Iron deposition at mineralization fronts and osteoid formation following chronic cadmium exposure in ovariectomized rats

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in distribution in the endometrium during the menstrual cycle in women. Likewise the extracellular matrix (ECM) ligands for these receptors are likely to play a role in the establishment of a receptive endometrium. To develop primate models to study the role of these molecules in the cascade of molecular events leading to implantation, integrin expression and associated changes in ECM were investigated during the menstrual cycle and in early pregnancy in the baboon. Antibodies specific for the integrins (alpha(1-6) and alpha(v); beta1, beta3, and beta4) and ECM (laminin, collagen IV, fibronectin) were utilized. In addition, cytokeratin and alpha-smooth muscle actin were used as epithelial, stromal, and smooth muscle cell markers, respectively. Endometrium was obtained in duplicate or triplicate during the menstrual cycle and early pregnancy. Changes observed during the natural menstrual cycle were confirmed using ovariectomized, steroid-treated animals. Constitutively expressed integrins on the endometrial epithelium included the collagen/laminin receptors: alpha2, alpha3, alpha6, and beta4. The pattern of expression correlated well with the distribution of ECM in this tissue. Collagen IV was confined to the basement membrane of glandular epithelium and blood vessels. Laminin immunostaining was found in the basement membrane, mostly in the stroma of the basal region, in the glandular endometrium and vasculature. Fibronectin was present throughout the stroma but not in the basement membrane. The collagen receptor alpha1 beta1 and fibronectin receptor alpha4 beta1 appeared in the glandular epithelium in the luteal phase. As in the human, alpha1 and alpha4 disappeared from the glandular epithelium with the establishment of pregnancy. In contrast, the alpha4 beta3 vitronectin receptor appeared in the glandular epithelium only in pregnancy or following long-term steroid treatment with estrogen and progesterone but not during the time of uterine receptivity associated with the initial period of embryo attachment. Osteopontin, an ECM ligand for alpha(v) beta3, was coexpressed with this integrin in invading cytotrophoblasts, glandular epithelium, and decidualizing stromal cells. Decidualization in the baboon was associated with changes in integrin expression similar to those found in humans: there was an increase in alpha1, alpha3, alpha6, beta1, and alpha(v) beta3 in the decidualized stromal cells. Laminin and collagen IV expression also increased at the implantation site and throughout the endometrium. In contrast, fibronectin expression was most evident at the implantation site and corresponded to alpha5 expression on the invading cytotrophoblasts. In summary, marked similarities were found in the expression of ECM and the integrin receptors between the baboon and the human endometrium throughout the menstrual cycle and in pregnancy. Cycle-specific integrins, alpha1, and alpha4, were present on epithelial cells during the secretory phase. Delayed expression of alpha(v) beta3 in baboon endometrial glands correlated closely with the time of enhanced glandular secretory activity in this primate. The baboon appears to be an excellent model for the investigation of the role of integrins and ECM leading to successful implantation.     

The receptive endometrium is characterized by apoptosis in the glands

Apoptosis in the human endometrium up to now has been detected during the mid to late luteal phase and therefore connected to the onset of the menstrual shedding. However, there is increasing evidence that regulated apoptosis may be important during decidualization and implantation. To investigate a possible role for apoptosis in the human endometrium and its regulation, we correlated the immunolocalization of the apoptosis regulatory protein bcl-2 and the proliferation marker Ki67 to the in-situ nuclear DNA fragmentation - a key feature of apoptosis - detected by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) method during the menstrual cycle. Whereas proliferation and bcl-2-expression were predominantly detected in the glandular compartment during the proliferative phase, only single apoptotic cells could be shown during this period. During the transformation of the endometrium (days 15-19) proliferation and bcl-2 expression decreased markedly and there was no sign of apoptosis. At the beginning of the implantation window (days 19-20) we could detect the first signs of apoptosis in the glandular epithelia in the basalis, which extended to the functionalis during the luteal phase. Proliferation and bcl-2 expression are limited to the stromal compartment comprising the large granular lymphocytes - during this time, and extend in parallel with apoptosis from the basal to the functional layers. Apoptosis therefore may be related to the loss of the protective effect of bcl-2 and may have significance for the establishment of an endometrium adequately prepared for successful implantation.     

Identification and characterization of cytosolic sulfotransferases in normal human endometrium

Understanding the factors which alter estrogen metabolism and activity in endometrial tissue is important because unopposed estrogen stimulation is an important risk factor in the development of endometrial carcinoma. The cyclic progression of the endometrium through proliferative and secretory phases is normally under the control of the ovarian hormones beta-estradiol (E2) and progesterone. One mechanism by which progesterone inhibits the activity of E2 in secretory endometrium is by elevating the degree of E2 sulfation, thereby reducing its ability to bind to the estrogen receptor and elicit a cellular response. Our laboratories have investigated the cytosolic sulfotransferases (STs) found in biopsies of both proliferative and secretory endometrium obtained from five normal pre-menopausal women who were not taking any drugs or steroids. Two of the human cytosolic STs were detected in human endometrial tissues. The phenol-sulfating form of phenol ST (P-PST) was found at varying levels in cytosol from both proliferative and secretory endometrium in all of the women studied but with no consistent correlation to the phase of the menstrual cycle. In contrast, estrogen ST (EST) was not detected in the proliferative endometrial cytosol of any of the women studied but was consistently found in all of the secretory endometrial cytosols. The presence and levels of these STs was confirmed by ST activity studies, immunoblot analysis and Northern blot analysis. These results indicate that the expression of EST in human endometrial tissues varies with the phase of the menstrual cycle and is most likely regulated by progesterone secreted from the ovaries.     

Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis

The human endometrium undergoes a complex process of vascular and glandular proliferation, differentiation, and regeneration with each menstrual cycle in preparation for implantation. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic protein that appears to play an important role in both physiological and pathological neovascularization. To investigate whether VEGF may regulate human endometrial angiogenesis, we examined VEGF messenger ribonucleic acid (mRNA) and protein throughout the menstrual cycle and studied the regulation of VEGF by reproductive steroids in isolated human endometrial cells. By ribonuclease protection analysis, VEGF mRNA increased relative to early proliferative phase expression by 1.6-,2.0-, and 3.6-fold in midproliferative, late proliferative, and secretory endometrium, respectively. In histological sections, VEGF mRNA and protein were localized focally in glandular epithelial cells and more diffusely in surrounding stroma, with greatest VEGF expression in secretory endometrium. Consistent with these in vivo results, the treatment of isolated human endometrial cells with estradiol (E2), medroxyprogesterone acetate (MPA), or E2 plus MPA significantly increased VEGF mRNA expression over the control value by 3.1-, 2.8-, and 4.7-fold, respectively. The VEGF response to E2 was rapid, with steady state levels of VEGF mRNA reaching 85% maximum 1 h after the addition of steroid. E2 also caused a 46% increase in secreted VEGF protein, and the combination of E2 and MPA caused an 18% increase. VEGF expression in endometriosis, an angiogenesis-dependent, estrogen-sensitive disease was similar to that seen in eutopic endometrium. Peritoneal fluid concentrations of VEGF were significantly higher in women with moderate to severe endometriosis than in women with minimal to mild endometriosis or no disease. VEGF, therefore, may be important in both physiological and pathological angiogenesis of human endometrium, as it is an estrogen-responsive angiogenic factor that varies throughout the menstrual cycle and is elevated in women with endometriosis.     

Placental protein 14 in endometrium during menstrual cycle and effect of early luteal phase mifepristone administration on its expression in implantation stage endometrium in the rhesus monkey

Placental protein 14 (PP14) is a glycoprotein which is secreted by secretory phase endometrium and decidua in women. Despite the suggestion that PP14 is involved in the process of endometrial maturation for blastocyst implantation, our understanding in this regard is poor. In the present study, the concentrations and distribution patterns of immunodetectable PP14 in the endometrium during proliferative and secretory phases of normal ovulatory menstrual cycles, as well as in implantation stage endometrium in naturally mated ovulatory cycles with or without early luteal phase mifepristone treatment, were investigated using the rhesus monkey as a primate model. Immunopositive PP14 was observed mainly in epithelial cells of glands and it was detected in one major immunopositive band at Mr 28 kDa in tissue homogenate and spent medium. The area of immunopositive precipitation of PP14 in glands was minimal in follicular phase endometrium, and was higher (P < 0.01) in early, mid- and late luteal phase endometrium compared with that in pre- and periovulatory phases of the cycle, but there was no change in its area profile in the glandular compartment throughout the luteal phase. Immunopositivity for PP14 in luminal contents of gland displayed an increasing profile from early to late secretory phases. Thus, the concentrations and the distribution of immunodetectable PP14 in luteal phase endometrium of the rhesus monkey showed marked similarity with those of human endometrium during the natural menstrual cycle. Although there was no marked change in the band characterstics for the protein in implantation stage endometrium following early luteal phase mifepristone treatment, it was markedly decreased (P < 0.01) in tissue homogenate and in vitro spent medium along with a lesser (P < 0.02) degree of immunoprecipitation in the glands in implantation stage samples of mifepristone treatment group compared with that in control group samples. Thus, the contragestional effect of early luteal phase mifepristone treatment appears to be associated with a decrease in the concentration of immunodetectable PP14 in implantation stage endometrial glands and its secretion in the rhesus monkey. It remains to be seen whether this decline is caused from direct antiprogesterone action on endometrial glands during progesterone dominance, or secondarily from associated retarded development of endometrium.     

Immunolocalization of inhibin and activin subunits in human endometrium across the menstrual cycle

Inhibin/activin alphaC/alphaN and betaA subunits were localized immunohistochemically in the human endometrium throughout the menstrual cycle using an affinity-purified sheep polyclonal antibody raised against the alphaC/alphaN subunit and an affinity-purified rabbit polyclonal antibody raised against the betaA subunit. The betaB subunit was below the level of detection in all human endometrial samples tested. Immunoreactive inhibin alphaC/alphaN subunit was localized in the luminal epithelium, glandular epithelium, stromal tissues and vascular endothelium with no significant variation across the normal menstrual cycle. Immunoreactive betaA subunit, common to inhibin A and activins AA and AB was localized in the luminal and glandular epithelium and in migratory cells while the endometrial stromal cells, decidua, vascular smooth muscle and endothelium were devoid of immunoreactivity. A significant variation of immunoreactive betaA subunit was observed in glandular and luminal epithelium across the normal menstrual cycle. In proliferative endometrium, only a very low level of betaA immunostaining was seen in luminal and glandular epithelium, while the luminal epithelial staining increased significantly in the early secretory phase and remained relatively constant over the rest of the menstrual cycle. A progressive increase in betaA immunoreactivity was observed also in the glandular epithelium during the secretory phase reaching a maximum in the late secretory phases, and decreasing at menstruation. Co-localization studies on serial sections suggested that the migratory cells expressing strong betaA immunoreactivity were macrophages and neutrophils but not eosinophils or mast cells. Thus, cells within the human endometrium are capable of expressing inhibin/activin molecules in vivo. The variation in the pattern of secretion of the betaA subunit across the menstrual cycle suggests that activin peptides may have a physiological role in endometrial function.     

Exchange of Ser-4 for Val, Leu or Asn in the sequon Asn-Ala-Ser does not prevent N-glycosylation of the cell surface glycoprotein from Halobacterium halobium

The aim of the present study was to gain a better understanding of the localization of apoptotic cells within the human endometrium during the menstrual cycle and to elucidate the relationships among the following for the human endometrium: apoptosis, p21 expression, and cell proliferation. Apoptosis and p21 expression were identified mainly in the glandular cells of the basal layer in the late secretory phase. In contrast, cells positive for Ki-67 were observed predominantly in the functional layer (in the proliferative phase in glandular cells and in the secretory phase in stromal cells). A very strong positive correlation (r = 0.81; P < 0.001) was demonstrated between the number of apoptotic cells and the number of p21-positive cells present among the glandular cells but, topographically, individual apoptotic cells were not coincident with p21-positive cells in serial sections. The results of this study suggest that the proliferation of the glandular cells of the basal layer is regulated by both apoptosis and p21 expression, particularly in the late secretory phase. Such regulation may be necessary to maintain a healthy population of glandular cells in the basal layer of the endometrium.     

Expression of telomerase activity in human endometrium is localized to epithelial glandular cells and regulated in a menstrual phase-dependent manner correlated with cell proliferation

Telomerase activity is observed in most malignant tumors and germ cells, whereas normal somatic cells usually do not express it. Human endometrium is composed of glandular and stromal components and exhibits dramatic changes in proliferative activity during the menstrual cycle, which is exquisitely regulated by estrogen function. We previously reported that normal human endometrium expresses telomerase activity. However, it remains unclear which of the above components are the major sources of telomerase activity and how levels of telomerase activity are regulated over the menstrual cycle. Quantitative analysis of telomerase activity revealed that it changes dramatically over the course of the menstrual cycle and is strictly regulated in a menstrual-phase-dependent manner. Maximal activity equivalent to that in endometrial cancer was present in late proliferative phase, and minimal activity in late secretory phase. Postmenopausal endometrium and endometrium treated with anti-estrogen drugs exhibited decreased telomerase activity. Testing isolated epithelial glandular cells and stromal cells, we found that telomerase activity was localized to epithelial glandular cells. In situ RNA hybridization analysis also revealed epithelial-specific expression of human telomerase RNA. In vitro analysis of cultured epithelial cells demonstrated that telomerase activity is correlated with epithelial proliferation but not affected by estrogen treatment. These findings suggest that expression of telomerase activity is specific to epithelial cells and linked to cell proliferative status. The involvement of estrogen in telomerase regulation remains to be elucidated.     

Compartmentalization and cyclic variation of immunoreactivity of renin and angiotensin converting enzyme in human endometrium throughout the menstrual cycle

Renin and angiotensin converting enzymes (ACE) are responsible for the generation of angiotensin II which regulates blood pressure and fluid/electrolyte homeostasis. The cellular localization and cyclic distribution of renin and ACE in human endometrium are demonstrated in this study. Immunohistochemical studies revealed that both renin and ACE were consistently localized in the endometrial glandular epithelia throughout the menstrual cycle; however, the immunostainings respectively for ACE and renin were weak and moderate in stromal cells of proliferative endometrium and negligible in secretory endometrium. No renin immunostaining was detected around endometrial blood vessels. Although endothelial cells consistently stained for ACE, no renin immunoreactivity was detected in these cells during the menstrual cycle. Western blot analysis using ACE antibody directed against human kidney identified a single protein band with a relative molecular mass of approximately 153 kDa. The intensity of this band showed cyclic variation during the menstrual cycle with the highest ACE expression during the late secretory phase and at menses suggesting that ACE plays a role in the initiation of menstruation. The differences in the cellular distribution patterns of these two enzymes further supports our previous proposition that angiotensin II has different functions at the different stages of the menstrual cycle.     

The gene encoding LERK-7 (EPLG7, Epl7), a ligand for the Eph-related receptor tyrosine kinases, maps to human chromosome 5 at band q21 and to mouse chromosome 17

The human uterine glandular epithelium undergoes a sequence of well characterized changes during the menstrual cycle that presumably play an important role in preparation for blastocyst implantation. The aim of this study was to measure objectively glandular volume over the entire menstrual cycle and compare the results with eight different clinical superovulation or hormone replacement therapy (HRT) subject groups. Endometrial biopsies were taken from control normal menstrual cycle subjects (n = 96), and eight other smaller groups of women who had received different in-vitro fertilization (IVF) related treatments. The total area of glandular epithelium was objectively measured from routine histological slides using computerized image analysis. Control menstrual cycle results showed a significantly greater gland area in the early secretory stage of the cycle than at any time between the early proliferative through to the mid-late proliferative stages (P < 0.05). IVF patients receiving clomiphene citrate and human menopausal gonadotrophin had a significantly smaller glandular area than those in the control groups at equivalent stages of the menstrual cycle. The use of progesterone supplementation removed this significant difference. Patients on the ?Flare' regime had the highest gland area, although this was not significantly different from controls. Buserelin down-regulation gave a gland area that was closest to the normal cycle controls. The three HRT groups showed high variability in gland volume between patients. The results from this study demonstrate that superovulation can cause significant alterations in endometrial gland volume, but that these do not necessarily preclude implantation.     

Attenuation of calcitonin gene expression in pregnant rat uterus leads to a block in embryonic implantation

The peptide hormone calcitonin plays a key role in calcium homeostasis in many tissues, such as bone and kidney. Our previous studies revealed that the expression of calcitonin is dramatically induced in the glandular epithelium of rat uterus between days 3-5 of pregnancy before the onset of blastocyst implantation on day 5. Calcitonin expression is switched off once implantation has progressed to day 6. The coincidence in timing suggested that calcitonin may function as a regulatory signal in the uterus during the early events leading to implantation. Here we report that the implantation stage-specific expression of calcitonin can be specifically attenuated by administering antisense oligodeoxynucleotides (ODNs) directed against exon IV of calcitonin messenger RNA into the uterine horns on day 2 of gestation. The loss of calcitonin messenger RNA and protein expression upon antisense ODN treatment is accompanied by a severe impairment in implantation of embryos. Based on the observations that 1) treatment with two different antisense ODNs possessing different base compositions produced similar phenotypes; and 2) treatment with the complementary sense ODNs did not affect either calcitonin expression or implantation, we conclude that the effects of antisense ODNs on calcitonin expression and implantation are specific and functionally linked. Our study strengthens the hypothesis that a transient expression of calcitonin in the preimplantation phase uterus is critical for blastocyst implantation.     

Involvement of cyclin-dependent kinase inhibitor p27Kip1 in growth inhibition of endometrium in the secretory phase and of hyperplastic endometrium treated with progesterone

A cyclin-dependent kinase (cdk) inhibitor, p27Kip1 (p27), binds to the cyclin E-cdk2 complex and functions as a suppressor of cell cycle promotion. Here, the involvement of p27 in the growth of normal human endometrium was immunohistochemically studied, and the findings were compared with those of Ki-67, cyclin E and cdk2. In addition, to elucidate the effect of progesterone on the expression of p27, tissues from patients with endometrial hyperplasia were examined before and after the administration of medroxyprogesterone acetate (MPA) for the treatment of this disease. In the glandular cells of the normal endometrium, p27 was negligible during the proliferative phase, whereas it was markedly increased in the secretory phase. The staining pattern of Ki-67 was the reverse. Cyclin E/cdk2-positive cells were observed throughout the menstrual cycle. In the secretory phase, the cyclin E/cdk2-positive cells were also positive for p27, suggesting an interaction between these molecules. Stromal cells, especially in the basalis, showed a consistent expression of p27 throughout the menstrual cycle. The expression of p27 in hyperplastic epithelia before the MPA treatment was negligible, whereas it was greatly increased after the treatment. The Ki-67 positivity decreased after the treatment. These findings suggest that p27 is involved in the progesterone-induced growth suppression of normal and hyperplastic endometria.     

Integrin expression in normal and out-of-phase endometria

Integrins have recently been proposed as having a major role in endometrial receptivity. Different patterns of integrin expression have been described during the normal endometrial cycle, and the co-expression of several integrins, mainly alpha1, alpha4 and beta3 has been considered as specific to the 'window of implantation'. In the present study 55 infertile patients underwent two endometrial biopsies during a single menstrual cycle. An early biopsy was done on postovulatory days 6-8, and a late biopsy was performed on postovulatory days 10 to 12. Histological dating as well as immunohistochemical evaluation of alpha1, alpha4, beta1, beta3, beta5, alpha(v)beta3 integrin expression and oestrogen and progesterone receptors were determined in all endometrial biopsies. Oestradiol and progesterone serum concentrations in serum were evaluated on the same days of the endometrial samplings. Nine out of the 55 midluteal biopsies (16.4%) showed out-of-phase endometria, but all biopsies were in phase in the late luteal phase. Differences in integrin expression between in- and out-of-phase biopsies were observed only for alpha(v)beta3 integrin glandular expression during the midluteal phase. Alpha(v)beta3 integrin glandular expression was found in all late luteal phase biopsies. Alpha(v)beta3 expression was closely correlated with histological maturation of the endometrium appearing suddenly at postovulatory day 6-7 and being expressed by all endometria dated as postovulatory day > or = 8, irrespective of midluteal endometrial biopsies being in phase or out of phase. No differences in integrin expression were detected between patients with or without endometriosis or between patients who became spontaneously pregnant and those who did not. In conclusion, further studies are necessary before patterns of integrin expression may offer an alternative to predict uterine receptivity and implantation potential.     

Response of the primate secretory endometrium to subchronic hypercortisolemia

OBJECTIVE: To assess the impact of subchronic and moderate hypercortisolism on the secretory endometrium of the cynomolgus monkey. METHODS: Osmotic pumps containing hydrocortisone phosphate (HP) were implanted subcutaneously in each monkey on the first day of the menstrual cycle; each monkey also received pumps containing saline in another cycle. Blood was obtained three times per week and urine was collected daily for hormone analyses. Endometriectomy was performed 13 +/- 1 days after the serum estradiol (E2) peak in each study cycle. RESULTS: Infusion of HP elevated serum cortisol levels by an average of 70%. Mean serum progesterone (P) levels were decreased by 50% during the secretory phase of HP-treatment cycles by comparison with self-control cycles (P < .01); as a result, the mean endometrial glycogen concentration was reduced by 30% (P < .05) and the activity of 17 beta-hydroxysteroid dehydrogenase was decreased by 70% (P < .05). Serum E2 levels were not consistently elevated by HP treatment, but cytosolic estrogen receptor levels of the endometrium were decreased by 50% (P < .01), indicating increased estrogenic stimulation. Histologic development of the secretory endometrium was retarded, but the length of the secretory phase was not affected by the treatment. CONCLUSION: A moderate elevation of serum cortisol levels over one menstrual cycle consistently produced a reduction in serum P and a hypoprogestogenic-hyperestrogenic response of the secretory endometrium in the cynomolgus monkey.     

Immunohistochemical characterization of proliferation, oestrogen receptor and progesterone receptor expression in endometriosis: comparison of eutopic and ectopic endometrium with normal cycling endometrium

Recent studies examining oestrogen and progesterone receptor status and the proliferative activity of endometriotic lesions have produced conflicting reports. This study aimed to clarify the receptor status and proliferative activity of eutopic and ectopic endometrium from women with endometriosis and endometrium from normal women. Progesterone and oestrogen receptor expression and proliferative activity were studied in eutopic and ectopic endometrium from 30 women with endometriosis and in endometrium from 30 normal cycling women using microwave-pretreated paraffin-embedded sections stained with an avidin-biotin peroxidase technique. Progesterone and oestrogen receptor expression in the control endometrium did not differ from that of eutopic endometrium from women with endometriosis. Oestrogen receptor expression in ectopic endometrium increased from the proliferative to the late secretory phase. Epithelial progesterone receptor expression decreased during the cycle. Oestrogen receptor expression in both epithelium and stroma of ectopic endometrium was significantly higher than in eutopic endometrium throughout the cycle. In contrast, stromal progesterone receptor expression tended to be reduced in ectopic endometrium compared with eutopic tissue. Epithelial progesterone receptor expression was increased in ectopic endometrium but only in the late secretory phase. Although proliferative activity in the epithelium of control and eutopic endometrium was reduced from the proliferative to the late secretory phase, stromal activity did not vary. The proliferative activity in ectopic endometrium remained low and constant throughout the cycle. In the proliferative and early secretory phases, the proliferative activity of eutopic endometrium was increased compared with ectopic endometrium, but in the late secretory phase, levels were comparable. These findings challenge previous reports which have suggested that oestrogen receptors are reduced in ectopic tissue. This may have clinical implications for the development of novel treatments for endometriosis.     

Hemorrhages in abnormal production of gonadotropins

A review of the organic and hormonal causes of irregular menstrual bleeding is presented. Menstrual bleeding irregularities are the most frequent gynecological ailment. Oligomenorrhea is most often caused by a prolonged proliferation phase in the menstrual cycle. In younger women this may be due to insufficient hormonal secretion of the pituitary gland; in older women it may be due to late follicular development in spite of normal glandular secretion. Polymenorrhea is usually caused in younger women by a shortening of the proliferation phase, usually because the ovaries are hypersensitive to pituitary hormones, which causes an aceleration of follicular development. Polymenorrhea in older women is usually due to a shortened secretory phase caused by early degeneration of the corpus luteum, and later may develop into oligomenorrhea or amenorrhea. Irregular corpus luteum function after ovulation can cause bleeding irregularities. When the corpus luteum fails to produce progesterone, the endometrium develops irregularly, causing premenstural spotting or increased volume of menstrual bleeding. Slow degeneration of the corpus luteum causes increased progesterone production and irregular discharge of the endometrium. Anovulation is the most frequent cause of bleeding irregularities, because there is no progesterone secretion. A quick increase in estrogen production in the proliferation phase in conjunction with anovulation causes an irregular discharging of the endometrium with prolonged or increased bleeding. A slow increase in estrogen production during the proliferation phase in conjunction with anovulation causes hyperplasia. In a few patients, anovulation in conjunction with a secretion of estrogen insufficient to maintain the endometrium can cause endometrial atrophy. Irregular bleeding is usually functional in younger women and organic in older women. To diagnose the cause of irregular menstrual bleeding, organic reasons should be explored 1st. A complete history should be taken, then abrasio, recording the basal temperature, and hematological, and hormonal analysis should be used to pinpoint the causes of irregular bleeding. Abrasio is the most common treatment for bleeding irregularities, and hormone treatment is the 2nd most common. Hysterectomy should be used only in extreme cases for treatment of bleeding irregularities.     

Isolation of rat chromosome-specific paint probes by bivariate flow sorting followed by degenerate oligonucleotide primed-PCR

Angiogenesis is an essential component of endometrial regeneration after menses in preparation for implantation. Vascular endothelial growth factor (VEGF) is a secreted angiogenic peptide with mitogenic activity specific for endothelial and trophoblast cells. VEGF-immunoreactivity was detected in glandular epithelium throughout the menstrual cycle by immunohistochemistry, but, showed cyclic variation in the stroma and the blood vessels. During the early proliferative phase, strong staining was seen in the glandular epithelial cells while staining in the stroma was confined to a subpopulation of stromal cells and endometrial blood vessels appeared negative. In contrast, very intense staining of the endometrial stromal cells was seen in the mid proliferative endometrium possibly due to increased synthesis of VEGF by oestrogen. In the late proliferative endometrium, staining was seen in the endothelial cells and the perivascular stromal cells around the endometrial blood vessels. The greatest degree of immunostaining of stromal cells was observed in the mid to late proliferative endometrium. Throughout the secretory phase no staining was seen around the endometrial blood vessels and staining of endometrial stromal cells was confined to early secretory endometrium. In the late secretory endometrium only the glands were positive to VEGF antibody. The observed increase in the immunostaining of stroma suggests increased production of VEGF from early to mid and late proliferative endometrium which parallels the increase in the oestradiol levels in the proliferative phase of the menstrual cycle. It is proposed that VEGF may serve as a paracrine mediator of the effects of ovarian steroids on endometrial vascular development.     

The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation: a potential role in human implantation

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein strongly associated with normal implantation in the mouse. We have recently determined that LIF is expressed in the human endometrium in a menstrual cycle dependent manner. Maximal expression is observed between days 19 and 25 of the menstrual cycle, coinciding with the time of human implantation. In this study we have utilized purified cultures of human cytotrophoblasts to examine the effects of LIF on several morphologic and biochemical markers of the trophoblastic differentiation. We purified human cytotrophoblasts from term placentae and cultured them with and without LIF (10 ng/mL). The secretion of human CG, oncofetal fibronectin, and progesterone were measured at 24, 48, 72, and 96 h. Northern blot analysis was used to assess messenger RNA (mRNA) expression of beta hCG and oncofetal fibronectin. We found that LIF markedly decreased trophoblast production of hCG protein at 72 and 96 h, as well as expression of beta hCG mRNA. LIF also significantly increased the expression of oncofetal fibronectin mRNA and secretion of the protein. LIF did not affect steroidogenic activity of cultured trophoblasts, as determined by progesterone production. These biochemical changes are characteristic of cytotrophoblast differentiation toward an anchoring extravillous phenotype. Thus, LIF appears to be an important regulator of human embryonic implantation by directly modulating trophoblast differentiation.