Infertility in men with cystic fibrosis.

The majority of men with cystic fibrosis (CF) have associated congenital bilateral absence of the vas deferens (CBVAD). This congenital defect results in the absence of the anatomic ducts through which spermatozoa pass from the testes to the urethra. No spermatozoa are found in the semen, a condition referred to as obstructive azoospermia. This abnormality of the genital-urinary system is associated with the same genetic mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR ), which leads to the classic presentation of a patient with CF. However, patients may have CBVAD and CFTR mutations without symptoms of CF. Screening for the common mutations may miss a milder rare gene alteration: a DNA variant in the 5T allele. With the advent of assisted reproductive technologies, fertility is now possible for these men. The National Institutes of Health recommend genetic counseling for any couple attempting assisted reproductive techniques when the man has CF or presents with obstructive azoospermia and is positive for a CF mutation.

The lack of influence of age on male fertility.

OBJECTIVE: This study was undertaken to determine the effect of male aging on sperm quality as determined by semen analysis, the fertilization rate of human oocytes in vitro, and live birth rates. STUDY DESIGN: Retrospective analysis correlating outcome measures with male age was performed for 558 oocyte donation cycles in 441 couples. The oocyte donation model was chosen because it controls for oocyte quality and endometrial receptivity, which allows variations in sperm quality as a function of male age to be the only dependent variable. Outcome measures analyzed were semen analysis, fertilization rates in vitro, pregnancy rates, live birth rates, and cumulative live birth rates by life-table analysis. RESULTS: There was a negative correlation between male age and total sperm count, but there was no correlation between male age and any of the other parameters in the semen analysis. There was no association between male age and the fertilization rate of donated oocytes in vitro, pregnancy rates, or live birth rates. Recipient couples were grouped by quartiles of male age, and cumulative live birth rates were the same in the 4 groups. CONCLUSION: Whereas male aging is associated with a significant decline in total sperm count, this change is not reflected in a decreased fertilization rate or a decreased live birth rate in the oocyte donation model.

Serum inhibin B levels in males with gonadal dysfunction.

OBJECTIVE: To determine whether inhibin B levels are reflective of the etiology of gonadal dysfunction. DESIGN: Institutional study. SETTING: A tertiary care university-affiliated infertility clinic. PATIENT(S): Forty-four men: 16 with primary testicular failure, 10 with partial idiopathic hypogonadotropic hypogonadism (IHH), 8 with primary germ cell failure, one with iatrogenic hypogonadotropic hypogonadism, one with untreated Kallmann's syndrome, and 8 healthy fertile controls. INTERVENTION(S): Three individuals (one each with IHH, hypogonadotropic hypogonadism [HH], and Kallmann's syndrome) underwent treatment with human chorionic gonadotropin. MAIN OUTCOME MEASURE(S): Baseline serum inhibin B, FSH, LH, total testosterone and estradiol levels, and sperm concentrations were measured. RESULT(S): Serum inhibin B concentrations were significantly higher in fertile controls (255 +/- 59 pg/mL) than in men presenting with primary testicular failure (75 +/- 46 pg/mL, P<.0001) or in those presenting with primary germ cell failure (73 +/- 31 pg/mL, P<.0001). Inhibin B levels were also lower in males with partial IHH (187 +/- 112 pg/mL, P<.05). The patient with iatrogenic HH had a level of 184 pg/mL, whereas the patient with Kallmann's syndrome had nondetectable levels (<10 pg/mL). Serum inhibin B levels correlated positively with sperm concentration (P=.0001), and negatively with FSH levels (P=.01) and LH levels (P<.05). Human chorionic gonadotropin therapy altered inhibin B levels. CONCLUSION(S): Inhibin B plays an important role as an endocrine regulator of FSH secretion, whereas gonadotropins are involved in the regulation of inhibin B secretion.

Evaluation of a large cohort of men presenting for a screening semen analysis.

OBJECTIVE: To determine: [1] what percentage of men in an infertile relationship will have a semen abnormality, [2] the average value for each semen parameter in this group of men, [3] the distribution of abnormal semen parameters in this group, and [4] if our data support the hypothesis that sperm concentration is declining. DESIGN: Retrospective cohort study. SETTING: County hospital university-based infertility clinic. PATIENT(S): Male partners of women presenting for an infertility evaluation. INTERVENTION(S): Semen specimens were collected after 2-5 days of abstinence. MAIN OUTCOME MEASURE(S): Sperm concentration, motility, and morphology. RESULT(S): Fifty-two percent of samples had at least one sperm abnormality based on World Health Organization criteria. Fifty-one percent had an abnormality in sperm motility, 18% in sperm concentration, and 14% in sperm morphology. Four percent of the patients were azoospermic. CONCLUSION(S): No decline in sperm density was revealed in semen collected by men presenting for an initial screening semen analysis.

Comparison of two methods for the measurement of sperm concentration.

OBJECTIVE: To determine the accuracy of both the hemocytometer and the MicroCell, to evaluate which method is the most reliable, and to confirm the accuracy of latex beads as an internal standard. DESIGN: Prospective procedural assessment. SETTING: University-based infertility clinic. PATIENT(S): One hundred sixty-five male sexual partners of women undergoing screening for infertility. INTERVENTION(S): Semen analysis. MAIN OUTCOME MEASUREMENT(S): Sperm and latex bead concentrations. RESULT(S): Sperm concentration values obtained with the hemocytometer were highly correlated with those obtained from the MicroCell (r = 0.88). The mean value of the latex beads concentration was closer to the standard value using the hemocytometer than the MicroCell. CONCLUSION(S): Both the hemocytometer and the MicroCell are suitable as screening techniques to measure sperm concentration. Fixed suspensions of latex beads serve as reliable internal quality control standards.

Normative reproductive indices for male and female adult Sprague-Dawley rats.

The Sprague-Dawley rat is traditionally used as the experimental model for the study of contraceptive agents and reproductive toxicants. Until recently, the normative values used to compare hormone levels after drug exposure were based on the values generated by radioimmunoassay methods developed 30 years ago. To ascertain normative reproductive indices for adult female and male Sprague-Dawley rats over a 6-month age period, we measured luteinizing hormone, testosterone, and estradiol using commercially available kits that employ updated assay techniques. In addition, sperm indices were correlated with reproductive hormones over the same time period. Animals were killed at 107, 128, 156, 212, and 268 days of age irrespective (for females) of cycle stage. Serum LH levels did not change with increasing age; however, the female rats had significantly higher LH values than did the males at comparable ages (p < 0.001). Testosterone levels and sperm parameters did not significantly change with increasing age. Estradiol levels were significantly higher in 107-day-old female rats than they were in female rats in all older age groups (p < 0.01). The values reported can be used in designing and interpreting data generated in ongoing, long term toxicological and contraceptive studies using the rat animal model.

Effects of lead exposure on GnRH and LH secretion in male rats: response to castration and alpha-methyl-p-tyrosine (AMPT) challenge.

Animal and clinical studies suggest that lead exposure disrupts the hypothalamic-pituitary axis. To define more precisely the toxic action of lead on the hypothalamic-pituitary unit, a series of in vivo and in vitro experiments were performed. The first experiment was designed to determine whether lead exposure exerts an inhibitory effect on GnRH secretion as reflected by an enhanced inhibition of luteinizing hormone (LH) secretion in response to the tyrosine hydroxylase inhibitor methyl-p-tyrosine (AMPT). In the control animals, the AMPT dose had no significant effect on LH secretion, whereas LH fell significantly in the lead-treated animals. In experiments designed to evaluate the effects of lead exposure on the pattern of pulsatile release of gonadotropins castrated control and lead-dosed animals were cannulated, and serial blood sampling was performed. Baseline LH and follicle-stimulating hormone values were not statistically different between the control and lead-treated group. There were no significant differences noted in pulsatile patterns when the data were analyzed as groups. Pituitary cells harvested from lead-treated animals released significantly more LH that did the control animals. These data are consistent with the hypothesis that the signals between the hypothalamus and pituitary gland are disrupted by lead exposure in the intact animal. However, the lead-exposed castrated rat's hypothalamic-pituitary unit is able to adapt to the toxic effects of lead.

Factors influencing sperm-zona pellucida binding in vitro using the intact zona model.

The zona pellucida binding assay assesses the ability of spermatozoa to bind to the zona pellucida. The present study investigated the influence of: (i) prior oocyte exposure to spermatozoa on subsequent sperm-zona pellucida binding in vitro; and (ii) cryopreservation of oocytes. Only oocytes obtained from fertile donors were used and the binding capacity of non-inseminated, cryopreserved oocytes was compared with both inseminated/unfertilized, cryopreserved oocytes and inseminated/unfertilized, non-cryopreserved oocytes recovered from in-vitro fertilization cultures on sperm-zona pellucida binding using an intact zona model. There was no statistically significant difference in sperm-zona binding between non-inseminated, cryopreserved oocytes (range 9.6-23.2), inseminated/unfertilized, cryopreserved oocytes (range 15.0-16.0) and inseminated/ unfertilized, non-cryopreserved oocytes (range 3.3-23.0). The coefficient of variation for sperm binding to all oocyte groups was very large (range 37-121%). We conclude that neither prior exposure of human oocytes to human spermatozoa nor cryopreservation of human oocytes influences the subsequent binding of a different population of spermatozoa to the zona pellucida. However, large oocyte-to-oocyte variation of sperm-zona binding may diminish the usefulness of this assay in clinical practice and as a research tool.

The diagnosis and treatment of male infertility.

Male factor infertility is a heterogeneous disorder, a factor that precludes the development of either a single laboratory test to predict sperm fertility potential or a single therapy to improve fertility. A better understanding of the pathophysiology of the disorder will ultimately lead to more clearly defined diagnostic categories and specific treatment regimens.

A method for quantification and correction of proteins after transfer to immobilization membranes.

A method is described for quantification of specific proteins after electrophoresis and transfer to immobilization membranes for Western blots. This method is analogous to methods used to correct the amounts of specific transcripts detected on Northern blots. Ponceau S staining of proteins bound to immobilization membranes is efficient and accurate compared to antibody binding in terms of time, effort and cost. Comparison of Ponceau S to other detection and staining methods for quantifying proteins and the basic chemistry of Ponceau S are summarized.

Effects of toxic levels of lead on gene regulation in the male axis: increase in messenger ribonucleic acids and intracellular stores of gonadotrophs within the central nervous system.

Lead is a male reproductive toxicant. Lead exposure results in a general suppression of the hypothalamic-pituitary-testicular (HPT) axis in male rats. The mechanism(s) for this disruption by lead is unknown. Toxic lead levels seem to disrupt central nervous system (CNS) control of the HPT system, resulting in a decrease in serum testosterone levels and sperm concentrations. A study designed to elucidate the mechanisms accounting for the disruption of the normal function of the male axis by toxic lead levels at the molecular level demonstrated a 2-3-fold enhancement of mRNA levels of GnRH and the tropic hormone LH. A 3-fold increase of intracellular stores of LH was also found. Because mRNA levels of LH and GnRH and pituitary levels of stored LH are proportional to blood levels of lead, we hypothesize that lead interferes with the normal release of tropic hormones and disrupts hormonal feedback mechanisms. The observed pleiotropic effects of lead upon the male axis and other systems may be explained by simple and unique competition by lead with normal metal ion binding sites that govern genetic control of specific genes.

Lead exposure in vivo alters the fertility potential of sperm in vitro.

Lead is a male reproductive toxicant. We previously reported that in vivo lead exposure results in a suppression of the hypothalamic-pituitary-testicular axis in male rats. This study was designed to assess if lead exposure in vivo alters (1) the ability of sperm to fertilize ova in vitro, (2) the morphology of the spermatozoa, and (3) the relationship of cell types in the testes as evaluated by DNA flow cytometry. Male Sprague-Dawley rats were given access to either lead-free (0.0%) or 0.3% lead acetate-containing water for 14, 30, or 60 days starting at 100 days of age. On the day of termination, sperm harvested from the caudae epididymis were incubated with eggs harvested from superovulated non-lead-treated females and were scored for stages of penetration. Sperm were also studied by electron microscopy. The testes of animals treated for 60 days were processed for DNA flow cytometry. The overall distribution of the stages of fertilization was significantly different between control and lead-treated animals. The lead-treated groups fertilized significantly fewer eggs than did sperm from the control group. Increased duration of exposure to lead acetate did not result in a more significant percentage of eggs not fertilized. No ultrastructural changes were noted in the spermatozoa of animals treated with lead compared to control animals. There were no differences in the histogram patterns of testicular cells collected from lead-treated animals and control animals. We conclude that lead alters sperm function by altering the hormonal control of spermatogenesis rather than by direct toxic action on spermatozoa.

Inclusion of synthetic DNA templates of similar length and base composition to PCR-amplified products in restriction enzyme digestions: an efficient aid in characterization of point mutations.

Because of a subtle anomaly we encountered upon an analytical gel while characterizing a point mutation in an exon of a patient, we decided to perform expensive and time-consuming procedures to characterize the anomaly. Although initial and subsequent Southern blots and PCR analyses of this patient's mutation suggested that his mutation lay directly within a TaqI recognition site, further characterization revealed that the mutation actually lay in a base immediately outside the recognition site. Had we included an appropriate double-stranded DNA control in the restriction enzyme digestion of this patient's PCR-amplified exon, we could have arrived at the correct conclusion as to the location of the mutation without incurring high costs and time loss. This brief report depicts the use of DNA controls of appropriate length and base composition as a means of avoiding erroneous conclusions and expense in routine mutational analyses in the clinical setting.

Comparison of two weeks versus one week of prenatal ethanol exposure in the rat on gonadal organ weights, sperm count, and onset of puberty.

Sprague-Dawley dams from Harlan Ind. (Indianapolis, IN) were administered a fortified ethanol liquid diet containing 35% ethanol derived calories for two weeks (E-2) beginning on day 7 or one week (E-1) beginning on day 13 of gestation and continuing through parturition. Control dams were pair-fed an isocaloric liquid diet containing no ethanol during these periods or remained on lab chow and water. E-2 dams consumed an average of 13.52 g ethanol/kg bwt during the first week of exposure (days 8-14) and 12.50 g ethanol/kg bwt the second week (days 14-20). E-1 dams consumed significantly less than E-2 dams during the second week (9.75 g/kg; p < 0.0001). Although the lower consumption in E-1 dams led to a significant decrease in maternal weight gained during the few days of pregnancy compared to E-2 dams, birthweights of E-1 offspring were significantly heavier than those of E-2 offspring (p < 0.05). No effect of ethanol was detected on anogenital distance at birth in either sex. Puberty was delayed in female offspring of both E-1 and E-2 dams (p < 0.01) as measured by age of vaginal opening. These data suggest that the primary teratogenic actions of ethanol in the rat on fetal growth, as well as delayed puberty in females, occur in the last week of gestation. In adult E-2 males, testis weight was significantly heavier than all other groups when indexed to body weight. No effect of prenatal ethanol exposure was observed on the indexed weights of prostate, epididymis, or seminal vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of azoospermia in normal men with combined Nal-Glu gonadotropin-releasing hormone antagonist and testosterone enanthate.

The effects of a combined GnRH antagonist and testosterone (T) replacement regimen on gonadotropins and spermatogenesis were examined to assess its potential as a male contraceptive regimen. The potent Nal-Glu GnRH antagonist ([Ac-D2-Nal1,D4-Cl-Phe2,D3-Pal3,Arg5, D4-p-methoxybenzoyl-2-amino butyric acid6,D-Ala10]GnRH) was administered daily (7.5 mg, sc) to eight normal men for 16 weeks. T enanthate was given im starting at week 2 and every 2 weeks thereafter through week 14 of the treatment phase. Serum LH, FSH, T, and estradiol concentrations were measured frequently during the 5-week control period, the 16-week treatment phase, and the 14-week recovery phase. Semen analyses were performed every week during the control phase and every 2 weeks during the treatment and recovery phases. Seven of eight subjects became azoospermic by 6-10 weeks of treatment; the eighth subject, who failed to achieve azoospermia, suppressed his sperm count to 7 million/mL by week 14 (from a mean baseline of 42 million/mL) before treatment was prematurely terminated because of localized swelling at each of his injection sites. Sperm counts returned to baseline 10-14 weeks after the end of Nal-Glu administration. Seven of the eight subjects showed suppression of LH to the limit of assay detection (less than 0.2 U/L), whereas the eighth subject showed incomplete suppression. Serum bioactive and immunoreactive LH concentrations showed concordant responses. Mean serum FSH concentrations were also markedly suppressed. Serum T and estradiol concentrations declined dramatically during the first 2 weeks of Nal-Glu GnRH treatment, but returned to the normal physiological range after T enanthate replacement was initiated. Libido and sexual potency were maintained. No systemic side-effects, other than erythema and induration at injection sites, were observed. These data demonstrate that combined GnRH antagonist plus T treatment can predictably and reversibly induce azoospermia in most men and has potential as a male contraceptive regimen.

PIP: This study examined the hypothesis that a gonadotropin-releasing hormone (GnRH) antagonist, given at a dose that will suppress gonadotropin secretion and combined with a replacement dose of androgen, will induce reversible azoospermia in normal men while maintaining sexual function. About 8 normal men aged 24-48 years participated in the study. Sperm concentration in all 8 subjects decreased during the treatment; 7 became azoospermic by 6-10 weeks of treatment. In the 8th subject, who failed to achieve azoospermia, the sperm count declined to 7 million/ml by week 14, when treatment was prematurely terminated because of localized inflammation and induration at each of the injection sites. Sperm count returned to baseline following the end of Nal-Glu administration. No statistically significant change was observed in the sperm function during the oligospermic phase of treatment from baseline. 7 subjects showed suppression of luteinizing hormone, while the 8th experienced edema and discomfort at the injection sites. Mean serum follicle stimulating hormone concentrations were suppressed. Serum testosterone and estradiol concentrations declined during the treatment but returned to normal following T enanthate replacement initiation. Main side effects of Nal-Glu GnRh treatment were local erythema and induration at the injection sites due to local histamine release. In conclusion, the combined GnHR antagonist and T treatment can induce azoospermia in most men and has potential as a male contraceptive regimen.

Prenatal lead exposure in the rat during the third week of gestation: long-term behavioral, physiological, and anatomical effects associated with reproduction.

Sprague-Dawley dams were administered lead acetate (0.1%) in their drinking water from Day 14 of gestation to parturition to determine whether exposure of the fetus to elevated lead (Pb) levels during a period of rapid differentiation of the hypothalamic-pituitary-gonadal (HPG) axis would disrupt HPG function in adulthood. At birth, offspring from 20 Pb-treated and 10 control dams were weighed and 2 litter representatives from each sex were fostered to untreated dams. Animals were weaned at 26 days of age and subsequently group housed by sex and treatment. Blood Pb levels in prenatally exposed pups were below the limits of detectability at weaning. Female offspring from Pb-treated dams were found to have a significant delay in the day of vaginal opening. In a sample of lead exposed females, 50% were found to exhibit prolonged and irregular periods of diestrous which was accompanied by an absence of observable corpora lutea when they were euthanized at 83 days of age. Male offspring from these dams were found to have decreased sperm counts at 70 and 160 days of age and to exhibit significantly less territorial scent marking and masculine sex behavior in adulthood compared to controls. Azoospermia was observed in 1 lead exposed animal at 70 days of age and 2 animals at 160 days. Enlarged prostates were observed in Pb-exposed males measured at 160 days, but other sex organ weights were normal. Volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus in adulthood was significantly reduced by approximately 35% in Pb-exposed males. Pulsatile release of gonadotropins, measured in castrated adult animals of both sexes, revealed irregular release patterns of both FSH and LH in some Pb animals which were not observed in controls. The overall pattern of results suggests that multiple levels of the HPG axis can be affected by exposure to Pb during a period of gestation when structures related to the HPG axis are undergoing rapid proliferation. These data indicate that lead exposure during this period places the exposed animal at significant risk for reduced reproductive capacity in adulthood.

The effect of age of exposure on lead-induced testicular toxicity.

The present study was designed to assess the significance of age of exposure on the expression of lead toxicity on the male gonad. Male Wistar rats, age 42 days, 52 days and 70 days were treated with lead acetate in their water for 30 days prior to sacrifice. The lead treated groups in all cases had blood lead values significantly greater than control animals. Blood lead levels in control animal groups were less than 7 micrograms/dl. Serum testosterone and sperm concentration and production rate were significantly suppressed in those animals that were exposed to lead acetate starting at age 52 days and 70 days, but not 42 days. These data indicate that prepubertal rats may be less sensitive to the toxic effects of lead than are rats whose exposure begins after puberty has been initiated.

The effect of duration of exposure on the expression of lead toxicity on the male reproductive axis.

This study assesses the significance of duration of exposure on the expression of lead toxicity on the male reproductive system. Male Wistar rats, 52 days old, were treated with 0.0% or 0.6% lead acetate in their water for 7, 14, 30, or 60 days prior to sacrifice. In all cases, the lead-treated groups had blood lead and free erythrocyte protoporphyrin (FEP) levels significantly higher than control animals (P less than 0.0001). Serum testosterone levels and spermatogenesis were suppressed in all lead-treated groups compared to the corresponding controls (P less than 0.05 and P less than 0.001, respectively), except for the group treated for 7 days. The data presented verify that exposure to lead acetate is toxic to the reproductive axis in male rats, but that increased duration of exposure after 14 days does not further suppress serum testosterone levels or spermatogenesis.

Prenatal exposure to a low-frequency electromagnetic field demasculinizes adult scent marking behavior and increases accessory sex organ weights in rats.

Pregnant Sprague-Dawley dams were exposed to a low-level, low-frequency pulsed electromagnetic (EM) field (15 Hz, 0.3 msec duration, peak intensity 8 gauss) for 15 min twice a day from day 15 through day 20 of gestation, a period in development that is critical for sexual differentiation of the male rat brain. No differences in litter size, number of stillborns, or body weight were observed in offspring from field-exposed dams. At 120 days of age, field-exposed male offspring exhibited significantly less scent marking behavior than controls. Accessory sex organ weights, including epididymis, seminal vesicles, and prostate, were significantly higher in field-exposed subjects at this age. However, circulating levels of testosterone, luteinizing hormone, and follicle-stimulating hormone, as well as epididymal sperm counts, were normal. These data indicate that brief, intermittent exposure to low-frequency EM fields during the critical prenatal period for neurobehavioral sex differentiation can demasculinize male scent marking behavior and increase accessory sex organ weights in adulthood.