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.

Health risk perceptions predict smoking-related outcomes in Greek college students.

Health risk perception in smoking behavior was prospectively evaluated in a cluster-randomized trial for smoking cessation in Greek college students. Perceived Vulnerability (PV), Precaution Effectiveness, Optimistic Bias, and smoking behavior measures (quit attempts and cessation) were assessed in college-aged Greek student smokers at baseline, end of treatment (3 months), and follow-up (6 months). Using generalized estimating equations, baseline risk perception variables and change in risk perception variables between baseline and end of treatment were examined as predictors of the dichotomous smoking outcome variables. Results revealed that higher baseline PV [OR = 1.42 (1.21, 1.68)] predicted a greater likelihood of a quit attempt (n = 267). An increased likelihood of cessation [OR = 1.41 (1.15, 1.72)] was also predicted by an increase in PV from baseline to end of treatment (n = 243). Overall results suggested that PV was the strongest predictor of smoking behavior change, supporting further examination of health risk perceptions in promoting smoking cessation among Greek college smokers.

Falsely elevated testosterone due to heterophile antibodies.

BACKGROUND: Falsely elevated testosterone is a rare phenomenon that may result from heterophile antibodies. Similar to patients with heterophile antibodies for ß-hCG, incorrect management may result in unnecessary testing or therapy. CASE: A previously healthy postmenopausal woman presented with a falsely elevated total testosterone level due to interference consistent with heterophile antibodies, with subsequent normal levels detected by liquid chromatography-mass spectrometry. CONCLUSION: Asymptomatic patients may present with an elevated hormone level due to heterophile antibody interference. Molecular studies for hormone levels can assist greatly when falsely elevated levels are suspected, but these tests are costly and time-intensive.

Addition of eosin to the aniline blue assay to enhance detection of immature sperm histones.

OBJECTIVES: To compare the aniline blue assay with and without eosin, and to correlate the results with pregnancy outcome after intracytoplasmic sperm injection (ICSI) procedure. DESIGN: A retrospective study. SETTING: University-based fertility center. PATIENT(S): One hundred thirty infertile patients. INTERVENTION(S): Left-over washed sperm after each ICSI procedure were fixed on glass slides and stained with aniline blue with and without eosin. MAIN OUTCOME MEASURE(S): Chromatin condensation, pregnancy, and age. RESULT(S): Percentage chromatin condensation assessed by aniline blue-eosin was higher compared with standard aniline blue (72.4 +/- 2.4% vs. 64.0 +/- 2.4% [mean +/- SEM]). Chromatin condensation was higher in pregnant (86.6 +/- 0.9%) versus nonpregnant (80.9 +/- 2.1%) women age 35 years or more. In younger women, chromatin condensation was not correlated with pregnancy outcome. There was no correlation between chromatin condensation and ICSI fertilization or male age. CONCLUSION(S): Adding eosin counterstain to aniline blue improved assessment of chromatin condensation, suggesting that the standard assay underestimated chromatin condensation. The association between chromatin condensation and pregnancy in older but not younger women suggests that oocytes of younger women had the capacity to compensate for the immature sperm shortcomings.

Male age and sperm necrosis in assisted reproductive technologies.

INTRODUCTION: Sperm apoptosis is well characterized but studies on the effect of male age and necrozoospermia are lacking. The objectives were: (a) to analyze percentages of apoptotic and necrotic sperm in ejaculates, and (b) to compare the results between younger and older age groups. MATERIALS AND METHODS: Routine semen analyses were carried out (n = 189 males) and sperm cells were analyzed by dual fluorescence assay Hoechst 33342 and propidium iodide, and the acridine orange test. RESULTS: The percentage of necrotic sperm in the ejaculate increased by 22% for males aged over 35. There was a positive correlation between age and necrosis (R = 0.30). Sperm apoptosis increased by 17% in males aged 45 and older. The population of DNA intact sperm declined in males aged 40 and over (R = -0.21). There were no age-related changes in strict normal morphology, sperm concentration and semen volume. A decrease in rapid progressive motility was correlated (R = -0.24) with male age and was significant after age 35. CONCLUSIONS: The study demonstrated increased necrosis, DNA damage and apoptosis while rapid progression and total motility declined with advancing age in the male beginning as early as age 35. The order of the observed changes was sequential, suggesting the involvement of different pathways in sperm necrosis after age 40.

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.

Influence of nanoparticles on morphological differentiation of mouse embryonic stem cells.

OBJECTIVE: To assess mouse embryonic stem (ES) cell viability, growth, and differentiated morphology after exposure to different concentrations of nanoparticles. DESIGN: Cell culture for 6 days. SETTING: University research laboratory. ANIMALS: Cryopreserved mouse ES-D3 (American Type Culture Collection, Manassas, VA) cells. INTERVENTION(S): ES cells were exposed to either 0 (control), 0.4, or 12.2 million/mL mixed-size fluorescent nanoparticles in culture (37 degrees C, 5% CO(2) in air) for 6 days. MAIN OUTCOME MEASURE(S): Cell viability and morphometric analysis were performed. RESULT(S): ES cells exposed to both concentrations of nanoparticles exhibited smaller cell surface area. The effect was not concentration dependent. In contrast, ES cell nucleus size was unaffected. The nanoparticles distributed into the cytoplasm, pseudopods, and the perinuclear region. ES cell viabilities were reduced 40% and 30% in the low versus high relative concentration, respectively. ES cells in low-concentration nanoparticles became mostly columnar and embryoid body shaped. However, in high-concentration nanoparticles, they differentiated toward fibroblast-like and less squamous types. CONCLUSION(S): The observed reduced ES cell surface area suggested disruption of cytoskeletal development but not nuclear organization by nanoparticles. The ring-like formation of nanoparticles around the nucleus and the resulting cell morphologies suggested nanoparticles may influence differentiation.

In vitro tagging of embryos with nanoparticles.

PURPOSE: To develop an in vitro method for tagging embryos and to compare the development of the embryos after nanoparticles injection versus externally-applied nanoparticles derived from either polystyrene or polyacrylonitrile. METHODS: Each mouse 1-cell embryo (the selected test-model) was either: (a) injected by intracytoplasmic injection or (b) co-incubated with different nanoparticles at 37 degrees C, 5% CO2 in air. The embryos were assessed after 2 and 6 days of culture. RESULTS: Embryo development was similar for externally-applied polystyrene nanoparticles and control (97.6 +/- 2.7 versus 100.0 +/- 0%) but different for polyacrylonitrile nanoparticles (90.0 +/- 2.8 %) on day 2. However, the results were similar on Day 6. Injected embryos were linked to lower percent development on Day 2. Few injected embryos reached blastocyst stage on Day 6 after a brief UV-fluorescence exposure. CONCLUSIONS: Tagging embryos by external polystyrene-based nanoparticles was the better method when compared with injected nanoparticles. Larger nanoparticles in microsphere range were easier to qualitate. Inhibited hatching limited their use beyond the blastocyst stage.

Human papilloma virus DNA exposure and embryo survival is stage-specific.

PURPOSE: Human papillomavirus (HPV) has been shown to disrupt late-stage implanting embryos. The objectives were (a) to assess the development of early embryos exposed to HPV DNA and (b) to analyze the blastocyst hatching process after HPV exposure. METHODS: The study involved exposing two-cell and 4-8-cell mouse embryos to DNA fragments from either HPV type 16, type 18 or DQA1 (control). The embryos were incubated for 120 h and assessed. RESULTS: HPV 16 and 18 inhibited two-cell embryo development. In contrast, delaying the exposure of HPV DNA until the 4-8-cell stage resulted in further embryonic development. There was 25.9% less blastocyst formed with HPV 16 exposure. Additionally, there were 25.9-31.8% more degenerated embryos with HPV 16 exposure. CONCLUSIONS: The study demonstrated embryo stage-specific effects of HPV on early development. The results suggested HPV exposure was linked to two-cell embryo demise and delaying the exposure of HPV until later embryo stages permitted embryo development. HPV 16 was shown to decrease blastocyst formation while HPV 18 inhibited the blastocyst hatching process.

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.

Mouse embryonic stem cells for quality control testing in assisted reproductive technology programs.

OBJECTIVE: To compare the mouse embryonic stem (ES) cell assay with the sperm motility test or 1-cell mouse embryo bioassay for embryotoxic materials. STUDY DESIGN: Cryo-preserved-thawed mouse ES-D3 cells, 1-cell mouse embryos and donor sperm were incubated for 1-4 days in culture medium exposed to a control and 4 different test materials. ES cell viability (eosin method), apoptosis (Sybr-Gold fluorescence), development of blastocysts and sperm motility parameters were measured. RESULTS: The initial viabilities of ES cell were determined to be 37.0 +/- 4.2% (n = 225) and 54.8 +/- 7.4% (n = 218) by the eosin and Sybr Gold methods, respectively. Reduced viability of ES cells in latex glove-treated medium (25.6 +/- 0.3% and 25.7 +/- 0.3% versus control, 32.8 +/- 0.2% and 33.5 +/- 1.0% by eosin or Sybr Gold, respectively, p < 0.05) was consistent with standard bioassays. However, toxicity in the syringe was detected only by the ES cell assay. The ES cell assay sensitivities were 33% and 67% (eosin and Sybr Gold methods, respectively), and specificities were 100% for both methods. CONCLUSION: Mouse ES cell assay based on Sybr-Gold asssessment was as effective as standard bioassays for detecting embryotoxicity. The results suggested that the mouse ES assay could be used for testing contact materials and DNA-modifying agents. More studies are needed to refine and enhance the sensitivity of the ES cell assay for routine use in assisted reproductive technology clinics.

Luteal phase serum cell-free DNA as a marker of failed pregnancy after assisted reproductive technology.

PURPOSE: DNA-damaging factors have been reported in patients that failed to achieve pregnancy after assisted reproductive technologies (ART). The hypothesis was that increased circulating cell-free DNA released by damaged cells could predict unfavorable conditions leading to failed ART treatment. The objective was to compare the relative concentrations of cell-free DNA in the luteal phase sera of nonpregnant versus pregnant patients. METHODS: Frozen-thawed sera (30 IVF cases) were obtained 1 week after embryo transfer. There were 16 pregnant and 14 nonpregnant cases and controls consisting of male sera (n = 8 cases). Modified isocratic capillary electrophoresis was performed and the images analyzed for cell-free DNA. RESULTS: Circulating cell-free DNA were identified in the sera of all patients. The serum concentrations of high (12 kb) and low (1 kb) molecular weight cell-free DNA were similar for both nonpregnant and pregnant patients. Male control sera had higher cell-free DNA concentrations compared with females. Evaluation of sera from a control case showed no fluctuations in cell-free DNA concentrations throughout specific days of the menstrual cycle. CONCLUSIONS: The results do not support the use of the luteal phase cell-free DNA concentration as a marker for failed pregnancies. The equal concentrations of high and low molecular weight cell-free DNA and ladder band-like gel patterns suggested cell apoptosis as the source of DNA.

Modified isocratic capillary electrophoresis detection of cell-free DNA in semen.

PURPOSE: The objectives were: i) to analyze semen for the presence of cell-free DNA and ii) to determine the association between sperm parameters and cell-free DNA. METHODS: Cell-free DNA in semen (N = 25 cases) were detected using the modified capillary gel electrophoresis (CE) procedure. SYBR-Gold was used to stain high (12 Kb) and low (1 Kb) molecular weight DNA fragments and the images analyzed. RESULTS: The quantity of low-molecular weight cell-free DNA was positively correlated to rapid progression, curvilinear velocity (>40 microm/s), normal strict morphology and capacitation index. High-molecular weight cell-free DNA intensity index was negatively correlated to post-wash hyperactivation. Sperm concentration was not related to cell-free DNA quantity. The sperm freezing process did not increase cell-free DNA but reduced the more labile low-molecular weight cell-free DNA. CONCLUSIONS: Cell-free DNA present in semen was correlated to important sperm parameters linked to normal sperm function. The data suggested the possible use of cell-free DNA as a marker of semen quality. This study reports on the novel finding of cell-free DNA released along with sperm during each ejaculation.

Dual fluorescence analysis of DNA apoptosis in sperm.

OBJECTIVE: The objective was to compare fluorochrome Hoechst 33342 (Ho342) with combined Ho342/propidium iodide (PI) stains for assessment of sperm quality. STUDY DESIGN: Washed donor sperm cells were incubated in either 0, 0.15, or 15 micromol/L camptothecin (CAM) or 0.37 or 3.7 mmol/L genistein (GEN) for 4 hours at 37 degrees C. The sperm cells were analyzed for cycle-independent apoptosis and necrosis by single- compared with dual-stained fluorescence microscopy to contrast the relative effectiveness of these two approaches. RESULTS: The single-stain procedure did not detect viability differences (overall 76.1% +/- 2.2% live). In contrast, the dual-stain procedure identified a dose-dependent decrease in viability and increased necrozoospermia for CAM and GEN treatments. Apoptosis was 2-fold higher with topoisomerase inhibitor treatment. CONCLUSION: The two topoisomerase inhibitors were associated with increased apoptosis and dose-dependent necrosis. The data suggested that the dual-stain combination Ho342/PI was more sensitive than the single Ho342 stain analysis and permitted quantifying the apoptosis and necrosis events in sperm.

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.

Effect of pentoxifylline on tumor suppressor and proto-oncogene apoptosis in sperm.

PURPOSE: Pentoxifylline (PTX), a methylxanthine phosphodiesterase inhibitor reduces superoxide anions responsible for DNA apoptosis. The null hypothesis was that PTX was equally effective in reducing damage to specific cell genes. The objective was to determine the DNA integrity of the BRCA1 tumor suppressor gene and the c-myc proto-oncogene after PTX. METHODS: Sperm (64 samples, 4 patients) were preincubated in either 0 (control) or 3.6 mM PTX (30 min), washed and incubated for 4 h at either 37 or 40 degrees C heat shock activation. Single primer polymerase chain reactions (PCR) were carried out on lysed sperm targeting either BRCA1 exon 11 or c-myc exon 1. Control single-stranded DNA (ssDNA) were stained with 9 microM Hoechst 33342 (blue) while PTX-treated ssDNA were stained with SYBR Gold (green). Nytran membrane discs with control ssDNA were hybridized to PTX-derived ssDNA. Fluorescent images stored in a microarray design were analyzed using ANOVA and Students' t-test for (P < 0.05) significance. RESULTS: BRCA1 integrity was higher with PTX pretreatment (93.3 + 10.4 vs. control 50.5 + 9.2; mean + SEM). In contrast, there was no difference in c-myc integrity (56.8 + 9.0 vs. 41.7 + 6.4). Sense or antisense primers gave similar DNA fragmentation results. CONCLUSIONS: The data showed PTX pretreatment protected BRCA1 but not c-myc suggesting that PTX did not equally protect different cell genes. A possible explanation was that proto-oncogenes had more fragile sites. The study involved the DNA disc chip assay to assess separate PCR-amplified sense and antisense strands. The results suggested that both strands were equally affected by PTX pretreatment.

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.