Tetrad of Hormonal and Biochemical Manifestations in Phenotypes of Polycystic Ovary Syndrome

J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 73-83. https://doi.org/10.47277/JIRB/8(4)/73

Tetrad of Hormonal and Biochemical Manifestations

in Phenotypes of Polycystic Ovary Syndrome

Sonal Agarwal , Deepika Krishna, Gautham Praneesh, Kamini Rao

Milann Fertility Centre, Bengaluru, India

Received: 21/08/2020

Accepted: 17/10/2020

Published: 20/12/2020

Abstract

A prospective observational case- control study was conducted to evaluate the relationship and prevalence of subclinical hypothyroidism,

hyperprolactenemia, impaired glucose metabolism and hyperhomocysteinemia as components of hormonal/biochemical manifestation tetrad

amongst four phenotypes of polycystic ovary syndrome (PCOS) with respect to controls. 200 women diagnosed as PCOS as per the

ESHRE/ASRM Rotterdam criteria were taken as cases (group I) and 200 as controls (group II).All women recruited came for subfertility

treatment to assure homogeneity in study population. Group I was further divided into four phenotypes.Records of demographic (age, BMI,

duration of infertility), biochemical(homocysteine,fasting blood sugar,HBA1C)and hormonal (thyroid profile and prolactin)parameters of

patients were taken. Both groups were comparable in age, ethnicity and marital status. Percentages of phenotypes A,B,C and D in our Indian

population were 25.5%,16%,35.5% and 23 % respectively. BMI was significantly more in PCOS as compared to non-PCOS with highest

mean in phenotype B subgroup. All parameters measured were significantly increased in PCOS group compared with non-PCOS group with

prolactin levels similar in all phenotypes. Impaired glucose tolerance and diabetes incidence was more in phenotype C and D. Hypothyroidism

and hyperhomocystenemia were higher in phenotypes B and C. Phenotypes are affected by hormonal and biochemical manifestations and if

followed for long term may be associated with metabolic syndrome in future. Thus non classical phenotypes should also be properly

monitored and treated as this hormonal imbalance and biochemical derangements add to the brunt of management of PCOS.

Keywords: Hyperhomocysteinemia , Hyperprolactinemia, Hypothyroidism, Impaired glucose metabolism

Introduction

a multidisciplinary approach to benefit the patient in a holistic

manner depending on evidence based medicine practice(7).We

have evaluated prevalence of various phenotypes and scrutinized

their hormonal and biochemical characteristics compared to non-

PCOS women presenting to our center.

Polycystic ovarian syndrome (PCOS) is the most common

multisystem endocrinopathy with epigenetic and environmental

influences affecting early pre-pubertal age to post-menopausal

age group with varied presentations in each(1). The variability in

etiopathogenesis and clinical presentation has made the

appropriate diagnosis of PCOS difficult (2). This endocrine-

metabolic disorder affects 5%–10% of women of reproductive

age with oligo-anovulation, menstrual disturbances, and/or

androgen excess as the main features (3).It has been affirmed by

various studies that hypothyroidism is also a state of insulin

resistance which is the crux of genesis of PCOS. Individually both

hypothyroidism and PCOS have changes in lipid metabolism

For all clinical manifestations, the root cause is

hyperandrogenemia and impaired insulin response (8).

Hypothyroidism leads to metabolic derangements such as

decrease in sex-hormone binding globulin causing increased

levels of free androgens in blood and decreased metabolic

clearance, defective glucose disposal and hyperlipidemia, thus

affecting gonadal function and fertility (9, 10). Increased TSH

levels along with hyperprolactenemia lead to deposition of

mucopolysaccharides in various organs such as ovaries affecting

its function resulting in anovulation and reproductive disruption.

Beta cell dysfunction and insulin resistance can lead to

development of type 2 diabetes mellitus. Insulin resistance may

lead to increase in homocysteine levels due to altered methionine

metabolism (11). High sensitivity C-reactive protein, adiponectin

and homocysteine are serum biomarkers of cardiovascular

disease and are found to be abnormal in women with PCOS.

There is clustering of obesity, impaired glucose tolerance,

dyslipidemia and hypertension as risk factors (12).

(

increase in total cholesterol and in low-density lipoprotein

cholesterol) and endothelial function leading to increased risk of

arterial hypertension and other cardiovascular problems(4).

Association of thyroid dysfunction and other hormonal and

biochemical parameters of PCOS are thus frequently studied to

establish a relationship between the two.

PCOS is a constellation of symptoms and signs having

significant implications on woman’s health and fertility (5).

Phenotypes are influenced by genetic, racial, geographic,

environmental and lifestyle factors like diet and over-

nutrition.This affects them causing more adverse effects in

respect to reproductive and metabolic outcome (6). PCOS is

characterized by vast diversity in manifestations, necessitating a

Measurement of hormonal and biochemical parameters in this

study will help in providing early interventions in women with

Corresponding author: Sonal Agarwall, Milann Fertility Centre, Bengaluru, India. Email: sonaljaipur28@gmail.com

J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 73-83. https://doi.org/10.47277/JIRB/8(4)/73

PCOS to prevent progression from pre-diabetic state to full

blown diabetes and other risk factors.

significant drug intake suspected to affect metabolic function

were not included in the study.Women with any renal, hepatic,

thyroid or cardiac dysfunction,Cushing’s syndrome, congenital

adrenal hyperplasia and adrenal tumors were excluded. All

women had to undergo detailed anthropometric assessment in

form of measurement of height,weight,waist and hip

circumference,blood pressure and systemic examination .Body

mass index (BMI) was calculated by the formula: weight in

kilograms/(height in meters)2 .Hirsutism was assessed using the

modified Ferriman-Gallwey score, with nine specified body areas

counted by a single observer.A score of more than/equal to 8 out

of 36 was considered significant. Biochemical hyperandrogenism

was defined as a serum total T level of >0.022 nmol/L. Trans-

vaginal ultrasonography was done by a single clinician to rule out

any inter-observer variation to see presence of more than 12

ovarian follicles 2-9 mm in any ovary and increased ovarian

volume > 10 cc(calculated using the formula 0.5 × length × width

Materials and methods

Study design and participants

This prospective observational case-control study was

conducted at Milann fertility center which is a tertiary care center.

Study was conducted from April 2017 to January 2018.Women

were divided into two groups according to European Society of

Human Reproduction & Embryology/American Society for

Reproductive Medicine(ESHRE/ASRM) Rotterdam criteria into

cases and controls. Rotterdam criteria for the diagnosis of PCOS

includes presence of any two of the following criteria:clinical or

biochemical hyperandrogenemia, anovulation/Oligomenorrhea

and polycystic ovaries on ultrasonography(13). They were

recruited at the time of diagnosis and who have not yet initiated

treatment for any biochemical and hormonal derrangements. All

participants before allocation signed an informed consent form.

Approval was obtained from the Institutional Ethical Committee.

Women of group I were divided into four phenotypes -

hyperandrogenism (HA) + oligo-/anovulation (OA) + polycystic

ovaries at ultrasound (PCO) (phenotype A,full-blown syndrome);

HA + OA (phenotype B, former National Institutes of Health

definition); HA + PCO (phenotype C) and OA + PCO (phenotype

D). Four different combinations of phenotypes have been

deciphered by Rotterdam/AEPCOS society based on clinical and

endocrinological findings (14).Consequently records of

demographic, hormonal and biochemical parameters of patients

were taken.

×thickness)suggestive

PCOM

(polycystic

ovary

morphology).Women were divided into 2 groups based on

ESHRE/ASRM Rotterdam criteria with group I as PCOS

women(cases) and group II as non-PCOS women(controls).

Thyroid stimulating hormone (TSH), free thyroxine (FT4),

glycosylated hemoglobin (HBA1C), fasting blood glucose (FBS),

prolactin (PRL) and plasma homocysteine (PH) were measured

in all women recruited in the study. The blood samples were

obtained from peripheral vein in early morning after a fasting

period of atleast 8 hours. TSH, FT4 and PRL and total T levels

were measured by electrochemiluminescence assay (Cobas e411-

Hitachi).Plasma homocysteine and fasting blood sugar were

measured by enzymatic method through Cobas c311.HBA1C was

measured through BioRad DIO method. Intra- and inter assay

variations were within the limits permitted by manufacturer

company.

Subclinical hypothyroidism is defined as serum TSH levels

of >2.5mIU/L with no clinical symptoms and signs having normal

FT4 levels according to ASRM guidelines. In our study a TSH

cutoff of 2.5 IU/L was taken to define subclinical hypothyroidism

to determine association of increased TSH levels in all four

phenotypes of PCOS and also in controls. Plasma homocysteine

cut off of 8umol/l was taken for hyperhomocystenemia.HBA1C

Patient population

Inclusion criteria:

All PCOS defined as per ESHRE/ASRM Rotterdam criteria

of 2003 for group I and non-PCOS in group II, aged 21-35 years,

BMI >18 and <30 kg/m2, no symptoms of hypothyroidism and

free thyroxine levels (t4) in range of 4.6-12ug/dl, proven

subfertility and willingness to participate in study.

Exclusion criteria:

Patients with chronicdiseases like overt hypothyroidism and

hyperthyroidism, kidney or liver failure, late-onset adrenal

hyperplasia and diabetes, severe endometriosis, severe male

factor infertility, multiple fibroids, previous IVF treatment failure

and those not willing to participate.

>5.3%, FBS >100 mg/dl and PRL >30 ng/dl cut off were taken

for deranged blood sugar levels (marker for insulin resistance)

and hyperprolactenemia respectively. The results were described

as mean ± standard deviation. Significance level was defined at

%, and the software used for the analysis was the SAS statistical

Recruitment and analysis

software package, version 9.1.

All patients who came to our center who were unable to

conceive were interviewed in detail. History taking comprised of

complaints,past treatment history,menstrual history related to age

of menarche, regularity, duration, and number of cycles per

year,marital history,contraceptive and obstetric history.Questions

regarding past medical, surgical history and family history were

also asked.Written informed consent was taken from all patients

recruited for the study.

Participant flow

The participant flow is shown in Figure I. Out of 221 PCOS

patients screened for study, only 200 followed up with blood tests

and thus were enrolled for study. Out of 214 NON-PCOS women

only 200 followed up with blood tests whowere enrolled for

study. Thus 200 women with PCOS in group I and 200 non-PCOS

women were included in group II for final analysis.

Oligomenorrhea was defined as an inter-menstrual interval of

more than 35 days or a total of eight or fewer menstrual cycles

per year. Duration and extent of abnormal hair growth, weight

gain, and development of acne or alopecia, along with family

history of hirsutism, menstrual disorders, and diabetes mellitus or

glucose intolerance was written in record file. Women giving

history of any use of hormonal preparation, androgens or any

Statistical analysis

Descriptive statistics were presented as means and standard

deviation for continuous variables .Frequencies and proportions

were used for categorical variables. Independent sample t test was

used for continuous variables which were normally distributed

J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 73-83. https://doi.org/10.47277/JIRB/8(4)/73

and Mann-Whitney U test for data not normally distributed. Chi-

and PH as associative factors with PCOS infertile women.

Histograms have been created to show association between

different variables amongst cases and controls.All tests were two-

sided with p-value of less than 0.05 considered as statistically

significant.

square test or Fisher’s exact test was used for categorical variables

where appropriate. Odds ratio (OR) with 95% confidence

intervals (CIs) was calculated. For post-hoc analysis one way

ANOVA test was used.

In addition, receiver operating

characteristic analysis was used to evaluate TSH, HBA1C, PRL

Figure 1. Participant flow chart

Results

to 5.605 ±2.99 in controls (group II). Table 3 illustrates various

hormonal and biochemical parameters such as plasma

homocysteine, AMH, TSH, PRL, FBS and HBA1C amongst

PCOS group and non-PCOS group. In the PCOS group, all

hormonal and biochemical parameters were observed to be higher

as compared to NON-PCOS group and were statistically

significant.

Both groups were divided into two categories based on their

HBA1C and FBS levels into pre-diabetic state and type 2 diabetes

mellitus.

Demographic profile of PCOS and non-PCOS women was

compared and different phenotypes of PCOS women were also

compared through sub-group analysis as illustrated in table 1 and

.In our study in PCOS group maximum prevalence was of

phenotype C (35.5%) followed by phenotype A (25.5%),

phenotype D (23%) and least for phenotype B (16%). Mean age

of women of phenotype A, B, C and D was 29.5 ±3.7, 28.9±3.7,

9.2±3.8 and 29.4±3.3 years respectively. Mean age of group II

women was 30.1±4.1 years. Age was similar in both groups and

sub-groups. Women with PCOS had a mean BMI of 25.9±4.3

Kg/m2 and non-PCOS women had BMI of 24.5±3.6 kg/m2.

Higher BMI was observed in all phenotypes of PCOS with respect

to controls (p=0.001).Obese and overweight women were seen

maximum in phenotype B, followed by phenotype D, A and C

and were least in controls. Both groups are similar regarding

number of women presenting with primary or secondary

infertility. Duration of infertility was noticed to be significantly

more in cases (group I) with mean of 6.065 ±3.045 as compared

CATEGORY 1 (PRE-DIABETES): HBA1C 6-6.4% AND

FBS 100-125 MG/DL

CATEGORY 2 (TYPE 2 DIABETES MELLITUS): HBA1C

>=6.45 AND FBS >=126 MG/DL

Population histogram was created for each category for both

groups.Our study has deciphered prevalence of IGT as 17.5% and

type 2 DM as 2% in PCOS women and 5% IGT and 1.5% T2DM

prevalence in non-PCOS population as depicted in table 4 which

J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 73-83. https://doi.org/10.47277/JIRB/8(4)/73

is quite similar to previous studies done across the world.

different phenotypes of PCOS and histogram was formed for sub-

group analysis and main group analysis both. 55 % (n=105)

women in group I had TSH>= 2.5 m IU/L and only 39.5% (n=79)

of group II had TSH>= 2.5 m IU/L. There was statistically

significant difference between the two groups (P=0.041).Our

study showed higher prevalence of SCH in women with PCOS

compared with non-PCOS women. Bar graph with standard error

of mean in figure 3 (C) depicts that TSH levels are highest in

women with phenotype B followed by phenotype C and D and

least in phenotype A. 6.5%(n=13) women of group I and

5.5%(n=11) women of group II had prolactin levels of more

than/equal to 30 ng/ml. There was a statistically significant

association of PRL with PCOS (p<0.001). It is portrayed in figure

3 (B) that all four phenotypes of PCOS have similar levels of

prolactin. 68 % (n=136) of PCOS women and only 49 %(n-98) of

NON-PCOS group of women had plasma homocysteine levels of

more than/equal to 8. The mean serum homocysteine

concentration was significantly higher in the PCOS group

(11.45±6.563 mmol/l) versus in NON-PCOS group (9.72±7.404

mmol/L) with p value <0.001. Our study has outlined higher

homocysteine levels in phenotype C followed by phenotype B.

Phenotype A and D has similar prevalence with similar

homocysteine levels as illustrated in figure 3 (D).

Association of pre-diabetes and T2DM was calculated with

homocysteine and prolactin levels. High HBA1C and increased

fasting glucose level is more prevalent in PCOS women with

hyperhomocystenemia.This shows association between these

biochemical factors in PCOS resulting in the acquisition of the

endocrinological tetrad. There is higher prevalence of

hyperprolactenemia in PCOS group as compared to non-PCOS

group as illustrated in population histogram in figure 2. 69.5%

(

n=139) of PCOS women had HBA1C levels of more than /equal

to 5.3 % as compared to 44.5% (n=89) of non-PCOS women.

With regard to glucose metabolism, mean HBA1C values were

found to be higher in women with PCOS with statistically

significant difference (p<0.001). Highest HBA1C levels was seen

in phenotype D,then phenotype C and B followed by least in

phenotype A as depicted in figure 2 (A).Fasting blood sugar was

found highest in phenotype D, then phenotype C and B with

similar levels and least in phenotype A as shown in figure 2 (B).

Measurement of anti-mullerian hormone (AMH) was also

assessed in different PCOS women as depicted in figure 2 (C)

which shows higher range in phenotype B, then phenotype D and

A, followed by phenotype C with least level.

We studied association of subclinical hypothyroidism in

Table 1: demographic data of PCOS and non-PCOS women

Variable

Age (years)

Group I (PCOS women)

29.3±3.6

Group II (non-PCOS women)

30.1±4.1

P value

0.025

BMI(kg/m2)

25.9±4.3

24.5±3.6

0.001

Duration of infertility (years)

Primary infertility(n)

Secondary infertility(n)

Data are expresses as mean± SD

6.065±3.045

144(72%)

56(28%)

5.605±2.99

138(69%)

62(31%)

Table 2: demographic data of different phenotypes of PCOS women (group I)

Variable

Age (years)

BMI(kg/m2)

Duration of infertility (years)

Data are expresses as mean± SD

Phenotype A

29.5±3.7

25.4±3.9

6.5±3.5

Phenotype B

28.9±3.7

27.3±4.8

5.9±3.3

Phenotype C

29.2±3.8

24.9±3.5

5.6±2.7

Phenotype D

29.4±3.3

26.4±4.4

6.3±2.8

Table 3: hormonal and biochemical parameters

Variable

Plasma homocysteine

AMH

TSH

PRL

HBA1C

FBS

Group I (PCOS women)

Group II (non-PCOS women)

9.72±7.404

P value

<0.001

0.041

<0.001

11.45±6.563

6.41±3.699

2.74± 1.496

17.49±6.723

5.50±0.628

87.78±19.02

2.51±2.270

2.57±1.580

14.59±7.794

5.31±0.458

77.40±13.61

Data are expresses as mean± SD

Table 4: prevalence of pre-diabetes and type 2 diabetes melllitus in PCOS and non-PCOS women

Variable

Pre0diabetes

Type 2 diabetes mellitus

Group I (PCOS women)

35(17.5%)

Group II (non-PCOS women)

10(5%)

3(1.5%)

4(2%)