FERTILI'I~' AND STERILITY ~
Vol. 67, N(I. 1, .January 1997
Copyright ' 1997 American St~ciety for Reproductive Medicine
Pritttt,d otl acid-free i m p e r ill U..~. A.
Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile men
Sheena E. M. Lewis, Ph.D.*¢ E. Samantha L. Sterling, M.Med.Sci* Ian S. Young, M.D.,~ William Thompson, M.D.* The Queen ~ University of Bel/bst, Belfast, Northern Ireland, United Kingdom
Objective: .To measure individual antioxidants in sperm and seminal plasma fl'om fertile and infertile men to determine if any particular antioxidant is reduced in infertile men. Design: Semen samples were prepared by a discontinuous Percoll gradient to separate sperm and seminal plasma, and the antioxidant concentrations of each were assessed. Samples also were screened for phorbol ester-induced reactive oxygen species ¢ROS) activity. Setting: Departments of Obstetrics and Gynaecolog-y, and Clinical Biochemistry, The Queen's University of Belfast, Northern h'eland. Patient(s): Fifty-nine male patients attending our infertility center: 18 men whose wives had ongoing pregnancies from IVF with normozoospermic semen profiles, 20 infertile men with normozoospermic and 21 men with asthenozoospermic semen profiles. Main Outcome Measure(s): Ascorbate, urate, sulphydryl groups, tocopherol and carotenoid concentrations were measured in sperm and seminal plasma from fertile and infertile men. Result(s): In seminal plasma, ascorbate contributes almost twice as much as urate and thiol levels are about one third of ascorbate. Ascorbate levels in seminal plasma of asthenozoospermic individuals I+ROS~ are significantly reduced. In sperm, thiols contributed most and ascorbate only a fraction of the total. Conclusion(s): In seminal plasma, ascorbate, urates, and thiols are the major antioxidants present. In contrast, within sperm, this group is the major contributor. In samples exhibiting ROS activity, ascorbate concentrations in the seminal plasma are significantly reduced, Fertil Steril "~ 1997;67:142-7 Key Words: Antioxidants, sperm, seminal plasma, asthenozoospermia, infertility
I n a d e q u a t e s p e r m function is a m a j o r c o n t r i b u t o r to m a l e infertility (11. Recent findings h a v e shown t h a t reactive oxygen species (ROS) can lead to deleterious effects on a r a n g e of s p e r m p a r a m e t e r s including morphology and motility (2). S p e r m m e m b r a n e s are p a r t i c u l a r l y susceptible to ROS d a m a g e because of t h e i r high content of p o l y u n s a t u r a t e d
Received February 5, 1996; revised and accepted August 20, 1996. * Department of Obstetrics and Gynaecology. t Reprint requests: Sheena E. M. Lewis, Ph.D., Department of Obstetrics and Gynaecology, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland, United Kingdom (FAX: 44-232-328247; ecs-mail:
[email protected]). $ Department of Clinical Biochemistry.
142
L e w i s e t al.
Antioxidants in semen and infertility
f a t t y acids a n d t h e i r lack of defensive m e c h a n i s m s (2). Production of ROS can be detected in s p e r m of 40c~ of infertile m e n , w h e r e a s none is detected in s e m e n from n o r m a l m e n (3, 4). To c o u n t e r a c t the effects of ROS, s e m e n is believed to possess a n u m b e r of a n t i o x i d a n t s y s t e m s including c h a i n - b r e a k i n g a n t i o x i d a n t s c a p a b l e of r e d u c i n g oxidant radical levels t h a t would o t h e r w i s e i n i t i a t e a n d p r o p a g a t e free radical chain reactions. N o n e t h e less, s u r p r i s i n g l y little i n f o r m a t i o n is a v a i l a b l e a b o u t the precise c o n c e n t r a t i o n s of individual antioxidants in s p e r m t h e m s e l v e s or in s e m i n a l p l a s m a . O u r p r e s e n t day knowledge still d e p e n d s on d a t a from m o r e t h a n a decade ago (5). It is now possible to review the data, u s i n g m o r e s o p h i s t i c a t e d techniques such as h i g h - p e r f o r m a n c e liquid c h r o m a t o g -
Fertility and Sterility '~"
raphy (HPLC) with fluorometric detection to determine even low concentrations of individual antioxidants accurately. In a recent study we showed that the total antioxidant capacity of seminal plasma was significantly higher in fertile men than in asthenozoospermic men (6). This subsequent study is the first, to our knowledge, to measure individual antioxidants in sperm and seminal plasma from the same semen sample of fertile and infertile men to find which particular antioxidant(s) is reduced in infertile men. MATERIALS AND METHODS
A total of 59 couples attending the Regional Fertility Centre in Belfast were included in the study. Semen samples were classified in four groups: 1. Normozoospermic fertile: >20 × 10/~ sperm/mL, >50% progressive motility and >60% normal morphology from men whose wives had an ongoing pregnancy resulting from IVF. 2. Normozoospermic infertile: (semen profile as above) the female partners had undergone extensive infertility evaluation and none showed a detectable gynaecological abnormality, and infertility was of unknown etiology. None of the normozoospe~nic samples showed phorbol ester (PMA)-induced ROS activity. 3. Asthenozoospermic infertile: >20 x I0 ~ sperm/ mL, 60c~ normal morphology, whose sperm showed PMA-induced ROS activity. 4. Asthenozoospermic infertile: >20 × 106 sperm/ mL, 60c~ normal morphology, whose sperm showed no PMA-induced ROS activity. Sperm Preparation
Semen samples were produced by masturbation after 48 to 96 hours of sexual abstinence. Samples were left for 30 minutes for liquefaction to occur, and then the sperm were prepared on a discontinuous Percoll gradient by the method of Krausz et a] (7). Two-step (90:45) discontinuous gradients were prepared by sequentially pipetting 1 mL of 90% and 45% Percoll into a Falcon tube so t h a t an interface was visible between the layers: 1 mL of semen was layered on top of the gradient and centrifuged at 1,500 rpm for 20 minutes. Seminal plasma was collected from the top of the column and spun again at 3,500 rpm for 10 minutes. The s u p e r n a t a n t was removed and aliquoted into 1.5 mL Eppendorftubes. The 50% Percoll fraction was discarded and the cells were collected from the base of the 100% Percoll layer. These cells were resuspended in 7 to 10 pL
Vol. 67, No. 1, January 1997
Biggers, Whitten, and Whittingham medium (BWW) and centrifuged at 1,500 rpm for 10 minutes. The final sperm concentration was measured and sperm samples were then aliquoted into Eppendorf tubes and subjected to rapid freeze-thawing three times to lyse the cells. A second semen sample was obtained from each man within 1 month of the first sample and prepared by a direct swim-up technique. The technique, which isolates a subpopulation of highly motile sperm, was used for two reasons. First, it is the method best suited to mimic the situation in vivo where the sperm most likely to fertilize the oocyte swim out of the seminal plasma. This is also the subpopulation of sperm used in m a n y assisted conception techniques. Second, this method facilitates the direct measurement of ROS activity from healthy sperm without the antioxidant effects of the protective seminal plasma or the pro-oxidative effects of abnormal sperm and contaminant leukocytes also present in unprepared semen. The leukocyte concentration of each sample was < 1 × l0 GmL-1 so its ROS contribution was considered small according to World Health Organization standards (8). We also measured the chemiluminescence of the sperm population left in the semen (4) after each swim-up and found where ROS activity was present in the motile subpopulation, it was also present but in much higher levels in the immotile subpopulation (data not shown). Five hundred microliters of 0.2 mM luminol were added to 400 pL sperm (to give a final concentration of 10 x 10G/mL) and 75 pL of BWW medium. Chemiluminescence
Determination of ROS activity was carried out on a Bio-Orbit 1250 luminometer (BioOrbit, Turku, Finland) over a 5-minute period; 25 pL of PMA (8 x 10-s M) was then added to the mixture of sperm and luminol in the cuvette and the luminescence recorded. Reactive oxygen species formation as considered detectable when the luminescence was > 1.0 mV/s per 106 sperm at peak value observed over a 15-minute period after addition of PMA. Antioxidant Assays Determination o f Urate
Urate was measured by a standard enzymatic technique. This was carried out on a Kodak Ektachem analyzer (Rochester, NY). Determination o1" Total Ascorbic Acid by H P L C With Fluorescence Detection
Total ascorbate was measured by HPLC as described by Speeke et al. (9). In this method, L-
Lewis et al. Aiztioxidalzts in semen and infertility
143
Table 1 Concentrations of Individual Antioxidants in Seminal Plasma of Fertile and Infertile Men* Antioxidant
Normozoospermic fertile (n = 18}
Normozoospermic infertile {n = 20}
Asthenozoospermic (Total)(n = 2D
Asthenozoospermic ( + R O S } I n = 6~
Asthenozoospermic ( R O S ~ ( n - 12)
Wnol / L
Urate Ascorbate Tocopherol SH groups
190 364 0.46 123
1125 to 290, (179 to 4811 10.32 to 0.52) 199 to 136)
235 292 0.49 144
I136 to 303) (223 to 403) (0.34 to 0.581 t l l 3 to 181)
* Values are medians with interquartile ranges in parentheses.
ascorbic acid is c o n v e r t e d to dehydro-L-ascorbic acid by e n z y m a t i c oxidation a n d condenses with o-phenyl e n d i a m i n e to its quinoxaline derivative. This derivative is s e p a r a t e d on a r e v e r s e d - p h a s e H P L C column and detected fluorometrically in c o n c e n t r a t i o n s as low as 0.2 pmoUL. Determination of Retinol, ~-Tocopherol, c~- and flCarotene, and Lycopene and With Tocopherol Acetate as Internal Standard
Retinol, tocopherol, lycopene, or- a n d /3-carotene were m e a s u r e d by H P L C u s i n g the m e t h o d of T h u r n h a m et al. (10). Retinol a c e t a t e a n d tocopherol acet a t e were used as i n t e r n a l s t a n d a r d s . Determination of Total Protein Sulphydl3,1 Groups
The thiol groups in both s p e r m a n d s e m i n a l p l a s m a are a l m o s t all p r o t e i n - a s s o c i a t e d (11) so an a s s a y to m e a s u r e total protein s u l p h y d r y l g r o u p s was used. In this a s s a y (12) the s u l p h y d r y l groups in s p e r m a n d s e m i n a l p l a s m a r e a c t with 5,51-dithiobis (2-nitrobenzoic acid) to form a colored product t h a t can be m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y . Statistics
In view of the n o n - G a u s s i a n distribution of d a t a the n o n p a r a m e t r i c M a n n - W h i t n e y U t e s t was employed to e x a m i n e differences b e t w e e n fertile and infertile groups. The a n a l y s i s was carried out u s i n g Arcus Pro II (Medical C o m p u t i n g , Aughton, U n i t e d Kingdom). A probability value o f P < 0.05 was considered significant. Values are given as m e d i a n s (25c~ and 75% quartiles). RESULTS
185 320 0.35 134
t l2S to 298, t218 to 486) (0.30 to 0.48) 1121 to 141~
250 121 0.46 134
1180 to 389) {86 to 180~(0.39 to 0.51) ( l l 6 to 139)
180 372 0.32 133
1115 (191 (0.27 1126
to 220J to 496) to 0.43) to 146)
¢ Group is sigmificantlydifferent fi'omnormozoospermicfertile g~'oup,P •< 0.05 Mann Whitney U test.
zoospermic [ - R O S , P > 0.051). H o w e v e r , t h e a s t h e n ozoospermic ( + R O S ) g r o u p h a d a r a n g e of 35 to 249 pmol/L, which w a s significantly lower t h a n the norm o z o o s p e r m i c fertile values. For s p e r m , a r o u n d h a l f the s a m p l e s in each g r o u p g a v e v a l u e s u n d e t e c t a b l e by the a s s a y ( < 1 p m o l / 10 "' cells), w h e r e a s a n u m b e r r e a c h e d 100 pmol/10 t° cells. T h e wide r a n g e indicates large v a r i a t i o n in the s p e r m values b e t w e e n i n d i v i d u a l s (Table 2). B e c a u s e of this, no significant v a l u e s were found b e t w e e n groups. M e a s u r e m e n t of U r a t e
S u b s t a n t i a l a m o u n t s of u r a t e w e r e detected in the s e m i n a l p l a s m a r a n g i n g from 70 to 490 pmol/L in fertile m e n a n d from 60 to 370 p m o l / L in the two infertile groups. T h e r e w e r e no significant (P > 0.05) differences b e t w e e n the groups. U r a t e levels in s p e r m were negligible at < 3 pmol/10 ~° cells (Table 2). M e a s u r e m e n t of P r o t e i n S u l p h y d r y l G r o u p s
S e m i n a l fluid protein s u l p h y d r y l c o n t e n t r a n g e d from 99 to 415 pmol/L in all the i n d i v i d u a l s (Table 1) a n d s p e r m c o n t e n t w a s f r o m 57 to 890 pmol/10 t° cells (Table 2). No significant differences were found b e t w e e n s e m i n a l p l a s m a s of different groups. However, in the s p e r m , the a s t h e n o z o o s p e r m i c (total) group h a d significantly h i g h e r (P < 0.05) thiol levels (Table 2). This elevation w a s m o s t l y due to the c o n t r i b u t i o n of the R O S - p r o d u c i n g s p e r m . M e a s u r e m e n t of a - T o c o p h e r o l , or- and fl-Carotene, L y c o p e n e , and R e t i n o l
M e a s u r e m e n t of A s c o r b a t e
S u b s t a n t i a l a m o u n t s of a s c o r b a t e were p r e s e n t in s e m i n a l p l a s m a with c o n c e n t r a t i o n s r a n g i n g from 106 to 566 pmol/L in fertile m e n (Table 1). In infertile m e n the r a n g e s were s i m i l a r (106 to 966 p m o l / L, normozoospermic; 110 to 785 pmol/L; a s t h e n o -
144
Lewis et al. Antioxidants in semen and infertility
a-Tocopherol w a s p r e s e n t in the s e m i n a l p l a s m a of each g r o u p a t low c o n c e n t r a t i o n s (0.08 to 0.69 pmoYL) (Table 1) a n d only t r a c e s of a - t o c o p h e r o l were detected in s p e r m from a n y group. N e i t h e r aor B-carotene, lycopene nor retinol w a s d e t e c t e d in s e m i n a l p l a s m a or s p e r m from a n y groups.
Fertility and Sterility"
Table
2
C o n c e n t r a t i o n s o f I n d i v i d u a l A n t i o x i d a n t s in S p e r m o f F e r t i l e a n d I n f e r t i l e M e n *
Antioxidant
Normozoospermic fertile(n = 18)
Normozoospermic infertileCn = 20)
Asthenozoaspermic (Total)(n = 21)
Asthenozoospermic (+ROS)(n 6)
Asthenozoospermic ( R O S ) ( n = 12)
