Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF

To assess the association of zona pellucida micromanipulation and subsequent development of monozygotic twins, cases of assisted embryo hatching (AH) and intracytoplasmic sperm injection (ICSI) were identified and related to treatment type, implantation and zygosity data. Embryology records from all patients undergoing in-vitro fertilization (IVF) at this centre from January 1995 to March 1998 were reviewed. In this study, 3546 transfer cycles were completed, with clinical pregnancy established in 1911 (54% per transfer) patients undergoing a single IVF cycle. These pregnancies occurred in 1674 (88%) IVF cycles, 120 (6%) donor oocyte cycles (DER), and 117 (6%) frozen embryo transfer (FET) cycles. During the study period, 23 cases of monozygotic (MZ) twins were identified, representing an overall frequency of 1.2%. Chorionicity was determined by transvaginal ultrasound at 7 weeks when the number of embryos transferred was less than the number of fetal heart-beats, or when >1 fetal heartbeat per gestational sac was seen. Zygosity was confirmed by placental evaluation at delivery, and corroborated the antenatal diagnosis in all cases. Among IVF study patients the frequency of MZ twinning was not statistically different between zona manipulated and zona intact subgroups. While this investigation is the largest to date describing the relationship between MZ twins and zona procedures, studies with even greater statistical power are needed to clarify it more precisely, particularly in DER and FET settings. A greater overall frequency of MZ twinning for IVF patients may be a function of the higher number of embryos transferred in IVF, rather than discrete zona manipulations.     

Monozygotic twinning after assisted reproductive techniques: a phenomenon independent of micromanipulation

A 3 year retrospective analysis was conducted of pregnancies achieved after various assisted reproductive treatment modalities in our infertility practice, to calculate and compare the rates of monozygotic twinning (MZT). A total of 731 pregnancies achieved after various assisted reproduction treatments were reviewed. Gonadotrophin therapy for induction of ovulation and controlled ovarian hyperstimulation (COH) yielded 129 clinical pregnancies. Conventional IVF yielded 139 pregnancies. IVF and intracytoplasmic sperm injection (ICSI) with or without assisted hatching (AH) yielded 463 pregnancies, all during the same time period. The rates of multiple pregnancy (monozygotic and dizygotic) twins and triplets were recorded. MZT was found in 1.5% of ovulation induction or COH pregnancies (2/129). The incidence of MZT after conventional IVF was 0.72% (1/139). After IVF-ICSI/AH, MZT was found in 0.86% (4/463). The overall rate of MZT was 0.95% (7/731). Five cases were dizygotic triplets and two cases were monozygotic twins. We found the rate of MZT after assisted reproduction treatment increased more than two-fold over the background rate in the general population. Dizygotic triplets were found more often than monozygotic twins. The rate of MZT was consistently increased, irrespective of treatment modality or micromanipulation. This may signify that the aetiology of increased MZT after assisted reproduction is the gonadotrophin treatment rather than in-vitro conditions, micromanipulation, or multiple embryo transfer.     

Laser zona pellucida thinning--an alternative approach to assisted hatching

BACKGROUND: The purpose of this study was to assess the efficacy and hatching characteristics of in-vitro cultured human embryos subjected to laser zona pellucida thinning. METHOD: Zona thinning was performed on 110 embryos using a non-contact 1.48 microm diode laser and the hatch rate in vitro was compared with 42 control embryos. Variation of zona thickness and degree of zona expansion was assessed. Scanning electron microscopy was performed on embryos entrapped during hatching to identify the site of hatching. RESULTS: The rate of hatching was significantly higher in laser thinned blastocysts compared with control embryos (68 versus 33% respectively, P < 0.01). Laser thinning increased the variation of zona thickness in embryos from 11.6-27.3%. Natural zona thinning occurred in 92% of laser thinned hatching blastocysts and 100% of control embryos. CONCLUSION: These results suggest that laser zona thinning is effective and may provide significant advantages over conventional assisted hatching techniques, which create holes.     

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.     

Fertilization and early embryology: Use of lasers in assisted fertilization and hatching

The erbium-yttrium-aluminium-garnet (Er: YAG) laser has beenapplied to micromanipulation in humans. It was used in the fertilizationprocess for both subzonal insemination (SUZI) and for partialzona dissection (PZD). Laser-assisted micromanipulation achievedsignificantly higher fertilization rates (34.8%) when comparedto mechanical SUZI (16.1%), but use of the laser did not improvethe PZD results (laser 14.8% versus mechanical 14%). The Er:YAG laser was used to assist hatching. In the mouse it significantlyimproved the hatching rate (80 versus 29.3%) 110 h after administrationof human chorionic gonadotrophin. This technique was appliedin two different centres to patients with previous in-vitrofertilization (IVF) failures. The implantation rate per embryo(14.4% laser-assisted hatching versus 6% control group) andthe pregnancy rate per transfer (40 versus 16.2%) were improved.     

Fertilization and early embryology: The zona reaction in human oocytes as seen with scanning electron microscopy

Using scanning electron microscopy we found differences in thefine structure of the zona pellucida between unfertilized andfertilized human pronuclear stage oocytes in an in-vitro fertilizationprogramme. In unfertilized oocytes, the zona pellucida appearedporous, comprising a large number of ring-shaped structures,called hoops, randomly superimposed in several layers. Superficialpores had a mean diameter of 4µm, with the diameter decreasingin more inner lying pores. In fertilized oocytes, the zona pellucidawas compact; the hoops appeared to melt and the pores to beobliterated by an amorphous material emerging from the innerzona. The micrographs provide ultrastructural evidence of thezona reaction in human oocytes and give insights into the morphologicaland mechanical aspects of the polyspermy-blocking mechanismin humans.     

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)     

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.     

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.     

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.     

Monozygotic twinning associated with day 5 embryo transfer in pregnancies conceived after IVF

BACKGROUND: This study examines the association between day of embryo transfer and monozygotic (MZ) twinning. METHODS: We used a population-based sample of 108,36 IVF/embryo transfer procedures in which the patients oocytes' were freshly fertilized (non-frozen; non-donor) and 39,98 resultant pregnancies from US clinics in 1999 and 2000. Cases were pregnancies for which the number of fetal hearts observed on ultrasound exceeded the number of embryos transferred. These pregnancies were considered to contain at least one set of MZ twins. A total of 226 MZ pregnancies were compared with two control groups: 23,880 singleton pregnancies (one fetal heart) and 15,092 other multiple-gestation pregnancies (> or = 2 fetal hearts but the number of fetal hearts on ultrasound was less than or equal to the number of embryos transferred). RESULTS: Cases of presumed MZ multiple-gestation pregnancies were more likely to have had a day 5 embryo transfer compared with day 3 embryo transfers than singleton pregnancies [adjusted odds ratio (AOR) = 3.92, 95% confidence interval (CI) = 2.97-5.17] or other multiple-gestation pregnancies (AOR = 3.91, 95% CI = 2.96-5.17) conceived with IVF/embryo transfer. CONCLUSIONS: Day 5 embryo transfer may be associated with increased MZ twinning.     

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     

Fertilization and early embryology: Scanning electron microscopy of the zona pellucida of human oocytes during intracytoplasmic sperm injection (ICSI)

During intracytoplasmic sperm injection (ICSI) approximately10% of all injected oocytes degenerate. The reason for thisprocess is unknown. It has been speculated that the mechanicalprocedure of the insertion of the ICSI needle induces injuriesto the zona pellucida which lead to the death of the cell. Byscanning electron microscope (SEM), it could be shown that thesurface structure of mature oocytes is extremely elastic sothat the injection needle penetrates the zona pellucida withoutdestroying the mesh-like or more compact surface. No tissuepieces or zona fragments were detectable. After a culture timeof 15 min the penetration site on the zona was no longer easilyvisible. We believe that oocyte degeneration is not caused bythe penetration of a glass needle into the ooplasm but by aninjury to the meiotic spindle or by an excessive dose of fluid[polyvinylpyrrolidone (PVP) or medium] during sperm injection.     

Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C

To facilitate our understanding of the role of zona pellucida glycoproteins during fertilization in humans, recombinant human zona pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of approximately 110, approximately 70-75 and approximately 65 kDa, respectively, as compared to approximately 80, approximately 65 and approximately 50 kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose alpha 1-3 or mannose alpha 1-6 residues) and Jacalin (alpha-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P=0.0005) and hZPC (P=0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase (P>0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway.     

Fertilization and early embryology: Zona opening of human embryos using a non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy

The aim of this study was to examine the safety and the efficiencyof a ‘non-contact’ UV laser to assist hatching throughzona opening of human embryos. Between January and November1995 we performed zona drilling for assisted hatching usinga new laser system (PALM UV Laser microbeam), operating in a‘non-contact’ mode to create a hole in the zonapellucida of human embryos. In a randomized study, laser zonaopening was applied on embryos from two groups of patients withrepeated in-vitro fertilization (IVF) failures (two to fourattempts): group A was composed of 107 patients who receivedmixed embryos (216 laser-treated and 223 not treated) and groupB of 72 patients who received 218 laser-treated embryos only.Both groups were compared with a control group of 98 patientswhose embryos were not laser treated (n = 407) (group C). Themean ages of all groups (38.1, 38.2 and 37.8 years respectively)and the number of IVF attempts (two to four attempts) were similar.The resulting clinical pregnancies were 39 (36.4%) in groupA, 32 (44.4%) in group B and 19 (19.3%) in group C. The implantationrates/embryo were 9.3% in A, 16% in B and 5.1% in the controlgroup. In total, 17 normal babies have been delivered (10 ingroup A and seven in group B). These results show that laserzona drilling increased the pregnancy and implantation ratesin all the treated patients. The increase was slight but significantin patients of group A (P < 0.01 and P < 0.02), it waseven higher in the patients of group B (P < 0.05).     

SHORT COMMUNICATION: Repeated sperm injection under the zona following initial fertilization failure

When fertilization fails following micromanipulative underzonainsemination, it is possible to repeat the procedure addingmore spermatozoa to achieve fertilization, embryonic developmentand pregnancy. We report on 18 human in-vitro fertilizationcycles where this approach was used. In nine cycles only late-fertilizedembryos were available for transfer, and these gave rise totwo viable pregnancies (22.2% per transfer). In six cycles,where a mixture of late- and timely fertilized embryos wereavailable for transfer, two viable pregnancies arose (33.3%per transfer). In three cycles no fertilization was achievedeven after reinsemination by repeated under-zona insemination.     

Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis.

Assisted hatching by zona drilling using acidic Tyrode's solution was performed during three randomized trials in 330 in-vitro fertilization patients. The trials were designed in order to study the overall effect of the procedure and whether characteristic patient [i.e. maternal age and basal levels of follicle stimulating hormone (FSH)] and embryonic features (i.e. zona pellucida thickness) are important for the decision to perform assisted hatching routinely. Couples (n = 137; Trial 1) in whom the female partner had normal basal FSH levels were randomized in a control group (without micromanipulation) and a zona drilling group (all embryos micromanipulated). The incidence of implantation (67/239; 28%) of zona-drilled embryos compared favourably with that of control embryos (49/229; 21%), but the difference was not significant. Retrospective analysis revealed that those embryos whose zonae were thicker than 15 microns were rescued. In order to test the validity of this finding, selective assisted hatching was performed on embryos with a poor prognosis in 163 other patients (Trial 2). The couples were randomized into a control group and a group in which embryos were selectively zona-drilled, based on zona thickness and other embryonic features. The rate of embryonic implantation in the selectively zona-drilled group was 25% (70/278), significantly (P less than 0.05) higher than that of control embryos (51/285; 18%). Although it was demonstrated retrospectively and prospectively that assisted hatching by zona drilling is effective in embryos with thick zonae (greater than or equal to 15 microns), patients whose embryos have thin (less than 13 microns) zonae may be jeopardized by the procedure.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 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.     

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.