Problem An association between serum prolactin (PRL) and peripheral blood natural killer (NK) cells has been described in healthy women. We explored for the first time the PRL response to the thyrotrophin-releasing hormone (TRH) test and the association between PRL and NK cells in women with reproductive failure. Methods A total of 130 women [31 primary infertility, 69 recurrent spontaneous abortion (RSA), and 30 fertile women] were evaluated by a TRH test to analyze the following: basal PRL (bPRL), peak-time PRL, PRL absolute and relative increase, decline-time PRL. Hyperprolactinaemia (HPRL) was defined as bPRL ≥ 15 ng/mL. NK cells were characterized by immunophenotyping. Results Significantly higher bPRL levels were found in the infertile women than in controls. Both the infertile and the RSA women showed significantly elevated NK levels. bPRL levels correlated with NK cells in HPRL-infertile women. Conclusions In patients with HPRL, an association between NK cell and bPRL results. The dynamic test in the infertile women would help in the management of the pregnancy impairment.