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Faecal Microbiota Changes in
Indian Patients with Postpartum
Depressive Disorder
A project proposal submitted to
The Department of Microbiology and Biotechnology Centre
The Maharaja Sayajirao University of Baroda, Vadodara
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TABLE OF CONTENTS
Sr
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Topic

Page no
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 Currently, it has been found that the bacterial population in gut influences the HPA
axis
...

 The extent of influence of gut microbiota is still under research
...

 The prevalence of postpartum depression in India women is quite high and
currently the efforts to ameliorate the condition of women suffering from PPD are
not enough
...

 Such study can help us further understand PPD with respect to gut microbiota and
help us create interventions that help such patients by designing their gut microbiota
to mimic that of a normal person
...


INTRODUCTION
Postpartum Depression (PPD)
...

(Batt et al
...

(Zhou et al
...

Factors

Remarks

Timing of onset

PPD occurs specifically during postpartum
(Childbirth to 4-8 weeks onset)
...


Women who experienced depression onset
within 8 weeks postpartum were almost four times
more likely to present with severe depression
compared with women who experienced depression
onset during pregnancy
...


Genetic factors

Genetics contribute more to PPD in the early
postpartum period (within 6–8 weeks postpartum)
than in the late postpartum period
...


Hormonal changes

PPD involves the hormonal changes specific to the
transition from pregnancy to postpartum
...


Psychosocial factors

Childbirth and infant care are psychosocial stressors
that are unique to the postpartum period
...
, 2020)

Prevalence of Postpartum depression in Indian women
...
3% of the total sample was found to be depressed
according to the Edinburgh Postpartum Depression Scale (EPDS)
...
, 2021)
 Another meta-analysis conducted to determine the prevalence of antenatal common
mental disorders among women in India found that Low-income countries such as
india has antenatal mental disorders prevalence of - 15
...
8%
...
, 2020)

Factors that affect the incidence of postpartum depression
...

◦ Higher percentage of women from joint family suffered from PPD than from
nuclear family
...
, 2021)
◦ Higher rates of depression with spouses having a difficult relationship with their
mother-in-law
...
, 2020)
 Partner support
...

▪ Individuals with low partner support are 5 times more likely to be affected by
PPD
...
, 2021)
◦ Intimate partner violence is positively associated with increased rate of antenatal
depression
...
, 2020)
 Social support
...

▪ Individuals with Moderate social support (MSS) are 3 times more likely to
have PPD than individuals with High social support (HSS)
...

(Lanjewar et al
...

(Kalra et al
...

◦ Increased education is associated with decreased incidence of PPD
...
, 2021)
 Child gender
...
, 2021), 15% of total women
who conceived a male child experienced PPD whereas only 11
...

(Lanjewar et al
...

(Kalra et al
...

◦ High attention shift was associated with increased incidence of PPD
...

(Lanjewar et al
...

 There is substantial evidence to show that postpartum depression adversely affects
mothers, infants, and their families
...

(Lanjewar et al
...

(Kalra et al
...

 Postpartum depression is often treated with psychotherapy – and rarely with
medications
...

(Mayo Clinic, 2018)
◦ Antidepressants – These are often provided in serious cases of Postpartum
depression
...

(Zhou et al
...

(Barandouzi et al
...


The gut microbiome and depressive disorders
...

(Cryan & Dinan, 2012)
 The gastrointestinal microbiota modulates host function from various aspects:
◦ They could produce short-chain fatty acids (SCFAs)
...

◦ They also affect hypothalamic–pituitary–adrenal (HPA) axis
...

(Zhou et al
...

(Kovtun et al
...

(Kovtun et al
...

(Kovtun et al
...


REVIEW OF LITERATURE
Literature

Relavant conclusion

 It was observed that the relative
Altered faecal microbiota composition in
patients with major depressive disorder
...
, 2015)

abundance of Bacteroidetes,
Proteobacteria, and Actinobacteria had
markedly increased in MDD patients
...


 Researchers showed that the diversity
Faecal Microbiota Changes in Patients With
Postpartum Depressive Disorder
(Zhou et al
...

 Lachnospiraceae and Faecalibacterium,
were found to be associated with levels
of sex hormones
...

(Zheng et al
...


 A randomised, double-blind, placeboEffect of Lactobacillus rhamnosus HN001 in
Pregnancy on Postpartum Symptoms of
Depression and Anxiety: A Randomised Doubleblind Placebo-controlled Trial
(Slykerman et al
...

 Women were randomised to receive
either placebo or HN001 daily from
enrolment until 6 months postpartum if
breastfeeding
...


 The researchers studied effects on
The preventive and curative effects of
lactobacillus reuteri NK33 and bifidobacterium
adolescentis NK98 on immobilization stressinduced anxiety/depression and colitis in mice
...
, 2019)

immobilization stress (IS)-induced
anxiety/depression and colitis in mice
...
A
...
R
...
A
...
, & Cong, X
...
(2020)
...
 Frontiers in
Psychiatry, 11
...
org/10
...
2020
...
M
...
A
...
M
...
A
...
N
...
Is
Postpartum Depression Different From Depression Occurring Outside of the Perinatal Period? A
Review of the Evidence
...
https://doi
...
1176/appi
...
20190045

 Cryan, J
...
, & Dinan, T
...
(2012)
...
 Nature Reviews Neuroscience, 13(10), 701–712
...
org/10
...
-M
...
-E
...
-H
...
The Preventive and Curative Effects of
Lactobacillus reuteri NK33 and Bifidobacterium adolescentis NK98 on Immobilization StressInduced Anxiety/Depression and Colitis in Mice
...

https://doi
...
3390/nu11040819

 Jiang, H
...
, Zhang, Y
...
, Ma, Z
...
, Wang, W
...
, Tan, Z
...
, Li,
L
...
(2015)
...
 Brain, Behavior, and Immunity, 48, 186–194
...
org/10
...
bbi
...
03
...
, Tran, T
...
, Romero, L
...
, & Fisher, J
...
Prevalence and determinants
of antenatal common mental disorders among women in India: a systematic review and metaanalysis
...
https://doi
...
1007/s00737-020-01024-0

 Kovtun, A
...
, Averina, O
...
, Angelova, I
...
, Yunes, R
...
, Zorkina, Y
...
, Morozova, A
...
,
Pavlichenko, A
...
, Syunyakov, T
...
, Karpenko, O
...
, Kostyuk, G
...
, & Danilenko, V
...

(2022)
...
 Biomedicines, 10(9), 2162
...
org/10
...
, Tisdale, M
...
, Liu, J
...
, Philip, C
...
, Reyes, F
...
P
...
A
...
, Connor, P
...
E
...
, Tribble, D
...
, & Riddle, M
...
(2018)
...
 PLOS ONE, 13(8),
e0202178
...
org/10
...
pone
...
, Nimkar, S
...
(2021)
...
 Asian Journal of
Psychiatry, 57, 102567
...
org/10
...
ajp
...
102567

 Mayo Clinic
...
 Postpartum depression - Diagnosis and treatment - Mayo Clinic
...
org
...
mayoclinic
...
F
...
, Wickens, K
...
M
...
, Barthow, C
...
, Kang, J
...
, Stone, P
...
, Stanley, T
...
, Purdie, G
...
, & Mitchell, E
...

(2017)
...
 EBioMedicine, 24, 159–165
...
org/10
...
ebiom
...
09
...
, Jin, G
...
, Liu, T
...
, Wang, B
...
(2020)
...
 Frontiers in Cellular and Infection
Microbiology, 10(151)
...
org/10
...
2020
...
, Zolnik, C
...
, Qiu, Y
...
, Wang, T
...
D
...
R
...
C
...
J
...
, & Burk, R
...
(2018)
...
 Frontiers in Cellular and Infection Microbiology, 8
...
org/10
...
2018
...
, Zeng, B
...
, Liu, M
...
, Xu, X
...
, Chen, J
...
, Du, X
...
, Yang, D
...
, Meng, H
...
, Melgiri, N
...
, Licinio, J
...
, & Xie, P
...
Gut microbiome remodeling induces depressive-like behaviors through a pathway
mediated by the host’s metabolism
...

https://doi
...
1038/mp
...
44

 Zhou, Y
...
, Yu, H
...
(2020)
...
 Frontiers in Cellular and Infection Microbiology, 10
...
org/10
...
2020
...
There are not enough
measures, awareness or understanding about PPD to help the patients that suffer
from postpartum depression
...

 Due to inefficiencies in upholding the proper mental care measures and the lack of
proper drugs to help people in stress, this study was conducted to provide a different
outlook to postpartum depression
...


OBJECTIVES
There are six main objectives to this study:
 OBJECTIVE I
...

 OBJECTIVE II
...

 OBJECTIVE III
...

 OBJECTIVE IV
...

 OBJECTIVE V
...

 OBJECTIVE VI
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 Ethics, Consent and Permission
...

◦ All procedures will be designed and conducted in accordance with the
Declaration of Helsinki
...

 Patients and healthy volunteers will be selected from:
◦ Inpatient and Outpatient from antenatal care facilities
...

 Inclusion criteria for patients:
◦ The patient should be married
...

◦ Patients should have been diagnosed with PPD by a psychiatrist
...

▪ EDPS will be provided in English/Hindi and in native language if possible
...

 Inclusion criteria for healthy volunteers:
◦ Healthy volunteers should have normal health examination after delivery
...

▪ EDPS will be provided in English/Hindi and in native language if possible
...

 Exclusion criteria for patients:
◦ Exclude patients that have bipolar disorder, serious mental disorder like
schizophrenia, etc
...


OBJECTIVE II
Analysis of Patient’s demographic and clinical data
...

◦ Sociodemographic parameters
...

▪ Body Mass Index (BMI)
...

▪ Duration of postpartum
...

▪ Ethnicity
...

▪ EPDS (Edinburgh Postnatal Depression Scale)
...

◦ Data was expressed in mean ± standard deviation
...


OBJECTIVE III
...

 Collection of fecal samples
...

◦ The stools were liquified using sterile distilled water and a thin smear (125 µl)
was applied to each of the four sample areas in GE Healthcare WB120205 NonIndicating FTA Classic Card
...
5 Inches) with few packs of
desiccant
...

◦ The FTA card was placed on a Cutting Mat (6 X 8)
...
00mm)
...

 PCR amplification
...

◦ PCR was carried out using Thermofisher Quantstudio 3 Real Time PCR
Machine
...

Components

Volume (µl)

Forward primer
...
(10µM)

1

Template DNA
...
(2
...


5

EasyPfu DNA Polymerase
...
5 U/µl)

1

Distilled water
...

◦ Thermal cycling consisted of an initial denaturation step at 95◦C for 5min,
followed by 30 cycles of denaturation at 94◦C for 30 s, annealing at 60◦C for 30
min, and extension at 72◦C for 40 s, with a final extension step at 72◦C for 4
min
...

◦ Amplicons were run for each sample on an agarose gel
...

◦ Expected band size for 515f-806r was ∼300–350 bp
...

◦ Amplicons were quantified with Quant-iT PicoGreen dsDNA Assay Kit
...

◦ The amplicon library for high-throughput sequencing was outsourced to Gujarat
Biotech Research Centre (GBRC) ILLUMINA MISEQ
...

 Bioinformatic analysis
...
0, the raw
sequences will be processed to concatenate reads into tags according to the
overlapping relationship
...

◦ The processed tags will be clustered into the Amplicon Sequence Variants
(ASVs)
...


OBJECTIVE IV
...

 “Vegan” package in R 3
...
1 software will be used for the analysis
...

◦ β-diversity will be undertaken using UniFrac that uses phylogenetic information,
and the principal coordinate analysis (PCoA) was employed to calculate the
distance matrixes
...

Elucidating the composition of Microbial Communities of
patients and healthy control
...

◦ The histograms of species will be made for the two groups at the levels of
phylum, class, order, family, and genus on the basis of the annotation results
...

 Comparing Differences in Bacterial Genus Between the Healthy

Control Group and Postpartum Depressive Disorder Group
...
4
...

▪ linear discriminant analysis effect size (LEfSe) will be applied through the
Huttenhower Lab Galaxy Server and be used to investigate the differences in

the abundance between the two groups and to explore the specific bacteria
associated with PPD (P < 0
...


OBJECTIVE VI
...

 Vegan” package in R 3
...
1 software will be used for the analysis
...


FUTURE PROSPECTS








Since India is a very diverse country, we aim to infer the
gut microbiome of PPD patients of Indian population
...

In future, we could design prebiotic and probiotic
treatments or interventions to ameliorate depressive
symptoms and help improve quality of life of both a
mother and her child
...

We can also compare other factors responsible for PPD
and the role of gut microbiome in either repressing or
encouraging the depressive symptoms
...


Objective II
Analysis of Patient’s demographic
and clinical data
...


2 months

24 months

Objective IV
Analysis of Microbial α- and βDiversity between patients and
healthy control
...


Objective VI
Associations of Gut Microbiota with
Clinical Indicators
...


BUDGET
Kits
Product

Price/Unit (INR) Total price (INR)

Amount needed

QIAamp PowerFecal Pro DNA Kit
(50 piece)

34,910/-

6,98,200/-

20

Quant-iT™ dsDNA Assay Kits,
High sensitivity (HS)
(1000 assays)

53,472/-

53,472/-

1

ProNex® NGS Library Quant Kit
(500 reactions)

57,270/-

1,14,540/-

2

Instruments
Product

Price/Unit (INR) Total price (INR)

Amount needed

PowerLyzer 24 Homogenizer

3,45,000/-

3,45,000/-

1

RM-03 Plus microcentrifuge

62,600/-

62,600/-

1

Vortex Adapter
24 (1
...
0 ml) tubes

15,930/-

15,930/-

1

Digital VORTEX GENIE 2-Mixer

1,03,716/-

1,03,716/-

1

Thermofisher Quantstudio 3
Real Time PCR Machine

13,25,000/-

13,25,000/-

1

Genei Mini Sub system
(Very large model)

35,080/-

35,080/-

1

GBRC ILLUMINA MISEQ
(1 Run)

1,000/-

10,00,000/-

1000

Extra cost

Amount INR

Travelling Expenses

30,000/-

Contingency

1,00,000/-

Non-recurrring items
Product

Price/Unit (INR) Total price (INR)

Amount needed

Zymo R1101-1-10 Feces catcher
(10 pack)

8,915/-

8,91,500/-

100

Stool Sample Container
(100 Pieces Per Packet)

325/-

3,250/-

10

GE Healthcare WB120205 NonIndicating FTA Classic Card
4 Sample Areas Per Card
500 µl Total Volume
(Pack of 100)

58,060/-

5,80,600/-

10

MBP for EC+ Card 4x4

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