Salpingectomy increases peri-implantation endometrial HOXA10 expression in women with hydrosalpinx

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1 Salpingectomy increases peri-implantation endometrial HOXA10 expression in women with hydrosalpinx Gaurang S. Daftary, M.D., Umit Kayisli, Ph.D., Emre Seli, M.D., Orhan Bukulmez, M.D., Aydin Arici, M.D., and Hugh S. Taylor, M.D. Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut Objective: To determine whether women with hydrosalpinx would have diminished endometrial HOXA10 expression and whether salpingectomy would reverse HOXA10 suppression. The homeobox gene HOXA10 is a transcription factor that is necessary for embryo implantation; its expression in human endometrium correlates with receptivity and implantation. Increased endometrial HOXA10 expression may be one mechanism by which salpingectomy results in increased implantation rates in IVF. Design: Prospective clinical trial. Setting: Academic medical center. Patient(s): Women with unilateral or bilateral hydrosalpinx. Intervention(s): Expression of HOXA10 was examined prospectively during the midluteal phase in endometrium obtained from infertile women (n 9) with hydrosalpinges before and after salpingectomy, as well as from fertile controls (n 6). Quantitative HOXA10 mrna expression was determined by real-time reverse-transcription polymerase chain reaction, and HOXA10 protein expression was determined by immunohistochemistry. Main Outcome Measure(s): Expression of HOXA10 mrna and protein. Result(s): Expression of HOXA10 mrna was significantly lower in infertile women with hydrosalpinges, compared with the case of fertile controls. Salpingectomy resulted in a statistically significant, 15-fold increase in endometrial HOXA10 expression. Immunohistochemical analysis confirmed the quantitative real-time reversetranscription polymerase chain reaction findings. Increased HOXA10 expression was evident in both glandular epithelial cells and endometrial stroma. Conclusion(s): HOXA10 is necessary for implantation. Here, we demonstrate decreased HOXA10 expression in response to hydrosalpinx fluid as a potential molecular mechanism for diminished implantation rates. Salpingectomy restores endometrial HOXA10 expression. This may be one mechanism by which salpingectomy results in augmented implantation rates in IVF. (Fertil Steril 2007;87: by American Society for Reproductive Medicine.) Key Words: HOXA10, hydrosalpinx, endometrium, implantation, IVF A hydrosalpinx is a distally blocked, dilated, fluid-filled fallopian tube that arises as a sequela of salpingitis, typically after pelvic inflammatory disease. Hydrosalpinges are known to be associated with diminished implantation and pregnancy rates and an increased spontaneous abortion rate in IVF cycles (1). Recent meta-analyses revealed that not only was there a 50% reduction in both implantation and pregnancy rates, the spontaneous abortion rate was doubled in the presence of a hydrosalpinx compared with the case of women with non-hydrosalpinx tubal-factor infertility (2, 3). Further, improved pregnancy and delivery rates have been described with laparoscopic salpingectomy for hydrosalpinges before IVF (4, 5). Received March 15, 2006; revised and accepted June 21, Supported by grants HD36887 and ES10610 from the National Institutes of Health (Bethesda, MD). Reprint requests: Hugh S. Taylor, M.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, P.O. Box , New Haven, Connecticut (FAX: ; hugh.taylor@yale.edu). Although the infundibular end of the tube may be partially or completely occluded, it generally is believed that the uterine ostium of the fallopian tube remains patent, exposing the uterine cavity to hydrosalpinx fluid. Hydrosalpinges increase in size with ovulation induction, and hydrosalpinx fluid accumulates in the uterine lumen (6 8). The adverse impact of exposure to hydrosalpinx fluid may affect both the endometrium and the implanting embryo. Microorganisms, debris, leukocytes, cytokines, prostaglandins, and leukotrienes in hydrosalpinx fluid all could affect implantation (9). In addition, hydrosalpinx fluid is comprised of lower calcium, phosphate, glucose, and total protein and is hypoosmolar compared with serum (10). It is likely that the altered concentrations of physiological constituents are less nutritive, whereas the pathological constituents exert deleterious and possibly toxic effects in the uterine environment at the time of embryo implantation. Mouse embryos cultured in hydrosalpinx fluid exhibit delayed and arrested maturation with a high rate of degeneration, compared with those cultured in standard culture media; however, hydrosalpinx fluid /07/$32.00 Fertility and Sterility Vol. 87, No. 2, February 2007 doi: /j.fertnstert Copyright 2007 American Society for Reproductive Medicine, Published by Elsevier Inc. 367

2 has not demonstrated a toxic effect on human embryo development (11 13). Aside from embryo toxicity and simple mechanical flushing of embryos from the uterine cavity, the presence of hydrosalpinx also may reduce endometrial receptivity by diminishing the expression of such molecules as HOXA10, V 3 integrin, and LIF (8, 14 18). HOXA10 is a transcription factor that is necessary for embryo implantation. Mice with a targeted mutation of the Hoxa10 locus are infertile as a result of failure of implantation (19). Although embryos obtained from these Hoxa10 ( / ) mice develop normally when transferred to the uterus of a wild-type surrogate mouse, embryos from wild-type mice fail to implant in the uteri of Hoxa10 ( / ) mice. Altering the endometrial expression levels of HOXA10 in murine endometrium results in a corresponding alteration of implantation rates and, therefore, of litter size (20). HOXA10 is expressed in human endometrium during the menstrual cycle, in which its expression is regulated by estrogen and progesterone (21). Endometrial epithelial and stromal HOXA10 expression levels are up-regulated in the midluteal phase, coincident with the time of implantation. We have shown elsewhere that increasing concentrations of hydrosalpinx fluid suppresses HOXA10 expression in vitro in endometrial cells in a dose-related manner (15). In this study, we evaluate the relationship between hydrosalpinx fluid and endometrial HOXA10 expression in vivo in women, as well as investigate the effect of salpingectomy on endometrial HOXA10 expression. MATERIALS AND METHODS Tissue Collection Recruitment was performed over a 2-year period; infertile women with unilateral or bilateral hydrosalpinges confirmed by hysterosalpingography were selected for evaluation. On hysterosalpingography, a hydrosalpinx was diagnosed by the presence of ampullary filling and distension in the absence of spillage of dye from the fimbrial end of the fallopian tube. All subjects were younger than 40 years of age and experienced regular menstrual cycles, with no evidence of ovulatory dysfunction, indicated by midluteal P levels of 10 ng/ml. In addition, male factor was ruled out by a normal semen analysis according to standard World Health Organization criteria (22). Tissue collection was performed at the Hacettepe University School of Medicine (Ankara, Turkey), at which the study had received prior approval of the institutional review board. Approval for subsequent sample analysis also was obtained from the institutional review board of the Yale University School of Medicine. The control group included six age-matched women with proven fertility who were undergoing sterilization by bilateral tubal ligation. These women were eumenorrheic, with no known medical problems, and had had at least one successful pregnancy (beyond 20 wk) in the past. None of these patients were using any medications. All included subjects were adequately counseled, and written consent was obtained. Endometrial samples were obtained if laparoscopy was scheduled during the midluteal phase, coincident with the putative window of implantation (cycle days 19 21) in eumenorrheic women. The day of the menstrual cycle was established from the subjects menstrual history. Subjects with hydrosalpinx underwent salpingectomies to remove the affected fallopian tube. These women underwent follow-up endometrial sampling on the corresponding menstrual cycle days of the fourth posttreatment cycle. The Pipelle device (Laboratoire CCD, Paris, France) was used for all biopsies. Real-Time Polymerase Chain Reaction Levels of HOXA10 mrna were evaluated by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and were normalized to -actin with the Roche LightCycler (Nutley, NJ). The reaction was performed by using the LightCycler RNA Master SYBR Green I kit. Reaction conditions included 1.0 g ofrna,2mmor3 mm Mn[OAc] 2 for HOXA10 and -actin, respectively, 150 nm of each primer, and 1 RNA Master SYBR Green, for a final reaction volume of 20 L. Primer sequences for each gene were as follows. HOXA10: 5=-AGGTGGACGCT- GCGGCTAATCTCTA-3= and 5=-GCCCCTTCCGAGAG- CAGCAAAG-3=. -Actin: 5=-CGTACCACTGGCATCGT- GAT-3= and 5=-GTGTTGGCGTACAGGTCTTTG-3=. Reverse transcription was performed for 30 minutes at 61 C, followed by initial denaturation at 95 C for 30 seconds and 45 cycles including denaturation at 95 C for 2 seconds; annealing at 65 C (HOXA10), 62 C ( -actin), or 58 C (EP4) for 5 seconds; and elongation at 72 C for 14 seconds. A melting curve was created after the amplification to observe the specificity of the primers or number of products amplified. A standard curve was constructed, and all HOXA10 values were normalized to -actin. Immunohistochemistry The endometrial samples were transported on ice, embedded in OCT (Tissue Tek, Torrance, CA), snap-frozen in liquid nitrogen, and kept at 80 C until use. Serial cryosections (thickness 5 m) were placed on poly-l-lysine coated microscope slides and fixed at 4 C in formaldehyde for 10 minutes. After a 5-minute rinse in distilled water, an antigenpresenting step was performed by steaming the slides in 0.01 M sodium citrate buffer for 20 minutes, followed by removal of the staining jar from the steam chamber and cooling for 20 minutes. Slides were rinsed for 5 minutes in phosphatebuffered saline with 0.1% Tween-20 (PBST), and sections were circumscribed with a hydrophobic pen. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 5 minutes, followed by a 5-minute PBST wash. Nonspecific binding was blocked with 1.5% normal horse serum in PBST for 1 hour at room temperature. Slides were incubated in goat polyclonal HOXA10 (sc-17159; Santa Cruz Biotech, Santa Cruz, CA) at 1:3,000 overnight at 4 C. Normal goat IgG (Santa Cruz) was used as a negative control. Slides were 368 Daftary et al. Salpingectomy increases HOXA10 expression Vol. 87, No. 2, February 2007

3 washed in 1 PBST. Biotinylated horse anti-goat (Vector Laboratories) was applied at 3.5 g/ml for 1 hour at 4 C. Slides were washed in 1 PBST, incubated in ABC Elite(Vector) for 15 minutes, washed in 1 PBST, and incubated for 5 minutes in 3,3-diaminobenzidine (DAB) (Vector). A 20-second exposure to hematoxylin was used as a counterstain. Slides were rehydrated through 3-minute ethanol and xylene washes and were mounted with Permount. A semiquantitative method incorporating both the number of staining cells and the intracellular staining intensity, the H-score, was used to analyze HOXA10 immunoreactivity (23, 24). Briefly, the staining intensity was classified as no staining (0), weak but detectable (1), distinct (2), or very strong (3). The H-score was calculated as follows: H-score Pi(i 1), where Pi is the percentage of stained cells in each intensity category, and i is the intensity score (0, 1, 2, or 3). The endometrial glandular epithelium and endometrial stroma were analyzed individually for the percentage of cells in each staining category to obtain an aggregate H-score for each tissue. FIGURE 1 Effect of salpingectomy on endometrial HOXA10 mrna expression in presalpingectomy (Pre), postsalpingectomy (Post) samples, and fertilecontrol (Ctl) endometrial samples. Real-time quantitative RT-PCR was used to determine levels of endometrial HOXA10 mrna expression. *P.049, Pre vs. Post. **P.011, Pre vs. Ctl. Statistical Analysis Because the data from immunohistochemistry and quantitative real-time RT-PCR were not normally distributed, comparisons of samples were analyzed with the nonparametric Wilcoxon signed rank test. Statistical calculations were performed by using SigmaStat for Windows, version 3.0 (Jandel Scientific, San Rafael, CA). Statistical significance was defined as P.05. RESULTS In this study, the mean ( SD) age of all subjects with hydrosalpinx was years, and that of fertile controls was years. Both groups were matched with respect to age. The study subjects had an average duration of infertility of years and had a mean day 3 FSH level of miu/ml. Five of 9 patients had bilateral hydrosalpinges. All salpingectomy specimens were confirmed on histology to have chronic salpingitis. To ensure comparability, histological dating of endometrial biopsies was performed. No out-of-phase endometrium was observed in either group. We have shown elsewhere that hydrosalpinx fluid diminishes HOXA10 expression in vitro in endometrial cells in a dose-dependent manner (15). To determine whether this effect also is seen in the presence of hydrosalpinx in vivo, quantitative real-time RT-PCR was used to determine HOXA10 mrna levels from endometrial samples obtained during the mid-luteal phase. Endometrial HOXA10 mrna expression levels obtained from infertile patients with hydrosalpinges (n 9) were compared with those obtained from age-matched fertile controls (n 6). Endometrial HOXA10 expression during the window of implantation was Daftary. Salpingectomy increases HOXA10 expression. Fertil Steril significantly lower in women with hydrosalpinges compared with fertile controls (P.011; Fig. 1). Further, presalpingectomy endometrial HOXA10 mrna expression tended to be higher in samples obtained from patients with unilateral compared with bilateral hydrosalpinx. To evaluate the effect of salpingectomy on endometrial HOXA10 expression in subjects with hydrosalpinx, preoperative and postoperative endometrial HOXA10 mrna expression levels on each subject were analyzed by quantitative real-time RT-PCR (Fig. 1). An increase in endometrial HOXA10 expression was seen in seven of the nine postsalpingectomy endometrial samples (Fig. 1, Post bar) compared with corresponding presalpingectomy endometrial samples (Fig. 1, Pre bar). HOXA10 levels were increased by 15-fold after salpingectomy after normalization with -actin (P.049; Fig. 1). In addition, no difference in HOXA10 mrna expression levels was observed when the postsalpingectomy values were compared with those of age-matched fertile controls, indicating that salpingectomy restores HOXA10 expression to physiological levels (Fig. 1). To determine the localization of HOXA10 expression, endometrial tissues sampled before and after salpingectomy were evaluated by immunohistochemistry. HOXA10 was expressed in stromal, luminal, and glandular epithelial cells (Fig. 2). Expression was maximal in stromal cells, in keeping with previously described patterns of endometrial HOXA10 expression (25). All specimens were evaluated by H-score to quantify observed HOXA10 expression pattern. The Fertility and Sterility 369

4 FIGURE 2 Immunolocalization of endometrial HOXA10 from infertile women with hydrosalpinges before (A) and after (B) salpingectomy. Representative ( 40) photomicrographs show the results of immunohistochemistry performed with a HOXA10 monoclonal antibody. Expression of HOXA10 was observed in both the endometrial epithelium and stroma, with predominant stromal expression. Daftary. Salpingectomy increases HOXA10 expression. Fertil Steril mean ( SEM) H-Score for HOXA10 staining was significantly increased, from to in endometrial stroma and from to in endometrial epithelium (both P.05) in postsalpingectomy endometrial samples compared with in presalpingectomy endometrial samples, indicating increased HOXA10 expression (Table 1). DISCUSSION Hydrosalpinges are associated with adverse reproductive outcomes in IVF, as a result of lower implantation and pregnancy rates and an increased rate of spontaneous abortions (1 3). Multiple studies, including a recent meta-analysis, have shown the overall benefit of salpingectomy before IVF in improving reproductive outcome; salpingectomy has been recommended as a therapeutic option for patients with hydrosalpinx (4, 5, 26 28). In this study, we have demonstrated that in women with hydrosalpinx, the expression of the endometrial receptivity marker HOXA10 is significantly lower during the midluteal phase than in matched fertile controls. In addition, in this study, we demonstrate that endometrial expression of HOXA10 is increased at the time of embryo implantation in patients who have undergone salpingectomy for hydrosalpinx, suggesting an improvement in endometrial receptivity. These findings support previous reports that suggest a detrimental effects of hydrosalpinx on implantation and the benefit of its surgical removal on endometrial receptivity. One mechanism by which hydrosalpinx fluid lowers implantation and pregnancy rates and increases abortion rates likely relates to leakage of fluid into the uterine cavity. Larger and bilateral hydrosalpinges impair fertility to a greater extent than do smaller and unilateral hydrosalpinges (29, 30). We have shown elsewhere (15) that increasing concentrations of hydrosalpinx fluid cause a corresponding, dose-responsive decrease in HOXA10 mrna expression. It is likely that the larger volume of fluid and increased concentration of toxic substances from larger or bilateral hydrosalpinges contribute further to the deleterious effect of hydrosalpinges on HOXA10 expression. Decreased endometrial HOXA10 expression has been shown to be associated with diminished implantation (19, 20). Not surprisingly, diminished HOXA10 is a common underlying defect that also is seen in other clinical conditions that are associated with low implantation rates, such as endometriosis and polycystic ovary syndrome (31, 32). One mechanism by which HOXA10 has a role in endometrial receptivity is through regulation of the cell-surface marker 3 integrin. 3 Integrin is a component of the vitronectin receptor that is expressed by endometrial epithelial cells during the window of receptivity to embryo implantation. 3 Integrin and its ligand osteopontin have been proposed to mediate initial embryo endometrial interactions. Defective endometrial expression of 3 integrin also has been demonstrated elsewhere in patients with hydrosalpinx (16). HOXA10 has been shown to directly up-regulate 3 integrin in human endometrium and thereby participates at the embryo maternal interphase during implantation (18). It will be interesting to assess the endometrial expression patterns of other markers of embryo TABLE 1 H-Score for HOXA10 expression. Cell type Before salpingectomy After salpingectomy Glands Stroma Note: For both cell types, P.05, comparing scores before and after salpingectomy. Daftary. Salpingectomy increases HOXA10 expression. Fertil Steril Daftary et al. Salpingectomy increases HOXA10 expression Vol. 87, No. 2, February 2007

5 receptivity in patients with hydrosalpinx as well as after salpingectomy. HOXA10 is expressed in both endometrial epithelial and stromal cells, where it likely subserves the different physiological functions of the sex steroids (33). In the mid luteal and late luteal phases, epithelial HOXA10 expression is sustained at high levels despite the decline in P-receptor concentrations. Endometrial epithelial HOXA10 expression is driven by both stromal paracrine factors and also HOXA10 auto-regulation; HOXA10 also regulates its own expression in endometrial epithelium (34, 35). In addition to demonstrating the deleterious effect of hydrosalpinx fluid on endometrial epithelial cells in vitro, in this study we observed that in vivo, both endometrial epithelial and stromal HOXA10 expression is diminished in patients with hydrosalpinx, and these defects are reversed with salpingectomy. Because salpingectomy restores both epithelial and stromal HOXA10 expression, hydrosalpinx fluid must affect both of these molecular pathways in both cell types. Although salpingectomy before IVF has been demonstrated to improve implantation rates, this approach entails the performance of a surgical procedure, with consequent risks and expenses (4, 5). It may be possible in the future to correct molecular defects in the endometrium, such as those resulting from exposure to hydrosalpinx fluid, by administration of specific gene therapy rather than surgery. In the case of HOXA10, murine litter size easily can be altered by increasing the local concentration of HOXA10 in the endometrium through administration of the HOXA10-expressing constructs (20). Recently, it has been shown that gene transfection of the human uterus can be accomplished easily and efficiently, and we speculate that in a subset of patients with hydrosalpinges, targeted intrauterine gene therapy with HOXA10 may be an additional therapeutic option besides salpingectomy (24). Further studies will be needed to clarify the relative importance of endometrial HOXA10 expression in assisted reproductive technology outcome and to determine whether HOXA10 effectively may be used to improve pregnancy rates in women undergoing IVF ET. REFERENCES 1. Strandell A, Waldenstrom U, Nilsson L, Hamberger L. Hydrosalpinx reduces in-vitro fertilization/embryo transfer pregnancy rates. Hum Reprod 1994;9: Zeyneloglu HB, Arici A, Olive DL. Adverse effects of hydrosalpinx on pregnancy rates after in vitro fertilization-embryo transfer. Fertil Steril 1998;70: Camus E, Poncelet C, Goffinet F, Wainer B, Merlet F, Nisand I, et al. Pregnancy rates after in-vitro fertilization in cases of tubal infertility with and without hydrosalpinx: a meta-analysis of published comparative studies. Hum Reprod 1999;14: Strandell A, Lindhard A, Waldenstrom U, Thorburn J. Hydrosalpinx and IVF outcome: cumulative results after salpingectomy in a randomized controlled trial. Hum Reprod 2001;16: Johnson NP, Mak W, Sowter MC. Laparoscopic salpingectomy for women with hydrosalpinges enhances the success of IVF: a Cochrane review. Hum Reprod 2002;17: Hill GA, Herbert CM, Fleischer AC, Webster BW, Maxson WS, Wentz AC. Enlargement of hydrosalpinges during ovarian stimulation protocols for in vitro fertilization and embryo replacement. Fertil Steril 1986;45: Schiller VL, Tsuchiyama K. Development of hydrosalpinx during ovulation induction. J Ultrasound Med 1995;14: Mansour RT, Aboulghar MA, Serour GI, Riad R. Fluid accumulation of the uterine cavity before embryo transfer: a possible hindrance for implantation. J In Vitro Fert Embryo Transfer 1991;8: Nackley AC, Muasher SJ. The significance of hydrosalpinx in in vitro fertilization. Fertil Steril 1998;69: Beyler SA, James KP, Fritz MA, Meyer WR. Hydrosalpingeal fluid inhibits in-vitro embryonic development in a murine model. Hum Reprod 1997;12: Mukherjee T, Copperman AB, McCaffrey C, Cook CA, Bustillo M, Obasaju MF. Hydrosalpinx fluid has embryotoxic effects on murine embryogenesis: a case for prophylactic salpingectomy. Fertil Steril 1996;66: Strandell A, Sjogren A, Bentin-Ley U, Thorburn J, Hamberger L, Brannstrom M. Hydrosalpinx fluid does not adversely affect the normal development of human embryos and implantation in vitro. Hum Reprod 1998;13: Granot I, Dekel N, Segal I, Fieldust S, Shoham Z, Barash A. Is hydrosalpinx fluid cytotoxic? Hum Reprod 1998;13: Bloechle M, Schreiner T, Lisse K. Recurrence of hydrosalpinges after transvaginal aspiration of tubal fluid in an IVF cycle with development of a serometra. Hum Reprod 1997;12: Daftary GS, Taylor HS. Hydrosalpinx fluid diminishes endometrial cell HOXA10 expression. Fertil Steril 2002;78: Meyer WR, Lessey BA, Castelbaum AJ. Hydrosalpinx and altered uterine receptivity. Fertil Steril 1997;68: Seli E, Kayisli UA, Cakmak H, Bukulmez O, Bildirici I, Guzeloglu- Kayisli O, et al. Removal of hydrosalpinges increases endometrial leukaemia inhibitory factor (LIF) expression at the time of the implantation window. Hum Reprod 2005;20: Daftary GS, Troy PJ, Bagot CN, Young SL, Taylor HS. Direct regulation of beta3-integrin subunit gene expression by HOXA10 in endometrial cells. Mol Endocrinol 2002;16: Satokata I, Benson G, Maas R. Sexually dimorphic sterility phenotypes in Hoxa10-deficient mice. Nature 1995;374: Bagot CN, Troy PJ, Taylor HS. Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation. Gene Ther 2000;7: Taylor HS, Arici A, Olive D, Igarashi P. HOXA10 is expressed in response to sex steroids at the time of implantation in the human endometrium. J Clin Invest 1998;101: World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. New York: Cambridge University Press, Budwit-Novotny DA, McCarty KS, Cox EB, Soper JT, Mutch DG, Creasman WT, et al. Immunohistochemical analyses of estrogen receptor in endometrial adenocarcinoma using a monoclonal antibody. Cancer Res 1986;46: Daftary GS, Taylor HS. Efficient liposome-mediated gene transfection and expression in the intact human uterus. Hum Gene Ther 2001;12: Sarno JL, Kliman H, Taylor HS. HOXA10, Pbx2, and Meis1 protein expression in the human endometrium: formation of multimeric complexes on HOXA10 target genes. J Clin Endocrinol Metab 2005;90: Garcia JE, Jones HW Jr, Acosta AA, Andrews MC. Reconstructive pelvic operations for in vitro fertilization. Am J Obstet Gynecol 1985; 153: Murray DL, Sagoskin AW, Widra EA, Levy MJ. The adverse effect of hydrosalpinges on in vitro fertilization pregnancy rates and the benefit of surgical correction. Fertil Steril 1998;69:41 5. Fertility and Sterility 371

6 28. Kassabji M, Sims JA, Butler L, Muasher SJ. Reduced pregnancy outcome in patients with unilateral or bilateral hydrosalpinx after in vitro fertilization. Eur J Obstet Gynecol Reprod Biol 1994;56: de Wit W, Gowrising CJ, Kuik DJ, Lens JW, Schats R. Only hydrosalpinges visible on ultrasound are associated with reduced implantation and pregnancy rates after in-vitro fertilization. Hum Reprod 1998; 13: Wainer R, Camus E, Camier B, Martin C, Vasseur C, Merlet F. Does hydrosalpinx reduce the pregnancy rate after in vitro fertilization? Fertil Steril 1997;68: Cermik D, Selam B, Taylor HS. Regulation of HOXA-10 expression by testosterone in vitro and in the endometrium of patients with polycystic ovary syndrome. J Clin Endocrinol Metab 2003;88: Taylor HS, Bagot C, Kardana A, Olive D, Arici A. HOX gene expression is altered in the endometrium of women with endometriosis. Hum Reprod 1999;14: Daftary GS, Taylor HS. Pleiotrophic effects of HOXA10 on the functional development of the peri-implantation endometrium. Mol Reprod Dev 2004;67: Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL, McCarty KS Jr. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metab 1988;67: Kelly M, Daftary GS, Taylor HS. An autoregulatory element maintains HOXA10 expression in endometrial epithelial cells. Am J Obstet Gynecol 2006;194: Daftary et al. Salpingectomy increases HOXA10 expression Vol. 87, No. 2, February 2007