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MP68-09 EULERIAN VIDEO MAGNIFICATION: A NOVEL TECHNIQUE FOR IMPROVED SPERM SELECTION IN MEN WITH SEVERE OLIGOASTHENOSPERMIA
THE JOURNAL OF UROLOGYâ
Vol. 191, No. 4S, Supplement, Tuesday, May 20, 2014
they present for sperm cryopreservation. Testosterone, which plays an essential role in spermatogenesis, also drives energy, mood, bone health, and overall sense of well-being, Each of these important clinical parameters can also be diminished in the setting of oncologic diagnosis. The objective of this study is to investigate the baseline androgen levels in newly diagnosed male cancer patients presenting for fertility preservation. METHODS: We performed an IRB-approved, single center, retrospective study at a tertiary care center by querying the institutional database for all male patients with a new oncologic diagnosis presenting for a fertility preservation consult. Demographic information, oncologic diagnosis, and serum LH and testosterone levels were queried. Hypogonadism was defined a serum total testosterone level < 300ng/dL. RESULTS: Our search identified 357 male patients with a new oncologic diagnosis presenting for fertility preservation consultation. The mean age of the population was 32.2 (range 18-55, median age 31). The predominant malignancies identified included 139 patients with lymphoproliferative malignancies (38.9%), 109 patients with testicular malignancies (30.5%), 22 patients with prostate malignancies (15.8%), 22 patients with gastrointestinal malignancies (15.8%), and 18 patients with brain malignancies (5.0%). Of the 357 patients, 198 (55%) had a serum testosterone level measured and 97 (27%) had a serum LH level measured. Of the 198 patients with a testosterone level measured, 95 (48%) were hypogonadal. Of the 97 patients with an LH level measured, 4 (4%) had elevated levels. There were no statistically significant differences in rates of hypogonadism amongst the cancer subtypes. CONCLUSIONS: Our study suggests that hypogonadism is highly prevalent in males presenting for fertility preservation consultation. Given the high rates of hypogonadism among all cancer types and the negative effects of low testosterone on men’s health and well being, physicians should consider assessing the androgen status of men newly diagnosed with cancer. Source of Funding: none
MP68-08 HUSP26 EXPRESSION IN HUMAN TESTIS AND RELATIONSHIP TO SPERM RETRIEVAL OUTCOME Matthew Wosnitzer*, Anna Mielnik, Ali Dabaja, Brian Robinson, Peter Schlegel, Darius Paduch, New York, NY INTRODUCTION AND OBJECTIVES: Human ubiquitin specific protease 26 (hUSP26), an X-linked gene, has been identified by in vitro, mouse and human SNP analysis studies to be associated with male infertility and low testosterone production. The aims of this study were to establish hUSP26 expression in human testis tissue, to characterize differences in hUSP26 and androgen receptor (AR, given known interaction) according to testicular histology, and to identify relationship to sperm retrieval outcome. METHODS: Testicular specimens from 15 patients with severely impaired sperm production (n¼13) or normal production (n¼2) were used for RNA extraction, cDNA synthesis, and quantitative realtime PCR (qRT-PCR). hUSP26 mRNA levels were measured using dual-color multiplex qRT-PCR with Roche LightCycler 480. TATAbinding protein (TBP) served as the housekeeping gene for relative quantification experiments. hUSP26/TBP and AR/TBP expression ratios were calculated using LightCycler 480 software (Roche Diagnostics) for relative quantification and corrected based on standard curves. Most testicular samples were obtained during microdissection TESE that was performed as previously described. Preoperative physical examination and serum lab values were collected. RESULTS: hUSP26 expression was verified by qRT-PCR. Specifically, hUSP26 expression was seen to be significantly increased in early maturation arrest testis with sequentially decreased expression in hypospermatogenesis and normal testis (p<0.03). No expression
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was identified in patients with complete Sertoli cell-only (SCO) on testicular histology. Further hUSP26 expression was found to have a negative trend with preoperative serum FSH (p<0.07). No association was identified with serum total testosterone, LH, estradiol, prolactin, CBC, BMI or testicular volume. An ROC curve model utilizing both hUSP26 and AR mRNA expression significantly predicted microdissection TESE sperm retrieval outcome (AUC 0.86, p<0.03). CONCLUSIONS: hUSP26 is expressed in testis, most highly during early maturation arrest and least in SCO, supporting the action of hUSP26 early during spermatogenesis in humans. Quantification of hUSP26 and AR mRNA expression may assist in predicting microdissection TESE outcomes. Further study of cellular localization, protein interaction, and predictive ability requires investigation with additional specimens. Source of Funding: Ferdinand Valentine Research Fellowship in Urology- NY Academy of Medicine
MP68-09 EULERIAN VIDEO MAGNIFICATION: A NOVEL TECHNIQUE FOR IMPROVED SPERM SELECTION IN MEN WITH SEVERE OLIGOASTHENOSPERMIA Gavin Langille*, James Dupree, Jason Kovac, Ranjith Ramasamy, Dolores Lamb, Larry Lipshultz, Houston, TX INTRODUCTION AND OBJECTIVES: The absence of sufficient motile sperm in samples used for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) can present a serious clinical problem for infertility specialists wishing to select viable sperm. Identification of optimal quality sperm can be especially challenging in men with oligoasthenospermia. Eulerian Video Magnification (EVM) is a technique that provides an opportunity to detect subtle movements in video feeds not appreciable to the unaided human eye. EVM is a computational algorithm that uses spatial and temporal variations present in video sources to enhance, in a spatially-multiscale manner, the variation of individual pixel values over time. In patients whose semen samples demonstrate significantly impaired motility by traditional light microscopy, EVM could be used to select viable motile sperm that would have been otherwise identified as non-motile using traditional light microscopic visualization. METHODS: Using traditional light microscopy with 400x magnification, semen analysis (SA) samples from men demonstrating severe oligoasthenospermia (density < 1 million/ml and motility < 20%) were investigated. A high definition (HD) video camera was used to record video footage from three high-powered fields (HPF) per SA. Each video recording was scored twice for motility, first using the raw HD footage, and second after the video was processed using EVM previously developed at the Massachusetts Institute of Technology (http://people.csail.mit.edu/mrub/vidmag). Quantitation of motile sperm in each video recording was compared between the raw and EVMenhanced images. RESULTS: Video from eleven SA samples with three recordings each were analyzed. A total of 442 sperm were recorded across all samples. After processing with EVM, the number of additionally recognized motile/viable sperm increased by 14% (p<0.05). CONCLUSIONS: EVM is a novel technique to identify viable sperm that would not have been recognized using traditional light microscopy. Using a micro-locator and micro-pipette, these sperm can then be retrieved for IVF-ICSI. Our data suggests that an additional 14% of retrieved ova can then be inseminated. Further studies are underway to refine this technique and to test the efficacy of the additional EVMidentified sperm in improving IVF-ICSI outcomes for the oligoasthenospermic patient. Source of Funding: None
Vol. 191, No. 4S, Supplement, Tuesday, May 20, 2014
they present for sperm cryopreservation. Testosterone, which plays an essential role in spermatogenesis, also drives energy, mood, bone health, and overall sense of well-being, Each of these important clinical parameters can also be diminished in the setting of oncologic diagnosis. The objective of this study is to investigate the baseline androgen levels in newly diagnosed male cancer patients presenting for fertility preservation. METHODS: We performed an IRB-approved, single center, retrospective study at a tertiary care center by querying the institutional database for all male patients with a new oncologic diagnosis presenting for a fertility preservation consult. Demographic information, oncologic diagnosis, and serum LH and testosterone levels were queried. Hypogonadism was defined a serum total testosterone level < 300ng/dL. RESULTS: Our search identified 357 male patients with a new oncologic diagnosis presenting for fertility preservation consultation. The mean age of the population was 32.2 (range 18-55, median age 31). The predominant malignancies identified included 139 patients with lymphoproliferative malignancies (38.9%), 109 patients with testicular malignancies (30.5%), 22 patients with prostate malignancies (15.8%), 22 patients with gastrointestinal malignancies (15.8%), and 18 patients with brain malignancies (5.0%). Of the 357 patients, 198 (55%) had a serum testosterone level measured and 97 (27%) had a serum LH level measured. Of the 198 patients with a testosterone level measured, 95 (48%) were hypogonadal. Of the 97 patients with an LH level measured, 4 (4%) had elevated levels. There were no statistically significant differences in rates of hypogonadism amongst the cancer subtypes. CONCLUSIONS: Our study suggests that hypogonadism is highly prevalent in males presenting for fertility preservation consultation. Given the high rates of hypogonadism among all cancer types and the negative effects of low testosterone on men’s health and well being, physicians should consider assessing the androgen status of men newly diagnosed with cancer. Source of Funding: none
MP68-08 HUSP26 EXPRESSION IN HUMAN TESTIS AND RELATIONSHIP TO SPERM RETRIEVAL OUTCOME Matthew Wosnitzer*, Anna Mielnik, Ali Dabaja, Brian Robinson, Peter Schlegel, Darius Paduch, New York, NY INTRODUCTION AND OBJECTIVES: Human ubiquitin specific protease 26 (hUSP26), an X-linked gene, has been identified by in vitro, mouse and human SNP analysis studies to be associated with male infertility and low testosterone production. The aims of this study were to establish hUSP26 expression in human testis tissue, to characterize differences in hUSP26 and androgen receptor (AR, given known interaction) according to testicular histology, and to identify relationship to sperm retrieval outcome. METHODS: Testicular specimens from 15 patients with severely impaired sperm production (n¼13) or normal production (n¼2) were used for RNA extraction, cDNA synthesis, and quantitative realtime PCR (qRT-PCR). hUSP26 mRNA levels were measured using dual-color multiplex qRT-PCR with Roche LightCycler 480. TATAbinding protein (TBP) served as the housekeeping gene for relative quantification experiments. hUSP26/TBP and AR/TBP expression ratios were calculated using LightCycler 480 software (Roche Diagnostics) for relative quantification and corrected based on standard curves. Most testicular samples were obtained during microdissection TESE that was performed as previously described. Preoperative physical examination and serum lab values were collected. RESULTS: hUSP26 expression was verified by qRT-PCR. Specifically, hUSP26 expression was seen to be significantly increased in early maturation arrest testis with sequentially decreased expression in hypospermatogenesis and normal testis (p<0.03). No expression
e801
was identified in patients with complete Sertoli cell-only (SCO) on testicular histology. Further hUSP26 expression was found to have a negative trend with preoperative serum FSH (p<0.07). No association was identified with serum total testosterone, LH, estradiol, prolactin, CBC, BMI or testicular volume. An ROC curve model utilizing both hUSP26 and AR mRNA expression significantly predicted microdissection TESE sperm retrieval outcome (AUC 0.86, p<0.03). CONCLUSIONS: hUSP26 is expressed in testis, most highly during early maturation arrest and least in SCO, supporting the action of hUSP26 early during spermatogenesis in humans. Quantification of hUSP26 and AR mRNA expression may assist in predicting microdissection TESE outcomes. Further study of cellular localization, protein interaction, and predictive ability requires investigation with additional specimens. Source of Funding: Ferdinand Valentine Research Fellowship in Urology- NY Academy of Medicine
MP68-09 EULERIAN VIDEO MAGNIFICATION: A NOVEL TECHNIQUE FOR IMPROVED SPERM SELECTION IN MEN WITH SEVERE OLIGOASTHENOSPERMIA Gavin Langille*, James Dupree, Jason Kovac, Ranjith Ramasamy, Dolores Lamb, Larry Lipshultz, Houston, TX INTRODUCTION AND OBJECTIVES: The absence of sufficient motile sperm in samples used for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) can present a serious clinical problem for infertility specialists wishing to select viable sperm. Identification of optimal quality sperm can be especially challenging in men with oligoasthenospermia. Eulerian Video Magnification (EVM) is a technique that provides an opportunity to detect subtle movements in video feeds not appreciable to the unaided human eye. EVM is a computational algorithm that uses spatial and temporal variations present in video sources to enhance, in a spatially-multiscale manner, the variation of individual pixel values over time. In patients whose semen samples demonstrate significantly impaired motility by traditional light microscopy, EVM could be used to select viable motile sperm that would have been otherwise identified as non-motile using traditional light microscopic visualization. METHODS: Using traditional light microscopy with 400x magnification, semen analysis (SA) samples from men demonstrating severe oligoasthenospermia (density < 1 million/ml and motility < 20%) were investigated. A high definition (HD) video camera was used to record video footage from three high-powered fields (HPF) per SA. Each video recording was scored twice for motility, first using the raw HD footage, and second after the video was processed using EVM previously developed at the Massachusetts Institute of Technology (http://people.csail.mit.edu/mrub/vidmag). Quantitation of motile sperm in each video recording was compared between the raw and EVMenhanced images. RESULTS: Video from eleven SA samples with three recordings each were analyzed. A total of 442 sperm were recorded across all samples. After processing with EVM, the number of additionally recognized motile/viable sperm increased by 14% (p<0.05). CONCLUSIONS: EVM is a novel technique to identify viable sperm that would not have been recognized using traditional light microscopy. Using a micro-locator and micro-pipette, these sperm can then be retrieved for IVF-ICSI. Our data suggests that an additional 14% of retrieved ova can then be inseminated. Further studies are underway to refine this technique and to test the efficacy of the additional EVMidentified sperm in improving IVF-ICSI outcomes for the oligoasthenospermic patient. Source of Funding: None