Frequency of Anticardiolipin Antibodies in Women with Recurrent Fetal Loss

Aim: To find out the frequency of Anticardiolipin Antibodies in women with recurrent fetal loss Study design & duration: One year descriptive study Methods: Seventy five females were included in the study with ages ranging from 20-40 years with minimum two miscarriages. Diabetic females and females with history of bacterial or viral diseases were excluded from the study. Results: In this group the mean age was 27.60±3.29 years. Mean number of fetal losses was 2.95±1.45 whereas mean duration of marriage was 5.79±4.11 years. Conclusion: It is suggested that the patient with history of recurrent miscarriages must be screened for Anticardiolipin Antibodies to bring them out of psychological and physical trauma. Keywords: Anticardiolipin Antibodies (aCL), Recurrent fetal loss (RFL), aPTT

P–365 Altered endometrial oestrogen-responsiveness and aberrant expression of cell-fate markers may contribute to the aetiology of recurrent pregnancy loss

Study question Do women with recurrent pregnancy loss (RPL) have an aberrant expression of oestrogen receptor-β(ERβ) and cell-fate markers during the window of implantation (WOI) endometrium? Summary answer Women with RPL are found to have significantly altered levels of ER βand Ki–67 in the WOI endometrium, possibly resulting in anti-proliferative and anti-angiogenic effects. What is known already RPL affects 1% of all women and has been associated with altered endometrial angiogenesis and proliferation when compared with the endometrium of healthy fertile women. RPL can be subcategorised into recurrent loss of anembryonic pregnancy, fetal loss (following evidence of a fetal heartbeat) and recurrent implantation failure (RIF). ERβis the only oestrogen-receptor (ER) known to be expressed in the vascular endothelium of the endometrium and is the dominant ER during the WOI. It has an important role in endometrial regeneration and is proposed to regulate the angiogenic and vascular changes that occur in embryo implantation. Study design, size, duration: This pilot case-control study took place at the Liverpool Women’s Hospital and included 38 women; 29 who suffered RPL and 9 controls with proven fertility (≥2 healthy pregnancies). Of the RPL group, 9 had recurrent loss of anembryonic pregnancy, 10 had recurrent fetal loss and 10 had RIF. Endometrial samples were collected during the WOI (cycle day 22+/–2). Participants/materials, setting, methods To determine whether markers of endometrial cell proliferation and oestrogen-responsiveness are associated with RPL, we assessed the immuno-staining for ER β, progesterone receptor (PR) and cell-fate marker Ki–67 in endometrial biopsies during the WOI using immunohistochemistry. A semi-quantitative immuno-staining score was used to assess the endometrial glands, stroma, luminal epithelium, perivascular and vascular endothelium compartments separately. Statistical differences between groups were calculated by non-parametric tests and significance level set at p < 0.05. Main results and the role of chance During the WOI, the endometrial epithelium of women with RIF and recurrent anembryonic pregnancy loss showed significantly higher levels of ER βwhen compared with fertile controls (p = 0.01 and p = 0.01, respectively). This may indicate an anti-proliferative process occurring at the site of implantation with very early pregnancy losses. In contrast, with women with recurrent fetal loss, a significantly lower level of ERβwas found within the vascular endothelium when compared with the fertile controls (p < 0.01). This supports the theory that increased oxygen levels may compromise trophoblastic invasion, thereby leading to fetal loss. The presence of Ki–67 (a marker of proliferation) was significantly lower within the vascular endothelium of all types of RPL: recurrent anembryonic loss (p = 0.02), RIF (p = 0.02) and recurrent fetal loss (p < 0.01). These findings suggest ineffective endometrial angiogenesis in RPL, resulting in a suboptimal endometrial microenvironment. PR was found to be significantly reduced (p < 0.01) in the perivascular area of women with RIF versus fertile controls. Since decidualisation and preparation of the endometrium for a successful implantation is controlled by critical target genes downstream of PR, this alteration in PR may be an important feature of their defective endometrial phenotype. Limitations, reasons for caution Samples analysed were taken from the functional endometrium and therefore the results do not reflect the basalis. The WOI was identified using history and histological appearance, rather than timing with ovulation. Although we detected statistical significance, generalisation of the results requires further studies with larger sample size. Wider implications of the findings: This data provides novel insight into the biological correlates of clinical types of RPL and suggests that specific alterations in the regulation of endometrial cell fate and oestrogen- responsiveness are associated with different types of RPL. This highlights possible new therapies for RPL, such as selective oestrogen receptor modulators (SERMs). Trial registration number Not applicable

P-365 Pre-selected for an award: Altered endometrial oestrogen-responsiveness and aberrant expression of cell-fate markers may contribute to the aetiology of recurrent pregnancy loss

Study question Do women with recurrent pregnancy loss (RPL) have an aberrant expression of oestrogen receptor-β (ERβ) and cell-fate markers during the window of implantation (WOI) endometrium? Summary answer Women with RPL are found to have significantly altered levels of ERβ and Ki-67 in the WOI endometrium, possibly resulting in anti-proliferative and anti-angiogenic effects. What is known already RPL affects 1% of all women and has been associated with altered endometrial angiogenesis and proliferation when compared with the endometrium of healthy fertile women. RPL can be subcategorised into recurrent loss of anembryonic pregnancy, fetal loss (following evidence of a fetal heartbeat) and recurrent implantation failure (RIF). ERβ is the only oestrogen-receptor (ER) known to be expressed in the vascular endothelium of the endometrium and is the dominant ER during the WOI. It has an important role in endometrial regeneration and is proposed to regulate the angiogenic and vascular changes that occur in embryo implantation. Study design, size, duration This pilot case-control study took place at the Liverpool Women’s Hospital and included 38 women; 29 who suffered RPL and 9 controls with proven fertility (≥2 healthy pregnancies). Of the RPL group, 9 had recurrent loss of anembryonic pregnancy, 10 had recurrent fetal loss and 10 had RIF. Endometrial samples were collected during the WOI (cycle day 22+/-2). Participants/materials, setting, methods To determine whether markers of endometrial cell proliferation and oestrogen-responsiveness are associated with RPL, we assessed the immuno-staining for ERβ, progesterone receptor (PR) and cell-fate marker Ki-67 in endometrial biopsies during the WOI using immunohistochemistry. A semi-quantitative immuno-staining score was used to assess the endometrial glands, stroma, luminal epithelium, perivascular and vascular endothelium compartments separately. Statistical differences between groups were calculated by non-parametric tests and significance level set at p < 0.05. Main results and the role of chance During the WOI, the endometrial epithelium of women with RIF and recurrent anembryonic pregnancy loss showed significantly higher levels of ERβ when compared with fertile controls (p = 0.01 and p = 0.01, respectively). This may indicate an anti-proliferative process occurring at the site of implantation with very early pregnancy losses. In contrast, with women with recurrent fetal loss, a significantly lower level of ERβ was found within the vascular endothelium when compared with the fertile controls (p < 0.01). This supports the theory that increased oxygen levels may compromise trophoblastic invasion, thereby leading to fetal loss. The presence of Ki-67 (a marker of proliferation) was significantly lower within the vascular endothelium of all types of RPL recurrent anembryonic loss (p = 0.02), RIF (p = 0.02) and recurrent fetal loss (p < 0.01). These findings suggest ineffective endometrial angiogenesis in RPL, resulting in a suboptimal endometrial microenvironment. PR was found to be significantly reduced (p < 0.01) in the perivascular area of women with RIF versus fertile controls. Since decidualisation and preparation of the endometrium for a successful implantation is controlled by critical target genes downstream of PR, this alteration in PR may be an important feature of their defective endometrial phenotype. Limitations, reasons for caution Samples analysed were taken from the functional endometrium and therefore the results do not reflect the basalis. The WOI was identified using history and histological appearance, rather than timing with ovulation. Although we detected statistical significance, generalisation of the results requires further studies with larger sample size. Wider implications of the findings This data provides novel insight into the biological correlates of clinical types of RPL and suggests that specific alterations in the regulation of endometrial cell fate and oestrogen- responsiveness are associated with different types of RPL. This highlights possible new therapies for RPL, such as selective oestrogen receptor modulators (SERMs). Trial registration number Not applicable

Seroprevalence and risk factors of Toxoplasma gondii infection in women with recurrent fetal loss from the province of Khyber Pakhtunkhwa, Pakistan

BACKGROUND: In women with a bad obstetric history (BOH), infection is an established cause of recurrent fetal loss. A common infecting agent is the protozoan parasite Toxoplasma gondii (T. gondii). The aim of this study was to measure the prevalence of toxoplasmosis in women with recurrent fetal loss from the Khyber Pakhtunkhwa province of Pakistan. METHODS: The study included 360 females aged 16–40 years, of which 180 had a bad obstetric history (study group) and the other 180 had no such history (control group). Blood serum samples were tested for toxoplasma IgM antibodies by Enzyme Linked Immunosorbent Assay and for toxoplasma IgG antibodies using an Immunochromatographic technique. RESULTS: The overall seroprevalence of toxoplasma infection in study group females was 40.6% and in control group females it was 7.2%. Specifically, IgM prevalence was 12.8% in the study group and 1.1% in the control group. IgG prevalence was 23.9% in the study group and 6.1% in the control group. IgM and IgG combined prevalence was 3.9% in the study group cases. There is a statistically significant association between BOH and seropositivity for T. gondii (p < 0.0001, Chi square test). Various risk factors associated with T. gondii seroprevalence in study and control groups were analyzed. CONCLUSION: The seroprevalence of toxoplasmosis was significantly higher in women with a bad obstetric history compared to those with no such history. Associated risk factors had no significant effects on the results.

Immunological Basis for Recurrent Fetal Loss and Pregnancy Complications

Pregnancy stimulates an elaborate assortment of dynamic changes, allowing intimate approximation of genetically discordant maternal and fetal tissues. Although the cellular and molecular details about how this works remain largely undefined, important clues arise from evaluating how a prior pregnancy influences the outcome of a future pregnancy. The risk of complications is consistently increased when complications occurred in a prior pregnancy. Reciprocally, a prior successful pregnancy protects against complications in a future pregnancy. Here, we summarize immunological perturbations associated with fetal loss, with particular focus on how both harmful and protective adaptations may persist in mothers. Immunological aberrancy as a root cause of pregnancy complications is also considered, given their shared overlapping risk factors and the sustained requirement for averting maternal–fetal conflict throughout pregnancy. Understanding pregnancy-induced immunological changes may expose not only new therapeutic strategies for improving pregnancy outcomes but also new facets of how immune tolerance works that may be applicable to other physiological and pathological contexts.

Do Current Antiphospholipid Antibody Syndrome Guidelines Effectively Target at-Risk Obstetric Patients?

Introduction: The incidence of spontaneous miscarriages is 1%, and up to 50% of recurrent fetal losses (RFL) have unclear etiology. Antiphospholipid syndrome (APS) has been reported in 5-15% of women with RFL. Current clinical criteria for APS include venous thromboembolism (VTE) and /or fetal loss. In addition to the clinical criteria, laboratory confirmation must be obtained using tests for lupus anticoagulant (LA), anticardiolipin (ACL) and beta-2 glycoprotein I antibodies (β2GPI). These tests are often provided by many laboratories as panels; moreover, given the relatively large number of tests, judicious ordering of these panels is warranted to contain costs and to avoid mislabeling patients who have clinically insignificant positive results. Some existing literature suggests that patients with recurrent fetal loss and ACL antibodies are over-tested and/or over-diagnosed. We aim to determine the frequency and likelihood of positive antiphospholipid antibody (APA) among patients with fetal loss. Materials and Methods: A retrospective search was conducted for APA panels using data from our laboratory information system from Jan 2014 to Jan 2015. Recorded variables included age, ordering physician subspecialty and indication for testing. For patients with fetal loss, the number of losses and gestational age at the time of loss were recorded. Specimens were classified as positive, indeterminate or negative. For positive and indeterminate tests, the results of LA, ACL, and β2GPI antibody tests and titers were noted. Results: Our search identified 430 panels ordered for APA. The median age was 42 (range: 1-92). The three highest ordering physician subspecialties were Ob/Gyn (20.2%), Internal Medicine (17.7%) and Heme/Onc (17.2%). The three most common indications for ordering the APA panel were venous thromboembolism (26.5%), fetal loss (18.4%), and autoimmune disease (15.4%). The largest percentage of positive results came from panels ordered by Heme/Onc 45% (20/44) and for the indication of VTE 40.9% (18/44). Fetal loss (n=74) accounted for 4.5% (2/44) of positive results. Further investigation of the fetal loss cases showed that 82.4 % (61/74) met criteria for appropriate APA testing. There was no statistically significant difference for gestational age < 10 weeks (1 positive, 4 indeterminate, and 30 negative specimens) or > 10 weeks (1 positive, 1 indeterminate, and 37 negatives; p=0.334); ordering physician subspecialty and the APA testing result. Both fetal loss patients that tested positive for APS had increased titers of ACL IgM and β2GPI IgM. Conclusions: Despite being the second most common indication for APA testing, fetal loss only accounted for 4.5% of positive results. Compared to historical data that report an incidence of 5-15% APS in patients with recurrent fetal loss, we found an incidence of only 2.7%. The reason for this discrepancy is not clear. Confounding factors such as inappropriate work-ups or incomplete testing are unlikely as 82.7% of patients meet standard testing criteria and our APA panels included multiple tests for LA, ACL and β2GPI. Larger studies are needed to confirm our findings, as this may call for redefinition of APA testing guidelines to better target at risk obstetric patients. Disclosures No relevant conflicts of interest to declare.

A Case Series of Antiphospholipid Syndrome Patients

Introduction: Antiphospholipid syndrome (APS) is a pro-thrombotic, autoimmune mediated clinical syndrome associated with increased propensity for venous and arterial thrombosis and recurrent fetal loss. A significant clinical heterogeneity exists in the condition ranging from minor easily managed venous thromboses and TIAs to unusual, catastrophic, or refractory thromboses. It is generally unknown what factors are useful in predicting the clinical course. Our goal was to characterize our cohort and to look for any other factors that may be associated with thrombosis. Methods: We analyzed the records of 979 patients seen in a single physician cohort over a 4-year period at an academic institution. 81 had some mention of APS. Out of 81 patients, 23 patients were included. Seven of these patients did not meet our criteria precisely, but were considered as having APS due to high clinical suspicion by the treating physician. We defined APS by the presence of a thrombotic event or recurrent fetal loss in association with one of the following laboratory criteria repeatedly positive at least 12 weeks apart: Lupus anticoagulant (LAC), Beta-2 Glycoprotein antibodies (aB2GP), Cardiolipin antibodies (aCL). Cardiolipin and Beta-2-Glycoprotein antibodies were defined as abnormal if levels were above 40 mpl/gpl; IgA antibodies for Beta-2-Glycoprotein were also allowed. The tests for LAC were as follows: dilute Russell Viper Venom time (dRVVT), hexagonal lipid neutralization, partial thromboplastin time (PTT) mixing study, and tissue thromboplastin inhibition (TTI) 1:50/1:500. The TTI could not be the only abnormal test and TTIs were excluded if the Prothrombin time (PT) was prolonged for any reason. LAC tests done with patients on dabigatran, apixiban, or rivaroxaban were considered uninterruptable. Results: Table 1 and Table 2 outline our findings. As we had so few double or triple positive patients we were unable to detect a difference in clinical severity related to the number of laboratory criteria met. 6/14 women were on hormonal therapy at time of diagnosis. 5/18 patients who were tested for thrombophilias were found to have one; three with prothrombin gene variant (PGM) and two with factor V Leiden (FVL). In carefully assessing for concurrent risk factors, 18/23 (78%) of our patients had at least one risk factor for thrombosis, 9 (39%) had at least two risk factors, and four (17%) had at least three risk factors. Notably all five (22%) of the patients with no preexisting risk factors had no recurrence of thrombosis. We also found that two (9%) of our patients normalized the APS laboratory abnormalities over time. Discussion: Thrombophilia testing in patients with thromboses has grown dramatically over the last several decades and there are questions as to the utility in managing patients. The presence of antiphospholipid antibodies that are associated with APS, can be seen in asymptomatic patients, but are also seen in patients with catastrophic thromboses, thromboses refractory to anticoagulation, and in thromboses in unusual locations. In our case series, most patients were single positive and most had concurrent risk factors for thrombosis. We did not see evidence that double or triple positive APS patients had a more severe clinical course as compared to single positive patients, but we had so few multiple positive APS patients to allow us to conclude this with any certainty. An interesting observation was how many of the patients in our cohort had other known risk factors for thromboses specifically FVL, PGM, and hormonal therapy. We noted that patients with no other known risk factor other than APS seemed to have a better clinical course, while those with multiple risk factors had a more severe clinical course. Lastly, we noted some patients, who had met the diagnostic criteria for LAC, had their laboratory testing normalize over time. We plan to extend this case series in a larger patient cohort at our institution. Disclosures No relevant conflicts of interest to declare.