Performance evaluation of sperm concentration, motility, and morphological analysis for GSA-810 series of sperm quality analysis system.

BACKGROUND: The performance evaluation of each computer-assisted sperm analysis (CASA) system may provide a basis for the interpretation of clinical results and further improvement of the CASA system. METHODS: The accuracy of the GSA-810 CASA system was evaluated by detecting latex bead quality control products. The precision of sperm concentration, morphology, and percentages of progressively motile sperm (PR) were evaluated by coefficient of variation (CV). Three samples with sperm concentration of about 100 × 106 /mL were diluted to evaluate the linear range. RESULTS: The detection values of latex beads were within the range of target values. The CVs of sperm concentration and PR were significantly and negatively correlated with sperm concentration (r = -0.561, p = 0.001) and PR value (r = -0.621, p < 0.001), respectively. The R2 values of the linear range of sperm concentration were ≥0.99. There was no significant difference in sperm motility and PR within 1-10 min at 36.5°C ± 0.5°C. The coincidence rates of sperm morphology and sperm head morphology for 36 semen samples analyzed by the GSA-810 system and manual method were 99.40% and 99.67%, respectively. The CVs of the percentage of sperm with abnormal morphology and percentage of sperm with abnormal head morphology were less than 5%. CONCLUSION: The GSA-810 system can accurately analyze normal semen samples, but the repeatability of the results is poor for oligozoospermia and asthenozoospermia samples. The future CASA system for analyzing sperm morphology should focus on recognizing the middle and tail segments of a spermatozoon.

Analysis of selected sperm samples by a computer-assisted system with high frame rate: A prospective study.

Background and Aims: Due to the rapid motility of the selected sperm, sperm parameters cannot be accurately determined by the manual method. So, the application of a computer-assisted sperm analysis system with a high frame rate (HFR-CASA, 85 Hz) in sperm selection is investigated. Methods: A total of 177 semen samples were collected for sperm selection with density gradient centrifugation. Then, the manual method and HFR-CASA method will be used to observe and analyze the sperm concentration, motility, and percentage of progressively motile sperm (PR) of the selected sperm samples. The quality control of sperm concentration was performed with microballoons. Two selected sperm samples were analyzed 10 times repeatedly to evaluate the accuracy of the HFR-CASA. Results: The results of microballoons analyzed with the HFR-CASA were in control. The coefficients of variation of sperm concentration, motility, and PR from two selected sperm samples were all below 10%. The results of 177 selected sperm samples analyzed by the manual method and HFR-CASA showed that although there were significant positive correlations in sperm concentration, motility, and PR between them (p < 0.001), the manual method significantly underestimated sperm concentration (p = 0.002) but overestimated sperm motility and PR (p < 0.001). When sperm concentration was below 50 × 106/mL, the manual method significantly overestimated sperm concentration (p < 0.05). However, when sperm concentration was more than 100 × 106/mL, the manual method significantly underestimated sperm concentration (p < 0.001). Regardless of sperm concentration, the manual method consistently overestimated sperm motility and PR (p < 0.001). When sperm concentration was more than 20 × 106/mL, there was no correlation in sperm PR between them (p > 0.05). When sperm concentration was below 50 × 106/mL, the correct rate of captured sperm by the HFR-CASA was more than 98%. Conclusion: The HFR-CASA method is more accurate than the manual method in analyzing the selected sperm samples.

Correlations of joint detection of 22 vaginal microbes with routine examination results of vaginal secretions and assisted reproductive outcomes.

The correlations of joint detection of 22 vaginal microbes with routine examination results of vaginal secretions and assisted reproductive outcomes were investigated. There were 37 samples with abnormal vaginal microecology in 107 vaginal secretion samples. The top 5 detection rates of microorganisms were Ureaplasma urealyticum (73.83%), Prevotella sp. (70.09%), Gardnerella vaginalis (53.27%), L. crispatus (52.34%) and L. inerts (51.40%). When the levels of Bacillus and hydrogen peroxide in vaginal secretions decreased or pH increased, the abnormal rates of vaginal microecology increased significantly (P < 0.01). The clinical pregnancy rate (53.66%, 22/41) in the women with normal vaginal microecology was higher than that (37.5%, 9/24) with abnormal vaginal microecology. In conclusions, the joint detection of 22 vaginal microbes can quickly and effectively determine whether the vaginal microecology is normal or not. The evaluation of vaginal microecology may be valuable in predicting the assisted reproductive outcomes of infertile women.

Effects of six kinds of sperm staining methods on human sperm size and evaluation of their staining effects.

BACKGROUND: Large- and small-headed sperm are common morphological abnormalities. If different sperm staining methods affect sperm size, they will make a difference in the accuracy of sperm morphological analysis results. In this case, the normal reference values of sperm head parameters for different staining methods should be established. METHODS: Six sperm staining methods, including Papanicolaou, Diff-Quik, Shorr, Hematoxylin-eosin (HE), Wright, and Wright-Giemsa staining, were used to stain the sperm smears of 25 semen samples, respectively. Sperm head parameter's length (L), width (W), area (A), perimeter, acrosomal area (Ac), and the derived values L/W and Ac/A of 2500 sperm (100 for each specimen) per staining method were measured by a computer-aided sperm morphological analysis system. RESULTS: The highest sperm head length and width were observed with the Wright-Giemsa and Wright staining, followed by the Diff-Quik. The lowest sperm head length and width were observed with the Papanicolaou staining, and the sperm head length and width of HE and Shorr staining were between those of Papanicolaou and Diff-Quik staining. There was the same trend in changes in sperm head area and perimeter. Diff-Quik and Shorr staining could clearly distinguish acrosome and nucleus, followed by HE staining, whereas the boundary between acrosome and nucleus was not evident in Papanicolaou, Wright, and Wright-Giemsa staining. CONCLUSION: Different staining methods influence sperm size, and the normal reference values of sperm head parameters of each staining method should be established. Diff-Quik and Shorr staining may be suitable methods for routine sperm morphological analysis.

Screening of sperm antigen epitopes by phage display technique and its preliminary clinical application.

BACKGROUND: At present, there is a lack of standardized preparation methods of sperm antigen for the detection of antisperm antibody (AsAb). To screen sperm antigen mimotopes from a phage display random peptide library and use them to establish an enzyme-linked immunosorbent assay (ELISA) for the detection of AsAb, immunoglobulins were extracted from the sera of rabbits with positive AsAb and negative AsAb, respectively, by the saturated ammonium sulfate method, and a phage display 12-mer peptide library was affinity panned by the extracted immunoglobins coated on the ELISA plate. Then, the obtained positive phage clones were identified by ELISA and sent for sequencing and peptides synthesis. Last, a diagnostic ELISA was established to detect clinical serum and seminal plasma samples. RESULTS: A total of sixty phage clones were chosen by affinity panning, and sixteen of them reacted positively with AsAb in indirect ELISA and sandwich ELISA. Following DNA sequencing and translation, the peptide sequences of the sixteen positive clones were obtained. By comparison in Blast database, four of sixteen positive clones were found to be closely related to male reproduction. Two (#1 and #25) of four mimotopes were synthesized, and an ELISA method was established using the two mimotopes as sperm specific antigens. One hundred and thirty-four serum samples and seventy-four seminal plasma samples from infertile couples were analyzed by the established ELISA with #1 and #25 mimotopes, respectively. The positive rates of AsAb in serum samples were 20.15% (27/134) for #1 and 11.19% (15/134) for #25, respectively, and the coincidence rate between them was 91.04% (122/134). The positive rates of AsAb in seminal plasma samples were 1.35% (1/74) for both #1 and #25, and the coincidence rate was 100%. CONCLUSION: Sperm antigen mimotopes can be obtained successfully by the phage display technique, and can be used as standard sperm specific antigens to establish an ELISA method for the detection of AsAb.

RéSUMé: CONTEXTE: À ce jour, il n'existe pas de méthodes normalisées de préparation d'antigènes spermatiques pour la détection des anticorps anti-spermatozoïdes (ACAS). Dans le but d'élaborer un tel test ELISA (enzyme-linked immunosorbent assay), nous avons extrait de sérum de lapins des anticorps anti-spermatozoïdes humains via la technique du sulfate d'ammonium saturé et en ayant recours à une librairie phagique de peptides (12-mer). Les clones positifs ont été identifiés par ELISA, séquencés à façon et les peptides correspondants ont été synthétisés. In fine, un test ELISA diagnostic a été conçu pour être utilisé avec des échantillons cliniques de sérum et de plasmas séminaux. RéSULTATS: Au total, soixante clones de phages ont été sélectionnés, et seize d'entre eux se sont avérés interagir avec les ACAS en ELISA indirect comme en ELISA sandwich. Les séquences peptidiques de ces seize clones positifs ont été obtenues. Par comparaison avec les bases de données (Blast), quatre de ces seize clones positifs se sont révélés être étroitement liés à la reproduction masculine. Deux des quatre mimotopes (#1 et #25) ont été synthétisés, et un test ELISA a été généré en utilisant ces deux mimotopes comme antigènes spécifiques des spermatozoïdes. Cent trente-quatre échantillons de sérum et soixante-quatorze échantillons de plasma séminal de patients de couples infertiles ont alors été analysés avec ce test ELISA. Respectivement, les échantillons sériques se sont révélés positifs à 20,15% (27/134) pour le mimotope #1 et à 11,19% (15/134) pour le mimotope #25, avec un taux de coïncidence de 91,04% (122/134). Seul un échantillon de plasma séminal (1/74, soit 1, 35%) s'est révélé positif à la fois pour le mimotope #1 et #25 (coïncidence 100%). CONCLUSION: La technique « phage display¼ nous a permis d'identifier des mimotopes d'antigènes spermatiques qui ont pu être utilisés afin de générer un test ELISA pour la détection d'anticorps anti-spermatozoïdes.

[Standardization and quality control for detection of sperm DNA damage by flow cytometry: A preliminary investigation].

OBJECTIVE: To investigate preliminarily the standardization and quality control for the detection of sperm DNA damage by flow cytometry. METHODS: Semen samples were randomly selected and observed for the effects of acid denaturation time, acridine orange (AO) staining time, semen sample refrigeration, freezing and repeated freezing-thawing on the results of sperm DNA fragmentation index (DFI). RESULTS: Sperm DFI increased gradually with the prolongation of acid denaturation time, significantly at 2 minutes in comparison with that at 30 seconds (P<0.05). There was no significant difference in sperm DFI after AO staining for 5, 20, 40, 60, 100 and 140 minutes. Sperm DFI also exhibited an evident increase with the prolongation of the refrigeration of the semen samples at 2℃－8℃, significantly at 2 days. The semen samples could be frozen directly at －20℃, and three times of repeated freezing and thawing produced little effect on the results of sperm DFI, except for some inadequate stability. Based on the data obtained from freezing the semen samples after sub-packed and tested 2 days for 1 month and simulation of inter-laboratory quality control, the calculated CV value was 7.13%. CONCLUSIONS: In detection of sperm DFI, it is very important to ensure the accuracy of acid denaturation time, which cannot exceed 1 minute at most. The time of or after AO staining does not significantly affect the results of sperm DFI. The samples for detection of sperm DFI should be fresh and not exceed 1 day in case of refrigeration. Directly frozen semen samples can be used as the materials for inter-laboratory quality control for detection of sperm DFI. Whether cryoprotectants can make frozen semen samples more stable and how to prepare and transport the materials for inter-laboratory quality control are the key problems to be solved in the future.

[Establishment and evaluation of a flow cytometry technique reflecting the severity of human sperm DNA damage].

OBJECTIVE: To establish a flow cytometry (FC) technique reflecting the severity of human sperm DNA and evaluate its performance. METHODS: We analyzed the red and green fluorescence peaks of normal sperm in 1 165 human semen samples, defined the average lower limit of 5% of green fluorescence and the average upper limit of 95% of red fluorescence as the red and green fluorescence limits of the four-quadrant gate, and established an FC technique for detection of the sperm DNA fragmentation index (DFI), analysis of its repeatability and linear range and determination of the reference value of normal fertile men. We also analyzed the correlations of the sperm DFI, mild DNA damage marker (DFIm) and severe DNA damage marker (DFIs) with sperm concentration and motility in 122 men from the Infertility Clinic of Zhongda Hospital. RESULTS: With the established FC technique based on the four-quadrant gate, the sperm DFI, DFIm and DFIs were clearly distinguished among different groups of males, and the coefficients of variation obtained in 10 repeated examinations of the semen samples with a high, medium or low DFI using the FC technique were all lower than 5%. The sperm DFI showed a very good correlation within the range of 8.93%－3.90% (r > 0.99). With the upper limit of 95% as the range of normal reference value, the sperm DFI of 274 of the normal fertile males was ≤ 25.50%. The sperm DFI was remarkably negatively correlated with sperm motility and the percentage of progressively motile sperm (PMS) but exhibited no significant correlation with sperm concentration. The DFIs showed significantly higher related coefficients with sperm motility and PMS (r = －0.592 and －0.543) than DFIm (r = －0.323 and －0.236). Both DFIs and DFIm were markedly higher in the patients with decreased sperm motility and PMS than in the normal fertile men, the former even more significantly (P < 0.01) than the latter (P < 0.05). CONCLUSIONS: Compared with the existing FC technique, ours can reflect the severity of sperm DNA damage and is more suitable for clinical application. DFIs may be more closely related to male fertility.

IFN-γ Induces Gastric Cancer Cell Proliferation and Metastasis Through Upregulation of Integrin ß3-Mediated NF-κB Signaling.

Interferon γ (IFN-γ), a multifunctional cytokine, was upregulated in the resected gastric cancer tissue. However, whether IFN-γ is involved in the regulation of gastric cancer has not been well elucidated. Herein, we aimed to investigate the effects and mechanism of IFN-γ on gastric cancer. In this study, we found a vital role of IFN-γ in enhancing proliferation, inhibiting apoptosis, and promoting cell migration and invasion in gastric cancer cells SGC-7901 and MGC-803. Additionally, IFN-γ activated nuclear factor κB (NF-κB) signaling pathway by upregulating the phosphorylation expression of p65 and IκBα, and induced the expression of integrin ß3 in vitro. Therefore, to further investigate the relationship between IFN-γ and integrin ß3, SGC-7901 cells were transfected with integrin ß3 siRNA. And then cells expressed lower cell viability, migration, and invasion rates, while cell apoptosis was significantly enhanced. Meanwhile, expression of integrin ß3, MMP-2, MMP-9, and NF-κB, including p65 and IκBα, and the nuclear translocation of NF-κB/p65 were dramatically repressed, whereas IFN-γ significantly improved the effects. Moreover, in vivo, the experiment of xenograft model and pulmonary metastasis model also retarded in integrin ß3 siRNA group. And the expression of integrin ß3, MMP-2, MMP-9, and NF-κB was repressed. However, the treatment with IFN-γ improved tumor volume, lung/total weight, tumor nodules, and the protein expression described above compared with integrin ß3 siRNA group. Overall, the results indicated that IFN-γ induces gastric cancer cell proliferation and metastasis partially through the upregulation of integrin ß3-mediated NF-κB signaling. Hence, the inhibition of IFN-γ or integrin ß3 may be the key for the treatment of gastric cancer.