Dr. S. Arefi Assistant Prof. , Affiliated researcher Evidence-based guidelines for the investigation of recurrent miscarriage ESHRE Special Interest Group for Early Pregnancy (SIGEP). Dr. S. Arefi Assistant Prof. , Affiliated researcher

Common Case علی و مریم 3 سال است که ازدواج کرده اند. بیمار دو بار سقط خودبخودی حدود 9-10 هفته داشته اند. پزشک جهت بیمار به طور empiric در حاملگی بعدی اسپیرین و سیکلوژست شروع کرد . بیمار مجددا در 7 هفتگی Blighted ovum شد. چه تصمیمی برای بیمار می گیرید؟

FAQ Do we need to postponed the workups till 3th abortion? Do we need to do all investigations in all patients with RSA?

Immunology , Inflammation implantation

Seminal fluid The seminal plasma introduced during intercourse in the mouse, pig and human female reproductive tract has been shown to induce local inflammatory changes . Robertson SA, et al . J Reprod Fertil. 1996 A considerable proportion of the transforming growth factor (TGF)-β synthesized in prostate in humans , secreted into the fluids that contribute to the ejaculate . The TGF-β are key regulators of several aspects of the immune responses including lymphocytes, macrophages and dendritic cells. Letterio J,Annu Rev Immunol. 1998 TGF-β also promotes conversion of CD16+ peripheral blood natural killer (NK) cells into CD16- NK cells in vitro . TGF-β derived from seminal plasma may affect the maternal immune response in pregnancy through promoting type 2 and/or Th3 immunity to paternal antigens from the outset of their introduction to the female genital tract. Robertson SA,J Reprod Immunol. 2002

Th1 response is mediated by pro-inflammatory cytokines such as interleukin 1 (IL1), IL2, IL6, IL12, IL15, IL18, interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), Elevated pro-inflammatory cytokine/ chemokine expression, as well as an increasing Th1 inflammatory response is necessary for the acquisition of uterine receptivity. Th2 response is characterized by the involvement of regulatory cytokines such as IL4, IL5, IL10, IL13, and granulocyte macrophage colony stimulating factor (GM-CSF) and is therefore known also as anti-inflammatory. Th2-humoral inflammation is required for pregnancy maintenance

local injury Mechanical endometrial injury (biopsy/scratch or hysteroscopy►inflammatory response and high level of endometrial proinflammatory cytokines, such as interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and tumor necrosis factor-α (TNF-α). The pro-inflammatory TNFα enhances the expression of other cytokines/chemokines that recruit, in turn, macrophages and DCs to the site of injury. These immune cells secrete different factors that on the one hand may affect uNK cell differentiation and on the other stimulate the luminal endometrial cells to produce adhesion molecules enabling the attachment of the embryo to the uterine lining, facilitating implantation . van Mourik MS et al, J Leukoc Biol. 2009.

Hysteroscopy : endometrial injury , implantation The value of hysteroscopy in patients with Repeated IVF Failure S.Arefi et al .TUMJ,2008 Findings on hysteroscopy in patients with in vitro fertilization by intra cytoplasmic single sperm injection and embryo transfer failures S. Arefi et al, SMJ 2008; Repeated IVF failure and impact of hysteroscopy S.Arefi et al ,IJRM,2008

Relation of Inflammation and Implantation: Serum CRP level at the day of ET Increasing Serum ratio of CRP level (as a sensitive marker in inflammatory reactions) in day of embryo transfer to the day of ovum pick up (≥1.23) can predict the success in patients undergoing IVF/ICSI. - S. Arefi, et al , C-reactive protein level and pregnancy rate in patients undergoing IVF/ICSI ,IJRM, 2010

The Contribution of the Endometrium to Embryo Implantation Compared with controls, RM was associated with significantly higher endometrial PROK1 mRNA levels and approximately 100-fold lower PRL levels Consecutive pregnancy rate is higher than predicted in recurrent miscarriage (RM) Predicted 1 month 3 months 6 months Fert patients 0.8% 8% 41% RPL patients 13% 68% RPL = recurrent pregnancy loss; FER = fertile INF = infertile without RPL Salker et al. PLoS One 2010 Key point: The embryo selective function has been shown to be defective in women with RM and fails to discriminate between high and low quality embryos Macklon et al. ESHRE 2013. Oral 098

The Contribution of the Endometrium to Embryo Implantation (Review) Excessive selectivity Insufficient selectivity RIF Decidual Phenotype RM Only the best embryo will do Poor embryos allowed to implant Key point: In recurrent implantation failure (RIF), the decidual phenotypical abnormalities may be opposite to those in recurrent miscarriage Macklon et al. ESHRE 2013. Oral 098

Does the endometrium “choose” the embryo? Weimar et al PLoS One 2012 Key point: Emerging evidence suggests that the endometrium “chooses” the embryo. Poor quality embryos cause less migration in fertile women, but not in women with recurrent miscarriage (RM) Macklon et al. ESHRE 2013. Merck Serono Satellite Symposium

The probable causes of Reduced endometrial receptivity classified as two headings: 1- Morphological: Thin endometrium, abnormalities within cavity like large Polyp, Myoma, Adhesions, septum 2- Molecular : -Altered expression of adhesion molecules , Integrines ,and immunological factors - Increase of natural killer cells activities - Imbalance of cytokine networks (balance between IL-12 and IL-18) - Auto antibodies like ASA, ACA,LAC,ANA, Anti DNA, Anti Zona* and Anti ovarian antibodies*, β2-glycoprotein-I antibodies, Antibodies to annexin-V * Arefi S,et al Intrafollicular fluid antigamete antibodies in infertile patient candidates for ICSI. IJRM,2006 summer; 7(2): 101-110 * Arefi S,et al . Antizona pellucida antibodies in follicular fluid and outcome of ICSI.MEFS Journal 2006; 11(1): 35-42 -

Immunologic investigations ESHRE Special Interest Group for Early Pregnancy (SIGEP). An excessive maternal immune response against paternal antigens resulting in abnormal immune cells and cytokine production (Laird et al., 2003) particular interest on Natural Killer (NK) cells: 1-phenotypic and functional differences between peripheral and uterine NK cells 2-percentage of CD56+ NK cells in peripheral blood of healthy individual varies from 5 to 29% and is affected by sex, stress, ethnicity and age. Tests to measure blood NK cells ,useful ?(Moffett et al., 2004). Recent data have shown that a high number of uterine NK cells is found in the endometrium of women with RM and this could be reduced by therapy (Quenby et al., 2005). Prospective trials are needed and endometrial sampling should only be offered to women within the context of research programs.

Other Investigations: Coagulation investigations Endocrinologic investigations Parental cytogenetic investigation Histopathological and Cytogenetic investigation of the product of conception Anatomical investigations Male investigation

Coagulation investigations ESHRE Special Interest Group for Early Pregnancy (SIGEP). History of three or more early pregnancy losses, before 10 weeks 1 or more unexplained deaths at ≥10 weeks of a morphologically normal fetus 1 or more premature births at ≤34 weeks with severe preeclampsia or placental insufficiency should be offered a testing for: LAC ,aCL, APA, to exclude an antiphospholipid syndrome (APS) wilson et al

Investigation Association of three polymorphisms of Coagulation Factor XIII and recurrent pregnancy Loss It has been shown no significant difference between case and control with regard to FXIII G103T ,But it has been seen significantly more polymorphism of A614T and C1694T in patients with recurrent pregnancy loss. Mahmood J.Tehrani, Raheleh Torabi, Soheila Arefi ,et al,Am J Reprod immunology,2010

Analysis of Plasminogen activator inhibitor -1,integrin beta 3,beta fibrinogen and MTHFR in Iranian women with recurrent pregnancy loss It has been shown mutation of PAI-1 4 G allele (specially homozygot) and beta fibrinogen increase the risk of RSA. Conversly integrin beta 3 polymorphism had a protective role for RSA as it was significantly more frequent in control group. MTHFR 677C/T polymorphism was significantly more frequent in patient with RSA.Also significant correlation between MTHFR 677C/T and 1298A/ C polymorphism,but no meaningful association between RSA and MTHFR 1298A/ C Mahmood J. Terani,Raheleh Torabi,Amir H. Zarnani, Soheila Arefi, et al Am J Reprod ,Immmunol ,2010

Among the genetic causes of this condition, a common one is polymorphism at position 677 in the methyl tetrahydrofolate reductase (MTHFR) gene, which in the homozygous form leads to a thermolabile enzyme variant Makris, 2000

Association of ACE,PAI-1,and coagulation factor XIII gene polymorphism and with recurrent abortionin Iranian patients Only Homozygosity for PAI-1 (4G/4G)has been shown significantly more in Patient with RSA in this study. Soltanghoraee H.,Memariani T.,Aarabi M.,Hantoushzadeh S.,Arefi S.,et al,Medical Journal of Reproduction and Infertility,2007

Anatomical investigations ESHRE Special Interest Group for Early Pregnancy (SIGEP). Diagnosis: Traditionally, laparoscopy, HSG and/or hysteroscopy have been used to these uterine malformations in women with RM. Ultrasound, and in particular 3D ultrasound, has become an accurate, reproducible, non-invasive, out-patient method for the diagnosis of congenital uterine anomalies (Salim et al., 2003). Using 3D ultrasound, it has been reported that women with a subseptate uterus have a higher incidence of first trimester loss, whereas women with an arcuate uterus have a greater proportion of second trimester loss and preterm delivery (Woelfer et al., 2001). With both arcuate and subseptate uteri, the length of the remaining cavity was shorter, and the size of the fundal distortion was higher in the RM group. Three-dimensional, extended field-of-view ultrasound method for estimating large strain mechanical properties of the cervix during pregnancy. House M 2012

Endocrinologic investigations ESHRE Special Interest Group for Early Pregnancy (SIGEP) Although hypothyroidism and well-controlled diabetes are not associated with RM (RCOG, 1998), thyroid function tests and HbA1C measurements are accurate and inexpensive and can still be considered as part of the evaluation (Christiansen et al., 2005). In hypothyroidism, infertility is more likely a problem than pregnancy loss. Obesity is associated with a statistically significant increased risk of first trimester and recurrent miscarriage [odds ratios 1.2 and 3.5 (Lashen et al., 2004) The association between polycystic ovary syndrome (PCOS) and RM could be secondary to the association between obesity and miscarriage ( Bellver et al., 2003). Hypersecretion of LH, high androgen levels, hyper-prolactinaemia and luteal phase defects (LPD) have been associated with RM.

Parental cytogenetic investigation ESHRE Special Interest Group for Early Pregnancy (SIGEP) Structural chromosome abnormalities, usually balanced translocation. Four factors increase the probability of carrier status: 1-low maternal age at second miscarriage 2-history of three or more miscarriages 3-history of two or more miscarriages in a brother or sister 4-history of two or more miscarriages in the parents of either partner (Franssen et al., 2005). After one miscarriage, it is generally accepted to refrain from karyotyping. The incidence of carrier status after one miscarriage is 2.2% (Braekeleer de and Dao, 1990). It is thus advised to refer for parental karyotype only when the probability of carrier status is ≥2.2%

Histopathological investigation of the product of conception ESHRE Special Interest Group for Early Pregnancy (SIGEP) Whilst it is routine practice to send products of conception for histological examination, mainly to exclude a gestational trophoblastic disorder, the usefulness of histopathogical investigation of placental and/or fetal tissue in RM on future pregnancy management for an individual couple remains to be determined (Jauniaux et al., 1996; Jauniaux and Burton, 2005). New morphologic classifications continue to be proposed (Hakvoort et al., 2006). In couples with RM, there are a few reports showing an increased incidence of thrombo-inflammatory lesions such as perivillous fibrin deposition, chronic villitis and deciduitis (Doss et al., 1995; Hustin et al., 1996), in particular, when the karyotype is normal (Redline et al., 1999). Although these lesions support the immunologic imbalance concept, the contribution of these histological findings to the management of RM is limited and has not been trialled prospectively.

Cytogenetic investigations of the product of conception ESHRE Special Interest Group for Early Pregnancy (SIGEP) The risk of live born trisomy following an aneuploidy in a sporadic early pregnancy failure is around 2% (Alberman, 1992). Chromosomal analyses of the products of conception in couples with RM indicate that a normal conceptus karyotype in a previous pregnancy is a predictor of subsequent miscarriage (Morikawa et al., 2004). Women <36 years of age with RM have a higher frequency of euploid miscarriage. The cost benefit of performing systematic karyotyping of products of conception after one miscarriage on the overall management of RM needs to be investigated prospectively in large populations.

Other investigations High level of homocysteine (hyperhomocysteinaemia) can be associated with RM Nelen et al., 2000 Cytokine gene polymorphisms in recurrent spontaneous abortions Choi YK, 2008 Polymorphisms in VEGF, progesterone receptor and IL-1 receptor genes in women with recurrent spontaneous abortion. Traina E ,2010 A lot of information is available about environmental toxins. The association between miscarriage and ionizing radiation, organic solvents, alcohol, mercury and lead is confirmed, whilst an association to caffeine, hyperthermia and cigarette smoking is suspected Gardella and Hill, 2000 Low plasma folate levels have been associated with an increased risk of first trimester miscarriage George, 2002

Male factor evaluation? Recurrent pregnancy loss results as a factor of a couple. It has been emphasized the contribution of the male to implantation failure, miscarriage, and congenital anomalies suggested by recent literature. With further investigation, evaluation of the male may be considered a routine part of the evaluation in the near future. Puscheck EE, 2007

A recent systematic review showed that sperm DNA damage is associated with lower pregnancy rate of natural, intrauterine insemination (IUI), and in vitro fertilization (IVF), and an increased risk of pregnancy loss in those couples undergoing IVF or intracytoplasmic sperm injection (ICSI). Zini A, Sigman M (2009)

conclusion The majority of RMs remain unexplained .Even though a cause of the RM can be found in up to 50% of the women, only for women with RMs resulting from antiphospholipid syndrome, a potentially effective treatment namely the use of anticoagulants is available. Supportive care is regularly offered to these women, after which live birth rates up to 85% are reported . Current guidelines from the European Society of Human Reproduction and Embryology (ESHRE) and the Royal College of Obstetricians and Gynaecologists ; (RCOG), 2011.

Thank you! Arefi S, Modaressi M.H, Jeddi Tehrani M, et al. Clinical significant of pericentric inversion of chromosome 9: A case report of recurrent abortion. Medical Journal of Reproduction & Infertility 2003 Summer; 4(3): 251-258 Mahmood Jeddi Tehrani,Raheleh Torabi,Amir Hassan Zarnani ,Soheila Arefi, et al, Analysis ofPlasminogen activator inhibitor -1,integrin beta 3 ,beta Fibrinogen and MTHFR polymorphism in Iranian women with recurrent pregnancy loss American Journal of Reproductive Immunology,1011 Memariani; Arefi; Aarabi; Hantoosh Zadeh; Akhondi; Modarressi ,Polymorphisms of plasminogen activator inhibitor-1, angiotensin converting enzyme and coagulation factor XIII genes in patients with recurrent spontaneous abortion, The Journal of Maternal-Fetal & Neonatal Medicine,2010 Haleh Soltanghoraee, Soheila Arefi, Afsaneh Mohammadzadeh1 , Arman Taheri., Hojat Zeraati, Seyed Behnam Hashemi, M.P.H., Mohammad Mehdi Akhondi. Thyroid autoantibodies in euthyroid women with recurrent abortions and infertility ,IJRM,vol 8,no 4,pp 15-156,Antumn 2010 Jeddi Tehrani,Torabi R ,Arefi S, et al ,Investigating Association of Three Polymorphisms of Coagulation Factor XIII and Recurrent Pregnancy Loss..AM J Reprod Immunol 2010,Aprl 12 Zarei,Saeed; Soltanghoraee, Haleh ;Mohammadzadeh, Afsaneh; Arefi Soheila; Zarnani, Amir Hassan ;Idali Farah ;Tavangar, Banafsheh; Savadi Shirazi,Elham;Moshref Behzad,Narges;Jeddi-Tehrani, Serum leptin levels in women with immunological recurrent abortion, Mahmood,Journal of reproduction &infertility,volume 11,issue 1,April/June 2010 Torabi R.Ostadkarampour m..Arefi S., Keramatipour M., Zarei S.,Jedi Tehrani M.The relationship between polymorphisms of blood coagulation factor V gene and recurrent pregnancy losses, ,Journal of reproduction and infertility ,Vol 9 Issue 4,Jaunary/March 2009 Soltan Gharaei H., Arefi S., Mohammadzadeh A., Zonobi M., Zeraati H.,Bahrami M.E., Prevalence of seven antibodies in women with recurrent abortion in Tehran, Journal of Tehran University of Medical Science. 2007; 65(7) Soltangharaee H, Memariani T, Aarabi M, Hantoushzadeh S, Arefi S, Aarabi M, Akhondi M.A, Modarresi M.H. Association of ACE, PAI-1 and coagulation factor XIII gene polymorphisms with recurrent spontaneous abortion in Iranian patient. Medical Journal of Reproduction and Infertility 2007 winter; 7(4): 324-330 Samira Hosseini, Amir Hassan Zarnani, Hossein Asgarian-Omran, Zohreh Vahedian-Dargahi, Mohammad Reza Eshraghian, Zahra Akbarzadeh-Pasha, Soheila Arefi, Mahmood Jeddi-Tehrani, Fazel ShokriComparative Analysis of NK Cell Subsets in Menstrual and Peripheral Blood of Patients with Unexplained Recurrent Spontaneous Abortion and Fertile Subjects,Journal of Reproductive Immunology 01/2014;