Anti-platelet therapy holds promises in treating adenomyosis: experimental evidence

Background: Recently emerging evidence indicates that endometriotic lesions are wounds undergoing repeated tissue injury and repair (ReTIAR), and platelets induce epithelial-mesenchymal transition (EMT), fibroblast-to- myofibroblast transdifferentiation (FMT), leading ultimately to fibrosis. Due to the commonality of cyclic bleeding as in endometriosis, adenomyotic lesions are also wounds that undergo ReTIAR, and we have recently provided evidence corroborating platelet-induced EMT, FMT and fibrogenesis in adenomyosis. This study sought to evaluate the effect of antiplatelet therapy in a mouse model of adenomyosis.

Adenomyosis was induced in 57 female ICR mice with neonatal dosing of tamoxifen, while another 12 (group C) were dosed with solvent only, serving as a blank control. Starting from 4 weeks after birth, hotplate test was administrated to all mice every 4 weeks. At the 16th week, all mice with induced adenomyosis were randomly divided into 6 groups: untreated, low- and high-dose Ozagrel, low- and high-dose anti-mouse GPIb α polyclonal IgG antibody to deplete platelets, and isotype-matched inert IgG non-immune antibody. Group C received no treatment. After 3 weeks of treatment, they were hotplate tested again, their uterine horns and brains were harvested, and a blood sample was taken to measure the plasma corticosterone level by ELISA. The left uterine horn was used for immunohistochemistry analysis. The brainstem nucleus raphe magnus (NRM) sections were subjected to immunofluorescence staining for GAD65. The depth of myometrial infiltration and uterine contractility were evaluated.

We found that both Ozagrel treatment and platelet depletion dose-dependently suppressed myometrial infiltration, improved generalized hyperalgesia, reduced uterine contractility, and lowered plasma corticosterone levels, improved the expression of some proteins known to be involved in adenomyosis and slowed down the process of fibrogenesis. It also elevated the number of GAD65-expressing neurons in the brainstem NRM, possibly boosting the GABAergic inhibition of pain due to adenomyosis.

This study further provides evidence that platelets play important roles in the development of adenomyosis. Anti-platelet treatment is efficacious in suppression of myometrial infiltration, improving generalized hyperalgesia, reducing uterine hyperactivity and systemic corticosterone levels. Collectively, these results demonstrate that anti-platelet therapy seems to be promising for treating adenomyosis.

Adenomyosis, Generalized hyperalgesia, Hotplate latency, Mouse, Ozagrel, Platelet, Uterine contractility * Correspondence: [email protected] †Equal contributors 2Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, Shanghai 200011, China 3Shanghai Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011, China Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 DOI 10.1186/s12958-016-0198-1

Adenomyosis is a common gynecologic disorder with a poorly understood pathogenesis [1]. As in endometriosis, it is characterized by the ectopic deposition and growth of endometrial glands and stroma deep and haphazardly into the myometrium [1]. It shares with endometriosis many similarities in terms of estrogen-dependency, pro- gesterone resistance, symptomology, and many molecu- lar aberrations but differs in risk factors, age at onset and, possibly, etiology [2]. Similar to endometriosis, our current knowledge of its pathophysiology is still woefully inadequate. Consequently, treatment of adenomyosis has been a challenge [3], with hysterectomy being the treat- ment of choice for severe symptomatic adenomyosis. Thus, medical treatment of adenomyosisis still an unmet medical need. Adenomyosis is first and foremost viewed as an estrogen-dependent disease, featuring increased local production of estrogen [4]. It also displays signs of inflammation, characterized by the constitutive activa- tion of NF-kB [5], increased macrophage infiltration [6], and elevated expression of COX-2, a rate-limiting enzyme in catalyzing prostaglandin (PG) E2 (PGE 2) [7], and increased production of proinflammatory cy- tokines and chemokines [8]. All existing therapeutics for adenomyosis are hormonal drugs. As with endometriosis, the ectopic endometrium in ade- nomyosis also experience cyclic bleeding. Yet bleeding, an indication of vascular injury, is a cardinal hallmark of a wound or tissue damage. Cons equently, a physiological process, called wound healing o r tissue repair, ensues. As such, platelets must be involved, as shown recently for endometriosis [9]. In fact, based on serial immunohisto- chemistry analyses of ectopic endometrium in a mouse model of adenomyosis, we recently report that activated platelets coincide with TGF-β1 release and the induction of TGF-β/Smad signaling pathway in adenomyosis, as well as evidence of epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibrobl ast transdifferentiation (FMT), resulting ultimately in fibrosis [10] and also smooth muscle metaplasia (Shen et al., unpublished data). These observations are confirmed in human adenomyosis [11]. Therefore, due to the commonality shared with endometriosis, i.e., cyclic bleeding, adeno- myotic lesions behave just like endometriotic lesions, which are essentially wounds that undergo repeated tissue injury and repair (ReTIAR) [9, 12, 13]. In light of the important roles that platelets play in the development of endometriosis [9, 14, 15] and adenomyosis [10, 11], one may wonder as whether anti-platelet therapy may have any potential in treating adenomyosis. This study was undertaken to test the hypo thesis that anti-platelet treatment, by either platelet de pletion or administration of Ozagrel, indeed has potential for therapeutic purposes.

Chemicals Ozagrel, a thrmboxane A2 (TXA 2) synthese inhibitor [16], was purchased from YaoDa Pharmacology Industry Company (Shenyang, China) and was dissolved in 0.9 % normal saline for intraperitoneal administration. The rat anti-mouse GPIb α polyclonal IgG antibody and its isotype-matched non-immune rat anti-mouse IgG anti- body were purchased from Emfret Analytics (Eibelstadt, Germany). Tamoxifen citrate was purchased from Fudan Forward Pharmaceutical Company (Shanghai, China). All other chemicals were purchased from Sigma unless stated otherwise. Animals and the procedure for induction of adenomyosis Four pregnant ICR mice with a gestational age of 15 –16 days were purchased from Shanghai Laboratory Animal Corporation (Shanghai, China) and each of them was housed in a single cage during the rest of the gestation period and the ensuing birth and nursing period. Their pups (1 day after birth) were sexed and the female pups were selected for use in this study. The same litter of pups and the dam were housed in the same cage until weaned. All mice were housed in an animal care facility under controlled conditions (20 °C, 12:12 light/dark cycle with lights on at 6:00 AM) and had free access to chow and fresh water. Following Parrott et al. [17, 18], and as reported previ- ously [2, 19, 20], adenomyosis was induced by orally dosing female neonatal mice with 1 mg/kg tamoxifen suspended in peanut oil/lecithin/condensed milk mix- ture (2:0.2:3, by volume) at a dose volume of 5 μl/g bodyweight from day 2 to day 5 after birth. Female con- trol neonatal mice, selected randomly, were fed similarly with the same amount of solvent, without tamoxifen. When these female mice reached 3 weeks of age, they were weaned and separated from the dams. All experiments were performed under the guidelines of the National Research Council ’s Guide for the Care and Use of Laboratory Animals [21] and approved by the institutional experimental animals review board of Shanghai OB/GYN Hospital, Fudan University. Experimental protocol This experiment was conducted side-by-side with another experiment evaluating the efficacy of epigallocatechin-3- gallate (EGCG) in treating adenomyosis in mice, as re- ported in [20]. Fifty-six female neonatal pups were orally dosed with tamoxifen from day 2 to day 5 after birth, while another 12 were dosed in similar fashion with the solvent only (control group, or group C). Starting from 4 weeks after birth, hotplate test was administered to all mice every 4 weeks, as described previously [2, 19] (see Additional file 1 for full description). At the 16th week Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 2 of 16 after birth, all mice dosed with tamoxifen were randomly divided into6 groups of roughly equal size, Group U ( n = 9), or the untreated group, received the vehicle only. Another two groups received, for 3 weeks, daily intraperi- toneal (i.p.) administration of either low-dose (12.5 μg/g) Ozagrel (Group L, n = 10) or of high-dose (25 μg/g) Ozagrel (Group H, n = 10). The Ozagrel doses were deter- mined based on the dosage given to an adult female and then converted from human to mouse based on body sur- face area and further adjusted based on each mouse ’s bodyweight measured every day before Ozagrel adminis- tration. The mice in group C received no treatment at all and served as a blank control. The other three groups were intravenously (i.v.) administrated with different doses of rat anti-mouse GPIb α polyclonal IgG and non-immune rat anti-mouse IgG: Group LD ( n = 10) received a low- dose (1 μg/g) rat anti-mouse GPIb α polyclonal IgG treat- ment; Group HD ( n = 9) received a high-dose (2 μg/g) rat anti-mouse GPIbα polyclonal IgG treatment; Group NI ( n = 8) received 1 μg/g non-immune (NI) rat anti-mouse IgG isotope-matched with the anti-GPIb α antibody; The mice in group C were treated the same as above. The dosages of rat anti-mouse GPIb α polyclonal IgG were determined based on instructions provided by Emfret Analytics. After the 3-week-long treatment period (at the 19th week), the final hotplate test was administered to all the mice with or without induced adenomyosis, after body- weight measurement. A 0.5 ml blood sample was taken between 9:00 and 15:00 of the dayfrom each mouse, and was used for the measurement of plasma corticosterone levels by enzyme linked immunosorbent assay (ELISA, see Additional file 1 for more details). After the blood samples were taken, all mice were sacrificed by perfusing the heart with formalin. For each mouse, both uterine horns were harvested and the uterine weight was re- corded. The left uterine horn was used for uterine con- tractility measurement (described in Additional file 1), and the right one was fixed in 4 % paraformaldehyde im- mediately after collection and then embedded in paraf- fin. The brains of all mice were harvested and analyzed (described below). The experiment design is shown schematically in Fig. 1. We evaluated the depth of myometrial infiltration of ec- topic endometrium following the criteria of Bird et al. [22], as reported previously [2]. Briefly, Grade 1 was de- fined to be the case where penetration of the ectopic endometrium into superficial myometrium; Grade 2, penetration into mid-myometrium; and Grade 3, penetra- tion beyond mid-myometrium. For ease of statistical ana- lysis, Grade 0 was recorded when there was a complete absence of any ectopic endometrium in the myometrium. For histological examination, serial 4- μm sections were obtained from each paraffin-embedded tissue block, and then 3 randomly selected sections were chosen for H&E staining to confirm pathologic diagnosis, as described previously [2, 19]. If endometrial glands and stroma were seen to be infiltrated into in myometrium, the diagnosis of adenomyosis was made. Histochemistry and immunohistochemistry analyses As described above, the right horn of uterus was fixed in 4 % formalin, and then embedded in paraffin. From each tissue block, serial 4- μm sections were obtained and subjected to H&E staining to confirm pathologic diagno- sis of adenomyosis, which was characterized by the pres- ence of endometrial glands and stroma that are completely enveloped by myometrium and discontinu- ous with the endometrial cavity [23, 24]. The rabbit polyclonal antibodies against progester- one receptor isoform B (PR-B, ab2765; Abcam, Hong Kong, China), phosphorylated-p65 (ab30623; Abcam), cyclooxygenase 2 (COX-2, #4842; CST, Boston, USA), oxytocin receptor (OTR, ab115664; Abcam), transient receptor potential cation channel, subfamily V, mem- ber 1 (TRPV1, ab31895;Abcam), collagen I (ab292; Abcam), collagen IV (ab6586; Abcam), and the rat monoclonal antibody against F4/80 (MCA497G; AbD- Serotec, Cambridge, England), CD41(ab33661), diluted to1:50,1:80,1:200, 1:100, 1: 1000,1:100, 1:500, 1:200 and 1:100, respectively, were used as primary antibodies. Serial 4- μm sections were made from paraffin- embedded tissue blocks. After routine deparaffinization and dehydration, some sections were randomly selected to be heat-retrieved with Tis-EDTA buffer (0.5 mol/L PH 9.0) over 98 °C for a total of 30 min for immuno- staining for PR-B, p-p65, COX-2, OTR and TRPV1, and the others were heat-retrieved with citric acid (0.01 mol/ L pH 6.0) over 98 °C for a total of 30 min for immuno- histochemistry analysis of collagen I, collagen IV, and F4/80. Then all sections were cooled naturally at room temperature, and then incubated with the primary anti- bodies at 4 °C overnight. After the sections were rinsed with PBS, they were incubated with the secondary anti- body (Sunpoly-HII, BioSunTechnclogy, Shanghai, China) for half an hour, or, for F4/80, with the goat anti-rat anti- body (AbDSerotec) for 1 hour. The bound antibody complexes were stained for 3 –5 minor until appropriate for microscopic examination with diaminobenzidine (DAB) (BioSunTechnclogy Co., Ltd) and then counter- stained with hematoxylin and mounted. Images were obtained with the microscope (Olympus BX51, Olympus, Tokyo, Japan) fitted with a digital camera (Olympus DP70, Olympus). Five randomly selected im- ages from 10 to 12 images on 2 –4 sections of each mouse were taken for each immunostaining marker to obtain a mean optional density value by Image Pro-Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA) as reported in [25]. Staining was defined via color intensity, and a color mask Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 3 of 16 was made. The mask was then applied equally to all im- ages, and measurement readings were obtained. Immuno- histochemical parameters assessed in the area detected included (a) integrated optical density (IOD); (b) total stained area (S); and (c) mean optical density (MOD), which is defined as MOD = IOD/S, equivalent to the mean intensity of stain in all positive cells. For F4/80, we counted the number of F4/80-positive macrophages from five randomly selected images and calculated their average. Myometrial OTR staining scores were calculated by multiplying the percentage of positive OTR cells per section (0 –100 %) by a semi- quantitative classifier for immunohistochemical staining intensity, which was scored as 0 if there was a complete absence of any staining, 1 for weak staining, 2 for mod- erate staining, and 3 for strong staining. Consequently, the staining scores ranged from a minimum of 0 to a maximum of 300. We used the mean score averaged over 5 randomly selected images. For all markers, the staining levels were scored on ec- topic endometrium in mice with induced adenomyosis. For mice in the control group or in mice without ectopic endometrium due to treatment, they were scored in the endometrium. We counted the number of macrophages (F4/80-positive) infiltrated into the ectopic endometrium in the entire focal field for mice with adenomyosis. For the mice in control group or mice that had none such lesion due to treatment, the number of F4/80-positive macrophages in endometrium was counted. The mouse spleen tissue was used for the positive im- munostaining of macrophages, while breast cancer tissue sections were used for positive control for other markers, Negative control sections were processed simi- larly, but using a non-immune rabbit or rat IgG instead of the primary antibody, or by omitting the primary anti- body from the incubation medium. No positive reaction was observed under these conditions. All sections were inspected a single investigator (BZ) without the know- ledge of the group identity. The positive and negative controls are shown in Additional file 1: Figure S2 of Supplemental Information. GAD65 immunofluorescence staining of neurons in brainstem nucleus raphe magnus The procedure has been reported previously in [26]. Briefly, the mouse brains containing the nucleus raphe magnus (NRM) were harvested and immediately embed- ded in O.C. T. compound in liquid phlegm after the mice were sacrificed. The NRM sections were between 5.68 and 6.48 mm to the bregma and the NRM was lo- cated 1.72 –2.68 mm interaurally, as described previously [26]. Serial 6- μm sections were performed on a cryostat for each block and stored at the temperature below −20 °C until use. The sections were incubated in goat anti mouse serum (BioSunTechnclogy) for 10 min and then incubated in mouse anti primary antibody against GAD65 (ab26113, Abcam; 1:1,000) at 4 °C overnight. GAD65 is expressed in the cytoplasm of presynaptic neurons. The sections were rinsed with PBS (pH 7.4) and incubated in secondary antibody mixed with DyLight649 (E032610, Earthox, San Francisco, USA) for one hour, and then rinsed with PBS (pH 7.4) again and mounted. Fig. 1 Schematic illustration of the experiment design of this study. Untreated: mice that received no treatment; Lo-dose Ozagrel (Oza): mice treated with 12.5 μg/g Oza (low-dose); Hi-dose Oza: mice treated with 25μg/g Oza (high-dose). Lo-dose PD: mice treated with 1μg/g rat anti-mouse GPIbα polyclonal IgG antibody for platelet depletion (PD); Hi-dose PD: mice treated with 2μg/g rat anti-mouse GPIbα polyclonal IgG antibody; Non- immune: received 1μg/g non-immune (NI) rat anti-mouse IgGisotope-matched with the anti-GPIbα antibody See text on experiment protocol for more details Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 4 of 16 Images were obtained with a microscope (Olympus BX51) fitted with a digital camera (Olympus DP70). Five randomly selected images out of 6 –7 sections of each mouse were taken for each immunostained parameter to count the numbers of GAD65-positive (red) cells in the NRM, located between 1.72 mm and 2.68 mm interau- rally, and the mean was calculated. Statistical analysis Comparison of the distributions among two or more groups of continuous variables was made using the Wil- coxon and Kruskal-Wallis tests, respectively, and the paired Wilcoxon test was used when the before-after comparison was made for the same group of subjects. Pearson’s or Spearman ’s rank correlation coefficient was used when evaluating correlations between two variables when both variables were continuous or when at least one variable was ordinal. To see whether Ozagrel treat- ment or platelet depletion and other possible factors were responsible for the change in hotplate latency be- fore and after the treatment, a multiple linear regression model was used. To see whether there is trend in immu- nostaining levels as a function of the depth of myome- trial infiltration, Jonckheere trend was used. To determine correlates of depth of myometrial infil- tration, we used the Cox proportional odds logistic re- gression model. This model assumes, implicitly, that the data were ordered categorical data, with an implicit underlying order (scale of severity) in the data [27], with 4 categories corresponding to Grade 0, I, II, and III infiltration. P values of less than 0.05 were considered statistically significant. All computations were made with R statistics software system version 3.3.1 [28].

Consistent with Parrott et al. [17, 18] and as previously reported [2, 19], we found that adenomyosis was suc- cessfully induced in all (100 %) mice dosed with tamoxi- fen but none in un-dosed mice. Ozagrel was well-tolerated, as no mice in either LO or HO group died, and we found nothing unusual in these mice. In HD group, however, 1 mouse died after it re- ceived the 4th injection of the depletion antibody, and 2 appeared to be lethargic. In the LD group, no mice died and nothing appeared unusual. There was no difference in platelet counts between the mice in groups UT, NI, LO, and HO at the end of the experiment. However, the platelet count in mice in both LD and HD groups was reduced by 99.6 and 99.7 % as compared with those in the NI group, demonstrating the effectiveness of platelet depletion in these two groups. Treatment effect on the depth of myometrial infiltration, hotplate latency, and uterine and bodyweight We first evaluated the effect of Ozagrel treatment or plate- let depletion on the depth of myometrial infiltration. We found that, compared with un treated mice, mice treated with either low- or high-dose O zagrel had significantly less infiltration (both p-values <0.001; Fig. 2a). Compared with NI mice, mice in either LD or HD group also had signifi- cantly less infiltration (both p-values <0.001; Fig. 2a). Mice in HO and HD groups appeared to have less infiltration than those in the LO or LD group (Fig. 2a). The multiple linear regression analysis suggested that both Ozagrel treatment and platelet depletion significantly and dose-dependently reduced the depth of myometrial infiltration (regression coefficient β = −0.956, p =3 . 2 × 1 0−7, and β = −0.627, p =1 . 5 × 1 0-6, respectively, R2 =0 . 6 2 ) , b u t NI mice had deeper infiltration ( β =0 . 8 5 6 ,p =0 . 0 1 5 ) . T h e Cox regression analysis yielded similar results (all three p- values <0.028). We found that there is a significant difference in uterine weight vs. bodyweight ratio among the 7 groups of mice (p < 0.001; Fig. 2b). Using the ratio as a dependent variable and the bodyweight after treatment, the induction of ade- nomyosis, dose of Ozagrel, the non-immune IgG injection or not, and the dose of antibody used in platelet depletion as covariates, we found, via a linear multiple regression, that the non-immune IgG was positively associated with the ratio ( p <0 . 0 1 ;R2 = 0.60; Fig. 2b) while both Ozagrel and anti-platelet doses were negatively associated with the ratio (both p-values <0.001). Similar results were obtained for the uterine weight (data not shown). The induction of adenomyosis was significantly asso- ciated with reduced latency just 4 weeks after birth or 23 days after the completion of tamoxifen dosing ( p < 0.05; Fig. 2c). At 8 weeks after birth, the difference in hotplate latency amount the four groups of mice be- came very pronounced ( p < 0.001; Fig. 2c), with the mice with induced adenomyosis all having reduced la- tency ( p < 0.001). At 12 and 16 weeks after birth, the mice with induced adenomyosis had further progres- sively reduced hotplate latency (both p-values <0.001; Fig. 2c). In all mice with induced adenomyosis, the la- tency evaluated at week 8, 12 and 16 was all significantly decreased as compared with the previously evaluated latency (all p-values <0.001; Fig. 2c). After platelet depletion or treatment with Ozagrel for 3 weeks, however, the hotplate latency was signifi- cantly improved in a dose-dependent fashion ( β = 4.008, p < 0.001, and β = 1.792, p < 0.001, respectively, in a multiple linear regression analysis, R2 = 0.73; Fig. 2c). In contrast, the presence of adenomyosis and the injection of the dummy antibody were associated with decrease in hotplate latency ( p <0 . 0 0 1 a n dp < 0.01, respectively). Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 5 of 16 While there was no difference in bodyweight among the 7 groups of mice at 4 and 8 weeks after birth (both p-values >0.05; Fig. 2d), the difference became statisti- cally and progressive significant starting from week 12 (p <0 . 0 5 a n dp < 0.01), and was significant at the end of the experiment (p < 0.001; Fig. 2d). The multiple linear re- gression analysis using pre-treatment bodyweight, pres- ence of adenomyosis, and the dose of Ozagrel, the non- immune IgG injection or not, and the dose of antibody used in platelet depletion as covariates, we found that only the induction of adenomyosis was negatively associated with the bodyweight ( p <0 . 0 0 1 ;R2 = 0.66) while the pre- treatment bodyweight was positively associated with the bodyweight ( p < 0.001). In other words, neither Ozagrel treatment nor platelet depletion had any impact on body- weight, but the induction of adenomyosis had a negative impact due, possibly, to adenomyosis-associated pain and/ or pain-induced suppression of appetite. Treatment effect on uterine contractility There was a significant difference in the amplitude of uter- ine contractility after drug treatment among the 7 groups (p < 0.001; Fig. 3a). In particular, both untreated and NI mice had a significantly higher amplitude as compared with the mice without adenomyosis (both p-values < 0.001; Fig. 3A). Regressing the amplitude on the Ozagrel dose, the presence of adenomyosis, the non-immune IgG injection or not, and the dose of antibody used in platelet depletion indicated that, while Ozagrel treat- ment and platelet depletion were both negatively asso- ciated with the amplitude in a dose-dependent fashion (both p-values < 0.001), the induction of adenomyosis and the IgG injection were positively and significantly associated with increased amplitude ( p < 0.001, and p <0 . 0 1 , r e s p e c t i v e l y ;R2 = 0.60). Similarly, there was a significant difference in the fre- quency of uterine contractility after drug treatment ( p < Fig. 2 Some summary results of the experiment. a Boxplot of the depth of myometrial infiltration among different groups of mice with induced adenomyosis. b Boxplot of uterine vs. body weight ratio at the end of 3-week-long RSV treatment among different groups of mice. c Kinetic changes in average hotplate latency among different treatment groups. d Kinetics of mean bodyweight among different treatment groups. UT: Untreated group; CT: Blank control group; LO: low-dose Ozagrel group; HO: high-dose Ozagrel group; LD: platelet depletion using low-dose anti- body; HD: platelet depletion group using high-dose antibody; NI: mock depletion using non-immune antibody; Tx: Treatment; Exp ’t: Experiment. The arrows showing different tests are the administrated hotplate tests. Theblue line in (c) and (d) indicates the duration of the treatment. In (a)a n d(b), the statistical significance of the difference between the testing group and the comparison group was indicated, and“***” means that the p-value is less than 0.001. In (d), the statistical significance was referring to the difference among the 7 groups of mice. *:p < 0.05; **: p < 0.01; ***: p < 0.001 Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 6 of 16 0.01; Fig. 3B). Regressing the frequency (log-transformed to enhance normality) on the uterine weight vs. body- weight ratio, Ozagrel dose, the presence of adenomyosis, the IgG injection or not, and the dose of antibody used in platelet depletion indicated that the induction of ade- nomyosis was positively associated with the contractile frequency ( p < 0.01; R2 = 0.30) while Ozagrel treatment and platelet depletion were both negatively associated with the frequency ( p < 0.001 and p < 0.01, respectively). The contractile amplitude correlated positively with the contractile frequency ( r = 0.51, p < 0.001). Both the amplitude and frequency were found to correlate posi- tively with the uterine vs. bodyweight ratio ( r = 0.79, p < 0.001, and r = 0.52, p < 0.001). Treatment effect on plasma level of CORT We found that there is a significant difference in plasma CORT levels among the 7 groups of mice ( p < 0.001; Fig. 3c). In particular, the untreated mice had a signifi- cantly elevated CORT levels as compared with mice without adenomyosis, so did the NI mice (both p-values <0.001; Fig. 3c). Regressing the plasma CORT level (log- transformed to enhance normality) on the Ozagrel dose, the presence of adenomyosis, the non-immune IgG in- jection or not, and the dose of antibody used in platelet depletion indicated that both the induction of adeno- myosis and the injection of the dummy antibody were positively associated with the CORT levels ( p < 0.001 and p < 0.05, respectively) while both Ozagrel treatment and platelet depletion were dose-dependently and nega- tively associated with the CORT levels (both p-values <0.001; R2 = 0.59). We also found that the CORT levels correlated negatively with the hotplate latency ( r = −0.90, p < 0.001; Fig. 3d), suggesting that pain severity may be positively associated with the severity of stress. Effect of antiplatelet treatment on platelet aggregation, macrophage infiltration, and select markers in ectopic endometrium We evaluated the immunoreactivity results for all mice. Figure 4 shows the extent of platelet aggregation and of macrophage infiltration and p-p65, PR-B, COX-2, and Fig. 3 Summary results on uterine contractility and plasma corticosterone levels. a Boxplot of the amplitude of uterine contractility among different groups of mice with induced adenomyosis. b Boxplot of the frequency of uterine contractility among different groups of mice. c Boxplot of the plasma corticosterone levels among different groups of mice. d Scatter plot showing the relationship between hotplate latency and the plasma corticosterone levels. In ( a), (b) and ( c), the statistical significance of the difference between the testing group and the comparison group was indicated. *: p < 0.05; **: p < 0.01; ***: p < 0.001. In ( d), each letter represents one mouse, and the alphabet indicates the group identity, which is the same as used in Fig. 2c, d. The correlation coefficient, with its statistical significance level, is shown in the figure Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 7 of 16 TRPV1 immunostaining in ectopic endometrium among different groups. For COX-2, the staining was predomin- antly localized in the cytoplasm of glandular epithelial cells in ectopic and eutopic endometrium. Both PR-B and p-p65 staining was localized primarily in the nuclei of glandular epithelial cells of eutopic and ectopic endo- metrium while TRPV1 staining was seen mainly in the cytoplasm and cell membranes of glandular epithelial cells; We found that there was a significant difference in im- munoreactivity to PR-B, p-p65, COX-2, and TRPV1 in ectopic/eutopic endometrium and to OTR in myome- trium among different groups (all p-values <0.01; Figs. 5 and 6, and Table 1). In particular, multiple linear regres- sion analyses (all immunoreactivity levels were square- root transformed to improve normality unless stated otherwise) indicated that while adenomyosis induction was associated with the increase (decrease for PR-B) while Ozagrel treatment or platelet depletion was associ- ated, in a dose-dependent manner, with a significant re- duction (increase for PR-B) of immunoreactivity to all these proteins or the extent of platelet aggregation/ macrophage infiltration (all p-values <0.01, with R2 ran- ging from 0.31 to 0.76; Table 1 and Fig. 5). In addition to these markers, we also performed im- munostaining of lesional OTR, a marker of SMM, and collagen I and IV, markers of fibrosis, in adenomyotic lesions, as well as OTR in myometrium, which was likely responsible for uterine hyperactivity. OTR staining was localized in both the cell membrane and the cytoplasm of glandular epithelial and stromal cells as well as myo- metrial smooth muscle cells (Fig. 6). We scored OTR staining levels in epithelial/stromal cells and myometrial muscle cells separately. No difference in OTR staining levels in the epithelial component was found (data not shown), and hence only the data in the stromal compo- nent were demonstrated. Both collagen I and collagen IV staining was seen nearly uniformly in extracellular matrix of the ectopic endometrial stromal tissues, irrespective of the proximity to the glandular epithelial cells or not. We found that for all these markers, the presence of adenomyosis and, in the case of lesional and myo- metrial OTR staining, the injection of non-immune antibodies were positively associated with the staining levels while Ozagrel treatment and platelet depletion were associated, in a dose-dependent manner, with a significant reduction of immunoreactivity to all these proteins (all p-values <0.001; Fig. 5; Table 1). We found that the immunostaining levels of these pro- teins were all highly correlated, with the positive correlation coefficients ranged from 0.69 to 0.95 (PR-B excluded; all p- values <0.001) and the negative correlation coefficients ranged from −0.72 to −0.93 (all p-values < 0.001 for PR-B vs. others). Fig. 4 Representative photomicrographs of indicated immunostaining in eutopic (for blank control group) or ectopic (all other groups) endometrium among different treatment groups, which are arranged in different columns. CD41: CD41-labeled platelets; F4/80: F4/80-labled macrophages (indicated by yellow arrows); p-p65: phosphorylated form of NF-kB p65 subunit; PR-B: progesterone receptor isoform B; COX-2: Cyclooxygenase 2; TRPV1: transient receptor potential cation channel, subfamily V, member 1. Blank: blank control endometrium; Hi-Oza: high-dose Ozagrel group; Lo-Oza: low-dose Ozagrel group; Hi-Dep: platelet depletion with high-dose antibody; Lo-Dep: platelet depletion withlow-dose antibody; IgG: non-immune mock antibody. Scale bar = 125μm Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 8 of 16 Fig. 5 Summary of immunohistochemistry results. Boxplot of immunoreactivity against CD41 ( a), the number of F4/80+ positive macrophages (b), p-p65 c, PR-B ( d), COX-2 ( e), TRPV1 ( f),OTR (g), myometrial OTR ( h), Collagen I ( i), and Collagen IV ( j) in ectopic/eutopic endometrium. The group labels are the same as used in Fig. 2 Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 9 of 16 We found that the extent of platelet aggregation and of the macrophage infiltration as well as the immunore- activity to PR-B, p-p65, COX-2 and TRPV1 were all highly correlated with the depth of myometrial infiltra- tion (all positive except PR-B, which was negative, and the Spearman ’s correlation coefficients ranged from 0.69 to 0.88 ( −0.87 for PR-B), all p-values <0.001). The Jonc- kheere trend test indicated that all these immunostain- ing levels were significantly associated with the depth of myometrial infiltration (all p-values <0.001; Fig. 7). A multiple linear regression analysis indicated that the OTR and PR-B staining levels in ectopic endometrium were the only 2 co-variables that are associated with the depth of myometrial infiltration (OTR, positive associ- ation, p = 6.6×10−15, PR-B, negative association, and p = 0.0020, respectively; R2 = 0.83). Effect of treatment on the number of GAD65-positive neurons in the brainstem nucleus raphe magnus (NRM) To see whether Ozagrel treatment and platelet depletion had any effect on the GABAergic inhibition system in the NRM, we performed an immunofluorescent staining of GAD65 in the NRM (Fig. 8a) and counted the number of GAD65-positive and synapsin I-positive neurons in the NRM. This number would be a measure of the number of GAD65-expressing neurons in the NRM. We found that there is a significant difference in the number of GAD65-positive neurons in the NRM among the seven groups ( p < 0 . 0 0 1 ;F i g .8 b ) .Am u l - tiple linear regression analysis (the number of cells was square-root transformed to improve normality) indicated that while adenomyosis induction was asso- ciated with the reduction in the number of GAD65- positive neurons in the NRM ( p < 0.001) as previously reported [26] while both Ozagrel treatment and plate- let depletion were associated dose-dependently with a significant increase of the number of GAD65-positive neurons (both p-values <0.001; R2 = 0.92). The number of GAD65-positive neurons (log-trans- formed) in the NRM was found to be positively corre- lated with the hotplate latency after treatment ( r = 0.88, p < 0.001; Fig. 8c). It also was found to be negatively cor- related with the plasma CORT levels ( r = −0.86, p < 0.001; Fig. 8D). Fig. 6 Representative immunohistochemisty staining of markers of smooth muscle metaplasia and fibrosis in ectopic and eutopic endometrium. Different rows indicate different proteins in different groups (arranged in different columns) with different doses of Ozagrel, different doses of rat anti-mouse GPIbα polyclonal IgG and non-immune rat anti-mouse IgG. For oxytocin receptor (OTR), M stands for staining in the myometrium, stromal and gland epithelium was separately evaluated. Magnification in all figures: ×400. Scale bar = 125 μm Table 1 Results from early/later platelet depletion experiment. All results were based on multiple regression analyses with the independent variable square-root transformed and dummy vari- ables indicating the presence or absence of adenomyosis, non- immune IgG antibody injection or not, the dosage of antibody to deplete platelets and the dosage of Ozagrel as co-variables Name Induction of adenomyosis Ozagrel treatment Platelet depletion R2 Extent of CD41+ platelet aggregation ↑ *** ↓ *** ↓ *** 0.54 Number of infiltrated F4/ 80+ macrophages ↑ *** ↓ ** ↓ * 0.30 Phosph-p65 ↑ *** ↓ ** ↓ ** 0.31 PR-B ↓ *** ↑ *** ↑ *** 0.73 COX-2 ↑ *** ↓ *** ↓ *** 0.48 TRPV1 ↑ *** ↓ *** ↓ *** 0.47 OTR ↑ *** ↓ *** ↓ *** 0.58 Collagen I ↑ *** ↓ *** ↓ * 0.29 Collagen IV ↑ *** ↓ ** ↓ *** 0.33 Myometrial OTR ↑ *** ↓ *** ↓ *** 0.77 ↓: Denotes that the immunoreactivity to this protein at hand was significantly decreased based on multiple linear regression analysis; ↑Denotes that the immunoreactivity to this protein of interest was significantly increased based on multiple linear regression analysis. The R2 value of the corresponding regression model is shown at the right-most column. Symbols of statistical sig- nificance levels: *: p<0.05; **: p<0.01; ***: p<0.001 Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 10 of 16 Fig. 7 (See legend on next page.) Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 11 of 16 Factors associated with the uterine contractility Both contractile amplitude and frequency correlated posi- tively with the myometrial OTR staining levels ( r =0 . 8 2 ,p < 0.001, and r = 0.35, p < 0.01, respectively; Additional file 1: Figure S3A, B of Supplemental Information) and also with the lesional OTR staining levels in the ectopic endomet- rium (r =0 . 9 4 ,p < 0.001, and r =0 . 5 0 ,p <0 . 0 0 1 ; A d d i t i o n a l file 1: Figure S3 c, d of Supplemental Information). For contractile amplitude, the multiple linear regres- sion incorporating uterine weight/bodyweight ratio, (See figure on previous page.) Fig. 7 Summary results on immunohistochemistry measures as a function of the depth of myometrial infiltration. Boxplot of the extent of platelet aggregation (a), extent of macrophage infiltration (b), immunoreactivity against p-p65 (c), PR-B (d), COX-2 (e), TRPV1 (f), OTR (g), myometrial OTR (h), collagen I (i), and collagen IV (j) in ectopic endometrium as a function of the depth of myometrial infiltration of endometrial tissues. The p-value shown in each figure is the statistical significance of the Jonckheere trend test Fig. 8 a Micrographs of immunofluorescent staining of GAD65 in the nucleus raphe magnus (NRM) in different groups of mice. Both GAD65- and Synapsin I-positive neurons were identified, as indicated by white arrows. To see the picture more closely, the area of interested was amplified three times. The original amplification:x400. The scale bar represents 125μm. b Boxplot showing the number of GAD65+ neurons in the NRM among different treatment groups. The dashed line represents the median value of all mice. Blank: blank control endometrium; Hi-Oza: high-dose Ozagrel group; Lo-Oza: low-dose Ozagrel group; Hi-Dep: platelet depletion with high-dose antibody; Lo-Dep: platelet depletion with low-dose antibody; IgG: non-immune mock antibody.c Scatter plot of hotplate latency vs. the log-transformed number of GAD65-positive neurons in the NRM for all groups of mice;d Scatter plot of plasma corticosterone levels vs. the log-transformed number of GAD65-positive neurons in the NRM for all groups of mice. Each alphabet in the figure represents one experimental observation, and thealphabets are the abbreviations of different treatment groups. C: Blank control; U: Untreated; o:Low- dose Ozagrel; O: High-dose Ozagrel; d: Platelet depletion using low-dose antibody; D: Platelet depletion using high-dose antibody; N: Non-immune IgG. The correlation coefficient and its statistical significance levels are shown in (c)a n d(d). ***: p < 0.001 Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 12 of 16 depth of myometrial infiltration (0 if no adenomyosis), extent of platelet aggregation and macrophage infiltra- tion and all immunostaining measurements in ectopic endometrium identified the depth of myometrial infiltra- tion ( p < 0.01), uterine weight/bodyweight ratio ( p < 0.001), the extent of platelet aggregation ( p < 0.001), and the number of infiltrating macrophages ( p < 0.01) as four covariates that were associated with the contractile amp- litude ( R2 = 0.94). For contractile frequency, we found, through multiple linear regression analysis, that only the uterine weight/bodyweight ratio ( p < 0.001), lesional and myometrial OTR staining levels (both p < 0.05), and PR- B staining levels ( p < 0.05) were associated with the con- tractile frequency ( R2 = 0.42). Determinants of thermal response latency after treatment We carried out a multiple linear regression analysis to identify which factors potentially determine the change in thermal response latency before and after drug treat- ment using the pre-treatment latency, bodyweight, depth of myometrial infiltration (grade = 0 if no adenomyosis), uterine weight vs. bodyweight ratio, amplitude and fre- quency of uterine contraction, and presence of adeno- myosis as covariates. We found that the uterine weight vs. bodyweight ratio ( p < 0.01), contractile amplitude ( p < 0.01), the presence of adenomyosis ( p < 0.001) and the depth of myometrial infiltration ( p < 0.001) were all negatively associate with the change in before-after hot- plate latency ( R2 = 0.90).

We have provided evidence that anti-platelet treatment, through either platelet depletion or Ozagrel treatment, resulted in the suppression of myometrial infiltration, improved generalized hyperalgesia, reduced uterine weight vs. bodyweight ratio and stress level, and reduced amplitude and frequency of uterine contraction in mice with induced adenomyosis. The anti-platelet treatment also improved the expression of some proteins known to be involved in adenomyosis and reduced the number of infiltrating macrophages. In particular, it reduced the lesional expression of OTR, a SMM marker [29], and of collagen I and IV, markers of extracellular matrix de- posits and thus fibrosis. Moreover, it increased the num- ber of GAD65-expressing neurons in the brainstem NRM, thus likely boosting the GABAergic inhibition of pain due to adenomyosis, which in turn helps pain relief and reduces the stress level. Our data are consistent with our finding that increased platelet aggregation and the extent of fibrosis in both mouse and human adenomyosis [10, 11]. They are also consistent with our previous report that anti-platelet therapy is effective in treating endometriosis in mouse [9, 14, 30] and that the expression of tissue factor in adenomyosis is elevated [5, 31]. Tissue factor plays a critical role in the initiation of platelet activation and co- agulation [32]. In addition, considerable experimental data [33, 34] and limited clinical data [35] support the involvement of hyperprolactinemia in adenomyosis, yet prolactin is a potent cofactor for platelet aggregation [36, 37]. These data, taken together, seem to suggest that patients with adenomyosis may be in a hypercoagulable state as those with endometriosis [38]. This may explain the report of cerebral infarcts associated with adeno- myosis [39] and increased mean platelet volume in women with adenomyosis [40]. Our data are also consistent with our previous reports that PR-B expression in adenomyosis is reduced [41] due possibly to PR-B promoter hypermethylation [42]. In addition, they are consistent with reported constitutive acti- vation of NF- κB [5], increased expression of COX-2 [7], TRPV1 [43] and OTR in adenomyosis [29, 43, 44]. The in- creased uterine contractility, and, in particular, its close as- sociation with the OTR expression and with the reduced hotplate latency as reported in this study are consistent with what we reported in human adenomyosis [44]. In other words, the mouse model used in this study recapitu- lates several important features of human adenomyosis, i.e., inflammation and angiogenesis as displayed by the consti- tutive activation of NF- κB and increased COX-2 but de- creased PR-B expression in adenomyotic lesions, increased uterine weight and presumably enlarged uterus, increased generalized hyperalgesia, and elevated uterine contractility, due possibly to elevated myometrial OTR expression. Re- markably, anti-platelet treatment either reversed or abro- gated these changes. We note that the anti-platelet treatment achieved the therapeutic effects very similar to EGCG [20, 26, 45] and resveratrol [46, 47] as we reported earlier. However, it is perhaps no coincidence that EGCG is anti-platelet [48] and so is resveratrol [46]. In fact, some compounds that are reported to be promising in treating adenomyosis in preclinical and clinical studies, such as andrographolide [2, 5, 49], valproic acid [19, 50, 51], and statins [52, 53], all turn out to be anti-platelet [54 –57]. Even danazol, a once- popular, FDA-approved drug for treating endometriosis, has long been reported to have anti-platelet effect [58, 59]. That said, we should emphasize that, despite promis- ing results of anti-platelet treatment by either platelet depletion or Ozagrel treatment as shown here, we are not advocating their use in clinical setting per se , even though Ozagrel is a prescription drug as of now. Adeno- myosis is a benign disease and certainly not life- threatening. As such, it places higher premium on drug safety as compared with other life-threatening diseases such as cancer. While Ozagrel is generally safe and holds promises in treating adenomyosis, the hemorrhage risk it entails deserves caution. This study was meant to be a Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 13 of 16 proof-of-concept study, demonstrating the therapeutic potential of anti-platelet therapy for adenomyosis. It is not intended to advocate Ozagrel per se for the treat- ment of human adenomyosis. More research is needed to determine which anti-platelet compound has the de- sirable benefit-to-risk ratio in treating adenomyosis. While the exact mechanisms of action for anti- platelet therapy remain to be investigated, it is possible that anti-platelet treatment suppresses the activation of the TGF- β1/Smad3 signaling pathway and the expres- sion of ER- β, both of which can be induced by activated platelets [15, 60]. In addition, the treatment suppresses the activation of NF- κB, which also can be induced by platelets (Zhang et al., unpublished data). Moreover, ac- tivated platelets express P-selectin (CD62P/GMP-140) on their cell surface [61 –63], which binds to its ligand, P-selectin glycoprotein ligand-1 (PSGL-1), that is expressed on the cell surface of most leukocytes, such as neutrophils, monocytes, Th1 lymphocytes, eosino- phils, and basophils, and facilitates inflammation, hemostasis, thrombosis, and the growth and metastasis of cancer [62, 64]. P-selectin interacts with PSGL-1, a transmembraine homodimer, to mediate the rolling of leukocytes on stimulated endothelial cells and the het- erotypic aggregation of activated platelets and leuko- cytes [65], and activate mitogen-activated protein kinases (MAPKs) [66] and β2 integrins [67]. Hence, anti-platelet treatment should abrogate or attenuate in- flammation caused by adenomyosis, as seen in reduced p - p 6 5e x p r e s s i o ni nt h i ss t u d y . Anti-platelet therapy can also suppress neurite outgrowth and thus hyperinnervation in adenomyosis [68, 69] since ectopic endometrial stromal cells secrete platelet inducers such as thrombin and thromboxane A2 (TXA2)[ 7 0 ] .T X A2 has been reported to stimulate neurite outgrowth in cere- bral cortical neurons [71], and we have also found that it can do so in dorsal ganglia root neurons [72]. Since TXA 2, PGH2,a n dP G I2 have been reported to be potent inducers of uterine contractility [73] and uterine contractility is doc- umented to be correlated with the severity of dysmenor- rhea in adenomyosis [44], the suppression of platelet activation and the resultant COX-2 down-regulation may suppress hyperinnervation and uterine hyperactivity, thus responsible for improved gene ralized hyperalgesia and re- duced plasma CORT levels. The reduced plasma CORT levels in mice with anti- platelet treatment is likely to result from the modulation of chronic stress (adenomyosis-induced pain) response through GABA receptors as in a chick model of acute stress [74]. Alternatively, adenomyosis-induced pain or hyperalgesia may result in synaptic dysfunction, for ex- ample, HDAC-mediated impairment of GABA synaptic inhibition in the brainstem NRM [75]. However, whether suppression of platelet activation may restore the GABA synaptic inhibition in NRM through the reduction of HDAC activity remains to be clarified.

This study further provides evidence that platelets play important roles in the development of adenomyosis. In addition, this study demonstrates that anti-platelet treat- ment is efficacious in suppressing myometrial infiltra- tion, improving generalized hyperalgesia, reducing both uterine hyperactivity and systemic CORT levels in mice with induced adenomyosis. Collectively, these re- sults demonstrate that anti-platelet therapy holds prom- ises as a non-hormonal treatment for treating adenomyosis. Additional file Additional file 1: Supplemental materials [76 –80]. (PDF 358 kb) Abbreviations CORT: Corticosterone; COX-2: Cyclooxygenase-2; EGCG: Epigallocatechin-3- gallate; EMT: Epithelial-mesenchymal transition; ER- β: Estrogen receptor β; FDA: FMT: fibroblast-to-myofibroblast transdifferentiation; GABA: γ- aminobutyric acid; GAD65: Glutamic acid decarboxylase 65; H&E: Hematoxylin and eosin; HD: Platelet depletion by high-dose rat anti- mouse GPIb α polyclonal IgG treatment; HDAC: Histone deacetylase; HO: High-dose Ozagrel treatment; LD: Platelet depletion by low-dose rat anti-mouse GPIb α polyclonal IgG treatment; LO: Low-dose Ozagrel treatment; MAPK: Mitogen-activated protein kinase; NI: Non-immune rat anti- mouse IgG isotope-matched with the anti-GPIb α antibody; NRM: Nucleus raphe magnus; OTR: Oxytocin receptor; PGH 2: Prostaglandin H2, PGI 2 prostaglandin I2; p-p65: Phosphorylated p65 subunit; PR-B: Progesterone receptor isoform B; PSGL-1: P-selectin glycoprotein ligand-1; ReTIAR: Repeated tissue injury and repair; SMM: Smooth muscle metaplasia; TGF-β1: Transforming growth factor β1; TRPV1: Transient receptor potential cation channel, subfamily V, member 1; TXA 2: Thromboxane A2 Acknowledgment The authors would like to thank the Administration of Wenzhou People ’s Hospital for its support and encouragement, and funding agencies for their financial support. This paper has been presented orally at the First Congress of the Society of Endometriosis and Uterine Disorders (SEUD) held in Paris on May 9, 2015. Funding This work was supported in part by grant Y14H040004 (YMC) from the National Science Foundation of Zhejiang Province, grants 81471434 (SWG), 81270676 (SWG), 81530040 (SWG), and 81370695 (XSL) from the National Science Foundation of China. None of the funders, however, has any role in the design of the study, and the collection, analysis, and interpretation of data and in writing the manuscript. Availability of data and materials The data used in this study are available upon request. Authors' contributions SWG conceived and designed the study, performed data analysis and data interpretation, and drafted the manuscript. BZ and YMC carried out most of the experiment, XLS and XSL provided assistance in immunohistochemistry analysis. All participated in writing up the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Zhu et al. Reproductive Biology and Endocrinology (2016) 14:66 Page 14 of 16 Consent for publication Not applicable. Ethics approval This study was approved by the institutional experimental animals review board of Shanghai OB/GYN Hospital, Fudan University. Acknowledgment of financial support This research was supported in part by grant Y14H040004 (YMC) from the Science Foundation of Zhejiang Province, grants 81471434 (SWG), 81270676 (SWG), 81530040 (SWG), 81370695 (XSL) and 81671436 (XSL) from the National Natural Science Foundation of China. Author details 1Department of Obstetrics and Gynecology, The People ’s Hospital, Wenzhou, Zhejiang 325800, China. 2Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, Shanghai 200011, China. 3Shanghai Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011, China. Received: 17 August 2016 Accepted: 23 September 2016

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