Quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels.

This study describes a digital technique for uterine morphometry and its application to endometrial structure during the bovine oestrous cycle. Neither the number nor the size of uterine gland ducts changed during the cycle but a reduction in total endometrial area from days 0 to 8 after oestrus led to an increase in the proportion of the endometrium occupied by gland ducts (gland duct density). This effect on day 8 was maintained to day 16. When endometrial morphology was related to circulating progesterone concentrations on days 5 and 8 of the luteal phase, no relationships were found on day 5, but on day 8, a high progesterone concentration was associated with an increased number of gland ducts. Furthermore, in animals slaughtered on day 8, a high progesterone concentration on day 5 was associated with decreased gland duct size, though a simultaneous decrease in endometrial area led to an increase in gland duct density. The results suggest that contrary to expectation, endometrial glands do not grow and regress during the oestrous cycle, although cyclic changes in endometrial area controlled by progesterone lead to changes in gland duct density.

Distribution patterns of uterine glands and embryo spacing in the mouse.

To clarify the mechanism of implantation, relationship between positioning of the mouse embryo in the uterus and distribution of uterine glands along the long axis of the uterine horn was examined by three-dimensional remodelling of the uterine endometrium. There were two unique regions in the endometrium. Uterine glands were distributed widely from mesometrial to anti-mesometrial side in one region. It was localized from lateral to anti-mesometrial side in another. These different regions were alternately aligned throughout the uterine horn. The number and position of embryos was consistent with that of the latter region. This study suggests that the type of distribution of uterine glands is closely related to the positioning of the embryo in mice.

The role of interleukin-11 in pregnancy involves up-regulation of alpha2-macroglobulin gene through janus kinase 2-signal transducer and activator of transcription 3 pathway in the decidua.

IL-11 expressed by endometrial stromal cells is crucial for normal pregnancy. IL-11 receptor alpha (IL-11Ralpha) null mice are infertile due to abnormal development of the placenta. In these mice, the mesometrial decidual tissue, which is the site of trophoblast invasion, thins and disappears at mid-pregnancy. Degeneration of the decidua is accompanied by uncontrolled trophoblast invasion. In this report, we show, using IL-11Ralpha null mice, that a defect in IL-11 signaling in the decidua leads to severe down-regulation of alpha(2)-macroglobulin (alpha(2)-MG), a metalloproteinase inhibitor crucial for limiting trophoblast invasion. We also present evidence, using uterine stromal cells that decidualize in culture, that IL-11 robustly stimulates the endogenous alpha(2)-MG expression and enhances alpha(2)-MG promoter activity. Serial 5' deletion and internal deletion of the promoter reveal two important signal transducer and activator of transcription (Stat) binding sites. Mutation of either one of these motifs decreases IL-11 stimulation, whereas double mutation prevents IL-11 action. We also found that IL-11 activates Janus kinase 2 (Jak2) and induces rapid phosphorylation, nuclear translocation, and promoter binding activity of Stat3 in decidual cells, whereas Jak1, Tyk2, and Stat5 activities are not affected. In addition, Jak2 inhibitor totally prevents alpha(2)-MG expression in decidual cells. Taken together, results of this investigation provide, at least in part, an explanation for the overinvasiveness of the trophoblast in IL-11Ralpha null mice and reveal, for the first time, that IL-11 signals through the Jak2/Stat3 pathway in decidual cells to stimulate the expression of alpha(2)-MG, a protease inhibitor essential for normal placentation in pregnancy.

Phenotypic analysis of the mouse placenta.

Placental development is a dynamic and complex process and much of our current understanding of the underlying molecular processes comes from analysis of targeted gene mutations in mice. There are more than 50 strains of mutant mice that have placental defects, and it has become widely appreciated that placental defects should be suspected in cases where embryonic lethality is observed. The degree to which these phenotypes are investigated is highly variable, owing to a general lack of expertise in the field. However, there has been considerable progress in developing techniques and reagents for analyzing placental phenotypes that are relatively simple to apply and that should be accessible to all investigators. This chapter provides a basic outline of the strategies for the general identification and then the subsequent detailed investigation of placental phenotypes.

Effects of interleukin-12 administration during the pre- and peri-implantation period on mouse embryofetal development.

PROBLEM: The immunological success of pregnancy is thought to depend upon the establishment of a balance between favorable and deleterious cytokines, the current paradigm viewing pregnancy as a T helper (Th)2 cytokine-dependent phenomenon. In this context, a particular attention should be directed to the potential role of interleukin (IL)-12, which promotes the development of Th1 responses, in the induction of adverse pregnancy-related phenomena. Indeed, very few data linked the Th1-inducer IL-12 to the event of abortion. METHODS: In this study, we have investigated the maternal and fetal effects of exogenous administration of IL-12 to CD1 (BR) ICR mice during the pre- and peri-implantation period (day 2-6 of pregnancy). Animals have been evaluated for parameters of reproductive performance, embryo and fetal developmental toxicity and maternal toxicity. RESULTS: Intraperitoneal administration of IL-12 at concentrations from 2.5 to 10 microg/kg daily did not result in an increase in the murine abortion rate. A statistically significant, although minimal, decrease in the number of somites were found in the embryos of animals treated with IL-12 at a dose of 10 microg/kg/day. However, developmental parameters at birth were similar between the two groups of animals suggesting that alteration of somites might be a transitory state during treatment. An increased body weight gains and reduced feed and water consumption were observed in the mothers treated with the cytokine. CONCLUSION: In the present experimental conditions and in this specific strain of mice, IL-12 does not exert adverse effects on reproductive performance and induces an only modest harmful action on mothers and embryos.

Mapping of zones of altered morphology and chorionic connective tissue cellular phenotype in human fetal membranes (amniochorion and decidua) overlying the lower uterine pole and cervix before labor at term.

OBJECTIVE: The purpose of this study was to determine the location, frequency, and extent of altered fetal membrane morphology before term labor and its relation to myofibroblast activation in their connective tissue layers. STUDY DESIGN: Fetal membranes that were obtained from 10 women who underwent prelabor cesarean delivery at 38 to 39 weeks of gestation underwent biopsy examination with respect to the internal os of the cervix. The thickness of their constituent layers was measured, and the numbers of alpha-smooth muscle actin immunoreactive cells (ie, marker of myofibroblast activation) within the reticular layer were counted. RESULTS: A region that measured 119+/-21cm(2), that exhibited altered morphology of the fetal membranes from the lower uterine pole, and that was characterized by increased connective tissue thickness and decreased thickness of the cellular layers was demonstrated in all patients. In 8 of 10 patients, this region was centered on the location of the Babcock tissue forceps. Within this region was an area of fetal membranes that exhibited alpha-smooth muscle actin immunoreactivity in the cells of the reticular layer and whose number correlated with parameters of altered morphology. CONCLUSION: All patients before labor at term possess an area of fetal membranes that are located in the lower uterine pole that exhibit altered morphology that is associated with myofibroblastic activation in the chorionic connective tissue.

Immunolocalization of proinflammatory cytokines in myometrium, cervix, and fetal membranes during human parturition at term.

Each of the proinflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF) alpha has been identified in reproductive tissues during labor. The cellular origin of these cytokines is unclear. The aim of this study was to localize these proinflammatory cytokines in myometrium (upper and lower segment), cervix, and fetal membranes at term. Biopsies were taken from women undergoing cesarean section either before or after the onset of labor. Immunohistochemistry was used to localize each of the cytokines IL-1beta, IL-6, IL-8, and TNFalpha. Leukocytes were localized using an antibody to CD45. In myometrium and cervix, immunostaining for IL-1beta was predominantly in leukocytes. In fetal membranes, IL-1beta localized to leukocytes and to the stromal cells of the decidua. In myometrium, IL-6, IL-8, and TNFalpha were restricted to leukocytes, which were present in greater numbers in tissue obtained during labor. In cervix, IL-6, IL-8, and TNFalpha localized to leukocytes and glandular and surface epithelium. IL-8 also localized to cervical stromal cells. In fetal membranes, IL-6 and TNFalpha were expressed by decidual stromal cells, infiltrating leukocytes, and extravillous trophoblasts. In membranes, IL-8 localized to leukocytes in the chorion but was not detected in the amnion. In fetal membranes collected at labor, IL-8 was expressed in decidual stromal cells. Infiltrating leukocytes are a major source of cytokines in uterine tissues during labor.

The ornithine decarboxylase gene is essential for cell survival during early murine development.

Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene. ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation.

Uterine natural killer cells in a three-dimensional tissue culture model to study trophoblast invasion.

The high numbers of CD56(+) cells with natural killer (NK) functions present in the uterine mucosa during the late secretory phase of the menstrual cycle and during early pregnancy have been considered to be implicated in implantation and in the regulation of trophoblast invasion. A three-dimensional organ culture model was used to study the interactions of these uterine NK cells with Jeg-3 and BeWo choriocarcinoma cells as a model of the invasive trophoblast. For this purpose, fragments of endometrial and decidual tissue were put in close contact with multicellular spheroids of choriocarcinoma cells in small silicon funnels. After the formation of stable contacts, the confrontation cultures were transferred to spinner flasks, cultivated for up to 6 days, and prepared for immunohistochemistry. During 2 days of cocultivation, the first cells started to move forward into the stromal component of the confrontation culture as demonstrated by staining of the choriocarcinoma cells using anti-human cytokeratin. Invasion advanced until, after a total of 6 days, some choriocarcinoma cells had already penetrated deeply into the host tissue. After a cultivation period of 1 week, both the endometrial and decidual tissue fragments still contained several CD56(+) uterine NK cells, and some of them expressed the proliferation-associated marker Ki-67 without any exogenous activation. A few CD56(+) cells were found directly at the invasion front, as well as between the choriocarcinoma cells. These cells also contained the cytolytic granule protein perforin indicating a migration of NK cells with cytolytic potential toward the potentially invasive cells. In conclusion, this human system closely resembles the in vivo conditions during trophoblast invasion and provides an appropriate in vitro model for studying dynamic processes involving various cell types during trophoblast invasion at the experimental level. Moreover, it enables us to study the effects of cytokines and growth factors that possibly regulate trophoblast invasion.

Efficacy and effects of short- and medium-term contraception in the common marmoset (Callithrix jacchus) using melengestrol acetate implants.

This study examines the effect of melengestrol acetate (MGA) implants on reproductive function and various biochemical parameters, ovarian activity, and uterine morphology in ten female common marmosets implanted for either 6-8 or 19-21 months. Measures of body weight, concentrations of urinary glucose and blood liver enzymes were taken. Ovarian activity was assessed by analysis of urinary progestin levels and ultrasound examinations of the ovaries. Ultrasonography was also used to evaluate uterine morphology. MGA was highly effective in preventing pregnancies in the study animals. No changes in biochemical parameters were found; however, seven females developed a substantial weight gain during the study. Follicular development was not suppressed, as indicated by the presence of antral follicles, luteinized structures, and elevated urinary progestin levels. The uteri of the MGA-treated subjects were moderately enlarged with a thickened endometrium that showed a marked change in structural appearance indicative of hypertrophy and decidualization. After implant removal these changes quickly disappeared and all females ovulated within 3 weeks and conceived within 4 months post-treatment. MGA appears to be an acceptable contraceptive in the marmoset, although non-steroidal methods should be evaluated as possible potential alternatives.

Fertility impairment in granulocyte-macrophage colony-stimulating factor-deficient mice.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified as a potentially important mediator of intercellular communication in the female reproductive tract, with principal target cells being the large populations of myeloid leukocytes in the cycling and pregnant uterus, the preimplantation embryo, and trophoblast cells of the developing placenta. To determine the physiological significance of this cytokine in reproduction, the fertility of genetically GM-CSF-deficient (GM-/-) mice was examined. Implantation rates were normal in GM-/- mice, and viable pups were produced. However, the mean litter sizes of GM-/- x GM-/- breeding pairs were 25% smaller at weaning than those of GM+/- x GM+/- pairs, due to fetal death late in gestation and early in postnatal life, with a disproportionate loss of male pups. On Day 17 of pregnancy, the mean number of resorbing and malformed fetuses was twice as high in pregnant GM-/- females (21%, vs. 11% in GM+/- females); the mean fetal weight and the mean fetal:placental ratio in surviving conceptuses were diminished by 7% and 6%, respectively; and the number of very small fetuses (< 500 mg) was 9-times as high (23% vs. 2.5%). Mortality during the first 3 wk of life was 4.5-times as high in pups born to GM-/- mothers (9%, vs. 2% in GM+/- females), and diminished size persisted in GM-/- pups, particularly males, into adulthood. The detrimental effect of maternal GM-CSF deficiency was less apparent when GM-/- females were mated with GM+/+ males; litter sizes at birth and at weaning were not significantly smaller than in GM+/- matings, and fetal weights and fetal:placental ratios were also comparable. When polymerase chain reaction was used to genotype embryonic tissue in heterozygote matings, GM-/- fetuses from GM-/- females were found to be smaller than their GM+/- littermates and smaller than GM-/- fetuses gestated in GM+/- females. The size and distribution of uterine granulocyte and macrophage populations were normal during the estrous cycle, during early pregnancy, and in midgestation. Analysis of placental structure revealed that the ratio of labyrinthine to spongiotrophoblast areas was reduced by approximately 28% in GM-/- placentae, and the proportion of vacuolated trophoblast "glycogen cells" in the spongiotrophoblast layer was diminished. Compromised placental function as a result of subtle developmental aberrations may therefore partially account for embryonic growth retardation in GM-CSF-deficient mice. Collectively, these studies show that fetal growth and viability are jeopardized in the absence of maternal GM-CSF. The detrimental effects are most clearly evident when the conceptus is also GM-CSF deficient, suggesting that GM-CSF of either maternal or fetal origin is required for optimal growth and survival of the fetus in mice.

Effect of inhibition of matrix metalloproteinases on endometrial decidualization and implantation in mated rats.

Implantation of the embryo into the endometrium is a highly regulated event that is critical for establishment of pregnancy. Molecules involved in this process provide potential targets for post-coital contraception. The aims of this study were to determine whether matrix metalloproteinases (MMPs) are present at implantation sites in rats and whether administration of a broad-based inhibitor of MMPs could inhibit embryo implantation. Uterine extracts from non-pregnant rats and from rats on days 3-9 of pregnancy were examined for the presence of MMPs. Doxycycline (5 or 15 mg day-1) was administered by gavage to rats from the day of mating (day 0) to day 7 of pregnancy and the uterus was examined for implantation sites. A number of MMPs were present in all uterine samples. MMP-2 reached a peak on day 3, whereas the highest expression of MMP-7 occurred on day 7. MMP-13 and MMP-3 were present in smaller amounts. MMP-9 was detectable only on day 9. Treatment of rats with doxycycline had no effect on the number of implantation sites or on the total uterine mass. However, in treated rats, the process of decidualization was impaired and both the width and length of the decidual zone was reduced, resulting in a decrease in total decidual area from 1.20 +/- 0.07 to 0.91 +/- 0.07 mm2 (mean +/- SEM, controls versus doxycycline treated, P < 0.02). It is concluded that administration of MMP inhibitors during early pregnancy retards decidual development, but does not block implantation.

The placenta and its significance in neonatal outcome.

The placenta should not be overlooked as a source of much valuable diagnostic information. Close evaluation of the placenta and its attached membranes may reveal further information. Essential data may be obtained from pathologic examination, and if questions exist, specimens should be retained with proper care.

Localization of the cellular expression of inhibin in trophoblastic tissue.

AIMS: Inhibin is a peptide hormone which is normally produced by ovarian granulosa cells and which inhibits the release of follicle stimulating hormone from the pituitary gland, thus acting as a modulator of folliculogenesis. Serum inhibin levels are higher during pregnancy than during the normal menstrual cycle and the placenta is thought to be a source of circulating inhibin. Previous studies have yielded conflicting results as to the cellular localization of inhibin in the placenta and the aim of the present study was to investigate the immunohistochemical localization of the hormone in placental tissue. We also wished to investigate whether inhibin could be demonstrated in choriocarcinoma and in non-gestational trophoblastic tissue. MATERIALS AND RESULTS: Immunohistochemical staining was performed using a monoclonal antibody against the alpha subunit of human inhibin. Specimens included in the study were intrauterine products of conception (n = 36), extrauterine products of conception (n = 4), decidualized endometrium (n = 15), extrauterine decidualized tissue (n = 3), hydatidiform mole (n = 5), uterine choriocarinoma (n = 2) and testicular embryonal carcinoma with syncytiotrophoblast giant cells (n = 6). In cases of products of conception, including hydatidiform mole, there was consistent strong positive staining of syncytiotrophoblast but no staining of cytotrophoblast with anti-inhibin. Staining with anti-inhibin highlighted trophoblastic cells within the placental bed. In a small number of cases there was focal weak positive staining of decidua. There was positive staining of the two cases of uterine choriocarcinoma and of syncytiotrophoblast giant cells in the six cases of testicular embryonal carcinoma. CONCLUSIONS: The study shows that immunohistochemically detectable inhibin alpha subunit in placental tissue is mainly localized within syncytiotrophoblast although in some cases there is also positive staining of decidua. Production of inhibin by these cells may account for raised serum levels during pregnancy. Inhibin can also be demonstrated in choriocarcinoma and in nongestational trophoblastic tissue. Inhibin is a sensitive marker of syncytiotrophoblast and staining with this antibody may prove useful in the diagnosis of choriocarcinoma and in the demonstration of trophoblastic cells in germ cell tumours.

Fetal death in mice lacking 5alpha-reductase type 1 caused by estrogen excess.

Female mice deficient in steroid 5alpha-reductase type 1 have a decreased litter size. The average litter in homozygous deficient females is 2.7 pups vs. 8.0 pups in wild type controls. Oogenesis, fertilization, implantation, and placental morphology appear normal in the mutant animals. Fetal loss occurs between gestation days 10.75 and 11.0 commensurate with a midpregnancy surge in placental androgen production and an induction of 5alpha-reductase type 1 expression in the decidua of wild type mice. Plasma levels of androstenedione and testosterone are 2- to 3-fold higher on gestation day 9, and estradiol levels are chronically elevated by 2- to 3-fold throughout early and midgestation in the knockout mice. Administration of an estrogen receptor antagonist or inhibitors of aromatase reverse the high rate of fetal death in the mutant mice, and estradiol treatment of wild type pregnant mice causes fetal wastage. The results suggest that in the deficient mice, a failure to 5alpha-reduce androgens leads to their conversion to estrogens, which in turn causes fetal death in midgestation. These findings indicate that the 5alpha-reduction of androgens in female animals plays a crucial role in guarding against estrogen toxicity during pregnancy.

Presence of urokinase plasminogen activator and plasminogen activator inhibitor-1 messenger ribonucleic acids in rat endometrium during decidualization in vivo.

Rat endometrial stromal cells undergoing decidualization in vitro secrete urokinase-type plasminogen activator (uPA), and this secretion is regulated by prostaglandin E2. The present study was undertaken to determine whether uPA and plasminogen activator inhibitor-1 (PAI-1) mRNAs are expressed in vivo in the decidua of pregnant rats and in the deciduoma of "pseudopregnant" rats. Total RNA was prepared from nondecidualized and decidualized endometrial tissues at various stages of early pregnancy and examined by Northern blot analysis using specific cDNA probes for rat uPA and PAI-1. There was little uPA mRNA in the endometrium during the first 5 days of pregnancy (Day 1 = the presence of sperm in the vagina). A high level of uPA mRNA was detected on Day 7, and it declined thereafter. There was a gradual increase in PAI-1 mRNA in the decidua from Day 7 of pregnancy, reaching a peak level on Day 15 when the decidua was transformed into the maternal placenta. (RNA was not analyzed beyond Day 15 of pregnancy in this study.) In situ hybridization studies verified that uPA mRNA was present in the decidua adjacent to the implanting embryo on Day 7. Plasminogen activator inhibitor-1 mRNA was scattered in the decidualized endometrium, but greater amounts of PAI-1 mRNA were found in the fetal tissue on Day 10 of pregnancy. Northern blot analysis of RNA from the deciduoma produced in ovariectomized, steroid-treated rats by intrauterine injection of oil demonstrated a similar temporal pattern of expression of uPA mRNA; i.e., the level of uPA mRNA was highest on Day 7 and decreased thereafter. The level of PAI-1 mRNA in deciduoma was not detectable by Northern blot technique during the first 10 days of pseudopregnancy. These findings confirm that uPA mRNA is present in vivo in rat decidual cells, independent of the presence of a conceptus. By contrast, the level of PAI-1 mRNA in the uterus is probably influenced by the presence of the conceptus.

Decidua and placenta in mice after treatment with a synthetic glucocorticoid.

To investigate a possible long-term effect of glucocorticoids on decidua and placenta of mice, a single dose of 24 mg kg-1 body weight triamcinolone acetonide in crystalline suspension was given subcutaneously to NMRI mice on gestational day (GD) 2. Deciduae and placentae, as well as corticosterone and triamcinolone concentrations in maternal plasma of GDs 10 and 17 were examined. NADPH-cytochrome P450 reductase involved in drug biotransformation was detected immunocytochemically and showed co-localization with NADPH diaphorase histochemistry in the decidua and placenta. Both reactions were higher in endothelial cells of decidual sinusoids on GD 10, but were lower on GD 17 in the trophoblast, spongiotrophoblast and extraplacental visceral yolk-sac epithelial cells of treated mice than in untreated animals. Histochemistry of 11 beta-hydroxysteroid dehydrogenase, an enzyme that metabolizes biologically active adrenocortical steroids and their synthetic congeners in the placenta, showed higher activity on GD 17 in enlarged labyrinthic trophoblast I cells of treated mice than in untreated animals. As corticosterone concentrations were still decreased on GD 17, when triamcinolone concentrations were no longer detectable, a long-term suppression of adrenal gland function seems obvious.

Epidermal growth factor--induced implantation and decidualization in the rat.

Effects of epidermal growth factor (EGF) on blastocyst implantation and experimentally-induced decidualization were examined in the rat. Intraluminal injection of EGF into each uterine horn induced implantation in the ovariectomized progesterone-treated delayed implanting rat in a dose-dependent manner. This induction was inhibited by indomethacin, an inhibitor of prostaglandin synthesis. Intraluminal injection of EGF on day 5 of pseudopregnancy elicited a greater decidual response when compared to the vehicle-injected contralateral uterine horn. These results suggest that EGF may play important roles in the process of implantation and decidualization in the rat.