Volume 4, Issue 1 (7-2006)                   IJRM 2006, 4(1): 7-11 | Back to browse issues page

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Salehnia M, Arianmanesh M, Beigi M. The impact of ovarian stimulation on mouse endometrium: a morphometrical study. IJRM. 2006; 4 (1) :7-11
URL: http://journals.ssu.ac.ir/ijrmnew/article-1-45-en.html
1- Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran , mogdeh@dr.com
2- Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran
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Implantation is a complex sequence of processes between the embryo and endometrium. The surface of embryo and endometrium undergoes a series of changes within a short time, which is considered as “implantation window”. During this time, the endometrium has high efficiency for receiving the embryo (1,2). These changes have been observed on the morphology, ultrastructure and molecular levels of endometrium (3,4). At the time of embryo adhesion, the microvilli are replaced with another fungi     form     cytoplasmic projections named as pinopodes. These swelling projections have been appeared for a short time (24-48 hours) at the endometrium surface and assumed as uterine receptivity markers in some mammals (3-5). The effect of progesterone on endometrium receptivity is clear. This hormone is needed   to   create   typical   luteal   changes  and   the secretary stage of the endometrium during the decidual reaction (6,7). The preparation of endometrium for embryo reception is dependent on the ovarian hormones which are affected by ovarian hyperstimulation procedure (8).
     There are some regimes for ovulation induction and hormones replacement therapy such as progesterone administration after human chorionic gonadotropic hormones (HCG) injection for the maintenance of corpus luteum and preparation of endometrium for embryo transfer. After the administration of exogenous gonadotropin hormone to obtain a large numbers of oocytes, the secretion of oestrogen and progesterone increases (9).  Investigations on human and experimental animals showed that after hyperstimulation, the implantation rates declined in comparison with the normal groups (8-11). Fossum et al (9) reported a significant decrease in the implantation rates after embryo transfer to ovarian stimulated mice using Pregnant Mare Stimulating Gonadotropin (PMSG) and HCG and suggested that this failure was caused by changes in uterine receptivity (9). In Karmer et al (12) study a high luteal phase oestradiol/progesterone ratio has been associated with implantation failure in mice. Basir et al (10) concluded that excessive high concentration of oestradiol leads to suboptimal endometrial environment for implantation and this may explain the findings regarding the decreased implantation and pregnancy rates in IVF.
     Since the surface and glandular epithelial thickness depends on the ovarian hormones, it is suggested that some morphometric indices of endometrium should be changed after ovarian induction regimes. Previous researches showed a delay in maturation of endometrium epithelium and stroma after ovarian stimulation in human and animals (10, 13-15).
     The main question is that if the high level of oestrogen and progesterone concentrations after ovarian hyperstimulation and progesterone injection (as replacement therapy) does influence the structure of endometrium at the peri-implantation period? The purpose of this study was to investigate the alterations in some morphological indices of mouse endometrium after hyperstimulation using HMG and HCG injections followed by the daily injections of progesterone at the implantation time.
Materials and Methods


     Female virgin NMRI mice, aged 6-10 weeks, were cared for and used according to the guide for the care and use of laboratory animals  and housted under 12h light: 12h dark condition. They were randomly divided into three groups:
     Group A: control group, which were rendered pseudopregnant by cervical stimulation (16).
     Group B: hyperstimulated mice, which were superovulated using an intraperitoneal injection of 10 i.u. HMG (Sereno) followed by another injection of 10 i.u. HCG (Organon) 48 hours later. On the evening of the second injection, the mice were rendered pseudopregnant the same as the control group.
     Group C: hyperstimulated mice with progesterone administration, which superovulation the same as group B, then daily subcutaneous injections of progestrone (1 mg/mouse) were performed (17) and the mice were rendered pseudopregnat the same as the other groups. 

          Tissue preparation

     Thirty mice from each group were sacrificed by cervical dislocation on 3 (pre implantation time in mice) and 4 (implantation time in mice) days after HCG injection. The samples were obtained from the middle 1/3 part of their uterine horns immediately and processed for the following studies.

         Morphometrical study

     Five tissues from each group, on third and forth day were fixed in formaldehyde, embedded in paraffin wax, sectioned at 6 micrometer and stained using hematoxyline and eosin technique.
     After preparation of the sections, 3 slides were chosen randomly from each sample and at least four fields of view were measured from each slide. The following endometrial parametres were measured in each field of view: (I) the surface epithelial cell thickness (μm) from the luminal border to its basement membrane; (II) the glandular epithelial cell thickness (μm) from the luminal border to its basement membrane; (III) the endometrial thickness (μm) from the luminal border of the epithelium to the upper layer of the myometrium and (IV) the  gland diameter (μm) (18) . The measurments on each slide were made using the 40 times objective of a Zeiss microscope with a calibrated eye piece.

     Statistical analysis

     Data were collected from each group and the mean±SD was calculated. Groups were compared using student t-test. Data were analysed using SPSS softwares.


     At the light microscopic levels, the morphology of the surface epithelium in the control, hyperstimulated and hyperstimulated-progesterone injected groups were simple columnar, pseudostratified columnar and simple low columnar, respectively.
     The morphometric data on three and four days after HCG injection (table I and II) showed that the surface epithelial cell thickness on the third and fourth days of HCG injection was decreased in the hyperstimulated groups (18.58± 3.5 μm, 23.67± 4.18 μm) compared with the non-stimulated group (23.57± 4.31 μm, 38.40± 2.88 μm) (p= 0.0001). The hyperstimulated-progesterone injected group had lower epithelial cell thickness on days three (17.16± 3.55 μm) or four (16.92± 4.24 μm) after pseudopregnancy in comparison with the control and hyperstimulated groups (p= 0.0001). These data demonstrated that the ovarian induction, which was followed by progesterone administration, influenced the endometrial thickness. Similarly, there were statistically significant differences between the glandular cell thickness in hyperstimulated (11.52± 2.65 μm, 9.4± 1.66 μm), hyperstimulated-progesterone injected (10.02± 2.6 μm, 12.06± 2.84 μm) and the control groups (14.58± 2.77 μm, 23.35± 4.3 μm) respectively (p= 0.0001).
     The mean diameter of glands, three days after HCG injection in the control, hyperstimulated and hyperstimulated-progesterone injected groups were 39.48± 7.85 μm, 33.88± 7.29 μm and 36.26± 7.57 μm, respectively, which showed no significant differences among these groups. But on the fourth day of HCG injection, the mean diameter of glands was greater in the control group (52.20± 9.11 μm) compared to the hyperstimulated (33.33± 8.14 μm) and hyperstimulated-progesterone injected groups (39± 8.7 μm) (p=0.0001).
     The endometrial thickness on the third day of  pseudopregnancy in the control, hyperstimulated and hyperstimulated-progesterone injected groups was 234.96± 49.95 μm, 238.56± 38.62 μm and 209.27± 54.33 μm respectively and there were no significant differences among these groups. Whereas, on the fourth day of pesudopregnancy, there was significant difference between the control (276.48± 41.21 μm) and the hyperstimulated-progesterone injected group (230.08± 65.52 μm; p=0.001) and also there was significant difference between the latter group and the hyperstimulated group (265.38± 59.98 μm; p= 0.013).
     The stroma of both hyperstimulated and progesterone injected groups were compact and their intercellular spaces were narrower than the control group (fig1).


   Our observation showed that in the hyperstimulated-progesterone injected group, the height of epithelium was decreased in comparison with the control and hyperstimulated groups. These changes may be due to the alteration in the ratio of progesterone to oestrogen, which caused a reduction in the cytoplasm and / or changes in the volume of the nucleus. Risek et al (19) showed that progesterone injection to immature rats decreased the height of endomerial epithelium. The elevated progesterone level may cause the decline in endometrial receptivity, which was previously showed after ovarian hyperstimulation (12,19).
     Dursum et al (20) showed exogenous administration of gonadotropins significantly affects the morphology of the endometrium and the mitotic index in the implantation period of the embryo.
     These morphological effects became more pronounced when the administrated dose of exogenous gonadotropins was increased.
     In addition, our results showed that in both hyperstimulated groups the stroma is compact therefore, the decidualizations were defective in hyperstimulated groups. In agreement with our results, Kramer (21) showed that in ovarian hyperstimulated rats no decidualization reaction was seen. He concluded that it was due to the decrease in vascular permeability (21). Also Stein and Kramer (18) showed stromal cells in hyperstimulated rats ovary failed to undergo decidualization. McRae and Heap (22) reported that in ovariectomised rats under progesterone treatment, the number of permeable vessels was decreased, whereas after the treatment of these animals with oestrogen, the permeability of vessels was increased. They concluded that progesterone controlls the permeability of these vessels. Kramer (21) showed that the ratio of progesterone to estrogen before implantation in the hyperstimulated groups was low which was probably due to a decrease in the permeability of the vessels.
     In contrast to our results, Kolb et al (23) speculated that high levels of progesterone in the early luteal phase of cycles, undergoing controlled hyperstimulation, caused premature endometrial luteinization and a premature appearance of the implantation window. In addition, our group reported previously (24) that the progesterone injection following ovarian induction could cause premature expression of endometrium pinopodes before implantation time. Thus, ovarian hyperstimulation with or without progesterone injection alter the thickness of the surface and glandular epithelium of endometrium, which could affect the endometrial receptivity.
Type of Study: Original Article |

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