Defining bioassay conditions to evaluate sperm/zona interaction: Inhibition of zona binding mediated by solubilized human zona pellucida

Objective: Our goal was to study the influence of solubilized human zonae pellucidae on zona binding potential and acrosome reaction. Materials and Methods: Zona pellucida (ZP) solutions were prepared by dissolving zona in acidic buffer, NaH₂PO₄ (pH 2.5), to obtain 0.1 and 0.5 zona pellucida/µl. Zona binding capacity was evaluated by the addition of oocytes (10-fold) to sperm/zona pellucida solution droplets. The number of sperm bound to each oocyte was recorded. Zona pellucida-mediated acrosome activity was evaluated after 60 min of coincubation of sperm and 0.5 ZP/µl. Results: The mean (±SE) number of sperm bound for control, 0.1 ZP/µl, and 0.5 ZP/µl was 181.2±12, 79.6±5, and 38.8±3, respectively. Zona pellucida-exposed sperm populations showed significant more acrosome-reacted sperm compared to control sperm, namely, 78 versus 32%, respectively (P=0.001). Conclusions: The observed zona binding inhibition might be ascribed to zona receptor blocking on the sperm surface or the inability of acrosome-reacted sperm to bind to the zona pellucida.     

Hemizona assay: Use of fresh versus salt-stored human oocytes to evaluate sperm binding potential to the zona pellucida

Salt-stored human oocytes (pH 7.0) showed sperm binding ability equal to that of fresh, living oocytes under hemizona assay (HZA) conditions.     

Enzymatic characterization of zona pellucida hardening in human eggs and embryos

Purpose To characterize possible hardening of the human zona pellucida (ZP) and evaluate the effect of culture duration, patient age, and ZP thickness, ZP of unfertilized eggs (experiment 1, n =367; experiment 2, n =174) and abnormal embryos (experiment 1, n=52) were randomly designated for -chymotrypsin treatment after 0, 24, 48, 72, 96, 120 h (experiment 1) and 48 h, 72 h, and 1 week (experiment 2) of in vitroculture in HTF medium supplemented with 0.5% human serum albumin. Mean ZP thickness was predetermined in experiment 2.Methods The dispersion of the ZP glycoproteins was assessed, and the duration of time for complete digestion was recorded as an index of ZP hardness.Results In experiment 1, enzyme digestion duration increased (P<0.05) in the first 24 h in vitrofrom 18.0±2.0 to 34.6±2.5 min, and tended to decrease over the next 4 days in culture (25.2±1.3, 29.4±0.9, 27.3±0.6, 26.6+1.1, and 20.7±1.5 min on Day 2– 6 ZP, respectively). Zona hardening of fertilized eggs was revealed by a longer (P<0.01) digestion time (32.2±1.8 vs 25.8±0.6 min).Conclusions There were significant patient-to-patient variations (16.4±0.7 to 39.6±2.2 min); however, age was not correlated to enzyme digestion duration. In experiment 2 we determined that ZP thickness (range 8.4–21.6 m; mean 14.6±0.2 m) was not correlated (r=0.09) to the digestion interval (mean 24.3 ± 0.8 min). Based on our enzymatic ZP digestion measurements, it is apparent that spontaneous zona hardening does occur within 24 h of in vitroculture, similar to levels achieved postfertilization. The data do not support, however, the concept that additional, abnormal hardening of the ZP occurs during extended culturing.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

Production of monoclonal antibodies to porcine zona pellucida and their inhibition of sperm penetration through human zona pellucida in vitro

Two hybridoma cell lines producing murine monoclonal antibodies to antigens common to the zona pellucida (ZP) of pigs and humans were obtained by immunization of mice with solubilized porcine zona antigen. Indirect immunofluorescence tests showed that both these monoclonal antibodies stained the entire layer of porcine ZP but stained different regions of human ZP, one staining the entire layer and the other only the outer surface. At high concentrations, these two monoclonal antibodies directed against antigens common to porcine and human ZP prevented sperm binding and penetration into human ZP in vitro, whereas a monoclonal antibody directed against an antigen restricted to porcine ZP did not have these inhibitory effects. It is concluded that human and porcine ZP share at least two antigens with different locations in the ZP, and that these influence or are essential for interaction of human sperm with the ZP. These results provide a rationale for using porcine ZP clinically as a vaccine for human immunocontraception.     

Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation rate

Two hundred eighteen consenting patients entered a randomized study of the application of chemical zona pellucida thinning on their day 3 embryos, prior to uterine transfer. Of those control patients (n =108), whose embryos remained unmanipulated, 40 (37.0%) have ongoing/delivered pregnancies, while in the experimental group (n =110), whose embryos had their zonae pellucidae chemically thinned, there are 49 patients (44.6%) who have ongoing/delivered pregnancies. Although this difference is not significant, clearly the application of this micromanipulative intervention has not been detrimental, and this bodes well for routine application of embryonic micromanipulation procedures in general. Certain patient subgroups were studied including older women, those with elevated basal follicle stimulating hormone levels, patients with embryos of differing zona thickness, and patients with embryos of differing uniformity of zona thickness. No significant influence of chemical removal of the outside of the zona on the implantation rate of embryos in any of these subgroups was observed other than a marginally significant (P =0.095) improvement of implantation of embryos with less than 4.0 µm variation in zona thickness when chemical zona thinning was applied. Failure of chemical zona thinning to enhance human embryo implantation significantly, compared to assisted hatching by complete zona drilling, strongly suggests that the bilayered human zona pellucida needs to be fully breached, unlike that of the mouse.     

Mechanical zona pellucida removal of vitrified-warmed human blastocysts does not affect the clinical outcome

Research QuestionDoes complete mechanical removal of the zona pellucida modify the outcome of transfer of vitrified–warmed human blastocysts?DesignIn a prospective randomized controlled study, 419 couples were allocated to either zona pellucida-free (n = 209) or zona intact (n = 210) vitrified–warmed embryo transfer. Main outcome measures included clinical pregnancy, implantation and ongoing pregnancy rates.ResultsTransfer of zona pellucida-free blastocysts resulted in clinical pregnancy, implantation and ongoing pregnancy rates (35,9%, 33,9% and 32,1%, respectively), similar to those achieved with zona intact control embryos (39%, 36,4% and 33,1%, respectively).ConclusionTotal mechanical removal of the zona pellucida did not affect the tested parameters of clinical outcomes.     

Analysis of the human zona pellucida during culture: Correlation with diagnosis and the preovulatory hormonal environment

Purpose: The objective of this study was to analyze sequentially the human zona pellucida changes in an in vitro fertilization program as it relates to several variables. Methods: The zona pellucida thickness was measured daily in zygotes and cleavage-stage embryos on a Nikon inverted microscope equipped with Hoffman modulation contrast optics, using an ocular micrometer. A total of 512 embryos from 96 patients was evaluated. Results: There was a highly significant direct correlation between zona thickness and preovulatory estradiol and basal day 3 FSH levels (P < 0.02 andP < 0.0006, respectively). This relationship showed a rapid reversal following 48 hr of culture; embryos from patients with the highest FSH levels had thinner zonae prior to transfer (P < 0.0007). The zonae from patients with unexplained infertility were thicker (19.4 ± 2.7 μm) than those from patients with endometriosis (17.7 ± 2.2 μm), tubal (17.5 ± 2.4 μm), or male-factor infertility (16.4 ± 2.7 μm) (P < 0.0001) on the first day of culture. Conclusions: We hypothesize that the thickness of the human zona pellucida is influenced by the preovulatory hormonal environment and diagnosis. These factors should be considered as part of the embryo quality evaluation prior to transfer or when assessing the possibility of using assisted hatching. More studies are needed to understand the factors regulating the thickness of the human zona pellucida.     

Sequence variations in human ZP genes as potential modifiers of zona pellucida architecture

To examine putative associations between zona pellucida (ZP) anomalies and sequence variations in genes expressing structural ZP glycoprotein components, sequence data of 31 volunteers participating in IVF treatments were obtained and analyzed together with morphologic data of the respective oocytes. Our results suggest that some of the most frequent zona anomalies may be at least partly explained by sequence variations in genes expressing the four human ZP proteins, especially those in ZP2 and ZP3.     

Infertility and Immunocontraception based on zona pellucida

The zona pellucida (ZP) is an extracellular matrix surrounding ovarian oocytes, ovulated eggs and preimplantation embryos. It plays several important roles at different stages of reproduction. Its constituent glycoproteins are expressed specifically in the ovary. It is thus possible to produce autoantibodies to ZP proteins that interfere with reproductive functions including folliculogenesis, fertilization and implantation. First, this article describes the history of anti-ZP antibodies detected in women with idiopathic infertility. Second, the current relationship between anti-ZP antibodies and infertility is discussed in relation to assisted reproductive medicine. Third, we introduce the latest studies of animal experiments involving the ZP. Finally, immunocontraceptive vaccine development using various ZP antigens is reviewed.     

Improved implantation rate after chemical removal of the zona pellucida

OBJECTIVE: To evaluate the implantation rate achieved after chemical removal of the zona pellucida from day 5 human in vitro-derived embryos. DESIGN: Prospective, randomized, controlled study. SETTING: A tertiary care infertility clinic. PATIENT(S): Two hundred fifty-seven patients undergoing IVF with transfer of morulas or blastocysts on day 5 after oocyte retrieval. All patients had had at least two previous implantation failures. INTERVENTION(S): Chemical removal of zona pellucida by using acidic Tyrode's solution vs. no removal (controls). MAIN OUTCOME MEASURES: Clinical pregnancy rate and implantation rate per transfer. RESULT(S): Embryos without zona pellucida implanted at nearly twice the rate of control embryos (15.7% vs. 27.5%). The pregnancy rate was also significantly higher in the zona pellucida-free group than the control group (31.0% vs. 46.1%). Removal of zona pellucida was most effective in embryos with delayed development, which reached the morula or early cavitating stage on day 5 of in vitro culture (implantation rate, 12.1% vs. 25.7%). CONCLUSION(S): Chemical removal of zona pellucida from day 5 in vitro cultured human embryos is an effective and safe method of significantly improving the implantation rate, especially of embryos with delayed development.     

Effects of aging on the zona pellucida surface of mouse oocytes

In a scanning microscopy study of mouse oocytes from immature, young, and aged females and of oocytes agedin vivo orin vitro, we have observed four types of zona pellucida, which we classify as types A, B, C, and D. Oocyte aging gives rise to a significant increase in predegenerative (type C) oocytes; this type of zona pellucida surface could result from a zona hardening effect and decrease the rate of fertilization of the oocytes affected.     

Relationship between zona pellucida-induced acrosome reaction,sperm morphology,sperm-zona pellucida binding,and in vitro fertilization

OBJECTIVE: To evaluate the possible relationships between sperm morphology, acrosome responsiveness to solubilized human zona pellucida, and sperm-zona binding potential among [1] consecutive andrology referrals and [2] randomly selected in vitro fertilization (IVF) cases. DESIGN: Prospective analytical study. SETTING: Academic training hospital.Randomly selected couples consulting for infertility. INTERVENTION(S): Acrosome reaction response to solubilized human zona pellucida was recorded. MAIN OUTCOME MEASURE(S): We determined the difference in the percentage of sperm that acrosome reacted after exposure to solubilized zona pellucida and spontaneous acrosome reaction. The results were expressed as percentage zona induced acrosome reaction (ZIAR). RESULT(S): Data were analyzed using correlation coefficients (r) and receiver operator characteristics (ROC curve analyses). The ROC curve analyses indicated ZIAR to be a sensitive indicator for fertilization failure during IVF therapy, with sensitivity and specificity of 81% and 75%, respectively. For andrology referrals, a positive and statistically significant correlation existed between ZIAR data and sperm morphology (r = 0.65) and sperm-zona binding (r = 0.57). CONCLUSION(S): ZIAR results provide further information regarding dysfunctional sperm and can be used as an additional diagnostic test. Our results predicted fertilization failure during IVF treatment.     

Genetic and physiological study of morphologically abnormal human zona pellucida

Objective

To investigate genetic, molecular and functional aspects of human zona pellucida (ZP) in oocytes with an abnormal appearance.

Study design

The study included three women with unexplained infertility whose oocytes had an abnormal ZP appearance and the mother and fertile sister of one of them. The coding exons and their flanking intron regions of the four ZP genes and the regulatory element for the ZP3 gene were sequenced. Immunofluorescence staining of discarded oocytes using monoclonal antibodies against recombinant human ZP glycoproteins and a hemizona assay were performed.

Results

No new mutations were observed in the ZP1 (12 exons), ZP2 (19 exons), ZP3 (9 exons), ZP4 (12 exons) genes or in the ZP3 regulatory element of the three studied women. Sequencing of the genes revealed eight synonymous and non-synonymous reported polymorphisms only in ZP1, ZP2 and ZP3. Immunofluorescence staining of the discarded oocytes of two women showed clear and strong staining of the ZP1, ZP2 and ZP4 proteins, but weak staining of the ZP3 protein, although their ZP displayed normal sperm binding ability in the hemizona assay. Intracytoplasmic sperm injection yielded good pregnancy outcomes, even though few injected oocytes developed normally up to day 3.

Conclusions

The abnormal oocyte ZP appearance in the three study women may not have been due to the genetic changes in the ZP genes. Moreover, sperm binding was normal despite low ZP3 staining observed, suggesting that ZP3 profile may play a subordinate role in the reported cases. Our findings support previous studies which claim that abnormal oocyte morphology is not associated with a decrease in fertilization rates or birth outcomes in couples undergoing intracytoplasmic sperm injection.     

A comparison of the effects of different degrees of zona pellucida damage followed by cryopreservation on the postthaw development of mouse embryos

Purpose: The totally intact zona pellucida is not essential for the development of embryos. It is still unclear how much effect the degree of damages to the zona pellucida will have on the developmental potential of postthaw embryos after cryopreservation. We compared the developmental potential of cryopreserved mouse embryos after induction of two degrees of mechanical damage to the zonae pellucidae by micromanipulation. Methods: In experiment I, the development of 124 cryopreserved ICR mouse embryos to the blastocyst stage after zona pellucida penetration of two-cell embryos as in the procedures of subzonal sperm insertion (SUZI) was compared with the development of zona-intact cryopreserved embryos. In experiment II, the zonae pellucidae of 93 two-cell mouse embryos were dissected as in the procedures of partial zonal dissection (PZD), following which the embryos were frozen. This postthaw development was also compared with that of zona-intact two-cell cryopreserved embryos. All the embryos were thawed and cultured to the blastocyst stage. Additional controls were provided by culturing zonaintact and zona-penetrated or zona-dissected embryos without cryopreservation. Results: The development of unfrozen mouse embryos was not affected by either zona penetration (P=0.433) or zona dissection (P=0.659). The developmental potential of cryopreserved mouse embryos was significantly affected after zona dissection (blastocyst rate, 31% ZD vs 72%, control; P<0.001) but not after zona penetration (blastocyst rate, 59% ZP vs 64% control; P=0.441). Conclusions: The quality of cryopreserved embryos was affected by a large hole on the zona pellucida created by zona dissection but not by simple zona penetration.     

胚胎透明带厚度与妊娠结局相关性的研究

目的:探讨新鲜胚胎移植前透明带厚度与妊娠结局的相关性。方法:以695个移植前新鲜胚胎为研究对象,按其透明带厚度分为A组(<15.00μm)、B组(≥15.00μm),比较胚胎种植率及妊娠率的差异。再按患者年龄分为a组(≥36岁)、b组(<36岁),分析年龄与透明带的关系及胚胎种植率、妊娠率的差异。结果:(1)A、B两组患者年龄、不孕年限、移植胚胎评分及移植胚胎数差异无统计学意义(P>0.05),两组患者的胚胎种植率、妊娠率差异有统计学意义(P<0.05),当胚胎透明带≥15.00μm时,其种植率及妊娠率明显下降(P<0.05);(2)a、b两组患者的透明带厚度(13.82±2.31 vs 13.66±2.62μm)差异无统计学意义(P>0.05),两组患者的胚胎种植率(11.3% vs 23.1%)、妊娠率(19.4% vs 56.7%)差异有统计学意义(P<0.05),当患者≥36岁时,胚胎种植率及妊娠率明显下降。结论:胚胎透明带厚度与妊娠结局直接相关。