A comparative study of Bacterial Vaginosis among Vitamin D deficient pregnant women and Normal pregnant women

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A Dissertation on

"A COMPARATIVE STUDY OF BACTERIAL VAGINOSIS AMONG VITAMIN –D DEFICIENT PREGNANT WOMEN AND NORMAL PREGNANT

WOMEN"

Dissertation Submitted to

THE TAMIL NADU Dr.M.G.R. MEDICAL UNIVERSITY CHENNAI- 600032

with partial fulfillment of the regulations for the award of the degree of

M.S.OBSTETRICS AND GYNAECOLOGY

COIMBATORE MEDICAL COLLEGE, COIMBATORE

MAY 2018

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CERTIFICATE

Certified that this is the Bonafide Dissertation in "A COMPARATIVE STUDY OF BACTERIAL VAGINOSIS AMONG VITAMIN –D DEFICIENT PREGNANT WOMEN AND NORMAL PREGNANT WOMEN" was a work done by Dr.SARANYAA.T and submitted in partial fulfillment of the requirements for the Degree of M.S. OBSTETRICS AND GYNAECOLOGY, of The Tamilnadu Dr.M.G.R Medical University, Chennai.

Date: Professor and Unit Chief

Department of Obstetrics and Gynaecology Coimbatore Medical College.

Date: Professor and HOD

Department of Obstetrics and Gynaecology Coimbatore Medical College.

Date: The DEAN

Coimbatore Medical College

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DECLARATION

I Solemnly declare that the Dissertation titled "A COMPARATIVE STUDY OF BACTERIAL VAGINOSIS AMONG VITAMIN –D DEFICIENT PREGNANT WOMEN AND NORMAL PREGNANT WOMEN" was done by me at Coimbatore Medical College during the academic year Oct 2016 – Sep 2017 under the guidance of Prof. Dr.R.Manonmani M.D., DGO., this Dissertation is submitted to the Tamilnadu Dr.M.G.R Medical University towards the fulfillment of the requirement for the award of M.S. Degree in Obstetrics and Gynaecology.

PLACE: Dr. SARANYAA.T

DATE:

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ACKNOWLEDGEMENT

I express my gratitude to Dr.B.Asokan, M.S, MCh, the Dean, Coimbatore Medical College Hospital for providing facilities to carry out my project work successfully.

I sincerely thank Prof. Dr.R.Manonmani M.D.,DGO., Chief and HOD, Department of Obstetrics and Gynaecology for her constant guidance and encouragement throughout the period of my study.

I wish to express my thanks to DR.D.Vatsaladevi M.D.D.G.O., DR.Murugalakshmi M.D.D.G.O., Dr.Premakumari M.D.D.G.O., DR.N.Geetha MD.,OG Professors, Department of Obstetrics and Gynaecology, Coimbatore Medical College Hospital, Coimbatore whose knowledge and experience has guided and inculcated in me a sense of confidence.

I also extend my thanks to all my Assistant professors who have constantly motivated me with their words of support and encouragement and entire postgraduate colleagues for their help and support. Last but not the least, I would like to thank all my patients without whose co operation this dissertation would never have been the light of the day.

Date : Signature of the candidate

Place : DR.SARANYAA.T.

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CERTIFICATE – II

This is to certify that this dissertation work titled "A COMPARATIVE STUDY OF BACTERIAL VAGINOSIS AMONG VITAMIN –D DEFICIENT PREGNANT WOMEN AND NORMAL PREGNANT WOMEN" of the candidate DR.SARANYAA.T with registration Number 221616302 for the award of M.S in the branch of OBSTETRICS AND GYNAECOLOGY, I personally verified the urkund.com website for the purpose of plagiarism Check. I found that the uploaded thesis from introduction to conclusion shows the result of 1%

(One) percentage of plagiarism in the dissertation.

Guide & Supervisor sign with Seal.

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INDEX

SR.NO CONTENT PAGE NO.

I INTRODUCTION 1

II AIMS AND OBJECTIVES 3

III REVIEW OF LITERATURE 4-64 IV MATERIALS AND

METHODS

65-67 V OBSERVATIONS AND

RESULTS

68-87

VI DISCUSSION 88-94

VII SUMMARY 95-96

VIII CONCLUSION 97

IX

ANNEXURES BIBLIOGRAPHY PROFORMA CONSENT FORM MASTER CHART

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LIST OF TABLES

I NUGENTS CRITERIA 28

2. VITAMIN D LEVELS 57

3. RDA OF VITAMIN D 60

4. MAX TOLERABLE DOSE OF VITAMIN D

61

5. AGE DISTRIBUTION 68

6. GESTATIONAL AGE 69

7. PARITY 70

8. BMI 72

9. SIGNS AND SYMPTOMS OF BV 74

10. GRAM STAIN 75

11. GRAM STAIN AND BV 76

12. VITAMIN D STATUS 78

13. VITAMIN D STATUS AND BV 79

14. MATERNAL OUTCOMES 82

15. FETAL OUTCOMES 84

16. FOLLOW UP 87

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LIST OF FIGURES

1. BV HPE SLIDE 10

2. BV PICTURE 18

3. GRAM STAIN PICTURE 26

4. WET MOUNT PICTURE OF BV 28

5. HAYS CRITERIA 30

6. BV OTHER METHODS 31

7. BV CULTURE 32

8. VITAMIN D CHEMICAL STRUCTURE 41 9. VITAMIN D METABOLISM 1 43 10. VITAMIN D METABOLISM 2 48

11. VDR RECEPTOR 50

12. FUNCTION OF VITAMIN D 51 13.

REPRODUCTIVE FUNCTION OF VITAMIN D

52

14. AGE DISTRIBUTION 68

15. BMI 72

16. SIGNS AND SYMPTOMS OF BV 74

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17. GRAM STAIN 75

18. GRAM STAIN AND BV 77

19. VITAMIN D STATUS 78

20. VITAMIN D STATUS AND BV 80

21. MATERNAL OUTCOMES 83

22. FETAL OUTCOMES 84

23. FOLLOW UP 87

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ABBREVIATONS

BV - Bacterial Vaginosis

Vit D - Vitamin -D

AN - Antenatal

PN - Postnatal

PP - Post Partum

BMI - Body Mass Index

GDM - Gestational Diabetes Mellitus

PIH - Pregnancy Induced Hypertension

PPROM - Preterm Premature Rupture of Membranes

PROM - Premature Rupture of Membranes

GA - Gestational Age

OCP - Oral Contraceptive pills

RTI - Reproductive tract infection

HPV - Human Papilloma Virus

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IUCD - Intra Uterine Contraceptive device

KOH - Potassium Hydroxide

HPE - Histopathological Examination

POC - Products of Conception

USG - Ultrasonogram

UVB - Ultraviolet Rays B

VDR - Vitamin D Receptor

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INTRODUCTION

This study emphasis on the prevalence of Bacterial Vaginosis among pregnant women and its association with Vitamin –D deficiency.

It also emphasis their impact on maternal and fetal health.

Bacterial Vaginosis a poly-microbial over growth of predominantly anaerobic bacteria such as GardnellaVaginalis, Mycoplasma hominis, Mobiluncus, Bacteroides, Peptostreptococcus over Normal vaginal flora of Lacto bacilli^1,2. This commonly affects the women of reproductive age group and also it is the most common infection among pregnant women^1,19.

Bacterial Vaginosis is associated with adverse pregnancy outcomes such as Preterm labour, PPROM, Spontaneous abortion, Recurrent pregnancy loss, Chorioamnonitis, Postpartum Endometritis, Post operative wound infections^19,20,21.

Bacterial Vaginosis is a sexually associated disease and also increases the prevalence of other sexually transmitted diseases⁴³.

Bacterial Vaginosis is curable disease, but it has high recurrence rate. Hence better evaluation, screening and treatment of this condition is necessary to reduce its prevalence and of adverse pregnancy outcomes.

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BV is associated with Vitamin – D deficiency hence better evaluation of this condition will prevent adverse fetal and maternal outcomes.

Vitamin –D is a fat soluble seco-steroid which plays an important role in Calcium and phosphate homeostasis, apart from this it has other non classical benefits like Immuno modulation, infection control^l6,8,53etc., Studies have shown that a there is a strong positive correlation between Vitamin –D deficiency with the prevalence of BV. Aim of this study is to identify their degree of association and their impact on maternal and fetal health^11,53.

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AIM OF THE STUDY

To identify the association between Bacterial Vaginosis and Vitamin –D deficiency among pregnant mothers.

OBJECTIVES:

1. To estimate the prevalence of Bacterial Vaginosis and Vitamin D deficiency among pregnant population

2. To identify the association between occurrence of bacterial vaginosis and Vitamin –D deficiency and their impact on maternal and fetal health.

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REVIEW OF LITERATURE

Anne L Dunlop et al investigated the association between serum 25 hydroxy vitamin D, folate, Omega 6 / Omega 3 ratio and bacterial vaginosis during pregnancy³. She reported that a strong association between vitamin D less than 12 ng/ml and folate deficiency less than 5 ng/ml with Bacterial Vaginosis (Nugent’s score more than or equal to 7) during pregnancy

Bodnar et al reported that maternal Vitamin D deficiency is associated with bacterial vaginosis in first trimester of pregnancy. In this cohort study he found a dose response association between 25 hydroxy vitamin D deficiency and prevalence of Bacterial Vaginosis and also the likelihood of Bacterial vaginosis decreases as vitamin D status improves⁴. There is a strong racial disparity among the prevalence of Bacterial vaginosis

Fichorova R N et al found that placental colonization are seen in Bacterial vaginosis resulted in adverse pregnancy outcome like preterm labour, PPROM, foetal distress⁵ etc.., He also reported that the association between vitamin D deficiency and bacterial vaginosis in pregnant women. He also states that vitamin D sufficiency reduces the risk of bacterial infections like bacterial vaginosis through induction of

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HenselK.J et al found an association between Vitamin D deficiency and bacterial vaginosis. He also states that Vitamin D deficiency was a modifiable risk factor for bacterial vaginosis among pregnant women⁶. Hence evaluation of Vitamin D supplementation for prevention of bacterial vaginosis in pregnancy and also use of vitamin D supplementation as adjunct therapy for bacterial vaginosis in pregnancy is warranted.

Turner et al also reported an association between increased prevalence of bacterial vaginosis among vitamin D deficient women⁷. Treatment of vitamin D deficiency in pregnant women reduced the recurrence of bacterial vaginosis, but it doesn’t reduce the recurrence of bacterial vaginosis in non pregnant women.

Joonjslk shin et al studied the importance of non classical role of vitamin D in pregnancy and in placenta. Placenta produces a response to vitamin D which functions as a modulator of implantation, cytokine production and immune response to infection⁸. Hence vitamin D deficiency in pregnancy is associated with bacterial infections like Bacterial Vaginosis, Pre eclampsia ,GDM etc.,

Lorraine et al showed that hcap18/LL-37 Human cathelicidin concentration is increased in cervico vaginal secretions from women with

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modulating immune response to invading infectious agents in the reproductive tract. This raises the possibility that vitamin D sufficiency may upregulate hcap18/LL-37 expression in the reproductive tract thereby affecting the susceptibility to Bacterial Vaginosis.

Dixon et al also identified a positive correlation between circulating hcap18/LL-37 (anti – microbial peptide gene) and serum 25 hydroxy vitamin D¹⁰, thereby depicting the role of Vitamin –D in immuno modulatory function.

Bhan et al also observed similar finding. He administrated high dose of vitamin D2 (ergocalciferol) and observed the changes in human cathelicidin (hcap18/LL-37) values, thereby suggesting positive correlation between bacterial infections and vitamin D deficiency^11,12.

Adrian F Gombart also discovered that Vitamin D induces cathelicidin antimicrobial peptide gene expression thereby regulating immune function and response against bacterial and viral infection¹³.

Kearns et al also reported positive correlation between Vitamin D deficiency and infectious diseases¹⁴.

Andrea almos et al found the non classical benefit of vitamin D like Immuno modulatory role as well as its capacity to regulate hormonogenesis¹⁵. Human placenta expresses CYP 27-B1 which

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catalyses local synthesis of calcitriol. This study recommends vitamin D supplementation during pregnancy is accessible and is a safe way to reduce adverse pregnancy outcome like preterm labor, PROM due to infection, Preeclampsia, GDM etc.,

Urrutia and Throp stated that vitamin D deficiency is related to many adverse pregnancy events like bacterial vaginosis, preterm labour, PROM, preeclampsia, GDM¹⁶etc.

Wagner, Taylor, Johnson and Hollis studied the role of vitamin D in pregnancy and lactation¹⁷. The study states that Vitamin D maintains calcium and bone metabolism in both mother and foetus, Immuno modulation in mother and also impact of maternal vitamin D deficiency on foetal health.

Abilasha Gupta et al stated that bacterial vaginosis in pregnancy has been associated with preterm labour, PROM, chorioamnionitis, spontaneous abortion, recurrent abortion, PP Endometritis¹⁸. They also suggested screening of bacterial vaginosis should start early in pregnancy in women, who are at high risk of preterm labour.

Xu et al also showed a strong positive correlation between bacterial vaginosis and preterm labour.

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Chitra Jayakrishnan et al studied in detail about the occurrence of bacterial vaginosis and fetal outcomes¹⁹, this study found a positive correlation between bacterial vaginosis and preterm labour.

Kiss et al (2004) states that presence of Bacterial Vaginosis at 26- 32 weeks of gestation has been shown to associated with preterm labour²⁰.

Flynn et al (1999) also shown that BV increases the risk of preterm labour²¹.

One study also found that risk appears double when detected early in pregnancy. (21%) when compared to later pregnancy (11 %) –Joesoef et al 1993 :Leitich et al 2003 )

Carey et al used oral Metronidazole to treat BV in pregnancy but did not find any reduction in incidence of bacterial Vaginosis²².

Okun et al women who treated with antibiotic for Bacterial Vaginosis found to have reduction in persistent and recurrent infections²³.

Main risk factor for PPROM is infection (Gomez et al 1997 : Mercer et al 2003)²⁴.

Bacterial proteases decrease the strength and elasticity of fetal membranes thereby causing PPROM.

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Infection with gonococcal, trichomonal, chlamydial, or colonization with GBS and BV (Bacterial Vaginosis) have an increased risk of PPROM , (Edwards et al 1978 :Minkoff et al 1984)²⁵.

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THEORETICAL BACKGROUND : Bacterial Vaginosis

Bacterial vaginosis also known as non specific vaginitis / anaerobic vaginositis/ vaginal bacteriosis/ gardnerella vaginitis.

Bacterial vaginosis is termed as vaginosis rather than vaginitis because it is associated with alteration in normal vaginal flora rather than due to any specific infections^1,2. It is a polymicrobial infection in which normal hydrogen peroxide producing lactobacillus is replaced by anaerobes thereby altering the vaginal microbiota.

There is a considerable decrease in number of lactobacilli in the vaginal secretion with hundred fold increase in the anaerobes. Some of the most common anaerobes are Gardnerella vaginalis, Mobiluncus, Mycoplasma hominis, Bacteroides, Peptostreptococcus, Hemophilus vaginalis etc.,

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VAGINAL SECRETIONS

Normal vaginal secretion composed of vulvar secretion from sebaceous gland, sweat gland, bartholins glands, Skene glands, transudate from vaginal wall, exfoliated cells from vagina and cervix, cervical mucus, endometrium, oviductal fluids, microorganisms and their metabolic products.

The vaginal desquamated tissue is made up of vaginal epithelial cells that are responsive to hormones like Oestrogen and Progesterone.

The vaginal epithelial composition varies with age and also by hormonal changes.

Superficial cells are the main type of cells in reproductive age group. These cells are the predominant cell type when there is excess oestrogen stimulation (as in proliferative phase of menstrual cycle).

Intermediate cells predominance during luteal phase of menstrual cycle which is under the control of progesterone. Parabasal cells predominates in post menopausal lady where there is low level of both oestrogen and progesterone.

Normal vaginal flora is predominantly aerobic consisting of different species of bacteria but the most common is hydrogen peroxide

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producing lactobacilli. This protects against vaginal infection by following ways:

1. Superficial cells under the influence of oestrogen laded with glycogen.

The vaginal epithelial cells breakdown glycogen to monosaccharides which can then be converted by lactobacilli to lactic acid. This lactic acid production lowers the pH of vagina to less than 4.5 (acidic pH)

2. The lactobacilli has unique ability to produce hydrogen peroxide thereby preventing the growth of other anaerobes.

ORGANISM NORMALLY OCCURING IN VAGINA^1,2

 Lactobaacillus

 S.Epidermidis

 S.Aureus

 GBS

 E.coli

 Klebseilla

 Proteus

 Clostridium

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 Propionibacterium

 Prevotella

 Bacteriods.

PHYSIOLOGICAL CAUSES OF LEUCORRHOEA

 During puberty

 During menstrual cycles - under the influence of hormones

 During intercourse

PATHOLOGICAL CAUSE OF LEUCORRHOEA Non infective causes like

Polyp, Ectopy, OCP use, Pelvic organ prolapse etc.

Infective causes like PID, Cervicitis, Vaginitis.

DEFENSE MECHANISM PREVENTING INFECTION

VULVAL DEFENSE

 Apposition of labia

 Bartholin gland secretion

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VAGINAL DEFENSE

 Apposition of vaginal walls

 Stratified squamous epithelium

 Lactic acid producing DODERLEIN‘S bacilli which reduces the vaginal PH in acidic range and prevents infection and inhibits the growth of other organism

 Bacteriocins–acidocins and lactacin.

CERVICAL DEFENSE

 Mucus plug.

 Racemose type of glands.

 Squamous epithelial lining of ectocervix.

UTERINE DEFENSE

 Closure of ostia which prevents any ascending infection to fallopian tubes.

 Cyclical shedding of endometrium.

TUBAL DEFENSE:

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INFECTIONS OF REPRODUCTIVE TRACT Divided into

1. Infection of lower genital tract 2. Infection of upper genital tract

INFECTION OF LOWER GENITAL TRACT

Lower genital tract infection includes infection of vulva, vagina and cervix, they are also referred to as RTI (reproductive tract infections).

some of the infections such as Trichomoniasis, Chlamydial infection, HPV and Gonococcal infection, Syphilis, Chancroid, LGV, Granuloma inguinale are sexually transmitted infections. Others such as Bacterial Vaginosis are sexually associated diseases^1,2,19.

PREVALENCE^1,2,19,21

Prevalence of lower genital tract infection in INDIA Based on

History alone -11-72 %

Clinical examination–17-40%

Lab confirmation –38%

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CHARACTERISTICS OF NORMAL VAGINAL FLORA ; White and odourless

Components are

Secretions from vulval glands Transudate from vaginal wall Cervical glands secretion Endometrial and tubal fluids Normal flora

Aerobic bacteria

 Lactobacilli

 Streptococcus and staphylococcus

 Gardenellavaginalis

 Anaerobic bacteria

 Peptococcus, peptostreptococcus and bacteriods.

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Normal PH 3.8–4.5 Microscopy

Desquamated epithelial cells White blood cells

Lactobacilli

CONDITIONS CAUSING VAGINAL DISCHARGE

 Bacterial Vaginosis

 Trichomonal vaginitis

 Candidal vaginitis

BACTERIAL VAGINOSIS :

Most common infection causing vaginitis, this is caused by shift in the normal vaginal smear. There is disequilibrium in the vaginal microbiota with decline in the number of lactobacilli, the lactobacilli prevents the growth of anaerobes by reducing the pH of the vaginal secretion and by its ability to produce hydrogen peroxide. But the

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disequilibrium increases the pH of the vaginal secretion and also decrease in the release of hydrogen peroxide favouring the growth of anaerobes.

Gardnerella form a biofilm which prevents the action of antibiotics which inturn favours the growth of anaerobes, hence infection is polymicrobial and also recurrence is more common

CLINICAL SYMPTOMS :

Bacterial vaginosis is a most common infection of reproductive age group. This is the most common infection affecting pregnant women.

The Prevalence of Bacterial Vaginosis is between 10 to 35 percent²¹, depending upon region and race

PREDISPOSING FACTORS :

The following are the risk factors of Bacterial Vaginosis,

 Multiple sexual partners

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 Poor personal hygiene

 Injudicious use of antibiotics

 IUCD

 Frequent vaginal douching

 Cigarette smoking

 Increased vaginal pH as after menstruation

 Black ethnic group

 Lesbians

SIGNS AND SYMPTOMS :

50 percent of women with Bacterial Vaginosis are asymptomatic , while others have

 Discharge per vaginum which is white, thin, homogenous discharge

 Typical unpleasant fishy vaginal odour

 There may be associated pruritis usually non pruritic

 Burning micturition , increased frequency of micturation ,urgency.

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 Lower abdominal pain

 Postcoital increase in discharge and dyspareunia DIAGNOSIS

Bacterial vaginosis is diagnosed by taking a thorough history, detailed clinical examination and investigations

History

History taking should include the following

 Age

 Parity

 Demographic details

 Socioeconomic status

 Chief complaints

 Presenting illness such as description of vaginal discharge in terms of onset, duration, precipitating factors, colour, consistency, amount, odour, associated pruritis, dysuria, dyspareunia, lower abdominal pain and constitutional symptoms.

 Abdominal pain

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 Menstrual history

 Obstetric history

 Marital history–number of sexual partner ,recent change in sexual partner, disease in sexual partner.

 Contraceptive history

 Any medication history

 Past medical /surgical history

 Previous similar complaints and antibiotic treatment

 Personal history.

 Occupational history

CLINICAL EXAMINATION :

 General examination

 BMI

 Systemic examination

 Abdominal examination

 Examination of external genitalia

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 speculum examination

 bimanual examination

Abdominal examination Look for areas of tenderness Organomegaly

Presence of mass Free fluid

Scars, sinuses, engorged veins ,hernia orifices.

Systemic examination

Other systems CVS, RS, CNS examined Examination of pelvis

Done in dorsal lithotomy position after emptying the bladder with proper lighting after getting consent.

Examination of external genitalia Inspection

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Vulva , hair distribution ,any skin lesion, introitus, uretheral meatus ,any vaginal discharge

Speculum examination

Using self retaining cuscos speculum with proper lighting 1. Vaginal discharge–colour, consistency ,amount , odour.

2. Bleeding per vaginum

3. Any other lesions, growth in cervix and vagina.

Bimanual examination or abdomino pelvic examination 1. Uterus–position (anteverted or retroverted )

2. Size 3. Shape

4. Consistency 5. Mobility 6. Tenderness

7. Adenexal fullness and tenderness 8. POD–fullness or mass.

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PROCEDURE :

Along with examination the following procedures done after getting informed written consent.

Cytological screening for cancer cervix –Pap smear Examination of vaginal discharge

Two High vaginal swab taken from the posterior fornix using sterile cotton tipped swab. One is spread on a slide and fixed and sent to microbiological lab for gram staining. Other KOH is added and look for fishy odour and microscopic examination done.

INVESTIGATIONS : Investigations include

1. complete blood count including Total count, differential count, 2. Erythrocyte sedimentation rate

3. Pap smear 4. Vaginal smear

DIAGNOSTIC CRITERIA FOR BV.

Two diagnostic criteria is used for diagnosis of Bacterial vaginosis

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AMSEL’SCRITERIA :

This criteria is proposed by Amsel in 1984 to establish the diagnosis of bacterial vaginosis

 Vaginal discharge –thin, homogenous white or greycolour

 WHIFF TEST : Addition of KOH results in fishy odour

 Vaginal pH more than 4.5

 Presence of clue cells on wet mount microscopy examination

Any 3 out of these 4 is required to establish the diagnosis of bacterial vaginosis.

GRAM STAIN

Gram staining of the vaginal secretion was introduced by Nugent²⁶ in the year 1991.

The technique of gram staining is described in brief

 Gram stain

Gram staining is a method of staining used to distinguish gram positive and gram negative bacteria. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls,

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Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decolouring process.

PROCEDURE :

The process involves the following steps:

1. Cells are stained with crystal violet dye. Next, a Gram's iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water.

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2. A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost.

3. A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. However, the decolorized Gram negative cells are stained red.

NUGENT’S CRITERIA

The vaginal secretion taken in a glass slide and is analysed by gram staining technique, diagnosis of Bacterial Vaginosis is by Nugents criteria²⁶.

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The below table represents nugents scoring system

LACTOBACILLUS

GARDNERLLA/

BACTEROIDES

MOBILUNCUS

QUANTITY SCORE QUANTITY SCORE QUANTITY SCORE

4+ 0 0 0 0 0

3+ 1 1+ 1 1+ or 2+ 1

2+ 2 2+ 2 3+ or 4+ 2

1+ 3 3+ 3

0 4 4+ 4

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A score of more than or equal to 7 indicates bacterial vaginosis A score of 4-6 indicates intermediate stage of bacterial vaginosis A score of less than 4 indicates no bacterial vaginosis

ADVANTAGES :

Nugent screening offer advantage over Amsel criteria by

 Better reliability

 Reproducibility

 Better sensitivity and specificity

It is the gold standard method in diagnosing Bacterial Vaginosis, hence can be used for screening tool for diagnosing Bacterial Vaginosis.

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HAY’S CRITERIA :

This criteria was devised by Hay in the year 2002

 GRADE 0 : Epithelial cells with no bacteria

 GRADE 1 : Normal bacterial vaginal flora (Lactobacilli morphotypes predominant)

 GRADE 2 : Reduced numbers of lactobacillus morphotypes with mixed bacterial flora

 GRADE 3 : Mixed bacterial flora only, few or absent lactobacillus morphotypes

 GRADE 4 : Gram positive cocci only

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OTHER METHODS :

Several newers methods are introduced the following are some of them,

 DNA probes have been developed to directly detect the presence of Gardnerella vaginalis and also the pathogenic level of Gardnerella. AFFRIMVP3 microbial identification system (BECTON DICKINSON) is a commercially available DNA probe office based test kit that simultaneously detects the presence of gardnerella, trichomonas and candida

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CULTURE :

Bacterial vaginosis grows on blood and chocolate agar. It forms hemolytic colonies on human and rabbit blood agar. It is catalase negative and oxidase negative

TREATMENT

Treatment will be recommended for non pregnant women with signs and symptoms of BV^27,28. In pregnant mothers treatment will be recommended in all mothers who are positive for BV, some of the recommended regimens are as follows.

 Tab .Metronidazole 500 mg orally twice daily for 7 days

 0.75 % metronidazole gel intravaginally once a day for a period of 5 days

 2 % clindamycin cream intravaginally at bedtime for a period of 7 days

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 Tab. Clindamycin 3 mg twice daily orally for a period of 7 days

Alcohol consumption should be avoided during treatment.

Alternative treatments includes (not approved to use in pregnant mothers)

 Tab. Tinidazole 2 gram orally once daily for 2 days

 Tab. Tinidazole one gram oral for 5 days

Newer studies for using intravaginal probiotics or lactobacilli is under study follow up after treatment is necessary because persistent or recurrence of disease is common³⁰. Re-treatment with same regimen for recurrence is recommended.

Treatment is recommended for all symptomatic and also asymptomatic pregnant women^27,28. Two studies demonstrating the efficiency of oral Metronidazole twice daily for a period of 5 days the study concluded 70% cure rates by using Nugent’s criteria to define cure.

Another study demonstrates 80% cure rates by using clindamycin regimen.

Multiple studies fails to demonstrate Association with metronidazole during pregnancy and teratogenic effect on foetus, hence it is safe to use in pregnancy²⁹. There is no difference between oral therapy

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and topical therapy hence symptomatic pregnant women can be treated with either vaginal or oral Metronidazole. Many adverse pregnancy outcomes such as preterm labour, PPROM, Spontaneous Abortions, postoperative infections are related to Bacterial Vaginosis. Meta analysis concluded antibiotic treatment does not prevent preterm labour. However in one study oral therapy reduces the late miscarriages and other study pointed out decrease in fetal morbidity such as low birth weight, IUGR etc.

Carey et al used oral metronidazole to treat BV in pregnancy but did not find any reduction in incidence of Bacterial Vaginosis²².

Okun et al states that women who treated with antibiotic for Bacterial Vaginosis found to have reduction in persistent and recurrent infections²³.

Data are inconsistent with use of antibiotics in asymptomatic women 7 trials were conducted among them two shows no benefits ,4 shows benefits ,one is inconclusive. Evidence is insufficient to recommend routine treatment in asymptomatic low risk women.

Treatment is recommended in those women high risk for Preterm labour.

Yet further studies are needed²⁹. Metronidazole is secreted in breast milk hence low dose used in lactating mother. No evidence of adverse effect in

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MATERNAL AND FETAL COMPLICATIONS ASSOCIATED WITH BACTERIAL VAGINOSIS^18,19,20

Some of the maternal complication occurring due to bacterial vaginosis are

 Spontaneous abortion

 Recurrent pregnancy loss

 Preterm labour

 PPROM

 Chorioamnionitis

 Low Birth weight

 PP Endometritis

 Wound infection

ABORTION

Abortion or miscarriage is defined as termination of pregnancy either induced or spontaneous before 20 weeks of gestation or fetus weight less than 500 grams¹⁸.

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RECURRENT PREGNANCY LOSSES (RPL)

 Couples with 3 or more clinically recognized pregnancy losses

< 20 weeks^18,19 ectopic and molar pregnancies not included

 ASRM defines RPL as “ a distinct disorder defined by 2 or more failed clinical pregnancies”.

 Clinical pregnancies–USG proven or by HPE of POC

 Incidence 1-2%.

PRETERM LABOUR

Labour before 37 completed weeks, it is defined as presence of uterine contraction of sufficient strength, also in frequency causing progressive effacement and dilatation of the cervix between 20 to 37 weeks of gestation^18,19.

Incidence around 10%

Around 25 % - 40% cause of preterm labour were result of intrauterine infection as stated by Cunningham et al 2010.Goldenberg et al proposed that IU infection trigger labour by activating innate immune system.

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Kiss et al (2004) states that presence of Bacterial Vaginosis at 26- 32 weeks of gestation has been shown to associated with preterm labour²⁰.

Flynn et al (1999) also shown that BV increases the risk of preterm labour²¹.

One study also found that risk appears double when detected early in pregnancy.(21%)when compared to later pregnancy (11 %) – Joesoef et al 1993 :Leitich et al 2003 )

Carey et al used oral metronidazole to treat BV in pregnancy but did not find any reduction in incidence of Bacterial Vaginosis²².

Okun et al states that women who treated with antibiotic for Bacterial Vaginosis found to have reduction in persistent and recurrent infections²⁴.

Studies are underway to analyse the benefits of screening and treating both high risk and low risk mothers, results are equivocal.

PPROM^19,20

Rupture of fetal membranes prior to onset of labour is termed PROM (prelabour rupture of membranes).

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PPROM defined as occurrence of rupture of membranes prior to 37weeks and also before onset of labour.

Incidence around - 3 -18.5 % ( Gunn et al 1970 )

Main risk factor for PPROM is infection (Gomez et al 1997 : Mercer et al 2003)

Bacterial proteases decrease the strength and elasticity of fetal membranes thereby causing PPROM .

Infection with Gonococcal, Trichomonal, Chlamydial, or colonization with GBS and BV (Bacterial Vaginosis) have an increased risk of PPROM, (Edwards et al 1978 : Minkoff et al 1984).

PP ENDOMETRITIS

It is defined as the occurrence of temperature of 38 (100.4 F) on any two days of the first ten days postpartum after first 24 hours.

Main cause is PP Endometritis, organism causing endometritis are commensals of lower genital tract, Bacterial vaginosis etc.,

WOUND INFECTION :

Many organism causes wound infection, one of them is Bacterial vaginosis.

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VITAMIN - D VITAMINS :

Vitamin may be regarded as organic compounds required in the diet in small amounts to perform specific biological functions for normal maintenance of optimum growth and health of organism. The usage of A B and C to vitamins was introduced in 1915 by McCollum and Davis.

CLASSIFICATION :

They first classified only two vitamins– 1. Fat soluble / Lipid soluble (A,D,E,K ) 2. Water soluble

There are about 15 vitamins essential for humans.

Fat soluble vitamins absorption is associated with fats, can be stored in liver and adipose tissue. Excess consumption of these vitamins particularly A and D leads to their accumulation and toxic effects

All the fat soluble vitamins are isoprenoid compounds, since they are made up of one or more of five carbon units namely isoprene units (-CH=CH.CH3-CH=CH-) Fat soluble vitamins perform diverse functions.

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VITAMIN - D

Vitamin D is a fat soluble vitamin. It resembles sterols in structure and functions like a hormone. Vitamin D is called as sunshine vitamin.

The synthesis of Vitamin D3 in the skin is proportional to the exposure of sunlight. Dark skin pigment (melanin) adversely influences the synthesis of cholecalciferol.

Vitamin D was isolated by Angus (1931) who named it calciferol.

CHEMISTRY :

Ergocalciferol (vitamin D3) is formed from ergosterol and is present in plants. Cholecalciferol (Vitamin D3) is found in animals. Both the sterols are similar in structure except that ergocalciferol has an additional methyl group and a double bond. Ergocalciferol and cholecalciferol are the sources for Vitamin D activity and are referred to as provitamins

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During the course of cholesterol synthesis 7 dehydrocholestrol is found as an intermediate. On exposure to sunlight, 7 dehydro cholesterol is converted to cholecalciferol in skin.

SOURCES :

1. Skin synthesize vitamin D when exposed to sun (UV B 290- 315nm)

2. Fish liver oil 3. Fatty fish 4. Egg yolk

5. Fortified food –milk cheese

SOURCES :

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TYPES :

Vitamin D is expressed by

1 Cholecalciferol (Vitamin D3)

2 Ergocalciferol Vitamin D2 (from olant fungi yeast) ABSORPTION TRANSPORT AND STORAGE :

Vitamin D is absorbed in the small intestine for which bile is essential. Though lymph, vitamin D enters the circulation bound to plasma alpha globulin and is distributed throughout the body. Liver and other tissues stores store small amounts of vitamin D.Vitamin D is incorporated into chylomicrons which convey the vitamins from lymph to systemic circulation. In lymph Vitamin D in chylomicron is transferred to DVP

The 7 dehydro cholesterol available in malphigian layer of epidermis. The UV B rays 290-315 nm breaks the bond between 9-10 of the steroid ring to form provitamin (secosteroid). The cis double bond between 5^th-6 carbon atom is then isomerized to trans double bond to give cholecalciferol (Vitamin D3) .ProvitaminD3 is converted photochemically to previtaminD3 which converted to Vitamin D3 by temperature dependent process.

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METABOLISM :

SYNTHESIS OF VITAMIN D :

Vitamin D2 and D3 are not biologically active. They metabolise in body and converted to active form Vitamin D can be synthesized in body by exposure to sunlight³².

SYNTHESIS IN SKIN :

The UVB rays (290-315 nm) from sun exposure coverts 7 dehydro cholesterol to cholecalciferol (vitamin D3)³².

FACTORS AFFECTING VITAMIN D3 PRODUCTION : 1. Colour of the skin :

METABOLISM :

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White skin people produces more vitamin D than dark skin people because melanin protects skin against damage from UVB exposure. So darker skin with more melanin allow less UVB to enter the skin. Less UVB , less Vitamin D is produced.

2. Area of skin exposed

More the skin is exposed, more vitamin D is produced. 1000-20000 IU of vitamin D can be produced within minutes. Some cultural practices in India like wearing purdah reduces Vitamin–D synthesis.

3. Altitude :

People live in high altitude and people living near equator produces more Vitamin D

4. Time of the day :

Vitamin D production peaks during the middle of the day.

5. Ageing :

As the skin gets aged, the capacity to produce Vitamin D decreases.

6. Use of sunscreen :

Use of Sunscreen decreases vitamin D production.

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SYNTHESIS IN LIVER :

The cholecalciferol synthesis from the skin transported to liver. In liver, it is hydroxylated at 25^th position to 25 hydroxycholecalciferol (25 OH-D3) by a specific hydroxylase (25-hydroxylase). It is called as calcidiol, which has minimal biological activity. Hepatic 25 hydroxylase is a microsomal monooxygenase and it is a product of gene CYP27A1.

This enzyme requires cytochrome P450 and NADPH.

25 hydroxy Cholecalciferol is the main storage form of vitamin D, so measurement of 25 hydroxy Cholecalciferol gives amount of Vitamin D in the body.

SYNTHESIS IN KIDNEY :

The 25 hydroxy Cholecalciferol is bound to vitamin D binding protein which is a alpha globulin (VDBP) in plasma. This is taken to kidney, where it is further hydroxylated at first carbon by 1 alpha hydroxylase to1, 25-dihydroxycholecalciferol. Since it contains 3 hydroxyl group, it is called as calcitriol.

This is the biologically active form of vitamin D and it is a hormone.

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The 1 alpha hydroxylase enzyme is located in mitochondria of proximal convoluted tubule and it is a product of gene CYP27B1. This requires cytochrome P450, NADPH and ferrodoxin.

The role of vitamin D is to maintain calcium and phosphate metabolism and also has many nonclassical benefits

FUNCTIONS OF VITAMIN D :

THE VITAMIN D functions are divided into 1. Classical/calcemic effect

2. Nonclassical / extra calcemic effect CLASSICAL ROLE OF VITAMIN D:

Calcitriol (biologically active form) regulates plasma calcium and phosphate homeostasis⁴⁶. Its effect is exerted at 3 different levels

1. ACTION OF CALCITRIOL ON INTESTINE :

Vitamin D3 promotes the absorption of calcium and phosphate from intestine. Calcium is absorbed passively on brush border surface.

From intestinal cell to blood, transport of calcium needs energy and this step is mediated by vitamin D3 Vitamin D3 acts like a steroid hormone, enters the cell by binding to cytoplasmic receptor and it binds to Vitamin

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D receptor (VDR) in nucleus. This inturn binds to VDRE which leads to gene activation by transcription, which codes for calbindin. Calbindin helps in calcium absorption.

2. ACTION OF CALCITRIOL ON BONES :

Calcitriol is essential for bone formation⁴⁶. D3 stimulates the activity of osteoblast increases the bone mineral density and helps in bone remodeling. Calcitriol along with parathoromone increases the mobilization of calcium and phosphate from bone, thereby maintaining serum calcium and phosphate level

3. ACTION OF CALCITRIOL ON KIDNEY :

Vitamin D3 increases the absorption of calcium and phosphate from renal tubule. It also minimizes the excretion of calcium and phosphate.

REGULATION OF VITAMIN–D SECRETION :

When the calcitriol concentration is adequate, the 24 hydroxylase enzyme in kidney gets activated and vitamin D2 is converted to 24,25 – cholecalciferol, which is less active form of vitamin D thereby preventing vitamin D toxicity³².

The formation of 1,25cholecalciferol is regulated by renal 1 alpha hydroxylase. 1 alpha hydroxylase activity is inhibited by excess 1,25

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dihydroxycholecalciferol (feedback inhibition). The 1 alpha hydroxylase regulates 1,25hydroxycholecalciferol. Its activity is increased by hypocalcemia / parathyroid hormone.

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NONCLASSICAL ROLES OF VITAMIN D :

1,25 dihydroxy Cholecalciferol receptors are found in almost all tissues of the body. Besides intestine and bone, the receptors are found in heart, brain, macrophages, prostate even in uterus^42,44. This shows possibility of vitamin D deficiency in many diseases and also supplementation of vitamin D will help in treating many disorders in near future.

The VDR – VITAMIN D RECEPTOR – is a transcriptional factor which regulates the expression of around 2000 genes directly or indirectly. Vitamin D affects cell growth, cell differentiation and immune modulation⁴².

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These actions of vitamin D explains the utility of Vitamin D in number of diseases. The endocrine, paracrine and intracrine functions of the vitamin D are under research.

Some of the functions are listed below : IN KIDNEY

1. Calcium homeostasis 2. Muscle health

3. Bone health

4. Blood pressure regulation

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5. Cardiovascular health 6. Neural development 7. Immunomodulation

IN MONOCYTES AND MACROPHAGES : 1. Immuno modulation

2. Control of invading pathogens 3. Prevention of autoimmune diseases

IN PROSTATE/ BREAST/ COLON/ LUNG¹⁶: 1. Inhibits cell proliferation

2. Regulates cell growth and differentiation

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Hence vitamin D deficiency plays an important role in rickets, osteomalacia /osteoporosis and also more likely to play a role in diabetes mellitus, systemic hypertension, cardiovascular diseases, infections like tuberculosis, bacterial vaginosis^16,44, etc.., and autoimmune diseases

ROLE OF VITAMIN D IN REPRODUCTIVE HEALTH :

Vitamin D is involved in the regulation of cell growth, differentiation and metabolic modulation⁵⁶. Sufficient vitamin D level is required for steroidogenesis, endometrial development and reproductive functions. It regulates the key gene associated with embryo implantation. Vitamin D deficiency is also linked with polycystic ovarian syndrome. It enables immunological adaptation by the mother to maintain pregnancy thereby preventing miscarriage.

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Maternal Vitamin D deficiency is associated with increased risk of gestation diabetes mellitus, eclampsia, bacterial vaginosis and low birth weight infant. The risk of auto and cellular immune abnormalities is increased in women with RPL and vitamin D deficiency.

Vitamin D source of infants result from transfer from stores of mother. (ref . S karras, H fakhoury et al) Low maternal vitamin D levels during pregnancy is associated with many adverse neonatal outcomes like low birth weight, preterm births, detrimental effects on child’s bone and teeth development and risk of infectious diseases. This finding is demonstrated by identifying nuclear Vitamin D receptors and vitamin D activating 1 alpha hydroxylase in deciduas and placenta. This was stated by Evans et al.

HUMAN CATHELICIDIN AND ITS ASSOCIATION WITH VITAMIN–D AND BACTERIAL VAGINOSIS^5,9,10,12:

Multifunctional host defense peptide ( HDP) plays a important role in innate immunity, Hcap18/LL-37 is the most important human cathelicidin this is expressed in Neutrophils and epithelial cells. It is produced by cervical epithelial cells altered expression of LL -37 causes infections like BV and other bacterial infections and by inflammatory stimuli.

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The Hcap/LL-37 is increased in the presence of bacterial Vaginosis as a protective mechanism against BV infection. Normalisation of the levels after antibiotics treatment for BV proves their association⁵.

CAMP expression is increased by exposure to Vitamin – D3 and CYP27B1 are expressed by END E6/E7 cell lines of cervix, this enzyme converts inactive form of Vitamin d to active form in autocrine manner, treatment with Vitamin D increases the expression of human cathelicidin Vitamin –D regulates the expression of cathelicidin thereby preventing BV^5,9.

This hypotheis is further proven by Lorraine et al and also Dixon et al also identified a positive correlation between circulating hcap18/LL-37 (anti–microbial peptide gene) and serum 25 hydroxy vitamin D^9,10.

Adrian F Gombart also discovered that Vitamin D induces cathelicidin antimicrobial peptide gene expression thereby regulating immune function and response against bacterial and viral infection¹³. PHARMACOKINETICS :

When vitamin D supplementation was given, only one fourth of vitamin D2 and D3 was absorbed and utilized. Remaining of which is absorbed and excreted through bile¹⁵. Among the vitamin D absorbed, very small amount is stored in fat and skeletal muscle. The remaining is

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converted to 25 hydroxy vitamin D and is excreted from the body. The elimination half life of vitamin D is 2 to 3 weeks and the concentration of vitamin D gradually increases and attains plateau around 40-90 days. In vitamin D sufficient persons, researchers proved supplementation of vitamin D will not increase the vitamin D levels in body. The most common circulating form of vitamin D is 25 hydroxyvitamin D. Its concentration is 1000 times more than 1,25 hydroxy vitamin D. 25 hydroxy vitamin D is converted to its active form, 1,25 dihydroxy vitamin D to perform its functions. 1,25dihydroxy vitamin D lasts in circulation for only 3 to 5 days.

ASSESMENT OF INDIVIDUALS VITAMIN D STATUS^45,48,49 : Accurate indicator of vitamin D status of person is by measuring the vitamin D levels.

Vitamin D status can be measured by measuring 25 hydroxy vitamin D levels or by measuring 1,25dihydroxy vitamin D levels. The most preferred analysis is measuring 25 hydroxy vitamin D levels⁴⁸.

This includes both D2 and D3 but not 1,25hydroxy vitamin D. The measurement of 1,25hydroxy vitamin D is generally not preferred because it cannot detect vitamin D levels accurately in vitamin D deficiency because of its feedback mechanism. This inturn increases the

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levels of 1,25dihydroxy vitamin D.So assessing 1,25 dihydroxy vitamin D levels may be inaccurate as it may be normal or increased mild to moderate in vitamin D deficiency⁴⁹. It becomes low in only very severe vitamin D deficiency. 1,25hydroxy vitamin D levels may be inaccurate when there is associated hepatic disease or renal disease or primary hyperparathyroidism.

Hence estimation of 25 hydroxy vitamin D is the most accurate method of assessing an individual’s vitamin D status. Analysing serum calcium, phosphate and ALP enzyme in a person to assess the vitamin D status of that person is also inaccurate. Among persons with vitamin D deficiency 25% can have normal levels of serum calcium and phosphate and ALP but PTH LEVELS will be elevated. Hence researchers are not in favour of measuring serum calcium and phosphate to assess vitamin D levels of a person.

VITAMIN D LEVELS

In accordance with Endocrinological Soceity

 Optimum levels of 25 hydroxy vitamin D is more than 30 ng/ ml.

 When the levels ranging from 20-29 ng/ml is seen, it is called insufficient.

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 When the vitamin D levels range less than 20 ng/ml it is called

‘’Vitamin D deficient’’

 Less than 8 ng/ml it is severe deficiency .

Different councils recommends the following cut offs,some of them are listed below.

Researchers proved that level of more than 30 nanograms/ml of 25 hydroxy vitamin D is essential for the normal absorption of calcium.

Hence the serum concentration is less than 20 nanograms per ml, symptoms of vitamin D deficiency develops. When the levels are less than 8 nanograms per ml, it is full blown vitamin D deficiency which requires high dose vitamin D supplementation

PREVALENCE OF VITAMIN D DEFECIENCY :

Vitamin D deficiency is a pandemic health issue and it prevails in VITAMIN–D

COUNCIL (ng/ml)

ENDOCRINE SOCEITY

FOOD AND NUTRITION

BOARD

TESTING LABS

DEFICIENT 0- 30 0 -20 0-11 0-31

INSUFFICIENT 31-39 21-29 12-20

SUFFICIENT 40 -80 30-100 >20 32-100

TOXIC >150

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the increased prevalence of vitamin D deficiency in India and is around 70-100%²¹.

Following are the factors which increase the vitamin D deficiency : 1. Obesity

2. Dark skinnedperson’

3. Reduced sunlight exposure 4. Use of sunscreens

5. Consumption of nondairy beverages 6. Lifestyle modification

7. Cultural practices like wearing purdah 8. Associated systemic diseases like

a. Pancreatic insufficiency b. Malabsorption syndromes c. Cystic fibrosis

d. Chronic kidney diseases e. Chronic liver diseases

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India, inspite of being a tropical country with adequate sunlight exposure, increase in prevalence of vitamin D deficiency is attributed to cultural practices, malabsorption and malnutrition.

TREATMENT OF VITAMIN D DEFICIENCY : 1. Awareness and health education programme.

2. Adequate sunlight exposure 3. Dietary modification

4. Food fortification–milk cereals 5. Vitamin D supplementation VITAMIN D SUPPLEMENTS : Treatment for vitamin D deficiency

Vitamin D deficiency is the most prevalent worldwide.Treatment of the same includes 1,25 hydroxy Cholecalciferol or calcitriol is the most biologically active form of Vitamin D. Vitamin D3 has four times more active potential than Vitamin D2. Hence Vitamin D3 supplements are more commonly used than Vitamin D2. Supplements includes combination of multivitamin strength of 50-100 IU. Vitamin –D can be safely supplemented in pregnancy in any trimester.

Aim is to achieve target level of more than 30 ng/ml .

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RECOMMENDED DAILY ALLOWANCE INSTITUTE OF MEDICINE 1997 recommends⁷⁸

1. 200 IU/day–Children and adults upto age of 50 2. 400 IU/day–Adults from 50-70 years

3. 600 IU/day–more than 70 years

But these levels are found to be inadequate hence they revised the guidelines :

INSTITUTE OF MEDICINE GUIDELINES 2005⁷⁸:

To achieve a level of 30-32 nanograms/ml a Vitamin D of 1000 IU/day is recommended. But even 1000IU/day–is not enough to achieve Vitamin D sufficiency in cases of severe deficiency, where large bolus

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doses of vitamin D is essential to correct the vitamin D deficiency^92,93,94. Studies have revealed that bolus vitamin D will not lead to equivalent increase in levels of 25(OH) as stated above the response to the vitamin D depends on the baseline Vitamin - D level.

TOLERABLE UPPER INTAKE LEVELS⁸⁵:

The following table shows the tolerable upper intake levels of Vitamin - D

Tolerable Upper Intake Levels (ULs) for Vitamin D [1]

Age Male Female Pregnancy Lactation 0–6 months 1,000 IU

(25 mcg)

1,000 IU (25 mcg) 7–12 months 1,500 IU

(38 mcg)

1,500 IU (38 mcg) 1–3 years 2,500 IU

(63 mcg)

2,500 IU (63 mcg) 4–8 years 3,000 IU

(75 mcg)

3,000 IU (75 mcg) 9–18 years 4,000 IU

(100 mcg)

4,000 IU (100 mcg)

4,000 IU (100 mcg) 19+ years 4,000 IU

(100 mcg)

4,000 IU (100 mcg)

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SAFETY PROFILE OF VITAMIN D :

Hypervitaminosis is rare. Toxicity occurs only when high doses of vitamin D is given for prolonged periods⁶¹. Single high dose bolus or weekly high dose bolus over short period doesn’t cause toxicity.

SIGNS AND SYMPTOMS OF VITAMIN D TOXICITY : 1. Weakness

2. Polyuria 3. Poor appetite 4. Intense thirst 5. Nausea 6. Vomiting 7. Hypertension 8. Weight loss

The toxicity is mainly due to hypercalcemia⁶³, which includes Hypercalciuria, nephrocalcinosis, nephrolithiasis, hyperphosphatemia.

Toxicity mainly depends on period of vitamin D supplementation rather than its dosage.

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DRUG INTERACTION

Vitamin D can interact with following drugs. Hence adequate precaution is advised while taking vitamin D with these following drugs

1. Antacids 2. Steroids

3. Anticonvulsants 4. Thiazides

5. Diuretics

6. Hydroxy Chloroquine 7. Barbiturates

8. Alcohol 9. Nicotine

CLINICAL IMPROVEMENT :

Clinical improvement after vitamin D supplementation takes upto forty to ninety days⁹². Vas quez et al reported clinical response 5-9 months after vitamin D supplementation.

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Vieth et al stated improvement occurs after 6 months of supplementation. A minimum period of atleast 2 months is required for clinical improvement.

Hence to find improvement Vitamin D levels are to be taken after 2 months and again after 6 months.

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MATERIALS AND METHODS:

SOURCE OF DATA:

100 AN mothers without any high risks of different ages, parity and BMI attending AN OPD clinic of the Department of Obstetrics and Gynaecology of Coimbatore Medical College Hospital for routine antenatal visit.

STUDY PERIOD:

October 2016 to September 2017 SAMPLE SIZE:

100 Antenatal Women.

STUDY DESIGN Prospective study Inclusion criteria:

1. Women >= 18 years

2. Singleton fetus - primigravida or multi gravida without any associated fetal anomaly

3. No associated Maternal co-morbid conditions

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4. BMI ranging from 19 to 35

5. ANC with and without white discharge per vaginum and other pelvic symptom.

Exclusion criteria:

1. Patient refusal

2. Presence of any pelvic pathology

3. Prior antibiotic treatment (within 2 weeks) 4. Bleeding per vaginum

5. GDM, PIH complicating pregnancies and other Obstetric high risk cases are excluded.

6. Medical disease complicating pregnancy

7. History of intake of drugs like calcium, vitamin D, Oral contraceptives.

8. Patient not able to give consent (psychiatric patients).

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METHOD OF RANDOMISATION:

Sealed Envelope method METHODOLOGY:

After obtaining written informed consent, 100 patients aged 21 to 35 years old adult females in mid gestation period (11 – 26 weeks ) attending the AN OPD clinics at Government Coimbatore medical college hospital ,Coimbatore enrolled for the study.

Each patient visiting our AN OPD enquired about their age, BMI, parity index, chief complaints, detailed menstrual history, obstetric history, any pelvic pathology, past medical and surgical history taken.

General, systemic and detailed pelvic examinations were done. Sterile high vaginal swabs were taken for microbiological examination and a blood sample for analysing serum 25(OH) vitamin D3 level. Patients were followed throughout their pregnancy and 6 weeks postpartum.

Patient are screened for Bacterial Vaginosis and Vitamin–D deficiency and their association were analyzed. Patient were treated for the same and are observed throughout the AN period and 6 weeks postpartum. The collected data were compiled and will be subjected to statistical analysis using statistical package for social services (SPSS)

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RESULTS AND ANALYSIS The study is under taken under following divisions

Table 1. AGE

The frequency of occurrence in accordance with age are listed below, 14% belongs to <20 years of age

62% belongs to 20-30 years of age 24% belongs to >30 years of age.

Age Frequency Percent

<20 yrs 14 14.0

20-30 yrs 62 62.0

>30 yrs 24 24.0

Total 100 100.0

Table 1. AGE

14

62 24

Age

<20 yrs 14 14.0

20-30 yrs 62 62.0

>30 yrs 24 24.0

Total 100 100.0

Table 1. AGE

<20 yrs 20-30 yrs

>30 yrs

<20 yrs 14 14.0

20-30 yrs 62 62.0

>30 yrs 24 24.0

Total 100 100.0

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Table 2. GESTATIONAL AGE :

Frequency Percent

<12 weeks 65 65.0

12-16 weeks 35 35.0

Total 100 100.0

Among the study population 65% belongs to <12 weeks of gestation 35% belongs to 12- 16 weeks of gestation.

Gestational Age * Signs and Symptoms of BV (Fishers exact test) Signs and

Symptoms of BV

Total

P value Absent Present

Gestational Age

<12 weeks No 48 17 65 0.263

% 73.8% 26.2% 100.0%

12-16 weeks

No 22 13 35

% 62.9% 37.1% 100.0%

Total No 70 30 100

% 70.0% 30.0% 100.0%

The above table shows there is no statistical significance in relation of

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Table 3. PARITY :

Frequency Percent

Primi 37 37.0

2nd Gravida 43 43.0

>=3rd gravida 20 20.0

Total 100 100.0

Among the study population 37% are Primigravida.

43% are 2^ndgravida 20% are Multi gravida

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Parity * Signs and Symptoms of BV (Chi-square test) Signs and Symptoms

of BV

Total P value Absent Present

Parity Primi No 28 9 37 0.247

% 75.7% 24.3% 100.0%

2nd Gravida No 31 12 43

% 72.1% 27.9% 100.0%

>=3rd gravida

No 11 9 20

% 55.0% 45.0% 100.0%

Total No 70 30 100

% 70.0% 30.0% 100.0%

The above results are analysed using chi square test and this shows there is no statistical significance between parity and the prevalence of Bacterial Vaginosis.