title: Grandmaternal smoke exposure reduces female fertility in a murine model, with great-grandmaternal smoke exposure unlikely to have an effect.
abstract: What effect does multigenerational (F2) and transgenerational (F3) cigarette smoke exposure have on female fertility in mice?
  
  
  
  Cigarette smoking has a multigenerational effect on female fertility.
  
  
  
  It has been well established that cigarette smoking decreases female fertility. Furthermore, a growing body of evidence suggests that smoking during pregnancy decreases the fertility of daughters and increases cancer and asthma incidence in grandchildren and great-grandchildren.
  
  
  
  Six-week-old C57BL/6 female mice were exposed nasally to cigarette smoke or room air (controls) for 5 weeks prior to being housed with males. Females continued to be exposed to smoke throughout pregnancy and lactation until pups were weaned. A subset of F1 female pups born to these smoke and non-smoke exposed females were bred to create the F2 grandmaternal exposed generation (multigenerational). Finally, a subset of F2 females were bred to create the F3 great-grandmaternal exposed generation (transgenerational). The reproductive health of F2 and F3 females was examined at 8 weeks and 9 months.
  
  
  
  Ovarian and oocyte quality was examined in smoke exposed and control animals. A small-scale fertility trial was performed before ovarian changes were examined using ovarian histology and immunofluorescence and/or immunoblotting analysis of markers of apoptosis (TUNEL) and proliferation (proliferating cell nuclear antigen (PCNA) and anti-Mullerian hormone (AMH)). Oocyte quality was examined using immunocytochemistry to analyze the metaphase II spindle and ploidy status. Parthenogenetic activation of oocytes was used to investigate meiosis II timing and preimplantation embryo development. Finally, diestrus hormone serum levels (FSH and LH) were quantified.
  
  
  
  F2 smoke exposed females had no detectable change in ovarian follicle quality at 8 weeks, although by 9 months ovarian somatic cell proliferation was reduced (P = 0.0197) compared with non-smoke exposed control. Further investigation revealed changes between control and smoke exposed F2 oocyte quality, including altered meiosis II timing at 8 weeks (P = 0.0337) and decreased spindle pole to pole length at 9 months (P = 0.0109). However, no change in preimplantation embryo development was observed following parthenogenetic activation. The most noticeable effect of cigarette smoke exposure was related to the subfertility of F2 females; F2 smoke exposed females displayed significantly increased time to conception (P = 0.0042) and significantly increased lag time between pregnancies (P = 0.0274) compared with non-smoke exposed F2 females. Conversely, F3 smoke exposed females displayed negligible oocyte and follicle changes up to 9 months of age, and normal preimplantation embryo development.
  
  
  
  None.
  
  
  
  This study focused solely on a mouse model of cigarette smoke exposure to simulate human exposure.
  
  
  
  Our results demonstrate that grandmaternal cigarette smoke exposure reduces female fertility in mice, highlighting the clinical need to promote cessation of cigarette smoking in pregnant women.
  
  
  
  This study was funded by the Australian Research Council, National Health and Medical Research Council, Hunter Medical Research Institute, Newcastle Permanent Building Society Charitable Trust, and the University of Newcastle Priory Research Centers in Chemical Biology, Healthy Lungs and Grow Up Well. The authors declare no conflict of interest.
authors.affiliation: School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.|Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: N J
authors.initials: NJ
authors.lastname: Camlin
authors.affiliation: School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia.|Priority Research Centre for Healthy Lungs, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: A G
authors.initials: AG
authors.lastname: Jarnicki
authors.affiliation: School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia.|Priority Research Centre for Healthy Lungs, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: R L
authors.initials: RL
authors.lastname: Vanders
authors.affiliation: School of Women's & Children's Health, University of New South Wales, Sydney, NSW2052, Australia.
authors.forename: K A
authors.initials: KA
authors.lastname: Walters
authors.affiliation: School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia.|Priority Research Centre for Healthy Lungs, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: P M
authors.initials: PM
authors.lastname: Hansbro
authors.affiliation: School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.|Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, NSW 2308, Australia.|School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: E A
authors.initials: EA
authors.lastname: McLaughlin
authors.affiliation: Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, NSW 2308, Australia.|School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: J E
authors.initials: JE
authors.lastname: Holt
mesh_terms: D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D000073865:Cigarette Smoking / Q000009:adverse effects*; D004477:Ectogenesis; D005260:Female; D047109:Fetal Development / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007247:Infertility, Female / Q000209:etiology* / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D007774:Lactation; D018811:Maternal Exposure / Q000009:adverse effects*; D008810:Mice, Inbred C57BL; D009865:Oocytes / Q000378:metabolism / Q000473:pathology*; D009866:Oogenesis; D010053:Ovary / Q000378:metabolism / Q000473:pathology*; D011247:Pregnancy; D011297:Prenatal Exposure Delayed Effects; D012720:Severity of Illness Index; D061685:Time-to-Pregnancy
chemical_list: D015415:Biomarkers
keywords: cigarette smoke; grandmaternal; great-grandmaternal; multigenerational; oocyte; ovary; subfertility; transgenerational