Viability assessment of fresh and frozen/thawed isolated human follicles: reliability of two methods (Trypan blue and Calcein AM/ethidium homodimer-1).

PURPOSE: To assess the reliability of trypan blue (TB) and calcein AM/ethidium homodimer-1 (CaAM/EthD-1) staining to evaluate the viability of fresh and thawed human ovarian follicles. METHODS: Isolated follicles from fresh and thawed cortex were stained using TB versus CaAM/EthD-1 methods (n = 10 patients). Measurements were performed by two independent observers. The reliability was evaluated by intraclass correlation coefficient (ICC) and the differences between paired measurements were tested by the Wilcoxon test. RESULTS: Inter-observer reliability was excellent for each method. Nevertheless, it was even better with the TB method (ICC = 0.83) compared with CaAM/EthD-1 (ICC = 0.75). Moreover, the ICCs for viability measurements using the two methods were good for each observer (observer 1: ICC = 0.49; observer 2: ICC = 0.40). CONCLUSION: Compared with CaAM/EthD-1, TB appears to be more reliable as a staining method for follicle viability evaluation. TB staining is a quick and useful method, complementary to histological analysis for quality control in ovarian tissue cryopreservation.

Activated caspases in thawed epididymal and testicular spermatozoa of patients with congenital bilateral absence of the vas deferens and intracytoplasmic sperm injection outcome.

OBJECTIVE: To analyze the expression of activated caspases and membrane permeability in thawed epididymal and testicular spermatozoa of patients with congenital bilateral absence of the vas deferens (CBAVD). DESIGN: Retrospective study. SETTING: Biology and medicine of reproduction in University hospital. PATIENT(S): Eight CBAVD patients. INTERVENTION(S): Staining of activated caspases and viability (propidium iodide, PI); intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Proportion of viable (Casp-/PI-) or dead (Casp-/PI+) spermatozoa without activated caspases, viable (Casp+/PI-) or dead (Casp+/PI+) spermatozoa with activated caspases. ICSI results. RESULT(S): Higher percentage of dead (Casp+/PI+; 84.0% vs. 57.5%) and viable (Casp+/PI-; 12.0% vs. 0) spermatozoa with activated caspases were observed in testicular than in epididymal samples. No significant difference was observed between the percentage of total testicular and epididymal spermatozoa permeant for PI. The outcome of ICSI fertilization (67.5% vs. 57.4%), good morphology embryo at day 2 (75.9% vs. 61.3%), clinical pregnancy (26.7% vs. 15.4%), and implantation (15.6% vs 9.5%) rates were better when ICSI were performed with epididymal sperm samples. CONCLUSION(S): These results support the hypothesis of an abortive apoptotic process and demonstrate that combined staining of the activated caspases and membrane permeability provide complementary measurements for the evaluation of viable and functional spermatozoa to better understand ICSI outcomes with epididymal and testicular spermatozoa.

Follicular growth and estradiol follow-up after subcutaneous xenografting of fresh and cryopreserved human ovarian tissue.

OBJECTIVE: To assess ovarian cortex surrounding benign ovarian cysts after cryopreservation and grafting to severe combined immunodeficient (SCID) mice. DESIGN: Animal study. SETTING: Academic research laboratories. PATIENT(S): Ovarian tissue obtained from 15 patients. INTERVENTION(S): Grafting of fresh and frozen/thawed ovarian tissue into the subcutaneous space of 22 SCID mice for 80 days. MAIN OUTCOME MEASURE(S): Histologic analysis before and after grafting. Serum E(2) measured before (after 37 days of grafting) and after FSH/LH supplementation (end of the study). RESULT(S): After grafting, follicular density had decreased for frozen/thawed tissue in all cases. The follicular distribution was modified in fresh tissue: Primordial follicles proportion was reduced (79% vs. 17%), whereas the primary and secondary ones were increased (21% vs. 57% and 0% vs. 23%, respectively). The same tendency was observed in frozen/thawed tissue. Significant E(2) secretion was obtained before and after FSH/LH supplementation in castrated mice, grafted with either fresh or frozen/thawed tissue. CONCLUSION(S): Fresh and cryopreserved ovarian cortex surrounding benign ovarian cysts grafted into the subcutaneous space of SCID mice is able to sustain ovarian tissue function, although follicular growth appears lower with frozen/thawed tissue.

In vitro alachlor effects on reactive oxygen species generation, motility patterns and apoptosis markers in human spermatozoa.

Due to its extensive production and application, the toxicity of chloracetanilide herbicide alachlor[2-chloro-2',6'-diethyl-N-(methoxymethyl)-acetanilide] should be evaluated to establish minimum effects. In this study, we have examined the in vitro effects of alachlor on human sperm motion using a computer-assisted sperm analyser (CASA). An evaluation of both reactive oxygen species (ROS) and markers of apoptosis was also performed to investigate the mechanism by which alachlor modifies the sperm movement. After exposure up to 2 h to alachlor (0, 0.18, 0.37, 0.90 and 1.85 mM), the percentage of viable, motile spermatozoa and sperm velocities were concentration and/or time dependently decreased. The most sensitive parameters were progressive motility, mean average path velocity and mean straight velocity. Alachlor (1.85 mM) induced an increase in ROS production. A decrease of mitochondrial membrane potential (DeltaPsi(m)), an increase of both phosphatidylserine (PS) externalization and DNA fragmentation, which were concentration and/or time dependent, were also observed. It is possible that toxic effects of alachlor result in an oxidative stress which could act as a mediator of apoptosis. Alachlor could also contribute to some hypofertility cases.

Human ovarian tissue from cortex surrounding benign cysts: a model to study ovarian tissue cryopreservation.

BACKGROUND: The scarcity of human ovarian tissue is a major problem in developing research on ovarian cryopreservation. We were interested in ovarian cortex surrounding benign ovarian cysts harvested during their requisite operations. METHODS: Ovarian tissue was collected from 25 women (mean age = 27.7 +/- 1.0 SEM) and frozen in serum-free cryoprotective medium. Histological and viability analysis were performed on fresh and frozen-thawed slices of tissue. RESULTS: Dermoid (n = 7), endometriosis (n = 13) and serous (n = 5) cysts were observed. Follicular densities (expressed per mm3) in ovarian cortex surrounding dermoid cysts were higher than in endometriosis and serous cysts for both histological (median of follicular densities: 13.04, 0.31 and 0.89 respectively) and viability analysis (2.93, 0.05 and 0.71 respectively). Freezing-thawing did not result in gross abnormality of follicle population either in number or morphology (80% of follicles preserved a normal pattern). However, a slight decrease of the density of living follicles (expressed per mm2) was reported. CONCLUSIONS: Ovarian cortex surrounding ovarian cysts, especially dermoid cysts, could be considered a source of ovarian tissue for future research. In our study, the cryopreservation procedure resulted in high follicular survival assessed by both histological and viability analysis. Nevertheless, further studies of in vivo and in vitro follicular maturation are needed to strengthen this model.