To the Editor:
Defects in motile cilia are associated with several human diseases (1), so understanding the regulatory mechanisms of cilia function in the disease stage is of great importance. The results of a retrospective cohort study on the effect of cigarette smoke exposure on ciliation and ciliogenesis in human oviductal epithelium in vivo are currently in press in Fertility and Sterility ( Pier et al., 2012). From a methodological standpoint, the quality of the evidence presented appears to be the best that is currently available on this topic in oviduct-related scientific research. However, some considerations in this study require attention.
In the experiments of Pier et al., no significant differences in cilia-specific protein expression or ciliogenesis-related transcription factor regulation were observed between the two groups of patients with or without smoking. Thus, the authors conclude that “cigarette smoking does not seem to result in quantitative differences in the density of ciliation nor expression of ciliogenesis factors.” The pathogenic mechanisms underlying cigarette smoking in human oviductal cells, however, is still not well understood. To my understanding, the paper only provides a snapshot of cilia-specific gene and protein expression profiles in the oviducts of women following chronic exposure to cigarette smoke. The effects of biomedical interventions on a biological system, however, are normally measured by steady-state or time-course experiments. Thus this study cannot exploit and describe the dynamic mechanisms of tubal gene and/or protein regulation affected by cigarette smoke exposure. The failure to detect differences in the expression of cilia-specific molecules in women with or without chronic cigarette smoke exposure may be due to (a) a length of time after cigarette smoke exposure that is not favorable to the processes of ciliation and ciliogenesis or (b) indirect effects of cigarette smoking, or both. I suggest that an in vitro experiment could address this issue for establishment of a cause-and-effect relationship between cigarette smoke exposure and ciliation and ciliogenesis factor expression in human tubal ciliated cells.
In the oviduct, ciliated epithelial cells push the gametes and embryo along the tube and appear to be largely responsible for the tubal transport because this transport continues even when muscle cell-dependent contractile activity is blocked by isoproterenol, a β-adrenergic agonist (2). There is a great deal of evidence that animals or oviductal explants exposed to tobacco smoke extract show decreases in cilia numbers, ciliary beat frequency, and cilia-dependent oocyte retrieval rates (3). This demonstrates that tobacco smoke could interrupt normal cilia-dependent tubal function and result in the development of tubal ectopic pregnancy. In the article by Pier et al., the authors state that “our findings suggest that pathophysiologic mechanisms other than ciliation account for the increased risk of ectopic pregnancy in women who smoke.” Because alterations of oviductal functions are mediated by different regulatory mechanisms (4, 5), I agree that multiple mechanisms of smoking-induced oviductal damage and dysfunction can possibly contribute to the initiation and development of tubal ectopic pregnancy in women who smoke. However, our current understanding of the underlying causes of tubal ectopic pregnancy in humans is based predominantly on descriptive observations rather than interventive experimentation. Using a retrospective cohort study protocol, as these authors have done, makes it difficult to determine whether the absence of smoking-induced ciliary protein homeostasis and ciliogenesis during the onset of tubal ectopic pregnancy is the whole truth or only part of it.
I would recommend that the authors add considerations of the developmental aspects of tubal ectopic pregnancy, and the cellular and molecular events of ciliation and ciliogenesis under in vivo conditions, to minimize the risk of reader misinterpretation.
Ruijin Shao, M.D., Ph.D.
Institute of Neuroscience and Physiology
The Sahlgrenska Academy at University of Gothenburg
1. Bettencourt-Dias M, Hildebrandt F, Pellman D, Woods G, Godinho SA. Centrosomes and cilia in human disease. Trends Genet 2011;27:307-15.
2. Halbert SA, Tam PY, Blandau RJ. Egg transport in the rabbit oviduct: the roles of cilia and muscle. Science 1976;191:1052-3.
3. Shao R, Zou S, Wang X, Feng Y, Brannstrom M, Stener-Victorin E, et al. Revealing the hidden mechanisms of smoke-induced fallopian tubal implantation. Biol Reprod 2012;86:131.
4. Shao R. Understanding the mechanisms of human tubal ectopic pregnancies: new evidence from knockout mouse models. Hum Reprod 2010;25:584-7.
5. Shaw JL, Horne AW. The paracrinology of tubal ectopic pregnancy. Mol Cell Endocrinol 2011;358:216-22.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2012.11.012
The authors respond:
We thank the readers for their interest in our study and for their insightful commentary. Human oviductal ciliated epithelium is a dynamic tissue of the female reproductive tract that undergoes cyclic changes in cell height and beat frequency in response to ovarian sex steroids, estrogen, and progesterone. Study of the effect(s) of toxins such as cigarette smoking on oviductal ciliation is complicated by multiple confounders such as menstrual cycle phase and exogenous hormone use, infection-mediated injury such as that induced by Chlamydia, and segmental differences in ciliation density. Admittedly, these confounders coupled with limited access to prospectively collected tubal segments render study of human oviductal pathophysiology challenging (1). Our study sought to control for these confounders in assessing not only the association between cigarette smoking and oviductal ciliation, but also the upstream effect of smoking on oviductal ciliogenesis using a prospectively collected and well curated tissue bank of human tubal segments (2).
A great deal of preclinical data has been generated regarding the effects of cigarette smoking on the oviduct. In the hamster model, ciliary beat frequency seems to be significantly altered when animals are exposed to mainstream and sidestream smoke. Few preclinical studies and no clinical studies, however, have evaluated the impact of cigarette smoke exposure on cilia quantity within the oviduct. Magers et al. used bright field microscopy to count ciliated cells in the hamster ampulla, and noted a modest decrease in the ciliated:secretory cell ratio in exposed animals (3). Whereas this preclinical finding was unconfirmed by orthogonal measurement, the histologic assessment of oviductal ciliation in our study was corroborated by quantitative immunohistochemistry and immunoblot.
Optimized in vitro models using animal or human oviductal explants are useful in the delineation of local cell-cell signaling pathways under pathophysiologic conditions. However, by their nature, in vitro models sacrifice systemic fidelity, particularly in the investigation of complex or indirect (i.e., endocrine disruptive) effects. Ultimately, in vitro findings require confirmation by studies using human oviductal specimens to validate their etiologic role in human pathology. Conclusive biologic evidence to explain the epidemiologically determined relationship between cigarette smoking and tubal pregnancy is lacking (1). Further well-designed studies are clearly needed to shed light on this relationship. By design a cross-sectional comparison of human tubal segments in active smokers and nonsmokers, our study provides insight on the cellular and subcellular anatomic impact(s) of smoking. The absence of an association between cigarette smoking and tubal ciliation or ciliogenesis in our study suggests that physiologic rather than anatomic etiologies underpin the relationship between smoking and ectopic pregnancy.
Bruce Pier, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology
Avedis Kazanjian, Ph.D., Department of Clinical Investigation
Laurie Gillette, M.Sc., Department of Clinical Investigation
Karen Strenge, M.D., Department of Pathology
Richard Owen Burney, M.D., M.Sc., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology
Madigan Healthcare System, Tacoma, Washington
1. Shao R, Zou S, Wang X, Feng Y, Brannstrom M, Stener-Victorin E, et al. Revealing the hidden mechanisms of smoke-induced fallopian tubal implantation. Biol Reprod 2012;86:131.
2. Pier B, Kazanjian A, Gillette L, Strenge K, Burney RO. Effect of cigarette smoking on human oviductal ciliation and ciliogenesis. Fertil Steril 2012;Epub ahead of print.
3. Magers T, Talbot P, DiCarlantonio G, Knoll M, Demers D, Tsai I, et al. Cigarette smoke inhalation affects the reproductive system of female hamsters. Reprod Toxicol 1995;9:513-25.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2012.11.011