Yonago Acta medica 1999;42:79–86

Effects of Unilateral Cryptorchidism on Contralateral Epididymal
Sperm Quality, Quantity and Fertilizing Capacity
Takayuki Kaki and Nikolaos Sofikitis
Department of Urology, Faculty of Medicine, Tottori University, Yonago 683-0826, JapanThe effect of unilateral cryptorchidism on ipsilateral testicular function has been wellstudied both in human and experimental animals. However, the effect of unilateralcryptorchidism on contralateral testicular function remains unclear. To evaluate theeffect of left cryptorchidism on right testicular function, 3 groups of immature malerats were used: group A, left cryptorchid rats; group B, sham operated rats and groupC, orchidopexed rats. Ten weeks post-cryptorchidism induction, right testicular versus
intraabdominal temperature difference (R∆T), bilateral epididymal sperm content andmotility, bilateral testicular weight, fertility rate in vivo and in vitro, and serum testosterone responses to human chorionic gonadotropin stimulation were tested. Most of
the above parameters were significantly lower in group A than in group B and significantly higher in group C than in group A. The current findings suggest that there isa detrimental effect of unilateral cryptorchidism on sperm fertilizing capacity in vitro
not only on the ipsilateral side but also on the contralateral side.
Key words: cryptorchidism; infertility; rat; spermatozoa

menses and delayed menarche (Alpert andKlein, 1983; David et al., 1992; Seppo et al.,1996).
By the 12th to 14th week of gestation thetestis migrates from the urogenital ridge to thelevel of the internal inguinal ring. The testis
begins its transinguinal descent during the 26 th
to 28 th week of gestation with simultaneous
gubernaculum swelling and processus vaginalis
extension into the scrotum. Testicular descentis believed to depend on an integration of factors: an increase in intraabdominal pressure,gubernaculum tension and the hormonal influence of high local concentrations of dihydrotestosterone.
Cryptorchidism is a preexisting factor in 3%
to 8% of infertile men and in 20% of men withazoospermia (Alpert and Klein, 1983; David etal., 1992; Seppo et al., 1996). Animal studies
also support the effect of cryptorchidism on fertility. It is generally accepted that a relatively

Male infertility is both a private and a social
problem. Cryptorchidism is a well describedclinical condition associated with male infertility (Alpert and Klein, 1983; David et al., 1992;
Seppo et al., 1996).
Cryptorchidism is a defect involving maldescent of the testicle. The objectives of therapy for cryptorchidism include preservation offertility, reduction of the risk of malignancy andalleviation of physiological stress. The incidence of cryptorchidism in the newborn is 2%
to 6%; however with spontaneous descent thisincidence drops to 1% by the age of 3 months(Alpert and Klein, 1983; David et al., 1992; Seppo
et al., 1996). Prematurity, low birth weight andmultiple gestations are predisposing factors
with the increase in incidence correlating to thedegree of prematurity. A family history ofcryptorchidism may be present in 5%. Mothersof children with cryptorchidism tend to havemild pituitary impairment manifested by short

Abbreviations: BWW, Biggers-Whitten-Whittingham; hCG, human chorionic gonadotropin; IU,
International Unit; IVF, in vitro fertilization; R∆T, right testicular versus intraabdominal temerature
difference79

T. Kaki and N. Sofikitis

low temperature is preferable in spermatogenesis in mammalian species (Harrison andWeiner, 1949). Actually, several studies indicate that the temperature in the scrotum is 4 to
5˚C lower than in the abdomen in most mammals (Harrison and Weiner, 1949). Waites andMoule (1961) measured the temperature of theabdominal aorta and testicular artery at various
points and found that the temperature of thetesticular artery was significantly smaller. A
major deleterious effect of high intratesticulartemperature on spermatogenesis has been demonstrated (David et al., 1981; Nagler et al.,1987). It is well known that the function of thecryptorchid testis is impaired by the rise intemperature (Alpert and Klein, 1983; Nagler etal., 1987; David et al., 1992; Seppo et al., 1996).
The effect of unilateral cryptorchidism on ipsilateral testicular function has been well studiedin humans and experimental animals (Rager etal., 1975; Keel et al., 1980; Jegoue et al., 1983).
However, the effect of unilateral cryptorchidism on contralateral testicular function has notbeen extensively studied. Ono and Sofikitis(1997) have held that the endocrine and exocrine function of the contralateral testis are impaired by a rise in temperature.
The present study focuses on the effects ofleft cryptorchidism on the right testicular andepididymal function and the overall sperm fertilizing capacity.

left inguinal incision, placement of the left
testis into the abdomen and replacement of thetestis into its normal position. Four weeks after
cryptorchidism induction, rats of group C had
undergone left orchidopexy. Ten weeks after
the initial operation, rats of groups A (n = 20), B(n = 10) and C (n = 10) were processed forevaluation of abdominal temperature, righttesticular temperature and fertility potential invivo. The concentration of testosterone wasthen determined in blood aspirated from the inferior vena cava by a midline abdominal incision. The incision was closed and all the rats
were administered human chorionic gonadotropin (hCG) (1500 units; Teikoku Zoki Co.,Tokyo, Japan) intraabdominally. Three hours
after hCG stimulation, testosterone responses inthe vena cava were evaluated. Then, all the rats
were sacrificed by an injection of saturated potassium chloride into the left venticle. Bilateralepididymal caudal sperm content and motility
and bilateral testicular weight were measured.
Furthermore in vitro fertilization (IVF) trials
were performed to appreciate epididymal
caudal sperm fertilizing potential both on theright and left side.

Testicular temperature
Right testicular temperature was assessed by
percutaneous insertion of a 29-gauge needle
probe attached to a digital thermometer (Unique
Medical, PTC201 model, Tokyo). Intraabdominal temperature was monitored with a rectal
probe and body temperature was maintained between 36.7 and 37.3˚C with radiant heat throughout the procedure. The difference between theintraabdominal and intratesticular temperatureon the right side (R∆T) was recorded as in themethod described previously (Sofikitis et al.,1992).

Materials and Methods
The Wistar rats used were 2-week-old males
obtained from the Shimizu Experimental Material Company (Kyoto, Japan). The rats were divided into 3 groups: A = rats which had undergone cryptorchidism induction; B = shamoperated rats; and C = rats which had undergone
cryptorchidism induction and subsequently
orchidopexy. To create left cryptorchidism(groups A and C) left inguinal incision was
performed . The left testis was sutured at theposterior abdominal wall with 6-0 nylon sutures
under sodium pentobarbital anesthesia (25 mg/
kg; Nembutal, Abott Laboratories, Chicago,
IL). The sham operation (group B) included a

Determination of the weight of testes
The testes and epididymes cauda were excised,dissected free of surrounding tissue, and thetestes were weighed on a Mettler Basbal scale(Delta Range, Tokyo).

80

Left cryptorchidism and right epididymal sperm fertilizing capacity

matozoa in their vagina by microscopic examination of the vaginal fluid. Females were thenchecked 3 times daily from day 21 for parturition (day 1 was designated as the day of mating). A male rat was considered fertile if its matings resulted in at least one pregnancy.

Epididymal caudal sperm content andquantitative sperm motility (% motility)
Each epididymis was separated carefully fromits testicle under a magnification of 10 times,
provided by a stereo zoom microscope (model
TL2, Olympus Corp., Tokyo). The epididymis
was divided into 3 segments: the head, the bodyand the tail. The epididymal tail was trimmed
and minced in 5 mL of Biggers-WhittenWhittingham (BWW) (Sofikitis et al., 1992)
medium adjusted to pH 7.4 at 37˚C with 6-mol/
L sodium hydroxide. The minced epididymal
tissue was then separated from the liberated
spermatozoa by filtration through a stainlesssteel wire mesh with a pore size of 60 µm. Six
droplets of the filtrate were used for assessing
the sperm count (number of spermatozoa/mL
BWW medium) and the average number was
calculated for each animal. The sperm count
was determined with a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel).
The chamber was placed on the slide of an
ordinary microscope, and a 20-power objective
and 10-power eyepiece were used. Ten dropletsof the filtrate were counted to calculate the percentage of motile spermatozoa immediately
after its preparation, so that the estimate of motility became more accurate.

Preparation of sperm suspension for IVF
Epididymal caudal suspensions were centrifuged at 300 × g for 20 min. Sperm pellets were transferred to Toyoda and Chang medium (Toyoda
and Chang, 1974). Finally sperm suspensions
containing 2 × 106 spermatozoa/mL were prepared and processed for IVF as previously described (Toyoda and Chang, 1974) .

Collection of oocytes and IVF
Immature female rats were injected subcutaneously with 25 IU pregnant mare serum gonadotropin (Sigma Chemical Company, St. Louis,
MO). Fifty-four hours later an intraabdominal
injection of 20 IU hCG (Teikoku Zoki Company, Tokyo) was done. Rats were killed 19 h
after hCG injection. The oviducts were
removed and the ampullar portion was put intoa plastic dish containing Toyoda and Chang
medium (Toyoda and Chang, 1974). The oocytes in cumulus masses were removed fromthe oviducts and introduced into the Toyoda
and Chang medium. A volume of 0.1 mL ofsperm suspension was introduced into 0.9 mL

Determination of serum testosterone concentration
Testosterone concentration was determined byradioimmunoassay using a kit from the Nihon
DPC Corporation (Tokyo) according to themethod of Coyotupa and coworkers (1992).
The intra- and inter-assay coefficients of variation were 5.5 and 9.2%, respectively. The sensitivity of the assay was 0.1 ng/mL.

Table 1. Difference (∆) between intraabdominal and right intratesticular temperature∆Temperature
(˚C)

Group

Fertility assessment in vivo
Two female Wistar rats (3 months old) in thefirst hours of estrus as determined by vaginal
smear examination, were placed in a single cagewith each male rat. Two hours later, the female
rats were checked after mating to detect sper-

A: Cryptorchid[20]

3.2 ± 0.3

B: Control[10]

4.3 ± 0.3

C: Orchiopexy[10]
3.7 ± 0.3
Values are expressed as mean ± SD.
[ ], number of samples.
*Significant differences; P < 0.05.

81

*
*
*

T. Kaki and N. Sofikitisof the Toyoda and Chang medium containing the oocytes within cumulus masses.
The dishes were kept within a CO2 incubator (5% CO2 in air). Ten oocytes wereinseminated with spermatozoa from the left
epididymis of each group. Furthermore, 10
oocytes were inseminated with spermatozoa from the right cauda epididymis ofeach rat in each group .

Table 2. Testicular weight (mg) in cryptorchid
rats
Group
B
Control[10]A
Cryptorchid[20]

C
Orchiopexy
[10]

*
*

*
Right 814.0 ± 91.7 1341.3 ± 135.4 1060.0 ± 149.7*

Examination of oocytes*

Twenty-four hours after insemination, thepercentage of oocytes with 2 pronuclei plus
a second polar body was evaluated via an
inverted microscope (Olympus IX-70,
Tokyo).

*Left 277.1 ± 70.7 1266.2 ± 123.5 631.2 ± 144.7
Values are expressed as mean ± SD.
[ ], number of samples.
*Significant differences; P < 0.05.

control group and the orchidopexy group (Table
1).

Statistical analysis
Values were expressed as mean ± SD. Statistical analysis was performed on all data using
Fisher’s PLSD test to analyze intergroup differences. A probability P < 0.05 was considered to
be statistically significant. Assessment of thevarious parameters was performed in a blindedfashion.

Testicular weight

Results

Left testicular weight was significantly lower
than right testicular weight in cryptorchid rats
(Table 2). Left testicular weight was significantly smaller in cryptorchid rats than in control
rats and rats which had undergone orchidopexy.
Right testicular weight in cryptorchid rats was
significantly lower than in control rats or rats
which had undergone orchidopexy.

Testicular temperature

Epididymal sperm content and motility

The cryptorchid group showed a significantly
smaller mean R∆T value compared with the

The epididymal caudal sperm content wassignificantly lower bilaterally in the cryptorchid

Table 3. Sperm content and motility in the cauda epididymis
Concentration (× 106/mL)
Right
Left

Group
A: Cryptorchid [20]
B: Control[10]27.9 ± 5.7
83.1 ± 10.3

C: Orchiopexy [10]57.0 ± 9.5
Values are expressed as mean ± SD.
[ ], number of samples.
*Significant differences; P < 0.05.

Motility (%)
Right

2.5 ± 0.9***

79.9 ± 12.3
19.5 ± 6.1

82

Left

29.8 ± 11.7***

72.7 ± 11.5
48.3 ± 9.7

5.4 ± 2.5***

73.8 ± 9.6
40.2 ± 10.1***

Left cryptorchidism and right epididymal sperm fertilizing capacity

rats than in the control group (Table 3).
The percentage of motile sperm was significantly lower bilaterally in cryptorchids
rats than in the control group (Table 3).
The epididymal caudal sperm content andmotility on the left and right side were significantly higher in rats which had undergone orchidopexy than in the cryptorchidrats.

Leydig cell function

Table 4. Testosterone responses to hCG stimulation
Group

Testosterone (ng/dL)
Pre-hCG
Post-hCG

A: Cryptorchid [20]174 ± 53488 ± 74

B: Control

199 ± 47819 ± 89

[10]

*
*

C: Orchiopexy [10]188 ± 62781 ± 87
Values are expressed as mean ± SD.
[ ], number of samples.
hCG, human chorionic gonadotropin.
*Significant differences; P < 0.05.

Peripheral serum basal testosterone levels
were not significantly different among the
3 groups. Peripheral serum testosterone responses to hCG stimulation were significantly
lower in cryptorchid rats than in rats which had
undergone orchidopexy or control rats (Table4). There was no significant difference intestosterone responses between control rats andrats which had undergone orchidopexy.

Discussion
Cryptorchidism is a preexisting factor in 3% to
8% of infertile men and in 20 % of men withazoospermia (Waites and Moule, 1961; Alpert
and Klein, 1983; Tellaioglus et al., 1994). The
incidence of infertility ranges from 10% to 20%
in patients with unilateral cryptorchidism(Tellaloglus et al., 1994). Animal studies indicate a detrimental effect of unilateral cryptorchidism on fertility potential (Tellaloglus et al.,1994). In congenital unilateral cryptorchid rats
the fertility rate is 0% compared with 100% in
control rats (Patkowski et al., 1992). In the congenital canine cryptorchid model all bilateralcryptorchid animals were azoospermic, whereas the total sperm count, sperm motility andpercentage of normal sperm morphology wereFertility potential in vivo and in vitro
The effects on fertility are summarized in Table
5. The proportion of fertile rats in thecryptorchid group was significantly lower thanin the control group. The percentage of oocyteswith 2 pronuclei plus a second polar body after
the IVF technique using left or right caudal
sperm was significantly lower in cryptorchidrats than in control rats or rats which had undergone orchidopexy.

Table 5. Effect of cryptorchidism on fertility in vivo and in vitro
Group

Numberoffertile rats

A: Cryptorchid [20]
B: Control

[10]

2

Number of
inseminatedoocytes†(10)10 (100)

200*

Number of 2-PN oocytes‡
Right caudal
Left caudal
epididymis
epididymis
23(11.5)

100

82

(82)

C: Orchiopexy [10]7 (70)
100( ), percentage; [ ], number of samples of the group.
†Inseminated oocytes by sperms from each side.
‡Oocyte with 2 pronuclei plus second polar body.
*Significant differences; P < 0.05.

68

(68)

*

83

*
*

0

(0)

84

(84)

47

(47)

*
*

T. Kaki and N. Sofikitis

diminished in the unilateral cryptorchid animals(Kawakami et al., 1995).
A model of cryptorchidism was created in
the present study in rats at 2 weeks of age. The
results of the present study confirm several
previous studies (Patkowski et al., 1992;
Tellaloglus et al., 1994; Kawakami et al., 1995)
indicating a detrimental effect of unilateralcryptorchidism on ipsilateral testicular function. Furthermore, the present study indicatesthat the R∆T, the right testicular weight, theright epididymal caudal sperm content, motility
and fertilizing capacity are significantly smaller
in rats with left cryptorchidism than in control
rats. In addition, the current study demonstrates
that right orchidopexy partially restores the influence of left cryptorchidism on right epididymal sperm quantitative parameters. Although
the effect of unilateral cryptorchidism on ipsilateral testicular function may be attributable tothe increase in ipsilateral testicular temperature,
there is much controversy concerning themechanism(s) that mediate the detrimental
effect of unilateral cryptorchidism on contralateral testicular function. Ono and Sofikitis(1997) have provided evidence that the consequences of left cryptorchidism on right testicular function may be due to an increase in righttesticular temperature affecting adversely theright testicular Leydig and Sertoli cell secretory
function. The above increase in right testiculartemperature is attributable to an increase inright testicular blood flow (Ono and Sofikitis,1997). Alterations in contralateral testicularblood flow in animals with unilateral cryptorchidism may be due to an increase in the number of testicular arterial microvessels or an extension of intravascular space (Ono andSofikitis, 1997). A detrimental effect of left
cryptorchidism on right testicular function isadditionally indicated by the significantly
smaller right testicular weight in animals withleft cryptorchidism since it is known that
testicular weight is positively and significantly
correlated with testicular function (Sofikitis etal., 1992).
The absence of significant difference inserum basal testosterone concentration amongthe 3 groups does not indicate an absence ofdifferences in total (left and right) Leydig
cellular secretory function since basal
testosterone concentration in serum is not areliable marker of the testosterone biosynthesis
rate (Steinberger et al., 1973).
Impaired secretory function of Leydig andSertoli cells on the right side due to a temperature increase in animals with left cryptorchidism will detrimentally affect spermatogenesis
and epididymal sperm maturation on the rightside. It is known that optimal intratesticular andintraepididymal testosterone (secreted by the
Leydig cells) and androgen-binding protein(secreted by the Sertoli cells) concentrations areimportant for activating and maintaining
spermatogenesis and the epididymal sperm
maturation process. In fact, a harmful effect ofleft cryptorchidism on right epididymal caudal
sperm content and motility was demonstrated in
the current study. Furthermore, the present
study reveals a detrimental effect of left cryptorchidism on the epididymal caudal sperm
function since the outcome of IVF trials using
spermatozoa from the right epididymal cauda
was significantly smaller in cryptorchid animals. This decrease in right epididymal caudal
sperm function may be attributable to a) the
smaller quantitative motility profiles of spermatozoa recovered from the right epididymis andb) the smaller acrosin content of right epididymal caudal spermatozoa in animals with left
cryptorchidism as a previous study tends to support (Ono and Sofikitis, 1997). Optimal sperm
motility and acrosin content are prerequisites
for sperm movement towards the oocyte, acrosomal reaction, and penetration of the zona
pellucida by the spermatozoon. Furthermore,considering that there is a defect in the right epididymal function in animals with left cryptorchidism (Ono and Sofikitis, 1997) and that theepididymal sperm maturation process involvesa cascade of multiple events influencing several
qualitative and quantitative parameters ofspermatozoa, the probability that additional
sperm characteristics important for the fertilization process are detrimentally affected in theright epididymis in rats with left cryptorchidism
can not be excluded.
The detrimental effect of unilateral cryptor84

Left cryptorchidism and right epididymal sperm fertilizing capacitychidism on contralateral testicular/epididymal
function is of great clinical importance. Further
studies are necessary to clarify whether thisdefect in contralateral epididymal sperm fertilizing capacity is progressive. If progressive damage occurs in the normally descended testis/
epididymis of subjects with unilateral cryptorchidism in whom orchidopexy cannot be performed, epididymal sperm aspiration and sperm
cryopreservation techniques should be performed on the contralateral side to the cryptorchid
testis-side. Such frozen sperm samples may be
used in assisted reproduction programs in the
future.
The present study additionally indicates thatorchidopexy of a cryptorchid testis partiallyalleviates not only ipsilateral testicular and epididymal function but also contralateral epididymal caudal sperm content, motility and fertilizing capacity. Therefore, orchidopexy of a unilaterally cryptorchid testis should be regarded
as a treatment for the contralateral, normallydescended testis, as well.
A defect in right epididymal caudal sperm
fertilizing capacity is revealed in rats with left
cryptorchidism in the current study. This defectmay represent a diminished sperm capacity tobind and/or penetrate the zona pellucida. Additional studies are necessary to investigate separately various events after the sperm’s entrance
into the oocyte (i.e., sperm nucleus decondensation, male pronucleus formation and oocyte
activation). Furthermore, the potential for implantation of embryos generated by the fertilization of oocytes with spermatozoa from theright epididymis of rats with left cryptorchidism needs to be evaluated. Such studies arein progress in our laboratory.

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(Received December 18, 1998, Accepted January 7,1999)

86