Seminal plasma round cells as an indicator of spermatogenic integrity

SPERM BIOLOGY O-289 Wednesday, October 22, 2014 11:15 AM SEMINAL PLASMA ROUND CELLS AS AN INDICATOR OF SPERMATOGENIC INTEGRITY. T. Cozzubbo, S. Cheung, Q. V. Neri, Z. Rosenwaks, G. D. Palermo. Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: To characterize the origin and meaning of round cells (RC) in human ejaculates and their relationship to spermatogenesis. DESIGN: In a prospective fashion, a total of 3,660 men undergoing male infertility screening in a period of 28 months were included in the study. Semen parameters were assessed according to the WHO criteria 2010. Cases displaying R2 million RC were screened for bacteria presence. The role of RC on clinical outcome was assessed in specimens used for ART. MATERIALS AND METHODS: Raw specimen containing RC were processed by peroxidase and identification of transmembrane glycoproteins. To identify and characterize eventual immature germ cells (IGC) specific stains for Sertoli-cell cytoplasmic remnants and protamines to assess their spermiogenic stage were used. DNA fragmentation (TUNEL) and aneuploidy (9 chromosome FISH) were carried out on IGC and spermatozoa. RESULTS: Of a total of 3660 men screened, 1.3% presented with RC. Men (n¼47) who consented to participate were 43.09yrs had a concentration of 32.327.5x106/mL, motility of 34.719.6%, and morphology of 2.01.7%. The actual proportion of WBC within those specimen was 30.0% with the remainder being IGC (range 0.79 – 24.90 million). All specimens screened were negative for uropathogens. The IGC were characterized by single/multiple nuclei as proven by their ploidy content. Ejaculates with IGC had higher incidence of aneuploidy in their spermatozoa (P¼0.0001). There was also a concordant DNA fragmentation rate between IGC and spermatozoa. Interestingly, IGC stained for vimentin indicating their encasement in Sertoli-cell. A subgroup of men (n¼14) underwent 33 cycles whose female partners were 37.34yrs. The fertilization was 59.4% resulting in a clinical pregnancy of 33.3% and a pregnancy loss of 9.1%. The presence of RC is clearly associated with impaired fertilization (P¼0.0001) and an apparent deleterious effect on pregnancy outcome when compared to a matched control void of RCs. CONCLUSION: The traditional perception of the seminal round cells has ranged from an infection marker to a ‘‘good Samaritan’’ cell. However, genetic and epigenetic assessments of these cells carried out in this study evidenced that they were largely byproducts of abnormal spermatogenesis proven by the observation of compacted chromatin nuclei engulfed in Sertoli-cell cytoplasm. This suggests that the presence of round cells in the ejaculate may serve as an indicator of an acute spermatogenic insult and may yield important information on compromised gamete competence. Supported by: Reproductive Medicine, Weill Cornell Medical College. O-290 Wednesday, October 22, 2014 11:30 AM COMPARISON OF OUTCOMES FROM CONVENTIONAL INTRACYTOPLASMIC SPERM INJECTION AND SELECTED HYALURONAN-BOUND SPERM INJECTION. J.-H. Tsai, E. C. Nestle, J. H. Lim, J. E. O’Brien, J. R. Graham, T. Han, M. J. Tucker. Shady Grove Fertility, Rockville, MD. OBJECTIVE: Mature sperm that bind hyaluronan, a major constituent of the cumulus matrix surrounding the oocyte, have been reported to have lower DNA fragmentation and potentially lower aneuploidy. The present study evaluated the use of selected hyaluronan-bound (HB) sperm in IVF-ICSI cycles. DESIGN: Retrospective study. MATERIALS AND METHODS: An IRB-approved review of 10,834 cycles receiving assisted reproductive therapy during 2010-2013 at a large private IVF center. HB sperm injection was used for patients who had previous poor treatment outcomes using conventional ICSI. HB sperm were selected by their ability to bind hyaluronan microdots while remaining motile for subsequent injection of oocytes. Fertilization rate and implantation rate (IR) were compared by t-test. Clinical pregnancy rate per transfer (CPR) and spontaneous abortion (Sab) rate were compared by chi-square and Fisher’s exact test. Statistical significance was defined as P <0.05 for comparisons between the two groups (conventional ICSI vs HB-ICSI). RESULTS: There were 10,511 cycles undergoing conventional ICSI and 304 cycles undergoing HB-ICSI. Fertilization rates were similar (73.4% vs 72.1%, NS). HB-ICSI did not improve IR (40.18% vs 29.34%) or CPR (46.95% vs 34.42%) compared to conventional ICSI. However, the Sab rate of HB-ICSI was lower than with conventional ICSI. Specifically, for cycles in patients younger than 36 years old, the Sab rates were 8.62% vs 22.16%.

FERTILITY & STERILITYÒ

All Ages

N

IR (%) CPR (%) Sab (%)

Conventional ICSI 10511 40.18 (age ¼ 36.1  5.3 years) HB-ICSI (age ¼ 36.2  4.9 years) 304 29.34* Age % 35 yrs Conventional ICSI 4143 45.94 HB-ICSI 131 36.83*

46.95

16.3

34.42*

12.61

52.13 43.70*

22.16 8.62*

*P < 0.05 CONCLUSION: HB-ICSI was associated with a decline in spontaneous abortions vs. conventional ICSI. This supports the suggestion that HB bound sperm may have increased levels of developmental maturity and sperm chromatin integrity, possibly resulting in more robust embryos. As HB-ICSI was selectively used for patients who had poor outcomes with conventional ICSI, HB-ICSI outcomes may be better among unselected patients. The weakness of this review of data is its uncontrolled retrospective nature. Prospective studies are needed to further evaluate the benefit of this technique. O-291 Wednesday, October 22, 2014 11:45 AM SPERM DNA METHYLATION PATTERNS ARE HIGHLY PREDICTIVE OF FERTILITY STATUS AND MAY BE PROGNOSTIC FOR IVF EMBRYO QUALITY. A. Smith,a K. I. Aston,b P. J. Uren,a T. G. Jenkins,b D. T. Carrell.b aMolecular and Computational Biology, University of Southern California, Los Angeles, CA; bSurgery (Urology), University of Utah Andrology and IVF Laboratories, Salt Lake City, UT. OBJECTIVE: To determine the relationship between genome-wide sperm DNA methylation patterns and fertility status, IVF embryo quality, and IVF outcome. DESIGN: Genome-wide sperm DNA methylation patterns were compared between 127 IVF patients and 36 normozoospermic fertile men. IVF patients were stratified into three groups: patients with generally high quality embryos and positive pregnancy (n ¼ 53), patients with generally poor quality embryos and positive pregnancy (n ¼ 42), and patients with generally poor quality embryos and negative pregnancy (n ¼ 31) following IVF. MATERIALS AND METHODS: Following somatic cell lysis and confirmation of the absence of potentially contaminating cells, sperm DNAwas extracted, bisulfite converted, and hybridized to Infinium 450HumanMethylation BeadChips to measure methylation at > 485,000 sites across the genome. Statistical comparisons were made between patients with good embryogenesis versus patients with poor embryogenesis and between all patients versus controls. A combination of feature-selection, via support-vector machine, and logistic regression under cross-validation was applied to construct a predictive model for infertility. Hierarchical clustering was applied to good and poor embryogenesis samples, using Euclidean distance between methylation profiles. RESULTS: Hierarchical clustering of samples based on DNA methylation levels identified two out-groups that accounted for close to half of the poor embryogenesis patients. These out-groups had high purity, consisting of more than 83% poor-embryogenesis samples, pointing to a clear link between sperm DNA methylation status and embryo quality. Further, predictive models constructed based on the current sperm DNA methylation dataset proved highly accurate in classifying samples as fertile or infertile with a sensitivity of 84.3%, a specificity of 92.1%, a positive predictive value of 97.3%, and a negative predictive value of 63.6%. CONCLUSION: Sperm DNA methylation patterns differ significantly and consistently between infertile men and fertile, normozoospermic controls. Methylation patterns may also be predictive of embryo quality following IVF.

O-292 Wednesday, October 22, 2014 12:00 PM A PROPOSED 360 SCORING METHOD TO REPLACE CONVENTIONAL BIDIMENSIONAL MOPRHOLOGICAL ANALYSIS. B. A. Levine,a J. Feinstein,b Q. V. Neri,a D. Goldschlag,a S. Belongie,c Z. Rosenwaks,a G. D. Palermo.a aThe Ronald O. Perelman & Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY; bDepartment of Computer Science, College of Engineering, Cornell University, New York, NY; cCornell Tech, Cornell University, New York, NY. OBJECTIVE: In a one-year time span, we noticed that only one-third of our semen analysis reports fit the normal morphology parameters. In this study we

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