in the scrotal testis in experimental unilateral cryptorchidism. J Pediatr Surg 25: 402±405. 18. Retif PJ (1977) Fertility in undescended testis. S Afr Med J 52: 610± ...
Pediatr Surg Int (1998) 13: 392±395
Ó Springer-Verlag 1998
ORIGINAL ARTICLE
M. Srinivas á S. Agarwala á S. Datta Gupta á S. N. Das C. Shaha á D. K. Mitra
Fertility and unilateral undescended testis in the rat model II
Accepted: 14 October 1997
Abstract Continuing experimental work on the eect of experimentally created unilateral undescended testis (UL-UDT) in neonatal rats, this study examined the fertility and correlated it to contralateral (CL) testicular morphology, seminiferous tubular diameter (STD), DNA ¯owcytometry, and the presence of serum antisperm antibodies (ASA) at 120±135 days of age. In our previous reported work, the fertility of rats with ULUDT at 65±80 days of age was the same as that of controls. In the present study the rats with UL-UDT had signi®cantly reduced fertility (P < 0.01) compared to controls, even though the Johnsen scores and mean STD of the CL testicular tissue were comparable. DNA ¯owcytometry demonstrated a signi®cant decrease (P < 0.001) in haploid cell population in the CL testicular tissue of rats with UL-UDT. Furthermore, the rats with UL-UDT who either received an immunosuppressive or in whom the UDT was excised early showed almost normal fertility and DNA histograms like those of controls. Signi®cantly high titres of serum ASA were detected only in the group with UL-UDT when tested at 135 days of age. From these results, in combination with earlier results on similar work, it may
be inferred that UL-UDT causes immunologically-mediated, progressive damage to the CL descended testis, leading to a decrease in fertility in rats. Key words Fertility á Unilateral undescended testis á DNA ¯owcytometry á Anti-sperm antibodies á Rats
Introduction
M. Srinivas á S. Agarwala á D.K. Mitra (&) Department of Paediatric Surgery, All India Institute of Medical Sciences, New Delhi, India
The fertility potential of patients with unilateral (UL) undescended testis (UDT) is a widely debated topic. In clinical and experimental work some authors have reported no change in fertility [2, 5, 11, 16], while others have demonstrated a decrease in fertility [6, 8, 10, 18]. In our previous experimental work on UL-UDT in rats [1] the fertility had been tested in early adulthood, at the age of 65 days, and no decline in fertility was observed. It was inferred that the fertility potential should be tested in later adulthood, and a decrease would indicate progressive histological changes resulting in a decline in fertility potential, akin to the human situation. The present study was designed on the lines of the previous work to study the fertility and correlate it to contralateral (CL) testicular histology, DNA ¯owcytometry, and serum anti-sperm antibodies (ASA) in rats with experimentally created UL-UDT in later adulthood, i.e., at 120±135 days of age.
S. Datta Gupta Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
Materials and methods
S.N. Das Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India C. Shaha Sperm Biotechnology Laboratory, National Institute of Immunology, New Delhi, India
Male newborn Wistar albino rats aged 2 days were operated upon according to the group assigned. After weaning, rats in all groups were provided with equal amounts of commercial feed at a scheduled time once every day. Water was made available ad libitum throughout the study period. The rats were housed in cages in the department's animal laboratory, which has central air-conditioning. To simulate natural conditions a 12-h light and dark cycle was maintained in the laboratory.
393 Two-day-old rat pups were anaesthetised with intraperitoneal ketamine (2 mg/kg body weight) and a single dose of ceftriaxone was administered (100 mg/kg) at the time of surgery. All instruments were sterilised in 2% glutaraldehyde. After positioning the anaesthestised rat on the board, the ventral surface was prepped with povidone iodine and under an operating microscope a transverse lower abdominal incision was made. Depending on the group, the allocated procedure was performed on the right testis as detailed below. The incision was then closed in layers with 6-0 polyglactin and the wound cleaned with povidone iodine and spirit. All groups were coded, and decoding was done at the end of the study. Each group comprised 10 rats. In group I (controls) the abdomen was opened and closed without disturbing the testis. In group II(UL-UDT) a right gubernaculectomy was done and the gubernaculum was anchored to the anterior abdominal wall with 10-0 polypropylene. In group III (UL orchidectomy) the Right testis was excised. In group IV (UL-UDT + CSA), in addition to the procedure as in group II all rats received cyclosporin (CSA) (10 mg/kg day) orally. In group V (UL-UDT + D30 orchidectomy) the initial procedure as in group II was followed by orchidectomy at 30 days of age. The left testis was not disturbed in any of the rats. At 120 days of age, each male rat was housed separately with two non-pregnant adult females of proven fertility for 15 days. The female rats were then segregated and followed to the end of pregnancy and their litters counted as a measure of fertility. At 135 days of age the male rats were killed; their blood was collected, centrifuged, and stored at )20 °C for ASA detection. Their left, normally-descended testis was harvested and divided into halves. One half was ®xed in Bouin's solution for 24 h and processed for histopathological examination for evaluating the Johnsen score and seminiferous tubular diameter (STD); the other was transported in Rosewell Park Memorial Institute solution for DNA ¯owcytometry. Specimens ®xed in Bouin's solution were processed routinely and embedded in paran. Thick sections (5 lm) were cut transversely and stained with haematoxylin and eosin (H & E) and periodic acid-Schi. Using a 25´ objective, 25 tubules in one section of the biopsy were evaluated and scored for assessment of the degree of testicular maturity according to the Johnsen criteria [7]. In every H & E-stained section a minimum of 25 circular tubules were measured in two axes drawn perpendicular to each other using a computerised image-analysis system comprising a Pentium processor-based computer (Celebris XL, Digital Corp., USA), an Olympus research microscope (Olympus, USA), a 10-bit digital camera (Correco, Canada), and Optimas 5.2 image analysis software (Optimas, USA). Freshly harvested testis was separated from tunica albuginea and minced in phosphate buer saline (PBS). The resultant single-cell suspension was washed twice in PBS and a 100-ll aliquot was ®xed in 70% ethanol in PBS. After centrifugation, the pellet was resuspended in propidium iodode and subjected to RNA digestion by RNAase (Sigma, USA). DNA histograms were then obtained on a ¯owcytometer (Becton-Dickinson FAC scan, California, USA). The data thus obtained were analysed by software Cell Fit.
Whole rat sperm as an antigen was used for the detection of ASA. A concentration adjusted to 5 ´ 109 sperm/l was coated onto a microtitre plate. Non-speci®c antigenic sites were blocked with polyvinyl alcohol. Rat serum was added to produce a solid-phasebound antigen-antibody complex. After washing, goat anti-rat immunoglobulin antibody (second antibody) conjugated with horseradish peroxidase was added. After washing, substrate Ophenylene diamine dihydrochloride was added. Absorbance values were then recorded by a photometer (Anthos Labtec HT 2,1.21E). Optical density readings were taken at 492 nm. Statistical analysis of the study was performed using the BMDP statistical package (BMDP Software, USA). Descriptive statistics and analysis of variance were performed.
Table 1 Mean values of fertility, Johnsen score (JSR), seminiferous tubular diameter (STD), and percentage of haploid cells (% Haploid) and serum anti-sperm antibody (ASA) estimation in the
various groups at 135 days age. (UL unilateral, UDT undescended testis, orchid orchidectomy, CSA cyclosporine, D30 day 30 of life)
Groups
Fertility
JSR
I (control) II (UL-UDT) III (UL orchid) IV (UL-UDT + CSA) V (UL-UDT + D30 orchidectomy)
8.3 6.5 7.9 7.6 7.8
9.27 9.00 9.00 8.88 9.33
a b
0.68 0.70a 0.90 0.50 0.63
P < 0.01 group II compared to group I P < 0.001 group II compared to group I
0.35 0.35 0.20 0.35 0.35
Results Fertility The mean fertility evaluated by litter size Fig. 1, Table 1) was 8.3 0.68 in group I (control) and 7.9 0.9 in group III (UL orchidectomy). This dierence was not signi®cant. The mean fertility in group II (UL-UDT) was 6.5 0.7, which was signi®cantly (P < 0.01) less than that of group I. The mean fertility in groups IV(UL-UDT + CSA) and V(UL-UDT + D30 orchi-
Fig. 1 Mean fertility in the various groups at 120±135 days of age
STD(lm)
% Haploid
ASA
316.5 310.9 342.1 313.7 310.3
70.20 1.80 60.89 3.96b not done 70.20 1.60 68.00 3.90
Negative Positive Negative Negative Negative
21.4 2.8 12.2 19.7 22.1
394
dectomy) was 7.6 0.51 and 7.8 0.63, respectively, both of which were not signi®cantly dierent (P > 0.05) from group I. Johnsen score The mean Johnsen testicular maturation score of 9.27 0.35 in group I was not signi®cantly dierent from that of group II (UL-UDT) 9.00 0.35 (Table 1). Seminiferous tubular diameter STD was not signi®cantly dierent in any of the groups (Table 1). DNA ¯owcytometry The mean percentage of haploid cells in group II (ULUDT) was 60.89 3.96 (Table 1), which was signi®cantly less than that of group I (P < 0.001). The mean percentage in group IV (UL-UDT + CSA) was 70.16 1.16 and that in group V (UL-UDT + D30 orchidectomy) was 68 3.9, both of which were not signi®cantly dierent from group I. Anti-sperm antibodies The ELISA test for the detection of ASA was strongly positive only for group II (UL-UDT), while it was negative for all the other groups (Table 1).
Discussion The fertility potential of patients with UL-UDT is an unresolved controversy. Mandat et al. [11] reported no change in fertility in patients who had undergone UL orchidopexy in childhood. A similar observation was made by Puri and O'Donnell [16]. Contrary to these ®ndings, abnormalities have been reported in semen samples of patients who have undergone orchidopexy for UL-UDT [10, 12, 22]. In addition to these observations, there is mounting experimental evidence that fertility is reduced in UL-UDT [2, 5, 9, 15]. Previous experimental work done at this laboratory [1] had shown no decrease in fertility in rats with UL-UDT when tested at 65±80 days of age, i.e., young adulthood. In the present work, the fertility of rats tested at 120±135 days of age was signi®cantly (P < 0.01) reduced. The rats in whom the UL-UDT was excised at 30 days of age showed no reduction in fertility. This also implies that the continuing presence of the UDT induces and propagates progressive CL testicular damage as a function of time. In this study rats with UL-UDT who simultaneously received an immunosuppressive (CSA) had fertility that
was not signi®cantly dierent from the controls. This indicates that immunosuppression was able to prevent the progressive deterioration in fertility even in the continuing presence of the UL-UDT. It is therefore inferred that the UL-UDT causes immunologically-mediated CL testicular damage. This is further substantiated by our ®nding of signi®cantly elevated levels of serum ASA in rats with UL-UDT and not in the other groups. Similar observations were made by Mengel et al. [14] in UL cryptorchid dogs. We were surprised to ®nd no signi®cant dierences amongst the various groups in Johnsen score and mean STD. The rat testis has 14 stages in the maturation cycle of the seminiferous epithelium [3], and it is likely that subtle changes may not become apparent by the Johnsen scoring system, which was originally standardised for human testicular biopsies [7]. It is possible that if this damage continues, then at a later age the histological changes would become more apparent on routine histology. DNA ¯owcytometry of testicular tissue is more sensitive and less prone to observer bias in determining the various changes in the germ-cell population [4, 17]. In the present study, the DNA histograms of the CL descended testicular tissue showed a signi®cant (P < 0.001) decrease in haploid cell population in the rats with UL-UDT. The administration of CSA or early excision of the UDT prevented this decrease, further rendering support to the concept of immunologicallymediated continuous CL testicular damage. In 1966 Shiari et al. [21] reported that spermatogenesis in the CL descended testis was suppressed in males with UL-UDT. Biopsies in patients with UL-UDT have revealed spermatogenic arrest [13, 20], degeneration, and various other histological abnormalities in the CL descended testis [9, 16]. Based on these observations and their own work, Salman et al. [19] suggested that orchidectomy for UL-UDT was helpful in improving CL morphology and that orchiopexy may also be helpful if performed early. Based on the present work, early removal of the UDT de®nitely preserves fertility, but whether orchiopexy will have a similar eect will need to be studied in future work.
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