1. Infertility Problem and Its Management
Infertility affects 1 in 10 couples who want children. Fertility declines in both sexes after the
ages of 30-35, and because more and more couples are delaying starting a family until their 30s,
infertility is becoming more common in the developed world. If conception has not occurred
after a year of unprotected, regular sex, one or both partners may be having a fertility problem.
Female infertility:
About half of couples who experience difficulties conceiving do so as a result of female
infertility. Fertility in women decreases with age and is generally lower by age 35, making
conception more difficult for women over this age.
For conception to occur, all of the following steps must take place: ovulation (the production
and release of a mature egg by an ovary), fertilization of the egg by a sperm, transport of the
fertilized egg along the fallopian tube to the uterus, and implantation of the fertilized egg in the
lining of the uterus. If any stage is interrupted or does not occur, conception cannot take place.
What are the causes?
There are a number of fertility problems in females that may affect one or more of the processes
required for conception. The problems can develop at different stages of conception.
Problems with ovulation:
A common cause of female infertility is the ovaries’ failure to release a mature egg during
every monthly cycle. Ovulation is controlled by a complex interaction of hormones produced by
the hypothalamus (an area of the brain), the pituitary and thyroid glands, and the ovaries. A
common and treatable cause of female infertility is polycystic ovary syndrome which may cause
a hormone imbalance that prevents ovulation from taking place. Disorders of the thyroid gland,
such as hypothyroidism, may also cause a hormonal imbalance that can affect the frequency of
ovulation. Pituitary gland disorders, such as prolactinoma, a non cancerous tumor, may cause a
similar imbalance. In some women, ovulation does not always occur, for reasons that are
unclear. In some cases, women who have been using oral contraceptives for a number of years
may take time to reestablish a normal hormonal cycle after discontinuing them. Excessive
exercise, stress and obesity or low body weight may affect hormone levels and cause temporary
infertility.

2. Premature menopause also results in a failure to ovulate. It can occur with no apparent cause or
may be the result of surgery, chemotherapy or radiation therapy. In rare cases, the ovaries do not
develop normally due to a chromosomal abnormality, such as Turner syndrome.
Problems with egg transport and fertilization:
The passage of the egg from the ovary to the uterus may be impeded by damage to one of the
fallopian tubes. This damage may be due to pelvic infection, which may in turn result from a
sexually transmitted disease such as chlamydial cervicitis. Such infections may exist with no
symptoms and may be detected only if you have difficulty conceiving.
Endometriosis, a condition that can lead to the formation of scar tissue and cysts within the
pelvis, may also damage the fallopian tubes, preventing the passage of an egg.
In some women, the egg cannot be fertilized because the mucus produced naturally by the cervix
contains antibodies that destroy the partner’s sperm before they reach the egg.
Problems with implantation:
If the lining of the uterus has been damaged by an infection, such as gonorrhea, the implantation
of a fertilized egg may not be possible. Hormonal problems may also result in the uterine lining
not being adequately prepared for successful implantation. Non cancerous tumors that distort the
uterus and rarely, structural abnormalities present from birth may make it impossible for a
fertilized egg to embed itself in the uterine lining.
What might be done?
Your doctor will ask you about your general state of health, your lifestyle, you’re medical and
menstrual history, and your sex life before recommending particular tests and treatments.
Most causes of female infertility can now be identified through testing. You can find out if
and when you ovulate by using an ovulation prediction kit, available over the counter, or by
recording your body temperature daily. If your doctor suspects that you are not ovulating
regularly, you may have repeated blood tests during your menstrual cycle to assess the level of
the hormone progesterone (which normally rises after ovulation).Repeated ultrasound scanning
of the ovaries during the cycle may also be done to check if and when ovulation occurs. In
addition, a tissue sample may be taken from the uterus and examined for abnormalities.
If tests show that you are not ovulating, you may need further blood tests to check the levels of
thyroid hormones and other hormones, and drugs may be prescribed to stimulate ovulation.
However, if you are ovulating, the next step is to find out whether your partner is producing
sufficient normal sperm by analyzing two or more semen samples.
If you are ovulating normally and your partner’s sperm are normal, your doctor will check if
there is a problem preventing the egg and sperm from meeting. For example, you and your
partner may be asked to have sex during the time that you are ovulating so that a sample of your
cervical mucus (collected within a few hours of intercourse) can be tested for antibodies to
sperm. If analysis of the sample shows antibodies to sperm, there are several methods of

3. treatment. Corticosteroids may be prescribed to suppress the production of antibodies, or your
partner’s semen may be injected directly into your uterus to avoid contact with the mucus. If
these steps are not successful, your doctor may recommend assisted conception.
If the cause of the infertility has still not been found, your doctor may arrange for further
investigations to look for a blockage in the fallopian tubes or an abnormality of the uterus. One
such test is laparoscopy, in which an endoscope containing a camera is inserted through the
abdomen. Another is hysterosalpingography, in which a dye is injected through the cervix and X-
rays are taken as the dye enters the reproductive organs. The treatment depends on the problem.
For example, a tubal blockage may be corrected by microsurgery, and endometriosis may be
treated with drugs.
What is the prognosis?
Treatments for female infertility have greatly increased the chance of pregnancy. Success rates
vary, depending on the cause of the infertility and the type of the treatment. Fertility drugs
stimulate ovulation in 1 in 3 women, but there is a risk of multiple pregnancies. Microsurgery to
clear obstructed fallopian tubes is sometimes successful but increases the risk of ectopic
pregnancy. Success rates for assisted conception methods range from 15 to 30 percent per
individual treatment.
MALE INFERTILITY: In about 1 in 3 couples who have difficulty conceiving, the problem
results from male infertility. In males, fertility depends partly on the production of enough
normal sperm to make it likely that one will fertilize an egg and partly on the ability to deliver
the sperm into the vagina during sexual intercourse. If either of these factors is adversely
affected, infertility may result.
What are the causes?
Unlike the causes of female infertility, which are more easily identifiable, the cause of infertility
can be difficult to find in some men. A cause is discovered in only 1 in 3 men investigated.
Problems with sperm production:
A low sperm count or the production of abnormal sperm may have various causes. Normally,
the testis has a temperature of about 4 degree F (2 degree C) lower than the rest of the body. Any
factors that raise the temperature of the testis can reduce the number of sperm produced.
Aspects of your lifestyle that may impair sperm production include smoking, drinking alcohol,
using certain medications and recreational drugs, and even wearing tight clothing.
Sperm production can be adversely affected by some long-term illness, such as chronic kidney
failure and by some infections, such as mumps, that occur after puberty. Conditions affecting
the urethra, such as hypospadias, or the scrotum, such as a varicocele, may also reduce fertility.
In addition, fertility problems may occur if the testis is damaged by medical procedures such as
surgery, chemotherapy, or radiation therapy for disorders such as cancer of the testis.

4. Low sperm production may also be due to a hormonal or chromosomal deficiency. Insufficient
production of the sex hormone testosterone by the testes can cause a low sperm count. Since the
pituitary gland controls testosterone secretion, pituitary disorders, such as a tumor, may also lead
to reduced sperm production. Rarely, low testosterone levels are due to a chromosomal
abnormality such as K line felter syndrome. The most common cause of a low sperm count is
idiopathic oligospermia, in which there is a reduced sperm count for no identifiable reason.
There is no effective treatment.
Problems with sperm delivery:
A number of factors may prevent sperm from reaching the vagina. The most easily identifiable
factor is impotence - the inability to achieve or maintain an erection. Other factors include
damage to the Epididymis and vas deferens (tubes that transport sperm).Damage is often due to a
sexually transmitted disease such as gonorrhea. It may also be caused by retrograde ejaculation,
in which semen flows back into the bladder when the bladder valves do not close properly. This
condition can occur after prostate surgery.
What might be done?
Your doctor will ask about your health, medical history, and sex life and give you a physical
examination, including an examination of your genitals. You may also need to provide semen
samples. If your sperm count is low or your sperm are abnormal, further investigations will be
done, such as blood tests to check hormone levels. Treatment depends on the diagnosis. Low
testosterone levels can be treated with hormone injections. Artificial insemination may be used
in cases of impotence or retrograde ejaculation; for the latter, the sperm may be taken from
urine. Damage to the epididymis or vas deferens may be treated by microsurgery.
If you produce only a few healthy sperm, a sample may be taken from an epididymis or testis by
microsurgery. A process called intracytoplasmic sperm injection (ICSI) may then be used to
fertilize an egg with a single sperm.
What is the prognosis?
If the infertility is treatable, the chance of regaining fertility is high. With artificial insemination
the chance of conceiving in one menstrual cycle is about 10-15 percent, and the treatment usually
works within 6 months. Each attempt at assisted conception is successful in 15-30 percent of
cases, depending on the technique.
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5. Evaluation of the Male for Infertility
Infertility is a major health concern for a large proportion of reproductive age patients. The
purpose of this article is to give a concise but practical overview of the evaluation of the infertile
man because approximately 40 % of infertility cases involve male factors. Significant medical
pathology can now be uncovered by a comprehensive infertility evaluation of the man. Advances
in the understandings and diagnoses of male factor infertility is advancing at a rapid pace. The
man must not be ignored, and the following information is a guide to his evaluation.
Evaluation
The initial workup begins whenever the patient presents. This is predicted by the fact that the
longer a couple remains infertile the less chance there is for cure. A rapid, noninvasive, cost-
effective evaluation is essential.
History
The cornerstone of the evaluation of the infertile man is the history and physical examination.
Table1 outlines the complete pertinent history. The sexual history is paramount. Some of the
problems most commonly encountered in this patient population are related to the timing of
intercourse, with it being too frequent or too infrequent. The history of an undescended testicle is
significant. In a patient with a history of unilateral cryptorchidism, regardless of the time of
orchidopexy, overall semen quality is considerably less than that found in normal men. Bilateral
cyrptorchidism is extremely important. Progressive damage occurs to the germinal epithelium if
the testicle is not in its proper position in the scrotum. It has been shown that orchidopexy should
be performed prior to 2 years of age to maintain a significant level of spermatogenic function.
Any previous surgery of the retroperitoneum, bladder neck (prostate), pelvis, inguinal region, or
scrotum should be assessed. Any surgery on the bladder neck may cause retrograde ejaculation.
Inguinal surgery such as herniorrhaphy, undertaken when an infant or an adult, may have caused
vassal occlusion or vascular insufficiency to the testicule. Fever can cause impaired testicular
function. The ejaculate may not be affected for more than 3 months after the event, as
spermatogenesis takes about 74 days. Postpubertal mumps may cause mumps orchitis, which

6. results in an atrophic testis. Fifty percent of patients with testicular cancer have subnormal sperm
densities prior to therapy. A history of diabetes or multiple sclerosis should raise questions about
potency and ejaculatory function. Exposure to drugs and toxins should be detailed. The routine
use of hot tubs or saunas should be discontinued, as elevated temperatures impair
spermatogenesis. A family history of cystic fibrosis is important owing to associated vassal
agenesis and epididymal abnormalities. Finally, a history of anosmia (lack of smell) indicates the
possibility of hypogonadotropic hypogonadism. Galactorrhea, head-aches, and impaired visual
fields suggest the presence of a central nervous system tumor.
TABLE NO. 1
Infertility History
1) SEXUAL HISTORY
3) PAST SURGICAL HISTORY
1. Duration of infertility
1. Orchietomy
2. Previous treatments
2. Orchidopexy
3. Potency surgery
3. Retroperitoneal surgery
4. Timing and frequency of
4. Pelvic, inguinal or scrotal surgery
intercourse
5. Herniorrhaphy
2) PAST MEDICAL HISTORY 4) MEDICATIONS AND GONADOTOXINS
1. Undescended testicles 1. Chemotherapeutic agents
2. Testicular torsion / trauma 2. Therapeutic drugs
3. Delayed puberty 3. Chemicals (pesticides)
4. Pelvic injury 4. Recreational drugs :smoking,
5. Diabetes marijuana,cocaine
6. Previous or current therapy 5. Androgenic steroids
7. Viral and febrile illness history 6. Thermal exposure (hot tubs)
8. Postpubertal mumps orchitis 7. Radiation
9. Sexually transmitted diseases
10. Urinary tract infections
11. Cystic fibrosis, or family history of
it
Physical Examination
The physical examination must be thorough, with special attention to the genitalia. The penile
curvature and location of the urethral meatus should be assessed, as abnormalities may result in
improper delivery of the ejaculate. Testicular size and consistency must be recorded, with the
length measured with calipers and the volume estimated with an orchidometer. Size is an
important indicator of spermatogenic capability, as more than 80 % of the testis is involved in

7. sperm production. When there is damage to the testicular tubules, loss of mass occurs. The
normal length of the testis is about 4 cm and the volume more than 20 ml. Epididymal induration
and irregularities should be noted. The presence of a vas must be documented, as 2 % of infertile
men have congenital absence of the vas. Varicoceles, that is, dilated spermatic veins that present,
as a “bag of worms” above the testicle in the scrotum must be identified. A varicocele can cause
abnormalities in gonadal function. The scrotal contents should be palpated with the patient in
both the supine and standing positions. Many varicoceles are not visible and may be discernible
only when the patient stands or performs a Valsalva maneuver. Varicoceles often result in a
smaller testis on that side. Ninety percent are left-sided, and any discrepancy in size between the
two testes should arouse suspicion of a varicocele. A rectal examination is essential to assess
prostate size, evidence of infection, and the presence of midline cysts. Look carefully for signs of
hypogonadism, such as decreased body hair, gynecomastia, infantile genitalia, and decreased
muscular development.
Laboratory Evaluation
The laboratory is an integral part of a full-service infertility center. If an on-site laboratory is not
available, specimens must be analyzed by a dedicated infertility laboratory. Data from a
reputable laboratory are critical . Unfortunately, the semen analysis must be done locally because
the specimen must be evaluated shortly after production. Most other studies can be sent out to
any reputable laboratory.
Semen Analysis
The primary laboratory test is the semen analysis. It must be emphasized that semen analysis is
not a test for fertility. It does not separate patients into sterile and fertile groups; it does give
diagnostic information and allows a directed evaluation and treatment. At least two semen
analyses must be obtained to establish a baseline. The standard semen analysis allows evaluation
of semen volume, pH, density (sperm per milliliter), motility, measurement of forward
progression of sperm, and sperm morphology. The semen is examined also for evidence of sperm
agglutination, hyperviscosity, and the presence of white blood cells. The World Health
Organisation (WHO) range of values for normal semen analysis is given in Table No. 2
TABLE NO. 2
WHO(1999) CRITERIA FOR NORMAL SEMEN ANALYSIS
SEMEN PARAMETER VALUE
Volume 2.0 –5.0 ml
Density > 20 million/ml
Motility > 50 %
Forward progression > 2 (scale 1 – 4)
Morphology > 30% normal forms
Leukocytes < 1 million / ml
Agglutination None
Hyperviscosity None

8. Some laboratories use computer-assisted semen analyses, which are of some value for measuring
sperm motility, however, they should be used only as a source of supplemental information.
Attention has been turned to a more accurate manual analysis of sperm morphology.
Leukocytes In The Semen
Leukocytes in semen have significant effects on sperm function. They modulate an autoimmune
response, adversely affect motility and fertilizing capacity, and deter sperm transport in the
female reproductive tract. The semen of most men contains some immature sperm forms (round
cells), which ordinarily cannot be distinguished form white blood cells (WBCs). This often leads
to an erroneous diagnosis of pyospermia or infection. Semen cultures are not indicated in
asymptomatic patients, as they are essentially always negative. Routine cultures for a typical
organisms are unwarranted because they are not always accurate, are labor- and cost-prohibitive,
and have not been shown to have a clinical impact. For the few patients with symptoms of
urinary or genital tract infections cultures should be prepared. The specific cultures obtained
depend on the individuals ‘symptoms’ and examination but should include cultures of urine,
expressed prostatic secretions, and a postprostatic massage urine sample. Common sexually
transmitted organisms such as Chlamydia, Mycoplasma and Ureaplasma have been implicated in
reproductive failure. Patients with active prostaititis or other urinary tract infections frequently
have decreased sperm count and motility.
Fructose In The Semen
With low-volume oligospermia or low-volume azoospermia, one should be concerned about
retrograde ejaculation and ejaculatory duct obstruction. The assessment for ejaculatory duct
obstruction may incorporate a test for seminal fructose, a sugar produced in the seminal vesicles.
Its absence may indicate the possible absence of the seminal vesicles or obstruction of the
ejaculatory ducts.
Anti-Sperm Antibodies
The incidence of anti-sperm antibodies in the infertile man range from 8 % to 21 %. In men only
antibodies present on the sperm surface are clinically important. Anti-sperm antibodies have
implications at various stages in the fertilization process, that is, due to poor sperm penetration
into cervical mucus; impaired acrosome reaction and zona binding. Risk factors for the
development of sperm-bound antibodies include previous testicular surgery, trauma, or infection,
as does a history of torsion, cryptorchidism and genitourinary infections. Additionally,
obstructive azoospermia (possibly due to obstruction from a previous hernia repair, congenital
absence of the vas deferens, or vasectomy) can induce sperm autoimmunity.
Sperm Function Tests
Hypo-Osmotic Swelling Test (Hos Test) : The integrity of sperm cell plasma membrane is
essential for endowing fertilizing capability to the sperm. The assessment of plasma membrane
function is therefore useful indicator of healthy sperm. The test is based on the principle of hypo-
osmotic solutions being passively transferred across intact cell membranes. Sperm with
functionally intact cell membranes swell and their tails undergo coiling when exposed to hypo-

9. osmotic conditions. There is a high degree of correlation between the results of swelling test and
fertilizing capacity as measured by the sperm penetration assay.
Acrosome Intactness Test: The acrosome contains a number of enzymes which help human
spermatozoa penetrate the outer investments of the ovum. Several functional and ultrastructural
acrosomal defects that lead to male infertility have been reported. Acrosome Intactness Test
evaluates the functional status of the acrosome and serves as a good indicator of sperm’s ability
to penetrate the oocyte’s investments. The test is based on the ability of the proteolytic enzymes
of the acrosome to dissolve gelatin when sperm are placed over a gelatin coated slide.
Sperm Nuclear Chromatin Decondensation Test: One of the early events of fertilization
following the sperm penetration with the egg is the decondensation of the sperm nuclear
chromatin. Sperm with defective heads don’t decondense and are dysfunctional. This test helps
in determining the incidence of sperm with defective heads. The test is based on the principle of
sodium dodecyl sulphate (SDS) and ethyl diamine tetra acetic acid’s (EDTA) ability to permeate
the sperm head membrane and chelate the zinc protecting the disulfide linkages between the
nuclear proteins. Exposure of sperm to these compounds facilitates the in vitro decondensation of
nuclear chromatin and thus aids in identifying sperm whose nuclei lack the ability to
decondense.
Sperm Mitochondrial Activity Indices (Smai): Respiratory enzyme present in the
mitochondria provide energy for sperm motility. The presence of these enzymes can be identified
by the Nitroblue Tetrazolium (NBT) reaction. This dye when exposed to mitochondrial enzymes,
gets reduced and precipitates to form a blue black compound called formazan. The intensity of
the reaction and distribution of formazan are used to determine SMAI which is indicative of the
functional status of the sperm mitochondria. Lack of mitochondrial enzymes impair sperm
motility and may cause infertility.
Sperm-Cervical Mucus Interaction: The postcoital test assesses the sperm in the partner’s
cervical mucus and the interaction between the two. The test is performed just prior to ovulation.
A specimen of cervical mucus, obtained within a few hours of intercourse, is examined under a
microscope. More than 10 sperm per high power field, most of which demonstrate progressive
motility, constitutes a normal study. Indications for postcoital testing include hyperviscous
semen, unexplained infertility and low-volume semen with good sperm density. This test is
contraindicated for patients with poor quality semen specimens. Inherent poor reproducibility
and the fact that there are specimens from both parties make the study difficule to interpret. If an
abnormal result is obtained, an in vitro cervical mucus penetration test may be performed. These
tests have been developed to standardize and isolate semen factors.
Sperm Penetration Assay: The sperm penetration assay is a sophisticated test that measures the
physiologic ability of the human sperm to enter a zona-free hamster egg and begin the
fertilization reaction. The zona pellucida is the barrier to cross-species fertilization. When
hamster eggs are rendered zona-free and penetrated by human spermatozoa in vitro, they serve as
a substitute for human ova in a preliminary assessment of fertilizing capacity. For successful
penetration, sperm must be able to undergo capacitation, the acrosome reaction, fusion with the

10. oolema and incorporation into the ooplasm. Scoring is based on the percentage of ova penetrated,
or number of penetrations per ovum. The lower limit of normal is 10 – 30 % of ova penetrated.
Hormonal Screening
A brief review of male reproductive endocrine physiology is essential. The testes are dual
organs. There is an endocrine (hormonal) component consisting of Leydig cells, Sertoli cells, and
germ (sperm) cells. This component is necessary for male sexual differentiation and maturation,
normal potency and ejaculatory capability, and spermatogenic maturation. Endocrine and
spermatogenic compartments are anatomically and functionally integrated. Proper hormone
balance is initiated by a pulsatile hypothalamine release of gonadotropin releasing hormone
(GnRH). This causes pituitary release of follicle-stimulating hormone (FSH) and luteinizing
hormone (LH), which have a direct action on the testis. FSH acts on Sertoli cells to provide a
favorable milieu for spermatogenesis. LH stimulates the Leydig cell to secrete testosterone,
providing the locally high concentration required for spermatogenesis. Serum testosterone
reflects Leydig cell function and provides an indication for intratesticular testosterone. Table
No.3 depicts the various hormonal patterns and their corresponding clinical entities.
TABLE NO.3
HORMONAL PATTERNS AND CORRESPONDING CLINICAL STATUS
CLINICAL STATUS FSH LH T
Normal Normal Normal Normal
Testicular failure Elevated Elevated Normal or Low
Germinal aplasia Elevated Normal Normal
Hypogonadotropic hypogonadism Low Low Low
Diagnostic Studies
Scrotal Ultrasonography
The use of ultrasonography to image organs and vessels and to measure blood flow is beneficial
during evaluation of the infertile man. Its principal application regarding male factor infertility is
for the diagnosis of varicoceles. The diagnosis is based on a venous diameter of 3.5 mm or more
with the patient at rest so he can be scanned in the supine position. Subclinical varicoceles are
approximately 3 mm in diameter. Color flow Doppler allows determination of the direction and
magnitude of blood flow. To detect the change in flow, or reflux, the patient must perform the
Valsalva maneuver and may require examination in the standing position. This positioning
allows adequate assessement of reflux in the testicular veins, although the accuracy and clinical
significance are not absolute. Scrotal ultrasonography and color duplex Doppler are excellent
adjuncts in patients with equivocal examinations.
Transrectal Ultrasonography
Transrectal ultrasonography (TRUS) is now being used to detect varying degrees of ejaculatory
duct obstruction. It is essentially a noninvasive, inexpensive office procedure that is readily

11. available. Ejaculatory duct obstruction is easily diagnosed, and the results are highly accurate
when using TRUS in azoospermic patients with low ejaculate volume. Obstruction is
documented by the presence of dilated seminal vesicles more than 1.5 cm in diameter seen on
transverse imaging. Additional findings indicating obstruction include midline intraprostatic
cystic structures and intraprostatic calcifications along the projected course of the ejaculatory
ducts. The absence of seminal vesicles and ampulla of the vas is diagnostic of congenital
abnormalities.
Postejaculate Urinalysis
A postejaculate urinalysis to detect retrograde ejaculation should be obtained in patients with
anejaculation (no antegrade ejaculate), those with low-volume azoospermia, and all others with
low-volume semen samples, including those with oligospermia and normal concentration. The
patient voids to completion, produces anejaculate and then immediately voids into a specimen
container. The unspun voided specimen is then evaluated. A diagnosis of retrograde ejaculation
is confirmed when more than 10 sperm are noted per high power field
Testis Biopsy
Testis biopsy is reserved for patients who have azoospermia, essentially normal-size testes,
palpable vas deferens and epididymis and a normal volume of semen. In these cases, testis
biopsy allows the differentiation between patients with microtubular obstructive disease who are
candidates for microsurgical repair and patients with disorders of sperm development. Testicular
biopsy can be performed in the office under local anesthesia. Plain I % lidocaine is used to
infiltrate the anterior scrotal skin and dartos layers. With the testicle firmly held in position and
the anterior surface up against the scrotal wall, a 1 cm incision is made down to the tunica
vaginalis. Holding stitches are placed in the tunica vaginalis and it is opened sharply the length
of the incision. An eyelid retractor is placed and additional 1 % lidocaine is dripped on the the
tunica albuginia. A holding stitch is placed in the tunica albuginia, which is then incised
approximately 0.5 – 1.0 cm in length. Testicular tubules extrude from the opening and are
excised with tenotomy scissor. The tissue is placed in support medium, such as human tubular
fluid or Ham’s F-10, for transport to the laboratory. The biopsy is best done where intravenous
sedation can be administered. Testicular biopsy remains the gold standard in regard to diagnosis
when one is searching for a small number of sperm. With the current use of the Johnson Scoring
Technique when reading testicular biopsies, it has become a method of good prognostic value as
it afforded a quantitative evaluation of germ cells and leydig cells over and above routine
microscopic study. Additionally it permits evaluation of vassal patency, the status of the
epididymis and surgical treatment of a varicocele if this was diagnosed per operatively.
Results Of Evaluation
Evaluation of the infertile male categorizes patients. Not only can diagnoses be made (see Table
No.4) treatment plans can be discussed and initiated. Both partners should be present during the
initial visit and any subsequent visit during which treatment decisions are made. Each case must
be individualized with male partner issues, female partner issues, success rates for treatment
options, costs, morbidities, and the couple’s expectations being addressed.

12. TABLE NO. 4
DIAGNOSES AFTER EVALUATION
DIAGNOSIS PATIENTS (%)
Varicocele 37 – 42
Idiopathic 20 - 25
Obstruction 6 - 14
Anti-sperm antibodies 3-9
Testicular failure 1-9
Pyospermia/infection 1-5
Ejaculatory dysfunction 1–3
Endocrinopathies 1
Evaluation of the female for infertility
The strategy for female infertility evaluation has evolved into an efficient, cost- effective
investigation that can usually be performed during two menstrual cycles. Invasive procedures
such as hysterosalpingogram and laparoscopy are scheduled during the proliferative phase of the
cycle to avoid the risk of a procedure during a concomitant pregnancy. Serum progesterone
measurements are timed for 6 –8 days before the onset of menses, and endometrial biopsy is
performed 2 – 3 days prior to the onset of menses. In general, hysterosalpingography, serum
progesterone, is recommended during the first month. A second visit is scheduled at the
conclusion of these studies to review the data and to advance to laparoscopy based on any
detected abnormalities. If no explanation is discovered after these initial studies, endoscopy
(hysteroscopy and laparoscopy) is performed to exclude endometriosis and nonobstructive tubal
adhesions (found in approximately 50 % of cases with negative basal studies) and occult
intrauterine lesions (fewer than 1 % of patients).

13. Introduction
Involuntary infertility affects about 10 – 15 % of couples of reproductive age. The causes of
infertility are equally distributed between the male and the female and often the physician
encounters multiple itiologies during the investigation. Most infertile couples have one or more
of three major causes – a male factor, ovulatory dysfunction and tubal – peritoneal disease.
Among the couple, the clinical evaluation of the female patient with reproductive dysfunction is
simplified by the fact that the patient serves as her own bioassay. In the female 15 –20 % of
causes of infertility are due to ovulatory dysfunction ; 30 – 40 % due to pelvic factors such as
endometriosis, adhesion, or tubal disease and 5 % due to cervical factors. The basic infertility
evaluation of the female is aimed at evaluating for these disorders. Thus, the evaluation of
women with infertility is an increasingly important part of strategy for infertility testing of the
couple. Most of the evaluation of the female usually requires two menstruating cycles. The
present article gives brief account of the evaluation work up of the female for infertility
Medical History
A thorough work up is based upon an extensive history and physical examination. The woman
should be asked about the timing of her pubertal development and menarche. Menstrual history
should include cycle length, duration and amount of bleeding, associated dysmenorrhea, and
premenstrual symptoms. A history of spontaneous, regular, cyclic, predictable menses is in
almost all women consistent with ovulation, while a history of amenorrhea or abnormal or
unpredictable bleeding suggests an ovulation or uterine pathology. Previous pregnancies,
abortions and birth control history should also be documented. The patient should be asked about
dyspareunia and dysmenorrhea that may be linked to endometriosis. A history of pelvic
inflammatory disease, sexually transmitted disease, and past use of an intrauterine device may be
associated with tubal disease. A history of galactorrhea may be an indication of elevated
prolactin levels, while a history of pubertal onset of progressive hirsutism associated with
oligomenorrhea may indicate polycystic ovarian disease or other disorders of androgen excess.
Excessive weight loss or gain, stress, and exercise are often associated with ovulatory disorders.
Sexual, social, and psychological issues should be explored. Any prior infertility evaluation,
surgery, or medical therapy is essential information and therefore records, films, or photos
should be sought and carefully re-evaluated.

14. Investigations
The investigation assesses two main areas – those of ovulation and pelvic viscera normality.
Ovulation is assessed first because it is less invasive and does not have the potential for
morbidity that can be associated with assessment of uterine, ovarian and tubal normality.
Assessment of Ovulation
The assessment of ovulation is complex, involving history, examination and investigation. Of
couples who present with infertility, 20 – 25 % will have disorders of ovulation. Pre-menstrual
mastalgia, mid-cycle pain in iliac fossa and clear, stretchy, lubricative, mid-cycle mucus are
some of the known symptoms of ovulation. Three procedures are used to confirm that ovulation
has occurred, and two additional procedures are used to determine that ovulation will occur in
the immediate future as shown in TABLE NO 1.
TABLE NO. 1
Assessment of Ovulatory Status
Test Timing
BBT Complete cycle
Sonography Late follicular
LH testing Late follicular
Serum progesterone Mid-luteal
Endometrial biopsy Late luteal
To determine that ovulation has occurred, most clinicians use basal body temperature (BBT)
chart monitoring, luteal phase serum progesterone measurements, or secretory phase endometrial
biopsy. BBT chart monitoring typically reveals a temperature below 98 degree F during the
follicular phase. After ovulation the temperature rises 0.2 degree – 0.6 degree F and is sustained
for 9 – 13 days during the luteal phase. Immediately before or coincident with the onset of
menses, the temperature falls below 98 degree F. This typical “biphasic” profile is demonstrated
repeatedly in ovulatory women. Use of BBT chart monitoring to determine dysfunctional
ovulation (in contrast to the absence of ovulation) is generally not helpful. Unfortunately,
parameters such as the number of temperature –elevated days and the magnitude of the
temperature rise have correlated with other measures of luteal function (progesterone,
endometrial biopsy) and have not been used reliably to initiate therapy. Thus, BBT chart
monitoring can establish ovulation but is unable to determine the presence or absence of
ovulatory disturbances. Serum progesterone concentrations are higher than 5 ng / ml during the
luteal phase. Most clinicians use the luteal phase progesterone level to establish both ovulation
and the quality of ovulation. If the serum progesterone is higher than 5 ng / ml, ovulation is
confirmed. This measurement can apply to any day of the luteal phase. When more rigorous
criteria are set for the time of progesterone measurement (6 – 8 days prior to the onset of menses
– typically day 20 –22 of the cycle), several investigators have reported that the “quality” of
ovulation can be determined. The precise threshold value of progesterone is controversial, but
most agree that a mid- luteal progesterone level of less than 10 ng / ml is consistent with luteal
dysfunction. Additionally, most authors agree that a serum progesterone level higher than 20 ng /
ml is consistent with adequate luteal function. There is no consensus on what a serum

15. progesterone level of 10 – 20 ng / ml indicates about luteal function, but it clearly means that
ovulation has occurred. Endometrial biopsy is typically performed during the late luteal phase
to classify the morphologic transformation of the secretory endometrium. The “luteal phase
defect” has been defined as endometrium that is more than 48 hours “out of phase” with the
cycle. The proper interpretation of this test requires three pieces of information:
1. The date the test was performed.
2. The date natural menstruation occurred.
3. The morphologic dating of the endometrial specimen.
For example, a specimen obtained on day 26 of a 28 – day cycle that was interpreted as
consistent with day 23 endometrium would be considered out of phase, but a specimen obtained
6 days before the onset of menstruation consistent with day 23 secretary endometrium would be
considered in phase. Finally, interest has emerged concerning adjunctive measurement of
endometrial peptides. In particular, some integrins are known to be expressed at unique times
during the secretory phase. Measurement of these factors may increase the accuracy of properly
classifying endometrial specimens. Unfortunately, there is a mixed degree of agreement
regarding serum progesterone measurements and endometrial maturity. Hence, these two tests
currently stand at the “discretion of the practitioner” as independent but not correlated tests.
Follicular measurements by sonography have been used to predict ovulation. In general, a
naturally growing follicle expands at approximately 2 –3 mm per day and ruptures after the
follicular diameter approaches 20 –22 mm. After rupture the follicle generally collapses, and
fluid collects in the cul-de-sac. Commonly, a luteal structure can be observed in the ovary.
Finally, urinary measurements of mid-cycle luteinizing hormone (LH) can detect the
preovulatory LH surge. Because urinary LH measurements are done infrequently (usually once
or twice daily), it is reasonable to estimate that ovulation will occur 24 –36 hours after detection
of the surge.
The abnormalities of ovulation must be assessed for their cause. The direction of assessment is
formulated from the history and examination findings. Polycystic ovarian disease (PCOD) may
be associated with abnormalities of ovulation and luteal phase defect. Diagnosis depends on an
ultrasound examination demonstrating greater than 10 to 15 follicles in each ovary, raised
androstenedione and testosterone levels, with an LH, FSH ratio greater than 2 : 1. In the presence
of the galactorrhoea, the prolactin level is almost invariably elevated, and if this elevation is
twice the upper limit of normal, both the follicular and luteal phases are usually abnormal and
often oligomenorrhoea or amenorrhoea is present.
Assessment of Pelvic Viscera
Clinical pelvic examination will alert the physician to gross abnormalities, by noting both
abnormal masses attached to or separate from the uterus or ovaries, or reduced mobility of these
structures. Abnormal discharge or undue tenderness may raise the question of pelvic
inflammatory disease, endometriosis or ovarian cysts. Two principal anatomic defects deserve
evaluation for couples with infertility. Uterine abnormalities (malformations, uterine fibroids,
endometrial polyps) and tuboperitoneal factors (pelvic scarring from infection or prior surgery,
endometriosis, congenital tubal abnormalities) can be evaluated by several techniques (Table
No.2).

16. (TABLE NO. 2)
Assessment of Tubopeitoneal / Anatomic Status
Test Comment
Hysterosalpingography High false-negative rate
Laparoscopy Confirms peritoneal disease
Hysteroscopy Confirms intrauterine disease
Sonohysterography lesions Visualizes mural and intrauterine
Sonography contour Identifies uterine and endometrial
A time- honored test for evaluating uterine and tubal factors is hysterosalpingography. It is a
contrast study performed under fluoroscopy, where radiopaque solutions are injected into the
uterus to define the outline of the uterus and fallopian tubes. If the hysterosalpingogram is
normal (normal uterine contour with bilateral tubal fill and spill), it has a negative predictive
value of only about 60%. This is principally because the infertility population has a high
incidence of endometriosis, which typically represents peritoneal disease but not tubal disease.
Therefore, the tubes appear open on the hysterosalpingogram, but the disease remains
unrecognized by this limited study. Similarly, proximal tubal occlusion has a 50 % positive
predictive value. At the time of further diagnostic studies proximal tubal occlusion cannot be
demonstrated, probably related to the presence of tubal spasm or another technical problem
associated with the procedure. However, the hysterosalpingogram has a high positive predictive
value if distal tubal occlusion is detected. When distal tubal occlusion is detected, untreated
patients are at increased risk for pelvic infection. Therefore, these patients should be treated with
an outpatient course of antibiotics. Sonography is a valuable tool for detecting uterine structural
lesions such as uterine fibroids and adnexal pathology. Although the role of sonography has been
limited by its inability to evaluate tubal status and subtle abnormalities of the endometrium
(polyps, small fibroids), it is emerging as a relatively simple OPD procedure that can provide the
clinician with extra details concerning the pelvic anatomy. The inclusion of sonohysterography
has explained our view of the endometrium to detect intrauterine abnormalities that may go
undetected. However, even with sonohysterography, demonstration of tubal patency is difficult.
Despite, the problems, sonography and sonohysterography currently are best applied to the
evaluation of the uterus and endometrium.
Endoscopy is currently considered the most thorough, comprehensive tool for evaluating pelvic
anatomy. Laparoscopy allows visualization of the peritomeum to detect endometriosis and to
assess tubal status. Hysteroscopy can be used to evaluate the endometrium and the tubal ostia.
Additionally, when disease is detected, therapy can be provided. These therapies include
adhesiolysis, neosalpingostomy, and adnexal surgery. Tubal lesions can be grouped in two major
groups : distal and proximal tubal diseases. Proximal tubal obstruction occurs less frequently that
distal obstruction and its etiology and treatment are more controversial as given in TABLE
NO.3.
TABLE NO.3
ETIOLOGIES OF PROXIMAL AND DISTAL TUBAL DISEASE

17. Distal Tubal Obstruction
Hydrosalpinx (thin / thick walled ; hydrosalpinx simplex / follicularis)
Pelvic inflammatory disease
Proximal Tubal Obstruction
Congenital atresia
Debris, viscous secretions
Muscular spasm, stromal edema
Obliterative fibrosis
Salpingitis isthmica nodosa
Chronic tubal inflammation
Intrinsic (intramural) endometriosis
Endosalpingiosis
Tubocornual polyps
Granulomatous salpingitis (genital tuberculosis, actinomycosis, schistomsomiasis, sarcoidosis,
foreign bodies)
Remnants of ectopic pregnancy
Tubal sterilization
Even after thorough investigation, 10 percent of couples will lack a distinct diagnosis of their
cause(s) of infertility. A complete review of the couple’s evaluation is warranted to assure no
oversights; a repeat history may reveal sexual dysfunction, nonproductive timing of intercourse,
or the use of spermicidal agents as lubricants. Borderline test results may need to be repeated.
Assuming satisfaction with the couple’s evaluation, aperiod of expectant management will result
in pregnancy in some couples. Artificial insemination techniques may then be elected. In vitro
fertilization and gamete intrafallopian transfer are the final options for the couple with
unexplained infertility.
Clinical Focus
• Routine screening of infertile women for anemia, blood grouping, antibody status though
not mandatory but are extremely helpful prior to diagnosis and therapy of their infertility.
• Basal body temperature charting, sonographic follicular monitoring, urinary LH testing,
and mid-luteal progesterone levels are important tests to evaluate ovulation and
subsequent corpus luteum function.
• Initial medical history of women compatible with polycystic ovarian disease (PCOD),
oligomenorrhea, or amenorrhea associated with weight loss or excessive obesity,
exercise, eating disorders and galactorrhea are helpful indicators of ovulatory
dysfunction.
• Hysterosalpingography (HSG) provides information regarding the shape of the uterine
cavity and patency of the fallopian tube whereas laproscopic intervention helps in
identifying infertility factors such as endometriosis, pelvic adhesions and uterine
leiomioma.

18. Conclusion
About half of couples who experience difficulties conceiving do so as the result of female
infertility. Most causes of female infertility can now be identified through testing. A general state
of health, lifestyle pattern, and medical and menstruation history is often helpful before specific
investigation for the diagnosis of female infertility is initiated. By measuring the levels of FSH
on day 3 of cycle, clinician can get an indication of how close a woman is to menopause and
whether she has any ovarian reserve. Pre-menstrual molimina, basal body temperature, mid-cycle
mucus changes, plasma progesterone, urinary LH levels and ultra sound follicle tracking are
some of the tests which can assess ovulation in women. Once preliminary tests show that the
woman is ovulating, a battery of more advanced tests is employed to identify the cause of
woman’s infertility. These tests investigate the condition and functioning of hormones, ovaries,
uterus and fallopian tubes of the woman. The use of laproscope and hysteroscope which enables
the direct visualization of the pelvic viscera helps identify uterine and tubal abnormalities of the
infertile woman.

19. In Vitro Fertilization And Embryo Transfer
Since the birth of first child conceived using the process of in-vitro fertilization (IVF) in 1978,
more than 1 million children have been born world-wide through this fascinating procedure.
Although originally developed to treat women with absent or irreparably damaged fallopian
tubes, IVF procedure have since been used as a treatment modality for a wide variety of
infertility problems. In IVF procedure the physiological process of fertilization takes place
outside the body of the female patient. In an artificially constructed environment. For this
purpose, one or more oocytes are recovered from preovulatory ovarian follicles and subsequently
co incubated in a defined culture medium together with motile spermatozoa previously separated
from the seminal plasma, immotile spermatozoa and non-spermatozoal cells. After identification
of successful fertilization, either at the pronuclear stage, at an early cleavage stage or at the later
blastocyst stage, the embryos are placed in the uterine cavity for nidation in the endometrium and
pregnancy.
Introduction
In vitro fertilization (IVF) means “in glass” or “in the laboratory”, hence the term “test-tube
baby” is a complex procedure for the treatment of infertility in a wide variety of couples who are
unable to conceive naturally. The IVF procedure at its very simplest involves removal of eggs
from the ovaries, fertilization with the sperm in the laboratory and transferred of the early
embryos into the uterus.The minimum requirements for IVF are that the female has a normal
uterine cavity, a source of oocytes and enough sperm in the male to achieve fertilization.
Although IVF can be performed with naturally matured oocyte, it has become wide spread
practice to carry out this treatment in combination with hormonal stimulation of the ovaries, so

20. that multiple oocyte become available for IVF. The improved pregnancy rate of IVF in
hormonally stimulated cycles results mainly from the replacement of several fertilized oocytes.
The indications for treatment through IVF have broadened over the years.
Indications For IVF:
As couples undergo evaluation and treatment of infertility, IVF is the first line of therapy in
certain situations. Some of these indications are given in table no. 1 given below :
TABLE NO.1
Major Indications for IVF
All the indications mentioned in the above table represent either an unfavorable environment for
egg – sperm interaction or an inability of an egg and sperm to unite and fertilize in-vivo. The use
of in-vitro fertilization technique allows the rescue of eggs and sperms from unfavorable
conditions, provides a more favorable environment for egg- sperm interaction, confirms
fertilization and ensures delivery of the embryo to the uterus.
An Outline of the IVF-ET Process
The work of the embryologist is the culmination of a series of events that were set in train the
day the couple presented to their infertility clinician. The embryologist’s main brief is to identify
and take responsibility for the oocytes aspirated by the IVF clinician at the oocyte recovery, with
the intention of providing optimal conditions for the creation of embryos which will be returned
to the patient or frozen for subsequent use by the patient. The process is summarized below.
• Pre OPU
o Follicular phase tracking
• Day 0
o Sperm Production ---> Sperm Preparation
o OOCYTE Pick up (OPU) and gamete
o Intrafallopian transfer (GIFT)
• Insemination
• Day 1
o Fertilization check
• Embryo Culture
• Day 2 or 3
o Embryo transfer
o Embryo cryopreservation
Ovulation Stimulation Protocol
The aim of ovulation stimulation protocol in IVF-ET procedures have always been to produce a

21. sufficient number of oocytes for maximizing the success of treatment. In gonadotropin only
stimulation protocols, the existence of elevated basal levels of LH were responsible for
cancellation of a large number of treatment cycles. The discovery that the tonic administration of
GnRHa induces a state of hypophyseal desensitization helped remove many of the ill effects of
previous stimulation regimes. Ovulation stimulation strategies using GnRH analogues are now
adopted in ART procedures to control endogenous secretion of LH. GnRHa administration down
regulates the pituitary gland and prevents the release of FSH and LH, resulting in ovarian
quiescence. Pituitary down regulation can be confirmed by endocrine and ultrasound testing.
Complete pituitary suppression shows following endocrine parameters.
1. LH < 5 IU/L
2. E-2 < 50 pg/ml
3. P < 1 ng/ml
4. Thereafter adequate superovulation can be achieved by daily administration of 150 / 225
IU of FSH.
The protocol of GnRha together with hMG is the bench mark for ovarian stimulation in all IVF
programmes. A number of protocols have been developed by various workers. Brief information
about the commonly used protocol called the long down regulation protocol is given step-wise
below :
1. In long protocol 500 ïg GnRHa administration is initiated on day 21 of previous cycle
and down regulation is normally complete after 7 days. There after the concentration of
GnRHa is reduced to 200 ug and continues till the day of hCG administration.
2. FSH (225 IU daily) started from day 2 of the menstrual cycle and continues till the day of
hCG administration when sufficient number of mature follicles are present. In normo-
ovulatory women, 10 days of gonadotropin stimulation is usually sufficient to achieve the
criteria for ovulation induction and ensures sufficient estrogen priming for the
endometrium
3. Ovulation induction done through administration of hCG (10,000 units) when leading
follicles are >18mm in diameter.
4. Oocyte retrieval done 34-39 hours after hCG administration.
Retrieval Of Oocytes
The retrieval of oocytes for assisted fertilization now-a-days is performed 34 –36 hours
following hCG administration by ultrasound guided transvaginal puncture of the follicles. The
oocyte retrieval is essentially an outpatient procedure performed under light (neurolept)
anesthesia. Usually a combination of Propofol, Midazolam and Fentanyl is administered
intravenously. The direct location of ovaries behind the vagina, its plasticity and insensitivity to
pain, makes transvaginal ultrasound guided follicle puncture oocyte retrieval procedure relatively
easy and comfortable. Once adequate sedation has been administered, a speculum is used to
expose and clean the vagina and cervix of the patient in dorsal lithotomy position in sterile
drapes. Thereafter, a transvaginal ultrasound probe with attached fixed needle guide assesses the
position of ovaries in relation to blood vessels, bowel and the uterus. Thereafter an appropriate
trajectory path for the follicular aspiration needle through the pelvic is selected on the ultrasound
screen.

22. In the next step, the aspirations system is checked to be operational by sucking a small amount of
culture medium through the needle into a culture tube. This tube is then discarded. A negative
suction pressure of 100 mm Hg is adjusted with the aspiration pump. The needle is then inserted
in the vaginal probe guide and the largest ovarian follicle is entered. Using the foot pedal, 100
mm Hg negative suction pressure is applied to the tubing equipped with a test tube trap
mechanism and the collapse of the follicle is visualized on the ultrasound screen. The follicular
fluid containing oocyte is aspirated into a round bottom test tube containing one ml modified
Human Tubal Fluid (HTF). In most cases it is not necessary to withdraw the needle from the
ovary but rather to change its position within the ovary to go from one follicle to the next. A
continuous negative pressure should be maintained throughout the oocyte aspiralum procedure.
After aspiration of all oocytes, the pressure is released and the needle is removed from the ovary
and the vagina. This entire process is then repeated on the contralateral ovary. Normally, oocyte
retrieval procedure on both ovaries can be completed in about 30 minutes time. Immediately
after oocyte retrieval procedure, the vaginal wall puncture sites are visually inspected for
hemostasis. If needed, pressure is applied or a suture placed at the bleeding site.
Oocyte With Embryologist
Surgically collected oocytes (collected as aspirated follicular fluid or follicular flushing) in a
culture tube in the operation theatre is handed over to the embryologist in the adjacent laboratory
which are immediately transferred into a large petri dish and examined under low power
magnification zoom stereomicroscope for the presence of oocytes along with surrounding
granulosa cells. Based on the appearance of oocyte cumulus mass (OCM) and the granulosa cells
and the presence or absence of germinal vesicle and first polar body. The oocyte maturity is
classified as shown in Table no.2 below :-
TABLE NO. 2
CLASSIFICATION OF OOCYTE MATURITY
• Mature Oocyte
o Expanded cumulus and corona layer.
o Presence of first polar body.
o Germinal vesicle absent.
• Immature oocyte
o Compact corona cumulus composed of only a few cell layers.
o Germinal vesicles present.
• Atretic oocytes
o Sparse unexpanded cumulus with a dark and irregularly shaped ooplasm.
Thereafter the embryologist removes the good quality mature oocytes from the follicular fluid
using a Pasteur pipette and puts it in buffered culture media vessel or microdrops into the
incubator equilibrated with 5% CO2 until the time of insemination some 3 to 6 hours later.
Semen Preparation
Two sperm preparation techniques, that is, swim up and density gradient centrifugation method
are currently used in IVF procedure. The goal is to remove both the immotile and

23. morphologically abnormal spermatozoa and to retain as many motile spermatozoa as possible. A
brief description of swim up method is given below :
• The semen sample is allowed to liquefied at 37 degree C for 10 – 20 minutes.
• An aliquots of 1ml of semen are placed in 5 ml labeled Falcon tubes and gently overlaid
with 2 ml of HTF medium.
• The tubes are incubated at 37 degree C for 45-60 minutes to allow progressively motile
sperm to swim into the overlaid medium.
• The medium (up to 90 % of the supernatant) is aspirated and placed into a labeled
collection tube and centrifuged at 300-400 g for 10 minutes.
• The supernatant is removed and the pellet is resuspended in 0.3 – 1.0 ml of fresh
medium.
• To calculate the insemination volume for IVF, sperm count, motility, and velocity are
assessed on a sample in the counting chamber of a standard haemocytometer.
• The sample is allowed to settle (>3 minutes) and motile sperm in a minimum of five
squares from the central 25 squares are counted to give a rough estimate of the motile
concentration.
• The insemination volume is adjusted to give a total of 1,00,000to 2,00,000 sperm / ml in
the medium containing the oocytes.
Insemination Of Oocytes
Approximately 4 – 6 hours after oocyte retrieval, place the sperm sample and the oocytes in the
Isolette. Verify the identity of the gametes and record on the patient’s verification form. Have a
second biologist verify the gametes prior to insemination to ensure proper identification.
Resuspend the spermatozoa, as some settling oocurs while sitting in the incubator. Add the
predetermined amount of spermatozoa to each oocyte and observe the culture drops
microscopically to confirm the approximate number of spermatozoa per oocyte. Return the
oocytes to the incubator until the fertilization check.
Evaluation Of Fertilization
Oocytes are examined between 16 to 20 hours after insemination by sperm to see whether
fertilization has occurred or not. The presence of two pronuclei and two polar bodies is indicative
of normal fertilization. Once the presence of normal fertilization is confirmed, the fertilized
oocytes are returned to the CO2 incubator in a fresh culture medium for another 24 hours.
Selection Of Best Embryos
The oocyte is well equipped with most of the developmental materials in the form of proteins,
mRNA etc to take it to 4 to 8 cell stage after fertilization. Thereafter human embryo genome
activates to take care of its growing needs. The quality of oocytes is therefore of paramount
importance to remain viable and initiate development immediately after fertilization. Many
clinics therefore have focussed attention to predict the embryo quality from pronuclear stage.
Various parameters have been employed to identify good quality zygotes and embryos. Zygotes
resulting in maximum implantation success rate firstly have nearly equal number of nucleolar
precursor bodies (NPB) in both male and female pronuclei and never differed by more than 3 in
number. Second, the NPB in both the pronuclei are either polarized or non-polarized but never
polarized in one and non-polarized in the other. Embryos showing these morphological criteria at

24. the pronuclear stage when transferred produced pregnancy rates of 50% as compared to
pregnancy rates of 9% with embryos not adhering to their criteria. More recently a multiple step
scoring system is in use to identify good quality embryos and the following plan could be
followed :-
18-19 hours after insemination
• The pronuclei are examined for
o Symmetry
o The presence of even number of NPB
o The positioning of the polar bodies
25-26 hours after insemination
• Embryos that have already cleaved to the 2 cell stage
• Zygotes that have progressed to nuclear membrane breakdown
42-44 hours after insemination
• Number of blastomeres should be greater than or equal to four
• Fragmentation of blastomeres should be less than 20%
• No multinucleated blastomeres
66-68 hours after insemination
• Number of blastomeres should be greater than or equal to eight
• Fragmentation of blastomeres should be less than 20%
• No multinucleated blastomeres.
Embryo Transfer
Embryo transfer (ET) is normally performed two days after oocyte pick-up or 44 to 48 hours
after insemination. Normally, ET procedure is performed without anesthesia. The patient is
placed in a slight Trendelenbarg position. After visualization of the cervix with a speculum, the
interior portion of the cervix is clamped with a forsal and a catheter is introduced into the
cervical canal. Once, the patient is properly prepared for transfer, the embryologist is asked to
load 2-3 embryos into the transfer catheter in about 20 ul volume. This fine catheter is then
inserted through the previously position broader cathether into the uterine cavity up-to about 6
cm depth. The embryos are released into the uterus by gental pressure on the plunger. It is
important to ensure that the embryos are located near the tip of the catheter to minimize the
volume of medium injected with the embryos. After the catheter has been withdrawn, the
embryologist examines it to ensure that no embryos have been retained.
Luteal Phase Support
As compared to natural cycles, the decline of serum E-2 and P hormone concentrations are more
abrupt in ART cycle and needs luteal phase support. Surveillance of the luteal phase may be
limited to a serum -hCG measurement two weeks after transfer. Some programs favour a

25. more extensive protocol for luteal monitoring with serial determinations of estradiol,
progesterone, and -hCG levels. Luteal phase hormonal support is considered to be beneficial
following adjunctive GnRH agonist therapy as the incidence of histological luteal phase defect is
increased in IVF cycles. Most infertility clinics routinely give exogenous luteal support to
patients undergoing ART procedures. Progesterone and hCG are most commonly used, although
the latter increases the risk of ovarian hyper stimulation syndrome (OHSS) in some patients.
More recently micronized P is preferred as luteal support agent due to its improved absorption
and can be given through oral or vaginal routes. The latter route however, offers several
advantages over the oral route and is thus preferred due to following reasons.
• It is convenient and acceptable to patients.
• It does not hurt or require any special equipment or training to administer.
• It rarely produces allergic reactions.
Intravaginal progesterone pessaries are also advised during the luteal phase of stimulated cycles
after IVF-ET procedure to overcome the undesirable ill effects of excessive estrogen action on
the uterine endometrium. Progesterone also increases endometrial receptivity and thus improve
the implantation rate of the embryos.
Complications
Although IVF is a safe procedure, some complication related to the stimulation, the procedure
and the outcome of the treatment are not uncommon. Some of the complications are given in the
table no.3 given below :
TABLE NO.3
COMPLICATIONS OF IVF-ET TREATMENT
• Stimulated related complications
o Ovarian hyper stimulation syndrome (OHSS).
• Procedure related complications
o Anesthesia reactions (nausea, vomiting, adverse drug reactions, airway
obstruction, airway injury & aspiration pneumonia.
o Tromatic organ injury (bowel perforation, blood vessel laceration & ovarian
hemorrhage.
o Post procedure complications (ovarian abscess, pyosalpinges, endomyometritis,
ovarian tortion, hydrosalpinges & pelvic hematome.
• Outcome related complications
o Multiple gestations
o Spontaneous abortion
o Premature labour
o Preeclampsia
o Ectopic gestation

26. Clinical Focus
• In order to maximize the chances of a successful pregnancy through IVF, ovaries are
stimulated using gonadotropins hormones to produce multiple eggs for fertilization.
• Transvaginal ultrasound guided oocytes retrieval is nearly an outpatient procedures which
can normally be performed without intensive anesthetics.
• Proper oocyte scoring for identification and selection of mature oocytes significant
enhances fertilization and pregnancy rates.
• Exposure of sperm, oocytes and embryos for temperature, pH and CO2 concentration and
changes causes irrepareable damage and needs to be strictly monitored for providing
optimal conditions.
• The embryology laboratory is a key component of any IVF programme where vital steps
of oocyte culture are undertaken, so the laboratory must be appropriately located, sized
and equipped.
Conclusion
The evolution of IVF has been rapid and significant. Once available to only a few couples around the
world, IVF is now a viable therapeutic option for thousands of couples. No longer considered
experimental, IVF has become a routine part of infertility treatment protocols. As advances in
technology continue to simplify IVF procedures, IVF is rapidly becoming a routine part of infertility
treatment in the office. Hormonal stimulation protocols are designed to mimic the natural events that
lead to production of many oocytes. These oocytes are aspirated from the ovarian follicles few hours
prior to ovulation and fertilized by sperm cells in the laboratory. Success rates continue to improve, and
costs continue to decrease.
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27. Infertility Treatments in India
India offers world class medical facilities for all types of infertility treatment and its options
available which can be comparable with any of the western countries.
India has state of the art Hospitals and the best qualified doctors which deal with all all types of
infertility treatment. With the best infrastructure, the best possible Medical facilities, accompanied
with the most competitive prices, you can get the treatment done in India at the lowest charges.