Human Reproduction, Vol. 19, No. 1, 157-159,
January 2004
© 2004 European Society of Human Reproduction and Embryology
The impact of cigarette smoking on zona pellucida thickness of oocytes and embryos prior to transfer into the uterine cavity
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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BACKGROUND: Smoking has been reported to promote infertility. The zona pellucida plays an important role in fertilization and implantation. We report, for the first time, the effect of cigarette smoking on zona pellucida thickness of oocytes and embryos as one of the factors that may interfere with fertility. METHODS: This study comprised 169 women, grouped according to their smoking habits: 31 active smokers, whose husbands do not smoke; 44 active smokers, whose husbands smoke; 65 passive smokers, because of smoking husbands and 29 non-smokers (women and husbands). Zona pellucida thickness was measured prospectively on printed photos of 903 oocytes and 456 embryos. RESULTS: The zona pellucida thickness of oocytes and embryos of non-smoking women was significantly thinner than those of active and passive smokers. However, no significant differences were observed in the natural ability of the zona pellucida to become thinner after 48 h in culture. CONCLUSIONS: Our study demonstrates that active and passive cigarette smoking increases the zona pellucida thickness of oocytes and embryos. Our findings also show that active and passive smoking has no significant effect on the thinning mechanism of the zona pellucida, which implies that it is independent of the initial zona pellucida thickness.
Key words: cigarette smoking/embryo/oocyte/zona pellucida
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Introduction |
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Smoking by male and/or female partners has been reported to compromise reproduction and success rates of assisted reproductive technology (Curtis et al., 1997
; Augood et al., 1998). Shiverick and Salafia (1999) reported on the anti-estrogenic effect of smoking in terms of direct effects of nicotine, cadmium and poly-aromatic hydrocarbons on estrogen synthesis and metabolism. Other studies have shown that cigarette smoking in women appears to significantly reduce their ovarian reserve (El-Nemr et al., 1998; Crha et al., 2001; Klonoff-Cohen et al., 2001), and number of total fertilization failures is higher (Rosevear et al., 1992; Augood et al., 1998). Recent models have proved evidence that smoking may alter uterine–Fallopian tube function (Knoll and Talbot, 1998; Saraiya et al., 1998). Zenzes (2000) determined that smoking alters the meiotic spindle, leading to chromosome errors. 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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Patients
This prospective study comprised 169 women aged 21–46 years. Each couple signed a consent form approved by the Committee for Research in Human Subjects of the Carmel Medical Center. Thirty-one women were classified as active smokers (>10 cigarettes per day, at least during the course of the previous year), whereas their husbands were non-smokers. Forty-four women and their husbands were active smokers; 65 were passive smokers (their husbands smoked >10 cigarettes per day), and 29 women and their husbands were non-smokers, and served as controls.
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 3–5 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 34–36 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 16–20 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 couple’s 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, Student’s t-test and Kruskal–Wallis 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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Zona pellucida measurements were obtained from 903 oocytes and 456 embryos 48 h after fertilization, and before their transfer into the uterine cavity. Patients were classified into four groups according to their exposure to cigarette smoking: group 1: 31 women were active smokers (>10 cigarettes per day), whereas their husbands were non-smokers; group 2: 44 women and their husbands were active smokers (>10 cigarettes per day); group 3: 65 women were passive smokers (their husbands smoked >10 cigarettes per day); and group 4: 29 couples both non-smokers, served as controls.
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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Thinning of zona pellucida occurred during the time interval between fertilization and 48 h. No significant difference was noted in the thinning of the zona pellucida in any of the embryos studied, regardless of their origin, i.e. from active, passive or non-smokers (Table I). The mean (± SD) age of the patients in each group was not significantly different (Table II).
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No significant difference was found in the total dose of hMG needed for ovarian stimulation, and the day of hCG administration, or in the mean ± SD of day 5 serum FSH levels in any of the groups studied (Table II).
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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Epidemiological studies have demonstrated that cigarette smoking is associated with decreased fertility. Thickness of the zona pellucida has been proposed to be one of the primary factors influencing embryonic hatching ability (Cohen et al., 1989
; Roux et al., 1995) and implantation and pregnancy rates (Shiverick and Salafia, 1999). Assisted hatching has been the most popular technique to treat embryos with thick zona pellucida and improve embryonic hatching (Cohen et al., 1992; Liu et al., 1993). 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, patient’s 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 3–5 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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The authors would like to acknowledge the contribution of Dr Ada Tamir from the Department of Epidemiology, Faculty of Medicine, Technion Israel Institute of Technology, to the statistical analysis of the data.
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Submitted on October 14, 2002; resubmitted on September 25, 2003; accepted on September 30, 2003.
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