In utero exposure to cigarette smoke dysregulates human fetal ovarian developmental signalling

Paul A Fowler, Andrew J Childs, Frédérique Courant, Alasdair MacKenzie, Stewart M Rhind, Jean-Philippe Antignac, Bruno Le Bizec, Panagiotis Filis, Fergus Evans, Samantha Flannigan, Abha Maheshwari, Siladitya Bhattacharya, Ana Monteiro, Richard A Anderson, Peter J O'Shaughnessy

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Abstract

STUDY QUESTION: How does maternal cigarette smoking disturb development of the human fetal ovary?

SUMMARY ANSWER: Maternal smoking increases fetal estrogen titres and dysregulates several developmental processes in the fetal ovary.

WHAT IS KNOWN ALREADY: Exposure to maternal cigarette smoking during gestation reduces human fetal ovarian cell numbers, germ cell proliferation and subsequent adult fecundity.

STUDY DESIGN, SIZE, DURATION: The effects of maternal cigarette smoking on the second trimester human fetal ovary, fetal endocrine signalling and fetal chemical burden were studied. A total of 105 fetuses were studied, 56 from mothers who smoked during pregnancy and 49 from those who did not.

PARTICIPANTS/MATERIALS, SETTING METHODS: Ovary, liver and plasma samples were collected from electively terminated, normally progressing, second trimester human fetuses. Circulating fetal hormones, levels of 73 fetal ovarian transcripts, protein localization, density of oocytes/primordial follicles and levels of 16 polycyclic aromatic hydrocarbons (PAHs) in the fetal liver were determined.

MAIN RESULTS AND THE ROLE OF CHANCE: Circulating fetal estrogen levels were very high and were increased by maternal smoking (ANOVA, P = 0.055-0.004 versus control). Smoke exposure also dysregulated (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.046-0.023) four fetal ovarian genes (cytochrome P450 scc [CYP11A1], NOBOX oogenesis homeobox [NOBOX], activator of apoptosis harakiri [HRK], nuclear receptor subfamily 2, group E, member 1 [NR2E1]), shifted the ovarian Inhibin βA/inhibin α ratio (NHBA/INHA) transcript ratio in favour of activin (ANOVA, P = 0.049 versus control) and reduced the proportion of dominant-negative estrogen receptor 2 (ERβ: ESR2) isoforms in half the exposed fetuses. PAHs, ligands for the aryl hydrocarbon receptor (AHR), were increased nearly 6-fold by maternal smoking (ANOVA, P = 0.011 versus control). A fifth transcript, COUP transcription factor 1 (nuclear receptor subfamily 2, group F, member 1: NR2F1, which contains multiple AHR-binding sites), was both significantly increased (ANOVA, P = 0.026 versus control) and dysregulated by (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.021) maternal smoking. NR2F1 is associated with repression of FSHR expression and smoke-exposed ovaries failed to show the normal increase in FSHR expression during the second trimester. There was a significantly higher number of DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4) VASA-positive (ANOVA, P = 0.016 versus control), but not POU domain, class 1, transcription factor 1 (POU5F1) OCT3/4-positive, oocytes in smoke-exposed fetuses and this matched with a significantly higher number of primordial follicles (ANOVA, P = 0.024 versus control).

LIMITATIONS, REASONS FOR CAUTION: The effects of maternal smoking on establishment of the maximum fetal primordial follicle pool cannot be reliably studied in our population since the process is not completed until 28 weeks of gestation and normal fetuses older than 21 weeks of gestation are not available for study. Our data suggest that some fetal ovaries are affected by smoke exposure while others are not, indicating that additional studies, with larger numbers, may show more significant effects.

WIDER IMPLICATIONS OF THE FINDINGS: Fetal exposure to chemicals in cigarette smoke is known to lead to reduced fecundity in women. Our study suggests, for the first time, that this occurs via mechanisms involving activation of AHR, disruption of inhibin/activin and estrogen signalling, increased exposure to estrogen and dysregulation of multiple molecular pathways in the exposed human fetal ovary. Our data also suggest that alterations in the ESR2 positive and dominant negative isoforms may be associated with reduced sensitivity of some fetuses to increased estrogens and maternal smoking.

STUDY FUNDING/COMPETING INTERESTS: The study was supported by grants from the Chief Scientist Office (Scottish Executive, CZG/1/109, and CZG/4/742), NHS Grampian Endowments (08/02), the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 212885, a Society for Reproduction & Fertility summer studentship, Medical Research Scotland (research grant 354 FRG) and the Medical Research Council (WBS: U.1276.00.002.00001 and G1100357). The authors declare they have no competing interests, be it financial, personal or professional.

Original languageEnglish
Pages (from-to)1471-1489
Number of pages19
JournalHuman Reproduction
Volume29
Issue number7
Early online date20 May 2014
DOIs
Publication statusPublished - Jul 2014

Keywords

  • ovary
  • fetal human
  • cigarette
  • polycyclic aromatic hydrocarbons
  • AhR

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    Fowler, P. A., Childs, A. J., Courant, F., MacKenzie, A., Rhind, S. M., Antignac, J-P., Le Bizec, B., Filis, P., Evans, F., Flannigan, S., Maheshwari, A., Bhattacharya, S., Monteiro, A., Anderson, R. A., & O'Shaughnessy, P. J. (2014). In utero exposure to cigarette smoke dysregulates human fetal ovarian developmental signalling. Human Reproduction, 29(7), 1471-1489. https://doi.org/10.1093/humrep/deu117