---
title: "Birth Control vs Inflammation over menses"
format:
  html:
    code-fold: true
execute:
  warning: false
  cache: true

---


# Study Description

This is an observational study to evaluate the relationship between birth control and vaginal inflammation In response to menstruation. Absolute abundance of bacteria was measured by 3 qPCR assays (for total Bacteria, L. crispatus, L. iners). Cytokine levels (in copies/ml of vaginal fluid) were also measured by Luminex. We also looked at the number and type of immune cells in the vagina using Flow Cytometry. Data was collected at four timepoints; before, at the start, the end, and after menstruation.


# Primary Aim

To investigate whether taking birth control is associated with vaginal inflammation throughout the menstrual cycle.

# Data processing

```{r}
library(tidyverse)
library(tableone)
library(cowplot) # load for the theme
theme_set(theme_cowplot(16) + background_grid()) # set cowplot theme

sample_map <- read_csv("data/00_sample_ids_period.csv")
metadata <- read_csv("data/01_participant_metadata_period.csv")
luminex <- read_csv("data/02_luminex_period.csv")
flow_cytometry <- read_csv("data/03_flow_cytometry_period.csv")
```

## Merging the dataset

```{r}

merged_dataset <- 
  sample_map %>%
  left_join(metadata) %>%
  left_join(luminex) %>%
  left_join(flow_cytometry) %>%
  mutate(cd4_ratio = cd4_t_cells / cd3_positive )  %>%
  mutate(time_point = fct_relevel(time_point,"week_prior","onset","end_bleeding", "week_post"))

```


Let's create table one

```{r}
processed_for_tableone <- merged_dataset %>%
  select(-limits, -sample_id) %>%
  pivot_wider(names_from = cytokine, values_from = conc) %>% # move cytokine to wide
  filter(time_point == "week_prior") # we just want baseline characteristics for table 1

CreateTableOne(
  data = processed_for_tableone,
  vars = c("age", "pcos_status", "period_product", "cd4_ratio", "IL-1a", "IL-1b", "IP-10", "MIG", "TNF-a" ),
  factorVars = c("pcos_status","period_product" ),
  strata = "arm",
  test = FALSE
) %>%
  print(
    nonnormal = c("cd4_ratio", "IL-1a", "IL-1b", "IP-10", "MIG", "TNF-a"),
    
  )

```

## Arm cytokines


```{r}
#| fig-width: 4
#| fig-height: 9
merged_dataset %>%
  filter(cytokine == "TNFa") %>%
  ggplot(aes(x=conc, y=arm)) +
  geom_boxplot() +
  geom_jitter(alpha = 0.5) +
  scale_x_log10(labels=scales::label_log()) +
  facet_grid(rows=vars(time_point), scales="free")

```

```{r}
#| fig-width: 12
#| fig-height: 9

merged_dataset %>%
  ggplot(aes(x=conc, y=arm)) +
  geom_boxplot() +
  geom_jitter(alpha = 0.5) +
  scale_x_log10(labels=scales::label_log()) +
  facet_grid(rows=vars(time_point), cols=vars(cytokine), scales="free")
```


## Time series visualization


```{r}
#| fig-width: 8
#| fig-height: 12

merged_dataset %>%
  ggplot(aes(y=conc, x=time_point)) +
  geom_boxplot() +
  geom_point(alpha = 0.5) +
  geom_line(alpha=0.5, aes(color=arm, group=pid)) +
  scale_y_log10(labels=scales::label_log()) +
  facet_grid(rows=vars(cytokine), scales="free")

```

## Cytokines vs pcos

How does PCOS associate with inflammation over menstruation? First, let's look at one of the cytokines at one of the timepoints.

```{r}
merged_dataset %>%
  filter(time_point == "week_prior", cytokine == "TNFa") %>%
  ggplot(aes(x = pcos_status, y=conc, color = arm)) +
  geom_boxplot() +
  geom_point(position = position_jitterdodge()) +
  scale_y_log10() +
  facet_grid(cols=vars(time_point), rows=vars(cytokine), scales="free_y")
```

Now, let's look at all the cytokines together with all time points.

```{r}
#| fig-width: 15
#| fig-height: 8

merged_dataset %>%
  ggplot(aes(x = pcos_status, y=conc, color = arm)) +
  geom_boxplot() +
  geom_point(position = position_jitterdodge()) +
  scale_y_log10() +
  facet_grid(cols=vars(time_point), rows=vars(cytokine), scales="free_y")
  
```

## Cytokines vs flow

Are there any relationships between cytokines and cd4 ratio?


```{r}

merged_dataset %>%
  ggplot(aes(x=conc, y=cd4_ratio, color=arm, )) +
  geom_point() +
  scale_x_log10(labels=scales::label_log()) +
  facet_wrap(~cytokine)
```