The Egyptian IVF-ET Centre, Cairo 11431, Egypt
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Abstract |
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Key words: acid Tyrode's/hatching/ICSI/poor prognosis/zona-free embryo transfer
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Introduction |
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Recently, successful pregnancies have been reported after transfer of zona-free blastocysts using pronase, with high pregnancy and implantation rates (Fong et al., 1997, 1998
). The aim of our study was to evaluate outcomes after transfer of day 3 zona-free embryos, using acid Tyrode's solution.
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Materials and methods |
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Ovarian stimulation, oocyte retrieval, and oocyte handling
All patient cycles were suppressed with a gonadotrophin-releasing hormone agonist (GnRHa) long protocol using 200 µg/8 h Suprefact nasal spray (Hoechst AG, Frankfurt am Main, Germany) in the luteal phase of the cycle until the day of human chorionic gonadotrophin (HCG) injection. When ovarian suppression was confirmed by low serum oestradiol concentration, human menopausal gonadotrophin (HMG) (Pergonal; E.I.P. Co. Industries, Cairo, Egypt) was given at a dose of 150 IU i.m. daily for 6 days. The dose was then modified according to the ovarian response. Monitoring was commenced after 6 days of HMG injection, using vaginal ultrasound (ultrasound) and serum oestradiol. Ten thousand units of HCG (Pregnyl; Nile Co., Cairo, Egypt) were given i.m. when two or more follicles reached 18 mm in mean diameter. Ovum retrieval using transvaginal ultrasound was scheduled 36 h after HCG injection.
The oocytes were denuded of their surrounding cumulus cells 2 h after retrieval, using hyaluronidase 80 IU/ml in HEPES-buffered Earle's balanced salt solution (Medicult, Copenhagen, Denmark) for 10-15 s. The oocytes were then transferred to G1 medium (Scandinavian IVF Science AB, Vitrolife AB, Gothenburg, Sweden) for complete removal of the corona cells by repeated aspiration in a finely pulled pipette. The oocytes were rinsed and incubated in G1 medium under mineral oil (Squibb, Princeton, NJ, USA) until the time of injection, which was done only for oocytes that extruded their first polar bodies. The microinjection procedure has been described previously (Mansour, 1998). Embryos were not grown to the blastocyst stage and zona measurements were not performed.
Embryo quality was assessed in all patients according to the following criteria: grade 1: embryos reaching the 4-cell stage by day 2, or the 8-cell stage by day 3 with equal regular blastomeres and no fragmentation; grade 2: embryos not reaching the 4-cell stage on day 2 or the 8-cell stage by day 3, and/or with 25% fragmentation; grade 3: embryos that did not reach the 4-cell stage by day 2 or the 8-cell stage by day 3 and/or with more than 25% fragmentation.
Zona pellucida removal
The zona was removed by exposing the embryos to acid Tyrode's solution (Irvine Scientific, Santa Ana, CA 92705, USA; pH 2.12.5) for a few seconds followed by immediate repeated rinsing in G1 medium. This technique required very quick handling in order to avoid unnecessary exposure of the embryo to the acidic solution. The zona dissolved on contact with the acid solution, so the embryo was removed immediately and rinsed several times to remove any traces of the acid Tyrode's solution. The embryos were incubated until the time of transfer, which was done within 2 h of zona removal.
Embryo transfer
This was done on day 3 after oocyte collection using the Wallace catheter (H.G.Wallace Ltd, West Sussex, UK) or the Labotect catheter (Labotect, Bonvender-Gottingen, Germany) if the Wallace catheter could not be introduced. Luteal phase support was given routinely in the form of 2500 IU of HCG every fourth day. Cases that were considered at high risk of developing ovarian hyperstimulation syndrome were given a daily progesterone injection (100 mg, progesterone; Steris, Phoenix, AZ, USA) in place of the HCG. A serum ß-HCG test was done 2 weeks after the transfer, and ultrasound examination was performed after a further 23 more weeks for patients with a positive test. Clinical pregnancy was diagnosed by the presence of a gestational sac with fetal echoes.
Statistical evaluation
Patient age and duration of infertility were compared between the zona-free and zona-intact cases within groups A and B using an unpaired t-test. Clinical pregnancy rates were compared using Fisher's exact test. Embryo quality was compared using a 2 test for trend. P values < 0.05 were considered to be significant. Data are presented as mean ± SD.
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Results |
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Discussion |
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Results of published studies in the literature vary, but different designs and patient selection criteria, often with low numbers of subjects, make interpretation of the findings difficult. Some studies show significant benefits of AZH in women with a poor prognosis due to older age and multiple prior IVF/ICSI cycle failures (Liu et al., 1993; Schoolcraft et al., 1994
, 1995
; Stein et al., 1995
; Chao et al., 1997
; Magli et al., 1998
; Meldrum et al., 1998
). Other studies reveal no improvement in outcome measures in unselected patients (Hellebaut et al., 1996
) and no advantage of AZH in patients of 36 years and over (Lanzendorf et al., 1998
).
There has been some concern that manipulation of the zona pellucida could lead to abnormalities in the hatching process, with an increased monozygotic twinning rate (Nijs et al., 1993; Alikani et al., 1994
). A case of conjoined twins in a triplet pregnancy after IVF and assisted hatching has been described (Skupski et al., 1995
). Trapping of the embryo as it hatches through a slit in the zona pellucida after using microdissection, may increase the risk of monozygotic pregnancy and abnormal pregnancy. This may be less likely with the application of acid Tyrode's solution, which produces a larger and more rounded opening in the zona. Further studies are needed to establish the true incidence of these complications, and patients should be counselled accordingly. The use of complete zona removal should decrease the risk.
The technique of AZH recently entered a new phase with the first reports of healthy pregnancies resulting from the transfer of zona-free blastocysts (Fong et al., 1997, 1998
). Enzymatic treatment of the zona pellucida with pronase was used on day 5 blastocysts in 19 women, with a pregnancy rate of 53%. A high multiple pregnancy rate of 40%, with two triplet pregnancies led the authors to conclude that the high implantation rates observed necessitate a reduction in the number of zona-free blastocysts transferred.
Our study compared the transfer of zona-free and zona-intact day 3 embryos in two groups of women. The complete removal of the zona pellucida before embryo transfer improved pregnancy rates in a selected group of poor prognosis women undergoing ICSI. The full explanation for this is not clear, but it is possible that multiple IVF failures and poor results in older women may be partly attributed to inadequate zona hatching and subsequent implantation failure. The transfer of zona-free embryos as performed by us completely removes this barrier to implantation.
There was no detrimental effect of zona removal on the pregnancy rate in either of the groups studied, implying that the technique itself did not adversely affect outcomes. Reassuringly, the multiple pregnancy rate did not appear to be raised in the zona-free group, although the numbers were too small to allow statistical analysis. The technique we describe using acid Tyrode's for zonal removal is relatively simple but requires very quick handling. It is efficient and fast, and can be performed directly before transfer, with minimal additional use of staff or laboratory time.
In conclusion, our technique of complete zona removal may be considered as an option to improve pregnancy rates after ICSI in women with a poor prognosis. To the best of our knowledge, this is the first report in the literature.
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Notes |
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References |
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Submitted on June 21, 1999; accepted on January 20, 2000.