Seminal plasma does not aid in the transport of phenolsulfonphthalein across the uterotubal junction in mares

Abstract

This study tested the hypothesis that the presence of prostaglandin E2 in seminal plasma would aid in the transport of phenolsulfonphthalein (PSP) across the uterotubal junction. Five mares in estrus were inseminated during estrus with PSP dissolved in phosphate-buffered saline and during the subsequent estrus with PSP added to a standard insemination dose. Serum and urine samples were obtained at hours 0, 1, 2, and 3 following treatment and examined for the presence of PSP. Phenolsulfonphthalein could not be detected in any of the urine samples collected from mares following either treatment. None of the serum samples collected following intrauterine installation of PSP in PBS contained PSP. Phenolsulfonphthalein was detected in serum samples from 1 mare following insemination with semen containing PSP. Components in seminal plasma such as PGE2 did not facilitate the transport of PSP across the uterotubal junction as had been hypothesized.

Introduction

Occlusion of the uterine tube has long been a suspected source of unexplained infertility in mares. Such occlusions can be due to fibrous adhesions, gelatinous masses, or cysts to neoplastic tumors and are theorized to interfere with or prevent the passage of sperm, and fertilization and/or migration of the embryo into the uterus (1,2). While numerous attempts have been made to design effective tests to diagnose uterine tube pathologies, these tests are highly invasive, unreliable, or inconclusive. One such test is the phenolsulfonphthalein (PSP) test, first proposed by George Speck in 1948 to assess uterine tube patency in women (3). This test has since seen moderate successful use as a diagnostic tool for uterine tube pathologies in cattle (4,5). Its use in horses, however, has seen limited study with poor success. The test involves the deposition of a solution of PSP (better known as phenol red) into the uterus, from which it travels up the uterine tube and into the peritoneal cavity. Absorption by the peritoneum and secretion via the kidneys allows for the presence of PSP to be detected in urine. The presence of PSP in a urine sample obtained 30 min following intrauterine PSP instillation indicates a positive result for patency of the uterine tube, while a negative result indicates an occlusion. Most difficulties recorded in using the PSP test on mares have been due to a high frequency of contamination leading to false positives: uterine contractions causing reflux of fluid into the vagina and vestibule resulting in contamination of the urethra during catheterization (6).

Given the limited use of the PSP test in mares, alternative approaches for assessing uterine tube patency have been explored. Deposition of starch granules onto the ovarian surface followed by vaginal and cervical washings proved a reliable way to determine uterine tube patency (6). Laparoscopic placement of fluorescent beads into the oviduct followed by uterine lavage 48 h later to retrieve the beads proved to have 71.4% sensitivity and 85.7% specificity in determining oviductal patency. In that study mares were euthanized following uterine lavage in order to examine the uterine tubes (7). Deposition of fluorescent microspheres onto the ovarian surface using an ultrasound-guided transvaginal approach followed by uterine lavage 48 h later had limited usefulness due to a lack of repeatable results (mares were subjected to the procedure twice at least 14 d apart) (8). Lastly, the insertion of a catheter into the oviduct under endoscopic guidance with the instillation of indigo carmin followed by abdominocentesis has been reported. In 75% of attempts the catheter could be inserted into the oviduct and in 8 out of 10 mares abdominal fluid was recovered (9). These diagnostic tests for uterine tube patency are all invasive, which precludes their routine use in clinical practice.

The uterotubal junction (UTJ) has a particularly well-developed tunica muscularis in the mare and therefore serves as a barrier to the passage of fluid under normal physiological conditions. The prominence of the UTJ necessitates production of prostaglandin E2 (PGE2) by the equine embryo to facilitate transport to and passage through the uterotubal junction (10,11). Consequently, due to the absence of PGE2 production, unfertilized oocytes are sequestered in the uterine tube (12). Likewise, mechanisms are in place to allow passage of spermatozoa into the uterine tube. The many components of seminal plasma indicate that it might play an important role in myometrial contractions that facilitate the passage of sperm through the uterus (13). Stallion semen contains PGE2 at a concentration averaging 43.73 ± 4.93 ng/mL as determined with a bioassay (14). Intrauterine instillation of PGE2 before artificial insemination improves pregnancy rates with good quality semen (15). Likewise, addition of PGE to the inseminate during hysteroscopic artificial insemination (AI) resulted in an increased number of sperm passing the UTJ in 2 of 3 mares (16). Studies on the effect of PGE2 on the contractility of rat and pig oviductal tissue reveal that PGE2 can have both stimulatory and inhibitory effects (17,18). The effects of PGE2 are mediated via 4 prostaglandin E2 receptors (PTGER1-4); PTGER2 and PTGER4 are responsible for smooth muscle relaxation, whereas PTGER1 and PTGER3 cause smooth muscle contraction (19).

The aim of the current study was to re-visit and validate the non-invasive PSP test in mares. Presumably, the significant barrier posed by the UTJ requires some adjustment to the PSP test in order for it to be effective in mares. Our proposed modification involves inseminating the mare with semen containing PSP, rather than PSP dissolved in saline or PBS as used in previous studies. Components of seminal plasma such as PGE2 are expected to act on the barrier imposed by the UTJ in mares. To address the contamination issues, we proposed to place a Foleystyle urinary catheter prior to intrauterine instillation of PSP and leave it in place for the duration of the study.

Materials and methods

Experimental design and sample collection

Semen was collected and extended 1:5 in E-Z Mixin-“BF” (Animal Reproduction Systems, Chino, California, USA) yielding sperm concentrations between 25 and 50 × 10⁶/mL. The effect of PSP (Sigma Aldrich, Oakville, Ontario) on sperm motility was assessed through adding increasing amounts (0.1%, 0.3%, 0.4%, 0.5%, and 1%) to extended semen, followed by evaluation of total and progressive motility using a light microscope with heated stage and computer-assisted sperm analysis (SpermVision; MOFA Global, Ingersoll, Ontario). Sperm motility was impaired when 0.3% or more of PSP was added; therefore, 0.1% of PSP was used for all experiments.

In a preliminary experiment the observation reported by Allen et al (6) was confirmed: when urine samples were obtained through repeated catheterization, contamination of the vestibule with PSP was noted in all mares when the 2 and 3 h urine samples were collected. Contamination of the vestibule with PSP was determined through the presence of bright red stain on the examination sleeve. It was therefore decided to insert a Foley-style catheter into the bladder before the start of the experiment and leave it in place for the duration of sample collection.

Five Warmblood mares (age ranging from 7 to 12 y) of proven fertility were recruited via convenience sampling for the purpose of this study under an approved Animal Use Protocol reviewed by the Animal Care Committee at the University of Calgary. Estrous cycles were monitored per rectal palpation and ultrasonography. Once a mare was confirmed to be in estrus (presence of a follicle ≥ 35 mm in size, pronounced uterine edema and relaxed cervix) a Foley-style catheter 26 FR (MOFA Global) was inserted into the urethra and the inflatable cuff was filled with air once inside the bladder. Gentle traction was applied to confirm the catheter was securely placed and anchored in the bladder. Urine and blood serum samples were obtained and 30 mL semen extender mixed with 0.1% PSP was instilled into the uterine body (control cycle). Urine samples were then collected at hours 1, 2, and 3 through the existing catheter. A blood sample for serum preparation was concurrently collected. During the next estrous period mares were subjected to the same procedure, except that PSP (0.1%) was added to 500 Mio motile sperm in semen extender yielding 30 to 35 mL total volume (treated cycle).

Analysis of urine and serum samples

Blood serum samples were frozen at −20°C pending further analysis. Following urine collection, each sample was divided into 3 aliquots of 5 mL and the pH was adjusted to i) > pH 8 using NaOH; ii) pH 7; and iii) < pH 6 using HCl. A pH meter was used to determine the pH readings. The presence of PSP was evident through a color change to red in an alkaline milieu and a color change to yellow in an acidic milieu.

Serum samples were processed as described by Marshall and Vickers (20) with slight modifications. In brief, 100 μL of serum was slowly added to 900 μL of 95% ethanol followed by centrifugation for 1 min at 1000 × g. The resulting supernatant was divided in halves and acidified with HCl or alkalinized with NaOH. Each sample was then analyzed in triplicate through spectrophotometry. Absorbance was measured at 430 nm and 560 nm (SpectraMax i3x; Molecular Devices, Sunnyvale, California, USA) as the absorbance of PSP in acidic milieu peaks at 430 nm and in alkaline milieu at 560 nm. Serum samples spiked with PSP (0.0005%) served as positive control. Serum samples collected from mares (n = 3) not exposed to PSP collected at hours 0, 1, 2, and 3 were included in the analysis to confirm that no spontaneous variations in the absorption spectrum of serum resembling the presence of phenol red existed. Samples were considered positive for PSP when a concurrent increase in absorbance at 430 and 560 compared to the sample at 0 hour was observed. Absorbance values for each wave length were compared using 1-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (GraphPad Prism 6; Graphpad Software, La Jolla, California, USA). Absorbance values for serum samples spiked with PSP were compared using a 2-tailed t-test. A P-value < 0.05 was considered significant for both analyses.

Results

Urine samples were collected successfully from all mares at all time points. Following adjustment of pH, resulting color changes were monitored. Phenolsulfonphthalein in solution is a pH indicator exhibiting a gradual transition from yellow to red over the pH range 6.8 to 8.2. Above pH 8.2, phenol red turns a bright pink (fuchsia) color. None of the urine samples collected from either the control or treatment group displayed a color change following the adjustment of pH.

Serum samples were collected successfully from all mares at all time points. A serum sample spiked with PSP served as positive control and displayed the expected increase in absorbance at 430 nm and 560 nm (P = 0.005; Figure 1). Serum samples collected from mares not exposed to PSP did not display significant variation in their absorbance spectrum in an acidic (430 nm) or alkaline (560 nm) environment over a period of 3 h (Figure 2).

Figure 1.

Spiking of serum samples with PSP resulted in a significant increase in absorbance in an acidic (430 nm) and in an alkaline (560 nm) environment (P = 0.005).

Figure 2.

Serum samples collected from mares not exposed to PSP did not display significant variation in their absorbance in an acidic (430 nm) or an alkaline (560 nm) environment.

Serum samples collected 2 and 3 h after AI from 1 of the mares inseminated with semen containing PSP had a significantly higher absorption at 430 and 560 nm compared with the serum sample collected before AI (P < 0.05; Figure 3, panel a). Neither serum samples from the remaining mares inseminated with semen containing PSP, nor serum samples from mares that received PSP in semen extender displayed a change in absorbance consistent with the presence of phenol red (representative absorbance values shown in Figure 3, panel b).

Figure 3.

Absorbance of serum samples collected from mares following artificial insemination with semen containing 0.1% PSP in an acidic (430 nm) or an alkaline (560 nm) environment. Panel a — serum samples collected from mare A at 2 and 3 h post AI displayed significant higher absorption at 430 and at 560 nm (P < 0.05). Panel b — serum samples collected from mare B at 1, 2, and 3 h after AI did not display significant higher absorption at 430 and 560 nm. *indicates a P < 0.05 compared with the 0 h sample.

Discussion

Causes of infertility (failure to achieve pregnancy) include uterine infections and endometrial cysts in some cases (21). However, there are unexplained cases of infertility in which mares fail to conceive despite the absence of detectable uterine or ovulatory abnormality or dysfunctions and optimal breeding management (good quality semen from more than 1 stallion). In recent years, the uterine tube has gained increasing attention as a cause for infertility. The uterine tube is the site of oocyte maturation, fertilization, and early embryo development. Given the importance of PGE2 in oviductal transport of the equine embryo, PGE2 has emerged as an empirical treatment for cases of unexplained infertility. Laparoscopic application of PGE2 onto the serosal surface of the uterine tube surface restored fertility in mares previously suffering from unexplained infertility (22). In that study, 7 of 8 mares became pregnant and delivered a live foal, and a viable embryo was flushed from the uterus of 17 of 20 embryo-donor mares (22). Therefore, it was concluded that blocked uterine tubes might have been the underlying cause for the infertility. Similar results had previously been reported by Allen et al (23), with 14 out of 15 mares conceiving following the laparoscopic application of PGE2 onto the oviducts. In abattoir-derived equine reproductive tracts, a high prevalence of tubal pathologies has been reported (24). Although at least some of these mares were presumably culled due to infertility, in the absence of clinical history, the extent to which these abnormalities affected fertility is unknown.

The aim of the current study was to develop a non-invasive diagnostic test assessing uterine tube patency. Because the PSP test has been used successfully in women and in cattle, but has not been effective in horses, it was modified to account for shortcomings that have been described when it was used in mares. The existing PSP test was modified in 2 ways. Preliminary experiments confirmed the previous observation that urine samples were contaminated with reflux of previously intrauterine instilled PSP solution when urine was obtained through repeated catheterization. We therefore placed a Foley-style catheter into the bladder before intrauterine administration of PSP, which successfully prevented the contamination. To address the hypothesis that components in seminal plasma, namely PGE2, would enable PSP suspended in semen to pass the uterotubal junction, mares were inseminated with semen containing 0.1% PSP and results were compared to intrauterine instillation of semen extender containing 0.1% PSP. To mimic a physiologic situation, during which seminal plasma provides sufficient PGE2 to allow for sperm passage across the UTJ, 500 million sperm cells were extended to a concentration between 25 and 50 × 10⁶/mL, yielding a total volume of 30 mL. Collection of serum and urine samples was extended to 3 h as sperm transport into the uterine tube is delayed in the mare with spermatozoa appearing in the oviduct 2 h following insemination (25).

Neither semen nor semen extender containing PSP resulted in the collection of urine that contained PSP. Only 1 of the 5 mares inseminated with semen containing PSP displayed changes in the absorbance spectrum of the serum samples collected 2 and 3 h following AI that were consistent with the presence of PSP. Our hypothesis that PGE2 present in seminal plasma would aid in the transport of PSP across the UTJ was therefore not proven. A non-invasive test assessing uterine tube patency remains elusive. CVJ