RECURRENT PREGNANCY LOSS Nitric oxide platelet production in spontaneous miscarriage in the first trimester Francesca Raffaelli, B.Sc., a Laura Nanetti, Ph.D., a Arianna Vignini, Ph.D., a Laura Mazzanti, Ph.D., a Stefano Raffaele Giannubilo, M.D., b Claudia Maria Curzi, M.D., b Angelo Turi, M.D., b Paola Vitali, M.D., b and Andrea Luigi Tranquilli, M.D. b a Department of Biochemistry, Biology and Genetics and b Department of Obstetrics and Gynaecology, Marche Polytechnic University, Ancona, Italy Objective: To investigate the role played by platelet nitric oxide (NO) metabolism in patients with spontaneous miscarriage (SM) and recurrent spontaneous miscarriage (RSM) compared with healthy pregnant women. Design: Retrospective case-control study. Setting: Patients and controls in an academic research environment. Intervention(s): None. Patient(s): Thirty singleton pregnant women who experienced SM, nine singleton pregnant women who presented with RSM, and 30 singleton healthy pregnant women matched for age, parity, and gestational age were enrolled. Main Outcome Measure(s): NO levels and peroxynitrite (ONOO - ) production; moreover, inducible NO synthase (iNOS), endothelial NO synthase (eNOS), and nitrotyrosine expression (N-Tyr) were observed in the same samples. Result(s): A significant increase was shown in platelet NO and ONOO - levels and in iNOS and N-Tyr both in SM and in RSM pregnant women compared with controls. Conclusion(s): The data herein reported imply that a modified NO pathway might play a key role in the physiological changes of advancing gestation but may also contribute to the pathophysiology of spontaneous miscarriage. Thus, any factors balancing NO metabolism might be useful in the treatment of miscarriage, thus reducing the substantial morbid- ity and associated mortality. (Fertil Steril Ò 2010;93:1976–82. Ó2010 by American Society for Reproductive Medicine.) Key Words: Spontaneous miscarriage, recurrent spontaneous miscarriage, platelets, nitric oxide, peroxynitrite, nitric oxide synthase Spontaneous miscarriages (SMs) occur when an embryo or fetus is lost before the twentieth week of development. Ap- proximately 10%–15% of clinically detected pregnancies end in SM depending on the age and health of the pregnant woman (1, 2). Recurrent SM (RSM), defined as three or more consecutive pregnancy losses before 20 weeks’ gesta- tion, affects 0.5%–2% of women (1, 2). The etiology of SM is often multifactorial; between 50% and 60% of these early pregnancy failures are linked to chro- mosomal abnormalities, mainly trisomies, while other cases are due to maternal diseases such as diabetes, acquired thrombophilia, and immune disorders as well as external fac- tors such as cigarette smoking and radiation exposure. How- ever, approximately 40% of early pregnancy failures remain unexplained (3). The understanding and prevention of SM is a major undertaking in human health and one of the primary goals of obstetric research (4). It is generally agreed that the unavailability of pharmacological interventions effective in preventing SM probably reflects the poor understanding of the molecular mechanisms behind the onset of labor (5). Nitric oxide (NO) contributes to maternal systemic vasodi- lation during pregnancy, regulates uterine and fetoplacental blood flow, and is involved in uterine quiescence before parturition (6). NO is a short-lived free-radical gas synthesized by a family of enzymes known as NO synthases (NOS), which catalyze the conversion of L-arginine to NO and citrulline. NOS enzymes are expressed in three isoforms: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS) (7). All of these isoforms are present in trophoblast cells of the first trimester and in various cells of the uterine cervix (8, 9). The expression of NOS isoforms and the release of NO in the cervix have been shown to increase with advancing gestational age and during cervical ripening (10). Previous studies have found that NO can be detectable in early placental tissue and that increased serum and urine levels have been found in women suffering from preterm Received September 3, 2008; revised and accepted December 17, 2008; published online February 12, 2009. F.R. has nothing to disclose. L.N. has nothing to disclose. A.V. has nothing to disclose. L.M. has nothing to disclose. S.R.G. has nothing to disclose. C.M.C. has nothing to disclose. A.T. has nothing to disclose. P.V. has nothing to disclose. A.L.T. has nothing to disclose. Reprint requests: Dr. Francesca Raffaelli, Marche Polytechnic University, School of Medicine, Via Tronto 10, Ancona, Italy (FAX: 39-071-220-6058; E-mail: raffi.3@virgilio.it). Fertility and Sterility â Vol. 93, No. 6, April 2010 0015-0282/10/$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2008.12.060 1976