Reproductive Health

The overall burden of reproductive ill health is very high and many women die every year due to the complications of pregnancy and child birth. Maternal mortality which continues to remain unacceptably high, glaringly reflects the inequity in women’s access to basic life saving interventions as well as inadequacy of the health care system. Most maternal deaths occur due to haemorrhage, sepsis, toxaemia, obstructed labour and unsafe abortions while safe and affordable technologies to prevent such deaths do exist. Equally important issues related to this are nutritional anaemia, adolescent pregnancies, and gender discrimination. The high population growth over-shadowing the health gains as reflected by increase in life expectancy and impressive decline in infant mortality, has resulted in increased demand on the already over stretched health care delivery system. The Council’s research relating to reproductive health therefore encompasses interrelated areas of family planning, safe motherhood, prevention and management of complications of abortions, reproductive tract infection (RTIs)/sexually transmitted diseases (STDs), infertility, adolescent health and gender perspective and are directed to address the issues related to reproductive health through basic and clinical research as well as operationalising the existing knowledge and available technologies. Several new initiatives have been launched during the year under report. These researches have been undertaken through the Council’s Institute for Research in Reproduction (IRR), Mumbai, the network of Human Reproduction Research Centres (HRRCs) located in different parts of the country and several non-ICMR institutes.

The IRR, Mumbai continued its focus on research in basic reproductive biology. A human sperm antigen, 80kDa, has been identified from human sperm extract and efforts are ongoing to develop this peptide as an immunogen for developing an antifertility vaccine. Efforts are also on to develop integrin as a clinical marker to assess the fertility potential in men. The nucleotide sequence of cDNA clone of bonnet monkey oviductal glycoprotein (OGP) synthesized last year was compared with cDNA nucleotide sequences of human, baboon and rhesus monkey OGP this year. Simultaneously a recombinant protein was also synthesized from cDNA clone of bonnet monkey OGP to raise antibodies against OGP.

Emergency contraception is useful in a situation where the prevalence of use of regular contraceptive is low, in couples using barrier methods or oral pills or in couples having infrequent sex. The wider use of this method can prevent unwanted pregnancies and abortions. Since this method is not widely available or known in the country, a study has been initiated in 31 HRRCs to assess the knowledge and demand of these methods.

Male participation in the Family Welfare Programme is very poor and acceptance of vasectomy particularly is low. Phase III clinical evaluation of indigenously developed male intravasal contraceptive RISUG (styrene maleic anhydride dissolved in dimethyl sulphoxide) has been initiated as a multicentre study.

Evaluation of the feasibility of using pregnancy kits for early detection of pregnancy (within 30 days of the missed period) at the field level, so that women could seek early antenatal or MTP services, revealed that paramedical workers can use the kits effectively in the field, however, there is a need to generate awareness about early detection of pregnancy for better reproductive outcome. A study on the management practices of meconium stained amniotic fluid and meconium aspiration syndrome in the newborn, a major obstetric complication during delivery, has been initiated in 31 HRRCs.

A study on feasibility of using syndromic approach for diagnosis and management of RTI/STDs at the PHC level has been initiated at 10 HRRCs. The training of paramedical workers in identification of symptoms indicative of RTI/STDs is complete and field work is ongoing.

An expert group on neonatal health has been constituted to address neonatal mortality and related issues. Project proposals on innovative approaches for home-based management of sick neonates and other thrust areas identified by the Group have been developed. An Indo–US agreement on bilateral collaboration in maternal and child health research has been finalised and the Council will be acting as a nodal agency to steer this programme.

FERTILITY REGULATION

CONTRACEPTION

Basic Research

Basic research in the area of male and female fertility regulation was carried out mainly at IRR, Mumbai.

Magainin Peptides

Earlier studies at IRR had indicated that Magainin-A (Mag-A) can be used as a safe and effective intravaginal contraceptive in rats and rabbits. In monkeys Mag-A (1 mg) introduced intravaginally on the day of ovulation (mating confirmed by the presence of sperm in the vaginal smears) prevented pregnancy in all five animals. These animals returned to regular cycling, suggesting the suitability of Mag-A for contra-ceptive use.

Role of Disulphide Bonds of the b-subunit in Receptor Binding of hCG and in Heterodimer Formation

The disulphide bond between cysteine (Cys) residues at 93 and 100 of hCG-b is the ‘determinant loop’ which specifies the activity of the heterodimer to be CG, LH, FSH or TSH. The synthesized disulphide loop peptide (Cys93-S-S-Cys100) 92-101 hCG-b was checked for its ability to inhibit radiolabelled hCG binding to its receptor on the sheep corpora lutea membrane. The results indicated that (i) the disulphide loop structure between Cys residues at 93 and 100 is probably not essential for receptor binding of hCG (ii) the net positive charge of the region between 93 and 100 alone is not sufficient for receptor binding, and (iii) the Cys residues at 93 and 100 along with the net positive charge seem to be the minimum requirement for receptor binding of hCG.

Studies on Mammalian Oviductal Glycoprotein

The earlier studies at IRR had demonstrated the ability of antibodies to glycoprotein secreted from epithelial cells of oviduct (OGP) to interact with ovulated eggs and sperms and inhibit fertilization, implying a critical role for OGP during fertilization and embryo development. The partial cDNA encoding bonnet monkey OGP has been successfully cloned. In the experiments carried out this year, the nucleotide sequence (AF 132 215) showed 96% homology to human, baboon and rhesus monkey estrogen induced OGP. The cDNA clone of bonnet monkey OGP was recloned into pet 20b vector to obtain recombinant protein and to raise antibodies to OGP. The protein could be immunostained with antibodies to human OGP, baboon OGP and antipeptide antibodies to the well conserved region of mammalian OGP. Rabbit antibodies to rOGP were developed and it immunostained enriched human OGP on Western blots and immunostained epithelial cells secreting OGP in murine oviductal section taken during early pregnancy. Ability of these antibodies to interfere in in vitro fertilization is being examined.

Evaluation of Antifertility Effect of FSH Binding Inhibitor

Human ovarian follicular fluid is being purified and characterized for follicle stimulating hormone binding inhibitor (FSHBI). The peptide inhibiting FSH binding to ovarian granulosa cells showed a single peak on reversed phase high performance liquid chromatography (RP-HPLC). The effect of this peptide on progesterone (P4) production by granulosa cells was studied in vitro. FSH at concentrations of 0.3-30 µg/well gave a dose dependent increase in P4. Partially purified peptide (hGF2 fraction) alone could not affect P4 production but in the presence of 10 ng FSH, it inhibited P4. The highly purified peptide inhibited P4 production at a dose of 1.0 µg. Thus FSHBI acts by regulating P4 production from granulosa cells (Fig.10&11).

Role of Integrins in Implantation

To date, the role of integrins (ITGs) in mediating embryo-endometrial interactions remains poorly understood. The studies at IRR in the murine model suggest that a4, a6, a1 and b3 are maximally expressed in the endometrium on day 3 or 4 after fertilization. The expression of a4, a6, a6 b1, a5 ITGs on oocytes and blastocysts is stage dependent. Down regulation of a1 and b3 ITG expression by interleukin-2 leads to failure of implantation. It is proposed that ITGs may have a role in implantation.

Expression of Endometrial Functional Markers in Fertile and Infertile Bonnet Monkeys

Studies on the expression of selected factors e.g. steroid receptors, cytokines and growth factors as well as identification of novel endometrial markers were carried out on the endometrial samples collected from bonnet monkeys during different phases of the menstrual cycle. Studies were also carried out to correlate the expression of these markers during the peri-implantation period with the fertility outcome in the animals treated with different doses of an antiprogestin, onapristone. Results obtained from the studies initiated last year were substantiated by carrying out similar studies on a larger number of animals. Expressions of progesterone receptor, transforming growth factor (TGFb) (Fig.12), and its receptor (TGFbR) (Fig.13) were downregulated in the endometria from infertile animals as shown by RT-PCR studies. No significant correlation was seen between expression of these factors and the fertility status of animals.

Several differentially expressed cDNA fragments were identified by DDRT-PCR studies. These fragments either showed upregulation or downregulation in the endometria from treated animals as compared to the control group. One of these cDNA fragments was cloned in pGEMT vector and sequenced using T7 and Sp6 primer. This cDNA fragment of 88 bp showed high expression during the peri-implantation period in the control bonnet monkeys while in the animals rendered infertile with onapristone, expression of this fragment was downregulated. Sequence homology search of this cDNA fragment did not reveal significant homology to any known endometrial factor. Further characterization of this and other differentially expressed fragments and their potential role in implantation may help us to identify the endometrial factors which if expressed aberrantly may lead to infertility. This may also facilitate identification of newer targets of contraception.

Development of an in vitro Screening System for Evaluating the Potencies of Progestins developed for Therapeutic and Contraceptive Applications

An estrogen independent, progesterone receptor (PR) reporter gene system using mammalian cells is being developed at IRR to relate the response of the test progestin to progesterone in a single assay to get the true potency estimates of the test compounds. To achieve this objective a progesterone responsive reporter gene construct pUGCAT was introduced into the mammalian cell line T47D which retained the PR expression in culture. The transfection of T47 cells with pUGCAT has been accomplished. Exposure of the transfected cells to 10-5 and 10-7M progesterone expressed the CAT enzyme. Standardization of the progesterone dose related response curve for the transfected UGCAT with six to eight doses of progesterone ranging from 10-4 to 10-12M gave two types of response patterns of CAT expression. To get consistent pattern of CAT expression optimization is being continued.

Purification and Cloning of an Epididymal Sperm Maturation Antigen

An epididymal sperm maturation antigen has been purified from sheep cauda using conventional biochemical techniques. The blue-sepharose bound fraction containing the 26 kDa protein was further subjected to chromatography on HPLC MonoQ column, resulting in a homogeneous preparation of 26 kDa protein as seen by SDS-PAGE electrophoresis. N-terminal sequencing of the purified protein is in progress.

The cDNA clone isolated from a lambda ZAP monkey epididymal cDNA library using monoclonal antibodies to the 26 kDa protein, was sequenced. Sequence analysis of 995 bases at the N-terminal and 1100 bases at the C-terminal have been read and attempts to complete sequencing are underway. No significant homology with sequences in the gene data bank has been obtained so far.

Human Seminal Plasma Inhibin (HSPI) as Sperm Vaccine for Fertility Regulation

The earlier studies at IRR demonstrated that active immunization of adult bonnet monkeys with R-17-BSA conjugate using Freund’s complete adjuvant (FCA) resulted in a significant decrease in sperm motility without altering sperm count or serum LH and testosterone levels. An inverse correlation between circulatory antipeptide antibody and sperm motility was observed. These results are promising but as FCA cannot be used as an adjuvant in humans it was decided to use muramyl dipeptide (MDP) as an adjuvant with squalene and arlacel for emulsification. Initial immunization studies with MDP in rabbits resulted in antibodies that were able to recognize native protein and in titres similar to those obtained when FCA was used as an adjuvant. Currently a new group of ten monkeys is being monitored for baseline data on semen parameters and will be immunized with R-17-DT using MDP as an adjuvant and the effects on fertility will be studied.

Studies with Human Sperm Antigen

A 80 kDa human sperm antigen (HSA) is one of the potential candidate antigens for immuno-contraception, as it has been demonstrated to be immunogenic, efficacious, conserved and specific to sperm. It was reported that chemically the antigen is unique as N-terminal amino acid sequence of the native protein and its fragments obtained by enzymatic digestion did not show sequence homology with any of the known proteins in the database. These peptide sequences were synthesized and characterized further. The passive administration of these antipeptide antibodies in male rats caused the agglutination of the epididymal spermatozoa along with almost complete loss of motility while in female rats the passive administration of antipeptide antibodies also rendered the animals infertile. Active immunization with the synthetic peptides evoked a better immune response in female rabbits than in male rabbits. Antifertility studies in female rabbits are in progress.

Clinical Research

Phase III Clinical Trial with Subdermal Implant NorplantR

NorplantR (6 capsules) was offered as one of the contraceptive choices at the Council’s 31 HRRCs in a study where all the women seeking family planning methods were given a balanced presentation of the existing (IUD, OC, condom, male and female sterilization) and new (NorplantR, once a week pill centchroman, vaginal pessary TODAY and diaphragm) methods of contraception during November 1994 to December 1996. The acceptors of NorplantR are being followed up for a period of 5 years of contraceptive use to evaluate the efficacy and continuation rates. A total of 2048 NorplantR acceptors have been observed for 70,744 women months of use, 154 women have completed 5 years of use so far. One method failure has been reported after 3 years of use. The continuation rates were 92.0, 79.4 , 66.4, 52.5 and 41.0 per 100 users at 1,2,3,4 and 5 years of use respectively. The main reason for discontinuation was menstrual irregularities. The cumulative discontinuation rates for this reason were 3.5, 9.0, 14.3, 20.3 and 25.9 per 100 users at 1, 2, 3,4 and 5 years of use respectively. The study is ongoing and women who are willing to continue with the method are being followed upto 5 years of use. Women who have discontinued the use of NorplantR due to some reason and are exposed to the risk of pregnancy are being followed up for two years for return of fertility as judged by pregnancy and outcome of pregnancy.

Emergency Contraception among Women

Emergency contraceptives are the methods to be used within a short time after unprotected intercourse, so as to avoid unplanned pregnancy. A study has been initiated in 31 HRRCs to determine whether awareness regarding emergency contraceptives exists among women of child bearing age and adolescents and to find out such behaviour, attitudes and opinions that would lead to the risk of unplanned pregnancy; to identify the most acceptable route of administration for emergency contraception and; to determine the existing awareness about emergency contraception among providers. A total of 1000 beneficiaries of family welfare services and 300 providers of these services have been interviewed through a detailed questionnaire.

Major ICMR Research Projects in Reproductive Health

Male Intravasal Contraceptive RISUG

The concept of injecting a bioactive compound into the lumen of the vas deferens to obtain a potentially reversible long-term contraceptive effect is a new dimension in the male contraceptive field. The animal studies and phase I and phase II clinical trials with the injectable contraceptive RISUG, a specific copolymer of styrene with maleic anhydride dissolved in 60 mg solvent of dimethyl sulphoxide, have indicated that the new technique is safe and efficacious. A phase III clinical trial with this preparation has been initiated to evaluate the safety and efficacy of the drug in a large number of subjects. All the subjects will be followed up for six months following treatment to study the immediate and long term adverse effects, semenology and pregnancy protection. A total of 16 subjects have been enrolled in the study.

INFERTILITY

Female Infertility

Role of Hyperinsulinaemia in Women with Polycystic Ovaries

A study was undertaken at IRR, Mumbai, to determine whether women presenting with polycystic ovaries (PCO) with or without hirsutism and/or obesity and those having polycystic ovaries only are at risk of developing insulin resistance or have an altered insulin pattern and their responses to insulin sensitizing agents. The study revealed that women who had PCO and obesity irrespective of their androgen status showed significantly enhanced insulin response compared to those who were obese with normal looking ovaries and normal androgen levels as well as those having polycystic ovaries only.

Induction of ovulation by clomiphene citrate in women desiring conception revealed negative response in 14 of the 18 women treated. Treatment with insulin sensitizing agent, metformin resulted in normalisation of menstrual cyclicity and reduction in weight in 8 of the 14 non responders. However, no spontaneous ovulation was observed. Simultaneous administration of clomiphene/ human menstrual gonadotrophin (hMG) with metformin resulted in ovulation in 50% of them with pregnancy in one.

The results suggest that obesity plays an important role in development of PCO leading to hyperinsulinaemia which can be a risk factor for cardiovascular disease and type II diabetes, if left untreated.

Male Infertility

Molecular Identity and Functional Significance of Progesterone Receptor and Expression of Integrin on Human Spermatozoa

A transcript of 536 bp was detected in human spermatozoa RNA using primers specific for uterine progesterone receptor (PR). The transcript was amplified, cloned in pGEMT vector and sequenced using both forward and reverse primers. The partial cDNA clone for the sperm PR showed complete homology to the classical PR in the regions from nucleotide number 2694 to 3230. The cDNA corresponding to this transcript contains the regions corresponding to the DNA binding domain as well as hormone binding domain. This was suggestive of the molecular similarity between the classical PR and sperm PR. The studies at IRR on the expression of progesterone binding sites in fertile and infertile males demonstrated lesser number of PR positive spermatozoa in infertile males. This indicated that the progesterone receptor on spermatozoa may have a predictive value in assessing the functional status of spermatozoa. A preliminary map of antigenic profiles based on SDS-PAGE/ immunoblot analysis has been constructed. The scanning of protein profiles showed the presence of @ 57 and = 44 kDa proteins unique to fertile samples. The 57 kDa protein was a glycoprotein localized on acrosomal region. Approximately 40% of spermatozoa expressed this protein which was involved in sperm egg interaction. The antigen is being sequenced.

Relevance and Efficacy of Clomiphene Citrate Therapy in Idiopathic Subfertile Males

Of the fifteen idiopathic subfertile men being administered clomiphene citrate 8 were put on an alternate day dose schedule while 7 were put on a daily dose schedule. The former group fared better than the latter in terms of improved sperm density, motility and morphology. There was improvement in two functional parameters related to fertilizing ability viz. hypo-osmotic swelling test and sperm mitochondrial activity index in the alternate day group. Two of these individuals with improved seminal and functional parameters could successfully impregnate their

wives. Two pregnancies ensued post- therapy – one each in the alternate-day and daily-dose regimen groups. Men who did not show any improvement in seminal status were associated with aberrant spermatogenesis at the testicular level. The study is ongoing

MATERNAL AND CHILD HEALTH

Management Practices of Meconium Stained Amniotic Fluid and Meconium Aspiration Syndrome

One of the major problems an obstetrician faces while conducting labour is meconium stained amniotic fluid (MSAF) which is present in 10-20% of all deliveries. Meconium aspiration syndrome (MAS) occurs in 2-4% of all deliveries (or 20-25% of MSAF cases) leading to a mortality of 8-48% and 20% of morbidity at various stages of growth. There are no data available in India on the management practices, outcome of pregnancy, morbidity and mortality of MAS babies. The study has been initiated at 31 HRRCs to assess the management practices for pregnant women with MSAF and to study the morbidity and mortality in newborns with MAS. A total of 4066 cases of MSAF have been enrolled so far; 431 (10.6%) babies born to these women developed MAS. The enrolment of MSAF cases and follow up of MAS babies is ongoing.

Feasibility of using Pregnancy Detection Kit in PHC/SC/Villages and Urban Slums

The early detection of pregnancy is essential for safe motherhood and reproductive health of women. At present no pregnancy kit is available in the National Family Welfare Programme. At the request of Ministry of Health and Family Welfare, ICMR initiated a study to assess the demand for early detection of pregnancy and; to evaluate the sensitivity and specificity of pregnancy tests kits when used under programme conditions [(at PHC/ sub-centre (SC) /Urban Health Centre and at field level)] by paramedical staff.

The study was carried out in two urban slum areas (in Delhi and Calcutta) and two rural areas (in Rajasthan and U.P.) covering one lakh population each. The sample size envisaged at each centre was 1500 subjects, 500 at HRRCs and 1000 in PHC/SC/urban slums. Extensive IEC campaign was launched by the participating centres by distribution of pamphlets and organizing group meetings. The medical and the paramedical staff of the HRRCs and the participating health centres were trained in the use of the kits and the method of confirmation of pregnancy. The interim data indicated sensitivity and specificity of these tests as confirmed by clinical findings at follow up visit to be about 90% when used by paramedical staff. The preliminary analysis indicated that while paramedical workers are able to use the pregnancy kit, there is a need to intensify the IEC compaign.

Assessment of Vaccine Wastage during Pulse Polio Immunization Programme

The requirement of oral polio vaccine (OPV) is presently determined by the guidelines laid down by WHO/UNICEF. Accordingly, a wastage multiplication factor (WMF) of 1.33 which corresponds to 25% wastage of OPV is assumed at present. Since no study of this nature has been conducted in India, the Ministry of Health & Family Welfare entrusted this job to the Council. The Council conducted the study through its network of HRRCs and its permanent institutes in 31 districts from 11 states and 3 Union Territories on 17th January 1999 during the Pulse Polio Immunisation (PPI) programme. All the booths were covered in a district amounting to 30,767 booths in 31 districts. The information on wastage was based on number of vials received at a booth, number of vials used and the number of children given vaccine. The study has shown an overall wastage of 14.7 % doses and a WMF of 1.17 of OPV which is well below the present assumption of 25% wastage and a multiplication factor of 1.33. The wastage during the immunisation should be minimised to make it more cost effective. At the request of Ministry of Health & Family Welfare, it is proposed to initiate a study to determine the amount of wastage during the routine immunization programme for all the six vaccines and suggest methods for reducing it.

Management of Reproductive Tract Infections / Sexually Transmitted Diseases

A study on the feasibility of using syndromic approach for diagnosis and management of RTI/STDs at PHC level where facilities for clinical examination or laboratory facilities are not available has been initiated at 10 HRRCs. A training programme was organized for the Research Officers of HRRCs on interview methodology and syndromic management including wet smear examination. The paramedical staff have been trained in interviewing the men/women in the selected villages and in eliciting information on symptoms indicative of RTI/STDs and high risk sex behaviour. The diagnosis made by paramedical workers will be validated by clinical and wet smear examination of all these men/women irrespective of whether they are symptomatic or asymptomatic. The first part of the study on validity of syndromic diagnosis has been initiated in all the centres. The interview survey is ongoing.

Involvement of Practitioners of Indian Systems of Medicine in the Delivery of RCH Services in Rural Areas

Despite a widespread network of the modern system of medicine and the services available through government and private sources, people in India have utmost faith in the traditional systems of medicine as the services are generally cheaper and available in small towns/ rural areas and the remedies are free from side effects. According to a recent estimate, India has 5.6 lakh formally trained practitioners of Indian Systems of Medicine (ISMPs). These practitioners provide the bulk of primary health care services to the rural population. As such, their services can be utilized for improving the delivery of reproductive and child health (RCH) services. The objective of this study is to involve ISMPs in improving the utilization of RCH services in rural areas within the existing infrastructure available at PHC/district level. The project was implemented in UP, MP and Rajasthan. In each of the selected PHCs, the identification of ISMPs was done and their inclusion in the study was based on their willingness for involvement in the study. The base line surveys of ISMPs and the community have been completed. In the base line community survey 403 ISMPs were identified in the experimental and 357 in the control area. The ISMPs had about 12-16 years in the field; the average area covered was from 8-20 kms. in different centres and the number of villages covered varied from 4-13. On an average every ISM practitioner treated about 292 paediatric cases annually. About 2-5% of ISMPs also provided services for infertility and RTI/STDs. The contraceptive services were provided on an average to around 58 cases annually.

In the experimental area 136 ISMPs were provided orientation training for 3 days on various aspects of RCH care before involving them in the study and were monitored for their involvement in RCH services. These ISMPs have developed close linkages with PHC staff in ensuring the delivery of the services. The data received so far have shown an upward increase in the referral for various RCH services by the ISM practitioners in experimental area by propagating the distribution of contraceptives, antenatal and natal care, MTP, provision of iron and folic acid and vitamin A tablets and ORS packets as well as referral of children for immunisation. In control area no training was provided.

Socio-behavioural Aspects of Menopause

A study was undertaken at IRR, Mumbai to assess the knowledge, attitude and perceptions of women regarding menopause and to study some aspects of sexuality during menopause. A total of 332 women from the low socio-economic strata with established menopause were interviewed personally. The mean age at menopause was 45.34 yr. Although most women had knowledge about the cause of menopause (i.e. menopause is a physiological process), a few (11%) stated that menopause can only occur after the removal of the uterus. Sixty three per cent of women had a positive attitude towards menopause. Majority of them (77%) felt that they were free to take decisions in household matters which were formerly restricted to men. Misconceptions about menopause were reported by 6.3% of women and the most common myth was its association with decrease in eye sight (5%). Overall sex desire was decreased in 40% of women during menopause and total loss of sexual desire was expressed by 18.8% women. The study is ongoing.

 

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