AIDS), Was Reported in 1981 in the United States (CDC, 2006)

CHAPTER ONE

INTRODUCTION

1.1 Background of study

The first case of what was later called acquired immunodeficiency syndrome (AIDS), was reported in 1981 in the United States (CDC, 2006). Since then, the human immunodeficiency virus (HIV) epidemic expanded world-wide with varying prevalence in different regions of the world. In the

United States; at the end of 2003, approximately 1,039,000-1,185,000 persons in the United States were living with HIV/AIDS, an estimated 24%-27% of whom were unaware of their infection. There has been various global interventions with varying outcomes including the decrease in overall AIDS incidence, the substantial increase in survival after AIDS diagnosis (especially since highly active antiretroviral therapy [HAART] became the standard of care in 1996), and the continued disparities among racial/ethnic minority populations. Comprehensive national surveillance system, expanding the use of new HIV-testing technologies, promoting knowledge of HIV sero-status, and improving access to care and prevention interventions have also been strongly advocated (CDC, 2006).

Since the beginning of the epidemic, almost 78 million people have been infected with the HIV virus and about 39 million people have died of HIV. Globally, 35.0 million [33.2–37.2 million] people were living with HIV at the end of 2013. An estimated 0.8% of adults aged 15–49 years worldwide are living with HIV, although the burden of the epidemic continues to vary considerably between countries and regions. Sub-Saharan Africa remains most severely affected, with nearly 1 in every 20 adults living with HIV and accounting for nearly 71% of the people living with HIV worldwide

(WHO, 2015).

In Nigeria, according to UNAID report (2015), about 3.4million Nigerians are living with HIV. In

2013 Nigeria continued her commitment towards meeting the vision of Millennium Development

Goal (MDG) to halt and reverse HIV and AIDS epidemic in the country and promote the achievement of universal access to HIV/AIDS prevention, treatment, care and support in line with global commitments. With the valuable support of local and international partners, the country has seen the epidemic profile change significantly from HIV prevalence of 5.8% in 2001 to 4.1% in

2010. (Nigeria GARPR, 2014).

HIV infection in pregnancy can be very challenging. The current prevalence rate of HIV infection among pregnant women in Nigeria ranges from 3% to 8% in different regions of the country (Sagay, et al., 2005; Egesie and Mbooh, 2008; FMOH-PMTCT Guideline, 2010; Umeononihu, et al., 2013)

The HIV prevalence following repeat testing in late pregnancy in Nnewi-Nigeria is 3.91%

(Umeononihu et al., 2013).

HIV infection has been reported to have little effect on pregnancy outcome or complications in the developed world. Adverse pregnancy outcomes have, however, been reported more commonly in a number of African studies including complications of both early and late pregnancy. HIV may be the direct cause or a marker of a complex interaction of related medical and social conditions that affect pregnancy (WHO/UNAID, 1998)

HIV infection has been linked to a higher rate of spontaneous abortion. Higher rates of ectopic pregnancy have been reported in HIV-positive women than in uninfected women, which may be related to the effects of other concurrent sexually transmitted diseases. Preterm labour may be more common in HIV-positive women, with rates as high as double those rates seen in uninfected women

(WHO/UNAID, 1998). Preterm rupture of membranes may also be increased in HIV-positive women and abruptio placentae has been described as more common in HIV-positive women. There

2 is little difference in the birth weight of babies born to HIV-positive mothers in developed countries.

In contrast, low birth weight has been reported in some studies in developing countries

(WHO/UNAID,1998).

High rates of infant and maternal mortality have been reported world-wide (Uzoigwe and John,

2004; Oladapo et al., 2006; Hill et al., 2007; Rasch, 2007). Considerable efforts in assessing fetal mortality are being made in certain parts of the developed countries (MacDorman and Kirmeyer,

2009). In the contrary, assessment of fetal mortality in Nigeria is far below expectation. Little is known about the biochemical investigation of intra-uterine fetal demise (IUFD), and threatened abortions in Nigeria in spite of their obvious occurrences (Oladapo et al., 2007; Chigbu et al., 2009).

Pregnancy loss admittedly occurring at various health care levels at various stages of pregnancy in

Nigeria are often unexplained, and at best grossly under investigated. This leaves a lot of information gap that need to be explored and will be very helpful if provided. The determination of a consistent biochemical index will elucidate potential threats to normal pregnancy, and will suggest precautionary obstetric measures in Nigeria. Already several mortality indices fail to favour the developing countries compared to the developed countries (Murray and Lopez, 1997). In addition, perinatal mortality has been shown to occur at a higher rate among black Americans than their white counterparts (MacDorman and Kirmeyer, 2009). Pregnancy usually runs a course of about 40 weeks in human starting with the fertilization of the ovum by spermatozoa in the fallopian tube of the woman. This is followed by implantation, sequential, rapid and systematic cell division, differentiation, specialization, and maturation usually resulting to a foetus. In obstetrics and gynaecology, normal pregnancy has been divided into three stages as first, second and third trimesters (Andelman, 1973; Ndububa, 2010). In normal pregnancy, the integrity of the foetus and its ability to develop normally is often dependent in part or in whole on certain physiological, biochemical, physical, and psychological state of the mother. At certain stage of gestation, the

3 mother undertakes some basic functions of life such as respiration, nutrition, excretion, and thermoregulation etc for the foetus. It is a common finding that the mothers nutritional, and biochemical needs are often compromised in favour of the foetus (Anetor et al., 2003; Ikaraoha et al., 2005; Idowu et al., 2005).The journey from conception to birth is fraught with danger. Pregnancy is often associated with some level of stress on the mother which invariably transcends to the foetus

(Ndububa, 2010). In maternal distress, foetal life is however threatened in varying degrees commensurate with the nature and extent of maternal distress. It has also been noted that in some situations, certain deleterious responses of the mother could pose a serious threat to the foetus.

Pregnancy constitutes a major challenge to the maternal immune system, which must tolerate foetal alloantigens encoded by paternal genes. Local factors at the maternal-foetal interface are required to maintain such tolerance and to assure foetal survival (Salmon, 2004).

Some serum autoantibodies have association with pregnancy outcomes. The anti-phospholipid antibodies (aPLa) are known to have pathogenic effects on pregnancy outcome. They may react with foetal trophoblast as well as with maternal decidual cells directly inducing a defective placentation.

In addition, the thrombogenic effect of aPL may cause placental infarctions with the consequent impairment of the blood exchange between maternal and foetal circulation (Vinatier, et al., 2001).

There is evidence that thyroid autoimmunity is an important risk factor for miscarriage and preterm birth (Girardi, 2008).The presence of thyroid autoantibodies is relatively common in women of reproductive age. The prevalence ranges from 6% to 20%,(Girard, 2008) Studies have reported an association between the presence of thyroid autoantibodies, particularly thyroid peroxidase antibodies and adverse pregnancy outcomes, including miscarriage, preterm birth, and adverse neurodevelopmental sequelae in children (Surgi et al., 2004). It is thought that the presence of

4 thyroid autoantibodies could be associated with a subtle deficiency of thyroid hormones required in pregnancy (McIntyre, 2003)

Cubillos et al., (1997) found that the incidence of ANAs was 31.8% in patients with a history of miscarriages , but only 5.7% in 35 healthy patients with proven fertility and no history of pregnancy loss or autoimmune disease.

Pregnancy associated plasma protein-A (PAPP-A) can be very useful indicator of present pathology or future complications of pregnancy. PAPP-A is a 400kDa secreted homodimeric glycoprotein that belong to the metzincin superfamily of metalloproteases. It is best known as a regulator of insulin- like growth factor II (IGF-II) activity and plays a crucial role in placental development (Qin et al.,

2007). The cells most associated with PAPP-A expression are endometrial stromal fibroblasts and extravillous trophoblasts (Glerup et al., 2007). During placental growth, IGF-II seems to be the dominant insulin-like growth factor. It promotes steroidogenesis, glucose transport, and trophoblast

(foetal tissue) invasion of the uterine endometrium (decidua). Pregnancy associated plasma protein-

A is raised in pre-eclamptic toxaemia, antepartum haemorrhage and premature labour.

Pregnancy involves a lot of hormonal interplay. Variations in the levels of these hormones might imply pregnancy risk of varying degree. For instance, total estriol is lowered in foetal growth retardation and the unconjugated steroid rise in pre-eclampsia and lowered in retarded foetal growth

(Lin, 1977; Lin and Halbert, 1978; Hughes et al., 1980). Estriol (E3) is the major estrogen formed by the fetoplacental unit during pregnancy. The measurement of Estriol (E3) in body fluids has routinely been used for the monitoring and management of fetal well-being, particularly in the high- risk pregnant patient. The concentration of E3 in plasma increases gradually during the first 20 weeks gestation and more rapidly during the third trimester. Consistently low levels or a sudden and continual decrease of serum E3 during the third trimester is highly indicative of foetal distress and

5 possibly intrauterine death (Gagnon et al., 2008; Jelloffe-Pawlowski et al., 2011; Sattiyanan et al.,

2016)

Poorutilization of biochemical testing in pregnancy may contribute to poor pregnancy outcome.

Biochemical tests have long been established as reliable index of health assessment (Hughs et al.,

1980; Okwara, et al., 2008). Already poor laboratory utilization has been reported in this region

(Ahaneku et al., 1999). Also there is paucity of record on pregnancy outcomes in relation to the mother‘s biochemical status during pregnancy.

Continued advances in perinatology and neonatology and the introduction of biochemical and biophysical methods for evaluating pregnancy are expected to substantially improve the perinatal outcome. Biochemical evaluation of placental function appears to play an important role in the management of complications of early and late pregnancy (Hughs et al., 1980; Weintrob et al.,

2006). Improvements in methodology and increased experience in the application of the various techniques have enhanced ability to diagnose normal and abnormal pregnancy. However this is yet to transcend to improvement in this field.

Biochemical tests of foetal well-being are falling into disfavour. Much attention is given to the pregnant mother, and later on to the mother and the new born child. Not much is done for the unborn child. However not all successful pregnancy gets to full term. Furthermore, not all full term pregnancies have their babies born alive. The aetiology of foetal demise is unknown in 25-60% of all cases in the United States. In cases where a cause is clearly identified, the cause of foetal death can be attributable to foetal, maternal, or placental pathology. Incidence of pregnancy losses in developed economies is quite alarming. Worst situation may be expected in developing nations such as Nigeria. In 2003, data from the National Centre for Health Statistics showed a US national average foetal mortality rate of 6.9 deaths per 1000 births (US Department of Health and Human

6 services, 2005). The situation is obviously worse in Nigeria; and grossly underreported (Oladapo et al., 2007; Chigbu et al., 2009). Worldwide, this rate varies considerably depending on the quality of medical care available in the country in question and the definitions used for classifying foetal deaths. Medicare in Nigeria remains an issue of great concern. Underreporting of pregnancy abnormalities in developing nations is common, which makes comparisons even more difficult. In our environment, the unfortunate mother usually retires to faith; whereas the attending clinician often does not investigate thoroughly to determine possible cause. It is equally worrisome to note that some cases of pregnancy losses occur weeks before they are noticed, even with regular antenatal attendance.

1.2 Statement of problem

There is poor utilization of biochemical testing in pregnancy, especially in developing countries like

Nigeria. Also there is poor biochemical investigation of pregnancy disorders such as IUFD, and invariably, IUFD may be associated with poor biochemical testing.

1.3 Justification for the study

High rates of infant and maternal mortality have been reported world-wide. But little is known about the prevalence and biochemical investigation of pregnancy losses in Nigeria in spite of their obvious occurrences. Pregnancy loss admittedly occurring at various health care levels at various stages of pregnancy in Nigeria are often unexplained, and at best grossly under investigated. This leaves a lot of information gap that will be very helpful if provided. This study will reveal some potential causes of pregnancy losses, and will suggest precautionary obstetric measures in Nigeria. This study will obviously improve pregnancy outcomes in Nigeria.

1. 4 Study aim and objective

This study aims to determine the relevance of serum levels of some pregnancy associated metabolites to pregnancy outcomes of HIV sero-positive pregnant women.

The objective of this study is to determine the following parameters among HIV sero-positive pregnant women and their controls at NAUTH.

1. To determine the serum levels of some autoantibodies such as; anti phospholipids antibodies

detectable by anti beta-2-glycoprotein 1 (β2GP1) assay, thyroid autoantibodies detectable by

thyroid peroxidase antibodies (TPO) assay, antinuclear antibodies (ANA).

2. To determine serum levels of some pregnancy associated proteins such as; PAPP-A, total

protein, and albumin.

3. To determine serum levels of some pregnancy associated hormones such as; progesterone,

and estriol.

4. To monitor some metabolites such as calcium, inorganic phosphate, magnesium.

5. To relate the above findings to pregnancy outcomes of HIV sero-positive pregnant subjects.

CHAPTER TWO

LITERATURE REVIEW

2.1.1 Pregnancy

This is the period from conception to birth. After the egg is fertilized by a sperm and then implanted in the lining of the uterus, it develops into the placenta and embryo, and later into a foetus.

Pregnancy usually lasts 40 weeks, beginning from the first day of the woman's last menstrual period, and is divided into three trimesters, each lasting three months.

Due to technological advances, pregnancy is increasingly occurring among older women in the

United States. The pregnancy rate in the USA is 26.6% (Ventura, et al., 2014). Africa has a higher rate of teenage pregnancy, birth and abortion rates (Sedge, et al., 2015) mainly due to poor pregnancy control measures, literacy level, religious, cultural believes, and social status (Thobejane,

2015). The pregnancy rate in Nigeria is 38.03% (Index Mundi, 2015) quite higher than the rate in

USA.

The different stages of pregnancy are marked with anatomical and physiological changes.

Physiologic changes in pregnancy induce profound alterations to the pharmacokinetic properties of many medications. These changes affect distribution, absorption, metabolism, and excretion of drugs, and thus may impact their pharmacodynamic properties during pregnancy (Costantine,2014).

At the end of the first month, the embryo is about a third of an inch long, and its head and trunk-plus the beginnings of arms and legs-have started to develop. The embryo receives nutrients and eliminates waste through the umbilical cord and placenta. By the end of the first month, the liver and

9 digestive system begin to develop, and the heart starts to beat. In second month, the heart starts to pump and the nervous system (including the brain and spinal cord) begins to develop. The 1 in (2.5 cm) long fetus has a complete cartilage skeleton, which is replaced by bone cells by month's end.

Arms, legs and all of the major organs begin to appear. Facial features begin to form.

Figure 1.1 The uterus as it changes in size over the duration of the trimesters

Source: www.corpshumain.ca

In the third month, the foetus has grown to 4 in (10 cm) and weighs a little more than an ounce (28 g). Now the major blood vessels and the roof of the mouth are almost completed, as the face starts to take on a more recognizably human appearance. Fingers and toes appear. All the major organs are now beginning to form; the kidneys are now functional and the four chambers of the heart are complete (Yanamandra and Chandraharan, 2015; Lowdermilk, 2015)

By the fourth month the foetus begins to kick and swallow, although most women still can't feel the baby move at this point. Now 4 oz (112 g), the foetus can hear and urinate, and has established sleep-wake cycles. All organs are now fully formed, although they will continue to grow for the next five months. The foetus has skin, eyebrows, and hair. The foetus weighs up to a 1 lb (454 g) and measuring 8-12 in (20-30 cm), and experiences rapid growth in the fifth month. At this point, the mother may feel her baby move, and she can hear the heartbeat with a stethoscope. Rapid growth continues up till the seventh month. In the eight month growth slows down as the baby begins to take up most of the room inside the uterus. Now weighing 4-5 lbs (1.8-2.3 kg) and measuring 16-18 in (40-45 cm) long, the foetus may at this time prepare for delivery next month by moving into the head-down position. Adding 0.5 lb (227 g) a week as the due date approaches, the foetus drops lower into the mother's abdomen and prepares for the onset of labor, which may begin any time between the 37th and 42nd week of gestation. Most healthy babies will weigh 6-9 lb (2.7-4 kg) at birth, and will be about 20 in. long (Yanamandra and Chandraharan, 2015; Lowdermilk, 2015)

In normal pregnancy implantation takes place in the uterus. Below are some descriptions of various types of abnormal pregnancy.

Abdominal pregnancy :ectopic pregnancy within the abdominal cavity.

Ampullar pregnancy :ectopic pregnancy in the ampulla of the uterine tube.

Cervical pregnancy :ectopic pregnancy within the cervical canal.

Combined pregnancy :simultaneous intrauterine and extrauterine pregnancies.

Cornual pregnancy :pregnancy in one of the horns of a bicornuate uterus.

Ectopic pregnancy :extrauterine pregnancy development of the embryo outside the uterine cavity.

False pregnancy :development of the signs of pregnancy without the presence of an embryo.

Heterotopic pregnancy :combined pregnancy.

Interstitial pregnancy :ectopic pregnancy in the part of the uterine tube within the uterine wall.

Intraligamentary pregnancy :intraligamentous pregnancy ectopic pregnancy within the broad ligament.

Molar pregnancy :conversion of the early embryo into a mole.

Multiple pregnancy :presence of more than one fetus in the uterus at the same time.

Mural pregnancy :interstitial pregnancy.

Ovarian pregnancy :ectopic pregnancy occurring in an ovary.

Phantom pregnancy :false pregnancy due to psychogenic factors.

Postterm pregnancy :one that has extended beyond 42 weeks from the onset of the last menstrual period or 40 completed weeks from conception.

Tubal pregnancy :ectopic pregnancy within a uterine tube.

Tuboabdominal pregnancy :ectopic pregnancy partly in the fimbriated end of a uterine tube and partly in the abdominal cavity.

Tubo-ovarian pregnancy :ectopic pregnancy occurring partly in the ovary and partly in a uterine tube.

Dorland's Medical Dictionary for Health Consumers (2007).

Figure 2.1 Diagram showing locations of ectopic (extrauterine) pregnancy.

Adapted from Dorland's Medical Dictionary for Health Consumers (2007).

2.1.2 Symptoms of pregnancy

Thefirstsign of pregnancy is usually a missedmenstrualperiod; althoughsomewomenbleed in thebeginning. A woman'sbreastsswellandmaybecometender as themammaryglandsprepareforeventualbreastfeeding.Nipplesbegin to enlargeandtheveinsoverthesurface of thebreastsbecomemorenoticeable (Yanamandra and

Chandraharan, 2015; Lowdermilk, 2015).

Nausea and vomitingareverycommonsymptomsandareusuallyworse in themorningandduringthefirsttrimester of pregnancy.Theyareusuallycaused by hormonalchanges, in particular,increasedlevels of progesterone.Womenmayfeelworsewhentheirstomach is empty.

Manywomenalsofeelextremelytiredduringtheearlyweeks.Frequenturination is common,andtheremay be a creamywhitedischargefromthevagina.Somewomencravecertainfoods,and an extremesensitivity to smellmayworsenthenausea.Weightbegins to increase (Yanamandra and Chandraharan, 2015;

Lowdermilk, 2015).

In thesecondtrimester(13-28weeks) a womanbegins to looknoticeablypregnantandtheenlargeduterus is easy to feel.Thenipplesgetbiggeranddarker,skinmaydarken,andsomewomenmayfeelflushedandwarm.Appetit emayincrease. By the22ndweek,mostwomenhavefeltthebabymove.Duringthesecondtrimester,nauseaandvomitingoftenf

14 adeaway,andthepregnantwomanoftenfeelsmuchbetterandmoreenergetic.Heartrateincreases as doesthevolume of blood in thebody.

By thethirdtrimester(29-40weeks),manywomenbegin to experience a range of commonsymptoms.Stretchmarksmaydevelop on abdomen,breasts,andthighs,and a darklinemayappearfromthenavel to pubichair. A thinfluidmay be expressedfromthenipples.Manywomenfeelhot,sweateasilyandoftenfind it hard to getcomfortable.Kicksfrom an activebabymaycausesharppains,andlowerbackachesarecommon. There will be evidence of addedstress of extraweight.BraxtonHickscontractionsmaygetstronger

(Yanamandra and Chandraharan, 2015; Lowdermilk, 2015)

At aboutthe36thweek in a firstpregnancy(later in repeatpregnancies),thebaby'sheaddropsdownlowintothepelvis.Thismayrelievepressure on theupperabdomenandthelungs,allowing a woman to breathemoreeasily.However,thenewpositionplacesmorepressure on thebladder.

On average,pregnantwomenneed an additional300calories a day. Generally,womenwillgainthree to fivepounds in thefirstthreemonths,addingone to twopounds a weekuntilthebaby is born. An average,healthyfull-termbaby at birthweighs7.5 lb

(3.4kg),andtheplacentaandfluidtogetherweighanother3.5lb.Theremainingweightthat a womangainsduringpregnancy is mostlydue to waterretentionandfatstores.

In addition to thetypical,commonsymptoms of pregnancy,somewomenexperienceotherproblemswhichusuallydisappearafterdelivery.Constipationma ydevelop as a result of foodpassingmoreslowlythroughtheintestine.Hemorrhoidsandheartburnarefairlycommonduringlatepre

15 gnancy.Gumsmaybecomemoresensitiveandbleedmoreeasily;eyesmaydryout,makingcontactlensesfeel painful.Swollenanklesandvaricose veinsmay be a problem in thesecondhalf of pregnancy,andchloasmamayappear on theface (Yanamandra and Chandraharan, 2015; Lowdermilk,

2015).

Chloasma,alsoknown as the"mask of pregnancy" or melasma, is caused by hormonalchangesthatresult in blotches of palebrownskinappearing on theforehead,cheeks,andnose.Theseblotchesmaymergeintoonedarkmask. It usuallyfadesgraduallyafterpregnancy,but it maybecomepermanent or recurwithsubsequentpregnancies.Somewomenalsofindthatthelinerunningfromthetop to thebottom of theirabdomendarkens.This is calledthelineanigra.

Whiletheabovesymptomsareallconsidered to be normal,therearesomesymptomsthatcould be a sign of a moredangerousunderlyingproblem. Such signs include;

1. abdominalpain

2. rupture of theamnioticsac or leaking of fluidfromthevagina

3. bleedingfromthevagina

4. no foetalmovementfor 24 hours(afterthefifthmonth)

5. continuousheadaches

6. marked,suddenswelling of eyelids,hands, or faceduringthelastthreemonths

7. dim or blurryvisionduringlastthreemonths

8. persistentvomiting (Yanamandra and Chandraharan, 2015; Lowdermilk, 2015)

2.1.3 Diagnosis of pregnancy

Manywomenfirstdiscovertheyarepregnantafter a positivehomepregnancytest.Pregnancyurinetestscheckforthepresence of humanchorionicgonadotropin(hCG),which is produced by a placenta.Homepregnancytestsaremorethan97%accurate if theresult is positive,andabout80%accurate if theresult is negative. If theresult is negativeandthere is no menstrualperiodwithinanotherweek,thepregnancytestshould be repeated.Whilehomepregnancytestsareveryaccurate,theyarelessaccuratethan a pregnancytestconducted in a laboratory. Bloodtests to determinepregnancyareusuallyusedonlywhen a veryearlydiagnosis of pregnancy is needed.Thismoreexpensivetest,whichalsolooksforhCG,canproduce a resultwithinnine to 12 daysafterconception.

Oncepregnancyhasbeenconfirmed,thereare a range of screeningteststhatcan be done to screenforbirth defects, whichaffectabout 1.4 to 5.2% of unbornchildren (Bhandari et el, 2015).

Othertestsarerecommendedforwomen at higherriskforhaving a childwith a birthdefect.Thiswouldincludewomenoverage35,whohadanotherchild or a closerelativewith a birthdefect, or whohavebeenexposed to certaindrugs or highlevels of radiation.Womenwithany of theseriskfactorsmaywant to consideramniocentesis, chorionicvillussampling(CVS) or ultrasound.

1. Amniocentisis — This test is performed between week 15 and week 20 of pregnancy.

Although not all women decide to have an amniocentesis, it is routinely performed on

women who are at risk for genetic disorders or are over age 35. During the procedure a small

sample of the amniotic fluid surrounding the fetus is obtained. The fluid is used to help

identify chromosomal and genetic disorders and certain birth defects. When done during the

third trimester, this procedure also can assess the maturity of the baby's lungs (Damron,

2014; Kamath-Rayne and DeFranco, 2014)

2. Chorionic Villus Sampling (CVS) — CVS is usually performed between week 10 and week

13 of pregnancy. Like amniocentisis, CVS is a prenatal test that can often detect genetic

abnormalities and chromosomal disorders. However, the main advantage of a CVS versus

amniocentesis is that it can be performed much earlier in pregnancy (Alfirevic et al., 2003).

3. Expanded Alpha-Fetoprotein Screening — This blood test is performed during 15 to 20

week of antenatal visit. It measures the levels of alpha-fetoprotein (AFP), a protein released

by the baby's liver and found in the blood, as well as hCG and estriol. Abnormal results on

the expanded AFP test may indicate foetal brain or spinal cord defects, multiple foetuses, a

miscalculated due date, or Down syndrome, a chromosomal abnormality that includes mental

retardation and distinct physical features.

4. Foetal Monitoring - This involves the monitoring of the foetal heart rate and uterine

activity. Fetal monitoring involves using an ultrasound transducer to measure the foetal heart

rate and a toco transducer to identify uterine activity.

5. Genetic Screening — Many genetic abnormalities, such as cystic fibrosis, sickle cell anemia

and hemophilia A, can be diagnosed before birth. This screening may be recommend during

pregnancy if the patient is over the age of 35, if there is a family history of genetic disorders,

or if there had been previous incidence of a foetus or baby with a genetic abnormality.

6. Glucose Tolerance Test — This test is usually performed during the fifth month of

pregnancy. It measures the levels of sugar (glucose) in the blood. Abnormal glucose levels

may indicate gestational diabetes, a form of diabetes that may develop during pregnancy and

requires monitoring.

7. Group B Strep Culture — Group B Streptococcus is an infection in the mother that can

lead to complications and sometimes death in the newborn if the infection is transmitted to

the baby, which can occur during delivery. Group B strep has become more prevalent in the

last two decades (Hadavanda et al., 2015)

8. Nuchal Translucency Screening (NT) — this is a new, non-invasive test performed early in

pregnancy to identify an increased risk for Down syndrome and other birth defects. NT

screening is performed between 11 and 14 weeks of pregnancy. It is offered to women of all

ages. The screening is done via a high-resolution ultrasound exam of the nuchal area — a

fold of skin at the back of the neck of the foetus. The results are combined with the mother's

age to determine an adjusted risk for Down syndrome. The rate of detection for Down

syndrome is about 80 percent. Based on the results, a woman has the option of undergoing

CVS or amniocentesis for diagnosis (Xu et al., 2014; Spencer, 2014)

9. Ultrasound— This machine generates a picture of the foetus using sound waves. By looking

at the image, your doctor can tell the age of the foetus and whether there are twins. A detailed

ultrasound also can detect certain birth defects.

2.1.4 Prenatal Care

Expert prenatal care ensures that both mother and baby are as healthy as possible throughout pregnancy. During prenatal visits, tests are performed on the mother and occasionally on the fetus to assess any potential risks, treat any complications, and to monitor the growth and development of your baby. Early antenatal booking is well recommended.

Additional prenatal care may be necessary if there are preexisting medical conditions, such as diabetes, or if complications arise during pregnancy, or due to bad previous history of pregnancy.

2.2.5 Disorders of pregnancy

2.2.5.1 Hypertensive disorders in pregnancy (HDP)

Hypertensive disorders in pregnancy (HDP) are associated with severe maternal obstetric complications and are a leading contributor to maternal mortality. Furthermore, HDP lead to preterm delivery, foetal intrauterine growth restriction, low birth weight and perinatal death.

Although the exact incidence is unknown, it has been estimated that 5-10% of US pregnancies are complicated by HDP. Data from the Nationwide Inpatient Sample of the Healthcare Cost and

Utilization Project and National Hospital Discharge Survey have shown marked increases in the incidence of gestational hypertension and preeclampsia in the past two decades, and more women entering pregnancy with chronic (preexisting) hypertension. Women with chronic (preexisting) hypertension have been shown to have a markedly increased risk of severe adverse outcomes, such as maternal cerebrovascular accidents and placental abruption, compared to normotensive women.

The prevalence of HDP is believed to be increasing due to obesity trends and childbearing in older aged women.

2.2.5.2 Classification of Hypertensive Disorders in Pregnancy (HDP)

Hypertensive Disorders in Pregnancy (HDP) are comprised of a spectrum of disorders typically classified into categories and stratified according to severity: chronic preexisting) hypertension,

20 gestational hypertension, preeclampsia (including chronic (preexisting) hypertension with superimposed preeclampsia) and eclampsia.

1. Chronic (preexisting) hypertension

Chronic (preexisting) hypertension is defined as hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg or both) that is present before 20 weeks of gestation or prior to pregnancy. Elevated readings should be documented on more than one occasion.

2. Gestational Hypertension

Gestational hypertension is defined as new hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg or both) presenting at or after 20 weeks gestation without proteinuria or other features of preeclampsia; this terminology replaces the term ―Pregnancy Induced

Hypertension (PIH).‖

3. Preeclampsia and chronic (preexisting) hypertension with superimposed preeclampsia

Preeclampsia is defined as hypertension plus significant proteinuria, specifically gestational hypertension plus new onset proteinuria, or chronic (preexisting) hypertension with new or worsening proteinuria. When preeclampsia develops in women with chronic (preexisting) hypertension, the classification of disease is chronic (preexisting) hypertension with superimposed preeclampsia. Preeclampsia can also occur without proteinuria, and manifest as hypertension plus other adverse conditions (with hepatic, hematopoietic, or other manifestations), reflecting the multi- organ processes that characterize this disorder. It has been reported that up to 20% of women with eclampsia have hypertension without proteinuria in the week preceding the onset of eclampsia

2.2.5.3 Chromosomal abnormalities of pregnancy

Abnormalities can involve the chromosomes themselves or can involve just one or more genes. Most gene abnormalities do not result in an abnormality in the baby unless an abnormal gene is inherited from both the mother and the father. Usually, if only one copy of a gene is defective, the ‗good copy‘ from the other parent will take over. Chromosome abnormalities are more common than is generally realized. More than 50 per cent of miscarriages in the early stages of pregnancy are due to abnormalities of the chromosomes (Liu, et al., 2015; Rabiega-Gmyret, et al., 2015). In less than 1 per cent of all live births, the baby is found to have a recognizable chromosome abnormality (Sipek, et al., 2013; Springett and Morris, 2014).

1. Down's syndrome

The most well-known type of chromosome abnormality is Down's syndrome. Instead of having two no. 21 chromosomes, individuals affected by the condition have three. Down's syndrome causes a low IQ and distinct features such as short limbs and a characteristic wrinkle around the eyes. Heart defects are also common and are present at birth.Down's syndrome is not common, but the chances of having an affected baby increase as you get older. The prevalence rate in a certain hospital in

Nigeria was 0.12% (Adeyokunnu,1982). In Croatia the incidence rate is about 7% (Glivetic, et al.,

2015). If a previous pregnancy has been affected by Down's syndrome, the risk is increased threefold. If a close family member is affected, it will be required to test for inherited chromosome abnormality that increases the risk of having an affected child (balanced translocation).

2. Other syndromes

Children with an extra chromosome no. 13 (Patau's syndrome) or no. 18 (Edward's syndrome) seldom survive beyond birth. These conditions are less common than Down's syndrome.

If an embryo has a 45X0 composition in the chromosomes, with the other sexual chromosome missing, it will be a girl with Turner's syndrome. These girls are short and cannot have children because their ovaries are underdeveloped. Their mental development is normal, but certain heart defects are common. Prenatal screening of cytogenetic anomalies by Sheth et al., (2015) showed abnormal karyotypes in 7.2% of the cases. Trisomy 21 was the most common abnormality detected in (2.7%) followed by trisomy 18(0.6%) and trisomy 13 (0.1%) samples. Besides, structural abnormalities such as reciprocal and Robertsonian translocation were detected in 1.2% cases.

Preimplantation genetic diagnosis is a procedure that involves the removal of one or more nuclei from oocytes (a polar body) or embryos (blastomeres or trophectoderm cells) in order to test for problems in genome sequence or chromosomes of the embryo prior to implantation. It provides new hope of having unaffected children, as well as avoiding the necessity of terminating an affected pregnancy for genetic parents who carry an affected gene or have balanced chromosomal status.

Polymerase chain reaction-based molecular techniques are the methods used to detect gene defects with a known sequence and X-linked diseases. The indication for using this approach has expanded for couples who are prevented from having babies because they carry a serious genetic disorder to couples with conditions that are not immediately life threatening, such as cancer predisposition genes and Huntington disease. In addition, fluorescent in situ hybridization (FISH) has been widely applied for the detection of chromosome abnormalities. FISH allows the evaluation of many chromosomes at the same time, up to 15 chromosome pairs in a single cell. Preimplantation genetic screening, defined as a test that screens for aneuploidy, has been most commonly used in situations of advanced maternal age, a history of recurrent miscarriage, a history of repeated implantation failure, or a severe male factor. Unfortunately, randomized controlled trials have as yet shown no benefit with respect to preimplantation genetic screening using cleavage stage biopsy, which is

23 probably attributable to the high levels of mosaicism at early cleavage stages and the limitations of

FISH. Recently, two main types of array-based technology combined with whole genome amplification have been developed for use in preimplantation genetic diagnosis; these are comparative genomic hybridization and single nucleotide polymorphism-based arrays. Both allow the analysis of all chromosomes, and the latter also allows the haplotype of the sample to be determined. The promising results of these two approaches will inspire further validation of these array platforms, even at the single-cell level. It remains to be decided which embryo stage is the best for biopsy. Moreover, if randomized controlled trials are confirmed to play a role in increasing delivery rates, this will be a major step forward for assisted reproductive technology patients around the world (Chenet al., 2014).

Non-invasive prenatal screening (NIPS) for fetal chromosome abnormalities using cell-free deoxyribonucleic acid (cfDNA) in maternal serum has significantly influenced prenatal diagnosis of fetal aneuploidies since becoming clinically available in the fall of 2011. High sensitivity and specificity have been reported using different approaches and varying positive predictive values

(Neufeld-Kaiser, et al., 2015)

Meiotic errors during oocyte maturation are considered the major contributors to embryonic aneuploidy and failures in human IVF treatment. Various technologies have been developed to screen polar bodies, blastomeres and trophectoderm cells for chromosomal aberrations. Array-CGH analysis using bacterial artificial chromosome (BAC) arrays is widely applied for preimplantation genetic diagnosis (PGD) using single cells. Recently, an increase in the pregnancy and live birth rate has been demonstrated using array-CGH to evaluate trophectoderm cells (Feichtinger, et al.,2015).

Study by Society of Obstetricians and Gynaecologists of Canada, and collaborators, (2014) showed multiple pregnancy as the most powerful predictive factor for adverse maternal, obstetrical, and

24 perinatal outcomes. Recurrent pregnancy loss (RPL) which is generally known as >3 consecutive pregnancy losses before 20 weeks' gestation is seen in 0.5-2% of women. Ocak et al., (2013) evaluated the association of parental and fetal chromosomal abnormalities with recurrent pregnancy loss in and showed the frequency of three types of hereditary thrombophilia's; (MTHFR C677T polymorphisms, FV Leiden G1691A mutation and Prothrombin (factor II) G20210A mutation) in female patients. Parental chromosomal abnormality was detected in 2.8% of all cases, 5.7% of the couples, most of which (92.9%) were structural abnormalities. All of the structural abnormalities were balanced chromosomal translocations. Chromosomal analysis performed from the abortion materials detected a major chromosomal abnormality in 31.9% of the cases. The most frequently observed alteration in the hereditary thrombophilia genes was heterozygote mutation for the MTHFR

C677T polymorphisms. Balanced translocations are the most commonly detected chromosomal abnormalities in couples being evaluated for recurrent pregnancy loss and these patients are the best candidates for offering prenatal genetic diagnosis by the help of which there is a possibility of obtaining a better reproductive outcome.

2.2.6 Adverse pregnancy outcome

2.2.6.1 Preterm birth

Preterm birth is defined as birth at less than 37 completed weeks of gestation and it accounts for approximately 75% of perinatal mortality and nearly half of the cases of long-term neurologic

25 morbidity (McCormick, 1985). The rate of preterm births in the United States was 12.8% in 2006, a rate that had increased by more than 20% between 1990 and 2006 (Martin et al.,2009). The estimated annual health care cost due to preterm birth had exceeded US $26 billion in 2005 or

$51,600 per infant (Behrmen et al., 2007). Preterm birth is a heterogeneous phenotype with many biological pathways (Behrmen et al., 2007) and is classified into two broad categories, spontaneous and indicated, based on the presence or absence of factors that place the mother or the fetus at risk

(Meis et al., 1987;1995;1998 ). Spontaneous preterm birth (sPTB) accounts for the majority of preterm births in developed countries and it occurs as a result of spontaneous onset of labor (40% to

45%) or preterm premature rupture of fetal membranes (25% to 30%) before 37 weeks of gestation.

Preterm births that are the result of conditions that directly threaten the health of the mother or fetus are categorized as indicated preterm births and account for the remaining 25% to 30% of preterm deliveries (Meis et al., 1987;1995;1998; Goldenberg et al., 2008).

The onset of preterm labor is thought to be brought on by multiple mechanisms or pathways that may have been initiated weeks to months before the actual presence of clinical symptoms (Behrmen et al., 2007;Romero et al., 2010). Various maternal or fetal demographic, behavioral, and clinical characteristics have been associated with preterm birth including maternal race/ethnicity (Fiscella,

1996; Goldenberg et al., 1996), younger maternal age (Hediger et al., 1997;Branum and Shoendorf,

2005), maternal age over 35 years (Cnattingius et al., 1992), cigarette smoking (Berkowitz and

Papienik, 1993), low prepregnancy weight (Kramer et al., 1995;Siega-Riz et al., 1996;Savtz, 1999), psychosocial stress (Hadegaard et al., 1996), previous preterm birth (Merer et al.,1999), and intrauterine infection (Goldenberg et al., 2005).The identification of risk factors for predicting preterm birth is advantageous because it allows for the initiation of risk-specific treatment for at-risk women and these risk factors may provide insights into a better understanding of the mechanisms

26 leading to preterm birth (Goldenberg et al., 2003;2005). However, methods for identifying women at risk of preterm birth by the reliance of demographic, behavioral, and biological risk factors have low sensitivities (Behrmen et al., 2007). Cervicovaginal fluids, amniotic fluid, urine, saliva, and serum or plasma are biologic fluids that have been used as a source for identifying biochemical markers for the prediction of preterm birth. For large-scale, population-based clinical and epidemiological studies, methodological and ethical considerations are critical as researchers choose to incorporate the sampling of biological specimens in otherwise asymptomatic women and such considerations include cost, ease of specimen collection and storage, and potential maternal and fetal risks. This paper summarizes the current literature on biochemical markers of sPTB in asymptomatic women, in which the focus is placed solely on markers that offer a great degree of acceptability to most pregnant women while minimizing maternal and fetal risks. Thereby, biochemical markers that are collected through invasive procedures such as amniocentesis are omitted from this review.

In Nigeria, over 900,000 children under the age of five years die every year. Early neonatal death is responsible for a little over 20% of these deaths. Prematurity remains a significant cause of these early neonatal deaths. In some series, it is reported to be responsible for 60–70% of these deaths. The prevalence of pre-term delivery was 120 per 1,000 live births. Factors significantly associated with pre-term delivery were low socio-economic class, previous pre-term delivery, antepartum hemorrhage, premature rupture of fetal membranes, urinary tract infection, pregnancy induced hypertension, induced labor, and booking elsewhere outside the teaching hospital (Olugbenga, et al.,

2010). Iyoke, et al., (2014) reported a higher prevalence rate of preterm birth of 176 per 1,000 live births in southern Nigeria. The adjusted perinatal mortality rate for preterm babies in the study center was 46.1% while the stillbirth rate for preterm babies was 22.0%. The adjusted early neonatal death rate was 24.0%.

In 2012, preterm birth affected more than 450,000 babies—that's 1 of every 9 infants born in the

United States. Preterm birth is the birth of an infant before 37 weeks of pregnancy. Preterm-related causes of death together accounted for 35% of all infant deaths in 2010, more than any other single cause. Preterm birth is also a leading cause of long-term neurological disabilities in children. Preterm birth costs the U.S. health care system more than $26 billion in 2005.

2.2.6.1.1 Causes of preterm birth

Many environmental factors have been independently associated with preterm birth (PTB).

However, exposure is not isolated to a single environmental factor, but rather to many positive and negative factors that co-occur (Rappazzo, et al., 2015). Preterm labour is now thought to be a syndrome initiated by multiple mechanisms, including infection or inflammation, uteroplacental ischaemia or haemorrhage, uterine overdistension, stress, and other immunologically mediated processes. Preterm birth have been associated with nutrition, body weight, employment, physical activity, douching, alcohol and illicit drug use before and during pregnancy.

2.2.6.2 Low birth weight.

Low birthweight has been defined by the World Health Organization (WHO) as weight at birth of less than 2,500 grams (5.5 pounds).There is an increasing rate of low birth weight (LBW) infants.

LBW data from national health surveys in Oman, and published reports from Oman's Ministry of

Health and the World Health Organization assessed between January and August 2014 showed

Oman‘s LBW rate has been increasing since the 1980s. It was approximately 4% in 1980 and had nearly doubled (8.1%) by 2000. Since then, it has shown a slow but steady rise, reaching 10% in recent times. High rates of consanguinity, premature births, number of increased pregnancies at an

28 older maternal age and changing lifestyles are some important factors related to the increasing rate of LBW (Islam, 2015).

2.2.6.3 Intra-Uterine Fetal Death (IUFD)

Fetal death at any point during gestation is a traumatic event not only to the family but also to the caregiver. Stillbirths generally account for half of all perinatal mortality, with an estimated 4 million occurring worldwide each year (WHO.2006). South Asia has the world‘s largest numerical stillbirth burden with rates ranging from 25 to 40/1000 births (Fikree et al., 2002). Recent estimates suggest that stillbirth rates greater than 30 per 1000 births are common among the least developed countries, especially in Sub-Saharan Africa and Southeast Asia. By comparison, rates of 3-5 per 1000 deliveries have been documented in the U.S. and other developed countries and rates of 10-15 per

1000 are reported in mid-level countries, such as those in South and Central America (Stanton et al.,

2006;McClureet al., 2006).

According to results of study by Jahanfar from Iran in 2000 the rate of fetal death in Iran was 1020 cases that 49.6% of them were male fetuses (Jahanfar et al.,2005). Maternal hypertension, diabetes mellitus, renal disease, and autoimmune disorders, as well as placentation abnormalities and congenital anomalies, are examples of conditions that can place the pregnancy at high risk of fetal compromise (Lalor et al., 2008).

2.2.7 Some pregnancy associated biochemical parameters

2.2.7.1 Pregnancy associated plasma protein-A (PAPP-A)

PAPP-A was initially found in the plasma of pregnant women among three other highly abundant proteins by Lin and colleagues in 1974. The molecules were detected with anti-third trimester pregnancy serum polyclonal antibodies not reactive with normal human serum and were alphabetically named (PAPP-A, PAPP-B, PAPP-C and PAPP-D) according to their precipitation order in the immunodiffusion method used (Lin et al., 1974). Later it was revealed that the molecule detectable in pregnancy plasma that had been named PAPP-A was actually a covalent complex of

PAPP-A and the proform of eosinophil major basic protein (proMBP) in equimolar amounts (Oxvig et al., 1993). More specifically, the molecule is a heterodimer consisting of two PAPP-A subunits and two proMBP subunits connected by disulfide bridges (PAPP-A/proMBP complex) (Oxviget al.,

1994b). However, PAPP-A produced in cell culture conditions is a homodimer of two PAPP-A subunits (Lawrence et al., 1999; Overgaard et al., 2000). In non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) PAPP-A/proMBP complex from pregnancy plasma migrates as a 500 kDa entity. Upon reduction PAPP-A subunit is seen at 200 kDa. However, proMBP forms a smear at 50-90 kDa most likely because of high and variable glycosylation of this subunit. (Oxvig et al.,1994a.) The calculated size of PAPP-A subunit is 199 kDa, when the determined protein and carbohydrate contents are taken into account. The calculated size of the

PAPP-A/proMBP complex is 474 kDa. (Oxviget al., 1994b.)

The gene of PAPP-A is found in human chromosome region 9q33.1 (Sinosich, 1990). PAPP-A is encoded by 22 exons (Overgaard et al., 2003) and the sequence of PAPP-A seems to be highly conserved between mammals (Mazerbourg et al., 2001; Hourvitz et al., 2002). For example, between mouse and human the sequence identity is 91.1% (Soe et al., 2002). Homology has also been noticed between human and zebrafish PAPP-A (Overgaard et al., 2003). PAPP-A is transcribed as an mRNA

30 of 8400 nucleotides of which 4881 nucleotides are translated into preproPAPP-A of 1627 amino acids (Haaning et al., 1996). Thus the PAPP-A transcript has an unusually long 5‘ untranslated region. The preproform includes a putative signal peptide of 22 residues and a highly basic

(calculated pI 12.9) propeptide of 58 residues (Haaning et al., 1996). The PAPP-A propeptide is not required for folding or secretion of the protein and the proform is most likely cleaved by a protease recognizing Arg-X-X-Arg motif, possibly furin, prior to secretion (Kristensen et al., 1994;

Overgaard et al., 2000). The mature PAPP-A monomer polypeptide contains 1547 amino acid residues representing the C-terminal part of the proform of PAPP-A (Kristens en et al., 1994).

In the amino acid sequence of the PAPP-A monomer there are 13 putative sites for N-glycosylation and 7 putative sites for attachment of glycosaminoglycan groups (Kristensenet al., 1994). Of the 13 potential sites for N-linked carbohydrate 11 are substituted. However, PAPP-A is most likely not O- glycosylated (Overgaard et al., 2003). In pregnancy, there are 44 N-acetylglucosamine monosaccharides for each PAPP-A polypeptide chain (Overgaard et al., 2000). In recombinantly produced PAPP-A, however, the degree of glycosylation has been reported to be lower (Overgaard et al., 2000). There are 82 Cys residues in the amino acid sequence of the mature PAPP-A monomer

(Kristensen et al., 1994). Cys-1130 is responsible for the covalent dimerization of PAPP-A monomers while Cys-381 and Cys-652 are involved in the complexation with proMBP and are free in PAPP-A dimer (Overgaard et al., 2003; Glerup et al., 2005). All other cysteines seem to form intrachain bridges within structural domains except Cys-563, for which the status is unknown

(Overgaard et al., 2003). Some proteins with sequence homology with PAPP-A have been identified.

A previously unknown protein found in 2001 in a search of public DNA sequence databases showed

46% homology with PAPP-A and was thus named PAPP-A2 (Overgaard et al., 2001). A splicing variant of PAPP-A with a highly basic 29-residue insert at an exon junction was detected in mice in

2002 and was accordingly named PAPP-Ai (Soe et al., 2002). Furthermore, an archaeal protein

31 resembling PAPP-A, ulilysin of Methanosarcina acetivorans, was found in a bioinformatic search in which structure prediction was utilized (Tallant et al., 2006). With a molecular size of 29-kDa this protein seems to consist only of a catalytic domain similar to that of PAPP-A. Although the sequence homology is not high, the key residues important for catalysis, substrate binding and structural stability are fully conserved between PAPP-A and ulilysin (Tallant et al., 2006).

Apart from these proteins, no homology has been detected with other proteins apart from certain structural module similarities which will be discussed in the following section. It is noteworthy that the sequence of PAPP-A is not related to -2-macroglobulin, although in some early reports published in the 1980s these proteins were assumed to be alike. This faulty assumption arose from certain similar physical properties including molecular weight, amino acid composition, isoelectric point and electrophoretic mobility (Sutcliffe et al., 1980).

In 1999, PAPP-A as a dimer of PAPP-A subunits was identified as the previously unknown insulin-like growth factor (IGF)-dependent IGFBP-4 protease produced by human fibroblasts in culture medium (EC 3.4.24.79) (Lawrence et al., 1999). Later PAPP-A was also confirmed to be responsible for similar activity in other systems such as follicular fluid of various mammals

(Mazerbourg et al., 2001), in human pregnancy, and in culture medium conditioned by human and porcine coronary artery vascular smooth muscle cells (VSMC) (Bayes-Genis et al., 2001b), luteinizing human granulosa cells (Iwashita et al., 1998), cells of mouse osteoblast cell line MC3T3-

E1 (Bunn et al., 2004), bovine mammary fibroblast cells (Fleming et al., 2005), human trophoblast and decidualized endometrial stromal cells (Giudice et al., 2002) and human osteoblasts (Qin et al.,

2000). In addition to IGFBP-4, PAPP-A has also been shown to degrade IGFBP-5 (Laursen et al.,

2001; Rivera and Fortune, 2003b) and IGFBP-2 (Monget et al., 2003; Gerard et al., 2004; Kumar et al., 2005).

2.2.7.1.1PAPP-A andFetal weight: Small for gestational age (SGA) and intra-Uterine growth retardation (IUGR).

Various studies of varied designs have investigated the relationship between low PAPP-A level and fetal weight or growth (Peterson et al., 2008; Spencer et al., 2008; Van-Ravenswaaij et al., 2010;

Marttala et al., 2010; Carbone et al., 2012; Saruhan et al., 2012). Four of the 6 studies, involving a total of 24 668 women, reported statistically significant relationships between PAPP-A levels below the fifth percentile or less than 0.4 multiples of the median (MoMs) and SGA or IUGR (P< .05)(

(Peterson et al., 2008; Spencer et al., 2008; Van-Ravenswaaij et al 2010; Marttala et al., 2010) One of these, a retrospective cohort study involving 3269 women, investigated the predictive value of

PAPP-A levels below the fifth percentile for SGA. Their results yielded a positive predictive value of 2.97 (95% CI 1.1 to 6.4) of a PAPP-A level below the fifth percentile for SGA. However, the authors believed that a positive predictive value of 2.97 was not strong enough to consider the use of

PAPP-A level to screen for SGA(Carbone et al 2012). Another 2 of the 6 recent studies included a

2012 retrospective case-control study, which found that a PAPP-A level below the 10th percentile was not significantly associated with SGA (Saruhan et al., 2012), and a 2011 retrospective cohort study involving 28 566 women, which found no predictive value of PAPP-A level below the fifth percentile for low birth weight (Van-Ravenswaaij et al 2010). The current evidence for an association between PAPP-A level below the fifth percentile and SGA remains mixed.

2.2.7.1.2 PAPP-A and preterm birth

Some studies investigated the relationship between low PAPP-A and preterm delivery (Van-

Ravenswaaij et al 2010;Goetzinger et al., 2010;Kirkegaard et al 2010;Saruhan et al., 2012). Of these,

2 retrospective cohort studies that included a combined total of 11 681 women found a statistically

33 significant association between low PAPP-A level (below the 10th percentile in one study and below the 5th percentile in another) and preterm delivery(Goetzinger et al., 2010;Kirkegaard et al 2010). It is notable that the study that found an association between PAPP-A level below the 10th percentile and preterm delivery did not find a strong enough association to endorse the use of PAPP-A level to screen for preterm delivery((Goetzinger et al., 2010) The other 2 studies included a retrospective case-control study of 663 women that did not find an association between PAPP-A level below the

10th percentile and preterm delivery (Saruhan et al., 2012) and a 2011 retrospective cohort study of

28 566 women that concluded a PAPP-A level below the fifth percentile was not predictive of preterm labour((Van-Ravenswaaij et al 2010) The current evidence for an association between low

PAPP-A levels and preterm labour remains equivocal, and no strong evidence exists to support the measurement of low PAPP-A level as a test for preterm delivery.

2.2.7.1.3 PAPP-A levels andhypertensive disorders of pregnancy

There have been studies investigating a link between hypertensive disorders of pregnancy— specifically preeclampsia—and low PAPP-A levels(Poon et al., 2009; Odibo et al 2011). Two of the prospective cohort studies found an inverse relationship between measured PAPP-A levels and preeclampsia. In a prospective cohort study of 45 women (20 in the control group and 25 with 1 or more of fetal growth restriction [n = 7], gestational hypertension [n = 7], and preeclampsia [n = 13]), placental abnormalities were found in all cases in the study group but not in the control group.

Abnormal placentation was found to be associated with a lower mean (SD) PAPP-A level of 0.7

(0.3) MoM in the test group versus 1.1 (0.5) in the control group (P = .03) (Odibo et al 2011). In a prospective screening study of 8051 women, median PAPP-A MoMs were 1.002 in normal pregnancies and 0.555 and 0.911 in pregnancies with early and late preeclampsia, respectively.

Logarithmic analysis of the median PAPP-A levels revealed significantly lower values in women

34 presenting with both early (P< .01) and late (P< .03) preeclampsia compared with the control group

((Poon et al). The remaining 2 studies were both retrospective cohort studies involving 663 and 28

566 women that investigated the association between a PAPP-A level lower than a specific cutoff point (10th percentile and 5th percentile, respectively) and the development of preeclampsia (Van-

Ravenswaaij et al 2010;Saruhan et al., 2012). These studies found no association with preeclampsia and no predictive value of a low PAPP-A level for preeclampsia.

2.2.7.1.4PAPP-A andspontaneous abortion

Two studies investigated the relationship between low PAPP-A levels and subsequent second- trimester spontaneous pregnancy loss (Van-Ravenswaaij et al 2010;Hanita et al., 2012). In 2012,

Hanita et al conducted a prospective cohort study of 42 women with threatened miscarriage and 40 controls. From the total of 82 women, the authors found that the mean PAPP-A level was significantly lower in the group with threatened miscarriages who subsequently progressed to spontaneous abortions compared with the control group (0.78 MoMs vs 1.00 MoMs; P< .05). In

2011, the cohort study of 28 566 women found a low PAPP-A level below the fifth percentile was predictive of subsequent miscarriage (odds ratio 14.53; 95% CI 10.44 to 20.22) (Van-Ravenswaaij et al 2010). These studies suggest that low PAPP-A levels might be associated with subsequent pregnancy loss before fetal viability.

2.2.7.1.5 PAPP-A levels and stillbirth

Three studies investigated the association between low PAPP-A level and the rate of stillbirth(proctor et al 2009;Marttala et al 2010; Van-Ravenswaaij et al 2010) In 2010, a retrospective cohort of 19 536 women found a statistically significant association between PAPP-A levels below the fifth percentile and rates of stillbirth (P< .002).( Marttala et al 2010) However, the

2011 large historical cohort study of 28 566 women found no predictive value of a low PAPP-A level (below the fifth percentile) for stillbirth (Van-Ravenswaaij et al 2010). A 2009 study found that among women with low PAPP-A levels (below 0.3 MoMs), a higher risk of stillbirths was found when an abnormal placenta was seen on ultrasound, potentially explaining the link between a low

PAPP-A level and rates of stillbirth (Proctor et al 2009). Current evidence regarding the relationship of low PAPP-A levels with stillbirth rates remains conflicting, and additional studies are required to elucidate the relationship between a low PAPP-A level and rates of stillbirth, and the mechanism that would lead to stillbirth in such cases.

2.2.7.1.6 PAPP-A and abnormal placental morphology

Although some evidence supports a positive association between low PAPP-A levels and adverse perinatal outcomes, there remains a relative paucity of high-quality population-based studies to determine the strength of the association and to understand which additional factors (such as multiple abnormal FTS or IPS values, clinical risk factors, or abnormal ultrasound findings) modify this risk. Therefore, it might be more helpful to shift our focus to study the likely pathogenesis behind these cases of adverse outcomes—abnormal placentation. Studies have investigated the relationship between low PAPP-A levels and placentation. A small study in 2011 used Doppler ultrasound to compare ―vascularization‖ in the placentas of 12 women with low PAPP-A levels

(lower than 0.3 MoMs) with the placentas of 11 women in a control group (Rizzo et al., 2011). This study found a statistically significant difference in placental vascularization that correlated with reduced number of capillary vessels per villus cross section (P = .005) and smaller capillary diameters (P = .041) in women with low PAPP-A levels. In 2009, a study showed that in women with low levels of PAPP-A, uterine artery Doppler ultrasound results (as a surrogate marker for uteroplacental vascular resistance) at 22 weeks‘ gestational age could be used to predict an increased

36 risk of preterm delivery, SGA, and low birth weight (Cooper et al., 2006). Two studies investigating the relationship between low PAPP-A levels and preeclampsia found that low PAPP-A levels were significantly associated with abnormal placental morphology (P = .03) and abnormal uterine artery

Doppler scan results (P = .001). A 2009 study evaluated ultrasound assessment of placental function at 19 to 22 weeks in 90 women with low PAPP-A levels (below 0.3 MoMs); abnormal placental morphology was found in 19 women (21%), which was significantly associated with IUGR, preterm delivery, and stillbirth, while abnormal uterine artery Doppler scan results were of less value (P<

.05) (Proctor et al 2009)

2.2.7.1.7 PAPP-A and HIV infection

There are no statistically significant differences between the HIV-positive and HIV-negative women in the median maternal levels of free β-hCG, PAPP-A (Spencer , 2011; Savvidou 2011)

2.2.7.2 Estriol

Estrogens are involved in development and maintenance of the female phenotype, germ cell maturation, and pregnancy. They are also important in many other gender non-specific functions in men and women. These include growth, nervous system maturation, bone metabolism, and endothelial responsiveness.

There are 3 major biologically active estrogens in humans: estrone (E1), estradiol (E2), and estriol

(E3) (Alonso and Rosenfield, 2002). Like all members of the steroid hormone family, they diffuse into cells and bind to specific nuclear receptors, which in turn alter gene transcription in a tissue specific manner. E2 is the most potent natural human estrogen, closely followed by E1, while E3

37 possess only 20% of E2's affinity for the estrogen receptor. In men and nonpregnant women, E1 and

E2 are formed from the androgenic steroids androstenedione and testosterone, respectively. E3 is derived largely through conversion of E2, and to a lesser degree from 16a-metabolites of E1. E2 and

E1 can also be converted into each other, and both can be inactivated via hydroxylation and conjugation.

During pregnancy E3 becomes the dominant estrogen. The fetal adrenal gland secretes dehydroepiandrosterone-sulfate (DHEAS), which is converted to E3 in the placenta and diffuses into the maternal circulation (Wood, 2014). The half-life of unconjugated E3 (uE3) in the maternal blood system is 20 to 30 minutes, since the maternal liver quickly conjugates E3 to make it more water soluble for urinary excretion. E3 levels increase throughout the course of pregnancy, peaking at term.

Measurement of serum E2 and E1 levels is an integral part of assessment of reproductive function in females (Mohan and Prakash, 2010), and also has applications in both men and women in osteoporosis risk assessment and monitoring of hormone replacement therapy (Fan et al., 2014).

Decreased 2nd trimester uE3 has been shown to be a marker for Down and trisomy-18 syndromes

(Shawn et al., 2010). It also is low in cases of gross neural tube defects such as anencephaly. Based on these observations, uE3 has become a part of multiple marker prenatal biochemical screening, together with alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and inhibin-A measurements (QUAD / Quad Screen (Second Trimester) Maternal, Serum). Low levels of uE3 also have been associated with pregnancy loss,Down syndrome, Edward's syndrome,Smith-Lemli-Opitz syndrome (defect in cholesterol biosynthesis), X-linked ichthyosis and contiguous gene syndrome

(placental sulfatase deficiency disorders), aromatase deficiency, and primary or secondary fetal adrenal insufficiency (Santolaya-Forgas et al., 1996; Hu et al., 2014)

High levels of uE3, or sudden increases in maternal uE3 levels, are a marker of pending labor. The rise occurs approximately 4 weeks before onset of labor. Since uE3 has been shown to be more accurate than clinical assessment in predicting labor onset, there is increasing interest in its use in assessment of pre-term labor risk (Ramsey and Andrew, 2003; Soghra et al., 2014).

High maternal serum uE3 levels may also be occasionally observed in various forms of congenital adrenal hyperplasia. It has been reported that a low or absent maternal serum unconjugated estriol

(uE3) level is associated with placental steroid sulfatase (STS) deficiency occasioned by deletion of the STS gene as assessed by fluorescence in situ hybridization (FISH) (Kashork, et al.,2002).

Undetectable maternal estriol values may indicate a severe fetal pathology and should lead to further investigations (Minsart, et al 2008).

Estriol is only produced in significant amounts during pregnancy as it is made by the placenta from

16-hydroxydehydroepiandrosterone sulfate (16-OH DHEAS),(Raju, et al., 1990) an androgen steroid made in the fetal liver and adrenal glands. The human placenta produces pregnenolone and progesterone from circulating cholesterol. Pregnenolone is converted in the fetal adrenal gland into dehydroepiandrosterone (DHEA), a C19 steroid, then subsequently sulfonated to dehydroepiandrosterone sulfate (DHEAS). DHEAS is converted to 16-OH DHEAS in the fetal liver.

The placenta converts 16-OH DHEAS to estriol, and is the predominant site of estriol synthesis.

Estriol can be measured in maternal blood or urine and can be used as a marker of fetal health and wellbeing. Because many pathological conditions in a pregnant woman can cause deviations in estriol levels, these screenings are often seen as less definitive of fetal-placental health than non- stress testing. Conditions which can create false positives and false negatives in estriol testing for fetal distress include preeclampsia, anemia, and impaired kidney function(Pagana and Pagana,

2009).

Elevated second trimester maternalserum unconjugated estriol is independently associated with a higher rate of spontaneous preterm birth (Olsen et al., 2014). The measurements of estriol from maternal saliva samples appear to correlate with maternal serum levels. Some studies demonstrated that elevated salivary estriol was a better marker for later sPTB in asymptomatic pregnant women, and the use of salivary estriol testing has potential advantages (i.e., noninvasive nature and ease of collection). However, one major limitation of the use of saliva as a biomarker is confounding by factors such as patient activity, posture, food consumption, and diurnal variations in estriol levels.

2.2.7.3 Auto antibodies and pregnancy

Certain autoantibodies which are found in autoimmune diseases including connective tissue diseases

(CTDs) can impair fertility. Reproductive failure may present as pregnancy loss, either as miscarriage, intrauterine fetal death or stillbirth. There are also late obstetric complications such as intrauterine growth restriction, pre-eclampsia and pre-term birth. However, no antibody appears to be singularly responsible (pathognomic) for pregnancy loss. It may be more appropriate to assess a combination of antibodies rather than one antibody. However, a large meta-analysis of published trials is required in order to determine the prevalence of each particular autoantibody and different combinations of antibodies in different forms of reproductive failure (Carp, et al., 2008)

2.2.7.3.1 Anti-phospholipid antibodies (aPL)

The aPLs are known to have pathogenic effects on pregnancy outcome in animal models (Blank, et al., 1991). They may react with fetal trophoblast as well as with maternal decidual cells so directly

40 affecting several cell functions pivotal for inducing a defective placentation (Meroni, et al., 2008).

Complement-mediated placental inflammation has also been reported to play a key role in an experimental model of fetal loss induced by passive injection of large amount of aPL after the implantation (Girardi, et al., 2008). However, the real importance of complement activation in human pathology is still a matter of research (Cavazzana, et al., 2007). In addition, the thrombogenic effect of aPL may cause placental infarctions with the consequent impairment of the blood exchange between maternal and fetal circulation (Meroni, et al., 2008). It has been reported that 5–51% of patients with recurrent spontaneous miscarriage have aCL and 0–20% have LA (Vinatier, et al.,

2001). Although aPLs are a risk factor for pregnancy loss, they are not pathognomic.

Attempts have been made to find the specific autoantibody responsible for pregnancy loss. There is evidence that the pathogenic antibodies are those directed towards β2GP1 (Meroni, et al., 2008).

Unfortunately, there are no cohort studies examining the natural history of untreated patients with aPL. There is a widespread belief, based on early studies that these antibodies are so pathogenic that they should always be treated. The nearest to a large study on the natural history is Empson et al.'s

(Empson, et al., 2002) meta-analysis of 95 patients comparing treatment with aspirin with placebo.

There was an 86% live birth rate in the placebo group. Hence, the PPV seems to be only 14%.

However, as false-negative rates were not quoted, it is impossible to arrive at a valid conclusion about the PPV. Marai et al.,(2004) compared the results of autoantibody testing on 38 women with recurrent miscarriages (at least three consecutive miscarriages) in the first trimester of pregnancy, compared with a control group of 28 parous women with normal fertility. There was no association with aPL. However, the small numbers may have masked any difference in the results. A recent meta-analysis study reported that LA, aCL and aβ2GP1 all were strong risk factors for pregancy loss

(Opatrny, et al., 2006). A larger study of 269 patients (Shoenfeld et al. 2006) showed an increased prevalence of aPL (11% compared with 2.5%), with a PPV of 75%.

2.2.7.3.1.1 Obstetric complications of aPL

In APS, defined as the persistent presence of aPL (LA, aCL, aβ2GPI antibodies) and the loss of three or more miscarriages prior to 10 weeks, or the loss of one or more fetuses above 10 weeks, there is a higher incidence of obstetric complications including IUGR, PET, stillbirth and pre-term labour.

Although there have been follow-up studies of APS, showing an increased incidence of late obstetric complications compared with a healthy control group, there have been no follow-up studies of the obstetric complications in aPL-associated recurrent miscarriage compared with recurrent pregnancy loss without aPL. The incidence of obstetric complications is higher in women with recurrent pregnancy loss than in the general population (Carp, 2007).

2.2.7.3.1.2 Treatment of obstetrical APS

The standard treatment of aPL in pregnancy consists of low-molecular weight heparin and low-dose aspirin (Empson, et al 2005; Miyakis, et al., 2006). However, there are no comparative studies with which to judge this regimen. Hence, no predictive value can be given about treatment using evidence-based medicine. There are only descriptive reports. Recently, doubt has been cast on the role of aspirin, as a meta-analysis comparing three trials of aspirin compared with placebo showed a non-statistically significant OR of 1.05 in favour of aspirin (Vinatier, et al., 2001). Low-molecular weight heparins may lower the fetal loss rate; however, there is no effect on the incidence of late obstetric complications. Hence, when APS presents with late obstetric complications rather than

42 pregnancy loss, no prediction can be made about prognosis with anti-coagulant treatment.

Intravenous immunoglobulin (IVIg), however, actually lowers the titre of autoantibodies. Although it is not an anticoagulant and does not prevent thrombosis, there is some evidence that IVIg may lower the incidence of late obstetric complications (Carp, et al., 2001). However, there is insufficient data to allow any predictive value to be given.

2.2.7.3.2 Anti-thyroid antibodies

Anti-thyroid antibodies (ATAs) have been suggested to be independent markers of ‗at-risk‘ pregnancy. Euthyroid women with recurrent miscarriage have increased levels of autoantibodies either against thyroglobulin (aTG) or thyroid peroxidase (TPO) while the probability of abortion in women with ATA has been shown to be greater than in controls (Matalon, et al., 2001). There are animal models in which active immunization of mice with thyroglobulin has raised antibodies to thyroglobulin, and leading to increased fetal wastage and lower fetal and placenta weights

(Tartkover-Matalon, et al., 2003). However, the pathophysiological role of these antibodies is still unclear. In recurrent pregnancy loss, the association between pregnancy loss and thyroid antibodies may be a result of: (i) a direct effect of ATAs on fetal tissue or (ii) the thyroid antibodies representing an underlying more generalized defect in autoimmunity. However, the prevalence of

ATA has been reported to be 15–20% in normal pregnant women, compared with 20–25% in women with recurrent miscarriages (Ghazeeri, et al., 2001). In Marai et al.'s (2004) study, anti-TPO antibodies were the only autoantibodies found to have a significant association with recurrent miscarriage. Similar study by IIjima et al (1997) showed similar outcome. The aTPOs were found in

21% of the women with recurrent miscarriages (8/38) as compared with 0% in women with

43 infertility and no miscarriages (0/20). However, in Shoenfeld et al.'s (2006) larger study, there was no association with recurrent miscarriage as a whole, but anti-TG antibodies were associated with late pregnancy loss compared with controls, (OR 8.44; 95% CI 1.6, 43.8), PPV = 40%. Study by

Mannisto et al (2009) showed that thyroid dysfunction and antibodies were not associated with pregnancy complications. Overt hypothyroidism was associated with subsequent maternal thyroid disease [hazard ratio (HR) (95% confidence interval), 17.7 (7.8–40.6)] and diabetes [6.0 (2.2–16.4)].

Subclinical hypothyroidism [3.3 (1.6–6.9)], TPO-Ab-positivity [4.2 (2.3–7.4)], and TG-Ab-positivity

[3.3 (1.9–6.0)] were also associated with later thyroid disease. No association was found between thyroid dysfunction/antibodies and hypertension or overall mortality. Thyroid dysfunction and antibodies during pregnancy seem to predict later thyroid disease. Thyroid dysfunction and antibodies during pregnancy seem to predict later maternal morbidity to thyroid diseases. Therefore, the prognostic value of ATA remains uncertain.

2.2.7.3.3 Anti-nuclear antibodies

Cubillos et al. (1997) found that the incidence of ANAs was 31.8% in patients with a history of miscarriages (110 patients), but only 5.7% in 35 healthy patients with proven fertility and no history of pregnancy loss or autoimmune disease.

In previous studies, a high prevalence of low-titre ANA have been reported in the sera of patients with both explained and unexplained pregnancy losses (Xu, et al., 1990). However, the significance of these findings is still unclear. ANAs were found with a higher prevalence in patients with autoimmune disease in Shoenfeld et al. (2006) study. However, they were not found to occur with a higher prevalence in patients with infertility or recurrent pregnancy loss.

2.2.7.4 Calcium

Calcium is the fifth most abundant element in the human body, with about 1000 g present in adults.

It plays a key role in skeletal mineralization, as well as a wide range of biologic functions. It is an essential element that is only available to the body through dietary sources. Current dietary calcium recommendations range from 1000 to 1500 mg/d, depending on age. In some individuals, particularly the elderly (McCabe et al., 2004), calcium supplements may be needed to achieve the recommended dietary calcium intake (Munro, 2010). Calcium requirement is dependent on the state of calcium metabolism, which is regulated by three main mechanisms: intestinal absorption, renal reabsorption, and bone turnover. These in turn are regulated by a set of interacting hormones, including parathyroid hormone (PTH), 1,25-dihydroxyvitamin D [1,25(OH)2D], ionized calcium itself, and their corresponding receptors in the gut, kidney, and bone (Munro, 2010). During pregnancy, total calcium declines in parallel with serum albumin, whereas free calcium is unchanged

(Vasudevan et al., 2010). The fetal circulation is relatively hypercalcemic as evidenced by higher total and free calcium in cord blood than in maternal serum. Calcium concerntrations decline after birth in healthy term neonates during the first few days but soon rise to concerntrations slightly greater than those observed in adults (Burtis et al., 2012).

Calcium is required for the optimal activity of several extracellular digestive enzymes, including proteases, phospholipases and nucleases. Along the length of the gastrointestinal tract a calcium ion sensing receptor is expressed, and it is thought that the expression of this receptor plays a role in gastric acid secretion (Theobald, 2005). Calcium is essential for blood coagulation (clotting), a process that involves two pathways: the intrinsic pathway and the extrinsic pathway. Calcium plays a role in both pathways. The process of clot formation involves a cascade of proteolytic reactions, whereby inactive enzymes or clotting factors are activated. Each activated enzyme or clotting factor

45 then, in turn, activates another inactive factor, with the response amplifying each time, ultimately resulting in the production of thrombin from prothrombin and the production of a fibrin clot

(Vasudevan et al., 2010; Burtis et al., 2012).

Calcium plays an important role in provoking neurotransmitter release from nerve cells and in muscle contraction. Both nerve and muscle cells are electrically excitable and their cell membranes contain calcium selective ion channels; these open when the membranes are depolarised (e.g. upon arrival of action potentials via nerves), causing an influx of calcium and an increase in cytosolic calcium concentration. In nerves, this results in the release of neurotransmitters (e.g. acetylcholine)

(Sembulingam and Prema, 2010). A similar series of events causes heart muscle contraction. The role of calcium in the contraction of smooth muscle (e.g. the muscle lining the lungs and digestive tract) differs from that of skeletal and heart muscle contraction as the thin filaments of smooth muscle do not contain troponin. Rather, in smooth muscle, an increase in intracellular calcium ion concentration brings about binding of calcium to calmodulin (which is structurally similar to troponin) in the cytosol. The resultant calcium–calmodulin complex brings about the phosphorylation of myosin, allowing myosin crossbridges to form with actin and, thus, the muscle to contract. In smooth muscle only phosphorylated myosin can form cross-bridges with actin.

Relaxation is induced by dephosphorylation (Sembulingam and Prema, 2010).

Figure 2.2 Calcium homeostasis

Source: www. cnx.org

2.2.7.4.1 Calcium metabolism in pregnancy

The effect of pregnancy on the maternal skeleton has preoccupied the scientific medical community for decades. Fetal calcium deposition has been shown to peak at 350 mg/day in the third trimester,

(Sparks, 1984) and maternal calcium absorption increases to meet that demand, with greater increases reported among women with low intakes (Heaney and Skillman 1971;Ritchieet al.,

1998;Vargas et al., 2004). Maternal bone turnover also increases, (Zeni et al., 2003; Hellmeyer et al., 2006) although women with low calcium intakes may respond differently to the additional demand (Ritchie et al., 1998).

Studies suggest that maternal calcium absorption increases significantly during the second and third trimesters (Cross et al., 1995).This increase in calcium absorption is directly related to maternal calcium intake. Ritchie et al. (1998) reported that women with a daily average calcium intake of

1,171 mg during pregnancy absorbed 57% during the second trimester and 72% during the third trimester. Several studies have reported that calcitriol [1,25(OH)2D] levels increase progressively each trimester, thereby influencing the increase in calcium absorption (Cross et al., 1995; Ritchie et al., 1998). Calcium absorption during pregnancy is mediated by changes in maternal calcitropic hormones. During the first trimester, parathyroid hormone (PTH) levels in Caucasian women consuming adequate amounts of calcium decrease to low-normal levels and then increase to the higher end of normal in the third trimester (Gallacher et al., 1994;Cooper et al., 2006) reflecting the increase in calcium transfer from mother to fetus. Although PTH levels typically do not increase above normal during pregnancy, levels of a prohormone, parathyroid hormone receptor protein

(PTHrP), do increase in maternal circulation (Kovacs et al., 1997). PTHrP is recognized by PTH receptors and therefore has PTH-like effects. This prohormone is produced by mammary and fetal tissues to stimulate placental calcium transport to the fetus. (Kovacs et al., 1997). PTHrP may also protect the maternal skeleton from bone resorption (Kovacs et al., 1997) by increasing both calcium absorption in the small intestine and tubular resorption in the kidney. PTHrP may also support mineralization of trabecular and cortical bone in the fetus (Cooper etal., 2006).

Other calcitropic hormones affecting maternal calcium metabolism are both the active [1,25

(OH)2D] and the inactive [25(OH)D] forms of vitamin D. Serum 25(OH)D levels do not change during pregnancy, but an increase in 1-a-hydroxylase and additional synthesis in the placenta allows for an increase in the conversion of 25(OH)D to 1,25(OH)2D. Maternal serum 1,25(OH)2D levels increase twofold during pregnancy, allowing the intestinal absorption of calcium also to double.-

(Ritchie et al., 1998;Zeni et al., 2003) Both free and protein-bound forms of calcitriol increase during pregnancy,( Wilson et al., 1990; Kent et al., 1993) as do concentrations of vitamin-D binding protein.(Bouillon et al., 1981) Due to the corresponding changes in vitamin-D-binding protein and

1,25(OH)2D, the index of free 1,25(OH)2D does not increase until the third trimester,( Ritchie et al., 1998;Wilson et al., 1990) which may explain the large increase in calcium absorption seen during late pregnancy. Because maternal 25(OH)D does cross the placenta and because there is a positive association between maternal serum 25(OH)D, cord blood 25(OH)D, and infant 25(OH)D levels at delivery,(Kovacs et al., 1997) it is thought that vitamin D plays a role in fetal bone development. However, there is a paucity of research in this area, and it is not clear what effect maternal vitamin D status has on maternal and/or fetal bone outcomes. A more in-depth review of the role of vitamin D during pregnancy is provided by Dror and Allen (2010).

2.2.7.4.2 Calcium level and preeclampsia

In the 1980s, it was reported that there is an inverse relationship between calcium intake and pregnancy-induced hypertension (PIH) defined as systolic blood pressure of >140 mmHg and/or

49 diastolic blood pressure of >90 mmHg that has occurred on at least two occasions at least 4 hours to

1 week apart ( Villar et al., 2006). PIH has been estimated to complicate 5% of all pregnancies and

11% of first pregnancies (Villar et al., 2004) often resulting in preterm birth (Ananth and Basso,

2010).

Preeclampsia is a condition in which hypertension (as defined above) occurs during the latter half of gestation and is associated with an increase in urinary protein. Low calcium intakes during pregnancy may 1) stimulate PTH secretion, increasing intracellular calcium and smooth musclecontractibility, and/or 2) release renin from the kidney, leading to vasoconstriction and retention of sodium and fluid. These physiological changes can lead to the development of

Pregnancy Induced Hypertension and preeclampsia (Theobald, 2005). A meta-analysis of the role of calcium supplementation during pregnancy in the prevention of gestational hypertensive disorders found a 45% reduction in the development of PIH in women receiving calcium versus placebo

(relative risk [RR] 0.55; 95% confidence interval [CI] 0.36–0.85) (Imdad et al., 2011). A Cochrane review of 13 trials involving 15,730 pregnant women reported that the average risk of preeclampsia was reduced in those receiving calcium supplements (RR 0.45) and that the effect was greatest in women with low baseline calcium intakes (RR 0.36) (Hofmeyr et al., 2003; 2006; 2007). The review concluded that pregnant women consuming low amounts of calcium could reduce their risk of preeclampsia by 31% to 65% if they consumed an additional 1,000 mg of calcium each day

(Hofmeyr et al., 2003). This review also reported that the risk of developing PIH could be reduced with calcium supplementation (RR 0.65; 95% CI 0.53–0.81), especially in women with low baseline dietary calcium intakes (RR 0.44; 95% CI 02.8–0.70) (Hofmeyr et al., 2010).

2.2.7.4.3 Calcium level and preterm birth (delivery)

Calcium supplementation has shown effectiveness in reducing the risk of preterm delivery in women with low calcium intakes. Among pregnant women who regularly consumed less than 600 mg of calcium per day and were supplemented with additional calcium (1,500 mg/day), decreases in the risk of preterm delivery, in maternal morbidity, and in the neonatal mortality index were observed

(Villar et al., 2006). A Cochrane review reported that women who were chronically low consumers of calcium but who took 1,000 mg of calcium supplement per day also reduced their risk of preterm birth by 24% (Hofmeyr et al., 2006).

2.2.7.4. Calcium transfer from mother to fetus

By the time of parturition, a fetus has formed 98% of its skeleton, accumulating approximately 30 g of calcium. Calcium is actively transported across the placenta, with the transfer from mother to fetus beginning by week 12 of gestation and peaking at week 36 (Forbes, 1976). Placental calcium transport is dependent upon transport proteins located in the syncytiotrophoblast, which forms a barrier between the mother and fetus (Martin et al., 2007). Ninety-nine percent of the flow of calcium is maternal-to-fetal, and this active, one-way process is under way by the third trimester, when the majority of calcium is transferred, with the fetus accumulating 250–350 mg/day (Kovacs and kronenberg, 1997; Prentice, 2003; Martin et al., 2007).

2.2.7.4.5 Hypocalcaemia

This is the presence of low serumcalcium levels in the blood. Physiologically, blood calcium is tightly regulated within a narrow range for proper cellular processes. (Armstrong and Cota, 1999)

Calcium in the blood exists in three primary states: bound to proteins (mainly albumin), bound to anions such as phosphate and citrate, and as free (unbound) ionized calcium (Armstrong and Cota,

1999). Only the ionized calcium is physiologically active. Normal blood calcium level is between

8.6 to 10.3 mg/dL (2.15 to 2.57 mmol/L) (Burtis et al., 2012) and that of ionized calcium is 4.6 to 5.3 mg/dL (1.15 to 1.33 mmol/L). Common causes of hypocalcemia includehypoparathyroidism, vitamin D deficiency, and chronic kidney disease (Armstrong and Cota, 1999). Symptoms of hypocalcemia include neuromuscular irritability (including tetany as manifested by Chvostek's sign or Trousseau's sign, bronchospasm), electrocardiographic changes, and seizures. Treatment is dependent upon the cause, but most commonly includes supplementation of calcium and some form of vitamin D or its analogues (Durlach, 1997).

2.2.7.4.6 Hypercalcaemia

Body calcium is under such close homeostatic control that an excessive accumulation in blood or tissues from over-consumption is virtually unknown. There are a number of conditions, however, that result from failure of the calcium control mechanisms either generally or locally. General failure of one or more control mechanisms results in hypercalcaemia (high blood calcium concentrations)

Local disturbances, usually related to impaired arterial supply and consequent tissue necrosis, result in the deposition of calcium salts. Hypercalcaemia is characterised by anorexia, nausea and, sometimes, vomiting, muscle weakness, generalised itchiness and excessive thirst and urination. If onset is acute, confusion or stupor may result (Theobald, 2005). Kidney failure usually occurs unless treatment is given. Hypercalcaemia occurs as a result of either increased mobilisation of calcium from bone or increased tubular reabsorption or decreased glomerular filtration in the kidneys. More than one mechanism is usually involved. The resultant increase in concentration of circulating calcium results in the deposition of calcium salts in many tissues including the heart and kidneys

(Theobald, 2005).

2.2.7.4.7 Calcium and cardiovascular disease

It has long been suggested that there is an association between cardiovascular mortality and hardness of water, with people in areas served by hard water being less prone to disease. This has been attributed to the calcium content of water and also magnesium. Calcium intake has been shown to be inversely associated with risk of cardiovascular disease in epidemiological studies whilst randomised controlled trials have shown that calcium supplementation may improve serum lipid concentrations.

High intakes of calcium (≥ 1000 mg/day) may have beneficial effects on lipoprotein profiles, i.e. reduce total cholesterol and LDL (low density lipoprotein) cholesterol concentrations and increase

HDL (high density lipoprotein) cholesterol concentration (Roqoveanu et al., 2015).

A low serum-ionised calcium concentration and a low dietary intake of calcium have been shown to be associated with elevated blood pressure and increased risk of hypertension in a number of epidemiological studies, suggesting an inverse relationship between calcium intake and blood pressure. Meta-analyses of randomized controlled trials involving calcium supplementation (from diet and supplements) suggest that calcium exerts a modest hypotensive effect at high intakes in both normotensive and hypertensive individuals, and that both SBP and diastolic blood pressure (DBP) are significantly reduced (Sallinenet al., 1996; Dazai et al., 1996). Dietary intervention studies in which individuals are, amongst other things, encouraged to increase calcium consumption through increased intake of reduced or low-fat dairy products have been shown to improve blood pressure.

There is some preliminary evidence to suggest that calcium may play a role in the fetal programming of blood pressure; with maternal calcium status influencing the blood pressure of offspring later in life. Pre-natal maternal intake of calcium has been shown to be associated with blood pressure in infants, and pre-natal calcium supplementation trials have shown that calcium intake is inversely associated with blood pressure in young children (Bergel and Belizan, 2002;

Bergel and Barros, 2007).

2.2.7.5 Magnesium

Magnesium, an abundant mineral in the body, is naturally present in many foods, added to other food products, available as a dietary supplement, and present in some medicines (such as antacids and laxatives). (IOM, 1997) Magnesium is a cofactor in more than 300 enzyme systems that regulate diverse biochemical reactions in the body, including protein synthesis, muscle and nerve function, blood glucose control, and blood pressure regulation (Rude, 2010;2012). Magnesium is required for energy production, oxidative phosphorylation, and glycolysis. It contributes to the structural development of bone and is required for the synthesis of DNA, RNA, and the antioxidant glutathione. Magnesium also plays a role in the active transport of calcium and potassium ions across cell membranes, a process that is important to nerve impulse conduction, muscle contraction, and normal heart rhythm (Rude, 2012).

An adult body contains approximately 25 g magnesium, with 50% to 60% present in the bones and most of the rest in soft tissues (Volpe, 2012). Less than 1% of total magnesium is in blood serum, and these levels are kept under tight control. Normal serum magnesium concentrations range between 0.75 and 0.95 millimoles (mmol)/L (IOM, 1997; Elin, 2010). Hypomagnesemia is defined as a serum magnesium level less than 0.75 mmol/L(Gibson, 2005) Magnesium homeostasis is largely controlled by the kidney, which typically excretes about 120 mg magnesium into the urine each day (Rude, 2010). Urinary excretion is reduced when magnesium status is low (IOM, 1997).

2.2.7.5.1 Magnesium and pregnancy

Magnesium is involved in many essential bodily functions. Magnesium helps build strong bones and teeth, regulates insulin and blood-sugar levels, and helps as activators certain enzymes function

54 properly. Examples include Hexokinase, Adenyl cyclase, Alkaline phosphatase etc. It also controls cholesterol and irregular heartbeats (Dahle et al., 1995).

The healthy growth and development of the fetus depend on a steady supply of nutrients from the mother. During pregnancy the body needs more of this mineral and RDA requires upward adjustments.Pregnancy is accompanied by increases in the dietary intake of vitamins minerals such as magnesium (Pinto et al., 2009)

Physical and emotional stress during pregnancy also increases magnesium requirements, which means that pregnant women who do not intake sufficient amount of magnesium are at a risk of becoming magnesium deficient. Magnesium deficiency during pregnancy can lead to many serious consequences for the mother and the baby. And a severe deficiency of magnesium during pregnancy may lead to preeclampsia, birth defects, infant mortality and pre-mature labor. Adequate magnesium during your pregnancy makes one feel healthier and help cope better with discomforts related to increased hormonal activity during pregnancy.

2.2.7.5.2 Magnesium deficiency in pregnancy

Magnesium plays an important role in pregnancy for the formation of new tissues (maternal and fetal). Pregnant to women require higher magnesium intake than the normal non-pregnant women of same age (Shaikh et al., 2011; Hashemi et al., 2012). Serum magnesiumlevels decline during pregnancy, because of increased demand and increase renal excretion of magnesium and inadequate water intake has been concluded that pregnancy is actually a state of extracellular magnesium depletion. Mg deficiency in pregnant women compared with non-pregnant women, they found, that

55 both total and ionized magnesium were significantly lower during normal pregnancy (Standley et al.,

1997). Since magnesium has an inhibitory role on myometrial contractions, it dilates blood vessels, improve cerebral blood flow. Attention has been paid to the role of magnesium deficiency in causing preterm labour, IUGR (Inter Uterine Growth Restriction), hypertension. Hypomagnesemia leads to neuromuscular hyper-excitability resulting in muscle cramp and uterine\ hyper-activity

(Hantoushzadeh et al., 2007).

Magnesium has an established element for foetal well-being. Its deficiency during pregnancy has been reported to be associated with pre-eclampsia, small for gestational age (IUGR) and preterm labour (Mercer and Merlino, 2009), leg cramps (Khalida et al., 2012). Magnesium supplementation during pregnancy may improve foetal and maternal outcome by decreasing incidence of preterm

Labor, for prevention and control of seizure (convulsions) (Naz et al., 2005) and reduce incidence of maternal and neonatal hospital admissions (Khalida et al., 2012).

2.2.7.5.3 Health risk of excessive magnesium

Too much magnesium from food does not pose a health risk in healthy individuals because the kidneys eliminate excess amounts in the urine (Musso, 2009). However, high doses of magnesium from dietary supplements or medications often result in diarrhea that can be accompanied by nausea and abdominal cramping (Institute of Medicine, 1997). Forms of magnesium most commonly reported to cause diarrhea include magnesium carbonate, chloride, gluconate, and oxide (Ranade,

2001). The diarrhea and laxative effects of magnesium salts are due to the osmotic activity of unabsorbed salts in the intestine and colon and the stimulation of gastric motility.

Very large doses of magnesium-containing laxatives and antacids (typically providing more than

5,000 mg/day magnesium) have been associated with magnesium toxicity (Kutsal, 2007), including

56 fatal hypermagnesemia in a 28-month-old boy (McGuire, 2000) and an elderly man (Onishi,2006).

Symptoms of magnesium toxicity, which usually develop after serum concentrations exceed 1.74–

2.61 mmol/L, can include hypotension, nausea, vomiting, facial flushing, retention of urine, ileus, depression, and lethargy before progressing to muscle weakness, difficulty breathing, extreme hypotension, irregular heartbeat, and cardiac arrest (Musso, 2009). The risk of magnesium toxicity increases with impaired renal function or kidney failure because the ability to remove excess magnesium is reduced or lost (IOM, 1997; Musso, 2009).

2.2.7.5.4 Magnesium calcium relationship.

The relationship between calcium and magnesium in the bodies is very complex. They work together in many functions, such as regulating heartbeat, muscle tone and contraction, and nerve conduction.

At other times, calcium and magnesium seem to compete by binding competitively to the same sites in the body (Walton, 2015).

The calcium and the magnesium are required for many metabolic reactions that take place inside of the body. The calcium is often called as the backbone mineral as it is an important mineral that aids in the formation of bones and teeth. Magnesium is called as natural tranquilizer as it is usually involved to relieve the tension by acting on the nerves and the muscles. The calcium and the magnesium are involved in the transmission of nerve impulse and also believed to involve in the regulation of hydrogen ion concentration (Power, 1999).

During pregnancy, there is a progressive decline in concentration of Ca and Mg in maternal serum possibly due to hemodilution, increased urinary excretion, and increased transfer of these minerals

57 from the mother to the growing fetus (Rayman, 2003). In addition, low dietary intake and accelerated metabolism might be other contributing factors.

2.2.7.5.5 Magnesium, hypertension and cardiovascular diseases

Hypertension is a major risk factor for heart disease and stroke. Studies to date, however, have found that magnesium supplementation lowers blood pressure, at best, to only a small extent. A meta- analysis of 12 clinical trials found that magnesium supplementation for 8–26 weeks in 545 hypertensive participants resulted in only a small reduction (2.2 mmHg) in diastolic blood pressure

(Dickinson et al., 2006). The dose of magnesium ranged from approximately 243 to 973 mg/day.

The authors of another meta-analysis of 22 studies with 1,173 normotensive and hypertensive adults concluded that magnesium supplementation for 3–24 weeks decreased systolic blood pressure by 3–

4 mmHg and diastolic blood pressure by 2–3 mmHg (Kass et al., 2012). The effects were somewhat larger when supplemental magnesium intakes of the participants in the nine crossover-design trials exceeded 370 mg/day. A diet containing more magnesium because of added fruits and vegetables, more low-fat or non-fat dairy products, and less fat overall was shown to lower systolic and diastolic blood pressure by an average of 5.5 and 3.0 mmHg, respectively (Champagne, 2006). However, this

Dietary Approaches to Stop Hypertension (DASH) diet also increases intakes of other nutrients, such as potassium and calcium, that are associated with reductions in blood pressure, so any independent contribution of magnesium cannot be determined.

Several prospective studies have examined associations between magnesium intakes and heart disease. The Atherosclerosis Risk in Communities study assessed heart disease risk factors and levels of serum magnesium in a cohort of 14,232 white and African-American men and women aged

45 to 64 years at baseline (Peacock et al., 2010). Over an average of 12 years of follow-up, individuals in the highest quartile of the normal physiologic range of serum magnesium (at least 0.88 mmol/L) had a 38% reduced risk of sudden cardiac death compared with individuals in the lowest quartile (0.75 mmol/L or less). However, dietary magnesium intakes had no association with risk of sudden cardiac death. Another prospective study tracked 88,375 female nurses in the United States to determine whether serum magnesium levels measured early in the study and magnesium intakes from food and supplements assessed every 2 to 4 years were associated with sudden cardiac death over 26 years of follow-up (Chiuve et al., 2011). Women in the highest compared with the lowest quartile of ingested and plasma magnesium concentrations had a 34% and 77% lower risk of sudden cardiac death, respectively. Another prospective population study of 7,664 adults aged 20 to 75 years in the Netherlands who did not have cardiovascular disease found that low urinary magnesium excretion levels (a marker for low dietary magnesium intake) were associated with a higher risk of ischemic heart disease over a median follow-up period of 10.5 years. Plasma magnesium concentrations were not associated with risk of ischemic heart disease (Joosten et al., 2013). A systematic review and meta-analysis of prospective studies found that higher serum levels of magnesium were significantly associated with a lower risk of cardiovascular disease, and higher dietary magnesium intakes (up to approximately 250 mg/day) were associated with a significantly lower risk of ischemic heart disease caused by a reduced blood supply to the heart muscle (Del

Gobbo et al., 2013).

Higher magnesium intakes might reduce the risk of stroke. In a meta-analysis of 7 prospective trials with a total of 241,378 participants, an additional 100 mg/day magnesium in the diet was associated with an 8% decreased risk of total stroke, especially ischemic rather than hemorrhagic stroke

(Larsson et al., 2012). One limitation of such observational studies, however, is the possibility of confounding with other nutrients or dietary components that could also affect the risk of stroke.

A large, well-designed clinical trial is needed to better understand the contributions of magnesium from food and dietary supplements to heart health and the primary prevention of cardiovascular disease (Song and Liu, 2012). Diets with higher amounts of magnesium are associated with a significantly lower risk of diabetes, possibly because of the important role of magnesium in glucose metabolism (Larsson, 2007) Hypomagnesemia might worsen insulin resistance, a condition that often precedes diabetes, or it might be a consequence of insulin resistance (Simmons, 2010).

Diabetes leads to increased urinary losses of magnesium, and the subsequent magnesium inadequacy might impair insulin secretion and action, thereby worsening diabetes control (Rude, 2012).

A 2011 meta-analysis of prospective cohort studies of the association between magnesium intake and risk of type 2 diabetes included 13 studies with a total of 536,318 participants and 24,516 cases of diabetes (Dong, 2011). The mean length of follow-up ranged from 4 to 20 years. Investigators found an inverse association between magnesium intake and risk of type 2 diabetes in a dose- responsive fashion, but this association achieved statistical significance only in overweight (body mass index [BMI] 25 or higher) but not normal-weight individuals (BMI less than 25). Again, a limitation of these observational studies is the possibility of confounding with other dietary components or lifestyle or environmental variables that are correlated with magnesium intake.

2.3.1 Disease burden of HIV/ AIDS

HIV/AIDS has been a major subject of public health discussion since the last two decades. By the end of 2008, 34.4million people worldwide were living with HIV/AIDS (WHO, 2010). Of this figure

31.3million are adults and 30million live in low and middle income countries. Thus, 87.2% of the world population living with HIV/AIDS lives in low and middle income countries. Sub-Saharan

Africa bears the largest chunk, 67%, of the world‘s HIV/AIDS burden (WHO, 2010). Nigeria is classified as a low income country. With a population of 148.093million people Nigeria bears

2.4million people living with HIV/AIDS (WHO/UNAIDS/UNICEF, 2008). Of this figure 198,000 received antiretroviral therapy in 2007 from 215 treatment sites (WHO/UNAIDS/UNICEF,

2008)even though 750,000 had need for the treatment. With an annual population growth of 2.1%

(2005-2010) and adult prevalence of HIV of 2.5% (WHO/UNAIDS/UNICEF, 2008) the national

HIV/AIDS burden will increase. More people may access antiretroviral treatment if the national coverage for antiretroviral treatment increases.

2.3.2 World Health Organization (Who) Clinical Staging For Hiv/Aids

The WHO (WHO, 2007) divided the progression of HIV/AIDS into four clinical stages. This revised clinical staging and immunological classification of HIV were designed to assist in the clinical management of patients, more especially in resource poor countries where laboratory facilities are very limited (WHO/UNAIDS/UNICEF, 2007).

HIV Associated Symptoms WHO Clinical Stage

Asymptomatic 1

Mild symptoms 2

Advanced symptoms 3

Severe symptoms 4

WHO Stage 1

The patient is asymptomatic but has persistent generalized lymphadenopathy (WHO, 2007).

WHO Stage 2

The patient may have moderate unexplained weight loss (less than 10% of presumed or measured body weight), recurrent respiratory tract infections such as sinusitis, tonsillitis, otitis media and pharyngitis, herpes zoster, angular stomatitis, recurrent oral ulceration, papular pruritic skin eruptions, seborrhoiec dermatitis, and fungal nail infections (WHO,2007).

WHO Stage 3

The patient may have unexplained severe weight loss (more than 10% of presumed or measured body weight), unexplained chronic diarrhoea lasting longer than 1 month, unexplained persistent fever, intermittent or persistent lasting over a month, persistent oral candidiasis, oral hairy luekoplakia, on-going pulmonary tuberculosis, severe bacterial infection such as pneumonia, empyema thoracis, pyomyositis, bone or joint infection, meningitis or septicaemia (WHO, 2007).

Other symptoms include acute necrotizing ulcerative stomatitis, gingivitis or periodontitis, unexplained anaemia (Hb less than 8g/dl), unexplained neutropeania (< 0.5 x 109 per litre), and unexplained chronic thrombocytopeania (< 50 x 109 per litre) (WHO, 2007).

WHO Stage 4

The patient may have HIV wasting syndrome, pnuemocystic pneumonia, recurrent severe bacterial pneumonia, chronic herpes simplex infection (orolabial, genital or anorectal of more than 1 month duration or visceral at any site), oesophageal, tracheal, bronchial or lung candidiasis(WHO,2007).

Others include extrapulmonary tuberculosis, extrapulmonary cryptococcosis including meningitis, karposis sarcoma, cytomegalovirus infection, central nervous system toxoplasmosis, HIV encephalopathy, disseminated non-tuberculous mycobacterial infection, disseminated endemic mycosis, chronic cryptosporidiosis, chronic isosporiasis, cerebral or B-cell non-Hodgkin lymphoma, progressive multifocal leukoencephalopathy, and symptomatic HIV-associated nephropathy or cardiopathy(WHO,2007).

The WHO (WHO, 2007) went further to set criteria for HIV staging events which are essentially practical ways of identifying clinical symptoms that will enable good clinical staging.

2.3.3 Antiretroviral Drugs

Antiretroviral drugs are antiviral drugs used in the treatment of HIV infection. Antiretroviral drugs are generally classified as:

-Protease (proteinase) inhibitors

-Neucloside reverse transcriptase inhibitors

-Non-neucloside reverse transcriptase inhibitors

The proteinase inhibitors inhibits HIV proteinase. The HIV proteinase cleaves polyprotein precursors to release mature proteins which includes the HIV proteinase itself, reverse trascriptase, integrase and various other structural proteins important in HIV replication (Iwe, 2005). They include indinavir, ritonavir, saquinavir, atazanavir, lopinavir, nelfnavir, etc. Their main side effect

63 include nephrolithiasis, peripheral neuropathy, gastrointestinal effects such as diarrhea and nausea, headache, abdominal obesity and hyperlipidaemia (Iwe, 2005).

The nucleoside reverse transcriptase inhibitors are pyrimidine nucleoside analogues. They are phosphorylated by a viral-induced enzyme and thereafter incorporated into the growing viral DNA thereby terminating further growth of the DNA chain. They are therefore DNA terminators. They include zidovudine, lamivudine, zalcitabine, didanosine, stavudine, etc. Their main side effects include pancreatitis, peripheral neuropathy, nausea, bone marrow depression, headache, insomnia and myalgia (Iwe, 2005).

The non-nucleoside reverse transcriptase inhibitors are not nucleoside analogues. They include nevirapine and loviride.

2.3.4 Antiretroviral Therapy

Standard antiretroviral therapy consists of the use of 3 antiretroviral drugs. This is also called highly active antiretroviral therapy (HAART). This minimizes the development of drug resistance, maximizes viral load reduction and minimizes transmission from person to person (Chukwuanukwu et al.,2003 ). The time of initiation of HAART is determined by a number of factors including CD 4 count, viral load and general clinical condition. New and compelling evidence show that earlier commencement of HAART is associated with increased survival and reduction of opportunistic infections (WHO, 2009). In the most recent guideline (WHO, 2009) on antiretroviral therapy the

World Health Organization made 8 key recommendations based on emerging evidences.

2.3.4.1 WHO Key Recommendations On Antiretroviral Therapy

Recommendation 1

When to Start HAART

a. Start treatment in all patients with CD4 count less than 350cells/ml irrespective of clinical

status.

b. CD4 testing is required to identify if patients with HIV and WHO clinical stage 1 or 2 disease

need to start antiretroviral therapy.

c. Start antiretroviral treatment in all patients with HIV and WHO clinical stage 3 or 4

irrespective of CD4 count.

In this recommendation the WHO placed more emphasis on avoiding death, disease progression

and HIV transmission over and above cost and feasibility. The prognosis of patients who started

treatment according to recommendation 1.a above is much better than those who started

treatment at CD4 count below 250cells/ml.

Recommendation 2

What to start

a. Start any of the following regimens in ART-naïve individuals eligible for treatment:

i. zidovudine (AZT) + lamivudine (3TC) + Efavirence (EFV)

ii. AZT + 3TC +nevirapine (NVP)

iii. (Tenofovir disoproxil fumarate) TDF + 3TC or emtricitabine(FTC) + EFV

iv. TDF + 3TC or FTC + NVP

In this recommendation the WHO placed emphasis on avoiding the disfiguring, unpleasant and potentially life threatening toxicity of stavudine (d4T), the need to select regimen suitable in most patient groups and the need of fixed dose combinations to improve compliance.

Recommendation 3

HAART for HIV/Tuberculosis co-infection

a. Start HAART on any HIV-infected individual with active tuberculosis irrespective of CD4

count.

b. Start Tuberculosis treatment first followed by HAART as soon as possible.

c. Use efavirence (EFV) as preferred non nucleoside reverse transcriptase inhibitor in patients

starting antiretroviral therapy while on Tuberculosis (TB) treatment.

The emphasis of the WHO here is in reducing early mortality from HIV/TB co-infection, reducing

TB transmission when HAART is started earlier in all individuals with TB and improved management of TB.

Recommendation 4

HAART for HIV/HBV (Hepatitis B Virus) co-infection

a. Start HAART for all patients with HIV/HBV co-infection who require treatment for HBV

infection irrespective of CD4 count or WHO clinical stage.

b. Start TDF and 3TC or FTC containing regimens in all HIV/HBV co-infected patients

requiring therapy.

The WHO placed emphasis on improved HIV/HBV management and HBV diagnosis; there is a 3 to

6-fold increase in developing chronic hepatitis, an increased risk of fibrosis and cirrhosis and a 17- fold increased risk of death in HBV/HIV patients compared with HIV patients without HBV.

Recommendation 5

HAART for Pregnant Women

a. Start ART in all pregnant women with HIV infection and CD4 count equal or less than

350cells/ml irrespective of clinical symptoms.

b. CD4 count testing is required to identify if pregnant women with HIV and WHO clinical

stage 1 or 2 disease will require ART or prophylaxis.

c. Start ART in all pregnant women with HIV and WHO clinical stage 3 or 4 irrespective of

CD4 count.

d. Start one of the following in ART-naïve pregnant women who are eligible for treatment:

i. AZT + 3TC + EFV

ii. AZT + 3TC + NVP

iii. TDF + 3TC or FTC + EFV

iv. TDF + 3TC or FTC + NVP

e. Do not start EFV during the first trimester of pregnancy.

The WHO placed high value in ensuring that treatment is started early in pregnant women with HIV to avoid mother to child transmission and improve child and maternal health outcomes over and above cost or feasibility of medication.

Recommendation 6

When to switch HAART

a. Where available use Viral Load (VL) to confirm treatment failure.

b. Where routinely available use VL every 6 months to detect viral replication.

c. A persistent VL above 5,000 copies/ml confirms treatment failure.

d. When VL is not available use immunological criteria to confirm clinical failure.

Here, the WHO was concerned about the limitations of clinical and immunological monitoring in

diagnosing treatment failure and placed high value on avoiding premature or unnecessary switching

to expensive second line HAART.

Recommendation 7

Second-line HAART

a. A boosted protease inhibitor plus two nucleoside analogues are recommended as second line

HAART.

b. Atazanavir + low dose ritonavir (ATV/r) and Lopinavir +low dose ritonavir (LPV/r) are

preferred boosted PI‘s for second-line HAART.

c. Simplification of second-line drugs is recommended.

d. If d4T or AZT has been used in the first-line drugs use TDF + 3TC or FTC as the

nucleoside backbone in the second-line drugs.

e. If TDF has been used in first-line use AZT + 3TC as nucleoside backbone in second-line.

In developing this recommendation the WHO placed priority on using simpler second-line regimen and the availability of heat-stable fixed dose combinations.

Recommendation 8

Third-line Regimens

a. National programmes should develop policies for third-line regimens that consider funding,

sustainability and provision of equitable access to HAART.

b. Third-line regimens should include new drugs with known anti-HIV activity such as

integrase inhibitors and second generation NNRTI‘s and PI‘s.

c. Patients on a failing second-line regimen with no new antiretroviral drug options should

continue with a tolerated regimen.

2. 4 Prevention of Mother to Child Transmission (PMTCT)

Despite the proven effectiveness of the prevention of mother to child transmission (PMTCT) of human immunodeficiency virus (HIV) program, Nigeria currently has the highest burden of vertical

69 transmission of HIV in the world due to poor coverage of the PMTCT program partly as a result of poor knowledge of PMTCT interventions amongst healthcare providers in the country (Nkwo, 2012)

Elimination of new HIV infections among children can be achieved through the Prevention of

Mother To Child Transmission (PMTCT). PMTCT is an intervention to ensure that no child is born with HIV and it is an essential step to ensuring an AIDS free generation. The PMTCT initiative provides drugs, counseling and psychological support to help mothers safeguard their infants against the virus.

Pregnant women infected with HIV are at high risk of transmitting HIV to their infants during pregnancy, during birth or through breastfeeding. Over 90% of new HIV infections among infants and young children occur through Mother to Child Transmission. Without any intervention, the risk of transmission of infection from the mother to the baby is 20-45 per cent. With an evidence-based set of comprehensive interventions, this transmission rate can be reduced to less than 2 per cent. The current WHO guidelines recommend two interventions:

1. providing lifelong ART to all pregnant and breastfeeding women living with HIV regardless

of CD4 count or clinical stage or

2. providing ART (ARV drugs) for pregnant and breastfeeding women with HIV during the

mother-to-child transmission risk period and then continuing lifelong ART for those women

eligible for treatment for their own health.

The current global goal is to accelerate progress towards the elimination of new child infections by

2015 and keeping their mothers alive. This can be achieved by ensuring that PMTCT interventions are made available to all women that need it.

The current global goal has 2 targets

1. Reduce the number of new childhood HIV infections by 90%.

2. Reduce the number of HIV-related maternal deaths by 50%. (PMTCT,2007)

2.5 Apgar score

This is a scoring system devised as a rapid method of assessing the clinical status of the newborn

infant at 1 minute of age. Virginia Apgar invented the Apgar score in 1952 as a method to quickly

summarize the health of newborn children (Apgar, 1953). Apgar was an anesthesiologist who

developed the score in order to ascertain the effects of obstetricanesthesia on babies.

This scoring system provided a standardized assessment for infants after delivery. The Apgar score

comprises five components: 1) color, 2) heart rate, 3) reflexes, 4) muscle tone, and 5) respiration,

each of which is given a score of 0, 1, or 2. The resulting Apgar score ranges from zero to 10. The

five criteria are summarized using words chosen to form a backronym (Appearance, Pulse, Grimace,

Activity, and Respiration).

Thus, the Apgar score quantitates clinical signs of neonatal depression such as cyanosis or pallor,

bradycardia, depressed reflex response to stimulation, hypotonia, and apnea or gasping respirations.

The score is reported at 1 minute and 5 minutes after birth for all infants, and at 5-minute intervals

thereafter until 20 minutes for infants with a score less than 7. The Apgar score provides an accepted

71 and convenient method for reporting the status of the newborn infant immediately after birth and the response to resuscitation if needed. Scores 7 and above are generally normal, 4 to 6 fairly low, and 3 and below are generally regarded as critically low.

A newly proposed Combined-Apgar score was proposed as a good predictor of neonatal mortality and morbidity in the admitted neonates, regardless of their gestational age and resuscitation status. It is also superior to the Conventional-Apgar in predicting adverse neonatal outcomes in very preterm, near term and term neonates (Dalili et.al., 2016).

A low score on the one-minute test may show that the neonate requires medical attention but does not necessarily indicate a long-term problem, particularly if the score improves at the five-minute test. An Apgar score that remains below 3 at later times—such as 10, 15, or 30 minutes—may indicate longer-term neurological damage, including a small but significant increase in the risk of cerebral palsy. However, the Apgar test's purpose is to determine quickly whether a newborn needs immediate medical care. It is not designed to predict long term health issues.

Low Apgar score may be caused by repeated late decelerations and prolonged second stage of labor and may be associated with increased risk of neonatal respiratory distress, need for mechanical ventilatory support, and hypoxic-ischemic-encephalopathy (Salustiana et. al., 2012)

Apgar score can be utilized as a predictor of neurological disability. Nelson and Ellenberg (1981) noted that low Apgar scores were risk factors for cerebral palsy, but 55% of children with later cerebral palsy had Apgar scores of 7 to 10 at one minute, and 73% scored 7 to 10 at five minutes. Of

99 children who had Apgar scores of 0 to 3 at ten, 15, or 20 minutes and survived, 12 (12%) had later cerebral palsy; 11 of the 12 were also mentally retarded (in ten, IQ less than 50) and half had seizure disorders. Eight children who survived after having very low late Apgar scores and who did

72 not have cerebral palsy had lesser but significant disabilities. Of the children who had Apgar scores of 0 to 3 at ten minutes or later and survived, 80% were free of major handicap at early school age.

Study by Svenviket.al., (2015) determined preterm birth as a prominent risk factor for low

Apgarscores. Whereas study by Viau et. al., (2015) identified high infant mortality rate among infants with Apgar score 0-3. Low Apgarscores was identified as significant perinatal risk factors for infantile seizures, especially in full-term and normal-birth weight infants, and have a strong negative linear relationship with EEG and brain MRI results in the seizure group (Eun et.al., 2016). There is also a correlation between risk of low Apgar score and maternal socioeconomic status (Odd et.al.,

2014). In a multivariate analysis by Pan et. al., (2016), the presence of reduced vision was associated with a low 5-min Apgar score at birth (<7 vs. 7-10; odds ratio [OR] = 1.66, 95 % CI 1.48-3.05) after adjusting for age, gender, parental history of myopia, maternal age, gestational age, and birth weight.

In addition, both myopia and amblyopia were associated with Apgar scores of less than 7 in multivariate analyses.

2.6 Body mass index(BMI)

Body Mass Index (BMI) is a simple index of weight-for-height that is commonly used to classify underweight, overweight and obesity in adults. It is defined as the weight in kilograms divided by the square of the height in metres (kg/m2) (WHO,2006).

Table 2.1 : The International Classification of BMI

Classification BMI(kg/m2)

Principal cut-off Additional cut-off

points points

Underweight <18.50 <18.50

Severe thinness <16.00 <16.00

Moderate thinness 16.00 - 16.99 16.00 - 16.99

Mild thinness 17.00 - 18.49 17.00 - 18.49

18.50 - 22.99 Normal range 18.50 - 24.99 23.00 - 24.99

Overweight ≥25.00 ≥25.00

25.00 - 27.49 Pre-obese 25.00 - 29.99 27.50 - 29.99

Obese ≥30.00 ≥30.00

30.00 - 32.49 Obese class I 30.00 - 34.99 32.50 - 34.99

35.00 - 37.49 Obese class II 35.00 - 39.99 37.50 - 39.99

Obese class III ≥40.00 ≥40.00

Source: Adapted from WHO, 1995, WHO, 2000 and WHO 2004.

BMI values are age-independent and the same for both sexes. However, BMI may not correspond to the same degree of fatness in different populations due, in part, to different body proportions. The health risks associated with increasing BMI are continuous and the interpretation of BMI gradings in relation to risk may differ for different populations.

In recent years, there was a growing debate on whether there are possible needs for developing different BMI cut-off points for different ethnic groups due to the increasing evidence that the associations between BMI, percentage of body fat, and body fat distribution differ across populations and therefore, the health risks increase below the cut-off point of 25 kg/m2 that defines overweight in the current WHO classification (WHO, 2006).

CHAPTER THREE

MATERIALS AND METHODS

3.1.1 Recruitment of participants

Three hundred and twenty three (323) antenatal women attending the antenatal clinic of the Nnamdi

Azikiwe University Teaching Hospital Nnewi were recruited for the study. These comprised of 136

HIV sero-positives (test group) and 187 sero-negatives (control group). This is a cross-sectional descriptive study. Every alternate consenting participants at different gestational ages were recruited.

The number of participants was determined based on Nigerian birth rate.

For a study design based on a simple random sampling, the sample size required can be calculated according to the following formula given by International Fund for Agricultural development

(IFAD, 2012). n = t2 x p(1-p) /m2

Where; n = required sample size t = confidence level at 95% (standard value of 1.96)

76 p = estimated prevalence of subject of interest in the study area m = margin of error at 5% (standard value of 0.05)

In this study, birth rate of 35.5births per 1000 Nigerian population (2011 estimate) was used in place of estimated prevalence.

Thus for p=0.35, n becomes 34.9

The study size was enlarged in order to accommodate adequate sub-grouping and cases of subject withdrawal from the study.

3.1.2 Sample collection

About 8mls of whole blood samples were collected into plain tubes. These were allowed to clot, retract, and centrifuged at 5000 revolution per minute for ten minutes. The supernatant was aspirated using Pasteur‘s pipette into sterile plain containers, labelled appropriately and stored frozen (-20Ċ) until analyzed. Samples were analysed for PAPP-A, estriol, progesterone, beta-2-glycoprotein-1, antinuclear antibody, thyroid peroxidase antibody, using ELISA technique. Total protein, albumin, calcium, and in-organic phosphorous were done using respective colorimetric methods.

3.1.3 Data collection

Questionnaires were administered on the subjects. Anthropometric data including body weight (kg), height (M), blood pressure (mmHg), and pulse rate (pulse/minute) were taken using weighing scale, stadiometer, mercury in glass sphygmomanometer, and stop watch respectively.

Also birth statistics including birth weight, mode of delivery (normal-SVD, Cesarean sectioning-CS, maternal HIV status, birth outcomes such as intra uterine fetal death (IUFD), breached presentations,

Apgar score were taken for a period of 32 months (May 2011- Dec 2013)

Some of the participants were monitored to delivery, and the birth statistics taken. One hundred and thirty did not report at the hospital for delivery.

3.1.4 Inclusion criteria

All subjects qualified for the study were pregnant women certified fit by attending gynecologist/obstetrician and who willingly consented to participate in the study. Subjects did not have disorders such as preeclampsia, overt diabetes mellitus, and hypertension.

3.1.5 Exclusion criteria

Non pregnant women.

3.1.6 Ethical considerations

Ethical approval was obtained from the Ethics Committee of the Nnamdi Azikiwe University

Teaching Hospital Nnewi. Informed consent was obtained from every participant. Questionnaire was administered on the participants. Participants exercise their free will and could withdraw from the study at any time.

Participants were not prone to hazard and were not paid. Furthermore, information obtained from the study was not divulged to unauthorized person, such that appropriate confidentiality was maintained. Ethical issues did not arise from the study.

3.2 Analytical procedures

3.2.1 Estimation of pregnancy associated plasma protein-A (PAPA-A)

PAPA-A was assayed using ELISA kit by Diagnostic Automation, Inc Clabasas USA

1. Principle of the assay

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for PAPP-A has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any PAPP-A present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for PAPP-A is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of PAPP-A bound in the initial step. The color development is stopped and the intensity of the color is measured.

The intensity of colour measured is directly proportional to the PAPA-A in the sample

2. Reagent preparation

Reagents were prepared according to manufactures instructions. Lyophilized standard were reconstituted with 150 µL of deionized water. The reconstituted standards are stable for two months at 2-8Ċ. Controls were also reconstituted with 150 µL of deionized water and let stand for 10 minutes. It was mixed well several times before use. The reconstituted controls are stable for two months at 2-8Ċ

Wash solution were prepared by diluting 30mL of the concentrated wash solution with 1170 mL of deionized water to final volume of 1,200 mL. The diluted wash solution is stable for two weeks at room temperature.

Enzyme conjugate was prepared thirty minutes before use by diluting 1.0 mL of concentrated enzyme conjugate with 10 mL of conjugate diluent. Enzyme conjugate has to be prepared fresh

30 minutes before assay.

3. Test procedure

All reagents and specimen were allowed to attend room temperature (20-28Ċ) before use. All reagent were mixed gently without foaming. All standards, samples, and controls were run concurrently. Standards were run in duplicates

Appropriate number of microtitre wells were secured in the frame holder. Ten microliters (µL) of each standard, control and samples were dispensed with new disposable tips into appropriate wells.

One hundred microliters of assay buffer was dispensed into each well and mixed thoroughly for 10 seconds. It is important to have a complete mixing in this step. This was incubated for 30 minutes at room temperature (20-28Ċ). The microplates was washed properly 3 times with 400µL of diluted wash solution. Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

One hundred microliters of 100µL of diluted enzyme conjugate was dispensed into each well and incubated for 30 minutes at room temperature (20-28Ċ). The microplates was washed againg properly 3 times with 400µL of diluted wash solution. Absorbent paper was used to remove residual water droplets.

This was followed by the addition of 100µL of enzyme substrate solution to each well and incubated for 15 minutes at room temperature (20-28Ċ) in the dark. The reaction was ended by adding 50µL of stop solution. Absorbance reading was at 450nm.

The concentration of the controls and samples were generated automatically by the automatic microplate reader from standard curve obtained from the average absorbance values of each set of standards plotted against their concentrations.

3.2.2 Estimation of progesterone

Progesterone was assayed using ELISA kit by Delta Biologicals S.R.L, Pomezia Rome; a surbsidiary of Erba Diagnostic, Inc USA

1. Principle of the assay

This assay employs the quantitative competitive enzyme immunoassay technique. A monoclonal antibody specific for progesterone (anti-progesterone) has been pre-coated onto a microplate (solid phase). Standards and samples are pipetted into the wells and any progesterone present is bound by the immobilized antibody. Progesterone in the sample (antigen) competes with horseradish- peroxidase(HRP)-progesterone (enzyme-labeled antigen) for binding onto the limited number of anti-progesterone coated on the microplates. After incubation, a simple washing is performed to wash away any unbound substances.this is followed by the addition of enzyme substrate solution to the wells and color develops. The color development is stopped and the intensity of the color is measured. The intensity of colour measured is inversely proportional to the progesterone in the sample.

Reagent preparation

1. Wash solution was prepared by diluting content of vail of the buffered wash solution

concentrate with deionized water to final volume of 500 mL. The diluted wash solution is

stable for 30 days at 2-8 Ċ.

2. The other reagents were ―ready for use‖

2. Test procedure

Appropriate number of microtitre wells were secured in the frame holder. Twenty microliters (µL) of each standard, control and samples were dispensed with new disposable tips into appropriate wells. Two hundred microliters of enzyme conjugate was dispensed into each well and mixed thoroughly for 10 seconds. It is important to have a complete mixing in this step. This was incubated for 60 minutes at 37 Ċ. The microplates was washed properly 3 times with 300µL of diluted wash solution. Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

3.2.3 Estimation of free estriol (also called oestriol)

Estriol was assayed using ELISA kit by Delta Biologicals S.R.L, Pomezia Rome; a subsidiary of

Erba Diagnostic, Inc.

1. Principle of the assay

This assay employs the quantitative competitive enzyme immunoassay technique. A monoclonal antibody specific for estriol (anti- estriol) has been pre-coated onto a microplate (solid phase).

Standards and samples are pipetted into the wells and any estriol present is bound by the immobilized antibody. Estriol in the sample (antigen) competes with horseradish-peroxidase (HRP)- estriol (enzyme-labeled antigen) for binding onto the limited number of anti- estriol coated on the microplates. After incubation, a simple washing is performed to wash away any unbound substances.

This is followed by the addition of enzyme substrate solution to the wells and color develops. The color development is stopped and the intensity of the color is measured. The intensity of colour measured is inversely proportional to the estriol in the sample.

2. Reagent preparation

Wash solution was prepared by diluting content of vail of the buffered wash solution concentrate with deionized water to final volume of 500 mL. The diluted wash solution is stable for 30 days at 2-

8 Ċ.

3. Test procedure

Appropriate number of microtitre wells were secured in the frame holder. Twenty five microliters

(µL) of each standard, control and samples were dispensed with new disposable tips into appropriate wells. One hundred microliters of enzyme conjugate was dispensed into each well and mixed thoroughly for 10 seconds. It is important to have a complete mixing in this step. This was incubated for 60 minutes at 37 Ċ. The microplates was washed properly 3 times with 300µL of diluted wash solution. Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

3.2.4 Anti-β2-Glycoprotein 1 antibodies (a-β2-GP1)

Anti-β2-Glycoprotein 1 antibodies was assayed using ELISA kit by Delta Biologicals S.R.L,

Pomezia Rome; a subsidiary of IVAX Diagnostics, Inc

1. Principle of the assay

This assay employs the quantitative indirect solid phase enzyme immunometric assay (ELISA) technique. This method is designed for quantitative measurement of IgA class outoantibodies directed against β2-Glycoprotein 1. The assay is based on micoplate coated with highly purified β2-

Glycoprotein 1. Standards, controls and prediluted samples are pipetted into the wells. Any present antibody binds to the immobilized antigen on the inner surface of the microplate wells. After 30 minutes incubation the micoplate is washed with wash buffer to remove non-reactive serum components. An antihuman-IgA horseradish-peroxidase (HRP) conjugate solution is pipetted into the wells to bind to the autoantibodies bound to the immobilized antigens. After a 15 minutes incubation a second washing is done to wash off excess enzyme conjugate.

This is followed by the addition of enzyme substrate solution to the wells and color develops. The color development is stopped and the intensity of the color is measured. The intensity of colour measured is directly proportional to the concentration of antibodies present in the sample.

Reagent preparation

Wash solution was prepared by diluting content of vail of the buffered wash solution concentrate with deionized water to final volume of 1L. The diluted wash solution is stable for 30 days at 2-8 Ċ.

Sample diluent was prepared by diluting content of vail of the diluent concentrate with deionized water to final volume of 100mL. The diluted diluent solution is stable for 30 days at 2-8 Ċ.

Test procedure

Appropriate number of microtitre wells were secured in the frame holder. Samples were diluted

1:100 with sample diluent before assay (10 µL of sample to 1000 µL of sample diluent). One hundred microliters (100µL) of each calibrators, control and prediluted samples were dispensed with new disposable tips into appropriate wells. This was incubated for 30 minutes at room temperature (20-28Ċ). The microplates was washed properly 3 times with 300µL of diluted wash solution. Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

One hundred microliters (100µL) of enzyme conjugate was added into each well, mixed thoroughly for 10 seconds and incubated for 15 minutes at room temperature (20-28Ċ).

The microplates was washed properly 3 times with 300µL of diluted wash solution. Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

3.2.5 Estimation of anti-thyroid peroxidase autoantibodies (anti-TPO)

Anti-thyroid peroxidase antibodies was assayed using ELISA kit by Diamedix Corporation, Inc,

Miami, USA; a subsidiary of IVAX Diagnostics, Inc

1. Principle of the assay

Purified TPO antigen from human thyroid is bound to micowells. Diluted patients sera, standards, controls are pipetted into the wells. Any present anti-TPO IgG antibody binds to the immobilized antigen on the inner surface of the microplate wells forming antigen-antibody complex. After 30 minutes incubation the microplate is washed with wash buffer to remove non-reactive serum components. An antihuman-IgG horseradish-peroxidase(HRP) conjugate solution is pipetted into the wells to bind to the autoantibodies bound to the immobilized antigens. After a 30 minutes incubation a second washing is done to wash off excess enzyme conjugate.

This is followed by the addition of enzyme substrate solution to the wells and color develops. The color development is stopped and the intensity of the color is measured. The intensity of colour measured is directly proportional to the concentration of antibodies present in the sample.

2. Reagent preparation

Wash solution

Wash solution was prepared by diluting content of vail of the buffered wash solution concentrate with deionized water to final volume of 1050mL. The diluted wash solution is stable for 30 days at

2-8 Ċ.

3. Test procedure

Appropriate number of microtitre wells were secured in the frame holder. Samples were diluted

1:101 with sample diluent before assay (10 µL of sample to 1000 µL of sample diluent). One hundred microliters (100µL) of each calibrators, control and prediluted samples were dispensed with new disposable tips into appropriate wells. This was incubated for 30 minutes at room temperature

(20-28Ċ). The microplates was washed properly 3 times with 300µL of diluted wash solution.

Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

One hundred microliters (100µL) of enzyme conjugate was added into each well, mixed thoroughly for 10 seconds and incubated for 30 minutes at room temperature (20-28Ċ).

This was followed by the addition of 100µL of enzyme substrate solution to each well and incubated for 30 minutes at room temperature (20-28Ċ) in the dark. The reaction was ended by adding 100µL of stop solution. Absorbance reading was at 450nm.

3.2.6 Estimation of Antinuclear autoantibodies (ANAs)

Anti-nuclear antibodies was assayed using ELISA kit by Diamedix Corporation, Inc, Miami, USA; a subsidiary of IVAX Diagnostics, Inc

1. Principle of the assay

Purified ANA antigens are bound to mircowells. Diluted patients sera, standards, controls are pipetted into the wells. Any present antibodies to these antigens in diluted sample will bind to the immobilized antigen on the inner surface of the microplate wells forming antigen-antibody complex.

Washing of the microwells removes unbound serum antibodies and non-reactive serum components.

An antihuman-IgG horseradish-peroxidase(HRP) conjugate solution is pipetted into the wells to bind to the autoantibodies bound to the immobilized antigens forming a ―conjugate-antibody-antigen‖ sandwich. A second washing washes off excess enzyme conjugate.

This is followed by the addition of enzyme substrate solution to the wells and color develops. The color development is stopped and the intensity of the color is measured. The intensity of colour measured is directly proportional to the concentration of antibodies present in the sample.

2. Reagent preparation

Wash solution

Wash solution was prepared by diluting content of vail (50ml) of the buffered wash solution concentrate with deionized water to final volume of 1000mL.

3. Test procedure

(1:101) before assay (5 µL of sample to 500 µL of sample diluent). Desired number of microtitre wells were secured in the frame holder. One hundred microliters of each diluted calibrators, cut-off, control and pre-diluted samples were transferred with new disposable tips into appropriate wells.

This was incubated uncovered for 30 minutes at room temperature (20-28Ċ). The microplates was washed properly 3 times with 300µL of diluted wash solution. Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

One hundred microliters (100µL) of enzyme conjugate was added into each well, mixed thoroughly for 10 seconds and incubated for 30 minutes at room temperature (20-28Ċ).

4. Calculation of results

Index value = Absorbance of sample Mean absorbance of cut-off calibrator

3.2.7 Estimation of Magnesium

1. Principle

Magnesium ions in an alkaline medium form a coloured complex with xylidyl blue. The absorbance increase is proportional to the magnesium concentration in the sample. Glycoletherdiamine-

N,N,N‘,N‘-tetraacetic acid (GEDTA) is used as masking agent for calcium ions.

2. Test procedure

Ten microliters each of sample, standard, control were dispensed into their respective test tubes.

Ten microliters of deionized water was dispensed into the blank test tube. This was followed by the addition of 1000µL of magnesium reagent into all tubes, mixed, and incubated at room temperature for ten minutes.

Absorbance of the sample, standard, and controls were measured against reagent blank within

60minutes at 546nm wavelength.

3. Result calculation

Concentration of magnesium in samples

= Absorbance of sample X concentration of standard (1.09mmol/L) Absorbance of standard

3.2.8 Estimation of Phosphorous

1. Principle

Phosphorous reacts with molybdate in strong acidic medium form a coloured complex . The absorbance of the complex in the ultra violet region is directly proportional to the phosphorous concentration in the sample.

Reaction principle

6- + 3- 7H3PO4 +12 (MO7024) +51H > 7[P(MO12O40)] +36H2O

2. Test procedure

Ten microliters each of sample, standard, control were dispensed into their respective test tubes.

Ten microliters of deionized water was dispensed into the blank test tube. This was followed by the addition of 1000µL of phosphorous reagent into all tubes, mixed, and incubated at room temperature for 5 minutes.

Absorbance of the sample, standard, and controls were measured against reagent blank within

10minutes at 340nm wavelength.

1. Result calculation

Concentration of phosphorous in samples

= Absorbance of sample X concentration of standard (3.2mmol/L) Absorbance of standard

3.2.9 Estimation of calcium

1. Principle

Calcium ions form a violet complex with O-Cresolphthalein complexone in an alkaline medium.

2. Test procedure

Twenty five microliters each of sample, standard, control were dispensed into their respective test tubes.

Twenty five microliters of deionized water was dispensed into the blank test tube. This was followed by the addition of 1000µL of calcium reagent into all tubes, mixed, and incubated at room temperature for 10 minutes.

Absorbance of the sample, standard, and controls were measured against reagent blank within

10minutes at 578nm wavelength.

1. Result calculation

Concentration of calcium in samples

= Absorbance of sample X concentration of standard (2.45mmol/L) Absorbance of standard

3.2.9 Estimation of total protein

1. Principle

Cupric ions in an alkaline medium interact with protein peptide bonds resulting in the formation of a coloured complex.

2. Test procedure

Twenty microliters each of sample, standard, control were dispensed into their respective test tubes.

Twenty microliters of deionized water was dispensed into the blank test tube. This was followed by the addition of 1000µL of total protein reagent into all tubes, mixed, and incubated at room temperature for 30 minutes.

Absorbance of the sample, standard, and controls were measured against reagent blank within

10minutes at 546nm wavelength.

3. Result calculation

Concentration of total protein in samples

= Absorbance of sample X concentration of standard (60g/l) Absorbance of standard

3.2.11 Estimation of albumin

1. Principle

The measurement of albumin is based on it quantitative binding to the indicator 3,3‘,5,5‘- tetrabromo-m cresol sulphonephthalein (bromocresol green, BCG). The albumin-BCG-complex absorbs maximally at 578nm, the absorbance being directly proportional to the concentration of albumin in the sample.

2. Test procedure

Ten microliters each of sample, standard, control were dispensed into their respective test tubes.

Ten microliters of deionized water was dispensed into the blank test tube. This was followed by the addition of 3000µL of albumin reagent into all tubes, mixed, and incubated at room temperature for

10 minutes.

Absorbance of the sample, standard, and controls were measured against reagent blank within

10minutes at 578nm wavelength.

3. Result calculation

Concentration of albumin in samples

= Absorbance of sample X concentration of standard (40g/l) Absorbance of standard

3.3 Statistical analysis

Data obtained from the study were presented as mean ± standard deviation. It was analysed using the

Statistical Package for Social Sciences (SPSS) version 20 using appropriate statistical tools. Level of significance was taken at P<0.05

CHARPTER FOUR

RESULTS

Table 4.1 shows the comparison of biochemical parameters, age, and body mass index between HIV sero-positive and HIV seronegative pregnant subjects. Progesterone was significantly higher in HIV sero-positives (59.43 ± 17.84) compared to HIV sero-negatives (54.89 ± 8.24) (p<0.05). Likewise thyroid peroxidase autoantibody was significantly higher in HIV sero-positive subjects (104.90 ±

51.06) compared to HIV sero-negatives (89.50 ± 33.51) (p<0.05). There were no significant differences in all other parameters compared (p>0.05)

Table 4.2 shows the comparison of biochemical parameters based on body mass index (normal, overweight, and obese) in HIV sero-positive pregnant subjects. There were no significant differences in all parameters compared (p>0.05)

Table 4.3 shows the comparison of biochemical parameters based on body mass index (normal, overweight, and obese) in HIV sero-negative pregnant subjects. Estriol, albumin, magnesium, and calcium showed significant BMI dependent variations. Post Hoc analysis in table 4.4 shows that estriol was significantly higher in overweight subjects (5.13 ± 3.43) (p<0.05) compared to normal subjects (3.34 ± 2.41). Also estriol was significantly lower in obese subjects (3.34 ± 2.41) (p<0.05) compared to overweight subjects (5.13 ± 3.43). Albumin was significantly higher in normal subjects

(42.63 ± 2.42) (p<0.05) compared to overweight subjects (40.47 ± 2.43). Magnesium was significantly higher in overweight subjects (1.07 ± 0.20) (p<0.05) compared to normal subjects (0.91

± 0.17). Calcium was significantly higher in obese (2.17±0.36) compared to overweight (1.98±0.22).

There were no significant differences in all other parameters compared (p>0.05)

Table 4.5 shows the relationship between Apgar score and some biochemical parameters of HIV sero-positive pregnant subjects. Post Hoc analysis in table 4.6 shows that pregnancy associated plasma protein-A level in subjects with good Apgar score (8-10) (23.58 ± 13.92) was significantly lower than the level from subjects with poor Apgar score (5-7) (45.13 ± 14.66) (p<0.05). Calcium level in subjects with good Apgar score (8-10) (2.18 ± 0.21) was significantly lower than the level from subjects with poor Apgar score (5-7) (2.44 ± 0.33) (p<0.05). Also calcium level was significantly higher (p<0.05) in subjects with Apgar score 5-7 (2.44 ± 0.33) compared to Apgar score 0-4 (2.11 ± 0.15). There were no significant differences in all other parameters compared

(p>0.05)

Table 4.7 shows the relationship between Apgar score and some biochemical parameters of HIV sero-negative pregnant subjects. There were no significant differences in all parameters compared

(p>0.05).

Table 4.8 shows the comparison of biochemical parameters in different trimesters of pregnancy among HIV sero-positive pregnant subjects. Post Hoc analysis in table 4.9 shows that estriol showed significant (p<0.05) progressive increases from first trimester to third trimester (0.35 ± 0.49; 3.63 ±

2.88; 6.51 ± 3.71, respectively). Progesterone and pregnancy associated plasma protein-A (PAPP-

A) were significantly lower in first trimester (37.34 ± 15.33; 12.36 ± 12.84) compared with second

(61.01 ± 17.82; 29.15 ± 21.76) respectively. There were no significant differences in progesterone and PAPP-A levels between second and third trimesters. There were no significant differences in all other parameters compared (p>0.05)

Table 4.10 shows the comparison of biochemical parameters in different trimesters of pregnancy among HIV sero-negative pregnant subjects. Post Hoc analysis in table 4.11 shows that progesterone showed significant (p<0.05) progressive increases from first trimester to third trimester (37.82 ±

4.95, 52.11 ± 7.20, 56.54 ± 7.11) respectively. Estriol was significantly higher in third trimester

(6.12 ± 3.10) compared with first (0.35 ± 0.33) and second trimesters (2.73 ± 1.62). There was no significant difference in estriol level between first and second trimesters. Albumin was significantly higher in first trimester (45.91 ± 2.27) compared to second (41.91 ± 2.41) and third trimester (40.58

± 2.59) (p<0.05). There was no significant difference in all other parameters compared (p>0.05)

Figures 4.1- 4.10 shows the correlation relationship between parameters. There were significant positive correlations between estriol, progesterone, PAPPA-A and gestational age (p<0.05).There were significant negative correlations between total protein, albumin, and GA, PAPP-A and Apgar score, PAPP-A and BMI, total protein and parity, blood pressure and calcium (p<0.05)

There was a significant positive correlation between estriol and gestational age (p<0.05)

Table: 4.1 Comparison of some biochemical parameters between HIV sero-negative and HIV seropositive antenatal subjects

Parameter HIV HIV positive t-value P-value negative (n=136) (n=187) Estriol(ng/ml) 4.78 ± 3.28 4.63 ± 3.72 0.372 0.7099 Progesterone (ng/ml) 54.89 ± 8.24 59.43 ± 17.84 3.062 0.0024* Total protein (g/L) 70.15 ± 5.68 71.05 ± 9.46 1.083 0.2796 Albumin (g/L) 41.27 ± 2.62 41.80 ± 4.22 1.398 0.1632 Globulin (g/L) 28.89 ± 5.26 29.25 ± 8.08 0.486 0.6276 Magnesium (mmol/L) 0.99 ± 0.19 1.00 ± 0.22 0.406 0.6848 Inorganic phosphorous (mmol/L) 1.84 ± 0.51 1.85 ± 0.84 0.1498 0.8810 Calcium (mmol/L) 2.06 ± 0.24 2.11 ± 0.33 1.591 0.1126

Anti-thyroid peroxidase antibodies (IU/ml) 89.50± 33.51 104.90 ± 51.06 3.270 0.0012* Pregnancy associated plasma protein-A 24.85 ± 9.47 27.60 ± 19.56 1.674 0.0951 (µg/ml) Anti-nuclear antibodies 0.84 ± 0.27 0.89 ± 0.31 1.648 0.1004 Anti-beta 2 glycoprotein 1 IgA (U/ml) 10.37 ± 9.67 12.94 ± 18.90 1.178 0.2404 Body mass index 26.96 ± 5.05 27.97 ± 6.17 1.190 0.2356 Age (years) 30.78 ± 5.10 31.42 ± 4.63 0.896 0.3714

Values are mean ± standard deviation.

*Values with p < 0.05 per row are statistically significant.

Table : 4.2 Influence of body mass index on some biochemical parameters of HIV positive pregnant subjects.

Parameters Body Mass Index (BMI) Based Groupings

Normal Overweight Obese F value P value

(n = 26) (n= 37) (n =23)

Estriol(ng/ml) 3.91 ± 3.66 5.18 ± 3.93 4.23 ± 4.04 0.9191 0.4029 Progesterone (ng/ml) 60.20 ± 17.12 59.46 ± 18.86 59.12±19.48 0.0224 0.9778 Total protein (g/L) 71.25 ± 7.70 71.11 ± 8.99 70.04±11.28 0.1254 0.8823 Albumin (g/L) 42.79 ± 3.35 41.91 ± 4.54 41.42 ± 4.86 0.6482 0.5256 Globulin (g/L) 28.47 ± 6.51 29.20 ± 9.01 28.62 ± 9.22 0.0670 0.9353 Magnesium (mmol/L) 1.05 ± 0.22 1.04 ± 0.24 0.95 ± 0.17 1.597 0.2087 Inorganic phosphorous 1.66 ± 0.56 2.06 ± 1.10 1.83 ± 0.86 1.542 0.2201 (mmol/L)

100

Calcium (mmol/L) 2.16 ± 0.44 2.09 ± 0.37 2.10 ± 0.37 0.2625 0.7698 Anti-thyroid peroxidase 112.0 ± 35.93 99.61 ± 38.12 94.62±30.03 1.615 0.2051 antibodies (IU/ml) Pregnancy associated 29.06 ± 23.02 30.40 ± 18.46 19.13±16.92 2.560 0.0834 plasma protein-A (µg/ml) Anti-nuclear antibodies 0.91 ± 0.37 0.97 ± 0.33 0.83 ± 0.21 1.391 0.2545 Anti-beta 2 glycoprotein 1 13.15 ± 14.61 12.78 ± 10.94 13.04±16.22 0.0061 0.9939 IgA (U/ml)

Values are mean ± standard deviation.

Values with p>0.05 are not statistically significant.

Table : 4.3Influence of body mass index on some biochemical parameters of HIV negative pregnant subjects.

Parameters Body Mass Index (BMI) Based Groupings F value P value

Normal Overweight Obese

(n = 35) (n = 32) (n = 22)

Estriol(ng/ml) 3.34 ± 2.41 5.13 ± 3.43 3.02 ± 2.13 4.998 0.0088* Progesterone (ng/ml) 53.37 ± 7.61 54.57 ± 6.91 51.71 ± 9.01 0.8899 0.4144 Total protein (g/L) 70.52 ± 4.91 68.49 ± 5.88 69.71 ± 5.08 1.225 0.2989 Albumin (g/L) 42.63 ± 2.42 40.47 ± 2.43 41.59 ± 2.59 6.412 0.0025* Globulin (g/L) 27.90 ± 5.17 28.02 ± 5.65 28.12 ± 5.01 0.012 0.9880 Magnesium (mmol/L) 0.91 ± 0.17 1.07 ± 0.20 0.98 ± 0.16 6.677 0.0020* Inorganic phosphorous (mmol/L) 1.71 ± 0.40 1.93 ± 0.53 1.82 ± 0.42 1.950 0.1485 Calcium (mmol/L) 2.07 ± 0.13 1.98 ± 0.22 2.17 ± 0.36 4.249 0.0174* Anti-thyroid peroxidase 93.78 ± 29.73 79.44 ± 30.48 86.36 ± 35.34 1.740 0.1816

101 antibodies (IU/ml) Pregnancy associated plasma 24.02 ± 8.42 26.03 ± 9.29 23.21 ± 9.59 0.7318 0.4840 protein-A (µg/ml) Anti-nuclear antibodies 0.87 ± 0.28 0.80 ± 0.18 0.80 ± 0.26 0.8791 0.4188 Anti-beta 2 glycoprotein 1 IgA 10.43 ± 8.75 10.34 ± 9.54 10.36 ± 6.01 0.0010 0.9990 (U/ml)

Values are mean ± standard deviation.

*Values with p<0.05 are statistically significant.

Table : 4.4 Post HOC analysis of influence of body mass index on some biochemical parameters of HIV sero negative pregnant subjects.

Parameters Normal Normal Overweight vs vs vs overweight obese Obese Estriol * * NS Albumin * NS NS Magnesium * NS NS Calcium NS NS *

NB:

Normal; n = 35, overweight; n=32, obese; n = 22

102

*Values with p<0.05 are statistically significant.

NS=not significant (p>0.05)

Table : 4.5The relationship between Apgar score and some biochemical parameters of HIV seropositive pregnant subjects.

Parameters Apgar Score Based Groupings

0 - 4 5 -7 8 - 10 F value P value

(n = 06) (n = 06) (n = 35)

Estriol (ng/ml) 2.50 ± 1.53 4.63 ± 2.15 3.65 ± 3.89 0.5477 0.5822 Progesterone (ng/ml) 55.50± 10.32 65.85± 11.58 53.44±15.94 1.764 0.1833 Total protein (g/L) 66.66 ± 8.13 67.34 ± 5.20 71.78 ± 8.55 1.532 0.2273 Albumin (g/L) 41.73 ± 2.13 41.39 ± 3.60 43.08 ± 3.07 1.132 0.3315 Globulin (g/L) 24.93 ± 8.00 25.95 ± 4.80 28.70 ± 8.49 0.7478 0.4793 Magnesium 0.87 ± 0.19 0.96 ± 0.09 0.91 ± 0.19 0.3725 0.6912 (mmol/L) Inorganic 1.92 ± 0.28 1.63 ± 0.42 1.68 ± 0.31 1.613 0.2109 phosphorous (mmol/L) Calcium (mmol/L) 2.11 ± 0.15 2.44 ± 0.33 2.18 ± 0.21 4.156 0.0222*

103

Anti-thyroid 96.23±22.65 84.53±17.64 108.40 ± 0.3554 0.7029 peroxidase 76.87 antibodies (IU/ml) Pregnancy 35.23 ± 8.77 45.13 ± 14.66 23.58 ± 13.92 7.536 0.0015* associated plasma protein-A (µg/ml) Anti-nuclear 0.74 ± 0.18 0.87 ± 0.30 0.83 ± 0.27 0.4007 0.6723 antibodies Anti-beta 2 8.43 ± 2.76 8.70 ± 6.25 17.15 ± 26.39 0.6062 0.5499 glycoprotein 1 IgA (U/ml) Values are mean ± standard deviation.

*Values with p<0.05 are statistically significant.

Table : 4.6 Post HOC analysis ofthe relationship between Apgar score and some biochemical parameters of HIV sero-positive pregnant subjects.

Parameters Apgar score Apgar score Apgar 0 – 4vs 5-7 0-4 vs 8 –10 score 5-7vs8 –10 Pregnancy associated NS NS * plasma protein-A Calcium * NS *

NB:

Apgar score 0 – 4; n = 06, 5 -7; n = 06, 8 – 10; n = 35

*Values with p<0.05 are statistically significant.

NS=not significant (p>0.05)

104

Table : 4.7The relationship between Apgar score and some biochemical parameters of HIV seronegative pregnant subjects.

Parameters Apgar Score Based Groupings

0 - 4 5 - 7 8 - 10 F value P value

(n = 04) (n = 10) (n = 82)

Estriol(ng/ml) 5.73 ± 2.53 5.99 ± 3.69 4.70 ± 3.03 0.7234 0.4878 Progesterone (ng/ml) 54.68 ± 5.86 59.61 ± 5.52 55.42 ± 8.04 1.342 0.2664 Total protein (g/L) 69.70 ± 4.05 70.98 ± 5.36 70.49 ± 5.12 0.0931 0.9112 Albumin (g/L) 38.89 ± 0.67 41.98 ± 2.60 41.29 ± 2.51 2.252 0.1109 Globulin (g/L) 30.81 ± 4.46 29.00 ± 7.39 29.19 ± 5.02 0.1909 0.8266 Magnesium (mmol/L) 1.12 ± 0.25 0.93 ± 0.11 1.00 ± 0.19 1.532 0.2215 Inorganic phosphorous (mmol/L) 1.53 ± 0.20 1.97 ± 0.58 1.75 ± 0.42 1.759 0.1780 Calcium (mmol/L) 1.85 ± 0.20 2.01 ± 0.36 2.05 ± 0.23 1.344 0.2659 Anti-thyroid peroxidase 115.95± 52.10 77.77± 38.27 91.05± 32.67 1.826 0.1668 antibodies (IU/ml) Pregnancy associated plasma 28.85 ± 20.38 27.36 ± 6.70 23.19 ± 8.00 1.182 0.3114 protein-A (µg/ml) Anti-nuclear antibodies 1.00 ± 0.27 0.69 ± 0.27 0.83 ± 0.27 2.100 0.1282 Anti-beta 2 glycoprotein 1 IgA 7.33 ± 3.32 7.16 ± 2.07 10.17 ± 6.90 1.242 0.2934 (U/ml)

105

Values are mean ± standard deviation.

Values with p>0.05 are not statistically significant.

Table : 4.8Comparison of some biochemical parameters in different trimester of HIV sero-positive pregnant subjects.

Parameters Trimester

First trimester Second Third F value P value trimester trimester (n = 12) (n = 44) (n = 49)

Estraiol(ng/ml) 0.35 ± 0.49 3.63 ± 2.88 6.51 ± 3.71 21.85 <0.0001* Progesterone (ng/ml) 37.34 ± 15.33 61.01 ± 17.82 65.61 ± 17.10 13.02 <0.0001* Total protein (g/L) 72.02 ± 11.31 72.02 ± 10.30 70.22 ± 9.15 0.4324 0.6502 Albumin (g/L) 44.24 ± 4.99 41.97 ± 4.01 41.15 ± 4.40 2.508 0.0864 Globulin (g/L) 27.78 ± 8.33 30.05 ± 9.61 29.07 ± 7.40 0.3806 0.6844 Magnesium (mmol/L) 0.88 ± 0.13 1.03 ± 0.26 1.03 ± 0.22 2.252 0.1104 Inorganic phosphorous 1.56 ± 0.39 1.83 ± 0.86 1.87 ± 0.72 0.8194 0.4436

106

(mmol/L) Calcium (mmol/L) 2.23 ± 0.15 2.08 ± 0.40 2.12 ± 0.36 0.8167 0.4448 Anti-thyroid peroxidase 119.59 ± 27.34 106.79 ± 36.20 99.23 ± 42.18 1.469 0.2351 antibodies (IU/ml) Pregnancy associated plasma 12.36 ± 12.84 29.15 ± 21.76 29.32 ± 20.12 3.713 0.0278* protein-A (µg/ml) Anti-nuclear antibodies 0.81 ± 0.19 0.88 ± 0.29 0.89 ± 0.28 0.4101 0.6647 Anti-beta 2 glycoprotein 1 6.80 ± 2.40 7.04 ± 2.02 7.33 ± 2.83 0.2937 0.7461 IgA (U/ml)

Values are mean ± standard deviation.

*Values with p<0.05 are statistically significant.

Table : 4.9 Post HOC analysis of the comparison of some biochemical parameters in different trimester of HIV sero-positive pregnant subjects.

Parameters First First Second trimester trimester Trimester vs vs vs Second third third trimester trimester trimester

Pregnancy associated * * NS plasma protein-A Estriol * * * Progesterone * * NS

107

*Values with p<0.05 are statistically significant.

First trimester; n=12, second trimester; n=44, third trimester; n=49

NS=not significant (p>0.05)

Table :4.10 Comparison of some biochemical parameters in different trimester of HIV sero-negative pregnant subjects.

Parameters Trimester

First Second Third F value P value trimester trimester trimester (n = 06) (n = 49) (n = 90) Estriol(ng/ml) 0.35 ± 0.33 2.73 ± 1.62 6.12 ± 3.10 35.07 <0.0001* Progesterone (ng/ml) 37.82 ± 4.95 52.11 ± 7.20 56.54 ± 7.11 23.13 <0.0001* Total protein (g/L) 74.70 ± 4.70 69.90 ± 6.02 69.03 ± 4.79 3.456 0.0342* Albumin (g/L) 45.91 ± 2.27 41.91 ± 2.41 40.58 ± 2.59 0.0000 <0.0001* Globulin (g/L) 28.79 ± 5.29 27.99 ± 5.50 28.44 ± 5.07 0.1448 0.8653 Magnesium (mmol/L) 0.96 ± 0.18 1.00 ± 0.24 0.99 ± 0.16 0.1315 0.8769 Inorganic phosphorous 1.67 ± 0.31 1.85 ± 0.48 1.81 ± 0.52 0.3736 0.6889 (mmol/L) Calcium (mmol/L) 2.24 ± 0.15 2.01 ± 0.20 2.06 ± 0.27 2.533 0.0830 Anti-thyroid peroxidase 68.78 ± 19.76 92.98 ± 36.53 86.32 ± 32.32 1.645 0.1968 antibodies (IU/ml) Pregnancy associated plasma 19.32 ± 16.22 23.65 ± 8.67 26.46 ± 9.39 2.602 0.0777

108 protein-A (µg/ml) Anti-nuclear antibodies 1.03 ± 0.30 0.83 ± 0.22 0.81 ± 0.28 1.992 0.1403 Anti-beta 2 glycoprotein 1 IgA 7.82 ± 2.60 9.38 ± 5.17 10.10 ± 9.57 0.3004 0.7410 (U/ml)

Values are mean ± standard deviation.

*Values with p<0.05 are statistically significant.

Table : 4.11 Post HOC analysis of the comparison of some biochemical parameters in different trimester of HIV sero-negative pregnant subjects.

Parameters First First Second trimester trimester trimester vs vs vs Second Third Third trimester trimester trimester n = 90 Progesterone * * * Estriol NS * * Albumin * * * Total protein NS * NS

109

*Values with p<0.05 are statistically significant.

First trimester; n=6, second trimester; n=49, third trimester; n=90

NS=not significant (p>0.05)

110

4 Free Estraiol (ng/ml) Free Estraiol 2 Y = -1.486X + 0.2252 ------95% CI r = 0.9131; p = 0.0000

0 10 20 30 40 50 Gestation Age (weeks)

-2

Figure: 4.1 The correlation between serum free estriol level of subjects and gestational age

111

40 Progesterone (ng/ml) Progesterone

20 Y = 35.56X + 0.7324 ------95% CI r = 0.7518; p = 0.0000

0 0 10 20 30 40 50 Gestation Age

Figure: 4.2 The correlation between serum progesterone level of subjects and gestational age

112

Y = 75.09X - 0.1775 ------95% CI 80 r = -0.4679; p = 0.0053

70 Total Protein (g/L)Protein Total

60 0 10 20 30 40 50 Gestation Age

Figure: 4.3 The correlation between serum total protein level of subjects and gestational age

113

Y = 45.37X - 0.1551 ------95% CI r = -0.6891; p = 0.0000

45 Albumin (g/L) Albumin

35 0 10 20 30 40 50 Gestation Age

Figure: 4. 4 The correlation between serum albumin level of subjects and gestational age

114

50 Y = 11.76X + 0.5093 ------95% CI r = 0.6020; p = 0.0002

g/ml) 

10 Pregnancy Associated Plasma Protein-A ( Protein-A Plasma Associated Pregnancy

0 0 10 20 30 40 50 Gestation Age

Figure: 4.5 The correlation between serum pregnancy associated plasma protein-A level of subjects and gestational age

115

Y = 34.28X - 1.150 ------95% CI

r = -0.230; p = 0.0074

g/ml)  60

20 Pregnancy Associated Plasma Protein- A ( Protein- A Plasma Associated Pregnancy

0 0 2 4 6 8 10 12 APGARS-1 Apgar score

Figure: 4.6 The correlation between serum pregnancy associated plasma protein-A level of subjects and Apgar score

116

80 Y = 46.42X - 0.6951 ------95% CI r = -0.215; p = 0.0470

70 g/ml)

 60

0 20 30 40 50 60 70 80

Pregnancy Associated Plasma Protein-A ( Protein-A Plasma Associated Pregnancy Body Mass Index -10

-20

-30

Figure: 4.7 The correlation between serum level of pregnancy associated plasma protein-A and body mass index of subjects

117

Y = 72.63X - 0.9465 ------95% CI r = -0.191; p = 0.0386

70 Total Protein (g/L)Protein Total

50 0 2 4 6 8 Parity

Figure: 4.8 The correlation between serum level of total protein and parity of subjects

118

Y = 3.059X - 0.0093 ------95% CI r = -0.402; p = 0.0021

2 Calcium concentration (mmol/l) concentration Calcium

0 80 90 100 110 120 130 140 Systolic BP

Figure: 4.9 The correlation between serum level of total calcium and systolic blood pressure of subjects

119

Y = 2.631X - 0.0084 ------95% CI r = -0.295; p = 0.0275

2 Calcium concentration (mmol/l) concentration Calcium 1

0 50 60 70 80 90 Diastolic BP

Figure: 4.10 The correlation between serum level of total calcium and diastolic blood pressure of subjects

120

Figure 4.11 shows some delivery outcomes at NAUTH Nnewi between June 2010 and December

2013. Normal delivery accounted for 62.69% of the outcomes. While Caesarian section and breached deliveries accounted for 35.12% and 2.20% respectively.

Figure 4.12 shows the incidence rate of intra-uterine fetal death (IUFD) at NAUTH Nnewi between

June 2010 and December 2013. The incidence rate of IUFD among HIV sero-positive pregnant subjects was 7.31%, while the incidence rate among HIV sero-negative pregnant subjects was 7.

47%. The over-all incidence rate among pregnant women was 7. 42%.

Figure 4.13 shows pattern of antenatal attendance of HIV sero-positive and HIV sero-negative pregnant subjects at NAUTH Nnewi between June 2010 and December 2013. Antenatal attendance by HIV sero-positive pregnant women constituted 25.39% while attendance by HIV sero-negative pregnant subjects constituted 74.61%

Figure 4.14 shows the comparison of pattern of antenatal attendance in different trimesters of HIV sero-positive and HIV sero-negative pregnant subjects at NAUTH Nnewi between June 2010 and

December 2013. Antenatal attendance in first and second trimesters by HIV sero-positive pregnant women (11.5%, 41.9%; respectively) was greater than the attendance by HIV sero-negative pregnant subjects (4.14%, 33.79% respectively). The trend changed in the third trimester with a decline in the attendance by HIV sero-positive pregnant women (6.86%) with respect to the attendance by HIV sero-negative pregnant subjects (62.07%)

121

100% 12

8 62.69%

6 Number of deliveries/month of Number 35.12% 2.20% 4

0 TD (X 10) ND (X 10) CS (X 10) Breached

Figure : 4.11 Some delivery outcomes at NAUTH Nnewi between June 2010 and December 2013.

122

7.55

7.5 7.47

7.45 7.42

7.4

7.35 7.31

7.3 percentage (%)percentage 7.25

7.2

7.15

7.1 HIV sero-negative HIV sero-positive Total incidence

Figure 4.12 Incidence of IUFD at NAUTH Nnewi

123

100% 120

110

100 74.61% 90

40 Number of deliveries/month of Number 25.39%

0 Total deliveries RVD Negative RVD positive

Figure : 4.13 Pattern of antenatal attendance of HIV sero-positive and HIV sero-negative subjects between June 2010 and December 2013.

124

70 62.07 60

50 46.86 41.9 40 33.79

20 11.5 10 Percentage antenatalattenance Percentage(%) 4.14

0 HIV seropositives HIV seronegatives HIV seropositives HIV seronegatives HIV seropositives HIV seronegatives -10 first trimester second trimester third trimester

Figure 4.14 Pattern of antenatal attendance in different trimesters at NAUTH-Nnewi

125

Figure 4.15 shows the pattern of weekly variation of serum estriol level (ng/ml) among HIV seronegative and seropositive pregnant subjects with good pregnancy outcome.

Estriol increased steadily from the early weeks of gestation on till term for the two groups that had good pregnancy outcome (live baby).

The level of increases was however higher at most stage in HIV sero negative subjects than in HIV seropositives. Towards term (at 32-34 and at 40 weeks) there were drops in estriol levels in HIV sero-positive subjects relative to HIV sero-negative pregnant subjects.

Figure 4.16 shows the pattern of weekly variation of serum progesterone level (ng/ml) among HIV seronegative and seropositive pregnant subjects with good pregnancy outcome. For the two groups studied, progesterone increased markedly from the early weeks of gestation. It attained a serum level of 45ng/ml at about 9weeks of gestation to levels of 65ng/ml towards term. At delivery (40 weeks), there were drop in serum progesterone level. The level of drop was greater in HIV sero positive subjects than in HIV seronegatives.

Figure 4.17 shows the pattern of weekly variation of total protein level (g/L) among HIV seronegative and seropositive pregnant subjects with good pregnancy outcome. Total protein showed similar pattern of gestational variation in HIV sero positive subjects and in HIV seronegatives.

Total protein varied between 80g/mL at early pregnancy and 85g/L towards term in both groups.

Figure 4.18 shows the pattern of weekly variation of albumin level (g/L) among HIV seronegative and seropositive pregnant subjects with good pregnancy outcome. Albumin showed similar pattern of gestational variation in HIV sero positive subjects and in HIV seronegatives. Serum albumin

126 level steadily declined from about 50g/mL at early pregnancy to about 40g/L towards term in both groups.

Figure 4.19 shows the pattern of weekly variation globulin level (g/L) among HIV seronegative and seropositive pregnant subjects with good pregnancy outcome. Globulin showed similar pattern of gestational variation in HIV sero positive subjects and in HIV seronegatives. Serum albumin level fluctuated between 25g/mL at early pregnancy and 35g/L towards term in both groups.

Figure 4.20 shows the pattern of weekly variation of serum magnesium level (mmol/L) among HIV seronegative and seropositive pregnant subjects with good pregnancy outcome. Magnesium did not change significantly during pregnancy. The serum level were however higher at most stage in HIV sero negative subjects than in HIV seropositives.

Figure 4.21 shows the pattern of weekly variation of serum inorganic phosphorous level (mmol/L) among HIV seronegative and seropositive pregnant subjects with good pregnancy outcome. The serum level did not vary significantly during pregnancy for both groups. The serum level were however higher at most stage in HIV sero negative subjects than in HIV seropositives.

Figure 4.22 shows the pattern of weekly variation of serum total calcium level (mmol/L) among HIV seronegative and seropositive pregnant subjects with good pregnancy outcome. The serum level did not vary significantly during pregnancy for both groups. The serum level were however lower at most stage in HIV sero negative subjects than in HIV seropositives.

127

10 HIV -VE HIV +VE

4 Estraiol Conc (ng/ml) Conc Estraiol

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.15 Weekly variation of serum estriol level (ng/ml) of HIV seronegative and seropositive pregnant subjects with life birth.

128

80 HIV -VE HIV +VE

40 Progesterone Conc (ng/ml) Conc Progesterone

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.16 Weekly variation of serum progesterone level (ng/ml) of HIV seronegative and seropositive pregnant subjects with life birth.

129

100 HIV -VE HIV +VE

40 Total(g/L) Protein

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.17 Weekly variation of serum total protein level (g/L) of HIV seronegative and seropositive pregnant subjects with life birth.

130

50 HIV -VE HIV +VE

Albumin (g/L) Albumin 20

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.18 Weekly variation of serum albumin level (g/L) of HIV seronegative and seropositive pregnant subjects with life birth.

131

60 HIV -VE HIV +VE

30 Globulin Conc (g/L)Conc Globulin

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.19 Weekly variation of serum globulin level (g/L) of HIV seronegative and seropositive pregnant subjects with life birth.

132

1.6

HIV -VE

1.4 HIV +VE

1.2

1.0

0.8

Mg Conc (mmol/l) Conc Mg 0.6

0.4

0.2

0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.20 Weekly variation of serum magnesium level (mmol/L) of HIV seronegative and seropositive pregnant subjects with life birth.

133

HIV -VE 3.0 HIV +VE

2.5

2.0

1.5

1.0 Inorganic phosphorous (mmol/L) phosphorous Inorganic

0.5

0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.21 Weekly variation of serum inorganic phosphorous level (mmol/L) of HIV seronegative and seropositive pregnant subjects with life birth

134

3.0 HIV -VE HIV +VE

2.5

2.0

1.5

1.0 Calcium concentration (mmol/l) concentration Calcium

0.5

0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.22 Weekly variation of serum total calcium level (mmol/L) of HIV seronegative and seropositive pregnant subjects with life birth

135

200 HIV -VE HIV +VE 180

160

140

120

100

60 Anti-thyroid peroxidase antibodies (IU/ml) antibodies peroxidase Anti-thyroid 40

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.23 Weekly variation of serum anti-thyroid peroxidase level (IU/ml) of HIV seronegative and seropositive pregnant subjects with life birth.

136

50 HIV -VE HIV +VE

10 Pregnancy associated plasma protein-A (µg/ml) protein-A plasma associated Pregnancy

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.24 Weekly variation of serum pregnancy associated plasma protein-A level (ng/ml) of HIV seronegative and seropositive pregnant subjects with life birth.

137

1.6 HIV -VE HIV +VE

1.4

1.2

1.0

0.8

0.6 Anti-nuclear antibody levels antibody Anti-nuclear

0.4

0.2

0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.25 Weekly variation of anti-nuclear antibody index value of HIV seronegative and seropositive pregnant subjects with life birth.

138

30 HIV -VE HIV +VE

10 Anti-beta 2 glycoprotein 1 IgA (U/ml) 1 IgA 2 glycoprotein Anti-beta

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.26 Weekly variation of serum anti-beta-2-glycoprotein 1 level (U/ml) of HIV seronegative and seropositive pregnant subjects with good pregnancy outcome.

139

PREGNANCIES WITH GOOD OUTCOME 12 PREGNANCIES WITH BAD OUTCOME

6 Estraiol Conc (ng/ml) Conc Estraiol 4

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.27 Weekly variation of serum estriol level (ng/ml) of pregnant subjects with good outcome compare with pregnant subjects with IUFD.

140

60 PREGNANCIES WITH GOOD OUTCOME PREGNANCIES WITH BAD OUTCOME

20 Pregnancy associated plasma protein-A (µg/ml) protein-A plasma associated Pregnancy 10

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.28 Weekly variation of serum pregnancy associated plasma protein-A level (µg/ml) of pregnant subjects with good outcome compared with pregnant subjects with IUFD.

141

1.6 PREGNANCIES WITH GOOD OUTCOME PREGNANCIES WITH BAD OUTCOME

1.4

1.2

1.0

0.8

0.6 Anti-nuclear antibody levels antibody Anti-nuclear

0.4

0.2

0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.29 Weekly variation of anti-nuclear antibody index value of pregnant subjects with good outcome compared with pregnant subjects with IUFD.

142

100 PREGNANCIES WITH GOOD OUTCOME PREGNANCIES WITH BAD OUTCOME

40 Progesterone Conc (ng/ml) Conc Progesterone

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.30 Weekly variation of serum progesterone level (ng/ml) of pregnant subjects with good outcome compared with pregnant subjects with IUFD.

143

100 PREGNANCIES WITH GOOD OUTCOME PREGNANCIES WITH BAD OUTCOME

40 Total(g/L) Protein

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.31 Weekly variation of serum total protein level (g/L) of pregnant subjects with good outcome compare with pregnant subjects with IUFD.

144

60 PREGNANCIES WITH GOOD OUTCOME PREGNANCIES WITH BAD OUTCOME

30 Albumin (g/L) Albumin

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.32 Weekly variation of serum albumin level (g/L) of pregnant subjects with good outcome compare with pregnant subjects with IUFD.

145

50 PREGNANCIES WITH GOOD OUTCOME PREGNANCIES WITH BAD OUTCOME

20 Globulin Conc (g/L)Conc Globulin

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.34 Weekly variation of serum globulin level (g/L) of pregnant subjects with good outcome compare with pregnant subjects with IUFD.

146

PREGNANCIES WITH GOOD OUTCOME 1.5 PREGNANCIES WITH BAD OUTCOME

1.0 Mg Conc (mmol/l) Conc Mg

0.5

0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.35 Weekly variation of serum magnesium level (mmol/L) of pregnant subjects with good outcome compare with pregnant subjects with IUFD.

147

3.0 PREGNANCIES WITH GOOD OUTCOME PREGNANCIES WITH BAD OUTCOME

2.5

2.0

1.5

1.0 Inorganic phosphorous (mmol/L) phosphorous Inorganic

0.5

0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.36 Weekly variation of serum phosphorous level (mmol/L) of pregnant subjects with good outcome compare with pregnant subjects with IUFD.

148

3.0 PREGNANCIES WITH GOOD OUTCOME PREGNANCIES WITH BAD OUTCOME

2.5

2.0

1.5

1.0 Calcium concentration (mmol/l) concentration Calcium

0.5

0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.37 Weekly variation of serum calcium level (ng/ml) of pregnant subjects with good outcome compare with pregnant subjects with IUFD.

149

PREGNANCIES WITH GOOD OUTCOME 200 PREGNANCIES WITH BAD OUTCOME

180

160

140

120

100

60 Anti-thyroid peroxidase antibodies (IU/ml) antibodies peroxidase Anti-thyroid 40

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.38 Weekly variation of serum anti-peroxidase antibody level (IU/ml) of pregnant subjects with good outcome compare with pregnant subjects with IUFD.

150

100 PREGNANCIES WITH GOOD OUTCOME PREGNANCIES WITH BAD OUTCOME

40 Anti-beta 2 glycoprotein 1 IgA (U/ml) 1 IgA 2 glycoprotein Anti-beta

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 GESTATIONAL AGE

Figure: 4.39 Weekly variation of serum anti-beta-2-glycoprotein-1 level (U/ml) of pregnant subjects with good outcome compare with pregnant subjects-with-IUFD.

151

CHAPTER FIVE

DISCUSSION, CONCLUSION, AND RECOMMENDATION

5.1 Discussion

Nigeria still remains among the nations ravaged by HIV infection and high rates of infant, neonatal, perinatal, and maternal mortality (Olapade and Lawoyin, 2008; Glob one,2012; Ogunjimi, et al

2012; Onwudiegwu and Awowole, 2012; Bello and Joseph, 2014) . Although Nigeria saw a 27% decline in maternal mortality rate between 2005 (820 per 100,000 live births) and 2010 (630 per

100,000 live births), the country is still among the top 13 countries with the highest maternal mortality rate in the world. Nigeria‘s current maternal mortality rate is 630 per 100,000 live births

(Ndep, 2014). Some researchers have related hemorrhage, puerperal sepsis (Bello et al., 2015;Umar and Kabamba, 2016), and background hospital factors (Soma-Pillay and Pattinson, 2016). Other causes of poor pregnancy outcomes could be, poor financial and geographic access to good quality healthcare delivery services, obstructed labour, malaria and complicated abortions (WHO, 2015).

Unavailability or lack of access to laboratory services also has long been identified as important indices contributory to poor health service delivery in Nigeria (Ahaneku et al.,1999). In countries with more robust health system, pre-pregnancy testing is encouraged. Also comprehensive laboratory testing is done as early as pregnancy is established. This practice is not applied in our own environment and possibly contributing to high rates of poor pregnancy outcomes.

Pregnancy although described as a physiological condition is burdened with biochemical alterations with untoward effects on the mother. The difficulty experienced in pregnancy may be further compounded by environmental, social, emotional factors, as well as the presence of pre-existing disease condition such as HIV infection. The current prevalence rate of HIV infection among

152 pregnant women in Nigeria ranges from 3% to 8% in different regions of the country (Sagay, et al.,

2005; Egesie and Mbooh, 2008; FMOH-PMTCT Guideline, 2010; Umeononihu, et al.,

2013).Although this study showed that 25.39% of antenatal visits were by HIV sero-positives, it is believed that a lot more are not accessing good health care. The proportion of HIV infected antenatal women obtaining good antenatal care is not commensurate with current expectation in view of the obvious benefits of the prevention of mother to child transmission (PMTCT)of HIV scheme. High maternal mortality has been explained by some researchers to be caused by a combination of individual factors such as attending antenatal clinics but choosing to deliver at home, at a church or by a traditional birth attendant (Igberase and Ebeigbe, 2007). Others identified women who never attended ante natal clinics but show up at the hospital as emergency cases with varied degrees of complications (Guerrier et al., 2013).

In this study, the pattern of antenatal attendance increased steadily all through the different trimesters for both HIV seropositive and seronegative subjects. The antenatal attendance in the first trimester by HIV seropositive subjects (11.2%) was however higher compared to antenatal attendance in the first trimester by HIV seronegative subjects (4.14%). Gill et al., (2015) reported similar earlier antenatal visits by HIV infected subjects. This could imply a perceived awareness of the benefits of early antenatal booking by the known HIV subjects who took advantage of the

PMTCT scheme. Although an earlier study by Beauclair, et. al., ( 2014) showed that the timing of a woman's first antenatal care (ANC) visit may not be an important determinant of stillbirths in isolation; further suggesting the possible role of quality of care, incorporating established, effective biomedical interventions (such as PMTCT scheme), as important factors influencing outcomes in this setting.

153

The comparison of pattern of antenatal attendance in different trimesters of HIV sero-positive and

HIV sero-negative pregnant subjects at NAUTH Nnewi between June 2010 and December 2013 shows disparity in antenatal attendance. Antenatal attendance in first and second trimesters by HIV sero-positive pregnant women (11.5%, 41.9%; respectively) was greater than the attendance by HIV sero-negative pregnant subjects (4.14%, 33.79% respectively). The trend changed in the third trimester with a decline in the attendance by HIV sero-positive pregnant women (6.86%) with respect to the attendance by HIV sero-negative pregnant subjects (62.07%). This may imply that the

HIV sero-positive subjects tend more to withdraw from antenatal care in tertiary health centres, with the likelihood of patronizing local birth attendants. This practice will negatively affect pregnancy outcomes, as well as increase HIV transmission rate from mother to child and to the care provider- who are in most cases not aware of the mothers HIV status.

Late antenatal booking has been widely reported in Nigeria and other African countries (Onoh, et al., 2012; Gudayu, 2015). And this relates directly with bad pregnancy outcome. Some suggest that most women book late because of a belief that there are no advantages in booking for antenatal care in the first three months of pregnancy. This seems to be because antenatal care is viewed primarily as curative rather than preventive in the studied population (Ndidi and Oseremen, 2010). Research is needed to determine the best approaches for health education programmes to correct the misconceptions about antenatal care. However, efforts toward maternal education, public health enlightenment campaigns, poverty reduction, and use of focused antenatal care model should be sustained as measures to encourage early initiation of antenatal care (ANC). Research has indicated that antenatal education for expectant mothers results in sustained improvement in knowledge of newborn care (Weiner,et al., 2011). Another study done in Enugu, Nigeria showed that the prevalence of anaemia in pregnancy at booking was high (40.4%) and recommended that early

154 antenatal booking and improved antenatal care are necessary for early diagnosis and treatment of the condition (Dim and Onah, 2007). These studies emphasized the importance of early antenatal care in ensuring good pregnancy outcome.

Several factors affecting the utilization of antenatal care in developing countries have been identified. These include: maternal education, husband's education, marital status, availability, cost, household income, women's employment, media exposure and having a history of obstetric complications. From a systematic review of the literature on factors that affect utilization of antenatal care, mothers who are educated, and those whose husbands are educated, are more likely to utilize antenatal care. Availability, affordability and easy accessibility of health units where antenatal care is offered increase utilization of antenatal care. Cultural beliefs and ideas about pregnancy also had an influence on antenatal care use, in that they may lead to mothers attending antenatal care late or not attending at all. Parity had a statistically significant negative effect on adequate attendance, where by women of a high parity tend not to attend antenatal care, attend late for the first antenatal visit or have few antenatal care visits. The quality of antenatal care might have an influence on utilization of antenatal care, leading to infrequent or late first visits to antenatal care. Whilst women of higher parity tend to use antenatal care less, this might be a result of an influence of women‘s age or religious beliefs (Ndidi and Oseremen, 2010)

Globally, during the period 2000–2010, about 53% of pregnant women attended the recommended minimum four times antenatal care. The proportion of pregnant women in developing countries who attended at least one antenatal care visit has increased from approximately 64% in 1990 to about

81% in 2009 but, in low-income countries, only 39% of pregnant women attended four times or more antenatal care during 2000–2010 (WHO, 2011). Clinical guidelines for normal

(uncomplicated) pregnancies recommend four routine antenatal care visits as follows: the first visit

155 between 12 weeks of pregnancy; the second visit around 26 weeks of pregnancy; the third antenatal care visit around 32 weeks; and fourth antenatal care visit between 36 and 38 weeks. The guidelines also recommend more frequent visits and early antenatal care visits for mothers with pregnancy complications, or those with identifiable risk factors for such complications, such as complications in a prior pregnancy, and in other conditions such as HIV infection (FMOH-Standard treatment guideline, 2008). In spite of the disadvantage of late antenatal booking, the practice is still common among pregnant Nigerian women, especially among known HIV seronegative subjects.

This study showed that the incidence rate of IUFD among HIV sero-positive pregnant subjects was

7.31% ; the rate among HIV sero-negative pregnant subjects was 7.47%,while the over-all incidence rate among pregnant women at the Centre was 7.42%. Higher rates have been reported in different

Centre‘s in Nigeria. This findings still put Nigeria among the leading countries with high rates of

IUFD worldwide. Intrauterine fetal death occurs in 2% of the pregnancies worldwide, and in about

0.5% of pregnancies in France (Quibel et. al., 2014)

The study bySamimi, et.al.,(2013) identified history of previous pregnancy loss, consanguinity and viral infections as risk factors for IUFD. Maternal haemoglobin concentration, serum creatinine levels, and blood sugar were also found to be important factors in IUFD. While as these factors were not considered in this study, their monitoring during antenatal care in developing countries like

Nigeria may enhance pregnancy outcome.

There is no significant difference in the incidence rate of IUFD between HIV infected pregnant women and the non-infected. This underscores the relevance of quality health care delivery especially to patients who may have such disorder such as HIV infection. The HIV infected subjects recruited for this study, were undergoing the prevention of mother to child transmission (PMTCT) of

156

HIV infection scheme of the hospital. The PMTCT scheme is an intervention to ensure that no child is born with HIV and it is an essential step to ensuring an AIDS free generation. The PMTCT initiative provides drugs, counselling and psychological support to help mothers safeguard their infants against the virus. Pregnant women infected with HIV are at high risk of transmitting HIV to their infants during pregnancy, during birth or through breastfeeding. Over 90% of new HIV infections among infants and young children occur through Mother to Child Transmission. Without any intervention, the risk of transmission of infection from the mother to the baby is 20-45 per cent.

With an evidence-based set of comprehensive interventions, this transmission rate can be reduced to less than 2 percent. Intervention applied in PMTCT invariably enhances general health of the mother, as is evident in this study.

Maternal serum progesterone level in this study was significantly higher (p<0.05) in HIV seropositive subjects (59. 43±17.84ng/ml) compared to control (54.89±8.24 ng/ml). Progesterone is an essential hormone in the process of reproduction. Although the pharmacokinetics and pharmacodynamics of progesterone have been well studied, its use in the pathophysiology of pregnancy remains controversial. One of these concerns is the way in which the hormone is administered. In obstetrics the most frequent uses of progesterone are in the treatment of threatened abortion, prevention of recurrent miscarriage, or in the support of the luteal phase in assisted reproduction programmes, and in threatened preterm labour. Randomized, controlled trials showed that women who received progesterone were statistically significantly less likely to have recurrent miscarriages before 34 weeks, to have an infant with birth weight of 2.5 kg or lower, or to have an infant diagnosed with intraventricular haemorrhage.Successful maintenance of pregnancy depends on maternal tolerance of the fetal semi-allograft (Szekeres-Bartho 2002).Progesterone, cortisol and

157 prolactin have strong immunomodulatory effects leading to immunotolerance during pregnancy

(Stites et al.,1983). Progesterone has been shown to be efficacious when continuation of pregnancy is hampered by immunological factors, luteinic and neuroendocrine deficiencies and myometrial hypercontractility (Ranzo, et. al., 2005). This suggest that elevation in serum progesterone level in pregnancy is protective. Thus the HIV seropositive subjects receiving ART may enjoy further protection from their elevated serum progesterone level. This may explain the similarity of pregnancy outcome such as incidence rate of IUFD observed in the two studied groups.

Serum progesterone level appears to play a protective role in HIV infection. Research is needed to verify the level of protection relative to the stage of the infection and the level of care being received. The increased progesterone level observed in this study tends to be an adaptive mechanism against HIV during pregnancy. It has been demonstrated that increase in levels of estrogen and progesterone during pregnancy may affect intra-uterine HIV-1 injection through their effect on maternal immunocompetent cells. These hormones at their physiological concentrations (0.1 μg-0.1 ng) demonstrated significant inhibition of HIV-1 release. The results suggest that in vivo low doses of female steroids may display specific antiviral activity in monocytes (Bourinbaiar, et el.,1992).

Study by Asin et al.,(2008) revealed that in HIV-1-infected cells, estradiol and progesterone regulate

HIV-1 replication most likely by directly altering HIV-1 transcriptional activation. It further suggested that an additional indirect mechanism of sex hormone regulation of cytokine and chemokine secretion cannot be excluded. Also variations of progesterone levels during the menstrual cycle could be involved in changes in susceptibility to infection, such as that caused by the human immunodeficiency virus (HIV). Progesterone could be involved in the acquisition and development of HIV disease, regulating infection susceptibility (Cabrera-Munoz et.al., 2010). Other studies demonstrated that progesterone inhibits HIV-1 replication in placental cells by reducing TNF levels,

158 which are required for optimal viral replication (Munoz, et al., 2007). Findings by Sheffield(2009) rather indicated the upregulation of HIV-1 co-receptor expression during certain clinical settings such as in pregnancy and other high progesterone states may predispose women to HIV-1 acquisition. The pattern of weekly variation of preogesterone in HIV seropositives and HIV seronegatives were similar. Although towards term, the drop in level of progesterone tended to be more pronounced in HIV sero positive subjects than in HIV seronegatives. Also there was no statistically significant difference in the maternal progesterone levels between live birth subjects and

IUFD subjects. This shows that in well managed cases of HIV infection in pregnancy, progesterone alteration is unlikely to be implicated in adverse pregnancy outcome.

There was no significant difference in level of estriol (E3) between HIV seropositive subjects and

HIV seronegative subjects. An earlier study by Spencer (2011) among HIV seropositive women in their first and second trimesters did not show any statistical difference in serum estriol level compared to normal. Although serum estriol along with bHCG, AFP, and PAPP-A can be utilized in the diagnosis of fetal abnormality in HIV seropositive women (Yudin et al., 2003), it has been shown that protease inhibitors used in the treatment of HIV infection do not affect plasma levels of estriol in HIV seropositive pregnant women (Einstein, et al., 2004). The findings on estriol level in this present study further supports proper antiretroviral therapy in pregnancy as a way of assuring good pregnancy outcome.

X-linked adrenal hypoplasia congenita (AHC) is a rare cause of adrenocortical insufficiency. Early postnatal diagnosis may prevent severe hypoglycemia, Addisonian crises and death. Low maternal estriol (E3) levels in the second trimester of pregnancy could indicate the possibility that the fetus suffers from a disorder that causes adrenal insufficiency. Suspicion is based on the fact that E3 originates from dehydroepiandrosterone (DHEA) synthesized in the fetal adrenals. In case of adrenal

159 insufficiency, the impaired production of fetal DHEA leads to a subsequent reduction of E3 concentrations in maternal serum (Durkovic, et al., 2014)

Estriol levels were significantly lower in subjects with IUFD compared to subjects that had live birth. Decreased 2nd trimester uE3 has been shown to be a marker for Down and trisomy-18 syndromes. It also is low in cases of gross neural tube defects such as anencephaly. Based on these observations, uE3 has become a part of multiple marker prenatal biochemical screening, together with alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and inhibin-A measurements

(QUAD / Quad Screen (Second Trimester) Maternal, Serum). Low levels of uE3 also have been associated with pregnancy loss, Smith-Lemli-Opitz syndrome (defect in cholesterol biosynthesis), X- linked ichthyosis and contiguous gene syndrome (placental sulfatase deficiency disorders), aromatase deficiency, and primary or secondary fetal adrenal insufficiency, and fetal growth restriction (Bick et al., 1999; Kim et al., 2000).

Estriol increased steadily from the early weeks of gestation till term in good pregnancy outcome

(live baby). The level of increases were however higher throughout the gestational period in HIV sero negative subjects than in HIV seropositives. Towards term (at 32-34 and at 40 weeks) there were drops in estriol levels in HIV sero-positive subjects relative to HIV sero-negative pregnant subjects. Although this late variation was not sufficient to cause significant difference in pregnancy

160 outcome, it could imply that HIV seropositive subjects may have higher risk of adverse pregnancy outcome towards term than their seronegative counterparts.

There was a significant positive correlation between PAPPA-A and gestational age. HIV seropositive subjects did not have significant difference in their plasma level of pregnancy associated plasma protein-A (PAPP-A) compared to controls. Again this could possibly be the result of good antenatal care coupled with proper antiretroviral therapy. Protease inhibitors used in the treatment of HIV infection do not affect plasma levels of PAPP-A in HIV seropositive pregnant women (Einstein, et al., 2004). Thus HIV seropositive subjects receiving ART, and who do not have other complication of pregnancy, will most likely have normal PAPP-A level. Sudden drop in plasma level of this parameter in such subject could be an indication of fetal abnormality. This study also showed that PAPP-A was significantly lower in HIV subjects with low Apgar score. Subjects who have lower than normal PAPP-A at a particular GA will likely have lower than normal Apgar score.

PAPPA-A was relatively higher at various GA in subjects with IUFD compared to subjects that had live birth. As at present there is no known obstetric complication associated with elevated PAPP-A.

This implies that the incidence of IUFD in these subjects were unconnected with their plasma PAPP-

A levels. The observed elevation in PAPP-A could be in response to intrinsic fetal abnormality.

PAPP-A elevation may be associated with pregnancy disorders. In pregnancy, melanoma migration, invasiveness and progression are promoted by Pregnancy-Associated Plasma Protein-A (PAPPA),

PAPPA is widely expressed by metastatic melanoma tumors and is elevated in melanoma cells exhibiting mesenchymal, invasive and label-retaining phenotypes( Prithvirajet al., 2015).

Among the autoantibodies studied, only thyroid peroxidase autoantibody was significantly higher in

HIV infected pregnant women. There were no significant differences in levels of antinuclear

161 antibody and beta 2 glycoprotein. Hoffmann and Brown (2007) observed that abnormal thyroid function test results are common among human immunodeficiency virus (HIV)–infected patients.

Although the prevalence of overt thyroid disease does not appear to be significantly increased in

HIV-infected patients, compared with the general population, specific patterns of abnormal thyroid function test findings are more frequently identified among HIV-infected patients. Among patients with advanced acquired immunodeficiency syndrome, nonthyroidal illness (i.e., euthyroid sick syndrome) is common. During antiretroviral therapy, the prevalence of subclinical hypothyroidism, which is characterized by isolated elevated thyroid-stimulating hormone levels, and isolated low free thyroxine levelsis increased. In addition, Graves disease, which is marked by low thyroid- stimulating hormone and elevated thyroxine levels, may occur during immune reconstitution. The findings of elevated TPO in the HIV seropositive subjects could be the result of immune restoration with ART. TPO antibodies (TPOAb) appear to be involved in the tissue destructive processes associated with the hypothyroidism observed in Hashimoto's and atrophic thyroiditis. The appearance of TPOAb usually precedes the development of thyroid dysfunction. Some studies suggest that TPOAb may be cytotoxic to the thyroid. Although TPOab may not necessarily cause hypothyroidism (Takasu and Yoshimura, 2008). Thyroid dysfunction can be associated with adverse pregnancy outcome (Argatska et al., 2015). Identification and treatment of overt and subclinical hypothyroidism lead to improvedpregnancy outcomes (Ma, et al., 2015; Bryant et al., 2015). It will thus be very beneficial to conduct elaborate thyroid assessment of pregnant subjects in our locality especially those with certain known disorders such as HIV infection.

Total protein, albumin and globulin showed similar patterns in their weekly gestational variation in

HIV sero positive subjects and in HIV seronegatives. Magnesium and phosphorous also did not

162 change significantly during pregnancy. These observations are also consistent with good antenatal care of the PMCTC scheme.

5.2 Conclusion:

This study shows variations in the biochemical parameters of pregnant women. Progesterone and thyroid peroxidase autoantibody were significantly higher in HIV sero-positive subjects than in the normal. This may significantly contribute to bad pregnancy outcome. There were no significant differences in the other biochemical parameters tested between these two groups. This could indicate efficiency of the PMTCT scheme. Pregnancy outcome assessed by the incidence rate of IUFD between the HIV sero positive pregnant women receiving ART compared to HIV sero negative pregnant women showed no significant difference. The incidence rate of IUFD in the studied population is high compared to findings in other countries. There were wide range weekly variations in biochemical parameters for both groups all through gestation period possibly indicating repeated threats to pregnancy loss. Biochemical monitoring of pregnancy will help reduce incidence of bad pregnancy outcome. Estriol increased progressively all through pregnancy. Lower levels were observed towards term in IUFD. Drop in level of estriol could be an early indicator of abnormality.Weekly serum estriol and PAPP-A determinations could be good indices of assessment of foetal risk.

5.3 Recommendation

Studies involving more biochemical parameters should be done with the scope covering longer duration. There is the need to establish modern prenatal and postnatal screening centres as these are unavailable in the country at the present. Appropriate screening is useful in prevention and early

163 diagnosis of congenital abnormalities. Record keeping is poor in our centre. The improvement of this will make prospective and retrospective studies easier and more meaningful.

5.4 Sponsorship

This work was funded by Tertiary Education Trust Fund (TETFUND)

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APPENDIX

CONSENT FORM Dear Ma,

I am a PhD student of Nnamdi Azikiwe University conducting a study on pregnant women in Nnewi, Anambra State.

My project topic is Measurements of Pregnancy Associated Plasma Protein-A (Papp-A), Nutritional, Immunological, and some other Biochemical Indices in Different Stages of Pregnancy in Nnewi - Eastern Nigeria.

The essence of the study is to find out the laboratory (biochemical) basis of some problems seen in pregnancy.

Your participation is this study will be voluntary. You are not being compelled. You are also free to withdraw from the study at any time without any repercussion. Participants will not be prone to hazard and will not be paid.

The study will require 8mls of blood sample collections in the different stages of pregnancy. A questionnaire will also be provided for you to fill.

Please accept my assurance of confidentiality on information obtained in the study.

It is my expectation that the result of this study will contribute positively to pregnancy outcome in this environment.

I thus appeal for your wilful participation in this study.

May you please sign bellow to indicate your approval to participate in this study?

……………………………………………………………………………………………….

Name of Participant Signature/Thumbprint DATE

Thank you.

OKWARA, JOHN E Principal investigator (08037109857) Lecturer 11 Chemical Pathology Department NAU

For enquiries please contact Dr G. UDIGWE Consultant, Obstetrics and Gynaecology Department, NAU.

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QUESTIONNAIRE FOR A STUDY TITLED:MEASUREMENTS OF PREGNANCY ASSOCIATED PLASMA PROTEIN-A (PAPP-A), NUTRITIONAL, IMMUNOLOGICAL, AND SOME OTHER BIOCHEMICAL INDICES IN DIFFERENT STAGES OF REGNANCY IN NNEWI -EASTERN NIGERIA. 1. HOSPITAL NO:……………………………………DATE…………..………………. 2. AGE:……………………………………………………………………………………. 3. HEIGHT:……………………………………………………………………………….. 4. WEIGHT:……………………………………………………………………………… 5. LMP:…………………………………………………………………………………… 6. GESTATIONAL AGE…………………………………………………..(WKS) 7. BP:…………………………………………………………………………………… 8. RACE (A) AFRICAN (B) WHITE (C) OTHERS…………………………….. 9. ETHINICITY (A) IBO (B) YORUBA (C) HAUSA (D) OTHERS………………. 10. OCCUPATION (A) HOUSE WIFE (B) CIVIL SERVANT (C) TRADER (D) OTHERS………………………………………………………………………. 11. PRESENCE OF SYSTEMIC DISEASE (A) DIABETES (B) HYPERTENSION (C)RENAL IMPAIREMENT (D) HEART DISODER (E) OTHERS…………………. 12. PRESENCE OF SOME PREGNANCY RELATED PROBLEMS (A) ANAEMIA (B) GDM (C) PRE- ENLAMPSIA (D) OTHERS…………………………………………... 13. ALCOHOL INTAKE (A) YES (B) NO 14. FREQUENCY OF ALCOHOL INTAKE (A) OCCASSIONALY (B) REGULARLY 15. CIGARETTE SMOKING (A) YES (B) NO 16. FREQUENCY OF CIGARETTE SMOKING (A) OCCASSIONALY (B) REGULARLY 17. TOBACCO (SNUFF) USAGE (A) YES (B) NO 18. FREQUENCY OF SNUFF USAGE (A) OCCASSIONALY (B) REGULARLY 19. HOW OLD IS YOUR LAST CHILD?...... 20. HOW MANY CHILDREN HAVE YOU?...... 21. DID YOU SUFFER PREGNANCY DELAY? (A) YES (B) NO 22. IF YES, FOR HOW LONG? 23. DID YOU LOSE ANY PREGNANCY? (A) BY MISCARRIAGE (B) ABORTION 24. DID YOU KNOW THE CAUSE? 25. AT WHAT MONTH OF PREGNANCY? 26. DID YOU SEE ANY DOCTOR? (A) YES (B) NO 27. DID YOU USE TRADITIONAL HERBS? (A) YES (B) NO IN YOUR PREVIOUS PREGNANCY 28. DID YOU TAKE ROUTINE DRUGS? 29. DID YOU NOTICE LEG SWELLINGS? 30. DID YOU HAVE PROTEIN IN URINE (A)YES (B) NO (C) DON‘T KNOW 31. DID YOU HAVE SUGAR IN URINE (A)YES (B) NO (C) DON‘T KNOW 32. WERE YOU AT ANY TIME ADMITTED IN HOSPITAL? (A)YES (B) NO 33. IF YES, FOR HOW LONG?

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