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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

July 2001
Volume V, Number 3
ISSN: 1537-6583
Pages: 176-190

Correlation Between Serum Levels Of 17b -Estradiol, Progesterone And b - Human Chorionic Gonadotropin And The Karyotype Of First Trimester Anembryonic And Embryonic Pregnancies

Bernardo Agudelo*, Carlos Mario Muñetón**, Gonzalo Vásquez**, José Luis Ramírez**

*Departament of Obstetrics and Gynecology,** Medical Genetics Unit, School of Medicine, University of Antioquia, Medellín, Colombia

Key words: Abortion, Anembryonic Pregnancy, Karyotype, Gonadotropins, Steroids, Ultrasound

Correspondence: C.M. Muñetón, Medical Genetic Unit, School of Medicine, University of Antioquia, Medellín, Colombia, AA 1226, Telephone 5106063, Fax (574) 2633509, Email: cmuneton@catios.udea.edu.co

Aknowledgements: This project received financial support from the Committee for Research Development (CODI Grant CIM-9906) and technical help from the Medical Research Center, School of Medicine, University of Antioquia. We are grateful to the participating institutions, Hospital San Vicente de Paúl, Medellín, and Hospital San Juan de Dios, Rionegro; also, to residents in the department of Obstetrics and Gynecology, University of Antioquia, who gathered the cases. We also thank Doctor Fernando Montoya, Professor of this medical school for his valuable assessment for statistical analysis and interpretation of results.

Abstract

Objectives
We aimed at establishing the correlation between karyotype of anembryonic and embryonic first trimester pregnancies and serum levels of  b-hCG, 17b -estradiol and progesterone.

Methods
Chromosomal analyses were performed on products of conceptions lower than 12 weeks, using standard-banding protocols. Measurements of 17b -estradiol and progesterone were carried out by immunoenzymatic assays and b -hCG by radioimmunoanalysis (RIA).

Results
Chromosomal analyses among 11 spontaneous abortions revelead 6 (54,5%) to be normal and 5 (45,5%) abnormal; of these latter, 4 were anembryonic pregnancies (44,4% total frequency). Serum b hCG and b -estradiol average levels were lower in the anembryonic pregnancies but did not reveal significant differences with normal levels. Hormonal levels were correlated with karyotype results and it was found that average b -hCG value was 3.8 times higher in cases with abnormal karyotype than in those in whom it was normal (p <0.05). Linear correlation analyses between hormonal measurements in anembryonic pregnancies were statistically significant (p <0.05). Correlations between b -hCG and progesterone, b -hCG and 17-b estradiol, and the latter with progesterone, proved to be significant in the group with abnormal karyotype (p < 0.05).

Conclusions
Anembryonic pregnancies present high frequency of chromosomal anomalies; such pregnancies with abnormal karyotype have a high serum level of b -hCG; this condition increased the rate of abortion.

Introduction

Human gestation loss occurs between 50% and 80% of conceptions (Roberts and Lowe, 1975; West, 1993). Spontaneous abortion is the most frequent complication of pregnancy; it is present in 20% to 31% of all conceptions if early pregnancy loss, the one that ocurrs between implantation and menstruation is taken into account (Wilcox et al., 1988; Ellish et al., 1996); besides, in all those pregnancies recognized by amenorrhea and detection of b -hCG the loss reaches 5%-20% as spontaneous abortions in the embryonic period (Boué et al., 1985; Goldstein, 1994; Ellish et al., 1996).

Ultrasound is an excellent non-invasive, high-sensitivity diagnostic tool for defining the characteristics of pregnancy and evaluating embryonic viability. Cardiac activity is regarded as an absolute criterion of embryo presence and it can be detected as early as 5.5 weeks (Robinson, 1975;Schaaps, 1992).

Anembryonic pregnancy (blighted ovum) is a frequent form of loss, that is present between 16% and 30% of all pregnancies (West, 1993; Coulam et al., 1997) and in up to 40% of ectopic pregnancies (Emmrich and Kopping, 1981). An empty sac very likely corresponds to an early embryonic death after implantation (Jauniaux et al., 1994; Jauniaux and Jaffe, 1997); consequently, the term anembryonic has been considered as an erroneous way to define this condition. Absence of embryonic structures at the seventh week supports the diagnosis of anembryonic pregnancy (Grudzinskas and Chard, 1992). Based on the available information it is not possible to deduct whether the lack of embryo results from its primary abnormal development or is secondary to its death near implantation with subsequent development of placental structures (Henderson et al., 1991; Jauniaux et al., 1994). Persistence of the exocoelomic cavity, without embryo and amniotic cavity development, corresponds to the empty gestational sac detected on ultrasound and is the potential source of biochemical signals in the early gestation (Chard et al., 1995). Presence of embryo and the membranes associated with its development, seems to be the determinant factor for an optimal hormonal response and the intricated relationship with endometrial and decidual factors (cytokines, growth factors, among others) that are necessary for normal pregnancy development (Barnea and Schurtzs-Svirsky, 1992; Giudice, (2) 1999).

Chromosomal anomalies are present in 60% of all first-trimester abortions, (Boué et al., 1985; Salafia et al., 1993; Hill, 1994; Coulam et al., 1997;Hassold and Hunt, 2001). Anembryonic pregnancy has been associated with chromosomal anomalies in up to 67% of cases (Minelli et al., 1993); this finding suggests a high probability of gestational loss.

Combined hormonal evaluation in the early weeks of pregnancy could be useful in the follow-up and for prognostic purposes (Johnson et al., 1993; Daily et al., 1994); several alternatives have been suggested: determination of Th1:Th2 cytokine pattern (Jenkins et al., 2000; Lim et al., 2000); progesterone measurement at the beginning of pregnancy (Azuma et al., 1993; Bopp and Shoupe, 1993); concentration of b -hCG follow-up (Kadar et al., 1993; Daily et al., 1994); measurement of early pregnancy factor (EPF) as predictor (Shahani et al., 1992); a -fetoprotein (AFP) measurement (Jauniaux and Jaffe, 1997), and cytogenetic analysis of the conception product.

Our purpose was to establish the possible correlation between karyotype of anembryonic and embryonic first trimester pregnancies and serum levels of b -hCG, 17b -estradiol and progesterone, at the moment of surgical termination of pregnancy from patients with spontaneous loss, that were neither under hormonal stimuli nor in assisted reproduction studies.

Materials and Methods

Pregnant women were included once they met the following criteria: pregnancy lower than 12 weeks, abortion threat and echographic confirmation of anembryonic pregnancy or early embryonic death; patients with induced or septic abortions were excluded. The study was carried out between May 1999 and June 2000 in two reference hospitals in Antioquia, Colombia, Hospital Universitario San Vicente de Paúl (Medellín) and Hospital San Juan de Dios (Rionegro).

After written informed consent, patients were asked to donate the product of conception, and to provide a peripheral blood specimen for hormonal measurements. Before uterine evacuation under analgesia with penthotal, 10 mL of peripheral blood were obtained without anticoagulant; serum was separated by centrifugation and stored at - 20ºC until hormonal levels measurement.

Chromosomal analyses were performed on the products of conception according to reported standard banding protocols (Verma and Babu, 1995; Muñetón et al., 1998). Measurements of 17b -estradiol and progesterone were carried out by immunoenzymatic assays (IMX-Abbott and AXSYM- Abbott, respectively) and b -hCG by radioimmunoanalysis (RIA). b -hCG was measured in mUI/mL, 17b -estradiol in pg/mL and progesterone in ng/mL.

Statistical Analysis

Variables were analysed from an Excel matrix from which they were transferred to the statistical package Statistica 6.0 (StatSoft). Analyses were done as independent variables and correlation matrixes. Differences were considered significant when p<0.05.

Results

Distribution of maternal and gestational ages and previous reproductive losses are listed in Table 1. Average maternal age was 26.2 years, and average gestational age at the moment of termination was 7.6 weeks, as determined by echography. Five of the 26 patients (19,2%) reported two or more previous abortions.

A total of 35 specimens were gathered on the basis of the inclusion criteria but nine of them were discarded either at the moment of processing or in the early stages of culture due to low quality or contamination. The final group was formed by 26 cases, of which 9 (34,6%) corresponded to embryonic and 17 (65,4%) to anembryonic pregnancies.

In 11 of the 26 specimens (42,3%) (9 anembryonic and 2 embryonic) a cytogenetic result was obtained (table 1); five of these (45,5%) had abnormal karyotype, while in 6 (54,5%) it was normal. The following chromosomal anomalies were found: three cases of X chromosome monosomy, one case of trisomy (47, XX + 21), and another of mosaicism (46, XX/45, X). Of these five cases, four (80%) were found in products of anembryonic pregnancies while the remaining one (20%) was embryonic. Of the 9 cases with anembryonic pregnancies, 4 (44,4%) had abnormal karyotype.

Results of hormonal measurements in embryonic and anembryonic pregnancies did not reveal significant differences (p < 0.05), although average values of b hCG and b -estradiol were lower in the anembyonic group (Table 2). In 11 of the 17 anembryonic pregnancies (64.7%), progesterone values were under 6 ng/mL (Figure 1), and in 12 cases of the same group (70,6%) 17b - estradiol values were under 400 pg/mL.

Hormonal measurements in patients with karyotype information revealed that average b -hCG level in the 5 cases with abnormal karyotype was significantly higher (p <0.05) than in the remaining 6 cases in which it was normal (proportion 3.8:1). (Table 3 and Figure 2).

Linear correlation analyses between hormonal measurements in the population with anembryonic pregnancy revealed that r value between b -hCG and 17b -estradiol was 0,88; the one between 17b -estradiol and progesterone was 0,86 , and that between b -hCG and progesterone was 0,80. All these correlation indexes were statistically significant (p <0.05). Correlation in the group with embryonic pregnancies was equally significant, with a value of r above 0,95 (p <0.05).

In the population with abnormal karyotype linear correlation of hormonal values showed an r of 0,95 between b -hCG and 17b -estradiol (figure 3); 0,98 between 17b -estradiol and progesterone (figure 4) and 0,98 between b -hCG and progesterone, all of which were statistically significant (p <0.05). In the group with normal karyotype, only the correlation between b -hCG and 17-b estradiol had a significant difference (r: 0,928; p <0.05).

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Table 1

Distribution and general information on the studied cases with karyotype result (n: 11)

*Gestational week calculated from echographic information.

Table 2

Average results of hormonal measurements, according to gestation type

*Average for each group

Table 3

Average results of hormonal measurements in the 11 cases with karyotype result

*Average for each group
**Statistically significant difference

Discussion

Women participating in this study consulted at emergency services of reference hospitals because of spontaneous abortions. Gestational age for analysis was the one reported by ultrasound which reflects the moment of embryo or chorioplacental membranes growth arrest. Period between embryonic death and obtainment of the specimen strongly influences the success of cell culture and therefore cytogenetic results. This is a limitation for chromosomal studies in groups similar to ours, namely, patients with no early pregnancy follow-up.

It was found that 45,5% of cases presented chromosomal anomalies; this result is similar to those reported in previous studies and to the one expected in general population, and is regarded as the main reason for early gestational loss (Boué et al., 1985; Simpson, 1997; Muñetón et al., 1998). Since in this study embryonic and anembryonic cases were compared, it must be emphasized that 44,4% of the latter revealed chromosomal anomalies, although the small number of anembryonic cases with positive cytogenetic result (n: 11) must be taken into account. Minelli et al., in 1993 found a 67% frequency of chromosomal anomalies in 52 spontaneous abortions of anembryonic pregnancies. Preliminary results of our study suggest that characterization of early gestational loss must be complemented with cytogenetic studies, as an important diagnostic tool for the clinician specially when an empty sac is detected.

Regarding hormonal measurements, b -hCG and sexual steroids (17-b estradiol and progesterone) were selected as criteria for evaluating the biosynthetic trophoblast activity and its interaction with the corpus luteum (Tulchinsky and Hobel, 1973; Falcone and Little (1), 1994; Falcone and Little (2), 1994). However, some authors have questioned the value of hormonal measurements for evaluating trophoblast quality due to lack of strict correlation between its biosynthesis and embryonic development (Jauniaux and Jaffe, 1997). We found that hormonal values at the moment of uterine evacuation (average by echography 7,6 weeks) were under the low levels that correspond to normal pregnancy (Barnea and Schurtz-Svirsky, 1992), but there was no significant difference between embryonic and anembryonic pregnancies.

Significantly lower levels of b -hCG, 17b -estradiol, progesterone and other hormones (PAPP-A and SP-1) have been reported in the literature in anembryonic abortions as compared to embryonic ones and to term pregnancies (Johnson et al., 1993). When an embryo does not develop, the amniotic cavity and the embryonic pole disappear which has been associated with lower b -hCG values, possibly because the interaction between embryo and chorial membranes interrupts (Jauniaux et al., 1994). Low level of plasmatic 17b -estradiol after six weeks of pregnancy has been associated with imminent gestational loss due to embryo death or absence (Soltes et al., 1993). In our study, lack of statistical difference in hormonal levels between the two groups can be explained by the influence of two particular cases (Table 1) namely, numbers 9 and 14. The former was an anembryonic pregnancy with abnormal karyotype (47, XX + 21), b -hCG 137.366 mUI/ mL, 17b - estradiol 1.574 pg/mL and progesterone 25,32 ng/mL; the latter (case 14, data not shown) corresponds to an embryonic pregnancy in which karyotype could not be obtained, b -hCG 169.182 mUI/mL, 17b -estradiol 1.654 pg/mL and progesterone 20,48 ng/mL. However, after exclusion of these two cases it was found that 17b -estradiol levels in the remaining 24 were significantly lower in the anembryonic group (p <0.05), (data not shown). This result emphasizes the importance of a healthy embryo during the early weeks of pregnancy in order to guarantee estrogen synthesis in the fetal-maternal placental interphase (Tulchinsky and Hobel, 1973; Falcone and Little, 1994). Production of adequate levels of 17b -estradiol, since implantation and throughout gestation, has been recognized as a sine qua non condition for ensuring synthesis and action of several hormones, growth factors, cytokines and immunomodulators that are necessary for supporting a successful pregnancy (Greco et al., 1993; Schuchard et al., 1993; Guidice, (1) 1995; Olsen and Kovacs, 1996; Valbuena et al., 1999). In both groups average progesterone (< 6.0 ng/mL) was below established optimal levels for the first trimester of pregnancy (Tulchinsky and Hobel, 1973; Daily et al., 1994). It has been reported that a progesterone value of 6.0 ng/mL or less associates with pregnancy loss in 81% of cases (Daily et al., 1994).

When hormonal levels were correlated with karyotype results (n: 11) it was found that average b -hCG value was 3.8 times higher in cases with abnormal karyotype than in those in whom it was normal (p <0.05). However, when independently considered, two cases were found that deserve further comment (Table 1): Case Nº 9 corresponds to a patient, with 6 weeks of gestation, empty sac, abnormal karyotype (47,XX + 21), b -hCG 137.366 mUI/mL, 17b -estradiol 1.574 pg/mL and progesterone 25.32 ng/mL. An independent analysis of hormonal values could suggest that gestation evolves "satisfactorily", but if the presence of an empty sac is correlated with high hormonal values, especially that of b -hCG, these findings can be associated with the existence of an abnormal karyotype (Bogart et al., 1987). This characteristic has been associated with molar pregnancies and trophoblastic gestational disease, which commonly present polyploidies (Kaufman, 1991). It can be suggested that b -hCG measurement as predictor of gestation normality should not be carried out isolatedly but in association with either progesterone or 17b - estradiol.

In both groups, correlation between hormonal levels was statistically significant (p <0.05) (data on anembryonic cases not shown); therefore, when an embryo is not detected in the sac, the simultaneous finding of low values in two of the studied hormones, can indicate with certainty an unavoidable gestational loss due to embryo absence or early death. This finding constitutes an useful diagnostic tool for the clinician dealing with patients with abortion threat during the first trimester, or in women at high reproductive risk.

Likewise, correlations between b -hCG and progesterone, b -hCG and 17-b estradiol, and the latter with progesterone, proved to be significant in the group with abnormal karyotype (p < 0.05). Soltes (1993) suggessted early and simultaneous measurements of b -hCG and 17-b estradiol as a predictive index of abnormal karyotype. In the group with normal karyotype the only significant correlation was the one between b -hCG and 17b -estradiol, which reinforces even more the concept that 17b -estradiol plays an important role in the regulation of placental biosynthesis.

From the analysed results it can be concluded that it is necessary to do chromosomal studies in every gestational loss, either embryonic or not; echographic evaluation of pregnancies with empty sacs should be complemented with simultaneous measurement of at least two of the three studied hormones (b -hCG, 17b -estradiol and progesterone); the finding of low hormonal levels during the first trimester of pregnancy, with absence of embryonic pole is a good indicator of gestational loss and of the unavoidable termination of pregnancy; the concept is supported of embryo`s important function in establishing its interaction with the placenta, the endometrium and the corpus luteum during early gestation; finally, it is once more corroborated that chromosomal anomalies play an important role in early pregnancy loss.

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Distribution of progesterone values according to type of gestation. Note that cases with anembryonic pregnancies tend toward levels under 6 ng/mL, in contrast with the embryonic ones.

b-hCG values according to karyotype in 11 cases. Levels were significantly higher in those with abnormal karyotype (p<0.05).

Lineal correlation between 17-b- estradiol and b-hCG in the group with abnormal katyotype (p<0.05).

Lineal correlation between progesterone and b-hCG in the group with abnormal karyotype (p<0.05).