Food intake diet and sperm characteristics in a blue zone: a Loma Linda Study.

OBJECTIVES: The study examined the effect the life-long vegetarian diet on male fertility and focused on vegetarians living in the Loma Linda blue zone, a demographic area known for life longevity. The objective was to compare sperm characteristics of vegetarian with non-vegetarian males. STUDY DESIGN: The cross-sectional observational study was based on semen analyses of 474 males from 2009 to 2013. Patients categorized themselves as either life-long lacto-ovo vegetarians (N=26; vegetable diet with dairy and egg products), vegans (N=5; strictly vegetables with no animal products) or non-vegetarians (N=443; no diet restrictions). Sperm quality was assessed using a computer-aided sperm analyzer and strict morphology and chromatin integrity were manually evaluated. RESULTS: Lacto-ovo vegetarians had lower sperm concentration (50.7±7.4M/mL versus non-vegetarians 69.6±3.2M/mL, mean±S.E.M.). Total motility was lower in the lacto-ovo and vegan groups (33.2±3.8% and 51.8±13.4% respectively) versus non-vegetarians (58.2±1.0%). Vegans had lowest hyperactive motility (0.8±0.7% versus lacto-ovo 5.2±1.2 and non-vegetarians 4.8±0.3%). Sperm strict morphologies were similar for the 3 groups. There were no differences in rapid progression and chromatin integrity. CONCLUSIONS: The study showed that the vegetables-based food intake decreased sperm quality. In particular, a reduction in sperm quality in male factor patients would be clinically significant and would require review. Furthermore, inadequate sperm hyperactivation in vegans suggested compromised membrane calcium selective channels. However, the study results are cautiously interpreted and more corroborative studies are needed.

Further evidence supporting the Diff-Quik stain for sperm chromatin integrity testing.

OBJECTIVE: To correlate intracytoplasmic sperm injection (ICSI) fertilization with chromatin status assessed by the Diff-Quik procedure modified with a one-minute soak step, and to determine the association of chromatin status with in vitro fertilization (IVF) pregnancy. STUDY DESIGN: This was a retrospective study of 81 IVF patients. Gradient-centrifuge washed sperm remaining after ICSI were fixed, stained by Diff-Quik, immersed in water for 1 minute, and analyzed under oil immersion light microscopy. Sperm nuclear coloration (types A-D), strict morphology, fertilization, and pregnancy status were determined. RESULTS: Sperm with light purple staining (type A) were correlated (R = 0.48, p < 0.05) with ICSI fertilization. The intraassay and interassay coefficients of variation were 5.9% and 4.1%, respectively. Sperm strict normal morphology was correlated neither with ICSI fertilization (R = 0.24, p > 0.05) nor with type A sperm (R = 0.35, p > 0.05). Sperm incubated in Fenton reagent that damaged DNA showed a time-dependent decrease in percent type A sperm. However, there was no correlation with IVF pregnancy status. CONCLUSION: This retrospective study showed that the inclusion of a one-minute soak step post-Diff-Quik staining enhanced the detection of sperm chromatin abnormalities related to ICSI fertilization. Fenton reagent-treated sperm suggested that the staining patterns correlated with DNA damage. A large prospective trial should be undertaken to confirm these findings.

A simple sperm DNA toroid integrity test and risk of miscarriage.

Current methods of analyzing sperm chromatin competency overlook the inner sperm compartment which is inaccessible to probes and reagents. By breaking the molecular protamine disulfide bridges, the DNA toroids are exposed to integrity analysis. The aim was to develop a simple nuclear toroid test and determine its association with fertilization, pregnancy, and miscarriage. The approach involved treating washed sperm remaining after ICSI procedures (N=35 cases) with acidified Triton X-100 and dithiothreitol (DTT) before Diff-Quik staining. Percentages of sperm with normal chromatin indicated by light-colored nuclei were assessed. The toroid integrity test showed more sperm with normal chromatin in the pregnant group (73.6±1.7%, mean±SEM) when compared with the miscarriage (51.2±6.6%) or nonpregnant groups (60.9±4.8%). Furthermore, the toroid results were correlated with ICSI fertilization (R=0.32, P=0.04) and pregnancy outcome (pregnant cases 73.6±1.7% versus nonpregnant 58.0±3.9%, P=0.001). ROC calculated cut-off was >70.0% for normal toroid integrity (sensitivity 0.98, specificity 0.33, and diagnostic accuracy 78.3%). An association between normal sperm toroid integrity and miscarriage was evident when the staining procedure included acidified detergent DTT pretreatment.

Retention of membrane charge attributes by cryopreserved-thawed sperm and zeta selection.

PURPOSE: Mature sperm can be selected based on their negative zeta electrokinetic potential. The zeta selection of cryopreserved sperm is unknown. The objective was to study the effect of zeta processing on the morphology and kinematic parameters of cryopreserved-thawed sperm. METHODS: Colloid-washed sperm (N = 9 cases) were cryopreserved for 24 h, thawed and diluted in serum-free medium in positive-charged tubes. After centrifugation, the tubes were decanted, serum-supplemented medium was added and the resuspended sperm were analyzed. Untreated sperm and fresh sperm served as the controls. RESULTS: There were improvements in strict normal morphology in fresh (11.8 +/- 0.3 versus control 8.8 +/- 0.3 %, mean +/- SEM) and thawed (8.7 +/- 0.2 versus control 5.4 +/- 0.2%) sperm after zeta processing. Percent sperm necrosis was reduced after zeta processing (66.0 +/- 0.6 versus unprocessed 74.6 +/- 0.3%). Progression decreased by 50% but not total motility after zeta processing of thawed sperm. CONCLUSIONS: The results suggested that the cryopreservation process did not impact the sperm membrane net zeta potential and higher percentages of sperm with normal strict morphology, acrosome integrity and reduced necrosis were recovered. The zeta method was simple and improved the selection of quality sperm after cryopreservation but more studies would be needed before routine clinical application.

A simple zeta method for sperm selection based on membrane charge.

OBJECTIVE: The objectives were: [1] to develop a simple zeta potential method for sperm isolation; and [2] to analyze the sperm maturity, morphology, kinematic, and DNA parameters. DESIGN: The phenomenon of sticky sperm adhering to slide surfaces was adapted for collecting charged sperm. SETTING: Clinical and academic research environment. PATIENT(S): Discarded colloid-washed sperm from routine laboratory testing (n = 8). INTERVENTION(S): Sperm were centrifuged in serum-free medium and collected for analyses. MAIN OUTCOME MEASURE(S): Kinematic parameters, DNA integrity, and maturity. RESULT(S): The percentages of mature (73.0% +/- 0.5% vs. control 63.5% +/- 0.5% SEM) and DNA intact sperm (85.0% +/- 0.3% vs. 69.5% +/- 0.5%) increased in the male factor subgroup. Strict normal morphology (19.3% +/- 0.1% vs. 10.0% +/- 0.1%), hyperactivation (7.0% +/- 0.1% vs. 3.6% +/- 0.1%), and progressive motility (29.1% +/- 0.1% vs. 19.9% +/- 0.1%) increased by twofold. CONCLUSION(S): The zeta method improved sperm parameters associated with increased fertilization and pregnancy after assisted reproduction procedures. Manipulation from the attaching-detaching process stimulated sperm metabolism without causing premature acrosome reactions. Total motility was unchanged suggesting a lack of association between total motility and zeta potential.

Cyclooxygenase (COX)-2 and granulosa cell apoptosis in vitro.

PURPOSE: C-myc was studied in cyclooxygenase (COX)-2 associated granulosa cell apoptosis, METHODS: Granulosa cells (N = 5 cases) were incubated for 24 h in either 1 or 50 microM COX-2 inhibitor, 1 or 50 microM COX-1/COX-2 inhibitor, negative or positive controls Single primer polymerase chain reaction of c-myc exon 1 were performed. Bisbenzimide-stained control single-stranded (ssDNA) were hybridized to SYBR Gold-stained ssDNA and fluorescent images analyzed. RESULTS: C-myc was disrupted by the high-dose COX-2 inhibitor. Cell viability decreased with COX-1 and COX-2 inhibition. However, cell viability was similar for the positive control and at low-dose COX-2 inhibition. CONCLUSIONS: Inhibition of both COX-1 and COX-2 initiated apoptosis without disrupting c-myc suggesting a protective effect on c-myc. The low dosage of the COX-2 inhibitor did not disrupt c-myc and cell viability. C-myc sensitization was not part of apoptosis.

Temperature variable and the efficiency of sperm mediated transfection of HPV16 DNA into cells.

AIM: To pretreat sperm at various temperatures before exposure to human papillomavirus (HPV) 16 DNA fragments and to assess the efficiency of HPV carrier sperm to transfect cumulus cells. METHODS: Cumulus cells from follicular aspirates were obtained, pooled and divided into culture dishes containing Sybr Gold-stained HPV DNA carrying sperm that were either pretreated at 4 degree C, 37 degree C or 40 degree C (n = 5). The cells were incubated in 5% CO(2) in air mixture at 37 degree C for 24 hours. The efficiency of sperm to take up fluorescent HPV DNA was determined at hour 0. After incubation, cumulus cell viability was assessed using the eosin method and the percentages of fluorescent cumulus cells determined. RESULTS: Over half of all the cumulus cells became fluorescent with the highest percentage in the 37 degree C group. Sperm pretreated at 4 degree C had the greatest amount of HPV DNA fragments. Total sperm motility was similar for the 3 pretreatment groups. There were no differences in cumulus viability among the groups. CONCLUSION: Sperm pretreated at 37 degree C transferred the greatest amount of fluorescent HPV DNA fragments to the cumulus cells. The HPV DNA was observed in the nuclear and cytoplasmic compartments. The data suggested the possibility of sperm as a vector for the transmission of HPV DNA to the cumulus cells surrounding ovulated oocytes, which might lead to early implantation failures.

A simple DNA disc chip in a microarray design based on modified comparative genomic hybridization for sperm DNA analysis.

OBJECTIVE: A DNA disc chip assay, based on comparative genomic hybridization, was designed to measure changes in sperm DNA intensities. The objective was to analyze the DNA integrity of hyperactive sperm cells after mild heat treatment. DESIGN: The assay based on a multiple cell comet assay was used to analyze changes in genomic DNA. Washed sperm DNA were tested on the assay and images stored in a microarray design. SETTING: Clinical and academic research environment. PATIENT(S): Frozen-thawed washed sperm from different donors (n = 7). INTERVENTION(S): Discarded sperm leftover from trial washes carried out at 37 degrees and 40 degrees C were frozen and processed for the DNA disc chip assay. MAIN OUTCOME MEASURE(S): Fluorescent intensities of DNA disc chips and sperm variables. RESULT(S): Heat treatment resulted in more than eightfold increase in sperm hyperactive motility with little degradation in DNA integrity. Sperm with low hyperactivation was associated with alterations in DNA after heat treatment. CONCLUSION(S): The DNA disc chip assay was simple, inexpensive, and permitted assisted reproduction technologies laboratories to use comparative genomic hybridization for cytogenotoxicity testing. However, the assay required manual processing, a fluorescent microscope, and computer. The data showed an association between sperm hyperactivation and DNA integrity suggesting that the hyperactivation marker may be used for selecting quality sperm for intracytoplasmic sperm injection. More studies are needed to examine temperature effects on ejaculated human sperm.