- Email: [email protected]
Transcriptome analysis of endometrial stroma-like organoids differentiated from human induced pluripotent stem cells
colonies were passaged by trypsinization. Male mESCs were separated from MEF cells by sedimentation. Approximately 1000 feeder-free mESCS were placed in 25 uL HD. Two days later, EBs were either transferred to HD containing retinoic acid (RA) or CD also supplemented with RA. On day 8, EBs were treated with collagenase IVand stained for GCAP/OCT4/DAZL or processed for gene expression analysis by NGS. RESULTS: Of a total of 260 EBs, 100 EBs were re-plated on day 2 to corresponding HD containing RA, while the remaining 160 EBs were transferred to CD supplemented with RA. On day 8, HD-cultured EBs reached approximately 150um in diameter, while EBs in CD reached 280-300um. EBs cultured in HD yielded an average of 77% GCAP positive cells, while those in CD only presented 22% GCAP positivity. EBs were also assessed for OCT4 activity to detect loss of stemness. In HD-cultured EBs, 40% of isolated cells were strongly positive for OCT4, compared to 60% of cells isolated from EBs in CD, indicating a higher level of cell differentiation in the former. To undoubtedly detect male germ cells, EBs were stained for DAZL. EBs cultured in HD and CD yielded 5% and 1% DAZL-positive cells, respectively. RNA sequencing revealed significant upregulation on Gdf9, Sox17, Kit, and Prm2 genes in HD-cultured EBs. These genes are critical for gametogenesis, confirming the presence of pre-meiotic and post-meiotic germ cells. CONCLUSIONS: Our attempt to induce the appearance and eventual maturation of male germ cells by the HD approach, by minimizing environment cell-surface interference, appears the most promising. Once the ability of these germ cells to progress through meiosis is confirmed, EB formation through HD may provide an inexpensive and reproducible model to experiment with in vitro gametogenesis. P-230 Tuesday, October 31, 2017 WITHDRAWN
P-231 Tuesday, October 31, 2017 HUMAN TROPHECTODERM (TE) CELL LINES: A NOVEL EXTENDED CELL CULTURE REPORT. O. Perez,a H. Adriaanse,a G. R. Navarrete,a a a b B. Tilley, A. Patel, R. Gada, M. Thomas,b S. J. Chantilis.b aDallas Fertility Center, Dallas, TX; bDallas-Fort Worth Fertility Associates, Dallas, TX. OBJECTIVE: The derivation of human TE cells or self-renewing progenitors has not been reported. This study is designed to demonstrate TE cell derivation during 590 hours of cell culture. DESIGN: Research ongoing study. MATERIALS AND METHODS: Study TE cells were obtained from embryos determined to be aneuploid after undergoing preimplantation genetic screening (PGS). Eight Patients consented to have subject embryos re-biopsied for this IRB-approved study. A total of 16 frozen-thawed aneuploid blastocysts were studied. An average of 5.6 cells was obtained from the rebiopsy of PGS tested blastocysts. TE cells were cultured in the presence of human fibroblast growth factor 4 in a time-lapse incubator (EmbryoScope). A controlled environment of 5% O2, 6% CO2 and 37 C was used to provide the cell growth conditions. All 16 specimens were cultured simultaneously using two time-lapse dishes with individual 30 ml of RPMI 1640 medium supplemented with 20% HSA. Culture media was changed every 24 hours. Cells were video monitored in 10-minute increments. A portion of more than 50% of the resulted number of cells were genetically analyzed by VeriSeq (high resolution NGS) to confirm the chromosomal analyses of the new cells. RESULTS: TE cell proliferation initiated within 20 minutes of culture for all tested cells. Individual small cells were released from the biopsied mass of cells. Cell activity was noted in all tested cells during the 590 hours of cell culture. Resulted TE cells were counted with a mean of 701 cells (range 450 to 1023 cells). Chromosomal analyses resulted genetic information in all 16 newly derived TE samples; 12 demonstrating aneuploidy, and 4 that were euploid. CONCLUSIONS: This novel study exposes the possibility to derive newly formed TE cells in vitro from a blastocyst. These results further develop our understanding of initial TE populations by demonstrating the capability to self-renew these cells in vitro. Perhaps this culture system of TE cell proliferation could be extended indefinitely, representing an option to verify the PGS outcome of non-viable genetically tested embryos and to establish human TE stem cell lines.
TE Biopsies (n) 16
# Abnormal # Normal # Abnormal Initial # Final # PGS PGS PGS Cells Cells Post-Culture Post-Culture Biopsies (n) (n) (Average) (Average) (n) 16
5.6
701
12
4
P-232 Tuesday, October 31, 2017 TRANSCRIPTOME ANALYSIS OF ENDOMETRIAL STROMA-LIKE ORGANOIDS DIFFERENTIATED FROM HUMAN INDUCED PLURIPOTENT STEM a,b M. T. Dyson,a J. S. Coon,a CELLS. K. Miyazaki, T. Maruyama,b S. Bulun.a aObstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, IL; bObstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan. OBJECTIVE: It is now appreciated that induced pluripotent stem cells (iPSCs) derived from skin biopsies or bone marrow aspirates can provide
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patient-specific pluripotent cells, which could be differentiated into various cells types. In fact, several bioengineered organs including kidney have recently generated through differentiation of iPSCs. We view uterine tissue engineering by using endometrial cells (ECs) differentiated from iPSCs can be considered an option for treatments in patients with uterine factor infertility or endometriosis; however, no model presently exists for generating ECs from iPSCs. The purpose of this study was to develop a chemically defined protocol for differentiating human iPSCs (hiPSCs) into ECs. DESIGN: Prospective experimental study MATERIALS AND METHODS: hiPSCs were purchased from ATCC. Embryoid bodies (EBs) were generated from hiPSCs following 1-day culture on EB formation plate and cultured with defined chemical cocktails for 13 days, and EBs were examined for stage-specific markers every other day. Day 14 (D14) EBs were then treated with 8-bromoadenosine 39,59-cyclic monophosphate (cAMP) and medroxyprogesterone acetate (MPA) for 8 days to determine the capacity of decidualization, which characterizes ESCs. RNAseq was performed on D4, D6, D8, D14, D22 EBs, hiPSCs, and normal endometrial stromal cells to determine transcriptome of each stage. RESULTS: Intermediate mesoderm-specific genes LHX1 and PAX2 were significantly upregulated in D4 EBs compared to hiPSCs as revealed by qPCR (N¼9, p<0.05). mRNA expression of Mullerian duct marker ISL1 was significantly higher in D8 EBs than in hiPSCs (N¼9, p<0.05). The expression of ESC-specific transcription factors HOXA10 and HOXA11 were upregulated in day 6 EBs and maintained thereafter. PGR, the steroid receptor predominantly expressed in ESCs, was upregulated progressively during the differentiation process until D14. After 8 days of treatment with cAMP and MPA, D22 EBs expressed significantly higher mRNA levels of decidualization markers such as FOXO1, IGFBP1, and PRL (N¼9, p<0.05) compared with cells treated with vehicle control (VC). ELISA (N¼9, p<0.05) confirmed the increased protein expression of FOXO1. Principal component analysis of RNAseq depicted a seamless transition of the transcriptional profiles during differentiation. Transcriptome of D14 EBs was closer to normal endometrial stromal cells compared with D8 EBs. D22 EBs shared 1404 differentially expressed genes with normal endometrial stromal cells when compared with VC. CONCLUSIONS: We have found that EBs differentiated from hiPSCs using our chemically defined protocol have specific characteristics resembling ESCs. These results suggest that hiPSCs can be an unlimited source of ESCs. We propose these results as the first step in advancing stem cell-based therapies for uterine factor infertility or endometriosis. Supported by: National Institutes of Health grant R37-HD36891 and R03HD082558, USA; Grant-in-Aid for Scientific Research (Young Scientists B), Japan; The Uehara Memorial Foundation, Japan; The Kanzawa Medical Research Foundation, Japan ENDOMETRIOSIS P-233 Tuesday, October 31, 2017 ENDOMETRIOSIS IS ASSOCIATED WITH RISK OF OVARIAN, ENDOMETRIAL, CERVICAL AND THYROID CANCER. G. Lai,a C. Tzeng.b aSchool of Public Health, Taipei Medical University, Taipei, Taiwan; bTaipei Medical University, Taipei, Taiwan. OBJECTIVE: The relationship between cancer and endometriosis was still unclear. Many reports suggested that endometriosis associated with increased risk of gynecological cancers, but the conclusion was still inconsistent. This study aimed to unravel the association between endometriosis and cancers. DESIGN: A retrospective population-based cohort study was performed by linking to National Health Insurance Research Database (NHIRD) in Taiwan. MATERIALS AND METHODS: A total of 19,378 women with newly identified endometriosis from 2000 to 2008 and 96,890 multivariablematched controls (1:5) were selected. Sociodemographic and other potential confounding factors were selected as adjusted variables from database. They were compared between the endometriosis and control cohort using a Chisquare test. The Cox regression model adjusted for potential confounders was used to assess the risk of cancers due to endometriosis. RESULTS: After adjusted potential confounders, the adjusted hazard ratio associated with endometriosis was 1.64 (95% confidence interval (CI) 1.491.81) for overall cancers, 4.77 (95% CI 3.40-6.70) for ovarian cancer, 4.19
FERTILITY & STERILITYÒ
(95% CI 2.91-6.04) for endometrial cancer, 2.67 (95% CI 1.93-3.70) for cervical cancers, and 1.64 (95% CI 1.15-2.35) for thyroid cancers. CONCLUSIONS: Estrogen stimulation and chronic inflammation might be as putative mechanisms to endometrial cancer. Besides, estrogen receptors participate in cellular processes contributing to enhanced mitogenic, migratory, and invasive properties of thyroid cells. The investigation of association between endometriosis and cancers would advance the exploration of endometriosis etiology. Supported by: This study was partially supported by a grant from Academia Sinica (BM102020946) P-234 Tuesday, October 31, 2017 LIPOPHILIC BUT NOT HYDROPHILIC STATINS INHIBIT GROWTH AND REDUCE INVASIVENESS OF HUMAN ENDOMETRIAL STROMAL CELLS. A. Sokalska,a A. Duleba.b aDepartment of Obstetrics and Gynecology, University of Pennsylvania, Division of Reproductive Endocrinology and Infertility, Philadelphia, PA; bDepartment of Reproductive Medicine, Division of Reproductive Endocrinology and Infertility, University of California, San Diego, La Jolla, CA. OBJECTIVE: Statins (competitive inhibitors of HMG-CoA reductase) reduce endometriosis burden in murine and baboon models of this disease. On the cellular level, statins decrease proliferation, induce apoptosis, and alter the cytoskeleton of human endometrial stromal (HES) cells. This study compared effects of lipid-soluble (simvastatin, lovastatin, atorvastatin) and water-soluble (pravastatin) statins on proliferation, apoptosis and invasiveness of HES cells. DESIGN: In vitro experiments conducted in the academic laboratory. MATERIALS AND METHODS: Endometrial biopsies were collected during proliferative phase from five volunteers. HES cells were cultured in the absence or in the presence of simvastatin, lovastatin, atorvastatin and pravastatin at different concentrations. Effects on DNA synthesis, apoptosis and invasiveness were evaluated using thymidine incorporation assay, caspases 3/ 7 activity assay and invasiveness assay, respectively. Each assay was repeated at least three times using cells from different participants. Statistical analysis was performed using analysis of variance, followed by post-hoc Dunnett’s test; when appropriate, values were logarithmically transformed. Differences were considered significant at P<0.05. RESULTS: The proliferation of HES cells was significantly decreased by simvastatin (by 47-89%), lovastatin (by 46-78%), and atorvastatin (by 2148%) in a concentration-dependent manner. Activity of executioner caspases 3/7 was significantly increased by simvastatin (by 10-25%), lovastatin (by 19%), and atorvastatin (by 7-10%) in a concentration-dependent manner. The greatest effects were observed in response to simvastatin. Accounting for the effects of statins on cell number, the invasiveness of HES cells was significantly decreased in cells treated with simvastatin (by 49%), lovastatin (by 54%), and atorvastatin (by 53%). Pravastatin had minimal effects on any of the tested end-points. CONCLUSIONS: Present findings demonstrate that only lipid-soluble statins are effective in inhibition of growth and invasiveness of HES cells. These findings may be clinically relevant in treatment of endometriosis and indicate that lipid-soluble statins should be considered for future clinical trials. Supported by: NIH Grant U54 HD052668 P-235 Tuesday, October 31, 2017 IVF/ET OUTCOMES FROM AFFECED AND UNAFFECTED OVARIES WITH UNILATERAL ENDOMETRIOMAS. A. Takashima, N. Takeshita. Obstetrics and Gynegology, Toho Medical Center Sakura Hospital, Chiba, Japan. OBJECTIVE: To assess whether the quantitative and qualitative IVF outcomes in ovaries with and without a small endometrioma with high average age of the participants in the same individual. DESIGN: In this retrospective analysis, we examined 336 infertile women with unilateral endometriomas < 40 mm who had undergone IVF or ICSI from April 2011 to December 2016. Single frozen embryo transfer cycles were performed with the separate data collections for both the affected and the unaffected ovaries, allowed for the evaluation of the implantation rate. MATERIALS AND METHODS: On day 3, the serum levels of AMH, FSH, and E2 were recorded. We evaluated and compared the following for the endometrioma-containing ovary and the contralateral intact ovary: the AFC, the degree of follicular flushing, the oocytes that were retrieved, the
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P-231 Tuesday, October 31, 2017 HUMAN TROPHECTODERM (TE) CELL LINES: A NOVEL EXTENDED CELL CULTURE REPORT. O. Perez,a H. Adriaanse,a G. R. Navarrete,a a a b B. Tilley, A. Patel, R. Gada, M. Thomas,b S. J. Chantilis.b aDallas Fertility Center, Dallas, TX; bDallas-Fort Worth Fertility Associates, Dallas, TX. OBJECTIVE: The derivation of human TE cells or self-renewing progenitors has not been reported. This study is designed to demonstrate TE cell derivation during 590 hours of cell culture. DESIGN: Research ongoing study. MATERIALS AND METHODS: Study TE cells were obtained from embryos determined to be aneuploid after undergoing preimplantation genetic screening (PGS). Eight Patients consented to have subject embryos re-biopsied for this IRB-approved study. A total of 16 frozen-thawed aneuploid blastocysts were studied. An average of 5.6 cells was obtained from the rebiopsy of PGS tested blastocysts. TE cells were cultured in the presence of human fibroblast growth factor 4 in a time-lapse incubator (EmbryoScope). A controlled environment of 5% O2, 6% CO2 and 37 C was used to provide the cell growth conditions. All 16 specimens were cultured simultaneously using two time-lapse dishes with individual 30 ml of RPMI 1640 medium supplemented with 20% HSA. Culture media was changed every 24 hours. Cells were video monitored in 10-minute increments. A portion of more than 50% of the resulted number of cells were genetically analyzed by VeriSeq (high resolution NGS) to confirm the chromosomal analyses of the new cells. RESULTS: TE cell proliferation initiated within 20 minutes of culture for all tested cells. Individual small cells were released from the biopsied mass of cells. Cell activity was noted in all tested cells during the 590 hours of cell culture. Resulted TE cells were counted with a mean of 701 cells (range 450 to 1023 cells). Chromosomal analyses resulted genetic information in all 16 newly derived TE samples; 12 demonstrating aneuploidy, and 4 that were euploid. CONCLUSIONS: This novel study exposes the possibility to derive newly formed TE cells in vitro from a blastocyst. These results further develop our understanding of initial TE populations by demonstrating the capability to self-renew these cells in vitro. Perhaps this culture system of TE cell proliferation could be extended indefinitely, representing an option to verify the PGS outcome of non-viable genetically tested embryos and to establish human TE stem cell lines.
TE Biopsies (n) 16
# Abnormal # Normal # Abnormal Initial # Final # PGS PGS PGS Cells Cells Post-Culture Post-Culture Biopsies (n) (n) (Average) (Average) (n) 16
5.6
701
12
4
P-232 Tuesday, October 31, 2017 TRANSCRIPTOME ANALYSIS OF ENDOMETRIAL STROMA-LIKE ORGANOIDS DIFFERENTIATED FROM HUMAN INDUCED PLURIPOTENT STEM a,b M. T. Dyson,a J. S. Coon,a CELLS. K. Miyazaki, T. Maruyama,b S. Bulun.a aObstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, IL; bObstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan. OBJECTIVE: It is now appreciated that induced pluripotent stem cells (iPSCs) derived from skin biopsies or bone marrow aspirates can provide
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Vol. 108, No. 3, Supplement, September 2017
patient-specific pluripotent cells, which could be differentiated into various cells types. In fact, several bioengineered organs including kidney have recently generated through differentiation of iPSCs. We view uterine tissue engineering by using endometrial cells (ECs) differentiated from iPSCs can be considered an option for treatments in patients with uterine factor infertility or endometriosis; however, no model presently exists for generating ECs from iPSCs. The purpose of this study was to develop a chemically defined protocol for differentiating human iPSCs (hiPSCs) into ECs. DESIGN: Prospective experimental study MATERIALS AND METHODS: hiPSCs were purchased from ATCC. Embryoid bodies (EBs) were generated from hiPSCs following 1-day culture on EB formation plate and cultured with defined chemical cocktails for 13 days, and EBs were examined for stage-specific markers every other day. Day 14 (D14) EBs were then treated with 8-bromoadenosine 39,59-cyclic monophosphate (cAMP) and medroxyprogesterone acetate (MPA) for 8 days to determine the capacity of decidualization, which characterizes ESCs. RNAseq was performed on D4, D6, D8, D14, D22 EBs, hiPSCs, and normal endometrial stromal cells to determine transcriptome of each stage. RESULTS: Intermediate mesoderm-specific genes LHX1 and PAX2 were significantly upregulated in D4 EBs compared to hiPSCs as revealed by qPCR (N¼9, p<0.05). mRNA expression of Mullerian duct marker ISL1 was significantly higher in D8 EBs than in hiPSCs (N¼9, p<0.05). The expression of ESC-specific transcription factors HOXA10 and HOXA11 were upregulated in day 6 EBs and maintained thereafter. PGR, the steroid receptor predominantly expressed in ESCs, was upregulated progressively during the differentiation process until D14. After 8 days of treatment with cAMP and MPA, D22 EBs expressed significantly higher mRNA levels of decidualization markers such as FOXO1, IGFBP1, and PRL (N¼9, p<0.05) compared with cells treated with vehicle control (VC). ELISA (N¼9, p<0.05) confirmed the increased protein expression of FOXO1. Principal component analysis of RNAseq depicted a seamless transition of the transcriptional profiles during differentiation. Transcriptome of D14 EBs was closer to normal endometrial stromal cells compared with D8 EBs. D22 EBs shared 1404 differentially expressed genes with normal endometrial stromal cells when compared with VC. CONCLUSIONS: We have found that EBs differentiated from hiPSCs using our chemically defined protocol have specific characteristics resembling ESCs. These results suggest that hiPSCs can be an unlimited source of ESCs. We propose these results as the first step in advancing stem cell-based therapies for uterine factor infertility or endometriosis. Supported by: National Institutes of Health grant R37-HD36891 and R03HD082558, USA; Grant-in-Aid for Scientific Research (Young Scientists B), Japan; The Uehara Memorial Foundation, Japan; The Kanzawa Medical Research Foundation, Japan ENDOMETRIOSIS P-233 Tuesday, October 31, 2017 ENDOMETRIOSIS IS ASSOCIATED WITH RISK OF OVARIAN, ENDOMETRIAL, CERVICAL AND THYROID CANCER. G. Lai,a C. Tzeng.b aSchool of Public Health, Taipei Medical University, Taipei, Taiwan; bTaipei Medical University, Taipei, Taiwan. OBJECTIVE: The relationship between cancer and endometriosis was still unclear. Many reports suggested that endometriosis associated with increased risk of gynecological cancers, but the conclusion was still inconsistent. This study aimed to unravel the association between endometriosis and cancers. DESIGN: A retrospective population-based cohort study was performed by linking to National Health Insurance Research Database (NHIRD) in Taiwan. MATERIALS AND METHODS: A total of 19,378 women with newly identified endometriosis from 2000 to 2008 and 96,890 multivariablematched controls (1:5) were selected. Sociodemographic and other potential confounding factors were selected as adjusted variables from database. They were compared between the endometriosis and control cohort using a Chisquare test. The Cox regression model adjusted for potential confounders was used to assess the risk of cancers due to endometriosis. RESULTS: After adjusted potential confounders, the adjusted hazard ratio associated with endometriosis was 1.64 (95% confidence interval (CI) 1.491.81) for overall cancers, 4.77 (95% CI 3.40-6.70) for ovarian cancer, 4.19
FERTILITY & STERILITYÒ
(95% CI 2.91-6.04) for endometrial cancer, 2.67 (95% CI 1.93-3.70) for cervical cancers, and 1.64 (95% CI 1.15-2.35) for thyroid cancers. CONCLUSIONS: Estrogen stimulation and chronic inflammation might be as putative mechanisms to endometrial cancer. Besides, estrogen receptors participate in cellular processes contributing to enhanced mitogenic, migratory, and invasive properties of thyroid cells. The investigation of association between endometriosis and cancers would advance the exploration of endometriosis etiology. Supported by: This study was partially supported by a grant from Academia Sinica (BM102020946) P-234 Tuesday, October 31, 2017 LIPOPHILIC BUT NOT HYDROPHILIC STATINS INHIBIT GROWTH AND REDUCE INVASIVENESS OF HUMAN ENDOMETRIAL STROMAL CELLS. A. Sokalska,a A. Duleba.b aDepartment of Obstetrics and Gynecology, University of Pennsylvania, Division of Reproductive Endocrinology and Infertility, Philadelphia, PA; bDepartment of Reproductive Medicine, Division of Reproductive Endocrinology and Infertility, University of California, San Diego, La Jolla, CA. OBJECTIVE: Statins (competitive inhibitors of HMG-CoA reductase) reduce endometriosis burden in murine and baboon models of this disease. On the cellular level, statins decrease proliferation, induce apoptosis, and alter the cytoskeleton of human endometrial stromal (HES) cells. This study compared effects of lipid-soluble (simvastatin, lovastatin, atorvastatin) and water-soluble (pravastatin) statins on proliferation, apoptosis and invasiveness of HES cells. DESIGN: In vitro experiments conducted in the academic laboratory. MATERIALS AND METHODS: Endometrial biopsies were collected during proliferative phase from five volunteers. HES cells were cultured in the absence or in the presence of simvastatin, lovastatin, atorvastatin and pravastatin at different concentrations. Effects on DNA synthesis, apoptosis and invasiveness were evaluated using thymidine incorporation assay, caspases 3/ 7 activity assay and invasiveness assay, respectively. Each assay was repeated at least three times using cells from different participants. Statistical analysis was performed using analysis of variance, followed by post-hoc Dunnett’s test; when appropriate, values were logarithmically transformed. Differences were considered significant at P<0.05. RESULTS: The proliferation of HES cells was significantly decreased by simvastatin (by 47-89%), lovastatin (by 46-78%), and atorvastatin (by 2148%) in a concentration-dependent manner. Activity of executioner caspases 3/7 was significantly increased by simvastatin (by 10-25%), lovastatin (by 19%), and atorvastatin (by 7-10%) in a concentration-dependent manner. The greatest effects were observed in response to simvastatin. Accounting for the effects of statins on cell number, the invasiveness of HES cells was significantly decreased in cells treated with simvastatin (by 49%), lovastatin (by 54%), and atorvastatin (by 53%). Pravastatin had minimal effects on any of the tested end-points. CONCLUSIONS: Present findings demonstrate that only lipid-soluble statins are effective in inhibition of growth and invasiveness of HES cells. These findings may be clinically relevant in treatment of endometriosis and indicate that lipid-soluble statins should be considered for future clinical trials. Supported by: NIH Grant U54 HD052668 P-235 Tuesday, October 31, 2017 IVF/ET OUTCOMES FROM AFFECED AND UNAFFECTED OVARIES WITH UNILATERAL ENDOMETRIOMAS. A. Takashima, N. Takeshita. Obstetrics and Gynegology, Toho Medical Center Sakura Hospital, Chiba, Japan. OBJECTIVE: To assess whether the quantitative and qualitative IVF outcomes in ovaries with and without a small endometrioma with high average age of the participants in the same individual. DESIGN: In this retrospective analysis, we examined 336 infertile women with unilateral endometriomas < 40 mm who had undergone IVF or ICSI from April 2011 to December 2016. Single frozen embryo transfer cycles were performed with the separate data collections for both the affected and the unaffected ovaries, allowed for the evaluation of the implantation rate. MATERIALS AND METHODS: On day 3, the serum levels of AMH, FSH, and E2 were recorded. We evaluated and compared the following for the endometrioma-containing ovary and the contralateral intact ovary: the AFC, the degree of follicular flushing, the oocytes that were retrieved, the
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