ICMR BULLETIN VOL. 26-No.-10-OCTOBER-1996.pdf

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ICMR
BULLETIN
Vol. 26, No. 10

October, 1996

RESEARCH IN INFERTILITY
It is recognised that infertility is multifactorial and
several male and female factors may co-exist contributing
to infertility in a given couple. It is therefore essential that
an integrated approach is developed for management of
infertile couples. Over the years the Institute for Research
in Reproduction (IRR), Mumbai has evolved a detailed
operational plan for evaluation and management of infertil­
ity with the main emphasis on male and female endocrine
infertility and reproductive disorders.

Table I. Male and female factors identified for infertility
in patients at the IRR, Mumbai

Male factors

Female factors

Idiopathic oligozoospennia

Non endocrine causes

Oligozoospennia due to
varicocele

Anovulation witliout
amenorrhoea

Oligozoospennia due to
infection

Amenorrhoea with normal
gonadotropins levels

The cause of infertility may vary from one geographic
and social area to another. Globally the major causes of
infertility in women are tubal occlusion and strictures, ovu­
latory disorders, sexually transmitted diseases and
endometriosis. The causes of infertility in men are impaired
spermatogenesis, disturbances in sperm-oocyte fusion, sexu­
ally transmitted diseases, agenesis of the epididymis and
other parts of the ductal system including the vas deferens,
anejaculation, etc. In the authors' experience male factors
contribute to almost 50 per cent of infertility in infertile
couples while the infertility in the remainders may be either
due to a female factor or a combination of male and female
factors. The factors frequently identified for infertility in
patients at the IRR are shown in Table I.

Primary testicular failure

Amenorrhoea with increased
gonadotropins levels

Obstnictive azoospermia

Amenorrhoea with decreased
gonadotropins levels

Idiopathic astlienozoospennia

Cervical infections

Asthenozoospennia due to
infection

Hyperprolactinaemia.

Anqaculation

Tubal factors

Hypospermia

Uterine factors

Abnormal sperm fbnns

Endometriosis

Hypothalamic pituitary failure

Corpus luteum dysfunction

In approximately 10-15 per cent of infertile couples no
apparently cause can be found and such cases are catego­
rized under Unexplained Infertility. Studies on female
infertility

were undertaken at IRR, Munibai to (i) elucidate the patho­
physiological mechanism in endocrine disorders viz.
hyperandrogenaemia, hyperprolactinaemia and hyper­
hypogonadism1; (ii) correlate clinical, morphological and

Impotency

Publication & Information, ICMR, New Delhi - I I 0 029

histological features with endocrine profiles2; (iii) evaluate
new drugs for induction or restoration of ovulation3; and
(iv) evaluate alternate diagnostic tools4-5. The experience
gained has enabled scientists at IRR to device algorithms
for primary and secondary amenorrhoea, polycystic ovarian
syndrome and recurrent abortions.
Reproductive Tract Infections
Reproductive tract infections, most of which are sexu­
ally transmitted diseases (STDs) can cause cervicitis,
endometritis, pelvic inflammation, tubal sterility, spontane­
ous, early or recurrent abortion, prematurity, perinatal mor­
bidity and mortality. The principal cause of inability to
conceive is pelvic infection which occludes the fallopian
tubes or leads to pelvic adhesions. Infection with Neisseria
gonorrhoea or Chlamydia trachomatis obstructs the Allopian tubes. The relative contribution of each of these organ­
isms to pelvic infection varies geographically and
polymicrobial etiology is possible. These infections are more
common in developing countries like India because of over­
crowding, illiteracy, unhygienic conditions and poor diag­
nostic and treatment facilities. Chronic infections can lead
to infertility, chronic pain and debility both in men and
women.
Chlamydia trachomatis is to-day one of the commonest
causes for STDs in the developing countries. Nearly half the
infections are asymptomatic causing damage to the fallopian
tubes leading to infertility and at times, life threatening
conditions like an ectopic pr军nancy. The prevalence of C.
trachomatis infection and its relationship to abnormal
laparoscopic fundings relating to tubal damage are being
studied in the infertile women attending the Infertility Clinic
at IRR. Of the 100 women enrolled C. trachomatis infection
was found in 30 per cent. A substantial number of C.
trachomatis positive and negative patients were infected
with other STD related organisms. The commonest amongst
then was Gardenerella vaginalis. Abnormal laparoscopic
findings were noted in 31 per cent of C. trachomatis positive
patients. Although, different microorganisms contribute to
infertility, the high prevalence of C. trachomatis infection
and its relationship to pelvic inflammatory diseases suggest
that in infertility management the routine screening for this
organism is mandatory.

The spouse of the infertile women who has pelvic infec­
tion and total blockage of the fallopian tubes frequently
presents serological evidence of prior chlamydial infection.
Upto 35 per cent of men with partners having cervicitis due

98

to C.trachomatis, are also positive for urethral chlamydial
cultures and of these 86 per cent are asymptomatic. With
increased societal permissiveness and tolerance and decline
in the mean age at first sexual intercourse, particularly of
male adolescents, increasing number of young men are
contracting chlamydial urethritis which remains
asymptomatic in a large number of cases. This group is
probably a major source of chlamydial genital tract infection
in adolescent or young adult female. Men with C.
trachomatis infection do not show any alteration in sperm
motility, morphology or count.
Evaluation of
Infertility

Probable Causes of

Unexplained

Unexplained infertility ie a failure to conceive for morel
than 2 years with both the partners having no major or minor
abnormalities, following basic investigations, was found in
10 per cent of the couples referred to the IRR s infertility
clinic. A study was therefore undertaken to determine the
probable causative factors of unexplained infertility (IRR
Annual Rq)ort 1988-1989) viz. corpus luteum insufficiency
(CLI), luteinized unruptured follicles (LUF), nidatory fail­
ure in women and sperm fertilizing capacity in men. In a
recently completed study (IRR unpublished observations)
39 couples were identified as having unexplained infertility.
Each 企male partner was studied fbr one to three menstrual
cycles using clinical parameters such as basal body tempera­
ture, cervical mucus and endometrial biopsy; serum and
urinary hormonal profiles and ultrasonographic scanning of
ovaries for fbllicuiar development and rupture. Corpus lu­
teum insufficiency was diagnosed if the urinary pregnandiol
glucuronide levels were below 20th centile of the values i\
the active follicular phase. Serial follicular scans were
utilised to detect LUF. Nidatory failure was determined by
detection of early pregnancy factor (EPF) in the serum of
fanale partners6. The male partners were evaluated fbr the
fertilizing potential of their sperms by hamster egg penetra­
tion assay or by hypo-osmotic swelling test (HOS).

A total of 64 cycles were studied in the women. Corpus
luteum insufficiency was diagnosed in 18 of the 64 cycles
(28%) and in 13 of these it was due to abnormal
fblliculogenesis as determined by poor estrone glucuronide
levels (< 20th centile ofactive follicular phase) and follicular
size at ultrasound ( < 18 mm). In the remaining five
increased preovulatory levels of prolactin were observed
which could have interferred with fblliculogenesis. Correc­
tion of fblliculogenesis by clomiphene citrate resulted in ohe

pregnancy while normalisation of prolactin in the individuals
with hyperprolactinaemia did not correct the CLI. The
phenomenon of LUF was found to repeat itself in only one
of two individuals in whom it was recognised. Early preg­
nancy Actor was detected in six cycles. This suggested
fertilization but, impaired implantation of the embryo prob­
ably due to defective endometrial development in spite of
adequate circulatory progesterone levels. The observation of
asynchrony of ovulation parameters in seven cycles sug­
gested an abnormality of hypothalamo-pituitary function or
a defect in the ovaries. Three cycles were found to be
anovulated. No obvious cause was detected in the remaining
28 cycles. The incidence therefore of abnormal cycles in
these "normal" infertile women was 51 per cent. Sperm
fertilizing capacity was found to be subnormal in 9 of 29
males. However, in these individuals variability in some of
seminal parameters viz. viscosity, motility was also noted.
In only 10 couples none of the above 治ctors were
identified and three of them conceived during the trial
whereas following discontinuation of trials, three more con­
ception occurred. Tb determine the frequency of successive
normal or abnormal cycles 15 individuals were studied for
three consecutive cycles. In five individuals all the three
cycles were found to be normal. In two others only two of
the three cycles were found to be normal while in the
remaining eight all the cycles were abnormal. However, in
five ofthem repeated CLI was observed. These observations
point to the inherent variability in spontaneous cycles and
also emphasise the need for an in depth evaluation of such
couples, as real unexplained infertility was detectable only
in 10 per cent of subjects.

In Vitro Fertilization and Embryo Transfer (IVF-ET)
In vitro fertilization of human eggs and implantation of
cleaving embryos after transfer into the uterus to establish
pregnancy had sparked a great deal of medical interest. The
birth of Louise Brown in England in 1978 and the subse­
quent number of births in England, Australia and the United
States, by this procedure had raised the hopes of many
infertile couples in India. The IRR, Mumbai played a pivotal
role in the advent of IVF-ET technology in India. Besides
treatment of infertility in women whose tubes were blocked
due to infection, another reason for developing this technique
was to ofifer a method of achieving pregnancy in women who
had unde电one tubectomy and were desirous of having
another child.泌th this in view, as early as in 1981-82
scientists at tiie IRR attempted collection of preovulatory

oocytes by laparoscopy from women who were infertile due
to tubal block and from those who had undergone tubal
sterilization. These oocytes were used for establishing the in
vitro fertilization technique.
Eighteen women aged below 35 years were initially
recruited for the IVF-ET programme. Except for blocked
tubes following sterilization there was no other cause for
their current infertility. The husband's senfcn was evaluated
by sperm penetration assay using zona free hamster eggs.

Since the number of embryos transferred increases the
chances of pregnancy per patient, the women were admin­
istered clomiphene citrate 50mg/day or human menopausal
gonadotropin (Pergonal) 150I.U. perdayorhFSH 10001.U.
per day fbr multiple follicular stimulation and oocyte re­
trieval. Therapy commenced from day 3 in women where the
cycle length was 28 + 3 days and from day 5 when cycle
length was 35 + 3 days.
Follicular growth was monitored by cervical mucus
scoring and/or ultrasound. When cervical scoring rose to 10/
12 and/or ultrasound scans indicated follicles ranging from
17-20 mm, 5000 l.U. hCG was administered. Laparoscopic
aspiration was done 36 hours later.

Out of the 26 follicles aspirated in 1983 at 14 aspira­
tions in 12 women, only 3 preovulatory oocytes could be
recovered. None of them fertilised in vitro. With change of
therapy to hFSH, and mcxiitoring with combination of clini­
cal and hormonal parameters, 13 preovulatory oocytes could
be obtained from 30 follicles aspirated at 13 aspirations in
12 women. Nine of these fertilized in vitro and 2 cleaved to
8 cell stage. Two embryo transfers were done but there was
no pregnancy.
The IRR continued its sustained efforts to improve the
IVF-ET programme and other assisted reproductive tech­
nologies. Various protocols for the induction of ovulation
were attempted. Couples with male factor infertility and
women with blocked tubes due to infection were also re­
cruited.
Until 1990,121 patients were treated fbr IVF-ET. Three
protocols fbr ovarian stimulation were used and the results,
in terms of number of oocytes available for retreival, fertili.zation rate, number of embryos transferred and pregnancy
rates, were compared7. In the first protocol (n=3 cycles), the
ovarian stimulating drugs (clomiphene citrate and human
menopausal gonadotropin) were given according to a rigid
dose regimen, the incidence of fertilization and pregnancy

99

were only about 10 per cent although the number of oocytes
available for retrieval was maximal. In the second protocol
(n=79 cycles) the dose regimen of the ovarian stimulating
drugs was individualized on the basis of the length of the
menstrual cycle; the results improved in terms of number of
pregnancies (n=7) and live births (n=5). In the third protocol
(n=30 cycles) where the day of initiating the ovarian stimu­
lation was programmed by pretreating the women with oral
contraceptives to suppress ovulation before starting ovarian
stimulation therapy, the results in terms of fertilization rate
were highest although the number of oocytes recovered was
reduced. The techniques of FVF-ET, intra uterine insemina­
tion and oocyte donation for premature ovarian failure were
successfully established resulting in 18 pregnancies by IVFET and 10 by gamete intrafallopian transfer (GIFT). Live
births by IVF-ET were 10 and 4 by GIFT.
FVF-ET occupies a central position in assisted repro­
ductive technologies (ARTs). Apart from the therapeutic
value, the FVF-ET procedure has yieded valuable informa­
tion about the responsiveness of the ovaries, normal and
abnormal fertilization, pre-embryo score, implantation and
pregnancy. ARTs beginning from simple intrauterine insemi­
nation combined with ovarian stimulation, gamete
intrafallopian transfer, zygote intrafallopian transfer (ZIFT)
have been used with variable success8.

Micromanipulations have added a new dimension to
IVF technology. Partial zona dissection (PZD), sub-zonal
insemination (SUZI) and intra-cytoplasmic sperm injection
(ICSI) have been used for improving fertilization. Men
whose sperm do not achieve fertilization or whose sperm
concentrations are too low have benefitted from SUZI where
a few spermatozoa are introduced between the zona pellucida and the oolemma. ICSI involves the injection ofa single
spermatozoon directly into the cytoplasm of the oocyte. ICSI
has been successful in patients with severe abnormal sperm
morphology and asthenozoospermia with the use of sperma­
tozoa retrieved from the epididymis. Trophoectoderm biopsy
and blastomere sampling have been used for the purpose of
pre-impiantation diagnosis of genetic disorders.

intracervical, intravaginal or by using cervical caps. Of late,
intrauterine insemination is being increasingly used fbr male
subfertility or cervical mucus hostility before the couple is
offered a more expensive and stressful programme of FVFET. The objective of such a regime is to deliver semen having
high sperm density and improved sperm motility directly into
the uterine cavity as close to the site of fertilization during
or immediately after oocyte release when decreased sperm
count, poor sperm motility, antisperm antibodies, cervical
infection/hostility hinder conception. In order to assist
couples with subfertility and provide higher chances fbr
conception, 70 couples over a period of 4 years were sub­
jected to intrauterine insemination (IUI) fbr a total of 120
cycles. Of these, 43 women had primary infertility and 27
had secondary infertility. In 53 of the 120 cycles, the chief
indication for this procedure was poor cervical mucus with
poor sperm motility in males. Majority of cycles (80%) were
anovulatory and ovulation was induced with- clomiphene
citrate, human menopausal gonadotropin or a combination
of both. Eighteen of the 70 couples conceived giving a
pregnancy rate of 25.5 per cent per couple and 15 per cent
per cycle. These results suggest that IUI with controlled
ovarian stimulation can serve as a usefill assisted reproduc­
tive procedure in individuals who have resistant factors fbr
infertility before offering them other expensive ARTs.

Human Semenology

Intrauterine Insemination in Management of Infertile
Couples

Semen analysis has generally been utilized in the evalu­
ation of male fertility. In order to establish the reference
norms for semen analysis in the infertility clinic at the IRR,
Mumbai, studies were undertaken fbr comparison of semen
characteristics in infertile couples with that of males with
recently proven fertility. The details of periconceptional
semen analysis in men of proven fertility and husbands of
infertile couples whose wives became pregnant while under
therapy, were obtained. In the former group semen analysis
was carried out within 6 to 36 weeks of pregnancy. The
volunteers were husbands of women who attended the
antenatal clinic or requested for medical termination of
pregnancy. In this group all the couples had proved fertility
prior to the current pregnancy. In the infertile group, semen
analysis was done during the 3 months periconceptional
period.

Artificial insemination, either homologous (AIH) or
donors has served an important role in the treatment of
infertile couples. Indications for this include physiological
and psychological dysfunction and abnormal semen charac­
teristics. AIH has been performed by various techniques viz.

The values for sperm count, total motility as well as
active forward progressive motility in this analysis showed
much lower values than normal fertile and in males where
no male factors could be detected. In 1979 the IRR labora­
tory norm was a count of20 million/ml. However, 14 of 105

100

subjects impregnated successfully with a count less than 20
million/ml. Moreover the laboratory norms for motility were
such that spermatozoa should show 30 per cent active
forward progressive motility. In this series of
periconceptional semen analysis, 60 of 105 subjects with
less than 30 per cent active forward motility impregnated
successfully. It was felt that for reference norms for semen
analysis, a very large series of representative, properly
selected subjects must be studied.

(iv) Sperm mitochondrial activity index (SMAI)13: SMAI
is a quantitative cytochemical test to determine
mito­
chondrial activity and thereby motility of spermatozoa.
Table DL Categorization of values of routine parameters in
semen samples from 3 groups of subjects.

Groups

Parameter
Fertile

Subfertile

Infertile

Liquefaction time (min.)
Volume (ml)

20-34
1.5-4.5

Viscosity
Particulate matter
Agglutination (%)
Motility (% RLP)
Vitality (%)
Density (mil/ml)
Morphology
Normal fbnns (%)
Headless spermatozoa

Normal
Nil-Mild
Nil
>25
>60
>20

34-45
4.6-5.0
1.2-1.4
Moderate
Moderate

10-24
40-59
10-19

>4.5
> 5.0
> 1.2
High
Severe
> 10
v 10
<40
v 10

> 30
>20

20-29
20-25

<20
>25 .

> 60 *
> 50
> 35
> 70
> 50

50-59
40-49
20-29
60-69
40-49

0.49
0-39
0.19
0-59
0.39

I. Routine

As a follow up of this study semen samples from fertile
individuals were continued to be analysed. Seminal fluid
smears were prepared, fixed and stained with Papanicolaou
stain, and semen morphology evaluated.
Efficiency ofroutine semen analysis to predictfunctional
and structural integrity of human spermatozoa

Routine analysis9 : The physical characteristics studied
were volume, liquefaction time, viscosity and pH according
to WHO Manual. Morphology was scored on smears stained
with Papanicolaou stain.
Scoring system : From the multitude of parameters studied,
10 were selected and a scoring system was applied. For the
purpose of scoring, semen samples were separated into 3
groups viz. fertile, subfertile and infertile, on the basis of
previously standardised normative values (Table II). Each
of these group was assigned an arbitrary score of 2, 1 and
0 respectively. Thus, any given sample could have a maxi­
mum score of 20 (fertile) and minimum score of zero (in­
fertile). The distribution of scores for the routine tests in the
fertile, subfertile and infertile groups was 20-16, 15-11 and
10-1 respectively, while that for the functional tests was 10,
9-5 and 4-0 respectively.

Functional analysis : Functional tests were done to mea­
sure the dynamic capability of spermatozoa. Each test was
directed against a specific organelle of spermatozoa.
(i)
Hypoosmotic swelling test (HOS test) : This test was
done to check the integrity of sperm plasma membranes and
was carried out according to the method of Jeyendran et al]0.

(ii) Test for acrosome intactness (TAI)11 : TAI was done
using a gelatin slide. The test is based on the principle that
acrosomal protease hydrolyses the high molecular weight
protein like gelatin.
(iii) Nuclear chromatin decondensation (NCD)12 : This
was done to check the compactness and the ability of sper­
matozoa to decondense in vitro.

M0

II. Functional
HOS (%)
Halo (%)
Halo diameter (pm)
NCD (%)
SMAI

、

RLP - Rapid linear progressive
HOS - Hypoosmotic swelling
NCD - Nuclear chromatin decondensation %
SMAI - Sperm mitochondrial activity index \

Thus the 5 parameters in functional analysis were
(i) HOS, TAI (ii) the halo percentage, (iii) halo diameter.
(iv) NCD and, (v) SMAI and the maximum score for func­
tional analysis was 10. The scoring system was uniform for
both routine and functional analysis. The group under which
each sample was categorized for routine and functional
analysis was then compared.
Incidence ofheadless spermatozoa in the semen offertile
and infertile men <

Spermatozoa exhibit certain forms of morphological
abnormality commonly referred to as 'Pinheads'. These
were found to be actually ’headless' sperms as determined
by phase contrast, light and electron microscopy. This
morphological defect was studied in the semen samples
assessed for routine characteristics. These sperms have only
a midpiece and tail. As these sperms do not have any of the
oiganelles essential for oocyte penetration and fertilization.
101

a correlation was sought between the incidence of'headless'
Sperms and infertility in men. A significant increase in the
incidence ofheadless spermatozoa ranging from 10 to 43 per
cent was found in semen samples obtained from infertile
men. Hence the per cent incidence of headless spermatozoa
could be used as a crude parameter to evaluate the quality
of semen samples. It provides a possible mechanism for
infertility since headless spermatozoa have virtually no fer­
tilizing potential14.
Role of bacteriological study in selection of cases for
IVF and embryo transfer

The influence of bacteria on sperm characteristics spe­
cially in asymptomatic males is not known. It is important
that the role of seminal bacteria in infertility and subfertility
be understood specially with the use of techniques like IVF
and GIFT. Bacteriological investigations were carried out on
50 semen samples from asymptomatic men to be enrolled fbr
the IVF-ET programme. Seminal parameters were corre­
lated with bacteriological findings. Samples were cultured
fbr both anaerobic and aerobic bacteria. Most semen
samples (n=44, 88%) contained two or more bacterial spe­
cies, the rest (n=6, 12%) did not show any bacterial growth.
The presence of bacteria in colony counts of 102/ml was
correlated with seminal characteristics. A colony count of
>102/ml was considered positive. A significant reduction in
count and motility was observed in all positive specimens.

Amorphous particulate matter as seen by wet smear was
moderate (+++) to severe (++++) in all bacteriologically
positive cases. The pH ranged between 7.2-8.1 and showed
no difference between the positive and negative cases. Vis­
cosity measurements showed high or abnormal viscosity to
be associated with bacteriologically positive cases and no
case with abnormal viscosity had negative culture. Majority
of the cases (81 %) with 'normal viscosity had. negative
bacterial culture. Percentage of abnormal morphology was
increased in positive cases (88%) along with abnormal HOS
test results.
The observation of amorphous particulate matter was
found to be a good indicator of culture positivity or nega­
tivity. The percentage of pus cells had no correlation with
the occurrence of subclinical infection. The results of the
HOS-test indicates that bacterial infection might possibly
alter sperm membrane integrity and thereby the fertilizing
ability. This study suggests that sperm characteristics are
severely altered in asymptomatic men with bacterial infec­
tion. The presence of a bacterial colony count of > 102/ml

102

in the semen sample, irrespective of whether it is a contami­
nation from urethera, skin or environment, is of importance.
It also establishes a causal relationship between
bacteriospermia and subfertility. Further, tlie results of this
study highlight the need fbr carrying out bacteriological
studies even in asymptomatic men prior to enrollment fbr
rVF-ET,5
Seminal viscosity as a predictor offertility status of the
sample

As viscosity of the seminal fluid is one of the important
parameters to determine the quality of semen, this was
studied in detail. A new needle- and syringe method was
developed which was validated with other viscometers. This
method is useful in quantitating viscosity. The seminal vis­
cosity readings were correlated witli other routine param­
eters like count, motility, morphology, particulate matter.
etc. A total of 208 cases were studied. Tliere was no cor­
relation of seminal viscosity with sperm count. Nonnal
viscosity was accompanied by normal motility and viability
whereas abnormal viscosity was partly associated with
abnormal motility and viability. Samples having high or
abnormal viscosity had high percentage of particulate debris
(86.6%) and high percentage of morphologically abnormal
forms (96.5%). In general increased viscosity was associ­
ated with a high per cent particulate matter; increased per
cent of morphologically abnormal forms; positive bacterial
culture; presence of sperm antibodies: increased occurrence
of amorphous particulate matter; and lower per cent motility
and viability.

Hence quantitation of seminal viscosity is a useful pre­
dictor of the quality of tlie sample.
Assessment of mitochondrial activity of human
spermatozoa with reference to motility and viability in
fertile and infertile men

Since motility is an important parameter to judge the
quality of seminal fluid, it was considered worthwhile to
develop a quantitative method of judging motility as com­
pared to routine subjective method.
The mitochondrial activity of human spermatozoa was
evaluated by a cytochemical method using nitrobkie tetrazolium salt13. The objective behind developing the test was to
quantitatively assess the mitochondrial activity and to check
whether there exists any correlation with the percentage of
motile spermatozoa in the sample.

A significant decrease in the mitochondrial activity was
observed in the infertile group. There was a significant
correlation between motility and mitochondrial activity.
There was no significant association between mitochondrial
activity and sperm viability. This is thus a simple, quanti­
tative, objective approach to judge the overall change in
sperm motility in fresh semen and is therefore of diagnostic
value and can be used as an indicator in various experimental
conditions.
Sperm function in asth en ozoospermia

With the understanding of routine semen analysis and
development of sperm function tests, a structure and func­
tion study on severe asthenozoospermia was undertaken11.
The study was done on 25 patients with severe sperm
motility problems. The parameters included routine analy­
sis, Rinctional tests, ultrastnictural study and scoring sys­
tem. In this study, eventhough Kartegener's syndrome was
suspected in 5 subjects (with history of recurrent cold, cough
and bronchitis) dynein arms of the flagella were absent in
only one case and that too not as an isolated defect. Both
inner and outer dynein arms were absent. Ultrastnictural
study revealed multiplicity of defects. Mitochondrial defect
may be one of the cause for the loss of spemi motility in this
population.

Factors Contributing to Repeated Pregnancy Wastage
Recurrent spontaneous abortion is a phenomenon of
varied etiology viz. genetic, immunological, endocrinologi­
cal, uterine anomalies and subclinical infection. The cyto­
toxic effects of infectious agents and the resultant inflamma­
tory responses may interfere with organogenesis or histogen­
esis in the foetus. In an attempt to assign either a high or low
risk fbr subsequent pregnancy losses, characteristics of etio­
logical factors such as uterine anomalies, endocrine dysfunc­
tion and subclinical infections in women were studied. The
interim results indicate that a significant number of women
are exposed to the risk of infectious agents. The infixtions
identified in the order of their frequency are toxoplasmosis
(32%), cytom钮alovinis (20%), rubella (16%), Chlamydia
trachomatis (16%) and syphilis (8%). Uterine anomalies
such as submucus polyp or incompetent os were seen in 14.2
per cent. Corpus luteum inadequacy was observed in 3
women only. These observations suggest that increasing
exposure of women to microbes leads to chronic maternal
genital infections.

Semen samples from males with partners having bad
obstetric history (BOH) have also been studied, so as to
identify the seminal characteristics and the 鱼nctional capac­
ity of spermatozoa in this group of men. The results indicate
increase in sperm head abnormalities along with nuclear
chromatin abnormalities. The study shows that apart from
a number of possible causes of BOH in the female viz.,
genetic, immunologic, endocrine, infection aftd uterine or­
ganic factors; contribution from the male could be consid­
ered as one of the possible etiological Actors. Hence, a
comprehensive evaluation of the male spouse is indicated.
Cytogenetic Abnorpialities as a Cause of Male Infertility

Chromosomal abnormalities have been known to be one
of the important causes of infertility. The frequency of
chromosomal aberrations has ranged from 1-3 per cent
amongst all infertile men. However, when karyotyping was
restricted to azoospennic men, this incidence rose to 15 per
cent. In several cytogenetic surveys in oligoazoospermie
males, the incidence ranged from 6-8 per cent.
Oligoazoospermic males (n=51), where infection and
surgical causes were excluded were karyotyped16. Of these,
six individuals had abnormal karyotype, giving an incidence
of 12 per cent. Three of them had 47, and one had 46, XY/
47, XXY mosaic Klinefelter syndrome. One had a deletion
of long arm of Y and one had a mosaicism for 46, XY/47,
XYY.
Significance ofminor cytogenetic abnormalities in male
infertility

The use of Giemsa banding technique fbr karyotyping
has revealed several minor abnormalities in individuals with
male infertility. These are abnormal length of Y chromo­
somes and enlarged satelites - acrocentric chromosomes (1315 and 21 -22). Family studies in these cases have been
infbmiative. Of the three cases where an enlarged Y was
found, paternal chromosomes could be studied in two. Both
these fathers had a long Y similar to the patients. These men
were normally fertile. It is felt that a large Y chromosome
may not compromise the reproductive function of an indi­
vidual. A short or a deleted Y chromosome, however, is more
likely to be the causal factor in male infertility. One patient
who had azoospennia was found to have Yq- with an
absence of the fluorescent part of the Y chromosome. This
indicates that the fluorescent part of Y may be crucial for
proper spermatogenesis. Recent reports also indicate

103

microdeletions in the Y chromosome to be a cause of male
infertility17.
Two large comprdiensive studies in male (oligozoospermia, 102 cases) and female (primary amenonhoea,
123 cases) reproductive disorders at IRR defined the selec­
tive population for which cytogenetic studies can be applied.
The study showed that though the occasional patient with
cytogenetic abnormality may have normal gonadotropins,
most often abnormality was detected in those who had
elevated serum FSH. It was clear that a study to determine
the frequency of cytogenetic abnormalities in male and
female reproductive disorders can be restricted only to the
latter group. The study also provided interesting observa­
tions like high frequency of isochromosomes for X in the
primary amenorrhoea group and rare abnormalities such as
familial metacentric Y and XY/XXY in persistent muilerian
structure syndrome in males.

basic understanding ofthe endocrine, genetic, psychological
aspects of both the partners.
References
1.

Vaidya, R.A., Shringi, M.S., Sheth, A.R., Mehetji, P.K.,
Raikar, R.S., and Pandya, S.K. The study of dopamine agonist
and serotonin antagonist in pathological hyperprolactinaemia.
Infertility 3 : 231, 1980.

2.

Haji, H.» Vaidya, R・,Meherji, P., Joshi, L., Sheth, A., Devi,
P.K. and Motashaw, N. Scope of laparoscopic ovarian biopsy
versus multiple serum gonadotropin estimations in the diag­
nosis of secondary amenorrhoea. J Obstet Gynecol India 27
:269, 1977.

3.

Meheiji* P.K. and Vaidya, R.A. A comparative evaluation
of centchroman and clomiphene citrate for the induction of
ovulation in infertile women - A multicentre project. J Ohstet
Gynecol India 34 : 323, 1984.

4.

Khatkhatay, M.I., Sankoli, G.M., Meheiji, P.K., Gogate, J..
Choudhary, V., and Joshi, U.M. Application of pencillinasc
linked ELISA of pregnanediol glucuronide for detection of
ovulation and assessment of corpus luteum function. Endocrinol
Jpn 34 : 465, 1987.

5.

Shahani, S.K., Moniz. C., Meheiji, P.K., Chitlange, S.M. and
Hinduja, I.N. Early pregnancy factors as a promising
immunobiomarker in human pregnancy. 1CMR Bulletin 21 :
13, 1991.

6.

Shahani, S.K.. Moniz, C.L., Chitlange, S.M. and Meheiji,
P.K. Early pregnancy factor as a marker for detecting in vivo
fertilization episodes in infertile women. Infertility 13 : 59.
1990.

7.

Hinduja, I.N., Mehta, R., Gopalkrishnan, K., Puri, C.P.,
Shah, J., Singh. V.P. and Anand Kumar, T.C. Comparison
of four different ovarian stimulation protocols in an in vitro
fertilization and embryo transfer programme. Indian J Med
Res 94 : 405, 1991.

8.

Fishel, S. The use of micro assisted fertilization in men. In:
Gamete Embryo Micromanipulaiion in Human Reproduction.
Eds. S. Fishel and M. Simond, Edwards Arnold, London
1993, p. 10.

9.

Gopalkrishnan, K., F^idwal, V. and Balaiah, D. Efficiency
of routine semen analysis to predict functional and structural
integrity of human spermatozoa. Indian J Expl Biol 33 : 652,
1995.'

Analysis of spermatozoa chromosomes

Analysis of human sperm chromosomes was made
possible by activating sperm within zona free hamster eggs.
This, technique is of immense value in evaluating the
normality of sperm chromosomes. The technique has been
standardized. At present, analysis of sperm chromosomes
from male partners of couples having a history of sponta­
neous abortions is being carried out.
Conclusion
Most of the studies have been done to gain an insight
into the causes of infertility more so of male infertility. The
basis of the studies on male factor infertility is to focus and
standardise simplified techniques and options of infertility
treatment and to make available to most people. Our data
give value to the first line of therapy such as intrauterine
inseminations in male factor subfertility before attempting
the use of sophisticated techniques such as IVF. In the
absence of an adequately supported ART programme these
simpler procedures should be developed for the economi­
cally poorer sections of the population. This will require
special emphasis on intracytoplasmic sperm injection which
will apart from offering treatment, provide good research
material. Infections like STDs present as the most important
and most preventable cause of infertility hence these should
be studied in detail. Apart from the purely economic reasons
one more reason for concern is the prevalence of multifac­
torial infertility. The diagnosis and treatment/managenient
of infertility being a multifactorial problem requires a lot of
104

10. Jeyendran, R.S., Vender Ven, H.H., Perez-Pelaez, M., Carbo,
B.G. and Zaneveld, I J.D. Development of an assay to assess
the functional integrity of the human sperm membrane and
its relation to other semen characteristics. J. Reprod FertU 70:
219, 1984.
11. Gopalkrishnan, K., Padwal, V.. D'Souza. S. and Shah, R.
Severe asthenozoospermia — A structural and functional
study. Jnt J Androl 18 (Suppl. 1) : 67, 1995.

12. Gopalkrishnan, K., Hinduja, I.N. and Anand Kumar, T.C. In
vitro decondensation of nuclear chromatin of human sperma­
tozoa: Assessing fertilizing potential. Arch Androl 27 : 43,
1991.
13. Gopalkrishnan, K., Hinduja, I.N. and Anand Kumar, T.C.
Assessment of mitochondrial activity of human spermatozoa
with reference to motility and viability in fertile and infertile
men. Mol Androl 3: 243, 1991.
14. Gopaikrishnan, K., Hinduja, I.N. and Anand Kumar, T.C.
Pin heads (headless) spermatozoa as markers in fertile and
infertile men. Adv Contract^ Deliv Syst 5 : 361, 1989.

15. Gopalkrishnan, K., Hinduja, I.N., Lau.'. P. and Mehta, A.P.
Role of microbial study in selection of su^iects for in vitro

fertilization and embryo transfer (IVF-ET). Indian J Med Rm
88 : 141, 1988.

16. Peter, J J., Viidya, R.A., Rege, N.R., Gokam, V.V., Phadke,
A.G. and Ambani, L.M. Cytogenetic abnormalities in male
infertility. Indian J Med Res 71 : 225, 1980.
17. R司o, R., Alagappan, R.K., Patrizio, P. and Page, D.C.
Severe oUgozoospermia resulting from deletions of azoospermia
factor gene on Y chromosome. Lancet 347 : 1290, 1996.

This write-up was contributed by Dr. Kamala Gopalkrishnan,
Dr.PervinK. Meheiji and Dr. HS. Juncja, Institute fbr Research
in Reproduction, Mumbai.

Ab.FACTS

Some Research Projects Completed Recently
Association of (Campylobacter pyloridis) Helicobacter
pylori in patients undergoing gastroscopy fbr benign
peptic and duodenal ulcers

Antral mucosal biopsy tissues of 100 patients each of
non-ulcers dyspepsia, benign gastric ulcer and with other
diseases of gastrointestinal tract were studied fbr analysing
the association of Helicobacter pylori with disease. The
biopsy samples obtained on endoscopy from apparently
healthy region of the antrum away from the ulcer site served
as control. The biopsy samples were subjected to urease tests
(indigenously standardised), culture and histopathology.
H. pylori was found to be significantly associated in
patients with gastritis and gastric, duodenal ulcers and gas­
tric carcinoma. Brain heart infusion agar with 7 per cent
horse serum, growth supplements and Butzler antibiotic
supplements supported the growth of77. pylori. SDS-PAGE
analysis revealed the presence of all the major proteins
except* the 120 kDa protein which was not present in all the
isolates. A liquid and a semi-solid urease test standardized
indigenously was found to be comparable and cost effective
than the commercially available CLO (Campylobactor Like
Organism) test.

Cytopathic effect was produced by broth-culture
filterates of H. pylori confirming the production of a cytotoxin in HeLa cell line. Intraperitoneal injections of cell
sonicates or live suspensions of H. pylori produced lethality
in Balb/c mice. However injections of heat treated suspen­
sion did not produce such effects confirming the production
of heat labile toxin by H. pylori.

Analysis of data on clinical history of patients &iled to
reveal the role of alcohol and smoking as associated risk
factors in the ulceration process in the patients with H. pylori
infection. Irr^ular dietary habits seemed to play a role in
the ulceration process.

PCR analysis showed the presence of similar DNA
sequences in both Campylobacter jejuni and H.pylori.
Usha Anand Rao
Department of Microbiology
Dr. ALM Postgraduate Institute
of Basic Medical Sciences
Madras.
Calcium homeostasis in isolated human parietal cells
in normal and altered physiological states

The study was carried out to ascertain the basal status
of intracellular free calcium and calcium transport across the
plasma membrane in an isolated parietal cell population
derived from patients with and without duodenal ulcer.

Multiple endoscopic gastric mucosal biopsies from the
corpus of the stomach in patients undergoing endoscopic
evaluation for epigastric pain/reevaluation of duodenal ulcer
were processed and isolated parietal cells were studied.
Gastric biopsy tissue taken 5 cm away from the lesion
in subjects with suspected gastric carcinoma but having
normal acid levels were used as controls. Patients taking
calcium antagonists and those concurrently taking % an­
tagonists were excluded. Relevant clinical details including

105

smoking habits, alcohol intake, and drug history were
recorded. Intracellular calcium was estimated in 21 patients
using fiira-2-acetoxymethyl ester. Calcium influx was
determined in 26 patients and efflux in 12 patients (in 10
patients both influx and efflux could be performed together)
by using radioactive calcium. Acridine orange retention
was used to assess acid production by parietal cells in 20
patients.
A homogeneous population of viable (>75%) parietal
cells was obtained with a yield of 1.75xl06 cells/ml. The
cells had characteristic eosinophitic cytoplasm and concen­
tric nucleus. The purity ofthe preparation was demonstrated
by a modest three-fbld rise in K+- dependent paranitrophenyl
phosphate activity from the "crude" to the pure after precoll
density gardient. Patients with an active duodenal ulcer had
a higher intracellular free calcium (239.75 nmol/106 cells)
as compared to patients who had nonspecific endoscopic
findings (mean 143.36 nmol/106 cells) and patients with
normal endoscopy (mean 145.6 nmol/106 cells). The acri­
dine orange retention as a measure of acid secretion was
higher but comparable in patients with active duodenal ulcer
(32.55%) and nonspecific endoscopic findings (35.32%) as
compared to individuals with normal endoscopy.

Only calcium influx at 20 min was significantly more
in patients with duodenal ulcers as compared to the control
group and those with nonspecific changes at endoscopy.
There was no difference between the groups (with active
duodenal ulcer, non-specific changes at endoscopy and
normal endoscopy) in calcium influx at 0 and 60 min;
calcium efflux at 0, 20 and 60 min. None of the parameters
tested showed consistent variations that could be attributed
to smoking, alcohol intake or presence of nonspecific
changes at endoscopy.
It is thus concluded that in the unstimulated state cal­
cium homeostasis in isolated parietal cells of patients with
duodenal ulcer shows only a minimal difference as compared
to controls.

Upjeet Kaur
Department of Experimental Medicine
Postgraduate Institute of Medical
Education and -Research
Chandigarh.
Publication
Kaur, U. Agnihotri, N., Kaur, S.t Khuller, M., Bambery, P., Sood, G.
and Singh, K. Duodenal ulcer: Calcium status in isolated parietal cells.
Dig Dis Sci 40: 887, 1995.

ICMR NEWS
Shri H.D. Deve Gowda, Hon'ble Prime Minister of
India,presented the ICMR Awards and Prizes to outstanding
Biomedical Scientists on October 15, 1996. He also
launched the concluding ceremony of the year long 85th
Anniversary celebrations of the ICMR. Shri Saleem I.
Shervani, Hon'ble Minister of State for Health and Family
Welfare and President ICMR, presided over the function.

***
The following meetings of various technical committees/groups of the Council were held:
Meetings of the Scientific Advisory Committees of the
ICMR Institutes/Centres:

Institute of Cytology and
Preventive Oncology,
New Delhi.

September 25, 1996

Institute of Immuno
haematolgy; Mumbai

September 26, 1996

Tuberculosis Research Centre,
Madras

September 27, 1996

106

Institute for Research in
Medical Statistics (both
Delhi and Madras chapters)
held at Madras
National Institute of Nutrition,
Hyderabad

Food and Drug Toxicology
Research Centre, Hyderabad
and
National Centre for Laboratory
Animal Sciences, Hyderabad

September 28, 1996

October 4, 1996

(October 5, 1996)

Meetings of the Scientific Advisory Groups (SAGs)/
Scientific Review Committees (SRCs)/ Project Review
Committees (PRCs)AVorking Groups/Task Forces (TFs)
held at New Delhi:
SRC of the Division of
Reproductive Health and
Nutrition

September 17-18, 1996

Working Group of' the
National Committee for
Research on Human
Reproduction (NCRHR)
Sub-committee on
Socio-behavioural Aspects
of Reproductive Health
Research

Working Group of
NCRHR Sub-committee
on Basic and Applied
Research in Human
Reproduction

September 19-20, 1996

the XXIV European Congress of Cytology at Ljubljana
(September 21-24, 1996).
Dr. T. Shanta Devi, Dy. Director, Tuberculosis Re­
search Centre (TRC), Madras, participated in the meeting
of the International Union Against Tuberculosis and Lung
Diseases (IUATLD) at Paris (October 2-5, 1996).

September 25-26, 1996

PRC for Projects in
Non-Communicable Diseases

September 27, 1996

SAG of the Division of
Publication and Information

September 30, 1996

Working Group of NCRHR
Sub-committee on Clinical
Research in Human
Reproduction

September 30 —
October 1, 1996

TF on Monitoring of Adverse
Drug Reactions in India

October 8, 1996

Working Group of NCRHR
Sub-committee on Operational
Research in Human Reproduction

Dr. C.N. Paramasivan, Dy. Director (Senior Grade),
TRC, Madras, participated in a joint WHO/IUATLD Glo­
bal Working Group Workshop on Antituberculosis Drug
Resistance Surveillance at Paris (October 6-7, 1966).
Dr. S.K. Bhattacharya, Director, National Institute of
Cholera and Enteric Diseases, Calcutta, delivered the
Alastair Ironside Memorial Lecture of 1996 at Manchester
(October 10, 1996).

Dr. M.D. Gupte, Officer-in-Chaige, CJIL Field Unit,
Avadi, Madras, participated in the meeting of the WHO
Task Force on the Monitoring and Evaluation of Elimination
of Leprosy, as also in the II International Conference on the
Elimination of Leprosy as a Public Health Problem at New
Delhi (October 10 and 11-13, 1996 respectively).

Oration:

October 11-12, 1996

Participation of ICMR Scientists in Scientific Events:

Dr. P.K. Nag, Asstt. Director, National Institute of
Occupational Health, Ahmedabad, participated in the Inter­
national Congress on Occupational Health，96 at Stockholm
(September 15-20, 1996).

Dr. D.K. Das, Officer-in-Charge, Institute of Cytology
and Preventive Oncology, New Delhi, participated in

Prof. V. Ramalingaswami, National Research Professor
and Professor Emeritus, All India Institute of Medical Sci­
ences, New Delhi, delivered the II Dr. B.R. Ambedkar
Centenary Award (1993-94) Oration of the ICMR on
"Changing Paradigms of Infectious Diseases in Developing
Countries" at Vigyan Bhavan, New. Delhi on October 15,
1996.
•••
The Council announces with deep r^ret the sad demise
of three of its scientists viz Dr. V Dhanda, Director, Vector
Control Research Centre, Pondicherry, on August 20,1996;
Dr P. Chattopadhyaya, Asstt. Director, National Institute of
Occupational Health, Ahmedabad on August 16, 1996; and
Dr. D.S. Chaudhury, formerly Deputy Director of the Ma­
laria Research Centre, Delhi on June 6, 1996.

107

ICMR AIDED SYMPOSIA/SEMINARS/WORKSHOPS/COURSES/CONFERENCES
Symposium/Seminar/Workshop/
Coune/Conference

Date & Place

Contact Address

XX National Congress of Indian Association of Medical
Microbiologists.

October 31 November 3, 1996;
(at Agra)

Dr. V.M. Katoch, Organising Secretary of the
Congress, Department of Microbiology, Central
Jalma Institute for Leprosy, Agra-282001.

Symposium on Arid Ecosystem of Thar Desert of
Rajasthan and Evolutionary Ecology and Logic of Life.

November 1-3, 1996;
(at Jodhpur)

Dr. A.R. Reddy, Convenor of the Symposium,
Defence Laboratory, Jodhpur-342011.

November 4-9, 1996;
(at Nauni-Solan)

Dr. P.L. Gautam, Organising Secretary, Dr. Y.S.
Parmar University of Horticulture and Forestry,
Nauni-Solan-173230.

Second Asian and Oceanic Congress of Andrology.

November 16-20, 1996;
(at Chandigarh)

Dr. N.R. Kalla, Chairman of Organising Commi­
ttee of the Congress, Department of Biophysics,
Panjab University, Chandigarh-160014.

XX National Conference of the Indian
Surgery of Hand.

December 7-8, 1996;
(at Agra)

Dr. S. Husain, Organising Secretary of the Con­
ference, Department of Plastic and Reconstnictive Surgery, Central Jalma Institute for Leprosy,
Agra-282001.

International Workshop
Plants.

on

Prospects

of Medicinal

Society for

COUNCIL'S TRAINING PROGRAMMES
Nutrition

Training Course for Laboratory Animal Supervisors
(September 2-November 29, 1996).

At the National Institute of Nutrition, Hyderabad:

Annual Training Course in Nutrition (December 1,
1996-February 28, 1997).

Haematology
At the Institute of Immunohaematology, Mumbai:

Laboratory Animal Technology

At the National Centre far Laboratory Animal Science,
National Institute of Nutrition, Hyderabad:

Training Course in Advanced Haematology and
Immunohaematology (September 17-October4,1996).

EDITORIAL BOARD
Dr. G.V. Satyavati

..Chairperson

Dr. Badri N. Saxena
Dr. Bela Shah

Member
Member

Dr. N. Medappa

Editor

Printed and Published by Shri J.N. Mathur for the Indian Council of Medical Research, New Delhi

at the ICMR Offset Press, New Delhi-110029
r'n, 21813/71

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