Showing papers in "Journal of Andrology in 1999"
TL;DR: Data from this study indicate that oxidative damage to sperm DNA may be important in the etiology of male infertility and that the assay of sperm 8-OHdG may have potential diagnostic value in the evaluation of sperm function and male fertility.
Abstract: Recently, there is increasing evidence suggesting that oxidative sperm DNA damage is closely associated with impaired sperm function and male infertility. 8-Hydroxydeoxyguanosine (8-OHdG) is considered to be a precise and sensitive biomarker of oxidative DNA damage. The present study was thus designed to evaluate the extent of oxidative DNA damage in sperm and its association with male infertility by assaying the 8-OHdG levels in human sperm samples. A total of 114 subjects (60 infertile patients and 54 age-matched healthy workers) participated in this study. The level of 8-OHdG in sperm DNA was determined by high-performance liquid chromatography with electrochemical detection, and the conventional seminal parameters were also measured according to World Health Organization guidelines. It was found that the level of sperm 8-OHdG in infertile patients was significantly higher than that in healthy subjects (10.03 vs. 4.79 8-OHdG/105 dG; geometric mean, P < 0.001). The correlation between sperm 8-OHdG levels and conventional seminal parameters were also analyzed. There is a significant positive correlation between 8-OHdG and sperm head defects (r = 0.38, P < 0.001), whereas significant inverse correlations were noted for 8-OHdG with sperm density (r = −0.42, P < 0.001), total sperm number (r = −0.42, P < 0.001), sperm motility (r = −0.24, P < 0.01), and normal sperm morphology (r = −0.39, P < 0.001). Data from this study thus indicate that oxidative damage to sperm DNA may be important in the etiology of male infertility and that the assay of sperm 8-OHdG may have potential diagnostic value in the evaluation of sperm function and male fertility.
158 citations
TL;DR: It is suggested that final Sertoli cell number per testis exerts an important effect on the circulating level of inhibin B (and FSH) in the rat.
Abstract: Sertoli cells undergo important changes in their number and function at different ages in the rat and may be the primary source of circulating inhibin B. The aims of this study were 1) to establish the profile of inhibin B levels from birth to adulthood in normal rats and 2) to identify whether experimental manipulation of Sertoli cell numbers was able to alter this profile. Levels of inhibin B, measured by a specific two-site assay, increased fivefold in normal Wistar rats between day 3 and days 10-15, plateaued, and then declined in late puberty to reach adult levels which were approximately 60% of those observed on days 10-15. The increase in inhibin B levels in the neonatal period coincided with the period of Sertoli cell multiplication as indicated by incorporation of bromodeoxyuridine. Neonatal treatment of rats with a GnRH antagonist (GnRHa) reduced Sertoli cell number and adult testis weight by 48% and significantly reduced plasma levels of inhibin B at all ages through to adulthood. Induction of neonatal hypothyroidism in Sprague-Dawley rats by administration of propylthiouracil (PTU) up to day 25 of age increased final testis weight by 41% (indicative of increased Sertoli cell numbers) and resulted in elevation of plasma levels of inhibin B at all ages beyond 7 days of age. The degree of change in inhibin B levels in adult rats in the two experimental treatment groups was approximately proportional to the change in final testis weight. Plasma follicle-stimulating hormone (FSH) showed changes opposite to inhibin B, with levels being lowered in PTU-treated rats and elevated (beyond day 25) in GnRHa-treated animals. The present results suggest that final Sertoli cell number per testis exerts an important effect on the circulating level of inhibin B (and FSH) in the rat. These findings are compared to the emerging data for the human male.
133 citations
TL;DR: Measurements show that in addition to the previously known secretion in seminal plasma, hK2 is secreted in amniotic fluid, breast milk, and saliva, suggesting that the two proteases might form a functional unit but not always as demonstrated by the sole presence of h kallikrein 2 in saliva.
Abstract: It has been demonstrated that prostate-specific antigen (PSA), in spite of its name, can be detected in body fluids and tumors from a variety of organs. Investigations have shown that human glandular kallikrein 2 (hK2), a related prostate-secreted protease, can activate the zymogen form of PSA, suggesting that the two enzymes might work as a functional unit, with hK2 as the activator molecule and PSA as the effector molecule. If this is true, then hK2 should be found together with PSA in body fluids other than seminal plasma, as well. Recently, a sensitive and specific assay was devised for hK2, enabling its measurement in picogram quantities. With this assay, the concentration of hK2 was determined in samples of seminal plasma, amniotic fluid, breast milk, and saliva. Simultaneously, the samples were assayed for molecular forms of PSA. In seminal plasma, the mean PSA concentration was 0.82 mg/ml, while the hK2 level was around two orders of magnitude lower: mean value, 6.4 microg/ml. Approximately the same ratio of PSA to hK2 as in seminal plasma was found in amniotic fluid and breast milk, but in most samples, the hK2 values were too low for direct measurements and had to be concentrated prior to analysis. Measurable levels of PSA, all in the free form, were detected in amniotic fluid at the thirteenth week of gestation and then gradually increased to levels around and over 1 microg/L from the twentieth week. Significant levels of PSA were detected in amniotic fluid collected at delivery, also. Measurable levels of mammary PSA were primarily detected in colostrum, with a range from less than 0.03 microg/L to 2.1 mg/L. Around half of the molecules were in complex with protease inhibitor. Most surprisingly, determinations on saliva samples showed that none of them had detectable PSA levels but had measurable concentrations of hK2 with a mean value, 0.09 microg/L. The presence in saliva suggests that hK2 can be the human equivalent to one of the mouse salivary kallikreins with important biological function, like the epidermal growth factor-binding protein or the gamma subunit of nerve growth factor. However, this was ruled out, as a phylogenetic analysis showed that the human and mouse glandular kallikreins evolved independently from a common precursor after the separation of the primate and rodent lineages. In conclusion, the measurements show that in addition to the previously known secretion in seminal plasma, hK2 is secreted in amniotic fluid, breast milk, and saliva. Furthermore, the concerted expression of PSA and hK2 in seminal plasma, amniotic fluid, and breast milk suggests that the two proteases might form a functional unit but not always as demonstrated by the sole presence of hK2 in saliva.
110 citations
TL;DR: A combination regimen of physiologic testosterone plus a low dosage of levonorgestrel offers great promise as a safe and effective male contraceptive regimen and suppresses sperm production comparably to T enanthate plus higher dosage LNG, while causing less weight gain and HDL cholesterol suppression.
Abstract: Studies using exogenous high-dosage testosterone (T) or a combination regimen of physiologic T plus high-dosage levonorgestrel (LNG) administration in normal men have shown that oligoazoospermia (<3 million/mL) or azoospermia can be achieved in the majority of the men. However, these hormonal regimens have been associated with significant weight gain and suppression of serum high-density lipoprotein (HDL) cholesterol levels. We hypothesized that a combination of physiologic exogenous testosterone and lower dosage LNG would result in uniform severe oligoazoospermia or azoospermia in normal men but would cause fewer adverse metabolic side effects. We conducted a randomized, placebo-controlled, single-blind trial comparing 6 months of T enanthate (100 mg IM, weekly) plus LNG, 125 microg by mouth, daily (LNG 125; n = 18) or LNG, 250 microg by mouth, daily (LNG 250; n = 18) and compared these regimens with our previous study of the same dosage of T enanthate combined with placebo LNG (LNG 0; n = 18) or with 500 mg of LNG (LNG= 500; n = 18). All three combination regimens of T enanthate and LNG suppressed spermatogenesis more rapidly and resulted in significantly more uniform severe oligoazoospermia (<1 million/mL) than the T-alone regimen. Severe oligoazoospermia was achieved in 89% of the LNG 125, 89% of the LNG 250, and 78% of the LNG 500 groups, respectively, versus 56% of the men in LNG 0 (P < 0.05 for the combination groups vs. LNG 0), but there were no significant differences between the combination regimens (P = NS). All four groups gained significant weight compared with their baselines, although the gain tended to be greater as the dosage of LNG increased (2.0+/-0.9, 2.9+/-1.1, 3.6+/-1.0, and 5.4+/-1.0 kg gained, compared with baseline in the LNG 0, 125, 250, and 500 groups respectively; P < 0.05 compared with baseline). Serum levels of HDL cholesterol decreased in all of the groups, but the effect was larger as the dosage of LNG increased (4+/-4% vs. 13+/-4%, 20+/-3%, and 22+/-4% decrease in HDL levels from baseline in the LNG 0, LNG 125, LNG 250, and LNG 500 groups respectively; P = 0.06 for LNG 125 compared with LNG 0, and P < 0.05 for LNG 250 and LNG 500 compared with LNG 0). We conclude that 1) the combination of physiologic exogenous T enanthate and LNG suppresses spermatogenesis more effectively than T enanthate alone and that 2) the combination regimen of T enanthate plus lower dosage LNG suppresses sperm production comparably to T enanthate plus higher dosage LNG, while causing less weight gain and HDL cholesterol suppression. A combination regimen of physiologic testosterone plus a low dosage of levonorgestrel offers great promise as a safe and effective male contraceptive regimen.
103 citations
TL;DR: A hypothetical model for spermatid adhesion and the control of that adhesion is proposed, thus providing a framework for hypotheses on the steps involved in the complex process of spermiation in rat testes.
Abstract: Spermiation, the process by which late spermatids separate from the Sertoli cell, is disrupted by a number of toxicants. In this study, we used immunohistochemistry (IHC) to identify some of the proteins associated with the spermatid-Sertoli junction. We confirmed the presence of tubulin, actin, and vinculin at the luminal edge of the seminiferous tubule, and we determined that paxillin is also present here. In other cell types, these proteins have been reported to colocalize with beta integrins. Numerous attempts to identify beta integrins by IHC and by use of Western blots were unsuccessful. Clear evidence was found for the presence of N-cadherin and its associated intracellular proteins: beta-catenin, pp120, desmoglein, pp60(src), and Csk. In addition, N-cadherin and desmoglein were found around spermatids retained by the epithelium. From these data and previous literature reports, we propose a hypothetical model for spermatid adhesion and the control of that adhesion, thus providing a framework for hypotheses on the steps involved in the complex process of spermiation in rat testes.
98 citations
TL;DR: It is suggested that varicocele-associated oxidative stress is evidenced both at the local and systemic levels, supported by results showing that urinary spontaneous chemiluminescence is also significantly increased in the patients.
Abstract: Varicocele is associated with an oxidative stress condition. We have measured the antioxidant defenses of varicocele patients both at the local (seminal plasma) and systemic (blood plasma) levels. The antioxidant defenses, as evaluated by the total reactive antioxidant potential parameter, decrease both in the seminal (controls = 676 ± 128; patients = 386 ± 186) and blood (controls = 519 ± 63; patients = 268 ± 110) plasma of varicocele patients. Compared with controls, patients with both normal spermiograms and spermiograms altered in motility or morphology demonstrated lower values. The results obtained suggest that varicocele-associated oxidative stress is evidenced both at the local and systemic levels. This conclusion is supported by results showing that urinary spontaneous chemiluminescence is also significantly increased in the patients.
94 citations
TL;DR: It is concluded that in young adult men, testosterone levels begin to rise on falling asleep, peak at about the time of first REM, and remain at the same levels until awakening.
Abstract: The relation between the pituitary-gonadal hormones' rhythm and sleep physiology in men is not fully elucidated. To examine whether the reproductive hormones are correlated with sleep architecture, we determined the nocturnal serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in six healthy young men. Serum hormone levels were obtained every 15 minutes from 1900 to 0700 hours with simultaneous polysomnographic sleep recordings. Hourly testosterone levels were lowest when subjects were awake (1900-2200 hours) than during sleep (2300-0700 hours). Testosterone nocturnal rise antedated the first REM by about 90 minutes. The rise in testosterone levels was slower when REM latency was longer. Mean nocturnal testosterone levels did not correlate with the number of rapid eye movement (REM) episodes. Also, pre-non-REM (NREM) testosterone levels were higher as compared with the pre-REM periods and lower during the first NREM period as compared with other nocturnal NREM periods. Serum LH levels disclosed a nocturnal rise that preceeded a similar rise in testosterone by about an hour. We conclude that in young adult men, testosterone levels begin to rise on falling asleep, peak at about the time of first REM, and remain at the same levels until awakening.
88 citations
83 citations
Journal Article•
TL;DR: In this paper, the role of FSH in the maintenance of adult rat spermatogenesis was explored by using immunoneutralization to selectively withdraw FSH action for periods of up to 8.5 days and then assessing the outcome by quantification of germ cell number.
Abstract: Follicle-stimulating hormone (FSH) plays an important part in testicular development. Its role in the regulation of spermatogenesis in the adult, however, remains controversial. This study aimed to explore the role of FSH in the maintenance of adult rat spermatogenesis by using immunoneutralization to selectively withdraw FSH action for periods of up to 8.5 days and then assessing the outcome by quantification of germ cell number. Adult rats received either an ovine polyclonal rat FSH antibody (FSHAb, 2 mg/ kg subcutaneous daily-a dosage known to neutralize >90% of FSH in serum) for 2, 4, 7, or 8.5 days or a control sheep immunoglobulin (ConAb, 2 mg/kg) for 8.5 days. Testes were perfusion fixed, and germ cell numbers per testis were quantified using the optical disector (sic) stereological method. The percentage of seminiferous tubules displaying apoptotic cells was determined by the in situ end labeling method (TUNEL). FSHAb treatment for 4, 7, or 8.5 days significantly reduced the number of type A/intermediate spermatogonia (∼74% of control values) associated with stages I-IV. Similar reductions were seen in type B spermatogonial and preleptotene spermatocyte numbers after 8.5 days of FSHAb treatment (∼69% of control values; P < 0.05). Decreases (P < 0.05) in the numbers of pachytene spermatocytes in stages I-III and VIII, round spermatids in stages I-III, VII, and VIII (-70% of control values), and step 19 elongated spermatids in stage VII (51% of control values) were achieved after 8.5 days of FSHAb treatment. Compared with control, FSHAb treatment increased the percentage of stage XIV-I tubules containing TUNEL-positive cells by about twofold after 7 days of FSHAb treatment (P < 0.05). This study supports a role for FSH in the maintenance of quantitatively normal adult rat spermatogenesis, specifically by regulating A 3 and A 4 spermatogonial subtypes. FSH may act on these spermatogonia by enhancing the stage-dependent survival of type A spermatogonia. Effects at other sites in spermatogenesis are suggested by the changes in spermatocyte and spermatid populations. However, to clarify these effects, selective FSH withdrawal would need to be prolonged until steady state had been achieved.
80 citations
TL;DR: The results indicate that TP1 is likely to be an important parameter in the histone-to-protamine exchange and in the initiation of spermatid elongation, and CREM is involved in the regulation of TNP1 gene expression and consequently plays a vital role in the correct differentiation step from round sperMatids to mature spermatozoa.
Abstract: During spermiogenesis, histones are replaced by transition proteins, which in turn are replaced by protamines. The TNP1 gene-encoding TP1 (transition protein 1) protein contains a cAMP-responsive element (CRE) that serves as binding site for the CRE modulator (CREM). To gain further insight into the complex regulation of nucleoprotein exchanges in haploid spermatids and its potential role for spermatogenic impairment, we studied the gene expression of testis-specific histone H1t, CREM, and TNP1 in testicular biopsies from men with normal spermatogenesis (n = 20) and with round spermatid maturation arrest (n = 16). During normal spermatogenesis, H1t messenger RNA (mRNA) was present in 86.2%+/-8.7% of pachytene spermatocytes (stages III-V), whereas H1t protein was synthesized in 83.5%+/-13.0% of pachytene spermatocytes (stages III-V) and persisted in 95.2%+/-3.1% of spermatids (steps 1-5). CREM mRNA was detectable in 74.2%+/-9.4% of pachytene spermatocytes (stages IV-V) and in 78.7%+/-10.0% of spermatids (steps 1-3). CREM protein was synthesized in 81.2%+/-14.2% of spermatids (steps 1-3). TNP1 mRNA was present in 80.0%+/-13.5% of spermatids (steps 2-4), whereas TP1 protein was synthesized in 89.7%+/-5.3% of spermatids (steps 3-4). In men with round spermatid maturation arrest, spermatids only develop to step 3 of differentiation. These spermatids were devoid of both CREM and TP1 but did contain H1t. These results indicate that TP1 is likely to be an important parameter in the histone-to-protamine exchange and in the initiation of spermatid elongation. CREM is involved in the regulation of TNP1 gene expression and consequently plays a vital role in the correct differentiation step from round spermatids to mature spermatozoa.
79 citations
TL;DR: Postthaw motility results from immediately after thawing and fertility results from experiments where intrauterine insemination was performed with semen frozen in proline- or glycine betaine-containing or HEPES- or Tris-based diluents were pooled and subjected to a pairwise correlation procedure.
Abstract: The fertility of ewes after artificial insemination and the relationship between fertility and motility characteristics assessed by a computerized motility analysis system were examined with ram semen frozen in diluents reported to improve postthaw motility. The percentages of motile and progressive spermatozoa were better when frozen in proline- or glycine betaine-containing or HEPES-based, rather than Tris-based, diluents (P < 0.01). The fertility of spermatozoa frozen in diluents containing proline or glycine betaine was slightly reduced, whereas when both compatible solutes were present, the reduction was more pronounced, in comparison with semen frozen in Tris- or HEPES-based diluents (9.5 versus 71.1 and 66.6%; P < 0.01). Fertility of frozen-thawed spermatozoa was higher after laparoscopic insemination than after cervical or transcervical insemination (P < 0.01). Similarly, higher fertility was obtained after cervical insemination with fresh than with frozen-thawed semen (32.4 versus 11.3%; P < 0.01). Furthermore, loss of embryos was lower after laparoscopic insemination of ewes with semen frozen in a Tris diluent than with semen frozen in proline diluents, in glycine betaine diluents, or in proline-plus-glycine betaine diluents (0.0 versus 26.0, 38.5, and 60.0%; P < 0.001). A wide variation in the postthaw percentage of motile (31.6-59.7%) and progressive (22.6-43.1%) spermatozoa and in the fertility of spermatozoa from individual rams was also observed after laparoscopic (29.2-59.7%) or cervical insemination (8.7-30.5%). Postthaw motility results from immediately after thawing and fertility results from experiments where intrauterine insemination was performed with semen frozen in proline- or glycine betaine-containing or HEPES- or Tris-based diluents were pooled and subjected to a pairwise correlation procedure. The correlation analysis showed relationships between some of the motility characteristics (P < 0.01), but there were no relationships between the motility characteristics and fertility.
TL;DR: A role for FSH is supported in the maintenance of quantitatively normal adult rat spermatogenesis, specifically by regulating A3 and A4 sperMatogonial subtypes and enhancing the stage-dependent survival of type A s permatogonia.
Abstract: Follicle-stimulating hormone (FSH) plays an important part in testicular development. Its role in the regulation of spermatogenesis in the adult, however, remains controversial. This study aimed to explore the role of FSH in the maintenance of adult rat spermatogenesis by using immunoneutralization to selectively withdraw FSH action for periods of up to 8.5 days and then assessing the outcome by quantification of germ cell number. Adult rats received either an ovine polyclonal rat FSH antibody (FSHAb, 2 mg/kg subcutaneous daily-a dosage known to neutralize >90% of FSH in serum) for 2, 4, 7, or 8.5 days or a control sheep immunoglobulin (ConAb, 2 mg/kg) for 8.5 days. Testes were perfusion fixed, and germ cell numbers per testis were quantified using the optical disector (sic) stereological method. The percentage of seminiferous tubules displaying apoptotic cells was determined by the in situ end labeling method (TUNEL). FSHAb treatment for 4, 7, or 8.5 days significantly reduced the number of type A/intermediate spermatogonia (approximately 74% of control values) associated with stages I-IV. Similar reductions were seen in type B spermatogonial and preleptotene spermatocyte numbers after 8.5 days of FSHAb treatment (approximately 69% of control values; P < 0.05). Decreases (P < 0.05) in the numbers of pachytene spermatocytes in stages I-III and VIII, round spermatids in stages I-III, VII, and VIII (approximately 70% of control values), and step 19 elongated spermatids in stage VII (51% of control values) were achieved after 8.5 days of FSHAb treatment. Compared with control, FSHAb treatment increased the percentage of stage XIV-III tubules containing TUNEL-positive cells by about twofold after 7 days of FSHAb treatment (P < 0.05). This study supports a role for FSH in the maintenance of quantitatively normal adult rat spermatogenesis, specifically by regulating A3 and A4 spermatogonial subtypes. FSH may act on these spermatogonia by enhancing the stage-dependent survival of type A spermatogonia. Effects at other sites in spermatogenesis are suggested by the changes in spermatocyte and spermatid populations. However, to clarify these effects, selective FSH withdrawal would need to be prolonged until steady state had been achieved.
TL;DR: Findings indicate that, in a physiological setting where the negative feedback control system governing the adult primate testis is operational, Sertoli cell number, rather than function, is the primary determinant of circulating inhibin B levels.
Abstract: The relationship between changes in Sertoli cell number and function and changes in circulating inhibin B concentrations was investigated following unilateral orchidectomy (UO) in the adult rhesus monkey. As expected, the 50% loss in Sertoli cells resulting from UO on day 0 was associated with a rapid and corresponding decline in plasma concentrations of inhibin B. The decrease in inhibin B levels was sustained until the remaining testis was removed on day 44, at which time a compensatory 50% increase (P < 0.05) in the number of round spermatids was evident in the absence of a change in Sertoli cell number. Moreover, Sertoli cell number and inhibin B levels among individual monkeys were highly correlated (r 2 = 0.65, P < 0.002). Round spermatid number and inhibin B, however, were poorly correlated (r 2 = 0.37, P < 0.04). These findings indicate that, in a physiological setting where the negative feedback control system governing the adult primate testis is operational, Sertoli cell number, rather than function, is the primary determinant of circulating inhibin B levels.
TL;DR: During aging, there is a breakdown of the blood-testis barrier, and there are striking changes in the appearance of Sertoli cells, suggesting a possible intrinsic limitation that prevents stem cells from renewing themselves, whether because of a degeneration of immunological origin orBecause of a lack of SERToli cell support.
Abstract: Steroidogenesis and spermatogenesis decrease in aging Brown Norway rats. We therefore hypothesized that there must be accompanying morphological changes taking place in the seminiferous tubules of the aging testis. The testes of Brown Norway rats ranging in age from 3 to 24 months were prepared for light and electron microscopy. To assess the integrity of the blood-testis barrier with age, a lanthanum nitrate study was done. The normal seminiferous tubules present in rats at 3 and 12 months of age were largely replaced at 24 months by fully regressed tubules that were virtually devoid of germ cells and contained large intercellular spaces. An electron-microscopic study of these regressed tubules showed a complete loss of cyclical variations of the organelles of the Sertoli cells. The nucleus was more irregularly shaped and was present at various levels in the epithelium. The endoplasmic reticulum was a loose, vesiculated network that was unlike the elaborate, tubular, anastomotic network noted in young animals. The lysosomes were large, oddly-shaped, and contained lipidic inclusions, in contrast to the distinct membrane-bound lysosomes and dense core bodies found in the young animals. Adjacent Sertoli cell processes encompassed large, empty intercellular spaces, possibly occupied previously by germ cells. The typical Sertoli-Sertoli junctions of the blood-testis barrier in the young animal were rarely seen at 24 months and were replaced by focal contact points, usually between three Sertoli cell processes. In the aged animals, lanthanum nitrate permeated the basal and adluminal compartments, extending between Sertoli cell processes and entering the intercellular spaces and lumen. In summary, during aging, there is a breakdown of the blood-testis barrier, and there are striking changes in the appearance of Sertoli cells. These results suggest a possible intrinsic limitation that prevents stem cells from renewing themselves, whether because of a degeneration of immunological origin or because of a lack of Sertoli cell support.
TL;DR: The results demonstrate that the 5alpha-reduction of testosterone is particularly important for progression through midspermiogenesis, because this phase of germ cell development is more sensitive to withdrawal of androgens.
Abstract: Germ cell development (spermiogenesis in particular) in the adult rat is known to be testosterone dependent. Recently we proposed a role for the 5alpha reduction of testosterone to dihydrotestosterone (DHT) in the short-term restoration of round spermatid maturation when testicular testosterone levels are experimentally lowered. The current study aimed to further characterize the involvement of 5alpha-reductase in the restoration of spermatogenesis by investigating the short- and long-term restoration of specific germ cell populations by testosterone in the presence or absence of a 5alpha-reductase inhibitor (L685,273). Spermatogenesis in adult rats was suppressed for 8 weeks using 3-cm testosterone and 0.4-cm estradiol silastic implants (testosterone-estradiol [TE] treatment); spermatogenesis was then restored by administration of increasing doses of testosterone with or without a competitive 5alpha-reductase inhibitor or with the androgen receptor antagonist flutamide. Animals were then killed after either 4 days or 6 weeks of treatment so that we could study the short- and long-term restorations of spermatogenesis. Stereological analysis showed that germ cell development between late pachytene spermatocytes to round spermatids in stage VII during either short- or long-term restoration was not affected by 5alpha-reductase inhibition, but it was affected by flutamide. The conversion of round spermatids between stages VII and VIII was restored by testosterone treatment, but this restoration was prevented by flutamide. Both the short- and long-term restorations of this midspermiogenic event were significantly decreased when 5alpha-reductase was inhibited. After long-term restoration of spermatogenesis, elongated spermatids were restored to 42% of control but were significantly suppressed to 20% of control by coadministration of the 5alpha-reductase inhibitor because of a reduction in the number of round spermatids progressing between stages VII and VIII. The results demonstrate that the 5alpha-reduction of testosterone is particularly important for progression through midspermiogenesis, because this phase of germ cell development is more sensitive to withdrawal of androgens. We suggest that testicular 5alpha-reductase activity is important for the restoration or maintenance of low levels of sperm production in a hormonally based contraceptive setting.
TL;DR: It is shown in this study that early reproductive aging in the BN rat began at around 15 months, and it is unlikely that hypothalamic-pituitary dysregulation is the major cause of testicular aging.
Abstract: Reproductive aging in the male Brown Norway (BN) rat is characterized by decreased Leydig cell steroidogenesis associated with seminiferous tubule dysfunction. This could be a result of a combination of a primary testicular defect and a secondary hypothalamic pituitary dysfunction. In the present study, we determined in the BN rat whether germ cell loss occurred via apoptosis. We then defined the age of onset of Leydig cell dysfunction and germ cell loss and examined whether chronic luteinizing hormone (LH) replacement would delay or prevent reproductive aging. Plasma hormone levels, testicular sperm concentrations, and germ cell apoptosis were studied in 6, 9, 12, 15, 18, and 21-month-old BN rats. Beginning at 15 months, testicular weight, sperm concentration, total sperm counts, plasma testosterone, LH, and inhibin decreased, whereas the proportion of regressed testes and plasma follicle-stimulating hormone (FSH) levels increased with aging. Accelerated germ cell apoptosis involving spermatogonia, preleptotene and pachytene spermatocytes, and spermatids was evident in some tubules of the relatively normal testes from 21-month-old rats. In the regressed testes, complete cessation of spermatogenesis occurred. The apoptotic index was higher in the testes of old (21-month-old) rats in particular at stages XII-XIV when compared with younger animals. Chronic LH replacement (0.5 microg i.p. twice per day) administered to 15-month-old BN rats for 6 months did not alter plasma hormone levels, testes weight, sperm concentration or content, or the germ cell apoptotic index. In the control group, 3 out of 10 testes were regressed, whereas in the LH-replaced group, only 1 out of 12 testes was regressed. We show in this study that early reproductive aging in the BN rat began at around 15 months. Germ cell loss associated with aging occurs via apoptosis. Replacement therapy with LH for 6 months does not decrease or delay the testicular dysfunction associated with aging. It is unlikely that hypothalamic-pituitary dysregulation is the major cause of testicular aging.
TL;DR: The existence of an as yet unknown testicular catecholaminergic neuronlike cell type in the human testis is shown and the increase of both "intrinsic" (neuronal cells) and "extrinsics" (nerve fibers) neuronal elements in pathological testicular biopsies suggests that the two parts of the humantesticular nervous system may be involved in pathogenesis and/or maintenance of GA and SCO syndromes.
Abstract: Neuronlike, catecholaminergic cells expressing tyrosine-hydroxylase (TH) have recently been found in the testis of a nonhuman primate species, the rhesus monkey. We examined whether neuronlike cells are present in the human testis. To this end, we first determined if the genes for TH and for a voltage-activated sodium channel (NaCh), a prerequisite for neuronal excitability, are expressed in normal adult testes. Using an RT-PCR approach, cDNA clones, identical to the sequences of human TH and to the alpha subunit of a NaCh type, were isolated. Immunohistochemical methods localized the corresponding proteins in testicular biopsies from adult men (age range, 28-44 years) without testicular pathologies and from infertile patients with either Sertoli cell only (SCO) syndrome or severe hypospermatogenesis and germ cell arrest (GA). TH and NaCh antibodies, as well as antibodies recognizing dopamine-transporter protein, identified immunoreactive cells of mainly bipolar or occasionally multipolar, elongated phenotype in most, but not all, biopsies of each group (12 out of 23). The results were corroborated by identification of TH gene expression by RT-PCR approaches in biopsies. Immunoreactive cell bodies, as well as nerve fibers, were more readily detected in SCO and GA biopsies. This was quantified after immunohistochemically visualizing all testicular neuronal elements, cell bodies, and fibers, with a neurofilament 200 (NF-200) monoclonal antibody in one set of randomly selected sections from all biopsies. We found significantly increased NF-200-immunoreactive cell bodies and fibers in SCO-syndrome and GA biopsies. These results show the existence of an as yet unknown testicular catecholaminergic neuronlike cell type in the human testis. This cell type may complement and act in concert with the well-known testicular sympathetic innervation. The increase of both "intrinsic" (neuronal cells) and "extrinsic" (nerve fibers) neuronal elements in pathological testicular biopsies suggests that the two parts of the human testicular nervous system may be involved in pathogenesis and/or maintenance of GA and SCO syndromes.
TL;DR: A high-quality basic semen analysis represents one cornerstone in the investigation of the infertile couple and the clinical value of traditional semen parameters in the diagnosis of male fertility is the subject of considerable debate.
Abstract: A high-quality basic semen analysis represents one cornerstone in the investigation of the infertile couple. However, the clinical value of traditional semen parameters in the diagnosis of male fertility is the subject of considerable debate. Some authorities clearly hold the view that apart from a diagnosis of azoospermia (or very severe oligozoospermia), a basic semen assessment is of little clinical value. A recent example of this is reflected by the opinion of McDonough (1997, p. 587), who wrote: “traditional sperm analysis as a clinical test may become nothing more than an ancestral heirloom. It may be performed spasmodically by those who know how to do it, like a 1940 airshow or laparotomy, to remind us of the good old days. We have come to the end of something. Surely someone will want to carve a headstone for traditional sperm analysis or perhaps a mausoleum would be more fitting.” Is this a correct view? Well, it does have some merit. For example, the diagnosis of an abnormal or normal semen sample according to the World Health Organization (WHO) guidelines (1992) is somewhat artificial, and this dichotomy is of limited diagnostic value. It is well documented that a significant proportion of fertile men are diagnosed as abnormal if such criteria, i.e., WHO (1992), are used (Ombelet et al, 1997). The question that needs to be asked is the following: what is the clinical value of
TL;DR: Biweekly intralesional injections of Peyronie's plaques with IFN-alpha-2B resulted in a significant improvement in penile curvature, diminished pain, and reduced plaque size, and resulted inA subjective improvement in sexual function.
Abstract: Intralesional therapy is a less invasive method for the treatment of Peyronie's disease. The objective of this study was to evaluate intralesional injections of interferon alpha 2B (IFN-alpha-2B) as an effective alternative to the surgical treatment of Peyronie's disease. Twenty-one patients with Peyronie's disease were evaluated by use of penile duplex Doppler ultrasonography for cavernosal blood flows, degree of penile curvature, and plaque size. A questionnaire was given to all patients to assess sexual function. Each patient then received biweekly intralesional injections of 1 x 10(6) units of IFN-alpha-2B in 10 ml of normal saline over a period of 6 months. There was no placebo control group in this study. At the conclusion of the study, penile duplex Doppler imaging was repeated. A questionnaire was completed by all patients to assess changes in sexual function after treatment. Twenty patients completed the study, with all men reporting subjective softening of their plaques. Nine of 10 patients initially reporting penile pain with erection (90%) had resolution of their phallalgia while on study protocol. Thirteen patients (65%) had significant improvement in curvature, ranging from 20 to 90%. Seventeen patients (85%) demonstrated an objective 10 to 80% decrease in plaque size. Biweekly intralesional injections of Peyronie's plaques with IFN-alpha-2B resulted in a significant improvement in penile curvature, diminished pain, and reduced plaque size, and resulted in a subjective improvement in sexual function.
TL;DR: DHT levels are lower in HIV-infected men than in healthy men but that neither DHT levels nor DHT-to-testosterone ratios correlate with weight loss, and the hypothesis that a defect in DHT generation contributes to weight loss independently of changes in testosterone levels is not supported.
Abstract: Weight loss is an important determinant of disease outcome in human immunodeficiency virus (HIV)-infected men. Others have suggested that a defect in dihydrotestosterone (DHT) generation contributes to weight loss in HIV-infected men. To determine whether DHT levels correlate with weight loss independently of changes in testosterone levels, we prospectively measured serum total- and free-testosterone and DHT levels in 148 consecutive HIV-infected men and 42 healthy men. Thirty-one percent of HIV-infected men had serum testosterone levels less than 275 ng/dL, the lower limit of the normal male range; of these, 81% had normal or low LH and FSH levels (hypogonadotropic), and 19% had elevated LH and FSH levels (hypergonadotropic). Overall, serum testosterone, free-testosterone, and DHT levels were lower in HIV-infected men than in healthy men, but serum DHT-to-testosterone ratios were not significantly different between the two groups. Serum total- and free-testosterone levels were lower in HIV-infected men who had lost 5 lb or more of weight in the preceding 12 months than in those who had not lost any weight. Serum DHT levels and DHT-to-testosterone ratios did not differ between those who had lost weight and those who had not. Serum testosterone and free-testosterone levels, but not DHT levels, correlated with weight change and with Karnofsky performance status. We also performed a retrospective analysis of data from a previous study in which HIV-infected men with serum testosterone levels less than 400 ng/dL had been treated with placebo or testosterone patches designed to nominally release 5 mg testosterone over 24 hours. Serum testosterone-to-DHT ratios did not change after testosterone treatment. Changes in fat-free mass were correlated with changes in both serum testosterone (r = 0.42, P = 0.018) and DHT (r = 0.35, P = 0.049) levels. Serum total- testosterone and DHT levels were highly correlated with one another, and when the change in serum testosterone was taken into account, serum DHT levels no longer showed a significant correlation with change in fat-free mass. We conclude that DHT levels are lower in HIV-infected men than in healthy men but that neither DHT levels nor DHT-to-testosterone ratios correlate with weight loss. During testosterone treatment, serum DHT levels increase proportionately, but the increments in serum testosterone correlate with the change in fat-free mass. Our data do not support the hypothesis that a defect in DHT generation contributes to weight loss in HIV-infected men independently of changes in testosterone levels; it is possible that such a defect might exist in HIV-infected men with more severe weight loss.
TL;DR: A three-generation genetic analysis of a family with a case of male factor infertility is reported, finding that the newborn male was found to bear a Y-chromosome microdeletion similar to that of his father, raising the possibility that the son will have the same fertility problem as his father.
Abstract: Sperm cells can be retrieved directly from the testis (testicular sperm extraction [TESE] procedure) and used for intracytoplasmic sperm injection (ICSI), circumventing underlying spermatogenetic defects. Thus, it is important that added information be available on the genetic defects in men undergoing TESE for the ICSI procedure and on the transmission of genetic factors associated with infertility to the offspring. We report a three-generation genetic analysis of a family with a case of male factor infertility. The proband, previously diagnosed as infertile, was physically examined and laboratory tested for gonadotrophic hormones, semen analysis, karyotype and Y-chromosome microdeletion screening in the blood and testis. The Y-chromosome microdeletion screening was performed by multiplex polymerase chain reaction with 20 Y-chromosome sequenced, tagged sites located at the Y chromosome. A microdeletion including the AZF-c region was detected in the azoospermic patient. His father, four brothers, and three offspring born after ICSI also underwent Y-chromosome microdeletion screening. The genetic analysis of the male members of the patient's family did not reveal similar microdeletions. The newborn male was found to bear a Y-chromosome microdeletion similar to that of his father. The fertilization capacity of the proband testicular microdeleted spermatozoa by the ICSI procedure is described. The transfer of the genetic defect raises the possibility that the son will have the same fertility problem as his father.
TL;DR: Results show that hK2 cleaves HMWK to produce bradykinin, not Lys-brady Kininin (like hK1), and the resultant heavy and light chains of H MWK show similar electrophoretic mobility to those cleaved by PK.
Abstract: Prostate-specific human glandular kallikrein (hK2) is an active enzyme in human seminal fluid. It is one of three serine proteases in the human kallikrein gene family, which includes hK1 (tissue kallikrein) and hK3 (prostate-specific antigen [PSA]). In order to examine kininogenase activity (i.e., production of kinin by these enzymes), we tested for bradykinin and/or Lys-bradykinin release upon incubation of hK2 and for other kallikreins with high-molecular weight kininogen (HMWK), which contains the nonapeptide bradykinin. Kinins are important regulatory peptides (especially for vascular permeability), and they may have a role in enhancing sperm motility. High-molecular weight kininogen is the substrate for plasma kallikrein (PK-a potent kinin-generating enzyme circulating in blood, not of the same gene family) and for hK1. Glandular kallikrein and protein-C inhibitor (PCI)-hK2 complex, a serpin protease inhibitor that binds hK2, were purified to homogeneity by affinity and size-exclusion chromatography. About one-half of the hK2 is found in complex with PCI. The kallikrein enzymes were incubated with HMWK, and the resulting cleavage products were analyzed for kinin activity using enzyme immunoassay, high-performance liquid chromatography and mass spectrometry, and in vitro bioassay. Our results show that hK2 cleaves HMWK to produce bradykinin, not Lys-bradykinin (like hK1 ), and the resultant heavy (56-kDa) and light (42-kDa) chains of HMWK show similar electrophoretic mobility to those cleaved by PK. Prostate-specific antigen (hK3) had no kinin-generating activity. We also identified three other intemal cleavage sites for hK2 in HMWK (Arg 427 , Arg 437 , and Arg 457 ) that yielded two peptides, one of which is identical to a PK-cleaved peptide. Glandular kallikrein is about 500-fold less active than is PK or tissue kallikrein, but it may play a physiologically important role in bradykinin release in seminal fluid.
TL;DR: Cytosolic PP1 and GSK-3 activities appear to be inversely related to the motility of monkey epididymal sperm and may have a regulatory role in the development of the potential for motility in epididcyal sperm.
Abstract: Human and monkey ejaculated sperm contain pro- tein phosphatase-1 (PP1), PP1 inhibitor 2 (12), and glycogen syn- thase kinase-3 (GSK-3). Inhibition of ejaculated human sperm pro- tein phosphatase (PP) activity with calyculin-a (CL-A) significantly stimulates motility, implicating protein dephosphorylation in motility regulation. The present experiments were conducted to character- ize and compare PP and GSK-3 activity in monkey caput and cau- dal epididymal sperm, to determine the cellular distribution of these enzymes, and to test the thesis that epididymal sperm PP activity is inversely related to motility. Caput epididymal sperm popula- tions, (8.8% motile) contained levels of PP activity that were >3 times as high as those of caudal spermatozoa. This PP activity was further identified by inhibitor response profiles as PP1. In both caput and caudal sperm, the majority of this PP1 activity was lo- calized in 100,000 x g soluble fractions. Western blot analysis indicated that a portion of this difference was the result of elevated amounts of PP1 in caput compared with caudal epididymal sperm. The presence of GSK-3 activity was undetectable in 100,000 X g insoluble fractions of epididymal sperm, whereas both caput and caudal sperm soluble fractions contained GSK-3 activity, which was approximately threefold higher in caput sperm compared with caudal populations. Treatment of caput epididymal sperm from the rhesus macaque with the PP inhibitor CL-A resulted in a significant, dose-dependent increase from 8 to 38% motile cells (without any effect on their path velocity). In contrast, CL-A had no significant influence on either percent motility or path velocity of caudal epi- didymal sperm. Cytosolic PP1 and GSK-3 activities appear to be inversely related to the motility of monkey epididymal sperm and may have a regulatory role in the development of the potential for motility in epididymal sperm.
TL;DR: With insemination of a limiting number of cryopreserved sperm, brief exposure of the thawed bull sperm to FertPlus peptide appeared to improve fertility dramatically, and should be confirmed with sperm from other bulls used in a more conventional manner.
Abstract: We evaluated the effect on fertility of in vitro exposure of thawed frozen bull sperm to synthetic FertPlus peptide prior to artificial insemination (AI). The peptide represented a 60-amino acid sequence within rat prosaposin. Commercial cryopreserved semen was from three Holstein bulls. Onset of estrus in groups of Holstein nulliparous heifers was synchronized via injection of prostaglandin F2-alpha, and heifers were scheduled for AI 8-24 hours after estrus was detected. Semen was thawed, diluted to 2.4 x 10(6) sperm/ml with buffer, and split to provide control and exposed aliquots (0 or 30 microM peptide) that were incubated at 37 degrees C for 10 minutes and then were held at 32 degrees C. The two aliquots of semen then were used on an alternate basis 2-65 minutes later to inseminate females. Each AI (one per female) involved the deposit of approximately 250,000 sperm into each uterine horn. This procedure for AI was used to reduce the pregnancy rate with control semen to below the maximum value for a given bull and to facilitate detection of any beneficial effect of the peptide. For each bull, approximately 32 heifers were inseminated with control semen, and approximately 32 heifers were inseminated with peptide-exposed semen. Pregnancy was evaluated ultrasonically approximately 60 days after AI. After excluding one group of heifers with unusually low fertility, averaged across all animals, a 29% increase in pregnancy rate resulted from exposure of sperm to peptide (P < 0.04; one-tailed chi-square test; means were 48 vs. 62%). Pregnancy rates for the three bulls for control and peptide-exposed semen, respectively, were 42 and 62%, 44 and 64%, and 56 and 61%; means in the first two pairs of values tended to differ (P approximately equal to 0.10). These observations should be confirmed with sperm from other bulls used in a more conventional manner. However, with insemination of a limiting number of cryopreserved sperm, brief exposure of the thawed bull sperm to FertPlus peptide appeared to improve fertility dramatically.
TL;DR: Clearly, human, boar, and bull sperm were beneficially modified by brief in vitro exposure to FertPlus peptide, so that for many samples a greater percentage of sperm was bound in vitro.
Abstract: We evaluated the effects of in vitro exposure of sperm to synthetic FertPlus peptide, which represents a 60-amino acid sequence within rat prosaposin, using a microwell sperm-binding assay (SBA), in which an extract of hen's egg served as the binding substrate. Sperm suspensions were incubated with FertPlus peptide (six to eight concentrations; 0 and 20-1,280 pM) at 37 degrees C for 10 minutes, diluted > or = 20 times, and placed onto SBA plates. After 60 minutes at 37 degrees C, unbound sperm were washed away and the DNA of bound sperm was quantified. Percentage of sperm bound was independent of the percentage of motile sperm, but immotile sperm did not bind. For fresh human sperm (25 ejaculates), the percentage of sperm bound was increased by exposure to 640 pM peptide (P or = 1.4 times the value for a 0 pM control aliquot. With frozen-thawed human sperm, for six of seven samples, binding was > or = 1.4 times greater after exposure to 640 pM peptide. For boar sperm held for approximately 24 hours at approximately 18 degrees C before use (28 ejaculates), there was a higher percentage of sperm bound for aliquots previously exposed to 1,280 pM peptide than there was for control aliquots (P or = 1.4 times. For frozen-thawed bull sperm, percentage of sperm bound was > or = 1.4 times greater for 4 of 10 samples that were briefly exposed to 160 pM peptide. Clearly, human, boar, and bull sperm were beneficially modified by brief in vitro exposure to FertPlus peptide, so that for many samples a greater percentage of sperm was bound in vitro. As presented in an accompanying paper, fertility of bull sperm was increased by brief exposure to FertPlus peptide.
TL;DR: In freshly ejaculated sperm from six dogs, P-BSA-FITC staining was observed in 13 +/- 1% of the viable, acrosome-intact cells, as characterized by bright fluorescence over the entire apical region, apparently, epididymal spermatozoa already possess the progesterone receptor.
Abstract: The aim of this study was to investigate whether the dog sperm acrosome reaction can be induced by progesterone and whether the action of progesterone is mediated by binding of pro- gesterone to a receptor on the sperm plasma. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living sper- matozoa. Ten mM progesterone increased the acrosome reaction in viable spermatozoa over time from 3 ± 1% at 0 hours to 69 ± 8% at 6 hours (six dogs). In freshly ejaculated sperm from six dogs, P- BSA-FITC staining was observed in 13 ± 1% of the viable, acro- Veterinary Faculty, Utrecht University, some-intact cells, as characterized by bright fluorescence over the entire apical region. The proportion of P-BSA-FITC-stained, viable, acrosome-intact cells increased to 84 ± 11% following 7 hours in- cubation in a low-calcium medium. In contrast, the majority (72 ± 3%) of fresh epididymal sperm already demonstrated bright P-BSA- FITC staining. Apparently, epididymal spermatozoa already possess the progesterone receptor. The receptor is masked at ejaculation and subsequently gradually exposed.
TL;DR: The human homologue for MSY2, Contrin, is described and it is predicted that inactivation of Contrin function in mammalian germ cells would prevent the formation of functional male gametes.
Abstract: Inactivation of germ-cell-specific molecules essential for the production of functional spermatozoa could lead to attractive new means for male contraception. The mouse protein MSY2 is the mammalian homologue of a class of Xenopus DNA/RNA-binding proteins needed for the transcription of testis-specific genes and for translational repression (masking) of paternal mRNAs. In this report, we describe the human homologue for MSY2, Contrin. Sequence analysis of Contrin cDNAs predicts a protein highly similar to its mouse and Xenopus germ-cell Y-box protein homologues with a cold shock domain and four basic/aromatic islands. Contrin is highly basic and is rich in the amino acids arginine and proline. It contains seven putative casein kinase 2 phosphorylation sites and three putative protein kinase C phosphorylation sites, suggesting that Contrin could be highly phosphorylated in vivo. The predicted protein sequence contains two nuclear localization signals, consistent with its predicted role of shuttling between nucleus and cytoplasm. Contrin maps to human chromosome 17p11.2-13.1. By the criteria of northern and western blotting, Contrin appears to be testis specific and distinct from other mammalian Y-box-binding proteins. We predict that inactivation of Contrin function in mammalian germ cells would prevent the formation of functional male gametes.
TL;DR: Age-stratified growth rates from men taking part in a longitudinal study of aging using magnetic resonance imaging of the prostate suggest that there is an age-related increase in prostate growth rate that peaks in men ages 56-65 and then declines.
Abstract: Men with prostatic enlargement are at highest risk of developing symptomatic lower urinary tract symptoms (LUTS) and related outcomes, such as acute urinary retention. The study of prostatic growth rate can identify the age range at which prostate growth peaks. Evaluation of the natural course of prostate growth requires repeated intraindividual volume measurements at time intervals sufficient to document growth. Our objective was to examine age-stratified prostate growth rates from men taking part in a longitudinal study of aging using magnetic resonance imaging (MRI) of the prostate. Sixty-four men (ages 30-71 years) enrolled in the Baltimore Longitudinal Study of Aging (BLSA) who had T2 pelvic MRIs taken approximately every 2 years were studied. Men were age stratified into four groups: 65 years old. Whole prostate and central gland (anatomically referred to as the transition zone) volumes were determined from the MRI images by a semi-automated image analysis program. Peripheral gland volumes were calculated as the difference between whole prostate and central gland volumes. Growth rates (cc per year) were calculated as change in volume divided by the time interval. On the basis of measurements from the T2 images (n = 128), we observed a linear trend between prostate volume and age. The overall prostate growth rate was 2.36 +/- 3.52 cc per year. Age-stratified growth rates revealed that prostate growth increased with age, peaked at 4.15 +/- 4.98 cc/year for the 56-65-year-old age group and then declined rapidly for the older-aged men. The central gland growth rates followed a trend similar to total prostate volume. These data suggest that there is an age-related increase in prostate growth rate that peaks in men ages 56-65 and then declines. Identification of this trend in prostate growth may aid physicians in targeting men for early diagnosis of LUTS and for possible early intervention. Future studies with a larger sample size are necessary to substantiate these findings.
TL;DR: It is argued that while ICSI iseffective incomparison with standard treatments and with Invitro fertilization (IVF) (when fertilization and Nancy are the outcomes measured), theefficacy and safety ofgamete micromanipulation inhuman IVF has not been thoroughly assessed and may beagenetic time bomb.
Abstract: LAMBFrom theScott Department ofUrology, Department ofCellBiology, Baylor College ofMedicine, Houston,Texas.AsDr.Schlegel haspresented, there isnodoubt thatin-tracytoplasmic sperm injection (ICSI) hasrevolutionizedthetreatment ofmale-factor infertility. Until thedevel-opment ofICSI, thetreatment ofidiopathic male infertil-ityhad remained essentially unchanged forthe last 30years. ICSI hasresulted inparenthood forpreviously in-fertile couples. Suboptimal semen specimens arecom-monly used toproduce conceptions following ICSI; nev-ertheless, thepotential fortransmitting defects infertilitytochildren asaresult ofthistypeofconception islargelyunknown. Inthis debate, Iwill argue thatwhile ICSI iseffective incomparison with standard treatments andwithinvitro fertilization (IVF) (when fertilization andpreg-nancy aretheoutcomes measured), theefficacy andsafetyofgamete micromanipulation inhuman IVFhasnotbeenthoroughly assessed. ICSI may beagenetic time bomb.Atbest, analysis oftheefficacy ofgamete microma-mpulation has proved difficult. Inmany studies, infor-mation concerning theevaluation ofthemale remainedincomplete orunclear. Different techniques were em-ployed toprocess thesperm and different criteria wereused toclassify semen samples. Some early studies re-ported pregnancies after amixed transfer ofembryos fer-tilized bygamete micromanipulation and IVF, and per-haps most importantly, few ofthestudies had adequatecontrols (reviewed inFishel etal,1993). Despite theseproblems, there isnodoubt that significantly improvedfertility results have been obtained through the use ofICSI inmale infertility patients.Amajor concern associated with theuseofICSI forthetreatment ofthemale-factor couple isthesafety ofthe procedure. Abnormal fertilization (three pronuclei,failure toextrude thesecond polar body) andegg acti-vation (parthenogenesis, one pronuclei) sometimes oc-curs, and these embryos arenot transferred. Agreater