Morphological and histochemical study of the endometrial effects of Ovral in the baboon

Summary In an effort to better understand changes induced by hormonal contraceptives, a group of female baboons were administered Ovral for a period of 9 months. During this time the endometrium was sampled by transcervical uterine biopsy from both the treated animals and from a control group. The biopsies were all obtained between 10 and 14 days of the treatment cycle or the normal menstrual cycle. The endometrial glandular cells from the treated animals exhibited an accelerated maturation compared with the controls. Ultrastructurally this was reflected by increased cell size, numerous long, slender microvilli on the apical membranes, and increased development of the Golgi complex. Differences were also observed in the predominant type of granule seen in the apical cytoplasm. After 3 and 6 months of treatment with Ovral, no significant differences were noted between groups or between animals within a group. However, after 9 months of treatment, the endometrium displayed differences from the earlier experimental groups as well as individual variations. The functional correlates of these observations are discussed and compared to human endometrium.     

Endometrial contraception: modulation of molecular determinants of uterine receptivity

Modulation of endometrial receptivity is a promising approach for fertility regulation since it allows a contraceptive to act specifically at the endometrium. This was corroborated by our previous observations that treatment with low doses of a pure progesterone antagonist (PA, antiprogestin), onapristone (ZK 98299), in bonnet monkeys inhibited fertility by selectively retarding endometrial development, without affecting the hypophyseal-hypothalamic function. In the present study, further investigations, undertaken to analyze the molecular repertoire of a nonreceptive primate endometrium, determined expression of: steroid hormone receptors, i.e. progesterone receptor (PR) and estrogen receptor (ER); cytokines, i.e. leukemia inhibitory factor (LIF): transforming growth factor beta (TGFbeta) and its receptor (TGFbetaR); and cell adhesion molecules, i.e. integrins (alpha(v)beta(3), alpha(1)beta(1)). These studies were conducted during the different phases of the normal menstrual cycle and following treatment with different doses of onapristone (2.5 mg, 5 mg, or 10 mg every third day for one cycle) in bonnet monkeys. The molecules were analysed collectively to explore the possibility of a correlation between expression of these markers and endometrial receptivity and to investigate whether there exists a regulatory link between expression of these molecules under in vivo conditions. Three types of expression patterns of endometrial factors were observed during the peri-implantation period following onapristone treatment: 1) LIF, alpha(v)beta(3), and alpha(1)beta(1) showed significant (P < 0.02) down regulation in glandular epithelium of endometria in animals treated with all three doses of onapristone as compared to the control group. This was indicative of their critical role in the progesterone-driven cascade leading to implantation. 2) PR, TGFbeta, and TGFbetaR remained unaffected in the endometria from 2.5 mg treated animals and showed down regulation in animals treated with 5 and 10 mg onapristone as compared to the control group, thereby suggesting that the expression of these markers may not truely reflect endometrial receptivity per se. However, their facilitatory role in preparing the endometrium for implantation can not be ruled out since continued perturbation in the expression of these molecules may affect endometrial growth, remodelling, and differentiation, which in turn may render the endometrium nonreceptive; 3) ER remained unaltered in endometria of animals rendered infertile with 2.5, 5, and 10 mg onapristone. This observation indirectly suggests that onapristone-induced endometrial changes are mediated via some specific mechanisms. The present study clearly demonstrates that endometrial non-receptivity induced at low doses of onapristone is associated with changes in the expression pattern of specific molecular markers. However, no direct correlation was observed between in vivo expression of TGFbeta, LIF, and integrins, thereby lending support to the concept that there exists redundancy or multiple pathways which regulate implantation events.     

TGF superfamily and MMP2, MMP9, TIMP1 genes expression in the endometrium of women with impaired reproduction

During the putative "implantation window", a period of maximal endometrial receptivity that spans 7-9 days after ovulation, a series of changes on the structural and molecular level occur that render the endometrium susceptible to implantation for the human embryo. Many members of the TGFbetas are expressed by human endometrium at different stages of menstrual cycle. Also studies regarding the MMP2 gene expression and activity of MMP2 in the implantation window have shown a higher expression and activity of MMP2 in women with impaired fertility. We have examined by RT-PCR the expression of TGFbeta2 and MMP2, MMP9 and TIMP1 in 28 patients with idiopathic infertility, 16 patients with unexplained recurrent miscarriage and 16 control women were enrolled in this study. Seven to nine days after ovulation endometrial biopsy by Pipelle or hysteroscopy was performed to assess the expression of TGFbeta2 , MMP2, MMP9 and TIMP1. We found that in endometria from women with idiopathic infertility TGFbeta2 expression was 2.8 fold higher than in endometria from control group and 2.1 fold higher in endometrial samples from women with unexplained recurrent miscarriage compared to the control group. The MMP2, MMP9 and TIMP1 expression in endometrial samples revealed no significant differences between the study groups and control group. There was a statistically significant negative correlation between TGFbeta2 and MMP9 expression in endometria from women in control group. The present investigations suggest that dysregulated TGFbeta2, MMP2, MMP9 and TIMP1 expression are associated with infertility and early pregnancy loss. However the exact mechanism of how overexpression of endometrial TGFbetaand MMPs interferes with implantation may be more complex.     

Patterns of inhibin and FSH localization in endometrium of baboon (Papio anubis) during menstrual cycle and early pregnancy.

Immunoreactive 10.5 KDa moiety of inhibin and hFSH was present in the baboon endometrium during menstrual cycle, early pregnancy and in castrated animals treated with steroid hormones, estrogen and/or progesterone. Endometrial differences during the menstrual cycle altered the intensity of immunostaining of inhibin and FSH. Maximum staining was observed in late luteal phase for both the hormones. In early pregnancy (35th day), the conceptus increased the staining for inhibin in the adjoining endometrial glands. Treatment of castrated animals with steroids for 14 days caused increased staining for inhibin. Maximum staining was observed when treated with estradiol or progesterone, whereas combination of estrogen and progesterone treatment decreased the staining reaction. In conclusion, both inhibin and FSH were localized in baboon endometrium and were under the influence of estrogen and progesterone.     

Ultrastructural cytochemical study of proteoglycans in the endometrium of pregnant mice using cationic dyes

Decidualization in rodents is accompanied by remarkable modifications of both fibrillar and non-fibrillar components of the endometrial extracellular matrix. Biochemical studies have shown that the levels of synthesis of hyaluronic acid and sulfated glycosaminoglycans change during decidualization in rodents. As the rodent decidua has regions containing cells in different stages of decidual transformation, we decided to analyse, by an ultrastructural cytochemical technique, the distribution of proteoglycans (PGs) in each region of the decidua of mice on different days of pregnancy. Endometria of mice on days 4, 5 and 7 of pregnancy were processed for electron microscopy in the presence of safranin O, a cationic dye which preserves most of the tissue PGs. The endometrium of non-pregnant mice was used as control. We observed evident differences in the arrangement and distribution of the network of PGs between non-pregnant and 4-day pregnant endometria, as well as between different regions of pregnant endometria. The possible relationship between these modifications and cell transformation that occurs during decidualization is discussed.     

Light, electron microscopic and immunohistochemical study of the effect of low-dose aspirin during the proestrus phase on rat endometrium in the preimplantation period

OBJECTIVE: To evaluate structural alterations in rat endometrium at preimplantation following treatment with aspirin beginning from proestrus by light microscopy, electron microscopy and immunohistochemical techniques. STUDY DESIGN: Twenty rats were divided into control (n = 10) and experimental (n = 10) groups. Experimental rats were treated with low-dose aspirin daily (2 mg/kg/day) during estrus, beginning from the proestrus phase, mated at end of cycle and treated with aspirin. Untreated pregnant rats were the control group. Rats in both groups were sacrificed at the 84th pregnancy hour; the uterus was rapidly removed and dissected free of surrounding adipose tissue. Uteri specimens from nonpregnant rats were transferred into fixative solution and processed for light, electron microscopic and immunohistochemical study. RESULTS: Light and electron microscopy of endometrium from control rats conformed to mid-diestrus phase; endometrial histology of the aspirin-treated group conformed to late diestrus phase. The endometrial layer was significantly thicker in the aspirin-treated group compared to the untreated control group (p <0.001). No significant difference was found in vessel number between groups. Staining with alphaV integrin was more dense in the aspirin-treated group. CONCLUSION: Based on histologic findings, we suggest low-dose aspirin has positive effects on preparing endometrium before implantation.     

Effects of an antiprogestin onapristone on the endometrium of bonnet monkeys: morphometric and ultrastructural studies

Our previous studies demonstrated the ability of low doses of antiprogestin ZK 98.299 (onapristone) to inhibit fertility in bonnet monkeys. In the present study cumulative effects of low doses of ZK 98.299 on the endometrial cytoarchitecture of bonnet monkeys were analyzed. Treatment with either the vehicle (n = 3) or onapristone at 2.5 mg (n = 4) or 5.0 mg (n = 3) was initiated on Day 5 of the first menstrual cycle and thereafter repeated every third day for four to seven consecutive cycles. The last treatment cycles were anovulatory in two animals treated with 2.5 mg and all animals treated with 5.0 mg. Endometrial biopsies were collected on Day 8 after the midcycle estradiol peak in ovulatory menstrual cycles and on Day 20 in anovulatory menstrual cycles during the last treatment cycle. Ultrathin sections of the fixed endometrium were stained with toluidine blue for morphometric analysis and uranyl acetate and lead citrate for ultrastructural analysis. The ZK 98.299-treated animals showed a dose-dependent endometrial atrophy as evident by a decrease in the height and diameter of the glands and early signs of compaction in the stroma. Ultrastructural analysis also revealed dose-dependent degenerative changes in the subcellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus. This suggests that long-term treatment with low doses of ZK 98.299 leads to the suppression of estrogen-dependent endometrial proliferation. However, this blockade operates independent of estradiol receptor (ER) and progesterone receptor (PR) concentrations as the expressions of these steroid receptors did not show any significant changes even after prolonged treatment. The study demonstrated an antiestrogenic effect of ZK 98.299 on endometrium after prolonged treatment in bonnet monkeys.     

Changes related to the oestrous cycle in the expression of endometrial and oviductal proteins of mice.

Soluble proteins extracted from the endometria and oviducts of normal sexually mature cycling Swiss Webster mice were analysed by two-dimensional high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Thirty endometrial and 25 oviductal proteins showed differential expression related to the oestrous cycle. In the endometrium, 19 proteins were maximally expressed in the oestrous phase, and significantly decreased or could not be detected in dioestrus. Eleven additional proteins were more prominent in dioestrus. Most of these endometrial cyclic proteins were acidic. In the oviduct, almost two-thirds of cycle-related, differentially expressed proteins were more strongly expressed in dioestrus and were significantly less prominent or could not be detected in the oestrous phase. In contrast to the endometrial proteins, most of the oviductal cyclic proteins were basic. Fourteen proteins appeared to be identical in both organs, and five of these showed the same cyclic pattern of expression. The remaining cyclic proteins were organ specific and showed uterus- or oviduct-specific changes during the oestrous cycle. Among the cyclic proteins, four endometrial and two oviductal proteins were restricted to oestrus, whereas two endometrial and seven oviductal proteins were restricted to dioestrus. These proteins could serve as markers for specific phases of the oestrous cycle. Our data show that the mouse oestrous cycle is associated with consistent and predictable changes in protein expression in both the endometrium and oviduct.     

Nucleolar organizer regions in the normal,hyperplastic and carcinomatous epithelium of endometrium

A silver colloid technique to identify nucleolar organizer region associated protein (AGNORs) has been applied to paraffin sections in a total of 43 endometrial hyperplasias (24 adenomatous and 19 adenocystic) 26 endometrial carcinomas and 22 normal endometria (11 of proliferative and 11 of secretory phase). A morphometric analysis of highly magnified photographic images of AGNORs in light microscopic preparations was performed. Malignant tumor cells showed significantly higher AGNOR numbers, maximum diameter and mean area compared with normal and hyperplastic endometrium, with the exception of adenocystic hyperplasia whose D_max and mean area were significantly larger. Regarding the distribution pattern of AGNOR dots in the cases studied, it was found that normal and hyperplastic endometrium had a mainly clustered distribution while endometrial adenocarcinomas revealed a scattered one. The significant differences observed in the number of AGNORs, their size and mean area between benign and malignant endometrial epithelia suggest that the AG-NOR staining technique is of diagnostic importance in distinguishing between these two groups.     

Dominant expression of progesterone receptor form B mRNA in ovarian endometriosis

This study was designed to examine the biological implications of progesterone receptor form A (PR-A) and B (PR-B) mRNA expressions in human ovarian endometriosis (ectopic endometrium). A high ratio of PR-B to PR-AB (PR-A+PR-B) mRNA expression was found in 8 of 14 cases of endometriosis, compared with the ratio in eutopic endometrium. The mean ratio in ectopic endometria was significantly (p < 0.01) higher than in eutopic endometria. The ratio in eutopic and ectopic endometria showed no significant change during the menstrual cycle. The mean ratio in ectopic endometria in the proliferative and secretory phases of the endometrium was significantly (p < 0.01) higher than in eutopic endometria. In conclusion, PR-B mRNA was relatively highly expressed in some endometriomas, which might lead to aberrations in the control of progestational effects involving responsiveness to sex steroidal growth regulation.     

Swine leukocyte antigen-DQ expression and its regulation by interferon-gamma at the maternal-fetal interface in pigs

Successful pregnancy requires an appropriate intrauterine immune response to the conceptus, which is a semiallograft within the uterus. We reported that swine leukocyte antigen-DQA (SLA-DQA), a major histocompatibility complex (MHC) class II gene, is expressed in the uterine endometrium at the time of conceptus implantation in pigs. Because MHC molecules play critical roles in the immune system, SLA-DQ was hypothesized to be involved in immune regulation during pregnancy. Therefore, we examined expression of SLA-DQ in uterine endometrial tissues obtained during the estrous cycle and pregnancy. SLA-DQA and SLA-DQB mRNAs were detected as 1.3-kb and 1.2-kb bands, respectively. Real-time RT-PCR analysis indicated that SLA-DQA and SLA-DQB mRNA expression was affected by day and pregnancy status, with the highest expression on Day 15 of pregnancy. SLA-DQ was localized primarily to subepithelial stromal cells and endothelial cells of the uterus. Using endometrial explant cultures from Day 12 of the estrous cycle, we determined that expression of SLA-DQA and SLA-DQB mRNAs increased in response to interferon-gamma (IFNG), which is produced by pig conceptus trophectoderm between Days 14 and 18 of pregnancy. The abundance of SLA-DQ protein was less in endometria from gilts with conceptuses resulting from somatic cell nuclear transfer compared with endometria from gilts with conceptuses resulting from natural mating. These results support our hypothesis that SLA-DQ is expressed in response to IFNG from the conceptus, and likely regulates immune response at the maternal-fetal interface to support the maintenance of pregnancy in pigs.     

Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus

Expression of the gene for prostaglandin synthase (PGS) was examined in whole endometrial tissue derived from ewes during the oestrous cycle (Days 4-14), on Day 15 of pregnancy and following ovariectomy and treatment with ovarian steroid hormones. Whilst no significant differences were seen in PGS mRNA concentrations analysed by Northern blot analysis in endometrial tissue during the oestrous cycle or in early pregnancy, treatment of ovariectomized (OVX) ewes with oestradiol-17 beta markedly reduced endometrial PGS mRNA concentration. There was no difference in PGS mRNA concentration in ewes treated with progesterone, either alone or in conjunction with oestrogen, from that in OVX controls. In contrast, differences in immunolocalization of PGS observed in uterine tissue from OVX-steroid-treated ewes were much more marked and reflected similar changes seen previously in the immunocytochemical distribution of endometrial PGS during the oestrous cycle. In OVX ewes and those treated with oestrogen, immunocytochemical staining for PGS was seen in stromal cells, but little immunoreactive PGS was located in the endometrial epithelial cells. However, in ewes treated with progesterone alone or with oestrogen plus progesterone, PGS was found in luminal and glandular epithelial cells and in stromal cells. Intensity of immunostaining for PGS in endothelial cells and myometrium did not differ between the treatments. Thus, whilst oestrogen lowers PGS mRNA in the endometrium, presumably in stroma, it may also increase the stability of the enzyme itself in the stromal cells. Although oestradiol-17 beta has no effect on PGS in endometrial epithelium, progesterone stimulates the production of PGS in endometrial epithelial cells without altering the overall abundance of PGS mRNA in the endometrium as a whole. Conceptus-induced changes in PGF-2 alpha release by ovine endometrium would not appear to be mediated via effects on PGS gene expression or protein synthesis.     

Nucleolar organizer regions in the normal, hyperplastic and carcinomatous epithelium of endometrium.

A silver colloid technique to identify nucleolar organizer region associated protein (AGNORs) has been applied to paraffin sections in a total of 43 endometrial hyperplasias (24 adenomatous and 19 adenocystic) 26 endometrial carcinomas and 22 normal endometria (11 of proliferative and 11 of secretory phase). A morphometric analysis of highly magnified photographic images of AGNORs in light microscopic preparations was performed. Malignant tumor cells showed significantly higher AGNOR numbers, maximum diameter and mean area compared with normal and hyperplastic endometrium, with the exception of adenocystic hyperplasia whose Dmax and mean area were significantly larger. Regarding the distribution pattern of AGNOR dots in the cases studied, it was found that normal and hyperplastic endometrium had a mainly clustered distribution while endometrial adenocarcinomas revealed a scattered one. The significant differences observed in the number of AGNORs, their size and mean area between benign and malignant endometrial epithelia suggest that the AGNOR staining technique is of diagnostic importance in distinguishing between these two groups.     

Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus)

Phase-dependent apoptotic changes in the human endometrium during an ovarian cycle imply a potential role of steroids in the regulation of apoptosis. The present study was undertaken to determine the direct role of hormones in endometrial apoptosis in marmosets (Callithrix jacchus), a primate species which shows similarity to humans in terms of the cycle length and pattern. Endometrial apoptosis was detected by 3'-end labeling (TUNEL) in various phases of ovarian cycle in naturally cycling healthy marmosets (n=14) and also in ovariectomized marmosets (n=13) treated with either estradiol alone (E) or progesterone alone (P) or estradiol followed by progesterone (E+P). Expressions of apoptosis associated genes such as Bcl-2 family members (Bax and Bcl-2), proliferating cell nuclear antigen (PCNA)--a proliferation marker and steroid receptors, ERalpha and PR A were analysed by immunohistochemical methods. Apoptosis was intense in the glandular epithelial cells of endometrium during the mid-luteal phase as compared to other phases in naturally cycling animals; in the E+P group as compared to other groups of ovariectomized animals (P<0.05). Pronounced apoptosis in the mid-luteal phase was accompanied by the increased expression of Bax in glandular epithelial cells; while Bcl-2 immunoreactivity remained unchanged. PCNA expression was higher in the naturally cycling animals in the follicular phase and in the E group of the ovariectomized animals as compared those in the other groups. Immunoreactive ERalpha and PR A in glandular epithelial cells were most abundant during early follicular phase in naturally cycling animals and in both E and E+P groups among the ovariectomized animals. The present study highlights the importance of apoptosis in endometrial remodeling during the ovarian cycle and secondly, the role of both estradiol and progesterone in the regulation of apoptosis.     

CA 125 is an excretory product of human endometrial glands

The present investigation was undertaken to study the cellular localization and kinetics of synthesis of CA 125 in the endometrium. CA 125 was localized by immunohistochemistry to the infranuclear region of epithelial cells during the proliferative phase and to the apical luminal border during the secretory phase. In gestational endometrium, both the cytoplasm and the apical luminal border of epithelial cells were intensely positive. No staining was seen in endometrial stromal cells during the normal cycle or in decidualized endometria. Results obtained from in vitro cultures of separated glandular and stromal cells were similar to those obtained by immunohistochemistry. That is, epithelial cells released between 5 and 25 times more CA 125 into the culture medium than did stromal cells. The release of CA 125 was highest in epithelial and stromal cells obtained during the early secretory phase. CA 125 concentrations were markedly elevated in endometrial aspirations obtained during the secretory phase or in endometria with crumbling stroma compared to plasma levels. Plasma levels of CA 125 were slightly elevated during menses. These results suggest that CA 125 is an exocrine product of endometrial epithelial cells. Plasma levels of CA 125 may be of endometrial origin only when the membrane barriers, which normally prevent its entry into the circulation, are damaged.