Isolation of germ cells from human testicular tissue for low temperature storage and autotransplantation.

OBJECTIVE: To develop a new protocol for conserving fertile potential in men undergoing sterilizing chemotherapy by low temperature banking of germ cells which can be returned to the patient's testes after thawing. DESIGN: Isolation of human and murine germ cells for comparing cellular viability after cooling to liquid nitrogen temperatures by the use of different cryoprotective agents and for infusion into the testis. SETTING: Laboratory research environment. PATIENT(S): Men undergoing routine surgery in a urology department. INTERVENTION(S): Testicular biopsy. MAIN OUTCOME MEASURE(S): Cellular viability and infusion of seminiferous tubules. RESULT(S): After isolation using a two-step enzymatic disaggregation protocol, 66% to 87% of germ cells from human and murine specimens, respectively, were still viable. Cell survival was similar in four commonly used cryoprotective agents after cooling to liquid nitrogen temperatures. Seminiferous tubules infused by back flow with dye solution via the rete testis were filled with an efficiency of 55%. CONCLUSION(S): Judging from the high viability of unfractionated germ cells, it is feasible to isolate germ cells from testicular biopsies for low temperature banking with the aim of attempting to restore fertility after iatrogenic sterilization.

Germ cell transfer into rat, bovine, monkey and human testes.

Germ cell transplantation is a potentially valuable technique offering oncological patients gonadal protection by reinitiating spermatogenesis from stem cells which were reinfused into the seminiferous tubules. In order to achieve an intratubular germ cell transfer, intratubular microinjection, efferent duct injections and rete testis injections were applied on dissected testes of four different species: rat, bull, monkey and man. Ultrasound-guided intratesticular rete testis injection was the best and least invasive injection technique with maximal infusion efficiency for larger testes. Deep infiltration of seminiferous tubules was only achieved in immature or partially regressed testes. This technique was applied in vivo on two cynomolgus monkeys. In the first monkey a deep infusion of injected cells and dye into the lumen of the seminiferous tubules was achieved. In the second, transplanted germ cells were present in the seminiferous epithelium 4 weeks after the transfer. These cells were morphologically identified as B-spermatogonia and located at the base of the seminiferous epithelium. In summary, this paper describes a promising approach for germ cell infusion into large testes. The application of this technique is the first successful attempt of a germ cell transfer in a primate.

Transplantation in reproductive medicine: previous experience, present knowledge and future prospects.

The use of transplantation in reproductive medicine has been considered by physicians and scientists alike for many years. Despite being side-tracked into futile pursuits of rejuvenation in the early days, the possibility of usefulness remains, particularly for preserving fertility in patients undergoing ablative chemo- or radiotherapy. These aims have been enhanced by advances in tissue cryopreservation. When isolated primordial follicles are transferred in the mouse, or ovarian tissue slices are grafted into sheep, it is possible to obtain follicular survival with subsequent maturation and oestrogen secretion and even restore fertility to sterilized hosts. For preservation of fertility, autografts avoid both the immunological problems of allografts and the ethical dilemmas when using donor tissue. In the male, the concept of spermatogonial cell transfer after isolation and frozen storage of cells recovered from a testicular biopsy is most attractive, since it may provide another option for rescuing fertility in cancer patients, and provide a much needed one in children. Recent results demonstrate that gonocytes from immature mice injected into the tubules of sterilized hosts restore spermatogenesis and produce fertile spermatozoa. Furthermore, the gonocytes can be stored frozen prior to transfer and still produce fertile tubules. This review presents a broad history of transplantation in the male and female genital tracts as well as attempts to anticipate possible future developments.

Measurement of intracellular pH in human spermatozoa by flow cytometry with the benzo[c]xanthene dye SNAFL-1: a novel, single excitation, dual emission, molecular probe.

In numerous animal species the acrosome reaction of spermatozoa has been linked to elevations in intracellular pH (pHi). However, whether or not this is merely a passive consequence of calcium ion influx is not known. Studies into the fluctuations in pHi in sperm cells have been hampered by the lack of a pH-sensitive probe that could be used in conjunction with flow cytometry. In this study, flow cytometric analysis of pHi in human spermatozoa was accomplished by using one of the new benzo[c]xanthene dyes (SNAFL-1). SNAFL-1 was then observed in situ with conventional fluorescent microscopy and was found to be located in the post-acrosomal cytoplasm of the head. It was then used to measure the differences in pHi between acrosome intact populations of spermatozoa, and populations that had been induced to acrosome-react with human follicular fluid or the calcium ionophore A23187 to mimic the calcium influx. It was concluded that the human sperm acrosome reaction is also accompanied by a rise in pHi and the natural agonist-induced rise could not be accounted for by calcium ion influx alone.

Endocervical mucus pH is inversely related to serum androgen levels and waist to hip ratio.

OBJECTIVE: To determine whether an inter-relationship exists between endocervical mucus pH, serum androgen levels, and waist to hip (W:H) ratio. SETTING: Donor insemination clinic, University of Sheffield, Jessop Hospital, Sheffield, United Kingdom. PATIENTS: One hundred patients receiving donor insemination. INTERVENTIONS: Waist, hip, height, and weight measurements were recorded for 100 patients receiving donor insemination. At the onset of the LH surge when patients attended for their first insemination, a sample of endocervical mucus and blood was taken. The Insler score of the mucus was recorded and the pH was measured with multirange pH paper. Of the 100 patients studied, 10 cases had an endocervical mucus pH < 6 despite Insler scores of > 10. Serum T, DHEAS, and androstenedione levels were measured in the 10 patients with pH < 6 and in 26 patients selected at random from the 90 patients with pH > or = 6. RESULTS: The W:H ratio was significantly higher in the patients with pH < 6 than pH > or = 6. No significant difference was seen in body mass index between the latter groups. All serum androgen levels were significantly higher in the patients with pH < 6 than pH > or = 7. CONCLUSION: A potential link (serum androgen levels) between a powerful predictive factor of fertility (W:H ratio) and a potential mechanism of subfertility (low endocervical mucus pH) has been demonstrated. This observation warrants further detailed evaluation particularly as it has been shown that a low endocervical mucus pH may be corrected by simple inexpensive treatment.

A morphometric comparison of the nuclear morphology of fresh and frozen-thawed human zona-bound and unbound sperm.

The role of sperm nuclear morphology in zona binding and the effect of cryopreservation on sperm nuclear morphology have been investigated using objective criteria. The nuclear dimensions of fresh and frozen-thawed sperm heads that had been prepared by the swim-up (SU) technique, and bound to human oocytes in vitro, were measured using a computer-assisted image analyzer. These were compared with each other and also to sperm in the SU samples and those that had not bound to the zona pellucida. The unbound (UB) sperm and SU sperm had very similar nuclear morphology. In contrast, the nuclear morphology of the zona-bound (ZB) sperm differed significantly (P < 0.05) from that of the SU and UB sperm. These differences were found specifically between area, breadth, and roundness in the SU and ZB sperm preparations, and between area and roundness in the UB and ZB sperm preparations; the ZB sperm had smaller but rounder nuclei than either the SU or UB sperm. In addition, the frozen-thawed sperm in the SU and UB groups showed a trend towards smaller nuclei than the corresponding fresh groups, whereas the frozen-thawed ZB sperm had significantly (P < 0.05) smaller nuclear dimensions than the fresh ZB sperm. From the present study it appears that the ZB sperm are a morphologically distinct population, although it is not yet clear whether these differences reflect functional events that occur in the sperm head during its maturation or changes at the level of the sperm's DNA after the initial stage of zona binding.(ABSTRACT TRUNCATED AT 250 WORDS)

The more accurate timing of insemination with regard to ovulation does not create a significant improvement in pregnancy rates in a donor insemination program.

OBJECTIVE: To determine if the accurate prediction of ovulation before artificial insemination of frozen donor semen was justified by increased pregnancy rates. DESIGN: A retrospective study of inseminations, over a 2-year period, administered to women who were enrolled on a donor insemination program and who were monitored using one of four ovulation timing regimens. Also, a study of the significance of follicle status at the time of insemination with relevance to pregnancy was carried out. SETTING: A university teaching hospital donor insemination program. PATIENTS: Patients entered in this study were undergoing insemination with thawed, cryopreserved, donor semen. MAIN OUTCOME MEASURES: Pregnancy and follicle status at the time of insemination. RESULTS: There was no statistically significant difference in pregnancy rates between the four methods of ovulation detection most commonly used for artificial insemination. Inseminations several hours either side of ovulation did not result in a significantly different pregnancy rate than inseminations carried out closer to ovulation. CONCLUSION: More accurate and expensive methods of detecting changes in ovulation result in no significant increase in fecundity than other less expensive timing techniques.