Vaginal epithelial dysfunction is mediated by the microbiome, metabolome, and mTOR signaling.

Bacterial vaginosis (BV) is characterized by depletion of Lactobacillus and overgrowth of anaerobic and facultative bacteria, leading to increased mucosal inflammation, epithelial disruption, and poor reproductive health outcomes. However, the molecular mediators contributing to vaginal epithelial dysfunction are poorly understood. Here we utilize proteomic, transcriptomic, and metabolomic analyses to characterize biological features underlying BV in 405 African women and explore functional mechanisms in vitro. We identify five major vaginal microbiome groups: L. crispatus (21%), L. iners (18%), Lactobacillus (9%), Gardnerella (30%), and polymicrobial (22%). Using multi-omics we show that BV-associated epithelial disruption and mucosal inflammation link to the mammalian target of rapamycin (mTOR) pathway and associate with Gardnerella, M. mulieris, and specific metabolites including imidazole propionate. Experiments in vitro confirm that type strain G. vaginalis and M. mulieris supernatants and imidazole propionate directly affect epithelial barrier function and activation of mTOR pathways. These results find that the microbiome-mTOR axis is a central feature of epithelial dysfunction in BV.

Upregulation of IFN-stimulated genes persists beyond the transitory broad immunologic changes of acute HIV-1 infection.

Chronic immune activation during HIV-1 infection contributes to morbidity and mortality in people living with HIV. To elucidate the underlying biological pathways, we evaluated whole blood gene expression trajectories from before, through acute, and into chronic HIV-1 infection. Interferon-stimulated genes, including MX1, IFI27 and ISG15, were upregulated during acute infection, remained elevated into chronic infection, and were strongly correlated with plasma HIV-1 RNA as well as TNF-α and CXCL10 cytokine levels. In contrast, genes involved in cellular immune responses, such as CD8A, were upregulated during acute infection before reaching a peak and returning to near pre-infection levels in chronic infection. Our results indicate that chronic immune activation during HIV-1 infection is characterized by persistent elevation of a narrow set of interferon-stimulated genes and innate cytokines. These findings raise the prospect of devising a targeted intervention to restore healthy immune homeostasis in people living with HIV-1.

Upregulation of PTPRC and Interferon Response Pathways in HIV-1 Seroconverters Prior to Infection.

Human immunodeficiency virus 1 (HIV-1) exposed seronegative (HESN) individuals may have unique characteristics that alter susceptibility to HIV-1 infection. However, identifying truly exposed HESN is challenging. We utilized stored data and biospecimens from HIV-1 serodifferent couple cohorts, in which couples' HIV-1 exposures were quantified based on unprotected sex frequency and viral load of the partner with HIV-1. We compared peripheral blood gene expression between 15 HESN and 18 seroconverters prior to infection. We found PTPRC (encoding CD45 antigen) and interferon-response pathways had significantly higher expression among individuals who went on to become seropositive and thus may be a signature for increased acquisition risk.

Alloimmunity to Class 2 Human Leucocyte Antigens May Reduce HIV-1 Acquisition - A Nested Case-Control Study in HIV-1 Serodiscordant Couples.

Enveloped viruses, including the Human Immunodeficiency Virus-1 (HIV), incorporate host proteins such as human leucocyte antigens (HLA) into their envelope. Pre-existing antibodies against HLA, termed HLA antibodies, may bind to these surface proteins and reduce viral infectivity. Related evidence includes macaque studies which suggest that xenoimmunization with HLA antigens may protect against simian immunodeficiency virus infection. Since HIV gp120 shows homology with class 2 HLA, including shared affinity for binding to CD4, class 2 HLA antibodies may influence HIV acquisition via binding to gp120 on the viral envelope. We conducted a nested case-control study on HIV serodiscordant couples, comparing the frequency of HLA antibodies among highly exposed persistently seronegative controls with those who went on to acquire HIV (HIV-seroconverters). We first performed low resolution HLA typing on 143 individuals who were HIV-infected at enrollment (index partners) and their corresponding sexual partners (115 highly exposed persistently seronegative individuals and 28 HIV-seroconverters). We then measured HLA class 1 and 2 antibodies in the highly exposed persistently seronegative individuals and HIV-seroconverters at early and late timepoints. We analyzed whether such antibodies were directed at HLA specificities of their HIV-infected index partners, and whether autoantibodies or complement-fixing class 2 HLA antibodies were present. Seventy-nine percent of highly exposed persistently seronegative individuals had HLA antibodies; 56% against class 1 and 50% against class 2 alleles. Half of the group of highly exposed persistently seronegative individuals, prior to seroconversion, expressed class 2 HLA antibodies, compared with only 29% of controls (p=0.05). HIV infection was a sensitizing event leading to de novo development of antibodies against HLA-A and HLA-B loci, but not against class 2 loci. HLA autoantibodies were present in 27% of highly exposed persistently seronegative individuals. Complement-fixing class 2 HLA antibodies did not differ significantly between highly exposed persistently seronegative individuals and seroconverters. In multivariable regression, presence of class 2 HLA antibodies at early timepoints was associated with reduced odds of HIV acquisition (odds ratio 0.330, confidence interval 0.112-0.976, p=0.045). These epidemiological data suggest that pre-existing class 2 HLA antibodies were associated with reduced odds of HIV acquisition.

Treatment with Commonly Used Antiretroviral Drugs Induces a Type I/III Interferon Signature in the Gut in the Absence of HIV Infection.

Tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are used for HIV treatment and prevention. Previously, we found that topical rectal tenofovir gel caused immunological changes in the mucosa. Here, we assess the effect of oral TDF/FTC in three HIV pre-exposure prophylaxis trials, two with gastrointestinal and one with cervicovaginal biopsies. TDF/FTC induces type I/III interferon-related (IFN I/III) genes in the gastrointestinal tract, but not blood, with strong correlations between the two independent rectal biopsy groups (Spearman r = 0.91) and between the rectum and duodenum (r = 0.81). Gene set testing also indicates stimulation of the type I/III pathways in the ectocervix and of cellular proliferation in the duodenum. mRNA sequencing, digital droplet PCR, proteomics, and immunofluorescence confirm IFN I/III pathway stimulation in the gastrointestinal tract. Thus, oral TDF/FTC stimulates an IFN I/III signature throughout the gut, which could increase antiviral efficacy but also cause chronic immune activation in HIV prevention and treatment settings.

Brief Report: Bacterial Vaginosis and Risk of HIV Infection in the Context of CD101 Gene Variation.

BACKGROUND: Whether bacterial vaginosis (BV) and CD101 immunoglobulin-like (Ig-like) variants independently increase HIV risk through mucosal inflammation is not well understood. We evaluated whether the impact of BV on HIV acquisition in women differs by the presence or absence of candidate CD101 Ig-like variants. METHODS: We used data from 2 studies of HIV serodiscordant couples in east (Kenya, Tanzania, and Uganda) and southern (Botswana, South Africa, and Zambia) Africa, which longitudinally assessed HIV acquisition (by ELISA) and BV (by Nugent score ≥7). We used previously generated CD101 sequence data for each case and control participant to create a binary variable indicating the presence/absence of any of 5 CD101 Ig-like variants. RESULTS: Confirming previously shown results in this cohort, Ig-like variants increased HIV-infection risk (adjusted hazard ratio [aHR], = 2.63; 95% confidence interval [CI], 1.41 to 4.89). BV was associated with 2.5-fold higher HIV-infection risk only in the absence of Ig-like variants (aHR = 2.47; 95% CI, 0.99 to 6.15; P = 0.052), whereas in the presence of Ig-like variants, BV was not associated with higher HIV-infection risk (aHR = 0.87; 95% CI, 0.35 to 2.15; P = 0.765); however, a test for interaction was nonsignificant (P = 0.116). CONCLUSIONS: We hypothesized that both BV and CD101 Ig-like variants facilitate HIV acquisition by augmenting similar genital inflammation pathways. Our findings indicate that inflammatory mucosal effects of Ig-like variants may influence the impact of BV on HIV risk. Host-defined inflammatory pathways may be useful targets for HIV prevention.

Schistosomiasis was not associated with higher HIV-1 plasma or genital set point viral loads among HIV seroconverters from four cohort studies.

BACKGROUND: Many regions of sub-Saharan Africa experience a high prevalence of both schistosomiasis and HIV-1, leading to frequent coinfection. Higher plasma HIV-1 viral loads are associated with faster disease progression and genital HIV-1 loads are a primary determinant of HIV-1 transmission risk. We hypothesized that schistosome infection would be associated with higher HIV-1 viral loads in plasma and genital samples. METHODS/PRINCIPAL FINDINGS: We utilized data from individuals who HIV-1 seroconverted while enrolled in one of four prospective cohort studies. Plasma and genital viral loads collected 4-24 months after the estimated date of HIV-1 acquisition, but prior to antiretroviral therapy initiation, were included. Detection of circulating anodic antigen in archived blood samples, collected prior to HIV-1 seroconversion, identified participants with active schistosomiasis; immunoblots determined the schistosome species causing infection. Our analysis included 370 HIV-1 seroconverters with plasma viral load results, of whom 82 (22%) had schistosomiasis. We did not find a statistically significant association between schistosomiasis and higher HIV-1 set point plasma viral loads (-0.17 log10 copies/ml, 95% CI -0.38 to 0.03); S. mansoni infection was associated with a lower set point (-0.34 log10 copies/ml, 95% CI -0.58 to -0.09). We found no association between schistosomiasis and cervical (+0.07 log10 copies/swab, 95% CI -0.20 to 0.34) or vaginal (+0.11 log10 copies/swab, 95% CI -0.17 to 0.39) set point viral loads; S. haematobium infection was associated with lower cervical viral loads (-0.59 log10 copies/swab, 95% CI -1.11 to -0.06). CONCLUSIONS/SIGNIFICANCE: These results do not support the hypotheses that schistosome coinfection increases plasma or genital HIV-1 viral loads.

Correction: Whole genome sequencing of extreme phenotypes identifies variants in CD101 and UBE2V1 associated with increased risk of sexually acquired HIV-1.

[This corrects the article DOI: 10.1371/journal.ppat.1006703.].

EPHA2 sequence variants are associated with susceptibility to Kaposi's sarcoma-associated herpesvirus infection and Kaposi's sarcoma prevalence in HIV-infected patients.

BACKGROUND: To determine if variations exist in the KSHV host receptor EPHA2's coding region that affect KSHV infectivity and/or KS prevalence among South African HIV-infected patients. METHODS: A retrospective candidate gene association study was performed on 150 patients which were randomly selected from a total of 756 HIV-infected patients and grouped according to their KS status and KSHV serodiagnosis; namely group 1: KS+/KSHV+; group 2: KS-/KSHV+; group 3: KS-/KSHV-. Peripheral blood DNA was used to extract DNA and PCR amplify and sequence the entire EPHA2 coding region, which was compared to the NCBI reference through multiple alignment. RESULTS: 100% (95% CI 92.9-100%) of the KS positive patients, and 31.6% (95% CI 28.3-35.1%) of the KS negative patients were found to be KSHV seropositive. Aggregate variation across the entire EPHA2 coding region identified an association with KS (OR = 6.6 (95% CI 2.8, 15.9), p = 2.2 × 10-5). This was primarily driven by variation in the functionally important protein tyrosine kinase domain (Pkinase-Tyr; OR = 4.9 (95% CI 1.9, 12.4), p = 0.001) and the sterile-α-motif (SAM; OR = 13.8 (95% CI 1.7, 111.6), p = 0.014). Mutation analysis revealed two novel, non-synonymous heterozygous variants (c.2254 T > C: OR undefined, adj. p = 0.02; and c.2990 G > T: OR undefined, adj. p = 0.04) in Pkinase-Tyr and SAM, respectively, to be statistically associated with KS; and a novel heterozygous transition (c.2727C > T: OR = 6.4 (95% CI 1.4, 28.4), adj. p = 0.03) in Pkinase-Tyr to be statistically associated with KSHV. CONCLUSIONS: Variations in the KSHV entry receptor gene EPHA2 affected susceptibility to KSHV infection and KS development in a South African HIV-infected patient cohort.

Toll-like receptor 9 polymorphism is associated with increased Epstein-Barr virus and Cytomegalovirus acquisition in HIV-exposed infants.

: Polymorphisms in the Toll-like receptor 9 1635 locus have been associated with HIV-1 acquisition and progression. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) acquisition were compared between Kenyan HIV-exposed infants by 1635 genotype. Having one or more copies of the 1635A allele was associated with increased CMV acquisition in HIV-infected infants (42 vs. 11%, P = 0.03) and increased risk of EBV acquisition in HIV-exposed uninfected infants (hazard ratio = 4.2, P = 0.02) compared with 1635GG. In addition, 1635A was associated with 0.4 log10 copies/ml lower median EBV levels in HIV-infected infants (P = 0.03). These data suggest a potentially important role for this locus in primary herpesvirus infection.

Whole genome sequencing of extreme phenotypes identifies variants in CD101 and UBE2V1 associated with increased risk of sexually acquired HIV-1.

Host genetic variation modifying HIV-1 acquisition risk can inform development of HIV-1 prevention strategies. However, associations between rare or intermediate-frequency variants and HIV-1 acquisition are not well studied. We tested for the association between variation in genic regions and extreme HIV-1 acquisition phenotypes in 100 sub-Saharan Africans with whole genome sequencing data. Missense variants in immunoglobulin-like regions of CD101 and, among women, one missense/5' UTR variant in UBE2V1, were associated with increased HIV-1 acquisition risk (p = 1.9x10-4 and p = 3.7x10-3, respectively, for replication). Both of these genes are known to impact host inflammatory pathways. Effect sizes increased with exposure to HIV-1 after adjusting for the independent effect of increasing exposure on acquisition risk. TRIAL REGISTRATION: ClinicalTrials.gov NCT00194519; NCT00557245.

Microbiome Composition and Function Drives Wound-Healing Impairment in the Female Genital Tract.

The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity.

HIV-1-Neutralizing IgA Detected in Genital Secretions of Highly HIV-1-Exposed Seronegative Women on Oral Preexposure Prophylaxis.

Although nonhuman primate studies have shown that simian immunodeficiency virus/simian-human immunodeficiency virus (SIV/SHIV) exposure during preexposure prophylaxis (PrEP) with oral tenofovir can induce SIV immunity without productive infection, this has not been documented in humans. We evaluated cervicovaginal IgA in Partners PrEP Study participants using a subtype C primary isolate and found that women on PrEP had IgA with higher average human immunodeficiency virus type 1 (HIV-1)-neutralizing magnitude than women on placebo (33% versus 7%; P = 0.008). Using a cutoff of ≥90% HIV-1 neutralization, 19% of women on-PrEP had HIV-1-neutralizing IgA compared to 0% of women on placebo (P = 0.09). We also estimated HIV-1 exposure and found that the proportion of women with HIV-1-neutralizing IgA was associated with the level of HIV-1 exposure (P = 0.04). Taken together, our data suggest that PrEP and high levels of exposure to HIV may each enhance mucosal HIV-1-specific humoral immune responses in sexually exposed but HIV-1-uninfected individuals. IMPORTANCE: Although there is not yet an effective HIV-1 vaccine, PrEP for at-risk HIV-1-uninfected individuals is a highly efficacious intervention to prevent HIV-1 acquisition and is currently being recommended by the CDC and WHO for all individuals at high risk of HIV-1 acquisition. We previously demonstrated that PrEP use does not enhance peripheral blood HIV-1-specific T-cell responses in HIV-exposed individuals. Here, we evaluate for cervicovaginal HIV-neutralizing IgA responses in genital mucosal secretions of HIV-exposed women, which is likely a more relevant site than peripheral blood for observation of potentially protective immune events occurring in response to sexual HIV-1 exposure for various periods. Furthermore, we assess for host response in the context of longitudinal quantification of HIV-1 exposure. We report that HIV-neutralizing IgA is significantly correlated with higher HIV-1 exposure and, furthermore, that there are more women with HIV-1-neutralizing IgA in the on-PrEP group than in the placebo group.

Dual contraceptive method use in HIV-serodiscordant Kenyan couples.

BACKGROUND: World Health Organization (WHO) guidelines recommend dual contraceptive method use with condoms and another contraceptive to reduce both incidence of HIV/sexually transmitted infection transmission and unintended pregnancies. This qualitative study assessed the barriers to and motivations for dual contraceptive use in Kenyan HIV-serodiscordant couples. METHODS: HIV-serodiscordant couples in Nairobi, Kenya, were recruited from two longitudinal cohorts. Qualitative semistructured interviews were conducted using a semistructured questionnaire. Twelve male and 12 female members of serodiscordant couples and 10 women with incident pregnancies during the cohort studies were included. RESULTS: Few couples reported using dual contraceptive methods, with men reporting more condom use than women. No HIV-seropositive men or HIV-seronegative women reported using non-condom contraception. Men and women agreed that men play a dominant role in decisions to use both condoms and contraception in HIV-serodiscordant couples. Participants reported that perceptions of side effects, male partner preference, and reproductive desire were critical factors in contraceptive decisions. Both men and women saw dual contraceptive method use as redundant and a sign of possible unfaithfulness. Many participants actively desired pregnancy, but few were able to accurately define monthly fertility windows. CONCLUSIONS: Dual contraceptive method use was low in these HIV-serodiscordant couples, with some couples finding it unnecessary while using condoms, and others being more focused on conceiving a child. Biomedical HIV prevention, including male circumcision, pre-exposure prophylaxis or antiretroviral therapy to reduce HIV transmission, may be more acceptable strategies to promote safer sexual relations among HIV-serodiscordant couples and safer conception when desired.

Toll-like receptor gene variants and bacterial vaginosis among HIV-1 infected and uninfected African women.

Bacterial vaginosis (BV) is a common vaginal syndrome associated with altered microflora that increases the risk of preterm delivery and acquisition of sexually transmitted diseases. The cause of BV is unknown although toll-like receptors (TLRs), that are central to innate immune responses, may be important. We evaluated associations between TLR SNPs and BV among HIV-1 infected and uninfected African women. Logistic regression was used to assess associations between SNPs (N=99) in TLRs 2-4, 7-9 and BV (as classified by Nugent's criteria). Among HIV-1 uninfected women, TLR7 rs5743737 and TLR7 rs1634323 were associated with a decreased risk of BV, whereas TLR7 rs179012 was associated with an increased risk. TLR2 SNP rs3804099 was associated with a decreased risk of BV among HIV-1 infected women. Our findings indicate that there may be differences in TLR association with BV among HIV-1 infected and HIV-1 uninfected women.

Host genetic and viral determinants of HIV-1 RNA set point among HIV-1 seroconverters from sub-saharan Africa.

UNLABELLED: We quantified the collective impact of source partner HIV-1 RNA levels, human leukocyte antigen (HLA) alleles, and innate responses through Toll-like receptor (TLR) alleles on the HIV-1 set point. Data came from HIV-1 seroconverters in African HIV-1 serodiscordant couple cohorts. Linear regression was used to determine associations with set point and R(2) to estimate variation explained by covariates. The strongest predictors of set point were HLA alleles (B*53:01, B*14:01, and B*27:03) and plasma HIV-1 levels of the transmitting partner, which explained 13% and 10% of variation in set point, respectively. HLA-A concordance between partners and TLR polymorphisms (TLR2 rs3804100 and TLR7 rs179012) also were associated with set point, explaining 6% and 5% of the variation, respectively. Overall, these factors and genital factors of the transmitter (i.e., male circumcision, bacterial vaginosis, and use of acyclovir) explained 46% of variation in set point. We found that both innate and adaptive immune responses, together with plasma HIV-1 levels of the transmitting partner, explain almost half of the variation in viral load set point. IMPORTANCE: After HIV-1 infection, uncontrolled virus replication leads to a rapid increase in HIV-1 concentrations. Once host immune responses develop, however, HIV-1 levels reach a peak and subsequently decline until they reach a stable level that may persist for years. This stable HIV-1 set point represents an equilibrium between the virus and host responses and is predictive of later disease progression and transmission potential. Understanding how host and virus factors interact to determine HIV-1 set point may elucidate novel mechanisms or biological pathways for treating HIV-1 infection. We identified host and virus factors that predict HIV-1 set point in people who recently acquired HIV-1, finding that both innate and adaptive immune responses, along with factors that likely influence HIV-1 virulence and inoculum, explain ∼46% of the variation in HIV-1 set point.

Variants in host viral replication cycle genes are associated with heterosexual HIV-1 acquisition in Africans.

OBJECTIVE: We evaluated genetic variants in 51 candidate genes encoding proteins that interact with HIV-1 during the virus life cycle for association with HIV-1 outcomes in an African cohort. METHODS: Using a nested case-control study within a cohort of heterosexual HIV-1-serodiscordant couples, we genotyped 475 haplotype-tagging single-nucleotide polymorphisms (tagSNPs) and 18 SNPs previously associated with HIV-1 transmission and/or progression (candidate SNPs) in 51 host genes. We used logistic and Cox proportional hazard regression with adjustment for sex, age, and population stratification to detect SNP associations with HIV-1 acquisition, plasma HIV-1 set point, and a composite measure of HIV-1 disease progression. Significant thresholds for tagSNP, but not candidate SNP, associations were subjected to Bonferroni correction for multiple testing. RESULTS: We evaluated 491 HIV-1-infected and 335 HIV-1-uninfected individuals for 493 SNPs, 459 of which passed quality control filters. Candidate SNP PPIA rs8177826 and tagSNP SMARCB1 rs6003904 were significantly associated with HIV-1 acquisition risk (odds ratio = 0.14, P = 0.03, and odds ratio = 2.11, Pcorr = 0.01, respectively). Furthermore, the TT genotype for CCR5 rs1799988 was associated with a mean 0.2 log10 copies per milliliter lower plasma HIV-1 RNA set point (P = 0.04). We also identified significant associations with HIV-1 disease progression for variants in FUT2 and MBL2. CONCLUSIONS: Using a targeted gene approach, we identified variants in host genes whose protein products interact with HIV-1 during the virus replication cycle and were associated with HIV-1 outcomes in this African cohort.

Toll-like receptor polymorphism associations with HIV-1 outcomes among sub-Saharan Africans.

OBJECTIVE: We evaluated Toll-like receptors (TLRs) single nucleotide polymorphisms (SNPs) for associations with HIV-1 acquisition, set-point and disease progression in African couples. METHODS: Seven candidate and 116 haplotype-tagging SNPs (tagSNPs) were genotyped in 504 HIV-1 infected cases, and 343 seronegative controls. RESULTS: TLR9 1635A/G was associated with reduced HIV-1 acquisition among HIV-seronegative controls with high but not low HIV-1 exposure (odds ratio [OR] = 0.7; P = .03 and OR = 0.9, P = .5, respectively). TLR7 rs179012 and TLR2 597C/T reduced set-point; the latter modified by time since HIV-1 acquisition. TLR8 1A/G reduced disease progression. CONCLUSIONS: TLR SNPs impact HIV-1 outcomes with epidemiologic factors modifying these relationships.

High rates of relationship dissolution among heterosexual HIV-serodiscordant couples in Kenya.

HIV-1 serodiscordant couples may experience increased risks of relationship dissolution; however, longitudinal stability of these relationships is poorly understood. We determined rates and correlates of separation among 469 serodiscordant couples in Nairobi and found that 113 (24 %) separated during 2 years of follow-up. Couples with a female HIV-1 infected partner (F+M-) and no income were more likely to separate than M+F- couples without income (HR = 5.0; 95 % CI 1.1-25.0), and F+M- and M+F- couples with income (HR = 2.4; 95 % CI 1.3-4.5 and HR = 2.3; 95 % CI 1.2-4.8, respectively). High separation rates may be important for couple support services and for conducting discordant couple studies.