Zona Pellucida Thinning Research Articles

Impact of the size of zona pellucida thinning area on vitrified-warmed cleavage-stage embryo transfers: a prospective, randomized study

The aim of this study was to determine if the size of zona pellucida thinning area by laser assisted hatching could affect the rates of pregnancy and implantation for vitrified-warmed embryo transfers at the cleavage-stage. A total of 120 vitrified-warmed cleavage-stage embryo transfers were randomly assigned to either quarter or half of zona pellucida thinning group. The rates of clinical pregnancy (46.7 versus 25.0%) and implantation (32.0 versus 16.2%) were significantly greater in the half thinning group than in the quarter thinning group (P = 0.0218 and P = 0.0090, respectively). The results of this investigation show that, in vitrified-warmed embryo transfers at the cleavage-stage, the size of zona pellucida thinning area by laser assisted hatching impacts the rate of clinical pregnancy and implantation and that half of zona pellucida thinning significantly increases both of these results compared with quarter of zona pellucida thinning.

Laser zona thinning in women aged ≤37 years: a randomized study

The objective of this prospective randomized double-blind clinical trial was to evaluate whether laser zona pellucida thinning of human embryos improves clinical outcomes in women <or=37 years old undergoing IVF-ET treatment. The study did not reveal any significant beneficial effect of laser zona thinning on clinical pregnancy (16 out of 45 vs. 18 out of 39) and live birth rates (13 out of 45 vs. 16 out of 39) between the laser-treated and nontreated groups of patients, although there was a trend toward an increased incidence of dichorionic multiple pregnancies in the study group compared with the control group (7 out of 13 vs. 4 out of 16).

Chemical zona pellucida thinning with acidified Tyrode’s solution: comparison between partial and circumferential techniques

To compare effectiveness of two different chemical zona thinning techniques. We studied 163 patients who had experienced IVF or ICSI failures in two or more cycles. Patients were assigned to one of three groups: zona intact (n=72), partial thinning (n=59), or circumferential thinning (n=73). Before transfer, the zonae pellucidae of embryos were thinned partially or circumferentially using acidified Tyrode's solution. Implantation rates were 8.9% in the intact zona group, 17.6% in the partial thinning group, and 11.9% in the circumferential thinning group: respective clinical pregnancy rates were 16.7% (12/72), 32.2% (19/59), and 27.4% (20/73). Both rates were significantly higher in the partial thinning group than the intact zona group. For circumferential thinning versus zona intact groups, differences fell short of significance. Following embryo transfer failure, partial thinning would be recommended over circumferential thinning for successful assisted hatching.

Randomized double-blind comparison of laser zona pellucida thinning and breaching in frozen–thawed embryo transfer at the cleavage stage

To compare the implantation and ongoing pregnancy rates of frozen-thawed embryo transfer (FET) using laser thinning with those of laser breaching of the zona pellucida (ZP). Double-blind randomized study. A tertiary assisted reproduction unit. Infertile patients undergoing FET cycles. In the thinning group, more than a quarter of the ZP's outer half-diameter was removed by using a 1,480-nm noncontact laser, whereas a hole about 30 microm in size was created in the ZP in the breaching group. Implantation and ongoing pregnancy rates. On the day of the FET, 180 patients were randomly divided into the thinning and breaching groups according to a computer-generated randomization list that was placed in sealed envelopes. The two groups were comparable in terms of demographic characteristics, ovarian response of the stimulated cycle, and quality of fresh and frozen-thawed embryos. The implantation and ongoing pregnancy rates of the thinning group were significantly higher than the corresponding rates of the breaching group but were similar to those of patients without ZP thinning or breaching. Laser ZP thinning is associated with significantly higher implantation and ongoing pregnancy rates in FET cycles compared with the case of laser ZP breaching.

A randomized double-blind controlled study on the efficacy of laser zona pellucida thinning on live birth rates in cases of advanced female age

It is conceivable that defective embryo hatching plays a part in the mechanisms involved in the decrease of embryo implantation rates with advancing age. In an effort to test this hypothesis, we tested the effectiveness of assisted hatching (AH) in women > or =37 years of age. We prospectively studied 103 IVF-embryo transfer patients undergoing 103 embryo transfers. All of them were > or =37 years of age and had <3 previous IVF-embryo transfer attempts. Laser-AH of transferred embryos was either performed (AH group, n = 49) or not (control group, n = 54) according to randomized and double-blind methodology. Primary outcome was live birth rate. Population characteristics were comparable in AH and control groups as well as the mean number of embryos transferred (2.7 +/- 0.6 versus 2.7 +/- 0.6) and the prevalence of top quality embryos transferred (65 versus 59%, respectively). We failed to find any statistically significant difference between AH and control groups with regard to implantation (16.1 versus 16.7%, respectively) and live birth rates (22.4 versus 29.6%, respectively). The present study indicates that AH does not improve IVF-embryo transfer outcome in women aged > or =37 years.

A Randomized Blinded Controlled Trial on the Effects of Laser Assisted Hatching on the Outcome of IVF Cycles in Women Under 38 Years of Age

Some studies have shown a beneficial effect of assisted hatching in older women undergoing IVF. There is almost no data evaluating the use of this technique in younger women. Zona thickness has been used as a criteria by some groups for the use of assisted hatching in all age groups. However, this measurement does not assess zona ‘hardness’. The objective of this study was to evaluate the effect of laser zona pellucida thinning on pregnancy outcome in an unselected group of women under 38 years of age undergoing IVF/ICSI procedures using a randomized blinded controlled trial.

Monochorionic triplet gestation after in vitro fertilization using donor oocytes: Case report and review

To report on a patient with a monochorionic triamnionic triplet pregnancy after IVF with donor oocytes. Case report. Academic tertiary care hospital. A 42-year-old woman who underwent IVF with donor oocytes. After failed IVF attempts, the patient chose to undergo treatment with donor oocytes. Her 23-year-old oocyte donor underwent standard controlled ovarian hyperstimulation. Retrieved oocytes were fertilized in vitro, and two embryos were transferred at the blastocyst stage. Intrauterine pregnancy with single gestational sac and three fetal poles with cardiac activity. After extensive counseling with perinatologists about pregnancy complications, the patient elected to terminate at 10 weeks of gestation. Several processes have been suggested to explain the increase in monozygotic twinning after IVF. These factors include advanced maternal age, superovulation, manipulation of the zona pellucida, and prolonged culture. It is possible that other factors may also play a role, especially in high-order monozygotic multiple pregnancies. All patients should be informed of the potential risk of a high-order multiple pregnancy after IVF, even when only one or two embryos are transferred.

Developmental and molecular aberrations associated with deterioration of oogenesis during complete or partial follicle-stimulating hormone receptor deficiency in mice.

Targeted disruption of the mouse FSH receptor gene (FSH-R) that mediates the action of the FSH results in a gene dose-related ovarian phenotype in the developing as well as the adult animal. While null females (FORKO) are sterile, the haplo-insufficient mice experience early reproductive senescence. The purpose of this study was to first record changes in oocyte development in the null FORKO and haplo-insufficient mice. Oocyte growth is significantly retarded in the null mutants with thinner zona pellucida in preantral follicles, but thicker zona pellucida in secondary follicles. This morphometric change indicates developmental aberrations in coordination of the germ cell (oocyte) and the somatic granulosa cell (GC) compartments. Markers for primordial germ cell proliferation and oocyte growth, such as the c-Kit/Kit-ligand and bone morphogenetic protein-15 (BMP-15) were downregulated in both null and +/- ovaries, suggesting disrupted communication between oocyte and GCs. Extensive changes in the expression of other oocyte-specific gene products like the zona pellucida glycoproteins (zona pellucida A, B, and C) indicate major alteration in the extracellular matrix surrounding the germ cells. This led to leaky germ cells that allowed infiltration of somatic cells. These results show that the loss of FSH-R signaling alters the follicular environment, where oocyte-granulosa interactions are perturbed, creating an out-of-phase germ cell and somatic cell development. We believe that these data provide an experimental paradigm to explore the mechanisms responsible for preserving the structural integrity and quality of oocytes at different ages.

Prospective Randomized Comparison of Two Forms of Assisted Hatching Using Laser in Thawed Embryos

Objectives: The process of embryo cryopreservation can affect the thickness and/or consistency of the zona pellucida (ZP), interfering with the embryo’s ability to perform the natural hatching process and consequently reducing embryo implantation rates (Cohen, 1999). In the present study, the efficacy of two forms of assisted hatching (AH) with laser applied to thawed embryos was compared: ZP thinning (TH) versus total ZP opening (TO). Design: A prospective and randomized comparison of two forms of assisted hatching. Materials and Methods: The thawed embryos of 117 patients were submitted to AH in a prospective and randomized manner and assigned to two groups: group I (TH), and group II (TO). In the AH procedure we used non-contact laser with a length of 1.48 μm (Fertilaser). Before manipulation, ZP thickness was measured at 4 different points using a scale in the microscope eyepiece. In group I, TH was performed in a single position reaching 60 to 80% of the ZP thickness using 1 to 2 irradiations of 10 ms. In group II, TO was performed using 3 irradiations of 10 ms. Embryo transfer was performed soon after the AH procedure. Results: Table I. ZP thinning (TH) versus total ZP opening (TO). Tabled 1Group I (TH)Group II (TO)pNo. of patients6156Age32.3 ± 3.831.6 ± 4.50.36Embryos transferred2.97 ± 0.92.96 ± 0.90.95ZP thickness (μm)17.3 ± 1.918 ± 2.60.22Pregnancy rate per transfer21.3%25%0.66Implantation rate11.6%13.2%0.74 Open table in a new tab Conclusion: There was no significant difference in AH efficacy when the procedure was executed with TH or TO of the ZP in thawed embryos. However, non-total opening (thinning) of the ZP may prevent the risk of early loss of blastomeres, embryo infections and the prophylactic use of antibiotics.

Intactness of zona pellucida does not affect the secretion of a trypsin-like protease from mouse blastocyst.

Assisted hatching (AH), which is known to improve the hatching potential of mammalian embryos, has been used to increase the pregnancy rate in in vitro fertilization cycles. However, the effect of AH on a trypsin-like protease, which is known to be associated with the hatching process, has not been studied. In this study, we evaluate whether the intactness of zona pellucida affects the secretion of a trypsin-like protease from mouse blastocyst. Four- to 8-cell stage mouse embryos were collected at 66- to 68 hr after hCG injection and divided into 3 groups according to the manipulation of zona pellucida. The groups are no treatment (control), drilling of zona pellucida (ZD) and thinning of zona pellucida (ZT). The activity of a trypsin-like protease, blastocyst development and hatching rate were compared among the three groups at 110 and 135 hr after hCG injection, respectively. The protease activity and blastocyst development were not significantly different among control, ZD and ZT groups at 110 and 135 hr after hCG injection, respectively. However, the hatching rate of ZD and ZT groups was significantly higher than that of control group at each time, respectively (p<0.001). Even in the zona pellucida removed embryos, the protease activity did not differ from the control group. In conclusion, the secretion of a trypsin-like protease from mouse blastocyst does not seem to be affected by the intactness of zona pellucida.

The hyaluronic acid receptor (CD44) is expressed in bovine oocytes and preimplantational stage embryos

A study involving 32 sexual mature females was conducted to characterize ovulation, fertilization and early embryonic development in vivo in raccoon dogs. Oocytes and embryos were collected from the oviducts and uteri, evaluated by stereomicroscopy. Ovulation occurred 25–32 h after a female first accepted mounting, regardless of copulation, when the females were paired with a male in the same cage. Ovulated oocytes were at the primary stage. The number of ovulated eggs in females with or without mating was 9.96 ± 2.65 and 9.00 ± 1.92, respectively. Embryos at 2–4 cell, 8–16 cell, morula, blastocyst, and hatched blastocyst stage were observed at 29–73, 48–100, 98–126, 169–198 and 217–268 h after first mating, respectively. Embryos were located in the oviduct prior to 4-cell stage and moved into the uterus after 16-cell stage. Embryos at different stages were often obtained from the same female. During the zygote underwent a series of cleavage divisions, the diameter of the embryo cell mass continuously increased through the 2-cell and 4-cell stage, then started to decrease and was the minimum size at the morula stage. At the blastocyst stage, embryos increased in volume, and finally developed into a hatching blastocyst with a thinner zona pellucida. This is the first full report of preimplantation embryonic development in the raccoon dog, which will facilitate the application of advanced assisted reproductive technology in canine species.

Relationship of subnormal semen parameters and subsequent zona pellucida thinning following in vitro fertilization.

Infertility due to male factor has been associated with reduced implantation rates despite normal fertilization. The mechanism responsible for lower pregnancy rates is not known. One cause of failure to achieve pregnancy despite transfer of embryos is impairment of zona pellucida (ZP) thinning, which inhibits the embryo from hatching despite initial cell cleavage. This study was designed to evaluate whether there is an association between the ability of the ZP to thin, as measured by ZP thickness on day of transfer, and subnormal semen parameters. Significant differences in ZP thickness (p < 0.05, Kruskal-Wallis) were noted according to median concentration of sperm, total motile sperm, and motile density in that these values were the highest in the group of embryos with the thinnest zona pellucida and lowest in the group with the thickest zona pellucida. Thus, some factor may be missing from sperm from subnormal specimens that normally assists in zona thinning.

Ovarian development in mice bearing homozygous or heterozygous null mutations in zona pellucida glycoprotein gene mZP3.

The plasma membrane of all mammalian eggs is surrounded by a thick extracellular coat, the zona pellucida (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZP1-3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the mZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous (mZP3 +/-) or homozygous (mZP3-/-) for the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female mZP3 +/- mice are as fertile as wild-type animals, but their eggs have a thin ZP (approximately 2.7 microns thick) as compared to the ZP (approximately 6.2 microns thick) of eggs from wild-type animals. On the other hand, female mZP3-/- mice are infertile and their eggs lack a ZP. The infertility apparently is due to the lack of a sufficient number of eggs in oviducts of superovulated mZP3-/- females. Light micrographs reveal that development of ovarian follicles is often retarded in mZP3-/- mice as compared to wild-type animals. This is manifested as reduced ovarian weights, reduced numbers of Graafian follicles, and reduced numbers of fully-grown oocytes in mZP3-/- females. It seems likely that the pleiotropic effects of the homozygous null mutation on ovarian development may be due, at least in part, to disruption of intercellular communication between growing oocytes and their surrounding follicle cells.

The effect of the oviduct, uterine, and in vitro environments on zona thinning in the mouse embryo

To evaluate the impact of the oviduct, uterine, and in vitro environments on zona pellucida thinning in the mouse embryo. Female mice were stimulated with pregnant mare serum gonadotropin and mated and hCG injection. Unilateral oviduct ligation was performed on day 2 of gestation using the dorsal approach. The mice were divided into equal groups and killed on days 2, 3, 4, 5, and 10 of gestation. In vitro incubated embryos served as controls. Average daily zona thickness measurements were subjected to analysis of variance and paired Student's t-test. The laboratory of the assisted reproductive program of Rush University Medical Center. Progressive daily decrease in average zona thickness. Zona measurements of embryos flushed out of uterine horns, ligated oviducts, and in vitro incubation demonstrated statistically significant decreases in zona thickness, from 9.6 +/- 1.6 microns (day 3) to 6.0 +/- 0.8 microns (day 5), from 11.6 +/- 2.2 microns (day 2) to 6.0 +/- 1.6 microns (day 5), and from 11.1 +/- 2.0 microns (day 2) to 6.0 +/- 1.6 microns (day 5), respectively. There were no differences in average zona thickness for embryos in the same cell stage and same protocol day in all three locations. Zona thinning seems to be induced primarily by the dividing embryo before implantation. A substantial tubal and uterine contribution to zona thinning was not detected in this mouse embryo model.

Mutant female mice carrying a single mZP3 allele produce eggs with a thin zona pellucida, but reproduce normally.

The mouse egg zona pellucida (ZP) is composed of three glycoproteins, called mZP1-3. Disruption of the mZP3 gene by targeted mutagenesis yields mice that are homozygous (mZP3-/-) for the null mutation; although the mutant mice are viable, females are infertile and their eggs lack a ZP. On the other hand, females heterozygous (mZP3+/-) for the mutation are fertile and their eggs have a ZP. Here, we examined fully grown oocytes from mZP3+/- females and found that, although they have a ZP, it is less than half the width (approximately 2.7 microns; volume, approximately 56 pl) of the ZP of oocytes from wild-type (mZP3+/+) mice (approximately 6.2 microns; volume, approximately 145 pl). Oocyte ZP were purified from ovarian homogenates by gradient centrifugation. Immunostaining of purified ZP on Western gels permitted an estimate to be made of the relative amounts of mZP3 and mZP2 present in the ZP of oocytes from mZP3+/+ and mZP3+/- mice. We found that the ZP from mZP3+/- mice contained, on average, 55 +/- 15% of the mZP3 and 44 +/- 8% of the mZP2 present in the ZP of mZP3+/+ mice; a result quite consistent with the observed widths and calculated volumes of the ZP. Despite the presence of a relatively thin ZP surrounding their eggs, reproduction of female mZP3+/- mice was indistinguishable from female mZP3+/+ mice. These results strongly suggest that, when a single mZP3 allele is present, approximately half the wild-type amount of mZP3 and approximately half the wild-type amount of mZP2 is assembled into a ZP. While this produces a relatively thin ZP, it apparently has no affect on reproduction. Furthermore, these results are consistent with the current molecular model for ZP structure.

Transcription in in vivo produced bovine zygotes and embryos

A study involving 32 sexual mature females was conducted to characterize ovulation, fertilization and early embryonic development in vivo in raccoon dogs. Oocytes and embryos were collected from the oviducts and uteri, evaluated by stereomicroscopy. Ovulation occurred 25–32 h after a female first accepted mounting, regardless of copulation, when the females were paired with a male in the same cage. Ovulated oocytes were at the primary stage. The number of ovulated eggs in females with or without mating was 9.96 ± 2.65 and 9.00 ± 1.92, respectively. Embryos at 2–4 cell, 8–16 cell, morula, blastocyst, and hatched blastocyst stage were observed at 29–73, 48–100, 98–126, 169–198 and 217–268 h after first mating, respectively. Embryos were located in the oviduct prior to 4-cell stage and moved into the uterus after 16-cell stage. Embryos at different stages were often obtained from the same female. During the zygote underwent a series of cleavage divisions, the diameter of the embryo cell mass continuously increased through the 2-cell and 4-cell stage, then started to decrease and was the minimum size at the morula stage. At the blastocyst stage, embryos increased in volume, and finally developed into a hatching blastocyst with a thinner zona pellucida. This is the first full report of preimplantation embryonic development in the raccoon dog, which will facilitate the application of advanced assisted reproductive technology in canine species.

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated ( 4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated ( 6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.

The oocyte of a new world marsupial, Monodelphis domestica: structure, formation, and function of the enveloping mucoid layer.

Ovulated oocytes of the gray short-tailed opossum Monodelphis domestica are surrounded by a thin zona pellucida and are devoid of a cumulus oophorus. In the ampulla of the oviduct, oocytes acquire a thick mucoid layer composed of concentrically arranged fibrillar material. Exocytosis by the secretory cells of the oviductal epithelium occurs in the region of the oviduct adjacent to the egg. This suggests that the oocyte-zona-mucus layer complex may influence the oviductal epithelium to secrete. During secretion, fibrillar contents of the secretion granules appear to be transformed into membranous material which presumably becomes fibrillar again as it is incorporated into the forming mucoid layer. Spermatozoa (which are known to pair in the cauda epididymis) are found in pairs and with intact acrosomes in the mucoid layer of fertilized eggs. This suggests that spermatozoa of Mondelphis remain paired until they reach the zona pellucida and that the acrosome functions in zona binding and/or penetration.

Characterization of oestrus and timed collection of oocytes in the grey short-tailed opossum, Monodelphis domestica

A system of test-pairing was used to detect reproductive behaviour in the grey short-tailed opossum. This enabled timing and characterization of the development of pro-oestrous and oestrous behaviour, and facilitated collection of ovulated and unovulated oocytes. Oestrus was induced 8.5 days (n = 80, 95% confidence limits 7.56-9.21) after the introduction of a male. Timed examination of the ovaries by laparotomy indicated that ovulation occurred 14-16 h after the first onset of oestrous behaviour. The development of follicles was linked to pro-oestrous behaviour, and ovulation occurred in the absence of copulation. Vaginal exfoliative cytology indicated that pro-oestrous behaviour was associated with an increasing number of keratinized epithelial cells, and at the time of maximum receptivity to males, a heavy infiltration of polymorphonuclear leucocytes was seen. Oocytes were typically marsupial: large (approximately 250 microns in diameter), with a yolky vitellus and thin zona pellucida. An average of 6 oocytes were ovulated per ovary.