Neutralization of vascular endothelial growth factor (VEGF) during controlled ovarian stimulation (COS) cycles alters production of angiogenic factors by rhesus macaque granulosa cells

pregnancy, live birth rate and miscarriage rates when compared to oocytes that did not have a visible spindle. Low ovarian reserve and excessive stimulation were also associated with lack of spindle and therefore lower pregnancy outcomes. O-138 Tuesday, October 18, 2016 12:30 PM SPECIAL RESEARCH PUBLICATION: GLOBAL TRANSCRIPTIONAL SILENCING IN THE OOCYTE IS MEDIATED BY AN J. Dumdie,a MRNA DECAY ACTIVATOR. H. Cook-Andersen,a M. Ramaiah,a K. Cho,b D. Skarbrevik,a M. Wilkinson.a aReproductive Medicine, University of California, San Diego, La Jolla, CA; bUniversity of California, San Diego, La Jolla, CA. OBJECTIVE: Global transcriptional silencing during the transition from the fully differentiated oocyte to the totipotent embryo is a highly conserved evolutionary event that is poorly understood despite its critical role in the earliest developmental stages of animals from worms to humans. Here, we report the unexpected finding that this germline-specific, genome-wide event depends on an mRNA decay activator. DESIGN: Laboratory research. MATERIALS AND METHODS: Oocyte-specific mouse knockout of Zfp36l2. RESULTS: We find that oocyte-specific loss of ZFP36L2, an RNAbinding protein critical for decay of a subset of cellular mRNAs, prevents global transcriptional silencing in oocytes during folliculogenesis. Female Zfp36l2 conditional knockout mice are infertile, and oocytes deficient in ZFP36L2 are developmentally incompetent, with defects in oocyte survival and maturation, as well as a complete failure to undergo normal fertilization. Single-cell RNAseq analysis of periovulatory oocytes revealed that ZFP36L2 regulates scores of genes that are candidates to mediate global transcriptional silencing, including factors with roles in histone modification, DNA methylation, transcription initiation, and transcription elongation. CONCLUSIONS: Our results define a critical role for an mRNA decay factor in the downregulation of transcription activators leading to germline global transcriptional silencing and developmental competence. These findings favor a model whereby global transcriptional silencing in the oocyte is mediated by RNA decay. Supported by: H. Cook-Andersen was supported by the Women’s Reproductive Health Research grant K12 HD001259 and by a grant from the American Society for Reproductive Medicine. OVARIAN STIMULATION O-139 Tuesday, October 18, 2016 11:15 AM THE CONCENTRATION OF COMMERCIAL HCG TRIGGER IS E. Davenport,a IMPRECISE AND INACCURATE. I. Woo,a S. A. Ingles,b F. Z. Stanczyk,c K. Chung,a K. Bendikson,a R. Paulson.a a University of Southern California, Los Angeles, CA; bPreventive Medicine, University of Southern California, Los Angeles, CA; cObstetrics & Gynecoogy, Keck School of Medicine of USC, Los Angeles, CA. OBJECTIVE: During controlled ovarian hyperstimulation for IVF, human chorionic gondatropin (HCG) is administered to induce oocyte maturation approximately 34-35 hours before oocyte aspiration. Post-trigger serum HCG levels have been shown to correlate with incidence of OHSS. One strategy used to prevent this complication is to reduce the HCG dose. Due to the variability in serum HCG post-trigger, we hypothesized serum HCG concentration from the manufacturer may not be consistent. The primary objective of this study was to measure the concentration of HCG in commercially available preparation. Secondary objectives were to determine if other factors were associated with serum HCG including: BMI, oocyte maturity, and risk for OHSS. DESIGN: Prospective observational study. MATERIALS AND METHODS: Each bottle of HCG trigger containing presumably 10,000 IU of HCG was mixed with 1mL of water and subsequently 0.1mL of each vial was analyzed for actual HCG concentration. Calculation was performed to determine the true dose patient received from the remaining 0.9ml. Patients returned the following day to measure serum HCG to ensure proper administration and absorption. All HCG concentration were measured with Immulite 2000. Descriptive statistics

e56

ASRM Abstracts

was performed along with a stepwise multiple regression analysis to predict whether dose, time from trigger injection, or BMI would influence serum HCG. RESULTS: 108 patients were identified between Nov 2015-March 2016. Patient’s HCG trigger concentration ranged from 6,591-24,766 IU/mL, with a mean 13,191
3,670 IU/mL. The dose patients actually received ranged from 7,361-22,289 IU/mL, instead of the expected 9,000IU. Serum HCG overall ranged from 76-637mIU/mL with mean 301
114mIU/mL. Patient’s weight was significantly correlated with serum HCG level (R 2 ¼0.27, p<0.01) after adjusting for dose and time from trigger. When stratifying BMI into normal, overweight, and obese categories, for each increasing BMI category, serum HCG levels were dropped by approximately 70mIU/mL (likelihood ratio test p ¼ 0.0001). Serum HCG was not correlated with number of oocytes retrieved, percent mature oocytes, pregnancy, or OHSS. OHSS occurred in 3 patients, with serum HCG ranging from 312544mIU/mL. CONCLUSIONS: Variability in serum HCG post-trigger is due to inconsistent HCG concentrations in commercial formulations and due to patient weight. The large variability in HCG concentrations in the vials makes it difficult to choose an optimal dose. Decreasing the dose of HCG to prevent OHSS may not be effective unless the actual concentration of HCG is calculated.

O-140 Tuesday, October 18, 2016 11:30 AM NEUTRALIZATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) DURING CONTROLLED OVARIAN STIMULATION (COS) CYCLES ALTERS PRODUCTION OF ANGIOGENIC FACTORS BY RHESUS MACAQUE GRANULOSA CELLS. C. V. Bishop. Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Beaverton, OR. OBJECTIVE: To determine if VEGF neutralization before hCG administration during COS cycles alters production of angiogenic factors by rhesus monkey granulosa cells (GCs) in vivo and in vitro. DESIGN: Randomized design; nonhuman primate model. MATERIALS AND METHODS: Rhesus macaque females (n¼13) were treated with a standard COS protocol [1], and then randomly assigned to treatment groups: Control (n¼7) or VEGF neutralizing agent Avastin 19
5 h (single IV bolus; 10mg/kg; n¼6) before hCG administration. The contents of one randomly chosen follicle/ ovary were aspirated [1] 36 h following hCG and collected for follicular fluid analyses. The remaining follicles were also aspirated and, following removal of the oocytes, GCs were isolated and cultured [2] with FSH (2.5 ng/ml) and LH (100 ng/ml) for 24 h. Production of free VEGF, angiopoietin 1 (ANGPT1), ANGPT2, and progesterone (P4) were analyzed in the follicular fluid and media samples as previously described [3]. All media samples were normalized by the number of viable GCs cultured, and all data were then analyzed by Linear Models function of SAS (v9.4). RESULTS: There were no differences in the total number of oocytes and MII-stage oocytes recovered between treatment groups (P>0.7). VEGF levels in follicular fluid were reduced 78-fold (P<0.009) by Avastin pre-treatment before hCG trigger. However, the intrafollicular levels of ANGPT1 and 2, and the ratio of ANGPT2/1 were not affected by VEGF neutralization (all P>0.3); levels of ANGPT2 were 36 to 48fold higher than levels of ANGPT1. After 24 h of culture media levels of VEGF were also reduced 4-fold (P<0.007) in GCs that were exposed to Avastin in vivo. In addition, media levels of P4 (P<0.02) and ANGPT1 (P<0.03) from cultured GCs were reduced following Avastin exposure in vivo. Levels of ANGPT2 were not altered (P>0.4), but the ratio of ANGPT2/1 increased (P<0.012) in Avastin-exposed GCs compared to control. CONCLUSIONS: Neutralization of VEGF in vivo alters the angiogenic potential of GCs in nonhuman primates during COS. These data confirm previous studies in primates that levels of ANGPT2 are elevated compared to ANGPT1 in stimulated follicles [4]. The elevated ANGPT 2/1 ratio observed in vitro in the absence of elevated VEGF both in vitro and in vivo suggests that the angiogenic signaling environment is altered to promote vessel degeneration [5] following VEGF neutralization. These data also compliment previous data demonstrating reduced ovarian blood volume and ovarian vascular permeability in rhesus macaques treated with Avastin prior to COS [6]. Further studies utilizing this nonhuman primate model could provide insights into mechanisms to prevent ovarian hyperstimulation syndrome during COS cycles in women.

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

References: 1. Hanna CB, Yao S, Ramsey CM, Hennebold JD, Zelinski MB, Jensen JT. Phosphodiesterase 3 (PDE3) inhibition with cilostazol does not block in vivo oocyte maturation in rhesus macaques (Macaca mulatta). Contraception 2015; 91:418-422. 2. Bishop CV, Hennebold JD, Kahl CA, Stouffer RL. Knockdown of Progesterone Receptor (PGR) In Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production. Biol Reprod 2016; PMID: 26985003. 3. Fisher TE, Molskness TA, Villeda A, Zelinski MB, Stouffer RL, Xu J. Vascular endothelial growth factor and angiopoietin production by primate follicles during culture is a function of growth rate, gonadotrophin exposure and oxygen milieu. Hum Reprod 2013; 28:3263-3270. 4. Molskness TA, Stouffer RL, Burry KA, Gorrill MJ, Lee DM, Patton PE. Circulating levels of free and total vascular endothelial growth factor (VEGF)-A, soluble VEGF receptors-1 and -2, and angiogenin during ovarian stimulation in non-human primates and women. Hum Reprod 2004; 19:822-830. 5. Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD. Angiopoietin-2, a Natural Antagonist for Tie2 That Disrupts in vivo Angiogenesis. Science 1997; 277:55-60. 6. Bishop C, Li X, Lee D. Neutralization of vascular endothelial growth factor (VEGF) before or after CG during COS cycles restores ovarian vascular parameters similar to natural menstrual cycles: a pilot study in nonhuman primates. Fertil Steril 2015; 104:e52. Supported by: R21 HD078819 (CVB), P51OD011092 (ONPRC).

O-141 Tuesday, October 18, 2016 11:45 AM CELL-FREE DNA IN SERUM AS PROGNOSTIC BIOMARKER FOR OVARIAN RESPONSE TO STIMULATION. E. Scalici,a A. Gala,b S. Belloc,c C. Vincens,b S. Hamamah.a aART-PGD Department, INSERM U1203, CHU Montpellier, Montpellier, France; bART-PGD Department, CHU Montpellier, Montpellier, France; cEylau-Unilabs Laboratory, Paris, France. OBJECTIVE: The aim of this study is to investigate if cell-free DNA (cfDNA) level in serum at day 3 of menstrual cycle could be clinically useful for prediction of patient’s ovarian response to stimulation during in vitro fertilization (IVF) procedure. DESIGN: This prospective study included 32 serum samples collected at day 3 of menstrual cycle from patients undergoing IVF/ICSI procedure. AntiM€ ullerian hormone (AMH) and cfDNA levels were measured in each serum sample in order to compare their predictive value for patient’s ovarian response to stimulation. MATERIALS AND METHODS: Serum samples were prepared in Proteinase K buffer and the total cfDNA was quantified by qPCR, using ALU 115 primers. CfDNA concentration was determined based on a standard curve obtained by successive dilution of genomic DNA. The P-values were calculated by using the unpaired t-test, Spearman correlation, multiple and logistic regressions and ROC curveanalysis on GraphPad and Medcalc softwares. RESULTS: Cell-free DNA concentrations (mean
SD¼152.02
160.48 ng/m, median¼92 ng/ml) were significantly and positively correlated with patient’s age (r¼0.18; p¼0.02). Indeed, cfDNA levels were significantly higher in serum from older women (R38 years) than those from young patients (<38 years) (p¼0.035). Very interestingly, cell-free DNA level was significantly related to the number of oocyte collected at

oocyte retrieval (p¼0.031). Furthermore, cfDNA levels predicted significantly a low ovarian response (<6 retrieved oocytes), independently of AMH concentrations [Adjusted Odd Ratio¼1.01 (1.0-1.02); p¼0.02]. The area under the ROC curve (AUC), which quantifies the low ovarian response prediction of cfDNA level was 0.79 [0.61-0.91] with higher sensitivity (73.33%) and specificity (82.35%) (p<0.001) than AMH level (AUC¼0.72 [0.54-0.87]; sensitivity¼60%; specificity¼88.2%, p¼0.02). CONCLUSIONS: Cell-free DNA levels in serum at day 3 of menstrual cycle can predict significantly the number of retrieved oocyte, independently of AMH concentrations. High cfDNA levels in serum could be significantly related to a low ovarian response. Cell-free DNA quantification at day 3 could be used as a supplemental tool to identify more accurately women with a risk of poor ovarian response, in order to develop a personalized care program for these patients. Supported by: This study was partially supported by the University Hospital of Montpellier and Ferring Pharmaceuticals.

O-142 Tuesday, October 18, 2016 12:00 PM THE EFFECT OF LUTEAL PHASE SUPPORT ON PREGNANCY RATES IN INTRAUTERINE INSEMINATION CYCLES FOLLOWING OVARIAN STIMULATION WITH GONADOTROPINS- A RANDOMIZED CONTROLLED TRIAL. J. Han,a T. Motan.b aObstetrics and Gynecology, University of Alberta, Edmonton, AB, Canada; bUniversity of Alberta, Edmonton, AB, Canada. OBJECTIVE: To investigate if luteal phase support with vaginal progesterone in controlled ovarian hyperstimulation (COH) using gonadotropins and intrauterine insemination (IUI) improves conception rates in couples with infertility. DESIGN: Single centre, open label randomized controlled trial. PARTICIPANTS: Couples with infertility meeting inclusion criteria underwent 298 cycles of COH with gonadotropins and IUI. MATERIALS AND METHODS: Couples meeting inclusion criteria had treatment at the Fertility and Women’s Endocrine Clinic at the Royal Alexandra Hospital using gonadotropin COH and IUI between December 2013 and December 2015. Inclusion criteria were: 1) More than 12 months of unprotected intercourse without conceiving, 2) Confirmed bilateral tubal patency, 3) More than 10 million motile sperm available for IUI. Randomization was done prior to the IUI with the study group receiving vaginal micronized progesterone (Endometrin 100 mg vaginally BID) from the day of IUI for at least 16 days until a serum pregnancy test. Control group participants received no luteal phase support. Clinic COH protocols were followed for all participants including baseline investigations and cycle monitoring. Ovulation was triggered with hCG when at least one follicle > 17 mm was seen, and IUI was performed approximately 36-hours later. Patient demographics, cycle characteristics and outcomes were analyzed. The primary outcome was clinical pregnancy rate (presence of a fetal heart beat after 6-weeks gestational age). Secondary outcomes were biochemical pregnancy rate, non-viable pregnancy rate and ongoing pregnancy rates (continuing past first trimester). RESULTS: There were no statistically significant differences (Fisher’s Exact) between the treatment and control groups in primary and secondary outcomes (table). There were no clinically significant differences in demographic, baseline characteristics and stimulation cycles between groups. CONCLUSIONS: This randomised control trial indicates that luteal phase support with vaginal progesterone does not affect the conception rate in intrauterine insemination cycles following ovarian stimulation with gonadotropins. Supported by: Ferring Pharmaceuticals subsidised the Endometrin used.

Table: Outcomes in progesterone supported and control groups with Odds Ratio

Outcome Clinical pregnancy Ongoing pregnancy Total pregnancy Biochemical pregnancy Spontaneous abortion

FERTILITY & STERILITYÒ

Control group N¼147 (%
standard deviation)

Progesterone group N¼ 151 (%
standard deviation)

OR (95% CI)

18.4
3.8 (27/147) 14.7
3.5 (22/147) 24.0
4.3 (36/147) 6.0
2.8 (9/147) 3.3
4.1 (5/147)

13.2
3.4 (20/151) 12.2
3.3 (18/151) 17.3
3.7 (24/151) 2.7
3.3 (4/151) 1.4
1.2 (2/151)

0.68 (0.36 - 1.26) 0.77 (0.39 - 1.48) 0.58 (0.33 - 1.03) 0.42 (0.13 - 1.31) 0.38 (0 - 1.74)

e57