Obstetric outcomes following frozen embryo transfer (FET) in patients with an endometrial thickness measuring <7mm

MATERIALS AND METHODS: Women with regular menses, proven fertility and exclusion criteria of endometriosis were enrolled at the time of elective tubal ligation. Patients underwent an endometrial biopsy and each sample was cultured for nine days to rid any endogenous hormone effect and plated into a control and decidualized group. The decidualized group were exposed to 1 mM medroxyprogesterone acetate and 0.5 mM cAMP for nine days. The mRNA expression levels of Prolactin (PRL) and Insulin Like Growth Factor Binding Protein 1 (IGFBP1) were measured by quantitative RT-PCR. The fold change in gene expression between control and decidualized samples was calculated. Patients were categorized into obese (BMI > 30) and lean (BMI <30). A Students t-test was used for statistical analysis and a stratified analysis was performed to control for race. Western blot analysis was then performed measuring accumulation of cytoplasmic LC3-II which determines autophagic flux. RESULTS: Thirty seven patients were enrolled with twenty three obese patients and fourteen lean patients. The fold change in gene expression of both markers of endometrial decidualization, PRL and IGFBP1, were significantly (p<0.05) lower in obese patients compared to lean. Western blot analysis comparing autophagic flux in obese vs lean race matched patients revealed impaired autophagic flux in obese patients compared to lean. CONCLUSIONS: By controlling the in vitro hormonal environment and measuring markers of endometrial decidualization we found that ESCs from obese women have lower mRNA expression levels of PRL and IGFBP1, indicating a reduced ability to undergo normal decidualization. Impaired autophagy may be one mechanism responsible for the decrease in decidualization and novel therapies that target improving this mechanism such as Niacin could alleviate poor decidualization and ultimately poor pregnancy outcomes in this population. The NICHD Grant R01 HD 065435. Supported by: ASRM/SREI Fellow Research Grant 2015.

P-285 Tuesday, October 18, 2016 A NEW WAY TO DETERMINE UTERINE RECEPTIVITY BY MOLECULAR PROFILING OF ENDOMETRIAL BIOPSIES. S. V. Dambaeva,a D. Katukurundage,a L. Wu,b N. Sung,b M. D. Salazar Garcia,b A. Skariah,b A. Gilman-Sachs,a J. Kwak-Kim,b K. Beaman.a aDept. Microbiology and Immunology, Clinical Immunology Laboratory, Rosalind Franklin University of Medicine and Science, North Chicago, IL; bDept. of Obstetrics and Gynecology, Reproductive Medicine Center, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL. OBJECTIVE: A unique gene expression pattern in the endometrium is vital for successful implantation and placentation. Aberrant gene expression, both increased and decreased, of uterine derived growth factors contributes to recurrent pregnancy loss (RPL) and infertility (IF). The objective of this study was to determine cellular and secreted factors involved in pathogenesis of RPL and IF by molecular analysis of endometrial biopsy samples. DESIGN: Patients with unexplained RPL and IF were enrolled in the Reproductive Medicine Center. Endometrial biopsies were collected in midluteal phase. MATERIALS AND METHODS: mRNA was extracted from endometrial samples, converted into cDNA and analyzed by quantitative RT-PCR (qRTPCR) for factors important for tissue homeostasis (IL-18, IL-15, IL-22, IL-22 receptor (IL-22R), fibroblast growth factor-inducible 14 (Fn14), serum glucocorticoid kinase 1 (SGK1)). The level of NK cells was quantitated by mRNA of CD56, CD57 and CD16b (also expressed by neutrophils). The ratio of CD16b/CD56 was used to determine relationship between CD56bright endometrial NK cells and CD16b positive cytotoxic NK cells and/or neutrophils. RESULTS: The initial evaluation of the endometrium of RPL (n¼11) and IF (n¼8) women revealed differential expression of Fn14. RPL samples had significantly higher levels of Fn14 mRNA than samples from women with IF (4.4+/-1.7 and 1.5+/-0.3 correspondingly). There was a direct correlation between expression of Fn14 and IL-18, IL-15, IL-22R in samples from women with IF. In all specimens, the CD16/CD56 ratio negatively correlated with CD56 mRNA (r¼ -0.792, p¼ 0.02). Interestingly, mRNA levels of CD16b paralleled the mRNA levels of CD57 (cytotoxic NK cell marker) in RPL samples but not in IF samples, indicating a measurable neutrophil increase in IF endometrium. High CD16/CD56 ratio correlated with low values of SGK1 mRNA expression and vice versa (r¼ -0.677, <0.01). CONCLUSIONS: The analysis of endometrial tissue by qRT-PCR can be important for the evaluation of uterine receptivity and for embryo

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ASRM Abstracts

implantation. The initial results show that specimens from women with RPL and IF have different patterns of cellular and growth factor expression. Support: Clinical Immunology Laboratory at Rosalind Franklin University, North Chicago, IL. Supported by: Clinical Immunology Laboratory at Rosalind Franklin University of Medicine and Science, North Chicago, IL. P-286 Tuesday, October 18, 2016 THE UTILITY OF ENDOMETRIAL AND UTERINE VASCULARITY MEASUREMENT BY TRANSVAGINAL ULTRASONOGRAPHY IN PREDICTING PREGNANCY OUTCOME DURING FROZENTHAWED EMBRYO TRANSFER CYCLES. K. Lee, J. Joo, S. Kim, S. Lee. Pusan National University Hospital, Busan, Korea, Republic of. OBJECTIVE: An appropriate endometrial condition and vascular supply are usually known as essential for implantation of embryo. This study was performed to assess the role of endometrial and uterine vascularity status measurement in predicting pregnancy outcome during frozen-thawed embryo transfer (FET) cycles. DESIGN: Total 70 infertile women were recruited with controlled ovarian stimulation (COS) followed by oocytes retrieval. After IVF or ICSI, embryos were cultured to blastocysts and blastocysts with good quality were selected for cryopreservation. MATERIALS AND METHODS: After endometrial preparation, vitrified blastocysts were thawed and assisted hatching by zona dissection was performed. On the day of embryo transfer (ET), endometrial thickness (EMT), resistance index (RI) and pulsatility index (PI) of sub-endometrial artery (SEA) and uterine artery (UA) were obtained by transvaginal sonography (TVS). All women were divided into pregnant or not groups, and these variables were compared between 2 groups. RESULTS: Patients’ general demographic characteristics weren’t different statistically between pregnant and nonpregnant groups. The overall implantation rate, clinical pregnancy rate, and ongoing pregnancy rate were 31.1%, 41.4% and 28.6%, respectively. 29 Patients who conceived had average EMT, RI of SEA, PI of SEA, RI of UA, and PI of UA values of 9.15mm, 0.91, 2.42, 0.95, and 3.37, respectively. 41 Patients who didn’t conceive had average EMT, RI of SEA, PI of SEA, RI of UA, and PI of UA values of 9.31mm, 1.01, 2.56, 0.94, and 3.00, respectively. In two groups, all variables were not statistically different (p > 0.05). CONCLUSIONS: Measurement of endometrial thickness and blood flow index of uterus and endometrium by transvaginal sonography during frozenthawed embryo transfer (FET) cycles can’t seem to predict the pregnancy outcome. But endometrial receptivity is thought to be still important factor in successful implantation of FET cycle and many studies agree that high degrees of endometrial perfusion shown by Doppler sonogram suggests more receptive endometrium. P-287 Tuesday, October 18, 2016 OBSTETRIC OUTCOMES FOLLOWING FROZEN EMBRYO TRANSFER (FET) IN PATIENTS WITH AN ENDOMETRIAL THICKNESS MEASURING<7MM. C. R. Juneau, J. M. Franasiak, S. J. Morin, T. A. Molinaro, R. T. Scott. Reproductive Medicine Associates of New Jersey, Basking Ridge, NJ. OBJECTIVE: While it is apparent that an endometrial thickness <7 mm is associated with diminished outcomes in assisted reproduction (lower implantation and pregnancy rates), we know that pregnancies do occur in patients with thin endometrial linings. There is no data, however, on obstetrical outcomes in these patients following IVF treatment. This analysis was performed to determine if endometrial thickness <7 mm is associated with adverse obstetric outcomes compared to patients with a thicker endometrium. DESIGN: Retrospective cohort MATERIALS AND METHODS: FETs from 2010-2016 at a single center were included. All transferred embryos were at the blastocyst stage of development. Cycles were divided into 2 groups based on endometrial thickness measured the day before transfer (<7 mm v. > 7 mm). Outcomes including live birth rate (LBR), maternal complications (abruption, bleeding, gestational diabetes, infection, pregnancy induced hypertension, placenta previa, retained products of conception), estimated gestational age (EGA) at delivery, birth weight (grams), and mode of delivery were compared between the 2 groups. Statistical analysis was performed using a c2 test of proportions or student t-test. Alpha error less than 0.05 was significant.

Vol. 106, No. 3, Supplement, September 2016

RESULTS: 392 FETs with an endometrial thickness <7 mm and 8057 FETs with an endometrial thickness >7mm were included. The mean number of embryos transferred was 1.4 blastocysts. 3888 transfers utilized preimplantation genetic screening (46.0%). The only patient demographic measured that was different between the 2 groups was body mass index (BMI) which was 24.75.0 kg/m2 in patients with a lining <7 mm compared to 25.85.8 kg/m2 in control patients (p¼0.0006). Adverse obstetric outcomes were compared between groups (table 1).

Table 1: Preterm delivery and low birth weight occur more often in FETs < 7mm.

Endometrial thickness

LBR Maternal complications EGA Birth weight (grams) Low birthweight (<2500 grams) Cesarean section rate

< 7 mm

R 7 mm

p-value

35.2% (138/392) 8.7% (12/138) 34.7+3.3 weeks 2948.1708.9 24.8% (34/137)

44.1% (3553/8057) 7.9% (279/3553) 35.5+3.2 weeks 3178.7722.6 15.9% (563/3539)

0.0005 0.7184 0.006 0.0003 0.0055

71.2% (79/110) 65.9% (1844/2799) 0.4857

CONCLUSIONS: In frozen embryo transfers, patients with an endometrial thickness <7 mm demonstrate a higher proportion of preterm delivery and low birth weight. In addition to counseling these patients on reduced pregnancy rates, patients should also be counseled on risk of preterm birth. P-288 Tuesday, October 18, 2016 HUMAN ENDOMETRIAL RECEPTIVITY-ASSOCIATED MIRNAS: BEYOND THE GENES. C. Innocenti,a D. Haouzi,b L. Drissennek,c Y. Antoine,d F. Entezami,e L. Delaroche,f C. Brunet,g S. Hamamah.h aUnitæ 1203 IRMB Montpellier, Montpellier, France; bU1203 - IRMB - CHRU Montpellier, Montpellier, France; cINSERM U1203, Human Early Embryonic Development and Pluripotency, Montpellier, France; dU1203-IRMB, Clermont-l’Hærault, France; eIVF Laboratory Eylau-UNILABS, Clinique de La Muette-Ramsay GDS, Paris, France; fEylau-Unilabs IVF Laboratory, Neuilly-sur-Seine, France; gEndocrinologist, Montpellier, France; hART/ PGD Department, Arnaud de Villeneuve Hospital, Montpellier, France. OBJECTIVE: Beyond the genes, do miRNAs are associated to endometrial receptivity status during the expected implantation windows? DESIGN: Endometrial biopsies were collected during the implantation windows under hormone replacement therapy (6 to 9 days after progesterone administration). Then RNAs were extracted for mRNA and miRNA purification to perform the Win-Test and the miRNA expression profile, respectively. The Win-test consists of measuring the expression level of 13 transcripts associated to the endometrial receptivity by RTqPCR. The miRNAs profile was evaluated with the AffymetrixÒ miRNA 4.1 Array Strips. MATERIALS AND METHODS: Endometrial biopsies were obtained from 20 patients with repeated implantation failure during IVF/ICSI cycles. The endometrial receptivity status was determined using the Win-Test allowing to classify endometrial samples as ‘receptive’ or ‘non-receptive’. Then, miRNA expression profiles between the two groups of patients, receptive (n¼15) and non-receptive (n¼5), were performed. RESULTS: Using 3 distinct statistical analyses, we identified five miRNAs differentially expressed between receptive and non-receptive endometrium [miR-1 (x-4.9, FDR<0.0001; miR-2 (x-2.5, FDR<0.0001), miR-3 (x-3.6, FDR<0.001), miR-4 (x-3.9, FDR<0.0001), miR-5 (x-2.1, FDR<0.0001)]. All of them were down-regulated in receptive endometrium tissues and associated with the over-expression of the 13 specific genes biomarkers of the Win-Test. These 5 miRNAs target 165 over-expressed mRNAs during the implantation windows, including the 13 specific genes biomarkers of the implantation window, that were reached to several biological functions that play a crucial role during implantation. CONCLUSIONS: The identification of miRNAs in endometrial tissues associated to endometrial receptivity opens new perspectives in poor implanted patient.

FERTILITY & STERILITYÒ

Supported by: This work was partially supported by a grant from the Ferring Pharmaceutical Company P-289 Tuesday, October 18, 2016 CDKN1C (P57): THE DETERMINANT OF HUMAN ENDOMETRIAL STROMAL CELL DECIDUALIZATION. L. Wang, L. Huang, H. Zhang, K. Qian. Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Reprodoctive Medical Center, Wuhan, China. OBJECTIVE: Uterine decidualization, characterized by human endometrial stromal cells (hESCs) differentiation into decidual cells with distinct morphological appearances, unique biosynthetic and particular secretory phenotypes, is of great significance to the establishment as well as the maintenance of pregnancy (1, 2). However, the exact cell cycle arrest mode and the exact molecular mechanism underlying hESCs decidualization are still not fully elucidated. We aim to confirm that the cyclin-dependent kinase inhibitors, p57, plays an important role in the decidualization of human hESCs. DESIGN: Basic experimental study using human sample. MATERIALS AND METHODS: Human uterine endometrial stromal cells were collected and decidualized by progesterone and cAMP. The cell cycle distribution during hESCs decidualization was analyzed by flow cytometry. Several cell cycle related genes were confirmed up- or down-regulated after decidualization by microarray technique and immunohistochemistry staining. And their roles in hESCs decidualization was analyzed by RNAi, flow cytometry, chemiluminescence assay and transmission electron microscope RESULTS: We confirmed a typical cell cycle distribution mode during hESCs decidualization characterized by a cell cycle arrest at G0/G1 phase. By microarray and immunohistochemistry staining, the decreased expression of CyclinD1, CDK2 and the CDC2 as well as the increased expression of p57 and p15 were observed during hESCs decidualization. We found that downregulation of p57 but not p15 significantly impaired the process of decidualization, manifesting in both cell morphological and secretory alteration of the hESCs as well as an inhibition of cell cycle arrest in G1/G0 phase. CONCLUSIONS: P57 plays a determinant role in the process of decidualization by promoting terminal withdraw of hESCs from cell cycle. References: 1. Qi QR, Zhao XY, Zuo RJ, Wang TS, Gu XW, Liu JL and Yang ZM. Involvement of atypical transcription factor E2F8 in the polyploidization during mouse and human decidualization. Cell Cycle 2015; 14: 1842-1858. 2. Gellersen B, Brosens IA and Brosens JJ. Decidualization of the human endometrium: mechanisms, functions, and clinical perspectives. Semin Reprod Med 2007; 25: 445-453. Supported by: National Natural Science Foundation of China (Grant No. 81571464 and Grant No. 81170583). P-290 Tuesday, October 18, 2016 ENDOMETRIAL VASCULARITY AND INCREASING ENDOMETRIAL THICKNESS CAN PREDICT LIVE BIRTH: A RETROSPECTIVE ANALYSIS OF 1575 FET CYCLES. H. Konar,a S. Sharma,b P. Chakraborty,c I. Saha,b R. Chattopadhyay,d S. Ghosh,e f a B. Chakravarty. Professor, Obstetrics & Gynecology, Kolkata, India; b ART, Consultant, Kolkata, India; cInfertility, Scientist, Kolkata, India; dReproductive Medicine, Embryologist, Kolkata, India; eAssisted Reproduction, Consultant, Kolkata, India; fReproductive Medicine, Director, Kolkata, India. OBJECTIVE: To evaluate the implication of increasing endometrial thickness and/or evidence of subendometrial blood flow in prediction of live birth in frozen embryo transfer (FET) cycles. DESIGN: Retrospective analysis. MATERIALS AND METHODS: 1575 FET cycles excluding donor oocyte recipients and gestational carriers were analysed. The endometrium was prepared using oral estradiol 6-12mg daily, started on day 2 or 3 of cycle Ultrasound was done from day 12 onwards to measure endometrial thickness. Once endometrial thickness was R8 mm, doppler study of endometrial blood flow was conducted. Embryo transfer (ET) was done once endometrial thickness R8 mm and or presence blood flow in the subendometrial region after 3days of progesterone administration. Patients were stratified into 3 groups (Group A (n¼929): have increasing endometrium and subendometrial blood

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