Serum and semen levels of immunoreactive prolactin, LH and FSH in normospermic, oligospermic and azoospermic men with obstructive infertility prior to and after vasoepididymostomy.

Attempts were made to validate RIA for prolactin (PRL), LH and FSH in semen from normospermic, oligospermic and azoospermic subjects. The RIA used to measure PRL and LH in semen fulfilled the criteria of reliability, whereas low levels of FSH in semen precluded the validation of FSH assay in semen. Semen levels of PRL and LH were significantly (P < 0.05) higher than serum levels in all groups of subjects investigated. Semen levels of FSH in azoospermic men after vasoepididymostomy (VEA), were significantly (P < 0.05) higher compared to azoospermic men prior to surgery. Serum levels of PRL were significantly higher (P < 0.05) in normospermic men compared to oligospermic and azoospermic men prior to and after surgery. Semen levels of PRL in normospermic men were comparable with oligospermic and azoospermic subjects prior to and after surgery. Serum levels of LH in oligospermic and azoospermic men who did not undergo surgery and in men reporting oligospermia after VEA were comparable to normospermic subjects but in men showing azoospermia post surgically, serum LH levels were significantly (P < 0.005) elevated. Semen levels of LH in men reporting azoospermia before surgery and in subjects showing oligospermia or azoospermia post surgically were significantly lower (P < 0.05) compared to men with normal sperm count. Serum levels of FSH were significantly elevated (P < 0.05) compared to semen levels in oligospermic men prior to surgery but this increase was not seen in post VEA subjects. These results were discussed.     

Sperm creatine phosphokinase activity as a measure of sperm quality in normospermic, variablespermic, and oligospermic men

We have found a significant inverse correlation between sperm concentrations and sperm creatine N-phosphotransferase (CPK) activities in oligospermic and normospermic human specimens. In the present work, we carried out serial CPK determinations to assess whether there is a relationship between fluctuating sperm concentrations and sperm quality in consistently oligospermic and variablespermic (sperm concentrations are occasionally in the greater than 20 million/ml range) husbands of 65 couples (23 normospermic men/51 samples, 25 consistently oligospermic men/80 samples, and 17 variablespermic men/68 samples). The sperm CPK activities were significantly lower in the normospermic vs. the oligospermic or variablespermic groups (p less than 0.001), but there were no differences between the latter two (p greater than 0.25). The mean CPK values of migrated sperm fractions in both the oligospermic and variablespermic populations were improved (at least 20% decline in CPK values) compared to those of the initial specimens (1.27 +/- 0.38 vs. 0.68 +/- 0.37 and 0.77 +/- 0.32 vs. 0.46 +/- 0.24 SEM U/100 million sperm, respectively, p less than 0.001 in both pairs) and the incidence of the "failed-to-improve" samples was also similar in the two groups (44/36 vs. 45/23, p greater than 0.2). The lack of differences in the mean CPK activities, in the distribution of CPK values under and over 0.250 U/100 million sperm level, and in the ratio of migrated samples with improved or with failed-to-improve CPK activities suggests that sperm quality is not different between men who are consistently oligospermic and those who occasionally produce normospermic specimens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between sperm creatine phosphokinase activity and sperm concentrations in normospermic and oligospermic men

Toward the development of biochemical probes for the assessment of sperm function we have measured the activities of sperm creatine-N-phosphotransferase (CPK). There was a highly significant inverse correlation (P < 0.001 in all comparisons) between sperm CPK activities and sperm concentrations in specimens of normospermic and oligospermic men with > 30 million sperm/ml (0.106 ± 0.01 SEM, Nequals;90, expressed as CPK U/100 million sperm), 20–30 million sperm/ml (0.333 ± 0.07 SEM, Nequals;30) and 10–20 million sperm/ml (0.583 ± 0.12 SEM, Nequals;30) when compared with the CPK values of the < 10 million/ml specimens (2.242 ± 0.46 SEM, Nequals;30). Furthermore, the distribution of CPK activities within these four groups showed that 96%, 67%, 43%, and 4% of the samples, respectively, were in the < 0.250 CPK U/100 million sperm normal range (mean + 2 SD of the > 30 million sperm/ml group). However, there was no relationship between sperm CPK activities and the values of sperm motility (P > 0.15) or morphology (Pequals;0.38) in the samples. The migrated sperm fractions (significantly improved in motility and velocity parameters) showed CPK activities lower than the initial semen specimens (P < 0.01, Nequals;150). In fact, in some oligospermic men the CPK activities of the migrated sperm fractions were within the range of normospermic samples. The data suggest that sperm CPK values in the initial specimens and the degree of improvement in the migrated sperm fractions reflect the relative concentrations of a “normal” sperm subpopulation. We propose that CPK activities and similar objective biochemical parameters may be important in predicting sperm quality and the fertilizing potential of oligospermic men.     

Long term effects of cyclophosphamide on testicular function.

Thirty men treated in childhood with cyclophosphamide for a mean of 280 days were assessed at a mean of 12.8 years after treatment for hormone concentrations and spermatogenesis. Four were azoospermic, nine oligospermic, and 17 normospermic. There was a significant inverse correlation of sperm density with cyclophosphamide dosage and duration of treatment. After a further mean follow up of 7.2 years three patients who were previously oligospermic and one who was azoospermic had normal sperm counts. All patients had normal sexual characteristics and libido. Serum androgen and prolactin concentrations did not differ significantly between patients and controls. Raised basal and stimulated follicle stimulating hormone concentrations were in keeping with impaired spermatogenesis. All patients had significantly raised luteinising hormone responses on stimulation with luteinising hormone releasing hormone. The results suggest compensated Leydig cell failure, and patients with this condition require long term evaluation of testicular function. Potential recovery of spermatogenesis with time requires appropriate counselling and contraceptive advice.     

Clinico-hormonal correlation of oligospermic patients in the below sea level environment (Jordan Valley)

A correlation between serum levels of luteinizing hormone (LH), total testosterone (T), free T and sex-hormone binding globulin (SHBG) in normospermic and in oligospermic male people was done. This study was designed to measure serum levels of these hormones and of SHBG in people living at different altitude environments relative to sea level: at 209-408 meters below (the Jordan Valley, JV) and at 620 meters above (Irbid city, IC). In addition, a clinical awareness study of oligospermia was done in the North of Jordan (IC). Seminal analysis in 287 male people (age range, 18 to 40 years old) during the period between 12/6/1999 and 12/2/2002 showed an oligospermia of 31.4%. Serum levels of LH, total T, free T and SHBG in normospermic subjects in IC were similar to those in normospermic of the JV (3.4 +/- 1.2 vs. 4.0 +/- 1.7 MIU/ml, 19.9 +/- 4.0 vs. 20.4 +/- 5.6 ng/ml, 53.9 +/- 15.6 vs. 47.9 +/- 10.7 pg/ml, 19.5 +/- 3.2 vs. 18.6 +/- 2.16 nmol/l, respectively). Oligospermia was associated with increase in total T at both IC (27.5 +/- 4.6 vs. 19.9 +/- 4.0 ng/ml) and the JV (30.7 +/- 3.4 vs. 20.5 +/- 5.6). The higher serum level of total T in oligospermic people in both IC and the JV was associated with higher levels of SHBG compared to those levels in normospermic subjects. On the other hand, oligospermic subjects have lower serum level of free T than in normospermic males (41.5 +/- 10.0 vs. 53.9 +/- 15.6) only in IC, while in the JV, serum free T level was similar (46.5 +/- 6.1 vs. 47.9 +/- 10.7). Taken together data for both locations, IC and the JV, suggest a clear correlation between total T and SHBG levels in both groups' normospermic and oligospermic subjects.     

Relationship between contents of lipocalin-type prostaglandin D synthase on the surface of infertility sperm and in seminal plasma

Lipocalin-type prostaglandin D synthase (L-PGDS) is localized in Leydig cells, sperm, and epithelial cells of the epididymis. The present study was to determine the correlation between content of this enzyme in seminal plasma and on the surface of sperm. We analyzed 90 semen samples. L-PGDS in seminal plasma was analyzed by an ELISA procedure. L-PGDS on sperm was analyzed by flow cytometry. The semen donors were categorized in three groups: normal, oligospermic, and azoospermic. According to results obtained, L-PGDS may have the ability to improve progressive motility of sperm, and L-PGDS in seminal plasma and on sperm surface may impact male fertility in the female reproductive tract. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 2, pp. 255–259.     

Intérêt du dosage de l’inhibine B et de l’AMH dans le plasma séminal: étude préliminaire

Objectives

The aim of this study was to establish reference values for seminal inhibin B and AMH concentrations in patients with normal and abnormal sperm parameters. Preliminary analysis was performed to evaluate the predictive value of these markers for retrieving testicular sperm in non-obstructive azoospermic men.

Methods

Seminal inhibin B and AMH concentrations were assayed by an enzyme-linked immunoassay in three groups of men: 47 patients with normal sperm parameters, 28 oligospermic men and 68 patients with azoospermia.

Results

Inhibin B and AMH concentrations varied considerably in the three groups, but were significantly higher in normospermic men (inhibin B: 714.36±522.66 ng/l, AMH: 97.08±135.15 pmol/l) than in oligospermic men (inhibin B: 417.5±386.9 ng/l, AMH: 62.02±93.33 pmol/l) and azoospermic men (59.61±2.65 ng/l et 13.12±31.94 pmol/l, respectively) (p<0.001). A significant correlation (p=0.0054) was observed between seminal inhibin B concentration and sperm production. Testicular biopsy allowed sperm retrieval in 11 out of 21 patients (52.3%). The predictive value of seminal inhibin B was analyzed using receiver operating characteristics (ROC) curve analysis. The best discriminating inhibin B concentration was 30 ng/l with an area under the curve (AUC) of 0.63.

Conclusion

This study confirms the correlation between seminal inhibin B and AMH concentrations and spermatogenesis. However, the significance of these two markers as predictors of the presence of testicular sperm in men with non-obstructive azoospermia is limited. This analysis shows that AMH and inhibin B, either alone or in combination with serum FSH, fail to predict the presence of sperm in men with non-obstructive azoospermia undergoing testicular sperm extraction.     

Seminal plasma zinc concentration and α-glucosidase activity with respect to semen quality

This study was conducted to examine the relationship between the concentration of zinc and neutral α-glucosidase (NAG) with semen quality. Semen samples from 75 male partners of couples who were attending the Obstetrics and Gynecology Department were analyzed for semen quality. Based on sperm count, the subjects were divided into three groups. Zinc and neutral α-glucosidase activity were estimated in seminal plasma. Results showed that mean the α-glucosidase activity was lowest among the azoospermic group with respect to oligozoospermic and normozoospermic groups. Mean zinc levels were also lower among azoospermics compared to oligozoospermic and normospermic groups. The difference was statistically significant (p<0.05). A significant positive correlation was observed between zinc levels and sperm count (r=0.29, p<0.05) and zinc and α-glucosidase activity (r=0.31, p<0.05) in seminal plasma. These results suggest that zinc and neutral α-glucosidase seem to play an important role in human reproduction.     

Spermatogenesis-related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa

We have demonstrated earlier that the per sperm creatine-N-phosphotransferase (CK) activity was increased in oligospermic vs. normospermic men. The increased sperm CK activity is related to higher concentrations of cellular CK, which may indicate a defect of cytoplasmic extrusion during spermatogenesis. In the present work, we examined whether in spermatozoa, similar to muscle, there is a change in the synthesis of B-CK and M-CK isoforms during cellular differentiation. In 109 normospermic and 50 oligospermic specimens (sperm concentrations 60.6 +/- 3.7 vs. 8.8 +/- 1.3 million sperm/ml; all values expressed as mean +/- SEM), the relative concentrations of the M-CK isoform (M-CK/M-CK + B-CK) were 27.2% +/- 2.1% vs. 6.7% +/- 0.9% (P less than 0.001). The per sperm CK activities showed comparable differences (0.21 +/- 0.02 vs. 0.89 +/- 0.1 CK IU/100 million sperm; P less than 0.001) in the two groups, and there was a close correlation between per sperm CK activities and M-CK concentrations (R = 0.69, P less than 0.001, N = 159). This indicates that the loss of cytoplasm and the commencement of M-CK isoform synthesis are related events during the last phase of spermatogenesis, also that the incidence of spermatozoa with incomplete cellular maturation is higher in oligospermic specimens. In characterizing the M-CK, we found that sperm (unlike muscle tissue) lack the MB hybrid of CK dimers. However, in the presence of muscle M-CK, the muscle-sperm MB-CK hybrid has formed. Thus in sperm and muscle the M-CK isoforms are structurally different, whereas the B-CKs are apparently homologous.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Centchroman administration in normospermic & oligospermic individuals

The effect of Centchroman, 3,4-trans-2,2-dimethyl-3-phenyl-4-para-(beta -pyrrolidinoethocy)-phenyl-7-methorychroman, administration was investigated in normospermic and oligospermic subjects. 3 normal volunteers, aged 32-40 years, were treated with increasing doses (30, 60, and 120 mg/day, each dose for 2 weeks). The sperm count was decreased in 1 volunteer but the percentages of nonmotile and abnormal spermatozoa were increased in all 3. There was no change in plasma testosterone and urinary 17-ketosteroid (17-KS) levels but the 17-ketogenic steroids (17-KGSs) were decreased in all of them. 3 out of 5 oligospermic subjects, aged 24-35 years, who received 30 mg/day for 6 weeks revealed increased sperm counts. Plasma testosterone levels were decreased in 4, urinary 17-KGSs were decreased in 2, and 17-KSs were decreased in 1 subject. Acid phosphatase, fructose, sialic acid and glycerylphosphoryl choline levels in semen, and serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, and urea in blood were not markedly altered in either group.     

Selenium status of idiopathic infertile Nigerian males

Selenium concentration in the sera and seminal plasma of 60 infertile males (40 oligospermia and 20 azoospermia) and 40 males with proven evidence of fertility (normospermia; control group) were estimated using atomic absorption spectrophotometry. Results were correlated with spermatogram and hormonal levels in order to determine their relationship and significance in male infertility. The mean serum concentrations of selenium was found to be significantly increased in oligospermic compared to azoospermic subjects and controls (p<0.01), whereas the seminal plasma level was significantly higher in azoospermic compared to oligospermic subjects and controls (p<0.001). Thus, the ratio of serum selenium to seminal plasma selenium was 1∶1 in controls, 4∶1 in oligospermia, and 1∶2 in azoospermic subject. A significant inverse correlation was observed between serum selenium level and sperm count (p<0.01). Similarly, seminal plasma selenium correlated with spermatozoa motility, viability, and morphology. Serum selenium level shows positive correlation with the serum testosterone level (p<0.01). In conclusion, there appears to be a physiological balance in the distribution of selenium in serum and seminal plasma compartment of control males. A disturbance in this balance has a significant influence on spermatogenesis. Selenium appears to have a positive influence on Leydig cells, thus influencing the secretion of testosterone.     

Sperm production and fertility of bonnet monkeys (Macaca radiata) following immunization with ovine follicle stimulating hormone.

Adult male bonnet monkeys were rendered oligospermic but not azoospermic following active immunization with ovine follicle stimulating hormone. The percentage of sperms in the semen having good motility was reduced with a concomitant increase in the sperm ATPase activity. Eight out of 10 immunized monkeys failed to impregnate females of proven fertility after mating for consecutive three cycles while the remaining two impregnated the cohabitated females during the third cycle at a time when the antibody titer was reduced. Active immunization with ovine follicle stimulating hormone may not produce complete azoospermia but renders adult male monkeys infertile provided sufficient antibody titer is maintained.     

Embryo outcome in Y-chromosome microdeleted infertile males after ICSI

A prospective study involving 118 infertile Japanese couples to assess the embryo outcomes in both azoospermic and oligoasthenoteratoazoospermic (OAT) patients with Y-chromosome microdeletion. The men were divided into two groups; azoospermia (n = 27), and OAT, sperm concentration <5 x 10(6)/ml (n = 91). They were investigated for Y-chromosome microdeletions by a polymerase chain reaction (PCR) amplification of the Y-chromosome-specific sequence tag site (STS). The embryo outcomes of patients found to have Y-microdeletion were determined. The frequency of microdeletion was 8.8% (9) and two had microdeletions distal to DAZ. The mean fertilization rate and the cleavage rate in the eight cycles of both azoospermic and oligospermic patients were 59.3 and 87.5%, respectively. The percentages of grade 1 & 2 embryos, > or =6 cells embryos, and blastocyts were 51.7, 65.6, and 45.3%, respectively. Three pregnancies resulted from the eight cycles (37.5%). CONCLUSION: in Y-chromosome microdeletion cycles in which sperm cells were available for intracytoplasmic sperm injection (ICSI), embryo outcome was comparable to conventional IVF.     

Testicular fine needle aspiration cytology for the diagnosis of azoospermia and oligospermia

OBJECTIVE: To evaluate qualitative and quantitative cytologic features on testicular fine needle aspiration biopsy in the diagnosis of azoospermia and oligospermia and to correlate cytologic and histologic diagnoses. STUDY DESIGN: In this prospective study, 50 infertile males selected from the infertility clinic of Guru Tegh Bahadur Hospital were studied. Fine needle aspiration cytology (FNAC) smears from both testes of 27 azoospermic and 23 oligospermic patients (sperm count < 10 million per milliliter) were stained with May-Grünwald-Giemsa and Papanicolaou stain. Differential counting of 500 spermatogenic cells was done, and the number of Sertoli cells per 500 germ cells was determined for calculating the spermatic index and Sertoli cell index, respectively. FNAC and testicular biopsy were performed under local anesthesia as a minor surgical procedure. RESULTS: Six groups were identified on FNAC smears from azoospermic patients: I. normal spermatogenesis (8), II. hypospermatogenesis (2), III. maturation arrest (2), IV. Sertoli cells only (6), V. atrophic pattern (7), and VI. Leydig cell predominance (2). In oligospermic patients two groups were identified: I. those with normal spermatogenesis (4), and II. those with subnormal spermatogenesis (19). Correlation with histopathologic examination was seen in 81.5% azoospermic and 65.2% oligospermic patients. CONCLUSION: Qualitative and quantitative evaluation of testicular FNAC provides useful information on both azoospermic and oligospermic patients. FNAC performed under local anesthesia is an acceptable outpatient procedure that consistently yields sufficient diagnostic material in all patients.     

Relationships between lipid peroxidation or cyclic nucleotides and motility in human asthenozoosperm and normosperm]

The relationships between sperm lipid peroxidation (LP) or cyclic nucleotides and sperm motility in normospermic and asthenozoospermic specimens were analyzed. Sperm motility was measured by the transmembrane migration method; LP was measured by thiobarbituric acid (TBA) method and the intracellular cAMP and cGMP contents was measured by radioimmunoassay in 20 fertile and 20 asthenozoospermic infertile human semen specimens. Results showed that in both fertile and infertile individual, there was a close negative correlation between sperm LP formation and motility (r = -0.76; P < 0.001 and r = -0.68; P < 0.001); there were significant positive correlations between intracellular cAMP (r = 0.64; P < 0.01 and r = 0.59; P < 0.01) or cGMP (r = 0.60; P < 0.01 and r = 0.55; P < 0.05) and sperm motility; and the correlation between LP and motility was the closest. These results suggest a causative role for LP in the aetiology of male infertility due to defective sperm motility, and confirmed that intra-cellular cyclic nucleotides likely also have influences on sperm motility.     

Short-term changes in levels of cyclic AMP, adenylate cyclase, and phosphodiesterase during the initiation of sperm motility in rainbow trout

In order to clarify the role of the system that generates and degrades cyclic AMP during the initiation of motility of trout sperm, short-term changes in levels of intraspermatozoal cyclic AMP, adenylate cyclase, and phosphodiesterase were measured. Levels of cyclic AMP and the activity of adenylate cyclase increased and reached a maximum level 1 sec after transfer of sperm to K+-free medium, where they became motile, and then decreased rapidly. However, there were no changes in either parameter in sperm which remained immotile in K+-rich medium. In addition, an increase in the activity of phosphodiesterase was observed 4 sec later than the increase in levels of cyclic AMP and adenylate cyclase. These findings suggest that a very rapid change in the level of intracellular cyclic AMP occurs within 1 sec, at the moment of spawning, by the activation of adenylate cyclase and phosphodiesterase, and regulates the initiation of trout sperm motility.     

Sperm concentration and the fertilization of human eggs in vitro

The effect of sperm concentration on the fertilization of preovulatory and immature human eggs was studied in the context of an ongoing in vitro fertilization-embryo transfer (IVF-ET) program. Fertilization success was independent of the follicular recruitment protocol used, and with preovulatory eggs, was inversely related to sperm concentration over the range of 2.5 - 50 X 10(4) motile sperm/ml. Maximum fertilization (80.8%) occurred at a concentration of 2.5 X 10(4) motile sperm/ml. The incidence of polyspermic fertilization was directly related to the sperm concentration, decreasing from 5.5% at 10 X 10(4) to 0% at 1-2.5 X 10(4) motile sperm/ml. Immature eggs cultured in vitro, then inseminated, also demonstrated an inverse relationship between fertilization and sperm concentration with a maximum fertilization rate of 66.6% at 5 X 10(4) motile sperm/ml. The percentage of motile sperm in the inseminating population had no influence on fertilization rates unless the value dropped below 40%. Fertilization success using sperm from oligospermic and polyzoospermic males was also examined. In contrast to males with normal semen parameters, oligospermic males demonstrated highest fertilization success at 50 X 10(4) motile sperm/ml. The IVF of preovulatory eggs using sperm from polyzoospermic males was comparable to that for males with normal semen parameters at equivalent sperm concentrations. The implications of these findings to the application of IVF-ET technology to the infertile couple is discussed.     

Prostaglandin levels in infertile patients affected by asthenozoospermia and prostatitis

19-hydroxy-prostaglandins and prostaglandins of the E series (19-OH PGEs) were estimated in the seminal plasma of asthenozoospermic patients (n = 15) and individuals affected by prostatitis (n = 10) and compared to controls (n = 13) and secretory azoospermic patients (n = 8). All of them were free from infections (except individuals affected by prostatitis), biochemical and ultrastructural problems. The results indicate that endogenous prostaglandin levels (19-OH PGEs and PGEs) bear no correlation either to motility or absence of spermatozoa. Significant increases of PGEs were observed in patients affected with prostatitis. Surprisingly PGE levels showed no correlation with the levels of 19-OH PGEs.     

Prostaglandin concentrations in the semen of fertile men

The PG concentrations in the semen of 23 fertile men were 73 microgram PGE/ml, 267 microgram 19-OH PGE/ml, 2.1 microgram PGF/ml and 18.3 microgram 19-OH PGF/ml. The wide ranges of concentrations found for the PGEs (2-272 microgram/ml) and for the 19-OH PGEs (53-1094 microgram/ml) throw some doubt on the previously established correlation between infertility and low prostaglandin concentrations.     

复方玄驹胶囊联合枸橼酸氯米芬和维生素E治疗男性肾阳虚型不育症的临床研究

摘要 目的：探讨复方玄驹胶囊联合枸橼酸氯米芬和维生素E对男性肾阳虚型不育症的治疗效果。方法：选择2017年1月-2019年12月于我院就诊并辨证为肾阳虚型男性不育症患者120例,采用随机数字表法分为研究组和对照组。对照组58例患者给予枸椽酸氯米芬和维生素E治疗,研究组62例患者在此基础上加用复方玄驹胶囊,比较两组临床疗效、治疗前后精子形态正常率、精液量、精子密度、前向运动率、精子液化时间、睾酮(testosterone,T)、黄体生成激素(luteotropic hormone,LH)、卵泡刺激素(follicle stimulatin hormone,FSH)及雌二醇(17β-estrodiol,E2)水平的变化。结果：治疗后,研究组患者的总有效率为85.48 %,显著高于对照组(72.41 %,P＜0.05)。与治疗前相比,两组患者精子形态正常率、精液量、精子密度和前向运动率均显著升高,而精子液化时间明显缩短(P＜0.05),研究组患者精子形态正常率、精液量、精子密度和前向运动率均显著高于对照组,而精子液化时间较对照组显著缩短(P＜0.05)。与治疗前相比,两组患者血清T、LH、FSH水平显著增加,E2水平无明显变化;与对照组相比,研究组血清LH水平显著增加(P＜0.05),T和FSH水平无显著差异。结论：复方玄驹胶囊联合枸橼酸氯米芬和维生素E对男性肾阳虚型不育症患者有较好的疗效,可显著提高精子质量,改善患者生殖激素水平。