Unprocessed spermatozoa 45 min after ejaculation and migratedspermatozoa did not undergo decondensation with SDS treatment,but decondensation occurred after treatment with SDS + EDTAor SDS + DTT. Spermatozoa incubated for 6 h under both non-capacitating(aged spermatozoa) and capacitating conditions (capacitatedspermatozoa) and reacted spermatozoa were decondensed only aftertreatment with SDS + DTT. In conclusion, the post-ejaculationand incubation time have to be taken into account when clinicalinterpretation of the effect of different treatments on spermchromatin condensation is made. chromatin/flow cytometry/spermatozoa     

The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (5 donors x 3 replicates) using Percoll gradients at 30–60 min post-ejaculation and preincubated in a complex ‘syntheitictubal fluid’ culture medium (STF) at 37° C under 5%CO₂ in air for 6h. Aliquots of these suspensions were then incubatedfor a further 2 h in STF containing 0, 5, 25, 50, 75 and 100%(v/v) pooled human follicular fluid (FF). Another aliquot wastreated with 10 µm A23187 in STF for 20min and then incubatedin fresh STF medium for a futher 2h to induce maximal acrosomeloss. Acrosome reactions were assessed using both the triple-staintechnique and fluorescent peanut agglutinin lectin-labelling.Sperm motility and movement characteristics were assessed fromvideorecordings using digital image analysis (CellSoft). Exposureto FF caused only relatively small proportions of the preincubatedspermatozoa to undergo acrosome reactions. The size of theseresponsive sub-populations was smaller than that capable ofresponding to a Ca²⁺ influx generated by A23187. Increased FFconcentrations induced a progressive loss of motility and trendsfor changes in movement characteristics that may have been relatedto reduced intracellular Ca²⁺. This interpretation of theseobervations is that while FF may act to stimulate or promotethe human sperm acrosome reaction it does not appear to be aspecific inducer of it. Consequently, a precise role for FFat the relatively low concentrations that would be expectedto be present in the tubal ampulla in the physiological regulationof human fertilization remains unproven     

Effect of pentoxifylline on sperm movement characteristics in normozoospermic and asthenozoospermic specimens.

Preliminary evidence has suggested that the phosphodiesterase inhibitor pentoxifylline augments the fertilizing potential of asthenozoospermic sperm samples, presumably by improving sperm movement. Here, we used computer-assisted sperm movement analysis to compare the effects of pentoxifylline in normozoospermic and asthenozoospermic specimens. The study focused on the following issues: the changes in individual movement characteristics in response to pentoxifylline, the rapidity of the response, the effect of sperm capacitation on the response, the persistence of the response after drug removal and the variability of responses among asthenozoospermic individuals. Data obtained show that (i) pentoxifylline increases the curvilinear velocity, path velocity, straight-line velocity, lateral head displacement, beat cross frequency and sperm hyperactivation in both normozoospermic and asthenozoospermic specimens, (ii) pentoxifylline does not modify the percentage of motile spermatozoa, (iii) the pentoxifylline effect reaches a maximum within 10 min of treatment in fresh semen as well as in capacitated sperm suspensions and persists for at least 2 h after drug removal and (iv) pentoxifylline improves the movement characteristics in most asthenozoospermic individuals. Results are discussed with regard to methods of therapeutic application of pentoxifylline as an enhancer of sperm movement in assisted reproductive technology.     

Andrology: Osmo-sensitivity of the human sperm acrosome reaction

The osmo-sensitivity of the human sperm acrosome reaction wasinvestigated by determining the effect of hyper- and hypo-osmolalconditions on the ionophore A23187- and dbcAMP-induced reactionof both capacitated and non-capacitated spermatozoa. Hyper-osmolalconditions inhibited the agonist-induced reactions of both typesof spermatozoa. Hypo-osmolal conditions caused a spontaneousloss of acrosomes from capacitated but not from non-capacitatedspermatozoa. The loss of acrosomes under hypo-osmolal conditionswas further enhanced by dbcAMP but not by ionophore A23187.Although significant decreases in sperm viability were occasionallyobserved at the high and low osmolalities, these decreases werenot consistent and could not account for the observed loss ofacrosomes. It is concluded that the human sperm acrosome reactionis osmo-sensitive. The acrosome reaction stimulated by ionophoreA23187 (raises intracellular Ca²⁺) and dbcAMP (activates proteinkinase A which causes protein phosphorylation) appears to involvewater entry downstream from the action of these agonists. Preincubationin albumin (capacitation) causes human spermatozoa to lose theiracrosomes under hypo-osmolal conditions. Finally, capacitationis not an essential prerequisite to the acrosome reaction aslong as agonists are used that by-pass certain membrane-relatedevents.     

Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction

BACKGROUND: Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. METHODS AND RESULTS: Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. CONCLUSIONS: Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.     

Different effects of the ionophore A-23187 and D-glucose on 45Ca2+ fluxes in isolated islets of ob/ob-mice.

Fluxes of 45Ca2+ were studied in beta-cell rich islets of non-inbred ob/ob-mice, using LaCl3 to wash out extra-cellular and superficially bound 45Ca2+. The ionophore A-23187 (10 microM) increased the 45Ca2+ uptake in islets both at 3 and 20 mM D-glucose, the effect being more pronounced after 10 min than after 120 min of incubation. In incubations for 120 min, 20 mM D-glucose induced a higher uptake of 45Ca2+ than did A-23187. The ionophore enhanced the unidirectional efflux of 45Ca2+ from preloaded islets. Pretreatment of islets with 20 mM D-glucose in non-radioactive medium inhibited the subsequent D-glucose-induced 45Ca2+ uptake. Similar pretreatment with A-23187 increased the subsequent ionophore-induced 45Ca2+ uptake. The results suggest that A-23187 acts by catalyzing Ca2+ fluxes across the beta-cell plasma membrane. The different effects of D-glucose and A-23187 on 45Ca2+ fluxes suggest that the two agents act through different mechanisms in the beta-cells.     

Objective identification of hyperactivated human spermatozoa by computerized sperm motion analysis with the Hamilton-Thorn sperm motility analyser

Sperm from 10 fertile donors were incubated for 8 h in a capacitating medium (BWW 3.5% HSA) and treated with 1.25 microliters of 2 mM Ca-ionophore solution. Sperm motion was analysed using the Hamilton-Thorn system before and after incubation and treatment. The acrosome reaction was detected with PSA-FITC labelling of the acrosome. Critical values of sperm movement parameters were defined for objective identification of hyperactive movement by the Hamilton-Thorn system. The incidence of hyperactive movement in the sperm suspensions was approximately 2%. No significant changes of hyperactive movement could be observed after the incubation period or the ionophore treatment, in contrast to a significant rise in the percentage of acrosome-reacted cells after ionophore treatment. The implications of these findings for monitoring hyperactive cells to test sperm function are discussed.     

Fertilization and early embryology: Factors affecting activation and fertilization of human oocytes following intracytoplasmic injection

Intracytoplasmic sperm injection (ICSI) has dramatically alteredthe treatment of severe male factor infertility, resulting inimproved fertilization and pregnancy rates. The purpose of thisstudy was to investigate oocyte activation and fertilizationin aged human oocytes following ICSI. Non-viable spermatozoawere injected into 24 h old human oocytes in the presence andabsence of calcium and were assessed for evidence of activationand fertilization 16–19 h after the injection procedure.Sham injections were also carried out to assess the effect ofthe injection procedure itself and the presence of calcium inthe injection medium on oocyte activation. Non-viable spermatozoainjected in the presence of 1.78 mM calcium were capable ofnormally fertilizing aged human oocytes and the resulting zygotesunderwent cleavage. None of the oocytes injected with non-viablespermatozoa in the absence of calcium were fertilized normally,although the rates of activation following all treatments weresimilar.     

Detection of apoptotic alterations in sperm in subfertile patients and their correlations with sperm quality

BACKGROUND: The aim of the present study was to define the effect of apoptosis on sperm quality and function. METHODS: The apoptotic features in sperm were assessed in 60 subfertile subjects, using Annexin-V staining for phosphatidylserine (PS) externalization and Tdt-mediated dUTP nick end labelling (TUNEL) assay for DNA fragmentation. RESULTS: On average, about 45% of the sperm were found to be apoptotic based on the results from Annexin-V staining, including both early (Annexin-V-positive, PI-negative) and late apoptosis (Annexin-V-positive, PI-positive). TUNEL-positive cells (median value 15%) significantly correlated to late apoptosis but not early apoptosis, indicating that DNA fragmentation only occurs at the later stage of sperm apoptosis. TUNEL-positive and late apoptotic cells (Annexin-V-positive, PI-positive) were found to be inversely correlated to sperm motility and vitality, and positively to abnormal sperm morphology. On the other hand, it is surprising to note that the apoptotic alterations in sperm positively correlated to sperm concentration or total sperm counts. CONCLUSIONS: Overall results from this study support the abortive apoptosis theory; apoptosis in mature sperm is initiated during spermatogenesis, after which some cells earmarked for elimination via apoptosis may escape the removal mechanism and contribute to poor sperm quality.     

Pregnancy following chemical activation of oocytes in a couple with repeated failure of fertilization using ICSI: case report

We report our attempts to achieve a successful pregnancy outcome with calcium ionophore A23187 and puromycin oocyte activation using sperm from a normozoospermic husband of a patient with previous repeated failed fertilization following ICSI. Oocytes from the female partner of a couple with a 4 year history of unexplained primary infertility with repeated failed fertilization following ICSI were used. In the latest ICSI attempt, oocytes were activated by treatment with calcium ionophore (5 min) and puromycin (5 h), then cultured. In this cycle, assisted oocyte activation with calcium ionophore and puromycin after ICSI resulted in a satisfactory fertilization rate (8/12; 66.7%); in prior cycles only one of 71 oocytes (1.4%) was fertilized. The outcome was a Caesarean section delivery of a healthy male infant without congenital abnormalities at 41 weeks, 2 days of gestation. In conclusion, the use of calcium ionophore and puromycin for oocyte activation was found to be a useful method in a case of repeated failed fertilization after ICSI.     

IVF within microfluidic channels requires lower total numbers and lower concentrations of sperm

BACKGROUND: Microfluidic technology has been utilized in numerous biological applications specifically for miniaturization and simplification of laboratory techniques. We sought to apply microfluidic technology to murine IVF. METHODS: Microfluidic devices measuring 500 microm wide, 180 microm deep, and 2.25 cm in length were designed and fabricated using poly(dimethylsiloxane) (PDMS). Controls were standard centre-well culture dishes with 500 microl of media, half of which also contained PDMS as a material control. Denuded mouse oocytes were placed into microchannels or centre-well dish controls in groups of 10, then co-incubated overnight with epididymal mouse sperm at various concentrations. Fertilization was assessed and Fisher's exact test was used for statistical analysis (P < 0.05 significant). RESULTS: Fertilization rates between the two control groups (42%, no PDMS; 41%, with PDMS; not significant) were similar. Fertilization rates for denuded oocytes at standard mouse insemination sperm concentration (1 degrees 10(6) sperm/ml) was poorer in microchannels (12%) than controls (43%; P < 0.001). As insemination concentrations decreased, fertilization rates improved in microchannels with a plateau between 8 degrees 10(4) and 2 degrees 10(4) sperm/ml (4000-1000 total sperm). At these concentrations, combined fertilization rate for denuded oocytes was significantly higher in microchannels than centre-well dishes (27 versus 10%, respectively; P < 0.001), and was not significantly different from corresponding controls with a sperm concentration of 1 degrees 10(6) (37%; P = 0.06). CONCLUSIONS: Murine IVF can be conducted successfully within microfluidic channels. Lower total numbers and concentrations of sperm are required. Microfluidic devices may ultimately be useful in clinical IVF.     

Pentoxifylline stimulates hyperactivation in human spermatozoa

The effects of pentoxifylline on the movement characteristicsof human spermatozoa incubated under capacitating conditionswere examined using automated digital image analysis. Washedspermatozoa from cryopreserved and fresh normozoospermic, andfresh oligozoospermic semen were incubated in the presence ofpentoxifylline (3.6 mM) for 30 min. In each group, pentoxifyllinecaused an increase in the average lateral head displacementand curvilinear velocity and a decrease in the linearity ofprogression. This related to a significant increase in the populationof spermatozoa developing hyperactivation. The effect was maximalat between 15 and 75 min incubation. No detrimental effect onsperm vitality was demonstrated. It was shown that hyper activationremained elevated compared with the control after washing pentoxifyllinefrom the suspension. This effect was maintained for 1 h, butbecame insignificant after 3 h. These data demonstrate thatpentoxifylline stimulates the development of hyperactivationin washed human spermatozoa, a phenomenon which may be of valuein the treatment of some forms of male factor infertility.