ICSI outcomes in men undergoing TESE for azoospermia and impact of maternal age.

This retrospective study compared clinical outcomes in men with obstructive and nonobstructive azoospermia after ICSI following testicular sperm extraction and the influence of maternal age. Fertilisation rates, embryo quality, pregnancy rates, miscarriage rates and live birth rates were evaluated. Men with obstructive azoospermia (OA) had significantly higher rates of diploid fertilisation and clinical pregnancy than men with nonobstructive azoospermia (NOA), but miscarriage rates and live birth rates were not significantly different. The higher rates of fertilisation, embryo quality and clinical pregnancy in men with OA were statistically significant when their female partners were <35 years but results were similar in both groups when female partners ≥35 years. Although the OA group had better overall quality embryos than the NOA group when maternal age was <35 years, embryologists can select the morphologically better embryos for transfer, eliminating the effect of embryo quality differences present in these two groups. Understanding more about factors that affect TESE/ICSI outcomes will not only help us predict patients' outcomes but it can help us educate and better counsel our patients.

Intravascular lipoma of the renal vein.

Lipomas are benign neoplasms composed of adipocytes encased in a fibrous capsule. Intravascular lipomas are rare and almost always incidental findings. In the published literature, the majority are described within the inferior vena cava (IVC) and less frequently reported in the superior vena cava, brachiocephalic vein, subclavian vein, internal jugular vein, external iliac vein and common femoral vein. We present the case of a 59-year-old male who presented with a symptomatic ureteral calculus and was found to have an intravascular lipoma of the right renal vein with extension into the IVC. To our knowledge, this is the first ever report of an intravascular lipoma in the renal vein. We discuss the imaging characteristics of intravascular lipomas and the differential diagnosis that should be considered.

Complete globozoospermia associated with PLCζ deficiency treated with calcium ionophore and ICSI results in pregnancy.

Globozoospermia is an infrequent pathology in which spermatozoa lack acrosomes. Patients are considered sterile without IVF augmented with intracytoplasmic sperm injection (ICSI), as fertilization is impaired due to absence of oocyte activation. As far as is known, this is the first study to report results of a comprehensive approach to the treatment of the semen parameters, sperm DNA fragmentation, aneuploidy, transmission electron microscopy, Western blotting and immunofluorescence for detection of phospholipase C zeta (PLCzeta), as well as ICSI outcome, of an affected patient. Morphological evaluation and transmission electron microscopy revealed complete globozoospermia with significant duplicate heads and tails. Analysis for DNA damage revealed fragmentation rates of approximately 80% in semen and 15-23% in swim-up fractions. PLCzeta was not detected by immunofluorescence or Western blotting. Aneuploidy rates were within normal ranges. ICSI followed by oocyte activation with calcium ionophore resulted in high rates of fertilization, and an ongoing pregnancy was established after transfer of cryopreserved-thawed embryos.

Differential laminar effects of amphetamine on prefrontal parvalbumin interneurons.

The increase in excitatory outflow from the medial prefrontal cortex is critical to the development of sensitization to amphetamine. There is evidence that psychostimulant-induced changes in dopamine-GABA interactions are key to understanding the behaviorally sensitized response. The objective of this study was to characterize the effects of different amphetamine paradigms on the Fos activation of GABAergic interneurons that contain parvalbumin in the medial prefrontal cortex. Although a sensitizing, repeated regimen of amphetamine induced Fos in all cortical layers, only layer V parvalbumin-immunolabeled cells were activated in the infralimbic and prelimbic cortices. Repeated amphetamine treatment was also associated with a loss of parvalbumin immunoreactivity in layer V, but only in the prelimbic cortex. An acute amphetamine injection to naive rats was associated with an increase in Fos, but in parvalbumin-positive neurons of the prelimbic cortex, where it was preferentially induced in layer III. These data indicate that distinct substrates mediate the response to repeated or acute amphetamine treatment. They also suggest that a sensitizing amphetamine regimen directs medial prefrontal cortex (mPFC) outflow, via changes in inhibitory neuron activation, toward subcortical centers important in reward.

Testis/sperm-specific histone 2B in the sperm of donors and subfertile patients: variability and relation to chromatin packaging.

BACKGROUND: The compaction of human sperm chromatin is the result of replacement of approximately 85% of histones with protamines. Germ-line testis/sperm-specific histone 2B (TSH2B) has been detected in only approximately 30% of mature spermatozoa. Its level in the semen of subfertile patients varies; its function is unknown. We evaluated TSH2B in the sperm samples of 23 donors and 49 subfertile patients and assessed its association with chromatin compaction status. METHODS: TSH2B level was measured using immunoblotting. Chromatin packaging quality was evaluated by staining with chromomycin A3 (CMA3) which marked spermatozoa with defective packaging. To assess both TSH2B and chromatin status in the same spermatozoon, CMA3 staining and TSH2B immunolocalization were performed sequentially. RESULTS: A significant correlation (r = 0.55, P = 0.0027) was found between TSH2B level and percentage of CMA3-positive sperm in patient and donor semen samples. When individual spermatozoa were assessed for these parameters, 92% of TSH2B-containing cells were also CMA3 positive. Variation in the total sperm TSH2B level was less in donors than in patients. CONCLUSIONS: CMA3 positive staining of TSH2B-containing individual spermatozoa and a significant correlation between the total TSH2B level and CMA3 percentage in semen samples suggest a structural role for TSH2B in sperm chromatin organization. Low variability of TSH2B level in donors implies a mechanism (however unknown) regulating this parameter.

Somatic cell apoptosis markers and pathways in human ejaculated sperm: potential utility as indicators of sperm quality.

In this study we extended earlier work to determine whether sperm respond to somatic cell apoptotic stimuli and whether apoptotic phenotypes are significant indicators of human sperm quality. We evaluated ejaculated sperm from fertile donors and subfertile patients following purification of fractions of high and low motility. In unstimulated conditions, caspase enzymatic activity was higher in motile fractions from subfertile patients than in donors, and was higher in low motility fractions from both groups. Staurosporine, but not a Fas ligand or H2O2, significantly increased caspase activity, but only in high motility fractions. Procaspase-3, -7 and -9 and low levels of active caspase-3, -7 and -9 were identified by immunoblot analysis. Apoptosis-inducing factor (AIF) was present in all samples but poly ADP-ribose polymerase-1 (PARP-1) was not detected. Phosphatidylserine translocation was significantly increased only with H2O2 treatment. In ejaculates of both subfertile and fertile men, we demonstrated the presence and activation of several proteins that are key constituents of apoptosis-related pathways in somatic cells, which may serve as markers for sperm quality.

Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study.

BACKGROUND: We aimed to investigate whether sperm DNA quality may predict intrauterine insemination (IUI) outcome. METHODS: The study was designed in a prospective cohort fashion, at a tertiary centre for reproductive medicine. A total of 119 patients underwent 154 cycles of IUI. Parameters related to demography, cycle management and semen sample used for IUI were evaluated. Conventional semen parameters, morphology (strict criteria), sperm DNA fragmentation and stability [evaluated by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and acridine orange staining under both acid and acid + heat denaturing conditions respectively] were measured. The main outcome measure was clinical pregnancy, defined as ultrasonographic visualization of intrauterine gestational sac(s). RESULTS: Logistic regression analyses were done on six sets of data, including all cycles combined, cycles with washed samples, first cycle of each couple, first cycle of each couple with washed samples, cycles stimulated with gonadotrophins and finally gonadotrophin-stimulated cycles with washed samples. The number of pre-ovulatory follicles on day of hCG, the age of the woman and the percentage of sperm with acid- + heat-resistant DNA were the parameters that predicted IUI outcome in most of these data subsets. For the gonadotrophin-stimulated cycles, age of the man appeared as a predictor as opposed to that of the woman; and for the cycles within this subgroup, where the semen sample was washed, sperm DNA fragmentation and age of the man were the only two parameters to predict IUI outcome. No samples with >12% of sperm having DNA fragmentation resulted in pregnancy. CONCLUSIONS: The number of follicles, age of the woman/man and sperm DNA quality may predict IUI outcome.

Global transcriptional response of Bacillus subtilis to heat shock.

In response to heat stress, Bacillus subtilis activates the transcription of well over 100 different genes. Many of these genes are members of a general stress response regulon controlled by the secondary sigma factor, sigma(B), while others are under control of the HrcA or CtsR heat shock regulators. We have used DNA microarrays to monitor the global transcriptional response to heat shock. We find strong induction of known sigma(B)-dependent genes with a characteristic rapid induction followed by a return to near prestimulus levels. The HrcA and CtsR regulons are also induced, but with somewhat slower kinetics. Analysis of DNA sequences proximal to newly identified heat-induced genes leads us to propose ~70 additional members of the sigma(B) regulon. We have also identified numerous heat-induced genes that are not members of known heat shock regulons. Notably, we observe very strong induction of arginine biosynthesis and transport operons. Induction of several genes was confirmed by quantitative reverse transcriptase PCR. In addition, the transcriptional responses measured by microarray hybridization compare favorably with the numerous previous studies of heat shock in this organism. Since many different conditions elicit both specific and general stress responses, knowledge of the heat-induced general stress response reported here will be helpful for interpreting future microarray studies of other stress responses.

Cryopreservation of fractionated, highly motile human spermatozoa: effect on membrane phosphatidylserine externalization and lipid peroxidation.

INTRODUCTION: This study investigated lipid peroxidation (LPO) and membrane integrity following cryopreservation-thawing. METHODS: Infertile men (study group) and donors (control group) were examined. Purified populations of highly motile spermatozoa were cryopreserved using TEST-yolk buffer and glycerol (TYB-G) followed by quick thaw. LPO was measured by a spectrophotometric assay, with and without a ferrous ion promoter. Annexin V binding was used to assess membrane translocation of phosphatidylserine (PS). RESULTS: Pre-freeze LPO was significantly higher in the study than in the control group (P = 0.03). In both groups, LPO measurements after thawing were significantly higher than the pre-freeze samples not exposed to TYB-G (P = 0.002 and P = 0.001 respectively). However, when the pre-freeze samples with TYB-G were compared with the post-thaw samples (all exposed to TYB-G), these differences were not significant. There was a significant increase in PS externalization following cryopreservation in both groups (P = 0.02 and P = 0.003 respectively). In donors, pre-freeze LPO concentrations had a significant positive correlation with thawed spermatozoa depicting PS externalization (r = 0.77, P = 0.04). CONCLUSION: Although patients had higher basal LPO than donors, LPO did not differ between fresh and cryopreserved-thawed fractionated motile spermatozoa. Freezing-thawing was associated with translocation of PS to the external membrane leaflet.

Outcome of intracytoplasmic sperm injection in azoospermic patients: stressing the liaison between the urologist and reproductive medicine specialist.

OBJECTIVES: To analyze the outcome of intracytoplasmic sperm injection (ICSI) cycles in infertile couples in whom the main diagnosis of infertility was azoospermia of obstructive and nonobstructive origin. METHODS: Eighty-three consecutive ICSI cycles were carried out with retrieved testicular or epididymal spermatozoa, 60 cycles in 32 patients with obstructive azoospermia and 23 cycles in 12 patients with nonobstructive azoospermia. Fifty-four testicular biopsies (testicular sperm extraction) and 18 epididymal aspirations (microepididymal sperm aspiration) were performed.Results. Motile spermatozoa were recovered in 65 cycles (90.3%). In another 3 (4.2%), nonmotile spermatozoa were retrieved. In 4 patients (5.5%), sperm could not be recovered. In 11 cycles, frozen sperm from a previous procedure were used. A significantly lower fertilization rate (64% versus 73%, P = 0.02), clinical pregnancy rate (13% versus 47%, P <0.001), and good embryo quality rates (35% versus 56%, P = 0.009) were observed in patients with nonobstructive azoospermia. In patients with obstructive azoospermia, no significant differences were observed when the outcome was analyzed on the basis of the sperm origin (ie, from testicular sperm extraction or microepididymal sperm aspiration). CONCLUSIONS: When combining testicular sperm extraction or microepididymal sperm aspiration with ICSI in patients with obstructive azoospermia, the results in terms of fertilization, implantation, and pregnancy rates were similar to those found in patients with nonazoospermic obstruction who underwent ICSI with ejaculated sperm. Patients with nonobstructive azoospermia had lower fertilization, embryo quality, and pregnancy rates than did those with obstructive azoospermia, probably because of severe defects in spermatogenesis, leading to poor gamete quality. The urologist and reproductive endocrinologist now have an excellent therapeutic option to offer men with previously intractable infertility.

Cryopreservation-Thawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation.

The objective of these studies was to evaluate the effect of cryopreservation-thawing of human spermatozoa on DNA fragmentation and membrane integrity. This was a prospective, controlled cohort study, performed at a university-based infertility center. Ejaculates were examined from 5 donors and 16 men undergoing infertility evaluation. Purified sperm populations were prepared by gradient centrifugation, cryopreserved using a manual method and TEST-yolk buffer and glycerol (TYB-G), followed by quick-thaw. Annexin V binding was used for assessing membrane translocation of phosphatidylserine, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was utilized for the evaluation of DNA fragmentation. The results were as follows: the percentage of live cells with intact membranes (annexin V-, live) was significantly reduced after cryopreservation-thawing. On the other hand, the percentages of live cells with phosphatidylserine translocation (annexin V-, live) and of necrotic (dead) cells increased significantly after thawing. TUNEL revealed percentages of cells with DNA fragmentation in the prefreeze and postthaw samples that were not significantly different. In a further attempt to examine differences in response to various cryoprotection protocols, experiments were carried out using no cryoprotection, glycerol alone, or TYB-G. Samples frozen with TYB-G demonstrated significantly higher percentages of live cells without phosphatidylserine translocation than the other conditions. We concluded that cryopreservation-thawing of human sperm from patients and donors was associated with membrane change, as revealed by membrane translocation of phosphatidylserine, while having no major impact on DNA fragmentation.

Cryopreservation-thawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine.

OBJECTIVE(S): [1] To evaluate sperm membrane damage during cryopreservation-thawing by the assessment of phosphatidylserine (PS) translocation and [2] to examine the relationship between reactive oxygen species (ROS) and cryopreservation-related alterations. DESIGN: Prospective cohort study. SETTING: University-based center. PATIENT(S): Men consulting for infertility and fertile donors (controls). INTERVENTION(S): Semen processing was performed by density gradient separation followed by cryopreservation and thawing. MAIN OUTCOME MEASURE(S): Membrane PS translocation was evaluated with annexin V binding, generation of ROS was detected by chemiluminescence, and motion parameters were assessed by computer analysis. RESULT(S): Annexin V binding was detected in the prefreeze fractions with high and low sperm motility. In the patient group, there were significantly higher postthaw levels of annexin V binding in both fractions when compared with prefreezing values. However, such induction of PS translocation was significantly higher in the fractions with high sperm motility. Significantly higher ROS levels were detected in prefreeze samples of the fractions with low sperm motility. CONCLUSION(S): In the population of men studied, [1] cryopreservation-thawing was associated with induction of membrane PS translocation; [2] postthaw ROS levels were lower than before freezing; and [3] neither annexin V binding results nor the generation of ROS were able to accurately predict sperm cryosurvival rates.

Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa.

OBJECTIVE: To evaluate the effects of oxidative stress on DNA and plasma membrane integrity of human spermatozoa. DESIGN: Prospective cohort study. SETTING: University-based, tertiary-care infertility center. PATIENT(S): Men (n = 10) undergoing infertility investigation. INTERVENTION(S): Purified populations of sperm with high motility were separated using Percoll density gradients. Then, spermatozoa were incubated with 0, 10, 100, and 200 microM hydrogen peroxide (H(2)O(2)) under capacitating conditions. MAIN OUTCOME MEASURE(S): Motion parameters were assessed by computer analysis. Genomic integrity was examined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay. Plasma membrane integrity was evaluated by the annexin V-binding assay, a measure of phosphatidylserine translocation. RESULT(S): Under basal conditions, there was a significant and negative relationship between sperm motility and the percentages of sperm with DNA fragmentation and membrane translocation of phosphatidylserine. After a 2-h incubation, there was a significant, dose-dependent effect of H(2)O(2) on motion parameters (decrease) and DNA fragmentation (increase). The percentage of annexin V(-) live (normal) cells declined significantly as the level of oxidative stress increased. Although the percentages of annexin V(+) live cells (sperm depicting translocation of phosphatidylserine) and necrotic cells increased at the highest H(2)O(2) levels, these changes were not significant. CONCLUSION(S): In vitro sperm incubation with H(2)O(2) induces DNA fragmentation in a dose-dependent fashion. The sublethal effects of oxidative stress on motion parameters were not significantly associated with membrane translocation of phosphatidylserine.

Semen treatment with progesterone and/or acetyl-L-carnitine does not improve sperm motility or membrane damage after cryopreservation-thawing.

OBJECTIVE: To assess the effects of progesterone and acetyl-L-carnitine used before semen cryopreservation-thawing on sperm motility parameters and plasma membrane integrity. DESIGN: Prospective cohort study. SETTING: Academic tertiary center. PATIENT(S): Subfertile men undergoing semen evaluation. INTERVENTION(S): Before cryopreservation, spermatozoa were incubated with water-soluble progesterone (1 and 10 microM), acetyl-L-carnitine (2.5, 5, 10, and 20 mM), or both (progesterone, 1 microM; and acetyl-L-carnitine, 5 mM). MAIN OUTCOME MEASURE(S): Postthaw change of motility parameters (computer-assisted measurements) and vitality-membrane integrity (examined with eosin-Y staining and annexin V-Cy3 binding assay). RESULT(S): There were no statistically significant differences between control samples and samples treated with progesterone and/or acetyl-L-carnitine for cryosurvival rate, motility parameters, or membrane integrity. The percentages of postthaw cells identified as live showed significantly different results with use of the eosin-Y staining and annexin V binding assay. CONCLUSION(S): Neither progesterone nor acetyl-L-carnitine seemed to prevent cryodamage assessed by motility changes or membrane integrity in human spermatozoa of subfertile men. Annexin V binding, a reflection of membrane translocation of phosphatidylserine, provided more distinct information about postfreezing membrane integrity changes than eosin-Y staining.

Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa.

UNLABELLED: The objectives of this cross-sectional observational study were: (i) to detect DNA damage and plasma membrane translocation of phosphatidylserine in purified sperm populations of high and low motility, and (ii) to analyse their relationship with the endogenous generation of reactive oxygen species. Ejaculates from infertile men were examined following gradient centrifugation. The main outcome measures were: sperm motion parameters (assessed with a computer analyser), generation of reactive oxygen species (measured by chemiluminescence), DNA damage (detected by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling and monoclonal antibody labelling of single-stranded DNA) and translocation of membrane phosphatidylserine (examined with annexin V staining). DNA fragmentation and membrane translocation of phosphatidyl-serine were observed in the fractions with low and high sperm motility in all patients. The fractions with low sperm motility had significantly higher proportion of cells with DNA damage and production of reactive oxygen species than the fractions with high sperm motility (P < 0.005). DNA fragmentation was significantly and positively correlated with the generation of reactive oxygen species (r = 0.42; P = 0.02). IN CONCLUSION: (i) spermatozoa from infertile men display translocation of membrane phosphatidylserine as diagnosed by annexin V positive staining; (ii) DNA damage (fragmentation and presence of single-stranded DNA) can be detected in ejaculated spermatozoa from infertile men in fractions with low and high sperm motility, and (iii) there is a relationship between DNA damage and oxidative stress.

Assessment of sperm cryodamage and strategies to improve outcome.

Sperm cryopreservation still represents a valuable clinical aid in the management of infertility. Its current principal indications include (1) donor sperm insemination; (2) freezing before cancer therapy to maintain reproductive capacity; (3) patient's convenience; and (4) because of the outstanding success with ICSI, even patients with different degrees of oligo-asthenoteratozoospermia can now be offered the use of frozen/thawed sperm for oocyte micromanipulation. Although sperm cryopreservation/thawing and results of insemination and IVF have been consistently good using donor semen, results of infertile men (with or without various degrees of oligoasthenoteratozoospermia) have yielded remarkably lower rates of survival and pregnancy. Freezing/thawing techniques have not been subjected to major changes in the last years, Furthermore, the exact nature of sperm cryodamage still remains to be elucidated. Various aspects of sperm freezing are revisited here (1) development of new technical approaches for cryopreservation; (2) analysis of the stimulatory effect of putative cryoprotectant additives; (3) the use of intrauterine insemination-ready processed samples; and (4) selection and optimization of end-points for analysis of cryodamage. It is expected that advances in such areas will improve significantly the cryopreservation/thawing outcome particularly as related to semen samples of subfertile men.

Gene expression changes associated with cytotoxicity identified using cDNA arrays.

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes.

Intra- and inter-laboratory variability in the assessment of sperm morphology by strict criteria: impact of semen preparation, staining techniques and manual versus computerized analysis.

We designed prospective studies to compare manual and computerized analysis of sperm morphology by strict criteria using different semen processing and staining techniques. A total of 54 semen samples were studied; slides were prepared from each subject from liquefied semen and after washing, and stained with Diff-Quik or Papanicolaou. An intra-laboratory, blind assessment was performed manually (two observers) and using a computerized analyser (two readings). This demonstrated a very good correlation between manual analysis of liquefied and washed samples with both staining techniques [intraclass coefficient (ICC) = 0.93 and 0.83]. Greater agreement was observed between computerized readings (washed samples) of Diff-Quik (ICC = 0.93) than of Papanicolaou-stained slides (ICC = 0.66). An excellent intra-laboratory correlation was observed for within-computer readings (ICC = 0.93). There was moderate agreement between inter-laboratory computer readings (two centres, ICC = 0.72). Although there was lower inter-laboratory agreement for manual and manual versus computer readings, overall results of all manual and computer analyses showed good agreement (ICC = 0.73). Diff-Quik staining is reliable for both manual (liquefied) and computer (washed) analysis of strict sperm morphology. Intra- and inter-computer analyses using this method reached satisfactory levels of agreement. There is still high inter-laboratory variability for the manual method.

Practical evaluation of standard-based low-cost video conferencing in telemedicine and epidemiological applications.

The results of the evaluation of use of low-cost video conferencing systems (VCSs) in telemedicine is presented. Applications sharing, a new feature of these systems, recently has allowed high-quality computer-supported collaborative work (CSCW). The video conferencing (VCing) equipment used was Intel ProShare 200 v2.0a. It is representative of other low-cost VCSs. The areas of application are epidemiology and telemedicine (orthopaedics and radiology). Potential end users filled out 58 evaluation questionnaires concerning user profiles, contents and benefits of the sessions, organizational aspects, user friendliness, user acceptance, cost effectiveness, technical and multipoint related aspects. Although the end users had a lot of computer experience, their knowledge in VCSs was rather limited. The users assessed the system capable of being integrated into routine work, despite a high organizational impact. The VCS is user friendly, application sharing being used in almost every session. Audio quality was not always sufficient. The remote video was sufficient, as was the quality of medical images such as CT, MRI or X-ray. The user acceptance of the system was high. Multipoint sessions require a structured protocol to be effective. Some technical problems with MCUs (Multipoint Control Units) occurred. The use of low-cost standard VCSs in telemedicine is advisable and is a good substitute for real meetings.