July 10, 1995
INTRODUCTION
PERSIAN GULF SYNDROME IS A CATCH-ALL DIGNOSIS FOR UNEXPLAINABLE
AILMENTS THAT INCLUDE HAIR LOSS, NAUSEA, RESPIRATORY PROBLEMS, FATIGUE,
LOSS OF MEMORY, MUSCLE AND JOINT PAINS, SHORTNESS OF BREATH AND
GASTROINTESTINAL COMPLAINTS. 1,2,3, THESE AILMENTS HAVE BEEN
REGISTERED BY APPROXIMATELY 30,000 VETERANS CLAIM TO HAVE DEVELOPED
SIMILAR SYMPTOMS.3
AN ADDITIONAL COMPLAINT HAS BEEN REPORTED.4 VETERANS OF THE
PERSIAN GULF WAR AND THEIR PARTNERS HAVE COMPLAINED OF A "BURNING
SENSATION" AFTER SEXUAL INTERCOURSE. THIS IS APPARENTLY FOLLOWED BY
VAGINAL IRRITATION IN THE FEMALE. AS YET, THIS ASPECT OF THE SYMDROME
HAS NOT BEEN INVESTIGATED.
THE CURRENT REPORT IS A PROSPECTIVE STUDY DESIGNED TO EVALUATE
SEMEN SAMPLES FROM MALES WHO HAVE RETURNED TO THE UNITED STATES FROM
THE PERSIAN GULF. THE AIM OF THE STUDY IS TO COMPARE VARIOUS SEMEN
PARAMETERS BETWEEN PERSIAN GULF VETERANS AND THE NORMAL POPULATION.
MATERIAL AND METHODS
OVERVIEW
BECAUSE AN EJACULATE IS COMPOSED OF TWO PARTS, THE SPERM AND THE
SEMINAL FLUIDS, BOTH PARTS WERE ANALYZED. THE STUDY COMPARED SUCH
PARAMETERS AS SPERM CONCENTRATION, SPERM MOTILITY AND SPERM MORPHOLOGY.
IN ADDITION, THE SEMINAL FLUIDS WERE EVALUATED FOR CHANGES IN PH
(HYDROGEN ION CONCENTRATION) AND OSMOLALITY (NUMBER OF PARTICLES IN
SOLUTION) OVER TIME, THE PRESENCE OF BACTERIAL AND FUNGAL
MICROORGANISMS AND PROTEIN CONTENT.
PROJECT APPROVAL
THIS STUDY PROPOSAL WAS PRESENTED TO, AND APPROVED BY, THE
INSTITUTIONAL REVIEW COMMITTEE OF THE GREENVILLE HOSPITAL SYSTEM.
STUDY POPULATION
THE STUDY POPULATION CONSISTED OF PERSIAN GULF VETERANS PRESENTING
TO REPRODUCTIVE ENCODRINOLOGY ASSOCIATES OF THE GREENVILLE HOSPITAL
SYSTEM FOR SEMEN ANALYSIS. ALL PATIENTS 1) SIGNED AND RECEIVED A COPY
OF A CONSENT FORM DESCRIBING THE AIM OF THE STUDY,2) RECEIVED A COPY OF
THE SEMEN SPECIMEN COLLECTION PROCEDURES AND 3) RECEIVED A
QUESTIONNAIRE AIMED AT EVALUATING THE VETERANS PRESENCE IN THE PERSIAN
GULF ARENA.
CONTROL POPULATION
SEMEN DATA FROM PATIENTS UNDERGOING IN VITRO FERTILIZATION WERE
USED AS CONTROLS. IN ORDER TO BE SELECTED AS A CONTROL, THE DATA SET
HAD TO MEET OR EXCEEDTHE MINUMUM SEMEN PARAMETER VALUES EXTABLISHED BY
THE WORLD HEALTH ORGANIZATION.5
EXPERIMENTAL PROTOCOL
UPON RECEIPT BY THE LABORATORY, SEMEN SPECIMENS WERE PLACED INTO A
37 DEGREE CELCIUS INCUBATOR FOR UP TO 30 MINUTES TO ALLOW LIQUEFACTION
TO TAKE PLACE. FOLLOWING LIQUEFACTION, SPECIMENS WERE GENTLY VORTEXED
AND EVALUATED FOR VOLUME, PH, OSMOLALITY, SPERM CONCENTRATION, MOTILITY
AND MORPHOLOGY, NUMBER OF ROUND CELLS PER HIGH POWER FIELD AND SPERM
AGGLUTINATION, (THESE PARAMETERS COMPRISE A ROUTINE SEMEN
ANALYSIS.6,7,8) SPECIMENS WERE ANLYZED AGAIN AT 1,2 AND 4 HOURS
FOLLOWING THE INITIAL SEMEN ANALYSES FOR PH, OSMOLALITY, SPERM
CONCENTRATION AND MOTILITY.
IF REQUIRED, SPECIMENS WERE DILUTED WITH PHOSPHATE-BUFFERED SALINE
TO BRING THEM INTO THE CHOSEN CONCENTRATIO RANGE OF APPROXIMATELY 35 TO
50 MILLION SPERMATOZOA PER MILLILITER. SPERM CONCENTRATION AD MOTILITY
WERE DETERMINED USING A COMPUTER-AUTOMATED SEMEN ANALYZER (CASA).
PARAMETER SETTINGS FOR CASA
THE IMPORTANCE OF REPORTING CASA PARAMETER SETTIGS HAS BEEN
ALLUDED TO IN A RECENT ARTICLE.9 THE PARAMETER SETTINGS WE USED FOR
SEMEN ANALYSIS ARE LISTED BELOW. NINE FIELDS A AND MINIMUM OF 200
SPERM CELLS WERE ANLYZED PER SPECIMEN.
THE PARAMETER SETTINGS USED WITH THE HAMILTON-THORM INTERNAL
VISUAL OPTICAL SYSTEM (IVOS) CASA WERE AS FOLLOWS: HIGH DENSITY;
FRAMES ACQUIRED - 7: FRAME RATE - 30/S; MINIMUM CONTRAST - 8; MINIMUM
SIZE - 6; LO/HI SIZE GATES - 0.6-1.6; LO/HI INTENSITY GATES - 0.6-1.6;
NON-MOTILE HEAD SIZE - 10; NON-MOTILE BRIGHTNESS (HEAD INTENSITY) - 20;
MEDIUM VAP VALUE - 25; LOW VAP VALUE - 10; SLOW CELLS MOTILE - YES; AND
TRESHOLD STRAIGHTNESS (STR) - 80.
MICROBIOLOGY
APPROXIMATELY 1.0 ML OF THE RAW SEMEN SPECIMEN WAS TRANSPORTED TO
THE MICROBIOLOGY LABORATORY LOCATED AT GREENVILLE MEMORIAL HOSPITAL FOR
FURTHER EVALUATION. THE FOLLOWING TESTS WERE PERFORMED: 1) ACID-FAST
BACTERIAL CULTURES AND SMEARS; 2) GRAM STAINS; 3) AEROBIC AND ANAEROBIC
BACTERIAL CULTURES ICLUDING CULTURES FOR NEISSERIA SP. ORGANISMS; AND
4) FUNGAL CULTURES.10 THE GRAM STAINS ALSO WERE EXAMINED FOR THE
PRESENCE OF WHITE BLOOD CELLS.
PROTEIN ANALYSIS
APPROXIMATELY 0.5 ML OF THE SEMINAL PLASMA WAS DILUTED WITH SPERM
WASHING MEDIUM (IRVINE SCIENTIFIC, IRVINE, CA) AND FROZEN IN LIQUID
NITROGEN FOR PROTEIN ANALYSIS. THE DILUTED 1 ML ALIQUOTS OF SEMINAL
PLASMA WERE THAWED AND CENTRIFUGED IN A CENTRICON r-10 CONCENTRATOR
TUBE (AMICON r INCL., BEVERLY, MA) IN ORDER TO CONCENTRATE THE PROTEINS
PRESENT. FOLLOWING CENTRIFUGATION, A 200 uL SAMPLE WAS USED TO
DETERMINE TOTAL PROTEIN USING THE BIORAD PROTEIN ASSAY (BIORAD
LABORATORIES, HERCULES, CA). THE REMAINIG 800 uL SAMPLE WAS
CENTRIFUGED IN A CENTRICON r-50 CONCENTRATOR TUBE TO SEPARATE THE
PROTEINS INTO TWO GROUPS (PROTEINS WITH MOLECULAR WEIGHTS (MW) BELOW
50,000 AND PROTEINS WITH MW ABOVE 50,000). MILLIPORE PRECAST
ELECTROPHORESIS GELS (MILLIPORE CO., BEDFOR, MA) WERE USED ON BOTH
GROUPS OF PROTEINS TO SEPARATE THE PROTEINS ON THE BASIS OF MW. BIO-RAD
SDS-PAGE MW MARKERS WERE USED AS STANDARDS WITH EACH GROUP OF
PROTEINS.11
DATA MANAGEMENT AND ANALYSES
DATA FOR THIS PROJECT WAS LIMITED TO 6 PATIENTS. THESE
PRELIMINARY DATA DO NOT ALLOW FOR STATISTICAL ANALYSES.
RESULTS
TABLE 1 CONTAINS THE COMPARATIVE THE SEMEN CHARACTERISTICS FOR THE
CONTROLS AND THE PERSIAN GULF VETERANS. WHILE THE VALUES ARE NOT THE
SAME FOR THE TWO GROUPS, THE ONLY VALUES THAT APPEAR TO BE
SIGNIFICANTLY DIFFERENT ARE THE PH AND THE TOTAL PROTEIN CONTENT.
TABLE 2 CONTAINS THE VARIOUS SPERM MOTION PARAMETERS ASSESSED
USING CASA. ALL OF THE VALUES OBTAINED, WITH THE EXCEPTION OF THE BEAT
CROSS FREQUENCY (BCF), WERE LOWER FOR THE PERSIAN GULF VETERANS. A
STATISTICALLY SIGNIFICAN DIFFERENCE I THE BCF OF THE TWO GROUPS COULD
NOT BE DETERMINED DUE TO THE LOW NUMBER OF PATIENTS IN EACH GROUP.
FIGURE 1 AND FIGURE 2 INDICATE "FUNGAL-LIKE" OR "PROTOZOAL-LIKE"
STRUCTURES THAT APPREARED IN THREE OF THE FIVE SEMEN SPECIMENS OF THE
PERSIAN GULF VETERANS FOR WHOM SEMEN SMEARS WERE PREPARED. NO OTHER
NOTABLE CONTAMINATES WERE DEMONSTRATED.
DISCUSSION
THIS PILOT STUDY WAS UNDERTAKEN TO EVALUATE SEMEN SPECIMENS FROM
SIX MALES WHO HAD RETURNED TO THE UNITED STATES FROM SERVICE IN THE
PERSIAN GULF. THE STUDY COMPARED VARIOUS SEMEN PARAMETERS BETWEEN
PERSIAN GULF VETERANS AND A CONTROL POPULATION WHO MET THE WORLD HEALTH
ORGANIZATION STANDARDS. THE SMALL NUMBER OF SAMPLESDOES NOT ALLOW FOR
ANY DEFINITIVE CONCLUSIOS. HOWEVER, RESULTS DO INDICATE POTENTIAL
DIFFERENCES AND THE NEED FOR FURTHER STUDIES.
IT APPEARS THAT THERE MAY BE A DECREASE IN SPERM MOTIO PARAMETERS
(TABLE 2) SIMILAR TO THAT WHICH HAS BEEN RECENTLY REPORTED IN THE
LITERATURE FOR OTHER PRIMATES EXPOSED TO ORGANIC PHOSPHATES.12 AN
INTERESTING SIDE BAR IS THE FACT THAT SOLDIERS WERE EXPOSED TO ORGANIC
PHOSPHATES IN THE FORM OF PESTICIDES WHILE IN THE PERSIAN GULF.13
(PERSIAN GULF VETERANS COORDINATING BOARD, 1995). FURTHERMORE, IT
APPEARS THAT THE SEMINAL FLUID OF THE VETERANS EXHIBITED AN INCREASED
PH AND DECREASED PROTEIN CONTENT (TABLE 2) AND CONTAINED A "FUNGAL-
LIKE" STRUCTURE (FIGURES 1 AND 2). INCREASED PH ENHANCES POLYAMINE
SYNTHESIS,14 WHICH IN TURN, COULD POSSIBLY RESULT IN AN INCREASE IN
AMMONIA. ELEVATED LEVELS OF AMMONIA COULD BE RESPONSIBLE FOR THE
"BURNING SENSATION" EXPERIENCED BY BOTH PARTNERS FOLLOWING INTERCOURSE.
THESE HYPOTHESES IMPLY THE NEED TO INVESTIGATE THE CONCENTRATION OF
PHOSPHATES AND POLYAMINES IN SEMEN AS WELL AS THE POSSIBLE PRESENCE OF
BACTERIA, FUNGUS OR OTHER ORGANISMS (IE. POSITIVE IDENTIFICATION OF
LEISHMANIA AS THE "PROTOZOAL-LIKE" STRUCTURE).
FUTURE DIRECTIONS
IT SHOULD BE NOTED THAT THIS PILOT STUDY WAS CONDUCTED BECAUSE OF
A REQUEST FROM THE COMITTEE ON VETERANS 'AFFAIRS OF THE UNITED STATES
SENATE TO THESE INVESTIGATORS.15 THE COMITTEE ON VETERANS'AFFAIRS HAD
RECEIVED A SERIES OF REPRODUCTIVE COMPLAINTS BY THE VETERANS OF THE
PERSIAN GULF WAR. IT SHOULD BE NOTED THAT THE FINDINGS FROM THIS PILOT
STUDY GENERATED SUFFICIENT DATE TO SUBMIT A LARGER PROPOSAL TO THE
GREENVILLE HOSPITAL SYSTEM/CLEMSON UNIVERSITY COOPERATIVE RESEARCH AND
EDUCATION PROGRAM. THIS PROPOSAL REQUESTED 1) A REVIEW OF THE CURRENT
LITERATURE CONCERNING PERSIAN GULF SYNDROME, 2) ANALYSES OF SEMEN
SPECIMENS FROM AN ADDITIONAL 20 PERSIAN GULF VETERANS AND 3) EVALUATION
OF THE FEMALE PARTNERS OF THESE 20 PERSIAN GULF VETERANS.
REFERENCES
1. MASCI D. PERSIAN GULF SYNDROME BILL APPROVED BY SENATE PANEL.
CONGRESSIONAL QUARTERLY WEEKLY REPORT. 1994;52(37):2698.
2. MASCI D. GULF SYNDROME COMPENSATION APPROVED BY HOUSE PANEL.
CONGRESSIONAL QUARTERLY WEEKLY REPORT. 1994;52(29):2057.
3. HORTON R. PERSIAN GULF SYNDROME PUZZLE. THE LANCET.1994;343:1150.
4. OPERATION DESERT STORM. TESTIMONY OF MS. KELLI ALBUCK, WIFE OF
PERSIAN GULF WAR VETERAN, BARRINGTON, IL. #9 TESTIMONY. IN:
POTENTIAL FOR REPRODUCTIVE DYSFUNCTION IS NOT BEING ADEQUATELY
MONITORED. GENERAL ACCOUNTING OFFICE. GAO/T-PEMD-94-31; AUGUST 5,
1994.
5. WORLD HEALTH ORGANIZATION. WHO LABORATORY MANUAL FOR THE
EXAMINATION OF HUMAN SEMEN AND SPERM-CERVICAL MUCUS INTERACTION.
CAMBRIDGE UNIVERSITY PRESS. CAMBRIDGE, ENGLAND. 1992.
6. JOHNSON JE, BOONE WR, BLACKHURST DW. MANUAL VERSUS COMPUTER
AUTOMATED SEMEN ANALYSES: PART I - COMPARISON OF COUNTING
CHAMBERS. SUBMITTED TO FERTIAL STERIL.
7. JOHNSON JE, BOONE WR, BLACKHURST DW. MANUAL VERSUS AUTOMATED SEMEN
ANALYSES: PART II - ACCURACY AND PRECISIO OF A COMPUTER AUTOMATED
SEMEN ANALYZER COMPARED TO THE HEMACYTOMETER. SUBMITTED TO FERTIL
STERIL.
8. JOHNSON JE, BOONE WR, BLACKHURST DW. MANUAL VERSUS COMPUTER
AUTOMATED SEMEN ANALYSES: PART III - COMPARISON OF OLD VERSUS NEW
DESIGN MICROCELL COUNTING CHAMBERS. SUBMITTED TO FERTIL STERIL.
9. DAVIS RO, KATZ DF. STANDARDIZATION AND COMPARABILITY OF CASA
INSTRUMENTS. J ANDROL 1992;13:81-86.
10. ISENBERG HD. CLINICAL MICROBIOLOGY PROCEDURES HANDBOOK. AMERICAN
SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC. 1992.
11. RICHARDSON ME, BODINE AB, FROMAN DP, THURSTON RJ. TURKEY ACROSIN.
I. ISOLATION, PURIFICATION, AND PARTIAL CHARACTERIZATION. BIOL
REPROD 1988; 38: 645-651.
12. BURLEIGH DW, KOLM P, HODGEN GD, MAHONY MC. EFFECT OF AN ORGANIC
PHOSPHATE (DIAZINON) ON CYNOMOLOGUS MONKEY SPERMATOZOA MOTION
PARAMETERS. BIOL REPROD. 1995;52(SUPPL.1):171.
13. PERSIAN GULF VETERANS COORDINATING BOARD. UNEXPLAINED ILLNESSES
AMONG DESERT STORM VETERANS: A SEARCH FOR CAUSES, TREATMENT, AND
COOPERATION. ARCH INTERN MED. 1995;155:262-268.
14. MCCANN PP, PEGG AE, SJOERDSMA A. INHIBITION OF POLYAMINE
METABOLISM: BIOLOGICAL SIGNIFICANCE AND BASIS FOR NEW THERAPIES.
ACADEMIC PRESS, INC. NEW YORK. 1987.
15. ROCKEFELLER IV, JD. PERSONAL COMMUNICATION VIA A LETTER DATED
JUNE 27, 1995.
SEMEN PARAMETERS AS THEY RELATE TO PERSIAN GULF WAR SYNDROME
WILLIAM R. BOONE 1,2 , M. ELAINE RICHARDSON 3, DEAN BENJAMIN 4,
JANE E. JOHNSON 2, MELISSA F. LAY 3, SHERI T. LEE 2, AND ROXANNE
S. BARNARD 3
2 REPRODUCTIVE ENDOCRINOLOGY ASSOCIATES, GREENVILLE HOSPITAL
SYSTEM, GREENVILLE, SC, 3 DEPARTMENT OF ANIMAL, DAIRY AND
VETERINARY SCIENCES, CLEMSON UNIVERSITY, CLEMSON, SC, 4
MICROBIOLOGY SECTION OF THE CLINICAL LABORATORY, GREENVILLE
HOSPITAL SYSTEM, GREENVILLE, SC.
TO WHOM CORRESPONDENCE SHOULD BE SENT : WILLIAM R. BOONE, PH.D.,
REPRODUCTIVE ENDOCRINOLOGY ASSOCIATES, 890 W. FARIS ROAD, SUITE
470, BOX2, GREENVILLE , SC 29605. TELEPHONE (803) 455-8488. FAX
(803) 455-8492.
TABLE 1. SEMEN CHARACTERISTICS OF CONTROL PATIENTS AND PERSIAN
GULF WAR VETERANS.
W.H.O. CONTROL PERSIAN GULF
PARAMETERS NORMAL VALUES PATIENTS VETERANS
***********************************************************************
CONC (X 10 6/M1) >20 129.9 +3.1 119+120
- _ _
MOTILITY (%) >50 65+ 12.6 48+8.0
_ _ _
% NORMAL >30 40.2+6.9 24.5+6.2
_ _ _
PH (LITMUS) 7.2-8.O 7.3+0.2 7.9+0.6
WBC (X 10 6/M1) <1 0 0
TOTAL PROTEIN (MG) ____ 770+770 127.5+70
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TABLE 2. SPERM MOTION PARAMETERS AS DETERMINED BY COMPUTER AUTOMATED
SEMEN ANALYZER (CASA).
MOTION CONTROL PERSIAN GULF
PARAMETERS PATIENTS VETERANS
***********************************************************************
VAP (U/SEC) 63.9+4.2 40.9+10
VSL (U/SEC) 53.6+4.4 35.7+7.5
VCL (U/SEC) 94.3+2.9 60.8+19.4
ALH (UM) 2.9+0.1 1.9+0.6
BCF (HZ) 16.3+1.7 20.3+4.4
STR (%) 78.4+2.O 49.0+26.0
LIN (%) 54.8+2.8 36.6+14.9
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