体外操作对人精子运动参数及细胞内活性氧簇的影响

目的：研究体外物理操作（离心以及微量加样器抽吸）对人精子运动参数以及细胞内活性氧簇（ROS）的影响,旨在优化人精子体外处理方法。方法：7例精液常规参数正常的标本,采用不同离心力（200 g,600 g）及离心时间（5 min,15 min）以及不同微量加样器抽吸次数（2,6,10次）进行处理,测试精子运动参数和细胞内ROS的变化。结果：①活动精子百分比以及活精子细胞内ROS水平主要受到离心时间的影响（P＜0.05）;前向运动精子百分比（PR）以及平均运动速率（VAP）均不受离心力及离心时间的影响（P＞0.05）。单因素方差分析多重比较结果提示,当离心时间为5 min以上时精子活动力显著下降,而ROS水平显著增加（P＜0.05）。②加样器抽吸2次以上可致人精子活动力显著下降（P＜0.05）,抽吸6次以上可导致人精子PR及VAP显著降低（P＜0.05）;但抽吸操作并未显著影响精子细胞内ROS的水平。结论：在人精子体外操作时应尽量缩短离心时间并控制加样器抽吸次数从而保证正常的精子运动参数,也应尽量缩短离心时间从而减少体外操作所致的细胞内ROS升高。     

体外操作对小鼠精子参数及内源性ROS的影响

目的：研究体外物理操作对小鼠精子回收率、活动精子百分比、膜完整率以及细胞内活性氧簇（DCFMFI）的影响,探索最佳的小鼠精子体外处理方法。方法：采用不同离心力及离心时间（200,400,600,800×g;5,10,15,20 min）,1 mL微量加样器不同抽吸次数（2,4,6,8,10次）以及不同Percoll密度梯度离心条件（600,800,1 000,1 200×g;15,30 min）处理小鼠精子,探讨导致各指标变化的原因以及最优的处理条件。结果：析因分析结果提示,离心力及离心时间双重因素影响离心操作时精子回收率以及Percoll密度梯度离心活动精子百分比（P＜0.05）;离心力单一因素影响离心操作时活动精子百分比以及Percoll密度梯度离心精子膜完整率（P＜0.05）;加样抽吸操作可影响精子膜完整率以及活动精子百分比（P＜0.05）。离心操作时,当离心力大于600×g时活动精子百分比下降（P＜0.05）;加样器抽吸小鼠精子4次以上,小鼠精子膜完整率、活动精子百分比均下降（P＜0.05）;Percoll密度梯度离心时间30 min,离心力达到800×g以上活动精子百分比与对照组比较升高（P＜0.05）,离心力达到1 000×g以上时精子膜完整性与对照组比较降低（P＜0.05）。各组存活精子DCFMFI无变化（P＞0.05）。结论：在小鼠精子离心时,离心力应控制在600×g以下;抽吸时次数应控制在4次以下;Percoll密度梯度离心最优条件为800×g 30 min。     

Effect of density gradient centrifugation on reactive oxygen species in human semen

Density gradient centrifugation can separate motile sperm from immotile sperm and other cells for assisted reproduction, but may also remove antioxidants from seminal plasma, resulting in oxidative stress. Therefore, we investigated reactive oxygen species (ROS) concentrations and distribution in semen before and after density gradient centrifugation. We assessed semen volume, sperm concentration, sperm motility, and ROS levels before and after density gradient centrifugation (300 x g for 20 minutes) in 143 semen samples from 118 patients. The ROS removal rate was evaluated in ROS-positive samples and ROS formation rate in ROS-negative samples. Thirty-eight of 143 untreated samples (26.6%) were ROS-positive; sperm motility was significantly lower in these samples than in ROS-negative samples (p < 0.05). After density gradient centrifugation, only seven of the 38 ROS-positive samples (18.42%) exhibited a ROS-positive lower layer (containing motile sperm) with a ROS removal rate of 81.58%, whereas the upper layer was ROS-positive in 24 samples (63.16%). In the ROS-negative group (n = 105), ROS was detected in 19 samples after centrifugation (18.10%, ROS generation rate), of which 18 were ROS-positive only in the upper layer or interface and the other was ROS-positive in both layers. Density gradient centrifugation can separate motile sperm from immotile sperm as well as remove ROS (including newly generated ROS). This data supports the view that density gradient centrifugation can select motile spermatozoa without enhancing oxidative stress.

Abbreviations: ROS: reactive oxygen species; SOD: superoxide dismutase; GPx: glutathione peroxidase; DNA: deoxyribonucleic acid; DGC: density gradient centrifugation; IUI: intrauterine insemination; IVF: in vitro fertilization; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA: ethylenediaminetetraacetic acid; HTF: HEPES-buffered human tubal fluid; IMSI: intracytoplasmic morphologically selected sperm injection; SMAS: sperm motility analyzing system; CASA: computer-assisted semen analyzer; WHO: World Health Organization     

Relationship of seminal plasma antioxidants and serum male hormones with sperm chromatin status in male factor infertility

We explored the relationship between sperm chromatin integrity, hormone levels, seminal plasma total antioxidant capacity (TAC), and routine sperm parameters in men with male factor (MF, n?=?81) and non-male factor (NMF, n?=?52) infertility. Semen and blood were collected and examined from men undergoing evaluation for infertility in the Avicenna Infertility Clinic. We have examined each patient for serum hormones (LH, FSH, E2, DHEA), sperm chromatin damage, level of protamination and seminal plasma TAC. Levels of FSH, LH, sperm chromatin damage, and abnormal protamination were significantly higher in MF vs. NMF groups (p?<?0.001). Sperm chromatin damage was correlated with percentage of CMA₃- positive sperm (r?=?0.64, p?<?0.001) and with sperm concentration (r?=??0.36, p?<?0.001), motility (r?=??0.21, p?<?0.05), and morphologically normal spermatozoa (r?=??0.29, p?<?0.001). Linear regression showed sperm chromatin damage was related to percentage of CMA₃- positive sperm (p?<?0.001) in ungrouped patients. It was related to both percentage of CMA₃- positive sperm and serum DHEA in the MF group (p?<?0.001 and p?<?0.05, respectively). Sperm chromatin maturity assessed by CMA₃ test was inversely related to sperm chromatin damage assessed by the toludine blue assay. Male factor infertility associated with sperm chromatin damage may be related to sperm protamination and to serum DHEA.     

Evaluation of the Sperm Motility Analyzer System (SMAS) for the Assessment of Sperm Quality in Infertile Men

Eighty-one fresh semen samples were analyzed to compare the sperm parameters obtained using the new Sperm Motility Analysis System (SMAS; version 1.0, Kaga Electronics, Tokyo, Japan) with the CellSoft^TM Series 3000 (CRYO Resources, New York, USA) computer-assisted semen analysis (CASA) system and conventional manual semen analysis, based on WHO guidelines.. Significant correlations of sperm concentration (p<0.0001) and sperm motility (p<0.0001) were observed between SMAS and manual semen analysis estimates. There were also significant correlations of sperm concentration (p=0.0003) and sperm motility (p<0.0001) between SMAS and CellSoft estimates. Significant correlations for motility-related parameters were demonstrated in sperm velocity (p<0.0001), and linearity (linear velocity (VSL) divided by curvilinear velocity (VCL)×100) (p<0.0001), amplitude of lateral head displacement (ALH) (p<0.0001), and beat/cross frequency (BCF) (p=0.0127), between SMAS and CellSoft estimates. In this study, we showed the usefulness of the new SMAS, which has high reliability in estimating sperm concentration, sperm motility, velocity and linearity compared with CellSoft. SMAS can be a promising alternative, providing cost-effective semen analysis with the utility of the CASA system.     

Comparative study of sperm washing and selection methods after cryopreservation and its influence on sperm subpopulational structure in a bovine model

Abstract

The effect of different sperm washing-selection methods on sperm morphometric characteristics as a study to detect differences in the subpopulational structure has been carried out in detail in a bovine model. Cryopreserved sperm samples from 5 bulls were thawed, pooled, and processed by TALP-washing centrifugation method (TWCM), selective Percoll discontinuous density-gradient centrifugation method (PDGM), and self-migration swim-up separation method (SUMM). Live-dead assay (SYBR-14/ethidium homodimer-1), chlortetracycline assay (CTC), and sperm motility were assessed, and aliquots of sperm were processed for automated sperm morphometry analysis (ASMA) simultaneously before (raw thawed sperm used as control, RTS) and after different sperm washing-selection techniques. Deleterious effects of different methods were evident, particularly on sperm membrane integrity (p?<?0.05) and capacitation status (p?<?0.05). Moreover, each cell was measured for four primary dimensional parameters, and three shape parameters. All sperm morphometric parameters evaluated were analyzed by principal component analysis (PCA) and multivariate clustering analyses. PCA revealed two principal components for each sperm washing or separation method explaining more than the 91% of the variance. The number of subpopulations found was the same for all methods (four) except for PDGM (three). However, irrespective of the number of subpopulations defined by PCA and clustering analyses, the sperm subpopulational structure was found to be different and strongly influenced by the sperm selection procedure due to statistical differences found regarding the sperm biophysical changes induced by each method used (p?<?0.001). It is concluded that different sperm washing-selection methods commonly used during IVF process, may lead to alterations in sperm morphometric characteristics, which might explain the different results seen after IVF, since an important influence of these methods on sperm subpopulational structure has been demonstrated.     

A novel sorting technology allows for highly efficient selection of sperm without chromatin damage

Sperm chromatin damage has been associated with male infertility, increased risk for spontaneous abortion, and poor embryo development. Available methods for detecting chromatin damage render the sperm no longer suitable for clinical use. Early apoptotic events resulting in chromatin damage are associated with increased permeability of the cell membrane to large ions. We propose the use of a large fluorescent organic cation, proprietary fluorochrome (PF-1), for fluorescence-activated cell sorting (FACS) for negative selection of sperm without chromatin damage. Sperm with chromatin damage are PF-1 positive. Performance of cell sorting by PF-1 was verified with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) after FACS on PF-1(+) and PF-1(-) subpopulations. Whereas 19.5% of PF-1 positive sperm were TUNEL positive only 1.5% sperm in the PF-1(-) fraction were TUNEL positive (p?<?0.00001). TUNEL values below 1.9% were considered background fluorescence. Post-sorting motility and vitality were 49.4% (SD: 12.5) and 65.0% (SD: 14.99), respectively. Proprietary fluorochrome activated sperm sorting may decrease or most likely eliminate all of TUNEL positive sperm without adverse effects on viability, providing a new therapeutic avenue for men with a high percentage of TUNEL positive sperm. Further research is needed to determine if the reduction in TUNEL positive sperm using PF-1 will improve in vitro fertilization (IVF) outcomes.     

Adding bovine seminal plasma prior to freezing improves post-thaw bull sperm kinematics but decreases mitochondrial activity

Variations in fertility between bulls with comparable sperm quality could be due to differences in their seminal plasma (SP). The aim of this study was to investigate the effect of adding bovine SP from bulls of known fertility to SP-free sperm samples. After removal of SP by Single Layer Centrifugation, resuspended sperm pellets were treated with SP from high or low fertility bulls at 0% (control), 1%, or 5% before freezing. Sperm quality was evaluated after thawing. Data were analyzed using Proc MIXED, SAS®. Bovine SP at 1% or 5% SP1 and SP5, respectively, decreased average path velocity, curvilinear velocity, and amplitude of lateral head displacement whereas wobble and linearity were increased. In addition, the proportion of spermatozoa with high mitochondrial membrane potential (MMP) was lowest for treatment with SP5 compared to SP1 and control. The proportion of SP did not affect other parameters of sperm quality. Thus, adding 5% bovine SP produced a favorable effect on some sperm velocity parameters but had an unfavorable effect on MMP. There were no differences in effect between SP from high and low fertility bulls.

Abbreviations: AI: artificial insemination; BCF: beat cross frequency; CASA: computer-assisted sperm analysis; IVF: in vitro fertilization; MMP: mitochondrial membrane potential; SLC: single layer centrifugation; SP: seminal plasma     

Sperm DNA Tests as Useful Adjuncts to Semen Analysis

Male infertility has traditionally been diagnosed by microscopic assessment of concentration, motility and morphology of sperm in the ejaculate. Most laboratories use sperm isolated by various methods such as density gradient centrifugation to enrich for subpopulations of sperm believed to have greater fertilization potential. These tests are essential to provide the fundamental information on which clinicians base their initial diagnosis. However, in the clinical setting, tests with superior prognostic value are needed. Tests showing much promise are those determining sperm DNA integrity, particularly the Comet, TUNEL, and Sperm Chromatin Structure assays.

Sperm nuclear DNA fragmentation has been positively correlated with lower fertilization rates in IVF, impaired implantation rates, an increased incidence of abortion and disease in offspring, including childhood cancer. The mitochondrial genome of sperm has also been shown to be a sensitive marker of sperm health. Although the usefulness of these tests is recognized, insufficient resources have been available to develop standardized tests and protocols that could lead to universally accepted clinical thresholds. Associated with the lack of useful prognostic tests is the lack of improvement in assisted conception success rates despite thirty years of worldwide use. International collaborations should be initiated to develop agreed protocols and establish clinical thresholds.     

Sperm DNA tests as useful adjuncts to semen analysis

Male infertility has traditionally been diagnosed by microscopic assessment of concentration, motility and morphology of sperm in the ejaculate. Most laboratories use sperm isolated by various methods such as density gradient centrifugation to enrich for subpopulations of sperm believed to have greater fertilization potential. These tests are essential to provide the fundamental information on which clinicians base their initial diagnosis. However, in the clinical setting, tests with superior prognostic value are needed. Tests showing much promise are those determining sperm DNA integrity, particularly the Comet, TUNEL, and Sperm Chromatin Structure assays. Sperm nuclear DNA fragmentation has been positively correlated with lower fertilization rates in IVF, impaired implantation rates, an increased incidence of abortion and disease in offspring, including childhood cancer. The mitochondrial genome of sperm has also been shown to be a sensitive marker of sperm health. Although the usefulness of these tests is recognized, insufficient resources have been available to develop standardized tests and protocols that could lead to universally accepted clinical thresholds. Associated with the lack of useful prognostic tests is the lack of improvement in assisted conception success rates despite thirty years of worldwide use. International collaborations should be initiated to develop agreed protocols and establish clinical thresholds.     

Sperm DNA damage or progressive motility: which one is the better predictor of fertilization in vitro?

Sperm progressive motility has been reported to be one of the key factors influencing in vitro fertilization rates. However, recent studies have shown that sperm DNA fragmentation is a more robust predictor of assisted reproductive outcomes including reduced fertilization rates, embryo quality, and pregnancy rates. This study aimed to compare the usefulness of sperm progressive motility and DNA damage as predictive tools of in vitro fertilization rates. Here, 136 couples provided 1,767 eggs with an overall fertilization rate of 64.2%. The fertilization rate in vitro correlated with both sperm progressive motility (r2?=?0.236; P?=?0.002) and DNA fragmentation (r2?=?-0.318; P?40%) compared with 2.6 times in sperm with poor motility (<40%). Further, sperm DNA fragmentation gave a higher specificity (93.3%) in predicting the fertilization rate than progressive motility (77.8%). Finally, the odds ratio to determine fertilization rate (>70%) was 4.81 (1.89-12.65) using progressive motility compared with 24.18 (5.21-154.51) using DNA fragmentation. This study shows that fertilization rates are directly dependent upon both sperm progressive motility and DNA fragmentation, but sperm DNA fragmentation is a much stronger test.     

Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa

In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved samples staining positive with propidium iodide, Hoechst H33258 and Trypan blue; these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1 : 6 or 1 : 20) with or without 0.1-5.0 mM theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800 g were associated with a significant reduction in motility (mean 56 +/- 3% decreasing to 27 +/- 3%), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures; osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates; and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mM theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.     

Ultraviolet damages sperm mitochondrial function and membrane integrity in the sea urchin Anthocidaris crassispina

Effects of ultraviolet A (UVA) and ultraviolet B (UVB) on mitochondrial function and membrane integrity of sea urchin sperm were investigated using flow cytometry and fluorescent probes. Both UVA and UVB impaired sperm mitochondrial function in a dose-dependent manner. Covariance analysis further showed that the slopes of change in mitochondrial function in relation to UVA and UVB were significantly different, suggesting that the modes of action were different. UVA did not affect membrane integrity, while membrane integrity showed a linear reduction with increasing UVB doses. Sperm mitochondria function showed significant positive correlations with sperm motility and subsequent fertilization success. Overall, our results showed that both UVA and UVB could decrease sperm motility and fertilization success through impairment of mitochondrial function, whereas UVB alone could cause additional damage through impairing the functional integrity of sperm membrane. Mitochondrial function of sperm may also offer a reliable ecotoxicological biomarker for predicting fertilization success in urchins.     

Impact of Sperm Collection Methods on Sperm Parameters in Spinal Cord Injured Men and Compared to Normal Controls in ICSI Program

Background To determine and compare the effect of electro ejaculation (EE), Masturbations, Penile vibratory stimulation (PVS), Percutaneous Epididymal Sperm Aspiration (PESA) on the quality and quantity of sperm parameters in the aspect of assisted reproduction.Methods A retrospective study on collected semen of 89 spinal cords injured (SCI) men (EE = 70, Mast = 3, PVS = 6, PESA = 10 individuals) and 49 neurologically intact men (EE = 5, Mast = 14, PVS = 30) with different methods were included and intracytoplasmic sperm injection (ICSI) with homologue oocytes were performed.Results The quality and quantity of semen in SCI was significantly lower than neurologically intact men (P < 0.01), and showed reduction of sperm parameters in compare with normal group including; 23.19% in motility, 14.56 % in normal morphology, and 41.51%in viability (P < 0.001). Within normal group the EE method has significantly influence on sperm parameters (P < 0.01). In SCI group the morphology was severely impaired. Hyper leukocytes significantly increased (P < 0.01). The fertilization rate of EE/ICSI was 60% which was lower than other methods of semen collection (71.7%).Conclusions The EE method of semen collection compared with PVS and other methods of normal ejaculatory stimulation showed significantly lower grade of sperm parameters. EE/ICSI is effective in treating an ejaculatory infertility. For ejaculatory stimulation it is recommended to Consider PVS as the first line of treatment in SCI men. PESA can be considered when EE and PVS both fail to cause seminal emission.     

吸烟对精子DNA完整性、精子参数的影响

目的:探讨吸烟对精子DNA完整性、精子参数的影响。方法:调查191例男性不育患者的吸烟情况,采用吖啶橙荧光染色后检测精子DNA完整性,计算机自动分析精子密度与活力,精子形态检测系统下人工修正方法进行精子形态分析。结果:吸烟组精子活力显著低于不吸烟组(P<0.05),精子密度和形态低于不吸烟组,但差异无统计学意义(P>0.05)。吸烟组精子DNA完整率异常例数显著高于不吸烟组(χ2=5.393,P<0.05),精子DNA完整率异常组精子密度显著低于精子DNA完整率正常组(P<0.05),精子DNA完整率异常组的精子活力、形态与正常组相比差异无统计学意义(P>0.05)。结论:吸烟影响精子DNA完整率;精子DNA完整率异常与精子参数异常相关;吸烟可能通过影响精子DNA完整性影响精子参数。     

Mechanisms of oligozoospermia: an oxidative stress perspective

Abstract

Infertile patients presenting with poor semen concentration, motility, and morphology have significantly higher levels of reactive oxygen species (ROS). In this cross-sectional study, our goal was to: 1) determine how semen parameters such as concentration, motility, morphology, as well as ROS are altered in oligozoospermic men alone and those in combination with poor sperm concentration, motility, morphology, and compare these with three control groups; unproven donors, donors with proven fertility <2 years, and proven donors with fertility established >2 years; 2) establish the cutoff, sensitivity, specificity for ROS, and see how it compared in the three donor groups compared to the patient groups, and 3) establish the reference range for the various semen parameters in these three donor groups compared to the oligozoospermic group by generating receiver operating characteristic (ROC) curves for each parameter. Fifty-six donors and 101 infertile men were included in this study. The patient group included: oligozoospermia: n?=?16; oligoasthenozoospermia (OA): n?=?12; oligoteratozoospermia (OT): n?=?19; oligoasthenoteratozoospermia (OAT): n?=?54. The patient group was compared with the three donor groups. Proven donors who had established a pregnancy in the past or recently (<2 years) had significantly improved semen motility and morphology compared to the OAT and OT groups (p?<?0.001). In the OAT group, normal morphology was positively correlated with concentration and negatively with levels of ROS. Compared to the three donor groups, oligozoospermic patients in the OAT, OT, and OA groups had significantly elevated ROS levels. The cutoff for ROS in the proven donors < 2 years was significantly lower with a higher sensitivity and specificity compared to the unproven donors and donors who had not established a recent pregnancy. These results indicate a positive association between semen parameters and ROS suggesting a common underlying mechanism in these infertile patient groups.     

PHOSPHOLIPID COMPOSITION OF HUMAN SPERM AND SEMINAL PLASMA IN RELATION TO SPERM FERTILITY

The phospholipid and fatty acid composition of sperm was studied in 8 healthy and 16 infertile men. Infertile men randomly formed from the patients with normal semen parameters according to WHO criterion. Therefore, all semen parameters of infertile patients were similar to the same characteristics of the semen of healthy men, except the abnormal forms. The amount of abnormal forms in infertile men was significantly higher than in healthy men. Sperm from infertile men show a drastic loss of phosphatidyl ethanolamine. At the same time, the significant increase of phosphatidyl serine in the sperm and seminal plasma of sterile patients was found. Lysophosphatidyl serine in the sperm of the infertile men was detected. Fatty acid composition of the semen of infertile men was altered. The levels of stearic and n-3 polyunsaturated fatty acids (eicosopentaenoic and docosahexaenoic acids) was dramatically lowered, but the values of some n-6 polyunsaturated fatty acids (linolenic and docosatetraenoic) acids increased. There was significant positive correlation between docosahexaenoic acid and sperm motility (r =. 82, p &lt;. 001) and negative correlation between linolenic acid and spermatozoa motility (r=-0.58, p&lt;.05). Infertility of men with normal semen quality can originate from the disorder of sperm lipid metabolism. The drastic loss of phosphatidyl ethanolamine and n-3 polyunsaturated fatty acids with simultaneous enhancement of phosphatidyl serine and some n - 6 polyunsaturated fatty acids in sperm could be an important cause of male infertility.     

Combination of Hypoosmotic Swelling/Eosin Y Test for Sperm Membrane Integrity Evaluation: Correlations with Other Sperm Parameters to Predict ICSI Cycles

The objective of our study was to evaluate the accuracy of the combination of hypoosmotic swelling (HOS) and eosin Y (Ey) exclusion tests to predict the ICSI cycles' outcome and its correlations with other sperm parameters.

The functional and structural integrity of sperm membrane was evaluated with the combined HOS/Ey test in 95 ICSI cycles and the results were correlated with other sperm parameters, including concentration, motility, strict morphology, and total motile sperm count. The combined HOS/Ey test was evaluated for the prediction of the ICSI cycles' outcome parameters including fertilization, cleavage, and pregnancy rates. The HOS/Ey test presented significant relationships with concentration, motility, and strict morphology (p < 0,0001) but it couldn't predict the fertilization, cleavage, and pregnancy outcomes of ICSI cycles. The combined HOS/Ey test has strong correlations with motility and strict morphology parameters of sperm samples but is not sufficiently sensitive to estimate the outcome of ICSI cycles.     

Shortening gametes co-incubation time improves live birth rate for couples with a history of fragmented embryos

Short gamete co-incubation (SGCO) consists in decreasing the duration of contact between oocytes and sperm from the standard overnight insemination (SOI) toward 2 hours. However, the effectiveness of this technique to improve in vitro fertilization and embryo transfer (IVF-ET) outcomes remains controversial. Our study was designed to evaluate the efficiency of SGCO in a poor prognosis population with a history of fragmented embryos defined by the presence of at least 50% of the embryos with more than 25% of cytoplasmic fragments. From January 2010 to January 2014, 97 couples were included in a SGCO protocol. We separated women into 2 subgroups: younger and older than 35 years. Compared to SOI, after SGCO, 2-cell stage embryos were higher in all women (p<0.001) and less fragmented in women over 35 years (p<0.05). On day 2, top quality embryos obtained and transferred were higher with SCGO than with SOI, independently of the age of the women (p<0.001). Moreover, the number of embryos with less than 25% of fragmentation was higher after SGCO than SOI (p<0.001) whereas the number of multinucleated embryos was lower (p<0.001). We observed that after fresh ET, independently of the age of the women, the clinical pregnancy rate was 3 times higher after SGCO than after SOI. However, the live-birth rate was 4 times higher with SGCO than with SOI in women above 35 years but 3 times higher with SGCO than with SOI in women younger than 35 years. The present results indicate that for a particular indication, reducing the time of oocytes and sperm co-incubation may improve IVF-ET outcomes in terms of live-birth rate.

Abbreviations: AMH: anti mullerian hormone; COC: cumulus-oocytes complex; E2: estradiol; ET: embryo transfer; FET: frozen embryo transfer; FSH: follicle stimulating hormone; GnRH: gonadotrophin releasing hormone; hCG: human chorionic gonadotropin hormone; hMG: human menopausal gonadotropin hormone; IRB: institutional review board; IVF: in vitro fertilization; IVF-ET: in vitro fertilization and embryo transfer; MNB: multinucleated blastomere; mRNA: messanger ribonucleic acid; OC: oocyte retrieval; O2: oxygen; ROS: reactive oxygen species; SGCO: short gamete co-incubation; SOI: standard overnight insemination.     

Concentration-dependent Sildenafil citrate (Viagra) effects on ROS production,energy status,and human sperm function

Abstract

Literature regarding the effects of sildenafil citrate on sperm function remains controversial. In the present study, we specifically wanted to determine if mitochondrial dysfunction, namely membrane potential, reactive oxygen species production, and changes in energy content, are involved in in vitro sildenafil-induced alterations of human sperm function. Sperm samples of healthy men were incubated in the presence of 0.03, 0.3, and 3?μM sildenafil citrate in a phosphate buffered saline (PBS)-based medium for 2, 3, 12, and 24 hours. Sperm motility and viability were evaluated and mitochondrial function, i.e., mitochondrial membrane potential and mitochondrial superoxide production were assessed using flow-cytometry. Additionally, adenosine triphosphate (ATP) levels were determined by high performance liquid chromatography (HPLC) analysis. Results show a decrease in sperm motility correlated with the level of mitochondria-generated superoxide, without a visible effect on mitochondrial membrane potential or viability upon exposure to sildenafil. The effect on both motility and superoxide production was higher for the intermediate concentration of sildenafil (0.3?µM) indicating that the in vitro effects of sildenafil on human sperm do not vary linearly with drug concentration. Adenosine triphosphate levels also decreased following sildenafil exposure, but this decrease was only detected after a decrease in motility was already evident. These results suggest that along with the level of ATP and mitochondrial function other factors are involved in the early sildenafil-mediated decline in sperm motility. However, the further decrease in ATP levels and increase in mitochondria-generated reactive oxygen species after 24 hours of exposure might further contribute towards declining sperm motility.