Atualizado - 27/10/2003
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INDICATIONS STRUCTURE AND FUNCTION OF ZONA PELLUCIDA The Zona Pellucida is a glycoprotein layer that involves the oocyte. It is believed that this non cellular structure is produced by the oocyte and by the cells that wrap the oocyte during its follicular development. It consists of two layers: one external, thick layer which is easily dissolved during AHA; and one internal thin layer which is harder to penetrate. During “in vitro” culture, the zona pellucida becomes fragile and loses its elasticity due to a process named zona hardening. This process causes a blockage against fertilization by polyspermy, creating a mechanism to protect the pre-embryo. FUNCTIONS OF ZONA PELLUCIDA
Abnormalities in structure and function of the glycoprotein structure complex may result in the impairment of fertilization, diminished viability of the pre-embryo and a poor prognostic for implantation. By creating an artificial hole on the oocyte superficies before implantation (AHA) it appears that natural hatching can be facilitated and make it easier for the pre-embryo with low potential for implantation to be retrieved. INDICATION FOR ASSISTED HATCHING 1 - Highly thick zona pellucida
2- Abnormal form of the zona pellucida/pre-embryo Oocytes and pre-embryos showing an oval form are not rare. However, these morules’ ability to become compact and develop to an “in vitro” hatching was slower than that ones having a normal, rounded form. This irregular pre-embryo form is usually associated with abnormal zona pellucida. AHA may help to promote an appropriate cell to cell contact, compactation and hatching of these abnormal pre-embryos. 3 - Abnormal zona pellucida color Dark (brown or yellow) zona pellucida is sometimes observed in vitro. It is noted that, in some oocytes specially those post-mature ones, the zona pellucida gets darkens and deteriorate progressively in prolonged culture (personal observation). When this morphological characteristic is present, the pre-embryo is considered a candidate for AHA. 4 - Cytoplasmic fragmentation Cytoplasmic fragments are cytoplasmatic membrane components expelled from the surfaces of fertilized oocytes and blastomeres of pre-embryos. The pre-embryos having excessive fragmentation (>20%) have a lower implantation rate than those ones without the fragmentation. In addition, the number of fragments can affect compactation and subsequent development of the blastocyst. Analyzing the embryos during a period of 72 hours, it has been verified that most cytoplasmic fragments developed between the stages of pronucleus and first or second cellular division. This precocious cytoplasmic fragmentation may indicate in vitro damage or intrinsic DNA abnormalities of the embryos. Fragmentation occurring after the first cleavage tends to be less excessive and may not impair subsequent embryo development. These fragments develop short before blastomere division, as multiple small bubbles in the cell membrane. While cleavage goes on, some bubbles reincorporate to other blastomeres, while others remain actual delimitated fragments. Occasionally, individual blastomeres may break into multiple fragments, and that indicates cell death. Why do fragments occur? Many factors might be responsible. Culture conditions, cytoplasmic competence, genetic integrity, unbalanced chromosomes and alterations in the kariocinesis and cytocinesis. Fragmented pre-embryo chromosomal analyses through FISH (Fluorescent In Situ Hybridization) indicate that more than 50% of them are abnormal, which can justify their low implantation rates. However, it is interesting to remark that there is no relationship between woman’s age cytoplasmic fragmentation, as is the case for the enhanced chromosomal abnormalities in women above 40. Light fragmentation (<10%) is usually characterized by small fragments, and may include polar body components, localized in a determined area. These embryos have a high inplantation rate and AHA is not indicated. Moderate fragmentation (10 – 20%) is associated to irregular blastomere form and size. If these fragments are localized in a determined area, they may cause the death of a determined blastomere. Also, these blastomeres may present excessive fragmentation during prolonged culture. AHA and fragment removal are indicated for these pre-embryos, which possess low implantation potential. Excessive fragmentation (> 20%) is correlated to low implantation rates. Many women posses this type of fragmentation in all of her embryos. Some of them may benefit from AHA associated to fragment removal. Embryos that possess multiple big irregular fragments have extremely low implantation rates. Big fragments are difficult to distinguish, they can only be differentiated from the cleaved blastomeres by visualization of a nucleus. It is true that blastomeres become smaller and smaller after successive mitoses. Pre-embryos having those abnormal characteristics have a poor prognostic for implantation, due to their blocked citocinesis and reduced vitality in the culture. An aggressive fragment removal may be done in those cases, but results are bad. 5- Poor morphology embryos and low growth rates Pre-embryos having slow growth (< 6 cells in 72 hours) have good implantation rates after treatment with AHA, comparing to non treated embryos. One explanation is that the “hole” in the ZP may promote easier nutrient transportation, which makes the in vivo hatching bettes. Another type of pre-embryo that can benefit from AHA is the one having irregular form and size blastomeres, which means abnormal kiticynesis, citoplasm granulation and vacuolization and other abnormalities visible in the optical microscope. 6 - Patient’s age The patient’s age is clearly associated with implantation and pregnancy success. Implantation rates fall drastically in patients over 40 submitted to IVF. That is why AHA can be indicated exclusively because of age (independently of the pre-embryo shape), for patients near or over 40. With the pasing of time oocytes lose their ability to implant, due to thickening of the zona or also endogenous systemic factors, in such a way that older women’s pre-embryos need AHA independently of morphology. In Cornell’s program there is a tendency to apply AHA routinely in patients above 40 (1/3 to ¼ Cornell patients will be 40 or over), and most their embryos are manipulated before transference. The clinical pregancy/ trensference rates for these patients (confirmed by ultrasonography) exceeds 35%. OTHER INDICATIONS High FSH (> 15 mUI/ml) on day three of the cycle indicates altered follicular reserve and ovarian physiology. This fact can affect the oocyte and its ZP inside the follicule. It has been demonstrated that these patients can benefit from AHA with doubling their implantation rates when compared to patients not treated by AHA. MOTHODOLOGY Techniques involving the use of Tyrode’s acidic solution (AT) with a 2.35 pH are more frequently applied to AHA. In this procedure we utilize a minimal quantity of acidified solution to disolve a small portion of the zona. If embryo exposition to this solution is short, there will be no negative effect for them. |
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The zona pellucida
is a physical barrier that serves the function of protecting the
oocytes and pre-embryos against antigens during the pre-implantation
period;
The
thicknes of the zona pellucida varies during the oocyte or pre-embryo
development. Thinning begins between the first and second cleavage,
and continues progressively during blastocyst formation. In the
absence of this thinning, possibly caused by absence of proteases,
the pre-embryo may remain caught inside the zona, effectively abolishing
any potential for implantation. As has been already described, the
creation of an artificial hole in the thick zona (> 15 mm) may
favour the natural hatching and the pre-embyo implantation.