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Placental villitis of unclear etiology during ovum donor in vitro fertilization pregnancy
Placental villitis of unclear etiology during ovum donor in vitro fertilization pregnancy Aaron K. Styer, MD,a Hannah J. Parker, MD,b Drucilla J. Roberts, MD,c Darryl Palmer-Toy, MD, PhD,c Thomas L. Toth, MD,d and Jeffrey L. Ecker, MDe Boston, Mass OBJECTIVE: Preliminary observations by a single pathologist at our institution revealed a 75% incidence of villitis of unexplained etiology in ovum donor in vitro fertilization pregnancies. Because the incidence of villitis of unexplained etiology in the general population is approximately 10%, we conducted a controlled study to compare the incidence of villitis of unexplained etiology in ovum donor in vitro fertilization pregnancies to that in in vitro fertilization pregnancies that do not use donated ova. STUDY DESIGN: Placental specimens of ovum donor in vitro fertilization pregnancies were matched randomly with pregnancies that resulted from both fresh and frozen/thawed native oocyte in vitro fertilization from March 5, 1995, to October 10, 2001, and examined in a blinded fashion by a single pathologist (D. J. R.) for villitis of unexplained cause. The incidence of villitis of unexplained etiology was analyzed in 27 patients who underwent ovum donor in vitro fertilization versus 37 patients who underwent native oocyte in vitro fertilization. RESULTS: Villitis of unexplained cause occurred in 22.2% of ovum donor in vitro fertilization pregnancies, 10.8% of native oocyte in vitro fertilization pregnancies (fresh and frozen/thawed combined), and 14.3% of frozen/thawed cycles (P = .21). CONCLUSION: Although the incidence was not statistically different than in in vitro fertilization that used native maternal oocytes, there was a 2-fold increase in villitis of unexplained cause in the ovum donor in vitro fertilization placentas, which suggests that immune-related disorders may be increased in ovum donor pregnancies. (Am J Obstet Gynecol 2003;189:1184-6.)
Key words: Ova donation, in vitro fertilization, villitis of unclear etiology, VUE
During mammalian pregnancy, inherited maternal and paternal genes govern embryo development. Remarkably, the maternal immune system tolerates the conceptus and does not reject products of paternal genes, which may be incompatible with the maternal immune system. Although several aspects of pregnancy immunobiology condition have been elucidated, many key components remain poorly understood. Maternal intolerance possibly due to zygotic expression of foreign antigens has been documented in the case of pathologic findings such as maternal floor infarct, massive chronic intervillositis, and villitis of unknown etiology (VUE).1-5 In these cases,
From the Department of Obstetrics and Gynecology, Vincent Center for Reproductive Biology,a the Department of Pathology,c the Divisions of Reproductive Endocrinology and Infertilityd and Maternal Fetal Medicine,e the Department of Obstetrics and Gynecology, Massachusetts General Hospital; and the Department of Obstetrics and Gynecology, Brigham and Women’s Hospital.b Received for publication January 14, 2003; revised March 25, 2003; accepted May 2, 2003. Reprint requests: Jeffrey L. Ecker, MD, Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Massachusetts General Hospital, 55 Fruit St, Boston, MA, 02114. E-mail: [email protected] Ó 2003, Mosby, Inc. All rights reserved. 0002-9378/2003 $30.00 + 0 doi:10.1067/S0002-9378(03)00577-5
1184
a maternal immune response is present in the absence of infection and may recur in subsequent pregnancies.6-8 Pregnancies that are conceived after ova donation are an interesting model to study the interactions of embryonic antigens with the maternal immune system. These pregnancies occur after in vitro fertilization (IVF) with ova donated by a relative, or more commonly an unrelated donor. As such, neither embryonic haplotype matches that of the gestational carrier. To date, there are no published studies that outline perinatal outcomes in this circumstance. One of us (D. J. R) examined 17 placental pathologic specimens that were identified as ovum donor sent for routine review between January 1, 1995, and December 31, 1998. A 75% incidence of chronic VUE was noted. This was higher than the reported 10% incidence of VUE in the general population.9 We hypothesized that the placental findings of an ovum donor pregnancy may show an increased incidence of immune-related findings, as manifested in placental disease. We organized a cohort to further investigate our preliminary finding. Material and methods Study population. The Partners Healthcare Institutional Board of Review for Human Subjects Research approved this study. To ensure similarity of preconcep-
Styer et al 1185
Volume 189, Number 4 Am J Obstet Gynecol
Table I. Demographic characteristics of ovum recipient and native oocyte IVF pregnancies Ovum recipient Age (y)y Race (No.) White African American Other Gestational age of delivery (wk)y Mode of delivery (No.) Vaginal Cesarean Previous deliveries (No.) 0 1 2 *Two-tail calculations. y Data are given as mean àt-test. §Fisher exact test.
44.4
Native oocyte
± 4.9
34.1
± 4.2
P value* 8.92 3 1013à
27 (100%) 0 (0%) 0 (0%) 37.7 ± 3.3
33 (89.2%) 3 (8.1%) 1 (2.7%) 35.9 ± 4.6
.13§
11 (40.7%) 16 (59.3%)
25 (67.6%) 12 (32.4%)
.043§
17 (63.0%) 9 (33.3%) 1 (3.7%)
28 (75.6%) 8 (21.6%) 1 (2.8%)
.08à
.29§ .39§ 1.0§
± SD.
tion hormonal stimulation, placentas of pregnancies that were the result of ovum donation IVF (OD-IVF) cases were compared randomly to patients who underwent IVF with fresh and frozen/thawed cryopreserved embryos that were the result of native maternal oocytes (control subjects) from March 5, 1995, to October 10, 2001, who were delivered at Massachusetts General Hospital (MGH). The ovum recipient patients and patients who underwent IVF with native maternal oocytes were identified through electronic medical record (EMR) pregnancy records at our institution with specific keywords (ie, ovum donor, infertility, donor egg), a manual search of all EMR pregnancy records with estimated date of confinement calculated from embryo transfer dating, and by a manual search of the computerized database of the IVF unit of the MGH Vincent Memorial Hospital with the same keywords. The additional group of fresh nondonor IVF cycles with subsequent delivery and placental records at MGH were selected randomly and matched with ovum donor cases. To select a control group of routine IVF cycle pregnancies/patients, the EMR records with embryo transfer dating were placed in ascending order by estimated date of confinement (EDC). For each case of OD-IVF, a control was chosen as the record with the next subsequent estimated date of confinement who met the following criteria: neither a donated ovum nor cryothaw cycle nor intrauterine insemination nor gamete intrafallopian tube transfer nor a previously chosen control and delivered at MGH after the second trimester with placental pathology on file. Furthermore, none of the original 17 cases that were examined initially were included in these cohorts. The incidence of VUE was compared between cases and control subjects with v2 analysis. Continuous and categoric variables were compared with the use of t test and Fisher exact test two-tailed analysis, with a probability value of <.05 considered statistically significant.
Diagnosis of VUE. The placental specimens of all selected patients were assigned sequential study numbers on the basis of ascending medical record number and were examined by the same pathologist (D. J. R.) in a blinded fashion. The pathologic criteria for VUE was based on previously published criteria that described regions of villi with increased villous stromal cellularity that were consistent with a mononuclear infiltrate, with >2 characteristic regions of villi, and with at least >3 villi located in the parenchyma, not only at the maternal floor.2 Exclusion criteria for study placentas included evidence of infectious organisms or inclusions on histologic examination, a clinical history that was suggestive of an infectious cause, and pregnancies that did not result in a live birth. Placental tissue is not saved from every delivery, and 3 specimens were not available from OD-IVF cases, and 10 specimens from the control subjects (routine fresh IVF and frozen/thawed combined), resulting in final cohorts of 27 and 37, respectively. Results Thirty-three patients who were undergoing OD-IVF, 14 frozen/thawed cycles, and 33 fresh IVF cycle were identified in the medical record. Patients who were eligible for the study with available pathologic specimens were 27 OD-IVF cycles and 37 control subjects. The average ages for OD-IVF cases and control subjects were 44.4 ± 4.9 years and 34.1 ± 4.2 years, respectively (P = 8.9210 3 1013, Table I). Most of the women in both groups were white. The gestational ages at delivery were also similar. There was a statistical difference in the mode of delivery between cases and control subjects. OD-IVF patients underwent a larger proportion of cesarean deliveries than control subjects (59.3% vs 32.4%, P = .043; Table I). VUE occurred in 22.2% of donor ova cycle pregnancies, 10.8% of nondonor IVF pregnancies (fresh and thawed embryo nondonor patients combined),
1186 Styer et al
October 2003 Am J Obstet Gynecol
Table II. Incidence of placental villitis of unexplained etiology (VUE)
OD-IVF IVF-F IVF-F/T
VUE (%)
Normal specimens
% VUE
6 (22.1) 3 (10.0) 1 (14.1)
27 27 6
22.1 10.0 14.1
v2 test, P = .21. OD-IVF, Patients undergoing IVF utilizing donated ovum; IVF-F, patients undergoing IVF with native ovum without cryopreservation; IVF-F/T, patients undergoing IVF with native ovum following cryopreservation.
and 14.3% of frozen/thawed embryo cycle patients. No statistical difference was observed between ovum recipient patients and all nondonor IVF cycle patients (v2 test, P = .21) (Table II). Comment Progressive advances in the field of assisted reproductive technologies have enabled many subfertile and infertile couples to conceive and deliver healthy children. Even in the case of severe male or female infertility factor, techniques such as intrauterine insemination, intracytoplasmic injection, and ovum donation IVF have made conception possible in the face of historically poor prognostic factors. As such, many patients wish to conceal the fact that their pregnancy was conceived by virtue of assisted reproductive technologies. Consequently, specific characteristics of these pregnancies, such as obstetric and perinatal outcome, have not been studied to date. The techniques of assisted reproductive technologies and the manipulation of gametes before fertilization has provided valuable insight into the mechanisms, biologic features, and immunology of pregnancy. Interestingly, pregnancy has evolved into an efficient process in which a developing embryo that is derived from maternal (host) and paternal (foreign) antigens avoid rejection by maternal immunosurveillance. Ovum donation pregnancy is an interesting model in that an embryo that is derived from another woman’s oocytes expresses antigens that are different than the maternal host. An initial observation of an increased rate of VUE that was made in a small group of donor egg IVF pregnancies compared to a baseline rate of approximately 10% was made. Although it was not statistically significant, we found a >200% increase in the diagnosis of VUE in the ova donor pregnancies than in the control subjects. Because the incidence of VUE in our control native oocyte IVF population was 10.8% and similar to the background rate of 10%, it is probably unlikely that the increased rate of VUE in OD-IVF cases is the result of an overdiagnosis of this pathologic condition. No effort to assess perinatal and
obstetric outcome was made because the number of cases of ova donor was small and we would not have the power to compare obstetric outcomes such as intrauterine restriction, preeclampsia, and intrauterine death. This specific study has several limitations. A significant limitation was sample size. Because this study demonstrated no difference in VUE among study and control groups and given a known VUE baseline incidence of 10% in the general population, our study only had limited power to exclude definitively a difference of VUE in 30% to 35% of patients (power analysis assuming a = .05 and b = .2). Patient confidentiality was also a limitation to our analysis. We feel our identification of ovum donor and native oocyte IVF pregnancies was complete, but we cannot exclude the possibility that some patients who desired confidentiality may have succeeded in concealing the fact that their pregnancy was conceived after ovum donation. Because ovum donation procedures still comprise the minority of IVF cases, misclassification (hidden ovum donors in our control group) is unlikely to explain our negative finding. Confidentiality also limited our ability to determine the identity of donors and to compare findings in related and unrelated donors. As such, a future larger study with available identifiers for related and unrelated donors and specifically reported obstetric outcomes would optimize the further study of ovum donor pregnancy on pregnancy immunosurveillance and clinical outcome and provide the needed power to exclude definitively an association between VUE and OD-IVF.
REFERENCES 1. Mandsager NT, Bendon R, Mostello D, Rosenn B, Miodovnik M, Siddiqi TA. Maternal floor infarction of the placenta: prenatal diagnosis and clinical significance. Obstet Gynecol 1994;83:750-4. 2. Gersell DJ. Chronic villitis, chronic chorioamnionitis, and maternal floor infarction. Semin Diagn Pathol 1993;10:251-66. 3. Andres RL, Kuyper W, Resnik R, Piacquadio KM, Benirschke K. The association of maternal floor infarction of the placenta with adverse perinatal outcome. Am J Obstet Gynecol 1990;163:935-8. 4. Naeye RL. Maternal floor infarction. Hum Pathol 1985;16:823-8. 5. Labarrere C and Mullen E. Fibrinoid and trophoblastic necrosis with massive chronic intervillositis: an extreme variant of villitis of unknown etiology. Am J Reprod Immunol Microbiol 1987;15: 85-91. 6. Doss BJ, Greene MF, Hill J, Heffner LJ, Bieber FR, Genest DR. Massive chronic intervillositis associated with recurrent abortions. Hum Pathol 1995;26:1245-51. 7. Redline RW and Abramowsky CR. Clinical and pathologic aspects of recurrent placental villitis. Hum Pathol 1985;16:727-31. 8. Boyd TK and Redline RW. Chronic histiocytic intervillositis: a placental lesion associated with recurrent reproductive loss. Hum Pathol 2000;31:1389-96. 9. Knox WF and Fox H. Villitis of unknown aetiology: its incidence and significance in placentae from a British population. Placenta 1984;5:395-402.
Key words: Ova donation, in vitro fertilization, villitis of unclear etiology, VUE
During mammalian pregnancy, inherited maternal and paternal genes govern embryo development. Remarkably, the maternal immune system tolerates the conceptus and does not reject products of paternal genes, which may be incompatible with the maternal immune system. Although several aspects of pregnancy immunobiology condition have been elucidated, many key components remain poorly understood. Maternal intolerance possibly due to zygotic expression of foreign antigens has been documented in the case of pathologic findings such as maternal floor infarct, massive chronic intervillositis, and villitis of unknown etiology (VUE).1-5 In these cases,
From the Department of Obstetrics and Gynecology, Vincent Center for Reproductive Biology,a the Department of Pathology,c the Divisions of Reproductive Endocrinology and Infertilityd and Maternal Fetal Medicine,e the Department of Obstetrics and Gynecology, Massachusetts General Hospital; and the Department of Obstetrics and Gynecology, Brigham and Women’s Hospital.b Received for publication January 14, 2003; revised March 25, 2003; accepted May 2, 2003. Reprint requests: Jeffrey L. Ecker, MD, Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Massachusetts General Hospital, 55 Fruit St, Boston, MA, 02114. E-mail: [email protected] Ó 2003, Mosby, Inc. All rights reserved. 0002-9378/2003 $30.00 + 0 doi:10.1067/S0002-9378(03)00577-5
1184
a maternal immune response is present in the absence of infection and may recur in subsequent pregnancies.6-8 Pregnancies that are conceived after ova donation are an interesting model to study the interactions of embryonic antigens with the maternal immune system. These pregnancies occur after in vitro fertilization (IVF) with ova donated by a relative, or more commonly an unrelated donor. As such, neither embryonic haplotype matches that of the gestational carrier. To date, there are no published studies that outline perinatal outcomes in this circumstance. One of us (D. J. R) examined 17 placental pathologic specimens that were identified as ovum donor sent for routine review between January 1, 1995, and December 31, 1998. A 75% incidence of chronic VUE was noted. This was higher than the reported 10% incidence of VUE in the general population.9 We hypothesized that the placental findings of an ovum donor pregnancy may show an increased incidence of immune-related findings, as manifested in placental disease. We organized a cohort to further investigate our preliminary finding. Material and methods Study population. The Partners Healthcare Institutional Board of Review for Human Subjects Research approved this study. To ensure similarity of preconcep-
Styer et al 1185
Volume 189, Number 4 Am J Obstet Gynecol
Table I. Demographic characteristics of ovum recipient and native oocyte IVF pregnancies Ovum recipient Age (y)y Race (No.) White African American Other Gestational age of delivery (wk)y Mode of delivery (No.) Vaginal Cesarean Previous deliveries (No.) 0 1 2 *Two-tail calculations. y Data are given as mean àt-test. §Fisher exact test.
44.4
Native oocyte
± 4.9
34.1
± 4.2
P value* 8.92 3 1013à
27 (100%) 0 (0%) 0 (0%) 37.7 ± 3.3
33 (89.2%) 3 (8.1%) 1 (2.7%) 35.9 ± 4.6
.13§
11 (40.7%) 16 (59.3%)
25 (67.6%) 12 (32.4%)
.043§
17 (63.0%) 9 (33.3%) 1 (3.7%)
28 (75.6%) 8 (21.6%) 1 (2.8%)
.08à
.29§ .39§ 1.0§
± SD.
tion hormonal stimulation, placentas of pregnancies that were the result of ovum donation IVF (OD-IVF) cases were compared randomly to patients who underwent IVF with fresh and frozen/thawed cryopreserved embryos that were the result of native maternal oocytes (control subjects) from March 5, 1995, to October 10, 2001, who were delivered at Massachusetts General Hospital (MGH). The ovum recipient patients and patients who underwent IVF with native maternal oocytes were identified through electronic medical record (EMR) pregnancy records at our institution with specific keywords (ie, ovum donor, infertility, donor egg), a manual search of all EMR pregnancy records with estimated date of confinement calculated from embryo transfer dating, and by a manual search of the computerized database of the IVF unit of the MGH Vincent Memorial Hospital with the same keywords. The additional group of fresh nondonor IVF cycles with subsequent delivery and placental records at MGH were selected randomly and matched with ovum donor cases. To select a control group of routine IVF cycle pregnancies/patients, the EMR records with embryo transfer dating were placed in ascending order by estimated date of confinement (EDC). For each case of OD-IVF, a control was chosen as the record with the next subsequent estimated date of confinement who met the following criteria: neither a donated ovum nor cryothaw cycle nor intrauterine insemination nor gamete intrafallopian tube transfer nor a previously chosen control and delivered at MGH after the second trimester with placental pathology on file. Furthermore, none of the original 17 cases that were examined initially were included in these cohorts. The incidence of VUE was compared between cases and control subjects with v2 analysis. Continuous and categoric variables were compared with the use of t test and Fisher exact test two-tailed analysis, with a probability value of <.05 considered statistically significant.
Diagnosis of VUE. The placental specimens of all selected patients were assigned sequential study numbers on the basis of ascending medical record number and were examined by the same pathologist (D. J. R.) in a blinded fashion. The pathologic criteria for VUE was based on previously published criteria that described regions of villi with increased villous stromal cellularity that were consistent with a mononuclear infiltrate, with >2 characteristic regions of villi, and with at least >3 villi located in the parenchyma, not only at the maternal floor.2 Exclusion criteria for study placentas included evidence of infectious organisms or inclusions on histologic examination, a clinical history that was suggestive of an infectious cause, and pregnancies that did not result in a live birth. Placental tissue is not saved from every delivery, and 3 specimens were not available from OD-IVF cases, and 10 specimens from the control subjects (routine fresh IVF and frozen/thawed combined), resulting in final cohorts of 27 and 37, respectively. Results Thirty-three patients who were undergoing OD-IVF, 14 frozen/thawed cycles, and 33 fresh IVF cycle were identified in the medical record. Patients who were eligible for the study with available pathologic specimens were 27 OD-IVF cycles and 37 control subjects. The average ages for OD-IVF cases and control subjects were 44.4 ± 4.9 years and 34.1 ± 4.2 years, respectively (P = 8.9210 3 1013, Table I). Most of the women in both groups were white. The gestational ages at delivery were also similar. There was a statistical difference in the mode of delivery between cases and control subjects. OD-IVF patients underwent a larger proportion of cesarean deliveries than control subjects (59.3% vs 32.4%, P = .043; Table I). VUE occurred in 22.2% of donor ova cycle pregnancies, 10.8% of nondonor IVF pregnancies (fresh and thawed embryo nondonor patients combined),
1186 Styer et al
October 2003 Am J Obstet Gynecol
Table II. Incidence of placental villitis of unexplained etiology (VUE)
OD-IVF IVF-F IVF-F/T
VUE (%)
Normal specimens
% VUE
6 (22.1) 3 (10.0) 1 (14.1)
27 27 6
22.1 10.0 14.1
v2 test, P = .21. OD-IVF, Patients undergoing IVF utilizing donated ovum; IVF-F, patients undergoing IVF with native ovum without cryopreservation; IVF-F/T, patients undergoing IVF with native ovum following cryopreservation.
and 14.3% of frozen/thawed embryo cycle patients. No statistical difference was observed between ovum recipient patients and all nondonor IVF cycle patients (v2 test, P = .21) (Table II). Comment Progressive advances in the field of assisted reproductive technologies have enabled many subfertile and infertile couples to conceive and deliver healthy children. Even in the case of severe male or female infertility factor, techniques such as intrauterine insemination, intracytoplasmic injection, and ovum donation IVF have made conception possible in the face of historically poor prognostic factors. As such, many patients wish to conceal the fact that their pregnancy was conceived by virtue of assisted reproductive technologies. Consequently, specific characteristics of these pregnancies, such as obstetric and perinatal outcome, have not been studied to date. The techniques of assisted reproductive technologies and the manipulation of gametes before fertilization has provided valuable insight into the mechanisms, biologic features, and immunology of pregnancy. Interestingly, pregnancy has evolved into an efficient process in which a developing embryo that is derived from maternal (host) and paternal (foreign) antigens avoid rejection by maternal immunosurveillance. Ovum donation pregnancy is an interesting model in that an embryo that is derived from another woman’s oocytes expresses antigens that are different than the maternal host. An initial observation of an increased rate of VUE that was made in a small group of donor egg IVF pregnancies compared to a baseline rate of approximately 10% was made. Although it was not statistically significant, we found a >200% increase in the diagnosis of VUE in the ova donor pregnancies than in the control subjects. Because the incidence of VUE in our control native oocyte IVF population was 10.8% and similar to the background rate of 10%, it is probably unlikely that the increased rate of VUE in OD-IVF cases is the result of an overdiagnosis of this pathologic condition. No effort to assess perinatal and
obstetric outcome was made because the number of cases of ova donor was small and we would not have the power to compare obstetric outcomes such as intrauterine restriction, preeclampsia, and intrauterine death. This specific study has several limitations. A significant limitation was sample size. Because this study demonstrated no difference in VUE among study and control groups and given a known VUE baseline incidence of 10% in the general population, our study only had limited power to exclude definitively a difference of VUE in 30% to 35% of patients (power analysis assuming a = .05 and b = .2). Patient confidentiality was also a limitation to our analysis. We feel our identification of ovum donor and native oocyte IVF pregnancies was complete, but we cannot exclude the possibility that some patients who desired confidentiality may have succeeded in concealing the fact that their pregnancy was conceived after ovum donation. Because ovum donation procedures still comprise the minority of IVF cases, misclassification (hidden ovum donors in our control group) is unlikely to explain our negative finding. Confidentiality also limited our ability to determine the identity of donors and to compare findings in related and unrelated donors. As such, a future larger study with available identifiers for related and unrelated donors and specifically reported obstetric outcomes would optimize the further study of ovum donor pregnancy on pregnancy immunosurveillance and clinical outcome and provide the needed power to exclude definitively an association between VUE and OD-IVF.
REFERENCES 1. Mandsager NT, Bendon R, Mostello D, Rosenn B, Miodovnik M, Siddiqi TA. Maternal floor infarction of the placenta: prenatal diagnosis and clinical significance. Obstet Gynecol 1994;83:750-4. 2. Gersell DJ. Chronic villitis, chronic chorioamnionitis, and maternal floor infarction. Semin Diagn Pathol 1993;10:251-66. 3. Andres RL, Kuyper W, Resnik R, Piacquadio KM, Benirschke K. The association of maternal floor infarction of the placenta with adverse perinatal outcome. Am J Obstet Gynecol 1990;163:935-8. 4. Naeye RL. Maternal floor infarction. Hum Pathol 1985;16:823-8. 5. Labarrere C and Mullen E. Fibrinoid and trophoblastic necrosis with massive chronic intervillositis: an extreme variant of villitis of unknown etiology. Am J Reprod Immunol Microbiol 1987;15: 85-91. 6. Doss BJ, Greene MF, Hill J, Heffner LJ, Bieber FR, Genest DR. Massive chronic intervillositis associated with recurrent abortions. Hum Pathol 1995;26:1245-51. 7. Redline RW and Abramowsky CR. Clinical and pathologic aspects of recurrent placental villitis. Hum Pathol 1985;16:727-31. 8. Boyd TK and Redline RW. Chronic histiocytic intervillositis: a placental lesion associated with recurrent reproductive loss. Hum Pathol 2000;31:1389-96. 9. Knox WF and Fox H. Villitis of unknown aetiology: its incidence and significance in placentae from a British population. Placenta 1984;5:395-402.