Human Reproduction vol.13 no.10 pp.2823–2827, 1998
The effects of twisted ischaemic adnexa managed by
detorsion on ovarian viability and histology: an
ischaemia–reperfusion rodent model*
Omur Taskin1,5, Mustafa Birincioglu2,
Abdullah Aydin3, Ali Buhur1, Feza Burak1,
Ismet Yilmaz2 and James M.Wheeler4
of Obstetrics and Gynaecology, 2Pharmacology and
Inonu University Medical School, Malatya, Turkey and
4Texas Women’s Hospital, Houston, TX, USA
Key words: free radicals/ischaemia/ovariectomy/reperfusion/
torsion
1Departments
3Pathology,
5To
whom correspondence should be addressed at: 7.cad. 70. Sok.
No:4/6, Ovecler, Ankara, Turkey
This prospective controlled follow-up study was designed to
examine the effects of adnexal torsion on long-term ovarian
histology and radical scavenger (FRS) activity, and subsequent viability following the detorsion of twisted ischaemic
adnexa, in a primate centre of a university clinic. Adnexal
torsion/occlusion was created by twisting the adnexa three
times and fixing on to the side wall or by applying vascular
clips in cycling female rats at 70 days of age. Following an
ischaemic period of 4 to 36 h, the twisted adnexas were
surgically removed and fixed. In the second group of rats,
following the above ischaemic periods, the torsion/occlusion
were relieved by detwisting or removing the vascular clips.
Then the animals were reperfused for a week and adnexas
were extirpated. After both ischaemia and reperfusion, the
removed adnexas were examined histologically and tissue
concentrations of glutathione peroxidase, superoxide dismutase, catalase and glutathione were determined. Regardless
of the ischaemia time, all the twisted adnexas were blackbluish in appearance. Despite the gross ischaemic–haemorrhagic features, histological sections revealed negligible
changes, with intact ovarian structure similar to controls in
4–24 h groups. Though decreased compared with controls,
the change in tissue concentrations of FRS was not significant
in 4–24 h groups. Only the 36 h group showed prominent
congestion on all sections and a significant decrease in all
radical scavenger concentrations studied. While no longterm reperfusion injury was observed histologically in 4–
24 h groups, the 36 h group ended with adnexal necrosis.
Our findings support the importance of early diagnosis and
conservative surgical management (detorsion) in adnexal
torsion. Lack of histological changes and unimpaired FRS
metabolism are consistent with the recent data that vascular
compromise is caused by venous or lymphatic stasis in early
torsion and that adnexal integrity is not correlated with gross
ischaemic appearance, thus providing evidence of adnexal
resistance against ischaemia.
*Presented in part, 1997 ASRM meeting, Cincinnati, USA.
© European Society for Human Reproduction and Embryology
Introduction
Adnexal torsion is an infrequent but well-recognized serious
cause of gynaecological surgical emergency with a prevalence
of 2.7% (Rock and Thomson, 1997). It may result from
pre-existing tubal/ovarian pathology or as a consequence of
hyperstimulation during ovulation induction (Mage et al.,
1989). Early diagnosis and immediate surgical intervention are
required to preserve fertility and to prevent peritonitis and loss
of the adnexa. Owing to non-specific clinical findings, delays
in diagnosis and surgical intervention are common. Definitive
diagnosis depends on direct visualization by laparotomy or
laparoscopy. Generally a twisted adnexa with a ‘black-bluish’
or necrotic appearance is encountered at surgery which supports
the traditional approach of salpingo-oophorectomy (Pryor
et al., 1995).
Despite the similarities between adnexal and testicular
torsion, testicular functional and histological changes, treatment
and its viability following management have been widely
studied compared with the ovary (Williamson et al., 1976).
Although the viability of the testis and its functionality
following torsion are discussed frequently, to our knowledge
there are no reports in women emphasizing adnexal integrity
during and following torsion. The degree and length of
torsion are important factors in management and prognosis.
A favourable outcome depends on prompt diagnosis and
treatment.
Although the removal of adnexa has been the traditional
surgery in torsion, recent reports have favoured conservative
surgery of performing detorsion and follow-up (Masciah et al.,
1990; Olsner et al., 1993). The above reports suggest that
‘black-bluish’ adnexas are not correlated to viability, and
unwinding of adnexas does not increase the risk of thromboembolism. Despite the above reports, to our knowledge there
is no study examining the ovarian resistance to ischaemia
and subsequent viability/tissue integrity nor the ovarian cell
antioxidant defence system following reperfusion. If reperfusion after ischaemic insult is not associated with ovarian
functional and structural damage, the performance of detorsion
would further be supported on a histological and biochemical
basis along with reported clinical experiences.
In the present study, we tried to demonstrate the effects of
adnexal torsion on long-term ovarian histology and radical
scavenger activity, and subsequent viability, following the
detorsion of twisted ischaemic adnexa in a rodent model.
2823
O.Taskin et al.
Materials and methods
This prospective controlled follow-up study was performed on female
Wistar rats at 70 days of age weighing 290–350 g in a primate centre
of a university clinic. The study protocol was reviewed and approved
by our Investigational Review Board. The surgical preparation and
procedures described below were performed in accordance with the
recommendations outlined in the National Institutes of Health Guide
(1985) for the care and use of laboratory animals.
Female Wistar rats (n 5 140) at 70 days of age weighing
approximately 300 g were housed and used in the study. On the day
of the surgical procedure, each rat was weighed and anaesthetized
with a single intraperitoneal injection of 9 mg/kg xylazine and 60 mg/
kg ketamine (both from Eczacibasi, Turkey), which was repeated
when needed. Rats were placed in dorsal recumbency and covered
with a sterile drape. A small 2.5-cm incision was used for laparotomy
and the uterine horns and adnexas were located. Sham operations
(laparotomy only) were performed in one group of animals as controls.
The ischaemic insult to the adnexas was performed and compared in
two different techniques in order to fully imitate torsion in humans
(vascular clip versus suture fixation). The stage of the cycle was
consistent in all of the animals operated. Adnexal torsion/occlusion
was created by twisting the adnexa three times and fixing it to the
side wall with 6-0 vicryl or by applying vascular clips just below the
ovary above the uterine horn in cycling reproductive age female rats.
Animals were randomly allocated into the study groups.
Following an ischaemia period of 4, 8, 12, 18, 24, 36 h (n 5 10
in each group), the twisted adnexas were surgically removed and
fixed for histopathological and biochemical examination. In the other
group of rats (n 5 10 in each group), following the above ischaemia
periods, the torsion/occlusion were relieved by detwisting or removing
the vascular clips. Then the animals were randomly allowed to
reperfuse for 8, 18, 36 h to 1 week, and adnexas were extirpated.
Both after ischaemia and reperfusion, the removed adnexas were
examined histologically and the tissue concentrations of radical
scavengers (FRS) [Se-dependent and Se-independent glutathione
peroxidase (GSH-Px), Cu-Zn superoxide dismutase (SOD), catalase
(CAT)] and glutathione (GSH) were determined in homogenized
ovarian tissues. All ovarian tissues were fixed in 10% buffered
formalin, embedded in paraffin, cut into 5 µm sections, and stained
with haematoxylin–eosin. For radical scavenger activity, samples
were transported in liquid nitrogen and kept frozen at –70°C.
Homogenates of adnexas were prepared by homogenization in
phosphate-buffered saline (1:5 w/v). Following homogenization,
samples were sonicated for 1.5 min in bursts of 30 s at 0°C and
centrifuged at 9600 g for 20 min (Nistico et al., 1992) Protein
concentrations were determined by the Lowry procedure (Lowry
et al., 1951) with bovine serum albumin as the standard. CAT activity
was assayed by the UV method. GSH-Px activity was determined by
the calorimetric method utilizing cumene hydroperoxide or hydrogen
peroxide as substrates (Lawrance and Burk, 1976). Cu-Zn SOD was
studied by the spectrophotometric method of McCord and Fridovich
(1969), while GSH concentrations were measured by the Anderson
procedure (Anderson, 1985; Ciriolo et al., 1991). Units of these
enzymes were expressed as µmol or nmol of substrates transformed/
min per mg of protein, respectively (Ciriolo et al., 1991).
All the samples were studied simultaneously in the same assay and
the laboratory was blinded to the treatment groups. The tissue
concentrations of the above radical scavengers were analysed between
and within groups by Kruskal–Wallis variance analysis, Mann–
Whitney U test and Wilcoxon-signed rank test where appropriate. A
P-value of , 0.05 denoted statistical significance.
2824
Figure 1. Eight hours of ischaemia; the photomicrograph
shows minimal congestion along with normal ovarian cortex and
follicles (haematoxylin–eosin, original magnification 3100). Scale
bar 5 15 µm.
Figure 2. Eighteen hours of ischaemia; the photomicrograph shows
congestion in ovarian sections (haematoxylin–eosin, original
magnification 3100). Scale bar 5 15 µm.
Figure 3. Thirty-six hours of ischaemia; apparent haemorrhagic
infarctus is seen in ovary (haematoxylin–eosin, original
magnification 3100). Scale bar 5 15 µm.
Results
There were no differences observed among the treatment
groups regarding age, weight, amount of anaesthetics used
Detorsion of twisted ischaemic ovarian adnexa
Figure 5. Ovarian Se-dependent and independent glutathione
peroxidase (GSH-Px) concentrations in control (0 h), 4–36 h
ischaemia and 7 days reperfusion (7DR) groups. (Mean 6 SD,
*P,0.05 and **not significant versus controls, 4–24 h).
Figure 4. Ovarian radical scavenger activity compared with
catalase, Cu-Zn superoxide dismutase (SOD) and total glutathione
concentrations in control (0 h), 4–36 h ischaemia and 7 days
reperfusion (7DR) groups. (Mean 6 SD, *P , 0.05 and **not
significant versus controls, 4–24 h.)
and postoperative loss rate. The mean operation time was
5.7 6 0.8 min and was similar between the groups. Both
ischaemic insults (torsion versus occlusion) used in the study
have revealed similar histopathological and biochemical
results. Regardless of the ischaemia time, all the twisted
adnexas were black-bluish in appearance compared with controls and the reperfusion group. Despite the gross ischaemic–
haemorrhagic features, histological sections revealed negligible
changes with an intact ovarian structure similar to controls in
4–24 h groups. The histopathological findings are depicted in
Figures 1–3. The 4–8 h groups showed intact structure with
normal ovarian cortex and follicles (Figure 1). With increased
ischaemic insult (12–24 h) the histology showed congestion,
haemorrhage and separation of parenchymal cells along with
normal ovarian cortex and follicles (Figure 2). Severe haemorrhage concealing ovarian parenchyma with infarct was prominent in 24–36 h groups (Figure 3). Despite the above changes
in ischaemic site, the histology of the contralateral adnexa was
unaffected and displayed normal ovarian cortex as observed
in controls.
When the tissue FRS numbers were compared, the observed
trend was similar and supported the above histological changes.
Although decreased compared with controls, the change in
tissue concentrations of GSH-Px, SOD, CAT, GSH was not
significant in 4–18 h groups (Figures 4 and 5). Despite a
prominent and consistent decrease, the change in FRS numbers
was not statistically significant in the 24 h group compared
with that of the other groups. The 36 h group showed
significantly lower tissue concentrations in all types of FRS
studied (Figure 4 and 5, P , 0.05), supporting the adverse
histological changes seen above. Apart from the 36 h group,
in which tissues became completely necrotic, all other groups
retained adnexal integrity with .70% of ovarian tissues
preserved.
In the second part of the experiment, following the above
periods of torsion/occlusion, the adnexas were detwisted and
allowed to reperfuse for up to 1 week to observe any reperfusion
changes in histological and biochemical basis. All the reperfusion groups revealed histological findings and tissue FRS
levels similar to those of sham-operated controls. No further
reperfusion injury was observed following ischaemia. While
the 4–24 h group had macroscopically and microscopically
normal-appearing intact ovaries, the 36 h ischaemic group
ended in adnexal necrosis with peritonitis following reperfusion
(Figure 6 and 7). The FRS levels increased after reperfusion
and were similar to control group denoting tissue integrity,
except in the 36 h group which showed prominent tissue injury
(Figures 4 and 5, P . 0.05).
Discussion
The traditional treatment of adnexal torsion has been salpingooophorectomy because of concerns regarding thromboembolic
phenomena and ‘black-bluish’ appearance of twisted adnexa,
which were assumed to be non-viable (Olsner et al., 1993).
The scarce data and knowledge on adnexal longevity following
torsion have further favoured the above intervention. Obvi2825
O.Taskin et al.
Figure 6. Eight hours of ischaemia and 7 days reperfusion group
showing normal ovarian follicles and stroma with no reperfusion
damage (haematoxylin–eosin, original magnification 3100). Scale
bar 5 15 µm.
Figure 7. Thirty-six hours of ischaemia and 12 h reperfusion;
haemorrhagic necrosis is seen in the ovarian sections
(haematoxylin–eosin, original magnification 3100). Scale bar 5
15 µm.
ously, this radical surgery may impair future fertility. In contrast
to traditional surgery, since most of the patients are in the
reproductive age group, conservative interventions have
become the preferred mode of treatment to salvage adnexa
(Hurwitz et al., 1983; Masciah et al., 1990; Olsner et al.,
1993; Chapron et al., 1996). There have been no reports of
embolic phenomena with conservative therapy but normalization of ovarian perfusion and subsequent function remain a
concern. However, there are case reports and series showing
preserved ovarian function following detorsion. Our findings
both histologically and biochemically support the validity of
the above conservative therapy.
Furthermore, our findings are consistent with recent reports
stating that macroscopic appearance is not correlated with the
degree of ischaemia. As we have shown, despite ‘black-bluish’
appearance, the adnexal structure was normal on histological
sections denoting that macroscopy was not a true indicator of
2826
the degree of ischaemia. In our study, similar histological and
macroscopic findings, both in occlusion and torsion groups,
support the theory that the ischaemic–necrotic appearance of
adnexa is the result of ovarian engorgement secondary to
venous stasis (Olsner et al., 1993; Gordon et al., 1994). This
provides evidence that complete arterial obstruction generally
does not occur in most cases.
The unique part of our study design was adnexal changes
following reperfusion and changes in FRS concentrations
which were related to the cell antioxidant defence system. The
tolerance of adnexa to ischaemia has not been fully outlined
yet. Besides, the ovary’s behaviour to reperfusion is not clear
and is based only on clinical reports favouring conservative
therapy in adnexal torsion. To our knowledge this study is the
first to examine the long-term effects of ischaemia–reperfusion
on the ovary. Reperfusion is known to further increase cellular
injury after ischaemic insult in some tissues like liver and
brain (Ozawa, 1982). In the ovaries our histological and
biochemical findings did not reveal any adverse effects of
reperfusion, thus supporting conservative surgery in adnexal
torsion. The scavenging system which normally counteracts
the actions of oxygen-derived free radicals in tissue injury
during ischaemia was found to be unchanged in our study
(Kunimoto et al., 1987). The scavenger system functionality
in the ovary further supports the validity of our histological
findings and the results of case reports, suggesting preservation
of the twisted adnexa by unwinding (Bernard et al., 1996).
Sugino et al. (1993) have reported that SOD and catalase
injections blocked the decrease in hormonal function during
ovarian ischaemia. These results provide further experimental
evidence for the scavenger system’s role in ovarian resistance
to ischaemia–reperfusion. More studies are needed to outline
the speculative role of FRS therapy in decreasing the susceptibility of the ovary to ischaemia, thus increasing its resistance
to permanent structural changes. The concern of contralateral
adnexal damage and loss of function, which was reported by
some investigators, was not supported by this study (Cakmak
et al., 1992). In the present study, normal contralateral ovaries
were not affected histologically and biochemically in the case
of unilateral adnexal torsion.
In a recent series, the average delay from admission to the
hospital to surgery in patients with adnexal torsion was reported
to be 15.5 h (Masciah et al., 1990). Since in our rodent model,
histologically and biochemically intact adnexas were observed
after 18–24 h of ischaemia, it is likely that conservative therapy
for adnexal torsion will preserve normal ovarian function in
spite of the ischaemic insult. Although our results in rodents
cannot be extrapolated in full to humans, they are consistent
with recent reports adopting an adnexal-sparing approach
instead of salpingo-oophorectomy.
In conclusion, our findings support the importance of early
diagnosis and conservative surgical management (detorsion)
in adnexal torsion. The lack of histological changes and
unimpaired radical scavenger metabolism are consistent with
recent data indicating that vascular compromise is caused by
venous or lymphatic stasis in early torsion and that adnexal
integrity is not correlated with gross ischaemic appearance. This
provides evidence for adnexal resistance against ischaemia.
Detorsion of twisted ischaemic ovarian adnexa
References
Anderson, M.E. (1985) Determination of glutathione and glutathione disulfide
in biological samples. In Meister, A. (ed), Methods in Enzymology. Academic
Press, New York, pp. 548–555.
Bernard, A., Rakoczi, I., Kosa, Z. et al. (1996) Laparoscopic management of
adnexal torsion during pregnancy following ovarian hyperstimulation. Hum.
Reprod. Update, 2, CD-ROM, item 3, video.
Cakmak, M., Mergen, K., Dindar, H. et al. (1992) Influence of unilateral
torsion to the contralateral ovary. Tokai. J. Exp. Clin. Med., 17, 105–108.
Chapron, C., Capella-Allouc, S. and Dubuisson, J.P. (1996) Treatment of
adnexal torsion using operative laparoscopy. Hum. Reprod., 11, 998–1003.
Ciriolo, M.R., Fiskin, K., Martino, A. et al. (1991) Age-related changes in
Cu,Zn superoxide dismutase, Se-dependent and independent glutathione
peroxidase and catalase in specific areas of rat brain. Mechanisms of Ageing
and Development, 61, 287–297.
Gordon, J.D., Hopkins, K.L., Jeffrey, R.B. and Giudice, L.C. (1994) Adnexal
torsion: color doppler diagnosis and laparoscopic treatment. Fertil. Steril.,
61, 383–385.
Hurwitz, A., Mildwidski, A., Yagel, S. and Adoni, A. (1983) Early unwinding
of torsion of an ovarian cyst as result of hyperstimulation syndrome. Fertil.
Steril., 40, 393–394.
Kunimoto, F., Morita, T., Ogawa, R. and Fujita, T. (1987) Inhibition of lipid
peroxidation improves survival rates of endotoxemic rats. Circulatory Shock,
21, 15–22.
Lawrance, R. and Burk, R.F. (1976) Glutathione peroxidase activity in
selenium-deficient rat liver. Biochem. Biophys. Res. Commun., 71, 952–958.
Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein
measurement with the folin phenol agent. J. Biol. Chem., 193, 1374–1378.
Mage, G., Canis, M., Mahnes, H. et al. (1989) Laparoscopic management of
adnexal torsion: a review of 35 cases. J. Reprod. Med., 34, 520–524.
Masciah, S., Bider, D., Moran, O. et al. (1990) Adnexal torsion of
hyperstimulated ovaries in pregnancies after gonadotropin therapy. Fertil.
Steril., 53, 76–80.
McCord, M.J. and Fridovich, I. (1969) Superoxide dismutase. An enzymic
function for erythrocuprein. J. Biol. Chem., 22, 6049–6055.
National Institutes of Health (1985) Guide for the Care and Use of Laboratory
Animals. National Institutes of Health publication. Vol. 86. Bethesda (MD).
Nistico, G., Ciriola, M.R., Fiskin, K. et al. (1992) NGF restores decrease
in catalase activity and increases superoxide dismutase and glutathione
peroxidase activity in the brain of aged rats. Free Rad. Biol. Med., 12,
177–181.
Olsner, G., Bider, D., Goldenberg, M. et al. (1993) Long-term follow-up of
the twisted ischemic adnexa managed by detorsion. Fertil. Steril., 60,
976–979.
Ozawa, K. (1982) Energy metabolism. In Cowley, R.A. and Trump, B.F.
(eds.), Pathophysiology of Shock, Anoxia, and Ischemia. William and
Wilkins, Baltimore, pp. 74–83.
Pryor, R., Wiczyk, H.P. and O’Shea, D.L. (1995) Adnexal infarction after
conservative surgical management of torsion of a hyperstimulated ovary.
Fertil. Steril., 63, 1344–1346.
Rock, J.A. and Thomson, J.D. (eds.) (1997) Operative Gynecology. LippincottRaven, Philadelphia.
Sugino, N., Nakamura, Y., Okuno, N. et al. (1993) Effects of ovarian ischemiareperfusion on luteal function in pregnant rats. Biol. Reprod., 49, 354–358.
Williamson, R.C.N. (1976) Torsion of the testis and allied conditions. Br.
J. Surg., 63, 465.
Received on December 1, 1998; accepted on July 14, 1998
2827