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Background: The maternal-fetal interface has a unique immunological response towards the implanting placenta. It is generally accepted that a T-helper type-2 (Th-2) cytokine prevailing environment is important in pregnancy. The proportion of Th-2 cells in the peripheral blood and decidua is significantly higher in pregnant women in the first trimester than in non-pregnant women. Glycodelin-A (GdA) is a major endocrine-regulated decidual glycoprotein thought to be related to fetomaternal defence. Yet the relationship between its immunoregulatory activities and the shift towards Th-2 cytokine profile during pregnancy is unclear., Methods: GdA was immunoaffinity purified from human amniotic fluid. T-helper, T-helper type-1 (Th-1) and Th-2 cells were isolated from the peripheral blood. The viability of these cells was studied by XTT assay. Immunophenotyping of CD4/CD294, cell death and GdA-binding were determined by flow cytometry. The mRNA expression, surface expression and secretion of Fas/Fas ligand (FasL) were determined by quantitative polymerase chain reaction, flow cytometry and ELISA, respectively. The activities of caspase-3, -8 and -9 were measured. The phosphorylation of extracellular signal-regulated kinases (ERK), p38 and, c-Jun N-terminal kinase was determined by western blotting., Results: Although GdA bound to both Th-1 and Th-2 cells, it had differential actions on the two cell-types. GdA induced cell death of the Th-1 cells but not the Th-2 cells. The cell death was mediated through activation of caspase -3, -8 and -9 activities. GdA up-regulated the expression of Fas and inhibited ERK activation in the Th-1 cells, which might enhance the vulnerability of the cells to cell death caused by a trophoblast-derived FasL., Conclusions: The data suggest that GdA could be an endometrial factor that contributes to the Th-2/Th-1 shift during pregnancy.

Background: Glycodelin-A interacts with spermatozoa before fertilization, but its role in modulating sperm functions is not known. Zona pellucida-induced acrosome reaction is crucial to fertilization and its dysfunction is a cause of male infertility. We hypothesized that glycodelin-A, a glycoprotein found in the female reproductive tract, potentiates human spermatozoa for zona pellucida-induced acrosome reaction., Methods: Glycodelin isoforms were immunoaffinity purified. The sperm intracellular cAMP concentration, protein kinase-A (PKA) and extracellular signal-regulated kinase (ERK) activities, and intracellular calcium were measured by ELISA, kinase activity assay kits and Fluo-4AM technique, respectively. The phosphorylation of inositol 1,4,5-trisphosphate type-1 receptor (IP3R1) mediated by ERK was determined by western blotting. Zona pellucida-induced acrosome reaction was detected by Pisum sativum staining., Results: Pretreatment of spermatozoa with glycodelin-A significantly up-regulated adenylyl cyclase/PKA activity and down-regulated the activity of ERK and its phosphorylation of IP3R1, thereby enhancing zona pellucida-induced calcium influx and zona pellucida-induced acrosome reaction. Glycodelin-F or deglycosylated glycodelin-A did not have these actions. Treatment of spermatozoa with a protein kinase inhibitor abolished the priming activity of glycodelin-A, whilst ERK pathway inhibitors mimic the stimulatory effect of glycodelin-A on zona pellucida-induced acrosome reaction., Conclusions: Glycodelin-A in the female reproductive tract sensitizes spermatozoa for zona pellucida-induced acrosome reaction in a glycosylation-specific manner through activation of the adenylyl cyclase/PKA pathway, suppression of extracellular signal-regulated kinase activation and up-regulation of zona pellucida-induced calcium influx. The action of glycodelin-A may be important in vivo to ensure full responsiveness of human spermatozoa to the zona pellucida.

Background: Ethical constraints limit the ability to study peri-implantation phase human endometrium. In this study, the donor oocyte model was used to study candidate endometrial markers of uterine receptivity., Methods: Archived, paraffin-embedded tissue obtained by endometrial biopsy during cycle days 21-23 of patients undergoing 'mock' hormonal treatment cycles were evaluated by standard histological criteria and immunohistochemical staining for alpha v beta 3 integrin and glycodelin. All of these patients (n = 101) had undergone a donor oocyte embryo transfer cycle utilizing the exact same hormonal protocol., Results: Histological evaluation revealed 62 (61.3%) in-phase, 34 (33.7%) dyssynchronous, 2 (2.0%) immature and 3 (3.0%) advanced endometria. The clinical outcomes of patients with either in-phase or dyssynchronous endometria were similar. Very strong correlations were noted between endometrial glandular dating and either alpha v beta 3 integrin or glycodelin immunostaining intensity (P < 0.001 for both). Glycodelin and alpha v beta 3 integrin immunostaining intensities were also highly correlated with each other (P < 0.001)., Conclusions: Throughout the time period corresponding to the putative window of maximal endometrial receptivity (cycle days 21-23) a dynamic process was observed in exogenous hormonal replacement cycles characterized by a rapid histological advancement of endometrial glandular elements as well as progressive alpha v beta 3 integrin and glycodelin expression.

The regulation of epithelial cell function and morphogenesis by the paracrine effectors from the mesenchyme or stroma has been well established using in-vivo studies. A more complete understanding of these relationships has been delayed due, in part, to a lack of appropriate co-culture models. In this study, we describe a co-culture model which demonstrates that normal paracrine relationships can be reconstituted in vitro and that human endometrial stromal cells regulate both growth and differentiation of primary human endometrial epithelial cells. Interesting differences in the proliferation of stromal and epithelial cells were noted in response to the basement membrane extract, Matrigel((R)). Exposure of stromal cells to Matrigel((R)) enhanced the paracrine capacity of these cells in vitro. When epithelial cells were co-cultured in contact with stromal cells embedded in Matrigel((R)), epithelial cell growth was inhibited by 65-80% compared to controls. Stromal cells in contact with Matrigel((R)) also regulated epithelial cell differentiation, as shown by induction of glycodelin expression. These co-culture studies show great promise as a method to investigate the cellular interactions between endometrial stromal and epithelial cells and their environment and to understand the molecular basis for the regulation of normal growth and differentiation of cells within complex tissues such as the endometrium.

This exploratory study was designed to determine whether treatment with the Yuzpe regimen of emergency contraception altered endometrial integrin expression or other markers of uterine receptivity. Nineteen parous women were followed for two menstrual cycles. In the second cycle, each participant took 100 mg ethinyl oestradiol and 1 mg norgestrel on the day of the urinary luteinizing hormone (LH) surge and repeated the dose 12 h later. In both cycles, endometrial biopsy, phlebotomy and vaginal sonogram were performed 8-10 days after the urinary LH surge. No significant difference was found between untreated and treated cycles in most measures of endometrial histology or in endometrial expression of beta3 integrin subunit, leukaemia inhibitory factor, glycodelin, or progesterone receptors assessed by immunohistochemical techniques. Five statistically significant changes were noted in treated cycles: a reduction in endometrial MUC-1 expression, an increase in endometrial oestrogen receptor, lower luteal phase serum oestrogen concentration, reduced endometrial thickness, and greater proportion of glandular supranuclear vacuoles. The relationship of these findings to the contraceptive action of the Yuzpe regimen is unclear.

Glycodelins are glycoproteins synthesized in various glands, with sequence homology to beta-lactoglobulins, and named according to their unique oligosaccharide structures. We purified, cloned and sequenced endometrium- and seminal plasma-derived glycodelins (GdA and GdS respectively) and found that they are involved in various types of cell-cell communications. These include interactions between the spermatozoon and the egg, and between immune cells and their targets. Endometrial GdA inhibits sperm-egg binding, whereas the differently glycosylated GdS in seminal plasma does not. These observation are of interest for reproductive physiology, detection of causes of infertility, and they also may have potential for contraceptive development.

Intrauterine devices (IUDs) exert contraceptive action by interfering with sperm transport, ovum development, fertilization and implantation. Glycodelin A (GdA) is a uterine glycoprotein that has local contraceptive activity by inhibiting sperm-egg binding. GdA is normally absent from endometrium during the fertile midcycle and it is not expressed until the fifth postovulatory day. The phase of menstrual cycle addressed in this study covers the phase when conception is most likely to follow an unprotected intercourse and when GdA is normally absent. We present here evidence that levonorgestrel-releasing IUD (LNg-IUD) is accompanied by 'inappropriate' expression of GdA in endometrium between days 7 and 16 of the menstrual cycle (six out of six cases). The same was also found in copper-releasing IUD (Cu-IUD)-wearing women, but less frequently (four out of 11 cases, P < 0.0345, Fisher's exact test). In-situ hybridization localized GdA mRNA into endometrial glands in the midcycle endometrium, confirming the cellular site of synthesis. Based on the potent inhibitory activity of GdA on sperm-egg binding, the presence of GdA in uterine glands of IUD wearers may lead to prior exposure of sperm to contraceptive GdA, thus contributing to the contraceptive activity of the IUD.

The effects of low daily doses of the antiprogestin mifepristone (RU 486) on ovarian and endometrial function were studied. The study included one control cycle, three treatment cycles and one follow-up cycle. During the treatment cycles, either 0.1 (n = 5) or 0.5 (n = 5) mg of mifepristone was administered once daily. Urine samples were collected three times weekly during the control and treatment cycles and pregnanediol glucuronide and oestrone glucuronide and luteinizing hormone (LH) were quantified by radioimmunoassay. Blood samples for cortisol measurement were collected once weekly and for serum glycodelin at the onset of menstruation. An endometrial biopsy was obtained in the mid-luteal phase in the control cycle and in the first and third treatment cycles and analysed by morphometric and histochemical methods. Binding of Dolichus biflorus agglutinin (DBA) lectin was measured and expression of progesterone and oestrogen receptors and glycodelin were analysed immunohistochemically. All cycles studied were ovulatory with an LH peak and elevated pregnanediol glucuronide concentrations. Follicular development seemed normal as judged by ultrasound examination. The length of the menstrual cycle and the menstrual bleeding were not significantly altered. Following administration of 0.5 mg mifepristone/day, endometrial development appeared to be slightly retarded and glandular diameter was significantly reduced. Furthermore, significant decreases in DBA lectin binding and endometrial expression of glycodelin were observed. Daily doses of 0.1 mg did not have any significant effect on the endometrium. No differences in oestrogen or progesterone receptor immunoactivity between control and treatment cycles were seen. This study provides further evidence that endometrial function is sensitive even to doses of antiprogestin that are low enough not to disturb ovulation. It remains to be established whether these effects are sufficient to prevent implantation.

The mechanisms underlying the protection of the human embryo/fetus from the maternal immune response are poorly understood. Substantial evidence indicates that carbohydrate recognition plays a primary role in the sequestration of leukocytes during inflammatory processes, lymphocyte homing, and initial gamete binding. Our previous studies suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Our more recent findings indicate that oligosaccharides participating in such processes are also associated with soluble glycoconjugates found in the human placenta, amniotic fluid, and decidua. We theorize that such glycoconjugates may abrogate the maternal immune/inflammatory response by blocking the primary adhesive interactions required for the expression of such activities. Foreign embryonic cells may also be protected by surface expression of oligosaccharide sequences that suppress immune effector cell action in a manner not dependent upon classical major histocompatibility (MHC) recognition. Glycoconjugates expressing selectin ligands may also manifest a potent contraceptive effect that may also be beneficial for both the mother and the developing embryo/fetus. This hypothesis provides a preliminary framework for understanding how temporally and spatially restricted immunosuppressive effects could be expressed in utero that protect the human embryo/fetus during this period of human development.

The effect of a low dose of mifepristone (RU486) on ovarian and endometrial function was studied in 14 healthy women. The study included one control and two treatment cycles. During the treatment cycles, either 2.5 mg (n = 9) or 5 mg (n = 5) of mifepristone was administered once weekly. The concentration of ovarian steroids and luteinizing hormone (LH) in urine was measured daily, cortisol in blood once weekly and glycodelin (placental protein 14; PP14) at the time of menstruation. Ovarian function was monitored by vaginal ultrasound. An endometrial biopsy was taken in each cycle in the mid-luteal phase, based on self-measurement of the LH peak, or on cycle day 22 if no LH peak could be detected. In the evaluation of the results, the outcome of the enzyme immunoassay of LH was used to date the biopsy. Endometrial progesterone and oestrogen receptors and Dolichus biflorus agglutinin (DBA) lectin binding were measured. Ovulation was not inhibited by treatment with mifepristone, and an LH peak could be determined in all control and treatment cycles. However, in four subjects (one with the higher and three with the lower dose) the follicular phase was prolonged by 6-13 days. The duration of the luteal phase and the concentrations of pregnanediol and oestrone glucuronide were not affected by treatment. A dose of 5 mg, and to a lesser extent 2.5 mg, mifepristone once weekly caused desynchronization of endometrial development. Endometrial progesterone receptor, but not oestrogen receptor, concentration was significantly increased by the higher dose. A significant reduction in DBA-lectin binding and in serum glycodelin concentrations was also found. Thus, low doses of mifepristone do not inhibit ovulation but delay endometrial development and impair secretory activity. Whether these effects are sufficient to prevent implantation remains to be established.

Products of the corpus luteum have targeted actions on the endometrium. Besides steroid hormones, the corpus luteum produces biologically active substances which may be either unique or shared by the endometrium and other tissues. Here we review selected markers of the corpus luteum and the endometrium as candidates for functional markers of the interplay between the two sites and relative to various treatment modalities. In clinical routine, the assessment of luteal phase is performed by morphological criteria. The timing of endometrial biopsy is important because specimens taken at different stages of the luteal phase give different results. After human menopausal gonadotrophin (HMG) superovulation, there is dyssynchrony in the morphological maturation of endometrial glands and stroma, and a marked difference has been found in endometrial development between progesterone-supplemented and non-supplemented cycles. The expression of steroid hormone receptors in endometrium is affected by ovarian stimulation regimens. After gonadotrophin-releasing hormone analogue/HMG superovulation, the progesterone receptor (PR) has been found less frequently in progesterone-supplemented cycles than in non-supplemented cycles. The relative distributions of oestrogen receptor and PR between epithelium and stroma have been reported to vary according to the number of days of progesterone exposure. Thus, the detection of PR in endometrial glands in the late luteal phase indicates that exposure of the endometrium to the action of progesterone is short. Certain biochemical changes in the uterus are not reflected in endometrial morphology. Under the influence of progesterone, secretory glandular epithelium synthesizes placental protein 14, more recently named glycodelin. Glycodelin inhibits the innate immune system and also has contraceptive actions. Endometrial glands secrete glycodelin into glandular lumen, uterine fluid and blood, where the concentrations rise during the last week of the secretory phase. The effects of various ovarian stimulation protocols on serum glycodelin concentrations are reviewed, along with recent studies on relaxin, prolactin and insulin-like growth factor binding protein 1, all products of the secretory endometrium.

Previous studies suggest that, in pregnancies after in-vitro fertilization (IVF) and embryo transfer following pituitary down-regulation with a gonadotrophin-releasing hormone analogue (buserelin) and ovulation induction with human gonadotrophins, the serum placental protein 14 (PP14) concentration is lower than in normally conceived pregnancies. We studied serum PP14 concentrations in two groups of women: (i) in 17 infertile women whose pregnancy followed IVF and embryo transfer using buserelin (long protocol) and human menopausal gonadotrophin for ovulation induction; (ii) in 15 women whose pregnancy followed transfer of frozen-thawed embryos. Similar PP14 concentrations were found in both groups on days 9-10, 14-15 and 70-77 after human chorionic gonadotrophin administration (buserelin, IVF/embryo transfer) or spontaneous luteinizing hormone surge (frozen-thawed embryo transfer). Our results show that PP14 secretion is not compromised by pituitary down-regulation with buserelin in infertile women with functional ovaries.

Human ovarian follicular fluid contains a number of insulin-like growth factor binding proteins (IGFBP) of which IGFBP-3 is the most abundant. IGFBP-3 synthesis is growth hormone-regulated. We studied the effect of prostaglandin F2 alpha (PGF2 alpha) on IGFBP-3 secretion by cultured human granulosa-luteal cells from follicular aspirates of women participating in an in-vitro fertilization programme. The IGFBP-3 concentration was measured using a specific monoclonal immunofluorimetric assay. Contrary to a previous report on unstimulated follicles, this study demonstrated a positive correlation between follicular fluid IGFBP-3 concentration and follicular size. PGF2 alpha was found to stimulate in a dose-dependent fashion the secretion of IGFBP-3. Significant (P < 0.05) effects were found at PGF2 alpha concentrations of 10(-8), 10(-7) and 10(-6) M. Because IGFBP-3 inhibits progesterone production stimulated by insulin-like growth factor (IGF)-I, the PGF2 alpha-induced stimulation of IGFBP-3 production may be one of the mechanisms whereby PGF2 alpha exerts its luteolytic effect via the IGF system.

A large number of proteins and peptides have been identified in the endometrium where they are likely to exert local biological effects. These substances may be enzymes, their inhibitors, proteinases, proteinase inhibitors, hormones, or bioactive peptides with diverse functions. Endometrial function and embryo-endometrial interactions require synchronized actions between endocrine and local factors. As examples of local factors recent studies on the insulin-like growth factor (IGF) system and placental protein 14 (PP14) are reviewed. IGF binding protein-1 (IGFBP-1) and PP14 are products of the secretory phase endometrium: IGFBP-1 is produced by decidualized stromal cells and PP14 by glandular epithelial cells. IGFBP-1 may inhibit the action of IGFs at the endometrial-trophoblastic interphase, and it may also have a role in stromal-epithelial interaction. PP14 has immunosuppressive properties, and recent findings indicate that it may play a part in the fertilization process by inhibiting binding of spermatozoa to the zona.

The purpose of this study was to investigate whether endometrium with retarded development differs, functionally, from endometrium with normal 'in-phase' development. Precisely timed endometrial biopsies were obtained from 24 women suffering from unexplained infertility at 4, 7, 10 and 13 days following the luteinizing hormone (LH) surge. Frozen sections were labelled with an anti-placental protein (PP) 14 monoclonal antibody using an avidin-biotin peroxidase technique and semi-quantification of endometrial PP14 was performed using a Quantimet 970 image analyser. Serum PP14 and saliva progesterone were measured for each patient. Data were analysed using one- and two-way analysis of variance. Normal and retarded endometrium were identified in 16 (group I) and eight (group II) women respectively. Both groups demonstrated a significant increase of the area of precipitate measured for PP14 from day LH + 4 to LH + 13. However, two-way analysis of variance showed that endometrial PP14 was significantly (P < 0.05) lower in the retarded endometrium group at LH + 10 and LH + 13. Serum PP14 was also significantly lower (P < 0.01) in women with retarded endometrial development at LH + 13. Women with normal endometrial development had a significantly higher (P < 0.05) concentration of cumulative saliva progesterone from LH + 3 to LH + 5. This study indicates that there are functional differences between normal and retarded endometrium. These differences may adversely affect uterine receptivity during implantation and the early placentation stage.

This study was designed to investigate the basic concentrations of different hormones in obese and non-obese patients with polycystic ovarian disease (PCOD). Eight women with PCOD, of whom four were obese with body mass index (BMI, kg/m2) of > 25 and four were non-obese with BMI < 25, volunteered to participate in this study. Serum samples were taken every 20 min over an 8 h period, starting at 2300 h, on day 5 of a spontaneous or gestagen-induced cycle. Basic insulin concentration was found to be significantly higher in the obese women compared with their non-obese counterparts (P < 0.0001). Serum concentrations of insulin-like growth factor binding protein (IGFBP-I) and sex hormone binding globulin (SHBG) were found to be significantly lower (P < 0.001 for both hormones) in the obese compared with the non-obese women. Serum concentrations of insulin-like growth factor I (IGF-I) did not differ between the two groups. The non-obese women had significantly higher serum concentrations of luteinizing hormone (LH) (P < 0.001) and of growth hormone (GH) (P < 0.002) than their obese counterparts. Based on these results, two models of the development of PCOD were suggested. In obese women, hyperinsulinaemia causes an excessive production of androgens through the enhancement of IGF-I receptors which, in synergism with LH, causes increased activity of cytochrome P-450c 17a. In non-obese patients, relative increase of GH concentration stimulates excessive ovarian IGF-I production. At this point synergism with LH results in excessive production of androgens by the same mechanism as in obese patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of RU 486 combined with tamoxifen and tamoxifen alone on hormonal parameters and endometrial development at the time of implantation were studied. Measurements of cytosolic oestrogen and progesterone receptors in endometrium and placental protein 14 (PP14) in plasma were also included. Three dosage schedules were used: single oral dose of 40 mg tamoxifen alone and in combination with 200 mg RU 486, and 40 mg tamoxifen for three consecutive days starting on the first day after the luteinizing hormone (LH) surge. The combined treatment prolonged the luteal phase (P < 0.05) and increased the plasma levels of progesterone. A single dose of tamoxifen did not affect the bleeding pattern and plasma hormone levels, but raised plasma oestradiol and LH with the 3-day treatment. The endometrium was retarded after the combined and the 3-day treatment with tamoxifen. Concentrations of cytosolic progesterone receptors were higher after the combined therapy, but were unaffected after tamoxifen only. PP14 levels were higher (P < 0.05) after repeated tamoxifen doses than in controls, but were lower with combined treatment. Progesterone and oestrogen are evidently necessary for endometrial maturation during the secretory phase of the menstrual cycle. PP14 levels in plasma cannot be used for clinical assessments of endometrial function because high levels coincide with disturbed endometrial development.

Uterine factors influence reproduction at the macro-anatomy level, and the effects of hormonal steroids on endometrial morphology are well recognized in the histopathological diagnosis of dysfunctional bleeding and infertility. During the past decade, attention has been paid to endometrial protein synthesis and secretion with respect to endocrine stimuli and implantation, and to the paracrine/autocrine effects of endometrial peptide growth factors, their binding proteins and other factors. The emphasis of this presentation is on protein secretion of the secretory endometrium, in which progesterone plays a pivotal role. Insulin-like growth factors have receptors on the endometrium, and IGF-binding proteins, stimulated by progesterone, modulate the effects of IGFs locally. Also other protein products of the secretory endometrium have been reviewed in this communication, with special emphasis on studies of a progesterone-associated endometrial protein which has many names in the literature, such as PEP, PP14, alpha 2-PEG and AUP. Extensive studies are ongoing in many laboratories to elucidate the regulation, function, interplay at tissue and cellular levels, and clinical significance of these proteins.

Placental protein 14 (PP14) and human chorionic gonadotrophin (HCG) were analysed in patients participating in an in-vitro fertilization-embryo transfer programme which did not include any kind of luteal support. Women with normal pregnancies, spontaneous abortions, ectopic pregnancies, biochemical pregnancies and non-pregnant women were compared. A combination of HCG and PP14 analyses distinguished between normal and abnormal implantation as early as 15 days after oocyte retrieval. The product of HCG (IU/l) and PP14 (micrograms/l) concentrations differed significantly between normal pregnancy, spontaneous abortion and ectopic pregnancy (P = 0.0248). It is concluded that both endometrial (PP14) and trophoblastic (HCG) markers, when used in combination, exhibit changes in abnormal implantation which may be clinically useful.

Localization and immunohistochemical staining patterns of endometrial protein PP14 were studied in 55 patients providing 86 histologically confirmed pelvic and ovarian endometriotic implants. The cellular localization of PP14 in endometriotic implants is comparable with that in the endometrium: epithelial cells express PP14, whereas stromal cells are negative. Positive immunostaining is restricted to apical secretory-like granules of the epithelial cells. In endometriotic implants with positive staining, PP14 is expressed by some, but not all, glandular epithelial cells. Furthermore, positive staining for PP14 is observed most frequently in foci with in-situ secretory differentiation, whereas implants with proliferative or atrophic implants only rarely express PP14. Finally, PP14 can be localized in endometriotic foci at different depths of infiltration, although positive labelling is seen more often in superficial implants. These data demonstrate that PP14 can be expressed by endometriotic glandular epithelial cells with secretory cellular differentiation and that the histological appearance of ectopic implants sometimes only poorly reflects their function.

Serum concentrations of insulin-like growth factor binding protein-1 (IGFBP-1) were measured in 42 patients with tubal infertility undergoing two different regimens of ovarian stimulation for in-vitro fertilization. The first group comprised 24 women given a luteinizing hormone-releasing hormone agonist analogue (buserelin, LHRHa) on cycle days 1-4 followed by follicle-stimulating hormone and human menopausal gonadotrophin (LHRHa group). The second group of 18 women received clomiphene citrate and gonadotrophins (CC group). On the day of human chorionic gonadotrophin administration, the sum of follicular diameters (P less than 0.05) and the number of follicles punctured in the LHRHa group (P less than 0.01) were significantly higher than in the CC group. In spite of the greater number of follicles, the oestrogen levels were similar in both groups, but women in the LHRHa group had significantly higher serum IGFBP-1 concentrations during the last 5 days of stimulation. These results suggest that the stimulated preovulatory follicles may contribute to the elevation of serum IGFBP-1.

In order to study the effects of insulin-like growth factor (IGF-I) and insulin-like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP-1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells.

A double-blind placebo-controlled study on bromocriptine administration during days 2-12 of ovarian hyperstimulation for in-vitro fertilization (IVF) showed that, in bromocriptine cycles, levels of the endometrial protein PP14 were higher in the late luteal phase. This was verified both by calculating forward from the day of human chorionic gonadotrophin (HCG) administration and backward from the onset of the next period. Bromocriptine had no effect on IVF performance. During bromocriptine treatment the serum prolactin levels declined and serum oestradiol levels were higher on day 9 of the cycle. There was a positive correlation (r = 0.55; P = 0.012) between the serum oestradiol levels on day 9 and the PP14 levels on days 22-23 of the cycle. No difference was found in the luteal phase progesterone levels between bromocriptine- and placebo-treated cycles. These results suggest that low prolactin and/or high oestradiol levels during the follicular phase have an influence on the subsequent secretory capacity of the endometrium as reflected by secretion of a specific endometrial protein.

Placental protein 14 isolated from the human placenta and its adjacent membranes, and progestagen-dependent endometrial protein (PEP) isolated from the endometrium have been described independently by two groups. We report results of the radioimmunological and immunodiffusion tests which show that the proteins are immunologically indistinguishable from each other.

Twenty-four women, 11 with endometriosis and 13 with fibrocystic mastopathy, were treated with a medium dose (300-400 mg daily) of Danazol for 6 months. The circulating level of progesterone, the 34K insulin-like growth factor-binding protein (IGF-bp) and endometrial protein PP14 (placental protein 14) were measured by specific radioimmunoassays before and during treatment. The serum progesterone concentration decreased significantly during Danazol treatment, as did the serum levels of 34K IGF-bp and PP14. By the third month of therapy amenorrhoea was observed in 22 out of 24 women and this was accompanied by a further decline in the IGF-bp and PP14 levels. In light of the previous observations on the IGF-bp and PP14 synthesis by secretory endometrium and the fact that Danazol causes endometrial atrophy, these results suggest that Danazol treatment has an effect on endometrial protein secretion.

Insulin-like growth factors IGF-I and IGF-II are bound to specific binding proteins in serum. The lower mol. wt binding protein (IGF-BP) has been detected in various tissues, including secretory endometrium and preovulatory follicles of the ovary. This group studied the circulating levels of IGF-BP in the serum of 23 patients with polycystic ovarian disease (PCOD) and found that one-third of them have a subnormal level. In comparison with PCOD patients with a normal level, those with a subnormal level had a higher degree of obesity and a tendency to be more hirsute. They also had a higher serum insulin concentration and testosterone/sex hormone-binding globulin (SHBG) ratio, but lower serum SHBG concentration than those with a normal IGF-BP level. PCOD is the second abnormal clinical condition, after insulinoma, in which subnormal serum IGF-BP concentrations have been reported. The significance of low serum IGF-BP levels to pathophysiology of PCOD remains to be elucidated by studies on local interaction between IGF-BP and insulin in the polycystic ovary.

The effect of cyclical oestrogen-progestogen replacement on the levels of endometrial secretory protein PP14 (placental protein 14) was studied in 39 post-menopausal women, 13 of whom had had a hysterectomy. In those women with an intact uterus, the average rise of the late luteal phase serum PP14 concentration was from 29 to 45 micrograms/l, a 55% rise (P less than 0.001). Only a slight rise (from 27 to 30 micrograms/l) was observed in hysterectomized women. Cyclical replacement with oestrogen and levonorgestrel caused a greater rise in serum PP14 level than did replacement with the same dose of oestrogen plus medroxyprogesterone acetate (P less than 0.05). Because PP14 is found also in the serum of hysterectomized post-menopausal women, the uterus cannot be the only source of circulating PP14. But, the difference between hysterectomized and non-hysterectomized women indicates that the quiescent post-menopausal uterus responds to oestrogen-progestogen replacement by increased PP14 secretion, which can be detected and quantified by a blood test. The measurement of PP14 in serum may also become useful for testing the effect of various progestogens.

Immunofluorescence staining was used to localize two 'endometrial' proteins, insulin-like growth factor-binding protein and human beta-lactoglobulin homologue, in cultured cells of chorionic villi and decidua. Both cultured cell types were positive for each protein. These results indicate that immunohistochemical detection of these two proteins cannot be used to distinguish between cultured chorionic villus and decidual cells.

The effect of luteal phase supplementation of micronized oral progesterone, 200 mg daily, on the serum levels of endometrial secretory placental protein 14 (PP14)/beta-lactoglobulin homologue was studied in a double blind/cross-over fashion on five infertile women with apparently normal ovulatory cycles. Blood samples were taken on cycle days 10-12, 20-22 and 24-27, and the serum progesterone and PP14 concentrations were measured by specific radioimmunoassays. In each progesterone cycle the serum progesterone levels were higher than in the corresponding placebo cycle. In each case, the serum concentration of endometrial PP14 was also higher during progesterone than placebo treatment, but this was seen in the late luteal phase only. In view of the previous demonstration of endometrial synthesis of PP14 and the immunological identity between PP14 and progestogen-dependent endometrial protein, these results indicate that endometrial protein secretion can be increased by micronized oral progesterone in infertile ovulatory women whose serum progesterone level is within the normal range.

Recent studies have shown that the human endometrium is capable of synthesizing a 34 kd insulin-like growth factor-binding protein (IGF-bp). Serum levels of immunoreactive IGF-bp were measured in non-pregnant women in order to find out whether the uterus contributes to the circulating level of 34 kd IGF-bp and whether these values would reflect the endometrial function. Serum samples were collected from 21 women of presumably fertile age before and after hysterectomy, from eight apparently healthy women at various phases of the menstrual cycle, and from 23 post-menopausal women. The serum concentrations of IGF-bp were estimated by radioimmunoassay (RIA), which measured both the unsaturated and the IGF-bound 34 kd IGF-bp. After hysterectomy, a significant decrease in serum IGF-bp levels was observed in samples taken one week post-operatively suggesting a uterine contribution to the circulating IGF-bp concentration. Five weeks after hysterectomy the serum IGF-bp level had returned to the pre-operative level indicating that other tissues can compensate for uterine IGF-bp. In healthy women of fertile age the serum IGF-bp level (94 samples from eight subjects) was 39 +/- 1.8 ng/ml (mean +/- SE) and, in post-menopausal women (23 subjects), it was 36 +/- 6.0 ng/ml. During the menstrual cycle, the average day-to-day variation of serum IGF-bp concentrations was 31%. No correlation was found between the levels of serum IGF-bp and oestradiol, progesterone, LH or FSH. These results indicate that, while the uterus is probably one of the sources of circulating 34 kd IGF-bp, other tissues must also contribute.(ABSTRACT TRUNCATED AT 250 WORDS)

The human endometrium has been reported to release CA 125 in tissue culture, and elevated levels have been found in patients with endometriosis and adenomyosis. The serum levels of CA 125 were measured in 22 women undergoing hysterectomy for adenomyosis (n = 11) or fibroids (n = 11) of the uterus. In 20 patients (91%) the pre-operative CA 125 level was normal (less than 35 U/ml). All patients with adenomyosis had a normal pre-operative serum CA 125 concentration. Five weeks after the operation the CA 125 levels did not differ from the pre-operative levels. Our results show that the uterine contribution to the serum CA 125 level is minimal, and do not confirm the initial enthusiasm concerning the possible use of levels as an aid in the diagnosis of adenomyosis.

Placental protein 5 (PP5), a serine protease inhibitor, was found in the endometrium and menstrual fluid of non-pregnant women. PP5 was present in all 16 endometrial samples taken at various phases of the menstrual cycle. In the secretory phase, the endometrial PP5 content was higher (median 17.4 micrograms/g protein; n = 8) than in the proliferative phase (median 3.8 micrograms/g protein; n = 8). In gel filtration, endometrial tissue homogenates yielded one immunoreactive peak corresponding to a mol. wt of 28,000 daltons, whereas placental PP5 eluted at 32,000 daltons. In sodium dodecyl sulphate polyacrylamide gel electrophoresis, endometrial PP5 comigrated with purified placental PP5 corresponding to a mol. wt of 30,000 daltons. All menstrual fluid samples (n = 14) contained immunoreactive PP5 at concentrations from 77 to 1150 micrograms/l. The mol. wt of menstrual fluid PP5-immunoreactivity was 13,000 daltons. The dose-response curves of purified PP5 standard and endometrial and menstrual fluid PP5 were parallel in the PP5 radioimmunoassay. The higher concentration of PP5 in secretory endometrium indicates association of PP5 with endocrine events of the menstrual cycle.