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. 2016 Jul 1;11(7):e0158304.
doi: 10.1371/journal.pone.0158304. eCollection 2016.

Presence of HHV-6A in Endometrial Epithelial Cells from Women with Primary Unexplained Infertility

Roberto Marci  1   2 , Valentina Gentili  3 , Daria Bortolotti  3 , Giuseppe Lo Monte  4 , Elisabetta Caselli  3 , Silvia Bolzani  3 , Antonella Rotola  3 , Dario Di Luca  3 , Roberta Rizzo  3
Affiliations

Presence of HHV-6A in Endometrial Epithelial Cells from Women with Primary Unexplained Infertility

Roberto Marci et al. PLoS One. .

Abstract

To elucidate the roles of human herpesvirus (HHV)-6 primary unexplained infertile women, a prospective randomized study was conducted on a cohort of primary unexplained infertile women and a cohort of control women, with at least one successful pregnancy. HHV-6 DNA was analyzed and the percentage and immune-phenotype of resident endometrial Natural Killer (NK) cells, as the first line of defense towards viral infections, was evaluated in endometrial biopsies. Cytokine levels in uterine flushing samples were analyzed. HHV-6A DNA was found in 43% of endometrial biopsies from primary unexplained infertile women, but not in control women. On the contrary, HHV-6B DNA was absent in endometrial biopsies, but present in PBMCs of both cohorts. Endometrial NK cells presented a different distribution in infertile women with HHV6-A infection compared with infertile women without HHV6-A infection. Notably, we observed a lower percentage of endometrial specific CD56brightCD16- NK cells. We observed an enhanced HHV-6A-specific endometrial NK cell response in HHV-6A positive infertile women, with a marked increase in the number of endometrial NK cells activating towards HHV-6A infected cells. The analysis of uterine flushing samples showed an increase in IL-10 levels and a decrease of IFN-gamma concentrations in infertile women with HHV6-A infection. Our study indicates, for the first time, that HHV-6A infection might be an important factor in female unexplained infertility development, with a possible role in modifying endometrial NK cells immune profile and ability to sustain a successful pregnancy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. HHV-6 analysis in endometrial biopsies.
a) HHV-6 DNA was searched by real time qPCR specific for U42 gene in endometrial biopsies. Results are expressed in viral copies/ug DNA and represent the mean copy number ± SD referred to duplicates of 2 independent assays. Infertile: primary unexplained infertile women; Control: women with at least one successful pregnancy. b) Cell fractions derived by immunomagnetic separation were characterized by PCR specific for HHV-6A U94 DNA after 3, 7, 14 and 20 days of culture. C+: HHV-6A infected J-Jahn cells. c) HHV-6A p41 (early protein) and gp116 (late protein) immunofluorescence (PE) (white arrow) in epithelial and stroma cells from endometrial biopsies of primary infertile women. CK18 and vimentin staining was used to confirm epithelial and stromal cell types, respectively. DAPI staining indicated DNA. Images were taken in bright field (left panels) or fluorescence (right panels) (Nikon Eclipse TE2000S) equipped with a digital camera. Original magnification 100×.
Fig 2
Fig 2. HHV-6A infection and menstrual cycle phases.
a) HHV-6 DNA was searched by real time qPCR specific for U42 gene in endometrial biopsies of infertile women during proliferative, ovulatory and secretory phases. Results are expressed in viral copies/ug DNA and represent the mean copy number ± SD referred to duplicates of 2 independent assays. b) HHV-6A IE2 (early protein) immunofluorescence (FITC) (white arrow) in epithelial cells from endometrial biopsies of infertile women during proliferative, ovulatory and secretory phases. DAPI staining indicated DNA. Images were taken in bright field (left panels) or fluorescence (right panels) (Nikon Eclipse TE2000S) equipped with a digital camera. Original magnification 100×.
Fig 3
Fig 3. NK cell status.
a) HeLa cells were infected with HHV-6A and virus antigen expression was analyzed by flow cytometry using a mouse mAb directed against HHV-6 IE2 early and gp116 late antigens. HeLa HHV-6A infected cells were co-cultured for 4 hours with NK cells, purified from endometrial biopsies and peripheral blood. NK cell activation status was evaluated after CD107a staining by flow cytometry. Percentage of CD107a NK cells are reported after co-culture with: b, d) HHV-6A infected HeLa cells; c, e) LPS, CpG-ODN or HSV-1 infected HeLa cells. Results are expressed in percentage and represent the mean copy number ± SD referred to duplicates of 2 independent assays. eNK cells: endometrial NK cells; pNK cells: peripheral NK cells; HHV-6A+: HHV-6A positive infertile women; HHV-6A-: HHV-6A negative infertile women; CTR: women with a previous successful pregnancy. * significant p value obtained by Student T test.
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Grants and funding

Funded by Regione Emila Romagna—Young researcher Liberati grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.