Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse.

Implantation rates remain low following human in-vitro fertilization (IVF). Suboptimal culture conditions may limit the ability of embryos to hatch as blastocysts, and artificial opening of the zona pellucida has been proposed as a means to promote subsequent hatching (assisted hatching). Such techniques must have minimal adverse effects on the embryos, while maximizing the potential for an embryo to hatch fully as a blastocyst. In a mouse model, we compared embryonic development after zona drilling, and cruciate thinning of the zona (CTOZ) intended to simulate the natural thinning of the zona pellucida. Using acidic Tyrode's solution both zona drilling and cruciate-thinning were performed on day 3 morulae. On day 4 the rates of complete hatching of blastocysts were 0/165, 24/172 and 72/175 in control, zona drilled and thinned groups respectively (P less than 0.0001). On day 5 the rates of complete hatching in the same groups were 20/165, 54/172 and 120/175 respectively (P less than 0.00001) and by day 6, 66/165, 74/172 and 130/175 respectively (P less than 0.00001). The rate of arrest at the morula stage was 24/172 versus 8/175 in the zona drilled and thinned groups respectively (P less than 0.005, whilst the rate of arrest at the blastocyst stage was 21/172 versus 14/175 respectively (NS). Hence cruciate thinning of the zona appears less detrimental at the morula stage than zona drilling, but eventual rates of arrest at the blastocyst stage were comparable. Both techniques significantly increased the rate of hatching, but zona drilling did not guarantee complete hatching.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of cooling on the properties of the zona pellucida of the mouse oocyte

Mouse oocytes were exposed to a variety of cooling regimes priorto insemination in vitro. Exposure to 4°C, but not to 25°C,was associated with a reduced fertilization rate, but developmentto the blastocyst stage of those oocytes that fertilized wasnot consistently different from that of non-cooled controls.The reduced fertilization rate seems to result from an effectof cooling on the zona pellucida, since it was not observedif the zona was removed prior to insemination, and since coolingrendered the zona pellucida resistant to the action of chymotrypsin.Using chymotrypsin resistance as an assay, the nature of thecooling-induced effect on the zona was investigated. It is suggestedthat rapid cooling to 4°C may promote release of corticalgranules and a premature zona reaction.     

Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilization and understanding implantation

It has been shown recently that delayed transfers improve implantationrates in assisted reproductive technology programmes. In a prospectivestudy, the pregnancy rates and safety of outcome were evaluatedin a group of patients after the transfer of day 5 blastocystswith enzymatic treatment of the zona pellucida. Nineteen womenwith a mean age of 32.6 ± 5.2 years and mean 2.1 ±2.2 repeated attempts had blastocyst transfers with a mean numberof 2.5 ± 0.7 embryos replaced per patient. The clinicalpregnancy rates per cycle/transfer and implantation rate were53% and 33%, respectively. The multiple pregnancy rate was 40%(two pregnancies were triplets). The pregnancy and implantationrates were very much higher than observed for most assistedreproduction technology centres. The ‘in-vitro implantation’rates of zona-free blastocysts on a variety of feeder monolayerswas 92%, offering some thoughts as to the role of the zona andinteraction of the inner cell mass and trophoectoderm with theendometrium in implantation. Based on the in-vitro studies andthe high multiple pregnancy rates, it appears that zona-manipulatedblastocysts implant relatively well and there would be a needto reduce the number of transferred embryos to one or two, thusreducing multiple pregnancies and having spare blastocysts availablefor cryopreservation. The results also suggest that using theembryo culture protocol and method of transfer in the presentstudy offers encouraging improvements to assisted reproductiontechnology, and enzymatic treatment of the zona may allow betteranchorage and dialogue of the embryo with the endometrium, helpingus to improve and understand implantation.     

Laser-assisted zona pellucida thinning prior to routine ICSI

BACKGROUND: In MII oocytes showing difficult oolemma breakage, ICSI can cause an increase in the degeneration rate. This may be overcome by laser-assisted ICSI using a 5-10 micro m opening in the zona pellucida for injection. However, such a small opening might impair the hatching process, especially if assisted hatching is applied in addition. In order to prevent this, the present study used routine injection through an area of zona pellucida in which laser zona thinning had been applied, providing for both a reduced mechanical stress to the oocyte and assisted hatching. METHODS: This prospective study involved 100 cycles with 1016 MII oocytes. Conventional ICSI (control group) was compared with a modified laser-assisted ICSI (study group) in sibling oocytes. In the latter group oocytes were injected through an extended area of zona thinning. RESULTS: Degeneration rate was significantly lower in the study group (P < 0.004). There were no differences in fertilization, or formation and quality of blastocysts. In the study group embryo quality on day 2 was significantly better (P = 0.004) and herniation of day 5 blastocysts was increased (P = 0.005). Rates of implantation and pregnancy were not affected. However, on day 3 laser-assisted ICSI proved beneficial (P = 0.038) in terms of clinical pregnancy rate. CONCLUSIONS: The new method combines a less invasive ICSI technique with assisted hatching. Our preliminary data indicate that in addition to an improved oocyte survival, this new approach increases the hatching rate in vitro, which may explain the increase in pregnancy rate, at least in day 3 transfers.     

Effect of damage to the zona pellucida on development of preimplantation embryos in the mouse

The effect on development of early mouse embryos of making ahair-line slit in the zona pellucida of approximately one-thirdits diameter was investigated. The rate of development to mid-gestationof operated zygotes and2-cell embryos transferred directly tothe oviduct was significantly lower than that of sham-operatedor unoperated controls. However, the operation had no discernibleeffect on the development of 2-cel embryos that were culturedfor 2 days prior to transfer to the uterus, or on embryos composedof 8 or more cells transferred directly to the oviduct. Zonaslit zygotes and 2-cell embryos exhibited a significantly higherrate of anomalous development to the morula or blastocyst stagethan controls following short-term transfer to the adult orimmature oviduct. Such anomalies could not be attributed todamage of the embryos by leucocytes or bacteria entering throughthe wound in the zona. Rather, the typically non-spherical shapeof slit zonae, together with the fact that some were empty onrecovery, was consistent with operated embryos having been damagedby compression during passage through the oviduct. This suggeststhat, providing it is intact, the zona pellucida protects theearly embryo from contraction of the oviductual musculaturewhich is sufficient to lyse, arrest or extrude blastomeres priorto the formation of intercellular junctions. Hence, in experimentalmanipulations entailing damage to the zonae of early embryos,there may be a case for allowing them to form morulae in vitroprior to transfer, rather than returning them directly to theoviduct     

High magnitude of light retardation by the zona pellucida is associated with conception cycles

BACKGROUND: Failures in expression of zona proteins correlate to subfertility in animals. Low expression of the zona proteins by the growing human oocyte may indicate reduced developmental potential. Therefore, we non-invasively analysed the thickness and the structure of the zona pellucida (ZP) of human oocytes with respect to embryo fate after ICSI. METHODS: Retardance magnitude and thickness of the inner, middle and outer layers of the ZP were quantitatively analysed by a Polscope in 166 oocytes selected for transfer after ICSI (63 patients; 32.8 +/- 4.4 years) on the basis of pronuclear score at day 1. Blastomere number was determined at day 2. Data were compared between conception cycles (CC; 65 oocytes/23 patients) and non-conception cycles (NCC; 101 oocytes/40 patients) and with respect to maternal age. RESULTS: The thickness was slightly elevated (P < 0.001), and the mean magnitude of light retardance was nearly 30% higher (P < 0.001) in the inner layer of the zona pellucida of oocytes contributing to CC compared to NCC. Embryos in the CC group tended to develop faster. CONCLUSIONS: The magnitude of light retardance by the zona pellucida inner layer appears to present a unique non-invasive marker for oocyte developmental potential.     

Four zona pellucida glycoproteins are expressed in the human

BACKGROUND: The zona pellucida (ZP) is an extracellular glycoprotein matrix which surrounds all mammalian oocytes. Recent data have shown the presence of four human zona genes (ZP1, ZP2, ZP3 and ZPB). The aim of the study was to determine if all four ZP proteins are expressed and present in the human. METHODS: cDNA derived from human oocytes were used to amplify by PCR the four ZP genes. In addition, isolated native human ZP were heat-solubilized, trypsin-digested and subjected to tandem mass spectrometry (MS/MS). RESULTS: All four genes were expressed and the respective proteins present in the human ZP. Moreover, a bioinformatics approach showed that the mouse ZPB gene, although present, is likely to encode a non-functional protein. CONCLUSIONS: Four ZP genes are expressed in human oocytes (ZP1, ZP2, ZP3 and ZPB) and preliminary data show that the four corresponding ZP proteins are present in the human ZP. Therefore, this is a fundamental difference with the mouse model     

A prospective randomized study to assess the benefit of partial zona pellucida digestion before frozen-thawed embryo transfers

BACKGROUND: Assisted hatching (AH) in fresh embryo transfer (ET) could be associated with increased implantation rates. However, very few prospective randomized studies have specifically addressed the issue of AH during frozen-thawed embryo transfers (FET) cycles, those that have reported controversial results. The aim of this study was to evaluate the benefit of an enzymatic zona pellucida treatment of frozen-thawed embryos before transfer. METHODS: This was a prospective study including 125 non-donor FET cycles from 125 infertile couples. FETs were randomly allocated into AH group (n = 61, embryos pretreated with pronase 5 IU/ml for 1 min at 37 degrees C) or control group (n = 64, untreated embryos). Zona pellucida thickness was measured for each transferred embryo. The main outcome parameters were clinical pregnancy and implantation rates. RESULTS: The two groups were comparable regarding mean women's age, duration and indications of infertility, IVF outcome after fresh ETs, numbers and quality of fresh and frozen embryos, frozen-thawed embryo survival rates and blastomeres survival indexes. Despite a statistically significant decrease of zona pellucida thickness after pronase treatment [(mean +/- SD) 18.5 +/- 2.25 versus 14.5 +/- 2.75 microm; P < 0.0001], implantation (9.6 versus 9.2%) and clinical pregnancy rates (18.0 versus 17.2%) were not statistically different after FETs, with a similar mean number of embryos transferred between AH and control groups, respectively. CONCLUSION: Within the constraints of our protocol, partial enzymatic digestion of zona pellucida by pronase was not related with any benefit of the FET outcome especially concerning the implantation ability of frozen-thawed embryos.     

Detection of zona pellucida proteins during human folliculogenesis

BACKGROUND: The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage. METHODS: Follicles (from n = 18 patients, 14-40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined. RESULTS: All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. CONCLUSIONS: ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.     

Cracking the egg: increased complexity in the zona pellucida

A functional zona pellucida is critical for both fertilization and the early stages of embryo development. Recent data from genomic and proteomic studies have questioned our simplistic view of the zona as being composed of three proteins whose functions are clearly defined. In the human, for example, the zona pellucida is composed of four proteins, not three. The increased complexity of the zona pellucida in humans and other species across the evolutionary tree now demands that we reconsider our reliance on the mouse model for understanding early fertilization events. Additionally, we are now well placed to examine, for the first time, potential defects in zona genes and their proteins associated with defined pathology.     

Fertilization and early embrology: Monozygotic twinning in the human is associated with the zona pellucida architecture

Six cases of identical twin pregnancies which occurred in 2163cycles of in-vitro fertilization during a 3 year period arereported. Monozygosity was confirmed when the number of fetusesexceeded the number of embryos replaced (n = 4) or when twoconcepti were observed in a single amniotic sac (n = 2). Eachof the reported pregnancies resulted from replacement of embryoseither with naturally thin zonae pellucida or embryos whosezonae had been breached during micromanipulation for assistedfertilization (subzonal sperminsertion) or assisted hatching.That such cases exclusively gave rise to monozygosity suggestsa link between the physical state of the zona pellucida, hatching,and generation of identical twins.     

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.     

The effect of pentoxifylline on sperm movement characteristics and zona pellucida binding potential of teratozoospermic men

This study investigates the effect of pentoxifylline on spermmovement and zona pellucida binding. Spermatozoa from nine teratozoospermicpatients (10.2 ± 6% normal forms) were included in thestudy. Test samples were diluted with a 4 mM solution of pentoxifylline,and control samples with Ham's F-10 culture medium only. Zonapellucida binding potential was measured under hemizona assayconditions (HZA). Sperm motility was evaluated at the start(0 h postswim-up) and end (4 h) of the HZA, under test-tubeconditions and under HZA conditions (50µ droplet underoil). Motilityparameters tested included the curvilinear velocity(VCL), straight line velocity (VSL) and linearity (LIN). Comparedto the controls, pentoxifylline-treated samples showed an immediatestimulation of sperm motility, under test-tube conditions, witha significant elevation of VCL at 0 h incubation (102.77 ±14.4 versus control value 84.60 ± 10.6 µm/s; P< 0.005), which however was reversed after 4 h incubation(73.16 ± 4.6 versus control value 85.47 ± 12.8µm/s; P < 0.005). A decline in sperm motility from0 to 4 h incubation was noted for all the pentoxifylline-treatedsamples, under both test tube conditions (VCL: 102.77 ±14.4 versus 73.16 ± 4.6 µm/s, P <<;<<0.005; VSL: 27.2 ± 10 versus 10.66 ± 2.2 µm/P <<; 0.005; LIN: 23.65 ± 7.1 versus 11.86 ±1.8%, P<<; 0.005) and HZA conditions (VCL: 100.04 ±13.1 versus 76.00 ± 7 µm/s, P <<; 0.005;VSL: 26.40 ± 8.7 versus 9.14 ± 4.5 µm/s,P <<; 0.005; LIN: 26.2 ± 12 versus 11.05 ±4.3%, P <<; 0.005). Pentoxifylline also showed no effecton the zona binding capacity of the sperm population, sinceno significant difference in zona binding capacity was seenbetween control and test samples. In conclusion, the effectof pentoxifylline on teratozoospermic spermatozoa is short-lived,and despite its stimulatory potential pentoxifylline shows nobeneficial use in enhancing zona pellucida binding of teratozoospermicspermatozoa.     

Factors associated with accidental fractures of the zona pellucida and multipronuclear human oocytes following in-vitro fertilization

A total of 101 multipronuclear oocytes (7.4% of fertilizations)were retrospectively identified in this in-vitro fertilizationprogramme. The use of a manual syringe suction system, insteadof an electric pump, to aspirate follicles, was associated witha significant increase in the proportion of oocytes with fracturedzonae pellucidae (P < 0.001), a lower normal fertilizationrate (P < 0.05) and a higher proportion of multipronuclearfertilizations (P < 0.001). Irrespective of the mode of follicularaspiration, significantly more multipronuclear fertilizationsoccurred following stimulation with a combination of clomipheneand human menopausal gonado-trophin, than after clomiphene alone(P < 0.05). It was concluded that the aspiration pressures,created by syringe suction, were more likely to rupture thezona pellucida of some oocytes, while in others it predisposedto an increased multipronuclear fertilization rate.     

The impact of cigarette smoking on zona pellucida thickness of oocytes and embryos prior to transfer into the uterine cavity

BACKGROUND: Smoking has been reported to promote infertility. The zona pellucida plays an important role in fertilization and implantation. We report, for the first time, the effect of cigarette smoking on zona pellucida thickness of oocytes and embryos as one of the factors that may interfere with fertility. METHODS: This study comprised 169 women, grouped according to their smoking habits: 31 active smokers, whose husbands do not smoke; 44 active smokers, whose husbands smoke; 65 passive smokers, because of smoking husbands and 29 non-smokers (women and husbands). Zona pellucida thickness was measured prospectively on printed photos of 903 oocytes and 456 embryos. RESULTS: The zona pellucida thickness of oocytes and embryos of non-smoking women was significantly thinner than those of active and passive smokers. However, no significant differences were observed in the natural ability of the zona pellucida to become thinner after 48 h in culture. CONCLUSIONS: Our study demonstrates that active and passive cigarette smoking increases the zona pellucida thickness of oocytes and embryos. Our findings also show that active and passive smoking has no significant effect on the thinning mechanism of the zona pellucida, which implies that it is independent of the initial zona pellucida thickness.     

Gonadotrophin-releasing hormone antagonists inhibit sperm binding to the human zona pellucida.

Previous work from our laboratory indicated that gonadotrophin-releasing hormone (GnRH) increases human sperm-zona pellucida binding. Here we present evidence that GnRH antagonists inhibit sperm-zona pellucida binding in humans. Motile spermatozoa (10(7) cells/ml) were incubated in modified Tyrode's medium at 37 degrees C, in 5% CO(2) in air. After 4.5 h, aliquots of spermatozoa were treated with saline (control) or with different concentrations of GnRH antagonists (test). Each sperm aliquot was then tested in the hemizona binding assay. In this assay, the control aliquot was incubated with half a human zona pellucida (hemizona) and the test aliquot was incubated with the matching half. After 20 min, the hemizonae were withdrawn and the number of zona-bound spermatozoa counted using phase-contrast microscopy. In addition, the effect of GnRH antagonists upon the pattern of sperm movement, frequency of sperm-zona pellucida collisions, and percentage of living and acrosome-reacted spermatozoa was determined. The results indicated that treatment with GnRH antagonists decreased the number of zona-bound spermatozoa and did not change the pattern of sperm movement, frequency of sperm-zona collisions, and percentage of acrosome-reacted spermatozoa. We suggest that this action of GnRH antagonists may be due to an effect on zona receptors on the sperm plasma membrane.     

Prospects of zona pellucida glycoproteins as immunogens for contraceptive vaccine

The zona pellucida (ZP) surrounding a mammalian oocyte mediatesthe initial recognition and binding of spermatozoon to oocytein a relatively species-specific manner and plays an importantrole in the subsequent activation events during the fertilizationprocess. The ZP comprises three biochemically and immunologicallydistinct glycoproteins termed ZP1, ZP2 and ZP3. The criticalrole of ZP glycoproteins in reproduction together with theirtissue-specific nature have led to their being considered aspotential candidate antigens for immunocontraception. Immunizationof females with ZP glycoproteins leads to a block of fertilityin several animal models. However, it is invariably associatedwith either a transient or an irreversible alteration in thecyclicity, hormonal profile and follicular development in theovary. To overcome these problems, attempts are being made todelineate relevant `B' cell epitopes on ZP proteins so as todesign immunocontraceptive vaccines based on synthetic peptidesdevoid of oophoritogenic `T' cell epitopes. Monoclonal antibodiescapable of inhibiting the gamete interaction are being employedto delineate such regions. Additionally, DNA-recombinant technologyhas made it feasible to obtain, in reasonably large quantities,the ZP glycoproteins from human and non-human primates. Availabilityof sequence information of these zona proteins and the availabilityof recombinant antigens (devoid of other ovarian-associatedproteins) will further help in understanding more preciselytheir functions during fertilization and make it feasible toundertake immunization studies to determine their prospectsas immunogens for fertility regulation.     

Fertilization and early embryology: The factors affecting sperm binding to the zona pellucida in the hemizona binding assay

Sperm binding to the zona pellucida is a prerequisite for fertilization.The hemizona binding assay (HZA) is commonly used to evaluatethe zona-binding capacity of spermatozoa. The present studyreports three factors that affect HZA. They were the base mediumused, the protein source and the size of pipette used for removingloosely bound spermatozoa during HZA. The number of spermatozoabound on the hemizona was compared between (1) Earle's balancedsalt solution (EBSS) and Ham's F-10, and (2) between human serumand bovine serum albumin (BSA). Results indicated that EBSSand human serum both significantly increase the number of boundspermatozoa when compared to Ham's F-10 (P < 0.0001, pairedt-test) and BSA (P < 0.05, paired t-test) respectively. Pipettesof different diameters were used to study the effect of sizein removing loosely bound spermatozoa on hemizona. Data showedthat the diameter of the pipette should be 200 mm, in ordernot to remove bound spermatozoa excessively. These results emphasizethe importance of standardization of the protocol of the hemizonaassay worldwide to be able to compare results between differentlaboratories.     

Carbohydrate analysis of the zona pellucida and cortical granules of human oocytes by means of ultrastructural cytochemistry

BACKGROUND: The zona pellucida (ZP), the mammalian oocyte coat, consists of a restricted number of highly glycosylated proteins. In vitro sperm binding studies suggest a higher binding affinity for the outer region of the ZP compared to its inner region in different species. However, the reason for this difference in binding distribution remains unresolved. Many studies suggest that the carbohydrate sequences linked to ZP glycoproteins act as ligands for sperm binding to this matrix. METHODS: Lectins and antibodies that recognize different carbohydrates were employed to perform an ultrastructural analysis of human ZP and cortical granule glycosylation. RESULTS: This study reveals variable glycosylation of the human ZP throughout its thickness, with pronounced differences between the most external and internal regions of this matrix. The binding studies also indicate that ZP glycoproteins express some carbohydrate sequences not previously detected in other species. Finally, cytochemical analysis of human cortical granules suggests similarities in glycosylation to ZP glycoproteins but not to cortical granules from other mammalian species. CONCLUSION: A heterogeneous carbohydrate composition was observed in the thickness of the human ZP that could be responsible for the different sperm binding affinity detected between the outer and inner regions of the ZP.     

Case report: pregnancy outcome following ICSI of oocytes with abnormal cytoplasm and zona pellucida

A couple with unexplained infertility was referred for routine IVF and ICSI treatment. Ovulation was induced by the GnRH analogue protocol combined with HMG and HCG. Preparation of denuded oocytes revealed a major disorder of the zona pellucida and abnormal oocytes. During preparation of ova for ICSI, 15 retrieved oocytes were denuded, 14 of which underwent ICSI treatment. Four of the oocytes collapsed and the remaining 10 appeared to have irregular, fragile zona pellucida. Nevertheless, following ICSI, seven low-quality embryos developed, three of which were transferred into the uterus. Two implantations were achieved, but only one embryo resulted in an uneventful pregnancy and delivery by Caesarean section of a normal female neonate with an Apgar score of 10. It is hypothesized that infertility was due to the abnormal oocyte structure and abnormal zona pellucida, which prevented natural conception. This condition was successfully resolved by the ICSI procedure.