- Email: [email protected]
Should endometriosis be an indication for intracytoplasmic sperm injection (ICSI) in fresh IVF cycles?
MATERIALS AND METHODS: Normal endometrial tissues were collected from 20 pre-menopausal women with no history of endometriosis. Additionally, matched eutopic endometrium and ectopic endometrial implants were obtained from 40 women with endometriosis and no recent history of hormonal therapy [deep infiltrating endometriosis (DIE), n¼20; endometrioma, n¼20]. Using the Histoscore method, a pilot immunohistochemical analysis of HOXB4 was performed on 9 normal endometrial tissues and 12 eutopic/ectopic endometrial tissue pairs. Kruskal-Wallis non-parametric test was used to compare multiple categories, Wilcoxon test was used for pair-wise comparisons. RESULTS: HOXB4 was immunolocalized in the nuclei of glandular epithelial cells in normal endometrium and eutopic/ectopic endometrial tissues from women with endometriosis. HOXB4 immunoreactivity was absent from endometrial stromal cells in all tissues examined. No significant difference in HOXB4 immunostaining intensity was found between controls and eutopic endometrium from endometriosis. Interestingly, HOXB4 immunoreactivity was significantly reduced in ectopic DIE compared to eutopic endometrium [P¼ 0.041] and endometrioma [P¼ 0.05] in women with endometriosis. Moreover, HOXB4 immunoreactivity in eutopic endometrium of disease and control groups combined was significantly higher in the proliferative phase than in the secretory phase [P¼ 0.026]. Conversely, HOXB4 immunoreactivity in ectopic implants was lower in proliferative phase than in the secretory phase [P¼ 0.021]. CONCLUSIONS: During the menstrual cycle the expression of HOXB4 in endometrial glandular epithelial cells is higher in the proliferative phase than the secretory phase. On the whole, HOXB4 expression does not appear to differ between normal endometrium and eutopic endometrium from endometriosis. However, its expression is reduced in DIE, but not endometrioma, and may be dysregulated in ectopic implants depending on the phase of the menstrual cycle. Supported by: Internal fund, Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada. Graduate Sponsorship Program,Princess Nourah bint Abdulrahman University,Riyadh, Kingdom of Saudi Arabia.
P-151 Tuesday, October 20, 2015 COULD THE UNILATERAL OVARIAN ENDOMETRIOSIS AFFECT THE CONTRALATERAL OVARY? NEW INSIGHTS FROM FOURIER TRANSFORM INFRARED (FTIR) E. Sereni,b C. Zaca’,b SPECTROSCOPY. G. Gioacchini,a E. Giorgini,c V. Notarstefano,a L. Vaccari,d O. Carnevali,a A. Borini.b a Universita Politecnica delle Marche, Ancona, Italy; bTecnobios Procreazione, Bologna, Italy; cPolytechnic University of Marche, Ancona, Italy; dElettra-Sincrotrone Trieste, Trieste, Italy. OBJECTIVE: Up to date, endometriosis’ possible impact on follicle cells metabolism has never been highlighted. In this light, the aim of this study was to evaluate, by FT-IR microspectroscopy, metabolic changes on granulosa cells (GCs) isolated from endometriosis affected ovaries and contralateral healthy ones DESIGN: This prospective non-randomized study has been conducted from January to September 2014 on women undergoing a controlled ovarian hyperstimulation for an IVF treatment. In particular, GCs were collected from both ovaries of 10 women with a diagnosis of unilateral ovarian endometrioma at the time of oocytes retrieval. 9 women with male, idiopathic or tubal-factor infertility diagnosis were selected for the control group. The three experimental groups matched for female age (36.24.1 vs 35.42.6) MATERIALS AND METHODS: GCs obtained from follicles aspirates were isolated from red blood cells and follicular fluid by density gradient centrifugation. FTIR analysis was performed by using a Bruker Vertex 70 Interferometer with a Hyperion 3000 Vis-IR microscope. QPCR analysis Supported FTIR results. . Data are presented as mean S.D. Two-Way ANOVA followed by Tukey test as Multiple comparisons test, was used for comparison among experimental groups. All statistical analyses were performed using the statistical software package Prism5 (Graphpad Software, Inc. USA) with significance accepted at P<0.05 RESULTS: Results are showed in table CONCLUSIONS: This preliminary study represents the first approach to evaluate the macromolecular and biochemical changes on GCs from both ovaries in patients with unilateral endometrioma. These findings pinpoint that this pathology causes relevant changes in the metabolism and amount of proteins, lipids, carbohydrates and nucleic acids, and increases peroxidative processes. It is impressive that the same biochemical alterations have been found both in ovaries with endometrioma and in the contralateral
e158
ASRM Abstracts
‘‘healthy’’ ones. All these results could be useful in developing new strategies to prevent the detrimental effects of endometriosis on fertility.
Control LIPIDS/CELL (%) PHOSPHOLIPIDS/ LIPIDS (%) LIPIDS PEROXIDATION(%) UNFOLDED PROTEIN (%) CARBOHYDRATES/ PROTEIN(%) RNA/DNA(%) PPARa PPARg FAN GLUT1 GLUT9
CONTRAL ATERAL OVARY WITH OVARY ENDOMETRIOMA SIGNIFICANCE
7.891.18a 11.550.27b 5.921.79a 12.500.24b
11.800.39b 12.020.55b
a vs b¼P<0.0001 a vs b¼P<0.0001
4.210.10a 7.470.73b
7.680.19b
a vs b¼P<0.0001
22.141.32a 30.034.02b
28.841.34c
a vs b¼P<0.05
16.062.37a 10.940.13b
10.390.92b
a vs b¼P<0.01
10.833.62a 1.130.22a 1.200.13a 1.130.41a 10.330.78a 9.080.44a
6.141.05b 8.700.97b 4.170.83b 6.231.29b 1.130.39c 1.190.47b
a vs b¼P<0.05 a vs b¼P<0.01 a vs b¼P<0.05 a vs b¼P<0.01 a vs b¼P<0.01 a vs b¼P<0.01
6.161.22b 7.000.88b 3.301.31b 4.130.12b 2.190.86b 1.510.80b
P-152 Tuesday, October 20, 2015 USE OF DEPOT GNRH ANTAGONIST (DEGARELIX) IN THE TREATMENT OF ENDOMETRIOMAS (OVARIAN ENDOMETRIOSIS) BEFORE IVF: A CONTROLLED TRIAL. M. Sbracia F. Scarpellini. CERM-Hungaria, Roma, Italy. OBJECTIVE: Endometriosis is a chronic condition affecting around 10% of women in reproductive age, and after surgery it recur in about 50% of cases. It is recognized that surgical treatment of ovarian endometriomas affects ovarian reserve. For this reason in case of ovarian endometriomas we tried a medical approach instead of the surgery using a long acting GnRH antagonist. The aim of the study was to evaluate the role Degarelix (Firmagon) in the treatment of women with ovarian endometriosis (endometriomas) before undergo IVF in order to reduce cyst volume and improve IVF outcome avoiding surgery. DESIGN: This randomized controlled study was conducted from the January 2014 to April 2015 on 58 women with ovarian endometriomas. MATERIALS AND METHODS: All patients were affected by ovarian endometriosis with endometriomas larger than 3.5cm in mean diameter. All patients din not undergo surgery for endometriosis and they underwent a subsequent IVF cycle in the next 4-8 months. The mean age of women was 35.1 + 2.0 (range 25/39). The women were assigned to the study group or to the control group by a computer generated sequence. The 31 women of the study group were treated with a single injection of Degarelix (Firmagon 120mg) subcutaneously, The 27 women of the control group were treated for 6 months with Leuprolide 3.75 mg/month alone. The women of the two groups were followed up during the treatment and after the treatment for IVF cycles. All patients underwent IVF within 3 months from the end of treatment. All patients were treated for the controlled ovarian hyperstimulation with the same protocol, a standard flexible GnRH antagonist protocol with recombinant FSH (Gonal-F) plus Cetrorelix (Cetrotide). Primary outcomes were time of endometriomas disappearance and IVF outcome. RESULTS: The two groups of patients did not show statistically significant differences for epidemiological data. The time of endometriomas disappearance was shorter in the group treated with Degarelix than in the control group (2.4+0.9months vs 4.1+1.2months, P<0.01). The pregnancy rate for IVF in the women treated with Degarelix was significantly higher than in the control group (45.1% vs 18.5%, P¼0.049). The implantation rate was 19.1 in Degarelix group versus 7.5 in controls (P¼0.0416). The number of mature oocyte collected was 8.6+3.4 in Degarelix group and 6.1+3.6 in controls (P¼0.0087). The number of embryo obtained was 4.7+1.2 in Degarelix group and 3.2+1.1 in controls (P<0.0001). CONCLUSIONS: Even though our data need to be validated in larger group of patients, Degarelix treatment seems to be a promising tool in patients with ovarian endometriosis, especially to avoid surgery in order to preserve ovarian reserve, improving IVF outcome. P-153 Tuesday, October 20, 2015 SHOULD ENDOMETRIOSIS BE AN INDICATION FOR INTRACYTOPLASMIC SPERM INJECTION (ICSI) IN FRESH IVF CYCLES?. J. Lekovich,a G. D. Palermo,b N. Pereira,c Z. Rosenwaks.a a Weill Cornell Medical College, New York, NY; bWeill Medical College of
Vol. 104, No. 3, Supplement, September 2015
Cornell University, New York, NY; cThe Ronald O. Perelman and Claudia Cohen Center, New York, NY. OBJECTIVE: Mechanism of infertility in endometriosis potentially involves oocyte/embryo defects and lower fertilization and implantation rates. Cumulus complex in endometriosis patients demonstrates mitochondrial dysfunction and higher oxidative stress. We sought to investigate if ICSI, which diminishes oxidative stress when compared to insemination, improves IVF outcomes in patients with endometriosis. DESIGN: Retrospective cohort MATERIALS AND METHODS: 221 patients who had surgically or sonographically confirmed endometriosis were compared with 150 patients with unexplained infertility. Patients with endometriosis as a sole cause of infertility were included. Exclusion criteria [1. > 41yo; 2. Male factor (sperm count < 15 x 106/cc, motility less than 40%, older than 55yo]. Primary outcomes: 1. Number of retrieved eggs; 2. Number of 2 pronuclear zygote (2PN) stage embryos; 3. Proportion of day 5 transfers; 4. Number of frozen blastocysts. Secondary outcomes: 1. Clinical intrauterine pregnancy [IUP] rate; 2. Live birth rate. Student’s t-test and ANOVA were used to continuous and Fisher’s exact and Chi-square tests for categorical variables. RESULTS: Within the endometriosis group, 124 patients utilized standard insemination, whereas 97 utilized ICSI. Endometriosis-insemination, endometriosis-ICSI and the control groups were matched for age, BMI, anti-mullerian hormone (AMH) and the number of eggs retrieved [11.95.3 vs. 13.13.1 vs. 13.1 5.9, P¼0.07]. The control and endometriosis-ICSI groups did not differ in terms of number of 2PNs, number of day 5 transfers, IUP and live birth rates (Table). Endometriosis-ICSI group demonstrated significantly higher percentage of 2PN embryos, percentage of day 5 transfers, number/proportion of clinical IUP as well as live birth rates when compared with the endometriosisinsemination group. Additionally, endometriosis-ICSI group had a lower proportion of cycles with no normal fertilization [0 (0%) vs 4 (3.2%), but this difference did not reach statistical significance. CONCLUSIONS: Our findings suggest that endometriosis might be an indication for ICSI, as patients with endometriosis who utilized ICSI achieved better outcomes (higher proportion of normal fertilization, higher number of day 5 transfers, clinical pregnancy and live birth rates) when compared to standard insemination cycles. Table 1 Endometriosis- EndometriosisInsemination(124) ICSI(97) Mean # eggs retrieved Total# of retrieved eggs(%) Mean # of 2PN Total# of 2PN (% of all retrieved) #cycles with no transfer(%) # Day 5 transfers (%) Mean # blastocysts frozen Clinical Intrauterine pregnancy (%) Live birth (%)
P1
Controls(150)
P2
P3
11.95.3 1301 6.93.9 736 (56.6)
13.13.1 1269 8.44.9 808 (63.7)
0.5
13.15.9 0.2 0.8 1954 0.01 8.85.1 <0.0010.8 <0.001 1311 (67.1) 0.001 0.4
4 (3.2) 14 (11.7) 1.21.9 63 (52)
0 (0) 22 (22.7) 2.12.7 62 (63.9)
0.3 0.003 0.01 0.04
2 (1.3) 41 (27.7) 2.31.7 96 (64.9)
0.5 <0.01 <0.01 0.03
49 (40.8)
53 (54.6)
0.04
82 (55.4)
<0.01 0.7
0.4 0.1 0.6 0.7
References: 1 Comparison between endometriosis-insemination and endometriosisICSI groups 2 Comparison between endometriosis-insemination and control groups 3 Comparison between endometriosis-ICSI and control groups Supported by: Institutional P-154 Tuesday, October 20, 2015 DIENOGEST ENHANCES AUTOPHAGY INDUCTION IN ENDOMETRIOTIC CELLS BY IMPAIRING ACTIVATION OF AKT, ERK1/2, AND MTOR. D. Choi,a M. Jo,b E. Lee,a D. Lee,a J. Choi.a aDepartment of Obstetrics and Gynecology, Seoul, Korea, Republic of; bSamsung Biochemical Research Institute, Seoul, Korea, Republic of. OBJECTIVE: To elucidate the therapeutic mechanisms of progestin, the effects of progesterone and progestin (dienogest) on autophagy induction and regulation in endometriotic cells were evaluated. Specifically, the effects of progesterone and dienogest on the PI3K-AKT and MEK1/2-ERK1/2 pathways were studied. These pathways activate mammalian target of rapamycin (mTOR), a major negative regulator of autophagy.
FERTILITY & STERILITYÒ
DESIGN: In vitro experimental study using human endometriotic cyst stromal cells (ECSCs). MATERIALS AND METHODS: ECSCs were treated with progesterone or dienogest. Autophagy was evaluated in vitro by measuring the expression of the microtubule-associated protein light chain 3-II (LC3-II) and autophagosome formation in ECSCs. The levels of AKT, ERK1/2, and mTOR activity were determined by quantifying the phosphorylated forms of AKT, ERK1/2, and S6K (the downstream target of mTOR). In addition, apoptosis was evaluated by measuring the cleavage of caspase-3 and by performing annexin-V and PI staining. RESULTS: Progesterone treatment did not have any significant effect on LC3-II expression, autophagosome formation, or phosphorylation of AKT, ERK1/2, or S6K1 in estrogen-treated ECSCs. However, dienogest treatment upregulated LC3-II expression and stimulated autophagosome formation. These effects were accompanied by decreased activation of AKT, ERK1/2, and S6K. Furthermore, incubation of ECSCs with AKT and ERK1/2 inhibitors, which mimicked dienogest-mediated suppression of AKT and ERK1/2 activity, increased LC3-II expression and autophagosome accumulation. In addition, to determine whether dienogest-mediated autophagy induction directly affects apoptosis, progestin-treated endometriotic cells were treated with or without 3-methyladenine (3-MA, autophagy inhibitor). Although dienogest treatment normally induced autophagy and promoted apoptosis, treatment with 3-MA blocked this proapoptotic effect. CONCLUSIONS: Our results suggest that dienogest treatment of endometriotic cells suppresses AKT and ERK1/2 activity, thereby in turn inhibiting mTOR, inducing autophagy, and promoting apoptosis.
P-155 Tuesday, October 20, 2015 ELEVATED PHOSPHATASE OF REGENERATING LIVER 3 (PRL-3) PROMOTES CYTOSKELETON REORGANIZATION, CELL MIGRATION AND INVASION IN ECTOPIC STROMAL CELLS FROM ENDOMETRIOMA. H. Zhan,a,b M. A. Bedaiwy,a B. Peng,a J. Lin.b aDepartment of Obstetrics & Gynaecology, The University of British Columbia, Child & Family Research Institute, Vancouver, BC, Canada; b Central Laboratory, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China. OBJECTIVE: To investigate the expression of Phosphatase of regenerating liver-3 (PRL-3) in endometrioma and its role in regulating cell motility in endometrial stromal cells from endometrioma. DESIGN: In vitro study on primary human endometrial and endometriotic cells from women with and without endometriosis. MATERIALS AND METHODS: Primary human endometrial stromal cells (ESCs) were isolated from eutopic endometrium of women with (EuEM, n¼19) or without (EuCo, n¼10) endometriosis, and ectopic biopsies of patients with endometrioma (OvEM, n¼26). Stromal cells from endometrioma were transfected with/without short-hairpin RNA (shRNA) or smallinterfering RNA (siRNA). The protein and mRNA levels of PRL-3 were examined by Western blot and RT-qPCR, respectively. Cell motility was evaluated by wound healing assay and transwell migration/invasion assay. The cellular distribution of F-actin and a-tubulin were examined by immunocytochemistry. RESULTS: PRL-3 are significantly elevated in ESCs from OvEM at the protein (P¼0.001) and the mRNA (P¼0.038) levels compared to EuCo. These relationships remain unchanged upon stratifying patients, according to the phase of the menstrual cycle. Knockdown of PRL-3 significantly modified cytoskeleton organization (F-actin, P¼0.025; a-tubulin,P¼0.046), inhibited cell migratory (P<0.001) and invasive capability (P<0.001), and attenuated the expression of RhoA (P¼0.001), RhoC (P¼0.007), ROCK1 (P¼0.023) and MMP9 (P¼0.047) but not MMP2 (P¼0.154) in stromal cells from endometrioma. CONCLUSIONS: The expression of PRL-3 is elevated in ectopic ESCs from endometrioma. PRL-3 plays important roles in regulating cytoskeleton organization, cell migration and invasion in these cells. Supported by: National Natural Science Foundation of China (No. 81170546).
P-156 Tuesday, October 20, 2015 INCREASED MIR-196A EXPRESSION REPRESSES PGR IN INFERTILE WOMEN WITH MINIMAL OR MILD ENDOMETRIOSIS BY INCREASED ACTIVATIONOF THE MEK/ERK SIGNAL. W. Huang, M. Zhou, Y. Song, L. Xiao. Department of
e159
P-151 Tuesday, October 20, 2015 COULD THE UNILATERAL OVARIAN ENDOMETRIOSIS AFFECT THE CONTRALATERAL OVARY? NEW INSIGHTS FROM FOURIER TRANSFORM INFRARED (FTIR) E. Sereni,b C. Zaca’,b SPECTROSCOPY. G. Gioacchini,a E. Giorgini,c V. Notarstefano,a L. Vaccari,d O. Carnevali,a A. Borini.b a Universita Politecnica delle Marche, Ancona, Italy; bTecnobios Procreazione, Bologna, Italy; cPolytechnic University of Marche, Ancona, Italy; dElettra-Sincrotrone Trieste, Trieste, Italy. OBJECTIVE: Up to date, endometriosis’ possible impact on follicle cells metabolism has never been highlighted. In this light, the aim of this study was to evaluate, by FT-IR microspectroscopy, metabolic changes on granulosa cells (GCs) isolated from endometriosis affected ovaries and contralateral healthy ones DESIGN: This prospective non-randomized study has been conducted from January to September 2014 on women undergoing a controlled ovarian hyperstimulation for an IVF treatment. In particular, GCs were collected from both ovaries of 10 women with a diagnosis of unilateral ovarian endometrioma at the time of oocytes retrieval. 9 women with male, idiopathic or tubal-factor infertility diagnosis were selected for the control group. The three experimental groups matched for female age (36.24.1 vs 35.42.6) MATERIALS AND METHODS: GCs obtained from follicles aspirates were isolated from red blood cells and follicular fluid by density gradient centrifugation. FTIR analysis was performed by using a Bruker Vertex 70 Interferometer with a Hyperion 3000 Vis-IR microscope. QPCR analysis Supported FTIR results. . Data are presented as mean S.D. Two-Way ANOVA followed by Tukey test as Multiple comparisons test, was used for comparison among experimental groups. All statistical analyses were performed using the statistical software package Prism5 (Graphpad Software, Inc. USA) with significance accepted at P<0.05 RESULTS: Results are showed in table CONCLUSIONS: This preliminary study represents the first approach to evaluate the macromolecular and biochemical changes on GCs from both ovaries in patients with unilateral endometrioma. These findings pinpoint that this pathology causes relevant changes in the metabolism and amount of proteins, lipids, carbohydrates and nucleic acids, and increases peroxidative processes. It is impressive that the same biochemical alterations have been found both in ovaries with endometrioma and in the contralateral
e158
ASRM Abstracts
‘‘healthy’’ ones. All these results could be useful in developing new strategies to prevent the detrimental effects of endometriosis on fertility.
Control LIPIDS/CELL (%) PHOSPHOLIPIDS/ LIPIDS (%) LIPIDS PEROXIDATION(%) UNFOLDED PROTEIN (%) CARBOHYDRATES/ PROTEIN(%) RNA/DNA(%) PPARa PPARg FAN GLUT1 GLUT9
CONTRAL ATERAL OVARY WITH OVARY ENDOMETRIOMA SIGNIFICANCE
7.891.18a 11.550.27b 5.921.79a 12.500.24b
11.800.39b 12.020.55b
a vs b¼P<0.0001 a vs b¼P<0.0001
4.210.10a 7.470.73b
7.680.19b
a vs b¼P<0.0001
22.141.32a 30.034.02b
28.841.34c
a vs b¼P<0.05
16.062.37a 10.940.13b
10.390.92b
a vs b¼P<0.01
10.833.62a 1.130.22a 1.200.13a 1.130.41a 10.330.78a 9.080.44a
6.141.05b 8.700.97b 4.170.83b 6.231.29b 1.130.39c 1.190.47b
a vs b¼P<0.05 a vs b¼P<0.01 a vs b¼P<0.05 a vs b¼P<0.01 a vs b¼P<0.01 a vs b¼P<0.01
6.161.22b 7.000.88b 3.301.31b 4.130.12b 2.190.86b 1.510.80b
P-152 Tuesday, October 20, 2015 USE OF DEPOT GNRH ANTAGONIST (DEGARELIX) IN THE TREATMENT OF ENDOMETRIOMAS (OVARIAN ENDOMETRIOSIS) BEFORE IVF: A CONTROLLED TRIAL. M. Sbracia F. Scarpellini. CERM-Hungaria, Roma, Italy. OBJECTIVE: Endometriosis is a chronic condition affecting around 10% of women in reproductive age, and after surgery it recur in about 50% of cases. It is recognized that surgical treatment of ovarian endometriomas affects ovarian reserve. For this reason in case of ovarian endometriomas we tried a medical approach instead of the surgery using a long acting GnRH antagonist. The aim of the study was to evaluate the role Degarelix (Firmagon) in the treatment of women with ovarian endometriosis (endometriomas) before undergo IVF in order to reduce cyst volume and improve IVF outcome avoiding surgery. DESIGN: This randomized controlled study was conducted from the January 2014 to April 2015 on 58 women with ovarian endometriomas. MATERIALS AND METHODS: All patients were affected by ovarian endometriosis with endometriomas larger than 3.5cm in mean diameter. All patients din not undergo surgery for endometriosis and they underwent a subsequent IVF cycle in the next 4-8 months. The mean age of women was 35.1 + 2.0 (range 25/39). The women were assigned to the study group or to the control group by a computer generated sequence. The 31 women of the study group were treated with a single injection of Degarelix (Firmagon 120mg) subcutaneously, The 27 women of the control group were treated for 6 months with Leuprolide 3.75 mg/month alone. The women of the two groups were followed up during the treatment and after the treatment for IVF cycles. All patients underwent IVF within 3 months from the end of treatment. All patients were treated for the controlled ovarian hyperstimulation with the same protocol, a standard flexible GnRH antagonist protocol with recombinant FSH (Gonal-F) plus Cetrorelix (Cetrotide). Primary outcomes were time of endometriomas disappearance and IVF outcome. RESULTS: The two groups of patients did not show statistically significant differences for epidemiological data. The time of endometriomas disappearance was shorter in the group treated with Degarelix than in the control group (2.4+0.9months vs 4.1+1.2months, P<0.01). The pregnancy rate for IVF in the women treated with Degarelix was significantly higher than in the control group (45.1% vs 18.5%, P¼0.049). The implantation rate was 19.1 in Degarelix group versus 7.5 in controls (P¼0.0416). The number of mature oocyte collected was 8.6+3.4 in Degarelix group and 6.1+3.6 in controls (P¼0.0087). The number of embryo obtained was 4.7+1.2 in Degarelix group and 3.2+1.1 in controls (P<0.0001). CONCLUSIONS: Even though our data need to be validated in larger group of patients, Degarelix treatment seems to be a promising tool in patients with ovarian endometriosis, especially to avoid surgery in order to preserve ovarian reserve, improving IVF outcome. P-153 Tuesday, October 20, 2015 SHOULD ENDOMETRIOSIS BE AN INDICATION FOR INTRACYTOPLASMIC SPERM INJECTION (ICSI) IN FRESH IVF CYCLES?. J. Lekovich,a G. D. Palermo,b N. Pereira,c Z. Rosenwaks.a a Weill Cornell Medical College, New York, NY; bWeill Medical College of
Vol. 104, No. 3, Supplement, September 2015
Cornell University, New York, NY; cThe Ronald O. Perelman and Claudia Cohen Center, New York, NY. OBJECTIVE: Mechanism of infertility in endometriosis potentially involves oocyte/embryo defects and lower fertilization and implantation rates. Cumulus complex in endometriosis patients demonstrates mitochondrial dysfunction and higher oxidative stress. We sought to investigate if ICSI, which diminishes oxidative stress when compared to insemination, improves IVF outcomes in patients with endometriosis. DESIGN: Retrospective cohort MATERIALS AND METHODS: 221 patients who had surgically or sonographically confirmed endometriosis were compared with 150 patients with unexplained infertility. Patients with endometriosis as a sole cause of infertility were included. Exclusion criteria [1. > 41yo; 2. Male factor (sperm count < 15 x 106/cc, motility less than 40%, older than 55yo]. Primary outcomes: 1. Number of retrieved eggs; 2. Number of 2 pronuclear zygote (2PN) stage embryos; 3. Proportion of day 5 transfers; 4. Number of frozen blastocysts. Secondary outcomes: 1. Clinical intrauterine pregnancy [IUP] rate; 2. Live birth rate. Student’s t-test and ANOVA were used to continuous and Fisher’s exact and Chi-square tests for categorical variables. RESULTS: Within the endometriosis group, 124 patients utilized standard insemination, whereas 97 utilized ICSI. Endometriosis-insemination, endometriosis-ICSI and the control groups were matched for age, BMI, anti-mullerian hormone (AMH) and the number of eggs retrieved [11.95.3 vs. 13.13.1 vs. 13.1 5.9, P¼0.07]. The control and endometriosis-ICSI groups did not differ in terms of number of 2PNs, number of day 5 transfers, IUP and live birth rates (Table). Endometriosis-ICSI group demonstrated significantly higher percentage of 2PN embryos, percentage of day 5 transfers, number/proportion of clinical IUP as well as live birth rates when compared with the endometriosisinsemination group. Additionally, endometriosis-ICSI group had a lower proportion of cycles with no normal fertilization [0 (0%) vs 4 (3.2%), but this difference did not reach statistical significance. CONCLUSIONS: Our findings suggest that endometriosis might be an indication for ICSI, as patients with endometriosis who utilized ICSI achieved better outcomes (higher proportion of normal fertilization, higher number of day 5 transfers, clinical pregnancy and live birth rates) when compared to standard insemination cycles. Table 1 Endometriosis- EndometriosisInsemination(124) ICSI(97) Mean # eggs retrieved Total# of retrieved eggs(%) Mean # of 2PN Total# of 2PN (% of all retrieved) #cycles with no transfer(%) # Day 5 transfers (%) Mean # blastocysts frozen Clinical Intrauterine pregnancy (%) Live birth (%)
P1
Controls(150)
P2
P3
11.95.3 1301 6.93.9 736 (56.6)
13.13.1 1269 8.44.9 808 (63.7)
0.5
13.15.9 0.2 0.8 1954 0.01 8.85.1 <0.0010.8 <0.001 1311 (67.1) 0.001 0.4
4 (3.2) 14 (11.7) 1.21.9 63 (52)
0 (0) 22 (22.7) 2.12.7 62 (63.9)
0.3 0.003 0.01 0.04
2 (1.3) 41 (27.7) 2.31.7 96 (64.9)
0.5 <0.01 <0.01 0.03
49 (40.8)
53 (54.6)
0.04
82 (55.4)
<0.01 0.7
0.4 0.1 0.6 0.7
References: 1 Comparison between endometriosis-insemination and endometriosisICSI groups 2 Comparison between endometriosis-insemination and control groups 3 Comparison between endometriosis-ICSI and control groups Supported by: Institutional P-154 Tuesday, October 20, 2015 DIENOGEST ENHANCES AUTOPHAGY INDUCTION IN ENDOMETRIOTIC CELLS BY IMPAIRING ACTIVATION OF AKT, ERK1/2, AND MTOR. D. Choi,a M. Jo,b E. Lee,a D. Lee,a J. Choi.a aDepartment of Obstetrics and Gynecology, Seoul, Korea, Republic of; bSamsung Biochemical Research Institute, Seoul, Korea, Republic of. OBJECTIVE: To elucidate the therapeutic mechanisms of progestin, the effects of progesterone and progestin (dienogest) on autophagy induction and regulation in endometriotic cells were evaluated. Specifically, the effects of progesterone and dienogest on the PI3K-AKT and MEK1/2-ERK1/2 pathways were studied. These pathways activate mammalian target of rapamycin (mTOR), a major negative regulator of autophagy.
FERTILITY & STERILITYÒ
DESIGN: In vitro experimental study using human endometriotic cyst stromal cells (ECSCs). MATERIALS AND METHODS: ECSCs were treated with progesterone or dienogest. Autophagy was evaluated in vitro by measuring the expression of the microtubule-associated protein light chain 3-II (LC3-II) and autophagosome formation in ECSCs. The levels of AKT, ERK1/2, and mTOR activity were determined by quantifying the phosphorylated forms of AKT, ERK1/2, and S6K (the downstream target of mTOR). In addition, apoptosis was evaluated by measuring the cleavage of caspase-3 and by performing annexin-V and PI staining. RESULTS: Progesterone treatment did not have any significant effect on LC3-II expression, autophagosome formation, or phosphorylation of AKT, ERK1/2, or S6K1 in estrogen-treated ECSCs. However, dienogest treatment upregulated LC3-II expression and stimulated autophagosome formation. These effects were accompanied by decreased activation of AKT, ERK1/2, and S6K. Furthermore, incubation of ECSCs with AKT and ERK1/2 inhibitors, which mimicked dienogest-mediated suppression of AKT and ERK1/2 activity, increased LC3-II expression and autophagosome accumulation. In addition, to determine whether dienogest-mediated autophagy induction directly affects apoptosis, progestin-treated endometriotic cells were treated with or without 3-methyladenine (3-MA, autophagy inhibitor). Although dienogest treatment normally induced autophagy and promoted apoptosis, treatment with 3-MA blocked this proapoptotic effect. CONCLUSIONS: Our results suggest that dienogest treatment of endometriotic cells suppresses AKT and ERK1/2 activity, thereby in turn inhibiting mTOR, inducing autophagy, and promoting apoptosis.
P-155 Tuesday, October 20, 2015 ELEVATED PHOSPHATASE OF REGENERATING LIVER 3 (PRL-3) PROMOTES CYTOSKELETON REORGANIZATION, CELL MIGRATION AND INVASION IN ECTOPIC STROMAL CELLS FROM ENDOMETRIOMA. H. Zhan,a,b M. A. Bedaiwy,a B. Peng,a J. Lin.b aDepartment of Obstetrics & Gynaecology, The University of British Columbia, Child & Family Research Institute, Vancouver, BC, Canada; b Central Laboratory, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China. OBJECTIVE: To investigate the expression of Phosphatase of regenerating liver-3 (PRL-3) in endometrioma and its role in regulating cell motility in endometrial stromal cells from endometrioma. DESIGN: In vitro study on primary human endometrial and endometriotic cells from women with and without endometriosis. MATERIALS AND METHODS: Primary human endometrial stromal cells (ESCs) were isolated from eutopic endometrium of women with (EuEM, n¼19) or without (EuCo, n¼10) endometriosis, and ectopic biopsies of patients with endometrioma (OvEM, n¼26). Stromal cells from endometrioma were transfected with/without short-hairpin RNA (shRNA) or smallinterfering RNA (siRNA). The protein and mRNA levels of PRL-3 were examined by Western blot and RT-qPCR, respectively. Cell motility was evaluated by wound healing assay and transwell migration/invasion assay. The cellular distribution of F-actin and a-tubulin were examined by immunocytochemistry. RESULTS: PRL-3 are significantly elevated in ESCs from OvEM at the protein (P¼0.001) and the mRNA (P¼0.038) levels compared to EuCo. These relationships remain unchanged upon stratifying patients, according to the phase of the menstrual cycle. Knockdown of PRL-3 significantly modified cytoskeleton organization (F-actin, P¼0.025; a-tubulin,P¼0.046), inhibited cell migratory (P<0.001) and invasive capability (P<0.001), and attenuated the expression of RhoA (P¼0.001), RhoC (P¼0.007), ROCK1 (P¼0.023) and MMP9 (P¼0.047) but not MMP2 (P¼0.154) in stromal cells from endometrioma. CONCLUSIONS: The expression of PRL-3 is elevated in ectopic ESCs from endometrioma. PRL-3 plays important roles in regulating cytoskeleton organization, cell migration and invasion in these cells. Supported by: National Natural Science Foundation of China (No. 81170546).
P-156 Tuesday, October 20, 2015 INCREASED MIR-196A EXPRESSION REPRESSES PGR IN INFERTILE WOMEN WITH MINIMAL OR MILD ENDOMETRIOSIS BY INCREASED ACTIVATIONOF THE MEK/ERK SIGNAL. W. Huang, M. Zhou, Y. Song, L. Xiao. Department of
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