Andrology: Pentoxifylline-enhanced acrosome reaction correlates with fertilization in vitro

To evaluate the possible effect of pentoxifylline on the acrosomereaction (AR) and its correlation with in-vitro fertilization(IVF), sperm samples obtained from 51 patients who underwentIVF treatment were studied. Acrosome reactions were evaluatedas spontaneous, pentoxifylline-treated and calcium ionophore(A23187) induced, before and after treatment. The correlationof AR with fertilization in vitro in spermatozoa pre-treatedwith pentoxifylline was sought. In cases with failure or verylow fertilization rate (10%) in their previous trials, spermatozoaafter swim-up were treated before insemination. Spontaneousacrosome loss remained low even after treatment (mean ±SD: 8.18 ± 1.74%). Response to A23187 was enhanced significantly(P < 0.001) by pre-treatment with pentoxifylline in 33 controlcases (group A) in which fertilization in vitro was previouslysuccessful without this treatment. Patients with at least twoepisodes of failed fertilization were divided into two groups.In 11 cases (group B), the IVF rate was improved significantly(P < 0.001) by the treatment. This was not observed in sevencases (group C) in which the treatment induced no increase inIVF rate. We achieved nine (27.3%) pregnancies in group A andfive (45.4%) pregnancies in group B. This study demonstratedthat pentoxifylline enhanced A23187 induced the acrosome reactionand this effect was correlated with improvement in IVF rate.     

Prediction of outcomes of assisted reproduction treatment using the calcium ionophore-induced acrosome reaction

BACKGROUND: Sperm concentration and motility are poor predictors of the outcome of intrauterine insemination (IUI), hysteroscopic intratubal insemination (HIT), or complete fertilization failure (CFF) in conventional IVF. We investigated whether the calcium ionophore-induced acrosome reaction (AR) constitutes an additional indicator of CFF and pregnancy that is independent of these semen parameters. METHODS: Infertile couples with no female factor (n=388) and women with tubal obstruction (n=32) were studied: IVF (n=133), ICSI (n=72), HIT (n=245) and IUI (n=61). The percentage of acrosome-reacted sperm in relation to viable sperm was calculated. Receiver operating characteristic curve and multiple logistic regression analyses were used to determine threshold values and the best predictor for CFF and pregnancy. RESULTS: Threshold values of AR for predicting CFF in IVF and pregnancy in IVF and HIT + IUI were 21, 26 and 22% respectively. These values were independent of the conventional semen analysis parameters. CFF was lower (2 versus 20%; P<0.01) and the pregnancy rate was higher (46 versus 24% P<0.05) for those with AR >21% in IVF. CFF and pregnancy rate in ICSI did not differ according to AR. Pregnancy rate was higher for those with an AR >22% for HIT + IUI (23 versus 11% P<0.01). CONCLUSIONS: Ionophore-induced AR appears to be a useful indicator in addition to routine semen analysis for selection of patients for treatment with appropriate assisted reproduction procedure.     

Flow cytometric light scattering analysis, acrosome reaction, reactive oxygen species production and leukocyte contamination of semen preparation in prediction of fertilization rate in vitro

The effects on fertilization of the morphology of spermatozoa, acrosome reaction, reactive oxygen species (ROS) production, leukocyte contamination and the light scattering in flow cytometry of semen preparation were investigated in 73 couples undergoing their first in- vitro fertilization treatment. All men had normal concentrations of spermatozoa with sufficient motility (> or = 50%) and yield (> or = 6 x 10(6)) in the semen preparation on the day of oocyte retrieval. The light scattering properties of all cells present in the semen preparation, as assessed with flow cytometry, were correlated with fertilization. The proportion of metaphase I oocytes was also correlated with the fertilization rate of all collected oocytes and of metaphase II oocytes. ROS production, leukocyte contamination, spontaneous or calcium ionophore-stimulated acrosome reaction, percentage of normal morphology, and multiple anomalies index had no independent contribution.      

Spontaneous and ionophore induced acrosome reaction in asthenozoospermic infertile semen.

Spontaneous and ionophore-induced ability of spermatozoa to acrosome-react was examined in asthenozoospermic infertile patients and fertile donors. Spermatozoa were washed free of seminal plasma and capacitated in B2 medium for 2 h at 37 degrees C. Subsequently 5, 10, 20 and 30 microM A23187 (final concentrations) were added to equal aliquots of these samples and incubated for an additional 30 min. The acrosome reaction was then determined by the triple stain technique. The percentage of spontaneous reaction (no ionophore) in asthenozoospermic samples was similar to that in fertile samples (4.2 and 3.8, respectively). However, the ionophore-induced reaction rate remained significantly lower in asthenozoospermic samples than in normozoospermic samples.     

Andrology: Incubation of spermatozoa from asthenozoospermic semen samples with pentoxifylline and 2-deoxyadenosine: variability in hyperactivation and acrosome reaction rates

We examined hyperactivation and acrosomal loss in asthenozoospermicpatients with a history of failed in-vitro fertilization (IVF).After selection by a Percoll gradient, spermatozoa were incubatedwith 3.6 mM pentoxifylline (PTX), 3.0 mM 2-deoxyadenosine (2-DXA)or both. Hyperactivation and ionophore A-23187-induced acrosomereaction were assessed immediately after sperm treatment andagain after 180 min. In all groups studied, the mean hyperactivationrates were found to be low. No significant differences werenoted between assessments immediately after treatment and 180min later, except after treatment with both PTX and 2-DXA. Themean hyperactivation rates were found not to improve as a resultof either PTX or 2-DXA, while the combination of both PTX and2-DXA revealed a significant enhancement of total hyperactivation.When individual hyperactivation rates between control and treatedsperm samples were compared, large differences in response wereobserved. Some sperm samples showed a marked increase in hyperactivationwith one treatment, while another treatment led to a decrease.Acrosome reaction rates assessed immediately after ionophoreA-23187 stimulation were found not to be significantly differentfrom those assessed 180 min later. No significant effect couldbe demonstrated for either treatment, although, here too, markedinterindividual variations were noted. It was concluded thatan unselective use of PTX, 2-DXA or both compounds together,may restore sperm function in certain of these patients, andperhaps improve fertilization in vitro, but in others it mayproduce no change or may even be detrimental to sperm function.     

Fertilization and early embryology: Determination of the acrosome reaction in human spermatozoa is predictive of fertilization in vitro

The acrosome reaction was determined in aliquots from ejaculatesof 74 patients undergoing in-vitro fertilization at the Universityof Giessen, Germany, by means of the triple-stain technique.The percentage of acrosome-reacted spermatozoa after low-temperatureinduction of the acrosome reaction was not significantly relatedwith the fertilization rate (H test, P = 0.693, SJ test, P =0.366). However, all patients showing < 13.0% acrosome-reactedspermatozoa had poor fertilization rates. Highly significantdifferences between patients could be detected by correlatingthe inducibility of the acrosome reaction with the fertilizationrate (H test, P = 0.018; SJ test, P = 0.004); patients withhigh fertilization rates showed a corresponding high inducibilityof acrosome reactions. From our results, it is evident thatpercentages of acrosome-reacted spermatozoa < 13.0% or aninducibility of the acrosome reaction of <7.5% are indicativeof subfertility.     

Fertilization and early embryology: Progesterone-induced acrosome reaction: potential role for sperm acrosome antigen-1 in fertilization

We have established a monoclonal antibody (mAb) AG7 defininga sperm acrosome antigen-1 (SAA-1) on spermatozoa from the humanand several mammalian species. MAb AG7 inhibits fertilizationof mouse eggs in vitro and in vivo. An important characteristicof mAb AG7 is its inhibition of the rise in intracellular calciuminduced by progesterone in human spermatozoa. Here we show that,following the acrosome reaction, SAA-1 is lost from the capof human spermatozoa but remains detectable in the equatorialregion. Acrosome reaction assays demonstrated a clear differencebetween progesterone- and A23187-induced acrosome reactions.For induction of the acrosome reaction with progesterone, aminimum capacitation time of 6 h was required. A23187 inducedthe acrosome reaction regardless of capacitation time. MAb AG7completely inhibited the progesterone-induced acrosome reaction,but not the A23187-induced acrosome reaction in human spermatozoa.Differences in the pattern of calcium flux induced by the twoagents might account for this phenomenon. The inhibition ofthe progesterone-induced acrosome reaction by mAb AG7 impliesa regulatory function of SAA-1 during the human sperm acrosomereaction.     

The spontaneous acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (10 donors x 3 replicates) using Percoll density gradientsat 30–60 min post-ejaculation. Sperm suspensions wereincubated in a complex ’synthetic tubal fluid‘ culturemedium (STF) at 37°C under 5% CO₂ in air for up to 36 h.Parallel aliquots were incubated with 50 µM A23187 toinduce maximum acrosome loss (AR_max). Acrosome reactions wereassessed using both the triple-stain (TS) technique and fluorescentpeanut agglutinin (PNA) lectin-labelling. During incubation,the proportion of TS acrosome reacted spermatozoa increasedfrom 9.1 to 54.3% with AR_max being 68.3%. Spermatozoa showingintact acrosomes by PNA labelling decreased from 68.4 to 26.1%over 36 h of incubation (AR_max = 13.8%). Simultaneously, spermatozoashowing complete acrosomal loss (no PNA labelling) increasedfrom 8.1 to 27.0% (AR_max= 46.3%). Therefore, while only 23.5%of cells were actually undergoing acrosomal changes at the startof incubation, this had increased to 46.9% after 36 h (AR_max=40.7%). These experiments clearly show that even in selectedpopulations, not all human spermatozoa are capable of undergoingan acrosome reaction. However, the incidence of acrosomal changesafter 36 h of incubation did approach the AR_max. These levelsof spontaneous occurrence of the human sperm acrosome reactionwere higher than those reported in many other in-vitro incubationstudies: an improvement that may be attributable to the morephysiological nature of the STF culture medium     

Cumulative pregnancy rates and selective drop-out of patients in in- vitro fertilization treatment

The validity of the cumulative pregnancy rate (CPR) calculated by life- table approach, obtained in a transport in-vitro fertilization (IVF) programme, was tested by the determination of possible influence of selective drop-out of patients with a poor treatment prognosis. A cohort of 1211 patients who had a first IVF cycle was followed, and the CPR after three IVF cycles was assessed. First cycles of patients who discontinued treatment after failed IVF, and of those who did not achieve a pregnancy but proceeded to a subsequent cycle, were compared for fertilization rate and for occurrence of prognosticators of poor treatment outcome: oocyte yield < or =2, and replacement of <2 embryos. The CPR after three cycles was 54.9%. No differences were found in the first and second cycles of patients who continued treatment and those who dropped out. Selective drop-out of patients with a poor treatment prognosis was not found. Therefore, although calculations of CPR using life-table analysis generally overestimate the real probability of pregnancy after successive IVF cycles, the calculated CPR after three IVF cycles gives a reliable indication of the chance of occurrence of a pregnancy for the population studied.      

Intracellular calcium increase and acrosome reaction in response to progesterone in human spermatozoa are correlated with in-vitro fertilization

In this study we have investigated responsiveness to progesteronein spermatozoa from a group of unselected male partners of couplesundergoing in-vitro fertilization (IVF). We evaluated progesterone-stimulatedintracellular Ca²⁺ ([Ca²⁺]_i) and percentage increase in acrosomereaction in the same sperm sample used for oocyte inseminations.[Ca²⁺]_i was measured with a fluorimetric method, while the acrosomereaction was assessed using a fluorescent probe (fluoresceinisothiocyanate-labelled peanut lectin). The average percentage[Ca²⁺]_i as well as the rate of increase in the frequency ofacrosome reaction following progesterone challenge were significantlylower (P < 0.005) in the group of patients with a fertilizationrate <50%. In addition, significant correlations betweenthe fertilization rate and the progesterone-stimulated [Ca²⁺]_iand acrosome reaction increases (r = 0.78 and r = 0.79 respectively)were observed. Furthermore, in cases of fertilization failure,no increase of [Ca²⁺]_i or acrosome reaction was observed inresponse to progesterone with the exception of one case. Ourresults indicate that [Ca²⁺]_i and acrosome reaction increasesin response to progesterone can be of value in the predictionof sperm fertilizing ability. As the two parameters were significantlycorrelated to each other (r = 0.86), the two assays have similarIVF predictive value and might be used interchangeably as adiagnostic tool in the assignment of male patients to the differentkinds of assisted fertilization techniques.     

Platelet activating factor enhances the acrosome reaction, fertilization in vitro by subzonal sperm injection and resulting embryonic development in the rabbit

This study was conducted to investigate the effect of plateletactivating factor (PAF) on the acrosome reaction and fertilizingcapacity of spermatozoa, and development of the resulting embryosin the rabbit. Rabbit spermatozoa were exposed to PAF, Iyso-PAF,or high ionic strength medium (HIS) prior to subzonal sperminjection (SUZI) into 326 mature oocytes, or morphological assessmentof the acrosome reaction. The rates of fertilization and blastocystformation were compared among the three treatment groups. Acrosomereaction was assessed by fluorescein isothiocyanate-conjugatedPisum sativum agglutinin (FITC–PSA) staining and electronmicroscopy. PAF-treated spermatozoa fertilized the oocytes ata significantly higher rate (56.1%) than did lyso-PAF-(36.8%,P< 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa.The embryos produced by PAF-treated spermatozoa showed significantlyhigher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%,P < 0.050) or HIS-(8.8%, P< 0.05) treated spermatozoa.FITC-PSA staining demonstrated a significantly higher incidenceof acrosome reaction in PAF-treated spermatozoa (45.8%) thanin Iyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01)treated spermatozoa. Acrosome reaction of PAF-treated spermatozoawas also confirmed by electron microscopy. PAF treatment ofspermatozoa enhances fertilizing capacity for SUZI possiblyby augmenting the acrosome reaction. Enhanced embryonic developmentwas also found in the oocytes fertilized by SUZI of PAF-treatedspermatozoa.     

Hyperactivation of capacitated human sperm correlates with the zona pellucida-induced acrosome reaction of zona pellucida-bound sperm

BACKGROUND: The aim of this study was to determine the relationship between human sperm hyperactivation (HA), sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction (AR) of ZP-bound sperm in vitro. METHODS: Sperm samples from 129 infertile men were studied. Motile sperm (2 x 10(6)) selected by Pure sperm were incubated with four oocytes in 1 ml human tubal fluid supplemented with 10% human serum. After 2-h incubation, the number of sperm bound to the ZP and the AR of ZP-bound sperm were examined. Velocities and HA of sperm in insemination medium were assessed by Hamilton-Thorn Sperm Analyzer. RESULTS: The HA was highly correlated with the ZP-induced AR in all the subjects (rho = 0.626, P < 0.001). In the 69 men with < or = 100 sperm bound/ZP, allowing accurate counts, HA was not significantly correlated with sperm-ZP binding. Men with <7% HA sperm were more likely to have very low ZP-induced AR. Only normal sperm morphology was significantly correlated with sperm-ZP binding (rho = 0.346 and 0.446 in semen and insemination medium, respectively, both P < 0.001). Sperm motility and velocities were significantly correlated with sperm morphology but not with either sperm-ZP binding or the ZP-induced AR. CONCLUSIONS: The correlation of HA with the ZP-induced AR of ZP-bound sperm suggests a mechanistic link between HA and the physiological AR in humans. Assessment of HA of capacitated sperm in vitro may be a useful clinical test for male infertility associated with defective ZP-induced AR that does not require the use of human oocytes.     

Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization

Capacitation is defined as the series of transformations that spermatozoa normally undergo during their migration through the female genital tract, in order to reach and bind to the zona pellucida, undergo the acrosome reaction, and fertilize the egg. During this process, extensive changes occur in all sperm compartments (head and flagellum; membrane, cytosol, cytoskeleton), factors originating from epididymal fluid and seminal plasma are lost or redistributed and membrane lipids and proteins are reorganized; ion fluxes induce biochemical modifications and controlled amounts of reactive oxygen species are generated; spermatozoa develop hyperactivated motility; and complex signal transduction mechanisms are initiated. The main purpose of capacitation is to ensure that spermatozoa reach the eggs at the appropriate time and in the appropriate state to fertilize these eggs, by finely-controlling the rate of the changes necessary to prime spermatozoa and by activating all the mechanisms needed for the subsequent acrosome reaction. The reversibility of some of the mechanisms leading to sperm capacitation may therefore be a very important aspect of the fine regulation and perfect timing of this process.      

Management of patients with antiphospholipid antibodies undergoing in vitro fertilization

Infertility affects 10-15% of all married couples of reproductive age in the United States and results in substantial emotional distress and medical investment. Though it is uncertain whether antiphospholipid syndrome (APS) is a cause of infertility, inevitably there is a small proportion of women who have both APS and infertility. In turn, some of these patients are candidates for ovulation induction, with or without assisted reproductive technologies, such as in vitro fertilization and embryo transfer, in an attempt to achieve successful pregnancy. The medications used for ovulation induction cause an increase in ovarian estrogen production beyond that typical of a normal menstrual cycle. Clinicians are appropriately concerned about the potential adverse effects of this estrogen surge on the clinical status of women with autoimmune disease. For APS, a primary concern would be that of thrombosis or embolism.     

The acrosome reaction in boar spermatozoa

In order to gain more insight into the molecular alterationsof the acrosome, boar spermatozoa were incubated in a calcium-containingmedium in the presence of the lonophore A23187. The time-courseof the acrosome reaction was assessed by phase-contrast microscopy.Different stages of the acrosome reaction were studied by immunoelectronmicroscopy using a specific antibody directed against the completeouter acrosomal membrane. The introduction of monoclonal antibodiesgenerated by immunization of Balb/c mice with the isolated outeracrosomal membrane permitted a study of the topography of distinctmembrane proteins during the acrosome reaction. The exposureof activation of a fucose-binding protein important for sperm—zonaattachment was studied using neo-glycoproteins labelled withcolloidal gold for transmission electron microscopy.     

Streptomycin-induced anaphylactic reaction during in vitro fertilization (IVF)

Indications for in vitro fertilization (IVF) have been cautiously extended over the years. IVF is usually considered to be a technically complex method with only minimal side-effects. We report the case of a woman who developed an anaphylactic reaction during IVF immediately after the embryo transfer.     

Frequency of disordered zona pellucida (ZP)-induced acrosome reaction in infertile men with normal semen analysis and normal spermatozoa-ZP binding

Results of zona pellucida (ZP)-induced acrosome reaction (AR) are reported for 186 normospermic men with unexplained infertility and compared with 34 normal fertile men and 54 patients with disordered ZP-induced AR (DZPIAR) diagnosed after failure of standard IVF. For each ZP-induced AR test, four oocytes that failed to fertilize in IVF were incubated for 2 h with 2x10(6)/ml motile spermatozoa. Spermatozoa tightly bound to the ZP were recovered by aspirating the oocytes with a pipette and the AR assessed using pisum sativum agglutinin labelled with fluorescein. The standard deviation of the difference was 5.2% for repeated tests for ZP-induced AR on different ejaculates from 54 men. The ranges for the ZP-induced AR were 3-98% for normospermic infertile men, 24-95% for fertile men and 0-16% for DZPIAR patients. In the normospermic group, there was a significant correlation between ZP-induced AR and sperm concentration (Spearman r = 0.238, P < 0.001). Using ZP-induced AR < or =16% as the threshold for diagnosis of DZPIAR, the frequency of this condition in normospermic infertile men would be 25%. Thus DZPIAR is common with normospermic idiopathic infertility and this condition should be diagnosed before assisted reproductive technology since it requires intracytoplasmic sperm injection.     

Modelling human sperm-egg interactions in vitro: signal transduction pathways regulating the acrosome reaction

Recent advances in characterizing sperm surface receptors and ion channels, when combined with the rapidly expanding knowledge of interactions among second messenger systems in somatic cells, permit formulation of a tentative molecular mechanism for the regulation of the human sperm acrosome reaction. As spermatozoa pass through the cumulus mass, progesterone binds to its sperm surface receptor, alkalinizes the sperm head cytosol and potentiates changes in intracellular ionized calcium. Primary binding of spermatozoa to egg involves receptors for mannosyl, N-acetylglucosaminyl and, possibly, fucosyl residues of the glycosylated zona protein, ZP3. These receptors aggregate on multivalent ligand binding, migrate to the equatorial region along an actin filament network formed between the plasma and acrosomal membranes during capacitation, and activate a G protein/protein kinase A/protein kinase C second messenger system and a secondary proteolysis signal. Binding of a receptor tyrosine kinase to ZP3 amino acid residues simultaneous with the sugar recognition event triggers tyrosine phosphorylation signalling. All signals combine to open a voltage-dependent calcium channel. The resulting elevated calcium signal depolymerizes the inter-membrane actin network and activates phospholipases, leading to an acrosome reaction.      

Intracytoplasmic sperm injection in mice: increased fertilization and development to term after induction of the acrosome reaction

A technique has been developed for intracytoplasmic sperm injection(ICSI) in the mouse with a relatively low rate of lysis of oocytes(range 5–25% across experiments) and pronuclear formationin around one-third of the intact oocytes (range 30–38%across experiments) for untreated spermatozoa. The treatmentof spermatozoa with calcium ionophore, to induce the acrosomereaction (increases acrosome-free spermatozoa from 28 to 58%)before ICSI, increased pronuclear formation to {small tilde}60%(range 59–627percnt; across experiments) in intact oocytes.The pronuclear oocytes developed to blastocysts in vitro andto term when transferred to recipient foster mothers at ratesequivalent to zygotes formed after insemination in vitro. Therewas no benefit for fertilization rates of activating oocyteswith 8% ethanol before or after ICSI, nor was there any evidenceof parthenogenetic activation by the sperm solution used forICSI. This technique adds to other in-vitro fertilization techniqueswhich can be used to explore gamete interactions and to recoverbreeding in infertile strains and reproductively unfit mice.