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Simplified and Objective Assessment of Spermatogenesis in Spinal Cord Injured Men by Flow Cytometry Analysis

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Simplified and Objective Assessment of Spermatogenesis in Spinal Cord Injured Men by Flow Cytometry Analysis Paraplegia 31 (1993) 785-792 © 1993 International Medical Society of Paraplegia Simplified and objective assessment of spermatogenesis in spinal cord injured men by flow cytometry analysis I H Hirsch MD,! D Kulp-Hugues MD,! J Sedor MS,! P McCue MD, 2 M B Chancellor MD,! WEStaas MD,3 Deparments of 1 Urology, 2 Pathology, 3 Physical Medicine and Rehabilitation, Regional Spinal Cord Injury Center of the Delaware Valley, Jefferson Medical College, Philadelphia, PA, USA. Deterioration of the germinal epithelium of the testis is a known sequela of spinal cord injury (SCI) that may influence the outcome of male reproductive rehabilitation efforts. Quantitative testicular biopsy, currently regarded as the standard of assessing the integrity of spermatogenesis, has not gained wide­ spread clinical use because of its invasive nature and relative technical complex­ ity. Alternatively, aspiration DNA flow cytometry analysis of the testis has offered a potential method of spermatogenic assessment that meets both the requirements of simplicity and objectivity. The objective of this study is to determine the capability of flow cytometry to assess spermatogenesis following SCI. Eleven SCI men underwent incisional testicular biopsy with the specimen simultaneously submitted for quantitative evaluation of the germinal epithelium by both quantitative histometry and DNA flow cytometry. The haploid percen­ tage of cells showed highly significant levels of correlation with key micrometric parameters of the quantitative testicular biopsy: spermatid/tubule (p < 0.002) and the spermatid/Sertoli cell ratio (p < 0.0005). Since tissue procurement is accomplished less invasively for flow cytometry analysis, we recommend this method as the modality of assuring integrity of the germinal epithelium in candidates for reproductive rehabilitation. Keywords: spermatogenesis; spinal cord injury; flow cytometry analysis. Introduction historically been considered virtually sterile The estimated prevalence of spinal cord with fertility rates ranging from 0% to 5% in various reports. Therefore, the introduction injury approaches 200,000 in the USA with most victims being males in their reproduct­ of electroejaculation has been one of the ive years of life.! Understandably, repro­ major achievements in reproductive medi­ ductive function ranks high among the cine during the past decade as successful priorities of global rehabilitation goals set semen recovery can now be accomplished in forth by newly injured SCI patients. There­ 80-90% of SCI men so treated. Among the fore, the Task Force on Medical Rehabilita­ urologic sequelae of SCI, neurogenic infer­ tion Research recently established, as a tility has received increasing attention with general priority, the investigation of me­ the application of new semen recovery chanisms that underlie infertility in SCI men techniques and assisted reproductive tech­ and the development of strategies for restor­ nologies. Despite these methods, only 10% of SCI men entering reproductive rehabili­ ing or improving fertility. 2 Because of the resultant ejaculatory failure in up to 90% of tation programs are able to achieve preg­ those affected, spinal cord injured men have nancy by intrauterine insemination3 and 30% by in vitro fertilization.4 In addition to Correspondence: Dr Hirsch, Department of Urology, enhancing male fertility potential following Jefferson Medical College, 1025 Walnut St (Room SCI, the availability of predictable semen 1112), Philadelphia, PA 19107, USA. recovery methodology has offered new 786 Hirsch et al Paraplegia 31 (1993) 785-792 avenues of clinical research in male repro­ Quantitative testicular biopsy ductive rehabilitation. A review of these Quantitative analysis of the seminiferous studies has shown that male reproductive tubules and epithelial parameters was per­ dysfunction following SCI is of multifactor­ formed on incisional testicular biopsies fixed ial origin with abnormalities demonstrable in Bouin's solution. The tubular diameter in the testis, the male conduction system, and tubular wall thickness were measured and the accessory sex glands. Of these by AO filar micrometer eye pieces and the factors, the one most widely studied has number of Sertoli cells and late spermatids been the problem of spermatogenic dysfunc­ per seminiferous tubule were counted under tion which has been reported in up to 90% 400x magnification. Using the Clermont's of men following SCJ.5 Therefore, failure of cytologic description of the human seminif­ sufficient sperm production may limit fertil­ erous epithelium, micrometric analysis was ity potential in candidates entering male performed counting only germinal cell ele­ reproductive rehabilitation programs. Ob­ ments in the later stages of spermatogenesis jective assessment of spermatogenesis can (Sc + Sd).7 For each patient, at least 10 most readily be achieved by the quantitative randomly selected round seminiferous testicular biopsy; however, this procedure is tubules were selected for analysis. The both invasive and labor intensive. Alternat­ mean concentration of Sertoli cells and ively, spermatogenic assessment by means mature spermatids per tubule was deter­ of DNA flow cytometry analysis provides a mined. The mean tubular wall thickness was potentially rapid and objective quantitative measured from photomicrographs obtained technique that has the advantage of tissue at a magnification of 400x with 40% en­ recovery by fine needle aspiration. While largement of the print. These parameters this method has shown good concordance were measured with a photomicrograph of a with the qualitative testicular biopsy, 6 it has micrometer at identical magnification and not as yet been studied in conjuction with enlargement. The following quantitative the quantitative testicular biopsy in spinal histometric values were assessed for each cord injured men. Therefore, the objective patient: mean concentration of late sperm­ of this study is to determine the accuracy of atids per tubule (spermatid/tubule), mean flow cytometry as a technique for assessing ratio of spermatids Sertoli cells per tubule spermatogenesis following SCI by compari­ (spermatid/Sertoli cell), and tubular wall son to the parameters of the quantitative thickness. testicular biopsy, the current standard of objective spermatogenesis assessment. DNA flow cytometry analysis Testicular specimens were mechanically dis­ Materials and methods sociated by forcefully injecting the tissue sample through a 22-gauge needle until the Entering a program of reproductive rehabil­ cells were completely dispersed in Hank's itation were 11 spinal cord injured men Balanced Salt Solution. a The resulting cell (ages 22-43) with lesions ranging from C5 suspension was then rinsed into a 50 ml to TlO. The mean duration of injury was 6.8 conical test tube and centrifuged at 400 x g years and, in 8/11 men, the degree of SCI for 10 minutes. Following removal of the was complete. These patients underwent supernatant, the cell pellet was resuspended unilateral incisional testicular biopsy under in citrate buffer solutionb and adjusted to a either local or no anesthesia as part of a concentration of 3 x 106 cells/m!. Polypro­ comprehensive assessment of fertility poten­ pylene freezing vialsc containing aliquots of tial and suitability for stimulated semen 0.2 ml were frozen at -80°C. Within 14 recovery. The 2-3 mm specimen of testicu­ days, an aliquot was thawed at room tem­ lar parenchyma was equally divided for perature, and the cell suspension trans­ parallel analysis by both standard quantitat­ ferred to fresh citrate buffer (2 ml) and ive micrometric techniques and DNA flow centrifuged at 400 x g for 10 minutes. All cytometry analysis. but 0.2 ml of the supernatant was removed, Paraplegia 31 (1993) 785-792 Spermatogenesis following SCI 787 the cell pellet was dispersed, and 1.8 ml of 240,---�---------------------, detergent buffer containing trypsind was added to the cell suspension and gently ill rocked for 10 minutes at room temperature. '" 1N u (J) Subsequently, a solution containing 1.5 ml - of trypsin inhibitor and Ribonuclease Ae .2 � - was added to the cell suspension and rocked c 2N :::> for 10 minutes. Finally, 1.5 ml of an iced 0 propidium iodide solutionf was added under U light-protected conditions and rocked. The cell suspension was filtered through a 53 . , I . I 1 j x micron nylon mesh into a 12 75 mm poly­ o 200 400 600 800 1000 styrene tubeg protected from light, and cooled on ice until analysis. Samples were FL2-A analyzed within 3 hours of the addition of Figure 1 DNA histogram demonstrating normal the propidium iodide solution. A minimum spermatogenesis with a haploid (IN) predomi­ of 10,000 cells were analyzed by a Becton nant cell population. Dickinson F ACScan using CellFIT sofware and doublet discrimination. Histogram 296 �------�----------------� analysis of the red fluorescence emitted by the propidium iodide was analyzed manu­ - � '" ally by setting markers around the IN, 2N u (J) and 4N peaks and calculating the relative - percentages of each ploidy compartment. .2 2N � - Known diploid controls were stained in the c :::> 1N same procedure as the sample cells to serve 0 u as a diploid reference. (For suppliers, see - Appendix I.) ). J.. I I I .. I • I Statistical analysis 0 200 400 600 800 1000 Flow cytometry analysis of incisional tes­ FL2-A ticular biopsies was carried out to determine the relative percentage of haploid (IN), Figure 2 DNA histogram demonstrating sperm­ diploid (2N), and tetraploid
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