Defective sperm-zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization

Sperm-zona pellucida binding and penetration were assessed on the oocytes that failed to fertilize from couples with >/=3 oocytes treated by standard in-vitro fertilization (IVF). There were four groups: fertilization rate 0% (n = 369), 1-25% (n = 194), 26-50% (n = 81) and 51-95% (n = 100). Of the couples with zero fertilization rate 70% had 相似文献   

Fertilization and early embryology: Does zona pellucida thickness influence the fertilization rate?

In human in-vitro fertilization (IVF), the cumulus oophorusis routinely removed to assess fertilization and hence the thicknessof the zona pellucida is measurable. This study aimed to measurethe thickness of the zona pellucida and to assess its influenceon fertilization rate in IVF programmes. The zona pellucidathickness varies from 10 to 31 µm with a mean of 17.5µm. One-way analysis of variance revealed that in IVFtrials performed with normal semen, the zona pellucida of fertilizedoocytes (16.6 ± 3.2 µm) was significantly thinnerthan the zona pellucida of unfertilized oocytes (18.9 ±4.0 µm; P < 0.001). As measured on micro-injected oocytes,the zona pellucida thickness did not change between ovulationand 16–20 h after fertilization. Zona pellucida thicknesswas not related to ooplasm diameter. In conclusion, zona pellucidathickness appears to be an additional factor that should betaken into account when interpreting the fertilization rate.Zona pellucida thickness influences sperm penetration, evenwhen the spermatozoa are considered normal. From a clinicalpoint of view, a thick zona pellucida (22 µm) could bean indicator for the use of micro-injection procedures.     

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.     

The role of cumulus cell-secreted proteins in the development of human sperm fertilizing ability: implication in IVF

Cumulus cells surrounding pre-ovulatory human oocytes were foundto secrete a variety of proteins which became firmly associatedwith the cumulus intercellular material. Antibodies raised againsthuman cumuli oophori completely blocked fertilization in vitroby impairing the sperm-zona pellucida interaction. A group ofglycoproteins of high mol. wt were Identified as the main cumuluscell secretory produds. These proteins showed a marked affinityfor human spermatozoa and were potent stimulators of the conversionof human and boar proacrosin into acrosin and of human spermacrosome reaction. Another fraction of proteins of human cumulusintercellular matrix with an apparent mol. wt of 25 000 daltonswas also found to stimulate significantly the acrosome reactionof human spermatozoa, although this fraction had no proacrosin-convertingactivity. These results indicate that proteins secreted by pre-ovulatoryhuman cumulus cells have an Indispensable role in the developmentof human sperm fertilizing ability. This effect seems to berealized by a concerted action of different types of cumulus-derivedproteins just prior to and during the sperm-zona pellucida interaction.Disorders of cumulus cell secretory activity may account forsome cases of idiophathic infertility and repeated IVF failures.     

Fertilization and early embryology: Timing of pronuclear development and first cleavages in human embryos after subzonal insemination: influence of sperm phenotype

The sequential transformations of human sperm nuclei in humaneggs after subzonal insemination (SUZI; n = 104) and the influenceof sperm defects on this timing were studied. This chronologywas compared to that of two control series of zygotes obtainedafter SUZI with normal spermatozoa (n = 35) and after in-vitrofertilization (IVF) with normal donor spermatozoa (D-IVF; n= 220). Pronuclear formation took place between 4.5 and 10.5h post-SUZI for 92.8% of the zygotes. They remained visiblefor 13 h and began to disappear 18.5 h post-SUZI. The time-spanbetween pronuclear disappearance and cleavage was 3 h. Zygotesobtained after D-IVF had a similar rate of pronuclear disappearancebut 4 h later. The second cell cycle was more rapid for zygotesobtained by D-IVF than by SUZI, but the developmental rate ofzygotes obtained by SUZI varied according to sperm phenotypes.For patients with previous unexplained IVF failures (controlgroup with normal spermatozoa), the developmental rate was lower,suggesting the influence of oocyte quality. In conclusion, theend of the first cell cycle of zygotes obtained by inseminationunder the zona pellucida appears 4 h earlier compared to zygotesobtained after insemination outside the zona pellucida.     

Andrology: The ability of the hemizona assay to predict human fertilization in different and consecutive in-vitro fertilization cycles

The objective of this prospective study was to examine the abilityof the hemizona assay (HZA) to predict fertilization outcomeof mature, pre-ovulatory oocytes under in-vitro fertilization(IVF) conditions. Since a large number of patients were evaluatedover a long period, the power of the HZA to prognosticate fertilizationresults in the same and subsequent (consecutive) IVF cyclesof those same patients was assessed. For IVF, only metaphaseII oocytes were used. For the HZA, both fresh oocytes donatedby patients at the time of IVF and oocytes recovered from surgicallyremoved ovarian tissue (and salt-stored) were used, and bisectedby micromanipulation techniques. Matching hemizonae were co-incubatedeither with spermatozoa from the patient (test) or from a fertileman (control) for 4 h. The number of spermatozoa tightly boundto the zona was counted. Patients (n = 112) were divided intotwo groups based on HZA results (expressed as HZA index or HZI):HZI 30% (n = 72) and <30% (n = 40). The patients with HZI<30% had significantly lower fertilization rates in boththe HZA—IVF cycle and in subsequent cycles compared topatients with HZI 30% (P < 0.03). Linear discriminant analysisindicated the HZA to have a sensitivity of 84%, and positiveand negative predictive values of 85 and 70% respectively, forprediction of fertilization outcome in a total of 233 cycles.It was concluded that the HZA is a good predictor of fertilizationrate in vitro, and can be used in the IVF setting to supplyadditional clinical information in malefactor patients.     

Acrosin activity in human spermatozoa in relation to semen quality and in-vitro fertilization

Acrosin, a sperm proteinase released during acrosomal exocytosis,facilitates penetration through the oocyte vestments. The purposeof this investigation was to determine if a correlation existsbetween the acrosin activity of ejaculated human spermatozoa,before motility enrichment techniques, and in-vitro fertilization(IVF) success using selected (glass wool or swim-up) spermatozoa.Since all the oocytes were retrieved from women receiving exogenoushormonal stimulation and a mixed population of mature and immatureoocytes were encountered, only cases with 50% mature oocyteswere analysed. Under these conditions, the acrosin activitywas significantly greater (P < 0.01) in the ejaculates inwhich spermatozoa ultimately fertilized <70% of the matureoocytes, than in the ejaculates in which spermatozoa ultimatelyfertilized 70% of the mature oocytes. Furthermore, a strongcorrelation (r = 0.962, P = 0.0001) was detected between pre-IVFacrosin activity and subsequent high (70%) IVF success. Acrosinactivity from normozoo-spermic and oligoasthenozoospermic menwas also compared and was significantly (P < 0.01) higherfor the normozoo-spermic group. These data suggest that measurementof acrosin activity may be a valuable clinical laboratory assayfor assessing the sperm fertilizing potential and that low acrosinactivity is associated with abnormal semen characteristics.     

Scanning electron microscopy analysis of the human zona pellucida: influence of maturity and fertilization on morphology and sperm binding pattern

Human oocytes from the same as well as from different patients have an extremely heterogeneous morphology of the zona pellucida surface as shown by scanning electron microscopy. For years it has been believed that this heterogeneous morphology plays an important part in the sperm-oocyte interaction. It was the aim of this investigation to analyse the morphology and the sperm binding patterns of the human zona pellucida. Oocytes were divided into four categories: mature, immature, fertilized and unfertilized. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between zona type and oocyte maturity or zona type and achieved fertilization. However, fertilized (polyploid) oocytes had a more compact and smooth zona surface than unfertilized ones. The analysis of the number and distribution patterns of bound spermatozoa on the zona pellucida revealed extremely variable patterns regardless of the zona morphology. Significant differences between mature and immature oocytes did not appear. In both groups there were oocytes with either no or numerous bound spermatozoa on the zona pellucida. Oocytes overloaded with spermatozoa could only be found in the mature group. Unfertilized oocytes had fewer bound spermatozoa on average than polyploid zygotes.     

Acrosome status and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro

It is known that only acrosome-reacted spermatozoa can fusewith the oolemma during normal fertilization with zona pellucida-intactoocytes. The aim of this study was to determine if the oolemmaof human zona pellucida-free oocytes selectively binds spermatozoawith normal morphology and a reacted acrosome. Oocytes thatfailed to fertilize in vitro because of severe sperm defectswere used. The zona pellucida was removed with acidic (pH 2–3)saline. Sperm samples were obtained from normal fertile donorsand normozoospermic men. Motile spermatozoa were selected witha swim-up technique and 2x10⁶/ml incubated with oocytes. Theresults from 23 experiments showed that at 2 h there was a significantlyhigher mean percentage of acrosomereacted spermatozoa boundto the zona pellucida (mean ±SD, 42±22) than inthe insemination medium (27 ± 12). In contrast, all spermatozoabound to the oolemma at 2h were acrosome reacted. Furthermore,each fresh zona pellucida had>100 spermatozoa bound comparedwith an average of 28 (range 4–81) spermatozoa bound perzona pellucida-free oocyte. There was no significant differencein the zona pellucida-induced acrosome reaction between fresh(45 ± 21)and salt-stored (35 ± 22) zonae pellucidae.The percentage with normal morphology was significantly higherfor spermatozoa bound to the zona pellucida (84 ± 13)and oolemma of zona pellucida-free oocytes (71 ± 25)than for spermatozoa in insemination medium (39 ± 11)(P<0.01). Extending the time of incubation of spermatozoawith zona pellucida-intact oocytes increased the proportionof spermatozoa undergoing the acrosome reaction (n=6, 2 h, 41± 23; 3 h, 53 ± 31; 4 h, 61 ± 34). However,there was a large variation in the percentage of acrosome reactionsamong spermatozoa bound to the zona pellucida between individualsperm samples. In conclusion, only acrosome-reacted spermatozoacan bind to the oolemma of zona pellucida-free oocytes. Bothfresh and salt-stored human zonae pellucidae are equally effectivein inducing the acrosome reaction. Both the zona pellucida andthe oolemma of zona pellucida-free oocytes selectively bindspermatozoa with normal morphology     

Enhancement of sperm-zona pellucida (ZP) binding capacity by activation of protein kinase A and C pathways in certain infertile men with defective sperm-ZP binding

BACKGROUND: Defective sperm–zona pellucida (ZP) binding (DSZPB) isa common cause of failure of fertilization in vitro. This studywas to determine if DSZPB is caused by defective pathways upstreamof protein kinase A (PKA) and C (PKC), or reduced protein tyrosinephosphorylation (TP). METHODS: Infertile men with DSZPB and either normal sperm morphology(NSM) 14% (n = 15) or 5% (n = 15) were studied. Sperm–ZPbinding test was performed by incubation of motile sperm withoocytes for 2 h with or without dibutyryl cyclic AMP (dbcAMP,PKA activator) or phorbol myristate acetate (PMA, PKC activator).TP of capacitated sperm in medium was assessed by immunofluorescencewith an anti-phosphotyrosine monoclonal antibody. RESULTS: For normal sperm with normal sperm–ZP binding, both PMAand dbcAMP significantly enhanced sperm–ZP binding ina dose–response manner. Only dbcAMP, but not PMA, significantlyincreased TP of capacitated sperm. In DSZPB men with severeteratozoospermia (NSM 5%), neither PMA nor dbcAMP enhancedsperm–ZP binding, despite dbcAMP significantly increasingthe TP of capacitated sperm for all samples. In contrast, forDSZPB with NSM 14%, PMA caused significantly increased spermbinding up to normal levels (40 sperm bound/ZP) in five men,and dbcAMP had a similar result in two men. Again TP was significantlyenhanced only by dbcAMP, but not by PMA. CONCLUSIONS: There is defective signalling in pathways upstream of PKC andPKA in some men with DSZPB and normal semen analysis. Stimulationof TP by dbcAMP does not enhance sperm–ZP binding capacityin DSZPB men with low TP, regardless of sperm morphology.     

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.     

Factors influencing sperm-zona pellucida binding in vitro using the intact zona model

The zona pellucida binding assay assesses the ability of spermatozoa to bind to the zona pellucida. The present study investigated the influence of: (i) prior oocyte exposure to spermatozoa on subsequent sperm-zona pellucida binding in vitro; and (ii) cryopreservation of oocytes. Only oocytes obtained from fertile donors were used and the binding capacity of non-inseminated, cryopreserved oocytes was compared with both inseminated/unfertilized, cryopreserved oocytes and inseminated/unfertilized, non-cryopreserved oocytes recovered from in- vitro fertilization cultures on sperm-zona pellucida binding using an intact zona model. There was no statistically significant difference in sperm-zona binding between non-inseminated, cryopreserved oocytes (range 9.6-23.2), inseminated/unfertilized, cryopreserved oocytes (range 15.0-16.0) and inseminated/ unfertilized, non-cryopreserved oocytes (range 3.3-23.0). The coefficient of variation for sperm binding to all oocyte groups was very large (range 37-121%). We conclude that neither prior exposure of human oocytes to human spermatozoa nor cryopreservation of human oocytes influences the subsequent binding of a different population of spermatozoa to the zona pellucida. However, large oocyte-to-oocyte variation of sperm-zona binding may diminish the usefulness of this assay in clinical practice and as a research tool.      

Human sperm--zona pellucida binding, sperm characteristics and in-vitro fertilization

The number of sperm bound to the zona pellucida (ZP) were countedon 660 oocytes that failed to fertilize in vitro from 162 patients.Oocytes from clutches in which some fertilized had significantlymore sperm bound to the ZP than did those from clutches in whichall oocytes failed to fertilize. Oocytes from patients in whomall were not fertilized and no sperm bound to the ZP were ableto bind normal fertile donor sperm after storage in ammoniumsulphate solution. The number of sperm bound to the ZP was significantlyrelated to the proportion of sperm with normal morphology andnormal intact acrosomes in semen, sperm concentration inseminated,sperm motility and viability. The number of sperm bound to theZP, sperm normal morphology, diagnosis of male infertility andsperm concentration in semen were significantly related to thefertilization rate by logistic regression analysis. Thus thenumber of sperm bound to the ZP is an indicator for IVF successand low binding appears to be more frequently associated withsperm defects than oocyte defects.     

Regulators of sperm function: Composition of the human zona pellucida and modifications following fertilization

The composition of individual human zonae pellucidae and modificationsto this extracellular coat both before and after fertilizationwere analysed using a rapid, sensitive, non-radioactive biotinylation-or lectin-based detection system; these assays use commerciallyavailable reagents and can be performed on fragments of individualzonae pellucidae. The zona pellucida from unfertilized eggsis composed of three glycoprotein species designated as huZP1,huZP2 and huZP3. Under non-reducing conditions, the molecularweights of these proteins are 150 kDa, 100 kDa, and 55–65kDa respectively. Following fertilization, huZP1 was not detectedunder either non-reducing or reducing conditions. In contrast,after fertilization huZP2 was detected under non-reducing conditions,but not under reducing conditions. The ability to detect pre-and postfertilization changes in a single human zona pellucidais discussed in relation to its value in assessing deficienciesin clinical and laboratory protocols used for in-vitro fertilization.     

A comparative analysis of embryo implantation potential in patients with severe teratozoospermia undergoing in-vitro fertilization with a high insemination concentration or intracytoplasmic sperm injection

The objective of this study was to assess fertilization, implantationand pregnancy rates in infertile patients with severe teratozoospermia[P (poor prognosis) pattern sperm morphology assessed by strictcriteria] treated by in-vitro fertilization (IVF) using a highinsemination concentration (HIC), or by intracytoplasmic sperminjection (ICSI). This was a retrospective cohort study performedin an academic tertiary institution. The outcome of 115 consecutiveICSI cycles was compared to that of a similar number of cyclesof IVF with HIC performed during a similar time frame and matchedby woman's age and basal serum (cycle day 3) follicle stimulatinghormone concentrations. The inclusion criteria were sperm morphology4% normal forms (P pattern) and 1 x106 total motile spermatozoaper ejaculate. The diploid fertilization rate in the HIC-IVFgroup was 86% and in the ICSI group 68% (P < 0.05). Importantly,an equal number of embryos was transferred to both groups ofpatients. The morphological quality of the embryos (proportionof transfers having superior morphology embryo scores) was significantlybetter in the ICSI group than in the patients receiving HIC-IVF.Although there was a clear trend for better implantation andpregnancy rates in the ICSI group, these differences were notstatistically significant We conclude that, although HIC-IVFresulted in a higher fertilization rate than ICSI in patientswith severe teratozoospermia, ICSI produced a significantlyhigher proportion of morphologically superior embryos with atendency towards a higher implantation potential. Therefore,teratozoospermic patients having adequate numbers of motilespermatozoa should be offered ICSI as an alternative to modified(HIC) IVF treatment.     

Clinical importance of zona pellucida-induced acrosome reaction and its predictive value for IVF

The study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. ZIAR tests were performed using 0.25 zona pellucida/microliter co-incubated with spermatozoa for 60 min. Acrosome reactions were measured with FITC-PSA staining, and expressed as the difference between stimulated and unstimulated (spontaneous) sperm populations. Results were compared with IVF rates of metaphase II oocytes. Interactive dot diagrams divided the patients into two groups, i.e. ZIAR <15% and ZIAR > 15%, with mean fertilization rates of 49 and 79% respectively. The sensitivity and specificity for ZIAR results versus fertilization were 93 and 100% respectively. The area under the curve was 99% and the 95% confidence interval did not include 0.5 which implies that the ZIAR test is able to predict fertilization failure among IVF patients. In conclusion, the ZIAR test has diagnostic potential since it can assist the clinician to identify couples that will benefit from intracytoplasmic sperm injection therapy.     

Immunology: Intravenous immunoglobulin for in-vitro fertilization failure

The aim of this study was to determine the effectiveness ofintravenous (i.v.) immunoglobulin (Ig) for treatment of individualsexperiencing failure after in-vitro fertilization (IVF) andembryo transfer. A total of 29 women with unexplained infertilitywho failed to become pregnant after IVF/embryo transfer weredivided into two groups based on performance in previous IVFcycles: 16 women had fertilization of 50%of oocytes retrievedand/or produced 3 embryos each cycle and 13 had fertilizationof<50% of oocytes retrieved and/or produced <3 embryoseach cycle. Each woman had received at least 12 transferredembryos (95th percentile for successful IVF patients) or hadexperienced two or more biochemical pregnancies without ultrasonicconfirmation of implantation during previous IVF/embryo transferattempts. All women received i.v. Ig 500 mg/kg prior to thenext embryo transfer. Only one of the 13 (8%) women with suboptimalfertilization and embryo yield became pregnant in the treatmentcycle. Of 16 women who had previously had fertilization of atleast 50% of oocytes retrieved and produced at least three embryos,nine (56%) became pregnant in the treatment cycle. The differencein pregnancy rates between the two groups is significant (P=0.02).Intravenous Ig is useful in the treatment of unexplained IVFfailure in women who have oocyte fertilization rates 50% andgenerate at least three embryos per cycle.     

A fucose-containing epitope potentially involved in gamete interaction on the human zona pellucida

The oligosaccharide moiety of human, porcine and bovine zonaepellucidae was studied with lectins and monoclonal antibodiesspecific for tri- or tetra-saccharidic epitopes containing atleast one terminal -L-fucose. Animal eggs were collected fromfollicular aspirates, human eggs were collected from in-vitrofertilization and embryo transfer programmes and pooled intosix groups. By direct immunofluorescence, the lectins reactivitywas detected for the animal or the human zonae pools in thesame way. Reactivity of Aleuria aurantia lectin demonstratedthe presence of –L-fucose terminal residues in the zonaefrom the three species studied. By indirect immunofluorescence,the 2–25 antibody reactivity was detected in every poolof human zonae whereas there was no evidence of any antibodyreactivity on animal zonae. Using an anti-Lewis-b blood groupantibody (2–25), we observed expression of this antigenas an intrinsic component of the human zona pellucida, independentlyof patients'Lewis red blood cell phenotypes. Antibody 2–25inhibited the sperm–atozoa-zona binding in a hemizonaassay, suggesting that this fucose-containing antigen couldbe part of a sperm-zona receptor.     

Fertilization and early embryology: Non-enzymatic formation of formaldehyde in mouse oocyte freezing mixtures

Three cryoprotectant solvents, dimethylsulphoxide, 1,2-propanedioland glycerol, were investigated for a non-enzymatic reactionproduct, formaldehyde. All three cryoprotectants demonstrateda direct relationship between increasing solvent molarity andincreasing formaldehyde concentration which was independentof temperature and protein (bovine serum albumin). Medium compositionsignificantly influenced the formaldehyde concentration withHTF T6 M16 = M2. The formaldehyde could be effectively removedby reduced glutathione, cysteine and dithiothreitol with cysteinebeing the most effective scavenging agent A reaction mechanismfor this scavenging is proposed. The combination of cysteineand cryoprotectant reduced the zona pellucida ‘hardening’effect in mouse oocytes.     

The presence of zonae pellucidae influences fusion rates between spermatozoa and zona free hamster oocytes

Human zonae pellucidae were added to suspensions of capacitatingspermatozoa. The zonae were prepared from oocytes that remainedunfertilized in our in-vitro fertilization (IVF) programme.After capacitation, zona free hamster oocytes were added andthe penetration rate of the hamster oocytes was determined.Controls without the addition of zonae were run in parallel.A distinct enhancement of the penetration rate was found whenzonne were present during capacitation, using sperm of fertiledonors. Sperm of several infertile men did not respond to thepresence of zonae with higher penetration rates It was condudedthat the zona peilucida is Involved in the induction of theacrosome-reaction of human spermatozoa. In addition the incubationsystem with zonae as described might be useful for the identificationof infertile semen.