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Genetic heterogeneity of endometriosis
OBJECTIVE: To compare the quality of human ovarian tissue after cryopreservation using original vitrification and slow freezing protocols. DESIGN: Retrospective study. MATERIALS AND METHODS: Cryopreservation of ovarian samples (n¼5) was both performed by our original vitrification and slow freezing protocols in parallel. Ovarian cortical samples were obtained from consenting patients (mean age 28.01.1 years) undergoing endoscopic surgery for benign cysts. Increasing concentrations of 1,2-propanediol (PROH), ethylene glycol (EG) and raffinose were used for vitrification. PROH and raffinose were used as cryoprotectants for slow freezing. The quality of fresh and thawed ovarian tissues was evaluated by histological analysis. Measurement of DNA fragmentation within follicles and stroma cells was assessed by TUNEL assay. RESULTS: Histological analysis showed a good preservation of follicle morphology whatever the cryopreservation protocol since the percentage of intact follicles were similar after vitrification (82.23.3%) and slow freezing (80.35.3%) compared with fresh tissue (98.91.1%; P>0.05). Moreover, the follicle density was not different in ovarian tissues after both vitrification (0.60.3 follicles/mm2) and slow freezing (0.50.2 follicles/ mm2) compared with the fresh tissues (0.70.3 follicles/mm2). The percentage of follicles with DNA fragmentation was not statistically different not only in the vitrified (20.89.0%) but also in slowly frozen (31.312.6%) tissues compared with the fresh control (8.83.9%; P>0.05). No difference was found regarding the percentage of stroma cells with DNA fragmentation in vitrified (8.93.5%) and slowly frozen (3.31.6%) tissues compared with fresh tissue (4.22.1%). CONCLUSION: The quality of ovarian tissue cryopreserved by our original protocols was similar for vitrification and slow freezing in terms of preservation of morphology and nuclear integrity of follicles and stroma cells. Supported by: Doctoral grant. GENETIC COUNSELING
P-66 Tuesday, October 23, 2012 GENETIC HETEROGENEITY OF ENDOMETRIOSIS. P. Farrington. Juneau Biosciences, LLC, Salt Lake City, UT. OBJECTIVE: We have recently developed a multiple marker DNA test to predict whether a symptomatic woman has endometriosis. Many women with endometriosis suffer from severe pelvic pain and dysmenorrhea with symptoms beginning near the onset of puberty. Others remain asymptomatic through their adolescence, and endometriosis is found at the time of an evaluation for refractory infertility. In the present study, we tested whether the genetic factors underlying endometriosis differ when a when an affected women experiences infertility. DESIGN: Descriptive cohort study. MATERIALS AND METHODS: DNA samples were collected from 541 women with documented infertility and endometriosis and 2802 women with endometriosis and no history of infertility. Endometriosis was confirmed through review of surgical and pathology reports. To reduce heterogeneity due to ancestry, all subjects were Caucasian. DNA samples were tested with 73 genetic markers previously associated with endometriosis in Caucasian patients. RESULTS: Five of the 73 DNA markers showed significant differences in observed allele frequencies in the subset of patients with infertility (see Table).
rs Number
Chromosome
rs11896599 rs721865 rs10971957 rs7721313 rs11657617
2 5 9 5 17
Nearest Gene PTH2R - MAP2 DVAF12 - UBAP1 H2AFY TMEM132E - CCT6B
Odds P Value Ratio 0.028 0.021 0.041 0.010 0.025
1.3 0.8 1.2 0.6 1.3
P-65 Tuesday, October 23, 2012 SINGLE NUCLEOTIDE POLYMORPHISM (SNP) MICROARRAYS (MA) MAY BE VALUABLE IN DETECTING SMALL GENOMIC IMBALANCES AND DISTINGUISHING BALANCED FROM NORMAL EMBRYOS FOR CARRIERS OF RECIPROCAL TRANSLOCATIONS. M. D. Werner, J. Campos, E. Forman, K. Hong, N. Treff, R. Scott. Reproductive Medicine Associates of NJ, Morristown, NJ. OBJECTIVE: In most circumstances, a balanced translocation is considered genomically equivalent to normal with clinical abnormalities limited to chromosomally imbalanced gametes. As such, PGD for translocations have focused on detecting imbalances and not with distinguishing balanced from normal. In fact, small insertions or deletions (indels) may occur at or near the chromosomal breakpoints. Rarely, such alternations can impact phenotype. Even when they do not, many couples would prefer karyotypically normal embryos to avoid passing the abnormality to future generations. This study determines if MA analysis can be used to detect small indels in carriers of balanced translocations and their embryos. DESIGN: Retrospective. MATERIALS AND METHODS: Genomic DNA from balanced translocation carriers was analyzed using a 260K SNP MA at the predicted sites of the translocation breakpoints. Embryos from these patients were similarly analyzed. RESULTS: SNP MA data were analyzed from 40 balanced translocation carriers and identified two cases with small indels. The 1st was a deletion of the JAG1 locus which may produce Alagille syndrome. 4 of 17 embryos were chromosomally normal with 2 being balanced (exhibiting the pathologic microdeletion). Two normals were transferred and a healthy infant delivered. The 2nd was an insertion on chromosome 15 which has no known phenotype. Of 4 embryos classified as normal or balanced, 3 were balanced due to the presence of the insertion and 1 was normal. The latter of these embryos was transferred and delivered. CONCLUSION: SNP MA analysis has been validated for screening embryos for translocations, but distinguishing normal from balanced has been difficult. This study demonstrates that it may be feasible to detect small indels which could distinguish normal and prioritize them for transfer. Ongoing research will determine if higher resolution MA or next-gen sequencing will increase sensitivity to these genetic imbalances allowing routine distinction of balanced from normal embryos.
FERTILITY & STERILITYÒ
CONCLUSION: Endometriosis is a polygenic multifactorial condition. The particular genes involved in any individual patient are likely to contribute to differences in clinical presentation. Discovering the genetic pathways involved may help in the effort to understand the pathophysiology and to ‘‘personalize’’ future treatments. Supported by: Juneau Biosciences. P-67 Tuesday, October 23, 2012 FMR1 GENE ALLELES IN INFERTILE WOMEN WITH PRIMARY OVARIAN INSUFFICIENCY, DIMINISHED OVARIAN RESERVE AND POOR RESPONSE TO OVARIAN STIMULATION. I. Streuli, S. Bouba, V. Gayet, C. Chapron, G. Viot, D. de Ziegler. Department of Obstetrics, Gynecology and Reproductive Medicine, APHP CHU Cochin, Universite Paris Descartes, Paris Sorbonne Cite, Paris, France. OBJECTIVE: Expansions in the number of CGG repeats in the fragile X mental retardation 1 (FMR1) gene are associated with increased mRNA levels that exert a toxic effect on ovarian follicles. Our aim is to determine whether expansions of the FMR1 gene between 35 – 54 repeats are more frequent in infertile women with an alteration of the ovarian function (primary ovarian insufficiency (POI), diminished ovarian reserve (DOR) and poor response (PR) to controlled ovarian stimulation (COS). DESIGN: Observational study. MATERIALS AND METHODS: We analyzed the files of 101 infertile women referred from the Infertility Unit to the genetic Unit of the Cochin Hospital for FMR1 gene testing and karyotpe between 01/08 and 08/11. We classified 85 women in whom FMR1 testing was performed in 5 groups according to the motive for genetic testing: 1) POI (FSH>11 IU/L with amenorrhea/irregular cycles) 2) DOR (regular cycles and FSH>11 UI/L, AMH<1ng/ml and/or antral follicle count<6), 3) PR (cancellation during COS or<6 follicles retrieved), 4) PR associated with DOR 5) family history only. RESULTS: Ten of the 85 (11.8%) women with an FMR1 testing had alleles between 35 - 54 repeats, and two women (2.4%) had alleles in the premutation range.
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P-66 Tuesday, October 23, 2012 GENETIC HETEROGENEITY OF ENDOMETRIOSIS. P. Farrington. Juneau Biosciences, LLC, Salt Lake City, UT. OBJECTIVE: We have recently developed a multiple marker DNA test to predict whether a symptomatic woman has endometriosis. Many women with endometriosis suffer from severe pelvic pain and dysmenorrhea with symptoms beginning near the onset of puberty. Others remain asymptomatic through their adolescence, and endometriosis is found at the time of an evaluation for refractory infertility. In the present study, we tested whether the genetic factors underlying endometriosis differ when a when an affected women experiences infertility. DESIGN: Descriptive cohort study. MATERIALS AND METHODS: DNA samples were collected from 541 women with documented infertility and endometriosis and 2802 women with endometriosis and no history of infertility. Endometriosis was confirmed through review of surgical and pathology reports. To reduce heterogeneity due to ancestry, all subjects were Caucasian. DNA samples were tested with 73 genetic markers previously associated with endometriosis in Caucasian patients. RESULTS: Five of the 73 DNA markers showed significant differences in observed allele frequencies in the subset of patients with infertility (see Table).
rs Number
Chromosome
rs11896599 rs721865 rs10971957 rs7721313 rs11657617
2 5 9 5 17
Nearest Gene PTH2R - MAP2 DVAF12 - UBAP1 H2AFY TMEM132E - CCT6B
Odds P Value Ratio 0.028 0.021 0.041 0.010 0.025
1.3 0.8 1.2 0.6 1.3
P-65 Tuesday, October 23, 2012 SINGLE NUCLEOTIDE POLYMORPHISM (SNP) MICROARRAYS (MA) MAY BE VALUABLE IN DETECTING SMALL GENOMIC IMBALANCES AND DISTINGUISHING BALANCED FROM NORMAL EMBRYOS FOR CARRIERS OF RECIPROCAL TRANSLOCATIONS. M. D. Werner, J. Campos, E. Forman, K. Hong, N. Treff, R. Scott. Reproductive Medicine Associates of NJ, Morristown, NJ. OBJECTIVE: In most circumstances, a balanced translocation is considered genomically equivalent to normal with clinical abnormalities limited to chromosomally imbalanced gametes. As such, PGD for translocations have focused on detecting imbalances and not with distinguishing balanced from normal. In fact, small insertions or deletions (indels) may occur at or near the chromosomal breakpoints. Rarely, such alternations can impact phenotype. Even when they do not, many couples would prefer karyotypically normal embryos to avoid passing the abnormality to future generations. This study determines if MA analysis can be used to detect small indels in carriers of balanced translocations and their embryos. DESIGN: Retrospective. MATERIALS AND METHODS: Genomic DNA from balanced translocation carriers was analyzed using a 260K SNP MA at the predicted sites of the translocation breakpoints. Embryos from these patients were similarly analyzed. RESULTS: SNP MA data were analyzed from 40 balanced translocation carriers and identified two cases with small indels. The 1st was a deletion of the JAG1 locus which may produce Alagille syndrome. 4 of 17 embryos were chromosomally normal with 2 being balanced (exhibiting the pathologic microdeletion). Two normals were transferred and a healthy infant delivered. The 2nd was an insertion on chromosome 15 which has no known phenotype. Of 4 embryos classified as normal or balanced, 3 were balanced due to the presence of the insertion and 1 was normal. The latter of these embryos was transferred and delivered. CONCLUSION: SNP MA analysis has been validated for screening embryos for translocations, but distinguishing normal from balanced has been difficult. This study demonstrates that it may be feasible to detect small indels which could distinguish normal and prioritize them for transfer. Ongoing research will determine if higher resolution MA or next-gen sequencing will increase sensitivity to these genetic imbalances allowing routine distinction of balanced from normal embryos.
FERTILITY & STERILITYÒ
CONCLUSION: Endometriosis is a polygenic multifactorial condition. The particular genes involved in any individual patient are likely to contribute to differences in clinical presentation. Discovering the genetic pathways involved may help in the effort to understand the pathophysiology and to ‘‘personalize’’ future treatments. Supported by: Juneau Biosciences. P-67 Tuesday, October 23, 2012 FMR1 GENE ALLELES IN INFERTILE WOMEN WITH PRIMARY OVARIAN INSUFFICIENCY, DIMINISHED OVARIAN RESERVE AND POOR RESPONSE TO OVARIAN STIMULATION. I. Streuli, S. Bouba, V. Gayet, C. Chapron, G. Viot, D. de Ziegler. Department of Obstetrics, Gynecology and Reproductive Medicine, APHP CHU Cochin, Universite Paris Descartes, Paris Sorbonne Cite, Paris, France. OBJECTIVE: Expansions in the number of CGG repeats in the fragile X mental retardation 1 (FMR1) gene are associated with increased mRNA levels that exert a toxic effect on ovarian follicles. Our aim is to determine whether expansions of the FMR1 gene between 35 – 54 repeats are more frequent in infertile women with an alteration of the ovarian function (primary ovarian insufficiency (POI), diminished ovarian reserve (DOR) and poor response (PR) to controlled ovarian stimulation (COS). DESIGN: Observational study. MATERIALS AND METHODS: We analyzed the files of 101 infertile women referred from the Infertility Unit to the genetic Unit of the Cochin Hospital for FMR1 gene testing and karyotpe between 01/08 and 08/11. We classified 85 women in whom FMR1 testing was performed in 5 groups according to the motive for genetic testing: 1) POI (FSH>11 IU/L with amenorrhea/irregular cycles) 2) DOR (regular cycles and FSH>11 UI/L, AMH<1ng/ml and/or antral follicle count<6), 3) PR (cancellation during COS or<6 follicles retrieved), 4) PR associated with DOR 5) family history only. RESULTS: Ten of the 85 (11.8%) women with an FMR1 testing had alleles between 35 - 54 repeats, and two women (2.4%) had alleles in the premutation range.
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