The secretory endometrial protein, placental protein 14, in women with ectopic gestation

FERTILITY AND STERILITY Copyright e 1992 The American Fertility Society

Vol. 57, No. 1, January 1992

Printed on acid-free paper in U.S.A.

The secretory endometrial protein, placental protein 14, in women with ectopic gestation

Susanne Ruge, M.D.*t Steen S0rensen, M.D.:j: Mogens Vejtorp, M.D.* Lars 0. Vejerslev, M.D.* Hvidovre University Hospital, Copenhagen, Denmark

Objective: To determine the serum level of the secretory endometrial protein, placental protein 14 (PP14) and progesterone (P) in women with ectopic gestation. Design: Blood samples were collected prospectively and preoperatively. Reference range was determined from a prospective population of 98 women with uncomplicated pregnancies and normal outcome. Setting: The women were admitted to a university hospital. Patients: Fifty-nine women with laparoscopically verified ectopic pregnancy entered the study. Intervention: At the time of diagnosis PP14 and P were measured. Main Outcome Measure: After observing the low serum levels of PP14 and P, a correlatio11 analysis was made and compared with the findings in normally pregnant women. Results: A significant positive correlation was found between the level ofPP14 and P (P < 0.00002), not found in normal intrauterine pregnancies. Conclusions: These findings suggest that the regulation of the PP14 production involves either a control mechanism from the ovary or is mediated by paracrine secretion. Fertil Steril 1992;57:102-6

A successful implantation depends on the timely interaction of the blastocyst with a receptive endometrium, which requires an adequate hormonal stimulation during the luteal phase of the cycle. 1 The steroid-primed endometrium synthesizes and secretes a secretory endometrial protein. This protein, also called placental protein 14 (PP14), is immunologically similar to progestagen-dependent endometrial protein2 and a 2 -pregnancy-associated endometrial globulin. 3 The synthesis and secretion of PP14 increases from the midluteal phase of the menstrual cycle, 4•5 and during the late luteal phase and early first trimester of pregnancy, it represents

Received April1, 1991; revised and accepted September 5, 1991. * Department of Obstetrics and Gynaecology.

t Reprint requests and present address: Susanne Ruge, M.D., Department of Obstetrics and Gynaecology, Gentofte Hospital, University of Copenhagen, DK-2900 Hellerup, Denmark. :j: Department of Clinical Chemistry. 102

Ruge et al. PP14 and EP

the primary secretory protein of the endometrium. 4 Because the secretory endometrium represents the major serum source, these changes are reflected in the serum PP14 concentration, which increases progressively from the late luteal phase to reach a peak between weeks 8 and 10 of pregnancy and declines gradually thereafter. 6 Although little information is available regarding the regulation of the synthesis and the secretion of PP14, its production is most likely associated with the progesterone (P)-dependent, histologically defined differentiation of the glandular epithelium. 7 Histologic studies of the endometrium in women with ectopic pregnancy (EP) most often show a secretory phase, and the changes have been found to be correlated with the P level. 8 The purpose of the present study was to examine the endometrial secretory activity in women with EP, evaluated by the serum concentration of PP14, and to compare the level of PP14 with that of P in women Fertility and Sterility

with ectopic and intrauterine pregnancies (IUPs), respectively. MATERIALS AND METHODS Subjects

During a 22-month period, 59 women with laparoscopically verified EP were studied prospectively. Maternal venous blood samples were obtained preoperatively. The gestational age was expressed as completed weeks from the last menstrual period. The mean age of the women was 29.0 years (range of 20 to 41 years). The reference range of PP14 and P in early pregnancy was determined using serum samples from a prospective population of 98 women with uncomplicated singleton pregnancies. Their gestational age was confirmed by an ultrasound (US) scan. All women delivered a living infant, normal for date (~lOth percentile), at term (~37 weeks of gestation). The mean age of the women was 26.3 years (range of 18 to 46 years). The blood samples were collected in plain tubes and allowed to clot. The serum was stored at -20°C until assayed. Assays

Five micrograms of purified PP14 (lot no. 136/154; Behringwerke AG, Marburg/Lahn, Germany) was iodinated in 0.12 mol/L Tris-HCl pH 7.6 in the presence of 0.5 mCi Na125l and 60 JLg of chloramineT as oxidant in a reaction volume of 90 JLL. The reaction was terminated after 45 seconds by addition of 150 JLg of sodium metabisulphite. The labeled PP14 was separated from iodide by Sephadex G-25 gel filtration. The assay consisted of 150 JLL of samples or PP14 standards (0 to 0.2 mg/L), 50 JLL of 125I-PP14 (10,000 cpm), and 100 JLL of anti-PP14 antiserum (1:10,000) (lot no. 201 ZA; Behringwerke AG) incubated at room temperature (RT) for 24 hours. Finally, 100 JLL of donkey antirabbit antibody coated cellulose suspension (Wellcome Diagnostics Ltd., Dartford, United Kingdom) was added and incubated at RT for 30 minutes. One milliliter of distilled water was then added, and separation of free and bound PP14 was carried out by centrifugation (2,000 X g for 10 minutes) and the supernatant decanted. The radioactivity of bound 125I-PP14 in standards and samples was counted, and the concentration of PP14 in a sample was estimated by reading off the percent bound precipitate in relation Vol. 57, No. 1, January 1992

to the standard curve by use of a spline function. All dilutions were performed in assay buffer, 75 mmol/L Tris-HCl pH 7.6, containing 1 g/L bovine albumin and 0.1 g/L NaN 3 • Detection limit was 0.005 mg/L, and interassay imprecision 7.5% (i = 0.069 mg/L, n = 35). Serum P was measured by a commercial kit (Farmos Diagnostica, Oulunsalo, Finland). Statistical analyses were performed by the Mann-Whitney Utest and Spearman's rank correlation test. RESULTS

The implantation site was in all cases the uterine tube. Fifteen (25%) women had a ruptured ectopic and 44 (75%) an unruptured tubal pregnancy. At the time of diagnosis the level of serum PP14 was far below the lOth percentile in all 59 women (Fig. 1). The level was significantly lower when compared with the level in women with IUP, (P < 0.00001). The lowest value measured was 0.04 7 mg/L, corresponding to the level in the late luteal phase in nonpregnant women,9•10 and the highest was 0.43 mgfL. Fifty-two (88%) women had serum P values< lOth percentile (Fig. 2) or even below the level observed in the midluteal phase for nonpregnant women (<25 nmol/L). The serum levels of PP14 and P were not influenced by the tubal status. The median values of PP14 and P in the women with ruptured ectopics

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Figure 1 Serum PP14levels in 59 women with EPs. The normal range is shown by the lines of best fit to the median, lOth and 90th percentiles.

Ruge et al.

PP14 and EP

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Figure 2 Serum P levels in 59 women with EPs. The normal range is shown by the lines of best fit to the median, lOth and 90th percentiles.

were not significantly different from the values observed in the women with intact tubes. One woman had a live extrauterine fetus demonstrated at US examination. The gestational age was 7 weeks. The value of PP14 and P was 0.39 mg/L and 35.6 nmol/L, respectively. A significant positive correlation (r = 0.52, P < 0.00002) was found between the level of PP14 and P in the women with EPs in all the gestational weeks 4 to 13 (Fig. 3). This correlation was not found in 98 women with normal IUPs in the gestational weeks 6 to 13 (P > 0.05).

elevation until the onset of the next period increased the circulating level of PP14. This in accordance with the report of Julkunen et al. 5who demonstrated higher rates of release of PP14 by secretory endometrium during in vitro incubation compared with release from a proliferative endometrium. No regulation by P was detected during short-term incubation.U Probably, the synthesis and secretion of PP14 represents a late event in the P-dependent differentiation of the secretory glandular epithelium because all correlates of synthesis are highest during the late luteal phase when steroid hormone levels are falling. 5·13 In pregnant women, however, the administration of RU38486 (Mifepristone; Roussel Laboratories Ltd., Broadwater Park, Uxbridge, United Kingdom), an antagonist to the effect of P, resulted in a rise in the PP14 level in association with falling P concentration, suggesting that the secretion of PP14 is independent of P in early pregnancy.15 In agreement with the work of Howell et al., 15 we found no correlation between PP14 and Pin women with normal IUPs. The level of PP14 was independent of the P level in all the gestational weeks 6 to 13, before as well as after the placenta takes over the P production. Consequently, the missing correlation cannot be explained by the luteoplacental shift of the P production. A controlling factor other than P may be present for the continuance of PP14 synthesis. In normally pregnant women the serum PP14 level continues to rise steeply until weeks 8 to 10 of pregnancy; thereafter it declines, possibly reflecting the presence and the subsequent involution of the secretory gland-containing decidual spongiosa, which involutes despite the continued P secretion.7

DISCUSSION

Human endometrium synthesizes several proteins, and specific patterns of protein secretion (endometrial fingerprints) characterize the endometrium at different stages of the cycle. The secretory endometrial protein or its synonym a 2-pregnancyassociated endometrial globulin is a dimeric glycoprotein whose synthesis and secretion increases from the midluteal phase ofthe menstrual cycle.4·11 During the late luteal phase and early first trimester of pregnancy, it represents the major product of the secretory glandular epithelium as evidenced by monoclonal and polyclonal-based immunohistology .12,13 The relationship between circulating PP14 and P is unclear. Seppala et al. 14 found that administration of oral P from the time of basal body temperature 104

Ruge et al.

PP14 and EP

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Figure 3 Scatter plot of the correlation between the PP14 and the P level in 59 women with EPs (r = 0.52, P < 0.00002).

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The factors that regulate PP14 production may therefore be linked with hormonal factors present during the late luteal phase and early first trimester of pregnancy, presumably derived from the corpus luteum (CL). In women with EPs, the histologic examination of the endometrium shows several histologic patterns of which the secretory type is the most frequently represented. A positive correlation between the level of P and the extent of the secretory pattern has been reported. 8 In our study, the women with EP had at the time of diagnosis serum PP14 values ranging from the same level as observed in the late luteal phase in nonpregnant women to values far beyond the lOth percentile for normally pregnant women. A significant positive correlation was found between the P and the PP14 level in the women with EPs, supporting the work of Guillaume et al.,8 who found that the mean P level was significantly higher in EPs with secretory type endometrium than those with proliferative. In accordance with earlier reports8·16 we found the P level depressed in the women with EPs and very close to the lower levels of P at luteal phase. The markedly low PP14 values and the positive correlation with P in the women with EPs may be the consequence of the abnormal CL function in EP 17·18 and in support of the view of a controlling factor from the ovary. This is in agreement with a study of ovarian failure in a woman, pregnant after frozen embryo transfer. The woman had low serum PP14 values in spite of physiological concentrations of P and estradiol during exogenous steroid replacement.19 Alternatively, the increase of PP14 levels could be mediated by paracrine secretion. This local regulatory mechanism may involve the dissemination of a stimulatory factor, synthesized from the site of intrauterine implantation to its target tissue, i.e., the miillerian duct. The human salpinx and the uterus are derived from the same embryonic source (the miillerian duct), and in 1986 Julkunen et al. 20 demonstrated the presence of PP14 in the secretory epithelial cells of the mucosa in all parts of the tube. In women with EPs the secretory area of the tube is small, and only a minor amount of PP14 is produced. This in contrast to the intrauterine implantation where the secretory area is great, which results in a higher production of PP14. This may account for the poor correlation between the P and the PP14 level in women with IUPs and for the low PP14 values found in women with EP. However, it may Vol. 57, No. 1, January 1992

be claimed that the lower serum levels of PP14 may reflect inefficient absorption into the circulation from the endometrial cavity or tube, rather than decreased production. The low serum levels of PP14 were independent of whether the uterine tube was ruptured or not. Theoretically, a ruptured EP has been considered more viable than a pregnancy with an intact tube. 21 This may support our earlier communication that viability of the EP seems of no importance for the secretion of PP14. 22 The clinical value of PP14 measurement as a new test in the diagnosis of EPs may be considered. Acknowledgments. We are indebted to Ms. Vibeke Myrh0j for technical assistance and to Dr. Hans Bohn, Research Laboratories, Behringwerke AG, P.O. Box, D-3550 Marburg/Lahn, Germany, for purified PP14 and anti-PP14 antiserum.

REFERENCES 1. W eitlauf HM: Biology of implantation. In Physiology of Reproduction, Edited by E Knobile, J Neill et a!. New York, Raven Press, 1988, p 231 2. Julkunen M, Raiker RS, Joshi SG, Bohn H, Seppala M: Placental protein 14 and progestogen dependent endometrial protein are immunologically indistinquistable. Hum Reprod 1:7, 1986 3. Bell SC, Bohn H: Immunochemical and biochemical relationship between human pregnancy-associated secreted endometrial a 1 - and a 2 -globulins (a 1 - and a 2 -PEG) and the soluble placental proteins 12 and 14 (PP12 and PP14). Placenta 7:283, 1986 4. Bell SC, Hales MW, Patel S, Kirwan PH, Drife JO: Protein synthesis and secretion by the human endometrium and decidua during early pregnancy. Br J Obstet Gynaecol 92:793, 1985 5. Julkunen M, Koistinen R, Sj0berg J, Rutanen E-M, Wahlstrom T, Seppala M: Secretory endometrium synthesizes placental protein 14. Endocrinology 118:1782, 1986 6. Julkunen M, Rutanen E-M, Koskiemies A, Bohn H, Seppala M: Distribution of placental protein 14 in tissues and body fluids during pregnancy. Br J Obstet Gynaecol 92:1145, 1985 7. Waites GT, Bell SC, Walker RA, Wood PL: Immunohistological distribution of the secretory endometrial protein, "pregnancy-associated endometrial a 2 -globulin," a glycosylated ,8-lactoglobulin homoloque, in the human fetus and adult employing monoclonal antibodies. Hum Reprod 5:105, 1990 8. Guillaume J, Benjamin F, Sicuranza B, Fu Wang C, Garcia A, Friberg J: Maternal serum levels of estradiol, progesterone and human chorionic gonadotropin in ectopic pregnancy and their correlation with endometrial findings. Surg Gynecol Obstet 165:9, 1987 9. Seppala M, Riitinen L, Julkunen M, Koistinen R, Wahlstrom T, Iino K, Alfthan H, Stenman U-H, Huhtala M-L: Structural studies, localization in tissue and clinical aspects of human endometrial proteins. J Reprod Fertil 36(Suppl): 127, 1988

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