Professional Documents
Culture Documents
1987 - Epididymal Markers in Human Infertility
1987 - Epididymal Markers in Human Infertility
Alpha-glucosidase, glycerophosphocholine, and From the Max Planck Clinical Research Unit
L-carnitine were measured in sperm-free seminal plasma for Reproductive Medicine and Institute of
to determine whether these markers reflected the epidi-
dymal function of men attending an infertility clinic. The Reproductive Medicine of the University,
putative markers correlated well with each other (r = MUnster, F.R. Germany
0.66 to 0.70) and in 92% of 283 cases were accurate in
categorizing semen as containing normal or subnormal
amounts of markers. Gtucosidase was considered the best
index of epididymal function and was used for a further The inclusion of indices of epididymal function in
306 samples. The ejaculate content of epididymal markers semen analysis has been recommended (Lewin, 1977;
was correlated with testicular volume and serum testos- Wetterauer, 1986) since the epididymis is intimately
terone below values of 30 ml and 30 nmol/l, respectively. involved in preparing spermatozoa for fertilization
Markers were also correlated with the concentration and (for review, see Cooper, 1986). To date, L-carnitine,
motility of spermatozoa in semen. Seventy-one of 425
patients (17%) displayed subnormal epididymal secre-
glycerophosphochotine, and 1,4-a-glucosidase (E.C.
tions, mainly in association with hypogonadism (Kline- 3.2.1.20) have been measured but there is stilt debate
fetter syndrome, Katlman syndrome, idiopathic hypo- over what information they can provide. Casano et at
gonadotropic hypogonadism) but also in cases of (1987) found that epididymat markers alone could
obstructed ducts, maldescended testicles, and local irradi-
not distinguish azoospermic men with blockage from
ation following hemicastration. Because azoospermic
those with spermatogenic arrest, because similar low
patients had reduced epididymal markers with both high
and low FSH levels and a large proportion of men with marker values were found in both groups. Guerin et
reduced glucosidase and normal FSH suffered from tes- at (1986), on the other hand, found that epididymal
ticular failure, it is suggested that other indices of testicu- markers taken together with serum FSH were rather
lar function are required for correct interpretation of reduced
good predictors of ductal blockage.
epididymal markers. Thirteen patients (3%) had low
Since it is known that carnitine is transported from
markers for which no cause was apparent; these may be
cases of infertility due to isolated epididymal dysfunc- blood into the epididymis (Brooks et at, 1973; Bohmer
tion. et at, 1979; Yeung et at, 1980), that glycerophos-
phocholine is synthesized from circulating (Ham-
Key words: human epididymis, infertility, glucosidase,
merstedt and Rowan, 1982) or tuminal (Wang et at,
carnitine, glycerophosphochotine, epididymal markers.
1981) precursors and that glucosidase is both syn-
J Androl 1988; 9:91-101. thesized and secreted (Grandmont et a!, 1983), each
of these substances may reflect a different facet of
Reprint requests: E. Nieschlag, M.D., Max Planck Clinical epididymat function. The markers may also provide
Research Unit for Reproductive Medicine, Steinfurterstra$e 107, information on the function of different regions of
D-4400 M#{252}nster, F.R. Germany.
Submitted for publication June 5,1987, revised version received the epididymis because most carnitine in human epi-
August 25, 1987; accepted for publication September 15, 1987. didymal tissue is found in the caput (Bohmer et at,
91
92 Journal of Androtogy . March/April 1988 Vol. 9
1978; Tomamichal and Bandhauer, 1986), most glyc- standard methods (WHO manual, 1987) and centrifuged
erophosphocholine in the corpus (Riar et al, 1973) within 2 hours at 1000 X g for 10 minutes at room
temperature to remove spermatozoa. Samples were stored
and most glucosidase in the cauda (Guerin et at, 1979)
at -20 C before analysis.
epididymidis.
In this study, the three epididymal markers are Assays of Epididymal Markers
compared and their content in semen is discussed in
Five hundred eighty-nine semen samples from the
relation to the disease of the patients attending an
patients and donors were analyzed. In initial studies, 283
infertility clinic. samples of normospermic, asthenospermic and azoos-
permic semen were assayed for glucosidase, carnitine and
Materials and Methods glycerophosphocholine. When a good correlation between
Patients and Volunteers the markers had been established, semen samples were
routinely assayed only for glucosidase.
Four hundred twenty-five patients (mean age 33 years, AtI spectrophotometric assays were performed with a
range 19 to 54) attending the infertility clinic of our insti- Pye Unicam PU8610 single beam, kinetics spectropho-
tute and 23 healthy donors, free from genital infections, tometer. All values represent the ejaculate content of the
with normospermic spermiograms and aged 22 to 32 (mean marker, to eliminate errors due to fluctuations in semen
age 26) were investigated. volume contributed by other accessory sex glands (Soufir
et al, 1984).
Classification of Subjects
Deproteinization of Semen Samples
Patients were placed into categories (Groups) defined
by the seminal parameters given in Table 1. If a patient for Carnitine and Glycerophosphocholine Assay
changed categories during the course of treatment, his Three hundred fifty zl thawed sperm-free seminal
semen vatues appear in each of the categories; where more plasma was centrifuged through Amicon filters (Centri-
than one appearance occurred within one group, however, con-30: molecular weight cut-off 30 kDa) at 2,500 X g for
a mean value was taken. 90 minutes at 10 C. Ultrafiltrates were kept on ice for the
Patients were additionally classified into 16 classes immediate assay of carnitine and the remainder was deep
according to their major ctinicat findings: patients who frozen (-20 C) for no more than 1 day before assay of
became fathers within the following 6 months (N = 10); glycerophosphocholine.
those with a palpably thickened epididymis (N = 37); those
with biopsy-proven seminiferous tubule damage (N = 8);
Glucosidase Assay
those with Ktinefetter’s syndrome (N = 3); those with
idiopathic hypogonadotropic hypogonadism before (N = This enzyme was measured by a modification of Chap-
8) or under (N 3) substitution therapy; those with high delaine et al (1978) on duplicate 8-itt aliquots of sperm-free
(>10 ng/ml) FSH but without testicular biopsy (N = 28); seminal plasma. Vatues were calculated per unit volume of
those with maldescended testicles (having had an orchido- semen and not per mg protein since protein is contributed
pexy: N 34); those with retractile testicles (N = 21); by other accessory organs.
those having received local irradiation, mostly with hemi- With Bil seminal plasma, the rate of product formation
castration (N = 10); those with varicocoele either as yet was linear up to 70 mU/mI and over 4 hours, where 1 unit
untreated (N = 56) or following surgical ligation of the v. is defined as the hydrolysis of 1 tmole of p-nitrophenyl-D-
spermatica (N = 25); those with a history of orchitis (N = 9); gtucopyranoside per minute. White glucosidase from Sigma
three patients with obstructed ducts, one with vasectomy, Chemical (M#{252}nchen) suffered a 25% loss in activity after
one with a failed vasovasostomy, and one case of surgically each freeze and thaw cycle, that in seminal plasma was
explored epididymal obstruction, and those with unex- stable, as shown by the controls included in each assay.
plained infertility, including five having previously had Inter- and intra-assay variations were 7.1% (N = 13) and
epididymitis (N = 170). 5.1% (N = 224), respectively. Sensitivity was 0.85 mU/rn!.
Carnitine Assay
L-carnitine was assayed by a modification of Ramsay Normozoosp.r,nic
and Tubbs (1975) in duplicate 50-til aliquots of deprote- (87)
inized seminal plasma. The accuracy of measurement of
carnitine standards estimated from the molar absorption
coefficient (see Yeung eta!, 1980) was 105 ± 17% (SD, N = 0-
10). Carnitine added to seminal ptasrna before ultrafiltra- 40-
tion was not lost on filters (recovery 95.8 ± 14.6% (SD), N
= 6). The standard curve was linear up to 1 mM and the
sensitivity was 13 1.M. Intra-assay variation was 6.9 ±
1.6% (N = 192) and inter-assay variations were 12.2, 16,
and 11.4% for high, medium and low pools (N = 25),
Oth.r caUgorl..s
respectively, in seven assays. Carnitine was stable to
(74)
freezing and thawing.
Statistical Analyses
0-
Statistical analysis was performed by analysis of var- 20 UUUoUo_O_D
iance after determining the nature of the distribution and 1 4zoosprrmic
making logarithmic transformation when appropriate. -I (59)
Differences between mean values were tested using 95%
confidence intervals.
0]_
0 100 200 300 400
Results
GLUCOSIDASE (mU PER EJACULATE)
Distribution of Markers
The overall populations of all three markers were Fig. 1. The distribution of a-glucosidase in semen from nor-
mospermic men (upper panel); oligo-, astheno- and terato-
togarithmicalty distributed and the distributions of
zoospermic men (middle panel) and azoospermic men (bottom
the markers for three groups of spermiograms are panel).
shown in Fig. 1. The mean glucosidase content of
semen from these patients is given in Table 1. The were considered to be deficient in markers, were
marker content of the normospermic population was derived from the geometric mean (az) and standard
used as the reference by which the others were deviation (s) at a probability level (z) of 5% from the
judged. The cut-off values (x), betow which samples equation z = - x)/s (Table 2).
TABLE 2. Epididymal Marker Content of Semen from with glycerophosphocholine (r = 0.31, P < 0.0001)
Normospermic Patients and carnitine (r = 0.20, P <0.001) but not glucosidase.
Glycerophos- With Sperm Concentration. In this analysis, only
Glucosidase phocholine Carnitine the concentrations of spermatozoa and the marker,
Units per mU mol mol rather than the semen content, were employed to
ejaculate avoid positive bias by the common factor of dilution,
Geometric and the azoospermic group was not included in the
mean 121.81 11.88 1.50
regression analysis. The tog sperm concentration
Cut-oft 52.9 (50)* 4.95 (5)* 0.64 (0.6)
Number of
was significantly correlated (P < 0.0001) with glyce-
samples 136 78 80 rophosphocholine (r = 0.63), carnitine (r = 0.58) and
g!ucosidase (r 0.45).
Number in parentheses are the rounded-up values used in
the clinical assessment of samples. With Testicular Volumes. There was a significant
(P < 0.0001) correlation between the log marker
content and paired testis volumes below 30 ml. Coef-
Variation of Epididymal Markers
ficients of correlation were 0.46 for carnitine, 0.40
within Individuals for glycerophosphocholine and 0.36 for glucosidase.
Three to seven semen samples from 14 patients Above this volume, an increase in testis size was not
collected over 3 to 7 months were assessed to gain related to a further increase in the ejaculate content
data on intra-individual variation. Coefficients of of the epididymal marker.
variation were 26.7, 34.6, and 33.5% for glycero- Testicular volume was also directly correlated with
phosphocholine, carnitine and glucosidase, respec- log total ejaculated spermatozoa (r = 0.32, P <
tively. 0.0001) and log serum T concentration (r = 0.26, P<
0.001) but inversely correlated with serum FSH (r =
Correlations between the Three Epididymal Markers -0.46, P < 0.0001).
There was a highly significant (P < 0.0001) corre- With Serum Testosterone. There were slight
lation between the concentration of the three markers correlations between tog serum T below 30 nmot/l
in semen. Coefficients of correlation were 0.70 and glycerophosphochotine (r = 0.25, P < 0.001) and
between glucosidase and carnitine, 0.68 between glucosidase (r = 0.23, P < 0.0001) but no correlation
glucosidase and glycerophosphochotine and 0.66 with the carnitine content of the ejaculate. There
between glycerophosphocholine and carnitine. Be- was no increase in the epididymal marker content of
cause of this, there was also good agreement in the semen with an increase in serum T above 30 nmolll.
classification of semen samples by each of the mar-
kers. For 283 samples, 76.3% were classified by alt Epididymal Markers and Azoospermia,
three markers as either above or below the respective Asthenospermia and Normosperinia
cut-off; in only 7.7% of cases was the classification The distribution of glucosidase in normospermic
based on glucosidase content of semen at variance semen clearly differed from the semen of azoosper-
with the prediction of the other two. On the basis of mic men, since the former group contained no sam-
these results, a-glucosidase appeared to be a repre- ples that fell in the very low category of a-glucosidase
sentative marker of human epididymal function. content. Distributions from oligo-, terato-and asthe-
Because its assay is cheaper, simpler and requires less nospermic patients were similar (Table 1) and are
seminal plasma than the others, it was chosen as the grouped together in Figs. 1, 2, and 3.
marker for initial screening of seminal plasma sam- In Azoospermia. Of the 64 azoospermic patients,
ples at our infertility clinic. 48 (75%) had high FSH levels (presumed testicular
failure). White the overall a-glucosidase content of
Correlation of Epididymal Markers
semen from azoospermic men was below the nor-
with other Seminal Parameters mospermic range, there was no difference in the
With Sperm Motility. There was a significant (P < mean a-glucosidase content when the azoospermic
0.0001) but weak correlation between the percentage semen was categorized by serum FSH levels (Table
of motile spermatozoa and the total ejaculate content 1). Carnitine (0.59 j.mot vs. 0.53 .imol) and glycero-
of gtycerophosphocholine (r = 0.37), carnitine (r = phosphocholine (4.47 imot vs. 5.4 imot) were similar
0.32) and glucosidase (r = 0.15, P<0.05). Sperm motil- in the two groups and were unrelated to circulating
ity ratings were also positively but weakly correlated FSH concentrations.
No.2 EPIDIDYMIS AND INFERTILITY. Cooper et al 95
IN 231 PATIENTS
IN 221 PATIEN
20-
No,,nozoospsiml.c
0 UUH (103)
Oth.r Ca*.gor.s
H (58)
HU 11DUUUo..
I
1_ 0
Azoosp.rm4c
(60)
fi
0-
0
Half of the patients with high FSH had normal, and maldescended testicles (6 of 8 cases) also had reduced
half reduced, a-glucosidase secretion. Where clinical glucosidase.
symptoms were known, high FSH associated with Of the remaining 16 patients with low FSH, 25%
normal glucosidase levels reflected clinical symptoms had normal glucosidase; these comprised idiopathic
of retractile testicles (5 cases), patients receiving hypogonadotropic hypogonadism patients receiving
treatment (4 cases), Sertoti cell only syndrome (2 testosterone therapy and other men possibly suffer-
cases) and seminoma with no irradiation therapy (1 ing from testicular failure insufficient to raise FSH or
case). Of men with biopsy-proven testis failure, three a proximal obstruction of the duct. The remaining 10
had normal and two reduced gtucosidase. Varicocoete patients with low glucosidase included four with
(2 cases and 3 cases) or testicular torsion (1 case and 1 biopsy- or hormonally-proven testicular failure. Of
case) were similarly associated with both normal and the remaining six likely to have blockage at some
reduced glucosidase secretion, respectively. All the level in the epididymis, three were known to have
azoospermic men who had suffered from orchitis (2 occluded ducts.
cases), received irradiation therapy for seminoma (3 In Asthenospermia. Of 86 samples, 10(12%) had re-
cases) or had a palpably thickened epididymis (2 duced epididymat markers (see Table 3), six in associa-
cases) had reduced gtucosidase and the majority of tion with vancocoete. Four of these had no clinical
those with Klinefelter’s disease (3 of 5 cases) and with evidence as to the cause of the reduced sperm motility.
96 Journal of Andrology March/April 1988 Vol. 9
Donors and Recent Fathers. There was no statisti- 0roIdt (9) ___________________
cally significant difference between the seminal
.rtoooosl. (untrsst.d) (66)[
marker content of donors and patients whose wives
became pregnant during the observation period. The
V.rtcooo.I. (tr.t.d) (25) [ -4-
comparisons below are made with the donor group. Un.xpl.lusd (170) -
hMG or hCG treatment had significantly higher One man with a retractile condition had normal tes-
epididymat secretion than their untreated counter- tis volume (17.5 ml) and T levels (15.6 nmol/l) but
parts. greatly reduced gtucosidase secretion (24.1 mU per
Epididymal Position in the Scrotum. Men who ejaculate) suggestive of blockage. Of interest in this
had previously been cryptorchid had tower epidid- group were two patients with hyperprotactinemia
ymal markers but this was not evident in men with and those remaining with no known pathology. Of
merely a retractile testis. this last group, three wives had fertility problems but
Varicocoele. Taken together, there was no reduc- four were normal; in the tatter cases, the infertility
tion in the secretion of glucosidase in men with any may have stemmed from epididymal dysfunction.
form of varicocoele. Semen from men who had
undergone treatment for varicocoele did not differ in
Discussion
marker content from untreated men.
Irradiation. Alt three markers were reduced in The present study shows that a-1,4-glucosidase,
patients having received irradiation therapy of the carnitine and glycerophosphocholine are all good
lymphatics following hemicastration for testicular indicators of the various secretory functions of the
cancer. Of three hemicastrates with large testicles epididymis, reflecting normal and reduced epididymal
(25 ml), a-glucosidase was reduced after irradiation, contributions to the ejaculate (see Introduction for
in two cases in association with reduced T levels. references). The three markers were welt correlated
Unexplained Infertility and Unexplained Low with each other, confirming similar relationships
Epididymal Markers. This group of 170 men could found by Tremblay et at (1982), Guerin et at (1986)
not be assigned to any of the previously mentioned and, Casano et al (1987). Paz et at (1977) speculated
categories because they had normal testicular size, that carnitine and glycerophosphocholine were
serum T and FSH and no relevant clinical history. Of secreted by different cell types, but the simitar corre-
55 men with normal glucosidase, 37 had wives with lations between a-glucosidase and glycerophos-
complications of their reproductive tracts, suggest- phocholine with carnitine that we found suggest that
ing that the male partner may not have been the the entire length of the human epididymis responds
cause of the couples’ infertility. equally to pathologic conditions and therapy or that
Thirteen patients in this group produced semen the site of secretion of each marker is similar. Apart
samples containing tow epididymat markers (Tables 3 from whole tissue content, these values are not
and 4). Three had marginally reduced glucosidase known for man. In the rat epididymis, high concen-
(47.3 to 48 mU per ejaculate), two having suffered trations of glycerophosphocholine are present more
testicular torsion and another with an inguinat testis, proximally than carnitine (Setchell and Hinton, 1981)
but all three had testis sizes (8, 12.5, and 15 ml) and and in the ram, luminal carnitine increases distally in
androgens (16 to 19 nmot/t) within the normat range. parallel with a-glucosidase (Besancon et at, 1985).
98 Journal of Andrology . March/April 1988 Vol. 9
The epididymal markers in semen from our donors, Tremblay, 1986; Tomamichel and Bandhauer, 1986)
selected for normospermic semen parameters, were and glycerophosphocholine (Naik et at, 1979; Kapur
similar to the upper range found in the normosper- et at, 1985; Giovenco et at, 1986). There are conflict-
mic population attending the clinic. Markers were ing reports on epididymat marker secretion during
reduced, as expected, when the epididymides were epididymitis itself (Table 5). Alternatively, inflamma-
obstructed and also when they had been exposed to tion or occlusion proximal to a site of secretion of the
abdominal temperature (maldescensus testis), irradiation marker would not be reflected in a decreased secre-
or when only one small testis was present. Here, tion, as has been reported for a-glucosidase (Guerin
small or nonfunctional organs could be responsible et at, 1986) and carnitine (Tomamichel and Band-
for the reduced secretion. hauer, 1986).
The failure to find a correlation between a palpably A reduction in semen a-glucosidase content below
swollen epididymis and epididymal markers was that of normospermic patients was found in 71
unexpected. The nature of this swelling, however, patients (17%) attending the infertility clinic. In most
remains to be established. If the duct has resealed cases this was associated with hypogonadism due to
after rupture, as can occur in experimental animals genetic damage (Klinefelter’s syndrome) or hypo-
after section of the vas deferens (Feller et at, 1986), or thalamo-pituitary dysfunction (idiopathic hypo-
if it involves the stroma rather than epithelium, epi- gonadotropic hypogonadism and Kailman’s syn-
didymat secretion would not be affected. A certain drome). In such cases, the changes reflect endocrino-
resistance of the epididymis to the stresses of disten- logic disorders. This association between the total
sion following vasectomy is shown by the return to semen content of epididymal markers and testicular
the ejaculate, after recanalization of the duct, of a- volumes was also found by Casano et at (1987) but
glucosidase (Tremblay, 1986), carnitine (Soufir, 1985; not by Wetterauer and Heite (1980), who used carni-
No.2 EPIDIDYMIS AND INFERTILITY . Cooper et a! 99
tine concentrations in their assessment. spermatozoa and motility rating with the ejaculate
In all other instances, the epididymal markers were content (epididymat output) of glucosidase, carnitine
maintained at normal levels. These included current and glycerophosphocholine rather than with marker
conditions of retractile or inguinat testicles, the pres- concentration. This suggests that the epididymal
ence of a varicocoele, palpably thickened epididymis markers reflect an aspect of epididymal function that
and past clinical histories of epididymitis, prostatitis may be related to the conferring of fertilizing ability
and mumps without orchitis. and motility upon the maturing spermatozoa. Corre-
The positive correlations between the three epidi- lations between sperm motility and epididymal mar-
dymal markers and sperm numbers, previously kers have been demonstrated for carnitine (Wetter-
reported for carnitine (Wetterauer and Heite, 1980; auer and Heite, 1978) and suggested for glucosidase
Soufir et al, 1984; Menchini-Fabris et al, 1984), sug- (Tremblay, 1986) and gtycerophosphocholine (Arrata
gested for a-gtucosidase (Tremblay et al, 1979) but et at, 1978; Sane et a!, 1982) but they have not always
disputed for glycerophosphochotine (Arrata et at, been confirmed (Soffer et at, 1981). Indeed, Johansen
1978; Sane et at, 1982), probably reflect both the fact and Bhmer (1979) found that the acetylcarnitine
that these semen constituents arise from the epidid- content of spermatozoa was better related to sperm
ymis as well as the influence of the testis on the motility than the free carnitine content of semen.
epididymis. A relationship between epididymat secre- However, as the intra-sperm acetylcarnitine content
tion and testis size (Casano et at, 1987) or number reflects not only the availability of carnitine (epidi-
(Abbaticchio et at, 1985) has previously been reported. dymat secretion) but also the ability of spermatozoa
The nature of the association between epididymat to take it up, this property may be independent of the
function and the testis may be a consequence of epididymis.
reduced blood flow (Casano et at, 1987), lowered Reduced epididymat secretion in cases of asthe-
circulating androgens (Lewin et at, 1981) or a direct nospermia with normal sperm counts may demon-
action of testicular exocrine secretion on the size of strate most clearly the rote of the epididymis in the
the attached epididymis (Cooper, 1986). In the bull, development of human sperm motility, but the
there is a relationship between scrotal circumference number of patients in which reduced epididymal
and epididymat histology (Veeramachaneni et a!, secretion appears to be the sole cause of poor motility
1986). is small.
In contrast to observations of Wetterauer and An interesting group of patients are the 13(24% of
Heite (1980) and Casano et at (1987), our study pre- those with tow markers) with no distinguishing clini-
sents evidence that circulating T concentrations are cal condition (Tables 3 and 4). Those with normal
related, although weakly, to the secretion of epidi- testis volume and serum T may represent men with
dymat markers, but only below a concentration of 30 deficient epididymat function.
nmot/l. This “threshold” could explain why T admin- The major finding in the present study is the utility
istration does not increase the carnitine content of of a-gtucosidase as a marker of epididymat function
semen when basal levels are already high (Lewin et and the dependence of human epididymal function
al, 1981) as welt as the inconsistent response to on the testis. While reduced a-gtucosidase with nor-
androgen therapy in cases of varicocoele (Tremblay, mal FSH is suggestive of epididymat blockage (Guerin
1986). Our results clearly show that raising serum T et at, 1986), in our study more than half of the
increases secretion of all markers in hypogonadat patients in this category had testicular lesions that
patients, confirming previous observations on gluc- could have been responsible for reduced epididymal
osidase (Gunaga et at, 1971; Tremblay, 1986) and function. Other indices of testicular function (volume,
carnitine (Menchini-Fabns et at, 1984). serum T and sperm count) are thus as important as
The correlation between sperm motility and epidid- serum FSH in assessing the significance of reduced
ymal markers is important if, as in other species, the epididymal secretions in a male infertility workup.
human epididymis promotes the motility of sperma-
tozoa. Motility patterns of maturing spermatozoa
taken from the human epididymis (Mooney et at, Acknowledgments
1972) differ from that of animal models (Acott et al, The authors thank Ms. K Nurmik for performing the assays, Ms.
1979), although increases in motility are observed C Kr#{252}semann, Ms. E Mrosek and Ms. I Upmann for performing
the semen analyses and Ms. S Baha for secretarial assistance, and
(Dacheux et al, 1984). It was found here that there acknowledge the help of Dr. med. M. Bals-Pratsch and Dr. med. U.
was a greater correlation of both percent motile A. Knuth in their investigation of the patients.
100 Journal of Andrology . March/April 1988 Vol. 9
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