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From the Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.
| Correspondence to: Dr Jie Qiao, Peking University Third Hospital, No. 49, North Garden Road, Haidian District, Beijing, China 100083 (e-mail: jie.qiao{at}263.net). |
| Received for publication January 14, 2009; accepted for publication August 14, 2009. |
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Abstract |
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Key words: Semen, infertility
In more recent decades, several studies have reported obvious declines in human sperm counts and overall quality, accompanied by an increase in male reproductive disorders. Exposure to environmental xenoestradiol has been suggested as one possible cause for these observations (Stillman, 1982; Sharpe and Skakkebaek, 1993; Rozati et al, 2002). Animal experimentation has also demonstrated these environmental estrogenic trends. Rats descended from a great-grandfather who was exposed to high levels of endocrine disruptors during fetal development positively correlated with low levels of sperm production (Anway et al, 2005; Anway and Skinner, 2006; Delbes et al, 2006). Based on these findings, some researchers have proposed a strong connection between local estradiol elevation and spermatogenic failure. Other investigations have revealed an obvious elevation of seminal estradiol in infertile men, particularly in oligozoospermic and oligoteratoasthenozoospermic patients (Bujan et al, 1993; Luboshitzky et al, 2002).
However, a variety of causes have been suggested to contribute to oligozoospermia and azoospermia in clinics; these include tract obstruction, inflammation, and spermatogenic failure, to name a few. More recent research has been careful to take into account the fact that most previous studies did not subclassify the patient's etiology into oligozoospermia or oligoteratoasthenozoospermia subtypes (Bujan et al, 1993; Luboshitzky et al, 2002). Strictly speaking, these studies were not critical enough to establish a relationship between the estradiol levels in seminal specimens and spermatogenesis. Given the circumstances, certain oligozoospermia cases caused by inflammation or obstruction are not equivalent to complete spermatogenic failure.
Apart from estradiol, androgenic influence on sperm production has also spawned much research (Voglmayr et al, 1980). Unfortunately, the relationship between intratesticular androgens and spermatogenesis remained unclear until very recently (Jarow and Zirkin, 2005). It was demonstrated that rat intratesticular testosterone could be reduced by 50%–60% without an adverse effect on spermatogenesis; however, there remained little known regarding the amount of testicular testosterone necessary to maintain normal spermatogenesis in the human. Prior to this investigation, the knowledge of human spermatogenesis in relation to testosterone levels was very narrow. It was known that testosterone could be converted into estrogen within the lumen of the male reproductive tract (Hess, 2000), and that human Leydig cells could express androgen receptors, estrogen receptors, and aromatase. This information suggested that the local balance between estrogenic and androgenic actions may be important for maintaining spermatogenesis (O'Donnell et al, 2001).
This study attempts to investigate the relationship between local estradiol/testosterone levels, their balance, and the spermatogenesis state. This was accomplished by separating specimens with a more strict grouping method. If the hypothesis is confirmed, these findings may provide a valuable biological indicator for predicting the spermatogenic state.
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Materials and Methods |
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Semen Analysis
Semen samples were collected by masturbation after 2–5 days of sexual
abstinence. Samples were allowed to liquefy at room temperature for
30–60 minutes and evaluated for sperm count and motility according to
World Health Organization
(1999) criteria. Following a
semen analysis, samples were centrifuged at 2500 x g for 10
minutes to sediment the spermatozoa. The resulting seminal plasma was
immediately separated and divided into 2 aliquots, then stored at
–20°C until assayed.
According to the results of the semen analysis, patients were divided into
three groups: normospermic, consisting of 103 healthy normospermic men, with
sperm concentrations 
20 x 106/mL and normal sperm
motility (a 25%, or a + b 25%); oligozoospermic, consisting of 94
patients with severe oligozoospermia, with sperm concentrations <0.2
x 106/mL; and azoospermic, containing 98 patients with
azoospermia, for whom no sperm were found in the ejaculate following
centrifugation at 2500 x g for 10 minutes.
Hormone Measurements
Seminal plasma samples were thawed at room temperature, sufficiently mixed,
then separated into 200-µl aliquots to determine the concentrations of
testosterone and estradiol by a competitive immunoassay, employing the
Immulite analyzer (Diagnostic Products Corporation, Los Angeles, California).
The intra-assay and interassay coefficients of variation were 5.5% and 6.8%
for estradiol, and 4.8% and 5.1% for testosterone, respectively. The
sensitivities of the assays were 73.4 pmol/L for estradiol and 0.7 nmol/L for
testosterone.
Following Up in All Azoospermic Patients
All azoospermic patients were subdivided into obstructive azoospermia (OA)
and nonobstructive azoospermia (NOA) groups. The OA group consisted of
patients with a clinical diagnosis of obstruction. Among these, 12 were
diagnosed with congenital absence of the vas deferens and seminal vesicles
(CAVD). For CAVD patients, semen analysis showed azoospermia with low seminal
volume of 0.4–1 mL and low pH of 6.5. Semen fructose tests were
negative. Transrectal ultrasonography demonstrated that both the seminal
vesicles and the vas deferens were critically absent. As such, the OA group
was further subdivided into 2 smaller groups: CAVD and non-CAVD groups.
Patients with incomplete spermatogenesis comprised the NOA group. Histological evidence showed maturation arrest (MA), Sertoli cell–only syndrome (SCOS), and tubular sclerosis/atrophy in these patients (Matsumiya et al, 1994). For NOA patients, at least 4 sites were randomly biopsied from each testis. Based on the biopsy results, the NOA group was further subdivided into 2 groups: NOA with sperm (+; NOA with successful sperm recovery, classified by the successful identification of at least 1 spermatozoon in the biopsy); and NOA without sperm (–; NOA with failed sperm recovery, in which no spermatozoon was found in the biopsy from either side of the testis).
Statistical Analysis
For this study, all statistical analyses were carried out using SPSS 10.0
for Windows; results were expressed as a mean ± SD. Statistically
significant differences between mean values were evaluated using a 1-way
analysis of variance; a post hoc test (LSD or Tamhane's T2) was used for
multiple comparisons. Statistically significant differences between subgroups
were compared using independent sample tests. Correlations between study
parameters were made by nonparametric Spearman's correlation coefficient. A
P value of <.05 was considered statistically significant for all
analyses.
Receiver operating characteristic (ROC) curves were constructed to examine the predictive value of seminal hormone levels and their ratios. Sensitivity (y-axis) against (1 – specificity) (x-axis) was plotted at each threshold level and the area under the curve (AUC) was computed by nonparametric Wilcoxon testing. The AUC value represents the predictive value for successful sperm recovery before a testicular biopsy among NOA patients. A value of <0.5 represents no increased chance of recovering spermatozoa following surgery.
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Results |
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Seminal plasma testosterone concentrations were significantly lower in the azoospermic group as compared with normospermic (P < .01) and oligozoospermic groups (P < .01). All three groups demonstrated significant differences in seminal plasma T/E2 ratio (P < .05).
Seminal Plasma Hormone Levels in Azoospermic Patients
After following up all azoospermic diagnoses, a total of 79 patients (23 OA
and 56 NOA) had either a testicular biopsy or epididymal aspiration performed.
In the NOA group, there were 13 patients with hypospermatogenesis, whereas the
other 43 men were diagnosed with MA or with SCOS (absence of sperm). The NOA
group had lower seminal testosterone concentrations than both the normospermic
group and the OA group (P < .01;
Table 2); all NOA patients with
either MA or SCOS also had lower testosterone levels and T/E2
ratios (P < .001).
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In the OA group, there were 12 patients with CAVD, and the other 11 men were without CAVD. The OA group demonstrated significantly higher estradiol levels and lower T/E2 ratios (P < .001) as compared with the normospermic and NOA groups. In addition, the CAVD group had much higher estradiol levels (691.6 ± 117.6 pmol/L) and lower T/E2 ratio values (0.015 ± 0.005) than other OA and NOA patients. After CAVD patients were excluded from the OA group, seminal estradiol levels were still significantly higher in the OA group vs the NOA group (283.7 ± 63.8 vs 239.2 ± 49.9, P < .001; Table 2; Figure 1).
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The predictive potency of seminal estradiol, testosterone, and their ratios was tested by ROC mapping. As displayed in Figure 2, the area under the ROC curve for testosterone peaked at a value of 0.771 (95% confidence interval [CI], 0.577–0.966), and the T/E2 ratio peaked at 0.886 (95% CI, 0.785–0.987). Several threshold values of seminal testosterone levels and T/E2 ratios were analyzed in terms of their specificity and sensitivity. Table 4 visualizes that the best compromise between specificity (81.8%, 84.8%) and sensitivity (72.7%, 72.7%) can be obtained with cutoff values of 4.10 nmol/L for seminal testosterone and 0.0161 for seminal T/E2 ratio, respectively.
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According to the established cutoff values for seminal testosterone and seminal T/E2 ratio (4.1 and 0.0161, respectively), 29 of 56 NOA patients (51.8%) were predicted to fail surgical sperm recovery; of these 29 predictions, 3 patients were found with spermatozoon in their testes. This data establishes the rate of missed diagnosis to be 10.3% (3 of 29). Additionally, of the 13 of 56 NOA patients (23.21%) predicted to have successful surgical sperm recovery, 3 were found without spermatozoa in their testes, setting the misdiagnosis rate at 23.08%. And for the other 14 patients, because of the mismatched values (one was less than the cutoff value, and another was more than the cutoff value), we could not predict their testicular biopsy. Finally, we found 7 men with spermatozoa and 7 men without spermatozoa in their testes.
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Discussion |
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Estrogen and androgen can be considered 2 sides of the same coin. On one hand, androgens can be converted into estrogens; on the other, Sertoli cells are known to secrete fluid under androgen control, and estrogen is involved in the resorption of the fluid in the efferent ducts (Hess et al, 1997; Sharpe, 1997). Despite a lack of knowledge about the specific mechanisms of how estrogen, androgen, or their balance affects spermatogenesis, this data clearly indicates that their local balance plays an important role in maintaining physiological spermatogenesis. Our results also suggest that seminal T/E2 ratios in OA patients, especially those diagnosed with CAVD, may be a good indicator for predicting azoospermia.
Although some studies have claimed that serum testosterone levels demonstrate no relationship to sperm concentration or testicular biopsy (Jackaman et al, 1977; Goulis et al, 2008), others suggest that testosterone may assume a critical role in both morphological development and reproductive function in the male (Holdcraft and Braun, 2004; Takada et al, 2008). Treatment with exogenous testosterone has been shown to fully rescue spermatogenesis in LH-R knockout mice (Lei et al, 2001). In this study, NOA patients without sperm (–) show very low levels of seminal testosterone, as compared with NOA patients with sperm (+); their biopsy tissues indicate severe seminiferous tubule atrophy, sclerosis, and Leydig cell hyperplasia. These findings imply that seminal testosterone levels may predict whether normal spermatogenesis is occurring within the seminiferous tubules of NOA patients.
In addition, serum hormone levels (prolactin [PRL], luteinizing hormone [LH], follicle-stimulating hormone [FSH], estradiol, and testosterone) were analyzed from NOA patients; the results suggest that FSH levels are significantly different between groups with sperm (+) and without sperm (–) (17.2 ± 15.7 IU/L vs 29.2 ± 18.3 IU/L, respectively). However, testosterone levels were not significantly deferent between the 2 groups (16.42 ± 8.69 nmol/L vs 12.98 ± 5.79 nmol/L, respectively; unpublished data). Therefore, FSH levels in serum may be considered as another indicator to predict normal spermatogenesis in NOA patients (Goulis et al, 2008).
In summary, these results demonstrated that 1) reduced T/E2 ratios and increased seminal estradiol levels may reflect the type of obstruction or CAVD, and 2) T/E2 ratios coupled with seminal testosterone levels may serve as good indicators for predicting the success of surgically retrieving spermatozoa from the testes from nonobstructive azoospermic patients. However, additional research encompassing a larger patient population is needed to confirm these predictive values in helping to avoid unnecessary testicular biopsies.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Anway MD, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors. Endocrinology. 2006; 147(6 Suppl): S43 –S49.[CrossRef][Medline]
Aquila S, Sisci D, Gentile M, Middea E, Siciliano L, Ando S. Human
ejaculated spermatozoa contain active P450 aromatase. J Clin
Endocrinol Metab. 2002; 87(7): 3385
–3390.
Bujan L, Mieusset R, Audran F, Lumbroso S, Sultan C. Increased
oestradiol level in seminal plasma in infertile men. Hum
Reprod. 1993; 8(1): 74
–77.
Carreau S, Lambard S, Delalande C, Denis-Galeraud I, Bilinska B, Bourguiba S. Aromatase expression and role of estrogens in male gonad: a review. Reprod Biol Endocrinol. 2003; 1: 35 .[CrossRef][Medline]
Delbes G, Levacher C, Habert R. Estrogen effects on fetal and
neonatal testicular development. Reproduction. 2006; 132(4): 527
–538.
Goulis DG, Tsametis C, Iliadou PK, Polychronou P, Kantartzi PD, Tarlatzis BC, Bontis IN, Papadimas I. Serum inhibin B and antimullerian hormone are not superior to follicle-stimulating hormone as predictors of the presence of sperm in testicular fine-needle aspiration in men with azoospermia. Fertil Steril. 2009; 91: 1279 –1284.[CrossRef][Medline]
Han Y, Feng HL, Sandlow JI, Haines CJ. Comparing expression of
progesterone and estrogen receptors in testicular tissue from men with
obstructive and nonobstructive azoospermia. J Androl. 2009; 30(2): 127
–133.
Hess RA. Oestrogen in fluid transport in efferent ducts of the male reproductive tract. Rev Reprod. 2000; 5(2): 84 –92.[Abstract]
Hess RA, Bunick D, Lee KH, Bahr J, Taylor JA, Korach KS, Lubahn DB. A role for oestrogens in the male reproductive system. Nature. 1997;390: 509 –512.[CrossRef][Medline]
Holdcraft RW, Braun RE. Hormonal regulation of spermatogenesis. Int J Androl. 2004; 27(6): 335 –342.[CrossRef][Medline]
Jackaman R, Ghanadian R, Ansell ID, McLoughlin PV, Chisholm GD. Relationships between spermatogenesis and serum hormone levels in subfertile men. Br J Obstet Gynaecol. 1977; 84(9): 692 –696.[Medline]
Janulis L, Bahr JM, Hess RA, Bunick D. P450 aromatase messenger
ribonucleic acid expression in male rat germ cells: detection by reverse
transcription-polymerase chain reaction amplification. J
Androl. 1996; 17(6): 651
–658.
Jarow JP, Zirkin BR. The androgen microenvironment of the human testis and hormonal control of spermatogenesis. Ann N Y Acad Sci. 2005;1061: 208 –220.[CrossRef][Medline]
Kelch RP, Jenner MR, Weinstein R, Kaplan SL, Grumbach MM. Estradiol and testosterone secretion by human, simian, and canine testes, in males with hypogonadism and in male pseudohermaphrodites with the feminizing testes syndrome. J Clin Invest. 1972; 51(4): 824 –830.[CrossRef][Medline]
Lei ZM, Mishra S, Zou W, Xu B, Foltz M, Li X, Rao CV. Targeted
disruption of luteinizing hormone/human chorionic gonadotropin receptor gene.
Mol Endocrinol. 2001; 15(1): 184
–200.
Luboshitzky R, Kaplan-Zverling M, Shen-Orr Z, Nave R, Herer P. Seminal plasma androgen/oestrogen balance in infertile men. Int J Androl. 2002; 25(6): 345 –351.[CrossRef][Medline]
Matsumiya K, Namiki M, Takahara S, Kondoh N, Takada S, Kiyohara H, Okuyama A. Clinical study of azoospermia. Int J Androl. 1994; 17(3): 140 –142.[Medline]
O'Donnell L, Robertson KM, Jones ME, Simpson ER. Estrogen and
spermatogenesis. Endocr Rev. 2001; 22(3): 289
–318.
Risbridger GP, Bianco JJ, Ellem SJ, McPherson SJ. Oestrogens and prostate cancer. Endocr Relat Cancer. 2003; 10(2): 187 –191.[Abstract]
Rozati R, Reddy PP, Reddanna P, Mujtaba R. Role of environmental estrogens in the deterioration of male factor fertility. Fertil Steril. 2002; 78(6): 1187 –1194.[CrossRef][Medline]
Sharpe RM. Do males rely on female hormones? Nature. 1997; 390(6659): 447 –448.[CrossRef][Medline]
Sharpe RM, Skakkebaek NE. Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet. 1993; 341(8857): 1392 –1395.[CrossRef][Medline]
Stillman RJ. In utero exposure to diethylstilbestrol: adverse effects on the reproductive tract and reproductive performance and male and female offspring. Am J Obstet Gynecol. 1982; 142(7): 905 –921.[Medline]
Takada S, Tsujimura A, Ueda T, Matsuoka Y, Takao T, Miyagawa Y, Koga M, Takeyama M, Okamoto Y, Matsumiya K, Fujioka H, Nonomura N, Okuyama A. Androgen decline in patients with nonobstructive azoospermia after microdissection testicular sperm extraction. Urology. 2008; 72(1): 114 –118.[CrossRef][Medline]
Voglmayr JK, Roberson C, Musto NA. Comparison of androgen levels in ram rete testis fluid, testicular lymph and spermatic venous blood plasma: evidence for a regulatory mechanism in the seminiferous tubules. Biol Reprod. 1980; 23(1): 29 –39.[Abstract]
World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interactions. 3rd ed. Cambridge, United Kingdom: Cambridge University Press; 1999 : 4–33.
Zondek B. Mass excretion of estrogenic hormone in the urine of the stallion. Nature. 1934; 33: 209 –210.
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, P < .01: NOA with sperm (–)
group vs OA without CAVD group or NOA with sperm (+) group; 
, P
= .000: NOA with sperm (–) group vs NOA with sperm (+) group;
