Roles of bovine Waddlia chondrophila and Chlamydia trachomatis in human preterm birth.

Waddlia chondrophila and Chlamydia trachomatis are intracellular bacteria associated with human miscarriage. We investigated their role in human preterm birth. Whereas presence of Chlamydia trachomatis DNA in genital tract was associated with human preterm birth, Waddlia was not, despite being present in women's genital tracts.

Introduction

In 2010, approximately 15 million babies were born preterm worldwide, and more than one million died due to complications from preterm births (PTB). Neonates that survive PTB exhibit an increased risk of neurodevelopmental impairments and respiratory complications [1]. The proportion of spontaneous PTB attributed to infection is approximately 50% [2]. However, a pathogen is identified in only one third of the cases, despite evidence of infection. Obligate intracellular bacteria, which do not grow on media used routinely to isolate human pathogens from clinical samples, might represent possible agents of PTB.

Chlamydia trachomatis, an obligate intracellular bacterium, is considered the world's most common sexually transmitted bacterial pathogen. Waddlia chondrophila is another member of the Chlamydiales order that has been shown to cause abortions in bovines [3]. Both of these intracellular bacteria have also been implicated in human adverse pregnancy outcomes [4-7]. In addition, C. trachomatis is known to cause premature rupture of the membranes and premature uterine activity, and growing evidence suggests a role for C. trachomatis in PTB [8]. In this study, we investigated the role of Waddlia and Chlamydia as emerging agents of PTB. We studied 407 women with PTBs or uneventful term pregnancies attending the University Hospital of Lausanne, Lausanne, Switzerland. In addition to serology, we also performed PCR to detect Waddlia and Chlamydia in the placenta and vaginal samples taken from these women, as well as histology on the placenta.

From 2006 to 2009, 407 women were enrolled into this study at the obstetrical ward of the University Hospital of Lausanne. The PTB group (n = 146) included women who spontaneously delivered before 37 weeks' gestation. The control group (n = 261) included women attending a labour ward with uneventful term pregnancies and no history of miscarriages, stillbirths or preterm labour. We compared demographic data and risk factors of patients with and without PTB or C. trachomatis infection by the Pearson χ2 test (or the Fisher exact test when indicated) for categorical variables. For continuous variables, medians were compared by the Wilcoxon-Mann-Whitney test. Multivariable analyses were performed to control for covariates. Statistical analyses were performed using the Stata software, version 13.0 (StataCorp, College Station, TX, USA).

Only positive urine cultures, gestational and maternal age were significantly different between control and PTB groups (Table 1). Other infectious causes were investigated in the PTB group, showing that a positive culture was found in the vagina, maternal or fetal side of the placenta in 32%, 37% and 17% of the PTB patients, respectively (Supplementary Table 1). Among all these bacterial species, genital mycoplasma and Gardnerella vaginalis, which have been previously associated with PTB [9], were recovered in some subjects from the vagina only (n = 25 and 3, respectively), never from the placenta.

TABLE 1

Characteristics of patients according to term history

Characteristic	Control (n = 261)	PTB (n = 146)	p
Gestational age at birth, weeks, ±SD	39.6 ± 1.1	32.6 ± 3.3	<0.001
Age, years, ±SD	31.5 ± 5.0	32.4 ± 5.9	0.057
<35 years	194 (74.3%)	94 (64.4%)	0.041
≥35 years	67 (25.7%)	52 (35.6%)
Parity, ±SD	0.5 ± 0.8	0.5 ± 0.8	0.950
0	160 (61.3%)	95 (65.1%)	0.228
1	72 (27.6%)	30 (20.6%)
>1	29 (11.1%)	21 (14.4%)
Origin
European	217 (83.1%)	113 (77.4%)	0.156
Non-European	44 (16.9%)	33 (22.6%)
Civil status
Married	201 (77.0%)	109 (74.7%)	0.739
Single	49 (18.8%)	32 (21.9%)
Divorced	11 (4.2%)	5 (3.4%)
Education
Nonuniversity studies	170 (65.1%)	106 (72.6%)	0.150
University studies	91 (34.9%)	40 (27.4%)
No. of lifelong sexual partners
1	58 (22.2%)	37 (25.3%)	0.393
2–3	43 (16.5%)	29 (19.9%)
4–6	45 (17.2%)	19 (13.0%)
>6	36 (13.8%)	13 (8.9%)
Not answered	79 (30.3%)	48 (32.9%)
Condom as previous contraceptive method	69 (26.4%)	33 (22.6%)	0.407
Smoking status
Nonsmoker	224 (85.8%)	129 (88.4%)	0.543
Smoker	37 (14.2%)	17 (11.6%)
Pets at home	82 (31.4%)	39 (26.7%)	0.366
Vegetarian	5 (1.9%)	5 (3.4%)	0.341
Chlamydia trachomatis serology
IgG positive	19 (7.3%)	13 (8.9%)	0.569
IgA positive	10 (3.8%)	9 (6.2%)	0.330
Both IgG and IgA positive	7 (2.7%)	7 (4.8%)	0.270
C. trachomatis PCR
Cervicovaginal swab	2 (0.7%)	7 (4.8%)	0.012
Placenta	2 (0.7%)	7 (4.8%)	0.012
At least one PCR positive	2 (0.7%)	9 (6.2%)	0.002
Waddlia serology
Total Ig ≥1/64	47 (18.0%)	31 (21.2%)	0.428
IgG ≥1/64	38 (14.6%)	22 (15.1%)	0.889
IgM ≥1/16	9 (3.5%)	11 (7.5%)	0.092
Waddlia PCR
Cervico-vaginal swab	11 (4.2%)	10 (6.9%)	0.252
Placenta	11 (4.2%)	4 (2.7%)	0.587
Other infections
Positive urine culture	7 (2.7%)	38 (26%)	<0.001
Streptococcus agalactiae	45 (18.0%)	22 (17.5%)	1.000
Brucella abortus	19 (7.3%)	11 (7.5%)	1.000
Parachlamydia acanthamoebae	2 (0.8%)	0 (0%)	0.539
Simkania negevensis	3 (1.2%)	1 (0.7%)	1.000

PTB, preterm birth.

Sera were tested for antibodies directed against C. trachomatis and W. chondrophila, respectively, by using the MOMP-R, CT pELISA (R-biopharm, Darmstadt, Germany) [10] and Waddlia-specific immunofluorescence as described elsewhere [6]. Isolated IgG mainly reflects past or chronic infection, whereas IgM and/or IgA reflect acute infection. Briefly, W. chondrophila strain ATCC VR-1470 was used as antigen, whereas Fluoline G or M (bioMérieux, Marcy l'Étoile, France) were used as secondary antibodies. An antibody titre of ≥1/64 for IgG and ≥1/32 for IgM were considered as positive, respectively [6]. There was no difference between control and PTB groups in terms of anti-Chlamydia IgG and IgA and anti-Waddlia IgG and IgM titres (Table 1). A total of 54 patients tested positive only for Waddlia IgG and 26 only for Chlamydia IgG, indicating the absence of serologic cross-reaction between both pathogens. Only six patients tested positive for both Waddlia and Chlamydia IgG (p 0.446).

C. trachomatis IgG seropositivity (Table 2) was associated with civil status (divorced vs. married, odds ratio (OR) 7.85; 95% confidence interval (CI) 2.61–23.62), education (OR 0.28; 95% CI 0.10–0.81) and number of previous sexual partners (>6 vs. 1: OR 13.12, 95% CI 1.53–112.32; “not answered” vs. 1: OR 13.55, 95% CI 1.76–104.09). Patients who used condoms as a previous contraceptive method show less C. trachomatis positive serologies, although this was not statistically significant. Of note, only six (55%) and one (9%) of the 11 patients positive for C. trachomatis DNA were also positive for C. trachomatis IgG and IgA, respectively. However, C. trachomatis IgG-positive patients exhibited significantly more histologic chorioamnionitis (50%) than C. trachomatis IgG-negative patients (28.3%, p 0.015).

TABLE 2

Characteristics of patients according to Chlamydia trachomatis serologic status

Characteristic	IgG negative (n = 375, 92.1%)	IgG positive (n = 32, 7.9%)	p
Age, years, ±SD	31.9 ± 5.3	30.9 ± 6.5	0.298
<35 years	263 (91.3%)	25 (8.7%)	0.421
≥35 years	112 (94.1%)	7 (5.9%)
Parity, ±SD	0.5 ± 0.8	0.5 ± 0.7	0.997
0	233 (91.4%)	22 (8.6%)	0.38
1	97 (95.1%)	5 (4.9%)
>1	45 (90%)	5 (10.0%)
Origin
European	307 (93.0%)	23 (7.0%)	0.164
Non-European	68 (88.3%)	9 (11.7%)
Civil status
Married	288 (92.9%)	22 (7.1%)	>0.001
Single	77 (95.1%)	4 (4.9%)
Divorced	10 (62.5%)	6 (37.5%)
Education
Nonuniversity studies	248 (89.9%)	28 (10.1%)	0.017
University studies	127 (97.0%)	4 (3.1%)
No. of lifelong sexual partners
1	94 (99.0%)	1 (1.0%)	0.006
2–3	66 (91.7%)	6 (8.3%)
4–6	61 (95.3%)	3 (4.7%)
>6	43 (87.8%)	6 (12.2%)
Not answered	111 (87.4%)	16 (12.6%)
Place of residence
Rural	126 (91.3%)	12 (8.7%)	0.699
City	249 (92.6%)	20 (7.4%)
Condom as previous contraceptive method
No	277 (90.8%)	28 (9.2%)	0.094
Yes	98 (96.1%)	4 (3.9%)
Smoking status
Nonsmoker	209 (95.0%)	11 (5.0%)	0.114
Smoker	48 (88.9%)	6 (11.1%)

Waddlia seropositivity was not associated with age, number of lifelong sexual partners, place of residence (rural vs. urban), smoking, pet ownership or meat consumption (data not shown). Interestingly, 4.9% of women of European heritage vs. 14.3% of those of non-European heritage had positive Waddlia IgG serologies of ≥1/256 (p 0.008). This correlation was also observed for Waddlia IgM of ≥1/32 (3.6% of European vs. 10.4% of non-European, p 0.034). Among non-European women, the highest Waddlia IgG titres (≥1/128 or ≥1/256) were more frequent for black ethnicity (20% and 14.3%, respectively). When total immunoglobulin (Ig ≥1/64) against Waddlia was considered, condoms as a previous contraceptive method protected against Waddlia infection (12.1% condom users vs. 21.4% of the noncondom users exhibited anti-Waddlia Ig reactivity, p 0.041).

After DNA extraction with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), all vaginal swabs and placenta samples were screened for C. trachomatis DNA by a TaqMan real-time PCR targeting the cryptic plasmid of C. trachomatis, as described earlier [11]. Samples were also tested by a 16S rRNA Waddlia-specific real-time PCR, as described previously [12]. No PCR inhibition was observed for either pathogen. Vaginal and placental samples from the PTB group were significantly more often C. trachomatis DNA positive than the control group (p 0.002, unadjusted OR 8.51; 95% CI 1.81–39.93). This association reminded significant even after adjustment for maternal age, origin, civil status, education, number of sexual partners and positive urine cultures (OR 7.93; 95% CI 1.34–46.76). Stepwise logistic regression allowed us to confirm that the presence of C. trachomatis DNA is an independent factor associated with PTB (Fig. 1). There was no difference between the control and PTB groups regarding Waddlia PCR results. Thirty-four patients were positive only for W. chondrophila, whereas ten other subjects were positive only for C. trachomatis by PCR. Only one patient was positive for both, demonstrating the absence of cross-amplification of these two obligate intracellular bacteria. Presence of Waddlia DNA was demonstrated in the genital tract or placenta of 13 PTB subjects (Table 3). Seven of them had more than one miscarriage in their medical history. Of these 13 patients, four exhibited both a positive Waddlia serology and presence of Waddlia DNA. Two had presence of Waddlia in multiple samples, including vaginal swab, urine and placenta. Only two of these 13 patients had either a positive C. trachomatis serology or PCR. Ten of these 13 patients had abnormal placenta histologies.

FIG. 1

Stepwise logistic regression showing independent association of positive Chlamydia trachomatis PCR with preterm birth.

TABLE 3

Clinical history, serology, PCR and placental histology of preterm patients with samples positive for Waddlia by real-time PCR

Patient no.	Maternal age, years	No. pregnancies	Parity	Gestational age at birth, weeks	Birth weight, g	Country of origin	Waddlia			Placental histology	Other possible etiologiesa
							IgG	IgM	PCR positive
17	37	2	0	35.3	2520	Switzerland	—	—	P	Calcifications
28	35	10	4	31.5	1870	Angola	—	—	P and VS	Subchorial fibrin, necrosis, lymphocytes in chorion and amnion, vasculitis
45	27	1	0	34.1	2340	Switzerland	—	—	VS	—
66	29	1	0	25.6	890	Portugal	—	—	VS	Subchorial fibrin, lymphocytes in chorion and amnion, oedema
133	40	2	1	30.5	1270	Togo	1/128	—	VS	Villous oedema
185	33	2	0	35.1	2060	Switzerland	—	—	VS	Villous oedema
223	32	2	0	36.3	2370	Portugal	—	—	VS	—	CT IgG and IgA positiveb
261	39	4	0	34.4	2540	Italy	1/128	—	P	Decidual fibrin
283	34	4	1	36.4	2560	Congo	1/256	—	VS	Subchorial fibrin
314	36	1	0	31.3	1310	Switzerland	—	—	VS	—
351	33	2	0	35.3	2280	Italy	1/128	1/32	VS	Marginal hemorrhage
476	35	2	1	30	1530	Italy	—	—	VS	Lymphocytes in decidua
572	26	1	0	35	1920	Switzerland	—	—	P and U	Intervillous hemorrhage	CT PCR positive placentac

Haematoxylin and eosin–stained histologic sections of all placenta specimens were examined for the type and degree of placentitis, endometritis and/or vasculitis by a pedopathologist.

P, placenta; VS, vaginal swab; U, urine; CT, C. trachomatis.

All patients were also tested for C. trachomatis, Brucella abortus, Streptococcus agalactiae, Parachlamydia acanthamoebae and Simkania negevensis.

Positive serology for C. trachomatis observed in this case reflects a possible coinfection because there is no serologic cross-reaction between C. trachomatis and Waddlia chondrophila, and because the W. chondrophila serology was negative.

C. trachomatis positive PCR reflects a likely coinfection because there is no cross-amplification with the PCRs we used.

Discussion

Both C. trachomatis and W. chondrophila have been implicated in adverse pregnancy outcomes [4,5,7,8]. However, there have been contradictory findings pertaining to the role of C. trachomatis infection in PTB. A few small studies have failed to demonstrate a risk of PTB associated with C. trachomatis infections [13,14], although several large, well-conducted studies have supported a role for C. trachomatis in PTB [15-17]. Thus, in a large study including 4055 subjects, about 15% of PTB was attributed to C. trachomatis [16].

We have previously confirmed an association between Waddlia antibodies and human miscarriage and have demonstrated its presence in placenta and/or genital tract [4-7]. In the present study, W. chondrophila was not associated with PTB despite inflammation, and abnormal histology of the placenta was observed among patients infected by Waddlia (positive PCR). Women of African descent were more likely to have positive Waddlia serology. Moreover, condom use was inversely associated with Waddlia seropositivity.

In contrast to Waddlia, presence of Chlamydia DNA in the genital tract and/or in the placenta was strongly associated with PTB. However, C. trachomatis seropositivity did not correlate with PTB, suggesting that acute rather than chronic C. trachomatis infection is associated with PTB. Interestingly, these results are opposite to our previous findings, in which we demonstrated that both chronic and acute C. trachomatis infections correlate with miscarriages [18]. Our results confirm those of a population-based prospective study recently published in the Generation R cohort in the Netherlands [16]. In this large study (>4000 pregnant women), Chlamydia DNA was strongly associated with PTB but not with miscarriage or perinatal death. Conflicting data between Chlamydia DNA, serology and pregnancy outcomes may reflect different pathophysiologic mechanisms, in which Chlamydia-induced miscarriages are the result of an immunologic process, whereas the direct impact of the bacteria results in Chlamydia-induced PTB.

A limitation of our study was the absence of investigation of other infectious etiology of PTB in all patients (PTB and control subjects). Some pathogens can reach the placenta by hematogenous spread or by an ascending route from the cervix. Among bacterial infections, Ureaplasma urealyticum, Mycoplasma hominis and bacterial vaginosis have been associated with PTB, but controversies regarding their true role during pregnancy persist [9,19].

Overall, these results strongly suggest a role of acute C. trachomatis infection in PTB, and we strongly recommend systematically testing for C. trachomatis in any woman at risk for or after preterm delivery.

Conflict of interest

None declared.