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

Expectant management of incomplete miscarriage, anembryonic pregnancy and early fetal demise: a comparative study

Background: Expectant management as first line management of early pregnancy miscarriages is less accepted due to failure and increased complications reported in few studies. Proper selection of cases improves outcome of expectant management. Aim of this study was to compare success rate and complications in expectant management in three groups of early pregnancy miscarriages- Incomplete miscarriage, anembryonic pregnancy and early fetal demise.Methods: Prospective observational study conducted in tertiary care centre for 3 years, including 107 patients with USG confirmed pregnancy miscarriage <13 weeks. Patients preferring expectant management were managed as outpatient without intervention for 2 weeks after which repeat USG was done to ascertain complete miscarriage. Failed expectant management patients underwent planned surgical uterine evacuation. Emergency admission and evacuation was done, if symptomatic during waiting period. Success rate and complications like emergency evacuation, vaginal bleeding, abdominal pain, limitation of physical activity and patient satisfaction were assessed and compared in subgroups of anembryonic pregnancy, early fetal demise and incomplete miscarriage. Statistical analysis was done by chi-square test.Results: Incomplete miscarriage group had highest success rate of 88.46%. followed by anembryonic pregnancy (72.5%) and EFD (47.83%) p value = 0.007. Complication rate was highest in EFD, followed by anembryonic and the least in incomplete miscarriage all of which was statistically significant except vaginal bleeding.Conclusions: Expectant management should be offered as first line choice for all types of early pregnancy miscarriages. Proper selection of case as to type of miscarriage especially incomplete miscarriage and selected cases of anembryonic pregnancy and EFD ensures higher success rate with lesser complications. Reserving medical and surgical management for unsuitable/failed cases.

KEHAMILAN ANEMBRIONIK PADA PRIMIGRAVIDA : SEBUAH TINJAUAN KASUS

Abstrak. Sebuah kasus, wanita berusia 22 tahun hamil 8-9 minggu dengan keluhan perdarahan pervaginam sejak 1 sebelum masuk rumas sakit (RS). Pemeriksaan ultrasonografi menunjukkan gambaran kantung gestasi tanpa pertumbuhan embrio dengan usia kehamilan 8 minggu. Terminasi kehamilan pada kasus ini dilakukan dengan metode dilatasi dan kuretase. Kehamilan anemebrionik merupakan salah satu bentuk kegagalan dalam kehamilan. Diperkirakan 10-15% hasil konsepsi tidak viabel dan akan mengalami abortus dan 3% diantaranya merupakan kehamilan anembrionik. Manifestasi klinis pada kasus ini meliputi riwayat amenorea, tanda-tanda kehamilan muda serta perdarahan pervaginam pada tahap akhir perjalananya. Berbagai faktor secara teoritis dikaitkan dengan kehamilan embrionik meliputi; faktor genetik paternal dan maternal, disfungsi hormonal serta infeksi dan kelainan imunologi. Kehamilan embrionik dapat ditegakkan melalui pemeriksaan ultrasonografi transabdominal maupun transvaginal dengan ditemukannnya kantung gestasi tanpa perkembangan embrio pada minggu 6-10 kehamilan. Kata Kunci: Kehamilan anembrionik, kuretaseAbstract. A case, 22-year-old woman with 8-9 weeks gestational aged with complaints of vaginal bleeding one day before hospital admission. Ultrasound examination showed a picture of gestational sac without embryo growth correspondent to 8 weeks gestational aged. Termination of pregnancy in this case was done by the method of dilation and curettage. Anembryonic Pregnancy is one form of failure in pregnancy. An estimated 10-15% of the pregnancy is not viable and will undergo abortion and 3% of it is anembryonic pregnancy. Clinical manifestations in this case includes a history of amenorrhea, signs of early pregnancy and vaginal bleeding at the final stage perjalananya. Various factors are theoretically associated with embryonic pregnancy include; paternal and maternal genetic factors, hormonal dysfunction, as well as infectious and immunological disorders. Embryonic Pregnancy can be enforced through a transabdominal or transvaginal ultrasound examination with detection of gestational sac without embryo development at 6-10 weeks gestation.Key words: Anembryonic pregnancy, curettage

Proteomics of the Human First Trimester Chorionic Villi Associated with Anembryonic Pregnancy

In this study, the proteomic approach based on high performance liquid chromatography connected with tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis were applied to identify differentially abundant proteins in chorionic villus samples (CVS) from women with blighted ovum and normal pregnancy. We identified about 600 proteins in the solubilized fraction of CVS. Comparative proteomic analysis revealed differences in the content (Average Normalized Abundances) of 187 proteins in blighted ovum. These included 134 down-regulated proteins and 53 up-regulated proteins. According to bioinformatics analysis these proteins participate in a variety of metabolic processes, including alcohol and tricarboxylic acid metabolism, response to endoplasmic reticulum stress, small molecular catabolic process, cellular respiration, and others. Proteins that demonstrated growing content in blighted ovum were mainly encoded by genes located on chromosomes 7 and 16 whereas proteins which demonstrated reducing abundance were mainly encoded by genes located on chromosomes 1, 2, and 11. We also revealed changes in the content of proteins encoded by genes located on the human chromosome 18; they are involved in apoptotic and drug metabolic processes with an important role in early pregnancy loss. Our pilot results demonstrate the efficiency of the LC-MS/MS approach for detecting the differences at the qualitative and semi-quantitative levels in the protein profiles of the CVS at anembryonic pregnancy compared to normal gestation. We conclude that globally profiled and differentially regulated proteins of CVS are helpful in obtaining molecular insights into biological processes of the pregnancy pathology.