Reproductive Medicine, Weill Cornell Medical College,. New York, NY. .... pital, Kitakyusyu, Japan; bFaunal Diversity Sciences, Graduate School of. Agriculture ...
DESIGN: A prospective study. MATERIALS AND METHODS: Sperm from individuals diagnosed as normozoospermia (n¼43) or asthenozoospermia (n¼32) were collected. Liquid chromatography-tandem mass spectrometry were used to detect levels of total 5-methylcytosine (5-mdC) and 5-hydroxymethylcytosine (5-hmdC) in sperm DNA as well as N6-methyl-adenosine (m6A) and 5-methylcytosine (5-mC) in RNA. Levels of the above four indicators were compared. Linear and logistic regression models were performed to determine the factors affecting IVF outcomes, adjusting for confounders. RESULTS: Demographic characteristics including fertilization rate (FR), cleavage rate (CR), good quality embryo rate (GQER) and pregnancy rate (PR) were similar between asthenozoospermia (AS) group and normozoospermia (NM) group, except for that sperm motility and concentration were decreased in AS group. The level of 5-mdC (representing global DNA methylation status) were found to be significantly decreased in AS group (3.430.65% versus 3.730.57% in NM group, P¼0.042). Linear regression analysis revealed that 5-mdC level was positively correlated with FR (b 8.11, 95% CI (1.02,15.20), P¼0.043) and GQER in AS group (b 16.18, 95% CI (2.294,30.067), P¼0.024), but was not related to cleavage rate (P¼0.084). In contrast, level of 5-hmdC (standing for DNA demethylation status) was negatively correlated with FR in AS group (b -25.12, 95% CI (-49.82,-0.42), P¼0.047)). The other two indicators were found to have no influence on IVF outcomes in our study. Either of the four indicators had significant influence on clinical pregnancy as logistic regression analysis revealed. CONCLUSIONS: Global sperm DNA methylation level was significantly decreased in asthenozoospermia samples. Besides, DNA methylation/demethylation status posed a potential influence on fertilization and embryo development in these patients. This indicates that even though current ART techniques help to select highly motile sperm in asthenozoospermia patients, epigenetic alterations may still exist in these sperm, which may affect the fertilization process and embryo development in IVF.
P-386 Wednesday, October 21, 2015 ASSESSING A COMPREHENSIVE CHROMOSOMAL ANALYSIS OF HUMAN SPERMATOZOA BY NEXT GENERATION SEQUENCING. S. Cheung, Q. V. Neri, Z. Rosenwaks, G. D. Palermo. Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: To carry out a complete molecular karyotype on sperm cells by unraveling, extracting and amplifying the compacted male genome in adequate quantity and quality in comparison to FISH. DESIGN: Following extraction and amplification of good quality sperm DNA copy number variations were compared between specimens to assess the occurrence of spermatogenetic meiotic errors. Aneuploidy outcome was compared to standard FISH assessment. MATERIALS AND METHODS: DNA extraction was achieved by a commercial kit. We processed 48 ejaculates where we extracted DNA and processed for DNA amplification. In 12 specimens where only few spermatozoa were processed, satisfactory DNA was obtained following PCR-based random hexamer amplification. Fifteen of these specimens were processed by NGS and the copy number variations were recorded and compared using CASAVA and VarScan2 software programs. FISH on chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22 was carried out on spermatozoa from fertile men as well as men with history of recurrent pregnancy loss. Following NGS, a patient sample was compared to that of a fertile anonymous donor’s. RESULTS: We extracted good quality DNA ranging from 500,000 to as low as 250 spermatozoa.Assessment of 9 chromosome FISH on spermatozoa obtained from fertile men (n¼7) the total aneuploidy rate was only 0.2%, whereas men with recurrent ART failure (n¼36) had a total aneuploidy of 3.92.5%. When we ranked the data according to paternal age, we established that the sperm aneuploidy rate progressively increased with advancing age to reach 10.4% at the average age of 6811yrs particularly for chromosomes 15 and 17.Following NGS, our data presented a total percentage of 21.47% of aneuploidy rate. The most represented was gonosomal with Y monosomy and disomy at 4.2%, followed by chromosome 15 at 3.3%, and chromosome 14 at 1.6%. On the other extreme chromosomes 18 and 21 had the lowest disomy at 0.10 and 0.15, respectively.When compared to standard FISH assessment, the NGS aneuploidy results depicted higher aneuploidy than FISH results overall. Moreover, NGS copy number variations
FERTILITY & STERILITYÒ
for individual chromosomes indicated higher aneuploidy levels for all chromosomes screened for by FISH, with the exception of chromosomes 13, 18, and 21. The remaining chromosomes X, Y, 15, 16, 17, and 22, were actually found to have more than twice the aneuploidy value when assessed by NGS. CONCLUSIONS: Sperm aneuploidy assessment help in counseling male factor infertile couples or those with recurrent pregnancy losses. FISH has been the procedure used for this assessment, however, it has a limited number of chromosomes evaluated and not to mention the inherent accuracy concerns related to the procedure and the inability when assessing haploid specimen to discern through nullisomy. NGS proved to be the procedure capable of assessing aneuploidy. It can assess a wide range of cells from 500,000 to 10million, it assess all chromosomes including nullisomy. In addition, there is the ability to assess the copy number variations. Supported by: WCMC. P-387 Wednesday, October 21, 2015 HIGH THROUGHPUT INTEGRATED PROTEOMIC ANALYSIS OF SPERMATOZOAL PROTEINS IN PATHOPHYSIOLOGY OF VARICOCELE ASSOCIATED MALE INFERTILITY. A. Agarwal,a D. Durairajanayagam,a,b R. Sharma,a L. Samanta,a,c R. F. Turki,d E. Sabanegh.e aCenter for Reproductive Medicine, Cleveland Clinic, Cleveland, OH; bPhysiology, Universiti Teknologi MARA, Sungai Buloh, Malaysia; cRedox Biology Laboratory, School of Life Sciences, Ravenshaw University, Orissa, India; dOb/Gyn Specialist, King AbdulAziz University, Mobil, AL; eUrology, Cleveland Clinic, Cleveland, OH. OBJECTIVE: Varicocele appears to affect later stages of spermatogenesis. It causes scrotal hyperthermia, hypoxia, hormonal imbalances, and re-flow of metabolites from renal and/or adrenal glands leading to oxidative stress. The objective was to study the major differences in the distribution of spermatozoal proteins in infertile men diagnosed with varicocele compared to fertile men. DESIGN: Prospective proteomic study. MATERIALS AND METHODS: Proteins were extracted from infertile men with unilateral and bilateral varicocele (n¼5) and men with proven fertility (n¼5). 1-D gel electrophoresis followed by LC/MS-MS (LTQ-Orbitrap Elite hybrid mass spectrometer) was used for protein identification. Mascot (Matrix Science, London, UK), SEQUEST (Thermo Fisher Scientific, San Jose, CA, USA) and X! Tandem (TheGPM, thegpm.org) were set up to search the human reference with database assuming trypsin as the digestion enzyme. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. RESULTS: Of the 99 proteins that were differentially expressed (DEP) in the varicocele group, 9 were uniquely expressed in the fertile group compared to 2 proteins that were unique to the varicocele groups. Over 87% of the DEP involved in major energy metabolism and key sperm functions were underexpressed in varicocele group. Key protein functions affected in the varicocele group were spermatogenesis, sperm motility (ACRBP, SPA17, AKA7), and mitochondrial dysfunction (NDUFS1, UQCRC2). CONCLUSIONS: We have identified proteins that are underexpressed in varicocele group. These proteins may be key players involved in the pathology of varicocele and in the onset of infertility. P-388 Wednesday, October 21, 2015 CLINICAL OUTCOME OF TREATMENTS FOR AZOOSPERMIA. A. Tanaka,a M. Nagayoshi,a I. Tanaka,a S. Ikuma,a T. Miki,a T. Yamaguchi,a H. Kusunoki,b S. Watanabe.c aSaint Mother Hospital, Kitakyusyu, Japan; bFaunal Diversity Sciences, Graduate School of Agriculture, Kobe University, Kobe, Japan; cAnatomical Science, Hirosaki University Graduate School of Medicine, Hirosaki, Japan. OBJECTIVE: Azoospermia is found in approximately one out of one hundred men and the ratio of obstructive azoospermia to non-obstructive one is about 3:7. The sole treatment for azoospermia is the collection of sperms or spermatids surgically. We analyzed the clinical outcome following each procedure over a period of 14 (1999-2013) years. Microsurgical epididymal sperm aspiration (MESA) for obstructive azoospermia and microscopic testicular sperm extraction (Micro-TESE) for non-obstructive azoospermia are generally recommended. However there has not been enough information available for the clinical data following each procedure.
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