1. Impact of Number of Pregnancies on Maternal Microchimerism Casey Trimmer York College Department of Biological Sciences Normally, every cell in an organism contains the exact same set of genetic material, as all of the cells of the body originate from one initial cell that copies its genetic material again and again. Chimerism is a condition in which an individual is composed of more than one set of genetic information; some cells contain one set, while other cells contain another. Microchimerism is a term used to describe this chimerism on a smaller scale: the genetic material in a small population of a person’s cells differs from the material in the rest of the cells (Turco and Bambara 2004). The phenomenon of cells passing back and forth between mother and fetus during pregnancy is termed fetomaternal cell trafficking (Scaletti et al. 2001). This exchange between mother and fetus is a cause of microchimerism, as a small population of fetal cells enters the body of the mother and vise versa. It had been assumed these foreign cells were quickly destroyed by the host’s immune system. However, recently it has been shown that these cells can persist for long periods of time (Nelson 2002, Bianchi et al. 1996), a condition termed persisting fetal microchimerism. This discovery of a foreign population of cells circulating though the bodies of mother and child has been the focus of recent research in autoimmune disorders. Research in microchimerism searches for the presence of male cells in women that had male pregnancies, as male DNA in a female is a clear indication of foreign DNA. Research has consistently shown that fetal cells persist in the mother’s body for an extended period of time—perhaps the mother’s entire life (Lee 2001). However, no research has been conducted on how the number of male pregnancies a woman has affects the frequency of autoimmune disorders or the frequency of finding foreign cells in her body. The objective of this study is to determine whether the number of pregnancies is related to the incidence of finding foreign cells in the body. I hypothesize that a greater number of male pregnancies would result in more cell exchanges between fetus and mother, thereby increasing the frequency of finding chimeric cells in the mother. REVIEW OF LITERATURERESEARCH DESIGN OBJECTIVE AND HYPOTHESIS Objective: Examine how the number of male pregnancies affects the frequency of finding male (foreign) DNA in the mother Hypothesis: A greater number of pregnancies would result in more cell exchanges between fetus and mother, thereby increasing the frequency of finding chimeric cells in the mother. LITERATURE CITED Ando, T., Imaizumi, M., Graves, P.M., Unger, P. and Davies, T.F. 2002. Intrathyroidal fetal microchimerism in Grave’s Disease. The Journal of Clinical Endrocrinology and Metabolism. [serial online] 87: 3315-3320. Available from HighWire. Arlett, C.M., Smith, B. and Jimenez, S.A. 1998. Identification of fetal DNA and cells in skin lesions from women with systemic sclerosis. The New England Journal of Medicine [serial online] 338: 1186-1191. Available from: http://content.nejm.org/cgi/content/full/338/17/1186.http://content.nejm.org/cgi/content/full/338/17/1186 Artlett, C.M., Cox, L.A. and Jimenez, S.A. 2000. Detection of cellular microchimerism of male or female origin in systemic sclerosis patients by polymerase chain reaction analysis of HLA-Cx antigens. Arthritis and Rheumatism [serial online] 43: 1062-1067. Available from: HighWire Evans, P.C., Lambert, N., Maloney, S., Furst, D.E., Moore, J.M. and Nelson, J.L. 1999. Long-term fetal microchimerism in peripheral blood mononuclear cell subsets in healthy women and women with schlerdoma. Blood [serial online] 93: 2033-2037. Available from: HighWire. Invervizzi, P., Andreis, C.D., Sirchia, S.M., Battezzati, P.M., Zuin, M., Rossella, F., Perego, F., Bignotto, M., Simoni, G. and Podda, M. 2000. Blood fetal microchimerism in primary biliary cirrhosis. Clin Exp Immunol [serial online] 122: 418-422. Available from HighWire. Klintschar, M., Schwaiger, P., Mannweiler, S., Regauer, S. and Kleiber, M. 2001. Evidence of fetal microchimerism in Hashimoto’s Thyroiditis. The Journal of Clinical Endocrinology and Metabolism [serial online] 86: 2494-2498. Available from HighWire. Lambert, N.C., Evans, P.C., Hashizumi, T.L., Maloney, S., Gooley, T., Furst, D.E. and Nelson, J.L. 2000. Cutting edge: persistent fetal microchimerism in T lymphocytes is associated with HLA-DQA1*0501: implications in autoimmunity. The Journal of Immunology [serial online] 164: 5545-5548. Available from: HighWire. Nelson, J.L., Furst, D.E., Maloney, S., Gooley, T., Evans, P.C., Smith, A., Bean, M.A., Ober, C. and Bianci, D.W. 1998. Microchimerism and HLA-compatible relationships of pregnancy in scleroderma. Lancet [serial online] 351: 559-562. Available from: Ebsco (Academic Search Premier). Srivatsa, B., Srivatsa, S., Johnson, K.L., Samura, O., Lee, S.L. and Bianchi, D.W. 2001. Microchimerism of presumed fetal origin in thyroid specimens from women: a case-control study. Lancet [serial online] 358: 2034. Available from: Ebsco (Academic Search Premier). Tanaka, A., Lindor, K., Gish, R., Batts, K., Shiratori, Y., Omata, M., Nelson, J.L., Ansari, A., Coppel, R., Newsome, M. and Gershwin, M.E. 1999. Fetal microchimerism alone does not contribute to the induction of primary biliary chirrhosis. Hepatology [serial online] 30: 833-838. Available from: Science Direct. Male cells have been found to exist in women’s blood for decades after pregnancy with a male child (Bianchi et al. 1996, Invernizzi et al. 2000). Presence of foreign cells in the body has been examined as a possible cause of autoimmune disorders. Research regarding autoimmune thyroid disorders (Table 1) shows presence of male cells in diseased thyroids and blood from patients with multiple disorders (Srivatsa et al. 2001, Klintschar et al. 2001, Ando et al. 2002). Research concerning the autoimmune liver disorder primary biliary cirrhosis (Table 2) found that male fetal cells persisted in the liver and blood of both control and disease women (Tanaka et al. 1999, Invernizzi et al. 2000) Research regarding the autoimmune disease scleroderma (Table 3) has shown male cells to be present in the blood of disease and control women (Nelson et al. 1998, Arlett et al. 1998, Evans et al. 1999, Artlett et al. 2000, and Lambert et al. 2000). Cells obtained from:Frequency of disease group w/ male cells Frequency of control group w/ male cells Blood 1 100% (n=17)70% (n=23) Skin Lesions 2 58% (n=19)0% (n=7) Blood 2 46% (n=69)4% (n=25) Blood 3 60% (n=20)31% (n=48) Blood 4 65% (n=63)28% (n=88) Blood 5 42% (n=12)47% (n=19) TABLE 3. Frequency of male cells in disease and control groups for scleroderma Cells obtained fromFrequency of disease group with male cells Frequency of control group w/ male cells Blood 1 36% (n=36)31% (n=36) Liver 2 70% (n=37)72% (n=31) TABLE 2. Frequency of male cells in disease and control groups for PBC Cells obtained fromDisorderFrequency of disease group w/ male cells Frequency of control group w/ male cells Thyroid 1 Various55% (n=29)0% (n=18) Thyroid 2 Hashimoto’s Thyroiditis 47% (n=17)4% (n=25) Blood 3 Grave’s Disease47% (n=17)29% (n=30) TABLE 1. Frequency of male cells in disease and control groups for thyroid disorders. 1 Srivatsa et al. (2001) 2 Klintschar et al. (2001) 3 Ando et al. (2002) 1 Invernizzi et al. (2000) 2 Tanaka et al. (1999) 1 Nelson et al. (1998)) 2 Artlett et al. (1998) 3 Evans et al. (1999) 4 Artlett et al. (2000 5 Lambert et al. (2000) EXPECTED RESULTS INTRODUCTION (Scaletti et al. 2001) Research Groups Chimeric cell groups N=20 women (30-45 yrs old) (1)4 women w/ 1 son (2)4 women w/ 2 sons (3)4 women w/ 3 sons (4)4 women w/ 4 sons (5)4 women w/ 5 sons 1. DNA extraction from blood samples 2. PCR Amplification of Y-chromosome specific sequence 3. Gel electrophoresis to visualize PCR results Control groups N=12 (30-45 yrs old) (1)4 women never pregnant (2)4 women only pregnant w/ females (3)4 men Expected gel: (Invernizzi et al. 2000) Base pair ladder No band  no male DNA Band  male DNA Male sample verification of PCR Possible relationships between number of male pregnancies and frequency of finding male DNA 1. Direct relationship: As number of male pregnancies increases, the frequency of finding male cells increases. 2. Saturation of cells: reach a certain number of male pregnancies where frequency of finding male cells will not increase 3. No relationship: The frequency of finding male cells is unrelated to number of male pregnancies—same frequency for all number of pregnancies 4. No relationship: The frequency of finding male cells is unrelated to number of male pregnancies—random frequency of finding male cells (affected by other factors) Possible other factors: older male siblings, unknown miscarriage, blood transfusion, organ transplant