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

Autologous Platelet-Rich Plasma Infusion to Improve Pregnancy Outcome in Suboptimal Endometrium: A Review

Over the past decade, platelet-rich plasma (PRP) has been used in several fields of medicine to promote cell growth and expedite wound healing for the treatment of arthritis, nerve injury, tendinitis, bone regeneration, cardiac muscle repair, and oral & plastic surgery. Recently, researchers have been applying autologous PRP to bolster the growth of endometrial lining in patients with a history of endometrium-related failed embryo transfers. Evidence reveals that PRP is a rich source of active cytokines and various growth factors, which come from an autologous source that can be easily attained from peripheral blood without risk of disease transmission to the patient. In this review, several studies were analyzed that involved patients 18–42 years of age undergoing hormone replacement therapy (HRT) in preparation for embryo transfer and serial transvaginal ultrasound in conjunction with PRP infusions into the endometrium via an intrauterine insemination (IUI) catheter. Exclusion criteria included patients with endometritis, polyps, or adhesions. Embryo transfers (ET) were performed when the endometrial lining achieved a thickness of >7 mm. The database indicates that PRP infusion therapy is a promising low-cost treatment for HRT patients that significantly increases endometrial thickness and improves pregnancy success in a previous suboptimal ET patient population

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

MFGE8 Regulates TGF-β-Induced Epithelial Mesenchymal Transition in Endometrial Epithelial Cells in vitro

This study investigated the role of milk fat globule-epidermal growth factor-factor 8 (MFGE8) in TGF-β-induced epithelial– mesenchymal transition (EMT) of endometrial epithelial cells. These were in vitro studies using human endometrial epithelial cells and mouse blastocysts. We investigated the ability of TGF-β to induce EMT in endometrial epithelial cells (HEC-1A) by assessment of cytological phenotype (by light and atomic force microscopy), changes in expression of the markers of cell adhesion/differentiation E- and N-cadherin, and of the transcription factor Snail (by immunofluorescence and immunoblotting), and competence to support embryo attachment in a mouse blastocyst outgrowth assay. We also studied the effects of E-cadherin expression in cells transfected by retroviral shRNA vectors specifically silencing MFGE8. Results demonstrated that TGF-β induced EMT as demonstrated by phenotypic cell changes, by a switch of cadherin expression as well as by upregulation of the expression of the mesenchymal markers Snail and Vimentin. Upon MFGE8 knockdown, these processes were interfered with, suggesting that MFGE8 and TGF-β together may participate in regulation of EMT. This study demonstrated for the first time that endometrial MFGE8 modulates TGF-β-induced EMT in human endometrium cells

Do GnRH analogues directly affect human endometrial epithelial cell gene expression?

We examined whether Gonadotrophin-releasing hormone (GnRH) analogues [leuprolide acetate (LA) and ganirelix acetate (GA)] modulate gene expression in Ishikawa cells used as surrogate for human endometrial epithelial cells in vitro. The specific aims were: (i) to study the modulatory effect of GnRH analogues by RT –PCR [in the absence and presence of E2 and P4, and cyclic adenosine monophosphate (cAMP)] on mRNA expression of genes modulated during the window of implantation in GnRH analogues/rFSH-treated assisted reproductive technology cycles including OPTINEURIN (OPTN), CHROMATIN MODIFYING PROTEIN (CHMP1A), PROSAPOSIN (PSAP), IGFBP-5 and SORTING NEXIN 7 (SNX7), and (ii) to analyze the 5′ -flanking regions of such genes for the presence of putative steroid-response elements [estrogen-response elements (EREs) and P4-response element (PREs)]. Ishikawa cells were cytokeratin+/vimentin2 and expressed ERa, ERb, PR and GnRH-R proteins. At 6 and 24 h, neither LA nor GA alone had an effect on gene expression. GnRH analogues alone or following E2 and/or P4 co-incubation for 24 h also had no effect on gene expression, but P4 significantly increased expression of CHMP1A. E2 + P4 treatment for 4 days, alone or followed by GA, had no effect, but E2 + P4 treatment followed by LA significantly decreased IGFBP-5 expression. The addition of 8-Br cAMP did not modify gene expression, with the exception of IGFBP-5 that was significantly increased. The GnRH analogues did not modify intracellular cAMP levels. We identified conserved EREs for OPN, CHMP1A, SNX7 and PSAP and PREs for SNX7. We conclude that GnRH analogues appear not to have major direct effects on gene expression of human endometrial epithelial cells in vitro.Fil: Zhang, Xiaomei. Eastern Virginia Medical School; Estados UnidosFil: Bocca, Silvina. Eastern Virginia Medical School; Estados UnidosFil: Franchi, Nilda Anahi. 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: Anderson, Sandra. Eastern Virginia Medical School; Estados UnidosFil: Kaur, Mandeep. King Abdullah University of Science and Technology; Arabia SauditaFil: Bajic, Vladimir B.. King Abdullah University of Science and Technology; Arabia SauditaFil: Oehninger, Sergio. Eastern Virginia Medical School; Estados Unido

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

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

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