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Serial plasma fibronectin levels in pre-eclamptic and normotensive women
International Journal of Gynecology & Obstetrics 66 Ž1999. 123]128
Article
Serial plasma fibronectin levels in pre-eclamptic and normotensive women S.S. Suda , I. Guptaa,U , L.K. Dhaliwal a , B. Kaur b, N.K. Ganguly b a
Department of Obstetrics and Gynaecology, Postgraduate Institute of Medical Education and Research, Chandigarh, India b Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Received 9 November 1998; received in revised form 12 April 1999; accepted 14 April 1999
Abstract Objecti¨ e: Endothelial cell damage has been put forward as an underlying factor for development of pre-eclampsia. This study was carried out to see if fibronectin, which is a marker of endothelial damage, could be used as a marker of pre-eclampsia. Methods: A longitudinal study was conducted on 100 normotensive primigravidae registered before 20 weeks of gestation. These subjects were followed until delivery and three blood samples were collected, first at registration, i.e. before 20 weeks, second around 28 weeks and third at 36 weeks or later till delivery. Fibronectin levels were assayed by ELISA and women observed for any signs of pre-eclampsia. Results: Fourteen subjects developed pre-eclampsia. Fibronectin levels were observed to rise as pregnancy advanced but the rise was significantly higher in subjects who developed pre-eclampsia. The fibronectin levels were also significantly higher in these 14 subjects even in the first sample, i.e. before 20 weeks of gestation when compared with normotensive subjects Ž P- 0.01.. Conclusions: Fibronectin levels could be used as an early valuable biomarker for the development of pre-eclampsia. Q 1999 International Federation of Gynecology and Obstetrics. Keywords: Plasma fibronectin; Pre-eclamptic; Normotensive
1. Introduction Hypertensive disorder is one of the commonest disorders complicating pregnancy and is a major U
Corresponding author. Tel.: q91-172-541032-39r421; fax: q91-172-540401.
cause of maternal and perinatal morbidity and mortality. In India the incidence of pre-eclampsia in referral institutes like the Postgraduate Institute of Medical Education and Research, Chandigarh is as high as 17.76% w1x. The exact aetiopathogenesis of pre-eclampsia has not been defined as yet. During the past decade, there have been significant advances to
0020-7292r99r$20.00 Q 1999 International Federation of Gynecology and Obstetrics. PII: S 0 0 2 0 - 7 2 9 2 Ž 9 9 . 0 0 0 7 2 - 7
124
S.S. Sud et al. r International Journal of Gynecology & Obstetrics 66 (1999) 123]128
suggest that pre-eclampsia is a complex clinical syndrome potentially involving all organ systems and hypertension is one of those manifestations. Recently, it has been shown that endothelial cell damage and altered mechanism of calcium magnesium balance contribute to the pathophysiology of pre-eclampsia w12,13,17x. It is postulated that the pre-eclampsia syndrome is a vasospastic disorder and probably has a placental origin. The changes in placental blood flow are related to stages of trophoblastic invasion of the spiral arteries during 14]20 weeks. When the trophoblastic invasion is over, the spiral arteries become a high resistance system. The defect observed in pre-eclampsia is lack of invasion of trophoblast to maternal arteries. The diminished placental perfusion causes endothelial damage w8x. Fibronectins are large adhesive glycoproteins Ž4]40 kDa. present in many tissues and in most body fluids and have been found to be a marker of endothelial damage w2x. Increased levels of fibronectin in plasma have been observed in established gestational proteinuric hypertension w3,14x. Fibronectin has been established as a better marker of endothelial damage than thrombomodulin, plasminogen activator inhibitor Type 1, angiotensinogen and Von-Williebrand factor w5,12,16x. It has been seen that fibronectin levels rise before signs and symptoms of pre-eclampsia develop w2,3,15x. Hence, it is established that endothelial damage occurs in pre-eclampsia and studies have been carried out to see if fibronectin levels are different in normotensive as compared to preeclamptic subjects w5,6x. Therefore a longitudinal study was planned to assess fibronectin levels in pre-eclampsia and normotensive subjects.
2. Subjects and method The study was conducted on 100 normotensive primigravidae recruited before 20 weeks of gestation from the antenatal clinic of the Department of Obstetrics and Gynaecology, Nehru Hospital, P.G.I., Chandigarh and were followed up until delivery. The first blood sample was collected at the first
visit before 20 weeks of gestation provided the subject was normotensive. Subsequently blood samples were collected around the 28th week and from 36 weeks until delivery. The subjects were followed up until delivery. The following criteria were used to diagnose a woman as having pre-eclampsia. 1. Blood pressure ) 140r90 mmHg on more than two occasions 6 h apart. 2. Proteinuria ) 0.3 gmrl in 24 h urine or traces to q1 by uristix Ži.e. G 300 mgrl.. 3. Pedal edema 1 q after 12 h of rest.
2.1. Exclusion criteria Women with diabetes mellitus, multiple pregnancies, any medical or surgical disorder complicating pregnancy, a history of blood or blood product transfusion in the last 6 months, a history of trauma or surgery and coagulation disorders were not recruited in the study. 2.2. Subjects Informed consent was obtained from all subjects before their inclusion in the study. A detailed history was taken. Clinical examination including blood pressure and investigations including haemoglobin, urine for protein and glucose, VDRL, screening test for glucose, Rh typing were done on all subjects. On all those subjects who developed pre-eclampsia, complete work up for hypertensive disorder of pregnancy including haemogram with platelets, renal function tests, 24 h urine protein estimation, coagulogram and collagen profile were carried out. After a detailed history and clinical examination 10 ml of venous blood was collected in a vial containing 1 ml of sodium citrate. Plasma was separated by centrifugation at 1500 g for 15 min at room temperature and stored in small aliquots at y708C. 2.3. Fibronectin estimation and extraction All the samples were processed at the same
S.S. Sud et al. r International Journal of Gynecology & Obstetrics 66 (1999) 123]128
125
Table 1 Comparison of fibronectin levels at different periods of gestation along with the statistical significance in the two groups
Pre-eclamptic women Ž N s 14. Normotensive women Ž N s 82. Statistical significance by Mann]Whitney test
1st sample - 20 weeks ŽMean " SEM.
2nd sample around 28 weeks ŽMean " SEM.
3rd sample ) 36 weeks ŽMean " SEM.
103.50" 21.29 40.95" 9.08 P- 0.01
772.80" 436.46 54.76" 6.82 P- 0.001
1843.57" 572.83 127.58" 19.11 P- 0.001
time and the fibronectin was estimated by ELISA. ELISA was performed by double antibody sandwich technique, using orthophenylene diamine as substrate w7x. Absorbance was read at 492 nm and plotted against standard fibronectin concentration as mgrml for fibronectin concentrations obtained from a standard curve w11x.
3. Statistical analysis The distribution of results was found to be non-normal on applying x 2 test for fitness Ž P) 0.05 .. Therefore, non-parametric test, i.e. Mann]Whitney w4x was applied on all samples of two groups and their statistical significance was obtained.
4. Results Out of the 100 subjects, 14 developed preeclampsia, i.e. 14%, and were managed per management protocol of Nehru Hospital, Postgraduate Institute of Medical Education and Research, Chandigarh. Mean age of pre-eclamptic subjects
was 23.14 years and 24.33 years in women who remained normotensive, which is comparable. In four subjects out of the normotensive group, one or the other value of plasma fibronectin was very erratic and these values were not considered when doing statistical analysis. The mean plasma fibronectin levels were higher in the pre-eclamptic group than the normotensive subjects in the first sample, i.e. - 20 weeks Ž P0.01.. Similarly mean plasma fibronectin levels were higher in the pre-eclamptic than the normotensive group in the second sample, i.e. around 28 weeks as well as the third sample, i.e. G 36 weeks Ž P- 0.001. ŽTable 1.. The same observations are shown as a bar diagram in Fig. 1. Tables 2 and 3 show serial levels of fibronectin in the pre-eclamptic group at - 20 weeks, around 28 weeks and after 36 weeks and normotensive group, respectively. For the purpose of statistical analysis by a Mann]Witney test in this group, all samples of - 20 weeks were labelled as subgroup a1, all samples around 28 weeks were labelled as subgroup a2, all samples atror after 36 weeks were labelled as subgroup a3 in the pre-eclampsia group and similarly b1, b2 and b3 in the normotensive group for the periods mentioned before. There was a statistically significant differ-
Table 2 Serial estimation of plasma fibronectin levels in pre-eclampsia and statistical significance between three samples a
Subgroup Number Mean " SEM a
1st sample - 20 weeks
2nd sample Around 28 weeks
3rd sample ) 36 weeks
a1 14 103.50" 21.29
a2 14 772.80" 436.48
a3 14 1843.57" 572.83
Note. a1 vs. a2, P - 0.001; a1 vs. a3, P- 0.001; and a2 vs. a3, P- 0.001.
126
S.S. Sud et al. r International Journal of Gynecology & Obstetrics 66 (1999) 123]128
Fig. 1. Mean fibronectin concentration in three samples of pre-eclamptic and normotensive subjects.
ence in values obtained in each of the subgroups. This shows that there is a statistically significant increase in levels of plasma fibronectin with increase in gestation but this increase is significantly higher in the pre-eclamptic group as compared to the normotensive group w9x. Fig. 2 shows the graphic presentation of all the 14 subjects developing pre-eclampsia and their respective three fibronectin levels w10x. It was also observed that the mean fibronectin levels even at - 20 weeks in subjects who developed preeclampsia later were significantly higher than those who remained normotensive throughout. Table 4 gives details of the 14 subjects who developed pre-eclampsia with their respective fibronectin levels in their three samples.
5. Discussion Gestational proteinuric hypertension Žpreeclampsia. is a primary cause of perinatal mor-
Fig. 2. Serial fibronectin levels in 14 pre-eclamptic subjects and mean fibronecting levels in three samples of normotensive subjects.
tality and morbidity. Early recognition and treatment will help to reduce this morbidity and mortality as well as prevent complication and therefore availability of simple predictors of the subsequent development of gestational hypertension might allow preventive treatment. A close correlation between increased fibronectin levels and gestational proteinuric hypertension Žpreeclampsia. has been reported by several authors but the sample size is small and most of the studies are cross-sectional studies w2,3,15x. Therefore we conducted a longitudinal study in 100 uncomplicated normotensive primigravidae, recruited before 20 weeks of gestation and followed them up until delivery. Serial plasma fibronectin levels were estimated. It was observed
Table 3 Serial estimation of fibronectin levels in normotensive group with the statistical significance a
Subgroup Number Mean " SEM a
1st sample - 20 weeks
2nd sample Around 28 weeks
3rd sample ) 36 weeks
b1 82 40.95" 9.8529
b2 82 54.76" 6.82
b3 82 127.58" 19.3
Note. On applying the Mann]Whitney test on all values of each subgroup. b1 vs. b2, P- 0.001; b1 vs. b3, P- 0.001; and b2 vs. b3, P - 0.001.
S.S. Sud et al. r International Journal of Gynecology & Obstetrics 66 (1999) 123]128
127
Table 4 Period of gestation at the time of development of pre-eclampsia and respective fibronectin levels at different periods of gestation Subject No.
Period of Gestation at which PIH diagnosed Žweeks.
BP range
3
33q2
14
39q1
15
34q1
17
36
19
39q5
20
36q5
21
36q2
46
37q1
50
36q4
51
36q3
56 61
37q2 36q2
72
36q6
98
36q4
150r90] 150r110 140r90] 150r110 160r100] 140r90 130r96] 140r100 140r100 130r90 164r100] 140r96 130r90] 140r98 140r90] 140r96 130r100] 140r100 150r90] 140r100 140r100 140r106] 140r90 140r100 140r90 160r100] 140r90
U.Alb. 24 hrs protein mgrl
I
II
II
Fibronectin levels Žmgrml. - 20 weeks
Around 28 weeks
) 36 Week
220
1400
6500
46
90
700
qqr900
204
100
204
qr500
208
209
700
qqr680
207
700
3000
qqr360
204
850
1250
qrnot done
39
90
1000
qqr620
22
83
260
qqr450
50
180
2100
qqr540
28
15
200
qqr350 qqr620
24 70
53 6500
300 7000
qr380
70
250
1400
qr840
70
300
1200
qqqr1 g qr480
that there was a statistically significant difference in serial plasma fibronectin in patients who developed pre-eclampsia when compared to those who did not. Thurnau et al. w14x, in a study of nine pre-eclamptic gravid women, nine normotensive gravid women and 10 non-gravid women, observed that the mean plasma fibronectin in pre-eclamptic gravidas was 1687 " 101 mgrml which was significantly higher than that of normotensive gravida 1129 " 94 mgrml. In the present study the values were 1843.57" 572.83 for the pre-eclamptic group and 127.58" 19.11 for the normotensive gravidae, respectively. Fibronectin was assayed by Thurnau et al. w14x by nephelometry and in the present study fibronectin levels were obtained by ELISA. The present study also showed that in 14 sub-
jects who developed pre-eclampsia later, the mean fibronectin levels, even in the first sample, were higher than the mean values at the same period of gestation in normotensive subjects. In fact, the mean firbonectin value in pre-eclamptic subjects before 20 weeks of gestation was 103.50" 21.29 mgrml which was higher than the second sample of normotensive subjects collected at around 28 weeks which was 54.76" 6.82 mgrml and matches more with the third sample of normotensive subjects, i.e. 127.58" 19.11 mgrml. The present study has also shown that in preeclamptic subjects fibronectin levels are elevated prior to 20 weeks of gestation because the underlying pathology of pre-eclampsia might start before 20 weeks. The study also shows that fi-
128
S.S. Sud et al. r International Journal of Gynecology & Obstetrics 66 (1999) 123]128
bronectin levels rise as pregnancy advances both in pre-eclamptic and normotensive subjects, which is in accordance with the observation made by Thuranu et al. w14x. However, the rise in the fibronectin level in pre-eclamptic women is much more than that in normotensive subjects as has been shown by Thuranu et al. w14x. From the present longitudinal study carried out in 100 antenatal subjects, it can be concluded that fibronectin could be used as an early marker of pre-eclampsia. The plasma fibronectin levels rise with the period of gestation but the rise is more marked in pre-eclampsia than in those who remain normotensive. However, to prove its sensitivity and specificity at a particular period of gestation, further studies on a very large number of antenatal women are needed with more frequent sampling for plasma fibronectin concentrations so that a cut off point could be utilized as a marker for pre-eclampsia. This could help in reducing maternal and perinatal morbidity and mortality even further. References w1x Annual Labour Room Statistics, Nehru Hospital, Postgraduate Institute of Medical Education and Research, Chandigarh, 1996. w2x Ballegeer V, Spitz B, Kietevls L, Moreau H, Assche AV, Collen D. Predictive value of increased plasma levels of fibronectin in gestational hypertension. Am J Obstet Gynecol 1989;161:432]436. w3x Chen CK, Lee CN et al. Fibronectin levels in normal pregnancy and preeclampsia. J Formos Med Assoc 1994;93:921]924. w4x Dannier WW. Mann]Whitney test. Biostatistics: a foundation for analysis in the health services. 3rd ed. New York: John Wily and Sons, 1983:388]432. w5x Friedman SA, de Groot CJ, Taylor RM, Golditch BD,
w6x
w7x w8x
w9x
w10x
w11x w12x
w13x w14x
w15x w16x w17x
Roberts JM. Plasma fibronectin as measure of endothelial involvement in preeclampsia and intrauterine growth retardation. Am J Obstet Gynecol 1994;170:838]841. Friedman SA, Schiff E, Emeis JJ, Dekker GA, Sibai BM. Biochemical collaboration of endothelial involvement in severe preeclampsia. Am J Obstet Gynecol 1995;172:202]203. Manu A, Taneja A. Extraction and assay of fibronectin from stored plasma. Ind J Med Res 1991;94:80]82. Noguera S, Sanchez MF, Aycela Barahana T, Arredondo Sober On F, Morgan MA. Observations on preeclampsia]eclampsia and advances in the evaluation of some laboratory tests. Ginecol Obstet Mex 1997;65:300]304. Paternoster DM, Stella A, Simioni S, Trovo S, Plebani P, Girolami A. Clotting inhibitors and fibronectin as potential markers in preeclampsia. Int J Gynecol Obstet 1995;47:263]268. Paternoster DM, Stella A, Simoni P, Girolami A, Plebani M. Fibronectin and antithrombin as markers of preeclampsia in pregnancy. Eur J Obstet Gynecol Reprod Biol 1996;70:33]39. Roberts JM, Taylor RM, Musci TJ, Rodger GM, Mubel RA, McLayhlin MK. Preeclampsia } an endothelial cell disorder. Am J Obstet Gynecol 1989;161:120]124. Shaarawy M, Didy HE. Thrombomodulin plasminogen activator inhibitor Type I and fibronectin as biomarkers of endothelial damage in preeclampsia and eclampsia. Int J Gynecol Obstet 1996;55:135]139. Speroff L, Dorfman GS. Prostaglandins and pregnancy hypertension. Clin Obstet Gynecol 1977;4:635]637. Thurnau GR, Morgan MA, Brubaker D, Romine M, Blackshaw C, Fishburne Jr. Plasma fibronectin levels in normal pregnancy and preeclampsia: a preliminary report. Int J Gynecol Obstet 1987;25:441]444. Yasumizu T, Koto J. Clinical evaluation of plasma fibronectin levels as biomarker of preeclampsia. J Obstet Gynecol Res 1996;22:221]227. Zang Z, Cheng W, Duan T. Investigation on causes of pregnancy induced hypertension. Chung-Hua Fu Ch’an Ho Tsa Chinh 1996;31:458]460. Zeeman GG, Dekker GA, Herman P, Van Geijn, Kraayenbrink AA. Endothelial function in a normal and preeclamptic pregnancy: a hypothesis. Eur J Obstet Gynecol Reprod Biol 1992;43:113]122.
Article
Serial plasma fibronectin levels in pre-eclamptic and normotensive women S.S. Suda , I. Guptaa,U , L.K. Dhaliwal a , B. Kaur b, N.K. Ganguly b a
Department of Obstetrics and Gynaecology, Postgraduate Institute of Medical Education and Research, Chandigarh, India b Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Received 9 November 1998; received in revised form 12 April 1999; accepted 14 April 1999
Abstract Objecti¨ e: Endothelial cell damage has been put forward as an underlying factor for development of pre-eclampsia. This study was carried out to see if fibronectin, which is a marker of endothelial damage, could be used as a marker of pre-eclampsia. Methods: A longitudinal study was conducted on 100 normotensive primigravidae registered before 20 weeks of gestation. These subjects were followed until delivery and three blood samples were collected, first at registration, i.e. before 20 weeks, second around 28 weeks and third at 36 weeks or later till delivery. Fibronectin levels were assayed by ELISA and women observed for any signs of pre-eclampsia. Results: Fourteen subjects developed pre-eclampsia. Fibronectin levels were observed to rise as pregnancy advanced but the rise was significantly higher in subjects who developed pre-eclampsia. The fibronectin levels were also significantly higher in these 14 subjects even in the first sample, i.e. before 20 weeks of gestation when compared with normotensive subjects Ž P- 0.01.. Conclusions: Fibronectin levels could be used as an early valuable biomarker for the development of pre-eclampsia. Q 1999 International Federation of Gynecology and Obstetrics. Keywords: Plasma fibronectin; Pre-eclamptic; Normotensive
1. Introduction Hypertensive disorder is one of the commonest disorders complicating pregnancy and is a major U
Corresponding author. Tel.: q91-172-541032-39r421; fax: q91-172-540401.
cause of maternal and perinatal morbidity and mortality. In India the incidence of pre-eclampsia in referral institutes like the Postgraduate Institute of Medical Education and Research, Chandigarh is as high as 17.76% w1x. The exact aetiopathogenesis of pre-eclampsia has not been defined as yet. During the past decade, there have been significant advances to
0020-7292r99r$20.00 Q 1999 International Federation of Gynecology and Obstetrics. PII: S 0 0 2 0 - 7 2 9 2 Ž 9 9 . 0 0 0 7 2 - 7
124
S.S. Sud et al. r International Journal of Gynecology & Obstetrics 66 (1999) 123]128
suggest that pre-eclampsia is a complex clinical syndrome potentially involving all organ systems and hypertension is one of those manifestations. Recently, it has been shown that endothelial cell damage and altered mechanism of calcium magnesium balance contribute to the pathophysiology of pre-eclampsia w12,13,17x. It is postulated that the pre-eclampsia syndrome is a vasospastic disorder and probably has a placental origin. The changes in placental blood flow are related to stages of trophoblastic invasion of the spiral arteries during 14]20 weeks. When the trophoblastic invasion is over, the spiral arteries become a high resistance system. The defect observed in pre-eclampsia is lack of invasion of trophoblast to maternal arteries. The diminished placental perfusion causes endothelial damage w8x. Fibronectins are large adhesive glycoproteins Ž4]40 kDa. present in many tissues and in most body fluids and have been found to be a marker of endothelial damage w2x. Increased levels of fibronectin in plasma have been observed in established gestational proteinuric hypertension w3,14x. Fibronectin has been established as a better marker of endothelial damage than thrombomodulin, plasminogen activator inhibitor Type 1, angiotensinogen and Von-Williebrand factor w5,12,16x. It has been seen that fibronectin levels rise before signs and symptoms of pre-eclampsia develop w2,3,15x. Hence, it is established that endothelial damage occurs in pre-eclampsia and studies have been carried out to see if fibronectin levels are different in normotensive as compared to preeclamptic subjects w5,6x. Therefore a longitudinal study was planned to assess fibronectin levels in pre-eclampsia and normotensive subjects.
2. Subjects and method The study was conducted on 100 normotensive primigravidae recruited before 20 weeks of gestation from the antenatal clinic of the Department of Obstetrics and Gynaecology, Nehru Hospital, P.G.I., Chandigarh and were followed up until delivery. The first blood sample was collected at the first
visit before 20 weeks of gestation provided the subject was normotensive. Subsequently blood samples were collected around the 28th week and from 36 weeks until delivery. The subjects were followed up until delivery. The following criteria were used to diagnose a woman as having pre-eclampsia. 1. Blood pressure ) 140r90 mmHg on more than two occasions 6 h apart. 2. Proteinuria ) 0.3 gmrl in 24 h urine or traces to q1 by uristix Ži.e. G 300 mgrl.. 3. Pedal edema 1 q after 12 h of rest.
2.1. Exclusion criteria Women with diabetes mellitus, multiple pregnancies, any medical or surgical disorder complicating pregnancy, a history of blood or blood product transfusion in the last 6 months, a history of trauma or surgery and coagulation disorders were not recruited in the study. 2.2. Subjects Informed consent was obtained from all subjects before their inclusion in the study. A detailed history was taken. Clinical examination including blood pressure and investigations including haemoglobin, urine for protein and glucose, VDRL, screening test for glucose, Rh typing were done on all subjects. On all those subjects who developed pre-eclampsia, complete work up for hypertensive disorder of pregnancy including haemogram with platelets, renal function tests, 24 h urine protein estimation, coagulogram and collagen profile were carried out. After a detailed history and clinical examination 10 ml of venous blood was collected in a vial containing 1 ml of sodium citrate. Plasma was separated by centrifugation at 1500 g for 15 min at room temperature and stored in small aliquots at y708C. 2.3. Fibronectin estimation and extraction All the samples were processed at the same
S.S. Sud et al. r International Journal of Gynecology & Obstetrics 66 (1999) 123]128
125
Table 1 Comparison of fibronectin levels at different periods of gestation along with the statistical significance in the two groups
Pre-eclamptic women Ž N s 14. Normotensive women Ž N s 82. Statistical significance by Mann]Whitney test
1st sample - 20 weeks ŽMean " SEM.
2nd sample around 28 weeks ŽMean " SEM.
3rd sample ) 36 weeks ŽMean " SEM.
103.50" 21.29 40.95" 9.08 P- 0.01
772.80" 436.46 54.76" 6.82 P- 0.001
1843.57" 572.83 127.58" 19.11 P- 0.001
time and the fibronectin was estimated by ELISA. ELISA was performed by double antibody sandwich technique, using orthophenylene diamine as substrate w7x. Absorbance was read at 492 nm and plotted against standard fibronectin concentration as mgrml for fibronectin concentrations obtained from a standard curve w11x.
3. Statistical analysis The distribution of results was found to be non-normal on applying x 2 test for fitness Ž P) 0.05 .. Therefore, non-parametric test, i.e. Mann]Whitney w4x was applied on all samples of two groups and their statistical significance was obtained.
4. Results Out of the 100 subjects, 14 developed preeclampsia, i.e. 14%, and were managed per management protocol of Nehru Hospital, Postgraduate Institute of Medical Education and Research, Chandigarh. Mean age of pre-eclamptic subjects
was 23.14 years and 24.33 years in women who remained normotensive, which is comparable. In four subjects out of the normotensive group, one or the other value of plasma fibronectin was very erratic and these values were not considered when doing statistical analysis. The mean plasma fibronectin levels were higher in the pre-eclamptic group than the normotensive subjects in the first sample, i.e. - 20 weeks Ž P0.01.. Similarly mean plasma fibronectin levels were higher in the pre-eclamptic than the normotensive group in the second sample, i.e. around 28 weeks as well as the third sample, i.e. G 36 weeks Ž P- 0.001. ŽTable 1.. The same observations are shown as a bar diagram in Fig. 1. Tables 2 and 3 show serial levels of fibronectin in the pre-eclamptic group at - 20 weeks, around 28 weeks and after 36 weeks and normotensive group, respectively. For the purpose of statistical analysis by a Mann]Witney test in this group, all samples of - 20 weeks were labelled as subgroup a1, all samples around 28 weeks were labelled as subgroup a2, all samples atror after 36 weeks were labelled as subgroup a3 in the pre-eclampsia group and similarly b1, b2 and b3 in the normotensive group for the periods mentioned before. There was a statistically significant differ-
Table 2 Serial estimation of plasma fibronectin levels in pre-eclampsia and statistical significance between three samples a
Subgroup Number Mean " SEM a
1st sample - 20 weeks
2nd sample Around 28 weeks
3rd sample ) 36 weeks
a1 14 103.50" 21.29
a2 14 772.80" 436.48
a3 14 1843.57" 572.83
Note. a1 vs. a2, P - 0.001; a1 vs. a3, P- 0.001; and a2 vs. a3, P- 0.001.
126
S.S. Sud et al. r International Journal of Gynecology & Obstetrics 66 (1999) 123]128
Fig. 1. Mean fibronectin concentration in three samples of pre-eclamptic and normotensive subjects.
ence in values obtained in each of the subgroups. This shows that there is a statistically significant increase in levels of plasma fibronectin with increase in gestation but this increase is significantly higher in the pre-eclamptic group as compared to the normotensive group w9x. Fig. 2 shows the graphic presentation of all the 14 subjects developing pre-eclampsia and their respective three fibronectin levels w10x. It was also observed that the mean fibronectin levels even at - 20 weeks in subjects who developed preeclampsia later were significantly higher than those who remained normotensive throughout. Table 4 gives details of the 14 subjects who developed pre-eclampsia with their respective fibronectin levels in their three samples.
5. Discussion Gestational proteinuric hypertension Žpreeclampsia. is a primary cause of perinatal mor-
Fig. 2. Serial fibronectin levels in 14 pre-eclamptic subjects and mean fibronecting levels in three samples of normotensive subjects.
tality and morbidity. Early recognition and treatment will help to reduce this morbidity and mortality as well as prevent complication and therefore availability of simple predictors of the subsequent development of gestational hypertension might allow preventive treatment. A close correlation between increased fibronectin levels and gestational proteinuric hypertension Žpreeclampsia. has been reported by several authors but the sample size is small and most of the studies are cross-sectional studies w2,3,15x. Therefore we conducted a longitudinal study in 100 uncomplicated normotensive primigravidae, recruited before 20 weeks of gestation and followed them up until delivery. Serial plasma fibronectin levels were estimated. It was observed
Table 3 Serial estimation of fibronectin levels in normotensive group with the statistical significance a
Subgroup Number Mean " SEM a
1st sample - 20 weeks
2nd sample Around 28 weeks
3rd sample ) 36 weeks
b1 82 40.95" 9.8529
b2 82 54.76" 6.82
b3 82 127.58" 19.3
Note. On applying the Mann]Whitney test on all values of each subgroup. b1 vs. b2, P- 0.001; b1 vs. b3, P- 0.001; and b2 vs. b3, P - 0.001.
S.S. Sud et al. r International Journal of Gynecology & Obstetrics 66 (1999) 123]128
127
Table 4 Period of gestation at the time of development of pre-eclampsia and respective fibronectin levels at different periods of gestation Subject No.
Period of Gestation at which PIH diagnosed Žweeks.
BP range
3
33q2
14
39q1
15
34q1
17
36
19
39q5
20
36q5
21
36q2
46
37q1
50
36q4
51
36q3
56 61
37q2 36q2
72
36q6
98
36q4
150r90] 150r110 140r90] 150r110 160r100] 140r90 130r96] 140r100 140r100 130r90 164r100] 140r96 130r90] 140r98 140r90] 140r96 130r100] 140r100 150r90] 140r100 140r100 140r106] 140r90 140r100 140r90 160r100] 140r90
U.Alb. 24 hrs protein mgrl
I
II
II
Fibronectin levels Žmgrml. - 20 weeks
Around 28 weeks
) 36 Week
220
1400
6500
46
90
700
qqr900
204
100
204
qr500
208
209
700
qqr680
207
700
3000
qqr360
204
850
1250
qrnot done
39
90
1000
qqr620
22
83
260
qqr450
50
180
2100
qqr540
28
15
200
qqr350 qqr620
24 70
53 6500
300 7000
qr380
70
250
1400
qr840
70
300
1200
qqqr1 g qr480
that there was a statistically significant difference in serial plasma fibronectin in patients who developed pre-eclampsia when compared to those who did not. Thurnau et al. w14x, in a study of nine pre-eclamptic gravid women, nine normotensive gravid women and 10 non-gravid women, observed that the mean plasma fibronectin in pre-eclamptic gravidas was 1687 " 101 mgrml which was significantly higher than that of normotensive gravida 1129 " 94 mgrml. In the present study the values were 1843.57" 572.83 for the pre-eclamptic group and 127.58" 19.11 for the normotensive gravidae, respectively. Fibronectin was assayed by Thurnau et al. w14x by nephelometry and in the present study fibronectin levels were obtained by ELISA. The present study also showed that in 14 sub-
jects who developed pre-eclampsia later, the mean fibronectin levels, even in the first sample, were higher than the mean values at the same period of gestation in normotensive subjects. In fact, the mean firbonectin value in pre-eclamptic subjects before 20 weeks of gestation was 103.50" 21.29 mgrml which was higher than the second sample of normotensive subjects collected at around 28 weeks which was 54.76" 6.82 mgrml and matches more with the third sample of normotensive subjects, i.e. 127.58" 19.11 mgrml. The present study has also shown that in preeclamptic subjects fibronectin levels are elevated prior to 20 weeks of gestation because the underlying pathology of pre-eclampsia might start before 20 weeks. The study also shows that fi-
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bronectin levels rise as pregnancy advances both in pre-eclamptic and normotensive subjects, which is in accordance with the observation made by Thuranu et al. w14x. However, the rise in the fibronectin level in pre-eclamptic women is much more than that in normotensive subjects as has been shown by Thuranu et al. w14x. From the present longitudinal study carried out in 100 antenatal subjects, it can be concluded that fibronectin could be used as an early marker of pre-eclampsia. The plasma fibronectin levels rise with the period of gestation but the rise is more marked in pre-eclampsia than in those who remain normotensive. However, to prove its sensitivity and specificity at a particular period of gestation, further studies on a very large number of antenatal women are needed with more frequent sampling for plasma fibronectin concentrations so that a cut off point could be utilized as a marker for pre-eclampsia. This could help in reducing maternal and perinatal morbidity and mortality even further. References w1x Annual Labour Room Statistics, Nehru Hospital, Postgraduate Institute of Medical Education and Research, Chandigarh, 1996. w2x Ballegeer V, Spitz B, Kietevls L, Moreau H, Assche AV, Collen D. Predictive value of increased plasma levels of fibronectin in gestational hypertension. Am J Obstet Gynecol 1989;161:432]436. w3x Chen CK, Lee CN et al. Fibronectin levels in normal pregnancy and preeclampsia. J Formos Med Assoc 1994;93:921]924. w4x Dannier WW. Mann]Whitney test. Biostatistics: a foundation for analysis in the health services. 3rd ed. New York: John Wily and Sons, 1983:388]432. w5x Friedman SA, de Groot CJ, Taylor RM, Golditch BD,
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Roberts JM. Plasma fibronectin as measure of endothelial involvement in preeclampsia and intrauterine growth retardation. Am J Obstet Gynecol 1994;170:838]841. Friedman SA, Schiff E, Emeis JJ, Dekker GA, Sibai BM. Biochemical collaboration of endothelial involvement in severe preeclampsia. Am J Obstet Gynecol 1995;172:202]203. Manu A, Taneja A. Extraction and assay of fibronectin from stored plasma. Ind J Med Res 1991;94:80]82. Noguera S, Sanchez MF, Aycela Barahana T, Arredondo Sober On F, Morgan MA. Observations on preeclampsia]eclampsia and advances in the evaluation of some laboratory tests. Ginecol Obstet Mex 1997;65:300]304. Paternoster DM, Stella A, Simioni S, Trovo S, Plebani P, Girolami A. Clotting inhibitors and fibronectin as potential markers in preeclampsia. Int J Gynecol Obstet 1995;47:263]268. Paternoster DM, Stella A, Simoni P, Girolami A, Plebani M. Fibronectin and antithrombin as markers of preeclampsia in pregnancy. Eur J Obstet Gynecol Reprod Biol 1996;70:33]39. Roberts JM, Taylor RM, Musci TJ, Rodger GM, Mubel RA, McLayhlin MK. Preeclampsia } an endothelial cell disorder. Am J Obstet Gynecol 1989;161:120]124. Shaarawy M, Didy HE. Thrombomodulin plasminogen activator inhibitor Type I and fibronectin as biomarkers of endothelial damage in preeclampsia and eclampsia. Int J Gynecol Obstet 1996;55:135]139. Speroff L, Dorfman GS. Prostaglandins and pregnancy hypertension. Clin Obstet Gynecol 1977;4:635]637. Thurnau GR, Morgan MA, Brubaker D, Romine M, Blackshaw C, Fishburne Jr. Plasma fibronectin levels in normal pregnancy and preeclampsia: a preliminary report. Int J Gynecol Obstet 1987;25:441]444. Yasumizu T, Koto J. Clinical evaluation of plasma fibronectin levels as biomarker of preeclampsia. J Obstet Gynecol Res 1996;22:221]227. Zang Z, Cheng W, Duan T. Investigation on causes of pregnancy induced hypertension. Chung-Hua Fu Ch’an Ho Tsa Chinh 1996;31:458]460. Zeeman GG, Dekker GA, Herman P, Van Geijn, Kraayenbrink AA. Endothelial function in a normal and preeclamptic pregnancy: a hypothesis. Eur J Obstet Gynecol Reprod Biol 1992;43:113]122.