Endometrial corticotropin-releasing hormone: expression, regulation, and potential physiological implications.

Our findings show that human and rat uterus express the CRH gene. Epithelial cells of both species are the main source of endometrial CRH, while stroma does not seem to express it, unless it differentiates to decidua. Immunoreactive CRH, produced by endometrial cells, has the chromatographic characteristics of authentic hypothalamic CRH, while the size of its mRNA in both human and rat uterus is similar to or identical with its counterpart, present in placenta and hypothalamus (1.3 kb). Estrogens and glucocorticoids inhibit and prostaglandin E2 stimulates the promoter of human CRH gene in transfected human endometrial cells, suggesting that endometrial CRH gene expression is under the control of these agents. Moreover, in rats, endometrial CRH expression is significantly higher at implantation sites, compared to that at interimplantation uterine regions. Given the proinflammatory/vasoregulatory properties of CRH, we hypothesize that endometrial CRH may participate in the regulation of intrauterine phenomena, such as blastocyst implantation, endometrial vascularization, and myometrial contractility.

Uterine pinopodes in peri-implantation human endometrium. Clinical relevance.

Current clinical studies indicate the existence in the human of an "implantation/nidation window," similar to that observed in animal models. During this short period of uterine receptivity, the formation of pinopodes on the apical cell membrane of the endometrial epithelium is a consistent morphological event, observed in a number of species studies including the human. In order to develop a specific marker of the implantation window in clinical practice, we have investigated the kinetics of pinopode formation through sequential endometrial sampling under various hormonal conditions. Our results show that the implantation window in humans, according to this marker, lasts less than 48 hours, and the timing of its opening is dependent on the hormonal treatment applied, occurring earlier in cycles following ovarian stimulation and later in cycles induced by hormone replacement treatment. Furthermore, the timing varies among different individuals under the same treatment. These findings suggest that examination for pinopodes in endometrial samples can be highly useful in infertility treatment and research for the assessment of the nidation window on an individual basis. Our preliminary data strongly support the value of this assessment for better timing of ovum transfer, leading to an increase in implantation rates. Studies are now in progress on the expression of other endometrial signals present in relation to the pinopodes.

Anti-STD vaginal contraceptive sponges.

PIP: Vaginal sponges offer women control over protection against both pregnancy and sexually transmitted diseases (STDs), including HIV. Spermicide-impregnated sponges combine the actions of a physical barrier that blocks the cervix with a material that absorbs the ejaculate and a spermicide. Commercially available spermicides contain 1-5% of nonoxynol-9, shown to inhibit organisms responsible for gonorrhea, chlamydia, candidiasis, genital herpes, syphilis, trichomoniasis, and HIV. On the other hand, nonoxynol-9 is associated with a significantly higher risk of vaginal colonization with bacterial agents, ulcerative genital diseases, and vulvitis. A lower dose of nonoxynol-9 appears to avert vaginal irritation without compromising contraceptive efficacy. Use of chlorhexidene, a spermicide less irritating to mucosal cells than nonoxynol-9 but active against HIV in vivo and in vitro, is under investigation. Also promising are initial findings regarding the Protectaid contraceptive sponge with F-5 gel. Epidemiologic studies and clinical trials should provide quantitative estimates of the level of protection offered by barrier methods and identify the method that combines the highest protection, ease of use, and user acceptability.^ieng

The role of prostaglandins in blastocyst implantation.

This review summarizes the information available on the involvement of prostaglandins in blastocyst implantation, and examines their interactions with three other inflammatory mediators, platelet-activating factor (PAF), interleukin (IL-1) and corticotrophin-releasing factor (CRF). Essential elements of this information, consistent with the assumption that prostaglandins play an important role in implantation, appear to be: (i) the burst of endometrial prostaglandin production, following the blastocyst signal(s) or an artificial stimulus; (ii) the main localization of this production at the luminal epithelium and release towards the stroma; and (iii) the presence at the stromal level of specific progesterone-dependent binding sites for prostaglandin E2. In addition, accumulated data indicate a paracrine interaction at the endometrial level between PAF and prostaglandin E2, which could serve, among others, to amplify the embryonic signal(s). Pro-inflammatory cytokines IL-1 alpha and IL-1 beta may also play a significant role in endometrial response via the modulation of prostaglandin E2 production. Prostaglandins and IL-1 induce the expression of CRF, which acts as an autocrine/paracrine inflammatory regulator. CRF exhibits a strong vasoactivity in skin tests, inducing a local increase of capillary permeability at a concentrations of 10(-10) M. Levels of CRF and its mRNA were found to be higher in rat implantation sites compared with those in the interimplantation regions. Stromal cells were found to be positive for immunoreactive CRF at the implantation sites only. It is suggested that CRF may be involved in the local increase of capillary permeability seen in implantation sites, and that its production by stromal cells may be the consequence of a paracrine action of epithelial prostaglandin, released under the effect of PAF and IL-1, derived from or produced by blastocysts in endometrial cells.

Hormonal anti-implantation agents: antiprogestins.

Oestrogens and progestogens separately or in combination are able to prevent implantation with high efficiency, thus acting as interceptive agents. Current interceptive medical regimens include high-dose oestrogens, the association of oestrogens with progestogens or progestogens alone. Compounds with antiprogesterone properties, such as RU 486 (mifepristone) or ZK 98734 (lilopristone), also exhibit a strong interceptive action which, as shown in animal models, is proportional to the dose and the day(s) of administration. Recent clinical studies show that RU 486 can be used successfully for postcoital interception. The regimen applied for this purpose consists of the intake of a single dose of 600 mg RU 486 within 72 h of a single act of unprotected intercourse. This treatment was found to be highly effective and to have a more favourably side-effect profile in comparison with the oestrogen-progestogen interceptive regimen. However, because of the induced irregularities of the cycle, the mifepristone regimen, as with the other hormonal methods, should not be used on a regular basis. Currently, all interceptive hormonal regimens are emergency methods. Their occasional use to prevent unwanted pregnancies may reduce the number of therapeutic abortions. However, the frequency and extent of their side-effects do not allow for a repeated postcoital use after every act of unprotected intercourse. Obviously, the development of an effective and safe 'morning after pill' requires further basic and clinical investigations.

Uterine pinopodes as markers of the 'nidation window' in cycling women receiving exogenous oestradiol and progesterone.

In 14 cycling women participating in an in-vitro fertilization (IVF) donation programme, we examined the timing of the 'nidation window' using as a stage-specific 'marker' the presence of fully developed pinopodes on the apical surface of the luminal uterine epithelium. Each woman received exogenous oestradiol from the second day of their cycle and progesterone starting on day 8 or day 15 of the oestrogenic treatment. The women underwent two biopsies during the same artificial cycle, on either days 6 and 9 or days 8 and 10 of the progesterone treatment. All patients to whom oestradiol was administered for 7 days prior to progesterone administration (n = 9), and two of the five treated with oestradiol for 14 days prior to the addition of progesterone, showed uterine pinopodes in either one or both biopsies. When present on a given day, pinopodes were at the same stage, developing, fully developed or regressing, showing that their total lifespan did not exceed 48 h. Fully developed pinopodes existed for 1 day only which may correspond to the short period of optimal endometrial receptivity observed in animal models. The timing of the presence of fully developed pinopodes varied from patient to patient, but these individual differences were not correlated with progesterone and oestradiol plasma concentrations. The brief duration of the nidation window and the observed individual variations in its timing suggest that the detection of uterine pinopodes could be a valuable tool for the prediction, on an individual basis, of the optimal date for successful egg replacement in IVF patients.

Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus.

We have shown previously that the epithelial cells of human endometrium produce CRH. The biological role of endometrial CRH is not yet known. Among other things, CRH appears to be involved in the inflammatory process, acting as an autocrine/paracrine proinflammatory regulator. Since the reaction of endometrium to the invading blastocyst has characteristics of an aseptic inflammatory reaction, we have hypothesized that endometrial CRH may participate in the inflammatory phenomena taking place at the implantation site of blastocyst. In the present study we demonstrate a higher content of immunoreactive (IR)-CRH and CRH mRNA in the implantation sites of early pregnant rat uterus compared to the inter-implantation regions. Specifically we have found that: a) rat uterus contained a 1.3 kb CRH transcript, similar or identical in size to that present in human placenta, b) the size of the IR-CRH present in uterine extracts was similar to authentic hypothalamic CRH, c) Northern blot analysis showed that the content of CRH mRNA in uterus at the implantation sites was 3.5 fold higher compared to that in the inter-implantation regions and finally, d) immunohistochemical localization of IR-CRH in early pregnant rat uterus revealed positive staining of the luminal epithelial cells in both implantation and inter-implantation uterine regions, while decidualized stromal cells were positive only at the implantation sites. Our data suggest that endometrial CRH may play a role in the implantation of blastocyst.

Effects of cholic acid and 'Protectaid' formulations on human sperm motility and ultrastructure.

Cholic acid (sodium cholate) and the other active ingredients of F-5 gel preparations in use for the impregnation of a new vaginal sponge (Protectaid) with contraceptive and anti-sexually transmitted disease properties, were assessed for their effects on human sperm motility and ultrastructure. Cholic acid (CA) produced an inhibition of motility which was both dose- and time-dependent. A complete suppression of motility was obtained at 30 s by a CA concentration of 1.25%. Nonoxynol-9 (NX9) compared with benzalkonium chloride (BZC) showed no significant difference at the concentration required (0.025%) to give a total inhibition of sperm motility after exposure for 30 s. The addition of F-5A gel containing 0.5% of each one of the spermicide ingredients (CA, NX9 and BZC) produced the total suppression of sperm motility within 30 s at a dilution of 1/50. Another preparation, F-5B gel, containing the spermicide ingredients at different concentrations (1.25% CA, 0.125% NX9 and 0.05% BZC) produced this same effect with a 1/10 dilution. Exposure of semen to a CA concentration of 1.25% or to 1/10 dilutions of F-5A gel for 30 s led to profound changes of sperm ultrastructure studied by scanning (SEM) and transmission (TEM) electron microscopy. SEM and TEM findings indicate that CA acts as a spermicide through its 'natural detergent' properties, damaging the outer plasma membrane of sperm cells. Protectaid formulations affect sperm motility and viability in a similar way.

The zona reaction in human oocytes as seen with scanning electron microscopy.

Using scanning electron microscopy we found differences in the fine structure of the zona pellucida between unfertilized and fertilized human pronuclear stage oocytes in an in-vitro fertilization programme. In unfertilized oocytes, the zona pellucida appeared porous, comprising a large number of ring-shaped structures, called hoops, randomly superimposed in several layers. Superficial pores had a mean diameter of 4 microns, with the diameter decreasing in more inner lying pores. In fertilized oocytes, the zona pellucida was compact; the hoops appeared to melt and the pores to be obliterated by an amorphous material emerging from the inner zona. The micrographs provide ultrastructural evidence of the zona reaction in human oocytes and give insights into the morphological and mechanical aspects of the polyspermy-blocking mechanism in humans.

Inhibition of progesterone receptor function results in loss of basic fibroblast growth factor expression and stromal cell proliferation during uterine remodelling in the pregnant rat.

Recent studies suggest that hormonal control of uterine cell proliferation may be moderated by polypeptide growth factors. It remains to be determined, however, whether growth factors cause or are the consequence of hormone action. Basic fibroblast growth factor (bFGF) has been shown to influence cell proliferation and differentiation of a variety of mesoderm-derived cells. To elucidate the regulatory mechanisms controlling stromal cell proliferation and differentiation required for embryo implantation further, immunohistochemical localization of the progesterone receptor and bFGF have been studied. The cell-specific distribution of these proteins was determined in the rat uterus during early pregnancy and after injection of the progesterone receptor antagonist mifepristone (RU 486) at days 1 and 2 post coitum (p.c.) to block implantation. Cell division was restricted to luminal and glandular epithelial cells in pregnant and RU 486-treated rats at day 3 p.c. At day 4 of pregnancy, cell proliferation switched from the epithelia to the stroma in pregnant rats, but after RU 486 treatment division of stromal cells was inhibited significantly (P < 0.05). Progesterone receptor distribution was altered and bFGF was absent in RU 486-blocked stromal cells. Expression of bFGF in luminal and glandular epithelial cells, however, was insensitive to the effects of progesterone receptor antagonism. bFGF content was stimulated in the luminal epithelium and in decidual cells by the implanting embryo. These results indicate that repression of progesterone receptor function in early pregnancy results in a cell-specific loss of bFGF from stromal cells and inhibition of their proliferation. The results further suggest that the regulation of endometrial cell bFGF content is modulated at the site of implantation by the embryo.

Physiological oestradiol and progesterone replacement cycles in women with ovarian failure: a model to study endometrial maturation and sex steroid receptor regulation by exogenous hormones.

High endometrial receptivity has been achieved with physiological oestradiol and progesterone replacement cycles in women with ovarian failure. To understand whether different protocols using the oral route or the transdermal route can influence the endometrial maturation and the regulation of sex steroid receptors, we studied 33 women with ovarian failure treated by two commonly used protocols and assessed endometrial receptivity using light microscopy, scanning electron microscopy and immunohistochemistry for oestrogen and progesterone receptors on biopsies taken to include different periods of the luteal phase. The morphology in these patients was similar to that observed in women with normal ovulatory cycles, indicating that the morphological response is not dependent on the type of oestradiol, oral or transdermal, in the replacement cycles as compared to the endogenous oestradiol in the menstrual cycle. The relative distribution of steroid receptors between the epithelium and stroma varies similarly to that observed during the luteal phase of the menstrual cycle. These results confirm the role of progesterone, especially the importance of the number of days of exposure to it, in the disappearance of steroid receptors from endometrial glands. These observations give a better understanding of endometrial receptivity around the time of presumed implantation and confirm clinical results concerning the best timing of oocyte transfer.

Spermicidal and antiviral properties of cholic acid: contraceptive efficacy of a new vaginal sponge (Protectaid) containing sodium cholate.

Cholic acid (sodium cholate) exhibits a strong spermicidal and antiviral [anti-human immunodeficiency virus (HIV)-1] activity. The same effects are observed for F-5 Gel, the active mixture of a new contraceptive sponge (Protectaid), which contains sodium cholate in association with low concentrations (0.5%) of nonoxynol-9 and benzalkonium chloride. Both cholic acid and the F-5 Gel exert a dose-dependent, in-vitro inhibitory effect (i) on the activity of HIV-1 associated reverse transcriptase in an acellular system and (ii) on the potential of HIV-1 efficiently to infect human lymphocytes. During 12 months use, the contraceptive efficacy of the 'Protectaid' sponge was 100% in 20 young women who had chosen this method for reasons of both contraception and anti-sexually transmitted disease. No side-effects were recorded throughout this period. Cervical cultures at 6-month intervals showed the presence of Mycoplasma hominis and Candida albicans in one or two cases. The combined spermicidal and anti-HIV properties of cholic acid reported in this paper and used in the 'Protectaid' sponge offer a new and modern protective method of contraception.

PIP: Cholic acid (sodium cholate) exhibits a strong spermicidal and antiviral [anti-human immunodeficiency virus (HIV)-1] activity. The same effects are observed for F-5 Gel, the active mixture of a new contraceptive sponge (Protectaid), which contains sodium cholate together with low concentrations (0.5%) of nonoxynol-9 and benzalkonium chloride. Both cholic acid and the F-5 Gel exerted a dose-dependent, in-vitro inhibitory effect 1) on the activity of HIV-1 associated reverse transcriptase in an acellular system (their 50% inhibitory dose was 7.2 mM and 0.8 x 10 -3 v/v, respectively, and 2) on the potential of HIV-1 to infect human lymphocytes efficiently. In the 3 semen samples examined, sperm motility was instantaneously inhibited by the addition of a 6 mM solution of sodium cholate or of a 1:10 dilution of F-5 Gel. Both cholic acid and F-5 Gel affected in a dose-dependent manner the viability of normal peripheral blood lymphocytes (NPBL) and CEM cells. The Protectaid contraceptive sponge impregnated with F-5 Gel was given to 20 young women aged 19-25 years for a period of 1 year who had chosen this method for both contraception and against sexually transmitted diseases. All women were instructed to insert the sponge within the 12 hours preceding each sexual intercourse and to remove it 4-6 hours afterwards. During 12 months of use with at least 3 intercourse per week, the contraceptive efficacy of the Protectaid vaginal sponge was 100%. Cervical cultures at 6-month intervals showed the presence of Mycoplasma hominis and Candida albicans in 1 and 2 cases, respectively. The combined spermicidal and anti-HIV properties of cholic acid reported and used in the Protectaid sponge offer a new and modern protective method of contraception. At the end of the study, cervical cultures revealed the presence of Escherichia coli and Candida albicans in 1 case each. No slide effects were recorded, and only 1 woman complained of discomfort.

Preovulatory effects of the progesterone antagonist mifepristone (RU486) in mice.

The progesterone antagonist mifepristone (RU486), was given in mice once on different days of pregnant mare's serum gonadotrophin-human chorionic gonadotrophin (PMSG-HCG) treatment and its action upon the induction of ovulation studied. RU486 administered on the day after PMSG significantly reduced the ovulation rate. Ovulation was completely inhibited when the progesterone antagonist was given simultaneously with HCG, but RU486 administered 4 h after HCG treatment remained ineffective. The development of two-cell zygotes harvested on day 2 post-coitum from mice treated with RU486 on the day after the PMSG treatment was followed in vitro and showed a significant decrease in the number of embryos developing to blastocysts. These results favour the involvement of progesterone in the ovulation process, indicating a direct effect of this hormone at the ovarian level via a progesterone receptor-mediated action.

Hormonal dependence of pinopode formation at the uterine luminal surface.

The uterine luminal epithelium during the period of receptivity for nidation displays characteristic protrusions of the apical surface named pinopodes. The effects of oestradiol and progesterone, singly or in combination, on the formation and regression of pinopodes were investigated using scanning electron microscopy. The appearance of pinopodes was found to be strictly progesterone dependent. When given together with progesterone, before the development of pinopodes, high doses of oestradiol (plasma level approximately 300 pmol/l) inhibited pinopode formation; on the contrary, low doses of oestradiol (nidatory doses) did not interfere with the process until the 4th day of treatment. When oestradiol was given as a single injection, after pinopode formation, both doses were equivalent in inducing their regression 48-72 h later. It appears that the hormonal conditioning for pinopode formation and for the development of uterine receptivity for egg implantation is the same. These observations support the hypothesis that pinopodes could be an extremely useful tool to estimate uterine receptivity. The experiments we describe here, together with observations made a few years ago, in stimulated cycles in the human, suggest that implantation failure as a result of a hormonal imbalance during the time intervening between ovulation and nidation, may be a general phenomenon.

Is cholic acid responsible for embryo-toxicity of the post-receptive uterine environment?

HPLC analysis of the embryo-toxic fraction of human uterine fluid, collected between the 22nd and 25th days of the menstrual cycle, revealed the presence of cholic acid at high concentrations. It is suggested that cholic acid could be responsible for the embryo-toxicity of the uterine environment, which follows the receptive period for implantation.

Effect of mifepristone (RU 486) on concentrations of prostaglandin E-2 binding sites in the rat endometrium.

Progesterone implants in ovariectomized rats increased endometrial concentrations of PGE-2 receptors. The increase was completely inhibited by simultaneous daily injection (7.5 mg/kg) of mifepristone (RU 486). A single injection of mifepristone on the morning of Day 1 of pseudopregnancy (day of oestrus) decreased the amount of PGE-2 receptors found in the endometrium on Day 5 by 64%. This inhibitory effect probably resulted from the antiprogesterone activity of this compound since it was not counteracted by simultaneous treatment with dexamethasone, shown to reverse totally the antiglucocorticoid action of mifepristone. The inhibition by mifepristone lasted only for 1 day; endometrial PGE-2 receptor levels on Day 6 of pseudopregnancy returned to the high values present in controls. Under these conditions, administration of the mifepristone did not affect the plasma oestradiol and progesterone concentrations during the 1st week of pseudopregnancy. The administration of mifepristone on Days 2 and 3 of pseudopregnancy kept the endometrial PGE-2 receptor levels low, even by 4 days after the end of treatment. We therefore concluded that, in the rat, progesterone priming leading to uterine receptivity can be delayed, at least by 1 day. In contrast, interruption of the progesterone action for a longer period later during the early pseudopregnant period resulted in an altered subsequent evolution of the endometrium, in terms of acquisition of the PGE-2 binding sites.

Hormonal dependence of the metrial gland: further studies on oestradiol and progesterone receptor levels in the rat.

The main objective of the present study was to analyse the hormonal dependence of the metrial gland formed in pseudopregnant animals following massive decidualization. On day 13 of pseudopregnancy (when the metrial gland reaches its maximal development) animals were ovariectomized and given s.c. implants of oestradiol and/or progesterone. A new implant technique for oestradiol delivery is described which provides circulating concentrations of oestradiol in the physiological range. In addition, we extended our previous work concerning oestradiol receptor and progesterone receptor concentrations in the metrial gland of pseudopregnant rats. The low oestradiol receptor concentration which we previously reported up to day 17 was maintained until the end of pseudopregnancy (day 21-1.5 fmol/micrograms DNA), whereas the progesterone receptor concentration remained raised (congruent to 3.5 fmol/micrograms DNA) from day 13 to day 19 and then decreased on day 21. The correlation of metrial gland weight and kinetics of the tissue oestradiol and progesterone receptors contents with the circulating oestradiol and progesterone concentrations lead to the following conclusions. First, the maintenance of the metrial gland is strictly progesterone-dependent. It is unlike the deciduoma which regresses spontaneously, even in the presence of progesterone. Secondly, the production of oestradiol receptor, but not of progesterone receptor, appears to be repressed in the metrial gland under the influence of progesterone. Thus, the tissue retains its ability to respond to progesterone because of a high concentration of progesterone receptor. It is difficult to attribute this high tissue progesterone receptor concentration to oestradiol stimulation since, even at low levels, oestradiol induces tissue regression. We suggest that the high progesterone receptor concentration could be due to constitutive (basal) progesterone receptor production.