Maternal Serum Tumor Necrosis Factor-alpha in Patients with Missed and Recurrent Miscarriage

Nadia Mudher Al-Hilli College of Medicine, Universityof Babylon, Hilla, Babylon, Iraq.

M J B Abstract Background: TNF-α is one of the cytokines produced by the immune system. Many studies have been done to investigate the role of the immune system in first trimester pregnancy loss. Objective: To evaluate the association of TNF-α with recurrent and missed miscarriage. Method: a case-control study involved 35 women, 15 with recurrent miscarriage (at least three consecutive spontaneous abortions) , 10 with missed miscarriage and 10 with normal first trimester pregnancy (control group). The study was conducted from the 1st of January to 30th of June 2008. Blood samples were taken from these women, TNF-α was measured in the sera by an immunoenzymometric assay using Biosource TNF-α EASIA kit (Biosource Europe S.A.). Results: student t-test was applied to analyze the difference in serum TNF-α level between the groups. The level of significance was defined at P value < 0.05. Significantly higher levels of TNF-α was found in women with recurrent miscarriage as compared to control group (68.94 pg/ ml versus 27.40 pg/ml). The difference was also significant when comparing women with missed miscarriage with the control group (54.39 pg/ml versus 27.40 pg/ml). The level of TNF-α was higher in women with recurrent miscarriage compared to women with missed miscarriage, however the difference between them was not statistically significant. Conclusion: TNF-α has a significant association with miscarriage, the level is significantly higher in patients with recurrent and missed miscarriage compared to normal pregnant women; the level is higher in those with recurrent miscarriage when compared to patients with missed miscarriage. الخلصة اشتملت الدراسة 35 امرأة, 15 امرأة يعانين من اسقاطات متكررة, 10 يعانين من اسقاط منسي و 10كن في الشهر الثلثة الولى من الحمل و يتمتعن بحمل طبيعي. تم سحب عينات الدم من المجموعة تحت الدراسة و قياس مستوى عامل تحلل الورم (Tumour Necrosis Factor TNF- α) أظهرت النتائج ان مستوى عامل تحلل الورم اعلى عند النساء اللئي يعانين من اسقاط متكرر أو اسقاط منسي مقارنة بالحوامل الطبيعيات. كما انه اعلى عند النساء ذوات السقاط المتكرر مقارنة بالنساء ذوات السقاط المنسي. مما يدل على ان عامل تحلل الورم له علقة وثيقة بحالت السقاط خصوصا المتكرر. usually the later refers to elective Introduction termination of pregnancy. Viability iscarriage implies spontaneous implies the ability of the fetus to survive Mloss of pregnancy before viability. extra-uterine life. This is generally Miscarriage replaces the term abortion as  Infections: Salmonella typhi, considered to occur at approximately 24 malaria, cytomegalovirus, weeks gestation. Rarely, fetuses born toxoplasmosis and others. before that gestation survive for short  Chemical agents: tobacco, time or at least show some signs of life. anaesthetic gases, pesticides and The World Health organization also others. included in this definition the fetus's  Immunological disorders: weight < 500 g [1]. antiphospholipid syndrome. Miscarriages are very common.  Thrombophilia (hereditary) [4]. Approximately 15 % of pregnancies end Between 1 and 2% of fertile women in miscarriage. However, it is likely that will experience recurrent miscarriage. As far more pregnancies are lost before they the majority of recurrent miscarriage are suspected, recognized or confirmed cases following investigations are [2]. There are different types of classified as idiopathic, it is generally miscarriages, these include: accepted that within the idiopathic group  Threatened: painless bleeding there is considerable heterogeneity and it from the uterus before 24 weeks is unlikely that one single pathological with the cervix not dilated and mechanism can be attributed to their the fetus alive. recurrent miscarriage history. Current  Inevitable: bleeding from the research is directed at theories related to uterus before 24 weeks with implantation, trophoblast invasion and pain and dilatation of the cervix. placentation [5].  Incomplete: part of the Immunity plays an important role at the conceptus has been expelled time of implantation. Many studies in with continuous bleeding due to animals and human indicate that some tissue retained. degree of systemic and uterine  Complete: the whole conceptus inflammation is necessary both for has been expelled. normal implantation and pregnancy.  Missed: pregnancy failure However, if the inflammation becomes (gestational sac containing dead too excessive it might cause pregnancy embryo or fetus before 24 complications such as fetal resorption / weeks) is identified before miscarriage. The main regulator of the expulsion of fetal and placental correct level of inflammation at the feto- tissue. maternal interface seems to be the  Recurrent: three or more uterine CD16 and CD56 bright natural consecutive miscarriages [3]. killer cells. Trophoblast debris, apoptotic Aetiology: Many factors have been cells and progesterone probably regulate implied in the causation of miscarriage. the production of inflammatory These include: cytokines from these cells. Miscarriage  Chromosomal abnormalities: of karyotypically normal embryos may trisomies, triploidy and occur when the level of inflammation monosomies. falls outside the optimal range, this may  Endocrine disorders: diabetes, be associated with high tumor necrosis thyroid disease, luteal phase factor (TNF)-α production [6]. deficiency, polycystic ovary TNF- was first identified as a syndrome. cytokine secreted by endotoxin-activated  Abnormalities of the uterus: macrophages that induced the necrosis of uterine anomalies, endometrial tumours. TNF- is now known as a adhesions and cervical pluripotent cell mediator and angiogenic incompetence. cytokine that promotes the production of

2 first trimester pregnancy were taken as other cytokines in various cells. The control group. human endometrium is characterized by Women were submitted to a a variety of cell types, including questionnaire including, age, gravidity fibroblasts, immune cells, vascular cells and parity, number of miscarriages, and epithelial cells, all of which express gestational age, medical illnesses, drug TNF- . Studies suggest a local role for history,and investigations done for the TNF- in a variety of normal cause of miscarriage. endometrial functions. Increased Exclusion criteria were: expression of this cytokine was shown to  Identified causes of miscarriage, cause pathophysiological effects e.g. infection or medical disease reflected by its involvement in of the mother. implantation failure, abortion and  Conditions associated with endometriosis [7]. increased TNF-α level, e.g. Another mechanism through which TNF- hyperemesis gravidarum. α may participate in the process of Blood samples were collected from miscarriage is that together with other women under study, sera isolated and cytokines like interferon gamma (INF-γ) TNF-α was measured by they initiate apoptosis of the corpus immunoenzymometric assay using luteum which is responsible for the Biosource TNF-α EASIA kit (Biosource maintenance of pregnancy via the Europe S.A.). TNF-α level was production of progesterone that is measured in picogrram / ml. The Kit required for the establishment of suitable providers recommend that each uterine environment during early laboratory establishes its own normal pregnancy. Inappropriate luteal function values. (luteal insufficiency) may contribute to Statistical Analysis: For statistical the high incidence of spontaneous analysis SPSS (version 10) program was abortion [8]. used. The results were represented through frequency, mean and standard Aim of Study To evaluate TNF-α deviation. Student t-test used to compare production in women with recurrent between means and study the pregnancy loss and compare it with those significance of the difference. P value with missed miscarriage and normal <0.05 was considered to be statistically pregnant women. significant. Patients and Methods Results The study was conducted in Babylon Table (1) show a comparison between Teaching Hospital for Gynecology and patients with recurrent miscarriage and Pediatrics from the first of January 2008 control group regarding the level of to 30th of June 2008. The study included TNF-α in maternal serum, the level was 35 women who attended emergency higher in those with recurrent department, out patient clinic or admitted miscarriage (68.94 versus 27.40 pg/ml) to gynecology ward. 25 women had and by using student t test we found that experienced recent miscarriage, 15 had the difference was highly significant (P recurrent miscarriage and 10 had missed value < 0.05). miscarriage. Ten women with normal

Table 1 show the difference in maternal serum TNF-α level in patients with recurrent miscarriage compared to normal pregnants. No. TNF-α level SD(±) Standard error df t P value (pg/ml) of the mean Recurrent 15 68.94 13.54 4.28 23 miscarriage 7.22 0.0005 control 10 27.40 26.43 8.363 19 missed miscarriage (54.39 versus 27.40 Table (2) compares patients with missed pg/ml ), this difference was statistically miscarriage with the control group significant ( P value < 0.05). regarding maternal serum TNF-α level, the level was higher in patients with

Table 2 show the difference in maternal serum TNF-α level in patients with missed miscarriage compared to normal pregnants. No. Mean TNF-α SD(±) Standard error df t P value level (pg/ml) of the mean Missed 10 54.39 39.69 12.35 19 miscarriage 2.62 0.02 control 10 27.40 26.43 8.36 19 miscarriage (68.94 versus 54.39 pg/ml). In table (3) we compare maternal serum However this difference between the two TNF-α level in patients with recurrent groups was not statistically significant (P and missed miscarriages. The level was value > 0.05). higher in patients with recurrent

Table 3 shows the difference in maternal serum TNF-α level between patients with recurrent and missed miscarriages. patients No. Mean TNF-α SD(±) Standard level (pg/ml) error of the df t P value mean Recurrent 15 68.94 13.54 4.28 23 miscarriage 1.36 0.9 Missed 10 54.39 39.69 12.55 19 miscarriage

4 pregnant women in the first significantly higher level of TNF-α in trimester. recurrent spontaneous abortion when 4. A difference is found in maternal compared to controls [10]. serum TNF-α level when we Regarding patients with missed compare patients with recurrent miscarriage, we find a significant miscarriage to those with missed difference in maternal serum TNF-α miscarriage. level when comparing them to control group. Recommendations A similar results obtained by Paradisi R. 1. Maternal serum TNF-α level et. al., who studied the proinflammatory should be considered as one of cytokines in women with threatened and the investigations done to women missed miscarriage and compared them with recurrent miscarriage. to normal non-pregnant women, he 2. Further studies are needed to found a significantly higher levels of identify the exact mechanism these cytokines in women with missed through which TNF-α is involved miscarriage, while no significant in the pathophysiology of difference was found in women with pregnancy loss. threatened miscarriage when compare 3. Studies are needed to evaluate the them to control group [11]. association of other inflammatory This mean that a similar mechanism may mediators in the process of exist in the pathophysiology of recurrent miscarriage. and missed miscarriage and to study the 4. Inhibitory factors for TNF-α may two groups together we compare the be used as a therapeutic agent in maternal serum TNF-α level in patient women with recurrent with recurrent and missed miscarriage, miscarriage who have elevated the level was higher in patients with serum TNF-α level. recurrent miscarriage though the difference was not statistically References significant. This may be explained by the 1. Cabill D. J, Wardle P. G. possibility that women with one or two Bleeding and Pain in Early missed miscarriages may have a Pregnancy. High Risk Pregnancy derangement in her immune system that Management Options. Saunders is not so great as in those with recurrent Elsevier 3rd edition 2005. miscarriage or that it is not the same Chapter 4, P 84-104 mechanism act each time as she may 2. Wilcox A. J. Incidence of early have successful pregnancies loss of pregnancy. New England Journal of Medicine. Volume Conclusions 319, number 4, July 1988, P 189- 1. There is an association between 194. elevated maternal serum TNF-α 3. Grudzinskas J. D, Miscarriage, level and early pregnancy loss. ectopic pregnancy and 2. The difference is highly trophoblastic disease. Dewhurst's significant when we compare Textbook of Obstetrics and patients with recurrent Gynaecology for Postgraduates. miscarriage with normal pregnant Blackwell Science 6th edition ladies in the first trimester. 1999. Chapter 7, p61-75. 3. A significant difference is found 4. Monga A. Disorders of Early in maternal serum TNF-α level Pregnancy. Gnaecology by Ten when we compare patients with Teachers. Hodder Arnold 18th missed miscarriage to normal

5 factor alpha- induced death of edition 2006. Chapter 8, P 89- microvascular endothelial cells 102. of the corpus luteum. Repro Biol 5. Raj Rai. Recurrent miscarriage. Endocrinol. Journal. 2003; 1:17. Dewhurst's Textbook of 9. Daher S, Camano L, Cytokines Obstetrics and Gynaecology for in recurrent pregnancy loss. Postgraduates. Blackwell Journal of Reproductive Science 7th edition 2007. Chapter Immunology. 2004 June; 62(1- 13, page 100-105. 2): 151-7. 6. Christiansen O., Nielsen H. 10. Kim HR, Park AJ, Lee MK. Inflammation and miscarriage. CD56/CD16 expression on Seminars in Fetal and Neonatal mononuclear cells and Medicine. 2003. volume 11, concentration of serum TNF- Issue 5, P 302-305. alpha in recurrent spontaneous 7. Morin M, Sengers V, Tumour abortion. Korean J Lab Med. necrosis factor- up-regulates 2006 Jun; 26(3): 198-203. macrophage migration inhibitory 11. Paradisi R. , Porcu E. Maternal factor expression in endometrial Serum Levels of Pro- stromal cells via the nuclear Inflammatory Cytokines in transcription factor NF- B. Missed and Threatened Oxford Journals, Human Abortion. Maternal Journal of Reproduction, 2006, Volume 21, Reproductive Immunology. Number 2, Pp. 421-428. January 2003. Volume 50, Issue 8. Pru J. K, Lynch M.P. Signaling 4, Pages 302 – 308. mechanism in tumor necrosis

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