Activation of the aryl hydrocarbon receptor by a component of cigarette smoke 1 reduces germ cell proliferation in the human fetal ovary

39 Fetal life is a critical time for female fertility, when germ cells complete proliferation, 40 initate meiosis and ultimately form the lifetime stock of primordial follicles. Female 41 fertility may be reduced by in utero exposure to cigarette smoke, which contains 42 ligands for the aryl hydrocarbon receptor (AhR). The AhR is a critical regulator of 43 ovarian germ cell survival in mice, thus activation of this receptor in the ovaries of 44 fetuses exposed to maternal cigarette smoke in utero may provide a mechanism by 45 which female fertility is reduced in later life. We have therefore investigated AhR 46 expression in the human fetal ovary, and examined the effects of an AhR ligand 47 present in cigarette smoke, on germ cells in human fetal ovaries cultured in vitro. 48 AHR mRNA expression increased 2-fold between first and late second trimester 49 (p=0.008). AhR protein was confined to germ cells at all gestations, but varied from 50 expression in most germ cells during the first trimester, to only patchy expression by 51 clusters of germ cells at later gestations. Culture of human fetal ovaries with the AhR 52 ligand 9,10-dimethyl-1,2-benzanthracene-3,4-dihydrodiol (DMBA-DHD; a 53 component of cigarette smoke) did not affect germ cell number in vitro, but 54 significantly reduced the proportion of proliferating germ cells by 29% (as assessed 55 by phospho-histone H3 staining (p=0.04)). Germ cell apoptosis was not significantly 56 affected. These results reveal that germ cells in the human fetal ovary express AhR 57 from the proliferative stage of development through entry into meiosis and beyond, 58 and demonstrate that AhR ligands found in cigarette smoke have the capacity to 59 impair human fetal ovarian germ cell proliferation. 60 61