Cryopreservation of a small number of fresh human testicular spermatozoa and testicular spermatozoa cultured in vitro for 3 days in an empty zona pellucida.

It is known that the motility of human testicular sperm can be improved when they are cultured in vitro for a few days. The purpose of this study was to determine whether it is better to freeze human testicular spermatozoa on the day of biopsy (fresh) or after they were cultured for 3 days. A modified, single-sperm freezing technique was used in this study. The study consisted of two parts: (1) ejaculated spermatozoa were used to examine the influence of different concentrations of glycerol and synthetic serum substitute (SSS) on the survival rate after cryopreservation, and (2) the survival rates between cryopreserved fresh testicular spermatozoa (Group 1) and testicular spermatozoa that were cultured for 3 days before freezing (Group 2) were compared. Empty zonae pellucidae were obtained from mouse eggs. Five to 10 motile spermatozoa were selected and injected into an empty zona pellucida. For freezing, the zona pellucida with spermatozoa was transferred into a HEPES-buffered human tubal fluid containing different concentrations of glycerol and kept at room temperature for 10 to 15 minutes, and then loaded into a 0.25-ml-plastic straw. The straws were exposed to liquid nitrogen vapor for 2 hours and then plunged into liquid nitrogen. For thawing, the straws were taken out of liquid nitrogen and placed into a 37 degrees C waterbath for 25 to 30 seconds. There was no statistically significant difference in survival rates between 3% and 10% SSS with different glycerol concentrations. There was no statistically significant difference in the survival rates of spermatozoa between Group 1 and Group 2 after cryopreservation. It appears that in vitro culture of testicular spermatozoa before freezing does not increase survival rate.

Feasibility study of repeated fluorescent in-situ hybridization in the same human blastomeres for preimplantation genetic diagnosis.

In order to increase the number of chromosomes examined in each blastomere, we have developed a repeated fluorescent in-situ hybridization (FISH) procedure by which six or more chromosomes can be analysed per blastomere of a human embryo. Three consecutive FISH procedures with directly-labelled fluorescent Vysis DNA probes were carried out for examination of chromosomes X, Y, 11, 13, 18 and 21 in the same blastomeres (n = 126) and lymphocytes (n = 164). Based on the initial number of nuclei, the percentages of nuclear loss and presence of signals were 3 and 92% respectively in blastomeres; 6 and 91% respectively in lymphocytes after the first FISH; 7 and 87% respectively in blastomeres and 10 and 86% respectively in lymphocytes, after the second FISH. These percentages were 13 and 78% respectively in blastomeres and 14 and 81% respectively in lymphocytes after the third FISH. The FISH procedure was repeated successfully in a couple for preimplantation genetic diagnosis of chromosomal aneuploidies in biopsied blastomeres of their embryos in our clinic. In conclusion, it is feasible to carry out repeated FISH procedures in the same blastomeres. Six or more chromosomes of a single blastomere may be examined using this procedure.

Ultrarapid detection of sex chromosomes with the use of fluorescence in situ hybridization with direct label DNA probes in single human blastomeres, spermatozoa, amniocytes, and lymphocytes.

OBJECTIVE: To assess the ultrarapid fluorescence in situ hybridization (FISH) procedure with a 1-minute hybridization time for gender determination. DESIGN: Fluorescence in situ hybridization with direct label fluorescence DNA probes for chromosomes X and Y were tested with the use of different hybridization times and different cell types. SETTING: Hospital-based IVF program. INTERVENTION(S): The efficiency of the FISH procedure with different hybridization times was compared with the use of male lymphocytes. The same FISH procedure, but with only 1-minute hybridization, was carried out in human blastomeres, spermatozoa, uncultured amniocytes, male lymphocytes, and female lymphocytes. MAIN OUTCOME MEASURE(S): Percentages of nuclei with positive signals. RESULT(S): The percentages of nuclei with positive signals in lymphocytes with hybridization times of 1, 3, 4, 10, 30, and 45 minutes were 97%, 97%, 98%, 98%, 98%, and 98%, respectively. The percentages of nuclei with positive signals after FISH with a 1-minute hybridization time in single blastomeres, spermatozoa, amniocytes, male lymphocytes, and female lymphocytes were 94%, 96%, 96%, 98%, and 97%, respectively. CONCLUSION(S): Chromosomes X and Y of human blastomeres. spermatozoa, uncultured amniocytes, and lymphocytes can be detected rapidly with the use of this ultrarapid FISH procedure with a 1-minute hybridization time.

Potential use of repeated fluorescence in situ hybridization in the same human blastomeres for preimplantation genetic diagnosis.

OBJECTIVE: To assess the feasibility of repeated fluorescence in situ hybridization (FISH) procedures in the same nucleus of a human blastomere. DESIGN: Three consecutive FISH procedures were performed in the same human blastomere by using direct label fluorescence CEP and WCP probes (Vysis). SETTING: Hospital-based private IVF program. PATIENT(S): Twenty-eight infertile couples who underwent conventional IVF in our center. INTERVENTION(S): Embryos from oocytes with three pronuclei after in vitro insemination were used in this study. MAIN OUTCOME MEASURE(S): The rates of nuclear loss, present signals, and absent signal were examined. RESULT(S): In group 1, the rates of presence of signals were 94% after the first FISH, 92% after the second FISH, and 88% after the third FISH. In group 2, the rates of presence of signals were 96% after the first FISH, 93% after the second FISH, and 87% after the third FISH. There was no statistically significant difference in the rates of nuclear loss, present signals, and absent signal between three consecutive FISH procedures and between CEP and WCP probes. CONCLUSION(S): Six or more chromosomes of a single blastomere may be examined with use of this repeated FISH procedure, which may be important for preimplantation genetic diagnosis.

Prolactin disorders.

OBJECTIVE: To review the pathophysiology, clinical manifestations, current diagnostic procedures, and treatment options for disorders involving PRL production. Common clinical dilemmas are discussed in a pragmatic fashion to guide the practitioner. DESIGN: A world literature search of basic sciences and medical articles from the last three decades was performed using computerized MEDLINE. Recent endocrine and reproductive endocrine textbooks also were reviewed. Studies were selected for their degree of contribution to the basic sciences and clinical understanding of the disorder and for the quality of their study design and content. The information was summarized and grouped according to its relevance and application to specific sections of the manuscript. Studies were evaluated and critically used to support the views of the authors and to suggest specific clinical management strategies. RESULT(S): Disorders derived from abnormal PRL production are relatively common in clinical practice. Infertility, menstrual disorders, and galactorrhea are the most frequent manifestations encountered in women. Although frequently benign, the disorder occasionally may have severe consequences. CONCLUSION(S): An understanding of the underlying physiology and pathophysiology coupled with the awareness of the heterogeneous presentation of this disorder should help the clinician to approach it successfully.

Enzyme activities and maturation in unstimulated and exogenous gonadotropin-stimulated human oocytes.

With the advent of new techniques of human in vitro fertilization (IVF), identifying parameters of oocyte quality to allow selection of those most likely to fertilize becomes crucial. Morphology of oocytes, which correlates positively with biological performance, is the currently utilized classification criterion. However, biological links between form and function are tenuous, and underlying mechanisms remain elusive. We investigated whether biochemical activation is quantitatively associated with the stages of maturation in ova obtained from patients undergoing gynecologic surgery during unstimulated cycles and women undergoing IVF after exogenous gonadotropin stimulation. Changes in selected enzymes from protein, lipid, and carbohydrate metabolism (hexokinase, phosphoglucomutase, glycogen synthetase, uridine diphosphoglucose pyrophosphorylase, glucose-6-phosphate dehydrogenase, cytosolic thiolase, beta-hydroxyacyl-CoA dehydrogenase, alanine aminotransferase, and aspartate aminotransferase) were determined simultaneously, in individual oocytes, utilizing a highly sensitive biochemical methodology. Several enzyme activities paralleled maturation grade and were higher in stimulated oocytes after correction for grade. These biochemical findings quantify metabolic and functional changes that increase as ova mature, possibly contributing to their reproductive performance.

Distribution of tritiated cocaine in selected genital and nongenital organs following administration to male mice.

STUDY OBJECTIVE: To determine the distribution of cocaine administered to male mice in selected extragenital and genital organs and to investigate its possible binding to sperm. DESIGN: Twenty-seven sexually mature virus-free albino male mice were used in various experiments whereby following intravenous injection of tritiated cocaine hydrochloride, radioactivity was determined in several extragenital and genital organs, as well as sperm. RESULTS: Radioactivity was detected in all of the organs that were tested, and the highest concentrations per milligram of tissue were found in the kidney and epididymis. Removal of the sperm from the epididymis significantly reduced the radioactivity of the organ. The spermatozoa that were isolated on glass filters showed a linear correlation vs radioactivity (r = .93). CONCLUSIONS: Radioactivity is distributed to several organs, including the genital tract, and is found in association with sperm after in vivo administration of tritiated cocaine. These results may explain the mechanism underlying a male-mediated teratogenesis, which has been observed in animals that were exposed to cocaine, and they raise a possibility that the spermatozoa may carry cocaine into the oocyte during fertilization.

Demonstration of specific binding of cocaine to human spermatozoa.

Exposure of males to cocaine has been linked to abnormal development of their offspring. To investigate the possible role of sperm, this study examined the interaction of cocaine with human spermatozoa. Washed sperm were incubated with tritiated cocaine (6.7 nmol/L) with or without unlabeled cocaine (670 mumol/L), and the samples were filtered and the remaining radioactivity quantitated. The specific binding was optimal at 20 minutes and 23 degrees C. Competition studies with tritiated cocaine (3.4 to 66.6 nmol/L) indicated the presence of approximately 3.6 x 10(3) binding sites per cell, with a high affinity receptor dissociation constant (Kd = 12.6 nmol/L). Cocaine concentrations as high as 670 mumol/L had no detectable effect on either the motility or viability of the cells. These results support the hypothesis that the sperm may act as a vector to transport cocaine into an ovum. This novel mechanism could be involved in the abnormal development of offspring of cocaine-exposed males.