International Journal of

     Introduction
Ovarian hormones are responsible for induction of endometrial receptivity. In all mammals, the uterus differentiates into receptivity state, and then blastocyst is capable to initiate the implantation.

     After this period the uterus is incapable of blastocyst reception (1). The maximal endometrial receptivity in the mouse is after day 5 of pregnancy and duration of window implantation is 24 hour (2).
     The endometrium in response to ovarian hormones undergoes the morphological and biochemical modifications (3). In the pseudopregnant mice, the hormonal milieu in the uterus is similar to normal pregnancy, thus in the pseudopregnant mice, implantation on the day 4 is quite similar to normal pregnancy (1).
     Estrogen activates epithelial cell proliferation and stromal inflammation while progesterone has antiproliferative role and antiinflamatory function (4). Lack of progesterone receptors in the mice led to infertility and sexual behavior alterations (5-7). Ablaition of progesterone by RU 486 caused the changes of endometrial cells and reduction of decidual reaction (8).
     Many ovarian hyperstimulation cycles are associated with failure in implantation. Hormone therapy affects the endometrial receptivity (9). Inadequate uterine receptivity is responsible for two- third of implantation failures whereas one – third of these failures are related to embryo (10). Fossum demonstrates that ovarian hyperstimulation with PMSG and HCG in the mouse decreases   the implantation rate (11).
Although ovarian hormones (E2, P4) are necessary for implantation in rat and mouse but absolute requirement for estrogen in some species is on debate.
     The recent data show that uterine receptivity remains open for an extended period at lower estrogen concentration and rapidly closes at higher levels (1). Endrwes et al have shown that estrogen injection in rat increased high of luminal and glandular epithelium and also increased the granulocytic infiltration in the stroma (12). The results suggest that careful regulation of estrogen concentration could improve implantation
 rate in IVF protocols (1).
     The roles of estrogen and estrogen + progesterone on the endometrial epithelium and different stages of endometrial cycle are known. The roles of these hormones to support endometrium after ovarian hyperstimulation at the luteal phase are unknown. There are reports that progesterone and HCG injection at luteal phase can not increase the implantation rate after ovarian hyperstimulation (13). In addition increasing of the E /P ratio in serum has high prognosis in ovarian hyperstimulation patients for prognosis of pregnancy rate (9)
     Although the progesterone is routinely used at luteal phase as support hormone for in IVF cycle, addition of estrogen to progesterone during luteal phase is controversial in implantation and in rising of pregnancy rate (14). There are some reports that the progesterone cannot supply suitable uterine for implantation (15,16). In this study the morphological and morphometrical alterations in the luminal and glandular epithelium as well as stromal changes after application of different hormones such as estrogen and progesterone were investigated.

Materials and methods

Animals
     The animals were obtained from animal house of Tabirz University of Medical Sciences. Adult male and female mice (8-10 weeks) were housed under temperature and light controlled condition with free access to food and water.

Preparation of animals

Male mice were vasectomized and after recovery were used for induction of psudopregnancy. Female mice were kept separately until the estrous cycles of mice become similar. The female mice based on superovulation were divided into two groups: control and experimental groups. Five mice were studied in each group.
     The female mice in the experimental groups were superovulated by injection of a single dose of 10 I.U. PMSG (pregnant mare serum gonadotropin) and after 48 hours, 10 I .U. HCG (human chorionic gonadotropin).  The mice were mated with the vasectomised mice to produce psudopregnancy.
     In the control group, psudopregnancy was induced in natural cycle without any superovulation. Female mice of control and experimental groups were housed over night with vasectomised males and the presence of vaginal plaque was checked in the following morning; a successful mating was considered to be the first day of psudopregnancy.
     Experimental group based on hormone therapy at luteal phase was divided into five groups:
1) Sham group: The superovulated mice that were induced for pseudopregnancy were not received any hormones for luteal phase .This group was received only vehicle (olive oil)
2) E group: The psudopregnant mice that were superovulated were received consecutive daily estrogen   (10ng in vehicle /mouse) injection until day 4 (17).
3) P group: The psudopregnant mice that were superovulated were received consecutive daily progesterone (1 mg in vehicle /mouse) injection until day 4 (15).
4) E +P group: The psudopregnant mice that were superovulated were received consecutive daily estrogen+progesterone (10 ng+1mg) injection until day 4.
5) RU 486+E group: The psudopregnant mice that were superovulated were received consecutive daily antiprogesterone + estrogen   (1mg+10 ng) injection until day 4.

Tissue preparation
     Animals in all groups were scarificed by cervical dislocation after 4.5 day of psudopregnancy. The samples were obtained from the 1/3 middle part of uterine horns and immediately were fixed in formaldehyde then were  embedded in paraffin wax .After preparation of 5 micrometer sections  , the sections were stained with H & E method and were studied by light microscopy.      For the assessment of morphometrical parameters the extracted uterine was divided into 4 pieces. The pieces were embedded in paraffin wax separeatly in defined direction. Five sections were provided from each pieces. In order to assess the high of luminal and glandular epithelium and stromal thickness each section were stained with H & E and measured by graded eye piece in four directions (18). Then the data were changed to micron by slide measurement and were analyzed by statistical method.
 
Statistical analysis
     The collected data from each group were analyzed by SPSS software with One Way ANOVA method.  
     The results of this study are presented into two parts; morphology and morphometry. 1) Control group: After 4.5 day of psudopregnancy the form of epithelium was columnar and nucleus was located in the base of cells.
     The height of glandular epithelium was decreased in comparison with luminal epithelium. The stromal cellular compaction was low and decidual reaction was seen. Luminal surface of endometrium was seen in normal folding (figure 1.a).
 
2) Experimental group:
A) Sham group: In this group the luminal epithelium was columnar and luminal surface of the epithelium was irregular with folding. The height of glandular epithelium was decreased in comparison to luminal epithelium. The number of gland and secretion was similar to control group. Decidual reaction was higher than control group (figure 1.b), (Table I).
B) Progesterone group: The shape of epithelium was cuboidal and the height of luminal epithelium was decreased in comparison to control group. The nucleus was located in the base of cells and occupied the main portion of the cytoplasems. The apical border of cells was regular and stroma was seen compacted. Stromal desidulization was lower than control group and their intercellular spaces were narrow. The endometrial folding was not seen in this group (figure 1.c), (figure 2 .a).
 
C) Estrogen group: The form of epithelium was pseudostratified and the epithelial height was higher than control group. The glands mainly contained secretions .The cellularity of stroma was low and the intercellular spaces were increased and the decidual reaction was higher than control group. The endometrial folding was severe (figure 1.d).
 
D) E+P group: The shape of epithelium was columnar and the nucleus was located in the middle of cells. The height of epithelium was increased in comparison to control group.
     Development of glands high and the majority of glands contained secretion. Cellularity of stroma was low and decidual reaction was higher than control group .The endometrial folding was severe (figure 1.e and 2.b).
 
E) RU 486+E group: The shape of epithelium was pseudostratified and the epithelial height was higher than control group. In comparison to control group, stroma in this group contained inflammatory and pyknotic cells that mainly were located in the subepithelium. The stromal thickness and decidual reaction were decreased in comparison to control group. The endometrial folding was moderate (figure 1.f), (Table I).      The morphometrical obtained data were prepared in the three parts: height of luminal epithelium, height of glandular epithelium and stromal thickness.
 
Height of luminal epithelium
     The height of luminal epithelium in the progesterone group was the lowest in comparison to other groups. The comparison of luminal epithelium in the control and sham groups showed that there was significant increase in the height of luminal epithelium in sham group. Comparison of luminal epithelium in the E, E+P and sham groups demonstrated that there were no significant differences among these groups, while they have shown significant differences in comparison to control group (Table II).The height of luminal epithelium in the RU 486 + E group was higher than other groups. The estrogen had a critical role in the increasing of height of the luminal epithelium.
 
Height of glandular epithelium 
     The lowest height of glandular epithelium was seen in the progesterone group. The comparison of the height of glandular epithelium in progesterone group with control group has not shown any differences, while the comparison of  progesterone group has  shown
 
significant differences with other groups.
 
Stromal thickness