1 Department of Obstetrics and Gynecology, Hacettepe University School of Medicine and 2 Department of Pathology, Ankara University School of Medicine, Ankara, Turkey
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Abstract |
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Key words:
vß3 integrin/endometrial receptivity/hydrosalpinx/salpingectomy
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Introduction |
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A direct embryotoxic effect of hydrosalpinx fluid (Mukherjee et al., 1996), physical interference due to the intrauterine accumulation of refluxed fluid (Mansour et al., 1991
), or altered endometrial receptivity (Lessey et al., 1994b
) have been among the proposed mechanisms by which a hydrosalpinx exerts an adverse influence on development and/or implantation of embryos.
Lessey et al. and Tabibzadeh evaluated the expression of integrin molecules in human endometrium throughout the menstrual cycle (Lessey et al., 1992, 1994a
; Tabibzadeh, 1992
). The vitronectin receptor,
vß3, was found to appear abruptly on cycle day 19 or 20, coincident with the opening of the putative window of implantation. The expression of
vß3 was absent in endometrial biopsies with maturational delay. Hence,
vß3 integrin appeared to be a consistent internal marker of luteal phase maturation and receptive endometrium (Lessey et al., 1992
, 1994a
). Lessey et al. also documented the presence of an endometrial dysfunction in patients with hydrosalpinges by means of histological delay and lower integrin expression (Lessey et al., 1994b
).
This prospective study was undertaken to assess whether surgical treatment of communicating hydrosalpinges resulted in a significant change in endometrial maturation and vß3 integrin expression.
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Materials and Methods |
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After all the patients were informed and written consent obtained, they were assigned to laparoscopy scheduled during the putative window of implantation (cycle day 1921). Following laparoscopic confirmation of hydrosalpinges and associated severe tubal disease, salpingectomy and endometrial sampling were performed in 10 patients. These women were subjected to post-treatment endometrial sampling on the corresponding days of the fourth consecutive cycle. The Pipelle device (Laboratoire C.C.D., Paris, France) was used for all biopsies. Transvaginal ultrasounds performed prior to surgery revealed visible hydrosalpinges in 5/10 women.
The endometrial samples were transported on ice and snap frozen in liquid nitrogen to be maintained at 70°C until cryo-sectioning. Serial cryo-sections 5 µm thick were made on poly-L-lysine-coated slides. These slides were then fixed in +4°C acetone for 10 min, and stained using immunofluorescent techniques. One slide for each case was also stained with haemotoxylin and eosin for endometrial histological dating, according to the criteria of Noyes et al. (Noyes et al., 1950).
Immunofluorescent staining
An indirect immunofluorescent technique was employed. The primary antibody, vß3 mouse monoclonal IgG1 (Santa Cruz Biotechnology, sc-7312, California, USA), was placed on cryosections after blocking with 1% bovine serum albumin in phosphate buffered saline (PBS) and incubated at room temperature for 1 h in a dark chamber. A PBS (pH = 7.27.4) rinse was followed by incubation with the secondary antibody (anti-mouse IgG-FITC, Santa Cruz Biotechnology, sc-2010) for 45 min at room temperature in a dark chamber. Subsequently, the samples were washed in PBS and mounted (Immu-mount, Shandon Inc, Pittsburgh, PA, USA). The staining was evaluated under a Nikon microscope by a single blinded observer. Photomicrographs were made using Kodak 400 ASA film.
An intestinal biopsy specimen known to express luminal and glandular epithelial vß3 was used as a positive control slide. As a negative control slide, a normal non-immune serum was used instead of the primary antibody.
Immunostaining was evaluated by the histological score (HSCORE). HSCORE was calculated using the following equation: HSCORE = Pi (i + 1); where i is the intensity of staining with a value of 1, 2 or 3 (weak, moderate or strong respectively), Pi is the percentage of stained epithelial cells for each intensity varying from 0 to 100%, and 1 is a correction for optical density. This yielded a range of results from 0 for no staining to 4 for maximal staining. Luminal and glandular epithelial HSCORE were assessed separately for a detailed evaluation of the endometrium. The HSCORE has been shown to yield low inter- and intra-observer variation (Budwit-Navotny et al., 1986
). Based on a previous receiver operator characteristic curve analysis (Lessey et al., 1994c
), HSCORE >0.7 was used as the threshold of a positive test for the evaluation of epithelial
vß3 immunostaining.
Statistical evaluation
Wilcoxon's signed rank test was used for the comparison of both luminal and glandular endometrial HSCORE of the pre- and post-treatment samples. The pre- and post-treatment HSCORE of ultrasound visible and non-visible hydrosalpinges were separately compared by the MannWhitney U-test. P < 0.05 was considered as statistically significant.
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Results |
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The conventional histological dating of endometrial biopsies was in concordance with the chronologic dating in all cases. Hence, no case of out-of-phase endometrium in either pre- or post-treatment samples was observed.
The immunofluorescent staining for vß3 integrin was observed on both the luminal and glandular epithelium. Diffuse cytoplasmic and membranous staining patterns were noticed. There was no
vß3 expression in the mesenchyme. Occasionally, vascular structures showed linear fluorescence.
The pre-treatment endometrial samples exhibited an average of 25% luminal epithelial vß3 staining with a median staining intensity of 1. The mean HSCORE for the luminal epithelium was 0.8. Eight out of 10 cases revealed an HSCORE <0.7, which was below the accepted cut-off for positive staining. The post-treatment samples revealed an average of 75% luminal epithelial immunostaining with a median staining intensity of 2. The corresponding mean HSCORE was 2.1. Nine out of 10 cases resulted in an improvement of HSCORE (P = 0.017) (Figure 1
). Finally, all of the post-treatment biopsies yielded a luminal epithelial HSCORE >0.7. Figure 2A
illustrates weak luminal epithelial
vß3 immunostaining prior to salpingectomy, and Figure 2B
shows increased staining after the treatment.
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All post-treatment endometrial biopsies of women with ultrasound visible hydrosalpinges (n = 5) resulted in an improved HSCORE. All but one post-treatment endometrial biopsy in ultrasound non-visible subjects (n = 5) also revealed a higher HSCORE. The post-treatment HSCORE for each group did not significantly differ from one another.
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Discussion |
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Following salpingectomy, endometrial luminal epithelial vß3 expression was significantly increased. Although an improvement was observed in the post-treatment glandular integrin expression, it did not reach statistical significance. The limited number of cases and a slower glandular response, which was not completed after a 3-cycle interval, might be possible explanations.
We decided to perform our post-treatment endometrial biopsies somewhat arbitrarily, in the fourth sequential cycle. This policy was employed according to some conventional knowledge. The normalization of endometrium has been accepted to take place following three cycles of continued treatment in certain disorders, such as dysfunctional uterine bleeding (Speroff, 1999). Likewise, a repeat biopsy has been recommended following 3 months of medical therapy in cases of endometrial hyperplasia (DiSaia, 1997). Ideally, the progressive change would have been shown by sequential monthly biopsies, but this was not possible due to ethical concerns. Hence, the timing of optimal improvement in endometrial receptivity markers following salpingectomy remains undetermined.
One of the challenges in the management of the hydrosalpinx is to determine who would benefit from salpingectomy. Vasquez et al. suggested salpingectomy in thick-walled hydrosalpinges with wall fibrosis and hydrosalpinges with mucosal adhesions (Vasquez et al., 1995). Puttemans et al. supported the use of tubal endoscopy in the decision-making process (Puttemans et al., 2000
). According to several authors, only the ultrasound visible hydrosalpinges would be responsible for the impaired implantation and pregnancy rates (Andersen et al., 1994
; de Witt et al., 1998). The findings of some studies (Meyer et al., 1997
), including this one, linked the possibly impaired endometrial receptivity by means of reduced expression of the
vß3 integrin to the presence of hydrosalpinx. Therefore, the evaluation of endometrial
vß3 integrin expression for the selection of salpingectomy candidates might be a promising alternative.
Since the transvaginal ultrasound was routinely employed in the pre-treatment evaluation of our patients, we were able to compare the influence of ultrasound visible and non-visible hydrosalpinges on endometrial integrin expression. Neither pre-treatment nor post-treatment integrin expression was different among cases with and without ultrasound visible hydrosalpinges. However, due to the limited number of cases in each group, the power is inadequate to reach strong conclusions as to whether ultrasound visibility of hydrosalpinx affects endometrial receptivity or not.
Ultrasound evaluation of hydrosalpinges has several potential limitations. A false negative interpretation is possible due to intermittent intrauterine drainage and on the other hand, an unrecognized hydrosalpinx may be stimulated during ovulation induction (Schiller and Tsuchiyama, 1995). Sonographic evaluation may not provide substantial information regarding endometrial receptivity and a possible toxicity of hydrosalpinx fluid, the latter of which is in fact a dilemma.
Mukherjee et al. reported embryotoxic effects of hydrosalpinx fluid on murine embryogenesis and suggested prophylactic salpingectomy (Mukherjee et al. 1996). However, several subsequent studies in mouse models failed to show obvious embryotoxicity (Beyler et al., 1997
; Murray et al., 1997
; Koong et al., 1998
; Spandorfer et al., 1999
). In addition, hydrosalpinx fluid was not found to exertjyany major negative effects on in-vitro development of human embryos (Granot et al., 1998
; Strandell et al., 1998
).
We conclude that the surgical treatment of communicating hydrosalpinges improves endometrial receptivity as assessed by immunofluorescent vß3 integrin staining. The initial impaired integrin expression and its improvement following salpingectomy is independent of the conventional histological dating of the endometrium. Therefore, women with communicating hydrosalpinges may undergo endometrial evaluation with the help of molecular markers of implantation, such as
vß3 integrin. This evaluation may be both decisive in determining the optimal management of cases, and used to assess the efficacy of the treatment. Finally, the expression of implantation markers should be correlated with implantation and clinical pregnancy rates in IVFembryo transfer programs.
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Notes |
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References |
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Submitted on April 24, 2001; accepted on July 20, 2001.