Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2015; 19: 3426-3432 Alpha (ALA) effects on subchorionic hematoma: preliminary clinical results

G. PORCARO 1, E. BRILLO 1, I. GIARDINA 1, R. DI IORIO 2

1Department of Obstetrics and Gynecology, University of Perugia, Perugia, Italy 2Department of Obstetrics, Gynecology and Urology, San Pietro Fatebenefratelli Hospital, Sapienza University, Rome, Italy

Abstract. – OBJECTIVE : The clinic use of al - Key Words: pha Lipoic Acid (ALA) is linked to its capability Threatened miscarriage, Alpha Lipoic Acid (ALA), to exert effects and, more interest - , Randomized controlled study, Subchori - ingly, to counteract the pathologic changes of onic hematoma. complex networks of cytokines, chemokines and growth factors, restoring their physiological state. The aim of this randomized controlled clin - ical trial was to test the contribution of oral sup - Introduction plementation of ALA to the standard treatment with Progesterone vaginal suppositories, in heal - Threatened miscarriage is characterized by ing subchorionic hematomas in patients with vaginal bleeding , other than spotting , and pelvic threatened miscarriage. Controls were adminis - pain within the first 20 weeks of gestation. It af - tered only Progesterone suppositories. fects around 20% of women, and spontaneous PATIENTS AND METHODS : Nineteen pregnant abortion occurs approximately in half of these women in the first trimester of gestation, with pregnancies 1. Up to 80% of these losses appear threatened miscarriage and ultrasound evidence 2 of subchorionic hematoma, were included in the to be caused by genetic abnormalities . Also in - trial and randomly divided in two groups: con - flammatory processes and immunologic disor - trols, treated with 400 mg Progesterone (200 mg ders can induce miscarriage 3-4 . The presence of a 2 times per day), given by vaginal suppositories, first-trimester subchorionic hematoma is a suit - and case study treated with the same Proges - able marker of patients at greater risk for adverse terone dosage, plus ALA, given orally at the pregnancy outcome 5. In particular, the presence dose of 600 mg (300 mg 2 times per day, DAV®, of a very large first-trimester subchorionic Lo.Li. Pharma srl, Italy). Sixteen patients com - hematoma has been suggested to be associated pleted the trial. Treatment was performed until complete resolution of the clinical picture. with a 46% risk of adverse pregnancy outcome RESULTS: In both groups, the subjects im - (spontaneous abortion and premature rupture of proved significantly but, in general, a better and membranes) 6. Therefore, its resorption represents faster evolution in the major signs of threatened a fundamental target to prevent preterm delivery. miscarriage was observed in the subjects treat - Th1, Th2, Th17 and regulatory T (Treg) cells 7 ed with ALA and Progesterone. In these patients, are essential in immunity 8 and pregnancy 9 and the speed of resorption of subchorionic are involved in boosting and/or dampening in - hematoma was significantly ( p ≤ 0.05) superior 10-12 compared to controls. The ALA and Proges - flammation . Th1 cells secrete inflammatory terone group showed a faster decrease or disap - cytokines, such as Tumor Necrosis Factor (TNF) pearance of all symptoms than that observed in and Interferon-gamma (IFN-gamma), but also In - the control group, however the difference was terleukine-10 (IL-10 ). Instead, Th2 cells produce not significant. several Interleukins, such as IL-4, IL-10, etc. 8 Fi - CONCLUSIONS: These preliminary results nally, Th17 cells , similarly involved in suggest that ALA supplementation significantly pregnancy 13 , produce the proinflammatory cy - contributes to speed up the process of restora - tokine IL-17 7,14-15 . On the other side, Treg cells tion of physiological conditions in threatened miscarriage and ameliorates the medical condi - play an essential role in the development of im - tions of both the mothers and the foetus, proba - munoregulation and induction of tolerance. IL-1 bly modulating the networks of cytokines, beta, a pro-inflammatory cytokine , mainly re - growth factors and other molecules . leased by monocytes and macrophages, is also

3426 Corresponding Author: Giusi Porcaro, MD; e-mail: [email protected] Alpha Lipoic Acid (ALA) effects on subchorionic hematoma: preliminary clinical results involved in gestation, exerting either an aborto - with pelvic pain and/or vaginal bleeding, and genic effect (probably prevailing) or a protection subchorionic hematomas, observed by ultrasound from miscarriage 16 . The maintenance of a healthy examination. The exclusion criteria were the gestation needs an adequate interplay between presence of multiple pregnancy, pre-pregnancy or Th1- and Th2-mediated immunity . Furthermore, gestation pathologies (arterial hypertension, ma - a broad spectrum of different molecules, such as ternal autoimmune diseases, antiphospholipid growth factors, cytokines and chemokines, are syndrome) , treatment with anti-hypertensive and implicated in acute wound and in its healing. anti-coagulant drugs, that could be confusing fac - TNF-alpha, IL-1 and IL-6 induce inflammation tors . The study was approved by the Bioethics in the acute wound (as well known, inflammation Committee SIFIOG (Italian Society of Phytother - is required for fighting infection ), and are in - apy and Dietary Supplements in Obstetrics and volved also in reepithelialization 17 . Vascular En - Gynecology ). All pregnant women were in - dothelial Growth Factor (VEGF ) stimulates ep - formed about the nature of the study and verbally ithelialization and collagen deposition in acceded to participate to it. The was wounds 18 , and Alpha-Smooth Muscle Actin (al - a randomized controlled study, with 1:1 alloca - pha-SMA ) takes part in fibrogenesis 19 , i.e. vascu - tion ratio. The study had two groups: controls , logenesis and angiogenesis. Inflammation, extra - with 400 mg Progesterone (200 mg 2 times per cellular matrix (ECM) degradation and remodel - day ), given by vaginal suppositories, and treated ing of new ECM are positively affected also by (case study) with the same Progesterone dosage , matrix metalloproteases (MMPs), though a selec - plus ALA (DAV ®, Lo.Li. Pharma srl , Rome, tive control of their activity is necessary to avoid Italy ), given orally at the dose of 600 mg (300 the wound worsening 20 . mg 2 times per day, in the morning and in the The use of progesterone in the managing of evening ,). In our study we used only these two threatened miscarriage, is prescribed by thera - groups of treatment , because it was impossible to peutic protocols, although, few evidences support add a third arm without treatment , owing to the its efficacy 21 . Bioethics Committee refusal to accept this possi - Our aim was to verify whether alpha lipoic bility. The protocol prescribed to administer all acid (ALA), a natural and safe molecule with an compounds until complete resolution of the clini - anti-inflammatory and antioxidant profile 22-24 , cal picture . can improve efficaciously the therapy with prog - Primary outcome: reduction/disappearance of esterone . The reason why ALA was used de - subchorionic hematoma detected by sonography , pends on its capability to counteract the patho - vaginal bleedings, pelvic pain and uterine con - logic changes of complex networks of cytokines, tractions . The size of the hematoma was com - chemokines and growth factors , restoring their pared with the size of the gestational sac during physiological state, and in consideration that the examination and classified as small (< 20% such factors are involved also in inducing threat - of the gestational sac), medium (20 %- 50% of the ened miscarriage . gestational sac), or large (> 50% of the gestation - The clinical trial has evaluated the innovative al sac) 6. In the case study the hematoma was therapeutic effects of ALA, by oral route, and medium-sized in five patients, and large in four Progesterone vaginal suppositories, compared cases, whereas three controls had a large with Progesterone alone, in reducing significant - hematoma and four a medium-sized one . Pro - ly the subchorionic hematomas and other symp - gressive hematoma resorption during treatment toms linked to spontaneous abortion . was calculated as percentage between two subse - quent time points for each patient and the medi - um value for each group has been obtained and Patients and Methods compared. With the aim of recording systemati - cally the changes in pain symptomatology for the Pregnant women with threatened miscarriage subsequent analysis and evaluation, patients re - were assessed for eligibility from March 2013 to ceived a sheet where they had to note the daily February 2014 at the Division of Obstetrics and number of contractions during the time period Gynecology, University of Perugia, Italy . We ap - between two medical examinations . plied the following inclusion criteria: the subjects Secondary outcomes : placental abruption, de - had to be between 20 and 40 years old, between crease of miscarriage incidence and preterm de - 6th and 13 th week of physiological pregnancy, livery .

3427 G. Porcaro, E. Brillo, I. Giardina, R. Di Iorio

The improvements have been monitored dur - At ultrasounds hematoma appears such as an ing all study, and evaluated respect to the base - anechoic area (i.e., echo free on a sonographic line (t = 0). image) of variable size, located between the uter - ine wall and the chorionic membrane (Figures 1A , B and C ). Statistical Analysis The improvement was general, but with a very Data analysis was performed using GraphPad different time trend in the healing, which was Prism software (GraphPad Software, Inc., La Jol - clearly much faster in patients treated with ALA la, CA, USA) . The M-estimator of regression plus Prog (Figure 2A). curve, used for the statistical analysis of signifi - cance, was calculated, for each patient, consider - ing only the first 100% value in the healing. The Mann-Whitney U -test was applied for determin - ing the significant difference between the two groups. The level of statistical significance was set below p = 0.05.

Results

Among twenty-nine assessed subjects, with threatened miscarriage, a total number of nine - teen pregnant women were included in the study. A The age range was from 26 to 39 years, with a mean value of 32.5 ± 3.9 years, and a mean body weight of 58.94 ± 7.01 kg . The mean age in con - trol group was 31 .7 ± 2.56 years, and in treated group was 33 .1 ± 4.75 years. The mean body weight was, respectively, of 58.7 ± 7.2 kg and 59.11 ± 7.3 kg . The average of gestational age was 8.7 ± 1.4 weeks in the case study and 8.5 ± 0.4 weeks in the control group. There was no sta - tistically significant difference between the case study and the control group in terms of mothers’ ages and gestational ages. There were three drop- B outs: two patients, in the Progesterone group, failed to complete the study owing to the in - creased vaginal bleeding and admission to hospi - tal at the beginning of the trial, whereas one pa - tient in the case study underwent termination of pregnancy because of fetal trisomy 21. Among the sixteen patients, who completed the study , there were eight nulliparous women in the case study, and one multigravida (three subjects had a previous miscarriage), while in the control group, there were six nulliparous, and one multigravida (two subjects had previously a miscarriage). No - body had had caesarean section. Patients were C controlled after a week from the baseline, and later every fifteen days until the resolution of the Figure 1. A, Subchorionic hematoma (9 th week of preg - nancy) in ALA plus Progesterone group. B, Subchorionic clinical picture. In no case, symptoms not no - th st hematoma reduction (10 week of pregnancy) in ALA plus ticed during the 1 medical examination, later af - Progesterone group. C, Complete subchorionic hematoma flicted the patient. Treatments did not cause any resorption (12 th week of pregnancy) in ALA plus Proges - adverse effect on mother or foetus. terone group.

3428 Alpha Lipoic Acid (ALA) effects on subchorionic hematoma: preliminary clinical results

The two groups were found significantly differ - ments show comparable results in the objective ent (Mann-Whitney U-Test: The U-value is 8. The resolution of uterine contractions (soft uterus), critical value of U at p ≤ 0.05 is 12. Therefore , the the results related to the subjective symptoms result is significant at p ≤ 0.05) (Figure 2B). (i.e. uterine contractions) highlighted that pa - Concerning the symptoms such as pelvic pain , tients in the case study showed a faster resolution uterine contractions , vaginal bleeding and soft of the symptoms, directly improving the life uterus, the changes of these parameters , from the quality of pregnant women. baseline (t 0) to the following medical controls (t 1 In particular, the results obtained for the t3), are shown in Table I . hematoma resorption, detected by scan , are of Only in soft uterus the changes have been the particular interest. In fact, the ALA plus Proges - same in both groups, whereas evident differ - terone group showed a clear improvement, statis - ences, but not statistically significant, can be ob - tically significant , whereas the other effects (on served in parameters such as pelvic pain , uterine vaginal bleeding, pelvic pain and uterine contrac - contractions and vaginal bleeding . In both tions) were not statistically significant . groups, episodes of threatened miscarriage, The use of progesterone in the management of preterm deliveries, placental abruption, were not threatened miscarriage is prescribed by therapeu - recorded . tic protocols, but the real extent of its efficacy in reducing miscarriages is debated 21,25-29 . Much more interesting and innovative is the therapeutic Discussion activity of ALA , a safe natural molecule . The bi - ological systems synthetize this compound, but it Our preliminary results indicate that patients can be also assimilated in food or nutritional sup - treated with ALA plus Progesterone have had a plements . ALA exerts a powerful anti-inflamma - better and faster evolution of the clinical signs tory and antioxidant activity 22-24 . It is important and symptoms related to threatened miscarriage . to highlight that ALA substantially decreases the The monitoring of subchorionic hematoma , secretion of inflammatory cytokines, such as vaginal bleeding, pelvic pain , and uterine con - TNF-alpha, IL-1beta and IL-17, whereas it stim - tractions showed that all signs and symptoms de - ulates the release of the anti-inflammatory cy - creased or disappeared in the group treated with tokine IL-10; it significantly suppresses the num - ALA plus Progesterone, before than in controls ber and percentage of encephalitogenic Th1 and (Progesterone alone ). Indeed, despite the treat - Th17 cells, increases splenic Treg-cells, inhibits ) % (

n e o i u t l p a r v

o r s o e t r

a a m i m t o s t e - a m M e H

A Time B

Figure 2. A, Progress of subchorionic hematoma resorption ( ∆ percentage of Mean ± SEM), detected by sonography at dif - ferent time points of treatment, in ALA plus Prog. group (n = 9) and Prog. group (n = 7). The size of the hematoma was com - pared (%) with the size of the gestational sac during the examination. Progressive hematoma resorption during treatment was calculated as ∆ percentage between two subsequent time points for each patient and the medium value for each group has been obtained and compared. B, Subchorionic hematoma resorption: statistical significance of the regression slopes (ALA plus Prog. group vs. Prog. group). Mean ± SEM of M-estimator for ALA plus Prog. Group (n = 9) and Prog. Group (n = 7). The M- estimator of regression curve, used for the statistical analysis of significance of hematoma resorption, was calculated, for each patient, considering only the first 100% value in the healing. The two groups were found significantly different (Mann-Whit - ney U-Test: The U-value is 8. The critical value of U at p ≤ 0.05 is 12. Therefore the result is significant at p ≤ 0.05*).

3429 G. Porcaro, E. Brillo, I. Giardina, R. Di Iorio

Table I. Effects of the treatments (ALA + Progesterone or Progesterone) on symptoms of threatened miscarriage.

Baseline 1st medical 2nd medical 3rd medical

med. exam. t 0 control t 1 control t 2 control t 3

Symptoms ALA+Pr Pr. ALA+Pr. Pr. ALA+Pr. Pr. ALA+Pr. Pr. (n = 9) (n = 7) (n = 9) (n = 7) (n = 9) (n = 7) (n = 9) (n = 7) Vag. bleed. 5 (55%) 4 (57%) 1 (11%) 2 (29%) 0000 Abd. pain 9 (100%) 7 (100%) 2 (22%) 4 (57%) 0 1 (14%) 00 Soft uterus 3 (33%) 4 (57%) 9 (100%) 7 (100%) 9 (100%) 7 (100%) 9 (100%) 7 (100%) Uter. contr. 8 (89%) 7 (100%) 4 (44%) 7 (100%) 0 5 (71%) 0 1 (14%)*

Data are given as sample size (number and percentage) of each group at different time points: t 0: baseline medical examination; t1: 1 week after baseline medical examination; t 2: 3 weeks after baseline medical examination; t 3: 5 weeks after baseline med - ical examination. *The symptom disappeared at the subsequent medical examination. the production of vascular and intracellular adhe - Conclusions sion molecules (VCAM-1 and ICAM-1) 30-35 . Moreover , ALA treatment also inhibits IL-6 pro - Our preliminary results suggest that ALA sup - duction and decreases T cell proliferation and ac - plementation may play a pivotal role in signifi - tivation 31 . These data suggest that ALA can en - cantly ameliorating the medical conditions of hance systemic Treg-cells which counteract the both the mothers and the foetus, reducing the inflammatory response. Acute treatments with complications linked to threatened miscarriage, ALA have been shown to directly decrease the and improving the pregnancy outcome. expression of NF- κB and matrix metallopro - teinase-9 (MMP-9) 20 . In an animal model, ALA –––––––––––––––– –-– –– was able in reducing the time of wound healing Conflict of Interest in uterine full thickness injury, increasing the Vascular endothelial growth factor (VEGF) and The Authors declare that there are no conflicts of interest. alpha-Smooth Muscle Actin (alpha-SMA) distri - 36 bution in tissues . ALA supplementation de - References creases prostaglandin E2 (PGE2) secretion 33 , and reduces pain and paraesthesia in patients with 1) YASSAEE F, S HEKARRIZ -F OUMANI R, A FSARI S, F ALLAHIAN sciatica, carpal tunnel syndrome and diabetic M. The Effect of progesterone suppositories on neuropathy 37-40 . In addition, other studies high - threatened abortion: a randomized clinical trial. J lighted its efficacy in contrasting TNF-induced Reprod Infertil 2014; 15:147-151. weakening of human fetal membranes 41,42 , and 2) KLIMAN HJ, M ILANO KM . The majority of miscar - the involvement of its deficiency in damages to riages are caused by genetic abnormalities. Fertil foetus development 43-45 . The therapeutic use of Steril 2013; 100: S306. ALA is safe and tolerable : in humans the oral 3) KWAK -K IM J, Y ANG KM, G ILMAN -S ACHS A. Recurrent pregnancy loss: a disease of inflammation and ALA supplementation at doses up to 2400 coagulation. J Obstet Gynaecol Res 2009; 35: 23 mg/day does not show any adverse effect . Sum - 609-622. marizing, our results could be explained by sev - 4) CHRISTIANSEN OB, N IELSEN HS, K OLTE AM . Inflamma - eral physiological actions above mentioned, and tion and miscarriage. Semin Fetal Neonatal Med mainly due to ALA activity in decreasing TNF, 2006; 11: 302-308. IL-1, IL-6, IL-17, and increasing splenic Treg- 5) NAGY S, B USH M, S TONE J, L APINSKI RH, G ARDÓ S. cells and IL-10 . Furthermore , ALA may speed up Clinical significance of subchorionic and retropla - the process of hematoma resorption, by enhanc - cental hematomas detected in the first trimester of pregnancy. Obstet Gynecol 2003; 102: 94-100. ing the levels of Vascular Endothelial Growth 6) LEITE J, R OSS P, R OSSI AC, J EANTY P. Prognosis of very Factor (VEGF) and alpha Smooth Muscle Actin large first-trimester hematomas. J Ultrasound (alpha-SMA) and decreasing the expression of Med 2006; 25: 1441-1445. NF- κB and MMP-9 . All these effects have also 7) PECK A, M ELLINS ED. Plasticity of T-cell phenotype as result the acceleration of tissue repair and an - and function: the T helper type 17 example. Im - giogenesis. munology 2009; 129: 147-153.

3430 Alpha Lipoic Acid (ALA) effects on subchorionic hematoma: preliminary clinical results

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