Routine application of partial zona dissection for male factor infertility

Partial zona dissection (PZD) increases the chances of fertilization by improving the access of spermatozoa to the perivitelline space (PVS) helping those spermatozoa unable to penetrate the zona pellucida (ZP) and possibly those poorly able to penetrate the oolemma. Problems arise in assessing semen to decide which parameters might indicate defects of this nature. PZD, by circumventing the ZP, may also increase the rate of polyspermy, especially in infertility where ZP and oolemmal penetration are not defective. Given these drawbacks, we performed PZD as routine treatment for male infertility in 70 in-vitro fertilization cycles. In three different groups, PZD proved to be either effective, ineffective or unnecessary. In the first group of 35 cycles, fertilization was 23% with initial PZD and 33% with PZD reinsemination (36% and 41% polyspermy respectively). No fertilization occurred following conventional insemination (CONV). Four pregnancies occurred in this group. In a second group of 19 cycles, fertilization did not occur with either PZD or CONV. In the final group of 16 cycles, fertilization was similar following both PZD and CONV, but polyspermy was 48% in the PZD category. Transfer of mixed PZD and CONV embryos in this group yielded 10 pregnancies. Assessment of all patient and seminal profiles, and those in an oligozoospermic subcategory, revealed no parameters of relevance to success or failure with PZD. However, one subgroup in the group of total failure to fertilize did have a significantly lower percentage of normal morphology (P less than 0.005), suggesting that degree of teratozoospermia may be a prognosticator of success using PZD.     

Experience with zona drilling and zona cutting to improve fertilization rates of human oocytes in vitro

Zona drilling (ZD) and zona cutting (ZC) were used in an IVF programme to assist fertilization in semen defect patients. Twenty-seven patients consented to ZD where acidified Tyrode's was used to create a hole in the zona pellucida. In 19 patients, ZD increased the fertilization rate to 29% compared with 8% (P less than 0.001) in their routine IVF cycles, and in eight patients precluded from routine IVF, a fertilization rate of 14% was achieved. Twenty-two patients consented to ZC where a slit in the zona is made mechanically. In 12 patients ZC increased the fertilization rate to 31% compared with 14% (P less than 0.01) from previous routine IVF cycles, and in 10 patients precluded from routine IVF, a fertilization rate of 34% was achieved. In 13 cycles, 68 uncut control oocytes were inseminated. In five cycles both control and ZC oocytes were fertilized (n.s.d.). In eight cycles no control oocytes were fertilized compared with 27% of ZC oocytes. The polyspermy rate was 4.6%. Twenty-four per cent of ZD and 12% of ZC (P less than 0.01) oocytes and embryos were degenerate after 42 h. Both ZD and ZC can increase the fertilization rate of sub-optimal semen, however, in our hands neither technique produced a pregnancy.     

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.     

IVF treatment of moderate male factor infertility: a comparison of mini-Percoll, partial zona dissection and sub-zonal sperm insertion techniques

In this study we examined various techniques of in-vitro fertilization(IVF) for treating couples in whom the male had subnormal semenparameters. We compared two sperm preparation methods (mini-Percolland conventional swim-up) for efficiency of recovery after preparationand for fertilization rates after IVF, and compared the suitabilityof partial zona dissection (PZD) and sub-zonal sperm insertion(SUZI) to patients with different types of male factor infertility.The mini-Percoll technique allowed the recovery of significantlymore motile spermatozoa from the same semen sample comparedto the swim-up method. More oocytes were fertilized after spermatozoawere prepared by the mini-Percoll technique. An increased numberof spermatozoa recovered from an ejaculate led to an improvementin the quality of spermatozoa in the insemination droplet. Subsequently,when using the PZD technique, the fertilization rate increasedwhen there was a higher number of spermatozoa in the patient'sejaculate. When comparing the two micromanipulation techniques,SUZI provided patients with oligoasthenzoo-spermia (i.e. <10 x 10⁶ spermatozoa/ml and 40% motility) with a higher chanceof obtaining 2-pronculeate eggs.     

Etiological implication of autoantibodies to zona pellucida in human female infertility.

PROBLEM: Autoantibodies to zona pellucida (ZP) have been implicated as a cause of infertility in woman by three lines of clinical and laboratory evidence. METHOD: First, positive anti-zona activities, as assessed by the passive hemagglutination reaction (PHAR) using bovine red blood cells sensitized with porcine zona antigen, were detected in 45 of 1,872 serum samples collected from infertile women, but in only three (two nonpregnant and one pregnant) of 592 serum samples from control subjects. Their incidence in 320 women with unexplained infertility (5.6%) was much higher than that in 1,552 women with infertility of known cause (1.7%, P < 0.01), which was comparable to the incidence observed in 193 age-matched fertile nonpregnant and pregnant women (1.5%, P < 0.05). None of the serum samples from 292 age-matched adult men and 100 children of between 5 and 10 years old gave a positive PHAR. Second, follow-up study for a minimum of 2 years with treatment revealed that no pregnancy occurred in 11 infertile women with a consistently positive PHAR, and only three pregnancies in 19 infertile women with a fluctuating positive PHAR. Third, three of seven serum samples from women who gave a consistently positive PHAR produced strong immunofluorescence reactions on human ZP even after adsorption with porcine and human AB blood cells. Pre-exposure of human ZP to the sera showing positive immunofluorescence, including that of one patient undergoing an in vitro fertilization program, greatly diminished the number of spermatozoa of normal quality that bound to and penetrated across human ZP. CONCLUSIONS: The observed high incidence of anti-zona activities and long-term resistancy to treatment in women with unexplained infertility may be closely correlated with inhibition of sperm-egg interaction by anti-zona autoantibodies produced in these women.     

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.     

Immunology: Assessment of the relevance of zona pellucida antibodies in follicular fluid of in-vitro fertilization (IVF) patients

The presence of anti-zona pellucida antibodies in the follicularfluid of 11 women who underwent in-vitro fertilization (IVF)and embryo transfer was analysed. Only infertile couples withtubal or unexplained pathologies were included in our study,which was aimed at investigating the relationship between anti-zonapellucida antibodies in follicular fluid and failed fertilization.Whether or not these antibodies were present in some or allfollicles in the same patient was also investigated. Out of55 follicular fluids analysed, 36.3% were positive to the testand no fertilization was observed in oocytes from these follicles,while 63.6% were negative, and the oocyte fertilization rateassociated with these was 51.4%. The presence of anti-zona pellucidaantibodies was positively correlated with the degree of fertilizationfailure (P <0.001 x² test).     

Effect of in-utero diethylstilboestrol exposure on human oocyte quality and fertilization in a programme of in-vitro fertilization.

Genital tract abnormalities and adverse pregnancy outcome are well known in women exposed in utero to diethylstilboestrol (DES). Data about adverse reproductive performance in women exposed to DES have been published, including controversial reports of menstrual dysfunction, poor responses after ovarian stimulation, oocyte maturation and fertilization abnormalities. We compared oocyte quality, in-vitro fertilization results and embryo quality for women exposed in utero to DES with a control group. Between 1989 and 1996, 56 DES-exposed women who had 125 in-vitro fertilization (IVF) attempts were retrospectively compared to a control group of 45 women with tubal disease, who underwent 73 IVF attempts. Couples suffering from male infertility were excluded. The parameters compared were oocyte quality (maturation abnormalities, immature oocyte, mature oocyte), fertilization and cleavage rate (per treated and metaphase II oocytes), and embryo quality (number and grade). We found no significant difference in oocyte maturational status, fertilization rates, cleavage rates, embryo quality and development between DES-exposed subjects and control subjects. These results suggest that in-utero exposure to DES has no significant influence on oocyte quality and fertilization ability as judged during IVF attempts.     

Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF

BACKGROUND: The zona pellucida (ZP) has multiple roles in reproductive processes, including oocyte maturation, fertilization and implantation. We used, for the first time, a genetic approach to study whether human ZP genes possess structural alterations in women with unsuccessful IVF trials. In theory, this may result in gradual reduction of sperm-zona interaction and eventually in total fertilization failure (TFF). METHODS: Eighteen infertile women (TFFs) whose IVF did not result in any fertilized oocytes, whereas fertilization by ICSI was successful, were screened for mutations in ZP genes by means of conformation-sensitive gel electrophoresis. Twenty-three fertilizers in IVF (FIVFs) and 68 women with proven fertility (WPFs) constituted the two control groups. RESULTS: Altogether, 20 sequence variations were found in the ZP genes. Two variations in ZP3, one in the regulatory region (c. 1-87 T --> G) and one in exon 6 [c. 894 G --> A (p. K298)] existed more frequently in TFFs than in FIVF and WPF groups (P-values 0.027 and 0.008, respectively). CONCLUSIONS: Our study on ZP genes of infertile women revealed a high degree of sequence variations. This may reflect gradual reduction of fertility among TFFs, but the putative roles and influences of single variations can only be hypothesized.     

Subzonal insemination, partial zona dissection or intracytoplasmic sperm injection? An easy decision?

This review aims to analyse and compare the results to dateof subzonal insemination (SUZI), partial zona dissection (PZD)and intracytoplasmic sperm injection (ICSI) to evaluate criticallywhether it is now possible to replace SUZI and PZD by ICSI.It appears that ICSI is a much more efficient assisted reproductiontechnique than SUZI and PZD for resolving cases of severe maleinfertility and/or repeated failure of conventional in-vitrofertilization (IVF). For ICSI compared with SUZI and PZD, fertilization(49.4, 17.7 and 16.8% respectively), percentage of patientsreaching embryo transfer (91.0, 55.1 and 23.3% respectively),percentage of transfers performed with two or three embryos(83.3% ICSI and 39.3% SUZI), pregnancy rate per embryo replacement(28.2, 18.7 and 16.5% respectively) and pregnancy rate per oocyteretrieval (24.8, 10.3 and 3.8% respectively) are all improved.In addition, cases of severely impaired semen characteristics,which were condemned to infertility for life with conventionalIVF, SUZI or PZD, can now be treated and resolved efficientlywith ICSI.     

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.     

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.     

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   

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.     

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.     

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.     

Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated

Partial zona dissection (PZD) of human oocytes facilitates spermpenetration through mechanically made holes in the zona pellucida.Only 1 of 69 eggs was damaged when sucrose was used to shrinkthe ooplasm during micromanipulation. The fertilization rateof micromanipulated oocytes in 18 couples with male factor infertilitywas 68% (34/50), which compared favourably with inseminationof non-micromanipulated controls (21/45, 47%). PZD was advantageousin oligozoospermic patients, but not in cases of asthenozoospermia,combined semen problems or immunological infertility. Threetwin and two singleton pregnancies resulted following replacementof 23 micromanipulated and eight control embryos in 14 patients.No differences in embryo morphology and development rates werefound between the micromanipulated and control groups. The incidenceof polyspermy in couples with abnormal semen analyses was relativelylow (<20%) possibly due to partial activation of the oocytesfollowing exposure to sucrose. Polyspermy was high (57%) innormozoospermic patients with either immunological infertility(n= 3) or failure of fertilization in previous cycles (n= 4).In the three immunological patients, nine of 11 hyaluronidaseand sucrose-exposed control embryos fertilized and six implanted,possibly indicating that cumulus and corona cells are contributingfactors inhibiting fertilization in such cases.     

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.     

Assisted fertilization in infertile women with patent Fallopian tubes. A comparison of in-vitro fertilization, gamete intra-Fallopian transfer and tubal embryo stage transfer

A comparison of the relative efficacy of in-vitro fertilizationwith uterine embryo transfer (TVF), tubal embryo stage transfer(TEST) and gamete intra-Fallopian transfer (GIFT) was performedin infertile patients with patent Fallopian tubes. A total of150 couples with unexplained infertility, peritoneal endometriosisor reduced semen quality were included in the study. The threegroups were comparable with regard to age distribution, indications,semen parameters, stimulation regimens, response to stimulationand numbers of oocytes retrieved. In the IVF and TEST groupsthere was no cleavage in 24% and a cleavage rate of only 47.6%.The highest cleavage rate was obtained in the endometriosispatients. The pregnancy rate was highest in the two groups inwhich in-vitro fertilization was performed, IVF = 45.7%, TEST= 37.9%, GIFT = 26.2%. To obtain one live intrauterine fetus,more oocytes had to be transferred in the GIFT group comparedtothe number of embryos in the IVF group, 14.4 versus 6.2, P <0.05. Due to a high success rate of IVF but at the same timea high frequency of no cleavage in cases of unexplained infertilityor male subfertility, we recommend FVF as the primary procedurein infertile couples with patent Fallopian tubes.     

Low proportions of sperm can bind to the zona pellucida of human oocytes

BACKGROUND: Sperm binding to the zona pellucida (ZP) is required for human fertilization. Under experimental conditions not limited by ZP binding sites, the cumulative numbers of sperm binding tightly to the ZP will asymptote with time to the total number of sperm in the insemination medium capable of binding. METHODS: Numbers of ZP-bound sperm were counted after groups of 10 oocytes were incubated with 2x10(4) motile sperm in 20 micro l droplets. The time-course of sperm binding was measured in three consecutive 2 h incubation periods using fresh oocytes for each period (n = 12). Using the kinetic theory of gases to model sperm-oocyte collision rates, the time-course results were extrapolated to give the total proportion of motile sperm capable of binding to the ZP. ZP binding of sperm after 4 h incubation was studied in 20 fertile and 20 normozoospermic subfertile men. RESULTS: The percentage of motile sperm capable of binding was for fertile men: mean 14% (range 8-25) and for the subfertile: 4.3% (range 0.1-13, P < 0.001). Sperm morphology correlated with the proportion of ZP-bound sperm. CONCLUSIONS: More than 75% of motile sperm from fertile men have no ability to bind to the ZP. This finding has important implications for improvement of semen analysis.