Victor M. Brugh

Isolation and Enrichment of Murine Spermatogonial Stem Cells Using Rhodamine 123 Mitochondrial Dye

Abstract Stem cells possess enormous therapeutic potential in tissue replacement. To study stem cells further, they must be isolated. Techniques are available for enrichment and study of hematopoietic stems cells, but thus far, techniques for purification of spermatogonial stem cells have not been described. Enrichment techniques for hematopoietic stem cells include the use of fluorescence-activated cell sorter analysis with Hoechst 33342 and rhodamine 123 (Rho) dyes. Use of Hoechst dye to isolate spermatogonial stem cells has been unsuccessful in our laboratory, and our results have conflicted with those from other laboratories. Taking advantage of the differential staining of the Rho dye, we report a novel method to enrich murine spermatogonial stem cells. Testicular cells are harvested from cryptorchid ROSA26 male mice. Populations of these cells are then stained with the Hoechst and Rho dyes, allowing them to be sorted by flow cytometry into a side population (SP) of Hoechst low-intensity cells and populations of low (Rholow) or high (Rhohi) fluorescent intensity. Sterile recipients, W/Wv mice, with an intrinsic germ cell deficiency were transplanted with the Hoechst SP cells, Rholow, Rhohi, and nonsorted donor cells. No spermatogonial stem cell colonies were derived from the Hoechst SP cells. The number of spermatogonial stem cell colonies from transplanted Rholow cells showed a 17- and 20-fold enrichment over those of Rhohi and nonsorted cells, respectively.

Male factor infertility

There is a male factor involved in up to half of all infertile couples. Potential causes of male factor infertility are many and require thorough evaluation for their accurate elucidation. A complete medical history in conjunction with a focused examination can allow for an appropriate choice of laboratory and imaging studies. The semen analysis is a crucial first step, but it is by no means sufficient to determine cause or dictate therapy. A systematic approach is necessary to help guide the evaluation and exclude less likely causes. The causes discussed within this article are broad, and the prognosis for any given couple depends, in large part, on the cause of the infertility. Without a firm understanding of the genetics, anatomy, physiology, and their interactions necessary to permit full functioning of the male reproductive system, the evaluation becomes an inefficient exercise that often fails to elucidate the precise cause of infertility. Treatment success relies not just on a clinical diagnosis but on a determination of the cause of the male factor infertility. Therefore, couples with a component of male factor infertility need a systematic evaluation directed at the male partner to maximize their reproductive potential.

Clinical and Consumer Trial Performance of a Sensitive Immunodiagnostic Home Test That Qualitatively Detects Low Concentrations of Sperm Following Vasectomy

PURPOSE Compliance with post-vasectomy semen analysis could be improved with the availability of a simple, rapid and accurate home test. SpermCheck Vasectomy, a highly sensitive lateral flow immunochromatographic diagnostic device, was designed to detect extreme oligospermia or azoospermia in men after vasectomy. We report the results of clinical and consumer testing of SpermCheck. MATERIALS AND METHODS A prospective, noncomparative observational study assessed the ability of SpermCheck Vasectomy to predict post-vasectomy sperm counts obtained using a hemacytometer procedure based on standard World Health Organization methodology. Consumer studies evaluated ease of use. RESULTS A cohort of 144 post-vasectomy semen samples was tested in the clinical trial. SpermCheck was 96% accurate in predicting whether sperm counts were greater or less than a threshold of 250,000 sperm per ml, a level associated with little or no risk of pregnancy. Sensitivity was 93% (95% CI 79% to 98%) and specificity was 97% (91% to 99%). The positive predictive value of the test was 93% (79% to 98%), and most importantly the negative predictive value was 97% (91% to 99%). The test gave a positive result 100% of the time at sperm concentrations of 385,000/ml or greater. Consumer studies with 109 lay volunteers showed that SpermCheck was easy to use. Volunteers obtained the correct or expected test result in every case and the correct response rate on a 20 question survey about the test was 97%. CONCLUSIONS SpermCheck Vasectomy, a simple and reliable immunodiagnostic test that can provide evidence of vasectomy success or failure, offers a useful alternative to improve compliance with post-vasectomy sperm monitoring. It is currently the only Food and Drug Administration approved test for this purpose.

Genetic disorders and infertility

Many aspects of male fertility are influenced by genetics. Over 150 genes have been shown to be associated with infertility in mouse models, although translation of these findings to human male infertility has been slow. Nevertheless, it is likely that a significant number of these gene deletions may be associated with human infertility. There is much that we do not understand about the molecular basis of human male infertility; patients should be advised of this caveat. Genetic disorders in humans can lead to impaired spermatogenesis, defective sperm function, and defects in delivery of sperm. It is critical for the urologist who evaluates and treats infertile couples to have a working knowledge of these disorders. An understanding of the genetic basis of male infertility allows for the appropriate counseling of patients about treatment options and risks to their potential offspring.

Y chromosome genes and male infertility

Normal spermatogenesis is a complex process that depends on many factors. Genetics plays a major role in many of these factors including providing a normal hormonal milieu, the development of the testis and ductal system, and control of the stepwise maturation of sperm in the testis. The Y chromosome plays a key role in testis determination and control of spermatogenesis. Understanding how these genes work together can elucidate of the exact cause of infertility in some patients once thought to have idiopathic infertility. It is not only important that patients understand the cause of their infertility. Using sperm from these men to attain pregnancies by assisted reproductive techniques will probably result in infertile male offspring. Additional consequences are currently unknown but are the topic of research investigations.

What the urologist should know about the female infertility evaluation.

As childbirth is delayed and divorce rates remain high, urologists will continue to see more and more infertile couples. It is imperative that urologists understand at least the basic aspects of the female evaluation. Fundamental information can be obtained from a simple, directed history. Risk factors for female infertility can be identified in a matter of minutes, and the reproductive endocrinologist can then carry out a complete female evaluation simultaneously with the male evaluation. Most female infertility problems can be classified as ovulatory, anatomic, or cervical. Signs of decreasing ovarian reserve with age can be ascertained easily with a history and perhaps the determination of a day-3 serum FSH level. Finally, a basic understanding of the female evaluation allows meaningful discourse between the urologist and the reproductive endocrinologist, leading to a more effective treatment plan for the infertile couple.