Zona Pellucida Changes Research Articles

Type 1 diabetes affects zona pellucida and genome methylation in oocytes and granulosa cells

Diabetes affects oocyte nuclear and cytoplasmic quality. In this study, we generated a type 1 diabetes (T1D) mouse model by STZ injection to study the effects of T1D on zona pellucida and genomic DNA methylation of oocytes and granulosa cells. T1D mice showed fewer ovulated oocytes, reduced ovarian reserve, disrupted estrus cycle, and significantly ruptured zona pellucida in 2-cell in vivo embryos compared to controls. Notably, diabetic oocytes displayed thinner zona pellucida and treatment of oocytes with high concentration glucose reduced the zona pellucida thickness. Differential methylation genes in oocytes and granulosa cells were analyzed by methylation sequencing. These genes were significantly enriched in GO terms by GO analysis, and these GO terms were involved in multiple aspects of growth and development. Most notably, the abnormal methylation genes in oocytes may be related to oocyte zona pellucida changes in diabetic mice. These findings provide novel basic data for further understanding and elucidating dysgenesis and epigenetic changes in type 1 diabetes mellitus.

Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida

Cryopreservation-induced modifications of zona pellucida (ZP) have been explored to a lesser extent compared to other oocyte compartments. Different methods have been applied to identify ZP changes, but most of them are invasive and measure only few properties of ZP.Raman microspectroscopy (RMS) is a powerful technique for studying the molecular composition of cells but to date few studies have been performed on the oocytes using this method. The aim of the present study is to investigate the structural modifications of ZP of vitrified/warmed in vitro matured ovine oocytes by means of RMS. Cumulus–oocyte complexes were recovered from the ovaries of slaughtered adult sheep, matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. ZPs of vitrified/warmed oocytes (VITRI), were exposed to vitrification solutions but not cryopreserved (CPA-exp) and untreated oocytes (CTR) were analyzed by RMS. We focused our analysis on the ZP protein and carbohydrate components by analyzing the 1230–1300cm−1 amide III region and the 1020–1140cm−1 spectral range in RMS spectra, respectively.The spectral profiles in the ranges of proteins and carbohydrates were comparable between CTR and CPA-exp ZPs, whereas VITRI ZPs showed a significantly altered protein secondary structure characterized by an increase in β-sheet content and a decrease in the α-helix content. A significant modification of the carbohydrate components was also observed. This study demonstrates that vitrification of ovine oocytes induces biochemical changes of ZP related to the secondary structure of proteins and carbohydrate residues. Cryoprotectants do not strongly alter the molecular composition of ZP which is affected mainly by cooling. Raman technology offers a powerful and non-invasive tool to assess molecular modifications induced by cryopreservation in oocytes.

Oviductal secretions: will they be key factors for the future ARTs?

A variety of evolutionary processes has led to the development of different organs to ensure that internal fertilization occur successfully. Fallopian tubes are a particularly interesting example of such organs. Some of the key events during fertilization and early embryo development occur in the oviduct. Knowledge of the different components described in the oviduct is extensive. Oviductal components include hormones, growth factors and their receptors that have important roles in the physiology of the oviduct and embryo development. Other oviductal factors protect the gamete and the embryos against oxidative stress and pathogens. Different proteins and enzymes are present in the oviductal fluid and have the ability to interact with the oocyte and the sperm before the fertilization occurs. Of special interest is the oviduct-specific glycoprotein (OVGP1), a glycoprotein that is conserved in different mammals, and its association with the zona pellucida (ZP). Interaction of the oocyte with oviductal secretions leads us to emphasize the concept of 'ZP maturation' within the oviduct. The ZP changes produced in the oviduct result in an increased efficiency of the in vitro fertilization technique in some animal models, contributing in particular to the control of polyspermy and suggesting that a similar role could be played by oviductal factors in human beings. Finally, attention should be given to the presence in the oviductal fluid of several embryotrophic factors and their importance in relation to the in vivo versus in vitro developmental ability of the embryos.

Monozygotic multiple gestation after intracytoplasmic sperm injection and preimplantation genetic diagnosis

To report a possible association between intracytoplasmic sperm injection (ICSI)-preimplantation genetic diagnosis (PGD) and monozygotic multiple gestation. Small case series. In vitro fertilization unit in an academic medical center. Three patients were treated with ICSI-PGD for sexing as well as selection against a known translocation. Transfer of day 4 embryos to the uterus. Clinical pregnancy. Two pairs of monozygotic twins and a triplet pregnancy. Repeated manipulation of the zona pellucida as well as extended embryo culture during ICSI-PGD treatments may result in monozygotic twin and triplet pregnancies.

Oviduct-specific glycoprotein and heparin modulate sperm-zona pellucida interaction during fertilization and contribute to the control of polyspermy.

Polyspermy is an important anomaly of fertilization in placental mammals, causing premature death of the embryo. It is especially frequent under in vitro conditions, complicating the successful generation of viable embryos. A block to polyspermy develops as a result of changes after sperm entry (i.e., cortical granule exocytosis). However, additional factors may play an important role in regulating polyspermy by acting on gametes before sperm-oocyte interaction. Most studies have used rodents as models, but ungulates may differ in mechanisms preventing polyspermy. We hypothesize that zona pellucida (ZP) changes during transit of the oocyte along the oviductal ampulla modulate the interaction with spermatozoa, contributing to the regulation of polyspermy. We report here that periovulatory oviductal fluid (OF) from sows and heifers increases (both, con- and heterospecifically) ZP resistance to digestion with pronase (a parameter commonly used to measure the block to polyspermy), changing from digestion times of approximately 1 min (pig) or 2 min (cattle) to 45 min (pig) or several hours (cattle). Exposure of oocytes to OF increases monospermy after in vitro fertilization in both species, and in pigs, sperm-ZP binding decreases. The resistance of OF-exposed oocytes to pronase was abolished by exposure to heparin-depleted medium; in a medium with heparin it was not altered. Proteomic analysis of the content released in the heparin-depleted medium after removal of OF-exposed oocytes allowed the isolation and identification of oviduct-specific glycoprotein. Thus, an oviduct-specific glycoprotein-heparin protein complex seems to be responsible for ZP changes in the oviduct before fertilization, affecting sperm binding and contributing to the regulation of polyspermy.

Monozygotic triplet pregnancy following egg donation and transfer of single frozen-thawed embryo

To describe a case of monozygotic triplet pregnancy following egg donation and the transfer of a single frozen-thawed embryo. Case report. District general hospital and regional department of fetal medicine in northeast England. A 38-year-old woman with a 2-year history of primary infertility due to severe endometriosis and poor ovarian reserve who conceived after egg donation and transfer of a single frozen-thawed embryo. Transfer of a single frozen-thawed embryo and delivery of three identical female infants by emergency caesarean section because of preterm labor at 32 weeks' gestation. Review of effect of assisted conception on monozygotic twinning rates. Healthy outcome for all three infants. Assisted reproductive treatments may lead to disturbances in zona pellucida architecture and an increase in monozygotic twinning rates. Couples need to be informed of this and the increased risks associated with these pregnancies before they begin with treatment.

Bypassing the effect of zona pellucida changes on embryo formation following cryopreservation of bovine oocytes

Objective: To evaluate the effects of zona pellucida changes following the cryopreservation of bovine oocytes using two different techniques. Design: Prospective randomised study. Setting: University research facility and IVF research laboratory, NURTURE, University of Nottingham, UK. Intervention: 508 bovine oocytes were collected from abattoir-derived ovaries and cryopreserved by slow freezing using standard straws and a new vitrification method using our self-constructed cryoloops. After thawing, the oocytes were inseminated by ICSI and standard IVF. Main outcome measures: Oocyte degeneration and oocyte cleavage. Results: Cleavage rates after IVF in fresh oocytes was higher (49.5%) than the cryopreservation groups (slow freezing: 15.4% and vitrification: 25.8%), whereas after ICSI, the rates were similar (fresh: 17.3%, slow freezing: 9.4%, vitrification: 16.0%). Conclusions: Zona pellucida changes after oocyte cryopreservation significantly reduce the embryo formation rates after IVF. ICSI bypasses this complication and in selected species it could be an appropriate method of insemination.

Correlation between the innermost layer of the Zona Pellucida (ZP) and age using computerized image analysis of polarized light microscope (Polscope)

The Human Zona Pellucida (ZP) is composed of three concentric layers that differ in the retardance and azimuth of ZP filaments and can be imaged by polarization microscopy. Retardance is an optical propriety of highly ordered molecules, which can be measured by polarization microscopy. Changes in the morphological characteristics of the ZP in relation to age have not yet been fully described, however previous reports suggest age effects on zona structure and function. The objective of this study was to test the hypothesis that the morphology and architecture of the ZP changes with age.

Assisted reproductive technologies and monozygous twins: implications for future study and clinical practice.

That the zona pellucida (ZP) plays a prominent role in the physiology of some human twinning is an attractive, albeit incompletely proven, medical hypothesis. Indeed, an association has been proposed between manipulation of the ZP and/or native ZP microarchitecture and monozygotic (MZ) twins. Ovulation induction also has been theoretically linked to in vivo ZP alterations facilitating MZ twin development. In vitro fertilization (IVF) relies on necessary (and, in some cases extended) embryo culture techniques potentially creating subtle ZP changes and subsequent MZ twinning. With growing experience in the assisted reproductive technologies and particularly IVF, some preliminary reports have noted an increased frequency of MZ twins after procedures that artificially breach the ZP (i.e., intracytoplasmic sperm injection [ICSI], or 'assisted hatching'). Such ZP manipulations ostensibly enhance oocyte fertilization or facilitate blastocyst hatching, thus improving pregnancy rates for couples undergoing fertility treatment. Evidence exists both to challenge and support the connection between these phenomena and MZ twins. This report outlines the fundamental embryological processes believed responsible for these conflicting observations; the current literature on the subject of human ZP micro-manipulation and MZ twins is also discussed.

Assisted reproductive technologies and monozygous twins: implications for future study and clinical practice

AbstractThat the zona pellucida (ZP) plays a prominent role in the physiology of some human twinning is an attractive, albeit incompletely proven, medical hypothesis. Indeed, an association has been proposed between manipulation of the ZP and/or native ZP microarchitecture and monozygotic (MZ) twins. Ovulation induction also has been theoretically linked to in vivo ZP alterations facilitating MZ twin development. In vitro fertilization (IVF) relies on necessary (and, in some cases extended) embryo culture techniques potentially creating subtle ZP changes and subsequent MZ twinning. With growing experience in the assisted reproductive technologies and particularly IVF, some preliminary reports have noted an increased frequency of MZ twins after procedures that artificially breach the ZP (i.e., intracytoplasmic sperm injection [ICSI], or ‘assisted hatching’). Such ZP manipulations ostensibly enhance oocyte fertilization or facilitate blastocyst hatching, thus improving pregnancy rates for couples undergoing fertility treatment. Evidence exists both to challenge and support the connection between these phenomena and MZ twins. This report outlines the fundamental embryological processes believed responsible for these conflicting observations; the current literature on the subject of human ZP micro-manipulation and MZ twins is also discussed. Twin Research (2000) 3, 217–223.

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

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. 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. There was a highly significant direct correlation between zona thickness and preovulatory estradiol and basal day 3 FSH levels (P < 0.02 and P < 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 microns) than those from patients with endometriosis (17.7 +/- 2.2 microns), tubal (17.5 +/- 2.4 microns), or male-factor infertility (16.4 +/- 2.7 microns) (P < 0.0001) on the first day of culture. 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.

Poor fertilization may be related to oocyte or zona pellucida recognition defects specific to certain hyperstimulation regimens and limited to some males but not others: a case report

To determine if the controlled ovarian hyperstimulation (COH) regimen may cause sperm to appear subfertile only to improve considerably by changing the COH protocol. Case report with retrospective review of previous fertilization rates according to COH protocol used. Fertilization and pregnancy rates (PRs) after IVF-ET. Known fertile donor sperm failed to fertilize any of 11 oocytes compared with 14 of 18 for processed retrograde ejaculate using the same oocyte pool. Retrospective analysis of other cycles for different female patients but same donor found 16.6% fertilization rate whenever luteal phase leuprolide acetate (LA)-hMG regimen was used compared with 70.6% with short-flare regimen. Some COH regimens may cause oocyte or zona pellucida changes that create recognition defects for some sperm but not others. Interestingly, the sperm with the binding defect with the luteal phase LA-hMG COH protocol exhibited good fertilization rates with oocytes prepared with the short-flare protocol and demonstrated high in vivo PRs after IUI.