Analysis of Human Sperm Chromatin Integrity

Determining potential maternal or paternal sources of abnormal chromosomal constitution gives opportunity for preconception genetic counseling. The most direct determination is achieved by analyzing the nuclear constitution of the gametes. The present study evaluated the integrity of human spermatozoal nuclear material in the two condensation stages of chromatin and chromosomes. Original semen samples (ORI) and their swim-up fractions (SW, selected for motility) from men of known (donors) and unknown (patients) fertility were analyzed. The extent of chromatin condensation was assessed by light microscopy and flow cytometry during the time course of a chemically-induced decondensation reaction. Motile spermatozoa were used to inseminate hamster oocytes for human sperm chromosomal analysis (original method). A modification of this technique was introduced in an attempt to overcome the motility barrier required for fertilization. Spermatozoa rendered immotile by cryodamage were directly microinjected into the perivitelline space of hamster oocytes in order to obtain fertilization and possible chromosomal development. The swim-up-selected spermatozoa showed a higher resistance to the chromatin decondensation assay (10.53% decondensed) than their corresponding nonselected whole semen samples (94.74%). Sperm chromosomal analysis by the traditional technique was restricted to donor samples (86.7% fertilization rate) since all the patients failed to achieve fertilization. Although a high number of chromosomal complements were obtained (2362) only 7.4% provided complete information (range 0-56 complements/donor). The observed X/Y relationship (39/44) was not significantly different than the expected 1/1 ratio. Ten spermatozoa (7.69%) carried structural and 3.08% carried numerical abnormalities. High rates of fertilization (64-86%) with low rates of polyspermy ($ Swim-up selected spermatozoa have a higher resistance to the in-vitro induced nuclear chromatin decondensation assay (NCDA) than their corresponding ORI samples, which may correlate with their greater nuclear stability. Although SW procedures are invaluable as an aid in infertility treatment due to their selectivity in motility, morphology, fertilizing ability, chromatin resistance, etc. they are not able to discriminate against spermatozoal carriers of genetic defects

Influence of Exposure to Benzo[a]pyrene on Mice Testicular Germ Cells during Spermatogenesis

The objective of this study was to assess the toxicological effect of exposure to benzo(a)pyrene, B[a]P, on germ cells during spermatogenesis. Mice were exposed to B[a]P at 1, 10, 50, and 100 mg/kg/day for 30 days via oral ingestion. Germ cells, including spermatogonia, spermatocytes, pachytene spermatocytes, and round spermatids, were recovered from testes of mice exposed to B[a]P, while mature spermatozoa were isolated from vas deferens. Reproductive organs were collected and weighed. Apoptotic response of germ cells and mature spermatozoa were qualified using the terminal deoxynucleotidyl transferase mediated deoxy-UTP nick end labeling (TUNEL) assay. B[a]P exposure a

Influence of Exposure to Benzo[a]pyrene on Mice Testicular Germ Cells during Spermatogenesis

The objective of this study was to assess the toxicological effect of exposure to benzo(a)pyrene, B[a]P, on germ cells during spermatogenesis. Mice were exposed to B[a]P at 1, 10, 50, and 100 mg/kg/day for 30 days via oral ingestion. Germ cells, including spermatogonia, spermatocytes, pachytene spermatocytes, and round spermatids, were recovered from testes of mice exposed to B[a]P, while mature spermatozoa were isolated from vas deferens. Reproductive organs were collected and weighed. Apoptotic response of germ cells and mature spermatozoa were qualified using the terminal deoxynucleotidyl transferase mediated deoxy-UTP nick end labeling (TUNEL) assay. B[a]P exposure at ≤10 mg/kg/day for 30 days did not significantly alter concentrations of germ cells and mature spermatozoa and apoptotic response in germ cells and mature spermatozoa. Exposure to B[a]P at 50 and 100 mg/kg/day induced testicular atrophy and yielded a significant reduction in the concentrations of spermatogonia, spermatocytes, pachytene spermatocytes, and round spermatid cells as compared with the control. Also, mature spermatozoa experienced decreased concentrations and viability. B[a]P-exposed mice experienced a significant increase in apoptotic germ cells as compared to the control mice. However, the mice dose concentrations were not relevant for comparison to human exposure

Influence of Exposure to Benzo[a]pyrene on Mice Testicular Germ Cells during Spermatogenesis

The objective of this study was to assess the toxicological effect of exposure to benzo(a)pyrene, B[a]P, on germ cells during spermatogenesis. Mice were exposed to B[a]P at 1, 10, 50, and 100 mg/kg/day for 30 days via oral ingestion. Germ cells, including spermatogonia, spermatocytes, pachytene spermatocytes, and round spermatids, were recovered from testes of mice exposed to B[a]P, while mature spermatozoa were isolated from vas deferens. Reproductive organs were collected and weighed. Apoptotic response of germ cells and mature spermatozoa were qualified using the terminal deoxynucleotidyl transferase mediated deoxy-UTP nick end labeling (TUNEL) assay. B[a]P exposure at ≤10 mg/kg/day for 30 days did not significantly alter concentrations of germ cells and mature spermatozoa and apoptotic response in germ cells and mature spermatozoa. Exposure to B[a]P at 50 and 100 mg/kg/day induced testicular atrophy and yielded a significant reduction in the concentrations of spermatogonia, spermatocytes, pachytene spermatocytes, and round spermatid cells as compared with the control. Also, mature spermatozoa experienced decreased concentrations and viability. B[a]P-exposed mice experienced a significant increase in apoptotic germ cells as compared to the control mice. However, the mice dose concentrations were not relevant for comparison to human exposure

Knowledge and attitudes regarding elective oocyte cryopreservation in undergraduate and medical students

Abstract Background To assess knowledge and attitudes regarding elective oocyte cryopreservation among female undergraduate students (UG) and medical students (MS) in Eastern Virginia. Methods An anonymous cross-sectional study surveying female UG at a local university and MS at our academic medical center in May of 2017. The survey contained questions on demographic information, interest in fertility preservation, and knowledge about age related changes in fertility. Results There were 74 of 102 female UG and 95 of 117 female MS who responded, for a response rate of 73 and 81% respectively. UG were significantly younger than MS (21.4 vs 26.8, p < 0.001). Further, UG generally planned on conceiving at a younger age than MS (age 26–30 vs 31–35), and favored younger ages to consider oocyte cryopreservation (age 26–30 vs 31–35). Only a minority of both UG and MS were willing to undergo egg freezing at the current price of approximately $10,000 (15% vs 26% respectively, p = 0.044). Moreover, 73% of students overall responded that they would be more likely to freeze oocytes if their employer paid. Notably, both UG and MS underestimated age of fertility decline. Conclusion Both UG and MS revealed a need for education on age-related changes in fertility. Most UG and MS would not undergo elective oocyte cryopreservation at the present cost but would consider it at a lower cost

Epithelial cell protein milk fat globule-epidermal growth factor 8 and human chorionic gonadotropin regulate stromal cell apoptosis in the human endometrium

To study the regulation of apoptosis in human endometrial cells. The specific aims were to determine whether milk fat globule-epidermal growth factor 8 (MFG-E8), a novel endometrial epithelial protein, modulates caspase activation and DNA fragmentation; and to examine whether hCG, an early embryonic product, regulates Bax and Bcl-2 equilibrium, as well as MFG-E8 expression. Design: Primary cultures of human endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Setting: Academic center. Patient(s): Ovulatory women aged 21-30 years. Intervention(s): Treatment with MFG-E8 and hCG. Main Outcome Measure(s): Apoptotic activity was quantified using a luciferase assay. Deoxyribonucleic acid fragmentation was detected by TUNEL assay. Bax, Bcl-2, and MFG-E8 messenger RNA expression levels were determined by quantitative reverse transcription-polymerase chain reaction. Immunocytochemistry was used to establish cell purity and presence of MFG-E8 and hCG-R (receptor) proteins. Result(s): Endometrial epithelial cells were cytokeratin+, vimentin-, MFG-E8+, and hCG-R+, whereas ESC were vimentin+, cytokeratin -, MFG-E8-, and hCG-R+. Treatment of ESC with MFG-E8 resulted in a 13-fold increase in caspase activity and a 30-fold increase in TUNEL. On the other hand, hCG decreased messenger RNA expression of Bax in ESC. Conclusion(s): Milk fat globule-epidermal growth factor 8 has proapoptotic activity, suggesting participation in endometrial remodeling via an epithelial-stromal cell paracrine effect. Conversely, pregnancy levels of hCG has opposite effects on stromal cells.Fil: Riggs, Ryan M.. Eastern Virginia Medical School; Estados UnidosFil: Bocca, Silvina. Eastern Virginia Medical School; Estados UnidosFil: Anderson, Sandra. Eastern Virginia Medical School; Estados UnidosFil: Franchi, Nilda Anahi. Eastern Virginia Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Rhavi, Bhaskara S.. Old Dominion University; Estados UnidosFil: Oehninger, Sergio. Eastern Virginia Medical School; Estados Unido

Milk fat globule epidermal growth factor 8 (MFG-E8): A novel protein in the mammalian endometrium with putative roles in implantation and placentation

MFG-E8 is a novel endometrial protein with conserved functions in tissue remodeling and angiogenesis in non-uterine tissues. Our aims were: 1. To examine the presence of MFG-E8 protein in the human endometrium during the window of implantation, in human endometrial cell lines, in human placental tissue at different gestational ages, and in murine implantation sites during early gestation; and 2. To study the regulation of MFG-E8 mRNA expression in mice implantation sites. Study design: MFG-E8 protein and its receptor integrin αvβ3 were detected by immunostaining in human endometrial biopsies obtained from normal volunteers, in human endometrial cell lines (epithelial: Ishikawa and HEC-1A, stromal: HESC, and endothelial: HEEC), in human products of conception from all trimesters of gestation, and in murine implantation and inter-implantation sites dissected on days 5 and 8 post-coitus. MFG-E8 gene expression was assessed by RT-PCR. Main outcome measures: Immunohistochemical determination of MFG-E8 in endometrium and products of conception as well as relative MFG-E8 mRNA expression in mice implantation sites. Results: MFG-E8 protein was present almost exclusively in the epithelial compartment of human endometrium. It was also expressed in the cytotrophoblasts and syncytiotrophoblasts outlining chorionic villi of the human placenta at all trimesters of gestation, and in murine implantation sites. MFG-E8 mRNA was significantly up-regulated in murine implantation sites and with increased gestational age. Conclusions: MFG-E8 expression in the endometrial epithelium as well as in chorionic villi suggests its possible role in endometrial reorganization during the receptive phase and in events related to normal pregnancy in mammals.Fil: Bocca, Silvina M.. Eastern Virginia Medical School; Estados UnidosFil: Anderson, Sandra. Eastern Virginia Medical School; Estados UnidosFil: Amaker, Barbara. Sentara Norfolk General Hospital; Estados UnidosFil: Swanson, R. James. Old Dominion Univeristy; Estados UnidosFil: Franchi, Nilda Anahi. Eastern Virginia Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Lattanzio, Frank A.. Eastern Virginia Medical School; Estados UnidosFil: Oehninger, Sergio Carlos. Eastern Virginia Medical School; Estados Unido