1 Obstetrics and Gynaecology, 2 Royal Victoria Hospital and 3 Anatomy, School of Medicine, Queens University Belfast, Institute of Clinical Science, Grosvenor Rd, Belfast BT12 6BJ, UK
4 To whom correspondence should be addressed. E-mail: c.mcvicar{at}qub.ac.uk
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Abstract |
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Key words: clinical pregnancy rate/ICSI/spermatids/sperm yield/vasectomy
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Introduction |
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For post-vasectomized men wishing to have second families, vasectomy reversal has been replaced in part by testicular biopsy via fine-needle testicular sperm aspiration (TESA) or percutaneous epididymal sperm aspiration, performed at an outpatient clinic and subsequently used in ICSI (Abdelmassih et al., 2002).
The aims of this study were to determine the effects of vasectomy on sperm yield and testicular histology. Secondly, we aimed to compare the fertility outcomes (cumulative embryo score and clinical pregnancy rates) after assisted conception of normal female partners of vasectomized men and men with obstructive azoospermia (OA).
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Materials and methods |
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Anaesthesia
The left spermatic cord was located, and 10 ml of 0.5% bupivacaine (without adrenalin) was injected on either side of it with use of a 21-guage needle. After 10 min, anaesthesia was confirmed by firm testicular palpation: if the patient was aware of pain or pressure, a further bolus was injected. The scrotal skin over the lower pole of the testicle was anaesthetized with 0.5 ml of 1% lignocaine.
Testicular biopsy
The skin and tunica albuginea of the testis were punctured with a disposable scalpel. Two biopsies were taken from the lower pole of the testis, opposite to the pole of the epididymis with the direction of the biopsy along the long axis of the testis. A 14-guage Trucut needle (Baxter Healthcare, Thetford, UK) was inserted through the puncture site into the testicle and advanced 1 cm, taking the tissue from the middle of the testis. The biopsy was taken by advancing the inner core of the needle and then closing the outer sheath rapidly over it. A core of tissue about the size of a grain of rice was trapped in the specimen notch. The tissue was then transferred into culture medium [BiggersWhittenWhittingham (BWW) medium] (Biggers et al., 1971). The procedure was repeated at a different site to obtain the second sample. The subject maintained pressure over the skin wound for 30 min before discharge from the clinic. Diclofenac sodium (100 mg) (Novartis Pharmaceuticals UK, Camberley, UK) was given at the time of the procedure and 12 h later for analgesia.
Sperm yield
The first biopsy was weighed before sperm were milked from the seminiferous tubules. Milking was performed by stabilizing the left end of each seminiferous tubule with forceps and drawing the tubule through a second pair of closed forceps in a left-to-right direction to milk the contents from the free end into the culture medium (BWW). The material obtained was centrifuged at 1000 g for 10 min. Subsequently the pellet was resuspended in 200 µl BWW, and 10 µl was placed on a haemocytometer to calculate sperm/g for the testis.
Spermatid and Sertoli cell number per unit area of biopsy
A second biopsy was not milked but was immersion-fixed intact without milking in Bouins fixative and stained with haematoxylin and eosin for histological assessment. From a transverse section of the intact biopsy, 10 tubules were assessed for each subject. The numbers of Sertoli cells, round spermatids and mature spermatids were counted per cm2 of tissue in round or slightly oval-shaped transverse tubules using a Leica IM measurement module (Northern Microsystems, Cambridge, UK). The spermatids were classified and assessed according to Clermont (1963), and divided into the following groups: the most immature generation of spermatids with spherical nuclei (Sa); darker round spermatids (Sb), also with spherical nuclei, typically pointed elongated spermatids (Sc); and spermatids with smaller, more compact structures (Sd) where the chromatin was condensed into a form similar to that in mature sperm (Figure 1). The ratio of the spermatid count per target area of biopsy to Sertoli cell count per unit area of biopsy was calculated.
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Seminiferous tunica propria thickness
As the thickness of the wall depends on the angle of sectioning we measured only the thinnest part of each tubule so that any difference in thickness in any group was not protocol induced. The tubule wall (tunica propria) included the lamina propria, peritubular myoid cells and the collagen layer. Each biopsy was also assessed by standard pathology techniques for inflammation or neoplasia.
Cumulative embryo score (CES)
A cumulative embryo score (CES) was used to calculate the embryo quality (Steer et al. 1992). Two days after oocyte recovery, prior to embryo transfer, the embryos were graded as follows: grade 4, equal-sized symmetrical blastomeres; grade 3, uneven blastomeres with <10% fragmentation; grade 2, 1015% blastomeric fragmentation; and grade 1, >50% blastomeric fragmentation or pronucleate single-cell embryos. The morphological grade of the embryo was then multiplied by the number of blastomeres to produce a quality score for each embryo. The scores of all embryos transferred per patient were summed to obtain the CES, to be used as an index of the total embryonic contribution to pregnancy.
Only partners of women less than 40 years of age with a normal endocrine profile and normal spontaneous ovulatory cycle were used in this study. The mean female age at the time of the ICSI cycle was 34.20 ± 0.75 years (range 2339) for the men who had been vasectomized 10 years and 32.90 ± 0.43 (range 2439) years for Group 2.
Assisted conception outcome
Clinical pregnancies per embryo transfer were calculated following pregnancy confirmation by ultrasonic fetal heart detection at 6 weeks.
Statistical analysis
Data were analysed using SPSS 10 for Windows. The non-parametric KruskalWallis test was employed to determine the differences in sperm yield between the men in each group and the CES value of the embryos. In addition, the non-parametric MannWhitney test was employed to determine the differences in spermatid number and the width of the tubule wall between fertile and post-vasectomized men. Differences in cumulative embryo scores and pregnancy rates in Groups 1 and 2 were also determined using the non-parametric MannWhitney test.
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Results |
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Effects of vasectomy on sperm yield
The sperm yields/g of both treated groups of men were significantly lower than those obtained from fertile men (P < 0.001, Table I).
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Effects of vasectomy on Sertoli and germ cells and the thickness of the tubule wall (tunica propria)
There was no significant reduction in the Sertoli cell number per unit area of biopsy in the men vasectomized for 10 years compared with previously fertile men. In contrast, there was a marked reduction in the number of early round spermatids (Sa) and dark mature spermatids (Sb) (P < 0.05) and in the number of mature spermatids (Sc and Sd) in the men vasectomized
10 years relative to the fertile men (P < 0.01). The ratio of round spermatids per target area of biopsy to Sertoli cell count per unit area was reduced from 3.46 to 2.86 and the elongated spermatids were reduced from 3.50 to 2.86 in the post-vasectomized men relative to the fertile men. A significant increase in the thickness of the tunica propria was also observed in the men vasectomized for
10 years compared with the fertile men (P < 0.001) (Table II). There was no evidence of an increase in the incidence of carcinogenesis or infection in the pathologists reports (data not included in Table II).
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Effects of vasectomy on the CES and clinical pregnancy rate
There was no difference between the age of the female partners in Groups 1 and 2 (P = 0.124) and no difference in the CES of the embryos transferred to the partners in Groups 1 and 3 (Table III). However, there was a significant reduction in the clinical pregnancy rate of partners of men vasectomized for 10 years relative to the men with non-surgically induced OA (P < 0.05, Table IV).
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Discussion |
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Such testicular dysfunction could be a result of endocrine disruption. Spermatogenesis is primarily controlled by the endocrine effects of FSH and the paracrine effects of testosterone on Sertoli cells. An increase in FSH has been observed in men following vasectomy (Mo et al., 1995), and Smith et al. (1976)
also found that, 1 year after vasectomy, levels of LH and testosterone were increased. However, this was not confirmed by Naik et al. (1976)
, who found that there were no significant changes in FSH, LH and testosterone after vasectomy. Further, Kothari and Gupta (1974)
reported that Leydig cell volumes increased after vasectomy.
Interstitial fibrosis may also affect the paracrine functions of the seminiferous tubules after vasectomy (Shiraishi et al. 2003), leading to a reduction in spermatogenesis. We noted a marked thickening in seminiferous tubule walls, in agreement with Gupta et al. (1975)
, who reported widespread degeneration of the germinal epithelium, including thickening of the basement membrane and interstitial fibrosis as early as 1 month after vasectomy. Howards and Johnson (1979)
reported an increase in testicular hydrostatic pressure 4 months after vasectomy using a guinea-pig model. Fibrosis may also be the result of inflammation, and inflammatory reactions have been reported after vasectomy in guinea-pigs (Alexander, 1973
). This is supported by the knowledge that up to 50% of men have antisperm antibodies in serum and/or semen within a year of vasectomy (Linnet, 1983
). However, no antibodies have been detected on the basement membranes of human or monkey testis after vasectomy (Alexander and Tung, 1979
; Bigazzi et al., 1979
). Therefore, it seems unlikely that an inflammatory reaction could account for the wall thickening observed in this study, although further studies are required to confirm or refute this hypothesis.
Spermatogenesis could also be impaired by cellular disturbances if electrolyte and transmembrane gradients are not maintained. Wang et al. (1994) extracted entire testis membranes from rabbits, including seminiferous tubules, Leydig cells, Sertoli cells, spermatocytes and spermatids. They reported decreases in Na, K+ and ATPase activities and also insufficient cAMP and ATP, correlating with the histological changes in the testis after vasectomy. They also observed that these effects were irreversible as vasectomy reversal did not restore levels to former baselines in either short-term (3 month) or long-term studies.
The role of reactive oxygen species (ROS) and oxidative stress in the sperm yield of post-vasectomy patients has been a subject of interest in recent years. We now know that the production of ROS is a necessary function of sperm (Aitken et al., 2003), but one that must be tightly regulated to prevent damage (Aitken and Krausz, 2001
). Testicular germ cells have been shown to generate hydrogen peroxide, a process that continues as sperm progress through the epididymis (Fisher and Aitken, 1997
). This group has also detected superoxide generation in epididymal sperm. Regulation occurs through a balance of ROS generation and the protective influence of preventative, chain-breaking and scavenger antioxidants, whose enzyme activity in testes has been well documented (Peltola et al., 1992
; Ochsendorf, 1999
; Mruk et al., 2002
; Fujii et al., 2003
). With this innate redox potential, an accumulation of sperm in the epididymis following vasectomy may lead to deregulation of ROS generation, causing oxidative stress. Semen after vasectomy reversal (Shapiro et al., 1998
) has been shown to have higher ROS levels than semen of fertile men. In further support, in another study in which testicular damage was induced in rats by unilateral torsion, levels of superoxide dismutase and catalase were reduced in the ipsilateral testis but were not altered in the contralateral testis (Saba et al., 1997
). Another study (Aydos et al., 1998
), on the generation of ROS in vasectomized rat testes and its relationship with the histological alterations in the testis following vasectomy, showed that output of malondialdehyde (a biomarker for oxidative stress) from vasectomized rats had a direct correlation with tissue destruction. This may be due to a reduced total antioxidant capacity (TAC), since Kolettis et al. (1999)
found that the seminal ROS-TAC score (a novel indicator of oxidative stress) was lower in men who were infertile after vasectomy reversal rather than in those who were fertile after vasectomy reversal. This is supported by a study reporting a reduction in key antioxidants in seminal plasma of infertile men from our group (Lewis et al. 1995
).
A final mechanism for spermatogenic impairment could be elevated rates of apoptosis. Apoptosis plays an important role in the normal testis (Lee et al., 1997; Rodriguez et al., 1997
), eliminating 5070% of germ cells at different stages of spermatogenesis prior to spermiogenesis (Billig et al., 1995
; Henriksen et al., 1995
). Apoptosis in the testis can be initiated through the Fas/FasL pathway (Lee et al., 1997
) and p53 upregulation (Yin et al., 2002
). It is regulated by many protein interactions, including those of the Bcl-2 family, before the cells commit to the process via the caspases and endonucleases. The p53Bax pathway has been shown to increase in the vasectomized rat, and the Bax:Bcl-2 ratio has also been shown to be significantly increased 8 weeks after vasectomy (Shiraishi et al., 2001
). Lue et al. (1997)
have even reported a significant increase in germ cell apoptosis 3 weeks after vasectomy when the spermatocytes are dividing (stage XIII) in hamsters.
In this study we observed no reduction in embryo quality at day 2 or day 3 in Groups 2 or 3 (CES) (Steer et al., 1992). This is to be expected as Braude et al. (1998)
have shown that the paternal genome does not influence embryo cleavage until after the third cleavage division. Further, sperm quality is almost irrelevant for fertilization when the ICSI procedure is used (Twigg et al., 1998
).
One confounding factor in this study might be the difference in age between groups: the mean age of men was 46 years in Group 1 and 37 in Group 2. However, we do not believe this to be the case. There is little in the literature, in terms of sperm quantity or quality, to suggest that men <50 years have impaired spermatogenesis. Limited data are available; most studies have chose greater or less than 50 years arbitrarily but the ranges in each group are usually very wide [3140 versus 4150 versus 5164 (Gallardo et al., 1996) and 3039 versus 4049 (Brzechffa et al., 1998
)]. In 2001, Kidd et al. (2001)
conducted a comprehensive review of all human age and fecundity status studies between 1980 and 1999. Of the 21 studies comparing sperm concentration with increasing age, five reported decreases, six found little or no association and eight reported an increase in sperm concentration with age. Few of the studies controlled for the duration of abstinence or other potential confounding factors. Thus Kidd et al. (2001)
concluded that the weight of evidence from the literature did not suggest that increased male age is associated with a significant reduction in sperm count.
This is also the first study to assess the clinical pregnancy rate of the partners of men who have undergone a vasectomy using TESA followed by ICSI. We found a significant reduction (from 28 to 9%) in the clinical pregnancy rate of the partners of the men who had had a vasectomy 10 years relative to those of men with OA from other aetiologies. Our work confirms that of Abdelmassih et al. (2002)
, who have recently reported a negative correlation between pregnancy rates after ICSI using epididymal sperm and an increasing time interval after vasectomy. This is supported by Borges et al. (2003)
, who used sperm retrieved from the epididymis followed by ICSI after vasectomies 14 years previously. They reported an increase in miscarriage rates. Further, Kubota (1969)
reported degenerative changes in the spermatids and sperm of vasectomized men. All these studies suggest that the quality of post-vasectomy sperm may be compromised and that they are less capable of achieving a pregnancy.
The results reported in this paper raise significant concerns about the effects of vasectomy on both testicular and spermatic function. We would conclude that the success of assisted conception is compromised when using sperm from men vasectomized more than 10 years previously. Further multicentre studies are needed to assess the appropriateness and efficiency of fertility treatments for this patient group.
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Acknowledgements |
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Submitted on September 3, 2004; resubmitted on February 17, 2005; accepted on May 6, 2005.
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