- Email: [email protected]
Before chemotherapy, cancer patients have lower serum AMH than age-matched healthy controls
and Gynecology, College of Medicine, University of Ulsan, Asan Medical Center, Seoul, Republic of Korea; bMedical Genetic Center, Asan Medical Center, Children’s Hospital, Seoul, Republic of Korea; cPediatrics, College of Medicine, University of Ulsan, Asan Medical Center, Seoul, Republic of Korea. OBJECTIVE: Citrullinemia type I (CTLN1) is an inherited metabolic error of the urea cycle in which citrulline is condensed with aspartate to form arginosuccinic acid due to a deficiency of argininosuccinic acid synthetase (ASS). Embryo transfer following preimplantation genetic diagnosis (PGD) for CTLN1 in day 3 embryos using polymerase chain reaction (PCR) was performed for live birth of healthy baby who is not affected with CTLN1 in a couple who has the birth history of the child with CTLN1. DESIGN: Case report. MATERIALS AND METHODS: A 38-year-old female patient had the birth history of the child affected with CTLN1 and she was a carrier of the mutation in ASS gene. For detection of the mutation carried by female patient, primers for DNA amplification and minisequencing were designed. Thirteen oocytes were retrieved following controlled ovarian stimulation (COS) using GnRH antagonist protocol. Eight oocytes were mature and suitable for intracytoplasmic sperm injection (ICSI). Eight oocytes were fertilized, of which five were suitable for blastomere bopsy in day 3 embryos. PCR assay and minisequencing were performed in biopsied cells. RESULTS: One embryo was found to be free of the mutation in ASS, three were found to be carriers of the maternal mutation, and one was affected with CTLN1. One unaffected blastocyst was transferred and this resulted in a clinical pregnancy. Transferred blastocyst were divided and developed to the identical twin boys. Healthy twin boys were born by cesarean section, and they were confirmed to be unaffected with CTLN1 by post-natal examination. CONCLUSION: PGD for CTLN1 using PCR provides the hopeful chance to have normal unaffected baby to the carriers of the mutation in ASS gene who have previous history of childbirth of baby with CTLN1.
O-361 Wednesday, October 16, 2013 05:30 PM DUAL TRIGGERING WITH GnRH AGONIST AND RECOMBINANT hCG IN GnRH ANTAGONIST IVF CYCLES. M. Sonmezer, B. Ozmen, C. S. Atabekoglu, E. Tolunay, O. Kan, B. Berker. Obstetrics and Gynecology, Ankara University, Ankara, Turkey. OBJECTIVE: To evaluate weather dual triggering with 500 mcg leuprolide acetate (LA) and 250 mcg recombinant hCG is beneficial in GnRH antagonist cycles. DESIGN: Prospective randomized Clinical Study. MATERIALS AND METHODS: Age and BMI-matched (range 25-32 years-old; 24-28 kg/m2) 90 patients diagnosed with a mild male factor and unexplained infertility were allocated to the study. All enrolled patients were randomly divided into two groups; group A (LA+ rec hCG) and B (rec hCG) according to triggering method on hCG day. The triggering was made when at least 3 follicles >17 mm were observed. RESULTS: Groups were comparable in terms of demographic features and the mean number of total collected oocytes, transferred embryos, fertilization rates.
The COH and Cycle Outcomes in the Groups
Parameters Total collected oocyte(s), n Collected MII oocyte(s), n Grade A embryo(s), n Transferred embryo(s), n Fertilization rates, (%) Cycle cancelation, (%)* Previous IVF failures, (%)# Pregnancy rates, (%)
Group A (n¼45)
Group B (n¼45)
P value
11.17.8 9.37 2.41.9 1.90.6 78 6.6 (3/45) 31 (14/45) 54.7 (23/42)
10.79.5 7.55 1.81.5 1.80.4 75 8.8 (4/45) 13 (6/45) 43.9 (18/41)
NS 0,02 0,02 NS NS NS 0,01 0,02
*
O-360 Wednesday, October 16, 2013 05:15 PM VENOPUNCTURE-FREE IVF: MEASUREMENT OF ESTROGEN IN CONTROLLED OVARIAN STIMULATION IVF CYCLES USING A ‘‘PATIENT-FRIENDLY’’ SALIVA-BASED ESTRADIOL ASSAY. A. Zimon,a B. Lannon,a S. Sheller,a D. Sakkas,a M. Ulrich,b M. Alper.a aBoston IVF Inc., Waltham, MA; bSalimetrics LLC, State College, PA. OBJECTIVE: Controlled ovarian stimulation (COH) during in vitro fertilization (IVF) cycles involves close monitoring of estradiol (E2) levels and measurements of follicle growth. E2 monitoring can occur 4-7 times during an IVF cycle, while repeated venopuncture adds to patient stress and anxiety. In contrast, sampling saliva is non-invasive and can be performed remotely. Here, we present a novel saliva-based E2 assay designed to serve infertility patient’s undergoing COH. The objective was to investigate whether a saliva based E2 assay could efficiently be used to monitor a COH cycle, in comparison to a standard serum based assay, and whether patient acceptance of a saliva based assay was greater. DESIGN: Prospective Study. MATERIALS AND METHODS: Concurrent serum and saliva E2 values were collected from patients who provided between 1 and 7 samples on different days of their COH IVF cycle. Serum E2 values were assessed routinely, while saliva values were measured and validated using an immunoassay developed in collaboration with Salimetrics LLC. RESULTS: The Salivary E2 assay was validated for intra- and inter-assay variability with a coefficient of variation of <10% ensuring accurate detection of estradiol. It was also validated for analytical and functional sensitivity and sample stability. Linear regression of serum and saliva E2 values of 20 patients who had >3 samples evaluated between Days 3 and 14 provided an R¼0.8. Saliva E2 values also correlated to follicle growth. Patient survey results showed that saliva sampling was the preferred method of analysis, associated with less anxiety and more likely to be recommended to friends undergoing IVF. CONCLUSION: Saliva based hormone testing provides an equivalent alternative to serum based assessment. The ease of saliva sampling allows a reduction in intervention and a patient friendly approach to IVF, which is associated with improved patient satisfaction and decreased stress. Saliva based hormone tests may become the preferred method of COH monitoring in the future.
S110
ASRM Abstracts
The cycle has been cancelled due to not available a transferable embryo. # The percantage of patients with at least 2 unsuccessful previous IVF cycles. The P value has been set at > 0.05. However the mean number of grade A embryos and of MII oocytes were significantly higher in dual triggered group. A higher rate of previous IVF failure has been observed in dual triggered patients. Pregnancy rate was statistically higher in dual triggered group than in standard group (54.7 vs. 43.9%, p¼0.02). One case of mild OHSS was observed in each group. CONCLUSION: This novel more physiological approach of dual triggering with 500 mcg of GnRH agonist plus 250 mcg recombinant hCG seem to be favorable in GnRH antagonist cycles. Dual triggering may increase the number of MII oocytes and grade A embryos and may enhance pregnancy rates. O-362 Wednesday, October 16, 2013 05:45 PM
BEFORE CHEMOTHERAPY, CANCER PATIENTS HAVE LOWER SERUM AMH THAN AGE-MATCHED HEALTHY CONTROLS. K. E. Palinska-Rudzka,a G. Hartshorne,a G. Lockwood,a T. Ghobara,a A. Eapen.a aMedicine, Univeristy of Warwick, Coventry, West Midlands, United Kingdom; bMidland Fertility Sevices, Aldridge, United Kingdom. OBJECTIVE: Is the ovarian reserve in young women with breast cancer or lymphoma different to that of age matched healthy controls prior to chemotherapy? DESIGN: Prospective longitudinal multicentre study: 198 female participants, aged 18-43 years, recruited between May 2010 and May 2012 from 13 centres in UK. Here, we present results of serum AMH at point of recruitment, in light of women’s reproductive history and lifestyle. MATERIALS AND METHODS: Serum AMH (2nd generation assay) concentrations were measured before the start of chemotherapy in cancer patients and compared with same age group of healthy volunteers. Cancer patients and volunteers known to have PCO, severe endometrosis, previous oophorectomy or infertility problems were excluded. RESULTS: Cancer patients (n¼65) had significantly lower serum AMH than age matched volunteers (n¼123), for the group as a whole. Mean age of breast cancer subgroup (n¼53) was 37.7 with mean AMH of 10.69 pmol/l. Mean age of hematology subgroup (n¼ 12) was 27.6. with mean
Vol. 100, No. 3, Supplement, September 2013
AMH of 13.39 pmol/l. Patients with breast cancer had significantly lower serum AMH than control group (p¼0.04). Lymphoma patients had serum AMH that appeared lower but was not significantly different from the volunteers (p¼0.67). Baseline characteristics including family history of early menopause, smoking status, BMI and contraception use were not significantly different in both groups. The ovarian reserve, assessed using serum AMH, is significantly lower in newly diagnosed breast cancer patients compared to healthy volunteers pior to chemotherapy. In our precition model, a cancer patient had serum AMH levels of an 6 year older volunteer. CONCLUSION: Reproductive age women with malignancy appear to have lower ovarian reserve than healthy volunteers even before any gonadotoxic treatment is commenced. In view of those findings routine measurement of AMH level before discussing and commencing fertility preservation treatment would be highly recommended even in young women. Supported by: University of Warwick; Midland Fertility Services. EARLY PREGNANCY II O-363 Wednesday, October 16, 2013 04:00 PM DIFFERENTIAL mIRNA PROFILES OF TROPHOBLAST TISSUE OBTAINED IN ECTOPIC PREGNANCIES. F. Dominguez,a T. Lozoya,b J. M. Moreno-Moya,a S. Martinez,a C. Simon,a A. Pellicer.b a Fundacion IVI/INCLIVA, Valencia, Spain; bHospital Universitario LaFe, Valencia, Spain. OBJECTIVE: The aim of this work is to describe and compare the expression profile of the first trimester miRNA trophoblastic tissue from Ectopic pregnancies (EP) and from Voluntary Termination of Pregnancy samples (VTOP) to deepen into the molecular regulation of this pregnancy complication. DESIGN: miRNA microarrays of first trimester trophoblast from ectopic pregnancies and VTOP (controls) were analyzed and validated with Real Time PCR. MATERIALS AND METHODS: RNA from eight first trimester VTOP and eight first trimester EP trophoblastic samples were extracted and miRNA presence were assessed by microarray technology. Global miRNAs expression was performed using Agilent Human miRNA Arrays. Data was normalized, log-transformed and analyzed using SAM for the differentially expressed miRNAs using Multiexperiment Viewer Software. Principal Component Analysis and Hierarchical clustering were done for the differentially expressed miRNAs, Real Time PCR was performed to confirm our results. RESULTS: Four miRNAs (hsa-miR-196b, hsa-miR-30a, hsa-miR-873, and hsa-miR-337-3p) were found to be downregulated in EP compared to healthy trophoblast tissue, and three miRNAs (hsa-miR-1288, hsa-miR451, and hsa-miR-223) were upregulated in EP compared to trophoblast control tissue samples. Differentially expressed miRNAs in ectopic pregnancy versus controls
miRNA
Fold Change
Q-value
-5.94 -2.72 -1.30 -1.24 1.21 3.44 9.56
<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
hsa-miR-196b hsa-miR-30a hsa-miR-873 hsa-miR-337-3p hsa-miR-1288 hsa-miR-451 hsa-miR-223
Using a supervised PCA and hierarchical clustering analysis, we were able to clearly separate both sample types in two populations. Further validation by Real Time PCR in a new cohort of samples (n¼17) confirmed our results. CONCLUSION: We found a differential miRNA expression pattern between ectopic and non pathological pregnancies in trophoblastic samples. Some of these miRNAs could be used as molecular targets to understand the early onset of this pregnancy complication. Supported by: Fundacion IVI and Hospital LaFe. O-364 Wednesday, October 16, 2013 04:15 PM RESCUE KARYOTYPING: A NOVEL APPROACH FOR RETROSPECTIVE CYTOGENETIC ASSESSMENT WITH DNA
FERTILITY & STERILITYÒ
EXTRACTED FROM ARCHIVED PARAFFIN-EMBEDDED MISCARRIAGE TISSUE. R. Kudesia,a M. Li,b J. Smith,b A. Patel,b Z. Williams.a aDepartment of Obstetrics, Gynecology & Women’s Health, Albert Einstein College of Medicine, Bronx, NY; bDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. OBJECTIVE: Determination of fetal aneuploidy is central to evaluation of recurrent pregnancy loss (RPL). However, for practical and technical reasons, this information is not always obtained. Here we report on the novel concept of ‘‘rescue karyotyping’’, wherein DNA extracted from archived paraffin-embedded miscarriage tissue obtained at the time of dilation and curettage is evaluated by array comparative genomic hybridization (aCGH). DESIGN: Retrospective case series. MATERIALS AND METHODS: Patients were included if they had unexplained RPL and a prior miscarriage with indication for karyotyping, which was either not requested or could not be performed. aCGH was performed on DNA extracted from slides produced from archived paraffin-embedded tissue. Statistical evaluation was performed using STATA v12.1 (College Station, TX). RESULTS: Rescue karyotyping was attempted on a total of 17 specimens resulting from miscarriages in 14 women. In 10 samples (58.8%), sufficient DNAwas extracted to enable further analysis. The interval from tissue collection to DNA extraction was 284 days403 days. There was no significant difference between the specimens that had sufficient DNA (mean¼272525 days) versus those that did not (mean¼296238 days), p¼0.13. There was also no significant difference in the gestational age at the time of miscarriage (p¼0.5). Of the 10 specimens tested, four were genetically normal, and six showed clinically relevant copy number variations including trisomy 11, trisomy 18 and a 20-Mb deletion including the region responsible for Williams syndrome. CONCLUSION: Rescue karyotyping using aCGH on DNA extracted from paraffin-embedded tissue provides the opportunity to obtain critical fetal cytogenetic information from prior miscarriages. Given the ease of obtaining results despite long loss-to-testing intervals or early gestational age at time of loss, this may provide a useful technique in the evaluation of couples with recurrent pregnancy loss. Supported by: Institutional and NIH (NICHD HD068546). O-365 Wednesday, October 16, 2013 04:30 PM ACYL-COA:LYSOCARDIOLIPIN ACYLTRANSFERASE (LYCAT): A POTENT PATHOGENIC FACTOR IN ANTIPHOSPHOLIPID SYNDROME (APS)-RELATED RECURRENT PREGNANCY FAILURE. W. Wang, Y. Tang, H.-C. Liu. The Ronald Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: To validate a novel model system to study the pathogenesis of recurrent miscarriages in APS patients. DESIGN: Experimental animal study. Elevated titers of antiphospholipid antibodies (aPL) have a close association with many obstetric disorders. Cadiolipin (CL), a major phospholipid of mitochondria, regulates membrane permeability and potential. Remodeling enzyme Lycat is required for both CL maturation and repair of damaged acyl chains. At present, APS animal models were generally created by immunization of exogenous antigens. This study presents the first genetic model in which elevated Lycat leads to a drastic rise of aPL, concomitant with a high rate of in utero fetal loss. MATERIALS AND METHODS: Mice over-expressing Lycat were generated by BAC transgenesis (Tg). Fertility were compared between Tg and WT mice. Mutant implantation sites were sectioned for histology, anti-phosphoH3 IF, TUNEL and in situ hybridization. Altered global gene expression of the mutant conceptus was first tested by NextGENe RNA-seq technology, followed by Q-PCR. Serum aPL were tested by ELISA. At least 3 samples were included in each assay for each genotype. Unpaired student’s t-test was performed between WT and Tg groups. RESULTS: All Tg female mice showed elevated aPL, whose levels are positively correlated to the severity of fetal loss. Pregnant Tg females produced fewer pups than the controls (Tg, 2.30.2, v.s. WT, 8.51.4; p<0.01). Degenerating conceptus revealed faulty extraembryonic structures involving trophoblast giant cells, chorion and amnion. As a result, the embryos developmentally arrested by E7.5. Absence of EOMES, Hand1, PL1 and Tpbp indicates defective trophoblast differentiation; while altered expression of Gas1 and Foxh1 suggests mis-instructed embryogenesis. Decreased synthesis of hemoglobins by decidual cells indicates severe oxygen starvation in the mutants.
S111
O-361 Wednesday, October 16, 2013 05:30 PM DUAL TRIGGERING WITH GnRH AGONIST AND RECOMBINANT hCG IN GnRH ANTAGONIST IVF CYCLES. M. Sonmezer, B. Ozmen, C. S. Atabekoglu, E. Tolunay, O. Kan, B. Berker. Obstetrics and Gynecology, Ankara University, Ankara, Turkey. OBJECTIVE: To evaluate weather dual triggering with 500 mcg leuprolide acetate (LA) and 250 mcg recombinant hCG is beneficial in GnRH antagonist cycles. DESIGN: Prospective randomized Clinical Study. MATERIALS AND METHODS: Age and BMI-matched (range 25-32 years-old; 24-28 kg/m2) 90 patients diagnosed with a mild male factor and unexplained infertility were allocated to the study. All enrolled patients were randomly divided into two groups; group A (LA+ rec hCG) and B (rec hCG) according to triggering method on hCG day. The triggering was made when at least 3 follicles >17 mm were observed. RESULTS: Groups were comparable in terms of demographic features and the mean number of total collected oocytes, transferred embryos, fertilization rates.
The COH and Cycle Outcomes in the Groups
Parameters Total collected oocyte(s), n Collected MII oocyte(s), n Grade A embryo(s), n Transferred embryo(s), n Fertilization rates, (%) Cycle cancelation, (%)* Previous IVF failures, (%)# Pregnancy rates, (%)
Group A (n¼45)
Group B (n¼45)
P value
11.17.8 9.37 2.41.9 1.90.6 78 6.6 (3/45) 31 (14/45) 54.7 (23/42)
10.79.5 7.55 1.81.5 1.80.4 75 8.8 (4/45) 13 (6/45) 43.9 (18/41)
NS 0,02 0,02 NS NS NS 0,01 0,02
*
O-360 Wednesday, October 16, 2013 05:15 PM VENOPUNCTURE-FREE IVF: MEASUREMENT OF ESTROGEN IN CONTROLLED OVARIAN STIMULATION IVF CYCLES USING A ‘‘PATIENT-FRIENDLY’’ SALIVA-BASED ESTRADIOL ASSAY. A. Zimon,a B. Lannon,a S. Sheller,a D. Sakkas,a M. Ulrich,b M. Alper.a aBoston IVF Inc., Waltham, MA; bSalimetrics LLC, State College, PA. OBJECTIVE: Controlled ovarian stimulation (COH) during in vitro fertilization (IVF) cycles involves close monitoring of estradiol (E2) levels and measurements of follicle growth. E2 monitoring can occur 4-7 times during an IVF cycle, while repeated venopuncture adds to patient stress and anxiety. In contrast, sampling saliva is non-invasive and can be performed remotely. Here, we present a novel saliva-based E2 assay designed to serve infertility patient’s undergoing COH. The objective was to investigate whether a saliva based E2 assay could efficiently be used to monitor a COH cycle, in comparison to a standard serum based assay, and whether patient acceptance of a saliva based assay was greater. DESIGN: Prospective Study. MATERIALS AND METHODS: Concurrent serum and saliva E2 values were collected from patients who provided between 1 and 7 samples on different days of their COH IVF cycle. Serum E2 values were assessed routinely, while saliva values were measured and validated using an immunoassay developed in collaboration with Salimetrics LLC. RESULTS: The Salivary E2 assay was validated for intra- and inter-assay variability with a coefficient of variation of <10% ensuring accurate detection of estradiol. It was also validated for analytical and functional sensitivity and sample stability. Linear regression of serum and saliva E2 values of 20 patients who had >3 samples evaluated between Days 3 and 14 provided an R¼0.8. Saliva E2 values also correlated to follicle growth. Patient survey results showed that saliva sampling was the preferred method of analysis, associated with less anxiety and more likely to be recommended to friends undergoing IVF. CONCLUSION: Saliva based hormone testing provides an equivalent alternative to serum based assessment. The ease of saliva sampling allows a reduction in intervention and a patient friendly approach to IVF, which is associated with improved patient satisfaction and decreased stress. Saliva based hormone tests may become the preferred method of COH monitoring in the future.
S110
ASRM Abstracts
The cycle has been cancelled due to not available a transferable embryo. # The percantage of patients with at least 2 unsuccessful previous IVF cycles. The P value has been set at > 0.05. However the mean number of grade A embryos and of MII oocytes were significantly higher in dual triggered group. A higher rate of previous IVF failure has been observed in dual triggered patients. Pregnancy rate was statistically higher in dual triggered group than in standard group (54.7 vs. 43.9%, p¼0.02). One case of mild OHSS was observed in each group. CONCLUSION: This novel more physiological approach of dual triggering with 500 mcg of GnRH agonist plus 250 mcg recombinant hCG seem to be favorable in GnRH antagonist cycles. Dual triggering may increase the number of MII oocytes and grade A embryos and may enhance pregnancy rates. O-362 Wednesday, October 16, 2013 05:45 PM
BEFORE CHEMOTHERAPY, CANCER PATIENTS HAVE LOWER SERUM AMH THAN AGE-MATCHED HEALTHY CONTROLS. K. E. Palinska-Rudzka,a G. Hartshorne,a G. Lockwood,a T. Ghobara,a A. Eapen.a aMedicine, Univeristy of Warwick, Coventry, West Midlands, United Kingdom; bMidland Fertility Sevices, Aldridge, United Kingdom. OBJECTIVE: Is the ovarian reserve in young women with breast cancer or lymphoma different to that of age matched healthy controls prior to chemotherapy? DESIGN: Prospective longitudinal multicentre study: 198 female participants, aged 18-43 years, recruited between May 2010 and May 2012 from 13 centres in UK. Here, we present results of serum AMH at point of recruitment, in light of women’s reproductive history and lifestyle. MATERIALS AND METHODS: Serum AMH (2nd generation assay) concentrations were measured before the start of chemotherapy in cancer patients and compared with same age group of healthy volunteers. Cancer patients and volunteers known to have PCO, severe endometrosis, previous oophorectomy or infertility problems were excluded. RESULTS: Cancer patients (n¼65) had significantly lower serum AMH than age matched volunteers (n¼123), for the group as a whole. Mean age of breast cancer subgroup (n¼53) was 37.7 with mean AMH of 10.69 pmol/l. Mean age of hematology subgroup (n¼ 12) was 27.6. with mean
Vol. 100, No. 3, Supplement, September 2013
AMH of 13.39 pmol/l. Patients with breast cancer had significantly lower serum AMH than control group (p¼0.04). Lymphoma patients had serum AMH that appeared lower but was not significantly different from the volunteers (p¼0.67). Baseline characteristics including family history of early menopause, smoking status, BMI and contraception use were not significantly different in both groups. The ovarian reserve, assessed using serum AMH, is significantly lower in newly diagnosed breast cancer patients compared to healthy volunteers pior to chemotherapy. In our precition model, a cancer patient had serum AMH levels of an 6 year older volunteer. CONCLUSION: Reproductive age women with malignancy appear to have lower ovarian reserve than healthy volunteers even before any gonadotoxic treatment is commenced. In view of those findings routine measurement of AMH level before discussing and commencing fertility preservation treatment would be highly recommended even in young women. Supported by: University of Warwick; Midland Fertility Services. EARLY PREGNANCY II O-363 Wednesday, October 16, 2013 04:00 PM DIFFERENTIAL mIRNA PROFILES OF TROPHOBLAST TISSUE OBTAINED IN ECTOPIC PREGNANCIES. F. Dominguez,a T. Lozoya,b J. M. Moreno-Moya,a S. Martinez,a C. Simon,a A. Pellicer.b a Fundacion IVI/INCLIVA, Valencia, Spain; bHospital Universitario LaFe, Valencia, Spain. OBJECTIVE: The aim of this work is to describe and compare the expression profile of the first trimester miRNA trophoblastic tissue from Ectopic pregnancies (EP) and from Voluntary Termination of Pregnancy samples (VTOP) to deepen into the molecular regulation of this pregnancy complication. DESIGN: miRNA microarrays of first trimester trophoblast from ectopic pregnancies and VTOP (controls) were analyzed and validated with Real Time PCR. MATERIALS AND METHODS: RNA from eight first trimester VTOP and eight first trimester EP trophoblastic samples were extracted and miRNA presence were assessed by microarray technology. Global miRNAs expression was performed using Agilent Human miRNA Arrays. Data was normalized, log-transformed and analyzed using SAM for the differentially expressed miRNAs using Multiexperiment Viewer Software. Principal Component Analysis and Hierarchical clustering were done for the differentially expressed miRNAs, Real Time PCR was performed to confirm our results. RESULTS: Four miRNAs (hsa-miR-196b, hsa-miR-30a, hsa-miR-873, and hsa-miR-337-3p) were found to be downregulated in EP compared to healthy trophoblast tissue, and three miRNAs (hsa-miR-1288, hsa-miR451, and hsa-miR-223) were upregulated in EP compared to trophoblast control tissue samples. Differentially expressed miRNAs in ectopic pregnancy versus controls
miRNA
Fold Change
Q-value
-5.94 -2.72 -1.30 -1.24 1.21 3.44 9.56
<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
hsa-miR-196b hsa-miR-30a hsa-miR-873 hsa-miR-337-3p hsa-miR-1288 hsa-miR-451 hsa-miR-223
Using a supervised PCA and hierarchical clustering analysis, we were able to clearly separate both sample types in two populations. Further validation by Real Time PCR in a new cohort of samples (n¼17) confirmed our results. CONCLUSION: We found a differential miRNA expression pattern between ectopic and non pathological pregnancies in trophoblastic samples. Some of these miRNAs could be used as molecular targets to understand the early onset of this pregnancy complication. Supported by: Fundacion IVI and Hospital LaFe. O-364 Wednesday, October 16, 2013 04:15 PM RESCUE KARYOTYPING: A NOVEL APPROACH FOR RETROSPECTIVE CYTOGENETIC ASSESSMENT WITH DNA
FERTILITY & STERILITYÒ
EXTRACTED FROM ARCHIVED PARAFFIN-EMBEDDED MISCARRIAGE TISSUE. R. Kudesia,a M. Li,b J. Smith,b A. Patel,b Z. Williams.a aDepartment of Obstetrics, Gynecology & Women’s Health, Albert Einstein College of Medicine, Bronx, NY; bDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. OBJECTIVE: Determination of fetal aneuploidy is central to evaluation of recurrent pregnancy loss (RPL). However, for practical and technical reasons, this information is not always obtained. Here we report on the novel concept of ‘‘rescue karyotyping’’, wherein DNA extracted from archived paraffin-embedded miscarriage tissue obtained at the time of dilation and curettage is evaluated by array comparative genomic hybridization (aCGH). DESIGN: Retrospective case series. MATERIALS AND METHODS: Patients were included if they had unexplained RPL and a prior miscarriage with indication for karyotyping, which was either not requested or could not be performed. aCGH was performed on DNA extracted from slides produced from archived paraffin-embedded tissue. Statistical evaluation was performed using STATA v12.1 (College Station, TX). RESULTS: Rescue karyotyping was attempted on a total of 17 specimens resulting from miscarriages in 14 women. In 10 samples (58.8%), sufficient DNAwas extracted to enable further analysis. The interval from tissue collection to DNA extraction was 284 days403 days. There was no significant difference between the specimens that had sufficient DNA (mean¼272525 days) versus those that did not (mean¼296238 days), p¼0.13. There was also no significant difference in the gestational age at the time of miscarriage (p¼0.5). Of the 10 specimens tested, four were genetically normal, and six showed clinically relevant copy number variations including trisomy 11, trisomy 18 and a 20-Mb deletion including the region responsible for Williams syndrome. CONCLUSION: Rescue karyotyping using aCGH on DNA extracted from paraffin-embedded tissue provides the opportunity to obtain critical fetal cytogenetic information from prior miscarriages. Given the ease of obtaining results despite long loss-to-testing intervals or early gestational age at time of loss, this may provide a useful technique in the evaluation of couples with recurrent pregnancy loss. Supported by: Institutional and NIH (NICHD HD068546). O-365 Wednesday, October 16, 2013 04:30 PM ACYL-COA:LYSOCARDIOLIPIN ACYLTRANSFERASE (LYCAT): A POTENT PATHOGENIC FACTOR IN ANTIPHOSPHOLIPID SYNDROME (APS)-RELATED RECURRENT PREGNANCY FAILURE. W. Wang, Y. Tang, H.-C. Liu. The Ronald Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: To validate a novel model system to study the pathogenesis of recurrent miscarriages in APS patients. DESIGN: Experimental animal study. Elevated titers of antiphospholipid antibodies (aPL) have a close association with many obstetric disorders. Cadiolipin (CL), a major phospholipid of mitochondria, regulates membrane permeability and potential. Remodeling enzyme Lycat is required for both CL maturation and repair of damaged acyl chains. At present, APS animal models were generally created by immunization of exogenous antigens. This study presents the first genetic model in which elevated Lycat leads to a drastic rise of aPL, concomitant with a high rate of in utero fetal loss. MATERIALS AND METHODS: Mice over-expressing Lycat were generated by BAC transgenesis (Tg). Fertility were compared between Tg and WT mice. Mutant implantation sites were sectioned for histology, anti-phosphoH3 IF, TUNEL and in situ hybridization. Altered global gene expression of the mutant conceptus was first tested by NextGENe RNA-seq technology, followed by Q-PCR. Serum aPL were tested by ELISA. At least 3 samples were included in each assay for each genotype. Unpaired student’s t-test was performed between WT and Tg groups. RESULTS: All Tg female mice showed elevated aPL, whose levels are positively correlated to the severity of fetal loss. Pregnant Tg females produced fewer pups than the controls (Tg, 2.30.2, v.s. WT, 8.51.4; p<0.01). Degenerating conceptus revealed faulty extraembryonic structures involving trophoblast giant cells, chorion and amnion. As a result, the embryos developmentally arrested by E7.5. Absence of EOMES, Hand1, PL1 and Tpbp indicates defective trophoblast differentiation; while altered expression of Gas1 and Foxh1 suggests mis-instructed embryogenesis. Decreased synthesis of hemoglobins by decidual cells indicates severe oxygen starvation in the mutants.
S111