1 Departments of Obstetrics and Gynaecology, 2 Pathology and 3 Paediatrics, University Hospital Nijmegen St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: abortion/CD34/chorionic vascularization/early pregnancy loss/homocysteine
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Homocysteine is a sulphur-containing intermediate in methionine metabolism, and can be catabolized in the trans-sulphuration pathway (vitamin B6-dependent) or remethylated to methionine (folate- and cobalamin-dependent). Mild hyperhomocysteinaemia can be caused by deficiencies of the relevant B-vitamins or by genetically determined reduction in the enzyme activities. Despite the high number of publications describing methioninehomocysteine metabolism in recent years, the pathogenic role of increased total homocysteine concentrations is still poorly understood. Methionine, involved in the formation of S-adenosylmethionine, is a substrate for DNA methylation. As hyperhomocysteinaemia may reflect reduced remethylation, one could postulate in relation to REPL that elevated total homocysteine is associated with an impaired DNA methylation and gene expression (Heby, 1995), possibly leading to disturbed chorionic vasculogenesis.
Recently, the development of the chorionic villous vascular system in normal human first-trimester pregnancies was investigated by quantitative CD34 immunohistochemistry (te Velde et al., 1997). This study showed a significant correlation between the menstrual age and the total number of vascular elements as well as the villous stromal area. In accordance with this was an earlier observation that embryonic death was associated with a defective chorionic villous vascularization, defined as lower vascular density (total number of blood vessels per eight villi) (Meegdes et al., 1988
).
However, few studies have been reported on chorionic villous vascularization in spontaneous early pregnancy loss (Meegdes et al., 1988; Rehder et al., 1989
; Rockelein et al., 1991
; Salafia et al., 1993
), usually based on a histological score in correlation with cytogenetic findings. There are no reports in which vascular profiles of single vascular elements, which can be assumed as more exact determinants of the vascular function, have been described in spontaneous miscarriage tissue. Quantitative analyses of vascular profiles can be made by a computerized image analysis system (e.g. Vidasplus system), which is frequently used in tumour angiogenesis research (Weidner, 1995
).
Therefore, to investigate the possible pathogenic role of elevated total homocysteine concentrations in REPL, we studied spontaneous miscarriage tissue of 19 women with REPL, in particular the chorionic villous vascularization, by both histopathology and the Vidasplus image analysis system (including CD34 immunohistochemistry) and correlated it with the maternal variables in homocysteine metabolism.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Early pregnancy loss was defined as the spontaneous ending of a pregnancy within 16 weeks of menstrual age, excluding ectopic and molar pregnancies. Recurrent early pregnancy loss was defined as at least two consecutive spontaneous abortions following conception from the same partner. Gestational age (in weeks) was calculated by both the menstrual age at the moment of the curettage or spontaneous expulsion, as well as by the maximum measured crownrump length (n = 17), collected from the clinical record. The Institutional Review Board of the University Hospital Nijmegen approved the study. Before participation, written informed consent was obtained from all subjects.
Methionine-loading test
Methioninehomocysteine metabolism was investigated by a standardized oral methionine-loading test (Wouters et al., 1993). At the time of measurement, none of the women was pregnant or lactating. Elevated total homocysteine was defined as a total homocysteine concentration fasting and/or after-load exceeding the 95th percentile of a group of 104 healthy parous controls (Nelen et al., 2000
). For the fasting total homocysteine concentration this was above 18.3 µmol/l, and for the after-load concentration was >61.5 µmol/l. Folate was considered to be decreased if the serum concentration was below 6.8 nmol/l (5th percentile).
Blood samples for measurement of plasma total homocysteine concentrations were drawn in ethylenediamine tetra-acetate (EDTA) vacutainer tubes of 4 ml and centrifuged within 30 min at 3000 gfor 10 min. The plasma was separated and stored at 20°C. Plasma total homocysteine concentrations were measured using a high-performance liquid chromatography (HPLC) technique and fluorimetric detection (Te Poele-Pothoff et al., 1995). Dry and heparinized vacutainer tubes of 10 ml were used for collecting venous blood samples to assay folate (serum and red cells), pyridoxal 5'-phosphate (an active form of vitamin B6) (whole blood) and vitamin B12 (serum) concentrations. Folate and vitamin B12 concentrations were measured simultaneously with Dualcount SPB (solid phase boil) Radioassay (Diagnostic Products Corporation, Los Angeles, CA, USA), as described previously (Mooij et al., 1991
). Determination of pyridoxal 5'-phosphate was performed using a HPLC technique (Schrijver et al., 1981
).
Histopathology
Formalin-fixed and paraffin-embedded miscarriage tissues of 19 women were used. Standard 5 µm-thick haematoxylin and eosin-stained microscopical sections were used for pathohistological scoring. The sections were scored on a scale for multiple features (villous oedema, fibrosis, vascularization and intervillous fibrin) (Figure 1a) by two authors (W.N., J.B.). The definitive scoring list was completed jointly. In cases of disagreement, the ultimate scoring was obtained by consensus with a third investigator (A.H.). During this procedure, the investigators were blinded to the clinical data and previous histological examinations.
|
Image analysis
Image analysis was performed using the Vidasplus system (Kontron GmbH, Eching, Germany). Microscope images were recorded using a three-chip CCD camera (DXC-325P; Sony, Tokyo, Japan) mounted on a conventional light microscope (Axioskop; Carl Zeiss, Jena, Germany) using a 20x objective (numerical aperture = 0.5). Resulting images, measuring a field of 200x212 µm, were stored on magneto-optical discs (Borsu Systema, Lelystad, The Netherlands) as true colour (24-bit RGB) images. For each case, 40 images with parts of intermediate villi were randomly recorded and evaluated by one author (W.N.) who was blinded to the clinical and histopathological data. Forty images were found to be sufficient to obtain stable running means (data not shown) for the different variables assessed as described below. Each villus was recorded only once. Image pixels were divided by the system into `object pixels' (pixels resulting from CD34-stained cells) and `background pixels', based on the intensities of the signals. Neighbouring object pixels were grouped together to form objects that possibly corresponded to vascular elements (Figure 1c).
Where necessary, interactive correction was performed, implying exclusion of field areas without chorionic villous tissue, closing of vessel lumina that were not completely surrounded by stained cells (Figure 1d), and erasing of falsely recognized non-vascular objects. The following vascularization variables calculated by the computerized system were used: (i) per image: % vascular area, vascular perimeter and vascular count; and (ii) per vascular element: the area, perimeter and diameter.
Statistics
The data were presented as medians (minimummaximum) because of the skewed distributions. For statistical analyses of the different field and vascular element parameters per woman, the median value for each parameter was chosen to be representative for that woman. Statistical comparisons between groups were performed using MannWhitney U-test. Correlation analyses were performed using Spearman's rank correlation tests. In the case of a significant correlation, a stepwise univariate analysis of variance with dummy variables was performed to estimate possible confounding. A two-sided P-value <0.05 was considered to be statistically significant. Statistical analyses were performed with SPSS for Windows Release 8.0 Standard Version (1997, Chicago, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Although women with normal total homocysteine concentrations seemed to have a better developed chorionic vascular system and less extensive fibrosis and intervillous fibrin depositions as scored by the two examiners (Figure 2), these differences failed to reach statistical significance.
In patients with elevated total homocysteine concentrations, between 88 and 410 vascular elements per woman were obtained compared with 162 to 531 vascular elements in normohomocysteinaemic women.
The total number of vascular elements measured per mm2 chorionic tissue, calculated by the Vidasplus image analysis system, was lower in women with elevated total homocysteine concentrations, but this difference failed to reach statistical significance (Table II). However, the median percentage vascular area and the median total vascular perimeter were significantly smaller in women with elevated total homocysteine concentrations. Comparing both total homocysteine groups for the parameters calculated per vascular element, significant smaller median areas, perimeters and diameters were observed in women with elevated total homocysteine concentrations.
Comparing women with decreased (n = 3) and normal (n = 16) serum folate concentrations, no significant differences were found in the histopathological scores or in the vascular parameters calculated by the image analysis system.
To evaluate differences in the vascular profiles between the two groups, a subdivision of the vascular elements was made on the basis of their diameter. As illustrated in Figure 3, women with recurrent early pregnancy loss and elevated total homocysteine concentrations were observed to have proportionally more vascular elements with a small diameter.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chorionic vasculogenesis comprises aggregates of haemangioblastic cells, which are subsequently dilated to form capillaries (Demir et al., 1989). In normal first-trimester pregnancies, this process of maturation has been characterized by an increase in the total number of vascular elements, but with a stable number of the haemangioblastic aggregates (te Velde et al., 1997
). As in our study the total number of vascular elements per mm2 chorionic tissue between both total homocysteine groups was not significantly different, a maturational rather than a constructional defect may be expected to be associated with elevated total homocysteine concentrations.
According to the postulated mechanism of reduced DNA methylation and gene expression, one may expect to find an impaired chorionic villous vascularization in cases of decreased serum folate concentrations, but this was not the case. One possible explanation may be the relatively small group size (n = 3) of folate-deficient individuals, although alternative pathogenic mechanisms (e.g. direct embryotoxic effects) (Obwegeser et al., 1999) should be considered. Moreover, in this study only the chorionic vascularization was investigated, but others have suggested that decidual angiogenic disturbances are also related to early pregnancy loss (Vailhe et al., 1999
).
The perimeter of a blood vessel may be accepted as representing the most functional variable of all calculated vascular parameters, because it best reflects the final fetalmaternal diffusion capacity. An impaired fetalmaternal diffusion is a plausible explanation for a disturbed fetal development or even fetal death, especially if one accepts the idea that fetalmaternal diffusion begins early in pregnancy (Valentin et al., 1996; Craven and Ward, 1999
). However, if a functional fetalmaternal circulation is not initiated before 12 weeks of pregnancyas has been suggested by some authors (Hustin and Schaaps, 1987
; Rodesch et al., 1992
)then the role of a defective chorionic vascularization in embryonic death remains to be elucidated. During these first weeks of pregnancy, embryonic nutrition is provided by the transfer of nutrients from the extra-embryonic coelomic cavity via the yolk sac to the embryo (Exalto, 1995
). In normal first-trimester pregnancies, low extra-embryonic coelomic total homocysteine concentrations and high folate and methionine concentrations have been reported (Campbell et al., 1993
; Steegers-Theunissen et al., 1997
). Furthermore, these extra-embryonic coelomic concentrations were positively correlated with maternal serum concentrations. Thus, we expect also higher total homocysteine and/or lower folate extra-embryonic coelomic concentrations in the pregnancies of women with elevated plasma total homocysteine concentrations.
We realize that the investigation of pathological changes in chorionic tissue after embryonic death is limited because of the uncertainty of whether vascular changes are caused by defective development, or occurred post mortem. However, as we also found no effect of the retention time on the vascular parameters in the regression analysiswhich is in agreement with observations by others (Meegdes et al., 1988)we consider that the data on vascular parameters do reflect embryonic development.
In conclusion, in recurrent early pregnancy loss, elevated maternal total homocysteine concentrations are associated with defective chorionic villous vascularization.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Campbell, J., Wathen, N., Perry, G. et al. (1993) The coelomic cavity: an important site of materno-fetal nutrient exchange in the first trimester of pregnancy. Br. J. Obstet. Gynaecol., 100, 765767.[ISI][Medline]
Coumans, A.B.C., Huijgens, P.C., Jakobs, C. et al. (1999) Haemostatic and metabolic abnormalities in women with unexplained recurrent abortion. Hum. Reprod., 14, 211214.
Craven, C.M. and Ward, K. (1999) Syncytiotrophoblastic fragments in first-trimester decidual veins: evidence of placental perfusion by the maternal circulation early in pregnancy. Am. J. Obstet. Gynecol., 181, 455459.[ISI][Medline]
Dekker, G.A., De Vries, J.I., Doelitzsch, P.M. et al. (1995) Underlying disorders associated with severe early-onset preeclampsia. Am. J. Obstet. Gynecol., 173, 10421048.[ISI][Medline]
Demir, R., Kaufmann, P., Castellucci, M. et al. (1989) Fetal vasculogenesis and angiogenesis in human placental villi. Acta Anat. (Basel), 136, 190203.[Medline]
Den Heijer, M., Koster, T., Blom, H.J. et al. (1996) Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N. Engl. J. Med., 334, 759762.
Exalto, N. (1995) Early human nutrition. Eur. J. Obstet. Gynecol. Reprod. Biol., 61, 36.[ISI][Medline]
Goddijn Wessel, T.A., Wouters, M.G.A.J., Van der Molen, E.F. et al. (1996) Hyperhomocysteinemia: a risk factor for placental abruption or infarction. Eur. J. Obstet. Gynecol. Reprod. Biol., 66, 2329.[ISI][Medline]
Graham, I.M., Daly, L.E., Refsum, H.M. et al. (1997) Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project. JAMA, 277, 17751781.[Abstract]
Heby, O. (1995) DNA methylation and polyamines in embryonic development and cancer. Int. J. Dev. Biol., 39, 737757.[ISI][Medline]
Hustin, J. and Schaaps, J.P. (1987) Echographic and anatomic studies of the maternotrophoblastic border during the first trimester of pregnancy. Am. J. Obstet. Gynecol., 157, 162168.[ISI][Medline]
Meegdes, B.H., Ingenhoes, R., Peeters, L.L. et al. (1988) Early pregnancy wastage: relationship between chorionic vascularization and embryonic development. Fertil. Steril., 49, 216220.[ISI][Medline]
Mooij, P.N., Thomas, C.M.G., Doesburg, W.H. et al. (1991) Multivitamin supplementation in oral contraceptive users. Contraception, 44, 277288.[ISI][Medline]
Nelen, W.L.D.M., Steegers, E.A.P., Eskes, T.K.A.B. et al. (1997a) Genetic risk factor for unexplained recurrent early pregnancy loss. Lancet, 350, 861.[ISI][Medline]
Nelen, W.L.D.M., Van der Molen, E.F., Blom, H.J. et al. (1997b) Recurrent early pregnancy loss and genetic-related disturbances in folate and homocysteine metabolism. Br. J. Hosp. Med., 58, 511513.[Medline]
Nelen, W.L.D.M., Blom, H.J., Thomas, C.M.G. et al. (1998) Methylenetetrahydrofolate reductase polymorphism affects the change in homocysteine and folate concentrations resulting from low dose folic acid supplementation in women with unexplained recurrent miscarriages. J. Nutr., 128, 13361341.
Nelen, W.L.D.M., Blom, H.J., Steegers, E.A.P. et al. (2000) Homocysteine and folate levels as risk factors for recurrent early pregnancy loss. Obstet. Gynecol. (in press).
Obwegeser, R., Hohlagschwandtner, M. and Sinzinger, H. (1999) Homocysteine a pathophysiological cornerstone in obstetrical and gynaecological disorders? Hum. Reprod. Update, 5, 6472.
Quere, I., Bellet, H., Hoffet, M. et al. (1998) A woman with five consecutive fetal deaths: case report and retrospective analysis of hyperhomocysteinemia prevalence in 100 consecutive women with recurrent miscarriages. Fertil. Steril., 69, 152154.[ISI][Medline]
Rehder, H., Coerdt, W., Eggers, R. et al. (1989) Is there a correlation between morphological and cytogenetic findings in placental tissue from early missed abortions? Hum. Genet., 82, 377385.[ISI][Medline]
Rockelein, G., Ulmer, R. and Schroder, J. (1991) Karyotype and placental structure of first-trimester spontaneous abortions: a morphometrical study. Eur. J. Obstet. Gynecol. Reprod. Biol., 38, 2532.[ISI][Medline]
Rodesch, F., Simon, P., Donner, C. et al. (1992) Oxygen measurements in endometrial and trophoblastic tissues during early pregnancy. Obstet. Gynecol., 80, 283285.[Abstract]
Salafia, C., Maier, D., Vogel, C. et al. (1993) Placental and decidual histology in spontaneous abortion: detailed description and correlations with chromosome number. Obstet. Gynecol., 82, 295303.[Abstract]
Schrijver, J., Speek, A.J. and Schreurs, W.H. (1981) Semi-automated fluorometric determination of pyridoxal-5'-phosphate (vitamin B6) in whole blood by high-performance liquid chromatography (HPLC). Int. J. Vitam. Nutr. Res., 51, 216222.[ISI][Medline]
Steegers-Theunissen, R.P.M., Boers, G.H.J., Blom, H.J. et al. (1992) Hyperhomocysteinaemia and recurrent spontaneous abortion or abruptio placentae. Lancet, 339, 11221123.[Medline]
Steegers-Theunissen, R.P.M., Boers, G.H.J., Trijbels, F.J.M. et al. (1994) Maternal hyperhomocysteinemia: a risk factor for neural-tube defects? Metabolism, 43, 14751480.[ISI][Medline]
Steegers-Theunissen, R.P.M., Wathen, N.C., Eskes, T.K.A.B. et al. (1997) Maternal and fetal levels of methionine and homocysteine in early human pregnancy. Br. J. Obstet. Gynaecol., 104, 2024.[Medline]
Te Poele-Pothoff, M.T., Van den Berg, M., Franken, D.G. et al. (1995) Three different methods for the determination of total homocysteine in plasma. Ann. Clin. Biochem., 32, 218220.[ISI][Medline]
te Velde, E.A., Exalto, N., Hesseling, P. et al. (1997) First trimester development of human chorionic villous vascularization studied with CD34 immunohistochemistry. Hum. Reprod., 12, 15771581.[Abstract]
Vailhe, B., Dietl, J., Kapp, M. et al. (1999) Increased blood vessel density in decidua parietalis is associated with spontaneous human first trimester abortion. Hum. Reprod., 14, 16281634.
Valentin, L., Sladkevicius, P., Laurini, R. et al. (1996) Uteroplacental and luteal circulation in normal first-trimester pregnancies: Doppler ultrasonographic and morphologic study. Am. J. Obstet. Gynecol., 174, 768775.[ISI][Medline]
Weidner, N. (1995) Intratumor microvessel density as a prognostic factor in cancer. Am. J. Pathol., 147, 919.[ISI][Medline]
Wouters, M.G.A.J., Boers, G.H.J., Blom, H.J. et al. (1993) Hyperhomocysteinemia: a risk factor in women with unexplained recurrent early pregnancy loss. Fertil. Steril., 60, 820825.[ISI][Medline]
Submitted on September 7, 1999; accepted on December 6, 1999.