Ultrastructural characterization of whole hydrosalpinx from infertile Chinese women

Cell Biology International 29 (2005) 849e856 www.elsevier.com/locate/cellbi

Ultrastructural characterization of whole hydrosalpinx from infertile Chinese women Louis Chukwuemeka Ajonuma a, Ernest Hung Yu Ng b, Lin Nga Chan a, Pak Ham Chow c, Lai Sin Kung c, Annie Nga Yin Cheung d, Lok Sze Ho a, Christine Briton-Jones e, Ingrid H. Lok e, Christopher J. Haines e, Hsiao Chang Chan a,* a

Department of Physiology, Faculty of Medicine, Epithelial Cell Biology Research Center, The Chinese University of Hong Kong, Hong Kong b Department of Obstetrics and Gynecology, Faculty of Medicine, University of Hong Kong, Hong Kong c Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong d Department of Pathology, Faculty of Medicine, University of Hong Kong, Hong Kong e Department of Obstetrics and Gynecology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Received 9 November 2004; revised 3 March 2005; accepted 30 May 2005

Abstract Hydrosalpinx (HSP) has been shown to be detrimental to the outcome of assisted reproduction, but little is known of its pathology. This prospective study examined and detailed ultrastructural characterization of HSP of infertile women presenting for assisted reproductive treatments. Both light and electron microscopies were used to characterize HSP. Hematoxylin and eosin staining of HSP showed areas without epithelial cell lining or with abnormalities such as flattening of the epithelial layer and exfoliation of epithelial cells with occasional normal columnar epithelial lining. HSP muscle fibers were atrophic and occasionally replaced by fibrous tissues, or separated by areas of severe edema. Inflammatory cells could be found in hydrosalpinx fluid (HF) in the lumen in areas with flattened to no epithelial cells, without epithelial lining, as well as in dilated blood vessels and/or lymph vessels. Scanning electron microscopy of the epithelial surface revealed epithelial denudation-severe loss of both cilia and microvilli and stomata exuding globular bodies on eroded ampulla surfaces. Severe chronic inflammation and damage to the epithelial lining and musculature of Fallopian tubes and the presence of inflammatory cells provides an explanation for HF formation, and thus for the detrimental effects of HF on reproductive processes and IVF outcome. Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Epithelial damage; Hydrosalpinx; Hydrosalpinx fluid; Infertility; Chronic inflammation

1. Introduction The fluid environment of the Fallopian tube, where sperm transport and capacitation, fertilization, early embryo development and embryo transport take place, is of immense importance in reproduction. Abnormal * Corresponding author. Tel.: C852 2609 6839; fax: C852 2603 5022. E-mail address: [email protected] (H.C. Chan).

volume and/or composition, such as in hydrosalpinx (HSP), are among the factors responsible for reduced fertility in IVF patients. The presence of HSP during in vitro fertilization and embryo transfer (IVF/ET) is associated with decreased implantation, pregnancy and increased rate of early pregnancy loss (Aboulghar et al., 1998; Zeyneloglu et al., 1998; Camus et al., 1999). Although toxic effects of hydrosalpinx fluid (HF) on mouse embryo development (Mukherjee et al., 1996; Beyler et al., 1997; Murray et al., 1997; Rawe et al., 1997;

1065-6995/$ - see front matter Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2005.05.012

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Sachdev et al., 1997; Koong et al., 1998; Spandorfer et al., 1999; Roberts et al., 1999; Carrasco et al., 2001; de Vantery Arrighi et al., 2001) and human spermatozoa in vitro (Ng et al., 2000) have been documented, the mechanisms underlying the adverse effects are far from understood (Ajonuma et al., 2002). Puttemans and Brosens (1996) and Vasquez et al. (1995) proposed three grades of HSP based on the appearance of salpingoscopy. HSP was clinically classified according to the extent of occlusion, ampullary dilatation and pertubal adhesions, and not on tubal morphology and mucosal appearance (Donnez and Casanas-Roux, 1986; Mage et al., 1986). There is paucity of information on the pathology of HSP. More than thee decades ago, David et al. (1969) reported foci of apparently normal ciliated and nonciliated epithelium, numerous cuboidal and flat epithelial cells, thin dilated blood vessels and capillaries in lamina propria of HSP. Other studies (Vasquez et al., 1981; Patton et al., 1984; Otubu et al., 1987; Li et al., 1996) looked at the ultrastructure of experimentally induced HSP in rodents and microbiopsy tissues from human Fallopian tube epithelium only (Vasquez et al., 1983; Donnez et al., 1984; Lescoat et al., 1995) and HSP simplex (Patek and Nilsson, 1977). The aim of the present study was to describe the pathology of whole HSP and its luminal epithelium from infertile patients presenting for assisted reproduction treatments, using both light and electron microscopy, comparing them with normal Fallopian tubes.

2. Materials and methods 2.1. Experimental design Women were recruited with ethical approval from both the University of Hong Kong and The Chinese University of Hong Kong. All patients gave written informed consent prior to their participation in this study. HSP (n Z 9) were obtained from patients undergoing laparoscopic bilateral salpingectomy. They were visible on transvaginal scanning (de Wit et al., 1998) during IVF cycles of infertile women who had tubal factor infertility only. Normal Fallopian tubes (n Z 6) were obtained from healthy women who agreed to have laparoscopic bilateral salpingectomy for tubal sterilization. The healthy women that donated their Fallopian tubes as controls were pre-menopausal and salpingectomy was performed during the follicular phase (proliferative stage) between days 7e12 of their uterine cycles. Immediately after surgical removal, HSP and normal Fallopian tubes were collected into sterile tubes containing cold minimum essential medium (MEM; Life

Technologies, Invitrogen, Grand Island, NY, USA) and placed on ice and transported to the cell culture laboratory. 2.2. Light microscopy HSP and normal Fallopian tubes were cut into small pieces after removal of fatty tissues and fixed in 4% paraformaldehyde overnight. Tissue samples were dehydrated in graded ethanol and embedded in paraffin wax. Sections (5 mm) were cut using a ReicherteJung Biocut Rotary Microtome 1130, and dried onto Superfrost microscope slides (Fisher brand, Fisher Scientific, Pittsburgh, PA, USA). For hematoxylin and eosin (H&E) staining, slides were dewaxed in xylene and dehydrated in graded alcohol and stained for light microscopy. Observation was performed under an inverted microscope (Leica, DMR BE, Wetzlar, Germany). 2.3. Electron microscopy HSP and normal Fallopian tubes were fixed in 2.5% glutaraldehyde (Electron Microscopy Science, Fort Washington, PA, USA) in 0.1 M PBS (pH 7.4) and rinsed in Sorensen’s buffer and post-fixed in 2% osmium tetroxide (Electron Microscopy Science, Fort Washington, PA, USA) for 1 h. Samples were further rinsed three times for 10 min each in Sorensen’s buffer. They were then dehydrated in graded alcohol, critical point dried in a LADD Critical Point Dryer (Burlington, VT, USA), coated with palladium gold and examined with a scanning electron microscope (JSM 6301FE, JEOL, Japan). For transmission electron microscopy (TEM), fixed tissues were embedded in Epon 812 and ultrathin sections were cut with a Leica Ultracut R (Zeiss, Germany), double stained with uranyl acetate and lead citrate. Sections were observed under an electron microscope (Hitachi H 7100, Tokyo, Japan).

3. Results Both HSP and control patients had similar median ages of 35 (range 25e40) and 36 (range 34e40) years, respectively. The median duration of infertility of HSP patients was 8 years (range 2e15) and all had primary infertility. All of the HSP tubes examined had lost their fimbriae, the fimbria was occluded and markedly dilated at the ampullary region, containing HF and gradually tapering towards the isthmus just before the utero-tubal junction. 3.1. Light microscopy H&E staining of HSP from the ampulla and isthmus portion showed epithelial linings with marked variations

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along the tubular tract. There were areas without epithelial cell linings. Epithelial cells were flattened; exfoliation of epithelial cells and occasional normal columnar epithelial lining could be seen. The lamina propria featured engorged blood vessels and fibrosis (Fig. 1AeD). Muscle fibers were reduced, atrophic and replaced by fibrous tissues. There were conspicuous areas of widening tissue spaces, which indicated severe edema (Fig. 2AeF), and extravasations of blood were common. Inflammatory cells were present in HF in the lumen in areas of flattened to no epithelium, as well as in dilated blood vessels and/or lymph vessels (Fig. 3A,B). No intraepithelial lymphocytes (IEL), that are present in normal epithelium, could be seen in areas of flattened to no epithelium (Fig. 4AeD).

3.2. Electron microscopy Under scanning electron microscopy (SEM), HSP epithelium showed marked abnormality. Most cells had no cilia and were devoid of microvilli, especially at the junction between the intact and eroded epithelial surface (Fig. 5A,B), compared with normal Fallopian tube epithelium that showed long cilia and dense microvilli (Fig. 6A,B). There were areas of denuded and necrotic epithelium in most samples (Fig. 5C). SEM of the eroded epithelial surface revealed stomata exuding globular bodies (Fig. 5D). SEM of Fallopian tube epithelium from normal patients showed long normal

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cilia and dense microvilli (Fig. 6A,B). TEM of HSP showed severe loss of cilia with focal cilia remnants, mitochondria, lipid droplets (Li), granular endoplasmic reticulum, basal laminar and collagenous stroma of lamina propria (not shown).

4. Discussion Our study documented marked loss of cilia and extensive loss of microvilli on HSP luminal epithelium. Atrophic muscle fibers, tissues fibrosis and distorted muscle fibers separated by severe edema seen in most of the Fallopian tube walls could impair muscle contractions and abolish tubal peristaltic movements necessary for transporting sperm to meet the oocyte or embryo from the ampulla to the uterus, thus affecting fertilization or implantation processes, respectively. Our results differ from those of Patek and Nilsson (1977), who showed that the epithelium of HSP simplex was only compressed to about one-sixth of its original height, had numerous cilia, HSP mucosa did not differ conspicuously from normal tubal mucosa and that SEM revealed no deciliation or differences from the epithelia of HSP and those of normal Fallopian tubes. It may be that the hydrosalpinx Patek and Nilsson (1977) studied were due to simple mechanical blockage of the tubes and not post infectious hydrosalpinx. Another possibility may be that their samples came from spots of intact cilia only, while this study examined the whole hydrosalpinx.

Fig. 1. Hematoxylin and eosin (H&E) staining of hydrosalpinx. Hydrosalpinx luminal epithelium showing flattened epithelial cells (arrows, A and B). Exfoliation of epithelial cells (arrowheads), reduced number of intraepithelial lymphocytes along the flattened epithelium as illustrated in C. Fibrosis of the lamina propria (thick arrows) (B), the lamina propria features engorged blood vessels and fibrosis (arrowheads, B and D) with occasional normal epithelium and lamina propria (C and D). L, lumen (!20; scale bar 30 mm).

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Fig. 2. (AeF) H&E staining of hydrosalpinx to show increased presence of fat cells (Li) (A), fibrosis, distortion and atrophy of HSP muscle fibers (AeF) and widening of tissue space which indicates severe fluid accumulation separating muscle fibers (BeF). Muscle fibers are reduced (C) and extravasations of blood is common (!40; scale bar 30 mm).

The present study also showed the extent of luminal epithelial and muscular damage in HSP that have not been previously reported in any detail. Damage to the luminal epithelium of HSP will affect transport of gametes, fertilization and early embryo development. The tubal fluid microenvironment, which is essential for a number of reproductive processes, depends on the integrity of the epithelium, damage to which will lead to abnormal secretion and absorption, hence the formation of HF. The observed damage to the epithelial lining of HSP suggests a possible cause of HF formation. The loss of membrane polarity and/or decreased expression of epithelial membrane transporters and ion channels of the fallopian tube may be responsible, at least in part, for the formation of HF (Ajonuma et al., 2002). Postinfectious HSP epithelial pathology-atrophy of mucosal folds and marked exfoliation of epithelial cells-may affect fluid transport leading to abnormal fluid secretion and reabsorption. David et al. (1969) suggested that damage to HSP luminal epithelium might

be due to the pressure of the HF. However, the presence of some areas of epithelial lining with normal columnar cells makes this unlikely. Interstitial fluid may leak from the damaged epithelial surface into the lumen. This may be one of the causes of HF formation and the decreased IVF outcome in the presence of HSP, especially in bilateral hydrosalpinges or those large enough to be seen on ultrasound that are associated with HF reflux into the uterus (Mansour et al., 1991; Andersen et al., 1996; Bloechle et al., 1997; Sharara and McClamrock, 1997). This study was the first to examine the whole intact HSP tube in humans and demonstrated on-going chronic inflammatory process in HSP and the presence of inflammatory cells in infertile patients presenting for assisted reproduction treatments. These inflammatory cells are thought to be involved in the release of cytokines and other compounds that are deleterious to intrauterine oocytes and spermatozoa (David et al., 1969). No pathogenic microorganisms or Chlamydia trachomatis have previously been reported in any of the

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Fig. 3. (A,B) H&E staining of hydrosalpinx luminal epithelium showing inflammatory cells (polymorphonuclear cells, P) present in HF in the lumen in areas of flattened (thin arrows) to no epithelium (thick arrows) as well as in dilated blood and/or lymph vessels. (!40; scale bar 30 mm). L, lumen; HF, hydrosalpinx fluid.

published studies of HF culture (Murray et al., 1997), but elevated endotoxin levels (Strandell et al., 1998) and vaginal commensals (Ng et al., 2000) have been reported. However, the presence of polymorphonuclear cells in HSP tissues and lumen in this study provides an explanation for long-suspected low-grade chronic infections in hydrosalpinges, especially by C. trachomatis. Damaged HSP epithelial cells may be the source of some bioactive substances, including cytokines such as tumor necrosis factor alpha (TNF-a) known to be toxic to embryos (David et al., 1969). The effect of Fallopian tube bacterial infections leading to HSP formation has been reviewed (Ajonuma et al., 2002).

However, another possibility is that low-grade chronic infections in HSP may induce differentiation of epithelial cells leading to enhanced expression and/or down-regulation of some ion channels and transporters responsible for transepithelial transport causing a shift in the secretion and absorption balance of the uterine tube epithelium (Ajonuma et al., 2005). This low-grade, silent but chronic infection may be the reason for the reported early and increased rate of differentiation of cultured HSP epithelial cells from epithelial to fibroblast-like cells in vitro and reduced secretory vacuoles that we observed in our previous study (Ajonuma et al., 2003). The transformation of cell nucleus to spindle-like

Fig. 4. (AeD) H&E staining of hydrosalpinx showing severe epithelial damage, dilated capillaries (C) and numerous chronic inflammatory cells (P) in both the lumen and lamina propria. Note the loss of epithelial lining (arrowhead), aggregation of leukocytes in HSP lumen (AeC) and muscle fibers (F) of the lamina propria (AeD) and extravasations of blood cells into stroma (!40; scale bar 30 mm). L, lumen.

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Fig. 5. (AeD) Scanning electron micrograph of HSP ampulla region showing secretory cells denuded of microvilli and the ciliated cells devoid of cilia (A and B) (!1500; bar 10 mm). Close up of a large field (B) (!3000; bar 10 mm). SEM of HSP ampullary epithelium at the junction between the denuded area and the eroded area (thin arrow) (C) (!2500; bar 10 mm) and SEM of the eroded epithelial surface of the ampulla region showing stomata (thick white arrows) exuding globular bodies (thin black arrows) (D) (!2500; bar 10 mm).

structures in the areas of abnormal epithelium and tissues may be the initiation of fibrosis in these tissues. The disappearance of IEL in the abnormal areas of the epithelium may suggest some immune involvement in the pathogenesis of Fallopian tube destruction in HSP. However, this remains to be elucidated. Although we did not assay for the presence of C. trachomatis in this study, the pattern of epithelial destruction in our samples is consistent with chlamydial infection. Demonstration of underlying chronic infection in hydrosalpinges in this study calls for more consideration in the use of antibiotics or counseling for salpingectomy in IVF patients with HSP. Sharara et al. (1996) and Hurst et al. (2001) treated IVF patients having HSP with doxycycline and both groups reported no significant difference between HSP and controls, demonstrating a treatment effect in the HSP group. What is more disturbing is that this low-grade silent chronic infection of the Fallopian tubes may also be going on in the endometrium, as evidenced by the inability of some of these patients to become pregnant cycles after salpingectomy. Gump et al. (1981), Wolner-

Hanssen et al. (1982) and Winkler et al. (1984) reported endometritis related to C. trachomatis infection, and Patton et al. (1994) also demonstrated it in the Fallopian tubes of women with postinfectious HSP. Although sperm come into contact with both cervical and uterine epithelial cells during passage through the female reproductive tract, spermeoviduct epithelial cell interactions are of particular importance (Smith, 1998). Studies have shown that interactions between sperm and Fallopian tube epithelium improves sperm motility characteristics, induces capacitation and increases sperm fertilizing ability (Barratt and Cooke, 1991; Kervancioglu et al., 1994, 2000). Possible explanations for this phenomenon may be that Fallopian tube secretions may induce sperm capacitation, reduce sperm oxidative stress and provide protective enzymes and glycoproteins. Spermatozoa that reach the oviduct have a tendency to attach to luminal epithelium and ciliated cells (Overstreet and Cooper, 1979; Katz and Yanagimachi, 1981; Suarez, 1987). However, damage to HSP epithelium will impair sperm, oocyte and embryo transport, thereby affecting fertilization and pregnancy rates. HSP epithelial cells may

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Fig. 6. Scanning electron micrograph of normal Fallopian tube taken from the ampulla to show epithelial cells with long cilia and dense microvilli (A) and SEM of normal secretory epithelium with microvilli (B) (!2500; scale bar 14 mm).

be producing a fluid milieu hostile to sperm that prevents sperm capacitation and acrosome reaction prerequisites for fertilization (Ajonuma et al., 2003). One of the interesting findings of this study was the demonstration of the presence of stomata on the surface of eroded HSP epithelium at the ampullary region secreting globular bodies (Fig. 5D). Although Li et al. (1996) reported small stomata located on the secretory cells of HSP that are of lymphatic origin, the exact nature of the stomata is unknown and the globular bodies are yet to be characterized. However, we speculate that these secretions could be abnormal protein(s) involved in mediating the toxicity of HF on spermatozoa and early embryo development. A protein of 54 kDa was identified in the normal human oviduct that could bind to the surface of entire spermatozoa (Lippes and Wagh, 1989; Wagh and Lippes, 1989) instead; HSP epithelium has been shown to be producing increased amounts of 57 kDa heat shock protein that is associated with less embryo development (Betty et al., 1993). In summary, the present study demonstrates chronic inflammation of the Fallopian tubes, severe epithelial pathology and extensive damage to the Fallopian tube musculature that may explain the mechanisms of HF formation and adverse effects of HF on implantation and pregnancy rates in the presence of HSP. More studies are needed to further characterize the stomata and globular bodies seen on the eroded HSP ampullary surface by SEM. Acknowledgment This study was supported by the Strategic Programme of the Chinese University of Hong Kong.

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