Andrology: The influence of sperm morphology and the acrosome reaction on fertilization outcome after sub-zonal injection (SZI) of human spermatozoa

The usefulness of sub-zonal injection (SZI) for the treatmentof severe male factor infertility has been restricted by lowand unpredictable fertilization rates and the high risk of polyspermyafter the injection of multiple spermatozoa. In this prospectivestudy, we have evaluated whether sperm morphology and the percentageof acrosome-reacted spermatozoa at the time of injection canbe used to predict SZI fertilization outcomes. Populations ofmotile spermatozoa equivalent to those injected were collectedfrom the medium/oil interface immediately after SZI of eachcohort of oocytes. Morphology was assessed using the World HealthOrganization 1987 criteria and the acrosomal status of spermatozoawas determined after staining with rhodamine-conjugated Pisumsativum agglutinin. A fertilization index (FI) was calculatedto express the actual fertilizing potential of the spermatozoainjected. In all, 67 patients underwent 72 SZI cycles. The overallfertilization and polyspermy rates were 36 and 47% respectively,and a clinical pregnancy rate per transfer of 22% was achieved.Linear regression analysis demonstrated a statistically significantrelationship between morphology and the FI (r = 0.506, P <0.0001). Patients with <10% normal morphology always hada FI < 10%, and this was reflected by low fertilization andpolyspermy rates and the high number (32%) of cycles with completefailure of fertilization in this group. In patients with >10% normal morphology, there were two patterns: low (10% FI)or high (>10% FI) fertility. This was evident in the fertilization(23 and 85%, respectively) and polyspermy (25 and 68%, respectively)rates of these two patient sub-groups. While the percentageof acrosome-reacted spermatozoa at the time of injection wasweakly correlated with the FI (r = 0.292, P < 0.05), it couldnot be used to predict differences in fertilization potentialbetween patient sub-groups. We conclude that sperm morphologyand acrosomal status at the time of injection are of limiteduse in predicting SZI fertilization outcomes, although patientswith poor morphology ( 10% normal) have lower fertilizationand polyspermy rates.     

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.     

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.     

Andrology: Acrosome reaction inducibility predicts fertilization success at in-vitro fertilization

We prospectively studied the ability of acrosome reaction (AR)inducibility to predict fertilization success in a group of232 infertile patients presenting sequentially for in-vitrofertilization (IVF). The median percentage of eggs fertilizedfor the overall patient population was 25% (interquartile range5–58%), with one to 29 oocytes available for insemination(median, five oocytes). The median percentage of eggs fertilizedat IVF increased as the percentage of spermatozoa able to undergoAR became greater: spermatozoa with a failed AR (5%) fertilizedonly 12% of eggs, while spermatozoa with AR values>9% fertilized50% of eggs. The assay had a specificity of 0.75, a sensitivityof 0.55 and an odds ratio of 2.9; thus, AR-positive patientsare 2.9 times more likely to achieve fertilization than patientswith a failed AR. Receiver operator characteristic (ROC) curveswere constructed for AR, sperm concentration and percentageof normal forms in semen. All three parameters proved to bepotentially useful in predicting the occurrence of fertilization,although AR and morphology appeared to be better than spermconcentration by ROC analysis. Patients were divided into fourclearly defined subgroups according to their traditional semencharacteristics, including morphology. The median percentageof eggs fertilized decreased as traditional semen characteristicsdeteriorated, from a median of 46% for patients with excellentsperm concentration, motility and morphology, to a median of29% for patients with suboptimal semen quality and a medianof 0% for patients with severely impaired semen. Within eachpatient subgroup, the median percentage of eggs fertilized was3-to 4-fold higher for individuals with a positive AR than forthose with a failed AR, indicating that AR has a greater effecton fertilization rate than traditional semen parameters includingmorphology. We now recognize that some men with good semen characteristicshave an unexpectedly poor AR and a markedly reduced fertilizationrate, while other men with poor traditional semen characteristicsunexpectedly retain AR and perform relatively well at IVF. Bycontrast to AR, morphology seemed to have little effect on fertilizationsuccess (two-way analysis of variance not significant). Thewife's age and oocyte quality were evenly distributed amongthe different patient subgroups, indicating that differencesin fertilization rate could not be attributed to either parameter.Our data indicate that AR has a much higher predictive valuefor IVF success than traditional semen parameters includingmorphology. We propose that AR assessment is a clinically usefuldiagnostic tool in determining a patient's likelihood of achievingfertilization at IVF.     

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.     

Andrology: Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro

It is believed that during the process of human fertilization,acrosome-intact spermatozoa bind to the surface of the zonapellucida which triggers the acrosome reaction and the enzymesreleased facilitate sperm penetration through the zona pellucida.We describe here reduced frequency of the acrosome reactionon the zona pellucida as a cause of infertility in 10 coupleswith long durations of infertility (average 6 years) and low(<15%, n= 3) or zero (n= 7) fertilization rates in vitro.Sperm concentration, motility, velocity (Hamilton-Thorn), morphologyand DNA normality were within the normal range in all the patients.Electron microscopy of spermatozoa did not reveal any specificultrastructural defects. All couples were negative for antispermantibodies by immunobead tests. Oocytes from other patientswhich failed to fertilize in in-vitro fertilization and normaldonor spermatozoa were used as controls for sperm-zona pellucidabinding and penetration experiments. Acrosome status of spermatozoabound to the zona pellucida was assessed with a fluorescentlectin and electron microscopy. The mean number of spermatozoabound to the zona pellucida was not significantly differentbetween patients and controls. However, the acrosome reactionof spermatozoa bound to the zona pellucida after 2 h incubationwas significantly lower (P< 0.001) in the patients (mean5%, range 0–16) than in the controls (mean 68%, range44–96). No zona pellucida (out of 40) was penetrated bypatient spermatozoa whereas most (39/40) zonae were penetratedby control spermatozoa (average 27 spermatozoa/four zonae pellucidae).The spontaneous acrosome reaction of spermatozoa in inseminationmedium was not different between patients (4%) and controls(3%), the acrosome reaction induced by calcium ionophore waslow (21 and 43% respectively) in six of the eight patients examined.In conclusion, these patients have spermatozoa with a disorderof the zona pellucida-induced acrosome reaction that resultsin failure of sperm-zona pellucida penetration and explainstheir infertility.     

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.     

Andrology: The acrosome reaction to ionophore challenge test: assay reproducibility, effect of sexual abstinence and results for fertile men

Since the human acrosome reaction is considered a prerequisitefor normal fertilization and the spontaneous acrosome reactionrate is low, laboratory tests using calcium ionophores to inducethe acrosome reaction have been devised and applied to the investigationof patients. The introduction of any new laboratory test intoroutine clinical practice is usually accompanied by the determinationof intra- and inter-subject variability within the normal population,and the derivation of reference values to distinguish betweenaffected and unaffected populations. The acrosome reaction toionophore challenge (ARIC) test was evaluated and found to have(i) intra- and inter-assay coefficients of variation of 10.8and 18.8% respectively, (ii) a high degree of intra-subjectvariability for three subjects studied over a 10 week period,(iii) a high degree of inter-subject variability when aliquotsof 20 ejaculates of donor semen of proven fertility were tested,and (iv) no effect of length of sexual abstinence on ARIC values.The results of this study suggest that the use of fresh semensamples from subjects of proven fertility for quality controlpurposes in the ARIC test may be inadequate due to the highdegree of intra-subject variability, and that this problem maybe overcome by utilizing a frozen quality control sample. Theresults also suggest that an isolated negative ARIC test isnot necessarily indicative of functionally incompetent spermatozoa,and highlight the importance of examination of the normal populationprior to the clinical application of such a test.     

Andrology: Characterization and frequency distribution of sperm acrosome reaction among normal and infertile men

We studied the acrosome reaction (AR) in human spermatozoa from58 normal fertile men and 232 infertile patients. The medianAR in the normal population was 17%; AR was >9% in 83% ofthe subjects studied and only 2% of fertile individuals hada failed AR (<5%). We calculated the within person variabilityusing multiple specimens from each subject; the 95% confidenceinterval for each subject was 4 AR units above and 4 AR unitsbelow his mean value. In contrast, the median AR for the infertilepopulation was 3%. AR values were below the normal range (<5%)in 60% of the patients and only 26% of the patients had AR values>9%. Because patients entered the study sequentially, withoutregard to the aetiology of infertility, the patient populationcomprised a wide variety of subjects ranging from individualswith good to extremely poor semen quality. There was a progressivelygreater number of individuals with a failed AR as semen qualitydeteriorated (P < 0.01). However, among patients with goodcount, motility and morphology, 20% unexpectedly had a failedAR, and among patients with severely impaired semen quality,29% had a positive AR. We also analysed the relationship ofAR values with other semen measurements. The frequency of acrosomalreactions was significantly correlated (Spearman, P < 0.001)with morphology (r = 0.509), concentration (r = 0.524), progression(r = 0.416) and percentage motility (r = 0.356), but not withthe percentage of sperm moving at a curvilinear velocity of<40 µm/s. When we constructed a quadrant analysis byplotting morphology (percentage normal forms) against the ARvalue for each subject, values of AR and morphology for fertilemen were uniformly distributed in all four quadrants, while83% of infertile patients fell in the quadrant characterizedby morphology < 10% and/or AR <20%. Our data show thatthe frequency distributions of AR among fertile men and infertilepatients differ markedly and that AR provides additional informationon the quality of a semen specimen, not derived from conventionalsemen analysis. We propose that AR measurement might be a clinicallyuseful diagnostic tool and address this issue in a companionpaper where we analyse prospectively the correlation of AR withfertilization success at in-vitro fertilization.     

Human sperm capacitation and the acrosome reaction.

A model is presented that describes the mechanism of human sperm capacitation and the acrosome reaction. The processes of capacitation and the acrosome reaction are proposed to function in control of the activation/release of acrosomal enzyme(s) involved in sperm penetration through the zona pellucida. During capacitation, the sperm head membranes are biochemically modified, allowing the acrosome reaction to take place when the spermatozoon approaches or reaches the zona pellucida, resulting in the localized activation and release of the appropriate enzyme(s). Further, capacitation is presented as a continuing process that occurs during sperm transport through the female genital tract and is physiologically not completed until the spermatozoon reaches the oocyte (unless the spermatozoa are kept at a particular genital tract site for prolonged periods). The biochemical alterations that occur during capacitation are discussed. It is suggested that extensive modifications in the lipid bilayer structure, e.g. in the cholesterol or phospholipid content, are not part of capacitation because such changes would prematurely destabilize the membranes. Rather, such changes occur during the acrosome reaction. It is also proposed that the human sperm acrosome reaction has many similarities to the somatic cell exocytotic events which occur during the regulated pathway of secretion. One or more oocyte stimuli result in the activation of protein kinases, likely (but not necessarily) via activation of G-protein coupled receptors on the sperm plasma membrane and the formation of second messengers. The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction. The role of other enzyme systems such as those involved in ion transport, proteolysis, phospholipid metabolism (including that of arachidonic acid) and other metabolic events, is discussed. Calcium ion influx as initiator of the acrosome reaction is reconsidered. The proposed model also takes into consideration the structural events of membrane fusion.     

Induction of acrosome reaction in human spermatozoa used for subzonal insemination.

Human spermatozoa were injected into the perivitelline space of oocytes from 43 couples (44 cycles) in whom fertilization had failed in conventional in-vitro fertilization (IVF). The spermatozoa were treated to enhance the percentage of acrosome-free spermatozoa either by incubation for 24 h in T6 medium with 50% follicular fluid (v/v) or by incubation for 24 h in T6 medium followed by electroporation and incubation for a few hours in T6 medium with 3.5 mM pentoxifylline. After these two procedures, the mean percentage of acrosome-free spermatozoa increased to 35.5 and 53.9% respectively. Up to three spermatozoa were injected into the perivitelline space of metaphase II oocytes; few oocytes were damaged during the injection procedure. The overall fertilization rate was 30.9% of the 433 oocytes that were intact after subzonal insemination. Only 3% of the injected oocytes had more than two pronuclei. The cleavage rate of the fertilized oocytes was 80%. There was no difference in the fertilization and cleavage rates between the two sperm treatment procedures. One, two or three embryos were replaced in 34 cycles and seven patients became pregnant. In three of the four ongoing pregnancies, prenatal diagnosis by amniocentesis indicated a normal karyotype.     

Andrology: Two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization

We have recently reported, in a small cohort of subjects, thatacrosome reaction (AR) and intracellular free calcium ([Ca²⁺]_i)increase in response to progesterone were significantly correlatedwith in-vitro fertilization (IVF) rate. In the present studywe extended these results to 90 subjects undergoing IVF. Weconfirm that both parameters were highly significantly correlatedwith the fertilization rate (P<0.001). In particular, significantlylower responses to progesterone were detected in subjects witha fertilization rate <50%, further enlightening the functionalsignificance of sperm responsiveness to progesterone with respectto the process of fertilization. Moreover, we report here thatboth tests are highly discriminant of fertilization success,with positive predictive values >90% for [Ca²⁺]_i values whichincrease by >1.2-fold and AR inducibility >7% (cutoffvalues). Conversely, AR following challenge with the calciumionophore A23187 was less significantly correlated with thepercentage fertilization rate (P<0.05), and showed lowerpredictive values than response to progesterone. All these tests([Ca²⁺]_i increase in response to progesterone, AR in responseto progesterone and to A23187) appear highly sensitive and moderatelyspecific The positive predictive value may rise to >95% whenthe combination of two tests ([Ca²⁺]_i and inducibility of ARin response to progesterone) is considered. No correlation withfertilization rate has been found for spontaneous AR or basal[Ca²⁺]_i. In conclusion, we propose that assessment of humansperm responsiveness to progesterone may be clinically usefulin predicting fertilizing ability in vitro.     

A new sperm collection method for in-vitro fertilization: collection at tail of epididymis.

We have developed a method for the collection of spermatozoa from the tail of the epididymis in patients with severe asthenozoospermia. The treatment was applied to 25 cycles, of which 16 cycles provided spermatozoa in good condition with concentration and motility greater than 50 x 10(4)/ml and greater than 50% respectively. Spermatozoa thus obtained were subjected to IVF in 11 cycles and to ZIFT in five cycles; five IVF cases reached cleavage and two of those cases resulted in pregnancy. Two of the five ZIFT cases also achieved a pregnancy. Despite limits on frequency, the sperm collection process is thought to be very useful for the treatment of severe asthenozoospermia.     

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.     

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.     

The acrosome reaction in human sperm from men of proven fertility

Suspensions of motile sperm were prepared from semen samplesdonated by 40 men of recent, proven fertility and incubatedunder capacitating conditions for 24 h. At selected time-pointsaliquots were removed, assessed for motility, fixed and examinedwith the electron microscope to determine the rate of acrosomeloss. Data indicate that acrosome loss increases significantlywith time, but absolute values are relatively low. After 24h a mean of 15.4% sperm had initiated the acrosome reaction;this figure included 9.7% which had completed it. The proportionof cells at intermediate stages was similar throughout incubation({small tilde}5%), indicating that initiation of the acrosomereaction occurs at a fairly constant rate. In four samples motilitydeclined over 24 h and in six, contaminating cells were observed.In the majority of these 10, acrosome loss was higher than thatobserved in the remaining 30 samples. Additionally, the assessmentof <25 000 cells during this study made it possible to evaluatespecific ultrastructural features of the normal acrosome reactionin human sperm. Six stages were identified, with the intermediateones involving loss of acrosomal matrix material while outermembranes appear to retain their integrity; this contrasts sharplywith the current view of the generalized mammalian sperm acrosomereaction.     

Teratozoospermia and in-vitro fertilization: a randomized prospective study

A prospective randomized study was conducted to assess the prognosticvalue of sperm morphology in an in-vitro fertilization (IVF)programme, using strict criteria. The first group (T, teratozoospermic)included 32 couples with an isolated teratozoospermia in themale partner (morphology <9% normal). The second group (C,control) contained 36 couples with normal semen parameters,including morphology (>9% normal, strict criteria). In bothgroups, 50 IVF cycles were performed. Patients were matchedfor indication for IVF. There was no difference between thetwo groups regarding age, duration of infertility, stimulationprotocol, catheter used for embryo transfer and different spermparameters. A statistically significant difference between theT and C groups respectively was observed regarding the fertilizationrate (69.2 and 79.4%P < 0.05), pregnancy rate per cycle (12.0and 42%, P < 0.001), the pregnancy rate per transfer (13.9and 42.0%, P< 0.01) and per embryo transferred (6.1 and 14.8%,P< 0.05). No pregnancy occurred in the poor prognosis group(morphology <5% normal). In cases of moderate teratozoospermia,the fertilization rate appeared normal (78.6%) but the conceptionrate remained low. We concluded that the use of strict criteriain the assessment of sperm morphology is useful in predictingfertilization and pregnancy rate in the human in-vitro model.     

Artificial induction of the acrosome reaction in human spermatozoa

This study investigated the use of human follicular fluid andpentoxifylline as inducers of the human sperm acrosome reactionin vitro. Motile sperm suspensions were prepared using a discontinuousPercoll gradient, preincubated for 3 h, divided into aliquotsand exposed to various concentrations of non-heart-inactivatedfollicular fluid for 1 and 24 h and pentoxifylline for 30 min.Detection of the acrosome reaction involved the combined useof a fluorescent vital stain, H33258 [GenBank] , and fluorescein isothiocyanate-conjugatedpeanut agglutinin (FITC-PNA). A short (1 h) exposure to follicularfluid at concentrations of 50% or more, did not compromise spermmotility and significantly increased the proportion of spermatozoahaving completed the acrosome reaction. Similarly, a 30 minexposure to pentoxifylline also significantly increased theproportion of spermatozoa having completed the acrosome reaction.     

Andrology: The effect of sperm preparation methods on the fertilization rate of oocytes micro-inseminated by subzonal sperm injection

Three methods were used to prepare spermatozoa for subzonalinjection into mature oocytes. In method A, the washed spermsuspension was incubated for 18 h in modified T6 culture medium.Method B consisted of incubating the sperm suspension for 6h in regular T6 culture medium. In method C, the sperm suspensionwas incubated for 6 h in regular T6 culture medium containing20% (v/v) follicular fluid. The percentages of acrosome-freespermatozoa and fertilization rates were compared for 42 treatmentcycles assigned randomly to the three sperm preparation methods.The sperm suspensions prepared by methods A and C each had significantlyhigher proportions of acrosome-free spermatozoa compared tosuspensions prepared by method B. The fertilization rates ofoocytes micro-injected with spermatozoa prepared by methodsA and C were significantly higher than for method B. Eight clinicalpregnancies resulted from 28 cycles in which embryo replacementoccurred. We conclude that the fertilization rate followingsubzonal sperm injection is related directly to the percentageof acrosome-free spermatozoa in the sperm suspension used formicro-injection     

Andrology: Subzonal sperm insertion with aged human oocytes from failed in-vitro fertilization attempts: fertilization results and some applications

This study was undertaken to assess the potential of aged humanoocytes from failed in-vitro fertilization attempts as a modelfor the study of fertilization events after subzonal sperm insertion(SUZI). Criteria of aged oocyte suitability for this purposewere (i) the absence of nuclei, (ii) the presence of a polarbody, (iii) the absence of cell division or fragmentation, (iv)marked ooplasmic contraction in hyperosmotic medium, and (v)rapid ooplasmic relaxation after returning into normo-osmoticmedium following the micromanipulation. Micro-injection techniqueswere essentially the same as for SUZI with fresh oocytes. Oocytesthat fused with the micro-injected spermatozoa developed pronucleiof typical internal structure. However, the number of pronucleiwas often higher than that theoretically expected if each spermnucleus incorporated into the oocyte gave rise to a single pronucleus.Thus, the use of aged oocytes implies the need for a specificmethod to assess the frequency of fusion in sperm samples examined.The results suggest that the method described here can be appliedin a preliminary diagnostic test before a therapeutic SUZI attemptand in studies aimed at the optimization of sperm treatmentprotocols to increase the fusion capacity of subzonally insertedspermatozoa.