- Email: [email protected]
Antioxidant improves oocyte metabolism and spindle alignment
differences in medical co-morbidities in women on HT compared to controls, including but not limited to osteoporosis, diabetes, hypertension, hyperlipidemia, coronary artery disease, fibrocystic breast disease, endometrial polyps, colonic polyps, endometrial cancer, ovarian cancer, mammogram BIRADS (breast imaging-reporting data system) 3, 4, or 5, or breast cancer. CONCLUSIONS: Evaluation by DXA of post-menopausal women receiving HT for up to 25 years demonstrates that long-term HT has no significant impact on body composition. It is notable that there was no increased prevalence of medical co-morbidities between the treated and control groups. These findings may inform the risk-benefit ratio when considering long-term HT for post-menopausal women. Supported by: Pfizer Corporation. REPRODUCTIVE ENDOCRINOLOGY: RESEARCH 1 O-13 Monday, October 19, 2015 11:15 AM ELEVATED SERUM ANTI-MULLERIAN HORMONE (AMH) STALLS OVARIAN FOLLICLE DEVELOPMENT BY DOWNREGULATING FSH- AND LH-RECEPTORS AND L. J. Williams,a INHIBIN-B PRODUCTION. L. Detti,a S. E. Osborne,a N. M. Fletcher,b G. M. Saed.b aObstetrics and Gynecology, University of Tennessee, Memphis, Memphis, TN; bWayne State University, Detroit, MI. OBJECTIVE: In granulosa cell cultures AMH inhibits the FSH-dependent follicular growth and the cyclic selection for dominance [1-3]. Women with polycystic ovary syndrome (PCOS) have high serum AMH levels from increased production [4] which are correlated to the follicle number and AMH levels [5]. We tested the hypothesis that administration of recombinant AMH to ovarian cortex fragments would inhibit follicular development by downregulating hormone receptors’ expression. DESIGN: Pilot experimental study with ovarian cortex obtained from 3 patients. MATERIALS AND METHODS: Immediately after explant the ovarian cortex specimens were divided into 5 equal fragments. One fragment was flash-frozen (untreated) and four were incubated for 48 hours 37 C in a pH-adjusted gamete buffer media with increasing AMH concentrations of 0-5-25-50 ng/ml. After incubation, all specimens were rinsed and flashfrozen for PCR analyses, which were executed in triplicates. We utilized real time RT-PCR to determine mRNA levels for FSH-R, LH-R and Inhibin-B in ovarian cortex tissue. We performed ANOVA with Tukey post hoc tests to evaluate changes in mRNA levels among the five different fragments. A p<0.05 defined significance. RESULTS: In ovarian cortex exposed to increasing AMH concentrations, FSH-R, LH-R and Inhibin-B mRNA levels followed a bell-shaped response with an initially increased, followed by a decreased, expression (Table). ANOVA analysis and post hoc tests showed significant differences among the treatment groups (p¼0.001 for FSH-R; p<0.001 for both, LH-R and inhibin B). CONCLUSIONS: Women with PCOS have increased AMH levels and their anovulation patterns relate to their AMH levels. Our results, at least in part, can explain anovulation as due to an AMH-mediated downregulation of ovarian cortex cells’ response to FSH and LH and decreased inhibin B production. FSH-R, LH-R and Inhibin-B mRNA expression in the different treatment groups.
Treatment Untreated Media Control 5 ng AMH 25 ng AMH 50 ng AMH
FSH-R expression (pg/mcg RNA)
LH-r expression (pg/mcg RNA)
Inhibin-B expression (pg/mcg RNA)
71.4 4.0 431 31.9 514.0 32.5 231.7 4.5 129.8 18.6
4.1 0.2 34.0 1.0 45.4 2.0 16.4 1.1 11.7 0.3
23.0 1.4 246.0 31.8 414.5 26.8 112.2 2.8 113.5 17.4
References: 1. Durlinger ALL, Kramer P, Karels B, et al. Control of primordial follicle recruitment by anti-Mullerian hormone in the mouse ovary. Endocrinol 1999;140:5789-96.
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ASRM Abstracts
2. Di Clemente N, Goxe B, Remy JJ, et al. Inhibitory effect of AMH upon aromatase activity and LH receptors of granulosa cells of rat and porcine immature ovaries. Endocrine 1994;2:553-8. 3. Durlinger ALL, Visser JA, Themmen APN. Regulation of ovarian function: the role of anti-Mullerian hormone. Reprod 2002;124:601- 9. 4. Lin YH, Chiu WC, Wu CH, Tzeng CR, Hsu CS, Hsu MI. Antimullerian hormone and 380 polycystic ovary syndrome. Fertil Steril 2011;96:230-235. 5. Detti L, Jeffries H, Williams JL, Diamond MP, Uhlmann RA. Fertility biomarkers can identify metabolic risks in women with PCOS: a crosssectional study. Submitted 2015. Supported by: UTHSC Department Funds. O-14 Monday, October 19, 2015 11:30 AM FSH REGULATES IGF2 EXPRESSION IN HUMAN GRANULOSA CELLS IN AN AKT-DEPENDENT MANNER. A. M. Zamah,a S. C. Baumgarten,b S. M. Convissar,b M. A. Fierro,a N. J. Winston,a C. Stocco,b H. Scoccia.a aObstetrics and Gynecology, University of Illinois, Chicago, IL; bPhysiology and Biophysics, University of Illinois, Chicago, IL. OBJECTIVE: IGF2 is highly expressed in the granulosa cells of human dominant ovarian follicles; however, little is known about the regulation of the IGF2 gene or the interaction of IGF2 and FSH during follicle development. Our objective is to examine the mechanisms involved in the regulation of the IGF2 gene by FSH and the interplay between FSH and IGF2 during granulosa cell differentiation. DESIGN: Human cumulus granulosa cells were separated from cumulusoocyte-complexes isolated from the follicular aspirates of IVF patients and cultured in triplicate for in vitro studies. MATERIALS AND METHODS: Protein and mRNA levels of IGF2 and CYP19A1 (aromatase) were quantified using Western blot and quantitative real-time PCR. IGF2 promoter-specific activation was determined by the amplification of alternative exons by PCR. Cell proliferation was assessed after treatment with FSH and/or IGF2. Data were analyzed with student t-tests and one-way ANOVA. P<0.05 was considered significant. RESULTS: FSH significantly (p<0.05) enhanced IGF2 expression after 8 hours of treatment and at low doses (1 ng/ml). Reciprocally, IGF2 synergized with FSH to increase cell proliferation and the expression of CYP19A1. When IGF2 activity was blocked, FSH was no longer able to stimulate CYP19A1 expression. Determination of IGF2 promoter usage in human cumulus cells showed that the IGF2 gene is driven by promoters P3 and P4. However, FSH exclusively increased P3 promoter derived transcripts. Moreover, the FSH-induced stimulation of P3-driven IGF2 transcripts was blocked by co-treatment with inhibitors of the serine/threonine protein kinase AKT or IGF1R (receptor). The inhibitory effect of the IGF1R inhibitor on FSH-induced IGF2 mRNA accumulation was reversed by overexpression of a constitutively active AKT construct. CONCLUSIONS: FSH is a potent enhancer of IGF2 expression in human granulosa cells. In return, IGF2 activation of the IGF1R and AKT is required for FSH to stimulate CYP19A1 expression and proliferation of granulosa cells. These findings suggest a positive loop interaction between FSH and IGF2 that is critical for human granulosa cell proliferation and differentiation. Supported by: This work was Supported by NIH grant RO1HD057110 (CS) and NIH training grant T32HL07692 (SCB, SMC). O-15 Monday, October 19, 2015 11:45 AM ANTIOXIDANT IMPROVES OOCYTE METABOLISM AND SPINDLE ALIGNMENT. C. E. Boots, A. Boudoures, W. Zhang, A. Thompson, K. H. Moley. Obstetric & Gynecology, Washington University, St. Louis, MO. OBJECTIVE: To determine whether maternal obesity and its resulting metabolic insults to the preconceptual oocyte are reversible after supplementation with an antioxidant, co-enzyme Q10 (CoQ10). DESIGN: Randomized controlled trial, Basic science animal model. MATERIALS AND METHODS: C57Bl6 mice were randomly assigned a high fat (HF) versus control (Con) chow diet for 12 weeks. After 6 weeks on the diet, CoQ10 or placebo injections were administered for the remaining 6 weeks. In order to assess oocyte metabolism, mice were injected with PMSG (10IU) at 16 weeks of age, sacrificed, and germinal vesicle (GV) oocytes
Vol. 104, No. 3, Supplement, September 2015
were collected. We used microanalytical assays to measure the levels of ATP and citrate in single oocytes. To evaluate spindle and chromosome alignment, mice were injected with PMSG and hCG, sacrificed and ovulated meiosis II (MII) oocytes were collected. Mature oocytes were stained with tubulin and DAPI. Imaging was performed on a Leica confocal microscope and analysis performed blindly with ImageJ software. ANOVA, students t-tests, and chisquare analysis were used in the statistical analysis as appropriate. RESULTS: HF mice weighed significantly more than mice on the control diet (28g vs 21.7g, p<0.001). There was no difference in weight between mice receiving CoQ10 or placebo. Overall, ATP and citrate levels were lower in HF mice compared to controls (p<0.001). CoQ10 significantly increased ATP and citrate levels in control diet mice but did not improve ATP or citrate in the HF mice. Overall, CoQ10 improved the rate of normal spindle and chromosome alignment (92.3% vs 80.2%, p¼0.039). However, this improvement did not reach statistical significance in the subanalysis by diet. HF fed mice receiving placebo injections had the lowest proportion of normal spindles (72.2%), whereas control diet mice receiving CoQ10 injections had the highest proportion of normal spindles (94.4%). Interestingly, there was no difference in oocyte size between HF and Con diets, but the cohorts receiving CoQ10 had significantly larger oocyte diameter (75.5mm vs 82.8mm, p<0.001). CONCLUSIONS: Supplementation with CoQ10 improves oocyte metabolism and spindle and chromosome positioning. The obese maternal environment impairs oocyte metabolism, and while CoQ10 increases metabolites in controls, there was no improvement in obese mice. CoQ10 did recover spindle abnormalities in obese mice, but these results did not reach statistical significance. We propose translational and clinical studies to further investigate this mechanism as a potentially therapeutic agent to improve both fertility and pregnancy outcomes in obese women. Supported by: T32 HD040135-13 (CEB), Scientific Advisory Board of Vivere Health (CEB), American Diabetes Association (KHM). O-16 Monday, October 19, 2015 12:00 PM GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GMCSF) IS CYCLE REGULATED IN HUMAN ENDOMETRIUM AND DYSREGULATED IN WOMEN WITH ENDOMETRIOSIS. H. S. Hoff,a B. A. Lessey,b S. L. Young.c aReproductive Endocrinology and Infertility, University of North Carolina, Chapel Hill, NC; bReproductive Endocrinology and Infertility, Greenville Health System, Greenville, SC; cUNC School of Medicine, Chapel Hill, NC. OBJECTIVE: Endometrial production of both colony stimulating factor (CSF1) and GMCSF are cycle regulated with significant increases during the window of implantation and further upregulation by hCG. Both cytokines can act on the embryo to promote implantation. Women with endometriosis and otherwise unexplained infertility are postulated to commonly have diminished endometrial receptivity due to altered eutopic gene expression, but, to our knowledge, no previous study has examined expression of the CSF gene family in these patients. The aims of this study were to more completely determine the normal, cyclic regulation of the CSF gene family and its receptors in human endometrium in a control population and to compare those findings in a population of women with endometriosis and infertility. DESIGN: Laboratory assessment of prospectively obtained endometrial specimens. MATERIALS AND METHODS: RNA was isolated from endometrial samples obtained after informed consent from normal subjects (n¼28) and those with endometriosis (n¼24), randomized to cycle phase using urine LH testing. Relative expression of CSF family ligands and receptors was determined by real-time RT-PCR in both whole endometrium and separated epithelium and stroma. Differences were assessed using Mann-Whitney and Kruskall-Wallis testing. RESULTS: Expression of GMCSF was minimal in the proliferative phase and demonstrated 4.9- and 17.5-fold median increases in the mid-secretory (MS, p¼0.03) and menstrual (p¼0.03) phases of normal controls. No cycle phase specific regulation was seen in women with endometriosis. Mid-secretory expression of GMCSF iwas 4.9-fold greater in normal controls versus women with endometriosis (p¼0.03). Cycle specific regulation in isolated primary stromal cells was similar to that seen in whole tissue, but epithelial expression was not cycle regulated. CSF1 expression did not differ significantly by cycle phase or disease state. CSF3 expression appeared to increase in the MS phase (p¼0.03), but not by disease state. However, very low levels of detection preclude confident interpretation. Endometrial expression of the two GMCSF receptors, a and b, did not vary by cycle phase, but a median
FERTILITY & STERILITYÒ
1.8-fold decrease in GMCSF receptor b was seen In women with endometriosis. CONCLUSIONS: Previous studies have demonstrated GMCSF to be cycle regulated, with increased expression noted during the mid-secretory phase. These data further characterize cycle regulation by noting a maximal rise at time of menses. The observed decrease in expression of GMCSF and its high affinity b receptor during the MS phase of women with endometriosis suggests the hypothesis that lack of GMCSF signaling may be one factor contributing to failed implantation and subfertility in women with endometriosis. References: 1. Srivastava A, Sengupta J, Kriplani A, Roy KK, Ghosh D. Profiles of cytokines secreted by isolated human endometrial cells under the influence of chorionic gonadotropin during the window of embryo implantation. Reprod Biol Endocrinol. 2013 Dec 17;11:116. http://dx.doi.org/ 10.1186/1477-7827-11-116. Zhao Y1, 2. Chegini N. The Expression of Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and Receptors in Human Endometrium. Am J Reprod Immunol. 1999 Nov;42(5):303-11. Supported by: Eunice Kennedy Shriver National Institute of Child Health and Human Development R01HD067721. O-17 Monday, October 19, 2015 12:15 PM BRCA1/2 GENE MUTATIONS ARE ASSOCIATED WITH DIMINISHED PRIMORDIAL FOLLICLE DENSITY AND INCREASED OOCYTE DNA DAMAGE IN THE HUMAN OVARY. W. Lin,a S. Titus,b E. Ginsburg,a K. H. Oktay.b aBrigham & Women’s Hospital, Boston, MA; bNY Medical College, Valhalla, NY. OBJECTIVE: BRCA1 and BRCA2 (BRCA1/2) genes are members of the ATM-mediated DNA repair pathway and are essential for DNA double strand break (DSB) repair. Multiple studies associated BRCA1/2 mutations with earlier natural menopause, lower serum AMH and low response to ovarian stimulation compared to BRCA1/2 mutation negative women, suggesting a role for BRCA1/2 genes in maintaining ovarian reserve. Additional evidence showed the age-related impairment in DSB repair might be a cause of normal oocyte aging and follicular depletion. We hypothesized that the deficient DNA DSB repair functions in BRCA1/2 mutation carriers, may lead to increased DNA DSBs and premature depletion of primordial follicles. DESIGN: Controlled laboratory study. MATERIALS AND METHODS: Under IRB approval, 230 BRCA1/2 carrier females with risk-reducing oophorectomies were identified from the pathology archive of a university hospital, and 25 met the inclusion criteria (<40 years old and absence of cancer diagnosis, ovarian pathology, prior chemotherapy or radiation exposure). These were age-matched with healthy ovaries harvested from organ donor cadavers (n¼11), which served as controls.The paraffin fixed ovarian specimens were sectioned at 5mm thickness, and stained with hematoxylin and eosin to calculate the primordial follicle density, as well as with an anti-gH2AX antibody to identify DNA DSBs in these follicles. From each ovary, ten 50mm apart serial sections were blindly evaluated. Primordial follicles were counted only in the sections nuclei present to avoid double counting. The mean and standard error values were compared by Student’s t-test. RESULTS: There were 17 cases with BRCA1 and 8 with BRCA2 mutations. All ovaries from BRCA1/2 mutation carriers and organ donor cadavers were disease free. Ovaries from BRCA1/2 mutation carriers (age 24-40 years, median 37) had significantly lower mean primordial follicle density compared to the controls (112 vs 236 follicles/mm3 ovarian cortex, p<0.05). When percentage of follicles that stain for gH2AX were compared, BRCA1/2 group showed significantly increased DNA DSBs in primordial follicles (62%7% vs 22%12%, p<0.05). There was no difference in primordial follicle density (112 vs 132, p¼0.71) or gH2AX staining (63% 8% vs 61%25%, p¼0.96) between the BRCA1 and BRCA2 subgroups. CONCLUSIONS: This is the first controlled study demonstrating diminished ovarian reserve in healthy BRCA1/2 mutation ovaries. Our results support and strengthen the previous studies that ovarian reserve is diminished in BRCA1/2 mutation carriers. As a novel finding, primordial follicles from these carriers have greater tendency for incurring DSBs. These results also vouch for the role of DNA DSB repair in maintaining ovarian reserve. The reduction in primordial follicle density may be due to premature attrition induced by lethal DNA damage. In addition to furthering our understanding of ovarian aging, our data may be useful when counseling BRCA1/2 mutation carriers about fertility planning.
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Treatment Untreated Media Control 5 ng AMH 25 ng AMH 50 ng AMH
FSH-R expression (pg/mcg RNA)
LH-r expression (pg/mcg RNA)
Inhibin-B expression (pg/mcg RNA)
71.4 4.0 431 31.9 514.0 32.5 231.7 4.5 129.8 18.6
4.1 0.2 34.0 1.0 45.4 2.0 16.4 1.1 11.7 0.3
23.0 1.4 246.0 31.8 414.5 26.8 112.2 2.8 113.5 17.4
References: 1. Durlinger ALL, Kramer P, Karels B, et al. Control of primordial follicle recruitment by anti-Mullerian hormone in the mouse ovary. Endocrinol 1999;140:5789-96.
e6
ASRM Abstracts
2. Di Clemente N, Goxe B, Remy JJ, et al. Inhibitory effect of AMH upon aromatase activity and LH receptors of granulosa cells of rat and porcine immature ovaries. Endocrine 1994;2:553-8. 3. Durlinger ALL, Visser JA, Themmen APN. Regulation of ovarian function: the role of anti-Mullerian hormone. Reprod 2002;124:601- 9. 4. Lin YH, Chiu WC, Wu CH, Tzeng CR, Hsu CS, Hsu MI. Antimullerian hormone and 380 polycystic ovary syndrome. Fertil Steril 2011;96:230-235. 5. Detti L, Jeffries H, Williams JL, Diamond MP, Uhlmann RA. Fertility biomarkers can identify metabolic risks in women with PCOS: a crosssectional study. Submitted 2015. Supported by: UTHSC Department Funds. O-14 Monday, October 19, 2015 11:30 AM FSH REGULATES IGF2 EXPRESSION IN HUMAN GRANULOSA CELLS IN AN AKT-DEPENDENT MANNER. A. M. Zamah,a S. C. Baumgarten,b S. M. Convissar,b M. A. Fierro,a N. J. Winston,a C. Stocco,b H. Scoccia.a aObstetrics and Gynecology, University of Illinois, Chicago, IL; bPhysiology and Biophysics, University of Illinois, Chicago, IL. OBJECTIVE: IGF2 is highly expressed in the granulosa cells of human dominant ovarian follicles; however, little is known about the regulation of the IGF2 gene or the interaction of IGF2 and FSH during follicle development. Our objective is to examine the mechanisms involved in the regulation of the IGF2 gene by FSH and the interplay between FSH and IGF2 during granulosa cell differentiation. DESIGN: Human cumulus granulosa cells were separated from cumulusoocyte-complexes isolated from the follicular aspirates of IVF patients and cultured in triplicate for in vitro studies. MATERIALS AND METHODS: Protein and mRNA levels of IGF2 and CYP19A1 (aromatase) were quantified using Western blot and quantitative real-time PCR. IGF2 promoter-specific activation was determined by the amplification of alternative exons by PCR. Cell proliferation was assessed after treatment with FSH and/or IGF2. Data were analyzed with student t-tests and one-way ANOVA. P<0.05 was considered significant. RESULTS: FSH significantly (p<0.05) enhanced IGF2 expression after 8 hours of treatment and at low doses (1 ng/ml). Reciprocally, IGF2 synergized with FSH to increase cell proliferation and the expression of CYP19A1. When IGF2 activity was blocked, FSH was no longer able to stimulate CYP19A1 expression. Determination of IGF2 promoter usage in human cumulus cells showed that the IGF2 gene is driven by promoters P3 and P4. However, FSH exclusively increased P3 promoter derived transcripts. Moreover, the FSH-induced stimulation of P3-driven IGF2 transcripts was blocked by co-treatment with inhibitors of the serine/threonine protein kinase AKT or IGF1R (receptor). The inhibitory effect of the IGF1R inhibitor on FSH-induced IGF2 mRNA accumulation was reversed by overexpression of a constitutively active AKT construct. CONCLUSIONS: FSH is a potent enhancer of IGF2 expression in human granulosa cells. In return, IGF2 activation of the IGF1R and AKT is required for FSH to stimulate CYP19A1 expression and proliferation of granulosa cells. These findings suggest a positive loop interaction between FSH and IGF2 that is critical for human granulosa cell proliferation and differentiation. Supported by: This work was Supported by NIH grant RO1HD057110 (CS) and NIH training grant T32HL07692 (SCB, SMC). O-15 Monday, October 19, 2015 11:45 AM ANTIOXIDANT IMPROVES OOCYTE METABOLISM AND SPINDLE ALIGNMENT. C. E. Boots, A. Boudoures, W. Zhang, A. Thompson, K. H. Moley. Obstetric & Gynecology, Washington University, St. Louis, MO. OBJECTIVE: To determine whether maternal obesity and its resulting metabolic insults to the preconceptual oocyte are reversible after supplementation with an antioxidant, co-enzyme Q10 (CoQ10). DESIGN: Randomized controlled trial, Basic science animal model. MATERIALS AND METHODS: C57Bl6 mice were randomly assigned a high fat (HF) versus control (Con) chow diet for 12 weeks. After 6 weeks on the diet, CoQ10 or placebo injections were administered for the remaining 6 weeks. In order to assess oocyte metabolism, mice were injected with PMSG (10IU) at 16 weeks of age, sacrificed, and germinal vesicle (GV) oocytes
Vol. 104, No. 3, Supplement, September 2015
were collected. We used microanalytical assays to measure the levels of ATP and citrate in single oocytes. To evaluate spindle and chromosome alignment, mice were injected with PMSG and hCG, sacrificed and ovulated meiosis II (MII) oocytes were collected. Mature oocytes were stained with tubulin and DAPI. Imaging was performed on a Leica confocal microscope and analysis performed blindly with ImageJ software. ANOVA, students t-tests, and chisquare analysis were used in the statistical analysis as appropriate. RESULTS: HF mice weighed significantly more than mice on the control diet (28g vs 21.7g, p<0.001). There was no difference in weight between mice receiving CoQ10 or placebo. Overall, ATP and citrate levels were lower in HF mice compared to controls (p<0.001). CoQ10 significantly increased ATP and citrate levels in control diet mice but did not improve ATP or citrate in the HF mice. Overall, CoQ10 improved the rate of normal spindle and chromosome alignment (92.3% vs 80.2%, p¼0.039). However, this improvement did not reach statistical significance in the subanalysis by diet. HF fed mice receiving placebo injections had the lowest proportion of normal spindles (72.2%), whereas control diet mice receiving CoQ10 injections had the highest proportion of normal spindles (94.4%). Interestingly, there was no difference in oocyte size between HF and Con diets, but the cohorts receiving CoQ10 had significantly larger oocyte diameter (75.5mm vs 82.8mm, p<0.001). CONCLUSIONS: Supplementation with CoQ10 improves oocyte metabolism and spindle and chromosome positioning. The obese maternal environment impairs oocyte metabolism, and while CoQ10 increases metabolites in controls, there was no improvement in obese mice. CoQ10 did recover spindle abnormalities in obese mice, but these results did not reach statistical significance. We propose translational and clinical studies to further investigate this mechanism as a potentially therapeutic agent to improve both fertility and pregnancy outcomes in obese women. Supported by: T32 HD040135-13 (CEB), Scientific Advisory Board of Vivere Health (CEB), American Diabetes Association (KHM). O-16 Monday, October 19, 2015 12:00 PM GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GMCSF) IS CYCLE REGULATED IN HUMAN ENDOMETRIUM AND DYSREGULATED IN WOMEN WITH ENDOMETRIOSIS. H. S. Hoff,a B. A. Lessey,b S. L. Young.c aReproductive Endocrinology and Infertility, University of North Carolina, Chapel Hill, NC; bReproductive Endocrinology and Infertility, Greenville Health System, Greenville, SC; cUNC School of Medicine, Chapel Hill, NC. OBJECTIVE: Endometrial production of both colony stimulating factor (CSF1) and GMCSF are cycle regulated with significant increases during the window of implantation and further upregulation by hCG. Both cytokines can act on the embryo to promote implantation. Women with endometriosis and otherwise unexplained infertility are postulated to commonly have diminished endometrial receptivity due to altered eutopic gene expression, but, to our knowledge, no previous study has examined expression of the CSF gene family in these patients. The aims of this study were to more completely determine the normal, cyclic regulation of the CSF gene family and its receptors in human endometrium in a control population and to compare those findings in a population of women with endometriosis and infertility. DESIGN: Laboratory assessment of prospectively obtained endometrial specimens. MATERIALS AND METHODS: RNA was isolated from endometrial samples obtained after informed consent from normal subjects (n¼28) and those with endometriosis (n¼24), randomized to cycle phase using urine LH testing. Relative expression of CSF family ligands and receptors was determined by real-time RT-PCR in both whole endometrium and separated epithelium and stroma. Differences were assessed using Mann-Whitney and Kruskall-Wallis testing. RESULTS: Expression of GMCSF was minimal in the proliferative phase and demonstrated 4.9- and 17.5-fold median increases in the mid-secretory (MS, p¼0.03) and menstrual (p¼0.03) phases of normal controls. No cycle phase specific regulation was seen in women with endometriosis. Mid-secretory expression of GMCSF iwas 4.9-fold greater in normal controls versus women with endometriosis (p¼0.03). Cycle specific regulation in isolated primary stromal cells was similar to that seen in whole tissue, but epithelial expression was not cycle regulated. CSF1 expression did not differ significantly by cycle phase or disease state. CSF3 expression appeared to increase in the MS phase (p¼0.03), but not by disease state. However, very low levels of detection preclude confident interpretation. Endometrial expression of the two GMCSF receptors, a and b, did not vary by cycle phase, but a median
FERTILITY & STERILITYÒ
1.8-fold decrease in GMCSF receptor b was seen In women with endometriosis. CONCLUSIONS: Previous studies have demonstrated GMCSF to be cycle regulated, with increased expression noted during the mid-secretory phase. These data further characterize cycle regulation by noting a maximal rise at time of menses. The observed decrease in expression of GMCSF and its high affinity b receptor during the MS phase of women with endometriosis suggests the hypothesis that lack of GMCSF signaling may be one factor contributing to failed implantation and subfertility in women with endometriosis. References: 1. Srivastava A, Sengupta J, Kriplani A, Roy KK, Ghosh D. Profiles of cytokines secreted by isolated human endometrial cells under the influence of chorionic gonadotropin during the window of embryo implantation. Reprod Biol Endocrinol. 2013 Dec 17;11:116. http://dx.doi.org/ 10.1186/1477-7827-11-116. Zhao Y1, 2. Chegini N. The Expression of Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and Receptors in Human Endometrium. Am J Reprod Immunol. 1999 Nov;42(5):303-11. Supported by: Eunice Kennedy Shriver National Institute of Child Health and Human Development R01HD067721. O-17 Monday, October 19, 2015 12:15 PM BRCA1/2 GENE MUTATIONS ARE ASSOCIATED WITH DIMINISHED PRIMORDIAL FOLLICLE DENSITY AND INCREASED OOCYTE DNA DAMAGE IN THE HUMAN OVARY. W. Lin,a S. Titus,b E. Ginsburg,a K. H. Oktay.b aBrigham & Women’s Hospital, Boston, MA; bNY Medical College, Valhalla, NY. OBJECTIVE: BRCA1 and BRCA2 (BRCA1/2) genes are members of the ATM-mediated DNA repair pathway and are essential for DNA double strand break (DSB) repair. Multiple studies associated BRCA1/2 mutations with earlier natural menopause, lower serum AMH and low response to ovarian stimulation compared to BRCA1/2 mutation negative women, suggesting a role for BRCA1/2 genes in maintaining ovarian reserve. Additional evidence showed the age-related impairment in DSB repair might be a cause of normal oocyte aging and follicular depletion. We hypothesized that the deficient DNA DSB repair functions in BRCA1/2 mutation carriers, may lead to increased DNA DSBs and premature depletion of primordial follicles. DESIGN: Controlled laboratory study. MATERIALS AND METHODS: Under IRB approval, 230 BRCA1/2 carrier females with risk-reducing oophorectomies were identified from the pathology archive of a university hospital, and 25 met the inclusion criteria (<40 years old and absence of cancer diagnosis, ovarian pathology, prior chemotherapy or radiation exposure). These were age-matched with healthy ovaries harvested from organ donor cadavers (n¼11), which served as controls.The paraffin fixed ovarian specimens were sectioned at 5mm thickness, and stained with hematoxylin and eosin to calculate the primordial follicle density, as well as with an anti-gH2AX antibody to identify DNA DSBs in these follicles. From each ovary, ten 50mm apart serial sections were blindly evaluated. Primordial follicles were counted only in the sections nuclei present to avoid double counting. The mean and standard error values were compared by Student’s t-test. RESULTS: There were 17 cases with BRCA1 and 8 with BRCA2 mutations. All ovaries from BRCA1/2 mutation carriers and organ donor cadavers were disease free. Ovaries from BRCA1/2 mutation carriers (age 24-40 years, median 37) had significantly lower mean primordial follicle density compared to the controls (112 vs 236 follicles/mm3 ovarian cortex, p<0.05). When percentage of follicles that stain for gH2AX were compared, BRCA1/2 group showed significantly increased DNA DSBs in primordial follicles (62%7% vs 22%12%, p<0.05). There was no difference in primordial follicle density (112 vs 132, p¼0.71) or gH2AX staining (63% 8% vs 61%25%, p¼0.96) between the BRCA1 and BRCA2 subgroups. CONCLUSIONS: This is the first controlled study demonstrating diminished ovarian reserve in healthy BRCA1/2 mutation ovaries. Our results support and strengthen the previous studies that ovarian reserve is diminished in BRCA1/2 mutation carriers. As a novel finding, primordial follicles from these carriers have greater tendency for incurring DSBs. These results also vouch for the role of DNA DSB repair in maintaining ovarian reserve. The reduction in primordial follicle density may be due to premature attrition induced by lethal DNA damage. In addition to furthering our understanding of ovarian aging, our data may be useful when counseling BRCA1/2 mutation carriers about fertility planning.
e7