Faculty Bibliography

INTRODUCTION: With the increasing success of advanced assisted reproductive technologies (AART), men with heretofore unbeatable male factor infertility are seeking treatment. However, we do not know the implications of using testicular or ejaculated sperm in AART, from patients with exposure to childhood chemotherapy or from untreated patients with germinal cell cancers. We report a morphological and cytogenetic study of the germinal cells in these patients. MATERIALS AND METHODS: This study involved four patients. Patient 1 had a seminoma with diffuse intraepithelial neoplasia. A biopsy of both testes and a post orchiectomy semen analysis were performed. Patient 2 had a pure embryonal cell carcinoma with intraepithelial neoplasia. An evaluation of the malignant testis and a post orchiectomy semen analysis were performed. Patients 3 and 4 had azoospermia as a result of chemotherapy for childhood leukemia; bilateral testis biopsies were performed to retrieve sperm for an AART cycle. On the biopsies and orchiectomy specimens, cytological analysis, eosin-negrosin viability assessment and morphological analysis by Testsimplets staining were performed. To ascertain presence of spermatids and chromosomal status of spermatozoa and spermatids, multiprobe fluorescence in situ hybridi-zation (FISH) was carried out on smears of the specimens. Electron microscopy and thin section microscopy were additionally performed. RESULTS: Semen analysis of patient 1 and 2 revealed oligo-asthenoteratozoospermia. The nonmalignant testicular biopsy in patient 1 revealed a sperm count of 200,000/ml with 0% motility and 32 % viability. In patient 2 the malignant testis revealed a sperm count of 10,000/ml with 7% motility and 60% viability; electron microscopy revealed a normal germinal cell ultrastructure. Both patients had chromosomally normal spermatozoa when biopsy tissue and semen was evaluated by FISH for chromosomes X, Y, and 18. Patients 3 and 4 had complete azoospermia on semen analysis and histologie exams of the!

Two women had infertility due to a symptomatic unicornuate uterus associated with rudimentary contralateral horn. Both carried successful pregnancies after laparoscopic resection of the horns

Although round spermatids have been studied extensively using staining techniques and electron microscopy, little information is available about their appearance in living conditions. We describe a method of collecting and identifying round spermatids from ejaculates and testicular biopsies. The validity of the selection procedure was confirmed by fluorescence in-situ hybridization. Based on cell size, morphological characteristics of nucleus and cytoplasm, and on the nucleus/cytoplasm ratio, we harvested a population of cells that was 84% haploid. This procedure can be applied to select spermatids for clinical or research purposes

PGD has been successfully used for several years. Over 40 babies have been born worldwide by use of these techniques. Unfortunately, a number of misdiagnoses have been made, a distressing consequence of a new frontier. Significant advances have been made to improve the efficiency and accuracy of PCR and FISH. The widespread use of this technology awaits further documentation of safety and accuracy. Other issues must also be addressed. First, the cost-effectiveness of the techniques relative to the traditional alternatives must be evaluated. A number of ethical issues regarding embryo screening must be addressed including what diseases are serious enough to warrant the procedure. Another concern is the use of this technology for nongenetic disorders such as gender selection. Finally, the experimental nature of these procedures must continually be discussed with patients, and long-term follow-up studies must be undertaken. Development of more accurate and less expensive assays coupled with improved IVF success rates may make PGD a more widely used clinical tool. The future awaits these developments

A young female carrier for incontinentia pigmenti underwent preimplantation genetic diagnosis to prevent the transfer of affected embryos. FISH diagnosis was performed using X, Y, 18 and 13/21 probes. Unexpectedly, 57% of the embryos were aneuploid for these chromosomes, a rate significantly higher (P < 0.005) than expected (9.3%). The patient achieved pregnancy but spontaneously aborted a trisomy 9 fetus.

The field of preimplantation genetic diagnosis has undergone significant advances since the report of the first birth from this method in 1990. The first birth in the USA was reported in 1992, as was the first successful diagnosis and delivery of a baby free of a single gene defect disorder (cystic fibrosis and then Tay Sachs). Investigators have now reported approximately 40 births worldwide from preimplantation genetic diagnosis using the polymerase chain reaction and fluorescent in-situ hybridization methods to analyze single cells removed from early cleavage stage preimplantation embryos. The International Working Group on Preimplantation Genetics meets annually to discuss progress and pitfalls in this field. Although preimplantation genetic diagnosis offers hope to patients at risk of transmitting disease, there are many technical hazards of this experimental procedure. Technical difficulties must be overcome in order for preimplantation genetic diagnosis to become a standard clinical tool. This review will highlight some of the recent advances and problems in the field of preimplantation genetic diagnosis

Proinflammatory cytokines and activated macrophages and T lymphocytes have been detected in peritoneal fluids of women with endometriosis and may impair fertility. Expression of the 60 kDa heat shock protein (hsp60) is one mechanism leading to a localized activation of macrophages and T lymphocytes and cytokine release. Peritoneal fluids, obtained from 68 women undergoing a diagnostic laparoscopy, were assayed for hsp60. As independent evidence of local immune activation, the fluids were analysed for interferon gamma (IFN gamma). Fluids were also tested for antibodies to Chlamydia trachomatis because a chronic asymptomatic infection by this organism may also release hsp60. At laparoscopy, 26 women were diagnosed with pelvic adhesions, 19 had endometriosis, 16 had a visibly normal pelvis, four had ovarian cysts while three had myomas. The prevalence of hsp60 was higher in peritoneal fluids from the women with endometriosis than in the other subjects (P = 0.005). Hsp60 was detected in seven (36.8%) of the endometriosis patients and in only one each of the women with adhesions, a normal pelvis or an ovarian cyst; all women with myomas were negative. Detection of IFN gamma in peritoneal fluids was highly correlated with the presence of hsp60 (P = 0.0003). IFN gamma was present in seven of nine (77.8%) women with hsp60 and in only five of 40 (12.5%) women lacking hsp60. Women with pelvic adhesions had an increased prevalence of immunoglobulin G antibodies to C.trachomatis compared with the other women (P = 0.01). There was no relationship between evidence of exposure to C.trachomatis and hsp60 in peritoneal fluids. These data suggest that hsp60 may be released into the peritoneal fluid as a consequence of implanted ectopic endometrium. Hsp60-mediated immune activation may be one mechanism leading to endometriosis-associated infertility