LINEBURG


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mechanism (Friedman et al., 1998).


VEGF Soluble Receptor
VEGF functions through a distinct membrane-spanning tyrosine kinase
receptor. The c-DNA encoding a soluble truncated form of one such receptor,
fms-like tyrosine kinase receptor, has been cloned from a human vascular
endothelial cell library (Kendall and Thomas, 1993). The m-RNA coding region
distinctive to this c-DNA has been con¬rmed to be present in vascular
endothelial cells. The recombinant soluble human receptor binds VEGF with
high af¬nity and inhibits its mitogenic activity for vascular endothelial cells.
This soluble receptor could potentially act as an ef¬cient speci¬c antagonist of
VEGF in vivo (Kendall and Thomas, 1993).
62 PATHOPHYSIOLOGY OF OVARIAN HYPERSTIMULATION SYNDROME




Free VEGF or Total VEGF
Alonso-Muriel et al. (2005) recently investigated the role of systemic total
VEGF and free VEGF as well as its physiologic inhibitors, such as soluble
receptor (sVEGFR-1) and a2-macroglobulin in the pathogenesis of OHSS.
Alonso-Muriel et al. (2005) concluded that free VEGF and not total VEGF is
related to the development of OHSS. The ratios of free VEGF to total VEGF and
free VEGF to a2-macroglobulin could be useful predictors of the development
of the syndrome.


Interleukins
Rizk et al. (1997) recently reviewed the role of interleukins (IL) in the
pathogenesis of OHSS. There is growing evidence for a role of the immune
system and, in particular, cytokines as mediators of pathophysiologic changes
in OHSS (Mathur et al., 1997). Cytokines are a family of low-molecular-weight
proteins that play important roles in the regulation of immunological and non-
immunological homeostatic responses (Mathur et al., 1997). They exert their
effects on cellular differentiation and activation.
Interleukins are a subset of cytokines originally thought to be lymphocyte
products involved in interactions between leukocytes. Interleukins have a
variety of actions on endothelium, ¬broblasts and granulosa and luteal cells
(Wang et al., 1991; Wang and Norman, 1992; Mathur et al., 1997).


Interleukin-2
Interleukin-2 is a glycoprotein with a molecular weight of 15 400 kDa. It is
released from activated T-lymphocytes and is rapidly cleared from circulation
with a half life of 3À22 minutes (Oppenheim et al., 1991). Interleukin-2 is not
central in the cascade of events leading to OHSS (Aboulghar et al., 1999; Rizk
and Aboulghar, 2005). Barak et al. (1992) studied the correlation between IL-2
and estradiol, progesterone and testosterone levels in periovulatory follicles
of IVF patients. There was no correlation between follicular ¬‚uid IL-2
concentration and follicular ¬‚uid estradiol and progesterone concentrations.
Furthermore, no correlation between follicular ¬‚uid IL-2 and serum estradiol
concentrations was observed. Orvieto et al. (1995) elected to use pooled
aspirated follicular ¬‚uid from each patient rather than to evaluate each follicle
separately. They demonstrated a signi¬cantly higher IL-2 concentration in
follicular ¬‚uid obtained at the time of oocyte recovery from patients who
developed OHSS as compared with the control group. A possible role for
follicular ¬‚uid IL-2 concentrations in the prediction of OHSS was suggested.
In a multicenter study, Revel et al. (1996) found undetectable IL-2 levels in all
samples of peritoneal ¬‚uid from patients with severe OHSS. Aboulghar et al.
(1999) found these con¬‚icting data for IL-2 levels in the peritoneal ¬‚uid
dif¬cult to explain. One possible explanation is the very short half-life of IL-2.
Based on these studies and their own data, Aboulghar et al. (1999) concluded
63
VEGF AS A CAPILLARY PERMEABILITY AGENT IN OHSS




that there is no solid evidence for the involvement of interleukin-2 as the major
mediator of vascular permeability in OHSS.


Interleukin-6
Circulating levels of IL-6 increase in a variety of acute illnesses including septic
shock (Damas et al., 1992). IL-6 mediates the acute phase response to injury,
a systemic reaction characterized by leukocytosis, increased vascular perme-
ability and increased levels of acute phase proteins synthesized by the liver
(Kishimoto, 1989). Interleukin-6 has been described in the follicular ¬‚uid in
women undergoing stimulation. A role for IL-6 in normal ovarian function has
been suggested by the observation that IL-6 mRNA is produced during the
neovascularization or angiogenesis that occurs in the development of ovarian
follicles. The rapid growth and luteinization of the stimulated ovary require
extensive angiogenesis.
Friedlander et al. (1993) examined the role of IL-6 and other cytokines
in four patients with OHSS. Five healthy women at the time of elective
laparoscopic tubal ligation served as controls. Control serum was also obtained
from healthy volunteers, and control peritoneal ¬‚uid was obtained from
patients on peritoneal dialysis. Both serum and ascitic ¬‚uid from women with
OHSS contained signi¬cantly greater levels of IL-6 than control serum and
peritoneal ¬‚uid. No signi¬cant differences in tumor necrosis factor (TNF) levels
in serum, ascitic ¬‚uid or peritoneal ¬‚uid could be found by enzyme-linked
immunosorbent assay (ELISA) or bioassay. The mechanism by which IL-6
might mediate the pathogenesis of this syndrome is not clear. However,
elevated levels of plasma IL-6 have been recorded in both acute pancreatitis
and acute alcoholic hepatitis, conditions in which ascites and hypotension are
common complications of severe disease. It was also found that the albumin
level was markedly lower than would be expected in two of the patients with
severe OHSS. This observation provides further clinical support for the
hypothesis that IL-6 plays a key pathophysiological role in OHSS, because IL-6
is a potent inhibitor of hepatic albumin production, switching the liver to
synthesis of acute-phase reactants.
Loret de Mola et al. (1996a) examined the production and immunolocal-
ization of IL-6 in patients with OHSS. Signi¬cantly higher serum and ascites
IL-6 levels were found in OHSS, compared with post-ovulatory serum and
peritoneal ¬‚uid from normal controls or serum after menotropin stimulation.
The same authors, having found a signi¬cant increase in cytokines in OHSS,
addressed the possibility of whether preovulatory cytokine levels could predict
the occurrence of OHSS. Preovulatory cytokine values were similar in OHSS
compared to controlled ovarian hyperstimulation. They therefore concluded
that cytokine measurement cannot be used to predict the occurrence of OHSS
prior to the administration of hCG (Loret de Mola et al., 1996b).
Abramov et al. (1996) studied the kinetics of four in¬‚ammatory cytokines
in the plasma of patients who developed severe OHSS after IVF. Higher
concentrations of IL-1, IL-6 and TNF were detected in all individuals upon
64 PATHOPHYSIOLOGY OF OVARIAN HYPERSTIMULATION SYNDROME




admission for severe OHSS. Concentrations dropped signi¬cantly along
with clinical improvements, with normal values recorded after complete
resolution. A statistically signi¬cant correlation was found between plasma
cytokine concentration and certain biological characteristics of the syndrome,
such as leukocytosis, increased hematocrit and elevated plasma estradiol
concentrations.


Interleukin-8
IL-8 is a chemoattractant and an activating cytokine to neutrophils and a potent
angiogenic agent (Rizk and Aboulghar, 1999, 2005). IL-8 is produced by a
number of cell types including monocytes, endothelial cells, ¬broblasts,
mesothelial cells and endometrial stromal cells. Signi¬cantly higher peritoneal
¬‚uid levels of IL-8 were found in 12 patients with severe OHSS compared with
20 controls. However, no statistical signi¬cance was observed in the serum levels
of patients and controls. This may imply a direct spill of IL-8 from the ovaries
to the peritoneal ¬‚uid. Chen et al. (2000) suggest that follicular ¬‚uid IL-6
concentrations at the time of oocyte retrieval and serum IL-8 concentrations
on the day of embryo transfer may serve as early predictors for this syndrome.


Interleukin-10
Manolopoulos et al. (2001) found high concentrations of IL-10 in peritoneal
¬‚uid and suggested a role for this anti-in¬‚ammatory cytokine during OHSS.
17 beta-estradiol and progesterone were elevated in peritoneal ¬‚uid and serum
during OHSS but no correlation with IL-10 concentrations was found.
Therefore, they assumed that IL-10 has a role in OHSS as a local mediator of
in¬‚ammation; however, it presents different aspects of the OHSS than the sex
steroids 17 b-estradiol and progesterone (Enskog et al., 2001b) (Figure III.5).


Interleukin-18
Barak et al. (2004) investigated the role of interleukin-18 (IL-18) in the
pathophysiology of severe OHSS, its potential use as a marker of OHSS
and correlation to capillary permeability. They studied 24 patients with severe
OHSS in a prospective controlled study. Two control groups were used. The
¬rst consisted of 40 age-matched women without ovulation induction
treatment, and the second group consisted of 19 women receiving the same
medication as a group of 19 women who did not develop OHSS. Signi¬cantly
higher levels of interleukin-18 were detected in the peritoneal and pleural ¬‚uids
as well as the serum of the women who developed severe OHSS in comparison
with the two control groups. Serum IL-18 dropped signi¬cantly when the
patient progressed to the diuretic phase and resolution of OHSS. A statistically
signi¬cant correlation was observed between serum IL-18 and capillary
permeability, as judged by the hematocrit and white blood cell count as well
as serum estradiol and IL-6 levels.
65
THE ROLE OF SELECTINS AND ICAM IN THE PATHOPHYSIOLOGY OF OHSS




Fig. III.5: Interleukin-10 concentrations in plasma of OHSS and control patients
Reproduced with permission from Enskog et al. (2001b). J Reprod Immunol 49:71À82



Angiogenin
Aboulghar et al. (1998) investigated the possible role of angiogenin in the
pathogenesis of OHSS. The study group consisted of ten healthy women
who developed severe OHSS, following ovarian stimulation by the long
GnRH-a/hMG protocols for IVF. A control group of ten patients underwent
stimulation according to the same protocol and did not develop OHSS. Blood
samples were taken from the OHSS group on the day of admission to hospital
for treatment and in the control group one week after oocyte retrieval. Ascitic
¬‚uid samples were aspirated during the routine aspiration of ascitic ¬‚uid
as treatment for severe OHSS, and peritoneal ¬‚uid samples were aspirated
transvaginally before oocyte retrieval in the control group.
In the OHSS group, the mean serum level of angiogenin, mean ascitic ¬‚uid
level of hCG administration and the mean hematocrit were 8390 + 6836 ng/ml,
2794 + 1024 ng/ml, 6300 + 2450 pg/ml and 46.6 + 4.4; as compared with
234 + 91 ng/ml, 254 + 105 ng/ml, 1850 + 1100 pg/ml and 36.8 + 4.6 in the
control group, respectively. The difference was highly signi¬cant between all
parameters. Angiogenin seems to play an important role in the formation of
neovascularization responsible for the development of OHSS.


THE ROLE OF SELECTINS AND ICAM IN THE
PATHOPHYSIOLOGY OF OHSS

The selectins, a group of cell adhesion molecules, are major mediators of
in¬‚ammatory, immunologic and angiogenic reactions (Rizk and Abdalla,
2006). Daniel et al. (1999) performed a prospective case-control study to
determine whether plasma and peritoneal ¬‚uid levels of vascular cell adhesion
66 PATHOPHYSIOLOGY OF OVARIAN HYPERSTIMULATION SYNDROME




molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule (sICAM-1)
are altered in women with OHSS. The study group consisted of 16 women with
severe OHSS and the control group consisted of ten women treated with
controlled ovarian hyperstimulation and eight women with normal ¬ndings
at diagnostic laparoscopy. The mean peritoneal ¬‚uid levels of sVCAM-1 and
sICAM-1 and the mean plasma levels sVCAM-1 were signi¬cantly higher in the
women with OHSS than the control group. However, the mean plasma levels
of sICAM-1 were comparable. The authors observed a positive correlation
between the levels of plasma estradiol at the time of hCG and sVCAM-1, and
between the number of oocytes retrieved and the levels of sICAM-1. Soluble
cell adhesion molecules may therefore have a role in the pathogenesis and
progression of OHSS (Daniel et al., 1999).
Daniel et al. (2001) carried out a prospective case-control study involving
16 women with OHSS. Ten matched women treated by ovarian stimulation and
eight women with normal diagnostic laparoscopy results served as controls.
Peritoneal ¬‚uid and serum were assayed for soluble endothelial selectin and
soluble platelet selectin by speci¬c enzyme-linked immunoabsorbent assay
(ELISA). Signi¬cantly higher levels of soluble endothelial selectin and soluble
platelet selectin were found in the peritoneal ¬‚uid of the women with OHSS
compared with the basal levels in unstimulated women. Women with OHSS
had signi¬cantly lower soluble serum endothelial selectin levels compared with
those treated by controlled ovarian stimulation who did not develop OHSS.
The serum soluble platelet selectin levels were similar in both groups. Daniel
et al. (2001) concluded that ascitic ¬‚uid of women with OHSS contains
appreciable amounts of soluble selectins, suggesting their ovarian origin and
possible involvement in the syndrome.
Abramov et al. (2001) studied the potential involvement of the soluble
endothelial cell-leukocyte adhesion molecules E-selectin and intercellular
adhesion molecule (ICAM-1) in the pathophysiology of capillary hyper-
permeability in the OHSS. Soluble ICAM-1 and soluble E-selectin are
potentially involved in the pathophysiology of capillary hyperpermeability in
severe OHSS.


ROLE OF THE IMMUNE SYSTEM IN THE PATHOGENESIS
OF OHSS

Several cytokines have important functions in reproductive physiology (Adashi,
1990; Ben-Rafael and Orvieto, 1992). Immunochemistry reveals the presence in
the ovary of T-lymphocytes that are capable of secreting IL-2. The human
corpus luteum cell population has a concentration of macrophages at the
junction of the theca, granulosa and lutein cells (Wang et al., 1992). Enskog
et al. (2001b) hypothesized that patients developing OHSS may have a
disturbed responsiveness or delayed activation of the immunosuppressive
cytokine system. They performed a prospective cohort study on 428 patients
undergoing IVF. Fifteen patients who developed severe OHSS were compared
67
ROLE OF ENDOTHELIAL CELLS IN THE PATHOGENESIS OF OHSS




with matched control patients. Levels of IL-4, IL-10 and IL-13, estradiol and
progesterone were measured throughout the stimulation and up to seven days
after embryo transfer and during hospitalization for OHSS. Signi¬cantly lower
levels of IL-10 levels were observed at the initiation of gonadotrophin therapy
in OHSS patients with an increase observed after OHSS development. In OHSS
patients, a negative correlation was observed between IL-10 levels and the
number of follicles at the time of oocyte retrieval. No correlation was observed
between IL-10 and steroid levels. Levels of IL-13 and IL-14 were low in both
groups and did not change during stimulation. Enskog et al. (2001b)
hypothesized that the lower levels of IL-10 at the initiation of stimulation in
OHSS patients, as compared with controls, might be of pathophysiological
signi¬cance by allowing for an enhanced Th-1 type immune response and
therefore an increased and generalized in¬‚ammation (Figure III.5). The
increase in IL-10 after the development of OHSS may suggest that IL-10 is
induced in a systemic attempt to suppress the in¬‚ammation of OHSS.


ROLE OF ENDOTHELIAL CELLS IN THE
PATHOGENESIS OF OHSS

The role of endothelial cells in the pathogenesis of OHSS was elegantly
investigated by Albert et al. (2002). The hypothesis of involvement of the
endolthelial cell was based upon their observation of high levels of VEGF in
serum and lower levels of VEGF in the follicular ¬‚uid in women at risk of
developing OHSS (Pellicer et al., 1999). This interesting observation suggested
that cells other than ovarian follicles could be a potential cellular source and
target of VEGF. To test this hypothesis, Albert et al. (2002) developed an
in-vitro model using high estradiol and hCG concentrations in human
microvascular endothelial cells. They investigated the ability of the micro-
vascular endothelial cells to express and secrete the mediators that could be
involved in the development of the syndrome.
The endothelium is a source of both VEGF (Banerje et al., 1997) and IL-6
(Van der Meeren et al., 1991). Once VEGF and IL-6 are produced, they may act
at the paracrine and autocrine level, inducing the vascular changes leading to
the syndrome. Receptors for both VEGF (Shweiki et al., 1993) and IL-6
(Mantovani et al., 1997) have been identi¬ed in luteal cells and have been
shown to increase capillary permeability in human (Goldsman et al., 1995) and
animal models (Rizk et al., 1997; Schenker, 1999).
Albert et al. (2002), in their study of the response of the endothelium,
observed that hCG induced an upregulation of the VEGF receptor KDR in
human endothelial cells. This receptor, the most functional receptor for human
VEGF, transduces signals for mitogenicity, angiogenesis and cytoskeletal
organization. On the basis of these ¬ndings, Albert et al. (2002) postulated
that hCG induces the secretion and reception of VEGF in the endothelial cells,
generating an acute response manifested by vascular permeability. The lack of
a role of estradiol in the pathogenesis of OHSS was demonstrated when the
68 PATHOPHYSIOLOGY OF OVARIAN HYPERSTIMULATION SYNDROME




presence of regulation of VEGF and hCG receptors was investigated.
Estradiol alone was unable to upregulate either type of receptor at the
messenger RNA or protein levels. Again, that it was the addition of hCG that
induced a cascade of events that resulted in a signi¬cant increase of the VEFG
KDR receptor.
The integrity of the endothelial cytoskeleton is important for the functional
competence of an endothelial barrier. Permeability to water and solutes depend
on the shape and con¬guration of the endothelial cells. Albert et al. (2002) used
confocal microscopy to analyze the monolayers. They observed an irregular
alignment and arrangement of the active ¬laments and morphological changes
of cell shape and gap formation of adjacent cells and in HUMEC-L treated with
estradiol and hCG, but not in endothelial cells treated with estradiol only. This
effect was reversed by anti-VEGF. The authors deduced three important
messages:

(1) that estradiol alone does not increase vascular permeability
(2) hCG acts through VEGF
(3) blocking VEGF action is a valid alternative to overcome the changes
induced in the endothelium by hCG.

Albert et al. (2002) concluded that the endothelium along with the ovary is a
primary target for hCG (Figure III.6). As a result, VEGF and its KDR receptor
are stimulated, resulting in an acute biological response in the capillaries
causing increased permeability. Blocking VEGF action with speci¬c antibodies
prevents the changes induced by hCG, providing the rationale for new
therapeutic approaches to prevent or treat OHSS.



Fig. III.6: The role of endothelial function in the pathophysiology of OHSS
Reproduced with permission from Albert et al. (2002). Mol Hum Reprod 8:409À18
69
SPONTANEOUS OHSS AND GONADOTROPH ADENOMAS




SPONTANEOUS OHSS AND GONADOTROPH
ADENOMAS

Gonadotroph adenomas are benign tumors that arise from the gonado-
troph cells of the anterior pituitary gland. They comprise approximately 10%
of all pituitary adenomas (Snyder, 1987). Unlike most pituitary adenomas,
such as corticotrope adenomas associated with Cushing™s syndrome and
somatotrope adenomas causing acromegaly, gonadotroph adenomas often do
not cause a recognizable clinical syndrome (Snyder, 1985). The majority of
patients will present with symptoms of intracranial masses (Kwekkboom et al.,
1989).
Some of the clinical presentations in patients with gonadotroph
adenomas resemble those with PCOS in several ways, such as infertility,
amenorrhea and oligorrhea, and enlarged ovaries with multiple cysts.
However, women with gonadotroph adenomas differ from those with
PCOS in several ways (Castelbaum et al., 2002). Several cases of spontaneous
OHSS associated with FSH secretion of pituitary adenomas have been
reported recently (Djerassi et al., 1995; Christin-Maitre et al., 1998; Catargi et
al., 1999; Valimaki et al., 1999; Pentz-Vidovic et al., 2000; Shimon et al., 2001;
Castelbaum et al., 2002; Roberts et al., 2005). Roberts et al. (2005) recently
reported a case of spontaneous OHSS caused by an FSH-secreting pituitary
adenoma (Figure III.7). No ascites or hematological abnormalities were
observed as in some of the reported cases, so, by de¬nition, the patient could
not be classi¬ed as having OHSS (Roberts et al., 2005). However, the ovaries
were hyperstimulated and it is interesting to hypothesize the reasons for the
absence of ascites in this case (Figure III.7), despite supraphysiologic serum
estradiol levels.



Fig. III.7: MRI of gonadotroph adenoma
Reproduced with permission from Roberts et al. (2005). Fertil Steril 83:208À10
70 PATHOPHYSIOLOGY OF OVARIAN HYPERSTIMULATION SYNDROME




REFERENCES

Aboulghar MA, Mansour RT, Serour GI & Rizk B (1996). Ovarian hyperstimulation
syndrome: modern concepts in pathophysiology and management. Middle East Fertil
Soc J 1:3À16.
Aboulghar MA, Mansour RT, Serour GI et al. (1998). Elevated levels of angiogenin in
serum and ascitic ¬‚uid from patients with severe ovarian hyperstimulation syndrome.
Hum Reprod 13:2068À71.
Aboulghar MA, Mansour RT, Serour GI et al. (1999). Elevated levels of interleukin-2,
soluble interleukin-2 receptor alpha, interleukin-6, soluble interleukin-6 receptor
and vascular endothelial growth factor in serum and ascitic ¬‚uid of patients
with severe ovarian hyperstimulation syndrome. Eur J Obstet Gynecol Reprod Biol
87:81À5.
Abramov Y, Schenker JG, Lewin A et al. (1996). Plasma in¬‚ammatory cytokines correlate

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