Faculty Bibliography

Barriers to assisted reproduction include clinical diagnoses that present the greatest challenge in achieving reproductive success and laboratory limitations that prevent the in vitro production of healthy, viable embryos with the highest likelihood for implantation. Although patients with various conditions are candidates for assisted reproductive technologies, advanced reproductive age, male factor infertility, and unexplained;infertility are the barriers that have most significantly affected assisted reproductive technologies success in the recent years. The success of in vitro fertilization is directly related to gamete quality. The goal of every ovarian stimulation protocol is the harvesting of an adequate number of mature preovulatory follicles. Several different stimulation protocols have been specifically designed to provide optimal ovarian response in patients with a history of unsuccessful attempts of in vitro fertilization. Subzonal sperm injection and partial zona dissection, techniques that were developed to improve fertilization in cases of severe male factor infertility, have for the most part given way to intracytoplasmic sperm injection, the process in which a single spermatozoon is injected into an oocyte. Intracytoplasmic sperm injection results in superior fertilization rates and does not appear to adversely affect embryo viability, a condition often encountered with subzonal sperm injection and partial zona dissection. Embryo morphologic characteristics provide the most accurate index for predicting implantation after in vitro fertilization. Embryo morphologic characteristics are directly related to in vitro culture conditions. Coculture techniques have been developed specifically to overcome the developmental arrests observed with standard in vitro fertilization techniques. Assisted hatching, the microsurgical removal of a small segment of the zona pellucida, has been shown to improve implantation, especially in patients with history of previous poor outcome with in vitro fertilization. Preimplantation genetic diagnosis is the technique of performing genetic testing on cell-stage embryos before initiation of pregnancy. Preimplantation genetic diagnosis involves single-blastomere biopsy of an early cleavage-stage preimplantation embryo, followed by genetic testing with polymerase chain reaction or fluorescent in situ hybridization. In the future, as implantation rates improve, fewer embryos will be transferred without fear of compromising in vitro fertilization success. Such new techniques as cytoplasmic and germinal vesicle transfer may prove beneficial as therapeutic alternatives to oocyte donation.

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

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

Introduction: Round spermatids have been studied extensively in stained and electron microscopy preparations. However, little information is available about their appearance in fresh specimens. We describe a method to identify and collect unstained round spermalids from ejaculates and testicular biopsies. Material and methods: Ejaculates (n=115) and testicular biopsies (n=7) were assessed for the presence of round cells by routine semen analysis and Testsimplets staining. With the aid of a micromanipulator, cells considered to be round spermatids according to cell size and shape, nuclear characteristics, presence of the acrosome, and cytoplasmic characteristics were collected in groups of identical diameter. They were then placed on separate microslides and analyzed by multiprobe fluorescence in situ hybridization (FISH) to test the validity of the selection process and in order to determine the range of spermatid diameter. When no spermatozoa were present in a testicular biopsy, the dissected tissue was smeared and processed by FISH to examine for hapioid cells (spermatids). If hapioid cells were observed selection of individual round cells and FISH analysis were performed. Results: Routine semen analysis and Testsimplets identified >1 × 106 round cells in 33 ejaculates: three of five azoospermic ejaculates had spermatids. Five biopsies contained spermatozoa; one had spermatids and the other showed spermatocytic arrest. The results of the smears examined by FISH confirmed the observations by the Testsimplets staining. Four ejaculates and six biopsies were processed with micromanipulation. Spermatoids chosen with the micromanipulator were 5-7.5 ?, in diameter with a spherical and homogeneous nucleus 4-6.5 ?. in diameter. An acrosome granule was occasionally observed as a light-gray spot; cytoplasm surrounded the nuclear as a thin halo. The surface of the spermalid was smooth or slightly irregular. The 1000 cells (10 slides containing 10 cells each) individually selected were analyzed by FISH!

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!