International Journal of Epidemiology

Murphy, Caitlin C; Cirillo, Piera M; Krigbaum, Nickilou Y; Singal, Amit G; Jones, Dean P; Zaki, Timothy; Cohn, Barbara A

2023 International Journal of Epidemiology

doi: 10.1093/ije/dyad004pmid: 36692207

Abstract Background Incidence rates of colorectal cancer (CRC) are increasing among younger adults and in mid-life, implicating exposures in early life as risk factors. We examined the association between in-utero exposure to antibiotics and risk of CRC in adult offspring. Methods The Child Health and Development Studies is a prospective cohort of women receiving prenatal care between 1959 and 1966 in Oakland, California, with deliveries through June 1967. Diagnosed conditions and all prescribed medications were abstracted from mothers’ medical records beginning 6 months prior to pregnancy through delivery. We identified mothers who received antibiotics in pregnancy, including penicillins, tetracyclines, short-acting sulfonamides and long-acting sulfonamides. Diagnoses of CRC in adult (age ≥18 years) offspring were ascertained through 2021 by linkage with the California Cancer Registry. Cox proportional models were used to estimate adjusted hazard ratios (aHR), with follow-up accrued from birth through cancer diagnosis, death or last contact. Results Of 18 751 liveborn offspring, about 15% (n = 2635) were exposed in utero to antibiotics: 5.4% (n = 1016) to tetracyclines, 4.9% (n = 918) to penicillins, 4.2% (n = 785) to short-acting sulfonamides and 1.5% (n = 273) to long-acting sulfonamides. Compared with offspring not exposed, associations between in-utero exposure and CRC in adult offspring were: aHR 1.03 (95% CI 0.32, 3.31) for tetracyclines; aHR 1.12 (95% CI 0.35, 3.58) for penicillins; aHR 0.83 (95% CI 0.20, 3.42) for short-acting sulfonamides; and aHR 4.40 (95% CI 1.63, 11.88) for long-acting sulfonamides. Conclusion Our findings support an association between in-utero exposure to long-acting sulfonamides and CRC in adulthood. Colorectal cancer, young adult, antimicrobial agent, risk factor Key Messages Incidence rates of colorectal cancer are increasing in younger adults and in mid-life, but the reason for this increase is not known. Early exposure to antibiotics has been suggested as a potential explanation. In this prospective study of more than 18 000 mother-child dyads, in-utero exposure to long-acting sulfonamides increased risk of colorectal cancer in adult offspring. There was no difference in risk associated with short-acting sulfonamides or other antibiotic classes, such as tetracyclines and penicillins. Although long-acting sulfonamides are no longer routinely prescribed in humans, their continued presence as environmental pollutants raise the possibility that long-term exposure may also increase risk of colorectal cancer. Introduction Incidence rates of colorectal cancer (CRC) have increased in young adults (age 18–49 years) in the USA, and more recently rates have increased in adults in their early 50s.1 The shifting epidemiology of CRC has sparked interest in identifying novel risk factors.2 Importantly, incidence rates of CRC have increased across birth cohorts,3 starting with persons born in the 1960s and implicating exposures in early life as risk factors. Early life, beginning in utero, represents a critical window of susceptibility,4 and exposures during this time can translate into large effects on risk of cancer in adulthood. A robust experimental and epidemiological literature supports the importance of gestation and infancy for several adult cancers.5 In the 1960s, antibiotics were frequently prescribed to pregnant women to treat urinary tract infections,6,7 upper respiratory infections, sexually transmitted infections and skin infections.8 Antibiotics cross the placental barrier via simple diffusion,9 and exposure in utero may directly affect the developing gastrointestinal tract of the fetus through alterations in maternal microbiota or immune response.10 Exposure in utero may also have prolonged antimicrobial effects in newborns.11 This is consistent with embryo-fetal toxicity of several antibiotic classes (e.g. tetracyclines12,13), as well as epidemiological studies demonstrating long-term consequences of antibiotic exposure in early life for childhood obesity,14–16 a well-established risk factor for CRC.17 We examined the association of in-utero exposure to antibiotics and CRC in adult offspring of the Child Health and Developments Studies (CHDS), a multi-generational cohort of more than 18 000 mother-child dyads followed for 60 years. The CHDS provides a unique opportunity to study impacts of early life by linking in-utero exposures with cancers ascertained from a population-based registry.18,19 Materials and methods Study population The CHDS was established in 1959 and recruited nearly all (98%) pregnant women receiving prenatal care from the Kaiser Foundation Health Plan (Oakland, CA) between June 1959 and September 1966, with deliveries through June 1967. Embedding the study in the Kaiser Foundation Health Plan provided several advantages: mothers received all care at centralized facilities; care was equally available; and medical services, including visits and prescribed medications provided by all physicians across specialties, were assembled for each mother into a single file. Additional details of the CHDS and methodology are available.20 CHDS participants are regularly monitored by linkage to the California Department of Motor Vehicles, California Department of Vital Statistics and California Cancer Registry. We supplement surveillance by additionally linking with the National Death Index. Mothers and their families are matched to these sources using an accumulated name and address history, successfully identifying more than 80% of families. Primary outcome We ascertained diagnoses of CRC in adult (age ≥18 years) offspring through 2021 by linkage with the California Cancer Registry (International Classification of Disease in Oncology, 3rd edition, codes C18.0–1, C19.9, C20.9). We used a rigorous protocol to verify cases, comparing fixed (e.g. birth date, sex, race) and changeable (e.g. address) identifiers by manual review. In-utero exposure to antibiotics Clinical information, including prenatal visits, diagnosed conditions and all prescribed medications, were abstracted from mothers’ medical records beginning 6 months prior to pregnancy through labour and delivery. We identified mothers who received antibiotics during pregnancy, including the timing (first trimester: Days 0–90; second trimester: Days 91–180; third trimester: Days ≥181), frequency and indication. Because the mechanism of action differs across antibiotic classes, we examined in-utero exposure to: (i) tetracyclines; (ii) penicillins; (iii) short-acting sulfonamides; and (iv) long-acting sulfonamides (Supplementary Table S1, available as Supplementary data at IJE online). We distinguished short- from long-acting sulfonamides because pharmacokinetics considerably differ.21 We did not examine other classes, including cephalosporins and macrolides, because they were not available or rarely prescribed in the late 1950s and 60s. Statistical analysis We estimated incidence rates of CRC and 95% confidence intervals based on the discrete probability distribution for a binomial parameter, separately for any in-utero exposure to tetracyclines, penicillins, short-acting sulfonamides and long-acting sulfonamides. We used Cox proportional hazards models to estimate hazard ratios (HR) and their 95% confidence intervals, accounting for correlation between observations from siblings (n = 4244) with robust sandwich estimators. Follow-up time was accrued from birth through date of cancer diagnosis, death or last contact. The majority of exposed offspring (87.6% of 2635 exposed, Supplementary Table S2, available as Supplementary data at IJE online) were exposed to only one antibiotic class. Therefore, we built separate models for any in-utero exposure to tetracyclines, penicillins, short-acting sulfonamides and long-acting sulfonamides, with offspring not exposed to any antibiotic (n = 16 116) as the reference category. Those exposed to more than one antibiotic class (12.4% of 2635 exposed) contributed exposed person-time to more than one model, as appropriate. For example, offspring exposed to penicillins and tetracyclines contributed exposed person-time to the penicillin and tetracycline models. We selected confounders a priori as maternal characteristics associated with both in-utero exposure to antibiotics and CRC in adult offspring,19 year of birth, maternal smoking (current vs else), maternal race (non-Hispanic Black vs else) and maternal body mass index (overweight or obese vs else). Maternal smoking and race were reported by mothers during in-person interviews at enrollment. We used height and weight reported by mothers during in-person interviews at enrollment or recorded at the first prenatal visit to measure body mass index. We did not adjust for mediators (e.g. gestational age, birthweight), as recommended in the literature.22 We assessed the proportional hazards assumption in adjusted models by including an interaction term of log(time) and in-utero exposure to antibiotics. The assumption was not violated in any model (tetracyclines: P = 0.55; penicillins: P = 0.72; short-acting sulfonamides: P = 0.66; long-acting sulfonamides: P = 0.45). Sensitivity analyses To address the possibility of confounding by indication, we examined the association between upper respiratory infection, urinary tract infection, sexually transmitted infection and skin infection in mothers and CRC in offspring, using Cox proportional hazards models as detailed above. We also built adjusted models of in-utero exposure that included the indicating condition. We calculated E-values23,24 to address unmeasured confounding. E-values provide an estimate of the minimum strength of association that an unmeasured confounder must have with both the exposure and outcome to explain away the observed association. We applied the formula RR + sqrt[RR x (RR-1)] and report the limit of the confidence interval closest to the null. Missingness ranged from 1.5% (maternal race) to 20.8% (maternal smoking) for variables included in adjusted models. We used multiple imputation by fully conditional specification to estimate associations with CRC in adult offspring, separately for any in-utero exposure to tetracyclines, penicillins, short-acting sulfonamides and long-acting sulfonamides. Fully conditional specification25 relaxes assumptions of joint multivariate normality and linearity and is well suited for imputation of both categorical and continuous variables. All analyses were conducted in SAS version 9.4 (SAS Institute, Cary, NC). Patient and public involvement The CHDS routinely engages its own cohort members in community-based participatory research. We meet quarterly with our Participant Advisory Council (PAC), a racially and sexually diverse representation of the cohort, to develop research questions, resolve ethical issues related to study participation, design innovative recruitment methods and improve dissemination of findings. At the beginning of this study, we met with PAC members to provide an overview of CRC and to discuss study concepts, rationale and approach; they asked questions and offered feedback.19 Meetings will be scheduled in the future to present and interpret ongoing results, as well as plans to disseminate findings to the larger cohort. Results Of 18 751 liveborn offspring, about 15% (n = 2635) were exposed in utero to antibiotics: 5.4% (n = 1016) to tetracyclines, 4.9% (n = 918) to penicillins, 4.2% (n = 785) to short-acting sulfonamides and 1.5% (n = 273) to long-acting sulfonamides (Figure 1). Table 1 shows characteristics of offspring by any in-utero exposure to tetracyclines, penicillins, short-acting sulfonamides and long-acting sulfonamides. Median and mean follow-up (in years) was similar by in-utero exposure to antibiotics [tetracyclines: median 50.5 (IQR 27.5–53.5), mean 40.4 (SD 18.5); penicillins: median 50.5 (IQR 29.5–52.5), mean 40.4 (SD 18.0); short-acting sulfonamides: median 49.5 (IQR 26.5–53.5), mean 39.0 (SD 18.7); long-acting sulfonamides: median 50.5 (IQR 27.5–53.5), mean 40.4 (SD 18.5); and not exposed: median 50.5 (IQR 26.5–53.5), mean 39.3 (SD 19.0)]. Figure 1 Open in new tabDownload slide Study flow diagram Table 1 Characteristics of 18 751 liveborn offspring of the Child Health and Development Studies, 1959–67, by in-utero exposure to antibiotics . No in-utero exposure (n = 16 116) . In-utero exposure to tetracyclines (n = 1016) . In-utero exposure to penicillins (n = 918) . In-utero exposure to short-acting sulfonamides (n = 785) . In-utero exposure to long-acting sulfonamides (n = 273) . Offspring characteristics, n (%) Sex  Male 8228 (51.1) 519 (51.1) 463 (50.4) 411 (52.4) 155 (56.8)  Female 7888 (49.0) 497 (48.9) 455 (49.6) 374 (47.6) 118 (43.2) Year of birth  1959–61 4878 (30.3) 245 (24.1) 252 (27.5) 206 (26.2) 110 (40.3)  1962–64 7730 (48.0) 609 (59.9) 392 (42.7) 371 (47.3) 134 (49.1)  1965–67 3508 (21.8) 162 (15.9) 274 (29.8) 208 (26.5) 29 (10.6) Race and ethnicity  Non-Hispanic White 10544 (66.4) 737 (73.6) 558 (61.5) 477 (61.5) 184 (67.9)  Non-Hispanic Black 3645 (23.0) 195 (19.5) 291 (32.1) 240 (30.9) 62 (22.9)  Hispanic 538 (3.4) 25 (2.5) 20 (2.2) 33 (4.3) 8 (3.0)  Asian 674 (4.3) 20 (2.0) 14 (1.5) 8 (1.0) 6 (2.2)  Other 471 (3.0) 24 (2.4) 25 (2.8) 18 (2.3) 11 (4.1)  Missing 244 15 10 9 2 Gestational age  <37 weeks 1239 (7.8) 76 (7.5) 76 (8.3) 76 (9.7) 29 (10.6)  ≥37 weeks 14579 (92.2) 940 (92.5) 842 (91.7) 709 (90.3) 244 (89.4)  Missing 298 Birthweight (grams)  <2500 913 (5.7) 61 (6.0) 57 (6.2) 55 (7.0) 23 (8.4)  2500–3999 13841 (85.9) 848 (83.5) 776 (84.5) 664 (84.6) 223 (81.7)  ≥4000 1362 (8.5) 107 (10.5) 85 (9.3) 66 (8.4) 27 (9.9) Maternal characteristics Maternal age at pregnancy (years)  <24 1433 (9.0) 70 (7.0) 94 (10.4) 91 (11.7) 27 (10.0)  20–24 4849 (30.4) 287 (28.5) 282 (31.1) 249 (32.1) 85 (31.5)  25–29 4640 (29.1) 290 (28.8) 245 (27.0) 227 (29.2) 76 (28.2)  30–34 2822 (17.7) 222 (22.1) 160 (17.6) 137 (17.6) 45 (16.7)  35–39 1661 (10.4) 116 (11.5) 100 (11.0) 58 (7.5) 27 (10.0)  ≥40 565 (3.5) 21 (2.1) 27 (3.0) 15 (1.9) 10 (3.7)  Missing 146 10 10 8 3 Parity at pregnancy  Primiparous 5056 (31.6) 227 (22.4) 245 (26.9) 240 (30.8) 75 (27.5)  Multiparous 10939 (68.4) 786 (77.6) 667 (73.1) 539 (69.2) 198 (72.5)  Missing 3 6 6 0 Body mass index (kg/m2)  Underweight or healthy (<25) 10508 (75.4) 654 (75.7) 573 (70.7) 532 (74.8) 183 (78.5)  Overweight or obese (≥25) 3428 (24.6) 210 (24.3) 237 (29.3) 179 (25.2) 50 (21.5)  Missing 913 152 108 74 40 Maternal education  Less than high school 2459 (17.9) 150 (17.4) 152 (19.7) 149 (22.1) 57 (24.7)  High school or trade school 5299 (38.5) 360 (41.8) 309 (40.1) 276 (41.0) 76 (32.9)  Some college or college degree 5995 (43.6) 351 (40.8) 309 (40.1) 249 (36.9) 98 (42.4)  Missing 2363 155 148 111 42 Annual family income4  <Median 4182 (36.7) 220 (31.6) 218 (35.7) 208 (38.0) 76 (39.8)  ≥Median 7221 (63.3) 477 (68.4) 393 (64.3) 339 (62.0) 115 (60.2)  Missing 4713 319 307 238 82 Smoking  Never 6203 (48.5) 306 (39.4) 297 (40.9) 283 (45.6) 96 (44.4)  Former 2190 (17.1) 133 (17.4) 110 (15.7) 107 (17.3) 35 (16.2)  Current 4404 (34.4) 337 (43.4) 304 (43.4) 230 (37.1) 85 (39.4)  Missing 3319 240 217 165 57 . No in-utero exposure (n = 16 116) . In-utero exposure to tetracyclines (n = 1016) . In-utero exposure to penicillins (n = 918) . In-utero exposure to short-acting sulfonamides (n = 785) . In-utero exposure to long-acting sulfonamides (n = 273) . Offspring characteristics, n (%) Sex  Male 8228 (51.1) 519 (51.1) 463 (50.4) 411 (52.4) 155 (56.8)  Female 7888 (49.0) 497 (48.9) 455 (49.6) 374 (47.6) 118 (43.2) Year of birth  1959–61 4878 (30.3) 245 (24.1) 252 (27.5) 206 (26.2) 110 (40.3)  1962–64 7730 (48.0) 609 (59.9) 392 (42.7) 371 (47.3) 134 (49.1)  1965–67 3508 (21.8) 162 (15.9) 274 (29.8) 208 (26.5) 29 (10.6) Race and ethnicity  Non-Hispanic White 10544 (66.4) 737 (73.6) 558 (61.5) 477 (61.5) 184 (67.9)  Non-Hispanic Black 3645 (23.0) 195 (19.5) 291 (32.1) 240 (30.9) 62 (22.9)  Hispanic 538 (3.4) 25 (2.5) 20 (2.2) 33 (4.3) 8 (3.0)  Asian 674 (4.3) 20 (2.0) 14 (1.5) 8 (1.0) 6 (2.2)  Other 471 (3.0) 24 (2.4) 25 (2.8) 18 (2.3) 11 (4.1)  Missing 244 15 10 9 2 Gestational age  <37 weeks 1239 (7.8) 76 (7.5) 76 (8.3) 76 (9.7) 29 (10.6)  ≥37 weeks 14579 (92.2) 940 (92.5) 842 (91.7) 709 (90.3) 244 (89.4)  Missing 298 Birthweight (grams)  <2500 913 (5.7) 61 (6.0) 57 (6.2) 55 (7.0) 23 (8.4)  2500–3999 13841 (85.9) 848 (83.5) 776 (84.5) 664 (84.6) 223 (81.7)  ≥4000 1362 (8.5) 107 (10.5) 85 (9.3) 66 (8.4) 27 (9.9) Maternal characteristics Maternal age at pregnancy (years)  <24 1433 (9.0) 70 (7.0) 94 (10.4) 91 (11.7) 27 (10.0)  20–24 4849 (30.4) 287 (28.5) 282 (31.1) 249 (32.1) 85 (31.5)  25–29 4640 (29.1) 290 (28.8) 245 (27.0) 227 (29.2) 76 (28.2)  30–34 2822 (17.7) 222 (22.1) 160 (17.6) 137 (17.6) 45 (16.7)  35–39 1661 (10.4) 116 (11.5) 100 (11.0) 58 (7.5) 27 (10.0)  ≥40 565 (3.5) 21 (2.1) 27 (3.0) 15 (1.9) 10 (3.7)  Missing 146 10 10 8 3 Parity at pregnancy  Primiparous 5056 (31.6) 227 (22.4) 245 (26.9) 240 (30.8) 75 (27.5)  Multiparous 10939 (68.4) 786 (77.6) 667 (73.1) 539 (69.2) 198 (72.5)  Missing 3 6 6 0 Body mass index (kg/m2)  Underweight or healthy (<25) 10508 (75.4) 654 (75.7) 573 (70.7) 532 (74.8) 183 (78.5)  Overweight or obese (≥25) 3428 (24.6) 210 (24.3) 237 (29.3) 179 (25.2) 50 (21.5)  Missing 913 152 108 74 40 Maternal education  Less than high school 2459 (17.9) 150 (17.4) 152 (19.7) 149 (22.1) 57 (24.7)  High school or trade school 5299 (38.5) 360 (41.8) 309 (40.1) 276 (41.0) 76 (32.9)  Some college or college degree 5995 (43.6) 351 (40.8) 309 (40.1) 249 (36.9) 98 (42.4)  Missing 2363 155 148 111 42 Annual family income4  <Median 4182 (36.7) 220 (31.6) 218 (35.7) 208 (38.0) 76 (39.8)  ≥Median 7221 (63.3) 477 (68.4) 393 (64.3) 339 (62.0) 115 (60.2)  Missing 4713 319 307 238 82 Smoking  Never 6203 (48.5) 306 (39.4) 297 (40.9) 283 (45.6) 96 (44.4)  Former 2190 (17.1) 133 (17.4) 110 (15.7) 107 (17.3) 35 (16.2)  Current 4404 (34.4) 337 (43.4) 304 (43.4) 230 (37.1) 85 (39.4)  Missing 3319 240 217 165 57 In-utero exposure to tetracyclines, penicillins, short-acting sulfonamides and long-acting sulfonamides not mutually exclusive, and 327 offspring were exposed to more than one class of antibiotics (see Supplementary Table S2, available as Supplementary data at IJE online). Offspring exposed to intermediate-acting sulfonamides or sulfonamides not otherwise specified are not included in the 785 or 273 offspring exposed to short- and long-acting sulfonamides, respectively. Open in new tab Table 1 Characteristics of 18 751 liveborn offspring of the Child Health and Development Studies, 1959–67, by in-utero exposure to antibiotics . No in-utero exposure (n = 16 116) . In-utero exposure to tetracyclines (n = 1016) . In-utero exposure to penicillins (n = 918) . In-utero exposure to short-acting sulfonamides (n = 785) . In-utero exposure to long-acting sulfonamides (n = 273) . Offspring characteristics, n (%) Sex  Male 8228 (51.1) 519 (51.1) 463 (50.4) 411 (52.4) 155 (56.8)  Female 7888 (49.0) 497 (48.9) 455 (49.6) 374 (47.6) 118 (43.2) Year of birth  1959–61 4878 (30.3) 245 (24.1) 252 (27.5) 206 (26.2) 110 (40.3)  1962–64 7730 (48.0) 609 (59.9) 392 (42.7) 371 (47.3) 134 (49.1)  1965–67 3508 (21.8) 162 (15.9) 274 (29.8) 208 (26.5) 29 (10.6) Race and ethnicity  Non-Hispanic White 10544 (66.4) 737 (73.6) 558 (61.5) 477 (61.5) 184 (67.9)  Non-Hispanic Black 3645 (23.0) 195 (19.5) 291 (32.1) 240 (30.9) 62 (22.9)  Hispanic 538 (3.4) 25 (2.5) 20 (2.2) 33 (4.3) 8 (3.0)  Asian 674 (4.3) 20 (2.0) 14 (1.5) 8 (1.0) 6 (2.2)  Other 471 (3.0) 24 (2.4) 25 (2.8) 18 (2.3) 11 (4.1)  Missing 244 15 10 9 2 Gestational age  <37 weeks 1239 (7.8) 76 (7.5) 76 (8.3) 76 (9.7) 29 (10.6)  ≥37 weeks 14579 (92.2) 940 (92.5) 842 (91.7) 709 (90.3) 244 (89.4)  Missing 298 Birthweight (grams)  <2500 913 (5.7) 61 (6.0) 57 (6.2) 55 (7.0) 23 (8.4)  2500–3999 13841 (85.9) 848 (83.5) 776 (84.5) 664 (84.6) 223 (81.7)  ≥4000 1362 (8.5) 107 (10.5) 85 (9.3) 66 (8.4) 27 (9.9) Maternal characteristics Maternal age at pregnancy (years)  <24 1433 (9.0) 70 (7.0) 94 (10.4) 91 (11.7) 27 (10.0)  20–24 4849 (30.4) 287 (28.5) 282 (31.1) 249 (32.1) 85 (31.5)  25–29 4640 (29.1) 290 (28.8) 245 (27.0) 227 (29.2) 76 (28.2)  30–34 2822 (17.7) 222 (22.1) 160 (17.6) 137 (17.6) 45 (16.7)  35–39 1661 (10.4) 116 (11.5) 100 (11.0) 58 (7.5) 27 (10.0)  ≥40 565 (3.5) 21 (2.1) 27 (3.0) 15 (1.9) 10 (3.7)  Missing 146 10 10 8 3 Parity at pregnancy  Primiparous 5056 (31.6) 227 (22.4) 245 (26.9) 240 (30.8) 75 (27.5)  Multiparous 10939 (68.4) 786 (77.6) 667 (73.1) 539 (69.2) 198 (72.5)  Missing 3 6 6 0 Body mass index (kg/m2)  Underweight or healthy (<25) 10508 (75.4) 654 (75.7) 573 (70.7) 532 (74.8) 183 (78.5)  Overweight or obese (≥25) 3428 (24.6) 210 (24.3) 237 (29.3) 179 (25.2) 50 (21.5)  Missing 913 152 108 74 40 Maternal education  Less than high school 2459 (17.9) 150 (17.4) 152 (19.7) 149 (22.1) 57 (24.7)  High school or trade school 5299 (38.5) 360 (41.8) 309 (40.1) 276 (41.0) 76 (32.9)  Some college or college degree 5995 (43.6) 351 (40.8) 309 (40.1) 249 (36.9) 98 (42.4)  Missing 2363 155 148 111 42 Annual family income4  <Median 4182 (36.7) 220 (31.6) 218 (35.7) 208 (38.0) 76 (39.8)  ≥Median 7221 (63.3) 477 (68.4) 393 (64.3) 339 (62.0) 115 (60.2)  Missing 4713 319 307 238 82 Smoking  Never 6203 (48.5) 306 (39.4) 297 (40.9) 283 (45.6) 96 (44.4)  Former 2190 (17.1) 133 (17.4) 110 (15.7) 107 (17.3) 35 (16.2)  Current 4404 (34.4) 337 (43.4) 304 (43.4) 230 (37.1) 85 (39.4)  Missing 3319 240 217 165 57 . No in-utero exposure (n = 16 116) . In-utero exposure to tetracyclines (n = 1016) . In-utero exposure to penicillins (n = 918) . In-utero exposure to short-acting sulfonamides (n = 785) . In-utero exposure to long-acting sulfonamides (n = 273) . Offspring characteristics, n (%) Sex  Male 8228 (51.1) 519 (51.1) 463 (50.4) 411 (52.4) 155 (56.8)  Female 7888 (49.0) 497 (48.9) 455 (49.6) 374 (47.6) 118 (43.2) Year of birth  1959–61 4878 (30.3) 245 (24.1) 252 (27.5) 206 (26.2) 110 (40.3)  1962–64 7730 (48.0) 609 (59.9) 392 (42.7) 371 (47.3) 134 (49.1)  1965–67 3508 (21.8) 162 (15.9) 274 (29.8) 208 (26.5) 29 (10.6) Race and ethnicity  Non-Hispanic White 10544 (66.4) 737 (73.6) 558 (61.5) 477 (61.5) 184 (67.9)  Non-Hispanic Black 3645 (23.0) 195 (19.5) 291 (32.1) 240 (30.9) 62 (22.9)  Hispanic 538 (3.4) 25 (2.5) 20 (2.2) 33 (4.3) 8 (3.0)  Asian 674 (4.3) 20 (2.0) 14 (1.5) 8 (1.0) 6 (2.2)  Other 471 (3.0) 24 (2.4) 25 (2.8) 18 (2.3) 11 (4.1)  Missing 244 15 10 9 2 Gestational age  <37 weeks 1239 (7.8) 76 (7.5) 76 (8.3) 76 (9.7) 29 (10.6)  ≥37 weeks 14579 (92.2) 940 (92.5) 842 (91.7) 709 (90.3) 244 (89.4)  Missing 298 Birthweight (grams)  <2500 913 (5.7) 61 (6.0) 57 (6.2) 55 (7.0) 23 (8.4)  2500–3999 13841 (85.9) 848 (83.5) 776 (84.5) 664 (84.6) 223 (81.7)  ≥4000 1362 (8.5) 107 (10.5) 85 (9.3) 66 (8.4) 27 (9.9) Maternal characteristics Maternal age at pregnancy (years)  <24 1433 (9.0) 70 (7.0) 94 (10.4) 91 (11.7) 27 (10.0)  20–24 4849 (30.4) 287 (28.5) 282 (31.1) 249 (32.1) 85 (31.5)  25–29 4640 (29.1) 290 (28.8) 245 (27.0) 227 (29.2) 76 (28.2)  30–34 2822 (17.7) 222 (22.1) 160 (17.6) 137 (17.6) 45 (16.7)  35–39 1661 (10.4) 116 (11.5) 100 (11.0) 58 (7.5) 27 (10.0)  ≥40 565 (3.5) 21 (2.1) 27 (3.0) 15 (1.9) 10 (3.7)  Missing 146 10 10 8 3 Parity at pregnancy  Primiparous 5056 (31.6) 227 (22.4) 245 (26.9) 240 (30.8) 75 (27.5)  Multiparous 10939 (68.4) 786 (77.6) 667 (73.1) 539 (69.2) 198 (72.5)  Missing 3 6 6 0 Body mass index (kg/m2)  Underweight or healthy (<25) 10508 (75.4) 654 (75.7) 573 (70.7) 532 (74.8) 183 (78.5)  Overweight or obese (≥25) 3428 (24.6) 210 (24.3) 237 (29.3) 179 (25.2) 50 (21.5)  Missing 913 152 108 74 40 Maternal education  Less than high school 2459 (17.9) 150 (17.4) 152 (19.7) 149 (22.1) 57 (24.7)  High school or trade school 5299 (38.5) 360 (41.8) 309 (40.1) 276 (41.0) 76 (32.9)  Some college or college degree 5995 (43.6) 351 (40.8) 309 (40.1) 249 (36.9) 98 (42.4)  Missing 2363 155 148 111 42 Annual family income4  <Median 4182 (36.7) 220 (31.6) 218 (35.7) 208 (38.0) 76 (39.8)  ≥Median 7221 (63.3) 477 (68.4) 393 (64.3) 339 (62.0) 115 (60.2)  Missing 4713 319 307 238 82 Smoking  Never 6203 (48.5) 306 (39.4) 297 (40.9) 283 (45.6) 96 (44.4)  Former 2190 (17.1) 133 (17.4) 110 (15.7) 107 (17.3) 35 (16.2)  Current 4404 (34.4) 337 (43.4) 304 (43.4) 230 (37.1) 85 (39.4)  Missing 3319 240 217 165 57 In-utero exposure to tetracyclines, penicillins, short-acting sulfonamides and long-acting sulfonamides not mutually exclusive, and 327 offspring were exposed to more than one class of antibiotics (see Supplementary Table S2, available as Supplementary data at IJE online). Offspring exposed to intermediate-acting sulfonamides or sulfonamides not otherwise specified are not included in the 785 or 273 offspring exposed to short- and long-acting sulfonamides, respectively. Open in new tab Timing and frequency of exposure was similar across antibiotic classes (Table 3). About 35% of exposed offspring were first exposed in the first trimester and most offspring were exposed to one prescription. There were some differences in indication by antibiotic class. For example, urinary tract infection was the most common indication for short-acting (84.8%) and long-acting (52.4%) sulfonamides, whereas upper respiratory infection was the most common indication for tetracyclines (66.4%) and penicillins (50.1%). Table 2 Characteristics of 80 adult offspring diagnosed with colorectal cancer . n . (%) . Sex  Male 39 48.8  Female 41 51.3 Year of birth  1959–61 32 40.0  1962–64 38 47.5  1965–67 10 12.5 Race and ethnicity  Non-Hispanic White 40 52.0  Non-Hispanic Black 26 33.8  Hispanic 5 6.5  Asian 3 3.9  Other 3 3.9  Missing 3 Year of diagnosis  1980–89  1990–99 2 2.5  2000–09 6 7.5  2010+ 26 32.5 Age at diagnosis (years) 46 57.5 Median (IQR) 50 (46–53)  18–29 2 2.5  30–39 3 3.8  40–49 17 21.3  50–59 58 72.5 Stage at diagnosis  Local 21 30.0  Regional 31 44.3  Distant 18 25.7  Missing 10 Tumour location  Proximal colon 21 26.9  Distal colon 33 42.3  Rectum 24 30.8  Missing 2 Family history of CRCa  No 68 85.0  Yes 12 15.0 . n . (%) . Sex  Male 39 48.8  Female 41 51.3 Year of birth  1959–61 32 40.0  1962–64 38 47.5  1965–67 10 12.5 Race and ethnicity  Non-Hispanic White 40 52.0  Non-Hispanic Black 26 33.8  Hispanic 5 6.5  Asian 3 3.9  Other 3 3.9  Missing 3 Year of diagnosis  1980–89  1990–99 2 2.5  2000–09 6 7.5  2010+ 26 32.5 Age at diagnosis (years) 46 57.5 Median (IQR) 50 (46–53)  18–29 2 2.5  30–39 3 3.8  40–49 17 21.3  50–59 58 72.5 Stage at diagnosis  Local 21 30.0  Regional 31 44.3  Distant 18 25.7  Missing 10 Tumour location  Proximal colon 21 26.9  Distal colon 33 42.3  Rectum 24 30.8  Missing 2 Family history of CRCa  No 68 85.0  Yes 12 15.0 CRC, colorectal cancer; IQR, interquartile range. a Family history of CRC defined as diagnosis in mother or father. Open in new tab Table 2 Characteristics of 80 adult offspring diagnosed with colorectal cancer . n . (%) . Sex  Male 39 48.8  Female 41 51.3 Year of birth  1959–61 32 40.0  1962–64 38 47.5  1965–67 10 12.5 Race and ethnicity  Non-Hispanic White 40 52.0  Non-Hispanic Black 26 33.8  Hispanic 5 6.5  Asian 3 3.9  Other 3 3.9  Missing 3 Year of diagnosis  1980–89  1990–99 2 2.5  2000–09 6 7.5  2010+ 26 32.5 Age at diagnosis (years) 46 57.5 Median (IQR) 50 (46–53)  18–29 2 2.5  30–39 3 3.8  40–49 17 21.3  50–59 58 72.5 Stage at diagnosis  Local 21 30.0  Regional 31 44.3  Distant 18 25.7  Missing 10 Tumour location  Proximal colon 21 26.9  Distal colon 33 42.3  Rectum 24 30.8  Missing 2 Family history of CRCa  No 68 85.0  Yes 12 15.0 . n . (%) . Sex  Male 39 48.8  Female 41 51.3 Year of birth  1959–61 32 40.0  1962–64 38 47.5  1965–67 10 12.5 Race and ethnicity  Non-Hispanic White 40 52.0  Non-Hispanic Black 26 33.8  Hispanic 5 6.5  Asian 3 3.9  Other 3 3.9  Missing 3 Year of diagnosis  1980–89  1990–99 2 2.5  2000–09 6 7.5  2010+ 26 32.5 Age at diagnosis (years) 46 57.5 Median (IQR) 50 (46–53)  18–29 2 2.5  30–39 3 3.8  40–49 17 21.3  50–59 58 72.5 Stage at diagnosis  Local 21 30.0  Regional 31 44.3  Distant 18 25.7  Missing 10 Tumour location  Proximal colon 21 26.9  Distal colon 33 42.3  Rectum 24 30.8  Missing 2 Family history of CRCa  No 68 85.0  Yes 12 15.0 CRC, colorectal cancer; IQR, interquartile range. a Family history of CRC defined as diagnosis in mother or father. Open in new tab Over 739 177.5 person-years of follow-up, 80 adult offspring were diagnosed with CRC (Table 2). Offspring were diagnosed between ages 23 and 59 years (median: 50.0 years, IQR: 46.0 –53.0 years); 15% had a family history of CRC. Table 3 Timing, frequency and indication of in-utero exposure to antibiotics . Tetracyclines (n = 1106) . Penicillins (n = 918) . Short-acting sulfonamides (n = 785) . Long-acting sulfonamides (n = 273) . . n . % . n . % . n . % . n . % . Timing of first exposure  Gestational week—median (IQR) 18.4 (8.4–28.5) 18.3 (8.1–29.1) 18.9 (8.4–27.9) 16.9 (6.7–26.3)  First trimester 372 (36.6) 342 (37.3) 285 (36.3) 109 (39.9)  Second trimester 325 (32.0) 279 (30.4) 265 (33.8) 92 (33.7)  Third trimester 319 (31.4) 297 (32.4) 235 (29.9) 72 (26.4) Number of prescriptions  1 846 (83.3) 596 (64.9) 663 (84.5) 248 (90.8)  ≥2 170 (16.7) 322 (35.1) 122 (15.5) 25 (9.2) Indication  Upper respiratory infection 675 (66.4) 460 (50.1) 43 (5.5) 74 (27.1)  Urinary tract infection 70 (6.9) 35 (3.8) 666 (84.8) 143 (52.4)  Sexually transmitted infection 3 (0.3) 99 (10.8) 0 – 0 –  Skin infection 94 (9.3) 68 (7.4) 12 (1.8) 35 (12.8)  Other infection 174 (17.1) 256 (27.9) 64 (8.2) 21 (7.7) . Tetracyclines (n = 1106) . Penicillins (n = 918) . Short-acting sulfonamides (n = 785) . Long-acting sulfonamides (n = 273) . . n . % . n . % . n . % . n . % . Timing of first exposure  Gestational week—median (IQR) 18.4 (8.4–28.5) 18.3 (8.1–29.1) 18.9 (8.4–27.9) 16.9 (6.7–26.3)  First trimester 372 (36.6) 342 (37.3) 285 (36.3) 109 (39.9)  Second trimester 325 (32.0) 279 (30.4) 265 (33.8) 92 (33.7)  Third trimester 319 (31.4) 297 (32.4) 235 (29.9) 72 (26.4) Number of prescriptions  1 846 (83.3) 596 (64.9) 663 (84.5) 248 (90.8)  ≥2 170 (16.7) 322 (35.1) 122 (15.5) 25 (9.2) Indication  Upper respiratory infection 675 (66.4) 460 (50.1) 43 (5.5) 74 (27.1)  Urinary tract infection 70 (6.9) 35 (3.8) 666 (84.8) 143 (52.4)  Sexually transmitted infection 3 (0.3) 99 (10.8) 0 – 0 –  Skin infection 94 (9.3) 68 (7.4) 12 (1.8) 35 (12.8)  Other infection 174 (17.1) 256 (27.9) 64 (8.2) 21 (7.7) IQR, interquartile range. Open in new tab Table 3 Timing, frequency and indication of in-utero exposure to antibiotics . Tetracyclines (n = 1106) . Penicillins (n = 918) . Short-acting sulfonamides (n = 785) . Long-acting sulfonamides (n = 273) . . n . % . n . % . n . % . n . % . Timing of first exposure  Gestational week—median (IQR) 18.4 (8.4–28.5) 18.3 (8.1–29.1) 18.9 (8.4–27.9) 16.9 (6.7–26.3)  First trimester 372 (36.6) 342 (37.3) 285 (36.3) 109 (39.9)  Second trimester 325 (32.0) 279 (30.4) 265 (33.8) 92 (33.7)  Third trimester 319 (31.4) 297 (32.4) 235 (29.9) 72 (26.4) Number of prescriptions  1 846 (83.3) 596 (64.9) 663 (84.5) 248 (90.8)  ≥2 170 (16.7) 322 (35.1) 122 (15.5) 25 (9.2) Indication  Upper respiratory infection 675 (66.4) 460 (50.1) 43 (5.5) 74 (27.1)  Urinary tract infection 70 (6.9) 35 (3.8) 666 (84.8) 143 (52.4)  Sexually transmitted infection 3 (0.3) 99 (10.8) 0 – 0 –  Skin infection 94 (9.3) 68 (7.4) 12 (1.8) 35 (12.8)  Other infection 174 (17.1) 256 (27.9) 64 (8.2) 21 (7.7) . Tetracyclines (n = 1106) . Penicillins (n = 918) . Short-acting sulfonamides (n = 785) . Long-acting sulfonamides (n = 273) . . n . % . n . % . n . % . n . % . Timing of first exposure  Gestational week—median (IQR) 18.4 (8.4–28.5) 18.3 (8.1–29.1) 18.9 (8.4–27.9) 16.9 (6.7–26.3)  First trimester 372 (36.6) 342 (37.3) 285 (36.3) 109 (39.9)  Second trimester 325 (32.0) 279 (30.4) 265 (33.8) 92 (33.7)  Third trimester 319 (31.4) 297 (32.4) 235 (29.9) 72 (26.4) Number of prescriptions  1 846 (83.3) 596 (64.9) 663 (84.5) 248 (90.8)  ≥2 170 (16.7) 322 (35.1) 122 (15.5) 25 (9.2) Indication  Upper respiratory infection 675 (66.4) 460 (50.1) 43 (5.5) 74 (27.1)  Urinary tract infection 70 (6.9) 35 (3.8) 666 (84.8) 143 (52.4)  Sexually transmitted infection 3 (0.3) 99 (10.8) 0 – 0 –  Skin infection 94 (9.3) 68 (7.4) 12 (1.8) 35 (12.8)  Other infection 174 (17.1) 256 (27.9) 64 (8.2) 21 (7.7) IQR, interquartile range. Open in new tab Incidence rates of CRC were 7.3 per 100 000 (95% CI 1.5, 21.4), 10.8 per 100 000 (95% CI 2.9, 27.6), 6.5 per 100 000 (95% CI 0.8, 23.6) and 54.4 per 100 000 (95% CI 20.0, 118.5) in offspring exposed to tetracyclines, penicillins, short-acting sulfonamides and long-acting sulfonamides, respectively (Table 4). By comparison, the incidence rate was 10.6 per 100 000 (95% CI 8.2, 13.4) in offspring not exposed to any antibiotics. Cumulative incidence of CRC is shown in Figure 2. Similar to incidence rates, cumulative incidence of CRC at age 55 years was 0.4%, 0.9%, 0.2% and 4.2% for in-utero exposure to tetracyclines, penicillins, short-acting sulfonamides and long-acting sulfonamides, respectively. Figure 2 Open in new tabDownload slide Cumulative incidence of colorectal cancer in adult offspring by in-utero exposure to antibiotics. X-axis begins at age 22 years to reflect youngest age at colorectal cancer diagnosis Table 4 Incidence rates (per 100 000 persons) and crude and adjusted hazard ratios for colorectal cancer in adult offspring with and without in-utero exposure to antibiotics . Person-years . n . Incidence rate per 100 000 . 95% CI . Crude HR . 95% CI . Adjusted HRa . 95% CI . Tetracyclines  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 41 025.0 3 7.3 1.5, 21.4 0.69 0.22, 2.19 1.03 0.32, 3.31 Penicillins  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 37 067.0 4 10.8 2.9, 27.6 1.09 0.40, 3.00 1.12 0.35, 3.58 Short-acting sulfonamides  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 30 641.5 2 6.5 0.8, 23.6 0.69 0.17, 2.80 0.83 0.20, 3.42 Long-acting sulfonamides  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 11 021.5 6 54.4 20.0, 118.5 4.63 2.01, 10.67 4.40 1.63, 11.88 . Person-years . n . Incidence rate per 100 000 . 95% CI . Crude HR . 95% CI . Adjusted HRa . 95% CI . Tetracyclines  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 41 025.0 3 7.3 1.5, 21.4 0.69 0.22, 2.19 1.03 0.32, 3.31 Penicillins  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 37 067.0 4 10.8 2.9, 27.6 1.09 0.40, 3.00 1.12 0.35, 3.58 Short-acting sulfonamides  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 30 641.5 2 6.5 0.8, 23.6 0.69 0.17, 2.80 0.83 0.20, 3.42 Long-acting sulfonamides  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 11 021.5 6 54.4 20.0, 118.5 4.63 2.01, 10.67 4.40 1.63, 11.88 Observations with missing values (n = 3965 for tetracyclines; n = 3938 for penicillins; n = 3886 for short-acting sulfonamides; and n = 3777 for long-acting sulfonamides) not included in adjusted models; see Supplementary Table S3 (available as Supplementary data at IJE online) for results of multiple imputation by fully conditional specification. HR, hazard ratio; CI, confidence interval; Ref., reference category. a Adjusted for year of birth, maternal race (Black v. else), maternal smoking (current vs else), and maternal body mass index (overweight or obese v. else). b Offspring not exposed in utero to any antibiotics (n = 16 116) are reference category for all models. Open in new tab Table 4 Incidence rates (per 100 000 persons) and crude and adjusted hazard ratios for colorectal cancer in adult offspring with and without in-utero exposure to antibiotics . Person-years . n . Incidence rate per 100 000 . 95% CI . Crude HR . 95% CI . Adjusted HRa . 95% CI . Tetracyclines  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 41 025.0 3 7.3 1.5, 21.4 0.69 0.22, 2.19 1.03 0.32, 3.31 Penicillins  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 37 067.0 4 10.8 2.9, 27.6 1.09 0.40, 3.00 1.12 0.35, 3.58 Short-acting sulfonamides  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 30 641.5 2 6.5 0.8, 23.6 0.69 0.17, 2.80 0.83 0.20, 3.42 Long-acting sulfonamides  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 11 021.5 6 54.4 20.0, 118.5 4.63 2.01, 10.67 4.40 1.63, 11.88 . Person-years . n . Incidence rate per 100 000 . 95% CI . Crude HR . 95% CI . Adjusted HRa . 95% CI . Tetracyclines  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 41 025.0 3 7.3 1.5, 21.4 0.69 0.22, 2.19 1.03 0.32, 3.31 Penicillins  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 37 067.0 4 10.8 2.9, 27.6 1.09 0.40, 3.00 1.12 0.35, 3.58 Short-acting sulfonamides  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 30 641.5 2 6.5 0.8, 23.6 0.69 0.17, 2.80 0.83 0.20, 3.42 Long-acting sulfonamides  Not exposed 633 985.0 67 10.6 8.2, 13.4 1.00 Ref.b 1.00 Ref.b  Any in-utero exposure 11 021.5 6 54.4 20.0, 118.5 4.63 2.01, 10.67 4.40 1.63, 11.88 Observations with missing values (n = 3965 for tetracyclines; n = 3938 for penicillins; n = 3886 for short-acting sulfonamides; and n = 3777 for long-acting sulfonamides) not included in adjusted models; see Supplementary Table S3 (available as Supplementary data at IJE online) for results of multiple imputation by fully conditional specification. HR, hazard ratio; CI, confidence interval; Ref., reference category. a Adjusted for year of birth, maternal race (Black v. else), maternal smoking (current vs else), and maternal body mass index (overweight or obese v. else). b Offspring not exposed in utero to any antibiotics (n = 16 116) are reference category for all models. Open in new tab Adjusted associations between in-utero exposure and CRC in adult offspring were as follows: aHR 1.03 (95% CI 0.32, 3.31) for tetracyclines; aHR 1.12 (95% CI 0.35, 3.58) for penicillins; aHR 0.83 (95% CI 0.20, 3.42) for short-acting sulfonamides; and aHR 4.40 (95% CI 1.63, 11.88) for long-acting sulfonamides (Table 4). Associations were similar in the sensitivity analysis using multiple imputation by fully conditional specification (Supplementary Table S3, available as Supplementary data at IJE online). Associations between CRC in offspring and conditions indicating antibiotics in mothers were as follows: HR 0.64 (95% CI 0.26, 1.56) for urinary tract infection; HR 1.02 (95% CI 0.64, 1.53) for upper respiratory infection; HR 1.50 (95% CI 0.37, 6.08) for sexually transmitted infection; and HR 0.85 (95% CI 0.45, 1.61) for skin infection (not shown). Similarly, the association between in-utero exposure to long-acting sulfonamides and CRC in offspring remained similar, although was less precise, in models additionally adjusted for urinary tract infection (aHR 8.40, 95% CI 2.76, 25.62), upper respiratory infection (aHR 4.81, 95% CI 1.82, 12.69) and skin infection (aHR 4.61, 95% CI 1.69, 12.62). For in-utero exposure to long-acting sulfonamides, applying the E-value formula produced E = 9.17 for the estimate and E = 3.62 for the lower confidence limit (not shown). Discussion Our findings support an association between in-utero exposure to long-acting sulfonamides and CRC in adulthood. Incidence rates of CRC were more than five times higher in offspring exposed to long-acting sulfonamides compared with offspring exposed to other antibiotic classes or not exposed. Cases exposed to long-acting sulfonamides were all exposed to sulfadimethoxine, and our findings may reflect a specific effect of sulfadimethoxine or an effect of long-acting sulfonamides as a drug class. Experimental studies will be critical to understand mechanisms of risk, and additional, well-conducted epidemiological studies in other pregnancy cohorts (e.g. Finnish Maternity Cohort26) may substantiate the association between in-utero exposure to long-acting sulfonamides and CRC which we have reported here. Exact mechanisms contributing to the higher risk of CRC in offspring exposed in utero to long-acting sulfonamides are not yet known, but our findings point to three possibilities. First, the pharmacokinetics of long-acting sulfonamides may play a role: long-acting sulfonamides have a serum half-life of up to 40 h, are highly protein bound and are slowly excreted.21,27 This is consistent with evidence that long-acting sulfonamides cross the placental barrier and quickly establish equilibrium; fetal blood levels are sufficient to cause both antimicrobial and toxic effects.28 Long-acting sulfonamides actively compete with bilirubin and can displace unconjugated bilirubin in the fetus. A small literature suggests unbound bilirubin is preferentially distributed to the gastrointestinal tract in newborns (vs to the liver in adults),29 and this process in utero may programme the sensitivity of developing fetal tissue in the colorectum. Second, long-acting sulfonamides bind and inhibit dihydropteroate synthase, an enzyme critical for bacterial synthesis of folate,21,30 suggesting that the folate metabolic pathway frequently implicated in adverse neonatal outcomes31 contributes to colorectal carcinogenesis later in life. Long-acting sulfonamides may additionally act as folic acid antagonists via independent effects on dihydrofolate reductase.30 Folic acid antagonists in early pregnancy may increase risk of congenital anomalies,31,32 and a recent meta-analysis33 suggests that sulfonamide antibiotics are associated with adverse pregnancy outcomes. Experimental studies have similarly shown long-acting sulfonamides in early pregnancy can produce a range of abnormalities in offspring.28,34 A large body of observational studies demonstrates that inadequate folate consumption is a risk factor for CRC,35 although effects of maternal consumption of folate during pregnancy on risk of CRC in offspring have not been studied in humans. Some studies suggest that folic acid supplementation protects against childhood cancers,36 and rodent models show maternal but not post-weaning folic acid supplementation reduces risk of CRC by two-thirds in offspring.37,38 Elsewhere, maternal serum levels of folate contribute to differential expression of APC2 in newborn cord blood.39 APC2 is functionally redundant of APC,40 and APC mutations are found in the very earliest stages of the adenoma–carcinoma sequence. Although these studies are not specific to long-acting sulfonamides, they support the concept that the fetus may be particularly susceptible to folate-related methylation during development.41 Third, the regulatory history of long-acting sulfonamides provides some insight into hypersensitivity-related mechanisms by which in-utero exposure may increase risk of CRC. Long-acting sulfonamides were first introduced in the USA in late 195742 for upper respiratory and urinary tract infections.43 Several case reports subsequently linked sulfadimethoxine and sulfamethoxypyridazine with Stevens–Johnson syndrome, a rare but often fatal inflammatory skin condition. In December 1965, the U.S. Food and Drug Administration required that all long-acting sulfonamides carry a warning label, and they withdrew their approval of sulfadimethoxine in March 1966.42,44 Other hypersensitivity reactions were later reported,45 such as toxic epidermal necrolysis,46 hypersensitivity vasculitis and granulomatous hepatitis.47 There is very little information on the regulatory history of long-acting sulfonamides in countries other than the USA, and although still currently used in Russia,48 it is not clear the extent to which these antibiotics may contribute to the growing burden of CRC worldwide.49 Incidence rates of CRC in offspring exposed in utero to tetracyclines or penicillins are suggestive of no association. It is possible that we were underpowered to detect modest associations with these antibiotic classes, as evidenced by the wide confidence intervals for the adjusted effect estimates. It is also possible that risk of CRC may be related to use of these antibiotics later in life rather than in-utero exposure. Several European studies demonstrate an association between oral antibiotic use in adulthood and CRC, and particularly use of penicillins.50–53 Importantly, these studies used national registries to establish links between antibiotic use and incident cancers. In the USA, a report from the Nurses’ Health Study suggests that long-term antibiotic use, starting in early adulthood (age 20–39 years), is associated with colorectal adenoma.54 A strength of our study is the robust follow-up of the cohort, with detailed information on both mothers and offspring. The prospective design of the CHDS offers a unique opportunity to study exposures in utero and cancers diagnosed in the 60 years after offspring were born. Antibiotic prescriptions were prospectively collected from mothers’ medical records, and we ascertained cancers in offspring by linkage with a high-quality, population-based cancer registry, minimizing the possibility of bias due to measurement error. Follow-up of offspring was similar by in-utero exposure to antibiotics, and it is unlikely that differential ascertainment of cancer in offspring explains our findings. There are some limitations of our study. We could not examine effects of timing of antibiotic exposure because exposed offspring were similarly first exposed in the first, second and third trimester. It is not known whether mothers took antibiotics as prescribed, a common limitation in studies of medication use.55 However, we do not expect this to be differential by antibiotic class. In observational studies of medication use, it is also possible that associations are related to the underlying medical conditions indicating the medication. Specifically, urinary tract infections accounted for about half of long-acting sulfonamides in our study, and infections during pregnancy may disrupt fetal development via effects of various metabolic byproducts, colonization or other bacterial activity.56 We addressed this possibility by examining associations with urinary tract infection and other conditions indicating antibiotics, providing some confidence that observed associations are not explained by indications for use. Associations between in-utero exposure to antibiotics and CRC in offspring may be confounded by factors not measured in the CHDS. We addressed unmeasured confounding by calculating E-values. These results suggest that the observed aHR of 4.40 for the association with in-utero exposure to long-acting sulfonamides can only be explained away by an unmeasured confounder associated with both the exposure and outcome by a risk ratio of 9-fold each, scenarios that are highly unlikely. Finally, we did not examine some antibiotic classes used in current clinical practice (e.g. cephalosporins) because they were not available or rarely prescribed in the late 1950s and 60s. In summary, we observed higher incidence rates of CRC in adult offspring exposed in utero to long-acting sulfonamides, but rates of CRC in offspring exposed to short-acting sulfonamides or other antibiotic classes, including tetracyclines and penicillins, were suggestive of no association. Although no longer routinely prescribed in humans, long-acting sulfonamides are used extensively in veterinary medicine and prophylactically in medicated feed for livestock.57,58 As a consequence, long-acting sulfonamide residues are frequently detected in milk and soft-cheese products,59 eggs60 and beef, pork and chicken meat,61,62 as well as in water and soil.63,64 Their continued presence as environmental pollutants raises the possibility that long-term exposure, albeit at lower levels, may also increase risk. Additional, well-conducted studies are needed to test for associations with long-term exposure from applications to livestock used for human consumption. Ethics approval Individual participants have not provided consent for this study. The requirement for informed consent was waived as the risk to participants was considered minimal. The Institutional Review Board at the Public Health Institute (#I92-004) and the University of Texas Health Science Center at Houston (#21–0271) approved this study. Data availability De-identified (anonymized) data are available upon request from Barbara A Cohn, PhD, Director of the Child Health and Development Studies. Requests will be reviewed by Dr Cohn, research staff and the Institutional Review Board at the Public Health Institute. Approval of requests for de-identified (anonymized) data requires execution of a data use agreement. Supplementary data Supplementary data are available at IJE online. Author contributions Study concept and design: C.C.M., B.A.C. Acquisition of data: B.A.C., P.M.C., N.Y.K. Analysis and interpretation of data: all authors. Statistical analysis: C.C.M., P.M.C. Drafting of manuscript: C.C.M. Critical revision: all authors. The authors affirm that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained. Funding Research reported in this publication was supported by the National Cancer Institute at the National Institutes of Health under award number R01CA242558 (C.C.M.) and by the National Institute of Child Health and Development at the National Institutes of Health under contract number HHSN275201100020C (B.A.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Mention herein of trade names, commercial products or organizations does not imply endorsement by the US government. Collection of cancer data used in this study was supported by the California Department of Public Health as part of the statewide cancer reporting programme mandated by California Health and Safety Code Section 103885; the National Cancer Institute’s Surveillance, Epidemiology, and End Results Program under Contract HHSN261201000140C (awarded to the Cancer Prevention Institute of California), Contract HHSN261201000035C (awarded to the University of Southern California) and Contract HHSN261201000034C (awarded to the Public Health Institute); and the Centers for Disease Control and Prevention’s National Program of Cancer Registries, under Agreement U58DP003862-01 (awarded to the California Department of Public Health). Acknowledgements A version of this manuscript was presented at Digestive Disease Week 2021, Virtual, 21–23 May 2021. 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Environ Sci Pollut Res Int 2010 ; 44 : 6591 – 600 . Google Scholar OpenURL Placeholder Text WorldCat © The Author(s) 2023; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/pages/standard-publication-reuse-rights)