pregnant rats (d19) and Group 3 of preeclampsia model rats via a well-estab- lished reduced uterine perfusion pressure (RUPP). AMH, AMH-R and Kiss.
pregnant rats (d19) and Group 3 of preeclampsia model rats via a well-established reduced uterine perfusion pressure (RUPP). AMH, AMH-R and Kiss mRNA levels were quantified by RT-PCR. Kruskall Wallis test was used. RESULTS: Compared to nonpregnant rats, the ovaries from late-pregnant rats had 47% [ in AMH-R mRNA levels (p¼0.04) and a trend towards an [ in AMH (57%) and Kiss mRNA levels (56%) (p¼0.1). Compared to latepregnant animals, the ovaries from RUPP animals had Y Kiss mRNA levels (33%), although this difference was not significant, most likely in view of the small n values (p¼0.1). CONCLUSION: These preliminary data suggest that in normal pregnancy there are changes in the ovarian physiology pertaining to genes related to follicular development and ovarian reserve, such as AMH. Similar to placental expression, Kiss gene expression in the ovary tended to be downregulated in preeclampsia model. Understanding the impact of pregnancy and preeclampsia on the ovaries may further the field of ovarian physiology and help understanding the mechanisms of ovulatory dysfunction. Supported by: Internal funds. P-561 Wednesday, October 16, 2013 SERUM IL-10 IS A PREDICTIVE MARKER OF POOR ENDOMETRIAL RECEPTIVITY IN WOMEN WITH IDIOPATHIC RECURRENT SPONTANEOUS MISCARRIAGE. A. Ganesh,a P. Banerjee,b B. Chakravarty,a K. Chaudhury.b aInstitute of Reproductive Medicine, Kolkata, West Bengal, India; bSchool of Medical Science and Technology, Indian Institute of Technology, Kharagpur, Kharagpur, West Bengal, India. OBJECTIVE: Dysregulation in the expression of endometrial cytokines, matrix metalloproteases, angiogenic and vasoactive factors during peri-implantation window are associated with unreceptive endometrium. The present study focuses on identifying the key molecules contributing towards poor endometrial receptivity during peri-implantation period in women with idiopathic recurrent spontaneous miscarriage (IRSM). DESIGN: A prospective case-controlled study carried out on 66 women with IRSM reporting for treatment at the Institute of Reproductive Medicine, Kolkata, India between January 2010to December 2012. 50 fertile women undergoing sterilization were included in the study as controls. All women were age below 35 years and having average BMI. MATERIALS AND METHODS: Blood samples were collected from all women during mid secretory phase and serum was extracted from it. Levels of Interleukin-10 (IL-10), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in serum were assessed by ELISA. Multivariate analysis was performed to identify the key factor contributing towards poor endometrial receptivity in IRSM women. RESULTS: Serum expression of IL-10, VEGF, eNOS and TIMP-1 were observed to be significantly lower and MMP-9 was significantly higher in women with IRSM compared to controls. IL-10 was identified as the key contributory factor for poor endometrial receptivity in women with IRSM. Further, 62.58 pg/ml is the serum cut-off level of IL-10 beyond which endometrial receptivity is impaired in women with IRSM. CONCLUSION: As part of routine clinical examination, IL-10 may be assessed in serum of women with IRSM. Adequate clinical measures for preventing early pregnancy loss can be taken if the serum IL-10 expression is found beyond the threshold level in these women. P-562 Wednesday, October 16, 2013 INFORMATICS-BASED MOLECULAR KARYOTYPING OF PRODUCTS OF CONCEPTION (POC): FOCUS ON MOLAR PREGNANCIES. M. K. Maisenbacher,a D. M. Clark,a E. Cheung,a B. Pettersen,a R. Lathi,b M. Rabinowitz.a aNatera, Inc., San Carlos, CA; b Stanford Fertility and Reproductive Medicine Center, Stanford University, Palo Alto, CA. OBJECTIVE: Report incidence of paternal triploidy and full paternal uniparental disomy (UPD) identified in >6,500 POC samples analyzed via single nucleotide polymorphism (SNP) microarray and a bioinformatics technique (Parental SupportÔ). A high correlation between dispermia or duplication of the male genome exists with complete or partial molar pregnancy (CMP or PMP). Both are common causes of miscarriage (affecting 1/1000 pregnancies in Caucasian women) and convey risk of gonadotrophoblastic disease/neoplasia (GTD/GTN). About 2/3 of cases of molar pregnancy miscarried in the first trimester are not diagnosed by ultrasound. Molecular karyotyping of POC using SNP microarray with Parental Support
FERTILITY & STERILITYÒ
allows for identification of the most common genetic causes of CMP (full paternal UPD) and PMP (paternal triploidy) which cannot be identified by traditional karyotyping or array CGH. DESIGN: Retrospective analysis. MATERIALS AND METHODS: Review of 6657 consecutive fresh POC samples sent to a reference lab. Maternal blood samples and miscarriage tissue were shipped overnight to the lab. Miscarriage samples were examined for chorionic villi and/or fetal tissue and prepped using a standardized technique. Genotyping was performed using Illumina CytoSNP-12b microarrays and Parental Support. RESULTS: Of the 6657 cases, 5391 had fetal results (81%). 141 cases (2.6%) had paternal triploidy, including 22 hypotriploidy and 13 hypertriploidy. Full paternal UPD was identified in 15 cases (0.3%). Of these, one was dispermic UPD and 14 were isospermic UPD (1 also had Monosomy X). CONCLUSION: SNP microarray with Parental Support provides an invaluable method for analysis of POC samples that diagnoses all forms of triploidy with parental origin and full paternal UPD with the underlying mechanism. In our cohort, these abnormalities account for 3% of miscarriages. Genetic identification of these losses as CMP or PMP can provide important information for correct medical management, prediction of the patient’s risk of GTD/GTN and recurrence risk. P-563 Wednesday, October 16, 2013 IMPLICATIONS OF PLACENTAL-FETAL MOSAICISM ON NONINVASIVE PRENATAL TESTING (NIPT). B. Levy,a K. Merrion,b M. Hill,b M. P. Hall,b Z. Demko,b R. Matthew.b aDepartment of Pathology and Cell Biology, Columbia University, New York, NY; bNatera, Inc., San Carlos, CA. OBJECTIVE: Report three NIPT cases with evidence of mosaicism. DESIGN: In an IRB-approved study validating the non-invasive prenatal Next-generation Aneuploidy Test Using SNPs (NATUS) algorithm, we tested the ploidy status of chromosomes 13, 18, 21, X and Y in cell-free DNA (cfDNA) isolated from plasma from pregnant women. MATERIALS AND METHODS: cfDNA was analyzed via sequencing post-amplification using multiplex PCR targeting 19,488 single-nucleotide polymorphisms (SNPs). NIPT results (not reported to patients) were compared to fetal chromosome results of amniocentesis, chorionic villus sampling (CVS), cord blood, or products of conception (POC) testing. RESULTS: Case 1: NATUS cfDNA analysis (19 weeks gestational age [GA]) indicated fetal Monosomy X. Mosaic 45,X diagnosis was made via amniocentesis. Case 2: NATUS cfDNA analysis (15 weeks GA) indicated fetal euploidy. Non-mosaic trisomy 18 diagnosis was made via amniocentesis. Fetal, placental, and cord blood samples obtained at elective termination (15 weeks GA) were tested via molecular karyotyping (SNP microarray and/or targeted SNP sequencing); the three cord blood samples, 25 fetal tissue samples, and 56 placental membrane samples were largely non-mosaic trisomy 18. However, 48 placental villi samples were found to be mosaic; most contained 40% euploid cells. Case 3: NATUS cfDNA analysis (14 weeks GA) indicated fetal Monosomy X. Mosaic 45,X diagnosis was made via targeted SNP sequencing analysis of POC. CONCLUSION: Fetal aneuploidy detection via NATUS analysis may depend on the proportion of abnormal cells contributing to the fetal cfDNA fraction. Since mosaicism levels vary in different tissues, analysis of DNA deriving from the placenta may not reflect the chromosomal status of the fetus (as in Case 2); this has long been recognized from CVS cytogenetic studies. This reaffirms placental origins of cfDNA, which is problematic if the chromosomal composition of the region contributing to the cfDNA population differs from that of the fetus. Supported by: NIH 4R44HD062114-02. P-564 Wednesday, October 16, 2013 ABSTRACT WITHDRAWN
P-565 Wednesday, October 16, 2013 TRIPLOIDY DETECTION VIA SINGLE NUCLEOTIDE POLYMORPHISM (SNP)-BASED NON-INVASIVE PRENATAL TESTING (NIPT): TWO CASE STUDIES. M. Hall, M. Hill, B. Zimmerman, S. Sigurjonsson, Z. Demko, M. Rabinowitz. Natera, Inc., San Carlos, CA.
S311
OBJECTIVE: Report on two NIPT cases with evidence of triploidy. DESIGN: Two triploid samples were analyzed using the non-invasive prenatal Next-generation Aneuploidy Test Using SNPs (NATUS) algorithm under a commercial protocol (reporting detection of Trisomies 13, 18, and 21, and Monosomy X) or as part of an IRB-approved research protocol. MATERIALS AND METHODS: Cell-free DNA (cfDNA) isolated from plasma from pregnant women was analyzed via sequencing after amplification using a multiplex PCR protocol targeting 19,488 SNPs. Sequencing results were analyzed with the NATUS algorithm. Laboratory personnel (and the NATUS algorithm) were blinded to sample karyotype. RESULTS: NATUS analysis identified multiple paternal (Case 1) or maternal (Case 2) haplotypes, indicating twins or triploidy. Case 1 (fetal fraction: 6.4%): multiple paternal haplotypes on multiple chromosomes (13, 18, 21, X) were detected, indicating either paternally-inherited triploidy or fraternal twins. Case 2 (fetal fraction: 3.2%): multiple maternal haplotypes on multiple chromosomes (13, 18, 21, X) were detected, similarly indicating either a maternally-inherited triploidy or fraternal twins. Ultrasound with chorionic villus sampling (Case 1) or amniocentesis (Case 2) confirmed single gestations affected with triploidy. CONCLUSION: This SNP-based approach examines the relative distributions of different alleles at polymorphic loci, thus not requiring a reference chromosome, and therefore offers the unique ability to detect triploidy. Here, this method correctly flagged both triploidy cases. This method is expected to distinguish twins from maternal triploidy based on the presence (in the case of twins) or absence (in the case of maternal triploidy) of an extra paternal haplotype, and similarly from paternal triploidy based on the presence or absence of an extra maternal haplotype. Studies are ongoing to differentiate all twin and triploidy cases; in the interim ultrasound will be required confirm which cases may be at risk of triploidy. Supported by: NIH 4R44HD062114-02. CONTRACEPTION/FAMILY PLANNING P-566 Wednesday, October 16, 2013 BLEEDING PROFILE OF A NOVEL ASCENDING-DOSE, EXTENDED-REGIMEN ETHINYL ESTRADIOL/LEVONORGESTREL COMBINATION ORAL CONTRACEPTIVE BASED ON WEIGHT, BODY MASS INDEX, AGE, SMOKING STATUS, AND RACE. J. Gersten,a H. Weiss,b N. Ricciotti,c J. Hsieh,d B. Howard.e a New Age Medical Research Corporation, Miami, FL; bGlobal Medical Affairs, Teva Women’s Health, Petach Tikva, Israel; cResearch and Development, Teva Pharmaceuticals, Inc, Frazer, PA; dGlobal Biostatistics, Teva Pharmaceuticals, Inc, Frazer, PA; eU.S. Medical Affairs, Teva Women’s Health, Frazer, PA. OBJECTIVE: To examine the bleeding profile of an ascending-dose, extended-regimen (ADER) combination oral contraceptive (COC) (QuartetteÒ) based on weight, body mass index (BMI), age, smoking status, and race. DESIGN: Multicenter, open-label, single-arm, phase 3 study. MATERIALS AND METHODS: Sexually active women 18-40 years of age received up to 4 cycles of a 91-day regimen consisting of ethinyl estradiol (EE) 20 mcg/levonorgestrel (LNG) 150 mcg for 42 days, EE 25 mcg/LNG 150 mcg for 21 days, EE 30 mcg/LNG 150 mcg for 21 days, and EE 10 mcg for 7 days. Women kept daily diaries recording bleeding and spotting. Bleeding analyses were evaluated after stratification by weight (
