Contents lists available at ScienceDirect Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jri Effects of Progesterone, Dydrogesterone and Estrogen on the Production of Th1/Th2/Th17 Cytokines by Lymphocytes from Women with Recurrent Spontaneous Miscarriage Ghadeer AbdulHussain a , Fawaz Azizieh b , Ma’asoumah Makhseed c , Raj Raghupathy a, * a Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait b Department of Mathematics and Biology, Gulf University of Science and Technology, Kuwait c Al Hakim Clinic, Kuwait ARTICLE INFO Keywords: Recurrent spontaneous miscarriage cytokines progesterone dydrogesterone estrogen immunomodulation ABSTRACT Anti-inflammatory Th2 cytokines have been shown to be associated with healthy, successful pregnancy while pro-inflammatory Th1 and Th17 cytokines are associated with pregnancy loss due to recurrent spontaneous miscarriage. This nexus between unexplained recurrent spontaneous miscarriage (uRSM) and maternal in- flammatory has led to the possibility of using pregnancy-related hormones to modify the maternal cytokine bias in a manner that is conducive to successful pregnancy. We investigated the ability of progesterone, dy- drogesterone and estrogen to modulate cytokine production by peripheral blood lymphocytes from women undergoing uRSM. Peripheral blood mononuclear cells (PBMC) from females with uRSM were stimulated in vitro with phytohemagglutinin (PHA) in the presence and absence of progesterone or dydrogesterone or 17β-estra- diol. Culture supernatants were assayed for IFN-α, TNF-γ, IL-2, IL-6, IL-10, IL-13, IL-17A, and IL-23 by ELISA. Progesterone and dydrogesterone significantly down-regulated the secretion of the Th1 cytokines IFN-α and TNF-γ, and the Th17 cytokine IL-17A, and IL-23. Additionally, the secretion of the Th2 cytokine IL-6 was up- regulated. Estrogen, on the other hand, decreased the production of IFN-α and IL-2, increased the production of IL-6 but did not affect IL-17A and IL-23 secretion. Progestogens and estrogen can decrease the production of some Th1/Th17 inflammatory cytokines secreted by lymphocytes from uRSM and upregulate the production of anti-inflammatory cytokines. These data support the notion that progestogens can be used for altering maternal cytokine profiles to manage pregnancy complications. 1. Introduction Recurrent spontaneous miscarriage (RSM), defined as two or more consecutive spontaneous miscarriages before 20 weeks of gestation, occurs in 0.5% - 1% of all pregnancies (Sugiura-Ogasawara et al., 2014). The etiology of RSM is multifactorial; well documented and widely accepted risk factors include antiphospholipid syndrome, ana- tomical uterine malformations, and parental chromosome anomalies (Saravelos and Regan, 2014). However, with a substantial proportion of RSM remaining idiopathic or of unexplained etiology, immunological factors have been proposed and explored (Wang et al., 2016). Cytokines are crucial messenger molecules of the immune system, but also contribute to the success of pregnancy at various stages, in- cluding successful implantation of the embryo and growth and differ- entiation of the trophoblast (Robertson and Moldenhauer, 2014). Women with unexplained RSM (uRSM) have been shown to have a greater proclivity towards a Th1-type or pro-inflammatory cytokine profile compared to healthy pregnant women (Hill and Choi, 2000; Saito et al., 2010). Peripheral blood cells from subjects with a history of uRSM stimulated with human trophoblast antigens produce higher le- vels of Th1 cytokines with embryotoxic activity as compared to healthy pregnant women (Hill and Choi, 2000). We have previously reported that higher levels of Th2-type cytokines are produced by mitogen-sti- mulated peripheral lymphocytes from women with a history of healthy pregnancy, while lymphocytes from women with uRSM secrete greater levels of the pro-inflammatory Th1-type cytokines (Raghupathy et al., 2000). Ratios of inflammatory cytokines to anti-inflammatory cytokines are also higher in uRSM than in healthy pregnancy, indicating a greater Th1-bias in uRSM as opposed to a stronger Th2-bias in normal preg- nancy (Makhseed et al., 1999). Clerici‘s group tested cytokine produc- tion in pregnant women 1-2 weeks before any upcoming pathology could be detected and found decreased production of IL-4 and IL-10, https://doi.org/10.1016/j.jri.2020.103132 Received 10 November 2019; Received in revised form 2 March 2020; Accepted 8 April 2020 ⁎ Corresponding author at: Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait. E-mail address: raj@hsc.edu.kw (R. Raghupathy). Journal of Reproductive Immunology 140 (2020) 103132 0165-0378/ © 2020 Published by Elsevier B.V. T