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OFFICE OF THE DEAN RESEARCH
UNIVERSITY OF KASHMIR

SYNOPSIS FOR INTEGRATED Ph. D PROGRAMME
NAME OF THE FACULTY:Biological Sciences
DEPARTMENT:Centre of Research for Development
NAME OF THE SCHOLAR: Shafat Ali
NAME OF THE SUPERVISOR:Dr. Md. Niamat Ali
Associate Professor
Centre of Research for Development
University of Kashmir, Srinagar- 190006
NAME OF CO- SUPERVISORS:
Dr. Sabhiya Majid
Head and Professor
Department of biochemistry
Govt. Medical College, Srinagar.

Dr. Shahnaz Taing
Head and Professor
Department of Obstetrics and Gynaecology
Lalla-Ded Hospital, Srinagar.

DATE OF REGISTRATION:
SCHOLARS UNIQUE ID:
PROPOSED TITLE: “Evaluation of allogenic factors (cytokines) in recurrent miscarriage among Kashmiri female population”
Summary of the proposed research work:
The scores of abnormal human conceptions end in miscarriage, the most common problem associated with pregnancy. Miscarriage is the abrupt loss of fetus before the 23rd week of gestation that influences about 30%-50% of pregnancies including preclinical cases. The loss of two or more consecutive pregnancies, primary or secondary is known as recurrent miscarriage (RM) or recurrent pregnancy loss (RPL) or recurrent spontaneous abortion (RSA), affecting about 1%-5% of conceptions. Different etiologies like parental chromosomal abnormalities, embryonic chromosomal aberrations, uterine anomalies, endocrinal disorders, thrombophilia, antiphospholipid antibody syndrome, immunological and environmental factors either singly or in combination have been postulated to contribute to recurrent pregnancy loss. Even after evaluation of these causes, about 50% etiologies of RPL are still unexplained. Immunological rejection of the fetus by the maternal immune system due to abnormal immune cells and the cytokine production is claimed to be one cause of unexplained pregnancy loss. A significant relationship between recurrent miscarriage and immune mechanisms has been found via researches on genetic and epigenetic polymorphisms related mainly to immune response and inflammatory mediators. Therefore, unexplained recurrent pregnancy loss may perhaps be due to an immune imbalance induced by pro-inflammatory and anti-inflammatory cytokines produced by T-helper cells (Th1, Th2, and Th17) and regulatory T (Treg) cells. IL-17, the signature cytokine produced by Th17 cells mediates inflammation, autoimmunity and immunological rejection of foreign tissue. Meanwhile, activated type 1 macrophages and dendritic cells produce IL-23 that helps to maintain and/ or expand Th17 cells. IL23 is absolutely required for disease pathogenesis in many models of Th17 cell- mediated diseases. The current study aims to evaluate the role of cytokines produced by Th-cells, type 1 macrophages and dendritic cells via their serum concentration and gene polymorphisms in women with recurrent miscarriage in Kashmiri population.
Objectives:
To study the incidence of recurrent miscarriage in Kashmiri women population.

To evaluate the serum concentration of cytokines (TNF-?, IL1?, IL6, IL10, IL17 and IL23) and to determine their relationship with recurrent miscarriage.

To study cytokine (IL17 and IL23) gene polymorphisms and their association with recurrent miscarriage.

Methodology:
To study the incidence of recurrent miscarriage in Kashmiri women population.

All the patient details will be collected from the department of Obstetrics and Gynecology, LD hospital, an associated hospital of Govt. Medical College, Srinagar. The study will be a prospective observational obtrusive study wherein the reported possible etiology of recurrent miscarriages includes genetic factors, uterine anatomical defects, infection, endocrine, and immunological factors among the women with at least two recurrent miscarriages will be taken into consideration in order to find out the incidence of recurrent miscarriages in Kashmiri women population. Clinical characteristics of patients will be studied. The clinical details will be taken following a questionnaire, either from patient, if the patient will be in condition to respond the questions or from the attendant.

To evaluate the serum concentration of cytokines (IFN-?,TNF-?,TNF-?,IL1,IL2,IL6,IL10,IL12, IL17 and IL23) and to determine their relationship in recurrent miscarriage patients.

The study will be a case control including two groups of women. One group will consist of women with a history of at least two recurrent miscarriages, primary or secondary. They will be clinically examined and investigated for different known causes of recurrent miscarriage. The diagnosis of unexplained RPL will be made after excluding any veri?able causes, such as abnormalities of the uterus or cervix, chromosomal abnormality, infection, endocrine and metabolic diseases, congenital thrombophilias and autoimmune disease. The other group will consist of women with at least one successful pregnancy and no history of recurrent miscarriage.
Inclusion criteria for patient:
Women with history of 2 or more spontaneous abortions, primary or secondary.

All patients will be ethnic Kashmiri women population.

Exclusion criteria for patients:
Women with history of only one spontaneous abortion.

Women with history of two or more induced abortions.
Inclusion criteria for control:
Women with at least one successful pregnancy and no history of recurrent miscarriage.

All subjects will be ethnic Kashmiri women population.

Peripheral blood samples (3 ml) each of patients with recurrent miscarriages and normal women with at least one successful pregnancy and no history of recurrent miscarriage will be drawn after proper consent. Whole blood will be collected in plain vials. These serum samples will be evaluated for different cytokines by Enzyme Linked Immunosorbent Assay (ELISA) using commercial kits according to the manufacturer’s instructions.

To study cytokines (IL17 and IL23) gene polymorphism and their association with recurrent miscarriage.

A case control study will be done. Peripheral blood samples (2 ml) of patients with recurrent miscarriages and normal women with at least one successful pregnancy and no history of recurrent miscarriage will be drawn after proper consent. Whole blood will be collected in EDTA vials. The collected blood samples will be used in the study of IL17 and IL23 single nucleotide polymorphism by Restriction Fragment Length Polymorphism (RFLP) wherein DNA will be extracted by phenol–chloroform method (Koons et al., 1994) with some modifications. The DNA fragments of interest will be amplified by PCR using specific primers. The amplified products will be digested by specific restriction enzymes. This will be followed by Agarose gel electrophoresis. The DNA bands will be observed by gel dock.

Statistical analysis:
The data collected will be analyzed using a Statistical Package for Social Sciences (SPSS), version 20.0 (Chicago, IL, USA), Mann-Whitney U test will be used to estimate the statistical significance of differences observed between the groups and student’s t- test will be used to examine the relation between qualitative variables.

State of Knowledge (Literature Survey)
National Level
Embryonic or foetal termination is the most common outcome of conception and observed as nature’s quality control for selecting genetically normal offspring. (Sudhir et al., 2016). Miscarriage is perhaps the most common obstetric complication of human conceptions (Silver and Warren 2006). Miscarriage is the spontaneous pregnancy loss without medical or mechanical means before the fetus is enough developed to survive or early pregnancy loss before 20th week of gestation, or 139 days (Li et al., 2014).The spontaneous miscarriage in India was reported to be approximately 10 % (Patki and Chauhan 2016). However, the recent studies revealed that the occurrence of spontaneous miscarriage among the Indian women is 32% (Patki and Chauhan 2016). The comparative prevalence of recurrent miscarriages is much lower than spontaneous miscarriages, (Ford and Schust 2009). Recurrent miscarriage is classically defined as the occurrence of three or more consecutive losses of clinical pregnancies prior to the 20th week of gestation. However, this definition is variable according to many investigators. The American Society for Reproductive Medicine defines recurrent miscarriage as two or more failed pregnancies (Practice Committee of the American Society for Reproductive Medicine 2012). Clinically apparent recurrent miscarriage among the Indian women is observed to be 7.46 % (Patk and Chauhan 2016).The well-known causes of RPL include genetic abnormalities, immunological factors, anatomic defects, endocrinal factors, certain thrombophilias and infections. The specific treatment improves pregnancy outcome. The identifiable causes accounted for 53% cases. Anatomical defects being commonest accounted for 21%, endocrinal factors for 20% and genetic factors contribute to 1% of recurrent miscarriages. They offered as first trimester abortions and history of Down’s syndrome. Immunological factors accounted for 7% and Antiphospholipid syndrome (APS or APLA) contribute to 5% of recurrent miscarriages. They offered as late first trimester abortion and late second trimester abortion. Systemic lupus erythematosus accounted for 2% and infection for 1%.Medical causes accounted for 3% of recurrent miscarriages in women with history of chronic hypertension in second trimester abortion. However, 47% of RPL are still unexplained. (Singh A et al., 2017). The frequency of chromosomal aberrations among Kashmiri couples with repeated fetal loss has been reported to be about 7.75% including numerical as well as structural chromosomal abnormalities. Males (2.12%) as well as females (5.63%) had chromosomal aberrations with balanced translocations (4.22%), duplications (0.70%), deletions (0.70%) and inversions (2.11%) (Zargar et al., 2015). Factor V Leiden (FVL) G1691A and prothrombin G20210A mutations are not associated with recurrent miscarriage in Kashmiri women population (Mahrukh et al., 2017). Maternal age is identified as independent risk factor for miscarriage (Patk and Chauhan 2016). Indian women with 31–49 years age at ?rst birth are found more vulnerable for spontaneous miscarriage than those with less age at ?rst birth (Patki and Chauhan 2016). The incidence of miscarriage is reported to be 10 % in women aged 20–24 years and 51 % in women aged 40–44 years (Larsen et al., 2013). A statistically signi?cant association has been found between age and spontaneous miscarriage. With the increased age of patients the incidence of more than or equal to three spontaneous miscarriages increases. Besides, the possibility of a subsequent miscarriage after the ?rst, second, and third miscarriage also increases. The percentage of patients with a subsequent miscarriage after the ?rst, second, and third miscarriage is reported as 24.97%, 34.04%, and 21.88 %, respectively (Patki and Chauhan 2016).However, earlier studies reported that the risk of a subsequent miscarriage after one spontaneous miscarriage is approximately 22–25 %; after two spontaneous miscarriages, the risk of another is 25 %; and after three spontaneous miscarriages, this risk further increases to 30 % (Simpson and Carson 2013).

International Level
Human reproduction is characterized by its inefficiency as large proportion of all conceptions fails to reach a live birth. Approximately 15-20% of all clinically recognized pregnancies worldwide result in spontaneous loss (Larsen et al., 2013; Hooker et al., 2014) and many pregnancies are lost before a woman realizes that she is pregnant (Ford and Schust 2009). The total pregnancy loss including preclinical cases is estimated to be 30–50% (Stephenson 2011). Approximately 5% of couples trying to conceive have two consecutive miscarriages and only 1% experience three or more (Bulletti et al., 1996). Recurrent pregnancy loss (RPL) is defined by more than three consecutive miscarriages prior to 20 gestational weeks (Ford and Schust 2009) but now-a-days, even two consecutive miscarriages are considered for further evaluations (Jaslow and Kutteh 2013). The risk of subsequent miscarriage after the ?rst two, three and four successive pregnancy losses is 30%, 33 % and 40% respectively, in women without a history of live birth (Ford and Schust 2009). The prevalence of recurrent pregnancy losses including only clinical miscarriages occurring before 21 weeks of gestation is 0.8–1.4 %. However, the prevalence of RPL taken as three consecutive pregnancy loses is high if biochemical losses are also included (Larsen et al., 2013). The recognized potential etiologies of recurrent pregnancy loss include genetic factors, uterine anatomical defects, infection, endocrine, and immunological factors. The majority of early pregnancy losses (50%–60%) are the outcome of chromosomal abnormalities, which can be of parental origin, or arise de novo in the embryo from parents with normal chromosomes. Balanced translocation are the most common parental abnormalities found in 2%–4% RPL cases as compared to 0.7% in the general population. Embryonic aneuploidy is established as the most common cause of early pregnancy loss. Uterine anomalies and chronic endometritis are reportedly found in up to 19% and 10%–27% women with RPL respectively. The prevalence of Antiphospholipid antibody syndrome (APS or APLA) in women with RPL varies however, it is generally accepted to be 5%–20%. Polycystic ovarian syndrome is associated with an increased risk of miscarriage (Hachem et al., 2017). Many studies have shown a relationship between inherited thrombophilia and pregnancy failure and complications (Lissalde?Lavigne et al., 2005). Maternal age and the number of prior spontaneous miscarriages are also associated with recurrent miscarriages (Li et al., 2002). Although a spectrum of causes is known for recurrent miscarriage but still etiology behind approximately 50% of RPL is unidentified. Such RPL is defined as unexplained recurrent miscarriage (Christiansen et al., 2008).However, the chances for a future successful pregnancy in couples with unexplained recurrent miscarriage could be as high as 50%–70% and depend mostly on maternal age and the number of previous losses (Lund et al., 2012; Kling et al., 2016). Preferably, epidemiological studies of recurrent pregnancy loss should begin after three or more losses whereas clinical evaluation may be carried out subsequent to a second miscarriage (Bulletti et al., 1996). Immune dysregulation has been proposed as a possible etiology in unexplained recurrent miscarriage. Indeed, maternal immune tolerance of the fetus is essential for normal implantation and pregnancy, and is characterized by an induction of regulatory T cells and an anti-inflammatory Th-2 profile. Thus, a disruption of the normal CD4 T-helper cell (Th) and uterine natural killer (NK) activity, and a Th imbalance in the endometrium could lead to implantation failure and pregnancy loss. (Mekinian et al.,2016). It is well recognized that at maternal-fetal interface equilibrium exists between Th1/ Th2 cells and the regulatory T (Treg)/Th17 cells where these cells secrete cytokines including IL-1?, IL-4, IL-10 and IL-17. These cytokines develop a complex network that maintains the pregnancy. An imbalance in these cytokines may result in spontaneous abortion (LeMaoult et al 2005). Recurrent pregnancy loss is associated with elevated level of pro-in?ammatory cytokine TNF-?. Recurrent pregnancy loss may be cured by blocking TNF-?. (Carpentier et al., 2011). New research studies correlate IL-23 and IL-17 with the pathogenesis of unexplained recurrent miscarriage (Sai?, et al., 2014).
Importance/Justification of Research Work
Recurrent miscarriage has been a mysterious problem and needs a lot of efforts to arrive at an appropriate solution. Recurrent miscarriage of unknown etiology presents a challenge to the clinician. The psychological impact for affected couples can be profound, with increased depression, anxiety and lowered self-esteem. The immunological aspect has been examined many times in the past. However, immunological abnormality has been suggested as the main cause of recurrent miscarriage. Even though genomic, transcriptomic and proteomic studies have already identified components of the immune response, thrombosis, steroid biosynthesis, apoptosis and angiogenesis-related genes that are associated with recurrent miscarriage. More screening studies are required for a better understanding of the underlying etiology of recurrent miscarriage. Therefore, the elucidation of the detailed functions of T helper cells and the cytokines they produce will shed light on the pathogenesis of recurrent miscarriage. It is hoped that a more comprehensive understanding of the molecular mechanisms for maintaining normal pregnancy will ultimately be of great benefit for developing efficient therapeutics for recurrent miscarriage patients. Even though various treatments, including hormonal and immunological therapies, have been applied to the treatment of women suffering from recurrent miscarriage but it is controversial whether proper evaluation of the cause of failed pregnancies has been performed. Therefore, it has been emphasized that proper evaluation and standardization for recurrent miscarriage patients is needed if the possible risks and appropriate therapeutic approaches are to be effectively assessed. More comprehensive studies at a molecular level are required in order to understand the causes of recurrent miscarriage and develop treatment protocols. Recent development in cytogenetics and immunogenetics along with greater understanding of implantation and maternal-embryo interactions has presented new insights into the possible causes of recurrent pregnancy loss and opened up new avenues for research into its prevention and treatment. The flux of these cytokines if observed in first and second trimester can serve as molecular marker for recurrent miscarriage and adequate treatment /precautionary measures can be taken to reduce incidence of miscarriages. Study in the flux of these cytokines and the underlying mechanistic basis can be suggestive of novel treatment regimens and drug development in future. The detection of the cytokine gene polymorphisms in women with recurrent miscarriage also will help in treating and managing the pregnancy of a carrier via immunotherapy. The population specific screening approach will also be developed that will help in establishing population / individual specific pharmacos-genomic and counseling approach.

WORK ELEMENTS AND DETAILED PLAN OF IMPLEMENTATION ALONG WITH TIME SCHEDULE:
Work elements Time period
Course work and preparation of the synopsis 0-12 months
Collection of data, Literature survey, Analysis of Data and lab work 13-18 months
Collection of data, Literature survey, Analysis of Data and lab work 19-24 months
Collection of data, Analysis of Data, Literature survey and lab work 25-30 months
Compilation of Data and presentation of thesis 31-36 months
REFERENCES
A. Patki, N. Chauhan, An epidemiology study to determine the prevalence and risk factors associated with recurrent spontaneous miscarriage in India. The Journal of Obstetrics and Gynecology of India, 66(5), 310-315 (2016)
A. Singh, A. Kujur, K. Rathore, An evaluation of recurrent pregnancy loss. International Journal of Reproduction, Contraception, Obstetrics and Gynecology 6(4),1332-1335 (2017)
A.B. Hooker, M. Lemmers, A.L. Thurkow, M.W. Heymans, B.C. Opmeer, H. A. Brölmann, and J. A. Huirne, Systematic review and meta-analysis of intrauterine adhesions after miscarriage: prevalence, risk factors and long-term reproductive outcome. Human Reproduction Update, 20(2), 262-278 (2014)
A.Mekinian, J. Cohen, J. Alijotas-Reig, L. Carbillon, P. Nicaise?Roland, G. Kayem, and M. Bornes. Unexplained recurrent miscarriage and recurrent implantation failure: is there a place for immunomodulation? American Journal of Reproductive Immunology, 76(1), 8-28 (2016)
B. Sai?, S.A. Rezaee, N. Tajik, M.E. Ahmadpour, M. Ashra?, R. Vakili, S. SoleimaniAsl, R. A?atoonian, M. Mehdizadeh, Th17 cells and related cytokines in unexplained recurrent miscarriage at the implantation window, Reproductive biomedicine online, 29(4), 481-489 (2014)
Bulletti, C. Flamigni, E. Giacomucci ,Reproductive failure due to spontaneous abortion and recurrent miscarriage, Human Reproduction Update, 2(2), 118-136 (1996)
C.R. Jaslow, W.H. Kutteh, Effect of prior birth and miscarriage frequency on the prevalence of acquired and congenital uterine anomalies in women with recurrent miscarriage: a crosssectional study, Fertility Sterilty, 99 (7), 1916-1922 (2013)
E.C. Larsen, O.B. Christiansen, A.M. Kolte, N. Macklon, New insights into mechanisms behind miscarriage, BMC Medicine 11(1), 154 (2013)
G. Lissalde?Lavigne, P. Fabbro?Peray, E. Cochery?Nouvellon, E. Mercier, S. Ripart?Neveu, J. P. Balducchi, P. Mares, IN FOCUS: Factor V Leiden and prothrombin G20210A polymorphisms as risk factors for miscarriage during a first intended pregnancy: the matched case–control ‘NOHA first’study. Journal of Thrombosis and Haemostasis, 3(10), 2178-2184 ( 2005)
H.B. Ford, D.J. Schust, Recurrent pregnancy loss: etiology, diagnosis, and therapy. Reviews in obstetrics and gynecology, 2(2),76–83 (2009)
J. LeMaoult, N. Rouas-Freiss, E.D. Carosella, Immuno-tolerogenic functions of HLA-G: relevance in transplantation and oncology, Autoimmunity reviews, 4(8), 503-509 (2005)
J. Simpson, S. Carson, Genetic and nongenetic causes of pregnancy loss. Women, 3(30),4 (2013)
Kling, J. Magez, J. Hedderich, S. von Otte, D. Kabelitz, Two-year outcome after recurrent first trimester miscarriages: prognostic value of the past obstetric history, Archives of gynecology and obstetrics, 293(5), 1113-1123 (2016)
L. Li, P.C. Leung, T.K. Chung, C. C Wang, Systematic review of Chinese medicine for miscarriage during early pregnancy, Evidence-based Complementary and Alternative Medicine, 2014, 1-16 (2014)
M. H. Zargar, T. M. Malla, F. A. Dar, Occurrence of Chromosomal Alterations in Recurrent Spontaneous Abortion Couples: A Case-Only Study from Kashmir, North India. Journal of Genetic Syndromes & Gene Therapy, 6(3), 1-4 (2015).

M. Lund, M. Kamper-Jørgensen, H.S. Nielsen, Lidegaard , A.M.N. Andersen, O.B. Christiansen, Prognosis for live birth in women with recurrent miscarriage: what is the best measure of success? Obstetrics and Gynecology,119(1),37–43 (2012)
M.D. Stephenson, Recurrent early pregnancy loss. Seminar Report Med;29:461-2 (2011)
N. Sudhir, T. Kaur, Beri, A. Kaur, Cytogenetic analysis in couples with recurrent miscarriages: a retrospective study from Punjab, north India, Journal of Genetics, 95 (4), 887–894 (2016)
O.B. Christiansen, R. Steffensen, H.S. Nielsen, K. Varming, Multifactorial etiology of recurrent miscarriage and its scientific and clinical implications, Gynecologic and obstetric investigation, 66(4), 257-267, (2008)
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R.M. Silver, J.E. Warren. Preconception councelling for women with Thrombophilia, Clinical obstetrics and gynecology, 49(4), 906-919 (2006)
S. Shafia, M. H. Zargar, R. Ahmad, N. Khan, Association assessment of thrombophilic gene mutations in Kashmiri women with recurrent miscarriages. World Journal of Pharmaceutical Research ,6(16), 1334-1344 (2017)
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DATE OF PRESENTATION OF THE SYNOPSIS BEFORE DRC:
DD MM YYYY
Recommendation of Departmental Research Committees (DRC)
Recommendation of DRC: -________________________________________________
_______________________________________________________________________
Signature with Name
1. _________________________________ 2._________________________________
_________________________________ 4._________________________________
5.___________________________________6._________________________________

7. 8._________________________________
II) Certificate of the supervisor and co-supervisors:
We certify that we have drafted the I-Ph.D synopsis entitled “Evaluation of allogenic factors (cytokines) in recurrent miscarriage among Kashmiri female population” in the consultation with the scholar concerned; viz.Mr. Shafat Ali, keeping in view his academic needs and no such research work has earlier been done on the above topic.
Signature with Name of the supervisor: Dr. Md. Niamat Ali…………………………………….

Signature with Name of the co- supervisors …………………………………
Dr. Sabhiya Majid
Head and Professor
Department of biochemistry
Govt. Medical College, Srinagar.

Dr. Shahnaz Taing
Head and Professor
Department of Obstetrics and Gynaecology
Lalla-Ded Hospital, Srinagar.

Forwarded to the Dean, Faculty of Biological science, for placing the above programme before the Board of Research Studies and for the approval of the Vice- chancellor:
Dated: …………………….

Director
(CORD)

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