In vitro initiated sperm forward motility in caput spermatozoa: weak and transient

Testicular spermatozoa during journey through epididymis acquire forward motility, which is essential for fertility. To understand the biochemistry of sperm motility initiation, various initiation media have been developed that permitted high level of motility induction (55-60%) in the immature caput-spermatozoa in presence of activating principles: theophylline, bicarbonate and epididymal plasma (EP) when analysed microscopically. Here, we show for the first time using caprine model that stability and quality of in vitro-induced motility in the caput spermatozoa is insignificant in contrast to naturally induced motility in mature cauda spermatozoa. In vitro-induced motility of the immature spermatozoa was lost completely upon the removal of these activators by centrifugation. Selective withdrawal of either EP or HCO(3) by dilution retains 50-60% of the in vitro-induced motility. Spectrophotometric analysis revealed that in vitro-induced vertical motility in immature spermatozoa is too little when compared to mature spermatozoa. In in vitro-initiated caput spermatozoa, cyclic adenosine monophosphate level becomes doubled but lesser than cauda spermatozoa. This revelation concludes that scientific knowledge generated over the years on the basis of in vitro initiation method is insignificant and needs improvisation to delineate biochemical regulation of sperm motility which in turn has remarkable potential in wide biological fields, especially in infertility treatment.     

Comparative analysis of in vitro contracture tests with ryanodine and a combination of ryanodine with either halothane or caffeine: a comparative investigation in malignant hyperthermia

BACKGROUND: The diagnosis of susceptibility to malignant hyperthermia (MH) is currently performed on muscle biopsies subjected to halothane-caffeine in vitro contracture tests (IVCTs). There is a consensus on our need to improve the diagnostic potential of IVCTs if we are to maximize the information available for research and diagnosis in MH. This study was designed as a pilot comparative study and we aimed at comparing the ryanodine test and new tests using a combination of ryanodine, halothane and caffeine. METHODS: One hundred and thirty-two subjects (52 MHS and 80 MHN) were included in this study and new IVCTs were performed in additional muscle biopsy specimens. The contracture time-course was compared considering the onset time of contracture (OT) and the time to reach a 10 mN contracture (10T). Cut-off values were determined using ROC analyses. RESULTS: For the ryanodine test, sensitivity and specificity calculated for OT were 84.6% and 90.4%, respectively, and were better than those obtained using 10T. Combined tests using either caffeine and ryanodine or halothane and ryanodine did provide higher sensitivities (from 85.3 to 93.9%). A better specificity was only observed for the IVC tests combining halothane (cumulated) and caffeine both with ryanodine (93.9% for both). The largest sensitivity was observed when halothane was used as a bolus and combined with ryanodine. The specificity was always larger with the combined tests as compared to the test using ryanodine alone (from 79.1 to 90.9%). This superiority was confirmed, at least in part, when comparing genetic investigations and the results of new tests in a subgroup of subjects. CONCLUSIONS: This pilot study showed a clear diagnostic potential for new IVC tests combining halothane, the triggering agent of MH, and ryanodine acting at the calcium release channel, and should be considered as a first step in the investigation of combined tests.     

In vitro sensitivity test of stomach cancer tissues by the use of metal grid method

Ninety-nine specimens obtained from 53 patients with stomach cancer were cultured for about 3 days by means of the stainless steel grid method. In vitro effects of antitumor drugs on the cancer cells were evaluated autoradiographically or biochemically using a liquid scintillation counter to measure the uptake of³H-thymidine. The radioactivity of the labeled tumor cells of both control fragments and fragments affected by drugs varied greatly among individual tumors. Therefore, the in vitro efficacy of antitumor drugs was represented as a comparison with that of control fragments. Positive correlation between in vitro tests and the clinical effects of antitumor drugs was observed in the specimens of 18 cases.     

In vitro effects of clomiphene citrate on sperm parameters and fertilisation rate in male NMRI mice

Clomiphene citrate (CC), as a medication in male infertility, improves the sperm parameters in oral consumption but various detrimental side effects have been reported including testicular tumours, gynaecomastia, skin allergic reactions and ocular symptoms. Therefore, this study was designed to evaluate the in vitro effects of CC on sperm parameters and fertilisation rate in IVF protocol. Sperm samples of NMRI adult mice were divided into six groups: group 1 received no treatment (control group), while groups of 2, 3, 4, 5 and 6 (experimental groups) were incubated with the doses of 0.001, 0.01, 0.1, 1 and 10 µg/ml of CC in culture medium respectively. Sperm parameters (viability, morphology and motility), DNA fragmentation levels and fertilisation rate in IVF were evaluated. The results demonstrated that the doses of 0.1 µg/ml (p = .000007 for viability and p = .00006 for fertilisation rate) and 1 µg/ml (p = .032 for viability and p = .005 for fertilisation rate) CC cause a significant improvements; also, the dose of 0.1 µg/ml CC found effective on sperm motility (p = .0003). In the field of IVF, the application of 0.1 and 1 µg/ml of CC in the culture medium may improve the sperm parameters in IVF protocol with no side effects.     

Evaluation of bovine cervical mucus penetration as a test of human spermatozoal function for an in vitro fertilization programme

Thirty-two couples participating in an in vitro fertilization (IVF) programme were evaluated as regards the prognostic value on fertilization of spermatozoal performance through flat capillary tubes filled with standardized midcycle bovine cervical mucus (Penetrak, Serono Diagnostics, Surrey, UK). A statistically significant correlation (P < 0.033) was observed between the distance travelled by the neat spermatozoa in the mucus and the % penetration of oocytes at IVF. There were also significant correlations between motility and progression (P < 0.004) and a borderline correlation between progression and the Penetrak results (P < 0.098). There was no significant difference between the Penetrak distances travelled between the 9 who conceived (33.4 mm) and the 23 who did not (29.9 mm). While the test does add to the knowledge of fertilization potential, the results extrapolated to a larger series would give false positive rates of 25% and false negative rates of 11%. The absence of a clear end point renders the Penetrak mucus penetration test insufficiently accurate to be used as a main measure of the male factor when advising for or against IVF therapy.     

The progesterone‐induced sperm acrosome reaction is a good option for the prediction of fertilization in vitro compared with other sperm parameters

Progesterone (P₄) is crucial for the physiological function of spermatozoa. In the study, we investigated the correlation between P₄‐induced sperm acrosome reaction (AR) and parameters including sperm progressive motility, normal morphology and sperm DNA fragmentation (SDF), and compared the in vitro fertilization (IVF) predictive values of these indicators based on the multivariate regressions analysis and receiver operator characteristics (ROC) curve analyses. The results demonstrated a negative correlation between P₄‐induced sperm AR and the SDF, with the correlation ?9.05 (?17.25 to ?0.84), p<0.05, n = 47). No relationship was found between the sperm progressive motility, normal morphology and the induced AR. The P₄‐induced AR and SDF were both significantly correlated to the fertilization rate. ROC curve analyses indicated that P₄‐induced AR was a better prognostic predictor for the fertilization rate compared with the SDF, with the areas under the curve 0.729 (0.580–0.849), p<0.01 and 0.637 (0.484–0.772), p=0.16 respectively. The cut‐off value for P₄‐induced AR to predict “50% fertilization rate” was 23.4% with sensitivity and specificity of 63.3% and 88.2% respectively. The overall results indicated that the assessment of P₄‐induced AR seemed to be a more sensitive indicator for fertilization rate in vitro compared with other sperm parameters.     

Evaluation of the percentage of sperm motility at 24 h and sperm survival ratio for prediction of in vitro fertilization

Summary. The present study was carried out to investigate the predictive value of the percentage of sperm motility after 24-h incubation and sperm survival ratio from semen and inseminated sperm suspension using grading of motility by WHO criteria with respect to the fertilization of oocytes in vitro. A total of 789 oocytes from 85 cases were inseminated and the mean fertilization rate obtained was 72.5%. There was no significant correlation between all of the sperm motility results with fertilization rate in vitro. All sperm motility results were not significantly different between the non-fertilizing group and the fertilizing group and also between the group of fertilization rate ≤ 25th percentile (fertilization rate ≤62.5%) and the group of fertilization rate >62.5%. However, the initial percentage of rapid progressive sperm motility and progressive motility in semen and inseminated sperm suspension at 24 h gave significant differences between the group of fertilization ≤ 50th percentile (fertilization rate ≤80%) and the group of fertilization rate >80%. Overall accuracy using these parameters for prediction of fertilization rate >80% was only about 60%. In conclusion, the percentage of sperm motility at 24 h and sperm survival ratio in both semen and inseminated sperm suspension have no practical value in predicting fertilization rate in vitro. Moreover, detailed motility grading cannot improve the predictivity of these sperm motility parameters.     

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos

Gamete co‐incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co‐incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.     

The effect of the antisperm auto-antibody-bound sperm on in vitro fertilization outcome

Summary.  To evaluate the effects of antisperm auto-antibody-bound sperm on the outcome of in vitro fertilization-embryo transfer (IVF-ET), 160 infertile couples undergoing treatment by in vitro fertilization were recruited in this study. In the study group (11 couples, 15 cycles), the male partners were positive for antisperm autoantibodies determined by immunobead test (IBT). In the control group (149 couples, 152 cycles), the men had no such antibodies. The percentages of fertilization rate, cleavage rate and pregnancy rate of the study group and control group were 75.0±5.2% vs. 69.3±2.4%; 82.8±3.7% vs. 89.8±1.2% and 6.7% vs. 11.8%, respectively. There were no significant differences in in vitro fertilization outcome between both groups. The region, type and/or percentage of sperm-bound antibodies also had no effect on the in vitro fertilization outcome. In conclusion, in vitro fertilization-embryo transfer is not significantly affected by antisperm autoantibody-bound sperm determined by immunobead test.     

In vitro model for the response to irradiation of different types of human intracranial tumours

Summary Twenty-seven human low and high grade gliomas and five meningiomas were cultured in vitro as tumour tissue and/or tumour cells. Cell survival or growth was taken as a measure of radiation response. Astrocytomas II–III and glioblastomas manifested individual patterns of radiosensitivity, ranging from 10 to 90 Gy. Meningiomas did not react. Our findings are consistent with the differences in radiosensitivity of human gliomas experienced clinically and corroborate the validity of the in vitro model.     

Semen analysis in 21st century medicine： the need for sperm function testing

Sperm function testing, once commonly performed for the infertile couple before employing assisted reproductive technology （ART）, has fallen out of favour in many reproductive medicine centers throughout the world. Indeed, the most recent addition of the ＇World Health Organisation （WHO） Laboratory Manual for the Examination and Processing of Hu- man Semen＇ now groups many of these procedures into a section termed Research Procedures. In large part, this reflects the current clinical practice of bypassing the in-depth evaluation of the male partner, while assuming that if a spermatozoon can be found for intracytoplasmic sperm injection （ICSI）, it must be a healthy cell capable of achieving fertilization. Never- theless, sperm function testing can provide valuable clinical insights into defects causing male infertility. Admittedly, in some cases, functional sperm deficiencies can be overcome using an ART. In other cases, couples will be empowered by the knowledge of the cause of their infertility, and for some couples, perhaps even the likelihood of ICSI success （relative to the spermatozoa）. The knowledge allows them to make truly informed reproductive decisions, including （perhaps） the de- cision to seek donor insemination, to adopt or to remain childless. Knowledge of the cause of their infertility may provide closure for couples and a sense of confidence regarding their choice of reproductive treatment.     

Human sperm movement assessed with the Hamilton-Thorn motility analyzer and in vitro fertilization

Summary. The relationship between sperm movement characteristics obtained by computerized analysis and the in vitro fertilization rates of human oocytes was studied. In 144 consecutive in vitro fertilization treatments a sample of prepared semen was analysed by a Hamilton-Thorn Motility Analyzer. In addition a visual estimation of sperm count and motility was made. Significant correlations with the fertilization rate were found for all visual parameters. Of the computerized measurements, the mean velocities of motile spermatozoa and the concentration of motile cells were significantly correlated. The average path velocity correlated best ( r = 0.42, P < 0.001). There was no relationship between the percentage of motile sperm showing hyperactivated movement and the fertilization rate. A forward stepwise logistic regression analysis selected the following variables of predictive value for fertilization: average path velocity, male factor infertility as indication for in vitro fertilization, motility and concentration, as measured by the Hamilton-Thorn analyzer. A logistic regression model to predict the cases with low (< 0.2) or high fertilization rates, included the average path velocity as a significant variable and classified the samples with 90% overall accuracy. In conclusion: movement characteristics of spermatozoa in culture medium, especially the average path velocity are of prognostic value in prediction of human oocyte fertilization rates.     

The predictability of clinical antitumor effects using two distinctive in vitro chemosensitivity tests: An analysis of true positive cases

The results of two types of in vitro chemosensitivity tests, namely, the human tumor clonogenic assay (HTCA) and the succinic dehydrogenase inhibition assay (SDIA), for solid tumors, including stomach, colorectal and lung cancers, were analyzed and their correlation with clinical effects evaluated. The anticancer agents employed were mitomycin C (MMC), 5-fluorouracil (5-FU), adriamycin (ADM) and cisplatin (DDP). The evaluability rates of the assays were 54.5% for HTCA and 89.0% for SDIA. Among the 29 cases with evaluable lesions subjected to HTCA, there were 4 true positives, 9 false positives, and 16 true negatives, whereas among the 32 cases subjected to SDIA, the corresponding numbers were 2, 6, and 24, respectively. There were no false negatives for either assay, the accuracy of prediction for HTCA being 69.0% and for SDIA, 81.3%. The true positives of both assays included one complete response (CR) and five partial responses (PR), although the eventual outcome was cancer death in all cases. Interestingly, in five out of the six true positive cases, the agent involved was either ADM or DDP, both the which are usually regarded as second line anticancer agents for gastrointestinal carcinomas.     

精子形态与体外受精胚胎移植临床妊娠结局的关系

目的:当前精子形态对体外受精(IVF)的影响尚存争议,本研究旨在评估正常形态精子百分率在体外受精-胚胎移植(IVF-ET)中的应用价值。方法:选择生殖遗传中心659对IVF-ET治疗夫妇,按正常形态精子百分率分为4组,A组(2%)112个周期,B组(≥2%~4%)180个周期,C组(≥4%~5%)74个周期,D组(≥5%)293个周期。比较各组间的受精率、正常受精率、卵裂率、优胚率以及新鲜移植周期的生化妊娠率、临床妊娠率、流产率及活产率等指标。结果:4组间的受精率、正常受精率和可移植胚胎率有差异。C组(71.90%)和D组(72.89%)的受精率均显著高于A组(57.97%)和B组(63.29%)(P均0.05),C组与D组间、A组和B组间均无显著差异(P均0.05)。D组的正常受精率(57.16%)显著高于A组(46.52%)和B组(50.89%)(P均0.05),C组正常受精率(54.67%)显著高于A组(P0.05),其余组间无显著差异(P均0.05)。D组的可移植胚胎率(55.62%)显著高于B组(45.75%)(P0.05),其余组间均无显著差异(P均0.05)。D组的无可移植胚胎患者比例(8.87%)显著低于A组(20.54%)和B组(18.89%)(P均0.05),C组(12.16%)与其余组间均无显著差异(P均0.05)。各组间新鲜移植周期的生化妊娠率、临床妊娠率、种植率、流产率和活产率均无显著差异(P均0.05)。结论:正常形态精子百分率对IVF-ET的受精率及胚胎形成有一定影响,用于评估IVF受精结局时,5%临界值略优于4%。     

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection （ICSI） using ejaculated and testicular spermatozoa

AIM: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). METHODS: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. RESULTS: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. CONCLUSION: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.     

卵胞浆内单精子注射在常规体外受精失败病例中的应用

目的评价卵胞浆内单精子注射(ICSI)对常规体外受精(IVF)失败病例的应用效果。方法对于常规IVF受精失败的病人在第2天进行补救ICSI(A组)21个周期,或在下一个周期直接应用ICSI技术治疗(B组)18个周期。分别与因严重少、弱精子症而行ICSI的243周期(对照组)的受精率、优质胚胎率和妊娠率等进行比较。结果A组的受精率、卵裂率、优质胚胎率分别为61.53%、81.73%、72.94%,均比B组的83.87%、97.69%、84.25%显著降低(P<0.05),而两组的多原核率差异无显著性(3.55%vs1.29%,P>0.05)。比较临床妊娠率、种植率和冷冻周期率,B组与对照组均无显著差异,而A组与对照组均有显著差异。结论常规IVF受精失败者可通过第2天补救ICSI或下一周期直接行ICSI而提高受精率和种植率,而后者比前者能获得更好的妊娠结局。     

An evaluation of the hypo-osmotic sperm swelling test as a predictor of fertilizing capacity in vitro

The ability of sperm to swell in hypo-osmotic conditions was examined in 211 semen samples from the partners of patients about to undergo oocyte retrieval for in-vitro fertilization (IVF). The test was performed using aliquots of semen, the remainder of which was then prepared for IVF. No significant difference was found, in either the percentage of swollen sperm or the type of swelling response, between samples that achieved fertilization in vitro and those that did not, or between any of the diagnostic categories of infertility (tubal damage, unexplained infertility, oligospermia). In samples which achieved fertilization in vitro there were correlations between sperm swelling and sperm motility (r = -0.51) and abnormal morphology (r = 0.33), but no such correlations were demonstrated in samples that failed to achieve fertilization. Moreover, there was no significant difference between the percentage of swollen sperm in semen (mean motility 64%), in samples immediately after preparation for IVF (mean motility 96%) or in capacitated sperm 24 h after preparation (mean motility 91%). These results demonstrate that the hypo-osmotic sperm swelling test does not assist in the prediction of the fertilizing capacity of human sperm in vitro.     

Measurement of induced acrosome reactions in human sperm using physiologic stimuli--relevance for the prediction of fertilization outcome

Fertilization failure following standard in vitro fertilization in couples with normozoospermic men is an as yet unexplained phenomenon. A wide range of gametic disorders as well as environmental factors might contribute to this pathologic condition. One crucial condition appears to be the inability of the spermatozoa to undergo the acrosome reaction (AR). A discriminative test to distinguish fertile from non-fertile spermatozoa would be of utmost interest. In a prospective study, semen samples from men with normal semen parameters and fertilization failure were compared with semen samples from men with normal semen parameters and normal fertilization as to their capacity to undergo the AR. AR was induced using calcium ionophore as well as the physiologic stimuli progesterone and prostaglandin E(1). Discriminance analyses were undertaken to help identify patients with probable fertilization failure. Our data show that in patients with fertilization failure, the capacity of spermatozoa to undergo induced AR is greatly reduced using both unphysiologic and physiologic stimuli. However, physiologic stimuli are more suitable to identify patients with fertilization failure. Using physiologic stimuli, a formula was established to identify patients likely to fail at fertilization.     

Correlation analysis of the progesterone‐induced sperm acrosome reaction rate and the fertilisation rate in vitro

In this study, we aimed to investigate whether progesterone‐induced acrosome reaction (AR) rate could be an indicator for fertilisation rate in vitro. Twenty‐six couples with unexplained infertility and undergoing in vitro fertilisation (IVF) treatment were involved. On the oocytes retrieval day after routine IVF, residual sperm samples were collected to receive progesterone induction (progesterone group) or not (control group). AR rate was calculated and fertilisation rate was recorded. The correlation between progesterone‐induced AR and fertilisation rate and between sperm normal morphology and 3PN (tripronuclear) were analysed using the Spearman correlation analysis. The AR rate of progesterone group was statistically higher than that of the control group (15.6 ± 5.88% versus 9.66 ± 5.771%, P < 0.05), but not significantly correlated with fertilisation rate (r = ?0.053, P > 0.01) or rate of high‐quality embryo development (r = ?0.055, P > 0.01). Normal sperm morphology also showed no significant correlation with the amount of 3PN zygotes (r = 0.029, P > 0.01), rate of 3PN zygotes production (r = 0.20, P > 0.01), rate of 3PN embryo development (r = ?0.406, P > 0.01), fertilisation rate (r = ?0.148, P > 0.01) or progesterone‐induced AR rate (r = 0.214, P > 0.01). Progesterone can induce AR in vitro significantly; however, the progesterone‐induced AR may not be used to indicate fertilisation rate.     

In vitro chemosensitivity test for human genito-urinary tumors using collagen gel matrix

BACKGROUND: Although the aim of chemosensitivity tests is to predict the efficacy of anticancer agents for individual patients, no generally accepted assay has been established. METHODS: A chemosensitivity test was conducted for solid tumors with an organ culture system using collagen gel matrix (CGM). Seventy-five samples of transitional cell carcinoma (TCC), 20 of germ cell tumor (GCT) and 13 of renal cell carcinoma (RCC) were used for the chemosensitivity test, and 20 patients were treated with anticancer drugs on the basis of the test results. RESULTS: Positive rates of anticancer drugs for the 75 TCC samples were 64.9% for carboplatin, 63.4% for cisplatin, 32.1% for etoposide, 19.7% for THP-adriamycin, 16.7% for vinblastine, and 12.3% for methotrexate, indicating that positive rates of the latter three agents consisting of an MVAC regimen were unexpectedly low. The GCT had higher positive rates than the other cancers while RCC had the lowest. In 20 eligible patients (seven patients with bladder tumors and 13 with GCT), the true positive and true negative rates were 42% (5/12) and 75% (6/8), respectively, and the sensitivity and specificity were 71% (5/7) and 46% (6/13), resulting in a 55% (11/20) accurate predictive value. CONCLUSION: Although predictive accuracy was moderate when combination chemotherapy was used, information about chemosensitivity may have some beneficial effect on the treatment of patients with invasive bladder cancer or advanced GCT, because insensitive drugs detected by the test could be deleted or replaced with more sensitive ones.