The increasing prevalence of gastroschisis, a congenital defect of the abdominal wall, has been documented over the past several decades in the U.S.¹ and around the world.^2-6 In the U.S., the greatest increase is observed among young women, especially those less than 20 years of age.¹ The cause for this increase and for the dramatic difference by maternal age are unknown. The rapidity of the increase suggests that environmental factors play a role. A study from the National Birth Defects Prevention Study reported that women delivering an infant with gastroschisis were four times more likely to report a genitourinary (GU) infection (i.e., urinary and sexually transmitted infections [STIs]) during the periconceptional period than control mothers.⁷ That study also suggested a specific role of Chlamydia trachomatis (CT) infection, which accounted for 43% of GU reported infections among case mothers compared to 18% among controls. GU infections were also disproportionately more frequent in women <25 years of age. Two additional studies reported an association between periconceptional GU infection and risk for gastroschisis.^8,9 Several factors suggest that CT infection should be further investigated as a potential risk factor for gastroschisis: 1) it is the most prevalent sexually transmitted bacterial infection; 2) prevalence is highest in women aged 15-24 years, and remains high despite a national chlamydia control program;¹⁰ and 3) the rising rate of CT infection among young women has paralleled the increasing prevalence of gastroschisis.

However, investigating CT infection as a risk factor for gastroschisis is challenging, for several reasons: 1) subclinical CT infections are frequent (up to 75% of CT-infected women are asymptomatic¹¹) which makes using maternal self-report as a marker for CT exposure less ideal due to the potential exposure misclassification, and 2) CT infections may resolve spontaneously (i.e., without treatment) and therefore a negative CT test (i.e., nucleic acid amplification test – NAAT) at a prenatal visit does not rule out a prior periconceptional CT exposure.¹² Hence, more objective and sensitive approaches are needed to evaluate CT exposure. One such proposed measure is a CT-specific antibody response. Geisler et al.¹³ recently reported that IgG1 and IgG3, detected by a CT elementary body (EB) ELISA, comprised the predominant anti-CT serum antibody response, and the seropositivity rate detected by CT EB ELISA was higher than a commercial CT ELISA (Medac). Our primary objective was to evaluate the association between gastroschisis and CT seropositivity, which was performed using the CT EB ELISA. Secondary objectives included: 1) assess study participation rates of pregnant women prenatally diagnosed with and without gastroschisis; 2) determine the utility of medical record review in documenting prenatal STI screening; and 3) evaluate associations of sexual history with gastroschisis.

Of 113 eligible pregnant women (34 cases; 81 controls) 99 (86%) participated:33 (97.1%) cases and 66 (81.5%) controls. Gestational age at enrollment was on average 27.3 weeks (range 16-37 weeks) for cases and 27.7 weeks (range 18-38.2 weeks) for controls.

Cases were significantly more likely to be younger, less educated, smoke cigarettes, report a previous STI, and among those with a previous pregnancy, report a different partner with their current pregnancy than the most recent pregnancy (Table 1). Compared to controls, cases reported a significantly lower median age at sexual debut (defined as first reported age at vaginal intercourse) (p=0.0002) and a greater number of sexual partners since their debut (p=0.058) (Figure 1a, 1b). Both the number of sexual partners since becoming sexually active (sexual debut) and a change in partner from the previous pregnancy were associated with an increased risk for gastroschisis (Table 2). Among women <25 years of age, cases reported more sexual partners than controls (p=0.006) compared to women ≥25 (p=.66). When specifically queried about previous STI diagnoses, cases more often reported a prior CT infection (5 [15.2%] vs. 1 [1.5%]), genital herpes (1 [3.0%] vs. 1 [1.5%]), and gonorrhea (1 [3%] vs. 0%) (any self-reported STI: aOR=6.7, 95%CI: 0.7, 58.8). Among women reporting illicit drug use during the periconceptional period, only marijuana use was reported.

All participating cases and controls consented to have their medical records reviewed. Evidence of a negative screening NAAT for chlamydia and gonorrhea at the first prenatal visit was documented in the prenatal medical records for 19 (57.6%) cases and 37 (56%) controls. The remaining medical records either did not document that a screening NAAT was performed (10 [30.3%] cases; 28 [42.4%] controls), or if performed, did not document the results (4 [12.1%] cases; 1 [1.5%] controls). No medical records included the screening method used to determine a negative or positive screen.

The findings show that maternal anti-CT IgG3 seropositivity was associated with a 3.9-fold increased risk for gastroschisis in the offspring. A study by Geisler et al.¹³ demonstrated that anti-CT IgG1 and IgG3 comprised the predominant anti-CT serum antibody responses. It is believed that IgG3 is involved early in response to infection and then is short-lived with IgG1 emerging as the major effector in eradicating infection.¹⁵ Geisler et al.¹³ reported some decline in the magnitude of the IgG3 response within 6 months of chlamydia treatment. It is plausible that detection of anti-CT IgG3 in this cohort of pregnant women suggests a more recent CT exposure, potentially in the periconceptional period where CT infection could contribute to the development of gastroschisis.

The mechanism by which CT infection could lead to gastroschisis is not known. One mechanism might be proinflammatory cytokines induced by chlamydia impairing normal abdominal wall development. CT infections can lead to salpingitis, infertility, ectopic pregnancy, reactive arthritis, and perihepatitis.¹⁶ The innate and adaptive immune response induces systemic inflammation leading to cytokine production which has the potential to damage cells.

The sexual history of participants was consistent with behaviors that are known to increase the likelihood of acquiring an STI.^11,17,18 Compared to controls, cases as a group were sexually active at a younger age, had more sexual partners, and more frequently reported a change in partner from their previous pregnancy. Sexually active adolescents and young women are much more susceptible to sexually transmitted infections. Unlike the squamous cells in the cervical epithelium of adult females, the genital tracts of adolescent and young women are lined by immature undifferentiated columnar epithelial cells.¹⁷ This immature epithelium is thought to be more susceptible to pathogens and is the preferred site for C. trachomatis and Neisseria gonorrhea.¹⁷ In epidemiologic studies, young maternal age has been the strongest and most consistent risk factor for gastroschisis.¹ The mechanism underlying this association is not understood but could be related to the combination of exposure (increased risk for sexually transmitted infections due to more risky behaviors) and susceptibility (the biology of the reproductive tract). Eight (24.2%) cases and 19 (28.8%) controls did not have any evidence (no IgG1 or IgG3 seropositivity) of prior CT infection, suggesting that other infections or environmental exposures are responsible for gastroschisis.

The study had several strengths. Participation in the study for both case and control women was high, demonstrating that women are willing to participate in a research study during pregnancy despite their new diagnosis of a fetal anomaly. We used a highly sensitive serologic test, CT EB ELISA, to maximize the detection of a prior known or subclinical CT infection; our study also confirmed that CT EB ELISA detects chlamydia antibodies more often than commercially available assays. The use of a sensitive test is crucial as it helps reduce (or eliminate) the potential for exposure misclassification which may influence the accuracy of detected associations between CT infection and risk for gastroschisis. This study was the first step to determine whether CT seropositivity increases the risk for gastroschisis. Whether it is the pathogen or the inflammatory response as a result of the pathogen requires further research. We also found that more than 40% of prenatal medical records lacked detailed information on whether screening for STIs was conducted during the first prenatal visit or if conducted, the results were not listed. There is also the possibility that some of the negative screens were false negatives since the immune system can clear a subclinical local chlamydia infection.¹¹

The findings from this study must be interpreted in the context of its limitations. An important limitation is that this was an exploratory pilot study to investigate the feasibility for the methodology with a small sample of eligible cases. The focus on prenatally diagnosed cases meant that we would have missed a woman whose affected pregnancy was not prenatally diagnosed. Among Utah residents, approximately 90% of cases are prenatally diagnosed and referred to the University of Utah for delivery. Only a small number of women (n=3 or 10%) would not have had the opportunity to participate in the study because they were not prenatally diagnosed with gastroschisis. The gestational age at enrollment was later than anticipated. We estimated initially that the majority of the diagnoses and enrollment would have occurred at 18 to 20 weeks gestation. In reality, the average gestational-age at enrollment was 27 weeks, which may have influenced our ability to detect active chlamydia infection by NAAT. Community obstetric providers may have referred their patients for a MFDC confirmation ultrasound weeks after the initial ultrasound diagnosing a gastroschisis. Another limitation of the study was that we were not able to test for other pathogens known to cause STIs. However, serum samples were banked for future studies on additional viral or bacterial pathogens. Finally, the questionnaire was administered during pregnancy, soon after the prenatal diagnosis of a fetal abnormality (gastroschisis) and 76% of the cases were less than 25 years of age compared to 44% of controls. Young women may feel more comfortable, in general, answering questions about their sexual encounters, potentially inflating the findings on sexual history.

GU infections, and CT infections specifically, could represent an intermediate step between risky sexual behaviors and increased risk for gastroschisis: these behaviors increase the likelihood to acquire one or more sexually transmitted infections, which in turn may be particularly harmful in pregnancies of young women because of the particular susceptibility of their reproductive system. Finding an increased risk for gastroschisis among women with evidence of a recent CT infection is the first step in understanding the association with this pathogen and the host's subsequent systemic inflammatory response. CT has the ability to induce a systemic inflammatory response, the magnitude of which may differ in cases compared to controls as a result of the individual's immunogenetic determinants. Future research should include evaluation of the influence of CT-specific cellular immune responses on gastroschisis risk. In addition, this study underscores the important role of biomarker-based studies in birth defect epidemiology. Because the majority of CT infections in women are subclinical, association can be easily missed or distorted if appropriate biomarkers such as serum antibodies are not used, and associations are exclusively based on self-reports of such challenging exposures.

Anti-CT IgG3 seropositivity was associated with more than a 3-fold risk for gastroschisis, which in combination with the association of risky sexual behavior suggests recent CT infection around or shortly after conception increases gastroschisis risk.