1 IVF Unit, Department of Obstetrics and Gynecology, Carmel Medical Center, 7 Michal St., Haifa 34362 and 2 Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer, Israel
3 To whom correspondence should be addressed. e-mail: jshiloh{at}rafael.co.il
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Abstract |
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Key words: cigarette smoking/embryo/oocyte/zona pellucida
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Introduction |
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The zona pellucida surrounding the oocyte plays an important role before and after fertilization, until hatching and implantation.
There is no information on the effect of cigarette smoking on zona pellucida thickness of oocytes and embryos, as one of the factors that may interfere with fertility.
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Materials and methods |
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All patients enrolled underwent ICSI procedure, either due to male factor, or previous failure of IVF cycles. The causes of infertility are categorized as follows: unexplained (n = 11); male factor only (n = 111); tubal factor + male factor (n = 24); endometriosis (n = 1); anovulation (n = 11); high levels of FSH (n = 11).
The FSH levels on days 35 of the cycle were determined for each patient. Induction of ovulation was similar in all patients: down-regulation, using a long protocol, followed by ovarian stimulation, a procedure as previously described by Dirnfeld et al. (1993). Briefly, GnRH analogue (Decapeptyl 3.75 mg; Ferring, Israel) or buserelin nasal spray (Suprefact® 1000 µg/day; Hoechst Pharmaceuticals, Germany) was administered in the mid-luteal phase, and continued until hCG administration (Chorigon, Teva Pharmaceuticals Ind. Ltd, Israel). Ovarian stimulation was performed by i.m. administration of hMG and/or FSH (Pergonal/Metrodin; Teva Pharmaceuticals).
Follicular growth monitoring was performed by transvaginal ultrasonography (TVS) and serum levels of 17-estradiol (E2), LH and progesterone. When three or more follicles reached a diameter of 18 mm, patients received hCG, 5000 IU i.m. Transvaginal-guided follicular aspiration was performed 3436 h after hCG administration. Luteal support was maintained by i.m administration of 100 mg/day progesterone in oil. Retrieved oocytes were incubated in human tubal fluid (HTF) medium, supplemented by 2.5% serum substitute (Irvine Scientific, USA). Semen samples were examined for concentration, motility and morphology. The samples were then washed twice in HTF medium, supplemented by 2.5% serum substitute. Swim-up procedure was used to select viable moving sperm.
Fertilization
Fertilization was achieved using the ICSI procedure, as described by Van Steirteghem et al. (1993). Denudation of the oocytes prior to ICSI could enable us to measure the zona pellucida thickness at the time of fertilization. Fertilization was confirmed 1620 h after the procedure.
Zona pellucida measurements
Zona pellucida thickness was measured prospectively in all oocytes at the time of fertilization, and in all embryos 48 h after fertilization, either before embryo transfer or before cryopreservation. Measurements were taken from printed photos using a Nikon inverted microscope, equipped with Nomarski contrast optics, at x200 magnification (Nikon Corporation, Japan). Thickness was measured at a minimum of six different points on the zona pellucida, at the same magnification. All measurements were taken by the same biologist, who was not aware of the couples smoking status. The error associated with the method of measurement was <1 µm. Intra- and inter-assay variations were <1 µm and 10% respectively.
Statistics
The calculated means of at least six zona pellucida thickness measurements of each oocyte and embryo are expressed as means ± SEM. Univariate analyses, including one-way analysis of variance, Students t-test and KruskalWallis test, were used to determine the differences between the groups. Multiple linear regression analysis was used to determine the effects of age, FSH and smoking on zona pellucida thickness.
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Results |
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Thickness of zona pellucida of oocytes and embryos was significantly increased in active and passive smokers than in non-smokers (Table I). There was no significant difference observed in zona pellucida thickness of oocytes or embryos in active compared with passive smokers (Table I).
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In the multiple linear regression analysis, only smoking significantly correlated (P < 0.0001) with zona pellucida thickness, but no significant correlation was found in regard to age or FSH.
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Discussion |
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The aims of this current study were to elucidate, for the first time, whether active or passive cigarette smoking is a contributing factor to zona pellucida thickness. Our results showed that zona pellucida thickness of oocytes and embryos in active as well as passive smokers is significantly increased compared with zona pellucida thickness of non-smokers.
In a previous study (Shiloh and Dirnfeld, 2000), a significant correlation was found between zona pellucida thickness of embryos, patients age and early follicular serum FSH levels. The present study confirms our previous findings; however, results are presented here according to smoking habits and not according to age and/or FSH levels. The mean age of the patients in each group studied was not significantly different, nor was there a significant difference in the mean levels of days 35 serum FSH between the groups. Because age and FSH levels were similar in each group, their impact on zona pellucida thickness was similar. Multiple linear regression of the effects of smoking, age and FSH levels on zona pellucida thickness in the current study revealed that only smoking significantly correlated with zona pellucida thickness.
After fertilization, the human zona pellucida undergoes hardening, and gradually becomes thinner. Thinning of the zona pellucida makes it possible for the embryo to hatch. Our findings also show that regardless of the smoking habits of the parents, in contrast with the effect of smoking on the initial zona pellucida thickness, the natural thinning of zona pellucida during the time interval between fertilization and 48 h later was not affected. We have not found any significant differences in the ability of the zona pellucida to become thinner in any of the groups studied. The above findings may imply that the mechanism of zona pellucida thinning is independent of its initial thickness, and probably results from a combination between mechanical pressure and the lysins produced by the cleaving embryo.
Implantation and pregnancy rates were not available in the present study because it would not be possible to analyse them unless a single embryo were transferred. However, in view of the reported association between zona pellucida thickness and its effect on implantation rates (Cohen et al., 1989), the observed smoking-related thickening of the zona pellucida may have a relevant impact in IVF/ICSI patients.
In summary, our study demonstrates that zona pellucida thickness of oocytes and embryos derived from active as well as passive cigarette smokers are increased compared with controls. Our findings also show that active and passive smoking had no significant effect on the thinning mechanism of the zona pellucida, which implies that the thinning mechanism is likely to be independent of the initial zona pellucida thickness.
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Acknowledgement |
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References |
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Submitted on October 14, 2002; resubmitted on September 25, 2003; accepted on September 30, 2003.