Transfer of zona-free embryos improves outcome in poor prognosis patients: a prospective randomized controlled study

R.T. Mansour1, C.A. Rhodes, M.A. Aboulghar, G.I. Serour and A. Kamal

The Egyptian IVF-ET Centre, Cairo 11431, Egypt


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Assisted zona hatching (AZH) has been used in IVF programmes for several years. Recently one group has reported successful pregnancies after transfer of zona-free blastocysts. The aim of our study was to evaluate outcomes after transfer of zona-free day 3 embryos. Two groups of women undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Group A consisted of 52 women under the age of 40 years undergoing their first ICSI attempt. They were alternately randomized to receive zona-free embryos (27 women) and zona-intact embryos (25 women). The second group (group B) included 71 women with a poor prognosis, as defined by age 40 years or more, and/or at least two previous failed IVF/ICSI attempts. They were randomized in a 3:4 ratio (30 zona-free, 41 zona-intact). Acid Tyrode's solution was used to remove the zona pellucida before embryo transfer on day 3 after oocyte collection. The pregnancy rate in group A was not significantly improved when the zona pellucida was removed. However, in the poor prognosis group B, zona removal resulted in a significantly higher pregnancy rate when compared with controls (23 versus 7.3%). We conclude that complete removal of the zona pellucida can improve pregnancy rates in women with poor IVF/ICSI prognosis.

Key words: acid Tyrode's/hatching/ICSI/poor prognosis/zona-free embryo transfer


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Successful hatching of the embryo from the zona pellucida is a prerequisite for implantation in the uterus. Assisted zona hatching was introduced in IVF programmes to breach the zona pellucida and promote the natural process of hatching (Cohen et al., 1992Go).

Recently, successful pregnancies have been reported after transfer of zona-free blastocysts using pronase, with high pregnancy and implantation rates (Fong et al., 1997Go, 1998Go). The aim of our study was to evaluate outcomes after transfer of day 3 zona-free embryos, using acid Tyrode's solution.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Two groups of patients were studied over a 6 month period from January to June 1998. Group A consisted of 52 women under the age of 40 years undergoing their first intracytoplasmic sperm injection (ICSI) attempt. Group B included 71 women with a poor prognosis for success after ICSI. Sixteen patients were having their first attempt at ICSI at age 40 or more years; 55 patients had had previous IVF/ICSI failures in two or more cycles, of whom eight were aged >=40 years. Cases were randomized to undergo embryo transfer on day 3 after oocyte retrieval with or without complete zona removal. In group A, alternate cases were randomized. In group B the randomization was in a 3:4 ratio.

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, 1998Go). 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 2.1–2.5) 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 2–3 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 {chi}2 test for trend. P values < 0.05 were considered to be significant. Data are presented as mean ± SD.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The patient characteristics for each group are shown in Table IGo. There was no statistical difference between the zona-free and zona-intact cases within groups A and B with regard to age and duration of infertility (Student's t-test). Details of oocytes retrieved and outcome data are also shown in Table IGo. In group A, the fertilization rate and clinical pregnancy rates were not statistically different between the study and control groups. In group B, the pregnancy rate was significantly higher when zona-free embryos were transferred: 23 versus 7.3% (P < 0.05, Fisher's exact test).


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Table I. Demographic data and results
 
Embryo quality assessment is shown in Table IIGo. There was no significant difference in embryo quality between the zona-free and zona-intact groups in group A or B ({chi}2 test). The overall multiple pregnancy rate in the women with zona-free embryos transferred was 15.8% (three twin pregnancies out of 19 pregnancies in total). This was comparable to the rate in the zona-intact group (15.4%: two twin pregnancies out of 13 pregnancies in total), although numbers were too low for meaningful statistical analysis.


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Table II. Embryo quality in zona-intact and zona-removed groups
 
There was no degeneration of any embryos as a result of the technique of zona removal using acid Tyrode's solution. Figure 1Go shows one of the study embryos before zona removal. Figure 2Go shows an embryo following removal of the zona pellucida with completely intact blastomeres.



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Figure 1. Embryo before exposure to acid Tyrode's solution.

 


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Figure 2. Embryo after removal of zona pellucida with completely intact blastomeres.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The relatively low implantation rates of embryos resulting from IVF have been attributed at least in part to impaired hatching (Cohen, 1991Go). In a prospective randomized study using acid Tyrode's solution for zona drilling of 3 day old embryos, Cohen et al. showed that assisted zona hatching (AZH) could improve the implantation rate in poor prognosis patients (Cohen et al., 1992Go). Several other techniques to facilitate the hatching process have been reported, including partial zona dissection using a sharp pipette (Hellebaut et al., 1996Go; Chao et al., 1997Go), and a laser (Obruca et al., 1994Go).

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., 1993Go; Schoolcraft et al., 1994Go, 1995Go; Stein et al., 1995Go; Chao et al., 1997Go; Magli et al., 1998Go; Meldrum et al., 1998Go). Other studies reveal no improvement in outcome measures in unselected patients (Hellebaut et al., 1996Go) and no advantage of AZH in patients of 36 years and over (Lanzendorf et al., 1998Go).

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., 1993Go; Alikani et al., 1994Go). A case of conjoined twins in a triplet pregnancy after IVF and assisted hatching has been described (Skupski et al., 1995Go). 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., 1997Go, 1998Go). 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.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alikani, M., Noyes, N., Cohen, J. et al. (1994) Monozygotic twinning in the human is associated with the zona pellucida architecture. Hum. Reprod., 9, 1318–1321.[Abstract]

Chao, K.H., Chen, S.U., Chen, H.F. et al. (1997) Assisted hatching increases the implantation and pregnancy rate of in vitro fertilization (IVF)–embryo transfer (ET), but not that of IVF–tubal ET in patients with repeated IVF failures. Fertil. Steril., 67, 904–908.[ISI][Medline]

Cohen, J. (1991) Assisted hatching of human embryos. J. In Vitro Fert. Embryo Transf., 8, 179–190.[ISI][Medline]

Cohen, J., Alikani, M., Trowbridge, J. et al. (1992) Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum. Reprod., 7, 685–691.[Abstract]

Fong, C.Y., Bongso, A., Ng, S.C. et al. (1997) Ongoing normal pregnancy after transfer of zona-free blastocysts: implications for embryo transfer. Hum. Reprod., 12, 557–560.[ISI][Medline]

Fong, C.Y., Bongso, A., Ng S.C. et al. (1998) Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilisation and understanding implantation. Hum. Reprod., 13, 2926–2932.[Abstract/Free Full Text]

Hellebaut, S., De Sutter, P., Dozortsev, D. et al. (1996) Does assisted hatching improve implantation rates after in vitro fertilisation or intracytoplasmic sperm injection in all patients? A prospective randomised study. J. Assist. Reprod. Genet., 13, 19–22.[ISI][Medline]

Lanzendorf, S.E., Nehchiri, F., Mayer, J.F. et al. (1998) A prospective, randomised, double-blind study for the evaluation of assisted hatching in patients with advanced maternal age. Hum. Reprod., 13, 409–413.[ISI][Medline]

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Meldrum, D.R., Wisot, A., Yee, B. et al. (1998) Assisted hatching reduces the age-related decline in IVF outcome in women younger than age 43 without increasing miscarriage or monozygotic twinning. J. Assist. Reprod. Genet., 15, 418–421.[ISI][Medline]

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Submitted on June 21, 1999; accepted on January 20, 2000.