Testicular Strain Elastography in Fertile and Infertile Men- a Comparative Cross Sectional Study

TESTICULAR STRAIN ELASTOGRAPHY IN FERTILE AND INFERTILE MEN- A COMPARATIVE CROSS SECTIONAL STUDY

Dissertation submitted to

THE TAMILNADU Dr.M.G.R. MEDICAL UNIVERSITY

In partial fulfillment of the requirements Of

M.D. DEGREE EXAMINATION BRANCH- VIII- RADIODIAGNOSIS

GOVT KILPAUK MEDICAL COLLEGE CHENNAI- 600010

THE TAMILNADU DR.M.G.R. MEDICAL UNIVERSITY CHENNAI- TAMILNADU, INDIA

MAY 2020 CERTIFICATE

This is to certify that the dissertation “TESTICULAR STRAIN

ELASTOGRAPHY IN FERTILE AND INFERTILE MEN- A

COMPARATIVE CROSS SECTIONAL STUDY” titled submitted by Dr.

AKASH KUMAR. B.Y appearing for M.D (RADIODIAGNOSIS) degree

examination in May 2020 is a bonafide record of work done by him under my

guidance and supervision in partial fulfillment of requirement of the TamilNadu

Dr.M.G.R. Medical University, Chennai. I forward this to the TamilNadu

Dr.M.G.R Medical University, Chennai.

Dr.J.DEVIMEENAL,MD.,DMRD.,DNB Dr. P. CHIRTRARASAN, MDRD Guide Guide, Professor & Head of Department, Associate Professor, Department of Radiodiagnosis, Department of Radiodiagnosis, Govt Kilpauk Medical College & Hospital, Govt. Kilpauk Medical College, Kilpauk, Chennai- 10. Chennai-600010

Prof.Dr. P. VASANTHAMANI, M.D., D.G.O.,MNAMS.,DCPSY.,MBA DEAN, Govt Kilpauk Medical College, Chennai-600010

DECLARATION

I, Dr. AKASH KUMAR. B.Y, solemnly declare that this dissertation

“TESTICULAR STRAIN ELASTOGRAPHY IN FERTILE AND

INFERTILE MEN- A COMPARATIVE CROSS SECTIONAL STUDY”is a bonafide work done by me at Government Kilpauk Medical College, under the supervision of Dr. J. Devimeenal, Professor and HOD, and Dr.

P.Chirtrarasan, Associate Professor, Dept. of Radiodiagnosis, Government

Kilpauk Medical College. This dissertation is submitted to the Tamil Nadu Dr.

M.G.R Medical University, towards partial fulfillment of requirement for the award of M.D. Degree Radiodiagnosis.

Place: Chennai Signature of the candidate Date: Dr. AKASH KUMAR. B.Y

CERTIFICATE – II

This is to certify that this dissertation work titled dissertation

“TESTICULAR STRAIN ELASTOGRAPHY IN FERTILE AND

INFERTILE MEN - A COMPARATIVE CROSS SECTIONAL STUDY” of the candidate Dr. AKASH KUMAR. B.Y with Registration Number

201718251 for the award of M.D degree in the branch of

RADIODIAGNOSIS. I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to conclusion pages and result shows 7% of plagiarism in this dissertation.

Guide & Supervisor sign with Seal. ACKNOWLEDGEMENT

I express my heartful gratitude to the Dean,

Prof.Dr.P.VASANTHAMANI,M.D.,D.G.O.,MNAMS.,DCPSY.,MBA Government

Kilpauk Medical College for permitting me to do this study.

I express my gratitude to my guides Prof Dr. J. Devimeenal, Professor and

Head of Department and Prof Dr. P. Chirtrarasan, Associate professor, Dept of Radiodiagnosis, Govt. Kilpauk medical college for their valuable guidance in doing the dissertation work .Their encouragement created an interest for me to pursue this study .Further their constant supervision and support, that made me possible to finish this study without much difficulty.

I am extremely thankful to my Professor Dr. K.GOPINATHAN, MD(RD),

Associate professor Dr. K. Geetha MD(RD) and other Assistant professors of

Department of Radiodiagnosis, Govt.Kilpauk Medical College, Chennai for their constant support, encouragement and advice during my study.

I also thank my past and present fellow postgraduates who helped me in carrying out my work and preparing this dissertation.

I thank all Radiology technicians, staff nurses, and all the paramedical staff members of our department for their co-operation in conducting the study.

I thank my family members for their understanding and co-operation for completion of this work.

Last but not the least; I owe my sincere gratitude to the patients and their relatives who co-operated for this study, without whom the study could not have been possible.

CONTENTS

6 CONTENTS PAGE

1. INTRODUCTION 1 2. REVIEW OF LITERATURE 3 3. AIMS AND OBJECTIVES 36 4. MATERIALS AND METHODS 37 5. CASES 40 6. STATISTICAL ANALYSIS AND RESULTS 48 7. DISCUSSION 76 8. CONCLUSION 79 9. BIBLIOGRAPHY Abbreviations Proforma Patient Consent Form Patient Information Sheet Master Chart Ethical Committee Certificate Plagiarism

INTRODUCTION

“Infertility is defined as the inability of a non-contracepting sexually active couple, to achieve spontaneous pregnancy within one year”. It affects one fifth to one sixth of couples in the reproductive age1.

When compared to other species, human beings are inefficient in terms of reproduction. The fertility rate per cycle is thought to be around 20% and the accumulated pregnancy rate in those couples with proven fertility is approximately 90% after 12 months and 94% after 2 years1. In approximately 20% of infertile couples, male infertility is the sole cause, and in about 30%–40% of these couples, male and female factors are the causes. Therefore, a condition involving the male partner contributes to approximately 50% of cases of infertility.

The diagnostic workup of male infertility should include a thorough medical and reproductive history, physical examination, and semen analysis, followed by imaging. Ultrasound is the first-line imaging modality which is used for the evaluation of male genital tract as it is noninvasive, safe and there is no exposure to radiation. In addition to physical examination and semen analysis, ultrasonography of scrotum may be helpful in demonstrating obstruction or testicular dysgenesis2. Its sensitivity and specificity increase even more by using Doppler. 2

Conventional ultrasonography has the limitation of only functional analysis of testicular tissue, whereas elastography is a promising technique in this field. New insights for the structural and functional evaluation of testicular tissue have been provided by the recent technical advances in ultrasound applications and post-processing developments3.

Elastography was first described by Ophir et al. It is a new imaging technique which displays the images of tissue stiffness. These images that are created by elastography are thought to be an extension of the ancient palpation techniques. It gives a better information regarding the spatial localization and is also less subjective.

Real time elastography, a method which shows stiffness of tissue under real time conditions demonstrates different values of elasticity dependent on volume and function of testis. Elastography assesses elasticity of testis. It is defined as the tendency of the tissue to resist deformation when a force is applied, or to resume its original shape after the removal of the force.

The principle of sonoelastography is to use repeated, slight pressure on the examined organ with the ultrasound transducer.

Ultrasound elastography techniques can be categorized as:

1) Strain imaging, and 2) Shear wave imaging.

Here in this study, we study only the Strain elastography and its

diagnostic value in male infertility. 3

REVIEW OF LITERATURE

ANATOMY OF MALE REPRODUCTIVE SYSTEM:

The male reproductive system consists of external (penis, scrotum, epididymis, and testes) and internal (accessory) organs.

Primary functions of the male reproductive organs are:

1. Production, maintenance, transportation, nourishment and protection

of the semen.

2. Discharge of sperm into the female genital tract.

3. Production and secretion of male sex hormones4.

SCROTUM:

It is a dual-chambered sac of skin and smooth muscle containing the testes, and is homologous to the labia majora in females.

It is located between the penis and anus as an extension of perineum. In humans, increased testosterone secretion during puberty causes skin darkening and pubic hair development on the scrotum.

The left testis is usually lower than the right, which functions to avoid compression when there is impact. There is also more cooling of testis due to this asymmetry4.

Functions of scrotum:

The function of the scrotum is to maintain the temperature of the testes slightly lower than that of the rest of the body. Higher temperatures causes damage to the sperm count.

The temperature is controlled by movement of the testis away or towards the body based on the temperature of environment. By moving the testes away from the abdomen and increasing the exposed surface area, there is faster dispersion of excess heat. This is achieved by the contraction and relaxation of the cremaster muscle and the dartos fascia in the scrotum.

Temperature regulation is not the only function of the scrotum. It has been also suggested that if testes were located within the abdominal cavity, they would be subjected to the changes in abdominal pressure exerted by the abdominal musculature, which would result in more rapid emptying of sperm from the testes and epididymis before the sufficient maturation of spermatozoa for fertilization4. 5

Fig.1: Image of the external, muscle and deep tissue views of the scrotum4.

TESTIS:

The testis is the male gonad. It is homologous to ovary in females. They

are components of both the reproductive system and the endocrine

system. The testes produce sperm (spermatogenesis) and androgens,

primarily testosterone. Luteinizing hormone results in testosterone

release. The presence of both testosterone and follicle-stimulating

hormone (FSH) is needed to support spermatogenesis.

Fig.2: A diagram of the major components of an adult human testis. 1. Tunica albuginea, 2. Septula testis, 3. Lobulus testis, 4. Mediastinum testis, 5. Tubuliseminifericortorti, 6. Tubuliseminiferi recti, 7. Rete testis, 8. Ductuli efferentestesti, 9a. Head of epididymis, 9b. Body of epididymis, 9c. Tail of epididymis, 10. Vas deferens, 11a. Tunica vaginalis (parietal lamina), 11b. Tunica vaginalis (visceral lamina), 12. Cavity of tunica vaginalis 4.

Each testis is covered by the tunica vaginalis, tunica albuginea, and tunica

vasculosa. The tunica vaginalis is the lower portion of the processus

vaginalis and is reflected from the testes on the inner surface of the

scrotum, thus forming the visceral and parietal layers5. Tunica albuginea

is a dense covering for the testes located beneath the visceral layer of the

tunica vaginalis.

Tunica albuginea is a tough membranous shell containing very fine coiled

tubes called seminiferous tubules. These are lined with a layer of germ

cells that develop into sperm cells (also known as spermatozoa). The

developing sperm travels through the seminiferous tubules to the rete 7 testis which is located in the mediastinum testis, to the efferent ducts, and then to the epididymis where newly-created sperm cells mature. The sperm moves into the vas deferens and is eventually expelled through the urethra, via the urethral orifice through contraction of the muscles.

Leydig cells is located between the seminiferous tubules. It functions to produce and secrete testosterone and other androgens important for sexual development and puberty, including secondary sexual characteristics. They also support libido, spermatogenesis, and erectile function. Also, testosterone controls testicular volume. The sertoli cells are necessary for testis development and spermatogenesis.

TESTIS DUCTS:

This includes the seminiferous tubules and vas deferens, which are involved in producing and transporting sperms.

Seminiferous tubules, located in the testes, are the site for creation of spermatozoa. The epithelium of the tubule consists of tall, columnar cells called Sertoli cells. Spermatogenic cells are located between the sertoli cells and they differentiate through meiosis to become sperm cells. There are two types of seminiferous tubules: convoluted, located toward the 8 lateral side, and straight, as the tubule comes medially to form ducts that will exit the testis.

The ductus (vas) deferens is the continuation of the epididymis. It measures 30-45-cm long and transports sperm to the ejaculatory ducts6,7.

As it travels posterior to the testis and medial to the epididymis, the convoluted portion of the ductus deferens becomes more straighter.

Subsequently, the ductus travels upwards on the posterior aspect of the spermatic cord until it reaches the deep inguinal ring, where it is invloved in the formation of the spermatic cord and loops over the inferior epigastric artery. At this point, the ductus travels along the lateral pelvic wall, medial to the distal ureter, along the posterior wall of the bladder until it reaches the seminal vesicles dorsal to the prostate.

Seminal vesicles: Sac-like pouches that attach near the bladder base to the vas deferens. The anterior surface is contiguous with the posterior wall of the bladder and the posterior surface is in contact with the rectovesical

(Denonvilliers) fascia. They produce fructose that serve as energy sources for sperm. The seminal vesicle fluid constitutes most of the volume of the ejaculate.

Ejaculatory ducts:The ejaculatory ducts measures 2 cm in length and is formed from the union of the seminal vesicle and the ampulla of the vas deferens. Each duct starts at the base of the prostate and ends at the seminal colliculus (verumontanum).

Prostate gland: A walnut-sized structure located below the urinary bladder anterior to rectum. It produces additional fluid to the ejaculate that serves as nourishment for sperm.

Bulbourethral (Cowper’s) glands: Pea-sized structures located on either sides of the urethra just below the prostate gland. They produce a clear, slippery fluid that empties directly into the urethra. Fluid produced by these glands lubricates the urethra and neutralizes acidity associated with residual urine.

Male factor is responsible for approximately 50% cases of infertility.

Impaired sperm production and function may be caused by congenital or acquired factors that act at the pretesticular, testicular or post-testicular level.

Factors reducing male fertility:

 Infections of urogenital tract

 Malignancies

 Urogenital abnormalities

 Increased temperature of scrotum

 Endocrine disturbances

 Genetic abnormalities8

Testicular deficiency:

Testicular deficiency due to primary spermatogenic failure is caused by a number of conditions. It is considered as the most common cause of reduced fertility in males.

Etiology:

Congenital

 Anorchia

 Testicular dysgenesis

 Genetic and chromosomal abnormalities

Acquired

 Torsion of testis

 Trauma

 Inflammatory diseases like mumps and orchitis

 Systemic diseases like renal and liver failure

 Exogenous factors (Medications, heat)

 Varicocele

 Tumors

 Testicular surgery8

Conclusions for testicular deficiency:

Impaired spermatogenesis has an association . with elevated concentration of FSH.

Spermatozoa are found in 50% of patients . with nonobstructive azoospermia.

Pregnancies and live births . Subsequently occur in 30-50% of couples with nonobstructive azoospermia, when testicular biopsy shows spermatozoa8.

Chromosomal abnormalities:

Chromosome abnormalities. may be numerical (ex. trisomy) or structural.

(ex. Inversions and translocations).

 Abnormalities of sex . chromosomes (Klinefelter’s syndrome and its

variants. 12

 Autosomal abnormalities like .robertsonian translocations,

reciprocal translocations.

 Abnormalities . of sperm chromosomes.

Genetic defects:

 Kallmann syndrome – patients have hypogonadotrophic

hypogonadism and anosmia along with cleft palate, facial

asymmetry, colour blindness ., deafness, undescended testis, and

renal aplasia.

 Androgen insensitivity. syndrome

 Cystic fibrosis and its mutations.

Obstructive azoospermia:

It is the absence of spermatozoa in . semen as a result of obstruction. It is less common than non-obstructive azoospermia. Seen in about 15-20% of men with azoospermia.

Patients present with enlarged epididymis, normal testis size and FSH values. Obstruction in men with primary infertility is often present at the epididymal level, affecting 30-67% of azoospermic men. Congenital form of epididymal obstruction include chronic sinopulmonary infection

(Young’s syndrome). 13

Obstruction of vas deferens isconsidered to be the most common cause of

acquired. obstruction following vasectomy.

It may also occur after hernia repair.

Obstruction of the ejaculatory duct is found in approximately 1-3% of

cases of obstructive azoospermia .and it may be classified as cystic or post

inflammatory. Cystic obstructions are often congenital . (like Mullerian

duct cyst or urogenital sinus/ejaculatory duct cysts) and are usually .

midline. In urogenital sinus abnormalities, the ejaculatory ducts . empty into the cyst9, while in Mullerian duct anomalies, the ejaculatory ducts

are .laterally displaced and are compressed by the cyst. Complete

obstructions of the ejaculatory ducts . are usually associated with

decreased volume of semen, decreased. fructose in semen, and acidic pH.

The seminal vesicles are also usually. dilated (anterior-posterior diameter

> 15 mm).

Varicocele:

Clinically, varicocele is classified as10:

 Subclinical: Not visible or palpable at rest or during . valsalva, but

can be demonstrated by doppler.

 Grade 1: Palpable only during valsalva.

 Grade 2: Palpable. at rest, but not visible.

 Grade 3: Visible and palpable . at rest. 14

Varicocele is diagnosed by clinical examination and confirmed by .colour doppler ultrasound.

Varicocele is present in 11.7% of adults and 25.4%. of men with abnormal semen analysis11.

The exact association between reduction in male fertility and varicocele is

not known, but a recent meta-analysis report has shown that improvement

12 of semen is observed after. surgical correction .

Simplified color Doppler ultrasound classification of varicoceles13

GRADE FEATURES

1 Reflux in the vessels of the inguinal canal that is observed only during the Valsalva maneuver and absence of varicosity on standard ultrasonography.

2 Small varicosities with reflux seen only during the Valsalva maneuver.

3 Enlarged vessels whose caliber increases during the Valsalva maneuver.

4 Obvious vessel enlargement with reflux that is present under basal conditions and does not increase during the Valsalva maneuver.

Testicular atrophy:

Testicular atrophy is significant when the volume of the testis is reduced to 50% of the volume of the unaffected testis. It is associated with decreased spermatogenesis and reduced fertility. It may occur due to infarction, inflammation (epididymo-orchitis), cryptorchidism, varicocele, trauma, or chronic mass effect. Other causes of testicular atrophy are liver cirrhosis, estrogen treatment, and hypopituitary disorders.

Epididymo-orchitis:

Infection and inflammation of the genitourinary tract are the commonest causes of male infertility.

This affects spermatogenesis and alter the number and quality of sperm by affecting the process of sperm storage, motility, development, and the maturation of the sperm membrane which is normally seen to occur in the epididymis.

Chronic epididymitis and epididymo-orchitis can also result in the atrophy of testis.

Testicular microlithiasis:

Calcium deposits develop either within the seminiferous tubules or may arise from the basement component of the tubules. 16

It is diagnosed when ultrasound depicts five or more echogenic foci smaller than 3 mm per field of view without presence of posterior acoustic shadowing.It is associated with infertility, testicular atrophy, cryptorchidism, Klinefelter syndrome, hypogonadism, and alveolar microlithiasis15,16.

20 to 60 percent of the seminiferous tubules are usually involved, resulting in oligospermia and reduced sperm motility, which explains the association of testicular microlithiasis with infertility.

Cryptorchidism:

It is considered as the most common . congenital abnormality of male genitalia, caused by delayed spontaneous descent. Spontaneous descent however becomes less likely after the age of 6 months. Most commonly, it is seen in the inguinal canal.

It can be a result of testicular dysgenesis, and can result . in maldescended testis, hypospadias, infertility, and a high risk for malignancy.

This results in impaired spermatogenesis due to the abnormal location of the testis outside of the scrotum and cause infertility.

The prevalence of azoospermia seen with unilateral cryptorchidism is about 13%; whereas in patients with untreated bilateral cryptorchidism, it is about 89%17.

MISCELLANEOUS CONDITIONS THAT CAUSE INFERTILITY:

Erectile dysfunction:

It is defined as the persistent inability of a person to achieve and/or maintain a penile erection which is sufficient to engage in sexual activity.

Approximately 52% of the males aged between 40–69 years have erectile dysfunction, and among these, about 10% are affected severely18. It can be due to organic or psychological causes, or both.

Ejaculatory dysfunction:

A male with normal semen parameters can be infertile if he is not able to deliver the ejaculate into the female partner. Normal erectile and ejaculatory functions are necessary for proper delivery of the semen into the female partner.

Ejaculatory dysfunctions that may interfere with male fertility are as follows:

Premature ejaculation – This is the most common form of sexual dysfunction. In this condition, ejaculation occurs too early and without self control, causing marked distress.

Anorgasmia – It is the inability to achieve the sensation of orgasm, which in turn results in semen transport failure. 18

Retrograde ejaculation – In this condition, the semen enters the male’s bladder instead of entering the vagina despite an erection.

Anejaculation – It is the inability to achieve antegrade or retrograde ejaculation due to failed emission or anorgasmia19.

The diagnostic workup in infertile men are done to:

1) Identify treatable, reversible, and/or health-threatening conditions

2) Select patients suitable for assisted reproduction techniques

3) Determine appropriate genetic counseling and measures for

prevention such as pre-implantation and prenatal diagnosis to

safeguard the health of future offspring20.

The infertility workup is usually started after the couple has attempted conception for more than 12 months. Potentially correctable causes of infertility, such as congenital abnormalities and obstructive disorders can be identified by imaging. Brief medical history, preferably obtained in the presence of the female partner, and physical examination with semen analysis should be performed. Relevant endocrine, genetic, and imaging examinations should be conducted20.

Evaluation of Male Infertility:

The initial diagnostic workup should include a brief medical history, physical examination, semen and hormone analyses, and imaging.

The medical history must focus to identify risk factors and behavior that could affect fertility. It includes duration of infertility, age of the patient and partner, any gynecologic factors involving the female partner, medications affecting the hypothalamic-pituitary-gonadal axis, cryptorchidism, sexual disorders, sexual intercourse frequency, history of smoking and alcohol intake, surgery, and pubertal development and disorders20.

Typical findings from the history and physical . examination of a testicular deficiency patient are:

 Cryptorchidism

 Infection of. genitourinary tract

 Torsion of testis

 Trauma. to testis

 Exposure to environmental toxins

 Anabolic drugs

 Radiation. exposure

 Cytotoxic agents 20

 Testicular tumors

 Abnormal secondary. sexual characteristics

 Gynecomastia

 Abnormal volume and consistency. of testis

Semen and Hormone Analyses:

Properties of normal semen:

 Volume greater than 1.5 mL

 Concentration greater than 15 million per milliliter

 Total progressive and non-progressive motility greater than 40%

 Progressive motility greater than 32%

 pH > 7.2

 Greater than 4% normal consistency21.

Abnormalities:

Azoospermia – Absence of sperm

Oligozoospermia – Sperm count <15 million per milliliter.

Asthenozoospermia – < 40% motile spermatozoa

Teratozoospermia – <4% normal forms

Hypospermia – Semen volume <1.5 ml

Hyperspermia – Semen volume >5.5 ml

Aspermia – Absence of semen 21

IMAGING

The major role of imaging is to identify the causes of infertility, such as congenital anomalies and obstructive disorders of sperm transport which are correctable. It can also be used as a guide to impregnate the female partner, such as aspiration of sperm from the epididymis or seminiferous tubules followed by in vitro fertilization or intracytoplasmic sperm injection.

The imaging modalities used for evaluating the male reproductive system are ultrasonography (US) and magnetic resonance (MR) imaging, as well as other invasive techniques such as venography and vasography20.

Ultrasonography: Scrotal ultrasound is the preferred modality because of its noninvasiveness, safety, and is inexpensive and allows multiplanar imaging.This examination can be used for evaluating potential testicular abnormalities, to calculate the testicular volume, and identify peritesticular abnormalities like varicocele, abnormalities of epididymis and prostate, and erectile dysfunction.

Transverse and longitudinal ultrasonography of the testes and color flow

Doppler ultrasound of testicular and spermatic cord vascularity are performed using a high-frequency linear-array transducer.

Testicular volume measurements, correlating with semen profiles, also should be obtained. Testicular volume is calculated by using the formula 22

(length × width × anteroposterior diameter) × π/6, and the normal value range is 15–20 ml.

Transrectal ultrasound can be used in addition to evaluate the prostate and identify more central sources of spermatic obstruction. A seminal vesicle diameter > 1.5 cm and an ejaculatory duct diameter > 2.3 mm are suggestive of ejaculatory duct obstruction, especially when associated with cysts or calcifications along the duct22.

MR Imaging: MR imaging is superior to transrectal ultrasound for examining the patients with infertility and can also serve as an alternative to traditional invasive vasography.

Due to its superior soft-tissue contrast and multiplanar capabilities, MR imaging can depict the detailed anatomy and pathophysiologic features of the reproductive tract,with the prostate, seminal vesicles, and ejaculatory ducts. It is the modality of choice for imaging the accessory sexual glands and their ducts and can also help in guiding diagnostic or corrective interventional procedures.

T2-weighted images depict the prostate, seminal vesicles, and structures surrounding it. Three-dimensional T2-weighted fast spin-echo MR imaging has some advantages over two-dimensional MR imaging: it allows imaging to be done with thinner sections without any intersection 23 gaps, it generates higher signal-to-noise ratios, and it also helps in reformatting the acquired images in any desired plane23.

Computed tomography:Computed tomography facilitates limited soft- tissue resolution and not used frequently for evaluating infertility.

Vasography: Also known as seminal vesiculography, was once the reference standard to evaluate male reproductive system. It involves cannulation of the vas deferens under anaesthesia. Owing to the widespread acceptance of MR imaging, this invasive technique is no longer frequently used to evaluate the male reproductive system20.

Conventional ultrasonography has a limitation of only functional. analysis

of testicular tissue, whereas elastography is a promising . technique in this field.

ELASTOGRAPHY:

Elastography was first described by Ophir et al. It is a relatively new imaging technique that displays the images of stiffness of tissues. The images that are created by elastography are thought as an extension of the ancient palpation techniques. Further development and refinement has been done recently to enable quantitative assessment of stiffness of tissues. 24

Elastography methods may take advantage of the change in elasticity of soft tissues due to specific physiological or pathological processes24. For example, there is a mechanical difference between many solid tumors and its surrounding healthy environment. Similarly, fibrosis seen with chronic liver disease causes the liver to become more stiffer than the adjacent normal tissues. Elastography provides additional information to conventional ultrasound by adding stiffness as another property to current available ultrasound imaging techniques.

The various studies published in the literature regarding the development and practical applications of ultrasound elastography methods are classified by considering the four key steps (Fig 3. )

Step (1) : Different kinds of stimuli can be adopted to deform a soft tissue. In the literature, static loads, external vibrators and acoustic radiation forces (ARF) have been applied to generate distinct responses in a soft tissue, leading to different static and dynamic elastography methods.

Step (2) :Accurately tracking the mechanical responses of various target soft tissues generated by various stimuli is considered a key step in an elastography method. Various different medical imaging methods have been used to this end, giving rise to ultrasound elastography, magnetic resonance elastography, optical elastography and so on. Motivated by this need, some dedicated imaging techniques have been introduced. For 25 example, apart from the measurement of axial displacements, techniques based on the ultrasound imaging have been presented to also obtain the lateral displacements and strains. Another example is the development of a method to image two – dimensional motion vectors by taking advantage of ultrafast ultrasound imaging techniques ( the frame rate can be upto

6000 Hertz or even higher).

Step (3) : Knowing the responses of soft tissues under given stimuli tracked with various medical imaging methods, it is possible to know the mechanical properties of soft tissues which has received significant attention from different disciplines. Along with linear elastic parameters, it has also been demonstrated that hyperelastic, viscoelastic and anisotropic elastic parameters of soft tissues may be inferred using various inverse methods.

Step (4) :The mechanical properties of living soft tissues which are inferred from the responses to imposed stimuli may provide some valuable information for diagnosing and treating some diseases. This step is the main interest of clinicians who use elastography, and most of the publications from clinical research focus on this aspect25. 26

Fig 3 : An illustration of the key steps involved in elastography26

PRINCIPLES AND TECHNIQUES OF ULTRASOUND

ELASTOGRAPHY:

Physics of ultrasound elastrography:

Elastography assesses elasticity of tissue, which is the tendency of tissue to resist deformation when a force is applied, or to resume the original shape after removal of the force.

Assuming that a material is entirely elastic and its deformation does not depend on time ( i.e. viscosity) , elasticity can be described by Hooke’s law :

Where stress (σ) is the force per unit area with kilopascals as unit ( i.e.

N/m) (Fig 4, top row), strain (ε) is the expansion per unit length which is 27 dimensionless (Fig 4 , second row), and the elastic modulus (Γ) relates stress to strain with kilopascals as unit (Fig 4, third row )27.

Fig.4: Ultrasound elastography physics, deformation models. Static deformations of entirely elastic materials can be described by stress σ (force per unit area, top row),

strain ε. (expansion per unit length, middle row), and elastic modulus Γ (stress

divided by strain, bottom row). This . is applied to normal (perpendicular to surface,

first column), shear. (tangential to surface, second column), and bulk (normal inward 27 or pressure, third column) forces . used in ultrasound elastography

Three types of elastic moduli (Γ) are defined by the method of deformation :

Young’s modulus (E) , shear modulus (G), and bulk modulus (K).

Young’s modulus E is derived by the following equation when a normal stress (σ ) produces a normal strain (ε ), where normal is perpendicular to the surface (Fig 4, first column):

Shear modulus G is derived by the following equation 28 when a shear stress (σ ) produces a shear strain (ε ), where shear is tangential to the surface. (Fig 4 , second column) :

Bulk modulus K is derived by the following equation when a normal inward force or pressure (σ ) produces a bulk strain or change in volume

(ε ). ( Fig 4 , third column):

As elastic modulus Γ is higher, the resistance of a material to deformation is more, which can be thought as increased stiffness27. In strain imaging, the normal strain is measured after applying normal stress to yield estimates of Young’s modulus E.

Fig.5: Ultrasound elastography physics, measurement. methods27. 29

In strain imaging (a), displacement of tissue is measured by correlating

RF echo signals between search windows (boxes) before and after

compression.

In shear wave imaging (b), particle motion is perpendicular to the

direction of wave propagation, with shear wave speed cSrelated to the

shear modulusG.

In B – Mode ultrasound (c), particle motion is parallel to the direction of

wave propagation, with longitudinal wave speed cL related to the bulk

modulus K. (Fig 5) [27].

Ultrasound elastography techniques:

From these principles, the different techniques of ultrasound elastography

can be classified by the measured physical quantity (Fig 6 ).

Fig.6: Ultrasound Elastography Techniques. Currently available techniques of ultrasound elastography can be categorized by the measured physical quantity: 1) strain . imaging (left), and 2) shear wave imaging (right). Excitations methods include quasi-static mechanically-induced

displacement via active external compression . or physiologic motion (orange), dynamic mechanically-induced compression via a “thumping” transducer at the tissue surface to produce

shear waves (green), and dynamic ultrasound-induced. tissue displacement and shear waves by 27 acoustic radiation force impulse. excitation (blue) . 30

Here in this study, we study only the strain elastography and its diagnostic value in male infertility.

Strain Imaging :

Strain elastography was the first technique to be introduced. There are two approaches for strain imaging : Strain elastrography ( SE) and

Acoustic radiation force impulse (ARFI) strain imaging.

Strain Elastography:

Strain elastography is further subdivided by the method of excitation :

1) In the first method, manual compression is given by the operator on

the tissue with ultrasound transducer28.

2) In the second excitation method, the ultrasound transducer is held

steady without moving, and internal physiologic motion is seen to

generate tissue displacement (E.g. cardiovascular, respiratory). Since

this method is independent of superficially applied compression, it

may be used commonly to assess organs located deep.

The displacement induced by the tissue in the same direction as the

applied stress is measured by various different methods dependent on

the manufacturer, including radiofrequency (RF) echo correlation –

based tracking, Doppler processing, or a combination of the above two

methods. 31

RF echo correlation-based tracking is the most common and simple methods. In this method, 2D RF – lines are measured along the axis of displacement. Correlation of the RF echo signal in different acquisitions allows for the measurement of the displacement of the tissue and estimation of the normal strain.

The stress which is applied manually or physiologically is not quantifiable, but by assuming uniform normal stress σ, the measured normal strain ε gives a qualitative measure of Young’s modulus E and thus tissue elasticity (Fig.2,first column, last row).

The strain measurements are displayed as a semitransparent color map known as an elastogram, overlaid on the B- mode image. Usually in most of the vendors, low strain (i.e. stiff tissue) is shown in blue colour, and high strain (i.e. soft tissue) is shown in red colour, although the color varies based on the ultrasound vendor29. A pseudo-quantitative measurement called the strain ratio is used , which is the ratio of strain measured in adjacent normal reference tissue region of interest (ROI) to the strain measured in a target lesion ROI. A strain ratio of >1 indicates that the target lesion compresses less than the normal reference tissue, which indicates lower strain and greater stiffness-30.

Acoustic radiation force impulse (ARFI) strain imaging

This is an alternative approach to measure strain. In this technique, short duration (0.1-0.5 medial segment) high intensity acoustic pushing pulse

(acoustic radiation force) is used for tissue displacement (displacement of approximately 10-20 micrometer) in the normal direction i.e. perpendicular to the surface31. The displacement within a specified ROI is subsequently measured in similar way as in strain elastography.

Technical limitations in ultrasound elastography:

There are several technical factors affecting ultrasound elastography.

Among the various elastography methods mentioned above, measurement are challenging to reproduce from methods using external stimuli, such as strain elastography.

 Measurements obtained in these methods are highly subjective,

since the extent of the stress applied is difficult to control with

manual compression being operator dependent and due to

inherent variability of physiologic motions.

 Selection of the ROI is also dependent on the operator.

 The magnitude of the stress induced by the operator can cause

some strain concentration artifacts around specific structures

which may then cause distortion of strain field and generate

artifacts in images or lead to erroneous measurement.

 SE methods allow only semi quantitative assessment of stiffness

which are longitudinally difficult to compare.

 Elastography is also susceptible to internal sources of stress ( i.e.

Cardiac, breathing). For example in liver, stiffness is usually

measured in the right lobe over the left lobe preferably to reduce

internal stimulations generated by the heart beat which can

subsequently result in erroneous measurements32,33. In

elastography modes utilizing internal sources of excitation stress

(i.e. Cardiac) which cannot be operator regulated, it is important

to note that these stresses are complex, not easily quantifiable and

are variable through time ( as a function of physiology) , thus can

tamper with measured strain responses.

Commercially available modes of ultrasound elastography rely on a set of assumptions about the examined tissue material, so as to make the analysis and interpretation of measurements/imaging simple.

 Linear; resulting strain increases in a linear fashion as a function of

incremental stress.

 Elastic; tissue deformation is independent of the rate of stress, and

tissue returns to original non deformed equilibrium state.

 Isotropic; the tissue is symmetrical/ homogeneous and it responds

in a similar way to stress in all directions. 34

 Incompressible; the overall volume of tissue remains the same and

there is no change under stress applied.

ELASTOGRAPHY OF TESTIS:

It is performed the same way as done at other sites:

Each testis is assigned an elasticity score based on a 5- point scale34:

 Score of 1: the entire testis is mostly shaded in red indicating

predominantly high strain pattern.

 Score of 2: the peripheral part of the testis is blue and the central

part is red, indicating central high strain with a peripheral low

strain pattern.

 Score of 3: the entire testis shows evenly distributed green color

and the peripheral part of the testis is blue, indicating average

strain pattern.

 Score of 4 : the entire testis is mostly blue and green, indicating

predominantly low strain pattern.

 Score of 5: entire testis is blue, indicating low strain pattern.

Strain elastography in testicular lesions is not routinely done, and is a relatively new approach.

Aigner et al. showed that benign and malignant testicular lesions can be differentiated with a high sensitivity rate using elastography.

In the evaluation of infertility, a study by Schurich et al. showed that strain elastography can be used to assess the structure of testicular parenchyma and therefore it can be used as an additional modality to detect pathological alterations of testicular tissue. They also noted that testicular volume and function were significantly affecting the elasticity pattern of the testicular tissue.

However, there are not much studies showing the importance of testicular strain elastography in the evaluation of infertility.

The hypothesis of this study is that abnormal semen parameters are associated with pathological alterations in the testicular parenchyma, and depiction of which cannot be done by conventional ultrasound.

Therefore we have done this study to show the differences in testicular elasticity as measured by strain elastography in infertile men with abnormal semen analysis when compared to the fertile men, and also to see the differences in elasticity patterns in various semen abnormalities.

AIMS AND OBJECTIVES

To determine and compare the diagnostic value of strain elastography in fertile and infertile men, and to correlate the results of strain elastography with semen parameters and hormone profiles of the patients.

MATERIALS AND METHODS

STUDY DESIGN:

Comparative cross sectional study.

STUDY PERIOD:

June 2018 – May 2019, for a period of 1 year

STUDY POPULATION:

 Infertile group - Men between 20–45 years of age, clinically.

diagnosed with primary infertility and with abnormal semen

analysis and did not receive any previous fertility treatment.

 Fertile group – Men between 20–45 years of age with normal

semen analysis.

SAMPLE SIZE: 82

INCLUSION CRITERIA:

 Infertile group - Men between 20–45 years of age, clinically

diagnosed with primary infertility and with abnormal semen

analysis and did not receive any previous fertility treatment.

 Fertile group – Men between 20–45 years of age with normal

semen analysis. 38

EXCLUSION CRITERIA:

 Patients who did not undergo semen analysis.

 Undescended testis.

 History of orchidectomy or previous testicular

biopsy.

 Atrophy of testis, acute. trauma changes, and prior surgical

interventions to testis.

 Testicular mass.

 Testicular microlithiasis, and infarct.

DATA COLLECTION:

Data collection was performed in the included study group using a standard questionnaire/ proforma that includes the basic patient details such as name, age, address, occupation, dietary habits and history of smoking/ alcohol, history of previous testicular surgery/ trauma, history of previous testicular malignancy/ infarct.

METHODOLOGY

 Patients were explained about the study.

 Informed consent was obtained.

 Study subjects were divided into 2 groups: Group 1 (Infertile)

and Group 2 (Fertile) having 41 patients each.. 39

 Gray scale ultrasonography was done first to look for the

echotexture and size of the testis.

 This was followed by Doppler of the intraparenchymal arteries

where the resistive index was measured.

 Strain elastography of testis was performed after this using GE-

Logic S7 machine, supplied with SE software and using a 7-

12MHz frequency transducer.

 The whole examination was performed in the supine position.

 Testicular volumes and the flow in the intraparenchymal

arteries were measured followed by strain elastography.

 The strain ratios were calculated by putting multiple equally

sized regions of interest(ROIs) on the testicular tissue (A) and

scrotal subcutaneous . fatty tissue (B).

 Strain ratio (SR) value was automatically calculated on the

sonography machine by comparing . A to B (B/A) for each

patient and mean values were obtained.

 Mean values in infertile and fertile men were then compared.

CASE 1

30 year old male came to the andrology clinic for complaints of primary infertility since 2 years.

Semen analysis showed asthenozoospermia.

The Testicular volume is 16.42 cc.The mean resistive index of intraparenchymal artery is 0.455.

The Strain value (SV) was 2.8 and the Strain ratio (SR) was 0.56.

CASE 2

32 year old male came to the andrology clinic for complaints of primary infertility since 4 years.

Semen analysis showed azoospermia.

The Testicular volume is 19.7 cc.

The mean resistive index of intraparenchymal artery is 0.455.

The Strain value (SV) was 2.13 and the Strain ratio (SR) was 0.463.

CASE 3

28 year old male came to the andrology clinic for complaints of primary infertility since 2 years.

Semen analysis was normal.

The Testicular volume is 13.31cc.

The mean resistive index of intraparenchymal artery is 0.45.

The Strain value (SV) was 1.06 and the Strain ratio (SR) was 0.20.

CASE 4

29year old male came to the andrology clinic for complaints of primary infertility since 3 years.

Semen analysis was normal.

The Testicular volume is 12.51cc.

The mean resistive index of intraparenchymal artery is 0.45.

The Strain value (SV) was 1.375 and the Strain ratio (SR) was 0.30.

STATISTICAL ANALYSIS & RESULTS

The Data was entered in a excel worksheet and double checked.

IBM SPSS version 22 software is used for statistical analysis.

Data were presented as mean±SD. Continuous variables were . evaluated by mean and SD, and compared by Student’s T test. Correlation of semen

parameters. with SE results were tested by One way ANOVA.

P value < 0.05 is considered statistically significant.

Table 1: Descriptive analysis for age in study population (N=82)

AGE GROUP * GROUP Crosstabulation

GROUP

Infertile Fertile Total

AGE <=30 Count 20 24 44 GROUP % within 48.8% 58.5% 53.7% GROUP

% of Total 24.4% 29.3% 53.7%

>30 Count 21 17 38

% within 51.2% 41.5% 46.3% GROUP

% of Total 25.6% 20.7% 46.3%

Group Statistics Std. Std. Error

GROUP N Mean Deviation Mean

AGE Infertile 41 31.68 3.778 .590

Fertile 41 29.68 3.182 .497

Figure 1: Age wise distribution of the study population in two groups

Independent Samples Test

t-test for Levene's Test for Equality of Equality of Variances Means

F Sig. t df

AGE Equal variances 1.093 .299 2.593 80 assumed

Equal variances 2.593 77.750

not assumed

Independent Samples Test

t-test for Equality of Means

Sig. (2- Mean Std. Error 95% tailed) Difference Difference Confidence Interval of the Difference

Lower

AGE Equal .011 2.000 .771 .465 variances assumed

Equal .011 2.000 .771 .464 variances not assumed

Table 2: Testicular volumes (TV) in Infertile and Fertile population

Group Statistics

Std. Std. Error

GROUP N Mean Deviation Mean

TV Infertile 41 13.869 3.506 .547

Fertile 41 14.996 1.9811 .309

Independent Samples Test

t-test for Levene's Test for Equality of Equality of Variances Means

F Sig. t df

Volume(ml) Equal variances .321 .573 -3.993 80 assumed

Equal variances -3.993 75.661

not assumed

Independent Samples Test

t-test for Equality of Means

Sig. (2- Mean Std. Error tailed) Difference Difference

Volume(ml) Equal variances .000 -.6390 .1600 assumed

Equal variances not .000 -.6390 .1600 assumed

Figure 2:Mean Testicular volume in Infertile and Fertile population

Testicular Volume

15.2 14.99

14.8

14.6

14.4

14.2 Mean Testicular volume 14 13.869 13.8

13.6 Mean Testicular volume Testicular Mean 13.4

13.2 Infertile Fertile Group

Table 3: Resistive index (RI) of Intraparenchymal artery in Infertile and Fertile population

Group Statistics

Std. Std. Error

GROUP N Mean Deviation Mean

RI Infertile 41 .44427 .056131 .008766

Fertile 41 .43854 .042430 .006626

Independent Samples Test

t-test for Levene's Test for Equality of Equality of Variances Means

F Sig. t df

RI Equal variances 1.905 .171 .522 80 assumed

Equal variances .522 74.461

not assumed

Independent Samples Test

t-test for Equality of Means

95% Confidence Interval of the Difference Sig. (2- Mean Std. Error tailed) Difference Difference Lower

RI Equal variances .603 .005732 .010989 -.016137 assumed

Equal variances .604 .005732 .010989 -.016162 not assumed

Figure 3: Mean Resistive index of Intraparenchymal artery

Intraparenchymal artery Resistive Index 0.442 0.44

0.44 0.438 0.436 0.434 0.432 0.43 Resistive Index 0.43 0.428

Mean Resitive Index Resitive Mean 0.426 0.424 Infertile Fertile Group

Table 4: Strain value (SV) of testis in Infertile and Fertile population

Group Statistics

Std. Std. Error

GROUP N Mean Deviation Mean

SV Infertile 41 1.6807 .378483 .059109

Fertile 41 1.3807 .525077 .082003

Independent Samples Test

t-test for Levene's Test for Equality of Equality of Variances Means

F Sig. t df

SV Equal variances 4.198 .044 2.968 80 assumed

Equal variances 2.968 72.730

not assumed

Independent Samples Test

t-test for Equality of Means

95% Confidence Interval of the Difference Sig. (2- Mean Std. Error tailed) Difference Difference Lower

SV Equal variances .004 .300000 .101086 .098832 assumed

Equal variances .004 .300000 .101086 .098523 not assumed

Figure 4: Mean Strain value of testis in Infertile and Fertile

population

Testicular Strain value 1.8 1.68

1.6 1.38 1.4 1.2 1 0.8 0.6 Strain value 0.4

Mean Strain valueStrain Mean 0.2 0 Infertile Fertile Group

Table 5: Strain ratio of testis in Infertile and Fertile population

Group Statistics

Std. Std. Error

GROUP N Mean Deviation Mean

SR Infertile 41 .366 .077 .012

Fertile 41 .220 .090 .014

Independent Samples Test

t-test for Levene's Test for Equality of Equality of Variances Means

F Sig. t df

SR Equal variances .924 .339 7.816 80 assumed

Equal variances 7.816 78.114

not assumed

Independent Samples Test

t-test for Equality of Means

95% Confidence Interval of the Difference Sig. (2- Mean Std. Error tailed) Difference Difference Lower

SR Equal variances .000 .146 .018 .108 assumed

Equal variances .000 .146 .018 .108 not assumed

Figure 5: Mean Strain ratio of testis in Infertile and Fertile

population

Testicular Strain Ratio

0.4

0.35 0.3 0.25 0.2 0.366 0.15 0.221 0.1

Mean Strain ratio Strain Mean 0.05 0 Infertile Fertile Group

Figure 6: Area under the ROC curve (AUC) for Testicular volume

TV 100

40 Sensitivity

0 0 20 40 60 80 100 100-Specificity

Table 6:Area under the ROC curve (AUC) for Testicular volume

Area under the ROC curve (AUC) 0.618

Standard Errora 0.0654

95% Confidence intervalb 0.490 to 0.746

z statistic 1.806

Significance level P (Area=0.5) 0.0709

Figure 7: Area under the ROC curve (AUC) for Resistive index of

intraparenchymal artery

RI 100

40 Sensitivity

0 0 20 40 60 80 100 100-Specificity

Table 7:Area under the ROC curve (AUC) for Resistive index of

intraparenchymal artery

Area under the ROC curve (AUC) 0.551

Standard Errora 0.0653

95% Confidence intervalb 0.423 to 0.679

z statistic 0.774

Significance level P (Area=0.5) 0.4390

Figure 8: Area under the ROC curve (AUC) for Strain value of testis

SV 100

40 Sensitivity

0 0 20 40 60 80 100 100-Specificity

Table 8:Area under the ROC curve (AUC) for Strain value of testis

Area under the ROC curve (AUC) 0.708

Standard Errora 0.0614

95% Confidence intervalb 0.587 to 0.828

z statistic 3.380

Significance level P (Area=0.5) 0.0007

Figure 9: Area under the ROC curve (AUC) for Strain ratio of testis

SR 100

40 Sensitivity

0 0 20 40 60 80 100 100-Specificity

Table 9: Area under the ROC curve (AUC) for Strain ratio of testis

Area under the ROC curve (AUC) 0.875

Standard Errora 0.0443

95% Confidence intervalb 0.788 to 0.962

z statistic 8.460

Significance level P (Area=0.5) <0.0001

Figure 10: Comparison of ROC curves of Testicular volume (TV),

Resistive index (RI), Strain value (SV) and Strain ratio (SR)

100

40 Sensitivity TV 20 RI SV SR 0 0 20 40 60 80 100 100-Specificity

Table 10: Pairwise comparison of ROC curves of Testicular volume

(TV), Resistive index (RI), Strain value (SV) and Strain ratio (SR)

TV ~ RI Difference between areas 0.0675 Standard Errorc 0.0926 95% Confidence Interval -0.114 to 0.249 z statistic 0.729 Significance level P = 0.4661 TV ~ SV Difference between areas 0.0895 Standard Errorc 0.0891 65

95% Confidence Interval -0.0850 to 0.264 z statistic 1.005 Significance level P = 0.3148 TV ~ SR Difference between areas 0.257 Standard Errorc 0.0796 95% Confidence Interval 0.101 to 0.413 z statistic 3.230 Significance level P = 0.0012 RI ~ SV Difference between areas 0.157 Standard Errorc 0.0987 95% Confidence Interval -0.0364 to 0.351 z statistic 1.591 Significance level P = 0.1116 RI ~ SR Difference between areas 0.325 Standard Errorc 0.0842 95% Confidence Interval 0.159 to 0.490 z statistic 3.853 Significance level P = 0.0001 SV ~ SR Difference between areas 0.167 Standard Errorc 0.0442 95% Confidence Interval 0.0808 to 0.254 z statistic 3.789 Significance level P = 0.0002

Table 11: Comparison of Mean Testicular volumes in various Sperm

disorders

95% Confidence Interval for Mean

Std. Std. Lower Upper Group N Mean Deviation Error Bound Bound

Oligozoospermia 6 15.220 2.299 .938 12.807 17.634

Azoospermia 9 15.111 3.529 1.176 12.397 17.824

Asthenozoospermia 14 14.304 3.096 .827 12.516 16.092

Both Oligozoospermia 12 11.756 3.812 1.100 9.334 14.179 and Asthenozoospermia

Total 41 13.869 3.506 .547 12.763 14.976

ANOVA

Sum of Mean Squares df Square F Sig.

Between 81.046 3 27.015 2.434 .080 Groups

Within 410.679 37 11.099

Groups

Total 491.725 40

Figure 11:Mean plot of Testicular volumes in various Sperm disorders

1 – Oligozoospermia

2 – Azoospermia

3- Asthenospermia

4 – Both Oligozoospermia and Asthenozoospermia

Table 12: Comparison of Mean Resistive index in various Sperm disorders

95% Confidence Interval for Group Mean

Std. Std. Lower Upper N Mean Deviation Error Bound Bound

Oligozoospermia 6 .404 .055 .022746 .34570 .46264

Azoospermia 9 .433 .060 .020207 .38674 .47993

Asthenozoospermia 14 .440 .040 .010773 .41708 .46363

Both 12 .477 .057 .016543 .44067 .51349 Oligozoospermia and Asthenozoospermia

Total 41 .444 .056 .008766 .42655 .46199

ANOVA

Sum of Mean Squares df Square F Sig.

Between .024 3 .008 2.880 .049 Groups

Within .102 37 .003

Groups

Total .126 40

Figure 12: Mean plot of Resistive index in various Sperm disorders

1 – Oligozoospermia

2 – Azoospermia 3- Asthenospermia

4 – Both Oligozoospermia and Asthenozoospermia

Table 13: Comparison of Mean Strain value of testis in various

Sperm disorders

95% Confidence Interval for Group Mean

Std. Std. Lower Upper N Mean Deviation Error Bound Bound

Oligozoospermia 6 1.639 .472 .192 1.14354 2.13479

Azoospermia 9 1.830 .360 .120 1.55265 2.10735

Asthenozoospermia 14 1.480 .303 .081 1.30500 1.65571

Both 12 1.823 .351 .101 1.60016 2.04651 Oligozoospermia and Asthenozoospermia

Total 41 1.680 .378 .059 1.56127 1.80020

ANOVA

Sum of Mean Squares df Square F Sig.

Between 1.017 3 .339 2.661 .062 Groups

Within 4.713 37 .127

Groups

Total 5.730 40

Figure 13: Mean plot of Strain value of testis in various Sperm disorders

1 – Oligozoospermia

2 – Azoospermia

3- Asthenospermia

4 – Both Oligozoospermia and Asthenozoospermia

Table 14: Comparison of Mean Strain ratio of testis in various Sperm disorders

95% Confidence Interval for Group Mean

Std. Std. Lower Upper N Mean Deviation Error Bound Bound

Oligozoospermia 6 .363 .068 .027 .291 .434

Azoospermia 9 .341 .061 .020 .294 .389

Asthenozoospermia 14 .348 .057 .015 .315 .381

Both 12 .409 .101 .029 .345 .473 Oligozoospermia and Asthenozoospermia

Total 41 .366 .077 .012 .342 .391

ANOVA

Sum of Mean Squares df Square F Sig.

Between .033 3 .011 1.917 .144 Groups

Within .209 37 .006

Groups

Total .242 40

Figure 14: Mean plot of Strain ratio of testis in various Sperm disorders

1 – Oligozoospermia

2 – Azoospermia

3- Asthenospermia

4 – Both Oligozoospermia and Asthenozoospermia

Among the total 82 patients, the mean age group in the infertile group

(Group 1) was 31.68 ± 3.78 and in the fertile group (Group 2) was 21.68

±3.18 (Table 1).

The mean testicular volume in Group 1was 13.87 ± 3.50 and Group 2 was

14.99 ± 1.98 (Table 2 and Figure 2).

The mean resistive index of the intraparenchymal artery inGroup 1was

0.44 ± 0.056 and in Group 2was 0.43 ± 0.042 (Table 3 and Figure 3).

The mean testicular strain value in Group 1 was 1.68 ± 0.37 and inGroup

2was 1.38 ± 0.525 (Table 4 and Figure 4).

The mean testicular strain ratio in Group 1 was 0.36 ± 0.07 and inGroup 2 was 0.22 ± 0.09 (Table 5 and Figure 5).

Area under the ROC curve (AUC) for testicular volume was 0.618 with a

P value of 0.0709(Table 6 and Figure 6).

Area under the ROC curve (AUC) for resistive index of the intraparenchymal artery was 0.551 with a P value of 0.439 (Table 7 and

Figure 7).

Area under the ROC curve (AUC) for testicular strain value was 0.708 with a P value of 0.0007 (Table 8 and Figure 8).

Area under the ROC curve (AUC) for testicular strain ratio was 0.875 with a P value of <0.0001 (Table 9 and Figure 9).

On comparing the various sperm disorders, the mean testicular volume in patients with oligozoospermia, azoospermia, asthenozoospermia and in 75 patients with both oligo and asthenozoospermia were 15.220 ± 2.299,

15.111 ± 3.529, 14.304 ± 3.096 and 11.756 ± 3.812 respectively (Table

11 and Figure 11).

The mean resistive index of intraparenchymal arteries in patients with oligozoospermia, azoospermia, asthenozoospermia and in patients with both oligo and asthenozoospermia were .40 ± .055, .43 ± .060, .44 ±

.040, .47 ± .057 respectively (Table 12 and Figure 12).

The mean strain value of testis in patients with oligozoospermia, azoospermia, asthenozoospermiaand in patients with both oligo and asthenozoospermia were 1.63 ± .472, 1.83 ± .360, 1.48 ± .303, 1.82 ±

.351 respectively (Table 13 and Figure 13).

The mean strain ratio of testis in patients with oligozoospermia, azoospermia, asthenozoospermia and in patients with both oligo and asthenozoospermia were .363 ± .068, .341 ± .061, .348 ± .057, .409 ±

.101 respectively (Table 14 and Figure 14).

DISCUSSION

The mean testicular volume was higher in Group 2 compared to Group 1.

This implies that higher the testicular volume, more the number of seminiferous tubules, which in turn produces more sperm. Therefore, the testicular volume is directly proportional to the sperm count. However the testicular volume was not significantly different between the groups.

A previous study by Pinggera et al. 35 showed that there was increased vascular resistance in those patients with abnormal sperm features compared to the normal patients. It also stated that there was no significant difference in the testicular volumes.

A study by Biogiotti et al.36 showed that the RI and peak systolic velocity

(PSV) were significantly higher in infertile men with varicocele and the

RI values were different between azoospermic and oligozoospermic patients.

Study by Ackar et al.37 showed that intra-testicular arterial resistance and testicular volume did not differ between infertile men with subclinical varicoceles and infertile men without varicoceles. 77

Present study showed that resistive index of the intraparenchymal arteries did not show any difference between the groups. Other studies also showed similar results38.

A study by Faruk et al.39 showed that testicular volumes were directly proportional with SR. But our study did not show a significant variation of the testicular volume with SR. It also showed that strain values are inversely related with the sperm concentration and sperm counts in infertile men, which was in agreement with the result of our study.

The testicular SV and SRs showed a significant difference between the groups. SR was higher in group 1 compared to group 2.

The ROC curve analysis showed that SR had the maximum AUC and showed a significant difference between the 2 groups with a P value of

<0.0001.

We also studied the different subgroups under group 1 and showed no significant difference in the elastography scores between different sperm abnormalities.

A study by Min Li et al. showed that SR were significantly different between nonobstructive and obstructive azoospermia patients. In our study, we did not categorize the patients as obstructive or nonobstructive azoospermia.

Ashok agarwal et al.40 stated that varicocele was associated with reduced sperm count, motility and morphology, but not the semen volume. In our study, we excluded all the patients with varicocele to eliminate this difference.

In our study, we could not find out any correlation between the elastography scores and the FSH values.

CONCLUSION

 Testicular volume was not significantly different between infertile

and fertile men.

 Resistive index of the intraparenchymal arteries did not show any

difference between infertile and fertile men.

 The strain value and strain ratios were significantly higher in

infertile men with abnormal semen parameters as compared to

those with normal semen analysis, and the strain elastography

results were found to be significantly different in patients with

abnormal sperm counts.

 This technique therefore proves to be a useful tool for the

evaluation of male infertility. But further large scale studies may be

needed to clarify the value of this imaging modality in the

assessment of male infertility.

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ABBREVIATIONS

FSH – Follicle stimulating hormone

MRI – Magnetic resonance imaging

RF – Radiofrequency

SE – Strain elastography

ARFI – Acoustic radiation force impulse

ROI – Region of interest

RI – Resistive index

SV – Strain value

SR – Strain ratio

TV – Testicular volume

ROC – Receiver operator characteristic

AUC – Area under curve

PROFORMA

 Name:

 Age:

 Address:

 Mobile number:

 Years of married life:

 Semen analysis: Sperm count:

Sperm motility:

Sperm morphology:

 Testicular volume:  Hormonal ( FSH) analysis:

 Findings in Elastography: Strain value:

Strain ratio:

PATIENT CONSENT FORM

Study detail :

“TESTICULAR STRAIN ELASTOGRAPHY IN FERTILE AND INFERTILE MEN- A COMPARATIVE CROSS SECTIONAL STUDY”

Study Centre : Govt. Kilpauk Medical College Hospital,

Govt Royapettah hospital, Chennai

Patients Name :

Patients Age :

Identification Number :

Patient may check ( ) these boxes

I confirm that I have understood the purpose of procedure for the above study. I had the opportunity to ask question and all my questions and doubts have been answered to my complete satisfaction.

I understand that my participation in the study is voluntary and that I am free to withdraw at any time without giving reason, without my legal rights being affected.

I understand that sponsor of the clinical study, others working on the sponsor’s behalf, the ethics committee and the regulatory authorities will not need my permission to look at my health records, both in respect of current study and any further research that may be conducted in relation to it, even if I withdraw from the study I agree to this access. However, I understand that my identify will not be revealed in any information released to third parties or published, unless as required under the law. I agree not to restrict the use of any data or results that arise from this study.

I hereby make known that I have fully understood the use of above procedure, the possible complications arising out of its use and the same was clearly explained to me.

I agree to take part in the above study and to comply with the instruction given during the study and faithfully cooperate with the study team and to immediately inform the study staff if I suffer from any deterioration in my health or well-being or any unexpected or unusual symptoms.

I hereby consent to participate in this study.

I hereby give permission to undergo complete clinical examination and diagnostic tests including hematological, biochemical, radiological tests.

Signature / thumb impression :

Patients Name and Address : Place Date

Signature of Investigator :

Study Investigator’s Name : Place Date

PATIENT INFORMATION LEAFLET

“TESTICULAR STRAIN ELASTOGRAPHY IN FERTILE AND INFERTILE MEN- A COMPARATIVE CROSS SECTIONAL STUDY”

Study place – Govt. Kilpauk Medical College Hospital, Govt. Royapettah hospital, Chennai.

We wish you to take part in a research study.

 Before you decide whether to take part, it is important for you to understand why research is being done and what it will involve.  Please take time to read the following information carefully. Discuss it with friends and relatives. Take time to decide whether you want to participate in the study or not.  You are free to decide whether or not to take part in this research study. If you choose not to participate, this will not affect the care you get from your doctors.  Ask us if there is anything that is not clear or if you want more information.  Thank you for reading this information. If you decide to take part you will be given a copy of this information sheet and your signed consent form.

Important things you need to know.

 We want to compare testicular elastography findings in fertile and infertile men.  This will not affect your treatment.  You can stop taking part in the study anytime you want, without giving a reason

Sperm _coun Morph t_milli Motili ology_i AG on_ml ty_% ndex_ Volum Viscosi NAME E _ _ %_ e_ml_ ty pH RT LT TV RT LT RI RT LT SV RT LT SR MUNUSAMY 28 5 45 20 2.5 Normal Alkaline 12.8 13.6 13.2 0.48 0.41 0.445 1.84 1.75 1.8 0.37 0.56 0.465 VISHAL 28 15 50 30 2.5 Normal Alkaline 16.7 14.9 15.8 0.39 0.42 0.405 1.4 0.9 1.15 0.42 0.37 0.395 RAVISHANKAR 29 10 55 25 2 Normal Alkaline 14.6 15.9 15.3 0.43 0.39 0.41 1.87 1.69 1.78 0.41 0.38 0.395 RAJA 30 9 50 60 1.6 Normal Alkaline 14.8 11.9 13.4 0.33 0.3 0.315 1.27 0.87 1.07 0.25 0.36 0.305 DILIPKUMAR 32 10 44 30 1.5 Normal Alkaline 16.9 11.7 14.3 0.36 0.39 0.375 2 2.7 2.35 0.32 0.24 0.28 ARUL 32 8 44 40 2.5 Normal Alkaline 20.6 18.3 19.4 0.54 0.41 0.475 2.48 0.9 1.69 0.43 0.25 0.34 MAGIZHSAMY 29 0 1.5 Normal Alkaline 10.1 9.5 9.8 0.42 0.41 0.415 1.94 1.86 1.9 0.32 0.24 0.28 SURESH 30 0 1.5 Normal Alkaline 20.4 16.2 18.3 0.23 0.41 0.32 2.35 2.15 2.25 0.43 0.4 0.415 RAMIZ 30 0 1 Normal Alkaline 18.7 18 18.3 0.41 0.42 0.415 1.67 1.98 1.83 0.43 0.32 0.375 GANESH 31 0 1.5 Normal Alkaline 12.6 11.8 12.2 0.38 0.36 0.37 1.96 2.16 2.06 0.38 0.28 0.33 SATHISH 32 0 1 Normal Alkaline 18 18.7 18.3 0.41 0.46 0.435 1.78 1.85 1.82 0.31 0.34 0.325 SUGUMAR 32 0 1 Normal Alkaline 14.1 12.5 13.3 0.51 0.47 0.49 1.3 1.9 1.6 0.23 0.29 0.26 ANTHONY 34 0 1.5 Normal Alkaline 20.7 16.5 18.6 0.51 0.43 0.47 1.87 2.12 2 0.32 0.29 0.305 IMAM 34 0 1.5 Normal Alkaline 10.5 11.8 11.2 0.51 0.45 0.48 0.8 1.2 1 0.24 0.43 0.335 SHANKAR 36 0 1 Normal Alkaline 16.3 15.6 16 0.56 0.45 0.505 1.4 2.65 2.03 0.25 0.65 0.45 MOHAN RAJ 24 42 7 20 1 Normal Alkaline 12.2 8.35 10.3 0.41 0.38 0.395 2.13 1.38 1.76 0.4 0.25 0.325 SUNDARA VADIVEL29 25 5 45 1 Normal Alkaline 20.2 16.9 18.6 0.33 0.58 0.455 1.4 1.34 1.37 0.42 0.35 0.385 RAMKI 29 52 18 10 2.5 Normal Alkaline 9.7 11.9 10.8 0.54 0.49 0.515 1.7 1.3 1.5 0.4 0.34 0.37 MEENAKSHI 29 20 15 20 1 Normal Alkaline 14.1 14.9 14.5 0.43 0.45 0.44 1.83 1.75 1.79 0.36 0.32 0.34 MANIKANDAN 30 50 15 10 1.5 Normal Alkaline 19.9 15.5 17.7 0.42 0.49 0.455 0.95 0.98 0.97 0.35 0.4 0.375 MUTHU 30 56 38 20 1.5 Normal Alkaline 19.5 13.3 16.4 0.49 0.42 0.455 1.3 1.7 1.5 0.45 0.36 0.405 NAVEEN 30 45 4 40 2 Normal Alkaline 21.4 20.5 20.9 0.52 0.44 0.48 1.99 1.86 1.93 0.42 0.36 0.39 LOGESH 30 55 4 40 3.5 Normal Alkaline 12.8 10.8 11.8 0.39 0.41 0.4 1.4 1.5 1.45 0.38 0.27 0.325 RAM 31 60 8.48 30 5.4 Normal Alkaline 11.5 13.2 12.3 0.45 0.44 0.445 1.52 1.8 1.66 0.3 0.3 0.3 PERIYASAMY 32 60 10 50 2.5 Normal Alkaline 14.1 15.5 14.8 0.39 0.32 0.355 1.3 0.9 1.1 0.23 0.36 0.295 YUVARAJ 33 80 30 30 3 Normal Alkaline 12.2 12.3 12.3 0.47 0.47 0.47 0.9 1.4 1.15 0.43 0.5 0.465 EZHUMALAI 33 48 8 40 1.5 Normal Alkaline 13.8 12.5 13.2 0.38 0.43 0.405 1.98 1.76 1.87 0.32 0.28 0.3 KARTHICK 34 50 8 50 2 Normal Alkaline 14.9 12.9 13.9 0.43 0.45 0.44 0.9 1.4 1.15 0.37 0.36 0.365 ASHIK 37 35 8 55 1.5 Normal Alkaline 12.2 13.5 12.9 0.53 0.38 0.455 0.9 2.18 1.54 0.23 0.24 0.235 DILLIBABU 26 9.54 29.5 60 2 Normal Alkaline 15.6 13.8 14.7 0.49 0.49 0.49 2.12 1.87 2 0.49 0.44 0.465 MUTHUKUMAR 28 3 11 50 1.5 Normal Alkaline 9.74 8 8.87 0.5 0.49 0.495 1.63 1.43 1.53 0.33 0.33 0.33 MANIKANDAN 28 10 30 40 2 Normal Alkaline 16.7 18.1 17.4 0.39 0.44 0.415 2.42 1.79 2.11 0.38 0.35 0.365 BHARATH 29 5 10 20 2 Normal Alkaline 12.8 15.2 14 0.42 0.39 0.405 2.1 2.33 2.22 0.41 0.36 0.385 ABDUL 29 15 10 20 2 Normal Alkaline 13.7 12.7 13.2 0.44 0.46 0.45 2.1 2.2 2.15 0.44 0.38 0.41 RAVI 33 11 5 40 1.5 Normal Alkaline 11.6 13.6 12.6 0.47 0.52 0.495 1.75 1.98 1.87 0.41 0.4 0.405 ANDRALEVIS 34 10 25 20 1.5 Normal Alkaline 13.9 12.1 13 0.45 0.45 0.45 1.43 1.3 1.37 0.25 0.33 0.29 MANIKATHAN 36 15 6 15 1.5 Normal Alkaline 7.2 10.9 9.05 0.38 0.44 0.41 1.86 1.54 1.7 0.46 0.4 0.43 MADHAN 37 10 5 10 2 Normal Alkaline 5.7 5.47 5.59 0.49 0.52 0.505 1.65 1.25 1.45 0.93 0.43 0.68 PARTHIBAN 38 15 5 20 1.5 Normal Alkaline 13.1 9.8 11.5 0.58 0.56 0.57 1.95 1.86 1.91 0.36 0.24 0.3 YUVENDRAN 41 15 10 20 1.5 Normal Alkaline 5.47 5.4 5.44 0.63 0.53 0.58 2.38 2.25 2.32 0.43 0.45 0.44 SATHISH KUMAR42 15 30 30 2 Normal Alkaline 19.5 12.1 15.8 0.45 0.47 0.46 0.95 1.63 1.29 0.23 0.6 0.415 Surya 21 45 42 40 2 Normal Alkaline 17.4 14.8 16.1 0.48 0.43 0.455 1 2.42 1.71 0.3 0.46 0.38 Sundar 21 42 42 40 3.2 Normal Alkaline 14.5 13.5 14 0.38 0.42 0.4 1.2 1.56 1.38 0.13 0.18 0.155 Manikandan 25 32.4 44 30 2 Normal Alkaline 15.6 14.6 15.1 0.35 0.37 0.36 1.38 0.8 1.09 0.23 0.15 0.19 Raj 26 36.7 56 40 2.5 Normal Alkaline 16.1 16.5 16.3 0.62 0.52 0.57 0.88 0.93 0.91 0.15 0.18 0.165 Isravel 26 43.8 47 55 1.5 Normal Alkaline 21.4 20.4 20.9 0.45 0.54 0.495 0.89 1.34 1.12 0.16 0.18 0.17 Raman 27 37.1 53 30 2 Normal Alkaline 12.6 14.7 13.7 0.37 0.46 0.415 1.34 0.76 1.05 0.24 0.15 0.195 Raja 27 44.3 51 30 2 Normal Alkaline 18.4 18.6 18.5 0.41 0.45 0.43 0.88 0.96 0.92 0.12 0.18 0.15 Baskar 28 50 48 55 3.5 Normal Alkaline 13.8 14.6 14.2 0.39 0.42 0.405 2.45 2.95 2.7 0.57 0.23 0.4 Vasu 28 44 53 30 2.5 Normal Alkaline 12.8 13.6 13.2 0.47 0.45 0.46 0.98 0.95 0.97 0.17 0.15 0.16 David 28 37.6 51 40 2 Normal Alkaline 12.8 16.1 14.5 0.37 0.45 0.41 2.1 2.45 2.28 0.34 0.13 0.235 Dinesh 28 52.6 42 50 3 Normal Alkaline 13.8 12.5 13.2 0.38 0.45 0.415 1.23 1.54 1.39 0.18 0.25 0.215 Dhanavel 28 42.6 48 40 4 Normal Alkaline 15.4 13.6 14.5 0.45 0.45 0.45 0.89 0.96 0.93 0.15 0.18 0.165 Veeramani 28 38.4 47 20 2.5 Normal Alkaline 15.3 14.7 15 0.39 0.4 0.395 0.88 1.35 1.12 0.21 0.14 0.175 Prabhakaran 29 37 51 60 2 Normal Alkaline 13.6 12.6 13.1 0.39 0.54 0.465 1.35 0.9 1.13 0.24 0.14 0.19 Ezhumalai 29 42 41 60 2.5 Normal Alkaline 16.4 16.9 16.7 0.39 0.45 0.42 1.34 1.82 1.58 0.15 0.16 0.155 Venkatesh 29 64 55 20 3 Normal Alkaline 15.4 17.3 16.4 0.42 0.39 0.405 2.12 2.34 2.23 0.45 0.43 0.44 Anandhan 29 48.5 52 25 2.5 Normal Alkaline 14.2 15.6 14.9 0.38 0.39 0.385 1.67 1.95 1.81 0.21 0.18 0.195 Rajendran 29 38.9 48 30 1.5 Normal Alkaline 16.2 18.1 17.2 0.43 0.47 0.45 0.98 1.25 1.12 0.14 0.21 0.175 Sabari 29 38.9 48 40 2 Normal Alkaline 15.8 15.4 15.6 0.52 0.51 0.515 1.34 1.12 1.23 0.17 0.25 0.21 Abdul 29 41.4 56 25 2 Normal Alkaline 10.2 10.3 10.3 0.43 0.38 0.405 2.45 2.16 2.31 0.48 0.35 0.415 Kuppusamy 30 44 49 40 3.5 Normal Alkaline 13.2 15.8 14.5 0.41 0.51 0.46 0.89 1.34 1.12 0.15 0.11 0.13 Sekar 30 54.8 47 40 1.5 Normal Alkaline 13.6 14.8 14.2 0.45 0.38 0.415 0.78 0.95 0.87 0.25 0.19 0.22 Annamalai 30 37.5 56 30 3.5 Normal Alkaline 14.8 12.4 13.6 0.38 0.45 0.415 0.95 0.79 0.87 0.15 0.24 0.195 Kishore 30 44.6 43 30 1.5 Normal Alkaline 13.5 12.8 13.2 0.45 0.45 0.45 0.79 0.88 0.84 0.15 0.18 0.165 Rajesh 31 55 40 60 2.5 Normal Alkaline 14.6 18.6 16.6 0.54 0.47 0.505 0.98 0.85 0.92 0.14 0.21 0.175 Karupusamy 31 46.1 48 40 2.6 Normal Alkaline 19.4 16.8 18.1 0.48 0.38 0.43 0.88 0.95 0.92 0.19 0.12 0.155 Subash 31 39.7 60 45 2 Normal Alkaline 14.5 13.6 14.1 0.42 0.53 0.475 0.96 0.89 0.93 0.15 0.11 0.13 Elangovan 31 42.1 50 35 2.5 Normal Alkaline 14.6 12.8 13.7 0.42 0.39 0.405 0.89 0.95 0.92 0.12 0.24 0.18 Nasar 31 56.8 51 60 2.5 Normal Alkaline 15.5 16.6 16.1 0.39 0.45 0.42 1.45 0.89 1.17 0.12 0.19 0.155 Mohammed 32 38 48 20 2.4 Normal Alkaline 13.5 14.2 13.9 0.46 0.45 0.455 1.65 1.98 1.82 0.32 0.23 0.275 Selvam 32 60 54 50 2.5 Normal Alkaline 15.5 15.8 15.7 0.48 0.38 0.43 1.23 0.96 1.1 0.15 0.12 0.135 Hariharan 32 45.7 40 50 3.5 Normal Alkaline 12.8 14.3 13.6 0.42 0.45 0.435 2.1 2.56 2.33 0.34 0.21 0.275 Kannan 32 46.7 48 35 3 Normal Alkaline 13.5 15.1 14.3 0.41 0.38 0.395 2.34 2.16 2.25 0.54 0.34 0.44 Ragunathan 32 44.8 52 40 2.5 Normal Alkaline 14.5 13.6 14.1 0.45 0.45 0.45 0.79 1.23 1.01 0.15 0.23 0.19 Babu 33 52.1 47 50 2 Normal Alkaline 13.5 14.6 14.1 0.51 0.54 0.525 1.38 1.32 1.35 0.15 0.17 0.16 Marthandam 33 39.5 41 50 3.5 Normal Alkaline 12.8 14.5 13.7 0.52 0.49 0.505 1.23 1.24 1.24 0.2 0.13 0.165 Arun 34 36 47 40 2 Normal Alkaline 17.6 15.4 16.5 0.39 0.45 0.42 0.95 1.1 1.03 0.3 0.15 0.225 Marimuthu 34 45.8 51 25 2.5 Normal Alkaline 16.5 15.8 16.2 0.41 0.42 0.415 2.32 2.12 2.22 0.42 0.35 0.385 Akhil 34 46.2 47 40 2 Normal Alkaline 13.6 14.1 13.9 0.41 0.39 0.4 1.45 1.25 1.35 0.17 0.23 0.2 Kumar 35 45 53 30 2 Normal Alkaline 19.4 19.6 19.5 0.41 0.44 0.425 2.1 2.15 2.13 0.43 0.32 0.375 Dhayanathan 35 51.2 61 20 3 Normal Alkaline 12.3 13.1 12.7 0.43 0.46 0.445 1.24 1.45 1.35 0.23 0.15 0.19