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The Alabama Preterm Birth Study: Intrauterine infection and placental histologic findings in preterm births of males and females less than 32 weeks
American Journal of Obstetrics and Gynecology (2006) 195, 1533–7
www.ajog.org
The Alabama Preterm Birth Study: Intrauterine infection and placental histologic findings in preterm births of males and females less than 32 weeks Robert L. Goldenberg, MD,a William W. Andrews, PhD, MD,a Ona M. Faye-Petersen, MD,b Alice R. Goepfert, MD,a Suzanne P. Cliver,a John C. Hauth, MDa Departments of Obstetrics and Gynecologya and Pathology,b University of Alabama at Birmingham, Birmingham, AL Received for publication December 8, 2005; revised May 4, 2006; accepted May 11, 2006
KEY WORDS Infant sex Preterm birth Placental histology Placental infection
Objective: The objective of the study was to determine whether there are differences in the placental histology and various markers of infection/inflammation between preterm male and female fetuses. Study design: The placentas and umbilical cords of 446 infants born at 23 to 32 weeks were examined histologically, cultured for aerobic and anaerobic bacteria and mycoplasmas, and the interleukin-6 levels in cord blood determined. Results: Male infants were significantly more likely to have positive placental cultures than female infants (63.4% versus 51.8%, P = .01, odds ratio 1.5, 1.0 to 2.4). Cord blood Mycoplasma hominis and Ureaplasma urealyticum infections were marginally more common in male than female fetuses (27.6% versus 19.2%, P = .06, odds ratio 1.7, 0.9 to 2.9), but cord blood interleukin-6 levels were not different between male and female fetuses. The only significant histologic difference between male and female placentas was in decidual lymphoplasmacytic cell infiltration (6.3% versus 0.9%, P = .003, odds ratio 8.3, 1.8 to 39.0). Males had a higher percentage of decidual lymphohistiocytic cell infiltration, but the differences were not significant (11.3% versus 7.4%, P = .160, odds ratio 1.6, 0.8 to 3.2). Conclusion: Male infants were significantly more likely to have positive placental membrane cultures than female infants. Decidual lymphoplasmacytic cell infiltrations were more common in male versus female placentas, confirming a previous observation and suggesting that a maternal immune reaction to fetal tissue may be more common in male fetuses. Ó 2006 Mosby, Inc. All rights reserved.
In nearly all studies published to date, perinatal mortality in male infants is higher than in female infants.1-5 At least part of this difference is accounted Reprints not available from the authors. Address correspondence to Robert L. Goldenberg, MD, Department of Obstetrics/Gynecology, 1500 6th Avenue South, CRWH 379, Birmingham, AL 35233-1602. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2006.05.023
for by the higher rate of preterm births in males.6-10 However, the reason for the disparity in preterm births between male and female fetuses is largely unexplained. Intrauterine infection/inflammation has been shown to be associated with, and possibly causative for, a majority of early preterm births.11,12 To determine whether differences in intrauterine infection/inflammation might explain some portion of the male/female differences in
1534
Goldenberg et al
Table I Demographic and obstetric factors in women with male and female fetuses Characteristics
Male n = 220
Maternal parity 0 53.9 R1 46.1 Race, % Black 58.2 White 39.5 Hispanic 1.4 Other 0.9 Maternal age, y, % !20 28.2 20-30 53.6 O30 18.2 Smoking status, % Yes 13.2 No 86.8 Diabetes, % Yes 3.9 No 96.1 Pre-eclampsia, % Yes 30.5 No 69.5 Type of PTB, % Indicated 31.4 Spontaneous 68.6 Gestational age, wks, % 21-24 7.7 25-28 42.3 29-32 50.0 Corticosteroid use, % 85.6 Antibiotic use, % 87.1 BWT (!) G SD 1190 G 409 GA (!) G SD 28.5 G 2.3
Female n = 226
P value
50.2 49.8
.440
54.0 42.5 2.6 0.9
.681
21.7 61.9 16.4
.180
11.5 88.5
.590
5.6 94.4
.432
37.6 62.4
.111
41.6 58.4
.025
7.1 42.9 50.0 87.8 89.2 1092 G 363 28.6 G 2.2
.964
.448 .484 .008 .783
PTB, Preterm birth; BWT, birth weight; GA, gestational age.
preterm birth, in this analysis we questioned whether markers of intrauterine infection/inflammation were different between male and female early preterm deliveries. Furthermore, in the only other comprehensive study of male/female differences in placental histology in very preterm infants that we could locate, Ghidini and Salafia13 found that male gender was associated with an increase in chronic decidual inflammation. They speculated that this finding may be associated with a maternal immune response against the invading interstitial trophoblast. In the current study, we specifically sought to confirm, and if possible expand on, the observations of Ghidini and Salafia13 regarding differences in chronic decidual inflammation involving male and female infants. To accomplish these goals, we performed a secondary analysis of data from the Alabama Preterm Birth Study, an investigation of 457 23- to 32-week deliveries in which various markers of intrauterine infection/inflammation were collected and the placental histology evaluated.14
Material and methods From December 5, 1996, to June 13, 2001, 457 consecutive deliveries of infants born between 23 and 32 weeks were evaluated.14 A chart review was performed by trained research nurses to gather demographic characteristics such as maternal race, age, parity, smoking status, diabetes, and pre-eclampsia and determine whether the delivery was spontaneous or indicated. Spontaneous preterm birth was defined as delivery after either spontaneous preterm labor or spontaneous preterm premature rupture of membranes. Indicated preterm birth was defined as delivery effected for maternal or fetal indications. Clinical chorioamnionitis was determined retrospectively by chart review and was considered present if diagnosed by the attending faculty obstetrician, usually for a combination of fever, abdominal pain, and an elevated maternal white count. Any use of corticosteroids for fetal maturation prior to delivery or the use of antibiotics within the 2 weeks prior to delivery was coded as positive for that variable. Birth weight, gestational age at delivery, and infant sex were noted. Placentas for 446 of the neonates (98%) were available for study. In each case, a minimum of 2 membrane rolls, 2 complete sections of umbilical cord (1 from the placental and 1 from the fetal end of the cord), and 2 to 4 transmural parenchymal sections were submitted for histological examination. All cases were evaluated histologically by a single pathologist (O. F. -P.), blinded to clinical outcome, using a placental evaluation protocol adapted from Bendon et al.15 For this study, the free membranes, fetal plate, villi, and umbilical cord were evaluated qualitatively for the presence of polymorphonuclear (PMN) infiltration.16-18 A PMN infiltration was considered present with PMNs in an essentially linear distribution. Specifically, scattered, entrapped, single, or sparsely distributed cells were not scored as an infiltration. The number of PMNs in an infiltration generally exceeded 20 per high-power field (!400) but 5 to 10 or more per high-power field were also scored as a PMN infiltration. Mononuclear infiltrates were divided into lymphohistiocytic and lymphoplasmacytic subtypes,19 and the free membranes, decidua basalis, and fetal plate were also qualitatively evaluated for the presence of both types of chronic inflammation.19 The villi were evaluated for both PMN and mononuclear inflammation. The decidua basalis was also evaluated for the presence or absence of diffuse decidual leukocytoclastic necrosis, a finding commonly associated with vascular insufficiency.20,21 The chorioamnionic interfaces of 445 placentas were cultured for aerobic and anaerobic organisms including Mycoplasma hominis and Ureaplasma urealyticum as previously described.22,23 At delivery, a sample of umbilical cord blood from 343 of the infants was collected by aseptic puncture of the umbilical cord and was cultured
Goldenberg et al Table II
1535
Placental histologic findings in male versus female infants
Placental histologic findings
Male (%) n = 220
Female (%) n = 226
P value
RR (95% CI)
Free membranes: PMN cell infiltration Fetal plate: PMN cell infiltration Cord: PMN cell infiltration Decidua basalis: DDLN Free membranes: lymphohistiocytic infiltration Free membranes: plasmacytic infiltration Decidua basalis: lymphohistiocytic infiltration Decidua basalis: lymphoplasmacytic infiltration Villi: acute inflammation Villi: chronic inflammation
49.8 40.1 29.2 25.6 10.1 0.9 11.3 6.3 0.5 1.0
46.8 39.7 31.8 28.6 9.6 0.9 7.4 0.9 1.4 0.0
.535 .938 .555 .456 .849 .989 .160 .003 .623 .241
1.06 1.01 0.92 0.89 1.06 1.01 1.54 6.7 0.34 NA
(0.88, (0.80, (0.69, (0.65, (0.60, (0.14, (0.84, (1.53, (0.04,
1.29) 1.27) 1.22) 1.21) 1.87) 7.13) 2.81) 29.41) 3.29)
DDLN, Diffuse decidual leukocytoclastic necrosis; RR, risk ratio; CI, confidence interval; NA, not applicable, zero denominator.
for M. hominis and U. urealyticum, as described previously.23 The remaining cord blood (n = 309) was then centrifuged, aliquoted, and stored at –70(C until the interleukin (IL)-6 assay was performed. IL-6 concentrations in the cord plasma were determined by enzymelinked immunosorbent assay kits (R&D Systems, Inc, Minneapolis, MN). The lower limit of sensitivity of the assay was 0.7 pg/mL. The intra-assay and interassay coefficients of variation were 2.6% and 4.5%, respectively. Data analyses were performed with SAS software (version 9, SAS Institute, Inc, Cary, NC). Frequencies and means between groups were compared with the use of c2 tests for discrete variables and t tests and Wilcoxon rank sum tests for continuous variables. A P value less than .05 was chosen to define statistical significance. Logistic regression analyses, adjusting for various maternal characteristics were used to determine the odds ratios (ORs) for placental and umbilical cord cultures and subtypes of chronic decidual inflammation to be associated with male sex. The study was approved by the University of Alabama at Birmingham Institutional Review Board.
Results Table I shows the characteristics of the mothers of the male and female infants enrolled in this study. Male infants were more likely to come from spontaneous rather than indicated deliveries, (P = .025) and weighed about 100 g more (P = .008). There were no differences in corticosteroid or antibiotic use between male and female infants. We next evaluated the relationship between infant sex and various markers of infection/inflammation. Male infants were significantly more likely to have a positive placental culture than were female infants (63.4% versus 51.8%, P = .01). They were also marginally more likely to have cord blood cultures positive for U. urealyticum or
M. hominis (27.6% versus 19.2%, P = .06). Adjusting for race, marital status, corticosteroid use, and birth weight and whether the delivery was indicated or spontaneous, the OR for a positive placental culture being associated with male sex was 1.5 (1.0 to 2.4) and for a positive cord blood mycoplasma or ureaplasma culture to be associated with male sex, 1.7 (0.9 to 3.0). Using a Wilcoxon rank sum test, the distributions of IL-6 levels were similar for male and female infants (median values 11.1 versus 11.3 pg/mL, P = .946). The rate of clinical chorioamnionitis was also similar in the mothers of male and female infants (11.6% versus 14.7%, P = .33). Table II shows the relationship between each of the placental histologic findings and infant sex. Only the relationship between infant sex and the presence of lymphoplasmacytic infiltrations was statistically significant, with the rates higher in placentas of male infants, compared with placentas of female infants (6.3% versus 0.9%, P = .003). Adjusting for race, marital status, corticosteroid use, and birth weight and whether the delivery was indicated or spontaneous, the OR for decidual lymphohistiocytic inflammation being associated with a male fetus was 1.6 (0.8 to 3.2) and for lymphoplasmacytic inflammation, 8.3 (1.8 to 39.0). The percent of placentas with lymphohistiocytic or lymphoplasmacytic infiltrations in other sites was not different between male and female infants. Of note, chronic villitis, a lesion thought to indicate a maternal host-versus-graft response,24 and often seen in association with chronic decidual inflammation,25,26 was present in less than 1% of cases and was not significantly different in placentas from male and female infants.
Comment In this study of 23- to 32-week deliveries, male infants were significantly more likely to be born following a spontaneous rather than indicated delivery and were also significantly more likely to have placental membrane
1536 bacterial infection. Because most early spontaneous preterm births are associated with, and probably caused by, placental membrane infection associated with acute placental inflammation,11,12,27 these data suggest that a portion of the increase in male versus female early preterm births may be due to intrauterine infection. However, the discordance between the significant increase in positive placental membrane cultures in males and the absence of any difference between male and female infant cord IL-6 levels, acute placental histologic inflammation, or clinical chorioamnionitis is unexplained. In this data set, there were slightly more female than male infants. From previous studies, we would have expected the reverse to be true, especially because we were studying early preterm births.6-9 Because these cases were recruited consecutively, selection bias does not explain the female predominance, and we can only conclude that these results occurred by chance. Nevertheless, this finding, plus the fact that this is a secondary analysis of an existing data set, provides further reason to be cautious about accepting infection as an important cause for male/female differences in preterm birth. Furthermore, although the difference was not significant, the fact that the mothers of female infants were more likely to have pre-eclampsia might partially explain the higher rate of indicated preterm births in these infants and thus the greater proportion of spontaneous preterm births in male fetuses. In any case, because of the lack of concordance between the placental culture results and other markers of inflammation, and the larger than expected proportion of female infants,6-9 as well as the higher (but not significantly different) percent of female infants with mothers with pre-eclampsia in this data set, further study is required before the hypothesis of an infection-related increase in male preterm birth can be accepted. As did Ghidini and Salafia,13 we found that in very preterm infants, male versus female gender is associated with higher rates of chronic decidual inflammation of both the lymphohistiocytic and lymphoplasmacytic variety, although in our study, only the difference in lymphoplasmacytic infiltrations was significant. Conversely, in male, compared with female infants, we did not observe an increase in acute inflammation at any site or chronic inflammation in sites other than the decidua basalis. The reason for this difference is not clear, and although it may be related to the increased infection seen in the male placentas, a PMN cell response to bacterial infection is what would generally be expected. Redline et al28 have stated that plasma cells in particular, and B-lymphocytes in general, are always abnormal in the endometrial cavity, and when present, they are an indicator of inappropriate antigenic exposure. Thus, a more aggressive maternal immune response against male versus female fetuses is a plausible explanation for these findings.
Goldenberg et al Supporting this hypothesis is the fact that chronic placental inflammation is increased in women with recurrent miscarriages and indicated preterm birth.29,30 The location of the chronic inflammation at the decidualchorionic interface also suggests that the mononuclear cells are of maternal origin, and this may signal a maternal response against the fetal trophoblast. Further studies that elucidate the character of the lymphohistiocytic and lymphoplasmacytic infiltrates and help define the nature of the maternal response are necessary. In summary, this study found that early preterm male infants were more likely to be born following spontaneous preterm birth and to have a higher percentage of positive intrauterine cultures. They were also more likely to have lymphoplasmacytic inflammation at the choriodecidual interface. Thus, there are differences in some markers of intrauterine infection and inflammation between male and female fetuses. Whether these differences explain the increased preterm birth rate or the higher mortality in male fetuses and newborns will require further study.
References 1. Ward WP. Perinatal mortality in Utrecht, The Netherlands, 1880-1940. Econ Hum Biol 2003;1:379-98. 2. Effer SB, Moutquin JM, Farine D, Saigal S, Nimrod C, Kelly E, et al. Neonatal survival rates in 860 singleton live births at 24 and 25 weeks gestational age. A Canadian multicentre study. BJOG 2002;109:740-5. 3. Khoury MJ, Marks JS, McCarthy BJ, Zaro SM. Factors affecting the sex differential in neonatal mortality: the role of respiratory distress syndrome. Am J Obstet Gynecol 1985;151:777-82. 4. Perelman RH, Palta M, Kirby R, Farrell PM. Discordance between male and female deaths due to the respiratory distress syndrome. Pediatrics 1986;78:238-44. 5. Naeye RL, Burt LS, Wright DL, Blanc WA, Tatter D. Neonatal mortality, the male disadvantage. Pediatrics 1971;48:902-6. 6. McGregor JA, Leff M, Orleans M, Baron A. Fetal gender differences in preterm birth: findings in a North American cohort. Am J Perinatol 1992;9:43-8. 7. James WH. Why are boys more likely to be preterm then girls? Plus other related conundrums in human reproduction. Hum Reprod 2000;15:2108-11. 8. Cooperstock M, Campbell J. Excess males in preterm birth: interactions with gestational age, race, and multiple birth. Obstet Gynecol 1996;88:189-93. 9. Ingemarsson I. Gender aspects of preterm birth. BJOG 2003; 110(Suppl 20):34-8. 10. James WH. Sex ratios of offspring and the causes of placental pathology. Hum Reprod 1995;10:1403-6. 11. Goldenberg RL, Hauth JC, Andrews WW. Intrauterine infection and preterm delivery. N Engl J Med 2000;342:1500-7. 12. Goldenberg RL, Culhane JF. Infection as a cause of preterm birth. Clin Perinatol 2003;30:677-700. 13. Ghidini A, Salafia CM. Gender differences of placental dysfunction in severe prematurity. BJOG 2005;112:140-4. 14. Goepfert A, Andrews W, Carlo W, Ramsey P, Cliver S, Goldenberg R, et al. Umbilical cord plasma interleukin-6 concentrations in preterm infants and risk of neonatal morbidity. Am J Obstet Gynecol 2004;191:1375-81.
Goldenberg et al 15. Bendon RW, Faye-Petersen O, Pavlova Z, Qureshi F, Elder N, Das A, et al. Histologic features of chorioamnion membrane rupture: development of methodology. Pediatr Pathol Lab Med 1997;17:27-42. 16. Zhang J, Kraus FT, Aquino TI. Chorioamnionitis: a comparative histologic, bacteriologic, and clinical study. Int J Gynecol Pathol 1985;4:1-10. 17. Smulian JC, Shen-Schwarz S, Vintzileos AM, Lake MF, Ananth CV. Clinical chorioamnionitis and the histology of placental inflammation. Obstet Gynecol 1999;94:1000-5. 18. Redline RW, Heller D, Keating S, Kingdom J. Placental diagnostic criteria and clinical correlation- a workshop report. Placenta 2005;26(Suppl):S114-7. 19. Khong TY, Bendon RW, Qureshi F, Redline RW, Gould S, Stallmach T, et al. Chronic deciduitis in the placental basal plate: definition and interobserver reliability. Hum Pathol 2000;31:292-5. 20. Stanek J, Al-Ahmadie HA. Laminar necrosis of placental membranes: a histologic sign of uteroplacental hypoxia. Pediatr Dev Pathol 2005;8:34-42. 21. Goldenberg RL, Faye-Petersen O, Andrews WW, Cliver SP, Hauth JC. Diffuse decidual leukocytoclastic necrosis and Pregnancy Outcome. Am J Obstet Gynecol 2006;193:S53, (Abstract 147). 22. Andrews WW, Hauth JC, Goldenberg RL, Gomez R, Romero R, Cassell GH. Amniotic fluid interleukin-6: correlation with upper genital tract microbial colonization and gestational age in women delivered after spontaneous labor versus indicated delivery. Am J Obstet Gynecol 1995;173:606-12.
1537 23. Waites KB, Rikihisa Y, Taylor-Robinson D. Mycoplasma and Ureaplasma. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology. 8th ed. Washington, DC: ASM Press; 2003. p. 972-90. 24. Redline RW, Patterson P. Villitis of unknown etiology is associated with major infiltration of fetal tissue by maternal inflammatory cells. Am J Pathol 1993;143:473-9. 25. Benirscke K, Kaufman P. Pathology of the human placenta. 4th ed. New York, New York: Springer; 2000. p. 653-9. 26. Benirscke K, Kaufman P. Pathology of the human placenta. 4th ed. New York (NY): Springer; 2000. p. 242-8. 27. Russell P. Inflammatory lesions of the human placenta. I. Clinical significance of acute chorioamnionitis. Am J Diagn Gynecol Obstet 1979;1:127-37. 28. Redline RW, Faye-Petersen O, Heller D, Qureshi F, Savell V, Vogler C, Society for Pediatric Pathology, Perinatal Section, Amniotic Fluid Infection Nosology Committee. Amnionic infection syndrome: nosology and reproducibility of placental reaction patterns. Pediatr Dev Pathol 2003;6:435-48. 29. Jacques SM, Qureshi F. Chronic chorioamnionitis: a clinicopathologic and immunohistochemical study. Hum Pathol 1998;29: 1457-61. 30. Kohut KG, Anthony MN, Salafia CM. Decidual and placental histologic findings in patients experiencing spontaneous abortions in relation to pregnancy order. Am J Reprod Immunol 1997;37: 257-61.
www.ajog.org
The Alabama Preterm Birth Study: Intrauterine infection and placental histologic findings in preterm births of males and females less than 32 weeks Robert L. Goldenberg, MD,a William W. Andrews, PhD, MD,a Ona M. Faye-Petersen, MD,b Alice R. Goepfert, MD,a Suzanne P. Cliver,a John C. Hauth, MDa Departments of Obstetrics and Gynecologya and Pathology,b University of Alabama at Birmingham, Birmingham, AL Received for publication December 8, 2005; revised May 4, 2006; accepted May 11, 2006
KEY WORDS Infant sex Preterm birth Placental histology Placental infection
Objective: The objective of the study was to determine whether there are differences in the placental histology and various markers of infection/inflammation between preterm male and female fetuses. Study design: The placentas and umbilical cords of 446 infants born at 23 to 32 weeks were examined histologically, cultured for aerobic and anaerobic bacteria and mycoplasmas, and the interleukin-6 levels in cord blood determined. Results: Male infants were significantly more likely to have positive placental cultures than female infants (63.4% versus 51.8%, P = .01, odds ratio 1.5, 1.0 to 2.4). Cord blood Mycoplasma hominis and Ureaplasma urealyticum infections were marginally more common in male than female fetuses (27.6% versus 19.2%, P = .06, odds ratio 1.7, 0.9 to 2.9), but cord blood interleukin-6 levels were not different between male and female fetuses. The only significant histologic difference between male and female placentas was in decidual lymphoplasmacytic cell infiltration (6.3% versus 0.9%, P = .003, odds ratio 8.3, 1.8 to 39.0). Males had a higher percentage of decidual lymphohistiocytic cell infiltration, but the differences were not significant (11.3% versus 7.4%, P = .160, odds ratio 1.6, 0.8 to 3.2). Conclusion: Male infants were significantly more likely to have positive placental membrane cultures than female infants. Decidual lymphoplasmacytic cell infiltrations were more common in male versus female placentas, confirming a previous observation and suggesting that a maternal immune reaction to fetal tissue may be more common in male fetuses. Ó 2006 Mosby, Inc. All rights reserved.
In nearly all studies published to date, perinatal mortality in male infants is higher than in female infants.1-5 At least part of this difference is accounted Reprints not available from the authors. Address correspondence to Robert L. Goldenberg, MD, Department of Obstetrics/Gynecology, 1500 6th Avenue South, CRWH 379, Birmingham, AL 35233-1602. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2006.05.023
for by the higher rate of preterm births in males.6-10 However, the reason for the disparity in preterm births between male and female fetuses is largely unexplained. Intrauterine infection/inflammation has been shown to be associated with, and possibly causative for, a majority of early preterm births.11,12 To determine whether differences in intrauterine infection/inflammation might explain some portion of the male/female differences in
1534
Goldenberg et al
Table I Demographic and obstetric factors in women with male and female fetuses Characteristics
Male n = 220
Maternal parity 0 53.9 R1 46.1 Race, % Black 58.2 White 39.5 Hispanic 1.4 Other 0.9 Maternal age, y, % !20 28.2 20-30 53.6 O30 18.2 Smoking status, % Yes 13.2 No 86.8 Diabetes, % Yes 3.9 No 96.1 Pre-eclampsia, % Yes 30.5 No 69.5 Type of PTB, % Indicated 31.4 Spontaneous 68.6 Gestational age, wks, % 21-24 7.7 25-28 42.3 29-32 50.0 Corticosteroid use, % 85.6 Antibiotic use, % 87.1 BWT (!) G SD 1190 G 409 GA (!) G SD 28.5 G 2.3
Female n = 226
P value
50.2 49.8
.440
54.0 42.5 2.6 0.9
.681
21.7 61.9 16.4
.180
11.5 88.5
.590
5.6 94.4
.432
37.6 62.4
.111
41.6 58.4
.025
7.1 42.9 50.0 87.8 89.2 1092 G 363 28.6 G 2.2
.964
.448 .484 .008 .783
PTB, Preterm birth; BWT, birth weight; GA, gestational age.
preterm birth, in this analysis we questioned whether markers of intrauterine infection/inflammation were different between male and female early preterm deliveries. Furthermore, in the only other comprehensive study of male/female differences in placental histology in very preterm infants that we could locate, Ghidini and Salafia13 found that male gender was associated with an increase in chronic decidual inflammation. They speculated that this finding may be associated with a maternal immune response against the invading interstitial trophoblast. In the current study, we specifically sought to confirm, and if possible expand on, the observations of Ghidini and Salafia13 regarding differences in chronic decidual inflammation involving male and female infants. To accomplish these goals, we performed a secondary analysis of data from the Alabama Preterm Birth Study, an investigation of 457 23- to 32-week deliveries in which various markers of intrauterine infection/inflammation were collected and the placental histology evaluated.14
Material and methods From December 5, 1996, to June 13, 2001, 457 consecutive deliveries of infants born between 23 and 32 weeks were evaluated.14 A chart review was performed by trained research nurses to gather demographic characteristics such as maternal race, age, parity, smoking status, diabetes, and pre-eclampsia and determine whether the delivery was spontaneous or indicated. Spontaneous preterm birth was defined as delivery after either spontaneous preterm labor or spontaneous preterm premature rupture of membranes. Indicated preterm birth was defined as delivery effected for maternal or fetal indications. Clinical chorioamnionitis was determined retrospectively by chart review and was considered present if diagnosed by the attending faculty obstetrician, usually for a combination of fever, abdominal pain, and an elevated maternal white count. Any use of corticosteroids for fetal maturation prior to delivery or the use of antibiotics within the 2 weeks prior to delivery was coded as positive for that variable. Birth weight, gestational age at delivery, and infant sex were noted. Placentas for 446 of the neonates (98%) were available for study. In each case, a minimum of 2 membrane rolls, 2 complete sections of umbilical cord (1 from the placental and 1 from the fetal end of the cord), and 2 to 4 transmural parenchymal sections were submitted for histological examination. All cases were evaluated histologically by a single pathologist (O. F. -P.), blinded to clinical outcome, using a placental evaluation protocol adapted from Bendon et al.15 For this study, the free membranes, fetal plate, villi, and umbilical cord were evaluated qualitatively for the presence of polymorphonuclear (PMN) infiltration.16-18 A PMN infiltration was considered present with PMNs in an essentially linear distribution. Specifically, scattered, entrapped, single, or sparsely distributed cells were not scored as an infiltration. The number of PMNs in an infiltration generally exceeded 20 per high-power field (!400) but 5 to 10 or more per high-power field were also scored as a PMN infiltration. Mononuclear infiltrates were divided into lymphohistiocytic and lymphoplasmacytic subtypes,19 and the free membranes, decidua basalis, and fetal plate were also qualitatively evaluated for the presence of both types of chronic inflammation.19 The villi were evaluated for both PMN and mononuclear inflammation. The decidua basalis was also evaluated for the presence or absence of diffuse decidual leukocytoclastic necrosis, a finding commonly associated with vascular insufficiency.20,21 The chorioamnionic interfaces of 445 placentas were cultured for aerobic and anaerobic organisms including Mycoplasma hominis and Ureaplasma urealyticum as previously described.22,23 At delivery, a sample of umbilical cord blood from 343 of the infants was collected by aseptic puncture of the umbilical cord and was cultured
Goldenberg et al Table II
1535
Placental histologic findings in male versus female infants
Placental histologic findings
Male (%) n = 220
Female (%) n = 226
P value
RR (95% CI)
Free membranes: PMN cell infiltration Fetal plate: PMN cell infiltration Cord: PMN cell infiltration Decidua basalis: DDLN Free membranes: lymphohistiocytic infiltration Free membranes: plasmacytic infiltration Decidua basalis: lymphohistiocytic infiltration Decidua basalis: lymphoplasmacytic infiltration Villi: acute inflammation Villi: chronic inflammation
49.8 40.1 29.2 25.6 10.1 0.9 11.3 6.3 0.5 1.0
46.8 39.7 31.8 28.6 9.6 0.9 7.4 0.9 1.4 0.0
.535 .938 .555 .456 .849 .989 .160 .003 .623 .241
1.06 1.01 0.92 0.89 1.06 1.01 1.54 6.7 0.34 NA
(0.88, (0.80, (0.69, (0.65, (0.60, (0.14, (0.84, (1.53, (0.04,
1.29) 1.27) 1.22) 1.21) 1.87) 7.13) 2.81) 29.41) 3.29)
DDLN, Diffuse decidual leukocytoclastic necrosis; RR, risk ratio; CI, confidence interval; NA, not applicable, zero denominator.
for M. hominis and U. urealyticum, as described previously.23 The remaining cord blood (n = 309) was then centrifuged, aliquoted, and stored at –70(C until the interleukin (IL)-6 assay was performed. IL-6 concentrations in the cord plasma were determined by enzymelinked immunosorbent assay kits (R&D Systems, Inc, Minneapolis, MN). The lower limit of sensitivity of the assay was 0.7 pg/mL. The intra-assay and interassay coefficients of variation were 2.6% and 4.5%, respectively. Data analyses were performed with SAS software (version 9, SAS Institute, Inc, Cary, NC). Frequencies and means between groups were compared with the use of c2 tests for discrete variables and t tests and Wilcoxon rank sum tests for continuous variables. A P value less than .05 was chosen to define statistical significance. Logistic regression analyses, adjusting for various maternal characteristics were used to determine the odds ratios (ORs) for placental and umbilical cord cultures and subtypes of chronic decidual inflammation to be associated with male sex. The study was approved by the University of Alabama at Birmingham Institutional Review Board.
Results Table I shows the characteristics of the mothers of the male and female infants enrolled in this study. Male infants were more likely to come from spontaneous rather than indicated deliveries, (P = .025) and weighed about 100 g more (P = .008). There were no differences in corticosteroid or antibiotic use between male and female infants. We next evaluated the relationship between infant sex and various markers of infection/inflammation. Male infants were significantly more likely to have a positive placental culture than were female infants (63.4% versus 51.8%, P = .01). They were also marginally more likely to have cord blood cultures positive for U. urealyticum or
M. hominis (27.6% versus 19.2%, P = .06). Adjusting for race, marital status, corticosteroid use, and birth weight and whether the delivery was indicated or spontaneous, the OR for a positive placental culture being associated with male sex was 1.5 (1.0 to 2.4) and for a positive cord blood mycoplasma or ureaplasma culture to be associated with male sex, 1.7 (0.9 to 3.0). Using a Wilcoxon rank sum test, the distributions of IL-6 levels were similar for male and female infants (median values 11.1 versus 11.3 pg/mL, P = .946). The rate of clinical chorioamnionitis was also similar in the mothers of male and female infants (11.6% versus 14.7%, P = .33). Table II shows the relationship between each of the placental histologic findings and infant sex. Only the relationship between infant sex and the presence of lymphoplasmacytic infiltrations was statistically significant, with the rates higher in placentas of male infants, compared with placentas of female infants (6.3% versus 0.9%, P = .003). Adjusting for race, marital status, corticosteroid use, and birth weight and whether the delivery was indicated or spontaneous, the OR for decidual lymphohistiocytic inflammation being associated with a male fetus was 1.6 (0.8 to 3.2) and for lymphoplasmacytic inflammation, 8.3 (1.8 to 39.0). The percent of placentas with lymphohistiocytic or lymphoplasmacytic infiltrations in other sites was not different between male and female infants. Of note, chronic villitis, a lesion thought to indicate a maternal host-versus-graft response,24 and often seen in association with chronic decidual inflammation,25,26 was present in less than 1% of cases and was not significantly different in placentas from male and female infants.
Comment In this study of 23- to 32-week deliveries, male infants were significantly more likely to be born following a spontaneous rather than indicated delivery and were also significantly more likely to have placental membrane
1536 bacterial infection. Because most early spontaneous preterm births are associated with, and probably caused by, placental membrane infection associated with acute placental inflammation,11,12,27 these data suggest that a portion of the increase in male versus female early preterm births may be due to intrauterine infection. However, the discordance between the significant increase in positive placental membrane cultures in males and the absence of any difference between male and female infant cord IL-6 levels, acute placental histologic inflammation, or clinical chorioamnionitis is unexplained. In this data set, there were slightly more female than male infants. From previous studies, we would have expected the reverse to be true, especially because we were studying early preterm births.6-9 Because these cases were recruited consecutively, selection bias does not explain the female predominance, and we can only conclude that these results occurred by chance. Nevertheless, this finding, plus the fact that this is a secondary analysis of an existing data set, provides further reason to be cautious about accepting infection as an important cause for male/female differences in preterm birth. Furthermore, although the difference was not significant, the fact that the mothers of female infants were more likely to have pre-eclampsia might partially explain the higher rate of indicated preterm births in these infants and thus the greater proportion of spontaneous preterm births in male fetuses. In any case, because of the lack of concordance between the placental culture results and other markers of inflammation, and the larger than expected proportion of female infants,6-9 as well as the higher (but not significantly different) percent of female infants with mothers with pre-eclampsia in this data set, further study is required before the hypothesis of an infection-related increase in male preterm birth can be accepted. As did Ghidini and Salafia,13 we found that in very preterm infants, male versus female gender is associated with higher rates of chronic decidual inflammation of both the lymphohistiocytic and lymphoplasmacytic variety, although in our study, only the difference in lymphoplasmacytic infiltrations was significant. Conversely, in male, compared with female infants, we did not observe an increase in acute inflammation at any site or chronic inflammation in sites other than the decidua basalis. The reason for this difference is not clear, and although it may be related to the increased infection seen in the male placentas, a PMN cell response to bacterial infection is what would generally be expected. Redline et al28 have stated that plasma cells in particular, and B-lymphocytes in general, are always abnormal in the endometrial cavity, and when present, they are an indicator of inappropriate antigenic exposure. Thus, a more aggressive maternal immune response against male versus female fetuses is a plausible explanation for these findings.
Goldenberg et al Supporting this hypothesis is the fact that chronic placental inflammation is increased in women with recurrent miscarriages and indicated preterm birth.29,30 The location of the chronic inflammation at the decidualchorionic interface also suggests that the mononuclear cells are of maternal origin, and this may signal a maternal response against the fetal trophoblast. Further studies that elucidate the character of the lymphohistiocytic and lymphoplasmacytic infiltrates and help define the nature of the maternal response are necessary. In summary, this study found that early preterm male infants were more likely to be born following spontaneous preterm birth and to have a higher percentage of positive intrauterine cultures. They were also more likely to have lymphoplasmacytic inflammation at the choriodecidual interface. Thus, there are differences in some markers of intrauterine infection and inflammation between male and female fetuses. Whether these differences explain the increased preterm birth rate or the higher mortality in male fetuses and newborns will require further study.
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