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Pathophysiology 19 (2012) 131–135

Progesterone and ovarian stimulation control endometrial pinopodeexpression before implantation in mice

Bahman Rashidi a,∗, Jafar Soleimani Rad b,c, Leila Roshangar b,c, Rafie Alizadeh Miran d

a Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iranb Departments of Anatomy and Histology, School of Medicine, Tabriz University of Medical Science, Tabriz, Iran

c Drugs Applied Research Center, Tabriz University of Medical Science, Tabriz, Irand Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Science, Tehran, Iran

Received 11 January 2011; accepted 11 March 2012

bstract

Endometrial epithelial pinopodes are considered as markers of endometrial receptivity and seem to be directly involved in the adhesion ofhe blastocysts to the endometrial surface. The aim of the study was to assess the effects of ovarian stimulation and progesterone injection oninopode expression, and compare morphological characteristics in the preimplantation stage in mice. Adult female mice (n = 30) were dividednto three groups: control, superovulated and superovulated-progesterone injected. In experimental groups the mice received 7.5 I.U human

enopausal gonadotropin (HMG) and then after 48 h 7.5 I.U human chorionic gonadotropic (HCG) hormone. After that every two femalesere put with one male in one cage for mating. Superovulated-progesterone group were injected with progesterone (1 mg/mouse) in 24, 48,2 h interval after the HMG injection. Animals were sacrificed in 96 h after HMG injection, and their uterines (the middle one-third) wererepared for transmission electron microscope studies. That demonstrated that all groups differed from each other. In most of the controls andyperstimulated-progesterone mice 4 days after HMG injection, long and short microvilli were seen, but they had no developed pinopodes,

hile in hyperstimulated mice, well developed pinopodes were expressed 4 days after HMG injection.In conclusion: hyperstimulated mice without progesterone injection may be useful for the studies of pinopodes and implantation.2012 Elsevier Ireland Ltd. All rights reserved.

eywords: Ovarian stimulation; Progesterone; Pinopodes; Transmission electron microscopy; Female mice

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. Introduction

Implantation failure is considered to be the major limit-ng factor in the success of infertility treatment including then vitro fertilization (IVF) [1]. Inadequate uterine receptivitys responsible for approximately two-thirds of implanta-ion failures, whereas the embryo itself is responsible fornly one-third of these failures [2,3]. Theoretically, reducedmplantation rates in IVF cycles could result from impairedr premature endometrial maturation, which may be accom-anied by alterations in pinopode expression [4].

Pinopodes are transient apical protrusions of the endome-rial epithelial surface that occur only during the receptivehase [5]. Their function in humans is still not fully

∗ Corresponding author. Tel.: +98 0311 7922440.E-mail address: b rashidi@med.mui.ac.ir (B. Rashidi).

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928-4680/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.pathophys.2012.03.005

nderstood. Because of their temporal and spatial expression,inopodes have been suggested as suitable morphologi-al markers of endometrial receptivity [6]. The surfaces ofinopodes may have receptors for adhesion molecules, whichre essential for embryo implantation [7].

Singh et al. (1996) have reported that pinopod devel-pment on uterine luminal epithelium is dependent onrogesterone alone [8]. The high concentration of estrogennterferes the normal formation of pinopodes [9,10]. Hor-

onal treatments have been shown to advance or retard theiming of pinopode formation [7]. In mice, ovarian hyper-timulation is known to have a detrimental effect on theormation and disappearance of pinopodes [11]. Some inves-igators have showed that ovarian stimulation did not have

armful effect on the endometrial receptivity [4,12].

A better understanding of the mechanisms regulating thembryo implantation may improve the ability of clinicians

1 physiology 19 (2012) 131–135

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Fig. 1. Transmission electron microscopy of the uterine luminal epithe-lmX

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o treat infertility, to prevent early pregnancy loss ando develop new contraceptive approaches [13]. Electron

icroscopy is the major tool used to observe endometriuminopodes [7,14]. Since the comparison of human specimensn ovulation- induced women with those at natural cycle islmost impossible, the animal studies are unavoidable.

The aim of the present study was to compare mor-hological characteristics of mice uterine endometrium byransmission electron microscopy in controls with those atuperovulated cycle with or without progesterone injection.

. Materials and methods

.1. Animals

We maintained 30 adult (3.5 months) Syrian female and0 adult Syrian male mice (mean weight, 25 ± 5 g) undertandard laboratory conditions. The mice were acclimatizedor 1 week under a 12 h:12 h light:dark cycle at room temper-ture of 22 ± 2 ◦C. Female mice were randomly divided intogroups.

.1.1. Group A: Non-stimulated control group10 mice were placed in the control group and for mating,

very 2 female mice with 1 male mouse put in one cage.

.2. Group B: Hyperstimulated group

The mice of this group (also n = 10) were superovulatedy intraperitoneal (i.p.) of 7.5 I.U HMG (Menogon, Ferring,harmaceuticals, Germany) and HCG (Pregnyl, Organon,etherlands) at an interval of 48 h. On the evening of the

econd injection they were mated with male mice as inroup B.

.2.1. Group C: Hyperstimulated androgesterone-injection group

In this group the mice (n = 10) were superovulated andated same as the previous group (group B), then they

eceived i.p. injections of progesterone (Saha, Mashhad, Iran)1 mg/mouse) at 24, 48, and 72 h interval after HMG injec-ion.

.3. Tissue preparation

Ninety-six hours after HMG injection, the mice in experi-ental groups together with control mice were sacrificed and

heir uterine were flashed for blastocysts. Uterine specimens,nly from those that their uterine contained blastocysts (ateast 10 in each group) was prepared (the middle one-third)or TEM as follows:

Samples were washed by normal saline and then threeimes with phosphate buffer for 10 min and then were cuto 1 mm × 1 mm × 1 mm by scalpel. All samples were takenrom the middle third of the uterus and mice from both

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ium cells (control mice). N, nucleus; G, granule; BL, basal lamina; M,itochondria. These organelles were almost similar in three groups (Mag:3592).

ides. Tissues were fixed with 2% glutaraldehyde in bufferedaraformaldehyde (Thuringowa, Australia) at room temper-ture for 30 min, and at 4 ◦C overnight, and then post-fixed in% osmium tetroxide for 2 h at dark room. Specimens wereashed with 0.1 M phosphate buffer (pH = 7.4) for 10 min

three times). The tissues were dehydrated in increasing con-entrations (30–100%) of ethanol followed by propylenexide and then were embedded in pure resin. Semithin sec-ions (500 nm thick) were stained with 2% toluidine blue.ltrathin sections (90–150 nm thick) (ultramicrotome, Zeiss,ermany) were stained with uranyl acetate and aqueous lead

itrate. The sections were examined using Zeiss transmissionlectron microscope (LEO 906, Germany) [15].

. Results

.1. Non-stimulated controls

In the group A without hyperstimulation, epithelial cellsluminal) of uterine were oval with euchromatin nuclei sur-ounded by thin marginal heterochromatin. Their cytoplasmas rich in organelles such as rough endoplasmic reticulum,olyribosomes and mitochondria. The microvilli of the uter-ne epithelium cells were abundant and large 96 h after matingFigs. 1 and 2A). In this group no developed pinopodes inuminal cell microvilli were observed.

.2. Hyperstimulated groups

In the group B with hyperstimulation, 96 h after HMGnjection the microvilli decreased in number and length.

B. Rashidi et al. / Pathophysiology 19 (2012) 131–135 133

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ig. 2. Transmission electron microscopy of the uterine luminal epitheliuyperstimulated mice; (D) hyperstimulated-progesterone injected mice. MV

mooth and slender membrane projections form, arisingrom the entire cell apex (developing pinopodes). Some cellsad no microvilli and were transformed to pinopodes. Inhis group the pinopodes were well developed and some ofhem had short microvilli in their surface (more pronouncedinopodes in the luminal cells and therefore less microvilli).he nuclei of this group like group A, were oval and mainlyuchromatic with a prominent nucleolus and heterochromatinispersed peripherally; and the cytoplasmic organelles wereound in the cells (Fig. 2B and C).

.3. Hyperstimulated and progesterone injected mice

In the hyperstimulated-progesterone injected mice inroup C 96 h after the HMG injection the short microvilliere seen. In this group the apical surface of cells were similar

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96 h after HMG injection. (A) Non-stimulated control mice; (B and C)villi; M, mitochondria; JC, junctional complex; P, pinopode (Mag: X4646).

o non-stimulated group and different from stimulated group.he euchromatic and oval nuclei with distinctive nucleolind double membranes were situated at the mid part of theells. The organelles of the samples from hyperstimulated-rogesterone injected mice were the same as control groupFig. 2D).

. Discussion

The endometrium is the sole uterine tissue that undergoesyclic restoration after each menstrual cycle in expectation

f pregnancy. Since the advances in the laboratory meth-ds of fertility IVF (In vitro fertilization), and regardlessf the progress in the field of ovulation, oocyte maturation,ertilization and embryo development have been successful,

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34 B. Rashidi et al. / Patho

he implantation rate in Assisted Reproductive TechnologyART) has been considerably less than expected. Embryomplantation is a crucial step in the of a pregnancy. Thendometrium is receptive to embryonic apposition, attach-ent, and invasion only during a defined window that is

emporally and spatially limited. Even though there is still notull conformity as to the accurate timing of embryo implan-ation in humans, clinical studies suggest that the window isemporally limited to days 0–24 of a normal ovulatory cycle16–18].

The importance of endometrial appearance as a predictorf outcome in patients treated with IVF is well established19]. Pinopodes seem to be useful markers for endome-rial receptivity in many species [20]. In cell cultures, thelastocyst adheres to the pinopode-presenting area on thendometrial cells [6]. The morphological changes leading tohe implantation are modulated by estradiol and progesteroneevels through which the ovary exerts in the control overhe endometrium. Pinopode formation and deterioration arelosely associated with the increases and decreases, respec-ively, in serum progesterone concentration. At pinopodeevelopment, levels progesterone receptors A and B in thelandular and luminal epithelial cells decrease; this effect isostly dependent on the lack of progesterone receptor B.erum estrogen levels and levels of estrogen receptor alphand beta did not correlate with pinopode formation [5]. Theigh level of hormones which produce during ovarian hyper-timulation may effect on the fine structure of endometrium21,22]. The duration of pinopode formation was limited toshort time and their appearance was delayed after ovar-

an hyperstimulation. The absence of pinopodes before themplantation time might be due to the high level of estrogenn hyperstimulating animals [23].

The pinopodes on the epithelial surface are visible in lighticroscopy. However, other structures may be mistaken for

inopodes in light microscopy [24]. Therefore, EM is bettero confirm the presence of pinopodes in the endometrial tis-ue [25]. Our ultrastructural studies showed that 96 h afterMG injection pinopodes were not visible in the control

nd superovulated-progesterone injected mice and all thepithelial cells had microvilli on the apical surface. But super-vulated mice had pinopodes at the same time.

In the present study, we showed that after the administra-ion of HMG & HCG, the well organized pinopodes werexpressed over the surface of mouse endometrium. It seemshat ovarian hyperstimulation of mice could cause prematurexpression of pinopodes on the pre implantation time.

Emadi and Salehnia reported that in the hyperstimulatedice without progesterone injection 4 days after HCG injec-

ion well-developed pinopodes were present and some wereollapsed [26]. Similar to our results have reported, Salehniabout the effects of progesterone on pinopodes. The proges-

erone may cause premature expression of pinopodes andhe implantation failure after ovarian induction may be dueo these timing changes [27]. The premature appearance ofinopodes after hyperstimulation has been reported in some

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gy 19 (2012) 131–135

tudies [28]. The average day of pinopode formation was 1–2ays earlier in ovarian stimulation, as compared to naturalycles studied in the present and in previous work [7,19].

A correlation between the number of pinopodes andmplantation rate after embryo transfer has been shown [29].his is in agreement with the fact that ovarian stimulationsing only gonadotropins leads to higher pregnancy rates30]. This finding is consistent with previous reports. Ghaemit al. reported that the progesterone injection without ovar-an induction improved the embryo survival and implantationates, but after superovulation it did not ameliorate the nega-ive effects of superovulation on the implantation rate [31].

This alteration in expression of pinopodes results in anlteration in the window of implantation. The alteration inmplantation rate following ovarian hyperstimulation in ani-

als may be due to these time changes [26]. On the otherand Nikas et al. [30] have shown that the ovarian stimula-ion did not affect the quantity and life span of the endometrialinopodes in humans.

In our study, the developing pinopodes were seen duringhe hyperstimulation. In a study, development of pinopodesollowed the progesterone peak, but absolute concentrationsf progesterone and pinopode development were not cor-elated [5]. Thus, our ultrastructural studies showed thatrogesterone injection after hyperstimulation did not haveny effect on pinopode developing and feature of surfaceells in this group is similar to control group. In contrast tour results, Salehnia et al. [21] have shown that after thedministration of progesterone, well-organized pinopodesere formed on the surface of the mouse endometrium before

mplantation, but the projections transformed to smaller onesnd some had collapsed on day 4 after HCG injection.hey reported that progesterone injection after hyperstim-lation could cause premature expression of pinopodesefore implantation time [25]. Also, Stavreus-Evers et al.eported that the increase in serum progesterone level andown-regulation of progesterone receptor B are important inevelopment of pinopodes [5]. Also, our results demonstratedhat the evaluation of endometrial maturation according toheir morphological characteristics indicates that the proge-trone injection in superovulated mice does not acceleratendometrial maturation.

These controversial reports show that although there areany studies about effects of ovarian hormones on pinopode

ormation in mice, more studies are still needed.In conclusion our study on mice endometrial sam-

les showed that ovarian stimulation by HMG/HCG andMG/HCG + progesterone altered or shifted endome-

rial pinopode formation. Ovarian stimulation byMG/HCG causes formation of pinopodes and better

han HMG/HCG + progesterone at preimplantation time inice. Evaluation of endometrial maturation according to

heir morphological characteristics (pinopodes) indicateshat superovulatory drugs in mice stimulate endometrial

aturation but progesterone injections do not accelerate it.t seems that hyperstimulated mice without progesterone

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njection (according to the proposed method for the applica-ion of assisted reproductive medicine) may be more helpfulnd increase the pregnancy rate.

cknowledgment

This study was supported by funding from the Drugpplied Research Center, Tabriz University of Medical Sci-

nces, Tabriz, Iran. Authors acknowledge their thankfulness.

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