Hum. Reprod. Advance Access published online on August 10, 2008
Human Reproduction, doi:10.1093/humrep/den299
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Effect of rising hCG levels on the human corpus luteum during early pregnancy
1 Department of Obstetrics and Gynecology, Oulu University Hospital, PO Box 5000, FIN-90014 Oulu, Finland 2 Department of Clinical Chemistry, Oulu University Hospital, FIN-90014 Oulu, Finland
3 Correspondence address. E-mail: ilkka.jarvela{at}oulu.fi
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Abstract |
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BACKGROUND: During early pregnancy, the most important task of the corpus luteum (CL) is to produce sufficient progesterone until the luteoplacental shift occurs. Progesterone production is closely related to the extensive vasculature surrounding and supplying the CL. The synthesis of both progesterone and factors controlling the vasculature in the CL is regulated by hCG, which is released initially at rising levels from the placenta. The primary aim of this research was to evaluate changes in the CL vasculature during early pregnancy.
METHODS: Twenty naturally conceived pregnancies were examined weekly from weeks 5 to 11. At each visit, blood samples were obtained to determine the concentrations of hCG, progesterone and 17-OH progesterone (17-OHP). The vasculature in the ovaries was assessed using three-dimensional power Doppler ultrasonography.
RESULTS: The vascular supply in the ovary containing the CL was greatest at week 5, and thereafter, declined continuously until week 11. The decrease in the vasculature correlated with the decrease in 17-OHP. Mean hCG levels reached a maximum at week 8, progesterone levels reached the nadir at week 7 and increased after that.
CONCLUSIONS: Vasculature in the CL appears to be created already by the fifth week of pregnancy and it does not enlarge despite rising hCG levels. The activity of the CL during pregnancy may be measured non-invasively by assessing its vasculature with three-dimensional ultrasonography.
Key words: estradiol/gestational sac/luteoplacental shift/pregnancy-associated plasma protein-A/placenta
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Introduction |
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Continuing rescue of the corpus luteum (CL) by hCG is essential, otherwise declining progesterone secretion is detrimental to maintaining pregnancy (Csapo et al., 1973
). The CL is the primary organ producing progesterone up to the seventh to eighth week of pregnancy, after which the placenta is capable of producing enough progesterone (Csapo et al., 1973; Csapo and Pulkkinen, 1978). This period during which the placenta is assuming the main responsibility of progesterone production from the CL is called the luteoplacental shift.
The capacity of the CL to produce progesterone is closely related to the extent of its vascular network (Niswender et al., 1976; Miyazaki et al., 1998; Niswender et al., 2000; Järvelä et al., 2007). The amount of vasculature accounts for more than 20% of the total volume of the CL, exceeding that of any other tissue (Dharmarajan et al., 1985; Niswender et al., 2000). The dense capillary network enables hormone-producing cells to obtain oxygen, nutrients and hormone precursors necessary to synthesize and release large amounts of progesterone.
CL angiogenesis appears to be controlled by local secretion of growth factors (Hazzard and Stouffer, 2000) such as the vascular endothelial growth factor (VEGF) (Sugino et al., 2000; Wulff et al., 2001a,b). The expression and synthesis of these factors is regulated by hCG (Fraser et al., 2005). Because hCG concentrations increase up to 8–9 weeks of pregnancy (Tulchinsky et al., 1972; Tulchinsky and Hobel, 1973), it could be expected that the volume of the CL vasculature increases as well, leading to rising systemic levels of progesterone. However, Tulchinsky et al. (1972) and Tulchinsky and Hobel (1973) observed that progesterone levels remained quite steady until the placental progesterone production increased (Tulchinsky et al., 1972; Tulchinsky and Hobel, 1973).
Knowledge of the status of the CL vasculature in humans during the first trimester of pregnancy is limited. Tissue samples from women with ectopic pregnancies and from women following treatment in vivo with increasing doses of hCG to simulate early pregnancy has been collected and analysed (Rodger et al., 1997; Gaytan et al., 1999; Wulff et al., 2001a,b). These methods excluded the possibility of following up, longitudinally, a single CL and its vasculature in vivo, especially during normal pregnancy. In addition, they did not enable quantification of the volume of the vascular network.
Three-dimensional power Doppler ultrasonography enables detection of the total vasculature in a whole and strictly restricted volume such as the ovary (Jokubkiene et al., 2006; Järvelä et al., 2007; Jayaprakasan et al., 2007) by mapping and quantifying the power Doppler signal in the entire region of interest. The power Doppler signal detected represents moving blood cells and the three-dimensional image created resembles microangiography in an organ. The primary advantages of using three-dimensional power Doppler ultrasonography are that the method is non-invasive and safe, the information obtained can be restricted to the organ of interest and the development of ovarian volume and vasculature can be tracked longitudinally. To date, three-dimensional power Doppler ultrasonography has not been used in clinical studies involving evaluation of CL function during pregnancy.
The aim of the study was to evaluate changes in the CL vasculature, and placental and CL hormone output during early pregnancy.
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Patients
Pregnant patients were recruited who had contacted the local health care centre in the city of Oulu to organize their follow-up during the pregnancy. Only patients with no known gynecological pathology (e.g. endometriosis, fibroids, any operation to gynecological organs) were included in the study. All the women had conceived naturally.
The aim was to recruit the patients as early as possible at the start of the pregnancy. In addition, the patients were examined following each completed week of pregnancy, from the 5th to the 11th week of pregnancy. At each visit, blood samples were collected for hormonal assays and an ultrasound examination was performed.
The estimated delivery date (EDD) was assessed according to the last menstrual bleeding and was ascertained at the last visit by measuring the crown-rump length (CRL). If the EDD, as assessed by the CRL, differed more than ±7 days from that calculated from the last menstrual bleeding, the date was changed.
Informed written consent was obtained from each subject, and the study was approved by the Ethics Committee of Oulu University Hospital, Oulu, Finland.
Assays
Serum concentrations of testosterone, progesterone and hCG were analysed using an automated chemiluminescence system (Advia Centaur; Bayer Healthcare LCC, Diagnostic Division, Tarrytown, NY, USA). Serum placental pregnancy-associated plasma protein-A (PAPP-A) concentrations were determined by fluoroimmunoassay (PerkinElmer, Turku, Finland). Radioimmuno assays were used for 17-OH progesterone (17-OHP), androstenedione (Diagnostic Products Corp., Los Angeles, CA, USA) and estradiol (E2) (Orion Diagnostica, Oulunsalo, Finland), following instructions provided by the manufacturers.
The intra-assay and inter-assay coefficients of variation were 5.0 and 5.4% for 17-OHP, 5.0 and 8.6% for androstenedione, 4.0 and 5.6% for testosterone, 5.7 and 6.4% for E2, 3.7 and 5.4% for progesterone, 2.0 and 3.5% for hCG and 3.5 and 4.9% for PAPP-A, respectively. External quality control of the hormone assays was organized by national (Labquality Ltd, Helsinki, Finland) and international (Bio-Rad Laboratories EQAS, Irvine, CA, USA) companies.
Ultrasonography
The patients were examined at each visit using three-dimensional ultrasonography (Voluson Expert 730; Kretz, Zipf, Austria) equipped with a transvaginal probe. This technique enabled determination of the state of the pregnancy. In addition, the ultrasound data of the uterus and the ovaries were stored in three-dimensional volumes for subsequent analysis.
The power Doppler ultrasound was used to locate the blood vessels in the ovaries and to identify the ovary containing the CL. The power Doppler mode was not applied at any time during the visit to investigate the pregnancy itself (the fetus or the trophoblastic tissue). Therefore, data concerning the fetus, gestational sac or the placenta does not include any power Doppler information.
Volume acquisition and storage were described in detail elsewhere (Järvelä et al., 2003a). Identical, pre-installed settings were used to acquire the ultrasound and the power Doppler information. The setting conditions for this study were as follows: frequency, mid; dynamic set, 2; balance, G > 170; smooth, 5/5; ensemble, 16; line density, 7; power Doppler map, 5; and the setting conditions for the subpower Doppler mode were: gain, –5.6; quality, normal; wall motion filter, low 1; and velocity range, 0.9 kHz.
Determination of ovarian volume and vascularization was performed using the built-in virtual organ, computer-aided analysis Imaging Program (VOCAL, GE Healthcare, Zipf, Austria) for three-dimensional power Doppler histogram analysis. The manual mode of the VOCAL Contour Editor was used to cover the entire three-dimensional volume of the ovary, with 30° rotation steps (Järvelä et al., 2007). Hence, six contour planes were analysed for each ovary to cover 180°. After obtaining the total ovarian volume, the programme calculated the ratio of coloured voxels to all voxels; this fraction of the total volume (%) was expressed as the vascularization index (VI). Vascularized volume (ml) in the ovary was calculated by multiplying the total ovarian volume by the VI.
Similarly, the three-dimensional ultrasound data of the uterus, including the fetus, gestational sac and placenta, were analysed. During the first weeks of pregnancy, the trophoblastic tissue surrounds entirely the gestational sac, giving an almost uniform diameter. By the end of first trimester, two-thirds of this primitive placenta disappear (Jauniaux et al., 2006) and the trophoblastic tissue (placenta) polarizes to a specific place inside the uterine cavity.
The volume of the gestational sac was first assessed by determining the outlines of the gestational sac in six contour planes (30° rotation steps) using the manual mode of the VOCAL Contour Editor. The volume of the gestational sac constituted the entire exocoelomic cavity including the amnionic cavity and secondary yolk sac. Similarly, the outlines of the trophoblastic tissue were determined in six contour planes (30° rotation steps). The volume obtained included not only the gestational sac but also all of the trophoplastic tissue (i.e. placenta), and therefore, after subtracting the volume of the gestational sac from this latter volume, the actual volume of the placenta was obtained.
Statistics
The Statistical Package for the Social Sciences (SPSS) for Windows 12.0.1 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The general linear model for repeated measures was used to measure significance within the study group. Departure from a normal distribution was assessed using the Kolmogorov–Smirnov test. To compare serum hormone levels between different time points, the paired samples t-test was used as a post hoc test for normally distributed variables, and the Wilcoxon test was used for variables with skewed distribution. A value of P < 0.05 was considered statistically significant. All values given are the mean ± SE.
The reproducibility of volume and VI measurements using transvaginal three-dimensional power Doppler ultrasonography was assessed by calculating the intraclass correlation coefficient and repeatability coefficient (r) separately for dominant and non-dominant ovaries at pregnancy week 9. To determine the intra-observer reproducibility, one observer (I.Y.J) analysed the same volume twice, 2 years apart, being unaware of the results of the first analysis when performing the second one. The coefficients were assessed for volume, VI and vascularity (=volume x VI/100). The statistical analysis for reproducibility has described earlier (Järvelä et al., 2003b).
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Results |
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The results of the statistical analysis for reproducibility (Table I) indicate good reproducibility of the technique and are in line with those observed earlier (Jokubkiene et al., 2006
; Järvelä et al., 2007).
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Altogether, 20 women with normal ongoing pregnancies were recruited. Ten of the patients were examined at the fifth week of pregnancy. At week 6, 15 women were examined; at weeks 7 and 8, 19 women were examined; at weeks 9 and 10, 20 women were examined; and, at week 11, 19 women were examined. The mean age of the patients was 25.4 ± 1.0 years (range 19.0–35.9). The mean number of pregnancies was 1.8 ± 0.3 (range 1–6) and the mean number of deliveries was 0.6 ± 0.2 (range 0–3). The mean BMI was 22.8 ± 0.8 (range 18.4–30.1).
Hormone levels
Mean hCG levels (Fig. 1) increased from 8825.6 ± 2338.2 IU/ml at week 5 to 118 254.8 ± 9491.6 IU/ml at week 8 (P < 0.001); thereafter, a non-significant decrease in the mean hCG levels was observed. At the final check-up (11 weeks of pregnancy), the mean hCG level (SE) was 96 786.2 (7933.2) IU/ml. The increase in hCG levels before 8 weeks of pregnancy did not correlate with the increase in placental volume during the same period.
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Mean PAPP-A levels (Fig. 1) exhibited a continuous increase throughout the follow-up period. The lowest value (5.2 ± 1.0 mIU/l) was observed at week 5 and the greatest value (1607.7 ± 249.4 mIU/l) at week 11. The increases in PAPP-A, and the placental volume between weeks 6 and 11 of pregnancy, correlated significantly (R = 0.644, P < 0.05).
Concentrations of progesterone (Fig. 1) exhibited a slight, but non-significant, decrease from weeks 5 to 7 of pregnancy. The lowest level (65.6 ± 6.0 nmol/l) during the follow-up period was observed at week 7. Subsequently, progesterone levels began to increase, reaching the maximum level (112.6 ± 7.4 nmol/l) at the end of the follow-up period at week 11 (week 7 versus week 11, P < 0.001).
Concentrations of 17-OHP (Fig. 1) were at the greatest level (21.3 ± 2.4 nmol/l) initially at week 5 of pregnancy. A steady decrease occurred in the concentrations during the follow-up period and the nadir (10.1 ± 0.5 nmol/l) was observed at week 10 (week 5 versus week 10, P < 0.001). The decrease in 17-OHP concentrations and dominant ovarian vascularization between weeks 6 and 11 of pregnancy correlated significantly (R = 0.606, P < 0.05). No differences were observed in androstenedione or testosterone levels during the study period (data not presented).
Similar to PAPP-A, levels of E2 increased throughout the follow-up period (data not shown). At 5 weeks of pregnancy, the mean level was 1.1 ± 0.1 and was significantly higher at 11 weeks, at 8.2 ± 0.7 nmol/l (P < 0.001). The increase in E2 levels between the 6th and 11th weeks of pregnancy did not correlate with the corresponding increase in placental volume.
Gestational sac and placental volume
The volume of the gestational sac increased steadily from 0.4 ± 0.1 ml at week 5 to 69.3 ± 4.6 ml at week 11 (P < 0.001) (Fig. 2). Similarly, the placental volume increased from 1.0 ± 0.3 ml at week 5 to 59.6 ± 6.3 ml at week 11 (P < 0.001) (Fig. 2).
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Ovarian volume and vascularization
Analysis of repeated measurements showed changes in the dominant ovarian volume (Fig. 2) during the follow-up period. At week 5, the volume was 13.5 ± 2.3 ml and increased to 15.5 ± 1.7 ml at week 7 (P < 0.05). Thereafter, the volume decreased, continuously, to 9.0 ± 0.5 ml at week 11 (P < 0.01, compared with week 7). At week 11, the volume was significantly lower than at week 5 (P < 0.01).
The data for VI and vascularized volume for both dominant and non-dominant ovaries during the follow-up period are presented in Table II. Vascularization in the dominant ovary containing the CL (Fig. 2) showed significant changes. The volume at week 11 was significantly lower than at week 6 or 7 (P < 0.05). The volume was at its highest, initially, at week 5 (3.4 ± 0.8 ml) and reached the nadir at week 11 (1.7 ± 0.2 ml).
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In the non-dominant ovary (Fig. 2, Table II), no changes either in the total volume or in the vascularized volume were observed during the follow-up period. The dominant ovary was larger and more completely vascularized than the non-dominant ovary at each measurement.
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Discussion |
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In this study, CL function in women who conceived naturally was followed, longitudinally, from weeks 5 to 11 of pregnancy. According to these results, despite rising hCG levels, CL activity declined after week 5 of pregnancy, confirming that the hCG-induced CL rescue is only temporary. Although CL activity decreased continuously during the follow-up period, at 11 weeks of pregnancy the CL was clearly still functioning, although its activity had diminished considerably.
The most important task of the CL is to produce sufficient progesterone up to the luteoplacental shift (Csapo et al., 1973). The vasculature in the CL is extensive and closely related to progesterone secretion (Niswender et al., 1976; Miyazaki et al., 1998; Niswender et al., 2000; Järvelä et al., 2007). CL activity was assessed in this research using two different methods. First, systemic hormone levels were assessed, which represented hormone secretion, not only from the CL but also from other endocrine organs, the most important of which is the placenta. Second, CL vascularity was assessed using three-dimensional power Doppler ultrasonographic equipment, which enabled quantification of the total volume and the vascularized volume of the dominant ovary containing the CL and to follow the pregnancies, longitudinally, throughout the study period. Because the patients were not recruited until their pregnancy test was positive, the period from ovulation to testing (i.e. pregnancy weeks 2–4) was excluded from the follow-up period.
A decline in progesterone levels was observed from pregnancy week 5 onwards and the nadir was achieved at pregnancy week 7, after which the levels increased. Secretion of 17-OHP, which is believed to be produced only in the CL during early pregnancy (Tulchinsky and Simmer, 1972), declined continuously from the fifth week of pregnancy onwards. These findings are consistent with those from earlier studies (Tulchinsky and Simmer, 1972, Tulchinsky et al., 1972, Tulchinsky and Hobel, 1973). Changes in CL vascularity were similar to 17-OHP in that both decreased steadily up to the 11th week of pregnancy. The close connection between CL hormone secretion and its vascularity was confirmed by the determination that the decline in 17-OHP secretion correlated with the decrease in CL vascularity.
According to earlier studies, it is not clear whether the hCG-induced rescue of the CL is accompanied by further angiogenesis and vessel stabilization (Fraser and Wulff, 2003). The production of angiogenic factors such as VEGF in the CL during pregnancy appears to be under the influence of hCG (Sugino et al., 2000; Wulff et al., 2000; Wulff et al., 2001a,b; Fraser and Wulff, 2003). Rodger et al. (1997) simulated early pregnancy by administrating increasing hCG doses during the luteal phase, and thereafter collecting CL samples for analysis. Endothelial cell proliferation was assessed using quantitative immunochemistry for CD34 and Ki-67. They observed unchanging levels of proliferation in the endothelial cells, suggesting that vascularization of the CL was already established during the luteal phase (Rodger et al., 1997). However, when luteal cell hypertrophy after hCG treatment was taken into consideration, the hCG-induced rescue of the CL was found to be associated with increased angiogenic activity (Wulff et al., 2001a,b). Previous studies were cross-sectional and the experimental design used did not enable a longitudinal follow-up of patients. According to the results of our study, serum hCG levels rose from weeks 5 to 8 during pregnancy as observed earlier (Tulchinsky et al., 1972, Tulchinsky and Hobel, 1973). The vascular network supplying the CL was formed already at week 5 of the pregnancy, and thereafter gradually decreased, reaching the nadir at the last follow-up visit, at pregnancy week 11. VEGF response to hCG was not measured in this study because systemic VEGF does not represent accurately local changes occurring in a single CL (Järvelä et al., 2007). Despite not measuring angiogenic factors, the results of our research suggest that rising hCG stimulation during early pregnancy is not accompanied by further neoangiogenesis or increase in the vasculature in the human CL.
The critical step in CL synthesis of progesterone is the delivery of cholesterol to cytochrome P450scc, which is governed by the steroidogenic acute regulatory protein, StAR (Devoto et al., 2002). According to Devoto et al. (2001), StAR protein levels in the CL are highly correlated with plasma progesterone levels (Devoto et al., 2001). During the luteal phase, expression of the StAR protein is under the control of LH and is greatest during the early and mid luteal phases and declines significantly in the late luteal phase (Devoto et al., 2002). Luteal rescue with hCG has been found to be associated with continued expression of the StAR protein (Duncan et al., 1999). The secretion pattern of progesterone in the present study was in agreement with earlier studies concerning early pregnancy (Tulchinsky et al., 1972; Tulchinsky and Hobel, 1973). Despite the rising hCG stimulus, progesterone levels decreased rather than increased from the fifth week of pregnancy onwards, and the nadir was achieved at the seventh week of pregnancy. Thereafter, the luteoplacental shift probably occurred and progesterone levels started to rise. This study did not allow assessment of corpus luteal StAR expression in response to rising hCG stimulation, and therefore, it is not possible to conclude whether changes in StAR protein levels were of any significance. Taking into account, however, the close relationship between the CL vasculature and the 17-OHP secretion observed here, it is likely that the primary reason for the decline in CL hormone secretion during the first weeks of pregnancy was the vanishing blood supply to the CL.
The dominant ovary enlarged slightly from the fifth to the seventh week of pregnancy whereas no change was observed in the non-dominant ovary, suggesting that the variation in the volume of the dominant ovary was due to changes in the CL. Determination of the exact position in the CL at which the increase in volume occurred was not possible. Because the CL vasculature started to decrease after the fifth week of pregnancy, the increase in volume of the endothelial cells and pericytes is excluded. One option is that the increase in volume was due to extravasation of blood and plasma into the cavity of the CL, which earlier was the dominant follicle. Another option is the enlargement of the hormonally active luteal cell volume. However, because progesterone or 17-OHP secretion decreased before the eighth week of pregnancy, the latter option is unlikely.
The blood supply of the non-dominant ovary remained unchanged during the first weeks of pregnancy. The vasculature in the non-dominant ovary was minimal in comparison with the dominant ovary, suggesting that the vascular network in the dominant ovary was predominantly supplying the CL, and therefore, closely related to CL activity. At 11 weeks of pregnancy, the dominant ovary was still larger than the non-dominant ovary, indicating that functional activity continued at some level.
We measured the volume of the gestational sac and trophoblastic tissue (i.e. placenta) and related this to hormone secretion. As far as is known, the volume of the placenta has not been measured as early during pregnancy as in this research. The volume of the gestational sac and placenta increased exponentially, the gestational sac becoming larger than the placenta at 9 weeks of pregnancy, just after the luteoplacental shift. The volume measurements concerning the gestational sac are in agreement with earlier studies using the same methodology (Lee et al., 2006).
The increase in the placental volume was correlated with the systemic levels of PAPP-A. PAPP-A primarily originates from placental syncytiotrophoblasts (Bischof et al., 1981; Sinosich et al., 1982) and has gained favour as a clinically useful first-trimester screening marker of trisomy 21 (Niemimaa et al., 2001). The PAPP-A levels in the current study were similar to those in previous reports (Bischof et al., 1981; Sinosich et al., 1982). Changes in mean hCG or E2 concentrations were not associated with enlargement of the placenta. According to current knowledge (Hay, 1988), hCG secretion is related directly to the mass of hCG-producing trophoblastic tissue. The decline in hCG levels after 8 weeks of pregnancy is explained by the reduction in the mass of syncytiocytotrophoblast and trophoblast tissue and mirrors the morphologic change of the placenta from an organ of invasion to an organ of transfer (Hay, 1988).
The vascular network of the CL appears to be formed by the fifth week of pregnancy. As observed by the decrease in both vascular supply and 17-OHP secretion, CL activity declines despite increasing stimulation by hCG. The reason for the inadequacy of the CL to respond to increasing stimulation during early pregnancy remains to be assessed. Because vascularity in the dominant ovary appears to reflect CL activity, three-dimensional power Doppler ultrasonography could be used as a non-invasive tool for evaluating CL function during early pregnancy.
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I.Y.J. was supported by a grant from The Finnish Medical Foundation.
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Submitted on March 10, 2008; resubmitted on July 1, 2008; accepted on July 10, 2008.
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