Preliminary results on the role of embryonic human chorionic gonadotrophin in corpus luteum rescue during early pregnancy and the relationship to abortion and ectopic pregnancy

Verónica Alam1,3, Elena Altieri2 and Fernando Zegers-Hochschild2

1 Unidad de Medicina Reproductiva, Clínica Las Nieves, Santiago and 2 Unidad de Medicina Reproductiva, Clínica Las Condes, Santiago, Chile.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The precise mechanisms by which corpus luteum (CL) function is modulated during early pregnancy are not known. Evidence in failed pregnancies (ectopic, abortions), shows that factors other than human chorionic gonadotrophin (HCG) could be involved in its regulation. The objective of this study was to investigate the dynamics of ß-HCG, progesterone and oestradiol production in early pregnancy and its relation to embryonic quality and topographic localization. Plasma concentrations of progesterone, oestradiol and ß-HCG were studied between days +12 and +21 after an in-vitro fertilization (IVF) embryo transfer in 11 intrauterine pregnancies, 10 intrauterine abortions and seven tubal pregnancies. Tubal pregnancies and abortions were grouped according to doubling time (DT) of HCG. Results showed that oestradiol concentrations were apparently reduced in both ectopic pregnancies and abortions compared with normal pregnancies. The fall in oestradiol concentrations was seen in ectopic pregnancies with an abnormal DT for HCG and in all abortions. When the ectopic pregnancy had a normal DT, oestradiol and progesterone concentrations were normal. In abortions, the fall in oestradiol and progesterone concentrations was less influenced by the DT of HCG. These findings suggest that corpus luteum function depends on an adequate DT of HCG more than an absolute value, and with normal trophoblastic tissue the site of implantation does not affect CL function.

Key words: corpus luteum/ectopic/implantation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is widely accepted that during the luteal phase of the menstrual cycle, luteinizing hormone (LH) is needed to maintain corpus luteum (CL) hormonal production. During the early stages of pregnancy, human chorionic gonadotrophin (HCG) is responsible for corpus luteum hormonal rescue and maintenance of luteal oestradiol and progesterone production (Csapo and Pulkkinen 1978Go; Grudzinskas et al., 1986Go).

Published data have shown that the increasing concentration of HCG during pregnancy is not followed by a proportional increment in ovarian steroid production. This finding suggests that following the rescue of the corpus luteum, ovarian steroidogenesis is not controlled by HCG alone (Norman et al., 1988Go; Kratzer and Taylor 1990Go; Johnson et al., 1993aGo). Recent studies have shown lower concentrations of oestradiol and progesterone in ectopic pregnancies when compared to normal intrauterine pregnancies (Norman et al., 1988Go; Johnson et al., 1993bGo; Lower et al., 1993Go). However, it has been demonstrated that trophoblastic tissue produced HCG with equal immuno- and bioactivity when implanted either in the Fallopian tube, as occurs in ectopic pregnancy, or in the uterine cavity, (Kratzer and Taylor, 1990Go). The rescue of the corpus luteum should then be unaffected by the site of implantation. However, the comparison of normal intrauterine pregnancies and failed intrauterine pregnancies (anembryonic pregnancies) have also shown different steroid production. This suggests that an impaired CL function could be the result of a failure in trophoblastic division (Norman et al., 1988Go; Johnson et al., 1993cGo). To date, most of the information published provides hormonal values for isolated samples, in late stages of pregnancy and once the trophoblastic tissue has failed to produce normal amounts of HCG.

The objective of this report was to provide information on CL production of progesterone and oestradiol in the first week of pregnancy and its relation to embryo quality.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of twenty eight women who had become pregnant following an in-vitro fertilization (IVF) and uterine transfer of embryos, was recruited for the study. Blood samples were obtained on days +12, +15, +18 and +21 after embryo transfer. All patients had a ß-HCG value less than 80 IU/l on day +12, to rule out multiple implantations (Zegers-Hochschild et al., 1994Go). Samples were allowed to clot at room temperature, centrifuged at 800g for 5–10 min and plasma was stored at –20°C for retrospective analysis. Samples were analysed for ß-HCG, progesterone and oestradiol using an enzyme linked immunosorbent assay (Enzimun Test System ES-300, Boehringer Mannheim, Roche, Santiago, Chile). The inter-assay coefficients of variation (CV) were 3–4%. The intra-assay CV were 10.8, 5.5 and 6.9% for oestradiol, progesterone and ß-HCG respectively.

All patients were under luteal support with injectable (i.m.) progesterone (Laboratorie Chile S.A., Santiago, Chile) 50 mg in oil per day from the day of transvaginal aspiration.

Patients were divided in three groups as follows: Group 1 (n = 11) pregnancies ended in a single normal live birth; Group 2 (n = 10) pregnancies ended in a spontaneous clinical abortion and Group 3 (n = 7) were ectopic pregnancies.

Doubling times (DT) for HCG were calculated using the following formula: DT = log 2x(time interval in days)/log (HCG2 at end of DT/HCG1 at beginning of DT) (Pittaway et al., 1985Go). Delayed DT was defined as an average HCG doubling time >=1.8 days, in the following time intervals days: 12–15, 15–18 and 18–21, or one absolute value of DT above 2 days in any of the three intervals.

The absolute values of plasma ß-HCG and oestradiol, in the three groups, were compared using one-way analysis of variance and Student's t-test. The level of significance was P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Doubling times in days for HCG in the intervals between days +12 and +21 after embryo transfer are as follows: 1.53 + 0.34 days for normal pregnancies (n = 11); 1.43 + 0.22 for intrauterine abortions with normal DT (n = 3) and 2.70 + 1.20 days for intrauterine abortions with delayed DT (n = 7); ectopic pregnancies with normal DT (n = 2) was 1.48 + 0.16 days and 2.06 + 0.15 days for delayed ectopic pregnancies (n = 5).

Absolute data for HCG, oestradiol and progesterone are shown in Table IGo.


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Table I. Human chorionic gonadotrophin (HCG), oestradiol and progesterone concentrations in early pregnancies with normal or delayed doubling times (DT) for HCG.
 
Figure 1Go shows oestradiol levels for abortions and ectopic pregnancies. The shaded area represents one standard deviation above and below the mean value for normal pregnancies. All abortions had lower concentrations of oestradiol compared with ectopic pregnancies. For ectopic pregnancies these lower levels were only seen in the group of patients with delayed DT for HCG. Patients with ectopic pregnancies and normal DT for HCG presented with normal oestradiol secretion (Figure 2Go).



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Figure 1. Mean ± SD oestradiol concentration (antilog transformed) for ectopic pregnancies and intrauterine abortions compared with normal pregnancies. The only significant difference (P < 0.05) occurred on day 18 after embryo transfer, between delayed abortions (972 ± 1129 pmol/l) and abortions with normal doubling time (1287 ± 1714 pmol/l). The apparent discrepancy on the figure at this point is due to the large error standard which occurs when absolute values are converted to antilog. DT = doubling time.

 


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Figure 2. Mean progesterone concentrations in ectopic pregnancies compared with normal pregnancies. DT = doubling time.

 
Pregnancies that ended with spontaneous abortions had lower oestradiol concentrations than normal pregnancies regardless of DT for HCG.

Progesterone production was not significantly disminished in both ectopic pregnancies and abortions. Analysis of ectopic pregnancies with normal and delayed DT, showed a trend for reduced progesterone production in delayed DT patients (Figure 2Go). In the abortion group there was no difference in progesterone secretion between normal and delayed patients.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study shows that the rate of duplication for HCG, between days +12 and +21 after embryo transfer, allowed the identification of ectopic and intrauterine pregnancies with normal and abnormal embryos.

Ectopic pregnancies with normal developing embryos and normal HCG production modulate a normal response of the CL, measured by the levels of oestradiol and progesterone. The site of implantation did not seem to affect this response. On the other hand, oestradiol levels were significantly diminished in ectopic pregnancies with delayed DT for HCG secretion. These data suggest that in ectopic pregnancies, two different populations can be differentiated. One population presents with normal DT HCG and normal steroid production, suggesting that the trophoblast and embryo are normal and had the potential to continue if they were properly implanted. The main limitation here seems to be related to anatomical limitations at the site of implantation. The other population of ectopic pregnancies, with delayed DT for HCG and low steroid levels, would have been aborted, regardless of whether it were implanted in the Fallopian tube or the uterine cavity.

Although the presence of multiple CL may affect the bioavailability of HCG, it is almost impossible to correct data by the number of CL. However all the patients have multiple follicles and CL after ovarian stimulation so the comparison between them would be valid.

It has been reported that lower steroid concentrations seen in ectopic pregnancies, despite apparently normal HCG values, suggest that the hormone acts in the ovary through intermediate substances produced by the endometrium (Liu et al., 1991Go). Further support in this direction has been published by Lower et al. who suggested that a metabolic or biochemical signal is triggered as a result of direct contact of the trophoblast with the endometrium (Lower et al., 1993Go). The results of this study do not support the theory that uterine implantation is a prerequisite for normal CL rescue. HCG duplication rate, and not its absolute value, seems to be a better sign of normal development and this is the main factor in maintaining CL function. The data in the current study demonstrated that ectopic pregnancies with normal DT behave like normal pregnancies in terms of oestradiol and progesterone secretion.

Oestradiol levels were apparantly lower in abortions, as compared with normal intrauterine and ectopic pregnancies, regardless of the DT of HCG but the differences were not significant. Nevertheless, this suggests that intrauterine abortions may constitute a more homogeneous population in which all the embryos implanted will fail to continue growing, in spite of having normal DT early in gestation, although for a limited period of time (Lenton et al., 1982Go). It is postulated that if the sampling is extended beyond day +21, most of these abortions will exhibit a delayed DT for HCG.

These data suggest that the trophoblast of intrauterine pregnancies that will end in abortion is not able to produce normal amounts of oestradiol regardless of whether or not it has enough HCG support. No genetic analysis was performed on these miscarriages that could explain, at least partially, the impaired trophoblast. Data was reported (Liu et al., 1991Go), that also showed that in 56.5% of spontaneous abortions with normal DT for HCG a drop in oestradiol concentrations was observed on day +15 after embryo transfer, suggesting that steroidogenesis in the CL is a better predictor of trophoblastic growth, and its impairment would end in a miscarriage. It should be noted that the lack of statistical significance in our data may have been due to the small number of cases examined.

Luteal dynamics can be affected by progesterone support in IVF luteal phases. Thus, progesterone may not represent an ideal indicator of luteal function. A more specific indicator could be relaxin which is produced mainly by the corpus luteum. A previous study (Emmi et al., 1991Go) demonstrated that pregnant patients with oocyte donation due to ovarian failure had no detectable levels of relaxin. However, the same authors conclude that this hormone is not necessary for pregnancy maintenance. Based on this observation, the significance of change on relaxin plasma levels between normal and ectopic pregnancies and abortions will need further investigation.

No associations have been found between other CL proteins, such as the pregnancy-associated plasma protein-A (PAPP-A) or Schwangerschaft protein-1 (SP-1) (Johnson et al., 1993bGo). However this can be explained by the relative lack of sensitivity of the assays more than the actual usefulness of the markers.

Progesterone levels are not significantly reduced in ectopic pregnancies and abortions as compared to normal pregnancies. This may be due to the supra-physiological exogenous support of the luteal phase with progesterone in oil. This exogenous support produces high levels of plasma progesterone, so subtle variations in endogenous production cannot be easily detected. However, progesterone levels are of relative value, an earlier study (Azuma et al., 1993Go) has shown that 2.3% (8/345) of pregnancies that resulted in normal birth had serum progesterone values below the fifth percentile of single IVF pregnancies at week 4 of gestation. Oestradiol values were also below the fifth percentile, but thereafter they rose to values just within normal limits. Evaluation of the minimal concentration of progesterone to maintain ongoing human pregnancy has not been established.

Finally, it has been reported that luteal oestradiol is not necessary in the establishment of a clinical pregnancy in the human and it was also demonstrated that embryos start to produce oestradiol as early as 18 days after embryo transfer (Zegers-Hochschild and Altieri, 1995Go). It is therefore likely that oestradiol and progesterone are indirect markers of CL recognition of a normal embryo rather than hormones responsible for the maintenance of pregnancy by themselves. It has been shown (Johnson et al., 1993bGo) that progesterone and oestradiol in normal pregnancies were derived from the CL but in ectopic pregnancies the CL failed and the main origin of these hormones was the trophoblast as early as week five of pregnancy.

The data presented here suggest that an ultrasonography with no intrauterine gestational sac between the 4th and 5th week of pregnancy, and normal oestradiol levels with normal rising of HCG represents a high risk of having an ectopic pregnancy. On the other hand, if HCG and oestradiol levels are decreasing a more expectant type of management would be acceptable.


    Notes
 
3 To whom correspondence should be addressed. E-mail: alam{at}entelchile.net Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Azuma, K., Calderon, I., Besanko, M. et al. (1993) Is the luteo-placental shift a myth? Analysis of low progesterone levels in successful ART pregnancies. J. Clin. Endocrinol. Metab., 77, 195–198.[Abstract]

Csapo, A.I. and Pulkkinen, M. (1978) Indispensability of the human corpus luteum in the maintenance of early pregnancy. Obstet. Gynecol. Survey, 33, 69–81.[Medline]

Emmi, A.M., Skurmick, J., Goldsmith, T. et al. (1991) Ovarian control of pituitary hormone secretion in early human pregnancy. J. Clin. Endocrinol. Metab., 72, 1359–1363.[Abstract]

Grudzinskas, J.G., Westergaard, J.G. and Teisner, B. (1986) Biochemical assessment of placental function: early pregnancy. Clin. Obstet. Gynaecol., 13, 553–69.[ISI][Medline]

Johnson, M.R., Riddle, A.F., Grudzinskas, J.G. et al. (1993a) Endocrinology of IVF pregnancies during the first trimester. Hum. Reprod., 8, :316–322.[Abstract]

Johnson, M.R., Riddle, A.F., Irvine, R. et al. (1993b) Corpus luteum failure in ectopic pregnancy. Hum. Reprod., 8, 1491–1495.[Abstract]

Johnson, M.R., Riddle, A.F., Sharma, V. et al. (1993c) Placental and ovarian hormones in anembryonic pregnancy. Hum. Reprod., 8, 112–115.[Abstract]

Kratzer, P.G. and Taylor, R.N. (1990) Corpus luteum function in early pregnancies is primarily determined by the rate of change of human chorionic gonadotropin levels. Am. J. Obstet. Gynecol., 163, 1497–1502.[ISI][Medline]

Lenton, E.A., Neal, L.M., and Sulaiman, R. (1982) Plasma concentrations of human chorionic gonadotropin from the time of implantation until the second week of pregnancy. Fertil. Steril., 37, 773.[ISI][Medline]

Liu, H.C., Davis, O., Grifo, J. et al. (1991) Rescue of the corpus luteum by embryonic HCG may be mediated by endometrial signals. Abstracts of the 47th Annual Meeting, American Fertility Society, S43-S44.

Lower, A.M., Yovich, J.L., Hancock, C. and Grudzinskas,J.G. (1993) Is luteal function maintained by factors other than chorionic gonadotrophin in early pregnancy. Hum. Reprod., 8, 645–648.[Abstract]

Norman, R.J., Buck, R.H., Kemp, M.A. and Joubert, S.M. (1988) Impaired corpus luteum function in ectopic pregnancy cannot be explained by altered human chorionic gonadotropin. J. Clin. Endocrinol. Metab., 66, 1166–1170.[Abstract]

Pittaway, D.E., Reisch, R.L. and Wentz, A.C. (1985) Doubling times of human chorionic gonadotropin increase in early viable intra-uterine pregnancies. Am. J. Obstet. Gynecol., 152, 299–302.[ISI][Medline]

Zegers-Hochschild, F. and Altieri, E. (1995) Luteal estrogen is not required for the establishment of pregnancy in the human. J. Assist. Reprod. Gen., 12, 1–5[ISI][Medline]

Zegers-Hochschild, F., Altieri, E., Fabres, C. et al. (1994) Predictive value of human chorionic gonadotrophin in the outcome of early pregnancy after in-vitro fertilization and spontaneous conception. Hum. Reprod., 9, 1550–1555.[Abstract]

Submitted on November 24, 1998; accepted on June 14, 1999.





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