- Email: [email protected]
Infertile men with non-obstructive azoospermia exhibit defects in the DNA mismatch repair pathway
O-15 Monday, October 20, 2014 06:00 PM GNRH AGONIST WITH LOW-DOSE HCG CO-TRIGGER IS ASSOCIATED WITH HIGHER RISK OF SEVERE OHSS THAN GNRH AGONIST ALONE. K. O’Neill, S. Senapati, I. Maina, C. Gracia, A. Dokras. Department of Obstetrics & Gynecology, University of Pennsylvania, Philadelphia, PA. OBJECTIVE: Use of a gonadotropin-releasing hormone (GnRH) antagonist protocol with GnRH agonist (GnRHa) trigger for final oocyte maturation is the most effective strategy to reduce the rate of ovarian hyperstimulation syndrome (OHSS) during IVF. However, concerns persist regarding GnRHa trigger and ART outcomes. Use of a combination GnRHa and low-dose human chorionic gonadotropin (hCG) co-trigger has been reported to reduce OHSS and preserve IVF outcomes. The objective of this study was to examine the impact of final oocyte maturation method (GnRHa v co-trigger) on OHSS, oocyte yield and maturity. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Autologous IVF cycles using a GnRH antagonist protocol at Penn Fertility Care from 1/2008 through 4/2014 with either GnRHa alone or GnRHa and low-dose hCG for oocyte maturation were included. Primary outcome was OHSS by Golan criteria; secondary outcomes were total oocytes retrieved and oocyte maturity. Logistic, linear and Poisson regression models were used to compare outcomes by trigger group and predict OHSS, adjusting for confounders. RESULTS: Demographics and ovarian reserve measures were similar in the groups. After adjusting for age, AFC and PCOS diagnosis, more mature oocytes were retrieved after co-trigger v GnRHa alone (RR 1.18 [CI 1.11.27], p<0.01 and 1.14 [CI 1.02-1.29], p¼0.03). Early OHSS was more common after co-trigger (8.6% v 0%, p<0.01); with the majority (4/6, 67%) developing severe OHSS. Age<35, BMI and AFC were predictive of OHSS. Clinical pregnancy rate after day 5 transfer did not differ. IVF Cycle Characteristics & Outcomes By Trigger Group
Co-Trigger (n¼71)
GnRHa Trigger (n¼106)
p value
Age* 34 (32-37) 32 (30-35) 0.02 Antral Follicle 18 (12-25) 23 (15-31) <0.01 Count (AFC)* Anti-Mullerian 3.2 (1.7-5.0) 4 (2.5-6.5) 0.04 Hormone Level [ng/mL]* PCOS(%) 13 (16) 41 (38) 0.05 History of OHSS(%) 4 (44) 6 (14) 0.04 Total Gonadotropin 2650 (1850-3700) 2350(1800-2963) 0.10 Use [IU]* Max E2 [pg/mL]* 3542 (2936-5014) 4333 (3501-5653) <0.01 Total Follicles* 27.5 (21-35) 31 (25-40) 0.01 Oocytes Retrieved* 18 (12-24) 17 (11-22) 0.22 % Maturity* 82 (73-91) 68 (55-86) 0.02 Any OHSS(%) 6 (8.6) 0 <0.01 Severe OHSS(%) 4 (6) 0 0.02 Freeze All for 4 (9.3) 1 (1) 0.03 OHSS(%) *Median(Interquartile Range), Mann-Whitney U test. Chi-square test CONCLUSION: Although use of a co-trigger is associated with greater oocyte yield and maturity compared to GnRHa trigger alone, it is accompanied by an elevated risk of severe OHSS. Predictors of OHSS were limited and therefore co-trigger should be used with caution. Supported by: T32 PD10032525 (KO), 5K12HD001265-14 (SS). SOCIETY FOR MALE REPRODUCTION AND UROLOGY TRAVELING SCHOLARS O-16 Monday, October 20, 2014 04:15 PM INFERTILE MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA EXHIBIT DEFECTS IN THE DNA MISMATCH REPAIR L. Gomez,a,b R. Ramasamy,a,c PATHWAY. A. D. Ridgeway,a,b L. Lipshultz,c D. Lamb.a,b,c,d aMolecular and Cellular Biology, Baylor Col-
FERTILITY & STERILITYÒ
lege of Medicine, Houston, TX; bCenter for Reproductive Medicine, Baylor College of Medicine, Houston, TX; cScott Department of Urology, Baylor College of Medicine, Houston, TX; dLester and Sue Smith Chair Urologic Research, Baylor College of Medicine, Houston, TX. OBJECTIVE: To identify MMR deficiencies and define the role of DNA methylation on MMR gene expression in NOA patients. DESIGN: We examined the global DNA methylation status of NOA patients and fertile controls using the Infinium HumanMethylation450 microarray. DNA was isolated from fibroblasts cultured from from the testicular biopsy of NOA patients (n¼21) and fertile controls (n¼5). MATERIALS AND METHODS: Genes with the most significant changes in DNA methylation (p<0.05) were identified in NOA patients vs. controls using the R statistical suite. Bisulfite clonal sequencing validated the microarray results and provided a platform to screen additional men (10 NOA and 15 controls). MMR gene expression was quantified using qPCR, immunofluorescence and Western blot. The functional consequences of MMR defects were tested using a cell proliferation assay of growth after exposure to the DNA alkylating agent N-Nitroso-N-methylurea (MNU) or the radiomimetic drug Neocarzinostatin (Neo). RESULTS: DNA methylation was increased in a cohort of NOA patients (6/31) at a meiosis-associated gene in the MMR family - MSH5. MSH5 is implicated in the repair of double-stranded breaks (DSB) and in the resolution of the holiday junction during meiosis. The NOA cohort also exhibited down-regulation of MSH5 gene and reduced MSH5 protein expression. Cells with methylated MSH5 ceased to proliferate in response to Neo and did not up-regulate MSH5 expression. This was in contrast to control cells that had a 3-fold increase after Neo treatment. Defects in MMR genes that did not display abnormal patterns of DNA methylation (potentially due to genetic causes) were defined using the Sn1-type alkylating agent MNU. A subset of testicular fibroblasts from 30 NOA men were resistant to the cytotoxic effects of MNU. This finding was in contrast to fertile control cells (n¼10) that were sensitive to MNU treatment. CONCLUSION: A cohort of NOA men with defects in an important DNA repair pathway for spermatogenesis -MMR were identified. Defects included the deleterious DNA methylation of MSH5 and an abnormal response of NOA fibroblasts to cytotoxic drugs. These defects may affect genomic stability and DNA recombination in germ cells and contribute to impaired spermatogenesis in NOA. Supported by: Funding Supported by a Male Reproductive Health Research Career (MHRH) Development Physician Scientist Award (K12) (HD073917-01) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program and by NIH grants P01HD36289 from the Eunice Kennedy Shriver NICHD and 1R01DK078121 from the National Institute of Kidney and Digestive Diseases.
O-17 Monday, October 20, 2014 04:30 PM A SPERM-DERIVED WW DOMAIN-BINDING PROTEIN INITIATES ZYGOTIC DEVELOPMENT IN HUMAN AND MOUSE AND DETERMINES MALE-FACTOR INFERTILITY. M. Aarabi,a,b H. Balakier,c S. Bashar,c S. I. Moskovtsev,c,d P. Sutovsky,e,f C. L. Librach,c,d R. Oko.a aDepartment of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada; bDepartment of Human Genetics, McGill University, Montreal, QC, Canada; cCReATe Fertility Centre, Toronto, ON, Canada; dDepartment of Obstetrics & Gynaecology, University of Toronto, Toronto, ON, Canada; eDepartment of Animal Sciences, University of Missouri, Columbia, MO; fDepartments of Obstetrics, Gynecology & Women’s Health, University of Missouri, Columbia, MO. OBJECTIVE: To examine whether sperm-derived postacrosomal WWbinding protein (PAWP) is required for initiation of calcium oscillations and zygotic development after human and mouse fertilization; to determine PAWP levels in the spermatozoa of men undergoing intracytoplasmic sperm injection (ICSI) and to correlate them with infertility treatment outcomes. DESIGN: Prospective clinical and laboratory study. MATERIALS AND METHODS: Following Research Ethics Board approvals, recombinant human PAWP protein or complementary RNA (cRNA) was injected into mouse and human oocytes. PAWP-induced calcium oscillations were measured and compared to sperm–induced oscillations in a time-lapse trial. Competitive peptides derived from the PAWP sequence were co-injected with sperm/PAWP for inhibition trials. Surplus sperm after use for ICSI cycle was collected from 110 men to determine the levels of PAWP by flow cytometry and its correlation to treatment outcomes.
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Co-Trigger (n¼71)
GnRHa Trigger (n¼106)
p value
Age* 34 (32-37) 32 (30-35) 0.02 Antral Follicle 18 (12-25) 23 (15-31) <0.01 Count (AFC)* Anti-Mullerian 3.2 (1.7-5.0) 4 (2.5-6.5) 0.04 Hormone Level [ng/mL]* PCOS(%) 13 (16) 41 (38) 0.05 History of OHSS(%) 4 (44) 6 (14) 0.04 Total Gonadotropin 2650 (1850-3700) 2350(1800-2963) 0.10 Use [IU]* Max E2 [pg/mL]* 3542 (2936-5014) 4333 (3501-5653) <0.01 Total Follicles* 27.5 (21-35) 31 (25-40) 0.01 Oocytes Retrieved* 18 (12-24) 17 (11-22) 0.22 % Maturity* 82 (73-91) 68 (55-86) 0.02 Any OHSS(%) 6 (8.6) 0 <0.01 Severe OHSS(%) 4 (6) 0 0.02 Freeze All for 4 (9.3) 1 (1) 0.03 OHSS(%) *Median(Interquartile Range), Mann-Whitney U test. Chi-square test CONCLUSION: Although use of a co-trigger is associated with greater oocyte yield and maturity compared to GnRHa trigger alone, it is accompanied by an elevated risk of severe OHSS. Predictors of OHSS were limited and therefore co-trigger should be used with caution. Supported by: T32 PD10032525 (KO), 5K12HD001265-14 (SS). SOCIETY FOR MALE REPRODUCTION AND UROLOGY TRAVELING SCHOLARS O-16 Monday, October 20, 2014 04:15 PM INFERTILE MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA EXHIBIT DEFECTS IN THE DNA MISMATCH REPAIR L. Gomez,a,b R. Ramasamy,a,c PATHWAY. A. D. Ridgeway,a,b L. Lipshultz,c D. Lamb.a,b,c,d aMolecular and Cellular Biology, Baylor Col-
FERTILITY & STERILITYÒ
lege of Medicine, Houston, TX; bCenter for Reproductive Medicine, Baylor College of Medicine, Houston, TX; cScott Department of Urology, Baylor College of Medicine, Houston, TX; dLester and Sue Smith Chair Urologic Research, Baylor College of Medicine, Houston, TX. OBJECTIVE: To identify MMR deficiencies and define the role of DNA methylation on MMR gene expression in NOA patients. DESIGN: We examined the global DNA methylation status of NOA patients and fertile controls using the Infinium HumanMethylation450 microarray. DNA was isolated from fibroblasts cultured from from the testicular biopsy of NOA patients (n¼21) and fertile controls (n¼5). MATERIALS AND METHODS: Genes with the most significant changes in DNA methylation (p<0.05) were identified in NOA patients vs. controls using the R statistical suite. Bisulfite clonal sequencing validated the microarray results and provided a platform to screen additional men (10 NOA and 15 controls). MMR gene expression was quantified using qPCR, immunofluorescence and Western blot. The functional consequences of MMR defects were tested using a cell proliferation assay of growth after exposure to the DNA alkylating agent N-Nitroso-N-methylurea (MNU) or the radiomimetic drug Neocarzinostatin (Neo). RESULTS: DNA methylation was increased in a cohort of NOA patients (6/31) at a meiosis-associated gene in the MMR family - MSH5. MSH5 is implicated in the repair of double-stranded breaks (DSB) and in the resolution of the holiday junction during meiosis. The NOA cohort also exhibited down-regulation of MSH5 gene and reduced MSH5 protein expression. Cells with methylated MSH5 ceased to proliferate in response to Neo and did not up-regulate MSH5 expression. This was in contrast to control cells that had a 3-fold increase after Neo treatment. Defects in MMR genes that did not display abnormal patterns of DNA methylation (potentially due to genetic causes) were defined using the Sn1-type alkylating agent MNU. A subset of testicular fibroblasts from 30 NOA men were resistant to the cytotoxic effects of MNU. This finding was in contrast to fertile control cells (n¼10) that were sensitive to MNU treatment. CONCLUSION: A cohort of NOA men with defects in an important DNA repair pathway for spermatogenesis -MMR were identified. Defects included the deleterious DNA methylation of MSH5 and an abnormal response of NOA fibroblasts to cytotoxic drugs. These defects may affect genomic stability and DNA recombination in germ cells and contribute to impaired spermatogenesis in NOA. Supported by: Funding Supported by a Male Reproductive Health Research Career (MHRH) Development Physician Scientist Award (K12) (HD073917-01) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program and by NIH grants P01HD36289 from the Eunice Kennedy Shriver NICHD and 1R01DK078121 from the National Institute of Kidney and Digestive Diseases.
O-17 Monday, October 20, 2014 04:30 PM A SPERM-DERIVED WW DOMAIN-BINDING PROTEIN INITIATES ZYGOTIC DEVELOPMENT IN HUMAN AND MOUSE AND DETERMINES MALE-FACTOR INFERTILITY. M. Aarabi,a,b H. Balakier,c S. Bashar,c S. I. Moskovtsev,c,d P. Sutovsky,e,f C. L. Librach,c,d R. Oko.a aDepartment of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada; bDepartment of Human Genetics, McGill University, Montreal, QC, Canada; cCReATe Fertility Centre, Toronto, ON, Canada; dDepartment of Obstetrics & Gynaecology, University of Toronto, Toronto, ON, Canada; eDepartment of Animal Sciences, University of Missouri, Columbia, MO; fDepartments of Obstetrics, Gynecology & Women’s Health, University of Missouri, Columbia, MO. OBJECTIVE: To examine whether sperm-derived postacrosomal WWbinding protein (PAWP) is required for initiation of calcium oscillations and zygotic development after human and mouse fertilization; to determine PAWP levels in the spermatozoa of men undergoing intracytoplasmic sperm injection (ICSI) and to correlate them with infertility treatment outcomes. DESIGN: Prospective clinical and laboratory study. MATERIALS AND METHODS: Following Research Ethics Board approvals, recombinant human PAWP protein or complementary RNA (cRNA) was injected into mouse and human oocytes. PAWP-induced calcium oscillations were measured and compared to sperm–induced oscillations in a time-lapse trial. Competitive peptides derived from the PAWP sequence were co-injected with sperm/PAWP for inhibition trials. Surplus sperm after use for ICSI cycle was collected from 110 men to determine the levels of PAWP by flow cytometry and its correlation to treatment outcomes.
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