The rationale for requesting a second consecutive sperm ejaculate for assisted reproductive technology.

We assessed the value of requesting a second semen sample provided within 1 hour of the first in cases of unacceptable ejaculate quality (compared to previous occasions) or very poor semen characteristics. The study population consisted of 109 males with severe oligo-terato-astenospermic (OTA) syndrome in an assisted reproduction program. Semen volume, sperm count and sperm motility were recorded in the first and second samples with a Makler Counting Chamber before sperm processing, and total motile sperm in the ejaculate was calculated. Differences in sperm parameters between the consecutive samples were determined by paired-sample t test. In 36 cases (33%) the second sample was found to be superior in quality to the first and was therefore used for the fertilization process. Of the 12/109 cases in which the first ejaculate demonstrated no motile sperm, five (41%) had detectable motile sperm in the second (total 0.1 x 10(6), 0.2 x 10(6), 0.3 x 10(6), 8.4 x 10(6) and 20.8 x 10(6)). We conclude that a request for second ejaculate immediately after the first in males with poor semen quality or no detectable motile sperm can yield a better sample in a significant percentage of cases. Using this method, clinicians can avoid the utilization of elaborate sperm processing techniques and the need for unnecessary micromanipulative fertilization.     

Pooled sequential ejaculates: a way to increase the total number of motile sperm from oligozoospermic men

We investigated the yield of total number of motile spermatozoa from oligozoospermic men by pooling two closely spaced sequential ejaculates. Semen characteristics were compared between sequential ejaculates (within a period of 1 to 4 hours) of 18 oligozoospermic males (sperm concentration less than 20 X 10(6)/mL and total sperm count less than 40 X 10(6) in the ejaculate) and a control group of 16 normozoospermic men. Whereas the median total number of motile sperm of normozoospermic males significantly decreased from 70 X 10(6) in the first ejaculate to 23 X 10(6) in the second sequential ejaculate, such a decrease was not detected in oligozoospermic males, 3.6 X 10(6) and 3.1 X 10(6), respectively. The percent of normozoospermic and oligozoospermic men who demonstrated a decreased (less than 50%), a comparable (50% to 150%), or an increased (greater than 150%) total motile sperm count in the second ejaculate in comparison with the first ejaculate were 69%, 31%, and 0 versus 39%, 28%, and 33%, respectively. Consequently, pooling of two sequential ejaculates significantly increased the median total number of motile sperm from normozoospermic males by 144% and from oligozoospermic males by 329%, (to 10.2 X 10(6]. We suggest that pooling of two sequential ejaculates from oligozoospermic males is a simple and cost effective method to increase significantly the total number of motile sperm for intrauterine insemination, in vitro fertilization, gamete intrafallopian transfer, or semen cryopreservation.     

Leukocytes in human seminal fluid

The peroxidase method is sufficient for quantification of granulocytes, but immunocytology is the standard for detection of white blood cells (WBC) in semen. Granulocytes are the most prevalent WBC in semen (50 to 60%) followed by macrophages (20 to 30%) and T-lymphocytes (2 to 5%). The frequency of leukocytospermia (> 106 WBC/ml) among male infertility patients is 29%. There are ample evidences for sperm damage by WBC: 1) Seminal WBC numbers were higher in infertile patients than among fertile men; 2) leukocytospermia was associated with decreased sperm numbers and impared sperm motility; 3) WBC damage sperm function and were an important prognostic factor for IVF-ET failure. Approximately 80% of leukocytospermic samples are microbiologically negative. In some cases Chlamydia trachomatis might have triggered a persistent inflammatory reaction. Genital tract inflammation facilitates the formation of sperm antibodies.     

Use of carnitine therapy in selected cases of male factor infertility: a double-blind crossover trial

OBJECTIVE: To determine the efficacy of L-carnitine therapy in selected cases of male factor infertility. DESIGN: Placebo-controlled, double-blind, crossover trial. SETTING: University tertiary referral center. PATIENT(S): One hundred infertile patients (ages 20-40 years) with the following baseline sperm selection criteria: concentration, 10-20 x 10(6)/mL; total motility, 10%-30%; forward motility, <15%; atypical forms, <70%; velocity, 10-30 micro/s; linearity, <4. Eighty-six patients completed the study. INTERVENTION(S): Patients underwent L-carnitine therapy 2 g/day or placebo; the study design was 2 months of washout, 2 months of therapy/placebo, 2 months of washout, and 2 months placebo/therapy. MAIN OUTCOME MEASURE(S): Variation in sperm parameters used in the patients selection criteria, in particular, sperm motility.Excluding outliers, a statistically significant improvement in semen quality, greater than after the placebo cycle, was seen after the L-carnitine therapy for sperm concentration and total and forward sperm motility. The increase in forward sperm motility was more significant in those patients with lower initial values, i.e., <5 x 10(6) or <2 x 10(6) of forward motile sperm/ejaculate or sperm/mL. CONCLUSION(S): Based on a controlled study of efficacy, L-carnitine therapy was effective in increasing semen quality, especially in groups with lower baseline levels. However, these results need to be confirmed by larger clinical trials and in vitro studies.     

Positive bacterial culture of semen from infertile men with asymptomatic leukocytospermia

OBJECTIVE: To examine results of semen culture in a population of infertile men with asymptomatic leukocytospermia, and to determine the correlation between culture results and sperm characteristics in these patients. METHODS: Semen samples were collected from a group of infertile men (n = 80) after 2-3 days of sexual abstinence. Standard semen analysis was performed according to World Health Organization (WHO) guidelines. Seminal leukocyte concentrations were determined by a myeloperoxidase staining technique. Culture of semen was performed on enriched and specific culture media. RESULTS: Based on the results of semen culture, the samples were classified into three groups: group 1 (enteric gram-negative bacilli [Enterobacteriaceae], n = 13), group 2 (bacteria other than Enterobacteriaceae, n = 15), and group 3 (negative cultures, n = 52). No correlation was found between positive semen culture and sperm characteristics (concentration, motility, and morphology). However, seminal leukocyte concentrations were negatively correlated with percentages of motile sperm and normal sperm morphology. CONCLUSION: Our data indicate that semen samples from infertile men with asymptomatic leukocytospermia may have bacterial contamination, evidenced by positive bacterial cultures. Lack of correlation between positive semen cultures and sperm characteristics may be indicative of early or mild (subclinical) infection.     

Correlations between seminal radioimmunoreactive prolactin, sperm count, and sperm motility in prevasectomy and infertility clinic patients.

Semen samples were collected from 50 prevasectomy patients and 43 infertility clinic patients. Various parameters were measured, including semen radioimmunoreactive prolactin (rPRL) levels. Linear correlations were tested between the measured parameters, and a significant correlation was found between semen rPRL concentration and sperm count for both the prevasectomy (r = 0.73, P less than 0.00002) and the clinic patients (r = 0.72, P less than 0.00002). For the clinic patients a correlation was also found between the amount of ejaculate rPRL and sperm motility (r = 0.46, P less than 0.002) and between ejaculate volume and motility (r = 0.36, P less than 0.02).     

Role of environmental estrogens in the deterioration of male factor fertility

OBJECTIVE: To evaluate the role of the environmental estrogens polychlorinated biphenyls (PCBs) and phthalate esters (PEs) as potential environmental hazards in the deterioration of semen parameters in infertile men without an obvious etiology. DESIGN: Randomized controlled study. SETTING: Tertiary care referral infertility clinic and academic research center. PATIENT(S): Twenty-one infertile men with sperm counts <20 million/mL and/or rapid progressive motility <25% and/or <30% normal forms without evidence of an obvious etiology and 32 control men with normal semen analyses and evidence of conception.Semen and blood samples were obtained as part of the treatment protocol. MAIN OUTCOME MEASURE(S): Evaluation of semen parameters such as ejaculate volume, sperm count, motility, morphology, vitality, osmoregulatory capacity, sperm chromatin stability, and sperm nuclear DNA integrity. RESULT(S): PCBs were detected in the seminal plasma of infertile men but not in controls, and the concentration of PEs was significantly higher in infertile men compared with controls. Ejaculate volume, sperm count, progressive motility, normal morphology, and fertilizing capacity were significantly lower in infertile men compared with controls. The highest average PCB and PE concentrations were found in urban fish eaters, followed by rural fish eaters, urban vegetarians, and rural vegetarians. The total motile sperm counts in infertile men were inversely proportional to their xenoestrogen concentrations and were significantly lower than those in the respective controls. CONCLUSION(S): PCBs and PEs may be instrumental in the deterioration of semen quality in infertile men without an obvious etiology.     

Semen quality before and after processing by a swim-up method: relationship to outcome of intrauterine insemination

Twenty-five couples with normal semen analyses and postcoital tests of less than or equal to 3 motile sperm/high-power field were treated by intrauterine insemination (IUI) with sperm prepared by a swim-up method. Eight women conceived (32%). Influence of semen parameters both before and after swim-up, patient age, and duration of infertility on outcome of IUI was assessed with logistic discriminant analysis. The percent motility of sperm after swim-up allowed significant differentiation of pregnant and nonpregnant patients (P = 0.0009). For patients with greater than 79% motility after swim-up, discrimination provided by post-swim-up motility (P = 0.0063) was enhanced by inclusion in the analysis of either total number of motile sperm used for insemination (P = 0.0021) or sperm concentration after swim-up (P = 0.0019), Predictions of nonpregnancy and pregnancy were correct at rates of 93.3 and 70%, respectively. There were no significant differences between semen parameters of conception and nonconception cycles of patients who conceived.     

The resazurin reduction test provides an assessment of sperm activity

OBJECTIVE: The objective of the study was to determine if reduction of the dye resazurin by semen could be correlated with the concentration of motile sperm. DESIGN: After assessment of sperm count and motility, specimens were incubated for 1 hour with resazurin (25 micrograms/mL of semen) and visual color changes indicative of dye reduction noted. SETTING: Specimens were obtained from men seeking care for infertility at one of four sites: (1) University of California, San Francisco (UCSF) In Vitro Fertilization Program; (2) UCSF Andrology Laboratory; (3) a gynecological practice in Maine; and (4) a private andrology laboratory in Southern California. PATIENTS: Individuals were self-selected by their desire to have a semen analysis in conjunction with the diagnosis or treatment of infertility. INTERVENTIONS: None. MAIN OUTCOME MEASURE: The reduction of the dye resazurin and its correlation with motile sperm density. RESULTS: When the motile sperm concentration was greater than or equal to 20 X 10(6)/mL, 86% of specimens produced a positive color change. Conversely, 86% of specimens with a motile sperm concentration of less than 20 X 10(6)/mL either did not change color or changed only over a narrow range. CONCLUSION: Reduction of resazurin offers an assessment of the active sperm in a specimen without the need to do a sperm count or evaluation of motility.     

Semen analysis data from fresh and cryopreserved donor ejaculates: comparison of cryoprotectants and pregnancy rates

Patients (155) were selected at random for fresh or cryopreserved semen and inseminated on the predicted day of ovulation. Semen analysis was performed using a microcomputerized multiple-exposure photography system. Frozen semen was used with either glycerol or TEST-yolk (TEST-buffered 20% egg yolk with 10% glycerol) as the cryoprotectant. Cryopreservation resulted in significant decreases in all semen parameters measured. Of these, velocity appeared to be the least effected. TEST-yolk provided significantly more protection against a reduction in velocity compared with glycerol. A total of 18, 17, and 27 patients conceived using fresh, glycerol, or TEST-yolk-preserved semen, respectively. For these same groups, a cumulative pregnancy rate of 52.9%, 27.1%, and 68.5%, respectively, was observed (not significant). The total number of motile sperm per insemination used for fresh artificial inseminations resulting in conception (132.4 X 10(6] was significantly greater than the number used for successful glycerol- and TEST-yolk-preserved semen (approximately 24 X 10(6]. These results demonstrate that although the number of motile sperm of cryopreserved ejaculates are dramatically reduced compared with the fresh counterparts, if a minimum criteria for ejaculate quality is established, the use of cryopreserved semen can offer a viable, effective, and relatively safe alternative to artificial insemination by donor with fresh semen.     

An estimate of the fertility potential of the fractions of the split ejaculate in terms of the motile sperm count.

The motile sperm count is a simple index which is equivalent to the product of sperm density (millions of sperm per milliliter) and percentage of motility (motile sperm per 100 sperm x 0.01). Since other studies have demonstrated a positive correlation between this index and the pregnancy rate, it has been hypothesized that the motile sperm count could be useful for expressing the potential fertility of individual semen specimens. Collection of semen by the split ejaculate technique and subsequent artificial insemination homologous (AIH) with the best split fraction has been used for the treatment of oligospermia. We calculated the motile sperm count for whole semen and the first fraction of split ejaculates from 45 men who were studied for infertility. Subsequently, 15 couples agreed to AIH with the first fraction of the split ejaculate. The data indicate that the motile sperm count may be a reasonable index for comparing the potential fertility of the semen specimens and thereby estimating the chances of successful AIH.     

The clinical value of conventional semen analysis

The objectives of this study were, firstly, to relate semen variables to treatment independent conception rates by life-table analysis after having accounted for known female factors; secondly, to assess the relationship between the length of involuntary infertility before investigation and the predictive value of semen parameters; and thirdly, to examine the relationship between the type of progressive spermatozoal motility and fertility outcome. Laboratory error in the assessment of semen variables was minimized by using one consistent observer. Seven hundred thirty-nine subjects were recruited to the study over a 34-month period, and a 96.5% follow-up rate was achieved. Where the female partner had regular spontaneous ovulation, no pelvic pathology, and more than 48 months' preceding infertility, the Grade 2 motile sperm density (the concentration of spermatozoa exhibiting slow or sluggish linear or nonlinear motility) was the variable that best predicted fertility outcome (X1(2) = 20.24, P less than 0.0001). Where the Grade 2 motile sperm density was below 5 X 10(6)/ml in the latter group (19%), no conceptions were reported at 32 months' follow-up. Semen variables were not of predictive value where there were fewer than 48 months' infertility before investigation, or where the female partner had ovulatory dysfunction or pelvic pathology.     

Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity

Purpose

To assess the effect of leukocytospermia and semen processing on sperm DNA and mitochondria.

Methods

Twenty-two patients with and 41 without leukocytospermia were included. Sperm DNA fragmentation was assessed by the Comet assay, and mitochondrial activity by a colorimetric method for active mitochondria. Semen was processed using Percoll, and motility, DNA fragmentation, and mitochondrial activity were analyzed pre- and post-processing.

Results

No differences were observed in age, abstinence, volume, sperm morphology, progressive motility, concentration, and vitality (p?>?0.10). Variables were grouped according to time (pre- vs post-processing) and group (leukocytospermia vs non-leukocytospermia) because no interactions could be observed. Leukocytospermia was associated to increased DNA fragmentation, while semen processing led to a decrease in DNA fragmentation and to increased mitochondrial activity.

Conclusion

While semen processing selects sperm with higher rates of DNA integrity independent of the presence or absence of leukocytes in semen, samples without leukocytospermia present more sperm without DNA fragmentation. Semen processing also selects sperm with higher mitochondrial activity.     

Seminal immune response in infertile men with leukocytospermia: effect on antioxidant activity.

OBJECTIVE: To investigate the incidence of leukocytospermia and relation to T helper cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-4 (IL-4), antisperm antibodies and antioxidant activity. Design: Semen samples from 176 infertile men and 24 fertile controls were investigated. METHODOLOGY: The protocol included tubal patency test, hysterosalpingography and laparoscopy and dye test and ovulation through mid-luteal phase progesterone for the wives. The husbands had semen analysis, cytomorphology evaluation and semen culture. Seminal TNFalpha and IL-4, antisperm antibodies, total antioxidant activity, superoxide dismutase and zinc were determined. RESULTS: Leukocytospermia occurred in 44.3% of the infertile men compared to 12.5% of the fertile men (P<0.01). Thirty-six (20.5%) men had pathogenic bacterial organisms which constituted 46.2% of those with leukocytospermia. Sperm parameters were worse with leukocytospermia in terms of sperm count (P<0.01), total motility progressive motility (P<0.01), morphology, asthenozoospermia, sperm membrane integrity and antisperm antibodies. TNFalpha and IL-4 had an inverse relationship; the expression of TNFalpha was higher with leukocytospermia and bacteriospermia (P<0.001), while IL-4 was higher in fertile controls (P<0.005). Incidence of antisperm antibodies was higher with leukocytospermia. Total antioxidant activity, superoxide dismutase and zinc were lower with leukocytospermia. CONCLUSION: Leukocytospermia impairs sperm function through reduced antioxidant activity and enhanced T helper 1 modulation.     

A placebo-controlled double-blind randomized trial of the use of combined l-carnitine and l-acetyl-carnitine treatment in men with asthenozoospermia

OBJECTIVE: To determine the efficacy of combined l-carnitine and l-acetyl-carnitine therapy in infertile males with oligo-astheno-teratozoospermia. DESIGN: Placebo-controlled double-blind randomized trial. SETTING: University tertiary referral center. PATIENT(S): Sixty infertile patients (aged 20-40 years) with the following baseline sperm selection criteria: concentration, 10 to 40 x 10(6)/mL; forward motility, <15%; total motility, 10% to 40%; and atypical forms, <80%. Fifty-six patients completed the study. INTERVENTION(S): Patients were submitted to a combined treatment of l-carnitine (2 g/d) and l-acetyl-carnitine (1 g/d) or of placebo; the study design was 2 months' wash-out, 6 months of therapy or of placebo, and 2 months' follow-up. MAIN OUTCOME MEASURE(S): Variation in the semen parameters that were used for patient selection. RESULT(S): Even though increases were seen in all sperm parameters after combined carnitine treatment, the most significant improvement in sperm motility (both forward and total) was present in patients who had lower initial absolute values of motile sperm (<4 x 10(6) forward or <5 x 10(6) total motile spermatozoa per ejaculate). CONCLUSION(S): Combined treatment with l-carnitine and l-acetyl-carnitine in a controlled study of efficacy was effective in increasing sperm motility, especially in groups with lower baseline levels.     

The relationship of sperm parameters to cycle fecundity in superovulated women undergoing intrauterine insemination

Semen parameters of raw and prepared (post-swim-up) specimens from 451 cycles of intrauterine insemination (IUI) were analyzed in relation to cycle fecundity in 232 patients undergoing ovarian stimulation with sequential clomiphene citrate/menotropin therapy. Pregnancy occurred in 42 cycles, resulting in an overall pregnancy rate of 17.7%, and a cycle fecundity of 9.3%. Cycle fecundity was positively correlated with the parameters of post-swim-up log sperm density (r = 0.994), and with log total motile sperm inseminated (r = 0.964; inseminates were limited to a maximum of 20 million total motile sperm). Post-swim-up motility did not correlate (r = 0.308) with cycle fecundity; however, most specimens had a motility of greater than 40% post-swim-up. Only one pregnancy occurred when less than 1 million motile sperm were inseminated (38 cycles), which resulted in a cycle fecundity of 2.6% for these cycles. This may represent the threshold of effectiveness for IUI in this setting. Highest cycle fecundity was obtained with an inseminate containing approximately 10 million or more motile sperm. Parameters of raw samples correlated less well with cycle fecundity than did prepared specimens. Analysis of post-swim-up semen parameters can provide useful prognostic information for women undergoing IUI with ovarian stimulation; this information is helpful in counseling patients regarding their chances of success with this therapy.     

Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients

OBJECTIVE: To investigate whether there is an impact of different sources of reactive oxygen species (ROS) on sperm functions. DESIGN: Prospective study. SETTING: Patients at the Center for Dermatology and Andrology, Giessen, Germany. PATIENT(S): Semen collected from 63 randomly collected patients attending the IVF unit of the University of Giessen, Germany. INTERVENTION(S): Only patients with nonleukocytospermia were included in this study. MAIN OUTCOME MEASURE(S): Sperm count and motility before and after sperm separation by swim-up, morphology, DNA fragmentation, and extrinsic (by leukocytes) and intrinsic ROS production (by spermatozoa) were evaluated. RESULT(S): Leukocytes correlated significantly with extrinsic ROS production (r = 0.576), but markedly less with intrinsic ROS production (r = 0.296). Sperm count, morphology, and motility in the ejaculate were markedly more affected by extrinsic than by intrinsic ROS. The DNA fragmentation was strongly positively correlated with intrinsic ROS production, whereas this correlation was weaker for extrinsic ROS production. No correlation was found between DNA fragmentation and the number of leukocytes, whereas the correlations with motility in the ejaculate and the motile sperm count after swim-up were highly significant. Moreover, significant differences were observed for extrinsic and intrinsic ROS production between groups of patients having a high (> or = 1 x 10(6)/mL) and a low number (<1 x 10(6)/mL) of leukocytes in the ejaculate. CONCLUSION(S): The origin of ROS seems to have an influence on the site of the damage. Because leukocyte counts <1 x 10(6)/mL caused a significant decrease of motility and DNA integrity, the threshold given by the World Health Organization (WHO) should be re-evaluated.     

Preparation of oligozoospermic and/or asthenozoospermic semen for intrauterine insemination using the SpermPrep semen filtration column.

OBJECTIVE: To improve the quality of oligozoospermic and/or asthenozoospermic semen by the SpermPrep (Fertility Technologies Inc., Natick, MA) semen filtration column. DESIGN: The SpermPrep column was applied for semen manipulation in oligozoospermia and/or asthenozoospermia (sperm count less than 20 x 10(6)/mL, sperm motility less than 40%). After concentration of motile sperm using a 40% Percoll density gradient centrifugation, the sperm suspension was filtered through the SpermPrep column. The percentage yield of motile sperm by the SpermPrep method was compared with those by a two-layer Percoll density gradient (Pharmacia, Uppsala, Sweden) centrifugation and a swim-up method. Infertile couples with poor quality semen were treated with intrauterine insemination (IUI) with motile sperm by the three preparations through three cycles. SETTING: Department of Obstetrics and Gynecology, Osaka University Hospital. PATIENTS: Twenty-one couples with long-standing infertility because of poor quality semen. MAIN OUTCOME MEASURE: Recovery of motile sperm, sperm motility, and outcome of IUI were evaluated among three semen preparations. RESULTS: Motility was improved by the SpermPrep method in 32 of 33 cases of oligozoospermia and/or asthenozoospermia. Percentage yield of motile sperm by the SpermPrep method was significantly greater than those by the two-layer Percoll density gradient and swim-up methods (42.7 +/- 4.6 versus 22.1 +/- 3.1 and 13.8 +/- 3.5), but there is no significant difference in the sperm motility among three semen preparations. After one treatment cycle for each preparation, 2 of 21 women conceived after IUI with motile sperm separated in the SpermPrep method. CONCLUSIONS: The SpermPrep method is an improved semen manipulation method for oligozoospermia and/or asthenozoospermia.     

分步射精法在人精子优选技术中的应用

目的:探讨分步射精法在人精子优选技术中提高前向运动精子回收率的有效性。方法:40例少弱精患者随机分为:完整精液组(20例)和部分精液组(20例)。禁欲4-5d,手淫连续完成射精和分步射精获取精液,应用上游法优选精子。比较二组精子密度、活动率、前向运动精子比率和上游后前向运动精子回收率等参数的差异。结果:部分精液组精子的密度、活动率、前向运动精子的比率及其回收率都明显高于完整精液组,差异具有显著性(P＜0．05)。结论:分步射精法在精子优选技术中能够明显提高前向运动精子的回收率。     

Inhibiting effect of artificial cryptorchidism on spermatogenesis

In order to provide a contraceptive method in man, an attempt at inhibition of spermatogenesis was made by means of thermogenesis induced by artificial cryptorchidism. This experimental protocol consists of pushing up the testicles into the inguinal canal and keeping them there, each day during waking hours, by means of an adapted athletic supporter. Fourteen men volunteered for this protocol during a 6- to 12-month period. They all had a normal semen analysis before starting the experiment. The total sperm count, the sperm count per milliliter, the motility, the motile sperm count per milliliter, and the total motile sperm count dropped significantly after the first or second month; they reached their lowest values after the sixth month and remained stable during the next 6 months. At that time the average values reached were a total sperm count of 12 to 34 X 10(6)/ejaculate, a sperm count of 3 to 10 X 10(6)/ml, a motility of 21% to 34%, a motile sperm count of 1 to 3 X 10(6)/ml, and a total motile sperm count of 4 to 12 X 10(6)/ejaculate.