Integrated immune monitoring of HCMV infection in pregnant women with complications and its association with adverse pregnancy outcomes

Abstract

Human Cytomegalovirus (HCMV) infection is associated with bad obstetric history (BOH) and adverse pregnancy outcomes (APO). Here, we characterized antiviral humoral profiles, systemic and virus specific cellular immune responses concurrently in pregnant women (n = 67) with complications including BOH and associated these signatures with pregnancy outcomes. Infection status was determined using nested blood PCR, seropositivity and IgG avidity by ELISA. Systemic and HCMV specific (pp65) cellular immune responses were evaluated by flow cytometry. Seropositivity was determined for other TORCH pathogens (n = 33) on samples with recorded pregnancy outcomes. This approach was more sensitive in detecting HCMV infection. Blood PCR positive participants, irrespective of their IgG avidity status, had higher cytotoxic potential in circulating CD8⁺ T cells (p < 0.05) suggesting that infection associated cellular dysfunction was uncoupled with avidity maturation of antiviral humoral responses. Also, impaired anamnestic degranulation of HCMV-pp65-specific T cells compared to HCMV blood PCR negative participants (p < 0.05) was observed. APO correlated with HCMV blood PCR positivity but not serostatus (p = 0.0039). Most HCMV IgM positive participants (5/6) were HCMV blood PCR positive with APO. None were found to be IgM positive for other TORCH pathogens. Multiple TORCH seropositivity however was significantly enriched in the APO group (p = 0.024). Generation of HCMV specific high avidity IgG antibodies had no bearing on APO (p = 0.9999). Our study highlights the utility of an integrated screening approach for antenatal HCMV infection in the context of BOH, where infection is associated with systemic and virus specific cellular immune dysfunction as well as APO.

Keywords: Adverse pregnancy outcomes; Bad obstetric history; HCMV infection; IgG avidity; PCR.

Fig. 1

Obstetric history of pregnant women with (A) pregnancy complications in primigravida (PC) and (B) Bad Obstetric History (BOH). N = Number of participants in which the complication was seen. SA: spontaneous abortion; IUFD: intrauterine fetal death; MA: missed abortion; FGR: fetal growth restriction.

Fig. 2. Distribution of HCMV seropositivity in pregnant women with BOH and PC.

(A) HCMV Serology status; n = 67 (B) HCMV IgG Avidity status; n = 59 (C) HCMV infection status; n = 59. +: Positive; -: Negative.

Fig. 3. Cytotoxic potential in PN (n = 14) and PP (n = 35) pregnant women with BOH and PC.

(A) % Granzyme-B expressing T cells (CD3+) (B) % Granzyme-B expressing CD8⁺ T cells (C) % Granzyme-B expressing CD4⁺ T cells Data points in red indicates samples for which ICCS Assay was performed. Comparisons between groups were evaluated by non-parametric Mann–Whitney U test (*p < 0.05; **p < 0.01; ^nsp ≥0.05). p < 0.05 was considered significant.

Fig. 4. Phenotypic and functional systemic immune response during second trimester in PN (n = 9) and PP (n = 15) pregnant women with BOH and PC.

(A) % Granzyme-B expressing T cells (CD3⁺) (B) % Granzyme-B expressing CD8⁺ T cells (C) % CD4⁺ T cells (D) % CD8⁺ T cells (E) CD4/CD8 Ratio. Data points in red indicates samples for which ICCS Assay was performed. Comparisons between groups were evaluated by non-parametric Mann–Whitney U test (*p < 0.05; **p < 0.01; ^nsp ≥0.05). p < 0.05 was considered significant.

Fig. 5. HCMV specific immune response in PN (n = 7) and PP (n = 6) pregnant women with BOH and PC.

(A) % CD107a expressing monofunctional CD4⁺ and CD8⁺ T cells. Data points in green indicates samples for which pregnancy outcome was available (B) Comparison of % CD107a expressing monofunctional CD4⁺ and CD8⁺ T cells between unstimulated (UN) and positive control (PMA) in PN and PP group. (C) CD107a expressing different monofunctional T cell subsets; TCM^: Central Memory, TN: Naive, TEM: Effector Memory, TTD: Terminally differentiated in unstimulated and stimulated condition (pp65 peptide pool). Comparisons between groups were evaluated by non-parametric Mann–Whitney U test (*p < 0.05; ^nsp ≥0.05). p < 0.05 was considered significant.