Testicular effect of mustard gas

ADULT UROLOGY

TESTICULAR EFFECT OF MUSTARD GAS M. R. SAFARINEJAD

ABSTRACT Objectives. To explain the testicular effect of mustard gas. Methods. Eighty-one patients who had been exposed to sulfur mustard and had the presenting symptom of infertility underwent evaluation of their reproductive system. Three semen analyses, serum hormonal determinations (luteinizing hormone, follicle-stimulating hormone, and testosterone), and genital examinations were completed for all patients, as were testicular biopsies in 24 patients. Results. Azoospermia and severe oligospermia were diagnosed in 42.5% and 57.5% of patients, respectively. Hormone studies revealed an elevated plasma follicle-stimulating hormone level and normal plasma luteinizing hormone and testosterone concentrations. Testicular biopsy showed selective atrophy of the germinal epithelium, intact Sertoli cells, and normal-appearing Leydig cells. Conclusions. Mustard gas can cause defective spermatogenesis years after exposure. UROLOGY 58: 90–94, 2001. © 2001, Elsevier Science Inc.

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ulfur mustard [bis (2-chloroethyl) sulfide] was initially used as a vesicant chemical warfare agent during World War I. Its use in the past 2 decades and the growing capacity for the manufacture of chemical weapons have increased the risk that soldiers or civilians may be exposed to sulfur mustard. Most of those exposed to sulfur mustard experience nonfatal but disabling skin, eye, and respiratory problems, often for a prolonged period before full recovery. These are the three key targets affected by exposure to sulfur mustard used as a chemical warfare agent. Sulfur mustard, like other alkylating agents, has systemic toxicity to the cells of bone marrow, lymphoid tissue, gut mucosa, and germinal epithelium. On the basis of our own experience treating mustard gas casualties in the Iran-Iraq War, we describe the characteristic hormonal and testicular histologic features in 81 infertile men who were injured by exposure to mustard gas. MATERIAL AND METHODS The study group comprised 81 men with infertility who had been exposed to mustard gas. The men were divided into three From the Department of Urology, Military University of Medical Sciences, Tehran, Iran Reprint requests: M. R. Safarinejad, M.D., Department of Urology, Military University of Medical Sciences, P.O. Box 193951849, Tehran, Iran Submitted: December 18, 2000, accepted (with revisions): March 1, 2001

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© 2001, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED

groups: 44 severely injured men, 20 moderately injured men, and 17 mildly injured men. These patients were injured with mustard gas during 1985 to 1988 in the Iran-Iraq War. Most of the men were exposed at the time of the attack; some were exposed when in a contaminated area several hours after the attack. They had mild to severe injuries of the skin, eyes, mouth, upper respiratory tract, and lungs. All these men had been referred for an infertility evaluation at least 3 years after exposure. Their ages ranged between 28 and 41 years (mean age 34.7). Thirty-one patients had fathered children before exposure. A detailed work history of each man, including work in areas with potential gonadotoxic agent exposure, was reviewed. Routine medical information and study-specific data were obtained at the time of the physical examination. This examination focused on the genitalia and included precise testicular measurements. The information recorded included birth dates of all biologic children, current and past birth control measures, libido, erectile ability, perceived infertility, results of previous semen analyses, the occurrence of any genitourinary tract malformations, infection, trauma, or surgery (especially vasectomy), the use of medications, and a history of other major diseases. None of the patients had undergone a previous vasectomy, vasovasostomy, or herniorrhaphy. Of the 81 patients, 12 (14.8%) had a previous history of a left-side varicocelectomy at another center. The proportion of patients with varicocele was almost similar to that of the normal population.1 Three semen analyses were requested following our standard procedure of a 48-hour interval of sexual abstinence. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were measured by radioimmunoassay. Values for normal adult males range from 3 to 10 mIU/mL for FSH, from 3 to 11 mIU/mL for LH, and from 4.0 to 12.0 ng/mL for testosterone. Other pertinent biochemical parameters of hepatic, renal, and hematopoietic function were re0090-4295/01/$20.00 PII S0090-4295(01)01085-8

TABLE I. Mean basal serum hormone levels and semen quality in three groups of subjects and sperm count in oligospermic patients with normal reference ranges provided Normal Reference Ranges

Variable LH (mIU/mL) FSH (mIU/mL) Testosterone (ng/mL) Sperm count in oligospermic men (106/mL) Sperm motility (%) Normal morphology (%)

Intensity of Exposure Mild ⫾ ⫾ ⫾ ⫾

0.8 1.8 1.8 1.8

Moderate

3–11 3–10 4–12 ⬎20

4.7 14.8 6.8 5.9

(3.7–5.8) (13.4–17) (4.7–9) (4.8–6.8)

⬎50 ⬎30

20.9 ⫾ 3.7 (26.6–56) 41.2 ⫾ 4.9 (18–71)

4.6 15.6 8.4 4.8

⫾ ⫾ ⫾ ⫾

0.9 2.0 1.8 2.4

(3.5–6) (13.4–18) (6.4–10.4) (3.2–5.5)

16.1 ⫾ 3.3 (16–48) 30.2 ⫾ 4.8 (16–68)

Severe 5.7 18 6.4 3.9

⫾ ⫾ ⫾ ⫾

0.9 (4.4–6.8) 2.8 (15–21.5) 2 (4.2–9) 1.8 (2.8–4.2)

14.6 ⫾ 4.1 (1–46) 8.4 ⫾ 3.8 (2–35)

Data presented as the mean ⫾ SD, with the range in parentheses. KEY: LH ⫽ luteinizing hormone; FSH ⫽ follicle-stimulating hormone.

viewed from the most recent serum chemistry studies and complete blood count studies of each patient. World Health Organization (WHO) criteria were used for the comparison of the semen variables. Using the WHO recommendation, we considered a semen sample as normal if it had a volume of more than 2.0 mL, with a sperm concentration of 2 ⫻ 106 spermatozoa/mL or more, 50% or more with forward progressive motility, and 30% or more with normal oval forms.2 Twenty-four men volunteered to undergo open testicular biopsies. A major problem, which was never well resolved, was the estimation of the individual exposure to sulfur mustard. For this reason, the exposure was coded qualitatively. This qualitative exposure estimate was based simply on the history of the intensity of exposure and the injuries recorded on the medical records and the remaining complications from previous mustard gas exposure. The baseline hormone levels and semen parameters are expressed as the mean ⫾ SD. The variables were compared by Student’s t test with the Bonferroni correction. Statistical analysis was performed using the computer statistical software Statistical Package of Social Sciences, version 4.0 (SPSS, Chicago, Ill) and Statistical Analysis System, version 6.04 (SAS Institute, Cary, NC).

RESULTS The assessment of the effects of sulfur mustard on the reproductive function of the men in this study was complicated by a lack of individual studies before sulfur mustard exposure. The testicular size was not appreciably different when the exposed groups were compared with normal fertile men. However, a decrease in testicular size did appear among those men found to be azoospermic when compared with the accepted norms of testicular diameter, that is 5 ⫻ 3 cm. No other genital abnormalities distinguished the normal and exposed populations. Routine screening of the renal, hepatic, and hematopoietic functions demonstrated no significant impairment in any of the men. Patients with a history of severe injury to the lungs had mild to moderate chronic obstructive pulmonary disease. UROLOGY 58 (1), 2001

HORMONE PROFILES AND INTENSITY OF INJURY The mean basal hormone levels of the subjects with the normal reference ranges are listed in Table I. The mean serum LH level was 5 ⫾ 0.9 mIU/mL. This mean is regarded as normal in adult men. The mean serum level of testosterone was 7.2 ⫾ 1.86 ng/mL, also regarded as normal in adult men. The serum levels of FSH were significantly higher than the upper limit of normal (P ⬍0.05). The mean FSH level was 16.1 ⫾ 2.2 mIU/mL. The serum levels of LH and testosterone were similar for all three groups. The mean values did not differ significantly from each other. In subjects with severe mustard gas injuries (n ⫽ 44) who had severe oligospermia (less than 2 ⫻ 106/mL) or azoospermia, the mean FSH level was significantly higher than the mean values for the moderately and mildly injured subjects. By Student’s t test, the difference between these groups was statistically significant (P ⬍0.02) after the Bonferroni correction. SEMEN QUALITY Sperm Count. The sperm count was calculated as the highest total number of sperm per milliliter. Of the 81 infertile men, 34 (42.5%) had azoospermia and 47 (57.5%) had oligospermia. Of the 12 men with varicocele, 5 (41.6%) were azoospermic. All the azoospermic subjects were in the severely injured group. The mean sperm count for the oligospermic men was 4.8 ⫾ 2.0 ⫻ 106 sperm/mL. The mean sperm count of the severely, moderately, and mildly injured oligospermic men was 3.9 ⫾ 1.8, 4.8 ⫾ 2.4, and 5.9 ⫾ 1.8 million/mL, respectively. Each of these means was significantly lower than the normal mean according to the WHO criteria (P ⬍0.05). The differences among the three groups were not statistically significant. Sperm Motility. The sperm motility was calculated as the highest percentage of motility on any of 91

the ejaculates. We found a highly significant difference in sperm motility between the infertile mustard gas-injured patients and the normal WHO criteria. The mustard gas-injured subjects had a mean motility of 17.2% ⫾ 3.7% (range 1% to 50%), which was significantly lower than the mean according to the WHO criteria for normal (P ⬍0.02). The mean motility for the severely, moderately, and mildly injured infertile patients was 14.6% ⫾ 4.1%, 16.1% ⫾ 3.3%, and 20.9% ⫾ 3.7%, respectively. The mean values of the three groups did not differ significantly from each other. Sperm Morphology. The sperm morphology was calculated as the highest percentage of normal forms on any of the ejaculates. A significant difference was found in the morphology of sperm between the infertile mustard gas-injured men and the normal WHO criteria. The infertile mustard gas-injured men had a mean of 26.6% ⫾ 4.5% normal forms (range 20% to 56%). It was significantly lower than that of the normal criteria (P ⬍0.05). The mean percentage of normal forms for the severely, moderately, and mildly injured subjects was 8.4% ⫾ 3.8%, 30.2% ⫾ 4.8%, and 41.2% ⫾ 4.9%, respectively. The mean for the severely injured patients was significantly lower than that of the mildly injured subjects. Separate multiple regression analyses were performed to ascertain the possible relationship of sperm count (transformed in this analysis to the natural logarithm of sperm count) to several other variables, including age, LH, testosterone, FSH, and intensity of exposure to sulfur mustard. The correlation matrix for these patients was also assessed. The most striking feature of this matrix was the pair-wise interdependence exhibited by exposure, FSH, and log-sperm count. Exposure was directly related to the FSH level and inversely related to the log-sperm count. The FSH level was also inversely related to each of these three variables. The regression of the five predictor variables on the log-sperm count showed that only exposure and FSH were related at P ⬍0.01 to the log-sperm count, since inclusion of any of the remaining variables into the regression equation did not significantly reduce the residual variation. The multiple correlation coefficient for exposure and FSH on the log-sperm count was R ⫽ 0.770, and these two variables explain 59.4% of the total variation observed in the log-sperm count. In men in whom both testes were biopsied, the histologic features were similar on both sides. In the most severe cases (azoospermia), generalized absence of all spermatogenic activity was found. The seminiferous tubules were devoid of spermatogonia. The pathologic picture resembled a Sertoli cell-only syndrome (Fig. 1). In the less se92

FIGURE 1. Testicular biopsy from an azoospermic patient. Testicular tubule lined by Sertoli cells only. Groups of Leydig cells are present in the interstitial tissue.

FIGURE 2. Testicular biopsy from an oligospermic patient. Atrophy of seminiferous epithelium with a significant decrease in the amount of cellularity is shown.

verely affected men (oligospermia), a significant decrease in the amount of cellularity within the seminiferous tubules was found (Fig. 2). No evidence of inflammation and minimal evidence of increased fibrosis and interstitial changes were present. COMMENT Mustard gas may occur in the form of sulfur mustard gas S (CH2CH2Cl)2 or nitrogen mustard gas. There are several types of nitrogen mustard gas, and N (CH2CH2Cl)3 is the most toxic. This form of the gas becomes a liquid on the skin. During the first 5 minutes of exposure, 50 ␮g/ cm2 causes blistering.3 Mustard gas is able to penetrate leather and normal clothing within a few minutes, but rubber material provides protection for several hours. The mustard gases are very persistent chemical agents. Chemicals capable of inUROLOGY 58 (1), 2001

ducing blisters, known as blistering or vesicating agents, have been widely known for more than 150 years. Mustard gas causes clinical symptoms after absorption from the conjunctiva, mucosa, and skin. Physicians familiar with other alkylating agents used in clinical medicine deal mainly with their systemic toxicity to cells of the bone marrow, lymphoid tissue, and gut mucosa. Owing to its alkylating and electrophilic properties, mustard gas is able to change the structure of nucleic acids, cellular membranes, and proteins. Thus, it can act with amine groups, carboxyl groups, SH groups, OH groups, and primary phosphate groups. The reaction with cellular DNA is important and particularly well understood. Mustard gas causes cross-linking of the two complementary strands in the DNA molecule and thus interferes with DNA synthesis and cellular division.4 Blistering agents were extensively used in chemical warfare during World War I, well before the development of the more deadly nerve agents 25 years later. Blistering agents continue to be used in present-day conflicts for several reasons. They are simple to produce; they cause a large number of incapacitating nonlethal and lethal injuries; and because of their persistence after dispersal, they can prevent an enemy from making use of contaminated areas. Sulfur mustard (2,2-dichlorodiethyl sulfide) and Lewisite (2-chlorovinyl dichloroarsine) are the best known vesicating chemical warfare agents. Sulfur mustard is the blistering agent most commonly associated with warfare. It has been documented in regional conflicts during the past 50 years, most recently in the Iran-Iraq War. Sulfur mustard is an oily liquid that is only slightly soluble in water. The reactivity of sulfur mustard in the target tissue requires a chemical cyclization step.4 When administered as either a vapor or liquid, sulfur mustard is rapidly absorbed into tissue. Exposure to sulfur mustard may produce pathologic changes in the lungs, eyes, bone marrow, and perhaps other organs. The testis appears to be especially susceptible to sulfur mustard. Infertility was the presenting symptom in all our patients. Sexual dysfunction was not the chief complaint. No abnormal findings were detected by the physical and genital examinations. The histologic findings were similar in all cases. Almost total atrophy of the seminiferous epithelium was found, with the interstitial cells intact. The most severely affected individuals appeared to have a Sertoli cellonly pattern. The hormonal data revealed no evidence of an alteration in the pituitary-testicular axis. We believe that in some mustard gas-injured paUROLOGY 58 (1), 2001

tients, the testicular damage is reversible. Our experiences demonstrated that of the severely injured sulfur mustard gas patients, about 40% exhibited fertility problems in the long term. Of the subjects who had had “mild” sulfur mustard toxicity initially and fully recovered after 3 months, up to 90% never exhibited oligospermia. When oligospermia developed, only 20% recovered from mild to moderate oligospermia. These data demonstrate the reversibility of testicular damage and a threshold limit of toxicity. Azizi et al.5 investigated the acute and chronic effects of mustard gas in young men. The serum total and free testosterone and dehydroepiandrosterone levels were markedly decreased in the first 5 weeks after exposure. FSH, LH, prolactin, and 17 alpha-OH progesterone were normal in the first week, LH increased by the third, and FSH and prolactin by the fifth week. All hormone levels had returned to normal by the 12th week after exposure. In 28 (66.6%) of 42 men examined 1 to 3 years after injury, the sperm count was less than 3 million cells/mL and the FSH level was increased compared with normal men. The findings of their study are consistent with those of the present study. Our experience with mustard gas-injured patients emphasizes the need for follow-up evaluation of reproductive function in men exposed to sulfur mustard gas. Our observations suggest that FSH alone is as good a predictor of the effects as FSH, LH, and testosterone together. With the use of log-sperm count rather than a numerical sperm count, this FSH predicting tool is somewhat more sensitive. Since sulfur mustard does appear to have a gonadotoxic effect in humans, one must inevitably address the issue of whether a threshold limit of toxicity exists. The length of exposure time, proximity to the source, intensity of exposure, and wearing of protective clothing and a mask all had great implications in testicular damage. Those casualties who had severe skin and respiratory injuries demonstrated significantly poorer semen qualities, and most of these severely injured men were azoospermic. The exact nature of this systemic sulfur mustard effect, either inhalation and/or skin contact, the possibility of a no-effect concentration, and even the possible reversibility of the lesions remain to be explored by experimental animal model studies. Similar experimental animal model studies have been previously performed for other possible gonadotoxic agents such as glycerol.6 – 8

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for the Examination of Human Semen and Semen-Cervical Mucus Interaction, 3rd ed. Cambridge, Cambridge University Press, 1992. 3. NATO Handbook on the Medical Aspects of NBC Defensive Operation (Amed). 1973, pp 6 –9. 4. Ross WCJ: Biological Alkylating Agents. Stoneham, Massachusetts, Butterworths, 1962, pp 142–145. 5. Azizi F, Keshvarz A, and Roshanzamir F: Reproductive function in men following exposure to chemical warfare with sulfur mustard. Med War 11: 34 – 44, 1995.

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6. Suleiman A, Igdoura SA, and Wiebe JP: Suppression of spermatogenesis by low-level glycerol treatment. J Androl 15: 234 –243, 1994. 7. Weinbauer GF, Galhorta MM, and Nieschlag E: Focal testicular destruction following intratesticular injection of glycerol in rats. Int J Androl 8: 365–375, 1985. 8. Wiebe JP, Barr KJ, and Buckingham KD: Sustained azoospermia in squirrel monkey, Saimiri sciureus, resulting from a single intratesticular glycerol injection. Contraception 39: 447– 457, 1989.

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