Hum. Reprod. Advance Access published August 19, 2014 Human Reproduction, Vol.0, No.0 pp. 1 –7, 2014 doi:10.1093/humrep/deu210
ORIGINAL ARTICLE Fertility control
Ulipristal acetate resembles mifepristone in modulating human Fallopian tube function 1
Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong 2Centre for Reproduction, Development and Growth, The University of Hong Kong, Hong Kong 3Department of Anatomy, The University of Hong Kong, 21 Sassoon Road, Hong Kong *Correspondence address. Tel: +852-22553914; Fax: +852-25173278; E-mail: [email protected]
Submitted on May 20, 2014; resubmitted on July 9, 2014; accepted on July 18, 2014
study question: Do ulipristal acetate (UPA) and mifepristone have an effect on ciliary beat frequency and muscular contractions in the human Fallopian tube? summary answer: UPA, in resemblance to mifepristone, inhibits ciliary beat and muscular contraction of the human Fallopian tube, probably through an agonistic effect on the tubal progesterone receptor.
what is known already: UPA, like mifepristone, acts as an emergency contraceptive mainly by inhibiting ovulation. Little is known about its effects on tubal function.
study design, size, duration: This was an in vitro experimental study using Fallopian tube samples collected from 11 women undergoing hysterectomy for benign non-tubal gynaecological conditions. participants/materials, setting, methods: The tubal epithelium and longitudinal smooth muscle fibres were isolated, cultured and treated with UPA at graded concentrations of 0, 20, 200 and 2000 ng/ml, and mifepristone at graded concentrations of 0, 300, 3000 and 30 000 ng/ml, respectively. After treatment, ciliary beat frequency was determined using a photometric method. Basal tone, amplitude and frequency of muscular contraction were recorded through a force transducer. The mRNA expression of progesterone receptor (total and PR-B isoform), glycodelin and adrenomedullin were determined by real-time quantitative PCR. main results and the role of chance: There was an overall dose-dependent suppressive effect on ciliary beat frequency (P , 0.0001) after treatment with UPA at all concentrations and with mifepristone at 3000 ng/ml or above. The basal tone, amplitude and frequency of muscular contractions were significantly reduced (P , 0.05) after treatment with UPA at 200 ng/ml or above, and with mifepristone at 3000 ng/ml or above. UPA treatment at 200 ng/ml or above significantly up-regulated the mRNA expression of progesterone receptor and glycodelin and down-regulated the mRNA expression of adrenomedullin in Fallopian tube tissue (P , 0.05).
limitations, reasons for caution: Whether or not the tubal effect may translate into additional mechanisms for contraceptive action in vivo is uncertain.
wider implications of the findings: The clinical relevance of UPA with regard to contraceptive activity is worthy of further exploration.
study funding/competing interest(s): The study was supported by a Seed Fund from the Centre of Reproduction, Development and Growth, Faculty of Medicine, the University of Hong Kong. All authors have no competing interest to declare. Key words: ulipristal acetate / mifepristone / human Fallopian tube / ciliary beat / muscular contraction
†
The authors consider that the first two authors should be regarded as joint First Authors.
& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://humrep.oxfordjournals.org/ at University of Melbourne Library on September 12, 2014
Hang Wun Raymond Li1,2,†,*, Su-Bin Liao 1,3,†, William Shu-Biu Yeung 1,2, Ernest Hung-Yu Ng 1,2, Wai-Sum O1,2,3, and Pak-Chung Ho1,2
2
Introduction
Materials and Methods Fallopian tube sample collection and preparation Fallopian tubes donated by volunteers and collected during hysterectomy for benign gynaecological conditions not involving the tubes (n ¼ 11) were studied in vitro. All the subjects did not receive any hormonal treatment within 3 months before the operation. Written informed consent was obtained from the subjects before sample collection. Ethics approval was obtained from the Institutional Review Board, The University of Hong Kong/Hospital Authority Hong Kong West Cluster. The isthmic part of the collected Fallopian tubes was isolated for the experiments. After collection, the samples were immersed immediately in ice-cold Dulbecco’s modified Eagle’s medium/F12 medium with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) and processed within 24 h. The samples were rinsed in the medium a few times to remove all visible blood, and incubated overnight in 100 pmol/l estradiol and 10 nmol/l progesterone. Since the collection of the samples were not timed to a particular phase of the menstrual cycle (five each at the preovulatory and post-ovulatory phases, respectively, while one subject was anovulatory, based on menstrual history), such hormonal incubation was to
prime and condition the tubal samples to a standardized hormonal environment simulating the early luteal phase before the actual experiment.
Effect of UPA and mifepristone on ciliary beat frequency Strips of the oviductal ciliated epithelium from seven subjects (at least six strips per subject) and cut into 1 – 2 mm, were treated with UPA (supplied by HRA Pharma, France) in graded concentrations of 0, 20, 200 and 2000 ng/ml (i.e. 0, 0.04, 0.4 and 4 mM) and incubated for 24 h. These treatment concentrations were based on the pharmacokinetic data of the UPA 30 mg oral tablet that the total peak plasma concentration of UPA (bound and unbound) 1 h after ingestion was 176 + 89 ng/ml (Snow et al., 2011). Our experimental doses covered a 10-times range both higher and lower than the peak plasma concentration. Ciliary beat frequency of the treated epithelial strips was measured using a photometric method as previously described (Tsang et al., 2000; Li et al., 2010). The frequency was conveyed by a photo-multiplier and translated digitally in Hz. The above experiments were repeated with mifepristone treatment at graded concentrations of 0, 300, 3000 and 30 000 ng/ml (i.e. 0, 0.7, 7 and 70 mM) in place of UPA. This was based on previously reported mean peak serum concentration of mifepristone after a single 25 mg oral dose being 2900 ng/ml (Kekkonen et al., 1996). Therefore, the experimental concentrations of mifepristone covered a 10-times range both higher and lower than the pharmacological level.
Effect of UPA and mifepristone on oviductal muscular contractility Fallopian tube samples from 11 subjects were used for the in vitro-contractility experiments. Strips of longitudinal smooth muscle fibres were isolated and tied to holder electrodes which were bathed in Kreb’s solution (118 mM NaCl, 4.8 mM KCl, 1 mM MgSO4, 1.15 mM NaH2PO4, 15 mM NaHCO3, 10.5 mM glucose) and connected to a force transducer coupled to a graphic recorder. After equilibration for 30 min, UPA was added to the medium at graded concentrations of 20, 200 and 2000 ng/ml at 2 min intervals. The contraction tracing was recorded and analysed subsequently. The frequency was expressed in number of contractions per minute, while the basal tone and amplitude were expressed as relative units compared with a pretreatment time-point. Details of the contractility experimental set-up have been previously reported (Liao et al., 2011). The above experiments were repeated with mifepristone treatment at graded concentrations of 0, 300, 3000 and 30 000 ng/ml in place of UPA.
Effect of UPA treatment on expression of progesterone receptors and glycodelin Portions of Fallopian tube tissue from 11 subjects were incubated with UPA in graded concentrations of 0, 20, 200 and 2000 ng/ml as described above. The treated tissue was frozen and kept at 2808C until further use. Total RNA was extracted from the frozen – thawed tissue using TRIZOL reagent (Invitrogen). Reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Foster City, CA, USA) according to the suggested protocol. Real-time PCR was performed using the iCyclerTM (Bio-Rad Laboratories, Hercules, CA, USA) under the following conditions: 958C for 10 min for denaturation, followed by 40 cycles of 958C for 30 s, 578C for 30 s and 728C for 30 s. The primers used were: PGR (generic progesterone receptor) (GenBank accession no. NM_000926): 5′ -GAG CAC TGG ATG CTG TTG CT-3′ , forward, 5′ -GGC TTA GGG CTT GGC TTT C-3′ , reverse; PR-B (specific B isoform of the progesterone receptor) (GenBank accession no. NM_000926): 5′ -TGG GAT CTG AGA TCT TCG GAG-3′ , forward, 5′ -GAA GGG TCG
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Emergency contraception (EC) is essential in family planning services as it serves as an important back-up method in cases of unprotected intercourse or unplanned failure of a regular contraceptive method. The oral pill containing levonorgestrel (LNG) 1.5 mg taken within 72 h of unprotected intercourse is currently one of the most popular and recommended EC methods (World Health Organisation, 2004; Cheng et al., 2012). Although mifepristone, an anti-progestogen, at a single oral dose of 25– 50 mg administered up to 120 h after unprotected intercourse may have an even higher efficacy (Cheng et al., 2012), the availability of mifepristone at such doses for EC is limited to mainland China and Russia. In the past few years, ulipristal acetate (UPA) has also been introduced as an EC. UPA is a selective progesterone receptor modulator. A single oral dose of 30 mg UPA taken within 120 h of unprotected intercourse has proved more effective than the LNG regimen in randomized trials and meta-analysis (Creinin et al., 2006; Glasier et al., 2010); this product has since been marketed in many countries worldwide. UPA is a synthetic selective progesterone receptor modulator derived from 19-norprogesterone, with mainly antagonistic but also partial agonistic effects on the progesterone receptor (Leo and Lin, 2008). Delay or inhibition of ovulation is believed to be the main mechanism by which hormonal EC methods act (Gemzell-Danielsson et al., 2013). However, a study has also suggested that both LNG and mifepristone can reduce the frequency and area under the curve of muscular contraction tracings in the human Fallopian tube (Wanggren et al., 2008). This may contribute to an additional post-ovulatory mechanism of their EC action. Whether UPA has similar effects on human tubal function is currently unknown. The aim of the current study was to investigate the biological effect of UPA on human Fallopian tube function, namely ciliary beat frequency and muscular contractions in human Fallopian tube by in vitro experiments. The effect of UPA on the expression of progesterone receptor, glycodelin and adrenomedullin, which are the potential hormonal mediators of the effect of UPA on the tubal function, was also studied.
Li et al.
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Effect of UPA and mifepristone on tubal function GAC TTC TGC TG-3′ , reverse; Glycodelin (GenBank accession no. NM_002571): 5′ - ATG CTG TGC CTC CTG CTC -3′ , forward, 5′ -CTA GAA ACG GCA CGG CTC TT-3′ , reverse; G6PDH (GenBank accession no. NM_000402): 5′ -ATC GAC CAC TAC CTG GGC AA-3′ , forward, 5′ -TTC TGC ATC ACG TCC CGG A-3′ , reverse. The relative quantities of gene expression were expressed as fold changes relative to the control set as the sample without UPA treatment, and normalized to the house-keeping G6PDH gene. Melting curve analysis for each primer set revealed only one peak for each product.
Effect of UPA treatment on expression of adrenomedullin
Statistics Data presented were shown as box-whisker charts where the boxes showed the median and interquartile range, with the full range shown by the whiskers. For data not normally distributed, Kruskal– Wallis test with Dunn’s post hoc analysis or Friedman’s test was used to compare different treatment groups. Statistical analyses were performed using the GraphPad Prism 6 software (GraphPad, San Diego, CA USA).
Results Effect of UPA and mifepristone on ciliary beat frequency There was an overall dose-dependent suppressive effect of UPA on ciliary beat frequency at all concentrations studied (P , 0.0001 for all
Effect of UPA and mifepristone on muscular contractility The basal tone, amplitude and frequency of smooth muscle contractions after treatment with UPA at 20 ng/ml or mifepristone 300 ng/ml did not differ from the control (P . 0.05), but were significantly reduced in a dose-dependent manner after treatment with UPA at 200 ng/ml or above, or with mifepristone at 3000 ng/ml or above (Fig. 2).
Effect of UPA treatment on expression of progesterone receptors and glycodelin Treatment with UPA at 200 ng/ml or above (P , 0.05), but not 20 ng/ ml (P . 0.05), resulted in significant dose-dependent up-regulation of mRNA expression of generic PGR, PR-B and glycodelin (Fig. 3).
Effect of UPA treatment on expression of adrenomedullin Treatment with UPA at 200 ng/ml or above (P , 0.05), but not 20 ng/ ml (P . 0.05), resulted in significant down-regulation of mRNA expression of adrenomedullin (Adm) in a dose-dependent manner (Fig. 4).
Discussion The Fallopian tube plays an important role in human conception. Fertilization normally takes place in the ampullary region of the tube. Transport of the early embryo through the isthmic region towards the uterus is then accomplished by the ciliary beating in the tubal
Figure 1 Effect of (a) UPA and (b) mifepristone on ciliary beat frequency (CBF) of human Fallopian tube. The treatment groups were compared using Kruskal – Wallis test with Dunn’s multiple comparison post hoc analysis. Dose – response correlation analyses are shown in the boxes.
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The above real-time PCR experiment was repeated for the Adm gene on the tubal epithelial tissue treated with various concentrations of UPA using the following primers: Adm (GenBank accession number NM 001124): 5′ -TGC CCA GAC CCT TAT TCG G-3′ , forward, 5′ -CCG GAG GCC CTG GAA GT-3′ , reverse.
treatment groups compared with control). The same dose-dependent suppressive effect was also observed with mifepristone at 3000 ng/ml or above (P , 0.0001 compared with control; Fig. 1).
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Figure 2 Effect of (a) UPA and (b) mifepristone on basal tone, amplitude and frequency of smooth muscle contraction of human Fallopian tube. The treatment groups were compared using Friedman test with Dunn’s multiple comparison post hoc analysis. A representative tracing of the muscular contraction recording is shown in (c). Dose – response correlation analyses are shown in the boxes. (*P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 compared with control; Friedman’s test with Dunn’s post hoc multiple comparison.)
mucosa together with spontaneous contractions of the smooth muscle layer, although the relative importance of ciliary beating, muscular contraction and tubal fluid secretion on embryo transport is uncertain. Factors that reduce ciliary beating or tubal contractility may interfere with conception by influencing embryo transit and altering the timing of the embryo arrival at the uterine cavity so that it is out of the optimal implantation window. This may potentially contribute to contraception.
To our knowledge, this is the first report on a significant suppressive effect of UPA on ciliary beat frequency and smooth muscle contraction in the human Fallopian tube at a concentration equivalent to the peak serum concentration after a pharmacological dose. A similar result was also observed with mifepristone, which was in line with a previous report also on muscular contractions (Wanggren et al., 2008), but in contrast to another report on ciliary beat frequency (Mahmood et al., 1998) in the human Fallopian tube, with the reason for the latter discrepancy being unclear. Earlier studies in mouse models have also suggested that mifepristone treatment has an effect on embryo retention and postcoital contraceptive effects (Roblero et al., 1987; Yang and Wu, 1990). Our results suggest an additional mechanism by which UPA may exert its action as an EC. UPA is thought to act as an EC mainly by delaying or inhibiting ovulation (Brache et al., 2013; Gemzell-Danielsson et al., 2013). An additional post-ovulatory mechanism, however, would potentiate its action especially if the drug is administered after ovulation has occurred which often happens in clinical practice. This may enhance the overall contraceptive efficacy of the method, and would be of great clinical benefit. There has been theoretical concern that inhibition of tubal function may imply an elevated risk of tubal ectopic pregnancy. A meta-analysis (Cleland et al., 2010), however, suggested no evidence of increased risk of ectopic pregnancy with the use of LNG and mifepristone for EC, probably because the EC reduces the overall risk of conception mainly by inhibiting or delaying ovulation. A recent post-marketing surveillance report also observed no additional risk of ectopic pregnancy after use of UPA (Levy et al., 2014). It should be noted that establishment of tubal ectopic pregnancy may not be solely explained by delayed embryo transport or embryo retention per se, but has to be accompanied by an altered tubal environment and/or embryo factors to result in tubal implantation (Shaw et al., 2010).
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Figure 2 Continued.
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Effect of UPA and mifepristone on tubal function
tube. Dose – response correlation analyses are shown in the boxes. (*P , 0.05, **P , 0.01, ***P , 0.001 compared with control; Kruskal – Wallis test with Dunn’s post hoc multiple comparison.)
The current study also reports for the first time the effect of UPA on several hormonal mediators which may relate to the effect of UPA onto tubal activity. Some studies have suggested that progesterone may suppress ciliary beating and muscular contraction in the Fallopian tube (Mahmood et al., 1998; Wanggren et al., 2008). Hence, it might seem surprising on first sight that UPA and mifepristone, both commonly regarded as anti-progestogens, actually inhibited tubal activity in line with what progesterone itself does. Based on our results, we postulated three possible explanations as described below. First, our results showed that UPA at pharmacological concentrations (200 ng/ml) or above up-regulated mRNA expression of the progesterone receptor (both generic and specific to PR-B). This may potentiate the progesterone signalling.
Secondly, UPA may exert mainly an agonistic instead of antagonistic effect on progesterone receptors in the Fallopian tube. The same has been speculated for mifepristone (Wanggren et al., 2008), although there is no direct evidence. It has been suggested that the drug has agonistic activity on the PR-A isoform, but partial agonist/antagonist activity on the PR-B isoform (Leo and Lin, 2008). Previous work in mice has suggested that both isoforms are expressed in the non-stimulated ovary, but PR-A expression is significantly reduced after FSH stimulation, until after hCG stimulation when expression of both are raised again. On the other hand, both PR-A and PR-B are expressed in both non-stimulated and stimulated oviducts, with increased expression post-hCG stimulation (Teilmann et al., 2006). This might explain the speculated agonistic action of UPA in the Fallopian tube in contrast to the antagonistic
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Figure 3 Effect of ulipristal on the mRNA expression of PGR and the specific progesterone receptor B (PR-B) isoform and glycodelin in human Fallopian
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condition. Hence, there is still uncertainty over the clinical relevance of our experimental results. Nonetheless our results did suggest a possible avenue for further studies of an EC mechanism through the Fallopian tube. In all, our results suggest that UPA, in resemblance to mifepristone, inhibits ciliary beat and muscular contraction of the human Fallopian tube at the pharmacological dose, probably through an agonistic effect on the tubal progesterone receptor. This may contribute additional mechanisms for its contraceptive action, although the actual clinical significance is yet to be determined.
Acknowledgements
Figure 4 Effect of ulipristal on the mRNA expression of adrenomedullin (Adm) in human Fallopian tube. Dose – response correlation analysis is shown in the box. (*P , 0.05, ***P , 0.001 compared with control; Kruskal – Wallis test with Dunn’s post hoc multiple comparison.)
action at the ovary. It has also been suggested that the activities of PR-A and PR-B could be differentially modulated by different progesterone receptor modulators in a gene-selective manner (Leo and Lin, 2008), and more work needs to be done in this regard to understand the roles played by PR-A and PR-B signalling on Fallopian tube function. Since the expression of glycodelin is mainly regulated by progesterone signalling, the up-regulation of glycodelin by UPA at pharmacological concentrations or above provided supportive evidence that UPA acts as agonist in the Fallopian tube. Thirdly, the suppression of adrenomedullin by UPA at pharmacological dose or above, as first demonstrated in this study, may provide an additional clue. Previous studies by our group have revealed that adrenomedullin plays an important role in enhancing the ciliary beat frequency and muscular contraction tone, amplitude and frequency in human Fallopian tube (Li et al., 2010). There is one report indicating that UPA can down-regulate adrenomedullin and its receptors in a dose-dependent manner in cultured human uterine leiomyoma cells (Xu et al., 2006). The relative importance of modulation through the progesterone and adrenomedullin signalling pathways would require further exploration. In our experiments and data analyses, we took the peak serum concentrations of the respective drugs as the pharmacological concentration at the end organ level. However, there is no data on whether the same drug level actually appears in the tubal fluid and tissue after ingestion of the oral tablet. Furthermore, the peak drug concentration is normally sustained only for a transient period after single-dose administration. Moreover, UPA is highly bound in the human blood circulation (4.9% to blood cells and 94.1% to plasma proteins), with a free fraction of just 1% (Gainer and Ulmann, 2003). Although the culture medium used in our in vitro experiments did contain protein from FBS, it is not certain whether this could resemble the actual pharmacological
Authors’ roles H.W.R.L. and P.C.H. conceived and designed the study. S.-B.L. developed the experimental protocols and conducted all the experimental work under supervision by W.S.-B.Y. and W.S.O.; H.W.R.L., S.-B.L., E.H.-Y.N. and P.-C.H. analysed the data. H.W.R.L. wrote the manuscript which was critically discussed, edited and approved by all co-authors.
Funding This work was supported by a Seed Fund from the Centre of Reproduction, Development and Growth, Faculty of Medicine, the University of Hong Kong.
Conflict of interest All authors have no conflict of interest to declare.
References Brache V, Cochon L, Deniaud M, Croxatto HB. Ulipristal acetate prevents ovulation more effectively than levonorgestrel: analysis of pooled data from three randomized trials of emergency contraception regimens. Contraception 2013;88:611– 618. Cheng L, Che Y, Gu¨lmezoglu AM. Interventions for emergency contraception. Cochrane Database Syst Rev 2012;8:CD001324. Cleland K, Raymond E, Trussell J, Cheng L, Zhu H. Ectopic pregnancy and emergency contraceptive pills: a systematic review. Obstet Gynecol 2010; 115:1263– 1266. Creinin MD, Schlaff W, Archer DF, Wan L, Frezieres R, Thomas M, Rosenberg M, Higgins J. Progesterone receptor modulator for emergency contraception: a randomized controlled trial. Obstet Gynecol 2006;108: 1089–1097. Gainer EE, Ulmann A. Pharmacologic properties of CDB(VA)-2914. Steroids 2003;68:1005 – 1011. Gemzell-Danielsson K, Berger C, Lalitkumar PGLL. Emergency contraception—mechanisms of action. Contraception 2013;87:300–3088.
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We are grateful to Ms Joyce Yuen and other staff members of the Gynecology Team, Queen Mary Hospital for their assistance in patient recruitment and sample collection. We thank Laboratoire HRA Pharma for the free supply of the UPA compound for experimental use in this study. We also thank Professor Kristina Gemzell-Danielsson for her comments on the study results.
Effect of UPA and mifepristone on tubal function
Shaw JL, Dey SK, Critchley HO, Horne AW. Current knowledge of the aetiology of human tubal ectopic pregnancy. Hum Reprod Update 2010; 16:432– 444. Snow SE, Melillo SN, Jarvis CI. Ulipristal acetate for emergency contraception. Ann Pharmacother 2011;45:780 – 786. Teilmann SC, Clement CA, Thorup J, Byskov AG, Christensen ST. Expression and localization of the progesterone receptor in mouse and human reproductive organs. J Endocrinol 2006;191:525 – 535. Tsang KW, Tipoe G, Sun J, Ho JC, Lam B, Zheng L, Ooi GC, Ip M, Lam WK. Severe bronchiectasis in patients with “cystlike” structures within the ciliary shafts. Am J Respir Crit CareMed 2000;161:1300– 1305. Wanggren K, Stavreus-Evers A, Olsson C, Andersson E, GemzellDanielsson K. Regulation of muscular contractions in the human Fallopian tube through prostaglandins and progestogens. Hum Reprod 2008; 23:2359–2368. World Health Organization. Selected Practice Recommendations for Contraceptive use, 2nd edn. Geneva: World Health Organization, 2004. Xu Q, Ohara N, Chen W, Liu J, Sasaki H, Morikawa A, Sitruk-Ware R, Johansson EDB, Maruo T. Progesterone receptor modulator CDB-2914 down-regulates vascular endothelial growth factor, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells. Hum Reprod 2006;21: 2408 – 2416. Yang YQ, Wu JT. RU 486 interferes with egg transport and retards the in vivo and in vitro development of mouse embryos. Contraception 1990; 41:551– 556.
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Glasier AF, Cameron ST, Fine PM, Logan SJS, Casale W, Van Horn J, Sogor L, Blithe DL, Scherrer B, Mathe H et al. Ulipristal acetate versus levonorgestrel for emergency contraception: a randomised non-inferiority trial and meta-analysis. Lancet 2010;375:555–562. Kekkonen R, Heikinheimo O, Mandelin E, Lahteenmaki P. Pharmacokinetics of mifepristone after low oral doses. Contraception 1996;54:229– 234. Leo JCL, Lin VCL. The activities of progesterone receptor isoform A and B are differentially modulated by their ligands in a gene-selective manner. Int J Cancer 2008;122:230 – 243. Levy DP, Jager M, Kapp N, Abitbol JL. Ulipristal acetate for emergency contraception: postmarketing experience after use by more than 1 million women. Contraception 2014;89:431 – 433. Li HWR, Liao SB, Chiu PCN, Tam WW, Ho JC, Ng EHY, Ho PC, Yeung WSB, Tang F, O WS. Expression of adrenomedullin in human oviduct, its regulation by the hormonal cycle and contact with spermatozoa, and its effect on ciliary beat frequency of the oviductal epithelium. J Clin Endocrinol Metab 2010;95:E18– E25. Liao SB, Ho JC, Tang F, O WS. Adrenomedullin increases ciliary beat frequency and decreases muscular contraction in the rat oviduct. Reproduction 2011;141:367 –372. Mahmood T, Saridogan E, Smutna S, Habib AM, Djahanbakhch O. The effect of ovarian steroids on epithelial ciliary beat frequency in the human Fallopian tube. Hum Reprod 1998;13:2991 – 2994. Roblero LS, Ferna´ndez O, Croxatto HB. The effect of RU486 on transport, development and implantation of mouse embryos. Contraception 1987; 36:549– 555.
7
ORIGINAL ARTICLE Fertility control
Ulipristal acetate resembles mifepristone in modulating human Fallopian tube function 1
Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong 2Centre for Reproduction, Development and Growth, The University of Hong Kong, Hong Kong 3Department of Anatomy, The University of Hong Kong, 21 Sassoon Road, Hong Kong *Correspondence address. Tel: +852-22553914; Fax: +852-25173278; E-mail: [email protected]
Submitted on May 20, 2014; resubmitted on July 9, 2014; accepted on July 18, 2014
study question: Do ulipristal acetate (UPA) and mifepristone have an effect on ciliary beat frequency and muscular contractions in the human Fallopian tube? summary answer: UPA, in resemblance to mifepristone, inhibits ciliary beat and muscular contraction of the human Fallopian tube, probably through an agonistic effect on the tubal progesterone receptor.
what is known already: UPA, like mifepristone, acts as an emergency contraceptive mainly by inhibiting ovulation. Little is known about its effects on tubal function.
study design, size, duration: This was an in vitro experimental study using Fallopian tube samples collected from 11 women undergoing hysterectomy for benign non-tubal gynaecological conditions. participants/materials, setting, methods: The tubal epithelium and longitudinal smooth muscle fibres were isolated, cultured and treated with UPA at graded concentrations of 0, 20, 200 and 2000 ng/ml, and mifepristone at graded concentrations of 0, 300, 3000 and 30 000 ng/ml, respectively. After treatment, ciliary beat frequency was determined using a photometric method. Basal tone, amplitude and frequency of muscular contraction were recorded through a force transducer. The mRNA expression of progesterone receptor (total and PR-B isoform), glycodelin and adrenomedullin were determined by real-time quantitative PCR. main results and the role of chance: There was an overall dose-dependent suppressive effect on ciliary beat frequency (P , 0.0001) after treatment with UPA at all concentrations and with mifepristone at 3000 ng/ml or above. The basal tone, amplitude and frequency of muscular contractions were significantly reduced (P , 0.05) after treatment with UPA at 200 ng/ml or above, and with mifepristone at 3000 ng/ml or above. UPA treatment at 200 ng/ml or above significantly up-regulated the mRNA expression of progesterone receptor and glycodelin and down-regulated the mRNA expression of adrenomedullin in Fallopian tube tissue (P , 0.05).
limitations, reasons for caution: Whether or not the tubal effect may translate into additional mechanisms for contraceptive action in vivo is uncertain.
wider implications of the findings: The clinical relevance of UPA with regard to contraceptive activity is worthy of further exploration.
study funding/competing interest(s): The study was supported by a Seed Fund from the Centre of Reproduction, Development and Growth, Faculty of Medicine, the University of Hong Kong. All authors have no competing interest to declare. Key words: ulipristal acetate / mifepristone / human Fallopian tube / ciliary beat / muscular contraction
†
The authors consider that the first two authors should be regarded as joint First Authors.
& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://humrep.oxfordjournals.org/ at University of Melbourne Library on September 12, 2014
Hang Wun Raymond Li1,2,†,*, Su-Bin Liao 1,3,†, William Shu-Biu Yeung 1,2, Ernest Hung-Yu Ng 1,2, Wai-Sum O1,2,3, and Pak-Chung Ho1,2
2
Introduction
Materials and Methods Fallopian tube sample collection and preparation Fallopian tubes donated by volunteers and collected during hysterectomy for benign gynaecological conditions not involving the tubes (n ¼ 11) were studied in vitro. All the subjects did not receive any hormonal treatment within 3 months before the operation. Written informed consent was obtained from the subjects before sample collection. Ethics approval was obtained from the Institutional Review Board, The University of Hong Kong/Hospital Authority Hong Kong West Cluster. The isthmic part of the collected Fallopian tubes was isolated for the experiments. After collection, the samples were immersed immediately in ice-cold Dulbecco’s modified Eagle’s medium/F12 medium with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) and processed within 24 h. The samples were rinsed in the medium a few times to remove all visible blood, and incubated overnight in 100 pmol/l estradiol and 10 nmol/l progesterone. Since the collection of the samples were not timed to a particular phase of the menstrual cycle (five each at the preovulatory and post-ovulatory phases, respectively, while one subject was anovulatory, based on menstrual history), such hormonal incubation was to
prime and condition the tubal samples to a standardized hormonal environment simulating the early luteal phase before the actual experiment.
Effect of UPA and mifepristone on ciliary beat frequency Strips of the oviductal ciliated epithelium from seven subjects (at least six strips per subject) and cut into 1 – 2 mm, were treated with UPA (supplied by HRA Pharma, France) in graded concentrations of 0, 20, 200 and 2000 ng/ml (i.e. 0, 0.04, 0.4 and 4 mM) and incubated for 24 h. These treatment concentrations were based on the pharmacokinetic data of the UPA 30 mg oral tablet that the total peak plasma concentration of UPA (bound and unbound) 1 h after ingestion was 176 + 89 ng/ml (Snow et al., 2011). Our experimental doses covered a 10-times range both higher and lower than the peak plasma concentration. Ciliary beat frequency of the treated epithelial strips was measured using a photometric method as previously described (Tsang et al., 2000; Li et al., 2010). The frequency was conveyed by a photo-multiplier and translated digitally in Hz. The above experiments were repeated with mifepristone treatment at graded concentrations of 0, 300, 3000 and 30 000 ng/ml (i.e. 0, 0.7, 7 and 70 mM) in place of UPA. This was based on previously reported mean peak serum concentration of mifepristone after a single 25 mg oral dose being 2900 ng/ml (Kekkonen et al., 1996). Therefore, the experimental concentrations of mifepristone covered a 10-times range both higher and lower than the pharmacological level.
Effect of UPA and mifepristone on oviductal muscular contractility Fallopian tube samples from 11 subjects were used for the in vitro-contractility experiments. Strips of longitudinal smooth muscle fibres were isolated and tied to holder electrodes which were bathed in Kreb’s solution (118 mM NaCl, 4.8 mM KCl, 1 mM MgSO4, 1.15 mM NaH2PO4, 15 mM NaHCO3, 10.5 mM glucose) and connected to a force transducer coupled to a graphic recorder. After equilibration for 30 min, UPA was added to the medium at graded concentrations of 20, 200 and 2000 ng/ml at 2 min intervals. The contraction tracing was recorded and analysed subsequently. The frequency was expressed in number of contractions per minute, while the basal tone and amplitude were expressed as relative units compared with a pretreatment time-point. Details of the contractility experimental set-up have been previously reported (Liao et al., 2011). The above experiments were repeated with mifepristone treatment at graded concentrations of 0, 300, 3000 and 30 000 ng/ml in place of UPA.
Effect of UPA treatment on expression of progesterone receptors and glycodelin Portions of Fallopian tube tissue from 11 subjects were incubated with UPA in graded concentrations of 0, 20, 200 and 2000 ng/ml as described above. The treated tissue was frozen and kept at 2808C until further use. Total RNA was extracted from the frozen – thawed tissue using TRIZOL reagent (Invitrogen). Reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Foster City, CA, USA) according to the suggested protocol. Real-time PCR was performed using the iCyclerTM (Bio-Rad Laboratories, Hercules, CA, USA) under the following conditions: 958C for 10 min for denaturation, followed by 40 cycles of 958C for 30 s, 578C for 30 s and 728C for 30 s. The primers used were: PGR (generic progesterone receptor) (GenBank accession no. NM_000926): 5′ -GAG CAC TGG ATG CTG TTG CT-3′ , forward, 5′ -GGC TTA GGG CTT GGC TTT C-3′ , reverse; PR-B (specific B isoform of the progesterone receptor) (GenBank accession no. NM_000926): 5′ -TGG GAT CTG AGA TCT TCG GAG-3′ , forward, 5′ -GAA GGG TCG
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Emergency contraception (EC) is essential in family planning services as it serves as an important back-up method in cases of unprotected intercourse or unplanned failure of a regular contraceptive method. The oral pill containing levonorgestrel (LNG) 1.5 mg taken within 72 h of unprotected intercourse is currently one of the most popular and recommended EC methods (World Health Organisation, 2004; Cheng et al., 2012). Although mifepristone, an anti-progestogen, at a single oral dose of 25– 50 mg administered up to 120 h after unprotected intercourse may have an even higher efficacy (Cheng et al., 2012), the availability of mifepristone at such doses for EC is limited to mainland China and Russia. In the past few years, ulipristal acetate (UPA) has also been introduced as an EC. UPA is a selective progesterone receptor modulator. A single oral dose of 30 mg UPA taken within 120 h of unprotected intercourse has proved more effective than the LNG regimen in randomized trials and meta-analysis (Creinin et al., 2006; Glasier et al., 2010); this product has since been marketed in many countries worldwide. UPA is a synthetic selective progesterone receptor modulator derived from 19-norprogesterone, with mainly antagonistic but also partial agonistic effects on the progesterone receptor (Leo and Lin, 2008). Delay or inhibition of ovulation is believed to be the main mechanism by which hormonal EC methods act (Gemzell-Danielsson et al., 2013). However, a study has also suggested that both LNG and mifepristone can reduce the frequency and area under the curve of muscular contraction tracings in the human Fallopian tube (Wanggren et al., 2008). This may contribute to an additional post-ovulatory mechanism of their EC action. Whether UPA has similar effects on human tubal function is currently unknown. The aim of the current study was to investigate the biological effect of UPA on human Fallopian tube function, namely ciliary beat frequency and muscular contractions in human Fallopian tube by in vitro experiments. The effect of UPA on the expression of progesterone receptor, glycodelin and adrenomedullin, which are the potential hormonal mediators of the effect of UPA on the tubal function, was also studied.
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Effect of UPA and mifepristone on tubal function GAC TTC TGC TG-3′ , reverse; Glycodelin (GenBank accession no. NM_002571): 5′ - ATG CTG TGC CTC CTG CTC -3′ , forward, 5′ -CTA GAA ACG GCA CGG CTC TT-3′ , reverse; G6PDH (GenBank accession no. NM_000402): 5′ -ATC GAC CAC TAC CTG GGC AA-3′ , forward, 5′ -TTC TGC ATC ACG TCC CGG A-3′ , reverse. The relative quantities of gene expression were expressed as fold changes relative to the control set as the sample without UPA treatment, and normalized to the house-keeping G6PDH gene. Melting curve analysis for each primer set revealed only one peak for each product.
Effect of UPA treatment on expression of adrenomedullin
Statistics Data presented were shown as box-whisker charts where the boxes showed the median and interquartile range, with the full range shown by the whiskers. For data not normally distributed, Kruskal– Wallis test with Dunn’s post hoc analysis or Friedman’s test was used to compare different treatment groups. Statistical analyses were performed using the GraphPad Prism 6 software (GraphPad, San Diego, CA USA).
Results Effect of UPA and mifepristone on ciliary beat frequency There was an overall dose-dependent suppressive effect of UPA on ciliary beat frequency at all concentrations studied (P , 0.0001 for all
Effect of UPA and mifepristone on muscular contractility The basal tone, amplitude and frequency of smooth muscle contractions after treatment with UPA at 20 ng/ml or mifepristone 300 ng/ml did not differ from the control (P . 0.05), but were significantly reduced in a dose-dependent manner after treatment with UPA at 200 ng/ml or above, or with mifepristone at 3000 ng/ml or above (Fig. 2).
Effect of UPA treatment on expression of progesterone receptors and glycodelin Treatment with UPA at 200 ng/ml or above (P , 0.05), but not 20 ng/ ml (P . 0.05), resulted in significant dose-dependent up-regulation of mRNA expression of generic PGR, PR-B and glycodelin (Fig. 3).
Effect of UPA treatment on expression of adrenomedullin Treatment with UPA at 200 ng/ml or above (P , 0.05), but not 20 ng/ ml (P . 0.05), resulted in significant down-regulation of mRNA expression of adrenomedullin (Adm) in a dose-dependent manner (Fig. 4).
Discussion The Fallopian tube plays an important role in human conception. Fertilization normally takes place in the ampullary region of the tube. Transport of the early embryo through the isthmic region towards the uterus is then accomplished by the ciliary beating in the tubal
Figure 1 Effect of (a) UPA and (b) mifepristone on ciliary beat frequency (CBF) of human Fallopian tube. The treatment groups were compared using Kruskal – Wallis test with Dunn’s multiple comparison post hoc analysis. Dose – response correlation analyses are shown in the boxes.
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The above real-time PCR experiment was repeated for the Adm gene on the tubal epithelial tissue treated with various concentrations of UPA using the following primers: Adm (GenBank accession number NM 001124): 5′ -TGC CCA GAC CCT TAT TCG G-3′ , forward, 5′ -CCG GAG GCC CTG GAA GT-3′ , reverse.
treatment groups compared with control). The same dose-dependent suppressive effect was also observed with mifepristone at 3000 ng/ml or above (P , 0.0001 compared with control; Fig. 1).
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Figure 2 Effect of (a) UPA and (b) mifepristone on basal tone, amplitude and frequency of smooth muscle contraction of human Fallopian tube. The treatment groups were compared using Friedman test with Dunn’s multiple comparison post hoc analysis. A representative tracing of the muscular contraction recording is shown in (c). Dose – response correlation analyses are shown in the boxes. (*P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 compared with control; Friedman’s test with Dunn’s post hoc multiple comparison.)
mucosa together with spontaneous contractions of the smooth muscle layer, although the relative importance of ciliary beating, muscular contraction and tubal fluid secretion on embryo transport is uncertain. Factors that reduce ciliary beating or tubal contractility may interfere with conception by influencing embryo transit and altering the timing of the embryo arrival at the uterine cavity so that it is out of the optimal implantation window. This may potentially contribute to contraception.
To our knowledge, this is the first report on a significant suppressive effect of UPA on ciliary beat frequency and smooth muscle contraction in the human Fallopian tube at a concentration equivalent to the peak serum concentration after a pharmacological dose. A similar result was also observed with mifepristone, which was in line with a previous report also on muscular contractions (Wanggren et al., 2008), but in contrast to another report on ciliary beat frequency (Mahmood et al., 1998) in the human Fallopian tube, with the reason for the latter discrepancy being unclear. Earlier studies in mouse models have also suggested that mifepristone treatment has an effect on embryo retention and postcoital contraceptive effects (Roblero et al., 1987; Yang and Wu, 1990). Our results suggest an additional mechanism by which UPA may exert its action as an EC. UPA is thought to act as an EC mainly by delaying or inhibiting ovulation (Brache et al., 2013; Gemzell-Danielsson et al., 2013). An additional post-ovulatory mechanism, however, would potentiate its action especially if the drug is administered after ovulation has occurred which often happens in clinical practice. This may enhance the overall contraceptive efficacy of the method, and would be of great clinical benefit. There has been theoretical concern that inhibition of tubal function may imply an elevated risk of tubal ectopic pregnancy. A meta-analysis (Cleland et al., 2010), however, suggested no evidence of increased risk of ectopic pregnancy with the use of LNG and mifepristone for EC, probably because the EC reduces the overall risk of conception mainly by inhibiting or delaying ovulation. A recent post-marketing surveillance report also observed no additional risk of ectopic pregnancy after use of UPA (Levy et al., 2014). It should be noted that establishment of tubal ectopic pregnancy may not be solely explained by delayed embryo transport or embryo retention per se, but has to be accompanied by an altered tubal environment and/or embryo factors to result in tubal implantation (Shaw et al., 2010).
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Figure 2 Continued.
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Effect of UPA and mifepristone on tubal function
tube. Dose – response correlation analyses are shown in the boxes. (*P , 0.05, **P , 0.01, ***P , 0.001 compared with control; Kruskal – Wallis test with Dunn’s post hoc multiple comparison.)
The current study also reports for the first time the effect of UPA on several hormonal mediators which may relate to the effect of UPA onto tubal activity. Some studies have suggested that progesterone may suppress ciliary beating and muscular contraction in the Fallopian tube (Mahmood et al., 1998; Wanggren et al., 2008). Hence, it might seem surprising on first sight that UPA and mifepristone, both commonly regarded as anti-progestogens, actually inhibited tubal activity in line with what progesterone itself does. Based on our results, we postulated three possible explanations as described below. First, our results showed that UPA at pharmacological concentrations (200 ng/ml) or above up-regulated mRNA expression of the progesterone receptor (both generic and specific to PR-B). This may potentiate the progesterone signalling.
Secondly, UPA may exert mainly an agonistic instead of antagonistic effect on progesterone receptors in the Fallopian tube. The same has been speculated for mifepristone (Wanggren et al., 2008), although there is no direct evidence. It has been suggested that the drug has agonistic activity on the PR-A isoform, but partial agonist/antagonist activity on the PR-B isoform (Leo and Lin, 2008). Previous work in mice has suggested that both isoforms are expressed in the non-stimulated ovary, but PR-A expression is significantly reduced after FSH stimulation, until after hCG stimulation when expression of both are raised again. On the other hand, both PR-A and PR-B are expressed in both non-stimulated and stimulated oviducts, with increased expression post-hCG stimulation (Teilmann et al., 2006). This might explain the speculated agonistic action of UPA in the Fallopian tube in contrast to the antagonistic
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Figure 3 Effect of ulipristal on the mRNA expression of PGR and the specific progesterone receptor B (PR-B) isoform and glycodelin in human Fallopian
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condition. Hence, there is still uncertainty over the clinical relevance of our experimental results. Nonetheless our results did suggest a possible avenue for further studies of an EC mechanism through the Fallopian tube. In all, our results suggest that UPA, in resemblance to mifepristone, inhibits ciliary beat and muscular contraction of the human Fallopian tube at the pharmacological dose, probably through an agonistic effect on the tubal progesterone receptor. This may contribute additional mechanisms for its contraceptive action, although the actual clinical significance is yet to be determined.
Acknowledgements
Figure 4 Effect of ulipristal on the mRNA expression of adrenomedullin (Adm) in human Fallopian tube. Dose – response correlation analysis is shown in the box. (*P , 0.05, ***P , 0.001 compared with control; Kruskal – Wallis test with Dunn’s post hoc multiple comparison.)
action at the ovary. It has also been suggested that the activities of PR-A and PR-B could be differentially modulated by different progesterone receptor modulators in a gene-selective manner (Leo and Lin, 2008), and more work needs to be done in this regard to understand the roles played by PR-A and PR-B signalling on Fallopian tube function. Since the expression of glycodelin is mainly regulated by progesterone signalling, the up-regulation of glycodelin by UPA at pharmacological concentrations or above provided supportive evidence that UPA acts as agonist in the Fallopian tube. Thirdly, the suppression of adrenomedullin by UPA at pharmacological dose or above, as first demonstrated in this study, may provide an additional clue. Previous studies by our group have revealed that adrenomedullin plays an important role in enhancing the ciliary beat frequency and muscular contraction tone, amplitude and frequency in human Fallopian tube (Li et al., 2010). There is one report indicating that UPA can down-regulate adrenomedullin and its receptors in a dose-dependent manner in cultured human uterine leiomyoma cells (Xu et al., 2006). The relative importance of modulation through the progesterone and adrenomedullin signalling pathways would require further exploration. In our experiments and data analyses, we took the peak serum concentrations of the respective drugs as the pharmacological concentration at the end organ level. However, there is no data on whether the same drug level actually appears in the tubal fluid and tissue after ingestion of the oral tablet. Furthermore, the peak drug concentration is normally sustained only for a transient period after single-dose administration. Moreover, UPA is highly bound in the human blood circulation (4.9% to blood cells and 94.1% to plasma proteins), with a free fraction of just 1% (Gainer and Ulmann, 2003). Although the culture medium used in our in vitro experiments did contain protein from FBS, it is not certain whether this could resemble the actual pharmacological
Authors’ roles H.W.R.L. and P.C.H. conceived and designed the study. S.-B.L. developed the experimental protocols and conducted all the experimental work under supervision by W.S.-B.Y. and W.S.O.; H.W.R.L., S.-B.L., E.H.-Y.N. and P.-C.H. analysed the data. H.W.R.L. wrote the manuscript which was critically discussed, edited and approved by all co-authors.
Funding This work was supported by a Seed Fund from the Centre of Reproduction, Development and Growth, Faculty of Medicine, the University of Hong Kong.
Conflict of interest All authors have no conflict of interest to declare.
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We are grateful to Ms Joyce Yuen and other staff members of the Gynecology Team, Queen Mary Hospital for their assistance in patient recruitment and sample collection. We thank Laboratoire HRA Pharma for the free supply of the UPA compound for experimental use in this study. We also thank Professor Kristina Gemzell-Danielsson for her comments on the study results.
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