Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2016; 20: 1656-1663 Resolution of subchorionic hematoma and symptoms of threatened using vaginal alpha lipoic acid or progesterone: clinical evidences

M. COSTANTINO 1, C. GUARALDI 2, D. COSTANTINO 3

1Department of Chemistry and Pharmaceutical Technologies, University of Ferrara, Ferrara, Italy 2U.O.C. Obstetrics and Gynecology, Valdagno Hospital, Vicenza, Italy 3Women's Health Center, Ferrara, Italy

Abstract. – OBJECTIVE : Alpha Lipoic Acid hematoma can appear during the first trimester of (ALA) is a safe natural molecule that exerts a se - (early pregnancy) and it is a typical lective immunomodulating activity with antioxi - anomaly of this gestational period. It is due to the dant and anti-inflammatory properties. This ran - domized controlled clinical trial (RCT) tested the partial separation of the from the under - effect of the vaginal administration with ALA or lying decidua. Around 18% of all cases of vagi - Progesterone, in subchorionic hematoma re - nal bleeding in the first trimester are caused by a sorption in women with threatened miscarriage. subchorionic hematoma 1, which is detected only PATIENTS AND METHODS: 400 mg of vaginal via scan. It shows a normal gestation - Progesterone or 10 mg of vaginal ALA were ad - al sac close to an anechoic area , or echo free on a ministered to sixty-two pregnant women, in the first trimester of gestation with threatened mis - sonographic image, of variable size, having the carriage and subchorionic hematoma. Controls typical form of a half-moon . The scan image were patients who chose not to receive any plays a pivotal role to analyze any improvement treatment. or worsening. Also the clinical examination is re - RESULTS: In the ALA group the subchorionic quired, because the sonographic results have to hematoma was reabsorbed more quickly in com - be related to clinical symptoms (i.e. bleeding, parison with the progression detected in Prog - 2 esterone group ( p ≤ 0.05). The other parameters pelvic pain ) . The manifestation of a first- checked (pelvic pain and ) did trimester subchorionic hematoma represents a not show any significant difference and a small - very appropriate marker for identifying patients er number of was recorded in the at greater risk for threatened miscarriage 3, a seri - ALA group, compared to Progesterone group. ous problem in the first 20 weeks of pregnancy, CONCLUSIONS: Our data provides the first evidence of the efficacy of ALA, administered by characterized by vaginal blood leaking, other vaginal route, in the healing process of patients than spotting, and pelvic pain. The presence of a with threatened miscarriage, thus supporting large first-trimester subchorionic hematoma was the normal course of pregnancy. Clinical trial related to a 46% risk of adverse pregnancy out - registration number: NCT02601898 (ClinicalTri - come, i.e. premature and als.gov registry). spontaneous 4. Consequently, its resorp - Key Words: tion is an essential goal to avoid early pregnancy Threatened miscarriage, Vaginal alpha lipoic acid loss. Among several causes of miscarriages, up (ALA), Vaginal progesterone, Randomized controlled to 80% are genetic 5, but also inflammatory study, Subchorionic hematoma. processes and immunologic disorders can be mentioned 6,7 . A complex pathophysiological mechanism underlies the event of threatened Introduction miscarriage, where T helper (Th) cells (both Th1 and Th2), Th 17 and regulatory T (Treg) cells 8,9 Subchorionic hematoma is a gathering of are involved in combination with numerous sig - blood in the subchorial area, between the mem - nal molecules, exerting pro- or anti-inflammatory branes of the and the chorion, deriving effects. Th1 cells are involved in cellular immu - from a subchorionic hemorrhage. This kind of nity, and Th2 in humoral immunity. This classifi -

1656 Corresponding Author: Demetrio Costantino, MD; e-mail: [email protected] Treatment with vaginal lipoic acid for threatened miscarriage cation reflects the type and prevalence of cy - anti-coagulant or anti-hypertensive drugs. Fur - tokines secreted by each subset of cells 10,11 . Th17 thermore, patients with previous miscarriage (no cells play a central role in giving rise to inflam - more than three miscarriages) underwent to ex - mation 9,12,13 , whereas Treg cells usually turn amination to exclude karyotype abnormalities down the immune response 9. Especially in the and any possible clinical factor linked to recur - first months of pregnancy, inflammatory process rent pregnancy loss. All the patients gave an in - is essential to protect the host from pathogens, formed consent before entering the study. The allowing the continuation of pregnancy 14 . Inter - protocol was approved by the Ethics Committee . estingly, both excessive inflammation and im - mune suppression can prompt resorp - Study Design and Treatment Regimen tion 8,14 . In general , an adequate balance between The study was a Randomized Controlled Trial all the elements needs to be kept or restored for (RCT) with allocation concealment of patients in the maintenance of a healthy gestation. two treatments groups (1:1 ratio ): in one group The therapeutic effects of Progesterone in preg - patients received 400 mg Progesterone (Progef - nancy, in condition of threatened miscarriage , is fik ®, Effik Italia srl, two vaginal soft gel per day, approved by therapeutic protocols, but its efficacy before sleeping), and in the second one (case is strongly put in doubt and criticized 15-20 . In this study) 10 mg of ALA (DAV ® vaginal capsules, context , Alpha Lipoic Acid (ALA), a multifunc - Lo.Li. Pharma srl, Rome, Italy, one vaginal cap - tional natural molecule, is revealing a very interest - sule per day, before sleeping ). Randomization ing profile . This compound, given orally or i.v., is did not involve a third group which was formed safe at therapeutic doses, and exerts worthwhile bi - by twenty-two patients who decided not to re - ological activity, that are beneficial also for modu - ceive any treatment and it was used as control lating several mechanisms underlying threatened (Figure 1). Treatments were given until the total miscarriage 21-23 . Vaginal administration of ALA is resolution of the clinical picture . a new approach which can provide a direct effect at vaginal and uterine level. Outcomes and Follow-up The aim of this pilot study was to preliminary The primary outcome was the resolution of compare the therapeutic efficacy of ALA vs . subchorionic hematoma linked to threatened mis - Progesterone, by vaginal administration, on sub - carriage. The evaluation of the hematoma signifi - chorionic hematoma resorption in women at the cance was done comparing its size with that one first trimester of pregnancy with threatened mis - of the during the ultrasound ex - carriage . Furthermore, also the effects on pelvic amination, according to the method reported by pain and vaginal bleeding were evaluated. Nagy et al 4 which allows one to classify the sub - chorionic hematoma as small (< 20% of the ges - tational sac), medium (20%-50% of the gesta - Patients and Methods tional sac), or large (> 50% of the gestational sac) . Changes in hematoma resorption (% im - Patients provement/ worsening) during the treatment Gravid women with threatened miscarriage were obtained for each patient by calculating the were enrolled from January 2015 to August 2015 Δ percentage between two subsequent time at the Women’s Health Center, Azienda USL points. Then the average values for each group Ferrara – Italy. The inclusion criteria were: pa - were compared. The secondary outcomes were tients age 24-40 years and in the 7th to 12 th week reduction/disappearance of the subjective (pelvic of physiological gestation, with pelvic pain and pain) and objective (vaginal bleeding) symptoms. with or without moderate vaginal bleeding (com - A sheet was given to all patients to record the parable to the heaviest normal menstrual flow), evolution of these symptoms, which later had to and subchorionic hematoma, observed by sonog - be statistically analysed. raphy. The exclusion criteria were: lack of , Follow -up checks were performed at twenty absence of fetal heart tone, uterine anomaly or days (t = 1), and sixty days (t = 2) from the begin - fetal anomaly, presence of multiple pregnancy, ning of the treatment (baseline , t = 0) and both pre-pregnancy or gestation pathologies (such as clinical signs and symptoms were recorded by in - maternal autoimmune diseases, antiphospholipid vestigators. The incidence of miscarriage was eval - syndrome, arterial hypertension), diagnosis of al - uated in both groups and the treatment was judged lergic reaction to progesterone, therapies with successful if pregnancy went over 20 weeks.

1657 M. Costantino, C. Guaraldi, D. Costantino

Figure 1. Flow chart of participants over the course of the trial (Progesterone: P).

Statistical Analysis Results Percentages and mean values of sign and symptoms were calculated excluding the number Among eighty-four evaluated subjects, with of patients who miscarried. Statistical analysis of threatened miscarriage, a total number of seven - the clinical data was performed by SPSS soft - ty-six pregnant women were included in the trial ware (SPSS Inc., Chicago, IL, USA) employing according to the inclusion criteria . Patients char - Wilcoxon test for nonparametric data . A Mann- acteristics at baseline were homogeneous in both Whitney U test, with SPSS software, was used to groups, as shown in Table I . make the comparison between the single groups. The outcomes were evaluated excluding the The difference was considered statistically sig - number of patients who miscarried during the nificant if the p-value was ≤ 0.05. study.

Table I. Baseline patients features in the three groups.

Parameter Group ALA n = 27 Group P n = 27 Group C n = 22 p-value

Age (years) 29.8 ± 0.7 31.2 ± 1.6 30.4 ± 1.3 n.s. (weeks) 8.8 ± 0.2 9.1 ± 0.3 8.9 ± 0.2 n.s. Nulliparous (%) 13 (48%) 11 (41%) 9 (41%) n.s. Multiparous (%) 000n.s. Previous C-section (%) 2 (7%) 3 (11%) 1 (4%) n.s. Previous miscarriages 12 (44%) 10 (37%) 10 (66%) n.s. -1 miscarriage 6 6 5 n.s. -2 miscarriages 5 3 3 n.s. -3 miscarriages 1 1 2 n.s. Vaginal bleeding (%) 13 (48%) 14 (52%) 14 (64%) n.s. Hematoma size n.s. -Medium 24 (89%) 25 (92%) 20 (94%) n.s. -Large 3 (11%) 2 (7%) 2 (9%) n.s.

Data are expressed as mean ± SE or as number and percentage (Progesterone: P).

1658 Treatment with vaginal lipoic acid for threatened miscarriage

A blind investigator controlled the patients af - ter twenty days (t = 1), and sixty days (t = 2) from the baseline (t = 0) by vaginal ultrasound scan to check and register the evolution of sub - chorionic hematoma . The ALA group was found to be statistically different from Progesterone and control groups. The result was significant at p ≤ 0.05 (Figures 2 and 3). Patients treated with Progesterone did not show any significant differ - ence vs . controls . According to the inclusion criteria, pelvic pain was present in all the patients at the baseline and it was recorded only in 3 subjects treated with Progesterone and in 2 subjects who did not re - ceive treatment at the first medical examination (t1) and nobody at the second one (t2) . Also re - garding vaginal bleeding , the effects due to the treatments were similar . Although not significantly different, a smaller number of miscarriages was registered in the Figure 2. The progress of subchorionic hematoma resorp - ALA group: this is an interesting , positive trend , tion was detected by ultrasound in ALA group (n = 24), which should be further verified in a following Progesterone (P) group (n = 21) and Control group (n=17) at different time points of treatment and the data are shown trial with a large cohort of patients (Table II ). as Δ percentage of Mean ± SEM. Percentages and mean val - No adverse effects on foetus were detected ues were calculated excluding the number of patients who during the treatments and until the final check-up miscarried. The size of the hematoma was compared (%) of the study. Four patients in the case study with the size of the gestational sac during the examination. group reported sporadic episodes of mild vaginal Progressive hematoma resorption during treatment was cal - culated as Δ percentage between two subsequent time points burning which did not require suspension or dis - for each patient and the medium value for each group has continuation of the therapy. been obtained and compared.

Figure 3. A, Statistical significance of Δ (% improvement ) at t = 1 for the subchorionic hematoma resorption: ALA, Proges - terone (P) and control groups (mean ± SEM). Wilcoxon and Mann-Whitney U test were used. The ALA group was found sig - nificantly different ( p ≤ 0.05) from Progesterone and control groups. Patients treated with Progesterone did not show any sig - nificant difference vs controls.

1659 M. Costantino, C. Guaraldi, D. Costantino

Table II. Effects of the treatment with ALA, Progesterone (P) or no treatment (C) on symptoms and incidence of miscarriage.

Baseline 1st Medical 2nd Medical

med. exam. t 0 control t 1 control t 2 Miscarriage

Symptoms ALA PCALA PCALA PCALA PC (n = 24) (n = 21) (n =17) (n = 24) (n = 21) (n =17) (n = 24) (n = 21) (n =17) (n = 27) (n = 27) (n = 22) Pelvic pain 24 (100%) 21 (100%) 17 (100%) 0 (0%) 3 (14%) 2 (12%) – 0 (0%) 0 (0%) 365 Vag. bleed 13 (54%) 12 (57%) 11 (65%) 0 (0%) 0 (0%) 2 (12%) ––0 (0%)

Data are given as sample size (number and percentage) of each group at different time points: t 0: baseline medical examina - tion; t 1: 20 days after baseline medical examination; t 2: 60 days after baseline medical examination. Percentages were calculat - ed excluding the number of patients who miscarried.

Discussion own tissues, making Th1 cells central players in autoimmune diseases and also when extraneous As shown by our data, the subchorionic cells develop 11 . hematoma has shown a faster resorption as result Th2 cells stimulate strong antibody responses of the vaginal administration with ALA, than and eosinophil accumulation and also inhibit sev - with Progesterone . These findings are in full eral functions of phagocytic cell 11 . agreement with a very recent paper where the au - Saito and his team 8 have suggested an intrigu - thors have found an interesting effect due to the ing explanation involving, Th1 , Th2 , Th17 and oral administration of ALA plus Progesterone by regulatory T (Treg) cells in pregnancy . Th17 vaginal route vs only Progesterone 24 . cells produce IL-17, an proinflammatory cy - Data obtained in previous researches and stud - tokine, and exert a pivotal role in giving rise to ies can help in explaining these clinical results, inflammation 9,13,14 . Th 17 cells can induce patho - focusing the role played by some cytokines and genetic mechanisms in autoimmunity and acute T helper 1 (Th1) and T helper 2 (Th2) cells in transplant rejection, affecting also the develop - subchorionic hematoma formation and miscar - ment of gestation. It is worth of note that Th17 riage . The cytokine network is deeply involved cells and their released cytokine, IL-17, might in the positive or negative development of the exert a dual action on pregnancy, like IL-1. In - ongoing 25 , even though the knowl - deed, increased Th17 cells in pregnancy decidua edge of the specific underlying mechanisms is in might be disadvantageous for the maintenance of evolution. Th1 cells are essential in cellular im - physiological process 28 . On the other side, Treg munity, whereas Th2 cells are involved in hu - cells usually turn down the immune response 9, moral immunity. There are some peculiarities playing a pivotal role in the processes of im - that distinguish them: IFN-gamma and TNF-beta munoregulation and in the induction of tolerance. are released only by Th1, whereas IL-4 and IL-5 They secrete anti-inflammatory cytokines: IL-10, only by Th2. Moreover, Th1 predominantly se - IL-35, TGF-beta, which, directly or indirectly, crete IL-2, TNF-alpha, TGF-beta, and other (e .g. inhibit the release or the activity of pro-inflam - IL-10) in small amounts. On the other hand, Th2 matory cytokines . Treg cells inhibit cytotoxic ac - release higher quantities of IL-3, IL-6, IL-9, IL- tivity of natural killer (NK) cells, immunoglobu - 10, IL-13, TGF-beta, but little TNF-alpha and lin production by B cells, and dendritic cells IL-2 26,27 . Th1 cells, through their cytokines, stim - (DCs) maturation 29,30 . Th1/Th2/Th17 and Treg ulate macrophages, lymphocytes, and PMNs to lineages are associated with each other, and, in destroy bacterial pathogens 11 . Furthermore, they some cases, they are able to convert to other lin - induce the development of cytotoxic T cells , eages 8. Now , it holds to be true that in normal which play an essential role in the cell-mediated development of pregnancy inflammation is nec - immune response against the aggression of for - essary for successful implantation 14 but excessive eign agents such as viruses and tumor cells. This inflammation can cause embryo resorption, a pivotal function exerted by Th1 cells in immune process that can be counteracted in the by system sometimes can give rise to their over acti - Treg cells. On the whole , we have to keep in vation or misdirected attacks against some of our mind both excessive inflammation and immune

1660 Treatment with vaginal lipoic acid for threatened miscarriage suppression can cause embryo resorption 8,14 . A lating this network, can play a pivotal role in healthy pregnancy needs an adequate balance be - ameliorating significantly the medical condi - tween all the elements, without any unidirection - tions of mothers and foetus. al overstimulation. Our results have shown that ALA vaginal ad - The treatment with progesterone , an immune ministration can efficiently improve the medical suppressant, to ward off the risk of miscarriage picture of women with threatened miscarriage, is approved by therapeutic protocols, but its real positively affecting the hematoma resorption. In efficacy in preventing spontaneous abortion is this context, a controlled inflammatory process disputed 15-20 . In this context, Alpha Lipoic Acid is indispensable in many steps of a healthy (ALA), a safe multifunctional molecule, is re - pregnancy, also in the first trimester. Proges - vealing an interesting and innovative therapeu - terone is known to exert an immunosuppressive tic activity . In addition to being synthesized in action, whereas ALA is able to finely modulate small amounts by humans, ALA can be assimi - the complex of cells and molecules involved lated in food, taken as dietary supplement and, without a unidirectional activity. The use of this much better, vaginally administered in order to careful modulation should provide the best ther - directly reach the site of action. It is a powerful apeutic choice to treat patients with threatened antioxidant (called also the “ultimate antioxi - miscarriage, reducing, also markedly, the main dant”) because of the wide range of its actions clinical sign and symptoms. Our preliminary in regulating oxidative stress pathways and the evidences support this new approach proving capability to pass through biological mem - the first example on the vaginal use of ALA in branes 31,32 . ALA is not an immune suppressant, the clinical practice. but an immune modulator, which regulates many parameters, such as the secretion of in - –––––––––––––––– –-– –– flammatory cytokines, increases Treg-cell num - Conflict of Interest ber, inhibits the production of vascular and in - The Authors declare that there are no conflicts of interest. tracellular adhesion molecules (VCAM-1 and ICAM-1), reduces the expression of CD4 on the surface of blood mononuclear cells, and blocks the activation and cytotoxicity of natural killer References (NK) cells 21,22,33,34 . Treatments with ALA in - hibits the expression of matrix metallopro - 1) SAUEBREI EE . Early pregnancy: pre-embrionic and 35 embryonic periods. In: Sauebrei EE, Nguyen teinase-9 (MMP-9) which is involved in the KT, Nolan RL, editors. A practical guide to ultra - degradation of the extracellular matrix , and it sound in obstetrics and gynecology. Philadel - also reduces the secretion prostaglandin E2 phia (PA): Lippincott-Raven Publishers, 1998; (PGE2) 36 . pp. 122-131. In particular, because subchorionic hematoma 2) PEDERSEN JF, M ANTONI M. Prevalence and signifi - appears to be due to immunological vasculitis in cance of subchorionic hemorrhage in threatened the decidual vessels, vaginal treatment with ALA abortion: a sonographic study. AJR Am J Roentgenol 1990; 154: 535.537. may strongly support its resorption through its 3) NAGY S, B USH M, S TONE J, L APINSKI RH, G ARDÓ S. immunomodulating activity. Clinical significance of subchorionic and retropla - In addition, several studies have highlighted cental hematomas detected in the first trimester its efficacy in contrasting the weakening of hu - of pregnancy. Obstet Gynecol 2003; 102: 94-10. 37,38 man fetal membranes . It is ascertained that 4) LEITE J, R OSS P, R OSSI AC, J EANTY P. Prognosis of ALA deficiency is responsible for the deficit in very large first-trimester hematomas. J Ultra - development of the foetus 39-41 . sound Med 2006; 25: 1441-1445. 5) KLIMAN HJ, M ILANO KM. The majority of miscar - riages are caused by genetic abnormalities. Fertil Conclusions Steril 2013; 100: S306. 6) KWAK -K IM J, Y ANG KM, G ILMAN -S ACHS A. Recurrent pregnancy loss: a disease of inflammation and In pregnancy there is cross talk among differ - coagulation. J Obstet Gynaecol Res 2009; 35: ent immune cells, which communicate by 609-622. means of messenger molecules (mainly cy - 7) CHRISTIANSEN OB, N IELSEN HS, K OLTE AM . Inflamma - tokines) strictly related in a network. In situa - tion and miscarriage. Semin Fetal Neonatal Med tion such as threatened abortion, ALA, modu - 2006; 11: 302-308.

1661 M. Costantino, C. Guaraldi, D. Costantino

8) SAITO S, N AKASHIMA A, S HIMA T, I TO M. Th1/Th2/Th17 7-month multicenter randomized controlled trial and Regulatory T-Cell paradigm in pregnancy. (ALADIN III study). Diabetes Care 1999; 22: Am J Reprod Immunol 2010; 63: 601-610. 1296-1301. 9) PECK A, M ELLINS ED . Plasticity of T-cell phenotype 24) PORCARO G, B RILLO E, G IARDINA I, D I IORIO R. Alpha and function: the T helper type 17 example. Im - Lipoic Acid (ALA) effects on subchorionic munology 2010; 129: 147-153. hematoma: preliminary clinical results. Eur Rev 10) CARTER LL, D UTTON RW . Type 1 and Type 2: a fun - Med Pharmacol Sci 2015; 19: 3426-3432. damental dichotomy for all T-cell subsets. Curr 25) WEGMANN TG, L IN H, G UILBERT L, M OSMANN TR . Bidi - Opin Immunol 1996; 8: 336-342. rectional cytokine interactions in the maternal-fe - 11) ROMAGNANI S. T-cell subsets (Th1 versus Th2). tal relationship: is successful pregnancy a TH2 Ann Allergy Asthma Immunol 2000; 85: 9-18. phenomenon?. Immunol Today 1993; 14: 353- 356. 12) CROME SQ, W ANG AY, L EVINGS MK. Translational mi - ni-review series on Th17 cells: function and regu - 26) RAPHAEL I, N ALAWADE S, E AGAR TN, F ORSTHUBER TG . T lation of human T helper 17 cells in health and cell subsets and their signature cytokines in au - disease. Clin Exp Immunol 2010; 159: 109-119. toimmune and inflammatory diseases. Cytokine 2015; 74: 5-17. 13) ROMAGNANI S, M AGGI E, L IOTTA F, C OSMI L. A NNUNZIA - TO F. Properties and origin of human Th17 cells, 27) GEGINAT J, P ARONI M, M AGLIE S, A LFEN JS, K ASTIRR I, Mol Immunol 2009; 47: 3-7. GRUARIN P, D E SIMONE M, P AGANI M, A BRIGNANI S. Plasticity of human CD4 T cell subsets. Front Im - 14) Mor G, Cardenas I, Abrahams V, Guller S. In - munol 2014; 16: 630. doi: 10.3389/fim - flammation and pregnancy: the role of the im - mu.2014.00630. eCollection 2014. mune system at the implantation site. Ann N Y Acad Sci 2011; 1221: 80-87. 28) SANTNER -N ANAN B, P EEK MJ, K HANAM R, R ICHARTS L, ZHU E, F AZEKAS DE ST GROTH B, N ANAN R. Systemic 15) PALAGIANO A, B ULLETTI C, P ACE MC, D E ZIEGLER D, increase in the ratio between Foxp3+ and IL-17- CICINELLI E, I ZZO A. Effects of vaginal proges - producing CD4+ T cells in healthy pregnancy but terone on pain and uterine contractility in pa - not in preeclampsia. J Immunol 2009; 183: 7023- tients with threatened abortion before twelve 7030. weeks of pregnancy. Ann N Y Acad Sci 2004; 1034: 200-210 . 29) SAKAGUCHI S. Naturally arising Foxp3-expressing CD25 + CD4 + regulatory T cells in immunologi - 16) TIEN JC, T AN TY . Non-surgical interventions for cal tolerance to self and non-self. Nat Immunol threatened and recurrent miscarriages. Singa - 2005; 6: 345-352. pore Med J 2007; 48: 1074-1090. 30) AKBAR AN, V UKMANOVIC -S TEJIC M, T AAMS LS, M ACALLAN 17) QURESHI NS. Treatment options for threatened DC . The dynamic co-evolution of memory and miscarriage. Maturitas 2009; 65 Suppl 1: S35-41. regulatory CD4+ T cells in the periphery. Nat Rev 18) DAYA S. Luteal support: progestogens for pregnan - Immunol 2007; 7: 231-237. cy protection. Maturitas 2009; 65 Suppl 1: S29- 31) SHAY KP, M OREAU RF, S MITH EJ, S MITH AR, H AGEN 34; TM . Alpha-lipoic acid as a dietary supplement: 19) DUAN L, Y AN D, Z ENG W, Y ANG X, W EI Q. Effect of molecular mechanisms and therapeutic poten - progesterone treatment due to threatened abor - tial. Biochim Biophys Acta 2009; 1790: 1149- tion in early pregnancy for obstetric and perinatal 1160. outcomes. Early Hum Dev 2010; 86: 41-43. 32) MILLER SL, W ALLACE EM, W ALKER DW . Antioxidant 20) NORMAN JE, M ARLOW N, M ESSOW CM, S HENNAN A, Therapies: A Potential Role in Perinatal Medicine. BENNETT PR, T HORNTON S, R OBSON SC, M CCONNACHIE Neuroendocrinology 2012; 96: 13-23. A, P ETROU S, S EBIRE NJ, L AVENDER T, W HYTE S, N ORRIE 33) WANG KC, T SAI CP, L EE CL, C HEN SY, L IN GJ, Y EN MH, J; OPPTIMUM STUDY GROUP . Vaginal progesterone SYTWU HK, C HEN SJ . Alpha-Lipoic acid enhances prophylaxis for (the OPPTIMUM endogenous peroxisome-proliferator-activated re - study): a multicentre, randomised, double-blind ceptor-gamma to ameliorate experimental au - trial. Lancet. 2016 Feb 23. pii: S0140- toimmune encephalomyelitis in mice. Clin Sci 6736(16)00350-0. doi: 10.1016/S0140-6736(16) (Lond) 2013; 125: 329-340. 00350-0. [Epub ahead of print]. 34) COSTANTINO M, G UARALDI C, C OSTANTINO D, D E GRAZIA 21) CREMER DR, R ABELER R, R OBERTS A, L YNCH B. Safety S, U NFER V. Peripheral neuropathy in obstetrics: evaluation of alpha-lipoic acid (ALA). Regul Toxi - efficacy and safety of alpha-lipoic acid supple - col Pharmacol 2006; 46: 29-41. mentation. Eur Rev Med Pharmacol Sci 2014; 18: 22) CREMER DR, R ABELER R, R OBERTS A, L YNCH B. Long- 2766-2771. term safety of alpha-lipoic acid (ALA) consump - 35) GORCA A, H UK -K OLEGA H, P IECHOTA A, K LENIEWSKA P, tion: a 2-year study. Regul Toxicol Pharmacol CIEJKA E, S KIBSKA B. Lipoic acid – biological activity 2006; 46: 193-201. and therapeutic potential. Pharmacol Rep 2011; 23) ZIEGLER D, H ANEFELD M, R UHNAU KJ, H ASCHE H, Lo - 63: 849-858. bisch M, Schütte K, Kerum G, Malessa R. 36) LI G, F U J, Z HAO Y, J I K, L UAN T, Z ANG B. Alpha- Treatment of symptomatic diabetic polyneu - lipoic acid exerts anti-inflammatory effects on ropathy with the antioxidant alpha-lipoic acid: a lipopolysaccharide-stimulated rat mesangial cells

1662 Treatment with vaginal lipoic acid for threatened miscarriage

via inhibition of Nuclear Factor Kappa B (NF- κB) 39) AL GHAFLI MH, P ADMANABHAN R, K ATAYA HH, B ERG B. signaling pathway. Inflammation 2015; 38: 510- Effects of alpha-lipoic acid supplementation on 519. maternal diabetes-induced growth retardation 37) MOORE RM, N OVAK JB, K UMAR D, M ANSOUR JM, Mer - and congenital anomalies in rat . Mol Cell cer BM, Moore JJ. Alpha-lipoic acid inhibits tumor Biochem 2004; 261: 123-135. necrosis factor-induced remodeling and weaken - 40) PADMANABHAN R, M OHAMED S, S INGH S. Beneficial ef - ing of human fetal membranes. Biol Reprod fect of supplemental lipoic acid on diabetes-in - 2009; 80: 781-787. duced pregnancy loss in the mouse. Ann N Y 38) MOORE RM, S CHATZ F, K UMAR D, M ERCER BM, A BDEL - Acad Sci 2006; 1084: 118-131. RAHIM A, R ANGASWAMY N, B ARTEL C, M ANSOUR JM, 41) ANTONIO AM, G ILLESPIE RA, D RUSE -M ANTEUFFEL MJ. LOCKWOOD CJ, M OORE JJ . Alpha-lipoic acid inhibits Effects of lipoic acid on antiapoptotic genes in thrombin-induced fetal membrane weakening in control and ethanol-treated fetal rhombencephalic vitro. Placenta 2010; 31: 886-892. neurons. Brain Res 2011; 1383: 13-21.

1663