Reproductive outcome in carrier couples of b-thalassemia disorders in a tertiary hospital in central India

The b-thalassemia syndromes and hemoglobin disorders are the major genetic and public health challenges in Central India. In view of dubious credit of the highest infant mortality rate in Madhya Pradesh (62 as against 47 per 1000 livebirths of India in 2011) it was presumed that carrier couples of b-thalassemia disorders might be one of the contributing factors to high mortality. A total of 280 couples including their offspring with at least one affected and/or suspected case of bthalassemia/sickle cell disorders referred to our Centre from a tertiary hospital, Jabalpur during March 2010 to February 2013 were consecutively studied as matched case controls. Out of 280 couples, 200 were found normal and 80 couples had different b-thalassemia disorders. b-thalassemia carrier couples had significantly higher relative fertility (mean number of conceptions, i.e. 2.457 versus 1.480), higher infant mortality (3.5% versus 1.3%), higher below 10 years mortality (7.0% versus 2.7%) and lower surviving offspring (925.9 versus 970.6) than of controls. Still-births were three times lower (12.3), neonatal deaths almost two folds higher (24.7), three folds higher infant mortality (37.0) and almost three times higher below 10 years mortality per 1000 live-births were observed in carriers of b-thalassemia major than in controls. The present study indicated that afflicted couples of these hereditary disorders are increasing the afflicted offspring, being 60.7% surviving against controls (39.3%). This increased production of afflicted (heterozygous and homozygous) offspring leads to increased morbidity and mortality and might be contributing towards increased neonatal/infant mortality in Madhya Pradesh of Central India. As a preventive measure, affected families were imparted genetic/marriage counseling.


Introduction
2][3][4] These disorders have a wide geographical spread throughout the country, the highest prevalence being in the Northwestern, followed by North-eastern, and the lowest being in Southern parts of India. 5The bthalassemia disorders are caused by point mutations or, more rarely, deletions in the bglobin gene on chromosome 11, leading to reduced (b + ) or absent (b 0 ) synthesis of the bchains of hemoglobin. 6Transmission is autosomal recessive.Individuals of thalassemia major (homozygous b-thalassemia) usually present a spectrum of clinical manifestations (signs and symptoms) within the first two years of life with severe anemia, requiring regular red blood corpuscle (RBC) transfusions.
Untreated or poorly transfused individuals of thalassemia major are seen in some developing countries with growth retardation, pallor, jaundice, poor musculature, hepatosplenomegaly, leg ulcers, development of masses from extra-medullary hematopoiesis, and skeletal changes that result from expansion of the bone marrow. 4In persons of homozygous b-thalassemia, low levels of hemoglobin lead to lack of oxygen in many parts of the body.Affected individuals especially the children develop life-threatening anemia or shortage of red blood cells, which can cause pale skin, weakness, fatigue, and more serious complications. 6Persons of homozygous b-thalassemia are at an increased risk of developing abnormal blood clots.They do not gain weight and grow at an expected rate (failure to thrive) and may develop yellowing of the skin and whites of the eyes (jaundice).Affected individuals may have an enlarged spleen, liver, and heart, and their bones are misshapen. 6A few adolescents of thalassemia major experience delayed puberty.Many people of homozygous b-thalassemia, except thalassemia intermedia, have such severe symptoms that they need frequent blood transfusions to replenish their RBC supply.Regular transfusion therapy in homozygous thalassemia leads to iron overloadrelated complications including endocrine (hormonal) complications (growth retardation, failure of sexual maturation, diabetes mellitus, and insufficiency of the parathyroid, thyroid, pituitary, and less commonly, adrenal glands), dilated myocardiopathy, liver fibrosis and cirrhosis. 6iagnosis of thalassemia is based on hematological, biochemical and/or molecular genetics testing. 7Hematological indices of RBC show microcytic anemia.Homozygous thalassemia is characterized by reduced Hb level (<7 g/dL), mean corpuscular volume (MCV) >50<70 fL and mean corpuscular Hb (MCH) >12<20 pg.Thalassemia minor (trait) is characterized by reduced MCV and MCH, with increased hemoglobin A 2 level. 6Peripheral blood smear in affected individuals show morphological changes in RBC such as microcytosis, hypochromia, anisocytosis, poikilocytosis (spiculated tear-drop and elongated cells), and nucleated RBC (i.e., erythroblasts).The number of erythroblasts is related to the degree of anemia, and markedly increases after splenectomy. 6Genetically susceptible individuals to thalassemia include: infants, growing children, adolescents, and a large number of ignorant people.Sometimes, inadequate availability of oxygen to fetus may also lead to abortion, miscarriage or stillbirth.Inherited hemoglobin disorders are responsible for high infant morbidity, mortality and fetal wastage due to no or minimal treatment in India. 8,9he primary purpose of screening is the identification of infants with b-thalassemia for whom early intervention has shown markedly reduced morbidity and mortality. 10nherited hemoglobin disorders are expected to contribute to infant mortality and childhood A great deal of literature is available regarding the clinical and hematological aspects of these disorders, but the details regarding the reproductive outcome in affected couples are scanty in India.In view of credit for the highest infant mortality rate in the state of Madhya Pradesh (62 per thousand live-births in 2011) in comparison to other states and the average of 47 for India; and the high prevalence of hemoglobin disorders in the State 11,12 it was presumed that b-thalassemia disorders might be one of the significantly contributing factors for infant/neonatal mortality in carrier couples in Madhya Pradesh, India.

Material and Methods
A total of 280 couples including their offspring with at least 1 suspected/confirmed case of anemia/hemoglobinopathies routinely referred by the experts (in Gynaecology, Pediatrics, and Blood Bank) for investigations/confirmation of diagnosis, attending Netaji Subhash Chandra Bose Medical College & Hospital, Jabalpur, Madhya Pradesh in Central India were included in the study.The ethical clearance was obtained from the Human Ethical Committee of Regional Medical Research Centre for Tribals (ICMR), Jabalpur.
Confirmed cases of b-thalassemia/sickle cell disorders formed our study group and the negatives, free of hematological disorders/anemia, after rigorous scrutiny, were taken as control group.The cases, suffering from other hemoglobinopathies and/or hematological disorders, and genetic abnormalities were excluded from the study.Those cases with iron deficiency anemia, malaria, accidental or induced abortion, were also excluded from the study.All the non-genetic confounding factors more or less were similar for both groups (matched case controls), being taken from the same population source.Detailed reproductive history of each couple was recorded retrospectively like total number of conceptions, abortions, miscarriages or still-births, live-births, surviving children, neonatal or infant deaths, etc.
In this study, genotypes of couples such as AA/AA stands for normal Intravenous 2-3 mL blood was taken under aseptical conditions from each individual after taking informed/written consent for screening of hemoglobinopathies.Hematological parameters were studied using an automated Blood Cell Counter (Model-MS 5 9, Melet Schloesing Lab., Cergy-Pontoise Cedex, France).Laboratory investigations were carried out following the standard procedures after cross checking for quality control from time to time.The sickling test was performed using 2% freshly prepared sodium metabisulphite solution as a reducing agent for the presence or absence of sickle cell hemoglobin. 13The routine hemoglobin lysate electrophoresis was carried out on cellulose acetate membrane in Tris-ethylenediaminetetraacetic acid-Borate buffer at pH 8.9 and quantification of A 2 fraction of adult hemoglobin was done by elution method. 13,14The value more than 3.5% of A 2 fraction of adult hemoglobin was taken as cut off point for determining the b-thalassemia trait.Those individuals having very high hemoglobin A 2 value, i.e. more than 10% were suspected to have A 2 plus hemoglobin E; and the test was confirmed by the investigations of other family members.Estimation of fetal hemoglobin was done according to technique described by Weatherall. 14he diagnosis of sickle cell-b-thalassemia was based on the findings of hemoglobin A, F, S and A 2 on electrophoresis under alkaline pH, elevated A 2 levels (>3.5%).All the doubtful cases were further subjected to hemoglobin variant analysis for detecting any discrepancy (Bio-Rad Diagnostics, Hercules, CA, USA).
Data results obtained were analyzed as per the standard statistical methods by performing a 2_2 contingency chi-square test to compare normal with each patient subgroup for the difference between the two independent proportions and significance, if any, is also indicated.For collected data, calculations were made for reproductive wastage (abortions, stillbirths, neonatal mortality, and infant mortality) in percentages and also per thousand live-births, etc. Results were given to parents for treatment and further clinical management by the concerned referring doctor.All the carrier/affected persons were imparted genetic/marriage counseling.

Results
Theoretically, it is true that mating of carriers of b-thalassemia has a 25% probability of getting affected offspring, but at the same time it is equally true that each conception has the same probability (25%) of getting a normal or affected offspring with 50% risk of having carrier offspring (as expected), regardless of the previous pregnancy, if any.This independence segregation (dispersal) of defective genes at each conception interferes with the expected outcome of getting a normal, carrier or affected offspring and leads to imbalance of progeny in terms of abortions and still-births and normal, carrier or affected in the family.Results of this study illustrate the actual and factual imbalances of progeny in the vulnerable couples.
There was statistically significantly (P<0.05)higher frequency of neonatal deaths in couples of AS/S-b-thal genotypes (11.1%); and still higher (P<0.001) in AA/b-thal trait genotypes (14.3%) as compared to controls (1.3%).However, the difference between the couples of b-thalassemia disorders as a whole N o n -c o m m e r c i a l u s e o n l y (4.0%) and controls (1.3%) was statistically not significant (<0.10 P>0.05) for neonatal deaths (Table 1).
The mortality below 10 years was observed to be higher in almost all the couples of b-thalassemia disorders (Table 1), specifically significantly higher in couples of AA/b-thal trait (17.8%) genotypes (P<0.001) and b-thalassemia disorders (8.1%) as a whole (P<0.01)than in normal controls (2.7%).
The conventional system of accurate and precise proportional evaluation of abortions and still-births, and neonatal, infant and childhood mortality is the calculation of 1000 livebirths.The number of abortions per 1000 livebirths was almost same in both carrier of thalassemia major (49.4) and control (44.1) couples (Table 2).However, the frequency of abortions in couples of b-thalassemia disorders as a whole was low (27.1).The frequency of still-births in carrier couples of thalassemia major (12.3) was almost three times lower than in controls (40.4) per 1000 live-births.
Neonatal and infant mortality per 1000 livebirths were almost doubled in carriers of thalassemia major (24.7 and 37.0, respectively) than in controls (14.7).However, neonatal and infant mortality per 1000 live-births were much higher in couples of AA/b-thal trait (160.0,respectively) and AS/S-b-thal (125.0,respectively) genotypes (Table 2) than in controls (14.7).
Below 10 years mortality per 1000 live-births (Table 2) was more than two-folds higher in carriers of thalassemia major (74.1) and almost three times higher in couples of b-thalassemia disorders as a whole (86.9) than in controls (29.4).The mortality below 10 years per 1000 live-births was many folds higher in couples of AA/b-thal trait (200.0),AS/S-b-thal

Discussion
The findings of the present study are interesting and revealing many practical aspects of those communities which are genetically susceptible to recessively inherited hemolytic disorders such as b-thalassemia disorders in Central India (Table 1).The present study strongly supports the contention that hereditary factors in the carrier couples, apart from concomitant non-genetic confounding factors, are responsible for the high fetal wastage in the form of abortions, still-births, neonatal and infant mortality, and mortality below 10 years in India.These disorders are the outcome of nonviable homozygosity due to inbreeding inadvertently taking place in the vulnerable communities of the region. 15This is the 1 st study carried out taking into consideration these causative aspects of high mortality in the state of Madhya Pradesh, India.
Neonatal mortality was almost doubled in carriers of thalassemia major (24.7) than in controls (14.7) per 1000 live-births in the present study (Table 2).These findings have been supported by the high neonatal mortality rate which was 44 and 33 for Madhya Pradesh and India, respectively for the year 2010.These results are consistent with the similar findings reported from Odisha state in India, where the study 8 has pointed that there could be several reasons for raised neonatal mortality such as birth asphyxia, pre-eclamptic toxemia, puerperal sepsis, prematurity, low birth-weight babies, maternal malnutrition, serious malarial or other urino-genital tract infections, diarrhea, immunological incompatibility (such as ABO and Rhesus blood groups) between mother and fetus (hemolytic disease of the newborn), congenital anomalies or hereditary hemolytic disorders such as glucose-6-phosphate dehydrogenase deficiency, physical injuries during intra-uterine period/delivery, etc. besides other abnormal genetics.
There are very few studies available on the subject of fetal wastage in India.The overall neonatal and infant mortality was recorded for carrier couples of thalassemia major to be 24.7 and 37.0, respectively per 1000 live-births against controls (14.7) in the present study (Table 3).Similarly, the infant mortality rate of 62 was the highest for Madhya Pradesh per 1000 live-births as compared to 47 of India for the year 2011.Further, the findings of the present study for carrier couples of b-thalassemia major per 1000 live-births are not at variance from that of Odisha with respect to neonatal (24.7) and infant (37.0) mortality in Madhya Pradesh.This consistency of findings shows almost similar pattern of consanguinity or inbreeding in Odisha in the communities possessing recessively inherited genetic characters such as b-thalassemias 16 that lead to increased homozygosity of deleterious genes, and subsequently to reproductive wastage, that is, abortions, still-births, neonatal and infant mortality or childhood mortality in Central India.
Women's poor reproductive health in India is affected by a variety of socio-cultural and biological factors. 10Underlying poor reproductive health among Indian women is due to their poor overall health status on the one hand, and an inadequate delivery system to cater to the needs of secluded, shy and de-valued women on the other.Thus, efforts to improve women's education, raise enrollment and attendance rates of girls in school and reduce the drop-out rate on the one hand, and enhance income autonomy for women on the other, are fundamental issues in the long run for improvements of women and their family health; no less important are the improvements in quality and breadth of services catering to reproductive health needs of the women in Central India.
Unsafe motherhood is still a reality in much of Central India and particularly in its rural areas.Few women have access to antenatal care, high risk cases go undetected, anemia is acute during pregnancy, deliveries are conducted largely by untrained attendants in unhygienic conditions and knowledge of health and nutrition needs during pregnancy and the post-natal period are poorly disseminated.Disparities in women's access to health care at delivery are apparent.The official programs for maternal and child health cares reach a few pregnant and lactating women.Maternal health activities are unbalanced, focused on immunization, and provisions of iron and folic acid rather than on sustained women care or on the identification and detection of genetic abnormalities that are detrimental to their health and do not provide refer-ral services to high risk pregnancies in rural Central India.
Reproductive health services thus imply the enabling of individuals, both males and females, to decide freely and responsibly, the number, spacing and timing of their children.For this they must have the information and the means to attain the highest standard of sexual and reproductive health.Reproductive care, therefore, includes family welfare counseling, contraceptive information and services; safe delivery and post-natal care; infant, and women's health care; abortion; treatment of reproductive tract infections and sexually transmitted diseases; and counseling on human sexuality, reproductive health and responsible parenthood, which is lacking in Central India.
Looking at the overall scenario of reproductive wastage and survival in different carrier couples of b-thalassemia disorders, the number of normal (free of any type of thalassemia) children (66/168; 39.3%) born to carrier couples was lower than the afflicted heterozygous and homozygous children (102/168; 60.7%), indicating the progressive increase of afflicted heterozygous and homozygous offspring in these families (Table 4).This trend shows the lower fitness of the carrier couples or affected families, and consequently of the vulnerable population(s).
Urgently needed is greater insight into underlying risk factors, into why women's reproductive health needs remain unmet.Equally important is the need to structure the reproductive health services to respond to the gynecological and obstetric conditions that women experience, and which take into consideration the social, cultural and economic constraints that women face in expressing these conditions and in accessing these services.What are the leading reproductive morbidities observed in community settings?What are the leading conditions observed for different age groups -those in the reproductive ages, adolescents, women  Thalassemia minor is clinically asymptomatic but some subjects may have moderate anemia.Genetic counseling is recommended and prenatal diagnosis may be offered. 17,18Treatment of thalassemia major includes regular RBC transfusions, iron chelation and management of secondary complications of iron overload.In some circumstances, spleen removal may be required.Bone marrow transplantation remains the only definitive cure currently available.Prognosis for individuals of b-thalassemia has improved substantially during the last 65 years following recent medical advances in transfusion, iron chelation and bone marrow transplantation therapy. 9,17,18However, cardiac disease remains the main cause of death in patients with iron overload in Central India.
To bring the reduction/prevention and control of b-thalassemia disorders/sickle cell disorders in affected families, all the referred and affected families were imparted genetic/marriage counseling to effectively prevent the birth of an abnormal child in their families 17,18 for the betterment of future generations.

Conclusions
The increased production of surviving afflicted carrier and diseased offspring (60.7%) than the normal children (39.3%), leads to increased morbidity and mortality; and perhaps may be contributing towards increased neonatal/infant mortality in Madhya Pradesh.This is the 1 st study that has revealed that hereditary causes also, apart from other concomitant non-genetic factors, are responsible for the high neonatal/infant mortality (reproductive wastage) in the vulnerable population.Further, the progeny of couples with b-thalassemia disorders contribute disproportionately to the high neonatal/infant mortality in Madhya Pradesh.Affected families were imparted genetic/marriage counseling.It was envisaged to bring awareness among these couples through genetic/marriage counseling about these genetic disorders and their causal effects on health.Their eradication is necessary because they are not curable but preventable through carrier detection, prenatal diagnosis and, education and genetic counseling.
i a l u s e o n l y mortality if left untreated.Unfortunately, in the state of Madhya Pradesh, there is a dearth of physicians specializing in the clinical management and treatment of blood disorders including the sickle cell disease and b-thalassemia syndromes.
husband and normal wife (control); AA/b-thal trait, denotes for normal husband and b-thalassemia trait wife or normal wife and b-thalassemia trait husband; AS/b-thal trait, for sickle cell carrier husband and b-thalassemia trait wife or vice versa; bthal trait/b-thal trait, denotes that both husband and wife are carrier for thalassemia major; AS/S-b-thal, means one partner is carrier for sickle cell disease and other partner is suffering from sickle cell-b-thalassemia (having compound disease, i.e. sickle cell and bthalassemia); b-thal trait/S-b-thal, stands for one partner being carrier for thalassemia major and the other counterpart is sickle cellb-thalassemia; the genotype AA/S-b-thal, stands for one partner being normal and the other partner is sickle cell-b-thalassemia; AE/b-thal trait, stands for one partner being carrier of hemoglobin E disease and the other partner is carrier of thalassemia major; SE/bthal trait, denotes that one partner is carrier of thalassemia major and the other partner is suffering from a compound disease of hemoglobin E and sickle cell trait; b-thal Disorders (b-thalassemia disorders), mean here all the above diagnostic genotypes (pooled together) except the normal controls.A total of 280 couples were referred during the period from March 2010 to February 2013.Out of 280 couples, 200 were found normal and 80 couples had different bthalassemia/sickle cell disorders.A total of 992 persons (474 males and 518 females) were investigated for b-thalassemia/sickle cell disorders including the controls.
/S-b-thal 125.0 125.0 b-thalassemia disorders (average) 43.5 54.3 Madhya Pradesh # 44.0 (2010) 62.0 (2011) India § 33.0 (2010) 47.0 (2011) *Birth to 28 days (neonatal mortality); °birth to 365 days or within 1 year (infant mortality); # neonatal mortality rate, # neonatal mortality rate; § infant mortality rate, taken from Registrar General of India, Sample Registration System (SRS Bulletin, December 2011).N o n -c o m m e r c i a l u s e o n l y beyond the reproductive ages?What are the socio-cultural constraints women face in acquiring good reproductive health and safe pregnancy and delivery?What kinds of interventions can be designed to respond to these needs?Health facilities at the community level are poorly equipped to deal with gynecological emergencies.Therefore, in depth explorations are required especially in Central India.