Estrogen-Induced Abnormally High Cystic Fibrosis Transmembrane Conductance Regulator Expression Results in Ovarian Hyperstimulation Syndrome

Louis Chukwuemeka Ajonuma, Lai Ling Tsang, Gui Hong Zhang, Connie Hau Yan Wong, Miu Ching Lau, Lok Sze Ho, Dewi Kenneth Rowlands, Chen Xi Zhou, Chuen Pei Ng, Jie Chen, Peng Hui Xu, Jin Xia Zhu, Yiu Wa Chung and Hsiao Chang Chan

Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territory, Hong Kong

Address all correspondence and requests for reprints to: Dr. Hsiao Chang Chan, Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territory, Hong Kong. E-mail: hsiaocchan{at}cuhk.edu.hk.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ovarian hyperstimulation syndrome (OHSS) remains one of the most life-threatening and potentially fatal complications of assisted reproduction treatments, arising from excessive stimulation of the ovaries by exogenous gonadotropins administrated during in vitro fertilization procedures, which is characterized by massive fluid shift and accumulation in the peritoneal cavity and other organs, including the lungs and the reproductive tract. The pathogenesis of OHSS remains obscure, and no definitive treatments are currently available. Using RT-PCR, Western blot, and electrophysiological techniques we show that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed in many epithelia, is involved in the pathogenesis of OHSS. Upon ovarian hyperstimulation, rats develop OHSS symptoms, with up-regulated CFTR expression and enhanced CFTR channel activity, which can also be mimicked by administration of estrogen, but not progesterone, alone in ovariectomized rats. Administration of progesterone that suppresses CFTR expression or antiserum against CFTR to OHSS animals results in alleviation of the symptoms. Furthermore, ovarian hyperstimulation does not induce detectable OHSS symptoms in CFTR mutant mice. These findings confirm a critical role of CFTR in the pathogenesis of OHSS and may provide grounds for better assisted reproduction treatment strategy to reduce the risk of OHSS and improve in vitro fertilization outcome.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INFERTILITY OCCURS IN about 15% of couples worldwide (1) with most of them seeking assisted reproduction treatments (ART). Ovarian hyperstimulation syndrome (OHSS) is one of the most life-threatening and potentially fatal complications of ART (2, 3). The increasing demand and access to infertility treatments worldwide due to the rising infertility rate have led to the increased prevalence of OHSS. The reported incidence of OHSS is as high as 23% for all forms and up to 5% for severe OHSS of all in vitro fertilization (IVF) treatment cycles (2, 3). The most prominent feature of OHSS is marked ovarian enlargement together with ascites (fluid accumulating in the peritoneal space) almost always associated with the administration of human chorionic gonadotropin (hCG) (2, 3, 4). Other clinical features of severe OHSS include hypovolemia, oliguria and renal failure, pleural effusion, hydrothorax, hemoconcentration and thromboembolic phenomena, and even death as a result of electrolyte and fluid imbalance (2, 3, 4). This syndrome has been recognized for the past 40 yr, and the exact mechanism(s) for the induction of OHSS remains obscure. Increased vascular permeability mediated by vascular endothelial growth factor (VEGF) has been suggested to be responsible for the massive fluid shift from the intravascular compartment into the peritoneal cavity, leading to the formation of ascites. Other substances, such as growth factors, angiotensin, and cytokines, have also been suggested to play major and definitive roles; however, none of these substances or factors, including VEGF, has been demonstrated to be directly involved in the pathogenesis of OHSS (5, 6, 7, 8).

Fluid movements across secretary epithelia are secondary to ion movements. The importance of ion movements across epithelia lies in their coupling with the movement of water, which is not actively transported, but moves in response to osmotic gradients, largely established by the transport of ions. Rapid passage of fluid into luminal spaces, as seen in OHSS, may be a consequence of abnormal ion transport across the epithelia. Several pathological conditions, such as cholera-induced diarrhea, in which there are massive fluid fluxes across epithelial membranes, are mediated by altered expression and function of transepithelial ion channels, particularly CFTR (9), mutations of which have been implicated in the pathogenesis of cystic fibrosis (10), the most common lethal genetic disease in Caucasians, with a hallmark defect in epithelial electrolyte and fluid transport throughout the body (11).

CFTR is expressed in most epithelia including the airways and gastrointestinal and reproductive tracts (12, 13), and its expression is known to be regulated by ovarian hormones (14, 15), up-regulated by estrogen, and down-regulated by progesterone (16). Cyclic changes in CFTR expression and its channel activity have been correlated with cyclic changes in uterine fluid volume (17, 18). It has also been well established that estrogen levels are highly elevated during ovarian hyperstimulation, with excessively high levels observed in OHSS (19, 20). We, therefore, hypothesize that increased estrogen levels during ovarian hyperstimulation may lead to up-regulated CFTR expression and channel activity, resulting in elevated epithelial secretory activity and thus excessive fluid accumulation, as seen in OHSS.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To test this hypothesis, we developed OHSS in the rat as previously described (21). For ovarian hyperstimulation, we used several combinations of gonadotropins known to produce OHSS, i.e. human menopausal gonadotropin (HMG), FSH, or pregnant mare serum gonadotropin (PMSG) in conjunction with hCG. As shown in Fig. 1Go, rats hyperstimulated by either one of the treatments mentioned had fluid accumulation in the peritoneal cavity and uterine horns, and dilated bowel loops (Fig. 1AGo) with watery stools (data not shown) in contrast to the saline-treated control rats (Fig. 1BGo). Increased wet weights were also observed in most major organs, such as liver and lungs (data not shown). Most prominently, the increase in ovarian and uterine wet weights in the hyperstimulated rats were significantly different from those in the controls (Fig. 1Go, C and D), with enhanced CFTR expression at both mRNA and protein levels (Fig. 1Go, E and F). Thus, up-regulated CFTR expression in OHSS is correlated with the fluid accumulation observed in the organs of OHSS animals.



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Fig. 1. Induction of OHSS Rat Model and Up-Regulation of CFTR Expression

The PMSG- and hCG-induced OHSS rat model (A) shows accumulation of fluid in the peritoneal cavity and uterine horns (U), enlarged ovaries, and dilated colon (C) in contrast to the controls (B), which show normal uterine horns and formed stools in the gastrointestinal tract. Hyperstimulated rats given different combinations of gonadotropins showed significantly enhanced ovarian and uterine wet weights compared with the controls (C and D; n = 8) with corresponding enhancement of CFTR mRNA expression by RT-PCR (E; normalized to that of the internal marker GAPDH) and protein expression by Western blots in PMSG- and hCG-induced OHSS rat uteri and corresponding diestrous stage controls (F; normalized to that of ß-tubulin). ***, P < 0.001; *, P < 0.05 (compared with the control).

 
It is well documented that OHSS patients have high plasma estrogen levels (11, 20). It is possible that hyperstimulation of ovaries leads to high production and release of estrogen, which are responsible for the up-regulation of CFTR observed. We measured estrogen levels in our PMSG-/hCG-induced OHSS rats as well as in the controls at both estrous and diestrous stages of the estrous cycle. A more than 8-fold increase in estrogen levels was found in the OHSS rats compared with that the controls (Fig. 2Go), consistent with the high levels of estrogen previously observed in OHSS animal models (21). We also tested whether elevation of estrogen alone may lead to the development of OHSS symptoms in ovariectomized rats. We found that hyperstimulation did not produce OHSS symptoms in these ovary-removed rats. However, treatment of ovariectomized rats with estrogen (10 µg/kg·d), but not progesterone (25 mg/d), for 5 d induced excessive uterine fluid accumulation and uterine vascularization compared with ovariectomized controls (P < 0.001; Fig. 3AGo) and increased CFTR expression (Fig. 3BGo) similar to the OHSS rat model. It should be noted that although estrogen drastically increased CFTR expression, progesterone completely suppressed it in ovariectomized rats (Fig. 3BGo), which is consistent with previous results obtained in ovary-intact animals showing up-regulation and down-regulation of CFTR by estrogen and progesterone, respectively (16). OHSS symptoms and CFTR up-regulation were also observed in ovary-intact rats treated with high levels of estrogen (100 µg/kg), but not progesterone (75 mg/kg; data not shown). The serum level of exogenously administrated estrogen was similar to the estrogen levels in OHSS rats (data not shown). These results suggest that the ovarian sex hormone, estrogen, but not progesterone, is responsible for the up-regulation of CFTR leading to OHSS. This perhaps explains the clinical observation that OHSS rarely develops in individuals undergoing ovarian hyperstimulation in the absence of high estrogen levels.



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Fig. 2. Comparison of Serum Levels of Estrogen in Normal and OHSS Rats

Serum estrogen concentrations in OHSS and control rats at both estrous and diestrous stages of the estrous cycle were measured by the automated Elecsys Immunoanalyzer (Roche). Serum estrogen levels in OHSS rats were significantly different from the control values. ***, P < 0.001 (n = 7, 7, and 6 for diestrus, estrus, and OHSS, respectively).

 


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Fig. 3. Effects of Ovariectomy and Hormone Treatment on Uterine Wet Weights and CFTR Expression

A, Comparison of rat uterine wet weight 30 d after ovariectomy (control) and after treatment with progesterone or estrogen for 5 d (n =5/group), showing significantly increased uterine wet weights by estrogen. B, Semiquantitative RT-PCR (upper panel) and Western blot (lower panel) results, showing reduced CFTR expression by progesterone and increased expression by estrogen in ovariectomized rats.

 
To confirm that up-regulation of CFTR indeed leads to enhancement of its channel activity, from which excessive epithelial fluid secretion derives, we compared functional CFTR activity in the freshly isolated uterine epithelia of normal and OHSS rats by the short-circuit current (Isc) measurement, a technique for measuring active ion transport across the epithelium (22), in conjunction with the use of a cAMP-evoking agent, forskolin, and specific inhibitors of CFTR. In the presence of an epithelial sodium channel blocker, amiloride, to exclude a possible contribution of Na+ absorption to the current observed, the forskolin-induced Isc from the uterine epithelia of OHSS rats (PMSG plus hCG) was substantially increased compared with that in untreated controls at the diestrous or estrous stage of the uterine cycle (Fig. 4Go), when CFTR is known to be minimally and maximally expressed, respectively. The cAMP-dependent Isc could be blocked by a specific CFTR inhibitor-172 (Calbiochem, La Jolla, CA; Fig. 4Go, A–C), which has been demonstrated to inhibit CFTR-mediated, cholera toxin-induced fluid secretion (23), confirming that the observed cAMP-activated Isc reflects CFTR channel activity. The effect of exogenously administrated estrogen and progesterone for 5 d on uterine CFTR channel activity was also examined, and the Isc measurements showed that forskolin-induced or CFTR-mediated Isc in estrogen-treated uteri was significantly higher than that in progesterone-treated ones (Fig. 5Go). Taken together, it appears that up-regulation of CFTR due to elevated estrogen levels with enhanced CFTR channel activity may be responsible for the excessive fluid transport and accumulation in OHSS.



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Fig. 4. Effect of OHSS on Uterine Epithelial CFTR Channel Activity

A–D, CFTR-mediated uterine anion secretion measured by Isc in the presence of an epithelial Na+ channel blocker, amiloride (10 µM), showing an elevated forskolin (10 µM) response in PMSG-/hCG-induced OHSS uterine epithelia (C) compared with the controls at diestrous (A) and estrous (B) stages of the uterine cycle. The CFTR-mediated Isc was completely blocked by 2 µM CFTR-specific inhibitor (B and C), but not by another channel blocker, diisothiocyanostilbene-2,2'-disulfuric acid (DIDS; 100 µM), in PMSG-/hCG-induced OHSS (C). D, Statistical analyses of forskolin-activated CFTR-mediated Isc response (or CFTR inhibitor-sensitive current), showing much enhanced CFTR activity in OHSS uterine epithelia. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (n =12).

 


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Fig. 5. Effect of Treatment with Ovarian Hormones on the Uterine Epithelial CFTR Channel Activity

Estrogen treatment (10 µg/kg body weight·d) of female rats for 5 d induced CFTR-mediated Isc in the uterine epithelium, but progesterone treatment (25 mg/d·rat) did not (A and B). C, Statistical analyses of forskolin-activated CFTR-mediated Isc response, showing significant CFTR activity in the uteri of estrogen-treated rats. ***, P < 0.001 (n =12).

 
The involvement of CFTR in the pathogenesis of OHSS suggests that it may be possible to alleviate OHSS symptoms by suppressing CFTR expression or inferring with CFTR function. To test this, we first used progesterone, because progesterone had been shown to suppress CFTR expression previously (16) and presently (above). Indeed, progesterone treatment (25 mg/rat·d) of PMSG/hCG-induced OHSS rats significantly reduced uterine, ovarian, and lung wet weights (Fig. 6Go, A–C). Progesterone treatment of OHSS rats also eliminated uterine and peritoneal fluid accumulation and significantly reduced CFTR mRNA and protein expression, as shown in Fig. 6DGo for the uterus. Furthermore, treatment of noncycling PMSG-/hCG-induced OHSS rats (to exclude any cyclic influence) with 300 µl CFTR antiserum (1:100 dilution, vol/vol, in buffer; Zymed Laboratories, San Francisco, CA) by ip injection to neutralize CFTR formation resulted in the elimination of both uterine and peritoneal fluid accumulation and significantly reduced uterine and ovarian wet weights (Fig. 6Go, E and F). The ability to alleviate OHSS symptoms by either down-regulating CFTR expression or interfering with CFTR function not only confirmed a critical role of CFTR in the pathogenesis of OHSS, but also suggested that these treatments may be a preventive measure for OHSS during ART. Interestingly, progesterone has been used in a few clinical situations for luteal support during ART, in which it was found to significantly lower the incidence of OHSS in IVF cycles, although no explanation for such an effect was offered (24, 25, 26).



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Fig. 6. Alleviation of OHSS Symptoms by Progesterone Administration and CFTR Antiserum

A–C, Statistical comparison of ovarian, uterine, and lung wet weight values in PMSG-/hCG-induced OHSS rats (treated with heat-inactivated normal rat serum as OHSS controls) and OHSS rats (treated with progesterone, 25 mg/d·rat; n =6/group). *, P < 0.05. D, Demonstration of suppression of CFTR mRNA and protein expression by progesterone in the OHSS uterus by RT-PCR (upper panel) and western blot (lower panel). Noncycling PMSG-/hCG-induced OHSS rats treated with 300 µl CFTR antiserum (1:100 dilution, vol/vol, in buffer) significantly reduced ovarian (E) and uterine (F) wet weights compared with the heat-inactivated normal rat serum (NRS)-treated controls. *, P < 0.05 (n =5).

 
To confirm the involvement of CFTR in the pathogenesis of OHSS, CFTR-mutant mice [CFTR tm1Unc, with cystic fibrosis (CF) phenotype similar to that produced by {Delta}F508] and their wide-type controls were used, because there is no CF rat model available yet. The results showed that upon hyperstimulation, the wild-type control, but not CF, mice had significantly increased body weights as well as uterine and ovarian wet weights compared with unstimulated mice (Fig. 7Go). Ovarian hyperstimulation also induced uterine edema and fluid accumulation in the uteri and peritoneal cavities of the wild-type, but not CF, mice. The inability to induce OHSS in mice with CFTR mutation confirms the critical role of CFTR in the pathogenesis of OHSS. Because VEGF is currently considered to play a role in the pathogenesis of OHSS, one could also argue that VEGF and its receptors, VEGF-R1 and VEGF-R2, might be somehow down-regulated or absent in CF mice, resulting in the inability to produce OHSS in these animals. However, RT-PCR results did confirm mRNA expression of VEGF, VEGF-R1, and VEGF-R2, in both CF mice and their wild-type controls, and there was no significant difference in their protein levels by Western blot (data not shown). Therefore, VEGF could not have been responsible for the inability to induce OHSS in CF mice. In addition, progesterone treatment reduced OHSS symptoms (above), but did not have a significant effect on VEGF mRNA expression, excluding the possible direct involvement of VEGF in OHSS (data not shown). This is consistent with recent studies showing no correlation between plasma VEGF concentrations and OHSS (5, 6, 7, 8).



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Fig. 7. Inability to Induce OHSS Symptoms in CFTR Mutant Mice

Body (A), ovarian (B), and uterine (C) wet weights, showing insignificant increase in hyperstimulated CF mice (n =4) in contrast to the much increased wet weights in hyperstimulated wild-type controls (n =7). ***, P < 0.001.

 
In this study we have demonstrated that up-regulation of CFTR by either excessive elevation of estrogen or ovarian hyperstimulation leads to the development of OHSS symptoms in rodents. Considering the observed high levels of estrogen, exceeding normal physiological levels, in patients undergoing ovarian hyperstimulation (19, 20) and regulation of CFTR expression by ovarian hormones in humans (12), the present findings in OHSS rodent models are of strong clinical relevance, suggesting the involvement of CFTR in the pathogenesis of OHSS. OHSS appears to be caused by abnormally up-regulated CFTR with increased channel activity leading to excessive fluid shift and accumulation in the peritoneal cavity and in different organs, a condition that could be life threatening. It should be noted that CFTR has also been implicated in the pathogenesis of CF. In contrast to OHSS, however, CF exhibits a hallmark defect in electrolyte and fluid transport in most exocrine glands throughout the body due to defective CFTR function caused by its gene mutations (11). Taken together, the present study confirms a critical role for CFTR in regulating body electrolyte and fluid secretion, an abnormality of which, in either expression or function, could result in lethal conditions, such as seen in both CF and OHSS. However, it should be noted that the CFTR gene has more than 1000 mutations identified to date, most of which exhibit different phenotypes. This may explain why OHSS may not occur or may become severe despite high estrogen levels in some individuals who could have CFTR mutations and defective CFTR function.

CFTR, in addition to being a chloride channel, is known to function as a regulator of a number of other channels, such as aquaporin water channels (channels that allow rapid flow of water across epithelial membranes) and epithelial sodium channels. Enhancement of aquaporin channel activity (27) and suppression of epithelial sodium channel-mediated absorptive activity (28) by CFTR with net increases in electrolyte and fluid secretion may also contribute to the pathogenesis of OHSS secondary to the up-regulation of CFTR expression.

To date, treatment of this extremely morbid and potentially fatal complication of ovarian hyperstimulation remains empirical due to the fact that its pathogenesis is unknown. The present finding of CFTR involvement in the pathogenesis of OHSS and alleviation of OHSS symptoms by suppression of CFTR expression or function may provide grounds for better treatment strategy for patients at risk of developing OHSS and may improve infertility treatment outcome.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Sprague-Dawley (S-D) rats, CFTR mutant mice (CFTR tm1Unc, with CF phenotype similar to that produced by {Delta}F508), and their wild-type controls were used in this study. Animals were kept in the Laboratory Animal Service Center of the Chinese University of Hong Kong before experiments and were fed laboratory chow and water ad libitum. Rats were maintained under controlled conditions of 12 h of light, 12 h of darkness, temperature of 21–23 C, and humidity of 70–80%. Ethics committee approval was obtained before this study, and all animal experiments were conducted in accordance with the university Laboratory Animals Service Center’s guidelines on animal experimentation.

Ovariectomy
Incisions of 1.5–2 cm were made on the dorsal surface of S-D rats, weighing about 250–300 g, observing aseptic techniques, after anesthesia was administered using ip injection of a mixture of 10% ketamine HCl and 2% xylazine HCl (Alfasan, Woerden, Holland; 70 mg/kg). Both oviducts were exposed, one at a time, after careful dissection and bilateral oviductal ligation were performed, and ovaries were removed. The uterine horns were gently put back into the peritoneal cavity. The muscle layers were approximated with absorbable suture, skin incisions were closed using surgical clips, and adequate postoperative care was given. Rats were kept for at least 30 d to recover and adjust before additional experiments.

OHSS Induction
We developed the OHSS rat model as previously reported (21) with slight modification by using HMG (Pergonal, Serono Laboratories, Aubonne, Switzerland), FSH (Puregon, Organon NV, Oss, The Netherlands), or PMSG (Sigma-Aldrich Corp., St. Louis, MO) in conjunction with hCG (Pregnyl, Organon NV) in 40- to 45-d-old CF and wild-type mice, and sexually mature S-D rats. Gonadotropins were administered by ip injections. PMSG (300 IU/kg body weight), HMG (75 IU/rat·d), and FSH (75 IU /rat/d) were injected for 4 d consecutively; on the fifth day, rats received 300 IU hCG. For mice, each received 10 IU gonadotropin for 4 d and 30 IU hCG on the fifth day. Control animals received PBS for the same period of time.

Semiquantitative RT-PCR
Total RNA was obtained from the organs of CF and their wild-type control mice and from ovarian and uterine tissues of S-D rats using TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA). RT-PCR was performed using specific oligonucleotide primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CFTR, VEGF, VEGF-R1, and VEGF-R2. The conditions for PCRs were denaturation at 94 C for 45 sec; annealing at 53, 58, 62, 53, and 59 C for 60 sec; and extension at 72 C for 60 sec with 25 cycles for GAPDH and 30 cycles for CFTR, VEGF, VEGF-R1, and VEGF-R2. Optimal amplification cycles are determined based on the linear relationship between the amount of PCR product detected and the number of amplification cycles. The PCR products were analyzed using 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized in an UV-illuminated imager (Alpha Innotech Corp., San Leandro, CA). The intensities of the bands of CFTR, VEGF, VEGF-R1, and VEGF-R2 were normalized to that of GAPDH, which was amplified simultaneously. Experiments in the absence of reverse transcriptase were conducted as negative control.

Western Blot Analysis
Snap-frozen ovarian and uterine tissues were homogenized with protease inhibitors. The membrane lysates were obtained by centrifugation at 1200 rpm for 30 min at 4 C. After the protein concentration was determined, the membrane lysates containing equal amounts of protein were loaded onto a 10% SDS-PAGE. Blots were incubated with anti-CFTR antibody (Zymed Laboratories) and ß-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA). Enhanced chemiluminescence was visualized by film development. The ODs of the bands were quantified using Meta Morph image analysis software version 6.0 (Universal Imaging Corp., Dowingtown, PA).

Isc Measurement
Isc measurement has previously been described (22). In brief, freshly removed endometrial epithelia from which the serosa and muscular layers had been removed were clamped vertically between two halves of the Ussing chamber. The epithelia were bathed on both sides with Krebs-Henseleit solution that was maintained at 37 C. The Krebs-Henseleit solution had the following composition: 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 24.8 mM NaHCO3, 1.2 mM KH2PO4, and 11.1 mM glucose and was bubbled with 95% O2 and 5% CO2 to maintain the pH at 7.4. Drugs were added directly to the apical or basolateral side of the epithelium. The transepithelial potential differences exhibited by the epithelia were measured by the Ag/AgCl electrodes (World Precision Instruments, Sarasota, FL) connected to a preamplifier, which was connected to a voltage clamp amplifier (DVC 1000, World Precision Instruments). The change in Isc was defined as the maximal rise in Isc after agonist stimulation and was normalized as current change per unit area of the uterine epithelium (microamperes per square centimeter).

Hormonal Measurement
Serum estrogen concentrations were measured in the University Hospital pathology services laboratory using an electrochemiluminescence immunoassay by means of the Elecsys Estradiol 11 kit with the automated Elecsys Immunoanalyzer (Roche, Mannheim, Germany).

Statistical Analysis
Data are presented as the mean ± SEM. Statistical analyses were carried out by ANOVA. Multiple comparisons and differences between groups were analyzed using the Newman-Keuls test, and paired data were analyzed by t test. P ≤ 0.05 (two-tailed) was considered statistically significant. Analyses were carried out with PRISM (GraphPad, Inc., San Diego, CA).


    ACKNOWLEDGMENTS
 
We thank Prof. Nirmal S. Panesar for his help in the hormonal assay.


    FOOTNOTES
 
This work was supported by the Strategic Program of Chinese University of Hong Kong supported this work.

First Published Online July 28, 2005

Abbreviations: ART, Assisted reproduction treatment; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; HMG, human menopausal gonadotropin; IVF, in vitro fertilization; Isc, short-circuit current; OHSS, ovarian hyperstimulation syndrome; PMSG, pregnant mare serum gonadotropin; S-D, Sprague-Dawley; VEGF, vascular endothelial growth factor; VEGF-R, vascular endothelial growth factor receptor.

Received for publication March 7, 2005. Accepted for publication July 20, 2005.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals