doi:10.1111/jog.12650
J. Obstet. Gynaecol. Res. Vol. 41, No. 6: 870–875, June 2015
Amniotic components in the uterine vasculature and their role in amniotic fluid embolism Hiroko Nakagami1, Takeshi Kajihara1, Yoshimasa Kamei1, Osamu Ishihara1, Hidekazu Kayano2, Atsushi Sasaki2 and Atsuo Itakura3 Departments of 1Obstetrics and Gynecology and 2Pathology, Saitama Medical University, Saitama and 3Department of Obstetrics and Gynecology, Juntendo University, Tokyo, Japan
Abstract Aim: To evaluate whether the presence of amniotic components in the maternal uterine vasculature could be a specific pathological indicator for amniotic fluid embolism (AFE). Methods: Medical records of patients treated between January 2006 and March 2013 were retrospectively examined to identify patients who underwent post-partum hysterectomy or autopsy due to maternal death. Three subjects with AFE with disseminated intravascular coagulation (DIC)-type post-partum hemorrhage (PPH), and 13 non-AFE subjects were included in analysis. Histochemical staining using hematoxylin–eosin (HE) and alcian blue, and immunohistochemical staining for sialyl-Tn were conducted to detect amniotic components in the maternal uterine vasculature. Results: Alcian blue was positive for amniotic components in the uterine vasculature of all subjects with AFE and of several subjects without AFE. Similarly, HE and sialyl-Tn were negative in some AFE subjects and positive in some non-AFE subjects. Conclusions: The presence of maternal intravascular fetal material is not a specific indicator for AFE with DIC-type PPH. Therefore, the presence of fetal components in the uterine vasculature is unlikely to be a definitive indicator for AFE. Key words: alcian blue, amniotic fluid embolism, disseminated intravascular coagulation, post-partum hysterectomy.
Introduction Amniotic fluid embolism (AFE) is a rare but potentially fatal maternal obstetric complication accompanied by acute circulatory failure and disseminated intravascular coagulation (DIC). The estimated incidence of AFE is between 1 in 8000 and 1 in 80 000 births, and the maternal mortality rate is extremely high.1 AFE can occur when fetal components of amniotic fluid enter the maternal circulation. AFE was previously believed to be the result of the physical obstruction of the maternal pulmonary circulation by amniotic fluid
components, but increasing evidence has indicated that physical obstruction of the pulmonary vasculature in AFE may not be the result of a simple embolism.1,2 Benson proposed that the pathophysiology of AFE involves a maternal immune response to fetal material within the amniotic fluid that enters the maternal circulation.2,3 Clinical diagnosis of AFE uses exclusion criteria, and autopsy can confirm diagnosis by demonstration of the presence of fetal squamous cells, debris, mucin, meconium, or lanugo in the maternal pulmonary vasculature.4 Antemortem diagnosis of AFE is very difficult in
Received: July 7 2014. Accepted: October 23 2014. Reprint request to: Dr Takeshi Kajihara, Department of Obstetrics and Gynecology, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 3500495, Japan. Email: [email protected]
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© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Amniotic components and AFE
the clinical environment. Laboratory test results may be helpful in some cases, but they will not provide a definitive diagnosis. Courtney proposed that amniotic fluid might become trapped in the uterine veins during labor contractions.5 It remains unclear, however, whether the presence of amniotic components in the maternal uterine vasculature is a pathological indicator of AFE. The presence of amniotic components can be identified on conventional hematoxylin–eosin (HE) staining, but the use of histochemical and immunohistochemical staining techniques provides a more reliable assessment of the histological diagnosis of AFE. Therefore, we investigated the presence and distribution of amniotic components in the maternal uterine vasculature using HE staining, alcian blue (Alc) staining, and antisialyl-Tn (STN) staining in patients who underwent post-partum hysterectomy or autopsy due to maternal death.
Methods We examined the medical records of patients who were treated at Saitama Medical University Hospital, Saitama, Japan, between January 2006 and March 2013, to identify all patients who underwent post-partum hysterectomy or autopsy due to maternal death. Subjects included patients who were cared for at the hospital throughout their gestation and patients whose care was transferred to the hospital from local clinics or hospitals. AFE diagnosis was made on the basis of the diagnostic criteria proposed by the Japanese criteria of AFE.6 The enrollment criteria were as follows: (i) currently pregnant or within 12 h of delivery; (ii) one or more of the following conditions sufficiently severe to require medical treatment: (a) cardiac arrest, (b) unknown cause of massive bleeding within 2 h of delivery (≥1500 mL), (c) DIC, (d) respiratory distress; and (iii) the absence of other medical explanations for clinical course. The details of each subject’s obstetric history, maternal background, course of delivery, subsequent complications, and management were noted. This study was reviewed and approved by the institutional review board of the Saitama Medical University. All stored pathological samples from the uterine wall used in this study were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5 μm serial sections for HE, Alc, and STN staining. We used Alc solution (Muto Pure Chemicals, Tokyo, Japan) for Alc staining. The number of histological blocks examined for each subject varied from 3 to 11.
The immunohistochemical staining for STN was conducted as described previously.7,8 In brief, deparaffinized sections were sequentially treated with methanol containing 0.3% H2O2 for 30 min and 10% normal goat serum for 10 min at room temperature to block non-specific immunoreactive sites. Sections were then incubated with STN (1:100; Cell Signaling Technology, Beverly, MA, USA) at room temperature for 2 h. After incubation of the sections with the primary antiserum, the labeled streptavidin–biotin method was used, with the Histofine SAB-PO(R) Kit (Nichirei, Tokyo, Japan). The peroxidase reaction was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB)/H2O2 (0.02% DAB and 0.005% H2O2 in 0.05 M Tris buffer, pH 7.6) for 5 min, and the sections were counterstained with Mayer’s hematoxylin. As a negative control, some sections were incubated with normal rabbit serum instead of the specific antiserum. Two board-certified pathologists (HK and AS), who were blinded to the clinical details of each subject, independently evaluated each specimen for the presence or absence of fetal components following HE, Alc, and STN staining. In the present study, histological evaluation was focused on identifying amniotic or fetal components within more than one vessel in the preserved sections of the uterine wall.
Results Sixteen cases were identified using the present search strategy, and the medical records of each case were examined for possible inclusion in the study. Among the cases included, three met the diagnostic criteria for AFE. Table 1 lists subject clinical features. Table 2 lists the histological examination findings of the two pathologists. In the AFE group, positive HE staining was infrequent (1/3 and 0/3 subjects), whereas in the non-AFE group, HE was positive in 5/13 and 2/13 subjects, respectively (Fig. 1a,b). In the AFE group, Alc was positive in 3/3 and 3/3 subjects, whereas in the non-AFE group, it was positive in 7/13 and 7/13 subjects (Fig. 1c,d). In the AFE group, STN was positive for 1/3 and 0/3 subjects, while in the non-AFE group, it was positive in 2/13 and 2/13 subjects (Fig. 1e,f).
Discussion This study has shown that the presence of amniotic components in the uterine vasculature can be histologically identified in the specimens of subjects both with
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
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H. Nakagami et al.
Table 1 Clinical subject characteristics Patient
Age (years)
Parity
Gestational period (weeks)
Delivery mode
Hysterectomy type
Clinical diagnosis
Maternal outcome
39
0
32
C/S
NA
Dead
2
28
3
38
C/S
Total hysterectomy
3
39
1
37
C/S
Supra-hysterectomy
AFE, NRFS, adenomyosis, DIC-type PPH AFE, placenta accreta, DIC-type PPH AFE, DIC-type PPH
Non-AFE group 4 38 5 34 6 36 7 31 8 28 9 33
0 1 0 0 2 1
39 36 33 36 36 22
C/S C/S C/S NVD C/S NVD
Supra-hysterectomy Total hysterectomy Total hysterectomy NA Total hysterectomy Supra-hysterectomy
AFE group 1
10 11 12 13 14
33 34 37 36 36
2 1 2 2 1
36 40 36 37 37
C/S C/S C/S C/S C/S
Total hysterectomy Total hysterectomy Total hysterectomy Total hysterectomy Total hysterectomy
15 16
34 40
3 0
37 39
C/S NVD
Total hysterectomy Supra-hysterectomy
NRFS, atonic bleeding Placenta previa accreta Placenta previa accreta Placental abruption Placenta previa accreta IUFD, placenta accreta, Intrauterine infection Placenta previa accreta Placenta previa accreta Placenta previa accreta Placenta previa accreta Twin pregnancy, atonic bleeding Placenta previa accreta Placenta accreta
Alive Alive Alive Alive Alive Dead Alive Alive Alive Alive Alive Alive Alive Alive Alive
AFE, amniotic fluid embolism; C/S, cesarean section; DIC, disseminated intravascular coagulation; IUFD, intrauterine fetal death; NA, not available; NRFS, non-reassuring fetal status; NVD, normal vaginal delivery; PPH, post-partum hemorrhage.
Table 2 Histological findings Patient HE
Pathologist 1 Alcian blue
STN
HE
Pathologist 2 Alcian blue
STN
AFE group 1 2 3
− − +
+ + +
+ − −
− − −
+ + +
− − −
Non-AFE group 4 5 6 7 8 9 10 11 12 13 14 15 16
− + − − − − + − + + − + −
− − + + − − + − + + − + +
− − − + − − − − − − − + −
− − − − − − + − − − − − +
− − + − − + + − + + − + +
− − − + − − − − − − − − +
AFE, amniotic fluid embolism; HE, hematoxylin–eosin; STN, anti-sialyl-Tn.
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Figure 1 Amniotic components in the uterine vasculature identified using various histochemical methods. (a,c,e) Amniotic fluid embolism (AFE); (b,d,f) nonAFE. (a,b) Fetal components were detected on hematoxylin– eosin (HE) staining in the uterine vasculature in (a) AFE subject 3 and (b) non-AFE subject 5. (c,d) Alcian blue staining was positive in the uterine vasculature of (c) AFE subject 2 and (d) non-AFE subject 15. (e,f) Anti-sialyl-Tn was positive in the uterine vasculature of (e) AFE subject 1 and (f) non-AFE subject 16.
a
b
c
d
e
f
and without AFE. These observations suggest that the presence of fetal material of amniotic fluid in maternal uterine vasculature is not an indicator for AFE but rather a common finding in the post-partum uterine myometrium. Although previous studies have reported that squamous cells and other fetal debris were detected in the pulmonary arteries of pregnant women without AFE,9–11 to our knowledge, this is the first report to demonstrate these findings in the uterine vasculature. The symptoms of AFE were previously believed to be the result of physical obstruction of maternal pulmonary circulation by the components of amniotic fluid, but AFE is now considered to be the result of a maternal immune response to fetal materials that have been introduced into the maternal circulation, which can occur whenever the barrier between the
amniotic fluid and maternal circulation is disrupted.2,3 At present, it is believed that the most common routes for the entry of amniotic fluid into the maternal circulation are endocervical veins,12,13 injured uterine sites,14 and the placental attachment site.15 Given the unpredictable nature and the rarity of AFE, however, its pathophysiology remains poorly understood. The presence of amniotic components can be identified on conventional HE staining, although the use of histochemical and immunohistochemical staining techniques provides a more reliable assessment of the histological diagnosis of AFE.16 In Japan, zinc coproporphyrin 1 (ZnCP-1)17 and STN in the maternal serum have been widely used as diagnostic markers for AFE.18,19 Both markers, however, were negative for all AFE subjects in present study. Kanayama and Tamura
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reported that serum markers of ZnCP-1 and STN are not sensitive for the DIC type of AFE.6 In the present study, all cases of AFE started with atonic bleeding/ DIC. This observation was consistent with the previous report by Kanayama and Tamura.6 ZnCP-1 and STN antigens are considered major characteristic components of meconium and amniotic fluid. In addition, given that immunohistochemical staining for STN has been shown to be a more sensitive method than HE or Alc staining for detecting meconium and amniotic fluid-derived mucin in subjects with AFE,18 we used immunohistochemical staining for STN to identify fetal components in the maternal uterine vasculature in the present study. Numerous cases of atypical AFE with isolated coagulopathy have been reported.20–22 In spite of the presence of amniotic fluid debris in pulmonary arteries, some of these subjects had DIC or uterine atony without accompanying cardiorespiratory symptoms. Post-partum hemorrhage (PPH) complicated by coagulopathy without manifestation of DIC has been reported as DIC-type PPH.23 Corresponding to the widespread clinical diagnosis of AFE on ZnCP-1 and STN staining in Japan, PPH complicated by coagulopathy has been attributed to DIC preceding AFE.24 We showed that Alc staining was positive not only in the uterine vasculature of all AFE subjects but also in some subjects without AFE. Similarly, HE and STN staining suggested the presence of amniotic components in some subjects in the non-AFE group. These observations suggest that the presence of amniotic components in the uterine vasculature is not a predictive finding for AFE; rather, it may be a common finding in various conditions including placenta previa, placenta accreta, and the normal post-partum state. To achieve hemostasis, hysterectomy is often performed for severe PPH complicated by coagulopathy. The present results, however, suggest that histological detection of amniotic components in the uterine vasculature does not provide conclusive findings to assist in AFE diagnosis. The present study had several limitations. First, this study was conducted at a single center, and the small sample size may have inaccurately reflected the prevalence of amniotic components in the maternal uterine vasculature. Second, although Alc was positive in the uterine vasculature of all AFE subjects, the presence of fetal material could not be confirmed on HE or STN staining in any of these AFE subjects. The time interval between the rupture of fetal membranes and subse-
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quent hysterectomy or autopsy varied dramatically between subjects, and therefore it is possible that the fetal components had been eliminated from the uterine vasculature in some AFE subjects before hysterectomy or autopsy was performed.
Conclusion The presence of maternal intravascular fetal components is not a specific finding in AFE with DIC-type PPH. Therefore, the fetal component in the uterine vasculature is unlikely to be a definitive indicator of AFE with DIC-type PPH.
Acknowledgments The authors thank Ms Satomi Uchino-Suzuki for her expert technical assistance.
Disclosure No financial support was received by any of the authors for this study and none of the authors have any relationships with companies that may have a financial interest in the information contained in the manuscript.
References 1. Clark SL, Hankins GD, Dudley DA, Dildy GA, Porter TF. Amniotic fluid embolism: Analysis of the national registry. Am J Obstet Gynecol 1995; 172: 1158–1167. 2. Benson MD. Current concepts of immunology and diagnosis in amniotic fluid embolism. Clin Dev Immunol 2012; 2012: 946576. 3. Benson MD. A hypothesis regarding complement activation and amniotic fluid embolism. Med Hypotheses 2007; 68: 1019– 1025. 4. Lau G. Amniotic fluid embolism as a cause of sudden maternal death. Med Sci Law 1994; 34: 213–220. 5. Courtney LD. Coagulation failure in pregnancy. BMJ 1970; 1: 691. 6. Kanayama N, Tamura N. Amniotic fluid embolism: Pathophysiology and new strategies for management. J Obstet Gynaecol Res 2014; 40: 1507–1517. 7. Kajihara T, Uchino S, Suzuki M, Itakura A, Brosens JJ, Ishihara O. Increased ovarian follicle atresia in obese Zucker rats is associated with enhanced expression of the forkhead transcription factor FOXO1. Med Mol Morphol 2009; 42: 216– 221. 8. Kajihara T, Okagaki R, Ishihara O. LPS-induced transient testicular dysfunction accompanied by apoptosis of testicular germ cells in mice. Med Mol Morphol 2006; 39: 203–208. 9. Clark SL, Pavlova Z, Greenspoon J, Horenstein J, Phelan JP. Squamous cells in the maternal pulmonary circulation. Am J Obstet Gynecol 1986; 154: 104–106.
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10. Lee W, Ginsburg KA, Cotton DB, Kaufman RH. Squamous and trophoblastic cells in the maternal pulmonary circulation identified by invasive hemodynamic monitoring during the peripartum period. Am J Obstet Gynecol 1986; 155: 999–1001. 11. Plauché WC. Amniotic fluid embolism. Am J Obstet Gynecol 1983; 147: 982–983. 12. Cheung AN, Luk SC. The importance of extensive sampling and examination of cervix in suspected cases of amniotic fluid embolism. Arch Gynecol Obstet 1994; 255: 101–105. 13. Bastien JL, Graves JR, Bailey S. Atypical presentation of amniotic fluid embolism. Anesth Analg 1998; 87: 124–126. 14. Thomson AJ, Greer IA. Non-haemorrhagic obstetric shock. Baillieres Best Pract Res Clin Obstet Gynaecol 2000; 14: 19–41. 15. Conde-Agudelo A, Romero R. Amniotic fluid embolism: An evidence-based review. Am J Obstet Gynecol 2009; 201: 445.e1– 445.13. 16. Kobayashi H, Ooi H, Hayakawa H et al. Histological diagnosis of amniotic fluid embolism by monoclonal antibody TKH-2 that recognizes NeuAc alpha 2-6GalNAc epitope. Hum Pathol 1997; 28: 428–433. 17. Furuta N, Yaguchi C, Itoh H et al. Immunohistochemical detection of meconium in the fetal membrane, placenta and umbilical cord. Placenta 2012; 33: 24–30.
18. Kobayashi H, Ohi H, Terao T. A simple, noninvasive, sensitive method for diagnosis of amniotic fluid embolism by monoclonal antibody TKH-2 that recognizes NeuAc alpha 2-6GalNAc. Am J Obstet Gynecol 1993; 168: 848–853. 19. Oi H, Kobayashi H, Hirashima Y, Yamazaki T, Kobayashi T, Terao T. Serological and immunohistochemical diagnosis of amniotic fluid embolism. Semin Thromb Hemost 1998; 24: 479– 484. 20. Awad IT, Shorten GD. Amniotic fluid embolism and isolated coagulopathy: Atypical presentation of amniotic fluid embolism. Eur J Anaesthesiol 2001; 18: 410–413. 21. Levy R, Furman B, Hagay ZJ. Fetal bradycardia and disseminated coagulopathy: Atypical presentation of amniotic fluid emboli. Acta Anaesthesiol Scand 2004; 48: 1214–1215. 22. Yang JI, Kim HS, Chang KH, Ryu HS, Joo HJ. Amniotic fluid embolism with isolated coagulopathy: A case report. J Reprod Med 2006; 51: 64–66. 23. Kobayashi T. Obstetrical disseminated intravascular coagulation score. J Obstet Gynaecol Res 2014; 40: 1500–1506. 24. Kanayama N, Inori J, Ishibashi-Ueda H et al. Maternal death analysis from the Japanese autopsy registry for recent 16 years: Significance of amniotic fluid embolism. J Obstet Gynaecol Res 2011; 37: 58–63.
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J. Obstet. Gynaecol. Res. Vol. 41, No. 6: 870–875, June 2015
Amniotic components in the uterine vasculature and their role in amniotic fluid embolism Hiroko Nakagami1, Takeshi Kajihara1, Yoshimasa Kamei1, Osamu Ishihara1, Hidekazu Kayano2, Atsushi Sasaki2 and Atsuo Itakura3 Departments of 1Obstetrics and Gynecology and 2Pathology, Saitama Medical University, Saitama and 3Department of Obstetrics and Gynecology, Juntendo University, Tokyo, Japan
Abstract Aim: To evaluate whether the presence of amniotic components in the maternal uterine vasculature could be a specific pathological indicator for amniotic fluid embolism (AFE). Methods: Medical records of patients treated between January 2006 and March 2013 were retrospectively examined to identify patients who underwent post-partum hysterectomy or autopsy due to maternal death. Three subjects with AFE with disseminated intravascular coagulation (DIC)-type post-partum hemorrhage (PPH), and 13 non-AFE subjects were included in analysis. Histochemical staining using hematoxylin–eosin (HE) and alcian blue, and immunohistochemical staining for sialyl-Tn were conducted to detect amniotic components in the maternal uterine vasculature. Results: Alcian blue was positive for amniotic components in the uterine vasculature of all subjects with AFE and of several subjects without AFE. Similarly, HE and sialyl-Tn were negative in some AFE subjects and positive in some non-AFE subjects. Conclusions: The presence of maternal intravascular fetal material is not a specific indicator for AFE with DIC-type PPH. Therefore, the presence of fetal components in the uterine vasculature is unlikely to be a definitive indicator for AFE. Key words: alcian blue, amniotic fluid embolism, disseminated intravascular coagulation, post-partum hysterectomy.
Introduction Amniotic fluid embolism (AFE) is a rare but potentially fatal maternal obstetric complication accompanied by acute circulatory failure and disseminated intravascular coagulation (DIC). The estimated incidence of AFE is between 1 in 8000 and 1 in 80 000 births, and the maternal mortality rate is extremely high.1 AFE can occur when fetal components of amniotic fluid enter the maternal circulation. AFE was previously believed to be the result of the physical obstruction of the maternal pulmonary circulation by amniotic fluid
components, but increasing evidence has indicated that physical obstruction of the pulmonary vasculature in AFE may not be the result of a simple embolism.1,2 Benson proposed that the pathophysiology of AFE involves a maternal immune response to fetal material within the amniotic fluid that enters the maternal circulation.2,3 Clinical diagnosis of AFE uses exclusion criteria, and autopsy can confirm diagnosis by demonstration of the presence of fetal squamous cells, debris, mucin, meconium, or lanugo in the maternal pulmonary vasculature.4 Antemortem diagnosis of AFE is very difficult in
Received: July 7 2014. Accepted: October 23 2014. Reprint request to: Dr Takeshi Kajihara, Department of Obstetrics and Gynecology, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 3500495, Japan. Email: [email protected]
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© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Amniotic components and AFE
the clinical environment. Laboratory test results may be helpful in some cases, but they will not provide a definitive diagnosis. Courtney proposed that amniotic fluid might become trapped in the uterine veins during labor contractions.5 It remains unclear, however, whether the presence of amniotic components in the maternal uterine vasculature is a pathological indicator of AFE. The presence of amniotic components can be identified on conventional hematoxylin–eosin (HE) staining, but the use of histochemical and immunohistochemical staining techniques provides a more reliable assessment of the histological diagnosis of AFE. Therefore, we investigated the presence and distribution of amniotic components in the maternal uterine vasculature using HE staining, alcian blue (Alc) staining, and antisialyl-Tn (STN) staining in patients who underwent post-partum hysterectomy or autopsy due to maternal death.
Methods We examined the medical records of patients who were treated at Saitama Medical University Hospital, Saitama, Japan, between January 2006 and March 2013, to identify all patients who underwent post-partum hysterectomy or autopsy due to maternal death. Subjects included patients who were cared for at the hospital throughout their gestation and patients whose care was transferred to the hospital from local clinics or hospitals. AFE diagnosis was made on the basis of the diagnostic criteria proposed by the Japanese criteria of AFE.6 The enrollment criteria were as follows: (i) currently pregnant or within 12 h of delivery; (ii) one or more of the following conditions sufficiently severe to require medical treatment: (a) cardiac arrest, (b) unknown cause of massive bleeding within 2 h of delivery (≥1500 mL), (c) DIC, (d) respiratory distress; and (iii) the absence of other medical explanations for clinical course. The details of each subject’s obstetric history, maternal background, course of delivery, subsequent complications, and management were noted. This study was reviewed and approved by the institutional review board of the Saitama Medical University. All stored pathological samples from the uterine wall used in this study were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5 μm serial sections for HE, Alc, and STN staining. We used Alc solution (Muto Pure Chemicals, Tokyo, Japan) for Alc staining. The number of histological blocks examined for each subject varied from 3 to 11.
The immunohistochemical staining for STN was conducted as described previously.7,8 In brief, deparaffinized sections were sequentially treated with methanol containing 0.3% H2O2 for 30 min and 10% normal goat serum for 10 min at room temperature to block non-specific immunoreactive sites. Sections were then incubated with STN (1:100; Cell Signaling Technology, Beverly, MA, USA) at room temperature for 2 h. After incubation of the sections with the primary antiserum, the labeled streptavidin–biotin method was used, with the Histofine SAB-PO(R) Kit (Nichirei, Tokyo, Japan). The peroxidase reaction was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB)/H2O2 (0.02% DAB and 0.005% H2O2 in 0.05 M Tris buffer, pH 7.6) for 5 min, and the sections were counterstained with Mayer’s hematoxylin. As a negative control, some sections were incubated with normal rabbit serum instead of the specific antiserum. Two board-certified pathologists (HK and AS), who were blinded to the clinical details of each subject, independently evaluated each specimen for the presence or absence of fetal components following HE, Alc, and STN staining. In the present study, histological evaluation was focused on identifying amniotic or fetal components within more than one vessel in the preserved sections of the uterine wall.
Results Sixteen cases were identified using the present search strategy, and the medical records of each case were examined for possible inclusion in the study. Among the cases included, three met the diagnostic criteria for AFE. Table 1 lists subject clinical features. Table 2 lists the histological examination findings of the two pathologists. In the AFE group, positive HE staining was infrequent (1/3 and 0/3 subjects), whereas in the non-AFE group, HE was positive in 5/13 and 2/13 subjects, respectively (Fig. 1a,b). In the AFE group, Alc was positive in 3/3 and 3/3 subjects, whereas in the non-AFE group, it was positive in 7/13 and 7/13 subjects (Fig. 1c,d). In the AFE group, STN was positive for 1/3 and 0/3 subjects, while in the non-AFE group, it was positive in 2/13 and 2/13 subjects (Fig. 1e,f).
Discussion This study has shown that the presence of amniotic components in the uterine vasculature can be histologically identified in the specimens of subjects both with
© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
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Table 1 Clinical subject characteristics Patient
Age (years)
Parity
Gestational period (weeks)
Delivery mode
Hysterectomy type
Clinical diagnosis
Maternal outcome
39
0
32
C/S
NA
Dead
2
28
3
38
C/S
Total hysterectomy
3
39
1
37
C/S
Supra-hysterectomy
AFE, NRFS, adenomyosis, DIC-type PPH AFE, placenta accreta, DIC-type PPH AFE, DIC-type PPH
Non-AFE group 4 38 5 34 6 36 7 31 8 28 9 33
0 1 0 0 2 1
39 36 33 36 36 22
C/S C/S C/S NVD C/S NVD
Supra-hysterectomy Total hysterectomy Total hysterectomy NA Total hysterectomy Supra-hysterectomy
AFE group 1
10 11 12 13 14
33 34 37 36 36
2 1 2 2 1
36 40 36 37 37
C/S C/S C/S C/S C/S
Total hysterectomy Total hysterectomy Total hysterectomy Total hysterectomy Total hysterectomy
15 16
34 40
3 0
37 39
C/S NVD
Total hysterectomy Supra-hysterectomy
NRFS, atonic bleeding Placenta previa accreta Placenta previa accreta Placental abruption Placenta previa accreta IUFD, placenta accreta, Intrauterine infection Placenta previa accreta Placenta previa accreta Placenta previa accreta Placenta previa accreta Twin pregnancy, atonic bleeding Placenta previa accreta Placenta accreta
Alive Alive Alive Alive Alive Dead Alive Alive Alive Alive Alive Alive Alive Alive Alive
AFE, amniotic fluid embolism; C/S, cesarean section; DIC, disseminated intravascular coagulation; IUFD, intrauterine fetal death; NA, not available; NRFS, non-reassuring fetal status; NVD, normal vaginal delivery; PPH, post-partum hemorrhage.
Table 2 Histological findings Patient HE
Pathologist 1 Alcian blue
STN
HE
Pathologist 2 Alcian blue
STN
AFE group 1 2 3
− − +
+ + +
+ − −
− − −
+ + +
− − −
Non-AFE group 4 5 6 7 8 9 10 11 12 13 14 15 16
− + − − − − + − + + − + −
− − + + − − + − + + − + +
− − − + − − − − − − − + −
− − − − − − + − − − − − +
− − + − − + + − + + − + +
− − − + − − − − − − − − +
AFE, amniotic fluid embolism; HE, hematoxylin–eosin; STN, anti-sialyl-Tn.
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© 2014 The Authors Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Amniotic components and AFE
Figure 1 Amniotic components in the uterine vasculature identified using various histochemical methods. (a,c,e) Amniotic fluid embolism (AFE); (b,d,f) nonAFE. (a,b) Fetal components were detected on hematoxylin– eosin (HE) staining in the uterine vasculature in (a) AFE subject 3 and (b) non-AFE subject 5. (c,d) Alcian blue staining was positive in the uterine vasculature of (c) AFE subject 2 and (d) non-AFE subject 15. (e,f) Anti-sialyl-Tn was positive in the uterine vasculature of (e) AFE subject 1 and (f) non-AFE subject 16.
a
b
c
d
e
f
and without AFE. These observations suggest that the presence of fetal material of amniotic fluid in maternal uterine vasculature is not an indicator for AFE but rather a common finding in the post-partum uterine myometrium. Although previous studies have reported that squamous cells and other fetal debris were detected in the pulmonary arteries of pregnant women without AFE,9–11 to our knowledge, this is the first report to demonstrate these findings in the uterine vasculature. The symptoms of AFE were previously believed to be the result of physical obstruction of maternal pulmonary circulation by the components of amniotic fluid, but AFE is now considered to be the result of a maternal immune response to fetal materials that have been introduced into the maternal circulation, which can occur whenever the barrier between the
amniotic fluid and maternal circulation is disrupted.2,3 At present, it is believed that the most common routes for the entry of amniotic fluid into the maternal circulation are endocervical veins,12,13 injured uterine sites,14 and the placental attachment site.15 Given the unpredictable nature and the rarity of AFE, however, its pathophysiology remains poorly understood. The presence of amniotic components can be identified on conventional HE staining, although the use of histochemical and immunohistochemical staining techniques provides a more reliable assessment of the histological diagnosis of AFE.16 In Japan, zinc coproporphyrin 1 (ZnCP-1)17 and STN in the maternal serum have been widely used as diagnostic markers for AFE.18,19 Both markers, however, were negative for all AFE subjects in present study. Kanayama and Tamura
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reported that serum markers of ZnCP-1 and STN are not sensitive for the DIC type of AFE.6 In the present study, all cases of AFE started with atonic bleeding/ DIC. This observation was consistent with the previous report by Kanayama and Tamura.6 ZnCP-1 and STN antigens are considered major characteristic components of meconium and amniotic fluid. In addition, given that immunohistochemical staining for STN has been shown to be a more sensitive method than HE or Alc staining for detecting meconium and amniotic fluid-derived mucin in subjects with AFE,18 we used immunohistochemical staining for STN to identify fetal components in the maternal uterine vasculature in the present study. Numerous cases of atypical AFE with isolated coagulopathy have been reported.20–22 In spite of the presence of amniotic fluid debris in pulmonary arteries, some of these subjects had DIC or uterine atony without accompanying cardiorespiratory symptoms. Post-partum hemorrhage (PPH) complicated by coagulopathy without manifestation of DIC has been reported as DIC-type PPH.23 Corresponding to the widespread clinical diagnosis of AFE on ZnCP-1 and STN staining in Japan, PPH complicated by coagulopathy has been attributed to DIC preceding AFE.24 We showed that Alc staining was positive not only in the uterine vasculature of all AFE subjects but also in some subjects without AFE. Similarly, HE and STN staining suggested the presence of amniotic components in some subjects in the non-AFE group. These observations suggest that the presence of amniotic components in the uterine vasculature is not a predictive finding for AFE; rather, it may be a common finding in various conditions including placenta previa, placenta accreta, and the normal post-partum state. To achieve hemostasis, hysterectomy is often performed for severe PPH complicated by coagulopathy. The present results, however, suggest that histological detection of amniotic components in the uterine vasculature does not provide conclusive findings to assist in AFE diagnosis. The present study had several limitations. First, this study was conducted at a single center, and the small sample size may have inaccurately reflected the prevalence of amniotic components in the maternal uterine vasculature. Second, although Alc was positive in the uterine vasculature of all AFE subjects, the presence of fetal material could not be confirmed on HE or STN staining in any of these AFE subjects. The time interval between the rupture of fetal membranes and subse-
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quent hysterectomy or autopsy varied dramatically between subjects, and therefore it is possible that the fetal components had been eliminated from the uterine vasculature in some AFE subjects before hysterectomy or autopsy was performed.
Conclusion The presence of maternal intravascular fetal components is not a specific finding in AFE with DIC-type PPH. Therefore, the fetal component in the uterine vasculature is unlikely to be a definitive indicator of AFE with DIC-type PPH.
Acknowledgments The authors thank Ms Satomi Uchino-Suzuki for her expert technical assistance.
Disclosure No financial support was received by any of the authors for this study and none of the authors have any relationships with companies that may have a financial interest in the information contained in the manuscript.
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