Biostatistics Part III: Statistical Analyses

We did biostats once upon a time in two previous episodes around the beginning of our podcast series. If you’d like to take a listen, here are the links: Part I and Part II.

Let’s do a quick review and discuss the different types of studies that we could do:

• Exposure (or intervention): risk factor whose effect is being studied 

  • Also can be referred to as the “independent” or “predictor” variable 

• Outcome: something that develops as a consequence of exposure (or intervention)

  • Also referred to as the predicted or dependent variable (or variables) 

• First study category: temporality: 

  • Retrospective studies 

    • Means that the outcomes or the dependent variables (and likely independent variables!) have already occurred, or you have that data already  

  • Prospective studies 

    • Means that the outcomes or dependent variables (or even independent variables) have not yet been measured 

• Second category: descriptive vs analytical

  • Descriptive studies - where you’re merely trying to describe data on one or more characteristics of a group of individuals; these types of studies don’t usually try to answer a question or establish a relationship between variables:

    • Case report

    • Case series 

    • Cross-sectional studies 

  • Analytical - attempt to test a hypothesis and establish causal relationships between variables:

    • Observational - studies where a researcher is documenting a naturally occurring relationship between exposure and outcome 

      • Case Control studies - first determine if the outcome is present (ie. cases of lung cancer vs. cases where there is no lung cancer) and then traces the presence of prior exposure to a risk factor (ie. tobacco use)

      • Cohort studies - first determine the exposure to a risk factor and then assesses whether the outcome occurs a future time point 

    • Experimental - research actively performs an intervention in some or all members of a group 

      • Remember: only experimental studies can establish a causal relationship; observational studies can show correlation, but not reliably show causation! 

• It is important to know that you can have both retrospective and prospective observational studies, but experimental studies are all prospective.

(c) CREOGS OVER COFFEE, 2022

The next step is, let’s say you have your study and you’ve collected your data… now… HOW DO I ANALYZE IT ALL? Dr. Rebecca Hamm at UPenn has shared with us this crazy but excellent flow chart to figure it out. While we won’t hit everything in the podcast, we’ll hit some of the more common tests and the first few questions of the flowchart.

Courtesy of rebecca hamm, md

First question: What type of data do you have? 

• Continuous (example: age, BMI, weight, etc.)

  • See second question  

• Categorical (Gender; Yes/No; Category 1, 2, or 3)

  • You can use a Chi-Square test!

    • In simple terms, a Chi-Square (or Pearson’s Chi Square test) is going to determine if there is a statistically significant difference between expected frequencies and the observed frequencies in one or more categories of a contingency table

    • In your contingency table, if any category has <5 observations, then you have to use a Fisher’s exact test

• Second question: If you have a continuous variable, do you have parametric or nonparametric data? 

  • Parametric basically means: 

    • You have independent, unbiased samples 

      • Independence (in statistics terms) basically means the occurrence of one thing does not affect the probability of the occurrence of another thing 

    • The data is normally distributed 

      • How do you figure that out? Easiest way - create a histogram to check 

    • Harder way: there are many statistical tests of skewness that we won’t describe here! 

    • Equal variances 

      • Basically, variance is a statistical measurement of the spread between numbers in a data set, or how far each number in the set is from the mean (average) 

      • The square root of variance is standard deviation (you’ve probably heard of that!) 

      • Therefore, equal variance means that in order for us to consider data parametric, we have to assume that the variance is the same for both populations we are comparing 

    • Third question: If you have a continuous variable, what type of question are you asking? 

      • I want to know about relationships:

        • If you have a true independent variable, you can use a regression analysis

          • Example: a linear regression, where you actually have an equation and an R^2 value

          • Doesn’t have to be linear relationship - other forms of regression exist.

        • If you don’t have a true independent variable, then we have to do a correlation analysis  

        1. If parametric: Pearsons’ r test  

        2. Nonparametric: Spearman’s Rank Correlation 

      • I want to know about differences between the means of my groups: 

        • How many treatment groups do you have? 

          • If two: 

            1. If parametric, can use student’s t-test (paired or unpaired) 

            2. If nonparametric, then can use Mann-Whitney U or Wilcoxon Rank sum test 

          • If more than two: 

            1. If parametric, can use an ANOVA 

            2. If nonparametric, can use Kruskal-Wallis test 

Examples

•  Let’s try and figure out what the best statistical test is for the following situations! 

  • What is the frequency of repeat hypertensive disease of pregnancy in patients who took low dose aspirin vs. those that did not take low dose aspirin?

    • Questions you’ll want to ask: is this categorical or continuous? 

      • Categorical! Hypertensive disease is a “yes” or “no” in this case 

      • Therefore, we will want to use a Chi-Square test.

  • What is the gestational age at which patients with short cervix delivered if they got a cerclage or not? 

    • Question you want to ask: is gestational age at delivery categorical or continuous? 

      • Continuous! 

    • Now… is gestational age at delivery going to give us a parametric data set? Let’s see!

      • Is it independent: yes – the gestational age at which one person delivers should not affect the gestational age at which another person delivers in this data set.

      • Is it normal? Nope! – just going to give this one to you, but gestational age at delivery is not normally distributed (lots of people will delivery right around 39-40 weeks, and then there is a long, skewed tail of those that delivery very early, like 24 weeks etc) 

      • So we have a continuous, non-parametric set of data 

      • Next question: do we want to know relationship or difference of means? Difference of means!

        • So we can use a Wilcoxon Rank Sum test  

  • Is there a difference in admission hemoglobin between patients who received iron supplementation during pregnancy or not?

    • Question: is Hgb a continuous or categorical variable - continuous

    • Question: Is Hgb a parametric data set - for our purposes, let’s say yes! 

    • Question: Do we want to know a relationship or a difference of means? Difference of means 

      • So we can use Student’s t-test  

Anatomic Meditation: Pelvic Vasculature

Genetic Carrier Screening

Additional Reading
CO 690: Carrier Screening in the Age of Genomic Medicine
CO 691: Carrier Screening for Genetic Conditions
CO 816: Consumer Testing for Disease Risk

Previously on the podcast, we have talked through aneuploidy screening. But we’ve not talked in depth about carrier screening, so today’s podcast is dedicated to the other form of prenatal genetics we often consider! 

What is carrier screening?

• Aneuploidy screening: looking at some biochemical marker in an already pregnant individual to understand risk of trisomy (typically).

• Carrier screening: looking at genetics of parental contributions to assess potential risk in a current or hypothetical pregnancy. 

  • So this tells you - do you carry a condition that you are not affected by?

  • Only needs to be performed once in a lifetime - as opposed to aneuploidy screening, which needs to be re-performed with every pregnancy. 

• ACOG recommends that “information about carrier screening should be provided to every pregnant individual.” 

• Carrier screening most commonly looks for autosomal recessive conditions - that is, both parents need to be carriers in order for there to be a 25% risk of fetus being affected.

  • Certain X-linked conditions (i.e., hemophilia, Fragile X) can also be screened.

    • Information can be used in pregnancy planning, understanding risk of fetal condition that may impact life/lifespan of fetus, and choice for IVF with PGT or invasive testing in pregnancy.

  • Some other conditions may be discouraged from carrier screening (i.e., Huntington’s disease, BRCA genes) because of ethical concerns with doing carrier screening on fetuses, given these are adult-onset conditions. 

  • No “official threshold” for carrier screening generally, but most panels select conditions with a carrier frequency of ~1/100 or greater → generally a disease incidence of 1 in 40,000.

• There always remains some residual risk for carriage state/disease, even after carrier screening.

What strategies have been suggested for carrier screening?

• Historically, carrier screening was considered on an ethnicity basis (i.e., ethnic-based screening)

  • However, multiple limitations to this approach:

    • Challenging for individuals to define ancestry

    • Ancestral “mixing” between partners of different ethnicities causing different risks

    • The “pretest” probability of a positive is difficult to predict given these limitations

    • Couples with consanguinity may be at higher risk of recessive conditions being expressed in offspring, regardless of ethnic background.

• Current approaches favor panethnic or expanded carrier screening 

  • Panel of disorder screening is offered to all individuals regardless of ancestry.

  • The cost of screening has come down significantly, allowing for screening for hundreds of conditions at reasonable cost to patient.

• If family history of mutations/conditions are known, targeted screening can be considered to look for specific mutations.

What limitations are there in carrier screening? What does “residual risk” after carrier screening mean?

• These carrier screening panels look for known mutations in a population, based on a reference genome.

  • These reference genomes are overwhelmingly represented by White populations, so:

    • Carrier screening may not detect all mutant variants of an allele → residual risk

    • Carrier screening does not recognize new, potentially disease-causing variants.

      • Carrier testing is not sequencing! 

What conditions are recommended by ACOG to be screened for with carrier screening?

• Spinal muscular atrophy

  • Autosomal recessive disease with spinal cord motor neuron degeneration due to biparental inheritance of an SMN1 mutation/deletion.

  • Leading genetic cause of infant death.

  • Incidence of disease around 1 in 6-10k; carrier frequency in most populations around 1:40 to 1:60.

    • 2% of cases are the result of a new gene mutation. 

  • SMA has an interesting genetic profile:

    • There is generally one copy of SMN1 per chromosome, and a deletion/abnormality in each parental contribution leads to disease (again, autosomal recessive).

    • However, some of the population have two copies of SMN1 on a chromosome, and 0 copies on the other – so they are technically carriers (because of the chromosome with 0 copies).

    • Carrier screening tests for SMA generally look for the number of copies of SMN1 - so a patient with this particular variation (2+0) would be missed.

      • This 2+0 variation is much more common in African Americans - lowering the carrier detection rate of SMA from 95% in White patients to 71% in African Americans.

      • This leads to a higher residual risk from these tests as they may miss the 2+0 mutation.

• Cystic fibrosis

  • Most common life-threatening AR condition in White population.

    • Incidence 1/2500 in White; considerably less common in other ethnic groups.

  • Two copies of CFTR mutations (chromosome 7) cause the disease.

  • Most carrier screening looks for one of the 23 most common mutations that exist – again, predominantly in White populations.

    • But there are over 1700 CFTR mutations identified that can lead to CF!

    • Performance ranges from 94% sensitivity in Ashkenazi Jewish populations to less than 50% in Asian populations. 

    • Because of the number of mutations, some have advocated for CFTR sequencing to supplant panel testing as a way to determine carrier status and reduce residual risk amongst all populations. 

• Hemoglobinopathies

  • We have talked about these on the show before - thalassemias and sickle cell disease.

  • CBC and RBC indices should be performed in all pregnant persons to assess for anemia and risk of hemoglobinopathy.

    • Hb electrophoresis can be considered in all patients with anemia, particularly if there is family history or ethnicity-based risk factor, to screen for hemoglobinopathy.

    • Alpha thalassemias, though, can only be detected with molecular genetic studies - so if the electrophoresis is not conclusive, DNA-based testing should be pursued to assess for alpha thal. 

• Fragile X Syndrome

  • Most common inherited form of intellectual disability; distinctive facial features in males, enlarged testicles, delay in fine and gross motor skills are some manifestations.

  • 1 in 3600 males; 1 in 4k-6k females. 

  • Carrier frequency in the US around 1 in 250 for no known risk factors, or 1 in 86 for those with a family history of intellectual disability.

  • X-linked disorder of mutation in FMR1 gene.

    • The mutation is characterized by expansion of a trinucleotide repeat sequence (CGG); the more repeats, the more significant the mutation:

      • Intermediate (45-54 repeats)

      • Premutation (55-200 repeats)

      • Full mutation (>200 repeats)

    • Females carrying a premutation or full-mutation X chromosome are also at risk for premature ovarian insufficiency. Females with full mutation may also have fragile X characteristics of disease like in males, though with variable expression. 

We hear a lot about “Ashkenazi Judaism” and carrier screening. What does that mean and what conditions should be screened?

• Ashkenazi Jewish is defined in the committee opinions as individuals of Eastern and Central European Jewish descent.

  • Not a super accurate or helpful designation, as most individuals with Jewish ancestry in the USA are descended from these areas.

• Recommendations for specific screening:

  • Tay Sachs Disease - severe, progressive neurodegenerative disease with functional deficiency in the gene encoding the hexosaminidase A enzyme. 

    • Carrier rate in Ashkenazi Jewish populations around 1 in 30.

  • Cystic fibrosis

  • Canavan disease - severe degenerative neurologic disease

  • Familial dysautonomia - severe disorder of sensory and autonomic nervous systems

  • Multiple others are also considered, including Gaucher disease, Joubert syndrome, maple syrup urine disease, Niemann-Pick disease, and a few others. 

    • The panels developed for this population are very ethnicity-specific - so while great for this population, residual risk discussion can be complicated in non-Jewish individuals (as the incidence of carriage is often very low).

What about the genetic screening tests advertised to patients online?

• There are a whole host of “carrier screens” that are direct-to-consumer, and even some of the more reputable companies in this space have direct-to-consumer options given the decreasing expense of this technology. 

• However, these companies have varying degrees of privacy protections for genetic data.

• They also may have implications on patient’s eligibility for disability and other types of insurance; long-term care considerations; and ownership of one’s own genetic data.

• Some direct-to-consumer testing uses different kinds of technology to develop a picture of risk for a patient, that may or may not be helpful in their context. Abnormal results of concern should always be reviewed with a genetic counselor.

  • If you have any concerns or need more time for your patients to discuss whether they want to have carrier screening, it’s worthwhile to send them to a GC! They can help patients navigate targeted vs expanded carrier screening and help make decisions that are right for each individual patient. 

Placental Pathology II: Examination and Future Pregnancies

Check out this article from Contemporary OB/GYN on placental pathology to augment your learning: https://www.contemporaryobgyn.net/view/placental-pathology-it-time-get-serious 

What do they look for on the placenta anyway? 

• Gross examination of the placenta - remember, we aren’t pathologists! 

  • Fresh examination is usually best

    • Why?  Because you can send cultures, cytogenetic studies, and injections of the vessels on fresh, but not fixed, specimen

  • Umbilical cord 

    • General appearance: will comment on color, nodules, strictures, edema, and coiling 

    • Will also discuss area of placental insertion (ie. velamentous, marginal, etc) 

    • Length + knots 

    • Vessels - single or two UAs? 

    • Other things: hematomas, neoplasms, cysts 

  • Membranes 

    • Color

    • Insertion of the umbilical cord in membrane or on placenta? 

  • Placental parenchyma 

    • Usually will comment on weight and percentile for gestational age

      • Some correlation with birth weight  

    • Dimension and appearance - also, is it bilobed? Are there succenturiate lobes? 

    • Maternal surface: should be complete when looking at it grossly 

      • Can also see areas of infarcts

    • Fetal surface: review if there are large vessels coursing near the edge, if there are cysts, subchorionic hematomas, etc 

So what are some common findings, and what should we look for on the report? 

• Placental weight 

  • Some type of chronic stress may lead to smaller placentas

    • Think chronic hypertension, diabetes, etc

    • Usually can product placentas that are <10th%ile  

  • Some pitfalls: there are conditions that lead to fetal stress that can also make placentas abnormally large, ie. hydrops 

• Infarctions, vessel artherosis

  • Often, these are findings that are related ot hypertensive disorders of pregnancy 

  • Can see things like fibrinoid necrosis of the vessel wall, perivascular infiltrates of WBC; also can see infarcts

  • Can also see areas of abruption: but remember, abruption is a clinical diagnosis! 

    • This is because there can be small area of bleeding, placenta infarct etc that are not clinically relevant 

    • Under the microscope, abruption would appear as diffuse retromembranous or intradecidual hemorrhage, irregular basal intervillous thrombi, and recent villous stromal hemorrhage 

    • However, this is not specific, and can be seen with normal delivery as well 

• Infection

  • I feel like I often see “chorioamnionitis” written all over the reports, even when the patient doesn’t have chorio! So what do the pathologists see? 

  • Histopathologic findings are neutrophilic inflammation of the chorion and amnion 

  • You can also see inflammatory infiltrate of the vascular portion of the umbilical cord or Wahrton’s jelly 

  • The pitfall: 

    • Clinically diagnosed chorio may not always been seen on histology and vice versa! Why is that? 

    • Clinically: it is possible that there was another inflammatory process going on, or chorio was diagnosed by maternal fever, which can be caused by many other things (ie. misoprostol, epidural use) 

    • Histologically: Remember that evidence of inflammation on histology does not always mean that there is microbiologic evidence of infection; cultures of amniotic fluid or membranes do not document a bacterial infection in 25-30% of placentas with histologic chorio 

So how does this affect our practice or the patient’s future pregnancies? 

• There is some data that suggests that some placental pathologies can lead to recurrence of poor outcomes in pregnancy 

  • For example, one study showed that inflammation in the placenta were associated with recurrent preterm birth and spontaneous preterm birth 

    • Ie. Villitis 

  • There is also some suggestion that chronic endometritis can lead to recurrent miscarriages 

  • The current issue is that while research has shown these associations, there isn’t anything currently that has proven to clinically improve outcomes

  • Though this does spark some interesting debate about tamping down inflammation: since there is some observation that the use of antenatal steroids seems to temporarily improve preeclampsia 

  • This is all just speculation though, and doesn’t mean we recommend using chronic steroids to prevent preeclampsia! 

  • So… not a super satisfying answer  

• Other predictions 

  • With regards to abnormal placentation such as placenta accreta spectrum, there is a 25-30% recurrence risk based off of findings of histological examination of the placenta 

  • I’m not convinced this is clinically useful, unless during delivery, there was not a diagnosis of accreta 

  • Certainly, if there is focal accreta diagnosed clinically, I think clinically, we would also counsel the patient about increased recurrence risk 

• What placental pathology can’t do 

  • The literature suggests that widespread pathologic examination of the placenta does not prognosticate adverse childhood and neurologic outcomes 

  • In some selected cohorts though, there can be some associations 

• Another thing to know is that findings on the placenta can give patients closure on things like poor outcomes

  • Placenta pathology can be very useful in determining the etiology of stillbirth, particularly after 24 weeks gestation 

  • Studies show that placental examination was useful in up to 64% of cases of stillbirth (compared to only 12% for karyotype and 0.4% for parvo testing) 

  • However, we need to recognize that while this may give patients closure, it is not necessarily predictive of future pregnancies 

• The medical-legal realm 

  • People may ask if we can refute a legal claim after examination of a placenta 

  • We are not lawyers 

  • However, a Green Journal article that looked broadly in the literature about placental examinations showed that there was anecdotal evidence at best about placental pathology refuting cases of adverse childhood neurologic status 

  • In one analysis of 209 malpractice claims, only 2 cases were claimed to have been successfully defended by evidence gained through placental examination alone 

Placental Pathology I: Basics for the OB/GYN

Reading: https://www.contemporaryobgyn.net/view/placental-pathology-it-time-get-serious 

• Great article for this! 

What do we hope to get by sending a placenta to pathology? 

We want to know what happened! 

The goals are to get: 

• Findings that are relevant to the immediate care of the mother or baby

• Findings predictive of possible recurrence that could guide pregnancy care in subsequent pregnancies 

• Diagnoses that explain adverse pregnancy events (probably the most common reason we send a placenta) 

• Findings that may be important in medico-legal investigation of perinatal mortality and long-term morbidity 

So… which placentas should we send? 

There are many reasons, and some of them may depend on your institution, but we have broken this down into three categories 

• Maternal reasons 

  • Preterm delivery

  • Unexpected or recurrent pregnancy complications 

  • Maternal systemic disorders (ie. preeclampsia, malignancy, diabetes, etc) 

  • Infection

  • Excessive third-trimester bleeding 

• Fetal reasons 

  • Stillbirth, neonatal death 

  • Unexpected NICU admission 

  • SGA or LGA 

  • pH <7.0. 5-minute Apgar <7, or birth depression 

  • Neonatal seizures 

  • Hydrops, severe oligo/poly 

  • Multiple gestation 

• Placental reasons 

  • Structural abnormalities or size abnormalities 

  • Possible incomplete placenta 

• We can see that some of these reasons may not present until after the first day of life, so would recommend holding the placenta for up to 7 days if needed (can be stored unfixed in a fridge for that long!) 

The Anatomy and Development of the Placenta 

www.placentalab.org

We are going to first discuss a full-term placenta and then go over embryology

• The placenta can be thought of as three layers:

  • Maternal side (basal plate) 

    • Contains trophoblastic cells and decidual cells and contain the decidua basalis 

    • From the basal plate, the placenta septa bulge into the intervillous space, creating a system of grooves 

    • Basal plate is also penetrated by endometrial arteries and venules 

  • Intervillous space - separates the maternal and fetal side 

    • Exchange between the fetal and maternal circulatory systems occur between the main stem villi and the maternal endometrial arteries and venules in this space 

    • Remember: fetal and maternal blood don’t mix! 

  • Fetal side (chorion plate)  

    • Made of connective tissue and contains the amnion, the main stem villi, and chorionic arteries and veins, which then coalesce at the cord insertion site → umbilical cord 

    • The chorionic arteries and veins → arterioles and venules of the main stem villi 

    • The main stem villi project into the intervillous space and are connected to the maternal basal plate by anchoring villi 

• Placental embryology - super basic 

  • After fertilization and implantation, around day 5, the blastocyst is formed 

  • The blastocyst will eventually implant, and will contain the blastocyst cavity, the inner cell mass, and the trophoblast (which becomes the placenta) 

  • During implantation, there are complex interactions between the endometrium and the embryo → apposition, adhesion, and invasion 

    • Any dysfunction in these 3 processes can lead to abnormal placentation that can lead to affected placental function