Hormonal contraception (HC), including the injectable progestin depot medroxyprogesterone acetate (DMPA) and combined oral contraceptives (COCs), and pregnancy have been associated with increased risk of HIV in some, but not all, large prospective studies. In February 2012, due to the conflicting epidemiologic study results and lack of sufficient mechanistic information, the World Health Organization held a technical consultation to evaluate the existing evidence on hormonal contraception and HIV infection. The WHO decided not to change the guidelines for access to HC for women at risk of HIV but recommended that women who specifically use progestin-only injectable contraception and seek to prevent unintended pregnancy and HIV should be strongly advised to also use condoms for dual protection. Policy makers and women's advocate groups have expressed concern that the WHO statement is insufficient and difficult to translate into effective counseling for women. In addition, genital HSV-2 infection is known to significantly increase a woman's risk of acquiring HIV, but the mechanism and the impact of hormones on this relationship are not understood. A more complete mechanistic understanding of how HC and HSV-2 may modify the female immune system to increase the risk of HIV is key to clarifying the results of the epidemiologic studies and to establishing more accurate and effective public health policy and practice. The aims of the proposed research are two-fold: 1) to understand the associations between systemic hormonal levels, circulating regulators of inflammation and immunity and soluble innate immunity mediators in the cervix, their relationship with HIV acquisition risk, and how these relationships are altered by exposure to hormonal contraception (DMPA and COC) and pregnancy; and 2) to define the effects of pregnancy and hormonal contraception on the innate immune system preceding, at the time of, and during established (> 6 months) HSV-2 infection to better understand the effect of hormonal contraception and pregnancy on HSV-2 associated HIV risk. We will use ~4,000 longitudinal paired serum and cervical specimens from 1,200 women from Uganda and Zimbabwe who used DMPA, COCs and no hormonal contraception and participated in the HC-HIV study with well-specified HIV and HSV-2 seroconversion dates and who experienced substantial HSV-2, HIV-1 and pregnancy incidence. We will use ELISA and the Meso Scale Discovery multiplex platform to measure levels of endogenous hormones and regulators of inflammation and immunity in cervical samples and paired blood serum. We will use cross-sectional and longitudinal analyses to determine how hormonal contraception, pregnancy and menstrual cycle modify innate immunity, how systemic regulators affect the cervical immune environment, and how the immune regulators are associated with HSV-2 infection (incident and prevalent) and subsequent HIV acquisition risk. This research provides a unique opportunity to elucidate and validate the complex associations between endogenous and exogenous hormones, HSV-2 and HIV-1 through analysis of underlying shared biological mechanisms.